Fusion Of Lysosomes Research Articles

Prodigiosin inhibits cholangiocarcinoma cell proliferation and induces apoptosis via suppressing SNAREs-dependent autophagy

BackgroundProdigiosin (PG), a natural red pigment produced by numerous bacterial species, has been a eye-catching research point in recent years for its anticancer activity. However, the role of PG in the cancer biology of cholangiocarcinoma (CCA) remains vague.MethodsThe proliferation of CCA cells was detected by Cell Counting Kit-8(CCK-8), Colony formation assay and 5-ethynyl-2′-deoxyuridine (EdU) assay. Cell apoptosis was evaluated by flow cytometry assay and western blot assay. The effects of PG or SNAREs on cell autophagy were measured by autophagy flux assay and western blot assay. Xenograft mouse models were used to assess the role of PG in CCA cells in vivo.ResultsPG could inhibit the proliferation and viability of CCA cells in a concentration- and time-dependent manner via suppressing the late stage of autophagy. Mechanistically, PG inhibits the fusion of autophagosomes and lysosomes by blocking STX17 and SNAP29, components of soluble N-ethyl-maleimide-sensitive factor attachment protein receptors (SNAREs)complex. When STX17 and SNAP29 were overexpressed, the inhibitory effect of PG on CCA cells autophagy was relieved. In addition, PG showed obvious inhibitory effects on cancer cell viability but no toxic effects on organs in xenotransplantation models.ConclusionTaken together, our results demonstrated that PG inhibits CCA cell proliferation via suppressing SNAREs-dependent autophagy, implying that PG could be a potential chemotherapy drug for advanced CCA.

Reactive oxygen species prevent lysosome coalescence during PIKfyve inhibition.

Lysosomes are terminal, degradative organelles of the endosomal pathway that undergo repeated fusion-fission cycles with themselves, endosomes, phagosomes, and autophagosomes. Lysosome number and size depends on balanced fusion and fission rates. Thus, conditions that favour fusion over fission can reduce lysosome numbers while enlarging their size. Conversely, favouring fission over fusion may cause lysosome fragmentation and increase their numbers. PIKfyve is a phosphoinositide kinase that generates phosphatidylinositol-3,5-bisphosphate to modulate lysosomal functions. PIKfyve inhibition causes an increase in lysosome size and reduction in lysosome number, consistent with lysosome coalescence. This is thought to proceed through reduced lysosome reformation and/or fission after fusion with endosomes or other lysosomes. Previously, we observed that photo-damage during live-cell imaging prevented lysosome coalescence during PIKfyve inhibition. Thus, we postulated that lysosome fusion and/or fission dynamics are affected by reactive oxygen species (ROS). Here, we show that ROS generated by various independent mechanisms all impaired lysosome coalescence during PIKfyve inhibition and promoted lysosome fragmentation during PIKfyve re-activation. However, depending on the ROS species or mode of production, lysosome dynamics were affected distinctly. H2O2 impaired lysosome motility and reduced lysosome fusion with phagosomes, suggesting that H2O2 reduces lysosome fusogenecity. In comparison, inhibitors of oxidative phosphorylation, thiol groups, glutathione, or thioredoxin, did not impair lysosome motility but instead promoted clearance of actin puncta on lysosomes formed during PIKfyve inhibition. Additionally, actin depolymerizing agents prevented lysosome coalescence during PIKfyve inhibition. Thus, we discovered that ROS can generally prevent lysosome coalescence during PIKfyve inhibition using distinct mechanisms depending on the type of ROS.

Human platelets display dysregulated sepsis-associated autophagy, induced by altered LC3 protein-protein interaction of the Vici-protein EPG5

