bims-auttor Biomed News
on Autophagy and mTOR
Issue of 2023–10–29
68 papers selected by
Viktor Korolchuk, Newcastle University



  1. Mol Med. 2023 10 25. 29(1): 142
      Autophagy is an explicit cellular process to deliver dissimilar cytoplasmic misfolded proteins, lipids and damaged organelles to the lysosomes for degradation and elimination. The mechanistic target of rapamycin (mTOR) is the main negative regulator of autophagy. The mTOR pathway is involved in regulating neurogenesis, synaptic plasticity, neuronal development and excitability. Exaggerated mTOR activity is associated with the development of temporal lobe epilepsy, genetic and acquired epilepsy, and experimental epilepsy. In particular, mTOR complex 1 (mTORC1) is mainly involved in epileptogenesis. The investigation of autophagy's involvement in epilepsy has recently been conducted, focusing on the critical role of rapamycin, an autophagy inducer, in reducing the severity of induced seizures in animal model studies. The induction of autophagy could be an innovative therapeutic strategy in managing epilepsy. Despite the protective role of autophagy against epileptogenesis and epilepsy, its role in status epilepticus (SE) is perplexing and might be beneficial or detrimental. Therefore, the present review aims to revise the possible role of autophagy in epilepsy.
    Keywords:  Autophagy; Autophagy inducers; Epilepsy; Neurodegeneration; Seizure
    DOI:  https://doi.org/10.1186/s10020-023-00742-2
  2. Biomolecules. 2023 Sep 22. pii: 1435. [Epub ahead of print]13(10):
      Parkinson's disease (PD) is a devastating disease associated with accumulation of α-synuclein (α-Syn) within dopaminergic neurons, leading to neuronal death. PD is characterized by both motor and non-motor clinical symptoms. Several studies indicate that autophagy, an important intracellular degradation pathway, may be involved in different neurodegenerative diseases including PD. The autophagic process mediates the degradation of protein aggregates, damaged and unneeded proteins, and organelles, allowing their clearance, and thereby maintaining cell homeostasis. Impaired autophagy may cause the accumulation of abnormal proteins. Incomplete or impaired autophagy may explain the neurotoxic accumulation of protein aggregates in several neurodegenerative diseases including PD. Indeed, studies have suggested the contribution of impaired autophagy to α-Syn accumulation, the death of dopaminergic neurons, and neuroinflammation. In this review, we summarize the recent literature on the involvement of autophagy in PD pathogenesis.
    Keywords:  Parkinson’s disease (PD); apolipoprotein E4 (apoE4); autophagy; endocytosis; lysosomal degradation; synuclein α
    DOI:  https://doi.org/10.3390/biom13101435
  3. Autophagy. 2023 Oct 24.
      Macroautophagy/autophagy is an essential pro-survival mechanism activated in response to nutrient deficiency. The proper fusion between autophagosomes and lysosomes is a critical step for autophagic degradation. We recently reported that RUNDC1 (RUN domain containing 1) inhibits autolysosome formation via clasping the ATG14-STX17-SNAP29 complex to hinder VAMP8 binding. We showed that RUNDC1 colocalizes with LC3 and associates with mature autophagosomes in cell lines and the zebrafish model. We utilized liposome fusion and in vitro autophagosome-lysosome fusion assays to demonstrate that RUNDC1 inhibits autolysosome formation. Moreover, we found that RUNDC1 clasps the ATG14-STX17-SNAP29 complex via stimulating ATG14 homo-oligomerization to inhibit ATG14 dissociation, which in turn prevents VAMP8 from binding to STX17-SNAP29. Our results demonstrate that RUNDC1 is a negative regulator of autophagy that restricts autophagosome fusion with lysosomes and is crucial for zebrafish survival in nutrient-deficient conditions. Here, we summarize our findings and discuss their implications for our understanding of autophagy regulation.
    Keywords:  ATG14-STX17-SNAP29; RUNDC1; autophagosomes; autophagy; lysosomes; zebrafish
    DOI:  https://doi.org/10.1080/15548627.2023.2274210
  4. Mol Cells. 2023 Oct 23.
      Autophagy dysfunction is associated with human diseases and conditions including neurodegenerative diseases, metabolic issues, and chronic infections. Additionally, the decline in autophagic activity contributes to tissue and organ dysfunction and aging-related diseases. Several factors, such as down-regulation of autophagy components and activators, oxidative damage, microinflammation, and impaired autophagy flux, are linked to autophagy decline. An autophagy flux impairment (AFI) has been implicated in neurological disorders and in certain other pathological conditions. Here, to enhance our understanding of AFI, we conducted a comprehensive literature review of findings derived from two well-studied cellular stress models: glucose deprivation and replicative senescence. Glucose deprivation is a condition in which cells heavily rely on oxidative phosphorylation for ATP generation. Autophagy is activated, but its flux is hindered at the autolysis step, primarily due to an impairment of lysosomal acidity. Cells undergoing replicative senescence also experience AFI, which is also known to be caused by lysosomal acidity failure. Both glucose deprivation and replicative senescence elevate levels of reactive oxygen species (ROS), affecting lysosomal acidification. Mitochondrial alterations play a crucial role in elevating ROS generation and reducing lysosomal acidity, highlighting their association with autophagy dysfunction and disease conditions. This paper delves into the underlying molecular and cellular pathways of AFI in glucose-deprived cells, providing insights into potential strategies for managing AFI that is driven by lysosomal acidity failure. Furthermore, the investigation on the roles of mitochondrial dysfunction sheds light on the potential effectiveness of modulating mitochondrial function to overcome AFI, offering new possibilities for therapeutic interventions.
    Keywords:  V-ATPase; autophagy; impairment of autophagy flux; lysosomal acidity; mitochondria; reactive oxygen species
    DOI:  https://doi.org/10.14348/molcells.2023.0121
  5. Autophagy. 2023 Oct 23. 1-2
      ATG16L1 is an essential component of the Atg8-family protein conjugation machinery, providing membrane targeting for the ATG12-ATG5 conjugate. Recently, we identified an alternative E3-like complex that functions independently of ATG16L1. This complex utilizes the autophagosome-lysosome tethering factor TECPR1 for membrane targeting. TECPR1 is recruited to damaged lysosomal membranes via a direct interaction with sphingomyelin. At the damaged membrane, TECPR1 assembles into an E3-like complex with ATG12-ATG5 to regulate unconventional LC3 lipidation and promote efficient lysosomal repair.
    Keywords:  ESCRT; TECPR1; lysophagy; lysosome; membrane repair
    DOI:  https://doi.org/10.1080/15548627.2023.2267414
  6. Plant J. 2023 Oct 21.
      Autophagy, a fundamental cellular process, plays a vital role in maintaining cellular homeostasis by degrading damaged or unnecessary components. While selective autophagy has been extensively studied in animal cells, its significance in plant cells has only recently gained attention. In this review, we delve into the intriguing realm selective autophagy in plants, with specific focus on its involvement in nutrient recycling, organelle turnover, and stress response. Moreover, recent studies have unveiled the interesting interplay between selective autophagy and epigenetic mechanisms in plants, elucidating the significance of epigenetic regulation in modulating autophagy-related gene expression and finely tuning the selective autophagy process in plants. By synthesizing existing knowledge, this review highlights the emerging field of selective autophagy in plant cells, emphasizing its pivotal role in maintaining nutrient homeostasis, facilitating cellular adaptation, and shedding light on the epigenetic regulation that governs these processes. Our comprehensive study provides the way for a deeper understanding of the dynamic control of cellular responses to nutrient availability and stress conditions, opening new avenues for future research in this field of autophagy in plant physiology.
    Keywords:  ATG8; cellular homeostasis; chlorophagy; environment stress conditions; epigenetic; macroautophagy; plants
    DOI:  https://doi.org/10.1111/tpj.16508
  7. Autophagy. 2023 Oct 24.
      The neuronal metastable proteome includes several aggregation-prone proteins related to neurodegeneration. The complex morphology of neurons with very thin processes and enhanced protein turnover therefore necessitates efficient local machinery to remove excessive protein. In recent work we revealed that chaperone-mediated autophagy (CMA) provides cargo for dendritic exocytic lysosomes, a mechanism that serves in the rapid removal of disease-relevant, supersaturated proteins such as TARDBP/TDP-43 (TAR DNA binding protein) and HTT (huntingtin). We found that lysosomal exocytosis requires docking of the lysosomal protein LAMP2B to the glutamatergic receptor scaffold DLG3/SAP102 and that it is regulated by GRIN/NMDA (N-methyl-D-aspartate)-receptor activity. Thus, the small caliber of dendritic processes might impose a need for local disposal of aggregation-prone proteins like TARDBP and HTT. Moreover, we observed that lysosomal exocytosis might serve in both protein removal and modulation of synaptic processes, and the latter might be an inevitable consequence of the necessity for local disposal of CMA clients in dendrites.
    Keywords:  CMA; DLG3/SAP102; GRIN/NMDAR; LAMP2; exocytosis; lysosomes
    DOI:  https://doi.org/10.1080/15548627.2023.2274256
  8. Methods Mol Biol. 2023 Oct 28.
      Autophagy is a key process that maintains cellular homeostasis. Autophagy contributes to various physiological and pathophysiological processes. Development of methodologies for autophagy detection has greatly facilitated the research on autophagy. Among these methodologies, GFP-LC3 reporter has been popularly used in the literature. In this chapter, we will detail step-by-step the GFP-LC3 reporter protocol we have adapted in our lab. This protocol begins with the generation of lentivirus expressing GFP-LC3. Then, the cells are transduced with titrated virus. After selecting the positive cells, single colonies are isolated, characterized, validated, and used in further study.
    Keywords:  Autophagosome; Autophagy; GFP; LC3; Lysosome
    DOI:  https://doi.org/10.1007/7651_2023_501
  9. Autophagy. 2023 Oct 24.
      ATG5 plays a pivotal role in membrane Atg8ylation, influencing downstream processes encompassing canonical autophagy and noncanonical processes. Remarkably, genetic ablation of ATG5 in myeloid cells leads to an exacerbated pathological state in murine models of tuberculosis, characterized by an early surge in mortality much more severe when compared to the depletion of other components involved in Atg8ylation or canonical autophagy. This study shows that in the absence of ATG5, but not other core canonical autophagy factors, endolysosomal organelles display a lysosomal hypersensitivity phenotype when subjected to damage. This is in part due to a compromised recruitment of ESCRT proteins to lysosomes in need of repair. Mechanistically, in the absence of ATG5, the ESCRT protein PDCD6IP/ALIX is sequestered by the alternative conjugate ATG12-ATG3, contributing to excessive exocytic processes while not being available for lysosomal repair. Specifically, this condition increases secretion of extracellular vesicles and particles, and leads to excessive degranulation in neutrophils. Our findings uncover unique functions of ATG5 outside of the autophagy and Atg8ylation paradigm. This finding is of in vivo relevance for tuberculosis pathogenesis as modeled in mice.
    DOI:  https://doi.org/10.1080/15548627.2023.2273703
  10. Autophagy. 2023 Oct 24.
      Mitophagy, the process of removing damaged mitochondria to promote cell survival, plays a crucial role in cellular functionality. However, excessive, or uncontrolled mitophagy can lead to reduced mitochondrial content that burdens the remaining organelles, triggering mitophagy-mediated cell death. FBXL4 mutations, which affect the substrate-binding adaptor of the CUL1 (cullin 1)-RING ubiquitin ligase complex (CRL1), have been linked to mitochondrial DNA depletion syndrome type 13 (MTDPS13) characterized by reduced mtDNA content and impaired energy production in affected organs. However, the mechanism behind FBXL4 mutation-driven MTDPS13 remain poorly understood. In a recent study, we demonstrate that the CRL1-FBXL4 complex promotes the degradation of BNIP3 and BNIP3L, two key mitophagy cargo receptors. Deficiency of FBXL4 results in a strong accumulation of BNIP3 and BNIP3L proteins and triggers high levels of BNIP3- and BNIP3L-dependent mitophagy. Patient-derived FBXL4 mutations do not affect its interaction with BNIP3 and BNIP3L but impair the assembly of an active CRL1-FBXL4 complex. Furthermore, excessive mitophagy is observed in knockin mice carrying a patient-derived FBXL4 mutation, and in cortical neurons generated from human patient induced pluripotent stem cells (hiPSCs). These findings support the model that the CRL1-FBXL4 complex tightly restricts basal mitophagy, and its dysregulation leads to severe symptoms of MTDPS13.
