Note: Descriptions are shown in the official language in which they were submitted.
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INHIBITION OF NMDA RECEPTOR SIGNALLING IN REDUCING
NEURONAL DAMAGE
FIELD OF THE INVENTION
This invention relates to methods of reducing the damaging effect of an injury
to mammalian cells by treatment with compounds which reduce the binding
between
N-methyl-D-aspartate receptors and neuronal proteins; pharmaceutical
compositions
comprising said compounds and methods for the preparation of said
pharmaceutical
compositions.
BACKGROUND TO THE INVENTION
Ischemic or traumatic injuries to the brain or spinal cord often produce
irreversible damage to central nervous system (CNS) neurons and to their
processes.
These injuries are major problems to society as they occur frequently, the
damage is
often severe, and at present there are still no effective treatments for acute
CNS
injuries. Clinically, ischemic cerebral stroke or spinal cord injuries
manifest
themselves as acute deteriorations in neurological capacity ranging from small
focal
defects, to catastrophic global dysfunction, to death. It is currently felt
that the final
magnitude of the deficit is dictated by the nature and extent of the primary
physical
insult, and by a time-dependent sequence of evolving secondary phenomena which
cause further neuronal death. Thus, there exists a theoretical time-window, of
uncertain duration, in which a timely intervention might interrupt the events
causing
delayed neurotoxicity. However, little is known about the cellular mechanisms
triggering and maintaining the processes of ischemic or traumatic neuronal
death,
making it difficult to devise practical preventative strategies. Consequently,
there
are currently no clinically useful treatments for cerebral stroke or spinal
cord injury.
In vivo, a local reduction in CNS tissue perfusion mediates neuronal death in
both hypoxic and traumatic CNS injuries. Local hypoperfusion is usually caused
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by a physical disruption of the local vasculature, vessel thrombosis,
vasospasm, or
luminal occlusion by an embolic mass. Regardless of its etiology, the
resulting
ischemia is believed to damage susceptible neurons by impacting adversely on a
variety of cellular homeostatic mechanisms. Although the nature of the exact
disturbances is poorly understood, a feature common to many experimental
models
of neuronal injury is a rise in free intracellular calcium concentration
([Ca2+]i).
Neurons possess multiple mechanisms to confine [Ca2+]; to the low levels,
about
100nM necessary for the physiological function. It is widely believed that a
prolonged rise in [Ca2+]; deregulates tightly-controlled Ca 2+-dependent
processes,
causing them to yield excessive reaction products, to activate normally
quiescent
enzymatic pathways, or to inactivate regulatory cytoprotective mechanisms.
This,
in-turn, results in the creation of experimentally observable measures of cell
destruction, such as lipolysis, proteolysis, cytoskeletal breakdown, pH
alterations
and free radical formation.
The classical approach to preventing Ca 2+ neurotoxicity has been through
pharmacological blockade of Ca2+ entry through Ca 2+ channels and/or of
excitatory
amino acid (EAA) - gated channels. Variations on this strategy often lessen
EAA -
induced or anoxic cell death in vitro, lending credence to the Ca2+-
neurotoxicity
hypothesis. However, a variety of Ca 2+ channel- and EAA- antagonists fail to
protect against neuronal injury in vivo, particularly in experimental Spinal
Cord
Injury (SCI), head injury and global cerebral ischemia. It is unknown whether
this is
due to insufficient drug concentrations, inappropriate Ca 2+ influx blockade,
or to a
contribution from non-Ca2+ dependent neurotoxic processes. It is likely that
Ca 2+
neurotoxicity is triggered through different pathways in different CNS neuron
types.
Hence, successful Ca2+-blockade would require a polypharmaceutical approach.
As a result of investigations, I have discovered methods of reducing the
damaging effect of an injury to mammalian cells by treatment with compounds to
reduce the binding between N-methyl-D-aspartate (NMDA) receptors and neuronal
proteins.
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PUBLICATIONS
1. A. Ghosh, M.E. Greenberg, Science 268, 239 (1995); T.V. Bliss, G.L.
Collingridge,
Nature 361, 31 (1993).
2. J.W. Olney, Kainic acid as a tool in neurobiology., E.G. McGeer, J.W. Olney
and
P.L. McGeer, Eds. (Raven Press, New York, 1978), p. 95.; S.M. Rothman, J.W.
