Publications by authors named "David C Rubinsztein"

262 Publications

Breakthroughs and bottlenecks in autophagy research.

Trends Mol Med 2021 Jul 11. Epub 2021 Jul 11.

Department of Medical Genetics, Cambridge Institute for Medical Research, Wellcome Trust/MRC Building, Cambridge, UK; UK Dementia Research Institute, Cambridge Biomedical Campus, Cambridge, UK. Electronic address:

The study of autophagy has grown exponentially over the past two decades, and significant progress has been made in our understanding of its mechanisms and physiological significance. However, its application to human diseases remains limited. Here, we summarize the current status of autophagy research, with a particular focus on human diseases.
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http://dx.doi.org/10.1016/j.molmed.2021.06.012DOI Listing
July 2021

Glucose starvation induces autophagy via ULK1-mediated activation of PIKfyve in an AMPK-dependent manner.

Dev Cell 2021 Jul 8;56(13):1961-1975.e5. Epub 2021 Jun 8.

Department of Medical Genetics, Cambridge Institute for Medical Research, University of Cambridge, Cambridge, UK; UK Dementia Research Institute, Cambridge, UK. Electronic address:

Autophagy is an essential catabolic process induced to provide cellular energy sources in response to nutrient limitation through the activation of kinases, like AMP-activated protein kinase (AMPK) and ULK1. Although glucose starvation induces autophagy, the exact mechanism underlying this signaling has yet to be elucidated. Here, we reveal a role for ULK1 in non-canonical autophagy signaling using diverse cell lines. ULK1 activated by AMPK during glucose starvation phosphorylates the lipid kinase PIKfyve on S1548, thereby increasing its activity and the synthesis of the phospholipid PI(5)P without changing the levels of PI(3,5)P. ULK1-mediated activation of PIKfyve enhances the formation of PI(5)P-containing autophagosomes upon glucose starvation, resulting in an increase in autophagy flux. Phospho-mimic PIKfyve S1548D drives autophagy upregulation and lowers autophagy substrate levels. Our study has identified how ULK1 upregulates autophagy upon glucose starvation and induces the formation of PI(5)P-containing autophagosomes by activating PIKfyve.
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http://dx.doi.org/10.1016/j.devcel.2021.05.010DOI Listing
July 2021

Lysosome positioning and mTOR activity in Lowe syndrome.

EMBO Rep 2021 Jul 27;22(7):e53232. Epub 2021 May 27.

Department of Medical Genetics, Cambridge Institute for Medical Research, University of Cambridge, Cambridge, UK.

Lowe syndrome is a rare, developmental disorder caused by mutations in the phosphatase, OCRL. A study in this issue of EMBO Reports shows that OCRL is required for microtubule nucleation and that mutations in this protein lead to an inability to activate mTORC1 signaling and consequent cell proliferation in the presence of nutrients. These defects are the result of impaired microtubule-dependent lysosomal trafficking to the cell periphery and are independent of OCRL phosphatase activity.
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http://dx.doi.org/10.15252/embr.202153232DOI Listing
July 2021

Cell type-specific YAP1-WWTR1/TAZ transcriptional responses after autophagy perturbations are determined by levels of α-catenins (CTNNA1 and CTNNA3).

Autophagy 2021 Jun 4:1-3. Epub 2021 Jun 4.

Department of Medical Genetics, Cambridge Institute for Medical Research, Wellcome Trust/MRC Building, Cambridge Biomedical Campus, Cambridge, UK.

The YAP1-WWTR1/TAZ transcription co-factors are key determinants of cell growth that are perturbed in many cancers. Previous studies have reported divergent responses in YAP1-WWTR1/TAZ activities after autophagy perturbations in different contexts. Recently, we identified that α-catenin levels determine whether YAP1-WWTR1/TAZ signaling will be increased or decreased after macroautophagy/autophagy inhibition/induction. CTNNA1/α-catenin can act as a switch in this pathway, as it is an autophagy substrate and a negative regulator of YAP1-WWTR1/TAZ. However, YAP1-WWTR1/TAZ are also directly degraded by autophagy and there is a feedback loop where YAP1-WWTR1/TAZ positively regulate autophagy. These features were integrated into a mathematical numerical model based on a set of differential equations in order to clarify the integrated output on YAP1-WWTR1/TAZ activity at different time-points after autophagy perturbation in cells with distinct initial levels of α-catenins (CTNNA1 and CTNNA3). Our theoretical and experimental data allow an understanding of cell-type specific and time-dependent responses to autophagy manipulations that may be relevant in many contexts, including different types of cancer.
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http://dx.doi.org/10.1080/15548627.2021.1934273DOI Listing
June 2021

Transient siRNA-mediated protein knockdown in mouse followed by feeding/starving cycle and liver tissue analysis.

