Note: Descriptions are shown in the official language in which they were submitted.
CA 02304108 2000-03-08
WO 99/12559 PCT/US98/19216
-1_
INHIBITION OF APOPTOSIS
USING PROSAPOSIN RECEPTOR AGONISTS
BACKGROUND OF THE INVENTION
1. Field of the Invention
This invention relates generally to apoptosis, and more specifically to the
use
of prosaposin receptor agonists to inhibit apoptosis upregulation of
downstream cellular
signaling molecules, such as Akt and Bcl-2 that act to inhibit caspase-
mediated
apoptosis.
2. Background
Prosaposin is the precursor of a group of four heat-stable glycoproteins that
are required for glycosphingolipid hydrolysis by lysosomal hydrolases.
Prosaposin, a 70
kilodalton (kDa) glycoprotein, is proteolytically processed to generate
saposins A, B, C,
and D. The saposins exist as 4 tandem domains in prosaposin before
proteolysis. All 4
saposins are structurally similar to each other, having a similar placement of
six
cysteines, a glycosylation site and conserved proline residues. Unprocessed
prosaposin
also exists as an integral membrane protein and as a secreted protein that is
present in
human milk, cerebrospinal fluid and seminal plasma.
Prosaposin, saposin C, and prosaposin-derived peptides (prosaptides) have
therapeutic applications in promoting functional recovery after toxic,
traumatic,
myocardial ischemic, degenerative and inherited lesions to the peripheral and
central
nervous system. See, U.S. Patent No. 5,571,787. Prosaposin and prosaptides can
also be
used to counteract the effects of demyelinating diseases by inducing neurite
outgrowth
stimulating myelination. The neurotrophic and myelinotrophic activity of
prosaposin has
been localized to amino acids 18-29 of saposin C.
Tumor necrosis factor a (TNFa) is a proinflammatory cytokine. TNFa
induces a proinflammatory response in many disorders, including rheumatoid
arthritis,
Crohn's disease, irritable bowel syndrome, asthma, stroke cardiac infarction,
and
CA 02304108 2000-03-08
WO 99/12559 PCT/US98/19216
-2-
congestive heart failure. After TNFa therapy was identified as a potential
therapeutic
target for rheumatoid arthritis, antibodies to TNFa were shown to be effective
in both
animal models and human patients. A similar approach was taken in animal
models of
inflammatory bowel disease. In another animal model of inflammatory pain,
injection of
TNFa into the subperineural space in the sciatic nerve immediately proximal to
the
sciatic notch produces neuropathic pain in vivo. The results of behavioral
testing of
either mechanical or thermal hyperalgesia showed that TNFa-injected animals
displayed
a significant hyperalgesia compared to vehicle-injected animals, in which
hyperalgesia
lasted for 5 days. Thus, the role of TNFa in various diseases has been
established.
TNFa also induces programmed cell death (apoptosis) in several neural cell
types, including cortical neurons, oligodendrocytes, and oligodendrocyte
precursor cells.
Apoptosis accounts for most of the programmed cell death in tissue remodeling
and for
the cell loss that accompanies atrophy of adult tissues following withdrawal
of endocrine
and other trophic factor stimuli. However, abnormal apoptosis is responsible
for many
human diseases after injury, including traumatic, chemical, myocardial
ischemic, and
genetic causes.
The proinflammatory cytokine interferon y (IFNy) is a potent inducer of
oligodendrocyte apoptosis. Oligodendrocyte apoptosis has been observed at the
advancing margins of chronic active multiple sclerosis (MS) plaques. IFN~y may
therefore be a factor in the pathogenesis of multiple sclerosis by activating
apoptosis in
oligodendrocytes. Generally, proinflammatory cytokines such as TNFa and IFNy
are
likely factors in the abnormal apoptosis underlying the pathogenesis in many
demyelination disorders.
There is currently no effective treatment for the many diseases associated
with
abnormal apoptosis due to various causes.
SUMMARY OF THE INVENTION
The present invention provides a method for using prosaposin receptor
agonists to inhibit apoptosis. Of particular interest is inhibition of
apoptosis associated
with caspase activation. Caspase activation resulting in apoptosis may be
induced, for
CA 02304108 2000-03-08
WO 99/I2559 PCT/US98/19216
-3-
example, by proinflammatory cytokines, as well as by Alzheimer's disease,
stroke,
myocardial ischemia, increased intracellular Ca++ levels, and increased levels
of the
neurotransmitter glutamate. The invention is thus useful for treating a
proinflammatory
cytokine-induced disease, such as multiple sclerosis, rheumatoid arthritis,
irritable bowel
S syndrome, AIDS neuropathy and encephalitis, progressive multifocal
leukoencephalitis,
chronic myocardial atrophy, Alzheimers disease, and cell death of any type due
to
cytokine-induced apoptosis. One mechanism whereby prosaposin receptor agonists
inhibit proinflammatory cytokine-induced apoptosis is by activation of the
serine/threonine protein kinase Akt. Akt dissociates complexes of Bcl-2 family
members,
such as BAD-Bcl-2, releasing Bcl-2 and its family members which inhibit
caspases,
thereby inhibiting apoptosis. Thus, the activation (phosphorylation) of Akt by
the action
of prosaposin receptor agonists is a key event in the prevention of caspase-
mediated
apoptosis. The inhibition of apoptosis by prosaposin receptor agonists is a
unique method
of inhibiting apoptosis, because many other inhibitors of apoptosis inhibit
caspase-
mediated apoptosis at stages of the caspase proteolytic cascade different from
the stage
influenced by prosaposin receptor agonists. Thus, the use of prosaposin
receptor agonists
to inhibit caspase-mediated apoptosis represents a significant new function
for these
compositions.
An additional mechanism whereby prosaposin receptor agonists inhibit
apoptosis is by blocking activation of JNK, a proapoptotic signaling
component. Within
several minutes after binding to the receptor, prosaposin receptor agonists
block JNK
activation induced by TNFa. The activation of JNK by TNFa is another well
known
mechanism for TNFa-induced, as well as other proinflammatory cytokine-induced,
apoptosis.
The invention provides a method for inhibiting JNK-mediated and caspase-
mediated apoptosis by contacting cells at risk of such apoptosis with an
apoptosis-
inhibiting amount of a prosaposin receptor agonist. The cells may be contacted
in vivo
or ex vivo. In one embodiment, the cells are oligodendrocytes, neurons,
Schwann cells,
or myocytes.
CA 02304108 2000-03-08
WO 99/12559 PCT/US98/19216
-4-
In another embodiment, the prosaposin receptor agonist has at least about 11
amino acids and comprises the amino acid sequence
LeulleXaa,AsnAsnXaa,ThrXaa ~aa ~aa ~aa ,,wherein Xaa ~s any amino acid; Xaa
His
a charged amino acid; and Xaa3 is optionally present and, when present, is a
charged
amino acid. In another embodiment, the prosaposin receptor agonist is a
peptide selected
from SEQ ID N0:2, SEQ D) N0:3, SEQ ID N0:4, SEQ ID NO:S, SEQ m N0:6, SEQ
ID N0:7, SEQ ID N0:8, SEQ ID N0:9, SEQ ID N0:10, SEQ ID NO:11, and SEQ ID
N0:12.
BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1 is the polynucleotide sequence of human prosaposin cDNA.
FIG. 2 is the polypeptide sequences of prosaposin and saposin C.
FIG. 3 is the polypeptide sequence of several prosaposin-derived peptides.
FIG. 4 illustrates that the survival factor-promoted activation of Akt
requires
PI 3-kinase. Survival factor binding to the cognate receptor activates PI 3-
kinase and
other kinases. PI 3-kinase activates the serine/threonine kinase Akt.
Subsequently, Akt
phosphorylates specific targets, including the Bcl-2 family member BAD.
Phosphorylation inactivates BAD, causing other BCL-2 family members to inhibit
cell
death (apoptosis) and allow cell survival. Survival factor binding to the
cognate receptor
also activates MAPK to promote cell survival.
FIG. 5 illustrates that prosaposin receptor agonist TX14(A) binding to
prosaposin receptor acts to inhibit caspace-mediated apoptosis.
Proinflammatory
cytokine TNFa binds to TNF-R to activate adaptor molecules, such as TRADD.
TRADD
activates the caspase proteolytic cascade, causing apoptosis. Prosaposin
receptor agonist
binding to prosaposin receptor activates PI 3-kinase and other kinases. PI 3-
kinase
activates the serine/threonine kinase Akt. Subsequently, Akt phosphorylates
specific
targets, including the Bcl-2 family member BAD. Phosphorylation inactivates
BAD,
causing other BCL-2 family members to inhibit cell death (apoptosis) and allow
cell
survival.
CA 02304108 2000-03-08
WO 99/12559 PCT/US98/19216
-5-
FIG. 6 shows that (A) prosaposin and (B) prosaposin receptor agonist
TX14(A) prevent TNFa-induced viability loss in NS20Y cells. NS20Y cells were
incubated for 48 hr in DMEM containing 0.5% fetal bovine serum {FBS) and 100
ng/ml
TNFa t 2-fold dilutions of prosaposin (0 - 5 nM) or prosaptide {0 - 50 r1M).
Cell
viability was assessed using MTT reduction. Results are mean ~ SEM. Asterisk
(*)
indicates that mean is significantly different to TNFa treated cells; p<0.05.
FIG. 7 shows the time course of TNFa-induced viability loss and prevention
by prosaposin receptor agonists. NS20Y cells were incubated in DMEM containing
0.5%
FBS without (solid bar) or with (hatched bar) 100 ng/ml TNFa and 5 nM
prosaposin
(grey bar) or 50 nM prosaptide (hollow bar) for 48-96 hours. MTT was used to
assess
cell viability. Results are mean ~ SEM. Asterisk (*) indicates that mean is
statistically
different to TNFa treated cells; p<0.05.
FIG. 8 shows that prosaposin receptor agonist TX14(A) prevents
TNFa-induced death of NS20Y cells. NS20Y cells were treated with TNFa in DMEM
containing 0.5% FBS with or without increasing doses of prosaptide. At 48 hr
cells were
stained with trypan blue to assess cell death. Results are mean t SEM.
C=control,
T=TNFa.
FIG. 9 shows that prosaposin receptor agonist TX14(A) does not cause
proliferation of NS20Y cells. NS20Y cells were seeded at 10,000/well in 96-
well plates
and grown in DMEM containing 0.5% FBS and 2-fold dilutions of prosaptide (P;
solid
bar) or in DMEM containing 2-fold dilutions of FCS (S; hatched bar). Cell
proliferation
was assessed at 48 hr by measuring BrdU incorporation. Data are mean ~ SEM.
FIG. 10 shows inhibition of JNK2 phosphorylation in primary Schumann cells
by a prosaposin derived peptide, TX 14(A). Schwann cells were stimulated for S
minutes
with TNFa +/- TX14(A). Equal amounts of proteins from cell lysates were
analyzed by
SDS-PAGE and inununoblotted using a polyclonal antibody that recognizes
phosphorylated JNK2 (Promega, Madison, WI). Proteins were detected by ECL
{Amersham, Arlington Heights). Autoradiographs were scanned using ImageQuantTM
(Molecular Dynamics, Sunnyvale, CA). Data are shown as representative data of
two
independent experiments.
CA 02304108 2000-03-08
WO 99/12559 PCT/US98/19216
-6-
FIG. 11 shows inhibition of p 110 poly(ADP-ribose), PARP, cleavage by
TX14(A). Primary Schwann cells were placed in low serum media (0.25% FBS) for
1
hour +/- TX14(A). Equal amounts of proteins from cell lysates were analyzed by
SDS-
PAGE and immunoblotted using a polyclonal antibody rhar recognizes PARP
(Upstate
Biotechnology, Lake Placid, Nl~. Proteins were detected by ECL (Amersham,
Arlington
Heights). Autoradiographs were scanned using ImageQuantTM (Molecular Dynamics,
Sunnyvale, CA). Data are expressed as a mean ratio of pl 10 to p85 PARP ~SEM
of two
independent experiments.
FIG. 12 shows the effect of various doses of TX14(A) peptide (prosaptide;
SEQ ID N0:7) on TNFa-induced Schwann cell death. The peptide concentration is
shown on the x-axis and the percentage of trypan blue-stained cells is shown
on the y-
axis.
FIG. 13 shows the effect of prosaposin and TX14(A) (SEQ ID N0:7) on
proinflammatory cytokine-induced cell death in undifferentiated CG4
oligodendrocytes.
FIG. 13A shows the effect of prosaposin and TX14(A) (SEQ ID N0:7) on TNFa-
induced cell death in undifferentiated CG4 oligodendrocytes. FIG. 13B shows
the effect
of prosaposin and TX14(A) on IFN~y-induced cell death in undifferentiated CG4
oligodendrocytes.
FIG. 14 shows that prosaposin receptor agonist TX14(A) inhibits
proinflammatory cytokine TNFa-induced apoptosis in L6 myoblasts. L6 myoblasts
cells
were incubated for 96 hours either in media (control); media with 10 ng/ml
TNFa (TNF
Category); or media with 10 ng/ml TNFa and 200 ng/ml TX14(A). Cell death was
measured by trypan blue assay.
FIG. 15 is a chart depicting the effect of prosaptide in vivo on thermal
hyperalgesia following endoneurial injection of TNFa. Prosaptide (200 ~.g/kg)
was
injected subcutaneously 3 hr before injection of 10 ul TNFa (2.5 pg/ml).
CA 02304108 2000-03-08
WO 99/12559 PCTlUS98/19216
_7_
DETAILED DESCRIPTION OF THE INVENTION
The present invention provides a method for inhibiting apoptosis. At a
fundamental level, the invention provides a method for inhibiting caspase-
mediated
apoptosis using a prosaptide, prosaposin, or saposin C. Caspases can be
activated by
several factors, including cytokines, anticancer drugs, growth factor
deprivation,
myocardial ischemia, metabolic toxins, and Ca++ toxicity. The method of the
invention
involves administering an apoptosis-inhibiting amount of a prosaposin receptor
agonist
to cells.
As used herein, the term "prosaposin receptor agonist" refers to a molecule
that binds to any site on a cell to which prosaposin can bind, and to thereby
alter the
cell's fiznction in the same manner to prosaposin. Examples of prosaposin
receptor
agonists include prosaposin, prosaptides, and saposin C. A receptor agonist is
a substance
that mimics the receptor ligand, is able to attach to that receptor, and
thereby produces
a same action that the ligand usually produces. Drugs are often designed as
receptor
agonists to treat diseases and disorders caused when the ligand, such as a
hormone, is
missing or depleted in a subject.
Prosaposin is a 70 kDa glycoprotein that is the precursor of a group of 4
small
heat-stable glycoproteins that are required for hydrolysis of
glycosphingolipids by
lysosomal hydrolases. Prosaposin is a 517 amino acid protein, originally
identified as the
precursor of4 sphingolipid activator proteins, as described in U.S. Patent No.
5,571,787.
Four adjacent tandem domains in prosaposin are proteolytically processed in
lysosomes
to generate saposins A, B, C, and D, that activate hydrolysis of
glycosphingolipids by
lysosomal hydrolases. The unprocessed form of prosaposin is found in high
concentrations in human and rat brain, where it is localized within neuronal
surface
membranes. During embryonic development, prosaposin mRNA is abundant in brain
and
dorsal root ganglia. Furthermore, prosaposin binds with high affinity to
gangliosides, to
stimulate neurite outgrowth, and promote transfer of gangliosides from
micelles to
membranes.
Prosaposin receptor agonists can be identified both structurally and
functionally. A prosaposin receptor agonist has a structure that is similar to
the region
CA 02304108 2000-03-08
WO 99/12559 PCT/US98/19216
_g_
of prosaposin that, when bound to the prosaposin receptor, induces a
prosaposin receptor
activity. For example, the prosaposin receptor agonist can have a structure
that is similar
to the amino acid sequence LeuIleXaa,AsnAsnXaa,ThrXaazXaa3XaazXaal, where Xaa~
is any amino acid; Xaa2 is a charged amino acid; and Xaa3 is optionally
present and,
when present, is a charged amino acid. Functionally, a prosaposin receptor
agonist
induces a prosaposin receptor activity, for example, second messenger
signaling, neurite
outgrowth or myelination, decreased neuropathic pain, inhibition of
proinflammatory
cytokine-induced apoptosis, or inhibition of apoptosis caused by other agents.
In one embodiment, the prosaposin agonist is prosaposin itself. The
prosaposin may be either prosaposin from native sources or prosaposin that is
produced
by recombinant methods, such as recombinant human prosaposin purified from
spent
media of Spodoptera frugiperda (Sf9) cells infected with a baculovirus
expression vector
containing full-length cDNA for human prosaposin. Human prosaposin has the
amino
acid sequence set forth in SEQ ID N0:2. The human cDNA sequence for prosaposin
is
IS SEQ ID NO:1. When the subject to be treated is human, human prosaposin and
saposin
sequence may more particularly be used.
In another embodiment, the prosaposin agonist is saposin C. The term
"saposin C" refers to the proteolytic cleavage product from the third tandem
domain of
prosaposin. Saposin C can be isolated in pure form from spleens of patients
with Gaucher
disease, a lysosomal storage disorder, by the method of Morimoto et al. (Proc.
Natl.
Acad. Sci. USA, 87: 3493-3497, 1990). Human saposin C has the amino acid
sequence
set forth in SEQ ID N0:3.
The prosaposin agonist is a peptide including amino acids 18-28 of saposin
C. The term "prosaptide" includes a peptide comprising amino acids 18-28 of
saposin C
(SEQ ID N0:4), peptides that have the activity of prosaptide comprising amino
acids
18-28 of saposin C, or conservative variations of these amino acid sequences
that retain
a bioactivity of amino acids 18-28 of saposin C. A "conservative variation,"
as used
herein, denotes the replacement of an amino acid residue by another,
biologically similar
residue. The term "conservative variation" also includes the use of a
substituted amino
acid in place of an unsubstituted parent amino acid provided that antibodies
raised to the
CA 02304108 2000-03-08
WO 99/12559 PCT/US98/19216
_g_
substituted polypeptide also immunoreact with the unsubstituted polypeptide.
