U.S. patent application number 12/197915 was filed with the patent office on 2009-10-01 for neurotrophic components of the adnf i complex.
This patent application is currently assigned to THE GOVERMENT OF THE UNITED STATES OF AMERICA AS REPRESENTED BY THE. Invention is credited to DOUGLAS E. BRENNEMAN, RAQUEL CASTELLON, IIIANA GOZES, JANET M. HAUSER, CATHERINE Y. SPONG.
Application Number | 20090247457 12/197915 |
Document ID | / |
Family ID | 26983587 |
Filed Date | 2009-10-01 |
United States Patent
Application |
20090247457 |
Kind Code |
A1 |
BRENNEMAN; DOUGLAS E. ; et
al. |
October 1, 2009 |
NEUROTROPHIC COMPONENTS OF THE ADNF I COMPLEX
Abstract
This invention relates to Activity Dependent Neurotrophic Factor
I complex (ADNF I complex) and polypeptides of this complex, which
produce their neurotrophic effects through multiple proteases
intrinsic to the ADNF I complex. The invention also relates to
pharmaceutical compositions comprising ADNF I complex polypeptides,
as well as methods for reducing neuronal cell death in vitro and in
vivo, methods for treating oxidative stress in a patient, methods
for reducing a condition associated with fetal alcohol syndrome in
a subject, and methods of enhancing learning and memory both pre-
and post-natally, all of which methods use the ADNF I complex
polypeptides of the invention.
Inventors: |
BRENNEMAN; DOUGLAS E.;
(NORTH WALES, PA) ; CASTELLON; RAQUEL; (NORWALK,
CA) ; SPONG; CATHERINE Y.; (ARLINGTON, VA) ;
HAUSER; JANET M.; (BETHESDA, MD) ; GOZES; IIIANA;
(RAMAT-HASHARON, IL) |
Correspondence
Address: |
TOWNSEND AND TOWNSEND AND CREW, LLP
TWO EMBARCADERO CENTER, 8TH FLOOR
SAN FRANCISCO
CA
94111
US
|
Assignee: |
THE GOVERMENT OF THE UNITED STATES
OF AMERICA AS REPRESENTED BY THE
ROCKVILLE
MD
SECRETARY OF THE DEPTARTMENT OF HEALTH AND HUMAN
SERVICES
TEL AVIV
RAMOT AT TEL-AVIV UNIVERSITY
|
Family ID: |
26983587 |
Appl. No.: |
12/197915 |
Filed: |
August 25, 2008 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10489515 |
Oct 14, 2004 |
7427590 |
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PCT/US02/29146 |
Sep 12, 2002 |
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12197915 |
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60322760 |
Sep 12, 2001 |
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60371961 |
Apr 10, 2002 |
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Current U.S.
Class: |
514/1.1 ;
435/377 |
Current CPC
Class: |
C07K 14/475 20130101;
A61P 25/28 20180101; A61P 25/00 20180101; A61P 15/00 20180101; A61K
38/00 20130101; A61P 25/32 20180101 |
Class at
Publication: |
514/12 ; 435/377;
514/13; 514/15; 514/14 |
International
Class: |
A61K 38/17 20060101
A61K038/17; C12N 5/00 20060101 C12N005/00; A61K 38/10 20060101
A61K038/10; A61K 38/08 20060101 A61K038/08; A61P 25/28 20060101
A61P025/28; A61P 25/32 20060101 A61P025/32; A61P 15/00 20060101
A61P015/00; A61P 25/00 20060101 A61P025/00 |
Claims
1-17. (canceled)
18. A method for reducing neuronal cell death, the method
comprising contacting the neuronal cells with an Activity Dependent
Neurotrophic Factor I complex (ADNF I) polypeptide in an amount
sufficient to prevent neuronal cell death, wherein the ADNF I
complex polypeptide comprises an amino acid sequence or a fragment
thereof selected from the group consisting of: TABLE-US-00005
WSDVGVSSGSAPDAFK (SEQ ID NO:1) NNSTTYAPISANVSTALGSTAALPTAAGPV (SEQ
ID NO:2) NFLTSHYSAANSVVGGTNPGK (SEQ ID NO:3) NPSGTDWLNTNNQANPFN
(SEQ ID NO:4) LVPLTPINR (SEQ ID NO:5)
VLQAVXGADSNVAFQGKVIYRSESSGTSELLTR (SEQ ID NO:6) GPTADITLTK (SEQ ID
NO:7) GTPTGXGPLIQ (SEQ ID NO:8) VDPASGYPIVGYT (SEQ ID NO:9)
PSGTDWLNT (SEQ ID NO:10) and SESSGTSELLTR. (SEQ ID NO:11)
19. The method of claim 18, wherein the ADNF I complex polypeptide
comprises at least one D-amino acid.
20. The method of claim 18, wherein the ADNF I complex polypeptide
comprises all D-amino acids.
21. The method of claim 18, wherein the neuronal cells are selected
from the group consisting of spinal cord neurons, hippocampal
neurons, cerebral cortical neurons and cholinergic neurons.
22. The method of claim 18, wherein the neuronal cell death is in a
patient infected with immunodeficiency virus.
23. The method of claim 22, wherein the immunodeficiency virus is a
human immunodeficiency virus.
24. The method of claim 18, wherein the neuronal cell death is
associated with excito-toxicity induced by N-methyl-D-aspartate
stimulation.
25. The method of claim 18, wherein the neuronal cell death is
induced by the beta-amyloid peptide in a patient afflicted with
Alzheimer's disease.
26. The method of claim 18, wherein the neuronal cell death is
induced by cholinergic blockade in a patient afflicted with
Alzheimer's disease, the cholinergic blockade resulting in learning
impairment.
27. A method for treating oxidative stress in a patient, the method
comprising administering to the patient an Activity Dependent
Neurotrophic Factor I complex (ADNF I) polypeptide in an amount
sufficient to reduce oxidative stress, wherein the ADNF I complex
polypeptide comprises an amino acid sequence or a fragment thereof
selected from the group consisting of: TABLE-US-00006
WSDVGVSSGSAPDAFK (SEQ ID NO:1) NNSTTYAPISANVSTALGSTAALPTAAGPV (SEQ
ID NO:2) NFLTSHYSAANSVVGGTNPGK (SEQ ID NO:3) NPSGTDWLNTNNQANPFN
(SEQ ID NO:4) LVPLTPINR (SEQ ID NO:5)
VLQAVXGADSNVAFQGKVIYRSESSGTSELLTR (SEQ ID NO:6) GPTADITLTK (SEQ ID
NO:7) GTPTGXGPLIQ (SEQ ID NO:8) VDPASGYPIVGYT (SEQ ID NO:9)
PSGTDWLNT (SEQ ID NO:10) and SESSGTSELLTR. (SEQ ID NO:11)
28. The method of claim 27, wherein the ADNF I complex polypeptide
comprises at least one D-amino acid.
29. The method of claim 27, wherein the ADNF I complex polypeptide
comprises all D-amino acids.
30. A method for reducing a condition associated with fetal alcohol
syndrome in a subject who is exposed to alcohol in utero, the
method comprising administering to the subject an ADNF I complex
polypeptide in an amount sufficient to reduce the condition
associated with fetal alcohol syndrome, wherein the ADNF I complex
polypeptide comprises an amino acid sequence or a fragment thereof
selected from the group consisting of: TABLE-US-00007
WSDVGVSSGSAPDAFK (SEQ ID NO:1) NNSTTYAPISANVSTALGSTAALPTAAGPV (SEQ
ID NO:2) NFLTSHYSAANSVVGGTNPGK (SEQ ID NO:3) NPSGTDWLNTNNQANPFN
(SEQ ID NO:4) LVPLTPINR (SEQ ID NO:5)
VLQAVXGADSNVAFQGKVIYRSESSGTSELLTR (SEQ ID NO:6) GPTADITLTK (SEQ ID
NO:7) GTPTGXGPLIQ (SEQ ID NO:8) VDPASGYPIVGYT (SEQ ID NO:9)
PSGTDWLNT (SEQ ID NO:10) and SESSGTSELLTR. (SEQ ID NO:11)
31. The method of claim 30, wherein the condition is selected from
the group consisting of: a decreased body weight of a subject; a
decreased brain weight of the subject; a decreased level of VIP
mRNA of a subject; and death of a subject in utero.
32. The method of claim 30, wherein the ADNF I complex polypeptide
comprises at least one D-amino acid.
33. The method of claim 30, wherein the ADNF I complex polypeptide
comprises all D-amino acids.
34-45. (canceled)
46. A method for improving learning and/or memory in a subject, the
method comprising the step of administering prenatally to the
subject an Activity Dependent Neurotrophic Factor I complex (ADNF
I) polypeptide in an amount sufficient to improve postnatal
learning and/or memory of the subject, wherein the ADNF I complex
polypeptide comprises an amino acid sequence or a fragment thereof
selected from the group consisting of: TABLE-US-00008
WSDVGVSSGSAPDAFK (SEQ ID NO:1) NNSTTYAPISANVSTALGSTAALPTAAGPV (SEQ
ID NO:2) NFLTSHYSAANSVVGGTNPGK (SEQ ID NO:3) NPSGTDWLNTNNQANPFN
(SEQ ID NO:4) LVPLTPINR (SEQ ID NO:5)
VLQAVXGADSNVAFQGKVIYRSESSGTSELLTR (SEQ ID NO:6) GPTADITLTK (SEQ ID
NO:7) GTPTGXGPLIQ (SEQ ID NO:8) VDPASGYPIVGYT (SEQ ID NO:9)
PSGTDWLNT (SEQ ID NO:10) and SESSGTSELLTR. (SEQ ID NO:11)
47. The method of claim 46, wherein the subject has normal mental
capacity.
48. The method of claim 46, wherein the subject has mental
retardation, a family history of mental retardation, Down's
syndrome, or a mother who is at least 35 years of age when pregnant
with the subject.
49. The method of claim 48, wherein mental retardation is not
caused by consumption of alcohol by the subject's mother during
pregnancy.
50. The method of claim 46, wherein the ADNF I complex polypeptide
is administered around the time of neural tube development and/or
closure of the neural tube.
51. The method of claim 46, wherein the ADNF I complex polypeptide
is intraperitoneally administered to the mother during
pregnancy.
52. The method of claim 46, wherein the ADNF I complex polypeptide
is orally or nasally administered to the mother during
pregnancy.
53. The method of claim 46, wherein the ADNF I complex polypeptide
is encoded by a nucleic acid which is administered to the
subject.
54. The method of claim 46, wherein the ADNF I complex polypeptide
comprises at least one D-amino acid.
55. The method of claim 46, wherein the ADNF I complex polypeptide
comprises all D-amino acids.
Description
CROSS-REFERENCES TO RELATED APPLICATIONS
[0001] This application is a continuation of U.S. National Phase
application Ser. No. 10/489,515, filed Oct. 14, 2004, which is a
National Phase Application under 35 U.S.C. .sctn. 371 of
Application No. PCT/US2002/29146, filed Sep. 12, 2002, which claims
priority to U.S. Provisional Application No. 60/322,760 filed Sep.
12, 2001 and U.S. Provisional Application No. 60/371,961, filed
Apr. 10, 2001. All of these applications are incorporated herein by
reference.
[0002] This application is related to U.S. Ser. No. 07/871,973
filed Apr. 22, 1992, now U.S. Pat. No. 5,767,240; U.S. Ser. No.
08/342,297, filed Oct. 17, 1994 (published as WO96/11948), now U.S.
Pat. No. 6,174,862; U.S. Ser. No. 60/037,404, filed Feb. 7, 1997
(published as WO98/35042); U.S. Ser. No. 09/187,330, filed Nov. 11,
1998 (published as WO00/27875); U.S. Ser. No. 09/267,511, filed
Mar. 12, 1999 (published as WO00/53217); U.S. Ser. No. 60/149,956,
filed Aug. 18, 1999 (published as WO01/12654); U.S. Ser. No.
60/208,944, filed May 31, 2000; U.S. Ser. No. 60/267,805, filed
Feb. 8, 2001; and PCT 01/17758, filed May 31, 2001, herein each
incorporated by reference in their entirety.
STATEMENT AS TO RIGHTS TO INVENTIONS MADE UNDER FEDERALLY SPONSORED
RESEARCH AND DEVELOPMENT
[0003] Not applicable.
FIELD OF THE INVENTION
[0004] This invention relates to Activity Dependent Neurotrophic
Factor I complex (ADNF I complex) and polypeptides of this complex,
which produce their neurotrophic effects through multiple proteases
intrinsic to the ADNF I complex. The invention also relates to
pharmaceutical compositions comprising ADNF I complex polypeptides,
as well as methods for reducing neuronal cell death in vitro and in
vivo, methods for treating oxidative stress in a patient, methods
for reducing a condition associated with fetal alcohol syndrome in
a subject, and methods of enhancing learning and memory both pre-
and post-natally, all of which methods use the ADNF I complex
polypeptides of the invention.
BACKGROUND OF THE INVENTION
[0005] Neuronal cell death has been associated with various
clinical conditions and diseases. These conditions and diseases
include, for example, neurodegenerative diseases such as
Alzheimer's disease, AIDS-related dementia, Huntington's disease,
and Parkinson's disease. Neuronal cell death has been also
associated with developmental retardation and learning impairments.
These diseases and conditions are severely debilitating and have a
lifelong impact on individuals diagnosed with such diseases and
conditions.
[0006] It has previously been reported that Activity Dependent
Neurotrophic Factor (ADNF) polypeptides can be used to prevent or
reduce neuronal cell death. Activity Dependent Neurotrophic Factor
I (ADNF I) polypeptide is secreted by astroglial cells in the
presence of vasoactive intestinal peptide (VIP). The ADNF I
polypeptide exhibits survival-promoting activity for neurons at
surprisingly low, femtomolar concentrations (Brenneman & Gozes,
J. Clin. Invest. 97:2299-2307 (1996)). Further studies identified
peptide fragments of ADNF I that mimic the neurotrophic and
neuroprotective properties of ADNF I. The shortest peptide (i.e.,
the active core site) that captured the survival-promoting activity
of ADNF I was the peptide SALLRSIPA, (SEQ ID NO:12) designated as
ADNF-9 or SAL (Brenneman et al., J. Pharm. Exp. Therp. 285:619-627
(1998)). Studies of related molecules to the ADNF I polypeptide
resulted in the discovery of Activity Dependent Neuroprotective
Protein (called ADNP or ADNF III interchangeably). This protein was
cloned (Bassan et al., J. Neurochem. 72:1283-1293 (1999)) and was
found to have an active peptide similar in biological activity to
SAL. This peptide (i.e., the active core site) was NAPVSIPQ (SEQ ID
NO:13), designated as NAP.
[0007] ADNF polypeptides have been shown to prevent neuronal cell
death both in vitro and in vivo. For example, ADNF polypeptides
have been shown to prevent neuronal cell death associated with
tetrodotoxin (electrical blockade), the .beta.-amyloid peptide (the
Alzheimer's disease neurotoxin), N-methyl-D-aspartate
(excitotoxicity), and the human immune deficiency virus envelope
protein. In addition, daily injections of ADNF polypeptides to
newborn apolipoprotein E-deficient mice accelerated the acquisition
of developmental reflexes and prevented short-term memory deficits.
See, e.g., Bassan et al., J. Neurochem. 72:1283-1293 (1999).
Moreover, pretreatment with ADNF polypeptides has been previously
shown to reduce numerous or various conditions associated with
fetal alcohol syndrome in a subject. See, U.S. Ser. No. 09/265,511,
filed Mar. 12, 1999.
[0008] Although ADNF polypeptides have unlimited potential as
neuroprotectants and/or therapeutic agents, it would be
advantageous to provide additional ADNF polypeptides that have
different properties from the known ADNF polypeptides. For example,
availability of a number of ADNF polypeptides with different
affinities for their receptors would allow targeting specific
receptors in different cell types. Furthermore, additional ADNF
polypeptides would aid in designing a drug treatment regime that
can be individually tailored for each patient affected by
neurodegenerative disorders. Finally, knowledge regarding the
mechanism of action of ADNF would be useful for aiding in drug
treatment for patients affected by neurodegenerative disorders.
SUMMARY OF THE INVENTION
[0009] The invention demonstrates for the first time that the
neuroprotective action of the ADNF I complex is associated with
protease activity. The ADNF I complex has at least three, distinct
survival promoting peaks or components isolated by N--CHO capillary
electrophoresis (see, e.g., FIG. 2). The survival promoting
activity of components I and III of the ADNF I complex are
inhibited by E-64, an irreversible cysteine protease inhibitor, and
the survival promoting activity of component II of the ADNF I
complex is inhibited by peptstatin A, an inhibitor of aspartyl
proteases. The experiments conducted herein demonstrate that
component II is a calcium-dependent protease. This component can be
detected with a cathepsin D fluorogenic substrate:
Bz-Arg-Gly-Phe-Phe-Pro-4m.beta.NA, HCL. Inactivation of ADNF is
mediated through an intrinsic subtilisin-like protease in the ADNF
I complex, which ADNF I lytic activity is activated at
pH>8.0.
[0010] The present invention is also based upon a surprising
discovery that new ADNF I complex polypeptides are effective for
reducing neuronal cell death, for reducing oxidative stress, for
reducing condition(s) associated with fetal alcohol syndrome in a
subject, for enhancing learning and memory, both pre- and
post-natally, and for other conditions. The ADNF I complex
polypeptides contain active sites and provide neuroprotective and
growth-promoting functions. These ADNF I complex polypeptides are
from subunits of ADNF I and/or accessory molecules of ADNF I, which
co-isolate with ADNF I from conditioned medium of VIP-stimulated
astrocyte cultures. Antiserum to these polypeptides blocks the
action of ADNF I and ADNF I complex polypeptides.
[0011] In one aspect, the present invention provides an Activity
Dependent Neurotrophic Factor I complex (ADNF I) polypeptide, the
ADNF I polypeptide comprising an amino acid sequence or a fragment
thereof selected from the group consisting of:
TABLE-US-00001 WSDVGVSSGSAPDAFK (SEQ ID NO:1)
NNSTTYAPISANVSTALGSTAALPTAAGPV (SEQ ID NO:2) NFLTSHYSAANSVVGGTNPGK
(SEQ ID NO:3) NPSGTDWLNTNNQANPFN (SEQ ID NO:4) LVPLTPINR (SEQ ID
NO:5) VLQAVXGADSNVAFQGKVIYRSESSGTSELLTR (SEQ ID NO:6) GPTADITLTK
(SEQ ID NO:7) GTPTGXGPLIQ (SEQ ID NO:8) VDPASGYPIVGYT (SEQ ID NO:9)
PSGTDWLNT (SEQ ID NO:10) and SESSGTSELLTR. (SEQ ID NO:11)
[0012] In one embodiment, the polypeptide is covalently linked to a
lipophilic moiety, e.g., fatty acyl groups and steroids.
[0013] In another embodiment, the polypeptide comprises at least
one D-amino acid. In another embodiment, either an N-terminal amino
acid or a C-terminal amino acid of the polypeptide is a D-amino
acid. In another embodiment, both N-terminal and C-terminal amino
acids of the polypeptide are D-amino acids. In another embodiment,
the polypeptide comprises all D-amino acids.
[0014] In another embodiment, the ADNF I polypeptide comprises up
to about 20 amino acids at each of an N-terminus and a C-terminus
of the polypeptide.
[0015] In another aspect, the present invention provides an
antibody that specifically binds to the ADNF I complex polypeptide
described above.
[0016] In another aspect, the present invention provides a nucleic
acid encoding an amino acid sequence comprising an ADNF I complex
polypeptide as described above.
[0017] In another aspect, the present invention provides a
pharmaceutical composition comprising a pharmaceutically acceptable
excipient and an ADNF I complex polypeptide as described above.
[0018] In another embodiment, the ADNF I complex polypeptide is
administered intranasally or orally. In another embodiment, the
ADNF I complex polypeptide is encoded by a nucleic acid which is
administered to the subject.
[0019] In one aspect, the present invention provides a method for
preventing neuronal cell death, the method comprising contacting
neuronal cells with at least one of the above described ADNF I
complex polypeptides. In one embodiment, the neuronal cell death is
in a patient infected with immunodeficiency virus. In another
embodiment, the neuronal cell death is associated with
excito-toxicity induced by N-methyl-D-aspartate stimulation. In yet
another embodiment, the neuronal cell death is induced by the
beta-amyloid peptide in a patient afflicted with Alzheimer's
disease. In yet another embodiment, the neuronal cell death is
induced by cholinergic blockade in a patient afflicted with
Alzheimer's disease, which results in learning impairment. In
another embodiment, the neuronal cells are selected from the group
consisting of spinal cord neurons, hippocampal neurons, cerebral
cortical neurons and cholinergic neurons.
[0020] In yet another aspect, the present invention provides a
method for reducing oxidative stress in a patient, the method
comprising administrating to the patient at least one of the ADNF I
complex polypeptides described above in an amount sufficient to
treat oxidative stress.
[0021] In yet another aspect, the present invention provides a
method for reducing a condition associated with fetal alcohol
syndrome in a subject who is exposed to alcohol in utero, the
method comprising administering to the subject at least one ADNF I
complex polypeptide described above in an amount sufficient to
reduce a condition associated with fetal alcohol syndrome.
[0022] In one embodiment, the condition is selected from the group
consisting of: a decreased body weight of a subject; a decreased
brain weight of the subject; a decreased level of VIP mRNA of a
subject; and death of a subject in utero.
[0023] In another aspect, the present invention provides a method
for improving learning and/or memory in a subject, the method
comprising the step of administering postnatally to the subject at
least one ADNF I complex polypeptide described above in an amount
sufficient to improve postnatal learning and/or memory of the
subject.
[0024] In one embodiment, the subject is afflicted with a
neuropathology. In another embodiment, the subject has Alzheimer's
disease. In another embodiment, the subject has Down's syndrome. In
another embodiment, the subject is normal. In another embodiment,
the subject is old.
[0025] In another embodiment, the polypeptide improves short term
or reference memory.
[0026] In another aspect, the present invention provides a method
for improving learning and/or memory in a subject, the method
comprising the step of administering prenatally to the subject at
least one ADNF I complex polypeptide described above in an amount
sufficient to improve prenatal learning and/or memory of the
subject.