    Keywords:  Lysosome; mitochondria; mitophagy; multi-system disorder; ubiquitination
    DOI:  https://doi.org/10.1080/15548627.2023.2274260
  11. Glia. 2023 Oct 23.
      Mitochondria support the energetic demands of the cells. Autophagic turnover of mitochondria serves as a critical pathway for mitochondrial homeostasis. It is unclear how bioenergetics and autophagy are functionally connected. Here, we identify an endolysosomal membrane protein that facilitates autophagy to regulate ATP production in glia. We determined that Drosophila tweety (tty) is highly expressed in glia and localized to endolysosomes. Diminished fusion between autophagosomes and endolysosomes in tty-deficient glia was rescued by expressing the human Tweety Homolog 1 (TTYH1). Loss of tty in glia attenuated mitochondrial turnover, elevated mitochondrial oxidative stress, and impaired locomotor functions. The cellular and organismal defects were partially reversed by antioxidant treatment. We performed live-cell imaging of genetically encoded metabolite sensors to determine the impact of tty and autophagy deficiencies on glial bioenergetics. We found that tty-deficient glia exhibited reduced mitochondrial pyruvate consumption accompanied by a shift toward glycolysis for ATP production. Likewise, genetic inhibition of autophagy in glia resulted in a similar glycolytic shift in bioenergetics. Furthermore, the survival of mutant flies became more sensitive to starvation, underlining the significance of tty in the crosstalk between autophagy and bioenergetics. Together, our findings uncover the role for tty in mitochondrial homeostasis via facilitating autophagy, which determines bioenergetic balance in glia.
    Keywords:  Drosophila; autophagy; bioenergetics; endolysosomes; mitochondria; tweety homologs
    DOI:  https://doi.org/10.1002/glia.24484
  12. Autophagy. 2023 Oct 27. 1-3
      ENDOG, a mitochondrial intermembrane space located endonuclease, participates in DNA fragmentation and apoptosis by translocating to the nucleus. ENDOG can also relocate to the mitochondrial matrix, where it regulates mitochondrial genome cleavage. However, the biological function of cytoplasm-translocated ENDOG remains unclear. Our previous study reported that starvation induces the release of ENDOG from mitochondria to the cytoplasm, promoting macroautophagy/autophagy in a process conserved across species. We demonstrate that ENDOG can be phosphorylated by GSK3B, which enhances ENDOG binding to YWHAG/14-3-3γ, and leads to the release of TSC2 and PIK3C3/VPS34 from YWHAG/14-3-3γ, followed by MTORC1 pathway suppression and autophagy initiation. Additionally, we recently reported that ENDOG can also activate the MTORC2-AKT-ACLY signaling axis by promoting the release of RICTOR and TSC2 from YWHAG/14-3-3γ, resulting in acetyl-CoA production. Furthermore, cytoplasmic ENDOG can translocate to the endoplasmic reticulum, where it binds with HSPA5/BIP to release ERN1/IRE1a-EIF2AK3/PERK to activate the endoplasmic reticulum stress response, eventually promoting lipid synthesis. Collectively, ENDOG will be released from the mitochondrial intermembrane space, and translocated to the mitochondrial matrix, cytoplasm, and nucleus during different stress stimulation, where it digests DNA or interacts with crucial proteins to regulate different biological functions, including apoptosis, autophagy, mitophagy, and lipid synthesis.
    Keywords:  Autophagy; ENDOG; MTORC1; MTORC2; endoplasmic reticulum
    DOI:  https://doi.org/10.1080/15548627.2023.2271750
  13. Cell. 2023 Oct 18. pii: S0092-8674(23)01081-4. [Epub ahead of print]
      Lysosomes serve dual antagonistic functions in cells by mediating anabolic growth signaling and the catabolic turnover of macromolecules. How these janus-faced activities are regulated in response to cellular nutrient status is poorly understood. We show here that lysosome morphology and function are reversibly controlled by a nutrient-regulated signaling lipid switch that triggers the conversion between peripheral motile mTOR complex 1 (mTORC1) signaling-active and static mTORC1-inactive degradative lysosomes clustered at the cell center. Starvation-triggered relocalization of phosphatidylinositol 4-phosphate (PI(4)P)-metabolizing enzymes reshapes the lysosomal surface proteome to facilitate lysosomal proteolysis and to repress mTORC1 signaling. Concomitantly, lysosomal phosphatidylinositol 3-phosphate (PI(3)P), which marks motile signaling-active lysosomes in the cell periphery, is erased. Interference with this PI(3)P/PI(4)P lipid switch module impairs the adaptive response of cells to altering nutrient supply. Our data unravel a key function for lysosomal phosphoinositide metabolism in rewiring organellar membrane dynamics in response to cellular nutrient status.
    Keywords:  catabolism; functional proteomics; live correlative light and electron microscopy; lysosomes; mTOR; myotubularin; nutrient signaling; nutrients; phosphoinositides
    DOI:  https://doi.org/10.1016/j.cell.2023.09.027
  14. Cells. 2023 Oct 23. pii: 2504. [Epub ahead of print]12(20):
      The mTOR signaling pathway integrates signaling inputs from nutrients, including glucose and amino acids, which are precisely regulated by transporters depending on nutrient levels. The L-type amino acid transporter 1 (LAT1) affects the activity of mTORC1 through upstream regulators that sense intracellular amino acid levels. While mTORC1 activation by LAT1 has been thoroughly investigated in cultured cells, the effects of LAT1 expression on the activity of mTORC2 has scarcely been studied. Here, we provide evidence that LAT1 recruits and activates mTORC2 on the lysosome for PMA-induced cell migration. LAT1 is translocated to the lysosomes in cells treated with PMA in a dose- and time-dependent manner. Lysosomal LAT1 interacted with mTORC2 through a direct interaction with Rictor, leading to the lysosomal localization of mTORC2. Furthermore, the depletion of LAT1 reduced PMA-induced cell migration in a wound-healing assay. Consistent with these results, the LAT1 N3KR mutant, which is defective in PMA-induced endocytosis and lysosomal localization, did not induce mTORC2 recruitment to the lysosome, with the activation of mTORC2 determined via Akt phosphorylation or the LAT1-mediated promotion of cell migration. Taken together, lysosomal LAT1 recruits and activates the mTORC2 complex and downstream Akt for PMA-mediated cell migration. These results provide insights into the development of therapeutic drugs targeting the LAT1 amino acid transporter to block metastasis, as well as disease progression in various types of cancer.
    Keywords:  LAT1; PMA; cell migration; mTORC2
    DOI:  https://doi.org/10.3390/cells12202504
  15. Chembiochem. 2023 Oct 23. e202300579
      Lipidation of the LC3 protein has frequently been employed as a marker of autophagy. However, LC3-lipidation is also triggered by stimuli not related to canonical autophagy. Therefore, characterization of the driving parameters for LC3 lipidation is crucial to understanding the biological roles of LC3. We identified a pseudo-natural product, termed Inducin, that increases LC3 lipidation independently of canonical autophagy, impairs lysosomal function and rapidly recruits Galectin 3 to lysosomes. Inducin treatment promotes Endosomal Sorting Complex Required for Transport (ESCRT)-dependent membrane repair and transcription factor EB (TFEB)-dependent lysosome biogenesis ultimately leading to cell death.
    Keywords:  Biological activity; Endolysosomal membrane damage; LC3 lipidation; Lysosomal membrane permeabilization; Small molecule
    DOI:  https://doi.org/10.1002/cbic.202300579
  16. Biochem Pharmacol. 2023 Oct 25. pii: S0006-2952(23)00485-9. [Epub ahead of print] 115892
      Cellular senescence is caused by diverse stimuli and contributes to cardiovascular diseases. Several studies have indicated that PPARγ acts as a key mediator of lipid metabolism and shown that it has a protective effect on vascular biology. Nevertheless, the mechanism responsible for the anti-aging effects of PPARγ has not been fully elucidated in vascular smooth muscle cell (VSMC). Furthermore, although mTOR complex 2 (mTORC2) is known to be involved in cellular senescence and autophagy, relatively few studies have investigated its effects as compared with mTOR complex 1 (mTORC1). Therefore, we focused on mTORC2 function and investigated the relationship between PPARγ and mTORC2, and the anti-aging mechanism in VSMC. We found PPARγ activation dose-dependently mitigated the hydrogen peroxide (H2O2)-induced senescence. Treatment of fisetin induced the translocation of PPARγ from cytosol to nuclear and inhibited VSMC senescence. Moreover, activated PPARγ increased PTEN transcription, leading to inhibition of the mTORC2 signaling pathway. We determined mTORC2 activation contributed to senescence by suppressing the FoxO3a-autophagy signaling pathway, and dual knockdown of mTORC1 and mTORC2 decreased cellular senescence and increased autophagy activation more than respective single knockdown. Finally, fisetin acted as a PPARγ activator and inhibited VSMC senescence through the mTORC2-FoxO3a-autophagy signaling pathway. These results demonstrate PPARγ is associated with cellular senescence and that fisetin has an anti-aging effect via PPARγ activation and mTORC2 inhibition in VSMC. These results demonstrate that the mTORC2 signaling pathway regulates autophagy and cellular senescence, which suggests mTORC2 should be considered a significant target for preventing cellular senescence and age-related diseases.
    Keywords:  Autophagy; Fisetin; PPARγ; Senescence; Vascular smooth muscle cell; mTORC2
    DOI:  https://doi.org/10.1016/j.bcp.2023.115892
  17. Cells. 2023 Oct 20. pii: 2496. [Epub ahead of print]12(20):
      Mitochondrial dysfunction in astrocytes has been implicated in the development of various neurological disorders. Mitophagy, mitochondrial autophagy, is required for proper mitochondrial function by preventing the accumulation of damaged mitochondria. The importance of mitophagy, specifically in the astrocytes of the optic nerve (ON), has been little studied. We introduce an animal model in which two separate mutations act synergistically to produce severe ON degeneration. The first mutation is in Cryba1, which encodes βA3/A1-crystallin, a lens protein also expressed in astrocytes, where it regulates lysosomal pH. The second mutation is in Bckdk, which encodes branched-chain ketoacid dehydrogenase kinase, which is ubiquitously expressed in the mitochondrial matrix and involved in the catabolism of the branched-chain amino acids. BCKDK is essential for mitochondrial function and the amelioration of oxidative stress. Neither of the mutations in isolation has a significant effect on the ON, but animals homozygous for both mutations (DM) exhibit very serious ON degeneration. ON astrocytes from these double-mutant (DM) animals have lysosomal defects, including impaired mitophagy, and dysfunctional mitochondria. Urolithin A can rescue the mitophagy impairment in DM astrocytes and reduce ON degeneration. These data demonstrate that efficient mitophagy in astrocytes is required for ON health and functional integrity.
    Keywords:  BCKDK; astrocytes; autophagy; lysosome; mitochondria; mitophagy; optic nerve; βA3/A1-crystallin
    DOI:  https://doi.org/10.3390/cells12202496
  18. Int J Mol Sci. 2023 Oct 11. pii: 15078. [Epub ahead of print]24(20):
      Diabetic cardiomyopathy is a critical diabetes-mediated co-morbidity characterized by cardiac dysfunction and heart failure, without predisposing hypertensive or atherosclerotic conditions. Metabolic insulin resistance, promoting hyperglycemia and hyperlipidemia, is the primary cause of diabetes-related disorders, but ambiguous tissue-specific insulin sensitivity has shed light on the importance of identifying a unified target paradigm for both the glycemic and non-glycemic context of type 2 diabetes (T2D). Several studies have indicated hyperactivation of the mammalian target of rapamycin (mTOR), specifically complex 1 (mTORC1), as a critical mediator of T2D pathophysiology by promoting insulin resistance, hyperlipidemia, inflammation, vasoconstriction, and stress. Moreover, mTORC1 inhibitors like rapamycin and their analogs have shown significant benefits in diabetes and related cardiac dysfunction. Recently, FDA-approved anti-hyperglycemic sodium-glucose co-transporter 2 inhibitors (SGLT2is) have gained therapeutic popularity for T2D and diabetic cardiomyopathy, even acknowledging the absence of SGLT2 channels in the heart. Recent studies have proposed SGLT2-independent drug mechanisms to ascertain their cardioprotective benefits by regulating sodium homeostasis and mimicking energy deprivation. In this review, we systematically discuss the role of mTORC1 as a unified, eminent target to treat T2D-mediated cardiac dysfunction and scrutinize whether SGLT2is can target mTORC1 signaling to benefit patients with diabetic cardiomyopathy. Further studies are warranted to establish the underlying cardioprotective mechanisms of SGLT2is under diabetic conditions, with selective inhibition of cardiac mTORC1 but the concomitant activation of mTORC2 (mTOR complex 2) signaling.