Olney, TINS 10, 299 (1987).; D.W. Choi, Ann NYAcad Sci 747, 162 (1994).
3. S.A. Lipton, P.A. Rosenberg, New EngJMed 330, 613 (1994).
4. R. Sattler, M.P. Charlton, M. Hafner, M. Tymianski, J Neurochem 71, 2349
(1998).; M. Tymianski, M.P. Charlton, P.L. Carlen, C.H. Tator, J Neurosci 13,
2085 (1993).
5. K.O. Cho, C.A. Hunt, M.B. Kennedy, Neuron 9, 929 (1992).
6. H.C. Kornau, L.T. Schenker, M.B. Kennedy, P.H. Seeburg, Science 269, 1737
(1995).; J.E. Brenman, K.S. Christopherson, S.E. Craven, A.W. McGee, D.S.
Bredt,
JNeurosci 16, 7407 (1996).; B.M. Muller, et al, Neuron 17, 255 (1996).
7. H. Dong, et al, Nature 386, 279 (1997).; P.R. Brakeman, et al, Nature 386,
284
(1997).
8. S.E. Craven, D.S. Bredt, Cell 93, 495 (1998).; M. Niethammer, et al, Neuron
20,
693 (1998).; J.H. Kim, D. Liao, L.F. Lau, R.L. Huganir, Neuron 20, 683
(1998).; T.
Tezuka, H. Umemori, T. Akiyama, Nakanishi, T. Yamamoto, Proc Natl Acad Sci
USA 96,435 (1999).
9. Hertz, E., Yu, A.C.H., Hertz, L., Juurlink, B.H.J. & Schousboe, A. in A
dissection
and tissue culture manual of the nervous system (eds Shahar, A., de Vellis,
J.,
Vernadakis, A. & Haber, B.) Vol.1, 183-186 (Alan R. Liss Inc., New York,
1989).
10. S.F. Altschul, et al, Nucleic Acids Research 25, 3389 (1997).
11. O.T. Jones, et al, JNeurosci 17, 6152 (1997).
12. V.L. Dawson, T.M. Dawson, E.D. London, D.S. Bredt, S.H. Snyder, Proc Natl
Acad Sci USA 88, 6368 (1991).; V.L. Dawson, T.M. Dawson, D.A. Bartley, G.R.
CA 02273622 1999-06-02
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Uhl, S.H. Snyder, J Neurosci 13, 2651 (1993).; T.M. Dawson, D.S. Bredt, M.
Fotuhi, P.M. Hwang, S.H. Snyder, Proc Natl Acad Sci USA 88, 7797 (1991).
13. Xiong, Z., Lu, W. & MacDonald, J.F. proc Natl Acad Sci USA 94, 7012-7017
(1997).
14. R. Sattler, M.P. Charlton, M. Hafner, M. Tymianski, J Cereb Blood Flow
Metab
17, 455 (1997).
15. N. Bumashev, Z. Zhou, E. Neher, B. Sakmann, JPhysiol 485, 403 (1995).
16. M. Migaud, et al, Nature 396, 433 (1998).
17. J.R. Brorson, P.T. Schumacker, H. Zhang, JNeurosci 19, 147 (1999).
18. S.R. Jaffrey, S.H. Snyder, Annual Review of Cell & Developmental Biology
11, 417
(1995).
19. S.R. Jaffrey, S.H. Snyder, Annual Review of Cell & Developmental Biology
11, 417
(1995).
SUMMARY OF THE INVENTION
It is a preferred object of the present invention to provide in its broadest
aspect a
method of reducing the damaging effect of an injury to mammalian cells.
In a further preferred object, the invention provides pharmaceutical
compositions for use in treating mammals to reduce the damaging effect of an
injury
to mammalian tissue.
The present invention is based on the discovery of a neuroprotective effect
against excitotoxic and ischemic injury by inhibiting the binding between N-
methyl-
D-aspartate (NMDA) receptors and neuronal proteins in a neuron.
Accordingly, in one aspect the invention provides a method of inhibiting the
binding between N-methyl-D-aspartate receptors and neuronal proteins in a
neuron
said method comprising administering to said neuron an effective inhibiting
amount
of a peptide replacement agent for the NMDA receptor neuronal protein
interaction
domineer or precursor therefor to effect said inhibition.