ABSTRACT Platelets mediate central aspects of host responses during sepsis, an acute profoundly systemic inflammatory response due to infection. Macroautophagy/autophagy, which mediates critical aspects of cellular responses during inflammatory conditions, is known to be a functional cellular process in anucleate platelets, and is essential for normal platelet functions. Nevertheless, how sepsis may alter autophagy in platelets has never been established. Using platelets isolated from septic patients and matched healthy controls, we show that during clinical sepsis, the number of autophagosomes is increased in platelets, most likely due to an accumulation of autophagosomes, some containing mitochondria and indicative of mitophagy. Therefore, autophagy induction or early-stage autophagosome formation (as compared to decreased later-stage autophagosome maturation or autophagosome-late endosome/lysosome fusion) is normal or increased. This was consistent with decreased fusion of autophagosomes with lysosomes in platelets. EPG5 (ectopic P-granules autophagy protein 5 homolog), a protein essential for normal autophagy, expression did increase, while protein-protein interactions between EPG5 and MAP1LC3/LC3 (which orchestrate the fusion of autophagosomes and lysosomes) were significantly reduced in platelets during sepsis. Furthermore, data from a megakaryocyte model demonstrate the importance of TLR4 (toll like receptor 4), LPS-dependent signaling for regulating this mechanism. Similar phenotypes were also observed in platelets isolated from a patient with Vici syndrome: an inherited condition caused by a naturally occurring, loss-of-function mutation in EPG5. Together, we provide evidence that autophagic functions are aberrant in platelets during sepsis, due in part to reduced EPG5-LC3 interactions, regulated by TLR4 engagement, and the resultant accumulation of autophagosomes. Abbreviations: ACTB: beta actin; CLP: cecal ligation and puncture; Co-IP: co-immunoprecipitation; DAP: death associated protein; DMSO: dimethyl sulfoxide; EPG5: ectopic P-granules autophagy protein 5 homolog; ECL: enhanced chemiluminescence; HBSS: Hanks’ balanced salt solution; HRP: horseradish peroxidase; ICU: intensive care unit; LPS: lipopolysaccharide; LAMP1: lysosomal associated membrane protein 1; MAP1LC3/LC3: microtubule associated protein 1 light chain 3; MTOR: mechanistic target of rapamycin kinase; MKs: megakaryocytes; PFA: paraformaldehyde; PBS: phosphate-buffered saline; PLA: proximity ligation assay; pRT-PCR: quantitative real-time polymerase chain reaction; RT: room temperature; SQSTM1/p62: sequestosome 1; SDS-PAGE: sodium dodecyl sulfate-polyacrylamide gel electrophoresis; TLR4: toll like receptor 4; TEM: transmission electron microscopy; WGA: wheat germ agglutinin.

Curcumin prevents As3+-induced carcinogenesis through regulation of GSK3\u03b2/Nrf2

BackgroundArsenic (As3+) is a carcinogen with considerable environmental and occupational relevancy. Its mechanism of action and methods of prevention remain to be investigated. Previous studies have demonstrated that ROS is responsible for As3+-induced cell transformation, which is considered as the first stage of As3+ carcinogenesis. The NF-E2 p45-related factor-2 (Nrf2) signaling pathway regulates the cellular antioxidant response, and activation of Nrf2 has recently been shown to limit oxidative damage following exposure to As3+Methods and resultsIn this study, molecular docking was used to virtually screen natural antioxidant chemical databases and identify molecules that interact with the ligand-binding site of Keap1 (PDB code 4L7B). The cell-based assays and molecular docking findings revealed that curcumin has the best inhibitory activity against Keap1-4L7B. Co-immunoprecipitation (Co-IP) results indicated that curcumin is a potent Keap1 Kelch domain-dependent Nrf2 activator that stabilizes Nrf2 by hindering its ubiquitination. The increased activation of Nrf2 and its target antioxidant genes by curcumin could significantly decrease As3+-generated ROS. Moreover, curcumin induced autophagy in As3+-treated BEAS-2B via inducing autophagy by the formation of a p62/LC-3 complex and increasing autophagic flux by promoting transcription factor EB (TFEB) and lysosome-associated membrane protein 1 (LAMP1) expression. Knockdown of Nrf2 abolished curcumin-induced autophagy and downregulated ROS. Further studies showed that inhibition of autophagosome and lysosome fusion with bafilomycin a1 (BafA1) could block curcumin and prevented As3+-induced cell transformation. These results demonstrated that curcumin prevents As3+-induced cell transformation by inducing autophagy via the activation of the Nrf2 signaling pathway in BEAS-2B cells. However, overexpression of Keap-1 showed a constitutively high level of Nrf2 in As3+-transformed BEAS-2B cells (AsT) is Keap1-independent regulation. Overexpression of Nrf2 in AsT demonstrated that curcumin increased ROS levels and induced cell apoptosis via the downregulation of Nrf2. Further studies showed that curcumin decreased the Nrf2 level in AsT by activating GSK-3β to inhibit the activation of PI3K/AKT. Co-IP assay results showed that curcumin promoted the interaction of Nrf2 with the GSK-3β/β-TrCP axis and ubiquitin. Moreover, the inhibition of GSK-3β reversed Nrf2 expression in curcumin-treated AsT, indicating that the decrease in Nrf2 is due to activation of the GSK-3β/β-TrCP ubiquitination pathway. Furthermore, in vitro and in vivo results showed that curcumin induced cell apoptosis, and had anti-angiogenesis and anti-tumorigenesis effects as a result of activating the GSK-3β/β-TrCP ubiquitination pathway and subsequent decrease in Nrf2.ConclusionsTaken together, in the first stage, curcumin activated Nrf2, decreased ROS, and induced autophagy in normal cells to prevent As3+-induced cell transformation. In the second stage, curcumin promoted ROS and apoptosis and inhibited angiogenesis via inhibition of constitutive expression of Nrf2 in AsT to prevent tumorigenesis. Our results suggest that antioxidant natural compounds such as curcumin can be evaluated as potential candidates for complementary therapies in the treatment of As3+-induced carcinogenesis.

