LAMP2 Expression Research Articles

The autophagic protein p62 is a target of reactive aldehydes in human and murine cholestatic liver disease.

Inflammatory cholestatic liver diseases, including Primary Sclerosing Cholangitis (PSC), are characterized by periportal inflammation with progression to cirrhosis. The objective of this study was to examine interactions between oxidative stress and autophagy in cholestasis. Using hepatic tissue from male acute cholestatic (bile duct ligated) as well as chronic cholestatic (Mdr2KO) mice, localization of oxidative stress, the antioxidant response and induction of autophagy were analyzed and compared to human PSC liver. Concurrently, the ability of reactive aldehydes to post-translationally modify the autophagosome marker p62 was assessed in PSC liver tissue and in cell culture. Expression of autophagy markers was upregulated in human and mouse cholestatic liver. Whereas mRNA expression of Atg12, Lamp1, Sqstm1 and Map1lc3 was increased in acute cholestasis in mice, it was either suppressed or not significantly changed in chronic cholestasis. In human and murine cholestasis, periportal hepatocytes showed increased IHC staining of ubiquitin, 4-HNE, p62, and selected antioxidant proteins. Increased p62 staining colocalized with accumulation of 4-HNE-modified proteins in periportal parenchymal cells as well as with periportal macrophages in both human and mouse liver. Mechanistically, p62 was identified as a direct target of lipid aldehyde adduction in PSC hepatic tissue and in vitro cell culture. In vitro LS-MS/MS analysis of 4-HNE treated recombinant p62 identified carbonylation of His123, Cys128, His174, His181, Lys238, Cys290, His340, Lys341 and His385. These data indicate that dysregulation of autophagy and oxidative stress/protein damage are present in the same periportal hepatocyte compartment of both human and murine cholestasis. Thus, our results suggest that both increased expression as well as ineffective autophagic degradation of oxidatively-modified proteins contributes to injury in periportal parenchymal cells and that direct modification of p62 by reactive aldehydes may contribute to autophagic dysfunction.

Zexie Decoction regulates Akt/TFEB signaling pathway to promote lipophagy in hepatocytes

Taking lipophagy as the breakthrough point, we explored the mechanism of Zexie Decoction(ZXD) in improving lipid metabolism in the hepatocyte model induced by palmitic acid(PA) and in the animal model induced by high-fat diet(HFD) on the basis of protein kinase B(Akt)/transcription factor EB(TFEB) signaling pathway. Co-localization was carried out for the microtubule-associated protein light chain 3(LC3) plasmid labeled with green fluorescent protein(GFP) and lipid droplets(LDs), and immunofluorescence co-localization for liver LC3 of HFD mice and perilipin 2(PLIN2). The results showed that ZXD up-regulated the expression of LC3, reduced lipid accumulation in hepatocytes, and increased the co-localization of LC3 and LDs, thereby activating lipo-phagy. Western blot results confirmed that ZXD increased autophagy-related protein LC3Ⅱ/LC3Ⅰ transformation ratio and lysosome-associated membrane protein 2(LAMP2) in vivo and in vitro and promoted the degradation of sequestosome-1(SQSTM1/p62)(P<0.05). The results above jointly explained that ZXD regulated lipophagy. Furthermore, ZXD activated TFEB expression(P<0.05) and reversed the PA-and HFD-induced decrease of TFEB nuclear localization in hepatocytes(P<0.05). Meanwhile, ZXD activated liver TFEB to up-regulate the expression of the targets Lamp2, Lc3 B, Bcl2, and Atg5(P<0.05). Additionally, ZXD down-regulated the protein level of p-Akt upstream of TFEB in vivo and in vitro. In conclusion, ZXD may promote lipophagy by regulating the Akt/TFEB pathway.

PMON12 Effects of Endocrine Disruptors on Autophagy Markers Gene Expression in Immortalized GnRH Neurons and Hypothalami of Adult Male Mice