[0027] In one embodiment, the subject has mental retardation, a
family history of mental retardation, Down's syndrome, or a mother
who is at least 35 years of age when pregnant with the subject. In
another embodiment, the ADNF I polypeptide is administered around
the time of neural tube development and/or closure of the neural
tube. In another embodiment, the ADNF I polypeptide is orally,
nasally, or intraperitoneally administered to the mother during
pregnancy.
BRIEF DESCRIPTION OF THE DRAWINGS
[0028] FIG. 1. Electropherogram of biochemically (Brenneman &
Gozes, J. Clin. Invest. 97:2299-2304 (1996)) isolated ADNF I. The
ADNF I peak is observed at 15.55 min and corresponds to the 14 kDa
region as determined by molecular weight standards with the
eCAP/SDS capillary (Beckman Instruments). Purified ADNF I was
diluted 1:3 with water. The running and sample buffer for these
analyses were with the eCAP/SDS kit (Beckmann Instruments).
Electrophoresis was conducted with 8.1 kV constant voltage and with
reversed polarity. The capillary consisted of sodium dodecyl
sulfate coating with a 100 .mu.m inside diameter, 47 cm in length
(300 V/cm). The pressure injection of the sample was 70 sec at 80
psi. The peak at 12.26 is orange G, a column reference marker which
acts a control to monitor column performance. The minor peak at
21.15 min corresponds to a peak of approximately 60 kD, which is
likely an aggregate form of ADNF I. Sample was monitored with
absorbance at 200 nm.
[0029] FIG. 2. Survival-promoting activity of 14 kDa peak isolated
by eCAP/SDS capillary electrophoresis. ADNF I was purified by
previously described chromatographic procedures (Brenneman et al.,
J. Clin. Invest. 97:2299-2304 (1996)). Salt was removed by multiple
centrifugations with the microcon filtration units (0.22.mu.) from
Millipore Corp. The exchange buffer was 10 mM monobasic phosphate,
pH 4.5). The ADNF preparation was then serially diluted in
phosphate buffered saline and tested in dissociated cerebral
cortical cultures from newborn rats. The culture conditions and
treatment schedule employed has been previously described
(Brenneman & Gozes, J. Clin. Invest. 97:2299-2304 (1996)) with
one exception: cultures assessed with cell counts had 5% horse
serum MEM/N3 growth medium during the treatment period, whereas
cultures assessed with CFDA on serum-free, MEM/N3 growth medium.
Cultures were treated with ADNF I and 1 .mu.M tetrodotoxin after 3
days in vitro, and then assessed 5 days later by two methodologies:
neuronal cell counts (shaded circles) and 5(6)-carboxyfluorescein
diacetate (CFDA, closed circles), a marker for neuronal survival
(Petroski & Geller, J. Neurosci. Method. 52:23-32 (1994)). Cell
counts were performed as previously described (Brenneman &
Gozes, J. Clin. Invest. 97:2299-2304 (1996)). The dotted horizontal
line represents the cell counts for control cultures. The error
bars are the standard error.
[0030] FIG. 3. Reverse phase high pressure liquid chromatography of
ADNF I complex digest peptides. Gel band was subjected to
proteolytic digestion with modified trypsin according to the method
of Moritz et al. (Techniques in Protein Chemistry VI, pp 311-319,
(1995)). Resulting digests were separated by RP-HPLC on a
narrowbore (2.1.times.250 mm) Vyda 218TP52 column. The gradient
described previously was employed (Fernandez et al., Anal. Biochem.
201:255-264 (1992)). Fractions were collected on a Biobrene-treated
glass fiber filter and dried prior to amino acid sequencing. The
letters above the peaks are abbreviations for the observed digest
peaks.
[0031] FIG. 4. Survival-promoting activity of peptide fractions
from the ADNF I complex digestion. The peptide fractions from the
reverse phase HPLC column described in FIG. 3 were tested in
dissociated cerebral cortical cultures as described in FIG. 2.
Peptide fractions were serially diluted in phosphate buffered
saline. The test cultures were co-treated with 1 .mu.M
tetrodotoxin. Significant increases in neuronal cell counts were
observed with the following peptides fractions: NNST, PSG+VDP, SES,
and GPT.
[0032] FIG. 5. Synthesized NNST peptide increases neuronal survival
in TTX-treated cerebral cortical cultures. The peptide
NNSTTYAPISANVSTAALGSTALLPTAAGPV (SEQ ID NO:2) (NNST) was
synthesized and treated as described in FIG. 2. Both neuronal cell
counts and CFDA were used to assess neuronal survival. The dotted
line represents the neuronal cell counts of control cultures. The
error bar is the standard error.
[0033] FIG. 6. Synthesized GPT peptide increases neuronal survival
in TTX-treated cerebral cortical cultures. The peptide GPTADITLTK
(SEQ ID NO:7) (GPT) was synthesized and treated as described in
FIG. 2. Both neuronal cell counts and CFDA were used to assess
neuronal survival. The dotted line represents the neuronal cell
counts of control cultures. The error bar is the standard
error.
[0034] FIG. 7. Synthesized peptide PSGTDWLNT (SEQ ID NO:10)
increases neuronal survival in TTX-treated cerebral cortical
cultures. The peptide PSGTDWLNT (SEQ ID NO:10) is a truncated
version of the digest peptide NPSGTDWLNTNNQANPFN (SEQ ID NO:4). The
shortened peptide PSG was synthesized and treated as described in
FIG. 2. Neuronal cell counts were used to assess neuronal survival.
Each point is the mean of two closely agreeing (<10%) values.
These data provide evidence that shortened version of the digest
peptide can exhibit potent protection and thus serves as an example
for claim for all truncated peptides of the digest peptides.
[0035] FIG. 8. Synthesized peptide SESSGTSELLTR (SEQ ID NO:11)
increases neuronal survival in TTX-treated cerebral cortical
cultures. The peptide SESSGTSELLTR (SEQ ID NO:11) (SES) was
synthesized and treated as described in FIG. 2. Both neuronal cell
counts and CFDA were used to assess neuronal survival. The dotted
line represents the neuronal cell counts of control cultures. The
error bar is the standard error.
[0036] FIG. 9. Effects of antiserum against NFL and WSD peptide on
neuronal survival in cerebral cortical cultures. IgG fractions of
anti-NFL and anti-WSD were obtained with a protein A column
utilizing a standard kit from Pierce Inc. The IgG antibodies were
incubated for 5 five days in dissociated cerebral cortical cultures
obtained from newborn mice. Neuronal cell counts were conducted the
conclusion on the test period. Cultures were fixed and preserved as
described previously (Brenneman & Gozes, J. Clin. Invest.
97:2299-2304 (1996)). Anti-NFL produced cell death.
[0037] FIG. 10. Effects of antiserum against NNST, GAD and PSG
peptide on neuronal survival in cerebral cortical cultures. IgG
fractions of anti-NNST, anti-GAD and anti-PSG were obtained with a
protein A column utilizing a standard kit from Pierce Inc. The IgG
antibodies were incubated for 5 five days in dissociated cerebral
cortical cultures obtained from newborn mice. Neuronal survival was
assessed with CFDA as described in FIG. 2 above. Anti-PSG increased
cell survival and anti-NNST produced cell death.
[0038] FIG. 11. Antiserum to NFL prevents ADNF J-mediated increases
in neuronal survival. ADNF I was isolated by a multiple
chromatographic steps as previously described. The purified ADNF I
was further purified on an eCAP/SDS capillary the peak
corresponding to 14 kDa was collected. The survival-promoting
activity of the 14 kDa ADNF I complex is shown in the closed
circles, with three peaks of activity evident. In sister cultures,
300 ng/ml of anti-NFL IgG was used to co-treat cultures given a
dose response to ADNF I. All cultures but controls were also
treated with 1 .mu.M tetrodotoxin to prevent the release of
endogenous ADNF I or the ADNF secretagogue YIP. Each point is the
mean of two closely agreeing (<10%) values.
[0039] FIG. 12. Antiserum to ADNF I-related peptides prevent ADNF
I-mediated increases in neuronal survival in cerebral cortical
cultures. The ADNF I preparation described in FIG. 11 was used to
co-treat cultures with various antiserum made against ADNF
J-derived peptides: NNST, GAD, WSD, and NFL. In addition, antiserum
to heat shock protein 60 (anti-hsp60) and to the ADNF I agonist
ADNF-14 (VLGGGSALLRSIPA (SEQ ID NO:14) (VGR)) were compared for
their ability to block ADNF-mediated increases in neuronal
survival. All antiserum were tested at 300 ng/ml. Abbreviations for
the peptides are shown in the key.
[0040] FIG. 13. Effect of a protease inhibitor cocktail on ADNF
I-like immunoreactivity isolated by affinity magnetic beads with
anti-NFL. For these experiments, the protease inhibitor cocktail #1
from Calbiochem was utilized. This cocktail inhibits a broad range
of proteases and contains 500 .mu.M AEBSF
{4-[2-aminoethyl]benzenesulfonylfluoride HCl}, 150 nM aprotinin, 1
.mu.M E-64, 0.5 M EDTA, 1 .mu.M leupeptin hemisulfate. The protease
cocktail was incubated with conditioned medium from VIP (0.1
nM)-stimulated astrocyte cultures for 3 hours at 4 degrees C.
Conditioned medium without the cocktail was incubated in parallel
at 4 degrees C. The ADNF I in the conditioned media was isolated
with anti-NFL attached to magnetic beads. The beads containing the
ADNF I were isolated by a magnet and the ADNF I from the two
samples were eluted from the antibodies with 0.5 M citrate (pH 2).
The survival-promoting activity of ADNF I immunoreactivity from CM
without protease inhibitor (closed circles) in comparison to ADNF I
immunoreactivity from CM with protease inhibitor (open inverted
triangle) is shown.
[0041] FIG. 14. SELDI analysis of ADNF I purified with an affinity
column with anti-ADNF-14. ADNF I was isolated from conditioned
medium from VIP-stimulated astrocyte cultures with an affinity
column containing an IgG antibody prepared from ADNF-14, a 14 amino
acid agonist of ADNF I that has been previously described
(Brenneman & Gozes, J. Clin. Invest. 97:2299-2304 (1996)). ADNF
I immunoreactivity was eluted with 0.5 M citrate (pH 2) and then
concentrated in macrosep filtration units (Pall Filtron Corp) and
microcon filtration units (Amicon corp). ADNF I complex was applied
to a SELDI (surface-enhanced laser desorption/ionization) reverse
phase chip (Ciphergen, Inc), washed three times with HEPES buffer
and then allowed to dry. The surface was then treated with
saturated sinapinic acid in 50% acetonitrile and 0.5%
trifluoroacetic acid. The masses were determined with a
time-of-flight mass spectrometer (Ciphergen Inc.).
[0042] FIG. 15. A and B SELDI analysis of ADNF I complex purified
as described in FIG. 14 with the exception of using an affinity
column containing anti-NFL. SELDI analysis was done as described in
FIG. 14.
[0043] FIG. 16. ADNF 1-like immunoreactivity in fiber-like
structures in the basal forebrain of the newborn rat brain.
Immunocytochemistry was performed with a rabbit anti-WSD. A
1:10,000 dilution of a 2 mg/ml IgG solution was used as the primary
antibody. A rabbit Elite Vectastain kit was used for the staining
(Vector Laboratories). Magnification: 20.times..
[0044] FIG. 17. ADNF I-like immunoreactivity in the para abducens
nucleus of the brainstem of the newborn rat brain.
Immunocytochemistry with anti-WSD was performed as described for
FIG. 16. Magnification: 20.times..
[0045] FIG. 18. ADNF I-like immunoreactivity in cells of the
reticular formation of the brainstem of the newborn rat brain.
Immunocytochemistry with anti-WSD was performed as described for
FIG. 16. Magnification: 40.times..
[0046] FIG. 19. Western analysis of immunoreactivity for NNST
peptide in astrocytes lysate. A 1:300 dilution of an affinity
purified antibody to NNST was used for the immunoblot analysis of a
lysate of cultured astrocytes. A comparisons was made between
lysates of VIP-stimulated versus control astrocytes cultures.
[0047] FIG. 20. N--CHO coated capillary electrophoretic analysis of
ADNF I. ADNF I was isolated by multiple chromatographic steps as
previously described. The samples was desalted in macrosep
filtration units (Pall Filtron Corp) and microcon filtration units
(Amicon corp). The replacement buffer was 10 mM monobasic phosphate
(pH 4.5). The conditions for N--CHO electrophoresis is as follows:
running buffer was 100 mM monobasic phosphate (pH 4.55). The
polarity was reversed to run to positive. The injection was 70 sec
at 80 psi. Samples were diluted 1:3 with water before injection.
Samples were monitored with absorbance at 200 nm. The distance to
the flow cell was 60 cm. The numeral above the peaks indicate the
number of the ADNF I complex component: I, II, IIIA and IIIB.
[0048] FIG. 21. Survival-promoting activity associated with ADNF I
complex components collected in peaks from N--CHO capillary
electrophoresis. ADNF I isolated by previously described means was
fractionated on N--CHO capillary electrophoresis as described in
FIG. 20. Five collections were made into 50 ul of running buffer
(100 mM monophosphate buffer, pH 4.55). Three components of ADNF I
complex were collected as follows: component I (shaded squares) was
obtained between 4.8 and 5.1 min; component II (open triangles) was
obtained tween 8.5 and 10.2 min; and components IIIA and IIIB were
collected between 12.5 and 13.5 min. All remaining time of
collections were made into a single tube (closed inverted
triangles) Each of the collections was serially diluted in
phosphate buffered saline and assessed for biological activity in
tetrodotoxin-treated cerebral cortical cultures. In sister
cultures, various IgG antibodies (300 ng) were tested for their
ability to block the biological activity of various ADNF I complex
components at dilutions of their peak efficacy. Summary of these
findings is shown in the legend. These data indicate that all of
the survival-promoting activity was confined to the time of
collection corresponding to the three peaks. No activity was
observed apart from these peaks. Each peak showed a distinct
pattern of interaction with the panel of antibodies and each
differed greatly in potency from the other ADNF I complex
components.
[0049] FIG. 22. Mixed cerebral cortical cultures composed of
neurons and glia were tested for the effects of protease inhibition
on ADNF J-mediated protection from neuronal cell death produced by
electrical blockade with tetrodotoxin (TTX). As previously
described (Brenneman & Gozes, J. Clin. Invest 97:2299, 1996),
purified ADNF I was added only once to the cultures three days
after seeding neurons onto the confluent feeder layer of
astrocytes. To measure and verify the purification of the ADNF I,
the ADNF I preparation was isolated on eCAP/SDS capillary
electrophoresis. The peak appearing in the 14 kDa region of the
capillary was isolated into 50 ul of running buffer. The running
and sample buffer for these analyses was provided by a kit provided
by Beckmann Instruments. Electrophoresis was conducted with 8.1 kV
constant voltage. The pressure injection time was 50 sec at 80 psi.
The concentration of the ADNF I preparation was estimated to be 1
.mu.M. Ribonuclease A was used as a standard for this analysis.
Serial dilutions of the ADNF I preparation were tested in the
presence of 1 .mu.M TTX. Prior to treatment and serial dilutions,
the ADNF I preparation was mixed with a cocktail of protease
inhibitors prepared by Calbiochem (La Jolla, Calif.). The mixture
of protease inhibitors included 500 .mu.M AEBSF--HCl, 150 nM
aprotinin, 1 .mu.M E-64, 05 mM EDTA and 1 .mu.M leupeptin
hemisulfate. The concentrations of inhibitor were the final
concentration after mixing with the ADNF I preparation. The
treatment period was 4 days. The termination, cultures were fixed
in glutaraldehyde as described previously (Brenneman & Gozes,
1996) and the neurons counted without knowledge of the treatment
group. Control studies indicate that the protease inhibitor
cocktail itself had no survival-promoting activity used at the
dilutions (1:10,000) employed in the present experiment.
[0050] FIG. 23. Effect of pepstatin A on ADNF J-mediated protection
from neuronal cell death produced by tetrodotoxin. Experiments were
conducted as described in FIG. 22, with the exception that 1 .mu.M
pepstatin A alone was used to inhibit protease activity rather than
the Calbiochem cocktail. Pepstatin A is a specific inhibitor of
aspartyl proteases. For these experiments, pepstatin A was
dissolved in methanol at 1 mg/ml. This was a 100.times. working
stock solution that was added to the ADNF I preparation.
Pre-treatment with pepstatin A blocked the survival-promoting
activity of only the peak appearing at 10.sup.-8 dilution.
[0051] FIG. 24. Effect of E-64 on ADNF J-mediated protection from
apoptotic neuronal cell death by treatment with tetrodotoxin.
Experiments were conducted as described in FIG. 22, with the
exception that 10 .mu.g/ml E-64 was used to irreversibly inhibit
cysteine proteases. E-64 was dissolved in ethanol at 1 mg/ml.
Pre-treatment with E-64 blocked the survival-promoting activity on
the first and third peak of the ADNF I complex appearing at
10.sup.-12 and 10.sup.-5 dilution.
[0052] FIG. 25. Effect of subtilisin V inhibitor
(Boc-Ala-Pro-Phe-NHO-Bz-pCl) on ADNF J-mediated protection from
apoptotic neuronal cell death produced by 1 .mu.M tetrodotoxin.
Experiments were conducted as described in FIG. 22, with the
exception that 0.1 mM subtilisin V inhibitor was used to treat the
ADNF I preparation. The 100.times. stock solution of the inhibitor
was 1 mg dissolved in 0.2 ml of ethanol. This inhibitor is an
irreversible cysteine and serine protease inhibitor. This inhibitor
enhanced the activity of components I and III, the peaks at
10.sup.-12 and 10.sup.-5 dilution.
[0053] FIG. 26. The N--CHO electropherogram of purified component 2
preparation from ADNF I is shown. This preparation was obtained by
immunopurification from an affinity (anti-VLGGGSALLRSIPA (SEQ ID
NO:14)) column followed by acetone precipitation. A single peak at
200 nm was observed. The conditions of the capillary
electrophoresis was: 100 mM monobasic phosphate buffer, pH, 4.55;
50 .mu.A constant current; 70 sec injection at 80 psi; 75 um i.d
capillary (Beckman instruments). The N--CHO column surface is
designed to separate carbohydrate moieties. The preparation
utilized for protease assays appeared to be a single peak with a
retention time consistent with that component 2 as previously
described (DHHS Ref No.: E-209-01/1-0). The protein concentration
of this preparation was estimated to 1 .mu.M based on standard
curves generated to glycoprotein alpha-1 acid analyzed under
identical conditions and detection at 280 nm.
[0054] FIG. 27. In the same component II preparation as described
in FIG. 26, an analysis by MALDI (matrix assisted laser
desorption/ionization) time-of-flight mass-spectrum was conducted.
These data indicated at mass of 13,757.9 Daltons. A single peak was
observed. This preparation was used for protease assays.
[0055] FIG. 28. A time course of component II (see FIGS. 26 and 27)
in a cell-free protease assay using
Bz-Arg-Gly-Phe-Phe-Pro-M.beta.NA, HCl (Cathepsin D substrate). The
conditions of the assay are as follows: 10 .mu.l of substrate (5 mg
dissolved in 100 .mu.l of DMSO (dimethyl sulfoxide), diluted to 1
ml of water), 10 .mu.l of 10 mM CaCl.sub.2; 50 .mu.l of component
II (10.sup.-8 dilution of the preparation characterized in FIGS. 26
and 27); and 30 .mu.l of buffer (0.5 M dibasic phosphate, pH 7.8).
For this assay, buffer, enzyme, calcium were added to 96 well
culture trays. Based on glycoprotein alpha-1 acid standards on NCHO
capillary, the optimum protease activity is observed at 1 fM
amounts of components II.
[0056] With the addition of the substrate, the generation of
fluorescence was measured automatically every 5 min in a Cytofluor
(Perseptive Biosystems). All data was expressed as a change in
arbitrary fluorescence units per 5 min. As shown in FIG. 28, in the
presence of component II from ADNF I, the increase in fluorescence
was linear for 50 min (closed circles). There was no detectable
change in fluorescence in the absence of component II (open
circles). Each point is the mean.+-.the standard error of three
values.
[0057] FIG. 29. Demonstration of linearity of component II
concentration using a cell-free protease assay and the blockade of
this activity with pepstatin A. The conditions for the protease
assay were the same as that described for FIG. 28. In culture wells
treated with pepstatin, 10 .mu.l of the stock solution (1 mg
dissolved in 1 ml of methanol, diluted 1:100 with water) was added.
In control wells, an addition 10 ul of methanol/water (1:100
dilution in water) alone was added. Fluorescence changes indicative
of peptide subtract cleavage were monitored as described in FIG.
28.
[0058] FIG. 30. Demonstration of calcium dependence for the
protease activity detected in component II of the ADNF I complex.
The conditions for the protease assay were the same as that
described for FIG. 28 with the exception of the substitution of 10
ul of water in some wells rather than 10 ul of calcium chloride (10
mM stock). Each measurement is the mean of at least 3
determinations.+-.standard error. Fluorescence changes indicative
of peptide substrate cleavage were monitored as described in FIG.
28.
[0059] FIG. 31. Comparison of Survival-Promoting Activity and
Protease Activity in the same preparation of component II of the
ADNF I complex. The protease assay is described in FIG. 28 and the
neuronal survival assay is described in FIG. 22. Excellent
correlation between the two assays are observed.
[0060] FIG. 32. MALDI time-of flight-mass spectrograph of
immunopurified ADNF I. The molecular weight of the ADNF I complex
is shown to be 14590 Daltons in a buffer of pH 7.0. This is the
peak that is observed prior to increasing the pH and re-analyzing
the preparation (see FIG. 33).
[0061] FIG. 33. MALDI time-of flight-mass spectrograph of
immunopurified ADNF I after raising the pH to 8.0 for 30 min and
then neutralizing back pH 7.0. The peak of ADNF I at 14590 is no
longer detectable, suggesting an instability of ADNF I at high pH.
This sensitivity to pH is thought to be due to the presence of a
subtilisin-like protease that is activated at basic pH. The
activation of this component of ADNF I results in the catalytic
degradation MALDI time-of flight-mass spectrograph of ADNF I of the
protein to amino acids and small peptides.
