U.S. patent application number 11/399840 was filed with the patent office on 2006-10-19 for methods for modulating neuronal responses.
Invention is credited to Lidong Liu, Yitao Liu, Anthony Phillips, Yu Tian Wang, Yushan Wang.
Application Number | 20060234912 11/399840 |
Document ID | / |
Family ID | 34421804 |
Filed Date | 2006-10-19 |
United States Patent
Application |
20060234912 |
Kind Code |
A1 |
Wang; Yu Tian ; et
al. |
October 19, 2006 |
Methods for modulating neuronal responses
Abstract
The invention provides, in part, methods and reagents for
modulating neuronal apoptosis and for modulating synaptic
plasticity.
Inventors: |
Wang; Yu Tian; (Vancouver,
CA) ; Wang; Yushan; (Medicine Hat, CA) ;
Phillips; Anthony; (Vancouver, CA) ; Liu; Lidong;
(Richmond, CA) ; Liu; Yitao; (Richmond,
CA) |
Correspondence
Address: |
BLACK LOWE & GRAHAM PLLC
Suite 4800
701 Fifth Avenue
Seattle
WA
98104
US
|
Family ID: |
34421804 |
Appl. No.: |
11/399840 |
Filed: |
April 6, 2006 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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PCT/CA04/01813 |
Oct 8, 2004 |
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11399840 |
Apr 6, 2006 |
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60509249 |
Oct 8, 2003 |
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Current U.S.
Class: |
514/17.7 ;
514/18.9; 514/21.6 |
Current CPC
Class: |
A61P 9/10 20180101; A61P
9/12 20180101; G01N 2500/04 20130101; G01N 2500/02 20130101; A61K
38/00 20130101; A61P 3/04 20180101; A61P 3/10 20180101; A61P 25/14
20180101; A61P 3/00 20180101; A61P 9/00 20180101; A61K 31/7088
20130101; A61P 25/20 20180101; A61P 25/16 20180101; A61P 25/22
20180101; A61P 9/04 20180101; C07K 14/705 20130101; A61P 25/08
20180101; A61P 25/00 20180101; A61P 25/30 20180101; A61P 25/18
20180101; A61P 9/02 20180101; A61P 25/34 20180101; C12N 9/1205
20130101; G01N 2333/705 20130101; A61P 25/28 20180101; A61P 25/24
20180101; A61P 25/36 20180101; C07K 7/06 20130101; A61P 25/04
20180101 |
Class at
Publication: |
514/002 |
International
Class: |
A61K 38/00 20060101
A61K038/00 |
Claims
1-48. (canceled)
49. A method of modulating NMDA-mediated neuronal apoptosis, the
method comprising contacting a neuronal cell with an inhibitor of
AMPA receptor endocytosis.
50. The method of claim 49, wherein the inhibitor is an inhibitor
of regulated AMPA receptor endocytosis.
51. The method of claim 49, wherein the inhibitor is a GluR2,
GluR3, or GluR4 polypeptide.
52. The method of claim 49, wherein the inhibitor comprises the
amino acid sequence set forth in Table I or conservative
substitutions thereof, Formula I, or Formula A, or homologous
sequences thereto found in the C-terminus of the GluR2, GluR3, or
GluR4 subunits of the AMPA receptor or a fragment or variant
thereof, or comprises an antibody that specifically binds the amino
acid sequence set forth in Table I or conservative substitutions
thereof, Formula I, or Formula A, or homologous sequences thereto
found in the C-terminus of the GluR2, GluR3, or GluR4 subunits of
the AMPA receptor.
53. The method of claim 52, wherein the fragment comprises the
amino acid sequence of YREGYNVYG, YKEGYNVYG, YREGYNVYGIE or
YKEGYNVYGIE.
54. The method of claim 49, wherein the inhibitor further comprises
the amino acid sequence YGRKKRRQRRR or a carrier peptide sequence
that facilitates translocation of the inhibitor across cell
membranes.
55. A method of modulating AMPA receptor endocytosis, the method
comprising contacting a cell expressing an AMPA receptor with a
modulatory compound comprising the amino acid sequence set forth in
Table I or conservative substitutions thereof, Formula I, or
Formula A, or homologous sequences thereto found in the C-terminus
of the GluR2, GluR3, or GluR4 subunits of the AMPA receptor or a
fragment or variant thereof, or comprising an antibody that
specifically binds the amino acid sequence set forth in Table I or
conservative substitutions thereof, Formula I, or Formula A, or
homologous sequences thereto found in the C-terminus of the GluR2,
GluR3, or GluR4 subunits of the AMPA receptor.
56. The method of claim 55, wherein the modulatory compound is an
inhibitor of regulated AMPA receptor endocytosis.
57. The method of claim 55, wherein the modulatory compound is a
GluR2, GluR3, or GluR4 polypeptide.
58. The method of claim 55, wherein the fragment comprises the
amino acid sequence of YREGYNVYG, YKEGYNVYG, YREGYNVYGIE or
YKEGYNVYGIE.
59. The method of claim 55, wherein the modulatory compound further
comprises the amino acid sequence YGRKKRRQRRR or a carrier peptide
sequence that facilitates translocation of the inhibitor across
cell membranes.
60. A method of treating or preventing neurological damage or
dysfunction in a subject, the method comprising administering an
effective amount of an inhibitor of AMPA receptor endocytosis to
the subject.
61. The method of claim 60, wherein the inhibitor is an inhibitor
of regulated AMPA receptor endocytosis.
62. The method of claim 60, wherein the inhibitor is a GluR2,
GluR3, or GluR4 polypeptide.
63. The method of claim 60, wherein the inhibitor comprises the
amino acid sequence set forth in Table I or conservative
substitutions thereof, Formula I, or Formula A, or homologous
sequences thereto found in the C-terminus of the GluR2, GluR3, or
GluR4 subunits of the AMPA receptor or a fragment or variant
thereof, or comprises an antibody that specifically binds the amino
acid sequence set forth in Table I or conservative substitutions
thereof, Formula I, or Formula A, or homologous sequences thereto
found in the C-terminus of the GluR2, GluR3, or GluR4 subunits of
the AMPA receptor.
64. The method of claim 63, wherein the fragment comprises the
amino acid sequence of YREGYNVYG, YKEGYNVYG, YREGYNVYGIE or
YKEGYNVYGIE.
65. The method of any one of claims 12 through 16, wherein the
inhibitor further comprises the amino acid sequence YGRKKRRQRRR or
a carrier peptide sequence that facilitates translocation of the
inhibitor across cell membranes.
66. The method of claim 60, wherein the neurological damage
comprises NMDA-induced neuronal apoptosis.
67. The method of claim 60, wherein the neurological damage occurs
as a result of excessive activation of NMDA receptors or due to
changes in AMPA receptor endocytosis.
68. The method of claim 60, wherein the neurological damage or
dysfunction occurs as a result of at least one of a disorder
selected from the group consisting of stress, anxiety, depression,
hypoglycemia, cardiac arrest, epilepsy, cerebral ischemia, brain
trauma, Alzheimer's disease, Parkinson's disease, Huntington's
disease; neuropathic pain; amyotrophic lateral sclerosis (ALS);
Hutchinson Gilford syndrome; diabetes; ataxia; mental retardation;
dementias, disorders associated with smoking or obesity, high blood
pressure, disorders associated with defects or dysfunction in
learning or memory, psychiatric disorders, autism, schizophrenia,
fragile X syndrome, and disorders associated with substance abuse
or addiction to a drug.
69. The method of claim 68, wherein the drug is selected from at
least one of the group consisting of nicotine, alcohol, opiates,
heroin, codeine, morphine pethidine, methadone, marijuana,
phenyclidene, psychostimulants, amphetamines, cocaine,
barbiturates, pentobarbitone, quinalbarbitone, benzodiazepines,
temazepam, diazepam and flunitrazepam.
70. A method of modulating synaptic plasticity in a subject, the
method comprising administering an effective amount of an inhibitor
of AMPA receptor endocytosis to the subject.
71. The method of claim 70, wherein the inhibitor is an inhibitor
of regulated AMPA receptor endocytosis.
72. The method of claim 70, further comprising enhancing synaptic
plasticity.
73. The method of claim 70, wherein the subject is normal.
74. The method of claim 70, wherein the inhibitor comprises the
amino acid sequence set forth in Table I or conservative
substitutions thereof, Formula I, or Formula A, or homologous
sequences thereto found in the C-terminus of the GluR2, GluR3, or
GluR4 subunits of the AMPA receptor or a fragment or variant
thereof, or comprises an antibody that specifically binds the amino
acid sequence set forth in Table I or conservative substitutions
thereof, Formula I, or Formula A, or homologous sequences thereto
found in the C-terminus of the GluR2, GluR3, or GluR4 subunits of
the AMPA receptor.
75. The method of claim 74, wherein the fragment comprises the
amino acid sequence of YREGYNVYG, YKEGYNVYG, YREGYNVYGIE or
YKEGYNVYGIE.
76. The method of claim 70, wherein the inhibitor further comprises
the amino acid sequence YGRKKRRQRRR or a carrier peptide sequence
that facilitates translocation of the inhibitor across cell
membranes.
77. A method of screening for a modulator of AMPA receptor
endocytosis, the method comprising: (a) providing a system
comprising (i) an AMPA receptor polypeptide or a
biologically-active fragment thereof; (ii) an inhibitor of AMPA
receptor endocytosis; (b) providing a test compound; (c) contacting
the system with the test compound; and (d) determining whether the
test compound modulates AMPA receptor endocytosis.
78. A method of screening for a modulator of AMPA receptor
endocytosis, the method comprising: (a) providing an AMPA receptor
polypeptide or a biologically-active fragment thereof; (b)
providing a test compound; (c) contacting the AMPA receptor
polypeptide or a biologically-active fragment thereof with the test
compound; and (d) determining whether the test compound modulates
AMPA receptor endocytosis.
79. The method of claim 78, further comprising providing an
inhibitor of AMPA receptor endocytosis, contacting the AMPA
receptor polypeptide or a biologically-active fragment thereof with
the inhibitor, and determining whether the test compound modulates
AMPA receptor endocytosis when compared to the inhibitor.
80. The method of claim 77, wherein the inhibitor is an inhibitor
of regulated AMPA receptor endocytosis.
81. The method of claim 77, wherein the inhibitor is a GluR2,
GluR3, or GluR4 polypeptide.
82. The method of claim 77, wherein the inhibitor comprises the
amino acid sequence set forth in Table I or conservative
substitutions thereof, Formula I, or Formula A, or homologous
sequences thereto found in the C-terminus of the GluR2, GluR3, or
GluR4 subunits of the AMPA receptor or a fragment or variant
thereof, or comprises an antibody that specifically binds the amino
acid sequence set forth in Table I or conservative substitutions
thereof, Formula I, or Formula A, or homologous sequences thereto
found in the C-terminus of the GluR2, GluR3, or GluR4 subunits of
the AMPA receptor.
83. The method of claim 82, wherein the fragment comprises the
amino acid sequence of YREGYNVYG, YKEGYNVYG, YREGYNVYGIE or
YKEGYNVYGIE.
84. The method of claim 77, wherein the inhibitor further comprises
the amino acid sequence YGRKKRRQRRR or a carrier peptide sequence
that facilitates translocation of the inhibitor across cell
membranes.
85. A polypeptide comprising an amino acid sequence substantially
identical to at least one of the sequences selected from the group
consisting of YREGYNVYGIE, YKEGYNVYGIE, YREGYNVYG, and
YKEGYNVYG.
86. A polypeptide comprising an amino acid sequence substantially
identical to at least one of the sequences set forth in Table
I.
87. The polypeptide of claim 85, further comprising the amino acid
sequence YGRKKRRQRRR or a carrier peptide sequence that facilitates
translocation of the inhibitor across cell membranes.
88. A nucleic acid molecule encoding the amino acid sequence of
claim 87.
89. An antibody that specifically binds the amino acid sequence of
claim 87.
90. A substantially pure compound comprising of Formula I:
Z.sub.1-X.sub.1-X.sub.2-E-G-X.sub.3-N-V-X.sub.4-G-Z.sub.2; (I)
wherein X.sub.1 is Y, D, E, S, or T; X.sub.2 is K or R; X.sub.3 is
Y, D, E, S, or T; X.sub.4 is Y, D, E, S, or T; Z, is H.sub.2N--,
RHN-- or, RRN--; Z.sub.2 is --C(O)OH, --C(O)R, --C(O)OR, --C(O)NHR,
--C(O)NRR; R at each occurrence is independently selected from
(C.sub.1-C.sub.6) alkyl, (C.sub.1-C.sub.6) alkenyl,
(C.sub.1-C.sub.6) alkynyl, substituted (C.sub.1-C.sub.6) alkyl,
substituted (C.sub.1-C.sub.6) alkenyl, or substituted
(C.sub.1-C.sub.6) alkynyl; wherein "--" is a covalent linkage, and
wherein the compound is an inhibitor of AMPA receptor
endocytosis.
91. The compound of claim 90, wherein any one or more of X.sub.1,
X.sub.3, or X.sub.4 is a Y.
92. The compound of claim 90, wherein the compound inhibits AMPA
receptor endocytosis with an affinity that is at least as great as
the affinity of a polypeptide comprising a sequence selected from
the group consisting of YREGYNVYGIE, YKEGYNVYGIE, YREGYNVYG, and
YKEGYNVYG.
93. The compound of claim 92, further comprising the amino acid
sequence YGRKKRRQRRR or a carrier peptide sequence that facilitates
translocation of the inhibitor across cell membranes.
94. A method of modulating NMDA-mediated neuronal apoptosis, the
method comprising contacting a neuronal cell with an inhibitor of
clathrin-mediated endocytosis.
Description
FIELD OF THE INVENTION
[0001] The invention is, in general, in the field of neurology.
More specifically, the invention provides, in part, methods and
reagents for modulating neuronal apoptosis or synaptic
plasticity.
BACKGROUND OF THE INVENTION
[0002] Synaptic transmission is the process by which neurons
communicate by excitatory (generation of an action potential) or
inhibitory (inhibition of an action potential following excitation)
mechanisms. Excitatory synaptic transmission often occurs by means
of the neurotransmitter L-glutamate and its cognate glutamate
receptors, which include the N-methyl-D-aspartate (NMDA) and
.alpha.-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA)
subtype glutamate receptors. Synaptic plasticity refers to the
use-dependent ability of post-synaptic neurons to modulate their
response to the release of neurotransmitters during synaptic
transmission, and is thought to be important in learning and memory
processes.
[0003] The excessive stimulation of post-synaptic neurons (a
phenomenon known as "excitotoxicity"), which can lead to neuronal
death or apoptosis, has been implicated in a variety of central
nervous system (CNS) disorders. Activation of the NMDA receptor may
induce programmed cell death (apoptosis) in cultured hippocampal
neurons, and may underlie the loss of neurons and neuronal function
in central nervous system disorders ranging from acute brain trauma
and stroke to neurogenerative diseases such as Huntington's,
Alzheimer's, and Parkinson's Diseases..sup.1-5
[0004] NMDA receptor activation may also lead to facilitation of
clathrin-mediated endocytosis of AMPA receptors, which mediate fast
synaptic transmission at excitatory synapses in the mammalian
CNS..sup.6,7 AMPA receptor function can be modified at the level of
open channel probability.sup.34, channel conductance.sup.27;33, and
the kinetics of desensitization..sup.52 Rapid redistribution of
AMPA receptors to and from the postsynaptic domain is also thought
to be a means of regulating the strength of AMPA receptor-mediated
synaptic transmission..sup.43;45;6 AMPA receptors undergo
functionally distinct constitutive and regulated clathrin-dependent
cycling between intracellular compartments and the plasma membrane
via vesicle-mediated plasma membrane insertion (exocytosis) and
internalization (endocytosis)..sup.22;30;20;24;41;14 Regulating
these processes can lead to rapid changes in the number of AMPA
receptors expressed in the postsynaptic membrane, thereby
contributing to the expression of certain forms of synaptic
plasticity, including hippocampal long term potentiation (LTP)
.sup.35;42;50 and long term depression (LTD) in the cerebellum and
hippocampus..sup.14;24;25;44 AMPA receptors may be subjected to
stimulated endocytosis by diverse stimuli including growth factors,
such as insulin/IGF-1 .sup.14;25, agonist binding.sup.22;21;20, and
LTD-producing protocols..sup.24;14;25
SUMMARY OF THE INVENTION
[0005] The invention provides, in part, methods and reagents for
modulating neuronal apoptosis. The invention also provides, in
part, methods and reagents for modulating synaptic plasticity.
[0006] In some aspects, the invention provides a method of
modulating NMDA-mediated neuronal apoptosis by contacting a
neuronal cell with an inhibitor of AMPA receptor endocytosis. In
alternative aspects, the invention provides a method of modulating
NMDA-mediated neuronal apoptosis by contacting a neuronal cell with
an inhibitor of clathrin-mediated endocytosis. In alternative
aspects, the invention provides a method of treating or preventing
neurological damage or dysfunction in a subject by administering an
effective amount of an inhibitor of AMPA receptor endocytosis to
the subject.
[0007] In alternative embodiments, the neurological damage may
include NMDA-induced neuronal apoptosis, or may occur as a result
of excessive activation of NMDA receptors or due to changes in AMPA
receptor endocytosis, or may occur as a result of at least one of a
disorder selected from the group consisting of stress, anxiety,
depression, hypoglycemia, cardiac arrest, epilepsy, cerebral
ischemia, brain trauma, Alzheimer's disease, Parkinson's disease,
Huntington's disease; neuropathic pain; amyotrophic lateral
sclerosis (ALS); Hutchinson Gilford syndrome; diabetes; ataxia;
mental retardation; dementias, disorders associated with smoking or
obesity, high blood pressure, disorders associated with defects or
dysfunction in learning or memory, psychiatric disorders, autism,
schizophrenia, fragile X syndrome, or disorders associated with
substance abuse or addiction to a drug (e.g., nicotine, alcohol,
opiates, heroin, codeine, morphine pethidine, methadone, marijuana,
phenyclidene, psychostimulants, amphetamines, cocaine,
barbiturates, pentobarbitone, quinalbarbitone, benzodiazepines,
temazepam, diazepam or flunitrazepam).
[0008] In alternative aspects, the invention provides a method of
modulating synaptic plasticity in a subject by administering an
effective amount of an inhibitor of AMPA receptor endocytosis to
the subject (e.g., a normal subject i.e. one not having or not
diagnosed with neurological damage or dysfunction). In alternative
embodiments, the method may further include enhancing synaptic
plasticity. In alternative acts, the invention provides a method of
treating or preventing substance abuse in a subject by
administering an effective amount of an inhibitor of AMPA receptor
endocytosis to the subject.
[0009] In some aspects, the invention provides a method of
modulating AMPA receptor endocytosis by contacting a cell or system
(for example, a lipid vehicle) expressing an AMPA receptor with a
peptide comprising an amino acid sequence selected from the group
consisting of YREGYNVYGIE, YKEGYNVYGIE, YREGYNVYG, or YKEGYNVYG, or
with an antibody that specifically binds an amino acid sequence
selected from the group consisting of YREGYNVYGIE, YKEGYNVYGIE,
YREGYNVYG, and YKEGYNVYG.
[0010] In some aspects, the invention provides a method of
modulating AMPA receptor endocytosis, by contacting a cell
expressing an AMPA receptor with a modulatory compound comprising
the amino acid sequence set forth in Table I or conservative
substitutions thereof. Formula I, or Formula A, or homologous
sequences thereto found in the C-terminus of the GluR2, GluR3, or
GluR4 subunits of the AMPA receptor or a fragment or variant
thereof, or comprising an antibody that specifically binds the
amino acid sequence set forth in Table I or conservative
substitutions thereof, Formula I, or Formula A, or homologous
sequences thereto found in the C-terminus of the GluR2, GluR3, or
GluR4 subunits of the AMPA receptor.
[0011] In alternative aspects, the invention provides a method of
screening for a modulator of AMPA receptor endocytosis, by
providing a system including an AMPA receptor polypeptide or a
biologically-active fragment thereof; an inhibitor of AMPA receptor
endocytosis; providing a test compound; contacting the system with
the test compound; and deter whether the test compound modulates
AMPA receptor endocytosis.
[0012] In alternative aspects, the invention provides a method of
screening for a modulator of AMPA receptor endocytosis, the method
including providing an AMPA receptor polypeptide or a
biologically-active fragment thereof; providing an inhibitor of
AMPA receptor endocytosis; providing a test compound; contacting
the AMPA receptor polypeptide or a biologically-active fragment
thereof with the test compound or the inhibitor; and determining
whether the test compound modulates AMPA receptor endocytosis
[0013] In alternative aspects, the invention provides a method of
screening for a modulator of AMPA receptor endocytosis, by
providing an AMPA receptor polypeptide or a biologically-active
fragment thereof; providing a test compound; contacting the AMPA
receptor polypeptide or a biologically-active fragment thereof with
the test compound; and determining whether the test compound
modulates AMPA receptor endocytosis. In alternative embodiments,
the method may further include providing an inhibitor of AMPA
receptor endocytosis, contacting the AMPA receptor polypeptide or a
biologically-active fragment thereof with the inhibitor, and
determining whether the test compound modulates AMPA receptor
endocytosis when compared to the inhibitor.
[0014] In alternative aspects, the invention provides a polypeptide
including an amino acid sequence substantially identical to the
sequence of YREGYNVYGIE, YKEGYNVYGIE, YREGYNVYG, or YKEGYNVYG, or a
nucleic acid molecule encoding any of these amino acid sequences,
or an antibody that specifically binds any of these amino acid
sequences.
[0015] In alternative aspects, the invention provides a
substantially pure compound including Formula I:
Z.sub.1-X.sub.1-X.sub.2-E-G-X.sub.3-N-V-X.sub.4-G-Z.sub.2; where
X.sub.1 may be Y, D, E, S, or T; X.sub.2 may be K or R; X.sub.3 is
Y, D, E, S, or T; X.sub.4 may be Y, D, E, S, or T; Z.sub.1 may be
H.sub.2N--, RHN-- or, RRN--; Z.sub.2 may be --C(O)OH, --C(O)R,
--C(O)OR, --C(O)NHR, --C(O)NRR; R at each occurrence may be
independently selected from (C.sub.1-C.sub.6) alkyl,
(C.sub.1-C.sub.6) alkenyl, (C.sub.1-C.sub.6) alkynyl, substituted
(C.sub.1-C.sub.6) alkyl, substituted (C.sub.1-C.sub.6) alkenyl, or
substituted (C.sub.1-C.sub.6) alkynyl; wherein "--" may be a
covalent linkage, and wherein the compound may be an inhibitor of
AMPA a endocytosis. In alternative embodiments, any one or more of
X.sub.1, X.sub.3, or X maybe a Y.
[0016] In alternative aspects, the invention provides a
substantially pure compound including Formula A:
Z.sub.1-X.sub.1-X.sub.2-X.sub.3-X.sub.4-X.sub.5-X.sub.6-X.sub.7-X.sub.8-X-
.sub.9-Z.sub.2, where X.sub.1 may be an amino acid having a
hydropathic index of -0.3 to 4.3 or of -1.3 to -3.3 or may be a
neutral or an acidic amino acid, or may Gly, Ser, Thr, Cys, Asn,
Gln, Tyr, Asp, Glu; X.sub.2 may be an amino acid having a
hydropathic index of +1.0 to +5.0 or of +2.0 to +4.0 or may be a
basic amino acid or may be Lys, Arg, His; X.sub.3 may be an amino
acid having a hydropathic index of +1.0 to +5.0 or of +2.0 to +4.0
or may be an acidic amino acid or may be Asp, Glu; X.sub.4 may be
an amino acid having a hydropathic index of -2.0 to +2.0 or of -1.0
to +1.0 to or may be a neutral amino acid or may be Gly, Ser, Thr,
Cys, Asn, Gln, Tyr, X.sub.5 may be an amino acid having a
hydropathic index of 0.3 to 4.3 or of -1.3 to -3.3 or may be a
neutral or an acidic amino acid or may be Gly, Ser, Thr, Cys, Asn,
Gln, Tyr, Asp, Glu; X.sub.6 may be an amino acid having a
hydropathic index of-1.8 to +2.2 or of -0.8 to +1.2 or may be a
neutral amino acid or may be Gly, Ser, Thy, Cys, Asn, Gln, Tyr;
X.sub.7 may be an amino acid having a hydropathic index of -3.5 to
0.5 or of -2.5 to -0.5 or nay be a non-polar amino acid or may be
Ala, Val, Leu, Ile, Phe, Trp, Pro, Met; X8 may be an amino acid
having a hydropathic index of -0.3 to -4.3 or of -1.3 to -3.3 or
may be a neutral or an acidic amino acid or may be Gly, Ser, 1 hr,
Cys, Asn, Gin, Tyr, Asp, Glu; X.sub.9 may be an amino acid having a
hydropathic index of -2.0 to +2.0 or of -1.0 to +1.0 to may be a
neutral amino acid or may be Gly, Ser, Thr, Cys, Asn, Gln, Tyr, Z,
is H.sub.2N--, RHN-- or, RRN--; Z.sub.2 may be C(O)OH, C(O)R,
--C(O)OR, --C(O)NHR, --C(O)NRR; R at each occurrence may be
independently selected from (C.sub.1-C.sub.6) alkyl,
(C.sub.1-C.sub.6) alkenyl, (C.sub.1-C.sub.6) alkynyl, substituted
(C.sub.1-C.sub.6) alkyl, substituted (C.sub.1-C.sub.6) alkenyl, or
substituted (C.sub.1-C.sub.6) alkynyl; wherein "--" is a covalent
linkage, and wherein the compound may be an inhibitor of AMPA
receptor endocytosis. In alternative embodiments, any one or more
of X.sub.1, X.sub.5, or X.sub.8 may be a Y.
[0017] In alternative embodiments, the compound of Formula I or A
may inhibit AMPA receptor endocytosis with an affinity that is at
least as great as the affinity when the compound is a polypeptide
including a sequence of YREGYNVYGIE, YKEGYNVYGIE, YREGYNVYG, or
YKEGYNVYG. In alternative embodiments, the compound of Formula I or
A may include a similarity score of over zero based on either of
the PAM or Blosum similarity matrices. In alternative embodiments,
the compound of Formula I or A may further include the amino acid
sequence YGRKKRRQRRR.
[0018] In alternative aspects, the invention provides the use of
any of the polypeptides, nucleic acid molecules, antibodies, or
compounds according to the invention for treating or preventing
neurological damage or substance abuse in a subject, or for
modulating NMDA-mediated-neuronal apoptosis, or for modulating AMPA
receptor endocytosis, or for modulating sync plasticity in a
subject.
[0019] In various embodiments of the aspects of the invention, the
inhibitor may include an inhibitor of regulated AMPA receptor
endocytosis. In various embodiments of the aspects of the
invention, the inhibitor may include a GluR2, GluR3, or GluR4
polypeptide. In various embodiments of the aspects of the
invention, the inhibitor of AMPA receptor endocytosis may include a
peptide including any of the amino acid sequences of YREGYNVYGIE,
YKEGYNVYGIE, YREGYNVYG, or YKEGYNVYG or a fragment or variant
thereof, or may be a GluR2, GluR3, or GluR4 polypeptide, or may
include an antibody that specifically binds any of the amino acid
sequences of YREGYNVYGIE, YKEGYNVYGIE, YREGYNVYG, and YKEGYNVYG. In
various embodiments of the aspects of the invention, the inhibitor
may include the amino acid sequence set forth in Table I or
conservative substitutions thereof, Formula I, or Formula A, or
homologous sequences thereto found in the C-terminus of the GluR2,
GluR3, or GluR4 subunits of the AMPA receptor or a fragment or
variant thereof or include an antibody that specifically binds the
amino acid sequence set forth in Table I or conservative
substitutions thereof, Formula L or Formula A, or homologous
sequences thereto found in the C-terminus of the GluR2, GluR3, or
GluR4 subunits of the AMPA receptor. In various embodiments of the
aspects of the invention, may further include the amino acid
sequence YGRKKRRQRRR.
[0020] .alpha.-amino-3-hydroxy-5-methylisoxazole-4-propionic acid
or "AMPA" receptors are glutamate-gated ion channel receptors that
are involved in transduction of the post-synaptic signal. Native
AMPA receptors may be heteromeric, e.g, heteropentameric, protein
complexes assembled from combinations of GluR subunits 1-4. When
transiently expressed in non-neuronal mammalian cells, individual
GluR subunits can form functional homomeric AMPA receptor channels,
and AMPA receptors in these heterologous expression systems can
undergo both constitutive and regulated clathrin-dependent
endocytosis. In some embodiments, an AMPA receptor includes a GluR2
subunit. GluR subunits may include without limitation the sequences
described in Accession numbers NP.sub.--113796; NPL032191;
NP.sub.--000818 for GluR1; NP_058957; NP.sub.--038568;
NP.sub.--000817; P23819 for GluR2; NP.sub.--116785 for GluR3; or
NP.sub.--058959 or NP.sub.--000820 for GluR4, and related
nucleotide sequences, for example, NM.sub.--000826. Other GluR
polypeptide or nucleotide sequences may be found in public
databases, such as GenBank.
[0021] A "phosphorylated" AMPA receptor includes polypeptide
subunits that are post-translationally modified on any amino acid
residue, for example, serine, threonine, or tyrosine, that is
capable of being phosphorylated in vivo. For example, a
phosphorylated AMPA receptor may include a GluR2 subunit that is
phosphorylated, for example, on any one or more of tyrosines 869,
873, and 876 of the sequence described in Accession number
NP.sub.--000817, or phosphorylated on any one or more of tyrosine
residues present in corresponding sequences in GluR subunits.
