bims-auttor 2022-11-06 papers

bims-auttor

Biomed News

on Autophagy and mTOR

Issue of 2022‒11‒06
39 papers selected by
Viktor Korolchuk, Newcastle University

The Role of Mitophagy in Various Neurological Diseases as a Therapeutic Approach.
Autophagy in striated muscle diseases.
The World's First (and Probably Last) Autophagy Video Game.
The emerging role of autophagy and mitophagy in tauopathies: From pathogenesis to translational implications in Alzheimer's disease.
Mitochondrial DNA quality control in the female germline requires a unique programmed mitophagy.
ER-mitochondrial contact protein Miga regulates autophagy through Atg14 and Uvrag.
Deubiquitylase PSMD14 inhibits autophagy to promote ovarian cancer progression via stabilization of LRPPRC.
Oxidative Stress Impairs Autophagy via Inhibition of Lysosomal Transport of VAMP8.
p62 bodies: phase separation, NRF2 activation, and selective autophagic degradation.
Disruption of mTORC1 rescues neuronal overgrowth and synapse function dysregulated by Pten loss.
Transient Systemic Autophagy Ablation Irreversibly Inhibits Lung Tumor Cell Metabolism and Promotes T-Cell Mediated Tumor Killing.
Defective autophagy triggered by arterial cyclic stretch promotes neointimal hyperplasia in vein grafts via the p62/nrf2/slc7a11 signaling pathway.
Genetic and Environmental Contributions to Autism Spectrum Disorder Through Mechanistic Target of Rapamycin.
A VPS15-like kinase regulates apicoplast biogenesis and autophagy by promoting PI3P generation in Toxoplasma gondii.
V-ATPase/TORC1-mediated ATFS-1 translation directs mitochondrial UPR activation in C. elegans.
Neuronal Puncta/Aggregate Formation by WT and Mutant UBQLN2.
A New Crosslinking Assay to Study Guanine Nucleotide Binding in the Gtr Heterodimer of S. cerevisiae.
The regulatory roles of circular RNAs via autophagy in ischemic stroke.
The telomeric protein TERF2/TRF2 impairs HMGB1-driven autophagy.
An inducible expression system for the manipulation of autophagic flux in vivo.
Strengthening the link between mitophagy and Parkinson's disease.
Aberrant regulation of autophagy linked to OA.
Phosphoproteomic Analysis Defines BABAM1 as mTORC2 Downstream Effector Promoting DNA Damage Response in Glioblastoma Cells.
Parkin regulates adiposity by coordinating mitophagy with mitochondrial biogenesis in white adipocytes.
SCF-SKP2 E3 ubiquitin ligase links mTORC1-ER stress-ISR with YAP activation in murine renal cystogenesis.
NRBF2-mediated autophagy contributes to metabolite replenishment and radioresistance in glioblastoma.
The Break-Fast study protocol: a single arm pre-post study to measure the effect of a protein-rich breakfast on autophagic flux in fasting healthy individuals.
Identification of active natural products that induce lysosomal biogenesis by lysosome-based screening and biological evaluation.
Distinct roles for different autophagy-associated genes in the virulence of the fungal wheat pathogen Zymoseptoria tritici.
Editorial: Autophagy in the central nervous system: Focus on neurons, glia and neuron-glia interactions.
Critical Reanalysis of the Mechanisms Underlying the Cardiorenal Benefits of SGLT2 Inhibitors and Reaffirmation of the Nutrient Deprivation Signaling/Autophagy Hypothesis.
Harnessing Dual-Fluorescence Lifetime Probes to Validate Regulatory Mechanisms of Organelle Interactions.
Pharyngeal pathology in a mouse model of oculopharyngeal muscular dystrophy is associated with impaired basal autophagy in myoblasts.
Challenges and Emerging Opportunities for Targeting mTOR in Cancer.
Inhibition of the AKT/mTOR pathway negatively regulates PTEN expression via miRNAs.
Allicin promotes autophagy and ferroptosis in esophageal squamous cell carcinoma by activating AMPK/mTOR signaling.
Electroacupuncture induces weight loss by regulating tuberous sclerosis complex 1-mammalian target of rapamycin methylation and hypothalamic autophagy in high-fat diet-induced obese rats.
Spatially resolved phosphoproteomics reveals fibroblast growth factor receptor recycling-driven regulation of autophagy and survival.
mTOR (Mammalian Target of Rapamycin): Hitting the Bull's Eye for Enhancing Neurogenesis After Cerebral Ischemia?

Cell Mol Neurobiol. 2022 Nov 03.

The Role of Mitophagy in Various Neurological Diseases as a Therapeutic Approach.

Simranjit Kaur, Neelam Sharma, Vishal Kumar, Deepali Sharma, Bhawna Devi, Lakshay Kapil, Charan Singh, Arti Singh.

  Mitochondria are critical to multiple cellular processes, from the production of adenosine triphosphate (ATP), maintenance of calcium homeostasis, synthesis of key metabolites, and production of reactive oxygen species (ROS) to maintain necrosis, apoptosis, and autophagy. Therefore, proper clearance and regulation are essential to maintain various physiological processes carried out by the cellular mechanism, including mitophagy and autophagy, by breaking down the damaged intracellular connections under the influence of various genes and proteins and protecting against various neurodegenerative diseases such as Parkinson disease (PD), amyotrophic lateral sclerosis (ALS), Alzheimer disease (AD), and Huntington disease (HD). In this review, we will discuss the role of autophagy, selective macroautophagy, or mitophagy, and its role in neurodegenerative diseases along with normal physiology. In addition, this review will provide a better understanding of the pathways involved in neuron autophagy and mitophagy and how mutations affect these pathways in the various genes involved in neurodegenerative diseases. Various new findings indicate that the pathways that remove dysfunctional mitochondria are impaired in these diseases, leading to the deposition of damaged mitochondria. Apart from that, we have also discussed the therapeutic strategies targeting autophagy and mitophagy in neurodegenerative diseases. The mitophagy cycle results in the degradation of damaged mitochondria and the biogenesis of new healthy mitochondria, also highlighting different stages at which a particular neurodegenerative disease could occur.

Keywords:  Alzheimer’s disease; Amyotrophic lateral sclerosis; Autophagy; Huntington’s disease; Mitochondrial damage; Mitophagy; Neurodegeneration; Oxidative stress; Parkinson’s disease

DOI:  https://doi.org/10.1007/s10571-022-01302-8
Front Cardiovasc Med. 2022 ;9 1000067

Autophagy in striated muscle diseases.

Haiwen Li, Lingqiang Zhang, Lei Zhang, Renzhi Han.

  Impaired biomolecules and cellular organelles are gradually built up during the development and aging of organisms, and this deteriorating process is expedited under stress conditions. As a major lysosome-mediated catabolic process, autophagy has evolved to eradicate these damaged cellular components and recycle nutrients to restore cellular homeostasis and fitness. The autophagic activities are altered under various disease conditions such as ischemia-reperfusion cardiac injury, sarcopenia, and genetic myopathies, which impact multiple cellular processes related to cellular growth and survival in cardiac and skeletal muscles. Thus, autophagy has been the focus for therapeutic development to treat these muscle diseases. To develop the specific and effective interventions targeting autophagy, it is essential to understand the molecular mechanisms by which autophagy is altered in heart and skeletal muscle disorders. Herein, we summarize how autophagy alterations are linked to cardiac and skeletal muscle defects and how these alterations occur. We further discuss potential pharmacological and genetic interventions to regulate autophagy activities and their applications in cardiac and skeletal muscle diseases.

Keywords:  autophagy; cardiomyopathy; gene therapy; heart disease; mitophagy; muscular dystrophy; myopathy; skeletal muscle disease

DOI:  https://doi.org/10.3389/fcvm.2022.1000067
Autophagy. 2022 Nov 02.

The World's First (and Probably Last) Autophagy Video Game.

Wayne D Hawkins, Emily R Grush, Daniel J Klionsky.

Macroautophagy/autophagy is the process by which portions of the cytoplasm are sequestered within a transient compartment and delivered to the degradative organelle of the cell, the vacuole or lysosome. Autophagy is a fundamental cytoprotective mechanism, and defects in this process are associated with many diseases. For example, the inability to degrade certain cargo such as mitochondria may lead to neurodegenerative disorders such as Parkinson disease. Autophagic cargo can be many different things including organelles such as mitochondria, but also proteins and protein aggregates, nucleic acids, and lipids. Much of our understanding of autophagy comes from studies in baker's yeast, Saccharomyces cerevisiae. In that organism, autophagy begins at the phagophore assembly site (PAS), which nucleates the initial sequestering compartment, referred to as a phagophore. With the help of autophagy-related (Atg) proteins and lipid addition, the phagophore membrane expands to enclose damaged or superfluous cytoplasmic components, eventually closing into a completed double-membrane vesicle called the autophagosome. The autophagosome is delivered to the degradative organelle where it fuses, releasing the encapsulated cargo into the interior of the organelle where it is broken down into macromolecular building blocks. The resulting building blocks are released back into the cytosol for reuse. Video games are modern expressions of art incorporating illustration, animation, and mechanistic design. While often underappreciated as a scientific art form, video games can beautifully express scientific topics in a way that is both intuitive and engaging, especially to a younger audience.