DETAILED DESCRIPTION OF THE INVENTION
Introduction
[0062] Proteases have major regulatory roles in the activation and
inactivation of critical proteins/peptides that are important to
cellular survival. Many neurodegenerative diseases are believed to
involve the inappropriate regulation of activated cell death:
apoptosis. The control of apoptotic death is now recognized to be
largely under the control of caspases, cysteine proteases that
regulate survival/death at checkpoints through intricate
interacting cascades of intracellular events. The extracellular
signals that regulate these pathways is an active area of research.
In addition, proteases are known to cleave pro-hormones to
activated forms. This body of knowledge indicates that hormones can
be released in a less active form and activated by proteolytic
cleavage. Importantly, the action of circulating proteins can also
be inactivated by the action of proteases. Thus, proteases are
recognized as major regulatory enzymes that control critical
pathways important to development, homeostasis and disease.
[0063] The present application demonstrates that ADNF I produces
its neurotrophic effects through multiple proteases intrinsic to
the ADNF I multiple-component protein complex. ADNF I has been
described as a complex of at least three components. The ADNF I
components were demonstrated by the isolation of three distinct,
survival-promoting peaks isolated by N--CHO capillary
electrophoresis (see, e.g. FIG. 2). These peaks had very different
EC50's in producing survival-promoting activity for tetrodotoxin
treated neurons and each was neutralized by a distinct group of
anti-peptides derived from ADNF I. In the present application, data
is presented which shows that the neurotrophic activities of the
ADNF components reside in their proteolytic actions.
[0064] In the present application, novel ADNF I peptides from the
ADNF I complex are also provided. The amino acid composition of the
ADNF I-related peptides were determined from trypsin digests of
biochemically purified ADNF I as described in U.S. Pat. No.
5,767,270. The newly discovered peptides exhibited neuroprotective
action from neuronal cell death produced by tetrodotoxin, a toxin
that blocks electrical activity and which produces death by
apoptosis. Inhibition of proteolytic activity or lowering the pH of
ADNF I revealed other survival-promoting components of ADNF I.
Novel peptides of the ADNF I complex were synthesized and confirmed
to exhibit survival-promoting activity. The components of the ADNF
I complex have been separated by N--CHO capillary electrophoresis
and this method can be used to characterize the components of ADNF
I.
[0065] Shorter peptides than that observed in the tryptic digests
of the ADNF I complex can increase neuronal survival. These data
indicate that core sequences reside within the digest peptides that
can mimic both the parent digest peptide and the parent protein
ADNF I. Furthermore, the addition of lipophilic moieties including
fatty acyl groups and steroids to the above peptide structures are
useful for therapeutic applications, as well as all D-amino acid
forms of the above peptides and mixtures of D- and L-amino acids
peptides.
[0066] Furthermore, antiserum made to some of these peptides
produced neuronal cell death in cerebral cortical cultures and the
anti-peptides from ADNF I complex blocked the survival-promoting
action of ADNF I. The antisera to the ADNF I complex peptides were
studied with immunohistochemistry to confirm their localization in
the central nervous system and to further establish the
characterization of the nature and uniqueness of these peptides and
ADNF I. Antiserum to the peptides were shown to produce neuronal
cell death in dissociated cerebral cortical cultures and all
antiserum listed below prevented ADNF 1-mediated-neuroprotection.
These antiserum and their peptide antigens are therefore useful as
diagnostic reagents to measure and distinguish ADNF I-like activity
from other survival-promoting agents
[0067] Because the above ADNF I complex peptides prevent apoptotic
death, these peptides can be used for prevention of cell death
produced by clinically relevant toxins where apoptosis is
documented as a pathophysiological mechanism for the associated
disease. These toxins include: beta amyloid peptide, excitatory
amino acids, oxidative stress, ethanol, and gp120, the external
envelope protein from the human immunodeficiency virus. Clinical
applications include: Alzheimer's disease, HIV-related dementia
complex, stroke, head trauma, cerebral palsy, fetal alcohol
syndrome and Parkinson's disease.
DEFINITIONS
[0068] The phrase "ADNF I complex polypeptide" or "ADNF I protein"
or "ADNF I polypeptide" or "ADNF I peptide" refers to one or more
polypeptides or acid or protease digest peptides from the ADNF I
complex, which includes ADNF I, ADNF I subunits, and ADNF I
co-factors that co-isolate with ADNF I from VIP-stimulated
astrocyte cultures, using the methods described herein and in
Brenneman & Gozes, J. Clin. Invest. 97:2299-2307 (1996). In one
embodiment, an ADNF I complex polypeptide comprises an active core
sequence having an amino acid sequence or a fragment thereof
selected from the group consisting of:
TABLE-US-00002 WSDVGVSSGSAPDAFK (SEQ ID NO:1)
NNSTTYAPISANVSTALGSTAALPTAAGPV (SEQ ID NO:2) NFLTSHYSAANSVVGGTNPGK
(SEQ ID NO:3) NPSGTDWLNTNNQANPFN (SEQ ID NO:4) LVPLTPINR (SEQ ID
NO:5) VLQAVXGADSNVAFQGKVIYRSESSGTSELLTR (SEQ ID NO:6) GPTADITLTK
(SEQ ID NO:7) GTPTGXGPLIQ (SEQ ID NO:8) VDPASGYPIVGYT (SEQ ID NO:9)
PSGTDWLNT (SEQ ID NO:10) and SESSGTSELLTR, (SEQ ID NO:11)
or conservatively modified variants thereof that have
neurotrophic/neuroprotective activity as measured with in vitro
cortical neuron culture assays described by, e.g., Brenneman et
al., J. Pharmacol. Exp. Therp. 285:629-627 (1998); Bassan et al.,
J. Neurochem. 72:1283-1293 (1999). An ADNF I complex polypeptide
includes alleles, polymorphic variants, or interspecies homolog, or
any subsequences thereof, that exhibit neuroprotective/neurotrophic
action on, e.g., neurons originating in the central nervous system
either in vitro or in vivo.
[0069] The term "ADNF I" refers to an activity dependent
neurotrophic factor polypeptide or complex having a molecular
weight of about 14,000 Daltons with a pI of 8.3.+-.0.25. In one
embodiment, ADNF I polypeptides have an active core site comprising
an amino acid sequence of Ser-Ala-Leu-Leu-Arg-Ser-Ile-Pro-Ala (also
referred to as "SALLRSIPA," "SAL," or "ADNF I-9"; SEQ ID NO:12).
See, Brenneman & Gozes, J. Clin. Invest. 97:2299-2307 (1996),
Glazner et al., Anat. Embryol. 200:65-71 (1999), Brenneman et al.,
J. Pharm. Exp. Ther. 285:619-27 (1998), Gozes & Brenneman, J.
Mol. Neurosci. 7:235-244 (1996), and Gozes et al., Dev. Brain Res.
99:167-175 (1997), all of which are herein incorporated by
reference. Unless indicated as otherwise, "SAL" refers to a peptide
having an amino acid sequence of
Ser-Ala-Leu-Leu-Arg-Ser-Ile-Pro-Ala (SEQ ID NO:12), not a peptide
having an amino acid sequence of Ser-Ala-Leu.
[0070] The terms "ADNF III" and "ADNP" refer to an activity
dependent neurotrophic factor polypeptide having a molecular weight
by western analysis of about 114 kDa (about 828 amino acid
residues), a calculated molecular weight of 123,562.8 daltons, and
a theoretical pI of about 6.97. ADNF III polypeptides have an
active core site comprising an amino acid sequence of
Asn-Ala-Pro-Val-Ser-Ile-Pro-Gln (also referred to as "NAPVSIPQ,"
"NAP," or "ADNF III-8"; SEQ ID NO:13). See, Zamostiano et al., J.
Biol. Chem. 276:708-714 (2001); Bassan et al., J. Neurochem.
72:1283-1293 (1999), incorporated herein by reference. Unless
indicated as otherwise, "NAP" refers to a peptide having an amino
acid sequence of Asn-Ala-Pro-Val-Ser-Ile-Pro-Gln (SEQ ID NO:13),
not a peptide having an amino acid sequence of Asn-Ala-Pro.
[0071] The phrase "reducing neuronal cell death" refers to
reduction, including prevention, of neuronal cell death. Reduction
is a change of a parameter by about 10% to about 100%, preferably
at least about 50%, and more preferably at least about 80% compared
to that of the control (e.g., without treatment with, e.g., ADNF I
complex polypeptides). The reduction of neuronal cell death can be
measured by any methods known in the art. For example, ADNF I
complex polypeptides that reduce neuronal cell death can be
screened using the various methods described in U.S. Ser. No.
60/037,404, filed Feb. 27, 1997 (published as WO98/35042) and U.S.
Ser. No. 09/187,330, filed Nov. 6, 1998, both of which are
incorporated herein by reference.
[0072] The phrase "oxidative stress" in cells or tissues refers to
enhanced generation of free radicals or reactive oxygen species
(ROS) (such as .alpha.-hydroxy ethyl radical, superoxide radical,
hydroxy radical, peroxy radical, and hydrogen peroxide) and/or a
depletion in antioxidant defense system causing an imbalance
between prooxidants and antioxidants. Enzymatic antioxidant system
includes, e.g., superoxide dismutase, catalase, glutathione
peroxidase, and glutathione reductase, and nonenzymatic
antioxidants include, e.g., reduced glutathione, vitamin A, C, and
E. See, Schlorff et al., Alcohol 17:97-105 (1999).
[0073] The phrase "reducing oxidative stress" refers to reduction,
including prevention, of oxidative stress in cells and tissues.
Reduction is a change of a parameter by about 10% to about 100%,
preferably at least about 50%, and more preferably at least about
80% compared to that of the control (e.g., without treatment with,
e.g., ADNF I complex polypeptides). The reduction in oxidative
stress can be measured by any methods known in the art. For
example, ADNF I complex polypeptides that reduce oxidative stress
can be screened by using primary neurons treated with FeSO.sub.4 in
vitro as described infra. Also, ADNF I complex polypeptides that
reduce oxidative stress can be screened using animals that ingested
ethanol which is known to cause oxidative stress in cells and
tissues. For example, the effects of ADNF I complex polypeptides on
lipid peroxidation in plasma and/or antioxidant system of rats that
ingested ethanol can be used. See, e.g., Schlorff et al., Alcohol
17:97-105 (1999).
[0074] The phrases "fetal alcohol syndrome" and "fetal alcohol
effects" relate to various physical and mental conditions of an
embryo, a fetus, or a subject who is exposed to alcohol in utero
(e.g., whose mother consumed alcohol during pregnancy) in an amount
sufficient to initiate the development of these conditions or to
cause these conditions in the absence of prevention treatment,
e.g., treatment with ADNF I complex polypeptides. Some of these
conditions include, but are not limited to, the following:
[0075] skeletal deformities: deformed ribs and sternum; curved
spine; hip dislocations; bent, fused, webbed, or missing fingers or
toes; limited movement of joints; small head; facial abnormalities:
small eye openings; skin webbing between eyes and base of nose;
drooping eyelids; nearsightedness; failure of eyes to move in same
direction; short upturned nose; sunken nasal bridge; flat or absent
groove between nose and upper lip; thin upper lip; opening in roof
of mouth; small jaw; low-set or poorly formed ears; organ
deformities: heart defects; heart murmurs; genital malformations;
kidney and urinary defects; central nervous system handicaps: small
brain; faulty arrangement of brain cells and connective tissue;
mental retardation--usually mild to moderate, but occasionally
severe; learning disabilities; short attention span; irritability
in infancy; hyperactivity in childhood; poor body, hand, and finger
coordination; and other abnormalities: brain weight reduction, body
weight reduction, a higher rate of death in utero, and a decrease
in the level of VIP (e.g., VIP mRNA).
[0076] The phrase "reducing a condition associated with fetal
alcohol syndrome" refers to reduction, including prevention, of
parameters associated with fetal alcohol syndrome. Reduction is a
change of a parameter by about 10% to about 100%, preferably at
least about 50%, and more preferably at least about 80% compared to
that of the control (e.g., exposed to alcohol in utero without any
treatment, e.g., treatment with ADNF I complex polypeptides). The
parameters can be any physical or mental condition listed above.
For example, they can be: (1) the percentage of fetus death, (2)
fetal weights and fetal brain weights, (3) the level of VIP (e.g.,
VIP mRNA) in embryos, (4) learning and/or memory, and (5) the
glutathione level.
[0077] The phrase "a subject with fetal alcohol syndrome" relates
to an embryo, a fetus, or a subject, in particular a human, who is
exposed to alcohol in utero and who has fetal alcohol syndrome or
who is at risk or in danger of developing, due to maternal alcohol
consumption, any of the conditions related to fetal alcohol
syndrome, such as the effects described above.
[0078] Various parameters can be measured to determine if an ADNF I
complex polypeptides or a mixture of ADNF I complex polypeptides
improves performance of a subject, when the peptide is administered
either pre- or post-natally (e.g., learning and memory). For
example, the degree of learning deficits can be compared between
the control (e.g., untreated with ADNF I complex polypeptides) and
a group pretreated with ADNF I complex polypeptides, either pre- or
post-natally. The phrase "improving learning and memory" refers to
an improvement or enhancement of at least one parameter that
indicates learning and memory. Improvement or enhancement is change
of a parameter by at least 10%, optionally at least about 20%, at
least about 30%, at least about 40%, at least about 50%, at least
about 60%, at least about 70%, at least about 80%, at least about
90%, at least about 100%, at least about 150%, at least about 200%,
etc. The improvement of learning and memory can be measured by any
methods known in the art. For example, ADNF I complex polypeptides
that improve learning and memory can be screened using Morris water
maze (see, also, Gozes et al., Proc. Natl. Acad. Sci. USA
93:427-432 (1996); Gozes et al., J. Pharmacol. Exp. Therap. 293:
1091-1098 (2000)). Memory and learning can also be screened using
any of the methods described herein or other methods that are well
known to those of skill in the art, e.g., the Randt Memory Test,
the Wechler Memory Scale, the Forward Digit Span test, or the
California Verbal Learning Test.
[0079] The term "memory" includes all medical classifications of
memory, e.g., sensory, immediate, recent and remote, as well as
terms used in psychology, such as reference memory, which refers to
information gained from previous experience, either recent or
remote (see, e.g. Harrison's Principles of Internal Medicine,
volume 1, pp. 142-150 (Fauci et al., eds., 1988).
[0080] Pathologies or neuropathologies that would benefit from
therapeutic and diagnostic applications of this invention include,
for example, the following:
[0081] diseases of central motor systems including degenerative
conditions affecting the basal ganglia (Huntington's disease,
Wilson's disease, striatonigral degeneration, corticobasal
ganglionic degeneration), Tourette's syndrome, Parkinson's disease,
progressive supranuclear palsy, progressive bulbar palsy, familial
spastic paraplegia, spinomuscular atrophy, ALS and variants
thereof, dentatorubral atrophy, olivo-pontocerebellar atrophy,
paraneoplastic cerebellar degeneration, and dopamine toxicity;
[0082] diseases affecting sensory neurons such as Friedreich's
ataxia, diabetes, peripheral neuropathy, retinal neuronal
degeneration;
[0083] diseases of limbic and cortical systems such as cerebral
amyloidosis, Pick's atrophy, Retts syndrome;
[0084] neurodegenerative pathologies involving multiple neuronal
systems and/or brainstem including Alzheimer's disease,
AIDS-related dementia, Leigh's disease, diffuse Lewy body disease,
epilepsy, multiple system atrophy, Guillain-Barre syndrome,
lysosomal storage disorders such as lipofuscinosis,
late-degenerative stages of Down's syndrome, Alper's disease,
vertigo as result of CNS degeneration;
[0085] pathologies associated with developmental retardation and
learning impairments, and Down's syndrome, and oxidative stress
induced neuronal death;
[0086] pathologies arising with aging and chronic alcohol or drug
abuse including, for example, with alcoholism the degeneration of
neurons in locus coeruleus, cerebellum, cholinergic basal
forebrain; with aging degeneration of cerebellar neurons and
cortical neurons leading to cognitive and motor impairments; and
with chronic amphetamine abuse degeneration of basal ganglia
neurons leading to motor impairments;
[0087] pathological changes resulting from focal trauma such as
stroke, focal ischemia, vascular insufficiency, hypoxic-ischemic
encephalopathy, hyperglycemia, hypoglycemia, closed head trauma, or
direct trauma;
[0088] pathologies arising as a negative side-effect of therapeutic
drugs and treatments (e.g., degeneration of cingulate and
entorhinal cortex neurons in response to anticonvulsant doses of
antagonists of the NMDA class of glutamate receptor).
[0089] The term "spatial learning" refers to learning about one's
environment and requires knowledge of what objects are where. It
also relates to learning about and using information about
relationships between multiple cues in environment. Spatial
learning in animals can be tested by allowing animals to learn
locations of rewards and to use spatial cues for remembering the
locations. For example, spatial learning can be tested using a
radial arm maze (i.e., learning which arm has food) a Morris water
maze (i.e., learning where the platform is). To perform these
tasks, animals use cues from test room (positions of objects,
odors, etc.). In human, spatial learning can also be tested. For
example, a subject can be asked to draw a picture, and then the
picture is taken away. The subject is then asked to draw the same
picture from memory. The latter picture drawn by the subject
reflects a degree of spatial learning in the subject.
[0090] The term "subject" refers to any mammal, in particular
human, at any stage of life. For example, the subject can refer to
an embryo, a fetus, a baby, a child, an adolescent or an adult.
[0091] A "normal" subject or a subject having "normal mental
capacity" refers to a subject whose intellectual functioning level
is around or above average (e.g., having an IQ above 75). A
"normal" subject can also refer to a subject, such as a fetus, who
does not appear to have any mental impairment (e.g., according to
an amniocentesis test) and/or has no risk factors (e.g., family
history of mental retardation or a mother who consumed alcohol in
excessive amount during pregnancy to cause fetal alcohol syndrome
in the fetus).
[0092] A subject is considered to have "mental retardation" based
on the following three criteria: intellectual functioning level
(IQ) is below 70-75; significant limitations exist in two or more
adaptive skill areas; and the condition is present from childhood
(defined as age 18 or less) (AAMR, 1992). Adaptive skill areas are
those daily living skills needed to live, work and play in the
community. They include communication, self-care, home living,
social skills, leisure, health and safety, self-direction,
functional academics (reading, writing, basic math), community use
and work.
[0093] The term "Down's syndrome" is a chromosome disorder and
occurs when, instead of the normal complement of 2 copies of
chromosome 21, there is a whole, or sometimes part of an additional
chromosome 21.
[0094] The term "contacting" is used herein interchangeably with
the following: combined with, added to, mixed with, passed over,
incubated with, flowed over, etc. Moreover, the ADNF I complex
polypeptides of the present invention can be "administered" by any
conventional method such as, for example, parenteral, oral,
topical, and inhalation routes. In certain embodiments, oral
administration is employed. In the context of methods related to
fetal alcohol syndrome, or enhanced learning and memory via
pre-natal treatment, ADNF I complex polypeptides can be
administered directly to an embryo, a fetus, or a subject in utero
or to the subject in utero indirectly, by administering the
polypeptide to the mother by any other methods described
herein.
[0095] "An amount sufficient" or "an effective amount" is that
amount of a given ADNF I complex polypeptide that reduces neuronal
cell death or reduces a condition, such as neuronal cell death,
fetal alcohol syndrome or oxidative stress as described herein, or
enhances learning and memory, as described herein. For example, in
the context of neuronal death, "an amount sufficient" or "an
effective amount" is that amount of a given ADNF I complex
polypeptide that reduces neuronal cell death in the assays of,
e.g., Hill et al., Brain Res. 603:222-233 (1993); Brenneman et al.,
Nature 335:639-642 (1988); or Brenneman et al., Dev. Brain Res.
51:63-68 (1990); Forsythe & Westbrook, J. Physiol. Lond.
396:515-533 (1988). In the context of reducing oxidative stress,
"an amount sufficient" or "an effective amount" is that amount of
ADNF I complex polypeptide that reduces or prevents, e.g., changes
in lipid peroxidation in plasma or changes in antioxidant system in
accordance with the assays described in Schlorff et al., Alcohol
17:97-105 (1999). In the context of reducing fetal alcohol
syndrome, "an amount sufficient" or "an effective amount" is that
amount of a given ADNF I complex polypeptide that reduces or
prevents, for example, (1) the percentage of fetus death, (2) a
reduction in fetal weights and fetal brain weights, or (3) a
reduction in the level of VIP mRNA in embryos. In the context of
improving learning and memory, "an amount sufficient" or "an
effective amount" is that amount of a given ADNF I complex
polypeptide that reduces the latency in finding a platform in a
watermaze test, either in the first daily test (indicative of
reference memory) or in the second daily test (indicative of short
term memory). The dosing range can vary depending on the ADNF I
complex polypeptide used, the route of administration and the
potency of the particular ADNF I complex polypeptide, but can
readily be determined using the foregoing assays.
[0096] The dosing range can vary depending on the ADNF I complex
polypeptide used, the route of administration and the potency of
the particular ADNF I complex polypeptide, but can readily be
determined using the foregoing assays.
[0097] The term "biologically active" refers to a peptide sequence
that will interact with naturally occurring biological molecules to
either activate or inhibit the function of those molecules in vitro
or in vivo. The term "biologically active" is most commonly used
herein to refer to ADNF I complex polypeptides or subsequences
thereof that exhibit neuroprotective/neurotrophic action on neurons
originating in the central nervous system either in vitro or in
vivo. The neuroprotective/neurotrophic action of ADNF I complex
polypeptides can be tested using, e.g., cerebral cortical cultures
treated with a neurotoxin (see, Gozes et al., Proc. Nat'l. Acad.
Sci. USA 93:427-432 (1996)).
