U.S. patent application number 10/269643 was filed with the patent office on 2003-05-29 for neurogenic compositions and methods.
Invention is credited to Berezin, Vladimir, Bock, Elisabeth Marianne, Lukanidin, Eugene.
Application Number | 20030100503 10/269643 |
Document ID | / |
Family ID | 23554687 |
Filed Date | 2003-05-29 |
United States Patent
Application |
20030100503 |
Kind Code |
A1 |
Lukanidin, Eugene ; et
al. |
May 29, 2003 |
Neurogenic compositions and methods
Abstract
The present invention has found that the Mts1 protein is
expressed in white matter astrocytes in the spinal cord. Such
expression is significantly increased following sciatic nerve
injury or dorsal root injury, particularly in astrocytes
surrounding dorsal funiculus containing the central processes of
the injured primary sensory neurons. The present invention has
further demonstrated that Mts1 proteins administered
extracellularly promote neurite outgrowth from neuronal cells.
Based on these surprising findings, the present invention provides
compositions and methods that are useful for the treatment of
various neurological conditions characterized by death,
degeneration or injury of neuronal cells.
Inventors: |
Lukanidin, Eugene;
(Copenhagen, DK) ; Bock, Elisabeth Marianne;
(Charlottenlund, DK) ; Berezin, Vladimir;
(Copenhagen N., DK) |
Correspondence
Address: |
SCULLY SCOTT MURPHY & PRESSER, PC
400 GARDEN CITY PLAZA
GARDEN CITY
NY
11530
|
Family ID: |
23554687 |
Appl. No.: |
10/269643 |
Filed: |
October 11, 2002 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10269643 |
Oct 11, 2002 |
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09781509 |
Feb 12, 2001 |
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09781509 |
Feb 12, 2001 |
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09393433 |
Sep 10, 1999 |
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Current U.S.
Class: |
514/8.4 ;
514/16.4; 514/17.8; 514/18.2; 514/8.6; 514/9.1; 530/350 |
Current CPC
Class: |
A61P 19/00 20180101;
A61P 25/02 20180101; Y02A 50/465 20180101; A61P 9/00 20180101; C07K
14/475 20130101; A61K 38/00 20130101; A61P 25/28 20180101; A61K
48/00 20130101; A61P 25/16 20180101; A61P 25/00 20180101; Y02A
50/30 20180101; A61P 27/02 20180101; C07K 14/4728 20130101 |
Class at
Publication: |
514/12 ;
530/350 |
International
Class: |
A61K 038/18; C07K
014/475 |
Claims
We claim:
1. an isolated functional derivative of an Mts protein.
2. An isolated Mts1-del75.
3. An isolated Mts1-4S.
4. An isolated multimeric Mts1 protein complex, comprising at least
three Mts1 protein molecules.
5. The isolated multimeric Mts1 protein complex of claim 4, having
a Mw in the range of about 30 kD to about 200 kD.
6. The isolated multimeric Mts1 protein complex of claim 4, wherein
the Mts1 protein molecule is wild type.
7. The isolated multimeric Mts1 protein complex of claim 4, wherein
the Mts1 protein molecule is Mts1-del75.
8. The isolated multimeric Mts1 protein complex of claim 4, wherein
the Mts1 protein molecule is of a mammalian origin.
9. A pharmaceutical composition comprising the isolated functional
derivative of an Mts1 protein of claim 1, and a pharmaceutically
acceptable carrier.
10. A pharmaceutical composition comprising the isolated complex of
claim 4, and a pharmaceutically acceptable carrier.
11. The pharmaceutical composition of claim 9 or 10, wherein said
pharmaceutically acceptable carrier is liquid, semi-solid, or
solid.
12. The pharmaceutical composition of claim 9 or 10, further
comprising a neurotropic factor.
13. The pharmaceutical composition of claim 12, wherein said
neurotropic factor is selected from the group consisting of bFGF,
aFGF, CNTF, NGF, BDNF, GDNF, NT3, NT4/5, IGF-1 and IGF-II.
14. A method of stimulating growth of neuronal cells, comprising
administering an Mts1 protein or a functional derivative thereof to
said neuronal cells.
15. A method of treating a neurological condition in a subject,
wherein said neurological condition is characterized by neuronal
degeneration, death or injury, comprising administering to the
subject a therapeutically effective amount of an Mts1 protein or a
functional derivative thereof and a pharmaceutically acceptable
carrier.
16. A method of treating a neurological condition in a subject,
wherein said neurological condition is characterized by neuronal
degeneration, death or injury, comprising administering to the
subject a therapeutically effective amount of an Mts1
protein-encoding nucleic acid sequence and a pharmaceutically
acceptable carrier.
17. The method of claim 16, wherein said nucleic acid sequence is
provided in an expression vector.
18. The method of claim 16, wherein said expression vector is a
plasmid, retroviral, adenoviral, herpes simplex viral,
adeno-associated viral, polio viral or a vaccinia vector.
19. The method of claims 15 or 16, wherein said neurological
condition is Parkinson's disease, Alzheimer's disease, Down's
Syndrome, stroke, cardiac arrest, sciatic crush, spinal cord
injury, injury to sensory neurons, or degenerative disease of the
retina.
20. The method of claim 19, further comprising administering
simultaneously a neurotropic factor.
21. The method of claim 20, wherein said neurotropic factor is
selected from the group consisting of bFGF, aFGF, CNTF, NGF, BDNF,
GDNF, NT3, NT4/5, IGF-1 and IGF-II.
22. The method of claim 19, wherein the administration is via an
oral, ophthalmic nasal, topical, transdermal, intravenous,
intraperitoneal, intradermal, subcutaneous or intramuscular,
intracranial, intracerebral, intraspinal, intravaginal,
intrauterine, or rectal route.
23. The method of claim 19, wherein the administration is via
implantation.
Description
FIELD OF INVENTION
[0001] The present invention relates to the discovery of the role
of the Mts1/S100A4 protein in the neural system. Compositions and
methods are provided that are useful for stimulating growth of
neuronal cells and treating neuronal damage caused by disease or
trauma.
BACKGROUND OF THE INVENTION
[0002] The S100 proteins comprise a large family of calcium-binding
proteins, some of which are expressed at high levels in the nervous
system. The S100 proteins have been implicated in a wide variety of
functions, such as modulation of enzyme function, alteration of
cytoskeletal dynamics, cell adhesion and control of cell cycle
progression (Schafer et al., Trends Biochem Sci 21: 134-140, 1996).
Expression of S100 protein has been shown to be associated with
invasive potential and metastatic spread of tumor cells (Inoue et
al., Virchows Arch A422:351-355, 1993).
[0003] The primary structure of S100 proteins is highly conserved
(Kligman et al., TIBS 13: 437-443, 1988; and Schaefer et al., TIBS
21: 134-140, 1996). In solutions S100 proteins easily form dimers
and cystein residues are not necessary for the noncovalent
dimerization of S100 (Mely et al., J. Neurochemistry 55: 1100-1106,
1990; Landar et al., Biochim. Biophys. Acta 1343: 117-129, 1997;
and Raftery et al., J Am. Soc. Mass Spectrom. 9: 533-539, 1988).
The tertiary structure of S100 proteins has been characterized
(Kilby et al., Structure 4: 1041-1052, 1996; Smith et al.,
Structure 6: 211-222, 1998; Sastry et al., Structure 15: 223-231,
1998; and Matsumura et al, Structure 6: 233-241, 1998). Each S100
monomer contains two EF-hand calcium binding domains (Schafer et
al., TIBS 21: 134-140, 1996). Calcium binding results in a
conformational alteration and exposure of a hydrophobic patch via
which S100 proteins interact with their targets (Smith et al,
Structure 6: 211-222, 1998; Sastry et al, Structure 15: 223-231,
1998; Matsumura et al, Structure 6: 233-241, 1998; and Kilby et
al., Protein Sci. 6: 2494-2503, 1997).
