bims-auttor Biomed News
on Autophagy and mTOR
Issue of 2019‒11‒03
eleven papers selected by
Viktor Korolchuk, Newcastle University

  1. J Mol Biol. 2019 Oct 22. pii: S0022-2836(19)30609-6. [Epub ahead of print]
      Neurons are electrically excitable, post-mitotic cells that perform sensory, relaying, and motor functions. Because of their unique morphological and functional specialization, cells of this type are sensitive to the stress caused by accumulation of misfolded proteins or damaged organelles. Autophagy is the fundamental mechanism that ensures sequestration of cytosolic material and its subsequent degradation in lysosomes of eukaryotic cells, thereby providing cell-autonomous nutrients and removing harmful cargos. Strikingly, mice and flies lacking functional autophagy develop early-onset progressive neurodegeneration. Like in human neurodegenerative diseases (NDDs) - Alzheimer's disease, Parkinson's disease, frontotemporal dementia, Huntingtin's disease, and amyotrophic lateral sclerosis - characteristic protein aggregates observed in autophagy-deficient neurons in the animal models are indicators of the ongoing neuronal pathology. A number of selective autophagy receptors (SARs) have been characterized that interact both with the cargo and components of the autophagic machinery, thus providing the molecular basis for selective degradation of sizable cytosolic components. Interference with autophagy in experimental models, but also during the pathological vagaries in neurons, will thus have far-reaching consequences for a range of selective autophagy pathways critical for the normal functioning of the nervous system. Here, we review the key principles behind the selective autophagy and discuss how the SARs may be involved in the pathogenesis of NDDs. Using recently published examples, we also examine the emerging role of less well studied selective autophagy pathways in neuronal health and disease. We conclude by discussing targeting selective autophagy as an emerging therapeutic modality in NDDs.
    Keywords:  LC3/GABARAPs; SAR; aggregates; neurodegeneration; selective autophagy
  2. Cell Res. 2019 Oct 30.
      Macroautophagy/autophagy defines an evolutionarily conserved catabolic process that targets cytoplasmic components for lysosomal degradation. The process of autophagy from initiation to closure is tightly executed and controlled by the concerted action of autophagy-related (Atg) proteins. Although substantial progress has been made in characterizing transcriptional and post-translational regulation of ATG/Atg genes/proteins, little is known about the translational control of autophagy. Here we report that Psp2, an RGG motif protein, positively regulates autophagy through promoting the translation of Atg1 and Atg13, two proteins that are crucial in the initiation of autophagy. During nitrogen starvation conditions, Psp2 interacts with the 5' UTR of ATG1 and ATG13 transcripts in an RGG motif-dependent manner and with eIF4E and eIF4G2, components of the translation initiation machinery, to regulate the translation of these transcripts. Deletion of the PSP2 gene leads to a decrease in the synthesis of Atg1 and Atg13, which correlates with reduced autophagy activity and cell survival. Furthermore, deactivation of the methyltransferase Hmt1 constitutes a molecular switch that regulates Psp2 arginine methylation status as well as its mRNA binding activity in response to starvation. These results reveal a novel mechanism by which Atg proteins become upregulated to fulfill the increased demands of autophagy activity as part of translational reprogramming during stress conditions, and help explain how ATG genes bypass the general block in protein translation that occurs during starvation.
  3. Immunity. 2019 Oct 17. pii: S1074-7613(19)30418-2. [Epub ahead of print]
      Regulatory T (Treg) cells are critical mediators of immune tolerance whose activity depends upon T cell receptor (TCR) and mTORC1 kinase signaling, but the mechanisms that dictate functional activation of these pathways are incompletely understood. Here, we showed that amino acids license Treg cell function by priming and sustaining TCR-induced mTORC1 activity. mTORC1 activation was induced by amino acids, especially arginine and leucine, accompanied by the dynamic lysosomal localization of the mTOR and Tsc complexes. Rag and Rheb GTPases were central regulators of amino acid-dependent mTORC1 activation in effector Treg (eTreg) cells. Mice bearing RagA-RagB- or Rheb1-Rheb2-deficient Treg cells developed a fatal autoimmune disease and had reduced eTreg cell accumulation and function. RagA-RagB regulated mitochondrial and lysosomal fitness, while Rheb1-Rheb2 enforced eTreg cell suppressive gene signature. Together, these findings reveal a crucial requirement of amino acid signaling for licensing and sustaining mTORC1 activation and functional programming of Treg cells.
