Neurodegenerative diseases (NDs) are characterized by the progressive loss of neurons in the human brain. Although the majority of NDs are sporadic, evidence is accumulating that they have a strong genetic component. Therefore, significant efforts have been made in recent years to not only identify disease-causing genes but also genes that modify the severity of NDs, so-called genetic modifiers. To date there exists no compendium that lists and cross-links genetic modifiers of different NDs.
In order to address this need, we present NeuroGeM, the first comprehensive knowledgebase providing integrated information on genetic modifiers of nine different NDs in the model organisms D. melanogaster, C. elegans, and S. cerevisiae. NeuroGeM cross-links curated genetic modifier information from the different NDs and provides details on experimental conditions used for modifier identification, functional annotations, links to homologous proteins and color-coded protein-protein interaction networks to visualize modifier interactions. We demonstrate how this database can be used to generate new understanding through meta-analysis. For instance, we reveal that the Drosophila genes DnaJ-1, thread, Atx2, and mub are generic modifiers that affect multiple if not all NDs.
As the first compendium of genetic modifiers, NeuroGeM will assist experimental and computational scientists in their search for the pathophysiological mechanisms underlying NDs. http://chibi.ubc.ca/neurogem.
Neurodegenerative diseases; Genetic modifiers; Database; Knowledgebase; Alzheimer’s disease; Parkinson’s disease; Huntington’s disease
Autophagosomes may derive membrane from diverse sources, including the plasma membrane, Golgi, endoplasmic reticulum and mitochondria. The plasma membrane contributes membrane to ATG12–ATG5-ATG16L1-positive phagophore precursor vesicles (LC3-negative) by both clathrin-dependent and -independent routes. We recently observed that ARF6 regulates autophagy and that this could be explained, at least in part, by its role in the generation of phosphatidylinositol 4,5-bisphosphate [PtdIns(4,5)P2], which influences endocytic uptake of plasma membrane into autophagosome precursors. The subsequent maturation of these small phagophore precursors into phagophores (ATG12–ATG5-ATG16L1-positive and LC3-positive), is assisted by SNARE-mediated homotypic fusion that increase their size and enhance their ability to acquire LC3-II. It appears that a plasma membrane-derived pool of VAMP7 is a key mediator of these fusion events. Thus, events at the plasma membrane may regulate distinct steps in the biogenesis of phagophores.
Arf6; ATG16L1; autophagy; phagophore; plasma membrane; SNARE
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.
•mATG9 traffics from the plasma membrane to recycling endosomes•mATG9 vesicles fuse with ATG16L1 vesicles in recycling endosomes•VAMP3, Rab11, myosin Vb, and starvation regulate mATG9-ATG16L1 vesicle fusion•mATG9-ATG16L1 vesicle fusions regulate autophagosome formation
Autophagosome membranes that originate in different cellular compartments follow distinct routes to recycling endosomes, where they fuse to form autophagosome precursors.
Autophagy, a major clearance route for many long-lived proteins and organelles, has long been implicated in cancer development. Myc is a proto-oncogene often found to be deregulated in many cancers, and thus is an attractive target for design of cancer therapy. Therefore, understanding the relationship between anti-Myc strategies and autophagy will be important for development of effective therapy. Here, we show that Myc depletion inhibits autophagosome formation and impairs clearance of autophagy substrates. Myc suppression has an inhibitory effect on autophagy via reduction of c-Jun N-terminal kinase 1 (JNK1) and B-cell lymphoma 2 (Bcl2) phosphorylation. Additionally, the decrease in JNK1 phosphorylation observed with Myc knockdown is associated with a reduction in ROS production. Our data suggest that targeting Myc in cancer therapy might have the additional benefit of inhibiting autophagy in the case of therapy resistance associated with chemotherapy-induced autophagy.
In response to toxic stimuli, BCL2L11 (also known as BIM), a BH3-only protein, is released from its interaction with dynein light chain 1 (DYNLL1 also known as LC8) and can induce apoptosis by inactivating anti-apoptotic BCL2 proteins and by activating BAX-BAK1. Recently, we discovered that BCL2L11 interacts with BECN1 (Beclin 1), and that this interaction is facilitated by DYNLL1. BCL2L11 recruits BECN1 to microtubules by bridging BECN1 and DYNLL1, thereby inhibiting autophagy. In starvation conditions, BCL2L11 is phosphorylated by MAPK8/JNK and this phosphorylation abolishes the BCL2L11-DYNLL1 interaction, allowing dissociation of BCL2L11 and BECN1, thereby ameliorating autophagy inhibition. This finding demonstrates a novel function of BIM beyond its roles in apoptosis, highlighting the crosstalk between autophagy and apoptosis, and suggests that BCL2L11’s dual effects in inhibiting autophagy and promoting apoptosis may have important roles in disease pathogenesis.
