bims-lysosi 2021-07-11 papers

bims-lysosi

Biomed News

on Lysosomes and signaling

Issue of 2021–07–11
29 papers selected by
Stephanie Fernandes, Max Planck Institute for Biology of Ageing

An mTORC1-Plin3 pathway is essential to activate lipophagy and protects against hepatosteatosis.
Monitoring TFEB translocation.
Measuring lysosomal size and frequency by electron microscopy.
Inborn errors of metabolism: Lessons from iPSC models.
High glucose-stimulated enhancer of zeste homolog-2 (EZH2) forces suppression of deptor to cause glomerular mesangial cell pathology.
MTORC1 inhibition drives crinophagic degradation of glucagon.
The role of endolysosomal trafficking in anticancer drug resistance.
The Akt-mTORC1 pathway mediates Axl receptor tyrosine kinase-induced mesangial cell proliferation.
The (pro)renin receptor (ATP6ap2) facilitates receptor-mediated endocytosis and lysosomal function in the renal proximal tubule.
Synthesis and Evaluation of Diphyllin β-Hydroxyl Amino Derivatives as Novel V-ATPase Inhibitors.
Autophagic flux assessment by immunoblot.
TFEB Links MYC Signaling to Epigenetic Control of Myeloid Differentiation and Acute Myeloid Leukemia.
Crosstalk between beta-adrenergic and insulin signaling mediates mechanistic target of rapamycin hyperactivation in liver of high-fat diet-fed male mice.
The Nutrient-Responsive Molecular Chaperone Hsp90 Supports Growth and Development in Drosophila.
Insights into VPS13 properties and function reveal a new mechanism of eukaryotic lipid transport.
SREBP-1c impairs ULK1 sulfhydration-mediated autophagic flux to promote hepatic steatosis in high-fat-diet-fed mice.
A new insight into the lens: cytosolic PLAAT phospholipases degrade organelles to make the lens transparent.
Structure of the TELO2-TTI1-TTI2 complex and its function in TOR recruitment to the R2TP chaperone.
Combined Inhibition of Akt and mTOR Is Effective Against Non-Hodgkin Lymphomas.
Visual system pathology in a canine model of CLN5 neuronal ceroid lipofuscinosis.
Splicing factor SRSF1 is indispensable for regulatory T cell homeostasis and function.
CYB561A3 is the Key Lysosomal Iron Reductase Required for Burkitt B-cell Growth and Survival.
Ferroptosis Regulation by Nutrient Signaling.
Combinatorial analysis of translation dynamics reveals eIF2 dependence of translation initiation at near-cognate codons.
Membrane perturbation by lipidated Atg8 underlies autophagosome biogenesis.
Rare manifestations and malignancies in tuberous sclerosis complex: findings from the TuberOus SClerosis registry to increAse disease awareness (TOSCA).
Recombinant SARS-CoV-2 envelope protein traffics to the trans-Golgi network following amphipol-mediated delivery into human cells.
Axon terminals control endolysosome diffusion to support synaptic remodelling.
RAB7 activity is required for the regulation of mitophagy in oocyte meiosis and oocyte quality control during ovarian aging.

Hepatology. 2021 Jul 07.

An mTORC1-Plin3 pathway is essential to activate lipophagy and protects against hepatosteatosis.

Marina Garcia-Macia, Adrián Santos-Ledo, Jack Leslie, Hannah L Paish, Amy L Collins, Rebecca S Scott, Abigail Watson, Rachel A Burgoyne, Steve White, Jeremy French, John Hammond, Lee A Borthwick, Jelena Mann, Juan P Bolaños, Viktor I Korolchuk, Fiona Oakley, Derek A Mann.

   BACKGROUND & AIMS: Non-alcoholic fatty liver disease (NAFLD) is the most common hepatic pathology in western countries and no treatment is currently available. NAFLD is characterized by the aberrant hepatocellular accumulation of fatty acids in the form of lipid droplets (LD). Recently, it was shown that liver LD degradation occurs via a process termed lipophagy; a novel form of autophagy. However, the molecular mechanisms governing liver lipophagy are elusive. Here, we aimed to ascertain the key molecular players that regulate hepatic lipophagy and their importance in NAFLD.
APPROACH & RESULTS: We analyzed the formation and degradation of LD in vitro (fibroblasts and primary mouse hepatocytes), in vivo and ex vivo (mouse and human liver slices) and focused on the role of the autophagy master regulator mammalian Target Of Rapamycin Complex 1 (mTORC1) and the LD coating protein Plin3 in these processes. We show that the autophagy machinery is recruited to the LD upon hepatic overload of oleic acid in all experimental settings. This led to activation of lipophagy, a process that was abolished by Plin3 knockdown using RNA interference. Furthermore, Plin3 directly interacted with the autophagy proteins Fip200 and Atg16L, suggesting that Plin3 functions as a docking protein or is involved in autophagosome formation to activate lipophagy. Finally, we show that mTORC1 phosphorylated Plin3 to promote LD degradation.
CONCLUSIONS: These results reveal that mTORC1 regulates liver lipophagy through a mechanism dependent on Plin3 phosphorylation. We propose that stimulating this pathway can enhance lipophagy in hepatocytes to help protect the liver from lipid-mediated toxicity, thus offering a new therapeutic strategy in NAFLD.

Keywords:  autophagy; fatty liver disease; hepatocytes; lipid droplets; perilipin

DOI:  https://doi.org/10.1002/hep.32048
Methods Cell Biol. 2021 ;pii: S0091-679X(20)30200-4. [Epub ahead of print]164 1-9

Monitoring TFEB translocation.

Guo Chen, Chenglong Mu, Yanfang Chen, Na An, Yushan Zhu, Oliver Kepp, Guido Kroemer.

  The transcription factor EB (TFEB) plays a critical role in autophagy induction and lysosomal biogenesis by orchestrating the expression of autophagy- and lysosome-related genes. In response to a series of stresses such as nutrient starvation, TFEB translocates from the cytoplasm to the nucleus, where it exerts its regulatory function. The activity of TFEB is tightly regulated by multiple phosphorylation and acetylation sites. Methods that rely on the analysis of posttranslational modification as a proxy for TFEB activation are often misleading. Here, we elaborate on protocols for monitoring nuclear translocation of TFEB by fluorescence microscopy.

Keywords:  Biosensor; High content; Lysosomal biogenesis; Macroautophagy; TFEB

DOI:  https://doi.org/10.1016/bs.mcb.2020.10.017
Methods Cell Biol. 2021 ;pii: S0091-679X(20)30202-8. [Epub ahead of print]164 47-61

Measuring lysosomal size and frequency by electron microscopy.

Michael W Hess, Lukas A Huber.

