Method of treating dopaminergic and GABA-nergic disorders

Abstract

It is shown here that hedgehog proteins possess novel activities beyond phenotype specification. Using cultures derived from the embryonic day 14.5 (E14.5) rat ventral mesencephalon, we show that hedgehog is also trophic for dopaminergic neurons. Interestingly, hedgehog not only promotes dopaminergic neuron survival, but also promotes the survival of midbrain GABA-immunoreactive (GABA-ir) neurons.

1 - 48 . (canceled)  
     
     
         49 . A method for treating or preventing Parkinson's disease or Huntington's disease comprising administering to a patient in need thereof a therapeutically effective amount of a ptc therapeutic.  
     
     
         50 . The method of  claim 50 , wherein the ptc therapeutic binds to patched and mimics hedgehog-mediated patched signal transduction.  
     
     
         51 . The method of  claim 50 , wherein the ptc therapeutic is a small organic molecule.  
     
     
         52 . The method of  claim 50 , wherein the ptc therapeutic mimics hedgehog-mediated patched signal transduction by altering the localization, protein-protein binding and/or enzymatic activity of an intracellular protein involved in a patched signal pathway.  
     
     
         53 . The method of  claim 50 , wherein the ptc therapeutic is an antisense construct which inhibits the expression of a protein which is involved in the signal transduction pathway of patched and the expression of which antagonizes hedgehog-mediated signals.  
     
     
         54 . The method of  claim 53 , wherein the antisense construct is an oligonucleotide of about 20-30 nucleotides in length and having a GC content of at least 50 percent.  
     
     
         55 . The method of  claim 54 , wherein the antisense oligonucleotide is selected from the group consisting of:  
       
         
           
                 
                 
                 
               
                     
                     
                 
                     
                   5′-GTCCTGGCGCCGCCGCCGCCGTCGCC; 
                     
                 
                     
                     
                 
                     
                   5′-TTCCGATGACCGGCCTTTCGCGGTGA; 
                 
                     
                   and 
                 
                     
                     
                 
                     
                   5′-GTGCACGGAAAGGTGCAGGCCACACT. 
                 
                     
                     
                 
             
                
                
                
                
                
                
                
                
               
            
           
         
       
     
     
         56 . The method of  claim 51 , wherein the ptc therapeutic is an inhibitor of protein kinase A.  
     
     
         57 . The method of  claim 56 , wherein the PKA inhibitor is a 5-isoquinolinesulfonamide.  
     
     
         58 . The method of  claim 56 , wherein the PKA inhibitor is represented in the general formula:  
       
         
           
           
               
               
           
         
       
       wherein, 
 R 1  and R 2  each can independently represent hydrogen, and as valence and stability permit a lower alkyl, a lower alkenyl, a lower alkynyl, a carbonyl (such as a carboxyl, an ester, a formate, or a ketone), a thiocarbonyl (such as a thioester, a thioacetate, or a thioformate), an amino, an acylamino, an amido, a cyano, a nitro, an azido, a sulfate, a sulfonate, a sulfonamido, —(CH 2 ) m , —R 8 , —(CH 2 ) m —OH, —(CH 2 ) m —O-lower alkyl, —(CH 2 ) n —O-lower alkenyl, —(CH 2 ) m —O—(CH 2 ) m —R 8 , —(CH 2 ) m —SH, —(CH 2 ) m —S-lower alkyl, —(CH 2 ) m —S-lower alkenyl, —(CH 2 ) n —S—(CH 2 ) m —R 8 , or  
 R 1  and R 2  taken together with N form a heterocycle (substituted or unsubstituted);  
 R 3  is absent or represents one or more substitutions to the isoquinoline ring such as a lower alkyl, a lower alkenyl, a lower alkynyl, a carbonyl (such as a carboxyl, an ester, a formate, or a ketone), a thiocarbonyl (such as a thioester, a thioacetate, or a thioformate), an amino, an acylamino, an amido, a cyano, a nitro, an azido, a sulfate, a sulfonate, a sulfonamido, —(CH 2 ) m , —R 8 , —(CH 2 ) m —OH, —(CH 2 ) m —O-lower alkyl, —(CH 2 ) m —O-lower alkenyl, —(CH 2 ) n —O—(CH 2 ) m —R 8 , —(CH 2 ) m —SH, —(CH 2 ) m —S-lower alkyl, —(CH 2 ) m —S-lower alkenyl, —(CH 2 ) n —S—(CH 2 ) m —R 8 ;  
 R 8  represents a substituted or unsubstituted aryl, aralkyl, cycloalkyl, cycloalkenyl, or heterocycle; and  
 n and m are independently for each occurrence zero or an integer in the range of 1 to 6.  
 
