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Rab5 modulates aggregation and toxicity of mutant huntingtin through macroautophagy in cell and fly models of Huntington disease

Rab5 modulates aggregation and toxicity of mutant huntingtin through macroautophagy in cell and... Research Article 1649 Rab5 modulates aggregation and toxicity of mutant huntingtin through macroautophagy in cell and fly models of Huntington disease 1 1,2 1 2 1, Brinda Ravikumar , Sara Imarisio , Sovan Sarkar , Cahir J. OʼKane and David C. Rubinsztein * Department of Medical Genetics, Cambridge Institute for Medical Research, Wellcome/MRC Building, Addenbrookeʼs Hospital, Hills Road, Cambridge, CB2 0XY, UK Department of Genetics, University of Cambridge, CB2 3EH, UK *Author for correspondence (e-mail: dcr1000@hermes.cam.ac.uk) Accepted 21 February 2008 Journal of Cell Science 121, 1649-1660 Published by The Company of Biologists 2008 doi:10.1242/jcs.025726 Summary Huntington disease (HD) is caused by a polyglutamine- endocytosis by various means suppressed autophagosome- expansion mutation in huntingtin (HTT) that makes the protein lysosome fusion (the final step in the macroautophagy pathway) toxic and aggregate-prone. The subcellular localisation of similar to bafilomycin A1. Thus, Rab5, which has previously huntingtin and many of its interactors suggest a role in been thought to be exclusively involved in endocytosis, has a endocytosis, and recently it has been shown that huntingtin new role in macroautophagy. We have previously shown that interacts indirectly with the early endosomal protein Rab5 macroautophagy is an important clearance route for several through HAP40. Here we show that Rab5 inhibition enhanced aggregate-prone proteins including mutant huntingtin. Thus, polyglutamine toxicity, whereas Rab5 overexpression attenuated better understanding of Rab5-regulated autophagy might lead toxicity in our cell and fly models of HD. We tried to identify to rational therapeutic targets for HD and other protein- a mechanism for the Rab5 effects in our HD model systems, conformation diseases. and our data suggest that Rab5 acts at an early stage of autophagosome formation in a macromolecular complex that Supplementary material available online at contains beclin 1 (BECN1) and Vps34. Interestingly chemical http://jcs.biologists.org/cgi/content/full/121/10/1649/DC1 or genetic inhibition of endocytosis also impeded macroautophagy, and enhanced aggregation and toxicity of Key words: Huntington disease, Autophagy, Rab5 mutant huntingtin. However, in contrast to Rab5, inhibition of Introduction which later fuse with lysosomes, where their contents are degraded Huntington disease (HD) is a late-onset inherited neurodegenerative (Klionsky and Ohsumi, 1999). It is believed that isolation condition caused by an expansion of CAG trinucleotide repeats (>35 membranes (phagophores) – which later give rise to repeats) in exon 1 of the IT15 gene. The mutation results in the autophagosomes – are formed at the phagophore-assembly site (also production of an abnormally long polyglutamine (polyQ) tract in referred to as pre-autophagosomal structures, PAS) in yeast (Suzuki the N-terminus of the huntingtin (HTT) protein (Rubinsztein, et al., 2001). PAS however have not been defined in mammalian 2002). Full-length mutant huntingtin undergoes several proteolytic autophagy. Several ‘Atg’ (autophagy) genes that regulate yeast cleavages, which give rise to N-terminal fragments comprising the autophagy have been identified, and many of these genes have first 100-150 residues containing the expanded polyQ tract. These mammalian orthologues. A protective role of autophagy has been N-terminal fragments are also the toxic species found in aggregates implicated in some neurodegenerative disorders, cancers and seen in HD mouse models and human brains (Lunkes et al., 2002). infectious diseases (Rubinsztein et al., 2007). Conditional knockouts Thus, HD pathogenesis is frequently modelled with fragments of of the key autophagy genes Atg5 or Atg7 in the brains of mice result exon 1 that contain expanded polyQ repeats, which cause toxicity in a neurodegenerative phenotype caused by aberrant accumulation and the formation of aggregates in cell models and in vivo of ubiquitinated proteins (Hara et al., 2006; Komatsu et al., 2006). (Rubinsztein, 2002). In addition to HD, where enhanced clearance of mutant huntingtin A key pathway that regulates the degradation of aggregate-prone fragments through induction of autophagy attenuates toxicity caused cytosolic proteins such as mutant huntingtin (both full-length and by mutant huntingtin in cell, fly and mouse models (Ravikumar et exon 1 forms) is macroautophagy – henceforth referred to simply al., 2002; Ravikumar et al., 2004), this pathway appears to be equally as autophagy (Ravikumar et al., 2002; Ravikumar et al., 2004; important for the clearance of several other intracellular proteins Shibata et al., 2006). Wild-type huntingtin (full-length or exon 1) that cause neurodegeneration, including the A53T point mutation has a very low dependence on autophagy for its clearance. in α-synuclein that causes autosomal-dominant Parkinson disease Autophagy, a process conserved from yeast to human, typically (Berger et al., 2006; Ravikumar et al., 2002; Webb et al., 2003). clears long-lived proteins and organelles by engulfing cytoplasmic The subcellular localisation of huntingtin, the nature of many of contents in double-membrane structures called autophagosomes, its normal interactors and its indirect interaction with Rab5 through Journal of Cell Science 1650 Journal of Cell Science 121 (10) HAP40 (Pal et al., 2006), suggested that it may have roles in of visible rhabdomeres in the double transgenic flies. Degeneration endocytosis. Rab5, a member of the small GTPase family, is a key of photoreceptors due to Q120 overexpression was greatly rescued regulator of the early endocytic pathway in mammalian cells (Bucci by overexpression of Rab5-EGFP (Fig. 1d). et al., 1992; Stenmark et al., 1994). Accordingly, we initially tested Since our previous studies suggest that aggregation of mutant the role of Rab5 in the regulation of toxicity of mutant huntingtin. huntingtin can be regulated by autophagy (Ravikumar et al., 2002), We show that Rab5 inhibition enhances toxicity induced by mutant we next tested whether the effects of Rab5 on Q74 aggregation are +/+ ) or autophagy- polyQ, whereas Rab5 overexpression attenuates mutant polyQ autophagy-dependent using either wild-type (Atg5 –/– ) mouse embryonic fibroblasts toxicity in HD cell and fly models. Our data suggest that Rab5, in deficient Atg5-knockout (Atg5 addition to its role in endocytosis, modifies aggregation and toxicity (MEFs) (Mizushima et al., 2001). As previously observed with of mutant huntingtin by having a novel role in the early stage of autophagy inhibitors (Ravikumar et al., 2002), Q74 aggregation was –/– +/+ compared with Atg5 autophagosome formation. Interestingly, inhibition of endocytosis higher in autophagy-incompetent Atg5 MEFs (Fig. 1e). We observed minimal Q74-induced cell death in by a variety of means also influenced the toxicity of mutant huntingtin by inhibiting autophagy at a downstream step the MEFs under the transfection conditions we used. As expected, (autophagosome-lysosome fusion) that is distinct from Rab5 overexpression of DN-Rab5 caused an increase and that of CA- inhibition. A possible therapeutic approach for HD and other Rab5 a decrease in Q74 aggregation in wild-type MEFs (Fig. 1f, proteinopathies would thus be the enhancement of Rab5 activity. left), similar to the results seen in COS-7 cells (Fig. 1b). However, overexpression of DN-Rab5 or CA-Rab5 had no effects on Q74 –/– MEFs (Mizushima Results and Discussion aggregation in autophagy-incompetent Atg5 Rab5 modifies polyglutamine toxicity and aggregation et al., 2001) (Fig. 1f, right). Furthermore, Rab5 inhibition abrogated We initially tested whether Rab5 can modify the toxicity of a mutant the ability of the autophagy enhancers, the mTOR inhibitor huntingtin exon 1 fragment with 74 polyQ repeats (Q74) (Narain rapamycin or the inositol monophosphatase inhibitor L-690,330, et al., 1999) in COS-7 cells. Dominant-negative inhibition of Rab5 which target mTOR-dependent or -independent pathways, by overexpression of a GTP-binding-defective Rab5 mutant carrying respectively, to clear Q74 aggregates (Fig. 1g; supplementary a S34N point mutation (DN-Rab5) (Stenmark et al., 1994) increased material Fig. S2) (Ravikumar et al., 2002; Sarkar et al., 2005). This the toxicity of Q74 (Fig. 1a), whereas overexpression of wild-type suggests that the effect of Rab5 on Q74 aggregation and toxicity (WT) Rab5 or of a constitutively active (CA) Rab5 mutant carrying were due to alterations in the autophagic pathway rather than its a Q79L mutation (CA-Rab5) (Stenmark et al., 1994) significantly role in endocytosis. decreased Q74-induced toxicity (Fig. 1a). Rab5 regulates autophagosome formation in mammalian cells We then examined the effect of Rab5 on aggregation of mutant Since the above data suggest a possible role of Rab5 on autophagy, huntingtin. Aggregation of mutant huntingtin correlates with its expression levels (Narain et al., 1999). Also, the proportion of cells we tested whether Rab5 influenced the formation of with inclusions formed by mutant huntingtin fragments generally autophagosomes. Two ubiquitin-like modifications are involved in correlates with its toxicity in cell culture models (although inclusions the expansion and completion of autophagosome formation. The may not themselves be as toxic as diffusely distributed mutant first involves conjugation of Atg12 to Atg5 in a reaction that requires huntingtin) (Arrasate et al., 2004; Ravikumar et al., 2002). DN- Atg7 (E1-like) and Atg10 (E2-like). Atg5-Atg12 conjugates are Rab5 increased Q74 aggregation, whereas CA-Rab5 and WT-Rab5 localised onto the PAS and dissociate upon completion of decreased the proportions of Q74-expressing cells with inclusions autophagosome formation. The second modification involves (Fig. 1b), mirroring the toxicity data (Fig. 1a). We next confirmed conjugation of microtubule-associated protein 1 light chain 3 that the effects of DN-Rab5 were mirrored by depletion of (hereafter referred to as LC3 and also known as MAP1LC3 or Atg8) endogenous Rab5 using RNA interference (RNAi). There are three to phosphatidylethanolamine (PE). LC3 (cytosolic) is cleaved at its isoforms of Rab5, namely Rab5A, Rab5B and Rab5C. Small C-terminus by Atg4 to form LC3-I. LC3-I is covalently conjugated interfering RNA (siRNA) that target individual Rab5 isoforms to PE to form LC3-II, a process requiring the activities of Atg7 and significantly increased Q74 aggregation, whereas simultaneous Atg3. LC3-II (membrane associated) is specifically targeted to Atg5- knockdown of all three isoforms increased Q74 aggregation even Atg12-associated, expanded phagophores and remains associated further (Fig. 1c, supplementary material Fig. S1). Thus, Rab5 with autophagosomes even after fusion with lysosomes, after which modulation affects aggregation and toxicity of the mutant huntingtin LC3-II can be delipidated and recycled. LC3 is the only known fragments. protein that specifically associates with autophagosomes and not We next tested whether Rab5 can modify the toxicity of mutant with other vesicular structures (Kabeya et al., 2000). Thus, LC3-II huntingtin in vivo using a Drosophila melanogaster HD model. Fly levels correlate with the numbers of autophagic vacuoles, which photoreceptors that express a mutant huntingtin fragment with 120 can also be assessed by counting LC3-positive vesicles (Kabeya et polyQ repeats (Q120) exhibit degeneration that is not observed in al., 2000). We first tested the effect of Rab5 on LC3. Whereas, flies that express the wild-type fragment with 23 polyQ repeats inhibition of Rab5 decreased the proportion of COS-7 cells with (Jackson et al., 1998). The Drosophila compound eye consists of >20 LC3-labelled autophagic vesicles (Fig. 2a), overexpression of many ommatidia, each comprising eight photoreceptor neurons with CA-Rab5 or WT-Rab5 significantly increased the proportion of cells light-gathering parts called rhabdomeres, seven of which can be with >20 LC3-positive autophagic vesicles (Fig. 2a). However, we visualised by light microscopy using the pseudopupil technique did not observe any change in the size or morphology of the LC3 (Franceschini and Kirschfeld, 1971). Neurodegeneration in the HD