Upregulation of the Autophagy Adaptor p62/SQSTM1 Prolongs Health and Lifespan in Middle-Aged Drosophila

Abstract

Autophagy, a lysosomal degradation pathway, plays crucial roles in health and disease. p62/SQSTM1 (hereafter p62) is an autophagy adaptor protein that can shuttle ubiquitinated cargo for autophagic degradation. Here, we show that upregulating the Drosophila p62 homolog ref(2)P/dp62, starting in midlife, delays the onset of pathology and prolongs healthy lifespan. Midlife induction of dp62 improves proteostasis, in aged flies, in an autophagy-dependent manner. Previous studies have reported that p62 plays a role in mediating the clearance of dysfunctional mitochondria via mitophagy. However, the causal relationships between p62 expression, mitochondrial homeostasis, and aging remain largely unexplored. We show that upregulating dp62, in midlife, promotes mitochondrial fission, facilitates mitophagy, and improves mitochondrial function in aged flies. Finally, we show that mitochondrial fission is required for the anti-aging effects of midlife dp62 induction. Our findings indicate that p62 represents a potential therapeutic target to counteract aging and prolong health in aged mammals.

Keywords: aging; autophagy; longevity; midlife; mitophagy; p62.

Figure 1.. Midlife dp62 Induction Extends Lifespan and Health Span

(A) qPCR analyses of dp62 mRNA levels on days 10, 28, 37, and 44 in daGS>UAS-dp62 females without RU486. n = 5 biological replicates with 3 flies per replicate; ***p < 0.001; one-way ANOVA/Bonferroni’s multiple-comparisons test. (B) qPCR analyses of dp62 mRNA levels on day 37 in daGS>UAS-dp62 females with or without RU486 mediated transgene induction for 7 days (day 30 to day 37). n = 5 biological replicates with 3 flies per replicate; ***p < 0.001; unpaired t test. (C) Western blot detection and quantification of dP62 levels in day 37 daGS>UAS-dp62 females with or without RU486-mediated transgene induction for 7 days (day 30 to day 37). n = 5 biological replicates with 5 flies per replicate. **p < 0.01; unpaired t test. (D) Survival curves of daGS>UAS-dp62 females with or without RU486-mediated transgene induction from day 30 onward. The shaded area indicates the duration of dp62 induction. p < 0.0001; log-rank test; n > 147 flies. (E) Capillary feeding assay (CAFE) of 37-day-old daGS>UAS-dp62 females with or without RU486-mediated transgene induction from day 30 to day 37. n = 10 vials of 10 flies per condition; p > 0.05 and is non-significant (n.s.); unpaired t test. (F) Climbing index as a measure of endurance of 37-day-old daGS>UAS-dp62 females with or without RU486-mediated transgene induction from day 30 to day 37. n = 3 biological replicates with 100 flies per replicate; ***p < 0.001 and *p < 0.05; unpaired t test. (G) Intestinal integrity during aging of daGS>UAS-dp62 females with or without RU486-mediated transgene induction since midlife (day 30) onward. n = 120 flies on day 10; **p < 0.01, *p < 0.05; one-way ANOVA/Bonferroni’s multiple-comparisons test. RU486 was provided in the media at a concentration of 25 μg/mL. Error bars represent SEM.

Figure 2.. Midlife dp62 Induction Improves Proteostasis in Aged Flies

(A-D″) Immunostaining of indirect flight muscles from day 10 (A, A′, and A″), 28 (B, B′, and B″), and 37 daGS>UAS-dp62 females with (D, D′, and D″) or without (C, C′, and C″) RU486-mediated transgene induction from day 30 to day 37, showing polyubiquitinated aggregates (green channel, anti-FK2); dP62 (red channel, anti-dP62); and muscles (blue channel stained with phalloidin/F-Actin). Scale bar is 10 μm. (E) Quantification of polyubiquitin aggregates in muscle as shown in (A)–(D). n > 8 flies; ***p < 0.001 and *p < 0.1; one-way ANOVA/Bonferroni’s multiple-comparisons test. (F) Western blot detection and densitometry of total ubiquitin-conjugated proteins from day 37 daGS>UAS-dp62 females with or without RU486-mediated transgene induction from day 30 to day 37. n = 5 replicates with 5 flies per replicate; **p < 0.01; unpaired t test. (G) Quantification of colocalization of dP62 with polyubiquitinated aggregates (anti-FK2) as shown in (C″) and (D″). n > 8 flies; **p < 0.01; unpaired t test. RU486 was provided in the media at a concentration of 25 μg/mL. Error bars represent SEM.

