U.S. patent application number 11/401670 was filed with the patent office on 2007-03-22 for acetylcholinesterase-derived peptides and uses thereof.
Invention is credited to Varda Deutsch, Amiram Eldor, Sofia Eldor, Dan Grisaru, Hermona Soreq.
Application Number | 20070065882 11/401670 |
Document ID | / |
Family ID | 26323845 |
Filed Date | 2007-03-22 |
United States Patent
Application |
20070065882 |
Kind Code |
A1 |
Soreq; Hermona ; et
al. |
March 22, 2007 |
Acetylcholinesterase-derived peptides and uses thereof
Abstract
The invention relates to a cell growth and/or differentiation
regulatory peptide comprising a sequence of about 9 to about 150
amino acids derived from acetylcholinesterase amino acid sequence,
preferably from the C-terminal region of acetylcholinesterase. The
invention also relates to pharmaceutical compositions comprising
the peptides, particularly for use in promoting survival of stem
cells, promoting differentiation of stem cells, promoting growth of
stem cells and/or promoting the growth-enhancing effect of a growth
factor on stem cells, alone, or in combination with other growth
factors. Of particular interest is the use of the peptides in the
treatment of thrombocytopenia, post-irradiation conditions,
post-chemotherapy conditions, or conditions following massive blood
loss and promotion of neural progenitors in use for cell therapies
aimed at restoring neural functions in diseased individuals.
Further, the invention relates to antibodies against the peptides,
inter alia for diagnostic use, for example, the diagnosis of
stress-induced male infertility. The invention also relates to in
vitro and in vivo methods for screening of drugs that affect the
central nervous system, and are potential modulators of
interactions between the "readthrough" form of
acetylcholinesterase, AChE-R, the intracellular receptor RACK1 and
the kinase PKC.
Inventors: |
Soreq; Hermona; (Jerusalem,
IL) ; Eldor; Amiram; (Tel Aviv, IL) ; Eldor;
Sofia; (Tel Aviv, IL) ; Deutsch; Varda;
(Jerusalem, IL) ; Grisaru; Dan; (Hertzlia,
IL) |
Correspondence
Address: |
COOPER & DUNHAM, LLP
1185 AVENUE OF THE AMERICAS
NEW YORK
NY
10036
US
|
Family ID: |
26323845 |
Appl. No.: |
11/401670 |
Filed: |
April 11, 2006 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
09998042 |
Nov 30, 2001 |
7067486 |
|
|
11401670 |
Apr 11, 2006 |
|
|
|
PCT/IL00/00311 |
May 31, 2000 |
|
|
|
09998042 |
Nov 30, 2001 |
|
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Current U.S.
Class: |
435/7.2 ;
530/388.26 |
Current CPC
Class: |
C12N 9/18 20130101; A61K
38/00 20130101; C07K 16/40 20130101; C07K 2319/00 20130101 |
Class at
Publication: |
435/007.2 ;
530/388.26 |
International
Class: |
G01N 33/567 20060101
G01N033/567; C07K 16/40 20060101 C07K016/40 |
Goverment Interests
STATEMENT AS TO FEDERALLY SPONSORED RESEARCH
[0002] This work was supported by the US Army Medical Research and
Material Command DAMD 17-99-9547 (July 1999-August 2004) and the
Defense Advance Research Project Agency DARPA N66001-01-C-8015 (May
2001-May 2004). The US Government has certain rights in this
invention.
Foreign Application Data
Date |
Code |
Application Number |
May 31, 1999 |
IL |
130224 |
Sep 2, 1999 |
IL |
131707 |
Claims
1. An antibody directed against the ARP peptide as denoted by SEQ
ID NO:1, which antibody is specific for any one of the AChE-R
variant of acetylcholinesterase and a C-terminal peptide derived
there from.
2. The antibody according to claim 2, for use in the diagnosis of
stress-induced male infertility.
3. A method for the diagnosis of stress-induced male infertility
comprising obtaining a sperm cell sample from said male; smearing
and drying said sample; contacting said dried sample with an
antibody specific for the AChE-R variant of acetylcholinesterase;
removing unbound antibody; detecting the extent of reaction between
said antibody and the AChE-R variant of acetylcholinesterase or a
fragment thereof present in said sample; determining the pattern of
expression of the AChE-R variant of acetylcholinesterase or a
fragment thereof, in said sperm cells; whereby the absence of
AChE-R from sperm heads together with intense presence in the
midpiece region indicates the presence of stress-induced male
infertility.
4. The method according to claim 3, for use in fertility
counseling.
5. A kit for the diagnosis of stress-induced male infertility,
comprising: (a) an antibody specific for the AChE-R variant; (b)
means for collecting a sperm sample; (c) slides for smearing the
sperm sample; (d) means for detecting the antibody; (e) negative
control slides.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application is a continuation-in-part (CIP) of U.S.
patent application Ser. No. 09/998,042, filed Nov. 30, 2001, which
is a continuation-in-part of PCT international application No.
PCT/IL00/00311, filed May 31, 2000, which claims priority of
Israeli Patent Application No. 130224, filed May 31, 1999 and
Israeli Patent Application No. 131707, filed Sep. 2, 1999,
incorporated herein by reference.
FIELD OF THE INVENTION
[0003] The invention is directed to the field of stem cell survival
and expansion. Specifically, the invention is directed at the stem
cell survival and expansion effects of peptides derived from
acetylcholinesterase. In addition, the invention relates to a
system for screening of nervous system drugs that are directed to
central nervous system conditions or disorders. More specifically,
the invention relates to the screening of modulators of the
AChE-R-PKC.beta.II-RACK1 complex.
BACKGROUND OF THE INVENTION
[0004] Stress insults evoke a plethora of responses in the
organism, affecting the functioning of various systems.
[0005] In the hematopoietic system, stress insults are associated
with rapid and significant changes in blood cell composition. For
example, following massive blood loss, or after surgery, the
hematopoietic system responds within hours, by an elevation of the
white blood cell and platelet counts. However, the mechanisms
responsible for initiating this adjustment are not fully
understood. Glucocorticoid hormones, known to be elevated under
stress, play a leading role in the adaptive reaction of the bone
marrow in response to stress. Glucocorticoid hormones induce
absolute increases in all hematopoietic lineages, especially
myeloid cells. This involves a cascade of events culminating in
changes in the proliferation, differentiation and apoptotic events
characteristic of each of the hematopoietic cell lineages [Lansdorp
(1995) Exp. Hematol. 23, 187-91]. Also, significant changes occur
under glucocorticoid hormones in the levels of hematopoietic growth
factors controlling the proliferation of stem cells from which
blood cells develop.
[0006] Hematopoietic stem cells (HSCs) are pluripotent, in that
they give rise to all blood cell lineages. These cells migrate
during ontogeny to settle in the bone marrow as a permanent
self-renewing source of blood cells. Under normal conditions the
vast majority of HSCs are non-dividing, but under conditions of
development or stress they can undergo clonal expansion and
self-renewal [Keller and Snodgrass (1990) J. Exp. Med. 171,
1407-18]. A large number of cytokines and growth factors, such as
stem cell factor (SCF), thrombopoietin (TPO), and FLT-3 ligand, are
thought to mediate the proliferative capacity of HSCs, through
specific receptors, c-kit, c-mpl and flt3/flk-2, respectively.
Alone, their capacity to stimulate proliferation is limited. For
example, SCF can maintain survival for a few days in vitro, but not
the self-renewal of HSCs (Li and Johnson, Blood 84, 408-14, 1994).
However, when used in combination, these growth factors acquire a
potent co-stimulatory effect. The early phase of adaptation of the
hematopoietic system to stress (first 24 hr), requires
coordinator(s), such as leu-enkephalin, which modulate the effects
of growth factors on stem cells. However, leu-enkephalin is present
in the circulation only immediately following the stress insult,
whereas the modulation of hematopoiesis continues long after that
phase. Therefore, additional long-acting modulators remain to be
identified.
[0007] The enzyme acetylcholinesterase (AChE) is expressed in brain
tissue, but also in most, if not all, of the mammalian
hematopoietic cell lineages. AChE is expressed in many parts of the
vertebrate embryo, with a developmentally regulated pattern in
specific cell types and tissues during the embryonic and adult
stages. AChE diversity is noted in several pathological states,
such as Alzheimer's disease, where AChE activity was shown to
decrease, not only in the primary site of the disease, the brain,
but also in the hematopoietic system.
[0008] It has now surprisingly been found that the C-terminal
peptides of AChE-S and AChE-R have independent biological
activities. Specifically, it has been found that these peptides
promote stem cell survival. It has also been found that these
peptides promote stem cell expansion, when used in combination with
growth factors. Further, it has been found that such peptides are
capable of augmenting hematopoiesis in vivo.
[0009] In the central nervous system (CNS), physiological stress
induces rapid and robust signaling processes in mammalian brain
neurons. These processes are known to suppress long term
potentiation (LTP) [Vereker, E. et al. (2000) J Neurosci, 20,
6811-9], augment long term depression (LTD) [Xu, L. et al. (1997)
Nature, 387, 497-500], and induce release of synaptic vesicles,
potentiating neurotransmission [Stevens, C. F. and Sullivan, J. M.
(1998) Neuron, 21, 885-93]. At the long term, stress-induced
signaling attenuates the stress response, enabling the organism to
be less excessively affected by a stressful event. This induces
neuronal dendrite branching [Sousa, N. et al. (2000) Neuroscience,
97, 253-66] and synapse re-organization [McEwen, B. S. (1999) Ann
Rev Neurosci, 22, 105-22]. However, the molecular pathway(s)
leading from short to long term processes and which enable the
adjustment to stressful stimuli, are not yet known.
[0010] Ample information suggests the involvement of specific
protein kinases in at least some of these stress-induced processes.
The enzymatic activity of certain subtypes of protein kinase C
(PKC) [Coussens, L. et al. (1986) Science, 233, 859-66] was shown
to be subject to changes (i.e. biochemical activation, membrane
translocation) under physiological [Hu, G. Y. et al. (1987) Nature,
328, 426-9], biochemical [Macek, T. A. et al. (1998) J Neurosci,
18, 6138-46] and cytoarchitectural [Tint, I. S. et al. (1992) Proc
Natl Acad Sci USA, 89, 8160-4] responses at the cellular and
organismal levels. A relevant mediator of the stress-related
changes in PKC activities is likely to be largely absent from brain
neurons under normal conditions, but should be induced rapidly and
for long periods following stress insults.
[0011] A relevant putative mediator of the stress-related changes
in PKC activities should be intracellular in its location and
capable of activating or translocating active PKC within neuronal
perikarya. The "readthrough" acetylcholinesterase variant AChE-R is
a promising candidate for this role [Soreq, H. and Seidman, S.
(2001) Nat Rev Neurosci, 2, 294-302]. Brain AChE-R is exceedingly
rare in the adult, non-stressed brain. Various stress insults
induce AChE-R overproduction through alternative splicing, creating
a different C-terminal domain from that of synaptic AChE (AChE-S).
AChE-R levels rise rapidly under acute psychological stress
[Kaufer, D. et al. (1998) Nature, 393, 373-7] or chemical
neurotoxication [Shapira, M. et al. (2000) Hum Mol Genet, 9,
1273-81] and stay elevated for over two weeks following head injury
[Shohami, E. et al. (2000) J Mol Med, 78, 228-36]. Being a
secretory protein, AChE-R fulfills the extracellular function of
reducing the stress-induced acetylcholine levels. In parallel, it
accumulates in neuronal cell bodies [Sternfeld, M. et al. (2000)
Proc Natl Acad Sci USA, 97, 8647-52], where acetylcholine
hydrolysis is unlikely. Transgenic mice overexpressing neuronal
AChE-R, but not the normally abundant synaptic variant AChE-S,
display reduced levels of stress-associated neuropathologies
[Sternfeld et al. (2000) id ibid.]. This suggests distinct
stress-related function(s) for the AChE-R protein. Intriguingly,
the unique C-terminal domain of AChE-R does not participate in
acetylcholine hydrolysis for which the core domain, common to all
of the AChE variants is sufficient [Duval, N. et al. (1992) J Cell
Biol, 118, 641-53].
[0012] Using a yeast two-hybrid screen, the inventors discovered
that the C-terminal domain of AChE-R forms a tight complex with
RACK1 [PCT/IL00/00311]. Interestingly, the inventors have shown
that PKC.beta.II is also part of this complex (FIG. 1), and all
three proteins can be co-immunoprecipitated.
[0013] In search for the marker of the transition between short and
long term processes following stress stimuli, the inventors have
demonstrated that interaction with AChE-R activates PKC.beta.II and
facilitates its translocation into densely packed neuronal clusters
(FIGS. 4 and 5), which may be causally involved with the
stress-protection capacity of overexpressed AChE-R.
[0014] In view of these unprecedented results, it this an object of
this invention to provide a method for screening of nervous system
drugs that modulate the trimeric complex AChE-R/PKC/RACK1
interactions.
SUMMARY OF THE INVENTION
[0015] This invention is directed at a cell growth and/or
differentiation regulatory peptide comprising a sequence of about 9
to about 150 amino acids derived from Acetylcholinesterase amino
acid sequence. The said sequence preferably contains a region
predicted to be rich in beta-pleated sheet structure and turns.
Also preferably, the said sequence contains a predicted amphipathic
helix structure. In another embodiment of the peptide of the
invention, the said sequence is derived from the C-terminal region
of acetylcholinesterase.
[0016] The said sequence is preferably derived from the readthrough
or synaptic variant of acetylcholinesterase, preferably from the
mature form thereof. The said sequence is preferably about 20 to
about 70 amino acids in length. More preferably, the said sequence
is SEQ ID: No. 1, SEQ ID: No. 2, SEQ ID: No. 3, SEQ ID: No.7 or SEQ
ID: No.8. Still more preferably, the said peptide is SEQ ID: No. 1,
SEQ ID: No. 2, SEQ ID: No. 3, SEQ ID: No.7 or SEQ ID: No.8.
[0017] A peptide of the invention which is a cyclic peptide is also
considered to be within the scope of the invention.
[0018] The peptide of the invention is preferably synthetic and
preferably comprises the amino acid sequence of SEQ ID: No. 1, SEQ
ID: No. 2, SEQ ID: No. 3, SEQ ID: No.7 or SEQ ID: No.8. More
preferably, the peptide has the amino acid sequence denoted by SEQ
ID: No. 1, 2, 3, 7 or 8. The peptide is preferably linear and
synthetic.
[0019] In one embodiment, the invention provides a peptide capable
of promoting cell survival and/or differentiation and comprising
the amino acid sequence denoted by SEQ ID: No. 1, SEQ ID: No. 2 or
SEQ ID: No. 3, and functional analogues and derivatives
thereof.
[0020] In another embodiment, the invention provides a peptide of
the invention which is a hematopoietic stem cell growth and/or
differentiation regulatory peptide. Preferably, said peptide is
capable of promoting stem cell survival and/or myeloid and
megakaryocytic differentiation and comprises the amino acid
sequence denoted by SEQ ID: No. 1, SEQ ID: No. 2 or SEQ ID: No. 3,
or functional analogues and derivatives thereof. Said peptide may
be either linear or cyclic, and is preferably synthetic.
[0021] In a still further embodiment, the invention provides a
peptide thereof for use in ex vivo or in vivo expansion of
hematopoietic stem cells and of neural progenitors.
[0022] The invention also provides a peptide thereof for use in ex
vivo or in vivo promotion of megakaryocytic differentiation of
hematopoietic stem cells.
[0023] The invention also relates to a pharmaceutical composition
comprising a synthetic peptide of any one of the preceding
embodiments of the invention.
[0024] The pharmaceutical composition preferably comprises a
synthetic peptide comprising the amino acid sequence of SEQ ID: No.
1, SEQ ID: No. 2, or SEQ ID: No. 3. More preferably, the
pharmaceutical composition comprises a synthetic peptide having the
amino acid sequence denoted by SEQ ID: No. 1, SEQ ID: No. 2, or SEQ
ID: No. 3. The peptide contained within the pharmaceutical
composition may be either linear or cyclic, and is preferably
synthetic.
[0025] The invention provides a pharmaceutical composition
according to the invention, for regulating hematopoietic stem cell
growth, promoting survival of stem cells, differentiation of stem
cells, promoting growth of stem cells, and/or promoting the
growth-enhancing effect a growth factor on stem cells. The growth
factor is preferably GM-CSF, SCF, TPO, EGF or bFGF.
[0026] In a preferred embodiment of the invention, the stem cells
are embryonic stem cells, nerve stem cells, epithelial stem cells,
mesenchymal stem cells, or hematopoietic stem cells.
[0027] The invention provides a pharmaceutical composition
comprising a peptide according to the invention for the treatment
of thrombocytopenia, post-irradiation condition, post-chemotherapy
condition, or condition following massive blood loss. The invention
also provides said pharmaceutical composition for use in inducing
synthesis of acetylcholinesterase mRNA and/or promoting the
formation of hematon bodies.
[0028] In a further embodiment, the invention provides an antibody
directed against a peptide of the invention. The antibody is
preferably provided for use in diagnosing elevated glucocorticoid
level; bone marrow stress, abnormality, dysfunction, or stressed
condition, or of increased platelet count or of brain infarct risk
in a mammal, or stress-induced male infertility. The antibody is
preferably directed at a peptide comprising SEQ ID: No. 1, SEQ ID:
No. 2, or SEQ ID: No. 3. More preferably, the antibody is directed
at a peptide which is selected from SEQ ID: No. 1, SEQ ID: No. 2,
or SEQ ID: No. 3.
[0029] In yet another embodiment, the invention provides a method
for the diagnosis of elevated glucocorticoid level; bone marrow
stress, abnormality, dysfunction or stressed condition, or of
increased platelet count or of brain infarct risk in a mammal,
comprising obtaining a sample from said mammal, contacting said
sample with an antibody of the invention, removing unbound
antibody, and detecting the extent of reaction between said
antibody and acetylcholinesterase or a fragment thereof present in
said sample. The said sample is preferably a serum or bone marrow
sample.
[0030] In a specifically preferred embodiment, the invention
provides a method for the diagnosis of stress-induced male
infertility comprising obtaining a sperm cell sample from said
male, smearing and drying said sperm cells, contacting said cells
with an antibody of the invention, removing unbound antibody and
detecting the extent of reaction between said antibody and
acetylcholinesterase or a fragment thereof present in said sperm
cell.
[0031] In another specifically preferred embodiment the invention
provides a method for the diagnosis of stress-induced male
infertility further comprising the step of determining the pattern
of expression of acetylcholinesterase or a fragment thereof in said
sperm cell.
[0032] In yet another embodiment, the invention provides a method
for the diagnosis of stress induced male infertility for use in
fertility counseling.
[0033] Another aspect of the invention relates to a method of
screening for a candidate drug or substance (hereinafter the "test
drug") that affects the central nervous system, wherein said test
drug is a modulator of the interaction between AChE-R/RACK1/PKC,
and which screening method comprises the steps of: [0034] a.
providing a reaction mixture comprising the AChE-R variant of AChE
or any functional fragment thereof, the cognate receptor for
activated kinase C (RACK1) and the protein kinase C .beta.II
(PKC.beta.II); [0035] b. contacting said mixture with a test drug
under suitable conditions for said interaction; and [0036] c.
determining the effect of the test drug on an end-point indication,
wherein said effect is indicative of modulation of said interaction
by the test drug.
[0037] In this screening method, the said modulator inhibits or
enhances the interaction between AChE-R/RACK1/PKC.
[0038] The reaction mixture may be a cell mixture or a cell-free
mixture, and may optionally further comprise solutions, buffers and
compounds which provide suitable conditions for interaction between
AChE-R/RACK1/PKC and the detection of an end-point indication for
said interaction. The modification of said end-point indicates
modulation of the interaction between AChE-R/RACK1/PKC by said test
drug.
[0039] In one embodiment of this screening method, the reaction
mixture is a cell-free mixture.
[0040] In this embodiment, the screening method comprises the steps
of: [0041] a. providing a cell-free mixture comprising the AChE-R
variant of AChE or any functional fragment thereof, RACK1 and
PKC.beta.II; [0042] b. contacting said mixture with the test drug
under conditions suitable for an in vitro interaction; and [0043]
c. determining the effect of the test drug on co-precipitation of
PKC.beta.II and RACK1 with the AChE-R or fragment thereof as an
end-point indication, whereby the absence or increase of said
co-precipitation indicates modulation of formation of a complex
between AChE-R/RACK1/PKC by the test drug.
[0044] The cell-free mixture may comprise any one of AChE-R variant
of AChE or any functional fragment thereof, RACK1 and PKC.beta.II,
which are provided as purified recombinant proteins or as a cell
lysate of cells expressing said proteins.
[0045] The said AChE-R variant of AChE may be a fusion protein
comprising AChE-R or functional fragment thereof and any one of GST
(Glutathion-S-Transferase) and GFP (Green Fluorescent Protein).
[0046] In another embodiment of this screening method, the said
reaction mixture is a cell mixture, particularly a transfected cell
culture, and more particularly a transfected mammalian cell
culture.
[0047] In this embodiment, the screening method comprises the steps
of: [0048] a. providing a transfected cell culture expressing the
AChE-R variant of AChE or functional fragment thereof, the cognate
receptor for activated kinase C (RACK1) and the PKC.beta.II; [0049]
b. contacting said transfected cell culture with the test drug;
[0050] c. detecting the interaction between AChE-R/RACK1/PKC in the
presence of the test drug by searching for an end-point indication,
whereby inhibition of said end-point indicates inhibition of
complex formation between AChE-R/RACK1/PKC by said test drug.
[0051] The transfected cell to be used may be transfected by:
[0052] a. an expression vector comprising a nucleotide sequence
coding for the AChE-R variant of AChE or a functional fragment
thereof; [0053] b. optionally, constructs comprising a nucleic acid
sequence coding for any one of the cognate receptor for activated
kinase C (RACK1) and the PKC.beta.II.
[0054] The end-point indication may be the sub-cellular
translocation of catalytically active PKC.beta.II, which can be
detected by a visually detectable signal.
[0055] Alternatively, the end-point indication may be
co-precipitation of PKC.beta.II and RACK1 with the AChE-R or
functional fragment thereof leading to a detectable signal, whereby
modification of said detectable signal in the presence of the test
drug indicates modulation of the formation of a complex between
AChE-R/RACK1/PKC by said test drug.
[0056] In a yet further embodiment of the screening method, the
modulator of the interaction between AChE-R/RACK1/PKC also
modulates the expression of RACK1 and/or PKC.beta.II.
[0057] A further aspect of the present invention is a method for
the in vivo screening of candidate drugs that affect the central
nervous system, wherein said drug is a modulator of an interaction
between AChE-R/RACK1/PKC, and which screening method comprises the
steps of: [0058] a. providing an AChE-R transgenic animal; [0059]
b. administering the test drug to said animal; [0060] c.
sacrificing the animal and dissecting its brain to give samples for
preparation of brain extracts or for immunohistochemistry; [0061]
d. detecting the expression of RACK1 or PKC.beta.II in said brain
samples; and [0062] e. determining the effect of the test drug on
an end-point indication, wherein said effect is indicative of the
in vivo modulation of said interaction by the test drug;
[0063] The end-point indication of this in vivo screening method is
the expression of RACK1 and PKC.beta.II in the brain, which can be
detected by a visually detectable signal.
[0064] Preferred transgenic animals are Xenopus and mammals, such
as mice, cows, goats, pigs and sheep. Most preferably, the
transgenic animal is an AChE-R transgenic mouse, which has been
described in Sternfeld et al. (2000) [Sternfeld et al. (2000) id
ibid.] herein incorporated by reference.
[0065] In one embodiment of the in vivo screening method, the RACK1
or PKC.beta.II expression can be detected by means that can detect
RNA or protein.
[0066] In one specific embodiment, the RNA detection is performed
by means appropriate for RNA detection, said means selected from
the group consisting of RT-PCR, Northern Blot, in situ
hybridization, RNAse protection and S1 nuclease analysis.
[0067] In another specific embodiment, the protein detection is
performed by means appropriate for protein detection, said means
selected from the group consisting of Western Blot and
immunohistochemistry.
[0068] The evaluation and screening methods of the invention are
suitable for assessing and screening for any test drug, e.g. test
drugs selected from protein based, carbohydrates based, lipid
based, nucleic acid based, natural organic based, synthetically
derived organic based, antibody based and metal based substances.
In preferred embodiments, the protein or antibody based substance
may products of combinatorial libraries.
[0069] The drugs to be evaluated by the methods of the invention
can be any candidate or known drugs, e.g. drugs for the treatment
of anxiety conditions, post-traumatic stress, Alzheimer's disease,
muscle malfunctioning, neurodegenerative disorders, damage
resulting from exposure to xenobiotics, panic, neuromuscular
disorders, Parkinson's disease, Huntington's chorea, muscle
fatigue, multiple chemical sensitivity, autism, multiple sclerosis
and Sjogren's disease.
[0070] In yet a further aspect, the invention provides for a method
for the treatment of stress-associated conditions or disorders, for
a subject in need of such treatment, said method comprising: [0071]
a. providing a composition comprising as active ingredient a
modulator of an interaction between AChE-R/RACK1/PKC; [0072] b.
administering a therapeutic effective amount of said composition to
said subject; wherein said modulator is selected by the drug
screening methods provided by the invention.
[0073] Lastly, the invention provides a kit for the diagnosis of
stress-induced male infertility, comprising: [0074] (a) an antibody
specific for the AChE-R variant; [0075] (b) means for collecting a
sperm sample; [0076] (c) slides for smearing the sperm sample;
[0077] (d) means for detecting the antibody; [0078] (e) negative
control slides.
[0079] All the above and other characteristics and advantages of
the invention will be further understood through the following
illustrative and non-limitative description of preferred
embodiments thereof, with reference to the appended drawings.
BRIEF DESCRIPTION OF THE FIGURES
[0080] FIG. 1A-D
[0081] FIG. 1A--Scheme of the AChE upstream gene sequence
[0082] Arrow indicates transcription start site, triangles,
conserved transcription factor binding motifs, boxes, exons 1, 5, 6
and intron 4' as indicated, white boxes, exons 2-4 and introns
2'-3- as indicated; GRE half site (hs).
[0083] FIG. 1B--Enrichment of UCB CD34.sup.+ cells
[0084] Flow cytometry of the recovered cells, using anti CD34 and
anti CD45 antibodies.
[0085] FIG. 1C--Cytochemical staining of enriched CD34.sup.+
cells
[0086] Cytochemical staining of enriched CD34.sup.+ cells for AChE
catalytic activity in the presence of inhibitors for BuChE and
AChE. The inhibitors are iso-OMPA (ISO) and BW284C51 (BW). Nuclear
staining (right) of the different forms of AChE was performed in
the presence of different concentrations of Hydrocortisone, the
AChEmRNA signal is shown as % of normal (N).
[0087] FIG. 1D--Effect of hydrocortisone on the expression of
AChEmRNA splicing variants (R, H and S) in UCB CD34.sup.+ cells
[0088] Upper panel shows cytochemical staining of enriched
CD34.sup.+ cells for AChE catalytic activity in the presence of
different concentrations of Hydrocortisone. The lower panel shows
in situ hybridization for detection of the different forms of AChE
under different concentrations of Hydrocortisone.
[0089] FIG. 2A-B: Expression of ARP in CD34 cells.
[0090] FIG. 2A--shows the expression of ARP in CD34.sup.+
hematopoietic cells as evaluated by flow cytometry in whole cord
blood.
[0091] FIG. 2B--shows the expression of ARP in CD34.sup.+
hematopoietic cells as evaluated by flow cytometry in bone marrow
from a patient with immune thrombocytopenic purpura (ITP).
[0092] Cells were fixed and permeabilized with Fix and Perm
(Caltag, Calif., US) and stained with monoclonal antibodies to CD34
conjugated to pycoerythrin indicated as FL-2 and with highly
specific rabbit anti-ARP antibodies followed by anti rabbit
antibodies conjugated to fluorescein isothiocyanate, indicated as
Fl-1. CD34.sup.+ cells were gated and 40,000 cells were analyzed
for ARP expression indicated as percentage of positive cells.
[0093] FIG. 3A-B: The spatiotemporal shifts in the intensity of
embryonic AChEmRNA transcripts through blood cell forming
tissues.
[0094] FIG. 3A--shows the analysis of blood cell-forming
organs--Top left: A sagital section of a human embryo showing the
hematopoietic organs--AGM, (aorta-gonad-mesonephros), LIV (liver),
SPL (spleen), and BM (bone marrow). Top right: Scheme of
gestational shifts in hematopoietic processes in yolk sac (YS).
[0095] FIG. 3B--shows ACHE gene expression in embryonic tissues. In
situ hybridization results and the average labeling intensities for
the AChE-S, AChE-E and AChE-R mRNA transcripts in AGM (triangles,
week 9), liver (diamonds), spleen (squares) and bone marrow
(triangles, weeks 20-25) of human fetuses at different gestational
ages (right side curves). The right side of the Figure shows
spatiotemporal changes in labeling intensity (int) for each probe
and organ as % of pixels (pi).
[0096] FIG. 4A-B: Structure of the peptides ARP and ASP, and their
effect on survival of CD34.sup.+ cells.
[0097] FIG. 4A--shows the amino acid sequence and predicted
secondary structure of the ARP and ASP peptides.
[0098] FIG. 4B--shows the effect of ARP (black bars) or ASP (gray
bars) peptide, compared to controls (white bars) on survival of
CD34.sup.+ cells, in combination with the indicated growth factors
(GF) or with no addition of growth factors--None (N), for 14 days
(d). The results are represented as Viable cells (fold of expansion
(Via.C. fexp).
[0099] FIG. 5: Effect of ARP and growth factors on transformed bone
marrow endothelial cell proliferation.
[0100] Transformed bone marrow endothelial cells were incubated in
a serum free medium (SFM) with 2nM of ARP, with or without
endothelial growth factors (bFGF 20 ng/ml and EGF 10 ng/ml), for 48
hrs. Cell proliferation was determined by the level of BrdU
incorporation measured by 5-Bromo-2'-deoxy-Uridine Labeling and
Detection Kit III. Each column shows the average value of four
wells +/- the standard error of the mean.
[0101] FIG. 6: ARP operates as an autologous inducer of ACHE gene
expression.
[0102] Left--In situ hybridization of representative CD34.sup.+
cells treated with ARP. Right--Average labeling densities of AChE
mRNA splice variants (S, E, R) versus ARP concentration (top), as
pixels (pi).times.10.sup.3.
[0103] FIGS. 7A-C: ARP induces stem cell proliferation as measured
by BrdU incorporation.
[0104] FIG. 7A--shows scheme of AChE and BuChE (BChE) genes and
AChE splice variants.
[0105] FIG. 7B--shows selective susceptibility of AChE-R mRNA in
CD34.sup.+ stem cells to AS-ODN destruction as parameter of pixels
(pi).times.10.sup.3.
[0106] FIG. 7C--shows stem cell proliferation in the presence of
the indicated antisense ODNs with GM-CSF and ARP added as indicated
for 16 hours (h) culture (cul), cell proliferation (c proli) is
shown.
[0107] FIGS. 8A-B: Redundant properties of ARP and SCF.
[0108] FIG. 8A--shows cell counts from long-term CD34.sup.+ liquid
cultures grown in the absence of growth factors (diamonds), in the
presence of early-acting cytokines (EAC: IL3, IL6, TPO and FLT3)
and SCF (squares), in the presence of EAC+ARP (triangles) or in the
presence of EAC+ARP+SCF (circles). Upper left, viable cell (Via c)
count as a parameter of fold of expansion (F exp), Upper right,
CD34+ cell count, Lower left, colony (Colo) forming unit count for
GM progenitors; Lower right, colony forming unit count for MK
progenitors.
[0109] FIG. 8B--shows representative photographs of the 28-day (D)
liquid cultures detailed in FIG. 8A. Upper left, control (Cont);
Upper right, cultures treated with EAC and SCF; Lower left,
cultures treated with EAC+SCF+ARP; Lower right, cultures treated
with EAC+ARP.
[0110] FIG. 9A-B: Migration of neurons to the perimeter of the
cortex in embryonic mouse brain.
[0111] FIG. 9A--shows labeling with anti-ARP that labels distinct
structures in embryonic cortex of mouse brain, or with anti-AChE
that labels neurons.
[0112] FIG. 9B--shows BrdU labeling in developing brain in order to
correlates the effects on mitotic activity with the treatment by
the C-terminal peptides: ARP, ASP, saline (sal), and an anti-AChE
oligodeoxynucleotide (ODN): inverse AS3 (in AS3) or AS3.
[0113] FIG. 10: Schematic illustration of the cortical plate.
