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Huntingtin facilitates selective autophagy Amir Gelman, Moran Rawet-Slobodkin and Zvulun Elazar Selective autophagy is essential for maintaining cellular homeostasis under different growth conditions. Huntingtin, mutated versions of which have been implicated in Huntington disease, is now shown to act as a scaffold protein that couples the induction of autophagy and the selective recruitment of cargo into autophagosomes. Macroautophagy (hereafter termed autophagy) is a catabolic process conserved in all eukaryotes that is responsible for the degradation of proteins and organelles under normal growth conditions and that is further induced in response to stress1. Research in recent years has revealed that maintaining a cell’s wellbeing under different growth conditions requires the operation of distinct autophagic processes that are selective for specific cellular constituents. In this issue of Nature Cell Biology, Rui et  al. report that Huntingtin (Htt) regulates autophagy by interacting with the autophagic cargo receptor p62 and with the autophagyinitiating kinase ULK1 (ref. 2). Htt is a large molecule that is highly conserved among vertebrates and invertebrates and is essential for human development and normal brain function3. Expansion of the polyglutamine (polyQ) tract in the Htt amino terminus leads to aggregation of the protein; this polyQ expansion and subsequent aggregate formation is the main cause of Huntington disease. Unlike in other neurodegenerative disorders, in which autophagy is recruited to remove protein aggregates, autophagy is often impaired in patients with Huntington disease4. In fact, wild-type Htt is thought to play a regulatory role in autophagy 5. Htt and its interacting protein HAP1 were reported to interact with dynein, promoting fast axonal retrograde trafficking of autophagosomes6. Moreover, a short Htt fragment that undergoes myristoylation (a lipidation that promotes membrane binding) positively regulates autophagy in multiple cell lines7. The observation of structural similarities between different Htt regions and the yeast proteins ATG23, Vac8 and ATG11 — factors that are essential for selective autophagy but not for starvation-induced autophagy, which is considered non-selective — led to the hypothesis that Htt directly regulates selective Amir Gelman, Moran Rawet-Slobodkin and Zvulun Elazar are in the Department of Biological Chemistry, The Weizmann Institute of Science, 76100 Rehovot, Israel. e-mail: [email protected]

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autophagy in mammals8. In fact, a recent publication reported9 the interaction of Htt with ULK1 and the autophagy-specific protein LC3. Both starvation-induced and selective autophagy involve the formation of autophagosomes — structures with double membranes enclosing cytoplasmic molecules that are marked for degradation10. The autophagosomes are then directed to the lysosome for degradation. Autophagic selectivity is achieved through autophagic receptors, such as p62, NBR1, NDP52 and optineurin, which recruit selected cargo molecules11. Formation of the autophagosome is initiated by the ULK1 kinase complex, the activity of which is regulated by the inhibitory kinase mTORC1 (ref. 1). Once initiated, autophagosome formation continues through the action of multiple other proteins, including LC3. Rui et al. began by assessing Drosophila fruit flies lacking the htt gene. Unlike in mammals, in which htt knockout is lethal to embryos, the authors found that homozygous htt-knockout flies are viable and display only mild agedependent mobility defects and reductions in viability. Absence of htt was found to be deleterious only in flies ectopically expressing a truncated form of Tau (Tau-ΔC), a mutation that is associated with some forms of Alzheimer disease and which causes Tau in mouse cortical neurons to be unstable and to undergo preferential degradation through autophagy. Loss of htt in these flies was accompanied by collapse of the thorax as well as an enhanced decline in mobility and a reduced life span. The authors next analysed heterozygotic flies with one allele of htt that ectopically express Tau-ΔC. In these flies, concurrent loss of the genes atg8a or atg1, which encode the fly orthologues of LC3 and ULK1, respectively, also led to loss of the thorax, indicating a strong genetic interaction between htt and autophagic factors. A similar result was observed in htt heterozygote flies lacking Ref(2)P, the fly orthologue of p62. Having established that expression of human Htt can restore the phenotypes observed in

these flies, the authors moved on to mammalian cell systems for mechanistic analyses. Knockdown of Htt under basal conditions inhibited autophagy-mediated protein degradation accompanied by accumulation of p62 and ‘empty’ autophagosomes. Starvationinduced autophagy was not affected by the loss of Htt, but the authors observed significant inhibition of autophagy in Htt-knockdown cells in response to proteotoxic, lipotoxic or mitochondrial stress induced by proteasome inhibitors, oleic challenge or mitochondrial depolarizing agents, respectively. These findings suggested that Htt plays a key part in multiple selective-autophagy responses. The authors next looked for possible interactions between Htt, p62 and ULK1 in mammalian cells. Expression of different truncated forms of Htt and analysis of co-immunoprecipitation between these factors identified two regions of interest: D6, which was observed to be essential for the interaction with p62, and D3, for the interaction with ULK1 (Fig. 1a). In principle, these interactions would allow the formation of a tertiary complex of Htt, p62 and ULK1, and thus for Htt to couple the induction of selective autophagy, regulated by ULK1, to p62-specific cargo recruitment. In support of this notion, the authors demonstrate that ULK1 is found in two mutually exclusive complexes, associated either with Htt or with the protein kinase mTORC1. Binding of Htt to ULK1 releases the latter from mTORC1, allowing induction of autophagy (Fig.  1b). Selectivity is then achieved by the binding of p62, associated with Lys63-ubiquitinated proteins, to Htt, leading to the recruitment of ULK1 to this complex. This exciting role for Htt in different forms of selective autophagy may also explain the pleiotropic nature of Huntington disease. Clearance of protein aggregates and cellular organelles, such as defective mitochondria, is impaired in this disease, suggesting that mutations in Htt not only lead to disease-causing protein aggregation, but also impair its ability to regulate autophagy. This represents