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In a further aspect, the invention provides a method of reducing the damaging
effect of ischemia or traumatic injury to the brain or spinal chord in a
mammal, said
method comprising treating said mammal with a non-toxic, damage-reducing,
effective amount of a peptide replacement agent for the NMDA receptor neuronal
5 protein interaction domain or precursor therefor.
The NMDA agent is, preferably, bindable with membrane associated guanylate
kinases, and most preferably, is selected from postsynaptic density-95
proteins,
PSD-95, PSD-93 and SAP102.
I have found that the replacement agent is a tSXV-containing peptide or
precursor therefor, preferably KLSSLESDV.
In a yet further aspect the invention provides a pharmaceutical composition
comprising a peptide replacement agent for the NMDA receptor neuronal protein
interaction domain or a precursor therefor in a mixture with a
pharmaceutically
acceptable carrier when used for reducing the damaging effect of an ischemic
or
traumatic injury to the brain or spinal chord of a mammal; preferably further
comprising antessapedia internalisation peptide.
In a further aspect, the invention provides a method of inhibiting the binding
between NMDA receptors and neuronal proteins in a neuron, said method
comprising administering to said neuron an effective inhibiting amount of an
antisense DNA to prevent expression of said neuronal proteins to effect
inhibition of
said binding. Preferably, this aspect provides a method wherein said antisense
DNA
reduces the expression of a membrane associated guanylate kinase bindable to
said
NMDA receptor. More preferably, the guanylate kinase is selected from PSD-95,
PSD-93 and SAP 102.
In the mammalian nervous system, the efficiency by which N-methyl-D-
aspartate receptor (NMDAR) activity triggers intracellular signaling pathways
governs neuronal plasticity, development, senescence and disease. I have
studied
excitotoxic NMDAR signaling by suppressing the expression of the NMDAR
scaffolding protein PSD-95. In cultured cortical neurons, this selectively
attenuated
NMDAR excitotoxicity, but not excitotoxicity by other glutamate or Ca 2+
channels.
NMDAR function was unaffected, as receptor expression, while NMDA-currents
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and 45Ca loading via NMDARs were unchanged. Suppressing PSD-95 selectively
blocked Ca2+ -activated nitric oxide production by NMDARs, but not by other
pathways, without affecting neuronal nitric oxide synthase (nNOS) expression
or
function. Thus, PSD-95 is required for the efficient coupling of NMDAR
activity to
nitric oxide toxicity and imparts specificity to excitotoxic Ca2+ signaling.
It is known that calcium influx through NMDARs plays key roles in mediating
synaptic transmission, neuronal development, and plasticity (1). In excess, Ca
influx
triggers excitotoxicity (2), a process that damages neurons in neurological
disorders
that include stroke, epilepsy, and chronic neurodegenerative conditions (3).
Rapid
Ca2+-dependent neurotoxicity is triggered most efficiently when Ca 2+ influx
occurs
through NMDARs, and cannot be reproduced by loading neurons with equivalent
quantities of Ca 2+ through non-NMDARs or voltage-sensitive Ca 2+ channels
(VSCCs) (4). This observation suggests that Ca 2+ influx through NMDAR
channels
is functionally coupled to neurotoxic signaling pathways.
Without being bound by theory, I believe that lethal Ca 2+ signaling by
NMDARs is determined by the molecules with which they physically interact. The
NR2 NMDAR subunits, through their intracellular C-terminal domains, bind to
PSD-95/SAP90 (5), chapsyn-110/PSD-93, and other members of the membrane-
associated guanylate kinase (MAGUK) family (6). NMDAR-bound MAGUKs are
generally distinct from those associated with non-NMDARs (7). I have found
that
the preferential activation of neurotoxic Ca2+ signals by NMDARs is determined
by
the distinctiveness of NIVIDAR-bound MAGUKs, or of the intracellular proteins
that
they bind. PSD-95 is a submembrane scaffolding molecule that binds and
clusters
NMDARs preferentially and, through additional protein-protein interactions,
may
link them to intracellular signaling molecules (8). Perturbing PSD-95 would
impact
on neurotoxic Ca 2+ signaling through NMDARs.