[0114] FIG. 11A-B: Suppression of ARP levels by antisense
treatment.
[0115] FIG. 11A--shows immunolabeling of ARP in treated brain.
[0116] FIG. 11B--shows in situ hybridization analysis in embryonic
brain.
[0117] FIGS. 12A-C: ARP has short- and long-term hematological
effects in vivo.
[0118] FIG. 12A--shows that ARP accumulates in the serum under
stress. Top: Poinceau-stained polyacrylamide gels; Bottom:
detection of ARP and AChE (arrows) in the immunoblot by anti-ARP
antibodies in Stress (Str) or control (cont).
[0119] FIG. 12B--shows that ARP facilitates the stress
(str)-induced hematopoietic responses in vivo by showing the number
of labeled cells (C) per 100 cells counted (Co) at .times.1000
magnification in 5 different fields. Bone-marrow (BM) labeling and
white blood cell (WBC).
[0120] Asterisks in FIGS. 12A and B denote statistical significance
(p.ltoreq., 0.05, ANOVA).
[0121] FIG. 12C--shows that persistent AChE-R overproduction
increases platelet (plt) and WBC counts (Cou) in a dose-dependent
manner. The upper panel shows the AChE activity (act) as a function
of nmol ATCh hydrolysis (hyd) per min and mg protein (prot).
[0122] FIG. 13A-C: AChE and ARP in human blood plasma.
[0123] FIG. 13A--shows plasma AChE activities (p1 AChE act) under
lipopolysaccharide exposure (8 ng/kg body weight). The level of
AChE activity in all samples was determined in the presence of
10.sup.-5 M iso-OMPA and for each individual was compared to the
placebo injection performed within 10 days as percents from the pre
injection levels (p inj lev) (* denotes statistical
significance).
[0124] FIG. 13B--shows immunodetection of ARP epitopes in human
blood. Plasma prepared from the blood of one volunteer was
electrophoresed by SDS-PAGE, and the gel immunoreacted with
anti-ARP-GST antibodies. The right lanes indicate the response to a
placebo (pla) injection; the next set, the response to injection of
lipopolysaccharide (LPS) as a parameter of hours post injection (h
po inj).
[0125] FIG. 13C--shows mass spectroscopy of gel-eluted band.
[0126] FIG. 14: Injected synthetic ARP (0.1 mg/Kg) induces slow
onset of LTP measured 24 hr post-injection.
[0127] Schaffer collaterals-CA1 synaptic pathway on hippocampal
slices from an injected mice with ARP (i.p. 0.1 mg/Kg body weight)
or with P-BAN as control, were tested after LTP induction. The
changes in the slope (sl) of the post synaptic field potential was
followed for 3 hrs (indicated by Time=T in minutes).
[0128] FIG. 15A-B: Repeated confined swim stress induces testicular
AChE-R overexpression
[0129] FIG. 15A--shows biochemical stress correlates. Shown are
average values and standard evaluation of the mean for serum
corticosterone concentrations and catalytic activities (act) of
AChE in testicular homogenates from untreated control (ct) and
stressed (str) mice as parameter of ng/ml serum (ser). Stars note
statistically significant differences (Wilcoxon-Mann-Whitney,
p<0.01).
[0130] FIG. 15B--shows elevated AChE-R production. Shown are
sections of testicular tubules from untreated FVB/N mice or from
FVB/N mice subjected to 4 consecutive daily treatments of confined
swim stress. Labeling was with antibodies selective for ARP, the
C-terminal peptide unique to AChE-R (top lane), or with an AChE-R
cRNA probe detecting AChE-R mRNA transcript (lower lane).
[0131] FIG. 16A-B: Differential distribution of sperm labeling with
antibodies targeted to AChE-R or AChES.
[0132] FIG. 16A--shows a scheme of mammalian sperm displaying its
various components.
[0133] FIG. 16B--shows ARP staining in mature spermatids. Shown are
compound confocal images of the most mature sperm cells in the
central space within testicular tubules. Analyzed sections were
labeled with antibodies targeted towards ARP. ARP labeling was
performed in mice subjected to confined swim stress (str) or ARP
injection as compared to untreated mice as control (Ct) or
transgenic mice expressing the AChE-R transgene.
[0134] FIG. 17A-B: Suppressed ARP labeling of sperm heads in
subjects with unexplained couple infertility.
[0135] FIG. 17A--shows representative staining examples. Shown are
compound confocal images of anti ARP-stained sperm cells from
healthy donors (cont) or from male partners from couples with
unexplained infertility (Cou inf).
[0136] FIG. 17B--shows cumulative fractions of sperm cells with
various stained domain. Shown are average .+-.SEM values of 3
analyzed populations from healthy donors (con) (top) or infertile
couples (Cou inf) (bottom) in sperm head (H), head+midpiece (HM),
midpiece (M), or unstained (none=N). Shown is the % of stained
sperms (sp st). Note example magnified cells in corresponding
columns.
[0137] FIG. 18A-B: The two hybrid system vectors.
[0138] FIG. 18A: Shows schematic model of the yeast two-hybrid
system.
[0139] FIG. 18B: Shows a scheme of the pGBKT7 that was used to
generate a fusion protein of the GAL4 DNA-BD and the bait protein,
ARP or ASP. pGADT7 is used to express the cDNA library as a fusion
to the GAL4 DNA-AD.
[0140] FIG. 19: Amino acid homology between RACK1 and the sequence
established from the rat neonatal aorta ARP.
[0141] Amino acid homology between RACK1 and the sequence
established from the rat neonatal aorta ARP- two-hybrid positive
clone (boxed), the synthetic peptides which inhibit PKC binding to
RACK1 and are therefore homologous, at least in part, to the
binding site of PKC to RACK1.
[0142] FIG. 20: Overlay assay for AChE-R-RACKL1 interaction. RACK1
was purified from E. coli as a fusion with maltose binding protein
(MBP), released from the fusion protein by proteolytic cleavage
with factor Xa. Both cleaved and uncleaved preparations were used
for the overlay assay (ove). RACK1 samples were electrophoresed on
a 4-10% denaturing polyacrylamide gel, blotted on an NC membrane,
which was then stained with Ponceau, and striped. Thus, membrane
protein blots were subjected to various labeling experiments:
[0143] FIG. 20A--Ponceau S staining of purified RACK1 fused to
bacterial maltose binding protein (MBP-RACK1,-) or the 36 kDa RACK1
protein released by factor Xa proteolysis (+). Maltose binding
protein (MBP) served as an internal control.
[0144] FIG. 20B--Horseradish peroxidase (HRP) immunolabeled RACK1
and its MBP complex and degradation products. Anti-RACK1 labeling
either in fusion with MBP or alone, but not with MBP itself,
demonstrated binding specificity.
[0145] FIG. 20C--RACK1-AChE-R complexes labeled by overlay with a
PC12 cell homogenate overproducing recombinant AChE-R, followed by
development with antibody to AChE N-terminus.
[0146] FIG. 20D--AChE N-terminus antibody showed no signal in
membranes that were not overlaid previously with AChE-R
overproducing cell homogenate (negative control).
[0147] Abbreviations: Prot.=protease; Ovl.=overlay; Detec.
Ab.=detection antibody; Ponc.=Ponceau; Hom.=homogenates;
N-ter.=N-terminus; n.=none.
[0148] FIG. 21: Accumulation of a RACK1-immunoreactivce protein in
the mouse post-stress brain.
[0149] Homogenates from mouse hippocampus (hip) and cortex (crt)
[composed of 3 stressed (str), 4-6 and 3 control (cont), 1-3 mice]
were separated on a denaturing gel and analyzed by immunoblot with
anti-RACK1 antibody. Jurkatt cells (Jc) homogenate was separated as
well.
[0150] FIG. 22: ARP1 promotes AChE-R/RACK1/PKC.beta.II triple
complex formation in transfected COS cells.
[0151] Top: CMV-based vector encoding pGARP, a GFP fusion protein
with ARP1. Bottom: The drawing on the side represents the
experimental concept. [0152] 1. Homogenates: Shown are
immunolabeled RACK1 and PKC.beta.II (but not ARP1) in
non-transfected COS cell homogenates (-). In the presence of
transfected GARP (+), COS cells show a band in the position
correspondent to ARP. [0153] 2. Anti-GFP: Immunoprecipitation with
anti-GFP antibodies precipitates PKC.beta.II, ARP and RACK1 in
pGARP transfected but not in non-transfected COS cells.
[0154] Abbreviations: Transfec.=transfection; Detec. Ab.=detection
antibody
[0155] FIG. 23: Immunoprecipitation of AChE-R/RACK1/PKC.beta.II
complexes.
[0156] RACK1 and PKC.beta.II co-immunoprecipitate with anti-AChE
antibodies. The schematic on the left represents the experimental
concept. [0157] 1. Homogenates: PKC.beta.II and RACK1 are
immunodetected in homogenates of COS cells, which do not express
AChE, and PKC.beta.II, RACK1 and AChE are detected in PC12 cell
homogenates. [0158] 2. Anti-AChE N-terminus: Dissolved
immunoprecipitation complexes created with antibodies to the
N-terminus of AChE display no signals in COS cells, but are
positive for all three partner proteins in PC12 cells,
demonstrating AChE requirement for the creation of these
complexes.
[0159] Abbreviations: Detec. Ab.=detection antibody;
Hom.=homogenates; Nter.=N-terminus; Prot.G=protein G.
[0160] FIG. 24A-J: RACK1 and AChE-R co-overexpression in parietal
cortex and CA1 neurons under stress.
[0161] Shown are parietal cortex sections stained with cresyl
violet or with anti-RACK1 or anti-AChE-R antibodies, in lower and
higher magnifications. Note uneven labeling patterns of both
proteins in the cytoplasm and proximal processes of individual
pyramidal neurons in the parietal cortex and hippocampus CA1
(insets). Note RACK1 and AChE-R expression increases in layers 5
(arrows) of the parietal cortex under stress.
[0162] FIG. 24A: Cresyl violet staining, lower magnification.
[0163] FIG. 24B: Cresyl violet staining, higher magnification.
[0164] FIG. 24C: No stress, anti-RACK1 staining, lower
magnification.
[0165] FIG. 24D: No stress, anti AChE-R staining, lower
magnification.
[0166] FIG. 24E: Stress, anti-RACK1 staining, lower
magnification.
[0167] FIG. 24F: Stress, anti-AChE-R staining, lower
magnification.
[0168] FIG. 24C: No stress, anti-RACK1 staining, lower
magnification.
[0169] FIG. 24D: No stress, anti AChE-R staining, lower
magnification.
[0170] FIG. 24E: Stress, anti-RACK1 staining, lower
magnification.
[0171] FIG. 24F: Stress, anti-AChE-R staining, lower
magnification.
[0172] FIG. 24G: No stress, anti-RACK1 staining, higher
magnification.
[0173] FIG. 24H: No stress, anti AChE-R staining, higher
magnification.
[0174] FIG. 241: Stress, anti-RACK1 staining, higher
magnification.
[0175] FIG. 24J: Stress, anti-AChE-R staining, higher
magnification.
[0176] Abbreviations: CV=cresyl violet; N. Str.=no stress;
Str.=stress.
[0177] FIG. 25: Transgenic AChE-R overexpression intensifies
neuronal RACK1 and PKC.beta.II labeling in hippocampal CA1
neurons.
[0178] FIG. 25A--Immunoblot analysis. The immunoblot shows the
bands corresponding to PKC.beta.II, AChE-R and RACK1 following gel
electrophoresis of clear hippocampal homogenates from two FVB/N
controls and two sex and age-matched AChE-R transgenic mice (Tg).
Note the intensified staining in transgenics and the fast migrating
additional PKC.beta.II band, which could not be detected in
controls. Representative results from five reproducible
experiments.
[0179] FIG. 25B-D--Partial overlaps in neuronal AChE-R accumulation
and PKC.beta.II distributions. Shown are selected brain sections
(posterior to Bregma 0.0-0.2 mm, 1.5-1.7 mm and 2.9-3.1 mm
respectively) and the corresponding subregions where AChE-R
accumulation (triangles) or PKC.beta.II co-labeling in AChE-R
accumulating neurons (circles) were detected. Staining intensity
was low (+), medium (++) or high (+++). The corresponding
subregions are numbered as follows: 1, Cortex upper layers; 2,
Cortex lower layers; 3, striatum; 4, lateral septum; 5, piriform
cortex; 6, hippocampus CA1; 7, hippocampus CA3; 8, hippocampus
dentate gyrus; 9, basolateral amygdala; 10, central amygdala; 11,
lateral hypothalamus; 12, ventromedial hypothalamus; 13, ventral
lateral thalamus; 14, Edinger-Westphal nucleus; 15, Red nucleus;
16, Pre-tectal area.
[0180] FIG. 25E: Hippocampal immunohistochemistry. Shown are
parallel CA1 regions from representative control and AChE-R
transgenics stained with antibodies toward PKC.beta.II, AChE-R or
RACK1, as indicated. Note the intensified non-homogeneous staining
of hippocampal neurons in the brain of transgenics for both AChE-R
and RACK1, the relatively high background staining of PKC.beta.II
and the microglia (arrows) positive for AChE-R.
[0181] Abbreviations: Cont.=control; Tg.=transgenic.
[0182] FIG. 26A-C: Double-labeling highlights the AChE-R modulation
of AChE-R/RACK1/PKC.beta.II complexes.
[0183] FIG. 26A--PKC activity is increased in AChE-R transgenics
PKC activity in brain homogenates from AChE-R transgenic was
measured using the PKC assay kit (Upstate Biotechnology). Note the
elevation of PKC activity in the different brain regions in the
AChE-R transgenics as compared to FVB/N controls.
[0184] FIG. 26B-C--Shown are merged confocal micrographs from
individual upper layer parietal cortex neurons of FVB/N and AChE-R
overexpressing transgenic mice, co-immunolabeled with AChE-R/RACK1
or AChE-R/PKC.beta.II. Staining was with antibodies to AChE-R
(green) and RACK1 or PKC.beta.II (red); merged micrographs show
yellow signals for overlap staining, with orange regions reflecting
high partner levels. Note the uneven, distinct distributions of the
analyzed antigens in cortical neurons, with AChE-R labeling
demonstrating perikaryal distribution, RACK1 more mobilized toward
the perikaryal region identified in top sections (No. B5) and
PKC.beta.II highlighted in dense clusters co-localized with both
RACK1 and AChE-R (No. C11).
[0185] FIG. 26B--co-immunolabeling with AChE-R and RACK1.
[0186] FIG. 26C--co-immunolabeling with AChE-R and PKC.beta.II.
[0187] Abbreviations: Cont.=control; Mrg.=merge;
Tg.=transgenic.
DETAILED DESCRIPTION OF THE INVENTION
[0188] A number of terms as used herein are defined herein below
[0189] AChE, Acetylcholinesterase; [0190] AChE-R, "readthrough"
form of AChE [0191] AChE-S, "synaptic" form of AChE [0192] AS,
antisense; [0193] AS-ODN, antisense oligodeoxynucleotide; [0194]
ARP, acetylcholinesterase "readthrough" peptide; [0195] ASP,
acetylcholinesterase "synaptic" peptide; [0196] BFU, burst-forming
units; [0197] B, erythroid bursts; [0198] Blast or bl, blast cell
(colonies); [0199] BuChE, Butyrylcholinesterase; [0200] CFU,
colony-forming units; [0201] CSF, colony-stimulating factor; [0202]
"derived from" acetylcholinesterase, the term "derived from", when
referring to acetylcholinesterase or to equivalent terms, in the
context of this application is intended to mean amino acid sequence
corresponding to amino acid sequence of acetylcholinesterase
protein, or to predicted amino acid sequence of the open reading
frame of an acetylcholinesterase splice variant mRNA. This includes
sequences identical to acetylcholinesterase sequence, and sequences
having one or more deletions, additions, and/or substitutions,
preferably of conservative nature, i.e., changes that are not
expected to change the overall structure of the peptide. However,
any change that does not affect the function or activity of the
peptide or that increases same is contemplated to be within the
scope of the invention; [0203] EAC, early-acting cytokines, these
are preferably IL-3, IL-6, Flt3, thrombopoietin, but may also
comprise others such as soluble IL-6 receptor; [0204] GEMM,
granulocyte-erythrocyte-macrophage-megakaryocyte (colonies); [0205]
GM, granulocyte-macrophage (colonies); [0206] GST,
glutathione-S-transferase; [0207] HSC, hematopoietic stem cells;
[0208] Mix, mixed hematopoietic colonies; [0209] MK, Megakaryocyte
(colonies); [0210] ODN, oligodeoxynucleotide; [0211] ORF, open
reading frame; [0212] PKC, protein kinase C [0213] RACK1, receptor
for the activated kinase C, member of the WD40 family protein
[0214] RT, room temperature; [0215] UCB, umbilical chord blood;
[0216] UTR, untranslated terminal region; [0217] WBC, white blood
cells.
[0218] This invention relates to peptides derived from the open
reading frame of the acetylcholinesterase mRNAs. The peptides of
the invention are useful, inter alia, in upregulating AChE mRNA,
enhancing growth and/or differentiation of stem cells. In addition,
this invention relates to the trimeric complex
AChE-R-PKC.beta.II-RACK1, and to methods of screening substances
that can affect the interactions between these three molecules both
in vitro and in vivo.
[0219] A number of methods of the art of molecular biology are not
detailed herein, as they are well known to the person of skill in
the art. Such methods include site-directed mutagenesis, PCR
cloning, expression of cDNAs, analysis of recombinant proteins or
peptides, transformation of bacterial and yeast cells, transfection
of mammalian cells, and the like. Textbooks describing such methods
are e.g., Sambrook et al., Molecular Cloning A Laboratory Manual,
Cold Spring Harbor Laboratory; ISBN: 0879693096, 1989, Current
Protocols in Molecular Biology, by F. M. Ausubel, ISBN: 047150338X,
John Wiley & Sons, Inc. 1988, and Short Protocols in Molecular
Biology, by F. M. Ausubel et al. (eds.) 3rd ed. John Wiley &
Sons; ISBN: 0471137812, 1995. These publications are incorporated
herein in their entirety by reference. Furthermore, a number of
immunological techniques are not in each instance described herein
in detail, as they are well known to the person of skill in the
art. See e.g., Current Protocols in Immunology, Coligan et al.
(eds), John Wiley & Sons. Inc., New York, N.Y.
[0220] AChE pre-mRNA undergoes alternative splicing, which
generates, at the translational level, three variants of the AChE
protein [Ben Aziz Aloya et al. (1993) Proc. Natl. Acad. Sci. USA
90, 2471-5]. The best known transcript is AChE-S mRNA, formed by
splicing of exon 4 to exon 6, which yields the principal "synaptic"
isoform, found in brain and muscle [Soreq et al. (1990) Proc. Natl.
Acad. Sci. USA 87, 9688-92]. Translation of this mRNA, results in a
C-terminal extension of the common core domain of 534 amino acid
residues by a 40 residue peptide, containing the cysteine that is
involved in dimerization. The second splicing option, which
generates the erythrocytic transcript (AChE-E), is based on
splicing of exon 4 to exon 5, which encodes a different 40 residue
peptide at the C-terminus. This latter peptide is subsequently
cleaved at residue 14 (number 557 from the N-terminus) and forms a
glycophospholipid linkage that may be integrated into erythrocyte
membranes [Kerem et al. (1993) J. Biol. Chem. 268, 180-184,] and
anchor AChE to their surfaces. The third splicing option involves
continuous transcription through pseudointron I4, to yield the
E1-E2-E3-E4-I4-E5 mRNA transcript. This transcript has been
detected in embryonic and tumor cells (AChE-R, the so called
"readthrough" form), as well as in stressed brain. Translation of
AChE-R mRNA results in a 26 residue hydrophilic C-terminal
extension devoid of cysteine [Li et al. (1991) J. Biol. Chem. 266,
23083-90]. In human AChE-transgenic Xenopus tadpoles, this isoform
is secreted largely as soluble monomers, unlike the AChE-S isoform
which accumulates in synapses.
[0221] All three variants share the same 534 residue core domain.
When recombinant AChE-R and AChE-S were produced in micro- injected
Xenopus oocytes and subjected to immunoblot analysis, a certain
degree of natural C-terminal truncation occurred which created
fast-migrating bands, with a molecular weight similar to that of
the core protein, i.e. without the C-terminus, in both preparations
[Sternfeld et al. (1998a) J. Neurosci. 18, 1240-9].
[0222] It has now surprisingly been found that a peptide derived
from Acetylcholinesterase has independent biological activities.
The invention is directed to a peptide comprising sequence derived
from the open reading frame of an acetylcholinesterase mRNA splice
variant. In a preferred embodiment of the invention, the splice
variant is AChE-R or AChE-S. The peptide of the invention comprises
preferably from about 9 to about 150, preferably about 100 amino
acids, of the AChE mRNA open reading frame. More preferably, the
peptide of the invention is derived from said AChE mRNA open
reading frame.
[0223] The peptide of the invention preferably comprises a major
region predicted to be rich in turns and .beta.-pleated sheets.
Also preferably, the peptide of the invention comprises an
amphipathic helix structure, preferably unilaterally hydrophobic,
which is preferably of a length of about 10 to about 30, more
preferably abut 17 amino acid residues. Further preferably, the
peptide of the invention is of low immunogenicity. This may be
tested by injecting the peptide to an experimental animal, in the
presence of adjuvants as known in the art. A peptide of low
immunogenicity will not elicit antibodies unless conjugated or
otherwise modified. Further details of antibody generation are
disclosed herein below, e.g., in the Experimental Procedures
section.
[0224] In a preferred embodiment of the invention, the peptide
comprises AChE sequence derived from the C-terminus thereof. The
peptide sequence is preferably derived from between about 9 to
about 150, preferably about 100 amino acids from the C-terminus of
the AChE translation product. More preferably, the peptide sequence
is derived from the C-terminus, preferably from between 9 to about
100 amino acids of the C-terminus, of the mature AChE protein.
[0225] Preferably, the peptide comprises amino acid sequence
corresponding to AChE intron 4, exon 4, exon 5, and/or exon 6
sequences. More preferably, the peptide comprises intron 4 and exon
5 sequences, or exon 4 and exon 6 sequence, or exon 5 and exon 6
sequences. Thus, in a preferred embodiment of the invention, the
peptide is derived from the AChE-R or AChE-S mRNA open reading
frames. Preferably, the peptide is derived from the C-terminus of
the protein coded by said open reading frames, and more preferably,
the peptide is derived from the C-terminus of the mature protein
coded by said AChE-R or AChE-S mRNAs. In a preferred embodiment of
the invention, the peptide comprises about 50, more preferably
about 30, and most preferably about 26 amino acids of the mature
C-terminus of AChE-R or AChE-S encoded protein.
[0226] The peptide of the invention comprises more preferably
between 20-70 amino acids of the C-terminus of a mature AChE
protein, which is preferably the protein encoded by the AChE-R or
AChE-S mRNAs.
[0227] In a more preferred embodiment, the peptide comprises the
C-terminal 26 amino acids of the mature translation product of
AChE-R mRNA, which is the amino acid sequence N-terminus
[0228] GMQGPAGSGWEEGSGSPPGVTPLFSP C-terminus, also denoted herein
as SEQ ID: No. 1. A peptide having said 26 C-terminal amino acids
is denoted herein as ARP, which is the most preferred embodiment of
the invention. Further preferred embodiment is the peptide having
53 amino acids comprising of the 26 amino acids derived from the
C-terminus of the protein encoded by the AChE-R mRNA and
consecutive sequence into the core domain, also denoted herein as
SEQ ID: No. 7.
[0229] In another preferred embodiment, the peptide of the
invention comprises the C-terminus between about 30 and about 70
amino acids of the protein encoded by the AChE-S mRNA. More
preferably, the peptide comprises about 40 amino acids of the
mature C-terminus of the protein encoded by the AChE-S mRNA. The
peptide having 40 amino acids derived from the C-terminus of the
protein encoded by the AChE-S mRNA is denoted herein as ASP, or SEQ
ID: No. 2. Another preferred embodiment is the non-helical region
of SEQ ID: No. 2, i.e., the 27 C-terminal amino acids thereof, also
denoted herein as SEQ ID: No. 3. Further preferred embodiment is
the peptide having 67 amino acids comprising of the 40 amino acids
derived from the C-terminus of the protein encoded by the AChE-S
mRNA and consecutive sequence into the core domain, also denoted
herein as SEQ ID: No. 8.
[0230] The peptide of the invention may comprise sequences derived
from sources other than the above-described, so long as the
function of said peptide is not substantially affected. The
additional sequences are generally zero to 300 amino acids in
length, and may comprise sequences derived from
acetylcholinesterase, growth factors, enzymes, structural proteins,
or non-natural sequences.
[0231] Acetylcholinesterase sequences that may be added may e.g.,
be taken from the above-described exons 4, 5, and/or 6, and/or
intron 4 sequences, for the purposes of creating an internally
linked peptide dimer. Dimers may possess enhanced activity
[Corcoran et al. (1998) Eur. Cytokine Netw. 9, 255-62]. Dimer
formation of the peptide of the invention may be achieved e.g., by
inclusion of cysteines in the peptide sequence. This may be
achieved either by choosing cysteines in the natural
acetylcholinesterase-derived sequence of the peptide, or by adding
cysteine residues to the peptide sequence, or by adding to the
peptide sequence such amino acid sequences that contain cysteine
residues, preferably amino acid sequences which are known to form
dimers.
[0232] Dimerization may also be achieved by fusing the desired
peptide sequence to an IgG backbone [Corcoran et al (1998) id
ibid.].
[0233] The peptide of the invention may comprise also growth factor
sequences. This may be useful e.g., when it is desired to enhance
survival and growth of hematopoietic stem cells. The growth factors
are in that case preferably selected from early-acting factors,
such as IL3, IL6, TPO and/or FLT3. When it is desired to enhance
expansion of hematopoietic stem cells, the amino acid sequence of
growth factors such as GM-CSF or preferably SCF, may be added to
the peptide sequence.
[0234] Alternatively, the peptide of the invention may be useful
when it is desired to enhance the growth of endothelial cells. The
growth factors are in that case preferably selected from
endothelial growth factors, such as EGF and bFGF.
[0235] Where known, the active peptide of such growth factors may
be used instead of the entire growth factor sequence. Of course,
dimerization of the peptide of the invention may also enhance
growth factor activity and/or binding affinity.
[0236] In some cases, it may be desired to promote differentiation
of a cell derived from a hematopoietic source. Such differentiation
may be especially desired if said cell has acquired growth-factor
independent uncontrolled growth characteristics. The peptide of the
invention may in such cases be fused to a toxin, which upon
entering the cell, may exert cytostatic effects, in addition to the
differentiation-promoting effect of the peptide of the invention.
Fusions of peptides to toxins have been described in many
researches and review articles, see e.g. [Pastan et al. (1991)
Science 254:1173-7].
[0237] One of the effects of the peptide of the invention is
targeted to bone marrow stem cells. Therefore, when it is desired
to provide the peptide of the invention for use in treating a
patient in need of such treatment, it may be desirable to target
the peptide of the invention to the cells desired. This may be
achieved by fusing the peptide to a sequence capable of binding to
a bone marrow stem cell surface marker. One example for such a
marker is the CD34 antigen. Consequently, the peptide of the
invention may comprise CD34-ligand sequence. Alternatively, the
peptide of the invention may comprise anti-CD34 single chain
sequences. The preparation of antibodies and single-chain
antibodies is well known in the art, see also below.
[0238] When it is desired to add amino acid sequences or domains to
the peptide of the invention, it may be desired to separate the
acetylcholinesterase-derived part of the sequences from the
additional sequences by way of a linker sequence. Linker sequences
may consist mainly of amino acids that do not provide spatial
constraints, such as glycine and preferably alanine. An example for
a flexible peptide linker sequence is described in e.g., [White et
al. (1999) J. Immunol. 162, 2671-6].
[0239] The peptide of the invention comprises most preferably SEQ
ID: No. 1 or 2. However, it is to be understood that the invention
pertains to any peptide comprising sequence structurally similar to
AChE sequence with substantially equal or greater activity. Changes
in the structure of the peptide comprise one or more deletions,
additions, or substitutions. The number of deletions or additions,
which may occur at any point in the sequence, including within the
acetylcholinesterase-derived sequence, will generally be less than
25%, preferably less than 10% of the total amino acid number. This
figure does not include additions as described above, e.g.,
addition of sequences coding for growth factor sequences.
[0240] Preferred substitutions are changes that would not be
expected to alter the secondary structure of the peptide, i.e.,
conservative changes. The following list shows amino acids that may
be exchanged (left side) for the original amino acids (right side).
TABLE-US-00001 Original Residue Exemplary Substitution Ala Gly; Ser
Arg Lys Asn Gln; His Asp Glu Cys Ser Gln Asn Glu Asp Gly Ala; Pro
His Asn; Gln Ile Leu; Val Leu Ile; Val Lys Arg; Gln; Glu Met Leu;
Tyr; Ile Phe Met; Leu; Tyr Ser Thr Thr Ser Trp Tyr Tyr Trp; Phe Val
Ile; Leu
[0241] Amino acids can also be grouped according to their essential
features, such as charge, size of the side chain, and the like. The
following list shows groups of similar amino acids. Preferred
substitutions would exchange an amino acid present in one group
with an amino acid from the same group. [0242] 1. Small aliphatic,
nonpolar: Ala, Ser, Thr Pro, Gly; [0243] 2. Polar negatively
charged residues and their amides: Asp, Asn, Glu, Gln; [0244] 3.
Polar positively charged residues: His, Arg, Lys; [0245] 4. Large
aliphatic nonpolar residues: Met, Leu, Ile, Val, Cys; [0246] 5.
Large aromatic residues: Phe, Tyr, Trp.
[0247] Further comments on amino acid substitutions and protein
structure may be found in additional references [Schulz et al.
(1987) Principles of Protein Structure, Springer-Verlag, New York,
N.Y.; Creighton, T. E. (1983) Proteins: Structure and Molecular
Properties, W.H. Freeman & Co., San Francisco].
[0248] The preferred conservative amino acid substitutions as
detailed above are expected to substantially maintain or increase
the function or activity of the peptide of the invention, as
detailed herein below. Of course, any amino acid substitutions,
additions, or deletions are considered to be within the scope of
the invention where the resulting peptide is a peptide of the
invention, i.e., a peptide which is substantially equal or superior
in terms of function to the preferred peptide of the invention.
[0249] The peptide of the invention may be further modified to
improve its function, affinity, or stability. For instance,
cyclization may be used to impart greater stability and/or overall
improved performance upon the peptide. A number of different
cyclization methods have been developed, including side chain
cyclization and backbone cyclization. These methods are well
documented in the prior art [Yu et al. (1999) Bioorg. Med. Chem. 7,
161-75; Patel et al. (1999) J. Pept. Res. 53, 68-74; Valero et al.
(1999) J. Pept. Res. 53, 56-67; Romanovskis et al. (1998) J. Pept.
Res. 52, 356-74; Crozet et al. (1998) Mol. Divers. 3, 261-76;
Rivier et al. (1998) J. Med. Chem. 41, 5012-9; Panzone et al.
(1998) J. Antibiot. (Tokyo) 51, 872-9; Giblin et al. (1998) Proc.
Natl. Acad. Sci. USA 95, 12814-8; Limal et al., (1998) J. Pept.
Res. 52:121-9; U.S. Pat. No. (USP) 5,444,150]
[0250] A preferred method of cyclization involves stabilization of
an amphipathic alpha-helix by using para-substituted amino acid
derivatives of a benzene ring [Yu et al. (1999) id ibid.]. Another
preferred method of cyclization is backbone cyclization [Reissmann
et al. (1994-95) Biomed. Pept. Proteins Nucleic Acids 1:51-6, and
references therein]. A relatively new method of cyclization which
involves backbone-to side chain connections may also be used
[Reissmann et al. (1994-95) id ibid.].
[0251] Other modifications as known in the art may be carried out.
For instance, it may be desirable to link polyethyleneglycol (PEG)
groups to the peptide. Such groups may impart enhanced stability
upon the peptide. Another effect of these groups may be lowered
immunogenicity. This feature of PEG-linked peptides may be
particularly desirable when the peptide of the invention is to be
used in vivo. Preparation of PEG-linked peptides has been described
[Guerra et al. (1998) Pharm. Res. 15:1822-7].
[0252] The present invention provides AChE-derived peptides. A
therapeutic or research-associated use of these tools may
necessitate their introduction into or interaction with tissue
cultured cells or cells of a living organism. For this purpose, or
for assisting in the penetrance of such peptides into the brain
through the blood-brain barrier, it is desired to improve membrane
permeability of the peptides. The principle of derivatization with
lipophilic structures may be used in creating peptides with
enhanced membrane permeability. For instance, the sequence of a
known membranotropic peptide may be added to the sequence of the
peptide of the invention [Soukchareun et al. (1998) Bioconjug.
