bims-auttor Biomed News
on Autophagy and mTOR
Issue of 2024‒11‒10
33 papers selected by
Viktor Korolchuk, Newcastle University
  1. HSP70 promotes amino acid-dependent mTORC1 signaling by mediating CHIP-induced NPRL2 ubiquitination and degradation.
  2. mTORC1 activity licenses its own release from the lysosomal surface.
  3. The dual role of the TSC complex in cancer.
  4. A switch from lysosomal degradation to secretory autophagy initiates osteogenic bone metastasis in prostate cancer.
  5. Autophagy as a critical driver of metabolic adaptation, therapeutic resistance, and immune evasion of cancer.
  6. USP14 inhibition enhances Parkin-independent mitophagy in iNeurons.
  7. Chondrocyte autophagy mechanism and therapeutic prospects in osteoarthritis.
  8. Retinol binding protein 4 restricts PCV2 replication via selective autophagy degradation of viral ORF1 protein.
  9. mTORC1 restricts TFE3 activity by auto-regulating its presence on lysosomes.
  10. Overexpression of SlATG8f gene enhanced autophagy and pollen protection in tomato under heat stress.
  11. Autophagy is essential for somatic embryogenesis in citrus through regulating amyloplast degradation and lipid homeostasis.
  12. The effect of rapamycin treatment on mouse ovarian follicle development in dehydroepiandrosterone-induced polycystic ovary syndrome mouse model.
  13. TMEM106B knockdown exhibits a neuroprotective effect in Parkinson's disease models via regulating autophagy-lysosome pathway.
  14. Evaluation of LC3-II Release via Extracellular Vesicles in Relation to the Accumulation of Intracellular LC3-positive Vesicles.
  15. Increased DNA damage in full-grown oocytes is correlated with diminished autophagy activation.
  16. AMFR-mediated Flavivirus NS2A ubiquitination subverts ER-phagy to augment viral pathogenicity.
  17. The cGAS-STING mediated crosstalk between innate immunity and autophagy in leishmaniasis using mathematical modeling: Uncovering new therapeutic avenues.
  18. Cytoglobin augments ferroptosis through autophagic degradation of ferritin in colorectal cancer cells.
  19. Regulation of mitochondrial autophagy by lncRNA MALAT1 in sepsis-induced myocardial injury.
  20. The IP3R inhibitor desmethylxestospongin B reduces tumor cell migration, invasion and metastasis by impairing lysosome acidification and β1-integrin recycling.
  21. Acute high-intensity muscle contraction moderates AChR gene expression independent of rapamycin-sensitive mTORC1 pathway in rat skeletal muscle.
  22. Endoplasmic reticulum stress-mediated autophagy in cancer and its interaction with apoptosis and ferroptosis.
  23. PINK1-mediated mitophagy attenuates pathological cardiac hypertrophy by suppressing the mtDNA release-activated cGAS-STING pathway.
  24. STIM1 promotes cervical cancer progression through autophagy activation via TFEB nuclear translocation.
  25. Activation of lysosomal Ca2+ channels mitigates mitochondrial damage and oxidative stress.
  26. Monocrotaline-mediated autophagy via inhibiting PI3K/AKT/mTOR pathway induces apoptosis in rat hepatocytes.
  27. PKCδ modulates SP1 mediated mitochondrial autophagy to exacerbate diacetylmorphine-induced ferroptosis in neurons.
  28. The USP12/46 deubiquitinases protect integrins from ESCRT-mediated lysosomal degradation.
  29. NLRX1 and STING alleviate renal ischemia-reperfusion injury by regulating LC3 lipidation during mitophagy.
  30. PINK1/Parkin-Mediated Mitophagy Ameliorates Mitochondrial Dysfunction in Lacrimal Gland Acinar Cells During Aging.
  31. GSK-3β inhibitor amplifies autophagy-lysosomal pathways by regulating TFEB in Parkinson's disease models.
  32. Critical role of ROCK1 in AD pathogenesis via controlling lysosomal biogenesis and acidification.
  33. Therapeutic potential of Parkin and its regulation in Parkinson's disease.
  1. FASEB J. 2024 Nov 15. 38(21): e70147
    HSP70 promotes amino acid-dependent mTORC1 signaling by mediating CHIP-induced NPRL2 ubiquitination and degradation.
    Jianfang Gao, Mingjun Lin, Jina Qing, Hongxia Li, Xiao Zeng, Wuzhou Yuan, Tingting Li, Shanping He.
      Mechanistic target of rapamycin complex 1 (mTORC1) is a master regulator of cell growth and its dysregulation leads to a variety of human diseases. Although NPRL2, an essential component of the GATOR1 complex, is reported to effectively suppress amino acid-induced mTORC1 activation, the regulation of NPRL2 protein stability is unclear. In this study, we show that chaperon-associated ubiquitin ligase CHIP interacts with NPRL2 and promotes its polyubiquitination and proteasomal degradation. Moreover, HSP70 mediates CHIP-induced ubiquitination and degradation of NPRL2. Consistently, overexpression of HSP70 enhances whereas HSP70 depletion inhibits amino acid-induced mTORC1 activation. Accordingly, knockdown of HSP70 promotes basal autophagic flux, and inhibits cell growth and proliferation. Taken together, these results demonstrated that HSP70 is a novel activator of mTORC1 through mediating CHIP-induced ubiquitination and degradation of NPRL2.
    Keywords:  CHIP; HSP70; NPRL2; amino acid; mTORC1; ubiquitination
    DOI:  https://doi.org/10.1096/fj.202401352R
  2. Mol Cell. 2024 Oct 25. pii: S1097-2765(24)00831-1. [Epub ahead of print]
    mTORC1 activity licenses its own release from the lysosomal surface.
    Aishwarya Acharya, Constantinos Demetriades.
      Nutrient signaling converges on mTORC1, which, in turn, orchestrates a physiological cellular response. A key determinant of mTORC1 activity is its shuttling between the lysosomal surface and the cytoplasm, with nutrients promoting its recruitment to lysosomes by the Rag GTPases. Active mTORC1 regulates various cellular functions by phosphorylating distinct substrates at different subcellular locations. Importantly, how mTORC1 that is activated on lysosomes is released to meet its non-lysosomal targets and whether mTORC1 activity itself impacts its localization remain unclear. Here, we show that, in human cells, mTORC1 inhibition prevents its release from lysosomes, even under starvation conditions, which is accompanied by elevated and sustained phosphorylation of its lysosomal substrate TFEB. Mechanistically, "inactive" mTORC1 causes persistent Rag activation, underlining its release as another process actively mediated via the Rags. In sum, we describe a mechanism by which mTORC1 controls its own localization, likely to prevent futile cycling on and off lysosomes.
    Keywords:  GATOR1; Rag GTPases; Rheb; TFE3; TFEB; Torin1; lysosomes; mTORC1; rapamycin
    DOI:  https://doi.org/10.1016/j.molcel.2024.10.008
  3. Trends Mol Med. 2024 Nov 01. pii: S1471-4914(24)00276-4. [Epub ahead of print]
    The dual role of the TSC complex in cancer.
    Josephine Hartung, Christine Müller, Cornelis F Calkhoven.
      The tuberous sclerosis complex (TSC1/TSC2/TBC1D7) primarily functions to inhibit the mechanistic target of rapamycin complex 1 (mTORC1), a crucial regulator of cell growth. Mutations in TSC1 or TSC2 cause tuberous sclerosis complex (TSC), a rare autosomal dominant genetic disorder marked by benign tumors in multiple organs that rarely progress to malignancy. Traditionally, TSC proteins are considered tumor suppressive due to their inhibition of mTORC1 and other mechanisms. However, more recent studies have shown that TSC proteins can also promote tumorigenesis in certain cancer types. In this review, we explore the composition and function of the TSC protein complex, the roles of its individual components in cancer biology, and potential future therapeutic targeting strategies.
    Keywords:  MYC; TSC; cancer; mTORC1; molecular glue degraders
    DOI:  https://doi.org/10.1016/j.molmed.2024.10.009
  4. J Extracell Vesicles. 2024 Nov;13(11): e70002
    A switch from lysosomal degradation to secretory autophagy initiates osteogenic bone metastasis in prostate cancer.
