Cell lines useful for assessing 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 . A method comprising the steps of:
 (A) screening an shRNA library using a test cell that expresses an autophagosome marker;   (B) subjecting the cell to metabolic stress;   (C) performing analysis on the cell to determine the localization of the autophagosome marker in the test cell; and   (D) comparing the localization of the marker in the test cell with that of a control cell;   
       wherein a lower level of localization of the marker in autophagosomes in the test cell compared to that demonstrated by the control cell indicates knockdown of a gene whose expression induces autophagy. 
     
     
         2 . A method for identifying inhibitors of autophagy comprising the steps of:
 (A) contacting a test cell that expresses an autophagosome marker with a compound;   (B) subjecting the test cell to metabolic stress;   (C) performing analysis on the test cell to determine the localization of the autophagosome marker in the cell; and   (D) comparing the localization of the marker in the cell with that of a control cell;   
       wherein a lower level of localization of the marker in autophagosomes in the test cell compared to that demonstrated by the control cell indicates that the compound is capable of inhibiting autophagy. 
     
     
         3 . The method of  claim 1  wherein the test cell is apoptosis-defective. 
     
     
         4 . The method of  claim 1  wherein the test cell is an immortalized baby mouse kidney cell. 
     
     
         5 . The method of  claim 1  wherein the autophagosome marker comprises EGFP-LC3. 
     
     
         6 . A method comprising:
 (A) screening an shRNA library using a test cell expressing a marker of protein aggregation;   (B) subjecting the test cell to metabolic stress;   (C) performing analysis on the test cell to determine the level of the marker; and   (D) comparing the level of the marker in the test cell with that of a control cell,   
       wherein a lower level of aggregates comprising the marker in the test cell compared to that demonstrated by the control cell indicates the lowered expression of a gene whose knockdown increases aggregate clearance. 
     
     
         7 . (canceled) 
     
     
         8 . (canceled) 
     
     
         9 . The method of  claim 6  wherein the test cell is autophagy-defective. 
     
     
         10 . (canceled) 
     
     
         11 . The method of  claim 6  wherein the marker comprises p62 protein linked to a label molecule. 
     
     
         12 .- 13 . (canceled) 
     
     
         14 . The method of  claim 2  wherein the test cell is apoptosis-defective. 
     
     
         15 . The method of  claim 2  wherein the test cell is an immortalized baby mouse kidney cell. 
     
     
         16 . The method of  claim 2  wherein the autophagosome marker comprises EGFP-LC3.