    Keywords:  RagA; RagB; Rheb; Treg cells; amino acids; autoimmunity; eTreg cells; mTOR; metabolism
  4. Cells. 2019 Oct 10. pii: E1233. [Epub ahead of print]8(10):
      Dysfunctional autophagy or ubiquitin-proteasome system (UPS) are suggested to underlie abnormal protein aggregation in neurodegenerative diseases. Frontotemporal dementia (FTD) and amyotrophic lateral sclerosis (ALS)-associated C9orf72 is implicated in autophagy, but whether it activates or inhibits autophagy is partially controversial. Here, we utilized knockdown or overexpression of C9orf72 in mouse N2a neuroblastoma cells or cultured neurons to elucidate the potential role of C9orf72 proteins in autophagy and UPS. Induction of autophagy in C9orf72 knockdown N2a cells led to decreased LC3BI to LC3BII conversion, p62 degradation, and formation of LC3-containing autophagosomes, suggesting compromised autophagy. Proteasomal activity was slightly decreased. No changes in autophagy nor proteasomal activity in C9orf72-overexpressing N2a cells were observed. However, in these cells, autophagy induction by serum starvation or rapamycin led to significantly decreased C9orf72 levels. The decreased levels of C9orf72 in serum-starved N2a cells were restored by the proteasomal inhibitor lactacystin, but not by the autophagy inhibitor bafilomycin A1 (BafA1) treatment. These data suggest that C9orf72 undergoes proteasomal degradation in N2a cells during autophagy. Lactacystin significantly elevated C9orf72 levels in N2a cells and neurons, further suggesting UPS-mediated regulation. In rapamycin and BafA1-treated neurons, C9orf72 levels were significantly increased. Altogether, these findings corroborate the previously suggested regulatory role for C9orf72 in autophagy and suggest cell type-dependent regulation of C9orf72 levels via UPS and/or autophagy.
    Keywords:  C9orf72; amyotrophic lateral sclerosis; autophagy; frontotemporal dementia; proteasomal degradation; ubiquitin-proteasome system
  5. Cell Mol Life Sci. 2019 Oct 29.
      Cholesterol accumulation in late endosomes is a prevailing phenotype of Niemann-Pick type C1 (NPC1) mutant cells. Likewise, annexin A6 (AnxA6) overexpression induces a phenotype reminiscent of NPC1 mutant cells. Here, we demonstrate that this cellular cholesterol imbalance is due to AnxA6 promoting Rab7 inactivation via TBC1D15, a Rab7-GAP. In NPC1 mutant cells, AnxA6 depletion and eventual Rab7 activation was associated with peripheral distribution and increased mobility of late endosomes. This was accompanied by an enhanced lipid accumulation in lipid droplets in an acyl-CoA:cholesterol acyltransferase (ACAT)-dependent manner. Moreover, in AnxA6-deficient NPC1 mutant cells, Rab7-mediated rescue of late endosome-cholesterol export required the StAR-related lipid transfer domain-3 (StARD3) protein. Electron microscopy revealed a significant increase of membrane contact sites (MCS) between late endosomes and ER in NPC1 mutant cells lacking AnxA6, suggesting late endosome-cholesterol transfer to the ER via Rab7 and StARD3-dependent MCS formation. This study identifies AnxA6 as a novel gatekeeper that controls cellular distribution of late endosome-cholesterol via regulation of a Rab7-GAP and MCS formation.
    Keywords:  Annexin A6; Cholesterol; Late endosomes; Membrane contact sites; NPC1; Rab7
  6. Science. 2019 Oct 31. pii: eaax0364. [Epub ahead of print]
      The tumor suppressor folliculin (FLCN) enables nutrient-dependent activation of the mechanistic target of rapamycin complex 1 (mTORC1) protein kinase via its guanosine triphosphatase (GTPase) Activating Protein (GAP) activity toward the GTPase RagC. Concomitant with mTORC1 inactivation by starvation, FLCN relocalizes from the cytosol to lysosomes. To determine the lysosomal function of FLCN, we reconstituted the lysosomal FLCN complex (LFC) containing FLCN, its partner FLCN-interacting protein 2 (FNIP2), the RagAGDP:RagCGTP GTPases as they exist in the starved state with their lysosomal anchor Ragulator complex, and determined its cryo-EM structure to 3.6 Å. The RagC-GAP activity of FLCN was inhibited within LFC, due to displacement of a catalytically required Arginine in FLCN from the RagC nucleotide. Disassembly of the LFC and release of the RagC-GAP activity of FLCN enabled mTORC1-dependent regulation of the master regulator of lysosomal biogenesis, transcription factor E3, implicating the LFC as a checkpoint in mTORC1 signaling.
  7. Cell Rep. 2019 Oct 29. pii: S2211-1247(19)31235-5. [Epub ahead of print]29(5): 1311-1322.e5
      The mechanistic target of rapamycin complex 2 (mTORC2) coordinates cell proliferation, survival, and metabolism with environmental inputs, yet how extracellular stimuli such as growth factors (GFs) activate mTORC2 remains enigmatic. Here we demonstrate that in human endothelial cells, activation of mTORC2 signaling by GFs is mediated by transmembrane cell adhesion protein CD146. Upon GF stimulation, the cytoplasmic tail of CD146 is phosphorylated, which permits its positively charged, juxtamembrane KKGK motif to interact with Rictor, the defining subunit of mTORC2. The formation of the CD146-Rictor/mTORC2 complex protects Rictor from ubiquitin-proteasome-mediated degradation, thereby specifically upregulating mTORC2 activity with no intervention of the PI3K and mTORC1 pathways. This CD146-mediated mTORC2 activation in response to GF stimulation promotes cell proliferation and survival. Therefore, our findings identify a molecular mechanism by which extracellular stimuli regulate mTORC2 activity, linking environmental cues with mTORC2 regulation.
