Quantitation of selective autophagic protein aggregate degradation in vitro and in vivo using luciferase reporters

Abstract

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.

Figure 1

A) U20S cells transiently transfected with polyQ19-luciferase (top panel) or polyQ80-luciferase (bottom panel) and immunostained with anti-luciferase (green) or anti-ubiquitin (red) antibodies. Note that polyQ80-luciferase forms small perinuclear ubiquitin positive inclusions while polyQ19-luciferase is diffuse throughout the cell. B) Immunoblot using anti-luciferase or anti-tubulin (loading control) of U20S lysates transfected with polyQ80-luciferase (top) and polyQ19-luciferase (bottom) following 6 treatment with nutrient deprivation (starvation) or rapamycin, chloroquine or bafilomycinA. Note that polyQ80-luciferase decrease in the starvation and rapamycin treated cell lysates. * is a non-specific band seen in non-transfected cells. C–D) Luciferase activity in U20S cells transfected with (C) polyQ19-luciferase or (D) polyQ80-luciferase and treated with nutrient deprivation for 4 hours or nutrient deprivation plus 20uM autophagy inhibitor 3-methyladenine (3MA). Only polyQ80-luciferase activity decreases with the induction of autophagy. ** denotes a p value of <.001. E) Representative epifluorescent image of U20S transfected with GFP-LC3 expression plasmid following no treatment, nutrient deprivation (starv) or nutrient deprivation plus bafilomycinA (starv +bafA) for 4 hours. Note the increase in GFP-LC3 positive puncta in nutrient deprived and nutrient deprived with bafilomycinA treated cells.

Figure 1

A) U20S cells transiently transfected with polyQ19-luciferase (top panel) or polyQ80-luciferase (bottom panel) and immunostained with anti-luciferase (green) or anti-ubiquitin (red) antibodies. Note that polyQ80-luciferase forms small perinuclear ubiquitin positive inclusions while polyQ19-luciferase is diffuse throughout the cell. B) Immunoblot using anti-luciferase or anti-tubulin (loading control) of U20S lysates transfected with polyQ80-luciferase (top) and polyQ19-luciferase (bottom) following 6 treatment with nutrient deprivation (starvation) or rapamycin, chloroquine or bafilomycinA. Note that polyQ80-luciferase decrease in the starvation and rapamycin treated cell lysates. * is a non-specific band seen in non-transfected cells. C–D) Luciferase activity in U20S cells transfected with (C) polyQ19-luciferase or (D) polyQ80-luciferase and treated with nutrient deprivation for 4 hours or nutrient deprivation plus 20uM autophagy inhibitor 3-methyladenine (3MA). Only polyQ80-luciferase activity decreases with the induction of autophagy. ** denotes a p value of <.001. E) Representative epifluorescent image of U20S transfected with GFP-LC3 expression plasmid following no treatment, nutrient deprivation (starv) or nutrient deprivation plus bafilomycinA (starv +bafA) for 4 hours. Note the increase in GFP-LC3 positive puncta in nutrient deprived and nutrient deprived with bafilomycinA treated cells.

Figure 2

A) Schematic representation of “autophagic flux” assay using the ratio of polyQ80-luciferase to polyQ19-luciferase activity. B) Raw data from differentiated C2C12 myotubes transfected with polyQ19 or polyQ80-luciferase constructs and treated with autophagy inducers rapamycin or nutrient deprivation for 18 hours. C) graphical representation of the data in B.

Figure 3

The ratio of polyQ80/polyQ19-luciferase activity in differentiated C2C12 myotubes transfected following (A) starvation for 4 or 18 hours (B) increasing concentrations of rapamycin for 18 hours or (C) increasing concentrations of autophagy inhibitors chloroquine (CQ) or bafilomycinA (Baf). * denotes a p value of <.01. ** denotes a p value of <.001. (D) U20S cells treated with proteasome inhibitor MG132 have a similar increase in both Q19 and Q80-luciferase activity after 4 hours of treatment. E) ATG5+/+ or ATG5−/− mouse embryonic fibroblasts (MEFs) were transfected with polyQ80 or polyQ19-luciferase and subjected to nutrient deprivation for 0 or 6 hours. The raw ratio of polyQ80/polyQ19-luciferase activity is presented. Note that the amount ratio of polyQ80/polyQ19-luciferase is elevated in ATG5−/− cells at time 0. In addition, whereas the ratio decreases in ATG5+/+ MEFs following nutrient deprivation, this does not occur in ATG5−/− MEFs suggesting impaired autophagic degradation.