  Changes in size and abundance of late endocytic and autophagic organelles are increasingly appreciated as highly indicative of the physiological or pathological conditions of cells. Electron microscopy (EM) is unsurpassed in high-resolution imaging of both ultrastructural and immunocytochemical features of subcellular compartments. EM-based morphometry permits precise quantitative analyses of organelles, especially after state-of-the-art cryopreparation. Here described step-by-step protocols cover (i) different approaches for sample preparation of almost any specimen, (ii) tools to identify and characterize classes or subpopulations of lysosomes and related organelles, and (iii) convenient, straightforward ways for manual, thus, non-automated measurements of globular or spheroid-shaped organelles.

Keywords:  Autophagolysosome; Cryofixation; Endolysosome; High-pressure freezing; Lysosome; Morphometry; Ultrastructure

DOI:  https://doi.org/10.1016/bs.mcb.2020.10.019
Rev Endocr Metab Disord. 2021 Jul 09.

Inborn errors of metabolism: Lessons from iPSC models.

Rubén Escribá, Raquel Ferrer-Lorente, Ángel Raya.

  The possibility of reprogramming human somatic cells to pluripotency has opened unprecedented opportunities for creating genuinely human experimental models of disease. Inborn errors of metabolism (IEMs) constitute a greatly heterogeneous class of diseases that appear, in principle, especially suited to be modeled by iPSC-based technology. Indeed, dozens of IEMs have already been modeled to some extent using patient-specific iPSCs. Here, we review the advantages and disadvantages of iPSC-based disease modeling in the context of IEMs, as well as particular challenges associated to this approach, together with solutions researchers have proposed to tackle them. We have structured this review around six lessons that we have learnt from those previous modeling efforts, and that we believe should be carefully considered by researchers wishing to embark in future iPSC-based models of IEMs.

Keywords:  CRISPR/Cas9; Disease modeling; Human induced pluripotent stem cells; Lysosomal storage disorders; Reprogramming; Targeted genome edition

DOI:  https://doi.org/10.1007/s11154-021-09671-z
Cell Signal. 2021 Jul 02. pii: S0898-6568(21)00161-3. [Epub ahead of print] 110072

High glucose-stimulated enhancer of zeste homolog-2 (EZH2) forces suppression of deptor to cause glomerular mesangial cell pathology.

Falguni Das, Amit Bera, Nandini Ghosh-Choudhury, Kavitha Sataranatarajan, Amrita Kamat, Balakuntalam S Kasinath, Goutam Ghosh Choudhury.

  Function of mTORC1 and mTORC2 has emerged as a driver of mesangial cell pathologies in diabetic nephropathy. The mechanism of mTOR activation is poorly understood in this disease. Deptor is a constitutive subunit and a negative regulator of both mTOR complexes. Mechanistic investigation in mesangial cells revealed that high glucose decreased the expression of deptor concomitant with increased mTORC1 and mTORC2 activities, induction of hypertrophy and, expression of fibronectin and PAI-1. shRNAs against deptor mimicked these pathologic outcomes of high glucose. Conversely, overexpression of deptor significantly inhibited all effects of high glucose. To determine the mechanism of deptor suppression, we found that high glucose significantly increased the expression of EZH2, resulting in lysine-27 tri-methylation of histone H3 (H3K27Me3). Employing approaches including pharmacological inhibition, shRNA-mediated downregulation and overexpression of EZH2, we found that EZH2 regulates high glucose-induced deptor suppression along with activation of mTOR, mesangial cell hypertrophy and fibronectin/PAI-1 expression. Moreover, expression of hyperactive mTORC1 reversed shEZH2-mediated inhibition of hypertrophy and expression of fibronectin and PAI-1 by high glucose. Finally, in renal cortex of diabetic mice, we found that enhanced expression of EZH2 is associated with decreased deptor levels and increased mTOR activity and, expression of fibronectin and PAI-1. Together, our findings provide a novel mechanism for mTOR activation via EZH2 to induce mesangial cell hypertrophy and matrix expansion during early progression of diabetic nephropathy. These results suggest a strategy for leveraging the intrinsic effect of deptor to suppress mTOR activity via reducing EZH2 as a novel therapy for diabetic nephropathy.

Keywords:  Diabetic renal hypertrophy; Kidney; Matrix expansion, EZH2, deptor; mTORC1

DOI:  https://doi.org/10.1016/j.cellsig.2021.110072
Mol Metab. 2021 Jul 02. pii: S2212-8778(21)00131-9. [Epub ahead of print] 101286

MTORC1 inhibition drives crinophagic degradation of glucagon.

Sangam Rajak, Sherwin Xie, Archana Tewari, Sana Raza, Wu Yajun, Boon-Huat Bay, Paul M Yen, Rohit A Sinha.

   OBJECTIVE: Crinophagy is a secretory granule-specific autophagic process that regulates hormone content and secretion in endocrine cells. However, despite being one of the earliest described autophagic processes, its mechanism of action and regulation in mammalian cells remains unclear.
METHODS AND RESULTS: Here, we examined mammalian crinophagy and its modulation regulate hormone secretion in a glucagon-producing mouse pancreatic α-cell line, alpha TC1 clone 9 (αTC9) and in vivo. Western blot, electron microscopy and immunofluorescence analyses were performed to study crinophagy and glucagon secretion in αTC9 cells and C57BL/6 mice, in response to the mammalian target of rapamycin complex 1 (MTORC1) inhibitor rapamycin. Amino acid depletion and pharmacological inhibition of MTORC1 increased the shuttling of glucagon-containing secretory granules into lysosomes for crinophagic degradation to reduce glucagon secretion via a macroautophagy-independent mechanism. Furthermore, MTORC1 inhibition reduced both intracellular and secreted glucagon in rapamycin-treated mice, in response to hypoglycaemia.
CONCLUSION: In summary, we have identified a novel crinophagic mechanism of intracellular glucagon turnover in pancreatic α-cells regulated by MTORC1 signaling.

Keywords:  Autophagy; Crinophagy; Diabetes; Glucagon; Lysosomes; MTORC1; Rapamycin

DOI:  https://doi.org/10.1016/j.molmet.2021.101286
Drug Resist Updat. 2021 Jul;pii: S1368-7646(21)00027-3. [Epub ahead of print]57 100769

The role of endolysosomal trafficking in anticancer drug resistance.

Noor A Hussein, Saloni Malla, Mariah A Pasternak, David Terrero, Noah G Brown, Charles R Ashby, Yehuda G Assaraf, Zhe-Sheng Chen, Amit K Tiwari.