vesicles under any of the above conditions (data not shown). The flies is progressive and is associated with a decrease in the number above results also correlated with decrease (upon DN-Rab5 of visible rhabdomeres in each ommatidium with time (Jackson et overexpression) or increase (upon CA-Rab5 or WT-Rab5 al., 1998). We crossed the HD flies with flies transgenic for wild- overexpression) with autophagosome-associated LC3-II levels on type Rab5 fused to EGFP (Rab5-EGFP) and looked at the number western blots when we blocked LC3-II clearance through inhibition Journal of Cell Science Rab5 regulates toxicity of mutant huntingtin via autophagy 1651 Fig. 1. Rab5 modulates the aggregation and toxicity of mutant huntingtin. (a) Quantification of GFP-expressing COS-7cells showing signs of cell death that had been transiently transfected with dominant-negative (DN), constitutive active (CA) or wild-type (WT) Rab5, or empty vector control, and the EGFP-tagged huntingtin exon 1 with 74 polyglutamine repeats (Q74) (at a 3:1 ratio) for 48 hours. ***P<0.0001, **P<0.001, *P<0.05. (b) Quantification of GFP-expressing COS-7 cells containing aggregates that were quantified for toxicity shown above. ***P<0.0001. (c) Quantification of GFP-expressing HeLa cells containing aggregates transiently transfected with siRNA targeting Rab5a, Rab5b, Rab5c or all three siRNA simultaneously (Rab5abc) for 72 hours, and also with EGFP-tagged huntingtin exon 1 with 74 polyglutamine repeats for the last 24 hours of the 72-hour siRNA transfection period. ***P<0.0001, *P<0.05. (d) Rab5 overexpression increases the numbers of rhabdomeres in ommatidia of mutant huntingtin-expressing flies. Frequency distribution of ommatidia with different numbers of rhabdomeres three days after eclosion (hatching) in progeny of flies that express mutant huntingtin exon 1 (gmrQ120) and that had been crossed to either a control stock (w ) (white minus; have huntingtin transgene only) or to Rab5- EGFP flies (have huntingtin and Rab5 transgenes). P0.001, t-test; P0.001, Mann-Whitney U test. The rhabdomere frequency of Rab5 flies crossed to a control stock is also shown. (e) Odds ratio of GFP-expressing cells with Q74 +/+ aggregates in wild-type (Atg5 ) vs –/– Atg5 knockout (Atg5 ) MEFs. ***P<0.0001. (f) Odds ratio of Q74- –/– expressing Atg5 (Atg5 knockout) or +/+ Atg5 (wild-type) MEF cells with aggregates, after transient transfection with dominant-negative Rab5 (DN- Rab5), constitutive active Rab5 (CA- Rab5) or empty vector control and EGFP-tagged huntingtin exon 1 with 74 polyglutamine repeats (Q74) (3:1 ratio) for 48 hours. ***P<0.0001. Odds ratios are given to compare pooled summary statistics across multiple independent experiments (see Materials and Methods). Control conditions are fixed at 1 in both cell lines to facilitate comparisons. (g) Quantification of GFP- expressing HeLa cells containing aggregates that had been transiently transfected with dominant-negative Rab5 (DN-Rab5) or empty vector (Cont) and huntingtin exon 1 with 74 polyglutamine repeats (Q74) (at a 3:1 ratio) for 48 hours and were either left untreated (–Rap) or treated with 0.2 μg/ml rapamycin (Rap) to induce autophagy. **P<0.001. Error bars in all graphs represent the s.e.m. of autophagosome-lysosome fusion using bafilomycin A1 (BafA1) autophagosome synthesis, rather than decreasing autophagosome- at 200 nM (Fig. 2b) (Sarkar et al., 2007). Further blockage of lysosome fusion and/or autophagosome degradation. autophagosome-lysosome fusion through a BafA1-independent Rab5 and its effector Vps34 regulate early steps of mechanism, using the dynein inhibitor erythro-9-[3-(2- autophagosome formation hydroxynonyl)] adenine (EHNA), together with this dose of BafA1, Autophagosome formation begins with a nucleation step during does not increase LC3-II compared with using BafA1 alone (Sarkar et al., 2007). Results shown in Fig. 2b suggest that Rab5 increased which membranes of unknown origin form phagophores; these then Journal of Cell Science 1652 Journal of Cell Science 121 (10) expand and fuse to form completed autophagosomes. The formation to LC3-I to form LC3-II. Atg5-Atg12 conjugates colocalise with K130R ) inhibits of autophagosome precursors is regulated by a macromolecular LC3-II on the PAS. Mutation of K130R in Atg5 (Atg5 complex that contains phosphoinositide 3-kinase class 3 (Vps34 or its conjugation with Atg12; as a result, membranes with K130R that do not colocalise with LC3-II PIK3C3), beclin 1 (BECN1, the human ortholog of yeast Atg6p), unconjugated Atg5 Atg14 and Vps15 (PIK3R4) (Kihara et al., 2001b). Vps34, which accumulate upon autophagy induction (Mizushima et al., 2001). The generates phosphatidylinositol-3-phosphate [PtdIns(3)P], directly Atg5-Atg12 conjugate only localises to phagophores and dissociates interacts with beclin 1 (Kihara et al., 2001a). 3-methyl adenine just before or after completion of autophagic vacuole formation (3MA) (Kovacs et al., 1998) or wortmannin (Blommaart et al., 1997) (Mizushima et al., 2001). So Atg5 and Atg12 are not associated inhibit PI 3-kinases, including Vps34, and block autophagy by with completed autophagosomes. preventing the formation of autophagosomes. Conjugation of Atg12 To investigate how Rab5 influenced early steps in autophagosome with Atg5 initiates the elongation process. This conjugation however formation, we investigated the distribution of GFP-tagged Atg5 is not required for membrane targeting of Atg5, but is necessary (GFP-Atg5), a marker for autophagosomal precursor structures for membrane elongation. This is followed by conjugation of PE (George et al., 2000; Mizushima et al., 2001). Cells were Fig. 2. (a) COS-7 cells were transiently transfected with empty vector (Control), DN- Rab5, CA-Rab5 or WT-Rab5 and GFP-LC3 or mRFP-LC3 (3:1 ratio) for 24 hours. GFP- positive or mRFP-positive cells with increased numbers of LC3-positive vesicles (>20 vesicles per cell) were counted. 29% of control cells had >20 vesicles per cell. ***P<0.0001. (b) Western blot analysis of COS-7 cells co-transfected with empty vector (C), DN-Rab5, CA-Rab5 or WT-Rab5 and Myc-LC3 for 24 hours in the presence of 200 nM bafilomycin A1 (treated for last 15 hours), using anti-Myc antibody. GFP was used as a transfection control. Representative image from three independent experiments; quantification of the band intensities from these experiments represented as LC3- II:GFP ratio is shown in the graph; *P<0.05. (c) Analysis of GFP-Atg5 structures (green) in HeLa cells transfected with control vector [either untreated or treated for 24 hours with 10 mM 3-methyladenine (3-MA)] or with dominant-negative Rab5 (DN-Rab5) and GFP-Atg5, after saponin extraction. Nuclei are shown in blue. An increased abundance of large punctate Atg5 structures can be noticed with DN-Rab5 and 3-MA treatment. (d) HeLa cells were transfected with siRNA targeting Vps34 or control siRNA for 48 hours after which GFP-Atg5 together with siRNA was transfected for further 24 hours. The cells were fixed following saponin extraction to visualise GFP-Atg5 (green) structures. Quantification of Q74-expressing HeLa cells with aggregates is shown in the graph. Cells were transiently transfected with control siRNA or siRNA targeting Vps34 for 48 hours and with HA-tagged huntingtin exon 1 containing 74 polyglutamine repeats for further 24 hours. ***P<0.0001. (e) Colocalisation of GFP- Atg5 structures (green) with Myc-tagged FYVE (red) in HeLa cells co-transfected with DN-Rab5, GFP-Atg5 and Myc-FYVE for 24 hours. (f) Colocalisation of GFP-Atg5 structures (green) with beclin 1 (red) in HeLa cells co-transfected with DN-Rab5, GFP- Atg5 and Flag-tagged wild-type (WT) beclin 1. (g) Colocalisation of endogenous Atg5 (red) and endogenous Rab5 (green) in HeLa cells treated with 3MA for 15 hours. In panels e-g we observed >30% colocalisation between GFP-Atg5 structures and saponin-extracted, membrane-associated, FYVE, beclin 1 or Rab5 in cells that expressed both of the respective proteins. (h) Colocalisation of GFP-Atg5 structures (green) with Atg12 (red) in HeLa cells co-transfected with DN-Rab5 and GFP-Atg5 and HA-tagged Atg12 for 24 hours. Nuclei labelled with DAPI are in blue. Journal of Cell Science Rab5 regulates toxicity of mutant huntingtin via autophagy 1653 permeabilised with saponin to remove soluble cytosolic contents or proteins associated with phagophores. A subset of the punctate and reveal membrane-associated GFP-Atg5. After Rab5 inhibition, Atg5 structures colocalised with a PtdIns(3)P marker, Myc-FYVE we noted an increased abundance of large punctate Atg5 structures (Fig. 2e) (Gaullier et al., 1998) and they also overlapped with (Fig. 2c). These contrast with the smaller and less abundant Atg5 beclin 1 (Fig. 2f) but did not colocalise with LC3 (supplementary puncta seen in the great majority of untreated cells (Fig. 2c). We material Fig. S5) suggesting that these were indeed early found similar results when we immunostained for endogenous Atg5 autophagic structures. We also did not observe any colocalisation (supplementary material Fig. S3). In control cells, however, these of the Atg5 structures with the Golgi markers p230 structures were rare (Fig. 2c; supplementary material Fig. S3). (supplementary material Fig. S6) (Derby et al., 2007) or golgin- Whereas saponin extraction precludes accurate quantitation (because 84 (Satoh et al., 2003) and the Atg5 in these structures was not it does not allow visualisation of transfected versus untransfected associated with the ER proteins BiP (Haas, 1994) or Grp94 (Argon cells), we saw these Atg5 structures in 3% of all control cells and and Simen, 1999) (data not shown). However, Rab5 associated in about 12% of Rab5-inhibited cells (an ~fourfold increase, with these Atg5-positive puncta, which is compatible with a role P<0.001 from triplicate slides). for Rab5 in early mammalian autophagy (Fig. 2g). We only Previous studies have shown that Rab5 interacts with and observed very rare colocalisation of Atg5 and Atg12 in such activates the PI 3-kinase Vps34 (Christoforidis et al., 1999). Thus, puncta (Fig. 2h). It should be noted that these experiments were we tested whether Vps34 inhibition had similar effects to Rab5 performed under constitutive autophagy conditions (full medium inhibition. As with Rab5 inhibition, inhibition of Vps34 with the containing serum and amino acids) in cells with Rab5 inhibition. PI 3-kinase inhibitors 3MA (Fig. 2c; supplementary material Fig. Rab5 inhibition or 3MA treatment was required to allow S3) or wortmannin (data not shown) (which are both established visualisation of sufficient numbers of these structures for blockers of autophagosome formation) or siRNA knockdown of characterisation. The accumulation of Atg5 structures by Vps34 Vps34 (Fig. 2d; supplementary material Fig. S4) resulted in or Rab5 inhibition might reflect a block in the progression of Atg5- increases of Atg5 structures. Similar to Rab5 inhibition, inhibition positive membranes to the formation of autophagic vacuoles, of Vps34 also increased the proportion of cells with Q74 which suggests a role of Rab5 in very early steps of autophagy. aggregates, consistent with autophagy inhibition (Fig. 2d) The observation that the Atg5 in these puncta was only rarely (Ravikumar et al., 2002). To further characterise the Atg5 associated with Atg12 led to the hypothesis that defective Atg5- structures we tested whether they colocalised with other markers Atg12 conjugation is causally related to puncta formation. Both Rab5 and Vps34 regulate Atg5- Atg12 conjugation The possibility that defective Atg5-Atg12 conjugation leads to defective autophagosome formation associated with an accumulation of phagophores enriched in unconjugated Atg5 was compatible with the similarity of the above results and the phenotypes seen in apg7 or apg12 yeast strains, which typically show one to five large Atg5-positive punctate structures per cell, (a phenomenon that is very rare in wild-type cells) (George et al., 2000). Thus, when we knocked down Atg7 (an E1-like enzyme crucial for Atg5-Atg12 conjugation; supplementary material Fig. S7) in mammalian cells, we observed increased GFP-Atg5 structures, consistent with those observed in yeast (Fig. 3a). These Atg5- positive structures also colocalised with beclin 1 (supplementary material Fig. S8). Accordingly, we tested whether the increase in Atg5 structures seen with Rab5 or Vps34 inhibition was associated with aberrant Atg5- Atg12 conjugation. Consistent with this hypothesis and with the only very rare colocalisation of Atg12 and Atg5 in the puncta that accumulated following inhibition of Rab5 (Fig. 2h), we found that the ratio of Atg5- Atg12 complex formed compared with the pool of unconjugated Atg12 was significantly lower in cells expressing DN-Rab5 (Fig. 3b). 