[0004] Intracellular and extracellular activities of S100 proteins
have also been described (McNutt, J Cutan. Pathol. 25: 521-529,
1988). Intracellular S100 proteins interact with numerous target
proteins and modulate multiple cellular processes regulating cell
growth, differentiation, metabolism and cytoskeletal structure
(Zimmer et al., Brain Res. Bulletin 37: 417-429, 1995; Schafer et
al., TIBS 21: 134-140, 1996; Donato, Cell Calcium 12: 713-726,
1991; and Lukanidin et al., In: Gunter U, Birchmeier W, eds.
Current Topics in Microbiology and Immunology: Attempts to
Understand Metastasis Formation II. Berlin, Heidelberg:
Springer-Verlag 213/II, 171-195, 1996). Extracellular
disulfide-linked dimers of S100B protein have been reported to
stimulate neurite outgrowth in primary cultures of cerebral cortex
neurons (Kligman et al., TIBS 13: 437-443, 1988). Such activity has
also been reported for oxidized form of the recombinant S100B
protein (Winningham-Major et al., J. Cell Biol. 109: 3063-3071,
1989).
[0005] The mts1/S100A4 gene, a member of the S100 gene family, was
isolated as a gene specifically expressed in metastatic murine
tumor cell lines (Ebralidze et al., Genes Dev. 3: 1086-1092, 1989).
Studies of Mts1-transfected non-metastatic murine cell lines and
Mts1 transgenic mice both indicate that Mts1 plays an important
role in tumor progression (Grigorian et al., Gene 135: 229-238,
1993; Takenaga et al., Oncogene 14: 331-337, 1997; Ambartsumian et
al., Oncogene 13: 1621-1630, 1996; and Davies et al., Oncogene 13:
1631-1637, 1996). Mts1 has also been shown to affect the
cytoskelton and cell motility (Takenaga et al., Jpn. J Cancer Res.
85: 831-839, 1994) via association with stress fibers (Gibbs et
al., J. Biol. Chem. 269: 18992-18999, 1994). The heavy chain of
non-muscle myosin (MHC) has been identified as a target for the
Mts1 protein (Kriajevska et al., J. Biol. Chem. 239: 19679-19682,
1994).
[0006] The present invention identifies, for the first time, the
neurogenic function of the Mts1 protein. Accordingly, the present
invention provides novel compositions and methods useful for
stimulating neurite growth in the treatment of neural damage caused
by disease or physical trauma.
SUMMARY OF THE INVENTION
[0007] One embodiment of the present invention provides an isolated
functional derivative of an Mts1 protein. A preferred functional
derivative of an Mts1 protein is Mts1 del75.
[0008] Another embodiment of the present invention provides an
isolated multimeric Mts1 protein complex. Such complex includes at
least three Mts1 protein molecules or functional derivatives
thereof.
[0009] In another embodiment, the present invention provides
pharmaceutical compositions which include an isolated functional
derivative of an Mts1 protein, or a multimeric Mts1 protein
complex, and a pharmaceutically acceptable carrier. The
pharmaceutical compositions can also include one or more
neurotropic factors.
[0010] In a further embodiment, the present invention provides
methods of stimulating growth of neuronal cells by administering an
Mts1 protein or a functional derivative thereof.
[0011] In a further embodiment, the present invention provides
methods of treating neurological conditions in a subject by
administering to the subject a therapeutically effective amount of
an Mts1 protein or a nucleotide sequence encoding an Mts1 protein.
The methods of the present invention can be employed in the
treatment of a variety of neurological conditions characterized by
neuronal degeneration, neuronal death or injury caused by disease,
physical trauma or ischemic conditions. Such neurological
conditions include Parkinson's disease, Down's Syndrome,
Alzheimer's disease, stroke, cardiac arrest, sciatic crush, spinal
cord injury, damaged sensory neurons in dorsal root ganglia and
other tissues, as well as degenerative diseases of the retina.
BRIEF DESCRIPTION OF DRAWINGS
[0012] FIG. 1 depicts Mts1-immunoreactivity (IR) (A,B,E) and
GFAP-IR (C,D,F) in the normal white matter of LA. (A) shows Mts1-IR
in the ventral and lateral funiculi, with exclusive expression in
white matter. Double labeling with antibodies to Mts1 and GFAP
shows that Mts1 is localized to astrocytes (B,D) and is
predominantly expressed in the cell bodies (B), while intense
GFAP-IR is observed in processes as well (D). Arrowheads indicate
cells that were labeled with anti-GFAP antibodies (D), but not with
antibodies to Mts1 (B). (E) shows a few Mts1-positive cell bodies
as well as Mts1-positive processes in paramedian septa of the
dorsal funiculus in C3 (E), despite widespread GFAP-IR (F). Bar=200
.mu.m (A,C), 50 .mu.m (B,D), 100 .mu.m (E,F).
[0013] FIG. 2 depicts Mts1-IR (A) and GFAP-IR (B) in the dorsal
funiculus and adjacent dorsal horn (DH) of L4 two days after
unilateral transection of dorsal roots L4 and L5. There was a
marked increase in Mts1-positive cell bodies and processes (A) in
the white matter, and a concomitant increased expression of GFAP
(B) on the operated side (right), but no Mts1-IR in the dorsal horn
(DH). Bar=200 .mu.m.
[0014] FIG. 3 depicts Mts1-IR (A,C) and GFAP-IR (B,D) in the dorsal
funiculus of L4 one week (A,B) and two months (C,D) after
unilateral transection of L4 and L5 dorsal roots. There was a
marked upregulation in the expression of Mts1 (A,C) and GFAP (B,D)
on the operated side (op). The dorsal horn (DH) was completely
devoid of Mts1 staining (C), despite a prominent increase in
GFAP-IR (D). Bar=100 .mu.m.
[0015] FIG. 4 depicts increased Mts1-IR (A,C) and GFAP-IR (B,D) in
the gracile funiculus (A,B) and the dorsal funiculus of C3 (C,D)
one week after ipsilateral injury to the L4 and L5 dorsal roots.
Op=operated side. Bar=100 .mu.m.
[0016] FIG. 5 depicts double labeling with antibodies to Mts1 and
GFAP (A), and double labeling with antibodies to Mts1 and the
microglia/macrophage marker ED1 (B) in the degenerating dorsal
funiculus two months after transection of the L4 and L5 dorsal
roots. Mts1-IR (A,B,green) is confined to GFAP-positive astrocytes
(A,red), but completely absent from ED1-positive cells (B,red).
Bar=50 .mu.m.
[0017] FIG. 6 depicts Mts1-IR (A,C) and GFAP-IR (B,D) in the dorsal
funiculus of L4 one week (A,B) and two months (C,D) after
unilateral transection of the sciatic nerve. There was an increased
expression of Mts1 at both postoperative survival times (A,C).
Mts1-IR was absent from the dorsal horn (C,DH). GFAP-IR was
increased two months (D), but not one week (B) after injury
compared to the unoperated side. Op=operated side. Bar=100
.mu.m.
[0018] FIG. 7A is a phase contrast micrograph of a 24 h low-density
culture of dissociated hippocampal cells of rat embryos (E18).
[0019] FIG. 7B is a phase contrast micrograph of a 24 h low-density
culture of dissociated hippocampal cells of rat embryos (E18) grown
in the presence of 5 .mu.M recombinant Mts1/S100A4 protein.
[0020] FIG. 7C is a phase contrast micrograph of a 24 h low-density
culture of dissociated hippocampal cells of rat embryos (E18) grown
in the presence of 5 .mu.M recombinant His-tagged 200aa C-terminal
peptide of myosin heavy chain.
[0021] FIG. 8A depicts the dose-dependent effect of Mts1/S100A4 on
neurite outgrowth in primary cultures of dissociated rat
hippocampal cells. Cultures were grown in the presence of various
amounts of the recombinant protein for 24 h, and neurite length per
cell was measured.