Generally,
only conservative amino acid alterations are undertaken, using amino acids
that have the
same or similar properties. Illustrative amino acid substitutions include the
changes of
alanine to serine; arginine to lysine; asparagine to glutamine or histidine;
aspartate to
S glutamate; cysteine to serine; glutamine to asparagine; glutamate to
aspartate; glycine to
proline; histidine to asparagine or glutamine; isoleucine to leucine or
valine; leucine to
valine or isoleucine; lysine to arginine, glutamine, or glutamate; methionine
to leucine
or isoleucine; phenylalanine to tyrosine, leucine or methionine; serine to
threonine;
threonine to serine; tryptophan to tyrosine; tyrosine to tryptophan or
phenylalanine;
valine to isoleucine or leucine. Further, deletion of one or more amino acids
can also
result in a modification of the structure of the resultant molecule without
significantly
altering its activity. This can lead to the development of a smaller active
molecule. Such
variations are encompassed by the present invention. An active octadecamer (18
amino
acid) peptide fragment is set forth as SEQ ID NO:S. An active docosanamer (22
amino
acid) peptide fragment is set forth as SEQ ID N0:6.
Thus, prosaptides of the invention have a length of at least about 11 amino
acid residues, for example, at least about 14 amino acid residues. Prosaptides
of the
invention comprise about 80 or fewer amino acid residues, for example, no more
than
about 40 amino acid residues or no more that about 22 amino acid residues.
In another embodiment, the prosaposin receptor agonist is a prosaptide
which has about 11 amino acids to about 80 amino acids (the full-length of
saposin C)
and the amino acid sequence LeuIleXaa,AsnAsnXaa,ThrXaa2Xaa3XaazXaa,Xaa,,
where Xaa, is any amino acid; Xaa2 is a charged amino acid; and Xaa3 is
optionally
present and, when present, is a charged amino acid. For example, the
prosaposin
receptor agonist may be a prosaposin-derived peptide. The prosaposin receptor
agonist may have the polypeptide sequence of SEQ ID N0:2, SEQ ID N0:3, SEQ ID
N0:4, SEQ ID NO:S, SEQ ID N0:6, or SEQ ID N0:7. The polypeptide sequence
LeuIleAspAsnAsnLysThrGluLysGluIleLeu (SEQ ID N0:4) corresponds to amino
acids 18 to 29 of saposin C. The polypeptide sequence:
CysGluPheLeuValLysGluValThrLysLeuIleAspAsnA.snLysThrGluLysGluIleLeu
CA 02304108 2000-03-08
WO 99/12559 PCT/US98/19216
- 10-
ID N0:6) corresponds to amino acids 8 to 29 of saposin C. The polypeptide
sequence
ThrDAlaLeuIleAspAsnAsnAlaThrGluGluIleLeuTyr (SEQ ID N0:7) corresponds to
amino acids 16 to 29 of saposin C rriodified by a D-alanine for lysine
substitution at
position 2; an alanine for lysine substitution at position 8; a deletion of
lysine at position
11 and the addition of a C-terminal tyrosine residue. See, TABLE 1. Such
modifications
can be useful for increasing peptide stability or uptake across the blood-
brain barrier as
described in EXAMPLE 6. As used herein, D-alanine can be represented by D-Ala
or X.
TABLE I
PEPTIDE SEQUENCE SEQ ID NO:
Prosaposin-derived 22-mer CEFLVKEVTKLIDNNKTEKEIL 6
Prosaposin-derived 14-mer TXLIDNNATE-EILY 7
where X=v-alanine
Prosaposin-derived 11-mer LIDNNKTEKEI I 4
I
The prosaposin receptor agonist can also be an active fragment derived from
another mammalian prosaposin. As used herein, the term "active fragment of
prosaposin"
is synonymous with "prosaptide." For example, an active fragment of mouse
prosaposin,
rat prosaposin, guinea pig prosaposin or bovine prosaposin such as SEQ ID NOS:
8
through 11 is a prosaposin receptor agonist.
The amino acid sequence of an active fragment of human prosaposin, that
corresponds to amino acids 8 to 29 of saposin C (docosanomer; SEQ ID N0:6), is
well
conserved among other species, as shown in TABLE 2. In particular, adjacent
asparagine
(1~ residues are conserved among human, mouse, rat, guinea pig and bovine
prosaposins.
In addition, a leucine (L) residue is conserved 3 to 4 residues toward the N-
terminus of
the 2 asparagine residues and one or more charged residues (aspartic acid (D),
lysine (K),
glutamic acid (E) or arginine (R)) are conserved 2 to 8 residues toward the C-
terminus
of the 2 asparagine residues. Each of these well-conserved residues is
underlined in
TABLE 2.
CA 02304108 2000-03-08
WO 99/12559 PCT/US98/19216
-11-
TABLE 2
SPECIES SEQUENCE SEQ ID NO:
Human CEFLVKEVTKLIDNNKTEKEIL 6
Mouse CQFVMNKFSE_LIVNNATE~ELLY 8
S Rat CQLVNRKLSELI1NNATE-EELL 9
Guinea Pig CEYVVKKVMLLIDNNRTEEKIT 10
Bovine CEFV VKEVAKLIDNNRTEEEIL 11
In another embodiment, the prosaposin receptor agonist is selected from a
population of peptides related in amino acid sequence to SEQ ID N0:6 by having
the
conserved asparagine residues, a leucine/isoleucine residue, and one or more
charged
residues at the positions corresponding to the positions in which these
residues are found
in SEQ ID N0:6, but also having one or more amino acids that differ from the
amino
acids of SEQ ID N0:6.
A prosaposin receptor agonist can be identified by screening a large
collection, or library, of random peptides or peptides of interest using
assays that detect
prosaposin receptor agonist function, for example, one of a number of animal
models of
apoptosis or inflammation known to those of skill in the art.
A prosaposin receptor agonist can be isolated or synthesized using methods
well known in the art. Such methods include recombinant DNA methods and
chemical
synthesis methods for production of a peptide. Recombinant methods of
producing a
peptide through expression of a nucleic acid sequence encoding the peptide in
a suitable
host cell are well known in the art and are described, for example, in
Sambrook et al.
(Molecular Cloning: A Laboratory Manual, 2nd Ed., Vols. 1 to 3, Cold Spring
Harbor
Laboratory Press, New York, 1989).
A prosaposin receptor agonist also can be produced by chemical synthesis,
for example, by the solid phase peptide synthesis method of Mernfield et al.
(J. Am.
Chem. Soc. 85:2149, 1964). Standard solution methods well known in the art
also can
be used to synthesize a peptide useful in the invention. See, for example,
Bodanszky
(Principles ofPeptide Synthesis, Springer-Verlag, Berlin, 1984) and Bodanszky
(Peptide
CA 02304108 2000-03-08
WO 99/12559 PCT/US98/1921b
- 12-
Chemistry, Springer-Verlag, Berlin, 1993). The chemically synthesized peptide
may be
prepared on an Applied Biosystems Model 430 peptide synthesizer using an
automated
solid-phase protocol provided by the manufacturer. Peptides may then be
purified by
high performance liquid chromatography (HPLC) on a Vydac C4 column to an
extent
S greater than 95%. A newly synthesized peptide can be purified, for example,
by high
performance liquid chromatography (HPLC), and characterized using, for
example, mass
spectrometry or amino acid sequence analysis.
A particularly useful modification of a prosaposin receptor agonist is one
that
confers, for example, increased stability, by incorporation of one or more D-
amino acids
or substitution or deletion of lysine can increase the stability of a
prosaposin receptor
agonist by protecting against peptide degradation. For example, as disclosed
herein, the
prosaposin-derived tetradecamer SEQ ID N0:7 has an amino acid sequence derived
from
amino acids 16 to 29 of saposin C, but which has been modified by substitution
or
deletion of each of the 3 naturally occurring lysines and the addition of a C-
terminal
tyrosine residue. In particular, the prosaposin-derived tetradecamer SEQ ID
N0:7 has
a D-alanine for lysine substitution at position 2; an alanine for lysine
substitution at
position 8 and a deletion of lysine at position 11. The D-alanine substitution
at position
2 confers increased stability by protecting the peptide from endoprotease
degradation,
as is well known in the art. See, for example, Partridge (Peptide Drug
Delivery to the
Brain, Raven Press, New York, 1991, page 247). The substitution or deletion of
a lysine
residue confers increased resistance to trypsin-like proteases, as is well
known in the art.
See, Partridge, supra. These substitutions increase stability and, thus,
bioavailability of
peptide SEQ ID N0:7, but do not affect activity in inhibiting apoptosis. The
prosaposin
receptor agonist can also be made as a cyclic peptide for increased stability.
A useful modification to a prosaposin receptor agonist can also be one that
promotes peptide passage across the blood-brain barrier, such as a
modification that
increases lipophilicity or decreases hydrogen bonding. For example, a tyrosine
residue
added to the C-terminus of the prosaposin-derived peptide (SEQ ID N0:7)
increases
hydrophobicity and permeability to the blood-brain barner. See, for example,
Banks et
al. (Peptides 13:1289-1294, 1992) and Partridge, supra. A chimeric
CA 02304108 2000-03-08
WO 99/12559 PCT/US98/19216
-13-
peptide-pharmaceutical that has increased biological stability or increased
permeability
to the blood-brain barrier, as described in EXAMPLE 6, for example, also can
be useful
in the method of the invention.
The term "prosaposin receptor" refers to a site on a cell to which prosaposin
or a prosaposin receptor agonist can bind, thereby acting to alter the cell's
function. The
prosaposin receptor is a G-protein-coupled cell surface receptor of 54-60 kDa,
isolated
from baboon brains, pig brains, whole rat brain, and mouse neuroblastoma
cells. This
receptor protein can be isolated from a P100 plasma membrane fraction by
affinity
purification using a neurite growth-inducing peptide contained within the
saposin C
sequence linked to a solid support. The 54-60 kDa protein crosslinks
irreversibly to
saposin C. The isolation of the putative prosaposin receptor is described in
EXAMPLES
6and7.
The term "apoptosis" refers to the cellular process of programmed cell death.
Apoptosis encompasses a group of characteristic structural and molecular
events, in
which a cell specifically and precisely controls its fate in a mixed cell
population.
Endogenous nucleases cleave chromatin between nucleosomes and reduce the
content
of intact DNA in cells undergoing apoptosis. Apoptosis accounts for most of
the
programrled cell death in tissue remodeling and for the normal cell loss that
accompanies
atrophy of adult tissues following withdrawal of endocrine and other growth
stimuli.
Thus, apoptosis is similar to proliferation in that both processes are tightly
regulated and
essential for the homeostasis of renewable tissues. Apoptosis is also
responsible,
however, for the abnormal cell death that occurs in many diseases.
Apoptosis can be recognized by a characteristic pattern of morphological,
biochemical and molecular changes in apoptotic cells. These changes can be
broadly
assigned to 3 stages: In the early stage, there is decreased cell size (cell
dehydration),
alterations in cell membranes, large (50 kilobase [kb]) DNA strand breaks, and
an
increase in cellular calcium levels. In the intermediate stage, DNA is cleaved
into
180-200 by fragments, giving the characteristic "laddering" on a DNA gel,
further
decrease in cell size, and a decreased cell pH. In the late stage, there is a
loss of
membrane function and the formation of apoptotic bodies.
CA 02304108 2000-03-08
WO 99/12559 PCTNS98/19216
-14-
Methods of detecting apoptosis can be based on the measurement of DNA
content, altered membrane permeability, or the detection of endonucleolysis as
characterized by DNA strand breaks. Such techniques are well known to those of
skill
in the art and can be readily performed without undue experimentation.
The apoptotic process can also be assayed by determining the activity of
prosaposin receptor, Akt, Bcl-2 family members, associated PI 3-kinase pathway
components, and JNK. Among the research tools that can be used are the well-
known
techniques involving antibodies and the various technologies (i.e.,
immunoprecipitation,
immunoblotting, and immunoaffinity chromatography) that use these molecular
probes.
See, Kohler et al. (Nature 256: 495, 1975); Current Protocols in Molecular
Biology
(Ausubel et al., ed., 1989); and Harlow and Lane {Antibodies: A Laboratory
Manual,
Cold Spring Harbor Laboratory, New York, 1997). When used appropriately, these
tools
provide the means to analyze the activity of enzymes, identify post-
translationally
modified proteins, quantitate protein and non-protein macromolecules, and
dissect the
biochemical events of the many phases of the cell cycle. Phosphorylation
assays, kinase
activity assays, immunoprecipitations, and immunoassays are provided in
EXAMPLE
1 and EXAMPLE 3.
The term "caspase" refers to any of the aspartate-specific cysteine proteases,
sharing a conserved active site that cleaves proteins at a highly specific
site, to induce
apoptosis. All caspases cleave their substrates after aspartate (Asp)
residues. Caspases
promote apoptosis through proteolytic degradation of cellular components, a
process
which is amplified by autocatalysis of the various caspases. Different members
of the
caspase superfamily (formerly known as the ICE family) have slightly different
substrate
specificities and rnay thus be involved in different aspects of the apoptotic
pathway.
Caspases generally function in the distal portions of the proteolytic cascades
involved
in apoptosis (see, FIG. 5).
Caspases are processed from a single-chain zymogen to a two-chain active
enzyme by cleavage at internal Asp residues. Caspases with large prodomains
are
generally regulatory caspases, whereas those with small prodomains are
generally
efFector caspases. Thus, active caspases can activate other caspases following
an initial
CA 02304108 2000-03-08
WO 99/12559 PCT/US98/19216
-15-
activating stimulus to form a proteolytic cascade, with the initial activation
of a
regulatory caspase serving to activate by proteolytic cleavage the downstream
effector
caspases.
During apoptosis, caspases break down cellular proteins, causing severe
morphological changes and cell shrinkage. Effector caspases, particularly
caspase-3,
cleave substrates such as poly(ADP-ribose) polymerase, actin, fodrin, and
lamin. In the
final stages of apoptosis, the chromosomal DNA is cleaved by a DNase enzyme.
The
enzyme caspase-3 activated DNase (CAD) cleaves chromosomal DNA. CAD does not
control apoptosis itself. Rather, the CAD inhibitor of caspase-3 activated
DNase (ICAD)
acts as a chaperone for CAD during CAD synthesis, remaining complexed with CAD
to
inhibit CAD DNase activity until the reactivity is triggered by appropriate
apoptotic
stimuli. Caspase-3, when activated by apoptotic stimuli, cleaves ICAD to
release the
DNase activity, allowing CAD, which carries a nuclear-localization signal, to
enter the
nucleus, and degrade chromosomal DNA in nuclei, causing the characteristic DNA
fragmentation. See, Sakahira et al. (Nature 391 (6662): 96-99, 1998); and
Enari et al.
(Nature 391 (6662): 43-50, 1998). Thus, activation of CAD downstream of the
caspase
cascade is responsible for characteristic DNA degradation during apoptosis.
The method of the invention involves administering an apoptosis-inhibiting
amount of a prosaposin receptor to cells. In one embodiment, the invention
provides a
method for inhibiting caspace-mediated apoptosis due to a proinflammatory
cytokine.
More particularly, the proinflammatory cytokine that induces apoptosis may be
TNFa,
one of the most intensively studied caspase activators. TNFa induces apoptosis
in many
cell types, including neurons, oligodendrocytes and oligodendrocyte precursor
cells.
While TNFa has been known to be important in proinflammatory responses for 20
years,
the particular biochemical steps have been incompletely understood until
recently. The
TNFa is now a paradigm that has been applied to the other activators of
caspace-
mediated apoptosis.
TNFa effects are mediated through binding of TNFa to two types of receptor,
the 75 kDa TNF-Rl and a 55 kDa TNF-R2. TNFa binding to a TNF-R initiates a
variety
of biological responses. For example, the cellular signaling in apoptosis
begins at the
CA 02304108 2000-03-08
WO 99/12559 PCTNS98/19216
- 16-
TNF-R1 and moves downstream in a series of biochemical reactions. Some
biological
responses, like cell proliferation and apoptosis, seem to be in opposition to
each other,
but TNF-Rl is known to control both kinds of biological responses. TNF-Rl has
3
separate responses: (1) apoptosis; (2) the activation of NF-xB, a
transcription factor that
inhibits apoptosis; and (3) the activation of JNK, a protein kinase.
The TNFa signal transduction pathway directly regulates caspase activation
through recruitment of adaptor molecules and caspases to the cytoplasmic
domain of
TNF-R. TNF-R contains a cytoplasmic death domain (DD) that activates the
apoptotic
process by interacting with the DD-containing adaptor proteins TNF-R-
associated DD
protein (TRADD) and Fas-associated DD protein (FADD/MORT1), leading to the
activation of cysteine proteases of the caspase family. The TRADD protein has
two
distinct functional domains. The protein has a DD body and a tail. The tail of
TRADD
binds to TRAF2, eventually resulting in activation of NF-xB. The body binds to
FADD,
another intracellular signaling protein, which then activates apoptosis.
Another DD-
containing protein that binds to TNF-R is caspase-8. Binding of TNFa
stimulates TNF-R,
leading to the formation of a receptor-bound death-inducing signaling complex
(DISC),
consisting of FADD and two different forms of caspase-8. As a result,
activation of the
caspase proteolytic cascade begins.
Another intensively studied caspase activator is Fas. Autoimmune disorders
are associated with defects in Fas pathway function. Inappropriate expression
of the Fas
ligand (Fast) can enable tumor cells to escape immune surveillance. The Fas
signal
transduction pathway also directly regulates caspase activation through
recruitment of
adaptor molecules and caspases to the cytoplasmic domain of the receptor. Fas
(Apo 1;
CD95) also contain a cytoplasmic death domain (DD) that activates the
apoptotic process
by interacting with TRADD and FADD, leading to caspase activation. Stimulation
of Fas
leads to the formation of a receptor-bound death-inducing signaling complex
{DISC),
consisting of FADD and two different forms of caspase-8. The discovery that
Fas-associated death domain protein {FADD) recruited caspase-8 to the Fas
signaling
complex by virtue of caspase-8 ability to bind the adapter molecule FADD
established
that this protease has a role in initiating the death pathway.
CA 02304108 2000-03-08
WO 99/12559 PCT/US98/19216
-17-
The method of the invention, using a prosaposin receptor agonist is effective
in inhibition of apoptosis in proinflammatory cytokine-susceptible cells that
contain the
prosaposin receptor, the downstream signaling elements of PI 3-kinase, Akt,
and Bcl-2,
and the caspase-mediated cell death mechanism (See, FIG. 4 and 5. See also,
Hemmings,
Science 275: 628-630, 1997; Franke et al., Nature 390: 116-117, 1997; Datta et
al., Cell
91: 23I-241, 1997).