    Xiaoyu Wei, Mengmeng Liang, Min Deng, Ji Zheng, Fei Luo, Qinyu Ma.
      The identification of both autophagy-related material degradation and unconventional secretion has paved the way for significant breakthroughs linking autophagy to a plethora of physiological processes and disease conditions. However, the mechanisms that coordinate these two pathways remain elusive. Here, we demonstrate that a switch from the lysosomal degradation to a secretory autophagy pathway is governed by protein tyrosine phosphatase 1B (PTP1B, encoded by PTPN1). Dephosphorylation at two tyrosine residues of syntaxin17 (STX17) by PTP1B reduces autophagosome-lysosome fusion while switching the cells to a secretory autophagy pathway. Both PTP1B overexpression and tumour-derived extracellular vesicles (EVs) can activate the secretory autophagy pathway in osteoblasts. Moreover, we demonstrate that osteoblastic LC3+ EVs, generated via the secretory autophagy pathway, are the primary contributor to tumour-associated bone remodelling in prostate cancer. Depletion of tumour-derived EVs secretion or genetic ablation of osteoblastic PTP1B rescues aberrant bone remodelling and lesions, highlighting the relevance between LC3+ EVs and the formation of bone metastatic niche. Our results reveal the significance of tumour-regulated PTP1B in the fate decision of autophagosomes, and propose a role ofLC3+ EVs in shaping the bone metastatic niche.
    Keywords:  autophagosome‐lysosome fusion; bone metastatic niche; extracellular vesicles; secretory autophagy; tumour‐associated bone remodelling
    DOI:  https://doi.org/10.1002/jev2.70002
  5. Curr Opin Biotechnol. 2023 Oct 27. pii: S0958-1669(23)00122-2. [Epub ahead of print]84 103012
    Autophagy as a critical driver of metabolic adaptation, therapeutic resistance, and immune evasion of cancer.
    Keisuke Yamamoto, Dosuke Iwadate, Eri Naito, Keisuke Tateishi, Mitsuhiro Fujishiro.
      Autophagy is a well-conserved intracellular degradation pathway. Besides its physiological role in normal cells, autophagy is activated in various cancer types and protects cancer cells from stresses such as nutrient deprivation, therapeutic insults, and antitumor immunity. Autophagy in cancer cells as well as normal cells in the host supports tumor metabolism, allowing for tumor growth under a nutrient-limited tumor microenvironment. Autophagy also protects cancer cells from treatments such as radiation therapy, cytotoxic chemotherapy, and targeted therapy. Though the roles of autophagy in antitumor immunity are complex and highly context-dependent, accumulating evidence now supports the role of autophagy in mediating immunotherapy resistance. Based on these preclinical findings, multiple clinical trials are currently ongoing to test the therapeutic efficacy of autophagy inhibition in cancer. Here, we review recent findings on the tumor-promoting roles of autophagy in cancer and discuss advances in therapeutic approaches that target autophagy in cancer.
    DOI:  https://doi.org/10.1016/j.copbio.2023.103012
  6. Pharmacol Res. 2024 Oct 30. pii: S1043-6618(24)00429-8. [Epub ahead of print] 107484
    USP14 inhibition enhances Parkin-independent mitophagy in iNeurons.
    Bernardo Greta, A Prado Miguel, Dashtmian Anna Roshani, Mariavittoria Favaro, Sofia Mauri, Alice Borsetto, Elena Marchesan, Joao A Paulo, Steve P Gygi, Daniel J Finley, Elena Ziviani.
      Loss of proteostasis is well documented during physiological aging and depends on the progressive decline in the activity of two major degradative mechanisms: the ubiquitin-proteasome system (UPS) and the autophagy-lysosomal pathway. This decline in proteostasis is exacerbated in age-associated neurodegenerative diseases, such as Parkinson's Disease (PD). In PD, patients develop an accumulation of aggregated proteins and dysfunctional mitochondria, which leads to ROS production, neuroinflammation and neurodegeneration. We recently reported that inhibition of the deubiquitinating enzyme USP14, which is known to enhance both the UPS and autophagy, increases lifespan and rescues the pathological phenotype of two Drosophila models of PD. Studies on the effects of USP14 inhibition in mammalian neurons have not yet been conducted. To close this gap, we exploited iNeurons differentiated from human embryonic stem cells (hESCs), and investigated the effect of inhibiting USP14 in these cultured neurons. Quantitative global proteomics analysis performed following genetic ablation or pharmacological inhibition of USP14 demonstrated that USP14 loss of function specifically promotes mitochondrial autophagy in iNeurons. Biochemical and imaging data also showed that USP14 inhibition enhances mitophagy. The mitophagic effect of USP14 inhibition proved to be PINK1/Parkin- independent, instead relying on expression of the mitochondrial E3 Ubiquitin Ligase MITOL/MARCH5. Notably, USP14 inhibition normalized the mitochondrial defects of Parkin KO human neurons.
    Keywords:  Autophagy; MARCH5/MITOL; Mitophagy; PINK1; Parkin; UPS; USP14
    DOI:  https://doi.org/10.1016/j.phrs.2024.107484
  7. Front Cell Dev Biol. 2024 ;12 1472613
    Chondrocyte autophagy mechanism and therapeutic prospects in osteoarthritis.
    Lan Li, Jie Li, Jian-Jiang Li, Huan Zhou, Xing-Wang Zhu, Ping-Heng Zhang, Bo Huang, Wen-Ting Zhao, Xiao-Feng Zhao, En-Sheng Chen.
      Osteoarthritis (OA) is the most common type of arthritis characterized by progressive cartilage degradation, with its pathogenesis closely related to chondrocyte autophagy. Chondrocytes are the only cells in articular cartilage, and the function of chondrocytes plays a vital role in maintaining articular cartilage homeostasis. Autophagy, an intracellular degradation system that regulates energy metabolism in cells, plays an incredibly important role in OA. During the early stages of OA, autophagy is enhanced in chondrocytes, acting as an adaptive mechanism to protect them from various environmental changes. However, with the progress of OA, chondrocyte autophagy gradually decreases, leading to the accumulation of damaged organelles and macromolecules within the cell, prompting chondrocyte apoptosis. Numerous studies have shown that cartilage degradation is influenced by the senescence and apoptosis of chondrocytes, which are associated with reduced autophagy. The relationship between autophagy, senescence, and apoptosis is complex. While autophagy is generally believed to inhibit cellular senescence and apoptosis to promote cell survival, recent studies have shown that some proteins are degraded by selective autophagy, leading to the secretion of the senescence-associated secretory phenotype (SASP) or increased SA-β-Gal activity in senescent cells within the damaged region of human OA cartilage. Autophagy activation may lead to different outcomes depending on the timing, duration, or type of its activation. Thus, our study explored the complex relationship between chondrocyte autophagy and OA, as well as the related regulatory molecules and signaling pathways, providing new insights for the future development of safe and effective drugs targeting chondrocyte autophagy to improve OA.
    Keywords:  apoptosis; autophagy; chondrocytes; osteoarthritis; senescence
    DOI:  https://doi.org/10.3389/fcell.2024.1472613
  8. Commun Biol. 2024 Nov 05. 7(1): 1438
    Retinol binding protein 4 restricts PCV2 replication via selective autophagy degradation of viral ORF1 protein.
    Qingbing Han, Hejiao Zhao, Meng Chen, Wenshuo Xue, Jun Li, Lei Sun, Yingli Shang.
      Autophagy is a highly conserved degradative process that has been linked to various functions, including defending host cells against pathogens. Although the involvement of autophagy in porcine circovirus 2 (PCV2) infection has become apparent, it remains unclear whether selective autophagy plays a critical role in PCV2 restriction. Here we show that retinol-binding protein 4 (RBP4), an adipokine for retinol carrier, initiates the autophagic degradation of PCV2 ORF1 protein. PCV2 infection increases RBP4 protein levels through MAPK-eIF4E axis in living cells. Ectopic expression of RBP4 or recombinant RBP4 treatment promotes the degradation of ORF1 protein. Mechanistically, RBP4 activates TRAF6 to induce K63-linked ubiquitination of ORF1, leading to SQSTM1/p62-mediated selective autophagy for degradation. Consequently, RBP4 deficiency increases viral loads and exacerbates the pathogenicity of PCV2 in vivo. Collectively, these results identify RBP4 as a key host restriction factor of PCV2 and reveal a previously undescribed antiviral mechanism against PCV2 in infected cells.