[0098] The terms "isolated," "purified," or "biologically pure"
refer to material that is substantially or essentially free from
components which normally accompany it as found in its native
state. Purity and homogeneity are typically determined using
analytical chemistry techniques such as polyacrylamide gel
electrophoresis or high performance liquid chromatography. A
protein that is the predominant species present in a preparation is
substantially purified. In particular, an isolated ADNF I complex
nucleic acid encoding an ADNF I complex polypeptide is separated
from open reading frames that flank the ADNF I complex gene(s) and
encode proteins other than ADNF I complex polypeptide. The term
"purified" denotes that a nucleic acid or protein gives rise to
essentially one band in an electrophoretic gel. Particularly, it
means that the nucleic acid or protein is at least 85% pure, more
preferably at least 95% pure, and most preferably at least 99%
pure.
[0099] "Nucleic acid" refers to deoxyribonucleotides or
ribonucleotides and polymers thereof in either single- or
double-stranded form. The term encompasses nucleic acids containing
known nucleotide analogs or modified backbone residues or linkages,
which are synthetic, naturally occurring, and non-naturally
occurring, which have similar binding properties as the reference
nucleic acid, and which are metabolized in a manner similar to the
reference nucleotides. Examples of such analogs include, without
limitation, phosphorothioates, phosphoramidates, methyl
phosphonates, chiral-methyl phosphonates, 2-O-methyl
ribonucleotides, peptide-nucleic acids (PNAs).
[0100] Unless otherwise indicated, a particular nucleic acid
sequence also implicitly encompasses conservatively modified
variants thereof (e.g., degenerate codon substitutions) and
complementary sequences, as well as the sequence explicitly
indicated. The term nucleic acid is used interchangeably with gene,
cDNA, mRNA, oligonucleotide, and polynucleotide.
[0101] The term "amino acid" refers to naturally occurring amino
acids in L-form and their enantiomers in D-form, amino acid
analogs, and amino acid mimetics. The two-mirror-image forms
(enantiomers) of amino acids are called the L-isomer and the
D-isomer, where L refers to levorotatory (left rotation of the
plane of polarization of light) and D refers to dextrorotatory
(right rotation of the plane of polarization). The term "amino
acid" also includes amino acids that are later modified, e.g.,
hydroxyproline, .gamma.-carboxyglutatmate, and O-phosphoserine.
Amino acid analogs refer to synthetic amino acids that have the
same basic chemical structure as naturally occurring amino acids in
L-form or their enantiomers in D-form, i.e., an .alpha. carbon that
is bound to a hydrogen, a carboxyl group, an amino group, and an R
group (e.g., homoserine, norleucine, methionine sulfoxide,
methionine methyl sulfonium). Such analogs have modified R groups
(e.g., norleucine) or modified peptide backbones, but retain the
same basic chemical structure as a naturally occurring amino acid.
Amino acid mimetics refer to chemical compounds that have a
structure that is different from the general chemical structure of
an amino acid, but that function in a manner similar to a naturally
occurring amino acid. Amino acid analogs and amino acids mimetics
can also be in L-form or in D-form.
[0102] Amino acids may be referred to herein by either their
commonly known three letter symbols or by the one-letter symbols
recommended by the IUPAC-IUB Biochemical Nomenclature Commission.
Nucleotides, likewise, may be referred to by their commonly
accepted single-letter codes.
[0103] "Conservatively modified variants" applies to both amino
acid and nucleic acid sequences. With respect to particular nucleic
acid sequences, conservatively modified variants refer to those
nucleic acids which encode identical or essentially identical amino
acid sequences, or where the nucleic acid does not encode an amino
acid sequence, to essentially identical sequences. Specifically,
degenerate codon substitutions may be achieved by generating
sequences in which the third position of one or more selected (or
all) codons is substituted with mixed-base and/or deoxyinosine
residues (Batzer et al., Nucleic Acid Res. 19:5081 (1991); Ohtsuka
et al., J. Biol. Chem. 260:2605-2608 (1985); Rossolini et al., Mol.
Cell. Probes 8:91-98 (1994)). Because of the degeneracy of the
genetic code, a large number of functionally identical nucleic
acids encode any given protein. For instance, the codons GCA, GCC,
GCG and GCU all encode the amino acid alanine. Thus, at every
position where an alanine is specified by a codon, the codon can be
altered to any of the corresponding codons described without
altering the encoded polypeptide. Such nucleic acid variations are
"silent variations," which are one species of conservatively
modified variations. Every nucleic acid sequence herein which
encodes a polypeptide also describes every possible silent
variation of the nucleic acid. One of skill will recognize that
each codon in a nucleic acid (except AUG, which is ordinarily the
only codon for methionine, and TGG, which is ordinarily the only
codon for tryptophan) can be modified to yield a functionally
identical molecule. Accordingly, each silent variation of a nucleic
acid which encodes a polypeptide is implicit in each described
sequence.
[0104] As to amino acid sequences, one of skill will recognize that
individual substitutions, deletions or additions to a nucleic acid,
peptide, polypeptide, or protein sequence which alters, adds or
deletes a single amino acid or a small percentage of amino acids in
the encoded sequence is a "conservatively modified variant" where
the alteration results in the substitution of an amino acid with a
chemically similar amino acid. Conservative substitution tables
providing functionally similar amino acids are well known in the
art. Such conservatively modified variants are in addition to and
do not exclude polymorphic variants, interspecies homologs, and
alleles of the invention.
[0105] The following groups each contain amino acids that are
conservative substitutions for one another:
1) Alanine (A), Glycine (G);
2) Serine (S), Threonine (T);
[0106] 3) Aspartic acid (D), Glutamic acid (E);
4) Asparagine (N), Glutamine (Q);
5) Cysteine (C), Methionine (M);
6) Arginine (R), Lysine (K), Histidine (H);
7) Isoleucine (I), Leucine (L), Valine (V); and
8) Phenylalanine (F), Tyrosine (Y), Tryptophan (W).
[0107] (see, e.g., Creighton, Proteins (1984)).
[0108] The terms "polypeptide," "peptide," and "protein" are used
interchangeably herein to refer to a polymer of amino acid
residues. The terms apply to amino acid polymers in which one or
more amino acid residue is a naturally occurring amino acid in
L-form or their enantiomers in D-form, an analog or mimetic of
amino acids in L-form or D-form, or combinations thereof.
[0109] The terms "identical" or percent "identity," in the context
of two or more nucleic acids or polypeptide sequences, refer to two
or more sequences or subsequences that are the same or have a
specified percentage of amino acid residues or nucleotides that are
the same (i.e., 70% identity, optionally 75%, 80%, 85%, 90%, or 95%
identity over a specified region), when compared and aligned for
maximum correspondence over a comparison window, or designated
region as measured using one of the following sequence comparison
algorithms or by manual alignment and visual inspection. Such
sequences are then said to be "substantially identical." This
definition also refers to the compliment of a test sequence.
Optionally, the identity exists over a region that is at least
about 50 amino acids or nucleotides in length, or more preferably
over a region that is 75-100 amino acids or nucleotides in
length.
[0110] For sequence comparison, typically one sequence acts as a
reference sequence, to which test sequences are compared. When
using a sequence comparison algorithm, test and reference sequences
are entered into a computer, subsequence coordinates are
designated, if necessary, and sequence algorithm program parameters
are designated. Default program parameters can be used, or
alternative parameters can be designated. The sequence comparison
algorithm then calculates the percent sequence identities for the
test sequences relative to the reference sequence, based on the
program parameters.
[0111] A "comparison window", as used herein, includes reference to
a segment of any one of the number of contiguous positions selected
from the group consisting of from 20 to 600, usually about 50 to
about 200, more usually about 100 to about 150 in which a sequence
may be compared to a reference sequence of the same number of
contiguous positions after the two sequences are optimally aligned.
Methods of alignment of sequences for comparison are well-known in
the art. Optimal alignment of sequences for comparison can be
conducted, e.g., by the local homology algorithm of Smith &
Waterman, Adv. Appl. Math. 2:482 (1981), by the homology alignment
algorithm of Needleman & Wunsch, J. Mol. Biol. 48:443 (1970),
by the search for similarity method of Pearson & Lipman, Proc.
Nat'l Acad. Sci. USA 85:2444 (1988), by computerized
implementations of these algorithms (GAP, BESTFIT, FASTA, and
TFASTA in the Wisconsin Genetics Software Package, Genetics
Computer Group, 575 Science Dr., Madison, Wis.), or by manual
alignment and visual inspection (see, e.g. Current Protocols in
Molecular Biology (Ausubel et al., eds. 1995 supplement)).
[0112] An example of algorithm that is suitable for determining
percent sequence identity and sequence similarity are the BLAST and
BLAST 2.0 algorithms, which are described in Altschul et al., Nuc.
Acids Res. 25:3389-3402 (1977) and Altschul et al., J. Mol. Biol.
215:403-410 (1990), respectively. Software for performing BLAST
analyses is publicly available through the National Center for
Biotechnology Information. This algorithm involves first
identifying high scoring sequence pairs (HSPs) by identifying short
words of length W in the query sequence, which either match or
satisfy some positive-valued threshold score T when aligned with a
word of the same length in a database sequence. T is referred to as
the neighborhood word score threshold (Altschul et al., supra).
These initial neighborhood word hits act as seeds for initiating
searches to find longer HSPs containing them. The word hits are
extended in both directions along each sequence for as far as the
cumulative alignment score can be increased. Cumulative scores are
calculated using, for nucleotide sequences, the parameters M
(reward score for a pair of matching residues; always >0) and N
(penalty score for mismatching residues; always <0). For amino
acid sequences, a scoring matrix is used to calculate the
cumulative score. Extension of the word hits in each direction are
halted when: the cumulative alignment score falls off by the
quantity X from its maximum achieved value; the cumulative score
goes to zero or below, due to the accumulation of one or more
negative-scoring residue alignments; or the end of either sequence
is reached. The BLAST algorithm parameters W, T, and X determine
the sensitivity and speed of the alignment. The BLASTN program (for
nucleotide sequences) uses as defaults a wordlength (W) of 11, an
expectation (E) or 10, M=5, N=-4 and a comparison of both strands.
For amino acid sequences, the BLASTP program uses as defaults a
wordlength of 3, and expectation (E) of 10, and the BLOSUM62
scoring matrix (see Henikoff & Henikoff, Proc. Natl. Acad. Sci.
USA 89:10915 (1989)) alignments (B) of 50, expectation (E) of 10,
M=5, N=-4, and a comparison of both strands.
[0113] The BLAST algorithm also performs a statistical analysis of
the similarity between two sequences (see, e.g., Karlin &
Altschul, Proc. Nat'l Acad. Sci. USA 90:5873-5787 (1993)). One
measure of similarity provided by the BLAST algorithm is the
smallest sum probability (P(N)), which provides an indication of
the probability by which a match between two nucleotide or amino
acid sequences would occur by chance. For example, a nucleic acid
is considered similar to a reference sequence if the smallest sum
probability in a comparison of the test nucleic acid to the
reference nucleic acid is less than about 0.2, more preferably less
than about 0.01, and most preferably less than about 0.001.
[0114] An indication that two nucleic acid sequences or
polypeptides are substantially identical is that the polypeptide
encoded by the first nucleic acid is immunologically cross reactive
with the antibodies raised against the polypeptide encoded by the
second nucleic acid, as described below. Thus, a polypeptide is
typically substantially identical to a second polypeptide, for
example, where the two peptides differ only by conservative
substitutions. Another indication that two nucleic acid sequences
are substantially identical is that the two molecules or their
complements hybridize to each other under stringent conditions, as
described below. Yet another indication that two nucleic acid
sequences are substantially identical is that the same primers can
be used to amplify the sequence.
[0115] A further indication that two polynucleotides are
substantially identical is if the reference sequence, amplified by
a pair of oligonucleotide primers or a pool of degenerate primers
that encode a conserved amino acid sequence, can then be used as a
probe under stringent hybridization conditions to isolate the test
sequence from a cDNA or genomic library, or to identify the test
sequence in, e.g., a northern or Southern blot. Alternatively,
another indication that the sequences are substantially identical
is if the same set of PCR primers can be used to amplify both
sequences.
[0116] The phrase "selectively (or specifically) hybridizes to"
refers to the binding, duplexing, or hybridizing of a molecule only
to a particular nucleotide sequence under stringent hybridization
conditions when that sequence is present in a complex mixture
(e.g., total cellular or library DNA or RNA).
[0117] The phrase "stringent hybridization conditions" refers to
conditions under which a probe will hybridize to its target
subsequence, typically in a complex mixture of nucleic acid, but to
no other sequences. Stringent conditions are sequence-dependent and
will be different in different circumstances. Longer sequences
hybridize specifically at higher temperatures. An extensive guide
to the hybridization of nucleic acids is found in Tijssen,
Techniques in Biochemistry and Molecular Biology--Hybridization
with Nucleic Probes, "Overview of principles of hybridization and
the strategy of nucleic acid assays" (1993). Generally, stringent
conditions are selected to be about 5-10.degree. C. lower than the
thermal melting point (T.sub.m) for the specific sequence at a
defined ionic strength pH. The T.sub.m is the temperature (under
defined ionic strength, pH, and nucleic concentration) at which 50%
of the probes complementary to the target hybridize to the target
sequence at equilibrium (as the target sequences are present in
excess, at T.sub.m, 50% of the probes are occupied at equilibrium).
Stringent conditions will be those in which the salt concentration
is less than about 1.0 M sodium ion, typically about 0.01 to 1.0 M
sodium ion concentration (or other salts) at pH 7.0 to 8.3 and the
temperature is at least about 30.degree. C. for short probes (e.g.,
10 to 50 nucleotides) and at least about 60.degree. C. for long
probes (e.g., greater than 50 nucleotides). Stringent conditions
may also be achieved with the addition of destabilizing agents such
as formamide. For selective or specific hybridization, a positive
signal is at least two times background, preferably 10 times
background hybridization. Exemplary stringent hybridization
conditions can be as following: 50% formamide, 5.times.SSC, and 1%
SDS, incubating at 42.degree. C., or, 5.times.SSC, 1% SDS,
incubating at 65.degree. C., with a wash in 0.2.times.SSC, and 0.1%
SDS at 65.degree. C.
[0118] Nucleic acids that do not hybridize to each other under
stringent conditions are still substantially identical if the
polypeptides which they encode are substantially identical. This
occurs, for example, when a copy of a nucleic acid is created using
the maximum codon degeneracy permitted by the genetic code. In such
cases, the nucleic acids typically hybridize under moderately
stringent hybridization conditions. Exemplary "moderately stringent
hybridization conditions" include a hybridization in a buffer of
40% formamide, 1 M NaCl, 1% SDS at 37.degree. C., and a wash in
1.times.SSC at 45.degree. C. A positive hybridization is at least
twice background. Those of ordinary skill will readily recognize
that alternative hybridization and wash conditions can be utilized
to provide conditions of similar stringency.
[0119] "Antibody" refers to a polypeptide comprising a framework
region from an immunoglobulin gene or fragments thereof that
specifically binds and recognizes an antigen. The recognized
immunoglobulin genes include the kappa, lambda, alpha, gamma,
delta, epsilon, and mu constant region genes, as well as the myriad
immunoglobulin variable region genes. Light chains are classified
as either kappa or lambda. Heavy chains are classified as gamma,
mu, alpha, delta, or epsilon, which in turn define the
immunoglobulin classes, IgG, IgM, IgA, IgD and IgE, respectively.
Typically, the antigen-binding region of an antibody will be most
critical in specificity and affinity of binding.
[0120] An exemplary immunoglobulin (antibody) structural unit
comprises a tetramer. Each tetramer is composed of two identical
pairs of polypeptide chains, each pair having one "light" (about 25
kD) and one "heavy" chain (about 50-70 kD). The N-terminus of each
chain defines a variable region of about 100 to 110 or more amino
acids primarily responsible for antigen recognition. The terms
variable light chain (V.sub.L) and variable heavy chain (V.sub.H)
refer to these light and heavy chains respectively.
[0121] Antibodies exist, e.g., as intact immunoglobulins or as a
number of well-characterized fragments produced by digestion with
various peptidases. Thus, for example, pepsin digests an antibody
below the disulfide linkages in the hinge region to produce
F(ab)'.sub.2, a dimer of Fab which itself is a light chain joined
to V.sub.H--C.sub.H1 by a disulfide bond. The F(ab)'.sub.2 may be
reduced under mild conditions to break the disulfide linkage in the
hinge region, thereby converting the F(ab)'.sub.2 dimer into an
Fab' monomer. The Fab' monomer is essentially Fab with part of the
hinge region (see Fundamental Immunology (Paul ed., 3d ed. 1993).
While various antibody fragments are defined in terms of the
digestion of an intact antibody, one of skill will appreciate that
such fragments may be synthesized de novo either chemically or by
using recombinant DNA methodology. Thus, the term antibody, as used
herein, also includes antibody fragments either produced by the
modification of whole antibodies, or those synthesized de novo
using recombinant DNA methodologies (e.g., single chain Fv) or
those identified using phage display libraries (see, e.g.
McCafferty et al., Nature 348:552-554 (1990))
[0122] For preparation of antibodies, e.g., recombinant,
monoclonal, or polyclonal antibodies, many techniques known in the
art can be used (see, e.g., Kohler & Milstein, Nature
256:495-497 (1975); Kozbor et al., Immunology Today 4: 72 (1983);
Cole et al., pp. 77-96 in Monoclonal Antibodies and Cancer Therapy,
Alan R. Liss, Inc. (1985); Coligan, Current Protocols in Immunology
(1991); Harlow & Lane, Antibodies, A Laboratory Manual (1988);
and Goding, Monoclonal Antibodies: Principles and Practice (2d ed.
1986)). The genes encoding the heavy and light chains of an
antibody of interest can be cloned from a cell, e.g., the genes
encoding a monoclonal antibody can be cloned from a hybridoma and
used to produce a recombinant monoclonal antibody. Gene libraries
encoding heavy and light chains of monoclonal antibodies can also
be made from hybridoma or plasma cells. Random combinations of the
heavy and light chain gene products generate a large pool of
antibodies with different antigenic specificity (see, e.g., Kuby,
Immunology (3.sup.rd ed. 1997)). Techniques for the production of
single chain antibodies or recombinant antibodies (U.S. Pat. No.
4,946,778, U.S. Pat. No. 4,816,567) can be adapted to produce
antibodies to polypeptides of this invention. Also, transgenic
mice, or other organisms such as other mammals, may be used to
express humanized or human antibodies (see, e.g., U.S. Pat. Nos.
5,545,807; 5,545,806; 5,569,825; 5,625,126; 5,633,425; 5,661,016,
Marks et al., Bio/Technology 10:779-783 (1992); Lonberg et al.,
Nature 368:856-859 (1994); Morrison, Nature 368:812-13 (1994);
Fishwild et al., Nature Biotechnology 14:845-51 (1996); Neuberger,
Nature Biotechnology 14:826 (1996); and Lonberg & Huszar,
Intern. Rev. Immunol. 13:65-93 (1995)). Alternatively, phage
display technology can be used to identify antibodies and
heteromeric Fab fragments that specifically bind to selected
antigens (see, e.g., McCafferty et al., Nature 348:552-554 (1990);
Marks et al., Biotechnology 10:779-783 (1992)). Antibodies can also
be made bispecific, i.e., able to recognize two different antigens
(see, e.g., WO 93/08829, Traunecker et al., EMBO J. 10:3655-3659
(1991); and Suresh et al., Methods in Enzymology 121:210 (1986)).
Antibodies can also be heteroconjugates, e.g., two covalently
joined antibodies, or immunotoxins (see, e.g., U.S. Pat. No.
4,676,980, WO 91/00360; WO 92/200373; and EP 03089).
[0123] Methods for humanizing or primatizing non-human antibodies
are well known in the art. Generally, a humanized antibody has one
or more amino acid residues introduced into it from a source which
is non-human. These non-human amino acid residues are often
referred to as import residues, which are typically taken from an
import variable domain. Humanization can be essentially performed
following the method of Winter and co-workers (see, e.g., Jones et
al., Nature 321:522-525 (1986); Riechmann et al., Nature
332:323-327 (1988); Verhoeyen et al, Science 239:1534-1536 (1988)
and Presta, Curr. Op. Struct. Biol. 2:593-596 (1992)), by
substituting rodent CDRs or CDR sequences for the corresponding
sequences of a human antibody. Accordingly, such humanized
antibodies are chimeric antibodies (U.S. Pat. No. 4,816,567),
wherein substantially less than an intact human variable domain has
been substituted by the corresponding sequence from a non-human
species. In practice, humanized antibodies are typically human
antibodies in which some CDR residues and possibly some FR residues
are substituted by residues from analogous sites in rodent
antibodies.
[0124] A "chimeric antibody" is an antibody molecule in which (a)
the constant region, or a portion thereof, is altered, replaced or
exchanged so that the antigen binding site (variable region) is
linked to a constant region of a different or altered class,
effector function and/or species, or an entirely different molecule
which confers new properties to the chimeric antibody, e.g., an
enzyme, toxin, hormone, growth factor, drug, etc.; or (b) the
variable region, or a portion thereof, is altered, replaced or
exchanged with a variable region having a different or altered
antigen specificity.
[0125] In one embodiment, the antibody is conjugated to an
"effector" moiety. The effector moiety can be any number of
molecules, including labeling moieties such as radioactive labels
or fluorescent labels, or can be a therapeutic moiety. In one
aspect the antibody modulates the activity of an ADNF I complex
polypeptide.
[0126] The phrase "specifically (or selectively) binds" to an
antibody or "specifically (or selectively) immunoreactive with,"
when referring to a protein or peptide, refers to a binding
reaction that is determinative of the presence of the protein,
often in a heterogeneous population of proteins and other
biologics. Thus, under designated immunoassay conditions, the
specified antibodies bind to a particular protein at least two
times the background and more typically more than 10 to 100 times
background. Specific binding to an antibody under such conditions
requires an antibody that is selected for its specificity for a
particular protein. For example, polyclonal antibodies raised to an
ADNF I complex polypeptide, polymorphic variants, alleles,
orthologs, and conservatively modified variants, or splice
variants, or portions thereof, can be selected to obtain only those
polyclonal antibodies that are specifically immunoreactive with
ADNF I complex polypeptide and not with other proteins. This
selection may be achieved by subtracting out antibodies that
cross-react with other molecules. A variety of immunoassay formats
may be used to select antibodies specifically immunoreactive with a
particular protein. For example, solid-phase ELISA immunoassays are
routinely used to select antibodies specifically immunoreactive
with a protein (see, e.g., Harlow & Lane, Antibodies, A
Laboratory Manual (1988) for a description of immunoassay formats
and conditions that can be used to determine specific
immunoreactivity).