    Keywords:  AMPK; SGLT2i; diabetes; diabetic cardiomyopathy; mTORC1/2
    DOI:  https://doi.org/10.3390/ijms242015078
  19. Sci Rep. 2023 Oct 21. 13(1): 18024
      Neurodegenerative disorders are characterized by the brain deposition of insoluble amyloidogenic proteins, such as α-synuclein or Tau, and the concomitant deterioration of cell functions such as the autophagy-lysosomal pathway (ALP). The ALP is involved in the degradation of intracellular macromolecules including protein aggregates. ALP dysfunction due to inherited defects in lysosomal or non-lysosomal proteins causes a group of diseases called lysosomal storage disorders (LSD) because of abnormal accumulation of lysosomal degradation substrates. Supporting the contribution of ALP defects in neurodegenerative diseases, deposition of amyloidogenic proteins occurs in LSD. Moreover, heterozygous mutations of several ALP genes represent risk factors for Parkinson's disease. The reciprocal contribution of α-synuclein accumulation and lysosomal dysfunction have been extensively studied. However, whether this adverse crosstalk also embraces Tau pathology needs more investigation. Here, we show in human primary fibroblasts that Tau seeds isolated from the brain of Alzheimer's disease induce Tau accumulation in acidic degradative organelles and lysosomal stress. Furthermore, inhibition of glucocerebrosidase, a lysosomal enzyme mutated in Gaucher's disease and a main risk for Parkinson's disease, causes lysosomal dysfunction in primary fibroblasts and contributes to the accumulation of Tau. Considering the presence of Tau lesions in Parkinson's disease as well as in multiple neurodegenerative disorders including Alzheimer's disease, our data call for further studies on strategies to alleviate ALP dysfunction as new therapeutic opportunity for neurodegenerative diseases and LSD.
    DOI:  https://doi.org/10.1038/s41598-023-44979-7
  20. Cell Mol Biol Lett. 2023 Oct 24. 28(1): 85
       BACKGROUND: Ubiquitin-proteasome-system-mediated clearance of misfolded proteins is essential for cells to maintain proteostasis and reduce the proteotoxicity caused by these aberrant proteins. When proteasome activity is inadequate, ubiquitinated proteins are sorted into perinuclear aggresomes, which is a significant defense mechanism employed by cells to combat insufficient proteasome activity, hence mitigating the proteotoxic crisis. It has been demonstrated that phosphorylation of SQSTM1 is crucial in regulating misfolded protein aggregation and autophagic degradation. Although SQSTM1 S403 phosphorylation is essential for the autophagic degradation of ubiquitinated proteins, its significance in proteasome inhibition-induced aggresome formation is yet unknown. Herein, we investigated the influence of SQSTM1 S403 phosphorylation on the aggresome production of ubiquitinated proteins during proteasome suppression.
    METHODS: We examined the phosphorylation levels of SQSTM1 S403 or T269/S272 in cells after treated with proteasome inhibitors or/and autophagy inhibitors, by western blot and immunofluorescence. We detected the accumulation and aggresome formation of ubiquitinated misfolded proteins in cells treated with proteasome inhibition by western blot and immunofluorescence. Furthermore, we used SQSTM1 phosphorylation-associated kinase inhibitors and mutant constructs to confirm the regulation of different SQSTM1 phosphorylation in aggresome formation. We examined the cell viability using CCK-8 assay.
    RESULTS: Herein, we ascertained that phosphorylation of SQSTM1 S403 did not enhance the autophagic degradation of ubiquitinated proteins during proteasome inhibition. Proteasome inhibition suppresses the phosphorylation of SQSTM1 S403, which facilitated the aggresome production of polyubiquitinated proteins. Interestingly, we found proteasome inhibition-induced SQSTM1 T269/S272 phosphorylation inhibits the S403 phosphorylation. Suppressing S403 phosphorylation rescues the defective aggresome formation and protects cells from cell death caused by unphosphorylated SQSTM1 (T269/S272).
    CONCLUSIONS: This study shows that inhibition of SQSTM1 S403 phosphorylation facilitates the aggresome formation of ubiquitinated proteins during proteasome dysfunction. SQSTM1 T269/S272 phosphorylation inhibits the S403 phosphorylation, boosting the aggresome formation of ubiquitinated protein and shielding cells from proteotoxic crisis.
    Keywords:  Aggrephagy; Aggresome; Cell death; Proteasome inhibition; SQSTM1 phosphorylation
    DOI:  https://doi.org/10.1186/s11658-023-00500-6
  21. Clin Exp Hypertens. 2023 Dec 31. 45(1): 2271186
      Objective: Excessive proliferation and migration of pulmonary arterial smooth muscle cell (PASMC) is a core event of pulmonary hypertension (PH). Regulators of G protein signaling 10 (RGS10) can regulate cellular proliferation and cardiopulmonary diseases. We demonstrate whether RGS10 also serves as a regulator of PH.Methods: PASMC was challenged by hypoxia to induce proliferation and migration. Adenovirus carrying Rgs10 gene (Ad-Rgs10) was used for external expression of Rgs10. Hypoxia/SU5416 or MCT was used to induce PH. Right ventricular systolic pressure (RVSP) and right ventricular hypertrophy index (RVHI) were used to validate the establishment of PH model.Results: RGS10 was downregulated in hypoxia-challenged PASMC. Ad-Rgs10 significantly suppressed proliferation and migration of PASMC after hypoxia stimulus, while silencing RGS10 showed contrary effect. Mechanistically, we observed that phosphorylation of S6 and 4E-Binding Protein 1 (4EBP1), the main downstream effectors of mammalian target of rapamycin complex 1 (mTORC1) as well as phosphorylation of AKT, the canonical upstream of mTORC1 in hypoxia-induced PASMC were negatively modulated by RGS10. Both recovering mTORC1 activity and restoring AKT activity abolished these effects of RGS10 on PASMC. More importantly, AKT activation also abolished the inhibitory role of RGS10 in mTORC1 activity in hypoxia-challenged PASMC. Finally, we also observed that overexpression of RGS10 in vivo ameliorated pulmonary vascular wall thickening and reducing RVSP and RVHI in mouse PH model.Conclusion: Our findings reveal the modulatory role of RGS10 in PASMC and PH via AKT/mTORC1 axis. Therefore, targeting RGS10 may serve as a novel potent method for the prevention against PH."
    Keywords:  AKT; RGS10; mTORC1; pulmonary arterial smooth muscle cell; pulmonary hypertension
    DOI:  https://doi.org/10.1080/10641963.2023.2271186
  22. Biochem Biophys Res Commun. 2023 Oct 10. pii: S0006-291X(23)01185-3. [Epub ahead of print]684 149101
      Tripartite motif (TRIM) family proteins as E3-ligases participate in various biological processes. TRIM21, as the first autoantibody protein, has been found to be associated with autophagy. However, the role of TRIM21 engaging in autophagy is still unclear. In this study, TRIM21 forms significate puncta in the cytoplasm and undergoes liquid-liquid phase separation in vitro. Furthermore, we identify phase separation of the coiled-coil (CC) domain is essential for autophagosome to mediate autophagy-related protein recruited. These findings show that phase separation of the CC domain of TRIM21 promotes autophagosome to impact cell fate.
    Keywords:  Autophagy; Coiled-coil domain; Phase separation; TRIM21
    DOI:  https://doi.org/10.1016/j.bbrc.2023.10.033
  23. Mol Autism. 2023 10 25. 14(1): 39
       BACKGROUND: Tuberous sclerosis complex (TSC) is an inherited neurocutaneous disorder caused by mutations in the TSC1 or TSC2 genes, with patients often exhibiting neurodevelopmental (ND) manifestations termed TSC-associated neuropsychiatric disorders (TAND) including autism spectrum disorder (ASD) and intellectual disability. Hamartin (TSC1) and tuberin (TSC2) proteins form a complex inhibiting mechanistic target of rapamycin complex 1 (mTORC1) signaling. Loss of TSC1 or TSC2 activates mTORC1 that, among several targets, controls protein synthesis by inhibiting translational repressor eIF4E-binding proteins. Using TSC1 patient-derived neural progenitor cells (NPCs), we recently reported early ND phenotypic changes, including increased cell proliferation and altered neurite outgrowth in TSC1-null NPCs, which were unaffected by the mTORC1 inhibitor rapamycin.
    METHODS: Here, we used polysome profiling, which quantifies changes in mRNA abundance and translational efficiencies at a transcriptome-wide level, to compare CRISPR-edited TSC1-null with CRISPR-corrected TSC1-WT NPCs generated from one TSC donor (one clone/genotype). To assess the relevance of identified gene expression alterations, we performed polysome profiling in postmortem brains from ASD donors and age-matched controls. We further compared effects on translation of a subset of transcripts and rescue of early ND phenotypes in NPCs following inhibition of mTORC1 using the allosteric inhibitor rapamycin versus a third-generation bi-steric, mTORC1-selective inhibitor RMC-6272.
    RESULTS: Polysome profiling of NPCs revealed numerous TSC1-associated alterations in mRNA translation that were largely recapitulated in human ASD brains. Moreover, although rapamycin treatment partially reversed the TSC1-associated alterations in mRNA translation, most genes related to neural activity/synaptic regulation or ASD were rapamycin-insensitive. In contrast, treatment with RMC-6272 inhibited rapamycin-insensitive translation and reversed TSC1-associated early ND phenotypes including proliferation and neurite outgrowth that were unaffected by rapamycin.
    CONCLUSIONS: Our work reveals ample mRNA translation alterations in TSC1 patient-derived NPCs that recapitulate mRNA translation in ASD brain samples. Further, suppression of TSC1-associated but rapamycin-insensitive translation and ND phenotypes by RMC-6272 unveils potential implications for more efficient targeting of mTORC1 as a superior treatment strategy for TAND.
    Keywords:  Autism spectrum disorder; Early neurodevelopment; Neural progenitor cells; Polysome profiling; RMC-6272; TSC1; Translatome; Tuberous sclerosis complex; mTORC1
    DOI:  https://doi.org/10.1186/s13229-023-00572-3
  24. Autophagy. 2023 Oct 24.
      Intervertebral disc degeneration (IDD) is the most critical pathological factor in the development of low back pain. The maintenance of nucleus pulposus (NP) cell and intervertebral disc integrity benefits largely from well-controlled mitochondrial quality, surveilled by mitochondrial dynamics (fission and fusion) and mitophagy, but the outcome is cellular context-dependent that remain to be clarified. Our studies revealed that the loss of NLRX1 is correlated with NP cell senescence and IDD progression, which involve disordered mitochondrial quality. Further using animal and in vitro tissue and cell models, we demonstrated that NLRX1 could facilitate mitochondrial quality by coupling mitochondrial dynamic factors (p-DNM1L, L-OPA1:S-OPA1, OMA1) and mitophagy activity. Conversely, mitochondrial collapse occurred in NLRX1-defective NP cells and switched on the compensatory PINK1-PRKN pathway that led to excessive mitophagy and aggressive NP cell senescence. Mechanistically, NLRX1 was originally shown to interact with zinc transporter SLC39A7 and modulate mitochondrial Zn2+ trafficking via the formation of an NLRX1-SLC39A7 complex on the mitochondrial membrane of NP cells, subsequently orchestrating mitochondrial dynamics and mitophagy. The restoration of NLRX1 function by gene overexpression or pharmacological agonist (NX-13) treatment showed great potential for regulating mitochondrial fission with synchronous fusion and mitophagy, thus sustaining mitochondrial homeostasis, ameliorating NP cell senescence and rejuvenating intervertebral discs. Collectively, our findings highlight a working model whereby the NLRX1-SLC39A7 complex coupled mitochondrial dynamics and mitophagy activity to surveil and target damaged mitochondria for degradation, which determines the beneficial function of the mitochondrial surveillance system and ultimately rejuvenates intervertebral discs.