3MA treatment and siRNA knockdown of Vps34 also had similar effects on Atg5-Atg12 Fig. 2e-h. See previous page for legend. conjugation compared with those seen with Journal of Cell Science 1654 Journal of Cell Science 121 (10) loss-of-function of Rab5 (DN-Rab5), a Vps34 activator (Fig. 3c, all Rab5 isoforms simultaneously (Fig. 3d, left). This effect was left and right respectively). We also found decreased levels of Atg5- similar to the conjugation defect we observed with Atg7 knockdown Atg12 complex compared with free Atg12 when we knocked down (Fig. 3d, right). Thus, Rab5 inhibition and loss of activity of Vps34 Fig. 3. Aberrant conjugation of Atg5 with Atg12 after Rab5 inhibition. (a) HeLa cells transfected with control or Atg7 siRNA for 48 hours were subjected to a second round of transfection with GFP-Atg5 together with the siRNA for another 24 hours. Then the cells were fixed following saponin extraction to visualise the GFP-Atg5 structures (green). (b) HeLa cells transfected with control vector or DN-Rab5 together with HA-Atg12 and Atg5 were subjected to western blot analysis with anti-HA antibody to detect free Atg12 and the Atg5-Atg12 complex. The ratio of Atg5-Atg12 versus free Atg12 is shown in the graph. Data are from four independent experiments; ***P<0.0001. (c) Atg5-Atg12:Atg12 ratio obtained from experiments similar to those shown in b, but performed in the presence or absence (control) of 3-methyladenine (3-MA; four independent experiments; left), or with control siRNA or siRNA targeting Vps34 (three independent experiments; right); ***P<0.0001, **P<0.001. (d) HeLa cells transfected simultaneously with siRNA targeting Rab5a, Rab5b and Rab5c (Rab5; left), Atg7 (right) or control siRNA for 48 hours were subjected to a second round of transfection with Atg5 and HA-Atg12 together with the Rab5 or Agt7 siRNA for another 24 hours. Western blot analysis was performed using anti-HA antibody to detect free Atg12 and the Atg5-Atg12 complex. Representative images from two independent, reproducible experiments are shown. (e) Immunostaining of HeLa cells transfected with either control (in the prescence or absence of 3-MA) or DN- Rab5 and HA-Atg12 for 24 hours after saponin extraction with anti-HA antibody (red). DAPI stained nuclei are in blue. (f) Immunostaining of HeLa cells transfected with control (with or without 3-MA) or DN-Rab5 for 24 hours after saponin extraction with anti-Atg12 antibody (red). DAPI stained nuclei are in blue. Arrows indicate punctate Atg12 structures in control cells. Quantification of cells with the punctate Atg12 structures in control, 3MA or DN-Rab5 in percent are given in the bar graph. ***P<0.0001. Journal of Cell Science Rab5 regulates toxicity of mutant huntingtin via autophagy 1655 (which is activated by Rab5) have a range of similar phenotypes: negative inhibition of Rab5 nor 3MA treatment significantly suppression of autophagy, increased numbers of Atg5-positive decreased the percentage of cells with these punctate endogenous structures and decreased Atg5-Atg12 conjugation. Atg12 structures (Fig. 3f). The significance of the Atg12 re- As both Rab5 and Vps34 are likely to regulate Atg5-Atg12 distribution is still a matter of ongoing research, but leads to the conjugation, which, in turn, regulates formation of autophagic hypothesis that Rab5 inhibition or 3MA treatment affects the vacuoles from Atg5-rich phagophores, we also looked at the accessibility of Atg12 for the conjugation process, probably by re- distribution of Atg12 in saponin-extracted cells with Rab5 distributing it away from its normal cellular localisation. inhibition. In control cells, overexpressed Atg12 was located in a Alternatively, the decrease in Atg5-Atg12 conjugation that we single large juxtanuclear domain (Fig. 3e), which did not change observed with Rab5 inhibition or 3MA treatment might result in with the presence or absence of Atg5 overexpression (data not aberrant accumulation of unconjugated Atg12. shown). However, with Rab5 inhibition, Atg12 was re-distributed Rab5 is found in a macromolecular complex containing Vps34 to several peripheral small punctate structures (Fig. 3e), a and beclin 1 phenomenon also seen with 3MA treatment (Fig. 3e). Similarly, Since a macromolecular complex containing Vps34 and beclin 1 when we looked at the distribution of endogenous Atg12, it was distributed in a single juxtanuclear domain (reminiscent of the yeast regulates the early nucleation step in autophagy, we next tested PAS) in wild-type cells (Fig. 3f). However, neither dominant- whether Rab5 was found in such a complex. We tested whether Rab5 interacted with beclin 1, because both proteins interact with Vps34 (Christoforidis et al., 1999; Kihara et al., 2001a), beclin 1 is associated with phagophores, and our data suggests that Rab5 acts at the autophagosome precursor stage and is also associated with such structures (Fig. 2g). In 3MA- treated cells, we observed colocalisation of endogenous Rab5 and beclin 1 (Fig. 4a), particularly in structures similar to those that accumulated when Rab5 or Vps34 were inhibited (e.g. Fig. 2). These putative autophagosome-precursors are precisely where we expected such colocalisation to occur. We also found strong colocalisation of beclin 1 with an activated Rab5 mutant (Fig. 4b). We used activated Rab5 because this forms large clear vesicles that allow unambiguous visualisation of membrane colocalisation of Rab5 with other proteins, a strategy frequently used for Rab5- interactor immunocytochemistry studies (Shin et al., 2005). We next looked for interaction of endogenous Rab5 with beclin 1 by immunoprecipitation. Rab5 interacted with beclin 1 (immunoprecipitated for beclin 1 and detected for Rab5) only in the presence of Vps34 suggesting that Rab5 is part of the macromolecular complex containing beclin 1 and Vps34 (Fig. 4c). Indeed, beclin 1 knockdown (supplementary material Fig. S9) enhances mutant huntingtin (Q74) aggregation (Fig. 4d) (Shibata et al., 2006), which is compatible with the knowledge that this will impair autophagosome formation. Crucially, beclin 1 knockdown led to decreased Atg5-Atg12 conjugation (Fig. 4e), which is compatible with the concept that both beclin 1 and Rab5 activity are required for Vps34 function in autophagosome formation. Thus, Rab5 activation of Vps34 probably regulates Fig. 4. Rab5 is part of a macromolecular complex containing Vps34 and beclin 1. (a) Colocalisation of endogenous Rab5 (green) and endogenous beclin 1 (red) in 3MA-treated HeLa cells after saponin extraction. DAPI-stained nuclei are in blue. (b) Colocalisation of constitutive active Rab5 (CA-Rab5; green) with wild-type beclin 1 (WT-Beclin, red). (c) COS-7 cells transfected with control vector alone (lane 1) or Flag-tagged wild-type beclin 1 alone (lane 2) or wild-type beclin 1 with wild-type Vps34 (lane 3) were immunoprecipitated using anti-Flag antibody (to immunoprecipitate beclin 1) and blotted for Rab5 using an anti-Rab5 antibody. (d) Q74-HA aggregation in HeLa cells transfected with control siRNA or siRNA targeting beclin 1 as described in Fig. 2d; P<0.005. (e) Ratio of Atg5-Atg12 conjugate to Atg12 (data from three independent experiments) in HeLa cells transfected with control siRNA or siRNA targeting beclin 1 for 72 hours. Atg5 and HA-Atg12 were transfected for the last 24 hours. P=0.0003. Journal of Cell Science 1656 Journal of Cell Science 121 (10) not only endosome maturation and multivesicular body formation we found decreased colocalisation of LC3-labelled vesicles with but also, through recruitment of beclin 1, initiation of autophagic the lysosomal marker lgp120, similar to colocalisation observed in vacuole formation. cells treated with BafA1 (Fig. 5e). Previous studies have shown that autophagosomes may fuse with endosomes to form intermediary The effect of Rab5 on autophagy is not due to a general compartments called amphisomes, which subsequently fuse with inhibition of the endocytic pathway lysosomes (Berg et al., 1998). Thus, it is possible that inhibition of Since Rab5 is an important regulator of the endocytic pathway, we the endocytic flux prevents the formation of amphisomes, thereby further tested whether the effects we saw with Rab5 inhibition are also inhibiting a subsequent step in the autophagic pathway, indirect consequences of endocytosis inhibition. We first perturbed amphisome-lysosome fusion. Again, unlike Rab5, we did not see the function of dynamin, a large GTPase essential for clathrin- any increase in the Atg5 structures (supplementary material Fig. mediated endocytosis, using dominant-negative dynamin (DN-Dyn). S10) nor could we observe any change in the ratio of Atg5-Atg12 DN-Dyn increased the percentage of COS-7 cells with aggregates complex to free Atg12 when dynamin was inhibited (Fig. 5f). and cell death similar to DN-Rab5 (Fig. 5a). However, unlike Rab5, These results suggest that DN-Dyn inhibits autophagy at the level overexpression of DN-Dyn increased the size and also the number of autophagic-lysosomal delivery, which is similar to what has been of LC3-positive autophagic vacuoles (Fig. 5b-d) similar to what we previously reported using Vps4, an AAA ATPase involved in and others observe after treatment with BafA1 (Bampton et al., trafficking through the endocytic pathway (Nara et al., 2002). 2005). This suggests that DN-Dyn inhibits autophagy at the level Overexpression of dominant-negative Vps4 showed defects in of autophagic-lysosomal delivery similar to BafA1. Accordingly, autophagy-dependent bulk protein degradation due to an impairment Fig. 5. (a) Quantification of GFP-expressing COS-7 cells with aggregates and abnormal nuclear morphology transiently transfected with dominant-negative dynamin II (DN-Dyn-II) or empty vector control and EGFP-tagged huntingtin exon 1 containing 74 polyglutamine repeats (3:1 ratio) for 48 hours. ***P<0.0001. (b) Distribution of GFP-LC3 vesicles in control (left) or DN-dynamin (red) transfected cells (right). (c) COS-7 cells were transiently transfected with empty vector (Control), DN-dynamin (DN-Dyn) and GFP-LC3 (3:1 ratio) for 24 hours. GFP-positive cells with an increased number of LC3-positive vesicles (>20 vesicles per cell) were quantified; ***P<0.0001. (d) Lysates from HeLa cells expressing empty vector control (Cont) or DN-dynamin (DN-Dyn) were blotted for endogenous LC3, and actin as control. Quantification of band intensities from four independent experiments is shown; **P<0.001. Under exposure conditions that allow endogenous LC3-II quantification in these cells, the LC3-I signal is frequently too low to be detected. (e) NRK cells were transiently transfected for 15 hours with either empty vector [control; with or without bafilomycin A1 (BafA1)] or DN-dynamin (DN-Dyn), and mRFP-LC3 and GFP-lgp120. mRFP-LC3 and GFP-lgp120 double-stained vesicles in individual cells are given in percent (Jahreiss et al., 2008). ***P<0.0001. (f) HeLa cells transfected with control vector or DN-dynamin (DN-Dyn) together with HA-Atg12 and Atg5 were subjected to western blot analysis using anti-HA antibody to detect free Atg12 and the Atg5-Atg12 complex. Atg5-Atg12:Atg12 ratio from three independent experiments is shown. (g) Quantification of GFP-expressing COS-7 cells with aggregates and abnormal nuclear morphology transiently transfected with dominant-negative (DN) Vps4 or empty vector control and EGFP-tagged huntingtin exon 1 containing 74 polyglutamine repeats (3:1 ratio) for 48 hours. ***P<0.0001. Journal of Cell Science Rab5 regulates toxicity of mutant huntingtin via autophagy 1657 in the formation of autolysosomes (Nara et al., 2002). Similar to of LC3-labelled autophagic vacuoles (Fig. 6b,c), did not increase our results for DN-Dyn we did not see an increase in Atg5 structures the number of Atg5 structures (data not shown) and had no effect (data not shown) or decrease in the levels of the Atg5-Atg12 on Atg5-Atg12 conjugation (Fig. 6d). Finally, we also looked at complex with DN-Vps4 (supplementary material Fig. S11) but, the effect of the endocytosis inhibitor methyl-β-cyclodextrin (β- similar to experiments using DN-Dyn (Fig. 5a) or BafA1 CD) on LC3-II levels both in the presence or absence of BafA1 (Ravikumar et al., 2002), we observed an increase in the aggregation (supplementary material