BIM; autophagy; apoptosis; BH-3 domain; BECN1
Autophagy is a rapidly expanding field in the sense that our knowledge about the molecular mechanism and its connections to a wide range of physiological processes has increased substantially in the past decade. Similarly, the vocabulary associated with autophagy has grown concomitantly. This fact makes it difficult for readers, even those who work in the field, to keep up with the ever-expanding terminology associated with the various autophagy-related processes. Accordingly, we have developed a comprehensive glossary of autophagy-related terms that is meant to provide a quick reference for researchers who need a brief reminder of the regulatory effects of transcription factors or chemical agents that induce or inhibit autophagy, the function of the autophagy-related proteins, or the role of accessory machinery or structures that are associated with autophagy.
autophagy; definitions; glossary; lexicon; terms
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.
Macroautophagy (in this paper referred to as autophagy) and the ubiquitin–proteasome system are the two major catabolic systems in cells. Autophagy involves sequestration of cytosolic contents in double membrane–bounded vesicles called autophagosomes. The membrane source for autophagosomes has received much attention, and diverse sources, such as the plasma membrane, Golgi, endoplasmic reticulum, and mitochondria, have been implicated. These may not be mutually exclusive, but the exact sources and mechanism involved in the formation of autophagosomes are still unclear. In this paper, we identify a positive role for the small G protein Arf6 in autophagosome formation. The effect of Arf6 on autophagy is mediated by its role in the generation of phosphatidylinositol 4,5-bisphosphate (PIP2) and in inducing phospholipase D (PLD) activity. PIP2 and PLD may themselves promote autophagosome biogenesis by influencing endocytic uptake of plasma membrane into autophagosome precursors. However, Arf6 may also influence autophagy by indirect effects, such as either by regulating membrane flow from other compartments or by modulating PLD activity independently of the mammalian target of rapamycin.
α-Synuclein and mutant huntingtin are the major constituents of the intracellular aggregates that characterize the pathology of Parkinson's disease (PD) and Huntington's disease (HD), respectively. α-Synuclein is likely to be a major contributor to PD, since overexpression of this protein resulting from genetic triplication is sufficient to cause human forms of PD. We have previously demonstrated that wild-type α-synuclein overexpression impairs macroautophagy in mammalian cells and in transgenic mice. Overexpression of human wild-type α-synuclein in cells and Drosophila models of HD worsens the disease phenotype. Here, we examined whether α-synuclein overexpression also worsens the HD phenotype in a mammalian system using two widely used N-terminal HD mouse models (R6/1 and N171-82Q). We also tested the effects of α-synuclein deletion in the same N-terminal HD mouse models, as well as assessed the effects of α-synuclein deletion on macroautophagy in mouse brains. We show that overexpression of wild-type α-synuclein in both mouse models of HD enhances the onset of tremors and has some influence on the rate of weight loss. On the other hand, α-synuclein deletion in both HD models increases autophagosome numbers and this is associated with a delayed onset of tremors and weight loss, two of the most prominent endophenotypes of the HD-like disease in mice. We have therefore established a functional link between these two aggregate-prone proteins in mammals and provide further support for the model that wild-type α-synuclein negatively regulates autophagy even at physiological levels.
Autophagy is an essential, conserved lysosomal degradation pathway that controls the quality of the cytoplasm by eliminating protein aggregates and damaged organelles. It begins when double-membraned autophagosomes engulf portions of the cytoplasm, which is followed by fusion of these vesicles with lysosomes and degradation of the autophagic contents. In addition to its vital homeostatic role, this degradation pathway is involved in various human disorders, including metabolic conditions, neurodegenerative diseases, cancers and infectious diseases. This article provides an overview of the mechanisms and regulation of autophagy, the role of this pathway in disease and strategies for therapeutic modulation.