NATURE CELL BIOLOGY VOLUME 17 | NUMBER 3 | MARCH 2015 © 2015 Macmillan Publishers Limited. All rights reserved

NEWS AND VIEWS a

ATG23 homology

Vac8 homology ULK1 binding region

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ATG11 homology p62 binding region

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mTORC1

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proteins and only then activate ULK1 to initiate the process? Is the interaction between p62 and Htt also essential for other selective autophagy processes, such as mitophagy and lipohagy, and does Htt interact with other autophagic receptors in addition to p62? It will be important to verify the existence of a tertiary p62–Htt–ULK1 complex, by testing whether the molecules interact directly or through other factors. It will also be interesting to investigate the role of LC3 in the formation of this complex and in directing it to the forming autophagosome. Multiple putative LC3interacting regions have been identified in Htt, suggesting that the proteins may interact directly 8,9. Studies addressing such questions will help to clarify the role of Htt in selective autophagic processes. COMPETING FINANCIAL INTERESTS The authors declare no competing financial interests

Figure 1 Huntingtin acts as a scaffold protein that regulates selective autophagy. (a) The mammalian Htt protein contains regions with structural similarities to the yeast proteins ATG23, Vac8 and ATG11 (ref. 8), which are essential factors for selective autophagy. Rui et al.2 show that Htt also contains regions that mediate its binding to the autophagy-initiating kinase ULK1 and the cargo receptor p62, allowing the protein to regulate the initiation of various forms of selective autophagy, including aggrephagy — autophagy induced in response to protein aggregation. (b) The authors propose that Htt facilitates the binding of p62 to protein aggregates tagged by polyubiquitin chains conjugated to Lys63. Simultaneously, Htt binds ULK1, thereby releasing it from its association with mTORC1, which is responsible for its inhibitory phosphrylation (red octagon). ULK1 then undergoes an activating phosphorylation event (green circle), and the now-active ULK1 induces the formation of autophagosomes from phagophores with associated LC3 molecules, and the p62-bound protein aggregates are delivered to these autophagosomes for degradation.

an important area for further investigation. However, the expression of key autophagyrelated factors such as ALFY (ref. 12) and PINK1 (ref. 13), which participate in these clearance processes, is also highly reduced in oligodendrocytes and neurons of patients with Huntington disease, suggesting that additional

pathways feed into the autophagy defects seen in Huntington disease14. Rui and colleagues2 also set the stage for future investigations. Open questions include: at precisely what stage of autophagosome formation does Htt participate? Does it first interact with p62-associated Lys63-ubiquitinated

NATURE CELL BIOLOGY VOLUME 17 | NUMBER 3 | MARCH 2015 © 2015 Macmillan Publishers Limited. All rights reserved

1. Abada, A. & Elazar, Z. EMBO Reports 15, 839–852 (2014). 2. Rui, Y‑N. et al. Nat. Cell Biol. 17, 262–275 (2015). 3. Cattaneo, E., Zuccato, C. & Tartari, M. Nat. Rev. Neurosci. 6, 919–930 (2005). 4. Martinez-Vicente, M. et al. Nat. Neurosci. 13, 567–576 (2010). 5. Martin, D. D., Ladha, S., Ehrnhoefer, D. E. & Hayden, M. R. Trends Neurosci. 38, 26–35 (2015). 6. Wong, Y. C. & Holzbaur, E. L. J. Neurosci. 34, 1293– 1305 (2014). 7. Martin, D. D. et al. Hum. Mol. Genet. 23, 3166–3179 (2014). 8. Steffan, J. S. Cell Cycle 9, 3401–3413 (2010). 9. Ochaba, J. et  al. Proc. Natl Acad. Sci. USA 111, 16889–16894 (2014). 10. Weidberg, H., Shvets, E. & Elazar, Z. Annu. Rev. Biochem. 80, 125–156 (2011). 11. Rogov, V., Dotsch, V., Johansen, T. & Kirkin, V. Mol. Cell 53, 167–178 (2014). 12. Isakson, P., Holland, P. & Simonsen, A. Cell Death Diff. 20, 12–20 (2013). 13. Scarffe, L. A., Stevens, D. A., Dawson, V. L. & Dawson, T. M. Trends Neurosci. 37, 315–324 (2014). 14. Kuhn, A., Thu, D., Waldvogel, H.  J., Faull, R.  L. & Luthi-Carter, R. Nat. Methods 8, 945–947 (2011).

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Huntingtin facilitates selective autophagy.

Selective autophagy is essential for maintaining cellular homeostasis under different growth conditions. Huntingtin, mutated versions of which have be...
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