David Rubinsztein, Cambridge University, UK
Ana Maria Cuervo, Albert Einstein College of Medicine, New York, USA
Anne Simonsen, Oslo University, Norway
Patrice Codogno, INSERM U984 Faculté de Pharmacie, Châtenay-Malabry, France
Richard Youle, NINDS, Bethesda, USA
Zhenyu Yue, Icahn School of Medicine at Mount Sinai, New York, USA
Autophagy is one of the two major intracellular neuronal pathways for clearing proteins and damaged organelles, such as mitochondria. The pathway has captured the interest of neuroscientists because it is uniquely able to clear aggregated protein that may not fit into the proteasome, and protein aggregation is a common feature of a variety of neurological diseases. In addition, autophagy has also been implicated in mental illnesses and may have wide applications in the central nervous system. This Advance Course will provide participants with a firm foundation into the biology of autophagy and the forms of autophagy that are specific to particular cargo, such as mitophagy. The participants will also familiarize themselves with the latest innovations to measure autophagy in biological systems, the links between autophagy and human disease, and strategies to target autophagy therapeutically.
The Course will be team taught by leaders in the field. Dr. Zhenyu Yue (Mount Sinai Hospital, USA) will update the class on the signaling pathways in neurons that regulate autophagy and their relationship to neurodegenerative diseases. Dr. David Rubinsztein (Cambridge University, U.K.) will review recent data elucidating upstream steps in the autophagy pathway and efforts to discover safe and effective ways to modulate it therapeutically. Dr. Ana Maria Cuervo (Albert Einstein College of Medicine, USA) will describe a specific form of autophagy called chaperone-mediated autophagy and its link to neurodegenerative diseases. Dr. Richard Youle (National Institutes of Health, USA) will teach about cargo selective autophagy, the role of mitophagy in some forms of Parkinson's disease and how to explore molecular mechanisms in animal models. Dr. Patrice Codogno (University of Paris, France) will describe fundamental cell biological underpinnings of autophagy, particularly the membrane biogenesis necessary to make autophagosomes. Dr. Anne Simonsen (University of Oslo, Norway) will focus on the specific lipids and lipid-binding proteins that mediate autophagosome biogenesis and govern cargo selection, and how these are affected in disease. Dr. Steven Finkbeiner (Gladstone Institutes and UCSF, USA) will serve as overall course director, and will describe innovative ways to measure autophagy and protein homeostasis in neurons, how proteostasis is affected in disease models, and efforts to discover and develop new regulators of autophagy that might lead to therapeutics.
Regulation of autophagy by VPS34-Beclin 1 and ULK kinase complexes in neurons and proteinopathies
Autophagy is a lysosomal degradation pathway involving autophagic vacuole (autophagosome) synthesis, trafficking, and fusion with endosomes and lysosomes. Autophagy is tightly regulated but can be activated in response to a variety of cellular stresses and injuries. Two evolutionarily conserved kinase complexes, ULK1/2 (Atg1) and VPS34-beclin1 (class III PI-3P kinase), are essential for the initiation of autophagosome formation. We have investigated the protein composition and function of multiple subunits of VPS34-beclin1 complexes in cells and neurons. Our data revealed different subcomplexes of the VPS34-beclin1 by combining Atg14L, UVRAG, Rubicon or NRBF2 that regulate PI3K III activity at different stages of the autophagic process and membrane trafficking pathways beyond autophagy. In addition, our investigation identified novel targets of ULK1 signaling through mTOR-dependent and -independent pathway. In neurons, autophagy occurs constitutively in the axons, dendrites and the soma. It has an important role in maintaining homeostasis of membrane/lipid and proteins. I will also review the evidence for the regulation of selective autophagy in the clearance of protein aggregates that are relevant to the proteinopathies, such as Huntington's disease. Our studies provide insight into the control mechanism for autophagy and suggest that ULK1 and VPS34-beclin 1 complexes are promising therapeutic targets for proteinopathies.
• Komatsu M, Wang Q, Holstein G, Kominami E, Chait C, Tanaka K, Yue Z (2007) Essential role for autophagic protein Atg7 in the maintenance of axonal homeostasis and prevention of axonal degeneration Proc. Natl. Acad. Sci. USA, 104 (36): 14489-14494.
• Zhong Y, Wang Q-J, Li X, Chait BT, Heintz N and Yue Z (2009) Distinct Regulation of Autophagic Activity by Novel Components Atg14L and Rubicon in Beclin 1-Vps34/phosphatidylinositol (PtdIns) 3-kinase complex. Nat. Cell Biol,. 11(4):468-76.
• Funderburk SF, Wang QJ, and Yue Z (2010) The Beclin 1-VPS34 complex - at the crossroads of autophagy and beyond. Trends in Cell Biol. 20(6):355-62.
