Autophagy initiation triggers p150Glued-AP-2ß interaction on the lysosomes and facilitates their transport.

The endocytic adaptor protein 2 (AP-2) complex binds dynactin as part of its noncanonical function, which is necessary for dynein-driven autophagosome transport along microtubules in neuronal axons. The absence of this AP-2-dependent transport causes neuronal morphology simplification and neurodegeneration. The mechanisms that lead to formation of the AP-2-dynactin complex have not been studied to date. However, the inhibition of mammalian/mechanistic target of rapamycin complex 1 (mTORC1) enhances the transport of newly formed autophagosomes by influencing the biogenesis and protein interactions of Rab-interacting lysosomal protein (RILP), another dynein cargo adaptor. We tested effects of mTORC1 inhibition on interactions between the AP-2 and dynactin complexes, with a focus on their two essential subunits, AP-2ß and p150Glued. We found that the mTORC1 inhibitor rapamycin enhanced p150Glued-AP-2ß complex formation in both neurons and non-neuronal cells. Additional analysis revealed that the p150Glued-AP-2ß interaction was indirect and required integrity of the dynactin complex. In non-neuronal cells rapamycin-driven enhancement of the p150Glued-AP-2ß interaction also required the presence of cytoplasmic linker protein 170 (CLIP-170), the activation of autophagy, and an undisturbed endolysosomal system. The rapamycin-dependent p150Glued-AP-2ß interaction occurred on lysosomal-associated membrane protein 1 (Lamp-1)-positive organelles but without the need for autolysosome formation. Rapamycin treatment also increased the acidification and number of acidic organelles and increased speed of the long-distance retrograde movement of Lamp-1-positive organelles. Altogether, our results indicate that autophagy regulates the p150Glued-AP-2ß interaction, possibly to coordinate sufficient motor-adaptor complex availability for effective lysosome transport.

Fusion with ARRDC1 or CD63: A Strategy to Enhance p53 Loading into Extracellular Vesicles for Tumor Suppression.

Small extracellular vesicles (sEVs) have emerged as promising therapeutic agents and drug delivery vehicles. Targeted modification of sEVs and their contents using genetic modification strategies is one of the most popular methods. This study investigated the effects of p53 fusion with arrestin domain-containing protein 1 (ARRDC1) and CD63 on the generation of sEVs, p53 loading efficiency, and therapeutic efficacy. Overexpression of either ARRDC1-p53 (ARP) or CD63-p53 (CDP) significantly elevated p53 mRNA and protein levels. The incorporation of ARRDC1 and CD63 significantly enhanced HEK293T-sEV biogenesis, evidenced by significant increases in sEV-associated proteins TSG101 and LAMP1, resulting in a boost in sEV production. Importantly, fusion with ARRDC1 or CD63 substantially increased the efficiency of loading both p53 fusion proteins and its mRNA into sEVs. sEVs equipped with ARP or CDP significantly enhanced the enrichment of p53 fusion proteins and mRNA in p53-null H1299 cells, resulting in a marked increase in apoptosis and a reduction in cell proliferation, with ARP-sEVs demonstrating greater effectiveness than CDP-sEVs. These findings underscore the enhanced functionality of ARRDC1- and CD63-modified sEVs, emphasizing the potential of genetic modifications in sEV-based therapies for targeted cancer treatment.

Burkholderia thailandensis uses a type VI secretion system to lyse protrusions without triggering host cell responses.

To spread within a host, intracellular Burkholderia form actin tails to generate membrane protrusions into neighboring host cells and use type VI secretion system-5 (T6SS-5) to induce cell-cell fusions. Here, we show that B. thailandensis also uses T6SS-5 to lyse protrusions to directly spread from cell to cell. Dynamin-2 recruitment to the membrane near a bacterium was followed by a short burst of T6SS-5 activity. This resulted in the polymerization of the actin of the newly invaded host cell and disruption of the protrusion membrane. Most protrusion lysis events were dependent on dynamin activity, caused no cell-cell fusion, and failed to be recognized by galectin-3. T6SS-5 inactivation decreased protrusion lysis but increased galectin-3, LC3, and LAMP1 accumulation in host cells. Our results indicate that B. thailandensis specifically activates T6SS-5 assembly in membrane protrusions to disrupt host cell membranes and spread without alerting cellular responses, such as autophagy.