  Multidrug resistance (MDR) remains a major obstacle towards curative treatment of cancer. Despite considerable progress in delineating the basis of intrinsic and acquired MDR, the underlying molecular mechanisms remain to be elucidated. Emerging evidences suggest that dysregulation in endolysosomal compartments is involved in mediating MDR through multiple mechanisms, such as alterations in endosomes, lysosomes and autophagosomes, that traffic and biodegrade the molecular cargo through macropinocytosis, autophagy and endocytosis. For example, altered lysosomal pH, in combination with transcription factor EB (TFEB)-mediated lysosomal biogenesis, increases the sequestration of hydrophobic anti-cancer drugs that are weak bases, thereby producing an insufficient and off-target accumulation of anti-cancer drugs in MDR cancer cells. Thus, the use of well-tolerated, alkalinizing compounds that selectively block Vacuolar H⁺-ATPase (V-ATPase) may be an important strategy to overcome MDR in cancer cells and increase chemotherapeutic efficacy. Other mechanisms of endolysosomal-mediated drug resistance include increases in the expression of lysosomal proteases and cathepsins that are involved in mediating carcinogenesis and chemoresistance. Therefore, blocking the trafficking and maturation of lysosomal proteases or direct inhibition of cathepsin activity in the cytosol may represent novel therapeutic modalities to overcome MDR. Furthermore, endolysosomal compartments involved in catabolic pathways, such as macropinocytosis and autophagy, are also shown to be involved in the development of MDR. Here, we review the role of endolysosomal trafficking in MDR development and discuss how targeting endolysosomal pathways could emerge as a new therapeutic strategy to overcome chemoresistance in cancer.

Keywords:  Autophagy; Cancer; Cathepsins; Endolysosomal trafficking; Lysosomes; Macropinocytosis; Multidrug resistance

DOI:  https://doi.org/10.1016/j.drup.2021.100769
J Leukoc Biol. 2021 Jul 04.

The Akt-mTORC1 pathway mediates Axl receptor tyrosine kinase-induced mesangial cell proliferation.

Yuxuan Zhen, Tracy L McGaha, Fred D Finkelman, Wen-Hai Shao.

  Glomerulonephritis (GN), an important pathologic feature of many renal diseases, is frequently characterized by mesangial cell proliferation. We and others have previously shown that the TAM family receptor tyrosine kinases Axl, Mer, and Tyro-3 contribute to cell survival, proliferation, migration, and clearance of apoptotic cells (ACs); that Axl contributes to GN by promoting mesangial cell proliferation; and that small molecule inhibition of Axl ameliorates nephrotoxic serum-induced GN in mice. We now show that stimulation of renal mesangial cell Axl causes a modest increase in intracellular Ca2+ and activates NF-κB, mTOR, and the mTOR-containing mTORC1 complex, which phosphorylates the ribosomal protein S6. Axl-induction of Akt activation is upstream of NF-κB and mTOR activation, which are mutually codependent. Axl-induced NF-κB activation leads to Bcl-xl up-regulation. Axl is more important than Mer at mediating AC phagocytosis by mesangial cells, but less important than Mer at mediating phagocytosis of ACs by peritoneal macrophages. Taken together, our data suggest the possibility that Axl mediates mesangial cell phagocytosis of ACs and promotes mesangial cell proliferation by activating NF-κB and mTORC1.

Keywords:  Axl; NF-κB; TAM receptors; apoptotic cell clearance; mTORC1; mesangial cell proliferation

DOI:  https://doi.org/10.1002/JLB.2A1220-850RRR
Pflugers Arch. 2021 Jul 06.

The (pro)renin receptor (ATP6ap2) facilitates receptor-mediated endocytosis and lysosomal function in the renal proximal tubule.

Marta Figueiredo, Arezoo Daryadel, Gabin Sihn, Dominik N Müller, Elena Popova, Anthony Rouselle, Genevieve Nguyen, Michael Bader, Carsten A Wagner.

  The ATP6ap2 (Pro)renin receptor protein associates with H+-ATPases which regulate organellar, cellular, and systemic acid-base homeostasis. In the kidney, ATP6ap2 colocalizes with H+-ATPases in various cell types including the cells of the proximal tubule. There, H+-ATPases are involved in receptor-mediated endocytosis of low molecular weight proteins via the megalin/cubilin receptors. To study ATP6ap2 function in the proximal tubule, we used an inducible shRNA Atp6ap2 knockdown rat model (Kd) and an inducible kidney-specific Atp6ap2 knockout mouse model. Both animal lines showed higher proteinuria with elevated albumin, vitamin D binding protein, and procathepsin B in urine. Endocytosis of an injected fluid-phase marker (FITC- dextran, 10 kDa) was normal whereas processing of recombinant transferrin, a marker for receptor-mediated endocytosis, to lysosomes was delayed. While megalin and cubilin expression was unchanged, abundance of several subunits of the H+-ATPase involved in receptor-mediated endocytosis was reduced. Lysosomal integrity and H+-ATPase function are associated with mTOR signaling. In ATP6ap2, KO mice mTOR and phospho-mTOR appeared normal but increased abundance of the LC3-B subunit of the autophagosome was observed suggesting a more generalized impairment of lysosomal function in the absence of ATP6ap2. Hence, our data suggests a role for ATP6ap2 for proximal tubule function in the kidney with a defect in receptor-mediated endocytosis in mice and rats.

Keywords:  Endocytosis; H+-ATPase; Low molecular weight proteins; Lysosome; Proximal tubule

DOI:  https://doi.org/10.1007/s00424-021-02598-z
Chem Biol Drug Des. 2021 Jul 07.

Synthesis and Evaluation of Diphyllin β-Hydroxyl Amino Derivatives as Novel V-ATPase Inhibitors.

Li Zhu, Yapeng Lu, Yu Li, Yong Ling, Yu Zhao.

  Natural diphyllin glycosides were identified as potent vacuolar H+ -ATPase (V-ATPase) inhibitors. A series of diphyllin β-hydroxyl amino derivatives were designed and synthesized as novel diphyllin derivatives. Most of these derivatives displayed potent cytotoxicity against six cancer cell lines with IC50 values in the submicromolar to nanomolar concentration range. Compounds 2b, 2c, 2l, 2m and 2n showed similar V-ATPase inhibitory potency to Bafilomycin A1. Compound 2l exhibited potent activity of modulation of lysosomal pH and cytoplasmic pH.

Keywords:  V-ATPase inhibitor; cytotoxicity; diphyllin; synthesis; β-hydroxyl amino derivatives

DOI:  https://doi.org/10.1111/cbdd.13920
Methods Cell Biol. 2021 ;pii: S0091-679X(20)30188-6. [Epub ahead of print]164 63-72

Autophagic flux assessment by immunoblot.

Qi Wu, Ai-Ling Tian, Hui Pan, Oliver Kepp, Guido Kroemer.

