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Sep 5, 2023
Astrovirus replication is dependent on induction of double-membrane vesicles through a PI3K-dependent, LC3-independent pathway
Theresa Bub, Virginia Hargest, Shaoyuan Tan, Maria Smith, Ana Vazquez-Pagan, Tim Flerlage, Pamela Brigleb, Victoria Meliopoulos, Brett Lindenbach, Harish N. Ramanathan, Valerie Cortez, Jeremy Chase Crawford, Stacey Schultz-Cherry
Human astrovirus is a positive-sense, single-stranded RNA virus. Astrovirus infection causes gastrointestinal symptoms and can lead to encephalitis in immunocompromised patients. Positive-strand RNA viruses typically utilize host intracellular membranes to form replication organelles, which are potential antiviral targets. Many of these replication organelles are double-membrane vesicles (DMVs). Here, we show that astrovirus infection leads to an increase in DMV formation through a replication-dependent mechanism that requires some early components of the autophagy machinery. Results indicate that the upstream class III phosphatidylinositol 3-kinase (PI3K) complex, but not LC3 conjugation machinery, is utilized in DMV formation. Both chemical and genetic inhibition of the PI3K complex lead to significant reduction in DMVs, as well as viral replication. Elucidating the role of autophagy machinery in DMV formation during astrovirus infection reveals a potential target for therapeutic intervention for immunocompromised patients.
Aug 24, 2023
Autophagy deficiency protects against ocular hypertension and neurodegeneration in experimental and spontaneous glaucoma mouse models
Angela Dixon, Myoung Sup Shim, April Nettesheim, Aislyn Coyne, Chien-Chia Su, Haiyan Gong & Paloma B. Liton
Glaucoma is a group of diseases that leads to chronic degeneration of retinal ganglion cell (RGC) axons and progressive loss of RGCs, resulting in vision loss. While aging and elevated intraocular pressure (IOP) have been identified as the main contributing factors to glaucoma, the molecular mechanisms and signaling pathways triggering RGC death and axonal degeneration are not fully understood. Previous studies in our laboratory found that overactivation of autophagy in DBA/2J::GFP-LC3 mice led to RGC death and optic nerve degeneration with glaucomatous IOP elevation. We found similar findings in aging GFP-LC3 mice subjected to chronic IOP elevation. Here, we further investigated the impact of autophagy deficiency on autophagy-deficient DBA/2J-Atg4bko and DBA/2J-Atg4b+/− mice, generated in our laboratory via CRISPR/Cas9 technology; as well as in Atg4bko mice subjected to the experimental TGFβ2 chronic ocular hypertensive model. Our data shows that, in contrast to DBA/2J and DBA/2J-Atg4b+/− littermates, DBA/2J-Atg4bko mice do not develop glaucomatous IOP elevation. Atg4b deficiency also protected against glaucomatous IOP elevation in the experimental TGFβ2 chronic ocular hypertensive model. Atg4 deletion did not compromise RGC or optic nerve survival in Atg4bko mice. Moreover, our results indicate a protective role of autophagy deficiency against RGC death and ON atrophy in the hypertensive DBA/2J-Atg4b+/− mice. Together, our data suggests a pathogenic role of autophagy activation in ocular hypertension and glaucoma.
Jul 20, 2023
Shear stress induces autophagy in Schlemm’s canal cells via primary cilia-mediated SMAD2/3 signaling pathway
Myoung Sup Shim, Angela Dixon, April Nettesheim, Kristin M. Perkumas, W. Daniel Stamer, Yang Sun and Paloma B. Liton
The Schlemm’s canal (SC) is a circular, lymphatic-like vessel located at the limbus of the eye that participates in the regulation of aqueous humor drainage to control intraocular pressure (IOP). Circumferential flow of aqueous humor within the SC lumen generates shear stress, which regulates SC cell behaviour. Using biochemical analysis and real-time live cell imaging techniques, we have investigated the activation of autophagy in SC cells by shear stress. We report, for the first time, the primary cilium (PC)-dependent activation of autophagy in SC cells in response to shear stress. Moreover, we identified PC-dependent shear stress-induced autophagy to be positively regulated by phosphorylation of SMAD2 in its linker and C-terminal regions. Additionally, SMAD2/3 signaling was found to transcriptionally activate LC3B, ATG5 and ATG7 in SC cells. Intriguingly, concomitant to SMAD2-dependent activation of autophagy, we also report here the activation of mTOR pathway, a classical autophagy inhibi-tor, in SC cells by shear stress. mTOR activation was found to also be dependent on the PC. Moreover, pharmacological inhibition of class I PI3K increased phosphorylation of SMAD2 at the linker and activated autophagy. Together, our data indicates an interplay between PI3K and SMAD2/3 signaling pathways in the regulation of PC-dependent shear stress-induced autophagy in SC cells.