Prosaposin receptor agonists stimulate several different signal transducers
after binding to the prosaposin receptor. These signal transducers include
mitogen-
activated protein kinase (MAPK), PI 3-kinase, and the non-receptor tyrosine
kinase
p60$". Signal transduction following prosaposin receptor agonist binding to
prosaposin
receptor has been shown in neuronal cells, Schwann cells, and myoblasts.
Prosaposin
receptor agonists utilize a pertussis toxin sensitive G-protein pathway to
activate MAPK
proteins. Furthermore, Akt is upregulated within minutes of cellular exposure
to
prosaposin receptor agonist. Akt activates an apoptosis-inhibiting Bcl-2
family member,
which then inhibits the action of caspases. Thus, prosaposin receptor agonists
prevent
caspase-mediated apoptosis.
Mitogen-activated protein kinase (MAPK) is a general name for a family of
serine/threonine kinases that play an important role in cell signaling by a
variety of
ligands and receptors, including receptor tyrosine kinases and G-protein
coupled
receptors. The extracellular signal-regulated protein kinases, ERK1 and ERK2,
are part
of the MAPK family. ERKI is the 44 kDa protein (p44''~"PK). ERK2 is the 42 kDa
protein
(p42"'"P"). Signaling proteins, such as phosphatidylinositol-3-kinase (PI 3-
kinase) and
protein kinase C (PKC) phosphorylate ERK proteins, either independently or in
association with the guanosine triphosphate (GTP)-binding protein Ras (p21 R~)
pathway.
In many cells, activation of the MAPK pathway by growth factors regulates gene
transcription associated with proliferation and differentiation. In
oligodendrocytes, ERK
proteins also are important for oligodendrocyte process extension.
Prosaposin receptor agonists bind to the prosaposin receptor with high
affinity
to activate ERK1 and ERKZ phosphorylation in PC12 cells, Schwann cells, and
CA 02304108 2000-03-08
WO 99/12559 PCT/US98/19216
-18-
oligodendrocytes. Prosaposin receptor agonists also activate ERK activity by a
pertussis
toxin-sensitive mechanism involving the adapter protein Shc, p60s", and PI 3-
kinase.
The survival of certain subsets of neurons of the peripheral nervous system
can be promoted by the activation of a pathway that includes Ras and protein
kinases
leading to mitogen-activated protein kinase (MAPK). The PI 3-kinase pathway is
also
important for the survival of several cell lines. Activation of PI 3-kinase
triggers the
activation of the serine-threonine kina.se Akt. Thus, Akt has a critical role
in the PI 3-
kinase pathway.
The activation of the serine/threonine protein kinase Akt is a key event in
apoptosis prevention. Modules made of protein kinases control cellular
processes,
including apoptosis. After growth factors bind to their cognate growth factor
receptor
tyrosine kinases, PI 3-kinases are recruited and activated. Inositol lipids
are
phosphorylated by PI 3-kinases to act as second messengers. The
serine/threonine protein
kinase Akt (protein kinase B; PKB) is one of the major targets of PI 3-kinase-
generated
signals. Akt dissociates a complex of Bcl-2 family members, activating an
apoptosis-
inhibiting Bcl-2 family member, which then inhibits caspases to prevent
apoptosis. Thus,
the activation of Akt is a key event in cell death prevention by the PI 3-
kinase pathway.
Akt is a proto-oncogene with a pleckstrin homology domain. The pleckstrin
homology domains can bind lipids, providing a mechanism linking the activation
of PI
3-kinase and Akt activity. PI 3-kinase activity can be inhibited by wortmannin
and by the
inhibitor LY294002. Both of these inhibitors inhibit the rapid activation of
Akt by
growth factors, such as platelet-derived growth factor (PDGF), epidermal
growth factor
(EGF), basic fibroblast growth factor (bFGF), insulin, and insulin-like growth
factor-1
(IGF-1). Activation of Akt by protein phosphatase inhibitors is, however,
relatively
insensitive to wortmannin and LY294002. Thus, the lipid kinase activity of PI
3-kinase
mediates Akt activation by growth factors, so Akt acts downstream of PI 3-
kinase (see,
FIG. 4 and 5).
T h a PI 3-kinase-derived second messengers,
phosphatidylinositol-3,4-bisphosphate (Ptdlns-3,4-P2) and
phosphatidylinositol-3,4,5-triphosphate {PtdIns-3,4,5-PI 3), promote
activation of Akt
CA 02304108 2000-03-08
WO 99/12559 PCT/US98/19216
-19-
in 3 steps: (i) the translocation of the kinase to the membrane, (ii) the
attachment to the
membrane by means of pleckstrin homology domain binding to phospholipid, and
(iii)
phosphorylation. The high-affinity association of Akt with PtdIns-3,4-P2 and
PtdIns-3,2,
promotes a conformational change leading to an increase of kinase activity.
Ptdlns-3,4-P2 and PtdIns-3,4-P3, which accumulate transiently upon cell
stimulation by
growth factors, also bind to the pleckstrin homology domain of Akt and promote
the
association of Akt with the membrane. PI 3-kinase activity further leads to an
increase
in Akt kinase by promoting Akt phosphorylation of Akt at 2 sites {Thr3o$ and
Ser4~3) by
an upstream kinase, known as PKBK. Both phosphorylation events can be
inhibited by
wortmannin in vivo.
The release of Akt from the membrane by inositol trisphosphate (IP3) is the
next regulatory step. IP3, generated from Ptdlns-4,5-P2 by phospholipase C,
releases
pleckstrin homology domain-containing proteins, including Akt, from membranes.
After
its release, Akt becomes available to phosphorylate downstream targets.
Akt is particularly important for the survival of neurons. For example, IGF-1
protects cerebellar neurons from apoptosis by activating Akt. Nerve growth
factor (NGF)
also promotes Akt activation in pheochromocytoma PC 12 cells, showing that
kinase
activation is also involved in the survival promoted by NGF. Thus, the Akt
signaling
pathway can prevent apoptosis of neurons.
The Akt signaling pathway for suppressing apoptosis then proceeds to the
phosphorylation of the Bcl-2 family member BAD, thereby inactivating BAD
promotion
of apoptosis and promoting cell survival. See, Datta et al. (Cell 91 (2): 231-
24, 1997). Akt
phosphorylates BAD in vitro and in vivo to inhibit BAD-induced apoptosis. The
inactivation of BAD allows other, apoptosis-inhibiting Bcl-2 family members to
inhibit
caspases.
The mammalian Bcl-2 family has members that are potent inhibitors of
programmed cell death and inhibit activation of caspases in cells (e.g., Bcl-
2, Bcl-xL, and
Bag). Other members of the Bcl-2 family promote apoptosis (e.g. Bax, Bcl-xs,
BAD, and
Bak). However, Bcl-2 family members have several different mechanisms of
function
which need not be mutually exclusive. Bcl-2 family proteins either suppress or
promote
CA 02304108 2000-03-08
WO 99112559 PCT/US98I19216
-20-
apoptosis by interacting with and functionally antagonizing each other. The
regulation
of apoptosis by Bcl-2 family members involves several regulatory processes
including
dimerization and phosphorylation. Members of the Bcl-2 family form homodimers
and
heterodimers to interact with one another, altering the balance between cell
survival and
apoptosis. Phosphorylation can also change the activity state of many Bcl-2
family
members. For example, phosphorylation of BAD by Akt causes BAD to be
sequestered
and inactivated.
All members of the Bcl-2 family share regions of homology termed BH (~Bcl
Homology) domains. The BH domains are (1) BHl and BH2, which in apoptosis
inhibitors allows heterodimerization with Bax to repress apoptosis; (2) BH3,
which in
the apoptosis promoters, Bax and Bak, allows heterodimerization with Bcl-xL
and Bcl-2
to promote apoptosis; and (3) BH4 which conserved in apoptosis inhibitors,
e.g. Bcl-xL,
but absent in apoptosis agonists except Bcl-xs. The BH4 domain allows
interaction with
apoptosis regulatory proteins such as Raf 1 and BAD. All members of the Bcl-2
family
(except BAD and Bid) contain a hydrophobic C-terminus (transmembrane, TM)
domain
which anchors the Bcl-2 protein to the cell membrane. BAD lacks this sequence
and is,
therefore, located throughout the cytoplasm. Bcl-2 and Bcl-xL localize
predominantly to
the outer mitochondria) membrane, but also to the nuclear and endoplasmic
reticulum
membranes. The GTP-binding protein Raf 1 translocates Bcl-2 family members to
the
mitochondria) membrane.
Dimerization of members of the Bcl-2 family regulates the cellular decision
to proceed to apoptosis. Apoptosis-inhibiting members such as Bcl-2 and Bcl-xL
form
dimers with the apoptosis-inducing activity of Bax and BAD to block Bax and
BAD
activity. Thus, the ratio of apoptosis-inhibiting Bcl-2 family members to
apoptosis-
inducing Bcl-2 family members is important in determining whether apoptosis
will
proceed. Excess apoptosis-inhibiting Bcl-2 family members promotes survival
whereas
excess apoptosis-inducing Bcl-2 family members promotes apoptosis. For
example,
Bcl-xL homodimers are required to actively suppress apoptosis or to actively
promote
survival. Therefore, Bcl-xL/Bax heterodimerization promotes apoptosis. By
contrast, Bax
homodimers are required to actively promote apoptosis or to actively inhibit
survival.
CA 02304108 2000-03-08
WO 99/12559 PCT/US98/19216
-21 -
Thus, Bcl-xLBax heterodimerization inhibits apoptosis. If Bcl-2 levels are
higher that
those of Bax, for example, then survival generally prevails, whereas the
opposite
circumstance is associated with cell death. These interactions either prevent
caspase
activation to inhibit apoptosis, or promote caspase activation to induce
apoptosis.
The Bcl-2 family member apoptosis inhibitors inhibit caspase activation. For
example, there is a direct interaction between caspases and Bcl-xL. The loop
domain of
Bcl-xL is cleaved by caspases in vitro and in cells induced to undergo
apoptotic death.
Interaction of Bcl-xL with caspases may be an important mechanism of
inhibiting cell
death. However, once Bcl-xL is cleaved, the C-terminal fragment of Bcl-~C
potently
induces apoptosis. Thus, the recognition/cleavage site of Bcl-x~ protects
against
apoptosis by acting at the level of caspase activation; cleavage of Bcl-xL
during the
execution phase of programmed cell death converts Bcl-xL from a protective to
a lethal
protein.
By inhibiting caspase activity in cells, prosaposin receptor agonists inhibit
the
apoptotic pathway and allow cell survival. Prosaposin receptor agonists
inhibit apoptosis
in proinflammory cytokine-susceptible cells that contain the prosaposin
receptor, the
downstream signaling elements of PI 3-kinase, Akt, and Bcl-2, and the caspase-
mediated
cell death mechanism. The inhibition of apoptosis by prosaposin receptor
agonists occurs
at the level of caspase activation, which is a unique method of inhibiting
apoptosis.
Known apoptosis inhibitors block caspace-mediated apoptosis at stages of the
caspase
proteolytic cascade different from the stage influenced by prosaposin
agonists.
Most therapies do not inhibit apoptosis by inhibiting caspase through the
activation of Akt and Bcl-2 falxiily members. For example, many therapies
involve the
inhibition of the early stages of the apoptotic pathway by modulation of the
binding of
proinflammatory cytokines to their cognate receptors and the receptor
activation. After
TNFa therapy was identified as a potential therapeutic target for rheumatoid
arthritis,
antibodies to TNFa were shown to have efficacy in both animal models and human
patients. See, Eigler et al. (Immunol Today 18(10): 487-492, 1997). Studies in
animals
and an open-label trial have suggested a role for antibodies to TNFa,
specifically the
chimeric monoclonal antibody cA2, in the treatment of Crohn's disease. See,
Targan et
CA 02304108 2000-03-08
W4 99/12559 PCTNS98/19216
-22-
al. (N. Engl. J. Med. 337(15): 1029-1035, 1997). Targan et al. found that
single infusion
of cA2 was an effective short-term treatment in many patients with moderate-to-
severe,
treatment-resistant Crohn's disease. Additionally, inhibiting proinflammatory
cytokines
(e.g., TNFa and IL-1) is an established rheumatoid arthritis therapy. See,
Maini et al.
(APMIS 105(4): 257-263, 1997). Clinical trials using monoclonal anti-TNFa
antibodies
have been particularly successful in controlling inflammation and markedly
reducing
acute phase proteins and cellular ingress. However, because disease invariably
relapses,
repeated therapy is necessary. Prosaposin receptor agonist treatment can be an
effective
alternative therapeutic agent for the treatment of Crohn's disease and
rheumatoid arthritis
because prosaposin receptor agonists are administered more easily than anti-
TNFa
antibodies, and animal studies demonstrate no antibodies against prosaptides
after
months of administration.
In certain therapies, activation of the transcription factor NF-xB inhibits
apoptotic signaling through the transcriptional activation of survival-
promoting genes.
1 S Prosaposin receptor agonists may also act through an alternative pathway
to additionally
promote survival.
In still other therapies, regulation of caspase-mediated apoptosis induction
can
be accomplished by expression of caspase inhibitors. Peptides that inhibit
caspases are
commercially available. For example, the peptides Caspase-3 Inhibitor I (REVD-
CHO;
a highly specific, potent, reversible, and cell-permeable inhibitor of caspase-
3);
Caspase-3 Inhibitor III (Ac-DEVD-CMK; a potent, cell-permeable and
irreversible
inhibitor of caspase-3); and Caspase-4 Inhibitor I (Ac-LEVD-CHO; a caspase-4
inhibitor) are available from Calbiochem (San Diego, CA). Prosaposin receptor
agonists
do not compete with these peptides.
Prosaposin receptor agonists may also act to inhibit apoptosis through
activation of a family of proteins known as Inhibitors of Apoptosis Proteins
(IAPs). IAPs
were first identified on the basis of sequence similarity to the insect
baculovirus which
infects cells and inhibits apoptosis. These molecules contain four conserved
regions
which have death antagonizing properties: (1) three baculovirus inhibitory
repeats (BIR);
and (2) a ring zinc forger domain. Both regions are likely involved in
mediating
CA 02304108 2000-03-08
WO 99/12559 PCT/US98/19216
-23-
protein-protein interactions. One IAP gene, Neuronal Apoptosis Inhibitor
Protein
(NAIP), is selectively expressed in surviving neurons. NAIP was discovered to
be the
gene deleted in spinal muscular atrophy, a genetic disorder which causes
spinal motor
neuron degeneration and muscular atrophy leading to the death of newborn
children. The
IAPs act by preventing the activity or activation of caspases.
The process of identifying cells that are susceptible to caspase-mediated
apoptosis may be accomplished in several ways, using laboratory methods known
to
those of skill in the art. For example, cells may be identified as susceptible
to
proinflammatory cytokine-induced apoptosis by one or a combination of the
following
analyses: identifying cells as undergoing apoptosis during events associated
with
contacting cells with proinflammatory cytokine; identifying proinflammatory
cytokine
during the events that cause cells to undergo apoptosis; preventing apoptosis
by
removing proinflammatory cytokine during the events that cause cells to
undergo
apoptosis; and inducing apoptosis by reintroducing proinflammatory cytokine or
ending
the removal of proinflammatory cytokine during the events that cause cells to
undergo
apoptosis. More particularly, cells may be identified as susceptible to
proinflammatory
cytokine-induced apoptosis by laboratory methods described by Vartanian et al.
(Molecular Medicine 1 (7): 732, 1995).
Expressly included as cells that are susceptible to proinflammatory cytokine-
induced apoptosis are oligodendrocytes, neurons, Schwann cells, or myocytes.
Since it
is known that TNFa induces apoptosis in several neural cell types, including
cortical
neurons, oligodendrocytes and vligodendrocyte precursor cells, an
identification of a cell
as a neural cell is also an identification of that cell as susceptible to TNFa-
mediated
apoptosis. Since it is known that IFN~y induces oligodendrocyte apoptosis, an
identification of a cell as an oligodendrocyte is also an identification of
that cell as
susceptible to IFNy-mediated apoptosis.
Prosaposin receptor agonists are useful in treating diseases that involve cell
death or that are mediated by proinflammatory cytokines. Prosaposin receptor
agonists
are therefore useful in treating degenerative diseases such as
neurodegenerative diseases
(e.g., Alzheimer's disease, post-polio syndrome, Parkinson's disease,
amyotrophic lateral
CA 02304108 2000-03-08
WO 99/12559 PCT/US98/19216
-24-
sclerosis, Huntington's disease), ischemic disease of the heart (e.g.,
myocardial
infarction), traumatic brain and spinal cord injury, pain syndromes, alopecia,
AIDS, and
toxin mediated liver disease. See, Nicholson (Nature Biotechnology 14: 297,
1996).
The identification of a subject having a proinflammatory cytokine-induced
apoptotic disease can be accomplished by various methods known to those of
skill in the
art. For example, a disease may be identified as being a proinflammatory
cytokine
induced disease by one or a combination of the following analyses: (1)
identifying the
disease as occurnng during events associated with the proinflammatory
cytokine; (2)
identifying proinflammatory cytokine during the apoptotic disease events that
cause
inflammation; (3) preventing apoptosis by removing proinflammatory cytokine
during
the disease events; and (4) inducing apoptosis by reintroducing
proinflammatory
cytokine or ending the removal of proinflammatory cytokine during the disease
events.
A subject to be treated according to the invention may be identified as being
at risk for having a proinflammatory cytokine-induced disease at a future
time. In
EXAMPLE 6, the experimental subjects were identified as having a TNFa-mediated
disease at the time of, or even before, the TNFa injection and therefore
before the onset
of hyperalgesia, a characteristic feature of experimental neuropathic pain
states. The
treatment method of the invention therefore includes both treating existing
proinflammatory cytokine-induced disease and prophylactically reducing the
severity of
future proinflammatory cytokine-induced disease.
Prosaposin receptor agonists are therefore useful in treating many disorders
in which TNFa is known to be involved, including rheumatoid arthritis (Feldman
et al.
(Annals of the New York Academy of Sciences 766: 272-278, 1995); Feldman et
al.
(Journal of Inflammation 47: 90-96, 1996)), Crohn's disease (Stokkers et al.