  Autophagy is one of the main adaptive mechanisms to maintain cellular homeostasis in response to multiple stresses. During autophagy diverse cellular components such as damaged organelles or superfluous proteins are targeted for lysosomal degradation. Importantly, during the initiation of autophagy MAP1LC3B (better known as LC3) lipidates into the membrane of the forming phagophore, which facilitates the formation and lengthening of autophagosomes. In addition, the autophagy receptor SQSTM1 (better known as p62) selectively recruits various cargos to autophagosomes for lysosomal degradation. Both, the conversion of LC3 as well as the degradation of p62 can be assessed as means of monitoring autophagy. Here we detail a protocol for assessing these key events of the autophagic flux via immunoblot.

Keywords:  Autophagic cargo; Drug discovery; LC3; Lipidation; Lysosomal degradation

DOI:  https://doi.org/10.1016/bs.mcb.2020.10.005
Cancer Discov. 2021 Mar;2(2): 162-185

TFEB Links MYC Signaling to Epigenetic Control of Myeloid Differentiation and Acute Myeloid Leukemia.

Seongseok Yun, Nicole D Vincelette, Xiaoqing Yu, Gregory W Watson, Mario R Fernandez, Chunying Yang, Taro Hitosugi, Chia-Ho Cheng, Audrey R Freischel, Ling Zhang, Weimin Li, Hsinan Hou, Franz X Schaub, Alexis R Vedder, Ling Cen, Kathy L McGraw, Jungwon Moon, Daniel J Murphy, Andrea Ballabio, Scott H Kaufmann, Anders E Berglund, John L Cleveland.

MYC oncoproteins regulate transcription of genes directing cell proliferation, metabolism, and tumorigenesis. A variety of alterations drive MYC expression in acute myeloid leukemia (AML), and enforced MYC expression in hematopoietic progenitors is sufficient to induce AML. Here we report that AML and myeloid progenitor cell growth and survival rely on MYC-directed suppression of Transcription Factor EB (TFEB), a master regulator of the autophagy-lysosome pathway. Notably, although originally identified as an oncogene, TFEB functions as a tumor suppressor in AML, where it provokes AML cell differentiation and death. These responses reflect TFEB control of myeloid epigenetic programs by inducing expression of isocitrate dehydrogenase-1 (IDH1) and IDH2, resulting in global hydroxylation of 5-methycytosine. Finally, activating the TFEB-IDH1/IDH2-TET2 axis is revealed as a targetable vulnerability in AML. Thus, epigenetic control by an MYC-TFEB circuit dictates myeloid cell fate and is essential for maintenance of AML. SIGNIFICANCE: Alterations in epigenetic control are a hallmark of AML. This study establishes that a MYC-TFEB circuit controls AML differentiation and epigenetic programs by inducing IDH1/IDH2 and hydroxylation of 5-methylcytosine, that TFEB functions as a tumor suppressor in AML, and that this circuit is a targetable vulnerability in AML.See related commentary by Wu and Eisenman, p. 116.

DOI: https://doi.org/10.1158/2643-3230.BCD-20-0029
Physiol Rep. 2021 Jul;9(13): e14958

Crosstalk between beta-adrenergic and insulin signaling mediates mechanistic target of rapamycin hyperactivation in liver of high-fat diet-fed male mice.

Sadia Ashraf, Nadia Ashraf, Gizem Yilmaz, Romain Harmancey.

  Nonalcoholic fatty liver disease (NAFLD) is the most common cause of chronic liver disease. While increased nutrient intake and sympathetic activity have been associated with the disease, the pathogenesis of NAFLD remains incompletely understood. We investigated the impact of the interaction of high dietary fat and sugar intake with increased beta-adrenergic receptor (β-AR) signaling on the activity of nutrient-sensing pathways and fuel storage in the liver. C57BL/6J mice were fed a standard rodent diet (STD), a high-fat diet (HFD), a high-fat/high-sugar Western diet (WD), a high-sugar diet with mixed carbohydrates (HCD), or a high-sucrose diet (HSD). After 6 week on diets, mice were treated with isoproterenol (ISO) and the activity of liver mTOR complex 1 (mTORC1)-related signaling analyzed by immunoblotting and correlated with tissue triglyceride and glycogen contents. ISO-stimulated AKT- and ERK-mediated activation of mTORC1 in STD-fed mice. Consumption of all four high-calorie diets exacerbated downstream activation of ribosomal protein S6 kinase beta-1 (S6K1) in response to ISO. S6K1 activity was greater with the fat-enriched HFD and WD and correlated with the presence of metabolic syndrome and a stronger activation of AKT and ERK1/2 pathways. Fat-enriched diets also increased triglyceride accumulation and inhibited glycogen mobilization under β-AR stimulation. In conclusion, crosstalk between β-AR and insulin signaling may contribute to HFD-induced hepatic steatosis through ERK1/2- and AKT-mediated hyperactivation of the mTORC1/S6K1 axis. The findings provide further rationale for the development of therapies aimed at targeting augmented β-AR signaling in the pathogenesis of NAFLD.

Keywords:  fatty liver; glycogen; high-fat diet; insulin; sympathetic nervous system

DOI:  https://doi.org/10.14814/phy2.14958
Front Physiol. 2021 ;12 690564

The Nutrient-Responsive Molecular Chaperone Hsp90 Supports Growth and Development in Drosophila.

Yuya Ohhara, Genki Hoshino, Kyosuke Imahori, Tomoya Matsuyuki, Kimiko Yamakawa-Kobayashi.

  Animals can sense internal nutrients, such as amino acids/proteins, and are able to modify their developmental programs in accordance with their nutrient status. In the fruit fly, Drosophila melanogaster, amino acid/protein is sensed by the fat body, an insect adipose tissue, through a nutrient sensor, target of rapamycin (TOR) complex 1 (TORC1). TORC1 promotes the secretion of various peptide hormones from the fat body in an amino acid/protein-dependent manner. Fat-body-derived peptide hormones stimulate the release of insulin-like peptides, which are essential growth-promoting anabolic hormones, from neuroendocrine cells called insulin-producing cells (IPCs). Although the importance of TORC1 and the fat body-IPC axis has been elucidated, the mechanism by which TORC1 regulates the expression of insulinotropic signal peptides remains unclear. Here, we show that an evolutionarily conserved molecular chaperone, heat shock protein 90 (Hsp90), promotes the expression of insulinotropic signal peptides. Fat-body-selective Hsp90 knockdown caused the transcriptional downregulation of insulinotropic signal peptides. IPC activity and systemic growth were also impaired in fat-body-selective Hsp90 knockdown animals. Furthermore, Hsp90 expression depended on protein/amino acid availability and TORC1 signaling. These results strongly suggest that Hsp90 serves as a nutrient-responsive gene that upregulates the fat body-IPC axis and systemic growth. We propose that Hsp90 is induced in a nutrient-dependent manner to support anabolic metabolism during the juvenile growth period.