[0022] FIG. 8B depicts the time-dependent effect of Mts1/S100A4 on
neurite outgrowth in primary cultures of dissociated rat
hippocampal cells. Hippocampal cells were seeded and allowed to
attach for 1 h after which recombinant Mts1/S100A4 was added to the
culture (time 0). At various time points afterwards, Mts1/S100A4
was removed by changing culture medium and neurite length per cell
was measured 24 h after addition of the protein.
[0023] FIG. 8C depicts the specificity of the Mts1/S100A4 effects
on neurite outgrowth in primary cultures of dissociated rat
hippocampal cells. Hippocampal cells were grown for 24 h in the
presence of 5 .mu.M Mts1/S100A4 and rabbit polyclonal anti-Mts1
antibodies at various dilutions. The length of neurites in treated
cultures is expressed as a percentage of the length of neurites in
control cultures.
[0024] FIG. 9A depicts the effects of Mts1/S100A4, S100.beta., NGF
and FGF on neurite outgrowth from hippocampal neurons. Cultures
were grown for 24 h in the absence or in the presence of
Mts1/S100A4, S100.beta..beta., NGF or FGF at indicated
concentrations. Results of a typical experiment are shown.
[0025] FIG. 9B depicts the effects of Mts1/S100A4, S100.beta., NGF
and FGF on neurite outgrowth from PC12-E2 cells. Four individual
experiments were performed. Results are given as mean .+-.SEM.
[0026] FIG. 10 depicts the neurogenic effects of the wild type and
mutatnt Mts1/S100A4 proteins. Hippocampal cells were grown for 24 h
in the presence of 5 .mu.M mouse recombinant Mts1/S100A4 or in the
presence of 5 .mu.M of the Mts1 mutated proteins. The length of
neurites in treated cultures is expressed as a percentage of the
length of neurites in control cultures. Four individual experiments
were performed. Results are given as mean .+-.SEM.
[0027] FIGS. 11A-11C depict the profiles of the recombinant wild
type (wt) Mts1 protein (11A) and two mutants, Y75F (11B) and del75
(11C) off size exclusion chromatography (SEC). One milliliter of
each protein (2 mg/ml) was chromatographed on a Superdex G75
column. The column was equilibrated with TND, eluted (1 ml/min)
with the same buffer and 3-ml fractions were collected. Results of
a typical experiment are shown. Relative positions of peak I, II
and III are indicated with respect to molecular weight markers:
thyroglobulin--670 kDa; bovine gamma globulin--158 kDa; chicken
ovalbumin--44 kDa; equine myoglobin--17 kDa. Inserts--fractions of
each peak were combined and assayed for neurite outgrowth activity
on hippocampal cells. The length of neurites in treated cultures is
expressed as a percentage of the length of neurites in control
cultures.
[0028] FIG. 11D depicts Commassie Blue staining of SDS-PAGE (a) and
Western blotting analysis (b) of peaks I, II and III of wt Mts1.
Immuno-staining was performed with affinity purified antibodies
against Mts1. Lanes 1-4--peak I (fractions 3-6); Lanes 5-7--peak II
(fractions 8-10); Lanes 8-10--peak III (fractions 14-16).
DETAILED DESCRIPTION OF EMBODIMENTS
[0029] The Mts1/S100A4 protein is known in the art to be involved
in the control of cell proliferation and metastasis of tumor cells.
The present inventor has surprisingly discovered a function of the
Mts1/S100A4 protein that is associated with the nervous system.
[0030] Specifically, it has been discovered by the present inventor
that the Mts1 protein is expressed in white matter astrocytes in
the spinal cord. In accordance with the present invention, it has
also been found that sciatic nerve injury as well as dorsal root
injury induces a marked and prolonged increase in the level of the
Mts1 protein, particularly in astrocytes surrounding dorsal
funiculus containing the central processes of the injured primary
sensory neurons. Additionally, the present invention demonstrates
that Mts1 proteins administered extracellularly promote neurite
outgrowth from neuronal cells.
[0031] Accordingly, the present invention employs the neurogenic
activity of the Mts1 protein and provides compositions and methods
that are useful for the treatment of various neurological
conditions characterized by the death, degeneration or injury of
neuronal cells.
[0032] By "neurogenic activity" is meant a biological activity that
induces, stimulates, or enhances the growth, maintains the
survival, or prevents the death of the neuronal cells of the
central and peripheral nervous system of a mammal. The activity can
manifest as differentiation of neurons, extension of neuritic
processes (i.e., outgrowth or elongation of neurites), or
innervation of neuritic processes into a tissue.
[0033] One embodiment of the present invention provides an isolated
functional derivative of an Mts1 protein.
[0034] "An Mts1 protein" as used herein, refers to a wild type Mts1
protein of a mammalian origin, such as human, murine and the like.
Preferred Mts1 proteins of the present invention include human Mts1
(SEQ ID NO: 1) and murine Mts1 (SEQ ID NO: 2), which are also
described in U.S. Pat. No. 5,801,142 and Ebralidze et al., Genes
Dev. 3: 1086-1092, 1989, respectively.
[0035] "A functional derivative of an Mts1 protein" refers to a
modified Mts1 protein having one or more amino acid substitutions,
deletions or insertions, which retains substantially the neurogenic
activity of a wild type Mts1 protein. By "substantially" is meant
at least about 35%, preferably, at least about 40%.
[0036] In accordance with the present invention, a preferred
functional derivative of a wild type Mts1 protein is Mts1-del75,
i.e., deletion of the Tyr residue at the position 75 in human or
murine Mts1 protein, or the corresponding Tyr in any other
mammalian Mts1 proteins. It has been determined by the present
inventor that Mts1-del75 is able to form polymers and confers about
70% neurogenic activity compared to a wild type Mts1 protein.
Another Mts1 mutant which has all four Cysteine residues mutated to
Serine (designated herein as "4S") retains about 40% of the
neurogenic activity of a wild type Mts1 protein.
[0037] Those skilled in the art can use any of the well-known
molecular cloning techniques to generate Mts1 derivatives having
one or more amino acid substitutions, deletions or insertions. See,
for example, Current Protocols in Molecular Cloning (Ausubel et
al., John Wiley & Sons, New York). Once a modified Mts1 protein
is made, such protein can be tested in functional assays to
determine whether such modified protein exhibits neurogenic
activity.
[0038] In accordance with the present invention, the neurogenic
activity of an Mts1 protein or protein complex can be determined by
a number of assays. A typical functional assay is described in
Example 2 hereinbelow. Briefly, an Mts1 protein is added in various
doses in the culture medium of neuronal cells, such as hippocampal
neuronal cells, or PC-12 cells. The cells can be kept exposed to
the protein for a certain period of time and the outgrowth of
neurites from the cultured cells are monitored. Parameters such as
the length of the longest neurite extension, the number of neurite
branches per cell, and the total neurite length per cell, are
measured. The determination as to whether a modified Mts1 protein
possesses neurogenic activity can be made by comparing these
parameters with those values of a wild type Mts1 protein and those
values of a control protein without neurogenic activity. Other
assays which can be employed for such determination include, e.g.,
the standard assay of endothelial cell motility in Boyden
Chamber.
[0039] Another embodiment of the present invention provides an
isolated multimeric Mts1 protein complex.
[0040] In accordance with the present invention, it has been found
that the neurogenic activity of Mts1 is associated with the
polymeric forms composed of three or more Mts1 protein molecules.
Not intending to be bound by any theory, it is proposed herein that
the Mts1 protein mediates its neurogenic effects via a cell surface
receptor which recognizes polymeric forms of the Mts1 protein.