A Review of APOE Genotype-Dependent Autophagic Flux Regulation in Alzheimer's Disease.

Autophagy is a basic physiological process maintaining cell renewal, the degradation of dysfunctional organelles, and the clearance of abnormal proteins and has recently been identified as a main mechanism underlying the onset and progression of Alzheimer's disease (AD). The APOE ɛ4 genotype is the strongest genetic determinant of AD pathogenesis and initiates autophagic flux at different times. This review synthesizes the current knowledge about the potential pathogenic effects of ApoE4 on autophagy and describes its associations with the biological hallmarks of autophagy and AD from a novel perspective. Via a remarkable variety of widely accepted signaling pathway markers, such as mTOR, TFEB, SIRT1, LC3, p62, LAMP1, LAMP2, CTSD, Rabs, and V-ATPase, ApoE isoforms differentially modulate autophagy initiation; membrane expansion, recruitment, and enclosure; autophagosome and lysosome fusion; and lysosomal degradation. Although the precise pathogenic mechanism varies for different genes and proteins, the dysregulation of autophagic flux is a key mechanism on which multiple pathogenic processes converge.

Polysiloxane-based ratiometric fluorescent probe for monitoring lysosomal autophagy pathway in three distinguishable channels

Autophagy is an essential process for the degradation of malfunctional organelles and the response to cancer immunotherapy. The occurrence of defective autophagy is correlated to obesity, aging, neurological diseases, inflammation and tumors. Therefore, it is urgent to accurately depict the autophagic process. We herein architecture a polysiloxane-based probe (PA-1) that was lysosome targeted and pH-responsive for discerning autophagy process. We have demonstrated that PA-1 enabled specific visualization of lysosomal autophagy. PA-1 also detected the down-regulation of the pH value that accompanied the autophagy process under starvation stimulation. During autophagy, the green fluorescence of some spots in the lysosomes gradually decreased along with the emergence of strong agglomerated red fluorescence, which might be caused by the membrane fusion of lysosomes and autophagosomes. The location of the autolysosome could be precisely displayed by the ratio image. In addition, we observed that the autophagy inhibitor CQ could increase the pH of the intracellular environment and inhibit autophagy. The design strategy will broaden the research field of polysiloxane in cellular physiological processes and serves as a candidate for basic research on autophagic processes and effective therapeutic strategies for autophagy-related diseases.

