Compositions and methods for detecting compounds to treat a neurological disorder
The present invention generally provides compositions and methods that can be used to detect compounds that modulate the activity of at least one of the DJ-1, Parkin and Pink-1 genes.
1 . A method of identifying a compound that increases DJ-1 gene expression in a cell, the method comprising:
(a) contacting a cell expressing a DJ-1 promoter with a candidate compound; and
(b) detecting an increase in DJ-1 gene expression, wherein an increase in the level of DJ-1 gene expression in the cell relative to a reference, identifies the candidate compound as increasing DJ-1 expression.
2 . The method claim 1 , wherein the DJ-1 promoter is a heterologous promoter operably linked to a detectable reporter present in an expression vector.
3 - 5 . (canceled)
6 . The method of claim 1 , wherein the DJ-1 promoter is endogenously expressed in the cell.
7 - 8 . (canceled)
9 . A method of identifying a compound that increases Parkin gene expression in a cell, the method comprising:
(a) contacting a cell expressing a Parkin promoter with a candidate compound; and
(b) detecting Parkin gene expression, wherein an increase in the level of Parkin gene expression in the cell relative to a reference, identifies the candidate compound as a compound that increases Parkin gene expression.
10 . (canceled)
11 . The method of claim 10 , wherein the Parkin promoter is present in an expression vector operably linked to a detectable reporter.
12 - 14 . (canceled)
15 . The method claim 14 , wherein Parkin gene expression is detected by assaying mRNA level or protein level.
16 . (canceled)
17 . A method of identifying a compound that increases Pink-1 gene expression in a cell, the method comprising:
(a) contacting a cell expressing a Pink-1 promoter with a candidate compound; and
(b) detecting Pink-1 gene expression, wherein an increase in the level of Pink-1 gene expression in the cell relative to a reference, identifies the candidate compound as a compound that increases Pink-1 gene expression.
18 . The method claim 17 , wherein the Pink-1 promoter is a heterologous promoter operably linked to a detectable reporter present in an expression vector.
19 - 25 . (canceled)
26 . A method for identifying a compound that treats or prevents a neurological disorder in a subject, the method comprising:
(a) contacting a cell comprising a DJ-1, Parkin, or Pink-1 promoter operably linked to a detectable reporter with a candidate compound; and
(b) detecting a change in the expression of the reporter sequence relative to a control, thereby identifying a compound that treats or prevents a neurological disorder.
27 - 35 . (canceled)
36 . The method of claim 26 , wherein the candidate compound is a histone deacetylase inhibitor (HDAC).
37 . The method of claim 26 , wherein the candidate compound is a short-chain fatty acid, hydroxamic acid, cyclic tetrapeptide, or benzamide.
38 . (canceled)
39 . The method of claim 26 , wherein the candidate compound is 4-phenylbutyrate, valproic acid, suberoylanilide hydroxamic acid (SAHA), pyroxamide, trochostatin A, oxamflatin, trapoxin A, apicidin, butyrate salt; or a derivative thereof.
40 . The method of claim 26 , wherein the method further comprises testing the compound in an animal model.
41 . The method of claim 40 , wherein the compound is administered to an animal before, during or after exposure to an amount of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) sufficient to cause symptoms associated with Parkinson's disease in the animal.
42 . The method of claim 40 , further comprising administering the compound to an animal before, during or after exposure to an amount of rotenone sufficient to cause symptoms associated with Parkinson's disease in the animal.
43 . The method of claim 40 , wherein further testing comprises administering the compound to a transgenic animal expressing α-synuclein.
44 . The method of claim 40 , wherein the animal is a rodent.
45 . The method of claim 44 , wherein Parkinson's disease is assayed by detecting degeneration of a nigrostriatal pathway, raphe nuclei, locus ceruleus, or motor nucleus of vagus.
46 . The method of claim 20 , wherein the method further comprises selecting compounds that treat or prevent at least one symptom of Parkinson's disease.
47 . (canceled)
48 . The method of claim 40 , wherein the method further comprises selecting a compound that reduces the severity of or delays the onset of a Parkinson's disease symptom in the animal by at least about 10% compared to a control.
49 . The method claim 48 , wherein the method is used to confirm that an HDAC inhibitor can prevent or treat Parkinson's Disease (PD).
50 . An expression vector comprising at least one of a DJ-1, Parkin or Pink-1 promoter sequence operably linked to at least one reporter sequence.
51 . The expression vector of claim 50 , wherein the DJ-1, Parkin or Pink-1 promoter sequence comprises about 2000 base pairs upstream of the DJ-1 Parkin or Pink-1 transcription start site.
52 . The expression vector of claim 50 , wherein the DJ-1, Parkin or Pink-1 promoter sequence comprises about 1500 base pairs upstream of the DJ-1, Parkin, or Pink-1 transcription start site.
53 - 69 . (canceled)
70 . The expression vector of claim 50 , wherein the DJ-1, Pink1, or Parkin promoter is operably linked in sequence to:
1) a polynucleotide encoding an ampicillin resistance gene or functional fragment thereof;
2) an f1 origin sequence;
3) an upstream synthetic poly(A) region;
4) a promoter,
5) a polynucleotide sequence encoding a luciferase derivative;
6) an SV40 late poly (A) signal; and
7) a polynucleotide encoding a neomycin resistance gene; or functional fragment thereof.
71 - 81 . (canceled)