Fig. S13). β-CD increased the levels of and associated cell death of huntingtin Q74 in the presence of DN- LC3-II in the absence of BafA1, but had no effect when BafA1 Vps4 (Fig. 5g). was present (Fig. 6e); which again suggests that inhibition of Results obtained using DN-Dyn were identical to those when endocytosis blocks autophagic-lysosomal delivery. DN-Dyn and using siRNA targeting clathrin heavy chain (supplementary material siRNA targeting clathrin heavy chain both had similar effects on Fig. S12), which is involved in formation of clathrin-coated vesicles. LC3-II levels in the presence of BafA1 similar to β-CD siRNA targeting clathrin heavy chain increased the percentage of (supplementary material Figs S14, S15). Thus, Rab5 inhibition, HeLa cells with aggregates (Fig. 6a), increased the number and size which impairs Atg5-Atg12 conjugation, autophagosome (and Fig. 6. (a) HeLa cells transfected with control siRNA or siRNA targeting clathrin heavy chain for 48 hours were subsequently transfected with EGFP-tagged huntingtin exon 1 containing 74 polyglutamine repeats (Q74) for 24 hours. Quantification of Q74 expressing cells with aggregates is shown in the graph. *P<0.01. (b) HeLa cells stably expressing GFP-LC3 were transfected with control or clathrin siRNA for 72 hours, numbers of LC3-positive vesicles were counted (>20 vesicles per cell); ***P<0.0001. (c) HeLa cells transfected with control siRNA or siRNA targeting clathrin heavy chain for 72 hours were subjected to western blot analysis using anti-LC3 and anti-actin antibodies. Under exposure conditions that allowed endogenous LC3-II quantification in these cells, the LC3-I signal was frequently too low to be detected. Quantification of band intensities from three independent experiments is shown. *P<0.05. (d) HeLa cells transfected with control siRNA (Cont) or siRNA targeting clathrin heavy chain (Cla) for 48 hours were subsequently transfected with HA-Atg12 and Atg5 for a further 24 hours. Western blot analysis was performed with anti- HA antibody to detect free Atg12 and the Atg5- Atg12 complex. The Atg5-Atg12:Atg12 ratio from three independent experiments is shown. (e) HeLa cells stably expressing GFP-LC3 were left untreated (Cont) or treated with 5 mM methyl-β-cyclodextrin (β-CD), in the presence (+) or absence (–) of bafilomycin A1 (BafA1) for 6 hours were subjected to western blot analysis using anti-GFP (to detect LC3) and anti-actin antibodies. (f) Lysates from HeLa cells transfected with empty vector (Cont), DN-Rab5 or CA-Rab5 were blotted for phosphorylated (S6-P) and total (S6-T) ribosomal protein S6. Lysate from cells treated with rapamycin (Rap) was used as a positive control. (g) HeLa cells transfected with control vector (Cont) or wild- type Rheb together with HA-Atg12 and Atg5 were subjected to western blot analysis with anti-HA antibody to detect free Atg12 and the Atg5-Atg12 complex. The Atg5-Atg12:Atg12 ratio from three independent experiments is shown. (h) Schematic hypothetical representation of how autophagy is regulated by Rab5. Our data suggest that the Atg5 structures are probably precursors of the pre- autophagosomal structures. The accumulation of Atg5 structures that were observed by us following inhibition of Rab5 or Vps34 might be owing to a block in the progression from early Atg5-positive autophagosomal structures to the formation of autophagic vacuoles. Journal of Cell Science 1658 Journal of Cell Science 121 (10) LC3-II) formation, and which is associated with an accumulation autophagosome formation) also resulted in increased numbers of of Atg5-positive structures, has different effects to a range of Atg5-positive autophagosome precursors. endocytosis inhibitors (such as β-CD, DN-Dyn, DN-Vps4 or siRNA Our data do not exclude the possibility that membranes for targeting clathrin) that do not affect Atg5-Atg12 conjugation but autophagosome biogenesis are derived from endosomes. However, block autophagic-lysosomal delivery and increase LC3-II levels. the effect of Rab5 inactivation on Atg5-Atg12 conjugation and (The specificity of the effect of Rab5 inhibition versus endocytosis autophagosome synthesis is not seen when using a wide range of inhibition on LC3-II levels has been clearly demonstrated in our molecules that inhibit endocytosis (β-CD, DN-Dyn, DN-Vps4 and experiments where we have treated cells with BafA1.) This argues siRNA targeting clathrin) that instead impede autophagic flux by that the effect on autophagosome formation owing to the loss of inhibiting autophagosome-lysosome fusion directly or by inhibiting Rab5 function is not due to effects on endocytosis. the autophagosome-endosome fusion step. This suggests that inhibition of endocytosis through different mechanisms will also Rab5 effect on autophagy is not due to mTOR signalling enhance aggregation and toxicity of polyglutamine by blocking Previous studies in Drosophila have shown that disruption of autophagy. It is possible that the loss of Rab5 activity has effects endocytosis can lead to changes in the target of rapamycin (TOR) on autophagy by perturbing other unrelated or unknown membrane signalling, a key process regulating autophagy (Hennig et al., 2006). trafficking pathways (distinct from endocytosis). However, it is Since Rab5 regulates endocytosis, we tested whether the conditions important to point out that overexpression of CA-Rab5 or WT-Rab5 of Rab5 inhibition or activation alters the mTOR-signalling pathway. enhanced autophagosome synthesis and suppressed aggregation and mTOR directly phosphorylates at least two effectors: S6 kinase-1 toxicity of mutant huntingtin in tissue culture cells and in vivo. and 4EBP1. S6 kinase-1 phosphorylates the ribosomal protein S6. We believe that our data suggest a sequential model in The levels of phosphorylation of any of these downstream targets mammalian cells, in which PtdIns(3)P generated by Vps34, in a are recognised indicators of mTOR activity in the cell (Jacinto and complex that comprises at least beclin 1 and active Rab5, is a key Hall, 2003). Accordingly, we tested whether DN-Rab5 or CA-Rab5 regulator of Atg12 conjugation to Atg5, a rate-limiting step in the altered the mTOR pathway by looking at the phosphorylation levels conversion of Atg5-positive autophagosome precursors to Atg5- of S6. We did not observe any changes in the phosphorylation of negative autophagosomes. On the one hand, inhibition of this S6 (Fig. 6f). We also did not see any changes in the phosphorylation putative cascade at a number of points will lead to impaired levels of S6 kinase-1 or 4EBP1 using DN-Rab5 or CA-Rab5 (data autophagy and enhance polyglutamine toxicity. On the other hand, not shown). Thus, the effects we observed with Rab5 were not owing better understanding of the initial rate-limiting steps of autophagy to alterations in the mTOR signalling. Furthermore, when mTOR may provide opportunities for the rational design of more specific signaling was activated by overexpressing Ras homolog enriched and safer autophagy-inducing therapeutic drugs than rapamycin in brain (Rheb; see supplementary material Fig. S16) we did not (which affects many pathways). This may be of relevance to HD observe the same effects we observed when expressing DN-Rab5; and also to a range of related neurodegenerative diseases caused we did not detect an increase in Atg5 structures (data not shown) by intracytosolic aggregate-prone proteins. and did not see any decrease in the Atg5-Atg12 conjugate compared with the pool of unbound Atg12 (Fig. 6g). Taken together, our results Materials and Methods suggest that the role of Rab5 on autophagy is independent of its Mammalian cell culture and transfection effects on endocytosis and is not because of perturbations in mTOR COS-7 and HeLa cells were grown in Dulbecco’s modified Eagle’s medium (DMEM) (Sigma) supplemented with 10% foetal bovine serum (FBS) (Sigma), 100 U/ml signalling. penicillin/streptomycin (Sigma), 2 mM L-glutamine (Sigma) at 37°C, 5% CO . DNA transfections were performed using lipofectamine reagent (Invitrogen). 20 nmol of Conclusion control (cat. no. 4611), Atg7 (ID 135756), Rab5a (ID 120372), Rab5b (ID 120273), Rab5c (ID 120809), clathrin heavy chain (ID 107565), Vps34 (ID 143802) or beclin In conclusion, we show that Rab5 can modify the toxicity of (ID 137200) siRNA (all from Ambion) were transfected using lipofectamine 2000 polyglutamine in cell and fly models. These modifying effects are according to manufacturer’s instructions. due to Rab5 regulating autophagy-dependent clearance of the toxic huntingtin mutant protein. Our data suggest that Rab5, previously Western blot analysis Western blot analysis was carried out applying standard techniques using the ECL considered as a specific endosome marker, also influences detection kit (Amersham). The primary antibodies used include anti-GFP (Clontech), mammalian autophagy. Inhibition of Rab5 resulted in a decrease anti-HA (Covance), anti-Myc and anti-actin (Sigma), anti-Atg7 (Rockland Inc.), anti- in LC3-positive vesicles suggesting a defect in the formation of Atg5 and anti-Rab5 (Abcam), anti-Vps34 (Zymed) and anti-beclin (Cell Signaling autophagic vacuoles. Rab5 is an activator of Vps34, a PI 3-kinase technology). Densitometry analysis was performed using Image J 1.36b or Scion Image Beta 4.02 softwares. For immunoprecipitation, cells were suspended in lysis essential for autophagy initiation, and we have shown that Rab5 is buffer (50 mM Tris HCl pH 7.4, 150 mM NaCl, 1 mM EDTA and 1% Triton X-100) a new member of a complex that contains Vps34 and beclin 1, and for 30 minutes on ice and supernatants were removed by centrifugation at 13,000 that is associated with autophagosome precursors. Thus, the most rpm in a tabletop centrifuge for 7 minutes at 4°C. 30 μl of anti-M2 affinity gel (Sigma) was added to the sample and incubated at 4°C for 2 hours with gentle rocking. After parsimonious explanation of our data is that Rab5 acts as an activator incubation, tubes were spun for 30 seconds at 4°C at less than 4000 g. Pellets were for Vps34 in autophagy as it is known to do in endocytosis. This washed four times with 1 ml of chilled buffer A (20 mM Tris HCl pH 7.2, 2 mM hypothesis is consistent with the various similarities we observed MgCl ,150 mM NaCl, 0.5% Nonidet P-40). Bound protein was eluted with 100 μl of 150 ng/μl 3FLAG peptide in TBS. in cells expressing loss-of-function of Rab5 or Vps34. Loss of either enzyme activity decreased Atg5-Atg12 conjugation, a crucial step Immunocytochemistry in early phagophore elongation. We speculate that accumulation of Immunocytochemistry was performed in HeLa cells transfected with GFP-Atg5, Atg5 structures concomitantly with Rab5 or Vps34 inhibition is extracted using 0.02% saponin and fixed using 4% paraformaldehyde. Saponin was prepared using PHEM buffer (60 mM NaPIPES, 25 mM NaHEPES, 10 mM EGTA owing to a block in their progression from early Atg5-positive 2 mM MgCl , pH 6.9) supplemented with 0.19 M NaCl. Primary antibodies used were autophagosomal structures to the formation of autophagosomes (Fig. anti-Myc, anti-Flag (Sigma), anti-Rab5 and anti-HA. Relevant negative controls without 6h). This block might be due to a defect in Atg12 recruitment. primary antibodies were performed alongside all experiments. Nuclei were stained Indeed, decreasing Atg5 conjugation (which is known to block with 46-diamidino-2-phenylindole (DAPI, 3 mg/ml; Sigma). Images were acquired Journal of Cell Science Rab5 regulates toxicity of mutant huntingtin via autophagy 1659 –/– using a Nikon Digital Camera DXM1200 and a Nikon Eclipse E600 fluorescence Japan) for HA-Atg12 and Atg5; Atg5 and WT-MEF cells; A. Sorkin microscope. We used Nikon ACT-1 version 2.12 acquisition software. Adobe (University of Colorado Health Sciences Center, Denver, CO) for Photoshop 6.0 (Adobe Systems, Inc.) was used for subsequent image processing. N34S and Q79L Rab5; J. Stankova (University of Sherbrooke, Québec, Canada) for K44A Dynamin; X. Wang (The University of Quantification of aggregate formation, abnormal nuclear Utah, Salt Lake City, UT) for DN-Vps4B; Eléonore Mayola for morphologies, LC3-positive vesicles technical support. 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Rab5 modulates aggregation and toxicity of mutant huntingtin through macroautophagy in cell and fly models of Huntington disease

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The Company of Biologists
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© 2021 The Company of Biologists. All rights reserved.
