bims-auttor Biomed News
on Autophagy and mTOR
Issue of 2021‒07‒18
forty-five papers selected by
Viktor Korolchuk
Newcastle University

  1. Adv Exp Med Biol. 2021 ;1332 51-66
      Autophagy is a dynamic process in which the eukaryotic cells break down intracellular components by lysosomal degradation. Under the normal condition, the basal level of autophagy removes damaged organelles, misfolded proteins, or protein aggregates to keep cells in a homeostatic condition. Deprivation of nutrients (e.g., removal of amino acids) stimulates autophagy activity, promoting lysosomal degradation and the recycling of cellular components for cell survival. Importantly, insulin and amino acids are two main inhibitors of autophagy. They both activate the mTOR complex 1 (mTORC1) signaling pathway to inhibit the autophagy upstream of the uncoordinated-51 like kinase 1/2 (ULK1/2) complex that triggers autophagosome formation. In particular, insulin activates mTORC1 via the PI3K class I-AKT pathway; while amino acids activate mTORC1 either through the PI3K class III (hVps34) pathway or through a variety of amino acid sensors located in the cytosol or lysosomal membrane. These amino acid sensors control the translocation of mTORC1 from the cytosol to the lysosomal surface where mTORC1 is activated by Rheb GTPase, therefore regulating autophagy and the lysosomal protein degradation.
    Keywords:  Amino acids; Arginine; Autophagosome; Autophagy; Calcium/calmodulin-dependent protein kinase kinase; Leucine; Lysosome; Mammalian target of rapamycin complex 1; Rag GTPase; Rheb
  2. Int Rev Cell Mol Biol. 2021 ;pii: S1937-6448(21)00024-1. [Epub ahead of print]362 141-170
      Lysosomal calcium is emerging as a modulator of autophagy and lysosomal compartment, an obligatory partner to complete the autophagic pathway. A variety of specific signals such as nutrient deprivation or oxidative stress can trigger lysosomal calcium-mediated nuclear translocation of the transcription factor EB (TFEB), a master regulator of global lysosomal function. Also, lysosomal calcium can promote the formation of autophagosome vesicles (AVs) by a mechanism that requires the production of the phosphoinositide PI3P by the VPS34 autophagic complex and the activation of the energy-sensing kinase AMPK. Additionally, lysosomal calcium plays a role in membrane fusion and fission events involved in cellular processes such as endocytic maturation, autophagosome-lysosome fusion, lysosomal exocytosis, and lysosomal reformation upon autophagy completion. Lysosomal calcium-dependent functions are defective in cellular and animal models of the non-selective cation channel TRPML1, whose mutations in humans cause the neurodegenerative lysosomal storage disease mucolipidosis type IV (MLIV). Lysosomal calcium is not only acting as a positive regulator of autophagy, but it is also responsible for turning-off this process through the reactivation of the mTOR kinase during prolonged starvation. More recently, it has been described the role of lysosomal calcium on an elegant sequence of intracellular signaling events such as membrane repair, lysophagy, and lysosomal biogenesis upon the induction of different grades of lysosomal membrane damage. Here, we will discuss these novel findings that re-define the importance of the lysosome and lysosomal calcium signaling at regulating cellular metabolism.
    Keywords:  Autophagy; Calcineurin; LSDs; Lysosomal calcium; Lysosome; MLIV; TFEB; TPCs; TRPML1; mTORC1
  3. Autophagy. 2021 Jul 12. 1-3
      Atg8-family protein lipidation is the most commonly used marker for monitoring autophagy. During macroautophagy, Atg8-family proteins are specifically conjugated to phosphatidylethanolamine (PE) in forming, double-membrane autophagosomes. A distinct, non-canonical autophagy pathway also operates, characterized by the Conjugation of ATG8s to endolysosomal Single Membranes (CASM). In our new study, we show that CASM is associated with the alternative conjugation of Atg8-family proteins to phosphatidylserine (PS), and PE, in response to various cellular stimuli. We also discover differences in the regulation of conjugation to PE and PS by ATG4s, and altered dynamics between the two species. The identification of alternative Atg8-family protein PS lipidation opens up exciting new questions on the roles, regulation and biology of Atg8-family proteins during non-canonical autophagy.
    Keywords:  ATG4; ATG8; LC3-associated phagocytosis; endolysosome; lipidation; non-canonical autophagy; phosphatidylserine
  4. Adv Exp Med Biol. 2021 ;1208 3-16
      Autophagy is a general term for the process of the lysosomal degradation of intracellular components, a process occurring exclusively in eukaryotic cells. Based on the way that intracellular substrates are transported to lysosomes, autophagy in mammalian cells can be divided into three main types: macroautophagy, microautophagy, and chaperone-mediated autophagy (CMA). Each type has its unique molecular machinery and is tightly regulated by various cellular signals, helping cells adapt to a changing environment. Autophagy can also be divided into two categories based on cargo selectivity: selective autophagy and nonselective autophagy. Nonselective autophagy refers to the bulk transport of organelles or other cytoplasmic components to lysosomes, while selective autophagy refers to the degradation of a specific substrate. Autophagy plays an essential role in maintaining cellular homeostasis, and dysregulation of it may participate in the pathological process of many human diseases.
    Keywords:  Autophagy; Basic process; Selective autophagy; Types
  5. Nat Commun. 2021 07 12. 12(1): 4245
      Tuberous Sclerosis Complex (TSC) is caused by TSC1 or TSC2 mutations, resulting in hyperactivation of the mechanistic target of rapamycin complex 1 (mTORC1). Transcription factor EB (TFEB), a master regulator of lysosome biogenesis, is negatively regulated by mTORC1 through a RAG GTPase-dependent phosphorylation. Here we show that lysosomal biogenesis is increased in TSC-associated renal tumors, pulmonary lymphangioleiomyomatosis, kidneys from Tsc2+/- mice, and TSC1/2-deficient cells via a TFEB-dependent mechanism. Interestingly, in TSC1/2-deficient cells, TFEB is hypo-phosphorylated at mTORC1-dependent sites, indicating that mTORC1 is unable to phosphorylate TFEB in the absence of the TSC1/2 complex. Importantly, overexpression of folliculin (FLCN), a GTPase activating protein for RAGC, increases TFEB phosphorylation at the mTORC1 sites in TSC2-deficient cells. Overexpression of constitutively active RAGC is sufficient to relocalize TFEB to the cytoplasm. These findings establish the TSC proteins as critical regulators of lysosomal biogenesis via TFEB and RAGC and identify TFEB as a driver of the proliferation of TSC2-deficient cells.