MCT4 and CD147 colocalize with MMP14 in invadopodia and support matrix degradation and invasion by breast cancer cells.

Expression levels of the lactate-H+ cotransporter MCT4 (also known as SLC16A3) and its chaperone CD147 (also known as basigin) are upregulated in breast cancers, correlating with decreased patient survival. Here, we test the hypothesis that MCT4 and CD147 favor breast cancer invasion through interdependent effects on extracellular matrix (ECM) degradation. MCT4 and CD147 expression and membrane localization were found to be strongly reciprocally interdependent in MDA-MB-231 breast cancer cells. Overexpression of MCT4 and/or CD147 increased, and their knockdown decreased, migration, invasion and the degradation of fluorescently labeled gelatin. Overexpression of both proteins led to increases in gelatin degradation and appearance of the matrix metalloproteinase (MMP)-generated collagen-I cleavage product reC1M, and these increases were greater than those observed upon overexpression of each protein alone, suggesting a concerted role in ECM degradation. MCT4 and CD147 colocalized with invadopodia markers at the plasma membrane. They also colocalized with MMP14 and the lysosomal marker LAMP1, as well as partially with the autophagosome marker LC3, in F-actin-decorated intracellular vesicles. We conclude that MCT4 and CD147 reciprocally regulate each other and interdependently support migration and invasiveness of MDA-MB-231 breast cancer cells. Mechanistically, this involves MCT4-CD147-dependent stimulation of ECM degradation and specifically of MMP-mediated collagen-I degradation. We suggest that the MCT4-CD147 complex is co-delivered to invadopodia with MMP14.

Comparative Analysis of Site-Specific N-glycosylation of LAMP1 from Breast Cancer Tissues.

Glycosylation changes in cancer proteins have been associated with malignant transformation. However, techniques for analyzing site-specific glycosylation changes in target proteins obtained from clinical tissue samples are insufficient. To overcome these problems, we developed a targeted N-glycoproteomic approach consisting of immunoprecipitation, glycopeptide enrichment, LC/MS/MS and structural assignment using commercially available analytical software followed by manual confirmation. This approach was applied to the comparative site-specific glycosylation analysis of lysosome-associated membrane glycoprotein 1 (LAMP1) between breast cancer (BC) tumors and normal tissues adjacent to tumors. Extensive determination of glycan heterogeneity from four N-glycosylation sites (Asn84/103/249/261) in LAMP1 identified 262 glycoforms and revealed remarkable diversity in tumor glycan structures. A significant increase in N-glycoforms with multiple fucoses and sialic acids at Asn84/249 and high-mannose-type glycans at Asn103/261 were observed in the tumor. Principal component analysis revealed that tumors of different subtypes have independent distributions. This approach enables site-specific glycopeptide analysis of target glycoprotein in breast cancer tissue and become a powerful tool for characterizing tumors with different pathological features by their glycan profiles.

Loss of RND3/RHOE controls entosis through LAMP1 expression in hepatocellular carcinoma.

Entosis is a process that leads to the formation of cell-in-cell structures commonly found in cancers. Here, we identified entosis in hepatocellular carcinoma and the loss of Rnd3 (also known as RhoE) as an efficient inducer of this mechanism. We characterized the different stages and the molecular regulators of entosis induced after Rnd3 silencing. We demonstrated that this process depends on the RhoA/ROCK pathway, but not on E-cadherin. The proteomic profiling of entotic cells allowed us to identify LAMP1 as a protein upregulated by Rnd3 silencing and implicated not only in the degradation final stage of entosis, but also in the full mechanism. Moreover, we found a positive correlation between the presence of entotic cells and the metastatic potential of tumors in human patient samples. Altogether, these data suggest the involvement of entosis in liver tumor progression and highlight a new perspective for entosis analysis in medicine research as a novel therapeutic target.

Discovery of a Small-Molecule Modulator of the Autophagy-Lysosome Pathway That Targets Lamin A/C and LAMP1, Induces Autophagic Flux, and Affects Lysosome Positioning in Neurons.