DOI: https://doi.org/10.1080/15548627.2022.2143212
Front Aging Neurosci. 2022 ;14 1022821

The emerging role of autophagy and mitophagy in tauopathies: From pathogenesis to translational implications in Alzheimer's disease.

Xiaolan Liu, Meng Ye, Liang Ma.

  Alzheimer's disease (AD) is the most prevalent neurodegenerative disease, affecting more than 55 million individuals worldwide in 2021. In addition to the "amyloid hypothesis," an increasing number of studies have demonstrated that phosphorylated tau plays an important role in AD pathogenesis. Both soluble tau oligomers and insoluble tau aggregates in the brain can induce structural and functional neuronal damage through multiple pathways, eventually leading to memory deficits and neurodegeneration. Autophagy is an important cellular response to various stress stimuli and can generally be categorized into non-selective and selective autophagy. Recent studies have indicated that both types of autophagy are involved in AD pathology. Among the several subtypes of selective autophagy, mitophagy, which mediates the selective removal of mitochondria, has attracted increasing attention because dysfunctional mitochondria have been suggested to contribute to tauopathies. In this review, we summarize the latest findings on the bidirectional association between abnormal tau proteins and defective autophagy, as well as mitophagy, which might constitute a vicious cycle in the induction of neurodegeneration. Neuroinflammation, another important feature in the pathogenesis and progression of AD, has been shown to crosstalk with autophagy and mitophagy. Additionally, we comprehensively discuss the relationship between neuroinflammation, autophagy, and mitophagy. By elucidating the underlying molecular mechanisms governing these pathologies, we highlight novel therapeutic strategies targeting autophagy, mitophagy and neuroinflammation, such as those using rapamycin, urolithin, spermidine, curcumin, nicotinamide, and actinonin, for the prevention and treatment of AD.

Keywords:  Alzheimer’s disease; autophagy; mitophagy; neuroinflammation; prevention and treatment; tau protein

DOI:  https://doi.org/10.3389/fnagi.2022.1022821
Cell Metab. 2022 Nov 01. pii: S1550-4131(22)00456-9. [Epub ahead of print]34(11): 1809-1823.e6

Mitochondrial DNA quality control in the female germline requires a unique programmed mitophagy.

Jonathan M Palozzi, Swathi P Jeedigunta, Anastasia V Minenkova, Vernon L Monteiro, Zoe S Thompson, Toby Lieber, Thomas R Hurd.

  Mitochondria have their own DNA (mtDNA), which is susceptible to the accumulation of disease-causing mutations. To prevent deleterious mutations from being inherited, the female germline has evolved a conserved quality control mechanism that remains poorly understood. Here, through a large-scale screen, we uncover a unique programmed germline mitophagy (PGM) that is essential for mtDNA quality control. We find that PGM is developmentally triggered as germ cells enter meiosis by inhibition of the target of rapamycin complex 1 (TORC1). We identify a role for the RNA-binding protein Ataxin-2 (Atx2) in coordinating the timing of PGM with meiosis. We show that PGM requires the mitophagy receptor BNIP3, mitochondrial fission and translation factors, and members of the Atg1 complex, but not the mitophagy factors PINK1 and Parkin. Additionally, we report several factors that are critical for germline mtDNA quality control and show that pharmacological manipulation of one of these factors promotes mtDNA quality control.

Keywords:  autophagy; germ line; germline; mitochondria; mitochondrial DNA; mitophagy; mtDNA; purifying selection; quality control

DOI:  https://doi.org/10.1016/j.cmet.2022.10.005
Cell Rep. 2022 Nov 01. pii: S2211-1247(22)01448-6. [Epub ahead of print]41(5): 111583

ER-mitochondrial contact protein Miga regulates autophagy through Atg14 and Uvrag.

Lingna Xu, Yunyi Qiu, Xufeng Wang, Weina Shang, Jian Bai, Kexin Shi, Hao Liu, Jun-Ping Liu, Liquan Wang, Chao Tong.

  Mitochondrial malfunction and autophagy defects are often concurrent phenomena associated with neurodegeneration. We show that Miga, a mitochondrial outer-membrane protein that regulates endoplasmic reticulum-mitochondrial contact sites (ERMCSs), is required for autophagy. Loss of Miga results in an accumulation of autophagy markers and substrates, whereas PI3P and Syx17 levels are reduced. Further experiments indicated that the fusion between autophagosomes and lysosomes is defective in Miga mutants. Miga binds to Atg14 and Uvrag; concordantly, Miga overexpression results in Atg14 and Uvrag recruitment to mitochondria. The heightened PI3K activity induced by Miga requires Uvrag, whereas Miga-mediated stabilization of Syx17 is dependent on Atg14. Miga-regulated ERMCSs are critical for PI3P formation but are not essential for the stabilization of Syx17. In summary, we identify a mitochondrial protein that regulates autophagy by recruiting two alternative components of the PI3K complex present at the ERMCSs.

Keywords:  CP: Cell biology; Drosophila; ER–mitochondrial contact; autophagy; lysosome; mitochondria

DOI:  https://doi.org/10.1016/j.celrep.2022.111583
Biochim Biophys Acta Mol Basis Dis. 2022 Oct 31. pii: S0925-4439(22)00265-4. [Epub ahead of print] 166594

Deubiquitylase PSMD14 inhibits autophagy to promote ovarian cancer progression via stabilization of LRPPRC.

Zitong Zhao, Heyang Xu, Yuan Wei, Li Sun, Yongmei Song.

  Autophagy is a key cellular process, which exists in many tumors and plays dual roles in tumor promotion and suppression. However, the role and mechanism of aberrant autophagy in ovarian cancer remains unclear. Ubiquitin-proteasome pathway is the most important pathway for specific protein degradation. Deubiquitinases (DUBs) have crucial roles in all the stages of tumorigenesis and progression. Herein, we explore the DUBs which contribute to aberrant autophagy in ovarian cancer. TCGA data analysis shows that the autophagy level is suppressed, and the selective autophagy receptor SQSTM1/p62 is abnormally high expressed in ovarian cancer. We screen and identify that the deubiquitinase PSMD14 negatively regulates autophagy level. Functional studies show that increased PSMD14 expression remarkably enhances ovarian cancer cells malignancy, whereas knockdown of PSMD14 has the opposite effect. Furthermore, in vivo assays show that knockdown of PSMD14 inhibits the growth, lung and abdominal metastasis of ovarian cancer. Mechanistically, PSMD14 directly interacts with LRPPRC and inhibits its ubiquitination, thereby inhibiting autophagy through LRPPRC/Beclin1-Bcl-2/SQSTM1 signaling pathway. Next, we demonstrate that PSMD14 is upregulated in ovarian cancer and high expression of PSMD14 positively correlates with LRPPRC. Taken together, we clarify the role of autophagy in regulating the ovarian cancer phenotype and provide insights into regulatory mechanism of autophagy in ovarian cancer.

Keywords:  Autophagy; Deubiquitylase; Metastasis; Ovarian cancer; Proliferation

DOI:  https://doi.org/10.1016/j.bbadis.2022.166594
Biol Pharm Bull. 2022 ;45(11): 1609-1615

Oxidative Stress Impairs Autophagy via Inhibition of Lysosomal Transport of VAMP8.

Yukiya Ohnishi, Daisuke Tsuji, Kohji Itoh.

  Autophagy is a highly conserved intracellular degrading system and its dysfunction is considered related to the cause of neurodegenerative disorders. A previous study showed that the inhibition of endocytosis transport attenuates soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) protein transport to lysosomes and block autophagy. The other studies demonstrated oxidative stress, one of the inducers of neurodegenerative diseases inhibits endocytosis transport. Thus, we hypothesized that oxidative stress-induced endocytosis inhibition causes alteration of SNARE protein transport to lysosomes and impairs autophagy. Here, we demonstrated that oxidative stress inhibits endocytosis and decreased the lysosomal localization of VAMP8, one of the autophagy-related SNARE proteins in a human neuroblastoma cell line. Moreover, this oxidative stress induction blocked the autophagosome-lysosome fusion step. Since we also observed decreased lysosomal localization of VAMP8 and inhibition of autophagosome-lysosome fusion in endocytosis inhibitor-treated cells, oxidative stress may inhibit VAMP8 trafficking by suppressing endocytosis and impair autophagy. Our findings suggest that oxidative stress-induced inhibition of VAMP8 trafficking to lysosomes is associated with the development of neurodegenerative diseases due to the blocked autophagosome-lysosome fusion, and may provide a new therapeutic target for restoring the autophagic activity.