The mammalian target of rapamycin (mTOR) is a well-conserved negative regulator of autophagy. Here we review our recent data describing how lysosomal positioning influences and coordinates mTOR activity, autophagosome biogenesis and autophagosome-lysosome fusion. In this way, lysosomal positioning regulates many diverse cellular responses to starvation and subsequent nutrient replenishment.
autophagy; lysosome; mTOR; intracellular pH; ARL8; kinesin
The study of autophagy is rapidly expanding, and our knowledge of the molecular mechanism and its connections to a wide range of physiological processes has increased substantially in the past decade. The vocabulary associated with autophagy has grown concomitantly. In fact, it is difficult for readers—even those who work in the field—to keep up with the ever-expanding terminology associated with the various autophagy-related processes. Accordingly, we have developed a comprehensive glossary of autophagy-related terms that is meant to provide a quick reference for researchers who need a brief reminder of the regulatory effects of transcription factors and chemical agents that induce or inhibit autophagy, the function of the autophagy-related proteins, and the roles of accessory components and structures that are associated with autophagy.
autophagy; lysosome; mitophagy; pexophagy; stress; vacuole
The accumulation of misfolded proteins in insoluble aggregates within the neuronal cytoplasm is one of the common pathological hallmarks of most adult-onset human neurodegenerative diseases. The clearance of these misfolded proteins may represent a promising therapeutic strategy in these diseases. The two main routes for intracellular protein degradation are the ubiquitin–proteasome and the autophagy–lysosome pathways. In this review, we will focus on the autophagic pathway, by providing some examples of how impairment at different steps in this degradation pathway is related to different neurodegenerative diseases. We will also consider that upregulating autophagy may be useful in the treatment of some of these diseases. Finally, we discuss how antioxidants, which have been considered to be beneficial in neurodegenerative diseases, can block autophagy, thus potentially compromising their therapeutic potential.
►Autophagy compromise occurs in different neurodegenerative diseases. ►Upregulating autophagy may be useful in the treatment of some neurodegenerative diseases. ►Many different reactive oxygen species scavengers impair autophagy
Autophagy; Neurodegeneration; Huntington's disease
Parkinson disease (PD) is the most common movement disorder affecting people. It is characterized by the accumulation of the protein α-synuclein in Lewy body inclusions in vulnerable neurons. α-Synuclein overexpression caused by gene multiplications is sufficient to cause this disease, suggesting that α-synuclein accumulation is toxic. Here we review our recent study showing that α-synuclein inhibits autophagy. We discuss our mechanistic understanding of this phenomenon and also speculate how a deficiency in autophagy may contribute to a range of pleiotropic features of PD biology.
Parkinson disease; alpha-synuclein; autophagy; Rab1a; Atg9
Bim is a proapoptotic BH3-only Bcl-2 family member. In response to death stimuli, Bim dissociates from the dynein light chain 1 (DYNLL1/LC8), where it is inactive, and can then initiate Bax/Bak-mediated mitochondria-dependent apoptosis. We found that Bim depletion increases autophagosome synthesis in cells and in vivo, and this effect is inhibited by overexpression of cell death-deficient Bim. Bim inhibits autophagy by interacting with Beclin 1, an autophagy regulator, and this interaction is facilitated by LC8. Bim bridges the Beclin 1-LC8 interaction and thereby inhibits autophagy by mislocalizing Beclin 1 to the dynein motor complex. Starvation, an autophagic stimulus, induces Bim phosphorylation, which abrogates LC8 binding to Bim, leading to dissociation of Bim and Beclin 1. Our data suggest that Bim switches locations between apoptosis-inactive/autophagy-inhibitory and apoptosis-active/autophagy-permissive sites.
► Bim negatively regulates autophagy in cell culture and in vivo ► Bim inhibits autophagosome formation by interacting with Beclin 1 ► Bim inhibits autophagy by mislocalizing Beclin 1 from the ER to microtubules ► Starvation induces autophagy and dissociates the Bim-Beclin1 interaction
The membrane origin of autophagosomes has long been a mystery and it may involve multiple sources. In this punctum, we discuss our recent finding that the plasma membrane contributes to the formation of pre-autophagic structures via clathrin-mediated endocytosis. Our study suggests that Atg16L1 interacts with clathrin heavy-chain/AP2 and is also localized on vesicles (positive for clathrin or cholera toxin B) close to the plasma membrane. Live-cell imaging studies revealed that the plasma membrane contributes to Atg16L1-positive structures and that this process and autophagosome formation are impaired by knockdowns of genes regulating clathrin-mediated endocytosis.
autophagy; plasma membrane; endocytosis; phagophore; origin
► The ubiquitin–proteasome system and autophagy are two main degradative pathways. ► Autophagy upregulation may protect against polyglutamine-expanded protein neurotoxicity. ► Autophagy compromise may occur in certain neurodegenerative diseases.