    Keywords:  Intervertebral disc degeneration; NLRX1; SLC39A7; mitochondrial dynamics; mitophagy; nucleus pulposus
    DOI:  https://doi.org/10.1080/15548627.2023.2274205
  25. Cancers (Basel). 2023 Oct 18. pii: 5029. [Epub ahead of print]15(20):
      Androgen receptor targeting remains the primary therapeutic strategy in prostate cancer, encompassing androgen biosynthesis inhibitors and androgen receptor antagonists. While both androgen-receptor-positive and "castration-resistant" prostate cancer are responsive to these approaches, the development of resistance is an almost inevitable outcome leading to the castration-resistant form of the disease. Given that "cytoprotective" autophagy is considered to be a predominant mechanism of resistance to various chemotherapeutic agents as well as to radiation in the cancer literature, the purpose of this review is to evaluate whether autophagy plays a central role in limiting the utility of androgen deprivation therapies in prostate cancer. Unlike most of our previous reports, where multiple functional forms of autophagy were identified, making it difficult if not impossible to propose autophagy inhibition as a therapeutic strategy, the cytoprotective form of autophagy appears to predominate in the case of androgen deprivation therapies. This opens a potential pathway for improving the outcomes for prostate cancer patients once effective and reliable pharmacological autophagy inhibitors have been developed.
    Keywords:  abiraterone; apalutamide; autophagy; bicalutamide; cytoprotective; enzalutamide
    DOI:  https://doi.org/10.3390/cancers15205029
  26. Front Neurosci. 2023 ;17 1235241
      Major depressive disorder is a global psychiatric condition characterized by persistent low mood and anhedonia, which seriously jeopardizes the physical and mental well-being of affected individuals. While various hypotheses have been proposed to explicate the etiology of depression, the precise pathogenesis and effective treatment of this disorder remain elusive. Mitochondria, as the primary organelles responsible for cellular energy production, possess the ability to meet the essential energy demands of the brain. Research indicated that the accumulation of damaged mitochondria is associated with the onset of depression. Mitophagy, a type of cellular autophagy, specifically targets and removes excess or damaged mitochondria. Emerging evidence demonstrated that mitophagy dysfunction was involved in the progression of depression, and several pharmacological interventions that stimulating mitophagy exerted excellent antidepressant actions. We provided an overview of updated advancements on the regulatory mechanism of mitophagy and the mitophagy abnormality in depressed patients and animals, as well as in cell models of depression. Meanwhile, various therapeutic strategies to restore mitophagy for depression alleviation were also discussed in this review.
    Keywords:  depression; drug therapy; mitochondria; mitophagy; regulatory mechanism
    DOI:  https://doi.org/10.3389/fnins.2023.1235241
  27. PLoS Biol. 2023 Oct 23. 21(10): e3002337
      The endoplasmic reticulum (ER) forms contacts with the lysosomal compartment, regulating lysosome positioning and motility. The movements of lysosomes are controlled by the attachment of molecular motors to their surface. However, the molecular mechanisms by which ER controls lysosome dynamics are still elusive. Here, using mouse brain extracts and mouse embryonic fibroblasts, we demonstrate that spatacsin is an ER-resident protein regulating the formation of tubular lysosomes, which are highly dynamic. Screening for spatacsin partners required for tubular lysosome formation showed spatacsin to act by regulating protein degradation. We demonstrate that spatacsin promotes the degradation of its partner AP5Z1, which regulates the relative amount of spastizin and AP5Z1 at lysosomes. Spastizin and AP5Z1 contribute to regulate tubular lysosome formation, as well as their trafficking by interacting with anterograde and retrograde motor proteins, kinesin KIF13A and dynein/dynactin subunit p150Glued, respectively. Ultimately, investigations in polarized mouse cortical neurons in culture demonstrated that spatacsin-regulated degradation of AP5Z1 controls the directionality of lysosomes trafficking. Collectively, our results identify spatacsin as a protein regulating the directionality of lysosome trafficking.
    DOI:  https://doi.org/10.1371/journal.pbio.3002337
  28. J Fungi (Basel). 2023 Oct 11. pii: 1003. [Epub ahead of print]9(10):
      Endocytosis and autophagy are the main pathways to deliver cargoes in vesicles and autophagosomes, respectively, to vacuoles/lysosomes in eukaryotes. Multiple positive regulators but few negative ones are reported to regulate the entry of vesicles and autophagosomes into vacuoles/lysosomes. In yeast, the Rab5 GTPase Vps21 and the ESCRT (endosomal sorting complex required for transport) are positive regulators in endocytosis and autophagy. During autophagy, Vps21 regulates the ESCRT to phagophores (unclosed autophagosomes) to close them. Phagophores accumulate on vacuolar membranes in both vps21∆ and ESCRT mutant cells under a short duration of nitrogen starvation. The vacuolar transport chaperon (VTC) complex proteins are recently found to be negative regulators in endocytosis and autophagy. Phagophores in vps21∆ cells are promoted to enter vacuoles when the VTC complex proteins are absent. Phagophores are easily observed inside vacuoles when any of these VTC complex proteins (Vtc1, 2, 4, 5) are removed. However, it is unknown whether the removal of VTC complex proteins will also promote the entry of phagophores into vacuoles in ESCRT mutant cells under the same conditions. Snf7 is a core subunit of ESCRT subcomplex III (ESCRT-III), and phagophores accumulate in snf7∆ cells under a short duration of nitrogen starvation. We used green fluorescence protein (GFP) labeled Atg8 to display phagophores and FM4-64-stained or Vph1-GFP-labeled membrane structures to show vacuoles, then examined fluorescence localization and GFP-Atg8 degradation in snf7∆ and snf7∆vtc4∆ cells. Results showed that Vtc4 depletion promoted the entry of phagophores in snf7∆ cells into vacuoles as it did for vps21∆ cells, although the promotion level was more obvious in vps21∆ cells. This observation indicates that the VTC complex proteins may have a widespread role in negatively regulating cargos to enter vacuoles in yeast.
    Keywords:  ESCRT; Snf7; VTC complex; Vps21; Vtc4; autophagy; phagophore; vacuole
    DOI:  https://doi.org/10.3390/jof9101003
  29. Biomolecules. 2023 Sep 27. pii: 1454. [Epub ahead of print]13(10):
      Autophagy is a highly conserved intracellular degradation pathway in eukaryotic organisms, playing an adaptive role in various pathophysiological processes throughout evolution. Inflammation is the immune system's response to external stimuli and tissue damage. However, persistent inflammatory reactions can lead to a range of inflammatory diseases and cancers. The interaction between autophagy and inflammation is particularly evident during viral infections. As a crucial regulator of inflammation, autophagy can either promote or inhibit the occurrence of inflammatory responses. In turn, inflammation can establish negative feedback loops by modulating autophagy to suppress excessive inflammatory reactions. This interaction is pivotal in the pathogenesis of viral diseases. Therefore, elucidating the regulatory roles of autophagy and inflammation in viral infections will significantly enhance our understanding of the mechanisms underlying related diseases. Furthermore, it will provide new insights and theoretical foundations for disease prevention, treatment, and drug development.
    Keywords:  NLRP3 inflammasome; autophagy; inflammation; viral infection; viral replication
    DOI:  https://doi.org/10.3390/biom13101454
  30. Eur J Med Res. 2023 Oct 25. 28(1): 456
      Insulin is a critical hormone that promotes energy storage in various tissues, as well as anabolic functions. Insulin resistance significantly reduces these responses, resulting in pathological conditions, such as obesity and type 2 diabetes mellitus (T2DM). The management of insulin resistance requires better knowledge of its pathophysiological mechanisms to prevent secondary complications, such as cardiovascular diseases (CVDs). Recent evidence regarding the etiological mechanisms behind insulin resistance emphasizes the role of energy imbalance and neurohormonal dysregulation, both of which are closely regulated by autophagy. Autophagy is a conserved process that maintains homeostasis in cells. Accordingly, autophagy abnormalities have been linked to a variety of metabolic disorders, including insulin resistance, T2DM, obesity, and CVDs. Thus, there may be a link between autophagy and insulin resistance. Therefore, the interaction between autophagy and insulin function will be examined in this review, particularly in insulin-responsive tissues, such as adipose tissue, liver, and skeletal muscle.
    Keywords:  Adipose tissue; Autophagy; Diabetes complications; Insulin resistance; Type 2 diabetes
    DOI:  https://doi.org/10.1186/s40001-023-01424-9
  31. Brain. 2023 Oct 26. pii: awad370. [Epub ahead of print]
      FTD and ALS are two untreatable neurodegenerative diseases that exist on a clinical, genetic, and pathological spectrum. The VCP gene is highly relevant, being directly implicated in both FTD and ALS. Here, we investigate the effects of VCP mutations on the cellular homoeostasis of hiPSC-derived cortical neurons, focusing on endo-lysosomal biology and tau pathology. We find that VCP mutations cause abnormal accumulation of enlarged endo-lysosomes accompanied with impaired interaction between nuclear FUS and SFPQ in human cortical neurons. The spatial dissociation of intra-nuclear FUS and SFPQ correlates with alternative splicing of the MAPT pre-mRNA and increased tau phosphorylation. Importantly, we show that increased 4R tau using antisense oligonucleotide technology is sufficient to drive toxic changes in control human neurons, which phenocopy VCP-mutant neurons. In summary, our findings demonstrate that tau hyperphosphorylation, endolysosomal dysfunction, lysosomal membrane rupture, endoplasmic reticulum stress and apoptosis are driven by a pathogenic increase in 4R tau.
    Keywords:  FUS; SFPQ; iPSC; lysosome; tauopathy
    DOI:  https://doi.org/10.1093/brain/awad370
  32. J Prev Alzheimers Dis. 2023 ;10(4): 661-668
      Age remains the largest risk factor in the development of neurodegenerative diseases such as Alzheimer's disease (AD). Numerous cellular hallmarks of aging contribute to the advancement of the pathologies associated with neurodegenerative disease. Not all cellular hallmarks of aging are independent and several fall into the broader category of cellular rejuvenation, which captures returning cells to a more youthful, improved functional state. Cellular rejuvenation is quickly becoming a hot topic in the development of novel therapeutic modalities for a range of diseases. Therapeutic approaches utilizing cellular rejuvenation technologies are rapidly advancing and will represent the next phase of AD therapeutics. This review focuses on two important processes, epigenetic reprogramming, and chaperone-mediated autophagy (CMA) that play a critical role in aging and in neurodegenerative diseases and the potential therapeutic approaches (gene therapy, small molecule) towards targeting these mechanisms. In aging and in AD, epigenetic changes on DNA (e.g., hypermethylation on CpG islands) lead to alterations in gene expression. Partial epigenetic reprogramming utilizes transcription factors to remove the epigenetic marks and to rejuvenate cells to a more youthful state. During aging and in neurodegenerative disorders, CMA becomes impaired resulting in a buildup of proteins known to be associated with neurodegenerative pathologies. The protein buildups lead to aggregates that preclude proteostasis leading to cell toxicity. Small-molecule CMA activators restore proteostasis and limit toxicity enabling cellular rejuvenation.
    Keywords:  Epigenetic reprogramming; LAMP2A; OSK; cellular rejuvenation; chaperone-mediated autophagy
    DOI:  https://doi.org/10.14283/jpad.2023.106
  33. bioRxiv. 2023 Oct 02. pii: 2023.10.02.560519. [Epub ahead of print]
      ApoE4 is the primary risk factor for Alzheimer's Disease. While apoE is primarily expressed by astrocytes, AD pathology including endosomal abnormalities and mitochondrial dysfunction first occurs in neurons. Lysosomes are poised at the convergence point between these features. We find that apoE4-expressing cells exhibit lysosomal alkalinization, reduced lysosomal proteolysis, and impaired mitophagy. To identify driving factors for this lysosomal dysfunction, we performed quantitative lysosomal proteome profiling. This revealed that apoE4 expression results in lysosomal depletion of Lgals3bp and accumulation of Tmed5 in both Neuro-2a cells and postmitotic human neurons. Modulating the expression of both proteins affected lysosomal function, with Tmed5 knockdown rescuing lysosomal alkalinization in apoE4 cells, and Lgals3bp knockdown causing lysosomal alkalinization and reduced lysosomal density in apoE3 cells. Taken together, our work reveals that apoE4 exerts gain-of-toxicity by alkalinizing the lysosomal lumen, pinpointing lysosomal Tmed5 accumulation and Lgals3bp depletion as apoE4-associated drivers for this phenotype.