Autophagy is a major catabolic degradation and recycling process that maintains homeostasis in cells and is especially important in postmitotic neurons. We implemented a high-content phenotypic assay to discover small molecules that promote autophagic flux and completed target identification and validation studies to identify protein targets that modulate the autophagy pathway and promote neuronal health and survival. Efficient syntheses of the prioritized compounds were developed to readily access analogues of the initial hits, enabling initial structure-activity relationship studies to improve potency and preparation of a biotin-tagged pulldown probe that retains activity. This probe facilitated target identification and validation studies through pulldown and competition experiments using both an unbiased proteomics approach and western blotting to reveal Lamin A/C and LAMP1 as the protein targets of compound RH1115. Evaluation of RH1115 in neurons revealed that this compound induces changes to LAMP1 vesicle properties and alters lysosome positioning. Dysfunction of the autophagy-lysosome pathway has been implicated in a variety of neurodegenerative diseases, including Alzheimer's disease, highlighting the value of new strategies for therapeutic modulation and the importance of small-molecule probes to facilitate the study of autophagy regulation in cultured neurons and in vivo.

LAMP1 targeting of the large T antigen of Merkel cell polyomavirus results in potent CD4 T cell responses and tumor inhibition.

Introduction: Most cases of Merkel cell carcinoma (MCC), a rare and highly aggressive type of neuroendocrine skin cancer, are associated with Merkel cell polyomavirus (MCPyV) infection. MCPyV integrates into the host genome, resulting in expression of oncoproteins including a truncated form of the viral large T antigen (LT) in infected cells. These oncoproteins are an attractive target for a therapeutic cancer vaccine. Methods: We designed a cancer vaccine that promotes potent, antigen-specific CD4 T cell responses to MCPyV-LT. To activate antigen-specific CD4 T cells in vivo, we utilized our nucleic acid platform, UNITE™ (UNiversal Intracellular Targeted Expression), which fuses a tumor-associated antigen with lysosomal-associated membrane protein 1 (LAMP1). This lysosomal targeting technology results in enhanced antigen presentation and potent antigen-specific T cell responses. LTS220A, encoding a mutated form of MCPyV-LT that diminishes its pro-oncogenic properties, was introduced into the UNITE™ platform. Results: Vaccination with LTS220A-UNITE™ DNA vaccine (ITI-3000) induced antigen-specific CD4 T cell responses and a strong humoral response that were sufficient to delay tumor growth of a B16F10 melanoma line expressing LTS220A. This effect was dependent on the CD4 T cells' ability to produce IFNγ. Moreover, ITI-3000 induced a favorable tumor microenvironment (TME), including Th1-type cytokines and significantly enhanced numbers of CD4 and CD8 T cells as well as NK and NKT cells. Additionally, ITI-3000 synergized with an α-PD-1 immune checkpoint inhibitor to further slow tumor growth and enhance survival. Conclusions: These findings strongly suggest that in pre-clinical studies, DNA vaccination with ITI-3000, using the UNITE™ platform, enhances CD4 T cell responses to MCPyV-LT that result in significant anti-tumor immune responses. These data support the initiation of a first-in-human (FIH) Phase 1 open-label study to evaluate the safety, tolerability, and immunogenicity of ITI-3000 in patients with polyomavirus-positive MCC (NCT05422781).

LAMP1 as a novel molecular biomarker to predict the prognosis of the children with autism spectrum disorder using bioinformatics approaches.

Autism spectrum disorder (ASD) is a neurodevelopmental disorder that usually manifests in childhood and is thought to be caused by a complex interaction of genetic, environmental, and immune factors. The majority of current ASD diagnostic methods rely on subjective behavioral observation and scale assessment, making early detection difficult. In this study, we confirmed that lysosomal-associated membrane protein 1 (LAMP1), a functional marker of immune cell activation and cytotoxic degranulation, was upregulated in ASD blood, brain cortex, and various genetic animal models or cells using bioinformatics approaches. The prognostic value of LAMP1 was investigated by correlating its expression with clinical ASD rating scales, and the receiver operating characteristic (ROC) curve analysis in ASD also revealed that it has a favorable diagnostic ability in distinguishing ASD from control cohort. According to gene set enrichment analysis (GSEA) results, LAMP1 correlated with genes that were enriched in natural kill and T cell immune function. Taking all of the evidence into account, we discovered that abnormal elevations of LAMP1 mRNA and protein in the blood of ASD children, may influence the development of ASD through its involvement in immune cell activity regulation. This report highlights a novel marker for ASD early detection as well as potential therapeutic targets.

LAMP1/2 as potential diagnostic and prognostic marker for brain lower grade glioma: A review.