    Keywords:  CD146; PI3K; Rictor; cell proliferation; mTORC1; mTORC2; signal transduction
  8. Nat Commun. 2019 Nov 01. 10(1): 5017
      Autophagy is an intracellular catabolic process prominent in starvation, aging and disease. Neuronal autophagy is particularly important, as it affects the development and function of the nervous system, and is heavily implicated in neurodegenerative disease. Nonetheless, how autophagy is regulated in neurons remains poorly understood. Using an unbiased proteomics approach, we demonstrate that the primary initiator of autophagy, the UNC-51/ULK kinase, is negatively regulated by the ubiquitin ligase RPM-1. RPM-1 ubiquitin ligase activity restricts UNC-51 and autophagosome formation within specific axonal compartments, and exerts effects broadly across the nervous system. By restraining UNC-51 activity, RPM-1 inhibits autophagosome formation to affect axon termination, synapse maintenance and behavioral habituation. These results demonstrate how UNC-51 and autophagy are regulated subcellularly in axons, and unveils a mechanism for restricting initiation of autophagy across the nervous system. Our findings have important implications beyond nervous system development, given growing links between altered autophagy regulation and neurodegenerative diseases.
  9. JCI Insight. 2019 Oct 29. pii: 129760. [Epub ahead of print]
      Mitochondrial quality control (MQC) is crucial for regulating central nervous system homeostasis and its disruption has been implicated in the pathogenesis of some of the most common neurodegenerative diseases. In healthy tissues, the maintenance of MQC depends upon an exquisite balance between mitophagy (removal of damaged mitochondria by autophagy) and biogenesis (de-novo synthesis of mitochondria). Here, we show that mitophagy is disrupted in diabetic retinopathy (DR) and decoupled from mitochondrial biogenesis during the progression of the disease. Diabetic retinas from human post-mortem donors and experimental mice exhibit a net loss of mitochondrial contents during the early stages of the disease process. Using novel diabetic mitophagy-reporter mice (mitoQC-Ins2Akita) alongside pMitoTimer (a molecular clock to address mitochondrial-age dynamics), we demonstrate that mitochondrial loss arose due to an inability of mitochondrial biogenesis to compensate for diabetes-exacerbated mitophagy. However, as diabetes duration increases, Pink1-dependent mitophagy deteriorates, leading to the build-up of mitochondria primed for degradation in DR. Impairment of mitophagy during prolonged diabetes is linked with the development of retinal senescence, a phenotype that blunted hyperglycaemia-induced mitophagy in mitoQC primary Müller cells. Our findings suggest that normalizing mitochondrial turnover may preserve MQC and provide novel therapeutic options for the management of DR-associated complications.
    Keywords:  Autophagy; Mitochondria; Neuroscience; Ophthalmology; Retinopathy
  10. Cell Death Differ. 2019 Oct 28.
      Autophagy regulates the degradation of unnecessary or dysfunctional cellular components. This catabolic process requires the formation of a double-membrane vesicle, the autophagosome, that engulfs the cytosolic material and delivers it to the lysosome. Substrate specificity is achieved by autophagy receptors, which are characterized by the presence of at least one LC3-interaction region (LIR) or GABARAP-interaction motif (GIM). Only recently, several receptors that mediate the specific degradation of endoplasmic reticulum (ER) components via autophagy have been identified (the process known as ER-phagy or reticulophagy). Here, we give an update on the current knowledge about the role of ER-phagy receptors in health and disease.
  11. Elife. 2019 Oct 30. pii: e50416. [Epub ahead of print]8
      Mutations that activate LRRK2 protein kinase cause Parkinson's disease. LRRK2 phosphorylates a subset of Rab GTPases within their Switch-II motif controlling interaction with effectors. An siRNA screen of all human protein phosphatases revealed that a poorly studied protein phosphatase, PPM1H, counteracts LRRK2 signaling by specifically dephosphorylating Rab proteins. PPM1H knockout increased endogenous Rab phosphorylation and inhibited Rab dephosphorylation in human A549 cells. Overexpression of PPM1H suppressed LRRK2-mediated Rab phosphorylation. PPM1H also efficiently and directly dephosphorylated Rab8A in biochemical studies. A 'substrate-trapping' PPM1H mutant (Asp288Ala) binds with high affinity to endogenous, LRRK2-phosphorylated Rab proteins, thereby blocking dephosphorylation seen upon addition of LRRK2 inhibitors. PPM1H is localized to the Golgi and its knockdown suppresses primary cilia formation, similar to pathogenic LRRK2. Thus, PPM1H acts as a key modulator of LRRK2 signaling by controlling dephosphorylation of Rab proteins. PPM1H activity enhancers could offer a new therapeutic approach to prevent or treat Parkinson's disease.
    Keywords:  biochemistry; chemical biology; human; mouse