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0021-9533
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0021-9533
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10.1242/jcs.025726
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Abstract

Research Article 1649 Rab5 modulates aggregation and toxicity of mutant huntingtin through macroautophagy in cell and fly models of Huntington disease 1 1,2 1 2 1, Brinda Ravikumar , Sara Imarisio , Sovan Sarkar , Cahir J. OʼKane and David C. Rubinsztein * Department of Medical Genetics, Cambridge Institute for Medical Research, Wellcome/MRC Building, Addenbrookeʼs Hospital, Hills Road, Cambridge, CB2 0XY, UK Department of Genetics, University of Cambridge, CB2 3EH, UK *Author for correspondence (e-mail: dcr1000@hermes.cam.ac.uk) Accepted 21 February 2008 Journal of Cell Science 121, 1649-1660 Published by The Company of Biologists 2008 doi:10.1242/jcs.025726 Summary Huntington disease (HD) is caused by a polyglutamine- endocytosis by various means suppressed autophagosome- expansion mutation in huntingtin (HTT) that makes the protein lysosome fusion (the final step in the macroautophagy pathway) toxic and aggregate-prone. The subcellular localisation of similar to bafilomycin A1. Thus, Rab5, which has previously huntingtin and many of its interactors suggest a role in been thought to be exclusively involved in endocytosis, has a endocytosis, and recently it has been shown that huntingtin new role in macroautophagy. We have previously shown that interacts indirectly with the early endosomal protein Rab5 macroautophagy is an important clearance route for several through HAP40. Here we show that Rab5 inhibition enhanced aggregate-prone proteins including mutant huntingtin. Thus, polyglutamine toxicity, whereas Rab5 overexpression attenuated better understanding of Rab5-regulated autophagy might lead toxicity in our cell and fly models of HD. We tried to identify to rational therapeutic targets for HD and other protein- a mechanism for the Rab5 effects in our HD model systems, conformation diseases. and our data suggest that Rab5 acts at an early stage of autophagosome formation in a macromolecular complex that Supplementary material available online at contains beclin 1 (BECN1) and Vps34. Interestingly chemical http://jcs.biologists.org/cgi/content/full/121/10/1649/DC1 or genetic inhibition of endocytosis also impeded macroautophagy, and enhanced aggregation and toxicity of Key words: Huntington disease, Autophagy, Rab5 mutant huntingtin. However, in contrast to Rab5, inhibition of Introduction which later fuse with lysosomes, where their contents are degraded Huntington disease (HD) is a late-onset inherited neurodegenerative (Klionsky and Ohsumi, 1999). It is believed that isolation condition caused by an expansion of CAG trinucleotide repeats (>35 membranes (phagophores) – which later give rise to repeats) in exon 1 of the IT15 gene. The mutation results in the autophagosomes – are formed at the phagophore-assembly site (also production of an abnormally long polyglutamine (polyQ) tract in referred to as pre-autophagosomal structures, PAS) in yeast (Suzuki the N-terminus of the huntingtin (HTT) protein (Rubinsztein, et al., 2001). PAS however have not been defined in mammalian 2002). Full-length mutant huntingtin undergoes several proteolytic autophagy. Several ‘Atg’ (autophagy) genes that regulate yeast cleavages, which give rise to N-terminal fragments comprising the autophagy have been identified, and many of these genes have first 100-150 residues containing the expanded polyQ tract. These mammalian orthologues. A protective role of autophagy has been N-terminal fragments are also the toxic species found in aggregates implicated in some neurodegenerative disorders, cancers and seen in HD mouse models and human brains (Lunkes et al., 2002). infectious diseases (Rubinsztein et al., 2007). Conditional knockouts Thus, HD pathogenesis is frequently modelled with fragments of of the key autophagy genes Atg5 or Atg7 in the brains of mice result exon 1 that contain expanded polyQ repeats, which cause toxicity in a neurodegenerative phenotype caused by aberrant accumulation and the formation of aggregates in cell models and in vivo of ubiquitinated proteins (Hara et al., 2006; Komatsu et al., 2006). (Rubinsztein, 2002). In addition to HD, where enhanced clearance of mutant huntingtin A key pathway that regulates the degradation of aggregate-prone fragments through induction of autophagy attenuates toxicity caused cytosolic proteins such as mutant huntingtin (both full-length and by mutant huntingtin in cell, fly and mouse models (Ravikumar et exon 1 forms) is macroautophagy – henceforth referred to simply al., 2002; Ravikumar et al., 2004), this pathway appears to be equally as autophagy (Ravikumar et al., 2002; Ravikumar et al., 2004; important for the clearance of several other intracellular proteins Shibata et al., 2006). Wild-type huntingtin (full-length or exon 1) that cause neurodegeneration, including the A53T point mutation has a very low dependence on autophagy for its clearance. in α-synuclein that causes autosomal-dominant Parkinson disease Autophagy, a process conserved from yeast to human, typically (Berger et al., 2006; Ravikumar et al., 2002; Webb et al., 2003). clears long-lived proteins and organelles by engulfing cytoplasmic The subcellular localisation of huntingtin, the nature of many of contents in double-membrane structures called autophagosomes, its normal interactors and its indirect interaction with Rab5 through Journal of Cell Science 1650 Journal of Cell Science 121 (10) HAP40 (Pal et al., 2006), suggested that it may have roles in of visible rhabdomeres in the double transgenic flies. Degeneration endocytosis. Rab5, a member of the small GTPase family, is a key of photoreceptors due to Q120 overexpression was greatly rescued regulator of the early endocytic pathway in mammalian cells (Bucci by overexpression of Rab5-EGFP (Fig. 1d). et al., 1992; Stenmark et al., 1994). Accordingly, we initially tested Since our previous studies suggest that aggregation of mutant the role of Rab5 in the regulation of toxicity of mutant huntingtin. huntingtin can be regulated by autophagy (Ravikumar et al., 2002), We show that Rab5 inhibition enhances toxicity induced by mutant we next tested whether the effects of Rab5 on Q74 aggregation are +/+ ) or autophagy- polyQ, whereas Rab5 overexpression attenuates mutant polyQ autophagy-dependent using either wild-type (Atg5 –/– ) mouse embryonic fibroblasts toxicity in HD cell and fly models. Our data suggest that Rab5, in deficient Atg5-knockout (Atg5 addition to its role in endocytosis, modifies aggregation and toxicity (MEFs) (Mizushima et al., 2001). As previously observed with of mutant huntingtin by having a novel role in the early stage of autophagy inhibitors (Ravikumar et al., 2002), Q74 aggregation was –/– +/+ compared with Atg5 autophagosome formation. Interestingly, inhibition of endocytosis higher in autophagy-incompetent Atg5 MEFs (Fig. 1e). We observed minimal Q74-induced cell death in by a variety of means also influenced the toxicity of mutant huntingtin by inhibiting autophagy at a downstream step the MEFs under the transfection conditions we used. As expected, (autophagosome-lysosome fusion) that is distinct from Rab5 overexpression of DN-Rab5 caused an increase and that of CA- inhibition. A possible therapeutic approach for HD and other Rab5 a decrease in Q74 aggregation in wild-type MEFs (Fig. 1f, proteinopathies would thus be the enhancement of Rab5 activity. left), similar to the results seen in COS-7 cells (Fig. 1b). However, overexpression of DN-Rab5 or CA-Rab5 had no effects on Q74 –/– MEFs (Mizushima Results and Discussion aggregation in autophagy-incompetent Atg5 Rab5 modifies polyglutamine toxicity and aggregation et al., 2001) (Fig. 1f, right). Furthermore, Rab5 inhibition abrogated We initially tested whether Rab5 can modify the toxicity of a mutant the ability of the autophagy enhancers, the mTOR inhibitor huntingtin exon 1 fragment with 74 polyQ repeats (Q74) (Narain rapamycin or the inositol monophosphatase inhibitor L-690,330, et al., 1999) in COS-7 cells. Dominant-negative inhibition of Rab5 which target mTOR-dependent or -independent pathways, by overexpression of a GTP-binding-defective Rab5 mutant carrying respectively, to clear Q74 aggregates (Fig. 1g; supplementary a S34N point mutation (DN-Rab5) (Stenmark et al., 1994) increased material Fig. S2) (Ravikumar et al., 2002; Sarkar et al., 2005). This the toxicity of Q74 (Fig. 1a), whereas overexpression of wild-type suggests that the effect of Rab5 on Q74 aggregation and toxicity (WT) Rab5 or of a constitutively active (CA) Rab5 mutant carrying were due to alterations in the autophagic pathway rather than its a Q79L mutation (CA-Rab5) (Stenmark et al., 1994) significantly role in endocytosis. decreased Q74-induced toxicity (Fig. 1a). Rab5 regulates autophagosome formation in mammalian cells We then examined the effect of Rab5 on aggregation of mutant Since the above data suggest a possible role of Rab5 on autophagy, huntingtin. Aggregation of mutant huntingtin correlates with its expression levels (Narain et al., 1999). Also, the proportion of cells we tested whether Rab5 influenced the formation of with inclusions formed by mutant huntingtin fragments generally autophagosomes. Two ubiquitin-like modifications are involved in correlates with its toxicity in cell culture models (although inclusions the expansion and completion of autophagosome formation. The may not themselves be as toxic as diffusely distributed mutant first involves conjugation of Atg12 to Atg5 in a reaction that requires huntingtin) (Arrasate et al., 2004; Ravikumar et al., 2002). DN- Atg7 (E1-like) and Atg10 (E2-like). Atg5-Atg12 conjugates are Rab5 increased Q74 aggregation, whereas CA-Rab5 and WT-Rab5 localised onto the PAS and dissociate upon completion of decreased the proportions of Q74-expressing cells with inclusions autophagosome formation. The second modification involves (Fig. 1b), mirroring the toxicity data (Fig. 1a). We next confirmed conjugation of microtubule-associated protein 1 light chain 3 that the effects of DN-Rab5 were mirrored by depletion of (hereafter referred to as LC3 and also known as MAP1LC3 or Atg8) endogenous Rab5 using RNA interference (RNAi). There are three to phosphatidylethanolamine (PE). LC3 (cytosolic) is cleaved at its isoforms of Rab5, namely Rab5A, Rab5B and Rab5C. Small C-terminus by Atg4 to form LC3-I. LC3-I is covalently conjugated interfering RNA (siRNA) that target individual Rab5 isoforms to PE to form LC3-II, a process requiring the activities of Atg7 and significantly increased Q74 aggregation, whereas simultaneous Atg3. LC3-II (membrane associated) is specifically targeted to Atg5- knockdown of all three isoforms increased Q74 aggregation even Atg12-associated, expanded phagophores and remains associated further (Fig. 1c, supplementary material Fig. S1). Thus, Rab5 with autophagosomes even after fusion with lysosomes, after which modulation affects aggregation and toxicity of the mutant huntingtin LC3-II can be delipidated and recycled. LC3 is the only known fragments. protein that specifically associates with autophagosomes and not We next tested whether Rab5 can modify the toxicity of mutant with other vesicular structures (Kabeya et al., 2000). Thus, LC3-II huntingtin in vivo using a Drosophila melanogaster HD model. Fly levels correlate with the numbers of autophagic vacuoles, which photoreceptors that express a mutant huntingtin fragment with 120 can also be assessed by counting LC3-positive vesicles (Kabeya et polyQ repeats (Q120) exhibit degeneration that is not observed in al., 2000). We first tested the effect of Rab5 on LC3. Whereas, flies that express the wild-type fragment with 23 polyQ repeats inhibition of Rab5 decreased the proportion of COS-7 cells with (Jackson et al., 1998). The Drosophila compound eye consists of >20 LC3-labelled autophagic vesicles (Fig. 2a), overexpression of many ommatidia, each comprising eight photoreceptor neurons with CA-Rab5 or WT-Rab5 significantly increased the proportion of cells light-gathering parts called rhabdomeres, seven of which can be with >20 LC3-positive autophagic vesicles (Fig. 2a). However, we visualised by light microscopy using the pseudopupil technique did not observe any change in the size or morphology of the LC3 (Franceschini and