[0041] According to the present invention, the terms "a multimeric
Mts1 protein complex" and "a polymeric Mts1 protein complex" as
used herein refer to a complex having at least three, i.e., three
or more, molecules of an Mts1 protein or a functional derivative of
an Mts1 protein. The complex can have a Mw of at least about 30 kd,
more preferably, at least about 100 kd, and up to about 200 kd, as
determined by, e.g., size-exclusion chromatography.
[0042] In accordance with the present invention, the Mts1 protein
molecules in the complex can be held together by covalent and/or
non-covalent interactions among Mts1 protein molecules. For
example, there are four Cys residues in both human and murine Mts1,
which can form intramolecular disulfide bonds under appropriate
conditions thereby leading to formation of polymeric Mts1
complexes. The present invention also contemplates polymeric Mts1
complexes formed by chemical cross-linking reagents. Chemical
cross-linking reagents and use thereof in making multimeric protein
complexes are well known in the art. In accordance with the present
invention, a Mts1 protein complex having neurogenic activity can be
formed through non-covalent interactions among Mts1 molecules as
well. For example, the present invention provides that Mts1-4S,
while unable to form any intramolecular or intermolecular disulfide
bonds, is able to form polymers and confers neurogenic activity at
a level of about 40% of that of a wild type Mts1 protein.
[0043] The Mts1 complexes of the present invention can be isolated
by a variety of methods. For example, an Mts1 protein can be
dissolved in solution under conditions that favor the formation of
polymers, e.g., a saline solution of about 0.15 M NaCl, pH7.5 with
a Mts1 concentration higher than, preferably, 1 mg/ml. Afterwards,
the solution can be subjected to an appropriate chromatography
procedure using, e.g., Size-Exclusion-Column euqilibrated with a
TND buffer (50 mM Tris-HCl, 150 mM NaCl, 1 mM DTT, pH 7.5). The
Mts1 protein can be eluted using the same TND buffer, and fractions
containing polymers can be collected and separated from the
fractions containing dimers. Such procedure is described in Example
3 hereinbelow. An Mts1 protein can also be subjected to chemical
cross-linking prior to chromatography or fraction procedures. Those
skilled in the art can make modifications when appropriate and
necessary.
[0044] In another embodiment, the present invention provides
pharmaceutical compositions which include a functional derivative
of an Mts1 protein, or an isolated multimeric Mts1 protein complex
composed of at least three Mts1 protein molecules.
[0045] The pharmaceutical compositions of the present invention can
be employed to promote neuronal cell growth or maintain the
survival of neuronal cells in the treatment of neurological
conditions characterized by the death, degeneration or injury of
neuronal cells.
[0046] The functional derivative or the protein complex of an Mts1
protein for use in the pharmaceutical compositions can be modified
according to procedures known in the art in order to enhance
penetration of the blood-brain barrier. For example, U.S. Pat. No.
5,604,198 discloses that a molecule can be conjugated to a
hydrophobic carrier which enhances the permeability of the blood
brain barrier (BBB). WO 90/14838 teaches chemical modifications of
a protein by increasing lipophilicity, altering glycosylation or
increasing the net positive charge in order to enhance the BBB
permeability of the protein.
[0047] According to the present invention, the pharmaceutical
compositions can also include one or more neurotropic factors.
[0048] Neurotropic factors are proteins which promote the survival
of neurons, some of which are also capable of promoting neurite
outgrowth and glial cell restoration or inducing cells to secrete
other neurotropic factors. Preferred neurotropic factors for use in
the present pharmaceutical compositions are those to which a broad
range of cell types respond. Examples of preferred neurotropic
factors include members of the BDNF/NGF family, such as bFGF (basic
fibroblast growth factor), aFGF (acidic fibroblast growth factor);
CNTF (ciliary neurotrophic factor), NGF (nerve growth factor), BDNF
(brain-derived neurotrophic factor), GDNF (glial cell line-derived
neurotrophic factor), NT-3 (neurotrophin-3), NT-4/5 (neurotrophin
4/5), IGF-1 (insulin growth factor-I), IGF-II (insulin growth
factor-II), and functional peptide fragments thereof. Human
neurotropic factors and functional derivatives are preferred.
[0049] The active ingredients of the pharmaceutical compositions
are preferably provided in a pharmaceutically acceptable carrier.
The carrier can be liquid, semi-solid, e.g. pastes, or solid
carriers. Except insofar as any conventional media, agent, diluent
or carrier is detrimental to the recipient or to the therapeutic
effectiveness of the active ingredients contained therein, its use
in the pharmaceutical compositions of the present invention is
appropriate. Examples of carriers include oils, water, saline
solutions, gel, lipids, liposomes, resins, porous matrices,
binders, fillers and the like, or combinations thereof. The carrier
can also be a controlled release matrix which allows a slow release
of the active ingredients mixed or admixed therein. Examples of
such controlled release matrix material include, but are not
limited to, sustained release biodegradable formulations described
in U.S. Pat. No. 4,849,141 to Fujioka et al., U.S. Pat. No.
4,774,091 to Yamashira, U.S. Pat. No. 4,703,108 to Silver et al.,
and Brem et al.(J. Neurosurg. 74: 441-446, 1991), all of which are
incorporated herein by reference.
[0050] In accordance with the present invention, a Mts1 functional
derivative or an Mts1 polymeric complex can be combined with the
carrier in solutions or in solid phase, preferably in a manner that
favors the stablization of the polymeric conformation of the Mts1
protein. If the mixing step is to be performed in liquid phase,
Mts1 proteins can be dissolved together with a carrier in solutions
such as saline (about 0.15M NaCl pH7.5) with an Mts1 concentration
of higher than, preferably, 1 mg/ml. If the mixing is to be
performed in solid phase, the Mts1 polymeric proteins can be
freeze-dried first to preserve the polymeric conformation, then
admixed with the carrier. The mixture can be made in formulations
suitable for injections, implantations, inhalations, ingestions and
the like.
[0051] In a further embodiment, the present invention provides
methods of stimulating growth of neuronal cells by administering an
Mts1 protein, a functional derivative of an Mts1 protein, or a
protein complex thereof, to such neuronal cells.
[0052] According to the present invention, an Mts1 protein or a
functional derivative or complex thereof, can be administered to
neuronal cells that are cultured in vitro. This aspect of the
invention is particularly useful in regeneration of neurons for
autotransplantation or neuron replacement as an alternative
treatment procedure to brains of patients with neurological
disorders. Techniques of culturing neurons in vitro fare known in
the art and are described in, e.g., U.S. Pat. Nos. 5,483,892,
5,753,506, 5,898,066, and 5,667,978, Mou et al. J. Comp. Neurol.
386: 529 (1997), and Tan et al. Cell Transplant 5: 577 (1996), the
teachings of which are incorporated herein by reference.
[0053] In a further embodiment, the present invention provides
methods of treating neurological conditions in a subject by
administering to the subject a therapeutically effective amount of
an Mts1 protein, a functional derivative thereof, or a nucleotide
sequence encoding an Mts1 protein.
[0054] The methods of the present invention can be employed in the
treatment of a variety of neurological conditions characterized by
neuronal degeneration, neuronal death or injury caused by disease,
physical trauma or ischemic conditions. Such neurological
conditions include Parkinson's disease, Alzheimer's disease, Down's
Syndrome, stroke, cardiac arrest, sciatic crush, spinal cord
injury, multiple sclerosis, peripheral neuropathies associated with
diabetes, motorneuron diseases, damaged sensory neurons in dorsal
root ganglia and other tissues, as well as degenerative diseases of
the retina.
[0055] By "treating" is meant prevent or inhibit neuronal
degeneration or neuronal death, promoting or stimulating neuronal
growth such that the symptoms of the disease condition are
prevented or alleviated.
[0056] In accordance with the methods of the present invention, an
Mts1 protein can be first treated to enrich the polymeric forms, or
can be used directly, as certain percentage of the molecules
spontaneously associate with each other to form polymers in
solution. An Mts1 protein or a functional derivative thereof can be
modified in order to enhance penetration of the blood-brain barrier
as described hereinabove.