Jul 18, 2023
Autophagy protein 5 controls flow-dependent endothelial functions
Pierre Nivoit, Thomas Mathivet, Junxi Wu, Yann Salemkour, Devanarayanan Siva Sankar, Véronique Baudrie, Jennifer Bourreau, Anne-Laure Guihot, Emilie Vessieres, Mathilde Lemitre, Cinzia Bocca, Jérémie Teillon, Morgane Le Gall, Anna Chipont, Estelle Robidel, Neeraj Dhaun, Eric Camerer, Pascal Reynier, Etienne Roux, Thierry Couffinhal, Patrick W. F. Hadoke, Jean-Sébastien Silvestre, Xavier Guillonneau, Philippe Bonnin, Daniel Henrion, Joern Dengjel, Pierre-Louis Tharaux & Olivia Lenoir
Dysregulated autophagy is associated with cardiovascular and metabolic diseases, where impaired flow-mediated endothelial cell responses promote cardiovascular risk. The mechanism by which the autophagy machinery regulates endothelial functions is complex. We applied multi-omics approaches and in vitro and in vivo functional assays to decipher the diverse roles of autophagy in endothelial cells. We demonstrate that autophagy regulates VEGF-dependent VEGFR signaling and VEGFR-mediated and flow-mediated eNOS activation. Endothelial ATG5 deficiency in vivo results in selective loss of flow-induced vasodilation in mesenteric arteries and kidneys and increased cerebral and renal vascular resistance in vivo. We found a crucial pathophysiological role for autophagy in endothelial cells in flow-mediated outward arterial remodeling, prevention of neointima formation following wire injury, and recovery after myocardial infarction. Together, these findings unravel a fundamental role of autophagy in endothelial function, linking cell proteostasis to mechanosensing.
Apr 4, 2023
ATG8-dependent LMX1B-autophagy crosstalk shapes human midbrain dopaminergic neuronal resilience
Natalia Jiménez-Moreno, Madhu Kollareddy, Petros Stathakos, Joanna J. Moss, Zuriñe Antón, Deborah K. Shoemark, Richard B. Sessions, Ralph Witzgall, Maeve Caldwell, Jon D. Lane
The LIM homeodomain transcription factors LMX1A and LMX1B are essential mediators of midbrain dopaminergic neuronal (mDAN) differentiation and survival. Here we show that LMX1A and LMX1B are autophagy transcription factors that provide cellular stress protection. Their suppression dampens the autophagy response, lowers mitochondrial respiration, and elevates mitochondrial ROS, and their inducible overexpression protects against rotenone toxicity in human iPSC-derived mDANs in vitro. Significantly, we show that LMX1A and LMX1B stability is in part regulated by autophagy, and that these transcription factors bind to multiple ATG8 proteins. Binding is dependent on subcellular localization and nutrient status, with LMX1B interacting with LC3B in the nucleus under basal conditions and associating with both cytosolic and nuclear LC3B during nutrient starvation. Crucially, ATG8 binding stimulates LMX1B-mediated transcription for efficient autophagy and cell stress protection, thereby establishing a novel LMX1B-autophagy regulatory axis that contributes to mDAN maintenance and survival in the adult brain.
Mar 1, 2023
Autophagy in the eye: from physiology to pathophysiology
Paloma B. Liton, Kathleen Boesze-Battaglia, Michael E. Boulton, Patricia Boya, Thomas A. Ferguson, Ian G. Ganley, Anu Kauppinnen, Gordon W. Laurie, Noboru Mizushima, Hideaki Morishita,j, Rossella Russo, Jaya Sadda, Rajalekshmy Shyam, Debasish Sinha, Debra A. Thompson and David N. Zacks
Autophagy is a catabolic self-degradative pathway that promotes the degradation and recycling of intracellular material through the lysosomal compartment. Although first believed to function in conditions of nutritional stress, autophagy is emerging as a critical cellular pathway, involved in a variety of physiological and pathophysiological processes. Autophagy dysregulation is associated with an increasing number of diseases, including ocular diseases. On one hand, mutations in autophagy-related genes have been linked to cataracts, glaucoma, and corneal dystrophy; on the other hand, alterations in autophagy and lysosomal pathways are a common finding in essentially all diseases of the eye. Moreover, LC3-associated phagocytosis, a form of non-canonical autophagy, is critical in promoting visual cycle function. This review collects the latest understanding of autophagy in the context of the eye. We will review and discuss the respective roles of autophagy in the physiology and/or pathophysiology of each of the ocular tissues, its diurnal/circadian variation, as well as its involvement in diseases of the eye.