Kirschfeld, 1971). Neurodegeneration in the HD vesicles under any of the above conditions (data not shown). The flies is progressive and is associated with a decrease in the number above results also correlated with decrease (upon DN-Rab5 of visible rhabdomeres in each ommatidium with time (Jackson et overexpression) or increase (upon CA-Rab5 or WT-Rab5 al., 1998). We crossed the HD flies with flies transgenic for wild- overexpression) with autophagosome-associated LC3-II levels on type Rab5 fused to EGFP (Rab5-EGFP) and looked at the number western blots when we blocked LC3-II clearance through inhibition Journal of Cell Science Rab5 regulates toxicity of mutant huntingtin via autophagy 1651 Fig. 1. Rab5 modulates the aggregation and toxicity of mutant huntingtin. (a) Quantification of GFP-expressing COS-7cells showing signs of cell death that had been transiently transfected with dominant-negative (DN), constitutive active (CA) or wild-type (WT) Rab5, or empty vector control, and the EGFP-tagged huntingtin exon 1 with 74 polyglutamine repeats (Q74) (at a 3:1 ratio) for 48 hours. ***P<0.0001, **P<0.001, *P<0.05. (b) Quantification of GFP-expressing COS-7 cells containing aggregates that were quantified for toxicity shown above. ***P<0.0001. (c) Quantification of GFP-expressing HeLa cells containing aggregates transiently transfected with siRNA targeting Rab5a, Rab5b, Rab5c or all three siRNA simultaneously (Rab5abc) for 72 hours, and also with EGFP-tagged huntingtin exon 1 with 74 polyglutamine repeats for the last 24 hours of the 72-hour siRNA transfection period. ***P<0.0001, *P<0.05. (d) Rab5 overexpression increases the numbers of rhabdomeres in ommatidia of mutant huntingtin-expressing flies. Frequency distribution of ommatidia with different numbers of rhabdomeres three days after eclosion (hatching) in progeny of flies that express mutant huntingtin exon 1 (gmrQ120) and that had been crossed to either a control stock (w ) (white minus; have huntingtin transgene only) or to Rab5- EGFP flies (have huntingtin and Rab5 transgenes). P0.001, t-test; P0.001, Mann-Whitney U test. The rhabdomere frequency of Rab5 flies crossed to a control stock is also shown. (e) Odds ratio of GFP-expressing cells with Q74 +/+ aggregates in wild-type (Atg5 ) vs –/– Atg5 knockout (Atg5 ) MEFs. ***P<0.0001. (f) Odds ratio of Q74- –/– expressing Atg5 (Atg5 knockout) or +/+ Atg5 (wild-type) MEF cells with aggregates, after transient transfection with dominant-negative Rab5 (DN- Rab5), constitutive active Rab5 (CA- Rab5) or empty vector control and EGFP-tagged huntingtin exon 1 with 74 polyglutamine repeats (Q74) (3:1 ratio) for 48 hours. ***P<0.0001. Odds ratios are given to compare pooled summary statistics across multiple independent experiments (see Materials and Methods). Control conditions are fixed at 1 in both cell lines to facilitate comparisons. (g) Quantification of GFP- expressing HeLa cells containing aggregates that had been transiently transfected with dominant-negative Rab5 (DN-Rab5) or empty vector (Cont) and huntingtin exon 1 with 74 polyglutamine repeats (Q74) (at a 3:1 ratio) for 48 hours and were either left untreated (–Rap) or treated with 0.2 μg/ml rapamycin (Rap) to induce autophagy. **P<0.001. Error bars in all graphs represent the s.e.m. of autophagosome-lysosome fusion using bafilomycin A1 (BafA1) autophagosome synthesis, rather than decreasing autophagosome- at 200 nM (Fig. 2b) (Sarkar et al., 2007). Further blockage of lysosome fusion and/or autophagosome degradation. autophagosome-lysosome fusion through a BafA1-independent Rab5 and its effector Vps34 regulate early steps of mechanism, using the dynein inhibitor erythro-9-[3-(2- autophagosome formation hydroxynonyl)] adenine (EHNA), together with this dose of BafA1, Autophagosome formation begins with a nucleation step during does not increase LC3-II compared with using BafA1 alone (Sarkar et al., 2007). Results shown in Fig. 2b suggest that Rab5 increased which membranes of unknown origin form phagophores; these then Journal of Cell Science 1652 Journal of Cell Science 121 (10) expand and fuse to form completed autophagosomes. The formation to LC3-I to form LC3-II. Atg5-Atg12 conjugates colocalise with K130R ) inhibits of autophagosome precursors is regulated by a macromolecular LC3-II on the PAS. Mutation of K130R in Atg5 (Atg5 complex that contains phosphoinositide 3-kinase class 3 (Vps34 or its conjugation with Atg12; as a result, membranes with K130R that do not colocalise with LC3-II PIK3C3), beclin 1 (BECN1, the human ortholog of yeast Atg6p), unconjugated Atg5 Atg14 and Vps15 (PIK3R4) (Kihara et al., 2001b). Vps34, which accumulate upon autophagy induction (Mizushima et al., 2001). The generates phosphatidylinositol-3-phosphate [PtdIns(3)P], directly Atg5-Atg12 conjugate only localises to phagophores and dissociates interacts with beclin 1 (Kihara et al., 2001a). 3-methyl adenine just before or after completion of autophagic vacuole formation (3MA) (Kovacs et al., 1998) or wortmannin (Blommaart et al., 1997) (Mizushima et al., 2001). So Atg5 and Atg12 are not associated inhibit PI 3-kinases, including Vps34, and block autophagy by with completed autophagosomes. preventing the formation of autophagosomes. Conjugation of Atg12 To investigate how Rab5 influenced early steps in autophagosome with Atg5 initiates the elongation process. This conjugation however formation, we investigated the distribution of GFP-tagged Atg5 is not required for membrane targeting of Atg5, but is necessary (GFP-Atg5), a marker for autophagosomal precursor structures for membrane elongation. This is followed by conjugation of PE (George et al., 2000; Mizushima et al., 2001). Cells were Fig. 2. (a) COS-7 cells were transiently transfected with empty vector (Control), DN- Rab5, CA-Rab5 or WT-Rab5 and GFP-LC3 or mRFP-LC3 (3:1 ratio) for 24 hours. GFP- positive or mRFP-positive cells with increased numbers of LC3-positive vesicles (>20 vesicles per cell) were counted. 29% of control cells had >20 vesicles per cell. ***P<0.0001. (b) Western blot analysis of COS-7 cells co-transfected with empty vector (C), DN-Rab5, CA-Rab5 or WT-Rab5 and Myc-LC3 for 24 hours in the presence of 200 nM bafilomycin A1 (treated for last 15 hours), using anti-Myc antibody. GFP was used as a transfection control. Representative image from three independent experiments; quantification of the band intensities from these experiments represented as LC3- II:GFP ratio is shown in the graph; *P<0.05. (c) Analysis of GFP-Atg5 structures (green) in HeLa cells transfected with control vector [either untreated or treated for 24 hours with 10 mM 3-methyladenine (3-MA)] or with dominant-negative Rab5 (DN-Rab5) and GFP-Atg5, after saponin extraction. Nuclei are shown in blue. An increased abundance of large punctate Atg5 structures can be noticed with DN-Rab5 and 3-MA treatment. (d) HeLa cells were transfected with siRNA targeting Vps34 or control siRNA for 48 hours after which GFP-Atg5 together with siRNA was transfected for further 24 hours. The cells were fixed following saponin extraction to visualise GFP-Atg5 (green) structures. Quantification of Q74-expressing HeLa cells with aggregates is shown in the graph. Cells were transiently transfected with control siRNA or siRNA targeting Vps34 for 48 hours and with HA-tagged huntingtin exon 1 containing 74 polyglutamine repeats for further 24 hours. ***P<0.0001. (e) Colocalisation of GFP- Atg5 structures (green) with Myc-tagged FYVE (red) in HeLa cells co-transfected with DN-Rab5, GFP-Atg5 and Myc-FYVE for 24 hours. (f) Colocalisation of GFP-Atg5 structures (green) with beclin 1 (red) in HeLa cells co-transfected with DN-Rab5, GFP- Atg5 and Flag-tagged wild-type (WT) beclin 1. (g) Colocalisation of endogenous Atg5 (red) and endogenous Rab5 (green) in HeLa cells treated with 3MA for 15 hours. In panels e-g we observed >30% colocalisation between GFP-Atg5 structures and saponin-extracted, membrane-associated, FYVE, beclin 1 or Rab5 in cells that expressed both of the respective proteins. (h) Colocalisation of GFP-Atg5 structures (green) with Atg12 (red) in HeLa cells co-transfected with DN-Rab5 and GFP-Atg5 and HA-tagged Atg12 for 24 hours. Nuclei labelled with DAPI are in blue. Journal of Cell Science Rab5 regulates toxicity of mutant huntingtin via autophagy 1653 permeabilised with saponin to remove soluble cytosolic contents or proteins associated with phagophores. A subset of the punctate and reveal membrane-associated GFP-Atg5. After Rab5 inhibition, Atg5 structures colocalised with a PtdIns(3)P marker, Myc-FYVE we noted an increased abundance of large punctate Atg5 structures (Fig. 2e) (Gaullier et al., 1998) and they also overlapped with (Fig. 2c). These contrast with the smaller and less abundant Atg5 beclin 1 (Fig. 2f) but did not colocalise with LC3 (supplementary puncta seen in the great majority of untreated cells (Fig. 2c). We material Fig. S5) suggesting that these were indeed early found similar results when we immunostained for endogenous Atg5 autophagic structures. We also did not observe any colocalisation (supplementary material Fig. S3). In control cells, however, these of the Atg5 structures with the Golgi markers p230 structures were rare (Fig. 2c; supplementary material Fig. S3). (supplementary material Fig. S6) (Derby et al., 2007) or golgin- Whereas saponin extraction precludes accurate quantitation (because 84 (Satoh et al., 2003) and the Atg5 in these structures was not it does not allow visualisation of transfected versus untransfected associated with the ER proteins BiP (Haas, 1994) or Grp94 (Argon cells), we saw these Atg5 structures in 3% of all control cells and and Simen, 1999) (data not shown). However, Rab5 associated in about 12% of Rab5-inhibited cells (an ~fourfold increase, with these Atg5-positive puncta, which is compatible with a role P<0.001 from triplicate slides). for Rab5 in early mammalian autophagy (Fig. 2g). We only Previous studies have shown that Rab5 interacts with and observed very rare colocalisation of Atg5 and Atg12 in such activates the PI 3-kinase Vps34 (Christoforidis et al., 1999). Thus, puncta (Fig. 2h). It should be noted that these experiments were we tested whether Vps34 inhibition had similar effects to Rab5 performed under constitutive autophagy conditions (full medium inhibition. As with Rab5 inhibition, inhibition of Vps34 with the containing serum and amino acids) in cells with Rab5 inhibition. PI 3-kinase inhibitors 3MA (Fig. 2c; supplementary material Fig. Rab5 inhibition or 3MA treatment was required to allow S3) or wortmannin (data not shown) (which are both established visualisation of sufficient numbers of these structures for blockers of autophagosome formation) or siRNA knockdown of characterisation. The accumulation of Atg5 structures by Vps34 Vps34 (Fig. 2d; supplementary material Fig. S4) resulted in or Rab5 inhibition might reflect a block in the progression of Atg5- increases of Atg5 structures. Similar to Rab5 inhibition, inhibition positive membranes to the formation of autophagic vacuoles, of Vps34 also increased the proportion of cells with Q74 which suggests a role of Rab5 in very early steps of autophagy. aggregates, consistent with autophagy inhibition (Fig. 2d) The observation that the Atg5 in these puncta was only rarely (Ravikumar et al., 2002). To further characterise the Atg5 associated with Atg12 led to the hypothesis that defective Atg5- structures we tested whether they colocalised with other markers Atg12 conjugation is causally related to puncta formation. Both Rab5 and Vps34 regulate Atg5- Atg12 conjugation The possibility that defective Atg5-Atg12 conjugation leads to defective autophagosome formation associated with an accumulation of phagophores enriched in unconjugated Atg5 was compatible with the similarity of the above results and the phenotypes seen in apg7 or apg12 yeast strains, which typically show one to five large Atg5-positive punctate structures per cell, (a phenomenon that is very rare in wild-type cells) (George et al., 2000). Thus, when we knocked down Atg7 (an E1-like enzyme crucial for Atg5-Atg12 conjugation; supplementary material Fig. S7) in mammalian cells, we observed increased GFP-Atg5 structures, consistent with those observed in yeast (Fig. 3a). These Atg5- positive structures also colocalised with beclin 1 (supplementary material Fig. S8). Accordingly, we tested whether the increase in Atg5 structures seen with Rab5 or Vps34 inhibition was associated with aberrant Atg5- Atg12 conjugation. Consistent with this hypothesis and with the only very rare colocalisation of Atg12 and Atg5 in the puncta that accumulated following inhibition of Rab5 (Fig. 2h), we found that the ratio of Atg5- Atg12 complex formed compared with the pool of unconjugated Atg12 was significantly lower in cells expressing DN-Rab5 (Fig. 3b). 