Huntingtin functions as a scaffold for selective macroautophagy

Abstract

Selective macroautophagy is an important protective mechanism against diverse cellular stresses. In contrast to the well-characterized starvation-induced autophagy, the regulation of selective autophagy is largely unknown. Here, we demonstrate that Huntingtin, the Huntington disease gene product, functions as a scaffold protein for selective macroautophagy but it is dispensable for non-selective macroautophagy. In Drosophila, Huntingtin genetically interacts with autophagy pathway components. In mammalian cells, Huntingtin physically interacts with the autophagy cargo receptor p62 to facilitate its association with the integral autophagosome component LC3 and with Lys-63-linked ubiquitin-modified substrates. Maximal activation of selective autophagy during stress is attained by the ability of Huntingtin to bind ULK1, a kinase that initiates autophagy, which releases ULK1 from negative regulation by mTOR. Our data uncover an important physiological function of Huntingtin and provide a missing link in the activation of selective macroautophagy in metazoans.

Figure 1. Drosophila huntingtin interacts genetically with the autophagy pathway

(a) Representative Scanning Electron Microscopy (SEM) images of the external thorax (a1–a4) and Phalloidin F-actin staining of the internal muscle structure underlying the thorax (a5–a8) in adult flies of the indicated genotype. Arrows: collapsed thorax. White dashed lines: missing dorsal longitudinal muscles. “ATau”: ectopic Tau expression from a single copy of UAS-Tau transgene driven by a single copy of A307-Gal4 line (A307-Gal4>UAS-Tau/+). Right: quantification of penetrance (top) or muscle loss (bottom) (n=3 independent experiments). (b,c) Phalloidin F-actin staining in fly mutants of the indicated genotypes either homozygous for atg8a^d4 or double heterozygous for dhtt^ko and other mutations: atg8a^d4, atg1^Δ3D, atg13^Δ81, atg7^d77 and Ref(2)P^c03993. Right: quantification of percentage of muscle loss (n=4 independent experiments). (d, e) Representative confocal images of whole-mount adult brains expressing mCherry-GFP-Atg8a reporter under a pan-neuronal driver (Appl-Gal4) (d4–d6). High-magnification view of boxed areas. (e) Quantification of the mCherry-Atg8a-positive puncta in the flies of the indicated genotypes. (n=15 fly brains (d1), n=18 (d2), n=19 (d3), pooled from 3 independent experiments). (f,g) Representative immnoblot (f) and quantification (g) of Ref(2)P protein from dhtt^ko−/− mutant flies and age-matched controls with the indicated genotypes. (n=3 independent experiments). (h–i) Representative immunoblot of Tau-ΔC in fly mutants homozygous for atg8a^d4 (h) or dhtt^ko (i). (j) Representative immunoblot of samples from flies co-expressing Tau-ΔC together with control LacZ (lanes 2 and 5) or with DTS7 (lanes 3 and 6), a temperature-sensitive dominant-negative mutant allele of a proteasome subunit. Inactivation of the proteasome by raising DTS7-expressing flies at non-permissive 28°C (lanes 4–6) did not cause higher level of accumulation of Tau-ΔC compared to control. All values are mean+s.e.m. and differences are significant for *P<0.05 using analysis of variance + Bonferroni test. Scale bars: 20 μm for d4–d6 and 100 μm for all the others. Uncropped images of blots are shown in Supplementary Figure 9.

Figure 2. Huntingtin is functionally conserved between the fly and humans and is required for effective autophagy in mammals