Chem. 9, 466-75]. Further, the peptide may be derivatized by partly
lipophilic structures such as Palmityl or Geraniol groups. For
instance, lauroyl derivatives of peptides have been described by
Muranishi et al., Pharm. Research 8, 649, 1991. Further
modifications of peptides comprise the oxidation of methionine
residues to thereby create sulfoxide groups [Zacharia et al. (1991)
Eur. J. Pharmacol. 203, p. 353]. Zacharia and coworkers also
describe peptide or derivatives wherein the relatively hydrophobic
peptide bond is replaced by its ketomethylene isoester
(COCH.sub.2). These and other modifications known to the person of
skill in the art of protein and peptide chemistry enhance membrane
permeability.
[0253] Another way of enhancing membrane permeability is the use of
receptors, such as virus receptors, on cell surfaces in order to
induce cellular uptake of the peptide or protein. This mechanism is
used frequently by viruses, which bind specifically to certain cell
surface molecules. Upon binding, the cell takes the virus up into
its interior. The cell surface molecule is called a virus receptor.
For instance, the integrin molecules CAR and AdV have been
described as virus receptors for Adenovirus [Hemmi et al. (1998)
Hum. Gene Ther. 9, 2363-73] and references therein. The CD4, GPR1,
GPR15, and STRL33 molecules have been identified as
receptors/co-receptors for HIV [Edinger et al. (1998) Virology 249,
367-78].
[0254] Thus, conjugating peptides, proteins or oligonucleotides to
molecules that are known to bind to cell surface receptors will
enhance membrane permeability of said peptides, proteins or
oligonucleotides. Examples for suitable groups for forming
conjugates are sugars, vitamins, hormones, cytokines, transferrin,
asialoglycoprotein, and the like molecules. Low et al., U.S. Pat.
No. 5,108,921, describe the use of these molecules for the purpose
of enhancing membrane permeability of peptides, proteins and
oligonucleotides, and the preparation of said conjugates. Of
course, as one type of the cells targeted by the peptide of the
invention are hematopoietic stem cells, it is advantageous to chose
a cell surface protein that will occur preferably on such cells,
such as a hematopoietic stem cell surface marker, preferably the
CD34 molecule.
[0255] Alternatively, membrane permeability may be enhanced
targeting the peptide to ubiquitous cell surface structures. Low
and coworkers (see above) teach that molecules such as folate or
biotin may be used to target a conjugate molecule to a multitude of
cells in an organism, because of the abundant and unspecific
expression of the receptors for these molecules.
[0256] The above use of cell surface proteins for enhancing
membrane permeability of a peptide of the invention may also be
used in targeting said peptide of the invention to certain cell
types or tissues. For instance, if it is desired to target cancer
cells, it is preferable to use a cell surface protein that is
expressed more abundantly on the surface of those cells. Examples
are the folate receptor, the mucin antigens MUC1, MUC2, MUC3, MUC4,
MUC5AC, MUC5B, and MUC7, the glycoprotein antigens KSA,
carcinoembryonic antigen, prostate-specific membrane antigen
(PSMA), HER-2/neu, and human chorionic gonadotropin-beta. Wang et
al. (1998) teaches the use of folate to target cancer cells [Wang
and Low (1998) J Control Release 53(1-3), 39-48] and Zhang et al.
(1998) teaches the relative abundance of each of the other antigens
noted above in various types of cancer and in normal cells [Zhang
et al. (1998) Clin. Cancer Res. 4, 2669-76]. As the peptide of the
invention preferably acts to promote differentiation of
hematopoietic stem cells, other markers may be used, as
advantageous in each particular case. The above-noted CD34 antigen,
or the CD41 and CD33 antigens, may be used as useful markers for
targeting a peptide of the invention to uncontrollably growing
cells that are derived from hematopoietic stem cells.
[0257] The protein, peptide or oligonucleotide of the invention may
therefore, using the above-described conjugation techniques, be
targeted to certain cell type as desired. For instance, if it is
desired to enhance differentiation in cells of the lymphocytic
lineage, a peptide of the invention may be targeted at such cells,
for instance, by using the MHC class II molecules that are
expressed on these cells. This may be achieved by coupling an
antibody, or the antigen-binding site thereof, directed against the
constant region of said MHC class II molecule to the protein or
peptide of the invention. Further, numerous cell surface receptors
for various cytokines and other cell communication molecules have
been described, and many of these molecules are expressed in more
or less tissue- or cell-type restricted fashion. Thus, for
instance, when it is desired to target a subgroup of T cells, the
CD4 T cell surface molecule may be used for producing the conjugate
of the invention. CD4-binding molecules are provided by the HIV
virus, whose surface antigen gp42 is capable of specifically
binding to the CD4 molecule.
[0258] The peptides of the invention may be introduced into cells
by the use of a viral vector. The use of vaccinia vector for this
purpose is detailed in Chapter 16 of the above-noted Current
Protocols in Molecular Biology. The use of Adenovirus vectors has
been described [Teoh et al. (1998) Blood 92, 4591-4601; Narumi et
al. (1998) Cell. Mol. Biol. 19, 936-941; Pederson et al. (1998) J.
Gastrointest. Surg. 2, 283-91; Guang-Lin et al. (1998) Transplant.
Proc. 30, 2923-4; Nishida et al. (1998) Spine 23, 2437-42;
Schwarzenberger et al. (1998) J. Immunol. 161, 6383-9; Cao et al.
(1998) J. Immunol. 161, 6238-44]. Retroviral transfer of antisense
sequences has been described [Daniel et al. (1998) J. Biomed. Sci.
5, 383-94].
[0259] When using viruses as vectors, the viral surface proteins
are generally used to target the virus. As many viruses, such as
the above Adenovirus, are rather unspecific in their cellular
tropism, it may be desirable to impart further specificity by using
a cell-type or tissue-specific promoter. Griscelli et al. (1998)
teach the use of the ventricle-specific cardiac myosin light chain
2 promoter for heart-specific targeting of a gene whose transfer is
mediated by Adenovirus [Griscelli et al. (1998) Hum. Gene Ther. 9,
1919-28]. The peptide of the invention is preferably targeted to
hematopoietic progenitor cells. Promoters and transcription factor
binding motifs involved in hematopoietic-specific transcription
have been described in a number of articles. It is also possible to
isolate and use in the practice of the invention a promoter of a
known gene which is specifically expressed in hematopoietic stem
cells, such as the SZF1 gene [Liu et al. (1999) Exp. Hematol. 27,
313-25], or the CD34 gene [U.S. Pat. No. 5,556,954]. Further
examples of transcription factor binding motifs and promoters
involved in hematopoietic stem cell specific transcription have
been described [Onyango et al. (1999) Exp. Hematol. 27, 313-25;
Meng et al. (1999) Blood 93, 500-8; Nony et al. (1998) J. Biol.
Chem. 273, 32910-9; Ye et al. (1998) Hum. Gene Ther. 9,
2197-205].
[0260] Isolation of 5' gene sequences and of the promoter contained
therein may be carried out by routine procedures, e.g., using
cosmid or P1 phage libraries, hybridization using cDNA sequences,
and isolation of genomic 5' gene fragments and testing same for
promoter activity, e.g., using the Chloramphenicolacetyltransferase
or luciferase genes as reporter genes. Such techniques have been
described [Ausubel et al. (eds.) Current Protocols in Molecular
Biology, John Wiley and Sons, New York; Sambrook et al., Molecular
Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, Cold
Spring Harbor; Jiang et al. (1999) J. Biol. Chem. 274, 7893-900;
Lennon et al. (1997) Immunogenetics 45, 266-73; Grombacher et al.
(1996) DNA Cell. Biol. 15, 581-8; Scherer et al. (1996) Hum. Genet.
97, 114-6; Dirks et al. (1989) J. Interferon Res. 9, 125-33].
[0261] Alternatively, or in addition, the viral vector may be
engineered to express an additional protein on its surface, or the
surface protein of the viral vector may be changed to incorporate a
desired peptide sequence. The viral vector may thus be engineered
to express one or more additional epitopes which may be used to
target said viral vector. For instance, cytokine epitopes, MHC
class II-, CD34-, CD33-, CD41-binding peptides, or epitopes derived
from homing molecules, may be used to target the viral vector in
accordance with the teaching of the invention.
[0262] The peptide of the invention possesses one or more of the
following activities: [0263] stem cell survival promoting activity;
[0264] stem cell expansion promoting activity; [0265] neural
progenitor expansion promoting activity; [0266] endothelial cell
expansion promoting activity; [0267] stem cell-derived cell
differentiation promoting activity; [0268] AChE mRNA inducing
activity in stem cells; [0269] SCF substitution activity for
hematopoietic stem cells; [0270] rescue of GM-CSF activity in
hematopoietic stem cells in the presence of anti-AChE antisense
oligonucleotides; [0271] induction of DNA synthesis activity in
GM-CSF treated hematopoietic stem cells; [0272] enhancement of
growth factor activity of GM-CSF, SCF, TPO EGF and bFGF. [0273]
interaction with the RACK1 protein, and therefore potential
involvement in intracellular signal transduction pathways.
[0274] The activity of the peptide according to the invention may
be investigated by assays involving stem cell survival and growth.
Such assays, which are known to the person of skill in the art, may
be conducted either in vivo or in vitro. The term "stem cells" as
used herein comprises stem cells of various origins and potency. It
includes the totipotent embryonic stem cells, but also nerve,
epithelial, and mesenchymal stem cells.
[0275] Preferred stem cells are embryonic stem cells, nerve stem
cells, epithelial stem cells, mesenchymal stem cells, and
hematopoietic stem cells. Particularly preferred embodiments of
assays for the activities of the peptide of the invention are given
in the Examples section herein under. It is to be understood that
the examples, which refer to hematopoietic stem cells, may be
carried out in a similar manner, following identical principles of
method and analysis, with other stem cells, as mentioned above.
[0276] Embryonic stem cells may be obtained from totipotent cells
of an embryo, according to procedures known in the art. A number of
publications describes embryonic stem cells of various origins and
their obtention [Robertson E. in: Teratocarcinoma and Embryonic
stem cells: A practical Approach, Robertson, E. (ed.), IRL Press,
Oxford, p. 71 (1987); Dushnik-Levinson and Benvenisty (1995) Biol.
Neonate 67, 77; Thomson et al. (1998) Science 282, 1145]. In vitro
aggregation of embryonic stem cells may result in the formation of
embryoid bodies. The cells of embryoid bodies are regionally
partially differentiated, including cells of mesoderm, ectoderm,
and endoderm lineages. The growth and differentiation of embryonic
stem cells, or cells derived from embryoid bodies, or the formation
of embryoid bodies, are processes that may be influenced by a
peptide of the invention. The preferred influence of a peptide of
the invention is stimulation of growth and/or differentiation of an
embryonic stem cell.
[0277] Nerve stem cells may be obtained and characterized as known
in the art [Morrison et al. (1999) Cell 96, 737-49]. Epithelial
stem cells may be obtained and characterized as known in the art
[Cotsarelis et al. (1999) Exp. Dermatol. 8, 80-8]. Mesenchymal stem
cells may be obtained and characterized as known in the art
[Pittenger et al. (1999) Science 284, 143-7; Horwitz et al. (1999)
Nat. Med. 5, 309-13; Ghilzon et al. (1998) Leuk. Lymphoma 32,
211-21; Bruder et al. (1998) Clin Orthop. 355 Suppl, S247-56].
[0278] Hematopoietic stem cells may be prepared from sources such
as bone marrow or umbilical chord blood. The procedures for
obtaining such cells have been described in a number of
publications known to those of skill in the art [U.S. Pat. Nos.
5,610,056, 5,728,581, 5,668,104, 5,199,942; and Ahmed et al. (1999)
Stem Cells 17:92-9]. Crude stem cell cultures are often
contaminated with non-pluripotent cells. It is therefore preferred
to purify these cells, using a stem cell marker. Any stem cell
marker, preferably a stem cell surface marker, as known in the art
may be used. The preferred stem cell marker is CD34. Cells are
purified using an agent capable of specifically binding the stem
cell marker. Such agents may be e.g., ligands, synthetic compounds
including peptides, or antibodies. A preferred agent is a specific
anti-stem cell marker antibody. A preferred antibody is the
anti-CD34 antibody. While antibodies may be prepared by methods
known well to those of skill in the art (see below), a preferred
anti-CD34 antibody is the CD34-PE, available from Becton Dickinson,
Immunocytometry System Inc., Mountain View, Calif., USA. Stem cell
marker enriched cells may be purified in a number of ways. For
instance, the stem cell marker carrying cells may be labeled, using
an antibody directed against the stem cell marker. The antibody is
either labeled itself or is reacted with a second, labeled
antibody. Examples of antibodies that may be used are the above
CD34-PE (Phycoerythrin-labeled), or a combination of anti-CD34
(e.g., the said CD34-PE) and FITC-RaMIg. FITC-RaMIgs are
Fluorescein-isothiocyanate-labeled rabbit anti-mouse IgG
antibodies. They should of course be used only if the first
antibody is a mouse (usually monoclonal) antibody. FITC-RaMIg or
similar labeled antibodies with the desired specificities are
available from several companies, including Sigma, St. Lois, Mo.,
USA, and PIERCE, Rockford, Ill. 61105, USA.
[0279] Using the above stem cell surface marker binding agent,
CD34.sup.+ cells may be purified in a variety of ways, including
panning, fluorescence-activated cell sorting, or by using magnetic
beads. Panning involves adsorbing cells binding a certain antibody
to a surface covered with this antibody, and thus enriching these
cells [Hoogenboom et al. (1999) Eur. J. Biochem. 260, 774-784]. The
use of magnetic beads involves magnetic beads that carry the
antibody. After the cells are bound to the bead via the antibody, a
magnetic force is applied to separate the beads from the rest of
the culture solution, thereby enriching the bound cells. Magnetic
beads are commercially available, e.g., from Dynal, Norway.
Antibodies and other agents may be bound to bead by a variety of
techniques known to the person of skill in the art, e.g., via
chemical cross-linking. Cross-linking agents and references
regarding the procedure of cross-linking are disclosed, e.g., in
the Life Science Products catalog of the above PIERCE.
[0280] Hematopoietic stem cells are then cultured as known in the
art, see e.g., Current Protocols in Immunology is published by John
Wiley & Sons. The concentration of the cells is kept initially
low, i.e., at about 50,000 to about 250,000 cells per ml. A
preferred medium is IMDM [Bruserud et al. (1999) J. Hematother. 8,
63-73]. The medium preferably comprises autologous plasma, in an
amount of between 5 and 30%, preferably 10%. Other additions to the
culture media are as known in the art, preferably 2 mM L-glutamine,
20 IU/ml Heparin, and antibiotics Penicillin, Streptomycin, and
Amphotericin B. Penicillin and Streptomycin are added to a final
concentration of preferably 5 .mu.g to 2 mg per ml, more preferably
100 .mu.g/ml. Amphotericin B is added at a final concentration of 1
to 100 .mu.M, preferably 20 .mu.M.
[0281] To the culture of hematopoietic stem cells may be added
early growth factors, including IL-3, IL-6, either alone or in
combination with soluble IL-6 receptor, Thrombopoietin, stem cell
factor, granulocyte-macrophage colony-stimulating factor, FLT-3 or
combinations thereof.
[0282] The preferred concentrations of these agents may be inferred
from the example section herein below, or alternatively, from the
above U.S. Pat. Nos. 5,610,056, 5,728,581, 5,668,104, 5,199,942,
and Ahmed et al., Stem Cells 17:92-9, 1999.
[0283] Alternatively, to the culture of endothelial cells there may
be added growth factors, including EGF and bFGF.
[0284] A peptide according to the invention is added to the stem
cell cultures, together with, or at different times than the above
growth factors. The preferred peptide concentration is about 1
ng/ml to about 1 500 ng/ml, more preferred about 50 to about 100
ng/ml.
[0285] Growth factors and the peptide according to the invention
may be supplemented preferably every 24 hours to about every ten
days, more preferably every 3-5 days. The cultures are grown for
about 14 hours to about three months, more preferably for about 24
hours to about one month.
[0286] Cultures of stem cells may then be analyzed by cytochemical
staining, by cell proliferation assay, viable cell count, cell
phenotyping, for CD34, CD33, and CD 41 markers, and growth of
progenitor colonies, using established techniques, preferably as
described herein below. Cell survival may also be evaluated by
determining dead cell counts, e.g., using apoptosis-specific
reagents. Such reagents in kit form are available from many
companies, including Clontech Laboratories UK Ltd., Hampshire RG24
8NE, UK, InterGen Energy Inc., Boston, Mass. 02114, USA, R&D
Systems, Minneapolis, Minn. 55413, USA, and Boehringer
Mannheim/Hoffmann-La Roche Ltd, 4070 Basel, Switzerland.
[0287] Stem cell growth may be quantified in a variety of ways,
such as viable cell count using trypan blue exclusion (see herein
below), or measuring DNA replication. A large number of reagents
and systems are commercially available for the purposes of
quantifying DNA replication. For instance, incorporation of the
nucleotide analog Bromo-deoxy-Uridine (BrdU) may serve as an
indicator of DNA replication (see herein below). However, also
radioactive nucleotide analogs, such as .sup.3H-Thymidine, may be
added to the culture medium and their incorporation into DNA
measured by liquid scintillation counting. Techniques for measuring
DNA replication are well known in the art and are described in many
textbooks and articles.
[0288] Peptides of the invention, when used either alone or in
combination with the above early cytokines, will promote stem cell
survival significantly. Preferably stem cell survival is promoted
by about 10%, more preferably by about 100%, and still more
preferably by about 1000 or more percent, when compared to control
cultures lacking the peptide of the invention.
[0289] It is understood by the skilled person that promotion of
stem cell survival by a peptide of the invention will usually be
highest in terms of percentage in the absence of other growth
factors. Further, the percentage of growth promotion by the peptide
of the invention depends upon the growth factor(s) used in
combination with the peptide of the invention, on the culture
conditions, and on the source and type of stem cells used for the
assay.
[0290] In another assay, the peptide of the invention may be tested
for its ability to promote stem cell differentiation. For instance,
hematopoietic stem cells expanded by growth in a medium comprising
a peptide of the invention will display increased ability to
differentiate into megakaryocytic (MK) and myeloid cells. The
differentiation of cells may be assayed from about three days in
culture to about three months, preferably from about one week in
culture to about three weeks, and more preferably at about two
weeks in culture medium comprising a peptide of the invention. The
differentiated morphology of the cells may be analyzed
microscopically, including analysis by light microscopy, electron
microscopy. Alternatively, the differentiation of stem cells may
conveniently be analyzed using cell surface differentiation
markers, such as cluster of differentiation (CD) markers. A
preferred CD marker for myeloid differentiation of hematopoietic
stem cells is the CD33 marker. A preferred marker for
megakaryocytic differentiation is the CD41 marker. Analysis of
these markers may be carried out as known in the art, e.g., using
antibodies specific for the desired marker, either labeled or in
conjunction with labeled second antibodies. The label may then be
detected by enzymatic reaction, fluorescence microscopy,
fluorescence-activated cell sorting, or the like techniques.
[0291] The activity of the peptide of the invention upon
differentiation of stem cells in culture may thus be determined by
the analysis of differentiation-associated markers. A peptide of
the invention having differentiation-promoting activity will
significantly enhance the number of cells in a culture that express
morphology of differentiated cells, or that express a
differentiation marker. The enhancement (as compared to control
cultures lacking the peptide of the invention) is preferably at
least about 30%, more preferably at least 150%, still more
preferably between at least 100 and at least 1000%.
[0292] The AChE production inducing activity of the peptide
invention may be tested by culturing stem cells or stem
cell-derived cells in the presence and in the absence of the
peptide of the invention. The amount of AChE in these cells may
then be quantified by techniques known in the art, such as
immunochemical or cytochemical staining, AChE enzyme assay, and the
like. For detection by immunochemical staining, anti-AChE
antibodies may be used; however, also AChE-specific substrates can
be employed (see herein below). The addition of AChE-specific
enzyme inhibitors such as iso-OMPA in this assay may serve as a
control reaction [Keymer et al. (1999) Eur. J. Neurosci. 11,
1049-57].
[0293] The above-detailed activities of the peptide of the
invention make it useful in the treatment of various disorders or
conditions in which growth enhancement of stem cells is desired. In
a preferred embodiment, the condition is thrombocytopenia or a
post-irradiation condition, or a chemotherapy condition, or a
condition of massive blood loss.
[0294] Thrombocytopenia is a severe complication encountered in
cancer patients treated with high dose chemotherapy followed by
autologous or allogenic transplantation of bone marrow
transplantation (BMT), peripheral blood stem cell (PBSC)
transplantation or cord blood transplantation (CBT).
[0295] While engraftment of the myeloid and erythroid lineages
occurs within 2-3 weeks, there is often a prolonged period of
profound thrombocytopenia due to insufficient platelet production.
Such patients require multiple platelet transfusions and
hospitalization and are at risk of life threatening hemorrhages for
a few weeks up to several months.
[0296] Megakaryocytopoiesis and platelet production depend on the
availability of adequate numbers of cytokine responsive progenitor
cells in the graft, while long term thrombopoiesis results from the
engraftment of multipotent stem cells. It was thought that the
recently discovered physiological regulator of thrombopoiesis,
thrombopoietin (TPO) or the truncated cloned MGDF (MK growth and
development factor) would enhance platelet recovery following
aggressive chemotherapy. Numerous studies showed that this exciting
new cytokine had potent early and late acting activities. However,
in phase II clinical trials, use of TPO before or after BMT had no
impact on the duration of severe thrombocytopenia nor did it reduce
the requirement for platelet transfusion post-transplant or in AML
patients post-chemotherapy. The lack of significant clinical
activity of TPO is probably related to the paucity of target cells,
the MK progenitors (MK-P). To complicate matters, MGDF has recently
been retracted due to immunogenicity in healthy donors who
developed antibodies and became severely thrombocytopenic.
[0297] IL-11, which has a limited positive effect on
thrombopoiesis, is currently being administered in cancer patients
to support platelet production following chemotherapy. IL-11 is not
MK lineage specific and not effective in BMT patients with
protracted thrombocytopenia. Insufficient MK precursor engraftment
and delayed platelet recovery may be due to BM stromal and
endothelial cell damage. These cells are responsible for the
production of cytokines and provide the appropriate environment for
megakaryocytopoiesis.
[0298] Currently, platelet transfusions, which have tripled in
number over the past 10 years, remain the only means to prevent
hemorrhage in severely thrombocytopenic patients. They are
extremely costly and provide only a limited mode of treatment, due
rapid allo-immunization and the development of refractoriness.
[0299] Expansion in vitro of hematopoietic stem cells and
precursors for the purpose of transplantation is a new and exciting
alternative approach to the treatment of thrombocytopenia. The
development of recombinant human hematopoietic growth factors and
sophisticated techniques for hematopoietic stem cell selection has
facilitated the use of cellular therapy by ex vivo manipulation of
hematopoietic cells before clinical use.
[0300] This novel modality, now referred to as "ex vivo expansion"
aims at the expansion of HSC and progenitors for transplantation.
Animal studies have shown shorter periods of cytopenias (including
thrombocytopenia) following lethal irradiation of animals
transplanted with BM cells that were expanded with cytokines IL-3,
IL-1 and GM-CSF or TPO, KL, IL-1a and IL-3.
[0301] The feasibility of expanding MK progenitors from BM with new
cytokine combinations and their subsequent transplantation to
shorten the period of protracted thrombocytopenia following BMT in
patients is starting to be explored. Facilitated platelet
engraftment in patients with received T cell depleted donor BM
pre-incubated with IL-3 and GM-CSF to stimulated the ex vivo
expansion of early and late MK precursors has been shown. Also the
peptides of the present invention may be used for inducing such
expansion.
[0302] Therefore, a preferred embodiment of the invention is the
use of a peptide of the invention in expansion of hematopoietic
stem cells. This may be particularly useful in medical
applications. As the amount of hematopoietic stem cells available
for purposes such as transplantation is typically very limited,
there is a need for the expansion of hematopoietic progenitor cells
for a number of clinical uses such as gene therapy, augmentation of
bone marrow transplantation (BMT) and replacement of BMT. Such
expansion may be either ex-vivo or in-vivo. U.S. Pat. No.
5,861,315, and references therein, which are incorporated herein in
their entirety by reference, describes methods for the expansion of
hematopoietic stem cells, using a combination of the cytokines IL-6
and soluble IL-6 receptor. The combination of IL-6 and soluble
IL-receptor is also said to sustain undifferentiated growth of
embryonic stem cells.
[0303] Thus, in a preferred embodiment of the invention, a peptide
of the invention is used for the ex uivo expansion of hematopoietic
stem cells. The isolation, culture, expansion, and transplantation
of such stem cell cultures has been described in many prior art
articles [U.S. Pat. No. 5,861,315; Contassot et al. (1998) Bone
Marrow Transplant. 22, 1097-102; Ahmed et al. (1999) id ibid.].
[0304] In a preferred embodiment, mononuclear cells (MNC) obtained
from human umbilical cord blood are separated by Ficoll-Hypaque
density gradient centrifugation after depletion of phagocytes with
Silica. CD34.sup.+ cells are purified as described hereinabove and
below, preferably using magnetic beads coated with anti-CD34
antibody. Purified CD34.sup.+ cells are then incubated in
suspension culture containing alpha-medium 20% fetal bovine serum
1% fraction V BSA. When cultured without serum, the culture
preferably contains 2% pure BSA, 10 .mu.g/ml insulin, 200 .mu.g/ml
transferrin, 10 .mu.M beta-mercaptoethanol, and 40 .mu.g/ml
low-density lipoprotein instead of FBS and BSA. At regular time
points, the distance of which is about three days to two weeks, the
culture is diluted with fresh medium in an about 1:1 ratio. For
assaying the number of hematopoietic progenitor cells, a sample is
removed from the culture and the cells cultured in a clonal
methylcellulose assay, as previously described [Nakahata et al.
(1982) J. Clin. Invest. 70, 1324-1328; U.S. Pat. No. 5,861,315].
The culture contained alpha-medium, 0.9% methylcellulose, 30% FBS,
1% BSA, 50 .mu.M beta-mercaptoethanol, and cytokines.
Methylcellulose cultures free of serum contain 1% BSA, 300 .mu.g/ml
human transferrin, 160 .mu.g/ml soybean lecithin, and 96 .mu.g/ml
cholesterol instead of BSA and FBS. Cytokines to be added to the
cultures include one or more of SCF, IL-1, IL-3, IL-6, soluble IL-6
receptor, IL-11, EPO, TPO, GM-CSF. Examples of preferred cytokines
or combinations thereof are: [0305] IL-3+IL-6+TPO+FLT3; [0306]
IL-6+sIL-6-R; [0307] IL-3+IL-6+sIL-6R+FLT3; [0308] IL-3+IL-11;
[0309] IL-3+IL-11+TPO; [0310] IL-1+IL-3+TPO; [0311]
SCF+IL-3+IL-6+TPO+FLT3; [0312] SCF+IL-3+IL-6+sIL-6R+FLT3; [0313]
SCF+IL-3+IL-11; [0314] SCF+IL-3+IL-11+TPO; [0315]
SCF+IL-1+IL-3+TPO; [0316] SCF+IL-6+sIL-6-R.
[0317] Examples of preferred concentrations are e.g., IL3, 5 ng/mL,
IL-6, 50 ng/mL, TPO 1 ng/mL, SCF, 10-100 ng/mL, FLT-3 ligand
(FLT3), 50 ng/mL, GM-CSF, 50 ng/mL, sIL-6R, 1280 ng/ml [U.S. Pat.
No. 5,861,315; Ahmed et al. (1999) id ibid.], see also Examples,
particularly Example 3, and "Experimental procedures" section
herein below. The peptide of the invention is added to the cultures
either concomitantly, before, or after the cytokines, preferably in
a concentration of about 50 ng/ml. However, it will be appreciated
by the person of skill in the art that the optimal concentration of
the peptide of the invention may vary according to its length,
structure, and modification, and according to the combination of
cytokines it is used with. Cytokines and the peptide of the
invention are replenished as described herein below, preferably
about every 2 to 7 days, more preferably about every 4 days. The
development of progenitors may be scored according to known
criteria [U.S. Pat. No. 5,861,315; Koike et al. (1998) Exp. Med.
168, 879-890; Tanaka et al. (1992) Blood 80, 1743-1749; Nakahata et
al. (1982) J. Clin. Invest. 70, 1324-1328]. The peptide of the
invention preferably has megakaryocyte-differentiation promoting
properties, as detailed in Example 3 herein below. The activity of
the peptide of the invention in promoting differentiation of stem
cells into megakaryocytes is particularly advantageous when ex-vivo
expanded stem cells are used for the purpose of bone marrow
transplantation, or when transplanting such cells after a bone
marrow transplantation. In these circumstances, thrombocytopenia is
a frequent and sometimes fatal occurrence [Ahmed et al. (1999) id
ibid].
[0318] In a further embodiment, the peptide of the invention can be
used for promotion of the expansion of committed neural progenitors
in vivo in a developing embryo. Furthermore, the peptide of the
invention can be used for promotion of the expansion of committed
neural progenitors ex vivo, using cultured embryonic stem cells
and/or embryoid bodies derived thereof.
[0319] The peptide of the invention may be used in free form or as
salt, e.g., as metal salt, including sodium, potassium, lithium or
calcium salt, or as a salt with an organic base, or as a salt with
a mineral acid, including sulfuric acid, hydrochloric acid or
phosphoric acid, or with an organic acid e.g., acetic acid or
maleic acid. Generally, any pharmaceutically acceptable salt of the
peptide of the invention may be used.
[0320] The peptide of the invention may be used as such or in the
form of a composition. A composition will generally contain salts,
preferably in physiological concentration, such as PBS
(phosphate-buffered saline), or sodium chloride (0.9% w/v), and a
buffering agent, such as phosphate buffer in the above PBS. The
preparation of pharmaceutical compositions is well known in the
art, see e.g., U.S. Pat. Nos. 5,736,519, 5,733,877, 5,554,378,
5,439,688, 5,418,219, 5,354,900, 5,298,246, 5,164,372, 4,900,549,
4,755,383, 4,639,435, 4,457,917, and 4,064,236. The peptide of the
present invention, or a pharmacologically acceptable salt thereof
is preferably mixed with an excipient, carrier, diluent, and
optionally, a preservative or the like pharmacologically acceptable
vehicles as known in the art, see e.g., the above US patents.
Examples of excipients include, glucose, mannitol, inositol,
sucrose, lactose, fructose, starch, corn starch, microcrystalline
cellulose, hydroxypropylcellulose, hydroxypropyl-methylcellulose,
polyvinyl-pyrrolidone and the like. Optionally, a thickener may be
added, such as a natural gum, a cellulose derivative, an acrylic or
vinyl polymer, or the like.
[0321] The pharmaceutical composition is provided in solid, liquid
or semi-solid form. A solid preparation may be prepared by blending
the above components to provide a powdery composition.
Alternatively, the pharmaceutical composition is provided as
lyophilized preparation. The liquid preparation is provided
preferably as aqueous solution, aqueous suspension, oil suspension
or microcapsule composition. A semi-solid composition is provided
preferably as hydrous or oily gel or ointment. About 0.001 to 60
w/v %, preferably about 0.05 to 25 w/v % of peptide is provided in
the composition.
[0322] A solid composition may be prepared by mixing an excipient
with a solution of the peptide of the invention, gradually adding a
small quantity of water, and kneading the mixture. After drying,
preferably in vacuo, the mixture is pulverized. A liquid
composition may be prepared by dissolving, suspending or
emulsifying the peptide of the invention in water, a buffer
solution or the like. An oil suspension may be prepared by
suspending or emulsifying the peptide of the invention or protein
in an oleaginous base, such as sesame oil, olive oil, corn oil,
soybean oil, cottonseed oil, peanut oil, lanolin, petroleum jelly,
paraffin, Isopar, silicone oil, fatty acids of 6 to 30 carbon atoms
or the corresponding glycerol or alcohol esters. Buffers include
Sorensen buffer (Ergeb. Physiol., 12, 393 1912), Clark-Lubs buffer
(J. Bact., 2, (1), 109 and 191, 1917), Macllvaine buffer (J. Biol.
Chem., 49, 183, 1921), Michaelis buffer (Die
Wasserstoffinonenkonzentration, p. 186, 1914), and Kolthoff buffer
(Biochem. Z., 179, 410, 1926).