    DOI:  https://doi.org/10.1038/s42003-024-07052-1
  9. Mol Cell. 2024 Oct 25. pii: S1097-2765(24)00832-3. [Epub ahead of print]
    mTORC1 restricts TFE3 activity by auto-regulating its presence on lysosomes.
    Susan Zwakenberg, Denise Westland, Robert M van Es, Holger Rehmann, Jasper Anink, Jolita Ciapaite, Marjolein Bosma, Ellen Stelloo, Nalan Liv, Paula Sobrevals Alcaraz, Nanda M Verhoeven-Duif, Judith J M Jans, Harmjan R Vos, Eleonora Aronica, Fried J T Zwartkruis.
      To stimulate cell growth, the protein kinase complex mTORC1 requires intracellular amino acids for activation. Amino-acid sufficiency is relayed to mTORC1 by Rag GTPases on lysosomes, where growth factor signaling enhances mTORC1 activity via the GTPase Rheb. In the absence of amino acids, GATOR1 inactivates the Rags, resulting in lysosomal detachment and inactivation of mTORC1. We demonstrate that in human cells, the release of mTORC1 from lysosomes depends on its kinase activity. In accordance with a negative feedback mechanism, activated mTOR mutants display low lysosome occupancy, causing hypo-phosphorylation and nuclear localization of the lysosomal substrate TFE3. Surprisingly, mTORC1 activated by Rheb does not increase the cytoplasmic/lysosomal ratio of mTORC1, indicating the existence of mTORC1 pools with distinct substrate specificity. Dysregulation of either pool results in aberrant TFE3 activity and may explain nuclear accumulation of TFE3 in epileptogenic malformations in focal cortical dysplasia type II (FCD II) and tuberous sclerosis (TSC).
    Keywords:  FCD IIb; NPRL2; Rag GTPases; Rheb; TFE3; TSC; amino acids; lysosomes; mTORC1
    DOI:  https://doi.org/10.1016/j.molcel.2024.10.009
  10. Sci Rep. 2024 11 06. 14(1): 26892
    Overexpression of SlATG8f gene enhanced autophagy and pollen protection in tomato under heat stress.
    Liu Song, Cen Wen, Zhuo He, Xingxue Zha, Qunmei Cheng, Wen Xu.
      Autophagy is a mechanism for the degradation of cellular components in eukaryotes and plays a critical role in plant responses to abiotic stress. As a core member of the autophagy process, ATG8's role in how plants respond to heat stress remains unclear. To investigate the response of the tomato autophagy core member ATG8f to heat stress, we studied the key gene ATG8f and generated tomato lines overexpressing SlATG8f using the recombinant expression vector pBWA(V)HS. We observed that under heat stress, SlATG8f overexpression (OE) plants exhibited decreased heat tolerance compared to wild-type (WT) plants. Specifically, OE plants showed increased relative electrolyte leakage, reduced soluble solid content, elevated chlorophyll content, and higher autophagosome numbers, with less damage to chloroplasts and mitochondria. Additionally, expression of some ATG8 family genes and heat shock protein-related genes was upregulated. Moreover, SlATG8f overexpressing plants had higher pollen vitality and more intact pollen morphology. These results suggest that while SlATG8f overexpression renders plants more sensitive to heat, it helps mitigate high-temperature damage to tomato pollen by maintaining chloroplast integrity and interacting with heat shock proteins to respond to heat stress.
    Keywords:   SlATG8f ; Autophagy; Heat stress; Tomato
    DOI:  https://doi.org/10.1038/s41598-024-77491-7
  11. New Phytol. 2024 Nov 04.
    Autophagy is essential for somatic embryogenesis in citrus through regulating amyloplast degradation and lipid homeostasis.
    Erlin Gao, Yunju Zhao, Mengxia Wu, Kun Wang, Qiwei Zheng, Yanlong Li, Xiaolu Qu, Xiaomeng Wu, Wenwu Guo, Pengwei Wang.
      Autophagy is a conserved degradation pathway that regulates the clearance of paternal substrate at the early embryogenesis stage of animals. However, its mode of action is likely different in plants, which can regenerate through apomixis without fertilisation. Somatic embryogenesis (SE) is a unique plant process widely used for plant propagation and germplasm utilisation. Here, we studied citrus as an example and found a higher autophagic activity after SE initiation. Interestingly, amyloplasts were frequently found inside autophagosomes, whereas the inhibition of autophagy blocks amyloplasts/starch degradation and hinders somatic embryo formation. Furthermore, the consumption of storage lipids was faster in autophagy mutants, suggesting lipid metabolism is activated when starch utilisation is blocked. Exogenous application of autophagy-inducing chemicals (e.g. spermidine) significantly promoted the formation of autophagosomes and increased SE efficiency, indicating a positive correlation between autophagy, energy metabolism, and somatic embryo formation in citrus. Taken together, our study unveils a pathway for the degradation of plant-specific organelles and provides an effective approach for plant propagation.
    Keywords:  amyloplasts; autophagy; citrus; somatic embryogenesis; storage lipids
    DOI:  https://doi.org/10.1111/nph.20242
  12. Zygote. 2024 Nov 05. 1-10
    The effect of rapamycin treatment on mouse ovarian follicle development in dehydroepiandrosterone-induced polycystic ovary syndrome mouse model.
    Ecem Yildirim, Tugce Onel, Sami Agus, Elif Gunalan, Bayram Yilmaz, Mehmet Serif Aydin, Aylin Yaba.
      Polycystic ovary syndrome (PCOS) is a complex reproductive and endocrine disorder affecting 5-10% of women of reproductive age, but the pathophysiology of PCOS still remains unknown. Here, the aim of our study was to analyze the effects of rapamycin treatment that may regulate impaired hormonal levels and folliculogenesis in dehydroepiandrosterone (DHEA)-treated PCOS mouse. We hypothesized that rapamycin may ameliorate the negative effects of PCOS in DHEA-induced PCOS mouse model. The target of rapamycin (TOR) gene product is a serine/threonine kinase that has been implicated in the control of cell growth, proliferation and autophagy, and rapamycin is a potent inhibitor of mTORC1 pathway. In this study, for the first time, mTORC1 and activation products are presented at protein and mRNA levels after rapamycin treatment in DHEA-induced PCOS mouse ovary. We showed that rapamycin treatment may regulate follicular development, hormonal levels and provide ovulation in DHEA-induced PCOS mouse. Additionally, we assessed decreased primordial follicle reserve, increased number of primary and secondary follicles, corpus luteum structure forms again after 10 days of rapamycin treatment. This study presented here suggests rapamycin treatment regulates hormonal phenotype and folliculogenesis in the ovary and also mTOR signalling pathway in granulosa cells of DHEA-induced PCOS mouse ovary which may have potential to attenuate understanding the mechanism of dominant follicle selection and anovulatory infertility.
    Keywords:  Ovulation; dehidroepiandrostenedion; folliculogenesis; mammalian target of rapamycin; polycystic ovary syndrome; rapamycin
    DOI:  https://doi.org/10.1017/S0967199424000388
  13. Biochim Biophys Acta Mol Basis Dis. 2024 Oct 25. pii: S0925-4439(24)00547-7. [Epub ahead of print] 167553
    TMEM106B knockdown exhibits a neuroprotective effect in Parkinson's disease models via regulating autophagy-lysosome pathway.
    Yumei Liu, Kunpeng Qin, Kaixin Dou, Jiwen Ren, Binghui Hou, Anmu Xie.
      BACKGROUND: TMEM106B, a lysosomal transmembrane protein, has been reported to be associated with Parkinson's disease (PD). However, the precise physiopathologic mechanism of TMEM106B in PD remains unclear.OBJECTIVE: This study aimed to explore the influence of TMEM106B on the autophagy-lysosome pathway (ALP) in PD.
    METHODS: 55 patients with PD and 40 healthy controls were enrolled. RT-qPCR and ELISA were employed to assess the levels of TMEM106B. In vitro and in vivo models of PD, Lentivirus-shTMEM106B and AAV-shTMEM106B were used to knockdown the expression of TMEM106B. Behavioral experiments, western blot, immunofluorescence, and immunohistochemistry were used to detect the effect of TMEM106B on the ALP process.