STAR Protoc 2021 Jun 27;2(2):100500. Epub 2021 Apr 27.

Department of Medical Genetics, Cambridge Institute for Medical Research, University of Cambridge, Hills Road, Cambridge, CB2 0XY, UK.

We present a protocol for in vivo siRNA-mediated knockdown of a gene of interest in mouse liver using systemic delivery via intravenous injection. We describe a step-by-step protocol for delivery of siRNA particles, with tips on how to optimize dosage. We detail steps for feeding/starving cycles as well as for liver tissue isolation, followed by gene expression analysis, measured at the mRNA and protein levels. For complete information on the generation and use of this protocol, please refer to Wrobel et al. (2020).
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http://dx.doi.org/10.1016/j.xpro.2021.100500DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8102171PMC
June 2021

α-Catenin levels determine direction of YAP/TAZ response to autophagy perturbation.

Nat Commun 2021 03 17;12(1):1703. Epub 2021 Mar 17.

Department of Medical Genetics, Cambridge Institute for Medical Research, Wellcome Trust/MRC Building, Cambridge, UK.

The factors regulating cellular identity are critical for understanding the transition from health to disease and responses to therapies. Recent literature suggests that autophagy compromise may cause opposite effects in different contexts by either activating or inhibiting YAP/TAZ co-transcriptional regulators of the Hippo pathway via unrelated mechanisms. Here, we confirm that autophagy perturbation in different cell types can cause opposite responses in growth-promoting oncogenic YAP/TAZ transcriptional signalling. These apparently contradictory responses can be resolved by a feedback loop where autophagy negatively regulates the levels of α-catenins, LC3-interacting proteins that inhibit YAP/TAZ, which, in turn, positively regulate autophagy. High basal levels of α-catenins enable autophagy induction to positively regulate YAP/TAZ, while low α-catenins cause YAP/TAZ activation upon autophagy inhibition. These data reveal how feedback loops enable post-transcriptional determination of cell identity and how levels of a single intermediary protein can dictate the direction of response to external or internal perturbations.
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http://dx.doi.org/10.1038/s41467-021-21882-1DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7969950PMC
March 2021

VCP/p97 modulates PtdIns3P production and autophagy initiation.

Autophagy 2021 04 9;17(4):1052-1053. Epub 2021 Mar 9.

Department of Medical Genetics, Cambridge Institute for Medical Research, Cambridge, UK.

VCP/p97 is an essential multifunctional protein implicated in a plethora of intracellular quality control systems, and abnormal function of VCP is the underlying cause of several neurodegenerative disorders. We reported that VCP regulates the levels of the macroautophagy/autophagy-inducing lipid phosphatidylinositol-3-phosphate (PtdIns3P) by modulating the activity of the BECN1 (beclin 1)-containing phosphatidylinositol 3-kinase (PtdIns3K) complex. VCP stimulates the deubiquitinase activity of ATXN3 (ataxin 3) to stabilize BECN1 protein levels and also interacts with and promotes the assembly and kinase activity of the PtdIns3K complex. Acute inhibition of VCP activity impairs autophagy induction, demonstrated by a diminished PtdIns3P production and decreased recruitment of early autophagy markers WIPI2 and ATG16L1. Thus, VCP promotes autophagosome biogenesis, in addition to its previously described role in autophagosome maturation.
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http://dx.doi.org/10.1080/15548627.2021.1898742DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8078685PMC
April 2021

Guidelines for the use and interpretation of assays for monitoring autophagy (4th edition).

Autophagy 2021 Jan 8;17(1):1-382. Epub 2021 Feb 8.

University of Crete, School of Medicine, Laboratory of Clinical Microbiology and Microbial Pathogenesis, Voutes, Heraklion, Crete, Greece; Foundation for Research and Technology, Institute of Molecular Biology and Biotechnology (IMBB), Heraklion, Crete, Greece.