[0323] A composition may be prepared as a hydrous gel, e.g. for
transnasal administration. A hydrous gel base is dissolved or
dispersed in aqueous solution containing a buffer, and the peptide
of the invention, and the solution warmed or cooled to give a
stable gel.
[0324] Preferably, the peptide of the invention is administered
through intravenous, intramuscular or subcutaneous administration.
Oral administration is expected to be less effective, because the
peptide may be digested before being taken up. Of course, this
consideration may apply less to a peptide of the invention which is
modified, e.g., by being cyclic peptide, by containing
non-naturally occurring amino acids, such as D-amino acids, or
other modification which enhance the resistance of the peptide to
biodegradation. Decomposition in the digestive tract may be
lessened by use of certain compositions, for instance, by confining
the peptide of the invention in microcapsules such as liposomes.
The pharmaceutical composition of the invention may also be
administered to other mucous membranes. The pharmaceutical
composition is then provided in the form of a suppository, nasal
spray or sublingual tablet. The dosage of the peptide of the
invention may depend upon the condition to be treated, the
patient's age, bodyweight, and the route of administration, and
will be determined by the attending physician. Doses ranging from
0.1 .mu.g/kg to 100 mg/kg, preferably from 0.5 .mu.g/kg to 5 mg/kg,
more preferably 0.1 .mu.g/kg to 1 mg/kg, most preferably about 100
.mu.g/kg.
[0325] The uptake of a peptide of the invention may be facilitated
by a number of methods. For instance, a non-toxic derivative of the
cholera toxin B subunit, or of the structurally related subunit B
of the heal-labile enterotoxin of enterotoxic Escherichia coli may
be added to the composition, see U.S. Pat. No. 5,554,378.
[0326] In another embodiment, the peptide of the invention is
provided in a pharmaceutical composition comprising a biodegradable
polymer selected from poly-1,4-butylene succinate,
poly-2,3-butylene succinate, poly-1,4-butylene fumarate and
poly-2,3-butylene succinate, incorporating the peptide of the
invention as the pamoate, tannate, stearate or palmitate thereof.
Such compositions are described e.g., in U.S. Pat. No.
5,439,688.
[0327] In another embodiment, a composition of the invention is a
fat emulsion. The fat emulsion may be prepared by adding to a fat
or oil about 0.1-2.4 w/w of emulsifier such as a phospholipid, an
emulsifying aid, a stabilizer, mixing mechanically, aided by
heating and/or removing solvents, adding water and isotonic agent,
and optionally, adjusting adding the pH agent, isotonic agent. The
mixture is then homogenized. Preferably, such fat emulsions contain
an electric charge adjusting agent, such as acidic phospholipids,
fatty acids, bilic acids, and salts thereof Acidic phospholipids
include phosphatidylserine, phosphatidylglycerol,
phosphatidylinositol, and phosphatidic acid. Bilic acids include
deoxycholic acid, and taurocholic acid. The preparation of such
pharmaceutical compositions is described in U.S. Pat. No.
5,733,877.
[0328] The invention also comprises anti-AChE antibodies and the
use thereof for the diagnosis of pathological conditions,
particularly hematopoietic pathological conditions.
[0329] The antibodies of the invention may also be used for
diagnosis of a stress-induced male infertility.
[0330] Polyclonal antibodies may be generated in rabbits, chicken,
mice, rats, sheep, or similar mammals. For generation of antibodies
against a peptide of the invention, the peptide is produced by
recombinant DNA technology in mammalian cells, as described in the
above general references for molecular biology. Alternatively, the
peptide may be synthetically produced by organic chemistry. The
peptide may also be produced in bacterial or insect cells as
detailed in the above-noted Current Protocols in Molecular Biology,
Chapter 16.
[0331] The peptide is purified from the cells in which it has been
produced. Peptide purification methods are known to the person of
skill in the art and are detailed e.g., in the above-noted Current
Protocols in Molecular Biology, Chapter 16, and in Current
Protocols in Protein Science, Wiley and Sons Inc. Chapters 5 and 6.
Advantageously, the peptide may be produced as a fusion with a
second protein, such as Glutathione-S-transferase or the like, or a
sequence tag, such as the histidine tag sequence. The use of fusion
or tagged proteins simplifies the purification procedure, as
detailed in the above-noted Current Protocols in Molecular Biology,
Chapter 16, and in the instructions for the His-tag protein
expression and purification kit, as available from Qiagen GmbH,
40724 Hilden, Germany.
[0332] If the protein or peptide has been expressed as a fusion
protein, it may be desirable to cleave the fusion partner before
using the protein for the generation of antibodies, in order to
avoid generation of antibodies against the fusion partner. The
cleavage of fusion partners and the isolation of the desired
protein are described in the above-noted Current Protocols in
molecular Biology, Chapter 16. Vectors, protocols and reagents for
expressing and purifying maltose-binding protein fused recombinant
proteins are also available commercially.
[0333] When producing a peptide of the invention, it may be
desirable not to remove the fusion partner, as the fusion protein
may stimulate the production of antibodies against the peptide.
Generally, this consideration may be relevant when generating
antibodies from peptides that are less than 50 amino acids in
length. In particular, it has been found that the ARP peptide, when
injected, is virtually non-immunogenic. A Keyhole Limpet hemocyanin
(KLH)-conjugated ARP peptide was found to elicit antibodies unable
to detect ARP or acetylcholinesterase. Antibodies capable of
detecting ARP were successfully generated using a
Glutathione-S-transferase-ARP fusion protein (detailed herein
below). Accordingly, in a preferred embodiment of the invention,
antibodies are elicited using a conjugate or fusion protein of the
peptide of the invention as antigen. A preferred fusion partner is
Glutathione-S-transferase.
[0334] As noted further above, the peptide may also be synthesized
by chemical methods known in the art of chemistry.
[0335] The generation of polyclonal antibodies against proteins is
described in Chapter 2 of Current Protocols in Immunology, Wiley
and Sons Inc. The generation of antibodies against peptides may
necessitate some changes in protocol, because of the generally
lower antigenicity of peptides when compared to proteins. The
generation of polyclonal antibodies against peptides is described
in the above-noted Current Protocols in Immunology, Chapter 9, and
exemplified herein below.
[0336] Monoclonal antibodies may be prepared from B cells taken
from the spleen or lymph nodes of immunized animals, in particular
rats or mice, by fusion with immortalized B cells under conditions
which favor the growth of hybrid cells. For fusion of murine B
cells, the cell line Ag-8 is preferred.
[0337] The technique of generating monoclonal antibodies is
described in many articles and textbooks, such as the above-noted
Chapter 2 of Current Protocols in Immunology. Chapter 9 therein
describes the immunization, with peptides, of animals. Spleen or
lymph node cells of these animals may be used in the same way as
spleen or lymph node cells of protein- immunized animals, for the
generation of monoclonal antibodies as described in Chapter 2
therein.
[0338] The techniques used in generating monoclonal antibodies are
further described in Kohler and Milstein, Nature 256, 495-497, 1975
and in U.S. Pat. No. 4,376,110.
[0339] In the preparation of antibodies from a gene bank of human
antibodies the hypervariable regions thereof are replaced by almost
random sequences [U.S. Pat. No. 5,840,479]. This method of antibody
generation is preferred if it is difficult to immunize an animal
with a given peptide or protein. The peptide of the invention may
be poorly immunogenic, even as a conjugate. The antibodies
described in U.S. Pat. No. 5,840,479 are further preferred if it is
desired to use antibodies with a structure similar to human
antibodies, for instance, when antibodies are desired that have low
immunogenicity in humans.
[0340] Once a suitable antibody has been identified, it may be
desired to change the properties thereof. For instance, a chimeric
antibody may achieve higher yields in production. Chimeric
antibodies wherein the constant regions are replaced with constant
regions of human antibodies are further desired when it is desired
that the antibody be of low immunogenicity in humans. The
generation of chimeric antibodies has been described in a number of
publications [Cabilly et al. (1984) Proc. Natl. Acad. Sci. USA 81,
3273; Morrison et al. (1984) Proc. Natl. Acad. Sci. USA 81, 6851;
Boulianne et al. (1984) Nature 312, 643; EP 125023; EP 171496; EP
173494; EP 184187; WO 86/01533; WO 87/02671; Harlow and Lane (1988)
Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory,
Cold Spring Harbor].
[0341] The term "antibody" is also meant to include both intact
molecules as well as fragments thereof, such as, for example, Fab
and F(ab').sub.2, which are capable of binding antigen. Fab and
F(ab').sub.2 fragments lack the Fc fragment of intact antibody,
clear more rapidly from the circulation, and may have less
non-specific tissue binding than an intact antibody [Wahl et al.
(1983) J. Nucl. Med. 24, 316-325].
[0342] It will be appreciated that Fab and F(ab').sub.2 and other
fragments of the antibodies useful in the present invention may be
used for the detection and quantitation of the peptide of the
invention and of intact AChE or its isoforms, according to the
methods disclosed herein for intact antibody molecules. Such
fragments are typically produced by proteolytic cleavage, using
enzymes such as papain (to produce Fab fragments) or pepsin (to
produce F(ab')2 fragments).
[0343] An antibody is said to be "capable of binding" a molecule if
it is capable of specifically reacting with the molecule to thereby
bind the molecule to the antibody. The term "epitope" is meant to
refer to that portion of any molecule capable of being bound by an
antibody that can also be recognized by that antibody. Epitopes or
"antigenic determinants" usually consist of chemically active
surface groupings of molecules such as amino acids or sugar side
chains, and have specific three-dimensional structural
characteristics as well as specific charge characteristics.
[0344] An "antigen" is a molecule or a portion of a molecule
capable of being bound by an antibody, which is additionally
capable of inducing an animal to produce antibody capable of
binding to an epitope of that antigen. An antigen may have one or
more than one epitope. The specific reaction referred to above is
meant to indicate that the antigen will react, in a highly
selective manner, with its corresponding antibody and not with the
multitude of other antibodies which may be evoked by other
antigens.
[0345] The antibodies, including fragments of antibodies, useful in
the present invention, may be used to quantitatively or
qualitatively detect the peptide of the invention, in a sample.
This can be accomplished by immunofluorescence techniques employing
a fluorescently or color-labeled antibody (see below) coupled with
light microscopic, flow cytometric, or fluorometric detection.
[0346] The antibodies. (or fragments thereof) useful in the present
invention may be employed histologically, as in immunofluorescence
or immunoelectron microscopy, for in situ detection of a peptide of
the invention. In situ detection may be accomplished by removing a
histological specimen from a mammal, and providing the labeled
antibody of the present invention to such a specimen. The antibody
(or fragment) is preferably provided by applying or by overlaying
the labeled antibody (or fragment) to a biological sample. Through
the use of such a procedure, it is possible to determine not only
the presence of the peptide, but also its distribution on the
examined tissue. Using the present invention, those of ordinary
skill will readily perceive that any of wide variety of
histological methods (such as staining procedures) can be modified
in order to achieve such in situ detection.
[0347] Such assays for the peptide of the invention typically
comprise incubating a biological sample, such as a biological
fluid, a tissue extract, freshly harvested cells such as
lymphocytes or leukocytes, cells which have been incubated in
tissue culture, or alternatively, human sperm cells, in the
presence of a labeled antibody capable of identifying the peptide,
and detecting the antibody by any of a number of techniques well
known in the art.
[0348] The biological sample may be treated with a solid phase
support or carrier such as nitrocellulose, or other solid support
or carrier which is capable of immobilizing cells, cell particles
or soluble proteins. The support or carrier may then be washed with
suitable buffers followed by treatment with a detectably labeled
antibody in accordance with the present invention, as noted above.
The solid phase support or carrier may then be washed with the
buffer a second time to remove unbound antibody. The amount of
bound label on said solid support or carrier may then be detected
by conventional means.
[0349] By "solid phase support", "solid phase carrier", "solid
support", "solid carrier", "support" or "carrier" is intended any
support or carrier capable of binding antigen or antibodies.
Well-known supports or carriers, include glass, polystyrene,
polypropylene, polyethylene, dextran, nylon amylases, natural and
modified celluloses, polyacrylamides, and magnetite. The nature of
the carrier can be either soluble to some extent or insoluble for
the purposes of the present invention. The support material may
have virtually any possible structural configuration so long as the
coupled molecule is capable of binding to an antigen or antibody.
Thus, the support or carrier configuration may be spherical, as in
a bead, cylindrical, as in the inside surface of a test tube, or
the external surface of a rod. Alternatively, the surface may be
flat such as a sheet, test strip, etc. Preferred supports or
carriers include polystyrene beads. Those skilled in the art will
know many other suitable carriers for binding antibody or antigen,
or will be able to ascertain the same by use of routine
experimentation.
[0350] The binding activity of a given lot of antibody, of the
invention as noted above, may be determined according to well known
methods. Those skilled in the art will be able to determine
operative and optimal assay conditions for each determination by
employing routine experimentation.
[0351] Other such steps as washing, stirring, shaking, filtering
and the like may be added to the assays as is customary or
necessary for the particular situation.
[0352] One of the ways in which an antibody in accordance with the
present invention can be detectably labeled is by linking the same
to an enzyme and used in an enzyme immunoassay (EIA). This enzyme,
in turn, when later exposed to an appropriate substrate, will react
with the substrate in such a manner as to produce a chemical moiety
which can be detected, for example, by spectrophotometric,
fluorometric or by visual means. Enzymes which can be used to
detectably label the antibody include, but are not limited to,
malate dehydrogenase, staphylococcal nuclease, delta-5-steroid
isomerase, yeast alcohol dehydrogenase, alpha-glycerophosphate
dehydrogenase, triose phosphate isomerase, horseradish peroxidase,
alkaline phosphatase, asparaginase, glucose oxidase,
beta-galactosidase, ribonuclease, urease, catalase,
glucose-6-phosphate dehydrogenase, glucoamylase and
acetylcholinesterase. The detection can be accomplished by
colorimetric methods which employ a chromogenic substrate for the
enzyme. Detection may also be accomplished by visual comparison of
the extent of enzymatic reaction of a substrate in comparison with
similarly prepared standards.
[0353] Detection may be accomplished using any of a variety of
other immunoassays. For example, by radioactive labeling the
antibodies or antibody fragments, it is possible to detect receptor
tyrosine phosphatase (R-PTPase) through the use of a
radioimmunoassay (RIA). A good description of RIA may be found in
Laboratory Techniques and Biochemistry in Molecular Biology, by
Work, T. S. et al., North Holland Publishing Company, NY (1978)
with particular reference to the chapter entitled "An Introduction
to Radioimmune Assay and Related Techniques" by Chard, T.,
incorporated by reference herein. The radioactive isotope can be
detected by such means as the use of a g counter or a scintillation
counter or by autoradiography.
[0354] It is also possible to label an antibody in accordance with
the present invention with a fluorescent compound. When the
fluorescently labeled antibody is exposed to light of the proper
wavelength, its presence can be then detected due to fluorescence.
Among the most commonly used fluorescent labeling compounds are
fluorescein isothiocyanate, rhodamine, phycoerythrine, pycocyanin,
allophycocyanin, o-phthaldehyde and fluorescamine.
[0355] The antibody can also be detectably labeled using
fluorescence emitting metals such as 1.sup.52E, or others of the
lanthanide series. These metals can be attached to the antibody
using such metal chelating groups as diethylenetriamine pentaacetic
acid (ETPA).
[0356] The antibody can also be detectably labeled by coupling it
to a chemiluminescent compound. The presence of the
chemiluminescent-tagged antibody is then determined by detecting
the presence of luminescence that arises during the course of a
chemical reaction. Examples of particularly useful chemiluminescent
labeling compounds are luminol, isoluminol, theromatic acridinium
ester, imidazole, acridinium salt and oxalate ester.
[0357] Likewise, a bioluminescent compound may be used to label the
antibody of the present invention. Bioluminescence is a type of
chemiluminescence found in biological systems in which a catalytic
protein increases the efficiency of the chemiluminescent reaction.
The presence of a bioluminescent protein is determined by detecting
the presence of luminescence. Important bioluminescent compounds
for purposes of labeling are luciferin, luciferase and
aequorin.
[0358] An antibody molecule of the present invention may be adapted
for utilization in an immunometric assay, also known as a
"two-site" or "sandwich" assay. In a typical immunometric assay, a
quantity of unlabeled antibody (or fragment of antibody) is bound
to a solid support or carrier and a quantity of detectably labeled
soluble antibody is added to permit detection and/or quantitation
of the ternary complex formed between solid-phase antibody,
antigen, and labeled antibody.
[0359] Typical, and preferred, immunometric assays include
"forward" assays in which the antibody bound to the solid phase is
first contacted with the sample being tested to extract the antigen
from the sample by formation of a binary solid phase
antibody-antigen complex. After a suitable incubation period, the
solid support or carrier is washed to remove the residue of the
fluid sample, including unreacted antigen, if any, and then
contacted with the solution containing an unknown quantity of
labeled antibody (which functions as a "reporter molecule"). After
a second incubation period to permit the labeled antibody to
complex with the antigen bound to the solid support or carrier
through the unlabeled antibody, the solid support or carrier is
washed a second time to remove the unreacted labeled antibody.
[0360] In another type of "sandwich" assay, which may also be
useful with the antigens of the present invention, the so-called
"simultaneous" and "reverse" assays are used. A simultaneous assay
involves a single incubation step as the antibody bound to the
solid support or carrier and labeled antibody are both added to the
sample being tested at the same time. After the incubation is
completed, the solid support or carrier is washed to remove the
residue of fluid sample and uncomplexed labeled antibody. The
presence of labeled antibody associated with the solid support or
carrier is then determined as it would be in a conventional
"forward" sandwich assay.
[0361] In the "reverse" assay, stepwise addition first of a
solution of labeled antibody to the fluid sample followed by the
addition of unlabeled antibody bound to a solid support or carrier
after a suitable incubation period is utilized. After a second
incubation, the solid phase is washed in conventional fashion to
free it of the residue of the sample being tested and the solution
of unreacted labeled antibody. The determination of labeled
antibody associated with a solid support or carrier is then
determined as in the "simultaneous" and "forward" assays.
[0362] The present invention provides an immunoassay for the
detection and quantification of a peptide of the invention. The
creation of immunoassays, such as RIA or ELISA, has been described
in many articles, textbooks, and other publications. Reference is
made to WO 97/03998, p. 48, line 4 to p. 52, line 27. Immunoassays
of the invention may be if two general types: Firstly, immunoassays
using an immobilized peptide of the invention, may be used.
Secondly, immunoassays using immobilized antibodies directed
against an epitope of a peptide of the invention may be used to
quantify a peptide of the invention.
[0363] In a preferred embodiment of the invention, the assay is an
immunoblot assay. The sample, e.g., a serum sample, is diluted,
e.g., 1:10, in order to avoid overloading. The sample is then
loaded onto a polyacrylamide gel, optionally a gradient gel, and
electrophoresed. Synthetic or recombinantly produced peptide of the
invention, preferably SEQ ID: No. 1, SEQ ID: No. 2, or SEQ ID: No.
3, may be added in separate lanes or spiked to the sample lanes, as
positive controls. The gel is then blotted, preferably onto a
Nitrocellulose or Nylon membrane. The blot is reacted with
antibodies against the peptide of the invention, preferably
antibodies reactive with SEQ ID: No. 1, 2 or 3. A more preferred
antibody is the rabbit anti-GST-ARP antibody as described herein.
Bound antibody may then be detected by antibodies reactive with the
antibody of the invention, e.g., anti-rabbit immunoglobulins. These
immunoglobulins are preferably labeled, e.g., by Peroxidase
conjugation. The detection of the label is then carried out
according to methods known in then art. Preferably,
peroxidase-conjugated immunoglobulins are detected using the
ECL.TM. detection system (Amersham Pharmacia Biotech, UK).
[0364] As described above, a preferred sample is serum. However,
other body fluids may be used, including cerebrospinal fluid,
liquor, saliva, and the like. Another specifically preferred sample
is sperm cells. Also, liquid extracts of body tissue may be
analyzed. Alternatively, body tissue may be analyzed without
extraction using cytochemical staining or immunostaining as
described herein.
[0365] A preferred body fluid is cerebrospinal fluid. For instance,
increased levels of AChE or of a peptide of the invention in
cerebrospinal fluid, may be indicative of elevated blood cortisol
levels, and may further be indicative of stress.
[0366] Such assays as hereinabove described may find use in
diagnostics, as the level of the peptide of the invention may need
to be evaluated in a number of conditions. For instance, such
assays may be useful in order to monitor the effect of treatment of
a patient with a peptide of the invention. Furthermore, such assays
may be used in determining psychological stress (see Example 8
herein below). Still further, such assays may be useful as an
indicator of stress to the bone marrow. Stressed bone marrow will
up-regulate ARP, which is a peptide of the invention, as detailed
herein under in the Examples section. Finally, such assays may be
useful in the diagnosis of a number of disorders where growth or
expansion of hematopoietic stem cells is adversely effected, or
alternatively, uncontrolled growth of hematopoietic stem cells
occurs.
[0367] Thus, in a preferred embodiment, the invention provides a
method for the diagnosis of elevated glucocorticoid level, bone
marrow stress, abnormality, dysfunction or stressed condition, or
of increased platelet count or of brain infarct risk in a mammal,
comprising obtaining a sample from said mammal, contacting said
sample with an antibody of the invention, removing unbound
antibody, and detecting the extent of reaction between said
antibody and acetylcholinesterase or a fragment thereof present in
said sample. The sample is preferably serum or a bone marrow
sample.
[0368] An additional aspect of the present invention relates to a
method for diagnosis of stress induced male infertility.
[0369] As demonstrated in Example 10, elevated serum corticosterone
levels and spermatid overexpression of AChE-R were observed in
FVB/N mice subjected to acute psychological stress or injected with
synthetic ARP of the present invention. Reduced seminal gland
weight and lower sperm counts and motility were observed in AChE-R
transgenic mice with massive AChE-R excess as compared with matched
controls. ARP immunostaining labeled mature mouse sperm in stressed
and ARP injected mice but meiotic pachytene spermatocytes in AChE-R
transgenics.
[0370] Most interestingly, in humans, ARP labeling covered sperm
head and midpiece in controls but was concentrated at the midpiece
in couple infertility specimens. Excess AChE-R and its C-terminal
peptide ARP may thus suppress male fertility through both
autonomous system regulation and direct sperm interactions.
[0371] The intensified midpiece labeling of sperm cells from
subjects with unexplained couple infertility emphasizes the
physiological relevance of the animal models of the invention.
Although stress in such patients may be treatment-induced
[Negro-Vilar et al. (1993) Enviromental Health Prespectives
Supplements 101(Suppl 2), 59-64], its effects may nevertheless be
significant. As exposure to anticholinesterases induces similar
AChE-R accumulation [Kaufer et al. (1998) id ibid], the findings of
the present invention may also explain the impaired sperm
properties and resultant male infertility that are associated with
exposure to agricultural insecticides [Rivier et al. (1986) Science
231, 607-609]. Moreover, the presumed co-localization with sperm
mitochondria may explain at least part of the impairments observed
in spermatogenesis and sperm motility through interference with
mitochondrial metabolism. Several potential means of preventing
such effects and improving fertility include transcriptional
suppression of stress-induced AChE overexpression, prevention of
the stress-associated shift in alternative splicing from AChE-S to
AChE-R (Kaufer et al. (1998) id ibid.) or antisense destruction of
the vulnerable AChE-R mRNA transcript [Grisaru et al. (1999) ibid;
Shohami et al. (2000) id ibid.]. AChE-R and ARP thus present
previously unrecognized targets for studying, analyzing and
treating stress-induced male infertility.
[0372] Thus, in a preferred embodiment the invention provides a
method for the diagnosis of stress induced male infertility,
comprising obtaining a sperm cell sample from said male, smearing
and air drying said sperm cell and contacting said sperm cell with
the antibody of the invention. Preferably, using the
immunohystochemical staining as described above.
[0373] Additionally, the invention provides a method for the
diagnosis of stress induced male infertility, further comprising
the step of determining the pattern of the AChE-R expression in
said sperm cell.
[0374] In yet another embodiment, the invention provides a method
for the diagnosis of stress induced male infertility for use in
fertility counseling.
[0375] Using the non-biased yeast two-hybrid screening approach,
the inventors found two potential links explaining the function of
intracellular, specially intraneural, AChE-R (see Example 13).
Tight, co-immunoprecipitable and naturally co-localized complexes
of AChE-R appeared to connect the C-terminal domain ARP1 with
PKC.beta.II and its intracellular shuttling protein RACK1 (Example
14). Intensified labeling and neuronal mobilization of both RACK1
and PKC.beta.II was observed in the stress-protected transgenic
mice constitutively overexpressing AChE-R (FIG. 21). Transgenic
AChE-R overexpression intensified PKC.beta.II clustering in
pyramidal neurons (FIG. 25) and translocated RACK1 to the
perikaryal circumference (FIG. 26), suggesting functional relevance
for AChE-R overexpression in the stress-suppressing cascades of
neuronal signal transduction.
[0376] A variety of RACK1 clones were identified with the capacity
to induce intense .beta.-gal staining in the yeast cell context,
strengthening the notion that AChE-R/RACK1 interactions are both
common and tight in the embryonic brain from which the screened
library originated (Example 13 and data not shown). The cell
biology tests point at neuronal accumulation and/or subcellular
mobilization as the outcome of these interactions under in vivo
stress or transgenic overexpression. The RACK1 region which was
found to be essential for AChE-R interaction consists of two
anti-parallel four strand "blades" which together cover ca. 30% of
the of the RACK1 perimeter. The large number of conserved WD domain
residues in this region of the protein may highlight the
requirement for correct blade folding of RACK1 as essential for
ARP1 interactions. The AChE-R-induced changes in the intensity of
RACK1/PKC.beta.II interactions, may also be physiologically
relevant.
[0377] In addition to its primary function of acetylcholine
hydrolysis, AChE was shown to initiate adhesive cell-cell
interactions through its core domain and promote mammalian neurite
extension in a manner similar to that of its non-enzyme membrane
protein homolog neuroligin [reviewed by Soreq and Seidman, (2001)
id ibid.]. Nevertheless, the neuritogenic activities of distinct
AChE variants appeared to depend on their unique C-termini, which
to date were not found to share sequence homologies with other
proteins. In hematopoietic cells, the 26 C-terminal residues of the
stress-induced AChE-R protein exert proliferative and growth factor
activities, as described in Examples 4, 7 and 8 of the present
application, and also in [Grisaru et al. (2001) Molecular Medicine,
7, 93-105]. However, it was not yet known whether these activities
depend on extra- or intracellular interactions. Also, neither the
molecular mechanism(s) nor the nervous system relevance of such
activities had yet been explored. The present invention
additionally reveals that, at least part of the C-terminus specific
non-catalytic effects of AChE-R, are intracellular and PKC.beta.II
mediated.
[0378] As described in Examples 16, 17 and 19,
co-immunoprecipitable and naturally co-localized complexes of
AChE-R appeared to connect the C-terminal domain ARP1 with
PKC.beta.II and its intracellular shuttling protein RACK1.
Intensified labeling and neuronal mobilization of both RACK1 and
PKC.beta.II was observed in the stress-protected transgenic mice
constitutively overexpressing AChE-R. Transgenic AChE-R
overexpression translocated RACK1 to the perikaryal circumference
and intensified PKC.beta.II clustering in pyramidal neurons, and
activated PKC suggesting functional relevance for AChE-R
overexpression in the stress-suppressing cascades of neuronal
signal transduction.
[0379] As shown by Examples 16 and 17, AChE-R interaction with
PKC.beta.II is indirect and is mediated by the PKC.beta.II
shuttling protein RACK1. RACK1 is a member of the WD family of
proteins. WD proteins can simultaneously bind different partners to
various regions in their multi-blade rings [Smith et al. (1999) id
ibid.], which provides flexibility and combinatorial diversity.
Thus, RACK1 is a scaffold for cell-cell interaction proteins such
as .beta.-integrin [Liliental and Chang (1998) J Biol Chem, 273,
2379-831, members of signaling cascades like cAMP phosphodiesterase
[Yarwood et al. (1999) J Biol Chem, 274, 14909-17], C2-containing
proteins such as phospholipase C-.gamma.l [Disatnik et al. (1994)
Proc Natl Acad Sci USA, 91, 559-63], src kinase [Chang et al.
(1998) Mol Cell Biol, 18, 3245-56] and/or PH domain-containing
proteins such as the .beta.-adrenergic receptor [Rodriguez et al.
(1999) Biochemistry, 38, 13787-94]. Interaction between RACK1 and
AChE-R would likely compete with other associations, changing the
subcellular balance between these variable complexes.
Alternatively, or in addition, AChE-R/RACK1 interactions may
promote the formation of additional triple complexes with other
proteins. Diverse links between AChE and other signaling molecules
may, in turn, explain its capacity to exert various non-catalytic
intracellular functions, a possibility which awaits further
investigation.
[0380] PKC.beta.II and RACK1 are readily available in neurons,
albeit in inactive and possibly, non-associated or, loosely
attached compositions. The present inventors have now found that
stress responses and the subsequent accumulation of AChE-R [Kaufer
et al. (1998) id ibid.] facilitate the formation of triple, tightly
bound AChE-R/RACK1/PKC.beta.II complexes.
[0381] Thus a further aspect of the invention relates to a method
for the screening of drugs for treatment of the nervous system.
According to this aspect, a test drug is selected by a screening
method for a candidate substance which is a modulator of the
interactions between AChE-R/RACK1/PKC. Of particular interest is a
drug which is specifically aimed at affecting CNS properties and
treating CNS-associated disorders. This screening method comprises
the steps of: (a) providing a reaction mixture comprising the
AChE-R variant of acetylcholinesterase or any functional fragment
thereof, the cognate receptor for activated kinase C (RACK1) and
the protein kinase C .beta.II (PKC.beta.II); (b) contacting said
mixture with a test drug under suitable conditions for said
interaction; and (c) determining the effect of the test drug on an
end-point indication. The effect of the test drug on the end-point
is indicative of modulation of said interaction by the test
drug.
[0382] In general, the present invention is directed to screening
methods for identifying modulators of particular signal pathways.
The interaction of the three complex participants
(AChE-R/RACK1/PKC) is observed in the presence and absence of a
candidate modulator. More particularly, the screening assay
according to the invention is conducted by assessing the
interaction between AChE-R, PKC and RACK1 either by measuring
binding directly or by measuring a physiological or metabolic
effect. The measurement is made in the presence and in the absence
of a candidate modulator. Successful candidates which agonize the
signal, effect an increase in a metabolic or physiologic output,
whereas antagonists effect a decrease in a selected metabolic or
physiological end point.
[0383] Depending on the assay system chosen, the interaction and
its modification can be observed in a variety of ways, including
intracellular binding assays affecting an observable parameter;
either a physiological readout, such as change in subcellular
distribution, or an in-vitro co-precipitation.
[0384] In one specific embodiment, the reaction mixture used by the
method of the invention may be a cell mixture or a cell-free
mixture.
[0385] It is known to the man of skill in the art that
protein-protein interactions are susceptible to conditions such as
pH, ion concentration, temperature, and any other factors that may
interfere with such interactions. Thus, according to a specific
embodiment, this reaction mixture may optionally further comprise
solutions, buffers and compounds which provide suitable conditions
for interaction between AChE-R/RACK1/PKC and the detection of an
end-point indication for said interaction.
[0386] In yet another specific embodiment the screened modulator
may either inhibit or enhance the interaction between
AChE-R/RACK1/PKC. Therefore, modification of the end-point
indicates modulation of the interaction between AChE-R/RACK1/PKC by
the test drug. For example, decrease or absence of the end point,
in the presence of the test drug, indicates inhibition of the
interaction between AChE-R/RACK1/PKC. Alternatively, presence or
any increase of the end-point indicates enhancement of the
interaction between AChE-R/RACK1/PKC by the test drug.
[0387] In a particular embodiment, the reaction mixture used by
this screening method of the present invention is a cell-free
mixture. According to this embodiment, the screening method
comprises the steps of: (a) providing a cell free mixture
comprising the AChE-R variant of acetylcholinesterase or any
functional fragment thereof, RACK1 and PKC.beta.II; (b) contacting
said mixture with a test drug under conditions suitable for an in
vitro interaction; and (c) determining the effect of the test
substance on co-precipitation of PKC.beta.II and RACK1 with the
AChE-R or fragment thereof as an end-point indication. Absence of
said co-precipitation indicates inhibition of formation of a
complex between AChE-R/RACK1/PKC by the test drug, whereas increase
in said co-precipitation indicates enhancement of this complex
formation.