Keywords:  autophagy; endocytosis; oxidative stress; soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) protein

DOI:  https://doi.org/10.1248/bpb.b22-00131
IUBMB Life. 2022 Nov 04.

p62 bodies: phase separation, NRF2 activation, and selective autophagic degradation.

Masaaki Komatsu.

  When p62/Sequestosome-1 binds to a ubiquitinated protein, it undergoes liquid-liquid phase separation and forms a membraneless organelle, p62 body. There are two major physiological functions of the p62 body. One is effective autophagic degradation of ubiquitinated proteins and the other is antioxidant stress response, both of which contribute to cellular homeostasis. In this review, I review the history of p62 research in relation to autophagy and outline the formation, degradation, and physiological functions of the p62 body. This article is protected by copyright. All rights reserved.

Keywords:  KEAP1; NRF2; autophagy; p62; phase separation

DOI:  https://doi.org/10.1002/iub.2689
Cell Rep. 2022 Nov 01. pii: S2211-1247(22)01435-8. [Epub ahead of print]41(5): 111574

Disruption of mTORC1 rescues neuronal overgrowth and synapse function dysregulated by Pten loss.

Kamran Tariq, Erin Cullen, Stephanie A Getz, Andie K S Conching, Andrew R Goyette, Mackenzi L Prina, Wei Wang, Meijie Li, Matthew C Weston, Bryan W Luikart.

  Phosphatase and tensin homolog deleted on chromosome 10 (PTEN) is a negative regulator of AKT/mTOR signaling pathway. Mutations in PTEN are found in patients with autism, epilepsy, or macrocephaly. In mouse models, Pten loss results in neuronal hypertrophy, hyperexcitability, seizures, and ASD-like behaviors. The underlying molecular mechanisms of these phenotypes are not well delineated. We determined which of the Pten loss-driven aberrations in neuronal form and function are orchestrated by downstream mTOR complex 1 (mTORC1). Rapamycin-mediated inhibition of mTORC1 prevented increase in soma size, migration, spine density, and dendritic overgrowth in Pten knockout dentate gyrus granule neurons. Genetic knockout of Raptor to disrupt mTORC1 complex formation blocked Pten loss-mediated neuronal hypertrophy. Electrophysiological recordings revealed that genetic disruption of mTORC1 rescued Pten loss-mediated increase in excitatory synaptic transmission. We have identified an essential role for mTORC1 in orchestrating Pten loss-driven neuronal hypertrophy and synapse formation.

Keywords:  CP: Cell biology; CP: Neuroscience; PTEN; Raptor; autism; dendrite; mTOR; rapamycin; synapse

DOI:  https://doi.org/10.1016/j.celrep.2022.111574
Autophagy. 2022 Oct 31.

Transient Systemic Autophagy Ablation Irreversibly Inhibits Lung Tumor Cell Metabolism and Promotes T-Cell Mediated Tumor Killing.

Kyle Nunn, Jessie Yanxiang Guo.

  Macroautophagy/autophagy is a highly conserved catabolic process pivotal to cellular homeostasis and support of tumorigenesis. Being a potential therapeutic target for cancer, we have worked to understand the implications of autophagy inhibition both systemically, and tumor-specifically. We utilized inducible expression of Atg5 shRNA to temporally control autophagy levels in a reversible manner to study the effects of tumor-intrinsic and systemic autophagic loss and restoration on established KrasG12D/+;trp53-/- (KP) lung tumor growth. We reported that transient systemic ATG5 loss significantly reduces KP lung tumor growth. Through in vivo isotope tracing and metabolic flux analyses, we noted that systemic ATG5 knockdown significantly reduces the uptake of glucose and lactate in lung tumors, leading to impaired TCA cycle metabolism and biosynthesis. Additionally, we observed an increased tumor T cell infiltration in the absence of systemic ATG5, which is essential for T cell-mediated tumor killing. Moreover, the impaired tumor metabolism and increased T cell infiltration are sustained when autophagy is restored in a short term. Finally, we found that intermittent systemic ATG5 knockdown, a mock therapy situation, significantly prolongs the lifespan of mice bearing KP lung tumors. Our findings lay the proof of concept for inhibition of autophagy as a valid approach to cancer therapy.

Keywords:  KRAS; autophagy; cancer metabolism; cancer therapy; immune evasion; lung tumor

DOI:  https://doi.org/10.1080/15548627.2022.2141534
J Mol Cell Cardiol. 2022 Oct 26. pii: S0022-2828(22)00534-X. [Epub ahead of print]173 101-114

Defective autophagy triggered by arterial cyclic stretch promotes neointimal hyperplasia in vein grafts via the p62/nrf2/slc7a11 signaling pathway.

Yi Chen, Min Bao, Ji-Ting Liu, Han Bao, Shou-Min Zhang, Yue Lou, Ying-Xin Qi.

  Autophagy is an adaptation mechanism to keep cellular homeostasis, and its deregulation is implicated in various cardiovascular diseases. After vein grafting, hemodynamic factors play crucial roles in neointimal hyperplasia, but the mechanisms are poorly understood. Here, we investigated the impacts of arterial cyclic stretch on autophagy of venous smooth muscle cells (SMCs) and its role in neointima formation after vein grafting. Rat jugular vein graft were generated via the 'cuff' technique. Autophagic flux in venous SMCs is impaired in 3-day, 1-week and 2-week grafted veins. 10%-1.25 Hz cyclic stretch (arterial stretch) loaded with FX5000 stretch system on venous SMCs blocks cellular autophagic flux in vitro and shows no significant impact on activity of mTORC1 and AMPK. Microtubule depolymerization but not lysosome dysfunction nor autophagosome/amphisome-lysosomal membrane fusion blockade is involved in the impairment of autophagic flux. Microtubule stabilization, induced by paclitaxel treatment and external stents intervention respectively, restores venous SMC autophagy and ameliorates neointimal hyperplasia in vivo. Moreover, autophagy impairment causes accumulation of the cargo receptor p62, which sequesters keap1 to p62 aggregates and results in the stabilization and nuclear translocation of nrf2 to modulate its target antioxidative gene SLC7A11. p62 silencing abrogates the increases of nrf2 and slc7a11 protein expression, glutathione level and venous SMC proliferation triggered by arterial cyclic stretch in vitro, and further hinders nrf2 nuclear translocation, reduces neointimal thickness after vein grafting in vivo. p62 (T349A) mutation also inhibited venous SMC proliferation and alleviated neointimal formation in vivo. These findings suggest that stabilization of microtubules to rescue autophagic flux or direct silencing of p62 are potential therapeutic strategies for neointimal hyperplasia.

Keywords:  Autophagy; Mechanical stretch; Microtubules; Neointimal hyperplasia; Vein graft; p62

DOI:  https://doi.org/10.1016/j.yjmcc.2022.10.001
Biol Psychiatry Glob Open Sci. 2022 Apr;2(2): 95-105

Genetic and Environmental Contributions to Autism Spectrum Disorder Through Mechanistic Target of Rapamycin.

Atsushi Sato, Kazutaka Ikeda.

  Autism spectrum disorder (ASD) is a neurodevelopmental disorder that affects an individual's reciprocal social interaction and communication ability. Numerous genetic and environmental conditions are associated with ASD, including tuberous sclerosis complex, phosphatase and tensin homolog hamartoma tumor syndrome, fragile X syndrome, and neurofibromatosis 1. The pathogenic molecular mechanisms of these diseases are integrated into the hyperactivation of mTORC1 (mechanistic target of rapamycin complex 1). Rodent models of these diseases have shown high mTORC1 activity in the brain and ASD-related behavioral deficits, which were reversed by the mTORC1 inhibitor rapamycin. Environmental stress can also affect this signaling pathway. In utero exposure to valproate caused ASD in offspring and enhanced mTORC1 activity in the brain, which was sensitive to mTORC1 inhibition. mTORC1 is a signaling hub for diverse cellular functions, including protein synthesis, through the phosphorylation of its targets, such as ribosomal protein S6 kinases. Metabotropic glutamate receptor 5-mediated synaptic function is also affected by the dysregulation of mTORC1 activity, such as in fragile X syndrome and tuberous sclerosis complex. Reversing these downstream changes that are associated with mTORC1 activation normalizes behavioral defects in rodents. Despite abundant preclinical evidence, few clinical studies have investigated the treatment of ASD and cognitive deficits. Therapeutics other than mTORC1 inhibitors failed to show efficacy in fragile X syndrome and neurofibromatosis 1. mTORC1 inhibitors have been tested mainly in tuberous sclerosis complex, and their effects on ASD and neuropsychological deficits are promising. mTORC1 is a promising target for the pharmacological treatment of ASD associated with mTORC1 activation.