    RESULTS: We found that the levels of TMEM106B were increased in the PD patients and PD models. TMEM106B knockdown markedly improved the motor deficits and tyrosine hydroxylase (TH) expression of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-lesioned mice. TMEM106B knockdown promoted α-syn clearance by regulating the ALP process in MPP+-induced SH-SY5Y cells and MPTP-treated mice. Further studies revealed that TMEM106B knockdown might activate ALP through activating AMPK-mTOR-TFEB axis. Furthermore, TMEM106B may play a vital role in the ALP by mediating the expression of TDP43.
    CONCLUSIONS: Taken together, our study suggests that TMEM106B knockdown mediates the ALP pathway, leading to a decrease in α-syn, providing a new direction and perspective for the regulation of autophagy in PD.
    Keywords:  Autophagy-lysosome pathway; Parkinson's disease; TDP43; TMEM106B
    DOI:  https://doi.org/10.1016/j.bbadis.2024.167553
  14. J Vis Exp. 2024 Oct 18.
    Evaluation of LC3-II Release via Extracellular Vesicles in Relation to the Accumulation of Intracellular LC3-positive Vesicles.
    Shun-Ichi Ito, Yoshinori Tanaka.
      (Macro)autophagy represents a fundamental cellular degradation pathway. In this process, double-membraned vesicles known as autophagosomes engulf cytoplasmic contents, subsequently fusing with lysosomes for degradation. Beyond the canonical role, autophagy-related genes also modulate a secretory pathway involving the release of inflammatory molecules, tissue repair factors, and extracellular vesicles (EVs). Notably, the process of disseminating pathological proteins between cells, particularly in neurodegenerative diseases affecting the brain and spinal cord, underscores the significance of understanding this phenomenon. Recent research suggests that the transactive response DNA-binding protein 43 kDa (TDP-43), a key player in amyotrophic lateral sclerosis and frontotemporal lobar degeneration, is released in an autophagy-dependent manner via EVs enriched with the autophagosome marker microtubule-associated proteins 1A/1B light chain 3B-II (LC3-II), especially when autophagosome-lysosome fusion is inhibited. To elucidate the mechanism underlying the formation and release of LC3-II-positive EVs, it is imperative to establish an accessible and reproducible method for evaluating both intracellular and extracellular LC3-II-positive vesicles. This study presents a detailed protocol for assessing LC3-II levels via immunoblotting in cellular and EV fractions obtained through differential centrifugation. Bafilomycin A1 (Baf), an inhibitor of autophagosome-lysosome fusion, serves as a positive control to enhance the levels of intracellular and extracellular LC3-II-positive vesicles. Tumor susceptibility gene 101 (TSG101) is used as a marker for multivesicular bodies. Applying this protocol, it is demonstrated that siRNA-mediated knockdown of syntaxin-6 (STX6), a genetic risk factor for sporadic Creutzfeldt-Jakob disease, augments LC3-II levels in the EV fraction of cells treated with Baf while showing no significant effect on TSG101 levels. These findings suggest that STX6 may negatively regulate the extracellular release of LC3-II via EVs, particularly under conditions where autophagosome-lysosome fusion is impaired. Combined with established methods for evaluating autophagy, this protocol provides valuable insights into the role of specific molecules in the formation and release of LC3-II-positive EVs.
    DOI:  https://doi.org/10.3791/67385
  15. Nat Commun. 2024 Nov 01. 15(1): 9463
    Increased DNA damage in full-grown oocytes is correlated with diminished autophagy activation.
    Fei Sun, Nourhan Nashat Ali, Daniela Londoño-Vásquez, Constantine A Simintiras, Huanyu Qiao, M Sofia Ortega, Yuksel Agca, Masashi Takahashi, Rocío M Rivera, Andrew M Kelleher, Peter Sutovsky, Amanda L Patterson, Ahmed Z Balboula.
      Unlike mild DNA damage exposure, DNA damage repair (DDR) is reported to be ineffective in full-grown mammalian oocytes exposed to moderate or severe DNA damage. The underlying mechanisms of this weakened DDR are unknown. Here, we show that moderate DNA damage in full-grown oocytes leads to aneuploidy. Our data reveal that DNA-damaged oocytes have an altered, closed, chromatin state, and suggest that the failure to repair damaged DNA could be due to the inability of DDR proteins to access damaged loci. Our data also demonstrate that, unlike somatic cells, mouse and porcine oocytes fail to activate autophagy in response to DNA double-strand break-inducing treatment, which we suggest may be the cause of the altered chromatin conformation and inefficient DDR. Importantly, autophagy activity is further reduced in maternally aged oocytes (which harbor severe DNA damage), and its induction is correlated with reduced DNA damage in maternally aged oocytes. Our findings provide evidence that reduced autophagy activation contributes to weakened DDR in oocytes, especially in those from aged females, offering new possibilities to improve assisted reproductive therapy in women with compromised oocyte quality.
    DOI:  https://doi.org/10.1038/s41467-024-53559-w
  16. Nat Commun. 2024 Nov 06. 15(1): 9578
    AMFR-mediated Flavivirus NS2A ubiquitination subverts ER-phagy to augment viral pathogenicity.
    Linliang Zhang, Hongyun Wang, Chao Han, Qi Dong, Jie Yan, Weiwei Guo, Chao Shan, Wen Zhao, Pu Chen, Rui Huang, Ying Wu, Yu Chen, Yali Qin, Mingzhou Chen.
      Flaviviruses strategically utilize the endoplasmic reticulum (ER) in their replication cycles. However, the role of ER autophagy (ER-phagy) in viral replication process remains poorly understood. Here, we reveal that prolonged Zika virus (ZIKV) infection results from the degradation of ER-phagy receptor FAM134B, facilitated by viral NS2A protein. Mechanistically, ER-localized NS2A undergoes K48-linked polyubiquitination at lysine (K) 56 by E3 ligase AMFR. Ubiquitinated NS2A binds to FAM134B and AMFR orchestrates the degradation of NS2A-FAM134B complexes. AMFR-catalyzed NS2A ubiquitination not only targets FAM134B degradation but also hinders the FAM134B-AMFR axis. Notably, a recombinant ZIKV mutant (ZIKV-NS2AK56R), lacking ubiquitination and ER-phagy inhibition, exhibits attenuation in ZIKV-induced microcephalic phenotypes in human brain organoids and replicates less efficiently, resulting in weakened pathogenesis in mouse models. In this work, our mechanistic insights propose that flaviviruses manipulate ER-phagy to modulate ER turnover, driving viral infection. Furthermore, AMFR-mediated flavivirus NS2A ubiquitination emerges as a potential determinant of viral pathogenecity.
    DOI:  https://doi.org/10.1038/s41467-024-54010-w
  17. Arch Biochem Biophys. 2024 Oct 31. pii: S0003-9861(24)00323-0. [Epub ahead of print]762 110201
    The cGAS-STING mediated crosstalk between innate immunity and autophagy in leishmaniasis using mathematical modeling: Uncovering new therapeutic avenues.
    Anil Tambekar, Vrushali Guhe, Shailza Singh.
      The present paper deals with the investigation into the cGAS-STING pathway, focusing on the signaling of interferons through mathematical modeling and identifying a significant positive feedback loop regulated by STING for activation of type 1 interferons (IFN-1). Cyclic GMP-AMP synthase (cGAS) is responsible for detecting cytosolic DNA and initiating the STING (stimulator of interferon genes) pathway, which in turn causes the synthesis of pro-inflammatory cytokines and type I interferons. In addition to being crucial for pathogen identification, this route interacts with autophagy, a cellular mechanism that is necessary for immunological homeostasis and pathogen removal. In the context of Leishmania infection, the cGAS-STING signaling axis has come to light as a critical mediator of the crosstalk between innate immunity and autophagy. Further, the protein-protein interaction studies underscored the significance of two distinct domains in mediating interactions with IRF3 and LC3. Importantly, our findings suggest the possibility of manipulating STING concomitantly to regulate IRF3 and LC3 independently. This study remarkably advances our understanding of STING's multifaceted roles, particularly in regulating IFN-1 and autophagy, highlighting its pivotal role as a cross-talk point in leishmaniasis.