[0022] An "unphosphorylated" AMPA receptor may be incapable of
being phosphorylated on an amino acid residue capable of being
phosphorylated in vivo, for example, by mutation of that residue to
an amino acid that is not capable of being phosphorylated. A
mutation of a tyrosine to an alamine in a polypeptide sequence, for
example, results in a protein that is not capable of being
phosphorylated at that particular position in the polypeptide
sequence. A GluR2 polypeptide that possesses an alanine or other
unphosphorylatable amino acid at positions 869, 873, and/or 876 of
the sequence described in Accession number NP.sub.--000817, instead
of a tyrosine, is an example of such an "unphosphorylated" AMPA
receptor. An unphosphorylated AMPA receptor may also be a protein
that is capable of being phosphorylated in vivo, but is not
phosphorylated due to, for example, the presence of an inhibitor,
for example, a kinase inhibitor, due to an antibody that interferes
with the phosphorylation site; due to the activity of a
phosphatase; or prevented from being phosphorylated by some other
means. A "constitutively phosphorylated" AMPA receptor is a protein
that possesses a mutation at an amino acid residue that is capable
of being phosphorylated in vivo, where the mutation mimics
phosphorylation at that residue, and the resultant polypeptide
possesses the biological activity of a phosphorylated polypeptide.
Generally, mutation of a phosphorylatable residue to a glutamic
acid or aspartic acid residue results in constitutive
phosphorylation.
[0023] A GluR CT polypeptide includes a peptide derived form, or
substantially identical to, the C-terminus of a GluR polypeptide
and that is capable of inhibiting AMPA receptor endocytosis, or
modulating neuronal apoptosis or synaptic plasticity. GluR CT
peptides include, without limitation, peptides including the
sequences set forth in Table I or conservative substitutions
thereof, Formula 1, or Formula A, or homologous sequences thereto
found in the C-terminus of the GluR2, GluR3, or GluR4 subunits of
the AMPA receptor. In some embodiments, a GluR CT peptide may
include other sequences (e.g, TAT PTD) in the form of for example a
fusion protein.
[0024] A "biologically-active fragment" of an AMPA receptor
includes an amino acid sequence found in a naturally ring AMPA
receptor that is capable of modulating apoptosis or cell death or
synaptic plasticity, or undergoing endocytosis, as described herein
or known to those of ordinary skill in the art. A "variant" of an
AMPA receptor includes a modification, for example, by deletion,
addition, or substitution, of an amino acid sequence found in a
naturally-occurring AMPA receptor that is capable of modulating
apoptosis or cell death, or synaptic plasticity, undergoing
endocytosis, as described herein or known to those of ordinary
skill in the art.
[0025] A "protein," "peptide" or "polypeptide" is any chain of two
or more amino acids, including naturally occurring or non-naturally
occurring amino acids or amino acid analogues, regardless of
post-translational modification (e.g., glycosylation or
phosphorylation). An "amino acid sequence", ""polypeptide",
"peptide" or protein" of the invention may include peptides or
proteins that have abnormal linkages, cross links and end caps,
non-peptidyl bonds or alternative modifying groups. Such modified
peptides are also within the scope of the invention. The term
"modifying group" is intended to include structures that are
directly attached to the peptidic structure (e.g., by covalent
coupling), as well as those that are indirectly attached to the
peptidic structure (e.g., by a stable non-covalent association or
by covalent coupling to additional amino acid residues, or
mimetics, analogues or derivatives thereof; which may flank the
core peptidic structure). For example, the modifying group can be
coupled to the amino-terminus or carboxy-terminus of a peptidic
structure, or to a peptidic or peptidomimetic region flanking the
core domain. Alternatively, the modifying group can be coupled to a
side chain of at least one amino acid residue of a peptidic
structure, or to a peptidic or peptido-mimetic region flanking the
core domain (e.g., through the epsilon amino group of a lysyl
residue(s), though the carboxyl group of an aspartic acid
residue(s) or a glutamic acid residue(s), through a hydroxy group
of a tyrosyl residue(s), a serine residue(s) or a threonine
residue(s) or other suitable reactive group on an amino acid side
chain). Modifying groups covalently coupled to the peptidic
structure can be attached by means and using methods well known in
the art for linking chemical structures, including, for example,
amide, alkylamino, carbamate or urea bonds. Peptides according to
the invention may include the sequences set forth in Table I or
conservative substitutions thereof, Formula I, or Formula A, or
homologous sequences thereto, found in the C-terminus of the GluR2,
GluR3, or GluR4 subunits of the AMPA receptor. In some embodiments,
the peptides may include other sequences (e.g, TAT PTD) in the form
of for example a fusion protein.
[0026] A "nucleic acid molecule" is any chain of two or more
nucleotides including naturally occurring or non-naturally
occurring nucleotides or nucleotide analogues. A nucleic acid
molecule is "complementary"to another nucleic acid molecule if it
hybridizes, under conditions of high stringency, with the second
nucleic acid molecule. Nucleic acid molecules according to the
invention include those molecules that encode the sequences set
forth in Table I or conservative substitutions thereof, Formula I,
or Formula A, or homologous sequences thereto, found in the
C-terminus of the GluR2, GluR3, or GluR4 subunits of the AMPA
receptor. In some embodiments, a nucleic acid molecule may include
other sequences (e.g., sequence coding for TAT PTD) to generate for
example a fusion protein.
[0027] A "substantially identical" sequence is an amino acid or
nucleotide sequence that differs from a reference sequence only by
one or more conservative substitutions, as discussed herein, or by
one or more non-conservative substitutions, deletion, or insertions
located at positions of the sequence that do not destroy biological
function as described herein. Such a sequence can be any integer
from 60% to 99%, or more generally at least 75%, 80%, 85%, 90%, or
95%, or as much as 96%, 97%, 98%, or 99% identical at the amino
acid or nucleotide level to the sequence used for comparison.
Sequence identity can be readily measured using publicly available
sequence analysis software (e.g., Sequence Analysis Software
Package of the Genetics Computer Group, University of Wisconsin
Biotechnology Center, 1710 University Avenue, Madison, Wis. 53705,
or BLAST software available from the National Library of Medicine,
USA). Examples of useful software include the programs, Pile-up and
PrettyBox. Such software matches similar sequences by assigning
degrees of homology to various substitutions, deletions,
substitutions, and other modifications. Substantially identical
sequences may for example be sequences that are substantially
identical to the amino acid sequences set forth in Table I or
conservative substitutions thereof, Formula I, or Formula A, or to
homologous sequences thereto found in the C-terminus of the GluR2,
GluR3, or GluR4 subunits of the AMPA receptor. In some embodiments,
a substantially identical sequence may further include sequences
substantially identical to other sequences (e.g, TAT PTD).
[0028] An antibody "specifically binds" an antigen when it
recognises and binds the antigen, for example, a GluR CT peptide,
but does not substantially recognise and bind other molecules in a
sample, for example, a GluR CT peptide that does not include such
sequences. Such an antibody has, for example, an affinity for the
antigen which is 10, 100, 1000 or 10000 times greater than the
affinity of the antibody for another reference molecule in a
sample.
[0029] "cell death" or "apoptosis," defines a specific execution of
programmed cell death that can be triggered by several
factors..sup.55 NMDA-mediated neuronal apoptosis is the neuronal
cell death observed upon activation of NMDA receptors.
[0030] "Endocytosis" is the process by which the plasma membrane of
a cell folds inward, to internalize components of the membrane as
well as other materials. Receptor endocytosis is typically mediated
by clathrin coated pits and vesicles.
[0031] An "inhibitor of chin mediated endocytosis" includes an
compound that is capable of specifically inhibiting clathrin
mediated endocytosis, without substantially inhibiting endocytosis
in general. An inhibitor of clathrin mediated endocytosis may
include, for example, myr -dyn, or inhibitors as described in
Jarousse and Kelly..sup.62 In some embodiments, an inhibitor of AMA
receptor endocytosis may also be an inhibitor of clathrin mediated
endocytosis.
[0032] An "inhibitor of AMPA receptor endocytosis" includes a
compound that may be in general capable of specifically inhibiting
endocytosis of the AMPA receptor, without substantially inhibiting
clathrin-mediated endocytosis in general, when compared with an
inhibitor of clathrin mediated endocytosis. In some embodiments, an
inhibitor of AMPA receptor endocytosis may include compounds that
do not affect basal levels of AMPA receptor endocytosis e.g.,
compounds that are inhibitors of "regulated" AMPA receptor
endocytosis. In some embodiments, an inhibitor of AMPA receptor
endocytosis may include compounds that are substantially identical
to the amino acid sequences set forth in Table I or conservative
substitutions thereof, Formula L or Formula A, or to homologous
sequences found in the C-terminus of the GluR2, GluR3, or GluR4
subunits of the AMPA receptor. In some embodiments, an inhibitor of
AMPA receptor endocytosis may include an antibody that mimics the
sequences set forth in Table I or conservative substitutions
thereof, Formula L or Formula A, or to homologous sequences found
in the C-terminus of the GluR2, GluR3, or GluR4 subunits of the
AMPA receptor, e.g., an anti-idiotypic antibody to an antibody that
specifically binds a GluR CT peptide.
[0033] "Synaptic plasticity" refer to the use-dependent changes
(long-term or short-term) in the efficiency of synaptic
transmission between neuronal cells. Synaptic plasticity is thought
to underlie the processes behind leaning and memory.
[0034] A "test compound" is any naturally-occurring or
artificially-derived chemical compound. Test compounds may include,
without limitation, peptides, polypeptides, synthesised organic
molecules, naturally occurring organic molecules, and nucleic acid
molecules. A test compound may "compete" with a known compound, for
example, an inhibitor of clathrin mediated endocytosis or an
inhibitor of AMPA receptor endocytosis, such as a GluR-CT peptide
or fragment thereof by, for example, interfering with modulation of
neuronal apoptosis or cell death or synaptic plasticity,
endocytosis, or protein phosphorylation, or other biological
response. Generally, a test compound can exhibit any value between
10% and 200%, or over 500%, modulation when compared to a GluR-CT
peptide or peptide analogue, or other reference compound. For
example, a test compound may exhibit at least any positive or
negative integer from 10% to 200% modulation, or at least any
positive or negative integer from 30% to 150% modulation, or at
least any positive or negative integer from 60% to 100% modulation,
or any positive or negative integer over 100% modulation. A
compound that is a negative modulator will in general decrease
modulation relative to a known compound, while a compound that is a
positive modulator will in general increase modulation relative to
a known compound.
[0035] A "sample" can be any organ, tissue, cell, or cell extract
isolated from a subject, such as a sample isolated form an animal
having neurological damage or neuronal dysfunction or a
neurological disorder. For example, a sample can include, without
limitation, hippocampal tissue or cells, cerebellar tissue or
cells, etc., or other neuronal or other tissue (e.g., from a biopsy
or autopsy), isolated from an animal with neurological damage,
dysfunction, or disorder, or from a normal animal i.e., not having
neurological damage, dysfunction, or disorder. A sample can also
include, without limitation, tissue such as neuronal cells,
peripheral blood, whole blood, red cell concentrates, platelet
concentrates, leukocyte concentrates, blood cell proteins, blood
plasma, platelet-rich plasma, a plasma concentrate, a precipitate
from any fractionation of the plasma, a supernatant from any
fractionation of the plasma, blood plasma protein fractions,
purified or partially purified blood proteins or other components,
serum, semen, mammalian colostrum, milk, urine, stool, saliva,
placental extracts, amniotic fluid, a cryoprecipitate, a
cryosupernatant, a cell lysate, mammalian cell culture or culture
medium, products of fermentation, ascitic fluid, proteins present
in blood cells, solid tumours isolated from a mammal with a
neuronal carcinoma, or any other specimen, or any extract thereof,
obtained from a patient (human or animal), test subject, or
experimental animal. A sample may also include, without limitation,
products produced in cell culture by normal cells or cells isolated
from a subject with neurological damage or neuronal dysfunction
(e.g., via recombinant DNA technology). A "sample" may also be a
cell or cell line created under experimental conditions, that are
not directly isolated from a subject. A sample can also be
cell-free, artificially derived or synthesised. In some
embodiments, samples refer to neuronal tissue or cells. In some
embodiments, the sample may be from a subject having neurological
damage or neuronal dysfunction; or from a normal subject i.e., not
diagnosed with or at risk for or suspected of having neurological
damage or neuronal dysfunction.
[0036] As used herein, a subject may be a human, non-human primate,
rat, mouse, cow, horse, pig, sheep, goat, dog, cat, Aplysia, etc.
The subject may be a clinical patient, a clinical trial volunteer,
an experimental animal, etc. The subject may be suspected of having
or at risk for having neurological damage or neuronal dysfunction,
be diagnosed with neurological damage or neuronal dysfunction, or
be a control subject that is confirmed to not have neurological
damage or neuronal dysfunction. Diagnostic methods for neurological
damage or neuronal dysfunction and the clinical delineation of
neurological damage or neuronal dysfunction diagnoses are known to
those of ordinary skill in the art.
[0037] By "contacting"is meant to submit an animal, cell, lysate,
extract, molecule derived from a cell or synthetic molecule to a
test compound.
[0038] By "determining"is meant analysing the effect of a test
compound on the test system. The means for analysing may include,
without limitation, antibody labelling, apoptosis assays,
immunoprecipitation, in vivo and in vitro phosphorylation assays,
cell death assays, immunofluorescence assays, ELISA,
ultrastructural analysis, histological analysis, animal models, or
any other methods described herein or known to those skilled in the
art.
[0039] "modulating" or "modulates" means changing, by either
increase or decrease. The increase or decrease may be a change of
any value between 10% and 90%, or of any value between 30% and 60%,
or may be over 100%, over 200%, over 300% or over 500% when
compared with a control or reference sample or compound.
[0040] Other features and advantages of the invention will be
apparent from the following description of the drawings and the
invention, and from the claims.
BRIEF DESCRIPTION OF THE DRAWINGS
[0041] FIGS. 1A-F. NMDA induces apoptosis in primary cultures of
rat hippocampal neurons. Mature hippocampal neurons were treated
with NMDA (100 .mu.M plus 10 .mu.M glycine; 1 h) and then returned
to normal media for 24 h. In this and the following Figs., all data
are expressed as mean.+-.SEM and analyzed using a non-paired
Student's t-test. A, B, NMDA treatment induces a time-dependent
increase in caspase-3 activity. A: western blot of cell lysates
using anti-cleaved caspase-3; B: ELISA assay detecting DEVD-pNA
cleavage. C, Agarose gel electrophoresis shows significant DNA
laddering after NMDA treatment. D, Cell Death ELISA assay for
apoptosis measuring histone-biotinylation shows that NMDA-induced
apoptosis is blocked by the competitive NMDA receptor antagonist,
APV. E, F, Cell Death ELISA assays for apoptosis show that
endocytosis inhibitors specifically block NMDA--but not
STS--induced apoptosis in cultured hippocampal neurons.
[0042] FIGS. 2A-D. Inhibitors of endocytosis disrupt NMDA
receptor-mediated activation of the cell death signaling pathway
without altering NMDA receptor function. A, B, Inhibition of
endocytosis has little effect on Ca2+ influx through activated NMDA
receptor channels. The upper trace (A) shows a record of NMDA
receptor activation-induced [Ca.sup.2+]fluctuations as measured by
ratiometric changes in Fura-2 fluorescence in a single hippocampal
neuron. Repetitive NMDA application (100 .mu.M) in the region of
the neuron under observation was accomplished using a pressure
ejection pipette at the time points indicated by the lower black
squares (500 ms each). Sucrose (400 mM) was applied to the bath as
indicated by the upper black bar. The histogram at the bottom (B)
summarizes [Ca.sup.2+]responses at indicated time points from three
individual neurons (mean.+-.SEM). C, A myristoylated
dynamin-derived peptide inhibits NMDA induced activation of
caspase-3. D, Endocytosis inhibition specifically disrupts the
NMDA-, but not the STS-induced reduction in Akt phosphorylation.
Cell lysates from neurons treated as indicated were first probed
with an antibody specific to Akt phosphorylated on serine 473, the
active form of the enzyme. Membranes were then stripped and
re-probed with an anti-Akt antibody. Blots from four individual
experiments were scanned and quantified. The histogram represents
Akt phosphorylation relative to total Akt. **, p<0.01 when
compared with the respective control group.
[0043] FIGS. 3A-B. NMDA induces AMPA receptor but not NMDA receptor
endocytosis, which is blocked by the membrane permeable
myristoylated dynamin peptide (Myr-Dyn) as well as a peptide
derived from GluR c-tail (R2-CT). A, ELISA-based cell-surface
receptor assay for NMDA receptor and AMPA receptor. NMDA treatment
induced a significant reduction in cell surface AMPA receptor but
not NMDA receptor, and AMPA receptor internalization was prevented
by pretreatment of neurons with the myristoylated, membrane
permeable dynamin inhibitor peptide (Myr-Dyn; 10 .mu.M), but not
the membrane impermeable control Dyn (** denotes p<0.01,
compared with control; n=36-72 wells from three separate
experiments for each group). B, NMDA-induced AMPA receptor
internalization is blocked by R2-CT , a peptide that specifically
blocked regulated AMPA receptor endocytosis.
[0044] FIGS. 4A-B. Blocking AMPA receptor endocytosis with R2-CT
prevents NMDA--but not STS-induced apoptosis in cultured
hippocampal neurons. A, Cell Death ELISA assay for apoptosis
showing that R2-CT blocks NMDA--but not STS-induced apoptosis. B,
Cell counting for apoptosis of PI stained cells after fixation,
showing that R2-CT blocked NMDA-induced apoptosis.
[0045] FIGS. 5A-B. Construction of GluR2 internal deletion or
carboxyl terminal truncation mutants and identification of a
tyrosine-based signal (GluR2-3Y). A. CT sequences of internal
deletion or truncation mutants of the full-length HA-tagged or
non-tagged GluR2 subunit. B. Quantification of cell-surface
expressed AMPARs containing the GluR2, or its various mutant
constructs, which were transiently transfected into HEK293 cells
and assayed by colorimetric cell-ELISA (n=6). Expression levels of
the constructs following transient transfection into HEK293 cells
were determined by cell-ELISA assays using an anti-HA antibody for
HA tagged constructs, or an anti-GluR2 subunit antibody for the
non-HA tagged construct, under permeabilized conditions. The level
of expression was normalized to the expression level of the
corresponding wild type construct (i.e. HA-GluR2 or GluR2). All
mutants were expressed at a level similar to the wild type
counterparts. Removing the tyrosine based signal prevents
insulin-induced depletion of cell surface AMPARs (filled bars)
without affecting the basal receptor level. Removing NSF-binding
domains affects basal, but not insulin-reduced receptor expression,
and that unlike neurons, both AP2 and PICK1-based endocytosis
signals are non-functional in HEK cells. *p<0.05,
**p<0.01.
[0046] FIGS. 6A-B. Effects of GluR2 CT mutations on endocytosis and
cell-surface expression of AMPA receptors. A. Quantitation of the
changes in constitutive (Basal) and regulated (insulin) endocytosis
of GluR2 and its various mutants using a colorimetric ELISA assay
with pre-labeled cells following the internalization of the
receptors over 30 min (% AMPAR endocytosis=100%-remaining
cell-surface receptors/total number of receptors; n=6). Control:
internalization measured in cells at 4 E C without any 37 E C
exposure (under these conditions, both constitutive and regulated
endocytosis is blocked). B. Cell-surface AMPA receptors in HEK293
cells transiently expressing GluR2 and its various mutants were
quantitated using colorimetric cell-ELISA based cell-surface
receptor assays (n=6). Statistical comparisons were made between
basal and insulin-treated conditions, except where indicated by
lines. *p<0.05, **p<0.01
[0047] FIGS. 7A-D. Insulin increases phosphorylation of tyrosine
residues within the GluR2 carboxyl terminal (CT) region. A. In
vitro tyrosine phosphorylation of the GluR2 CT. GST fusion proteins
of the GluR1 CT (GST-GluR1CT), the GluR2 CT (GST-GluR2CT), residues
869-876 (YKEGYNVYG) of the GluR2 CT (GST-GluR23Y), which contains a
cluster of three tyrosine residues (Y869, Y873, and Y876) and the
same amino acid stretch of the GluR2 CT with its tyrosine residues
replaced by alanines (GST-GluR23A), along with the GST back bone
(GST) as control were incubated in the absence (-) or presence (+)
of active recombinant pp60 c-Src. Phosphorylation products were
immunoblotted using an anti-phosphotyrosine antibody (top panel).
Ponceau S staining of the same blot showed that a similar amount of
GST fusion protein was used in each of the reactions (lower panel).
B. Expression levels of HA-GluR2 and HA-GluR23Y-3A (where tyrosines
869, 873 and 876 were mutated to alanines) 48 h after transient
transfection into HEK293 cells were determined by a cell ELISA
assay using permeabilized cells. C. HEK293 cells transiently
transfected with HA-GluR1, HA-GluR2 or HA-GluR23Y-3A, along with
empty vector (mock transfection) as control. Forty-eight hours
later, the cells were treated with or without 0.5 .mu.M insulin for
10 min. The lysates were then subjected to immunoprecipitation with
an anti-HA antibody under denaturing conditions and immunoblotting
with an anti-phosphotyrosine antibody (Top blot; IB: PY). The same
blot was stripped and re-immunoblotted with the anti-HA antibody to
ensure similar immunoprecipitation efficiency in all individual
experiments (lower blot; IB: HA). D. Mutation of individual
tyrosines of the Glu23Y CT peptide to alanines.
[0048] FIGS. 8A-B. The tyrosine cluster in the GluR2 CT is required
for regulated, but not constitutive, AMPA receptor endocytosis in
HEK293 cells. A. Colorimetric cell-ELISA receptor endocytosis
assays were performed with (Insulin) or without (Control)
stimulation (see FIG. 2) on HEK293 cells transiently transfected
with wild type HA-GluR2 subunit or HA-GluR23Y-3A, in which tyrosine
residues Y869, Y873 and Y876 were mutated into alanines. B.
Colorimetric cell-ELISA cell-surface receptor assay results of
HEK293 cells transfected and treated as in (A). Results were
obtained from 6 experiments for each individual group.
**p<0.01
[0049] FIGS. 9A-D. Insulin stimulates tyrosine phosphorylation of
GluR2 and long-lasting depression of AMPA receptor-mediated
synaptic transmission. A. Tissue homogenates from hippocampal
slices treated with (Basal) or without insulin (INS; 0.5 .mu.M, 10
min) were immunoprecipitated with anti-GluR1 or GluR2 antibodies
under denaturing conditions (IP: GluR1 or GluR2).
Immunoprecipitates were the immunoblotted using an
anti-phosphotyrosine antibody (IB: PY). The blot was sequentially
stripped and re-probed with anti-GluR2 (IB: GluR2) and anti-GluR1
(IB: GluR1) antibodies. B. Densitometric quantitation expressed as
the ratio of phosphorylated GluR2 to total GluR2 from three
separate experiments is summarized in the histogram on the right.
** p<0.01 C. EPSCs were recorded in CA1 neurons from hippocampal
slices using whole-cell recordings under the voltage-clamp mode at
a holding potential of -60 mV. Normalized EPSCs (EPSC/EPSC0) are
plotted from neurons recorded with pipettes containing standard
intracellular solution (Control, n=7) or intracellular solution
supplemented with GST-Y869KEGY873NVY876G (GluR23Y; n=5) or
GST-A869KEGA873NVA876G (GluR3A; n=6). Time zero is defined as the
time point at which the amplitudes of EPSCs were stabilize
(typically 5-10 min after the start of whole-cell recording), and
at t=10 min, insulin (0.5 .mu.M was applied in the bath as
indicated by the horizontal black bar-D. Representative EPSCs
averaged from four individual recordings before (Basal) or 10 min
following application of insulin (INS) are shown on the left.
[0050] FIGS. 10A-E. Tyrosine phosphorylation of the GluR2 subunit
is required for LFS-induced hippocampal CA1 long-term depression
(LTD). A. Homogenates of control or LFS-treated hippocampal slices
were immunoprecipitated with anti-GluR1 or GluR2 antibodies and
sequentially probed with anti-phosphotyrosine (PY), anti-GluR1
(GluR1) and anti-GluR2 antibodies (GluR2) as described herein. The
lane marked M contains molecular weight standards. B. The results
of three individual experiments are summarized in the bar graph. **
p<0.01 C. Representative responses are shown on the left D. The
graphs on the right, and in E, depict normalized EPSCs
(EPSCt/EPSC0) from neurons recorded as described with pipettes
containing standard intracellular solution (Control, n=7) or
intracellular solution supplemented with GluR23Y (B; n=6), GluR23A
(B; n=7) or GluR2834-843 (C; n=5). The LFS was delivered during the
time period indicated by the black horizontal bar.
[0051] FIGS. 11A-B. GluR2-CT peptide prevents ishemia-induced AMA
receptor endocytosis and neuronal apoptosis in a neuronal culture
model of stroke. A. Colorimetric (Cell-ELISA) assay shows that OGD
facilitates AMPA receptor endocytosis, thereby decreasing their
expression on the plasma membrane surface and pre-incubation of the
GluR2-CT peptide reduced the OGD-induced decrease in cell-surface
AMPA receptor expression. (n=6; *: P<0.05, Student's test,
compared with Control). B. Quantitative apoptosis assay 24 hr after
OGD using the Cell Death Detection ELISAplus kit (Roche, Cat# 1 774
425) demonstrates that OGD produces neuronal death that is largely
prevented by pretreatment of neurons with GluR2-CT. (n=6; **:
P<0.01, Student's t test, compared with OGD.
[0052] FIG. 12A-D. Systemic application of Tat-GlurR2.sub.3Y
peptide blocks the expression of behaviourl sensitization to the
abusive drug d-amphetemine in an animal model of drug addiction
GluR2-3Y or GluR2-3A peptide was fused to a Tat transduction domain
(Tat-GluR2-3Y or GluR2-3A) to facilitate membrane permeability.
Intravenous administration (IV; 1.5 nM/g or direct microinjection
into the nucleus accumbens (NAc),with the interference peptide
GluR2-3Y, but not by the control peptide GluR2-3A, blocks
D-amphetamine (D-Amph)-induced behavioural sensitization of
stereotypy. A. Stereotypy scores assessed at various time points
shows blockade of of sensitization following IV injection of
Tat-GluR2-3Y. Points represent mean stereotypy scores (+S.E.M for
each group of rats tested over the 2 hour session. Chronic
saline-treated rats served as control subjects. B. Summary of the
changes in stereotypy scores across the 2 hr test session converted
to the Area Under The Curve (AUC) for individual groups depicted in
graph A. C. Intracranial microinjection of GluR2-3Y into the NAc
also blocks D-Amph-induced sensitization. D. Intracranial
microinjection of the GluR2-3Y peptide into the ventral tegmental
area (VTA) does not block D-Amph-induced behavioural sensitization.
(*=P<0.05, relative to acute amphetamine group.)
[0053] FIG. 13. Tat-GluR2-3Y blocks NMDA-induced AMPAR endocytosis.
Day 12-13 in vitro Wistar cortical neurons were pretreated for 60
min with either saline or 1 .mu.M Tat-GluR2-3Y or Tat-GluR2-3A
followed by a 30 min 50 .mu.M NMDA treatment. The percentage AMPAR
expression as measured by cellular ELISA was defined as the amount
of surface expression (non-permeabilized) divided by the total
expression (permeabilized). Data are representative of either 1 or
4 separate experiments, each with 4 replicate measurements and are
expressed as mean.+-.SEM. * p<0.05, ** p<0.05, Tukey-Kramer
Test.
[0054] FIG. 14. Tat-GluR2-3Y attenuates neuronal apoptosis in
response to oxygen and glucose deprivation. Day 12-13 in vitro
Wistar cortical neurons were pretreated with either Tat-GluR2-3Y or
saline for 60 min, followed by 60 min of OGD or incubation at
37.degree. C. (control). At 24 h, apoptosis was quantified using an
ELISA targeted to free nucleosomes. The data were normalized to the
control and are expressed as mean.+-.SEM of 3 repeat experiments. *
OGD group was significantly different from all other groups
p<0.05, Tukey-Kramer Test.
[0055] FIG. 15. Dose tolerance curve to serial doses of
Tat-GluR2-3Y. Two adult male Sprague-Dawley rats were given serial
doses of Tat-GluR2-3Y and the basic vital parameters were
monitored. Doses of up to 6 nmoles/g evoked little response in the
parameters monitored; however, higher doses resulted in a large
decrease in mean arterial pressure and a concurrent increase in
breathing rate. Both animals showed no sign of altered behaviour
after coming out of anesthesia.
[0056] FIG. 16. Transient middle cerebral artery occlusion results
in increased apoptosis. Two adult male Sprague-Dawley rats were
subjected to either 90 min of MCA occlusion or surgery without MCA
occlusion (sham). At 24 h, the rats were sacrificed, and 12 .mu.m
brain slices were TUNEL stained. The number of TUNEL positive
nuclei was counted for 3 visual fields and are presented as
mean.+-.SEM (B). * p<0.01, Student's t-test.
[0057] FIGS. 17A-B. The effect Tat-GluR2-3Y on apoptosis in a rat
model of transient focal ischemia. Adult male Sprague-Dawley rats
were pretreated for 1 h with either saline, or 3 nmol/g of
Tat-GluR2-3Y or Tat-GluR2-3A and then subjected to 60 min of MCA
occlusion. The rats were given a neurological exam before sacrifice
at 24 h (A). 12 .mu.m coronal brain slices were TUNEL stained and
the number of TUNEL positive cells were counted for each section
(B). Data are normalized to a sham surgery control and are
expressed as mean values.+-.SEM. The peptide reduced apoptosis by
55% with respect to the control.
