bims-auttor Biomed News
on Autophagy and mTOR
Issue of 2022‒02‒13
forty-four papers selected by
Viktor Korolchuk, Newcastle University

  1. Acta Pharm Sin B. 2022 Jan;12(1): 33-49
      Metabolic homeostasis requires dynamic catabolic and anabolic processes. Autophagy, an intracellular lysosomal degradative pathway, can rewire cellular metabolism linking catabolic to anabolic processes and thus sustain homeostasis. This is especially relevant in the liver, a key metabolic organ that governs body energy metabolism. Autophagy's role in hepatic energy regulation has just begun to emerge and autophagy seems to have a much broader impact than what has been appreciated in the field. Though classically known for selective or bulk degradation of cellular components or energy-dense macromolecules, emerging evidence indicates autophagy selectively regulates various signaling proteins to directly impact the expression levels of metabolic enzymes or their upstream regulators. Hence, we review three specific mechanisms by which autophagy can regulate metabolism: A) nutrient regeneration, B) quality control of organelles, and C) signaling protein regulation. The plasticity of the autophagic function is unraveling a new therapeutic approach. Thus, we will also discuss the potential translation of promising preclinical data on autophagy modulation into therapeutic strategies that can be used in the clinic to treat common metabolic disorders.
    Keywords:  AIM, Atf8 interacting motif; ATGL, adipose triglyceride lipase; ATL3, Atlastin GTPase 3; ATM, ATM serine/threonine kinase; Autophagy; BA, bile acid; BCL2L13, BCL2 like 13; BNIP3, BCL2 interacting protein 3; BNIP3L, BCL2 interacting protein 3 like; CAR, constitutive androstane receptor; CCPG1, cell cycle progression 1; CLN3, lysosomal/endosomal transmembrane protein; CMA, chaperonin mediated autophagy; CREB, cAMP response element binding protein; CRY1, cryptochrome 1; CYP27A1, sterol 27-hydroxylase; CYP7A1, cholesterol 7α-hydroxylase; Cryptochrome 1; DFCP1, double FYVE-containing protein 1; FAM134B, family with sequence similarity 134, member B; FFA, free fatty acid; FOXO1, Forkhead box O1; FUNDC1, FUN14 domain containing 1; FXR, farnesoid X receptor; Farnesoid X receptor; GABARAPL1, GABA type A receptor associated protein like 1; GIM, GABARAP-interacting motif; LAAT-1, lysosomal amino acid transporter 1 homologue; LALP70, lysosomal apyrase-like protein of 70 kDa; LAMP1, lysosomal-associated membrane protein-1; LAMP2, lysosomal-associated membrane protein-2; LD, lipid droplet; LIMP1, lysosomal integral membrane protein-1; LIMP3, lysosomal integral membrane protein-3; LIR, LC3 interacting region; LXRa, liver X receptor a; LYAAT-1, lysosomal amino acid transporter 1; Liver metabolism; Lysosome; MCOLN1, mucolipin 1; MFSD1, major facilitator superfamily domain containing 1; NAFLD, non-alcoholic fatty liver disease; NBR1, BRCA1 gene 1 protein; NCoR1, nuclear receptor co-repressor 1; NDP52, calcium-binding and coiled-coil domain-containing protein 2; NPC-1, Niemann-Pick disease, type C1; Nutrient regeneration; OPTN, optineurin; PEX5, peroxisomal biogenesis factor 5; PI3K, phosphatidylinositol-4,5-bisphosphate 3-kinase; PINK1, phosphatase and tensin homolog (PTEN)-induced kinase 1; PKA, protein kinase A; PKB, protein kinase B; PLIN2, perilipin 2; PLIN3, perilipin 3; PP2A, protein phosphatase 2a; PPARα, peroxisomal proliferator-activated receptor-alpha; PQLC2, PQ-loop protein; PXR, pregnane X receptor; Quality control; RETREG1, reticulophagy regulator 1; ROS, reactive oxygen species; RTN3, reticulon 3; RTNL3, a long isoform of RTN3; S1PR2, sphingosine-1-phosphate receptor 2; S6K, P70-S6 kinase; S6RP, S6 ribosomal protein; SCARB2, scavenger receptor class B member 2; SEC62, SEC62 homolog, preprotein translocation factor; SIRT1, sirtuin 1; SLC36A1, solute carrier family 36 member 1; SLC38A7, solute carrier family 38 member 7; SLC38A9, sodium-coupled neutral amino acid transporter 9; SNAT7, sodium-coupled neutral amino acid transporter 7; SPIN, spindling; SQSTM1, sequestosome 1; STBD1, starch-binding domain-containing protein 1; Signaling proteins; TBK1, serine/threonine-protein kinase; TEX264, testis expressed 264, ER-phagy receptor; TFEB/TFE3, transcription factor EB; TGR5, takeda G protein receptor 5; TRAC-1, thyroid-hormone-and retinoic acid-receptor associated co-repressor 1; TRPML1, transient receptor potential mucolipin 1; ULK1, Unc-51 like autophagy activating kinase 1; UPR, unfolded protein response; V-ATPase, vacuolar-ATPase; VDR, vitamin D3 receptor; VLDL, very-low-density lipoprotein; WIPI1, WD repeat domain phosphoinositide-interacting protein 1; mTORC1, mammalian target of rapamycin complex 1
  2. Cell Calcium. 2022 Feb 05. pii: S0143-4160(22)00028-8. [Epub ahead of print]103 102553
      Neurodegenerative diseases (ND) pose a serious health burden to society and healthcare systems alike, with increasing incidence rates especially within aging populations. Alzheimer's disease (AD) is the most prevalent type of ND or dementia, followed by Parkinson's disease (PD), multiple sclerosis, amyotrophic lateral sclerosis, and Huntington's disease. Progressive neurological dysfunction and regional neuronal loss constitute the common characteristics of ND. Many ND are accompanied by accumulation of protein aggregates such as extracellular amyloid-β (in AD), intraneuronal hyper-phosphorylated tau (in AD), or α-synuclein (in PD). Two main systems are responsible for the clearance of damaged, dysfunctional or senescent proteins inside cells: the autophagy-lysosomal pathway and the ubiquitin-proteasome system. The importance of lysosomes in neurodegenerative processes is further highlighted by clinical phenotypes of lysosomal storage disorders (LSDs), comprising more than 70 inheritable diseases caused by mutations in lysosomal enzymes or lysosomal membrane proteins, often resulting in severe neurodegeneration. Dysfunctional lysosomal proteins and enzymes result in the lysosomal accumulation of undigested macromolecules, e.g. lipids, glycoproteins, glycosaminoglycans, or gangliosides. Defects in intracellular transport pathways involving endosomes and lysosomes are increasingly recognized as drivers of neurodegenerative disease pathology including AD and PD. Thus, accumulation of damaged proteins and organelles (e.g. mitochondria) in neurons and glial cells overwhelms the capacity of intracellular recycling and degradation mechanisms, exacerbating disease pathology. Endolysosomal ion channels have recently been established as important regulators of lysosomal exocytosis, ion homeostasis/pH, endolysosomal trafficking, fusion and fission, and autophagy. In particular two non-selective endolysosomal cation channel families, the mucolipin/TRPML/MCOLN channels and the two-pore channels/TPCs will be discussed here as potential pharmacological targets for LSD/ND treatment.
    Keywords:  Alzheimer; Dementia; Endosome; LSD; Lysosomal storage disorder; Lysosome; Parkinson; TPC1; TPC2; TRPML; TRPML1; TRPML2; TRPML3
  3. Autophagy. 2022 Feb 08. 1-3
      RB1CC1/FIP200 is a subunit of the ULK1 complex in more complex eukaryotes. This large polypeptide was proposed to be a functional homolog of the Atg17 and Atg11 scaffolding proteins in yeast. Previous studies showed that RB1CC1 can bind to various proteins of the macroautophagy/autophagy machinery, where the RB1CC1 Claw domain directly interacts with a short linear segment of its interactors. A mechanistic insight into how the small globular RB1CC1 Claw domain can interact with such an array of structurally variable proteins has been elusive. The recent study by Zhou et al., discussed here, yields structural data that not only provide a unifying mechanistic explanation of these interactions, but also reveals previously unknown RB1CC1 interactors and opens a new field for exploration of autophagy regulation.Abbreviations: FIR: FIP200-interacting region; LIR: LC3-interacting region; pS/p-S: phosphorylated serine.
