This article was downloaded by: [New York University] On: 27 May 2015, At: 00:27 Publisher: Taylor & Francis Informa Ltd Registered in England and Wales Registered Number: 1072954 Registered office: Mortimer House, 37-41 Mortimer Street, London W1T 3JH, UK
Autophagy Publication details, including instructions for authors and subscription information: http://www.tandfonline.com/loi/kaup20
HTT/Huntingtin in Selective Autophagy and Huntington Disease: A foe or a friend within? ac
ac
bc
Yan-Ning Rui , Zhen Xu , Bindi Patel , Ana Maria Cuervo a
The Brown Foundation Institute of Molecular Medicine
b
Department of Development and Molecular Biology
c
Equal contributions
d
Department of Anatomy and Structural Biology
bde
& Sheng Zhang
afg
e
Institute for Aging Studies, Albert Einstein College of Medicine, Bronx, New York 10461
f
Department of Neurobiology and Anatomy
g
Click for updates
Programs in Human and Molecular Genetics and Neuroscience, The University of Texas Graduate School of Biomedical Sciences, The University of Texas Medical School at Houston, The University of Texas Health Science Center at Houston (UTHealth), 1825 Pressler Street, Houston, TX, 77030 Accepted author version posted online: 18 May 2015.
To cite this article: Yan-Ning Rui, Zhen Xu, Bindi Patel, Ana Maria Cuervo & Sheng Zhang (2015): HTT/Huntingtin in Selective Autophagy and Huntington Disease: A foe or a friend within?, Autophagy, DOI: 10.1080/15548627.2015.1039219 To link to this article: http://dx.doi.org/10.1080/15548627.2015.1039219
Disclaimer: This is a version of an unedited manuscript that has been accepted for publication. As a service to authors and researchers we are providing this version of the accepted manuscript (AM). Copyediting, typesetting, and review of the resulting proof will be undertaken on this manuscript before final publication of the Version of Record (VoR). During production and pre-press, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal relate to this version also.
PLEASE SCROLL DOWN FOR ARTICLE Taylor & Francis makes every effort to ensure the accuracy of all the information (the “Content”) contained in the publications on our platform. However, Taylor & Francis, our agents, and our licensors make no representations or warranties whatsoever as to the accuracy, completeness, or suitability for any purpose of the Content. Any opinions and views expressed in this publication are the opinions and views of the authors, and are not the views of or endorsed by Taylor & Francis. The accuracy of the Content should not be relied upon and should be independently verified with primary sources of information. Taylor and Francis shall not be liable for any losses, actions, claims, proceedings, demands, costs, expenses, damages, and other liabilities whatsoever or howsoever caused arising directly or indirectly in connection with, in relation to or arising out of the use of the Content. This article may be used for research, teaching, and private study purposes. Any substantial or systematic reproduction, redistribution, reselling, loan, sub-licensing, systematic supply, or distribution in any
Downloaded by [New York University] at 00:27 27 May 2015
form to anyone is expressly forbidden. Terms & Conditions of access and use can be found at http:// www.tandfonline.com/page/terms-and-conditions
HTT/Huntingtin in Selective Autophagy and Huntington Disease: A foe or a friend within?
Yan-Ning Rui1,3, Zhen Xu1,3, Bindi Patel2,3, Ana Maria Cuervo2,4,5,8, and Sheng Zhang1,6,7,8 2
Department of Development and Molecular Biology Department of Anatomy and Structural Biology 5 Institute for Aging Studies Albert Einstein College of Medicine Bronx, New York 10461
Downloaded by [New York University] at 00:27 27 May 2015
4
1
The Brown Foundation Institute of Molecular Medicine, Department of Neurobiology and Anatomy 7 Programs in Human and Molecular Genetics and Neuroscience The University of Texas Graduate School of Biomedical Sciences The University of Texas Medical School at Houston The University of Texas Health Science Center at Houston (UTHealth) 1825 Pressler Street, Houston, TX, 77030 6
3
Equal contributions
8
Corresponding authors
[email protected] [email protected]
Abbreviations: ATG13, autophagy related 13; ATG101, autophagy related 101; KO, knockout; MAP1LC3A, microtubule-associated protein 1 light chain 3 alpha; MAPT/Tau, microtubule-associated
protein tau;
MTOR,
mechanistic
target
of rapamycin
(serine/threonine kinase); MTORC1, MTOR complex 1; PRKAA/AMPK, protein kinase,
1
AMP-activated, alpha catalytic subunit; RB1CC1, RB1-inducible coiled-coil 1; SQSTM1, sequestosome 1; ULK1, unc-51 like autophagy activating kinase 1
Key words: aggrephagy, cargo recognition, Huntingtin, Huntington disease, lipophagy, mitophagy,
Downloaded by [New York University] at 00:27 27 May 2015
MTORC1, nonselective autophagy, selective autophagy, SQSTM1/p62, ULK1.