[0058] FIGS. 18 A-B. Control experiments to confirm that GluR2-3Y
does not have non-specific effects on learned behaviours reinforced
by food or drug-reward stimuli. These experiments also demonstrate
that this interference peptide does not disrupt sensory motor or
memory functions related to performance of operant behaviour on two
different schedules of reinforcement A. Rats maintained on a
restricted feeding schedule were trained to lever-press for food
pellets (45 mg) on a fixed-ratio 2 (FR2) schedule during 2-hour
test sessions. Rats received IV injections of saline, GluR2-3A, or
GluR2-3Y, in a counterbalanced order, 60 min prior to the test
session. There were no significant differences in total number of
responses for food reward, between the three conditions. B. Rats
were first trained to self-administer d-amphetamine (0.2
mg/infusion) via a jugular catheter on an FR2 schedule of
reinforcement. Once responding in the 3 hour test sessions had
stabilized, the rats were then trained on a Progressive Ratio
Schedule in which successively more responses were required to
obtain each successive reinforcement. The ratio at which rats
failed to perform the appropriate number of responses in a 1 hour
period is called the beak point and this test is a sensitive
measure of the unconditional reward value of a specific reward
stimulus. Once stable Break point values were established, rats
received IV injections of saline, GluR2-3A, or GluR2-3Y, in a
counterbalanced order, 60 min prior to the test session. There were
no significant differences in the Break Point measures for
drug-reward, between the three conditions.
[0059] FIG. 19 GluR23y peptide blocked stress induced anxiety in a
rat model of stress. Rats (n=2) were injected with either 10 nM/g
GluR2-3Y or equal volume of vehicle ACSF (IP). They were given 30
minutes in a dark room post injection. After that they were placed
on an elevated platform for 30 minutes as a stressor. After that 30
minutes they were placed on the elevated plus maze for 5 minutes.
The GluR2-3Y injected rats spent more time on the open arms than
the ACSF rats. The ACSF rats spent most of their time in the
corners of the closed arms or rearing to look over the walls. Thus,
GluR23Y peptide blocked stress induced anxiety. These results
strongly suggest that facilitated AMPAR endocytosis and hence the
ession of LTD play an indispensable role in the expression of
stress-induced behaviors and that LTD blocker such as the GluR23Y
may be used therapeutics to treat stress-related brain
disorders.
DETAILED DESCRIPTION OF THE INVENTION
[0060] The invention provides, in part, methods and reagents for
modulating neuronal apoptosis. The invention also provides, in
part, methods and reagents for modulating synaptic plasticity. For
example, compounds according to the invention may be used as
neuroprotective agents that are capable of modulating AMPA receptor
endocytosis. In some embodiments, such compounds can modulate AMPA
receptor endocytosis and block neuronal apoptosis without affecting
NMDA receptor function, and therefore may bypass the negative
effects of blocking NMDA receptor function.
[0061] Alternative embodiments and examples of the invention are
described herein. These embodiments and examples are illustrative
and should not be construed as limiting the scope of the
invention.
Assays
[0062] Various assays, as described herein or known to one of
ordinary skill in the art, may be performed to determine the
modulatory activity of a compound according to the invention. For
example, modulation of synaptic plasticity, AMPA receptor
endocytosis, NMDA-induced neuronal apoptosis, or AMPA receptor
phosphorylation, may be tested as described herein or as known to
one of ordinary skill in the art. In some embodiments, assays may
be performed to test compounds for ability to inhibit AMPA receptor
endocytosis. Such assays include without limitation nucleic acid,
polypeptide, small molecule etc. based assays, such as
immunoassays, hybridization assays, small molecule binding assays,
peptide binding assays, antibody binding assays, competition
assays, endocytosis assays, phosphorylation assays, apoptosis and
cell death assays, histochemistry, animal and in vitro model
assays, etc.
[0063] AMPA receptor polypeptides may be provided in neuronal or
non-neuronal cells, or cell lysates. Cells and cell lines may be
obtained from commercial sources, for example, ATCC, Manassas, Va.,
USA. Suitable animal models for neurological disorders may be
obtained from, for example, The Jackson Laboratory, Bar Harbor,
Me., USA or from other sources. Suitable animal models include
models for stroke.sup.87-94, drug addiction .sup.101-106, 112,
schizophrenia.sup.107-111, Huntington's Disease.sup.112,
Epilepsy.sup.115, neurocomplication of AIDS.sup.116, mental
retardation (e.g., Fragile X retardation, Rett
syndrome).sup.117,118, and multiple sclerosis.sup.119,120.
[0064] The assays may be conducted using detectably labelled
molecules, i.e., any means for marking and identifying the presence
of a molecule, e.&, an oligonucleotide probe or primer, a gene
or fragment thereof, a peptide, or a cDNA molecule. Methods for
detectably-labeling a molecule are well known in the art and
include, without limitation, radioactive labelling (e.&, with
an isotope such as .sup.32P or .sup.35S) and nonradioactive
labelling such as, enzymatic labelling (for example, using
horseradish peroxidase or alkaline phosphatase), chemiluminescent
labeling, fluorescent labeling (for example, using fluorescein),
bioluminescent labeling, or antibody detection of a ligand attached
to the probe. Also included in this definition is a molecule that
is detectably labelled by an indirect means, for example, a
molecule that is bound with a first moiety (such as biotin) that
is, in turn, bound to a second moiety that may be observed or
assayed (such as fluorescein-labeled streptavidin). Labels also
include digoxigenin, luciferases, and aequorin.
Disorders and Conditions
[0065] Any disorder or condition which includes neural dysfunction,
for example due to neurological damage or behavioural sensitization
due to the excessive activation of NMDA receptors or due to changes
in AMPA receptor endocytosis may be treated, prevented, or studied
according to the methods and compounds of the invention. Therefore,
disorders associated with other conditions ranging from
hypoglycemia, hypoxia, and cardiac arrest to epilepsy are
considered neurological damage disorders according to the
invention. Disorders according to the invention include without
limitation cerebral ischemia, occurring for example after stroke
(ischemic stroke due to for example atherothrombotic disease of
e.g., extracranial arteries, or to emboli from the heart or lacunar
infarcts) or brain trauma (e.g., intracerebral hemorrhage or
subarachnoid hemorrhage); head injury, neurodegenerative disorders
in which compromised neurons become sensitive to excitotoxic
damage; Alzheimer's, Parkinson's, or Huntington's disease;
epilepsy, neuropathic pain; amyotrophic lateral sclerosis (ALS);
Hutchinson Gilford syndrome; diabetes; ataxia; mental retardation;
or dementias. Major risk factors for stroke include smoking,
diabetes, obesity, and high blood pressure. Accordingly, subjects
having any of these conditions or behaviours may be considered as
having a disorder according to the invention.
[0066] Disorders according to the invention also include those
disorders associated with defects or dysfunction in learning or
memory; psychiatric disorders, such as autism, schizophrenia or
fragile X syndrome; or disorders associated with substance abuse or
addition to drugs, including nicotine, alcohol, opiates such as
heroin, codeine and morphine, including derivatives such as
pethidine and methadone, nicotine, marijuana, phenyclidene,
psychostimulants such as amphetamines and cocaine, barbiturates
such as pentobarbitone and quinalbarbitone, and benzodiazepines
such as temazepam, diazepam and flunitrazepam.
Antibodies
[0067] The compounds of the invention can be used to prepare
antibodies to GluR2-CT peptides or analogues thereof, for example,
the sequences set forth in Table I or conservative substitutions
thereof, Formula I, or Formula A, or to homologous sequences found
in the C-terminus of the GluR2, GluR3, or GluR4 subunits of the
AMPA receptor, using standard techniques of preparation as, for
example, described in Harlow and Lane.sup.56, or known to those
skilled in the art. Antibodies can be tailored to minimise adverse
host immune response by, for example, using chimeric antibodies
contain an antigen binding domain from one species and the Fc
portion from another species, or by using antibodies made from
hybridomas of the appropriate species. In alternative embodiments
of the invention, antibodies may be raised, for exile, against a
phosphorylated GluR-CT peptide that is phosphorylated one or more
tyrosines or serines or threonines. In alternative embodiments of
the invention, antibodies may be raised, for example against a
constitutively phosphorylated GluR-CT peptide that replaces
existing tyrosines or seines or threonines with glutamates and
aspartates. In some embodiments, anti-idiotypic antibodies may be
raised, for example, against to an antibody that specifically binds
a GluR CT peptide or analogue thereof.
Polypeptides And Test Compounds
[0068] In one aspect, compounds according to the invention include
GluR2, GluR3, or GluR4 peptides and analogues and variants thereof
including, for example, the peptides described herein that are
phosphorylated or unphosphorylated at any one of the three
tyrosines, including polypeptides that are constitutively
phosphorylated, or that are unphosphorylatable, as well as homologs
and fragments thereof. For example, compounds according to the
invention include peptides including the sequences set forth in
Table I or analogues or variants thereof. TABLE-US-00001 TABLE I
YKEGYNVYG YKEGYNVDG YKEGYNVEG YKEGYNVSG YKEGYNVTG YKEGDNVYG
YKEGDNVDG YKEGDNVEG YKEGDNVSG YKEGDNVTG YKEGENVYG YKEGENVDG
YKEGENVEG YKEGENVSG YKEGENVTG YKEGSNVYG YKEGSNVDG YKEGSNVEG
YKEGSNVSG YKEGSNVTG YKEGTNVYG YKEGTNVDG YKEGTNVEG YKEGTNVSG
YKEGTNVTG DKEGYNVYG DKEGYNVDG DKEGYNVEG DKEGYNVSG DKEGYNVTG
DKEGDNVYG DKEGDNVDG DKEGDNVEG DKEGDNVSG DKEGDNVTG DKEGENVYG
DKEGENVDG DKEGENVEG DKEGENVSG DKEGENVTG DKEGSNVYG DKEGSNVDG
DKEGSNVEG DKEGSNVSG DKEGSNVTG DKEGTNVYG DKEGTNVDG DKEGTNVEG
DKEGTNVSG DKEGTNVTG EKEGYNVYG EKEGYNVDG EKEGYNVEG EKEGYNVSG
EKEGYNVTG EKEGDNVYG EKEGDNVDG EKEGDNVEG EKEGDNVSG EKEGDNVTG
EKEGENVYG EKEGENVDG EKEGENVEG EKEGENVSG EKEGENVTG EKEGSNVYG
EKEGSNVDG EKEGSNVEG EKEGSNVSG EKEGSNVTG EKEGTNVYG EKEGTNVDG
EKEGTNVEG EKEGTNVSG EKEGTNVTG SKEGYNVYG SKEGTNVDG SKEGYNVEG
SKEGYNVSG SKEGYNVTG SKEGDNVYG SKEGDNVDG SKEGDNVEG SKEGDNVSG
SKEGDNVTG SKEGENVYG SKEGENVDG SKEGENVEG SKEGENVSG SKEGENVTG
SKEGSNVYG SKEGSNVDG SKEGSNVEG SKEGSNVSG SKEGSNVTG SKEGTNVYG
SKEGTNVDG SKEGTNVEG SKEGTNVSG SKEGTNVTG TKEGYNVYG TKEGYNVDG
TKEGYNVEG TKEGYNVSG TKEGYNVTG TKEGDNVYG TKEGDNVDG TKEGDNVEG
TKEGDNVSG TKEGDNVTG TKEGENVYG TKEGENVDG TKEGENVEG TKEGENVSG
TKEGKNVTG TKEGSNVYG TKEGSNVDG TKEGSNVEG TKEGSNVSG TKEGSNVTG
TKEGTNVYG TKEGTNVDG TKEGTNVEG TKEGTNVSG TKEGTNVTG YEEGYNVYG
YREGYNVDG YREGYNVEG YREGYNVSG YREGYNVTG YREGDNVYG YREGDNVDG
YREGDNVEG YREGDNVSG YREGDNVTG YREGENVYG YREGENVDG YREGENVEG
YPEGENVSG YREGENVTG YREGSNVYG YREGSNVDG YREGSNVEG YREGSNVSG
YREGSNVTG YREGTNVYG YREGTNVDG YREGTNVEG YREGTNVSG YREGTNVTG
DREGYNVYG DREGYNVDG DREGYNVEG DREGYNVSG DREGYNVTG DREGDNVYG
DREGDNVDG DREGDNVEG DREGDNVSG DREGDNVTG DREGENVYG DREGENVDG
DREGENVEG DREGENVSG DREGENVTG DREGSNVYG DREGSNVDG DREGSNVEG
DREGSNVSG DREGSNVTG DREGTNVYG DREGTNVDG DREGTNVEG DREGThVSG
DREGTNVTG EREGYNVYG EREGYNVDG EREGYNVEG EREGYNVSG EREGYNVTG
EREGDNVYG EREGDNVDG EREGDNVEG EREGDNVSG EREGDNVTG EREGENVYG
EREGENVDG EREGENVEG EREGENVSG EREGENVTG EREGSNVYG EREGSNVDG
EREGSNVEG EREGSNVSG EREGSNVTG EREGTNVYG EREGTNVDG EREGTNVEG
EREGTNVSG EREGTNVTG SREGYNVYG SREGYNVDG SREGYNVEG SREGYNVSG
SREGYNVTG SREGDNVYG SREGDNVDG SREGDNVEG SRSGDNVSG SREGDNVTG
SREGENVYG SREGENVDG SREGENVEG SRSGENVSG SREGENVTG SREGSNVYG
SREGSNVDG SREGSNVEG SREGSNVSG SREGSNVTG SREGTNVYG SREGTNVDG
SREGTNVEG SREGTNVSG SREGTNVTG TREGYNVYG TREGYNVDG TREGYNVEG
TREGYNVSG TREGYNVTG TREGDNVYG TREGDNVDG TREGDNVEG TREGDNVSG
TREGDNVTG TREGENVYG TREGENVDG TREGENVEG TREGENVSG TREGENVTG
TREGSNVYG TREGSNVDG TREGSNVEG TREGSNVSG TREGSNVTG TREGTNVYG
TREGTNVDG TREGTNVEG TREGTNVSG TREGTNVTG YKEGYNVYGIE YKEGYNVDGIE
YKEGYNVEGIE YREGYNVSGIE YKEGYNVTGIE YKEGDNVYGIE YREGDNVDGIE
YKEGDNVEGIE YKEGDNVSGIE YKEGDNVTGIE YKEGENVYGIE YKEGENVDGIE
YKEGENVEGIE YKEGENVSGIE YKEGENVTGIE YKEGSNVYGIE YKEGSNVDGIE
YKEGSNVEGIE YKEGSNVSGIE YKEGSNVTGIE YKEGTNVYGIE YKEGTNVDGIE
YKEGTNVEGIE YKEGTNVSGIE YKEGTNVTGIE DKEGYNVYGIE DKEGYNVDGIE
DKEGYNVEGIE DKEGYNVSGIE DKEGYNVTGIE DKEGDNVYGIE DKEGDNVDGIE
DKEGDNVEGIE DKEGDNVSGIE DKEGDNVTGIE DKEGENVYGIE DKEGENVDGIE
DKEGENVEGIE DREGENYSGIE DKEGENVTGIE DKEGSNVYGIE DKEGSNVDGIE
DKEGSNVEGIE DKEGSNVSGIE DKEGSNVTGIE DKEGTNVYGIE DKEGTNVDGIE
DKEGTNVEGIE DKEGTNVSGIE DKEGTNVTGIE EKEGYNVYGIE EREGYNVDGIE
EKEGYNVEGIE EKEGYNVSGIE EKEGYNVTGIE EKEGDNVYGIE EKEGDNVDGIE
EKEGDNVEGIE EKEGDNVSGIE EKEGDNVTGIE EKEGENVYGIE EREGENVDGIE
EREGENVEGIE EKEGENVSGIE EKEGENVTGIE EKEGSNVYGIE EKEGSNVDGIE
EKEGSNVEGIE EKEGSNVSGIE EKEGSNVTGIE EKEGTNVYGIE EKEGTNVDGIE
EKEGTNVEGIE EKEGTNVSGIE EKEGTNVTGIE SKEGYNVYGIE SKEGYNVDGIE
SKEGYNVEGIE SKEGYNVSGIE SKEGYNVTGIE SKEGDNVYGIE SKEGDNVDGIE
SKEGDNVEGIE SKEGDNVSGIE SKEGDNVTGIE SKEGENVYGIE SKEGENVDGIE
SKEGENVEGIE SKEGENVSGIE SKEGENVTGIE SKEGSNVYGIE SKEGSNVDGIE
SKEGSNVEGIE SKEGSNVSGIE SKEGSNVTGIE SKEGTNVYGIE SKEGTNVDGIE
SKEGTNVEGIE SKEGTNVSGIE SKEGTNVTGIE TKEGYNVYGIE TKEGYNVDGIE
TREGYNVEGIE TKEGYNVSGIE TKEGYNVTGIE TKEGDNVYGIE TKEGDNVDGIE
TKEGDNVEGIE TKEGDNVSGIE TKEGDNVTGIE TKEGENVYGIE TKEGENVDGIE
TKEGENVEGIE TKEGENVSGIE TKEGENVTGIE TKEGSNVYGIE TKEGSNVDGIE
TKEGSNVEGIE TKEGSNVSGIE TKEGSNVTGIE TKEGTNVYGIE TKEGTNVDGIE
TKEGTNVEGIE TKEGTNVSGIE TKEGTNVTGIE YREGYNVYGIE YREGYNVDGIE
YREGYNVEGIE YREGYNVSGIE YREGYNVTGIE YREGDNVYGIE YREGDNVDGIE
YREGDNVEGIE YREGDNVSGIE YREGDNVTGIE YREGENVYGIE YREGENVDGIE
YREGENVEGIE YREGENVSGIE YREGENVTGIE YREGSNVYGIE YREGSNVDGIE
YREGSNVEGIE YREGSNVSGIE YREGSNVTGIE YREGTNVYGIE YREGTNVDGIE
YREGTNVEGIE YREGTNVSGIE YREGTNVTGIE DREGYNVYGIE DREGYNVDGIE
DREGYNVEGIE DREGYNVSGIE DREGYNVTGIE DREGDNVYGIE DREGDNVDGIE
DREGDNVEGIE DREGDNVSGIE DREGDNVTGIE DREGENVYGIE DREGENVDGIE
DREGENVEGIE DPEGENVSGIE DREGENVTGIE DREGSNVYGIE DREGSNVDGIE
DREGSNVEGIE DREGSNVSGIE DREGSNVTGIE DREGTNVYGIE DREGTNVDGIE
DREGTNVEGIE DREGTNVSGIE DREGTNVTGIE EREGYNVYGIE EREGYNVDGIE
EREGYNVEGIE EREGYNVSGIE EREGYNVTGIE EREGDNVYGIE EREGDNVDGIE
EREGDNVEGIE EREGDNVSGIE EREGDNVTGIE EREGENVYGIE EREGENVDGIE
EREGENVEGIE EREGENVSGIE EREGENVTGIE EREGSNVYGIE EREGSNVDGIE
EREGSNVEGIE EREGSNVSGIE EREGSNVTGIE EREGTNVYGIE EREGTNVDGIE
EREGTNVEGIE EREGTNVSGIE EREGTNVTGIE SREGYNVYGIE SREGYNVDGIE
SREGYNVEGIE SREGYNVSGIE SREGYNVTGIE SREGDNVYGIE SREGDNVDGIE
SREGDNVEGIE SREGDNVSGIE SREGDNVTGIE SREGENVYGIE SREGENVDGIE
SREGENVEGIE SREGENVSGIE SREGENVTGIE SREGSNVYGIE SREGSNVDGIE
SREGSNVEGIE SREGSNVSGIE SREGSNVTGIE SREGTNVYGIE SREGTNVDGIE
SREGTNVEGIE SREGTNVSGIE SREGTNVTGIE TREGYNVYGIE TREGYNVDGIE
TREGYNVEGIE TREGYNVSGIE TREGYNVTGIE TREGDNVYGIE TREGDNVDGIE
TREGDNVEGIE TREGDNVSGIE TREGDNVTGIE TREGENVYGIE TREGENVDGIE
TREGENVEGIE TREGENVSGIE TREGENVTGIE TREGSNVYGIE TREGSNVDGIE
TREGSNVEGIE TREGSNVSGIE TREGSNVTGIE TREGTNVYGIE TREGTNVDGIE
TREGTNVEGIE TREGTNVSGIE TREGTNVTGIE
[0069] In some embodiments, compounds according to the invention do
not have, or have to a lesser extent, the negative side effects
associated with the use of other neuroprotective agents. For
example, compounds according to the invention may exhibit any value
from between 10% to 100% reduction in psychotomimesis, respiratory
depression, cardiovascular disregulation, or any other adverse side
effect when compared to a NMDA receptor antagonist or glutamate
release blocker (such as Selfotel, Gavestinel, Aptinagel,
memantine. etc..sup.75-78,95-99).
[0070] In some embodiments, compounds according to the invention
are similarly efficacious or more efficacious than existing
neuroprotective agents such as NMDA receptor antagonists (e.g.,
Gavestinel or Aptinagel) or other neuroprotective agents (e.g.,
Kappa opiod peptide R antagonist such as Cervene; NOS inhibitors
such as Lubeluzole; Na.sup.+ channel blockers such as
Lubeluzole;cell membrane stabilizers such as Citicoline; Ca.sup.2+
channel antagonists; anti-ICAM antibodies such as Enlimornab; GABAA
receptor modulators such as Clomethiazole; glutamate release
inhibitors such as Riluzole)..sup.79-84,100 For example, compounds
according to the invention may exhibit any value from between 0% to
100% or greater than 100% efficacy when compared with other
neuroprotective agents.
[0071] In alternative embodiments, one or more of the compounds
described herein may be specifically excluded from one or more
aspects of the invention.
[0072] Compounds can be prepared by, for example, replacing,
deleting, or inserting an amino acid residue at any position of a
GluR peptide or peptide analogue, for example, a GluR2-CT peptide
sequence as set forth in Table I, Formula I, or Formula A, or to
homologous sequences found in the C-terminus of the GluR2, GluR3,
or GluR4 subunits of the AMPA receptor, as described herein, with
other conservative amino acid residues, i.e., residues having
similar physical, biological, or chemical properties, and screening
for the ability of the compound to inhibit endocytosis of the AMPA
receptor. In some embodiments of the invention, compounds of the
invention include antibodies that specifically bind to a GluR
polypeptide, for example, a GluR2-CT peptide, which may be
phosphorylated, unphosphorylated, unphosphorylatable, or
constitutively phosphorylated. In some embodiments of the
invention, compounds of the invention include antibodies that bind
to antibodies that specifically bind GluR CT peptides.
[0073] It is well known in the art that some modifications and
changes can be made in the structure of a polypeptide without
substantially altering the biological function of that peptide, to
obtain a biologically equivalent polypeptide. For example, in some
embodiments, compounds according to the invention may be adapted or
modified for oral administration such that they are resistant to
digestion by stomach acids. In one aspect of the invention,
polypeptides of the present invention also extend to biologically
equivalent peptides that differ from a portion of the sequence of
the polypeptides of the present invention by conservative amino
acid substitutions. As used herein, the term "conserved amino acid
substitutions" or "conservative substitution" refers to the
substitution of one amino acid for another at a given location in a
GluR CT peptide (e.g., as set forth in Table I, Formula I, or
Formula A, or to homologous sequences found in the C-terminus of
the GluR2, GluR3, or GluR4 subunits of the AMPA receptor), where
the substitution can be made without substantial loss of the
relevant function. In making such changes, substitutions of like
amino acid residues can be made on the basis of relative similarity
of side-chain substituents, for example, their size, charge,
hydrophobicity, hydrophilicity, and the like, and such
substitutions may be assayed for their effect on the function of
the peptide by routine testing.
[0074] As used herein, the term "ammo acids" means those L-amino
acids commonly found in naturally occurring proteins, D-amino acids
and such amino acids when they have been modified. Accordingly,
amino acids of the invention may include, for example:
2-Aminoadipic acid; 3-Aminoadipic acid; beta-Alanine;
beta-Aminopropionic acid; 2-Aminobutyric acid; 4-Aminobutyric acid;
piperidinic acid; 6-Aminocaproic acid; 2-Aminoheptanoic acid;
2-Aminoisobutyric acid, 3-Aminoisobutyric acid; 2-Aminopimelic
acid; 2,4 Diaminobutyric acid; Desmosine; 2,2'-Diaminopimelic acid;
2,3-Diamiopropionic acid; N-Ethylglycine; N-Ethylasparagine;
Hydroxylysine; allo-Hydroxylysine; 3-Hydroxyproline;
4-Hydroxyproline; Isodesmosine; allo-Isoleucine; N-Methylglycine;
sarcosine; N-Methylisoleucine; 6-N-methyllysine; N-Methylvaline;
Norvaline; Norleucine; and Ornithine,
[0075] In some embodiments, conserved amino acid substitutions may
be made where an amino acid residue is substituted for another
having a similar hydrophilicity value (e.g., within a value of plus
or minus 2.0, or plus or minus 1.5, or plus or minus 1.0, or plus
or minus 0.5), where the following may be an amino acid having a
hydropathic index of about -1.6 such as Tyr (-1.3) or Pro (-1.6)
are assigned to amino acid residues (as detailed in U.S. Pat. No.
4,554,101, incorporated herein by reference): Arg (+3.0); Lys
(+3.0); Asp (+3.0); Glu (+3.0); Ser (+0.3); Asn (+0.2); Gln (+0.2);
Gly (0); Pro (-0.5); Thr (-0.4); Ala (-0.5); His (-0.5); Cys
(-1.0); Met (-1.3); Val (-1.5); Leu (-1.8); Ile (-1.8); Tyr (-2.3);
Phe (-2.5); and Trp (-3.4).
[0076] In alternative embodiments, conservative amino acid
substitutions may be made where an amino acid residue is
substituted for another having a similar hydropathic index (e.g.,
within a value of plus or minus 2.0, or plus or minus 1.5, or plus
or minus 1.0, or plus or minus 0.5). In such embodiments, each
amino acid residue may be assigned a hydropathic index on the basis
of its hydrophobicity and charge characteristics, as follows: Ile
(+4.5); Val (+4.2); Leu (+3.8); Phe (+2.8); Cys (+2.5); Met (+1.9);
Ala (+1.8); Gly (-0.4); Thr (-0.7); Ser (-0.8); Trp (-0.9); Tyr
(-1.3); Pro (-1.6); His (-3.2); Glu (-3.5); Gln (-3.5); Asp (-3.5);
Asn (-3.5); Lys (-3.9); and Arg (-4.5).
[0077] In alternative embodiments, conservative amino acid
substitutions may be made using publicly available families of
similarity matrices..sup.63-69 The PAM matrix is based upon counts
derived from an evolutionary model, while the Blosum matrix uses
counts derived from highly conserved blocks within an alignment. A
similarity score of above zero in either of the PAM or Blosum
matrices may be used to make conservative amino acid
substitutions.
[0078] In alternative embodiments, conservative amino acid
substitutions may be made where an amino acid residue is
substituted for another in the same class, where the amino acids
are divided into non-polar, acidic, basic and neutral classes, as
follows: non-polar: Ala, Val, Leu, Ile, Phe, Trp, Pro, Met; acidic:
Asp, Glu; basic: Lys, Arg, His; neutral: Gly, Ser, Thr, Cys, Asn,
Gln, Tyr.
[0079] Conservative ammo acid changes can include the substitution
of an L-amino acid by the corresponding D-amino acid, by a
conservative D-amino acid, or by a naturally-occurring,
non-genetically encoded form of amino acid, as well as a
conservative substitution of an L-amino acid. Naturally occurring
non-genetically encoded amino acids include beta-alanine,
3-amino-propionic acid, 2,3-diamino propionic acid,
alpha-aminoisobutyric acid, 4-amino-butyric acid, N-methylglycine
(sarcosine), hydroxyproline, ornithine, citrulline, t-butylalanine,
t-butylglycine, N-methylisoleucine, phenylglycine,
cyclohexylalanine, norleucine, norvaline, 2-napthylalanine,
pyridylalanine, 3-benzothienyl alanine, 4-chlorophenylalanine,
2-fluorophenylalanine, 3-fluorophenylalanine,
4-fluorophenylalanine, penicillamine,
1,2,3,4-tetrahydro-isoquinoline-3 carboxylix acid,
beta-2-thienylalanine, methionine sulfoxide, homoarginine, N-acetyl
lysine, 2-amino butyric acid, 2-amino butyric acid, 2,4-diamino
butyric acid, p-aminophenylalanine, N-methylvaline, homocysteine,
homoserine, cysteic acid, epsilon-amino hexanoic acid, delta-amino
valeric acid, or 2,3-diaminobutyric acid.
[0080] In alternative embodiments, conservative amino acid changes
include changes based on considerations of hydrophilicity or
hydrophobicity, size or volume, or charge. Amino acids can be
generally characterized as hydrophobic or hydrophilic, depending
primarily on the properties of the amino acid side chain. A
hydrophobic amino acid exhibits a hydrophobicity of greater than
zero, and a hydrophilic amino acid exhibits a hydrophilicity of
less than zero, based on the normalized consensus hydrophobicity
scale of Eisenberg et al..sup.57 Genetically encoded hydrophobic
amino acids include Gly, Ala, Phe, Val, Leu, Ile, Pro, Met and Trp,
and genetically encoded hydrophilic amino acids include Thr, His,
Glu, Gin, Asp, Arg, Ser, and Lys. Non-genetically encoded
hydrophobic amino acids include t-butylalanine, while
non-genetically encoded hydrophilic amino acids include citrulline
and homocysteine.
[0081] Hydrophobic or hydrophilic amino acids can be further
subdivided based on the characteristics of their side chains. For
example, an aromatic amino acid is a hydrophobic amino acid with a
side chain containing at least one aromatic or heteroaromatic ring,
which may contain one or more substituents such as --OH, --SH,
--CN, --F, Cl, --Br, --I, --NO.sub.2, --NO, --NH.sub.2, --NHR -NRR,
--C(O)R, --C(O)OH, --C(O)OR, --C(O)NH.sub.2, --C(O)NHR C(O)NRR,
etc., where R is independently (C.sub.1-C.sub.6) alkyl, substituted
(C.sub.1-C.sub.6) alkyl, (C.sub.1-C.sub.6) alkenyl, substituted
(C.sub.1-C.sub.6) alkenyl, (C.sub.1-C.sub.6) alkynyl, substituted
(C.sub.1-C.sub.6) alknyl, (C.sub.5-C.sub.20) aryl, substituted
(C.sub.5-C.sub.20) aryl, (C.sub.6-C.sub.26) alkaryl, substituted
(C.sub.6-C.sub.26) alkaryl, 5-20 membered heteroaryl substituted
5-20 membered heteroaryl, 6-26 membered alkheteroaryl or
substituted 6-26 membered alkheteroaryl. Genetically encoded
aromatic amino acids include Phe, Tyr, and Trp, while
non-genetically encoded aromatic ammo acids include phenylglycine,
2-napthylalanine, beta-2-thienylalanine, 12,3,4
tetrahydro-isoquinoline-3-carboxylic acid, 4-chlorophenylalanine,
2-fluorophenylalanine3-fluorophenylalanine, and
4-fluorophenylalanine.