  6. J Biol Chem. 2021 Jul 07. pii: S0021-9258(21)00742-0. [Epub ahead of print] 100942
      TBK1 responds to microbes to initiate cellular responses critical for host innate immune defense. We found previously that TBK1 phosphorylates mTOR (mechanistic target of rapamycin) on S2159 to increase mTOR complex 1 (mTORC1) signaling in response to the growth factor EGF and the viral dsRNA mimetic poly(I:C). mTORC1 and the less well studied mTORC2 respond to diverse cues to control cellular metabolism, proliferation, and survival. While TBK1 has been linked to Akt phosphorylation, a direct relationship between TBK1 and mTORC2, an Akt kinase, has not been described. By studying MEFs lacking TBK1, as well as MEFs, macrophages, and mice bearing an Mtor S2159A knock-in allele (MtorA/A) using in vitro kinase assays and cell-based approaches, we demonstrate here that TBK1 activates mTOR complex 2 (mTORC2) directly to increase Akt phosphorylation. We find that TBK1 and mTOR S2159 phosphorylation promote mTOR-dependent phosphorylation of Akt in response to several growth factors and poly(I:C). Mechanistically, TBK1 co-immunoprecipitates with mTORC2 and phosphorylates mTOR S2159 within mTORC2 in cells. Kinase assays demonstrate that TBK1 and mTOR S2159 phosphorylation increase mTORC2 intrinsic catalytic activity. Growth factors failed to activate TBK1 or increase mTOR S2159 phosphorylation in MEFs. Thus, basal TBK1 activity cooperates with growth factors in parallel to increase mTORC2 (and mTORC1) signaling. Collectively, these results reveal crosstalk between TBK1 and mTOR, key regulatory nodes within two major signaling networks. As TBK1 and mTOR contribute to tumorigenesis and metabolic disorders, these kinases may work together in a direct manner in a variety of physiological and pathological settings.
    Keywords:  Akt; TBK1; mTOR; mTORC2; phosphorylation
  7. Aging Cell. 2021 Jul 12. e13431
      The mechanistic target of rapamycin (mTOR) has gathered significant attention as a ubiquitously expressed multimeric kinase with key implications for cell growth, proliferation, and survival. This kinase forms the central core of two distinct complexes, mTORC1 and mTORC2, which share the ability of integrating environmental, nutritional, and hormonal cues but which regulate separate molecular pathways that result in different cellular responses. Particularly, mTORC1 has been described as a major negative regulator of endosomal biogenesis and autophagy, a catabolic process that degrades intracellular components and organelles within the lysosomes and is thought to play a key role in human health and disease. In contrast, the role of mTORC2 in the regulation of autophagy has been considerably less studied despite mounting evidence this complex may regulate autophagy in a different and perhaps complementary manner to that of mTORC1. Genetic ablation of unique subunits is currently being utilized to study the differential effects of the two mTOR complexes. RICTOR is the best-described subunit specific to mTORC2 and as such has become a useful tool for investigating the specific actions of this complex. The development of complex-specific inhibitors for mTORC2 is also an area of intense interest. Studies to date have demonstrated that mTORC1/2 complexes each signal to a variety of exclusive downstream molecules with distinct biological roles. Pinpointing the particular effects of these downstream effectors is crucial toward the development of novel therapies aimed at accurately modulating autophagy in the context of human aging and disease.
    Keywords:  AKT; FOXOs; SGK-1; autophagy; mTORC2
  8. Trends Mol Med. 2021 Jul 11. pii: S1471-4914(21)00179-9. [Epub ahead of print]
      The study of autophagy has grown exponentially over the past two decades, and significant progress has been made in our understanding of its mechanisms and physiological significance. However, its application to human diseases remains limited. Here, we summarize the current status of autophagy research, with a particular focus on human diseases.
    Keywords:  ATG genes; autophagic flux; macroautophagy; neurodegenerative disease; tumor growth
  9. Adv Exp Med Biol. 2021 ;1208 79-98
      Autophagy is a lysosome-dependent degradation process. During autophagy, cytoplasmic components are sequestered and catabolized to supply nutrition and energy under starvation conditions. Recent work has demonstrated that many cargos can be specifically recognized and then eliminated via the core mechanism of autophagy which is termed as selective autophagy. The cargo recognition program provides the basis for the specific degradation of selective autophagy; thus, the exploration of the interaction between the cargo and the receptor is the key for revealing the underlying mechanism. Also, receptor protein complexes are required in various selective autophagy subtypes which process and guide the cargo to the core mechanism. Ubiquitination and phosphorylation are the main methods to modulate the affinity of the receptor toward cargo. Although many key processes of selective autophagy subtypes have been discovered and intensively studied, the precise ways in which the mechanisms of cargo recognition function remain mostly elusive. A fuller mechanistic understanding of selective autophagy will be important for efforts to promote disease treatment and drug development.
  10. Mol Cell. 2021 Jul 05. pii: S1097-2765(21)00497-4. [Epub ahead of print]
      Cells communicate with their environment via surface proteins and secreted factors. Unconventional protein secretion (UPS) is an evolutionarily conserved process, via which distinct cargo proteins are secreted upon stress. Most UPS types depend upon the Golgi-associated GRASP55 protein. However, its regulation and biological role remain poorly understood. Here, we show that the mechanistic target of rapamycin complex 1 (mTORC1) directly phosphorylates GRASP55 to maintain its Golgi localization, thus revealing a physiological role for mTORC1 at this organelle. Stimuli that inhibit mTORC1 cause GRASP55 dephosphorylation and relocalization to UPS compartments. Through multiple, unbiased, proteomic analyses, we identify numerous cargoes that follow this unconventional secretory route to reshape the cellular secretome and surfactome. Using MMP2 secretion as a proxy for UPS, we provide important insights on its regulation and physiological role. Collectively, our findings reveal the mTORC1-GRASP55 signaling hub as the integration point in stress signaling upstream of UPS and as a key coordinator of the cellular adaptation to stress.
    Keywords:  ECM; GORASP2; GRASP55; Golgi; MMP2; Rapamycin; Tuberous Sclerosis Complex (TSC); cellular stress response; mTORC1; unconventional protein secretion (UPS)
  11. Adv Exp Med Biol. 2021 ;1208 17-42
      Autophagosome formation is a regulated membrane remodeling process, which involves the generation of autophagosomal membrane precursors (vesicles), the assembly of the autophagosomal membrane precursors to form the phagophore, and phagophore elongation to complete the autophagosome. The sources of the autophagosomal membrane precursors are endomembrane compartments, such as the endoplasmic reticulum (ER), the ER-Golgi intermediate compartment (ERGIC), ER-exit sites (ERES), and endosomes. In response to stress, these structures are remodeled, to generate the early autophagosomal membrane precursors. The phagophore assembly site (PAS), which mainly localizes on the ER, harbors the site for autophagosomal membrane assembly, elongation, and completion. ATG proteins, membrane remodeling factors, and autophagic membranes follow a precise choreography to complete the overall process. In this chapter, we briefly discuss our current knowledge on the membrane origins of the autophagosome, as well as autophagosomal precursor generation, assembly, and expansion.