[0057] Nucleic acid sequences encoding an Mts1 protein can also be
employed in the methods of the present invention. Such sequences
are preferably provided in an expression vector. Expression vectors
for use in the present methods include any appropriate gene therapy
vectors, such as nonviral (e.g., plasmid vectors), retroviral,
adenoviral, herpes simplex viral, adeno-associated viral, polio
viruses and vaccinia vectors. Examples of retroviral vectors
include, but are not limited to, Moloney murine leukemia virus
(MoMuLV), Harvey murine sarcoma virus (HaMuSV), murine mammary
tumor virus (MuMTV), and Rous Sarcoma Virus (RSV)-derived
recombinant vectors. Multiple teachings of gene therapy are
available to those skilled in the art, e.g., W. F. Anderson (1984)
"Prospects for Human Gene Therapy" Science 226: 401-409; S. H.
Hughes (1988) "Introduction" Current Communications in Molecular
Biology 71: 1-12; T. Friedman (1989) "Progress Toward Human Gene
Therapy" Science 244: 1275-1281 and W. F. Anderson (1992) "Human
Gene Therapy" Science 256: 608-613. Preferred vectors include
neurotropic vectors such as herpes simplex viral vectors (U.S. Pat.
No. 5,673,344 to Kelly et al. and adenoviral vectors (Barkats et
al., Prog. Neurobiol. 55: 333-341, 1998).
[0058] Mts proteins or Mts1-encoding nucleic acid molecules can be
used alone or in conjunction with one or more neurotropic factors
described hereinabove, including members of the BDNF/NGF family
such as bFGF, aFGF, CNTF, NGF, BDNF, GDNF, NT3, NT4/5, IGF-1 and
IGF-II, as well as the functional peptide fragments identified
thereof. Human neurotropic factors are preferred for treating a
human subject.
[0059] The therapeutically active ingredients, i.e., Mts1 proteins
or nucleic acid molecules, alone or in conjunction with neurotropic
factors, can be combined with a pharmaceutically acceptable carrier
and prepared in formulations suitable for injections,
implantations, inhalations, ingestions and the like.
Pharmaceutically acceptable carriers are described hereinabove and
include oils, water, saline solutions, gel, lipids, liposomes,
resins, porous matrices, binders, fillers and the like, or
combinations thereof.
[0060] According to the present invention, these therapeutic
compositions can be administered to the subject being treated by
standard routes, including the oral, ophthalmic nasal, topical,
transdermal, parenteral (e.g., intravenous, intraperitoneal,
intradermal, subcutaneous or intramuscular), intracranial,
intracerebral, intraspinal, intravaginal, intrauterine, or rectal
route. Depending on the condition being treated, one route may be
preferred over others, which can be determined by those skilled in
the art. For example, topical route can be chosen when the target
area includes tissues or organs readily accessible by topical
application, such as neurological conditions of the eye or the
facial tissue. For certain conditions, direct injection or surgical
implantation in the proximity of the damaged tissues or cells may
be preferred in order to avoid the problems presented by BBB.
Successful delivery to CNS (Central Nervous System) by direct
injection or implantation has been documented. See, e.g., Otto et
al., J. Neurosci. Res. 22: 83-91 (1989); Goodman & Gilman's The
Pharmacological Basis of Therapeutics, 6.sup.th ed, pp244; Williams
et al., Proc. Natl. Acad. Sci. USA 83: 9231-9235 (1986); and Oritz
et al., Soc. Neurosci. Abs. 386: 18 (1990).
[0061] According to the present invention, the therapeutic
ingredients are preferably administered to the subject in need
thereof as early as possible after the neuronal injury or death
occurs in order to achieve the best therapeutic efficacy.
[0062] The amount of an Mts1 protein, a functional derivative, or
an Mts1-encoding nucleic acid molecule to be therapeutically
effective depends on the disease state or condition being treated
and other clinical factors, such as weight and physical condition
of the subject, the subject's response to the therapy, the type of
formulations and the route of administration. The precise dosage to
be therapeutically effective and non-detrimental to the subject can
be determined by those skilled in the art. As a general rule, the
therapeutically effective amount of Mts1 protein can be in the
range of about 0.01 mg to about 10 mg per kilogram of body weight;
preferably, in the range of about 0.1 mg to about 5 mg per kilogram
of body weight. The therapeutically effective dosage of an Mts1
protein can be in the range of about 0.5 .mu.g to about 2 mg per
unit dosage form. A unit dosage form refers to physically discrete
units suited as unitary dosages for mammalian treatment: each unit
containing a pre determined quantity of the active material
calculated to produce the desired therapeutic effect in association
with any required pharmaceutical carrier. The methods of the
present invention contemplate single as well as multiple
administrations, given either simultaneously or over an extended
period of time.
[0063] This invention is further illustrated by the following
examples, which are not to be construed in any way as imposing
limitations upon the scope thereof. The terms and expressions which
have been employed in the present disclosure are used as terms of
description and not of limitation, and there is no intention in the
use of such terms and expressions of excluding any equivalents of
the features shown and described or portions thereof. It is to be
understood that various modifications are possible within the scope
of the invention. All the publications mentioned in the present
disclosure are incorporated herein by reference.
EXAMPLE 1
Mts1 Expression is Up-Regulated After Peripheral or Dorsal Root
Injury
[0064] Introduction of the Experimental Model
[0065] The primary sensory neurons of the spinal cord with their
cell bodies located peripherally, send out dichotomizing processes,
one branch projecting peripherally to innervate peripheral tissues
and organs, the other branch entering the CNS via spinal dorsal
roots. Dorsal root axons terminate in a specific pattern in the
gray matter of the dorsal horn. In addition, collaterals of
myelinated primary sensory axons ascend in the dorsal funiculus of
the white matter to the lower brainstem where they terminate in the
dorsal column nuclei.
[0066] Injury to the dorsal root (rhizotomy) and injury to the
peripheral branches produce markedly different morphological and
molecular changes in the affected neurons. However, both injuries
are associated with prominent responses in surrounding non-neuronal
cells in the CNS, particularly astrocytes and
microglia/macrophages. Injury to the peripheral branches, e.g. by
section of the sciatic nerve, induces degenerative as well as
growth-associated changes (transganglionic changes) in the central
terminals and axons of the injured neurons (Aldskogius et al.,
Oxford Univ Press. pp 363-383, 1992; Woolf et al., Neurosci 34:
465-4678, 1990; and Woolf et al., J Comp Neurol 360: 121-134,
1995). concomitantly, microglial cells proliferate (Gehrmann et
al., Restor Neurol Neurosci 2: 181-198, 1991; Eriksson et al., Exp
Brain Res 114: 393-404, 1993; and Persson et al., Primary Sensory
Neuron 1: 47-64, 1995), and express various inflammatory mediators
(Liu et al., Neurosci 68: 167-179, 1995), while astrocytes
upregulate the expression of their major intermediate filament,
glial fibrillary acidic protein (GFAP) (Gilmore et al., Glia
3:342-349, 1990) but do not proliferate. Injury to the central
primary sensory process by section of the dorsal root, results in
complete disintegration (Wallerian degeneration) of the segment of
the axon no longer in continuity with the parent cell body. The
non-neuronal response to this degeneration includes proliferation
of microglia, that gradually develops into macrophages, as well as
proliferation of astrocytcs and a rapid increase in the expression
of GFAP in astrocytes (Liu et al., Glia 23:221-238, 1998).
[0067] Materials and Methods
[0068] Thirty-two adult, female, Sprague-Dawley rats (160-180 g
body weight) were used for the study. Prior to surgery and
perfusion, animals were anaesthetized with chloral hydrate (35
mg/kg body weight i.p.).