Abstract Benzophenones (BP) and Bisphenol A (BPA) are endocrine disrupting chemicals (EDC). Previously, we showed that the in-vitro exposure to EDC inhibited Kiss-induced gene expression in GT1-7 and GN11 cells (donated by Pamela Mellon, UCSD), and altered expression of inflammatory markers in GT1-7 cells and hypothalami of adult male mice.In the present study, we evaluated the effects of the in-vivo exposure to BP2 and BP3 (250 µg/kg/day, orally, for 5 days) on the expression of autophagy markers in hypothalamic nuclei in C57Bl/6 adult male mice, and the in-vitro exposure to BPA, BP2 or BP3 in immortalized GnRH neurons. Animals were sacrificed, brains rapidly dissected and conserved -80°C until processed. Micropunches containing Anteroventral Periventricular (AVPV) or Arcuate (ARC) nucleus were obtained. Effects of the in-vitro exposure for 12 or 24 hours to BPA, BP2 or BP3 (1×10-9M, Sigma), or DMSO as control, were evaluated in GT1-7 and GN11 cell lines, mature and immature GnRH neurons. ARN was obtained using Tri-Reagent (Molecular Research Center, Inc), reverse-transcribed and gene expression analyzed by qPCR, using Ppib as control gene. Results were presented as Media±SE and analyzed by ANOVA (Statistica, StatSoft Inc, USA).In-vivo, five-day exposure to BP2 or BP3 did not alter Lamp2 or p62 gene expression in the AVPV or ARC in adult male mice (ANOVA: ns). In GT1-7 cells, Ulk1 was not significantly altered by any of the stimuli (ANOVA: ns; n=5), whereas in GN11 cells, BP3 significantly increased Ulk1 gene expression after 12 and 24 h exposure [DMSO-24h: 0.93±0.21, DMSO-12h: 0.73± 0.19, BPA-24h: 1.78±0.31, BPA-12h: 1.47±0.33, BP2-24h: 1.51±0.49, BP2-12h: 1.06±0.23, BP3-24h: 2.41±0.89, BP3-12h: 2.45±0.82, Repeated Measures ANOVA: Main effect "TREATMENT" p<0.05, BP3 different from DMSO, p<0.05, n=4]. Our results reinforce the notion that the exposure to EDC have different effects depending on the experimental model and time of exposure, with developing cells being more susceptible to disruption that mature cells. We need more experiments to fully understand if exposure to EDC alters other markers of the autophagy process in mouse hypothalamus and GnRH neurons.Funding: CONICET, ANPCYT, International Society for Neurochemistry, Fund. Williams, Fund. R. Barón. Presentation: Monday, June 13, 2022 12:30 p.m. - 2:30 p.m.

Calycosin Ameliorates Bleomycin-Induced Pulmonary Fibrosis via Suppressing Oxidative Stress, Apoptosis, and Enhancing Autophagy.

Calycosin (CA) is a flavonoid extracted from the root of Astragalus membranaceus and has antioxidant, anti-inflammation, and antiapoptosis properties. The objective of this study was to investigate the efficacy of CA in protecting against pulmonary fibrosis. CA (14 mg/kg) and SB216763 (20 mg/kg) were administrated to bleomycin-induced pulmonary fibrosis mice for 3 weeks. The results concluded that CA alleviated the inflammation and collagen deposition in pulmonary fibrosis. In addition, CA reduced MDA level, enhanced SOD and TAC activities, and increased the activity of the Nrf2/HO-1 pathway. CA also regulated the expressions of apoptosis-related proteins. Moreover, CA enhanced autophagy via upregulating LC3, beclin1, PINK1, and reducing p62. CA also increased expression of LAMP1 and TFEB, and inhibited the release of lysosome enzymes from ruptured lysosomes. These results provide new evidence that CA protects against pulmonary fibrosis through inhibiting oxidative stress and apoptosis. In addition, autophagy abnormality and lysosome dysfunction are restored by CA.

Imatinib Mesylate Induces Necroptotic Cell Death and Impairs Autophagic Flux in Human Cardiac Progenitor Cells.

The receptor tyrosine kinase inhibitor imatinib improves patient cancer survival but is linked to cardiotoxicity. This study investigated imatinib’s effects on cell viability, apoptosis, autophagy, and necroptosis in human cardiac progenitor cells in vitro. Imatinib reduced cell viability (75.9 ± 2.7% vs. 100.0 ± 0.0%) at concentrations comparable to peak plasma levels (10 µM). Imatinib reduced cells’ TMRM fluorescence (74.6 ± 6.5% vs. 100.0 ± 0.0%), consistent with mitochondrial depolarisation. Imatinib increased lysosome and autophagosome content as indicated by LAMP2 expression (2.4 ± 0.3-fold) and acridine orange fluorescence (46.0 ± 5.4% vs. 9.0 ± 3.0), respectively. Although imatinib increased expression of autophagy-associated proteins and also impaired autophagic flux, shown by proximity ligation assay staining for LAMP2 and LC3II (autophagosome marker): 48 h of imatinib treatment reduced visible puncta to 2.7 ± 0.7/cell from 11.3 ± 2.1 puncta/cell in the control. Cell viability was partially recovered by autophagosome inhibition by wortmannin, with the viability increasing 91.8 ± 8.2% after imatinib-wortmannin co-treatment (84 ± 1.5% after imatinib). Imatinib-induced necroptosis was associated with an 8.5 ± 2.5-fold increase in mixed lineage kinase domain-like pseudokinase activation. Imatinib-induced toxicity was rescued by RIP1 inhibition: 88.6 ± 3.0% vs. 100.0 ± 0.0% in the control. Imatinib applied to human cardiac progenitor cells depolarises mitochondria and induces cell death through necroptosis, recoverable by RIP1 inhibition, with a partial role for autophagy.