• Friedman L, Lachenmayer L, Wang J, He L, Poulose S, Komatsu M, Holstein G and Yue Z (2012) Disrupted autophagy leads to dopaminergic axon and dendrite degeneration and promotes presynaptic accumulation of -synuclein and LRRK2 in the brain. J Neurosci. 32 (22): 7585-7593
• Lu J, He L, Wang Q-J, Behrends C, Chait BT, Araki M, Araki K, Friedman SL, Fiel MI, Li M, Yue Z (2014) NRBF2 Regulates Autophagy and Prevents Liver Injury by Modulating Beclin 1/Atg14L-Linked Class III Phosphatidylinositol-3 Kinase Activity Nature Communications 22;5:3920.
• Yamamoto A and Yue Z (2014) Autophagy and its Normal and Pathogenic States in the Brain Annual Reviews of Neuroscience vol 37, July 8th.
• Lim JM, Lachenmayer L, Liu W, Kundu M, Wang R, Komatsu M, Oh YJ, Zhao Y and Yue Z (2015) Proteotoxic Stress Induces Phosphorylation of p62/SQSTM1 by ULK1 to Regulate Selective Autophagic Clearance of Protein Aggregates PLoS Genetics 11(2):e1004987.
David C. Rubinsztein
Macroautophagy: a guardian against neurodegeneration
Intracellular protein aggregation is a feature of many late-onset neurodegenerative diseases, including Parkinson's disease, tauopathies, and polyglutamine expansion diseases (e.g., Huntington's disease (HD)). Many of these mutant proteins, such as huntingtin in HD, cause disease via toxic gain-of-function mechanisms. Therefore, the factors regulating their clearance are crucial for understanding disease pathogenesis and for developing rational therapeutic strategies.
We showed that the autophagy inducer, rapamycin, reduced the levels of mutant huntingtin and attenuated its toxicity in cells, and in Drosophila and mouse HD models. We extended the range of intracellular proteinopathy substrates that are cleared by autophagy to other related neurodegenerative disease targets and have provided proof-of-principle in cells, Drosophila and mice. To induce autophagy long term, we have been striving to identify safer alternatives to the mTOR inhibitor, rapamycin. To this end, we have been trying to discover novel components of the autophagy machinery and new signaling pathways and drugs that induce autophagy.
While autophagy induction is protective in models of various neurodegenerative diseases, certain other conditions, including lysosomal storage disorders, are associated with compromised autophagy. I will review these data and describe how our recent appreciation of the membrane trafficking events preceding autophagosome formation has helped the understanding of the roles of disease-associated variants.
• Williams et al (2008) Novel targets for Huntington's disease in an mTOR-independent autophagy pathway. Nature Chemical Biology 4: 295-305
• Moreau et al (2011) Autophagosome precursor maturation requires homotypic fusion. Cell 146: 303-317
• Puri et al (2013) Diverse autophagosome membrane sources coalesce in recycling endosomes. Cell 154: 1285-1299
• Vicinanza et al (2015) PI(5)P regulates autophagosome biogenesis. Molecular Cell 57:219-234.
• Menzies et al (2105) Compromised autophagy and neurodegenerative diseases. Nature Reviews Neuroscience 16:345-357
Ana Maria Cuervo
Chaperone-mediated autophagy: fighting neurodegeneration one protein at a time
Cells count on surveillance systems to handle protein alterations and organelle damage. Malfunctions in these systems contribute in large extent to the abnormal accumulation of those altered components in cells and tissues in numerous diseases and in aging. Our studies have focused primarily on the degradation of proteins in lysosomes through a selective form of autophagy, known as chaperone-mediated autophagy (CMA).
Cytosolic protein substrates for CMA are delivered to the lysosomal lumen for degradation by directly crossing the lysosomal membrane. This process is mediated by a set of cytosolic and lysosomal chaperones and by a receptor protein at the lysosomal membrane, the lysosome-associated membrane protein type 2A (LAMP-2A). In this type of autophagy, the limiting step is the binding of substrates to LAMP-2A. We found that changes in the levels and organization of LAMP-2A at the lysosomal membrane underlie the molecular basis for the regulation of CMA. This pathway is active in most cell types in mammalians, but its activity depends on cellular conditions. In recent years, the better molecular characterization of CMA has considerably advanced our understanding of the physiological role of this pathway and the consequences of its malfunctioning in the pathogenesis of detrimental human pathologies, such as neurodegenerative diseases.
I will describe our recent findings on the molecular effectors and regulators of CMA and our studies in support of a reciprocal interplay between pathogenic proteins, such as alpha-synuclein or tau and CMA. I will comment on the consequences of the functional decline of CMA with age and in age-related disorders and some of our current efforts to chemically modulate CMA activity to enhance the cellular response against proteotoxicity.
