The mechanistic target of rapamycin (mTOR) coordinates eukaryotic cell growth and metabolism with environmental inputs, including nutrients and growth factors. Extensive research over the past two decades has established a central role for mTOR in regulating many fundamental cell processes, from protein synthesis to autophagy, and deregulated mTOR signaling is implicated in the progression of cancer and diabetes, as well as the aging process. Here, we review recent advances in our understanding of mTOR function, regulation, and importance in mammalian physiology. We also highlight how the mTOR signaling network contributes to human disease and discuss the current and future prospects for therapeutically targeting mTOR in the clinic.

mTOR Signaling in Growth, Metabolism, and Disease

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

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

https://www.cell.com/cell/pdf/S0092-8674(20)31606-8.pdf

Hallmarks of health.

In 2008 we published the first set of guidelines for standardizing research in autophagy. Since then, research on this topic has continued to accelerate, and many new scientists have entered the field. Our knowledge base and relevant new technologies have also been expanding. Accordingly, it is important to update these guidelines for monitoring autophagy in different organisms. Various reviews have described the range of assays that have been used for this purpose. Nevertheless, there continues to be confusion regarding acceptable methods to measure autophagy, especially in multicellular eukaryotes. A key point that needs to be emphasized is that there is a difference between measurements that monitor the numbers or volume of autophagic elements (e.g., autophagosomes or autolysosomes) at any stage of the autophagic process vs. those that measure flux through the autophagy pathway (i.e., the complete process); thus, a block in macroautophagy that results in autophagosome accumulation needs to be differentiated from stimuli that result in increased autophagic activity, defined as increased autophagy induction coupled with increased delivery to, and degradation within, lysosomes (in most higher eukaryotes and some protists such as Dictyostelium) or the vacuole (in plants and fungi). In other words, it is especially important that investigators new to the field understand that the appearance of more autophagosomes does not necessarily equate with more autophagy. In fact, in many cases, autophagosomes accumulate because of a block in trafficking to lysosomes without a concomitant change in autophagosome biogenesis, whereas an increase in autolysosomes may reflect a reduction in degradative activity. Here, we present a set of guidelines for the selection and interpretation of methods for use by investigators who aim to examine macroautophagy and related processes, as well as for reviewers who need to provide realistic and reasonable critiques of papers that are focused on these processes. These guidelines are not meant to be a formulaic set of rules, because the appropriate assays depend in part on the question being asked and the system being used. In addition, we emphasize that no individual assay is guaranteed to be the most appropriate one in every situation, and we strongly recommend the use of multiple assays to monitor autophagy. In these guidelines, we consider these various methods of assessing autophagy and what information can, or cannot, be obtained from them. Finally, by discussing the merits and limits of particular autophagy assays, we hope to encourage technical innovation in the field.

Guidelines for the use and interpretatoin of assays for monitoring autophagy

Metabolic reprogramming, including enhanced biosynthesis of macromolecules, altered energy metabolism, and maintenance of redox homeostasis, is considered a hallmark of cancer, sustaining cancer cell growth. Multiple signaling pathways, transcription factors and metabolic enzymes participate in the modulation of cancer metabolism and thus, metabolic reprogramming is a highly complex process. Recent studies have observed that ubiquitination and deubiquitination are involved in the regulation of metabolic reprogramming in cancer cells. As one of the most important type of post-translational modifications, ubiquitination is a multistep enzymatic process, involved in diverse cellular biological activities. Dysregulation of ubiquitination and deubiquitination contributes to various disease, including cancer. Here, we discuss the role of ubiquitination and deubiquitination in the regulation of cancer metabolism, which is aimed at highlighting the importance of this post-translational modification in metabolic reprogramming and supporting the development of new therapeutic approaches for cancer treatment.

/pdf/the-role-of-ubiquitination-and-deubiquitination-in-cancer-359wzbrd1z.pdf

The role of ubiquitination and deubiquitination in cancer metabolism.