[0069] Twelve animals were subjected to section of the left sciatic
nerve at midthigh level. Two animals (n=2) were analyzed at each
postoperative survival time (1 day, 2 days, 3 days, 7 days, 1 month
and 2 months). In 18 animals, the left lumbar dorsal roots L4 and
L5 were exposed via a partial laminectomy and sectioned close to
the corresponding dorsal root ganglia (n=3 for each postoperative
survival time). At the indicated postoperative survival time, the
animals were perfused via the left ventricle first with saline
(37.degree. C.) followed by a solution of 4% formaldehyde (w/v) and
14% saturated picric acid (v/v) in a 0.15M phosphate buffer (pH
7.4, 4.degree. C.). Two intact control animals were perfused in the
same way. The L4-LS and C3 spinal cord segments as well as the
brainstem were removed, postfixed for about one and half hours, and
subsequently stored overnight in refrigerator. Serial, 14 .mu.m
transverse sections were cut on a cryostat and processed for
immunofluorescence. In addition, sets of sections were cut at 5
.mu.m to provide material for optimal microphotography.
[0070] Sections were briefly air-dried and washed in phosphate
buffer for 5-10 mins prior to incubation in BSA and 0.3% Triton
X100 (Sigma, USA).for one hour at room temperature. Sections were
incubated overnight at 4.degree. C. with antibodies against Mts1
(rabbit polyclonal, 1:1000). The immune complex was visualized with
FITC-conjugated sheep anti-rabbit IgG (Jackson, 1:40). For double
labeling experiments, anti-Mts1 antibodies were combined with one
of the following antibodies: (1) anti-GFAP (astrocytes, mouse
monoclonal (Serotec, U.K.), 1:3), (2) Ox42 (microglia, mouse
monoclonal (Serotec, U.K.), 1:600), or (3) ED1 (phagocytic
microglia/macrophages, mouse monoclonal (Serotec, U.K.), 1:400).
The cell marker antibodies were visualized with rhodamine
(TRITC)-conjugated anti-mouse IgG. Sections were viewed and
photographed in a Nikon Eclipse fluorescence microscope equipped
with filter for simultaneous examination of FITC and TRITC
fluorescence.
[0071] Intact Control Animals
[0072] Mts1 immunoreactivity (IR) was observed in the white matter
of the L4 and C3 segments of the spinal cord as well as in the
brainstem. The most prominent staining appeared in the ventral and
lateral funiculi as processes radiating from the subpial region and
towards the gray matter, leaving, however, its immediate white
matter surroundings free from Mts1-IR (FIGS. 1, A and B). Mts1-IR
cell bodies were typically located in the subpial region as well as
about midway between this region and the gray matter. Double
labeling with glial cell markers showed colocalization between Mts1
and anti-GFAP (FIGS. 1, B and D), but a minority of GFAP-positive
cells was not labeled with Mts1. However, astrocytes which did
express Mts1, showed a more complete labeling of their cell bodies
with anti-Mts1 than with anti-GFAP. Conversely, GFAP-IR processes
were usually only partially labeled with anti-Mts1 (FIGS. 1, B and
D).
[0073] The levels Of Mts1-IR were considerably lower in the dorsal
funiculus of L4-L5 and C3 as well as in the dorsal white matter of
the brainstem compared to the ventral and lateral funiculi. Only
some Mts1-positive profiles were observed (FIG. 1, E); there was no
apparent difference in GFAP staining (FIG. 1, F).
[0074] Dorsal Root Injury
[0075] Since the uninjured and injured sides of the spinal cord
were next to each other, changes in Mts1-IR as a result of sciatic
nerve or dorsal root transaction could be unambiguously identified.
The first sign of an upregulation of Mts1-IR in the L4 dorsal
funiculus was observed two days after dorsal rhizotomy (FIG. 2, A).
This was paralleled by an increased staining for GFAP in the same
area (FIG. 2, B). At this state, large Mts1-positive cells appeared
in the area occupied by the injured primary sensory axons in the
dorsal funiculus. The difference between the degenerating zone in
the dorsal funiculus and the uninjured white matter gradually
became stronger with increasing postoperative survival time (FIGS.
3, A and B), and was very intense at two months after injury (FIGS.
3, C and D). Importantly, the gray matter, including the dorsal
horn termination area of the injured primary afferents, was always
Mts1 negative, despite a marked up-regulation of GFAP-IR in the
termination sites of the injured primary afferent fibers (FIGS. 3,
B and D).
[0076] Increased immunoreactivity for Mts1 and GFAP also appeared
along the central processes of the injured lumbar primary sensory
afferents in the dorsal column of C3 and in the gracile nucleus. At
one week after rhizotomy Mts1-IR was up-regulated concomitantly
with GFAP-IR in the gracile funiculus and nucleus in the lower
brainstem (FIGS. 4, A and B) and in C3 in the circumscribed area of
the dorsal funiculus containing the degenerating ascending primary
sensory afferents (FIGS. 4, C and D).
[0077] Double labeling with markers for Mts1 and for astrocytes
(GFAP) or for microglia/macrophages (antibodies OX42 or ED1),
showed overlap between Mts1-IR and GFAP-IR in the dorsal funiculus
(FIG. 5, A), but none between Mts1- and OX42 or ED1-IR (FIG. 5,
B).
[0078] Sciatic Nerve Injury
[0079] Mts1-IR in the ipsilateral dorsal funiculus was upregulated
first at one week after sciatic nerve injury (FIG. 6, A) and showed
a gradually increasing expression with longer survival times.
However, at this postoperative time there was no increase in
GFAP-IR (FIG. 6, B) in the dorsal funiculus, although there was an
upregulation in the dorsal horn. The extent of Mts1-IR was never as
great after sciatic nerve injury as after dorsal root lesions, even
at the longest postoperative survival time of two months, when it
coincided with an increased GRAP-IR (FIGS. 6, C and D). The
upregulation of Mts1 was always confined to the somatotopically
appropriate area for sciatic nerve afferents in the dorsal
funiculus, and did not include its most dorsomedial part, occupied
by uninjured ascending sacral primary afferents, nor its
ventralmost part occupied by the corticospinal tract. The gray
matter was always free from Mts1-IR, despite an upregulation of
GFAP-IR (FIGS. 6, C and D). Double labeling with antibodies to Mts1
and with glial cell markers showed colocalization only with
antibodies to GFAP (cf. FIGS. 6, C and D).
EXAMPLE 2
Recombinant Mts1 Protein Stimulates Neurite Outgrowth in vitro
[0080] Murine Mts1 protein sequence was described by (Ebralidze et
al., Genes Dev. 3, 1086-1092, 1989). cDNA fragments encoding the
murine Mts1 protein and mutant Mts1 proteins containing a single
mutation Y75F, a tyrosine deletion (del75) or cysteine/serine
substitutions (4S) were cloned into pQE30 expression vector
(QIAGEN, Inc., CA) and partially sequenced. Expression of
recombinant His.sub.6-tagged proteins was induced by
isopropy-1-thio-.beta.-D-galactopyranoside, and bacterial lysates
were used for isolation of proteins according to the the
manufacturer's protocol. Proteins were separated on SDS-PAGE,
followed by Western blot analysis as described by Kriajevska et al.
(J. Biol. Chem. 273: 9852-9856, 1998).
[0081] Hippocampus was isolated from Wistar rat embryos at
gestational day 18 and dissociated cells were obtained as descried
by Maar et al. (J. Neurosci.Res. 47: 163-172, 1997). Briefly,
hippocampal tissue was homogenized, trypsinized and washed in the
presence of DNAse I and trypsin inhibitor. Hippocampal cells were
seeded in 8-well LabTek coverslides at a density of
5.times.10.sup.3 cells/CM.sup.2, maintained in neurobasal medium
supplemented with B27 supplement, 4 mg/ml bovine serum albumin
(BSA), penicillin (100 U/ml) and streptomycin (100 .mu.g/ml). Cells
were grown for 24 h in a humidified atmosphere with 5%
C0.sub.2.