    DOI:  https://doi.org/10.1101/2023.10.02.560519
  34. Int J Biol Macromol. 2023 Oct 19. pii: S0141-8130(23)04463-X. [Epub ahead of print] 127566
      The serine/threonine kinase PINK1 is responsible for phosphorylating a ubiquitin (Ub)-like domain in an E3 Ub ligase Parkin protein and a Parkin-bound Ub. PINK1 works as a mitochondrial quality control by phosphorylating and activating the E3 ubiquitin ligase Parkin. Recent medicinal study has reported that mutations of Parkin and PINK1 cause defects in mitophagy and induce early-onset Parkinson's disease (EOPD). In this study, we conducted molecular dynamics simulations to investigate the structural discrepancy caused by a clinical G409V mutation in PINK1 kinase domain's A-loop. The Ub phosphorylation begins with PINK1 D362 deprotonating the hydroxyl group of the substrate Ub's S65' and PINK1's A-loop is responsible for coordinating S65'. On contrary to G409 offering structural plasticity, the replaced, bulky V409 interferes with the alignment of D362-S65', seriously hampering Ub phosphorylation, leading to the accumulation of damaged mitochondria, and ultimately EOPD. In this study, we predicted the hPINK1WT-UbWT binding mode and detected the structural impact brought by G409V replacement. It is expected the concluded remarks to be beneficial for developing cures to alleviate structural interference and restore PINK1 function.
    Keywords:  Early-onset Parkinson's disease; Molecular dynamics simulations; PINK1
    DOI:  https://doi.org/10.1016/j.ijbiomac.2023.127566
  35. Autophagy. 2023 Oct 26.
      In neurons, autophagosome biogenesis occurs mainly in distal axons, followed by maturation during retrograde transport. Autophagosomal growth depends on the supply of membrane lipids which requires small vesicles containing ATG9, a lipid scramblase essential for macroautophagy/autophagy. Here, we show that ATG9-containing vesicles are enriched in synapses and resemble synaptic vesicles in size and density. The proteome of ATG9-containing vesicles immuno-isolated from nerve terminals showed conspicuously low levels of trafficking proteins except of the AP2-complex and some enzymes involved in endosomal phosphatidylinositol metabolism. Super resolution microscopy of nerve terminals and isolated vesicles revealed that ATG9-containing vesicles represent a distinct vesicle population with limited overlap not only with synaptic vesicles but also other membranes of the secretory pathway, uncovering a surprising heterogeneity in their membrane composition. Our results are compatible with the view that ATG9-containing vesicles function as lipid shuttles that scavenge membrane lipids from various intracellular membranes to support autophagosome biogenesis.
    Keywords:  ATG9; RAB26; autophagy; synapse; synaptic vesicles; vesicle proteome
    DOI:  https://doi.org/10.1080/15548627.2023.2274204
  36. Elife. 2023 Oct 24. pii: e87255. [Epub ahead of print]12
      Leucine-rich repeat kinase 2 (LRRK2) variants associated with Parkinson's disease (PD) and Crohn's disease lead to increased phosphorylation of its Rab substrates. While it has been recently shown that perturbations in cellular homeostasis including lysosomal damage can increase LRRK2 activity and localization to lysosomes, the molecular mechanisms by which LRRK2 activity is regulated have remained poorly defined. We performed a targeted siRNA screen to identify regulators of LRRK2 activity and identified Rab12 as a novel modulator of LRRK2-dependent phosphorylation of one of its substrates, Rab10. Using a combination of imaging and immunopurification methods to isolate lysosomes, we demonstrated that Rab12 is actively recruited to damaged lysosomes and leads to a local and LRRK2-dependent increase in Rab10 phosphorylation. PD-linked variants, including LRRK2 R1441G and VPS35 D620N, lead to increased recruitment of LRRK2 to the lysosome and a local elevation in lysosomal levels of pT73 Rab10. Together, these data suggest a conserved mechanism by which Rab12, in response to damage or expression of PD-associated variants, facilitates the recruitment of LRRK2 and phosphorylation of its Rab substrate(s) at the lysosome.
    Keywords:  cell biology; human
    DOI:  https://doi.org/10.7554/eLife.87255
  37. Int J Mol Sci. 2023 Oct 12. pii: 15116. [Epub ahead of print]24(20):
      The aryl hydrocarbon receptor (AHR) is a ligand-activated transcription factor and a substrate protein of a Cullin 4B E3 ligase complex responsible for diverse cellular processes. In the lung, this receptor is responsible for the bioactivation of benzo[a]pyrene during tumorigenesis. Realizing that the AHR function is affected by its expression level, we are interested in the degradation mechanism of AHR in the lung. Here, we have investigated the mechanism responsible for AHR degradation using human lung epithelial A549 cells. We have observed that the AHR protein levels increase in the presence of chloroquine (CQ), an autophagy inhibitor, in a dose-dependent manner. Treatment with 6-aminonicotinamide (6-AN), a chaperone-mediated autophagy (CMA) activator, decreases AHR protein levels in a concentration-dependent and time-dependent manner. This decrease suppresses the ligand-dependent activation of the AHR target gene transcription, and can be reversed by CQ but not MG132. Knockdown of lysosome-associated membrane protein 2 (LAMP2), but not autophagy-related 5 (ATG5), suppresses the chloroquine-mediated increase in the AHR protein. AHR is resistant to CMA when its CMA motif is mutated. Suppression of the epithelial-to-mesenchymal transition in A549 cells is observed when the AHR gene is knocked out or the AHR protein level is reduced by 6-AN. Collectively, we have provided evidence supporting that AHR is continuously undergoing CMA and activation of CMA suppresses the AHR function in A549 cells.
    Keywords:  6-AN; A549; CQ; LAMP2; aryl hydrocarbon receptor; chaperone-mediated autophagy
    DOI:  https://doi.org/10.3390/ijms242015116
  38. Cells. 2023 Oct 19. pii: 2486. [Epub ahead of print]12(20):
      Modulation of autophagy as an anticancer strategy has been widely studied and evaluated in several cell models. However, little attention has been paid to the metabolic changes that occur in a cancer cell when autophagy is inhibited or induced. In this review, we describe how the expression and regulation of various autophagy-related (ATGs) genes and proteins are associated with cancer progression and cancer plasticity. We present a comprehensive review of how deregulation of ATGs affects cancer cell metabolism, where inhibition of autophagy is mainly reflected in the enhancement of the Warburg effect. The importance of metabolic changes, which largely depend on the cancer type and form part of a cancer cell's escape strategy after autophagy modulation, is emphasized. Consequently, pharmacological strategies based on a dual inhibition of metabolic and autophagy pathways emerged and are reviewed critically here.
    Keywords:  Warburg effect; aerobic glycolysis; autophagy; autophagy-related (ATGs) genes/proteins; cancer cell metabolism; cancer plasticity; fatty acid oxidation (FAO); tumor microenvironment
    DOI:  https://doi.org/10.3390/cells12202486
  39. Mol Cancer Res. 2023 Oct 26.
      mTOR is a serine/threonine kinase that controls prostate cancer (PCa) cell growth in part by regulating gene programs associated with metabolic and cell proliferation pathways. mTOR-mediated control of gene expression can be achieved via phosphorylation of transcription factors, leading to changes in their cellular localization and activities. mTOR also directly associates with chromatin in complex with transcriptional regulators, including the androgen receptor (AR). Nuclear mTOR (nmTOR) has been previously shown to act as a transcriptional integrator of the androgen signaling pathway in association with the chromatin remodeling machinery, AR, and FOXA1. However, the contribution of cytoplasmic mTOR (cmTOR) and nmTOR and the role played by FOXA1 in this process remains to be explored. Herein, we engineered cells expressing mTOR tagged with nuclear localization and export signals dictating mTOR localization. Transcriptome profiling in AR-positive PCa cells revealed that nmTOR generally downregulates a subset of the androgen response pathway independently of its kinase activity, while cmTOR upregulates a cell cycle-related gene signature in a kinase-dependent manner. Biochemical and genome-wide transcriptomic analyses demonstrate that nmTOR functionally interacts with AR and FOXA1. Ablation of FOXA1 reprograms the nmTOR cistrome and transcriptome of androgen responsive PCa cells. This works highlights a transcriptional regulatory pathway in which direct interactions between nmTOR, AR and FOXA1 dictate a combinatorial role for these factors in the control of specific gene programs in PCa cells. Implications: The finding that canonical and nuclear mTOR signaling pathways control distinct gene programs opens therapeutic opportunities to modulate mTOR activity in PCa cells.
    DOI:  https://doi.org/10.1158/1541-7786.MCR-23-0087
  40. PLoS Pathog. 2023 Oct 23. 19(10): e1011748
      Acquired stress resistance (ASR) enables organisms to prepare for environmental changes that occur after an initial stressor. However, the genetic basis for ASR and how the underlying network evolved remain poorly understood. In this study, we discovered that a short phosphate starvation induces oxidative stress response (OSR) genes in the pathogenic yeast C. glabrata and protects it against a severe H2O2 stress; the same treatment, however, provides little benefit in the low pathogenic-potential relative, S. cerevisiae. This ASR involves the same transcription factors (TFs) as the OSR, but with different combinatorial logics. We show that Target-of-Rapamycin Complex 1 (TORC1) is differentially inhibited by phosphate starvation in the two species and contributes to the ASR via its proximal effector, Sch9. Therefore, evolution of the phosphate starvation-induced ASR involves the rewiring of TORC1's response to phosphate limitation and the repurposing of TF-target gene networks for the OSR using new regulatory logics.
    DOI:  https://doi.org/10.1371/journal.ppat.1011748
  41. Commun Biol. 2023 Oct 24. 6(1): 1080
      Stimulation of autophagy could provide powerful therapies for multiple diseases, including cancer and neurodegeneration. An attractive drug target for this purpose is Bcl-2, which inhibits autophagy by binding to the Beclin 1 BH3-domain. However, compounds that preclude Beclin 1/Bcl-2 binding might also induce apoptosis, which is inhibited by binding of Bcl-2 to BH3-domains of pro-apoptosis factors such as Bax. Here we describe the NMR structure of Bcl-2 bound to 35, a compound that we recently found to inhibit Beclin 1/Bcl-2 binding more potently than Bax/Bcl-2 binding. The structure shows that 35 binds at one end of the BH3-binding groove of Bcl-2. Interestingly, much of the 35-binding site is not involved in binding to Bcl-2 inhibitors described previously and mediates binding to Beclin 1 but not Bax. The structure suggests potential avenues to design compounds that disrupt Beclin 1/Bcl-2 binding and stimulate autophagy without inducing apoptosis.
    DOI:  https://doi.org/10.1038/s42003-023-05467-w
  42. Int J Mol Sci. 2023 Oct 20. pii: 15386. [Epub ahead of print]24(20):
      In recent years, efforts have been made to identify new anti-cancer therapies. Various types of nanomaterials, including silver nanoparticles (AgNPs), are being considered as an option. In addition to its well-known antibacterial activity, AgNPs exhibit cytotoxic potential in both physiological and cancer cells by inducing stress-mediated autophagy and apoptotic cell death. A rapidly growing collection of data suggests that the proper regulation of autophagic machinery may provide an efficient tool for suppressing the development of cancer. In this light, AgNPs have emerged as a potential anti-cancer agent to support therapy of the disease. This review summarizes current data indicating the dual role of AgNP-induced autophagy and highlights factors that may influence its protective vs. its toxic potential. It also stresses that our understanding of the cellular and molecular mechanisms of autophagy machinery in cancer cells, as well as AgNP-triggered autophagy in both normal and diseased cells, remains insufficient.
    Keywords:  AgNPs; ER stress; apoptosis; autophagy defect; cancer; cell death; cytotoxicity; lysosomes
    DOI:  https://doi.org/10.3390/ijms242015386
  43. Adv Med Sci. 2023 Oct 20. pii: S1896-1126(23)00044-5. [Epub ahead of print]68(2): 417-425
      Programmed cell death plays a crucial role in maintaining the homeostasis and integrity of multicellular organisms, and its dysregulation contributes to the pathogenesis of many diseases. Programmed cell death is regulated by a range of macromolecules and low-molecular messengers, including ceramides. Endogenous ceramides have different functions, that are influenced by their localization and the presence of their target molecules. This article provides an overview of the current understanding of ceramides and their impact on various types of programmed cell death, including apoptosis, anoikis, macroautophagy and mitophagy, and necroptosis. Moreover, it highlights the emergence of dihydroceramides as a new class of bioactive sphingolipids and their downstream targets as well as their future roles in cancer cell growth, drug resistance and tumor metastasis.