    Keywords:  ATG proteins; Autophagosome; Endoplasmic reticulum; Endosome; Membrane remodeling; Microfilament; Mitochondria; Phagophore; Plasma membrane
  12. Adv Exp Med Biol. 2021 ;1208 55-66
      The autophagosome delivers engulfed substrates to the lysosome for degradation via membrane fusion between the autophagosome and the lysosome. The process of membrane fusion is highly conserved in evolution. It is widely accepted that membrane fusion in general is driven by the zippering of the SNARE complex to form a four-helix bundle. Besides SNAREs, other proteins are required to complete fusion efficiently, including tethering proteins, Rab GTPases, and SM proteins (Sec1/SM family proteins). This chapter will summarize the current knowledge of the key machinery involved in autophagosome-lysosome fusion, including autophagic SNAREs, involved ATG proteins, the HOPS complex, Rab GTPase, and other relevant aspects.
  13. Adv Exp Med Biol. 2021 ;1208 265-287
      Biomarkers (short for biological markers) are biological measures of a biological state. Autophagy biomarkers play an important role as an indicator of autophagy during normal physiological processes, pathogenic processes or pharmacological responses to drugs. In this chapter, some biomarkers of different types of autophagy, including macroautophagy, selective autophagy, chaperone-mediated autophagy, and microautophagy, as well as the lysosomal biomarkers are introduced. The described biomarkers may be used to detect the level of autophagy in cells or tissues in a dynamic, real-time, and quantitative manner. However, each biomarker has its specific significance and limitation. Therefore, the analysis of the autophagy level in cells or tissues through the detection of autophagy biomarkers should be carried out carefully.
  14. Adv Exp Med Biol. 2021 ;1208 357-371
      Autophagy is an important intracellular lysosomal degradation process in cells, which is highly conserved from yeast to mammals. The process of autophagy is roughly divided into the following key steps: the formation of a membrane structure called ISM (isolated membrane) after stimulation, the biogenesis and maturation of autophagosomes, and finally the degradation of autophagosomes. A number of proteins are required to function in the whole process of autophagy. Since the initial genetic screening in yeast cells, multiple genes that play pivotal roles in autophagy have been discovered. These molecules have been named ATG genes (AuTophaGy related genes). The screening for new key molecules involved in autophagy has greatly promoted the characterization of the mechanism of the autophagy machinery and provides multiple targets for the development of autophagy-based regulatory drugs.
  15. Mol Brain. 2021 Jul 12. 14(1): 112
      Memory and long term potentiation require de novo protein synthesis. A key regulator of this process is mTORC1, a complex comprising the mTOR kinase. Growth factors activate mTORC1 via a pathway involving PI3-kinase, Akt, the TSC complex and the GTPase Rheb. In non-neuronal cells, translocation of mTORC1 to late endocytic compartments (LEs), where Rheb is enriched, is triggered by amino acids. However, the regulation of mTORC1 in neurons remains unclear. In mouse hippocampal neurons, we observed that BDNF and treatments activating NMDA receptors trigger a robust increase in mTORC1 activity. NMDA receptors activation induced a significant recruitment of mTOR onto lysosomes even in the absence of external amino acids, whereas mTORC1 was evenly distributed in neurons under resting conditions. NMDA receptor-induced mTOR translocation to LEs was partly dependent on the BDNF receptor TrkB, suggesting that BDNF contributes to the effect of NMDA receptors on mTORC1 translocation. In addition, the combination of Rheb overexpression and artificial mTORC1 targeting to LEs by means of a modified component of mTORC1 fused with a LE-targeting motif strongly activated mTOR. To gain spatial and temporal control over mTOR localization, we designed an optogenetic module based on light-sensitive dimerizers able to recruit mTOR on LEs. In cells expressing this optogenetic tool, mTOR was translocated to LEs upon photoactivation. In the absence of growth factor, this was not sufficient to activate mTORC1. In contrast, mTORC1 was potently activated by a combination of BDNF and photoactivation. The data demonstrate that two important triggers of synaptic plasticity, BDNF and NMDA receptors, synergistically power the two arms of the mTORC1 activation mechanism, i.e., mTORC1 translocation to LEs and Rheb activation. Moreover, they unmask a functional link between NMDA receptors and mTORC1 that could underlie the changes in the synaptic proteome associated with long-lasting changes in synaptic strength.
    Keywords:  BDNF; Endo-lysosomes; NMDA receptors; Optogenetics; Synaptic plasticity; mTOR
  16. Cell Death Differ. 2021 Jul 13.
      As a core kinase of antiviral immunity, the activity and stability of TANK-binding kinase 1 (TBK1) is tightly controlled by multiple post-translational modifications. Although it has been demonstrated that TBK1 stability can be regulated by ubiquitin-dependent proteasome pathway, it is unclear whether another important protein degradation pathway, autophagosome pathway, can specifically affect TBK1 degradation by cargo receptors. Here we report that E3 ubiquitin ligase NEDD4 functions as a negative regulator of type I interferon (IFN) signaling by targeting TBK1 for degradation at the late stage of viral infection, to prevent the host from excessive immune response. Mechanically NEDD4 catalyzes the K27-linked poly-ubiquitination of TBK1 at K344, which serves as a recognition signal for cargo receptor NDP52-mediated selective autophagic degradation. Taken together, our study reveals the regulatory role of NEDD4 in balancing TBK1-centered type I IFN activation and provides insights into the crosstalk between selective autophagy and antiviral signaling.
  17. Autophagy. 2021 Jul 12. 1-3
      ATG16L1 is a critical mediator of macroautophagy/autophagy required for LC3 lipidation and autophagosome formation. However, ATG16L1 has a C-terminal domain including 7 WD40-type repetitions (WD40 domain, WDD) that is unnecessary for the conventional autophagic pathway. Instead, this domain mediates unconventional activities where LC3 is lipidated in atypical subcellular localizations unrelated to canonical double-membrane autophagosomes. The WDD provides a docking surface for molecules including a specific amino acid motif, thus engaging the LC3 lipidation capabilities of ATG16L1 in single-membrane structures. The physiological implications of such atypical activities are poorly characterized. In a recent report we described the improvement of the WDD-binding motif and the identification of transmembrane molecules that harbor this element in their intracellular region. One of them, IL10RB (interleukin 10 receptor subunit beta), binds the WDD after IL10 activation to facilitate endocytosis, early trafficking and signaling of IL10-IL10R complexes without influencing their degradation rate. These results reveal a novel unconventional role of ATG16L1 in cytokine signaling that does not entail a degradative purpose, thus contributing to catalog the physiological roles played by unconventional activities of the autophagic machinery.