Effects of neuron autophagy induced by arsenic and fluoride on spatial learning and memory in offspring rats

There are numerous studies showing that exposure to arsenic (As) or fluoride (F) damages the nervous system, but there is no literature investigating the effects of combined As and F exposure to induce autophagy on neurotoxicity in the offspring. In this study, we developed a rat model of As and/or F exposure through drinking water from before pregnancy to 90 days postnatal. The offspring rats were randomly divided into nine groups. Sodium arsenite (NaAsO2) (0, 35, 70 mg/L) and Sodium fluoride (NaF) (0, 50, 100 mg/L) were designed according to 3 × 3 factorial design. Our results suggested that the presence of F might antagonize the excretion of total As in urine, and As–F co-exposure led to severe pathological damage in brain tissue and reduced spatial learning and memory ability. At the same time, the experiments showed that As and F increased Beclin1 expression and LC3B ratio to activate autophagy; both P62 and Lamp2 expression were increased, suggesting that autophagy lysosomal degradation was blocked; SYN and JIP1 expression were significantly decreased, disrupting synaptic structure and function. Axonal autophagosome reverse transport regulation might be affected by combined As–F exposure, exacerbating neuronal synaptic damage and inducing neurotoxicity. Further analysis showed that there was an interaction between As and F exposure-induced changes in autolysosome-related proteins in the hippocampus, which showed antagonism, and the antagonism of the high As combined exposure groups were stronger than that of the low As combined exposure groups. In conclusion, our study showed that combined As and F exposure might induce reverse transport impairment of autophagy on axons, leading to autophagy defects, which in turn led to disruption of synaptic morphology and function, induced neurotoxicity, and there was an interaction between As and F, the type of its combined effect was antagonism.

Lomitapide ameliorates middle cerebral artery occlusion-induced cerebral ischemia/reperfusion injury by promoting neuronal autophagy and inhibiting microglial migration.

Stroke has a high incidence and is a disabling condition that can lead to severe cognitive, motor, and sensory dysfunction. In this study, we employed a drug repurposing strategy to investigate the neuroprotective effect of lomitapide on focal ischemic brain injury and explore its potential mechanism of action. Experimental cerebral ischemia was induced by middle cerebral artery occlusion (MCAO) in adult male C57BL/6 mice and simulated by oxygen-glucose deprivation in N2a-BV2 cells in co-cultivation. Lomitapide significantly increased the survival rate, reduced the neuronal tissue loss, and improved the neurological function after MCAO. Furthermore, lomitapide could increase the expression of LC3-II, reduce the expression of P62 and LAMP2, promote autophagic flux, and inhibit apoptosis by increasing and inhibiting the expression of the apoptosis-associated proteins Bcl-2 and Bax, respectively. In addition, lomitapide inhibited the migration of pro-inflammatory microglia. Lomitapide is a lipid-lowering drug, and this is the first study to explore its protective effect on ischemic nerve injury in vitro and in vivo. Our results suggest that lomitapide can be repositioned as a potential therapeutic drug for the treatment of stroke.

Mechanism of Atractylodes macrocephala against Alzheimer's disease via regulating lysophagy based on LKB1-AMPK-TFEB pathway