Lysosomal membrane permeabilization (LMP) is observed under many pathological conditions, leading to cellular dysfunction and death. However, the mechanisms by which lysosomal membranes become leaky in vivo are not clear. Our data demonstrate that LMP occurs in neurons following controlled cortical impact induced (CCI) traumatic brain injury (TBI) in mice, leading to impaired macroautophagy (autophagy) and neuronal cell death. Comparison of LC-MS/MS lysosomal membrane lipid profiles from TBI and sham animals suggested a role for PLA2G4A/cPLA2 (phospholipase A2, group IVA [cytosolic, calcium-dependent]) in TBI-induced LMP. Activation of PLA2G4A caused LMP and inhibition of autophagy flux in cell lines and primary neurons. In vivo pharmacological inhibition of PLA2G4A attenuated TBI-induced LMP, as well as subsequent impairment of autophagy and neuronal loss, and was associated with improved neurological outcomes. Inhibition of PLA2G4A in vitro limited amyloid-β-induced LMP and inhibition of autophagy. Together, our data indicate that PLA2G4A -mediated lysosomal membrane damage is involved in neuronal cell death following CCI-induced TBI and potentially in other neurodegenerative disorders.Abbreviations: AACOCF3, arachidonyl trifluoromethyl ketone; ACTB/β-actin, actin, beta; AD, Alzheimer disease; ATG5, autophagy related 5; ATG7, autophagy related 7; ATG12, autophagy related 12; BECN1, beclin 1, autophagy related; C1P, ceramide-1-phosphate; CCI, controlled cortical impact; CTSD, cathepsin D; CTSL, cathepsin L; GFP, green fluorescent protein; IF, immunofluorescence; LAMP1, lysosomal-associated membrane protein 1; LAMP2, lysosomal-associated membrane protein 2; LC-MS/MS, liquid chromatography-tandem mass spectrometry; LMP, Lysosomal membrane permeabilization; LPC, lysophosphatidylcholine; LPE, lysophosphatidylethanolamine; MAP1LC3/LC3, microtuble-associated protein 1 light chain 3; NAGLU, alpha-N-acetylglucosaminidase (Sanfilippo disease IIIB); PC, diacyl glycerophosphatidylcholine; PE, diacyl glycerophosphatidylethanolamine; PE-O, plasmanyl glycerophosphatidylethanolamine; PE-P, plasmenyl glycerophosphatidylethanolamine; PLA2G4A/cPLA2, phospholipase A2, group IVA (cytosolic, calcium-dependent); RBFOX3, RNA binding protein, fox-1 homolog (C. elegans) 3; RFP, red fluorescent protein; ROS, reactive oxygen species; SQSTM1, sequestosome 1; TUBA1/α-tubulin, tubulin, alpha; TBI, traumatic brain injury; TFEB, transcription factor EB; ULK1, unc-51 like kinase 1.

https://tandfonline.com/doi/pdf/10.1080/15548627.2019.1628538

PLA2G4A/cPLA2-mediated lysosomal membrane damage leads to inhibition of autophagy and neurodegeneration after brain trauma.

Bi-allelic GBA1 mutations cause Gaucher's disease (GD), the most common lysosomal storage disorder. Neuronopathic manifestations in GD include neurodegeneration, which can be severe and rapidly progressive. GBA1 mutations are also the most frequent genetic risk factors for Parkinson's disease. Dysfunction of the autophagy-lysosomal pathway represents a key pathogenic event in GBA1-associated neurodegeneration. Using an induced pluripotent stem cell (iPSC) model of GD, we previously demonstrated that lysosomal alterations in GD neurons are linked to dysfunction of the transcription factor EB (TFEB). TFEB controls the coordinated expression of autophagy and lysosomal genes and is negatively regulated by the mammalian target of rapamycin complex 1 (mTORC1). To further investigate the mechanism of autophagy-lysosomal pathway dysfunction in neuronopathic GD, we examined mTORC1 kinase activity in GD iPSC neuronal progenitors and differentiated neurons. We found that mTORC1 is hyperactive in GD cells as evidenced by increased phosphorylation of its downstream protein substrates. We also found that pharmacological inhibition of glucosylceramide synthase enzyme reversed mTORC1 hyperactivation, suggesting that increased mTORC1 activity is mediated by the abnormal accumulation of glycosphingolipids in the mutant cells. Treatment with the mTOR inhibitor Torin1 upregulated lysosomal biogenesis and enhanced autophagic clearance in GD neurons, confirming that lysosomal dysfunction is mediated by mTOR hyperactivation. Further analysis demonstrated that increased TFEB phosphorylation by mTORC1 results in decreased TFEB stability in GD cells. Our study uncovers a new mechanism contributing to autophagy-lysosomal pathway dysfunction in GD, and identifies the mTOR complex as a potential therapeutic target for treatment of GBA1-associated neurodegeneration.

mTOR hyperactivity mediates lysosomal dysfunction in Gaucher's disease iPSC-neuronal cells.

Recent studies indicate a causative relationship between defects in autophagy and dopaminergic neuron degeneration in Parkinson disease (PD). However, it is not fully understood how autophagy is regulated in the context of PD. Here we identify USP24 (ubiquitin specific peptidase 24), a gene located in the PARK10 (Parkinson disease 10 [susceptibility]) locus associated with late onset PD, as a novel negative regulator of autophagy. Our data indicate that USP24 regulates autophagy by affecting ubiquitination and stability of the ULK1 protein. Knockdown of USP24 in cell lines and in human induced-pluripotent stem cells (iPSC) differentiated into dopaminergic neurons resulted in elevated ULK1 protein levels and increased autophagy flux in a manner independent of MTORC1 but dependent on the class III phosphatidylinositol 3-kinase (PtdIns3K) activity. Surprisingly, USP24 knockdown also improved neurite extension and/or maintenance in aged iPSC-derived dopaminergic neurons. Furthermore, we observed elevated levels of USP24 in the substantia nigra of a subpopulation of idiopathic PD patients, suggesting that USP24 may negatively regulate autophagy in PD.Abbreviations: Bafilomycin/BafA: bafilomycin A1; DUB: deubiquitinating enzyme; iPSC: induced pluripotent stem cells; MTOR: mechanistic target of rapamycin kinase; MTORC1: MTOR complex 1; nt: non-targeting; PD: Parkinson disease; p-ATG13: phospho-ATG13; PtdIns3P: phosphatidylinositol 3-phosphate; RPS6: ribosomal protein S6; SNPs: single nucleotide polymorphisms; TH: tyrosine hydroxylase; USP24: ubiquitin specific peptidase 24.

https://www.tandfonline.com/doi/pdf/10.1080/15548627.2019.1598754

The PARK10 gene USP24 is a negative regulator of autophagy and ULK1 protein stability.