Ajugol enhances TFEB-mediated lysosome biogenesis and lipophagy to alleviate non-alcoholic fatty liver disease

Lipophagy is the autophagic degradation of lipid droplets. Dysregulated lipophagy has been implicated in the development of non-alcoholic fatty liver disease (NAFLD). Ajugol is an active alkaloid isolated from the root of Rehmannia glutinosa which is commonly used to treat various inflammatory and metabolic diseases. This study aimed to investigate the effect of ajugol on alleviating hepatic steatosis and sought to determine whether its potential mechanism via the key lysosome-mediated process of lipophagy. Our findings showed that ajugol significantly improved high-fat diet-induced hepatic steatosis in mice and inhibited palmitate-induced lipid accumulation in hepatocytes. Further analysis found that hepatic steatosis promoted the expression of LC3-II, an autophagosome marker, but led to autophagic flux blockade due to a lack of lysosomes. Ajugol also enhanced lysosomal biogenesis and promoted the fusion of autophagosome and lysosome to improve impaired autophagic flux and hepatosteatosis. Mechanistically, ajugol inactivated mammalian target of rapamycin and induced nuclear translocation of the transcription factor EB (TFEB), an essential regulator of lysosomal biogenesis. siRNA-mediated knockdown of TFEB significantly abrogated ajugol-induced lysosomal biogenesis as well as autophagosome-lysosome fusion and lipophagy. We conclude that lysosomal deficit is a critical mediator of hepatic steatosis, and ajugol may alleviate NAFLD via promoting the TFEB-mediated autophagy-lysosomal pathway and lipophagy.

Activation of the purinergic receptor P2X7 improves hepatosteatosis by promoting lipophagy.

Nonalcoholic fatty liver disease (NAFLD) is a global health problem that develops through unclear molecular mechanisms. The P2X7 purinergic receptor (P2RX7) is an ATP-gated ion channel that belongs to the P2XR family. Thus far, studies on P2RX7 in NAFLD have been largely contradictory. Integrating experiments and modeling, we elucidate the dynamic processes of lipid droplet fusion and degradation following regulation of P2RX7. We show that activation of P2RX7 can activate the AMPK/ULK1 pathway to promote autophagosome generation and lysosomal degradation of autophagosomes. Inhibiting P2RX7 has the opposite effect. Notably, we find that lipid droplets become larger by the fusion of dysfunctional lysosomes but cannot be degraded by them following P2RX7 inhibition. Our study provides evidence that P2RX7 activation improves NAFLD by promoting lipophagy.

Resveratrol prevents haloperidol-induced mitochondria dysfunction through the induction of autophagy in SH-SY5Y cells

BackgroundHaloperidol is a commonly used antipsychotic drug and may increase neuronal oxidative stress associated with the side effects, including tardive dyskinesia and neurite withdraw. Autophagy plays a protective role in response to the accumulated reactive oxygen species (ROS) induced mitochondria damage. Resveratrol is an antioxidant compound having neuroprotective effects; however, it is unknown if resveratrol may stimulate autophagy and decrease mitochondria damage induced by haloperidol. HypothesisWe hypothesis that resveratrol stimulates the autophagic process and protects mitochondria lesion induced by haloperidol. MethodsMitoSOX™ Red Mitochondrial Superoxide Indicator and MitoTracker™ Green FM staining were used to measure the amount of the mitochondria ROS production and mitochondria mass in human SH-SY5Y cells treated with haloperidol and/or resveratrol. Autophagic related dyes and Western blot were applied to study the autophagic process and related protein expression. Besides, tandem monomeric mRFP-GFP-LC3 was used to investigate the fusion of autophagosome and lysosome. Transmission electron microscopy was used to investigate the mitochondrial and autophagic ultrastructures with or without haloperidol and resveratrol treatment. ResultsHaloperidol administration significantly increased mitochondria ROS and mitochondrial mass, indicating the increase of mitochondria dysfunction. Although haloperidol increased the autophagosomes and lysosome formation, the autophagosome-lysosome fusion and degradation were impaired. This was because we found an increased p62 after haloperidol treatment, an indication of autophagy incompletion. Importantly, resveratrol promoted the degradation of p62, upregulated the formation of autophagolysosome, and reversed haloperidol-induced mitochondria damage. ConclusionThese results collectively suggest that resveratrol may be introduced as a protective compound against haloperidol-induced mitochondria impairment and aberrant autophagy.