ADNF I Complex Polypeptides
[0127] Any suitable ADNF I complex polypeptide can be administered
in embodiments of the invention. For example, the polypeptide can
be an ADNF I complex polypeptide or a mixture thereof. In some
embodiments, ADNF I complex polypeptides may comprise all L-amino
acids, all D-amino acids, or a combination thereof. When ADNF I
complex polypeptides are to be orally administered, preferably an
ADNF I complex polypeptide comprises at least one D-amino acid
within its active core site, more preferably at the N-terminus
and/or the C-terminus of the active core site, and even more
preferably at the entire active core site or over the length of the
molecule. Alternatively, the D-amino acid can be at any suitable
position in the polypeptide sequence. Since D-enantiomers of
polypeptides are enzymatically more stable than their
L-enantiomers, particularly in the gastrointestinal tract, an ADNF
I complex polypeptide comprising D-amino acids are particularly
useful for oral administration.
[0128] ADNF I complex polypeptides can be synthesized using both
recombinant DNA methods, as well as being isolated from naturally
occurring sources, or synthesized using chemical methods, as known
to those of skill in the art and as described herein. Expression
vectors containing a nucleic acid encoding an ADNF I complex
polypeptide can be introduced into host cells, and then the
expressed ADNF I complex polypeptide can be purified.
[0129] Moreover, one of skill will recognize that other
modifications can also be made to the ADNF I complex polypeptides
without diminishing their biological activity. For example,
modifications can be made to avoid cleavage by enzymes in the
stomach or intestines. In another example, modifications can be
made to aid the purification process.
[0130] It will be readily apparent to those of ordinary skill in
the art that the biologically active ADNF I complex polypeptides of
the present invention can readily be screened for
neuroprotective/neurotrophic activity using a number of methods
known in the art. For example, a cerebral cortical cell culture
assay can be used. In cerebral cortical cell culture assays,
cerebral cortical cell cultures are prepared using the techniques
described by Forsythe & Westbrook, J. Physiol. Lond.
396:515-533 (1988) with the following modifications. Cerebral
cortex are used instead of hippocampus, and newborn rats are used
instead of E16 mice. After nine days growth in vitro, the cultures
are given a complete change of medium and treated with the ADNF I
complex polypeptide of interest (dissolved in phosphate buffered
saline) for an additional five days. To terminate, the cells are
fixed for immunocytochemistry and neurons identified with
antibodies against NSE (i.e., neuron specific enolase, a neuronal
specific marker). Cell counts are performed on 30 fields, with
total area of about 15 mm.sup.2. Neurons are counted without
knowledge of treatment. Control counts not treated with any drugs
should run for purposes of comparison. Furthermore, assays
described by, e.g., Hill et al., Brain Res. 603:222-233 (1993).
[0131] Using these assays, one of ordinary skill in the art can
readily prepare a large number of ADNF I complex polypeptides in
accordance with the teachings of the present invention and, in
turn, screen them using the foregoing assay to find ADNF I complex
polypeptides, in addition to those set forth herein, which possess
neuroprotective/neurotrophic activity.
Chemical Synthesis of ADNF I Complex Polypeptides
[0132] ADNF I complex polypeptides, including ADNF I complex
polypeptides comprising at least one D-amino acid, can be prepared
via a wide variety of well-known chemical synthesis techniques.
Polypeptides are typically synthesized in solution or on a solid
support in accordance with conventional techniques (see, e.g.,
Merrifield, Am. Chem. Soc. 85:2149-2154 (1963)). Various automatic
synthesizers and sequencers are commercially available and can be
used in accordance with known protocols (see, e.g. Stewart &
Young, Solid Phase Peptide Synthesis (2nd ed. 1984)). Solid phase
synthesis in which the C-terminal amino acid of the sequence is
attached to an insoluble support followed by sequential addition of
the remaining amino acids in the sequence is the preferred method
for the chemical synthesis of the polypeptides of this invention.
Using solid phase synthesis methods, one or more D-amino acids can
be inserted, instead of L-amino acids, into an ADNF I complex
polypeptide at any desired location(s). Techniques for solid phase
synthesis are described by Barany & Merrifield, Solid-Phase
Peptide Synthesis; pp. 3-284 in The Peptides: Analysis, Synthesis,
Biology. Vol. 2: Special Methods in Peptide Synthesis, Part A;
Merrifield et al., J. Am. Chem. Soc. 85:2149-2156 (1963); and
Stewart et al., Solid Phase Peptide Synthesis (2nd ed. 1984).
[0133] After chemical synthesis, the polypeptide(s) may possess a
conformation substantially different than the native conformations
of the constituent polypeptides. In this case, it is helpful to
denature and reduce the polypeptide and then to cause the
polypeptide to re-fold into the preferred conformation. Methods of
reducing and denaturing polypeptides and inducing re-folding are
well known to those of skill in the art (see Debinski et al., J.
Biol. Chem. 268:14065-14070 (1993); Kreitman & Pastan,
Bioconjug. Chem. 4:581-585 (1993); and Buchner et al., Anal.
Biochem. 205:263-270 (1992)). Debinski et al., for example,
describe the denaturation and reduction of inclusion body
polypeptides in guanidine-DTE. The polypeptide is then refolded in
a redox buffer containing oxidized glutathione and L-arginine.
Isolation of Nucleic Acids Encoding ADNF I Complex Polypeptides
[0134] This invention relies on routine techniques in the field of
recombinant genetics. Basic texts disclosing the general methods of
use in this invention include Sambrook et al., Molecular Cloning, A
Laboratory Manual (2nd ed. 1989); Kriegler, Gene Transfer and
Expression: A Laboratory Manual (1990); and Current Protocols in
Molecular Biology (Ausubel et al., eds., 1994)).
[0135] ADNF I complex polypeptides, nucleic acids, polymorphic
variants, orthologs, and alleles that are substantially identical
to an amino acid sequence of an ADNF I complex polypeptide can be
isolated using ADNF I complex polypeptide nucleic acid probes and
oligonucleotides under stringent hybridization conditions, by
screening libraries. Alternatively, expression libraries can be
used to clone ADNF I complex polypeptides, polymorphic variants,
orthologs, and alleles by detecting expressed homologs
immunologically with antisera or purified antibodies made against
human ADNF I complex polypeptides or portions thereof.
[0136] To make a cDNA library, one should choose a source that is
rich in ADNF I complex polypeptide-encoding RNA, such as
astrocytes, neuroblastomas, or fibroblasts. The mRNA is then made
into cDNA using reverse transcriptase, ligated into a recombinant
vector, and transfected into a recombinant host for propagation,
screening and cloning. Methods for making and screening cDNA
libraries are well known (see, e.g., Gubler & Hoffman, Gene
25:263-269 (1983); Sambrook et al., supra; Ausubel et al.,
supra).
[0137] For a genomic library, the DNA is extracted from the tissue
and either mechanically sheared or enzymatically digested to yield
fragments of about 12-20 kb. The fragments are then separated by
gradient centrifugation from undesired sizes and are constructed in
bacteriophage lambda vectors. These vectors and phage are packaged
in vitro. Recombinant phage are analyzed by plaque hybridization as
described in Benton & Davis, Science 196:180-182 (1977). Colony
hybridization is carried out as generally described in Grunstein et
al., Proc. Natl. Acad. Sci. USA., 72:3961-3965 (1975).
[0138] An alternative method of isolating ADNF I complex nucleic
acid and its orthologs, alleles, mutants, polymorphic variants, and
conservatively modified variants combines the use of synthetic
oligonucleotide primers and amplification of an RNA or DNA template
(see U.S. Pat. Nos. 4,683,195 and 4,683,202; PCR Protocols: A Guide
to Methods and Applications (Innis et al., eds, 1990)). Methods
such as polymerase chain reaction (PCR) and ligase chain reaction
(LCR) can be used to amplify nucleic acid sequences encoding human
ADNF I complex polypeptides directly from mRNA, from cDNA, from
genomic libraries or cDNA libraries. Degenerate oligonucleotides
can be designed to amplify ADNF I complex nucleic acids and
homologs using the sequences provided herein. Restriction
endonuclease sites can be incorporated into the primers. Polymerase
chain reaction or other in vitro amplification methods may also be
useful, for example, to clone nucleic acid sequences that code for
proteins to be expressed, to make nucleic acids to use as probes
for detecting the presence of ADNF I complex polypeptide-encoding
mRNA in physiological samples, for nucleic acid sequencing, or for
other purposes. Genes amplified by the PCR reaction can be purified
from agarose gels and cloned into an appropriate vector.
[0139] Gene expression of ADNF I complex polypeptides can also be
analyzed by techniques known in the art, e.g., reverse
transcription and amplification of mRNA, isolation of total RNA or
poly A.sup.+ RNA, northern blotting, dot blotting, in situ
hybridization, RNase protection, high density polynucleotide array
technology, e.g., and the like.
[0140] Nucleic acids encoding ADNF I complex polypeptides can be
used with high density oligonucleotide array technology (e.g.,
GeneChip.TM.) to identify ADNF I complex polypeptides, orthologs,
alleles, conservatively modified variants, and polymorphic variants
in this invention. In the case where the homologs being identified
are linked to modulation of T cell activation, they can be used
with GeneChip.TM. as a diagnostic tool in detecting the disease in
a biological sample, see, e.g. Gunthand et al., AIDS Res. Hum.
Retroviruses 14: 869-876 (1998); Kozal et al., Nat. Med. 2:753-759
(1996); Matson et al., Anal. Biochem. 224:110-106 (1995); Lockhart
et al., Nat. Biotechnol. 14:1675-1680 (1996); Gingeras et al.,
Genome Res. 8:435-448 (1998); Hacia et al., Nucleic Acids Res.
26:3865-3866 (1998).
[0141] The gene for ADNF I complex polypeptides is typically cloned
into intermediate vectors before transformation into prokaryotic or
eukaryotic cells for replication and/or expression. These
intermediate vectors are typically prokaryote vectors, e.g.,
plasmids, or shuttle vectors.
Expression in Prokaryotes and Eukaryotes
[0142] To obtain high level expression of a cloned gene, such as
those cDNAs encoding ADNF I complex polypeptides, one typically
subclones ADNF I complex nucleic acids into an expression vector
that contains a strong promoter to direct transcription, a
transcription/translation terminator, and if for a nucleic acid
encoding a protein, a ribosome binding site for translational
initiation. Suitable bacterial promoters are well known in the art
and described, e.g., in Sambrook et al., and Ausubel et al, supra.
Bacterial expression systems for expressing the ADNF I complex
polypeptides are available in, e.g., E. coli, Bacillus sp., and
Salmonella (Palva et al., Gene 22:229-235 (1983); Mosbach et al.,
Nature 302:543-545 (1983). Kits for such expression systems are
commercially available. Eukaryotic expression systems for mammalian
cells, yeast, and insect cells are well known in the art and are
also commercially available. In one preferred embodiment,
retroviral expression systems are used in the present
invention.
[0143] Selection of the promoter used to direct expression of a
heterologous nucleic acid depends on the particular application.
The promoter is preferably positioned about the same distance from
the heterologous transcription start site as it is from the
transcription start site in its natural setting. As is known in the
art, however, some variation in this distance can be accommodated
without loss of promoter function.
[0144] In addition to the promoter, the expression vector typically
contains a transcription unit or expression cassette that contains
all the additional elements required for the expression of the ADNF
I complex polypeptide-encoding nucleic acid in host cells. A
typical expression cassette thus contains a promoter operably
linked to the nucleic acid sequence encoding ADNF I complex
polypeptides and signals required for efficient polyadenylation of
the transcript, ribosome binding sites, and translation
termination. Additional elements of the cassette may include
enhancers and, if genomic DNA is used as the structural gene,
introns with functional splice donor and acceptor sites.
[0145] In addition to a promoter sequence, the expression cassette
should also contain a transcription termination region downstream
of the structural gene to provide for efficient termination. The
termination region may be obtained from the same gene as the
promoter sequence or may be obtained from different genes.
[0146] The particular expression vector used to transport the
genetic information into the cell is not particularly critical. Any
of the conventional vectors used for expression in eukaryotic or
prokaryotic cells may be used. Standard bacterial expression
vectors include plasmids such as pBR322 based plasmids, pSKF,
pET23D, and fusion expression systems such as MBP, GST, and LacZ.
Epitope tags can also be added to recombinant proteins to provide
convenient methods of isolation, e.g., c-myc. Sequence tags may be
included in an expression cassette for nucleic acid rescue. Markers
such as fluorescent proteins, green or red fluorescent protein,
.beta.-gal, CAT, and the like can be included in the vectors as
markers for vector transduction.
[0147] Expression vectors containing regulatory elements from
eukaryotic viruses are typically used in eukaryotic expression
vectors, e.g., SV40 vectors, papilloma virus vectors, retroviral
vectors, and vectors derived from Epstein-Barr virus. Other
exemplary eukaryotic vectors include pMSG, pAV009/A.sup.+,
PMTO10/A.sup.+, pMAMneo-5, baculovirus pDSVE, and any other vector
allowing expression of proteins under the direction of the CMV
promoter, SV40 early promoter, SV40 later promoter, metallothionein
promoter, murine mammary tumor virus promoter, Rous sarcoma virus
promoter, polyhedrin promoter, or other promoters shown effective
for expression in eukaryotic cells.
[0148] Expression of proteins from eukaryotic vectors can be also
be regulated using inducible promoters. With inducible promoters,
expression levels are tied to the concentration of inducing agents,
such as tetracycline or edysone, by the incorporation of response
elements for these agents into the promoter. Generally, high level
expression is obtained from inducible promoters only in the
presence of the inducing agent; basal expression levels are
minimal.
[0149] In one embodiment, the vectors of the invention have a
regulatable promoter, e.g., tet-regulated systems and the RU-486
system (see, e.g., Gossen & Bujard, Proc. Nat'l Acad. Sci. USA
89:5547 (1992); Oligino et al., Gene Ther. 5:491-496 (1998); Wang
et al., Gene Ther. 4:432-441 (1997); Neering et al., Blood
88:1147-1155 (1996); and Rendahl et al., Nat. Biotechnol.
16:757-761 (1998)). These impart small molecule control on the
expression of the candidate target nucleic acids. This beneficial
feature can be used to determine that a desired phenotype is caused
by a transfected cDNA rather than a somatic mutation.
[0150] Some expression systems have markers that provide gene
amplification such as thymidine kinase and dihydrofolate reductase.
Alternatively, high yield expression systems not involving gene
amplification are also suitable, such as using a baculovirus vector
in insect cells, with a ADNF I complex polypeptide-encoding
sequence under the direction of the polyhedrin promoter or other
strong baculovirus promoters.
[0151] The elements that are typically included in expression
vectors also include a replicon that functions in E. coli, a gene
encoding antibiotic resistance to permit selection of bacteria that
harbor recombinant plasmids, and unique restriction sites in
nonessential regions of the plasmid to allow insertion of
eukaryotic sequences. The particular antibiotic resistance gene
chosen is not critical, any of the many resistance genes known in
the art are suitable. The prokaryotic sequences are preferably
chosen such that they do not interfere with the replication of the
DNA in eukaryotic cells, if necessary.
[0152] Standard transfection methods are used to produce bacterial,
mammalian, yeast or insect cell lines that express large quantities
of ADNF I complex polypeptide, which are then purified using
standard techniques (see, e.g., Colley et al., J. Biol. Chem.
264:17619-17622 (1989); Guide to Protein Purification, in Methods
in Enzymology, vol. 182 (Deutscher, ed., 1990)). Transformation of
eukaryotic and prokaryotic cells are performed according to
standard techniques (see, e.g. Morrison, J. Bact. 132:349-351
(1977); Clark-Curtiss & Curtiss, Methods in Enzymology
101:347-362 (Wu et al., eds, 1983).
[0153] Any of the well-known procedures for introducing foreign
nucleotide sequences into host cells may be used. These include the
use of calcium phosphate transfection, polybrene, protoplast
fusion, electroporation, biolistics, liposomes, microinjection,
plasma vectors, viral vectors and any of the other well known
methods for introducing cloned genomic DNA, cDNA, synthetic DNA or
other foreign genetic material into a host cell (see, e.g.,
Sambrook et al., supra). It is only necessary that the particular
genetic engineering procedure used be capable of successfully
introducing at least one gene into the host cell capable of
expressing ADNF I complex polypeptides.
[0154] After the expression vector is introduced into the cells,
the transfected cells are cultured under conditions favoring
expression of ADNF I complex polypeptides, which is recovered from
the culture using standard techniques identified below.
Purification of ADNF 1 Complex Polypeptides
[0155] Either naturally occurring or recombinant ADNF I complex
polypeptides can be purified for use in functional assays, as well
as chemically synthesized ADNF I complex polypeptides. Naturally
occurring ADNF I complex polypeptides can be purified, e.g., from
human tissue. Recombinant ADNF I complex polypeptides can be
purified from any suitable expression system.
[0156] The ADNF I complex polypeptide may be purified to
substantial purity by standard techniques, including selective
precipitation with such substances as ammonium sulfate; column
chromatography, immunopurification methods, and others (see, e.g.
Scopes, Protein Purification: Principles and Practice (1982); U.S.
Pat. No. 4,673,641; Ausubel et al., supra; and Sambrook et al.,
supra).
[0157] A number of procedures can be employed when recombinant ADNF
I complex polypeptide is being purified. For example, proteins
having established molecular adhesion properties can be reversible
fused to the ADNF I complex polypeptide. With the appropriate
ligand, ADNF I complex polypeptide can be selectively adsorbed to a
purification column and then freed from the column in a relatively
pure form. The fused protein is then removed by enzymatic activity.
Finally, ADNF I complex polypeptide could be purified using
immunoaffinity columns.
[0158] A. Purification of ADNF I Complex Polypeptides from
Recombinant Bacteria
[0159] Recombinant proteins are expressed by transformed bacteria
in large amounts, typically after promoter induction; but
expression can be constitutive. Promoter induction with IPTG is one
example of an inducible promoter system. Bacteria are grown
according to standard procedures in the art. Fresh or frozen
bacteria cells are used for isolation of protein.
[0160] Proteins expressed in bacteria may form insoluble aggregates
("inclusion bodies"). Several protocols are suitable for
purification of ADNF I complex polypeptide inclusion bodies. For
example, purification of inclusion bodies typically involves the
extraction, separation and/or purification of inclusion bodies by
disruption of bacterial cells, e.g., by incubation in a buffer of
50 mM TRIS/HCL pH 7.5, 50 mM NaCl, 5 mM MgCl.sub.2, 1 mM DTT, 0.1
mM ATP, and 1 mM PMSF. The cell suspension can be lysed using 2-3
passages through a French Press, homogenized using a Polytron
(Brinkman Instruments) or sonicated on ice. Alternate methods of
lysing bacteria are apparent to those of skill in the art (see,
e.g., Sambrook et al., supra; Ausubel et al., supra).
[0161] If necessary, the inclusion bodies are solubilized, and the
lysed cell suspension is typically centrifuged to remove unwanted
insoluble matter. Proteins that formed the inclusion bodies may be
renatured by dilution or dialysis with a compatible buffer.
Suitable solvents include, but are not limited to urea (from about
4 M to about 8 M), formamide (at least about 80%, volume/volume
basis), and guanidine hydrochloride (from about 4 M to about 8 M).
Some solvents which are capable of solubilizing aggregate-forming
proteins, for example SDS (sodium dodecyl sulfate), 70% formic
acid, are inappropriate for use in this procedure due to the
possibility of irreversible denaturation of the proteins,
accompanied by a lack of immunogenicity and/or activity. Although
guanidine hydrochloride and similar agents are denaturants, this
denaturation is not irreversible and renaturation may occur upon
removal (by dialysis, for example) or dilution of the denaturant,
allowing re-formation of immunologically and/or biologically active
protein. Other suitable buffers are known to those skilled in the
art. Human ADNF I complex polypeptides are separated from other
bacterial proteins by standard separation techniques, e.g., with
Ni-NTA agarose resin.
[0162] Alternatively, it is possible to purify ADNF I complex
polypeptides from bacteria periplasm. After lysis of the bacteria,
when the ADNF I complex polypeptide is exported into the periplasm
of the bacteria, the periplasmic fraction of the bacteria can be
isolated by cold osmotic shock in addition to other methods known
to skill in the art. To isolate recombinant proteins from the
periplasm, the bacterial cells are centrifuged to form a pellet.
The pellet is resuspended in a buffer containing 20% sucrose. To
lyse the cells, the bacteria are centrifuged and the pellet is
resuspended in ice-cold 5 mM MgSO.sub.4 and kept in an ice bath for
approximately 10 minutes. The cell suspension is centrifuged and
the supernatant decanted and saved. The recombinant proteins
present in the supernatant can be separated from the host proteins
by standard separation techniques well known to those of skill in
the art.
[0163] B. Standard Protein Separation Techniques for Purifying ADNF
I Complex Polypeptides
[0164] Solubility Fractionation
[0165] Often as an initial step, particularly if the protein
mixture is complex, an initial salt fractionation can separate many
of the unwanted host cell proteins (or proteins derived from the
cell culture media) from the recombinant protein of interest. The
preferred salt is ammonium sulfate. Ammonium sulfate precipitates
proteins by effectively reducing the amount of water in the protein
mixture. Proteins then precipitate on the basis of their
solubility. The more hydrophobic a protein is, the more likely it
is to precipitate at lower ammonium sulfate concentrations. A
typical protocol includes adding saturated ammonium sulfate to a
protein solution so that the resultant ammonium sulfate
concentration is between 20-30%. This concentration will
precipitate the most hydrophobic of proteins. The precipitate is
then discarded (unless the protein of interest is hydrophobic) and
ammonium sulfate is added to the supernatant to a concentration
known to precipitate the protein of interest. The precipitate is
then solubilized in buffer and the excess salt removed if
necessary, either through dialysis or diafiltration. Other methods
that rely on solubility of proteins, such as cold ethanol
precipitation, are well known to those of skill in the art and can
be used to fractionate complex protein mixtures.