[0082] An apolar amino acid is a hydrophobic amino acid with a side
chain that is uncharged at physiological pH and which has bonds in
which a pair of electrons shared in common by two atoms is
generally held equally by each of the two atoms (i.e., the side
chain is not polar). Genetically encoded apolar amino acids include
Gly, Leu, Val, Ile, Ala, and Met, while non-genetically encoded
apolar amino acids include cyclohexylalanine . Apolar amino acids
can be further subdivided to include aliphatic amino acids, which
is a hydrophobic amino acid having an aliphatic hydrocarbon side
chain. Genetically encoded aliphatic amino acids include Ala, Leu,
Val, and Ile, while non-genetically encoded aliphatic amino acids
include norleucine.
[0083] A polar amino acid is a hydrophilic amino acid with a side
chain that is uncharged at physiological pH, but which has one bond
in which the pair of electrons shared in common by two atoms is
held more closely by one of the atoms. Genetically encoded polar
amino acids include Ser, Thr, Asn, and Gln, while non-genetically
encoded polar amino acids include citrulline, N-acetyl lysine, and
methionine sulfoxide. An acidic amino acid is a hydrophilic amino
acid with a side chain pKa value of less than 7. Acidic amino acids
typically have negatively charged side chains at physiological pH
due to loss of a hydrogen ion. Genetically encoded acidic amino
acids include Asp and Glu. A basic amino acid is a hydrophilic
amino acid with a side chain pKa value of greater than 7. Basic
amino acids typically have positively charged side chains at
physiological pH due to association with hydronium ion. Genetically
encoded basic amino acids include Arg, Lys, and His, while
non-genetically encoded basic amino acids include the non-cyclic
amino acids ornithine, 2,3,-diaminopropionic acid,
2,4-diaminobutyric acid, and homoarginine.
[0084] It will be appreciated by one skilled in the art that the
above classifications are not absolute and that an amino acid may
be classified in more than one category. In addition, amino acids
can be classified based on known behaviour and or characteristic
chemical, physical, or biological properties based on specified
assays or as compared with previously identified amino acids. Amino
acids can also include bifunctional moieties having amino acid-like
side chains.
[0085] Conservative changes can also include the substitution of a
chemically derivatised moiety for a non-derivatised residue, by for
example, reaction of a functional side group of an amino acid.
Thus, these substitutions can include compounds whose free amino
groups have been derivatised to amine hydrochlorides, p-toluene
sulfonyl groups, carbobenzoxy groups, t-butyloxycarbonyl groups,
chloroacetyl groups or formyl groups. Similarly, free carboxyl
groups can be derivatized to form salts, methyl and ethyl esters or
other types of esters or hydrazides, and side chains can be
derivatized to from O-acyl or O-alkyl derivatives for free hydroxyl
groups or N-im-benzylhistidine for the imidazole nitrogen of
histidine, Peptide analogues also include amino acids that have
been chemically altered, for example, by methylation, by amidation
of the C-terminal amino acid by an alkylamine such as ethylamine,
ethanolamine, or ethylene diamine, or acylation or methylation of
an amino acid side chain (such as acylation of the epsilon amino
group of lysine). Peptide analogues can also include replacement of
the amide linkage in the peptide with a substituted amide (for
example, groups of the formula --C(O)--NR, where R is
(C.sub.1-C.sub.6) alkyl, (C.sub.1-C.sub.6) alkenyl,
(C.sub.1-C.sub.6) alkynyl, substituted (C.sub.1-C.sub.6) alkyl,
substituted (C.sub.1-C.sub.6) alkenyl, or substituted
(C.sub.1-C.sub.6) alkynyl) or isostere of an amide linkage (for
example, --CH.sub.2NH--, --CH.sub.2S, --CH.sub.2CH.sub.2--,
--CH.dbd.CH-- (cis and trans), --C(O)CH.sub.2----CH(OH)CH.sub.2--,
or --CH.sub.2SO--).
[0086] The compound can be covalently linked, for example, by
polymerisation or conjugation, to form homopolymers or
heteropolymers. Spacers and linkers, typically composed of small
neutral molecules, such as amino acids that are uncharged under
physiological conditions, can be used. Linkages can be achieved in
a number of ways. For example, cysteine residues can be added at
the peptide termini, and multiple peptides can be covalently bonded
by controlled oxidation. Alternatively, heterobifunctional agents,
such as disulfide/amide forming agents or thioether/amide forming
agents can be used. The compound can also be linked to a another
compound that can modulate neuronal apoptosis, AMPA receptor
endocytosis, synaptic plasticity, learning or memory, or substance
abuse or addiction etc. The compound can also be constrained, for
example, by having cyclic portions.
[0087] Peptides or peptide analogues can be synthesised by standard
chemical techniques, for example, by automated synthesis using
solution or solid phase synthesis methodology. Automated peptide
synthesisers are commercially available and use techniques well
known in the art. Peptides and peptide analogues can also be
prepared using recombinant DNA technology using standard methods
such as those described in, for example, Sambrook, et al..sup.58 or
Ausubel et al..sup.59 In general, candidate compounds are
identified from large libraries of both natural products or
synthetic (or semi-synthetic) extracts or chemical libraries
according to methods known in the art. Those skilled in the field
of drug discovery and development will understand that the precise
source of test extracts or compounds is not critical to the
method(s) of the invention. Accordingly, virtually any number of
chemical extracts or compounds can be screened using the exemplary
methods described herein. Examples of such extracts or compounds
include, but are not limited to, plant-, fungal-, prokaryotic- or
animal-based extract, fermentation broths, and synthetic compounds,
as well as modification of existing compounds. Numerous methods are
also available for generating random or dire synthesis (e.g.,
semi-synthesis or total synthesis) of any number of chemical
compounds, including, but not limited to, saccharide-, lipid-,
peptide-, and nucleic acid-based compounds. Synthetic compound
libraries are commercially available. Alternatively, libraries of
natural compounds in the form of bacterial, fungal plant, and
animal ex are commercially available from a number of sources,
including Biotics (Sussex, UK), Xenova (Slough, UK), Harbor Branch
Oceanographic Institute (FL Pierce, FL, USA), and PharmaMar, MA,
USA. In addition, natural and synthetically produced libraries of,
for example, neuronal polypeptides, are produced, if desired,
according to methods known in the art, e.g., by stand extraction
and fractionation methods. Furthermore, if desired, any library or
compound is readily modified using standard chemical, physical, or
biochemical methods.
[0088] When a crude extract is found to modulate neuronal
apoptosis, AMPA receptor endocytosis, synaptic plasticity, learning
or memory, or substance abuse or addiction etc., further
fractionation of the positive lead extract is necessary to isolate
chemical constituents responsible for the observed effect. Thus,
the goal of the extraction, fractionation, and purification process
is the careful characterization and identification of a chemical
entity within the crude extract having neuronal apoptosis, AMPA
receptor endocytosis, synaptic plasticity, etc., modulatory
activities. The same assays described herein for the detection of
activities in mixtures of compounds can be used to purify the
active component and to test derivatives thereof. Methods of
fractionation and purification of such heterogeneous extracts are
known in the art. If desired, compounds shown to be useful agents
for treatment are chemically modified according to methods known in
the art. Compounds identified as being of therapeutic value may be
subsequently analyzed using a mammalian model, or any other animal
model for neuronal damage, neural dysfunction, synaptic plasticity,
learning or memory, or substance abuse or addiction.
Pharmaceutical Compositions, Dosages, And Administration
[0089] Compounds of the invention can be provided alone or in
combination with other compounds (for example, nucleic acid
molecules, small molecules, peptides, or peptide analogs), in the
presence of a liposome, an adjuvant, or any pharmaceutically
acceptable carrier, in a form suitable for administration to
humans. If desired, treatment with a compound according to the
invention may be combined with more traditional and existing
therapies for neurological damage, synaptic plasticity, learning or
memory, or substance abuse. For example, compounds according to the
invention may be administered as combination therapy with other
treatments such as free-radical inhibitors to maximise neuronal
survival; as complementary therapy to anti-coagulant prophylaxis in
subjects undergoing atrial fibrillation or are considered to be at
risk for stroke..sup.86 In some embodiments, the compounds may be
administered at specific therapeutic windows. For example, in some
embodiments, the compounds may be administered approximately 3
hours after onset of ischemia.
[0090] In some embodiments, compounds according to the invention
may be provided in fusion with a heterologous peptide to facilitate
translocation of the compounds across cell membranes, as for
example, described in U.S. Pat. No. 6,348,185; issued to
Piwnica-Worms; U.S. Patent Publication US 2003/0229202 (Guo et
al.), or PCT publication WO 00/62067 (Dowdy), Becker-Hapak et
al..sup.85, or Kabouridis.sup.114. In some embodiments, compounds
according to the invention may be provided in combination with a
carrier peptide, e.g., PEP 1.
[0091] In some embodiments, compounds according to the invention
may be provided in stem cells, e.g., neuronal stem cells, modified
to express the peptide. Suitable cells and vectors for such
delivery include viral vectors such as adenovirus, adeno-associated
virus, or Herpes Simplex Virus.sup.121,122.
[0092] Conventional pharmaceutical practice may be employed to
provide suitable formulations or compositions to administer the
compounds to patients suffering from or presymptomatic for
neurological damage or neural dysfunction. Compounds may be
administered systemically or may be administered directly to the
CNS or other region of neurological damage. In some embodiments,
compounds according to the invention may be provided in a form
suitable for delivery across the blood brain barrier. Any
appropriate route of administration may be employed, for example,
parenteral, intravenous, subcutaneous, intramuscular, intracranial,
intraorbital, ophthalmic, intraventricular, intracapsular,
intraspinal, intracisternal, intraperitoneal, intranasal, aerosol,
or oral administration. Therapeutic formulations may be in the form
of liquid solutions or suspensions; for oral administration,
formulations may be in the form of tablets or capsules; and for
intranasal formulations, in the form of powders, nasal drops, or
aerosols.
[0093] Methods well known in the art for making formulations are
found in, for example, "Remington's Pharmaceutical Sciences"
(19.sup.th edition), ed. A. Gennaro, 1995, Mack Publishing Company,
Easton, Pa Formulations for parenteral administration may, for
example, contain excipients, sterile water, or saline, polyalkylene
glycols such as polyethylene glycol, oils of vegetable origin, or
hydrogenated napthalenes. Biocompatible, biodegradable lactide
polymer, lactide/glycolide copolymer, or
polyoxyethylene-polyoxypropylene copolymers may be used to control
the release of the compounds. Other potentially useful parenteral
delivery systems for modulatory compounds include ethylene-vinyl
acetate copolymer particles, osmotic pumps, implantable infusion
systems, and liposomes. Formulations for inhalation may contain
excipients, for example, lactose, or may be aqueous solutions
containing, for example, polyoxyethylene-9-lauryl ether,
glycocholate and deoxycholate, or may be oily solutions for
administration in the form of nasal drops, or as a gel.
[0094] For therapeutic or prophylactic compositions, the compounds
are administered to an individual in an amount sufficient to stop
or slow cell degeneration or apoptosis, or to enhance or maintain
synaptic plasticity, depending on the disorder. An "effective
amount" of a compound according to the invention includes a
therapeutically effective amount or a prophylactically effective
amount. A "therapeutically effective amount" refers to an amount
effective, at dosages and for periods of time necessary, to achieve
the desired therapeutic result, such as reduction of cell
degeneration or apoptosis, or to enhance synaptic plasticity. A
therapeutically effective amount of a compound may vary according
to factors such as the disease state, age, sex, and weight of the
individual, and the ability of the compound to elicit a desired
response in the individual. Dosage regimens may be adjusted to
provide the optimum therapeutic response. A therapeutically
effective amount is also one in which any toxic or detrimental
effects of the compound are outweighed by the therapeutically
beneficial effects. A "prophylactically effective amount"refers to
an amount effective, at dosages and for periods of time necessary,
to achieve the desired prophylactic result, such as inhibition of
call degeneration or apoptosis, or to enhance synaptic plasticity.
Typically, a prophylactic dose is used in subjects prior to or at
an earlier stage of disease, so that a prophylactically effective
amount may be less than a therapeutically effective amount. A
preferred range for therapeutically or prophylactically effective
amounts of a compound maybe 0.1 nM-0.1M, 0.1 nM-0.05M 0.05 nM-15
.mu.M or 0.01 nM-10 .mu.M.
[0095] It is to be noted that dosage values may vary with the
severity of the condition to be alleviated or with the route of
administration selected. For example, for oral administration,
dosage values may be higher than for intravenous or intraperitoneal
administration. For any particular subject, specific dosage
regimens may be adjusted over time according to the individual need
and the professional judgement of the person administering or
supervising the administration of the compositions. Dosage ranges
set forth herein are exemplary only and do not limit the dosage
ranges that may be selected by medical practitioners. The amount of
active compound in the composition may vary according to factors
such as the disease state, age, sex, and weight of the individual.
Dosage regimens may be adjusted to provide the optimum therapeutic
response. For example, a single bolus may be administered, several
divided doses may be administered over time or the dose may be
proportionally reduced or increased as indicated by the exigencies
of the therapeutic situation. It may be advantageous to formulate
parenteral compositions in dosage unit form for ease of
administration and uniformity of dosage.
[0096] In the case of vaccine formulations, an immunogenically
effect amount of a compound of the invention can be provided, alone
or in combination with other compounds, with an adjuvant, for
example, Freund's incomplete adjuvant or aluminum hydroxide. The
compound may also be linked with a carrier molecule, such as bovine
serum albumin or keyhole limpet hemocyanin to enhance
immunogenicity.
[0097] In general compounds of the invention should be used without
causing substantial toxicity. Toxicity of the compounds of the
invention can be determined using standard techniques, for example,
by testing in cell cultures or experimental animals and determining
the therapeutic index, i.e., the ratio between the LD50 (the dose
lethal to 50% of the population) and the ID100 (the dose lethal to
100% of the population). In some circumstances however, such as in
severe disease conditions, it may be necessary to administer
substantial excesses of the compositions.
EXAMPLE 1
Materials And Methods
Primary Cultures of Hippocampal Neurons
[0098] Hippocampi were rapidly removed from embryonic E18 Sprague
Dawley rats and pooled prior to trituration. Hippocampal cell
suspensions were plated onto poly-D-lysine coated culture dishes or
glass coverslips and grown in Neurobasal.TM. media (Invitrogen) for
14 days in vitro (DIV). The media from mature 14 DIV neurons was
removed and replaced with 100 .mu.M NMDA plus 10 .mu.M glycine for
1 h at 37 .mu.C prior to restoring neurons to the defined growth
media. Twenty four hours after NMDA/glycine application, neurons
were processed using cell death assays. NMDA-induced
[Ca.sup.2+]responses were evoked and measured using methods
described previously.sup.26.
Cell Death Assays
[0099] Apoptosis quantification: NMDA-induced apoptosis was
quantified either using a Cell Death Detection Elisa Plus Kit
(Roche Applied Sciences), which is based on the in vitro
determination of cytoplasmic histone-associated DNA fragments, or
using TdT mediated addition of biotinylated 11-dUTP to the free
3'-OH ends of DNA. Absorbance readings for both assays were cared
out using a microplate reader.
[0100] Propidium Iodide (PI) staining of nuclei: After the
induction of apoptosis, cells were fixed with 4%
paraformaldehyde/4% sucrose for 10 min followed by ice cold acetone
for 1 min, and then stained with 20 mg/ml PI in Dulbecco's PBS for
30 min. Stained coverslips were mounted onto glass slides and
viewed with a Leica fluorescence microscope to identify condensed
nuclei. Cells with condensed nuclei were counted as apoptotic and
the percentage of apoptotic cells to the total number of cells was
calculated to give a semi-quantitative analysis, expressed as
percentage of apoptosis.
Treatment of Cells With Peptides
[0101] A short peptide (YKEGYNVYGIE) corresponding to amino acid
residues from 869 to 879 of the C-terminus of GluR2 (R2-CT) was
synthesised and incubated with a carrier protein (Pep-1).sup.23 at
a ratio of 1:20 in Dulbecco's modified Eagle's medium (DMEM, Gibco)
at 37.degree. C. in a humidified atmosphere containing 5% CO.sub.2
for 30 min to allow the formation of R2-CT/Pep-1 complex.
Hippocampal neurons (DIV 12-14) were then overlaid with the
preformed complex to reach a final R2-CT concentration of 1 .mu.M a
and further incubated for 1 h before experiments commenced.
Receptor Trafficking Assays
[0102] Cell ELISA assay: Quantification of cell-surface AMPA or
NMDA receptors was performed by a colorimetric cell-ELISA assay
essentially as described previously.sup.14. Briefly, hippocampal
neurons were treated with 100 .mu.M NMDA plus 10 .mu.M glycine for.
1 h and then fixed with 4% paraformaldehyde/4% sucrose in PBS for
10 min. Half of the cells in each treatment condition were then
permeabilized with 0.1% Triton-X 100 for 5 min. Receptors on the
plasma membrane surface and the total cellular pool were then
determined by incubating the cells with monoclonal antibodies
against the extracellular domains of GluR2 or NR1 (Chemicon, 1
.mu.g/ml) overnight at 4.degree. C., followed by incubation with
HRP-conjugated anti-mouse IgG secondary antibody (1:1000, Amerisham
Life Sciences) for 1 h at room temperature. Following extensive
washing with PBS, cells were incubated with OPD substrate (Sigma)
for approximately 10 min. Reactions were stopped with 0.2 volumes
of 3N HCl, and absorbance at 492 nm was read using a
spectrophotometric microplate reader.
[0103] Transferrin receptor endocytosis assay: To assess the effect
of endocytosis inhibitors on transferrin receptor endocytosis,
hippocampal neurons were incubated with 2 mg/ml Alexa-A488
conjugated transferrin (Molecular Probes) for 30 min at 37.degree.
C. in the presence or absence of endocytosis inhibitors.
Internalized receptors were then viewed with a Leica fluorescence
microscope.
cDNA Plasmids and Cell Transfection
[0104] Rat HA-tagged GluR1 and GluR2 receptor subunit cDNAs have
been described previously.sup.14. Constructs of HA-GluR2 carboxyl
internal deletion or truncation mutants were made by standard PCR
methods. The HA-GluR3Y-3A mutant was made using a Quick-Change Site
Directed Mutagenesis Kit (Stratagene). HEK293 cells (ATCC) were
using the calcium phosphate precipitation method. Thirty six to
forty eight hours after transfection, cells were washed with
extracellular recording solution (ECS in mM: 140 NaCl, 33 glucose,
25 HEPES, 5.4 KCl, 1.3 CaCl2; pH 7.4, 320 mOsm) and incubated in
ECS for at least one hour (serum starvation). For insulin
treatment, cells were incubated with ECS supplemented with 0.5
.mu.M human recombinant insulin (Sigma) for 10 min, after which the
cells were processed for immunocytochemistry and colorimetric
assays or lysed in RIPA buffer (50 mM Tris-HCl, 150 .mu.M NaCl and
0.1% triton X-100) for immunoprecipitation as described below.
Cloning, Expression, and Purification of GST Fusion Proteins
[0105] GST-GluR23Y and GST-GluR23A were constructed by subcloning
corresponding PCR fragments into pGEX 4T-1 vectors. GST fusion
proteins were expressed in DH5.alpha. E. coli and purified from
bacterial lysates according to the man s protocol (Pharmacia).
Products were dialyzed in PBS and concentrated using Microcon-10
columns (Amicon) for intracellular application during whole-cell
recordings.
Immunofluorescent Confocal Microscopy
[0106] HEK293 cells were plated onto poly-D-lysine coated glass
cover slips set in 35 mm culture dishes and transfected with 2
.mu.g of the plasmid of interest. For cell-surface receptor
expression assays, cells at 48 h post-transfection were fixed with
4% paraformaldehyde in PBS for 10 min. Surface AMPA receptors were
first labeled with monoclonal anti-HA antibody (1:2000, Babco,
Berkeley, Calif.) and visualized with an FITC-conjugated anti-mouse
IgG antibody (1:500, Sigma). For the surface AMPA receptor
internalization assay, HEK293 cells transfected with HA-tagged
GluR2 constructs were incubated live at 4.degree. C. with
monoclonal anti-HA antibody (10:g/ml) for 1 h to label surface AMPA
receptors. Cells were then incubated at 37.degree. C. in ECS
supplemented with or without 0.5 .mu.M insulin for 10 min before an
additional 20 min incubation in ECS to allow for constitutive or
regulated internalization of labeled receptors. Following a 10 min
fixation with 4% paraformaldehyde without permeabilization,
receptors remaining on the plasma membrane surface were stained
with FITC-conjugated anti-mouse IgG antibodies. The internalized
cell-surface receptors were subsequently labeled with
Cy3-conjugated anti-mouse IgG antibodies following cell
permeabilization as described by Man et al..sup.14
Colorimetric Assays
[0107] Colorimetric assays were performed essentially as previously
reported..sup.14
Immunoprecipitation and Western Blotting
[0108] Immunoprecipitation and Western blotting were carried out
essentially as previously reported..sup.14 Proteins from cerebral
cortex, hippocampal slices, cultured hippocampal neurons or
transfected HEK 293 cells were solubilzed in RIPA buffer containing
either 1% SDS (pha 5 min boiling; denaturing conditions) or 1% DOC
(non-denaturing conditions). For immunoprecipitation, 500 .mu.g of
protein from these tissue lysates was incubated with their
respective antibodies in 500 .mu.l of RIPA buffer for 4 h at
4.degree. C. Protein A-sepharose was added to the mixture and
incubated for an additional 2 h. The complex was isolated by
centrifugation and washed three times. Proteins eluted from the
sepharose beads were subjected to SDS-PAGE and immunoblotting using
their respective antibodies. For sequential re-probing of the same
blots, the membranes were stripped of the initial primary and
secondary antibodies and subjected to immunoblotting with another
antibody. Blots were developed using enhanced chemiluminescence
detection. (Amersham). Band intensities were quantified using Scion
Image PC software.
[0109] Hippocampal Neuron Cultures, Transfection, and
Fluorescence-Based Internalization Assays
[0110] As in Lee et al., 2002.sup.40 Pa et al., 2001..sup.49
Electrophysiological Recording
[0111] Hippocampal slices (400 .mu.m thickness) were prepared from
Sprague-Dawley rats aged 16-26 postnatal days and perfused at room
temperature with artificial cerebrospinal fluid containing (mM):
126 NaCl, 26 NaHCO3, 10 glucose, 3 KCl, 1.2 KH2PO4, 1 MgCl2, and 1
CaCl.sub.2, saturated with 95% O2/5% CO.sub.2.sup.14. The recording
pipettes (4-5 M.OMEGA.) were filled with solution containing (mM):
135 CsCl, 10 HEPES, 5 QX-314, 4 Mg-ATP, 2 MgCl.sub.2, 0.5 EGTA, 0.2
GTP and 0.1 CaCl.sub.2, pH 7.4, 310 mOsm. Whole-cell recording of
CA1 neurons and the induction of LFS-LTD were performed as
previously described..sup.14
Statistical Analysis
[0112] Student's t-tests were used whenever intra-experiment
samples were compared. For cross comparisons or analysis of data
between experiments all values were first subjected to a one-way
ANOVA and all groups were compared against control basal values.
Values were not statistically significant at F<0.5. Groups that
were found to be statistically significant were individually
compared using Dunnett's t-test. All analysis was done using
normalized values in the Statistica statistics package
(Statsoft).
Primary Neuronal Culture
[0113] The cortex was dissected from 18 days in utero Wistar
embryos and was treated with trypsin-EDTA for 15 min at 37.degree.
C. The cells were then washed 3 times and triturated to a single
cell suspension. The neurons with glia were then seeded at a
density of .about.2.5.times.10.sup.5 neurons/well in 12 well tissue
culture plates coated with poly-D lysine. The cells were then
cultured for 24-48 h in plating media (Gibco Neurobasal.TM., 1%
FBS, 2% B-27 supplement, 0.5 mM L-glutamate, and 25 .mu.M glutamic
acid), after which the cells were treated with Neurobasal.TM.
maintenance media (NMM: Gibco Neurobasal.TM. Media+.SmM
L-glutamate, 2% B27 supplement) with 10 .mu.M
5-Fluoro-2'-deoxyuridine (FDU) to enrich the culture for neurons
(.about.85%). After 24 h-48 h culture in FDU, the cells were
maintained on NMM changed every 4 days.
[0114] Peptide Generation
[0115] Tat-GluR2-3Y, Tat-GluR2-3A, and dansyl-conjugated
Tat-GluR2-3Y were all synthesized on an ABI 433A peptide
synthesizer (NAPS).
Neuronal Uptake of Dansyl-Labeled Tat-GluR2-3Y Peptide
[0116] Day in vitro (DIV) 13 primary cortical neurons at a density
of 2.5.times.10.sup.5/well in 12 well plates were washed once with
extracellular solution (ECS: 140 mM NaCl, 5.4 mM KCl, 1.3 mM
CaCl.sub.2, 10 mM HEPES, 33 mM D-glucose, pH 7.4) and then 1 mL
containing either no peptide (control) or 1 .mu.M dansyl
labeled-Tat-GluR2-3Y was added to the wells. After 5 min, 10 min,
30 min, or 60 min incubation at 37.degree. C. the wells were washed
twice with ECS and imaged using fluorescence microscopy using an
excitation wavelength of 550 nm.
Quantification of AMPAR Endocytosis in Response to NMDA
Treatment
[0117] Using cellular ELISA, the amount of intracellular versus
extracellular AMPAR expression was measured allowing quantification
of AMPAR endocytosis in response to NMDA insult. DIV 12-13 neurons
were washed once with room temperature ECS. 1 mL of NMM with or
without 1 .mu.M Tat-GluR2-3Y or Tat-GluR2-3A peptide was added to
the wells and the cells were incubated for 1 h at 37.degree. C. The
media was then aspirated and 1 mL of ECS with different
combinations of peptide (1 .mu.M Tat-GluR2-3Y or Tat-GluR2-3A) and
NMDA-glycine treatment (50 .mu.M NMDA+10 .mu.M glycine) was added
to the wells and the cells were incubated at room temperature for
30 min. The wells were then washed once with ECS and then
immediately fixed with 0.5 mLs of cold fixative (4%
paraformaldehyde, and 4% sucrose in PBS) for 10 min with shaking.
The cells were then washed 3 times with 1 mL of PBS. Half of the
wells for each treatment group were left unpermeabilized
(representing the extracellular AMPAR expression) and half were
permeabilized (representing total intracellular and extracellular
AMPAR expression) with 0.5 mLs of 0.2% Triton X 100 in PBS for 10
min with shaking followed by 3 PBS washes. The wells were then
blocked with 2% goat serum in PBS for 1 h. After blocking the
blocking buffer was aspirated and either 400 .mu.L of 1 ug/mL of
mouse anti-rat GluR2 N-terminus antibody in 2% goat serum (clone:
6C4, Chemicon) or 400 .mu.L of blocking buffer (no primary antibody
controls) was added to the wells and the plates were incubated
overnight with shaking at 4.degree. C. The plates were then washed
3 times with PBS and 400 .mu.L of 1/1000 horseradish
peroxidase-conjugated sheep anti-mouse IgG2a antibody in 2% goat
serum was added and the plates were incubated for 1 h at room
temperature with shaking. The plates were then washed 3 times with
PBS, then 1 mL of OPD solution (0.4 mg/mL o-phenylenediamine, 0.4
mg/mL urea hydrogen peroxide, and 50 mM phosphate-citrate buffer,
Sigma) was added and the plates were incubated for 5-10 min at room
teat with shaking. The peroxidase reaction was terminated by the
addition of 200 .mu.L of 3N HCl. The absorbance at 492 nm was read
using a .mu.Quant plate reader (Bio-Tek Instruments Inc.). The data
were analyzed by first subtracting the absorbance values for the
no-primary controls from the other samples. The percentage AMPAR
surface expression was then expressed as a ratio of the
non-permeabilized samples to the permeabilized samples. The
individual repeat experiments were then normalized and treatment
groups were compared using ANOVA followed by the Tukey-Kramer test,
(p=0.05).
[0118] Quantification of Neuronal Apoptosis in Oxygen and Glucose
Deprivation (OGD)
[0119] Neurons were subjected to 60 min of oxygen and glucose
deprivation and the apoptosis was quantified using a mono- and
oligonucleosome ELISA. DIV 13 neurons seeded at a density of
2.5.times.10.sup.5/well in 12 well plates were washed once with
ECS, and the cells were pretreated for 60 min with or without 1
.mu.M Tat-GluR2-3Y in NMM. The cells were then washed twice with
either OGD buffer (121 mM NaCl, 5 mM KCl, 1 mM Na-private, 1.8mM
CaCl.sub.2, 25 mM NaHCO.sub.3, 0.01 mM glycine; pH 7.4) for the OGD
samples, or with ECS for the non-OGD samples. The non-OGD samples
were then incubated for 25 h at 37.degree. C. in NMM and the OGD
samples were incubated in OGD buffer with or without Tat-GluR-3Y in
an anaerobic chamber at 37.degree. C. for 60 min. The OGD samples
were then incubated for 24 h at 37.degree. C. The neuronal
apoptosis was then quantified using a Cell Death Detection
ELISA.sup.PLUS kit (Roche Applied Science) as per the
manufacturer's instructions. The absorbance at 405 nm (reference
wavelength, 490 nm) was read using a .mu.Quant plate reader
(Bio-Tek Instruments Inc.). The individual repeat experiments were
then normalized and treatment groups were compared using ANOVA
followed by the Tukey-Kramer test, (p=0.05).