     
     
         59 . The method of  claim 50 , wherein patient is being treated prophylactically.  
     
     
         60 . An isolated and/or recombinantly produced polypeptide comprising an amino acid sequence which is at least 95 percent identical to a sequence represented by SEQ ID. NO. 16 or 17, or a bioactive extracellular fragment thereof.  
     
     
         61 . The polypeptide of  claim 60 , formulated in a pharmaceutically acceptable carrier.  
     
     
         62 . The polypeptide of  claim 60 , wherein the polypeptide is purified to at least 80% by dry weight.  
     
     
         63 . An isolated nucleic acid encoding a polypeptide comprising a hedgehog amino acid sequence which is at least 95 percent identical to a hedgehog protein selected from the group consisting of SEQ ID No:16 and SEQ ID No:17, and fragments thereof, which hedgehog amino acid sequence (i) binds to a patched protein, (ii) regulates differentiation of neuronal cells, (iii) regulates survival of differentiated neuronal cells, (iv) regulates proliferation of chondrocytes, (v) regulates proliferation of testicular germ line cells, or (vi) functionally replaces drosopholia hedgehog in transgenic  drosophila  fly, or a combination thereof.  
     
     
         64 . An isolated nucleic acid encoding a polypeptide having a hedgehog amino acid sequence encoded by a nucleic acid which hybridizes under stringent conditions to a nucleic acid sequence selected from the group consisting of SEQ ID No:7 and SEQ ID No:8, which hedgehog amino acid sequence of the polypeptide corresponds to a natural proteolytic product of a hedgehog protein and (i) binds to a patched protein, (ii) regulates differentiation of neuronal cells, (iii) regulates survival of differentiated neuronal cells, (iv) regulates proliferation of chondrocytes, (v) regulates proliferation of testicular germ line cells, or (vi) functionally replaces drosopholia hedgehog in transgenic  drosophila  fly, or a combination thereof.  
     
     
         65 . An expression vector, capable of replicating in at least one of a prokaryotic cell and eukaryotic cell, comprising the nucleic acid of  claim 64 .  
     
     
         66 . A host cell transfected with the expression vector of  claim 65  and expressing said recombinant polypeptide.  
     
     
         67 . A method of producing a recombinant hedgehog polypeptide comprising culturing the cell of  claim 66  in a cell culture medium to express a hedgehog polypeptide and isolating said hedgehog polypeptide from said cell culture.  
     
     
         68 . A probe/primer comprising a substantially purified oligonucleotide, said oligonucleotide containing a region of nucleotide sequence which hybridizes under stringent conditions to at least 10 consecutive nucleotides of sense or antisense sequence of SEQ ID No. 7 or 8, or naturally occuring mutants thereof.  
     
     
         69 . The probe/primer of  claim 68 , wherein the probe/primer further comprises a label group attached thereto and able to be detected.  
     
     
         70 . A test kit for detecting cells which contain a hedgehog mRNA transcript, comprising a probe/primer of  claim 69 .  
     
     
         71 . A purified preparation of an antisense nucleic acid which specifically hybridizes to and inhibits expression of a gene encoding a human Ihh or Dhh hedgehog protein under physiological conditions, which nucleic acid is at least one of (i) a synthetic oligonucleotide, (ii) single-stranded, (iii) linear, (iv) 20 to 50 nucleotides in length, and (v) a DNA analog resistant to nuclease degradation.  
     
     
         72 . The preparation of  claim 71 , which antisense nucleic acid is a DNA analog resistant to nuclease degradation.  
     
     
         73 . A method for treating or preventing a disorder selected from any of ischemic injury to the brain, stroke and cerebral ischemia, and amyotrophic lateral sclerosis (ALS), comprising administering an effective amount of a therapeutic comprising a polypeptide encoded by a nucleic acid that hybridizes under stringent conditions, including a wash step of 0.2×SSC at 65° C., to a nucleic acid represented in any one of SEQ ID Nos: 1-9.  
     
