Methods for modulating neuronal responses

Abstract

The invention provides, in part, methods and reagents for modulating neuronal apoptosis and for modulating synaptic plasticity.

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 1 -X 1 -X 2 -E-G-X 3 -N-V-X 4 -G-Z 2 ;  (I)  
       wherein 
 X 1  is Y, D, E, S, or T;  
 X 2  is K or R;  
 X 3  is Y, D, E, S, or T;  
 X 4  is Y, D, E, S, or T;  
 Z, is H 2 N—, RHN— or, RRN—;  
 Z 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 1 -C 6 ) alkyl, (C 1 -C 6 ) alkenyl, (C 1 -C 6 ) alkynyl, substituted (C 1 -C 6 ) alkyl, substituted (C 1 -C 6 ) alkenyl, or substituted (C 1 -C 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 1 , X 3 , or X 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.