Keywords:  Animal models; Autism spectrum disorder; Autophagy; Mechanistic target of rapamycin; Metabotropic glutamate receptor; Protein synthesis

DOI:  https://doi.org/10.1016/j.bpsgos.2021.08.005
PLoS Pathog. 2022 Nov;18(11): e1010922

A VPS15-like kinase regulates apicoplast biogenesis and autophagy by promoting PI3P generation in Toxoplasma gondii.

Rahul Singh Rawat, Priyanka Bansal, Pushkar Sharma.

Phosphoinositides are important second messengers that regulate key cellular processes in eukaryotes. While it is known that a single phosphoinositol-3 kinase (PI3K) catalyses the formation of 3'-phosphorylated phosphoinositides (PIPs) in apicomplexan parasites like Plasmodium and Toxoplasma, how its activity and PI3P formation is regulated has remained unknown. Present studies involving a unique Vps15 like protein (TgVPS15) in Toxoplasma gondii provides insight into the regulation of phosphatidyl-3-phosphate (PI3P) generation and unravels a novel pathway that regulates parasite development. Detailed investigations suggested that TgVPS15 regulates PI3P formation in Toxoplasma gondii, which is important for the inheritance of the apicoplast-a plastid like organelle present in most apicomplexans and parasite replication. Interestingly, TgVPS15 also regulates autophagy in T. gondii under nutrient-limiting conditions as it promotes autophagosome formation. For both these processes, TgVPS15 uses PI3P-binding protein TgATG18 and regulates trafficking and conjugation of TgATG8 to the apicoplast and autophagosomes, which is important for biogenesis of these organelles. TgVPS15 has a protein kinase domain but lacks several key residues conserved in conventional protein kinases. Interestingly, two critical residues in its active site are important for PI3P formation and parasitic functions of this kinase. Collectively, these studies unravel a signalling cascade involving TgVPS15, a novel effector of PI3-kinase in T. gondii and possibly other Apicomplexa, that regulate critical processes like apicoplast biogenesis and autophagy.

DOI: https://doi.org/10.1371/journal.ppat.1010922
J Cell Biol. 2023 Jan 02. pii: e202205045. [Epub ahead of print]222(1):

V-ATPase/TORC1-mediated ATFS-1 translation directs mitochondrial UPR activation in C. elegans.

Terytty Yang Li, Arwen W Gao, Xiaoxu Li, Hao Li, Yasmine J Liu, Amelia Lalou, Nagammal Neelagandan, Felix Naef, Kristina Schoonjans, Johan Auwerx.

To adapt mitochondrial function to the ever-changing intra- and extracellular environment, multiple mitochondrial stress response (MSR) pathways, including the mitochondrial unfolded protein response (UPRmt), have evolved. However, how the mitochondrial stress signal is sensed and relayed to UPRmt transcription factors, such as ATFS-1 in Caenorhabditis elegans, remains largely unknown. Here, we show that a panel of vacuolar H+-ATPase (v-ATPase) subunits and the target of rapamycin complex 1 (TORC1) activity are essential for the cytosolic relay of mitochondrial stress to ATFS-1 and for the induction of the UPRmt. Mechanistically, mitochondrial stress stimulates v-ATPase/Rheb-dependent TORC1 activation, subsequently promoting ATFS-1 translation. Increased translation of ATFS-1 upon mitochondrial stress furthermore relies on a set of ribosomal components but is independent of GCN-2/PEK-1 signaling. Finally, the v-ATPase and ribosomal subunits are required for mitochondrial surveillance and mitochondrial stress-induced longevity. These results reveal a v-ATPase-TORC1-ATFS-1 signaling pathway that links mitochondrial stress to the UPRmt through intimate crosstalks between multiple organelles.

DOI: https://doi.org/10.1083/jcb.202205045
Methods Mol Biol. 2023 ;2551 561-573

Neuronal Puncta/Aggregate Formation by WT and Mutant UBQLN2.

Nathaniel Safren, Lisa M Sharkey, Sami J Barmada.

  Protein aggregates are a common feature of nearly all neurodegenerative diseases, including Alzheimer's disease, Parkinson's disease, and amyotrophic lateral sclerosis (ALS). Here we describe a method to quickly and accurately measure protein aggregation in cells expressing a fluorescently tagged aggregation-prone protein. This unbiased method obviates the need for manual scoring and facilitates the identification of factors governing protein self-assembly and its downstream consequences for cell heath.

Keywords:  Longitudinal microscopy; Neurodegeneration; Protein aggregation; Proteostasis; Single cell

DOI:  https://doi.org/10.1007/978-1-0716-2597-2_34
Small GTPases. 2022 Jan;13(1): 327-334

A New Crosslinking Assay to Study Guanine Nucleotide Binding in the Gtr Heterodimer of S. cerevisiae.

Dylan D Doxsey, Kristen Veinotte, Kuang Shen.

  The mechanistic target of rapamycin (mTOR) complex is responsible for coordinating nutrient availability with eukaryotic cell growth. Amino acid signals are transmitted towards mTOR via the Rag/Gtr heterodimers. Due to the obligatory heterodimeric architecture of the Rag/Gtr GTPases, investigating their biochemical properties has been challenging. Here, we describe an updated assay that allows us to probe the guanine nucleotide-binding affinity and kinetics to the Gtr heterodimers in Saccharomyces cerevisiae. We first identified the structural element that Gtr2p lacks to enable crosslinking. By using a sequence conservation-based mutation, we restored the crosslinking between Gtr2p and the bound nucleotides. Using this construct, we determined the nucleotide-binding affinities of the Gtr heterodimer, and found that it operates under a different form of intersubunit communication than human Rag GTPases. Our study defines the evolutionary divergence of the Gtr/Rag-mTOR axis of nutrient sensing.

Keywords:  GTPase; Gtr GTPase; Rag GTPase; crosslinking; enzyme kinetics; guanine nucleotide; mTOR; nutrient sensing

DOI:  https://doi.org/10.1080/21541248.2022.2141019
Front Neurol. 2022 ;13 963508

The regulatory roles of circular RNAs via autophagy in ischemic stroke.

Xiaoqin Li, Lingfei Li, Xiaoli Si, Zheng Zhang, Zhumei Ni, Yongji Zhou, Keqin Liu, Wenqing Xia, Yuyao Zhang, Xin Gu, Jinyu Huang, Congguo Yin, Anwen Shao, Lin Jiang.

  Ischemic stroke (IS) is a severe disease with a high disability, recurrence, and mortality rates. Autophagy, a highly conserved process that degrades damaged or aging organelles and excess cellular components to maintain homeostasis, is activated during IS. It influences the blood-brain barrier integrity and regulates apoptosis. Circular RNAs (circRNAs) are novel non-coding RNAs involved in IS-induced autophagy and participate in various pathological processes following IS. In addition, they play a role in autophagy regulation. This review summarizes current evidence on the roles of autophagy and circRNA in IS and the potential mechanisms by which circRNAs regulate autophagy to influence IS injury. This review serves as a basis for the clinical application of circRNAs as novel biomarkers and therapeutic targets in the future.

Keywords:  autophagy; biomarkers; circRNA; ischemic stroke; therapeutic targets

DOI:  https://doi.org/10.3389/fneur.2022.963508
Autophagy. 2022 Oct 30. 1-12

The telomeric protein TERF2/TRF2 impairs HMGB1-driven autophagy.

Sara Iachettini, Fabio Ciccarone, Carmen Maresca, Carmen D' Angelo, Eleonora Petti, Serena Di Vito, Maria Rosa Ciriolo, Pasquale Zizza, Annamaria Biroccio.