Drosophila Rab39 Attenuates Lysosomal Degradation

Lysosomal degradation, the common destination of autophagy and endocytosis, is one of the most important elements of eukaryotic metabolism. The small GTPases Rab39A and B are potential new effectors of this pathway, as their malfunction is implicated in severe human diseases like cancer and neurodegeneration. In this study, the lysosomal regulatory role of the single Drosophila Rab39 ortholog was characterized, providing valuable insight into the potential cell biological mechanisms mediated by these proteins. Using a de novo CRISPR-generated rab39 mutant, we found no failure in the early steps of endocytosis and autophagy. On the contrary, we found that Rab39 mutant nephrocytes internalize and degrade endocytic cargo at a higher rate compared to control cells. In addition, Rab39 mutant fat body cells contain small yet functional autolysosomes without lysosomal fusion defect. Our data identify Drosophila Rab39 as a negative regulator of lysosomal clearance during both endocytosis and autophagy.

P2X4 Receptors Mediate Ca2+ Release from Lysosomes in Response to Stimulation of P2X7 and H1 Histamine Receptors.

The P2X4 purinergic receptor is targeted to endolysosomes, where it mediates an inward current dependent on luminal ATP and pH. Activation of P2X4 receptors was previously shown to trigger lysosome fusion, but the regulation of P2X4 receptors and their role in lysosomal Ca2+ signaling are poorly understood. We show that lysosomal P2X4 receptors are activated downstream of plasma membrane P2X7 and H1 histamine receptor stimulation. When P2X4 receptors are expressed, the increase in near-lysosome cytosolic [Ca2+] is exaggerated, as detected with a low-affinity targeted Ca2+ sensor. P2X4-dependent changes in lysosome properties were triggered downstream of P2X7 receptor activation, including an enlargement of lysosomes indicative of homotypic fusion and a redistribution of lysosomes towards the periphery of the cell. Lysosomal P2X4 receptors, therefore, have a role in regulating lysosomal Ca2+ release and the regulation of lysosomal membrane trafficking.

Restriction factor compendium for influenza A virus reveals a mechanism for evasion of autophagy.

The fate of influenza A virus (IAV) infection in the host cell depends on the balance between cellular defence mechanisms and viral evasion strategies. To illuminate the landscape of IAV cellular restriction, we generated and integrated global genetic loss-of-function screens with transcriptomics and proteomics data. Our multi-omics analysis revealed a subset of both IFN-dependent and independent cellular defence mechanisms that inhibit IAV replication. Amongst these, the autophagy regulator TBC1 domain family member 5 (TBC1D5), which binds Rab7 to enable fusion of autophagosomes and lysosomes, was found to control IAV replication in vitro and in vivo and to promote lysosomal targeting of IAV M2 protein. Notably, IAV M2 was observed to abrogate TBC1D5-Rab7 binding through a physical interaction with TBC1D5 via its cytoplasmic tail. Our results provide evidence for the molecular mechanism utilised by IAV M2 protein to escape lysosomal degradation and traffic to the cell membrane, where it supports IAV budding and growth.

Amino acids and mechanistic target of rapamycin regulate the fate of live engulfed cells.

Metabolic stress contributes to the regulation of cell death in normal and diseased tissues. While different forms of cell death are known to be regulated by metabolic stress, how the cell engulfment and killing mechanism entosis is regulated is not well understood. Here we find that the death of entotic cells is regulated by the presence of amino acids and activity of the mechanistic target of rapamycin (mTOR). Amino acid withdrawal or mTOR inhibition induces apoptosis of engulfed cells and blocks entotic cell death that is associated with the lipidation of the autophagy protein microtubule‐associated protein light chain 3 (LC3) to entotic vacuoles. Two other live cell engulfment programs, homotypic cell cannibalism (HoCC) and anti‐CD47 antibody‐mediated phagocytosis, known as phagoptosis, also undergo a similar vacuole maturation sequence involving LC3 lipidation and lysosome fusion, but only HoCC involves mTOR‐dependent regulation of vacuole maturation and engulfed cell death similar to entosis. We further find that the regulation of cell death by mTOR is independent of autophagy activation and instead involves the 4E‐BP1/2 proteins that are known regulators of mRNA translation. Depletion of 4E‐BP1/2 proteins can restore the mTOR‐regulated changes of entotic death and apoptosis rates of engulfed cells. These results identify amino acid signaling and the mTOR‐4E‐BP1/2 pathway as an upstream regulation mechanism for the fate of live engulfed cells formed by entosis and HoCC.