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BRIEF DESCRIPTION OF THE DRAWINGS
In order that the invention may be better understood preferred embodiments
will
now be described by way of example only with reference to the accompanying
drawings wherein:
Fig.1 a is an immunublot;
Fig. lb is a bar chart providing densitometric analysis of PSD-95 expression;
Fig. I c represents representative phase contrast and propidium fluorescence
images;
Fig.1 d is a bar chart of NMDA concentration against fraction of dead cells;
Fig. 1 e is a bar chart of NMDA concentration against Calcium accumulation.
Fig.2a1-b2 represent bar charts of selective activations of AMPA/Kainate
receptors
with Kainate (2a1 and 2-a2); and loadings with Vscc's (2-bl) and calcium
loading
(2-b2).
Fig.3a-d represent: immunoblots (3a); NMSa dose-response curves (3b); NMDA
current density measurements (3c); and current/time graph (3d) dialyzed with
hucifer yellow; and
Fig.4 bar charts (4a; 4c-4f) and immublot of effect on nNOS expression in
cultures;
hereinafter better described and explained.
DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS
METHODS:
Cultured cortical neurons were prepared by standard techniques (4,9) and
switched to serum-free media at 24h [Neurobasal with B27 supplement (Gibco)].
The AS ODN corresponded to nucleotides 435-449 (5'-GAATGGGTCACCTCC-3')
of mouse PSD-95/SAP90 mRNA (GeneBank Acc. No. D50621). Filter-sterilized
phosphodiester AS (5'-GAATGGGTCACCTCC-3'), SE, and MS (5'-
CCGCTCTATCGAGGA-3') ODNs (5 M) were added in culture medium during
feedings at 4,6,8 and 10 days after plating. Cultures were used for all
experiments
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(figs. 1-4) on day 12. ODN sequences exhibited no similarity to any other
known
mammalian genes (BLAST search (10) ).
Immunoblotting was done as described in ref "26". Tissue was harvested and
pooled from 2 cultures/lane. The blotted proteins were probed using a
monoclonal
anti-PSD-95 mouse IgGI (Transduction Labs, 1:250 dilution), polyclonal anti
PSD-
93 (1:1000 dilution) and anti SAP-102 (1:2000 dilution) rabbit serum
antibodies
(Synaptic Systems GmbH), a monoclonal anti NR1 mouse IgG2a (PharMingen
Canada, 1:1000 dilution) or a monoclonal anti nNOS (NOS type I) mouse IgG2a
(Transduction Labs, 1:2500 dilution). Secondary antibodies were sheep anti-
mouse,
or donkey anti-rabbit Ig conjugated to horseradish peroxidase (Amersham).
Immunoblots for PSD-95 were obtained for all experiments (Figs 1-4) from
sister
cultures, and all gels quantified using an imaging densitometer (Bio-Rad GS-
670).
cGMP determinations were performed 10 min after challenging the cultures
with NMDA, kainate, or high-K (Figs. 4c-e) with the Biotrak* cGMP
enzymeimmunoassay system according to the kit manufacturer's instructions
(Amersham). Staining for NADPH diaphorase (Fig 4b) was done as described in
ref.
12.
Electrophysiology. Whole cell patch-clamp recordings in the cultured neurons
were performed and analyzed as described in ref. 13. During each experiment a
voltage step of -10 mV was applied from holding potential and the cell
capacitance
was calculated by integrating the capacitative transient. The extracellular
solution
contained (in mM): 140 NaCl, 5.4 KCI, 1.3 CaCI2, 25 HEPES, 33 glucose.. 0.01
glycine, and 0.001 tetrodotoxin (pH =7.3-7.4, 320 - 335 mOsm). A multi-barrel
perfusion system was employed to rapidly exchange NNIDA containing solutions.
The pipette solution contained (in mM): 140 CsF, 35 CsOH, 10 HEPES, 11 EGTA,
2 tetraethylammonium chloride (TEA), 1 CaCl2, 4 MgATP, pH 7.3 at 300 mOsm.
Lucifer yellow (LY; 0.5% w/v) was included in the pipette for experiments in
figure
3d.