[0082] The neurogenic effect of Mts1 was analyzed-by
computer-assisted morphometry. The embryonic hippocampal neurons of
18-day rats were cultured with and without the Mts1 protein at low
cell density in serum free defined medium. Cells were then fixed in
4% paraformaldehyde and stained for 20 min in Commassie blue R250
(4 g/l in 45% v/v ethanol and 45% v/v acetic acid). Coverslides
were observed in a Nikon Diaphot 300 inverted microscope using
phase contrast optics (Nikon Plan 20.times.). Video recording was
made with a CCD video camera (Burle, USA). 512.times.512 pixel
images were stored in a computer using the PRIGRA software package
(Protein Laboratory, University of Copenhagen). To measure neurite
outgrowth from hippocampal neurons a simple procedure developed at
the Protein Laboratory and based on stereological principles was
used. Briefly, by means of the software package "ProcessLenghth"
(Protein Laboratory, University of Copenhagen), an unbiased
counting frame containing a grid with a certain number of
test-lines was superimposed on images of the cell cultures. The
number of intersections of cellular processes with the test-lines
was counted and related to the number of cell bodies, thereby
allowing qualification of the total neurite length per cell by
means of the equation, L=.pi./2.times.d.times.- J, in which L is
the neuritic length in micrometers, d is the vertical distance
between two test lines and J is the number of intersections between
the test lines and the neurites.
[0083] It was observed that hippocampal neurons cultured without
the Mts1 protein did not differentiate by extending processes (FIG.
7A). Treatment of hippocampal neurons with the recombinant
His-tagged wt Mts1 protein of 5 .mu.M for 12 hours had a robust
effect on their differentiation (FIG. 7B). Neurons extended
multiple, long branching processes. Cell cultures treated with the
recombinant His-tagged 200aa C-terminal peptide of the myosin heavy
chain (Kriajevsta et al., J. Biol. Chem 273: 9852-56, 1998) for 24
h, revealed minimal morphological changes in comparison to control
cultures (FIG. 7C).
[0084] The stimulation of neurites outgrowth by the recombinant
Mts1 protein was time- and dose-dependent. Mts1 was effective in
the micromolar concentration range, with the maximal
growth-stimulatory activity being 5-10 .mu.M (FIG. 8A). Mts1
treatment increased the total length of neurite per cell when
compared to the control, as well as the number of neurites (7
fold), the length of the longest neurite (14 fold) and the number
of branches (25 fold) per cell (Table 1).
1TABLE 1 Neurite Induction in Hippocampal Neurons in Vitro
Following Treatment with the Recombinant Mouse Mts1/S100A4 Protein
Total neurite Length of the length per longest neurite Neurite
Neurites cell per cell branches per cell (.mu.M) (.mu.M) per cell
Control 0.29 .+-. 0.06 12.6 .+-. 1.3 3.43 .+-. 0.5 0.013 .+-. 0.01
Mtsl 2.12 .+-. 0.3 93 .+-. 17 49.5 .+-. 1.5 0.36 .+-. 0.08 (5
.mu.M)
[0085] The duration of the Mts1 protein treatment required for
hippocampal cells to extend neurites was also determinaed. In these
experiments, Mts1 was added at the time (time 0) when seeded cells
were allowed to attach for 1 h. At various time points Mts1 was
removed by changing culture medium, and neurite outgrowth was
measured 24 h later. Cells exposed to the Mts1 protein for 15-30
min already displayed a 4-fold increase in the total length of
neurites when compared to control cells. The response of cells
exposed to Mts1 for more that 1.5 h was obvious and
indistinguishable after further incubation for 4, 6, 16 or 24 h,
respectively (FIGS. 8B). These data indicate that continuous
exposure of cells to Mts1 for 24 h is not required and that there
is an early period, approximately 1-1.5 hour, when the presence of
Mts1 is essential for the maximal neurite outgrowth.
[0086] The specificity of Mts1 neurogenic activity was tested by
examining the activity of the Mts1 protein after incubation with
antibodies to Mts1. The Mts1 protein was mixed with serial
dilutions of polyclonal anti-Mts1 antibodies in growth medium,
incubated for 1 h and applied to hippocampal cells. FIG. 8C shows
that incubation of Mts1 with antibodies directed against Mts1
reduced the neurite extension in a reverse proportion to the
antibodies dilutions. Incubation of Mts1 with control IgG, anti
nonmuscle myosin or normal rabbit serum, did not reduce the
response.
[0087] The neurogenic activity of Mts1 was compared with the
activities of other neurotrophic growth factors, including FGF
(Fibroblast Growth Factor), NGF (Nerve Growth Factor) and members
of S100 Ca.sup.2+-binding protein--S100.alpha. and S100.beta..
Neurite outgrowth from hippocampal neurons was not stimulated by
FGF, NGF or S100.beta. (FIGS. 9A). Treatment with S100.alpha. did
not affect hippocampal cultures either. Moreover, NGF actually
inhibited neurite outgrowth at high concentrations (5-10
.mu.M).
[0088] To assess the possibility that lack of responsiveness of
hippocampal cells to FGF, NGF, and S100.beta. reflected cell
specific activity of these neurotropic factors, PC-12 cells were
tested. As shown in FIG. 9B, Mts1 and S100.beta. showed equal
neurite outgrowth stimulatory activity in the PC-12 cells. As shown
in FIG. 9B, Mts1 and S100.beta. showed equal neurite outgrowth
stimulatory activity in the PC-12 cell system that was twice as
high compared with that in the hippocampal cells. In contrast,
neurite extension effect of FGF and NGF on cultured PC-12 cells was
significantly higher that on hippocampal cells. The data indicate
that the stimulatory effects of different neurotrophic factors are
cell specific, and Mts1 is a potent activator of neurites outgrowth
of hippocampal cells.
EXAMPLE 3
Structural Requirements for the Mts1 Neurite Outgrowth Promoting
Activity
[0089] To determine the structural elements in the Mts1 protein
that are required for promoting neurite outgrowth, three Mts1
mutatant proteins were tested. In one of the mutants, Tyrosine75
was substituted to Phenylalanine (Y75F). In the other mutant
Tyrosine75 was deleted (del75). It was found that Del75 could not
form dimers in the yeast two-hybrid system, while the Y75F mutant
formed perfect dimers in the yeast with an efficiency even higher
than wt Mts1.
[0090] When these two mutant Mts1 proteins were tested in the in
vitro system of cultured hippocampal cells, it was found that Y75F
did not stimulate neurite outgrowth from hippocampal cells. In
contrast, cells incubated with del75 for 24 h displayed abundant
neurites, although the degree of neurite outgrowth was generally
lower than that obtained with the wild type Mts1 (FIG. 10).
[0091] To examine whether disulfide bonds contribute to the
neurogenic activity of the Mts1 protein, the Mts1 mutant termed 4S
was used, in which all four cysteins (at positions 76,81,86 and 93)
of the Mts1 protein were changed to serines. It was found that 4S
was able to form dimers in the yeast two hybrid system, but unable
to interact with the heavy chain of myosin in a gel overlay assay.
When tested for the ability to stimulate neurite outgrowth, the 4S
mutant showed 40% of the neurogenic activity of that of wt Mts1
(FIG. 10).