Brain lower grade glioma (LGG) is a common type of glioma. The current treatment methods still have some limitations, and some LGG patients will inevitably continue to deteriorate after treatment. We found the value of lysosomal associated membrane proteins (LAMPs) in the diagnosis and prognosis of LGG, which helps to enhance the clinical understanding of LGG treatment and improved prognosis. We assess the role of LAMPs in LGG, via the publicly available TCGA database. We explored expression levels of LAMPs in LGG using GEPIA2, cBioPortal, and UALCAN databases. The correction of LAMPs expression levels with immune cell infiltration in LGG patient was assessed by TIMER database. The Lysosomal associated membrane protein 1 (LAMP1)/2/4 mRNA levels were significantly higher in LGG patients than in healthy controls. Morover, high mRNA expressions of LAMP1/2/Lysosomal associated membrane protein 3 were associated with poor overall survival. We found that the immune invasion of LGG was almost significantly correlated with the expression of LAMPs. The results suggested that mRNA expressions of LAMP1 and LAMP4 were significantly associated with histological subtypes in LGG patients. lysosomal associated membrane protein 2 and LAMP5 were significantly down-regulated expression in samples of TP53 mutant in LGG compared to TP53 wild type. In addition, Lysosomal associated membrane protein 3 and LAMP4 were significantly overexpressed in samples of TP53 mutant in LGG Enrichment analysis applied to each component indicated that biological function was primarily associated with series of pathways in synapse and immunity.

Quantitative Immunofluorescence to Study Phagosome Maturation and Resolution.

Cells such as macrophages and neutrophils can internalize a diverse set of particulate matter, illustrated by bacteria and apoptotic bodies through the process of phagocytosis. These particles are sequestered into phagosomes, which then fuse with early and late endosomes and ultimately with lysosomes to mature into phagolysosomes, through a process known as phagosome maturation. Ultimately, after particle degradation, phagosomes then fragment to reform lysosomes through phagosome resolution. As phagosomes change, they acquire and divest proteins that are associated with the various stages of phagosome maturation and resolution. These changes can be assessed at the single-phagosome level by using immunofluorescence methods. Typically, we use indirect immunofluorescence methods that rely on primary antibodies against specific molecular markers that track phagosome maturation. Commonly, progression of phagosomes into phagolysosomes can be determined by staining cells for Lysosomal-Associated Membrane Protein I (LAMP1) and measuring the fluorescence intensity of LAMP1 around each phagosome by microscopy or flow cytometry. However, this method can be used to detect any molecular marker for which there are compatible antibodies for immunofluorescence.

Thyroid hormone upregulates LAMP2 expression and lysosome activity.

Thyroid hormone (T3)-induced autophagy and its biological significance have been extensively investigated in recent years. However, limited studies to date have focused on the important role of lysosomes in autophagy. In this study, we explored the effects of T3 on lysosomal protein expression and trafficking in detail. Our findings showed that T3 activates rapid lysosomal turnover and expression of numerous lysosomal genes, including TFEB, LAMP2, ARSB, GBA, PSAP, ATP6V0B, ATP6V0D1, ATP6V1E1, CTSB, CTSH, CTSL, and CTSS, in a thyroid hormone receptor-dependent manner. In a murine model, LAMP2 protein was specifically induced in mice with hyperthyroidism. T3-promoted microtubule assembly was significantly disrupted by vinblastine, resulting in accumulation of the lipid droplet marker PLIN2. In the presence of the lysosomal autophagy inhibitors bafilomycin A1, chloroquine and ammonium chloride, we observed substantial accumulation of LAMP2 but not LAMP1 protein. T3 further enhanced the protein levels of ectopically expressed LAMP1 and LAMP2. Upon knockdown of LAMP2, cavities of lysosomes and lipid droplets accumulated in the presence of T3, although the changes in LAMP1 and PLIN2 expression were less pronounced. More specifically, the protective effect of T3 against ER stress-induced death was abolished by knockdown of LAMP2. Our collective results indicate that T3 not only promotes lysosomal gene expression but also LAMP protein stability and microtubule assembly, leading to enhancement of lysosomal activity in digesting any additional autophagosomal burden.

Haploinsufficiency of the lysosomal sialidase NEU1 results in a model of pleomorphic rhabdomyosarcoma in mice.