    Keywords:  Interferons; Leishmaniasis; Mathematical model; Positive feedback loop; STING
    DOI:  https://doi.org/10.1016/j.abb.2024.110201
  18. Mol Cell Biochem. 2024 Nov 06.
    Cytoglobin augments ferroptosis through autophagic degradation of ferritin in colorectal cancer cells.
    Chengjiang Fan, Ziyang Luo, Qingfang Zheng, Yuhang Xu, Yao Xu, Jianing Chen, You Meng, Haizhong Jiang, Kaitai Liu, Yang Xi.
      Autophagy has gained importance in the context of ferroptosis. Nevertheless, a deeper understanding of the regulatory mechanism governing autophagy-dependent ferroptosis is necessary. Cytoglobin (CYGB), a member of the globin family, exhibits antifibrotic effects, regulates cellular reactive oxygen species, and stimulates tumor inhibition. Herein, we present further insights into the role of CYGB in ferroptosis regulation. Our investigation confirmed that CYGB impedes cell proliferation and migration. Furthermore, a significant association between CYGB and the lysosomal pathway was suggested based on the RNA sequencing data analysis. Elevated lysosomal signal and colocalization of CYGB with lysosome-associated membrane glycoprotein 1 (LAMP1) were observed. Moreover, upregulated autophagy and augmented ferroptosis induced by RSL3 were confirmed in CYGB-overexpression cells with an obviously increased colocalization of nuclear receptor coactivator 4 (NCOA4) and LC3B. The autophagy inhibitor bafilomycin or chloroquine alleviated autophagy-dependent degradation of ferritin protein under RSL3 treated condition. Additionally, a colocalization of CYGB with the transferrin receptor (TFR) was confirmed. Our results demonstrate an important functional pathway by which CYGB regulates ferroptosis through TFR-binding and autophagic degradation of ferritin, and provide a potential pathway for the treatment of colorectal cancer.
    Keywords:  Colorectal cancer; Cytoglobin; Ferritin; Ferroptosis
    DOI:  https://doi.org/10.1007/s11010-024-05148-0
  19. Eur J Med Res. 2024 Nov 01. 29(1): 524
    Regulation of mitochondrial autophagy by lncRNA MALAT1 in sepsis-induced myocardial injury.
    Guangqing Huang, Xu Zhao, Yong Bai, Jie Liu, Wei Li, Yongquan Wu.
      BACKGROUND: Sepsis-induced myocardial injury (SIMI) is a severe complication of sepsis, contributing significantly to mortality. Mitochondrial dysfunction and dysregulated autophagy are implicated in SIMI pathogenesis. Long non-coding RNA MALAT1 has been associated with various diseases, including sepsis, but its role in SIMI remains unclear.OBJECTIVE: This study aimed to investigate the role of lncRNA MALAT1 in SIMI, specifically in the regulation of mitochondrial autophagy.
    METHODS: A sepsis-induced cardiomyopathy model was established in mice, and the cardiac tissues were analyzed. The expression of lncRNA MALAT1 was modulated and its effects on mitochondrial autophagy, myocardial injury, inflammation, and apoptosis were assessed. Furthermore, the interaction between MALAT1 and miR-146a was explored, as well as the involvement of the TLR4/NF-kB/MAPK signaling pathway.
    RESULTS: Activation of mitochondrial autophagy by urolithin A (UA) alleviated SIMI, inflammation, and cardiac dysfunction. Downregulation of MALAT1 enhanced mitochondrial autophagy, stabilized the mitochondrial membrane potential, and inhibited mitochondrial reactive oxygen species (ROS) production, leading to improved cell viability and reduced myocardial injury. Furthermore, MALAT1 interacted with miR-146a, and their modulation influenced mitochondrial autophagy, myocardial injury, and inflammation. The TLR4/NF-kB/MAPK signaling pathway was implicated in these processes.
    CONCLUSION: Our findings suggest that lncRNA MALAT1 plays a crucial role in SIMI by modulating miR-146a-mediated mitochondrial autophagy and the TLR4/NF-kB/MAPK signaling pathway. These results provide new insights into the pathogenesis of SIMI and potential therapeutic targets.
    Keywords:  LncRNA MALAT1; MiR-146a; Mitophagy; Myocardial injury; Sepsis
    DOI:  https://doi.org/10.1186/s40001-024-02098-7
  20. Biochim Biophys Acta Mol Basis Dis. 2024 Oct 31. pii: S0925-4439(24)00551-9. [Epub ahead of print]1871(1): 167557
    The IP3R inhibitor desmethylxestospongin B reduces tumor cell migration, invasion and metastasis by impairing lysosome acidification and β1-integrin recycling.
    Galdo Bustos, Ulises Ahumada-Castro, Eduardo Silva-Pavez, Hernán Huerta, Andrea Puebla, Camila Quezada, Pablo Morgado-Cáceres, César Casanova-Canelo, Natalia Smith-Cortinez, Maša Podunavac, Cesar Oyarce, Alvaro Lladser, Paula Farias, Alenka Lovy, Jordi Molgó, Vicente A Torres, Armen Zakarian, J César Cárdenas.
      Cancer is the second leading cause of death worldwide. >90 % of cancer-related deaths are due to metastasis, a process that depends on the ability of cancer cells to leave the primary tumor, migrate, and colonize different tissues. Inositol 1,4,5-trisphosphate receptor (IP3R)-mediated Ca2+ signaling plays an essential role in maintaining the homeostasis of cancer cells and the sustained proliferation. Desmethylxestospongin B (dmXeB) is a specific inhibitor of the IP3R that selectively arrests cell proliferation and promotes cancer cell death at high concentrations. However, whether migration, invasion and metastasis can be affected by this drug is unknown. Here, by using the highly metastatic triple negative breast cancer (TNBC) cell line MDA-MB-231, we demonstrate that a prolonged inhibition of IP3R-mediated Ca2+ signals with dmXeB significantly reduces cell migration and invasion in vitro and metastasis in vivo. We found that this phenomenon was independent of the bioenergetic control of IP3R over the mitochondria and AMPK activation. Furthermore, employing a tandem LC3-GFP-mcherry assay, we found that prolonged inhibition of IP3R with dmXeB leads to diminished autophagic flux. This reduction can be attributed to impaired lysosomal acidification, as evidenced by assessments using DQ-BSA and pHrodo. Since cell migration requires appropriate assembly and disassembly of focal adhesions, along with the internalization and recycling of integrins via autophagy, we explored the dependency of integrin recycling from autophagosomes, finding that IP3R inhibition with dmXeB impaired the recycling of β1-integrins, which accumulated within autophagosomes. Our findings reveal an unexpected effect of IP3R inhibition with dmXeB in cancer cells that could represent a novel therapeutic strategy for the treatment of cancer metastasis.
    Keywords:  Breast cancer cell migration; Calcium signaling; Inositol 1,4,5-trisphosphate receptors; Lysosomes; Metastasis
    DOI:  https://doi.org/10.1016/j.bbadis.2024.167557
  21. Exp Physiol. 2024 Nov 05.
    Acute high-intensity muscle contraction moderates AChR gene expression independent of rapamycin-sensitive mTORC1 pathway in rat skeletal muscle.
    Yuhei Makanae, Satoru Ato, Karina Kouzaki, Yuki Tamura, Koichi Nakazato.
      The relationship between mechanistic target of rapamycin complex 1 (mTORC1) activation after resistance exercise and acetylcholine receptor (AChR) subunit gene expression remains largely unknown. Therefore, we aimed to investigate the effect of electrical stimulation-induced intense muscle contraction, which mimics acute resistance exercise, on the mRNA expression of AChR genes and the signalling pathways involved in neuromuscular junction (NMJ) maintenance, such as mTORC1 and muscle-specific kinase (MuSK). The gastrocnemius muscle of male adult Sprague-Dawley rats was isometrically exercised. Upon completion of muscle contraction, the rats were euthanized in the early (after 0, 1, 3, 6 or 24 h) and late (after 48 or 72 h) recovery phases and the gastrocnemius muscles were removed. Non-exercised control animals were euthanized in the basal state (control group). In the early recovery phase, Agrn gene expression increased whereas LRP4 decreased without any change in the protein and gene expression of AChR gene subunits. In the late recovery phase, Agrn, Musk, Chrnb1, Chrnd and Chrne gene expression were altered and agrin and MuSK protein expression increased. Moreover, mTORC1 and protein kinase B/Akt-histone deacetylase 4 (HDAC) were activated in the early phase but not in the late recovery phase. Furthermore, rapamycin, an inhibitor of mTORC1, did not disturb changes in AChR subunit gene expression after muscle contraction. However, rapamycin addition slightly increased AChR gene expression, while insulin did not impact it in rat L6 myotube. These results suggest that changes in the AChR subunits after muscle contraction are independent of the rapamycin-sensitive mTORC1 pathway.