    Keywords:  Analytical gel filtration chromatography; FIR motif; LIR motif; NMR spectroscopy; autophagy receptor; crystal structure; fluorescence polarization-based assay
  4. Neuron. 2022 Jan 31. pii: S0896-6273(22)00056-3. [Epub ahead of print]
      The term autophagy encompasses different pathways that route cytoplasmic material to lysosomes for degradation and includes macroautophagy, chaperone-mediated autophagy, and microautophagy. Since these pathways are crucial for degradation of aggregate-prone proteins and dysfunctional organelles such as mitochondria, they help to maintain cellular homeostasis. As post-mitotic neurons cannot dilute unwanted protein and organelle accumulation by cell division, the nervous system is particularly dependent on autophagic pathways. This dependence may be a vulnerability as people age and these processes become less effective in the brain. Here, we will review how the different autophagic pathways may protect against neurodegeneration, giving examples of both polygenic and monogenic diseases. We have considered how autophagy may have roles in normal CNS functions and the relationships between these degradative pathways and different types of programmed cell death. Finally, we will provide an overview of recently described strategies for upregulating autophagic pathways for therapeutic purposes.
  5. Cardiovasc Res. 2022 Feb 12. pii: cvac014. [Epub ahead of print]
      The Hippo pathway, an evolutionarily conserved signaling mechanism, controls organ size and tumorigenesis. Increasing lines of evidence suggest that autophagy, an important mechanism of lysosome-mediated cellular degradation, is regulated by the Hippo pathway, which thereby profoundly affects cell growth and death responses in various cell types. In the heart, Mst1, an upstream component of the Hippo pathway, not only induces apoptosis but also inhibits autophagy through phosphorylation of Beclin 1. YAP/TAZ, transcription factor co-factors and the terminal effectors of the Hippo pathway, affect autophagy through transcriptional activation of TFEB, a master regulator of autophagy and lysosomal biogenesis. The cellular abundance of YAP is negatively regulated by autophagy and suppression of autophagy induces accumulation of YAP, which, in turn, acts as a feedback mechanism to induce autophagosome formation. Thus, the Hippo pathway and autophagy regulate each other, thereby profoundly affecting cardiomyocyte survival and death. This review discusses the interaction between the Hippo pathway and autophagy and its functional significance during stress conditions in the heart and the cardiomyocytes therein.
    Keywords:  Heart; Hippo pathway; Mst1; YAP; autophagy
  6. Drug Deliv Transl Res. 2022 Feb 11.
      Autophagy is a catabolic process in which an organism responds to its nutrient or metabolic emergencies. It involves the degradation of cytoplasmic proteins and organelles by forming double-membrane vesicles called "autophagosomes." They sequester cargoes, leading them to degradation in the lysosomes. Although autophagy acts as a protective mechanism for maintaining homeostasis through cellular recycling, it is ostensibly a cause of certain cancers, but a cure for others. In other words, insufficient autophagy, due to genetic or cellular dysfunctions, can lead to tumorigenesis. However, many autophagy modulators are developed for cancer therapy. Diverse nanoparticles have been documented to induce autophagy. Also, the highly stable nanoparticles show blockage to autophagic flux. In this review, we revealed a general mechanism by which autophagy can be induced or blocked via nanoparticles as well as several studies recently performed to prove the stated fact. In addition, we have also elucidated the paradoxical roles of autophagy in cancer and how their differential role at different stages of various cancers can affect its treatment outcomes. And finally, we summarize the breakthroughs in cancer disease treatments by using metallic, polymeric, and liposomal nanoparticles as potent autophagy modulators.
    Keywords:  Apoptosis; Autophagosomes; Autophagy; Cancer; Chemotherapeutic agents; Nanoparticles
  7. J Nutr Biochem. 2022 Feb 05. pii: S0955-2863(22)00026-2. [Epub ahead of print] 108955
      Autophagy is a dynamic process and critical for cellular remodeling and organelle quality control. In response to altered nutritional status (e.g., fasting and feeding), autophagic activity is finely tuned by transcriptional, posttranslational, and epigenetic regulations via various signaling pathways, including energy sensors (e.g., mTOR/AMPK-ULK1, mTORC1-WIPI2, mTORC1-Tfeb, PLIN5-Sirt1, and Sirt1-mediated deacetylation of autophagy proteins), fasting or feeding induced hormones (e.g., FGF21-PKA-JMJD3, FGF21-DREAM-Mid1-Tfeb, FGF19-SHP-LSD1, insulin-IRS-AKT-FoxO, glucagon-PKA-CREB), and lysosomal enzymes (e.g., CTSB and CTSL). In contrast to fasting that induces autophagy and health benefits, nutrient oversupply (overfeeding or feeding on high energy diets) dysregulates autophagy, which has been increasingly observed in animal models of human chronic diseases such as obesity, diabetes, non-alcoholic fatty liver disease, and cardiovascular disease. Studies have revealed multifaceted effects of high energy diets on autophagy, being either an inhibitor or enhancer autophagy. The conundrum may arise from the variations in methods for autophagy analysis, components of high energy diets and control diets for treatments, treatment durations, and the ages of genetic backgrounds of laboratory animals. In this article, we reviewed the evidence from both human and animal studies, presenting the molecular mechanism of autophagic response to altered nutritional status and discussing the contributing factors of and possible solution to the current conundrum concerning the exact role of high energy diets in autophagic regulation.
    Keywords:  autophagy; epigenetics; fasting; high energy diet; metabolism; nutritional status; overfeeding
  8. Proc Natl Acad Sci U S A. 2022 Feb 15. pii: e2113454119. [Epub ahead of print]119(7):
      Autophagy is a fundamental cellular process of protein degradation and recycling that regulates immune signaling pathways via multiple mechanisms. However, it remains unclear how autophagy epigenetically regulates the immune response. Here, we identified TRIM14 as an epigenetic regulator that reduces histone H3K9 trimethylation by inhibiting the autophagic degradation of the histone demethylase KDM4D. TRIM14 recruited the deubiquitinases USP14 and BRCC3 to cleave the K63-linked ubiquitin chains of KDM4D, which prevented KDM4D from undergoing optineurin (OPTN)-mediated selective autophagy. Tripartite motif-containing 14 (TRIM14) deficiency in dendritic cells significantly impaired the expression of the KDM4D-directed proinflammatory cytokines interleukin 12 (Il12) and Il23 and protected mice from autoimmune inflammation. Taken together, these findings highlight the cross-talk between epigenetic regulation and autophagy and suggest TRIM14 is a potential target of therapeutic intervention for inflammation-related diseases.
    Keywords:  KDM4D; TRIM14; autophagy; epigenetic regulation; inflammation
  9. Curr Biol. 2022 Feb 01. pii: S0960-9822(22)00093-8. [Epub ahead of print]
      Autophagy targets cytoplasmic materials for degradation and influences cell health. Organelle contact and trafficking systems provide membranes for autophagosome formation, but how different membrane systems are selected for use during autophagy remains unclear. Here, we report a novel function of the endosomal sorting complex required for transport (ESCRT) in the regulation of endoplasmic reticulum (ER) coat protein complex II (COPII) vesicle formation that influences autophagy. The ESCRT functions in a pathway upstream of Vps13D to influence COPII vesicle transport, ER-Golgi intermediate compartment (ERGIC) assembly, and autophagosome formation. Atg9 functions downstream of the ESCRT to facilitate ERGIC and autophagosome formation. Interestingly, cells lacking either ESCRT or Vps13D functions exhibit dilated ER structures that are similar to cranio-lenticulo-sutural dysplasia patient cells with SEC23A mutations, which encodes a component of COPII vesicles. Our data reveal a novel ESCRT-dependent pathway that influences the ERGIC and autophagosome formation.
    Keywords:  Atg9; Drosophila; ESCRT; Vps13D; autophagy
  10. Chem Commun (Camb). 2022 Feb 08.
      Autophagy is a process for the degradation and recycling of intracellular components and dysfunctional organelles. We developed an indole-embedded fluorescent naphthalimide for the selective imaging of autophagosomes in live cells. It was shown as intense puncta in the fluorescence confocal images and co-localizes with an autophagosome marker, LC3-RFP. In addition, it was applied to cellular autophagic models based on ER stress and starvation to verify its capability.