  TERF2/TRF2 is a pleiotropic telomeric protein that plays a crucial role in tumor formation and progression through several telomere-dependent and -independent mechanisms. Here, we uncovered a novel function for this protein in regulating the macroautophagic/autophagic process upon different stimuli. By using both biochemical and cell biology approaches, we found that TERF2 binds to the non-histone chromatin-associated protein HMGB1, and this interaction is functional to the nuclear/cytoplasmic protein localization. Specifically, silencing of TERF2 alters the redox status of the cells, further exacerbated upon EBSS nutrient starvation, promoting the cytosolic translocation and the autophagic activity of HMGB1. Conversely, overexpression of wild-type TERF2, but not the mutant unable to bind HMGB1, negatively affects the cytosolic translocation of HMGB1, counteracting the stimulatory effect of EBSS starvation. Moreover, genetic depletion of HMGB1 or treatment with inflachromene, a specific inhibitor of its cytosolic translocation, completely abolished the pro-autophagic activity of TERF2 silencing. In conclusion, our data highlighted a novel mechanism through which TERF2 modulates the autophagic process, thus demonstrating the key role of the telomeric protein in regulating a process that is fundamental, under both physiological and pathological conditions, in defining the fate of the cells.Abbreviations: ALs: autolysosomes; ALT: alternative lengthening of telomeres; ATG: autophagy related; ATM: ATM serine/threonine kinase; CQ: Chloroquine; DCFDA: 2',7'-dichlorofluorescein diacetate; DDR: DNA damage response; DHE: dihydroethidium; EBSS: Earle's balanced salt solution; FACS: fluorescence-activated cell sorting; GFP: green fluorescent protein; EGFP: enhanced green fluorescent protein; GSH: reduced glutathione; GSSG: oxidized glutathione; HMGB1: high mobility group box 1; ICM: inflachromene; IF: immunofluorescence; IP: immunoprecipitation; NAC: N-acetyl-L-cysteine; NHEJ: non-homologous end joining; PLA: proximity ligation assay; RFP: red fluorescent protein; ROS: reactive oxygen species; TIF: telomere-induced foci; TERF2/TRF2: telomeric repeat binding factor 2.

Keywords:  Autophagy; HMGB1; ROS; TERF2/TRF2; cancer; cell biology; oxidative stress

DOI:  https://doi.org/10.1080/15548627.2022.2138687
Autophagy. 2022 Oct 30. 1-14

An inducible expression system for the manipulation of autophagic flux in vivo.

Lars Schlotawa, Ana Lopez, Gentzane Sanchez-Elexpuru, Sylwia D Tyrkalska, David C Rubinsztein, Angeleen Fleming.

  Much of our understanding of the intracellular regulation of macroautophagy/autophagy comes from in vitro studies. However, there remains a paucity of knowledge about how this process is regulated within different tissues during development, aging and disease in vivo. Because upregulation of autophagy is considered a promising therapeutic strategy for the treatment of diverse disorders, it is vital that we understand how this pathway functions in different tissues and this is best done by in vivo analysis. Similarly, to understand the role of autophagy in the pathogenesis of disease, it is important to study this process in the whole animal to investigate how tissue-specific changes in flux and cell-autonomous versus non-cell-autonomous effects alter disease progression. To this end, we have developed an inducible expression system to up- or downregulate autophagy in vivo, in zebrafish. We have used a modified version of the Gal4-UAS expression system to allow inducible expression of autophagy up- or downregulating transgenes by addition of tamoxifen. Using this inducible expression system, we have tested which transgenes robustly up- or downregulate autophagy and have validated these tools using Lc3-II blots and puncta analysis and disease rescue in a zebrafish model of neurodegeneration. These tools allow the temporal control of autophagy via the administration of tamoxifen and spatial control via tissue or cell-specific ERT2-Gal4 driver lines and will enable the investigation of how cell- or tissue-specific changes in autophagic flux affect processes such as aging, inflammation and neurodegeneration in vivo.Abbreviations: ANOVA: analysis of variance; Atg: autophagy related; Bcl2l11/Bim: BCL2 like 11; d.p.f.: days post-fertilization; Cryaa: crystallin, alpha a: DMSO: dimethyl sulfoxide; Elavl3: ELAV like neuron-specific RNA binding protein 3; ER: estrogen receptor; ERT2: modified ligand-binding domain of human ESR1/estrogen receptor α; Gal4: galactose-responsive transcription factor 4; GFP: green fluorescent protein; h.p.f.: hours post-fertilization; HSP: heat-shock protein; Map1lc3/Lc3: microtubule-associated protein 1 light chain 3; RFP: red fluorescent protein; SD: standard deviation; SEM: standard error of the mean; UAS: upstream activating sequence; Ubb: ubiquitin b.

Keywords:  ATG4B; ATG5; LC3-II; autophagy; neurodegeneration; tamoxifen; zebrafish

DOI:  https://doi.org/10.1080/15548627.2022.2135824
Brain. 2022 Nov 02. pii: awac405. [Epub ahead of print]

Strengthening the link between mitophagy and Parkinson's disease.

Ian G Ganley.

DOI: https://doi.org/10.1093/brain/awac405
Nat Rev Rheumatol. 2022 Nov 04.

Aberrant regulation of autophagy linked to OA.

Jessica McHugh.

DOI: https://doi.org/10.1038/s41584-022-00872-7
J Proteome Res. 2022 Oct 31.

Phosphoproteomic Analysis Defines BABAM1 as mTORC2 Downstream Effector Promoting DNA Damage Response in Glioblastoma Cells.

Nuttiya Kalpongnukul, Rungnapa Bootsri, Piriya Wongkongkathep, Pornchai Kaewsapsak, Chaiyaboot Ariyachet, Trairak Pisitkun, Naphat Chantaravisoot.

  Glioblastoma (GBM) is a devastating primary brain cancer with a poor prognosis. GBM is associated with an abnormal mechanistic target of rapamycin (mTOR) signaling pathway, consisting of two distinct kinase complexes: mTORC1 and mTORC2. The complexes play critical roles in cell proliferation, survival, migration, metabolism, and DNA damage response. This study investigated the aberrant mTORC2 signaling pathway in GBM cells by performing quantitative phosphoproteomic analysis of U87MG cells under different drug treatment conditions. Interestingly, a functional analysis of phosphoproteome revealed that mTORC2 inhibition might be involved in double-strand break (DSB) repair. We further characterized the relationship between mTORC2 and BRISC and BRCA1-A complex member 1 (BABAM1). We demonstrated that pBABAM1 at Ser29 is regulated by mTORC2 to initiate DNA damage response, contributing to DNA repair and cancer cell survival. Accordingly, the inactivation of mTORC2 significantly ablated pBABAM1 (Ser29), reduced DNA repair activities in the nucleus, and promoted apoptosis of the cancer cells. Furthermore, we also recognized that histone H2AX phosphorylation at Ser139 (γH2AX) could be controlled by mTORC2 to repair the DNA. These results provided a better understanding of the mTORC2 function in oncogenic DNA damage response and might lead to specific mTORC2 treatments for brain cancer patients in the future.

Keywords:  BABAM1; DNA damage response; glioblastoma; mTORC2; phosphoproteomics

DOI:  https://doi.org/10.1021/acs.jproteome.2c00240
Nat Commun. 2022 Nov 04. 13(1): 6661

Parkin regulates adiposity by coordinating mitophagy with mitochondrial biogenesis in white adipocytes.

Timothy M Moore, Lijing Cheng, Dane M Wolf, Jennifer Ngo, Mayuko Segawa, Xiaopeng Zhu, Alexander R Strumwasser, Yang Cao, Bethan L Clifford, Alice Ma, Philip Scumpia, Orian S Shirihai, Thomas Q de Aguiar Vallim, Markku Laakso, Aldons J Lusis, Andrea L Hevener, Zhenqi Zhou.

Parkin, an E3 ubiquitin ligase, plays an essential role in mitochondrial quality control. However, the mechanisms by which Parkin connects mitochondrial homeostasis with cellular metabolism in adipose tissue remain unclear. Here, we demonstrate that Park2 gene (encodes Parkin) deletion specifically from adipose tissue protects mice against high-fat diet and aging-induced obesity. Despite a mild reduction in mitophagy, mitochondrial DNA content and mitochondrial function are increased in Park2 deficient white adipocytes. Moreover, Park2 gene deletion elevates mitochondrial biogenesis by increasing Pgc1α protein stability through mitochondrial superoxide-activated NAD(P)H quinone dehydrogenase 1 (Nqo1). Both in vitro and in vivo studies show that Nqo1 overexpression elevates Pgc1α protein level and mitochondrial DNA content and enhances mitochondrial activity in mouse and human adipocytes. Taken together, our findings indicate that Parkin regulates mitochondrial homeostasis by balancing mitophagy and Pgc1α-mediated mitochondrial biogenesis in white adipocytes, suggesting a potential therapeutic target in adipocytes to combat obesity and obesity-associated disorders.

DOI: https://doi.org/10.1038/s41467-022-34468-2
J Clin Invest. 2022 Nov 03. pii: e153943. [Epub ahead of print]

SCF-SKP2 E3 ubiquitin ligase links mTORC1-ER stress-ISR with YAP activation in murine renal cystogenesis.