Rhabdomyosarcoma, the most common pediatric sarcoma, has no effective treatment for the pleomorphic subtype. Still, what triggers transformation into this aggressive phenotype remains poorly understood. Here we used Ptch1+/-/ETV7TG/+/- mice with enhanced incidence of rhabdomyosarcoma to generate a model of pleomorphic rhabdomyosarcoma driven by haploinsufficiency of the lysosomal sialidase neuraminidase 1. These tumors share mostly features of embryonal and some of alveolar rhabdomyosarcoma. Mechanistically, we show that the transforming pathway is increased lysosomal exocytosis downstream of reduced neuraminidase 1, exemplified by the redistribution of the lysosomal associated membrane protein 1 at the plasma membrane of tumor and stromal cells. Here we exploit this unique feature for single cell analysis and define heterogeneous populations of exocytic, only partially differentiated cells that force tumors to pleomorphism and promote a fibrotic microenvironment. These data together with the identification of an adipogenic signature shared by human rhabdomyosarcoma, and likely fueling the tumor's metabolism, make this model of pleomorphic rhabdomyosarcoma ideal for diagnostic and therapeutic studies.

Human LAMP1 accelerates Lassa virus fusion and potently promotes fusion pore dilation upon forcing viral fusion with non-endosomal membrane.

Lassa virus (LASV) cell entry is mediated by the interaction of the virus glycoprotein complex (GPC) with alpha-dystroglycan at the cell surface followed by binding to LAMP1 in late endosomes. However, LAMP1 is not absolutely required for LASV fusion, as this virus can infect LAMP1-deficient cells. Here, we used LASV GPC pseudoviruses, LASV virus-like particles and recombinant lymphocytic choriomeningitis virus expressing LASV GPC to investigate the role of human LAMP1 (hLAMP1) in LASV fusion with human and avian cells expressing a LAMP1 ortholog that does not support LASV entry. We employed a combination of single virus imaging and virus population-based fusion and infectivity assays to dissect the hLAMP1 requirement for initiation and completion of LASV fusion that culminates in the release of viral ribonucleoprotein into the cytoplasm. Unexpectedly, ectopic expression of hLAMP1 accelerated the kinetics of small fusion pore formation, but only modestly increased productive LASV fusion and infection of human and avian cells. To assess the effects of hLAMP1 in the absence of requisite endosomal host factors, we forced LASV fusion with the plasma membrane by applying low pH. Unlike the conventional LASV entry pathway, ectopic hLAMP1 expression dramatically promoted the initial and full dilation of pores formed through forced fusion at the plasma membrane. We further show that, while the soluble hLAMP1 ectodomain accelerates the kinetics of nascent pore formation, it fails to promote efficient pore dilation, suggesting the hLAMP1 transmembrane domain is involved in this late stage of LASV fusion. These findings reveal a previously unappreciated role of hLAMP1 in promoting dilation of LASV fusion pores, which is difficult to ascertain for endosomal fusion where several co-factors, such as bis(monoacylglycero)phosphate, likely regulate LASV entry.

Intermittent fasting activates markers of autophagy in mouse liver, but not muscle from mouse or humans.

OBJECTIVES: Intermittent fasting (IF) activates autophagy in cardiac muscle and pancreatic islets. We examined the effect of IF on markers of autophagy in liver and skeletal muscle in mice and in humans. METHODS: Ten-wk-old C57 BL/6 J male mice were ad libitum (AL) fed a high-fat diet (HFD) or chow diet for 8 wk, before randomization to AL or IF (24-h fast, 3 non-consecutive days per week) for 8 wk (8-16 per group). Tissue was collected in the fed or 22-h fasted state. Fifty women (51 ± 2 y, 31.8 ± 4.3 kg/m2) were randomly assigned to one of two IF protocols (24-hfast, 3 non-consecutive days per week) and fed at 70% (IF70) or 100% (IF100) of energy requirements for 8 wk. Vastus lateralis muscle was collected at 0800 after 12- and 24-h fasts. Microtubule-associated protein light chain 1 (Map1 lc3 b), Beclin1 (Becn1), Sequestosome 1 (Sqstm1/p62), and Lysosomal associated membrane protein 2 (Lamp2) were assessed by quantitative polymerase chain reaction and LC3, BECLIN1 and LAMP1 protein content by immunoblotting. RESULTS: Fasting increased hepatic LC3 I protein and Map1 lc3 b mRNA levels in IF mice fed chow or HFD. LAMP1 protein and Beclin1 mRNA levels in liver were also increased by fasting, but only in chow-fed mice. IF did not activate markers of autophagy in mouse muscle. In humans, a 24-h fast increased SQSTM1. BECLIN1, SQSTM1 and LAMP2 mRNA levels were decreased in IF70 after a 12-h overnight fast . CONCLUSION: Markers of autophagy in liver, but not in muscle, were elevated in response to IF in mice. In humans, autophagy markers in muscle were reduced, likely in response to weight loss.