  11. Autophagy. 2022 Feb 07. 1-3
      Failed recognition and clearance of damaged mitochondria contributes to memory loss as well as Aβ and MAPT/Tau pathologies in Alzheimer disease (AD), for which there is an unmet therapeutic need. Restoring mitophagy to eliminate damaged mitochondria could abrogate metabolic dysfunction, neurodegeneration and may subsequently inhibit or slow down cognitive decline in AD models. We have developed a high-throughput machine-learning approach combined with a cross-species screening platform to discover novel mitophagy-inducing compounds from a natural product library and further experimentally validated the potential candidates. Two lead compounds, kaempferol and rhapontigenin, induce neuronal mitophagy and reduce Aβ and MAPT/Tau pathologies in a PINK1-dependent manner in both C. elegans and mouse models of AD. Our combinational approach provides a fast, cost-effective, and highly accurate method for identification of potent mitophagy inducers to maintain brain health.
    Keywords:  Aging; Alzheimer’s disease; autophagy; machine learning; mitophagy
  12. Sci Adv. 2022 Feb 11. 8(6): eabm6393
      The most common genetic risk factors for Parkinson's disease (PD) are a set of heterozygous mutant (MT) alleles of the GBA1 gene that encodes β-glucocerebrosidase (GCase), an enzyme normally trafficked through the ER/Golgi apparatus to the lysosomal lumen. We found that half of the GCase in lysosomes from postmortem human GBA-PD brains was present on the lysosomal surface and that this mislocalization depends on a pentapeptide motif in GCase used to target cytosolic protein for degradation by chaperone-mediated autophagy (CMA). MT GCase at the lysosomal surface inhibits CMA, causing accumulation of CMA substrates including α-synuclein. Single-cell transcriptional analysis and proteomics of brains from GBA-PD patients confirmed reduced CMA activity and proteome changes comparable to those in CMA-deficient mouse brain. Loss of the MT GCase CMA motif rescued primary substantia nigra dopaminergic neurons from MT GCase-induced neuronal death. We conclude that MT GBA1 alleles block CMA function and produce α-synuclein accumulation.
  13. mSystems. 2022 Feb 08. e0146321
      Autophagy is a conserved intracellular degradation mechanism in eukaryotes and is initiated by the protein kinase autophagy-related protein 1 (Atg1). However, except for the autophosphorylation activity of Atg1, the target proteins phosphorylated by Atg1 are largely unknown in filamentous fungi. In Beauveria bassiana (a filamentous insect-pathogenic fungus), Atg1 is indispensable for autophagy and is associated with fungal development. Comparative omics-based analyses revealed that B. bassiana Atg1 (BbAtg1) has key influence on the proteome and phosphoproteome during conidiogenesis. In terms of its physiological functions, the BbAtg1-mediated phosphoproteome is primarily associated with metabolism, signal transduction, cell cycle, and autophagy. At the proteomic level, BbAtg1 mainly regulates genes involved in protein synthesis, protein fate, and protein with binding function. Furthermore, integrative analyses of phosphoproteomic and proteomic data led to the identification of several potential targets regulated by BbAtg1 phosphorylation activity. Notably, we demonstrated that BbAtg1 phosphorylated BbAtg3, an essential component of the ubiquitin-like conjugation system in autophagic progress. Our findings indicate that in addition to being a critical component of the autophagy initiation, Atg1 orchestrates autophagosome elongation via its phosphorylation activity. The data from our study will facilitate future studies on the noncanonical targets of Atg1 and help decipher the Atg1-mediated phosphorylation networks. IMPORTANCE Autophagy-related protein 1 (Atg1) is a serine/threonine protein kinase for autophagy initiation. In contrast to the unicellular yeast, the target proteins phosphorylated by Atg1 are largely unknown in filamentous fungi. In this study, the entomopathogenic fungus Beauveria bassiana was used as a representative of filamentous fungi due to its importance in the applied and fundamental research. We revealed that Atg1 mediates the comprehensive proteome and phosphoproteome, which differ from those revealed in yeast. Further investigation revealed that Atg1 directly phosphorylates the E2-like enzyme Atg3 of the ubiquitin-like conjugation system (ULCS), and the phosphorylation of Atg3 is indispensable for ULCS functionality. Interestingly, the phosphorylation site of Atg3 is conserved among a set of insect- and plant-pathogenic fungi but not in human-pathogenic fungi. This study reveals new regulatory mechanisms of autophagy and provides new insights into the evolutionary diversity of the Atg1 kinase signaling pathways among different pathogenic fungi.
    Keywords:  autophagy-related kinase; conidiogenesis; filamentous fungus; fungal development; phosphoproteomic analysis; phosphorylation activity; ubiquitin-like conjugation system
  14. Cell Death Dis. 2022 Feb 08. 13(2): 132
      Autophagy is a conserved method of quality control in which cytoplasmic contents are degraded via lysosomes. Lipophagy, a form of selective autophagy and a novel type of lipid metabolism, has recently received much attention. Lipophagy is defined as the autophagic degradation of intracellular lipid droplets (LDs). Although much remains unknown, lipophagy appears to play a significant role in many organisms, cell types, metabolic states, and diseases. It participates in the regulation of intracellular lipid storage, intracellular free lipid levels (e.g., fatty acids), and energy balance. However, it remains unclear how intracellular lipids regulate autophagy. Impaired lipophagy can cause cells to become sensitive to death stimuli and may be responsible for the onset of a variety of diseases, including nonalcoholic fatty liver disease and metabolic syndrome. Like autophagy, the role of lipophagy in cancer is poorly understood, although analysis of specific autophagy receptors has helped to expand the diversity of chemotherapeutic targets. These studies have stimulated increasing interest in the role of lipophagy in the pathogenesis and treatment of cancer and other human diseases.
  15. J Cell Biol. 2022 Feb 10. pii: e202111077. [Epub ahead of print]221(3):
      Lysosomes serve as degradation hubs for the turnover of endocytic and autophagic cargos, which is essential for neuron function and survival. Deficits in lysosome function result in progressive neurodegeneration in most lysosomal storage disorders and contribute to the pathogenesis of aging-related neurodegenerative diseases. Given their size and highly polarized morphology, neurons face exceptional challenges in maintaining cellular homeostasis in regions far removed from the cell body where mature lysosomes are enriched. Neurons therefore require coordinated bidirectional intracellular transport to sustain efficient clearance capacity in distal axonal regions. Emerging lines of evidence have started to uncover mechanisms and signaling pathways regulating endolysosome transport and maturation to maintain axonal homeostasis, or "axonostasis," that is relevant to a range of neurologic disorders. In this review, we discuss recent advances in how axonal endolysosomal trafficking, distribution, and lysosomal functionality support neuronal health and become disrupted in several neurodegenerative diseases.
  16. Am J Cancer Res. 2022 ;12(1): 327-336
      Six Transmembrane Protein of Prostate 2 (STAMP2) is critical for prostate cancer (PCa) growth. We previously showed that STAMP2 regulates the expression of stress induced transcription factor ATF4, which is implicated in starvation-induced autophagy. We therefore investigated whether STAMP2 is involved in the regulation of autophagy in PCa cells. Here we show that STAMP2 suppresses autophagy in PCa cells through modulation of the integrated stress response axis. We also find that STAMP2 regulates mitochondrial respiration. These findings suggest that STAMP2 has significant metabolic effects through mitochondrial function and autophagy, both of which support PCa growth.