    Keywords:  ATG16L1; IL10R endocytosis and signaling; WD40 domain; cytokine receptor trafficking; cytokine signaling; endocytosis; unconventional autophagy
  18. Adv Exp Med Biol. 2021 ;1208 117-130
      Thanks to the advances in optical microscope technology and our knowledge of autophagic biomarkers, single-molecule events of autophagy are now accessible to human eyes. Different proteins are involved hierarchically in the biogenesis and maturation of autophagosomes. Detecting these autophagy-related proteins either by immunostaining or fluorescent protein labelling makes the dynamic autophagic process visible. However, low antibody specificity and weak endogenous expression of autophagy-related proteins in certain tissues limit the applicability of immunostaining in autophagy detection. To cope with this, live-cell imaging combined with various fluorescent probes has been developed and employed in monitoring autophagy. As the most widely used autophagic biomarker, LC3 can be used to visualize autophagosomes, and fluorescent probes targeting LC3, i.e., RFP/mCherry-GFP-LC3, and GFP-LC3-RFP-LC3ΔG, can examine autophagy flux dynamically and quantitatively. In addition, the application of novel fluorophores such as Keima helps to detect the temporal and spatial characteristics of autophagy. Furthermore, selective autophagy can be clarified by labelling corresponding substrates and autophagosomes or lysosomes simultaneously. With the help of two-photon microscopy, the process of autophagy in live animals has been uncovered. Here, we summarize the methods for observing autophagy by optical microscopy and the selection of fluorescent markers.
    Keywords:  Autophagy; Immunostaining; Live-animal imaging; Live-cell imaging; Selective autophagy
  19. FEBS J. 2021 Jul 16.
      Autophagy is a catabolic process that captures cellular waste and degrades them in the lysosome. The main function of autophagy is quality control of cytosolic proteins and organelles, and intracellular recycling of nutrients in order to maintain cellular homeostasis. Autophagy is upregulated in many cancers to promote cell survival, proliferation and metastasis. Both cell-autonomous autophagy (also known as tumor autophagy) and non-cell autonomous autophagy (also known as host autophagy) supports tumorigenesis through different mechanisms, including inhibition of p53 activation, sustaining redox homeostasis, maintenance of essential amino acids levels in order to support energy production and biosynthesis, and inhibition of anti-tumor immune responses. Therefore, autophagy may serve as a tumor-specific vulnerability and targeting autophagy could be a novel strategy in cancer treatment.
    Keywords:  Autophagy; Cancer; Cancer Metabolism; Immune Response; Metastasis; p53
  20. Neural Regen Res. 2022 Feb;17(2): 246-250
      Neuroinflammation and neurodegeneration are key components in the establishment and progression of neurodegenerative diseases including Alzheimer's Disease (AD). Over the past decade increasing evidence is emerging for the use of components of the canonical autophagy machinery in pathways that are characterized by LC3 lipidation yet are distinct from traditional macro-autophagy. One such pathway that utilizes components of the autophagy machinery to target LC3 to endosomes, a process termed LC3-associated endocytosis (LANDO), has recently been identified and regulates neuroinflammation. Abrogation of LANDO in microglia cells results in a propensity for elevated neuroinflammatory cytokine production. Using the well-established 5xFAD model of AD to interrogate neuroinflammatory regulation, impairment of LANDO through deletion of a key upstream regulator Rubicon or other downstream autophagy components, exacerbated disease onset and severity, while deletion of microglial autophagy alone had no measurable effect. Mice presented with robust deposition of the neurotoxic AD protein β-amyloid (Aβ), microglial activation and inflammatory cytokine production, tau phosphorylation, and aggressive neurodegeneration culminating in severe memory impairment. LANDO-deficiency impaired recycling of receptors that recognize Aβ, including TLR4 and TREM2. LANDO-deficiency alone through deletion of the WD-domain of the autophagy protein ATG16L, revealed a role for LANDO in the spontaneous establishment of age-associated AD. LANDO-deficient mice aged to 2 years presented with advanced AD-like disease and pathology correlative to that observed in human AD patients. Together, these studies illustrate an important role for microglial LANDO in regulating CNS immune activation and protection against neurodegeneration. New evidence is emerging that demonstrates a putative linkage between pathways such as LANDO and cell death regulation via apoptosis and possibly necroptosis. Herein, we provide a review of the use of the autophagy machinery in non-canonical mechanisms that alter immune regulation and could have significant impact in furthering our understanding of not only CNS diseases like AD, but likely beyond.
    Keywords:  Alzheimer’s disease; LC3-associated endocytosis; aging; autophagy; inflammation; microglia; neurodegeneration; neuroinflammation
  21. Adv Exp Med Biol. 2021 ;1208 175-190
      Macroautophagy (referred to as autophagy hereafter) is a highly conserved catabolic process in eukaryotic cells. Autophagy is essential for cellular homeostasis through elimination and recycling of large cytoplasmic components, such as abnormal protein aggregates and damaged organelles, via lysosomal degradation. Since being originally identified by genetic screening in yeast, autophagy-related (ATG) genes have played a central role in autophagy research in different organisms, including plants, worms, flies, and mammals. Mouse models for monitoring autophagic activity or clarifying its biological functions have also been established. These mice are powerful tools to investigate roles of autophagy in vivo. Owing to the rapid technological advances in molecular biology, it is ever more efficient and simpler to manipulate autophagy-associated genes. Herein, we will introduce some commonly used approaches of gene silencing in mammalian cells, including CRIPSR/Cas9-mediated gene knockout and siRNA- and shRNA-mediated gene knockdown. We also summarized the common mouse models used for assessing autophagy. We hope to bring the researchers some useful information as they study autophagy.
  22. Adv Exp Med Biol. 2021 ;1208 131-173
      Macroautophagy is an important biological process in eukaryotic cells by which longevity proteins, misfolded proteins, and damaged organelles are degraded. The autophagy process consists of three key steps: (1) the formation of autophagosomes; (2) the fusion of the autophagosomes with lysosomes; and (3) the degradation of the contents of autolysosomes. If any of the three steps is impaired, autophagy will not be able to complete its biological function. Dysfunctional or blocked autophagy is closely involved in the pathogenesis of a variety of diseases. The accurate determination of the autophagy activity in vivo and in vitro has become a challenge in the field of autophagy research. At present, the most widely used detection method to determine autophagy activity in mammalian cells is to quantify LC3B in the cells by Western blot, or to observe the formation and changes of autophagosomes and autolysosomes by immunofluorescence and electron microscopy. However, ignoring the dynamic characteristics of autophagy and only evaluating the number of autophagosomes or the presence of LC3B cannot completely reflect the activation or a blockage of the autophagy system, and objectively analyze its real role in the occurrence and development of a disease. For example, the accumulation of autophagosomes and autolysosomes can occur through an increase in substrate to be degraded after the activation of autophagy, or it may be caused by the partial obstruction or blockage of autophagy. In this chapter, new and familiar ways to detect the autophagic flux are methodically summarized to provide researchers with a multi-angled viewpoint.