Mitochondrial dysfunction is believed to be a major contributor to Parkinson’s disease (PD) pathogenesis. It was first linked to PD through the study of MPTP (1methyl-4-phenyl-1,2,3,6-tetrahydropyridine), a toxin that caused an outbreak of acute degenerative parkinsonism among intravenous drug users in the early 1980s. Metabolites of MPTP inhibit mitochondrial complex I (CI), the entry point to oxidative phosphorylation (OXPHOS) through NADH oxidation, and severe CI deficiency from chemical or genetic inhibition is sufficient to cause parkinsonism in animal models. Notably, CI expression and activity is (moderately) decreased in idiopathic PD, perhaps because of decreased expression of CI (and other OXPHOS subunits) from mitochondrial DNA (mtDNA). A second link between mitochondrial dysfunction and PD stems from functional studies of recessive PD genes PRKN (coding for Parkin) and PINK1. Their gene products form an evolutionarily conserved mitochondrial quality control pathway that selectively isolates and then degrades impaired mitochondria in lysosomes through autophagy (reviewed in Narendra). Prkn knockout (KO) mice have mild OXPHOS dysfunction, but do not develop parkinsonism. However, crossing Prkn KO mice to a model with excessive mtDNA mutations results in DA neuron loss and parkinsonism. This points to a synergistic relationship between loss of the PINK1/Parkin mitochondrial pathway quality control pathway and mtDNA mutations—and suggests mtDNA as a potential point of convergence between these lines of investigation. In this issue of Movement Disorders, Wasner et al attempt to bridge findings in PRKN KO model systems with induced pluripotent stem cell iPSC derived DA (iDA) neurons and single nuclei transcriptomics from PRKN-PD patients, exploring the connections among NADH reductive stress, mtDNA dyshomeostasis, and neuroinflammation. The authors first observed a moderate decrease in mitochondrial biogenesis in the absence of PRKN function. Levels of PGC1-α and NRF1, which transcriptionally regulate mitochondrial biogenesis, were decreased, as were their targets, the mitochondrial transcription factors TFAM and TFB2M. Consistently, mtDNA transcript levels were also decreased, and a biomarker of mtDNA expression, the 7S DNA:MT-ND1 ratio, was lower in PRKN-deficient cell lines and the midbrain of a patient with PRKN-PD. Wasner et al next explored the mechanism of decreased mitochondrial biogenesis in the absence of Parkin function. Parkin was previously suggested to activate PGC1-α through degradation of a proposed PGC1-α negative regulator, ZNF746. However, Wasner et al was not able to replicate this finding. Instead, they suggest that PGC1-α is regulated indirectly by Parkin through its effects on the metabolite NAD. Lowered NAD suppresses PGC1-α signaling by decreasing the activity of SIRT1, an NADdependent deacetylase. Although Wasner et al do not directly measure NAD and NADH, a recent study found that the NAD/NADH ratio is decreased in PRKN KO and patient iDA neurons, and both studies found an increase in the lactate/pyruvate ratio, which is metabolically coupled to the NAD/NADH ratio. Based on these findings, the authors suggest that Parkin deficiency may decrease mitochondrial biogenesis through lowered NAD, via the SIRT1–PGC1-α/NRF1–TFAM signaling axis. The source of the low NAD/NADH ratio in PRKNdeficient cells is not directly addressed experimentally by Wasner et al, but is plausibly a moderate decrease in OXPHOS resulting from Parkin deficiency. Notably, Wasner et al observed decreased CI activity in PRKN KO SH-SY5Y cells, consistent with an earlier study that found decreased CI activity in the Prkn KO mouse brain. CI is primarily responsible for oxidizing NADH to NAD in mitochondria and so maintains the high NAD/NADH ratio needed to drive metabolic © 2022 International Parkinson and Movement Disorder Society.

Julia A. Thayer

Papers

PLA2G4A/cPLA2-mediated lysosomal membrane damage leads to inhibition of autophagy and neurodegeneration after brain trauma.

mTOR hyperactivity mediates lysosomal dysfunction in Gaucher's disease iPSC-neuronal cells.

The PARK10 gene USP24 is a negative regulator of autophagy and ULK1 protein stability.

Midbrain on Fire: mtDNA Ignites Neuroinflammation in PRKN‐P