* Trade-mark
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Excitotoxicity and Ca2+ accumulation measurements were performed identically
to the methods described and validated in refs. 4 and 14. We used measurements
of
propidium iodide fluorescence as an index of cell death, and of radiolabelled
45Ca2+
accumulation for Ca2+ load determinations in sister cultures on the same day.
Experimental solutions were as previously described (4). Ca 2+ influx was
pharmacologically channeled through distinct pathways as follows: To NMDARs by
applying NMDA (x60 min) in the presence of both CNQX (Research Biochemicals
Inc) and nimodipine (Miles Pharmaceuticals), to non-NMDARs by applying kainic
acid (x60 min or 24h) in the presence of both MK-801 (RBI) and nimodipine, and
to
VSCCs using 50 mM K+ solution (x60 min) containing 10mM Ca2+ and S(-)-Bay K
8644, an L-type channel agonist (300-500nM; RBI), MK-801 and CNQX.
Antagonist concentrations were (in M): MK-801 10, CNQX 10, nimodipine 2. All
three antagonists were added after the 60 min agonist applications for the
remainder
of all experiments (24 h). A validation of this approach in isolating Ca 2+
influx to the
desired pathway in our cortical cultures has been published (4).
Whole cell patch-clamp recordings in the cultured neurons were performed and
analyzed as described in Z. Xiong, W. Lu, J.F. MacDonald, Proc Natl Acad Sci
USA 94, 7012 (1997). During each experiment a voltage step of -10 mV was
applied
from holding potential and the cell capacitance was calculated by integrating
the
capacitative transient. The extracellular solution contained (in mM): 140
NaCl, 5.4
KCI, 1.3 CaC12, 25 HEPES, 33 glucose, 0.01 glycine, and 0.001 tetrodotoxin (pH
=7.3-7.4, 320 - 335 mOsm). A multi-barrel perfusion system was employed to
rapidly exchange NMDA containing solutions. The pipette solution contained (in
mM): 140 CsF, 35 CsOH, 10 HEPES, 11 EGTA, 2 tetraethylammonium chloride
(TEA), 1 CaCl2, 4 MgATP, pH 7.3 at 300 mOsm. Lucifer yellow (LY; 0.5% w/v)
was included in the pipette for experiments in figure 3D.
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Data analysis: data in all figures were analyzed by ANOVA, with a post-hoc
Student's t-test using the Bonferroni correction for multiple comparisons. All
means
are presented with their standard errors.
In greater detail:
5 Figure 1, shows increased resilience of PSD-95 deficient neurons to NMDA
toxicity in spite of Ca 2+ loading. A. Immunoblot showing representative
effects of
sham (SH) washes, and PSD-95 AS, SE and MS ODNs, on PSD-95 expression. PC:
positive control tissue from purified rat brain cell membranes. Asterisk: non-
specific
band produced by the secondary antibody, useful to control for protein loading
and
10 blot exposure times. B. Densitometric analysis of PSD-95 expression pooled
from N
experiments. Asterisk: different from other groups, one-way ANOVA, F = 102,
p<0.0001. ODNs were used at 5 M except where indicated (AS 2 M). C.
Representative phase contrast and propidium iodide fluorescence images of PSD-
95
deficient (AS) and control (SE) cultures 24 h after a 60 min challenge with 30
M
NMDA. Scale bar: 100 m. D. Decreased NMDA toxicity at 24h in PSD-95
deficient neurons following selective NMDAR activation x 60 min (n=16
cultures/bar pooled from N=4 separate experiments). Asterisk: differences from
SE,
MS and SH (Bonferroni t-test, p<0.005). Death is expressed as the fraction of
dead
cells produced by 100 M NMDA in sham-ODN-treated controls (validated in 4,14).
E. No effect of PSD-95 deficiency on NMDAR-mediated Ca 2+ loading (n = 12/bar,
N = 3; reported as the fraction of 45Ca2+ accumulation achievable over 60 min
in the
sham controls by 100 M NMDA, which maximally loads the cells with calcium (4).
Figure 2, shows thatPSD-95 deficiency does not affect toxicity and Ca 2+
loading
produced by activating non-NMDARs and Ca 2+ channels. Cultures were treated
with
SH washes or AS or SE ODNs as in Fig. 1. A. Selective activation of
AMPA/kainate receptors with kainate in MK-801 (10 M) and nimodipine (NIM;
2 M) produces toxicity over 24h (Al) irrespective of PSD-95 deficiency, with
minimal 45Ca2+ loading (A2). B. Selective activation of VSCCs produces little
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toxicity (B 1), but significant 45Ca2+ loading (B2) that is also insensitive
to PSD-95
deficiency. n = 4 cultures/bar in all experiments.