[0166] Size Differential Filtration
[0167] The molecular weight of the ADNF I complex polypeptides can
be used to isolate them from proteins of greater and lesser size
using ultrafiltration through membranes of different pore size (for
example, Amicon or Millipore membranes). As a first step, the
protein mixture is ultrafiltered through a membrane with a pore
size that has a lower molecular weight cut-off than the molecular
weight of the protein of interest. The retentate of the
ultrafiltration is then ultrafiltered against a membrane with a
molecular cut off greater than the molecular weight of the protein
of interest. The recombinant protein will pass through the membrane
into the filtrate. The filtrate can then be chromatographed as
described below.
[0168] Column Chromatography
[0169] The ADNF I complex polypeptides can also be separated from
other proteins on the basis of its size, net surface charge,
hydrophobicity, and affinity for ligands. In addition, antibodies
raised against proteins can be conjugated to column matrices and
the proteins immunopurified. All of these methods are well known in
the art. It will be apparent to one of skill that chromatographic
techniques can be performed at any scale and using equipment from
many different manufacturers (e.g., Pharmacia Biotech).
Pharmaceutical Compositions and Administration
[0170] ADNF I complex polypeptides and nucleic acids encoding ADNF
I complex polypeptides can be pre-natally or post-natally
administered to the subject using any suitable methods known in the
art. For example, ADNF I complex polypeptides or nucleic acids can
be formulated as pharmaceutical compositions with a
pharmaceutically acceptable diluent, carrier or excipient. Suitable
formulations for use in the present invention are found in
Remington's Pharmaceutical Sciences (17th ed. 1985)), which is
incorporated herein by reference. A brief review of methods for
drug delivery is also described in, e.g., Langer, Science
249:1527-1533 (1990), which is incorporated herein by reference. In
addition, the pharmaceutical compositions comprising peptides and
proteins are described in, e.g., Therapeutic Peptides and Proteins
Formulations, Processing, and Delivery Systems, by Banga, Technomic
Publishing Company, Inc., Lancaster, Pa. (1995).
[0171] In one embodiment, ADNF I complex polypeptides are
formulated for oral or nasal administration, e.g., to the subject,
or for prenatal administration, to the subject's mother. In this
embodiment, it is preferred that ADNF I complex polypeptides
comprising one or more D-amino acids are used. A pharmaceutically
acceptable nontoxic composition is formed by incorporating any of
normally employed excipients, and generally 10-95% of active
ingredient and more preferably at a concentration of 25%-75%.
Furthermore, to improve oral or nasal absorption of ADNF I complex
polypeptides, various carrier systems, such as nanoparticles,
microparticles, liposomes, phospholipids, emulsions, erythrocytes,
etc. can be used. The oral or nasal agents comprising ADNF I
complex polypeptides of the invention can be in any suitable form
for oral or nasal administration, such as liquid, tablets,
capsules, or the like. The oral or nasal formulations can be
further coated or treated to prevent or reduce dissolution in
stomach. See, e.g. Therapeutic Peptides and Proteins, Formulation,
Processing, and Delivery Systems, by A. K. Banga, Technomic
Publishing Company, Inc., 1995.
[0172] Furthermore, the ADNF I complex polypeptides can be
formulated for parenteral, topical, nasal, sublingual, gavage, or
local administration. For example, the pharmaceutical compositions
are administered parenterally, e.g., intravenously, subcutaneously,
intradermally, or intramuscularly, or intranasally. Thus, the
invention provides compositions for parenteral administration that
comprise a solution of a mixture of ADNF I complex polypeptides,
dissolved or suspended in an acceptable carrier, preferably an
aqueous carrier. A variety of aqueous carriers may be used
including, for example, water, buffered water, 0.4% saline, 0.3%
glycine, hyaluronic acid and the like. These compositions may be
sterilized by conventional, well known sterilization techniques, or
they may be sterile filtered. The resulting aqueous solutions may
be packaged for use as is or lyophilized, the lyophilized
preparation being combined with a sterile solution prior to
administration. The compositions may contain pharmaceutically
acceptable auxiliary substances as required to approximate
physiological conditions including pH adjusting and buffering
agents, tonicity adjusting agents, wetting agents and the like,
such as, for example, sodium acetate, sodium lactate, sodium
chloride, potassium chloride, calcium chloride, sorbitan
monolaurate, triethanolamine oleate, etc. In one embodiment, a
nucleic acid encoding an ADNF I complex polypeptide is administered
as a naked DNA.
[0173] For aerosol administration, ADNF I complex polypeptides are
preferably supplied in finely divided form along with a surfactant
and propellant. The surfactant must, of course, be nontoxic, and
preferably soluble in the propellant. Representative of such agents
are the esters or partial esters of fatty acids containing from 6
to 22 carbon atoms, such as caproic, octanoic, lauric, palmitic,
stearic, linoleic, linolenic, olesteric and oleic acids with an
aliphatic polyhydric alcohol or its cyclic anhydride. Mixed esters,
such as mixed or natural glycerides may be employed. A carrier can
also be included, as desired, as with, e.g., lecithin for
intranasal delivery.
[0174] For solid compositions, conventional nontoxic solid carriers
may be used. Solid carriers include, for example, pharmaceutical
grades of mannitol, lactose, starch, magnesium stearate, sodium
saccharin, talcum, cellulose, glucose, sucrose, magnesium
carbonate, and the like.
[0175] The present invention also provides for therapeutic
compositions or medicaments comprising a mixture of one or more of
the ADNF I complex polypeptides described herein above in mixture
with a pharmaceutically acceptable excipient, wherein the amount of
a mixture the ADNF I complex polypeptides is sufficient to provide
a desirable therapeutic effect.
[0176] Small polypeptides such as ADNF I complex polypeptides cross
the blood brain barrier. For longer polypeptides that do not the
cross blood brain barrier, methods of administering proteins to the
brain are well known. For example, proteins, polypeptides, other
compounds and cells can be delivered to the mammalian brain via
intracerebroventricular (ICV) injection or via a cannula (see,
e.g., Motta & Martini, Proc. Soc. Exp. Biol. Med. 168:62-64
(1981); Peterson et al., Biochem. Pharamacol. 31:2807-2810 (1982);
Rzepczynski et al., Metab. Brain Dis. 3:211-216 (1988); Leibowitz
et al., Brain Res. Bull 21:905-912 (1988); Sramka et al.,
Stereotact. Funct. Neurosurg. 58:79-83 (1992); Peng et al., Brain
Res. 632:57-67 (1993); Chem et al., Exp. Neurol. 125:72-81 (1994);
Nikkhah et al., Neuroscience 63:57-72 (1994); Anderson et al., J.
Comp. Neurol. 357:296-317 (1995); and Brecknell & Fawcett, Exp.
Neurol. 138:338-344 (1996)). In particular, cannulas can be used to
administer neurotrophic factors to mammals (see, e.g., Motta &
Martini, Proc. Soc. Exp. Biol. Med. 168:62-64 (1981) (neurotensin);
Peng et al., Brain Res. 632:57-67 (1993) (NGF); Anderson et al., J.
Comp. Neurol. 357:296-317 (1995) (BDNF, NGF, neurotrophin-3).
[0177] Alternatively, longer ADNF I complex polypeptides that do
not cross blood brain barrier can be coupled with a material which
assists the ADNF I complex polypeptides to cross the blood brain
barrier and to traverse the plasma membrane of a cell, or the
membrane of an intra-cellular compartment such as the nucleus.
Cellular membranes are composed of lipid-protein bilayers that are
freely permeable to small, nonionic lipophilic compounds and are
inherently impermeable to polar compounds, macromolecules, and
therapeutic or diagnostic agents. However, proteins and other
compounds such as liposomes have been described, which have the
ability to translocate polypeptides such as ADNF I complex
polypeptides across a cell membrane.
[0178] For example, "membrane translocation polypeptides" have
amphiphilic or hydrophobic amino acid subsequences that have the
ability to act as membrane-translocating carriers. In one
embodiment, homeodomain proteins have the ability to translocate
across cell membranes. The shortest internalizable peptide of a
homeodomain protein, Antennapedia, was found to be the third helix
of the protein, from amino acid position 43 to 58 (see, e.g.
Prochiantz, Current Opinion in Neurobiology 6:629-634 (1996)).
Another subsequence, the hydrophobic domain of signal peptides, was
found to have similar cell membrane translocation characteristics
(see, e.g. Lin et al., J. Biol. Chem. 270:1 4255-14258 (1995)).
[0179] Examples of peptide sequences which can be linked to a ADNF
I complex polypeptides of the invention, for facilitating uptake of
ADNF I complex polypeptides s into cells, include, but are not
limited to: an 11 animo acid peptide of the tat protein of HIV (see
Schwarze et al., Science 285:1569-1572 (1999)); a 20 residue
peptide sequence which corresponds to amino acids 84-103 of the p16
protein (see Fahraeus et al., Current Biology 6:84 (1996)); the
third helix of the 60-amino acid long homeodomain of Antennapedia
(Derossi et al., J. Biol. Chem. 269:10444 (1994)); the h region of
a signal peptide such as the Kaposi fibroblast growth factor
(K-FGF) h region (Lin et al., supra); or the VP22 translocation
domain from HSV (Elliot & O'Hare, Cell 88:223-233 (1997)).
Other suitable chemical moieties that provide enhanced cellular
uptake may also be chemically linked to ADNF I complex
polypeptides.
[0180] Toxin molecules also have the ability to transport
polypeptides across cell membranes. Often, such molecules are
composed of at least two parts (called "binary toxins"): a
translocation or binding domain or polypeptide and a separate toxin
domain or polypeptide. Typically, the translocation domain or
polypeptide binds to a cellular receptor, and then the toxin is
transported into the cell. Several bacterial toxins, including
Clostridium perfringens iota toxin, diphtheria toxin (DT),
Pseudomonas exotoxin A (PE), pertussis toxin (PT), Bacillus
anthracis toxin, and pertussis adenylate cyclase (CYA), have been
used in attempts to deliver peptides to the cell cytosol as
internal or amino-terminal fusions (Arora et al., J. Biol. Chem.,
268:3334-3341 (1993); Perelle et al., Infect. Immun., 61:5147-5156
(1993); Stenmark et al., J. Cell Biol. 113:1025-1032 (1991);
Donnelly et al., Proc. Nat'l Acad. Sci. USA 90:3530-3534 (1993);
Carbonetti et al., Abstr. Annu. Meet. Am. Soc. Microbiol. 95:295
(1995); Sebo et al., Infect. Immun. 63:3851-3857 (1995); Klimpel et
al., Proc. Nat'l Acad. Sci. USA 89:10277-10281 (1992); and Novak et
al., J. Biol. Chem. 267:17186-17193 1992)).
[0181] Such subsequences can be used to translocate ADNF I complex
polypeptides across a cell membrane. ADNF I complex polypeptides
can be conveniently fused to or derivatized with such sequences.
Typically, the translocation sequence is provided as part of a
fusion protein. Optionally, a linker can be used to link the ADNF I
complex polypeptides and the translocation sequence. Any suitable
linker can be used, e.g., a peptide linker.
[0182] The ADNF I complex polypeptides and nucleic acids encoding
ADNF I complex polypeptides can also be introduced into an animal
cell, preferably a mammalian cell, via a liposomes and liposome
derivatives such as immunoliposomes and lipid:nucleic acid
complexes. The term "liposome" refers to vesicles comprised of one
or more concentrically ordered lipid bilayers, which encapsulate an
aqueous phase. The aqueous phase typically contains the compound to
be delivered to the cell, i.e., an ADNF I complex polypeptide.
[0183] The liposome fuses with the plasma membrane, thereby
releasing the ADNF I complex polypeptides into the cytosol.
Alternatively, the liposome is phagocytosed or taken up by the cell
in a transport vesicle. Once in the endosome or phagosome, the
liposome either degrades or fuses with the membrane of the
transport vesicle and releases its contents.
[0184] In current methods of drug delivery via liposomes, the
liposome ultimately becomes permeable and releases the encapsulated
compound (in this case, an ADNF I complex polypeptide) at the
target tissue or cell. For systemic or tissue specific delivery,
this can be accomplished, for example, in a passive manner wherein
the liposome bilayer degrades over time through the action of
various agents in the body. Alternatively, active drug release
involves using an agent to induce a permeability change in the
liposome vesicle. Liposome membranes can be constructed so that
they become destabilized when the environment becomes acidic near
the liposome membrane (see, e.g., Proc. Nat'l Acad. Sci. USA
84:7851 (1987); Biochemistry 28:908 (1989)). When liposomes are
endocytosed by a target cell, for example, they become destabilized
and release their contents. This destabilization is termed
fusogenesis. Dioleoylphosphatidylethanolamine (DOPE) is the basis
of many "fusogenic" systems.
[0185] Such liposomes typically comprise an ADNF I complex
polypeptide and a lipid component, e.g., a neutral and/or cationic
lipid, optionally including a receptor-recognition molecule such as
an antibody that binds to a predetermined cell surface receptor or
ligand (e.g., an antigen). A variety of methods are available for
preparing liposomes as described in, e.g., Szoka et al., Ann. Rev.
Biophys. Bioeng. 9:467 (1980), U.S. Pat. Nos. 4,186,183, 4,217,344,
4,235,871, 4,261,975, 4,485,054, 4,501,728, 4,774,085, 4,837,028,
4,235,871, 4,261,975, 4,485,054, 4,501,728, 4,774,085, 4,837,028,
4,946,787, PCT Publication No. WO 91/17424, Deamer & Bangham,
Biochim. Biophys. Acta 443:629-634 (1976); Fraley, et al., Proc.
Nat'l Acad. Sci. USA 76:3348-3352 (1979); Hope et al., Biochim.
Biophys. Acta 812:55-65 (1985); Mayer et al., Biochim. Biophys.
Acta 858:161-168 (1986); Williams et al, Proc. Nat'l Acad. Sci. USA
85:242-246 (1988); Liposomes (Ostro (ed.), 1983, Chapter 1); Hope
et al., Chem. Phys. Lip. 40:89 (1986); Gregoriadis, Liposome
Technology (1984) and Lasic, Liposomes: from Physics to
Applications (1993)). Suitable methods include, for example,
sonication, extrusion, high pressure/homogenization,
microfluidization, detergent dialysis, calcium-induced fusion of
small liposome vesicles and ether-fusion methods, all of which are
well known in the art.
[0186] In certain embodiments of the present invention, it is
desirable to target the liposomes of the invention using targeting
moieties that are specific to a particular cell type, tissue, and
the like. Targeting of liposomes using a variety of targeting
moieties (e.g., ligands, receptors, and monoclonal antibodies) has
been previously described (see, e.g., U.S. Pat. Nos. 4,957,773 and
4,603,044). Standard methods for coupling targeting agents to
liposomes can be used. These methods generally involve
incorporation into liposomes lipid components, e.g.,
phosphatidylethanolamine, which can be activated for attachment of
targeting agents, or derivatized lipophilic compounds, such as
lipid derivatized bleomycin. Antibody targeted liposomes can be
constructed using, for instance, liposomes which incorporate
protein A (see Renneisen et al., J. Biol. Chem., 265:16337-16342
(1990) and Leonetti et al., Proc. Nat'l Acad. Sci. USA 87:2448-2451
(1990).
[0187] Alternatively, nucleic acids encoding an ADNF I complex
polypeptide can also be used to provide a therapeutic dose of an
ADNF I complex polypeptide. These nucleic acids can be inserted
into any of a number of well-known vectors for the transfection of
target cells and organisms. For example, nucleic acids are
delivered as DNA plasmids, naked nucleic acid, and nucleic acid
complexed with a delivery vehicle such as a liposome. Viral vector
delivery systems include DNA and RNA viruses, which have either
episomal or integrated genomes after delivery to the cell. For a
review of gene therapy procedures, see Anderson, Science
256:808-813 (1992); Nabel & Felgner, TIBTECH 11:211-217 (1993);
Mitani & Caskey, TIBTECH 11:162-166 (1993); Dillon, TIBTECH
11:167-175 (1993); Miller, Nature 357:455-460 (1992); Van Brunt,
Biotechnolog 6(10):1149-1154 (1988); Vigne, Restorative Neurology
and Neuroscience 8:35-36 (1995); Kremer & Perricaudet, British
Medical Bulletin 51(1):31-44 (1995); Haddada et al., in Current
Topics in Microbiology and Immunology Doerfler and Bohm (eds)
(1995); and Yu et al, Gene Therapy 1: 13-26 (1994).
[0188] Methods of non-viral delivery of nucleic acids include
lipofection, microinjection, biolistics, virosomes, liposomes,
immunoliposomes, polycation or lipid:nucleic acid conjugates, naked
DNA, artificial virions, and agent-enhanced uptake of DNA.
Lipofection is described in, e.g., U.S. Pat. No. 5,049,386, U.S.
Pat. No. 4,946,787; and U.S. Pat. No. 4,897,355) and lipofection
reagents are sold commercially (e.g., Transfectam.TM. and
Lipofectin.TM.). Cationic and neutral lipids that are suitable for
efficient receptor-recognition lipofection of polynucleotides
include those of Felgner, WO 91/17424, WO 91/16024. Delivery can be
to cells (ex vivo administration) or target tissues (in vivo
administration).
[0189] In therapeutic applications, ADNF I complex polypeptides of
the invention are administered to a patient (either pre- or
post-natally) in an amount sufficient to reduce neuronal cell death
associated with various disorders, to reduce oxidative stress in a
patient, to reduce a condition associated with fetal alcohol
syndrome in a subject in utero, or to improve a subject's
performance (e.g., learning and/or memory). An amount adequate to
accomplish this is defined as "therapeutically effective dose."
Amounts effective for this use will depend on, for example, the
particular ADNF I complex polypeptide employed, the manner of
administration, the weight and general state of health of the
patient, and the judgment of the prescribing physician. For
example, an amount of ADNF I complex polypeptide falling within the
range of a 1 .mu.g to 50 .mu.g, preferably 1 .mu.g to 10 .mu.g dose
given intranasally or orally once a day per mouse (e.g., in the
evening) would be a therapeutically effective amount. This dose is
based on the average body weight of a mouse. Therefore, an
appropriate dose can be extrapolated for a human body.
[0190] For pre-natal administration, ADNF I complex polypeptides
can be pre-natally administered to the subject directly or
indirectly through the subject's mother. ADNF I complex
polypeptides can be administered at any time during the pregnancy.
Preferably, ADNF I complex polypeptides are administered to the
subject during the first trimester (i.e., first 12 weeks) of the
pregnancy when organs and the nervous system of the subject are
actively developing. More preferably, ADNF I complex polypeptides
are administered during the time of neural tube development (which
begins around 22 days post-conception) and prior to its closure.
ADNF I complex polypeptides can be administered as a single dose,
preferably during the critical period of neural tube development,
or can be administered as multiple doses throughout the
pregnancy.
Methods for Reducing Neuronal Cell Death
[0191] In another aspect, the present invention provides a method
for reducing neuronal cell death, the method comprising contacting
neuronal cells with an ADNF I complex polypeptide in an amount
sufficient to reduce neuronal cell death. In one embodiment, the an
ADNF I complex polypeptide comprises at least one D-amino acid,
preferably at the N-terminus and/or the C-terminus.
[0192] ADNF I complex polypeptides of the present invention can be
used in the treatment of neurological disorders and for the
prevention of neuronal cell death. For example, an ADNF I complex
polypeptide of the present invention can be used to prevent the
death of neuronal cells including, but not limited to, spinal cord
neurons, hippocampal neurons, cerebral cortical neurons and
cholinergic neurons. More particularly, ADNF I complex polypeptides
of the present invention can be used in the prevention of cell
death associated with (1) gp120, the envelope protein from HIV; (2)
N-methyl-D-aspartic acid (excito-toxicity); (3) tetrodotoxin
(blockage of electrical activity); and (4) .beta.-amyloid peptide,
a substance related to neuronal degeneration in Alzheimer's
disease.
[0193] As such, the ADNF I complex polypeptides of the present
invention can be used to reduce gp120-induced neuronal cell death
by administering an effective amount of an ADNF I complex
polypeptide of the present invention to a patient infected with the
HIV virus. The ADNF I complex polypeptides of the present invention
can also be used to reduce neuronal cell death associated with
excito-toxicity induced by N-methyl-D-aspartate stimulation, the
method comprising contacting neuronal cells with an ADNF I complex
polypeptide of the present invention in an amount sufficient to
prevent neuronal cell death. The ADNF I complex polypeptides of the
present invention can also be used to reduce cell death induced by
the .beta.-amyloid peptide in a patient afflicted or impaired with
Alzheimer's disease, the method comprising administering to the
patient an ADNF I complex polypeptide of the present invention in
an amount sufficient to prevent neuronal cell death. The ADNF I
complex polypeptides can also be used to alleviate learning
impairment produced by cholinergic blockage in a patient afflicted
or impaired with Alzheimer's disease. For example, ADNF I complex
polypeptides can be used to improve short-term and/or reference
memory in Alzheimer's patients.
[0194] Similarly, it will be readily apparent to those of skill in
the art that the ADNF I complex polypeptides of the present
invention can be used in a similar manner to prevent neuronal cell
death associated with a number of other neurological diseases and
deficiencies. Pathologies that would benefit from therapeutic and
diagnostic applications of this invention include conditions
(diseases and insults) leading to neuronal cell death and/or
sub-lethal neuronal pathology including, for example, the
following:
[0195] diseases of central motor systems including degenerative
conditions affecting the basal ganglia (Huntington's disease,
Wilson's disease, striatonigral degeneration, corticobasal
ganglionic degeneration), Tourette's syndrome, Parkinson's disease,
progressive supranuclear palsy, progressive bulbar palsy, familial
spastic paraplegia, spinomuscular atrophy, ALS and variants
thereof, dentatorubral atrophy, olivo-pontocerebellar atrophy,
paraneoplastic cerebellar degeneration, and dopamine toxicity;
[0196] diseases affecting sensory neurons such as Friedreich's
ataxia, diabetes, peripheral neuropathy, retinal neuronal
degeneration;
[0197] diseases of limbic and cortical systems such as cerebral
amyloidosis, Pick's atrophy, Retts syndrome;
[0198] neurodegenerative pathologies involving multiple neuronal
systems and/or brainstem including Alzheimer's disease,
AIDS-related dementia, Leigh's disease, diffuse Lewy body disease,
epilepsy, multiple system atrophy, Guillain-Barre syndrome,
lysosomal storage disorders such as lipofuscinosis,
late-degenerative stages of Down's syndrome, Alper's disease,
vertigo as result of CNS degeneration;
[0199] pathologies associated with developmental retardation and
learning impairments, and Down's syndrome, and oxidative stress
induced neuronal death;
[0200] pathologies arising with aging and chronic alcohol or drug
abuse including, for example, with alcoholism the degeneration of
neurons in locus coeruleus, cerebellum, cholinergic basal
forebrain; with aging degeneration of cerebellar neurons and
cortical neurons leading to cognitive and motor impairments; and
with chronic amphetamine abuse degeneration of basal ganglia
neurons leading to motor impairments;
[0201] pathological changes resulting from focal trauma such as
stroke, focal ischemia, vascular insufficiency, hypoxic-ischemic
encephalopathy, hyperglycemia, hypoglycemia, closed head trauma, or
direct trauma;
[0202] pathologies arising as a negative side-effect of therapeutic
drugs and treatments (e.g., degeneration of cingulate and
entorhinal cortex neurons in response to anticonvulsant doses of
antagonists of the NMDA class of glutamate receptor).