Jan 15, 2023
Defective Mitochondrial Quality Control during Dengue
Infection Contributes to Disease Pathogenesis
Bharati Singh, Kiran Avula, Shamim Akhtar Sufi, Nahid Parwin, Sayani Das, Mohd Faraz Alam, Subhashish Samantaray, Leelakrishna Bankapalli, Alankrita Rani, Kokavalla Poornima, Biswajita Prusty, Tareni P. Mallick, Shubham K. Shaw,
Hiren Dodia, Shobhitendu Kabi, Trupti T. Pagad, Sriprasad Mohanty, Gulam Hussain Syed
Mitochondrial fitness is governed by mitochondrial quality control pathways comprising mitochondrial dynamics and mitochondrial-selective autophagy (mitophagy). Disruption of these processes has been implicated in many human diseases, including viral infections. Here, we report a comprehensive analysis of the effect of dengue infection on host mitochondrial homeostasis and its significance in dengue disease pathogenesis. Despite severe mitochondrial stress and injury, we observed that the pathways of mitochondrial quality control and mitochondrial biogenesis are paradoxically downregulated in dengue-infected human liver cells. This leads to the disruption of mitochondrial homeostasis and the onset of cellular injury and necrotic death in the infected cells. Interestingly, dengue promotes global autophagy but selectively disrupts mitochondrial-selective autophagy (mitophagy). Dengue downregulates the expression of PINK1 and Parkin, the two major proteins involved in tagging the damaged mitochondria for elimination through mitophagy. Mitophagy flux assays also suggest that Parkin-independent pathways of mitophagy are also inactive during dengue infection. Dengue infection also disrupts mitochondrial biogenesis by downregulating the master regulators PPARγ and PGC1α. Dengue-infected cells release mitochondrial damage-associated molecular patterns (mtDAMPs) such as mitochondrial DNA into the cytosol and extracellular milieu. Furthermore, the challenge of naive immune cells with culture supernatants from dengue-infected liver cells was sufficient to trigger proinflammatory signaling. In correlation with our in vitro observations, dengue patients have high levels of cell-free mitochondrial DNA in their blood in proportion to the degree of thrombocytopenia. Overall, our study shows how defective mitochondrial homeostasis in dengue-infected liver cells can drive dengue disease pathogenesis.
Dec 6, 2022
USP32-regulated LAMTOR1 ubiquitination impacts mTORC1 activation and autophagy induction
Alexandra Hertel, Ludovico Martins Alves, Henrik Dutz, Georg Tascher, Florian Bonn, Manuel Kaulich, Ivan Dikic, Stefan Eimer, Florian Steinberg, Anja Bremm
The endosomal-lysosomal system is a series of organelles in the endocytic pathway that executes trafficking and degradation of proteins and lipids and mediates the internalization of nutrients and growth factors to ensure cell survival, growth, and differentiation. Here, we reveal regulatory, non-proteolytic ubiquitin signals in this complex system that are controlled by the enigmatic deubiquitinase USP32. Knockout (KO) of USP32 in primary hTERT-RPE1 cells results among others in hyperubiquitination of the Ragulator complex subunit LAMTOR1. Accumulation of LAMTOR1 ubiquitination impairs its interaction with the vacuolar H+-ATPase, reduces Ragulator function, and ultimately limits mTORC1 recruitment. Consistently, in USP32 KO cells, less mTOR kinase localizes to lysosomes, mTORC1 activity is decreased, and autophagy is induced. Furthermore, we demonstrate that depletion of USP32 homolog CYK-3 in Caenorhabditis elegans results in mTOR inhibition and autophagy induction. In summary, we identify a control mechanism of the mTORC1 activation cascade at lysosomes via USP32-regulated LAMTOR1 ubiquitination.