In 2008, we published the first set of guidelines for standardizing research in autophagy. Since then, this topic has received increasing attention, and many scientists have entered the field. Our knowledge base and relevant new technologies have also been expanding. Thus, it is important to formulate on a regular basis updated guidelines for monitoring autophagy in different organisms. Despite numerous reviews, there continues to be confusion regarding acceptable methods to evaluate autophagy, especially in multicellular eukaryotes. Here, we present a set of guidelines for investigators to select and interpret methods to examine autophagy and related processes, and for reviewers to provide realistic and reasonable critiques of reports that are focused on these processes. These guidelines are not meant to be a dogmatic set of rules, because the appropriateness of any assay largely depends on the question being asked and the system being used. Moreover, no individual assay is perfect for every situation, calling for the use of multiple techniques to properly monitor autophagy in each experimental setting. Finally, several core components of the autophagy machinery have been implicated in distinct autophagic processes (canonical and noncanonical autophagy), implying that genetic approaches to block autophagy should rely on targeting two or more autophagy-related genes that ideally participate in distinct steps of the pathway. Along similar lines, because multiple proteins involved in autophagy also regulate other cellular pathways including apoptosis, not all of them can be used as a specific marker for autophagic responses. Here, we critically discuss current methods of assessing autophagy and the information they can, or cannot, provide. Our ultimate goal is to encourage intellectual and technical innovation in the field.
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http://dx.doi.org/10.1080/15548627.2020.1797280DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7996087PMC
January 2021

VCP/p97 regulates Beclin-1-dependent autophagy initiation.

Nat Chem Biol 2021 04 28;17(4):448-455. Epub 2021 Jan 28.

Department of Medical Genetics, Cambridge Institute for Medical Research, The Keith Peters Building, Cambridge Biomedical Campus, Cambridge, UK.

Autophagy is an essential cellular process that removes harmful protein species, and autophagy upregulation may be able to protect against neurodegeneration and various pathogens. Here, we have identified the essential protein VCP/p97 (VCP, valosin-containing protein) as a novel regulator of autophagosome biogenesis, where VCP regulates autophagy induction in two ways, both dependent on Beclin-1. Utilizing small-molecule inhibitors of VCP ATPase activity, we show that VCP stabilizes Beclin-1 levels by promoting the deubiquitinase activity of ataxin-3 towards Beclin-1. VCP also regulates the assembly and activity of the Beclin-1-containing phosphatidylinositol-3-kinase (PI3K) complex I, thus regulating the production of PI(3)P, a key signaling lipid responsible for the recruitment of downstream autophagy factors. A decreased level of VCP, or inhibition of its ATPase activity, impairs starvation-induced production of PI(3)P and limits downstream recruitment of WIPI2, ATG16L and LC3, thereby decreasing autophagosome formation, illustrating an important role for VCP in early autophagy initiation.
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http://dx.doi.org/10.1038/s41589-020-00726-xDOI Listing
April 2021

Autophagy regulation by acetylation-implications for neurodegenerative diseases.

Exp Mol Med 2021 Jan 22;53(1):30-41. Epub 2021 Jan 22.

Department of Medical Genetics, University of Cambridge, Cambridge, UK.

Posttranslational modifications of proteins, such as acetylation, are essential for the regulation of diverse physiological processes, including metabolism, development and aging. Autophagy is an evolutionarily conserved catabolic process that involves the highly regulated sequestration of intracytoplasmic contents in double-membrane vesicles called autophagosomes, which are subsequently degraded after fusing with lysosomes. The roles and mechanisms of acetylation in autophagy control have emerged only in the last few years. In this review, we describe key molecular mechanisms by which previously identified acetyltransferases and deacetylases regulate autophagy. We highlight how p300 acetyltransferase controls mTORC1 activity to regulate autophagy under starvation and refeeding conditions in many cell types. Finally, we discuss how altered acetylation may impact various neurodegenerative diseases in which many of the causative proteins are autophagy substrates. These studies highlight some of the complexities that may need to be considered by anyone aiming to perturb acetylation under these conditions.
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http://dx.doi.org/10.1038/s12276-021-00556-4DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8080689PMC
January 2021

mTORC2 Assembly Is Regulated by USP9X-Mediated Deubiquitination of RICTOR.

Cell Rep 2020 12;33(13):108564

Department of Medical Genetics, Cambridge Institute for Medical Research, University of Cambridge, Hills Road, Cambridge CB2 0XY, UK; UK Dementia Research Institute, Cambridge, UK. Electronic address:

The mechanistic target of rapamycin complex 2 (mTORC2) controls cell metabolism and survival in response to environmental inputs. Dysregulation of mTORC2 signaling has been linked to diverse human diseases, including cancer and metabolic disorders, highlighting the importance of a tightly controlled mTORC2. While mTORC2 assembly is a critical determinant of its activity, the factors regulating this event are not well understood, and it is unclear whether this process is regulated by growth factors. Here, we present data, from human cell lines and mice, describing a mechanism by which growth factors regulate ubiquitin-specific protease 9X (USP9X) deubiquitinase to stimulate mTORC2 assembly and activity. USP9X removes Lys63-linked ubiquitin from RICTOR to promote its interaction with mTOR, thereby facilitating mTORC2 signaling. As mTORC2 is central for cellular homeostasis, understanding the mechanisms regulating mTORC2 activation toward its downstream targets is vital for our understanding of physiological processes and for developing new therapeutic strategies in pathology.
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http://dx.doi.org/10.1016/j.celrep.2020.108564DOI Listing
December 2020

Developing Therapies for Neurodegenerative Disorders: Insights from Protein Aggregation and Cellular Stress Responses.