Keywords:  Drosophila; fat body; heat shock protein 90; insulin-like peptide; target of rapamycin

DOI:  https://doi.org/10.3389/fphys.2021.690564
Biochim Biophys Acta Mol Cell Biol Lipids. 2021 Jun 30. pii: S1388-1981(21)00131-1. [Epub ahead of print] 159003

Insights into VPS13 properties and function reveal a new mechanism of eukaryotic lipid transport.

Marianna Leonzino, Karin M Reinisch, Pietro De Camilli.

  The occurrence of protein mediated lipid transfer between intracellular membranes has been known since the late 1960's. Since these early discoveries, numerous proteins responsible for such transport, which often act at membrane contact sites, have been identified. Typically, they comprise a lipid harboring module thought to shuttle back and forth between the two adjacent bilayers. Recently, however, studies of the chorein domain protein family, which includes VPS13 and ATG2, has led to the identification of a novel mechanism of lipid transport between organelles in eukaryotic cells mediated by a rod-like protein bridge with a hydrophobic groove through which lipids can slide. This mechanism is ideally suited for bulk transport of bilayer lipids to promote membrane growth. Here we describe how studies of VPS13 led to the discovery of this new mechanism, summarize properties and known roles of VPS13 proteins, and discuss how their dysfunction may lead to disease.

Keywords:  Chorein domain; Lipid channels; Membrane expansion

DOI:  https://doi.org/10.1016/j.bbalip.2021.159003
Mol Cell. 2021 Jun 30. pii: S1097-2765(21)00450-0. [Epub ahead of print]

SREBP-1c impairs ULK1 sulfhydration-mediated autophagic flux to promote hepatic steatosis in high-fat-diet-fed mice.

Thuy T P Nguyen, Do-Young Kim, Yu-Geon Lee, Young-Seung Lee, Xuan T Truong, Jae-Ho Lee, Dae-Kyu Song, Taeg Kyu Kwon, So-Hyun Park, Chang Hwa Jung, Changjong Moon, Timothy F Osborne, Seung-Soon Im, Tae-Il Jeon.

  A metabolic imbalance between lipid synthesis and degradation can lead to hepatic lipid accumulation, a characteristic of patients with non-alcoholic fatty liver disease (NAFLD). Here, we report that high-fat-diet-induced sterol regulatory element-binding protein (SREBP)-1c, a key transcription factor that regulates lipid biosynthesis, impairs autophagic lipid catabolism via altered H2S signaling. SREBP-1c reduced cystathionine gamma-lyase (CSE) via miR-216a, which in turn decreased hepatic H2S levels and sulfhydration-dependent activation of Unc-51-like autophagy-activating kinase 1 (ULK1). Furthermore, Cys951Ser mutation of ULK1 decreased autolysosome formation and promoted hepatic lipid accumulation in mice, suggesting that the loss of ULK1 sulfhydration was directly associated with the pathogenesis of NAFLD. Moreover, silencing of CSE in SREBP-1c knockout mice increased liver triglycerides, confirming the connection between CSE, autophagy, and SREBP-1c. Overall, our results uncover a 2-fold mechanism for SREBP-1c-driven hepatic lipid accumulation through reciprocal activation and inhibition of hepatic lipid biosynthesis and degradation, respectively.

Keywords:  SREBP-1c; ULK1; autophagy; hydrogen sulfide; steatosis; sulfhydration

DOI:  https://doi.org/10.1016/j.molcel.2021.06.003
Autophagy. 2021 Jul 07. 1-3

A new insight into the lens: cytosolic PLAAT phospholipases degrade organelles to make the lens transparent.

Hideaki Morishita, Tomoya Eguchi, Noboru Mizushima.

  All membrane-bound organelles are degraded during the terminal differentiation of lens fiber cells. How these organelles are degraded has been a long-standing question in biology. We recently revealed that PLAAT (phospholipase A and acyltransferase)-family phospholipases degrade organelles in the lens independently of macroautophagy. Here, we discuss the mechanism and physiological relevance of this new mode of intracellular degradation.

Keywords:  Autophagy; HRASLS; PLA2G16; PLAAT; lens; mice; organelle degradation; phospholipase; zebrafish

DOI:  https://doi.org/10.1080/15548627.2021.1950372
Cell Rep. 2021 Jul 06. pii: S2211-1247(21)00693-8. [Epub ahead of print]36(1): 109317

Structure of the TELO2-TTI1-TTI2 complex and its function in TOR recruitment to the R2TP chaperone.

Mohinder Pal, Hugo Muñoz-Hernandez, Dennis Bjorklund, Lihong Zhou, Gianluca Degliesposti, J Mark Skehel, Emma L Hesketh, Rebecca F Thompson, Laurence H Pearl, Oscar Llorca, Chrisostomos Prodromou.

  The R2TP (RUVBL1-RUVBL2-RPAP3-PIH1D1) complex, in collaboration with heat shock protein 90 (HSP90), functions as a chaperone for the assembly and stability of protein complexes, including RNA polymerases, small nuclear ribonucleoprotein particles (snRNPs), and phosphatidylinositol 3-kinase (PI3K)-like kinases (PIKKs) such as TOR and SMG1. PIKK stabilization depends on an additional complex of TELO2, TTI1, and TTI2 (TTT), whose structure and function are poorly understood. The cryoelectron microscopy (cryo-EM) structure of the human R2TP-TTT complex, together with biochemical experiments, reveals the mechanism of TOR recruitment to the R2TP-TTT chaperone. The HEAT-repeat TTT complex binds the kinase domain of TOR, without blocking its activity, and delivers TOR to the R2TP chaperone. In addition, TTT regulates the R2TP chaperone by inhibiting RUVBL1-RUVBL2 ATPase activity and by modulating the conformation and interactions of the PIH1D1 and RPAP3 components of R2TP. Taken together, our results show how TTT couples the recruitment of TOR to R2TP with the regulation of this chaperone system.

Keywords:  HSP90 chaperone; PIKK; R2TP; RUVBL1; RUVBL2; TELO2; TTI1; TTI2; TTT; mTOR

DOI:  https://doi.org/10.1016/j.celrep.2021.109317
Front Oncol. 2021 ;11 670275

Combined Inhibition of Akt and mTOR Is Effective Against Non-Hodgkin Lymphomas.

Ricardo Rivera-Soto, Yi Yu, Dirk P Dittmer, Blossom Damania.