Tat-GluR2-3Y Infiltration of Brain Tissue
[0120] Two adult male C57-Black/6 mice weighing .about.22 g were
given an intraperitoneal injection of either saline or 30 nmol/g of
dansyl-labeled Tat-GluR2-3Y. The mice were sacrificed at 2 h and
the brains were immediately removed and frozen at -80.degree. C. 40
micron coronal sections were cut with a cryostat and visualized
with fluorescence microscopy.
Transient Focal Ischemia Model
[0121] The procedure was performed essentially as described
previously (70). Briefly, adult male Sprague-Dawley rats between
280 and 320 g (20 h fasted weight) were anesthetized with an
inhaled mixture of 4% isofluorane, in 30% oxygen balanced nitrous
oxide, and maintained on 1.5% isofluorane. Bronchial secretions
were minimized by administering 0.5 mg/kg of atropine
intraperitoneally. Either, 3 nmoles/g of Tat-GluR2-3Y in saline, 3
mmoles/g Tat-GluR2-3A in saline, or saline only was administered 1
h before middle cerebral artery (MCA) occlusion, via a femoral vein
PE-50 catheter. The experimenter was blinded to the identity of the
treatment groups for all surgeries and down-stream experiments.
Under a dissection microscope, the common carotid artery (CCA),
external carotid artery (ECA), and internal carotid artery (ICA)
were exposed and dissected. The terminal lingual and maxillary
arteries were then cauterized and the pterygopalitine artery was
then ligated with 5-0 silk suture. After this point the ICA was the
only remaining extracranial branch of the CCA. The ECA was then
partially cut close to the rostral ligature and a 30 mm 3-0 nylon
monofilament with a heat rounded tip was inserted into the ECA and
advanced past the CCA bifurcation. The ECA was then completely cut,
mobilizing the ECA stamp containing the nylon suture. The nylon
suture was then flipped so that its tip was facing the ICA and the
nylon suture was then gently advanced approximately 20 mm until
resistance was felt. At this point the suture reached the origin of
the MCA and the anterior cerebral artery completely blocking the
blood flow to the MCA territory. The wound was then stitched closed
with silk suture and the animal was awoken by turning off the
isoflurane. Rectal temperature, and blood pressure measured with a
tail cuff were measured before treatment, 15 min post injection, 50
min post injection, and 15 min post MCA occlusion. The plasma pH,
O2, and CO.sub.2 were measured with a Rapidlab.TM. 348 blood gas
analyzer (Bayer Diagnostics) in some animals to ensure that the gas
flow rates used were appropriate and yielded reproducible blood
gases. The animal was then given a neurological examination after
45 min of MCA occlusion. This exam was used to exclude any animal
that did not experience significant occlusion of the MCA. The
examination consisted of 10 tests with a maximum deficit score of
23 (71). The individual tests are summarized in Table II.
TABLE-US-00002 TABLE II Summary of neurological scoring. Test
Description Score Postural reflex: Degree of twisting Degree of
body rotation towards parietic side when 0-2 held by tail. Degree
of forelimb flexion Degree of forelimb flexion when held by tail.
0-2 Gate disturbances Circling or walking towards parietic side, or
other 0-5 gate disturbances. Tail pull Biased movement towards one
side when tail is 0-2 pulled. Lateral resistance to push Degree of
lateral resistance to push. 0-2 Visual placing: Forward Presence of
a forelimb placing reflex in response to 0-2 a forward visual cue.
Lateral Presence of a forelimb placing reflex in response to 0-2 a
lateral visual cue. Tactile placing: Forward Presence of a forelimb
placing reflex in response to 0-2 a tactile stimulus on dorsal
surface of paw. Lateral Presence of a forelimb placing reflex in
response to 0-2 a tactile stimulus on lateral surface of paw.
Proprioceptive placing Presence of a forelimb placing reflex in
response to 0-2 being held by hind quarters above surface. Total
score 0-23
[0122] The animal was induced again after the neurological
examination and the nylon monofilament was withdrawn at 60 min
after the onset of occlusion returning blood flow to the MCA
territory. The neurological examination was performed again at the
time of sacrifice (-24 h). The sham surgery was performed as the
MCA occlusion, however, the nylon monofilament was not
inserted.
TTC Staining
[0123] Rats were sacrificed 3 days post MCA occlusion by deep
anesthesia followed by decapitation. The brain was removed
immediately after sacrifice and placed in an acrylic rat brain
matrix (Harvard Apparatus) and incubated at -80C. for 5 min. 1 mm
coronal slices were then cut with razor blades and placed in 37C.
solution of 2% 2,3,5-triphenyltetrazolium chloride (TTC, Sigma) in
PBS. The slices were then incubated for approximately 15 min until
sufficient colour developed.
TUNEL Staining
[0124] At day 1 post MCAo rats were anesthetized with 1.5 mL of 25%
urethane and were perfused with 100 mL of 0.9% saline followed by
120 mLs of 4% paraformaldehyde in PBS. The brains were then removed
and stored overnight at 4.degree. C. in 4% paraformaldehyde. The
brains were then transferred to a 30% sucrose and 0.1% sodium
azide, in PBS solution and stored at 4.degree. C. until the brains
completely sunk. The brains were then frozen in dry ice and 12
micron coronal slices were cut with a cryostat at -0.8 mm with
respect to the bregma using a free floating method (72). The slices
were then mounted on glass slides and stained with TMR-TUNEL
(terminal deoxyribonucleotide transferase [TdT]-mediated dUTP nick
end labeling) (Roche Applied Science) as per the manufacturer's
instructions. The slices were scored for number of cells that
stained positive for TMR-TUNEL per field of view at 10.times.
magnification. For each section the same 3 fields along the lateral
portion of the cortex on the affected hemisphere were scored (the
affected hemisphere was defined as the side with the greatest
amount of apoptosis).
EXAMPLE 2
NMDA-Induced Apoptosis Requires AMPA Receptor Endocytosis
[0125] In order to induce apoptosis in mature cultures of rat
hippocampal neurons (14 DIV+) we treated cells with a mild NMDA
insult of 100 .mu.M NMDA with 10 .mu.M glycine for 1 h followed by
recovery of the cells in normal media for periods of up to 24 h. As
shown in FIG. 1A, B, NMDA treatment induced a time-dependent
increase in caspase-3 activity, a biochemical indicator of neuronal
apoptosis, as detected by ELISA assay of DEVD-pNA cleavage. This
increase in caspase-3 activity peaked between 12-24 h after the
treatment, at which time the majority of neurons were either dying
or dead, exhibiting the hallmarks of apoptotic cell death,
including DNA laddering demonstrated by gel electrophoresis of
extracted DNA (FIG. 1C), and nuclear condensation with
disintegrating processes shown by nucleous staining with propidium
iodide or intercalating DNA dye, Hoechst 33258 (bisbenzimide). The
degree of neuronal apoptosis was also quantified by measuring
internucleosomal cleavage of DNA with both 11-dUTP (FIG. 1E) and
histone biotinylation assays FIG. IF). In contrast, in non-treated
cultures there was little apoptosis detectable either biochemically
or morphologically (FIG. 1A-F). Furthermore, the NMDA-induced
apoptosis was a result of specific activation of NMDA receptors, as
it was fully blocked by the NMDA receptor antagonist, APV (50
.mu.M; FIG. 1D). Therefore, NMDA treatment produced neuronal
apoptosis.
[0126] In order to determine the role of NMDA-induced endocytosis
in mediating neuronal apoptosis, we first examined the effect of
hypertonic sucrose, a well-characterized clathrin-dependent
endocytosis inhibitor that inhibits the assembly of clathrin-coated
pits.sup.13;14. AS shown in FIG. 1E, when cell were treated with
hypertonic sucrose (0.4 M), prior to the application of NMDA and in
its presence for 1 h, we found that apoptosis was dramatically
reduced. While hypertonic sucrose has been widely used as an
effective inhibitor of clathrin-mediated endocytosis, it may have
many actions other than inhibiting endocytosis. To further
establish an essential role of stimulated endocytosis in
NMDA-induced apoptosis, we also examined the effect of another
specific inhibitor for clathrin-dependent endocytosis. The
inhibitor is a short, dynamin-derived, myristoylated peptide that
is membrane permeable (myr-Dyn). It blocks the recruitment of
dynamin to clathrin-coated pits by amphiphysin, thereby inhibiting
clathrin-mediated endocytosis.sup.15. Indeed, incubation of neurons
with myr-Dyn (100 .mu.M) was found to be as effective as hypertonic
sucrose in reducing NMDA-induced apoptosis (FIGS. 1E and 1F). In
contrast, control Dyn peptides, both non-myristoylated
(non-membrane permeant) Dyn (Dyn; FIG. 1E) and scrambled myr-Dyn
(s-myr-Dyn; Fig 1F), had little effect. Thus, facilitated
clathrin-dependent endocytosis is necessary for NMDA
receptor-mediated apoptosis. In order to test whether the effects
of endocytosis inhibition were specific to NMDA-induced apoptosis,
we next tested the effect of these inhibitors on a
well-characterized neutral apoptosis model that is induced by
treating neurons with the kinase inhibitor staurosporine (STS; 100
nM, 1 h).sup.12. As shown in FIG. 1E, we found that both
endocytosis inhibitors failed to significantly alter the
STS-induced neuronal apoptosis. Therefore, clathrin-mediated
endocytosis is specifically required for neuronal apoptosis induced
by NMDA receptor activation. To rule out the possibility that these
endocytosis inhibitors may have prevented neuronal apoptosis by
interfering with NMDA receptor channel function, and hence
Ca.sup.2+ influx through the activated channel, we loaded
hippocampal neurons with the intracellular Ca2+ dye, Fura-2, and
then monitored the calcium influx evoked by repetitive local `puff`
application of NMDA (100 .mu.M; 500 ms) to neurons before and
during hypertonic sucrose treatment. As summarized in FIG. 2A, B,
sucrose at concentrations that inhibited endocytosis and apoptosis
did not significantly alter NMDA evoked [Ca.sup.2+].sub.i
responses. The fact that inhibition of endocytosis blocked
NMDA-induced apoptosis without affecting its [Ca.sup.2+ ].sub.i
responses indicates that intracellular increases in
[Ca.sup.2+].sub.i concentrations, although necessary.sup.3;4, may
not be sufficient to produce NDMA apoptosis.
[0127] Activation of certain forms of caspases, such as caspase-3
and -7.sup.16 (also FIG. 1A, B) has been implicated in NMDA-induced
neuronal apoptosis. We therefore investigated the effects of
inhibiting endocytosis on NMDA dependent activation of caspase-3.
NMDA treatment dramatically increased the level of the activated
form of caspase-3 as demonstrated by Western blots using an
antibody that specifically recognizes only activated/cleaved
caspase-3 (FIG. 2C). The membrane permeable myr-Dyn, at the
concentration that inhibits NMDA receptor-mediated apoptosis,
efficiently inhibited NMDA-mediated caspase-3 activation (FIG.
2C).
[0128] The serine/threonine kinase Akt/PKB has been implicated in
protecting neurons from apoptotic cell death.sup.17 and inhibition
of this kinase activity has been suspected to be involved in NMDA
receptor-mediated apoptosis.sup.18. We investigated whether the
endocytosis process plays a critical role in the inhibition of Akt
activity by determining the level of Akt phosphorylation at serine
473, a residue whose phosphorylation is required for full
activation of Akt.sup.19. As shown in FIG. 2D, treatment of neurons
with NMDA resulted in a significant reduction in S473
phosphorylated Akt and hence Akt activity, without altering levels
of total Akt. This reduction in Akt activity was largely prevented
by the inhibition of endocytosis with hypertonic sucrose. In
contrast, sucrose treatment had no effect on the reduction of Akt
phosphorylation following STS treatment, further supporting the
specific involvement of endocytosis in NMDA-induced apoptosis (FIG.
2D). Thus, stimulated endocytosis appears an obligatory step that
is down stream of rising in [Ca.sup.2+].sub.i and upstream of
caspase activation and Akt inhibition in NMDA-induced neuronal
apoptosis.
[0129] A significant reduction of cell-surface AMPA, but not NMDA,
receptors was observed following NMDA treatment and this reduction
was a result of facilitated receptor endocytosis as it was blocked
by endocytosis inhibitor myr-Dyn, but not the control peptide, Dyn
(FIG. 3A). To investigate whether there was a direct link between
the NMDA-induced AMPA receptor endocytosis and apoptosis, a peptide
derived from the short amino acid sequence between residues
tyrosine 869 and glutamic acid 879 within the carboxyl terminal
(CT) region of the GluR2 subunit of the AMPA receptor (YKEGYNVYGIE;
termed R2-CT) was delivered into cultured neurons by mixing it with
a carrier peptide (Pep1).sup.23 one hour prior to and during the
NMDA treatment. The results indicated that the NMDA-induced
reduction of cell-surface AMPA receptors was prevented (FIG.
3B).
[0130] In order to be sure that the blockade by this peptide was
not due to non-specific effects on the endocytotic process, we
examined its effect on transferrin receptor endocytosis, a
well-characterized clathrin-mediated receptor endocytosis.sup.13.
Incubation of hippocampal neurons with fluorescently-labeled
transferrin for 30 min resulted in an accumulation of the
fluorescently-labeled transferrin in the intracellular compartment.
This was a result of clathrin-mediated transferrin receptor
endocytosis as it was eliminated when 0.4 M sucrose was also
present during the period of transferrin incubation. In contrast,
R2-CT+Pep-1, applied to these neurons one hour prior to and during
the transferrin incubation, failed to prevent transferrin receptor
endocytosis. Thus, the R2-CT peptide is a dominant inhibitor that
can specifically block NMDA-induced AMPA receptor endocytosis, but
not non-specifically affect clathrin-mediated endocytotic
processes.
[0131] Furthermore, pre-treatment of the neurons with R2-CT+Pep-1
significantly reduced NMDA-induced apoptosis as quantified by the
histone biotinylation assay (FIG. 4A), and by PI nuclear staining
(FIG. 4B). PI staining after fixation showed that P2-CT blocked
NMDA-induced apoptosis. In this particular example, neither R2-CT
nor Pep-1 alone had any detectable effect on NMDA-induced
apoptosis. Similar to the general blockade of the clathrin-mediated
endocytotic process with either sucrose or myr-Dyn, interfering
with AMPA receptor endocytosis by R2-CT did not alter STS-induced
neuronal apoptosis (FIG. 4A). Taken together, our results have
provided strong evidence for an obligatory requirement for AMPA
receptor endocytosis in mediating NMDA-induced neuronal
apoptosis.
[0132] Therefore, a clathrin-dependent AMPA receptor endocytosis is
specifically required for NMDA-, but not STS-, induced apoptosis of
hippocampal neurons maintained in primary culture. Blocking
endocytosis has no effect on NMDA-induced Ca.sup.2+ responses, but
prevents both NMDA-induced activation of caspase-3 and inhibition
of Akt phosphorylation. Thus, AMPA receptor endocytosis may be a
critical link between NMDA-induced [Ca.sup.2+].sub.i overload and
intracellular cascades leading to apoptosis.
[0133] Thus, stimulation of NMDA receptor activates intracellular
signaling cascades leading to apoptosis, and facilitates
dynamin-dependent internalization on of the AMPA subtype glutamate
receptors. Blocking the dynamin-dependent internalization
specifically ameliorated NMDA (but not staurosporine)-activated
apoptotic cascades, without affecting NMDA-induced rises in
[Ca2+].sub.i. Specific inhibition of NMDA-induced AMPA receptor
endocytosis by a GluR2-derived peptide prevents NMDA induced
apoptosis, without affecting that produced by staurosporine. These
results demonstrate that AMPA receptor endocytosis may be required
in linking NMDA receptor activation to neuronal apoptosis, and
thereby suggests that AMPA receptor endocytosis plays an essential
role in reducing synaptic strength, and also actively mediates
other important intracellular pathways, including apoptotic cell
death.
EXAMPLE 3
Distinct Sequences within the GluR2 Carboxyl Terminus are Required
for Constitutive and Regulated AMPA Receptor Endocytosis
[0134] To identify sequence determinants for constitutive and
insulin-stimulated AMPA receptor
[0135] endocytosis, we made six GluR2 mutants containing various
deletions of the GluR2 CT (FIG. 5B). All constructs, except
GluR2.DELTA.854, were HA-tagged in the extracellular amino-terminal
region. Following transient transfection into HEK293 cells, these
constructs were expressed at a level comparable to their wild-type
counterparts, HA-GluR2 or GluR2, as determined by a colorimetric
cell-ELISA assay under permeabilized cell conditions (FIG. 5C).
[0136] The ability of these mutants to undergo both constructive
and regulated endocytosis was assayed as described
previously..sup.14 Surface receptors in live cells were
pre-labelled with an anti-HA antibody (or an antibody against the
extracellular N-terminal domain of GluR2 in the case of
GluR2.DELTA.854) at 4.degree. C. (which blocks endocytosis).
Surface labelled cells were then incubated at 37.degree. C. for 30
min to allow endocytosis to resume both in the absence and presence
of insulin (0.5 .mu.M) to determine changes in constitutive (basal)
and regulated (insulin-stimulated) AMPA receptor endocytosis,
respectively (FIG. 6A, B). Internalized receptors were then
visualized by confocal microscopy and quantitated by colorimetric
cell-ELISA-based receptor internalization assays (FIGS. 6A).
Representative confocal images of HEK293 cells were pre-labeled
with HA-tagged GluR2 or GluR2 mutants were obtained. Transfected
cells were pre-labeled with anti-HA antibody and then receptor
endocytosis was evaluated under basal conditions (constitutive
endocytosis) or following insulin stimulation (0.5 .mu.M, 10 min;
regulated endocytosis). Cell surface receptors were stained with
FITC under non-permeant conditions and internalized receptors were
subsequently stained with Cy3 after cell permeabilization. In order
to determine whether changes in internalization produced by these
mutations were able to alter surface receptor numbers, we also
measured the steady-state level of cell-surface AMPA receptors
using colorimetric cell-ELISA-based cell-surface receptor assays
(FIG. 6C).
[0137] As shown in FIGS. 6A, wild type GluR2 receptors underwent
both constitutive and insulin stimulated endocytosis. Thus, in the
absence of insulin, approximately 25% of the cell-surface receptors
were endocytosed within 30 min and this proportion was increased to
48% following brief insulin stimulation (0.5:M, 10 min). This
facilitated endocytosis was associated with a significant reduction
in the level of AMPA receptors expressed on the cell surface (FIG.
6B). Truncation of the last four amino acids (GluR.DELTA.880),
which form the PDZ binding motif, did not have any observable
effects on either constitutive or regulated endocytosis. However,
truncation of the last 30 (GluR2.DELTA.854) or 15 residues
(GluR2.DELTA.869) completed abolished the insulin-induced AMPA
receptor endocytosis, and the reduction in its cell-surface
expression (FIGS. 6A-B). Neither truncation altered the degree of
constitutive AMPA receptor endocytosis (FIG. 6A) or the basal level
of receptor expression on the cell surface (FIG. 6B). A significant
decease in the rate of constitutive internalisation of
GluR2.DELTA.834-843, in which the first 10 amino acids of the GluR2
CT were deleted, was observed (FIGS. 6A). However, this internal
deletion did not alter the steady-state number of AMPA receptors
expressed on the cell surface (FIG. 6B). Nor did it alter the
responsiveness to insulin, as GluR2.DELTA.834-843 showed enhanced
internalization similar in magnitude to wild-type GluR2 (FIGS. 6A
and 6B). On the other hand, the internal deletion mutant
GluR2.DELTA.844-853 showed no significant change in the degree of
constitutive endocytosis (FIG. 6A), but exhibited a small decrease
in insulin-stimulated endocytosis (FIG. 6A) and a reduction in the
steady-state receptor level on the cell surface (FIG. 6B).
EXAMPLE 4
GhuR2 CT Tyrosine Phosphorylation is Required for Insulin
Stimulated AMPA Receptor Endocytosis
[0138] The R2-CT sequence contains three tyrosine residues. To
determine whether these tyrosine residues are substrates of certain
tyrosine kinases, we performed in vitro kinase assays using active
recombinant Src and glutathione S-transferase (GST)-fusion proteins
of the carboxyl tails of GluR1 (GST-GluR1CT) and GluR2
(GST)-GluR2CT) (FIG. 7A). GST-GluR2CT, but not GST-GluR1CT or GST
alone, is specifically phosphorylated by Src kinase. Consistent
with the hypothesis that one or more of the tyrosine residues is
the substrate(s) for the Src phosphorylation, we found that the
recombinant Src kinase phosphorylated a GST fusion protein
containing the nine amino-acid stretch including all three
GluR2-unique tyrosine residues (GST-Y869KEGY873NVY876G).
Src-mediated phosphorylation was abolished when these tyrosine
residues were mutated into alanines (GST-A869KEGA873NVA876G).
[0139] To determine whether these GluR2 CT tyrosine residues are
phosphorylated in situ by endogenous tyrosine kinase activity in
response to insulin stimulation, we generated a GluR2 subunit
mutant in which tyrosine residues Y869, Y873 and Y876 were mutated
into alanines (HA-GluR23Y-3A). When transiently expressed in HEK293
cells, the mutant was expressed at the same level as its wild type
GluR2 counterpart (FIG. 7B). We first examined the potential
phosphorylation of these tyrosine residues in situ in cells
transiently expressing HA-GluR2, HA-GluR23Y-3A, or HA-GluR1. Cells
were treated with or without insulin (0.5 .mu.M, 10 min) and then
homogenized as detailed in the methods section. The expressed AMPA
receptor complexes were immunoprecipitated using an anti-HA
antibody under denaturing conditions and then immunoblotted for
their level of tyrosine phosphorylation using an
anti-phosphotyrosine antibody. The results demonstrate that there
was a detectable level of basal tyrosine phosphorylation of wild
type GluR2 and that the level of phosphorylation increased
following brief treatment with insulin (FIG. 7C). The triple Y-to-A
mutation strongly deceased both basal and insulin-induced tyrosine
phosphorylation of HA-GluR2. In contrast, there was almost no
detectable tyrosine phosphorylation on of GluR1 under either basal
or insulin-stimulated conditions (FIG. 7C). These results suggest
that tyrosine phosphorylation of GluR2 CT occurs in a cellular
context under basal conditions, and is enhanced by insulin.
[0140] Mutation of tyrosine residues of GluR2-CT prevents
insulin-induced reduction of cell-surface AMPA receptors. HEK cells
expressing wild type GluR2 or GluR2 Y-A mutants were treated with
insulin (0.5 .mu.M) for 10 min and with an additional 20 min
incubation period in ECS. Level of cell-surface receptors were
assayed using colorimetric assay. Mutation of any one of the
tyrosine residues was sufficient to prevent the insulin-induced
reduction in cell-surface AMPA receptor expression (FIG. 7D).
Without wishing to be bound by any hypothesis, these results may
suggest that all three tyrosine residues are substrates of tyrosine
phosphorylation, or that they are all involved in substrate
recognition by the kinase or some other aspect of the catalyzed
phosphorylation such that mutation of a particular tyrosine could
prevent phosphorylation even if it is not the direct target of
phosphorylation. Thus, in the latter case, mutating any of the
non-substrate tyrosine residues would affect substrate-kinase
interaction and hence be able to prevent phosphorylation of the
substrate tyrosine residue, thereby reducing stimulated receptor
endocytosis.
[0141] The functional significance of GluR2 CT tyrosine
phosphorylation with respect to insulin-stimulated endocytosis was
tested by assaying internalization on of HA-GluR2 and HA-GluR23Y-3A
in HEK293 cells (FIG. 8A, B). While mutation of these tyrosine
residues did not alter the steady-state level of GluR2 expressed on
the cell surface (FIG. 8B), it did block the insulin-induced
endocytosis (FIG. 8A) and insulin-induced reduction in the level of
cell-surface AMPA (FIG. 8B).
EXAMPLE 5
Insulin Increases Tyrosine Phosphorylation of GluR2, and Depresses
AMPA Receptor-Mediated Synaptic Transmission in Hippocampal
Slices
[0142] We next examined whether insulin stimulation could change
the level of tyrosinephosphorylation of AMPA receptors in intact
hippocampus, as it does in HEK293 cells expressing GluR2 subunits
(FIG. 7A-D), and whether this might be important for
insulin-mediated depression of AMPA receptor-mediated synaptic
transmission. Hippocampal slices were treated with insulin (0.5
.mu.M; 10 min), and GluR1 and GluR2 subunits were then
immunoprecipitated under denaturing conditions (as detailed herein)
and immunoblotted with an anti-phosphotyrosine antibody (FIG. 9A,
B). Consistent with results from cell culture, the GluR2 subunit
exhibited a clearly appreciable level of tyrosine phosphorylation
under basal conditions; moreover, the level of phosphorylation was
increased following insulin stimulation (FIG. 9A, B). In contrast,
the tyrosine phosphorylation levels of GluR1 were barely detectable
under both basal and insulin-treated conditions. These results
further substantiate the tyrosine phosphorylation of GluR2 in the
hippocampus and demonstrate that GluR2 tyrosine phosphorylation can
be stimulated by insulin.
[0143] The effect of postsynaptic application of GST-GluR23Y
(GST-YKEGYNVYG), and its mutant counterpart, GST-GluR23A
(GST-AKEGANVAG), as a control, during whole-ell recording of CA1
neurons in hippocampal slices was investigated, to determine the
correlation, if any, of the insulin-stimulated tyrosine
phosphorylation of AMPA receptors to persistent depression of
receptor-mediated excitatory postsynaptic currents (EPSCs). As
shown in FIG. 7A, the GST-GluR23Y, but not the GST-GluR23A, is a
good tyrosine phosphorylation substrate. Bath application of
insulin resulted in a persistent decrease in the AMPA component of
EPSCs (FIG. 9C, D). The insulin-induced EPSC depression was
prevented when wild-type GST-GluR23Y peptide (100 .mu.g/l) was
included in the recording pipette, whereas the same amount of
mutant peptide, GST-GluR23A, had no effect (FIG. 9C, D). Thus, the
wild type tyrosine-containing peptide, but not its mutant
counterpart, is sufficient to block insulin-induced persistent
depression of AMPA receptor-mediated EPSCS.
EXAMPLE 6
Tyrosine Residues in the GluR2 CT Mediate LTD
[0144] The level of GluR2 tyrosine phosphorylation was assayed
following low-frequency stimulation (LYS) of hippocampal slices (1
Hz for 15 min, which reliably induces LID under our experimental
conditions), to determine whether tyrosine phosphorylation of GluR2
CT may be required for LFS-induced long term depression (LTD).
Slices were homogenized in denaturing buffer 10 mm after the
stimulation and GluR subunits were immunoprecipitated and probed
for phosphotyrosine. As shown in FIG. 10A, there was basal tyrosine
phosphorylation of GluR2, but not GluR1, and LTD-inducing
stimulation increased the level of tyrosine phosphorylation of
GluR2 without affecting that of GluR1 (FIG. 10A). Induction of LTD
by LFS was blocked by postsynaptic application of GST-GluR23Y (100
.mu.g/ml ), but not by the mutant peptide GST-GluR23A (100
.mu.g/ml; FIG. 10B) or by GST-GluR2834-843 (FIG. 10C).
EXAMPLE 7
GluR2 CT Peptide Prevents Ischemia-Induced AMPA Receptor
Endocytosis and Neuronal Apoptosis in a Neuronal Culture Model of
Stroke
[0145] Ischemia-like insult was mimicked by oxygen and glucose
deprivation (OGD) for one hour in cultured cortical neurons (DIV
12-14). OGD is a well-characterized cell structure model of
ischemia. GluR2CT peptide (1 mM) was delivered into neurons by
mixing it with the carrier peptide PEP-1 and incubating neurons
with the mixture for one hour before OGD challenge. FIG. 11A shows
a colorimetric (Cell-ELISA) assay indicating that OGD facilitates
AMPA receptor endocytosis, thereby decreasing their expression on
the plasma membrane surface and pre-incubation of the GluR2-CT
peptide reduced the OGD-induced decrease in cell-surface AMPA
receptor expression. (n=6; *: P<0.05, Student's test, compared
with Control). FIG. 11B is a quantitative apoptosis assay 24 hr
after OGD using the Cell Death Detection ELISAplus kit (Roche, Cat#
1 774 425), demonstrating that OGD produces neuronal death that is
largely prevented by pre-treatment of neurons with GluR2-CT. (n=6;
** : P<0.01, Student's t test, compared with OGD. Together,
these results indicate that like NMDA receptor overactivation,
ischemia-like insults also produces neuronal death by facilitating
AMPA receptor endocytosis and as such, AMPA receptor
endocytosis-blocking peptides, such as GluR2-CT peptide can be used
in stoke treatment to reduce neuronal damage.
EXAMPLE 8
Systemic Application of Tat-GluR2.sub.3Y Peptide Blocks the
Expression of Behavioural Sensitization to the Abusive Drug
d-Amphetamine in an Animal Model of Drug Addiction
[0146] Behavioral sensitization is defined as an increase in the
psychomotor response to treatment with many classes of addictive
drugs (i.e. amphetamine, cocaine, nicotine, heroin) and can be
parsed into induction and expression phases. Behavioral
sensitization is a well accepted model of neural and behavioural
adaptations that are hypothesized to form the bases of addiction,
specifically dug-induced changes in the mesocorticolimbic dopamine
system that underlie the motivation to engage in drug-seeking
behavior.sup.60,61.