Annu Rev Cell Dev Biol 2020 10;36:165-189

UK Dementia Research Institute at the University of Cambridge, Cambridge CB2 0AH, United Kingdom; email:

As the world's population ages, neurodegenerative disorders are poised to become the commonest cause of death. Despite this, they remain essentially untreatable. Characterized pathologically both by the aggregation of disease-specific misfolded proteins and by changes in cellular stress responses, to date, therapeutic approaches have focused almost exclusively on reducing misfolded protein load-notably amyloid beta (Aβ) in Alzheimer's disease. The repeated failure of clinical trials has led to despondency over the possibility that these disorders will ever be treated. We argue that this is in fact a time for optimism: Targeting various generic stress responses is emerging as an increasingly promising means of modifying disease progression across these disorders. New treatments are approaching clinical trials, while novel means of targeting aggregates could eventually act preventively in early disease.
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http://dx.doi.org/10.1146/annurev-cellbio-040320-120625DOI Listing
October 2020

Autophagy in Neuronal Development and Plasticity.

Trends Neurosci 2020 10 13;43(10):767-779. Epub 2020 Aug 13.

Department of Medical Genetics, University of Cambridge, Cambridge Institute for Medical Research, The Keith Peters Building, Cambridge Biomedical Campus, Hills Road, Cambridge CB2 0XY, UK; UK Dementia Research Institute, University of Cambridge, Cambridge Institute for Medical Research, The Keith Peters Building, Cambridge Biomedical Campus, Hills Road, Cambridge CB2 0XY, UK. Electronic address:

Autophagy is a highly conserved intracellular clearance pathway in which cytoplasmic contents are trafficked to the lysosome for degradation. Within neurons, it helps to remove damaged organelles and misfolded or aggregated proteins and has therefore been the subject of intense research in relation to neurodegenerative disease. However, far less is understood about the role of autophagy in other aspects of neuronal physiology. Here we review the literature on the role of autophagy in maintaining neuronal stem cells and in neuronal plasticity in adult life and we discuss how these contribute to structural and functional deficits observed in a range of human disorders.
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http://dx.doi.org/10.1016/j.tins.2020.07.003DOI Listing
October 2020

Huntingtin-lowering strategies for Huntington's disease.

Expert Opin Investig Drugs 2020 Oct 14;29(10):1125-1132. Epub 2020 Aug 14.

Department of Medical Genetics, Cambridge Institute for Medical Research , Cambridge, UK.

Introduction: Huntington's disease (HD) is an incurable, autosomal dominant neurodegenerative disease caused by an abnormally long polyglutamine tract in the huntingtin protein. Because this mutation causes disease via gain-of-function, lowering huntingtin levels represents a rational therapeutic strategy.

Areas Covered: We searched MEDLINE, CENTRAL, and other trial databases, and relevant company and HD funding websites for press releases until April 2020 to review strategies for huntingtin lowering, including autophagy and PROTACs, which have been studied in preclinical models. We focussed our analyses on oligonucleotide (ASOs) and miRNA approaches, which have entered or are about to enter clinical trials.

Expert Opinion: ASO and mRNA approaches for lowering mutant huntingtin protein production and strategies for increasing mutant huntingtin clearance are attractive because they target the cause of disease. However, questions concerning the optimal mode of delivery and associated safety issues remain. It is unclear if the human CNS coverage with intrathecal or intraparenchymal delivery will be sufficient for efficacy. The extent that one must lower mutant huntingtin levels for it to be therapeutic is uncertain and the extent to which CNS lowering of wild-type huntingtin is safe is unclear. Polypharmacy may be an effective approach for ameliorating signs and symptoms and for preventing/delaying onset and progression.
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http://dx.doi.org/10.1080/13543784.2020.1804552DOI Listing
October 2020

Deadly Encounter: Endosomes Meet Mitochondria to Initiate Apoptosis.