  Non-Hodgkin lymphoma (NHL) are a diverse group of hematological malignancies comprised of over 60 subtypes. These subtypes range from indolent to aggressive. The PI3K/Akt/mTOR pathway has been shown to contribute to cell survival and proliferation and is constitutively active in most NHL. MK-7075 (miransertib) and MK-4440 are small molecules that effectively inhibit Akt and have entered clinical development. Using in vitro and in vivo models of NHL, we explored targeting the kinase Akt with miransertib and MK-4440 alone or in combination with the mTORC1 inhibitor, rapamycin (sirolimus). Both Akt inhibitors inhibited the pathway and NHL proliferation in a subtype-dependent manner. However, these compounds had a minimal effect on the viability of primary B-cells. Importantly, the combination of miransertib and sirolimus synergistically reduced cell proliferation in NHL, including in one indolent subtype, e.g., follicular lymphoma (FL), and two aggressive subtypes, e.g., diffuse large B-cell lymphoma (DLBCL) and primary effusion lymphoma (PEL). To establish in vivo efficacy, we used several xenograft models of FL, DLBCL, and PEL. The results obtained in vivo were consistent with the in vitro studies. The FL xenograft was highly sensitive to the inhibition of Akt alone; however, the tumor burden of PEL xenografts was only significantly reduced when both Akt and mTORC1 were targeted. These data suggest that targeting the PI3K/Akt/mTOR pathway with Akt inhibitors such as miransertib in combination with mTOR inhibitors serves as a broadly applicable therapeutic in NHL.

Keywords:  MK-4440; miransertib; non-Hodgkin lymphomas; sirolimus; viral lymphomas

DOI:  https://doi.org/10.3389/fonc.2021.670275
Exp Eye Res. 2021 Jun 30. pii: S0014-4835(21)00252-9. [Epub ahead of print] 108686

Visual system pathology in a canine model of CLN5 neuronal ceroid lipofuscinosis.

Grace Robinson Kick, Elizabeth J Meiman, Julianna C Sabol, Rebecca E H Whiting, Juri Ota-Kuroki, Leilani J Castaner, Cheryl A Jensen, Martin L Katz.

  CLN5 neuronal ceroid lipofuscinosis is a hereditary neurodegenerative disease characterized by progressive neurological decline, vision loss and seizures. Visual impairment in children with CLN5 disease is attributed to a progressive decline in retinal function accompanied by retinal degeneration as well as impaired central nervous system function associated with global brain atrophy. We studied visual system pathology in five Golden Retriever littermates homozygous for the CLN5 disease allele previously identified in the breed. The dogs exhibited signs of pronounced visual impairment by 21-22 months of age. Electroretinogram recordings showed a progressive decline in retinal function primarily affecting cone neural pathways. Altered visual evoked potential recordings indicated that disease progression affected visual signal processing in the brain. Aside from several small retinal detachment lesions, no gross retinal abnormalities were observed with in vivo ocular imaging and histologically the retinas did not exhibit apparent abnormalities by 23 months of age. However, there was extensive accumulation of autofluorescent membrane-bound lysosomal storage bodies in almost all retinal layers, as well as in the occipital cortex, by 20 months of age. In the retina, storage was particularly pronounced in retinal ganglion cells, the retinal pigment epithelium and in photoreceptor cells just interior to the outer limiting membrane. The visual system pathology of CLN5-affected Golden Retrievers is similar to that seen early in the human disease. It was not possible to follow the dogs to an advanced stage of disease progression due to the severity of behavioral and motor disease signs by 23 months of age. The findings reported here indicate that canine CLN5 disease will be a useful model of visual system disease in CLN5 neuronal ceroid lipofuscinosis. The baseline data obtained in this investigation will be useful in future therapeutic intervention studies. The findings indicate that there is a fairly broad time frame after disease onset within which treatments could be effective in preserving vision.

Keywords:  Autophagy; Dog; Lysosomal storage; Phagocytosis; Photoreceptor cells; Retinal degeneration; Retinal pigment epithelium

DOI:  https://doi.org/10.1016/j.exer.2021.108686
Cell Rep. 2021 Jul 06. pii: S2211-1247(21)00715-4. [Epub ahead of print]36(1): 109339

Splicing factor SRSF1 is indispensable for regulatory T cell homeostasis and function.

Takayuki Katsuyama, Vaishali R Moulton.

  The ability of regulatory T (Treg) cells to control the immune response and limit the development of autoimmune diseases is determined by distinct molecular processes, which are not fully understood. We show here that serine/arginine-rich splicing factor 1 (SRSF1), which is decreased in T cells from patients with systemic lupus erythematosus, is necessary for the homeostasis and proper function of Treg cells, because its conditional absence in these cells leads to profound autoimmunity and organ inflammation by elevating the glycolytic metabolism and mTORC1 activity and the production of proinflammatory cytokines. Our data reveal a molecular mechanism that controls Treg cell plasticity and offer insights into the pathogenesis of autoimmune disease.

Keywords:  SRSF1; T cells; Treg; autoimmunity; cytokines; immune homeostasis; immune regulation; inflammation; mTOR pathway; splicing factor

DOI:  https://doi.org/10.1016/j.celrep.2021.109339
Blood. 2021 Jul 07. pii: blood.2021011079. [Epub ahead of print]

CYB561A3 is the Key Lysosomal Iron Reductase Required for Burkitt B-cell Growth and Survival.

Zhonghao Wang, Rui Guo, Stephen J Trudeau, Emma Wolinsky, Tsliil Ast, Jin-Hua Liang, Chang Jiang, Yijie Ma, Mingxiang Teng, Vamsi Mootha, Ben Gewurz.

Epstein-Barr virus (EBV) causes endemic Burkitt lymphoma, the leading childhood cancer in sub-Saharan Africa. Burkitt cells retain aspects of germinal center B-cell physiology with MYC-driven B-cell hyperproliferation, yet little is presently known about their iron metabolism. CRISPR/Cas9 analysis highlighted the little studied ferrireductase CYB561A3 as critical for Burkitt proliferation, but not for that of closely related EBV-transformed lymphoblastoid cells or nearly all other Cancer Dependency Map cell lines. Burkitt CYB561A3 knockout induced profound iron starvation, despite ferritinophagy and plasma membrane transferrin upregulation. Elevated concentrations of ascorbic acid, a key CYB561 family electron donor or the labile iron source ferrous citrate rescued Burkitt CYB561A3 deficiency. CYB561A3 knockout caused catastrophic lysosomal and mitochondrial damage and impaired mitochondrial respiration. By contrast, lymphoblastoid B-cells with the transforming EBV latency III program were instead dependent on the STEAP3 ferrireductase. These results highlight CYB561A3 it as an attractive therapeutic Burkitt lymphoma target.

DOI: https://doi.org/10.1182/blood.2021011079
Nutr Res Rev. 2021 Jul 08. 1-40

Ferroptosis Regulation by Nutrient Signaling.