Myloid beta(Aβ) is produced by cleavage of amyloid precursor protein(APP), which is a main reason for Alzheimer's disease(AD) occurrence and development. This study preliminarily investigated the mechanism of Atractylodes macrocephala(AM) against AD based on LKB1-AMPK-TFEB pathway. The effect of AM on memory ability of AD transgenic Caenorhabditis elegans CL2241 was detected, and then the APP plasmid was transiently transferred to mouse neuroblastoma(N2 a) cells in vitro. The mice were divided into the blank control group, APP group(model group), positive control group(100 μmol·L~(-1) rapamycin), and AM low-, medium-and high-dose groups(100, 200 and 300 μg·mL~(-1)). The content of Aβ_(1-42) in cell medium, the protein level of APP, the fluorescence intensity of APP, the transcriptional activity of transcription factor EB(TFEB), the activity of lysosomes in autophagy, and autophagy flux were determined by enzyme-linked immunosorbent assay(ELISA), Western blot, fluorescence microscope, luciferase reporter gene assay, RLuc-LC3 wt/RLuc-LC3 G120 A, and mRFP-GFP-LC3, respectively. The protein expression of TFEB, LC3Ⅱ, LC3Ⅰ, LAMP2, Beclin1, LKB1, p-AMPK and p-ACC was detected by Western blot. Immunofluorescence and reverse transcription-polymerase chain reaction(RT-PCR) were used to detect the fluorescence intensity of TFEB and the mRNA expression of TFEB and downstream target genes, respectively. The results showed that AM reduced the chemotactic index of transgenic C. elegans CL2241, and decreased the content of Aβ in the supernatant of cell culture medium at different concentrations. In addition, AM lowered the protein level of APP and the fluorescence intensity of APP in a dose-dependent manner. Transcriptional activity of TFEB and fluorescence intensity of mRFP-GFP-LC3 plasmid were enhanced after AM treatment, and the value of RLuc-LC3 wt/RLuc-LC3 G120 A was reduced. AM promoted the protein levels of TFEB, LAMP2 and Beclin1 at different concentrations, and increased the protein expression ratio of LC3Ⅱ/LC3Ⅰ in a dose-dependent manner. Immunofluorescence results revealed that AM improved the fluorescence intensity and nuclear expression of TFEB, and RT-PCR results indicated that AM of various concentrations elevated the mRNA expression of TFEB in APP transfected N2 a cells and promoted the transcription level of LAMP2 in a dose-dependent manner, and high-concentration AM also increased the mRNA levels of LC3 and P62. The protein levels of LKB1, p-AMPK and p-ACC were elevated by AM of different concentrations. In summary, AM regulating lysophagy and degrading APP are related to the activation of LKB1-AMPK-TFEB pathway.

Ral GTPases are critical regulators of spinal cord myelination and homeostasis.

Efficient myelination supports nerve conduction and axonal health throughout life. In the central nervous system, oligodendrocytes (OLs) carry out this demanding anabolic duty in part through biosynthetic pathways controlled by mTOR. We identify Ral GTPases as critical regulators of mouse spinal cord myelination and myelin maintenance. Ablation of Ral GTPases (RalA, RalB) in OL-lineage cells impairs timely onset and radial growth of developmental myelination, accompanied by increased endosomal/lysosomal abundance. Further examinations, including transcriptomic analyses of Ral-deficient OLs, were consistent with mTORC1-related deficits. However, deletion of the mTOR signaling-repressor Pten in Ral-deficient OL-lineage cells is unable to rescue mTORC1 activation or developmental myelination deficiencies. Induced deletion of Ral GTPases in OLs of adult mice results in late-onset myelination defects and tissue degeneration. Together, our data indicate critical roles for Ral GTPases to promote developmental spinal cord myelination, to ensure accurate mTORC1 signaling, and to protect the healthy state of myelin-axon units over time.

The Degradation of TMEM166 by Autophagy Promotes AMPK Activation to Protect SH-SY5Y Cells Exposed to MPP.