     
         74 . A method for treating or preventing a disorder selected from any of ischemic injury to the brain, stroke and cerebral ischemia, and amyotrophic lateral sclerosis (ALS), comprising administering an effective amount of a ptc therapeutic, wherein said ptc therapeutic is an inhibitor of protein kinase A.  
     
     
         75 . The method of  claim 74 , wherein the inhibitor of protein kinase A is a 5-isoquinolinesulfonamide.  
     
     
         76 . The method of  claim 74 , wherein the inhibitor of protein kinase A is represented in the general formula:  
       
         
           
           
               
               
           
         
       
       wherein, 
 R 1  and R 2  each can independently represent hydrogen, and as valence and stability permit a lower alkyl, a lower alkenyl, a lower alkynyl, a carbonyl (such as a carboxyl, an ester, a formate, or a ketone), a thiocarbonyl (such as a thioester, a thioacetate, or a thioformate), an amino, an acylamino, an amido, a cyano, a nitro, an azido, a sulfate, a sulfonate, a sulfonamido, —(CH 2 ) m , —R 8 , —(CH 2 ) m —OH, —(CH 2 ) m —O-lower alkyl, —(CH 2 ) m —O-lower alkenyl, —(CH 2 ) n —O—(CH 2 ) m —R 8 , —(CH 2 ) m —SH, —(CH 2 ) m —S-lower alkyl, —(CH 2 ) m —S-lower alkenyl, —(CH 2 ) n —S—(CH 2 ) m —R 8 , or  
 R 1  and R 2  taken together with N form a heterocycle (substituted or unsubstituted);  
 R 3  is absent or represents one or more substitutions to the isoquinoline ring such as a lower alkyl, a lower alkenyl, a lower alkynyl, a carbonyl (such as a carboxyl, an ester, a formate, or a ketone), a thiocarbonyl (such as a thioester, a thioacetate, or a thioformate), an amino, an acylamino, an amido, a cyano, a nitro, an azido, a sulfate, a sulfonate, a sulfonamido, —(CH 2 ) m , —R 8 , —(CH 2 ) m —OH, —(CH 2 ) m —O-lower alkyl, —(CH 2 ) m —O-lower alkenyl, —(CH 2 ) n —O—(CH 2 ) m —R 8 , —(CH 2 ) m —SH, —(CH 2 ) m —S-lower alkyl, —(CH 2 ) m —S-lower alkenyl, —(CH 2 ) n —S—(CH 2 ) m —R 8 ;  
 R 8  represents a substituted or unsubstituted aryl, aralkyl, cycloalkyl, cycloalkenyl, or heterocycle; and  
 n and m are independently for each occurrence zero or an integer in the range of 1 to 6.  
 
     
     
         77 . The method of  claim 74 , wherein the inhibitor of protein kinase A is a cyclic AMP analog.  
     
     
         78 . The method of  claim 74 , wherein the inhibitor of protein kinase A is selected from N-[2-((p-bromocinnamyl)amino)ethyl]-5-isoquinolinesulfonamide, 1-(5-isoquinoline-sulfonyl)-2-methylpiperazine, KT5720, 8-bromo-cAMP, dibutyryl-cAMP, and PKA Heat Stable Inhibitor isoform a.  
     
     
         79 . The method of  claim 74 , wherein the ptc therapeutic binds to patched and mimics hedgehog-mediated patched signal transduction.  
     
     
         80 . The method of  claim 74 , wherein the binding of the ptc therapeutic to patched results in upregulation of patched and/or gli expression.  
     
     
         81 . The method of  claim 74 , wherein the ptc therapeutic mimics hedgehog mediated patched signal transduction by altering the localization, protein-protein binding and/or enzymatic activity of an intracellular protein involved in a patched signal pathway.  
     
     
         82 . The method of  claim 74 , wherein the ptc therapeutic alters the level of expression of a hedgehog protein, a patched protein or a protein involved in the intracellular signal transduction pathway of patched.  
     
     
         83 . The method of  claim 74 , wherein a patient is being treated prophylactically.