Dev Cell 2020 06;53(6):619-620

Cambridge Institute for Medical Research, Department of Medical Genetics, The Keith Peters Building, Cambridge Biomedical Campus, Hills Road, Cambridge CB2 0XY, UK; UK Dementia Research Institute, Cambridge BioMedical Campus, The Keith Peters Building, Hills Road, Cambridge CB2 0XY, UK. Electronic address:

Mitochondrial outer membrane permeabilization (MOMP) is a crucial event enabling apoptotic cell death. In this issue of Developmental Cell, Wang et al. reveal an interaction contributing to full MOMP execution, which depends on endosomes accumulating on apoptotic mitochondria. This causes mitochondrial lipid alterations that may contribute to functional pore assembly.
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http://dx.doi.org/10.1016/j.devcel.2020.05.030DOI Listing
June 2020

Leucine regulates autophagy via acetylation of the mTORC1 component raptor.

Nat Commun 2020 06 19;11(1):3148. Epub 2020 Jun 19.

Department of Medical Genetics, Cambridge Institute for Medical Research (CIMR), University of Cambridge, Cambridge, UK.

Macroautophagy ("autophagy") is the main lysosomal catabolic process that becomes activated under nutrient-depleted conditions, like amino acid (AA) starvation. The mechanistic target of rapamycin complex 1 (mTORC1) is a well-conserved negative regulator of autophagy. While leucine (Leu) is a critical mTORC1 regulator under AA-starved conditions, how Leu regulates autophagy is poorly understood. Here, we describe that in most cell types, including neurons, Leu negatively regulates autophagosome biogenesis via its metabolite, acetyl-coenzyme A (AcCoA). AcCoA inhibits autophagy by enhancing EP300-dependent acetylation of the mTORC1 component raptor, with consequent activation of mTORC1. Interestingly, in Leu deprivation conditions, the dominant effects on autophagy are mediated by decreased raptor acetylation causing mTORC1 inhibition, rather than by altered acetylation of other autophagy regulators. Thus, in most cell types we examined, Leu regulates autophagy via the impact of its metabolite AcCoA on mTORC1, suggesting that AcCoA and EP300 play pivotal roles in cell anabolism and catabolism.
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http://dx.doi.org/10.1038/s41467-020-16886-2DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7305105PMC
June 2020

cGMP via PKG activates 26S proteasomes and enhances degradation of proteins, including ones that cause neurodegenerative diseases.

Proc Natl Acad Sci U S A 2020 06 8;117(25):14220-14230. Epub 2020 Jun 8.

Department of Cell Biology, Blavatnik Institute, Harvard Medical School, Boston, MA 02115;

Because raising cAMP enhances 26S proteasome activity and the degradation of cell proteins, including the selective breakdown of misfolded proteins, we investigated whether agents that raise cGMP may also regulate protein degradation. Treating various cell lines with inhibitors of phosphodiesterase 5 or stimulators of soluble guanylyl cyclase rapidly enhanced multiple proteasome activities and cellular levels of ubiquitinated proteins by activating protein kinase G (PKG). PKG stimulated purified 26S proteasomes by phosphorylating a different 26S component than is modified by protein kinase A. In cells and cell extracts, raising cGMP also enhanced within minutes ubiquitin conjugation to cell proteins. Raising cGMP, like raising cAMP, stimulated the degradation of short-lived cell proteins, but unlike cAMP, also markedly increased proteasomal degradation of long-lived proteins (the bulk of cell proteins) without affecting lysosomal proteolysis. We also tested if raising cGMP, like cAMP, can promote the degradation of mutant proteins that cause neurodegenerative diseases. Treating zebrafish models of tauopathies or Huntington's disease with a PDE5 inhibitor reduced the levels of the mutant huntingtin and tau proteins, cell death, and the resulting morphological abnormalities. Thus, PKG rapidly activates cytosolic proteasomes, protein ubiquitination, and overall protein degradation, and agents that raise cGMP may help combat the progression of neurodegenerative diseases.
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http://dx.doi.org/10.1073/pnas.2003277117DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7321992PMC
June 2020

A location, location, location mutation impairs DNM2-mediated release of nascent autophagosomes from recycling endosomes.

Autophagy 2020 07 26;16(7):1353-1354. Epub 2020 May 26.

Department of Medical Genetics, Cambridge Institute for Medical Research , Cambridge, UK.

Elucidation of the membranes contributing to autophagosomes has been a critical question in the field, and an area of active research. Recently, we showed that key events in autophagosome formation, from PtdIns3P formation/WIPI2 recruitment to LC3-GABARAP membrane conjugation, occur on the RAB11A-positive compartment (recycling endosomes). This observation raised the question of how the LC3-positive autophagosome precursors detach from the recycling endosome. We recently observed that DNM2 (dynamin 2) mediates this step, and described how the DNM2 mutation that causes centronuclear myopathy (CNM) leads to the accumulation of autophagic structures on recycling endosomes, thereby stalling macroautophagy/autophagy. This physiologically important step highlights the importance of understanding release of nascent autophagosomes from the recycling endosomes as part of the autophagy itinerary.
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http://dx.doi.org/10.1080/15548627.2020.1764210DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7469587PMC
July 2020

A DNM2 Centronuclear Myopathy Mutation Reveals a Link between Recycling Endosome Scission and Autophagy.