    Keywords:  acetylcholine receptor; mTORC1; muscle contraction
    DOI:  https://doi.org/10.1113/EP091006
  22. Biochim Biophys Acta Mol Cell Res. 2024 Oct 26. pii: S0167-4889(24)00212-X. [Epub ahead of print] 119869
    Endoplasmic reticulum stress-mediated autophagy in cancer and its interaction with apoptosis and ferroptosis.
    Qiang Ma, Haitang Liao, Shuang Liu, He Huang, Arul Goel, Pedram Torabian, Chakrabhavi Dhananjaya Mohan, Chenyang Duan.
      The endoplasmic reticulum (ER) is a dynamic organelle that is a site of the synthesis of proteins and lipids and contributes to the regulation of proteostasis, lipid metabolism, redox balance, and calcium storage/-dependent signaling events. The disruption of ER homeostasis due to the accumulation of misfolded proteins in the ER causes ER stress which activates the unfolded protein response (UPR) system through the activation of IRE1, PERK, and ATF6. Activation of UPR is observed in various cancers and the tumor cells effectively utilize the UPR system to overcome ER stress. Also, ER stress and autophagy are the stress response mechanisms that operate together to maintain cellular homeostasis. In cancers, ER stress-mediated autophagy can function as either pro-survival or pro-death in a context-dependent manner. ER stress-mediated autophagy can have crosstalk with other types of cell death pathways including apoptosis and ferroptosis. In this article, we have reviewed the role of ER stress in the regulation of autophagy-mediated tumorigenesis and its interactions with other cell death mechanisms such as apoptosis and ferroptosis. We have also comprehensively discussed the effect of ER stress-mediated autophagy on cancer progression and chemotherapeutic resistance.
    Keywords:  Autophagy; Cancer; Cell death; Endoplasmic reticulum stress; Ferroptosis
    DOI:  https://doi.org/10.1016/j.bbamcr.2024.119869
  23. Cardiovasc Res. 2024 Nov 05. pii: cvae238. [Epub ahead of print]
    PINK1-mediated mitophagy attenuates pathological cardiac hypertrophy by suppressing the mtDNA release-activated cGAS-STING pathway.
    Haobin Zhou, Xiao Wang, Tianyu Xu, Daojing Gan, Zhuang Ma, Hao Zhang, Jian Zhang, Qingchun Zeng, Dingli Xu.
      AIMS: Sterile inflammation is implicated in the development of heart failure (HF). Mitochondria plays important roles in triggering and maintaining inflammation. Mitophagy is important for regulation of mitochondrial quality and maintenance of cardiac function under pressure overload. The association of mitophagy with inflammation in HF is largely unclear. As PINK1 is a central mediator of mitophagy, our objective was to investigate its involvement in cardiac hypertrophy, and the effect of PINK1-mediated mitophagy on cGAS-STING activation during cardiac hypertrophy.METHODS AND RESULTS: PINK1 knockout and cardiac-specific PINK1-overexpressing transgenic mice were created and subsequently subjected to transverse aortic constriction (TAC) surgery. In order to explore whether PINK1 regulates STING-mediated inflammation during HF, PINK1/STING (stimulator of interferon genes) double-knockout mice were created. Pressure overload was induced by TAC. Our findings indicate a significantly decline in PINK1 expression in TAC-induced hypertrophy. Cardiac hypertrophic stimuli caused the release of mitochondrial DNA (mtDNA) into the cytosol, activating the cGAS-STING signaling, which in turn initiated cardiac inflammation and promoted the progression of cardiac hypertrophy. PINK1 deficiency inhibited mitophagy activity, promoted mtDNA release, and then drove the overactivation of cGAS-STING signaling, exacerbating cardiac hypertrophy. Conversely, cardiac-specific PINK1 overexpression protected against hypertrophy thorough inhibition of the cGAS-STING signaling. Double-knockout mice revealed that the effects of PINK1 on hypertrophy were dependent on STING.
    CONCLUSIONS: Our findings suggest that PINK1-mediated mitophagy plays a protective role in pressure overload-induced cardiac hypertrophy via inhibiting the mtDNA-cGAS-STING pathway.
    Keywords:  Mitophagy; PINK1; STING; cardiac hypertrophy; inflammation
    DOI:  https://doi.org/10.1093/cvr/cvae238
  24. Cell Signal. 2024 Nov 01. pii: S0898-6568(24)00475-3. [Epub ahead of print]125 111500
    STIM1 promotes cervical cancer progression through autophagy activation via TFEB nuclear translocation.
    Xi Luo, Mengchan Jian, Ping Wu, Yahua Wu, Yulan Ma, Na Feng, Min Lu, Dandan Shi, Rui Liu, Yan Ding, Wenjun Zhang, Li Fan, Xiju He.
      BACKGROUND: Autophagy plays an important role in maintaining the stability of intracellular environment, abnormal autophagy is associated with the occurrence and progression of cancer, the role of STIM1 in regulating cancer autophagy remains controversial, and its clinical relevance is unclear. Our study aimed to investigate the effect and mechanism of STIM1 on cervical cancer, thus to provide new molecular therapeutic targets for cervical cancer in clinic.METHODS: We collected CIN III, FIGO IB and IIA fresh Specimens without chemotherapy from patients in Renmin Hospital of Hubei University of Medicine (n = 10). STIM1, TFEB and autophagy related proteins of different stage tissues were detected. In vitro, SKF96365 and AncoA4 were used to inhibit STIM1-administrated Ca2+ entry of SiHa cells, Cyclosporine A (calcineurin inhibitors) were used to inhibit CaN/TFEB pathway, Ad-mCherry-GFPLC3B was used to detect autophagy flux, shSTIM1 was used to knockdown STIM1 expression.
    RESULTS: The expression levels of STIM1, TFEB and autophagy related proteins were positively correlated with the progression of cervical cancer. Inhibition of STIM1-mediated SOCE can decrease proliferation and migration, and promoted the apoptosis of cervical cancer cells. Knockdown STIM1 can inhibit autophagy and TFEB nuclear translocation.
    CONCLUSION: STIM1 can promote autophagy and accelerate cervical cancer progression by increasing TFEB nuclear translocation of cervical cancer cells.
    Keywords:  Autophagy; Calcium; Cervical cancer; STM1; Transcription factor EB
    DOI:  https://doi.org/10.1016/j.cellsig.2024.111500
  25. J Cell Biol. 2025 Jan 06. pii: e202403104. [Epub ahead of print]224(1):
    Activation of lysosomal Ca2+ channels mitigates mitochondrial damage and oxidative stress.
    Xinghua Feng, Weijie Cai, Qian Li, Liding Zhao, Yaping Meng, Haoxing Xu.
      Elevated levels of plasma-free fatty acids and oxidative stress have been identified as putative primary pathogenic factors in endothelial dysfunction etiology, though their roles are unclear. In human endothelial cells, we found that saturated fatty acids (SFAs)-including the plasma-predominant palmitic acid (PA)-cause mitochondrial fragmentation and elevation of intracellular reactive oxygen species (ROS) levels. TRPML1 is a lysosomal ROS-sensitive Ca2+ channel that regulates lysosomal trafficking and biogenesis. Small-molecule agonists of TRPML1 prevented PA-induced mitochondrial damage and ROS elevation through activation of transcriptional factor EB (TFEB), which boosts lysosome biogenesis and mitophagy. Whereas genetically silencing TRPML1 abolished the protective effects of TRPML1 agonism, TRPML1 overexpression conferred a full resistance to PA-induced oxidative damage. Pharmacologically activating the TRPML1-TFEB pathway was sufficient to restore mitochondrial and redox homeostasis in SFA-damaged endothelial cells. The present results suggest that lysosome activation represents a viable strategy for alleviating oxidative damage, a common pathogenic mechanism of metabolic and age-related diseases.