The FACT complex facilitates expression of lysosomal and antioxidant genes through binding to TFEB and TFE3.

TFEB (transcription factor EB) and TFE3 (transcription factor binding to IGHM enhancer 3) orchestrate the cellular response to a variety of stressors, including nutrient deprivation, oxidative stress and pathogens. Here we describe a novel interaction of TFEB and TFE3 with the FAcilitates Chromatin Transcription (FACT) complex, a heterodimeric histone chaperone consisting of SSRP1 and SUPT16H that mediates nucleosome disassembly and assembly, thus facilitating transcription. Extracellular stimuli, such as nutrient deprivation or oxidative stress, induce nuclear translocation and activation of TFEB and TFE3, which then associate with the FACT complex to regulate stress-induced gene transcription. Depletion of FACT does not affect TFEB activation, stability, or binding to the promoter of target genes. In contrast, reduction of FACT levels by siRNA or treatment with the FACT inhibitor curaxin, severely impairs induction of numerous antioxidant and lysosomal genes, revealing a crucial role of FACT as a regulator of cellular homeostasis. Furthermore, upregulation of antioxidant genes induced by TFEB over-expression is significantly reduced by curaxin, consistent with a role of FACT as a TFEB transcriptional activator. Together, our data show that chromatin remodeling at the promoter of stress-responsive genes by FACT is important for efficient expression of TFEB and TFE3 targets, thus providing a link between environmental changes, chromatin modifications and transcriptional regulation.Abbreviations: ADNP2, ADNP homeobox 2; ATP6V0D1, ATPase H+ transporting V0 subunit d1; ATP6V1A, ATPase H+ transporting V1 subunit A; ATP6V1C1, ATPase H+ transporting V1 subunit C1; CSNK2/CK2, casein kinase 2; CLCN7, chloride voltage-gated channel 7; CTSD, cathepsin D; CTSZ, cathepsin Z; EBSS, earle's balanced salt solution; FACT complex, facilitates chromatin transcription complex; FOXO3, forkhead box O3; HEXA, hexosaminidase subunit alpha; HIF1A, hypoxia inducible factor 1 subunit alpha; HMOX1, heme oxygenase 1; LAMP1, lysosomal associated membrane protein 1; MAFF, MAF bZIP transcription factor F; MAFG, MAF bZIP transcription factor G; MCOLN1, mucolipin TRP cation channel 1; MTORC1, mechanistic target of rapamycin kinase complex 1; NaAsO2, sodium arsenite; POLR2, RNA polymerase II; PPARGC1A, PPARG coactivator 1 alpha; PYROXD1, pyridine nucleotide-disulfide oxidoreductase domain 1; RRAGC, Ras related GTP binding C; SEC13, SEC13 homolog, nuclear pore and COPII coat complex component; SLC38A9, solute carrier family 38 member 9; SSRP1, structure specific recognition protein 1; SUPT16H, SPT16 homolog, facilitates chromatin remodeling subunit; TFEB, transcription factor EB; TFE3, transcription factor binding to IGHM enhancer 3; TXNRD1, thioredoxin reductase 1; UVRAG, UV radiation resistance associated; WDR59, WD repeat domain 59.

CTRP3 alleviates myocardial ischemia/reperfusion injury in mice through activating LAMP1/JIP2/JNK pathway.