[0092] In order to determine which conformational forms of Mts1
were active with regard to neurogenic activity, size-exclusion
chromatography (SEC) of the recombinant Mts1 and the Mts1 mutants
were performed. A Superdex75 column (1.5 cm.sup.2.times.90.0 cm)
was equilibrated with a TND buffer (50 mM Tris-HCI, 150 mM NaCl, 1
mM DTT, pH 7.5) with and without 5 mM CaCl.sub.2. The column was
calibrated for molecular weight determinations using gel filtration
chromatography standard (Bio-Rad). The standard proteins included
Vitamin B-12 (MW 1.35 kDa), equine myoglobin (MW 17.0 kDa), chicken
ovalbumin (MW 44.0 kDa), bovine gamma globulin (MW 158.0 kDa),
thyroglobulin (MW 670.0 kDa). 1 ml of the mixed proteins standard
(2 mg/ml) was loaded onto the column and 3 ml fractions were
collected and monitored with A.sub.280 readings. Dextran blue was
applied to the column to determine its void volume. The K.sub.av
values were determined for each protein and plotted versus the log
of the molecular weight of the standard
K.sub.av=(V.sub.e-V.sub.0)/(V.sub.t-V.su- b.o) (V.sub.e is the
elution volume at the peak apex, V.sub.o is the void volume, and
V.sub.t is the total column volume; see Landar et al., Biochim.
Biophys. Acta 1343: 117-129, 1997).
[0093] 1ml samples of the Mts1 protein or mutants were applied onto
the column and a K.sub.av value was determined in each case. The
molecular weight of Mts1 was determined by comparing its K.sub.av
value to those found for the standard proteins. Gel filtration
chromatography experiments were performed under different
conditions: presence of reducing agent, 2 mM calcium or 2 mM EDTA,
0.5M or 0.15M NaCl. The fractions were assayed by both SDS-PAGE and
the neurite outgrowth test. Under either condition, the eluted
material showed a broad profile of distribution with molecular
masses ranging approximately from 30 to 200 kDa. (FIG. 11A).
Approximately half of the recombinant wild type Mts1 protein was
eluted as a high molecular weight complex. The distinct peak of a
dimer was consistently detected among different batches of freshly
prepared recombinant Mts1, whereas the elution profile of a higher
molecular mass material was less reproducible and varied in
different Mts1 preparations.
[0094] The elution profile of the Y75F mutant was different as
shown in FIG. 11B. 85% of the Y75F protein was eluted from gel
filtration columns as a single peak with a molecular weight of a
dimer, and 15% as materials of higher molecular weights ranging
from 30 to 100 kDa (FIG. 11B). The elution profile of the mutant
del75 was different from either the wild type Mts1 or the Y75F
mutant protein. Major part of the del75 protein was eluted as
materials of high molecular weights ranging approximately from 40
kDa to 200 kDa.
[0095] It was further found that the elution profiles of all
proteins were not influenced by alterations in the Ca++
concentration, nor by changes from reducing to non-reducing
conditions, nor by changes in ionic strength.
[0096] Different fractions eluted from the column, named peaks I,
II and III for all three tested proteins, were analyzed for the
presence of Mts1 by Coomassie staining and Western blot analysis
(FIG. 11D). The Mts1 protein under reducing condition yielded one
11 kDa band in all analyzed fractions. Western blot analysis with
affinity purified antiserum confirmed the Mts1 origin of the bands
described as monomer. SDS gel patterns of the two mutant proteins
were similar to Mts1.
[0097] The relative contribution to the neurogenic activity of
different forms of the Mts1 protein, eluted from the SEC column as
peaks I, II and III, was tested. Inserts in FIGS. 11A-11C show that
high molecular weight complexes (100-200 kDa ) of wt Mts1 as well
as del75 mutant stimulated neurite outgrowth. Peak I demonstrated
the highest activity. The neurogenic activity of the protein in
peak II was less reproducible and accounted for 30% of the activity
observed in the peak I. Dimeric forms (peak III) of wt Mts1 and two
Mts1 mutants (Y75F and 4S) showed no activity at any dose tested.
The data indicate that the ability to stimulate neurite extension
is attributed to the polymeric fraction of the Mts1 molecules with
unidentified structural conformations.
[0098] In order to monitor the polymerization of Mts1 and to
determine the molecular weight of the polymers more precisely, the
recombinant Mts1 protein was analyzed by Dynamic Light Scattering,
a standard technique for determination of the molecular weight of
globular proteins (Berne et al., Dynamic Light Scattering, Chap.5,
Wiley, New York, 1976). The diffusion coefficient (D.sub.t) and
calculated molecular weight were determined with DLS using Dyna Pro
801 Molecular Sizing Instrument (Protein Solutions Inc.). All
readings were recorded at 18.degree. C. All samples were filtrated
through a 0.02 .mu.m membrane (Whatman) before measurements.
Protein solutions were injected into a 25 .mu.l cell (cuvette) and
illuminated by a 25W 750 nm wave length laser. Data were fitted
with the Dynamics Version 4.0 software package. The molecular
weight (M.W.) was calculated by two alternative models. According
to the first model, M.W. was estimated from the hydrodynamic radius
(R.sub.h) using an empirically derived relationship between the
R.sub.h, and M.W. values for a number of well-characterized
globular proteins in a buffered aqueous solution, assuming that the
protein holds a standard globular shape and density. In the second
model, the volume-shape-hydration relationship was used, in which
model the calculation required the values of the hydrodynamic size,
partial specific volume, and frictional ratio. (The value of
partial specific volume (V) is 0.707 in the absence of Ca.sup.2+
and V increases when Ca.sup.2+ is added (Mani et al., FEBS Lett.
166, 258-262, 1984). The value of frictional ratio (f) is 1.45 and
f decreases when Ca.sup.2+ is added (Matsuda et al., Biochem, and
Mol. Biol. International 30, 419-424, 1993). In Table 2 it can be
seen that the recombinant Mts1 protein at a concentration 1.5 mg/ml
had a broad spectrum of molecular weights ranging from 28.9 kDa for
dimer, 47.2 kDa for tetramer, and up to 143.0-200.0 kDa for
polymeric molecules.
2TABLE 2 Dynamic Light Scattering Parameters D.sub.t R.sub.h M.W
(kDa) M.W (kDa) Oligomeric State (le-9*cm/s{circumflex over ( )}2)
nm First Model Second Model Dimers 785 2.56 28.9 nd Tetramers 636
3.16 47.2 nd Oligomers 398 4.99 143.0 200.0
[0099]
Sequence CWU 1
1
2 1 101 PRT Homo sapiens 1 Met Ala Cys Pro Leu Glu Lys Ala Leu Asp
Val Met Val Ser Thr Phe 1 5 10 15 His Lys Tyr Ser Gly Lys Glu Gly
Asp Lys Phe Lys Leu Asn Lys Ser 20 25 30 Glu Leu Lys Glu Leu Leu
Thr Arg Glu Leu Pro Ser Phe Leu Gly Lys 35 40 45 Arg Thr Asp Glu
Ala Ala Phe Gln Lys Leu Met Ser Asn Leu Asp Ser 50 55 60 Asn Arg
Asp Asn Glu Val Asp Phe Gln Glu Tyr Cys Val Phe Leu Ser 65 70 75 80
Cys Ile Ala Met Met Cys Asn Glu Phe Phe Glu Gly Phe Pro Asp Lys 85
90 95 Gln Pro Arg Lys Lys 100 2 101 PRT Mus musculus 2 Met Ala Arg
Pro Leu Glu Glu Ala Leu Asp Val Ile Val Ser Thr Phe 1 5 10 15 His
Lys Tyr Ser Gly Lys Glu Gly Asp Lys Phe Lys Leu Asn Lys Thr 20 25
30 Glu Leu Lys Glu Leu Leu Thr Arg Glu Leu Pro Ser Phe Leu Gly Lys
35 40 45 Arg Thr Asp Glu Ala Ala Phe Gln Lys Val Met Ser Asn Leu
Asp Ser 50 55 60 Asn Arg Asp Asn Glu Val Asp Phe Gln Glu Tyr Cys
Val Phe Leu Ser 65 70 75 80 Cys Ile Ala Met Met Cys Asn Glu Phe Phe
Glu Gly Cys Pro Asp Lys 85 90 95 Glu Pro Arg Lys Lys 100
* * * * *