Dev Cell 2020 04;53(2):154-168.e6

Department of Medical Genetics, Cambridge Biomedical Campus, Hills Road, Cambridge CB2 0XY, UK; Cambridge Institute for Medical Research, The Keith Peters Building, Cambridge Biomedical Campus, Hills Road, Cambridge CB2 0XY, UK; UK Dementia Research Institute, Cambridge BioMedical Campus, The Keith Peters Building, Hills Road, Cambridge CB2 0XY, UK. Electronic address:

Autophagy involves engulfment of cytoplasmic contents by double-membraned autophagosomes, which ultimately fuse with lysosomes to enable degradation of their substrates. We recently proposed that the tubular-vesicular recycling endosome membranes were a core platform on which the critical early events of autophagosome formation occurred, including LC3-membrane conjugation to autophagic precursors. Here, we report that the release of autophagosome precursors from recycling endosomes is mediated by DNM2-dependent scission of these tubules. This process is regulated by DNM2 binding to LC3 and is increased by autophagy-inducing stimuli. This scission is defective in cells expressing a centronuclear-myopathy-causing DNM2 mutant. This mutant has an unusual mechanism as it depletes normal-functioning DNM2 from autophagosome formation sites on recycling endosomes by causing increased binding to an alternative plasma membrane partner, ITSN1. This "scission" step is, thus, critical for autophagosome formation, is defective in a human disease, and influences the way we consider how autophagosomes are formed.
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http://dx.doi.org/10.1016/j.devcel.2020.03.018DOI Listing
April 2020

IFNB/interferon-β regulates autophagy via a -TBC1D15-RAB7 pathway.

Autophagy 2020 04 20;16(4):767-769. Epub 2020 Jan 20.

Development and Stem Cells Program, Monash Biomedicine Discovery Institute and Department of Anatomy and Developmental Biology, Monash University, Melbourne, Australia.

Loss of IFNB/interferon-β in mice causes a Parkinson disease-like phenotype where many features, including SNCA/α-synuclein and MAPT/tau accumulation, can be attributed to a late-stage block in autophagic flux. Recently, we identified a mechanism that can explain this phenotype. We found that IFNB induces expression of , a microRNA that can reduce the levels of TBC1D15, a RAB GTPase-activating protein. Induction of this pathway decreases RAB7 activity and thereby stimulates macroautophagy/autophagy. The relevance of these key players is deeply conserved from humans to , highlighting the importance of this ancient autophagy regulatory pathway.
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http://dx.doi.org/10.1080/15548627.2020.1718384DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7138209PMC
April 2020

Autophagy, Cellular Aging and Age-related Human Diseases.

Exp Neurobiol 2019 Dec;28(6):643-657

Department of Anatomy, Yonsei University College of Medicine, Seoul 03722, Korea.

Macroautophagy/autophagy is a conserved degradation system that engulfs intracytoplasmic contents, including aggregated proteins and organelles, which is crucial for cellular homeostasis. During aging, cellular factors suggested as the cause of aging have been reported to be associated with progressively compromised autophagy. Dysfunctional autophagy may contribute to age-related diseases, such as neurodegenerative disease, cancer, and metabolic syndrome, in the elderly. Therefore, restoration of impaired autophagy to normal may help to prevent age-related disease and extend lifespan and longevity. Therefore, this review aims to provide an overview of the mechanisms of autophagy underlying cellular aging and the consequent disease. Understanding the mechanisms of autophagy may provide potential information to aid therapeutic interventions in age-related diseases.
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http://dx.doi.org/10.5607/en.2019.28.6.643DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6946111PMC
December 2019

Autophagy Induction as a Therapeutic Strategy for Neurodegenerative Diseases.

J Mol Biol 2020 04 27;432(8):2799-2821. Epub 2019 Dec 27.