    Keywords:  Anoikis; Apoptosis; Autophagy; Ceramide; Mitophagy; Necroptosis
    DOI:  https://doi.org/10.1016/j.advms.2023.10.004
  44. Biomedicines. 2023 Oct 05. pii: 2706. [Epub ahead of print]11(10):
      Lung cancer accounts for the highest number of deaths among men and women worldwide. Although extensive therapies, either alone or in conjunction with some specific drugs, continue to be the principal regimen for evolving lung cancer, significant improvements are still needed to understand the inherent biology behind progressive inflammation and its detection. Unfortunately, despite every advancement in its treatment, lung cancer patients display different growth mechanisms and continue to die at significant rates. Autophagy, which is a physiological defense mechanism, serves to meet the energy demands of nutrient-deprived cancer cells and sustain the tumor cells under stressed conditions. In contrast, autophagy is believed to play a dual role during different stages of tumorigenesis. During early stages, it acts as a tumor suppressor, degrading oncogenic proteins; however, during later stages, autophagy supports tumor cell survival by minimizing stress in the tumor microenvironment. The pivotal role of the IL6-IL17-IL23 signaling axis has been observed to trigger autophagic events in lung cancer patients. Since the obvious roles of autophagy are a result of different immune signaling cascades, systems biology can be an effective tool to understand these interconnections and enhance cancer treatment and immunotherapy. In this review, we focus on how systems biology can be exploited to target autophagic processes that resolve inflammatory responses and contribute to better treatment in carcinogenesis.
    Keywords:  IL17; IL23; IL6; NSCLC; autophagy; cytokines; therapeutics
    DOI:  https://doi.org/10.3390/biomedicines11102706
  45. Autophagy. 2023 Oct 24.
      Phase transitions (PT) of biomolecules are heavily involved in neurodegenerative disorders. Almost all previous studies were focusing on the PT of misfolded proteins whereas RNA molecules containing expanded repeats such as the CAG repeats are also able to undergo PT in vitro, a process called RNA gelation. Meanwhile, the expanded CAG repeat (eCAGr) RNA forms condensates that are largely observed only in the nuclei and exhibit liquid-like properties without obvious gelation. Thus, whether eCAGr RNA gelation occurs in cells and what function it is involved in remained elusive. We recently discovered that eCAGr RNA forms solid-like RNA gels in the cytoplasm, but they are rapidly cleared by the lysosomes via an autophagy-independent but LAMP2C-depdent pathway, making their presence in the cytoplasm difficult to be observed. We further revealed that these RNA gels sequester EEF2 in the cells and thus suppress global protein synthesis. In vivo expression of eCAGr RNA alone without detectable protein expression in the mouse model led to neurodegeneration-relevant electrophysiological and behavioral phenotypes, demonstrating its possible pathogenic roles.
    Keywords:  Expanded CAG repeat RNA; huntington’s disease; phase transitions; translation
    DOI:  https://doi.org/10.1080/15548627.2023.2274208
  46. JCI Insight. 2023 Oct 23. pii: e165817. [Epub ahead of print]8(20):
      Albuminuria and podocyte injury are the key cellular events in the progression of diabetic nephropathy (DN). Acetyl-CoA synthetase 2 (ACSS2) is a nucleocytosolic enzyme responsible for the regulation of metabolic homeostasis in mammalian cells. This study aimed to investigate the possible roles of ACSS2 in kidney injury in DN. We constructed an ACSS2-deleted mouse model to investigate the role of ACSS2 in podocyte dysfunction and kidney injury in diabetic mouse models. In vitro, podocytes were chosen and transfected with ACSS2 siRNA and ACSS2 inhibitor and treated with high glucose. We found that ACSS2 expression was significantly elevated in the podocytes of patients with DN and diabetic mice. ACSS2 upregulation promoted phenotype transformation and inflammatory cytokine expression while inhibiting podocytes' autophagy. Conversely, ACSS2 inhibition improved autophagy and alleviated podocyte injury. Furthermore, ACSS2 epigenetically activated raptor expression by histone H3K9 acetylation, promoting activation of the mammalian target of rapamycin complex 1 (mTORC1) pathway. Pharmacological inhibition or genetic depletion of ACSS2 in the streptozotocin-induced diabetic mouse model greatly ameliorated kidney injury and podocyte dysfunction. To conclude, ACSS2 activation promoted podocyte injury in DN by raptor/mTORC1-mediated autophagy inhibition.
    Keywords:  Diabetes; Metabolism; Molecular biology; Nephrology
    DOI:  https://doi.org/10.1172/jci.insight.165817
  47. Sci Rep. 2023 Oct 26. 13(1): 18327
      Scribble is a master scaffold protein in apical-basal polarity. Current knowledge about the biological function of Scribble in colonic epithelial plasticity/regeneration during intestinal inflammation is limited. Here, we showed that the level of Scribble is decreased in inflammatory bowel disease (IBD) patients and mice with DSS-induced colitis. ScribΔIEC mice develops severe acute colitis with disrupted epithelial barrier integrity and impaired crypt stem cell's function. Mechanistically, Scribble suppressed the process of autophagy by modulating the stability of caspase-dependent degradation of Atg16L1 by directly interacting with Atg16L1 in a LRR domain-dependent manner in IECs and led to an accumulation of ROS both in intestinal stem cells and epithelial cells. In addition, further study indicates that dietary sphingomyelin alleviates DSS-induced colitis by increase the expression of Scribble, which suggests that Scribble may be the critical marker of IBD. Our study shows that Scribble deficiency is associated with the dysregulated autophagy and impaired maintenance of colonic stemness, and it may be a target for diagnosis and treatment of IBD.
    DOI:  https://doi.org/10.1038/s41598-023-45176-2
  48. Cancer Res Commun. 2023 Oct 24.
      Lipid droplets (LDs) are dynamic organelles that serve as hubs of cellular metabolic processes. Emerging evidence shows that LDs also play a critical role in maintaining redox homeostasis and can mitigate lipid oxidative stress. In multiple cancers, including prostate cancer (PCa), LD accumulation is associated with cancer aggressiveness, therapy resistance, and poor clinical outcome. PCa arises as an androgen receptor (AR) driven disease. Among its myriad roles, AR mediates the biosynthesis of LDs, induces autophagy, and modulates cellular oxidative stress in a tightly regulated cycle that promotes cell proliferation. The factors regulating the interplay of these metabolic processes downstream of AR remain unclear. Here, we show that Sigma1/SIGMAR1, a unique ligand-operated scaffolding protein, regulates LD metabolism in PCa cells. Sigma1 inhibition triggers lipophagy, an LD selective form of autophagy, to prevent accumulation of LDs which normally act to sequester toxic levels of reactive oxygen species (ROS). This disrupts the interplay between LDs, autophagy, buffering of oxidative stress and redox homeostasis, and results in the suppression of cell proliferation in vitro and tumor growth in vivo. Consistent with these experimental results, SIGMAR1 transcripts are strongly associated with lipid metabolism and reactive oxygen species pathways in prostate tumors. Altogether, these data reveal a novel, pharmacologically responsive role for Sigma1 in regulating the redox homeostasis required by oncogenic metabolic programs that drive PCa proliferation.
    DOI:  https://doi.org/10.1158/2767-9764.CRC-22-0371
  49. Viruses. 2023 Sep 27. pii: 2012. [Epub ahead of print]15(10):
      Every year, dengue virus (DENV) affects millions of people. Currently, there are no approved drugs for the treatment of DENV infection. Autophagy is a conserved degradation process that was shown to be induced by DENV infection and required for optimal DENV replication. The modulation of autophagy is, therefore, considered an attractive target to treat DENV infection. This study carried out a high-content image screen analysis using Crispr-Cas9 GFP-LC3 knocked-in HeLa cells of a compound library synthesized from or inspired by natural products and their biocongener precursors to discover novel autophagy inhibitors. The screen identified Ka-003 as the most effective compound for decreasing the number of autophagic vacuoles inside cells upon autophagy induction. Ka-003 could inhibit autophagy in a dose-dependent manner at low micromolar concentrations. More importantly, Ka-003 demonstrated the concentration-dependent inhibition of DENV production in Crispr-Cas9 GFP-LC3 knocked-in THP-1 monocytes. The core structure of Ka-003, which is a methyl cyclohexene derivative, resembles those found in mulberry plants, and could be synthetically prepared in a bioinspired fashion. Taken together, data indicate that Ka-003 hampered autophagy and limited DENV replication. The low cytotoxicity of Ka-003 suggests its therapeutic potential, which warrants further studies for the lead optimization of the compound for dengue treatment.
    Keywords:  autophagy; dengue virus; drug discovery; methyl cyclohexene; mulberry
    DOI:  https://doi.org/10.3390/v15102012
  50. J Clin Invest. 2023 Oct 24. pii: e171430. [Epub ahead of print]
      Reactivation and dysregulation of the mTOR signaling pathway is a hallmark of aging and chronic lung disease, however the impact on microvascular progenitor cells (MVPC), capillary angiostasis and tissue homeostasis is unknown. While the existence of an adult lung vascular progenitor has long been hypothesized, these studies show that Abcg2 enriches for a population of angiogenic tissue resident MVPC present in both adult mouse and human lungs using functional, lineage and transcriptomic analyses. These studies link human and mouse MVPC specific mTORC1 activation to decreased stemness, angiogenic potential, disruption of p53 and Wnt pathways, with consequent loss of alveolar-capillary structure and function. Following mTOR activation these MVPC adapt a unique transcriptome signature and emerge as a venous subpopulation in the angiodiverse microvascular endothelial subclusters. Thus, our findings support a significant role for mTOR in the maintenance of MVPC function, microvascular niche homeostasis as well as a cell-based mechanism driving loss of tissue structure underlying lung aging and the development of emphysema.
    Keywords:  Adult stem cells; Endothelial cells; Microcirculation; Stem cells; Vascular Biology
    DOI:  https://doi.org/10.1172/JCI171430
  51. Int J Mol Sci. 2023 Oct 10. pii: 15036. [Epub ahead of print]24(20):
      The Atg12 protein in yeast is an indispensable polypeptide in the highly conserved ubiquitin-like conjugation system operating in the macroautophagy/autophagy pathway. Atg12 is covalently conjugated to Atg5 through the action of Atg7 and Atg10; the Atg12-Atg5 conjugate binds Atg16 to form an E3 ligase that functions in a separate conjugation pathway involving Atg8. Atg12 is comprised of a ubiquitin-like (UBL) domain preceded at the N terminus by an intrinsically disordered protein region (IDPR), a domain that comprises a major portion of the protein but remains elusive in its conformation and function. Here, we show that the IDPR in unconjugated Atg12 is positioned in proximity to the UBL domain, a configuration that is important for the functional structure of the protein. A major deletion in the IDPR disrupts intactness of the UBL domain at the unconjugated C terminus, and a mutation in the predicted α0 helix in the IDPR prevents Atg12 from binding to Atg7 and Atg10, which ultimately affects the protein function in the ubiquitin-like conjugation cascade. These findings provide evidence that the IDPR is an indispensable part of the Atg12 protein from yeast.
    Keywords:  autophagy; crosslinking mass spectrometry; intrinsically disordered protein region; ubiquitin-like conjugation system
    DOI:  https://doi.org/10.3390/ijms242015036
  52. bioRxiv. 2023 Oct 03. pii: 2023.10.02.560611. [Epub ahead of print]
      Synapse maintenance is essential for generating functional circuitry and decrement in this process is a hallmark of neurodegenerative disease. While we are beginning to understand the basis of synapse formation, much less is known about synapse maintenance in vivo . Cysteine string protein α (CSPα), encoded by the Dnajc5 gene, is a synaptic vesicle chaperone that is necessary for synapse maintenance and linked to neurodegeneration. To investigate the transcriptional changes associated with synapse maintenance, we performed single nucleus transcriptomics on the cortex of young CSPα knockout (KO) mice and littermate controls. Through differential expression and gene ontology analysis, we observed that both neurons and glial cells exhibit unique signatures in CSPα KO brain. Significantly all neurons in CSPα KO brains show strong signatures of repression in synaptic pathways, while upregulating autophagy related genes. Through visualization of synapses and autophagosomes by electron microscopy, we confirmed these alterations especially in inhibitory synapses. By imputing cell-cell interactions, we found that neuron-glia interactions were specifically increased in CSPα KO mice. This was mediated by synaptogenic adhesion molecules, including the classical Neurexin1-Neuroligin 1 pair, suggesting that communication of glial cells with neurons is strengthened in CSPα KO mice in an attempt to achieve synapse maintenance. Together, this study reveals unique cellular and molecular transcriptional changes in CSPα KO cortex and provides new insights into synapse maintenance and neurodegeneration.
    Significance statement: Synapse maintenance is important for maintaining neuronal circuitry throughout life. However, little is known about molecules that affect synapse maintenance in vivo . CSPα, encoded by the Dnajc5 gene, is a synaptic vesicle chaperone that is linked to synapse maintenance and neurodegeneration. Here, we show by performing single nucleus transcriptomics of CSPα KO cortex that synapse instability is related to repression in synaptic pathways and elevation of autophagy in neurons. However, we find a heterogeneity of glial responses. Additionally, interactions between neurons and glia are increased in CSPα KO, mediated by synaptogenic adhesion molecules. This study provides a novel perspective on into synapse maintenance and reveals unique cellular and molecular transcriptional changes in CSPα KO brains.