(Journal
of Inflammation 47: 97-103, 1996)), irritable bowel syndrome, asthma, stroke
cardiac
infarction, and congestive heart failure. See, Eigler et al. (Immunol Today
18(10):
487-492, 1997); MacLellan et al. (Circ. Res. 81 (2): 137-144, 1997). TNFa
induces
apoptosis in several neuronal cell types, including cortical neurons,
oligodendrocytes and
oligodendrocyte precursor cells. The use of prosaposin receptor agonist for
the treatment
of any of these disorders is within the scope of the present invention. These
agonists can
CA 02304108 2000-03-08
WO 99/12559 PCT/US98/19216
-25-
be administered either alone or as an adjunct to conventional anti-
inflammatory therapies
such as steroid administration.
The proinflammatory cytokine IFNy is also a potent inducer of
oligodendrocyte apoptosis. Oligodendrocyte apoptosis has been observed at the
advancing margins of chronic active multiple sclerosis (MS) plaques (Vartanian
et al.,
Molecular Medicine 1 (7): 732,1995). IFNy may therefore be a factor in the
pathogenesis
of multiple sclerosis by activating apoptosis in oligodendrocytes. The method
of the
invention may be used for halting or slowing the progress of the IFNy-mediated
diseases
associated with neural or myelin degeneration in neural tissue, by contacting
neuronal
tissue susceptible to such degradation with a prosaposin receptor agonist.
There are
several diseases that result in inflammatory demyelination of nerve fibers
including
multiple sclerosis, Guillain-Barre disease, AIDS neuropathy, and AIDS cortical
demyelination. These diseases can be treated by administration of prosaposin
receptor
agonists to the cells affected by the disease. Because the molecular weight of
the active
docosanamer (22 amino acid; SEQ m N0:6) is approximately 2600, and an
octadecamer
(18 amino acid; SEQ )D NO:S) contained within this sequence will cross the
blood-brain
barrier, the docosanamer will also cross and enter the central nervous system.
TNFa and
IFNy may further be factors in the death of oligodendrocyte cells which
underly the
pathogenesis in many demyelination disorders. A patient diagnosed as having a
demyelination disease would also be expressly identified as having a
proinflammatory
cytolcine-induced disease that may be treated by the method of the invention.
Prosaposin receptor agonists can also be used in the treatment of Alzheimer's
disease. Caspase inhibition is relevent to neurodegenerative disease and
inhibition of
caspase activity may have an impact on the clinical course of
neurodegenerative diseases,
such as Alzheimer's disease. See, Holtzman et al. (Nature Medicine 3(9): 954-
955,
1997). Prosaposin receptor agonists, especially prosaposin receptor agonists
that cross
the blood-brain barrier, treatment can be a therapeutically effective agent
for treating
neurodegenerative diseases of the central nervous system.
Administration of a prosaposin receptor agonist can provide an effective
therapy for treatment of heart disease by inhibiting the effects of associated
CA 02304108 2000-03-08
W4 99/12559 PCT/US98/19216
-26-
proinflammatory cytokines. Apoptosis is a contributing cause of cardiac
myocyte loss
in ischemia/reperfusion injury, myocardial infarction, and long-standing heart
failure.
See, MacLellan et al. (Circ. Res. 81 (2): 137-144, 1997). Insights into the
molecular
circuitry controlling apoptosis suggest the potential to protect heart muscle
from
apoptosis through one or more of these pathways by pharmacological means.
Cytokines
that are expressed within the myocardium in response to environmental injury,
such as
TNFa, IL,-1, IL,-6, are important for initiating and integrating homeostatic
responses
during cardiovascular disease. See, Mann (Cytokine Growth Factor Rev. 7(4):
341-354,
1996). For example, the failing human heart expresses TNFa. See, Kubota et al.
(Circ.
Res. 81 (4): 627-635, 1997) in the development of congestive heart failure.
The ability of myocardium to successfully compensate for, and adapt to, stress
ultimately determines whether the heart will decompensate and fail, or whether
it will
maintain preserved function. Thus, the myocardial response to environmental
stress is
very important to heart function. See, Mann (Cytokine Growth Factor Rev. 7(4):
341-354, 1996). Cytokines that are expressed within the myocardium in response
to
environmental injury, i.e., TNFa, IL-1, and IL,-6, are important for
initiating and
integrating homeostatic responses within the heart. However, these
proinflammatory
cytokines all can produce cardiac decompensation when expressed at
sufficiently high
concentrations. Accordingly, the short-term expression of proinflammatory
cytokines
within the heart may provide the heart with an adaptive response to stress,
whereas
long-term expression of proinflammatory cytokines are maladaptive by producing
cardiac decompensation.
The arthritogenic activities of TNFa and its p55 TNF-R have been well
documented in experimental animal models of arthritis and in transgenic mice
expressing
wild-type or mutant transmembrane human TNFa proteins in their joints. See,
Alexopoulou et al. (Eur. J. Immunol. 27(10): 2588-2592, 1997). Prosaposin
receptor
agonist administration can provide an effective therapy for treatment of
arthritis, because
prosaposin receptor agonists inhibit the effects of proinflammatory cytokines
downstream of the interactions between TNFa and TNF-R.
CA 02304108 2000-03-08
WO 99/12559 PCTlUS98/19216
-27-
In summary, administering prosaposin receptor agonists to inhibit caspase-
mediated apoptosis, includes the use of such agonists in the treatment of
diseases such
as rheumatoid arthritis, Crohn's disease, irritable bowel syndrome, asthma,
cardiac
infarction, congestive heart failure, multiple sclerosis, acute disseminated
inflammatory
(AIDS) leukoencephalitis, Alzheimer's disease, Parkinson's disease,
amyotrophic lateral
sclerosis, post-polio syndrome, Huntington's disease, ischemic heart disease,
traumatic
brain injury, traumatic spinal cord injury, alopecia, AIDS dementia, cerebral
malaria,
HTLV neuropathy, Guillain-Barre disease, AIDS neuropathy, inflammatory
neurodegenerative diseases, and toxin-induced liver disease.
The term "apoptosis-inhibiting amount" means the amount of prosaposin
receptor agonist needed to inhibit apoptosis in a target cell. The amount of
prosaposin
receptor agorist that inhibits apoptosis can easily be determined by one of
skill in the art
using standard methods for assaying apoptosis. The activity of a prosaposin
receptor
agonist in inhibiting apoptosis can correlate with neurotrophic activity and
activity in
alleviating neuropathic pain or inducing neurotrophic activity. For example,
the
prosaposin-derived docosanomer (SEQ ID N0:6) and the prosaposin-derived
tetradecamer (SEQ ID N0;7) alleviate neuropathic pain and have neurotrophic
activity.
The prosaposin-derived dodecamer peptide (SEQ ID N0:4), which has the
conserved
adjacent asparagines, leucine and charged residues described above, is active
as a
neurotrophic factor. A typical minimum amount of prosaposin for the
neurotrophic factor
activity in cell growth medium is usually about 1.4 x 10-" M, or about 10
ng/ml. This
amount or more of prosaposin receptor agonists may also be used to inhibit
apoptosis or
reduce inflammation. Typically concentrations in the range of 0.1 ~g/ml to
about 10
~g/ml of any of these materials will be used.
The contact between the prosaposin receptor agonist and the cells may be
performed ex vivo or in vivo. Cells can be treated ex vivo by directly
administering
prosaposin receptor agonists to the cells. For example, cells can be treated
ex vivo by
culturing the cells in growth medium suitable for the particular cell type
followed by
addition of the agonist to the medium. Such ex vivo-treated cells can then be
administered
to a patient.
CA 02304108 2000-03-08
WO 99/12559 PCTNS98/19216
-28-
Cells are treated in vivo by administering the agonist by any effective method
that will result in contact between the prosaposin receptor agonist and the
cell. The
method of administration of an apoptosis-inhibiting amount of prosaposin
receptor
agonist may be by conventional modes of administration, including intravenous,
intramuscular, intradermal, pulmonary, nasal, mucosal, subcutaneous, epidural,
intraocular, topical in a biologically compatible carrier and oral
administration. The
composition may be injected directly into the blood in sufficient quantity to
give the
desired in vivo concentration. Direct intracranial injection or injection into
the
cerebrospinal fluid also may be used provided sufficient quantities can be
given such that
the desired local concentration is achieved. A pharmaceutically acceptable
injectable
carrier of well known type can be used. Such carriers include, for example,
phosphate
buffered saline (PBS) or lactated Ringer's solution. Alternatively, the
composition can
be administered to peripheral neural tissue by direct local injection or by
systemic
administration.
One skilled in the art can readily assay the ability of a prosaposin receptor
agonist to cross the blood-brain barner in vivo, for example, as disclosed in
EXAMPLE
6. In addition, an active fragment of prosaposin can be tested for its ability
to cross the
blood-brain barrier using an in vitro model of the blood-brain barrier based
on a brain
microvessel endothelial cell culture system, for example such as that
described by
Bowman et al. (Ann. Neurol. 14:396-402, 1983) or Takahura et al. (Adv.
Pharmacol.
22:137-165,1992). It had long been believed that in order to reach neuronal
populations
in the brain, neurotrophic factors would have to be administered
intracerebrally, since
these proteins do not cross the blood-brain barrier. However, the active
octadecamer (18
amino acid; SEQ ID NO:S) will cross and the active docosanamer (22 amino acid;
SEQ
ID N0:6) likely crosses this barner and would thus contact the brain cells
following
intravenously administration. An octadecamer (18 amino acid; SEQ ID NO:S)
peptide
consisting of amino acids 12-29 of the docosanamer (SEQ ID N0:6) with a
substitution
of tyrosine for valine at amino acid 12 (with a molecular weight of 2 kDa)
crosses the
blood-brain barner and enters the central nervous system. Conditions under
which a
CA 02304108 2000-03-08
WO 99/12559 PCT/US98/19216
-29-
peptide can cross the blood-brain barrier and enter the nervous system are
described by
Banks et al. (Peptides 13: 1289-1294, 1992).
Other neuronal populations, such as motor neurons, also can be treated by
intravenous injection, although direct injection into the cerebrospinal fluid
is also
envisioned as an alternate route.
Oral administration often is desirable, provided the prosaposin receptor
agonist is resistant to gastrointestinal degradation and readily absorbable.
The
substitution, for example, of one or more D-amino acids can confer increased
stability to
a prosaposin receptor agonist useful in the invention. Retroinverso
peptidomimetics that
are stable and retain bioactivity can also be devised, as described by
Brugidou et al.
(Biochem. Biophys. Res. Comm. 214(2): 685-693, 1995) and Chorev et al. (Trends
Biotechnol. 13(10): 438-445, 1995).
The prosaposin receptor agonists can be packaged and administered in unit
dosage form such as an injectable composition or local preparation in a dosage
amount
equivalent to the daily dosage administered to a patient or as a controlled
release
composition. A septum sealed vial containing a daily dose of the active
ingredient in
either phosphate-buffered saline or in lyophilized form is an example of a
unit dosage.
Appropriate daily systemic dosages of agonist based on the body weight for
treatment
of caspase-mediated apoptosis are in the range of from about 10 to about 100
~g/kg,
although dosages from about 0.1 to about 1,000 ~g/kg are also contemplated.
Thus, for
the typical 70 kg human, a systemic dosage can be between about 7 and about
70,000 p.g
daily and, alternatively, between about 700 and about 7,000 ~,g daily. A daily
dosage of
locally administered material will be about an order of magnitude less than
the systemic
dosage.
A prosaposin receptor agonist also can be administered in an inhalant form.
Inhalant drug delivery has been successfully used for (3-agonist and
corticosteroid drugs
of emphysema. See, Pingleton (JAMA, June 19, 1996). Mask ventilation is now a
first-line therapy for patients who have an exacerbation of chronic
obstructive pulmonary
disease. Similar methods of inhalant drug delivery can be used to deliver
prosaposin
receptor agonists.
CA 02304108 2000-03-08
WO 99/12559 PCT/US98/19216
-30-
A prosaposin receptor agonist also can be administered in a sustained release
form. The sustained release of a prosaposin receptor agonist has the advantage
of
inhibiting apoptosis over an extended period of time without the need for
repeated
administrations of the active fragment. Sustained release can be achieved, for
example,
with a sustained release material such as a wafer, an immunobead, a micropump
or other
material that provides for controlled slow release of the prosaposin receptor
agonist.
Such controlled release materials are well known in the art and available from
commercial sources (Alza Corp., Palo Alto CA; Depotech, La Jolla CA. See also,
Pardoll
(Ann. Rev. Immunol. 13: 399-415, 1995)). In addition, a bioerodible or
biodegradable
material that can be formulated with a prosaposin receptor agonist, such as
polylactic
acid, polygalactic acid, regenerated collagen, liposomes, or other
conventional depot
formulations, can be implanted to slowly release the active fragment of
prosaposin. The
use of infusion pumps, matrix entrapment systems, and transdermal delivery
devices also
are contemplated in the present invention.
The invention also provides a method for inhibiting apoptosis or alleviating
inflammation in a subject by transplanting into the subject a cell genetically
modified to
express and secrete a prosaposin receptor agonist. Transplantation can provide
a
continuous source of a prosaposin receptor agonist and, thus, sustained
alleviation of
neuropathic pain. For a subject suffering from prolonged apoptosis, such a
method has
the advantage of obviating or reducing the need for repeated administration of
an active
fragment of prosaposin.
Using methods well known in the art, a cell can be readily recombinantly
modified, such as by transfection with an expression vector containing a
nucleic acid
encoding a prosaposin receptor agonist. See, Chang (Somatic Gene Therapy, CRC
Press,
Boca Raton,1995). Following transplantation into the brain, for example, the
transfected
cell expresses and secretes a prosaposin receptor agonist and, thus, inhibits
apoptosis.
Such a method can be useful to alleviate neuropathic pain as described for the
transplantation of cells that secrete substances with analgesic properties.
See, ,f'or
example, Czech and Sagen (frog. Neurobiol. 46:507-529, 1995). In practice, the
transfected cell should be immunologically compatible with the subject.
Consequently,
CA 02304108 2000-03-08
WO 99/12559 PCT/US98/19216
-31-
autologous cells are particularly useful for recombinant modification. Non-
autologous
cells also can be useful if protected from immune rejection using, for
example,
microencapsulation or immunosuppression. Useful microencapsulation membrane
materials include alginate-poly-1.-lysine alginate and agarose (See, for
example, Goosen
(Fundamentals of Animal Cell Encapsulation and Immobilization, CRC Press, Boca
Raton,1993); Tai and Sun (FASEB J. 7:1061, 1993); Liu et al. (Hum. Gene Ther.
4:291,
1993); and Taniguchi et al. (Transplant. Proc. 24: 2977, 1992)).
For treatment of a human subject, the cell can be a human cell, although a
non-human mammalian cell also can be useful. In particular, a human
fibroblast, muscle
cell, filial cell, neuronal precursor cell or neuron can be transfected with
an expression
vector to express and secrete an active fragment of prosaposin such as SEQ ID
N0:4. A
primary fibroblast can be obtained, for example, from a skin biopsy of the
subject to be
treated and maintained under standard tissue culture conditions. A primary
muscle cell
also can be useful for transplantation. Considerations for neural
transplantation are
described, for example, in Chang, supra.
A cell derived finm the central nervous system can be particularly useful for
transplantation to the central nervous system, since the survival of such a
cell is enhanced
within its natural environment. A neuronal precursor cell is particularly
useful in the
method of the invention since a neuronal precursor cell can be grown in
culture,
transfected with an expression vector and introduced into an individual, where
it is
integrated. The isolation of neuronal precursor cells that are capable of
proliferating and
differentiating into neurons and filial cells is described in Renfranz et al.
(Cell
66: 713-729, 1991 ).
Methods of transfecting cells ex vivo are well known in the art. See, Kriegler
(Gene Transfer and Expression: A Laboratory Manual, W.H. Freeman & Co., New
York, 1990). For the transfection of a cell that continues to divide, such as
a fibroblast,
muscle cell, filial cell or neuronal precursor cell, a retroviral vector is
preferred. For the
transfection of an expression vector into a postmitotic cell such as a neuron,
a
replication-defective herpes simplex virus type 1 (HSV-1 ) vector is useful.
See, During
CA 02304108 2000-03-08
WO 99/12559 PCT/US98/19216
-32-
et al. (Soc. Neurosci. Abstr. 17:140, 1991 ) and Sable et al. (Soc. Neurosci.
Abstr. 17: 570,
1991).
A nucleic acid encoding an active fragment of prosaposin can be expressed
under the control of one of a variety of promoters well known in the art,
including a
constitutive promoter or inducible promoter. See, for example, Chang, supra. A
particularly useful constitutive promoter for high level expression is the
Moloney marine
leukemia virus long-terminal repeat (NILV-LTR), the cytomegalovirus immediate-
early
(CMV-IE) or the simian virus 40 early region (SV40).
The invention provides a method of alleviating neuropathic pain by
administering a neuropathic pain-alleviating amount of a prosaposin receptor
agonist to
a subject who is suffering from neuropathic pain caused by proinflammatory
cytokine.
The invention is therefore useful for treating neuropathic pain component of
inflammatory disease, although the pain relief is not due to an anti-
inflammatory effect.
The prosaposin receptor agonist activation of Akt described supra for
inhibition of
caspace-mediated apoptosis is relevant to the alleviation of neuropathic pain.
In an animal model, injection of TNFa into the subperineural space in the
sciatic nerve immediately proximal to the sciatic notch produces neuropathic
pain in
vivo. See, Wagner et al. (NeuroReport 7: 2897-2901, 1996). Using behavioral
testing of
either mechanical or thermal hyperalgesia, TNFa-injected animals suffer
significant
hyperalgesia compared to vehicle-injected animals, whose algesia lasted for 5
days. The
pain is due to nerve damage. Administration of prosaposin receptor agonist
prevents the
thermal hyperalgesia that occurs upon injection of TNFa into the sciatic
nerve. An
example of the alleviation of neuropathic pain by prosaposin receptor agonist
in the rat
TNFa-injection model is provided in EXAMPLE 6.
A reduction in pain can be determined by behavioral measurements assessing
the response to thermal or mechanical stimulii. When the subject is human, the
subject
can report a reduction in pain. Reduction in pain can also be detrmined in
animal models.
