Therapeutic modulation of autophagy

Abstract

Methods for screening for modulators of autophagy are disclosed. Methods for identifying genes whose expression inhibits autophagy, as well as genes whose expression promotes autophagy, are disclosed. Also disclosed are methods for identifying compounds that stimulate autophagy, as well as compounds that inhibit autophagy. Cell lines that may be used in the methods of identification are also disclosed.

1 .- 13 . (canceled) 
     
     
         14 . A method of identifying; a gene whose expression modulates autophagy comprising:
 (A) infecting a test cell expressing a marker of protein aggregation with shRNA from a lentivirus library, wherein lentiviral infection introduces an shRNA directed to a target gene into the test cell and wherein expression of the shRNA in the test cell causes lowered expression of the target gene:   (B) subjecting the infected test cell to metabolic stress and recovery therefrom;   (C) performing analysis on the infected test cell to determine the level of protein aggregates comprising the marker; and   (D) comparing the level of protein aggregates comprising the marker in the test cell infected with shRNA to a target gene with the level protein aggregates comprising the marker in a cell infected with control shRNA:   wherein a higher or lower level of aggregates comprising the marker in the test cell infected with an shRNA to a target gene compared to the level of aggregates observed in the cell infected with the control shRNA indicates that the target gene encodes an inducer or inhibitor of autophagy.   
     
     
         15 . The method of  claim 14  wherein the marker comprises p62/Sequestosome 1 protein linked to a label molecule. 
     
     
         16 . The method of  claim 14  wherein the test cell is autophagy-defective. 
     
     
         17 . The method of  claim 16  wherein the autophagy-defective cell haploinsufficient for an autophagy gene. 
     
     
         18 . The method of  claim 16  wherein the autophagy-defective cell is mill for an autophagy gene. 
     
     
         19 . The method of  claim 14  wherein the test cell is apoptosis-defective. 
     
     
         20 . The method of  claim 14  wherein the test cell is an immortalized baby mouse kidney cell. 
     
     
         21 . The method of  claim 14  further comprising the step of validating lowered expression of the target gene following shRNA infection to ensure knockdown:phenotype correlation of the candidate autophagy modulator. 
     
     
         22 . A method for identifying an inducer or inhibitor of autophagy comprising the steps of:
 (A) contacting a test cell expressing a marker of protein aggregation with a compound;   (B) subjecting the test cell to metabolic stress and recovery therefrom;   (C) performing analysis on the test cell to determine the level of protein aggregates comprising the marker; and   (D) comparing the level of protein aggregates comprising the marker in the test cell with that of a control cell not contacted with the compound, wherein either a higher or lower level of aggregates comprising the marker in the test cell indicates that the compound is capable of modulating autophagy.   
     
     
         23 . The method of  claim 22  wherein the marker comprises p62/Sequestosome 1 protein linked to a label molecule. 
     
     
         24 . The method of  claim 22  wherein the test cell is autophagy-defective. 
     
     
         25 . Them method of  claim 24  wherein the autophagy-defective cell is haploinsufficient for an autophagy gene. 
     
     
         26 . The method of  claim 24  wherein the autophagy-defective cell is mill for an autophagy gene. 
     
     
         27 . The method of  claim 22  wherein the test cell is apoptosis-defective. 
     
     
         28 . The method of  claim 22  wherein the test cell is an immortalized baby mouse kidney cell.