Figure 3, shows that there is no effect of perturbing PSD-95 on receptor
function. A. Immunoblots of PSD-95 ODN-treated cultures probed for PSD-95,
NR1, PSD-93, and SAP-102 using specific antibodies. PC: positive control
tissue
from purified rat brain cell membranes. B. NMDA dose-response curves and
representative NMDA currents (inset) obtained with 3-300 M NMDA. C. NMDA
current density measurements elicited with 300 M NMDA (AS: n = 18; SE: n =19;
SH: n = 17; one-way ANOVA F=1.10, p=0.34), and analysis of NMDA current
desensitization. Iss = steady-state current; Ipeak = peak current. AS: n=15;
SE: n = 16;
SH: n = 16 (ANOVA,, F=0.14, p=0.87). Time constants for current decay were AS:
1310 158 ms; SE, 1530 185 ms; SH: 1190 124 ms (ANOVA, F= 1.22, p=
0.31). D. Currents elicited with 300 M NMDA in neurons dialyzed with LY
(insert)
and 1 mM tSXV or control peptide.
Figure 4, shows the effect of coupling of NMDAR activation to nitric oxide
signaling by PSD-95. A. L-NAME protects against NMDA toxicity (n = 4, N = 2).
Asterisk: difference from 0 M L-NAME (Bonferroni t-test, p<0.05). B. No
effect of
SH and of PSD-95 AS and MS ODNs on nNOS expression in cultures (immunoblot)
and on NADPH diaphorase staining in PSD-95 AS and SE-treated neurons. PC:
positive control tissue from purified rat brain cell membranes. C. Effect of
isolated
NMDAR activation on cGMP formation (n=12 cultures/bar pooled from N=3
separate experiments) D,E. Effects of VSCC activation (n = 8/bar, N = 2), and
AMPA/kainate receptor activation (n = 4/bar, N= 1) on cGMP
formation. Data in C-E are expressed as the fraction of cGMP produced in SE-
treated cultures by 100 M NMDA. Asterisk: differences from both SH and SE
controls (Bonferroni t-test, p<0.0001). F. Sodium nitroprusside toxicity is
similar in
PSD-95 AS, SE and SH treated cultures.
PSD-95 expression was suppressed in cultured cortical neurons to < 10% of
control levels, using a 15-mer phosphodiester antisense (AS)
oligodeoxynucleotide
(ODN) (Fig. IA,B) Sham (SH) washes, sense (SE) and missense (MS) ODNs (9)
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had no effect. The ODNs had no effect on neuronal survivability and morphology
as
gauged by viability assays, herein below, and phase-contrast microscopy (not
shown).
To examine the impact of PSD-95 on NMDAR-triggered excitotoxicity, ODN-
treated cultures were exposed to NMDA (10-100 .tM) for 60 min, washed, and
either used for 45Ca2+ accumulation measurements, or observed for a further 23
h.
Ca 2+ influx was isolated to NMDARs by adding antagonists of non-NMDARs and
Ca2+ channels (4). NMDA toxicity was significantly reduced in neurons
deficient in
PSD-95 across a range of insult severities (Figs. 1C,D; EC50: AS: 43.2 4.3;
SE:
26.3 3.4, Bonferroni t-test, p <0.005). Concomitantly however, PSD-95
deficiency
had no effect on Ca2+ loading into identically treated sister cultures (Fig.
1E).
Therefore, PSD-95 deficiency induces resilience to NMDA toxicity despite
maintained Ca 2+ loading.
I next examined whether the increased resilience to Ca2+ loading in PSD-95
deficient neurons was specific to NMDARs. Non-NMDAR toxicity was produced
using kainic acid (30-300 M), a non-desensitizing AMPA/kainate receptor
agonist
(15), in the presence of NMDAR and Ca 2+ channel antagonists (4). Kainate
toxicity
was unaffected in PSD-95 deficient in neurons challenged for either 60 min
(not
shown) or 24 h (Fig. 2A1). Non-NMDAR toxicity occurred without significant
45Ca2+ loading (Fig. 2A2), as >92% of neurons in these cultures express
impermeable AMPA receptors (4). However, Ca 2+ loading through VSCCs, which is
non-toxic (4) (Fig. 2B1), was also unaffected by PSD-95 deficiency (Fig. 2B2).