Myocardial ischemia reperfusion (I/R) injury is an important complication of myocardial infarction reperfusion therapy, and no effective treatment has been identified. Based on preexisting evidence, C1q/tumor necrosis factor-related protein 3 (CTRP3) has been reported to be closely associated with myocardial dysfunction. In this study, we found that CTRP3 was downregulated in acute coronary syndrome (ACS) patients and myocardial I/R mice. Silence of CTRP3 aggravated cardiac systolic function due to I/R of mice, while CTRP3 overexpression ameliorated cardiac function. Moreover, overexpression of CTRP3 improved I/R inhibitory effects on the levels of creatinine phosphokinase (CPK), lactate dehydrogenase (LDH) and cardiac troponin-I (cTn-I), myocardial infarction area, the intensity of the 3-nitrotyrosine (3-NT), apoptosis and protein levels of LAMP1, JNK-Interacting Protein-2 (JIP-2) and JNK, while these effects could be exacerbated by downregulation of CTRP3. Co-IP experiments could identify physical interactions between CTRP3 and lysosomal-associated membrane protein 1 (LAMP1) and Numb and JIP2. LAMP1 silence aggravated the inhibition effects of I/R on JIP2 and JNK protein expression, CPK, LDH and cTn-I levels and caspase-3 activity, while overexpression of LAMP1 recovered these inhibition effects of I/R. JNK inhibitor (SP600125) could reverse the inhibitory effects of CTRP3 overexpression on CPK, LDH, cTn-I, myocardial infarction, strong positive staining for 3-NT and apoptosis. These findings demonstrated that CTRP3 protected against injury caused by myocardial I/R through activating LAMP1/JIP2/JNK pathway to attenuate myocardial injury, improve left ventricular function, decrease myocardial infarction, and reduce myocardial apoptosis.

Lysosomal Changes in Mitosis.

The recent discovery demonstrating that the leakage of cathepsin B from mitotic lysosomes assists mitotic chromosome segregation indicates that lysosomal membrane integrity can be spatiotemporally regulated. Unlike many other organelles, structural and functional alterations of lysosomes during mitosis remain, however, largely uncharted. Here, we demonstrate substantial differences in lysosomal proteome, lipidome, size, and pH between lysosomes that were isolated from human U2OS osteosarcoma cells either in mitosis or in interphase. The combination of pharmacological synchronization and mitotic shake-off yielded ~68% of cells in mitosis allowing us to investigate mitosis-specific lysosomal changes by comparing cell populations that were highly enriched in mitotic cells to those mainly in the G1 or G2 phases of the cell cycle. Mitotic cells had significantly reduced levels of lysosomal-associated membrane protein (LAMP) 1 and the active forms of lysosomal cathepsin B protease. Similar trends were observed in levels of acid sphingomyelinase and most other lysosomal proteins that were studied. The altered protein content was accompanied by increases in the size and pH of LAMP2-positive vesicles. Moreover, mass spectrometry-based shotgun lipidomics of purified lysosomes revealed elevated levels of sphingolipids, especially sphingomyelin and hexocylceramide, and lysoglyserophospholipids in mitotic lysosomes. Interestingly, LAMPs and acid sphingomyelinase have been reported to stabilize lysosomal membranes, whereas sphingomyelin and lysoglyserophospholipids have an opposite effect. Thus, the observed lysosomal changes during the cell cycle may partially explain the reduced lysosomal membrane integrity in mitotic cells.

Defective autophagy contributes to endometrial epithelial-mesenchymal transition in intrauterine adhesions.

Intrauterine adhesions (IUA), characterized by endometrial fibrosis, is a common cause of uterine infertility. We previously demonstrated that partial epithelial-mesenchymal transition (EMT) and the loss of epithelial homeostasis play a vital role in the development of endometrial fibrosis. As a pro-survival strategy in maintaining cell and tissue homeostasis, macroautophagy/autophagy, conversely, may participate in this process. However, the role of autophagy in endometrial fibrosis remains unknown. Here, we demonstrated that autophagy is defective in endometria of IUA patients, which aggravates EMT and endometrial fibrosis, and defective autophagy is related to DIO2 (iodothyronine deiodinase 2) downregulation. In endometrial epithelial cells (EECs), pharmacological inhibition of autophagy by chloroquine (CQ) promoted EEC-EMT, whereas enhanced autophagy by rapamycin extenuated this process. Mechanistically, silencing DIO2 in EECs blocked autophagic flux and promoted EMT via the MAPK/ERK-MTOR pathway. Inversely, overexpression of DIO2 or triiodothyronine (T3) treatment could restore autophagy and partly reverse EEC-EMT. Furthermore, in an IUA-like mouse model, the autophagy in endometrium was defective accompanied by EEC-EMT, and CQ could inhibit autophagy and aggravate endometrial fibrosis, whereas rapamycin or T3 treatment could improve the autophagic levels and blunt endometrial fibrosis. Together, we demonstrated that defective autophagy played an important role in EEC-EMT in IUA via the DIO2-MAPK/ERK-MTOR pathway, which provided a potential target for therapeutic implications.Abbreviations: ACTA2/α-SMA: actin alpha 2, smooth muscle; AMPK: adenosine 5'-monophosphate-activated protein kinase; AKT/protein kinase B: AKT serine/threonine kinase; ATG: autophagy related; CDH1/E-cadherin: cadherin 1; CDH2/N-cadherin: cadherin 2; CQ: chloroquine; CTSD: cathepsin D; DIO2: iodothyronine deiodinase 2; DEGs: differentially expressed genes; EECs: endometrial epithelial cells; EMT: epithelial-mesenchymal transition; FN1: fibronectin 1; IUA: intrauterine adhesions; LAMP1: lysosomal associated membrane protein 1; LPS: lipopolysaccharide; MAP1LC3/LC3: microtubule associated protein 1 light chain 3; MAPK: mitogen-activated protein kinase; MTOR: mechanistic target of rapamycin kinase; Rapa: rapamycin; SQSTM1/p62: sequestosome 1; T3: triiodothyronine; T4: tetraiodothyronine; TFEB: transcription factor EB; PBS: phosphate-buffered saline; TEM: transmission electron microscopy; TGFB/TGFß: transforming growth factor beta.