    Keywords:  Autophagic flux; Cargo sequestration assay; LC3B; SQSTM1/p62; TEM
  23. Adv Exp Med Biol. 2021 ;1208 289-309
      Autophagy is a catabolic process that removes aggregated proteins and damaged organelles via lysosomal degradation. Increasing evidence suggests that dysfunction of autophagy is associated with a variety of human pathologies, including aging, cancer, neurodegenerative diseases, heart diseases, diabetes, and other metabolic diseases. Current research suggests that the regulation of autophagy may be a novel target for the treatment of these diseases. For this purpose, it is essential to have a deep understanding on the molecular details of autophagy and its regulatory network in each of the disease contexts. Over the years, a variety of chemical autophagy inducers and inhibitors has been developed. The application of these autophagy regulators can assist us in the exploration of the mechanism and therapeutic potential of autophagy regulation. In this chapter, we summarize the recent advances in chemical autophagy regulators to provide methodological support for autophagy research.
  24. Adv Exp Med Biol. 2021 ;1208 333-356
      Autophagy is a highly conserved cellular process that delivers cellular contents to the lysosome for degradation. It not only serves as a bulk degradation system for various cytoplasmic components but also functions selectively to clear damaged organelles, aggregated proteins, and invading pathogens (Feng et al., Cell Res 24:24-41, 2014; Galluzzi et al., EMBO J 36:1811-36, 2017; Klionsky et al., Autophagy 12:1-222, 2016). The malfunction of autophagy leads to multiple developmental defects and diseases (Mizushima et al., Nature 451:1069-75, 2008). Drosophila and zebrafish are higher metazoan model systems with sophisticated genetic tools readily available, which make it possible to dissect the autophagic processes and to understand the physiological functions of autophagy (Lorincz et al., Cells 6:22, 2017a; Mathai et al., Cells 6:21, 2017; Zhang and Baehrecke, Trends Cell Biol 25:376-87, 2015). In this chapter, we will discuss recent progress that has been made in the autophagic field by using these animal models. We will focus on the protein machineries required for autophagosome formation and maturation as well as the physiological roles of autophagy in both Drosophila and zebrafish.
  25. Biochem Biophys Res Commun. 2021 Jul 06. pii: S0006-291X(21)01027-5. [Epub ahead of print]569 118-124
      The mammalian target of rapamycin complex 1 (mTORC1) is a crucial regulator of adipogenesis and systemic energy metabolism. Its dysregulation leads to a diversity of metabolic diseases, including obesity and type 2 diabetes. DEP-domain containing 5 (DEPDC5) is a critical component of GATOR1 complex that functions as a key inhibitor of mTORC1. So far, its function in adipose tissue remains largely unknown. Herein we evaluated how persistent mTORC1 activation in adipocyte via Depdc5 knockout modulates adiposity in vivo. Our data indicated that adipocyte-specific knockout of Depdc5 in aged mice led to reduced visceral fat, aggravated insulin resistance and enhanced adipose tissue inflammation. Moreover, we found that Depdc5 ablation resulted in upregulation of adipose triglyceride lipase (ATGL) in adipocytes and elevated levels of serum free fatty acids (FFAs). Intriguingly, rapamycin treatment did not reverse insulin resistance but alleviated adipose tissue inflammation caused by Depdc5 deletion. Taken together, our findings revealed that mTORC1 activation caused by Depdc5 deletion promotes lipolysis process and further exacerbates insulin resistance and adipose tissue inflammation in mice.
    Keywords:  Adipocyte; Depdc5; Inflammation; Insulin resistance; mTORC1
  26. Sci Adv. 2021 Jul;pii: eabg4574. [Epub ahead of print]7(28):
      The role of ubiquitin carboxyl-terminal hydrolase L1 (UCHL1; also called PARK5) in the pathogenesis of Parkinson's disease (PD) has been controversial. Here, we find that the loss of UCHL1 destabilizes pyruvate kinase (PKM) and mitigates the PD-related phenotypes induced by PTEN-induced kinase 1 (PINK1) or Parkin loss-of-function mutations in Drosophila and mammalian cells. In UCHL1 knockout cells, cellular pyruvate production and ATP levels are diminished, and the activity of AMP-activated protein kinase (AMPK) is highly induced. Consequently, the activated AMPK promotes the mitophagy mediated by Unc-51-like kinase 1 (ULK1) and FUN14 domain-containing 1 (FUNDC1), which underlies the effects of UCHL1 deficiency in rescuing PD-related defects. Furthermore, we identify tripartite motif-containing 63 (TRIM63) as a previously unknown E3 ligase of PKM and demonstrate its antagonistic interaction with UCHL1 to regulate PD-related pathologies. These results suggest that UCHL1 is an integrative factor for connecting glycolysis and PD pathology.
  27. Adv Exp Med Biol. 2021 ;1208 67-77
      Autophagy is a major intracellular degradation/recycling system that ubiquitously exists in eukaryotic cells. Autophagy contributes to the turnover of cellular components through engulfing portions of the cytoplasm or organelles and delivering them to the lysosomes/vacuole to be degraded. The trafficking of autophagosomes and their fusion with lysosomes are important steps that complete their maturation and degradation. In cells such as neuron, autophagosomes traffic long distances along the axon, while in other specialized cells such as cardiomyocytes, it is unclear how and even whether autophagosomes are transported. Therefore, it is important to learn more about the processes and mechanisms of autophagosome trafficking to lysosomes/vacuole during autophagy. The mechanisms of autophagosome trafficking are similar to those of other organelles trafficking within cells. The machinery mainly includes cytoskeletal systems such as actin and microtubules, motor proteins such as myosins and the dynein-dynactin complex, and other proteins like LC3 on the membrane of autophagosomes. Factors regulating autophagosome trafficking have not been widely studied. To date the main reagents identified for disrupting autophagosome trafficking include: 1. Microtubule polymerization reagents, which disrupt microtubules by interfering with microtubule dynamics, thus directly influence microtubule-dependent autophagosome trafficking 2. F-actin-depolymerizing drugs, which inhibit autophagosome formation, and also subsequently inhibit autophagosome trafficking 3. Motor protein regulators, which directly affect autophagosome trafficking.
  28. Adv Exp Med Biol. 2021 ;1208 99-114
      Autophagy, a highly conserved metabolic process in eukaryotes, is a widespread degradation/recycling system. However, there are significant differences (as well as similarities) between autophagy in animals, plants, and microorganisms such as yeast. While the overall process of autophagy is similar between different organisms, the molecular mechanisms and the pathways regulating autophagy are different, which is manifested in the diversity and specificity of the genes involved. In general, the autophagy system is much more complicated in mammals than in yeast. In addition, there are some differences in the types of autophagy present in animals, plants, and microorganisms. For example, there is a unique type of selective autophagy called the cytoplasm-to-vacuole targeting (Cvt) pathway in yeast, and a special kind of autophagy, chloroplast autophagy, exists in plants. In conclusion, although autophagy is highly conserved in eukaryotes, there are still many differences between autophagy of animals, plants, and microorganisms.