Figure 3.. Midlife dp62 Induction Requires Autophagy to Improve Proteostasis and Lifespan

(A-D) Immunostaining of indirect flight muscles from day 10 (A), 28 (B), and 37 daGS>UAS-dp62 females with (D) or without (C) RU486-mediated transgene induction from day 30 to day 37, showing Atg8a/LC3 foci (green channel, anti-Atg8a); and muscles (red channel, stained with phalloidin/F-actin). Scale bar is 10 μm. (E) Quantification of Atg8a/LC3 foci in muscle as shown in (A)–(D). n = 8 flies; ***p > 0.001; one-way ANOVA/Bonferroni’s multiple-comparison tests. (F and G) Staining of indirect flight muscles from day 37 daGS,mCherry-Atg8a>UAS-dp62 females with (G) or without (F) RU486-mediated transgene induction from day 30 to day 37, showing mCherry-Atg8a foci (red channel, mCherry). Scale bar is 10 μm. (H) Quantification of mCherry-Atg8a foci in muscle as shown in (F) and (G). n = 14 flies; *p < 0.05; unpaired t test. (I and J) Western blot detection (I) and quantification (J) of Atg8a levels in thorax extracts from day 10, 28, and 37 daGS>UAS-dp62 females with or without RU486-mediated transgene induction from day 30 to day 37. n > 5 replicates with 5 thoraces per replicate; ***p < 0.001 and **p < 0.01; one-way ANOVA/Bonferroni’s multiple-comparisons test. (K) Survival curves of daGS>UAS-Atg1RNAi,UAS-dp62 females with or without RU486-mediated transgene induction from day 30 onward. The shaded area indicates the duration of Atg1RNAi and dp62 induction. p > 0.05 and is non-significant (n.s.); log-rank test; n > 205 flies. (L) Quantification of ubiquitin aggregates in muscle as shown in (M) and (N). n = 16 flies; p > 0.5 and is non-significant (n.s.); unpaired t test. (M–M″ and N–N″) Immunostaining of indirect flight muscles from day 37 daGS>UAS Atg1RNAi, UAS-dp62 females with (N, N′, and N″) or without (M, M′, and M″) RU486-mediated transgene induction from day 30 to day 37, showing polyubiquitinated aggregates (green channel, anti-FK2); dP62 protein aggregates (red channel, anti-dP62) and muscles (blue channel, stained with phalloidin/F-actin). Scale bar is 10 μm. RU486 was provided in the media at a concentration of 25 μg/mL. Error bars represent SEM.

Figure 4.. Midlife dp62 Induction Improves Mitochondrial Function

(A and B) Immunostaining of indirect flight muscles from day 37 daGS>UAS-dp62 females with (B) or without (A) RU486-mediated transgene induction from day 30 to day 37, showing mitochondrial morphology (green channel, anti-ATP5a); muscles (red channel, stained with phalloidin/F-actin). Scale bar is 5 μm. (C) Quantification of mitochondrial area in muscle as shown in (A) and (B). n = 8 flies; **p < 0.01; unpaired t test. (D and E) Staining of indirect flight muscles from day 37 daGS>UAS-dp62 females with (E) or without (D) RU486-mediated transgene induction from day 30 to day 37 showing TMRE fluorescence. Scale bar is 10 μm. (F) Quantification of mitochondrial membrane potential measured by TMRE staining as shown in (D) and (E). n > 15 flies; **p < 0.01; unpaired t test. (G–H′) Staining of indirect flight muscles from 37-day-old daGS>UAS-dp62 females with (H and H′) or without (G and G′) RU486-mediated transgene induction from day 30 to day 37, showing mitochondria (green channel, Mitotracker green staining) and levels of superoxide radicals (red channel, staining with MitoSOX reagent). Scale bar is 10 μm. (I) Quantification of free superoxide radicals as shown in (G′) and (H′); n = 14 biological replicates; *p < 0.05; unpaired t test. RU486 was provided in the media at a concentration of 25 μg/mL. Error bars represent SEM.