Several animal models of neuropathic pain have been developed, including the
Chung
rat model, the streptozotocin-induced insulin-deficient diabetic rat model,
the Seltzer rat
model, the neuroma model, and several primate models. See, Myers (NIH Workshop
on
CA 02304108 2000-03-08
WO 99/12559 PCT/US98/19216
-33-
Low Back Pain (J. Weinstein, S. Gordon (Eds), American Academy of Orthopaedic
Surgeons, 1995)); Myers (Regional Anesthesia 20(3): 173-184, 1995) and Bennett
(Muscle & Nerve 16:1040-1048, 1993). The scientific literature on nerve root
injury has
expanded recently with the introduction of new models of cauda equina
compression.
Because common pathogenic mechanisms of nerve and nerve root injury are
associated
with the development of chronic pain states, an alleviation of neuropathic
pain by
administration of prosaposin receptor agonist that is successful in any one
animal model
of neuropathic pain may be extrapolated to all models and to all types of
human
neuropathic pain, as prosaposin receptor agonists operate at a fundamental
convergent
step in the pathogenesis of pain arising from nerve injury. An effective
concentration of
prosaposin receptor agonist may also be determined by comparison with the
concentrations of prosaposin receptor agonist recommended for other
conditions.
Activation of immune cells by pathogens also induces the release of a
proinflammatory cytokines. See, Watkins et al. (Brain Res. 692(1-2): 244-250,
1995).
1 S The activated immune system communicates to the brain by release of
proinflammatory
cytokines. See, Watkins et al. (Pain 63(3): 289-302, 1995). Proinflammatory
cytokines
mediate a variety of common neuropathic pain states. Illness responses in the
brains of
those suffering from neuropathic pain cause dramatic changes in neural
functioning. For
example, IL-1 ~3 can alter brain function, resulting in a variety of illness
responses
including increased sleep, decreased food intake, fever, etc. IL-1 (3 also
produces
neuropathic pain. This IL-1 (3-induced neuropathic pain is mediated by
activation of
subdiaphragmatic vagal afferents in the brain.
The physiological basis of IL-1 ~3-induced neuropathic pain is representative
of a general physiological basis for proinflammatory cytokine-induced
neuropathic pain.
For example, TNFa produces dose-dependent neuropathic pain as measured by the
tailflick test. This TNFa-induced neuropathic pain is further mediated by the
induced
release of IL-1 ~3. Furthermore, this TNFa-induced hyperalgesia (as well as
most illness
responses) is also mediated by activation of subdiaphragmatic vagal afferents.
The
effects of subdiaphragmatic vagotomy cannot be explained by a generalized
depression
of neural excitability. See, Watkins et al. (Brain Res. 692(1-2): 244-250,
1995).
CA 02304108 2000-03-08
WO 99/12559 PCT/US98/19216
-34-
Proinflammatory cytokines and the neural circuits that they activate are
therefore
involved in the neuropathic pain states produced by irritants, inflammatory
agents, and
nerve damage.
Thus, apparently diverse neuropathic pain states converge in the central
S nervous system and activate similar or identical neural circuitry.
Prosaposin receptor
agonists that cross the blood-brain barrier are especially useful for
treatment of illness
response and other proinflammatory cytokine-induced neuropathic pain
components in
the central nervous system.
The following examples are illustrative and are not intended to limit the
scope
of the present invention.
EXAMPLE 1
PROSAPOSIN RECEPTOR AGONISTS PREVENT TNFA-INDUCED DEATH
OF A NEURONAL CELL LINE
The purpose of this EXAMPLE was show that prosaposin and a peptide
derived from prosaposin could prevent TNFa neurotoxicity. TNFa treatment for
48 hr
or more caused up to 50% loss of viability in a neuronal cell line, NS20Y, as
demonstrated by MTT reduction. Prosaposin and prosaptide TX14(A) prevented the
loss
of viability dose dependently, with maximal protection seen at 5 nM and 50 nM,
respectively. Trypan blue exclusion and BrdU incorporation assays showed that
prosaptide increased viability by preventing cell death and did not cause cell
proliferation. The prevention of TNFa-induced death by prosaposin receptor
agonists
was not inhibited by pertussis toxin. Thus, the results of this EXAMPLE show
that
prosaposin and the prosaptide TX14(A) prevented the death of a neuronal cell
line
induced by TNFa by a pertussis toxin-insensitive pathway.
Materials and Methods. Prosaposin was purified from human milk as
previously described by FIiraiwa et al. (Arch. Biochem. Biophys. 304: 110-116,
1993).
CA 02304108 2000-03-08
WO 99/12559 PCT/US98/19216
-35-
Prospatide (TX14(A), was provided by Anaspec (San Jose, CA) at greater than
95%
purity. TNFa was purchased from R&D Systems (Minneapolis, MN) and pertussis
toxin
(PT) was from Calbiochem (San Diego, CA). Cell culture reagents were purchased
from
Gibco-BRL (Grand Island, NY).
The mouse neuroblastoma cell line, NS20Y, was a gift from Drs. T. Taketomi
and K. Uemura (Shinshu University, Matsumoto, Japan). Cells were maintained in
DMEM (high glucose) containing 10% fetal calf serum (FCS), 100 U/ml
penicillin, 100
~cg/ml streptomycin and 1.1 mg/ml sodium pyruvate, at 37°C under
humidified S% CO2.
For cell viability assays, cells were seeded at 1x104/well in 96-well plates
in
complete media and allowed to grow overnight. The next day, TNFa and
prosaposin or
prosaptide were applied to cells in DMEM containing penicillin, streptomycin,
sodium
pyruvate and 0.5% FCS. Cells were then incubated for 24-96 hr. Cell viability,
as
indicated by reduction of a tetrazolium salt (MTT) to a purple formazan
product, was
assessed using the CellTiter 96TM kit {Promega, Madison, WI] according to the
manufacturer's instructions. Standard curves were constructed to ensure that
optical
density measurements were within a linear range and to allow optical density
readings
to be converted to cell number. To assess the pertussis toxin sensitivity of
prosaposin
receptor agonist erects, cells were incubated in 10 ng/ml pertussis toxin for
24 hr; TNFa
in the presence or absence of prosaposin or prosaptide was then added and cell
viability
assessed at 48 hr. This regimen of pertussis toxin treatment has previously
been shown
to inhibit prosaptide-induced ERK phosphorylation in iSC cells by Campana
(unpublished observation) and thrombin-induced proliferation of CCL39 cells by
Chambard et al. (1987).
For trypan blue studies, cells were seeded at Sx104/well in 6-well plates in
complete media and grown overnight. Treatments were then added in DMEM
containing
penicillin, streptomycin, sodium pyruvate and 0.5% FCS and cells grown for a
further
48 hr. Cells were stained with trypan blue and viable (unstained) and non-
viable cells
(stained blue) were scored. Duplicate wells were prepared for each treatment
and within
each well two groups of 100 cells were scored.
CA 02304108 2000-03-08
WO 99/12559 PGT/US98/19216
-36-
Proliferation of NS20Y cells, as indicated by BrdU incorporation, was
measured using the Cell Proliferation ELISA, BrdU colorimetric kit from
Boehringer-Mannheim (Indianapolis, IN) according to the manufacturers
directions.
Cells were seeded as for MTT assays. Prosaptide was added to media containing
0.5%
FCS or FCS was added to serum-free media in 2-fold dilution series. Cells were
then
incubated for 24 - 96 hr.
All experiments were performed in duplicate or triplicate and in each FIG.
6-9, the mean t S.E.M. of a representative experiment is presented; nz2.
Pooled data
were analyzed using one-way ANOVA and the source of significance (p<0.05) was
determined using Scheffe's posthoc analysis.
Results. Treatment of NS20Y cells with TNFa resulted in a loss of viability
as demonstrated by a decrease in MTT reduction. The effect of TNFa was dose
dependent with maximal diminuition at 100 ng/ml TNFa. No loss of viability was
seen
at 24 hr. Thus, TNFa was administered at 100 ng/ml for 48 or more hours.
FIG. 6A shows that cultures which received TNFa for 48 hr demonstrated
a 35% reduction in the number of viable cells as compared to controls.
Prosaposin,
applied to cells as a single dose at t = 0 hr, partially prevented the loss of
viability in a
dose dependent manner with greatest protection seen at 5 nM. Similarly,
prosaptide
administered at t = 0 hr prevented the loss of viability caused by TNFa in a
dose-dependent manner. See, FIG. 6B. Maximal protection was seen when cells
were
treated with 50 nM prosaptide with greater than 90% of the cells maintained by
50 nM
of the peptide.
A single application of prosaposin at t = 0 hr prevented loss of viability at
all
time points studied, however, the potency of the effect decreased dramatically
over time.
See, FIG. 7. At 48 hr untreated cultures were 65% as viable as control
cultures whereas
cells treated with 5 nM prosaposin were 85% as viable. At time points longer
than 48 hr,
TNFa continued to result in decreased cell viability and at 96 hr viability
was
approximately 40% as compared to controls whereas prosaposin treated cells
were
approximately 54% as viable. In contrast, the protective effect of prosaptide
was
CA 02304108 2000-03-08
WO 99/12559 PCT/US98/19216
- 37 -
maintained at its maximum at all time points. At 72 and 96 hr, prosaptide-
treated cells
were as viable as controls.
The increase in viability of TNFa-insulted cells treated with prosaposin
receptor agonist is due to a decrease in cell death. To show this, trypan blue
exclusion
and BrdU incorporation experiments were conducted. FIG. 8 shows that 25% of
cells
treated for 48 hr with 100 ng/ml TNFa were trypan blue positive (dead) as
compared to
7% positive in control cultures; the increase in cell death was completely
prevented in
a dose-dependent manner by prosaptide at a maximal concentration of lnM. FIG.
9
shows that at 48 hr prosaptide did not induce cell proliferation at any dose.
The
proliferative capacity of the cells was confirmed by demonstrating a dose-
dependent
stimulation of proliferation by serum. Additionally, prosaptide did not
stimulate
proliferation at 24, 72 or 96 hr.
Hiraiwa et al. (Proc. Natl. Acad. Sci. USA 94: 4778-4781, 1997) have
demonstrated that prosaposin receptor agonists stimulate ERK phosphorylation
and
enhance sulfatide content in Schwann cells {Campana et al., FASEB J. 12: 307-
3I4,
1998) demonstrated that both of these prosaposin effects are inhibited by
pertussis toxin.
To show that the neuroprotective action of prosaposin is mediated by the same
mechanism, cells were incubated with pertussis toxin at 10 ng/ml overnight and
then
treated the cells with TNFa in the presence or absence of prosaposin receptor
agonists.
FIG. 9 shows that pertussis toxin alone caused a decrease of approximately 6%
in
viability of the cells. Similarly, there was 12% enhancement of the TNFa-
induced
viability loss when pertussis toxin was added. Treatment of cultures with
prosaposin at
5 nM or prosaptide at 50 nM largely prevented the loss of cell viability
caused by TNFa.
Addition of pertussis toxin caused a 7% and 10% decrease in the protective
effect of
prosaposin and TX14(A), respectively.
This EXAMPLE demonstrates that prosaposin and a prosaposin-derived
peptide of 14 amino acids, TX14(A), prevented the TNFa-induced death of a
neuronal
cell line. The neuroprotective action of prosaposin receptor agonists was dose-
dependent.
At the maximally effective doses the protection was almost complete.
Prosaposin was
able to protect cells from TNFa-induced death at a 10-fold lower molar
concentration
CA 02304108 2000-03-08
WO 99/I2559 PCT/US98/19216
-38-
than TX14{A). This 'is likely due to a difference in the binding affinity of
the two ligands
for the putative prosaposin receptor. The Kd of prosaposin binding to PC 12
cells was 2.5
nM while the Kd of prosaptide (TX14(A)) binding was 18.3nM.
Protection of neuronal cells from TNFa neurotoxicity was achieved by a
single dose of prosaposin or prosaptide given together with TNFa; there was no
pretreatment and no supplementation of the dose during the experiment. The
magnitude
of protection by prosaposin was greatly reduced at 96 hr as compared to that
seen at 48
hr. However, prosaptide maintained 100% protective capacity as long as 96 hr;
the
longest time point examined. This difference in efficacy may be due to a
difference in
stability of the two compounds. Prosaposin has been reported by Hiraiwa et al.
(Proc.
Natl. Acad. Sci. USA 94: 4778-4781, 1997) to be rapidly cleaved by cathepsin
D. Bn
contrast, in 50% human serum prosaptide has a half life of greater than 24 hr.
Using trypan blue exclusion and BrdU incorporation assays, the
TNFa-induced loss of viability seen using the MTT assay was confirmed to be
due to an
increase in cell death and that prosaptide prevented this death. A complete
prevention of
death was seen at 0.5 nM when using trypan blue. This effective concentration
is
100-fold less than that observed in the MTT assay. The reason for this
discrepancy is
unclear, however, it is possibly due to a difference in the sensitivity of the
2 assays.
Jabbar and colleagues (1996) also demonstrated discrepancies between results
obtained
with MTT and results obtained by cell counting. They showed that MTT
underestimated
the growth inhibition of COR-L23 cells by IFN~y. Similarly, an underestimation
of the
amount of TNFa induced death was observed when using the MTT assay. There was
a
SO% reduction in cell viability when the MTT assay was used, whereas the
trypan blue
assay revealed a 3-fold increase in the number of dead cells when cultures
were treated
with TNFa. This underestimation may cause a masking of the protective effects
of
prosaposin receptor agonists at lower concentrations and hence explain the
difference
between the results obtained using the two assays. The reduction of MTT is not
due
simply to mitochondria) reductases. While the assay does effectively measure
cell
viability, the mechanism by which it does so makes it vulnerable to many
influences and
this may explain discrepancies between the MTT assay and other viability
assays.
CA 02304108 2000-03-08
WO 99/12559 PCT/US98/19216
-39-
Despite the discrepancy, TNFa induced cell death and that prosaposin receptor
agonists
prevented that death.
Prosaposin receptor agonists can stimulate tyrosine phosphorylation in
NS20Y cells, iSC cells and primary Schwann cells. In primary Schwann cells,
prosaptide-induced ERK phosphorylation is inhibited by pertussis toxin
suggesting that
the putative prosaposin receptor is linked to a heterotrimeric G-protein
containing GoH or
G;8 subunits. Hiraiwa et al. (Proc. Natl. Acad. Sci. USA 94: 4778-4781, 1997)
have
recently presented data to suggest that the association is with Goa.
Prosaposin receptor
agonist-induced enhancement of sulfatide levels has been demonstrated in
Schwann cells
(See, Campana et al., FASEB J. 12: 307-314, 1998) and neurite outgrowth in
NS20Y cells
(See, Misasi et al., 1998).
Campana et al. (FASEB J. 12: 307-314, 1998) have shown that prosaptide
stimulates the phosphorylation of PI3-kinase in Schwann cells. PI3-kinase is
known to
play an integral role in the prevention of neuronal cell death by neurotrophic
factors
including BDNF, IGF, and NGF and the prevention of death of other cell types.
TNFa can be neurotoxic. This multifunctional cytokine can also be
neuroprotective. Whether TNFa acts in a protective or toxic capacity may well
be
determined by which neuronal cell line is being studied or the regimen of TNFa
treatment used. Under similar conditions NS20Y, PC12 and SK-N-MC cells are
susceptible to the cytotoxic effects of TNFa, whereas SH-SYSY and Neuro2A
cultures
do not lose viability when treated with 100 ng/ml TNFa for up to 96 hr.
Furthermore, the
susceptibility of SK-N-MC cells to TNFa changes with their differentiation
state. When
they are differentiated they display a TNFa-induced loss of viability within
48 hr
whereas when they are undifferentiated there is no apparent loss of viability
until 96 hr.
Prosaposin receptor agonists prevent the TNFa-induced cell death of a
neuronal cell. This will have important therapeutic benefits in the treatment
of
neurodegeneration.
CA 02304108 2000-03-08
WO 99/12559 PCT/US98/19216
-40-
EXAMPLE 2
PROSAPOSIN RECEPTOR AGONISTS INHIBITS JNK2
PHOSPHORYLATION IN SCHWANN CELLS
This EXAMPLE shows that prosaposin receptor agonist TX14(A) inhibits
JNK2 phosphorylation in Schwann cells after a 5 minute treatment (see, FIG.
10).
Additionally, prosaposin receptor agonist TX14(A) enhances Schwann cell
production
of p 1 OOP"' after one hour in low serum media (see, FIG. 11 ).
EXAMPLE 3
PROSAPTIDE ACTIVATES THE MAPK PATHWAY BY
A G-PROTEIN-DEPENDENT MECHANISM ESSENTIAL FOR ENHANCED
SULFATIDE SYNTHESIS BY SCHWANN CELLS
This EXAMPLE shows that treatment of primary Schwann cells and an
immortalized Schwann cell line, iSC, with a 14-mer prosaptide, TX14(A), (10
nM)
enhanced phosphorylation of mitogen-activated kinases, ERK1 (p44'''~nK;
extracellular
signal-regulated kinase 1 ) and ERK2 (p42''~~''K; extracellular signal-
regulated kinase 2)
within 5 minutes that was blocked by 4 hour pretreatment with pertussis toxin.
Furthermore, incubation of Schwann cells with the non-hydrolyzable GDP analog,
GDP-~iS, inhibited TX14(A)-induced ERK phosphorylation. TX14(A) enhanced the
sulfatide content of primary Schwann cells 2.5-fold which was inhibited by
pretreatment
with pertussis toxin or the synthetic MEK inhibitor, PD098059. In addition,
TX14(A)
increased the tyrosine phosphorylation of all 3 isoforms of the adapter
molecule, Shc,
which coincided with the association of p60s'~ and PI 3-kinase. Inhibition of
PI 3-kinase
by wortmannin blocked TX14(A)-induced ERK phosphorylation. This EXAMPLE
demonstrates that TX14(A) uses a pertussis toxin sensitive G-protein pathway
to activate
ERKs that is essential for enhanced sulfatide synthesis in Schwann cells.
Materials and Methods. TX14(A) (SEQ ID N0:7} was synthesized
commercially to 98% purity (AnaSpec, San Jose, CA). Platelet Derived Growth
Factor
CA 02304108 2000-03-08
WO 99/12559 PCT/US98/19216
-41 -
(PDGF) was purchased from Genzyme (Cambridge, MA ). GDP-X35, PD098059,
wortmannin (WT) and pertussis toxin (PT) were purchased from CalBiochem (San
Diego, CA). Anti-phosphotyrosine monoclonal Ab, anti-Src monoclonal Ab, anti-
PI
3-kinase polyclonal Ab and anti-Shc polyclonal Ab were purchased from Upstate
Biotechnology Incorporated (Lake Placid, New York).