  29. Nat Commun. 2021 07 09. 12(1): 4227
      Glycine decarboxylase (GLDC) is a key enzyme of glycine cleavage system that converts glycine into one-carbon units. GLDC is commonly up-regulated and plays important roles in many human cancers. Whether and how GLDC is regulated by post-translational modifications is unknown. Here we report that mechanistic target of rapamycin complex 1 (mTORC1) signal inhibits GLDC acetylation at lysine (K) 514 by inducing transcription of the deacetylase sirtuin 3 (SIRT3). Upon inhibition of mTORC1, the acetyltransferase acetyl-CoA acetyltransferase 1 (ACAT1) catalyzes GLDC K514 acetylation. This acetylation of GLDC impairs its enzymatic activity. In addition, this acetylation of GLDC primes for its K33-linked polyubiquitination at K544 by the ubiquitin ligase NF-X1, leading to its degradation by the proteasomal pathway. Finally, we find that GLDC K514 acetylation inhibits glycine catabolism, pyrimidines synthesis and glioma tumorigenesis. Our finding reveals critical roles of post-translational modifications of GLDC in regulation of its enzymatic activity, glycine metabolism and tumorigenesis, and provides potential targets for therapeutics of cancers such as glioma.
  30. Free Radic Biol Med. 2021 Jul 08. pii: S0891-5849(21)00412-3. [Epub ahead of print]
      Alzheimer's disease (AD) is a progressive neurodegenerative disease characterized by memory loss and multiple cognitive impairments. AD is marked by multiple cellular changes, including deregulation of microRNAs, activation of glia and astrocytes, hormonal imbalance, defective mitophagy, synaptic degeneration, in addition to extracellular neuritic amyloid-beta (Aβ) plaques, phosphorylated tau (P-tau), and intracellular neurofibrillary tangles (NFTs). Recent research in AD revealed that defective synaptic mitophagy leads to synaptic degeneration and cognitive dysfunction in AD neurons. Our critical analyses of mitochondria and Aβ and P-tau revealed that increased levels of Aβ and P-Tau, and abnormal interactions between Aβ and Drp1, P-Tau and Drp1 induced increased mitochondrial fragmentation and proliferation of dysfunctional mitochondria in AD neurons and depleted Parkin and PINK1 levels. These events ultimately lead to impaired clearance of dead and/or dying mitochondria in AD neurons. The purpose of our article is to highlight the recent research on mitochondria and synapses in relation to Aβ and P-tau, focusing on recent developments.
    Keywords:  Alzheimer's disease; Amyloid beta; Amyloid precursor protein; Autophagy; Mitophagy; Oxidative phosphorylation; Phosphorylated tau
  31. Adv Sci (Weinh). 2021 Jul 11. e2101614
      Under conditions of starvation, normal and tumor epithelial cells can rewire their metabolism toward the consumption of extracellular proteins, including extracellular matrix-derived components as nutrient sources. The mechanism of pericellular matrix degradation by starved cells has been largely overlooked. Here it is shown that matrix degradation by breast and pancreatic tumor cells and patient-derived xenograft explants increases by one order of magnitude upon amino acid and growth factor deprivation. In addition, it is found that collagenolysis requires the invadopodia components, TKS5, and the transmembrane metalloproteinase, MT1-MMP, which are key to the tumor invasion program. Increased collagenolysis is controlled by mTOR repression upon nutrient depletion or pharmacological inhibition by rapamycin. The results reveal that starvation hampers clathrin-mediated endocytosis, resulting in MT1-MMP accumulation in arrested clathrin-coated pits. The study uncovers a new mechanism whereby mTOR repression in starved cells leads to the repurposing of abundant plasma membrane clathrin-coated pits into robust ECM-degradative assemblies.
    Keywords:  MT1-MMP; breast cancer; clathrin-mediated endocytosis; extracellular matrix; invadopodia; mTOR; starvation
  32. Front Cell Dev Biol. 2021 ;9 691826
      Accompanying the precipitous age-related decline in human female fertility is an increase in the proportion of poor-quality oocytes within the ovary. The macroautophagy pathway, an essential protein degradation mechanism responsible for maintaining cell health, has not yet been thoroughly investigated in this phenomenon. The aim of this study was to characterize the macroautophagy pathway in an established mouse model of oocyte aging using in-depth image analysis-based methods and to determine mechanisms that account for the observed changes. Three autophagy pathway markers were selected for assessment of gene and protein expression in this model: Beclin 1; an initiator of autophagosome formation, Microtubule-associated protein 1 light chain 3B; a constituent of the autophagosome membrane, and lysosomal-associated membrane protein 1; a constituent of the lysosome membrane. Through quantitative image analysis of immunolabeled oocytes, this study revealed impairment of the macroautophagy pathway in the aged oocyte with an attenuation of both autophagosome and lysosome number. Additionally, an accumulation of amphisomes greater than 10 μm2 in area were observed in aging oocytes, and this accumulation was mimicked in oocytes treated with lysosomal inhibitor chloroquine. Overall, these findings implicate lysosomal dysfunction as a prominent mechanism by which these age-related changes may occur and highlight the importance of macroautophagy in maintaining mouse pre-ovulatory oocyte quality. This provides a basis for further investigation of dysfunctional autophagy in poor oocyte quality and for the development of therapeutic or preventative strategies to aid in the maintenance of pre-ovulatory oocyte health.
    Keywords:  amphisome; autophagosome; autophagy; lysosome; oocyte quality; protein degradation; reproductive system
  33. Aging (Albany NY). 2021 Jul 12. 13(undefined):
      Aberrant expression and denaturation of Tau, amyloid-beta and TDP-43 can lead to cell death and is a major component of pathologies such as Alzheimer's Disease (AD). AD neurons exhibit a reduced ability to form autophagosomes and degrade proteins via autophagy. Using genetically manipulated colon cancer cells we determined whether drugs that directly inhibit the chaperone ATPase activity or cause chaperone degradation and endoplasmic reticulum stress signaling leading to macroautophagy could reduce the levels of these proteins. The antiviral chaperone ATPase inhibitor AR12 reduced the ATPase activities and total expression of GRP78, HSP90, and HSP70, and of Tau, Tau 301L, APP, APP692, APP715, SOD1 G93A and TDP-43. In parallel, it increased the phosphorylation of ATG13 S318 and eIF2A S51 and caused eIF2A-dependent autophagosome formation and autophagic flux. Knock down of Beclin1 or ATG5 prevented chaperone, APP and Tau degradation. Neratinib, used to treat HER2+ breast cancer, reduced chaperone levels and expression of Tau and APP via macroautophagy, and neratinib interacted with AR12 to cause further reductions in protein levels. The autophagy-regulatory protein ATG16L1 is expressed as two isoforms, T300 or A300: Africans trend to express T300 and Europeans A300. We observed higher basal expression of Tau in T300 cells when compared to isogenic A300 cells. ATG16L1 isoform expression did not alter basal levels of HSP90, HSP70 or HSP27, however, basal levels of GRP78 were reduced in A300 cells. The abilities of both AR12 and neratinib to stimulate ATG13 S318 and eIF2A S51 phosphorylation and autophagic flux was also reduced in A300 cells. Our data support further evaluation of AR12 and neratinib in neuronal cells as repurposed treatments for AD.
