Kevin Moreau

Regulation of Mammalian Autophagy in Physiology and Pathophysiology

(Macro)autophagy is a bulk degradation process that mediates the clearance of long-lived proteins and organelles. Autophagy is initiated by double-membraned structures, which engulf portions of cytoplasm. The resulting autophagosomes ultimately fuse with lysosomes, where their contents are degraded. Although the term autophagy was first used in 1963, the field has witnessed dramatic growth in the last 5 years, partly as a consequence of the discovery of key components of its cellular machinery. In this review we focus on mammalian autophagy, and we give an overview of the understanding of its machinery and the signaling cascades that regulate it. As recent studies have also shown that autophagy is critical in a range of normal human physiological processes, and defective autophagy is associated with diverse diseases, including neurodegeneration, lysosomal storage diseases, cancers, and Crohns disease, we discuss the roles of autophagy in health and disease, while trying to critically evaluate if the coincidence between autophagy and these conditions is causal or an epiphenomenon. Finally, we consider the possibility of autophagy upregulation as a therapeutic approach for various conditions.

Plasma membrane contributes to the formation of pre-autophagosomal structures

Autophagy is a catabolic process in which lysosomes degrade intracytoplasmic contents transported in double-membraned autophagosomes. Autophagosomes are formed by the elongation and fusion of phagophores, which derive from pre-autophagosomal structures. The membrane origins of autophagosomes are unclear and may involve multiple sources, including the endoplasmic reticulum and mitochondria. Here we show in mammalian cells that the heavy chain of clathrin interacts with Atg16L1 and is involved in the formation of Atg16L1-positive early autophagosome precursors. Atg16L1 associated with clathrin-coated structures, and inhibition of clathrin-mediated internalization decreased the formation of both Atg16L1-positive precursors and mature autophagosomes. We tested and demonstrated that the plasma membrane contributes directly to the formation of early Atg16L1-positive autophagosome precursors. This may be particularly important during periods of increased autophagosome formation, because the plasma membrane may serve as a large membrane reservoir that allows cells periods of autophagosome synthesis at levels many-fold higher than under basal conditions, without compromising other processes.

Autophagosome Precursor Maturation Requires Homotypic Fusion

Summary Autophagy is a catabolic process in which lysosomes degrade intracytoplasmic contents transported in double-membraned autophagosomes. Autophagosomes are formed by the elongation and fusion of phagophores, which can be derived from preautophagosomal structures coming from the plasma membrane and other sites like the endoplasmic reticulum and mitochondria. The mechanisms by which preautophagosomal structures elongate their membranes and mature toward fully formed autophagosomes still remain unknown. Here, we show that the maturation of the early Atg16L1 precursors requires homotypic fusion, which is essential for subsequent autophagosome formation. Atg16L1 precursor homotypic fusion depends on the SNARE protein VAMP7 together with partner SNAREs. Atg16L1 precursor homotypic fusion is a critical event in the early phases of autophagy that couples membrane acquisition and autophagosome biogenesis, as this step regulates the size of the vesicles, which in turn appears to influence their subsequent maturation into LC3-positive autophagosomes.

Diverse Autophagosome Membrane Sources Coalesce in Recycling Endosomes

Summary Autophagic protein degradation is mediated by autophagosomes that fuse with lysosomes, where their contents are degraded. The membrane origins of autophagosomes may involve multiple sources. However, it is unclear if and where distinct membrane sources fuse during autophagosome biogenesis. Vesicles containing mATG9, the only transmembrane autophagy protein, are seen in many sites, and fusions with other autophagic compartments have not been visualized in mammalian cells. We observed that mATG9 traffics from the plasma membrane to recycling endosomes in carriers that appear to be routed differently from ATG16L1-containing vesicles, another source of autophagosome membrane. mATG9- and ATG16L1-containing vesicles traffic to recycling endosomes, where VAMP3-dependent heterotypic fusions occur. These fusions correlate with autophagosome formation, and both processes are enhanced by perturbing membrane egress from recycling endosomes. Starvation, a primordial autophagy activator, reduces membrane recycling from recycling endosomes and enhances mATG9-ATG16L1 vesicle fusion. Thus, this mechanism may fine-tune physiological autophagic responses.