    Keywords:  ATF4; Prostate cancer; STAMP2; autophagy; eIF2α; integrated stress response; mitochondria
  17. Int J Biol Macromol. 2022 Feb 07. pii: S0141-8130(22)00236-7. [Epub ahead of print]
      The definition for autophagy holds a 'single' meaning as a conserved cellular process that constitutes a recycling pathway for damaged organelles and long-lived proteins to maintain nutrient homeostasis and mediate quality control within the cell. But this process of autophagy may behave ambiguously depending on the physiological stress as the stress progresses in the cellular microenvironment; the 'single' meaning of the autophagy changes from the 'cytoplasmic turnover process' to 'tumor suppressive' and a farther extent, 'tumor promoter' process. In a tumorigenic state, the chemotherapy-mediated resistance and intolerance due to upregulated autophagy in cancer cells have become a significant concern. This concern has provided insight to the scientific community to enter into the arena of cross-talk between autophagy and apoptosis. Recent findings and ongoing research have provided insights on some of the key regulators of this cross-talk; one of them is Beclin1 and their involvement in the physiological and the pathophysiological processes; however, reconciliation of these two forms of death remains an arena to be explored extensively. This review sheds light on the interplay between autophagy and apoptosis, emphasizing one of the key players, Beclin1, and its importance in health and diseases.
    Keywords:  Apoptosis; Autophagy; Bcl-2; Beclin1; Tumor promoter; Tumor suppressor
  18. Nat Commun. 2022 Feb 10. 13(1): 805
      T follicular helper (Tfh) cells provide signals to initiate and maintain the germinal center (GC) reaction and are crucial for the generation of robust, long-lived antibody responses, but how the GC microenvironment affects Tfh cells is not well understood. Here we develop an in vivo T cell-intrinsic CRISPR-knockout screen to evaluate Tfh and Th1 cells in an acute viral infection model to identify regulators of Tfh cells in their physiological setting. Using a screen of druggable-targets, alongside genetic, transcriptomic and cellular analyses, we identify a function of HIF-1α in suppressing mTORC1-mediated and Myc-related pathways, and provide evidence that VHL-mediated degradation of HIF-1α is required for Tfh development; an expanded in vivo CRISPR screen reveals multiple components of these pathways that regulate Tfh versus Th1 cells, including signaling molecules, cell-cycle regulators, nutrient transporters, metabolic enzymes and autophagy mediators. Collectively, our data serve as a resource for studying Tfh versus Th1 decisions, and implicate the VHL-HIF-1α axis in fine-tuning Tfh generation.
  19. J Appl Physiol (1985). 2022 Feb 10.
      Exercise is critical for improving metabolic health and putatively maintains or enhances mitochondrial quality control in metabolic tissues. While previous work has shown exercise elicits hepatic mitochondrial biogenesis, it is unknown if acute exercise activates hepatic mitophagy, the selective degradation of damaged or low-functioning mitochondria. We tested if an acute bout of treadmill running increased hepatic mitophagic flux both immediately after and 2 hours post-exercise in 15-24-week-old C57BL/6J female mice. Acute exercise did not significantly increase markers of autophagic flux, however, mitophagic flux was activated 2 hours post-treadmill running as measured by accumulation of both LC3-II and p62 in isolated mitochondria in the presence of leupeptin, an inhibitor of autophagosome degradation. Further, mitochondrial associated ubiquitin, which recruits the autophagy receptor protein p62, was also significantly increased at 2 hours. Further examination via western blot and proteomics analysis revealed acute exercise elicits a time-dependent, dynamic activation of mitophagy pathways. Moreover, the results suggest that exercise induced hepatic mitophagy is likely mediated by both poly-ubiquitination and receptor mediated signaling pathways. Overall, we provide evidence that acute exercise activates hepatic mitophagic flux while also revealing specific receptor-mediated proteins by which exercise maintains mitochondrial quality control in the liver.
    Keywords:  Exercise; Liver; Mitochondria; Mitophagic Flux; Mitophagy
  20. Physiol Rep. 2022 Feb;10(3): e15181
      The mechanisms by which lower limb ischemia/reperfusion induces acute kidney injury (AKI) remain largely uncharacterized. We hypothesized that tourniquet-induced lower limb ischemia/reperfusion (TILLIR) would inhibit mitochondrial function in the renal cortex. We used a murine model to show that TILLIR of the high thigh regions inflicted time-dependent AKI as determined by renal function and histology. This effect was associated with decreased activities of mitochondrial complexes I, II, V and citrate synthase in the kidney cortex. Moreover, TILLIR reduced mRNA levels of a master regulator of mitochondrial biogenesis PGC-1α, and its downstream genes NDUFS1 and ATP5o in the renal cortex. TILLIR also increased serum corticosterone concentrations. TILLIR did not significantly affect protein levels of the critical regulators of mitophagy PINK1 and PARK2, mitochondrial transport proteins Tom20 and Tom70, or heat-shock protein 27. TILLIR had no significant effect on mitochondrial oxidative stress as determined by mitochondrial ability to generate reactive oxygen species, protein carbonylation, or protein levels of MnSOD and peroxiredoxin1. However, TILLIR inhibited classic autophagic flux by increasing p62 protein abundance and preventing the conversion of LC3-I to LC3-II. TILLIR increased phosphorylation of cytosolic and mitochondrial ERK1/2 and mitochondrial AKT1, as well as mitochondrial SGK1 activity. In conclusion, lower limb ischemia/reperfusion induces distal AKI by inhibiting mitochondrial function through reducing mitochondrial biogenesis. This AKI occurs without significantly affecting PINK1-PARK2-mediated mitophagy or mitochondrial oxidative stress in the kidney cortex.
    Keywords:  autophagy; ischemia; mitochondrial complex; mitochondrial oxidative stress; mitophagy
  21. Cancer Res. 2022 Feb 11. pii: canres.1168.2021. [Epub ahead of print]
      MYC family oncoproteins are regulators of metabolic reprogramming that sustains cancer cell anabolism. Normal cells adapt to nutrient-limiting conditions by activating autophagy, which is required for amino acid (AA) homeostasis. Here we report that the autophagy pathway is suppressed by Myc in normal B cells, in premalignant and neoplastic B cells of Eμ-Myc transgenic mice, and in human MYC-driven Burkitt lymphoma. Myc suppresses autophagy by antagonizing the expression and function of transcription factor EB (TFEB), a master regulator of autophagy. Mechanisms that sustained AA pools in MYC-expressing B cells include coordinated induction of the proteasome and increases in AA transport. Reactivation of the autophagy-lysosomal pathway by TFEB disabled the malignant state by disrupting mitochondrial functions, proteasome activity, amino acid transport, and amino acid and nucleotide metabolism, leading to metabolic anergy, growth arrest and apoptosis. This phenotype provides therapeutic opportunities to disable MYC-driven malignancies, including AA restriction and treatment with proteasome inhibitors.
  22. J Biochem. 2022 Feb 04. pii: mvac011. [Epub ahead of print]
      An important feature of several neurodegenerative diseases is the formation of pathological structures containing aggregated proteins. The autophagy receptor optineurin/OPTN is frequently observed in these structures. The role played by optineurin in these aggregates is not clear. In this study, we explored whether optineurin has a cytoprotective role in the cells having mutant protein aggregates. We overexpressed mutant huntingtin having 97 glutamine repeats (mHtt), and mutant ataxin-3 having 130 glutamine repeats (mAtax-3) in wild-type and optineurin-deficient neuronal (N2A) and non-neuronal cells (Optn-/- mouse embryonic fibroblasts), and determined the percentage of dead cells with mutant protein aggregates. Optineurin-deficient cells having mHtt or mAtax-3 aggregates showed higher cell death as compared to wild-type cells having mutant protein aggregates. Confocal microscopy revealed that optineurin formed a shell around mHtt and mAtax-3 aggregates through its C-terminal domain. The C-terminal domain of optineurin, which lacks LC3-interacting region required for autophagy, was necessary and sufficient to reduce cytotoxicity of mHtt and mAtax-3 aggregates. Our results show that in the absence of optineurin, mutant protein aggregates are highly toxic, revealing an autophagy-independent cytoprotective function of optineurin, which is mediated by its C-terminal domain.