• Cuervo AM (2011) Chaperone-mediated autophagy: Dice's wild idea about lysosomal selectivity. Nat. Rev. Mol. Cell Biol., 12: 535–541 (Timeline)
• Kaushik S, Cuervo AM (2012) Chaperone-mediated autophagy: a unique way to enter the lysosome world. Trends Cell Biol., 22: 407–417
• Orenstein SJ, Kuo SH, Tasset-Cuevas I, Arias E, Koga H, Fernandez-Carasa I, Cortes, E., Honig, L.S., Dauer, W., Consiglio A, Raya A, Sulzer, D, Cuervo AM (2013) Interplay of LRRK2 with chaperone-mediated autophagy. Nat. Neurosci., 16: 394–406
• Anguiano J, Gaerner T, Daas B, Gavathiotis E, Cuervo AM (2013) Chemical modulation of Chaperone-mediated autophagy by novel retinoic acid derivatives. Nat. Chem. Biol., 9: 374–382
• Schneider S, Villarroya J, Diaz A, Patel B, Urbanska AM, Thi MM, Villarroya F, Santambrogio L, Cuervo AM (2015) Loss of hepatic chaperone-mediated autophagy accelerates proteostasis failure in aging. Aging Cell, 14: 249–264
Autophagy of mitochondria, Parkinson's disease and ALS
Two genes mutated in autosomal recessive forms of Parkinson's disease have been identified in Drosophila to work in the same pathway to maintain dopaminergic neurons. In biochemical and cell biology studies, the products of these two genes, PINK1 and Parkin, normally work together in the same pathway to govern mitochondrial quality control, bolstering previous evidence that mitochondrial damage is involved in Parkinson's disease. PINK1 is a kinase located on mitochondria, and Parkin is an E3 ubiquitin ligase normally located in the cytosol. When mitochondria are damaged, Pink1 recruits cytosolic Parkin to the mitochondria to mediate mitophagy revealing a cell biology pathway in mammalian cells where Pink1 works in the same pathway and upstream of Parkin. PINK1 accumulates on the outer membrane of damaged mitochondria where it phosphorylates ubiquitin chains. These phosphorylated ubiquitin chains on the outer mitochondrial membrane bind to cytosolic Parkin and activate Parkin's E3 ubiquitin ligase activity yielding a feedback amplification loop that drives mitophagy to completion. Downstream of Parkin the machinery that mediates autophagosome recognition of damaged mitochondria links this pathway to genes mutated in ALS. Knocking out a series of autophagy receptors, including p62, NBR1, NDP52, Tax1BP1 and Optineurin, reveals the hierarchy of autophagy receptors involved in mitophagy. Optineurin and the kinase TBK1, both mutated in familial ALS cases, participate in mitophagy in addition to NDP52. Optineurin and NDP52 bind to ubiquitin chains on mitochondria and also recruit autophagy machinery proteins, including the upstream kinase Ulk1 and the downstream autophagoaome marker, LC3, to induce autophagosome engulfment of the damaged mitochondria. Animal models that display mitochondrial damage require endogenous Parkin to prevent domaminergic neuron loss and movement disorders. Interestingly, in a murine model of mitochondrial damage, the product of the kinase PINK1 (phospho-S65 ubiquitin) is detected to increase in the cortex, representing a biomarker of PINK1 activity. Students attending these lectures will learn about the molecular and cellular mechanisms involved in cargo selective autophagy, the role of mitophagy in some forms of Parkinson's disease and how to explore molecular mechanisms in animal models.
Autophagy and plasma membrane domains
Macroutophagy (hereafter referred to as autophagy) is a tightly regulated intracellular degradation pathway essential for cellular homeostasis regulation. It is initiated by the formation of autophagosomes that engulf portions of the cytoplasm and organelles and fuse with the lysosome for material degradation. The origin of autophagosomes is still unclear: multiple membrane sources, including the endoplasmic reticulum (ER), mitochondria, endosomes and the plasma membrane (PM) have been suggested to contribute to autophagosomal membrane biogenesis. In mammals, a platform for autophagosome biogenesis likely exists on the ER, and recent studies suggest that mitochondrial membrane contact sites are the putative precise sites. In parallel, ER-PM contact sites, principally linked to regulation of calcium homeostasis and lipid trafficking, may play more general physiologic function(s) via membrane dynamics regulation. In higher eukaryotes, three ER-localized proteins, the extended synaptotagmins (E-Syt1, 2 and 3), play a crucial role in tethering the ER to the PM, notably via phosphoinositides local metabolism. Here I will discuss the implication of ER-PM contact sites in the early stage of autophagosome biogenesis. In a second part of the talk, I will discuss the role of the interplay between autophagy and the primary cilium, a microtubule-based structure that is continuous with the plasma membrane, in response to stress situations (starvation, shear stress). Autophagy is important in the regulation of cell size downstream of the primary cilium in response to fluid flow. The functional dialog between autophagy and the primary cilium has an important physiological function. The primary cilium-dependent control of autophagy could be exploited for the development of novel treatments for diseases associated with primary cilium defects (ciliopathies). Overall, these findings highlight the importance of the plasma membrane domains in the autophagosome biogenesis and in the regulation of the autophagic pathway.