[0203] ADNF I complex polypeptides (including their alleles,
polymorphic variants, species homologs and subsequences thereof)
that reduce neuronal cell death can be screened using the various
methods described in U.S. Ser. No. 60/037,404, filed Feb. 7, 1997
(published as WO98/35042), and U.S. Ser. No. 09/187,330 filed Nov.
6, 1998, both of which are incorporated herein by reference. For
example, it will be readily apparent to those skilled in the art
that using the teachings set forth above with respect to the design
and synthesis of ADNF I complex polypeptides and the assays
described herein, one of ordinary skill in the art can identify
other biologically active ADNF I complex polypeptides. For example,
Brenneman et al., Nature 335:639-642 (1988), and Dibbern et al., J.
Clin. Invest. 99:2837-2841 (1997), incorporated herein by
reference, teach assays that can be used to screen ADNF I complex
polypeptides that are capable of reducing neuronal cell death
associated with envelope protein (gp120) from HIV. Also, Brenneman
et al., Dev. Brain Res. 51:63-68 (1990), and Brenneman & Gozes,
J. Clin. Invest. 97:2299-2307 (1996), incorporated herein by
reference, teach assays that can be used to screen ADNF I complex
polypeptides which are capable of reducing neuronal cell death
associated with excito-toxicity induced by stimulation by
N-methyl-D-aspartate. Other assays described in, e.g., WO98/35042
can also be used to identify other biologically active ADNF I
complex polypeptides, optionally comprising at least one D-amino
acid.
[0204] Moreover, ADNF I complex polypeptides that reduce neuronal
cell death can be screened in vivo. For example, the ability of
ADNF I complex polypeptides that can protect against learning and
memory deficiencies associated with cholinergic blockade can be
tested. For example, cholinergic blockade can be obtained in rats
by administration of the cholinotoxin AF64A, and ADNF I complex
polypeptides can be administered intranasally and the water maze
experiments can be performed (Gozes et al., Proc. Natl. Acad. Sci.
USA 93:427-432 (1996), the teachings of which are incorporated
herein by reference). Animals treated with efficacious ADNF I
complex polypeptides would show improvement in their learning and
memory capacities compared to the control.
[0205] Furthermore, the ability of ADNF I complex polypeptides that
can protect or reduce neuronal cell death associated with
Alzheimer's disease can be screened in vivo. For these experiments,
apolipoprotein E (ApoE)-deficient homozygous mice can be used
(Plump et al., Cell 71:343-353 (1992); Gordon et al., Neuroscience
Letters 199:1-4 (1995); Gozes et al., J. Neurobiol. 33:329-342
(1997)), the teachings of which are incorporated herein by
reference.
Methods for Reducing Oxidative Stress
[0206] In yet another aspect, the present invention provides
methods for treating oxidative stress in a patient by administering
to the patient an ADNF I complex polypeptide in an amount
sufficient to prevent or reduce oxidative stress, wherein the ADNF
I complex polypeptide optionally comprises at least one D-amino
acid, preferably at the N-terminus and/or the C-terminus. Oxidative
stress has been implicated in several neurodegenerative diseases in
humans (Cassarmno & Bennett, Brain Res. Reviews 29:1-25
(1999)). Moreover, oxidative stress produced from alcohol
administration has been associated with fetal death and
abnormalities (e.g., conditions associated with fetal alcohol
syndrome). See, e.g. Henderson et al., Alcoholism: Clinical and
Experimental Research 19:714-720 (1995). By using the ADNF I
complex polypeptides of the present invention, oxidative stress
associated with various clinical conditions can be reduced.
[0207] ADNF I complex polypeptides (including their alleles,
polymorphic variants, species homologs and subsequences thereof)
that are effective in reducing oxidative stress can be screened
using primary neurons. For example, cultured embryonic neurons
(E18) rat hippocampal neurons can be treated with, e.g., 0.5 .mu.M
FeSO.sub.4 to induce oxidative stress. The degree of oxidative
stress can be quantified by cell counting and/or morphological
criteria. Furthermore, apoptosis induced by oxidative stress
results in nuclear condensation and DNA fragmentation. Apoptotic
nuclei can be measured by counting cells in culture stained with
the fluorescent DNA-binding dye, e.g., Hoescht 33342. See Glazner
et al., Society for Neuroscience 27.sup.th Annual Meeting,
Abstracts vol. 23, part 2 (1997). To screen ADNF I complex
polypeptides that can reduce oxidative stress in vitro, FeSO.sub.4
treated neurons can be contacted with various ADNF I complex
polypeptides for sufficient time (e.g., 24 hours). Cells with
apoptotic nuclei can be quantified as described above. ADNF I
complex polypeptides that reduce the quantity of apoptotic nucleic
compared to control (e.g., cells untreated with ADNF I complex
polypeptides) can be used to treat oxidative stress in a
patient.
[0208] ADNF I complex polypeptides that are effective in reducing
oxidative stress can also be screened using in vivo assays. For
example, ethanol consumption is known to cause oxidative stress in
vivo. In the human body, ethanol is metabolized into cytotoxic
acetaldehyde by alcohol dehydrogenase enzyme in the liver and
acetaldehyde is oxidized to acetate by aldehyde oxidase or xanthine
oxidase giving rise to free radicals or reactive oxygen species
(ROS). See, e.g. Schlorff et al., Alcohol 17:95-105 (1999). Thus,
ethanol consumption can be used to induce oxidative stress in in
vivo animal models (e.g., rat, mouse, human, etc.). Thereafter,
animals suffering from ethanol induced oxidative stress can be used
as models to screen ADNF I complex polypeptides that can reduce the
level of oxidative stress.
[0209] The level of oxidative stress of cells and tissues of in
vivo animal models can be measured using a number of assays known
in the art. For example, protocols described in Schlorff et al.
(1999), supra, can be used to measure effects of rat ethanol
ingestion on lipid peroxidation in plasma (e.g., plasma
malondialdehyde) and changes in antioxidant system (e.g.,
superoxide dismutase, catalase, glutathione peroxidase, glutathione
reductase, etc.). Effective ADNF I complex polypeptides are those
that prevent or reduce changes in lipid peroxidation in plasma or
on antioxidant system in ethanol ingested animal models compared to
control (e.g., animal models untreated with ADNF I complex
polypeptides). In another example, fetal death and abnormalities
(e.g., conditions associated with fetal alcohol syndrome) are
considered a severe form of oxidative stress produced from alcohol
administration (Henderson et al., Alcoholism: Clinical and
Experimental Research 19:714-720 (1995)). Therefore, a well
established model (e.g., mice) for fetal alcohol syndrome can also
be used to screen for ADNF I complex polypeptides that can reduce
oxidative stress.
Methods for Reducing a Condition Associated with Fetal Alcohol
Syndrome
[0210] In yet another aspect, the present invention provides a
method for reducing a condition associated with fetal alcohol
syndrome in a subject who is exposed to alcohol in utero, the
method comprising administering to the subject an ADNF I complex
polypeptide in an amount sufficient to reduce the condition
associated with fetal alcohol syndrome, wherein the ADNF I complex
polypeptide optionally comprises at least one D-amino acid,
preferably at the N-terminus and/or the C-terminus.
[0211] Treatment of a well-characterized model for FAS (e.g.,
C57B1/6J mouse strain) with an ADNF I complex polypeptide reduces
or prevents alcohol induced fetus death, body and brain weight
reduction, and VIP mRNA reduction. Similarly, the human embryo,
fetus, or subject can be protected from alcohol induced effects by
administering an ADNF I complex polypeptide directly to the embryo,
fetus, or subject, or by administering the ADNF I complex
polypeptide indirectly to the fetus by administering it to the
mother. Preferably, ADNF I complex polypeptides are orally
administered.
[0212] ADNF I complex polypeptides (including their alleles,
polymorphic variants, species homologs and subsequences thereof)
that reduce a condition associated with fetal alcohol syndrome can
be screened using a well-characterized animal model for FAS. For
example, the C57B1/6J mouse strain can be used. Previous work with
this strain has defined the effects of dosage and embryonic timing
on maternal serum alcohol levels and embryonic effects (Webster et
al., Neurobehav. Tox., 2:227-34 (1980), incorporated herein by
reference). Intra-peritoneal treatment allows for defined and
reproducible dosages. Acute (single) dosages of alcohol can
reproduce the phenotype of FAS (Webster et al., (1980), supra).
Since treatment on E8 results in the highest rate of fetal
anomalies and demises, and vasoactive intestinal peptide's growth
regulating effects on the embryo are limited to the early
post-implantation period of embryogenesis, E8 can be chosen as a
test for screening neuroprotective ADNF I complex polypeptides. The
mice can be injected with 25% ethyl alcohol in saline (v/v) or
vehicle alone at, e.g., 0.030 ml/g maternal body weight at, e.g.,
9:00 a.m. on E8 (embryonic gestation day 8). Effective ADNF I
complex polypeptides can be screened by pretreating the mice 30
minutes prior to alcohol administration. In one embodiment, the
dose for nasal administration for an ADNF I complex polypeptide is
about 1 .mu.g-50 .mu.g, preferably about 1 .mu.g-10 .mu.g/mouse.
This dose is based on the average body weight of mice, and an
appropriate dose for human can be extrapolated based on the average
body weight of human.
[0213] Various parameters can be measured to determine if an ADNF I
complex polypeptide reduces a condition associated with fetal
alcohol syndrome. For example, a number of fetal demises (i.e.,
death) can be compared between the control (e.g., untreated with
ADNF I complex polypeptides) and a group treated with ADNF I
complex polypeptides. In another example, the fetal weight and
fetal brain weight in the surviving E18 fetuses can be compared. In
another example, the level of VIP mRNA can be compared between the
control and a group treated with ADNF I complex polypeptides. In
another example, the degree of learning deficits can be compared
between the control and a group treated with ADNF I complex
polypeptides. In another example, the glutathione level in the
control and the treated group can be compared.
Methods for Enhancing Learning and Memory
[0214] In yet another aspect, the present invention provides a
method of enhancing learning and memory, the method comprising
administering either pre- or post-natally to the subject an ADNF I
complex polypeptide in an amount sufficient to enhance learning and
memory, wherein the ADNF I complex polypeptide optionally comprises
at least one D-amino acid, preferably at the N-terminus and/or the
C-terminus.
[0215] Various parameters can be measured to determine if ADNF I
complex polypeptides improve performance (e.g., learning and
memory) in vivo. For example, the hidden platform test of the
Morris water maze, which is described in the example section below,
can be used to test spatial learning and memory. Generally, mice
that are treated with ADNF I complex polypeptides and control mice
(that are not treated with ADNF I complex polypeptides) are trained
to escape the swimming task by learning the position of a hidden
platform and climbing on it. The time it takes them to complete
this task is defined as the escape latency. This test can be
conducted one or more times daily for a number of days. One
parameter that is indicative of improved learning and memory is the
reduction in latency in escaping the swimming task by climbing onto
the hidden platform (see the example section below). See, also,
methods described in Gozes et al., Proc. Natl. Acad. Sci. USA
93:427-432 (1996), incorporated herein by reference. Animals
treated with suitable ADNF I complex polypeptides show improvement
in their learning and memory capacities compared to the controls
that are not treated with ADNF I complex polypeptides. Embodiments
of the invention are not limited by examples of the test used to
measure performance. Any suitable test methods can be used to
measure performance, such as learning and memory.
[0216] Other methods known in the art can be used in human subjects
to determine if an ADNF I complex polypeptide or a combination of
ADNF I complex polypeptides improves performance (e.g., learning
and memory) in vivo. For example, these methods include assessment
of memory or learning over time by the Randt Memory Test (Randt et
al., Clin. Neuropsychol., 1980, 2:184), Wechsler Memory Scale (J.
Psych. 19:87-95 (1945), Forward Digit Span test (Craik, Age
Differences in Human Memory, in: Handbook of the Psychology of
Aging, Birren, J., and Schaie, K. (Eds.), New York, Van Nostrand
(1977), Mini-Mental State Exam (Folstein et al., J. of Psych. Res.
12:189-192 (1975), or California Verbal Learning Test (CVLT). See,
also, U.S. Pat. No. 6,030,968. In these tests, factors unrelated to
effects of ADNF I complex polypeptides (e.g., anxiety, fatigue,
anger, depression, confusion, or vigor) are controlled for. See,
U.S. Pat. No. 5,063,206. Methods for assessing and controlling for
subjective factors is known in the art and determined by such
standard clinical tests such as the BECK Depression Scale,
Spielberger Trait State Anxiety test, and POMS test (Profile of
Mood State).
[0217] Spatial learning can also be tested in human. For example, a
subject can be asked to draw a picture, and then the picture is
taken away. The subject is then asked to draw the same picture from
memory. The latter picture drawn by the subject reflects a degree
of spatial learning in the subject.
[0218] Various parameters can be measured to determine if ADNF I
complex polypeptides improve learning and memory of a subject. For
example, the degree of learning and memory improvement can be
compared between the control (e.g., untreated with ADNF I complex
polypeptides) and a group pretreated with ADNF I complex
polypeptides. Learning and memory improvement can be assessed
using, for example, a Morris water maze for rodents (see, e.g., the
Example section) or any suitable tests such as those described
above for humans.
[0219] If any one or more of these parameters are changed for the
group treated with ADNF I complex polypeptides by, e.g., about 10%,
optionally at least about 20%, at least about 30%, at least about
40%, at least about 50%, at least about 60%, at least about 70%, at
least about 80%, at least about 90%, at least about 100%, at least
about 150%, at least about 200%, etc., compared to control, then it
can be said that the ADNF I complex polypeptides improved learning
and memory of the subject. Alternatively, statistical analysis
using ANOVA for continuous variables, Mann-Whitney U for
nonparametic data, Chi square for categorical variables or Fisher's
exact test with p<0.05 is considered significant.
EXAMPLES
[0220] The following examples are offered to illustrate, but not to
limit the claimed invention.
Example I
Demonstration of Additional Subunits or Accessory Components of
ADNF I Complex
[0221] Evidence is presented that purified ADNF I, when analyzed
under acidic conditions or when treated with a protease inhibitor
cocktail (see Example III), exhibited a complex dose response for
survival-promoting activity that indicates the existence of labile
and novel components that were not previously identified. These
data are consistent with the existence of labile components of the
ADNF I complex that are only apparent when a serine protease
inhibitor is present or when the pH is lowered to 4.5.
[0222] ADNF I was isolated as previously described (see, e.g.,
Brenneman & Gozes, J. Clin. Invest. 97:2299-2307 (1996)) and
then desalted and stored in 10 mM phosphate buffer (pH 4.5). The
purified ADNF I was diluted 1:3 with water and analyzed on eCAP/SDS
capillary electrophoresis (CE). The running and sample buffers for
these analyses were components of the eCAP/SDS kit (Beckmann
Instruments). Electrophoresis was conducted with 8.1 kV constant
voltage and with reversed polarity. The capillary consisted of
sodium dodecyl sulfate coating with a 100 .mu.m inside diameter, 47
cm in length (300 V/cm). The pressure injection time was 70 sec at
80 psi.
[0223] As shown in FIG. 1, this analysis revealed two peaks that
were not present in the buffer/marker blank: peaks at approximately
14 and 60 kDa. The 14 kDa peak was isolated and concentrated with 5
consecutive collections, and then tested for survival-promoting
activity in cerebral cortical cultures co-treated with 1 .mu.M
tetrodotoxin (see, e.g., Brenneman & Gozes, J. Clin. Invest.
97:2299-2307 (1996)). As shown in FIG. 2, the 14 kDa CE peak
exhibited three peaks of activity as assessed by neuronal cell
counts (see, e.g., Brenneman & Gozes, J. Clin. Invest.
97:2299-2307 (1996)) and by 5(6)-carboxyfluorescein diacetate
(CFDA), a marker for neuronal survival (Petroski & Geller, J.
Neurosci. Method. 52:23-32 (1994)).
Example II
Demonstration of Tryptic Digest Peptides from ADNF I Complex and
their Neuroprotective Activity in CNS Cultures
[0224] Digest peptides were obtained after treatment of purified
ADNF I with trypsin as previously described (Williams & Stone,
Techniques in Protein Chemistry VI. pp 79-90 (1997)). As shown in
FIG. 3, the peptides were separated by reverse phase HPLC. The
peptide identified for each of the peaks is given as the first
three amino acids of the peptide. Aliquots of the isolated peptides
were sequenced by Edman degradation or tested for
survival-promoting activity in TTX-treated cerebral cortical
cultures. As shown in FIG. 4, three digest peptide fractions
exhibited survival-promoting activity in vitro. The active peptides
were:
[0225] 1. NNSTTYAPISANVSTALGSTAALPTAAGPV (SEQ ID NO:2) (retention
time: 62 min) Abrev.: NNST
[0226] 2. GPTADITLTK (SEQ ID NO:7) (retention time: 43 min) Abrev.:
GPT
[0227] 3. NPSGTDWLNTNNQANPFN (SEQ ID NO:4) (retention time: 71 min)
Abrev. PSG
[0228] One additional digest fraction containing SESSGTSELLTR (SEQ
ID NO:11) was not available for testing, so this peptide was
synthesized. To confirm the activity of the three peptides above
plus SESSGTSELLTR (SEQ ID NO:11) (SES), the peptides were
synthesized and tested for survival-promoting activity in the
TTX-treated cortical cultures. As shown in FIG. 5, the synthesized
NNST (peptide #1 above) exhibited neuroprotection as assayed by
neuronal cell counts and the CFDA. In comparison to cultures
treated with TTX alone, both assays indicated that an increase in
neuronal survival was observed between 10.sup.-16 M and 10.sup.-14
M; with another peak of activity observed at 10.sup.-8 M treatment
with NNST. Similarly, an increase in neuronal survival was observed
with GPT (FIG. 6), PSG (FIG. 7) and with SES (FIG. 8).
[0229] Because the PSG fraction from the trypsin digests contained
two peptides, the similar action of the synthesized PSG peptide in
the cortical cultures strongly indicates that the biological action
observed in the digest fraction was due to PSG. Importantly, a
truncated form of PSG (PSGTDWLNT (SEQ ID NO:10)) exhibited potent
survival-promoting activity which mimicked that activity of the
full length digest PSG peptide (PSGTDWLNTNNQANPFN (SEQ ID NO:15)).
Thus the survival-promoting activity of the digest peptides
observed in part II were confirmed by the demonstration of
neuroprotective action of the corresponding synthesized peptides.
The peptides produce femtomolar-acting, neuroprotective activity in
cerebral cortical cultures.
Example III
Antiserum to ADNF I Complex Peptides: Biological Activity, Affinity
Chromatography and Immunohistochemical Localization
[0230] A. Biological Activity
[0231] To characterize the biological importance of newly
discovered ADNF I-related peptides, antisera were generated to
KLH-conjugated peptides in rabbits by standard protocols. As shown
in FIG. 9, the IgG fraction of anti-NFL produced neuronal cell
death in cerebral cortical cultures; whereas anti-WSD had little
effect on neuronal survival. The pre-immune serum of the rabbit
utilized for the anti-NFL antiserum production had no effect on
neuronal survival. Other anti-peptides from ADNF I were tested in
the CFDA assay. As shown in FIG. 10, a CFDA analysis that compared
the effects of various anti-peptides from ADNF I demonstrated that
antiserum to GAD (C-GADSNVAFQGKVIYRSESSGTSELLTR (SEQ ID NO:16)) or
anti-NNST produced a small decrease in neuronal survival. In
contrast, anti-PSG (C-PSGTDWLNTNNQANPFN (SEQ ID NO:15)) produced a
small increase in neuronal survival as measured in the CFDA assay.
Although only the anti-NFL peptide had marked effects on neuronal
survival, all the antiserum made to ADNF I peptides prevented ADNF
I-stimulated neuronal survival. Examples of this effect are shown
in FIG. 11. Biochemically isolated ADNF I was injected on an
eCAP/SDS capillary column and the single 14 kDa peak was collected
and confirmed to exhibit 3 peaks of survival-promoting activity. As
shown in FIG. 11, co-treatment with anti-NFL (300 ng/ml) prevented
the ADNF J-mediated increases in neuronal survival in TTX-treated
cerebral cortical cultures. Similar studies were conducted with all
antiserum to ADNF I peptides.
[0232] A summary of these studies is shown in FIG. 12. Antiserum
(IgG: 300 ng/ml) to the following peptides prevented ADNF
1-mediated increases in neuronal survival:
TABLE-US-00003 A) C-WSDVGVSSGSAPDAFK (SEQ ID NO:1) B)
C-NNSTTYAPISANVSTALGSTAALPTAAGPV (SEQ ID NO:2) C) C-NFL
TSHYSAANSVVGGTNPGK (SEQ ID NO:3) D) C-PSGTDWLNTNNQANPFN (SEQ ID
NO:15) E) C-GADSNVAFQGKVIYRSESSGTSELLTR (SEQ ID NO:16) F)
C-VLGGGSALLRSIPA (SEQ ID NO:14) G) Anti-heat shock protein 60
[0233] These data demonstrate that epitopes interacting with these
antibodies either are present on ADNF I (e.g., as subunits) or on
accessory proteins of ADNF I that are important for ADNF I
activity. These data further establish the immunological and
biochemical characteristics of ADNF I and the relevance of the
newly disclosed peptide structures.