2
Huntington disease (HD) is caused by a unique mutation, an abnormal expansion of a polyglutamine (polyQ) tract in the HTT (huntingtin) protein. It has long been hypothesized that in addition to gained toxicity from the expanded polyQ, loss of HTT’s normal cellular functions also contributes to HD pathogenesis. Consistently, wild-type HTT has a well-documented neuronal protective activity and is essential for postnatal
Downloaded by [New York University] at 00:27 27 May 2015
neuronal survival. However, despite its relatively simple genetic cause, the etiology of HD remains unclear, due in part to the poor understanding of the physiological cellular functions of HTT.
The perplexing HTT is a large ~3,144 amino acid-long protein with ubiquitous localization, but lacks any known functional domain that could provide clues about its cellular activities. The phenotypes of HTT knockout (KO) mice are equally puzzling: homozygote HTT KO mice die by day 7.5, but this early embryonic lethality is due to a critical role of HTT in extraembryonic membranes, not in the embryo per se, as it can be rescued if wildtype HTT is provided in extraembryonic tissues. HTT has a vast number of reported interacting partners that have been used to infer some of the growing list of cellular pathways that HTT could participate in. However, it is still uncertain how HTT achieves its well-documented neuroprotective role. Using both Drosophila and mammalian experimental models, we recently demonstrated that HTT has an essential function in selective autophagy where it serves as a scaffold by modulating the activities of the cargo receptor SQSTM1/p62 and the autophagy initiation kinase ULK1. We previously created a null deletion mutant (htt-ko)
3
of the single HTT homolog (htt) in Drosophila and showed that in contrast to mouse, httko flies, which develop ex utero, are fully viable with only mild aging-related defects. Unexpectedly, ectopic expression of a truncated form of the mammalian MAPT induced severe defects in htt-ko flies, suggesting that htt protects against pathogenic MAPT toxicity. Further, in genetic screens using this MAPT-induced phenotype as a functional readout, we detected dosage-sensitive genetic interaction between htt-ko and several
Downloaded by [New York University] at 00:27 27 May 2015
components of the autophagy pathway, including atg8a (MAP1LC3A homolog), atg1 (ULK1 homolog) and ref(2)P (SQSTM1/p62 homolog), thus unveiling a functional link between HTT and autophagy. HTT is not a foreigner to the autophagy field, although the main emphasis has been on the toxic effect of mutant HTT on this cellular clearance mechanism. In fact, our earlier work in HD demonstrated that reduced autophagy in the disease context, also reported by many other groups, originated from diminished ability to “trap” cytosolic cargo. We attributed the “empty autophagosomes” phenotype in HD cells to defective cargo recognition due to the presence of mutant polyQ-HTT in the inner part of the closing autophagosomes. However, our genetic studies ablating the wild-type HTT made us reconsider this proposed gain-of-toxicity function. In this recent study, we have found that in both htt-ko flies and HTT-depleted mammalian cells, autophagic abnormalities are present but surprisingly, starvationinduced autophagy is largely normal. However, loss of HTT compromised autophagic induction when challenged by several other stresses, including proteotoxicity, lipotoxicity and mitochondria damage. In all these instances, mammalian cells depleted of HTT also
4
showed an “empty autophagosomes” defect, suggesting that wild-type HTT is required for efficient cargo recognition by the autophagosome. We found that HTT and SQSTM1/p62, as well as their Drosophila counterparts Htt and Ref(2)P, physically interact and that proteotoxic stress promotes this interaction. Depletion of HTT reduces the association of SQSTM1 with MAP1LC3A and also compromises the binding of SQSTM1 with proteins with lysine-63-linked ubiquitin
Downloaded by [New York University] at 00:27 27 May 2015