Figure 5.. Midlife dp62 Induction Facilitates Mitophagy

(A and B) Immunostaining of indirect flight muscles from day 37 daGS>UAS-dp62 females with (B) or without (A) RU486-mediated transgene induction from day 30 to day 37, showing dsDNA (green channel, anti-dsDNA); muscles (red channel, stained with phalloidin/F-actin). Scale bar is 5 μm. (C) Quantification of mitochondrial DNA in muscle as shown in (A) and (B). n = 8 flies; **p < 0.01; unpaired t test. (D and E) Western blot detection and densitometry of Atg8a (D) and dP62 (E) levels in isolated mitochondria from day 37 daGS>UAS-dp62 females with or without RU486-mediated transgene induction from day 30 to day 37. n = 5 replicates, 5 flies per replicate; *p < 0.05; **p < 0.01; unpaired t test. (F–J′) mito-QC of brains from day 37 (F, G, and F′, G′) and day 44 (I, J, and I′, J′) females. Genotypes analyzed were daGS>UAS-dp62, UAS-mito-QC, and daGS>UAS-mito-QC females with RU486-mediated transgene induction from day 30 to day 37 or from day 30 to day 44, showing merged image of GFP and mCherry (F, G, and I, J) and punctate mCherry-only foci (resulting from merged image where GFP has been quenched; mitolysosomes) (F′, G′, I′, and J′). Scale bar is 10 μm. (H, K, and L) Quantification of mitolysosomes in brains as shown in (F′) and (G′), and (I′) and (J′) at day 37 (H), day 44 (K), and at day 37 and 44 (L). n > 10 flies; **p < 0.01; ***p < 0.001; unpaired t test (H-K). ***p < 0.001 and *p < 0.05; one-way ANOVA/Bonferroni’s multiple-comparisons test (L). (M and N) Immunostaining of indirect flight muscles from day 37 daGS>UAS-Atg1RNAi,UAS-dp62 females with (N) or without (M) RU486-mediated transgene induction from day 30 to day 37, showing mitochondrial morphology (green channel, anti-ATP5a); muscles (red channel, stained with phalloidin/F-actin). Scale bar is 5 μm. (O) Quantification of mitochondrial area in muscle as shown in (M) and (N). n = 9 flies; ***p < 0.001; unpaired t test. (P and Q) Staining of indirect flight muscles from day 37 daGS>UAS-Atg1RNAi,UAS-dp62 females with (Q) or without (P) RU486-mediated transgene induction from day 30 to day 37 showing TMRE fluorescence. Scale bar is 10 μm. (R) Quantification of mitochondrial membrane potential measured by TMRE staining as shown in (P) and (Q). n > 10 flies; p > 0.05 and is non-significant (n.s.); unpaired t test. RU486 was provided in the media at a concentration of 25 μg/mL. Error bars represent SEM.

Figure 6.. Mitochondrial Fission Is Required for Beneficial Effects of Midlife dp62 Induction

(A and B) Immunostaining of indirect flight muscles from day 37 daGS>UAS-dp62,UAS-Drp1^K38A females with (B) or without (A) RU486-mediated transgene induction from day 30 to day 37, showing mitochondrial morphology (green channel, anti-ATP5a) and muscles (red channel, stained with phalloidin/F-actin). Scale bar is 5 μm. (C) Quantification of mitochondrial area in muscle as shown in (A) and (B). n > 11 flies; p > 0.05 and is n.s.; unpaired t test. (D and E) Staining of indirect flight muscles from day 37 daGS>UAS-dp62,UAS-Drp1^K38A females with (E) or without (D) RU486-mediated transgene induction from day 30 to day 37 showing TMRE fluorescence. Scale bar is 10 μm. (F) Quantification of mitochondrial membrane potential measured by TMRE staining as shown in (D) and (E). n = 13 flies; p > 0.05 and is nonsignificant (n.s.); unpaired t test. (G) Survival curves of daGS>UAS-dp62,UAS-Drp1^K38A females with or without RU486-mediated transgene induction from day 30 onward. The shaded area indicates the duration of dp62 and Drp1^K38A induction. p < 0.0001, log-rank test; n > 186 flies. (H) Survival curves of daGS>UAS-dp62,UAS-ParkinRNAi females with or without RU486-mediated transgene induction from day 30 onward. The shaded area indicates the duration of dp62 and ParkinRNAi induction. p = 0.183 and is non-significant (n.s.), log-rank test; n > 125 flies. RU486 was provided in the media at a concentration of 25 μg/mL. Error bars represent SEM.