Neuronal oxidative stress caused by mitochondrial dysfunction plays a crucial role in the development of Parkinson’s disease (PD). Growing evidence shows that autophagy confers neuroprotection in oxidative-stress-associated PD. This work aims to investigate the involvement of TMEM166, an endoplasmic-reticulum-localized autophagy-regulating protein, in the process of PD-associated oxidative stress through the classic cellular PD model of neuroblastoma SH-SY5Y cells exposed to 1-methyl-4-phenylpyridinium (MPP+). Reactive oxygen species (ROS) production and mitochondrial membrane potential were checked to assess the oxidative stress induced by MPP+ and the cellular ATP generated was determined to evaluate mitochondrial function. The effect on autophagy induction was evaluated by analyzing p62 and LC3-II/I expression and by observing the LC3 puncta and the colocalization of LC3 with LAMP1/ LAMP2. The colocalization of mitochondria with LC3, the colocalization of Tom20 with LAMP1 and Tom20 expression were analyzed to evaluate mitophagy. We found that TMEM166 is up-regulated in transcript levels, but up-regulated first and then down-regulated by autophagic degradation in protein levels upon MPP+-treatment. Overexpression of TMEM166 induces mitochondria fragmentation and dysfunction and exacerbates MPP+-induced oxidative stress and cell viability reduction. Overexpression of TMEM166 is sufficient to induce autophagy and mitophagy and promotes autophagy and mitophagy under MPP+ treatment, while knockdown of TMEM166 inhibits basal autophagic degradation. In addition, overexpressed TMEM166 suppresses AMPK activation, while TMEM166 knockdown enhances AMPK activation. Pharmacological activation of AMPK alleviates the exacerbation of oxidative stress induced by TMEM166 overexpression and increases cell viability, while pharmacological inhibition mitophagy aggravates the oxidative stress induced by MPP+ treatment combined with TMEM166 overexpression. Finally, we find that overexpressed TMEM166 partially localizes to mitochondria and, simultaneously, the active AMPK in mitochondria is decreased. Collectively, these findings suggest that TMEM166 can translocate from ER to mitochondria and inhibit AMPK activation and, in response to mitochondrial oxidative stress, neuronal cells choose to up-regulate TMEM166 to promote autophagy/mitophagy; then, the enhancing autophagy/mitophagy degrades the TMEM166 to activate AMPK, by the two means to maintain cell survival. The continuous synthesis and degradation of TMEM166 in autophagy/mitochondria flux suggest that TMEM166 may act as an autophagy/mitochondria adaptor.

Modulating Chaperone-Mediated Autophagy and Its Clinical Applications in Cancer.

Autophagy is a central mechanism for maintaining cellular homeostasis in health and disease as it provides the critical energy through the breakdown and recycling of cellular components and molecules within lysosomes. One of the three types of autophagy is chaperone-mediated autophagy (CMA), a degradation pathway selective for soluble cytosolic proteins that contain a targeting motif related to KFERQ in their amino acid sequence. This motif marks them as CMA substrate and is, in the initial step of CMA, recognised by the heat shock protein 70 (Hsc70). The protein complex is then targeted to the lysosomal membrane where the interaction with the splice variant A of the lysosomal-associated membrane protein-2 (LAMP-2A) results in its unfolding and translocation into the lysosome for degradation. Altered levels of CMA have been reported in a wide range of pathologies including many cancer types that upregulate CMA as part of the pro-tumorigenic phenotype, while in aging a decline is observed and associated with a decrease of LAMP-2 expression. The potential of altering CMA to modify a physiological or pathological process has been firmly established through genetic manipulation in animals and chemical interference with this pathway. However, its use for therapeutic purposes has remained limited. Compounds used to target and modify CMA have been applied successfully to gain a better understanding of its cellular mechanisms, but they are mostly not specific, also influence other autophagic pathways and are associated with high levels of toxicity. Here, we will focus on the molecular mechanisms involved in CMA regulation as well as on potential ways to intersect them, describe modulators successfully used, their mechanism of action and therapeutic potential. Furthermore, we will discuss the potential benefits and drawbacks of CMA modulation in diseases such as cancer.

Glyphosate exposure deteriorates oocyte meiotic maturation via induction of organelle dysfunctions in pigs

BackgroundRecently, defects in mammalian oocytes maturation induced by environmental pollution results in the decreasing animal reproduction. Animal exposed to glyphosate is largely unavoidable because glyphosate is one of the most widely used herbicide worldwide due to its high-efficiency and broad-spectrum effects, which causes glyphosate an environmental contaminant found in soil, water and food. During the last few years, the growing and wider use of glyphosate has raised great concerns about its effects of reproductive toxicity. In this study, using porcine models, we investigated effects of glyphosate on organelle functions during oocyte meiosis.ResultsThe results showed glyphosate exposure disrupted porcine oocyte maturation. Expression levels of cumulus expansion-related genes were interfered, further indicating the meiotic defects. The damaging effects were mediated by destruction of mitochondrial distribution and functions, which induced ROS accumulation and oxidative stress, also indicated by the decreased mRNA expression of related antioxidant enzyme genes. We also found an interference of endoplasmic reticulum (ER) distribution, disturbance of Ca2+ homeostasis, as well as fluctuation of ER stress, showing with the reduced ER stress-related mRNA or protein expression, which could indicate the dysfunction of ER for protein processing and signal transduction in glyphosate-exposed oocytes. Moreover, glyphosate exposure induced the disruption of lysosome function for autophagy, showing with the decrease of LAMP2 expression and autophagy-related genes mRNA expression. Additionally, our data showed the distribution of Golgi apparatus and the functions of ribosome were disturbed after glyphosate exposure, which might affect protein synthesis and transport.ConclusionsCollectively, our study showed that exposed to glyphosate could affect animal reproduction by compromising the quality of oocytes through its wide toxic effects on organelle functions.