[0147] To induces behaviour sensitization to addictive drugs that
lead to substance abuse, four separate groups of adult rats were
given repetitive injections of d-amphetamine (2 mg/kg,
intraperitoneally (IP)) or saline, every other day for a total of
10 injections. On days 1, 5 and 10 of the injection regimen, the
rats were placed in 2-level locomotor boxes for 30 min before the
amphetamine injection to habituate to the boxes, and for an
additional 2 hours following the injection, and stereotypy scores
(drug-induced behaviours) were assessed at 1 minute intervals every
10 minutes for the duration of the 2 hour session. After the
10.sup.th injection of d-amphetamine, the rats were given 21 days
off, and chronically indwelling catheters were implanted into the
jugular vein under anaesthesia.
[0148] In order to deliver GluR2-CT peptide into neurons in the
brain following intravenous (IV) injection, the wild GluR2-CT
peptide containing 3Y residues or the corresponding peptide
sequence in which the 3 tyrosines were replaced with alanines was
fused to the cell-membrane transduction domain of the human
immunodeficiency virus-type 1 (HIV-1) Tat protein (YGRKKRRQRRR),
which is capable of crossing the blood brain barrier (BBB).sup.85,
to obtain Tat-GluR2-3Y (YGRKKRRQRRR-YKEGYNVYGIE) or Tat-GluR2-3A
(YGRKKRRQRRR-AKEGANVAGIE) peptides.
[0149] On day 21, the rats were pretreated with 1.5
nM/grTat-GluR2-3Y, or Tat-GluR2-3A or saline by either IV
injection, or intracranial microinjection into the nucleus
accumbens (Nac), and returned to their home cages for 60 min. The
rats were then placed in the locomotor boxes (observation chambers)
for 30 min and then treated with a challenge dose of d-amphetamine
(2 mg/kg, IP). Stereotypy scores were then assessed as described
(FIG. 12A). Points represent mean stereotypy scores (.+-.S.E.M) for
groups of rats over the 2 hour test session. Pretreatment with
Tat-GluR2-3Y completely blocked the acute expression of
d-amphetamine induced stereotypy, while Tat-GluR2-3A was
ineffective in this regard (F(2,31)=4.22, p<0.01). FIG. 12B
shows the peak effect of stereotypy, which occurred at
approximately 50 minutes after d-amphetamine pretreatment, which is
represented for each group. * indicates p<0.05 compared with the
saline treated group. One hour intravenous pre-treatment with
GluR23Y peptide, but not the control GluR23A, abolished the
expression of behavioural sensitization to a challenging dose of
amphetamine, without any notable side effects in rats (FIG. 12A,
B). The blockade of sensitization is due to specific action in the
NAc as, in a subsequent experiment, direct microinfusion of GluR23Y
into the NAc, but not the VTA, mimicked IV administration,
preventing the expression of the behaviour sensitization (FIG. 12C,
D). Systemic treatment with the effective wild-type peptide failed
to disrupt a leaned operant response for food reward delivered on
an FR-2 schedule (FIG. 18A). Further evidence for the high degree
of specificity of the peptide is its lack of effect on the
unconditional reward effect of D-amp (FIG. 18B). These data provide
the first evidence that LTD in the NAc is required for the
expression of behavioural sensitization, a behavioural correlate of
craving, and most significantly, that a membrane permeant short
"interference peptide"that blocks LTD can prevent the expression of
this behavioural sensitization without notable side effects. Thus,
the ability of treatment with Tat-GluR 2-3Y peptide to block the
emotion of behavioural sensitization is consistent with the use of
such peptides in the treatment of substance abuse and addiction to
classes of drug that induce behavioural sensitization.
EXAMPLE 9
Treatment of Ischemic Brain Damage by Blocking AMPA Receptor
Endocytosis
[0150] We investigate whether a peptide that can block AMPAR
endocytosis can function as a neuroprotective agent by preventing
glutamate induced neuronal apoptosis. First, in order to ensue that
the peptide was able to permeate neurons, primary Wistar cortical
neuron cultures were exposed to a dansyl-labeled Tat-GluR-3Y
peptide and the cells were then visualized by fluorescence
microscopy. DIV 13 neurons were treated with either saline
(control) or 1 .mu.M dansyl-labeled GluR23Y peptide for 10, 20, 30,
or 60 min. The peptide was able to permeate the cells in a time
dependent manner. The neurons took up the dansylated Tat-GluR2-3Y
in a time dependent manner with significant fluorescence visible by
10 min with a maximum at approximately 30 min.
[0151] Once it was known that the peptide could enter cortical
neurons, the ability of Tat-GluR2-3Y to block NMDA-induced AMPAR
endocytosis was examined. Primary Wistar cortical neurons
pretreated with or without Tat-GluR2-3Y were ejected to NMDA insult
and the surface expression of AMPARs was quantified using a cellar
ELISA assay. Baseline levels of AMPAR surface expression were
approximately 70%, with a corresponding intracellular pool of 30%.
NMDA-glycine treatment resulted in a significant decrease in AMPAR
surface expression with reference to the control from 69% to 55%
(p<0.05, Tukey-Kramer Test), that was completely blocked by
pretreatment with Tat-GluR2-3Y (73% surface expression, p<0.05
compared to NMDA group, Tukey-Kramer Test) (FIG. 13). Furthermore,
Tat-GluR2-3A, a muted version of Tat-GluR2-3Y was unable to block
NMDA-induced AMPAR endocytosis. It should also be noted that in
this example, each peptide alone had no effect on AMPAR surface
expression.
[0152] Since Tat-GluR2-3Y was able to block NMDA induced AMPAR
endocytosis, the ability of the peptide to protect cultured neurons
against oxygen and glucose deprivation (OGD)-induced apoptosis was
investigated. DIV 12-13 neurons were pretreated with either saline
or Tat-GluR2-3Y for 60 min, followed by 60 min of OGD at 37.degree.
C. or incubation at 37.degree. C. in media (control). The amount of
apoptosis was qualified using an ELISA assay targeted to free
nucleosomes which are characteristic of apoptosis. OGD induced
significant apoptosis compared with the control that was
substantially blocked by pretreatment with Tat-GluR2-3Y (p<0.05)
(FIG. 14).
[0153] For study of the peptide in vivo we first investigated
whether the peptide could pass the blood brain barrier (BBB) and
infiltrate neuronal tissue. Either dansyl-labeled Tat-GluR-3Y or
saline was administrated to male C57-Black/6 mice and 40 .mu.m
coronal brain slices were cut with a cryostat and visualized with
fluorescence microscopy. More specifically, two adult male
C57-Black/6 mice were given an intraperitoneal injection of either
30 mmoles/g of dansyl-labeled Tat-GluR2-3Y or saline. The mice were
sacrificed 2 h following injection and 40 micron coronal sections
were cut with a cryostat and visualized with fluorescence
microscopy. The results indicated that the dansyl-labeled peptide
brain sections exhibited a greater fluorescence intensity than the
control, confirming entry of the peptide into the brain, and that
dansyl-labeled Tat-GluR2-3Y crosses the blood brain barrier and
enters neural tissue.
[0154] In order to qualitatively describe the location and size of
the infarct produced by the intraluminal suture method of MCA
occlusion, 4 male Sprague Dawley rats were subjected to the
procedure, sacrificed at day 3 post MCA occlusion, and 1 mm brain
slices were stained with 2,3,5-triphenyltetrazolium chloride (TTC).
More specifically, adult male Sprague-Dawley rats of .about.300 g
body weight were subjected to 60 min of MCA occlusion using an
intraluminal 3-0 nylon monofilament. The rats were then sacrificed
at 3 days post MCA occlusion and the brains were sliced into 1 mm
sections and stained with TTC. The transient ischemia method
resulted in significant infarct volume with the maximum coronal
cross-sectional involvement at .about.-1.5 mm with respect to the
bregma. The infarct volume was reproducible with significant
cortical involvement in each rat. From the TTC staining, 0.8 mm
with respect to the bregma was chosen or apoptosis staining using
terminal deoxyribonucleotide transferase [TdT]-mediated dUTP nick
end labeling (TUNEL).
[0155] In order to determine the maximum dose that could be
administered without adverse reaction, two male Sprague Dawley rats
were injected with serial doses of Tat-GluR2-3Y ranging from 0.5
nmoles/g to 30 nmoles/g and basic vital parameters were monitored.
It was found that the drug was tolerated up to a dose of .about.12
nmoles/g after which there was a large decline in blood pressure
concurrent with an increase in breathing rate (FIG. 15) and
corresponding changes in pO2, and pCO.sub.2. Both animals were
revived following the dose response curve and showed no signs of
mental depression or other behavioural changes. Based on these
results the dose of 3 nmoles/g was chosen for subsequent in vivo
experiments. Assuming complete dispersion of the peptide in the
animal, this dose corresponds roughly to a concentration of 3
.mu.M.
[0156] As the proposed mechanism of neuroprotection for
Tat-GluR2-3Y is the prevention of apoptosis, it was first necessary
to demonstrate and quantify apoptosis in the model of transient
focal ischemia. Two male Sprague-Dawley rats were subjected to
either 90 min of MCA occlusion, or on surgery without occlusion.
Using TUNEL staining of brain slices obtained 24 h after surgery,
MCA occlusion was shown to cause significant apoptosis (FIG.
16).
[0157] Given the evidence that Tat GluR-3Y pretreatment was able to
block AMPAR receptor endocytosis and reduce OGD-induced apoptosis
in vitro, the ability of peptide pretreatment to prevent
neurological deficit and penumbral apoptosis in transient focal
ischemia was investigated. 15 male Sprague-Dawley rats were
pretreated with either 3 nmoles/g of Tat-GluR2-3Y or Tat-GluR2-3A
or saline for 60 min, after which, the right MCA was occluded for
60 min. The rats were given a neurological examination 45 min into
the MCA occlusion and at sacrifice (.about.24 h). No significant
difference was noted in the neurological scores at 24 h (FIG. 17A)
or during occlusion. Following sacrifice, 12 .mu.m coronal sections
were stained with TUNEL and the number of TUNEL positive cells in
the cortex of the affected hemisphere was scored (FIG. 17B).
Pretreatment with Tat--GluR2-3Y resulted in a .about.55% decrease
in apoptosis with resect to the saline control, while pretreatment
with Tat-GluR2-3A resulted in a .about.22% increase in apoptosis,
however, due to the small sample size and high variability, these
differences did not reach statistical significance. It was noted
during the surgery that pretreatment with Tat-GluR2-3Y and
Tat-GluR2-3A versus saline resulted in significantly lower mean
arterial blood pressure (MABP) 10 min prior to MCA occlusion;
p<0.05 Tukey-Kramer Test
EXAMPLE 10
Treatment of Stress-Related Disorders using the Glu R2-CT
peptide
[0158] Stress is known to prime the induction of LTD.sup.123 and to
results in stress-related disorders such as memory
impairment.sup.124, anxiety and depression.sup.125. Thus, the
GluR2-3Y peptide, by blocking regulated endocytosis and hence LTD,
may have therapeutic effects for these stress-related disorders. As
an example, we have therefore tested the effect of the peptide
against stress-induced anxiety using a well-established animal
anxiety model.sup.126. Rats (n=4) were injected with either 10 nM/g
GluR2-3Y or equal volume of vehicle ACSF (IP). They were given 30
minutes in a dark room post injection. After that they were placed
on an elevated platform for 30 minutes as a stressor and then
placed on the elevated plus maze for 5 minutes.sup.74. The GluR2-3Y
injected rats spent more time on the open arms than the ACSF rats.
The ACSF rats spent most of their time in the corners of the closed
arms or rearing to look over the walls. Thus, GluR2.sub.3Y peptide
blocked stress induced anxiety (FIG. 19). These results strongly
suggest that facilitated AMPAR endocytosis and hence the expression
of LTD play an indispensable role in the expression of induced
behaviors and that LTD blockers such as the GluR2.sub.3Y peptide
may be used as therapeutics to treat stress-related brain
disorders, including anxiety, post-traumatic syndrome and
depression.
EXAMPLE 11
Prevention of Drug Addiction Relapse and Treatment of psychotic
disorders using GluR2-CT peptides
[0159] Relapse induced by presentation of a priming dose of drug or
conditional stimuli paired previously with amphetamine or heroin
infusions is a critical phase of addictive behaviour. A rat model
of intravenous drug self administration is used, coupled with
extinction of drug-seeking behaviour prior to tests of
relapse.sup.73. The Tat-GluR 23Y pride, the mutated control peptide
GluR23A, and vehicle is injected intravenously prior to tests of
relapse. After demonstration of success in preventing relapse, a
battery of behavioural control experiments are conducted to ensure
that treatment with the Tat-GluR2 peptides does not produce
generalized deficits in learning and memory. This protocol uses
tests of recognition and spatial and temporal-order memory used
routinely, along with a standard neurological test battery to
ensure normal sensory and motor function (FIG. 18A-B). The effects
of the GluR23Y peptide on specific tests in rats that model
psychotic symptoms in humans including prepulse inhibition,
PCP-induced hyperactivity and social interaction is also examined.
As blockade of the sensitization occurs without affecting AMPAR
function and basal synaptic transmission, the adverse consequences
of blocking transmitter receptors often associated with other
currently available anti-psychotic drugs does not occur.
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OTHER EMBODIMENTS
[0287] Although various embodiments of the invention are disclosed
herein, many adaptations and modifications may be made within the
scope of the invention in accordance with the common general
knowledge of those skilled in this art. Such modifications include
the substitution of known equivalents for any aspect of the
invention in order to achieve the same result in substantially the
same way. Accession numbers, as used herein, refer to Accession
numbers from multiple databases, including GenBank, the European
Molecular Biology Laboratory (EMBL), the DNA Database of Japan
(DDBJ), or the Genome Sequence Data Base (GSDB), for nucleotide
sequences, and including the Protein Information Resource (PIR),
SWISSPROT, Protein Research Foundation (PRF), and Protein Data Bank
(PDB) (sequences from solved structures), as well as from
translations from annotated coding regions from nucleotide
sequences in GenBank, EMBL, DDBJ, or RefSeq, for polypeptide
sequences. Numeric ranges are inclusive of the numbers defining the
range. In the specification, the word "comprising" is used as an
open-ended term, substantially equivalent to the phrase "including,
but not limited to", and the word "comprises"has a corresponding
meaning. Citation of references herein shall not be construed as an
admission that such references are prior art to the present
invention. All publications are incorporated herein by reference as
if each individual publication were specifically and individually
indicated to be incorporated by reference herein and as though
fully set forth herein. The invention includes all embodiments and
variations substantially as hereinbefore described and with
reference to the examples and drawings.
Sequence CWU 1
1
510 1 11 PRT Artificial Synthetic Peptide 1 Tyr Arg Glu Gly Tyr Asn
Val Tyr Gly Ile Glu 1 5 10 2 11 PRT Artificial Synthetic Peptide 2
Tyr Lys Glu Gly Tyr Asn Val Tyr Gly Ile Glu 1 5 10 3 9 PRT
Artificial Synthetic Peptide 3 Tyr Arg Glu Gly Tyr Asn Val Tyr Gly
1 5 4 9 PRT Artificial Synthetic Peptide 4 Tyr Lys Glu Gly Tyr Asn
Val Tyr Gly 1 5 5 11 PRT Artificial Synthetic Peptide 5 Tyr Gly Arg
Lys Lys Arg Arg Gln Arg Arg Arg 1 5 10 6 12 PRT Artificial
Synthetic Peptide 6 Gly Ser Thr Tyr Lys Glu Gly Tyr Asn Val Tyr Gly
1 5 10 7 12 PRT Artificial Synthetic Peptide 7 Gly Ser Thr Ala Lys
Glu Gly Ala Asn Val Ala Gly 1 5 10 8 9 PRT Artificial Synthetic
Peptide 8 Tyr Lys Glu Gly Tyr Asn Val Asp Gly 1 5 9 9 PRT
Artificial Synthetic Peptide 9 Tyr Lys Glu Gly Tyr Asn Val Glu Gly
1 5 10 9 PRT Artificial Synthetic Peptide 10 Tyr Lys Glu Gly Tyr
Asn Val Ser Gly 1 5 11 9 PRT Artificial Synthetic Peptide 11 Tyr
Lys Glu Gly Tyr Asn Val Thr Gly 1 5 12 9 PRT Artificial Synthetic
Peptide 12 Tyr Lys Glu Gly Asp Asn Val Tyr Gly 1 5 13 9 PRT
Artificial Synthetic Peptide 13 Tyr Lys Glu Gly Asp Asn Val Asp Gly
1 5 14 9 PRT Artificial Synthetic Peptide 14 Tyr Lys Glu Gly Asp
Asn Val Glu Gly 1 5 15 9 PRT Artificial Synthetic Peptide 15 Tyr
Lys Glu Gly Asp Asn Val Ser Gly 1 5 16 9 PRT Artificial Synthetic
Peptide 16 Tyr Lys Glu Gly Asp Asn Val Thr Gly 1 5 17 9 PRT
Artificial Synthetic Peptide 17 Tyr Lys Glu Gly Glu Asn Val Tyr Gly
1 5 18 9 PRT Artificial Synthetic Peptide 18 Tyr Lys Glu Gly Glu
Asn Val Asp Gly 1 5 19 9 PRT Artificial Synthetic Peptide 19 Tyr
Lys Glu Gly Glu Asn Val Glu Gly 1 5 20 9 PRT Artificial Synthetic
Peptide 20 Tyr Lys Glu Gly Glu Asn Val Ser Gly 1 5 21 9 PRT
Artificial Synthetic Peptide 21 Tyr Lys Glu Gly Glu Asn Val Thr Gly
1 5 22 9 PRT Artificial Synthetic Peptide 22 Tyr Lys Glu Gly Ser
Asn Val Tyr Gly 1 5 23 9 PRT Artificial Synthetic Peptide 23 Tyr
Lys Glu Gly Ser Asn Val Asp Gly 1 5 24 9 PRT Artificial Synthetic
Peptide 24 Tyr Lys Glu Gly Ser Asn Val Glu Gly 1 5 25 9 PRT
Artificial Synthetic Peptide 25 Tyr Lys Glu Gly Ser Asn Val Ser Gly
1 5 26 9 PRT Artificial Synthetic Peptide 26 Tyr Lys Glu Gly Ser
Asn Val Thr Gly 1 5 27 9 PRT Artificial Synthetic Peptide 27 Tyr
Lys Glu Gly Thr Asn Val Tyr Gly 1 5 28 9 PRT Artificial Synthetic
Peptide 28 Tyr Lys Glu Gly Thr Asn Val Asp Gly 1 5 29 9 PRT
Artificial Synthetic Peptide 29 Tyr Lys Glu Gly Thr Asn Val Glu Gly
1 5 30 9 PRT Artificial Synthetic Peptide 30 Tyr Lys Glu Gly Thr
Asn Val Ser Gly 1 5 31 9 PRT Artificial Synthetic Peptide 31 Tyr
Lys Glu Gly Thr Asn Val Thr Gly 1 5 32 9 PRT Artificial Synthetic
Peptide 32 Asp Lys Glu Gly Tyr Asn Val Tyr Gly 1 5 33 9 PRT
Artificial Synthetic Peptide 33 Asp Lys Glu Gly Tyr Asn Val Asp Gly
1 5 34 9 PRT Artificial Synthetic Peptide 34 Asp Lys Glu Gly Tyr
Asn Val Glu Gly 1 5 35 9 PRT Artificial Synthetic Peptide 35 Asp
Lys Glu Gly Tyr Asn Val Ser Gly 1 5 36 9 PRT Artificial Synthetic
Peptide 36 Asp Lys Glu Gly Tyr Asn Val Thr Gly 1 5 37 9 PRT
Artificial Synthetic Peptide 37 Asp Lys Glu Gly Asp Asn Val Tyr Gly
1 5 38 9 PRT Artificial Synthetic Peptide 38 Asp Lys Glu Gly Asp
Asn Val Asp Gly 1 5 39 9 PRT Artificial Synthetic Peptide 39 Asp
Lys Glu Gly Asp Asn Val Glu Gly 1 5 40 9 PRT Artificial Synthetic
Peptide 40 Asp Lys Glu Gly Asp Asn Val Ser Gly 1 5 41 9 PRT
Artificial Synthetic Peptide 41 Asp Lys Glu Gly Asp Asn Val Thr Gly
1 5 42 9 PRT Artificial Synthetic Peptide 42 Asp Lys Glu Gly Glu
Asn Val Tyr Gly 1 5 43 9 PRT Artificial Synthetic Peptide 43 Asp
Lys Glu Gly Glu Asn Val Asp Gly 1 5 44 9 PRT Artificial Synthetic
Peptide 44 Asp Lys Glu Gly Glu Asn Val Glu Gly 1 5 45 9 PRT
Artificial Synthetic Peptide 45 Asp Lys Glu Gly Glu Asn Val Ser Gly
1 5 46 9 PRT Artificial Synthetic Peptide 46 Asp Lys Glu Gly Glu
Asn Val Thr Gly 1 5 47 9 PRT Artificial Synthetic Peptide 47 Asp
Lys Glu Gly Ser Asn Val Tyr Gly 1 5 48 9 PRT Artificial Synthetic
Peptide 48 Asp Lys Glu Gly Ser Asn Val Asp Gly 1 5 49 9 PRT
Artificial Synthetic Peptide 49 Asp Lys Glu Gly Ser Asn Val Glu Gly
1 5 50 9 PRT Artificial Synthetic Peptide 50 Asp Lys Glu Gly Ser
Asn Val Ser Gly 1 5 51 9 PRT Artificial Synthetic Peptide 51 Asp
Lys Glu Gly Ser Asn Val Thr Gly 1 5 52 9 PRT Artificial Synthetic
Peptide 52 Asp Lys Glu Gly Thr Asn Val Tyr Gly 1 5 53 9 PRT
Artificial Synthetic Peptide 53 Asp Lys Glu Gly Thr Asn Val Asp Gly
1 5 54 9 PRT Artificial Synthetic Peptide 54 Asp Lys Glu Gly Thr
Asn Val Glu Gly 1 5 55 9 PRT Artificial Synthetic Peptide 55 Asp
Lys Glu Gly Thr Asn Val Ser Gly 1 5 56 9 PRT Artificial Synthetic
Peptide 56 Asp Lys Glu Gly Thr Asn Val Thr Gly 1 5 57 9 PRT
Artificial Synthetic Peptide 57 Glu Lys Glu Gly Tyr Asn Val Tyr Gly
1 5 58 9 PRT Artificial Synthetic Peptide 58 Glu Lys Glu Gly Tyr
Asn Val Asp Gly 1 5 59 9 PRT Artificial Synthetic Peptide 59 Glu
Lys Glu Gly Tyr Asn Val Glu Gly 1 5 60 9 PRT Artificial Synthetic
Peptide 60 Glu Lys Glu Gly Tyr Asn Val Ser Gly 1 5 61 9 PRT
Artificial Synthetic Peptide 61 Glu Lys Glu Gly Tyr Asn Val Thr Gly