(K63-Ub) chains, which are preferential substrates of autophagy. However, loss of HTT does not affect self-polymerization of SQSTM1 or its association with K48-Ub-modified proteins mainly cleared by the proteasome. Collectively, these findings support the idea that HTT facilitates cargo recognition by modulating the assembly of the cargo receptors and autophagy proteins. We also found that the dependence on HTT for the initiation of stress-induced selective autophagy is related to its physical interaction with ULK1, whose kinase activity is essential for autophagy initiation. HTT depletion does not affect starvationinduced activation of ULK1 kinase, but compromises ULK1 activation upon proteotoxicity challenge, revealing a contribution of HTT to differentially regulating autophagosome biogenesis under basal or stress conditions. We found that the regulation of HTT on ULK1 is at the level of its interaction with the MTORC1 complex, another main regulator of ULK1 that binds to and inactivates ULK1 under nutrient-rich conditions. We showed that (1) ULK1 exists in 2 mutually exclusive complexes, ULK1HTT and ULK1-MTORC1; (2) HTT does not affect the kinase activity of MTORC1; (3) HTT competes with MTORC1 for binding with ULK1; (4) stresses that induce selective autophagy (e.g., proteotoxicity, lipotoxicity or mitophagy), but not starvation, result in an
5
increased association of ULK1 with HTT at the expense of MTOR, thus freeing ULK1 from its inhibition by MTORC1. Collectively, HTT promotes selective autophagy by activating and bringing together SQSTM1/p62 and ULK1 to assure spatial proximity between the cargo and autophagy initiation components, thereby orchestrating 2 major autophagy steps: cargo recognition and autophagy induction (Fig. 1).
Downloaded by [New York University] at 00:27 27 May 2015
Increasing evidence supports a tangled relationship between HTT, HD and autophagy. Thus, after the first report of altered autophagy in HD by DiFiglia, the Holzbaur lab showed that HTT facilitates axonal trafficking of autophagosomes, a function that is compromised by polyQ-HTT; Hayden’s group reported an autophagyinducing domain within HTT; Steffan with Thompson and colleagues also recently reported physical interactions between HTT and several autophagy proteins including SQSTM1 and ULK1. Our finding places HTT in the core of the autophagic process, bringing together components of the cargo recognition and autophagy initiation complexes, and raising many new interesting questions. How is HTT differentially activated upon different stress challenges? Does polyQ expansion directly compromise the endogenous activities of HTT, as implicated by the very similar “empty autophagosomes” phenotypes in both HD and HTT-depleted cells, and if so, will this effect be partially responsible for the toxicity of mutant polyQ-HTT in long-lived neurons? Last, how can we exploit HTT’s role in selective autophagy to modulate this process in the fight against this devastating brain disease called HD?
6
Downloaded by [New York University] at 00:27 27 May 2015
Figure 1. Model of HTT in promoting selective autophagy. HTT serves as scaffolding for selective autophagy by bringing together cargo bound through SQSTM1 and an initiator of autophagy, ULK1 kinase. Basal autophagy: under basal conditions, binding of HTT to the cargo receptor SQSTM1 promotes the selective recognition of K63-Ub-modified cargos. The absence of HTT leads to reduced selectivity in cargo recognition required for quality control autophagy, but it does not affect autophagy induction/autophagosome biogenesis, because basal ULK1 is sufficient to sustain ‘in bulk’ autophagy and basal quality control autophagy. 7
Induced autophagy: maximal activation of autophagy in response to stresses requires the release of MTORC1 inhibition over ULK1. Upon starvation stress, inactivation of MTORC1 promotes release and activation of ULK1, leading to induction of nonselective “bulk” autophagy. In response to nonstarvation stresses such as proteotoxicity, organelle toxicity and lipotoxicity, HTT become more active in competing ULK1 away from the MTORC1 inhibitory complex, resulting in
Downloaded by [New York University] at 00:27 27 May 2015
activation of selective autophagy.