    Keywords:  Optineurin; autophagy; mutant ataxin-3; mutant huntingtin; mutant protein aggregates; neurodegeneration
  23. Neurobiol Dis. 2022 Feb 07. pii: S0969-9961(22)00044-4. [Epub ahead of print] 105653
      Investigations into the pathogenetic mechanisms underlying amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD) have provided significant insight into the disease. At the cellular level, ALS and FTD are classified as proteinopathies, which is motor neuron degeneration and death characterized by pathological protein aggregates or dysregulated proteostasis. At both the clinical and molecular level there are common signaling pathways dysregulated across the ALS and FTD spectrum (ALS/FTD). Sequestosome-1/p62 is a multifunctional scaffold protein with roles in several signaling pathways including proteostasis, protein degradation via the ubiquitin proteasome system and autophagy, the antioxidant response, inflammatory response, and apoptosis. Notably these pathways are dysregulated in ALS and FTD. Mutations in the functional domains of p62 provide links to the pathogenetic mechanisms of p62 and dyshomeostasis of p62 levels is noted in several types of ALS and FTD. We present here that the dysregulated ALS and FTD signaling pathways are linked, with p62 converging the molecular mechanisms. This review summarizes the current literature on the complex role of p62 in the pathogenesis across the ALS/FTD spectrum. The focus is on the underlying convergent molecular mechanisms of ALS and FTD-associated proteins and pathways that dysregulate p62 levels or are dysregulated by p62, with emphasis on how p62 is implicated across the ALS/FTD spectrum.
    Keywords:  ALS; Aggregation; Amyotrophic lateral sclerosis; Autophagy; Cell signaling; FTD; Frontotemporal dementia; Protein degradation; SQSTM1/p62
  24. Oxid Med Cell Longev. 2022 ;2022 4906434
      Traumatic brain injury (TBI) contributes to death, and disability worldwide more than any other traumatic insult and damage to cellular components including mitochondria leads to the impairment of cellular functions and brain function. In neurons, mitophagy, autophagy-mediated degradation of damaged mitochondria, is a key process in cellular quality control including mitochondrial homeostasis and energy supply and plays a fundamental role in neuronal survival and health. Conversely, defective mitophagy leads to the accumulation of damaged mitochondria and cellular dysfunction, contributing to inflammation, oxidative stress, and neuronal cell death. Therefore, an extensive characterization of mitophagy-related protective mechanisms, taking into account the complex mechanisms by which each molecular player is connected to the others, may provide a rationale for the development of new therapeutic strategies in TBI patients. Here, we discuss the contribution of defective mitophagy in TBI, and the underlying molecular mechanisms of mitophagy in inflammation, oxidative stress, and neuronal cell death highlight novel therapeutics based on newly discovered mitophagy-inducing strategies.
  25. Cancer Res. 2022 Feb 07. pii: canres.2161.2021. [Epub ahead of print]
      Chaperone-mediated autophagy (CMA) is a homeostatic process essential for the lysosomal degradation of a selected subset of the proteome. CMA activity directly depends on the levels of LAMP2A, a critical receptor for CMA substrate proteins at the lysosomal membrane. In glioblastoma (GBM), the most common and aggressive brain cancer in adulthood, high levels of LAMP2A in the tumor and tumor-associated pericytes have been linked to temozolomide resistance and tumor progression. However, the role of LAMP2A, and hence CMA, in any cancer stem cell type or in glioblastoma stem cells (GSC) remains unknown. In this work, we show that LAMP2A expression is enriched in patient-derived GSCs, and its depletion diminishes GSC-mediated tumorigenic activities. Conversely, overexpression of LAMP2A facilitates the acquisition of GSC properties. Proteomic and transcriptomic analysis of LAMP2A-depleted GSCs revealed reduced extracellular matrix (ECM) interaction effectors in both analyses. Moreover, pathways related to mitochondrial metabolism and the immune system were differentially deregulated at the proteome level. Furthermore, clinical samples of GBM tissue presented with overexpression of LAMP2, which correlated with advanced glioma grade and poor overall survival. In conclusion, these results identify a novel role of CMA in directly regulating GSCs activity via multiple pathways at the proteome and transcriptome levels.
  26. Sci Total Environ. 2022 Feb 04. pii: S0048-9697(22)00721-5. [Epub ahead of print] 153629
      Nanoparticles (NPs) have been widely used for various purposes due to their unique physicochemical properties. Such widespread applications greatly increase the possibility of human exposure to NPs in various ways. Once entering the human body, NPs may interfere with cellular homeostasis and thus affect the physiological system. As a result, it is necessary to evaluate the potential disturbance of NPs to multiple cell functions, including autophagy. Autophagy is an important cell function to maintain cellular homeostasis, and minimizing the disturbance caused by NP exposures to autophagy is critical to nanosafety. Herein, we summarized the recent research progress in nanotoxicity with particular focuses on the perturbation of NPs to cell autophagy. The basic processes of autophagy and complex relationships between autophagy and major human diseases were further discussed to emphasize the importance of keeping autophagy under control. Moreover, the most recent advances on perturbation of different types of NPs to autophagy were also reviewed. Last but not the least, we also discussed major research challenges and potential coping strategies and proposed a safe-by-design strategy towards safer applications of NPs.
    Keywords:  Autophagy; Biosafety; Molecule mechanism; Nanotoxicity; Signaling pathway
  27. J Cell Sci. 2022 Feb 01. pii: jcs248534. [Epub ahead of print]135(3):
      Recent advances have revealed common pathological changes in neurodegenerative diseases, such as Alzheimer's disease, Parkinson's disease and amyotrophic lateral sclerosis with related frontotemporal dementia (ALS/FTD). Many of these changes can be linked to alterations in endoplasmic reticulum (ER)-mitochondria signaling, including dysregulation of Ca2+ signaling, autophagy, lipid metabolism, ATP production, axonal transport, ER stress responses and synaptic dysfunction. ER-mitochondria signaling involves specialized regions of ER, called mitochondria-associated membranes (MAMs). Owing to their role in neurodegenerative processes, MAMs have gained attention as they appear to be associated with all the major neurodegenerative diseases. Furthermore, their specific role within neuronal maintenance is being revealed as mutant genes linked to major neurodegenerative diseases have been associated with damage to these specialized contacts. Several studies have now demonstrated that these specialized contacts regulate neuronal health and synaptic transmission, and that MAMs are damaged in patients with neurodegenerative diseases. This Review will focus on the role of MAMs and ER-mitochondria signaling within neurons and how damage of the ER-mitochondria axis leads to a disruption of vital processes causing eventual neurodegeneration.
    Keywords:  Endoplasmic reticulum; MAMs; Mitochondria; Neurodegenerative diseases; Neurons; Tethers
  28. Curr Biol. 2022 Jan 28. pii: S0960-9822(22)00027-6. [Epub ahead of print]
      Mutations in the tumor-suppressor Hippo pathway lead to activation of the transcriptional coactivator Yorkie (Yki), which enhances cell proliferation autonomously and causes cell death non-autonomously. While Yki-induced cell proliferation has extensively been studied, the mechanism by which Yki causes cell death in nearby wild-type cells, a phenomenon called supercompetition, and its role in tumorigenesis remained unknown. Here, we show that Yki-induced supercompetition is essential for tumorigenesis and is driven by non-autonomous induction of autophagy. Clones of cells mutant for a Hippo pathway component fat activate Yki and cause autonomous tumorigenesis and non-autonomous cell death in Drosophila eye-antennal discs. Through a genetic screen in Drosophila, we find that mutations in autophagy-related genes or NF-κB genes in surrounding wild-type cells block both fat-induced tumorigenesis and supercompetition. Mechanistically, fat mutant cells upregulate Yki-target microRNA bantam, which elevates protein synthesis levels via activation of TOR signaling. This induces elevation of autophagy in neighboring wild-type cells, which leads to downregulation of IκB Cactus and thus causes NF-κB-mediated induction of the cell death gene hid. Crucially, upregulation of bantam is sufficient to make cells to be supercompetitors and downregulation of endogenous bantam is sufficient for cells to become losers of cell competition. Our data indicate that cells with elevated Yki-bantam signaling cause tumorigenesis by non-autonomous induction of autophagy that kills neighboring wild-type cells.