In polyglutamine diseases, an abnormally elongated polyglutamine tract results in protein misfolding and accumulation of intracellular aggregates. The length of the polyglutamine expansion correlates with the tendency of the mutant protein to aggregate, as well as with neuronal toxicity and earlier disease onset. Although currently there is no effective cure to prevent or slow down the progression of these neurodegenerative disorders, increasing the clearance of mutant proteins has been proposed as a potential therapeutic approach. The ubiquitin–proteasome system and autophagy are the two main degradative pathways responsible for eliminating misfolded and unnecessary proteins in the cell. We will review some of the studies that have proposed autophagy as a strategy to reduce the accumulation of polyglutamine-expanded protein aggregates and protect against mutant protein neurotoxicity. We will also discuss some of the currently known mechanisms that induce autophagy, which may be beneficial for the treatment of these and other neurodegenerative disorders.
HD, Huntington's disease; SCA, spinocerebellar ataxia; DRPLA, Denatorubral-pallidoluysian atrophy; SBMA, spinal and bulbar muscular atropy; Htt, Huntingtin; UPS, ubiquitin–proteasome system; HDL-2, Huntington's disease-like 2; IBs, inclusion bodies; RNAi, RNA interference; Atg, autophagy-related genes; ER, endoplasmic reticulum; PI3K, phosphatidylinositol 3-kinase; JNK1, c-Jun N-terminal protein kinase 1; PE, phosphatidylethanolamine; SNAREs, soluble N-ethylmaleimide-sensitive factor attachment protein receptors; mTOR, mammalian target of rapamycin; PI-3-P, phosphatidylinositol-3-phosphate; ROS, reactive oxygen species; IP3, inositol-1,4,5-triphosphate; IP3R, IP3 receptors; cAMP, cyclic AMP; IMPase, inositol monophosphatase; GSK3β, glycogen synthase kinase-3 β; I1R, imidazoline-1-receptor; SMERs, small molecule enhancers of rapamycin; SMIRs, small molecule inhibitors of rapamycin; Polyglutamine diseases; Autophagy; Neurodegeneration; Huntington's disease
Macroautophagy, a constitutive process in higher eukaryotic cells, mediates degradation of many long-lived proteins and organelles. The actual events occurring during the process in the dynamic system of a living cell have never been thoroughly investigated. We aimed to develop a live-cell assay in which to follow the complete itinerary of an autophagosome. Our experiments show that autophagosomes are formed randomly in peripheral regions of the cell. They then move bidirectionally along microtubules, accumulating at the microtubule-organizing centre, in a similar way to lysosomes. Their centripetal movement is dependent on the motor protein dynein and is important for their fusion with lysosomes. Initially, autophagosomes dock on to lysosomes, independent of lysosomal acidification. Two kinds of fusion then occur: complete fusions, creating a hybrid organelle, or more often kiss-and-run fusions, i.e. transfer of some content while still maintaining two separate vesicles. Surprisingly, the autophagolysosomal compartment seems to be more long lived than expected. Our study documents many aspects of autophagosome behaviour, adding to our understanding of the mechanism and control of autophagy. Indeed, although the formation of autophagosomes is completely different from any other vesicular structures, their later itinerary appears to be very similar to those of other trafficking pathways.
autophagosome; autophagy; dynein; fusion; lysosome
Mammalian target of rapamycin (mTOR) signalling and macroautophagy (henceforth autophagy) regulate numerous pathological and physiological processes including cellular responses to altered nutrient levels. However, the mechanisms regulating mTOR and autophagy remain incompletely understood. Lysosomes are dynamic intracellular organelles 1, 2 intimately involved both in the activation of mTOR complex 1 (mTORC1) signalling and in degrading autophagic substrates 3-8. Here we report that lysosomal positioning coordinates anabolic and catabolic responses to changes in nutrient availability by orchestrating early plasma membrane signalling events, mTORC1 signalling and autophagy. Activation of mTORC1 by nutrients correlates with its presence on peripheral lysosomes that are physically close to the upstream signalling modules, while starvation causes perinuclear clustering of lysosomes, driven by changes in intracellular pH (pHi). Lysosomal positioning regulates mTORC1 signalling, which, in turn, influences autophagosome formation. Lysosome positioning also influences autophagosome-lysosome fusion rates, and thus controls autophagic flux by acting both at the initiation and termination stages of the process. Our findings provide a fundamental physiological role for the dynamic state of lysosomal positioning in cells as a coordinator of mTORC1 signalling with autophagic flux.