1 5 62 9 PRT Artificial Synthetic Peptide 62 Glu Lys Glu Gly Asp
Asn Val Tyr Gly 1 5 63 9 PRT Artificial Synthetic Peptide 63 Glu
Lys Glu Gly Asp Asn Val Asp Gly 1 5 64 9 PRT Artificial Synthetic
Peptide 64 Glu Lys Glu Gly Asp Asn Val Glu Gly 1 5 65 9 PRT
Artificial Synthetic Peptide 65 Glu Lys Glu Gly Asp Asn Val Ser Gly
1 5 66 9 PRT Artificial Synthetic Peptide 66 Glu Lys Glu Gly Asp
Asn Val Thr Gly 1 5 67 9 PRT Artificial Synthetic Peptide 67 Glu
Lys Glu Gly Glu Asn Val Tyr Gly 1 5 68 9 PRT Artificial Synthetic
Peptide 68 Glu Lys Glu Gly Glu Asn Val Asp Gly 1 5 69 9 PRT
Artificial Synthetic Peptide 69 Glu Lys Glu Gly Glu Asn Val Glu Gly
1 5 70 9 PRT Artificial Synthetic Peptide 70 Glu Lys Glu Gly Glu
Asn Val Ser Gly 1 5 71 9 PRT Artificial Synthetic Peptide 71 Glu
Lys Glu Gly Glu Asn Val Thr Gly 1 5 72 9 PRT Artificial Synthetic
Peptide 72 Glu Lys Glu Gly Ser Asn Val Tyr Gly 1 5 73 9 PRT
Artificial Synthetic Peptide 73 Glu Lys Glu Gly Ser Asn Val Asp Gly
1 5 74 9 PRT Artificial Synthetic Peptide 74 Glu Lys Glu Gly Ser
Asn Val Glu Gly 1 5 75 9 PRT Artificial Synthetic Peptide 75 Glu
Lys Glu Gly Ser Asn Val Ser Gly 1 5 76 9 PRT Artificial Synthetic
Peptide 76 Glu Lys Glu Gly Ser Asn Val Thr Gly 1 5 77 9 PRT
Artificial Synthetic Peptide 77 Glu Lys Glu Gly Thr Asn Val Tyr Gly
1 5 78 9 PRT Artificial Synthetic Peptide 78 Glu Lys Glu Gly Thr
Asn Val Asp Gly 1 5 79 9 PRT Artificial Synthetic Peptide 79 Glu
Lys Glu Gly Thr Asn Val Glu Gly 1 5 80 9 PRT Artificial Synthetic
Peptide 80 Glu Lys Glu Gly Thr Asn Val Ser Gly 1 5 81 9 PRT
Artificial Synthetic Peptide 81 Glu Lys Glu Gly Thr Asn Val Thr Gly
1 5 82 9 PRT Artificial Synthetic Peptide 82 Ser Lys Glu Gly Tyr
Asn Val Tyr Gly 1 5 83 9 PRT Artificial Synthetic Peptide 83 Ser
Lys Glu Gly Tyr Asn Val Asp Gly 1 5 84 9 PRT Artificial Synthetic
Peptide 84 Ser Lys Glu Gly Tyr Asn Val Glu Gly 1 5 85 9 PRT
Artificial Synthetic Peptide 85 Ser Lys Glu Gly Tyr Asn Val Ser Gly
1 5 86 9 PRT Artificial Synthetic Peptide 86 Ser Lys Glu Gly Tyr
Asn Val Thr Gly 1 5 87 9 PRT Artificial Synthetic Peptide 87 Ser
Lys Glu Gly Asp Asn Val Tyr Gly 1 5 88 9 PRT Artificial Synthetic
Peptide 88 Ser Lys Glu Gly Asp Asn Val Asp Gly 1 5 89 9 PRT
Artificial Synthetic Peptide 89 Ser Lys Glu Gly Asp Asn Val Glu Gly
1 5 90 9 PRT Artificial Synthetic Peptide 90 Ser Lys Glu Gly Asp
Asn Val Ser Gly 1 5 91 9 PRT Artificial Synthetic Peptide 91 Ser
Lys Glu Gly Asp Asn Val Thr Gly 1 5 92 9 PRT Artificial Synthetic
Peptide 92 Ser Lys Glu Gly Glu Asn Val Tyr Gly 1 5 93 9 PRT
Artificial Synthetic Peptide 93 Ser Lys Glu Gly Glu Asn Val Asp Gly
1 5 94 9 PRT Artificial Synthetic Peptide 94 Ser Lys Glu Gly Glu
Asn Val Glu Gly 1 5 95 9 PRT Artificial Synthetic Peptide 95 Ser
Lys Glu Gly Glu Asn Val Ser Gly 1 5 96 9 PRT Artificial Synthetic
Peptide 96 Ser Lys Glu Gly Glu Asn Val Thr Gly 1 5 97 9 PRT
Artificial Synthetic Peptide 97 Ser Lys Glu Gly Ser Asn Val Tyr Gly
1 5 98 9 PRT Artificial Synthetic Peptide 98 Ser Lys Glu Gly Ser
Asn Val Asp Gly 1 5 99 9 PRT Artificial Synthetic Peptide 99 Ser
Lys Glu Gly Ser Asn Val Glu Gly 1 5 100 9 PRT Artificial Synthetic
Peptide 100 Ser Lys Glu Gly Ser Asn Val Ser Gly 1 5 101 9 PRT
Artificial Synthetic Peptide 101 Ser Lys Glu Gly Ser Asn Val Thr
Gly 1 5 102 9 PRT Artificial Synthetic Peptide 102 Ser Lys Glu Gly
Thr Asn Val Tyr Gly 1 5 103 9 PRT Artificial Synthetic Peptide 103
Ser Lys Glu Gly Thr Asn Val Asp Gly 1 5 104 9 PRT Artificial
Synthetic Peptide 104 Ser Lys Glu Gly Thr Asn Val Glu Gly 1 5 105 9
PRT Artificial Synthetic Peptide 105 Ser Lys Glu Gly Thr Asn Val
Ser Gly 1 5 106 9 PRT Artificial Synthetic Peptide 106 Ser Lys Glu
Gly Thr Asn Val Thr Gly 1 5 107 9 PRT Artificial Synthetic Peptide
107 Thr Lys Glu Gly Tyr Asn Val Tyr Gly 1 5 108 9 PRT Artificial
Synthetic Peptide 108 Thr Lys Glu Gly Tyr Asn Val Asp Gly 1 5 109 9
PRT Artificial Synthetic Peptide 109 Thr Lys Glu Gly Tyr Asn Val
Glu Gly 1 5 110 9 PRT Artificial Synthetic Peptide 110 Thr Lys Glu
Gly Tyr Asn Val Ser Gly 1 5 111 9 PRT Artificial Synthetic Peptide
111 Thr Lys Glu Gly Tyr Asn Val Thr Gly 1 5 112 9 PRT Artificial
Synthetic Peptide 112 Thr Lys Glu Gly Asp Asn Val Tyr Gly 1 5 113 9
PRT Artificial Synthetic Peptide 113 Thr Lys Glu Gly Asp Asn Val
Asp Gly 1 5 114 9 PRT Artificial Synthetic Peptide 114 Thr Lys Glu
Gly Asp Asn Val Glu Gly 1 5 115 9 PRT Artificial Synthetic Peptide
115 Thr Lys Glu Gly Asp Asn Val Ser Gly 1 5 116 9 PRT Artificial
Synthetic Peptide 116 Thr Lys Glu Gly Asp Asn Val Thr Gly 1 5 117 9
PRT Artificial Synthetic Peptide 117 Thr Lys Glu Gly Glu Asn Val
Tyr Gly 1 5 118 9 PRT Artificial Synthetic Peptide 118 Thr Lys Glu
Gly Glu Asn Val Asp Gly 1 5 119 9 PRT Artificial Synthetic Peptide
119 Thr Lys Glu Gly Glu Asn Val Glu Gly 1 5 120 9 PRT Artificial
Synthetic Peptide 120 Thr Lys Glu Gly Glu Asn Val Ser Gly 1 5 121 9
PRT Artificial Synthetic Peptide 121 Thr Lys Glu Gly Glu Asn Val
Thr Gly 1 5 122 9 PRT Artificial Synthetic Peptide 122 Thr Lys Glu
Gly Ser Asn Val Tyr Gly 1 5 123 9 PRT Artificial Synthetic Peptide
123 Thr Lys Glu Gly Ser Asn Val Asp Gly 1 5 124 9 PRT Artificial
Synthetic Peptide 124 Thr Lys Glu Gly Ser Asn Val Glu Gly 1 5 125 9
PRT Artificial Synthetic Peptide 125 Thr Lys Glu Gly Ser Asn Val
Ser Gly 1 5 126 9 PRT Artificial Synthetic Peptide 126 Thr Lys Glu
Gly Ser Asn Val Thr Gly 1 5 127 9 PRT Artificial Synthetic Peptide
127 Thr Lys Glu Gly Thr Asn Val Tyr Gly 1 5 128 9 PRT Artificial
Synthetic Peptide 128 Thr Lys Glu Gly Thr Asn Val Asp Gly 1 5 129 9
PRT Artificial Synthetic Peptide 129 Thr Lys Glu Gly Thr Asn Val
Glu Gly 1 5 130 9 PRT Artificial Synthetic Peptide 130 Thr Lys Glu
Gly Thr Asn Val Ser Gly 1 5 131 9 PRT Artificial Synthetic Peptide
131 Thr Lys Glu Gly Thr Asn Val Thr Gly 1 5 132 9 PRT Artificial
Synthetic Peptide 132 Tyr Arg Glu Gly Tyr Asn Val Asp Gly 1 5 133 9
PRT Artificial Synthetic Peptide 133 Tyr Arg Glu Gly Tyr Asn Val
Glu Gly 1 5 134 9 PRT Artificial Synthetic Peptide 134 Tyr Arg Glu
Gly Tyr Asn Val Ser Gly 1 5 135 9 PRT Artificial Synthetic Peptide
135 Tyr Arg Glu Gly Tyr Asn Val Thr Gly 1 5 136 9 PRT Artificial
Synthetic Peptide 136 Tyr Arg Glu Gly Asp Asn Val Tyr Gly 1 5 137 9
PRT Artificial Synthetic Peptide 137 Tyr Arg Glu Gly Asp Asn Val
Asp Gly 1 5 138 9 PRT Artificial Synthetic Peptide 138 Tyr Arg Glu
Gly Asp Asn Val Glu Gly 1 5 139 9 PRT Artificial Synthetic Peptide
139 Tyr Arg Glu Gly Asp Asn Val Ser Gly 1 5 140 9 PRT Artificial
Synthetic Peptide 140 Tyr Arg Glu Gly Asp Asn Val Thr Gly 1 5 141 9
PRT Artificial Synthetic Peptide 141 Tyr Arg Glu Gly Glu Asn Val
Tyr Gly 1 5 142 9 PRT Artificial Synthetic Peptide 142 Tyr Arg Glu
Gly Glu Asn Val Asp Gly 1 5 143 9 PRT Artificial Synthetic Peptide
143 Tyr Arg Glu Gly Glu Asn Val Glu Gly 1 5 144 9 PRT Artificial
Synthetic Peptide 144 Tyr Arg Glu Gly Glu Asn Val Ser Gly 1 5 145 9
PRT Artificial Synthetic Peptide 145 Tyr Arg Glu Gly Glu Asn Val
Thr Gly 1 5 146 9 PRT Artificial Synthetic Peptide 146 Tyr Arg Glu
Gly Ser Asn Val Tyr Gly 1 5 147 9 PRT Artificial Synthetic Peptide
147 Tyr Arg Glu Gly Ser Asn Val Asp Gly 1 5 148 9 PRT Artificial
Synthetic Peptide 148 Tyr Arg Glu Gly Ser Asn Val Glu Gly 1 5 149 9
PRT Artificial Synthetic Peptide 149 Tyr Arg Glu Gly Ser Asn Val
Ser Gly 1 5 150 9 PRT Artificial Synthetic Peptide 150 Tyr Arg Glu
Gly Ser Asn Val Thr Gly 1 5 151 9 PRT Artificial Synthetic Peptide
151 Tyr Arg Glu Gly Thr Asn Val Tyr Gly 1 5 152 9 PRT Artificial
Synthetic Peptide 152 Tyr Arg Glu Gly Thr Asn Val Asp Gly 1 5 153 9
PRT Artificial Synthetic Peptide 153 Tyr Arg Glu Gly Thr Asn Val
Glu Gly 1 5 154 9 PRT Artificial Synthetic Peptide 154 Tyr Arg Glu
Gly Thr Asn Val Ser Gly 1 5 155 9 PRT Artificial Synthetic Peptide
155 Tyr Arg Glu Gly Thr Asn Val Thr Gly 1 5 156 9 PRT Artificial
Synthetic Peptide 156 Asp Arg Glu Gly Tyr Asn Val Tyr Gly 1 5 157 9
PRT Artificial Synthetic Peptide 157 Asp Arg Glu Gly Tyr Asn Val
Asp Gly 1 5 158 9 PRT Artificial Synthetic Peptide 158 Asp Arg Glu
Gly Tyr Asn Val Glu Gly 1 5 159 9 PRT Artificial Synthetic Peptide
159 Asp Arg Glu Gly Tyr Asn Val Ser Gly 1 5 160 9 PRT Artificial
Synthetic Peptide 160 Asp Arg Glu Gly Tyr Asn Val Thr Gly 1 5 161 9
PRT Artificial Synthetic Peptide 161 Asp Arg Glu Gly Asp Asn Val
Tyr Gly 1 5 162 9 PRT Artificial Synthetic Peptide 162 Asp Arg Glu
Gly Asp Asn Val Asp Gly 1 5 163 9 PRT Artificial Synthetic Peptide
163 Asp Arg Glu Gly Asp Asn Val Glu Gly 1 5 164 9 PRT Artificial
Synthetic Peptide 164 Asp Arg Glu Gly Asp Asn Val Ser Gly 1 5 165 9
PRT Artificial Synthetic Peptide 165 Asp Arg Glu Gly Asp Asn Val
Thr Gly 1 5 166 9 PRT Artificial Synthetic Peptide 166 Asp Arg Glu
Gly Glu Asn Val Tyr Gly 1 5 167 9 PRT Artificial Synthetic Peptide
167 Asp Arg Glu Gly Glu Asn Val Asp Gly 1 5 168 9 PRT Artificial
Synthetic Peptide 168 Asp Arg Glu Gly Glu Asn Val Glu Gly 1 5 169 9
PRT Artificial Synthetic Peptide 169 Asp Arg Glu Gly Glu Asn Val
Ser Gly 1 5 170 9 PRT Artificial Synthetic Peptide 170 Asp Arg Glu
Gly Glu Asn Val Thr Gly 1 5 171 9 PRT Artificial Synthetic Peptide
171 Asp Arg Glu Gly Ser Asn Val Tyr Gly 1 5 172 9 PRT Artificial
Synthetic Peptide 172 Asp Arg Glu Gly Ser Asn Val Asp Gly 1 5 173 9
PRT Artificial Synthetic Peptide 173 Asp Arg Glu Gly Ser Asn Val
Glu Gly 1 5 174 9 PRT Artificial Synthetic Peptide 174 Asp Arg Glu
Gly Ser Asn Val Ser Gly 1 5 175 9 PRT Artificial Synthetic Peptide
175 Asp Arg Glu Gly Ser Asn Val Thr Gly 1 5 176 9 PRT Artificial
Synthetic Peptide 176 Asp Arg Glu Gly Thr Asn Val Tyr Gly 1 5 177 9
PRT Artificial Synthetic Peptide 177 Asp Arg Glu Gly Thr Asn Val
Asp Gly 1 5 178 9 PRT Artificial Synthetic Peptide 178 Asp Arg Glu
Gly Thr Asn Val Glu Gly 1 5 179 9 PRT Artificial Synthetic Peptide
179 Asp Arg Glu Gly Thr Asn Val Ser Gly 1 5 180 9 PRT Artificial
Synthetic Peptide 180 Asp Arg Glu Gly Thr Asn Val Thr Gly 1 5 181 9
PRT Artificial Synthetic Peptide 181 Glu Arg Glu Gly Tyr Asn Val
Tyr Gly 1 5 182 9 PRT Artificial Synthetic Peptide 182 Glu Arg Glu
Gly Tyr Asn Val Asp Gly 1 5 183 9 PRT Artificial Synthetic Peptide
183 Glu Arg Glu Gly Tyr Asn Val Glu Gly 1 5 184 9 PRT Artificial
Synthetic Peptide 184 Glu Arg Glu Gly Tyr Asn Val Ser Gly 1 5 185 9
PRT Artificial Synthetic Peptide 185 Glu Arg Glu Gly Tyr Asn Val
Thr Gly 1 5 186 9 PRT Artificial Synthetic Peptide 186 Glu Arg Glu
Gly Asp Asn Val Tyr Gly 1 5 187 9 PRT Artificial Synthetic Peptide
187 Glu Arg Glu
Gly Asp Asn Val Asp Gly 1 5 188 9 PRT Artificial Synthetic Peptide
188 Glu Arg Glu Gly Asp Asn Val Glu Gly 1 5 189 9 PRT Artificial
Synthetic Peptide 189 Glu Arg Glu Gly Asp Asn Val Ser Gly 1 5 190 9
PRT Artificial Synthetic Peptide 190 Glu Arg Glu Gly Asp Asn Val
Thr Gly 1 5 191 9 PRT Artificial Synthetic Peptide 191 Glu Arg Glu
Gly Glu Asn Val Tyr Gly 1 5 192 9 PRT Artificial Synthetic Peptide
192 Glu Arg Glu Gly Glu Asn Val Asp Gly 1 5 193 9 PRT Artificial
Synthetic Peptide 193 Glu Arg Glu Gly Glu Asn Val Glu Gly 1 5 194 9
PRT Artificial Synthetic Peptide 194 Glu Arg Glu Gly Glu Asn Val
Ser Gly 1 5 195 9 PRT Artificial Synthetic Peptide 195 Glu Arg Glu
Gly Glu Asn Val Thr Gly 1 5 196 9 PRT Artificial Synthetic Peptide
196 Glu Arg Glu Gly Ser Asn Val Tyr Gly 1 5 197 9 PRT Artificial
Synthetic Peptide 197 Glu Arg Glu Gly Ser Asn Val Asp Gly 1 5 198 9
PRT Artificial Synthetic Peptide 198 Glu Arg Glu Gly Ser Asn Val
Glu Gly 1 5 199 9 PRT Artificial Synthetic Peptide 199 Glu Arg Glu
Gly Ser Asn Val Ser Gly 1 5 200 9 PRT Artificial Synthetic Peptide
200 Glu Arg Glu Gly Ser Asn Val Thr Gly 1 5 201 9 PRT Artificial
Synthetic Peptide 201 Glu Arg Glu Gly Thr Asn Val Tyr Gly 1 5 202 9
PRT Artificial Synthetic Peptide 202 Glu Arg Glu Gly Thr Asn Val
Asp Gly 1 5 203 9 PRT Artificial Synthetic Peptide 203 Glu Arg Glu
Gly Thr Asn Val Glu Gly 1 5 204 9 PRT Artificial Synthetic Peptide
204 Glu Arg Glu Gly Thr Asn Val Ser Gly 1 5 205 9 PRT Artificial
Synthetic Peptide 205 Glu Arg Glu Gly Thr Asn Val Thr Gly 1 5 206 9
PRT Artificial Synthetic Peptide 206 Ser Arg Glu Gly Tyr Asn Val
Tyr Gly 1 5 207 9 PRT Artificial Synthetic Peptide 207 Ser Arg Glu
Gly Tyr Asn Val Asp Gly 1 5 208 9 PRT Artificial Synthetic Peptide
208 Ser Arg Glu Gly Tyr Asn Val Glu Gly 1 5 209 9 PRT Artificial
Synthetic Peptide 209 Ser Arg Glu Gly Tyr Asn Val Ser Gly 1 5 210 9
PRT Artificial Synthetic Peptide 210 Ser Arg Glu Gly Tyr Asn Val
Thr Gly 1 5 211 9 PRT Artificial Synthetic Peptide 211 Ser Arg Glu
Gly Asp Asn Val Tyr Gly 1 5 212 9 PRT Artificial Synthetic Peptide
212 Ser Arg Glu Gly Asp Asn Val Asp Gly 1 5 213 9 PRT Artificial
Synthetic Peptide 213 Ser Arg Glu Gly Asp Asn Val Glu Gly 1 5 214 9
PRT Artificial Synthetic Peptide 214 Ser Arg Glu Gly Asp Asn Val
Ser Gly 1 5 215 9 PRT Artificial Synthetic Peptide 215 Ser Arg Glu
Gly Asp Asn Val Thr Gly 1 5 216 9 PRT Artificial Synthetic Peptide
216 Ser Arg Glu Gly Glu Asn Val Tyr Gly 1 5 217 9 PRT Artificial
Synthetic Peptide 217 Ser Arg Glu Gly Glu Asn Val Asp Gly 1 5 218 9
PRT Artificial Synthetic Peptide 218 Ser Arg Glu Gly Glu Asn Val
Glu Gly 1 5 219 9 PRT Artificial Synthetic Peptide 219 Ser Arg Glu
Gly Glu Asn Val Ser Gly 1 5 220 9 PRT Artificial Synthetic Peptide
220 Ser Arg Glu Gly Glu Asn Val Thr Gly 1 5 221 9 PRT Artificial
Synthetic Peptide 221 Ser Arg Glu Gly Ser Asn Val Tyr Gly 1 5 222 9
PRT Artificial Synthetic Peptide 222 Ser Arg Glu Gly Ser Asn Val
Asp Gly 1 5 223 9 PRT Artificial Synthetic Peptide 223 Ser Arg Glu
Gly Ser Asn Val Glu Gly 1 5 224 9 PRT Artificial Synthetic Peptide
224 Ser Arg Glu Gly Ser Asn Val Ser Gly 1 5 225 9 PRT Artificial
Synthetic Peptide 225 Ser Arg Glu Gly Ser Asn Val Thr Gly 1 5 226 9
PRT Artificial Synthetic Peptide 226 Ser Arg Glu Gly Thr Asn Val
Tyr Gly 1 5 227 9 PRT Artificial Synthetic Peptide 227 Ser Arg Glu
Gly Thr Asn Val Asp Gly 1 5 228 9 PRT Artificial Synthetic Peptide
228 Ser Arg Glu Gly Thr Asn Val Glu Gly 1 5 229 9 PRT Artificial
Synthetic Peptide 229 Ser Arg Glu Gly Thr Asn Val Ser Gly 1 5 230 9
PRT Artificial Synthetic Peptide 230 Ser Arg Glu Gly Thr Asn Val
Thr Gly 1 5 231 9 PRT Artificial Synthetic Peptide 231 Thr Arg Glu
Gly Tyr Asn Val Tyr Gly 1 5 232 9 PRT Artificial Synthetic Peptide
232 Thr Arg Glu Gly Tyr Asn Val Asp Gly 1 5 233 9 PRT Artificial
Synthetic Peptide 233 Thr Arg Glu Gly Tyr Asn Val Glu Gly 1 5 234 9
PRT Artificial Synthetic Peptide 234 Thr Arg Glu Gly Tyr Asn Val
Ser Gly 1 5 235 9 PRT Artificial Synthetic Peptide 235 Thr Arg Glu
Gly Tyr Asn Val Thr Gly 1 5 236 9 PRT Artificial Synthetic Peptide
236 Thr Arg Glu Gly Asp Asn Val Tyr Gly 1 5 237 9 PRT Artificial
Synthetic Peptide 237 Thr Arg Glu Gly Asp Asn Val Asp Gly 1 5 238 9
PRT Artificial Synthetic Peptide 238 Thr Arg Glu Gly Asp Asn Val
Glu Gly 1 5 239 9 PRT Artificial Synthetic Peptide 239 Thr Arg Glu
Gly Asp Asn Val Ser Gly 1 5 240 9 PRT Artificial Synthetic Peptide
240 Thr Arg Glu Gly Asp Asn Val Thr Gly 1 5 241 9 PRT Artificial
Synthetic Peptide 241 Thr Arg Glu Gly Glu Asn Val Tyr Gly 1 5 242 9
PRT Artificial Synthetic Peptide 242 Thr Arg Glu Gly Glu Asn Val
Asp Gly 1 5 243 9 PRT Artificial Synthetic Peptide 243 Thr Arg Glu
Gly Glu Asn Val Glu Gly 1 5 244 9 PRT Artificial Synthetic Peptide
244 Thr Arg Glu Gly Glu Asn Val Ser Gly 1 5 245 9 PRT Artificial
Synthetic Peptide 245 Thr Arg Glu Gly Glu Asn Val Thr Gly 1 5 246 9
PRT Artificial Synthetic Peptide 246 Thr Arg Glu Gly Ser Asn Val
Tyr Gly 1 5 247 9 PRT Artificial Synthetic Peptide 247 Thr Arg Glu
Gly Ser Asn Val Asp Gly 1 5 248 9 PRT Artificial Synthetic Peptide
248 Thr Arg Glu Gly Ser Asn Val Glu Gly 1 5 249 9 PRT Artificial
Synthetic Peptide 249 Thr Arg Glu Gly Ser Asn Val Ser Gly 1 5 250 9
PRT Artificial Synthetic Peptide 250 Thr Arg Glu Gly Ser Asn Val
Thr Gly 1 5 251 9 PRT Artificial Synthetic Peptide 251 Thr Arg Glu
Gly Thr Asn Val Tyr Gly 1 5 252 9 PRT Artificial Synthetic Peptide
252 Thr Arg Glu Gly Thr Asn Val Asp Gly 1 5 253 9 PRT Artificial
Synthetic Peptide 253 Thr Arg Glu Gly Thr Asn Val Glu Gly 1 5 254 9
PRT Artificial Synthetic Peptide 254 Thr Arg Glu Gly Thr Asn Val
Ser Gly 1 5 255 9 PRT Artificial Synthetic Peptide 255 Thr Arg Glu
Gly Thr Asn Val Thr Gly 1 5 256 11 PRT Artificial Synthetic Peptide
256 Tyr Lys Glu Gly Tyr Asn Val Asp Gly Ile Glu 1 5 10 257 11 PRT
Artificial Synthetic Peptide 257 Tyr Lys Glu Gly Tyr Asn Val Glu
Gly Ile Glu 1 5 10 258 11 PRT Artificial Synthetic Peptide 258 Tyr
Lys Glu Gly Tyr Asn Val Ser Gly Ile Glu 1 5 10 259 11 PRT
Artificial Synthetic Peptide 259 Tyr Lys Glu Gly Tyr Asn Val Thr
Gly Ile Glu 1 5 10 260 11 PRT Artificial Synthetic Peptide 260 Tyr
Lys Glu Gly Asp Asn Val Tyr Gly Ile Glu 1 5 10 261 11 PRT
Artificial Synthetic Peptide 261 Tyr Lys Glu Gly Asp Asn Val Asp
Gly Ile Glu 1 5 10 262 11 PRT Artificial Synthetic Peptide 262 Tyr
Lys Glu Gly Asp Asn Val Glu Gly Ile Glu 1 5 10 263 11 PRT
Artificial Synthetic Peptide 263 Tyr Lys Glu Gly Asp Asn Val Ser
Gly Ile Glu 1 5 10 264 11 PRT Artificial Synthetic Peptide 264 Tyr
Lys Glu Gly Asp Asn Val Thr Gly Ile Glu 1 5 10 265 11 PRT
Artificial Synthetic Peptide 265 Tyr Lys Glu Gly Glu Asn Val Tyr
Gly Ile Glu 1 5 10 266 11 PRT Artificial Synthetic Peptide 266 Tyr
Lys Glu Gly Glu Asn Val Asp Gly Ile Glu 1 5 10 267 11 PRT
Artificial Synthetic Peptide 267 Tyr Lys Glu Gly Glu Asn Val Glu
Gly Ile Glu 1 5 10 268 11 PRT Artificial Synthetic Peptide 268 Tyr
Lys Glu Gly Glu Asn Val Ser Gly Ile Glu 1 5 10 269 11 PRT
Artificial Synthetic Peptide 269 Tyr Lys Glu Gly Glu Asn Val Thr
Gly Ile Glu 1 5 10 270 11 PRT Artificial Synthetic Peptide 270 Tyr
Lys Glu Gly Ser Asn Val Tyr Gly Ile Glu 1 5 10 271 11 PRT
Artificial Synthetic Peptide 271 Tyr Lys Glu Gly Ser Asn Val Asp
Gly Ile Glu 1 5 10 272 11 PRT Artificial Synthetic Peptide 272 Tyr
Lys Glu Gly Ser Asn Val Glu Gly Ile Glu 1 5 10 273 11 PRT
Artificial Synthetic Peptide 273 Tyr Lys Glu Gly Ser Asn Val Ser
Gly Ile Glu 1 5 10 274 11 PRT Artificial Synthetic Peptide 274 Tyr
Lys Glu Gly Ser Asn Val Thr Gly Ile Glu 1 5 10 275 11 PRT
Artificial Synthetic Peptide 275 Tyr Lys Glu Gly Thr Asn Val Tyr
Gly Ile Glu 1 5 10 276 11 PRT Artificial Synthetic Peptide 276 Tyr
Lys Glu Gly Thr Asn Val Asp Gly Ile Glu 1 5 10 277 11 PRT
Artificial Synthetic Peptide 277 Tyr Lys Glu Gly Thr Asn Val Glu
Gly Ile Glu 1 5 10 278 11 PRT Artificial Synthetic Peptide 278 Tyr
Lys Glu Gly Thr Asn Val Ser Gly Ile Glu 1 5 10 279 11 PRT
Artificial Synthetic Peptide 279 Tyr Lys Glu Gly Thr Asn Val Thr
Gly Ile Glu 1 5 10 280 11 PRT Artificial Synthetic Peptide 280 Asp
Lys Glu Gly Tyr Asn Val Tyr Gly Ile Glu 1 5 10 281 11 PRT
Artificial Synthetic Peptide 281 Asp Lys Glu Gly Tyr Asn Val Asp
Gly Ile Glu 1 5 10 282 11 PRT Artificial Synthetic Peptide 282 Asp
Lys Glu Gly Tyr Asn Val Glu Gly Ile Glu 1 5 10 283 11 PRT
Artificial Synthetic Peptide 283 Asp Lys Glu Gly Tyr Asn Val Ser
Gly Ile Glu 1 5 10 284 11 PRT Artificial Synthetic Peptide 284 Asp
Lys Glu Gly Tyr Asn Val Thr Gly Ile Glu 1 5 10 285 11 PRT
Artificial Synthetic Peptide 285 Asp Lys Glu Gly Asp Asn Val Tyr
Gly Ile Glu 1 5 10 286 11 PRT Artificial Synthetic Peptide 286 Asp
Lys Glu Gly Asp Asn Val Asp Gly Ile Glu 1 5 10 287 11 PRT
Artificial Synthetic Peptide 287 Asp Lys Glu Gly Asp Asn Val Glu
Gly Ile Glu 1 5 10 288 11 PRT Artificial Synthetic Peptide 288 Asp
Lys Glu Gly Asp Asn Val Ser Gly Ile Glu 1 5 10 289 11 PRT
Artificial Synthetic Peptide 289 Asp Lys Glu Gly Asp Asn Val Thr
Gly Ile Glu 1 5 10 290 11 PRT Artificial Synthetic Peptide 290 Asp
Lys Glu Gly Glu Asn Val Tyr Gly Ile Glu 1 5 10 291 11 PRT
Artificial Synthetic Peptide 291 Asp Lys Glu Gly Glu Asn Val Asp
Gly Ile Glu 1 5 10 292 11 PRT Artificial Synthetic Peptide 292 Asp
Lys Glu Gly Glu Asn Val Glu Gly Ile Glu 1 5 10 293 11 PRT
Artificial Synthetic Peptide 293 Asp Lys Glu Gly Glu Asn Val Ser
Gly Ile Glu 1 5 10 294 11 PRT Artificial Synthetic Peptide 294 Asp
Lys Glu Gly Glu Asn Val Thr Gly Ile Glu 1 5 10 295 11 PRT
Artificial Synthetic Peptide 295 Asp Lys Glu Gly Ser Asn Val Tyr
Gly Ile Glu 1 5 10 296 11 PRT Artificial Synthetic Peptide 296 Asp
Lys Glu Gly Ser Asn Val Asp Gly Ile Glu 1 5 10 297 11 PRT
Artificial Synthetic Peptide 297 Asp Lys Glu Gly Ser Asn Val Glu
Gly Ile Glu 1 5 10 298 11 PRT Artificial Synthetic Peptide 298 Asp
Lys Glu Gly Ser Asn Val Ser Gly Ile Glu 1 5 10 299 11 PRT
Artificial Synthetic Peptide 299 Asp Lys Glu Gly Ser Asn Val Thr
Gly Ile Glu 1 5 10 300 11 PRT Artificial Synthetic Peptide 300 Asp
Lys Glu Gly Thr Asn Val Tyr Gly Ile Glu 1 5 10 301 11 PRT
Artificial Synthetic Peptide 301 Asp Lys Glu Gly Thr Asn Val Asp
Gly Ile Glu 1 5 10 302 11 PRT Artificial Synthetic Peptide 302 Asp
Lys Glu Gly Thr Asn Val Glu Gly Ile Glu 1 5 10 303 11 PRT