Thus, suppressing PSD-95 expression affects neither toxicity nor Ca2+ fluxes
triggered through pathways other than NMDARs.
Immunoblot analysis (11) of PSD-95 deficient cultures revealed no alterations
in the expression of the essential NMDAR subunit NR1, nor of two other NMDAR-
associated MAGUKs, PSD-93 and SAP-102 (Fig. 3A). This indicated that altered
expression of NN DARs and their associated proteins was unlikely to explain
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reduced NMDA toxicity in PSD-95 deficiency (Fig. 1 C,D). Therefore, I examined
the possibility that PSD-95 modulates NMDAR function. NMDA currents were
recorded using the whole-cell patch technique (16) (Fig. 3B). PSD-95
deficiency had
no effect on passive membrane properties, including input resistance and
membrane
capacitance [Capacitance: AS 55.0 2.6 pF (n =18 ); SE 52.7 3.2 pF (n=19);
SH
48.1 3.4 pF (n = 17; ANOVA, F=1.29, p=0.28)]. Whole-cell currents elicited
with
3-300 M NMDA were also unaffected. Peak currents were AS: 2340 255 pA
(n=18); SE: 2630 276 (n=19); SH: 2370 223 (n=17) (Fig. 3B, inset; one-way
ANOVA, F = 0.43, p = 0.65). NMDA dose-response relationships also remained
unchanged (Fig. 3B; EC50 AS: 16.1 0.8 M (n=7); SE: 15.5 2.1 (n=6); SH:
15.9
2.9; one-way ANOVA, F= 0.02, p = 0.98), as were NMDA current density and
desensitization (Figs. 3C).
To further examine the effect of PSD-95 binding on NMDAR function, a 9 as
peptide (KLSSIESDV) corresponding to the C-terminal domain of the NR2B
subunit characterized by the tSXV motif (6) was injected into the neurons. At
0.5mM, this peptide competitively inhibited the binding of PSD-95 to GST-NR2B
fusion proteins (6), and was therefore predicted to uncouple NMDARs from PSD-
95. Intracellular dialysis of 1mM tSXV or control peptide (CSKDTMEKSESL) (6)
was achieved through patch pipettes (3-5 MCI) also containing the fluorescent
tracer
Lucifer Yellow (LY). This had no effect on NMDA currents over 30 min despite
extensive dialysis of LY into the cell soma
and dendrites (Fig. 3D). Peak current amplitudes were tSXV: 2660 257 pA (n=
9),
control: 2540 281 pA (n= 10; t(17) = 0.31, p = 0.76).
The data is consistent with that obtained from recently generated mutant mice
expressing a truncated 40K PSD-95 protein that exhibited enhanced LTP and
impaired learning (17). Hippocampal CAI neurons in PSD-95 mutants exhibited no
changes in NMDAR subunit expression and stoichiometry, cell density, dendritic
cytoarchitecture, synaptic morphology, or NMDAR localization using NR1
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immunogold labeling of asymmetric synapses. NMDA currents, including synaptic
currents, were also unchanged (16). I also found no effects of PSD-95
deficiency on
NMDAR expression, on other NMDAR associated MAGUKs, nor on NMDA-
evoked currents. In addition, NMDAR function gauged by measuring NMDA-
evoked 45Ca2+-accumulation was unaffected. Thus, the neuroprotective
consequences of PSD-95 deficiency must be due to events downstream from
NMDAR activation, rather than to altered NMDAR function.
The second PDZ domain of PSD-95 binds to the C-terminus of NR2 subunits
and to other intracellular proteins (8). Among these is nNOS (18), an enzyme
that
catalyzes the production of nitric oxide (NO), a short-lived signaling
molecule that
also mediates Cat+-dependent NMDA toxicity in cortical neurons (12). Although
never demonstrated experimentally, the NMDAR/PSD-95/nNOS complex was
postulated to account for the preferential production of NO by NMDARs over
other
pathways (8). To determine whether NO signaling plays a role in NMDA toxicity
in
the present cultures, we treated the cells with 1VG-nitro-L-arginine methyl
ester (L-
NAME), a NOS inhibitor (12). L-NAME protected the neurons against NMDA
toxicity (Fig. 4A), indicating the possibility that suppressing PSD-95 might
perturb
this toxic signaling pathway.