Dibyendu K Panda, Xiuying Bai, Yan Zhang, Nicholas A Stylianesis, Antonis E Koromilas, Mark L Lipman, Andrew C Karaplis.

  The Hippo pathway nuclear effector Yes-associated protein 1 (YAP) potentiates the progression of polycystic kidney disease (PKD) arising from ciliopathies. The mechanisms underlying the increase in YAP expression and transcriptional activity in PKD remain obscure. We observed that in kidneys from mice with juvenile cystic kidney (jck) ciliopathy, the aberrant hyperactivity of mechanistic target of rapamycin complex 1 (mTORC1) driven by ERK1/2 and PI3K/AKT cascades induced endoplasmic reticulum (ER) proteotoxic stress. To reduce it by reprogramming translation, the protein kinase R-like ER kinase (PERK)-eukaryotic initiation factor 2α (eIF2α) arm of the integrated stress response (ISR) was activated. PERK-mediated phosphorylation of eIF2α drove the selective translation of activating transcription factor 4 (ATF4), potentiating YAP expression. In parallel, YAP underwent K63-linked polyubiquitination by SCF-S-phase kinase-associated protein 2 (SKP2) E3 ubiquitin ligase, a Hippo-independent, nonproteolytic ubiquitination that enhances YAP nuclear trafficking and transcriptional activity in cancer cells. Defective ISR cellular adaptation to ER stress in eIF2α-phosphorylation-deficient jck mice further augmented YAP-mediated transcriptional activity and renal cyst growth. Conversely, pharmacological tuning down of ER stress-ISR activity and SKP2 expression in jck mice by administration of tauroursodeoxycholic acid (TUDCA) or tolvaptan, impeded these processes. Restoring ER homeostasis, and/or interfering with the SKP2-YAP interaction represent novel potential therapeutic avenues for stemming the progression of renal cystogenesis.

Keywords:  Chronic kidney disease; Nephrology

DOI:  https://doi.org/10.1172/JCI153943
Exp Mol Med. 2022 Nov 04.

NRBF2-mediated autophagy contributes to metabolite replenishment and radioresistance in glioblastoma.

Jeongha Kim, Hyunkoo Kang, Beomseok Son, Min-Jung Kim, JiHoon Kang, Kang Hyun Park, Jaewan Jeon, Sunmi Jo, Hae Yu Kim, HyeSook Youn, BuHyun Youn.

Overcoming therapeutic resistance in glioblastoma (GBM) is an essential strategy for improving cancer therapy. However, cancer cells possess various evasion mechanisms, such as metabolic reprogramming, which promote cell survival and limit therapy. The diverse metabolic fuel sources that are produced by autophagy provide tumors with metabolic plasticity and are known to induce drug or radioresistance in GBM. This study determined that autophagy, a common representative cell homeostasis mechanism, was upregulated upon treatment of GBM cells with ionizing radiation (IR). Nuclear receptor binding factor 2 (NRBF2)-a positive regulator of the autophagy initiation step-was found to be upregulated in a GBM orthotopic xenograft mouse model. Furthermore, ATP production and the oxygen consumption rate (OCR) increased upon activation of NRBF2-mediated autophagy. It was also discovered that changes in metabolic state were induced by alterations in metabolite levels caused by autophagy, thereby causing radioresistance. In addition, we found that lidoflazine-a vasodilator agent discovered through drug repositioning-significantly suppressed IR-induced migration, invasion, and proliferation by inhibiting NRBF2, resulting in a reduction in autophagic flux in both in vitro models and in vivo orthotopic xenograft mouse models. In summary, we propose that the upregulation of NRBF2 levels reprograms the metabolic state of GBM cells by activating autophagy, thus establishing NRBF2 as a potential therapeutic target for regulating radioresistance of GBM during radiotherapy.

DOI: https://doi.org/10.1038/s12276-022-00873-2
BMC Nutr. 2022 Nov 01. 8(1): 120

The Break-Fast study protocol: a single arm pre-post study to measure the effect of a protein-rich breakfast on autophagic flux in fasting healthy individuals.

Julien Bensalem, Leonie K Heilbronn, Jemima R Gore, Amy T Hutchison, Timothy J Sargeant, Célia Fourrier.

  BACKGROUND: Autophagy is a cellular process that cleanses cells and is particularly important during ageing. Autophagy has been extensively studied in vitro and in animal models and is known to be sensitive to nutrition. However, human data are limited because autophagic flux (autophagic degradative activity) has been challenging to measure in humans. This protocol paper describes the Break-Fast study, in which autophagic flux will be measured using a recently developed blood test, before and after ingestion of whey protein. This aims to determine whether an acute nutritional intervention can change autophagy in humans.METHODS: A minimum of forty healthy participants (both male and female) aged 20-50 years, BMI 18.5-29.9 kg/m2 will be recruited into this single arm pre-post study. Participants will visit the clinic after an overnight fast for a first blood collection after which they will consume a whey protein-rich drink. A second blood collection will be performed 60 minutes after consumption of the drink. The primary outcome is the change in autophagic flux at 60 minutes post drink. Secondary outcomes include changes in blood glucose, autophagy-related proteins and mRNA, plasma hormones (e.g. insulin, C-peptide, adiponectin, GLP-1, GIP, ghrelin), cytokines, amino acids and lipids, protein synthesis, and correlation between molecular cell damage and autophagic flux.
DISCUSSION: This study will provide information about whether autophagy responds to nutrients in humans, and if nutritional strategies could be used to treat or prevent autophagy-related diseases such as Alzheimer's disease or cancer.
TRIAL REGISTRATION: Australian New Zealand Clinical Trials Registry (ANZCTR), anzctr.org.au ACTRN12621001029886. Registered on 5 August 2021.

Keywords:  Amino acids; Autophagy; LC3; Macronutrient; Nutrition; Protein; Whey protein isolate; mTORC1

DOI:  https://doi.org/10.1186/s40795-022-00617-5
Heliyon. 2022 Oct;8(10): e11179

Identification of active natural products that induce lysosomal biogenesis by lysosome-based screening and biological evaluation.

Xiao Ding, Xu Yang, Yueqin Zhao, Yinyuan Wang, Jimin Fei, Zhenpeng Niu, Xianxiang Dong, Xuenan Wang, Biao Liu, Hongmei Li, Xiaojiang Hao, Yuhan Zhao.

  Lysosomal biogenesis is an essential adaptive process by which lysosomes exert their function in maintaining cellular homeostasis. Defects in lysosomal enzymes and functions lead to lysosome-related diseases, including lysosomal storage diseases and neurodegenerative disorders. Thus, activation of the autophagy-lysosomal pathway, especially induction of lysosomal biogenesis, might be an effective strategy for the treatment of lysosome-related diseases. In this study, we established a lysosome-based screening system to identify active compounds from natural products that could promote lysosomal biogenesis. The subcellular localizations of master transcriptional regulators of lysosomal genes, TFEB, TFE3 and ZKSCAN3 were examined to reveal the potential mechanisms. More than 200 compounds were screened, and we found that Hdj-23, a triterpene isolated from Walsura cochinchinensis, induced lysosomal biogenesis via activation of TFEB/TFE3. In summary, this study introduced a lysosome-based live cell screening strategy to identify bioactive compounds that promote lysosomal biogenesis, which would provide potential candidate enhancers of lysosomal biogenesis and novel insight for treating lysosome-related diseases.

Keywords:  Autophagy; Lysosomal biogenesis; Lysosome-based screening; Natural products; TFEB and TFE3

DOI:  https://doi.org/10.1016/j.heliyon.2022.e11179
Fungal Genet Biol. 2022 Oct 26. pii: S1087-1845(22)00093-7. [Epub ahead of print]163 103748

Distinct roles for different autophagy-associated genes in the virulence of the fungal wheat pathogen Zymoseptoria tritici.

Harry T Child, Michael J Deeks, Ken Haynes, Jason J Rudd, Steven Bates.

  The fungal wheat pathogen Zymoseptoria tritici causes major crop losses as the causal agent of the disease Septoria tritici blotch. The infection cycle of Z. tritici displays two distinct phases, beginning with an extended symptomless phase of 1-2 weeks, before the fungus induces host cell death and tissue collapse in the leaf. Recent evidence suggests that the fungus uses little host-derived nutrition during asymptomatic colonisation, raising questions as to the sources of energy required for this initial growth phase. Autophagy is crucial for the pathogenicity of other fungal plant pathogens through its roles in supporting cellular differentiation and growth under starvation. Here we characterised the contributions of the autophagy genes ZtATG1 and ZtATG8 to the development and virulence of Z. tritici. Deletion of ZtATG1 led to inhibition of autophagy but had no impact on starvation-induced hyphal differentiation or virulence, suggesting that autophagy is not required for Z. tritici pathogenicity. Contrastingly, ZtATG8 deletion delayed the transition to necrotrophic growth, despite having no influence on filamentous growth under starvation, pointing to an autophagy-independent role of ZtATG8 during Z. tritici infection. To our knowledge, this study represents the first to find autophagy not to contribute to the virulence of a fungal plant pathogen, and reveals novel roles for different autophagy-associated proteins in Z. tritici.