[0234] B. ADNF I Affinity Isolation
[0235] A dose response to ADNF I measuring survival-promoting
activity exhibited multiple peaks. Initial descriptions of activity
indicated two peaks of activity; however, in the present case, ADNF
I was shown to exhibit three peaks of activity. In FIG. 13, the
effect of protease inhibition was observed. ADNF I-like activity
was obtained by the use of anti-NFL IgG attached to magnetic beads.
The eluted material (closed circles) indicated two peaks of
activity. However, if a cocktail of protease inhibitors
(Calbiochem. protease inhibitor cocktail #1) is added before the
affinity capture and concentration, three peaks of activity were
observed in the ADNF I-like protein eluted from the affinity bead.
Furthermore, if the ADNF I preparation is maintained and stored
under acidic conditions (pH 4.5), three peaks of ADNF I activity
are also observed (see FIG. 2). These data support the observation
that ADNF I exhibits a complex dose response and that one or more
of the components is labile to a protease that either exists in
conditioned medium or is a part of the ADNF I complex. These data
further demonstrate that lowering the pH of the ADNF I complex
prevented the loss of the third peak of activity for ADNF I.
[0236] For ADNF I, 14-amino acid peptide (VLGGGSALLRSIPA (SEQ ID
NO:14)) was sequenced and deduced from biochemically purified ADNF
I. The 14 amino acid peptide (abbreviated: VGR) was used to prepare
antiserum. Affinity columns utilizing anti-NFL and anti-VGR were
used to extract ADNF I from conditioned medium from VIP-stimulated
astrocytes. The proteins eluted from these ADNF I affinity columns
were analyzed by Surface Enhanced Laser Desorption/Ionization
(SELDI). For these studies, a reverse phase protein chip (Ciphergen
Biosystems) was used to characterize ADNF I-like proteins eluted
from the affinity columns. As shown in FIG. 14, the anti-VGR column
isolated a single major peak at approximately 14,714 Daltons. As
shown in FIGS. 15 A and 15 B, the eluted material from NFL affinity
column showed a more complex pattern with major peaks at 14,744,
11480, 8292 and 4867. Thus the NFL column interacts with proteins
not observed with the VGR column. Eluted material from both the VGR
and NFL columns exhibited the three activity peaks of
survival-promoting activity characteristic of ADNF I. In addition,
multiples of 14,714 peak were observed (29608 and 58777),
suggesting aggregations of the ADNF I complex. Thus, with either of
these ADNF I antisera, 14 kDa ADNF I-like proteins can be isolated
in a single step from a complex mixture of proteins. Applications
of this technology include an assay for ADNF I and associated
molecules.
[0237] C. Immunocytochemical Localization of ADNF I Peptide WSD
[0238] To further characterize the ADNF I complex, the IgG
antibodies to C-WSDVGVSSGSAPDAFK (SEQ ID NO:1) (WSD) were utilized
to localize this epitope in the newborn brain. These data further
describe the ADNF I complex and provide another means to delineate
uniqueness from other proteins. Immunoreactivity was localized
throughout the brain including the forebrain structures of the
cortex and hippocampus. In anterior regions, immunoreactivity was
most evident in the fiber-like structures in the basal forebrain as
shown in FIG. 16. The most intense immunoreactivity was found in
the hindbrain and brain stem structures where cells and/or fibers
were identified in the spinal trigeminal nuclei, parabrachial
nuclei, para abducens nuclei (FIG. 17), cerebellar nuclei and
numerous cells within the reticular formation (FIG. 18).
Furthermore, ADNF I-like immunoreactivity was also detected in the
newborn rat brain and E9 mouse decidua with rabbit antibodies to
the peptides NNST and GAD.
[0239] D. Western Blot Analysis of Anti-Peptides from ADNF I
[0240] To further delineate the nature of the ADNF I complex and
peptides, a Western analysis was performed on cell extracts of
astrocytes. As shown in FIG. 19, anti-NNST detected an
intracellular band at 48 kDalton. This band was increased in the
lysate of astrocyte cultures stimulated with 0.1 nM VIP in
comparison to lysates from control cultures. These data suggest
that the NNST peptide exists as is a higher molecular protein or a
prepro-form before secretion.
Example IV
Isolation of Components of ADNF I and their Characterization:
N--CHO Capillary Electrophoretic Analyses
[0241] A. Capillary Methodology
[0242] To establish the locus of the newly discovered peptide
elements within the ADNF I complex, a procedure was devised to
separate the ADNF I components and then antiserum interaction
studies were conduced on isolated components of the ADNF I complex
to establish their characteristics. Five other chromatographic
procedures (anion exchange, cation exchange, size exclusion,
hydrophobic interaction and reverse phase) did not provide any
separation of these components. As shown in the electropherogram in
FIG. 20, biochemically purified ADNF I was separated into four
peaks: I, II and two closely migrating peaks: IIIa and IIIb. One
additional peak was observed and also appeared in the blank. For
this separation, an N--CHO capillary (75 .mu.m i.d.) obtained from
Beckman Instruments was used. A constant current of 50 .mu.A was
employed using a 100 mM monobasic phosphate buffer, pH 4.55. The
injection times were 70 sec at 80 psi. UV absorbance at 200 nm was
used to detect the peaks. The N--CHO column is based on polyvinyl
alcohol which minimizes the effects of electroosmotic force and has
a very hydrophobic surface. The N--CHO column surface is designed
to separate carbohydrate moieties, these data strongly suggest that
the components of ADNF I complex are glycosylated and that
disruption of the glycopeptides is necessary for separation of the
ADNF I complex. These conditions reveal the components of ADNF I
complex and can be used in an assay for ADNF I complex for
potential diagnostic purposes.
[0243] B. Biological Activity of ADNF I Complex Components
[0244] To further establish the characteristics of the ADNF I
complex, the peaks corresponding to component I (5.1 min) component
II (8.5 min) and a combination of components IIIa and IIIb
(12.3-12.6 min) were isolated into individual fractions. These
fractions were tested in TTX-treated cerebral cortical cultures
over a broad range of dilutions as shown in FIG. 21. Each of the
isolated CE peaks exhibited a monotonic peak of biological activity
over a wide range of concentrations. Each of the components showed
similar efficacy, but differed greatly in their relative potency.
Component I exhibited the most potent activity, with component II
having a potency of 10,000 times less than component I. Component
III was the least potent, with a relative potency 100,000,000 times
less than component I.
[0245] The antibodies to the newly discovered peptides, as well as
antiserum previously described to interact with ADNF I (Brenneman
& Gozes, J. Clin. Invest. 97:2299-2307 (1996)) were compared
for their ability to block the biological activity of each of the
three ADNF I complex components isolated by N--CHO-- capillary
electrophoresis.
[0246] Component I was blocked by antibodies to NFL, WSD, PSG, NNST
and NAP. Component II showed a different pattern of blockade with
activity blocked by WSD, PSG, VGR and GAD. Component III, the least
potent of the ADNF I complex components, was blocked only by GAD,
VGR and Hsp60. These data are consistent with the model that ADNF I
is complex of peptides that have separable survival-promoting
activity. Importantly, no pre-immune serum had any effect on any of
the three components of ADNF I.
Example V
Homology Between ADNF I Amino Acid Sequence and Known Proteases
[0247] The rationale for investigating the hypothesis that ADNF I
components may be proteases resides in the recognition of amino
acid sequence homology among ADNF I peptides and known proteases.
Evidence is presented for three types of proteases: a subtilisin,
furin-type protease, an aspartyl protease and cysteine
proteases.
TABLE-US-00004 TABLE 1 A. Subtilisin active site: GTSAALPTAAG (SEQ
ID NO:17) KEX1_KLULA P09231 A portion of NNST Peptide from
GSTAALPTAAG component I: (SEQ ID NO:18) 9/11 identical 11/11
identical or conserved Subtilisin from Bacillus: TSHPDLKNQIIGGKN
(SEQ ID NO:19) A portion of NFL Peptide from TSHYSAANSVVGGTN
component I: (SEQ ID NO:20) 7/14 identical B. Eukaryotic aspartyl
protease VDVDSGSAPIVGF active site: (SEQ ID NO:21) A portion of WSD
peptide from VGVSSGSAPDAF component II: (SEQ ID NO:22) 9/12
identical C. Cysteine protease of early leaf VATCSSYPVVA
senescence: (SEQ ID NO:23) A portion of VDP peptide VDPASGYPIVG
(component unknown) (SEQ ID NO:24) 5/11 identical
Example VI
Effect of Protease Inhibitors and the Survival-Promoting Activity
of the ADNF I Complex
[0248] As shown in FIG. 22, pre-treatment of the 14 kDa purified
ADNF I complex with a protease inhibitor cocktail prevented the
neuroprotective action of ADNF I against apoptotic death produced
by 1 .mu.M tetrodotoxin. In the dissociated cerebral cortical
cultures, the ADNF I complex-produced three peaks of protective
activity. Pre-treatment with the protease inhibitor cocktail
prevented the action of all three peaks. The protease cocktail was
composed of: 500 .mu.M AEBSF
[4-(2-aminoethyl)benzenesulfonylfluoride HCl], 150 nM aprotinin, 1
.mu.M E-64 (loxastatin), 0.5 mM EDTA and 1 .mu.M leupeptin. The
protease inhibitor cocktail was mixed with purified ADNF I complex
prior to diluting and treating the cultures. No biological activity
was observed after treatment with protease inhibitor cocktail alone
when tested at the same dilutions as that done with ADNF I. These
data indicate that the neuroprotective activity of the ADNF I
complex is blocked by protease inhibitors, indicating that the ADNF
I complex is composed of protease(s) and/or that it interacts with
protease(s).
[0249] As shown in FIG. 23, pre-treatment of the 14 kDa purified
ADNF I complex with an aspartyl protease inhibitor (Pepstatin A)
prevented the peak of activity observed at 10-8 dilution. These
data suggested that component II of the ADNF I complex (peak
observed at 10-8 dilution) was an aspartyl protease; whereas
component III (peak observed at 10-5 dilution) was not affected.
Component I (peak observed at 10.sup.-12 dilution) showed a small
decrease with Pepstatin A pre-treatment. These data indicate that
the neuroprotective activity of component II can be blocked by an
aspartyl protease inhibitor and demonstrated that component II is
an aspartyl protease. Evidence in cell-free enzyme assays will be
shown for component II.
[0250] As shown in FIG. 24, pre-treatment of the 14 kDa purified
ADNF I complex with a cysteine protease inhibitor (E-64) prevented
the peaks of activity observed at 10.sup.-12 and 10.sup.-5
dilution. These data suggested that component I of the ADNF I
complex (peak observed at 10.sup.-12 dilution) was a cysteine
protease, as well as component III (peak observed at 10.sup.-5
dilution). The protective activity of Component II was not
diminished by E-64. These data indicate that the neuroprotective
activity of components I and III were blocked by a cysteine
protease inhibitor and demonstrated that component I and III were
serine proteases.
[0251] As shown in FIG. 25, pre-treatment of the 14 kDa purified
ADNF I complex with a subtilisin V inhibitor enhanced the peaks of
activity observed at 10.sup.-12 and 10.sup.-5 dilution. These data
suggest that a subtilisin-like protease degrades portions of the
ADNF I complex. These experiments were done at physiological pH.
More data suggesting the presence of a subtilisin-like protease
intrinsic to the ADNF I complex is found in part 5 below. These
data suggest that even at physiological pH, components I and II of
the ADNF I complex may be degraded by an intrinsic subtilisin-like
protease in the ADNF I complex.
Example VII
The Direct Demonstration of Protease Activity of ADNF Component II
with a Fluorogenic Peptide Substrate
[0252] To demonstrate the protease activity in an ADNF I component,
cell-free, enzyme assays utilizing a fluorogenic peptide substrate
were performed. To demonstrate this principal, component II of ADNF
I was chosen. Several assessments of the component II were made to
establish the concentration, purity and identity of the protein
preparation used for the protease assay. As shown in FIG. 26, an
electropherogram of isolated component II was shown to be a single
peak migrating at approximately 10 min in an N--CHO capillary
electrophoresis column. This elution pattern is consistent with the
10 min peak being component II as described in our previous patent
application. As shown in FIG. 27, this preparation of component II
was shown to have a molecular weight of 13,757 Daltons as measured
by matrix-assisted laser desorption/ionization mass spectrometry.
Thus, the preparation of component II was shown to be a single peak
of protein by both capillary electrophoresis and mass
spectrometry.
[0253] As shown in FIG. 28, incubation of the component II
preparation was shown to produce proteolytic activity with the use
of the aspartyl protease substrate:
[0254] Bz-Arg-Gly-Phe-Phe-Pro-4M.beta.NA HCl. Component II produced
a linear increase in fluorescence for 50 min at 35 degrees C.
Control assays without added component II had no activity. To
further characterize component II protease activity, a
concentration effect study was conducted (FIG. 29). A linear
increase in protease activity was observed from 10.sup.-11 to
10.sup.-8 dilution; however, a dramatic decrease in the activity
was shown for all component II concentrations >10.sup.-8
dilution. To demonstrate the specificity of the protease activity,
pepstatin A (an aspartyl protease inhibitor) was shown to prevent
the component II-mediated increases in protease activity. These
data indicate that component II is a pepstatin A-sensitive
protease, consistent with this component being an aspartyl
protease. Further studies indicated that component II required 1 mM
calcium to produce protease activity (FIG. 30). No protease
activity was observed with component II without the addition of 1
mM calcium to the assay. As shown in FIG. 31, the concentration
effect curve of component was very similar in comparing its
survival-promoting effects and its protease activity. Both dose
response curves show high potency and abrupt attenuation at high
concentrations of component II. These data give additional support
to the conclusion that the survival-promoting activity of component
II and ADNF I are mechanistically defined by protease activity.
Example VIII
The Demonstration that a Subtilisin-Like Protease Exists that
Serves to Inactivate the ADNF I Complex
[0255] The ADNF I complex is highly unstable at pH>7.4, making
this protein difficult to analyze. As suggested by the amino acid
sequence homology to subtilisin (See section 1 above), the ADNF I
complex could be inactivated by the action of an intrinsic
protease. To address this possibility, a MALDI mass spectrometric
analysis was performed before and after raising the pH. For these
experiments, affinity purified ADNF I complex was shown to be a
broad protein peak with a molecular weight of 14,590 Daltons (FIG.
32) before pH manipulation. The ADNF I preparation was increased to
pH 8 for 30 min, and then neutralized to pH 7 prior to performing
MALDI analysis. As shown in FIG. 33, the pH manipulation resulted
in the disappearance of the 14 kDa peak and the appearance of small
peptides <800 Daltons. These data indicate the sensitivity of
ADNF I to increased pH and provide evidence for an intrinsic
protease that inactivates ADNF I by rapid proteolytic cleavage to
small peptides.
[0256] All publications and patent applications cited in this
specification are herein incorporated by reference as if each
individual publication or patent application were specifically and
individually indicated to be incorporated by reference.
[0257] Although the foregoing invention has been described in some
detail by way of illustration and example for purposes of clarity
of understanding, it will be readily apparent to one of ordinary
skill in the art in light of the teachings of this invention that
certain changes and modifications may be made thereto without
departing from the spirit or scope of the appended claims. For
example, any one or more of the features of the previously
described embodiments can be combined in any manner with one or
more features of any other embodiments in the present invention.
The scope of the invention should, therefore, be determined not
with reference to the above description, but instead should be
determined with reference to the appended claims along with their
full scope of equivalents.
Sequence CWU 1
1
24116PRTArtificial SequenceDescription of Artificial
SequenceActivity Dependent Neurotrophic Factor I (ADNF I) complex
polypeptide active core sequence from component II, WSD 1Trp Ser
Asp Val Gly Val Ser Ser Gly Ser Ala Pro Asp Ala Phe Lys 1 5 10
15230PRTArtificial SequenceDescription of Artificial
SequenceActivity Dependent Neurotrophic Factor I (ADNF I) complex
polypeptide active core sequence, ADNF I complex tryptic digest
peptide from component I, NNST 2Asn Asn Ser Thr Thr Tyr Ala Pro Ile
Ser Ala Asn Val Ser Thr Ala 1 5 10 15Leu Gly Ser Thr Ala Ala Leu
Pro Thr Ala Ala Gly Pro Val 20 25 30321PRTArtificial
SequenceDescription of Artificial SequenceActivity Dependent
Neurotrophic Factor I (ADNF I) complex polypeptide active core
sequence from component I, NFL 3Asn Phe Leu Thr Ser His Tyr Ser Ala
Ala Asn Ser Val Val Gly Gly 1 5 10 15Thr Asn Pro Gly Lys
20418PRTArtificial SequenceDescription of Artificial
SequenceActivity Dependent Neurotrophic Factor I (ADNF I) complex
polypeptide active core sequence, ADNF I complex tryptic digest
peptide, PSG 4Asn Pro Ser Gly Thr Asp Trp Leu Asn Thr Asn Asn Gln
Ala Asn Pro 1 5 10 15Phe Asn59PRTArtificial SequenceDescription of
Artificial SequenceActivity Dependent Neurotrophic Factor I (ADNF
I) complex polypeptide active core sequence, GPT 5Leu Val Pro Leu
Thr Pro Ile Asn Arg 1 5633PRTArtificial SequenceDescription of
Artificial SequenceActivity Dependent Neurotrophic Factor I (ADNF
I) complex polypeptide active core sequence 6Val Leu Gln Ala Val
Xaa Gly Ala Asp Ser Asn Val Ala Phe Gln Gly 1 5 10 15Lys Val Ile
Tyr Arg Ser Glu Ser Ser Gly Thr Ser Glu Leu Leu Thr 20 25
30Arg710PRTArtificial SequenceDescription of Artificial
SequenceActivity Dependent Neurotrophic Factor I (ADNF I) complex
polypeptide active core sequence, ADNF I complex tryptic digest
peptide, GPT 7Gly Pro Thr Ala Asp Ile Thr Leu Thr Lys 1 5
10811PRTArtificial SequenceDescription of Artificial
SequenceActivity Dependent Neurotrophic Factor I (ADNF I) complex
polypeptide active core sequence, GTP 8Gly Thr Pro Thr Gly Xaa Gly
Pro Leu Ile Gln 1 5 10913PRTArtificial SequenceDescription of
Artificial SequenceActivity Dependent Neurotrophic Factor I (ADNF
I) complex polypeptide active core sequence, VDP 9Val Asp Pro Ala
Ser Gly Tyr Pro Ile Val Gly Tyr Thr 1 5 10109PRTArtificial
SequenceDescription of Artificial SequenceActivity Dependent
Neurotrophic Factor I (ADNF I) complex polypeptide active core
sequence, ADNF I complex tryptic digest peptide, truncated form of
PSG 10Pro Ser Gly Thr Asp Trp Leu Asn Thr 1 51112PRTArtificial
SequenceDescription of Artificial SequenceActivity Dependent
Neurotrophic Factor I (ADNF I) complex polypeptide active core
sequence, ADNF I complex tryptic digest peptide, SES 11Ser Glu Ser
Ser Gly Thr Ser Glu Leu Leu Thr Arg 1 5 10129PRTArtificial
SequenceDescription of Artificial SequenceActivity Dependent
Neurotrophic Factor I (ADNF I) active core site, ADNF-9 or SAL
12Ser Ala Leu Leu Arg Ser Ile Pro Ala 1 5138PRTArtificial
SequenceDescription of Artificial SequenceActivity Dependent
Neuroprotective Protein (ADNP or ADNF III) active core site, NAP
13Asn Ala Pro Val Ser Ile Pro Gln 1 51414PRTArtificial
SequenceDescription of Artificial SequenceADNF I agonist ADNF-14
14Val Leu Gly Gly Gly Ser Ala Leu Leu Arg Ser Ile Pro Ala 1 5
101517PRTArtificial SequenceDescription of Artificial SequenceADNF
I complex tryptic digest peptide, full length PSG peptide 15Pro Ser
Gly Thr Asp Trp Leu Asn Thr Asn Asn Gln Ala Asn Pro Phe 1 5 10
15Asn1627PRTArtificial SequenceDescription of Artificial
SequenceADNF I complex peptide, GAD 16Gly Ala Asp Ser Asn Val Ala
Phe Gln Gly Lys Val Ile Tyr Arg Ser 1 5 10 15Glu Ser Ser Gly Thr
Ser Glu Leu Leu Thr Arg 20 251711PRTArtificial SequenceDescription
of Artificial Sequencesubtilisin, furin-type protease active site
17Gly Thr Ser Ala Ala Leu Pro Thr Ala Ala Gly 1 5
101811PRTArtificial SequenceDescription of Artificial
Sequenceportion of NNST peptide from component I 18Gly Ser Thr Ala
Ala Leu Pro Thr Ala Ala Gly 1 5 101915PRTArtificial
SequenceDescription of Artificial Sequencesubtilisin from Bacillus,
furin-type protease 19Thr Ser His Pro Asp Leu Lys Asn Gln Ile Ile
Gly Gly Lys Asn 1 5 10 152015PRTArtificial SequenceDescription of
Artificial Sequenceportion of NFL peptide from component I 20Thr
Ser His Tyr Ser Ala Ala Asn Ser Val Val Gly Gly Thr Asn 1 5 10
152113PRTArtificial SequenceDescription of Artificial
Sequenceeukaryotic aspartyl protease active site 21Val Asp Val Asp
Ser Gly Ser Ala Pro Ile Val Gly Phe 1 5 102212PRTArtificial
SequenceDescription of Artificial Sequenceportion of WSD peptide
from component II 22Val Gly Val Ser Ser Gly Ser Ala Pro Asp Ala Phe
1 5 102311PRTArtificial SequenceDescription of Artificial
Sequencecysteine protease of early leaf senescence 23Val Ala Thr
Cys Ser Ser Tyr Pro Val Val Ala 1 5 102411PRTArtificial
SequenceDescription of Artificial Sequenceportion of VDP peptide,
component unknown 24Val Asp Pro Ala Ser Gly Tyr Pro Ile Val Gly 1 5
10
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