(a–e) Human Huntingtin (hHtt) rescued dhtt^ko-associated phenotypes. (a) Thorax pictures (top) and Phalloidin F-Actin staining (bottom) of flies. Arrows: collapsed thorax. White dashed lines: muscle loss. Scale bar: 100 μm Right: quantification. (b) Representative confocal images of adult brains from GFP-mCherry-Atg8a expressing flies (scale bar: 100 (top) and 20 (bottom) μm. (c) Quantification. n=12 fly brains (b1), n=10 (b2), n=9 (b3), pooled from 3 independent experiments. (d) Representative Ref(2)P immunoblot in mutants flies of the indicated ages and genotypes. (e) Quantification. (n=3 independent experiments). (f–o) Autophagic activity in NIH3T3 mouse fibroblasts control (Ctr; transduced with empty vector) or knockdown for Htt (Htt(−)). (f) Immunoblot for Htt and quantification; (n=8). (g) Long half-life protein degradation rates (g) in cells cultured in serum-supplemented medium without additions (continuous line) or upon addition of lysosomal proteolysis inhibitors (ammonium chloride and leupeptin (N/L)) (discontinuous line) (n=6 plates in 6 independent experiments (with triplicate wells per condition). (h) Effect of N/L or treatment with 3-methyladenine (3MA) to inhibit macroautophagy, (n=6 plates in 6 independent experiments (with triplicate wells per condition). (i) Representative immunoblot of p62 in cells untreated (−) or treated for the indicated times with lysosomal protease inhibitors (PI). (j) Immunofluorescence for polyubiquitinated proteins. Arrows: protein aggregates. Scale bar: 10μm. (k,l) LC3-II flux in cells treated as in (i); representative immunoblot (k) and quantification (l) of LC3-II steady-state levels (left), net flux (middle) and synthesis (difference between 2 and 4 hr) (right) (n=6 plates in 6 independent experiments (with triplicate wells per condition).; (m) Representative electronmicrographs of cells maintained in serum-supplemented media. Bottom: higher magnification images of the boxed areas; (n) Quantification (from left to right) of AV number per section, relative cytosolic area occupied, percentage containing cargo and number per section containing single (selective) or multiple (in bulk) cytosolic content or an empty lumen, (n=12 micrographs for each condition from 3 independent experiments (4 micrographs/experiment)). (o) Representative images of immunogold labeling for LC3. Insets: Boxed areas at higher magnification. Scale bars: 2 μm. All values are mean+s.e.m. and differences are significant for *P<0.05 using either analysis of variance + Bonferroni test (e,c) or student’s t-test (f,h,l,n) Uncropped images of blots are shown in Supplementary Figure 9.

Figure 3. Huntingtin functions in selective macroautophagy

(a) Htt loss reduced autophagy-dependent degradation in response to different stressors. Long half-life protein degradation rates in NIH3T3 cells control (Ctr) or knockdown for Htt (Htt(−)) cultured in serum deprived (−) or supplemented (+) medium without additions or with the indicated stressors. (n=6 plates in 3 independent experiments (with triplicate wells per condition)). (b,c) GST-BHMT assay. Representative immunoblot for GST-BHMT in HEK293T cells treated as indicated (n=3 independent experiments). (d,e) Tau-ΔC degradation. Representative immunoblot of the time-course of Tau-ΔC levels in HeLa cells (d) and quantification of Tau-ΔC protein (e) (n=3 independent experiments). (f–l) Htt knockdown in NIH3T3 fibroblasts reduces lipophagy. (f) Higher susceptibility of Htt(−) cells to lipotoxicity (oleic or palmitic acid) but not to genotoxicity (etoposide), ) (n=6 plates in 3 independent experiments (with triplicate wells per condition).. (g,h) Accumulation of lipid droplets (LD) in oleic-treated cells. Representative images of Bodipy493/503-stained cells (g) and quantification of LD number per cell and average LD area (h). Scale bar: 10 μm. (n=3 independent experiments where a total of 100 cells were analyzed per condition). (i) Reduced increase in oxygen consumption rates (OCR) upon oleic (O) challenges, ) (n=6 plates in 4 independent experiments (with 4 wells per condition). (j) Reduced beta-oxidation measured as release of [¹⁴C]-carbon dioxide in [¹⁴C]-oleate-loaded cells, (n=4). (k,l) LC3 immunostaining in BODIPY493/503-stained cells. (k) Representative images (insets: higher magnification of boxed areas with colocalization pixels in white). Scale bar: 10 μm. (l) Quantification: percentage of LC3/BODIPY colocalization. (n=3 independent experiments a total of 110 cells were analyzed per condition). (m–p) Htt knockdown in NIH3T3 fibroblasts reduces mitophagy. (m,n) Htt(−) cells have higher content of depolarized mitochondria (MitoTracker-positive and MitoTracker-Red-CMXROS-negative). (m) Representative images (insets: higher magnification images). Arrows: depolarized mitochondria. Scale bar: 10 μm. (n) Quantification: percentage of depolarized mitochondria. (n=3 independent experiments a total of 85 cells were analyzed per condition). (o,p) LC3 immunostaining in MitoTracker-stained cells. (o) Representative images in both channels and colocalization pixels in white (insets: higher magnification images). Scale bar: 10 μm. (p) Quantification of LC3/MitoTracker colocalization. (n=3 independent experiments where a total of 110 cells were analyzed per condition). In all studies with lentivirus-mediated shRNA (f–p) control cells were transduced with viral particles carrying the empty vector. All values are mean+s.e.m. and differences are significant for *P<0.05 using student’s t-test (a,f–p) or analysis of variance + Bonferroni test (e). Uncropped images of blots are shown in Supplementary Figure 9.