[0388] In a particular embodiment, the cell-free mixture used by
the method of the present invention comprises any one of AChE-R
variant of acetylcholinesterase or any functional fragment thereof,
RACK1 and PKC.beta.II. These proteins may be provided as purified
recombinant proteins or alternatively, as a cell lysate of cells
expressing these proteins.
[0389] According to a specifically preferred embodiment, the AChE-R
variant of acetylcholinesterase comprised within the cell free
mixture may be a fusion protein. A "fusion protein" as used herein
is a recombinant protein made of segments, which are naturally not
normally fused in the same manner. As a non limiting example such
fusion protein may comprise AChE-R or functional fragment thereof
and any one of GST (Glutathion-S-Transferase) and GFP (Green
Fluorescent Protein, as exemplified in Example 14). Fusion protein
of any of the three complex precipitant proteins may alternatively
comprise a protein such as MBP (maltose-binding protein). Thus, a
fusion product of the AChE-R molecule with any one of GFP and GST,
is a continuous protein molecule having sequences fused by a
typical peptide bond, typically made as a single translation
product and exhibiting properties derived from each source
peptide.
[0390] In yet another alternative embodiment, the reaction mixture
used by the present invention is a cell mixture. More particularly,
the cell mixture may be a transfected cell culture. According to
this embodiment, the screening method comprises the steps of: (a)
providing transfected cell culture expressing the AChE-R variant of
acetylcholinesterase or functional fragment thereof, the cognate
receptor for activated kinase C (RACK1) and the PKC.beta.II; (b)
contacting said transfected cell culture with a test substance; (c)
detecting the interaction between AChE-R/RACK1/PKC in the presence
of the test drug by searching for an end-point indication, whereby
modification of said end-point indicates modulation of complex
formation between AChE-R/RACK1/PKC by the test drug.
[0391] According to a particular embodiment, the transfected cell
may be transfected by: (a) an expression vector comprising a
nucleotide sequence coding for the AChE-R variant of AChE or
functional fragment thereof; and/or optionally (b), constructs
comprising a nucleic acid sequence coding for any one of the
cognate receptor for activated kinase C (RACK1) and the
PKC.beta.II.
[0392] "Expression Vectors", as used herein, encompass vectors such
as plasmids, viruses, bacteriophage, integratable DNA fragments,
and other vehicles, which enable the integration of DNA fragments
into the genome of the host. Expression vectors are typically
self-replicating DNA or RNA constructs containing the desired gene
or its fragments, and operably linked genetic control elements that
are recognized in a suitable host cell and effect expression of the
desired genes. These control elements are capable of effecting
expression within a suitable host. Generally, the genetic control
elements can include a prokaryotic promoter system or a eukaryotic
promoter expression control system. Such system typically includes
a transcriptional promoter, an optional operator to control the
onset of transcription, transcription enhancers to elevate the
level of RNA expression, a sequence that encodes a suitable
ribosome binding site, RNA splice junctions, sequences that
terminate transcription and translation and so forth. Expression
vectors usually contain an origin of replication that allows the
vector to replicate independently of the host cell.
[0393] The term "operably linked" is used herein for indicating
that a first nucleic acid sequence is operably linked with a second
nucleic acid sequence when the first nucleic acid sequence is
placed in a functional relationship with the second nucleic acid
sequence. For instance, a promoter is operably linked to a coding
sequence if the promoter affects the transcription or expression of
the coding sequence. Generally, operably linked DNA sequences are
contiguous and, where necessary to join two protein-coding regions,
in the same reading frame.
[0394] Plasmids are the most commonly used form of vector but other
forms of vectors which serve an equivalent function and which are,
or become, known in the art are suitable for use herein. See, e.g.,
Pouwels et al. (1985 and supplements) Cloning Vectors: a Laboratory
Manual, Elsevier, N.Y.; and Rodriquez, et al. (eds.) (1988)
Vectors: a Survey of Molecular Cloning Vectors and their Uses,
Buttersworth, Boston, which are incorporated herein by
reference.
[0395] Accordingly, the term control and regulatory elements
includes promoters, terminators and other expression control
elements. Such regulatory elements are described in Goeddel
[Goeddel et al. (1990) Gene Expression Technology: Methods in
Enzymology 185, Academic Press, San Diego, Calif.]. For instance,
any of a wide variety of expression control sequences that control
the expression of a DNA sequence when operatively linked to it may
be used in these vectors to express DNA sequences encoding any of
the three desired proteins used by the screening method of this
invention.
[0396] In general, such vectors contain in addition specific genes,
which are capable of providing phenotypic selection in transformed
cells. A variety of selectable markers can be incorporated into any
construct. For example, a selectable marker which confers a
selectable phenotype such as drug resistance, nutritional
auxotrophy, resistance to a cytotoxic agent or expression of a
surface protein, can be used. The expression vector of the
invention may further comprise a tag sequence. Such sequences
enable the detection and isolation of the recombinant protein. As a
non-limiting example such tag sequences may be any one of HA,
c-myc, GST, GFP and His-6.
[0397] The use of prokaryotic and eukaryotic viral expression
vectors to express the genes coding for the AChE-R and optionally
for the PKC.beta.II and the RACK1 according to the present
invention, is also contemplated.
[0398] The vector is introduced into a host cell by methods known
to those of skilled in the art. Introduction of the vector into the
host cell can be accomplished by any method that introduces the
construct into the cell, including, for example, calcium phosphate
precipitation, microinjection, electroporation or transformation.
See, e.g., Ausubel, F. M. ed. (1989) Current Protocols in Molecular
Biology, John Wiley & Sons, New York.
[0399] "Cells" or "transfected cells" are terms used in the present
invention. It is understood that such terms refer not only to the
particular subject cells but to the progeny or potential progeny of
such a cell. Because certain modification may occur in succeeding
generations due to either mutation or environmental influences,
such progeny may not, in fact, be identical to the parent cell, but
are still included within the scope of the term as used herein.
[0400] "Transfected cell" as used herein refers to cells which can
be stably or transiently transfected with vectors constructed using
recombinant DNA techniques. A drug resistance or other selectable
marker is intended in part to facilitate the selection of the
transformants. Additionally, the presence of a selectable marker,
such as drug resistance marker may be of use in keeping
contaminating microorganisms from multiplying in the culture
medium. Such a pure culture of the transfected cell would be
obtained by culturing the cells under conditions which require the
induced phenotype for survival.
[0401] As used herein, the term "transfection" means the
introduction of a nucleic acid, e.g., an expression vector, into
recipient cells by nucleic acid-mediated gene transfer.
[0402] It is to be appreciated that the cell or the transfected
cell used by the screening method of the present invention may
further contain endogenously or exogenously expressible possible
interacting molecules essential for activation of said pathway or
said complex formation.
[0403] One preferred end-point indication for the "cell mixture"
and preferably the transfected cell based screening method, may be
the subcellular translocation of catalytically active PKC.beta.II.
Subcellular distribution of PKC.beta.II can be detected by a
visually detectable signal, generated for example, by using in situ
hybridization or immunohystochemistry. A visually detectable signal
may be for example a fluorescent signal (FIG. 26, using confocal
microscopy).
[0404] In general, PKC is mainly referred to as a morphologically
active kinase. However, PKC.beta.II has been shown to be associated
with oxidative [Paola et al., (2000) Biochem Biophys Res Commun,
268, 642-6] and ischemic stresses [Cardell and Wieloch, (1993) J
Neurochem, 61, 1308-14] and essential for fear conditioning [Weeber
et al., (2000) J Neurosci, 20, 5906-14]. Intriguingly, genomic
disruption of the glucocorticoid receptor, which upregulates AChE-R
production [Grisaru et al. (2001) ibid] abolished anxiety responses
[Tronche et al. (1999) Nat Genet, 23, 99-103]. The current findings
of the present invention therefore propose a chain of events that
may assist in overcoming traumatic stress responses. This cascade
initiates with glucocorticoid hormone release, proceeds with
transcriptional activation and alternative splicing to elevate
AChE-R levels and results in RACK1 and PKC.beta.II mobilization.
AChE-R thus emerges as a modulator, and PKC.beta.II as an initiator
of long-term morphological responses to stress. Relevant processes
include the reported PKC-induced inhibition of pre-synaptic
metabotropic glutamate receptors [Macek et al. (1998) J Neurosci,
18, 6138-46], coupling to the cAMP response element binding protein
in CA1 neurons [Roberson et al. (1999) J Neurosci, 19, 4337-48] and
the PKC-regulated release of vesicle pools [Stevens and Sullivan,
(1998) Neuron, 21, 885-93]. The increase in neuronal PKC.beta.II in
AChE-R transgenic mice further proposes that when such changes
become permanent they can confer stress protection, whereas the
PKC.beta.II disruption study indicates that chronic impairments in
this cascade may be detrimental. Nevertheless, excessive AChE-R
production may also be detrimental, at least for recovery from
closed head injury [Shohami et al. (2000) id ibid.].
[0405] Similarly to the "cell-free mixture"-based screening method,
another preferred end-point indication for the "cell mixture" based
screening method, may be co-precipitation of PKC.beta.II and RACK1
with the AChE-R or functional fragment thereof. Co-precipitation of
these interacting molecules leads to a detectable signal, whereby
modification of said detectable signal in the presence of the test
drug indicates modulation of the formation of a complex between
AChE-R/RACK1/PKC by said test drug.
[0406] In yet another specifically preferred embodiment, the cell
or the transfected cell used by the screening method of the
invention may be a prokaryotic or eukaryotic cell, particularly a
bacterial cell, yeast cell, an insect cell, a plant cell or
preferably a mammalian cell. Most preferred are cells selected from
the group consisting of COS and PC12 cells.
[0407] Based on the results described in Examples 18 to 22, AChE-R
and/or PKC.beta.II levels should be also tested in patients with
post-traumatic stress disorder [McEwen, (1999) Annu Rev Neurosci,
22, 105-22], post-stroke phenomena and inherited susceptibility to
processes in which PKC.beta.II plays a major role, e.g. panic
attacks [Gorman et al., (2000) Am J Psychiatry, 157, 493-505],
where fear conditioning is intimately involved. Likewise, the
dissociation between RACK1 and PKC.beta.II under ethanol exposure
[Ron et al. (2000) J Biol Chem, 274, 27039-46] may be relevant to
the stress-suppressing effect of alcohol. In addition, it would be
interesting to test PKC.beta.II levels and subcellular localization
in patients hypersensitive to anticholinesterases (e.g Alzheimer's
disease drugs) which also induce AChE-R overproduction [Kaufer et
al. (1998) ibid; Shapira et al. (2000) Hum Mol Genet, 9,
1273-81].
[0408] Therefore, a candidate drug which modulates the interaction
between AChE-R/RACK1/PKC.beta.II, may affect fear conditions, panic
and traumatic stress responses.
[0409] In a yet further embodiment of the screening method, the
modulator of the interaction between AChE-R/RACK1/PKC also
modulates the expression and intracellular distribution of RACK1
and/or PKC.beta.II.
[0410] Triple AChE-R/RACK1/PKC.beta.II complexes are likely
involved with the neuronal redistribution of PKC.beta.II in brain
development [Gallicano et al., (1997) Bioassays 19, 29-36], aging
[Battaini et al. (1999) Exp Neurol 159, 559-64] and
neurodegeneration [McNamara et al. (1999) J Neurochem 72, 1735-43],
all of which involve considerable modulations in AChE-R levels.
AChE-R is further expressed in other RACK1/PKC.beta.II producing
tissues, including epithelial, muscle, hematopoietic, and germ
cells [Soreq and Seidman, (2001) id ibid] where its capacity to
induce PKC.beta.II-mediated changes should be examined.
[0411] As shown by Examples 19-21, AChE-R/RACK1/PKC.beta.II
complexes are mobilized from their resting state intracellular
location, translocating RACK1 to the perikaryal circumference and
PKC.beta.II into densely packed clusters. Modified properties and
location of PKC.beta.II in stress-responding neurons may possibly
change the stress-induced kinase activation, phosphodiesterase
mobilization, or other processes. Several RACK1 reports have
addressed the effect of PKC-RACK interactions on PKC activity.
Addition of RACK1 to PKC in the presence of PKC activators did not
significantly change PKC activity [Chang et al. (1998) id ibid.],
yet PKC activators induced RACK1 interaction with PKC, Src [Chang
et al. (2001) id ibid.] and integrin .beta.1 [Liliental and Chang
(1998) id ibid.], presumably by inducing a conformational change in
RACK1 which exposes it to the binding of other proteins. PKC
activation is also known to induce the MAPK pathway associated with
stress responses [Gil et al. (2000) id ibid; Kaneki et al. (1999)
id ibid.].
[0412] The presence of PKC.beta.II in the deep cortical layers and
in the stratum oriens and stratum radiatum of CA1 in hippocampus
(FIG. 25) may reflect signal transmission across synapses between
axons coming from outside the region. Staining in axon bundles such
as the nigro-striatal tract may indicate that PKC.beta.II is
distributed along the entire axon of some neurons. Finally, the
currently observed perikaryal punctiform pattern (FIG. 26), is
compatible both with RACK1 interactions and with different
functions, as considered below.
[0413] A yet further aspect of the present invention is a method
for the in vivo screening of candidate drugs that affect the
central nervous system, wherein said drug is a modulator of an
interaction between AChE-R/RACK1/PKC. This screening method
comprises the steps of: (a) providing an AChE-R transgenic animal;
(b) administering the test drug to said animal; (c) sacrificing the
animal and dissecting its brain to give samples for preparation of
brain extracts or for immunohistochemistry; (d) detecting the
expression of RACK1 or PKC.beta.II in said brain samples; and (e)
determining the effect of the test drug on an end-point indication,
wherein said effect is indicative of the in vivo modulation of said
interaction by the test drug. The end-point indication of this
screening method is the expression of RACK1 and PKC.beta.II in the
brain.
[0414] The preferred transgenic animals are Xenopus and mammals,
such as mice, cows, goats, pigs and sheep. In said transgenic
animals, the transgene is a recombinant expression vector
containing a promoter controlling the transcription of the sequence
encoding the "readthrough" form of AChE, and wherein said promoter
is selected from the group of eukaryotic host cell compatible
promoters consisting of CMV, CMV-like, AChE and AChE-like promoter.
Most preferably, the transgenic animal is an AChE-R transgenic
mouse, which has been described in Sternfeld et al. (2000)
[Sternfeld et al. (2000) id ibid].
[0415] The brain of the transgenic animal should be dissected in a
manner suitable for either RNA or protein analysis, either in
tissue sections or in solution. Method for appropriate dissection
of tissues for sections and immunohistochemistry or RNA staining
are known to the man of skill in the art, and have been described
in numerous publications, e.g. Wilkinson and Nieto (1993) Methods
in Enzymology, 222, 361-372; Marti et al. (1995) Development 121,
2537-2547. Similarly, methods for dissection in order to prepare
tissue extracts have been described by e.g. Sternfeld et al. (2000)
[Sternfeld et al. (2000) id ibid].
[0416] In the above-described method, RACK1 and PKC.beta.II
expression can be detected through procedures specific for
detecting the expression of RNA or protein.
[0417] In one specific embodiment, the RNA detection is performed
by means appropriate for RNA detection, said means selected from
the group consisting of RT-PCR, Northern Blot, in situ
hybridization, RNAse protection and S1 nuclease analysis. These and
other techniques for RNA detection have been described in [Sambrook
et al. (1989) id ibid.], herein incorporated by reference.
[0418] In another specific embodiment, the protein detection is
performed by means appropriate for protein detection, said means
selected from the group consisting of Western Blot and
immunohistochemistry. These and similar methods for protein
detection have been described in Current Protocols in Immunology,
Coligan et al. (eds), John Wiley & Sons. Inc., New York.
[0419] Transgenic AChE-R -filled neurons with both punctiform
PKC.beta.II and RACK1 labeling are mostly relevant to
stress-response inhibitory pathways [Herman and Cullinan, (1997)
Trends Neurosci, 20, 78-84]. These were located in cortical upper
layers, the hippocampal CA1 region, the lateral septum and the
basolateral amygdala. PKC.beta.II punctiform patterns also appeared
in a subset of basolateral amygdala neurons, which are generally
considered excitatory to psychological stress. However, the
existence in this region of stress-inhibitory neurons, has been
discussed with regards to the regulation of fearful behavior [Davis
et al., (1994) Trends Neurosci, 17, 208-14]. These neurons
presumably suppress other basolateral amygdala neurons that are
stress-excitatory, consistent with the limited stress-related
neuropathology hallmarks in the brain of AChE-R transgenic mice
[Sternfeld et al. (2000) id ibid.]. The neuronal location of
AChE-R/RACK1/PKC.beta.II complexes further suggests relevance to
the PKC-activated down regulation of transient K.sup.+ channels in
dendrites of hippocampal CA1 pyramidal neurons [Hoffman and
Johnston, (1998) J Neurosci, 18, 3521-8]. Such downregulation may
contribute to the reduced stress overload of AChE-R overexpressing
mice.
[0420] It is to be understood that the term modulator is used
throughout this specification as any substance, being it a drug or
a compound, capable of enhancing or diminishing the interactions
within the complex AChE-R/RACK1/PKC. The modulator may be able to
interact with the complex, or with each separate component of the
complex, or even with any combination of two components of the
complex, in such a way that it may strengthen or weaken its
interactions. Alternatively, the modulator may affect, i.e., induce
or repress, augment or diminish, the expression of at least one of
the components of the complex. In addition, another effect of the
modulator may be to trigger a change in the intracellular
localization of at least one of the components of the complex. In
all of the above-exemplified situations, the component of the
complex might be in the complex or not, to be affected by the
competence of the modulator. Thus the modulator may be able to
affect each molecule when not in the complex, and thereby inhibit
complex formation.
[0421] It is noteworthy that each of the components in the triple
AChE-R/RACK1/PKC.beta.II complexes represents one out of several
options. Thus, other PKC isoforms most likely interact with
different shuttling proteins, with distinct and different effects
on the complex physiological phenomena that follow traumatic
experiences. Likewise, RACK1 operates as a shuttling vector for
many other proteins, and at least part of these interactions (e.g.
phosphodiesterase) are likely to compete with the currently
described one and ameliorate its consequences. Finally, the AChE-R
protein is one out of three diverse AChE isoforms, each with its
own C-terminal peptide and possibly different interactions.
[0422] The test drug for the screening and evaluation methods of
the invention may be any substance selected from the group
consisting of protein based, carbohydrates based, lipid based,
nucleic acid based, natural organic based, synthetically derived
organic based, antibody based and metal based substances.
[0423] As used herein, the term "nucleic acid" refers to
polynucleotides such as deoxyribonucleic acid (DNA), and, where
appropriate, ribonucleic acid (RNA). The terms should also be
understood to include, as equivalents, analogs of either RNA or DNA
made from nucleotide analogs, and, as applicable to the embodiment
being described, double-stranded polynucleotides and
single-stranded such as sense or anti-sense. More particularly,
anti-sense directed against the AChE-R variant may attenuate said
complex formation.
[0424] Among successful candidate drugs or substances will be
peptides which mimic regions on either of the three complex forming
proteins, as well as non-peptidic small molecules. Due to their
ease of identification, these peptides are particularly useful in
alternate forms of the screening assays that detect modification
and modulation of the interaction between AChE-R/RACK1/PKC.
Although the assay methods disclosed may not all be suitable for
direct screening of large chemical libraries, they do enable a
sophisticated screening of candidates that can be combined with
other techniques for selecting leads.
[0425] In a particular embodiment a protein or antibody based
substance may be a product of a combinatorial library. Thus, the
invention is also directed to methods to screen libraries of
candidate modulators using the above-described methods and to
peptides representative of sites on any of the three complexed
proteins, which are themselves useful in these assays as well as in
other applications involving the relevant interaction.
[0426] The drugs to be evaluated by the methods of the invention
can be any candidate or known drugs, e.g. drugs for the treatment
of anxiety conditions, post-traumatic stress, Alzheimer's disease,
muscle malfunctioning, neurodegenerative disorders, damage
resulting from exposure to xenobiotics, panic, neuromuscular
disorders, Parkinson's disease, Huntington's chorea, muscle
fatigue, multiple chemical sensitivity, autism, multiple sclerosis
and Sjogren's disease.
[0427] In yet a further aspect, the invention provides for a method
for the treatment of stress-associated conditions or disorders, for
a subject in need of such treatment, said method comprising: [0428]
a. providing a composition comprising as active ingredient a
modulator of an interaction between AChE-R/RACK1/PKC; [0429] b.
administering a therapeutic effective amount of said composition to
said subject; wherein said modulator is selected according to the
drug screening methods provided by the invention.
[0430] "Treatment" refers to therapeutic treatment. Those in need
of treatment include those already with a disease or disorder,
whether at clinical or pre-clinical stage.
[0431] Disclosed and described, it is to be understood that this
invention is not limited to the particular examples, process steps,
and materials disclosed herein as such process steps and materials
may vary somewhat. It is also to be understood that the terminology
used herein is used for the purpose of describing particular
embodiments only and not intended to be limiting since the scope of
the present invention will be limited only by the appended claims
and equivalents thereof.
[0432] It must be noted that, as used in this specification and the
appended claims, the singular forms "a", "an" and "the" include
plural referents unless the content clearly dictates otherwise.
[0433] Throughout this specification and the claims which follow,
unless the context requires otherwise, the word "comprise", and
variations such as "comprises" and "comprising", will be understood
to imply the inclusion of a stated integer or step or group of
integers or steps but not the exclusion of any other integer or
step or group of integers or steps.
[0434] The following examples are representative of techniques
employed by the inventors in carrying out aspects of the present
invention. It should be appreciated that while these techniques are
exemplary of preferred embodiments for the practice of the
invention, those of skill in the art, in light of the present
disclosure, will recognize that numerous modifications can be made
without departing from the spirit and intended scope of the
invention.
EXAMPLES
Experimental Procedures
[0435] Cell source: UCB was collected, following informed consent
of the parents and with the approval of the Sourasky Medical Center
Ethics Committee, as previously described [Grisaru et al. (1999a)
Am. J. Obstet. Gynecol, 180, 1240-1243]. Following 1:1 (v/v)
dilution in Iscove's modified Dulbecco medium (IMDM, Beit Haemek,
Israel), mononuclear cells were separated using 3% gelatin (Difco,
Detroit, Mich.) and Ficoll-Hypaque gradients (<1.077 g/ml;
Pharmacia, Uppsala, Sweden) [Pick et al. (1998) Br. J. Haematol.
103, 639-50]. CD34.sup.+ cells were enriched using CD34
immunoglobulin-coated magnetic beads (CD34 progenitor cell
selection system, Dynal, Norway). CD34.sup.+ cells analysis was
performed by flow cytometry (Becton Dickinson Immunocytochemistry
System Inc., San Jose, Calif.), using CD34-PE (Becton Dickinson
Immunocytometry System, Inc.) and CD45-FITC (Dako, Glostrup,
Denmark) monoclonal antibodies. May-Grunwald-Giemsa staining
revealed stem cell morphology.
[0436] Liquid cultures: UCB CD34.sup.+ cells were set for liquid
cultures at a concentration of 10.sup.5/mL in IMDM, containing 10%
autologous plasma, 2 mM L-glutamine (Sigma Chemical Co., St Louis,
Mo.), penicillin (100 mg/mL), streptomycin (100 mg/mL),
amphotericin B (2.times.10.sup.-5 M) (Sigma Chemical Co.), and
heparin (20 IU/mL, Gibco, Grand Island, N.Y.), all in a fully
humidified atmosphere at 37.degree. C. and 5% CO.sub.2. The
following elements were added where noted: [0437] 1. Hematopoietic
growth factors: Interleukin 3 (IL3, 5 ng/mL, Immunex, Seattle,
Wash.), interleukin 6 (IL6, 50 ng/mL, R&D Systems, Minneapolis,
Minn.), TPO (1 ng/mL, R&D Systems), stem cell factor (SCF, 10
ng/mL, R&D Systems), FLT-3 ligand (FLT3, 50 ng/mL, R&D
Systems), granulocyte-macrophage colony stimulating factor (GM-CSF,
50 ng/mL, Biogenesis Ltd, Bournemouth, UK) and combinations of the
above, for 24 hr to 28 days (supplemented every 4 days). [0438] 2.
Endothelial Growth factors: Epidermal growth factor (EGF 10 ng/ml,
Sigma Chemical Co.), basic Fibroblast growth factor (bFGF 20 ng/ml,
Sigma Chemical Co.) incubated in a serum free medium (SFM) for
48hr. [0439] 3. Stress mimicking conditions: Hydrocortisone sodium
succinate (Abic Ltd., Netanya, Israel) at concentrations equivalent
to normal, intermediate and stress serum cortisol levels (0.1, 0.6
and 1.2 .mu.M, respectively (De Vroede et al., Arch. Des. Child 78,
544-7, 1998), for 24 hr. [0440] 4. Antisense oligonucleotides:
3'-terminal 2'-O-Methylated 15- and 20-mer oligodeoxynucleotides in
the antisense (AS) orientation, targeted against the common
sequence domain in human AChEmRNA and BuChE mRNA, as control, were
used for 24 hr, as detailed elsewhere [Grisaru et al. (1999b) Mol.
Cell. Biol. 19, 788-95]. [0441] 5. AChE C-terminal peptides: The
following C-terminal peptides of the AChE synaptic (ASP, also
denoted SEQ ID: No. 2) and readthrough (ARP, also denoted SEQ ID:
No. 1) isoforms were synthesized using a 433A peptide synthesizer
(PE Applied Biosystems, Inc., Norwalk, Conn.).
[0442] ASP:1-DTLDEAERQWKAEFHRWSSYMVHWKNQFDHYSKQDRCSDL-40, also
denoted as SEQ ID: No. 2.
[0443] ARP: 1-GMQGPAGSGWEEGSGSPPGVTPLFSP-26, also denoted as SEQ
ID: No. 1.
[0444] Length and integrity of the peptide preparations were
ensured following purification by HPLC, using a D-6000
chromatography data station (Hitachi Instruments, Inc., San Jose,
Calif.). The working concentrations were 50 and 100 ng/mL
(supplemented every 4 days) for liquid cultures grown between 24 hr
to 28 days.
[0445] Culture analyses: Twenty-four hr liquid cultures served for
cytochemical staining, in situ hybridization, and cell
proliferation assay by 5-bromo-2'-deoxy-Uridine (BrdU)
incorporation [Grisaru et al. (1999b) id ibid.]. Twenty-eight day
liquid cultures were sampled every 6-8 days for viable cell
counting (using trypan blue dye exclusion), cell phenotyping
(CD34.sup.+, CD33.sup.+ and CD41.sup.+ quantification using flow
cytometry, with CD34-PE, CD33-FITC (Immunotech/Coulter, Hialeah,
Fla.), CD41-FITC (Immunoquality Products, Groningen, Netherlands)
and CD45-FITC monoclonal antibodies), and growth of progenitor
colony (granulocyte-macrophage and megakaryocytic), using
previously described techniques (Pick et al. (1998) id ibid.).
[0446] Cytochemical staining: Staining of AChE activity was
essentially as detailed elsewhere [Grisaru et al. (1999b) id ibid],
on non-fixed liquid cultures following 300.times.g centrifugation
on collagen-coated cover slips placed on the bottom of the culture
well, in the presence of 10.sup.-5 M iso-OMPA (ISO) or BW284C51
(BW), selective inhibitors of BuChE and AChE, respectively [see
also Keymer et al.(1999) Eur. J. Neurosci. 11, 1049-57]. Nuclear
staining was with 4',6-diamidino-2-phenylindole [DAPI, see e.g.,
Peterson et al. (1999) Genetics 152, 427-439].
[0447] Animals and tissue collection: Male FVB/N mice (2-6 months
old) were sacrificed 24 hr following 4 successive days of a forced
swim session as detailed [Kaufer et al. (1998) id ibid.], or a
single injection of ARP (34 nmol/kg weight), or phosphate buffer
(PB) for control. ARP, a 26 amino acid residue peptide synthesized
according to the C-terminal sequence of human AChE-R [Grisaru et
al. (1999b) id ibid.] was HPLC purified and mass-spectrometry
analyzed for purity. Control naive mice and hAChE-R transgenics
were sacrificed with no prior treatment.
[0448] Blood samples were allowed to clot 1 hr at room temperature
and overnight at 4.degree. C., followed by centrifugation and serum
collection.
[0449] Testes and seminal vesicles were excised and weighed, fixed
in 4% paraformaldehyde or Bouin's fixative for histological
staining or kept at -70.degree. C. for protein extraction.
[0450] Sperm cells were collected from one cauda epididymis
shredded in 1 ml saline.
[0451] Evaluation of serum corticosterone levels: concentrations
were determined by radioimmunoassay.
[0452] Epididymal sperm motility was assessed by visually
determining percent motile sperm. Sperm concentration was measured
using the Makler chamber (company, city, state).
[0453] In situ hybridization: In situ hybridization procedures,
were performed on cultured cells and human fetal tissues, as
detailed elsewhere [Grisaru et al. (1999b) id ibid; Kaufer et al.
(1998) id ibid.]. Cultured cells were centrifuged at 300.times.g
and fixed, using 4% paraformaldehyde, to collagen-coated cover
slips placed on the bottom of the culture well. Tissues from fetal
hematopoietic organs (AGM, liver, spleen and bone marrow) were
obtained in each of the selected gestational stages, from 2-3
normal aborted human fetuses. The project was approved by the
Sourasky Medical Center Ethics Committee, and written informed
consent was obtained from the parents. 5'-Biotinylated,
2'-O-methylated AChEcRNA probes complementary to 3'-alternative
human AChE exons were employed. Detection and quantification of the
various AChEmRNA transcripts in fetal tissues were performed as
previously described [Grisaru et al. (1999b) id ibid.]. Confocal
microscopy scans of the culture-derived cells were obtained using a
MRC-1024 Bio-Rad confocal microscope (Hemel Hempsted Herts., UK). A
projection was built from each cell image and specific criteria
were set for size and intensity of the Fast Red fluorescence.
Image-Pro 3.0 software (Media Cybernetics, Silver Spring, Md., USA)
was used to analyze the signals obtained. ANOVA (Analysis of
Variance) test was used for calculation of p values.
[0454] Human sperm smears: Air dried sperm smears of ejaculates
collected from male donors or infertility patients were stained
with the anti-ARP antibody as mentioned above.
[0455] Confocal microscopy: An MRC-1024 Bio-Rad confocal microscope
equipped with an inverted microscope and a 63.times./2.4 oil
immersion objective was used to scan the fast red precipitate used
for ARP and ASP immunodetection. Fast red was excited at 488 nm,
emission was measured using a 580df32 filter. Sections were scanned
every 0.35 .mu.m, and a three-dimensional projection was created
from all sections.
[0456] For imaging of AChE-R, RACK1 and PKC, brain slices were
scanned using a Bio-Rad MRC-1024 scanhead (Hemel Hempsted
Herts.,UK) coupled to an inverted Zeiss Axiovert 135M microscope
with a 40.times. oil immersion objective (N.A. 1.3). Excitation
wavelength was 488 nm (using 10% of a 100 mW laser power).
Fluorescence emission was measured using a 580df32 bandpass
interference filter (580 nm.+-.16 nm) for detecting
tetra-methyl-rhodamine and a 525/40 filter for detecting
fluorescein. The confocal iris was set to 3 mm. Conditions of
scanning took into consideration the overlap of fluorescein
fluorescence into the rhodamine filter (as were determined by
control experiments). Images were then further processed using
Image pro Plus 4.01 program (version 4.0, Media Cybernetics, Silver
Spring, Md.).
[0457] DNA sequence analysis: The reverse sequence of the 7q22
cosmid (accession no. AF002993) containing the human ACHE gene and
its upstream sequences, was searched for consensus motifs for
binding transcription factors which regulate hematopoietic
expression, using the MatInspector program with core similarity of
1, or the Findpatterns program of the University of Wisconsin GCG
software package (Quandt et al., Nucleic Acids Res. 23, 4878-84,
1995).
[0458] Immunoblot: Mouse serum was diluted 1:10. ARP, ASP,
recombinant AChE-S (Sigma Chemical Co.) and recombinant AChE-R
extracted from transfected COS cells served as positive controls.
Protein electrophoresis in SDS gradient (4-20%) polyacrylamide gels
(Bio-Rad Laboratories, Hercules, Calif.) was followed by
immunodetection using the rabbit anti-GST-ARP antibodies,
Peroxidase-conjugated anti-rabbit immunoglobulins and ECL.TM.
detection (Amersham Pharmacia Biotech, UK).
[0459] Methods for Culturing Murine Hematopoietic Cells:
Cell Collection
[0460] Blood: 1 ml of murine blood is collected by cardiac puncture
and immediately deposited into pediatric vaccutainer tubes
containing Na citrate. Blood counts are performed using the AcT
diff Coulter counter (Coulter-Beckman).