A recent genome-wide association (GWA) study of late-onset Alzheimer's disease (LOAD) identified 15 novel single nucleotide polymorphisms (SNPs) independent of ApoE. We hypothesized that variants associated with LOAD are also associated with poor cognitive function in elderly populations. We measured additive associations between the five most strongly associated LOAD SNPs and grouped Mini Mental State Examination (MMSE) scores. Variants were genotyped in respondents (mean age 79yrs) from the Oxford Healthy Aging project (OHAP) and other sites of the MRC Cognitive Function and Aging Study (MRC-CFAS). In adjusted ordinal logistic models, two variants were associated with poorer cognitive function: rs11622883 (OR=1.14, 95%CI: 1.01 to 1.28, p=0.040) and rs505058 (OR=1.29, 95% CI: 1.02 to 1.64, p=0.036). These SNPs are close to a SERPINA gene cluster and within LMNA respectively. The mechanisms underlying the associations with cognitive impairment and LOAD require further elucidation, but both genes are interesting candidates for involvement in age-related cognitive impairment.
Late-onset Alzheimer's disease; dementia; cognitive function; cognitive impairment; gene; single nucleotide polymorphism; ApoE; LMNA
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.
► Homotypic fusion of Atg16L1 vesicles enables their maturation into autophagosomes ► VAMP7 regulates Atg16L1 vesicle homotypic fusion and autophagosome formation ► Plasma membrane is a likely source of SNAREs required for Atg16L1 vesicle fusion ► Starvation, which induces autophagy, triggers Atg16L1 vesicle homotypic fusion
Autophagy, a major degradation process for long-lived and aggregate-prone proteins, affects various human processes, such as development, immunity, cancer, and neurodegeneration. Several autophagy regulators have been identified in recent years. Here we show that nitric oxide (NO), a potent cellular messenger, inhibits autophagosome synthesis via a number of mechanisms. NO impairs autophagy by inhibiting the activity of S-nitrosylation substrates, JNK1 and IKKβ. Inhibition of JNK1 by NO reduces Bcl-2 phosphorylation and increases the Bcl-2–Beclin 1 interaction, thereby disrupting hVps34/Beclin 1 complex formation. Additionally, NO inhibits IKKβ and reduces AMPK phosphorylation, leading to mTORC1 activation via TSC2. Overexpression of nNOS, iNOS, or eNOS impairs autophagosome formation primarily via the JNK1–Bcl-2 pathway. Conversely, NOS inhibition enhances the clearance of autophagic substrates and reduces neurodegeneration in models of Huntington's disease. Our data suggest that nitrosative stress-mediated protein aggregation in neurodegenerative diseases may be, in part, due to autophagy inhibition.
► NO inhibits autophagy by independently inhibiting JNK1 and IKKβ ► NO inhibits autophagic flux via mTOR and mTOR-independent routes ► NOS overexpression impairs autophagosome synthesis via JNK1–Bcl-2 pathway ► NOS inhibition induces autophagy and protects against neurodegeneration
Huntington's disease (HD) is a neurodegenerative disorder caused by an expanded polyglutamine (polyQ) tract in the huntingtin (htt) protein. Mutant htt toxicity is exposed after htt cleavage by caspases and other proteases release NH2-terminal fragments containing the polyQ expansion. Here, we show htt interacts and colocalizes with cdk5 in cellular membrane fractions. Cdk5 phosphorylates htt at Ser434, and this phosphorylation reduces caspase-mediated htt cleavage at residue 513. Reduced mutant htt cleavage resulting from cdk5 phosphorylation attenuated aggregate formation and toxicity in cells expressing the NH2-terminal 588 amino acids (htt588) of mutant htt. Cdk5 activity is reduced in the brains of HD transgenic mice compared with controls. This result can be accounted for by the polyQ-expanded htt fragments reducing the interaction between cdk5 and its activator p35. These data predict that the ability of cdk5 phosphorylation to protect against htt cleavage, aggregation, and toxicity is compromised in cells expressing toxic fragments of htt.
In the past decade, the zebrafish (Danio rerio) has become a popular model system for the study of vertebrate development, since the embryos and larvae of this species are small, transparent and undergo rapid development ex utero, allowing in vivo analysis of embryogenesis and organogenesis. These characteristics can also be exploited by researchers interested in signaling pathways and disease processes and, accordingly, there is a growing literature on the use of zebrafish to model human disease. This model holds great potential for exploring how autophagy, an evolutionarily conserved mechanism for protein degradation, influences the pathogeneses of a range of different human diseases and for the evaluation of this pathway as a potential therapeutic strategy. Here we summarize what is known about the regulation of autophagy in eukaryotic cells and its role in neurodegenerative disease and highlight how research using zebrafish has helped further our understanding of these processes.
► Zebrafish has become a popular model system for the study of vertebrate development. ► There is a growing literature on the use of zebrafish to model human disease. ► This model has potential for exploring how autophagy affects disease pathogenesis. ► This review considers how zebrafish may help further understanding of these processes.
Zebrafish; Autophagy; Neurodegeneration
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.