Method for Identiflying Modulators of Rufy2 Useful for Treating Alzheimer's Disease
Compositions and methods for identifying modulators of RUFY2 are described. The methods are particularly useful for identifying analytes that antagonize RUFY2's effect on processing of amyloid precursor protein to Aβ peptide and thus useful for identifying analytes that can be used for treating Alzheimer disease.
1 . An isolated polynucleotide encoding a RUFY2 polypeptide selected from the group consisting of:
a) a polypeptide comprising the amino acid sequence of SEQ ID NO: 2; and
b) a polypeptide comprising an amino acid sequence at least 95% identical to the amino acid sequence of SEQ ID NO: 2.
2 . An isolated polynucleotide of claim 1 comprising SEQ ID NO:1.
3 . A probe, vector or recombinant nucleic acid comprising the sequence set forth as SEQ ID NO: 1.
4 . An isolated cell comprising the probe, vector or recombinant nucleic acid of claim 3 .
5 . A method of making an isolated polypeptide comprising the amino acid sequence set forth as SEQ ID NO:2, said method comprising the steps of:
a) introducing the vector or recombinant nucleic acid of claim 4 into a host cell or cellular extract,
b) incubating said host cell or cellular extract under conditions whereby said polypeptide is expressed; and
c) isolating said polypeptide.
6 . A method for screening for analytes that antagonize processing of amyloid precursor protein (APP) to Aβ peptide, comprising:
(a) providing recombinant cells, which ectopically expresses RUFY2 and the APP;
(b) incubating the cells in a culture medium under conditions for expression of the RUFY2 and APP and which contains an analyte;
(c) removing the culture medium from the recombinant cells; and
(d) determining the amount of at least one processing product of APP selected from the group consisting of sAPPβ and Aβ peptide in the medium wherein a decrease in the amount of the processing product in the medium compared to the amount of the processing product in medium from recombinant cells incubated in medium without the analyte indicates that the analyte is an antagonist of the processing of the APP to Aβ peptide.
7 . The method of claim 6 wherein the recombinant cells each comprises a first nucleic acid that encodes RUFY2 operably linked to a first heterologous promoter and a second nucleic acid that encodes an APP operably linked to a second heterologous promoter.
8 . The method of claim 7 wherein the APP is APP NFEV .
9 . The method of claim 6 wherein a control is provided which comprises providing recombinant cells which ectopically express the APP but not the RUFY2.
10 . A method for screening for analytes that antagonize processing of amyloid precursor protein (APP) to amyloid β (Aβ) peptide, comprising:
(a) providing recombinant cells, which ectopically express RLTFY2 and a recombinant APP comprising APP fused to a transcription factor that when removed from the APP during processing of the APP produces an active transcription factor, and a reporter gene operably linked to a promoter inducible by the transcription factor,
(b) incubating the cells in a culture medium under conditions for expression of the RUFY2 and recombinant APP and which contains an analyte; and
(c) determining expression of the reporter gene wherein a decrease in expression of the reporter gene compared to expression of the reporter gene in recombinant cells in a culture medium without the analyte indicates that the analyte is an antagonist of the processing of the APP to Aβ peptide.
11 . A method for treating Alzheimer's disease in an individual comprising providing to the individual an effective amount of an antagonist of RUFY2 activity.
12 . A method for identifying an individual who has Alzheimer's disease or is at risk of developing Alzheimer's disease comprising obtaining a sample from the individual and measuring the amount of RUFY2 in the sample.
13 . The use of an antagonist of RUFY2 for the manufacture of a medicament for the treatment of Alzheimer's disease.
14 . The use of an antibody specific for RUFY2 for the manufacture of a medicament for the treatment of Alzheimer's disease.
15 . A vaccine for preventing and/or treating Alzheimer's disease in a subject, comprising an antibody raised against an antigenic amount of RUFY2 wherein the antibody antagonizes the processing of APP to Aβ peptide.