[0461] Bone marrow: The tibia and femurs are surgically removed,
cleaned and both ends of the bone cut open. The bones are placed in
small tissue culture plates containing 2-5 ml of medium composed of
RPMI, antibiotics and 10% heat inactivated fetal calf serum
(complete medium) supplemented with heparin (5 U/ml) to prevent
clotting. The BM contents are flushed out into the medium using a
25 gauge needle. Cells are passed up and down through the syringe
three times to guarantee a single cell suspension.
[0462] Spleen: The spleen is surgically removed and cleaned and
placed in 5 ml of medium composed of complete RPMI with heparin (5
U/ml) as described above. Both ends of the spleen are cut open and
the spleen cells are expressed from the organ capsule by squeezing
down on the spleen with the back barrel of a 5 cc sterile syringe.
A single cell suspension is prepared by passing the cells up and
down three times through a 5 ml syringe.
Cryopreseruation
[0463] The hematopoietic cells are washed by the addition of an
additional 5 ml of medium composed of complete RPMI with heparin,
counted by manual hemocytometry and pelleted at 1500 RPM for 10
minutes. The cell pellets are resuspended at a concentration of
5.times.10.sup.6-10.sup.-7 cells/ml in ice cold freezing medium
containing 50% DMSO and 30% RPMI (as above). Cells are allowed to
remain on ice for up to 5 minutes and subsequently placed at
-20.degree. C. for 30 minutes. Cells are then transferred to
-80.degree. C. for a period of up to 6 months; for longer storage,
cells are transferred to -180.degree. C.
Hematopoietic Progenitor Cultures--
[0464] For all colony assays duplicate samples of
1-2.times.10.sup.5 cells are placed into 1 ml of medium plus the
appropriate supplements for each cell lineage in small round tissue
culture dishes (2.5 cm). These small dishes are placed into a
larger TC dish (10 cm) together with a third small dish containing
sterile water to prevent evaporation in the cultures. Cells are
cultured at 37.degree. C. in a fully humidified atmosphere
containing 5% CO.sub.2.
BFU-E
[0465] Two hundred thousand (2.times.10.sup.5) cells are placed
into 1 ml of Alpha medium containing antibiotics, 30% methyl
cellulose, 10% FCS and 2 ng/ml recombinant murine (r-mu)
erythropoietin, and both BFU-E and the smaller CFU-E are counted
after 10 days.
CFU-GM and CFU-GEMM
[0466] One hundred thousand (1.times.10.sup.5) cells are placed
into 1 ml of Alpha medium containing antibiotics, 3% agar, 10% FCS
and 5 ng/ml or both r-mu IL-3 and r-mu GM-CSF. CFU-GM, CFU-GEMM and
CFU-bl are counted after 12 days.
CFU-MK
[0467] Two hundred thousand (2.times.10.sup.5) cells are placed
into 1 ml of Alpha medium containing antibiotics, 30% methyl
cellulose, 10% FCS and 5 ng/ml. r-mu thrombopoietin and 10 ng/ml
stem cell factor. CFU-MK and BFU-MK are counted by staining the
cells for AChE after 12 days.
Animal Models and In Vivo Experiments--transgenic--
[0468] FVB/N mouse pedigrees expressing human AChE variants were
described elsewhere, as were the biochemical methods for measuring
AChE activity [Sternfeld et al. (1998b) J. Physiol. Paris, 92,
249-55]. The confined swim protocol for exerting acute
psychological stress was performed as detailed [Kaufer et al.(1998)
id ibid.]. Immediately following the stress, the treated mice were
injected intraperitoneally with 100 ng ARP or 0.03 ng AS1, both per
gram body weight. Another group of non-stressed mice were injected
either with normal saline or ARP. Twenty-four hours later, the
animals were sacrificed and peripheral blood was collected in EDTA
covered tubes (Becton Dickinson Immunocytochemistry System, Inc.,
San Jose, Calif.) prepared with 25 units of heparin sodium USP
(Kamada LTD, Kibbutz Beit-Kama, Israel). Whole blood AChE activity
was analyzed, and WBC and platelet counts determined, using an Ac-T
diff hematology analyzer (Beckman Coulter, Inc., Fullerton,
Calif.).
Cytochemical and Immunohistochemical Staining
[0469] Staining of AChE activity was as detailed above. For
immunohistochemistry, murine bone marrow smears were fixed with 4%
paraformaldehyde (10 minutes, room temperature, RT); permeabilized
with buffer containing 20 mM HEPES (pH 7.4), 300 mM sucrose, 50 mM
NaCl, 3 mM MgCl.sub.2 and 0.5% Triton X-100 (4 minutes on ice);
washed twice with PBS (5 min each, RT); incubated in 1%
H.sub.2O.sub.2 in methanol (15 min RT); and washed twice in PBS.
Non-specific sites were blocked by incubating in 5% horse serum in
PBS (20 min, RT). Labeling was in a humidified chamber with 1:50
dilution of affinity purified rabbit antiserum prepared against
GST-fused recombinant ARP (1 hr, RT). Following 3 washes with PBS,
smears were incubated with 1:100 biotinylated goat anti-rabbit Ig
(Amersham Pharmacia Biotech UK Ltd., Buckinghamshire, UK) (30 min,
RT). After 3 PBS washes, a mixture of biotin and avidin-peroxidase
was added (30 min, ABC Elite Kit, Vector Labs, Burlingame, Calif.)
and reacted with diaminobenzidine-hydrogen peroxide mixture (Sigma
Chemical Co., St. Louis, Mo., 10 min), followed by counterstaining
with Meyer's hematoxylin mixture (Sigma Chemical Co., St. Louis,
Mo.), and immunomounting.
Immunohistochemistry and Nuclear Staining in Testis Sections
[0470] Detection of the AChE core protein and/or its variant
C-terminal peptides was performed on 7 .mu.m thick paraffin
embedded testis sections with either anti-ASP (C-16; Santa Cruz
Biotechnology, Santa Cruz, Ca) or anti-ARP polyclonal antibodies
(Sternfeld et al. (2000) id ibid.]. PCNA was detected using a
dedicated staining kit (Zymed Laboratories, San-Francisco, Calif.);
nuclear staining and counterstaining was with DAPI and hematoxylin,
respectively (Sigma, St. Louis Mo.).
Expression of Recombinant ARP
[0471] The sequence, coding the C-terminal region of I4 (i.e., the
"readthrough" variant of acetylcholinesterase, comprising the ARP
peptide sequence), was amplified by PCR using the following
oligonucleotide primers: [0472] GCT GGA TCC ATC GAG GGG CGA GGT ATG
CAG GGG CCA GCG GGC (14-up), also denoted as SEQ ID: No. 4, [0473]
and TAT AAG CTT CTA GGG GGA GAA GAG AGG GGT (I4-down), also denoted
as SEQ ID: No. 5, and introduced into pGEX-KG (ATCC accession No.
ATCC77103, see also Anal. Biochem. 192:262-267, 1991) plasmid.
Antibody Production
[0474] GST and I4-GST fusion protein were purified from the
supernatant of E. coli lysate by affinity chromatography on
glutathione-Sepharose (Pharmacia), eluted with 10 mM reduced
glutathione in 50 mM Tris-HCl, pH 8.0, dialyzed to 0.1 M ammonium
acetate buffer, pH 7.0, aliquoted and lyophilized. The stability
and identity of the protein was confirmed by SDS-PAGE. The
following protease inhibitors were used during the preparation:
aprotinin (10 microgram/ml), benzamidine (5 mM), Pefabloc SC (0.2
mM), and EDTA (1 mM). Prior to affinity chromatography, the E. coli
lysate was incubated for 20 min at 37.degree. C. with 0.2 mM Mg-ATP
in order to dissociate the fusion proteins from contamination of
bacterial proteins. The procedure was performed according to
Pharmacia recommendations.
[0475] Two New Zealand female rabbits were immunized subcutaneously
with 0.3 mg fusion protein in complete Freund's adjuvant, and then
reimmunized monthly with 0.2 mg fusion protein in incomplete
Freund's adjuvant. Blood samples were taken 10 days after the
immunization. The specific antibodies in the sera were detected by
ELISA on immobilized fusion protein, in the presence of excess of
soluble GST (20 microgram/ml). The reacting sera were chosen for
antibody purification. The immobilized I4-GST, GST and E. coli
lysate were prepared using Affigel 10 (Bio-Rad) according to the
manufacturer's recommendations.
[0476] Crude IgG fraction was prepared from the serum by 50%
saturation (NH.sub.4).sub.2SO.sub.4 precipitation and dialyzed in
100 mM Tris-HCl, pH 8.0. In order to get rid of anti-GST
antibodies, the IgG fraction was incubated with GST beads (Affigel
10, Bio-Rad) overnight at 4.degree. C. The bound material was
eluted with 4.5 M MgCl.sub.2. The procedure was repeated with the
unbound material several times, until no antibodies were eluted
from GST beads. In order to get rid of antibodies against possible
contamination of bacterial proteins, the same procedure was
performed with immobilized heat-shocked E. coli lysate
proteins.
[0477] The unbound material was then applied to I4-GST beads
(Affigel 10, Bio-Rad), incubated 2 hr at room temperature or
overnight at 4.degree. C., and the bound material was eluted with
3.5 M MgCl.sub.2. The eluted antibodies were dialyzed against 10 mM
Tris-HCl, pH 8.0, and then against PBS, containing 0.025%
NaN.sub.3.
[0478] Two-hybrid Screen
Vectors
[0479] A fragment of human AChE-R cDNA (nt 1796-1865 of hAChE,
accession number M55040, followed by nt 1-111 from the genomic
hAChE I4-E5 domain (Accession No. S71129, stop codon in position
86) was used as "bait" for the two-hybrid screen. Cloning into the
EcoR1/SmaI sites of pGBK-T7 (Clontech, Palo Alto, Calif.), yielding
the plasmid pGBK-ARP1. Cloning of the "bait" sequence into the
Bspl2OI/XbaI sites of pEGFP-C2 (Clontech) yielded the pGARP vector.
The AChE-R expressing plasmid used for transfections has been
described in detail [Seidman et al.(1995) Mol Cell Biol., 15,
2993-3002].
Screening
[0480] The "bait" EcoRI/HpaI fragment of AChE-R cDNA encodes the 51
amino acid long C-terminal fragment of AChE-R fused to the
DNA-binding domain (BD) (amino acids 1-147) of the yeast GAL4
transcriptional activator. An amplified and CsCl gradient-purified
human fetal brain cDNA library cloned into the AD vector [Chien et
al. (1991) Proc. Natl. Acad. Sci USA 88, 9578-9582] encodes for a
fusion protein with the yeast GAL4 activation domain AD, (amino
acids 768-881). The AH109 yeast strain (Clontech) was sequentially
transformed with the pGBK-ARP1 plasmid, and with 10-25 .mu.g of the
library DNA, using the Yeastmaker transformation system (Clontech).
A total number of 240,000 independent clones were screened.
[0481] Preparation of Recombinant RACK1
[0482] A plasmid overexpressing MBP-RACK1 in E. coli pDEM31, a
derivative of pMAL-c2 (New England Biolabs, Beverly, Mass.)
[Rodriguez et al. (1999) id ibid.], was a kind gift from Dr. Daria
Mochly-Rosen, Stanford. The pDEM31 vector expresses in E. coli
recombinant RACK1 fused to the maltose binding protein, which was
purified on an amylose affinity column (New England Biolabs). The
36 kDa RACK1 protein was released by proteolysis with factor Xa
(New England Biolabs).
[0483] Cell Transfection Experiments
[0484] PC12 cells were transiently transfected with the plasmid
encoding AChE-R, using Lipofectamine Plus (Life Technologies,
Paisley, UK). Cells were lysed 24 hours following transfection in
lysis buffer (0.1M phosphate buffer pH 7.4, 1% Triton X-100, and
Complete mini protease inhibitor cocktail (Roche, Mannheim,
Germany)). Cell debris was removed by centrifugation at
12,000.times.g for 10 min.
[0485] Overlay Assay
[0486] Protein samples containing recombinant RACK1 were separated
by SDS-PAGE. Following blotting, the nitrocellulose membrane was
incubated in a blocking solution (3% non fat dried milk, 2% BSA,
0.2% Tween-20 in Tris buffered saline (TBS, 0.1M tris pH 7.4, 1.7M
NaCl)) for 1 hour. Overlay was in 6 ml of 1:20 diluted clear
supernatant from homogenates of PC12 cells expressing either human
AChE-R [Grifman et al. (1998) Proc Natl Acad Sci USA 95, 13935-40].
The final protein concentration was 2mg/mL, in 50 mM Tris-HCl, pH
7.5, 0.2 M NaCl, 0.1% BSA, 0.1% polyethylene glycol (PEG), 12 mM
beta-mercaptoethanol and Complete mini protease inhibitors cocktail
(Roche), in a final concentration of 0.05% Triton X-100. Following
incubation (lh, room temperature), unbound material was removed by
3 brief washes and three 5 min washes in 0.05% Tween-20 in TBS.
Following fixation with 4% paraformaldehyde (30 min, at 4.degree.
C.), bound AChE was detected using goat polyclonal antibodies
targeted to the N-terminal domain of hAChE (Cat. No. sc-6431, Santa
Cruz Biotechnology, Santa Cruz, Calif.; dilution 1:500).
[0487] Co-immunoprecipitation
[0488] Clear supernatants of PC12 or COS cell homogenates (200
.mu.L, 1.5 mg protein/mL) were prepared by manual homogenization,
followed by 30 min centrifugation at 12,000.times.g, 4.degree. C.
Supernatants were diluted 5-fold with NET buffer (50 mM Tris-HCl,
pH 7.4, 150 mM NaCl, 1 mM EDTA, 0.25% gelatin and Complete mini
protease inhibitors cocktail (Roche)), in a final concentration of
0.05% Triton X-100). Goat polyclonal antibodies (Santa Cruz)
targeted to the N-terminal domain of hAChE (10 .mu.L, 200 .mu.g/mL)
were added for overnight rotation at 4.degree. C. 75 .mu.L of
Protein G MicroBeads (Miltenyi Biotec, Bergisch Galdbach, Germany)
was added and incubation continued for another h. Mixtures were
loaded on MACS magnetic separation columns (Miltenyi Biotec),
washed 3 times with 200 .mu.L of TBS buffer containing 0.05%
Tween-20 and eluted with gel loading buffer. Elutes were separated
by sodium dodecyl sulfate polyacrylamide gel electrophoresis
(Bio-Rad, Hercules, Calif.), blotted and incubated with the noted
detection antibodies. For immunoprecipitation, dissected mouse
brain regions were homogenized in nine volumes of the lysis buffer.
Homogenates were passed several times through a 21 G needle.
Insoluble debris was removed by a 30 min 12,000.times.g
centrifugation. Homogenates were kept frozen at -70.degree. C.
until use.
[0489] Laboratorv Animals and Stress Experiments
[0490] Male 6-8 weeks old FVB/N mice were subjected to saline
injection (0.2 ml, intraperitoneal) which induces mild
psychological stress in this stress-sensitive strain. Stressed
mice, control and AChE-R transgenic mice [Sternfeld et al. (2000)
id ibid] were sacrificed 24 h post-injection. To prepare brain
sections, four mice from each line were deeply anesthetized with
Pental (pentobarbitone sodium 200 mg/ml, CTS Chemical industries,
Petach Tikva, Israel) at a dose of 100 mg/Kg and transcardially
perfused with 4% (vol/vol) paraformaldehyde. Brains were post-fixed
by immersion in 4% (vol/vol) paraformaldehyde (overnight,
2-8.degree. C.) and incubated in 12% (vol/vol) sucrose in 0.1M
phosphate buffered saline (PBS). Coronal cryostat sections (30
.mu.m) were floated in PBS and kept at -20.degree. C. in 40%
(vol/vol) ethylene glycol and 1% polyvinylpyrrolidone in 0.1 M
potassium acetate (pH 6.5) until staining.
[0491] Antibodies and Working Dilutions
[0492] Immunohistochemical analyses were essentially as previously
described [Shoham and Ebstein (1997) Exp. Neurol. 147, 361-76;
Sternfeld et al. (2000) id ibid.], using rabbit anti-ARP [Sternfeld
et al. (2000) id ibid.] 1:100, rabbit anti PKC.beta.II (Cat. No.
sc-210, Santa Cruz) 1:100, rabbit anti PKC.beta.II (Sigma-Rehovot,
Israel) 1:250 and mouse anti RACK1 (Cat. No. R20620, Transduction
Laboratories, San Diego, Calif.) 1:200. Immunoblot analyses were
with rabbit anti-N-terminus AChE antibodies (Cat. No. N-19, Santa
Cruz), 1:500; mouse monoclonal antibody against all isoforms of PKC
of mouse, rat and human origin (Cat. No. sc-80, Santa Cruz),
dilution 1:100, or mouse monoclonal antibody against RACK1
(R20620,Transduction Laboratories), dilution 1:2500.
[0493] Sections were incubated with the primary antibody and then
with biotin-conjugated donkey anti-rabbit antibody (Cat No. AP132B,
Chemicon, Temecula, Calif.; 1 hour, room temp., overnight at
2-8.degree. C.) and extravidin-peroxidase (Sigma). RACK1 staining
was further preceded by trypsin type II treatment (Sigma), 1
.mu.g/ml with calcium chloride 0.001% for 2 min, at room temp.,
which required the addition of 0.001% soybean trypsin inhibitor
(Sigma) during staining. Detection was with horseradish
peroxidase-conjugated goat anti-mouse antibody (1:100 dilution,
Sigma). Pre-incubation of anti-RACK1 with 10 .mu.M RACK1 for 1 h at
room temp totally eliminated staining with anti-RACK1,
demonstrating specificity. For all antibodies, staining was
intensified with 0.075% diaminobenzidine and 0.05% nickel ammonium
sulfate.
[0494] Fluorescence Double Labeling of RACK1, ARP and
PKC.beta.II
[0495] RACK1 and ARP: Primary staining solutions contained 0.001%
trypsin inhibitor (Sigma type IIS), 0.3% Triton X100, 0.05% Tween
20, 2% normal goat serum, 2% normal donkey serum, rabbit anti-ARP1
(1:100) and mouse anti-RACK1 (1:100). The secondary antibody
solution contained 0.3% Triton X100, 0.05% Tween 20, 2% normal goat
serum, 2% normal donkey serum, donkey-anti-rabbit conjugated to
fluorescein (Chemicon, AP182F) diluted 1:100 and goat-anti-mouse
conjugated with tetra-methyl-rhodamine (Sigma, T7782) diluted
1:800. Sections were mounted on SuperFrost slides (Menzel Glaser,
Freiburg, Germany), air-dried, covered in ImmuMount (Shandon,
Pittsburgh, Pa.) and covered for microscopy.
[0496] PKC.beta.II and ARP1: The primary staining solution
contained 0.3% Triton X100, 0.05% Tween 20, 2% normal goat serum,
2% normal donkey serum, rabbit anti-ARP (1:100) and
mouse-anti-PKC.beta.II (Sigma, P8083), diluted 1:500. Secondary
antibody solutions and preparation for microscopy were as specified
above for ARP and RACK1.
Example 1
Hydrocortisone Elevates ACHE Gene Expression in Hematopoietic Stem
Cells
[0497] This example relates to ACHE gene expression in
hematopoietic stem cells and the influence of hydrocortisone
thereon. The inventors have searched the extended promoter of the
human ACHE gene (cosmid accession no. AF002993) for consensus
motifs that may bind stress-associated and hematopoietic
transcription factors. Two such clusters, one located 17 Kb
upstream from the transcription start site, and another positioned
at the first intron, were found to include motifs for AP1, NFkB,
EGR-1 (as identified by a matrix search against the TransFac
database, Heinemeyer et al., Nucleic Acids Res. 26, 364-370, 1998),
interleukin-6 (IL6), with the consensus sequence CTGGG/AAA,
glucocorticoid responsive element (GRE) half palindromic site,
TGTTCT, and Stat-5, TTCCCAGAA or TT(C/A)(C/T)N(A/G) (G/T)AA (FIG.
1A). Of these, the latter two motifs are known to be actively
involved in hematopoiesis (Darnell et al., Science 264, 1415-21,
1994) and cellular stress responses (Tronche et al., Curr. Opin.
Genet. Dev. 8, 532-8, 1998). Moreover, they act synergistically in
enhancement of .beta.-casein gene expression in hematopoietic cells
(Lechner et al., Immunobiology 198, 112-23, 1997) but have not yet
been studied in the context of AChE involvement in
hematopoiesis.
[0498] FIG. 1A is a scheme of the upstream human ACHE sequence
including clusters of hematopoietic and stress-related motifs.
Depicted is a scheme of the reverse sequence of the cosmid insert
(accession No. AF002993) of the human ACHE promoter. The arrow
represents the position of a transcription start site. Two
potentially relevant regions are shown, one beginning at nucleotide
5267 and one following the first exon (black box). Fully conserved
consensus sequences are marked by triangles. These include AP-1,
NF-.kappa.B, EGR-1, IL-6, glucocorticoid responsive element (GRE)
half-palindromic site, and Stat-5.
[0499] The functional effects of the glucocorticoid-binding motifs
in the ACHE upstream sequence (FIG. 1A) were investigated in human
UCB CD34.sup.+ stem cells isolated by anti-CD34-coated immunobeads
to yield a 85.+-.3% pure population, as confirmed by flow
cytometry. CD34.sup.+ cells were enriched from human UCB cells
using bead-attached antibodies to the CD34 protein.
[0500] FIG. 1B shows a representative flow cytometry of the
recovered cells, demonstrating that 89% of them express the CD34
antigen. The inset in FIG. 1B shows an example photograph of
enriched CD34.sup.+ cells stained by May-Grunwald-Giemsa. Note the
large nuclei surrounded by thin rims of cytoplasm, characteristic
of stem cells.
[0501] Enriched CD34.sup.+ cells were subjected to cytochemical
staining for AChE catalytic activity in the presence of 10.sup.-5 M
iso-OMPA (FIG. 1C, ISO) or BW284C51 (FIG. 1C, BW), selective
inhibitors of BuChE and AChE, respectively. Nuclear staining (FIG.
1C, right) was with DAPI. Note the selective appearance of brown
precipitates of AChE, but not BuChE reaction products. The data
shown in FIG. 1C demonstrate that CD34.sup.+ cells contain
cytochemically detectable levels of catalytically active AChE. The
identity of their cholinesterase as AChE was verified by its
sensitivity to the AChE-specific inhibitor BW284C51 and its
resistance to the butyryl-cholinesterase (BuChE) inhibitor iso-OMPA
(FIG. 1C).
[0502] The assumption that hematopoietic ACHE gene expression is
modulated under stress was tested in CD34.sup.+ cells cultured for
24 hr with increasing doses of hydrocortisone. Treated cells were
subjected to cytochemical staining for AChE activity as well as to
high resolution in situ hybridization followed by confocal
microscopic quantification of labeling density. This method
provides an accurate and credible tool for the examination of
transcriptional responses in the heterogeneous population of
primary HSCs from different individuals. Hybridizations were
performed for each of the three transcripts of human AChEmRNA
presented in FIG. 1A (S, E and R). Because each cRNA probe has its
own characteristic hybridization affinity, each transcript was
quantified separately. Individual CD34.sup.+ cells were treated
with the noted doses of hydrocortisone at levels equivalent to
physiologically normal, intermediate and stress conditions (0.1,
0.6 and 1.2 .mu.M, respectively; see De Vroede et al., ibid, 1998).
Cells were subjected to in situ hybridization with the noted
AChEcRNA probes, followed by confocal microscopy, projection of
image slices, quantification and color-coding of the labeling
signals.
[0503] FIG. 1D presents cytochemically stained cells (top) and
representations of 3-dimensional projections created from
confocally scanned sections of CD34.sup.+ cells following in situ
hybridization with 5'-biotinylated AChEcRNA probes selective for
the "synaptic" AChE-S mRNA variant, the "erythrocytic" AChE-E mRNA
variant encoding for glycophospholipid-anchored AChE-E and the
"readthrough" AChE-R mRNA form associated with stress. Detection
was by appearance of Fast Red precipitates [Grisaru et al. (1999b)
id ibid.]. Note increasing cytoplasmic labeling under high
hydrocortisone levels. Each photograph represents one out of 10-20
analyzed cells with deviations in labeling of less than 6%. FIG.
1D, left, bottom shows the relative increases in percent above
control for each of the analyzed transcripts under stress-relevant
concentrations. Note the accumulation of AChE-R mRNA transcripts
under moderate hydrocortisone concentrations.
[0504] FIG. 1D demonstrates that subtle elevation of hydrocortisone
concentration to 0.60 .mu.M induced a 40% selective increase in the
"readthrough" AChE-R mRNA transcript above the level observed under
non-stress hydrocortisone concentration (0.10 .mu.M) [De Vroede et
al. (1998) Arch. Dis. Child 78, 544-7]. However, at 0.60 .mu.M
hydrocortisone, no change was observed in enzyme activity of
CD34.sup.+ cells. In contrast, stress-associated hydrocortisone
levels (1.2 .mu.M) enhanced the labeling of all 3 AChEmRNA
transcripts and intensified the catalytic activity of the stem
cell-associated enzyme. The AChE-R mRNA-specific in situ
hybridization, therefore detects a clear increase in this variant
against a low background, while total cell-bound enzyme activity
registers no deviation from background at a sub-stress
hydrocortisone level.
Example 2
Expression of ARP in CD34.sup.+ Cells
[0505] The expression of ARP in CD34.sup.+ hematopoietic cells was
evaluated by flow cytometry in whole cord blood and bone marrow
from a patient with immune thrombocytopenic purpura (ITP), as
demonstrated in FIG. 2A and FIG. 2B respectively. Bone marrow from
ITP patients was chosen to study ARP expression in hematopoietic
progenitor cells due to the high turnover of normal CD34.sup.+ in
these patients. Cells were fixed and permeabilized with Fix and
Perm (Caltag, Calif., US) and stained with monoclonal antibodies to
CD34 conjugated to pycoerythrin (Beckton Dickinson, California, US)
indicated as FL-2 and with highly specific rabbit anti-ARP
antibodies followed by anti rabbit antibodies conjugated to
fluorescein isothiocyanate, expression indicated as percentage of
positive cells.
[0506] These findings demonstrate higher expression of AChE-R in
proliferating hematopoietic progenitors from either newborns or
individuals suffering from over-proliferation of blood cells.
Example 3
Readthrough AChE is Overproduced in the Myeloidogenic Mid-gestation
Liver
[0507] To study the relevance of each of the AChEmRNA transcripts
during development of the hematopoietic organs, in situ
hybridization was performed on paraffin-embedded sections taken
from human fetuses at different gestational ages. Consistent with
the embryonic spatiotemporal shifts in blood cell forming tissues,
we observed changes in the labeling intensity with the various
probes used, in the aorta-gonad-mesonephric region (AGM), liver,
spleen and bone marrow cells. FIG. 3A schematically presents the
migration of hematopoiesis between the various blood cell forming
tissues during fetal development. The top left of the figure
represents a sagital section of a human embryo showing the
hematopoietic organs--AGM (aorta-gonad-mesonephros), LIV (liver),
SPL (spleen), and BM (bone marrow). The top right of the figure is
a scheme of gestational shifts in hematopoietic processes which
shows the relative intensity of blood cell formation in the various
hematopoietic organs throughout human gestation. (according to
Tavassoli et al., Blood Cells 17, 269-81, 1991, Tavian et al.
Development 126, 793-803, 1999). Ages of embryos on which in situ
hybridization was performed are marked by gray columns.
[0508] FIG. 3B presents in situ hybridization results and the
average labeling intensities for the AChE-S, AChE-E and AChE-R mRNA
transcripts in AGM (triangles, week 9), liver (diamonds), spleen
(squares) and bone marrow (triangles, weeks 20-25) of human fetuses
at different gestational ages (right side curves). The figure shows
representative in situ hybridization micrographs from the noted
tissues of human fetuses at the noted gestational ages, using
selective probes for each of the above alternative human AChEmRNA
transcripts. The right side of the figure shows spatiotemporal
changes in labeling intensity for each probe and organ. Note that
AChEmRNA expression increases parallel to active hematopoiesis in
the examined organs.
[0509] For the AChE-S and AChE-E probes, expression levels were
distributed similarly in liver and spleen. For example, labeling
intensity for both these probes was high in mid-gestation liver and
spleen, when the principal hematopoietic activity was
erythropoiesis, and labeling of both decreased steadily from the
9th week onward, as myelopoiesis became more prevalent. In
contrast, the AChE-R transcript was detected only during the 16
week transition from erythro- to myelopoiesis in the mid-gestation
liver and not in the spleen (Porcellini et al., Int. J. Cell
Cloning 1, 92-104, 1983). The unique expression pattern of AChE-R
mRNA and its apparent correlation with myeloidogenesis demonstrated
that AChE-R acts as a selective hematopoietic element.
Example 4
ARP Sustains Cell Expansion and Differentiation
[0510] The predicted secondary structure of peptides ARP and ASP
was analyzed. FIG. 4A presents the amino acid sequences of ARP and
ASP (26 and 40 residues, respectively). Secondary structure
predicted using the peptide structure program of the GCG software
package (University of Wisconsin) was based on the Chou-Fasman
method. Depicted below the sequences are the secondary structures
predicted: T, turn, B, .beta.-sheet and H, .alpha.-helix, with
lower case letters representing lower predicted probability. Note
the predicted helix structure for the first 17 residues of ASP,
drawn using the Helicalwheel program of the GCG software package.
The amphipathic nature of this region is postulated based on the
unilateral positioning of hydrophobic residues (F, L, W, W, A).
[0511] Both the "synaptic" exon 6-derived and the "readthrough"
pseudointron 4-derived peptides (ASP, ARP) include a major region
predicted to be rich in turns and .beta.-pleated sheets; in
addition, the longer ASP peptide is predicted to contain a
unilaterally hydrophobic .alpha.-helical domain with amphipathic
properties (FIG. 4A). To test whether either of these peptides has
biological activity, the inventors added HPLC-purified synthetic
peptides (in 50 and 100 ng/ml final concentrations) once every 4
days to the growth medium in which isolated HSCs (CD34.sup.+) were
cultured for 2 weeks. FIG. 4B shows fold expansion values of viable
cells, based on trypan blue exclusion (average of 4-5
experiments.+-.standard error of the mean, SEM) grown for 2 weeks
in the presence of the noted growth factor or mixtures of factors
and, where marked, 50 ng/ml ASP or ARP. Asterisks note statistical
significance of the measured increases in cell counts as compared
to cultures without the peptide (p.ltoreq.0.05).
[0512] The effect of ARP on cell proliferation was further analyzed
using BrdU incorporation assay. BrdU incorporation was measured by
5-Bromo-2'-deoxy-Uridine Labeling and Detection Kit III, Roche.
[0513] As shown in Table 1, ARP, but not ASP, facilitates BrdU
incorporation into cord blood progenitors. ARP induces similar
effects on BrdU incorporation into CD34.sup.+ progenitors from the
peripheral blood of adult donors. TABLE-US-00002 TABLE 1 The effect
of ARP on BrdU incorporation to CD34.sup.+ cells hrs post- P
plating ARP, 2 nM ASP, 2 nM None 16 0.110 .+-. 0.005 0.095 .+-.
0.004 0.135 .+-. 0.005 0.05 24 0.275 .+-. 0.045 0.182 .+-. 0.018
0.212 .+-. 0.026 0.018 36 0.423 .+-. 0.099 0.260 .+-. 0.022 0.246
.+-. 0.035 Shown are BrdU incorporation values following the noted
incubation times.
[0514] In addition, the effect of ARP was examined in transformed
bone marrow endothelial cells (Schweitzer et al., Lab Invest vol.
76, 5-36:1997). Cells were incubated in a serum free medium (SFM)
with 2 nM of ARP, with or without endothelial growth factors (bFGF
20 ng/ml and EGF 10 ng/ml), for 48 hrs. As shown in FIG. 5, BrdU
uptake increased in ARP presence. The effect was more pronounced
when ARP was combined with bFGF and EGF.
[0515] Thus, ARP may serve as a proliferating factor for
endothelial cells, having the most dramatic effect when working in
synergy with the "classical" endothelial growth factors.
[0516] Addition of ARP alone increased the number of viable cells
more than 10-fold (FIG. 4B; p<0.008). ARP further improved
expansion of viable cells when it was administered in combination
with SCF, granulocyte-macrophage colony stimulating factor
(GM-CSF), bFGF and EGF or thrombopoietin (TPO). However, its growth
factor-accessory effect reached statistical significance only with
TPO (p<0.01). Similar doses of ASP were less effective than ARP
in promoting cell expansion, alone or when added with any
combination of these cytokines for 2 weeks (FIG. 4B and Table
1).