Targeting Autophagy Triggers Apoptosis and Complements the Action of Venetoclax in Chronic Lymphocytic Leukemia Cells.

Simple SummaryVenetoclax is an antagonist of the antiapoptotic protein Bcl-2, and is currently approved for treatment of chronic lymphocytic leukemia (CLL). Recently, clinical use has shown that patients develop resistance to venetoclax. Therefore, the demand for novel targets for treatment of CLL remains high. One such target is autophagy, an evolutionarily old system for degradation of long-lived proteins and organelles that recovers the energy for normal cellular functions. Here, the antileukemic potential of different autophagy inhibitors was evaluated in patient-derived CLL cells. Among these, inhibitors of the AMPK/ULK1 pathway and late-stage autophagy were the most potent, with selective cytotoxic activities seen. They also show activity against CLL cells with unfavorable genetic characteristics. These inhibitors complement the cytotoxic action of venetoclax. In conclusion, targeting autophagy shows potential as a novel approach for treatment of patients with CLL.Continuous treatment of patients with chronic lymphocytic leukemia (CLL) with venetoclax, an antagonist of the anti-apoptotic protein Bcl-2, can result in resistance, which highlights the need for novel targets to trigger cell death in CLL. Venetoclax also induces autophagy by perturbing the Bcl-2/Beclin-1 complex, so autophagy might represent a target in CLL. Diverse autophagy inhibitors were assessed for cytotoxic activities against patient-derived CLL cells. The AMPK inhibitor dorsomorphin, the ULK1/2 inhibitor MRT68921, and the autophagosome–lysosome fusion inhibitor chloroquine demonstrated concentration-dependent and time-dependent cytotoxicity against CLL cells, even in those from hard-to-treat patients who carried del(11q) and del(17p). Dorsomorphin and MRT68921 but not chloroquine triggered caspase-dependent cell death. According to the metabolic activities of CLL cells and PBMCs following treatments with 10 µM dorsomorphin (13% vs. 84%), 10 µM MRT68921 (7% vs. 78%), and 25 µM chloroquine (41% vs. 107%), these autophagy inhibitors are selective toward CLL cells. In these CLL cells, venetoclax induced autophagy, and addition of dorsomorphin, MRT68921, or chloroquine showed potent synergistic cytotoxicities. Additionally, MRT68921 alone induced G2 arrest, but when combined with venetoclax, it triggered caspase-dependent cytotoxicity. These data provide the rationale to target autophagy and for autophagy inhibitors as potential treatments for patients with CLL.

Metformin promotes microglial cells to facilitate myelin debris clearance and accelerate nerve repairment after spinal cord injury.

Spinal cord injury (SCI) is one kind of severe trauma for central nervous system. Myelin debris clearance and axon regeneration are essential for nerve regeneration after SCI. Metformin, a glucose-lowering drug, has been demonstrated to promote the locomotor functional recovery after SCI. In this study, we investigated the role and molecular mechanism of metformin on myelin preservation in a rat SCI model. SCI was induced in rats by compression at T9 level using a vascular clip. We showed that administration of metformin (50 mg·kg-1·d-1, ip) for 28 days significantly improved locomotor function in SCI rats. Metformin also ameliorated SCI-induced neuronal apoptosis and promoted axon regeneration in the spinal cord. Using co-immunofluorescence of IBa-1 and MBP, and luxol fasting blue (LFB) staining, we demonstrated that metformin promoted the transformation of M1 to M2 phenotype polarization of microglial cells, then greatly facilitated myelin debris clearance and protected the myelin in SCI rats. Furthermore, metformin ameliorated SCI-induced blockade of autophagic flux in the spinal cord, and enhanced the fusion of autophagosome and lysosome by inhibiting the AMPK-mTOR signaling pathway. Moreover, metformin significantly attenuated inflammatory responses in the spinal cord. In LPS-treated BV2 cells, pretreatment with metformin (2 mM) significantly enhanced autophagy level, suppressed inflammation and cell apoptosis. The protective effects were blocked in the presence of an autophagy inhibitor 3-methyladenine (3-MA, 5 mM), suggesting that the effect of metformin on autophagy in microglial cells is essential for the myelin preservation during nerve recovery. This study reveals a novel therapeutic effect of metformin in SCI recovery by regulating the activation of microglial cells and enhancing its autophagy level.