8
Autophagy Publication details, including instructions for authors and subscription information: http://www.tandfonline.com/loi/kaup20
HTT/Huntingtin in Selective Autophagy and Huntington Disease: A foe or a friend within? ac
ac
bc
Yan-Ning Rui , Zhen Xu , Bindi Patel , Ana Maria Cuervo a
The Brown Foundation Institute of Molecular Medicine
b
Department of Development and Molecular Biology
c
Equal contributions
d
Department of Anatomy and Structural Biology
bde
& Sheng Zhang
afg
e
Institute for Aging Studies, Albert Einstein College of Medicine, Bronx, New York 10461
f
Department of Neurobiology and Anatomy
g
Click for updates
Programs in Human and Molecular Genetics and Neuroscience, The University of Texas Graduate School of Biomedical Sciences, The University of Texas Medical School at Houston, The University of Texas Health Science Center at Houston (UTHealth), 1825 Pressler Street, Houston, TX, 77030 Accepted author version posted online: 18 May 2015.
To cite this article: Yan-Ning Rui, Zhen Xu, Bindi Patel, Ana Maria Cuervo & Sheng Zhang (2015): HTT/Huntingtin in Selective Autophagy and Huntington Disease: A foe or a friend within?, Autophagy, DOI: 10.1080/15548627.2015.1039219 To link to this article: http://dx.doi.org/10.1080/15548627.2015.1039219
Disclaimer: This is a version of an unedited manuscript that has been accepted for publication. As a service to authors and researchers we are providing this version of the accepted manuscript (AM). Copyediting, typesetting, and review of the resulting proof will be undertaken on this manuscript before final publication of the Version of Record (VoR). During production and pre-press, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal relate to this version also.
PLEASE SCROLL DOWN FOR ARTICLE Taylor & Francis makes every effort to ensure the accuracy of all the information (the “Content”) contained in the publications on our platform. However, Taylor & Francis, our agents, and our licensors make no representations or warranties whatsoever as to the accuracy, completeness, or suitability for any purpose of the Content. Any opinions and views expressed in this publication are the opinions and views of the authors, and are not the views of or endorsed by Taylor & Francis. The accuracy of the Content should not be relied upon and should be independently verified with primary sources of information. Taylor and Francis shall not be liable for any losses, actions, claims, proceedings, demands, costs, expenses, damages, and other liabilities whatsoever or howsoever caused arising directly or indirectly in connection with, in relation to or arising out of the use of the Content. This article may be used for research, teaching, and private study purposes. Any substantial or systematic reproduction, redistribution, reselling, loan, sub-licensing, systematic supply, or distribution in any
Downloaded by [New York University] at 00:27 27 May 2015
form to anyone is expressly forbidden. Terms & Conditions of access and use can be found at http:// www.tandfonline.com/page/terms-and-conditions
HTT/Huntingtin in Selective Autophagy and Huntington Disease: A foe or a friend within?
Yan-Ning Rui1,3, Zhen Xu1,3, Bindi Patel2,3, Ana Maria Cuervo2,4,5,8, and Sheng Zhang1,6,7,8 2
Department of Development and Molecular Biology Department of Anatomy and Structural Biology 5 Institute for Aging Studies Albert Einstein College of Medicine Bronx, New York 10461
Downloaded by [New York University] at 00:27 27 May 2015
4
1
The Brown Foundation Institute of Molecular Medicine, Department of Neurobiology and Anatomy 7 Programs in Human and Molecular Genetics and Neuroscience The University of Texas Graduate School of Biomedical Sciences The University of Texas Medical School at Houston The University of Texas Health Science Center at Houston (UTHealth) 1825 Pressler Street, Houston, TX, 77030 6
3
Equal contributions
8
Corresponding authors
[email protected] [email protected]
Abbreviations: ATG13, autophagy related 13; ATG101, autophagy related 101; KO, knockout; MAP1LC3A, microtubule-associated protein 1 light chain 3 alpha; MAPT/Tau, microtubule-associated
protein tau;
MTOR,
mechanistic
target
of rapamycin
(serine/threonine kinase); MTORC1, MTOR complex 1; PRKAA/AMPK, protein kinase,
1
AMP-activated, alpha catalytic subunit; RB1CC1, RB1-inducible coiled-coil 1; SQSTM1, sequestosome 1; ULK1, unc-51 like autophagy activating kinase 1
Key words: aggrephagy, cargo recognition, Huntingtin, Huntington disease, lipophagy, mitophagy,
Downloaded by [New York University] at 00:27 27 May 2015
MTORC1, nonselective autophagy, selective autophagy, SQSTM1/p62, ULK1.