    Keywords:  Alzheimer's; GRP78; autophagy; chaperone; neratinib
  34. Elife. 2021 Jul 13. pii: e69047. [Epub ahead of print]10
      Autophagy acts as a pivotal innate immune response against infection. Some virulence effectors subvert the host autophagic machinery to escape the surveillance of autophagy. The mechanism by which pathogens interact with host autophagy remains mostly unclear. However, traditional strategies often have difficulty identifying host proteins that interact with effectors due to the weak, dynamic and transient nature of these interactions. Here, we found that Enteropathogenic Escherichia coli (EPEC) regulates autophagosome formation in host cells dependent on effector NleE. The 26S Proteasome Regulatory Subunit 10 (PSMD10) was identified as a direct interaction partner of NleE in living cells by employing genetically incorporated crosslinkers. Pairwise chemical crosslinking revealed that NleE interacts with the N-terminus of PSMD10. We demonstrated that PSMD10 homodimerization is necessary for its interaction with ATG7 and promotion of autophagy, but not necessary for PSMD10 interaction with ATG12. Therefore, NleE-mediated PSMD10 in monomeric state attenuates host autophagosome formation. Our study reveals the mechanism through which EPEC attenuates host autophagy activity.
    Keywords:  biochemistry; cell biology; chemical biology; human
  35. Cell Death Dis. 2021 Jul 13. 12(7): 697
      The tripartite motif-containing protein 21 (TRIM21) plays important roles in autophagy and innate immunity. Here, we found that HECT and RLD domain containing E3 ubiquitin protein ligase 5 (HERC5), as an interferon-stimulated gene 15 (ISG15) E3 ligase, catalyzes the ISGylation of TRIM21 at the Lys260 and Lys279 residues. Moreover, IFN-β also induces TRIM21 ISGylation at multiple lysine residues, thereby enhancing its E3 ligase activity for K63-linkage-specific ubiquitination and resulting in increased levels of TRIM21 and p62 K63-linked ubiquitination. The K63-linked ubiquitination of p62 at Lys7 prevents its self-oligomerization and targeting to the autophagosome. Taken together, our study suggests that the ISGylation of TRIM21 plays a vital role in regulating self-oligomerization and localization of p62 in the autophagy induced by IFN-β.
  36. J Cancer Prev. 2021 Jun 30. 26(2): 145-152
      Cryptotanshinone is known for its inhibitory activity against tumorigenesis in various human cancer cells. However, exact mechanisms underlying the anticancer effects of cryptotanshinone are not fully elucidated. Here, we propose a plausible molecular mechanism, wherein cryptotanshinone represses rapamycin-sensitive mTORC1/S6K1 mediated cancer cell growth and cell transformation. We investigated the various effects of cryptotanshinone on the mTORC1/S6K1 axis, which is associated with the regulation of cell growth in response to nutritional and growth factor signals. We found that cryptotanshinone specifically inhibited the mTORC1-mediated phosphorylation of S6K1, which consequently suppressed the clonogenicity of SK-Hep1 cells and the neoplastic transformation of JB6 Cl41 cells induced by insulin-like growth factor-1. Finally, we observed that cryptotanshinone prevented S6K1 from binding to the Raptor/mTOR complex, rather than regulating mTOR and its upstream pathway. Overall, our findings provide a novel mechanism underlying anti-cancer effects cryptotanshinone targeting mTORC1 signaling, contributing to the development of anticancer agents involving metabolic cancer treatment.
    Keywords:  Raptor protein; cryptotanshinone; mTORC1; neoplastic cell transformation; p70S6K
  37. Nat Cell Biol. 2021 Jul 12.
      The integral membrane protein ATG9A plays a key role in autophagy. It displays a broad intracellular distribution and is present in numerous compartments, including the plasma membrane (PM). The reasons for the distribution of ATG9A to the PM and its role at the PM are not understood. Here, we show that ATG9A organizes, in concert with IQGAP1, components of the ESCRT system and uncover cooperation between ATG9A, IQGAP1 and ESCRTs in protection from PM damage. ESCRTs and ATG9A phenocopied each other in protection against PM injury. ATG9A knockouts sensitized the PM to permeabilization by a broad spectrum of microbial and endogenous agents, including gasdermin, MLKL and the MLKL-like action of coronavirus ORF3a. Thus, ATG9A engages IQGAP1 and the ESCRT system to maintain PM integrity.
  38. Adv Exp Med Biol. 2021 ;1208 191-264
      Social and economic impacts of neurodegenerative diseases (NDs) become more prominent in our constantly aging population. Currently, due to the lack of knowledge about the aetiology of most NDs, only symptomatic treatment is available for patients. Hence, researchers and clinicians are in need of solid studies on pathological mechanisms of NDs. Autophagy promotes degradation of pathogenic proteins in NDs, while microRNAs post-transcriptionally regulate multiple signalling networks including autophagy. This chapter will critically discuss current research advancements in the area of microRNAs regulating autophagy in NDs. Moreover, we will introduce basic strategies and techniques used in microRNA research. Delineation of the mechanisms contributing to NDs will result in development of better approaches for their early diagnosis and effective treatment.
  39. Adv Exp Med Biol. 2021 ;1332 1-15
      Amino acids have pleiotropic roles in animal biology including protein and glucose synthesis, cellular metabolism, antioxidant reactions, immune enhancers, and inducers or suppressors of gene expression. Recent studies have revealed important roles of amino acids in the regulation of gene expression in animals. Discoveries of cellular amino acid sensors and their mechanistic pathways have broadened our understanding of how the body responds to the deprivation of nutrients and amino acids in particular. Alterations in concentrations of extracellular amino acids can modulate transcription, translation, posttranscriptional modifications, and epigenetic regulation of genes and proteins. Cells have intracellular amino acid sensors, for example, Sestrin2 for leucine and CASTOR2 for arginine, that respond to sufficiency or deficiency in amino acids, thereby inhibiting or activating downstream signals for gene expression, respectively. The sufficiency of an amino acid in cells ensures its binding to cognate sensors and suppression of inhibitors of MTOR, leading to increased global protein synthesis. On the other hand, deprivation of amino acids activates the amino acid response pathway (GCN2-eIF2a-ATF4), leading to increased selective translation of the activating transcription factor 4 (ATF4). Deficiency of an amino acid itself or via the action of ATF4 suppression of MTORC1 activity limits global protein synthesis. ATF4, in response to low concentrations of cellular amino acids, mediates the transcription of groups of genes such as those for amino acid transport and biosynthesis (ASNS, CAT-1, SNAT2), autophagy (ATG3, ATG10, ATG12), and serine-glycine synthesis (PHGDH, PSAT1, PSPH, MTHFD2). Long-term amino acid starvation has a pronounced effect on cells: suppressed expression and translation of genes required for normal cell growth and metabolism and enhanced expression of genes required for cell adaptation and survival. Levels of amino acids also affect the posttranslational modifications of proteins through mechanisms such as acetylation, ADP-ribosylation, disulfide bond formation, glutamylation, and hydroxylation.