    Keywords:  Yorkie; autophagy; bantam; cell death; supercompetition; tumorigenesis
  29. Autophagy. 2022 Feb 07. 1-18
      Zaire ebolavirus (EBOV) causes a severe hemorrhagic fever in humans and non-human primates with high morbidity and mortality. EBOV infection is dependent on its structural glycoprotein (GP), but high levels of GP expression also trigger cell rounding, detachment, and downregulation of many surface molecules that is thought to contribute to its high pathogenicity. Thus, EBOV has evolved an RNA editing mechanism to reduce its GP expression and increase its fitness. We now report that the GP expression is also suppressed at the protein level in cells by protein disulfide isomerases (PDIs). Although PDIs promote oxidative protein folding by catalyzing correct disulfide formation in the endoplasmic reticulum (ER), PDIA3/ERp57 adversely triggered the GP misfolding by targeting GP cysteine residues and activated the unfolded protein response (UPR). Abnormally folded GP was targeted by ER-associated protein degradation (ERAD) machinery and, unexpectedly, was degraded via the macroautophagy/autophagy-lysosomal pathway, but not the proteasomal pathway. PDIA3 also decreased the GP expression from other ebolavirus species but increased the GP expression from Marburg virus (MARV), which is consistent with the observation that MARV-GP does not cause cell rounding and detachment, and MARV does not regulate its GP expression via RNA editing during infection. Furthermore, five other PDIs also had a similar inhibitory activity to EBOV-GP. Thus, PDIs negatively regulate ebolavirus glycoprotein expression, which balances the viral life cycle by maximizing their infection but minimizing their cellular effect. We suggest that ebolaviruses hijack the host protein folding and ERAD machinery to increase their fitness via reticulophagy during infection.Abbreviations: 3-MA: 3-methyladenine; 4-PBA: 4-phenylbutyrate; ACTB: β-actin; ATF: activating transcription factor; ATG: autophagy-related; BafA1: bafilomycin A1; BDBV: Bundibugyo ebolavirus; CALR: calreticulin; CANX: calnexin; CHX: cycloheximide; CMA: chaperone-mediated autophagy; ConA: concanamycin A; CRISPR: clusters of regularly interspaced short palindromic repeats; Cas9: CRISPR-associated protein 9; dsRNA: double-stranded RNA; EBOV: Zaire ebolavirus; EDEM: ER degradation enhancing alpha-mannosidase like protein; EIF2AK3/PERK: eukaryotic translation initiation factor 2 alpha kinase 3; Env: envelope glycoprotein; ER: endoplasmic reticulum; ERAD: ER-associated protein degradation; ERN1/IRE1: endoplasmic reticulum to nucleus signaling 1; GP: glycoprotein; HA: hemagglutinin; HDAC6: histone deacetylase 6; HMM: high-molecular-mass; HIV-1: human immunodeficiency virus type 1; HSPA5/BiP: heat shock protein family A (Hsp70) member 5; IAV: influenza A virus; IP: immunoprecipitation; KIF: kifenesine; Lac: lactacystin; LAMP: lysosomal associated membrane protein; MAN1B1/ERManI: mannosidase alpha class 1B member 1; MAP1LC3/LC3: microtubule associated protein 1 light chain 3; MARV: Marburg virus; MLD: mucin-like domain; NHK/SERPINA1: alpha1-antitrypsin variant null (Hong Kong); NTZ: nitazoxanide; PDI: protein disulfide isomerase; RAVV: Ravn virus; RESTV: Reston ebolavirus; SARS-CoV: severe acute respiratory syndrome coronavirus; SBOV: Sudan ebolavirus; sGP: soluble GP; SQSTM1/p62: sequestosome 1; ssGP: small soluble GP; TAFV: Taï Forest ebolavirus; TIZ: tizoxanide; TGN: thapsigargin; TLD: TXN (thioredoxin)-like domain; Ub: ubiquitin; UPR: unfolded protein response; VLP: virus-like particle; VSV: vesicular stomatitis virus; WB: Western blotting; WT: wild-type; XBP1: X-box binding protein 1.
    Keywords:  Autophagy; ER-phagy; ERAD; ERp57; EVD; ebola; filoviruses; glycoproteins; lysosomes; reticulophagy
  30. J Inherit Metab Dis. 2022 Feb 12.
      Inactivating mutations in the PPT1 gene encoding palmitoyl-protein thioesterase-1 (PPT1) underlie the CLN1 disease, a devastating neurodegenerative lysosomal storage disorder. The mechanism of pathogenesis underlying CLN1 disease has remained elusive. PPT1 is a lysosomal enzyme, which catalyzes the removal of palmitate from S-palmitoylated proteins (constituents of ceroid lipofuscin) facilitating their degradation and clearance by lysosomal hydrolases. Thus, it has been proposed that Ppt1-deficiency leads to lysosomal accumulation of ceroid lipofuscin leading to CLN1 disease. While S-palmitoylation is catalyzed by palmitoyl acyltransferases (called ZDHHCs), palmitoyl-protein thioesterases (PPTs) depalmitoylate these proteins. We sought to determine the mechanism by which Ppt1-deficiency may impair lysosomal degradative function leading to INCL pathogenesis. Here we report that in Ppt1-/- mice, which mimic CLN1 disease, low level of inositol 3-phosphate receptor-1 (IP3R1) that mediates Ca++ -transport from the ER to the lysosome dysregulated lysosomal Ca++ homeostasis. Intriguingly, the transcription factor NFATC4, which regulates IP3R1-expression, required S-palmitoylation for trafficking from the cytoplasm to the nucleus. We identified two palmitoyl acyltransferases, ZDHHC4 and ZDHHC8, which catalyzed S-palmitoylation of NFATC4. Notably, in Ppt1-/- mice, reduced ZDHHC4 and ZDHHC8 levels markedly lowered S-palmitoylated NFATC4 (active) in the nucleus, which inhibited IP3R1-expression, thereby, dysregulating lysosomal Ca++ homeostasis. Consequently, Ca++ -dependent lysosomal enzyme activities were markedly suppressed. Impaired lysosomal degradative function impaired autophagy, which caused lysosomal storage of undigested cargo. Importantly, IP3R1-overexpression in Ppt1-/- mouse fibroblasts ameliorated this defect. Our results reveal a previously unrecognized role of Ppt1 in regulating lysosomal Ca++ -homeostasis and suggest that this defect contributes to pathogenesis of CLN1 disease. This article is protected by copyright. All rights reserved.
    Keywords:  Batten disease; Infantile neuronal ceroid lipofuscinosis; Lysosomal storage disease; Neurodegeneration; Neuronal ceroid lipofuscinosis; Palmitoyl-protein thioesterase-1; S-palmitoylation
  31. Nutr Metab Cardiovasc Dis. 2021 Dec 30. pii: S0939-4753(21)00599-8. [Epub ahead of print]
      BACKGROUND AND AIMS: High glucose and its byproducts are important factors causing dysfunction of endothelial cells. Autophagy is critical for endothelial cellular homeostasis. However, the specific molecular mechanism of how autophagy is regulated in endothelial cells under high-glucose condition remains unknown. We aim to explore the role Sirt6 plays in regulating autophagy in AGE-treated endothelial cells and how this function is exerted via KLF4.METHODS AND RESULTS: Our results indicate that autophagy level increased in AGE-treated endothelial cells alongside with higher Sirt6 and KLF4 expression level. What's more, knock-in of Sirt6 by adenovirus led to augmented autophagy level while knockdown of Sirt6 led to the opposite. We also verified that Sirt6 affected KLF4 expression positively but KLF4 didn't influence Sirt6 expression level while knocking out of KLF4 impaired Sirt6-enhanced autophagy. Finally we found that STZ-induced diabetic mice showed more autophagosomes in endothelium and Sirt6 knockdown by adeno-associated virus reduced the number of autophagosomes. Knockdown of Sirt6 also caused impaired endothelium integrity but echocardiography indicated there were no significant functional differences.
    CONCLUSION: Our research reveals more about how Sirt6 regulates autophagy in endothelial cells under high-glucose simulated condition and provides further insight into the relationships between Sirt6 and KLF4.