Figure 4. Ultrastructure of cells knockdown for Huntingtin upon induction of selective types of autophagy

NIH3T3 fibroblasts control (Ctr) or knockdown for Htt (Htt(−)) were exposed to lactacystin (a), oleic acid (b) or mitochondria depolarizing agent FCCP (c) and processed for electron microscopy. Images show representative cellular areas and insets show examples of autophagic vacuoles (AV) at higher magnification to appreciate cargo content. Red arrows: protein aggregates inside AV (a), AV containing lipid material (b) or AV containing mitochondria (c). Yellow arrows: protein aggregates free in the cytoplasm. LD: lipid droplets. Note that induction of proteotoxicity with lactacystin favors autophagic sequestration of proteinaceous aggregate material over sequestration of LD that remain intact under these conditions (a), whereas induction of lipophagy with oleic, results in higher content of AV in close proximity of the LD (b). LD are also preserved from autophagic sequestration in the case of FCCP treatment, where membranous structures compatible with mitochondria undergoing degradation are detected in AV (c). Quantification of the number, average size and percentage of cytosolic area covered by AV is shown on the right (a) or at the bottom (b,c). (n=9 (in a), 9 (in b) ad 6 (in c) micrographs pooled from 3 independent experiments ). Scale bars: 0.5 μm. All values are mean+s.e.m. and differences are significant for *P<0.05 using student’s t-test.

Figure 5. Role of Huntingtin in selective autophagy in different mammalian cell types

Defective selective autophagy in different types of mammalian cells with reduced Htt levels. (a,d) Representative immunoblots to illustrate increased levels of p62 in total cellular lysates of mouse embryonic fibroblasts (MEFs) from mouse knock-out for Htt (HttKO) (a) or N2a cells knockdown (KD) for Htt (Htt(−)) untreated (−) or treated with BafA1 (d). Analysis of lipophagy and mitophagy as the colocalization of LC3 with BODIPY493/503-stained lipid droplets (b,g,i) or with MitoTracker®-labeled mitochondria (c,f,j) in MEFs from wildtype (WT) or Htt-KO mice (b,c), in N2a neuroblastoma cells (f,g) and mouse striatal derived cells (i,j) control or knocked-down for Htt (Htt(−)) upon exposure to oleic acid (for lipophagy) or FCCP (for mitophagy). Efficiency of knockdown was confirmed by immunoblot (e,h). Quantification of the percentage of colocalization in shown at the right. Images show merged channels and inserts show colocalization mask (as white pixels). Scale bars: 10 μm. The number of independent experiments (n) was 3 (in b, c,i,j) and 4 (in f,g) where the number of total cells counted for each experimental condition were 80 (b), 75 (c), 80 (f), 60(g), 80 (i) and 70 (j). All values are mean+s.e.m. and differences are significant for *P<0.05 using student’s t-test.

Figure 6. Huntingtin modulates autophagic induction and physically interacts with p62 and ULK1 proteins through two non-overlapping conserved regions

(a) Representative immunoblot for LC3 in HEK293T cells treated as indicated. Bottom: quantification of LC3-II levels and net LC3-II flux (lateral numbers shown net differences in LC3-II levels upon addition of BafA1) normalized against loading control actin. (n=3 independent experiments) (b) Immunostaining for LC3 in HeLa cells treated as indicated. (c–d) Quantification of LC3-positive puncta plotted by different inhibitor treatments (c) or by Htt genotype (d). Note that although the ratio of LC3 in cells treated or not with BafA1 is comparable in control and Htt(−) cells, the net changes in LC3 puncta content upon addition of BafA1 (shown in the lateral numbers) are markedly lower in these cells (n=5 wells, pooled from pooled from 3 independent experiments, >150 cells per experiment). Scale bar: 10 μm. (e) Representative immunoblot to show co-immunoprecipitation (co-IP) experiments using whole-animal extract from dhtt^ko−/− as negative control or a genome-tagging MiMIC fly line expressing 3XHA-tagged dHtt. (n=3 independent experiments). (f,g). Representative immunoblot to show co-IP experiments between Htt and p62 or ULK1 in HEK293T cells (f) and MEF cells (g) in comparison with negative controls si-Htt-treated (f) or MEF-Htt-KO (g) cells. Input lines show the Triton X100-soluble fraction from the whole cell lysate used for co-IP upon normalization of soluble p62 levels across samples. (n=3 independent experiments). (h) Representative confocal images of immunofluorescent stained cells for Htt (green), p62 (red) and Draq5 (blue). Bottom: high-magnification view of boxed areas. Scale bar: 5 μm (top) or 2 μm (bottom) (n=3 independent experiments). (i–k) Mapping of p62 and ULK1-interacting regions in Htt. (i) Schematics of fly and human conserved regions (blue) in Htt (top) and of the Htt deteltions (green) generated in this study (bottom). (j,k) Representative immunoblots for co-IP assays using the HA-tagged Htt deletions to pulldown Myc-p62 (j) or Myc-ULK1 (k). Whole cell extracts (WCE) are shown in the right. Myc-p62 was pulled down by the C-terminal CD and D6 fragments in Htt and Myc-ULK1 by the middle MD and D3 fragments in Htt. All values are mean+s.e.m. and differences are significant for *P<0.05 using analysis of variance + Bonferroni test. Uncropped images of blots are shown in Supplementary Figure 9.