Artificial Synthetic Peptide 303 Asp Lys Glu Gly Thr Asn Val Ser
Gly Ile Glu 1 5 10 304 11 PRT Artificial Synthetic Peptide 304 Asp
Lys Glu Gly Thr Asn Val Thr Gly Ile Glu 1 5 10 305 11 PRT
Artificial Synthetic Peptide 305 Glu Lys Glu Gly Tyr Asn Val Tyr
Gly Ile Glu 1 5 10 306 11 PRT Artificial Synthetic Peptide 306 Glu
Lys Glu Gly Tyr Asn Val Asp Gly Ile Glu 1 5 10 307 11 PRT
Artificial Synthetic Peptide 307 Glu Lys Glu Gly Tyr Asn Val Glu
Gly Ile Glu 1 5 10 308 11 PRT Artificial Synthetic Peptide 308 Glu
Lys Glu Gly Tyr Asn Val Ser Gly Ile Glu 1 5 10 309 11 PRT
Artificial Synthetic Peptide 309 Glu Lys Glu Gly Tyr Asn Val Thr
Gly Ile Glu 1 5 10 310 11 PRT Artificial Synthetic Peptide 310 Glu
Lys Glu Gly Asp Asn Val Tyr Gly Ile Glu 1 5 10 311 11 PRT
Artificial Synthetic Peptide 311 Glu Lys Glu Gly Asp Asn Val Asp
Gly Ile Glu 1 5 10 312 11 PRT Artificial Synthetic Peptide 312 Glu
Lys Glu Gly Asp Asn Val Glu Gly Ile Glu 1 5 10 313 11 PRT
Artificial Synthetic Peptide 313 Glu Lys Glu Gly Asp Asn Val Ser
Gly Ile Glu 1 5 10 314 11 PRT Artificial Synthetic Peptide 314 Glu
Lys Glu Gly Asp Asn Val Thr Gly Ile Glu 1 5 10 315 11 PRT
Artificial Synthetic Peptide 315 Glu Lys Glu Gly Glu Asn Val Tyr
Gly Ile Glu 1 5 10 316 11 PRT Artificial Synthetic Peptide 316 Glu
Lys Glu Gly Glu Asn Val Asp Gly Ile Glu 1 5 10 317 11 PRT
Artificial Synthetic Peptide 317 Glu Lys Glu Gly Glu Asn Val Glu
Gly Ile Glu 1 5 10 318 11 PRT Artificial Synthetic Peptide 318 Glu
Lys Glu Gly Glu Asn Val Ser Gly Ile Glu 1 5 10 319 11 PRT
Artificial Synthetic Peptide 319 Glu Lys Glu Gly Glu Asn Val Thr
Gly Ile Glu 1 5 10 320 11 PRT Artificial Synthetic Peptide 320 Glu
Lys Glu Gly Ser Asn Val Tyr Gly Ile Glu 1 5 10 321 11 PRT
Artificial Synthetic Peptide 321 Glu Lys Glu Gly Ser Asn Val Asp
Gly Ile Glu 1 5 10 322 11 PRT Artificial Synthetic Peptide 322 Glu
Lys Glu Gly Ser Asn Val Glu Gly Ile Glu 1 5 10 323 11 PRT
Artificial Synthetic Peptide 323 Glu Lys Glu Gly Ser Asn Val Ser
Gly Ile Glu 1 5 10 324 11 PRT Artificial Synthetic Peptide 324 Glu
Lys Glu Gly Ser Asn Val Thr Gly Ile Glu 1 5 10 325 11 PRT
Artificial Synthetic Peptide 325 Glu Lys Glu Gly Thr Asn Val Tyr
Gly Ile Glu 1 5 10 326 11 PRT Artificial Synthetic Peptide 326 Glu
Lys Glu Gly Thr Asn Val Asp Gly Ile Glu 1 5 10 327 11 PRT
Artificial Synthetic Peptide 327 Glu Lys Glu Gly Thr Asn Val Glu
Gly Ile Glu 1 5 10 328 11 PRT Artificial Synthetic Peptide 328 Glu
Lys Glu Gly Thr Asn Val Ser Gly Ile Glu 1 5 10 329 11 PRT
Artificial Synthetic Peptide 329 Glu Lys Glu Gly Thr Asn Val Thr
Gly Ile Glu 1 5 10 330 11 PRT Artificial Synthetic Peptide 330 Ser
Lys Glu Gly Tyr Asn Val Tyr Gly Ile Glu 1 5 10 331 11 PRT
Artificial Synthetic Peptide 331 Ser Lys Glu Gly Tyr Asn Val Asp
Gly Ile Glu 1 5 10 332 11 PRT Artificial Synthetic Peptide 332 Ser
Lys Glu Gly Tyr Asn Val Glu Gly Ile Glu 1 5 10 333 11 PRT
Artificial Synthetic Peptide 333 Ser Lys Glu Gly Tyr Asn Val Ser
Gly Ile Glu 1 5 10 334 11 PRT Artificial Synthetic Peptide 334 Ser
Lys Glu Gly Tyr Asn Val Thr Gly Ile Glu 1 5 10 335 11 PRT
Artificial Synthetic Peptide 335 Ser Lys Glu Gly Asp Asn Val Tyr
Gly Ile Glu 1 5 10 336 11 PRT Artificial Synthetic Peptide 336 Ser
Lys Glu Gly Asp Asn Val Asp Gly Ile Glu 1 5 10 337 11 PRT
Artificial Synthetic Peptide 337 Ser Lys Glu Gly Asp Asn Val Glu
Gly Ile Glu 1 5 10 338 11 PRT Artificial Synthetic Peptide 338 Ser
Lys Glu Gly Asp Asn Val Ser Gly Ile Glu 1 5 10 339 11 PRT
Artificial Synthetic Peptide 339 Ser Lys Glu Gly Asp Asn Val Thr
Gly Ile Glu 1 5 10 340 11 PRT Artificial Synthetic Peptide 340 Ser
Lys Glu Gly Glu Asn Val Tyr Gly Ile Glu 1 5 10 341 11 PRT
Artificial Synthetic Peptide 341 Ser Lys Glu Gly Glu Asn Val Asp
Gly Ile Glu 1 5 10 342 11 PRT Artificial Synthetic Peptide 342 Ser
Lys Glu Gly Glu Asn Val Glu Gly Ile Glu 1 5 10 343 11 PRT
Artificial Synthetic Peptide 343 Ser Lys Glu Gly Glu Asn Val Ser
Gly Ile Glu 1 5 10 344 11 PRT Artificial Synthetic Peptide 344 Ser
Lys Glu Gly Glu Asn Val Thr Gly Ile Glu 1 5 10 345 11 PRT
Artificial Synthetic Peptide 345 Ser Lys Glu Gly Ser Asn Val Tyr
Gly Ile Glu 1 5 10 346 11 PRT Artificial Synthetic Peptide 346 Ser
Lys Glu Gly Ser Asn Val Asp Gly Ile Glu 1 5 10 347 11 PRT
Artificial
Synthetic Peptide 347 Ser Lys Glu Gly Ser Asn Val Glu Gly Ile Glu 1
5 10 348 11 PRT Artificial Synthetic Peptide 348 Ser Lys Glu Gly
Ser Asn Val Ser Gly Ile Glu 1 5 10 349 11 PRT Artificial Synthetic
Peptide 349 Ser Lys Glu Gly Ser Asn Val Thr Gly Ile Glu 1 5 10 350
11 PRT Artificial Synthetic Peptide 350 Ser Lys Glu Gly Thr Asn Val
Tyr Gly Ile Glu 1 5 10 351 11 PRT Artificial Synthetic Peptide 351
Ser Lys Glu Gly Thr Asn Val Asp Gly Ile Glu 1 5 10 352 11 PRT
Artificial Synthetic Peptide 352 Ser Lys Glu Gly Thr Asn Val Glu
Gly Ile Glu 1 5 10 353 11 PRT Artificial Synthetic Peptide 353 Ser
Lys Glu Gly Thr Asn Val Ser Gly Ile Glu 1 5 10 354 11 PRT
Artificial Synthetic Peptide 354 Ser Lys Glu Gly Thr Asn Val Thr
Gly Ile Glu 1 5 10 355 11 PRT Artificial Synthetic Peptide 355 Thr
Lys Glu Gly Tyr Asn Val Tyr Gly Ile Glu 1 5 10 356 11 PRT
Artificial Synthetic Peptide 356 Thr Lys Glu Gly Tyr Asn Val Asp
Gly Ile Glu 1 5 10 357 11 PRT Artificial Synthetic Peptide 357 Thr
Lys Glu Gly Tyr Asn Val Glu Gly Ile Glu 1 5 10 358 11 PRT
Artificial Synthetic Peptide 358 Thr Lys Glu Gly Tyr Asn Val Ser
Gly Ile Glu 1 5 10 359 11 PRT Artificial Synthetic Peptide 359 Thr
Lys Glu Gly Tyr Asn Val Thr Gly Ile Glu 1 5 10 360 11 PRT
Artificial Synthetic Peptide 360 Thr Lys Glu Gly Asp Asn Val Tyr
Gly Ile Glu 1 5 10 361 11 PRT Artificial Synthetic Peptide 361 Thr
Lys Glu Gly Asp Asn Val Asp Gly Ile Glu 1 5 10 362 11 PRT
Artificial Synthetic Peptide 362 Thr Lys Glu Gly Asp Asn Val Glu
Gly Ile Glu 1 5 10 363 11 PRT Artificial Synthetic Peptide 363 Thr
Lys Glu Gly Asp Asn Val Ser Gly Ile Glu 1 5 10 364 11 PRT
Artificial Synthetic Peptide 364 Thr Lys Glu Gly Asp Asn Val Thr
Gly Ile Glu 1 5 10 365 11 PRT Artificial Synthetic Peptide 365 Thr
Lys Glu Gly Glu Asn Val Tyr Gly Ile Glu 1 5 10 366 11 PRT
Artificial Synthetic Peptide 366 Thr Lys Glu Gly Glu Asn Val Asp
Gly Ile Glu 1 5 10 367 11 PRT Artificial Synthetic Peptide 367 Thr
Lys Glu Gly Glu Asn Val Glu Gly Ile Glu 1 5 10 368 11 PRT
Artificial Synthetic Peptide 368 Thr Lys Glu Gly Glu Asn Val Ser
Gly Ile Glu 1 5 10 369 11 PRT Artificial Synthetic Peptide 369 Thr
Lys Glu Gly Glu Asn Val Thr Gly Ile Glu 1 5 10 370 11 PRT
Artificial Synthetic Peptide 370 Thr Lys Glu Gly Ser Asn Val Tyr
Gly Ile Glu 1 5 10 371 11 PRT Artificial Synthetic Peptide 371 Thr
Lys Glu Gly Ser Asn Val Asp Gly Ile Glu 1 5 10 372 11 PRT
Artificial Synthetic Peptide 372 Thr Lys Glu Gly Ser Asn Val Glu
Gly Ile Glu 1 5 10 373 11 PRT Artificial Synthetic Peptide 373 Thr
Lys Glu Gly Ser Asn Val Ser Gly Ile Glu 1 5 10 374 11 PRT
Artificial Synthetic Peptide 374 Thr Lys Glu Gly Ser Asn Val Thr
Gly Ile Glu 1 5 10 375 11 PRT Artificial Synthetic Peptide 375 Thr
Lys Glu Gly Thr Asn Val Tyr Gly Ile Glu 1 5 10 376 11 PRT
Artificial Synthetic Peptide 376 Thr Lys Glu Gly Thr Asn Val Asp
Gly Ile Glu 1 5 10 377 11 PRT Artificial Synthetic Peptide 377 Thr
Lys Glu Gly Thr Asn Val Glu Gly Ile Glu 1 5 10 378 11 PRT
Artificial Synthetic Peptide 378 Thr Lys Glu Gly Thr Asn Val Ser
Gly Ile Glu 1 5 10 379 11 PRT Artificial Synthetic Peptide 379 Thr
Lys Glu Gly Thr Asn Val Thr Gly Ile Glu 1 5 10 380 11 PRT
Artificial Synthetic Peptide 380 Tyr Arg Glu Gly Tyr Asn Val Asp
Gly Ile Glu 1 5 10 381 11 PRT Artificial Synthetic Peptide 381 Tyr
Arg Glu Gly Tyr Asn Val Glu Gly Ile Glu 1 5 10 382 11 PRT
Artificial Synthetic Peptide 382 Tyr Arg Glu Gly Tyr Asn Val Ser
Gly Ile Glu 1 5 10 383 11 PRT Artificial Synthetic Peptide 383 Tyr
Arg Glu Gly Tyr Asn Val Thr Gly Ile Glu 1 5 10 384 11 PRT
Artificial Synthetic Peptide 384 Tyr Arg Glu Gly Asp Asn Val Tyr
Gly Ile Glu 1 5 10 385 11 PRT Artificial Synthetic Peptide 385 Tyr
Arg Glu Gly Asp Asn Val Asp Gly Ile Glu 1 5 10 386 11 PRT
Artificial Synthetic Peptide 386 Tyr Arg Glu Gly Asp Asn Val Glu
Gly Ile Glu 1 5 10 387 11 PRT Artificial Synthetic Peptide 387 Tyr
Arg Glu Gly Asp Asn Val Ser Gly Ile Glu 1 5 10 388 11 PRT
Artificial Synthetic Peptide 388 Tyr Arg Glu Gly Asp Asn Val Thr
Gly Ile Glu 1 5 10 389 11 PRT Artificial Synthetic Peptide 389 Tyr
Arg Glu Gly Glu Asn Val Tyr Gly Ile Glu 1 5 10 390 11 PRT
Artificial Synthetic Peptide 390 Tyr Arg Glu Gly Glu Asn Val Asp
Gly Ile Glu 1 5 10 391 11 PRT Artificial Synthetic Peptide 391 Tyr
Arg Glu Gly Glu Asn Val Glu Gly Ile Glu 1 5 10 392 11 PRT
Artificial Synthetic Peptide 392 Tyr Arg Glu Gly Glu Asn Val Ser
Gly Ile Glu 1 5 10 393 11 PRT Artificial Synthetic Peptide 393 Tyr
Arg Glu Gly Glu Asn Val Thr Gly Ile Glu 1 5 10 394 11 PRT
Artificial Synthetic Peptide 394 Tyr Arg Glu Gly Ser Asn Val Tyr
Gly Ile Glu 1 5 10 395 11 PRT Artificial Synthetic Peptide 395 Tyr
Arg Glu Gly Ser Asn Val Asp Gly Ile Glu 1 5 10 396 11 PRT
Artificial Synthetic Peptide 396 Tyr Arg Glu Gly Ser Asn Val Glu
Gly Ile Glu 1 5 10 397 11 PRT Artificial Synthetic Peptide 397 Tyr
Arg Glu Gly Ser Asn Val Ser Gly Ile Glu 1 5 10 398 11 PRT
Artificial Synthetic Peptide 398 Tyr Arg Glu Gly Ser Asn Val Thr
Gly Ile Glu 1 5 10 399 11 PRT Artificial Synthetic Peptide 399 Tyr
Arg Glu Gly Thr Asn Val Tyr Gly Ile Glu 1 5 10 400 11 PRT
Artificial Synthetic Peptide 400 Tyr Arg Glu Gly Thr Asn Val Asp
Gly Ile Glu 1 5 10 401 11 PRT Artificial Synthetic Peptide 401 Tyr
Arg Glu Gly Thr Asn Val Glu Gly Ile Glu 1 5 10 402 11 PRT
Artificial Synthetic Peptide 402 Tyr Arg Glu Gly Thr Asn Val Ser
Gly Ile Glu 1 5 10 403 11 PRT Artificial Synthetic Peptide 403 Tyr
Arg Glu Gly Thr Asn Val Thr Gly Ile Glu 1 5 10 404 11 PRT
Artificial Synthetic Peptide 404 Asp Arg Glu Gly Tyr Asn Val Tyr
Gly Ile Glu 1 5 10 405 11 PRT Artificial Synthetic Peptide 405 Asp
Arg Glu Gly Tyr Asn Val Asp Gly Ile Glu 1 5 10 406 11 PRT
Artificial Synthetic Peptide 406 Asp Arg Glu Gly Tyr Asn Val Glu
Gly Ile Glu 1 5 10 407 11 PRT Artificial Synthetic Peptide 407 Asp
Arg Glu Gly Tyr Asn Val Ser Gly Ile Glu 1 5 10 408 11 PRT
Artificial Synthetic Peptide 408 Asp Arg Glu Gly Tyr Asn Val Thr
Gly Ile Glu 1 5 10 409 11 PRT Artificial Synthetic Peptide 409 Asp
Arg Glu Gly Asp Asn Val Tyr Gly Ile Glu 1 5 10 410 11 PRT
Artificial Synthetic Peptide 410 Asp Arg Glu Gly Asp Asn Val Asp
Gly Ile Glu 1 5 10 411 11 PRT Artificial Synthetic Peptide 411 Asp
Arg Glu Gly Asp Asn Val Glu Gly Ile Glu 1 5 10 412 11 PRT
Artificial Synthetic Peptide 412 Asp Arg Glu Gly Asp Asn Val Ser
Gly Ile Glu 1 5 10 413 11 PRT Artificial Synthetic Peptide 413 Asp
Arg Glu Gly Asp Asn Val Thr Gly Ile Glu 1 5 10 414 11 PRT
Artificial Synthetic Peptide 414 Asp Arg Glu Gly Glu Asn Val Tyr
Gly Ile Glu 1 5 10 415 11 PRT Artificial Synthetic Peptide 415 Asp
Arg Glu Gly Glu Asn Val Asp Gly Ile Glu 1 5 10 416 11 PRT
Artificial Synthetic Peptide 416 Asp Arg Glu Gly Glu Asn Val Glu
Gly Ile Glu 1 5 10 417 11 PRT Artificial Synthetic Peptide 417 Asp
Arg Glu Gly Glu Asn Val Ser Gly Ile Glu 1 5 10 418 11 PRT
Artificial Synthetic Peptide 418 Asp Arg Glu Gly Glu Asn Val Thr
Gly Ile Glu 1 5 10 419 11 PRT Artificial Synthetic Peptide 419 Asp
Arg Glu Gly Ser Asn Val Tyr Gly Ile Glu 1 5 10 420 11 PRT
Artificial Synthetic Peptide 420 Asp Arg Glu Gly Ser Asn Val Asp
Gly Ile Glu 1 5 10 421 11 PRT Artificial Synthetic Peptide 421 Asp
Arg Glu Gly Ser Asn Val Glu Gly Ile Glu 1 5 10 422 11 PRT
Artificial Synthetic Peptide 422 Asp Arg Glu Gly Ser Asn Val Ser
Gly Ile Glu 1 5 10 423 11 PRT Artificial Synthetic Peptide 423 Asp
Arg Glu Gly Ser Asn Val Thr Gly Ile Glu 1 5 10 424 11 PRT
Artificial Synthetic Peptide 424 Asp Arg Glu Gly Thr Asn Val Tyr
Gly Ile Glu 1 5 10 425 11 PRT Artificial Synthetic Peptide 425 Asp
Arg Glu Gly Thr Asn Val Asp Gly Ile Glu 1 5 10 426 11 PRT
Artificial Synthetic Peptide 426 Asp Arg Glu Gly Thr Asn Val Glu
Gly Ile Glu 1 5 10 427 11 PRT Artificial Synthetic Peptide 427 Asp
Arg Glu Gly Thr Asn Val Ser Gly Ile Glu 1 5 10 428 11 PRT
Artificial Synthetic Peptide 428 Asp Arg Glu Gly Thr Asn Val Thr
Gly Ile Glu 1 5 10 429 11 PRT Artificial Synthetic Peptide 429 Glu
Arg Glu Gly Tyr Asn Val Tyr Gly Ile Glu 1 5 10 430 11 PRT
Artificial Synthetic Peptide 430 Glu Arg Glu Gly Tyr Asn Val Asp
Gly Ile Glu 1 5 10 431 11 PRT Artificial Synthetic Peptide 431 Glu
Arg Glu Gly Tyr Asn Val Glu Gly Ile Glu 1 5 10 432 11 PRT
Artificial Synthetic Peptide 432 Glu Arg Glu Gly Tyr Asn Val Ser
Gly Ile Glu 1 5 10 433 11 PRT Artificial Synthetic Peptide 433 Glu
Arg Glu Gly Tyr Asn Val Thr Gly Ile Glu 1 5 10 434 11 PRT
Artificial Synthetic Peptide 434 Glu Arg Glu Gly Asp Asn Val Tyr
Gly Ile Glu 1 5 10 435 11 PRT Artificial Synthetic Peptide 435 Glu
Arg Glu Gly Asp Asn Val Asp Gly Ile Glu 1 5 10 436 11 PRT
Artificial Synthetic Peptide 436 Glu Arg Glu Gly Asp Asn Val Glu
Gly Ile Glu 1 5 10 437 11 PRT Artificial Synthetic Peptide 437 Glu
Arg Glu Gly Asp Asn Val Ser Gly Ile Glu 1 5 10 438 11 PRT
Artificial Synthetic Peptide 438 Glu Arg Glu Gly Asp Asn Val Thr
Gly Ile Glu 1 5 10 439 11 PRT Artificial Synthetic Peptide 439 Glu
Arg Glu Gly Glu Asn Val Tyr Gly Ile Glu 1 5 10 440 11 PRT
Artificial Synthetic Peptide 440 Glu Arg Glu Gly Glu Asn Val Asp
Gly Ile Glu 1 5 10 441 11 PRT Artificial Synthetic Peptide 441 Glu
Arg Glu Gly Glu Asn Val Glu Gly Ile Glu 1 5 10 442 11 PRT
Artificial Synthetic Peptide 442 Glu Arg Glu Gly Glu Asn Val Ser
Gly Ile Glu 1 5 10 443 11 PRT Artificial Synthetic Peptide 443 Glu
Arg Glu Gly Glu Asn Val Thr Gly Ile Glu 1 5 10 444 11 PRT
Artificial Synthetic Peptide 444 Glu Arg Glu Gly Ser Asn Val Tyr
Gly Ile Glu 1 5 10 445 11 PRT Artificial Synthetic Peptide 445 Glu
Arg Glu Gly Ser Asn Val Asp Gly Ile Glu 1 5 10 446 11 PRT
Artificial Synthetic Peptide 446 Glu Arg Glu Gly Ser Asn Val Glu
Gly Ile Glu 1 5 10 447 11 PRT Artificial Synthetic Peptide 447 Glu
Arg Glu Gly Ser Asn Val Ser Gly Ile Glu 1 5 10 448 11 PRT
Artificial Synthetic Peptide 448 Glu Arg Glu Gly Ser Asn Val Thr
Gly Ile Glu 1 5 10 449 11 PRT Artificial Synthetic Peptide 449 Glu
Arg Glu Gly Thr Asn Val Tyr Gly Ile Glu 1 5 10 450 11 PRT
Artificial Synthetic Peptide 450 Glu Arg Glu Gly Thr Asn Val Asp
Gly Ile Glu 1 5 10 451 11 PRT Artificial Synthetic Peptide 451 Glu
Arg Glu Gly Thr Asn Val Glu Gly Ile Glu 1 5 10 452 11 PRT
Artificial Synthetic Peptide 452 Glu Arg Glu Gly Thr Asn Val Ser
Gly Ile Glu 1 5 10 453 11 PRT Artificial Synthetic Peptide 453 Glu
Arg Glu Gly Thr Asn Val Thr Gly Ile Glu 1 5 10 454 11 PRT
Artificial Synthetic Peptide 454 Ser Arg Glu Gly Tyr Asn Val Tyr
Gly Ile Glu 1 5 10 455 11 PRT Artificial Synthetic Peptide 455 Ser
Arg Glu Gly Tyr Asn Val Asp Gly Ile Glu 1 5 10 456 11 PRT
Artificial Synthetic Peptide 456 Ser Arg Glu Gly Tyr Asn Val Glu
Gly Ile Glu 1 5 10 457 11 PRT Artificial Synthetic Peptide 457 Ser
Arg Glu Gly Tyr Asn Val Ser Gly Ile Glu 1 5 10 458 11 PRT
Artificial Synthetic Peptide 458 Ser Arg Glu Gly Tyr Asn Val Thr
Gly Ile Glu 1 5 10 459 11 PRT Artificial Synthetic Peptide 459 Ser
Arg Glu Gly Asp Asn Val Tyr Gly Ile Glu 1 5 10 460 11 PRT
Artificial Synthetic Peptide 460 Ser Arg Glu Gly Asp Asn Val Asp
Gly Ile Glu 1 5 10 461 11 PRT Artificial Synthetic Peptide 461 Ser
Arg Glu Gly Asp Asn Val Glu Gly Ile Glu 1 5 10 462 11 PRT
Artificial Synthetic Peptide 462 Ser Arg Glu Gly Asp Asn Val Ser
Gly Ile Glu 1 5 10 463 11 PRT Artificial Synthetic Peptide 463 Ser
Arg Glu Gly Asp Asn Val Thr Gly Ile Glu 1 5 10 464 11 PRT
Artificial Synthetic Peptide 464 Ser Arg Glu Gly Glu Asn Val Tyr
Gly Ile Glu 1 5 10 465 11 PRT Artificial Synthetic Peptide 465 Ser
Arg Glu Gly Glu Asn Val Asp Gly Ile Glu 1 5 10 466 11 PRT
Artificial Synthetic Peptide 466 Ser Arg Glu Gly Glu Asn Val Glu
Gly Ile Glu 1 5 10 467 11 PRT Artificial Synthetic Peptide 467 Ser
Arg Glu Gly Glu Asn Val Ser Gly Ile Glu 1 5 10 468 11 PRT
Artificial Synthetic Peptide 468 Ser Arg Glu Gly Glu Asn Val Thr
Gly Ile Glu 1 5 10 469 11 PRT Artificial Synthetic Peptide 469 Ser
Arg Glu Gly Ser Asn Val Tyr Gly Ile Glu 1 5 10 470 11 PRT
Artificial Synthetic Peptide 470 Ser Arg Glu Gly Ser Asn Val Asp
Gly Ile Glu 1 5 10 471 11 PRT Artificial Synthetic Peptide 471 Ser
Arg Glu Gly Ser Asn Val Glu Gly Ile Glu 1 5 10 472 11 PRT
Artificial Synthetic Peptide 472 Ser Arg Glu Gly Ser Asn Val Ser
Gly Ile Glu 1 5 10 473 11 PRT Artificial Synthetic Peptide 473 Ser
Arg Glu Gly Ser Asn Val Thr Gly Ile Glu 1 5 10 474 11 PRT
Artificial Synthetic Peptide 474 Ser Arg Glu Gly Thr Asn Val Tyr
Gly Ile Glu 1 5 10 475 11 PRT Artificial Synthetic Peptide 475 Ser
Arg Glu Gly Thr Asn Val Asp Gly Ile Glu 1 5 10 476 11 PRT
Artificial Synthetic Peptide 476 Ser Arg Glu Gly Thr Asn Val Glu
Gly Ile Glu 1 5 10 477 11 PRT Artificial Synthetic Peptide 477 Ser
Arg Glu Gly Thr Asn Val Ser Gly Ile Glu 1 5 10 478 11 PRT
Artificial Synthetic Peptide 478 Ser Arg Glu Gly Thr Asn Val Thr
Gly Ile Glu 1 5 10 479 11 PRT Artificial Synthetic Peptide 479 Thr
Arg Glu Gly Tyr Asn Val Tyr Gly Ile Glu 1 5 10 480 11 PRT
Artificial Synthetic Peptide 480 Thr Arg Glu Gly Tyr Asn Val Asp
Gly Ile Glu 1 5 10 481 11 PRT Artificial Synthetic Peptide 481 Thr
Arg Glu Gly Tyr Asn Val Glu Gly Ile Glu 1 5 10 482 11 PRT
Artificial Synthetic Peptide 482 Thr Arg Glu Gly Tyr Asn Val Ser
Gly Ile Glu 1 5 10 483 11 PRT Artificial Synthetic Peptide 483 Thr
Arg Glu Gly Tyr Asn Val Thr Gly Ile Glu 1 5 10 484 11 PRT
Artificial Synthetic Peptide 484 Thr Arg Glu Gly Asp Asn Val Tyr
Gly Ile Glu 1 5 10 485 11 PRT Artificial Synthetic Peptide 485 Thr
Arg Glu Gly Asp Asn Val Asp Gly Ile Glu 1 5 10 486 11 PRT
Artificial Synthetic Peptide 486 Thr Arg Glu Gly Asp Asn Val Glu
Gly Ile Glu 1 5 10 487 11 PRT Artificial Synthetic Peptide 487 Thr
Arg Glu Gly Asp Asn Val Ser Gly Ile Glu 1 5 10 488 11 PRT
Artificial Synthetic Peptide 488 Thr Arg Glu Gly Asp Asn Val Thr
Gly Ile Glu 1 5 10 489 11 PRT Artificial Synthetic Peptide 489 Thr
Arg Glu Gly Glu Asn Val Tyr Gly Ile Glu 1 5 10 490 11 PRT
Artificial Synthetic Peptide 490 Thr Arg Glu Gly Glu Asn Val Asp
Gly
Ile Glu 1 5 10 491 11 PRT Artificial Synthetic Peptide 491 Thr Arg
Glu Gly Glu Asn Val Glu Gly Ile Glu 1 5 10 492 11 PRT Artificial
Synthetic Peptide 492 Thr Arg Glu Gly Glu Asn Val Ser Gly Ile Glu 1
5 10 493 11 PRT Artificial Synthetic Peptide 493 Thr Arg Glu Gly
Glu Asn Val Thr Gly Ile Glu 1 5 10 494 11 PRT Artificial Synthetic
Peptide 494 Thr Arg Glu Gly Ser Asn Val Tyr Gly Ile Glu 1 5 10 495
11 PRT Artificial Synthetic Peptide 495 Thr Arg Glu Gly Ser Asn Val
Asp Gly Ile Glu 1 5 10 496 11 PRT Artificial Synthetic Peptide 496
Thr Arg Glu Gly Ser Asn Val Glu Gly Ile Glu 1 5 10 497 11 PRT
Artificial Synthetic Peptide 497 Thr Arg Glu Gly Ser Asn Val Ser
Gly Ile Glu 1 5 10 498 11 PRT Artificial Synthetic Peptide 498 Thr
Arg Glu Gly Ser Asn Val Thr Gly Ile Glu 1 5 10 499 11 PRT
Artificial Synthetic Peptide 499 Thr Arg Glu Gly Thr Asn Val Tyr
Gly Ile Glu 1 5 10 500 11 PRT Artificial Synthetic Peptide 500 Thr
Arg Glu Gly Thr Asn Val Asp Gly Ile Glu 1 5 10 501 11 PRT
Artificial Synthetic Peptide 501 Thr Arg Glu Gly Thr Asn Val Glu
Gly Ile Glu 1 5 10 502 11 PRT Artificial Synthetic Peptide 502 Thr
Arg Glu Gly Thr Asn Val Ser Gly Ile Glu 1 5 10 503 11 PRT
Artificial Synthetic Peptide 503 Thr Arg Glu Gly Thr Asn Val Thr
Gly Ile Glu 1 5 10 504 22 PRT Artificial Synthetic Peptide 504 Tyr
Gly Arg Lys Lys Arg Arg Gln Arg Arg Arg Tyr Lys Glu Gly Tyr 1 5 10
15 Asn Val Tyr Gly Ile Glu 20 505 22 PRT Artificial Synthetic
Peptide 505 Tyr Gly Arg Lys Lys Arg Arg Gln Arg Arg Arg Ala Lys Glu
Gly Ala 1 5 10 15 Asn Val Ala Gly Ile Glu 20 506 40 PRT Artificial
Mutant HA-GluR2 delta 834-843 506 Lys Arg Met Lys Val Ala Lys Asn
Pro Gln Asn Ile Asn Pro Ser Ser 1 5 10 15 Ser Gln Asn Ser Gln Asn
Phe Ala Thr Tyr Lys Glu Gly Tyr Asn Val 20 25 30 Tyr Gly Ile Glu
Ser Val Lys Ile 35 40 507 41 PRT Artificial Mutant HA-GluR2 delta
844-853 507 Ile Glu Phe Cys Tyr Lys Ser Arg Ala Glu Ala Asn Ile Asn
Pro Ser 1 5 10 15 Ser Ser Gln Asn Ser Gln Asn Phe Ala Thr Tyr Lys
Glu Gly Thr Asn 20 25 30 Val Tyr Gly Ile Glu Ser Val Lys Ile 35 40
508 21 PRT Artificial Mutant GluR2 delta 854 508 Ile Glu Phe Cys
Tyr Lys Ser Arg Ala Glu Ala Lys Arg Met Lys Val 1 5 10 15 Ala Lys
Asn Pro Gln 20 509 36 PRT Artificial Mutant HA-GluR2 delta 869 509
Ile Glu Phe Cys Tyr Lys Ser Arg Ala Glu Ala Lys Arg Met Lys Val 1 5
10 15 Ala Lys Asn Pro Gln Asn Ile Asn Pro Ser Ser Ser Gln Asn Ser
Gln 20 25 30 Asn Phe Ala Thr 35 510 47 PRT Artificial Mutant
HA-GluR2 delta 880 510 Ile Glu Phe Cys Tyr Lys Ser Arg Ala Glu Ala
Lys Arg Met Lys Val 1 5 10 15 Ala Lys Asn Pro Gln Asn Ile Asn Pro
Ser Ser Ser Gln Asn Ser Gln 20 25 30 Asn Phe Ala Thr Tyr Lys Glu
Gly Tyr Asn Val Tyr Gly Ile Glu 35 40 45
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