Department of Medical Genetics, Cambridge Institute for Medical Research (CIMR), University of Cambridge, Cambridge, UK; UK Dementia Research Institute, Cambridge Institute for Medical Research (CIMR), University of Cambridge, Cambridge, UK. Electronic address:

Autophagy is a major, conserved cellular pathway by which cells deliver cytoplasmic contents to lysosomes for degradation. Genetic studies have revealed extensive links between autophagy and neurodegenerative disease, and disruptions to autophagy may contribute to pathology in some cases. Autophagy degrades many of the toxic, aggregate-prone proteins responsible for such diseases, including mutant huntingtin (mHTT), alpha-synuclein (α-syn), tau, and others, raising the possibility that autophagy upregulation may help to reduce levels of toxic protein species, and thereby alleviate disease. This review examines autophagy induction as a potential therapy in several neurodegenerative diseases-Alzheimer's disease, Parkinson's disease, polyglutamine diseases, and amyotrophic lateral sclerosis (ALS). Evidence in cells and in vivo demonstrates promising results in many disease models, in which autophagy upregulation is able to reduce the levels of toxic proteins, ameliorate signs of disease, and delay disease progression. However, the effective therapeutic use of autophagy induction requires detailed knowledge of how the disease affects the autophagy-lysosome pathway, as activating autophagy when the pathway cannot go to completion (e.g., when lysosomal degradation is impaired) may instead exacerbate disease in some cases. Investigating the interactions between autophagy and disease pathogenesis is thus a critical area for further research.
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http://dx.doi.org/10.1016/j.jmb.2019.12.035DOI Listing
April 2020

Interferon-β-induced miR-1 alleviates toxic protein accumulation by controlling autophagy.

Elife 2019 12 4;8. Epub 2019 Dec 4.

Development and Stem Cells Program, Monash Biomedicine Discovery Institute and Department of Anatomy and Developmental Biology, Monash University, Melbourne, Australia.

Appropriate regulation of autophagy is crucial for clearing toxic proteins from cells. Defective autophagy results in accumulation of toxic protein aggregates that detrimentally affect cellular function and organismal survival. Here, we report that the microRNA miR-1 regulates the autophagy pathway through conserved targeting of the orthologous re-2/ub2/DC16 (TBC) Rab GTPase-activating proteins TBC-7 and TBC1D15 in and mammalian cells, respectively. Loss of miR-1 causes TBC-7/TBC1D15 overexpression, leading to a block on autophagy. Further, we found that the cytokine interferon-β (IFN-β) can induce miR-1 expression in mammalian cells, reducing TBC1D15 levels, and safeguarding against proteotoxic challenges. Therefore, this work provides a potential therapeutic strategy for protein aggregation disorders.
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http://dx.doi.org/10.7554/eLife.49930DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6914338PMC
December 2019

Latest advances in aging research and drug discovery.

Aging (Albany NY) 2019 11 21;11(22):9971-9981. Epub 2019 Nov 21.

Center for Healthy Aging, Department of Cellular and Molecular Medicine, University of Copenhagen, Copenhagen, Denmark.

An increasing aging population poses a significant challenge to societies worldwide. A better understanding of the molecular, cellular, organ, tissue, physiological, psychological, and even sociological changes that occur with aging is needed in order to treat age-associated diseases. The field of aging research is rapidly expanding with multiple advances transpiring in many previously disconnected areas. Several major pharmaceutical, biotechnology, and consumer companies made aging research a priority and are building internal expertise, integrating aging research into traditional business models and exploring new go-to-market strategies. Many of these efforts are spearheaded by the latest advances in artificial intelligence, namely deep learning, including generative and reinforcement learning. To facilitate these trends, the Center for Healthy Aging at the University of Copenhagen and Insilico Medicine are building a community of Key Opinion Leaders (KOLs) in these areas and launched the annual conference series titled "Aging Research and Drug Discovery (ARDD)" held in the capital of the pharmaceutical industry, Basel, Switzerland (www.agingpharma.org). This ARDD collection contains summaries from the 6 annual meeting that explored aging mechanisms and new interventions in age-associated diseases. The 7 annual ARDD exhibition will transpire 2-4 of September, 2020, in Basel.
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http://dx.doi.org/10.18632/aging.102487DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6914421PMC
November 2019

New factors for protein transport identified by a genome-wide CRISPRi screen in mammalian cells.

J Cell Biol 2019 11 5;218(11):3861-3879. Epub 2019 Sep 5.

University of Cambridge Metabolic Research Laboratories, Wellcome Trust-Medical Research Council Institute of Metabolic Science, University of Cambridge, Cambridge, UK

Protein and membrane trafficking pathways are critical for cell and tissue homeostasis. Traditional genetic and biochemical approaches have shed light on basic principles underlying these processes. However, the list of factors required for secretory pathway function remains incomplete, and mechanisms involved in their adaptation poorly understood. Here, we present a powerful strategy based on a pooled genome-wide CRISPRi screen that allowed the identification of new factors involved in protein transport. Two newly identified factors, TTC17 and CCDC157, localized along the secretory pathway and were found to interact with resident proteins of ER-Golgi membranes. In addition, we uncovered that upon TTC17 knockdown, the polarized organization of Golgi cisternae was altered, creating glycosylation defects, and that CCDC157 is an important factor for the fusion of transport carriers to Golgi membranes. In conclusion, our work identified and characterized new actors in the mechanisms of protein transport and secretion and opens stimulating perspectives for the use of our platform in physiological and pathological contexts.
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http://dx.doi.org/10.1083/jcb.201902028DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6829651PMC
November 2019

LC3-positive structures are prominent in autophagy-deficient cells.