The CACNA1A Mutant Disrupts Lysosome Calcium Homeostasis in Cerebellar Neurons and the Resulting Endo-Lysosomal Fusion Defect Can be Improved by Calcium Modulation.

Mutations in P/Q type voltage gated calcium channel (VGCC) lead severe human neurological diseases such as episodic ataxia 2, familial hemiplegic migraine 1, absence epilepsy, progressive ataxia and spinocerebellar ataxia 6. The pathogenesis of these diseases remains unclear. Mice with spontaneous mutation in the Cacna1a gene encoding the pore-forming subunit of P/Q type VGCC also exhibit ataxia, epilepsy and neurodegeneration. Based on the previous work showing that the P/Q type VGCC in neurons regulates lysosomal fusion through its calcium channel activity on lysosomes, we utilized CACNA1A mutant mice to further investigate the mechanism by which P/Q-type VGCCs regulate lysosomal function and neuronal homeostasis. We found CACNA1A mutant neurons have reduced lysosomal calcium storage without changing the resting calcium concentration in cytoplasm and the acidification of lysosomes. Immunohistochemistry and transmission electron microscopy reveal axonal degeneration due to lysosome dysfunction in the CACNA1A mutant cerebella. The calcium modulating drug thapsigargin, by depleting the ER calcium store, which locally increases the calcium concentration can alleviate the defective lysosomal fusion in mutant neurons. We propose a model that in cerebellar neurons, P/Q-type VGCC maintains the integrity of the nervous system by regulating lysosomal calcium homeostasis to affect lysosomal fusion, which in turn regulates multiple important cellular processes such as autophagy and endocytosis. This study helps us to better understand the pathogenesis of P/Q-type VGCC related neurodegenerative diseases and provides a feasible direction for future pharmacological treatment.

Hypoxia-stimulated ATM activation regulates autophagy-associated exosome release from cancer-associated fibroblasts to promote cancer cell invasion.

Cancer‐associated fibroblasts (CAFs) as a predominant cell component in the tumour microenvironment (TME) play an essential role in tumour progression. Our earlier studies revealed oxidized ATM activation in breast CAFs, which is independent of DNA double‐strand breaks (DSBs). Oxidized ATM has been found to serve as a redox sensor to maintain cellular redox homeostasis. However, whether and how oxidized ATM in breast CAFs regulates breast cancer progression remains poorly understood. In this study, we found that oxidized ATM phosphorylates BNIP3 to induce autophagosome accumulation and exosome release from hypoxic breast CAFs. Inhibition of oxidized ATM kinase by KU60019 (a small‐molecule inhibitor of activated ATM) or shRNA‐mediated knockdown of endogenous ATM or BNIP3 blocks autophagy and exosome release from hypoxic CAFs. We also show that oxidized ATM phosphorylates ATP6V1G1, a core proton pump in maintaining lysosomal acidification, leading to lysosomal dysfunction and autophagosome fusion with multi‐vesicular bodies (MVB) but not lysosomes to facilitate exosome release. Furthermore, autophagy‐associated GPR64 is enriched in hypoxic CAFs‐derived exosomes, which stimulates the non‐canonical NF‐κB signalling to upregulate MMP9 and IL‐8 in recipient breast cancer cells, enabling cancer cells to acquire enhanced invasive abilities. Collectively, these results provide novel insights into the role of stromal CAFs in promoting tumour progression and reveal a new function of oxidized ATM in regulating autophagy and exosome release.

Progranulin associates with Rab2 and is involved in autophagosome-lysosome fusion in Gaucher disease.