    DOI:  https://doi.org/10.1083/jcb.202403104
  26. Front Pharmacol. 2024 ;15 1499116
    Monocrotaline-mediated autophagy via inhibiting PI3K/AKT/mTOR pathway induces apoptosis in rat hepatocytes.
    Yazhou Guo, Yang Yuan, Ruibo Wang, Jun Bai, Yanqing Jia, Xinxin Qiu, Huafeng Niu, Long Li, Yan Luo, Baoyu Zhao, Zhencang Zhang.
      Monocrotaline (MCT), a major pyrrolizidine alkaloid, is well-known for its high liver toxicity. Dysregulation of autophagy induced apoptosis can lead to various liver diseases, including those induced by chemical compounds. Therefore, we aim to explore whether autophagy might serve as a potential strategy for addressing liver apoptosis caused by MCT. In primary rat hepatocytes (PRHs), MCT significantly increased the number of autophagosomes and the expression levels of LC3II, Becline-1, and Atg5, while it decreased the expression of p62 in a concentration-dependent manner at doses of 100, 200, 300, and 400 μM. Western blot assays revealed MCT inhibited the phosphorylation levels of the PI3K/AKT/mTOR pathway. To elucidate the role of autophagy in mediating MCT-induced apoptosis, we further pretreated PRHs with the autophagy agonist Rapamycin and the inhibitors Bafilomycin A1 and Chloroquine, respectively, and assessed the apoptosis of PRHs induced by MCT. The results displayed that Rapamycin increased the apoptosis rate and the expression of cleaved caspase-3, whereas Bafilomycin A1 and Chloroquine reduced the apoptosis and the expression of cleaved caspase-3 in PRHs. This study confirms that autophagy enhances PRHs apoptosis induced by MCT. In summary, this study demonstrates that MCT-induced autophagy via inhibition of the PI3K/AKT/mTOR pathway can lead to apoptosis in PRHs.
    Keywords:  PI3K/Akt/mTOR signaling pathway; apoptosis; autophagy; hepatotoxicity; monocrotaline
    DOI:  https://doi.org/10.3389/fphar.2024.1499116
  27. Int Immunopharmacol. 2024 Oct 25. pii: S1567-5769(24)01990-8. [Epub ahead of print]143(Pt 2): 113468
    PKCδ modulates SP1 mediated mitochondrial autophagy to exacerbate diacetylmorphine-induced ferroptosis in neurons.
    Mengjie Zhuang, Sensen Zhu, Liping Su, Li Liu, Min Ji, Chenlu Dai, Jingyu Liu, Wei Zhang, Hongwei Pu.
      Diacetylmorphine (DA) is widely implicated in neuronal injury; however, the underlying mechanisms remain unclear. We investigated the role of iron metamorphosis in DA-induced neurotoxicity using Sprague-Dawley rats and PC12 and SH-SY5Y cells. Tandem mass tag proteomics analysis showed that the upregulation of protein kinase C delta (PKCδ) and iron metabolism-related protein transferrin receptor (TFRC) significantly the enriched iron metabolism pathway. Subsequent experiments showed that DA exposure significantly upregulated PKCδ in PC12 cells, which increased the nuclear translocation of specificity protein 1 (SP1), and the intracellular free iron and lipid peroxide levels. In addition, silencing of PKCδ in rats improved behaviour and restored the expression level of glutathione peroxidase 4 (GPX4). In addition, DA exposure activated mitochondrial autophagy in PC12 cells, leading to a decrease in the mitochondrial membrane potential, accumulation of reactive oxygen species (ROS), elevation of LC3 (which plays a key role in autophagy), and a decrease in p62 expression. Following the inhibition of autophagy, the mitochondrial membrane potential and ROS were restored, as was the expression of voltage-dependent anion channel 1 (VDAC1) and GPX4. In conclusion, the present study suggests that PKCδ regulates SP1, further exacerbating DA-induced neuronal ferroptosis. Therefore, inhibition of PKCδ and mitochondrial autophagy or ferroptosis may be a key therapeutic target to ameliorate neurotoxicity following DA exposure.
    Keywords:  Diacetylmorphine; Mitophagy; Neuronal ferroptosis; PKCδ; SP1
    DOI:  https://doi.org/10.1016/j.intimp.2024.113468
  28. EMBO Rep. 2024 Nov 06.
    The USP12/46 deubiquitinases protect integrins from ESCRT-mediated lysosomal degradation.
    Kaikai Yu, Guan M Wang, Shiny Shengzhen Guo, Florian Bassermann, Reinhard Fässler.
      The functions of integrins are tightly regulated via multiple mechanisms including trafficking and degradation. Integrins are repeatedly internalized, routed into the endosomal system and either degraded by the lysosome or recycled back to the plasma membrane. The ubiquitin system dictates whether internalized proteins are degraded or recycled. Here, we use a genetic screen and proximity-dependent biotin identification to identify deubiquitinase(s) that control integrin surface levels. We find that a ternary deubiquitinating complex, comprised of USP12 (or the homologous USP46), WDR48 and WDR20, stabilizes β1 integrin (Itgb1) by preventing ESCRT-mediated lysosomal degradation. Mechanistically, the USP12/46-WDR48-WDR20 complex removes ubiquitin from the cytoplasmic tail of internalized Itgb1 in early endosomes, which in turn prevents ESCRT-mediated sorting and Itgb1 degradation.
    Keywords:  DUB; ESCRT; Integrin; USP12/USP46; Ubiquitination
    DOI:  https://doi.org/10.1038/s44319-024-00300-9
  29. Exp Cell Res. 2024 Nov 04. pii: S0014-4827(24)00414-2. [Epub ahead of print] 114323
    NLRX1 and STING alleviate renal ischemia-reperfusion injury by regulating LC3 lipidation during mitophagy.
    Yinping Liao, Pei Li, Qing Hang, Yang Chong, Wei Long, Xingji Wei, Dong Sun, Ya Liu.
      Mitophagy significantly influences renal ischemia/reperfusion (I/R) injury and recovery. NLRX1 is recognized for its regulatory role in governing mitochondrial damage, autophagy, and the expression of pro-inflammatory factors. Despite the acknowledged involvement of NLRX1 in these crucial cellular processes, its specific function in renal I/R injury remains unclear. We detected the expression of NLRX1, the cGAS-STING pathway, and autophagy-related proteins using Western Blot analysis. RT-qPCR was utilized to measure the expression of NLRX1 mRNA and cytokines, and changes in mitochondrial DNA (mtDNA) within the cytoplasm. Immunofluorescence was applied to observe alterations in DNA distribution within the cytoplasm. The EtBr drug, which depletes mtDNA, and the Mdivi-1 mitophagy inhibitor, were used to verify the promotion of mitophagy by NLRX1. The results demonstrated that NLRX1 was downregulated after H/R injury, and there was an increase in cytoplasmic DNA. NLRX1 overexpression not only reduced IL-1β and IL-6 levels, but also decreased mtDNA in the cytoplasm. Additionally, NLRX1 further increases mitochondrial LC3 lipidation after H/R injury, and this effect is inhibited by Mdivi-1 drugs. The activation of the cGAS-STING pathway after H/R injury is inhibited by EtBr drugs and NLRX1. Co-immunoprecipitation results showed that NLRX1 could bind to STING. Moreover, inhibiting STING reversed NLRX1-induced mitochondrial LC3 lipidation. Our study reveals that NLRX1 can bind to STING to promote mitophagy and inhibits inflammation caused by mtDNA/cGAS/STING signaling.
    Keywords:  Acute kidney injury; Mitophagy; NLRX1; STING
    DOI:  https://doi.org/10.1016/j.yexcr.2024.114323
  30. Invest Ophthalmol Vis Sci. 2024 Nov 04. 65(13): 12
    PINK1/Parkin-Mediated Mitophagy Ameliorates Mitochondrial Dysfunction in Lacrimal Gland Acinar Cells During Aging.
    Han Zhao, Yue Zhang, Yujie Ren, Wanpeng Wang.
