Quantitation of selective autophagic protein aggregate degradation in vitro and in vivo using luciferase reporters.
The analysis of autophagy in cells and tissue has principally been performed via qualitative measures. These assays identify autophagosomes or measure the conversion of LC3I to LC3II. However, qualitative assays fail to quantitate the degradation of an autophagic substrate and therefore only indirectly measure an intact autophagic system. "Autophagic flux" can be measured using long-lived proteins that are degraded via autophagy. We developed a quantifiable luciferase reporter assay that measures the degradation of a long-lived polyglutamine protein aggregate, polyQ80-luciferase. Using this reporter, the induction of autophagy via starvation or rapamycin in cells preferentially decreases polyQ80-luciferase when compared with a nonaggregating polyQ19-luciferase after four hours of treatment. This response was both time- and concentration-dependent, prevented by autophagy inhibitors and absent in ATG5 knockout cells. We adapted this assay to living animals by electroporating polyQ19-luciferase and polyQ80-luciferase expression constructs into the right and left tibialis anterior (TA) muscles of mice, respectively. The change in the ratio of polyQ80-luciferase to polyQ19-luciferase signal before and after autophagic stimulation or inhibition was quantified via in vivo bioluminescent imaging. Following two days of starvation or treatment with intraperitoneal rapamycin, there was a approximately 35% reduction in the ratio of polyQ80:polyQ19-luciferase activity, consistent with the selective autophagic degradation of polyQ80 protein. This autophagic response in skeletal muscle in vivo was abrogated by co-treatment with chloroquine and in ATG16L1 hypomorphic mice. Our study demonstrates a method to quantify the autophagic flux of an expanded polyglutamine via luciferase reporters in vitro and in vivo.
Ju
Jeong-Sun
JS
Department of Neurology, Washington University School of Medicine, 660 S. Euclid Avenue; Saint Louis, MO 63110, USA.
Miller
Sara E
SE
Jackson
Erin
E
Cadwell
Ken
K
Piwnica-Worms
David
D
Weihl
Conrad C
CC
D000818:Animals; D001343:Autophagy / Q000187:drug effects; D001681:Biological Assay / Q000379:methods*; D002454:Cell Differentiation / Q000187:drug effects; D045744:Cell Line, Tumor; D017930:Genes, Reporter; D006801:Humans; D015151:Immunoblotting; D008156:Luciferases / Q000378:metabolism*; D051379:Mice; D008810:Mice, Inbred C57BL; D018485:Muscle Fibers, Skeletal / Q000166:cytology / Q000187:drug effects; D010455:Peptides / Q000737:chemistry* / Q000378:metabolism*; D020836:Protein Structure, Quaternary; D020123:Sirolimus / Q000494:pharmacology; D014020:Tissue Extracts; D014162:Transfection
D010455:Peptides; D014020:Tissue Extracts; C097188:polyglutamine; D008156:Luciferases; D020123:Sirolimus