Progranulin (PGRN) is a key regulator of lysosomes, and its deficiency has been linked to various lysosomal storage diseases (LSDs), including Gaucher disease (GD), one of the most common LSD. Here, we report that PGRN plays a previously unrecognized role in autophagy within the context of GD. PGRN deficiency is associated with the accumulation of LC3-II and p62 in autophagosomes of GD animal model and patient fibroblasts, resulting from the impaired fusion of autophagosomes and lysosomes. PGRN physically interacted with Rab2, a critical molecule in autophagosome-lysosome fusion. Additionally, a fragment of PGRN containing the Grn E domain was required and sufficient for binding to Rab2. Furthermore, this fragment significantly ameliorated PGRN deficiency-associated impairment of autophagosome-lysosome fusion and autophagic flux. These findings not only demonstrate that PGRN is a crucial mediator of autophagosome-lysosome fusion but also provide new evidence indicating PGRN's candidacy as a molecular target for modulating autophagy in GD and other LSDs in general. KEY MESSAGES : PGRN acts as a crucial factor involved in autophagosome-lysosome fusion in GD. PGRN physically interacts with Rab2, a molecule in autophagosome-lysosome fusion. A 15-kDa C-terminal fragment of PGRN is required and sufficient for binding to Rab2. This PGRN derivative ameliorates PGRN deficiency-associated impairment of autophagy. This study provides new insights into autophagy and may develop novel therapy for GD.

Lysosomal membrane protein Sidt2 deletion impairs autophagy in human hepatocytes

To study the effect of lysosomal membrane protein Sidt2 deletion on autophagy in human hepatocytes. Crispr-Cas9 technology was used to construct a human hepatocyte (HL7702) model of Sidt2 knockout (Sidt2-/-), and the expression levels of the key autophagy proteins LC3II/I, P62 and autophagy-related proteins Atg5, Atg7, and Atg12 were detected.The co-localization of LC3B and P62 in the cells were analyzed with immunofluorescence assay to assess the identification and storage of P62 cargo proteins by the autophagosomes and the degradation of the autophagolysosomes.The co-localization of LC3B and LAMP1 was also determined with immunofluorescence assay to detect the fusion of the autophagosomes with the lysosomes, and LysoTracker was used to trace the acidic lysosomes. We successfully constructed a HL7702 cell model of Sidt2+/+ and Sidt2-/-, and compared with Sidt2+/+ cells, the Sidt2-/- cell model showed significantly increased expressions of LC3-II/I and P62 (P < 0.01).Immunofluorescence assay showed a significant increase of LC3B and P62 expressions (P < 0.001) and obviously lowered expressions of Atg5, Atg7, and Atg12 in Sidt2-/- cells (P < 0.05).The co-localization of LC3B and P62 and that of LC3B and LAMP1 were both reduced and the number of acidic lysosomes was significantly lowered in Sidt2-/- cells (P < 0.05). Sidt2 gene deletion disturbs the recognition and sequestration of P62 cargo protein by autophagosomes in human hepatocytes.At the same time, the decreased number of acidic lysosomes and the dysfunction of autophagosome and lysosome fusion cause the block of the autophagy-lysosome pathway, leading eventually to LC3B and P62 accumulation and impaired autophagy in the hepatocytes.

Cathepsin L, a Target of Hypoxia-Inducible Factor-1-α, Is Involved in Melanosome Degradation in Melanocytes.

Hypoxic conditions induce the activation of hypoxia-inducible factor-1α (HIF-1α) to restore the supply of oxygen to tissues and cells. Activated HIF-1α translocates into the nucleus and binds to hypoxia response elements to promote the transcription of target genes. Cathepsin L (CTSL) is a lysosomal protease that degrades cellular proteins via the endolysosomal pathway. In this study, we attempted to determine if CTSL is a hypoxia responsive target gene of HIF-1α, and decipher its role in melanocytes in association with the autophagic pathway. The results of our luciferase reporter assay showed that the expression of CTSL is transcriptionally activated through the binding of HIF1-α at its promoter. Under autophagy-inducing starvation conditions, HIF-1α and CTSL expression is highly upregulated in melan-a cells. The mature form of CTSL is closely involved in melanosome degradation through lysosomal activity upon autophagosome–lysosome fusion. The inhibition of conversion of pro-CTSL to mature CTSL leads to the accumulation of gp100 and tyrosinase in addition to microtubule-associated protein 1 light chain 3 (LC3) II, due to decreased lysosomal activity in the autophagic pathway. In conclusion, we have identified that CTSL, a novel target of HIF-1α, participates in melanosome degradation in melanocytes through lysosomal activity during autophagosome–lysosome fusion.