Keywords:  Autophagy; Plant pathogen; Virulence; Wheat; Zymoseptoria tritici

DOI:  https://doi.org/10.1016/j.fgb.2022.103748
Front Cell Dev Biol. 2022 ;10 1036587

Editorial: Autophagy in the central nervous system: Focus on neurons, glia and neuron-glia interactions.

Maria Jimenez-Sanchez, Olatz Pampliega, Sandra-Fausia Soukup.



Keywords:  autophagy; central nervous system; glia; neurodegeneration; neurons

DOI:  https://doi.org/10.3389/fcell.2022.1036587
Circulation. 2022 Nov;146(18): 1383-1405

Critical Reanalysis of the Mechanisms Underlying the Cardiorenal Benefits of SGLT2 Inhibitors and Reaffirmation of the Nutrient Deprivation Signaling/Autophagy Hypothesis.

Milton Packer.

  SGLT2 (sodium-glucose cotransporter 2) inhibitors produce a distinctive pattern of benefits on the evolution and progression of cardiomyopathy and nephropathy, which is characterized by a reduction in oxidative and endoplasmic reticulum stress, restoration of mitochondrial health and enhanced mitochondrial biogenesis, a decrease in proinflammatory and profibrotic pathways, and preservation of cellular and organ integrity and viability. A substantial body of evidence indicates that this characteristic pattern of responses can be explained by the action of SGLT2 inhibitors to promote cellular housekeeping by enhancing autophagic flux, an effect that may be related to the action of these drugs to produce simultaneous upregulation of nutrient deprivation signaling and downregulation of nutrient surplus signaling, as manifested by an increase in the expression and activity of AMPK (adenosine monophosphate-activated protein kinase), SIRT1 (sirtuin 1), SIRT3 (sirtuin 3), SIRT6 (sirtuin 6), and PGC1-α (peroxisome proliferator-activated receptor γ coactivator 1-α) and decreased activation of mTOR (mammalian target of rapamycin). The distinctive pattern of cardioprotective and renoprotective effects of SGLT2 inhibitors is abolished by specific inhibition or knockdown of autophagy, AMPK, and sirtuins. In the clinical setting, the pattern of differentially increased proteins identified in proteomics analyses of blood collected in randomized trials is consistent with these findings. Clinical studies have also shown that SGLT2 inhibitors promote gluconeogenesis, ketogenesis, and erythrocytosis and reduce uricemia, the hallmarks of nutrient deprivation signaling and the principal statistical mediators of the ability of SGLT2 inhibitors to reduce the risk of heart failure and serious renal events. The action of SGLT2 inhibitors to augment autophagic flux is seen in isolated cells and tissues that do not express SGLT2 and are not exposed to changes in environmental glucose or ketones and may be related to an ability of these drugs to bind directly to sirtuins or mTOR. Changes in renal or cardiovascular physiology or metabolism cannot explain the benefits of SGLT2 inhibitors either experimentally or clinically. The direct molecular effects of SGLT2 inhibitors in isolated cells are consistent with the concept that SGLT2 acts as a nutrient surplus sensor, and thus, its inhibition causes enhanced nutrient deprivation signaling and its attendant cytoprotective effects, which can be abolished by specific inhibition or knockdown of AMPK, sirtuins, and autophagic flux.

Keywords:  TOR serine-threonine kinases; autophagy; heart failure; sirtuins; sodium-glucose transporter 2 inhibitors

DOI:  https://doi.org/10.1161/CIRCULATIONAHA.122.061732
J Am Chem Soc. 2022 Nov 01.

Harnessing Dual-Fluorescence Lifetime Probes to Validate Regulatory Mechanisms of Organelle Interactions.

Yuping Zhao, Hyeong Seok Kim, Xiang Zou, Ling Huang, Xing Liang, Zihong Li, Jong Seung Kim, Weiying Lin.

Organelles are dynamic yet highly organized to preserve cellular homeostasis. However, the absence of time-resolved molecular tools for simultaneous dual-signal imaging of two organelles has prevented scientists from elucidating organelle interaction regulatory mechanisms on a nanosecond timescale. To date, the regulatory mechanisms governing the interaction between endoplasmic reticulum (ER) and autophagosomes are unknown. In this study, we propose a strategy for developing dual-fluorescence lifetime probes localized to the endoplasmic reticulum and autophagosomes to investigate their interaction regulatory mechanisms. Using the robust probe CF2, we investigated the regulatory mechanisms between ER and autophagosomes and discovered the following: (i) motile autophagosome in ER tips drives the ER tubule to grow and slide; (ii) the ER reticulate tubule forms a three-way junction centered on the autophagosome; (iii) ER autophagy is a type of cell damage index during drug-induced apoptosis. Thus, this study advances our knowledge of organelle interaction regulatory mechanisms, shedding light on the identification of therapeutic targets for neurodegenerative diseases.

DOI: https://doi.org/10.1021/jacs.2c08966
Front Cell Dev Biol. 2022 ;10 986930

Pharyngeal pathology in a mouse model of oculopharyngeal muscular dystrophy is associated with impaired basal autophagy in myoblasts.

Yu Zhang, Christopher Zeuthen, Carol Zhu, Fang Wu, Allison T Mezzell, Thomas J Whitlow, Hyojung J Choo, Katherine E Vest.

  Oculopharyngeal muscular dystrophy (OPMD) is a late-onset dominant disease that primarily affects craniofacial muscles. Despite the fact that the genetic cause of OPMD is known to be expansion mutations in the gene encoding the nuclear polyadenosine RNA binding protein PABPN1, the molecular mechanisms of pathology are unknown and no pharmacologic treatments are available. Due to the limited availability of patient tissues, several animal models have been employed to study the pathology of OPMD. However, none of these models have demonstrated functional deficits in the muscles of the pharynx, which are predominantly affected by OPMD. Here, we used a knock-in mouse model of OPMD, Pabpn1 +/A17 , that closely genocopies patients. In Pabpn1 +/A17 mice, we detected impaired pharyngeal muscle function, and impaired pharyngeal satellite cell proliferation and fusion. Molecular studies revealed that basal autophagy, which is required for normal satellite cell function, is higher in pharynx-derived myoblasts than in myoblasts derived from limb muscles. Interestingly, basal autophagy is impaired in cells derived from Pabpn1 +/A17 mice. Pabpn1 knockdown in pharyngeal myoblasts failed to recapitulate the autophagy defect detected in Pabpn1 +/A17 myoblasts suggesting that loss of PABPN1 function does not contribute to the basal autophagy defect. Taken together, these studies provide the first evidence for pharyngeal muscle and satellite cell pathology in a mouse model of OPMD and suggest that aberrant gain of PABPN1 function contributes to the craniofacial pathology in OPMD.

Keywords:  PABPN1; autophagy; craniofacial muscles; dysphagia; muscular dystrophy; oculopharyngeal muscular dystrophy; satellite cells

DOI:  https://doi.org/10.3389/fcell.2022.986930
Cancer Res. 2022 Nov 02. 82(21): 3884-3887

Challenges and Emerging Opportunities for Targeting mTOR in Cancer.

Kris C Wood, J Silvio Gutkind.

The mechanistic target of rapamycin (mTOR) plays a key role in normal and malignant cell growth. However, pharmacologic targeting of mTOR in cancer has shown little clinical benefit, in spite of aberrant hyperactivation of mTOR in most solid tumors. Here, we discuss possible reasons for the reduced clinical efficacy of mTOR inhibition and highlight lessons learned from recent combination clinical trials and approved indications of mTOR inhibitors in cancer. We also discuss how the emerging systems level understanding of mTOR signaling in cancer can be exploited for the clinical development of novel multimodal precision targeted therapies and immunotherapies aimed at achieving tumor remission.

DOI: https://doi.org/10.1158/0008-5472.CAN-22-0602
Acta Biochim Biophys Sin (Shanghai). 2022 Oct 25.

Inhibition of the AKT/mTOR pathway negatively regulates PTEN expression via miRNAs.

Linyan Wan, Yanan Wang, Jie Li, Yani Wang, Hongbing Zhang.