    DOI:  https://doi.org/10.1101/2023.10.02.560611
  53. Exp Ther Med. 2023 Nov;26(5): 538
      Osteoporosis is a systemic bone metabolic disorder that plagues the health and quality of life of the elderly. Autophagy plays an important role in bone formation while maintaining the homeostasis of the body. Trehalose is a mTOR-independent autophagy inducer, but to the best of our knowledge, there is no rat model of postmenopausal osteoporosis. The present study found that trehalose can delay postmenopausal osteoporosis in rats, which may be achieved by inducing and enhancing AKT/transcription factor EB pathway-dependent autophagy flow. The specific mechanism of its occurrence needs to be further studied. Trehalose-containing drugs are promising for delaying postmenopausal osteoporosis. Hematoxylin and eosin (H&E) staining, western blotting, micro computerized tomography (CT) scanning and Transmission electron microscopy were used to investigate the role of trehalose in postmenopausal osteoporosis rat model at protein, cell and histology aspects. According to the H&E staining results, the bone trabecular histological structure of the trehalose group was superior to that of the model group. The Micro CT scanning indicated the imaging structure of bone trabeculae in the trehalose group was superior to than that in the model group. Western blotting indicated the activation of autophagic flow in trehalose group, the autophagy degree of the trehalose group is greater than that of the model group; Transmission electron microscopy indicated the autophagy degree of the Trehalose group was greater than that of the model group under electron microscopy. Trehalose can delay postmenopausal osteoporosis in rats, which may be achieved by inducing and enhancing Akt/TFEB pathway-dependent autophagy flow.
    Keywords:  autophagy; osteoporosis; rat; trehalose
    DOI:  https://doi.org/10.3892/etm.2023.12237
  54. Exp Lung Res. 2023 ;49(1): 178-192
       PURPOSE/AIM: Bronchopulmonary dysplasia (BPD) is associated with poor survival in preterm infants. Intrauterine infection can aggravate the degree of obstruction of alveolar development in premature infants; however, the pathogenic mechanism remains unclear. In this study, we sought to determine whether pyroptosis could be inhibited by downregulating mammalian target of rapamycin (mTOR) activation and inducing autophagy in BPD-affected lung tissue.
    MATERIALS AND METHODS: We established a neonatal rat model of BPD induced by intrauterine infection via intraperitoneally injecting pregnant rats with lipopolysaccharide (LPS). Subsequently, mTOR levels and pyroptosis were evaluated using immunohistochemistry, immunofluorescence, TUNEL staining, and western blotting. The Shapiro-Wilk test was employed to assess the normality of the experimental data. Unpaired t-tests were used to compare the means between two groups, and comparisons between multiple groups were performed using analysis of variance.
    RESULTS: Pyroptosis of lung epithelial cells increased in BPD lung tissues. After administering an mTOR phosphorylation inhibitor (rapamycin) to neonatal rats with BPD, the level of autophagy increased, while the expression of autophagy cargo adaptors, LC3 and p62, did not differ. Following rapamycin treatment, NLRP3, Pro-caspase-1, caspase-1, pro-IL-1β, IL-1β, IL-18/Pro-IL-18, N-GSDMD/GSDMD, Pro-caspase-11, and caspase-11 were negatively regulated in BPD lung tissues. The opposite results were observed after treatment with the autophagy inhibitor MHY1485, showing an increase in pyroptosis and a significant decrease in the number of alveoli in BPD.
    CONCLUSIONS: Rapamycin reduces pyroptosis in neonatal rats with LPS-induced BPD by inhibiting mTOR phosphorylation and inducing autophagy; hence, it may represent a potential therapeutic for treating BPD.
    Keywords:  Bronchopulmonary dysplasia; autophagy; intrauterine infection; pyroptosis; rapamycin
    DOI:  https://doi.org/10.1080/01902148.2023.2266236
  55. Proc Natl Acad Sci U S A. 2023 Oct 31. 120(44): e2313010120
      Inter-organelle contact sites between mitochondria and lysosomes mediate the crosstalk and bidirectional regulation of their dynamics in health and disease. However, mitochondria-lysosome contact sites and their misregulation have not been investigated in peripheral sensory neurons. Charcot-Marie-Tooth type 2B disease is an autosomal dominant axonal neuropathy affecting peripheral sensory neurons caused by mutations in the GTPase Rab7. Using live super-resolution and confocal time-lapse microscopy, we showed that mitochondria-lysosome contact sites dynamically form in the soma and axons of peripheral sensory neurons. Interestingly, Charcot-Marie-Tooth type 2B mutant Rab7 led to prolonged mitochondria-lysosome contact site tethering preferentially in the axons of peripheral sensory neurons, due to impaired Rab7 GTP hydrolysis-mediated contact site untethering. We further generated a Charcot-Marie-Tooth type 2B mutant Rab7 knock-in mouse model which exhibited prolonged axonal mitochondria-lysosome contact site tethering and defective downstream axonal mitochondrial dynamics due to impaired Rab7 GTP hydrolysis as well as fragmented mitochondria in the axon of the sciatic nerve. Importantly, mutant Rab7 mice further demonstrated preferential sensory behavioral abnormalities and neuropathy, highlighting an important role for mutant Rab7 in driving degeneration of peripheral sensory neurons. Together, this study identifies an important role for mitochondria-lysosome contact sites in the pathogenesis of peripheral neuropathy.
    Keywords:  Charcot–Marie–Tooth disease; inter-organelle contact site; lysosome; mitochondria; peripheral neuropathy
    DOI:  https://doi.org/10.1073/pnas.2313010120
  56. J Asian Nat Prod Res. 2023 Oct 27. 1-13
      Morinda officinalis is a traditional Chinese tonic herb, and have been used in the treatment of multiple diseases. Here, three iridoid glycosides isolated from M. officinalis were evaluated for their roles in the autophagy-lysosomal pathway. All three iridoid glycosides could induce TFEB/TFE3-mediated lysosomal biogenesis and trigger autophagy. Interestingly, they promoted the nuclear import of TFEB/TFE3 without affecting their nuclear export, suggesting their role in the maintenance of lysosomal homeostasis. The results from this study shed light on the identification of autophagy activators from M. officinalis and provide a basis for developing them in the treatment of oxidative stress-involved diseases.
    Keywords:  Iridoid glycosides; Morinda officinalis; TFEB/TFE3; antioxidant activity; lysosomal biogenesis
    DOI:  https://doi.org/10.1080/10286020.2023.2269370
  57. Curr Issues Mol Biol. 2023 Sep 30. 45(10): 8040-8052
      Metabolic stress in skeletal muscle cells causes sustained metabolic changes, but the mechanisms of the prolonged effects are not fully known. In this study, we tested C2C12 cells with the AMP-activated protein kinase (AMPK) stimulator AICAR and measured the changes in the metabolic pathways and signaling kinases. AICAR caused an acute increase in the phosphorylation of the AMPK target ULK1, the mTORC1 substrate S6K, and the mTORC2 target Akt. Intriguingly, prior exposure to AICAR only decreased glucose-6 phosphate dehydrogenase activity when it underwent three-hour recovery after exposure to AICAR in a bicarbonate buffer containing glucose (KHB) instead of Dulbecco's Minimum Essential Medium (DMEM). The phosphorylation of the mTORC1 target S6K was increased after recovery in DMEM but not KHB, although this appeared to be specific to S6K, as the phosphorylation of the mTORC1 target site on ULK1 was not altered when the cells recovered in DMEM. The phosphorylation of mTORC2 target sites was also heterogenous under these conditions, with Akt increasing at serine 473 while other targets (SGK1 and PKCα) were unaffected. The exposure of cells to rapamycin (an mTORC1 inhibitor) and PP242 (an inhibitor of both mTOR complexes) revealed the differential phosphorylation of mTORC2 substrates. Taken together, the data suggest that prior exposure to AICAR causes the selective phosphorylation of mTOR substrates, even after prolonged recovery in a nutrient-replete medium.
    Keywords:  5-aminoimidazole-4-carboxamide ribonucleoside; C2C12 cells; mammalian target of rapamycin; mechanistic target of rapamycin; unc-like kinase 1
    DOI:  https://doi.org/10.3390/cimb45100508
  58. J Virol. 2023 Oct 26. e0143423
      Mitochondrial antiviral signaling protein (MAVS) and stimulator of interferon (IFN) genes (STING) are crucial factors of IFN induction in RNA and DNA viral infections. However, unrestricted IFN was harmful to the host by causing cytokine storm. Here, we report that zebrafish transmembrane protein 47 (TMEM47), an IFN-negative regulator in fish, represses IFN expression stimulated by both spring viremia of carp virus (SVCV) and cyprinid herpesvirus 2, which represent RNA and DNA viruses, respectively. The mRNA level of tmem47 was rapidly upregulated during RNA and DNA virus infection. Overexpression of TMEM47 significantly blocked SVCV- and CyHV-2-mediated IFN induction, whereas knockdown of tmem47 promoted ifn transcription under these viral infections. Furthermore, TMEM47 interacted with MAVS and STING, which are considered as key factors for IFN induction during RNA and DNA viral infection. Mechanistically, both MAVS and STING were degraded by TMEM47 in an autophagy-lysosome-dependent manner, and the autophagy factor autophagy-related gene 5 (ATG5) was essential for this process. Finally, at the cellular level, MAVS- and STING-mediated antiviral capacities were significantly suppressed by TMEM47. Taken together, our results demonstrate that zebrafish TMEM47 acts as a brake to inhibit IFN production in both RNA and DNA viral infections. IMPORTANCE Mitochondrial antiviral signaling protein (MAVS) and stimulator of interferon (IFN) genes (STING) are key adaptor proteins required for innate immune responses to RNA and DNA virus infection. Here, we show that zebrafish transmembrane protein 47 (TMEM47) plays a critical role in regulating MAVS- and STING-triggered IFN production in a negative feedback manner. TMEM47 interacted with MAVS and STING for autophagic degradation, and ATG5 was essential for this process. These findings suggest the inhibitory function of TMEM47 on MAVS- and STING-mediated signaling responses during RNA and DNA virus infection.
    Keywords:  MAVS; STING; TMEM47; autophagy-lysosome-dependent manner
    DOI:  https://doi.org/10.1128/jvi.01434-23
  59. EMBO Mol Med. 2023 Oct 26. e18242
      Missense mutations in the uromodulin (UMOD) gene cause autosomal dominant tubulointerstitial kidney disease (ADTKD), one of the most common monogenic kidney diseases. The unknown impact of the allelic and gene dosage effects and fate of mutant uromodulin leaves open the gap between postulated gain-of-function mutations, end-organ damage and disease progression in ADTKD. Based on two prevalent missense UMOD mutations with divergent disease progression, we generated UmodC171Y and UmodR186S knock-in mice that showed strong allelic and gene dosage effects on uromodulin aggregates and activation of ER stress and unfolded protein and immune responses, leading to variable kidney damage. Deletion of the wild-type Umod allele in heterozygous UmodR186S mice increased the formation of uromodulin aggregates and ER stress. Studies in kidney tubular cells confirmed differences in uromodulin aggregates, with activation of mutation-specific quality control and clearance mechanisms. Enhancement of autophagy by starvation and mTORC1 inhibition decreased uromodulin aggregates. These studies substantiate the role of toxic aggregates as driving progression of ADTKD-UMOD, relevant for therapeutic strategies to improve clearance of mutant uromodulin.
    Keywords:  ADTKD-UMOD; aggregates; gain-of-function; kidney fibrosis; unfolded protein response
    DOI:  https://doi.org/10.15252/emmm.202318242
  60. Life (Basel). 2023 Oct 08. pii: 2024. [Epub ahead of print]13(10):
      Autophagy is a highly conserved catabolic process in eukaryotic cells. Reactive nitrogen species play roles as inductors and signaling molecules of autophagy. A key mechanism of NO-mediated signaling is S-nitrosylation, a post-translational modification (PTM) of proteins at cysteine residues. In the present work, we analyzed the patterns of protein S-nitrosylation during the induction of autophagy in Triticum aestivum roots. The accumulation of S-nitrosylated proteins in the cells during autophagy induced with KNO2 and antimycin A was visualized using monoclonal antibodies with a Western blot analysis, and proteins were identified using a standard bottom-up proteomics approach. Protein S-nitrosylation is a labile and reversible PTM, and therefore the SNO group can be lost during experimental procedures. A subsequent bioinformatic analysis using predictive algorithms and protein-ligand docking showed that identified proteins possess hypothetical S-nitrosylation sites. Analyzing protein-protein interaction networks enabled us to discover the targets that can directly interact with autophagic proteins, and those that can interact with them indirectly via key multifunctional regulatory proteins. In this study, we show that S-nitrosylation is a key mechanism of NO-mediated regulation of autophagy in wheat roots. A combination of in silico predictive algorithms with a mass spectrometry analysis provides a targeted approach for the identification of S-nitrosylated proteins.