[0517] The compatibility of the ARP-supported expansion with
differentiation was tested by quantifying the myeloid (CD33+) and
megakaryocytic (MK, CD41+) cells after 2 weeks in liquid culture
(Table 2). Cells expanded under the influence of ARP displayed
increased ability to differentiate into MK and myeloid progeny.
Moreover, ARP potentiated the effect of TPO to enhance the number
of MKs. Surprisingly, the TPO-potentiating effect of ARP was found
to be more pronounced than the TPO-potentiating effect of stem cell
factor (SCF). SCF is a known TPO potentiating agent for stem cells
(Deutsch et al., Med. Oncol. 13, 31-42, 1996). ARP also facilitated
the capacity of GM-CSF and SCF to support myelopoiesis. Thus, the
effects of ARP over MK (p=0.08, paired Student's t-Test) and
myeloid (p=0.04) expansion are independent. Further, the effect of
ARP on the megakaryocytopoietic capacity of TPO and SCF (p=0.03) is
synergistic. The myeloid potentiation capacity of ARP over that of
GM-CSF and SCF is additive. TABLE-US-00003 TABLE 2 ARP potentiates
TPO, GM-CSF and SCF effects on megakaryocytic and myeloid
lineages.sup.a cell type cytokine MK (CD41.sup.+) Myeloid
(CD33.sup.+) None 0.03 .+-. 0.02 1.50 .+-. 0.79 ARP 8.20 .+-. 4.50
2.90 .+-. 0.65 TPO 3.93 .+-. 3.56 3.51 .+-. 2.23 TPO + ARP 48.43
.+-. 34.94 2.95 .+-. 0.64 GM-CSF 14.50 1.35 GM-CSF + ARP 7.80 1.98
SCF 0.30 .+-. 0.21 3.90 SCF + ARP 0.90 .+-. 0.27 6.49 SCF + TPO
20.60 .+-. 20.04 0.68 .sup.aPresented are fold expansions (and,
where noted, SEMs) of 2-week primary cultures of CD34.sup.+ cells
from 1 to 3 individuals grown with the noted cytokines. ARP, AChE
C-terminal "readthrough" peptide; TPO, thrombopoietin; GM-CSF,
granulocyte-macrophage colony stimulating factor; SCF, stem cell
factor; MK, megakaryocyte. Potentiated expansion values are
highlighted in bold letters.
Example 5
Autoregulatory Effect of ARP
[0518] ARP was added in concentrations of 50 or 100 ng/ml to
cultured CD34.sup.+ cells and the levels of the various AChEmRNA
transcripts after 24 hr were examined, by in situ hybridization
combined with confocal microscopy analysis. FIG. 6 (left) shows
representative individual CD34.sup.+ cells treated for 24 hr with
the noted doses of ARP in the absence of other growth factors and
subjected to in situ hybridization with probes selective for each
of the alternatively spliced variants of AChEmRNA. The right side
of the figure shows average labeling densities for 10-20 cells in
each case. FIG. 6 demonstrates similar increases for all 3
transcripts (S, E ,R) with peak activity at 50 ng/ml ARP. Note the
concomitant increases in all transcripts, and the uniform nature of
this response in all of the analyzed cells. This suggests that ARP
stimulates transcriptional enhancement of the AChE gene.
Autoregulatory continuation of AChE-R production could sustain the
ARP effect long after the initial ARP signal has been
terminated.
Example 6
ARP Retrieves the Antisense-suppressed Cell Proliferation Effect of
GM-CSF
Antisense Suppression of AChE-R Production
[0519] The ARP-induced enhancement of ACHE gene expression
suggested that the AChE-R protein and not necessarily ARP, may be
responsible for the sustained viability and the significant
expansion of HSCs. To distinguish between these two possibilities,
the inventors employed antisense oligodeoxynucleotides (AS-ODN) to
selectively suppress AChE-R mRNA levels, reduce the intracellular
production of AChE-R and test, under these conditions, the
proliferative effects of GM-CSF with or without ARP. AChE-R mRNA
includes a 1,094 bp 3'-untranslated region (UTR), with 62% G,C
content. This marks it as a more vulnerable molecule to nucleolytic
degradation than AChE-S mRNA, which includes a 219 bp UTR with 66%
G,C. A. FIG. 7A shows AS-ODNs targeted to the common sequence
domain of mRNA transcripts with variable UTRs. Shown are schematic
structures of the two human cholinesterase genes, ACHE and BCHE.
Exons are colored or gray, introns are shown in white. The open
reading frame (ORF) is drawn above each gene, and the positions and
predicted structures of the AS-ODNs that were employed are drawn
below. Also marked are the UTRs for the two AChEmRNA transcripts,
AChE-S (UTR=219 bp, 66% G, C) and AChE-R (UTR=1,094 bp, 62% G,
C).
[0520] To selectively reduce AChE-R mRNA levels in HSCs, extremely
low doses (20 pM) of anti-AChEmRNA AS-ODNs [Grisaru et al. (1999b)
id ibid.] were employed. AS1 and AS3 are 2'-O-methyl-protected
AS-ODNs targeted to ACHE exon 2, which is common for AChE-S and
AChE-R mRNA. An irrelevant AS-ODN (ASB) targeted to BuChE mRNA
served as a control (FIG. 7A and [Grisaru et al. (1999b) id ibid.].
FIG. 7B shows selective susceptibility of AChE-R mRNA to AS-ODN
destruction. CD34.sup.+ stem cells were treated for 24 hr at
37.degree. C. with 20 pM 2'-O-methylated AS-ODNs targeted to
AChEmRNA or BuChEmRNA. Shown are DAPI and AChE activity stainings
(left) and confocal images of in situ hybridizations for the AChE-S
and AChE-R transcripts (right) with 20 pM of the ASB, AS1 or AS3
AS-ODNs. Columns show average levels of staining efficiencies for
10-20 cells hybridized with each of the transcript-specific probes.
Note maintenance of cell-associated AChE activities and stable
levels of AChE-S mRNA under all treatments as opposed to selective
reduction of AChE-R mRNA under AS1 treatment. Thus, AS1, but not
AS3 reduced the in situ detected AChE-R mRNA levels in CD34.sup.+
cells under conditions where AChE-S mRNA levels remained unchanged
(FIG. 7B). The irrelevant ASB ODN was ineffective, demonstrating
sequence specificity of the AS1 effect.
ARP Retrieves the Antisense-suppressed Cell Proliferation Effect of
GM-CSF
[0521] Cell proliferation was evaluated by measuring BrdU
incorporation following 16 hr incubation in the presence of 20 pM
of the noted AS-ODNs with or without 50 ng/ml ARP and/or GM-CSF.
FIG. 7C shows average results of 3-6 reproducible experiments
.+-.SEM. Consistent with its expansion effect, incubation with
GM-CSF increased the incorporation of bromodeoxyuracil (BrdU) into
CD34.sup.+ cells over 16 hr (FIG. 7C). Addition of 50 ng/ml ARP
together with GM-CSF significantly potentiated this incorporation
(p<0.03), whereas ARP, AS1, AS3 or ASB did not affect BrdU
incorporation when added alone to the cells (FIG. 7C). The capacity
of GM-CSF to enhance BrdU incorporation was totally suppressed when
it was added together with 20 pM AS1. The suppressive effect of AS3
on GM-CSF-induced enhancement of BrdU incorporation, was much
weaker than that of AS1, consistent with its inability to suppress
AChE-R mRNA levels in CD34.sup.+ cells. To examine whether ARP
alone was required and sufficient to facilitate the cell
proliferation effect of GM-CSF, the inventors incubated the cells
with GM-CSF and ARP together with the suppressive AS1. ARP
completely reversed the AS1-induced suppression in BrdU
incorporation, retrieving the full capacity of GM-CSF to enhance
cell proliferation (FIG. 7C). Thus, the data show that ARP enhances
the GM-CSF-supported increases in cell proliferation, AS1 reduces
this enhancement far more effectively than AS3, and that ARP
retrieves the AS1-suppressed proliferation.
Example 7
ARP can Substitute for Stem Cell Factor
[0522] To determine whether the ARP expansion effects could replace
any of the known growth factors, the inventors tested ARP alone or
combined with known growth factors, on long-term CD34.sup.+ cell
cultures. FIG. 8A shows cell counts from long-term CD34.sup.+
liquid cultures grown in the absence of growth factors (diamonds),
in the presence of early-acting cytokines (EAC: IL3, IL6, TPO and
FLT3) and SCF (squares), or in the presence of EAC+ARP with SCF
(circles) or in the presence of EAC+ARP without SCF (triangles).
Viable cell counts are depicted in the upper left part of FIG. 8A.
CD34+ cell counts are presented in the upper right part of the
figure. The lower left and right parts are graphs of the number of
Granulocyte-Macrophage (GM) or Megakaryocyte (MK) progenitor colony
forming units.
[0523] FIG. 8A, upper left, shows that early-acting cytokines (a
mixture of IL3, IL6, TPO and FLT3) promote linear expansion of
CD34.sup.+ cells for up to 28 days. In the absence of this mixture,
there was no proliferation. SCF, although devoid of proliferative
activity by itself, enhances significantly the proliferation
induced by the above growth factors (Li and Johnson, Blood 84,
408-14, 1994). Addition of ARP, with or without SCF resulted in an
enhanced cellular proliferation, leading in both cases to a greater
than 2000-fold expansion within 28 days (FIG. 8A, upper left). This
demonstrates that the activity of ARP was additive to that of the
early-acting cytokines and that it could replace SCF.
[0524] FIG. 8A, upper right, shows that ARP operates as a
CD34.sup.+ survival factor. Note that CD34.sup.+ cell numbers reach
a plateau at 21 days in the presence of EAC (squares), and that ARP
facilitates further increases in CD34.sup.+ counts up to at least
28 days, regardless of the presence of SCF (triangles, circles).
Thus, the conclusions drawn above from the results of FIG. 8A,
upper left, are supported by the finding that ARP with or without
SCF promoted, with similar efficacy, the survival of CD34.sup.+
cells within the expanded cultures as compared with survival in the
absence of growth factors (FIG. 8A, upper right).
[0525] FIG. 8A, lower part, show that ARP increases the number of
GM and MK progenitors. Shown are counts of colony forming units for
GM (left) or MK (right) colonies grown from progenitors removed at
13, 21 and 28 days of the primary expansion phase detailed under
FIG. 8A. The numbers of colonies grown after EAC, EAC+SCF+ARP or
EAC+ARP treatment are very similar, suggesting redundant expansion
properties for SCF and ARP.
[0526] FIG. 8B shows that ARP facilitates development of hematon
bodies. Representative photographs of the 28-day liquid cultures
detailed in FIG. 8A above are shown. In the absence of growth
factors, sparse hematopoietic cells and many fibroblasts are seen
(control, upper left). Addition of EAC increases the density of
small, round hematopoietic stem cells and sparse MKs (FIG. 8B,
upper right, white arrow). FIG. 6B, lower half, demonstrates that
EAC +ARP facilitate the formation of hematon bodies (insets)
without (right) or with SCF (left). FIG. 8A, lower part, shows that
ARP increases the number of GM and MK progenitors. Shown are counts
of colony forming units for GM (left) or MK (right) colonies grown
from progenitors removed at 13, 21 and 28 days of the primary
expansion phase detailed under FIG. 8A. The numbers of colonies
grown after EAC, EAC+SCF+ARP or EAC+ARP treatment are very similar,
suggesting redundant expansion properties for SCF and ARP.
[0527] In summary, CD34.sup.+ cultures grown without growth factors
for 28 days displayed typical fibroblast morphology (FIG. 8B, Upper
left). In contrast, a dense population of small, round cells, with
characteristic stem cell morphology, was observed in cultures grown
for the same period in the presence of the early-acting cytokines
(FIG. 8B, Upper right). The addition of ARP, in the presence or
absence of SCF, sustained this stem cell morphology (FIG. 8B, Lower
left and right). Interestingly, floating "hematons", which are
independent hematopoietic units rich in myeloid, erythroid and
megakaryocyte progenitor cells (Blazsek et al., Exp. Hematol. 23,
309-19, 1995) were found in the ARP-containing cultures,
demonstrating the differentiation potential of this peptide (FIG.
8B, lower part, insets).
Example 8
ARP-treated Cells Maintain Multipotent Progenitor Properties
[0528] To test the number of progenitors and differentiation routes
available to ARP-treated cells, the inventors subjected the above
cultures to a second expansion phase. Cells removed once a week
from the primary liquid cultures were grown in the absence of ARP
in a semi-solid substrate. In the absence of the growth factor
mixture, there was no secondary expansion. IL3 and GM-CSF were used
to induce granulocyte-macrophage (GM) expansion and TPO and SCF was
used for megakaryocyte (MK) expansion. During this second expansion
phase, blood cell progenitors that had previously been treated with
early-acting cytokines developed into either GM or MK colonies
(FIG. 8B, lower part), depending upon the added growth factor. The
numbers of GM and MK colonies peaked by 3 weeks and were
essentially the same in cultures that were previously treated with
all of the early acting cytokines, with or without ARP.
ARP-supported hematopoiesis thus appeared to maintain normal growth
of differentiated myeloid and megakaryocyte colonies.
[0529] These tests provide evidence for maintenance of all type of
progenitors in ARP-treated cultures. TABLE-US-00004 TABLE 3 The
effect of various conditions on cultured cell count.sup.a
CD33.sup.+ and Total CD34.sup.+ CD33.sup.+ CD15.sup.+ viable (early
(early (total CD41.sup.+ Treatment cells progenitors) myeloids)
myeloids) (megakaryocytes) Control 61.0 1.0 7.2 12.3 30.9 ARP, 2 nM
570.0 87.2 329.0 530.0 42.3.sup.b cortisol, 1.2 .mu.M 80.0 13.0
45.0 73.0 10.1.sup.b ASP, 2 nM 100.0 7.2 10.0 13.0 4.6.sup.b SCF,
50 ng/ml 118.0 6.3 69.0 72.0 2.6.sup.b AS1, 20 pM 81.2 1.4 2.4 5.0
30.9 PBAN, 2 nM 105.0 1.7 1.6 3.1 52.9 .sup.aCultures were seeded
at 50,000 cells/well. Shown are cells per culture .times. 10.sup.-3
on day 14; 1 of 3 reproducible experiments as in the figure above.
.sup.bThese are also CD34.sup.+ positive early cells with expansion
potential.
Example 9
In vivo Effects of ARP and ASP on Embryonic Brain Development
[0530] In order to study the possible involvement of ARP in the
embryonic development process, the anti-ARP and anti-ASP antibodies
have been used to label structures in the embryonic mouse cortex.
These antibodies can label either cell types that produce AChE-R
and AChE-S, or cells that have binding sites for the ARP and ASP
peptides.
[0531] In the dorsolateral neurocortex, neurons migrate from the
lateral cortical stream toward the outer perimeter of the brain.
FIG. 9A shows that at embryonic day 14, the neuron bodies, which
lie toward the perimeter of the brain, have AChE-S, whereas, the
entire region, from the subventricular zone to the perimeter,
expresses AChE-R.
BrdU Labeling in Developing Brain
[0532] In order to study the effect of ARP on mitotic activity of
developing mouse brain, a BrdU incorporation analysis was
performed. The C-terminal peptides ARP or ASP were injected 0.1
mg/Kg into a pregnant mouse; 24 hr later, the BrdU was injected and
after 1 hr the embryo was isolated and fixed for examination. To
show specificity of the results for the peptides, the experiments
were performed in the presence of antisense oligonucleotides. The
AS3 ODN, 2 nM, was injected into a pregnant mouse and 5 hr later
the BrdU. After 1 hr the embryo was isolated and fixed. Controls
were saline injection or an ODN with the inverse sequence of the
AS3. Labeled neurons (positive for BrdU incorporation) were counted
to assess neuronal proliferation.
[0533] Embryonic neuroepithelial cells have one of two fates: (1)
to continue proliferation and migration up and down from the
ventricular zone up to the cortical plate, or (2) to quit the
proliferative cycle and initiate terminal differentiation. The
balance between these two processes determines the number of
proliferating neuronal progenitors as well as the thickness of the
cortical plate where post-mitotic neurons accumulate (see scheme at
FIG. 10).
[0534] As shown in FIG. 9B, ASP has minor positive effect on
proliferating neurons, evidenced in the increased number of BrdU
labeled nuclei. ARP enhances neuronal proliferation, yet more
significantly, while reducing the thickness of the cortical region
harboring differentiating post-mitotic neurons.
[0535] AS3, an ODN directed toward a sequence common to both AChE-S
and AChE-R, much more effectively suppresses the mRNA of AChE-R
than that of AChE-S [Shohami et al. (2000) J. Mol. Med. 78,
228-36]. The suppression of AChE-R is correlated with an increased
thickness of the layer of post-mitotic neurons compare to control
sections (reproducible outcome from one of three animals used for
each treatment).
Immunolabeling of ARP in Treated Brain
[0536] To confirm that the antisense treatment suppresses the level
of ARP production and that this occurs through destruction of
AChE-R mRNA, in situ hybridization and immunolabeling were
performed.
[0537] FIG. 11A presents labeling pattern of the embryonic brain
stained with the anti-ARP antibody after AS3 treatment (right) or
in controls (left). This Figure demonstrates that the AChE protein
is suppressed by the AS-ODN treatment, and that in the developing
cortex, it was concentrated mainly in post-mitotic neurons but may
also be visualized along the migratory pathway leading to this
layer from the ventricular zone.
[0538] Furthermore, FIG. 11B presents an in situ hybridization
analysis showing antisense suppression of AChE-R mRNA in the
embryonic brain. Also here, post-mitotic neurons are the only cells
to express AChE-R mRNA and this expression is completely suppressed
following AS3 treatment.
Example 10
In vivo ARP Effects
ARP Accumulates in the Serum Under Stress and Facilitates the
Stress-induced Hematopoietic Responses in vivo
[0539] To find out whether the ARP peptide occurs naturally in
blood and if its levels increase under psychological stress, FVB/N
mice (n=12) were subjected to confined swim protocol for exerting
acute psychological stress as detailed elsewhere [Kaufer et
al.(1998) id ibid.]. Serum samples removed 24 hr later were
subjected to gradient gel electrophoresis. FIG. 11A, top, shows a
Poinceau-stained polyacrylamide gradient gel (4-20%, Bio-Rad)
loaded with: (1) protein extract from COS cells transfected with
AChE-R encoding plasmid (Ben Aziz-Aloya et al., Proc. Natl. Acad.
Sci. USA 90, 2471-5, 1993, Seidman et al., Mol. Cell.- Biol. 15,
2993-3002, 1995) and mixed with synthetic ARP (ARP+AChE-R); (2)
recombinant AChE-S (Sigma), mixed with synthetic ASP (ASP+AChE-S);
(3) serum (2 .mu.L) from a saline-injected mouse, removed 24 hr
post-treatment (Control); (4) serum from a mouse subjected to
confined-swim stress as described above, removed 24 hr
post-treatment (Stress). Positions of molecular weight markers are
shown on the left. The gel was then electroblotted and
immunodetected (see "immunoblot" in the Experimental Procedures
section for details) with affinity-purified rabbit antibodies
elicited toward a recombinant GST-ARP fusion protein (FIG. 11A,
bottom). A 67 KDa protein, consistent with the expected size of
AChE-R, is detected in the serum (upper arrow). Furthermore,
selective labeling of synthetic ARP (but not AChE-S or ASP) by this
antibody is detected. Accumulation of ARP in the serum of stressed
mice is evident from the intense labeling of native ARP in the
stressed mouse serum (lower arrow).
[0540] To determine the in viuo capacity of ARP to affect
hematopoietic expansion under acute psychological trauma, mice were
injected immediately after the stress protocol with 0.1 mg/kg ARP
or 30 ng/kg AS1. Another group of mice were not subjected to stress
and were injected intraperitoneally with normal saline (n=6) or ARP
(n=4). 24 hours later, the animals were sacrificed and whole blood
obtained for AChE activity and white blood cells. Bone marrow
smears were subjected to immunohistochemical labeling with an
affinity purified rabbit antiserum prepared against GST-fused
recombinant ARP. FIG. 12B shows the number of labeled cells per 100
cells counted at .times.1000 magnification in 5 different fields.
Bone-marrow labeling and white blood cell (WBC) count were similar
in non-stressed mice regardless of ARP injection. In contrast, ARP
intensified labeling and increased the number of small positive
cells in the bone marrow of stressed mice, indicating that it
enhances AChE expression and increases stem cell expansion in vivo.
AS1 reduced the number of cells labeled with anti-ARP antibodies
(FIG. 12B). In peripheral blood, WBC counts revealed similar
ARP-dependent enhancement and AS1 suppression.
Persistent AChE-R Overproduction Increases Platelet and WBC Counts
in a Dose-dependent Manner
[0541] A series of AChE transgenic mouse pedigrees [Sternfeld et
al. (1998b) id ibid.] was employed, to reveal if chronic increases
in AChE-R would confer persistent changes in blood cell
composition. Blood AChE levels, platelet and WBC counts were
determined in FVB/N mice (Control, n=22) as compared to transgenic
FVB/N mice carrying the AChE-S (TG-S, n=12), AChE-R (TG-R70 and
TG-R45, n=9 and 6, respectively) or inert-inactivated AChE-S
(AChE-Sin, n=3) transgene. FIG. 12C shows results expressed as
average+standard error of the mean (SEM). The transgenic lines
expressing AChE-S variants indicated no increases in blood AChE and
no significant deviations from a normal blood cell composition. In
contrast, increases of 2.5 and 130-fold catalytic AChE activities
were observed in two pedigrees (TG-R45 and TG-70R), whereas WBC
counts were only increased in the more efficiently overproducing
line, suggesting a gene dose dependent effect for ARP over the
hematopoietic balance also under chronic excess conditions (FIG.
12C).
ARP Accumulation in the Serum Under Stress
[0542] The intense labeling of ARP in the unfractionated mouse
serum removed 24 hr following stress treatment revealed more
pronounced increases in this peptide than in its native protein
AChE-R. This may reflect elevated proteolytic activity under
stress. Combined with the absence of cleavage sites for common
proteases within the ARP sequence, this further explains the
reproducible series of proteolytic degradation products of serum
AChE-R which were intensified in the stressed serum samples. The
physiological implications of this finding are that AChE catalytic
activity measurements are underestimates of the extent of its
overproduction in the blood under stress. Likewise, measuring
acetylcholine hydrolysis may underestimate the actual amounts of
the AChE protein and its degradation products in the brain or
muscle. The reported decreases of AChE activity in Alzheimer's
disease may hence mislead researchers and clinicians alike by
masking the accumulation of morphologically active AChE-derived
peptides with long-term effects.
ARP Accumulation in Human Blood Plasma Under Lipopolysaccharide
Exposure
[0543] To test whether ARP accumulation can be observed in
different stress causing situations (as demonstrated above), ARP
expression and AChE activity were analyzed in human serum following
exposure to bacterial lipopolysaccharide (LPS) as a model for
bacterial infection.
[0544] Twenty volunteers, ages 19 to 30 years old were i.v.
injected with a placebo or endotoxin (Salmonella abortus equi., 0.8
ng/Kg body weight). Blood was collected at baseline, and at hourly
intervals up to 10 hr post-injection.
[0545] ARP levels and AChE activity were analyzed, as shown
hereunder. In addition, the emotional and behavioral states of
those twenty volunteers were assessed, as well as rectal
temperature, heart rate and plasma levels of cytokines and cortisol
(not shown).
[0546] FIG. 17A demonstrates analysis of the plasma AChE
activities. The level of AChE activity in all samples was
determined in the presence of 10.sup.-5M iso-OMPA and for each
individual was compared to the placebo injection performed within
10 days (* denotes statistical significance). Significant increase
in the AChE activity in the LPS exposed samples is shown. Blood
samples were taken from one volunteer at the noted time points
following injection of saline or a lipopolysaccharide. Plasma
prepared from these blood samples was electrophoresed by SDS-PAGE,
and the gel was immunoreacted with anti-ARP-GST antibodies (FIG.
13B). The right lanes indicate the response to a placebo injection
and the next set represents the response to injection of LPS. The
two right lanes show the reaction with recombinant AChE-R but not
with AChE-S, respectively. This significant increase in ARP
accumulation in the LPS exposed serum indicates increase in the ARP
cleavage, suggesting that it may also increase under bacterial
infection.
[0547] Interestingly during this experiment, it has been found by
the inventors that in conjunction with the accumulation of ARP,
endotoxin induced a significant increase in rectal temperature and
elevation in cortisol and cytokines levels (data not shown).
[0548] Moreover, a significant endotoxin-induced increase in
anxiety level was observed at 1-2 hr post-injection but not later
as well as significant increase in depressed mood, which was
evident at 3-4 hr post injection.
[0549] These endotoxin-induced emotional and cognitive disturbances
in healthy volunteers were associated with increased plasma levels
of AChE-R and cortisol (data not shown).
[0550] However, the observed correlations between depressed mood
and cortisol secretion, as well as between depressed mood and
cytokine secretion, suggesting that AChE-R and cortisol are
independently associated with endotoxin-induced increase in
depressed mood.
Mass Spectroscopy of Gel-eluted Band
[0551] To verify the identity of the plasma-accumulated short
peptide that immunoreacted with the anti-ARP antibodies, larger
plasma samples (180 .mu.g/lane) were electrophoresed. A
Poinceau-stained band that co-migrated with the ARP Ab-positive
band was cut out of the gel and subjected to electron spray mass
spectrometry. As shown in FIG. 13C, this analysis verified the
existence of a peptide having a molecular mass of 3611 in the
excised band. Calculation of predicted masses presents the presumed
proteolytic cleavage site 36 residues from the C-terminus of
AChE-R, between asparagine and arginine residues:
N.dwnarw.RFLPKLLSATGMQGPAGSGWEEGSGSPPGVTPLFSP, also denoted as SEQ
ID: No 6.
ARP Modulations Potentiate the in vivo Hematopoietic Responses to
Stress
[0552] While ARP alone did not exert immediate effects on mouse
blood cell composition, its injection under stress enhanced ARP
labeling in bone marrow cells and induced an elevation in WBC
counts within 24 hr. This suggests that acute stress modifies the
number and/or state of ARP-responsive elements on hematopoietic
cells. Anti-ARP antibodies labeled primarily small cells in
ARP-treated stressed animals, whereas the limited labeling in
untreated stressed animals and in AS1-treated stressed animals only
appeared in relatively larger cells. This indicates labeling of the
stem cells which expanded during the 24 hr post-stress. The similar
patterns of the in viuo effects on bone-marrow ARP labeling and WBC
counts with the ex vivo expansion effects on CD34.sup.+ cells
implies that stress-induced increases in AChE-R may be causally
related to the post-stress elevation in WBC counts (Goldberg et
al., Folia Biol. 36, 319-31, 1990).
[0553] Transgenic animal models used here provide an opportunity
for testing the chronic effects of elevations of different AChE
variants. While AChE-S had no apparent effect on either platelet or
WBC counts, AChE-R modulations exerted dose dependent changes:
2.5-fold excess in blood AChE-R activity, similar to the AChE-R
elevation noted in the mouse brain under stress [Kaufer et al.
(1998) id ibid] sufficed to significantly elevate platelet counts.
The more dramatic 130-fold excess in blood AChE-R levels of the
robust-producing transgenic pedigree [Sternfeld et al. (1998b) id
ibid.] elevated both platelet and WBC counts. This finding, and the
in vivo accumulation of ARP under stress, raise the possibility
that the increased risk for brain infarcts following acute stress
or exposure to anticholinesterases (Harmsen et al., Stroke 21,
223-9, 1990, Schultz et al., Anesthesiology 79, 114-21, 1993) is
associated with the increased platelet counts due to AChE-R
overproduction. This calls for a search for AChE-R overproduction
in Alzheimer's disease patients, where ARP may increase platelet
counts and cause the cerebral infarcts, characteristic of this
disease (Inestrosa et al., Neurosci Lett 163, 8-10, 1993, Snowdon
et al., Jama 277, 813-7, 1997). Anti-ARP antibodies provide a novel
diagnostic tool for testing this option (and for risk assessment)
and AS-ODN treatment may offer an attractive protocol for
prevention of such adverse responses.
[0554] The significance of ARP extends beyond the hematopoietic
system. There is evidence for cross-talk between hematopoietic
cells at different stages of differentiation and bone-marrow
stromal or endothelial cells. Stroma influences cytokine production
and is responsible for maintaining steady-state hematopoiesis and
its adjustment under stress (Gupta et al., Blood 91, 3724-33,
1998). It has been proposed that primitive CD34.sup.+ progenitors
provide a soluble positive feedback signal to induce cytokine
production by either stromal or endothelial cells (Jazwiec et al.,
Leukemia 12, 1210-20, 1998). ARP may play such a role, with
important implications for ex vivo stem cell expansion, cancer
treatment and gene therapy. In the mammalian brain, ARP may further
affect the stress-associated plasticity of neuron and glia
properties, consistent with previous findings of the inventors of
morphogenic activities for AChE-R in transfected glia (Karpel et
al., J. Neurochem. 66, 114-23, 1996).
[0555] The stem cell survival and proliferative effects of ARP
denote a previously unforeseen activity that is particular to the
AChE-R protein yet distinct from the acetylcholine hydrolysis and
cell-cell adhesion capacity characteristic of the core domain
common to all AChE isoforms. The pronounced expression of AChE-R
during early embryogenesis, further demonstrate the involvement of
ARP in inducing the proliferation of other embryonic stem cells.
Moreover, neural stem cells were shown to produce a variety of
blood cell types in vivo (Bjornson et al. Science 283, 534-7,
1999).
[0556] The findings presented here suggest that ARP is involved in
the induction of growth and expansion capacities of pluripotent
stem cells from multi-tissue origins. The unique properties of this
peptide and equivalent peptides can contribute toward the
development of diverse human differentiating cell sources for
biomedical and research purposes.
Example 11
AChE-R Effects on Hippocampal LTP Suggest Causal Involvement in
Neuronal Stress Responses
[0557] At the molecular level, psychological stress notably leads
to fast yet long lasting modulation of gene expression. As for the
genes concerning the cholinergic system, it has been shown that
within one hour from acute stress, long lasting changes in
cholinergic gene expression are facilitated [Kaufer et al. (1998)
id ibid.]. This particularly refers to drastic elevation in the
levels of the normally rare "readthrough" variant of
acetylcholinesterase (AChE-R), coupled with down-regulation of
acetylcholine synthesizing and packaging proteins, the enzyme ChAT
and the associated vesicular acetylcholine transporter (vAChT).
This feedback response presumably contributes to reduce ACh levels
following stress. Another outcome of stress responses involves a
sudden increase in proteolytic activities. This leads, among other
effects, to the cleavage of the C-terminal peptide (ARP) from the
"readthrough" core enzyme. Immunodetection using anti-ARP
antibodies reveals an increase in AChE-R degradation products in
the cerebrospinal fluid of patients under stress [Kaufer (2000) PhD
thesis, Hebrew University of Jerusalem, Jerusalem]. Moreover as
shown above, the injection of synthetic ARP by itself induces
proliferation of hematopoietic progenitor cells and over-expression
of bone marrow AChE-R within 24 hr [Grisaru et al. (2001) Molecular
Medicine, 7, 93-105]. These recent observations raised the
intriguing possibility that ARP also possesses physiological and
behavioral functions. To test this working hypothesis, the effects
on LTP of confined swim stress (1 hr after induction), were
compared with those induced by ARP injection (24 hr post-treatment)
and with transgenic mice over-expressing AChE-R.
Differential Properties of AChE Variants in Synaptic
Plasticity--Stress Effects
[0558] The "readthrough" AChE variant is the sole AChE variant that
is up-regulated under psychological stress. Therefore, the
possibility that the immediate recovery from psychological stress,
in light of the over-expression of the AChE "readthrough" form will
affect the pattern of LTP, was explored.
[0559] Stress was induced by forcing mice to swim twice for 4 min,
with 4 min interval, and 1 hr later slices were taken for LTP
experiments. The Schaffer collaterals-CA1 synapse pathway was
tested. Basal field potentials were recorded for 15 min at 0.033
Hz. LTP was then induced by 3 consecutive tetanic stimulations,
each of 1-sec duration, at 50 Hz with 20 sec inter-stimulus
intervals. After tetanization, the change in the slope of the
post-synaptic field potential (PSP) was followed for up to 3
hrs.