3MA treatment and siRNA knockdown of Vps34 also had similar effects on Atg5-Atg12 Fig. 2e-h. See previous page for legend. conjugation compared with those seen with Journal of Cell Science 1654 Journal of Cell Science 121 (10) loss-of-function of Rab5 (DN-Rab5), a Vps34 activator (Fig. 3c, all Rab5 isoforms simultaneously (Fig. 3d, left). This effect was left and right respectively). We also found decreased levels of Atg5- similar to the conjugation defect we observed with Atg7 knockdown Atg12 complex compared with free Atg12 when we knocked down (Fig. 3d, right). Thus, Rab5 inhibition and loss of activity of Vps34 Fig. 3. Aberrant conjugation of Atg5 with Atg12 after Rab5 inhibition. (a) HeLa cells transfected with control or Atg7 siRNA for 48 hours were subjected to a second round of transfection with GFP-Atg5 together with the siRNA for another 24 hours. Then the cells were fixed following saponin extraction to visualise the GFP-Atg5 structures (green). (b) HeLa cells transfected with control vector or DN-Rab5 together with HA-Atg12 and Atg5 were subjected to western blot analysis with anti-HA antibody to detect free Atg12 and the Atg5-Atg12 complex. The ratio of Atg5-Atg12 versus free Atg12 is shown in the graph. Data are from four independent experiments; ***P<0.0001. (c) Atg5-Atg12:Atg12 ratio obtained from experiments similar to those shown in b, but performed in the presence or absence (control) of 3-methyladenine (3-MA; four independent experiments; left), or with control siRNA or siRNA targeting Vps34 (three independent experiments; right); ***P<0.0001, **P<0.001. (d) HeLa cells transfected simultaneously with siRNA targeting Rab5a, Rab5b and Rab5c (Rab5; left), Atg7 (right) or control siRNA for 48 hours were subjected to a second round of transfection with Atg5 and HA-Atg12 together with the Rab5 or Agt7 siRNA for another 24 hours. Western blot analysis was performed using anti-HA antibody to detect free Atg12 and the Atg5-Atg12 complex. Representative images from two independent, reproducible experiments are shown. (e) Immunostaining of HeLa cells transfected with either control (in the prescence or absence of 3-MA) or DN- Rab5 and HA-Atg12 for 24 hours after saponin extraction with anti-HA antibody (red). DAPI stained nuclei are in blue. (f) Immunostaining of HeLa cells transfected with control (with or without 3-MA) or DN-Rab5 for 24 hours after saponin extraction with anti-Atg12 antibody (red). DAPI stained nuclei are in blue. Arrows indicate punctate Atg12 structures in control cells. Quantification of cells with the punctate Atg12 structures in control, 3MA or DN-Rab5 in percent are given in the bar graph. ***P<0.0001. Journal of Cell Science Rab5 regulates toxicity of mutant huntingtin via autophagy 1655 (which is activated by Rab5) have a range of similar phenotypes: negative inhibition of Rab5 nor 3MA treatment significantly suppression of autophagy, increased numbers of Atg5-positive decreased the percentage of cells with these punctate endogenous structures and decreased Atg5-Atg12 conjugation. Atg12 structures (Fig. 3f). The significance of the Atg12 re- As both Rab5 and Vps34 are likely to regulate Atg5-Atg12 distribution is still a matter of ongoing research, but leads to the conjugation, which, in turn, regulates formation of autophagic hypothesis that Rab5 inhibition or 3MA treatment affects the vacuoles from Atg5-rich phagophores, we also looked at the accessibility of Atg12 for the conjugation process, probably by re- distribution of Atg12 in saponin-extracted cells with Rab5 distributing it away from its normal cellular localisation. inhibition. In control cells, overexpressed Atg12 was located in a Alternatively, the decrease in Atg5-Atg12 conjugation that we single large juxtanuclear domain (Fig. 3e), which did not change observed with Rab5 inhibition or 3MA treatment might result in with the presence or absence of Atg5 overexpression (data not aberrant accumulation of unconjugated Atg12. shown). However, with Rab5 inhibition, Atg12 was re-distributed Rab5 is found in a macromolecular complex containing Vps34 to several peripheral small punctate structures (Fig. 3e), a and beclin 1 phenomenon also seen with 3MA treatment (Fig. 3e). Similarly, Since a macromolecular complex containing Vps34 and beclin 1 when we looked at the distribution of endogenous Atg12, it was distributed in a single juxtanuclear domain (reminiscent of the yeast regulates the early nucleation step in autophagy, we next tested PAS) in wild-type cells (Fig. 3f). However, neither dominant- whether Rab5 was found in such a complex. We tested whether Rab5 interacted with beclin 1, because both proteins interact with Vps34 (Christoforidis et al., 1999; Kihara et al., 2001a), beclin 1 is associated with phagophores, and our data suggests that Rab5 acts at the autophagosome precursor stage and is also associated with such structures (Fig. 2g). In 3MA- treated cells, we observed colocalisation of endogenous Rab5 and beclin 1 (Fig. 4a), particularly in structures similar to those that accumulated when Rab5 or Vps34 were inhibited (e.g. Fig. 2). These putative autophagosome-precursors are precisely where we expected such colocalisation to occur. We also found strong colocalisation of beclin 1 with an activated Rab5 mutant (Fig. 4b). We used activated Rab5 because this forms large clear vesicles that allow unambiguous visualisation of membrane colocalisation of Rab5 with other proteins, a strategy frequently used for Rab5- interactor immunocytochemistry studies (Shin et al., 2005). We next looked for interaction of endogenous Rab5 with beclin 1 by immunoprecipitation. Rab5 interacted with beclin 1 (immunoprecipitated for beclin 1 and detected for Rab5) only in the presence of Vps34 suggesting that Rab5 is part of the macromolecular complex containing beclin 1 and Vps34 (Fig. 4c). Indeed, beclin 1 knockdown (supplementary material Fig. S9) enhances mutant huntingtin (Q74) aggregation (Fig. 4d) (Shibata et al., 2006), which is compatible with the knowledge that this will impair autophagosome formation. Crucially, beclin 1 knockdown led to decreased Atg5-Atg12 conjugation (Fig. 4e), which is compatible with the concept that both beclin 1 and Rab5 activity are required for Vps34 function in autophagosome formation. Thus, Rab5 activation of Vps34 probably regulates Fig. 4. Rab5 is part of a macromolecular complex containing Vps34 and beclin 1. (a) Colocalisation of endogenous Rab5 (green) and endogenous beclin 1 (red) in 3MA-treated HeLa cells after saponin extraction. DAPI-stained nuclei are in blue. (b) Colocalisation of constitutive active Rab5 (CA-Rab5; green) with wild-type beclin 1 (WT-Beclin, red). (c) COS-7 cells transfected with control vector alone (lane 1) or Flag-tagged wild-type beclin 1 alone (lane 2) or wild-type beclin 1 with wild-type Vps34 (lane 3) were immunoprecipitated using anti-Flag antibody (to immunoprecipitate beclin 1) and blotted for Rab5 using an anti-Rab5 antibody. (d) Q74-HA aggregation in HeLa cells transfected with control siRNA or siRNA targeting beclin 1 as described in Fig. 2d; P<0.005. (e) Ratio of Atg5-Atg12 conjugate to Atg12 (data from three independent experiments) in HeLa cells transfected with control siRNA or siRNA targeting beclin 1 for 72 hours. Atg5 and HA-Atg12 were transfected for the last 24 hours. P=0.0003. Journal of Cell Science 1656 Journal of Cell Science 121 (10) not only endosome maturation and multivesicular body formation we found decreased colocalisation of LC3-labelled vesicles with but also, through recruitment of beclin 1, initiation of autophagic the lysosomal marker lgp120, similar to colocalisation observed in vacuole formation. cells treated with BafA1 (Fig. 5e). Previous studies have shown that autophagosomes may fuse with endosomes to form intermediary The effect of Rab5 on autophagy is not due to a general compartments called amphisomes, which subsequently fuse with inhibition of the endocytic pathway lysosomes (Berg et al., 1998). Thus, it is possible that inhibition of Since Rab5 is an important regulator of the endocytic pathway, we the endocytic flux prevents the formation of amphisomes, thereby further tested whether the effects we saw with Rab5 inhibition are also inhibiting a subsequent step in the autophagic pathway, indirect consequences of endocytosis inhibition. We first perturbed amphisome-lysosome fusion. Again, unlike Rab5, we did not see the function of dynamin, a large GTPase essential for clathrin- any increase in the Atg5 structures (supplementary material Fig. mediated endocytosis, using dominant-negative dynamin (DN-Dyn). S10) nor could we observe any change in the ratio of Atg5-Atg12 DN-Dyn increased the percentage of COS-7 cells with aggregates complex to free Atg12 when dynamin was inhibited (Fig. 5f). and cell death similar to DN-Rab5 (Fig. 5a). However, unlike Rab5, These results suggest that DN-Dyn inhibits autophagy at the level overexpression of DN-Dyn increased the size and also the number of autophagic-lysosomal delivery, which is similar to what has been of LC3-positive autophagic vacuoles (Fig. 5b-d) similar to what we previously reported using Vps4, an AAA ATPase involved in and others observe after treatment with BafA1 (Bampton et al., trafficking through the endocytic pathway (Nara et al., 2002). 2005). This suggests that DN-Dyn inhibits autophagy at the level Overexpression of dominant-negative Vps4 showed defects in of autophagic-lysosomal delivery similar to BafA1. Accordingly, autophagy-dependent bulk protein degradation due to an impairment Fig. 5. (a) Quantification of GFP-expressing COS-7 cells with aggregates and abnormal nuclear morphology transiently transfected with dominant-negative dynamin II (DN-Dyn-II) or empty vector control and EGFP-tagged huntingtin exon 1 containing 74 polyglutamine repeats (3:1 ratio) for 48 hours. ***P<0.0001. (b) Distribution of GFP-LC3 vesicles in control (left) or DN-dynamin (red) transfected cells (right). (c) COS-7 cells were transiently transfected with empty vector (Control), DN-dynamin (DN-Dyn) and GFP-LC3 (3:1 ratio) for 24 hours. GFP-positive cells with an increased number of LC3-positive vesicles (>20 vesicles per cell) were quantified; ***P<0.0001. (d) Lysates from HeLa cells expressing empty vector control (Cont) or DN-dynamin (DN-Dyn) were blotted for endogenous LC3, and actin as control. Quantification of band intensities from four independent experiments is shown; **P<0.001. Under exposure conditions that allow endogenous LC3-II quantification in these cells, the LC3-I signal is frequently too low to be detected. (e) NRK cells were transiently transfected for 15 hours with either empty vector [control; with or without bafilomycin A1 (BafA1)] or DN-dynamin (DN-Dyn), and mRFP-LC3 and GFP-lgp120. mRFP-LC3 and GFP-lgp120 double-stained vesicles in individual cells are given in percent (Jahreiss et al., 2008). ***P<0.0001. (f) HeLa cells transfected with control vector or DN-dynamin (DN-Dyn) together with HA-Atg12 and Atg5 were subjected to western blot analysis using anti-HA antibody to detect free Atg12 and the Atg5-Atg12 complex. Atg5-Atg12:Atg12 ratio from three independent experiments is shown. (g) Quantification of GFP-expressing COS-7 cells with aggregates and abnormal nuclear morphology transiently transfected with dominant-negative (DN) Vps4 or empty vector control and EGFP-tagged huntingtin exon 1 containing 74 polyglutamine repeats (3:1 ratio) for 48 hours. ***P<0.0001. Journal of Cell Science Rab5 regulates toxicity of mutant huntingtin via autophagy 1657 in the formation of autolysosomes (Nara et al., 2002). Similar to of LC3-labelled autophagic vacuoles (Fig. 6b,c), did not increase our results for DN-Dyn we did not see an increase in Atg5 structures the number of Atg5 structures (data not shown) and had no effect (data not shown) or decrease in the levels of the Atg5-Atg12 on Atg5-Atg12 conjugation (Fig. 6d). Finally, we also looked at complex with DN-Vps4 (supplementary material Fig. S11) but, the effect of the endocytosis inhibitor methyl-β-cyclodextrin (β- similar to experiments using DN-Dyn (Fig. 5a) or BafA1 CD) on LC3-II levels both in the presence or absence of BafA1 (Ravikumar et al., 2002), we observed an increase in the aggregation (supplementary material Fig. S13). β-CD increased the levels of and associated cell death of huntingtin Q74 in the presence of DN- LC3-II in the absence of BafA1, but had no effect when BafA1 Vps4 (Fig. 5g). was present (Fig. 6e); which again suggests that inhibition of Results obtained using DN-Dyn were identical to those when endocytosis blocks autophagic-lysosomal delivery. DN-Dyn and using siRNA targeting clathrin heavy chain (supplementary material siRNA targeting clathrin heavy chain both had similar effects on Fig. S12), which is involved in formation of clathrin-coated vesicles. LC3-II levels in the presence of BafA1 similar to β-CD siRNA targeting clathrin heavy chain increased the percentage of (supplementary material Figs S14, S15). Thus, Rab5 inhibition, HeLa cells with aggregates (Fig. 6a), increased the number and size which impairs Atg5-Atg12 conjugation, autophagosome (and Fig. 6. (a) HeLa cells transfected with control siRNA or siRNA targeting clathrin heavy chain for 48 hours were subsequently transfected with EGFP-tagged huntingtin exon 1 containing 74 polyglutamine repeats (Q74) for 24 hours. Quantification of Q74 expressing cells with aggregates is shown in the graph. *P<0.01. (b) HeLa cells stably expressing GFP-LC3 were transfected with control or clathrin siRNA for 72 hours, numbers of LC3-positive vesicles were counted (>20 vesicles per cell); ***P<0.0001. (c) HeLa cells transfected with control siRNA or siRNA targeting clathrin heavy chain for 72 hours were subjected to western blot analysis using anti-LC3 and anti-actin antibodies. Under exposure conditions that allowed endogenous LC3-II quantification in these cells, the LC3-I signal was frequently too low to be detected. Quantification of band intensities from three independent experiments is shown. *P<0.05. (d) HeLa cells transfected with control siRNA (Cont) or siRNA targeting clathrin heavy chain (Cla) for 48 hours were subsequently transfected with HA-Atg12 and Atg5 for a further 24 hours. Western blot analysis was performed with anti- HA antibody to detect free Atg12 and the Atg5- Atg12 complex. The Atg5-Atg12:Atg12 ratio from three independent experiments is shown. (e) HeLa cells stably expressing GFP-LC3 were left untreated (Cont) or treated with 5 mM methyl-β-cyclodextrin (β-CD), in the presence (+) or absence (–) of bafilomycin A1 (BafA1) for 6 hours were subjected to western blot analysis using anti-GFP (to detect LC3) and anti-actin antibodies. (f) Lysates from HeLa cells transfected with empty vector (Cont), DN-Rab5 or CA-Rab5 were blotted for phosphorylated (S6-P) and total (S6-T) ribosomal protein S6. Lysate from cells treated with rapamycin (Rap) was used as a positive control. (g) HeLa cells transfected with control vector (Cont) or wild- type Rheb together with HA-Atg12 and Atg5 were subjected to western blot analysis with anti-HA antibody to detect free Atg12 and the Atg5-Atg12 complex. The Atg5-Atg12:Atg12 ratio from three independent experiments is shown. (h) Schematic hypothetical representation of how autophagy is regulated by Rab5. Our data suggest that the Atg5 structures are probably precursors of the pre- autophagosomal structures. The accumulation of Atg5 structures that were observed by us following inhibition of Rab5 or Vps34 might be owing to a block in the progression from early Atg5-positive autophagosomal structures to the formation of autophagic vacuoles. Journal of Cell Science 1658 Journal of Cell Science 121 (10) LC3-II) formation, and which is associated with an accumulation autophagosome formation) also resulted in increased numbers of of Atg5-positive structures, has different effects to a range of Atg5-positive autophagosome precursors. endocytosis inhibitors (such as β-CD, DN-Dyn, DN-Vps4 or siRNA Our data do not exclude the possibility that membranes for targeting clathrin) that do not affect Atg5-Atg12 conjugation but autophagosome biogenesis are derived from endosomes. However, block autophagic-lysosomal delivery and increase LC3-II levels. the effect of Rab5 inactivation on Atg5-Atg12 conjugation and (The specificity of the effect of Rab5 inhibition versus endocytosis autophagosome synthesis is not seen when using a wide range of inhibition on LC3-II levels has been clearly demonstrated in our molecules that inhibit endocytosis (β-CD, DN-Dyn, DN-Vps4 and experiments where we have treated cells with BafA1.) This argues siRNA targeting clathrin) that instead impede autophagic flux by that the effect on autophagosome formation owing to the loss of inhibiting autophagosome-lysosome fusion directly or by inhibiting Rab5 function is not due to effects on endocytosis. the autophagosome-endosome fusion step. This suggests that inhibition of endocytosis through different mechanisms will also Rab5 effect on autophagy is not due to mTOR signalling enhance aggregation and toxicity of polyglutamine by blocking Previous studies in Drosophila have shown that disruption of autophagy. It is possible that the loss of Rab5 activity has effects endocytosis can lead to changes in the target of rapamycin (TOR) on autophagy by perturbing other unrelated or unknown membrane signalling, a key process regulating autophagy (Hennig et al., 2006). trafficking pathways (distinct from endocytosis). However, it is Since Rab5 regulates endocytosis, we tested whether the conditions important to point out that overexpression of CA-Rab5 or WT-Rab5 of Rab5 inhibition or activation alters the mTOR-signalling pathway. enhanced autophagosome synthesis and suppressed aggregation and mTOR directly phosphorylates at least two effectors: S6 kinase-1 toxicity of mutant huntingtin in tissue culture cells and in vivo. and 4EBP1. S6 kinase-1 phosphorylates the ribosomal protein S6. We believe that our data suggest a sequential model in The levels of phosphorylation of any of these downstream targets mammalian cells, in which PtdIns(3)P generated by Vps34, in a are recognised indicators of mTOR activity in the cell (Jacinto and complex that comprises at least beclin 1 and active Rab5, is a key Hall, 2003). Accordingly, we tested whether DN-Rab5 or CA-Rab5 regulator of Atg12 conjugation to Atg5, a rate-limiting step in the altered the mTOR pathway by looking at the phosphorylation levels conversion of Atg5-positive autophagosome precursors to Atg5- of S6. We did not observe any changes in the phosphorylation of negative autophagosomes. On the one hand, inhibition of this S6 (Fig. 6f). We also did not see any changes in the phosphorylation putative cascade at a number of points will lead to impaired levels of S6 kinase-1 or 4EBP1 using DN-Rab5 or CA-Rab5 (data autophagy and enhance polyglutamine toxicity. On the other hand, not shown). Thus, the effects we observed with Rab5 were not owing better understanding of the initial rate-limiting steps of autophagy to alterations in the mTOR signalling. Furthermore, when mTOR may provide opportunities for the rational design of more specific signaling was activated by overexpressing Ras homolog enriched and safer autophagy-inducing therapeutic drugs than rapamycin in brain (Rheb; see supplementary material Fig. S16) we did not (which affects many pathways). This may be of relevance to HD observe the same effects we observed when expressing DN-Rab5; and also to a range of related neurodegenerative diseases caused we did not detect an increase in Atg5 structures (data not shown) by intracytosolic aggregate-prone proteins. and did not see any decrease in the Atg5-Atg12 conjugate compared with the pool of unbound Atg12 (Fig. 6g). Taken together, our results Materials and Methods suggest that the role of Rab5 on autophagy is independent of its Mammalian cell culture and transfection effects on endocytosis and is not because of perturbations in mTOR COS-7 and HeLa cells were grown in Dulbecco’s modified Eagle’s medium (DMEM) (Sigma) supplemented with 10% foetal bovine serum (FBS) (Sigma), 100 U/ml signalling. penicillin/streptomycin (Sigma), 2 mM L-glutamine (Sigma) at 37°C, 5% CO . DNA transfections were performed using lipofectamine reagent (Invitrogen). 20 nmol of Conclusion control (cat. no. 4611), Atg7 (ID 135756), Rab5a (ID 120372), Rab5b (ID 120273), Rab5c (ID 120809), clathrin heavy chain (ID 107565), Vps34 (ID 143802) or beclin In conclusion, we show that Rab5 can modify the toxicity of (ID 137200) siRNA (all from Ambion) were transfected using lipofectamine 2000 polyglutamine in cell and fly models. These modifying effects are according to manufacturer’s instructions. due to Rab5 regulating autophagy-dependent clearance of the toxic huntingtin mutant protein. Our data suggest that Rab5, previously Western blot analysis Western blot analysis was carried out applying standard techniques using the ECL considered as a specific endosome marker, also influences detection kit (Amersham). The primary antibodies used include anti-GFP (Clontech), mammalian autophagy. Inhibition of Rab5 resulted in a decrease anti-HA (Covance), anti-Myc and anti-actin (Sigma), anti-Atg7 (Rockland Inc.), anti- in LC3-positive vesicles suggesting a defect in the formation of Atg5 and anti-Rab5 (Abcam), anti-Vps34 (Zymed) and anti-beclin (Cell Signaling autophagic vacuoles. Rab5 is an activator of Vps34, a PI 3-kinase technology). Densitometry analysis was performed using Image J 1.36b or Scion Image Beta 4.02 softwares. For immunoprecipitation, cells were suspended in lysis essential for autophagy initiation, and we have shown that Rab5 is buffer (50 mM Tris HCl pH 7.4, 150 mM NaCl, 1 mM EDTA and 1% Triton X-100) a new member of a complex that contains Vps34 and beclin 1, and for 30 minutes on ice and supernatants were removed by centrifugation at 13,000 that is associated with autophagosome precursors. Thus, the most rpm in a tabletop centrifuge for 7 minutes at 4°C. 30 μl of anti-M2 affinity gel (Sigma) was added to the sample and incubated at 4°C for 2 hours with gentle rocking. After parsimonious explanation of our data is that Rab5 acts as an activator incubation, tubes were spun for 30 seconds at 4°C at less than 4000 g. Pellets were for Vps34 in autophagy as it is known to do in endocytosis. This washed four times with 1 ml of chilled buffer A (20 mM Tris HCl pH 7.2, 2 mM hypothesis is consistent with the various similarities we observed MgCl ,150 mM NaCl, 0.5% Nonidet P-40). Bound protein was eluted with 100 μl of 150 ng/μl 3FLAG peptide in TBS. in cells expressing loss-of-function of Rab5 or Vps34. Loss of either enzyme activity decreased Atg5-Atg12 conjugation, a crucial step Immunocytochemistry in early phagophore elongation. We speculate that accumulation of Immunocytochemistry was performed in HeLa cells transfected with GFP-Atg5, Atg5 structures concomitantly with Rab5 or Vps34 inhibition is extracted using 0.02% saponin and fixed using 4% paraformaldehyde. Saponin was prepared using PHEM buffer (60 mM NaPIPES, 25 mM NaHEPES, 10 mM EGTA owing to a block in their progression from early Atg5-positive 2 mM MgCl , pH 6.9) supplemented with 0.19 M NaCl. Primary antibodies used were autophagosomal structures to the formation of autophagosomes (Fig. anti-Myc, anti-Flag (Sigma), anti-Rab5 and anti-HA. Relevant negative controls without 6h). This block might be due to a defect in Atg12 recruitment. primary antibodies were performed alongside all experiments. Nuclei were stained Indeed, decreasing Atg5 conjugation (which is known to block with 46-diamidino-2-phenylindole (DAPI, 3 mg/ml; Sigma). Images were acquired Journal of Cell Science Rab5 regulates toxicity of mutant huntingtin via autophagy 1659 –/– using a Nikon Digital Camera DXM1200 and a Nikon Eclipse E600 fluorescence Japan) for HA-Atg12 and Atg5; Atg5 and WT-MEF cells; A. Sorkin microscope. We used Nikon ACT-1 version 2.12 acquisition software. Adobe (University of Colorado Health Sciences Center, Denver, CO) for Photoshop 6.0 (Adobe Systems, Inc.) was used for subsequent image processing. N34S and Q79L Rab5; J. Stankova (University of Sherbrooke, Québec, Canada) for K44A Dynamin; X. Wang (The University of Quantification of aggregate formation, abnormal nuclear Utah, Salt Lake City, UT) for DN-Vps4B; Eléonore Mayola for morphologies, LC3-positive vesicles technical support. 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Published: May 15, 2008

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