    Keywords:  Triticum aestivum; autophagy; nitric oxide; protein S-nitrosylation
    DOI:  https://doi.org/10.3390/life13102024
  61. Hepatology. 2023 Oct 23.
       BACKGROUND AIMS: The hallmark of nonalcoholic fatty liver disease (NAFLD) or hepatic steatosis is characterized by lipid droplet (LD) accumulation in hepatocytes. Autophagy may have profound effects on lipid metabolism and innate immune response. However, how innate immune activation may regulate the autophagic degradation of intracellular LDs remains elusive.
    APPROACH RESULTS: A mouse model of a high-fat diet (HFD)-induced NASH was used in the myeloid-specific STING knockout (STINGM-KO) or STING/YAP double knockout (STING/YAPDKO) mice. Liver injury, lipid accumulation, lipid droplet proteins, autophagic genes, chromatin immunoprecipitation coupled with massively parallel sequencing (ChIP-Seq), and RNA-Seq were assessed in vivo and in vitro. We found that HFD-induced oxidative stress activates STING and YAP pathways in hepatic macrophages. Macrophage STING deficiency (STINGM-KO) enhances nuclear YAP activity, reduces lipid accumulation, and increases autophagy-related protein ATG5, ATG7, and LC3B but diminishes LD protein perilipin 2 (PLIN2) expression. However, disruption of STING and YAP (STING/YAPM-DKO) increases serum ALT and TG levels, reduces β-fatty acid oxidation gene expression but augments PLIN2 levels, exacerbating HFD-induced lipid deposition. ChIP-Seq reveals that macrophage YAP targets transmembrane protein 205 (TMEM205) and activates AMPKα, which interacts with hepatocyte mitofusin 2 (MFN2) and induces protein disulfide isomerase (PDI) activation. PDI activates hypoxia-inducible factor-1α (HIF-1α) signaling, increases autophagosome colocalization with LDs, promotes the degradation of PLIN2 by interacting with chaperone-mediated autophagy (CMA) chaperone HSC70.
    CONCLUSION: Macrophage STING-YAP axis controls hepatic steatosis by reprogramming lipid metabolism in a TMEM205/MFN2/PDI-dependent pathway. These findings highlight the regulatory mechanism of macrophage STING-driven YAP activity on lipid control.
    DOI:  https://doi.org/10.1097/HEP.0000000000000638
  62. Nano Lett. 2023 Oct 27.
      Dysregulated lipid metabolism contributes to neurodegenerative pathologies and neurological decline in lysosomal storage disorders as well as more common neurodegenerative diseases. Niemann-Pick type A (NPA) is a fatal neurodegenerative lysosomal storage disease characterized by abnormal sphingomyelin accumulation in the endolysosomal lumen. The ability to monitor abnormalities in lipid homeostasis intracranially could improve basic investigations and the development of effective treatment strategies. We investigated the carbon nanotube-based detection of intracranial lipid content. We found that the near-infrared emission of a carbon nanotube-based lipid sensor responds to lipid accumulation in neuronal and in vivo models of NPA. The nanosensor detected lipid accumulation intracranially in an acid sphingomyelinase knockout mouse via noninvasive near-infrared spectroscopy. This work indicates a tool to improve drug development processes in NPA, other lysosomal storage diseases, and neurodegenerative diseases.
    Keywords:  Niemann−Pick; biosensor; carbon nanomaterials; neurodegenerative disease; sphingomyelin
    DOI:  https://doi.org/10.1021/acs.nanolett.3c02502
  63. Clin Exp Rheumatol. 2023 Oct 19.
       OBJECTIVES: The deletion of chondrocyte autophagy seems to play a key role in the pathogenesis of osteoarthritis (OA). Patients with OA often have vitamin D (VD) deficiency, and VD supplementation can improve pain and alleviate the progression of joint structures in patients. In this study, we aimed to investigate whether VD could enhance autophagy by activating the adenosine monophosphate activated protein kinase (AMPK)/mammalian target of rapamycin (mTOR) signalling pathway and protect against OA.
    METHODS: In this study, the levels of target proteins and genes were examined by western blot and qRT-PCR. Apoptotic cells were detected using TUNEL staining. Characteristics of autophagy were observed by LysoTracker red staining, mRFP-GFP-LC3 adenovirus transfection, and transmission electron microscopy. siRNA-mediated AMPK and mTOR knockdown were used to investigate the role of the AMPK/ mTOR signalling pathway in VD-induced autophagy. Haematoxylin and eosin and safranin-O/fast green staining were used detect cartilage alterations.
    RESULTS: We suggested that VD significantly reduced chondrocyte death and alleviated extracellular matrix degradation. Further studies showed that VD promoted the expression of the autophagy-related protein LC3II through the AMPK/mTOR signalling pathway in chondrocytes, activated lysosome activity, promoted the formation of autophagy-associated lysosomes, which played a crucial role in the degradation of intracellular organelles and maintained homeostasis. The anti-apoptotic effect of VD on chondrocytes was associated with the activation of autophagy. The group of AMPK-normal and mTOR-knockdown in the presence of VD inhibited chondrocyte apoptosis by promoting autophagy.
    CONCLUSIONS: This study highlights that VD can activate chondrocyte autophagy through the AMPK/mTOR signalling pathway.
    DOI:  https://doi.org/10.55563/clinexprheumatol/chmuts
  64. Int J Biochem Cell Biol. 2023 Oct 24. pii: S1357-2725(23)00119-X. [Epub ahead of print] 106480
      The occurrence of autophagy dysregulation is vital in the development of myelodysplastic syndrome and its transformation to acute myeloid leukemia. However, the mechanisms are largely unknown. Here, we have investigated the mechanism of the bcl6 corepressor mutation in myelodysplastic syndrome development and its transformation to acute myeloid leukemia. We identified a novel pathway involving histone deacetylase 6 and forkhead box protein O1, which leads to autophagy defects following the bcl6 corepressor mutation. And this further causes apoptosis and cell cycle arrest. The bcl6 corepressor-mutation-repressed autophagy resulted in the accumulation of damaged mitochondria, DNA, and reactive oxygen species in myelodysplastic syndrome cells, which could then lead to genomic instability and spontaneous mutation. Our results suggest that the bcl6 corepressor inactivating mutations exert pro-carcinogenic effects through survival strike, which is only an intermediate process. These findings provide mechanistic insights into the role of the bcl6 corepressor gene in myelodysplastic syndrome.
    Keywords:  Autophagy dysregulation; BCOR mutation; Forkhead box protein O1; Histone deacetylase 6; Myelodysplastic syndrome; Myelodysplastic syndrome progression
    DOI:  https://doi.org/10.1016/j.biocel.2023.106480
  65. Alzheimers Res Ther. 2023 Oct 27. 15(1): 184
       BACKGROUND: Autophagy is vital in the pathogenesis of neurodegeneration. Thus far, no studies have specifically investigated the relationship between pituitary adenylate cyclase-activating polypeptide (PACAP) and autophagy, particularly in the context of Alzheimer's disease (AD). This study used in vitro and in vivo models, along with clinical samples, to explore interactions between PACAP and autophagy in AD.
    METHODS: AD model mice were administered 6 μl of 0.1 mg/ml PACAP liquid intranasally for 4 weeks, then subjected to behavioral analyses to assess the benefits of PACAP treatment. The underlying mechanisms of PACAP-induced effects were investigated by methods including real-time quantitative polymerase chain reaction, RNA sequencing, immunofluorescence, and western blotting. Exosomes were extracted from human serum and subjected to enzyme-linked immunosorbent assays to examine autophagy pathways. The clinical and therapeutic implications of PACAP and autophagy were extensively investigated throughout the experiment.
    RESULTS: Impaired autophagy was a critical step in amyloid β (Aβ) and Tau deposition; PACAP enhanced autophagy and attenuated cognitive impairment. RNA sequencing revealed three pathways that may be involved in AD progression: PI3K-AKT, mTOR, and AMPK. In vivo and in vitro studies showed that sirtuin3 knockdown diminished the ability of PACAP to restore normal autophagy function, resulting in phagocytosis dysregulation and the accumulation of pTau, Tau, and Aβ. Additionally, the autophagic biomarker MAP1LC3 demonstrated a positive association with PACAP in human serum.
    CONCLUSIONS: PACAP reverses AD-induced cognitive impairment through autophagy, using sirtuin3 as a key mediator. MAP1LC3 has a positive relationship with PACAP in humans. These findings provide insights regarding potential uses of intranasal PACAP and sirtuin3 agonists in AD treatment.
    TRIAL REGISTRATION: NCT04320368.
    Keywords:  AKT; Alzheimer’s disease; Autophagy; PACAP; PI3K; Sirt3; mTOR
    DOI:  https://doi.org/10.1186/s13195-023-01334-2
  66. Cell Rep. 2023 Oct 24. pii: S2211-1247(23)01341-4. [Epub ahead of print]42(11): 113329
      Development is regulated by various factors, including protein methylation status. While PRMT5 is well known for its roles in oncogenesis by mediating symmetric di-methylation of arginine, its role in normal development remains elusive. Using Myod1Cre to drive Prmt5 knockout in embryonic myoblasts (Prmt5MKO), we dissected the role of PRMT5 in myogenesis. The Prmt5MKO mice are born normally but exhibit progressive muscle atrophy and premature death. Prmt5MKO inhibits proliferation and promotes premature differentiation of embryonic myoblasts, reducing the number and regenerative function of satellite cells in postnatal mice. Mechanistically, PRMT5 methylates and destabilizes FoxO1. Prmt5MKO increases the total FoxO1 level and promotes its cytoplasmic accumulation, leading to activation of autophagy and depletion of lipid droplets (LDs). Systemic inhibition of autophagy in Prmt5MKO mice restores LDs in myoblasts and moderately improves muscle regeneration. Together, PRMT5 is essential for muscle development and regeneration at least partially through mediating FoxO1 methylation and LD turnover.
    Keywords:  CP: Developmental biology; CP: Molecular biology; PRMT; PTM; SCs; autophagy; myogenesis; posttranslational modification; protein arginine methyltransferase; satellite cells
    DOI:  https://doi.org/10.1016/j.celrep.2023.113329
  67. J Lipid Res. 2023 Oct 25. pii: S0022-2275(23)00138-4. [Epub ahead of print] 100465
      Accurate intracellular cholesterol traffic plays crucial roles. Niemann Pick type C (NPC) proteins NPC1 and NPC2, are two lysosomal cholesterol transporters that mediate the cholesterol exit from lysosomes. However, other proteins involved in this process remain poorly defined. Here we find that the previously unannotated protein TMEM241 is required for cholesterol egressing from lysosomes through amphotericin B-based genome-wide CRISPR-Cas9 knockout screening. Ablation of TMEM241 caused impaired sorting of NPC2, a protein utilizes the mannose-6-phosphate (M6P) modification for lysosomal targeting, resulting in cholesterol accumulation in the lysosomes. TMEM241 is a member of solute transporters 35 (SLC35) nucleotide sugar transporters family and localizes on the cis-Golgi network. Our data indicate that TMEM241 transports UDP-N-acetylglucosamine (UDP-GlcNAc) into Golgi lumen and UDP-GlcNAc is used for the M6P modification of proteins including NPC2. Furthermore, Tmem241-deficient mice display cholesterol accumulation in pulmonary cells and behave pulmonary injury and hypokinesia. Taken together, we demonstrate that TMEM241 is a Golgi-localized UDP-GlcNAc transporter and loss of TMEM241 causes cholesterol accumulation in lysosomes because of the impaired M6P-dependent lysosomal targeting of NPC2.
    Keywords:  Genome-wide CRISPR screen; Golgi apparatus; LDL; cholesterol trafficking; lipid transfer proteins; lipids; lysosomal protein sorting; nucleotide sugar transporter
    DOI:  https://doi.org/10.1016/j.jlr.2023.100465