Treatments targeting autophagy ameliorate the age-related macular degeneration phenotype in mice lacking APOE (apolipoprotein E).

Age-related macular degeneration (AMD) is a leading cause of vision loss with recent evidence indicating an important role for macroautophagy/autophagy in disease progression. In this study we investigate the efficacy of targeting autophagy for slowing dysfunction in a mouse model with features of early AMD. Mice lacking APOE (apolipoprotein E; B6.129P2-Apoetm1UncJ/Arc) and C57BL/6 J- (wild-type, WT) mice were treated with metformin or trehalose in the drinking water from 5 months of age and the ocular phenotype investigated at 13 months. Control mice received normal drinking water. APOE-control mice had reduced retinal function and thickening of Bruch's membrane consistent with an early AMD phenotype. Immunohistochemical labeling showed reductions in MAP1LC3B/LC3 (microtubule-associated protein 1 light chain 3 beta) and LAMP1 (lysosomal-associated membrane protein 1) labeling in the photoreceptors and retinal pigment epithelium (RPE). This correlated with increased LC3-II:LC3-I ratio and alterations in protein expression in multiple autophagy pathways measured by reverse phase protein array, suggesting autophagy was slowed. Treatment of APOE-mice with metformin or trehalose ameliorated the loss of retinal function and reduced Bruch's membrane thickening, enhancing LC3 and LAMP1 labeling in the ocular tissues and restoring LC3-II:LC3-I ratio to WT levels. Protein analysis indicated that both treatments boost ATM-AMPK driven autophagy. Additionally, trehalose increased p-MAPK14/p38 to enhance autophagy. Our study shows that treatments targeting pathways to enhance autophagy have the potential for treating early AMD and provide support for the use of metformin, which has been found to reduce the risk of AMD development in human patients.Abbreviations:AMD: age-related macular degeneration; AMPK: 5' adenosine monophosphate-activated protein kinase APOE: apolipoprotein E; ATM: ataxia telangiectasia mutated; BCL2L1/Bcl-xL: BCL2-like 1; DAPI: 4'-6-diamidino-2-phenylindole; ERG: electroretinogram; GAPDH: glyceraldehyde-3-phosphate dehydrogenase; GCL: ganglion cell layer; INL: inner nuclear layer; IPL: inner plexiform layer; IS/OS: inner and outer photoreceptor segments; LAMP1: lysosomal-associated membrane protein 1; MAP1LC3B/LC3: microtubule-associated protein 1 light chain 3 beta; MTOR: mechanistic target of rapamycin kinase; OCT: optical coherence tomography; ONL: outer nuclear layer; OPs: oscillatory potentials; p-EIF4EBP1: phosphorylated eukaryotic translation initiation factor 4E binding protein 1; p-MAPK14/p38: phosphorylated mitogen-activated protein kinase 14; RPE: retinal pigment epithelium; RPS6KB/p70 S6 kinase: ribosomal protein S6 kinase; SQSTM1/p62: sequestosome 1; TP53/TRP53/p53: tumor related protein 53; TSC2: TSC complex subunit 2; WT: wild type.