Neuronal autophagy and protein homeostasis in health and disease
The appearance of abnormal protein deposits in a variety of neurodegenerative diseases suggests an underlying mismatch between the production of aggregation-prone proteins and the capacity to clear them that contributes to pathogenesis. In this context, the autophagic clearance pathway has attracted considerable interest. Deficits in autophagy lead to neurodegeneration, and autophagy can clear aggregated proteins that may be indigestible by the ubiquitin proteasome system.
In this course, methods to measure autophagy and mitophagy will be discussed, including new methods based on photoswitchable proteins and longitudinal single-cell analysis. Evidence will be presented from studies of neuronal protein homeostasis in models of Parkinson's disease, motor neuron disease, Huntington's disease, and other neurodegenerative diseases that implicates autophagy as a critical pathway. Finally, efforts to pursue autophagy as a therapeutic target, including the discovery of new targets from genetic screens, the development of novel small-molecule inducers, and strategies for developing target engagement biomarkers/companion diagnostics, will be discussed. At the end of these lectures, students should have a better understanding of scientific foundation that supports the study of autophagy in neurodegenerative disease, methods to measure autophagy experimentally, and strategies to pursue autophagy therapeutically.
• Finkbeiner, S., Frumkin, M. & Kassner, P.D. Cell-based screening: Extracting meaning from complex data. Neuron 86, 160–174 (2015).
• Skibinski, G., Nakamura, K., Cookson, M.R. & Finkbeiner, S. Mutant LRRK2 toxicity in neurons depends on LRRK2 levels and synuclein but not kinase activity or inclusion bodies. J. Neurosci. 34, 418–433 (2014).
• Tsvetkov, A.S. et al. Proteostasis of polyglutamine varies among neurons and predicts neurodegeneration. Nat. Chem. Biol. 9, 586–592 (2013b).
• Barmada, S.J. et al. Autophagy induction enhances TDP43 turnover and survival in neuronal ALS models. Nat. Chem. Biol. 10, 677–685 (2014).
• Tsvetkov, A.S. et al. A small-molecule scaffold induces autophagy in primary neurons and protects against toxicity in a Huntington disease model. Proc. Natl. Acad. Sci. U.S.A. 107, 16982–16987 (2010).
Lipid-binding proteins in autophagy
Characterization of the molecular mechanisms of autophagy is a topic of intense investigation. Such insight may pave the way for the development of specific autophagy-modulating drugs that could effectively treat or even cure many devastating diseases. A large number of autophagy-related proteins have been identified as essential for non-selective and selective types of autophagy, but relatively little is known about the specific lipids and lipid-binding proteins involved in the membrane modeling events responsible for regulation of autophagosome biogenesis and cargo selection, and how these are regulated under various metabolic conditions and in disease.
My laboratory focuses on the interplay between lipids and proteins in autophagy. We found that the large phosphatidylinositol-3-phosphate (PI3P)-binding protein ALFY is important for sequestration of ubiquitinated protein aggregates for degradation by autophagy. ALFY interacts with the ubiquitin-binding autophagy receptors p62 and NBR1, and facilitates recruitment of the autophagic membrane through its binding to ATG5, GABARAP and PI3P (see references below). ALFY is mainly localized to the nucleus in non-stressed cells, and becomes recruited to ubiquitin-positive cytoplasmic structures upon proteotoxic stress. I will discuss our recent progress in understanding the function of ALFY in selective autophagy and neurodegenerative disease. I will also discuss other lipid-binding proteins identified in my lab as involved in autophagosome biogenesis.
• Simonsen et al. (2004) Alfy, a novel FYVE-domain-containing protein associated with protein granules and autophagic membranes. J. Cell Sci., 117: 4239–4251
• Filimoneko et al. (2010) The selective macroautophagic degradation of aggregated proteins requires the PIeP-binding protein Alfy. Mol. Cell, 38: 265–279
• Clausen et al. (2010) p62/SQSTM1 and ALFY interact to facilitate the formation of p62 bodies/ALIS and their degradation by autophagy. Autophagy, 6: 330–344
• Lystad et al. (2014) Structural determinants in GABARAP required for the selective binding and recruitment of ALFY to LC3B-positive structures. EMBO Rep., 15: 557–565