The effect of suppressing PSD-95 expression on NO signaling and toxicity was
examined using cGMP formation as a surrogate measure of NO production by Cat+-
activated nNOS (20,21). PSD-95 deficiency had no impact on nNOS expression
(Fig. 4B), nor on the morphology (Fig. 4B) or counts of NADPH diaphorase-
staining
(12) neurons (SH: 361 60, SE: 354 54, AS: 332 42 staining neurons /10mm
coverslip, 3 coverslips/group). However, in neurons lacking PSD-95 challenged
with
NMDA under conditions that isolated Ca2+ influx to NMDARs (4), cGMP
production was markedly attenuated (>60%; Fig. 4C, one-way ANOVA, p<0.0001).
Like inhibited toxicity (Figs. 1,2), inhibited cGMP formation in neurons
lacking
PSD-95 was only observed in response to NMDA. It was unaffected in neurons
CA 02273622 1999-06-02
loaded with Ca2+ through VSCCs (Fig. 4D), even under high neuronal Ca 2+ loads
matching those attained by activating NMDARs (compare Figs. 1E and 2B2) (4).
nNOS function therefore, was unaffected by PSD-95 deficiency. AMPA/kainate
receptor activation failed to load the cells with Ca2+ (Fig. 2A2), and thus
failed to
5 increase cGMP levels (Fig. 4E). Our findings indicate that suppressing PSD-
95
selectively reduces NO production efficiency by NMDAR-mediated Ca 2+ influx,
but
preserves NO production by Ca 2+ influx through other pathways.
Bypassing nNOS activation with NO donors restored toxicity in neurons lacking
PSD-95. The NO donors sodium nitroprosside (12) (Fig. 4F; EC50 300 M) and S-
10 nitrosocysteine (17) (not shown) were highly toxic, irrespective of PSD-95
deficiency. Thus, reduced NMDA toxicity in PSD-95 deficient cells was unlikely
to
be caused by altered signaling events downstream from NO formation.
Suppressing PSD-95 expression uncoupled NO formation from NMDAR
activation (Fig. 4C), and protected neurons against NMDAR toxicity (Fig. 1
C,D)
15 without affecting receptor function (Figs I E, 3A-D), by mechanisms
downstream
from NMDAR activation, and upstream from NO-mediated toxic events (Fig. 4F).
Therefore, PSD-95 imparts NMDARs with signaling and neurotoxic specificity
through the coupling of receptor activity to critical second messenger
pathways. Our
results have broader consequences, as NMDAR activation and NO signaling are
also
critical to neuronal plasticity, learning, memory, and behavior (1,18,19).
Thus, our
report provides experimental evidence for a mechanism by which PSD-95 protein
may govern important physiological and pathological aspects of neuronal
functioning.
Although this disclosure has described and illustrated certain preferred
embodiments of the invention, it is to be understood that the invention is not
restricted to those particular embodiments. Rather, the invention includes all
embodiments which are functional or mechanical equivalence of the specific
embodiments and features that have been described and illustrated.
CA 02273622 2005-02-01
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SEQUENCE LISTING
1. GENERAL INFORMATION
(i) APPLICANT:
(A) : TYMIANSKI, Michael
(ii) TITLE OF INVENTION: METHOD OF REDUCING INJURY TO MN-1MALIAN CELLS
(iii) NUMBER OF SEQUENCES: 3
(iv) CORRESPONDENCE ADDRESS:
(A) NAME: DEETH WILLIAMS WALL LLP
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(A) APPLICATION NO.: 2273622
(B) FILING DATE: June 2, 1999
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(A) NAME: DEETH WILLIAMS WALL LLP
(B) REFERENCE NUMBER: 4438 0005
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(A) OTHER INFORMATION: Description of Unknown Organism: peptide
CA 02273622 2005-02-01
- 17 -
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(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 9
(B) TYPE: PRT
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