  PI3K/AKT/mTOR pathway plays important roles in cancer development, and the negative role of PTEN in the PI3K/AKT/mTOR pathway is well known, but whether PTEN can be inversely regulated by PI3K/AKT/mTOR has rarely been reported. Here we aim to investigate the potential regulatory relationship between PTEN and Akt/mTOR inhibition in MEFs. AKT1 E17K and TSC2 -/- MEFs were treated with the AKT inhibitor MK2206 and the mTOR inhibitors rapamycin and Torin2. Our results reveal that inhibition of AKT or mTOR suppresses PTEN expression in AKT1 E17K and TSC2 -/- MEFs, but the transcription, subcellular localization, eIF4E-dependent translational initiation or lysosome- and proteasome-mediated degradation of PTEN change little, as shown by the real time PCR, nucleus cytoplasm separation assay and immunofluorescence analysis. Moreover, mTOR suppression leads to augmentation of mouse PTEN-3'UTR-binding miRNAs, including miR-23a-3p, miR-23b-3p, miR-25-3p and miR-26a-5p, as shown by the dual luciferase reporter assay and miRNA array analysis, and miRNA inhibitors collaborately rescue the decline of PTEN level. Collectively, our findings confirm that inhibition of mTOR suppresses PTEN expression by upregulating miRNAs, provide a novel explanation for the limited efficacy of mTOR inhibitors in the treatment of mTOR activation-related tumors, and indicate that dual inhibition of mTOR and miRNA is a promising therapeutic strategy to overcome the resistance of mTOR-related cancer treatment.

Keywords:  PI3K/AKT/mTOR; PTEN; miRNA

DOI:  https://doi.org/10.3724/abbs.2022159
Heliyon. 2022 Oct;8(10): e11005

Allicin promotes autophagy and ferroptosis in esophageal squamous cell carcinoma by activating AMPK/mTOR signaling.

Zhanfang Guo, Yanjiao Zhang.

  The antitumor effects of allicin have been demonstrated in various cancers. However, whether allicin improves esophageal squamous cell carcinoma (ESCC) has not yet been explored. The present study aimed to explore the function and underlying mechanism of action of allicin in ESCC treatment. Our data showed that allicin significantly suppressed ESCC cell proliferation in a dose- and time-dependent manner. A green fluorescent protein-light chain 3 (LC3) transfection assay showed that autophagosomes were elevated in ESCC cells treated with allicin compared with control ESCC cells and that 3-methyladenine (an autophagy inhibitor) reversed allicin-induced LC3 puncta. Furthermore, allicin significantly elevated the ratio of LC3II/LC3I but decreased p62 expression in ESCC cells. Allicin also increased adenosine monophosphate-activated protein kinase (AMPK) phosphorylation but decreased that of the mechanistic target of rapamycin kinase (mTOR), which then induced the elevation of autophagy-related 5 and autophagy-related 7 proteins in ESCC cells. Furthermore, allicin treatment increased the expression of nuclear receptor coactivator 4 (a selective cargo receptor) but suppressed the expression of ferritin heavy chain 1 (the major intracellular iron-storage protein) in ESCC cells and elevated malondialdehyde and Fe2+ production levels. In vivo assays showed that allicin significantly decreased tumor weight and volume. In summary, allicin may induce cell death in ESCC cells by activating AMPK/mTOR-mediated autophagy and ferroptosis. Therefore, allicin may have excellent potential for use in the treatment of ESCC.

Keywords:  AMPK/mTOR signaling; Autophagy; Esophageal squamous cell carcinoma; Ferroptosis

DOI:  https://doi.org/10.1016/j.heliyon.2022.e11005
Front Pharmacol. 2022 ;13 1015784

Electroacupuncture induces weight loss by regulating tuberous sclerosis complex 1-mammalian target of rapamycin methylation and hypothalamic autophagy in high-fat diet-induced obese rats.

Junpeng Yao, Xiangyun Yan, Xianjun Xiao, Xi You, Yanqiu Li, Yuqing Yang, Wei Zhang, Ying Li.

  Background: Obesity can be caused by abnormalities of hypothalamic autophagy, which is closely regulated by the epigenetic modification of TSC1-mTOR. However, whether the weight-reducing effect of EA may relate to the modification of TSC1-mTOR methylation and hypothalamic autophagy remain unclear. This study was conducted to reveal the possible mechanism by which EA reduces BW by measuring the levels of TSC1-mTOR methylation and hypothalamic autophagy-related components. Methods: The weight-reducing effect of EA was investigated in high-fat diet (HFD)-induced obese (DIO) rats by monitoring the BW, food consumption, and epididymal white adipose tissue (eWAT)/BW ratio. Hematoxylin and eosin staining was performed for morphological evaluation of eWAT. Immunofluorescence was utilized to observe the localization of LC3 in the hypothalamus. The expressions of autophagy components (Beclin-1, LC3, and p62) and mTOR signaling (mTOR, p-mTOR, p70S6K, and p-p70S6K) were assessed by western blot. The methylation rate of the TSC1 promoter was detected by bisulfite genomic sequencing. Results: Treatment with EA significantly reduced the BW, food consumption, and eWAT/BW ratio; attenuated the morphological alternations in the adipocytes of DIO rats. While HFD downregulated the expression levels of Beclin-1 and LC3 and upregulated those of p62, these changes were normalized by EA treatment. EA markedly decreased the methylation rate of the TSC1 gene promoter and suppressed the protein expressions of mTOR, p-mTOR, p70S6K, and p-p70S6K in the hypothalamus. Conclusion: EA could reduce BW and fat accumulation in DIO rats. This ameliorative effect of EA may be associated with its demethylation effect on TSC1-mTOR and regulation of autophagy in the hypothalamus.

Keywords:  autophagy; electroacupuncture; mammalian target of rapamycin; methylation; obesity; tuberous sclerosis complex 1

DOI:  https://doi.org/10.3389/fphar.2022.1015784
Nat Commun. 2022 Nov 03. 13(1): 6589

Spatially resolved phosphoproteomics reveals fibroblast growth factor receptor recycling-driven regulation of autophagy and survival.

Joanne Watson, Harriet R Ferguson, Rosie M Brady, Jennifer Ferguson, Paul Fullwood, Hanyi Mo, Katherine H Bexley, David Knight, Gareth Howell, Jean-Marc Schwartz, Michael P Smith, Chiara Francavilla.

Receptor Tyrosine Kinase (RTK) endocytosis-dependent signalling drives cell proliferation and motility during development and adult homeostasis, but is dysregulated in diseases, including cancer. The recruitment of RTK signalling partners during endocytosis, specifically during recycling to the plasma membrane, is still unknown. Focusing on Fibroblast Growth Factor Receptor 2b (FGFR2b) recycling, we reveal FGFR signalling partners proximal to recycling endosomes by developing a Spatially Resolved Phosphoproteomics (SRP) approach based on APEX2-driven biotinylation followed by phosphorylated peptides enrichment. Combining this with traditional phosphoproteomics, bioinformatics, and targeted assays, we uncover that FGFR2b stimulated by its recycling ligand FGF10 activates mTOR-dependent signalling and ULK1 at the recycling endosomes, leading to autophagy suppression and cell survival. This adds to the growing importance of RTK recycling in orchestrating cell fate and suggests a therapeutically targetable vulnerability in ligand-responsive cancer cells. Integrating SRP with other systems biology approaches provides a powerful tool to spatially resolve cellular signalling.

DOI: https://doi.org/10.1038/s41467-022-34298-2
Stroke. 2022 Nov 02.

mTOR (Mammalian Target of Rapamycin): Hitting the Bull's Eye for Enhancing Neurogenesis After Cerebral Ischemia?

Jiale Gao, Mingjiang Yao, Dennis Chang, Jianxun Liu.

  Ischemic stroke remains a leading cause of morbidity and disability around the world. The sequelae of serious neurological damage are irreversible due to body's own limited repair capacity. However, endogenous neurogenesis induced by cerebral ischemia plays a critical role in the repair and regeneration of impaired neural cells after ischemic brain injury. mTOR (mammalian target of rapamycin) kinase has been suggested to regulate neural stem cells ability to self-renew and differentiate into proliferative daughter cells, thus leading to improved cell growth, proliferation, and survival. In this review, we summarized the current evidence to support that mTOR signaling pathways may enhance neurogenesis, angiogenesis, and synaptic plasticity following cerebral ischemia, which could highlight the potential of mTOR to be a viable therapeutic target for the treatment of ischemic brain injury.

Keywords:  angiogenesis; cerebral ischemia; mTOR; neurogenesis; synaptic plasticity

DOI:  https://doi.org/10.1161/STROKEAHA.122.040376