Sci Rep 2019 07 12;9(1):10147. Epub 2019 Jul 12.

Department of Medical Genetics, Cambridge Institute for Medical Research, The Keith Peters Building, Cambridge, CB2 0XY, UK.

Autophagy is an evolutionarily conserved process across eukaryotes that degrades cargoes like aggregate-prone proteins, pathogens, damaged organelles and macromolecules via delivery to lysosomes. The process involves the formation of double-membraned autophagosomes that engulf the cargoes destined for degradation, sometimes with the help of autophagy receptors like p62, which are themselves autophagy substrates. LC3-II, a standard marker for autophagosomes, is generated by the conjugation of cytosolic LC3-I to phosphatidylethanolamine (PE) on the surface of nascent autophagosomes. As LC3-II is relatively specifically associated with autophagosomes and autolysosomes (in the absence of conditions stimulating LC3-associated phagocytosis), quantification of LC3-positive puncta is considered as a gold-standard assay for assessing the numbers of autophagosomes in cells. Here we find that the endogenous LC3-positive puncta become larger in cells where autophagosome formation is abrogated, and are prominent even when LC3-II is not formed. This occurs even with transient and incomplete inhibition of autophagosome biogenesis. This phenomenon is due to LC3-I sequestration to p62 aggregates, which accumulate when autophagy is impaired. This observation questions the reliability of LC3-immunofluorescence assays in cells with compromised autophagy.
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http://dx.doi.org/10.1038/s41598-019-46657-zDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6625982PMC
July 2019

ULK1-mediated phosphorylation of ATG16L1 promotes xenophagy, but destabilizes the ATG16L1 Crohn's mutant.

EMBO Rep 2019 07 24;20(7):e46885. Epub 2019 May 24.

Department of Cellular and Molecular Medicine, University of Ottawa, Ottawa, ON, Canada.

Autophagy is a highly regulated catabolic pathway that is potently induced by stressors including starvation and infection. An essential component of the autophagy pathway is an ATG16L1-containing E3-like enzyme, which is responsible for lipidating LC3B and driving autophagosome formation. ATG16L1 polymorphisms have been linked to the development of Crohn's disease (CD), and phosphorylation of CD-associated ATG16L1 T300A (caATG16L1) has been hypothesized to contribute to cleavage and autophagy dysfunction. Here we show that ULK1 kinase directly phosphorylates ATG16L1 in response to infection and starvation. Phosphorylated ATG16L1 localizes to the site of internalized bacteria and stable cell lines harbouring a phospho-dead mutant of ATG16L1 have impaired xenophagy, indicating a role for ATG16L1 phosphorylation in the promotion of anti-bacterial autophagy. In contrast to wild-type ATG16L1, ULK1-mediated phosphorylation of caATG16L1 drives its destabilization in response to stress. In summary, our results show that ATG16L1 is a novel target of ULK1 kinase and that ULK1 signalling to ATG16L1 is a double-edged sword, enhancing the function of the wild-type ATG16L1, but promoting degradation of caATG16L1.
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http://dx.doi.org/10.15252/embr.201846885DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6607016PMC
July 2019

Author Correction: Felodipine induces autophagy in mouse brains with pharmacokinetics amenable to repurposing.

Nat Commun 2019 Jun 4;10(1):2530. Epub 2019 Jun 4.

Department of Medical Genetics, Cambridge Institute for Medical Research, University of Cambridge, The Keith Peters Building, Cambridge Biomedical Campus, Hills Road, Cambridge, CB2 0XY, UK.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.
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http://dx.doi.org/10.1038/s41467-019-10536-yDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6547739PMC
June 2019

Coincidence detection of RAB11A and PI(3)P by WIPI2 directs autophagosome formation.

Oncotarget 2019 Apr 5;10(27):2579-2580. Epub 2019 Apr 5.

David C. Rubinsztein: Department of Medical Genetics, Cambridge Institute for Medical Research, Cambridge, UK; UK Dementia Research Institute, The Keith Peters Building, Cambridge Biomedical Campus, Cambridge, UK.

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http://dx.doi.org/10.18632/oncotarget.26829DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6498992PMC
April 2019
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