2
Huntington disease (HD) is caused by a unique mutation, an abnormal expansion of a polyglutamine (polyQ) tract in the HTT (huntingtin) protein. It has long been hypothesized that in addition to gained toxicity from the expanded polyQ, loss of HTT’s normal cellular functions also contributes to HD pathogenesis. Consistently, wild-type HTT has a well-documented neuronal protective activity and is essential for postnatal
Downloaded by [New York University] at 00:27 27 May 2015
neuronal survival. However, despite its relatively simple genetic cause, the etiology of HD remains unclear, due in part to the poor understanding of the physiological cellular functions of HTT.
The perplexing HTT is a large ~3,144 amino acid-long protein with ubiquitous localization, but lacks any known functional domain that could provide clues about its cellular activities. The phenotypes of HTT knockout (KO) mice are equally puzzling: homozygote HTT KO mice die by day 7.5, but this early embryonic lethality is due to a critical role of HTT in extraembryonic membranes, not in the embryo per se, as it can be rescued if wildtype HTT is provided in extraembryonic tissues. HTT has a vast number of reported interacting partners that have been used to infer some of the growing list of cellular pathways that HTT could participate in. However, it is still uncertain how HTT achieves its well-documented neuroprotective role. Using both Drosophila and mammalian experimental models, we recently demonstrated that HTT has an essential function in selective autophagy where it serves as a scaffold by modulating the activities of the cargo receptor SQSTM1/p62 and the autophagy initiation kinase ULK1. We previously created a null deletion mutant (htt-ko)
3
of the single HTT homolog (htt) in Drosophila and showed that in contrast to mouse, httko flies, which develop ex utero, are fully viable with only mild aging-related defects. Unexpectedly, ectopic expression of a truncated form of the mammalian MAPT induced severe defects in htt-ko flies, suggesting that htt protects against pathogenic MAPT toxicity. Further, in genetic screens using this MAPT-induced phenotype as a functional readout, we detected dosage-sensitive genetic interaction between htt-ko and several
Downloaded by [New York University] at 00:27 27 May 2015
components of the autophagy pathway, including atg8a (MAP1LC3A homolog), atg1 (ULK1 homolog) and ref(2)P (SQSTM1/p62 homolog), thus unveiling a functional link between HTT and autophagy. HTT is not a foreigner to the autophagy field, although the main emphasis has been on the toxic effect of mutant HTT on this cellular clearance mechanism. In fact, our earlier work in HD demonstrated that reduced autophagy in the disease context, also reported by many other groups, originated from diminished ability to “trap” cytosolic cargo. We attributed the “empty autophagosomes” phenotype in HD cells to defective cargo recognition due to the presence of mutant polyQ-HTT in the inner part of the closing autophagosomes. However, our genetic studies ablating the wild-type HTT made us reconsider this proposed gain-of-toxicity function. In this recent study, we have found that in both htt-ko flies and HTT-depleted mammalian cells, autophagic abnormalities are present but surprisingly, starvationinduced autophagy is largely normal. However, loss of HTT compromised autophagic induction when challenged by several other stresses, including proteotoxicity, lipotoxicity and mitochondria damage. In all these instances, mammalian cells depleted of HTT also
4
showed an “empty autophagosomes” defect, suggesting that wild-type HTT is required for efficient cargo recognition by the autophagosome. We found that HTT and SQSTM1/p62, as well as their Drosophila counterparts Htt and Ref(2)P, physically interact and that proteotoxic stress promotes this interaction. Depletion of HTT reduces the association of SQSTM1 with MAP1LC3A and also compromises the binding of SQSTM1 with proteins with lysine-63-linked ubiquitin
Downloaded by [New York University] at 00:27 27 May 2015