    Keywords:  AGE; Autophagy; Diabetes; Endothelial cells; KLF4; Sirt6
  32. Adv Exp Med Biol. 2021 ;1349 275-301
      Lysosomal ion channels mediate ion flux from lysosomes and regulate membrane potential across the lysosomal membrane, which are essential for lysosome biogenesis, nutrient sensing, lysosome trafficking, lysosome enzyme activity, and cell membrane repair. As a cation channel, the transient receptor potential mucolipin 1 (TRPML1) channel is mainly expressed on lysosomes and late endosomes. Recently, the normal function of TRPML1 channels has been demonstrated to be important for the maintenance of cardiovascular and renal glomerular homeostasis and thereby involved in the pathogenesis of some cardiovascular and kidney diseases. In arterial myocytes, it has been found that Nicotinic Acid Adenine Dinucleotide Phosphate (NAADP), an intracellular second messenger, can induce Ca2+ release through the lysosomal TRPML1 channel, leading to a global Ca2+ release response from the sarcoplasmic reticulum (SR). In podocytes, it has been demonstrated that lysosomal TRPML1 channels control lysosome trafficking and exosome release, which contribute to the maintenance of podocyte functional integrity. The defect or functional deficiency of lysosomal TRPML1 channels has been shown to critically contribute to the initiation and development of some chronic degeneration or diseases in the cardiovascular system or kidneys. Here we briefly summarize the current evidence demonstrating the regulation of lysosomal TRPML1 channel activity and related signaling mechanisms. We also provide some insights into the canonical and noncanonical roles of TRPML1 channel dysfunction as a potential pathogenic mechanism for certain cardiovascular and kidney diseases and associated therapeutic strategies.
    Keywords:  Atherosclerosis; Autophagy; Chronic kidney disease; Exosomes; Lysosome; NAADP; TRPML1 channel
  33. Brain. 2022 Feb 08. pii: awac044. [Epub ahead of print]
      Mutations in nitrogen permease regulator-like 3 (NPRL3), a component of the GATOR1 complex within the mechanistic target of rapamycin (mTOR) pathway, are associated with epilepsy and malformations of cortical development. Little is known about the effects of NPRL3 loss on neuronal mTOR signaling and morphology, or cerebral cortical development and seizure susceptibility. We report the clinical phenotypic spectrum of a founder NPRL3 pedigree (c.349delG, p.Glu117LysFS; n = 133) among Old Order Mennonites dating to 1727. Next, as a strategy to define the role of NPRL3 in cortical development, CRISPR/Cas9 Nprl3 knockout in Neuro2a cells in vitro and in fetal mouse brain in vivo was used to assess effects of Nprl3 knockout on mTOR activation, subcellular mTOR localization, nutrient signaling, cell morphology and aggregation, cerebral cortical cytoarchitecture, and network integrity. The NPRL3 pedigree exhibited an epilepsy penetrance of 28% and heterogeneous clinical phenotypes with a range of epilepsy semiologies i.e., focal or generalized onset, brain imaging abnormalities i.e., polymicrogyria, focal cortical dysplasia, or normal imaging, and EEG findings, e.g., focal, multi-focal, or generalized spikes, focal or generalized slowing. Whole exome analysis comparing a seizure-free group (n = 37) to those with epilepsy (n = 24) to search for gene modifiers for epilepsy did not identify a unique genetic modifier that explained the variability in seizure penetrance in this cohort. Nprl3 knockout in vitro caused mTOR pathway hyperactivation, cell soma enlargement, and the formation of cellular aggregates seen in time-lapse videos that were prevented with the mTOR inhibitors rapamycin or torin1. In Nprl3 KO cells, mTOR remained localized on the lysosome in a constitutively active conformation, as evidenced by phosphorylation of S6 and 4E-BP1 proteins, even under nutrient starvation (amino acid free) conditions, demonstrating that Nprl3 loss decouples mTOR activation from neuronal metabolic state. To model human malformations of cortical development associated with NPRL3 variants, we created a focal Nprl3 KO in fetal mouse cortex by in utero electroporation and found altered cortical lamination and white matter heterotopic neurons, effects which were prevented with rapamycin treatment. EEG recordings showed network hyperexcitability and reduced seizure threshold to pentylenetetrazol treatment. NPRL3 variants are linked to a highly variable clinical phenotype which we propose result from mTOR-dependent effects on cell structure, cortical development, and network organization.
    Keywords:  GATOR1; cortical malformations; epilepsy; focal cortical dysplasia; mTOR
  34. Circ Res. 2022 Feb 09. CIRCRESAHA121320047
      RATIONALE: Atherosclerosis is characterized by an accumulation of foam cells within the arterial wall, resulting from excess cholesterol uptake and buildup of cytosolic lipid droplets (LDs). Autophagy promotes LD clearance by freeing stored cholesterol for efflux, a process that has been shown to be atheroprotective. While the role of autophagy in LD catabolism has been studied in macrophage-derived foam cells, this has remained unexplored in vascular smooth muscle cell (VSMC)-derived foam cells that constitute a large fraction of foam cells within atherosclerotic lesions.OBJECTIVE: We performed a comparative analysis of autophagy flux in lipid-rich aortic intimal populations to determine whether VSMC-derived foam cells metabolize LDs similarly to their macrophage counterparts.
    METHODS AND RESULTS: Atherosclerosis was induced in GFP-LC3 transgenic mice by PCSK9 (proprotein convertase subtilisin/kexin type 9)-adeno-associated viral injection and Western diet feeding. Using flow cytometry of aortic digests, we observed a significant increase in dysfunctional autophagy of VSMC-derived foam cells during atherogenesis relative to macrophage-derived foam cells. Using cell culture models of lipid-loaded VSMC and macrophage, we show that autophagy-mediated cholesterol efflux from VSMC foam cells was poor relative to macrophage foam cells, and largely occurs when HDL (high-density lipoprotein) is used as a cholesterol acceptor, as opposed to apoA-1 (apolipoproteinA-1). This was associated with the predominant expression of ABCG1 in VSMC foam cells. Using metformin, an autophagy activator, cholesterol efflux to HDL was significantly increased in VSMC, but not in macrophage, foam cells.
    CONCLUSIONS: These data demonstrate that VSMC and macrophage foam cells perform cholesterol efflux by distinct mechanisms, and that autophagy flux is highly impaired in VSMC foam cells, but can be induced by pharmacological means. Further investigation is warranted into targeting autophagy specifically in VSMC foam cells, the predominant foam cell subtype of advanced atherosclerotic plaques, to promote reverse cholesterol transport and resolution of the atherosclerotic plaque.
    Keywords:  atherosclerosis; autophagy; cholesterol; flow cytometry; lipase
  35. Sci Transl Med. 2022 Feb 09. 14(631): eabh3763
      The Wolfram syndrome is a rare autosomal recessive disease affecting many organs with life-threatening consequences; currently, no treatment is available. The disease is caused by mutations in the WSF1 gene, coding for the protein wolframin, an endoplasmic reticulum (ER) transmembrane protein involved in contacts between ER and mitochondria termed as mitochondria-associated ER membranes (MAMs). Inherited mutations usually reduce the protein's stability, altering its homeostasis and ultimately reducing ER to mitochondria calcium ion transfer, leading to mitochondrial dysfunction and cell death. In this study, we found that activation of the sigma-1 receptor (S1R), an ER-resident protein involved in calcium ion transfer, could counteract the functional alterations of MAMs due to wolframin deficiency. The S1R agonist PRE-084 restored calcium ion transfer and mitochondrial respiration in vitro, corrected the associated increased autophagy and mitophagy, and was able to alleviate the behavioral symptoms observed in zebrafish and mouse models of the disease. Our findings provide a potential therapeutic strategy for treating Wolfram syndrome by efficiently boosting MAM function using the ligand-operated S1R chaperone. Moreover, such strategy might also be relevant for other degenerative and mitochondrial diseases involving MAM dysfunction.
  36. Am J Cancer Res. 2022 ;12(1): 108-122
      As a member of the catenin family, δ-catenin is overexpressed in many cancers, including prostate cancer, and the role of δ-catenin in prostate tumor growth has been reported. However, the involvement of δ-catenin in the migration and invasion of prostate cancer has rarely been studied. In this study, we innovatively proposed that δ-catenin would enhance the migration and invasion ability of prostate cancer cells. It is worth noting that the molecular mechanism underlying the effect involved the downregulation of autophagy. We demonstrated that δ-catenin could suppress autophagy by Bcl-2-regulated disruption of the Beclin1-Vps34 autophagosome complex. Furthermore, the effect of δ-catenin on promoting cell migration and invasion was dependent upon β-catenin-mediated Bcl-2 transcription. Finally, using rapamycin and bafilomycin, we largely confirmed that the degradation of Snails by autolysosomes may be related to δ-catenin regulated migration and invasion. Overall, our results indicated that δ-catenin promoted cell migration and invasion of prostate cancer cells via Bcl-2-regulated autophagy suppression.