Figure 3

The ratio of polyQ80/polyQ19-luciferase activity in differentiated C2C12 myotubes transfected following (A) starvation for 4 or 18 hours (B) increasing concentrations of rapamycin for 18 hours or (C) increasing concentrations of autophagy inhibitors chloroquine (CQ) or bafilomycinA (Baf). * denotes a p value of <.01. ** denotes a p value of <.001. (D) U20S cells treated with proteasome inhibitor MG132 have a similar increase in both Q19 and Q80-luciferase activity after 4 hours of treatment. E) ATG5+/+ or ATG5−/− mouse embryonic fibroblasts (MEFs) were transfected with polyQ80 or polyQ19-luciferase and subjected to nutrient deprivation for 0 or 6 hours. The raw ratio of polyQ80/polyQ19-luciferase activity is presented. Note that the amount ratio of polyQ80/polyQ19-luciferase is elevated in ATG5−/− cells at time 0. In addition, whereas the ratio decreases in ATG5+/+ MEFs following nutrient deprivation, this does not occur in ATG5−/− MEFs suggesting impaired autophagic degradation.

Figure 3

The ratio of polyQ80/polyQ19-luciferase activity in differentiated C2C12 myotubes transfected following (A) starvation for 4 or 18 hours (B) increasing concentrations of rapamycin for 18 hours or (C) increasing concentrations of autophagy inhibitors chloroquine (CQ) or bafilomycinA (Baf). * denotes a p value of <.01. ** denotes a p value of <.001. (D) U20S cells treated with proteasome inhibitor MG132 have a similar increase in both Q19 and Q80-luciferase activity after 4 hours of treatment. E) ATG5+/+ or ATG5−/− mouse embryonic fibroblasts (MEFs) were transfected with polyQ80 or polyQ19-luciferase and subjected to nutrient deprivation for 0 or 6 hours. The raw ratio of polyQ80/polyQ19-luciferase activity is presented. Note that the amount ratio of polyQ80/polyQ19-luciferase is elevated in ATG5−/− cells at time 0. In addition, whereas the ratio decreases in ATG5+/+ MEFs following nutrient deprivation, this does not occur in ATG5−/− MEFs suggesting impaired autophagic degradation.

Figure 4

A) Diagram of imaging paradigm. Mice were electroporated with polyQ80 and polyQ19-luciferase constructs into the right and left TA muscle respectively. Two days post-electroporation (day 0 or pre-treatment group) the ratio of polyQ80/polyQ19-luciferase activity was determined for each animal. Each cohort of animals was subsequently treated with autophagic stimulus for one and two days (post-treatment). After treatment, the ratio of polyQ80/polyQ19-luciferase activity was again determined for each animal and the change was plotted. B) Sample images of control or ATG16L1 hypomorphic mice before (pre-treatment) and after food deprivation (post-treatment). (C) Six animals in each of 4 groups were electoporated and then were fed, starved, starved plus treated with intraperitoneal chloroquine or treated with chloroquine alone for two days. Only the starved animal group had a significant decrease of ~34% in the ratio of polyQ80/polyQ19-luciferase activity. (D) 12 animals were electroporated and half received no treatment or were treated with rapamycin for one and two days. Only the rapamycin treated animals had a significant decrease of ~30%. (E) Immunoblot analysis of skeletal muscle lysates (tibialis anterior, gastrocnemius, or triceps) from mice fed (F) or following 2 days of nutrient deprivation (S), using a p62 antibody. α-tubulin is shown as a loading control. (F) Tibialis anterior skeletal muscle was isolated from fed or animals nutrient deprived or nutrient deprived and treatment with chloroquine for 2 days and flash frozen in liquid nitrogen cooled isopentane. 8µM sections were immunostained with anti-p62 antibody. All images are taken at the same exposure. Inset shows magnification of boxed region.