Two Schwann cell cultures were used; (1 ) a spontaneously transformed cell
line, iSC, from rat primary Schwann cells, as described by Bolin et al. (J.
Neurosci. Res.
33: 231-238,1992), and primary Schwann cells that were prepared from neonatal
rats as
described by Assouline et al. (In A Dissection and Tissue Culture Manual of
the Nervous
System (Shahar et al., eds), Wiley-Liss, New York, 1989) pp. 247-250. At the
first
passage, Schwann cells were fiirther selected from fibroblasts using an anti-
fibronectin
antibody and rabbit complement. This resulted in approximately 99% pure
Schwann cell
cultures as assessed by 5100 and fibronectin immunoflourescence. iSC cells
were
maintained in DME/F 12 containing 10% horse serum and P/S ( 100 U/mL
penicillin and
100 ,ug/mL streptomycin). Primary Schwann cells were maintained in DMEM
containing
10% fetal bovine serum (FBS), P/S, 21 ,ug/mL bovine pituitary extract and 4 mM
forskolin. All cells were incubated at 37°C under humidified 7.5% COz.
Primary Schwann cells and iSC cells were grown to 85% confluency in
maintenance media and changed to serum free media (SFM) 6 hours (primary
Schwann
cells) or 16-18 hours (iSC cells) before experimentation. Experiments
involving the non-
hydrolyzable GDP analogue, GDP-(3S, were performed by permeabilizing serum
starved
cells with saponin (20 ,ug/mL) for 3 minutes in the presence of GDP-~iS. Cells
were then
rinsed twice with SFM and reincubated at 37°C with GDP-(3S for 20
minutes prior to the
addition of effectors. Cells were pretreated with either pertussis toxin,
PD098059 or
wortmannin. In all experiments, cells were stimulated with effectors for 5
minutes,
washed 3 times with ice cold PBS containing 1mM sodium vanadate and lysed on
ice in
Iysis buffer as previously described by Campana et al. (Biochem. Biophys. Res.
Commun
229: 706-712, 1996). Protein content of each sample was determined using the
bicinchonic acid method (Sigma Chemical Ca., St Louis, MO). Western
immunoblotting
and densitometry were performed as described by Campana et al. (Biochem.
Biophys.
CA 02304108 2000-03-08
WO 99/12559 PCT/US98/19216
-42-
Res. Commun 229: 706-712, 1996), except that nitrocellulose membranes were
used
instead of PVDF membranes. Differences in treatments were analyzed by ANOVA
and
treatment means were analyzed by the Student's-Newman-Keuls Multiple
Comparisons
Test.
ERK activity was assessed using a MAP kinase activity kit (New England
Biolabs, Cambridge, MA) with minor modifications. Briefly, Schwann cells were
prepared as described above, stimulated with effectors for 5 minutes and lysed
in 20 mM
Tris (pH 7.5), 150 mM NaCI, 1 mM EDTA, 1 mM EGTA, I % Triton X-100, 2.5 mM
sodium pyrophosphate, 1mM (3-glycerolphosphate, 1 mM sodium vanadate, l,ug/mL
leupeptin and 1 mM PMSF. Protein content of each sample was determined as
above.
Primary Schwann cell lysates (100 fig) and iSC cell lysates (200 fig) were
incubated with
1:200 phospho-MAP kinase antibody overnight at 4°C. Immunoprecipitates
were
obtained by adding 20 ~cl (50% slurry) protein A sepharose CL-4B (Sigma, St
Louis,
MO) and incubating at 4°C for 4 hours or overnight. Beads were washed
twice in lysis
I S buffer and subsequently washed in kinase buffer containing 25 mM Tris (pH
7.5), 5 mM
(3-glycerolphosphate, 2 mM DTT, 0.1 mM sodium vanadate and 10 mM MgCl2.
Immunoprecipitates were incubated at 30°C for 30 minutes in kinase
buffer containing
1 ~g ELK-1 fusion protein and 100 ~M ATP. Reactions were terminated by the
addition
of 25 ~cl 3X SDS sample buffer. Samples were boiled for 5 minutes and proteins
were
resolved by SDS-PAGE). Proteins were electroblotted onto nitrocellulose
membrane and
ERK activity was identified by immunoblotting with a phospho-ELK-1 antibody
followed by detection with ECL (Amersham, Arlington Heights, IL).
iSC cells (approximately 2.0 x 10') were incubated in DMEM/F12 without
serum 18 hours prior to stimulation with TX14(A) for 5 minutes at 37°C.
Cells were then
lysed and immunoprecipitated as previously described by Lanfrancone et al.
(Oncogene
10: 907-917, 1995). Protein concentrations were determined by bicinchoninic
acid
method (Sigma Chemical Co., St Louis, MO). Immunoprecipitates containing equal
amounts of protein were resolved by SDS-PAGE and electrotransferred onto
nitrocellulose membranes (Bio-Rad Laboratories, Hercules, CA). After blocking
with 3%
BSA and 0.05% Tween 20, membranes were probed with specific antibodies at
4°C
CA 02304108 2000-03-08
WO 99/12559 PG"T/US98/19216
- 43 -
overnight in 1 % BSA diluted in T-TBS (20 mM Tris-HCL pH 7.6 150 mM NACI and
0.05% Tween 20). After extensive washing, proteins from the immunocomplexes
were
detected by horseradish-peroxidase conjugated species specific secondary
antibodies
(Bio-Rad, Hercules, CA) followed by ECL (Amersham, Arlington Heights, II,).
S Primary Schwann cells were incubated in DMEM containing 0.5% FBS with
and without effectors for 48 hours. Cells that were treated with pertussis
toxin (50
ng/mL) were preincubated for 4 hours in 0.5% FBS containing media before the
addition
of effectors. Cells treated with the synthetic inhibitor of MEK, PD098059,
were
preincubated at 37°C for 30 minutes prior to the addition of effectors.
Cells were rinsed
with PBS, harvested and sonicated in 100 ul distilled water. An aliquot of
cell lysate was
removed for protein analysis and the remainder was extracted with 5 mL of
chloroform/methanol, 2:1 (v/v). Schwann cell lipid extracts were
chromatographed and
immunostained with an anti-sulfatide monoclonal antibody that is highly
specific for
sulfatide as described by Hiraiwa et al. (Proc. Natl. Acad. Sci. USA 94: 4778-
4781,
1997). The effect of treatment changes in sulfatide synthesis were tested by
comparing
the differences by ANOVA and treatment means by the Student's-Newman-Keuls
Multiple Comparisons Test.
Results. TX14(A) increased both ERKI and ERK2 phosphorylation in
Schwann cells. There was a larger increase in the ratio of ERK1
phosphorylation to total
ERK1 protein (18-fold that of controls) than that of ERK2 (3-fold greater than
controls).
When iSC cells were preincubated with pertussis toxin, which catalyzes the ADP-
ribosylation of G,/G°a subunits of G-proteins, TX14(A)-induced ERK
phosphorylation
was inhibited. Similar results were also observed in primary Schwann cells. By
contrast,
PDGF, which binds to a tyrosine kinase receptor and stimulates proliferation
of Schwann
cells, stimulated ERKl (4-6 fold) and ERK2 (2 fold) phosphorylation but was
not
inhibited by pertussis toxin pretreatment. To further confirm that ERK
phosphorylation
by TX14(A) involved G-proteins, the iSC cells were incubated with GDP-(3S.
This
treatment also blocked TX14{A)-induced ERK phosphorylation.
ERK protein kinases are activated by phosphorylation of tyrosine and
threonine residues and both are required for full protein kinase activity.
Because the
CA 02304108 2000-03-08
WO 99/12559 PCTNS9$119216
antibody used only recognized the phosphorylated tyrosine residue on ERKs, the
TX14(A)-induced phosphorylation of ERK was correlated with ERK catalytic
activity.
Kinase activity was also increased in both primary Schwann cells and iSC cells
after
treatment with PDGF and TX14(A).
The activation of the adapter protein, Shc, was also examined in TX 14(A)
signaling. iSC cells expressed all 3 isoforms: p46s''°, p$~' , and
p66s"~.
Immunoprecipitation of iSC cell lysates with a polyclonal antibody to all 3
isoforms of
Shc, followed by Western blotting with an anti-phosphotyrosine antibody,
demonstrated
that TX14(A) greatly enhanced tyrosine phosphorylation of all 3 Shc isoforms.
Furthermore, 2 unidentified tyrosine phosphorylated proteins were observed in
the Shc
immunoprecipitates of approximately 60 kDa and 85 kDa in size. Western
blotting of Shc
immunoprecipitates with an antibody to p60s~° revealed that the 60 kDa
tyrosine
phosphorylated protein was p60s'~ and an antibody to pBSP' 3-kin~ revealed
that the 85 kDa
phosphorylated protein was indeed the p85 subunit of PI 3-kinase. Moreover,
after
TX14(A) treatment there was more PI3K associated with Shc immunoprecipitates
than
controls. Blots were reprobed with anti-Shc to demonstrate that equal amounts
of
unphosphorylated Shc proteins were loaded onto the gel. Subsequently, iSC cell
lysates
were immunoprecipitated with an antibody to p60$'~ and Western blotted with an
antibody to phosphotyrosine; this showed enhanced tyrosine phosphorylation of
PI
3-kinase after treatment with TX14(A). Furthermore, preincubation of iSC cells
with
wortmannin completely blocked TX14(A)-induced ERK phosphorylation. In
unstimulated cells, wortmannin treatment reduced ERK phosphorylation below
control
levels.
TX14(A) stimulates synthesis of sulfatide in Schwann cells. To determine
whether G-protein mediated ERK phosphorylation was involved in synthesis of
sulfatide,
primary Schwann cells were preincubated with either pertussis toxin or the
synthetic
inhibitor of MEK, PD098059, before TX14(A) stimulation. The anti-sulfatide
monoclonal antibody identified only sulfatide that had the same mobility as
purified
sulfatide in all samples. In addition, TX14(A) treatment increased the
sulfatide content
2.5-fold over controls. Pretreatment with either pertussis toxin or PD098059
inhibited
CA 02304108 2000-03-08
WO 99/12559 PCT/US98/19216
-45-
TX14(A)-induced sulfatide synthesis. The viability of Schwann cells treated
with either
PD098059 or pertussis toxin after 48 hours did not differ from controls as
determined by
trypan blue exclusion.
To confnm that the dose of PD098059 used to inhibit sulfatide synthesis also
inhibited ERK phosphorylation in primary Schwann cells, ERK phosphorylation
experiments in cells pretreated with PD098059 were performed. TX14(A)
increased the
phosphorylation of ERKs, however, the magnitude of the increase was less than
what
was observed in iSC cells. The same dose of PD098059 (SO ~cM) used in the
sulfatide
experiments blocked TX14(A)-induced phosphorylation of ERK in primary Schwann
cells. In addition PD098059 decreased ERK1 and ERK2 phosphorylation below
control
levels. Timecourse experiments of TX14(A)-induced phosphorylation of ERKs in
iSC
cells demonstrated that TX14(A) rapidly activates ERK1 and ERK2 within 5
minutes and
returned to baseline levels by 30 minutes.
Identification of a G protein dependent mechanism for TX14(A) signaling.
TX14(A) dose-dependently stimulates ERK phosphorylation in both iSC and
primary
Schwann cells. After quantification and expression of the data as a ratio of
phosphorylated ERKs to total ERK proteins, TX14(A) preferentially
phosphorylated
ERK1, although Schwann cells contained a greater amount of immunoreactive ERK2
protein. The same phenomenon has been observed in PC12 cells and ERK1 is
preferentially activated in oligodendrocytes. In this EXAMPLE, TX14(A)-
stimulated
ERK phosphorylation was blocked by pertussis toxin treatment which indicated
that the
primary mechanism of activation involved one or more pertussis toxin sensitive
G
proteins such as G; or Go, both of which are abundantly expressed in Schwann
cells.
ERK activation is associated with pertussis toxin-sensitive G-protein
signaling in COS-7 cells, CHO cells) and Swiss 3T3 cells. The mechanism of MAP
kinase activation by G-coupled receptors involves G~3 subunits. Both
prosaptides and
prosaposin specifically bind to PC12 cells in a dose dependent saturatable
manner with
high affinity (Kd=2.5 nM and 18 nM, respectively)). Similarly, cell surface
binding
assays using radio-labeled TX14(A) gave a single high affinity constant for
binding to
iSC cells with a Kd of 10 nM. These findings demonstrate that prosaposin and
TX14(A)
CA 02304108 2000-03-08
WO 99/12559 PCTNS98/19216
-46-
bind to a putative receptor which associates with pertussis toxin-sensitive G-
protein to
mediate signal transduction. Pertussis toxin-sensitive ERK signaling is known
for the
insulin-like growth factor receptor tyrosine kinase, as well as the more
common 7
transmembrane G-protein coupled receptors.
The pathways of signal transduction which underlie myelination have not
been clearly defined. In oligodendrocytes, the initial stages of myelination
involves non-
receptor tyrosine kinases of the Src family and ERK activation play an
important role in
process extension. In the peripheral nerve, tissue concentrations of ERKs have
been
shown to increase after peripheral nerve injury (day 3). ERKs have been
localized to
activated Schwann cells and increased concomitant with remyelination.
This EXAMPLE demonstrates that inhibition of MEK by PD098059
completely blocked TX14(A)-enhanced synthesis of sulfatide, an essential
myelin lipid
component of both central and peripheral nervous system myelin, in Schwann
cells. This
concentration of PD098059 (SO ~cM) specifically inhibits MEK and not other
kinases
such as PKC, PI 3-kinase or p38 MAP kinase. PD098059 did decrease ERK
phosphorylation below controls suggesting that primary Schwann cells in
culture contain
autocrine regulated ERKs.
In addition, the timecourse of ERK activation by TX 14(A) showed that only
5 minutes of stimulation is sufficient for enhanced sulfatide synthesis
observed 48 hours
later. Transient activation of EIRKs in PC 12 cells with growth factors, such
as EGF does
not lead to pronounced nuclear translocation, so that in Schwann cells, TX
14(A)-induced
ERK acts in the cytosol to contribute to myelin lipid synthesis. Thus, signal
transduction
through the ERK pathway is an essential signaling pathway responsible for
myelination
by Schwann cells.
TX14(A) signaling involved the adapter protein, Shc, and the non-receptor
tyrosine kinase, p60s'~. This EXAMPLE demonstrates that Shc associated with
p6(~"
following TX14(A) stimulation, which coincided with increased tyrosine
phosphorylation of Shc. The association of p605" and Shc has been observed
previously
in COS-7 cells after lysophosphatidate (LPA) stimulation and has been proposed
to be
-...
CA 02304108 2000-03-08
WO 99/12559 PCT/US98/19216
-47-
involved in early activation of ERKs by pertussis toxin-sensitive G-protein
coupled
receptors.
The results of this EXAMPLE with the MEK inhibitor, PD098059, showed
that ERK activation by TX14(A) is due to the p21~ mediated signaling cascade
in
Schwann cells. Howeverthis EXAMPLE also demonstrates that PI 3-kinase plays a
role
in ERK activation in response to TX14{A) based on the ability of wortmannin to
block
TX 14(A~induced ERK phosphorylation and the observation that TX 14(A) induced
a
larger amount of p85 P' 3-~'n~' in Shc immunoprecipitates coincident with Shc
tyrosine
phosphorylation. The concentration of wortmannin used in this EXAMPLE has been
shown previously to specifically inhibit PI 3-kinase activity in Swiss 3 T3
fibroblasts and
L6 rat myoblasts. PI 3-kinase has been shown to activate ERKs by a p21'~-
independent
mechanism and by linkage with G-protein coupled receptors showing that TX14(A)
signaling involves multiple and perhaps novel pathways leading to ERK
activation.
TXl4(A) Role in Myelination. Prosaposin is not only a neurotrophic factor,
but an essential factor for events involved in myelination, including
prevention of
Schwann cell and oligodendrocyte death and synthesis of a myelin lipid,
sulfatide.
Moreover, prosaposin-deficient transgenic mice have severe hypomyelination in
both the
central and peripheral nervous system which was apparently due to failure of
myelin
synthesis, rather than demyelination. The deficiency of myelin in these
animals and in
prosaposin deficient humans is due to the lack of a myelinotropic effect of
prosaposin
during development. This EXAMPLE shows that TX14(A), encompassing the
neurotrophic region of prosaposin, appeared to exert its trophic effect by
binding to a
high affinity receptor which activated a pertussis toxin-sensitive G-protein
and signaled
through ERKs to up regulate the synthesis of sulfatide in Schwann cells.
Inhibition of
ERK activation blocked enhanced synthesis of sulfatide implicating ERKs as a
key
signaling component in myelin lipid synthesis.
CA 02304108 2000-03-08
WO 99/12559 PCTNS98/19216
-48-
EXAMPLE 4
EFFECT OF PROSAPOSIN AND TX14(A~ON
PROINFLAMMATORY CYTOHINE-INDUCED
OLIGODENDROCYTE CELL DEATH
This EXAMPLE demonstrates that prosaposin or the prosaposin-derived
peptide TX14(A) (SEQ ID N0:7), can inhibit proinflammatory cytokine-induced
apoptosis. Undifferentiated CG4 oligodendrocytes were grown in DMEM containing
10% fetal calf serum. Cells were removed with trypsin and plated in 30 mm
petri dishes
onto glass coverslips in 0.5% fetal bovine serum for 2 days in the presence of
absence
of the following effectors: 200 ng/ml TNFa alone or in the presence of 1 nM
prosaposin,
5 nM prosaposin, 10 nM TX14(A) or 50 nM TX14(A). The same experiment was also
performed using 200 ng/ml IFNy alone or in the presence of 1 nM prosaposin, S
nM
prosaposin, 10 nM TX14(A) or 50 nM TX14(A). The MTT cell death assay was then
performed using a kit (Promega, Madison, WI). This assay measures the MTT dye
reduced by mitochondria. In the presence of TNFa and IFNy, the MTT absorbance
decreases due to increased cell death and greater reduction of the MTT dye by
mitochondria that are released from lysed cells.
As shown in FIG. 13A, TNFa-induced apoptosis is completely reversed by
prosaposin (1 nM and 5 nM) and TX14(A) (10 nM and SO nM). An inhibitory effect
was
also observed with IFNy, albeit not as strong as that obtained with TNFa. See,
FIG. 13B.
Therefore, prosaposin and TX14(A) inhibit TNFa and IFN~y-induced apoptosis in
oligodendrocytes.