    Keywords:  Bcl-2; autophagy; invasion; migration; prostate cancer; δ-catenin
  37. Commun Biol. 2022 Feb 10. 5(1): 124
      Statins play a major role in reducing circulating cholesterol levels and are widely used to prevent coronary artery disease. Although they are recently confirmed to up-regulate mitophagy, little is known about the molecular mechanisms and its effect on endothelial progenitor cell (EPC). Here, we explore the role and mechanism underlying statin (pitavastatin, PTV)-activated mitophagy in EPC proliferation. ApoE-/- mice are fed a high-fat diet for 8 weeks to induce atherosclerosis. In these mice, EPC proliferation decreases and is accompanied by mitochondrial dysfunction and mitophagy impairment via the PINK1-PARK2 pathway. PTV reverses mitophagy and reduction in proliferation. Pink1 knockout or silencing Atg7 blocks PTV-induced proliferation improvement, suggesting that mitophagy contributes to the EPC proliferation increase. PTV elicits mitochondrial calcium release into the cytoplasm and further phosphorylates CAMK1. Phosphorylated CAMK1 contributes to PINK1 phosphorylation as well as mitophagy and mitochondrial function recover in EPCs. Together, our findings describe a molecular mechanism of mitophagy activation, where mitochondrial calcium release promotes CAMK1 phosphorylation of threonine177 before phosphorylation of PINK1 at serine228, which recruits PARK2 and phosphorylates its serine65 to activate mitophagy. Our results further account for the pleiotropic effects of statins on the cardiovascular system and provide a promising and potential therapeutic target for atherosclerosis.
  38. Inflammation. 2022 Feb 07.
      Dendritic cells (DCs) are vital antigen-presenting cells (APCs) in the immune system, whose apoptosis is closely related to the development of sepsis. Mitophagy is one of the necessary forms of selective autophagy that removes damaged or dysfunctional mitochondria to regulate immunity and inflammation. However, its effect on the apoptosis of DC in sepsis remains unknown. Here, we showed that sepsis activated the apoptosis and mitophagy of DC, and mitophagy had an anti-apoptotic effect on sepsis-induced DC apoptosis. In this study, we used cecal ligation and puncture (CLP) to simulate the pathophysiological state of sepsis. Apoptosis and mitophagy of DC were significantly enhanced in CPL mice compared with controls, and in the Pink1-KO (Pink1-knockout) mice CLP model, the level of apoptosis in DC was further increased while the level of mitophagy was decreased. In addition, more severe mitochondrial dysfunction was exhibited in DC of Pink1-KO mice CLP model compared to wild-type (WT) mice. The results suggest that Pink1/Parkin-mediated mitophagy is activated during sepsis and has an anti-apoptotic effect on DC, which regulates immune functions.
    Keywords:  E3 ubiquitin ligases/Parkin; PTEN-induced putative kinase 1/Pink1; apoptosis; dendritic cell; mitophagy; sepsis.
  39. Diabetes. 2022 Feb 08. pii: db210983. [Epub ahead of print]
      Mitochondria-associated endoplasmic reticulum membrane (MAM) is emerging as a novel insight into tubular injury in diabetic nephropathy (DN), but the precise mechanism remains unclear. Here, we demonstrate that the expression of phosphofurin acidic cluster sorting protein 2 (PACS-2), a critical regulator of MAM formation, is significantly decreased in renal tubules of patients with DN, which is positively correlated with renal function and negatively correlated with degrees of tubulointerstitial lesions. Conditional deletion of Pacs-2 in proximal tubules (PT) aggravates albuminuria and tubular injury in streptozotocin (STZ)-induced diabetic mice. Mitochondrial fragmentation, MAM disruption and defective mitophagy accompanied by altered expression of mitochondrial dynamics and mitophagic protein including DRP1 and BECN1 are observed in tubules from diabetic mice, while these changes are more pronounced in PT-specific Pacs-2 knockout mice. In vitro, overexpression of PACS-2 in HK-2 cells alleviates excessive mitochondrial fission induced by high glucose through blocking mitochondrial recruitment of DRP1, and subsequently restores MAM integrity and enhances mitophagy. Mechanistically, PACS-2 binds to BECN1 and mediates the relocalization of BECN1 to MAM where it promotes the formation of mitophagosome. Together, these data highlight an important but previously unrecognized role of PACS-2 in ameliorating tubular injury in DN by facilitating MAM formation and mitophagy.
  40. Trends Cell Biol. 2022 Feb 02. pii: S0962-8924(22)00001-0. [Epub ahead of print]
      Lysosomes play major roles in growth regulation and catabolism and are recognized as critical mediators of cellular remodeling. An emerging theme is how the lysosome is itself subjected to extensive remodeling in order to perform specific tasks that meet the changing demands of the cell. Accordingly, lysosomes can sustain physical damage and undergo dramatic changes in composition following pathogen infection, accumulation of protein aggregates, or cellular transformation, necessitating dedicated pathways for their repair, remodeling, and restoration. In this review, we focus on emerging molecular mechanisms for piecemeal remodeling of lysosomal components and wholesale repair and discuss their implications in physiological and pathogenic challenges such as cancer, neurodegeneration, and pathogen infection.
    Keywords:  cancer; infection; lysosome; membrane damage; neurodegeneration; repair
  41. Cell Syst. 2022 Feb 07. pii: S2405-4712(22)00040-0. [Epub ahead of print]
      Huntington disease (HD) is a monogenic neurodegenerative disorder with one causative gene, huntingtin (HTT). Yet, HD pathobiology is multifactorial, suggesting that cellular factors influence disease progression. Here, we define HTT protein-protein interactions (PPIs) perturbed by the mutant protein with expanded polyglutamine in the mouse striatum, a brain region with selective HD vulnerability. Using metabolically labeled tissues and immunoaffinity purification-mass spectrometry, we establish that polyglutamine-dependent modulation of HTT PPI abundances and relative stability starts at an early stage of pathogenesis in a Q140 HD mouse model. We identify direct and indirect PPIs that are also genetic disease modifiers using in-cell two-hybrid and behavioral assays in HD human cell and Drosophila models, respectively. Validated, disease-relevant mHTT-dependent interactions encompass mediators of synaptic neurotransmission (SNAREs and glutamate receptors) and lysosomal acidification (V-ATPase). Our study provides a resource for understanding mHTT-dependent dysfunction in cortico-striatal cellular networks, partly through impaired synaptic communication and endosomal-lysosomal system. A record of this paper's Transparent Peer Review process is included in the supplemental information.
    Keywords:  AMPA receptors; Arp2/3; D. melanogaster; LuTHy; SNARE; immunoaffinity purification-mass spectrometry; label-free quantification; metabolic labeling; protein interactions; synaptic biology; vesicular trafficking
  42. Proc Natl Acad Sci U S A. 2022 Feb 15. pii: e2120404119. [Epub ahead of print]119(7):
      Transient receptor potential mucolipin 1 (TRPML1) is a Ca2+-permeable, nonselective cation channel ubiquitously expressed in the endolysosomes of mammalian cells and its loss-of-function mutations are the direct cause of type IV mucolipidosis (MLIV), an autosomal recessive lysosomal storage disease. TRPML1 is a ligand-gated channel that can be activated by phosphatidylinositol 3,5-bisphosphate [PI(3,5)P2] as well as some synthetic small-molecule agonists. Recently, rapamycin has also been shown to directly bind and activate TRPML1. Interestingly, both PI(3,5)P2 and rapamycin have low efficacy in channel activation individually but together they work cooperatively and activate the channel with high potency. To reveal the structural basis underlying the synergistic activation of TRPML1 by PI(3,5)P2 and rapamycin, we determined the high-resolution cryoelectron microscopy (cryo-EM) structures of the mouse TRPML1 channel in various states, including apo closed, PI(3,5)P2-bound closed, and PI(3,5)P2/temsirolimus (a rapamycin analog)-bound open states. These structures, combined with electrophysiology, elucidate the molecular details of ligand binding and provide structural insight into how the TRPML1 channel integrates two distantly bound ligand stimuli and facilitates channel opening.
    Keywords:  PI(3,5)P2; TRPML1; lysosomal channel; rapamycin