Yingao Qi, Xiaoli Zhang, Zhihui Wu, Min Tian, Fang Chen, Wutai Guan, Shihai Zhang.

  Tremendous progress has been made in the field of ferroptosis since this regulated cell death process was first named in 2012. Ferroptosis is initiated upon redox imbalance and driven by excessive phospholipid peroxidation. Levels of multiple intracellular nutrients (iron, selenium, vitamin E, and coenzyme Q10) are intimately related to the cellular antioxidant system and participate in the regulation of ferroptosis. Dietary intake of monounsaturated fatty acids (MUFAs) and polyunsaturated fatty acids (PUFAs) regulates ferroptosis by directly modifying the fatty acid composition in cell membranes. In addition, amino acids and glucose (energy stress) manipulate the ferroptosis pathway through the nutrient-sensitive kinases mechanistic target of rapamycin complex 1 (mTORC1) and AMP-activated protein kinase (AMPK). Understanding the molecular interaction between nutrient signals and ferroptosis sensors might help in the identification of the roles of ferroptosis in normal physiology and in the development of novel pharmacological targets for the treatment of ferroptosis-related diseases.

Keywords:  AMPK; Fe; Ferroptosis; Nutrient sensing; Se; mTORC1

DOI:  https://doi.org/10.1017/S0954422421000226
Nucleic Acids Res. 2021 Jul 06. pii: gkab549. [Epub ahead of print]

Combinatorial analysis of translation dynamics reveals eIF2 dependence of translation initiation at near-cognate codons.

Kazuya Ichihara, Akinobu Matsumoto, Hiroshi Nishida, Yuki Kito, Hideyuki Shimizu, Yuichi Shichino, Shintaro Iwasaki, Koshi Imami, Yasushi Ishihama, Keiichi I Nakayama.

Although ribosome-profiling and translation initiation sequencing (TI-seq) analyses have identified many noncanonical initiation codons, the precise detection of translation initiation sites (TISs) remains a challenge, mainly because of experimental artifacts of such analyses. Here, we describe a new method, TISCA (TIS detection by translation Complex Analysis), for the accurate identification of TISs. TISCA proved to be more reliable for TIS detection compared with existing tools, and it identified a substantial number of near-cognate codons in Kozak-like sequence contexts. Analysis of proteomics data revealed the presence of methionine at the NH2-terminus of most proteins derived from near-cognate initiation codons. Although eukaryotic initiation factor 2 (eIF2), eIF2A and eIF2D have previously been shown to contribute to translation initiation at near-cognate codons, we found that most noncanonical initiation events are most probably dependent on eIF2, consistent with the initial amino acid being methionine. Comprehensive identification of TISs by TISCA should facilitate characterization of the mechanism of noncanonical initiation.

DOI: https://doi.org/10.1093/nar/gkab549
Nat Struct Mol Biol. 2021 Jul 08.

Membrane perturbation by lipidated Atg8 underlies autophagosome biogenesis.

Tatsuro Maruyama, Jahangir Md Alam, Tomoyuki Fukuda, Shun Kageyama, Hiromi Kirisako, Yuki Ishii, Ichio Shimada, Yoshinori Ohsumi, Masaaki Komatsu, Tomotake Kanki, Hitoshi Nakatogawa, Nobuo N Noda.

Autophagosome biogenesis is an essential feature of autophagy. Lipidation of Atg8 plays a critical role in this process. Previous in vitro studies identified membrane tethering and hemi-fusion/fusion activities of Atg8, yet definitive roles in autophagosome biogenesis remained controversial. Here, we studied the effect of Atg8 lipidation on membrane structure. Lipidation of Saccharomyces cerevisiae Atg8 on nonspherical giant vesicles induced dramatic vesicle deformation into a sphere with an out-bud. Solution NMR spectroscopy of Atg8 lipidated on nanodiscs identified two aromatic membrane-facing residues that mediate membrane-area expansion and fragmentation of giant vesicles in vitro. These residues also contribute to the in vivo maintenance of fragmented vacuolar morphology under stress in fission yeast, a moonlighting function of Atg8. Furthermore, these aromatic residues are crucial for the formation of a sufficient number of autophagosomes and regulate autophagosome size. Together, these data demonstrate that Atg8 can cause membrane perturbations that underlie efficient autophagosome biogenesis.

DOI: https://doi.org/10.1038/s41594-021-00614-5
Orphanet J Rare Dis. 2021 Jul 06. 16(1): 301

Rare manifestations and malignancies in tuberous sclerosis complex: findings from the TuberOus SClerosis registry to increAse disease awareness (TOSCA).

TOSCA investigators

   BACKGROUND: Tuberous sclerosis complex (TSC) is a rare multisystem autosomal dominant disorder caused by pathogenic variants in either the TSC1 or TSC2 gene. Common manifestations of TSC have been grouped into major and minor clinical diagnostic criteria and assessed in clinical routine workup. However, case studies point towards the existence of rare disease manifestations and to the potential association of TSC with malignant tumors. In this study we sought to characterize rare manifestations and malignancies using a large cohort of patients.
METHODS: TuberOus SClerosis registry to increAse disease awareness (TOSCA) is a multicenter, international disease registry collecting clinical manifestations and characteristics of patients with TSC, both retrospectively and prospectively. We report rates and characteristics of rare manifestations and malignancies in patients with TSC who had enrolled in the TOSCA registry. We also examined these manifestations by age, sex, and genotype (TSC1 or TSC2).
RESULTS: Overall, 2211 patients with TSC were enrolled in the study. Rare manifestations were reported in 382 (17.3%) study participants and malignancies in 65 (2.9%). Of these rare manifestations, the most frequent were bone sclerotic foci (39.5%), scoliosis (23%), thyroid adenoma (5.5%), adrenal angiomyolipoma (4.5%), hemihypertrophy and pancreatic neuroendocrine tumors (pNET; both 3.1%). These rare manifestations were more commonly observed in adults than children (66.2% vs. 22.7%), in females versus males (58.4% vs. 41.6%; except for scoliosis: 48.9% vs. 51.1%), and in those with TSC2 versus TSC1 (67.0% vs. 21.1%; except for thyroid adenoma: 42.9% vs. 57.1%). In the 65 individuals with reported malignancies, the most common were renal cell carcinoma (47.7%), followed by breast (10.8%) and thyroid cancer (9.2%). Although malignancies were more common in adult patients, 26.1% were reported in children and 63.1% in individuals < 40 years. TSC1 mutations were over-represented in individuals with malignancies compared to the overall TOSCA cohort (32.1% vs. 18.5%).
CONCLUSION: Rare manifestations were observed in a significant proportion of individuals with TSC. We recommend further examination of rare manifestations in TSC. Collectively, malignancies were infrequent findings in our cohort. However, compared to the general population, malignant tumors occurred earlier in age and some tumor types were more common.