      Purpose: Aging alters the function of the lacrimal gland and disrupts the balance of the microenvironment on the ocular surface, eventually leading to aqueous-tear-deficient dry eye. Mitophagy has been reported to play an important role in aging, but the underlying mechanism remains unclear.Methods: The young (6 weeks) and middle-aged (12 months) male C57BL/6J mice were used in this study, and mitophagy agonist rapamycin and inhibitor Mdivi-1 were used in in vivo experiments. Hematoxylin and eosin, Masson, Oil Red O, and reactive oxygen species (ROS) staining were used to detect histological changes and lipids in lacrimal gland. Changes in the expression of proteins were identified by Western blotting of lacrimal gland lysates. Transmission electron microscopy and immunofluorescence staining were used to assess mitophagy. The single-cell RNA sequencing (scRNA-seq) and bioinformatics analyses were used to detect transcription signature changes during aging.
    Results: In this study, we discovered that aging increased oxidative stress, which increased apoptosis, and generated ROS in acinar epithelial cells. Furthermore, activation of PINK1/Parkin-mediated mitophagy by rapamycin reduced lacrimal gland ROS concentrations and prevented aging-induced apoptosis of acinar cells, thereby causing histological alterations, microstructural degradation, and increasing tear secretion associated with ROS accumulation. By contrast, Mdivi-1 aggregates mitochondrial function and thereafter leads to lacrimal gland function impairment by inhibiting mitochondrial fission and giving rise to mitophagy.
    Conclusions: Overall, our findings suggested that aging could impair mitochondrial function of acinar cells, and age-related alterations may be treated with therapeutic approaches that enhance mitophagy while maintaining mitochondrial function.
    DOI:  https://doi.org/10.1167/iovs.65.13.12
  31. Exp Neurol. 2024 Oct 25. pii: S0014-4886(24)00359-5. [Epub ahead of print] 115033
    GSK-3β inhibitor amplifies autophagy-lysosomal pathways by regulating TFEB in Parkinson's disease models.
    Jiahong Zhong, Xihui Yu, Yunming Zhong, Liya Tan, Fayou Yang, Jialan Xu, Jianlin Wu, Zhuomiao Lin.
      Parkinson's disease (PD), a common neurodegenerative disorder characterized by degeneration of the substantia nigra and a marked increase in Lewy bodies in the brain, primarily manifests as motor dysfunction. Glycogen synthase kinase-3 beta (GSK-3β) is known to play a critical role in various pathological processes of neurodegenerative diseases. However, the impact of GSK-3β inhibitors on PD progression and the underlying molecular mechanisms responsible for the effects have not been fully elucidated. Using in vitro and mouse models of 1-methyl-4-phenylpyridine (MPP+)-or methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-induced PD, we found that inhibition of GSK-3β activity alleviated mitochondrial damage, cell apoptosis, and neuronal cell loss by promoting the nuclear translocation of transcription factor EB (TFEB), thereby amplifying the autophagy-lysosomal pathway (ALP). Importantly, siRNA silencing of the TFEB gene impaired the GSK-3β inhibitor-mediated activation of the ALP pathway, thus negating the metabolic support required for neuronal functional improvement. Short-term treatment with the GSK-3β inhibitor significantly ameliorated motor dysfunction and improved motor coordination in model mice with MPTP-induced PD. GSK-3β inhibition increased the ALP and TFEB activities in the mice, thereby reducing α-synuclein aggregation and neuronal damage. In conclusion, our study demonstrates that inhibition of GSK-3β activity can delay the pathological processes of PD via promotion of the TFEB-ALP pathway, potentially providing a novel therapeutic target for this neurodegenerative disorder.
    Keywords:  Autophagy-lysosomal pathway; GSK-3β inhibitor; MPP(+); Parkinson's disease; TFEB
    DOI:  https://doi.org/10.1016/j.expneurol.2024.115033
  32. Transl Neurodegener. 2024 Nov 04. 13(1): 54
    Critical role of ROCK1 in AD pathogenesis via controlling lysosomal biogenesis and acidification.
    Chenghuan Song, Wanying Huang, Pingao Zhang, Jiyun Shi, Ting Yu, Jing Wang, Yongbo Hu, Lanxue Zhao, Rui Zhang, Gang Wang, Yongfang Zhang, Hongzhuan Chen, Hao Wang.
      BACKGROUND: Lysosomal homeostasis and functions are essential for the survival of neural cells. Impaired lysosomal biogenesis and acidification in Alzheimer's disease (AD) pathogenesis leads to proteolytic dysfunction and neurodegeneration. However, the key regulatory factors and mechanisms of lysosomal homeostasis in AD remain poorly understood.METHODS: ROCK1 expression and its co-localization with LAMP1 and SQSTM1/p62 were detected in post-mortem brains of healthy controls and AD patients. Lysosome-related fluorescence probe staining, transmission electron microscopy and immunoblotting were performed to evaluate the role of ROCK1 in lysosomal biogenesis and acidification in various neural cell types. The interaction between ROCK1 and TFEB was confirmed by surface plasmon resonance and in situ proximity ligation assay (PLA). Moreover, we performed AAV-mediated ROCK1 downregulation followed by immunofluorescence, enzyme-linked immunosorbent assay (ELISA) and behavioral tests to unveil the effects of the ROCK1-TFEB axis on lysosomes in APP/PS1 transgenic mice.
    RESULTS: ROCK1 level was significantly increased in the brains of AD individuals, and was positively correlated with lysosomal markers and Aβ. Lysosomal proteolysis was largely impaired by the high abundance of ROCK1, while ROCK1 knockdown mitigated the lysosomal dysfunction in neurons and microglia. Moreover, we verified ROCK1 as a previously unknown upstream kinase of TFEB independent of m-TOR or GSK-3β. ROCK1 elevation resulted in abundant extracellular Aβ deposition which in turn bound to Aβ receptors and activated RhoA/ROCK1, thus forming a vicious circle of AD pathogenesis. Genetically downregulating ROCK1 lowered its interference with TFEB, promoted TFEB nuclear distribution, lysosomal biogenesis and lysosome-mediated Aβ clearance, and eventually prevented pathological traits and cognitive deficits in APP/PS1 mice.
    CONCLUSION: In summary, our results provide a mechanistic insight into the critical role of ROCK1 in lysosomal regulation and Aβ clearance in AD by acting as a novel upstream serine kinase of TFEB.
    Keywords:  Alzheimer’s disease; Lysosomal acidification; Lysosomal biogenesis; ROCK1; TFEB
    DOI:  https://doi.org/10.1186/s40035-024-00442-9
  33. Biochem Pharmacol. 2024 Nov 03. pii: S0006-2952(24)00600-2. [Epub ahead of print] 116600
    Therapeutic potential of Parkin and its regulation in Parkinson's disease.
    N Safreena, Indu C Nair, Goutam Chandra.
      Parkinson's disease (PD) is a debilitating neurodegenerative disorder characterized by the progressive loss of dopaminergic neurons in the midbrain substantia nigra, resulting in motor and non-motor symptoms. While the exact etiology of PD remains elusive, a growing body of evidence suggests that dysfunction in the parkin protein plays a pivotal role in the pathogenesis of the disease. Parkin is an E3 ubiquitin ligase that ubiquitinates substrate proteins to control a number of crucial cellular processes including protein catabolism, immune response, and cellular apoptosis.While autosomal recessive mutations in the PARK2 gene, which codes for parkin, are linked to an inherited form of early-onset PD, heterozygous mutations in PARK2 have also been reported in the more commonly occurring sporadic PD cases. Impairment of parkin's E3 ligase activity is believed to play a pathogenic role in both familial and sporadic forms of PD.This article provides an overview of the current understanding of the mechanistic basis of parkin's E3 ligase activity, its major physiological role in controlling cellular functions, and how these are disrupted in familial and sporadic PD. The second half of the manuscript explores the currently available and potential therapeutic strategies targeting parkin structure and/or function in order to slow down or mitigate the progressive neurodegeneration in PD.
    Keywords:  Dopaminergic neurodegeneration; E3 ubiquitin ligase; Mitochondria; PARK2; Proteostasis
    DOI:  https://doi.org/10.1016/j.bcp.2024.116600
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