Figure 7. Huntingtin facilitates p62-mediated cargo recognition efficiency

(a) Representative immunoblot of Triton X-100 soluble and insoluble p62 in HEK293T cells treated as indicated (n=3 independent experiments). (b) Confocal images of HeLa cells immunostained for endogenous p62 (green) and Draq5 (blue). Scale bar: 8 μm. (c) Quantification of large p62 bodies. n=3 independent experiments>150 cells (d–e) Representative immunoblots of endogenous proteins with Ub-K63 or Ub-K48 modifications immunoprecipitated in MEFs cells from wildtype (WT) or Htt knock-out (Htt-KO) mice using anti-Ub-K63 (d) or anti-Ub-K48 (e) antibodies. Co-immunoprecipiated p62 is also shown. (f) Tau-ΔC-GFP and HA-tagged Ub-K63 or Ub-K48 were co-transfected into HEK293T cells, followed by in vitro ubiquitination assay with anti-GFP antibody and probed with anti-HA antibody. (g) Representative immunoblots of endogenous p62 co-immunoprecipitated with an anti-GFP antibody in stable Tau-ΔC-GFP expressing HeLa cells control or treated with siRNA against Htt (n=3 independent experiments). (h–j) Htt knockdown compromised p62/LC3 interaction in NIH3T3 fibroblasts. (h) Representative immunoblot of Co-IP experiments in NIH3T3 cells for LC3. Inp: input, IP: immunoprecipitated, FT: flow-through. (i,j) Co-immunostaining for p62 and LC3 in confluent NIH3T3 cells. (i) Representative images of merged channels of full fields (top) and high magnification of boxed areas (bottom). Scale bar: 10 μm. (j) Quantification: percentage of colocalization. (n=4 independent experiments a total of 80 cells were analyzed per condition). All values are mean+ s.e.m. and differences are significant for *P<0.05 using analysis of either variance + Bonferroni test (c) or student’s test (j). Uncropped images of blots are shown in Supplementary Figure 9.

Figure 8. Huntingtin-ULK1 and mTOR-ULK1 complexes are mutually exclusive

(a) Representative autoradiograph to show in vitro ULK1 kinase activity monitored by the phosophorylation level of myelin binding protein (p-MBP). Equal input of ULK1 protein was verified by immunoblot. (n=3 independent experiments). (b) Representative immunoblot of co-immunoprecipitation (co-IP) experiments using anti-FLAG antibody in HEK293T cells co-transfected with FLAG-tagged ULK1 and Myc-tagged Htt, Raptor, or mTOR, as indicated (n=3 independent experiments). (c, d) Representative immunoblot of Co-IP experiments using anti-FLAG (c) or anti-Myc (d) in in HEK293T cells co-transfected with tagged Htt, ULK1, mTOR and Raptor, as indicated. (e, f) Representative immunoblots of endogenous co-IP experiments in MEFs using anti-ULK1 (e) or anti-Htt antibodies (f). (g). Representative immunoblot to show reciprocal co-IP experiments in MEFs using anti-ULK1 or anti-mTOR antibodies, as indicated. (h) Representative immunoblot to show in vitro ULK1 kinase assay in HEK293T cells treated as indicated (i) Representative immunoblot of co-IP assays using anti-FLAG in HEK293T cells transfected with FLAG-ULK1 and HA-Htt as indicated to analyze association between ULK1 and endogenous Raptor or mTOR proteins (n=3 independent experiments). (j) A schematic model of Htt regulation of selective autophagy. Htt serves as scaffolding for selective autophagy by bringing together cargo bound through p62 and the initiator of autophagy ULK1. Basal autophagy: under basal conditions the absence of Htt leads to reduced selectivity in cargo recognition required for quality control autophagy, but it does not affect autophagy induction/autophagosome biogenesis, since basal ULK1 is sufficient to sustain “in bulk” autophagy and basal quality control autophagy. Induced autophagy: maximal activation of autophagy in response to stress requires the release of mTORC1 inhibition over ULK1. In starvation-induced autophagy inactivation of mTORC1 promotes release and activation of ULK1. Selective autophagy induced in response of different stressors requires Htt to actively compete away ULK1 from the mTORC1 inhibitory complex.