Mutation in VPS35 associated with Parkinson’s disease impairs WASH complex association and inhibits autophagy

Endosomal protein sorting controls the localization of many physiologically important proteins and is linked to several neurodegenerative diseases. VPS35 is a component of the retromer complex, which mediates endosome-to-Golgi retrieval of membrane proteins such as the cation-independent mannose 6-phosphate receptor. Furthermore, retromer is also required for the endosomal recruitment of the actin nucleation promoting WASH complex. The VPS35 D620N mutation causes a rare form of autosomal-dominant Parkinson’s disease (PD). Here we show that this mutant associates poorly with the WASH complex and impairs WASH recruitment to endosomes. Autophagy is impaired in cells expressing PD-mutant VPS35 or lacking WASH. The autophagy defects can be explained, at least in part, by abnormal trafficking of the autophagy protein ATG9A. Thus, the PD-causing D620N mutation in VPS35 restricts WASH complex recruitment to endosomes, and reveals a novel role for the WASH complex in autophagosome formation.

Cytoprotective roles for autophagy.

Macroautophagy (referred to as autophagy in this review) is a genetically regulated bulk degradation program conserved from yeast to humans, in which cytoplasmic substrates, such as damaged organelles and long-lived proteins, are delivered to lysosomes for degradation. In this review, we consider recent data that highlight possible mechanisms whereby autophagy mediates cytoprotective effects. These include the ability of autophagy to buffer against starvation, protect against apoptotic insults and clear mitochondria, aggregate-prone proteins and pathogens. These effects are pertinent to the roles of autophagy in normal human physiology, including the early neonatal period and ageing, as well as a variety of diseases, including cancer, neurodegenerative conditions and infectious diseases.

Protein misfolding disorders and macroautophagy

A large group of diseases, termed protein misfolding disorders, share the common feature of the accumulation of misfolded proteins. The possibility of a common mechanism underlying either the pathogenesis or therapy for these diseases is appealing. Thus, there is great interest in the role of protein degradation via autophagy in such conditions where the protein is found in the cytoplasm. Here we review the growing evidence supporting a role for autophagic dysregulation as a contributing factor to protein accumulation and cellular toxicity in certain protein misfolding disorders and discuss the available evidence that upregulation of autophagy may be a valuable therapeutic strategy.

PICALM modulates autophagy activity and tau accumulation

Genome-wide association studies have identified several loci associated with Alzheimer’s disease (AD), including proteins involved in endocytic trafficking such as PICALM/CALM (phosphatidylinositol binding clathrin assembly protein). It is unclear how these loci may contribute to AD pathology. Here we show that CALM modulates autophagy and alters clearance of tau, a protein which is a known autophagy substrate and which is causatively linked to AD, both in vitro and in vivo. Furthermore, altered CALM expression exacerbates tau-mediated toxicity in zebrafish transgenic models. CALM influences autophagy by regulating the endocytosis of SNAREs, such as VAMP2, VAMP3 and VAMP8, which have diverse effects on different stages of the autophagy pathway, from autophagosome formation to autophagosome degradation. This study suggests that the AD genetic risk factor CALM modulates autophagy, and this may affect disease in a number of ways including modulation of tau turnover.

Connections between SNAREs and autophagy

Autophagy involves the sequestration of portions of cytoplasm by double-membraned autophagosomes, which are then trafficked to lysosomes. After autophagosome-lysosome fusion, the contents of the autophagosomes are degraded by lysosomal hydrolases. SNAREs [soluble N-ethylmaleimide-sensitive fusion (NSF) attachment protein receptors] are molecules that mediate vesicular fusion events. Here, we review recent data implicating SNAREs as having key roles both in the genesis of autophagosomes, as well as in autophagosome-lysosome fusion, and we discuss the implications of these findings in the context of a long-standing mystery: the origin of autophagosomes.

Arf6 promotes autophagosome formation via effects on phosphatidylinositol 4,5-bisphosphate and phospholipase D

Arf6 positively regulates autophagosome membrane biogenesis by inducing PIP2 generation and PLD activation, which together may influence endocytic uptake of plasma membrane into autophagosome precursors.