Keywords:  Malignancy; Rare manifestation; TOSCA; TSC; Tuberous sclerosis complex

DOI:  https://doi.org/10.1186/s13023-021-01917-y
J Biol Chem. 2021 Jul 05. pii: S0021-9258(21)00740-7. [Epub ahead of print] 100940

Recombinant SARS-CoV-2 envelope protein traffics to the trans-Golgi network following amphipol-mediated delivery into human cells.

James M Hutchison, Ricardo Capone, Dustin D Luu, Karan H Shah, Arina Hadziselimovic, Wade D Van Horn, Charles R Sanders.

  The SARS-CoV-2 envelope protein (S2-E) is a conserved membrane protein that is important for coronavirus assembly and budding. Here, we describe the recombinant expression and purification of S2-E in amphipol-class amphipathic polymer solutions, which solubilize and stabilize membrane proteins, but do not disrupt membranes. We found that amphipol delivery of S2-E to pre-formed planar bilayers results in spontaneous membrane integration and formation of viroporin cation channels. Amphipol delivery of the S2-E protein to human cells results in plasma membrane integration, followed by retrograde trafficking to the trans-Golgi network (TGN) and accumulation in swollen perinuclear LAMP1-positive vesicles, likely lysosomes. Coronavirus envelope proteins have previously been proposed to manipulate the luminal pH of the TGN, which serves as an accumulation station for progeny coronavirus particles prior to cellular egress via lysosomes. Delivery of S2-E to cells will enable chemical biological approaches for future studies of SARS-CoV-2 pathogenesis and possibly even development of "Trojan Horse" anti-viral therapies. Finally, this work also establishes a paradigm for amphipol-mediated delivery of membrane proteins to cells.

Keywords:  COVID; CoV-2; E protein; SARS; amphipol; coronavirus; envelope; insertion; ion channel; lysosomes Golgi; membrane; planar bilayer; trans-Golgi network

DOI:  https://doi.org/10.1016/j.jbc.2021.100940
Life Sci Alliance. 2021 Aug;pii: e202101105. [Epub ahead of print]4(8):

Axon terminals control endolysosome diffusion to support synaptic remodelling.

Beatrice Terni, Artur Llobet.

Endolysosomes are acidic organelles formed by the fusion of endosomes with lysosomes. In the presynaptic compartment they contribute to protein homeostasis, the maintenance of vesicle pools and synaptic stability. Here, we evaluated the mobility of endolysosomes found in axon terminals of olfactory sensory neurons of Xenopus tropicalis tadpoles. F-actin restricts the motion of these presynaptic acidic organelles which is characterized by a diffusion coefficient of 6.7 × 10-3 μm2·s-1 Local injection of secreted protein acidic and rich in cysteine (SPARC) in the glomerular layer of the olfactory bulb disrupts the structure of synaptic F-actin patches and increases the presence and mobility of endolysosomal organelles found in axon terminals. The increased motion of endolysosomes is localized to the presynaptic compartment and does not promote their access to axonal regions for retrograde transportation to the cell body. Local activation of synaptic degradation mechanisms mediated by SPARC coincides with a loss of the ability of tadpoles to detect waterborne odorants. Together, these observations show that the diffusion of presynaptic endolysosomes increases during conditions of synaptic remodelling to support their local degradative activity.

DOI: https://doi.org/10.26508/lsa.202101105
Autophagy. 2021 Jul 07. 1-18

RAB7 activity is required for the regulation of mitophagy in oocyte meiosis and oocyte quality control during ovarian aging.

Xin Jin, Kehan Wang, Lu Wang, Wenwen Liu, Chi Zhang, Yuexin Qiu, Wei Liu, Huiyu Zhang, Dong Zhang, Zhixia Yang, Tinghe Wu, Jing Li.

  There is increasing evidence that mitophagy, a specialized form of autophagy to degrade and clear long-lived or damaged mitochondria, is impaired in aging and age-related disease. Previous study has demonstrated the obesity-exposed oocytes accumulate and transmit damaged mitochondria due to an inability to activate mitophagy. However, it remains unknown whether mitophagy functions in oocyte and what's the regulatory mechanism in oocyte aging. In the study, when fully grown oocytes were treated with CCCP, an uncoupling agent to induce mitophagy, we found the activation of the PRKN-mediated mitophagy pathway accompanied the blockage of meiosis at metaphase I stage. Our result then demonstrated its association with the decreased activity of RAB7 and all the observed defects in CCCP treated oocytes could be effectively rescued by microinjection of mRNA encoding active RAB7Q67L or treatment with the RAB7 activator ML098. Further study indicated PRKN protein level as a rate-limiting factor to facilitate degradation of RAB7 and its GEF (guanine nucleotide exchange factor) complex CCZ1-MON1 through the ubiquitin-proteasome system. In GV oocytes collected during ovarian aging, we found the age-related increase of PINK1 and PRKN proteins and a significant decrease of RAB7 which resulted in defects of mitophagosome formation and the accumulation of damaged mitochondria. The age-related retardation of female fertility was improved after in vivo treatment of ML098. Thus, RAB7 activity is required to maintain the balance between mitophagy and chromosome stability and RAB7 activator is a good candidate to ameliorate age-related deterioration of oocyte quality.Abbreviations: ATG9: autophagy related 9A; ATP: adenosine triphosphate; CALCOCO2/NDP52: calcium binding and coiled-coil domain 2; CCCP: carbonyl cyanide 3-chlorophenylhydrazone; CCZ1: CCZ1 vacuolar protein trafficking and biogenesis associated; GAPDH: glyceraldehyde-3-phosphate dehydrogenase; GAPs: GTPase-activating proteins; GEF: guanine nucleotide exchange factor; GV: germinal vesicle; GVBD: germinal vesicle breakdown; LAMP1: lysosomal-associated membrane protein 1; MI: metaphase I stage of meiosis; MII: metaphase II stage of meiosis; Mito: MitoTracker; mtDNA: mitochondrial DNA; MON1: MON1 homolog, secretory trafficking associated; OPTN: optineurin; PINK1: PTEN induced putative kinase 1; PRKN: parkin RBR E3 ubiquitin protein ligase; RAB7: RAB7, member RAS oncogene family; ROS: reactive oxygen species; TEM: transmission electron microscopy; TOMM20/TOM20: translocase of outer mitochondrial membrane 20; TUBB: tubulin, beta; UB: ubiquitin.

Keywords:  Aging; PRKN; RAB7; meiosis; mitophagy; oocyte

DOI:  https://doi.org/10.1080/15548627.2021.1946739