(K63-Ub) chains, which are preferential substrates of autophagy. However, loss of HTT does not affect self-polymerization of SQSTM1 or its association with K48-Ub-modified proteins mainly cleared by the proteasome. Collectively, these findings support the idea that HTT facilitates cargo recognition by modulating the assembly of the cargo receptors and autophagy proteins. We also found that the dependence on HTT for the initiation of stress-induced selective autophagy is related to its physical interaction with ULK1, whose kinase activity is essential for autophagy initiation. HTT depletion does not affect starvationinduced activation of ULK1 kinase, but compromises ULK1 activation upon proteotoxicity challenge, revealing a contribution of HTT to differentially regulating autophagosome biogenesis under basal or stress conditions. We found that the regulation of HTT on ULK1 is at the level of its interaction with the MTORC1 complex, another main regulator of ULK1 that binds to and inactivates ULK1 under nutrient-rich conditions. We showed that (1) ULK1 exists in 2 mutually exclusive complexes, ULK1HTT and ULK1-MTORC1; (2) HTT does not affect the kinase activity of MTORC1; (3) HTT competes with MTORC1 for binding with ULK1; (4) stresses that induce selective autophagy (e.g., proteotoxicity, lipotoxicity or mitophagy), but not starvation, result in an
5
increased association of ULK1 with HTT at the expense of MTOR, thus freeing ULK1 from its inhibition by MTORC1. Collectively, HTT promotes selective autophagy by activating and bringing together SQSTM1/p62 and ULK1 to assure spatial proximity between the cargo and autophagy initiation components, thereby orchestrating 2 major autophagy steps: cargo recognition and autophagy induction (Fig. 1).
Downloaded by [New York University] at 00:27 27 May 2015
Increasing evidence supports a tangled relationship between HTT, HD and autophagy. Thus, after the first report of altered autophagy in HD by DiFiglia, the Holzbaur lab showed that HTT facilitates axonal trafficking of autophagosomes, a function that is compromised by polyQ-HTT; Hayden’s group reported an autophagyinducing domain within HTT; Steffan with Thompson and colleagues also recently reported physical interactions between HTT and several autophagy proteins including SQSTM1 and ULK1. Our finding places HTT in the core of the autophagic process, bringing together components of the cargo recognition and autophagy initiation complexes, and raising many new interesting questions. How is HTT differentially activated upon different stress challenges? Does polyQ expansion directly compromise the endogenous activities of HTT, as implicated by the very similar “empty autophagosomes” phenotypes in both HD and HTT-depleted cells, and if so, will this effect be partially responsible for the toxicity of mutant polyQ-HTT in long-lived neurons? Last, how can we exploit HTT’s role in selective autophagy to modulate this process in the fight against this devastating brain disease called HD?
6
Downloaded by [New York University] at 00:27 27 May 2015
Figure 1. Model of HTT in promoting selective autophagy. HTT serves as scaffolding for selective autophagy by bringing together cargo bound through SQSTM1 and an initiator of autophagy, ULK1 kinase. Basal autophagy: under basal conditions, binding of HTT to the cargo receptor SQSTM1 promotes the selective recognition of K63-Ub-modified cargos. The absence of HTT leads to reduced selectivity in cargo recognition required for quality control autophagy, but it does not affect autophagy induction/autophagosome biogenesis, because basal ULK1 is sufficient to sustain ‘in bulk’ autophagy and basal quality control autophagy. 7
Induced autophagy: maximal activation of autophagy in response to stresses requires the release of MTORC1 inhibition over ULK1. Upon starvation stress, inactivation of MTORC1 promotes release and activation of ULK1, leading to induction of nonselective “bulk” autophagy. In response to nonstarvation stresses such as proteotoxicity, organelle toxicity and lipotoxicity, HTT become more active in competing ULK1 away from the MTORC1 inhibitory complex, resulting in
Downloaded by [New York University] at 00:27 27 May 2015
activation of selective autophagy.
8