    Keywords:  ATF4; Amino acids; Gene expression; MTOR; Protein synthesis
  40. Am J Physiol Renal Physiol. 2021 Jul 12.
      Primary cilia are widely regarded as specialized sensors in differentiated cells that have been implicated in the regulation of cell proliferation, differentiation, and viability. We previously showed that shortening of primary cilia sensitizes cultured kidney tubular cells to cisplatin-induced apoptosis. IFT88 is an essential component for ciliogenesis and maintenance. Here, we have further examined the effect of proximal tubule-specific IFT88 ablation on cisplatin-induced acute kidney injury (AKI). In the study, more severe AKI occurred in IFT88 knockout mouse than age- and sex-matched wild type mice. Mechanistically, cisplatin stimulated autophagy in kidney tubular cells as an intrinsic protective mechanism. However, renal autophagy was severely impaired in IFT88 knockout mouse. In cultured HK-2 cells, cisplatin induced more apoptosis when IFT88 was knocked down. Tat-beclin 1 peptide, a specific autophagy activator, could partially prevent IFT88-associated cell death during cisplatin treatment, although cilium length was not improved significantly. Re-expression of IFT88 partially restored autophagy in IFT88-knockdown cells and suppressed apoptosis during cisplatin treatment. Taken together, these results indicate that defective autophagy in IFT88-deficient kidney cells and tissues contributes to the exaggerated AKI following cisplatin exposure.
    Keywords:  IFT88; acute kidney injury; autophagy; cisplatin; kidney
  41. Adv Exp Med Biol. 2021 ;1208 373-386
      Autophagy is an evolutionarily conserved intracellular degradation process. Autophagy is closely involved in human health and diseases. In recent years, mass spectrometry-based proteomic methods have become important and powerful tools for autophagy studies. These types of techniques have been especially helpful to reveal the range of degradation substrates of autophagy through large-scale, unbiased analysis of cellular proteomes. At present, a variety of mass spectrometry-based proteomics methods have been successfully applied to autophagy research.
  42. J Cell Mol Med. 2021 Jul 10.
      Traumatic brain injury (TBI) provokes primary and secondary damage on endothelium and brain parenchyma, leading neurons die rapidly by necrosis. The mammalian target of rapamycin signalling pathway (mTOR) manages numerous aspects of cellular growth, and it is up-regulated after moderate to severe traumatic brain injury (TBI). Currently, the significance of this increased signalling event for the recovery of brain function is unclear; therefore, we used two different selective inhibitors of mTOR activity to discover the functional role of mTOR inhibition in a mouse model of TBI performed by a controlled cortical impact injury (CCI). Treatment with KU0063794, a dual mTORC1 and mTORC2 inhibitor, and with rapamycin as well-known inhibitor of mTOR, was performed 1 and 4 hours subsequent to TBI. Results proved that mTOR inhibitors, especially KU0063794, significantly improved cognitive and motor recovery after TBI, reducing lesion volumes. Also, treatment with mTOR inhibitors ameliorated the neuroinflammation associated with TBI, showing a diminished neuronal death and astrogliosis after trauma. Our findings propose that the involvement of selective mTORC1/2 inhibitor may represent a therapeutic strategy to improve recovery after brain trauma.
    Keywords:  KU0063794; apoptosis; astrogliosis; behavioural performance; mTOR; mTORC1; mTORC2; neuroinflammation; neuronal death; rapamycin; traumatic brain injury
  43. Nat Commun. 2021 07 12. 12(1): 4258
      The maintenance of constant karyoplasmic ratios suggests that nuclear size has physiological significance. Nuclear size anomalies have been linked to malignant transformation, although the mechanism remains unclear. By expressing dominant-negative TER94 mutants in Drosophila photoreceptors, here we show disruption of VCP (valosin-containing protein, human TER94 ortholog), a ubiquitin-dependent segregase, causes progressive nuclear size increase. Loss of VCP function leads to accumulations of MDC1 (mediator of DNA damage checkpoint protein 1), connecting DNA damage or associated responses to enlarged nuclei. TER94 can interact with MDC1 and decreases MDC1 levels, suggesting that MDC1 is a VCP substrate. Our evidence indicates that MDC1 accumulation stabilizes p53A, leading to TER94K2A-associated nuclear size increase. Together with a previous report that p53A disrupts autophagic flux, we propose that the stabilization of p53A in TER94K2A-expressing cells likely hinders the removal of nuclear content, resulting in aberrant nuclear size increase.
  44. Adv Exp Med Biol. 2021 ;1332 17-33
      Amino acids are the main building blocks for life. Aside from their roles in composing proteins, functional amino acids and their metabolites play regulatory roles in key metabolic cascades, gene expressions, and cell-to-cell communication via a variety of cell signaling pathways. These metabolic networks are necessary for maintenance, growth, reproduction, and immunity in humans and animals. These amino acids include, but are not limited to, arginine, glutamine, glutamate, glycine, leucine, proline, and tryptophan. We will discuss these functional amino acids in cell signaling pathways in mammals with a particular emphasis on mTORC1, AMPK, and MAPK pathways for protein synthesis, nutrient sensing, and anti-inflammatory responses, as well as cell survival, growth, and development.
    Keywords:  AMPK; Functional amino acids; MAPK; Sensor; Transceptor; mTORC1
  45. Brain. 2021 Jul 15. pii: awab268. [Epub ahead of print]
      Seizure risk is 10-fold higher in Alzheimer's disease patients than the general population, yet the mechanisms underlying this susceptibility and the effects of seizures on Alzheimer's disease are poorly understood. To elucidate our proposed bidirectional relationship between Alzheimer's disease and seizures, we studied Alzheimer's disease human brain samples (n = 34) and found that patients with a history of seizures (n = 14) had increased β-amyloid and tau pathology, and upregulation of the mechanistic target of rapamycin (mTOR) pathway compared to cases without known seizure history (n = 20). To establish whether seizures could accelerate Alzheimer's disease progression, we induced chronic hyperexcitability in the 5XFAD Alzheimer's disease mouse model by kindling with the chemoconvulsant pentylenetetrazol and observed that 5XFAD mice displayed higher seizure severity compared to wild type. Furthermore, kindled seizures exacerbated later cognitive impairment, Alzheimer's disease neuropathology and mTORC1 activation. Finally, we demonstrate that administration of the mTOR inhibitor rapamycin following kindled seizures rescued enhanced remote and long-term memory deficits associated with earlier kindling and prevented the seizure-induced increases in Alzheimer's disease neuropathology. These data demonstrate an important link between chronic hyperexcitability and progressive Alzheimer's disease pathology and suggest a mechanism whereby rapamycin may serve as an adjunct therapy to attenuate Alzheimer's disease progression.
    Keywords:  Alzheimer’s disease; cognition; mTOR; neuropathology; seizures