EXAMPLE 5
L6 MYOBLAST RESCUE
This EXAMPLE demonstrates that TX14(A) inhibits proinflammatory
cytokine TNFa-induced apoptosis in L6 myoblasts. L6 myoblasts cells were
incubated
for 96 hours either in media (control); media with 10 ng/ml TNFa ('I'NF
Category); or
CA 02304108 2000-03-08
WO 99/12559 PCT/US98/19216
-49-
media with 10 ng/ml TNFa and 200 ng/ml TX14(A). See, FIG. 14. Cell death was
measured by trypan blue assay as described in EXAMPLE 3. Cell death was
inhibited
in L6 myoblasts incubated with TNFa and TX 14(A), as compared to L6 myoblasts
incubated with TNFa only (approximately 60% cell death). Therefore, prosaposin
receptor agonist treatment inhibits TNFa-induced apoptosis in myoblasts.
EXAMPLE 6
EFFECT OF TX14(A) ON THERMAL HYPERALGESIA
FOLLOWING ENDONEURIAL INJECTION OF TNFa
This EXAMPLE demonstrates that a prosaposin-derived peptide, TX14(A)
peptide (prosaptide; SEQ ID N0:7), was effective in treating TNFa-induced
inflammation. The inflammatory component of peripheral nerve injury may affect
the
development of local neuropathologic changes as well as the onset of
hyperalgesia, the
characteristic features of experimental neuropathic pain states. See, Wagner
et al.
(NeuroReport 7: 2897-2901, 1996).
TNFa (2.5 pg/ml) was injected directly into the endoneurial space of normal
rat nerves. In a parallel experiment, TX14(A) {200 ~g/kg) was injected
subcutaneously
prior to injection of TNF-a. The resulting effects on behavior were monitored
for 1 week.
Behavioral measurements assessed the response to thermal stimuli and were
expressed
as a difference score (ipsilateral minus contralateral paw).
As shown in FIG. 15, the TX14(A) peptide dramatically reduced TNFa-
induced thermal hyperalgesia in the rat model. Therefore, TX14{A) peptide
(prosaptide)
inhibits TNFa-induced neuropathic pain in the endoneurial space of normal rat
nerves.
EXAMPLE 7
IN VIVO UPTAKE OF PROSAPOSIN-DERIVED PEPTIDES
BY THE CENTRAL NERVOUS SYSTEM
This EXAMPLE demonstrates that prosaposin-derived peptides cross the
blood-brain barrier.
CA 02304108 2000-03-08
WO 99/12559 PCTNS98/19216
-50-
An octadecamer (SEQ ID NO:S) consisting of amino acids 12-29 of saposin C
with a tyrosine substituted for valine at position 12 was chemically
synthesized on an
Applied Biosystems Model 430 peptide synthesizer. The peptide was then
radioiodinated
by the lactoperoxidase method; 20 x 106 cpm radiolabeled peptide were injected
into the
S auricles of rats. The animals were sacrificed after 1 hr and 24 hr, and the
hearts were
perfused with isotonic saline in order to remove the blood from the brain.
In order to determine the percentage of peptide uptake, the brain was then
counted in a gamma counter. In addition, the brain was homogenized and
fractionated
into a capillary rich fraction (pellet) and a parenchyma) brain fraction
(supernatant) after
dextran centrifugation. See, Triguero et al. (J. Neurochem., 54:1882-1888,
1990). This
method allows for the discrimination between radiolabeled peptide within blood
vessels
and that within the brain. After 24 hr, 0.017% of the injected peptide (SEQ ID
NO:S) was
detected in whole brain; 75% of the label was in the parenchyma) fraction and
25% was
in the capillary fraction. At 1 hr, 0.03% of the injected dose was present in
whole brain.
The prosaposin-derived peptide SEQ ID N0:7 also was assayed for ability
to cross the blood-brain barrier as follows. A female Sprague-Dawley rat was
anesthesized with methoxyflurane, and approximately 20 p.g peptide SEQ ID N0:2
(3.2
x 10g cpm) was injected into the tail vein. After 40 minutes, the rat was
sacrificed by
ether anesthesia and perfused with about 250 ml PBS through the heart. The
total amount
of peptide in brain, liver and blood was calculated as a percentage of the
injected material
as shown in TABLE 3. In order to determine the localization in brain, the
capillary
depletion method of Triguero (J. Neurochem. 54:1882, 1990) was used to
separate brain
tissue into a parenchyma fraction and a brain capillary fraction. The
fractionation results
showed that 87% of the SEQ ID N0:7 peptide present in brain was localized to
brain
parenchyma while 13% was found in brain capillary.
CA 02304108 2000-03-08
WO 99/12559 PCT/US98/19216
-51-
TABLE 3
TISSUE WEIGHT TOTAL CPM IN TISSUE PERCENTAGE OF
INITIAL CPM
Brain 1.3 g 161,000 0.050
Liver 8.8 g 5.2 x 106 1.625
Blood about 22 1.01 x 1 O8 31.6
pl
In a similar experiment in which rats were sacrificed after 3 hr treatment
with
SEQ ID N0:7, 0.06% of the peptide was evident in brain, of which 85% was in
the
parenchyma. These results demonstrate that at least some of the prosaposin-
derived
peptide SEQ ID N0:7 crossed the blood brain barner and was concentrated in the
brain
parenchyma rather than the vascular endothelium (blood vessels). The
percentage of
peptide that crossed the blood brain barrier is in the mid-range of peptides
that cross the
barrier as set forth in Banks, supra.
In order to determine the percentage of intact material in the brain, liver
and
blood, radiolabeled material (SEQ ID N0:7) isolated from the tissues was
analyzed by
high pressure liquid chromatography. To normalize for degradation during
processing
of tissue homogenates, peptide SEQ ID N0:7 was added to tissue homogenates.
The
extent of degradation observed with the added peptide material was used to
normalize
for degradation during tissue processing. After normalization, the results
were as follows:
SEQ ID N0:2 was about 60% intact in brain; about 80% intact in liver and about
40%
intact in blood. In a second experiment, peptide SEQ ID N0:7 was about 68%
intact in
brain. These results indicate that the peptide SEQ ID N0:7 crosses the blood
brain
barrier and is largely intact in brain.
CA 02304108 2000-03-08
WO 99/12559 PCT/US98/19216
-52-
EXAMPLE 8
ISOLATION OF PROSAPOSIN RECEPTOR
FROM WHOLE RAT BRAIN
A 54 kDa protein has been identified as the receptor for prosaposin as
S described in this EXAMPLE: A prosaposin receptor protein was isolated from
whole rat
brain, rat cerebellum and mouse neuroblastoma cells using the plasma membrane
P-100
fraction. Briefly, cells or tissues were solubilized and centrifuged at 14,000
rpm to
remove debris. The supernatant was centrifuged at 40,000 rpm for 1 hr at
4°C. The
pellet, enriched in plasma membrane, was solubilized in RIPA buffer (10 mM
MOPS,
pH 7.5, 0.3 M sucrose, 5 mM EDTA, 1% Trasylol, 10 ~M leupeptin and 10 ~M
pepstatin). This P-100 fraction was applied to an amity column containing the
bound,
active 14-mer fragment of saposin C, TX14(A). The column was washed with 0.05
M
NaCI to elute loosely-bound proteins followed by 0.25 M NaCI that eluted the
putative
54 kDa prosaposin receptor. In addition, it was determined that the 54-60 kDa
protein
could be eluted using a 100-fold excess of unbound peptide thus demonstrating
specific
elution. The 54 kDa protein was approximately 90% pure as judged by SDS-PAGE.
The
protein was purified to homogeneity using HPLC and eluted at SO% acetonitrile
in an
acetonitrile/water gradient on a Vydac C4 column. After treatment with the
cross-linking
reagent disuccinimidyl suberate (DSS; Pierce, Rockford, IL), the 54 kDa
protein bound
irreversibly to'ZSI labeled saposin C as evidenced by the 66 kDa molecular
weight of the
complex (54 kDa + 12 kDa).
EXAMPLE 9
ISOLATION OF PROSAPOSIN RECEPTOR;
EVIDENCE FOR A G-PROTEIN ASSOCIATED RECEPTOR
In this EXAMPLE, the prosaposin receptor was partially purified from
baboon brain membranes by affinity chromatography using a saposin C-column.
The
purified preparation gave a single major protein band with an apparent
molecular weight
CA 02304108 2000-03-08
WO 99/12559 PCT/US98/19216
-53-
of 54 kDa on SDS-PAGE. Affinity cross-linking of 11 kDa'ZSI-saposin C
demonstrated
the presence of a 66 kDa product, indicative of an apparent molecular weight
of 55 kDa
for the receptor. A GTPyS-binding assay using cell membranes from SHSYSY
neural
cells demonstrated agonist stimulated binding of [35S]GTPyS upon treatment
with
prosaptide TX14(A) a peptide from the neurotrophic region; maximal binding was
obtained at 2 nM. TX14(A) stimulated binding was abolished by prior treatment
of
SHSYSY cells with pertussis toxin and by a scrambled and an all D-amino acid-
derivative of the 14-mer. A 14-mer mutant prosaptide competed with T'X14(A)
with a
Ki of 0.7 nM. Immunoblot analysis using an antibody against the G°a
subunit
demonstrated that the purified receptor preparation contained a 40 kDa
reactive band
consistent with association of G°a and the receptor. The results of
this EXAMPLE show
that the signaling induced by prosaposin and TX14(A) is generated by binding
to a G°-
protein associated receptor.
TX14(A) also bound to PC12 cells and iSC Schwann cells with Kd values of
18.3 nM and 10 nM and increased phosphorylation of MAP kinase { i 5,16). These
findings suggested the presence of a specific receptor for prosaposin which
triggers a
MAP kinase cascade. In this EXAMPLE, a prosaposin receptor is characterized
from
baboon brain membranes and SHSYSY cells as a G-protein associated receptor.
Materials and Methods. Baboon brains were frozen in liquid nitrogen
immediatelly after death and stored at -70°C until use. Chemically
synthesized peptides
including TX14(A) (SEQ ID N0:7) and a 14-mer mutant prosaptide, 14M1, with a
single
amino acid substitution with aspartic acid replacing asparagine 6 in TX14(A)
were from
AnaSpec, Inc. (San Jose, CA), and were more than 97% pure. A saposin C-column
was
prepared by conjugation of carbohydrate-free chemically synthesized saposin C
with
Affigel-10 (Bio-Rad, Hercule, CA) by the manufacture's instruction. An
antibody against
human saposin C was purified by the procedure described by Hiraiwa et al.
(Arch.
Biochem. Biophys. 341: 17-24, 1997). Saposin C purified from Gaucher's spleen
(19)
was labeled with 'ZSI-NaI (New England Nuclear Biolabs, Cambridge, MA) using
Iodobeads (Pierce, Rockford, IL) and desalted by Sephadex G-10 column. The
labeled
saposin C gave a single band of 11 kDa on autoradiography after Tricine/SDS-
PAGE
CA 02304108 2000-03-08
WO 99/12559 PCTNS98/19216
-54-
{18). Protease inhibitors were from Sigma Chemical Ltd. (St Louis, MO). Triton
X-100
and sodium deoxycholate were purchased from Calbiochem. (La Jolla, CA). A
polyclonal
antibody specific for the human Goa subunit was obtained from Santa Cruz
Biotechnology (Santa Cruz, CA). All other chemicals and reagents were highest
grade
available.
Affinity purification of the receptor. All purification procedures were
carried
out at 4°C unless specified. Baboon brains ( 1.6 kg) from Southwest
Research Institute
(San Antonio, TX) were washed in chilled 10 mM MOPS, pH 7.5, containing 0.3 M
sucrose and a cocktail of protease inhibitors (5 mM EDTA, 1 mM PMSF, and 5
~cg/ml
of leupeptin, aprotinin, and pepstatin) (Buffer I). The brains were
homogenized in a
teflon-glass homogenizer in 3 volumes of Buffer I and centrifuged at I,S00 X g
for 20
min. The supernatant was then centrifuged at 100,000 X g for 60 min and the
pellet was
washed with Buffer I. Then, the pellet was suspended in lysis buffer (10 mM
Tris-HCI,
pH 7.5, containing 1 % sodium deoxycholate, 1 % Triton X-100, and the same
protease
inhibitor cocktail as in Buffer I) and incubated for 60 min on ice with
shaking. After
removal of the insoluble materials by centrifugation, the supernatant was
mixed with 20
ml of saposin C-beads and rotated for 12 hr. The beads were packed into a
column and
washed with 1 mM sodium phosphate buffer, pH 7.5, containing 0.1 % Triton X-
100 until
protein was not detected in the eluate. The proteins bound to the beads were
eluted with
0.1 M glycine-HCl buffer, pH 3.0, at room temperature (affinity purified
preparation).
Amity cross-1 inking. The affinity purified preparation (4 ,ug of protein) was
dialyzed against 4 changes of 1 liter of 1 mM sodium phosphate buffer, pH 7.5,
containing 0.1% Triton X-100 and then concentrated to 1.4 ml by
ultrafiltration using
Amicon PM-10 membrane. Cross-linking was performed by incubating the
concentrated
samples (0.3 ~cg protein) with'ZSI_saposin C (12.4 ng, 103 cpm/pg) in a final
volume of
200 ~cl of 0.25 M MOPS, pH 7.5, in the presence or absence of 1,000-fold
excess of
unlabeled saposin C. After 90 min-incubation at room temperature, 20 ~cl of 30
mM
disuccinimidyl suberate (Pierce, Rockford, IL) was added to the reaction
mixtures and
incubated for further 20 min. The reaction was terminated by the addition of
12 /.cl of 1
M Tris-HCI, pH 7.5, and the mixtures were left for 20 min at room temperature.
The
CA 02304108 2000-03-08
WO 99/12559 PCTNS98/19216
-55-
product cross-linked with saposin C was immunoprecipitated by an affinity
purified anti
saposin C antibody (2 fig), recovered by Protein A-insoluble (Sigma) then
subjected to
SDS-PAGE. After protein staining, the gel was dried and then exposed to a
Kodak film,
BioMax, at -$0°C.
GTP yS binding. The SHSYSY assay was essentially carried out as described
by Campana et al. (Biochem. Biophys. Res. Commun 229: 706-712, 1996), using
SHSYSY cell membrane preparations. The reaction was performed by incubation of
the
membrane preparations (50-100,ug protein) with 125 ~cCi of [35S]-GTP~yS (New
England
Nuclear Biolabs, 1250 Ci/mmol). In our experiments, a concentration of 3 ,uM
GDP was
added to amplify the difference between ligand stimulated and background
binding.
Unlabelled GTPyS (10 nM) was also added to define non-specific binding and
this value
was subtracted from specific binding. All assays were performed in duplicate.
Results. A putative prosaposin receptor was partially purified from baboon
brain membranes. A solubilized membrane preparation was purified by affinity
chromatography using a saposin C-column. From 1.6 kg of baboon brain, about 25
,ug
of the purified preparation was obtained. The purified preparation gave one
major band
with a molecular weight of 54 kDa on SDS-PAGE. Similar electrophoretic
patterns were
also observed in purified preparations from membrane fractions of human brain,
pig
brain and PC12 cells. Cross-linking experiment using 'ZSI-saposin C and the
purified
preparation demonstrated the presence of a 66 kDa band. On the other hand, no
band was
observed in the sample cross-linked in the presence of unlabeled saposin C.
Since
saposin C has a molecular weight of 11 kDa, a molecular weight of the putative
receptor
was calculated as 55 kDa.
MAP kinase phosphorylation induced by prosaposin receptor agonists in
primary Schwann cells is blocked by treatment with pertussis toxin. To
investigate
whether prosaptides interacted with a G-protein coupled receptor, a GTPyS-
binding
assay was performed using TX14(A) and membrane preparations from SHSYSY cells.
As shown in FIG. 13A, agonist-stimulated binding was increased by 50-60% above
control values in a dose-dependent manner at a maximal concentration of 2 nM.
Activation peaked at 2 nm and was bimodal similar to MAP kinase activation in
PC12
CA 02304108 2000-03-08
WO 99/I2559 PCT/US98/19216
-56-
cells. Other agonists which were active included saposin C (0.3 nM), and a 14-
mer
derived from the neurotrophic sequence of rat saposin C (SELIINNATEELLY; SEQ
)D
N0:12). A mutant peptide 14M1 (TXLIDDNATEEILY; where X=D-alanine) inhibited
the stimulation of TX14(A) in a dose-dependent manner with maximal inhibition
at a
concentration of 0.5 nM. Pretreatment of SHSYSY cells for 4 hr with pertussis
toxin (100
ng/ml) prior to membrane preparation abolished the agonist-stimulated binding
of
TX14(A). An all D-amino acid-derivative of TX14(A) and a scrambled peptide
derivative of TX14(A) were inactive. Furthermore, the purified receptor
preparation was
analyzed for G-proteins by western blotting using an antibody against Goa. The
purified
preparation contained cross-reacting material of 40 kDa indicating that Goa
copurified
with the receptor.
This EXAMPLE shows that the prosaposin receptor has a molecular weight
of 55 kDa and is a G° protein-associated receptor. Saposin C also
interacts with a 56 kDa
lysosomal protein, glucocerebrosidase, to stimulate the enzymic hydrolysis of
glucocerebroside. Western blot analysis using an antibody against purified
human
glucocerebrosidase gave no cross-reacting material in the purified receptor
preparation.
Prosaposin receptor agonists induced MAP kinase phosphorylation in Schwann
cells, and
this phosphorylation was blocked both by the treatment with pertussis toxin
and a non-
hydrolyzable GDP analog, GDP[3S. GTPyS-binding to cell membranes has been
utilized
to characterize agonist-promoted activation of several G-protein associated
receptors
including opioid receptors and 5-hydroxy tryptophane receptors. The assay
relies upon
agonist-promoted GDP/GTP exchange occurring at the G-protein level within the
receptor/G-protein complex; [35S]GTPyS binding is used to assess receptor
activation
since GTPyS is only slowly hydrolyzed by the intrinsic GTPase activity of the
G-protein.
Using SHSYSY membrane preparations, agonist stimulated binding by ,u-opioid
agonists
was about twice the control level whereas we obtained about 50-70%
augmentation using
TX14(A) and saposin C. Stimulation was dose dependent, saturable and inhibited
by a
mutant peptide. These results demonstrated that saposin C and prosaptides were
active
as ligands in a functional measure of receptor-associated G-protein
activation. In addition
CA 02304108 2000-03-08
WO 99/12559 PCT/US98/19216
-57-
the evidence that Goa copurifies with the putative prosaposin receptor
indicated that this
pertussis toxin-sensitive G protein was associated with the receptor.