Figure 4

A) Diagram of imaging paradigm. Mice were electroporated with polyQ80 and polyQ19-luciferase constructs into the right and left TA muscle respectively. Two days post-electroporation (day 0 or pre-treatment group) the ratio of polyQ80/polyQ19-luciferase activity was determined for each animal. Each cohort of animals was subsequently treated with autophagic stimulus for one and two days (post-treatment). After treatment, the ratio of polyQ80/polyQ19-luciferase activity was again determined for each animal and the change was plotted. B) Sample images of control or ATG16L1 hypomorphic mice before (pre-treatment) and after food deprivation (post-treatment). (C) Six animals in each of 4 groups were electoporated and then were fed, starved, starved plus treated with intraperitoneal chloroquine or treated with chloroquine alone for two days. Only the starved animal group had a significant decrease of ~34% in the ratio of polyQ80/polyQ19-luciferase activity. (D) 12 animals were electroporated and half received no treatment or were treated with rapamycin for one and two days. Only the rapamycin treated animals had a significant decrease of ~30%. (E) Immunoblot analysis of skeletal muscle lysates (tibialis anterior, gastrocnemius, or triceps) from mice fed (F) or following 2 days of nutrient deprivation (S), using a p62 antibody. α-tubulin is shown as a loading control. (F) Tibialis anterior skeletal muscle was isolated from fed or animals nutrient deprived or nutrient deprived and treatment with chloroquine for 2 days and flash frozen in liquid nitrogen cooled isopentane. 8µM sections were immunostained with anti-p62 antibody. All images are taken at the same exposure. Inset shows magnification of boxed region.

Figure 4

A) Diagram of imaging paradigm. Mice were electroporated with polyQ80 and polyQ19-luciferase constructs into the right and left TA muscle respectively. Two days post-electroporation (day 0 or pre-treatment group) the ratio of polyQ80/polyQ19-luciferase activity was determined for each animal. Each cohort of animals was subsequently treated with autophagic stimulus for one and two days (post-treatment). After treatment, the ratio of polyQ80/polyQ19-luciferase activity was again determined for each animal and the change was plotted. B) Sample images of control or ATG16L1 hypomorphic mice before (pre-treatment) and after food deprivation (post-treatment). (C) Six animals in each of 4 groups were electoporated and then were fed, starved, starved plus treated with intraperitoneal chloroquine or treated with chloroquine alone for two days. Only the starved animal group had a significant decrease of ~34% in the ratio of polyQ80/polyQ19-luciferase activity. (D) 12 animals were electroporated and half received no treatment or were treated with rapamycin for one and two days. Only the rapamycin treated animals had a significant decrease of ~30%. (E) Immunoblot analysis of skeletal muscle lysates (tibialis anterior, gastrocnemius, or triceps) from mice fed (F) or following 2 days of nutrient deprivation (S), using a p62 antibody. α-tubulin is shown as a loading control. (F) Tibialis anterior skeletal muscle was isolated from fed or animals nutrient deprived or nutrient deprived and treatment with chloroquine for 2 days and flash frozen in liquid nitrogen cooled isopentane. 8µM sections were immunostained with anti-p62 antibody. All images are taken at the same exposure. Inset shows magnification of boxed region.

Figure 5

A) Immunoblot of skeletal muscle lysate (gastrocnemius or tibialis anterior) from wild-type and ATG16L1 hypomorphic mice using an ATG16L1 antibody. B) A similar experiment as above was performed using 11 control or 7 ATG16L1 hypomorphic mice. The average polyQ80-luciferase/polyQ19-luciferase ratio for each animal is plotted. ATG16L1 mice have higher ratio and have a persistently elevated ratio following one day of starvation (6% increase from day 0). Whereas control animals had a 25% decrease in polyQ80/polyQ19-luciferase activity with starvation.