[0560] As shown in FIG. 14A, while slices from control mice exhibit
a stepwise potentiation of 235.+-.27% (n=3), the slices from
stressed mice demonstrate a different pattern. LTP had a slow onset
delayed by 5 to 20 min and reached a plateau of 238.+-.18% (n=8)
potentiation, similar in that respect to control levels.
[0561] Therefore, stress changes the onset of LTP, lagging the
early phase, yet achieving a subsequent stable potentiation.
AChE-R Effects
[0562] Transgenic mice over-expressing the "readthrough" isoform
AChE-R enabled direct examination of the question whether stress
affects LTP, via elevation of AChE-R. Slices were prepared from
adult control and transgenic mice, 3 to 5 months old, and LTP
experiments were performed as described above.
[0563] As shown in FIG. 14B, LTP in slices from transgenic mice
over-expressing AChE-R shows the same pattern of slow onset as in
the stress-induced mice (compare to FIG. 14A).
Injected ARP Effects
[0564] The option that ARP (the AChE Readthrough Peptide) serves as
a stress signal was next examined. In case that ARP participates in
signaling stress conditions by elevating the "readthrough" isoform
in the CNS, as in the hematopoietic system, it would be expected to
induce the LTP pattern that was observed under stress conditions or
in the AChE-R transgenic mice (FIGS. 14A and 14B respectively).
Therefore, mice were injected with ARP (i.p. 0.1 mg/Kg body weight)
or with P-BAN, an irrelevant insect peptide of similar size. As
shown in FIG. 14C, 24 hr later, the LTP pattern of 15 min slow
onset repeated itself in the slices from the ARP injected animals
but not from those injected with the control peptide.
[0565] In conclusion, these findings point at the proteolytic
cleavage of ARP as a causally involved step in the synaptic
responses to stress and suggesting existence of ARP binding sites
in the hippocampus.
[0566] Moreover, these findings point at the possible mechanisms by
which ARP might mediate such effects.
Example 12
Testicular Overproduction of the Stress-associated "Readthrough"
Acetylcholinesterase Variant Impairs Sperm Properties
[0567] Suppressed male fertility is often attributed to stressful
lifestyle, however, the protein(s) mediating such impairments are
not yet known. Since ARP accumulation was observed under different
stress situations, the contribution and the involvement of AChE-R
in stress-induced infertility was next examined.
Testicular AChE-R is Overexpressed in Psychologically Stressed
Mice
[0568] The corticosterone levels and AChE activities were examined
in testicular homogenates from FVB/N mice that were subjected to
repeated acute psychological stress (4 successive daily sessions of
confined swim). As shown in FIG. 15A, samples obtained from
stressed mice displayed drastically elevated serum corticosterone
levels and mildly increased AChE activities.
[0569] To study the pattern of AChE-R expression in stressed vs.
untreated mice testis, in situ hybridization using a cRNA probe
selective for the AChE-R mRNA transcript was performed on sections
of testicular tubules from untreated FVB/N mice or from FVB/N mice
subjected to 4 constitutive daily treatments of confined swim
stress. As shown in FIG. 15B lower lane, the results revealed mild
circumference labeling in testicular tubules from untreated FVB/N
mice. Twenty-four hr after the last swim session, AChEmRNA labeling
intensified and extended into several central cell layers, where
spermatogonia are localized.
[0570] Similarly, Immunolabeling with an antibody selective for
ARP, displayed no detectable staining in control mice, yet stained
internal spermatid cell layers in tubuli of stressed mice (FIG.
15B--top lane).
Impaired Sperm Qualities Under AChE-R Excess Suggest Functional
Significance to ARP
[0571] To determine the in vivo capacity of ARP to affect different
biochemical and physiological male fertility properties, the effect
of injection of ARP or chronic expression of AChE-R (AChE-R
transgenic mice) on different parameters was examined.
[0572] Twenty-four hours following injection of mice with 85
.mu.g/kg ARP (but not PBS), blood corticosterone levels were
doubled as compared with FVB/N mice or AChE-R transgenics (Table
4). While the mechanism(s) for such short-term glucocorticoid
increases are yet unknown, this finding suggested that ARP might be
independently involved in activating peripheral stress responses.
Seminal gland weight was substantially reduced in AChE-R
transgenics, but not in ARP injected mice, reinforcing the
distinction between ARP and AChE-R effects; however, sperm counts
were lower both in ARP-injected and in AChE-R transgenics than in
untreated FVB/N mice. This did not reflect changes in cell division
as the numbers of PCNA-positive cells in testicular tubules
remained unchanged (Table 4). Intriguingly, sperm cells displayed
significantly reduced motility in both ARP injected mice and AChE-R
transgenics (Table 4) suggesting that ARP exerts rapid yet long
lasting impairments of sperm properties. TABLE-US-00005 TABLE 4
AChE-R over-expression impairs biochemical and physiological male
fertility correlates Animals general ARP AChE-R properties
Control.sup.a injection.sup.b transgenics.sup.c blood
corticosterone, ng/ml 31.6 .+-. 7.5 (3) 58.1 .+-. 4.1 (3) 31.7 .+-.
3.9 (3) AChE activity, 0.2 .+-. 0.06 (5) 0.3 .+-. 0.06 (3) 70.3
.+-. 1.1 (3) nmol ATCh/min/ mg prot..sup.d seminal gland wt., 10.56
.+-. 0.95 (5) 10.79 .+-. 1.08 (3) 8.32 .+-. 0.28 (5) mg/gr weight
sperm counts, 7.12 .+-. 0.35 (5) 5.3 .+-. 1.2 (3) 3.9 .+-. 0.4 (3)
cells/epididimis .times. 10.sup.-6 sperm, motile, 12 .+-. 3.0 (5) 5
.+-. 1.7 (3) 9 .+-. 2.3 (3) % of total sperm cells anti-ARP None
1.3 .+-. 0.5 (10) 14.1 .+-. 1.7 (10) area immuno stained/tubule
perimeter, pixels.sup.d PCNA 26 .+-. 0.004 (20) 28 .+-. 0.005 (12)
28 .+-. 0.005 (20) positive cells/tubule perimeter,
no./pixels.sup.e .sup.aAll tested animals were FVB/N adult mice, 2
to 4 month-old males (numbers shown in parentheses). Controls were
untreated and PBS injected. .sup.b24-hr post i.p. injection of 34
nmol/Kg ARP. .sup.cLine 45 [Sternfeld et al. (1998a) id ibid].
.sup.dAverage labeled cells or labeled areas for the noted (in
parentheses) number of tubular sections and the identified
antibodies. Asterisks note significant difference (p < 0.05,
Wilcoxon-Mann-Whitnety) from controls.
[0573] Corticosterone elevation initiates at the
hypothalamic--pituitary-adrenal (HPA) axis, activated by calcium
increases under psychological stress [Kaufer et al. (1999) Current
Opinion in Neurology 12, 739-743]. The observed corticosterone and
AChE-R overexpression following ARP injection suggest an
HPA-activating, auto-regulatory function for ARP. AChE-R
accumulation would induce the cell-cell or cell-substrate signaling
capacities established for AChE [Grisaru et al.(1999b) id ibid.].
The normally rare AChE-R isoform differs from the major synaptic
AChE-S variant in such properties, for example in cultured glia
(Karpel et al. J. Neurochem., 66, 114-123, 1996), supporting causal
involvement for ARP in morphogenic functions. This calls for
identifying the yet unknown brain protein partner(s) of ARP and the
signal transduction mechanisms it activates.
Both Stress and ARP Injection Enhance ARP Immunolabeling of
Spermatid Heads
[0574] To subcellularly localize the AChE-R isoform, immunolabeled
mature spermatids from the central cavity of testicular tubules
were subjected to confocal microscopy (FIG. 16). Anti-ARP
antibodies failed to label spermatids from control mice, either
naive or PBS-injected (FIG. 16A and data not shown). In contrast,
either repeated acute stress (FIG. 16B) or a single ARP injection
(FIG. 16C) induced clear intracellular punctuated labeling that was
limited to spermatid heads and left their tails essentially
unlabeled. The spermatids observed in the central cavity of AChE-R
transgenics, with lower sperm cell counts, were only faintly
labeled, again in heads but not tails.
Loss of ARP Immunolabeling in Human Sperm Heads from Subjects with
Unexplained Couple Infertility
[0575] To test the validity of the predictions based on the mice
results, for human sperm properties, the inventors performed
immunolabeling of ARP in smeared sperm cells from individuals with
reported unexplained couple infertility. Healthy cells from sperm
donations served as controls. Normal specimens were primarily
co-stained in sperm head and midpiece regions. In contrast, sperm
samples from male partners of couples with unexplained infertility
displayed large fractions of cells labeling limited to the midpiece
and unlabeled heads (FIG. 17A). Cumulative analysis demonstrated
that these differences were statistically significant in that the
midpiece alone was stained in 55% of sperm from couple infertility
samples but only in 15% of normal donor sperm (FIG. 17B). Thus,
alterations in ARP labeling patterns spanned both human and mouse
sperm from subjects with impaired (or potentially impaired) sperm
properties, as compared to their controls.
[0576] While reduced seminal gland weight could probably be
attributed to the long-lasting effects of stress, the intensified
labeling of developing sperm cells with anti-ARP antibodies
suggests direct ARP effects on spermatogenesis and/or sperm
properties. Focal perinuclear labeling of pachytene spermatocytes
from AChE-R transgenics was associated with an apparent suppression
of spermatogenesis manifested in reduced sperm counts. Transient
excess, such as that induced following repeated acute stress or ARP
injection, caused more limited impairments in spermatogenesis yet
impaired sperm functioning most effectively. That this was
associated with ARP accumulation was indicated from the modified
ARP labeling of sperm heads in mice or midpiece and men.
[0577] Anti-ARP antibodies could label both ARP binding and AChE-R
production sites. In stressed or ARP-affected mouse spermatids, the
punctuated head labeling appeared reminiscent of the mitochondrial
distribution in the region surrounding spermatid heads. This
assumption was supported by the intriguing labeling patterns in
human sperm from infertile couples, where staining was most intense
in the midpiece region which is enriched in mitochondria in primate
sperm.
[0578] In summary, these results significantly indicate impaired
sperm properties under overproduction of the stress-associated
"readthrough" isoform of acetylcholin- esterase, AChE-R and its
naturally cleaved C-terminal peptide ARP.
[0579] Thus, excess AChE-R and its C-terminal peptide ARP may
suppress male fertility through both autonomous system regulation
and direct sperm interactions.
Example 13
Detection of ARP Binding Proteins by Using the Yeast Two-hybrid
System
[0580] In order to study the possible signaling pathway through
which ARP can exert its intracellular signaling leading to the
observed proliferation and differentiation, screening for detection
of ARP binding proteins was performed using the yeast 2-hybrid
system.
[0581] The yeast 2-hybrid system is based on that transcription
factors, such as GAL4, consist of two discrete modular domains: the
DNA-binding domain (DNA-BD) and the activation domain (AD). A
"bait" gene is expressed as a fusion to the DNA-BD, while a cDNA
library is expressed as a fusion to the AD (Chien et al. (1991) id
ibid; Fields et al. Trends Genet 10, 286-92, 1994). When the fusion
proteins interact, the DNA-BD and AD brought into close proximity,
thus reconstituting GAL4 and activating transcription of a reporter
gene (FIG. 18A).
[0582] The bait is cloned into the DNA-BD vector where it is
expressed as a fusion to amino acids 1-147 of the yeast GAL4
protein. A second gene or cDNA library is cloned into the AD
vector, where it is expressed as a fusion to amino acids 768-881 of
the yeast GAL4 protein. When the fusion proteins interact, the
DNA-BD and AD domains are brought into close proximity and can
activate transcription of reporter genes.
[0583] In order to identify AChE C-terminal peptide interacting
proteins, the GAL4-based two-hybrid system was used. Sequences that
encode AChE C-terminal peptides ARP and ASP were cloned into the
DNA-BD pGBKT7 vector (Clonetech), (FIG. 18B) to serve as the bait.
Three different cDNA libraries were cloned into the AD pGADT7
vectors and screened; adult and neonatal rat aorta and human fetal
brain (Clontech).
Summary of the Yeast 2-hybrid Preliminary Screens
[0584] Four preliminary yeast two-hybrid screens were performed,
the outcomes of which are summarized below several points suggest
that certain positive clones are meaningful. [0585] 1. The number
of ARP positives in the developing rat aorta seems to be
considerably higher than in the adult tissue, this is in line with
the embryonic expression pattern of AChE-R. [0586] 2. Several
positive clones appear more then once, representing independent
cDNA chains of variable lengths.
[0587] 3. In certain cases, the positives are logical candidates
for AChE interactions (see below). TABLE-US-00006 TABLE 5 Screening
for Binding Partners Number of Independent positives on - clones in
the Transfection Trp/-Leu/- Bait Library library efficiency
Ade/-His ASP rat neonatal aorta 2.6 .times. 10.sup.6 500,000 5 (SN)
r rat adult aorta 2 .times. 10.sup.6 1,140,000 3 (AR) (ARP) human
fetal brain 1 .times. 10.sup.7 .about.40,000 12 (RB) ARP rat
neonatal aorta 2.6 .times. 10.sup.6 880,000 29 (RN) ASP human fetal
brain 1 .times. 10.sup.7
Candidate Partners:
[0588] During the first library screenings, 8 candidate partners
emerged. Of these, a literature survey pointed to 2 candidates as
most promising.
For ARP:
[0589] Fragment of AChE-ARP used for the two-hybrid screen
[0590] Underlined is the actual ARP-peptide
[0591] PLEVRRGLRAQACAFWNRFLPKLLSATGMQGPAGSGWEEGSGSPPGVTPLFSP, also
denoted as SEQ ID: No. 7.
[0592] Receptor for Activated Protein Kinase C (RACK)--2 clones
[0593] H. sapiens melanoma antigen, family D, 1 (MAGED1)--2
clones
[0594] H. sapiens guanine nucleotide-binding protein g(i)/g(s)/g(t)
.beta. subunit 2 (transducin .beta. chain 2)
[0595] H. sapiens duplicate spinal muscular atrophy--2 clones
[0596] H. sapiens peptidase D (PEPD)
[0597] M. musculus Eph receptor A6 (Epha6)
[0598] H. sapiens succinate dehydrogenase iron-protein subunit
(sdhB) gene
[0599] H. sapiens mitogen-activated protein kinase 7 (MAPK7)
[0600] H. sapiens HLA-B associated transcript-3 (D6S52E)
[0601] mitochondrial intermediate peptidase (MIP)
[0602] 12-lipoxygenase
For ASP:
[0603] Fragment of AChE-ASP used for the two-hybrid screen
[0604] Underlined is the actual ASP-peptide:
PLEVRRGLRAQACAFWNRFLPKLLSATDTLDEAERQWKAEFHRWSSYMV
HWKNQFDHYSKQDRCSDL, also denoted as SEQ ID NO 8.
[0605] Receptor for Activated Protein Kinase C (RACK)--2 clones
(also showed up in the ARP screen, probably binds to the common
part).
[0606] H. sapiens C-terminal binding protein 2 (CTBP2).
[0607] H. sapiens activating signal cointegrator 1.
[0608] H. sapiens colon carcinoma laminin-binding protein.
[0609] H. sapiens clusterin (complement lysis inhibitor, SP-40,40,
sulfated glycoprotein 2, testosterone-repressed prostate message 2,
apolipoprotein J) (CLU).
[0610] H. sapiens mRNA for silencer element.
[0611] H. sapiens heme-regulated initiation factor 2-.beta. kinase
(HRI).
Identification of RACK1 as ARP and ASP Interacting Molecule
[0612] One of the isolated partners, RACK1, was further analyzed
for its ARP interactions. RACK1 is a cytoplasmic G protein
homologue, which serves as a protein kinase C receptor.
[0613] Interaction between RACK1 and AChE may therefore be the link
between AChE and other signaling molecules through which it exerts
its non-catalytic intracellular functions.
[0614] Amino acid sequence alignment of RACK1 with the sequence
obtained from the two-hybrid positive clone shows close to 100%
homology (FIG. 19 and SEQ ID: No. 9). Furthermore, only part of the
protein was expressed, which narrows the search to this part.
Interestingly, the isolated sequence of this part of RACK includes
peptides that were reported to be the binding sites of PKC to RACK1
(Ron et al., Proc Natl Acad Sci USA 91, 839-843, 1994)
Example 14
Overlay Assay Demonstrating the AChE-R-RACK1 Interaction
[0615] An in vitro overlay assay was combined with protein blot
analysis to test for RACK1 interaction with the full AChE-R
protein. RACK1 was expressed in E. coli and purified by affinity
chromatography from E. coli as a fusion with maltose binding
protein (MBP), and subsequently released from the fusion protein by
proteolytic cleavage using factor Xa. Both cleaved and uncleaved
preparations were used for the overlay assay. RACK1 samples were
submitted to electrophoresis on a 4-10% denaturing polyacrylamide
gel, blotted on a NC membrane, which was then stained with Ponceau
(FIG. 20A), and stripped. Three identical strips were used for
parallel experiments. Anti-RACK1 antibodies recognized the fusion
protein, proteolytically-released RACK1 and fragments thereof, but
not MBP (FIG. 20B). Parallel membranes were overlaid with a
homogenate obtained from rat pheochromocytoma PC12 cells
transfected with a plasmid encoding AChE-R [Seidman, S. et al.
(1995) id ibid.]. Antibodies to the N-terminus of human AChE (Santa
Cruz) detected specific binding of AChE to the intact RACK1 protein
on the overlaid membrane, but not to RACK1 degradation products or
to MBP (FIG. 20C). The non-overlaid membrane did not reveal any
interaction when incubated with these antibodies, demonstrating
AChE-R dependence (FIG. 20D).
Example 15
Accumulation of a RACK1-immunoreactivee Protein in the Mouse
Post-stress Brain
[0616] In order to study the expression of RACK1 in stress
situation, homogenates from mouse hippocampus and cortex (composed
of 3 stressed, 4-6 and 3 control, 1-3 mice) were separated on a
denaturing gel and analyzed by immunoblot with anti-RACK1 antibody
(FIG. 21). Surprisingly, a 50 kDa (apparent molecular weight)
immunopositive band, which is much larger than normal RACK1 (36
kDa), was observed to accumulate following stress in both
hippocampus and cortex. In parallel with the accumulation of this
band, the normal sized RACK1 observed to be diminishing in the
cortex. The intensity of the 50 kDa band strongly correlated with
the plasma corticosterone levels of these mice. Thus, RACK1
increases in quantity in the post-stress mammalian brain, where it
forms stable complex with yet unidentified protein(s).
Example 16
ARP1 Promotes Triple Complex Formation with RACK1 and
PKC.beta.II
[0617] pGARP, a vector that encodes a fusion protein between green
fluorescent protein (GFP) and ARP1 under the CMV promoter, was used
to test whether ARP1 further promotes triple complex formation with
PKC.beta.II and RACK1 in mammalian cells (FIG. 22). When
transfected into COS cells, which do not express AChE, anti-ARP
antibodies immunodetected GARP expression in cell homogenates.
Anti-GFP antibodies were ineffective in non-transfected cells but
immunoprecipitated GARP, RACK1 and PKC.beta.II from homogenates of
GARP transfected COS cells (FIG. 22).
Example 17
AChE-R Promotes Triple Complexes with RACK1 and PKC.beta.II in
Native PC12 Cells
[0618] Both COS and PC12 cells express RACK1 and PKC.beta.II
constitutively, whereas only PC12 expresses AChE-R, as observed in
immunoblots of the soluble fraction of cell homogenates (FIG. 23A).
Antibodies targeted to the N-terminal domain of AChE
co-immunoprecipitated both PKC.beta.II and RACK1 in PC12 but not
COS cells, supporting the notion of tight binding for
AChE-R/RACK1/PKC.beta.II in these PC12 cell complexes (FIG.
23B).
Example 18
Stress Induces Neuronal Accumulation of Immunoreactive AChE-R and
RACK1
[0619] The in uivo relevance of AChE-R/RACK1 interactions was
explored in normal and post-stress mouse brain. Immunoreactive
RACK1 was observed in the cytoplasm and closely proximal processes
of pyramidal neurons, in layers 3 and 5 of the frontal and parietal
cortex, in both superficial and deep layers of the piriform cortex,
and in regions CA1 and CA3 of the hippocampus. A subset of these
neurons also overexpresses AChE-R under acute psychological stress
(FIG. 24 and data not shown). Stress-induced increase of RACK1 was
seen in parietal cortex layer 5 (compare FIG. 24, C-E and G-I).
Unlike RACK1, AChE-R antibodies also stained cells with glial
morphology. Also, in some regions, such as hippocampal CA1, RACK1
staining formed an almost continuous pattern, whereas AChE-R was
localized to a subset of pyramidal neurons. For both AChE-R and
RACK1, uneven perikarial accumulation and increased neurite
labeling were observed under stress (FIG. 24).
Example 19
Transgenic AChE-R Overexpression Elevates Brain RACK1 Levels and
Intensifies the Formation of Neuronal PKC.beta.II Clusters
[0620] Hippocampal homogenates from AChE-R overexpressing
transgenics [Sternfeld et al. (2000) id ibid.] were tested to
investigate whether AChE-R overproduction would modulate the
levels, properties and/or neuronal localization of its partner
proteins RACK1 and PKC.beta.II. The results show that the
hippocampal homogenates displayed significant increases (compared
to the levels in control FVB/N stress-prone mice) of neuronal
AChE-R and RACK1, as well as a faster migrating PKC.beta.II band
that was only faintly detected in the hippocampus of a control
animal (FIG. 25A).
[0621] Of the three target proteins, RACK1 and AChE-R appeared more
widely distributed and could be detected in numerous brain regions
(FIG. 25B and data not shown). In the brain of AChE-R transgenics,
AChE-R overexpression was particularly conspicuous in neuron groups
showing punctuated PKC.beta.II staining (FIG. 25B-D). PKC.beta.II
labeling, in contrast, appeared higher than control levels in only
a fraction of the AChE-R overexpressing subregions. Finally, RACK1
staining was intensified in the AChE-R expressing hippocampal CA1
and dentate gyrus neurons, and less prominent in the parietal
cortex. This result suggested that the AChE-R/RACK1/PKC.beta.II
interactions facilitated the intracellular retention of the
secretory AChE-R protein.
Example 20
Diverse Subcellular Distributions of PKC.beta.II
[0622] In control mice, PKC.beta.II antibodies displayed diffuse
staining [Weeber et al. (2000) id ibid.] in sub-regions of layers
5,6 in the cortex, in the stratum oriens and stratum radiatum
layers of the hippocampus CA1 field, in the striatum matrix, and in
the substantia nigra pars reticulata. Axonal bundles including the
nigro-striatal tract were also labeled (data not shown). Another
and novel staining pattern consisted of dense PKC.beta.II clusters
in neuronal perikaria and in the axonal stems. This punctiform
pattern appeared in upper layers of the parietal, temporal and
piriform cortex, dorsal striatum, basolateral amygdala, hippocampal
CA1 and lateral septum. In general, cells in AChE-R transgenic mice
that displayed prominent AChE-R labeling were positive for RACK1
and presented PKC.beta.II punctiform staining (FIG. 25B-E). AChE-R
labeling in cells where in control mice included AChE-R, RACK1 and
punctuated PKC.beta.II, was not intensified in AChE-R transgenic
mice. These included neurons in the globus pallidus, substantia
nigra, superior culliculus, medial septum and diagonal band (FIG.
25B-E and data not shown). Other neurons were positive for AChE-R
staining in the control mice, and yet more so in AChE-R transgenic
mice, but had no PKC.beta.II punctiform staining. These resided in
the lateral and ventro-medial hypothalamus, central nucleus of the
amygdala, the hippocampal dentate gyrus, ventro-lateral thalamus,
and the Edinger-Westphal nucleus (FIG. 25C, D). C57B6J mice were
tested for the punctuated staining pattern, and a weaker but
discernible punctiform signal was observed in the same cell
populations as in the stress-prone FVB/N strain, used as control
(data not shown).
Example 21
Inter-related AChE-R/RACK1/PKC.beta.II Distributions
[0623] In samples obtained from AChE-R transgenic mice,
anti-PKC.beta.II antibodies detected diffuse and axonal staining
patterns similar to those observed in the parental FVB/N strain.
However, the punctuated pattern was altered. Stronger and denser
clusters of PKC.beta.II staining were located on the perikaryal
circumference of a larger fraction of hippocampal CA1 neurons (FIG.
25E2). Transgenic mice overexpressing the major synaptic isoform of
AChE [Beeri, R. et al. (1995) Curr Biol, 5, 1063-71] did not show
such changes in PKC.beta.II expression (data not shown), suggesting
that this in vivo effect depended on chronic AChE-R excess and/or
that it was prevented by AChE-S excess. AChE-R staining in the
transgenic brain was prominent in the cell bodies and proximal
processes of many, but not all CA1 hippocampal neurons, suggesting
that a specific subset of these neurons was especially amenable for
such accumulation (FIG. 25E-3,4). Sparse cells with morphology
reminiscent of microglia were also positive for AChE-R staining,
both in control and transgenic animals (FIG. 25E-5, 6). Intensified
labeling of perikaria and closely proximal neurites of CA1
pyramidal neurons was also observed by staining with RACK1
antibodies (FIG. 256E-3, 4).
Example 22
Subcellular Distributions of AChE-R, RACK1 and PKC.beta.II was
Overlapping
[0624] Confocal micrographs of upper layer neurons from the
parieto-temporal cortex double labeled with antibodies against
AChE-R and RACK1 or PKC.beta.II displayed distinct yet overlapping
distributions for the three partner proteins within neuronal
perikarya in compound field projections. As expected, AChE-R
labeling was conspicuously more intense in AChE-R transgenics than
in FVB/N controls (compare in FIG. 26B-1 to 4 and 26C-7 to 10). The
overexpression and the associated overlapping increases in the two
partner proteins were reflected in different colors (FIG. 26B and
C).
[0625] Proteins destined to be secreted are initially concentrated
near the nucleus, where their processing takes place, whereas
proteins that are associated with the perikaryal cytoskeleton,
plasma membrane and/or proximal process structures are distributed
more peripherally in the cell. In cortical neurons from control
mice, both AChE-R and RACK1 immunostaining formed perinuclear
accumulations (compare in FIG. 26B-1 to 3). In contrast, the
intensity of RACK1 staining in AChE-R transgenic mice was
especially high around the perikaryal circumference (compare in
FIG. 26B-2 and 5), suggesting subcellular translocation under
AChE-R excess. PKC.beta.II staining clusters were also removed from
the peri-nuclear domain in the control mice (FIG. 26C-8), and
showed uneven distribution in larger cellular spaces in transgenic
mice (FIG. 26C-11). In control mice, PKC.beta.II patterns differed
from both those of AChE-R and RACK1 in that they demonstrated both
diffuse staining and punctuated clusters of protein complexes,
compatible with the parallel light microscopy patterns.
Constitutive AChE-R overexpression further enlarged the perikaryal
space occupied by AChE-R, RACK1 and PKC.beta.II and seemed to
increase the intracellular density of their complexes.
Sequence CWU 1
1
9 1 26 PRT HOMO SAPIENS 1 Gly Met Gln Gly Pro Ala Gly Ser Gly Trp
Glu Glu Gly Ser Gly Ser 1 5 10 15 Pro Pro Gly Val Thr Pro Leu Phe
Ser Pro 20 25 2 40 PRT HOMO SAPIENCE 2 Asp Thr Leu Asp Glu Ala Glu
Arg Gln Trp Lys Ala Glu Phe His Arg 1 5 10 15 Trp Ser Ser Tyr Met
Val His Trp Lys Asn Gln Phe Asp His Tyr Ser 20 25 30 Lys Gln Asp
Arg Cys Ser Asp Leu 35 40 3 27 PRT HOMO SAPIENS 3 Phe His Arg Trp
Ser Ser Tyr Met Val His Trp Lys Asn Gln Phe Asp 1 5 10 15 His Tyr
Ser Lys Gln Asp Arg Cys Ser Asp Leu 20 25 4 42 DNA Artificial
Sequence Description of Artificial Sequence5' primer for intron 4
of human acetylcholinesterase 4 gctggatcca tcgaggggcg aggtatgcag
gggccagcgg gc 42 5 30 DNA Artificial Sequence Description of
Artificial Sequence3' primer for intron 4 of human
acetylcholinesterase 5 tataagcttc tagggggaga agagaggggt 30 6 37 PRT
homo sapiens 6 Asn Arg Phe Leu Pro Lys Leu Leu Ser Ala Thr Gly Met
Gln Gly Pro 1 5 10 15 Ala Gly Ser Gly Trp Glu Glu Gly Ser Gly Ser
Pro Pro Gly Val Thr 20 25 30 Pro Leu Phe Ser Pro 35 7 53 PRT
Artificial Sequence Description of Artificial Sequence arp-two
hybrid screen peptide 7 Pro Leu Glu Val Arg Arg Gly Leu Arg Ala Gln
Ala Cys Ala Phe Trp 1 5 10 15 Asn Arg Phe Leu Pro Lys Leu Leu Ser
Ala Thr Gly Met Gln Gly Pro 20 25 30 Ala Gly Ser Gly Trp Glu Glu
Gly Ser Gly Ser Pro Pro Gly Val Thr 35 40 45 Pro Leu Phe Ser Pro 50
8 67 PRT Artificial Sequence Description of Artificial Sequence ASP
- peptide for two-hybrid screen 8 Pro Leu Glu Val Arg Arg Gly Leu
Arg Ala Gln Ala Cys Ala Phe Trp 1 5 10 15 Asn Arg Phe Leu Pro Lys
Leu Leu Ser Ala Thr Asp Thr Leu Asp Glu 20 25 30 Ala Glu Arg Gln
Trp Lys Ala Glu Phe His Arg Trp Ser Ser Tyr Met 35 40 45 Val His
Trp Lys Asn Gln Phe Asp His Tyr Ser Lys Gln Asp Arg Cys 50 55 60
Ser Asp Leu 65 9 317 PRT Artificial Sequence Description of
Artificial Sequence homology of ARP and RACK 9 Met Thr Glu Gln Met
Thr Leu Arg Gly Thr Leu Lys Gly His Asn Gly 1 5 10 15 Trp Val Thr
Gln Ile Ala Thr Thr Pro Gln Phe Pro Asp Met Ile Leu 20 25 30 Ser
Ala Ser Arg Asp Lys Thr Ile Ile Met Trp Lys Leu Thr Arg Asp 35 40
45 Glu Thr Asn Tyr Gly Ile Pro Gln Arg Ala Leu Arg Gly His Ser His
50 55 60 Phe Val Ser Asp Val Val Ile Ser Ser Asp Gly Gln Phe Ala
Leu Ser 65 70 75 80 Gly Ser Trp Asp Gly Thr Leu Arg Leu Trp Asp Leu
Thr Thr Gly Thr 85 90 95 Thr Thr Arg Arg Phe Val Gly His Thr Lys
Asp Val Leu Ser Val Ala 100 105 110 Phe Ser Ser Asp Asn Arg Gln Ile
Val Ser Gly Ser Arg Asp Lys Thr 115 120 125 Ile Lys Leu Trp Asn Thr
Leu Gly Val Cys Lys Tyr Thr Val Gln Asp 130 135 140 Glu Ser His Ser
Glu Trp Val Ser Cys Val Arg Phe Ser Pro Asn Ser 145 150 155 160 Ser
Asn Pro Ile Ile Val Ser Cys Gly Trp Asp Lys Leu Val Lys Val 165 170
175 Trp Asn Leu Ala Asn Cys Lys Leu Lys Thr Asn His Ile Gly His Thr
180 185 190 Gly Tyr Leu Asn Thr Val Thr Val Ser Pro Asp Gly Ser Leu
Cys Ala 195 200 205 Ser Gly Gly Lys Asp Gly Gln Ala Met Leu Trp Asp
Leu Asn Glu Gly 210 215 220 Lys His Leu Tyr Thr Leu Asp Gly Gly Asp
Ile Ile Asn Ala Leu Cys 225 230 235 240 Phe Ser Pro Asn Arg Tyr Trp
Leu Cys Ala Ala Thr Gly Pro Ser Ile 245 250 255 Lys Ile Trp Asp Leu
Glu Gly Lys Ile Met Val Asp Glu Leu Lys Gln 260 265 270 Glu Val Ile
Ser Thr Ser Ser Lys Ala Glu Pro Pro Gln Cys Thr Ser 275 280 285 Leu
Ala Trp Ser Ala Asp Gly Gln Thr Leu Phe Ala Gly Tyr Thr Asp 290 295
300 Asn Leu Val Arg Val Trp Gln Val Thr Ile Gly Thr Arg 305 310
315
* * * * *