Dec 5, 2022
Excessive protein accumulation and impaired autophagy in the hippocampus of Angelman syndrome modeled in mice
Francesca Aria, Kiran Pandey, Cristina M. Alberini
Angelman syndrome (AS), a neurodevelopmental disorder caused by abnormalities of the 15q11.2-q13.1 chromosome region, is characterized by impairment of cognitive and motor functions, sleep problems, and seizures. How the genetic defects of AS produce these neurological symptoms is unclear. Mice modeling AS (AS mice) accumulate activity-regulated-cytoskeleton-associated protein (ARC/ARG3.1), a neuronal immediate early gene (IEG) critical for synaptic plasticity. This accumulation suggests an altered protein metabolism.
Nov 18, 2022
Chaperone-mediated autophagy regulates adipocyte differentiation
Susmita Kaushik, Yves R Juste, Kristen Lindenau, Shuxian Dong, Adrián Macho-González, Olaya Santiago-Fernández, Mericka McCabe, Rajat Singh, Evripidis Gavathiotis, Ana Maria Cuervo
Adipogenesis is a tightly orchestrated multistep process wherein preadipocytes differentiate into adipocytes. The most studied aspect of adipogenesis is its transcriptional regulation through timely expression and silencing of a vast number of genes. However, whether turnover of key regulatory proteins per se controls adipogenesis remains largely understudied. Chaperone-mediated autophagy (CMA) is a selective form of lysosomal protein degradation that, in response to diverse cues, remodels the proteome for regulatory purposes. We report here the activation of CMA during adipocyte differentiation and show that CMA regulates adipogenesis at different steps through timely degradation of key regulatory signaling proteins and transcription factors that dictate proliferation, energetic adaptation, and signaling changes required for adipogenesis.
Nov 16, 2022
Chloroquine induces transitory attenuation of proliferation of human lung cancer cells through regulation of mutant P53 and YAP
Heena Saini, Mahima Choudhary, Harshita Sharma, Shibasish Chowdhury, Sudeshna Mukherjee & Rajdeep Chowdhury
In this study, we identify key molecular factors de-regulated in NSCLCs. Analyze their expression by real-time PCR and immunoblot; map their localization by immuno-fluorescence microscopy. We further propose an FDA approved drug, chloroquine (CQ) that affects the function of the molecular factors and hence can be repurposed as a therapeutic strategy against NSCLCs. Available NSCLC mutation data reflects a high probabilistic chance of patients harboring a p53 mutation, especially a gain of function (GOF)-R273H mutation. The GOF-P53 mutation enables the P53 protein to potentially interact with non-canonical protein partners facilitating oncogenesis. In this context, analysis of existing transcriptomic data from R273H-P53 expressing cells shows a concomitant up-regulation of Yes-associated protein (YAP) transcriptional targets and its protein partner TEAD1 in NSCLCs, suggesting a possible link between R273H-P53 and YAP. We therefore explored the inter-dependence of R273H-P53 and YAP in NSCLC cells. They were found to co-operatively regulate NSCLC proliferation. Genetic or pharmacological inhibition of YAP and GOF-P53 resulted in sensitization of NSCLC cells. Further analysis of pathways controlled by GOF-P53 and YAP showed that they positively regulate the cellular homeostatic process- autophagy to mediate survival. We hence postulated that a modulation of autophagy might be a potent strategy to curb proliferation. In accordance to above, autophagy inhibition, especially with the FDA-approved drug- chloroquine (CQ) resulted in cytoplasmic accumulation and reduced transcriptional activity of GOF-P53 and YAP, leading to growth arrest of NSCLC cells.
Jun 10, 2022
Emerging roles of mitotic autophagy
Eugenia Almacellas, Caroline Mauvezin
Lysosomes exert pleiotropic functions to maintain cellular homeostasis and degrade autophagy cargo. Despite the great advances that have boosted our understanding of autophagy and lysosomes in both physiology and pathology, their function in mitosis is still controversial. During mitosis, most organelles are reshaped or repurposed to allow the correct distribution of chromosomes. Mitotic entry is accompanied by a reduction in sites of autophagy initiation, supporting the idea of an inhibition of autophagy to protect the genetic material against harmful degradation. However, there is accumulating evidence revealing the requirement of selective autophagy and functional lysosomes for a faithful chromosome segregation. Degradation is the most-studied lysosomal activity, but recently described alternative functions that operate in mitosis highlight the lysosomes as guardians of mitotic progression. Because the involvement of autophagy in mitosis remains controversial, it is important to consider the specific contribution of signalling cascades, the functions of autophagic proteins and the multiple roles of lysosomes, as three entangled, but independent, factors controlling genomic stability. In this Review, we discuss the latest advances in this area and highlight the therapeutic potential of targeting autophagy for drug development.
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