It should be noted that the present invention is not limited to only those
embodiments described in the DETAILED DESCRIPTION. Any embodiment that
retains the spirit of the present invention should be considered to be within
its scope.
However, the invention is only limited by the scope of the following claims.
CA 02304108 2000-03-08
WO 99/12559 PCT/US9$/19216
-58-
SEQUENCE LISTING
<110> O'Brien, John S
<120> Inhibition of Apoptosis Using Prosaposin Receptor
Agonists
<130> 07256/027001
<140>
<141>
<160> 12
<170> PatentIn Ver. 2.0
<210>1
<211>2749
<212>DNA
<213>Homo sapiens
<220>
<221> CDS
<222> (1)..(1572)
<400> 1
atgtacgccctc ttcctcctggcc agcctcctg ggcgcgget ctagcc 48
MetTyrAlaLeu PheLeuLeuAla SerLeuLeu GlyAlaAla LeuAla
1 5 10 15
ggcccggtcctt ggactgaaagaa tgcaccagg ggctcggca gtgtgg 96
GlyProValLeu GlyLeuLysGlu CysThrArg GlySerAla ValTrp
20 25 30
tgccagaatgtg aagacggcgtcc gactgcggg gcagtgaag cactgc 144
CysGlnAsnVal LysThrAlaSer AspCysGly AlaValLys HisCys
35 40 45
ctgcagaccgtt tggaacaagcca acagtgaaa tcccttccc tgcgac 192
LeuGlnThrVal TrpAsnLysPro ThrValLys SerLeuPro CysAsp
50 55 60
atatgcaaagac gttgtcaccgca getggtgat atgctgaag gacaat 240
IleCysLysAsp ValValThrAla AlaGlyAsp MetLeuLys AspAsn
65 70 75 80
CA 02304108 2000-03-08
WO 99/12559 PCTNS98/19216
-59-
gccact gaggaggag atccttgtt tacttggag aagacctgt gactgg 288
AlaThr GluGluGlu IleLeuVal TyrLeuGlu LysThrCys AspTrp
85 90 95
cttccg aaaccgaac atgtctget tcatgcaag gagatagtg gactcc 336
LeuPro LysProAsn MetSerAla SerCysLys GluIleVal AspSer
100 105 110
tacctc cctgtcatc ctggacatc attaaagga gaaatgagc cgtcct 384
TyrLeu ProValIle LeuAspIle IleLysGly GluMetSer ArgPro
115 120 125
ggggag gtgtgctct getctcaac ctctgcgag tctctccag aagcac 432
GlyGlu ValCysSer AlaLeuAsn LeuCysGlu SerLeuGln LysHis
130 135 140
ctagca gagctgaat caccagaag cagctggag tccaataag atccca 480
LeuAla GluLeuAsn HisGlnLys GlnLeuGlu SerAsnLys IlePro
145 150 155 160
gagctg gacatgact gaggtggtg gcccccttc atggccaac atccct 528
GluLeu AspMetThr GluValVal AlaProPhe MetAlaAsn IlePro
165 170 175
ctcctc ctctaccct caggacggc ccccgcagc aagccccag ccaaag 576
LeuLeu LeuTyrPro GlnAspGly ProArgSer LysProGln ProLys
180 185 190
gataat ggggacgtt tgccaggac tgcattcag atggtgact gacatc 624
AspAsn GlyAspVal CysGlnAsp CysIleGln MetValThr AspIle
195 200 205
cagact getgtacgg accaactcc acctttgtc caggccttg gtggaa 672
GlnThr AlaValArg ThrAsnSer ThrPheVal GlnAlaLeu ValGlu
210 215 220
catgtc aaggaggag tgtgaccgc ctgggccct ggcatggcc gacata 720
HisVal LysGluGlu CysAspArg LeuGlyPro GlyMetAla AspIle
225 230 235 240
tgcaag aactatatc agccagtat tctgaaatt getatccag atgatg 768
CysLys AsnTyrIle SerGlnTyr SerGluIle AlaIleGln MetMet
245 250 255
atg cac atg caa ccc aag gag atc tgt gcg ctg gtt ggg ttc tgt gat 816
Met His Met Gln Pro Lye Glu Ile Cys Ala Leu Val Gly Phe Cys Asp
260 265 270
gag gtg aaa gag atg ccc atg cag act ctg gtc ccc gcc aaa gtg gcc 864
Glu Val Lys Glu Met Pro Met Gln Thr Leu Val Pro Ala Lys Val Ala
275 280 285
tcc aag aat gtc atc cct gcc ctg gaa ctg gtg gag ccc att aag aag 912
Ser Lys Asn Val Ile Pro Ala Leu Glu Leu Val Glu Pro Ile Lys Lys
290 295 300
cac gag gtc cca gca aag tct gat gtt tac tgt gag gtg tgt gaa ttc 960
His Glu Val Pro Ala Lys Ser Asp Val Tyr Cys Glu Val Cys Glu Phe
305 310 315 320
CA 02304108 2000-03-08
WO 99/12559 PCT/US98/19216
-60-
ctg gtg aag gag gtg acc aag ctg att gac aac aac aag act gag aaa 1008
Leu Val Lys Glu Val Thr Lys Leu Ile Asp Asn Asn Lys Thr Glu Lys
325 330 335
gaa ata ctc gac get ttt gac aaa atg tgc tcg aag ctg ccg aag tcc 1056
Glu Ile Leu Asp Ala Phe Asp Lys Met Cys Ser Lys Leu Pro Lys Ser
340 345 350
ctg tcg gaa gag tgc cag gag gtg gtg gac acg tac ggc agc tcc atc 1104
Leu Ser Glu Glu Cys Gln Glu Val Val Asp Thr Tyr Gly Ser Ser Ile
355 360 365
ctg tcc atc ctg ctg gag gag gtc agc cct gag ctg gtg tgc agc atg 1152
Leu Ser Ile Leu Leu Glu Glu Val Ser Pro Glu Leu Val Cys Ser Met
370 375 380
ctg cac ctc tgc tct ggc acg cgg ctg cct gca ctg acc gtt cac gtg 1200
Leu His Leu Cys Ser Gly Thr.Arg Leu Pro Ala Leu Thr Val His Val
385 390 395 400
act cag cca aag gac ggt ggc ttc tgc gaa gtg tgc aag aag ctg gtg 1248
Thr Gln Pro Lys Asp Gly Gly Phe Cys Glu Val Cys Lys Lys Leu Val
405 410 415
ggt tat ttg gat cgc aac ctg gag aaa aac agc acc aag cag gag atc 1296
Gly Tyr Leu Asp Arg Asn Leu Glu Lys Asn Ser Thr Lys Gln Glu Ile
420 425 430
ctg.gct get ctt gag aaa ggc tgc agc ttc ctg cca gac cct tac cag 1344
Leu Ala Ala Leu Glu Lys Gly Cys Ser Phe Leu Pro Asp Pro Tyr Gln
435 440 445
aag cag tgt gat cag ttt gtg gca gag tac gag ccc gtg ctg atc gag 1392
Lys Gln Cys Asp Gln Phe Val Ala Glu Tyr Glu Pro Val Leu Ile Glu
450 455 460
atc ctg gtg gag gtg atg gat cct tcc ttc gtg tgc ttg aaa att gga 1440
Ile Leu Val Glu Val Met Asp Pro Ser Phe Val Cys Leu Lys Ile Gly
465 470 475 480
gcc tgc ccc tcg gcc cat aag ccc ttg ttg gga act gag aag tgt ata 1488
Ala Cys Pro Ser Ala His Lys Pro Leu Leu Gly Thr Glu Lys Cys Ile
485 490 495
tgg ggc cca agc tac tgg tgc cag aac aca gag aca gca gcc cag tgc 1536
Trp Gly Pro Ser Tyr Trp Cys Gln Asn Thr Glu Thr Ala Ala Gln Cys
500 505 510
aat get gtc gag cat tgc aaa cgc cat gtg tgg aac taggaggagg 1582
Asn Ala Val Glu His Cys Lys Arg His Val Trp Asn
515 520
aatattccat cttggcagaa accacagcat tggttttttt ctacttgtgt gtctggggga 1642
atgaacgcac agatctgttt gactttgtta taaaaatagg gctcccccac ctcccccatt 1702
tctgtgtcct ttattgtagc attgctgtct gcaagggagc ccctagcccc tggcagacat 1762
agctgcttca gtgccccttt tctctctgct agatggatgt tgatgcactg gaggtctttt 1822
agcctgccct tgcatggcgc ctgctggagg aggagagagc tctgctggca tgagccacag 1882
CA 02304108 2000-03-08
WO 99/12559 PCT/US98/19216
-61-
tttcttgact ggaggccatc aaccctcttg gttgaggcct tgttctggcc ctgacatgtg 1942
cttgggcact ggtgggcctg ggcttctgag gtggcctcct gccctgatca gggaccctcc 2002
ccgctttcct gggcctctca gttgaacaaa gcagcaaaac aaaggcagtt ttatatgaaa 2062
gattagaagc ctggaataat caggcttttt aaatgatgta attcccactg taatagcata 2122
gggattttgg aagcagctgc tggtggcttg ggacatcagt ggggccaagg gttctctgtc 2182
cctggttcaa ctgtgatttg gctttcccgt gtctttcctg gtgatgcctt gtttggggtt 2242
ctgtgggttt gggtgggaag agggcaatct gcctgaatgt aacctgctag ctctccgaag 2302
gccctgcggg cctggcttgt gtgagcgtgt ggacagtggt ggccgcgctg tgcctgctcg 2362
tgttgcctac atgtccctgg ctgttgaggc gctgcttcag cctgcacccc tcccttgtct 2422
catagatgct ccttttgacc ttttcaaata aatatggatg gcgagctcct aggcctctgg 2482
cttcctggta gagggcggca tgccgaaggg tctgctcggt gtggattgga tgctggggtg 2542
tgggggttgg aagctgtctg tggcccactt gggcacactt gggcacccac gcttctgtcc 2602
acttctggtt gccaggagac agcaagcaaa gccagcagga catgaagttg ctattaaatg 2662
gacttcgtga tttttgtttt gcactaaagt ttctgtgatt taacaataaa attctgttag 2722
ccagaaaaaa aaaaaaaaaa aaaaaaa 2749
<210> 2
<211> 524
<212> PRT
<213> Homo sapiens
<400> 2
Met Tyr Ala Leu Phe Leu Leu Ala Ser Leu Leu Gly Ala Ala Leu Ala
1 5 10 15
Gly Pro Val Leu Gly Leu Lys Glu Cys Thr Arg Gly Ser Ala Val Trp
20 25 30
Cys Gln Asn Val Lys Thr Ala Ser Asp Cys Gly Ala Val Lys His Cys
35 40 45
Leu Gln Thr Val Trp Asn Lys Pro Thr Val Lys Ser Leu Pro Cys Asp
50 55 60
Ile Cys Lys Asp Val Val Thr Ala Ala Gly Asp Met Leu Lys Asp Asn
65 70 75 80
Ala Thr Glu Glu Glu Ile Leu Val Tyr Leu Glu Lys Thr Cys Asp Trp
85 90 95
Leu Pro Lys Pro Asn Met Ser Ala Ser Cys Lys Glu Ile Val Asp Ser
100 105 110
CA 02304108 2000-03-08
WO 99/12559 PCTNS98/19216
-62-
Tyr Leu Pro Val Ile Leu Asp Ile Ile Lys Gly Glu Met Ser Arg Pro
115 120 125
Gly Glu Val Cys Ser Ala Leu Asn Leu Cys Glu Ser Leu Gln Lys His
130 135 140
Leu Ala Glu Leu Asn Hie Gln Lys Gln Leu Glu Ser Asn Lys Ile Pro
145 150 155 160
Glu Leu Asp Met Thr Glu Val Val Ala Pro Phe Met Ala Asn Ile Pro
165 170 175
Leu Leu Leu Tyr Pro Gln Asp Gly Pro Arg Ser Lys Pro Gln Pro Lys
180 185 190
Asp Asn Gly Asp Val Cys Gln Aap Cys Ile Gln Met Val Thr Asp Ile
195 200 205
Gln Thr Ala Val Arg Thr Asn Ser Thr Phe Val Gln Ala Leu Val Glu
210 215 220
His Val Lys Glu Glu Cys Asp Arg Leu Gly Pro Gly Met Ala Asp Ile
225 230 235 240
Cys Lys Asn Tyr Ile Ser Gln Tyr Ser Glu Ile Ala Ile Gln Met Met
245 250 255
Met His Met Gln Pro Lys Glu Ile Cys Ala Leu Val Gly Phe Cys Asp
260 265 270
Glu Val Lys Glu Met Pro Met Gln Thr Leu Val Pro Ala Lys Val Ala
275 280 285
Ser Lys Asn Val Ile Pro Ala Leu Glu Leu Val Glu Pro Ile Lys Lys
290 295 300
His Glu Val Pro Ala Lys Ser Asp Val Tyr Cys Glu Val Cys Glu Phe
305 310 315 320
Leu Val Lys Glu Val Thr Lys Leu Ile Asp Asn Asn Lys Thr Glu Lys
325 330 335
Glu Ile Leu Asp Ala Phe Asp Lys Met Cys Ser Lys Leu Pro Lys Ser
340 345 350
Leu Ser Glu Glu Cys Gln Glu Val Val Asp Thr Tyr Gly Ser Ser Ile
355 360 365
Leu Ser Ile Leu Leu Glu Glu Val Ser Pro Glu Leu Val Cys Ser Met
370 375 380
Leu His Leu Cys Ser Gly Thr Arg Leu Pro Ala Leu Thr Val His Val
385 390 395 400
Thr Gln Pro Lys Asp Gly Gly Phe Cys Glu Val Cys Lys Lys Leu Val
405 410 415
Gly Tyr Leu Asp Arg Asn Leu Glu Lys Asn Ser Thr Lys Gln Glu Ile
420 425 430
Leu Ala Ala Leu Glu Lys Gly Cys Ser Phe Leu Pro Asp Pro Tyr Gln
435 440 445
CA 02304108 2000-03-08
WO 99/12559 PCT/US98/19216
-63-
Lys Gln Cys Asp Gln Phe Val Ala Glu Tyr Glu Pro Val Leu Ile Glu
450 455 460
Ile Leu Val Glu Val Met Asp Pro Ser Phe Val Cys Leu Lys Ile Gly
465 470 475 480
Ala Cys Pro Ser Ala His Lys Pro Leu Leu Gly Thr Glu Lys Cys Ile
485 490 495
Trp Gly Pro Ser Tyr Trp Cys Gln Asn Thr Glu Thr Ala Ala Gln Cys
500 505 510
Asn Ala Val Glu His Cys Lys Arg His Val Trp Asn
515 520
<210> 3
<211> 80
<212> PRT
<213> Homo sapiens
<400> 3
Ser Asp Val Tyr Cys Glu Val Cys Glu Phe Leu Val Lys Glu Val Thr
1 5 10 15
Lys Leu Ile Asp Asn Asn Lys Thr Glu Lys Glu Ile Leu Asp Ala Phe
20 25 30
Asp Lys Met Cys Ser Lys Leu Pro Lys Ser Leu Ser Glu Glu Cys Gln
35 40 45
Glu Val Val Asp Thr Tyr Gly Ser Ser Ile Leu Ser Ile Leu Leu Glu
50 55 60
Glu Val Ser Pro Glu Leu Val Cys Ser Met Leu His Leu Cys Ser Gly
65 70 75 80
<210> 4
<211> 12
<212> PRT
<213> Homo sapiens
<400> 4
Leu Ile Asp Asn Asn Lys Thr Glu Lys Glu Ile Leu
1 5 10
CA 02304108 2000-03-08
WO 99/12559 PCT/US98/19216
-64-
<210> 5
<211> 18
<212> PRT
<213> Homo sapiens
<400> 5
Tyr Lys Glu Val Thr Lys Leu Ile Asp Asn Asn Lys Thr Glu Lys Glu
1 5 10 15
Ile Leu
<210> 6
<211> 22
<212> PRT
<213> Homo sapiens
<400> 6
Cys Glu Phe Leu Val Lys Glu Val Thr Lys Leu Ile Asp Asn Asn Lys
1 5 10 15
Thr Glu Lys Glu I1e Leu
<210> 7
<211> 14
<212> PRT
<213> Artificial Sequence
<220>
223> Description of Artificial Sequence:Tetradecamer
TX14A
<220>
<221> VARIANT
<222> (2)
<223> The alanine at position 2 is a D amino acid.
<400> 7
Thr Ala Leu Ile Asp Asn Asn Ala Thr Glu Glu Ile Leu Tyr
1 5 10
CA 02304108 2000-03-08
WO 99/12559 PCT/US98/19216
-65-
<210>8
<211>22
<212>PRT
<213>Mus musculus
<400> 8
Cys Gln Phe Val Met Asn Lys Phe Ser Glu Leu Ile Val Asn Asn Ala
1 5 10 15
Thr Glu Glu Leu Leu Tyr
20
<210> 9
<211> 21
<212> PRT
<213> Rattus sp.
<400> 9
Cys Gln Leu Val Asn Arg Lys Leu Ser Glu Leu Ile Ile Asn Asn Ala
1 5 10 15
Thr Glu Glu Leu Leu
20
<210>10
<211>22
<212>PRT
<213>Cavia guianae
<400> 10
Cys Glu Tyr Val Val Lys Lys Val Met Leu Leu Ile Aap Asn Asn Arg
1 5 10 15
Thr Glu Glu Lys Ile Ile
20
<210> 11
<211> 22
<212> PRT
<213> Bos sp.
<220>
CA 02304108 2000-03-08
WO 99/12559 PCT/US98/19216
-66-
<223> Description of Unknown Organism: bovine
<400> 11
Cys Glu Phe Val Val Lys Glu Val Ala Lys Leu Ile Asp Asn Asn Arg
1 5 10 15
Thr Glu Glu Glu Ile Leu
<210> 12
<211> 14
<212> PRT
<213> Rattus sp.
<400> 12
Ser Glu Leu Ile Ile Asn Asn Ala Thr Gln Gln Leu Leu Tyr
1 5 10