Phenotypic and genetic spectrum of ATP6V1A encephalopathy: a disorder of lysosomal homeostasis.

Vacuolar-type H+-ATPase (V-ATPase) is a multimeric complex present in a variety of cellular membranes that acts as an ATP-dependent proton pump and plays a key role in pH homeostasis and intracellular signalling pathways. In humans, 22 autosomal genes encode for a redundant set of subunits allowing the composition of diverse V-ATPase complexes with specific properties and expression. Sixteen subunits have been linked to human disease. Here we describe 26 patients harbouring 20 distinct pathogenic de novo missense ATP6V1A variants, mainly clustering within the ATP synthase α/β family-nucleotide-binding domain. At a mean age of 7 years (extremes: 6 weeks, youngest deceased patient to 22 years, oldest patient) clinical pictures included early lethal encephalopathies with rapidly progressive massive brain atrophy, severe developmental epileptic encephalopathies and static intellectual disability with epilepsy. The first clinical manifestation was early hypotonia, in 70%; 81% developed epilepsy, manifested as developmental epileptic encephalopathies in 58% of the cohort and with infantile spasms in 62%; 63% of developmental epileptic encephalopathies failed to achieve any developmental, communicative or motor skills. Less severe outcomes were observed in 23% of patients who, at a mean age of 10 years and 6 months, exhibited moderate intellectual disability, with independent walking and variable epilepsy. None of the patients developed communicative language. Microcephaly (38%) and amelogenesis imperfecta/enamel dysplasia (42%) were additional clinical features. Brain MRI demonstrated hypomyelination and generalized atrophy in 68%. Atrophy was progressive in all eight individuals undergoing repeated MRIs. Fibroblasts of two patients with developmental epileptic encephalopathies showed decreased LAMP1 expression, Lysotracker staining and increased organelle pH, consistent with lysosomal impairment and loss of V-ATPase function. Fibroblasts of two patients with milder disease, exhibited a different phenotype with increased Lysotracker staining, decreased organelle pH and no significant modification in LAMP1 expression. Quantification of substrates for lysosomal enzymes in cellular extracts from four patients revealed discrete accumulation. Transmission electron microscopy of fibroblasts of four patients with variable severity and of induced pluripotent stem cell-derived neurons from two patients with developmental epileptic encephalopathies showed electron-dense inclusions, lipid droplets, osmiophilic material and lamellated membrane structures resembling phospholipids. Quantitative assessment in induced pluripotent stem cell-derived neurons identified significantly smaller lysosomes. ATP6V1A-related encephalopathy represents a new paradigm among lysosomal disorders. It results from a dysfunctional endo-lysosomal membrane protein causing altered pH homeostasis. Its pathophysiology implies intracellular accumulation of substrates whose composition remains unclear, and a combination of developmental brain abnormalities and neurodegenerative changes established during prenatal and early postanal development, whose severity is variably determined by specific pathogenic variants.

A nonstructural protein encoded by a rice reovirus induces an incomplete autophagy to promote viral spread in insect vectors.

Viruses can hijack autophagosomes as the nonlytic release vehicles in cultured host cells. However, how autophagosome-mediated viral spread occurs in infected host tissues or organs in vivo remains poorly understood. Here, we report that an important rice reovirus, rice gall dwarf virus (RGDV) hijacks autophagosomes to traverse multiple insect membrane barriers in the midgut and salivary gland of leafhopper vector to enhance viral spread. Such virus-containing double-membraned autophagosomes are prevented from degradation, resulting in increased viral propagation. Mechanistically, viral nonstructural protein Pns11 induces autophagy and embeds itself in the autophagosome membranes. The autophagy-related protein 5 (ATG5)-ATG12 conjugation is essential for initial autophagosome membrane biogenesis. RGDV Pns11 specifically interacts with ATG5, both in vitro and in vivo. Silencing of ATG5 or Pns11 expression suppresses ATG8 lipidation, autophagosome formation, and efficient viral propagation. Thus, Pns11 could directly recruit ATG5-ATG12 conjugation to induce the formation of autophagosomes, facilitating viral spread within the insect bodies. Furthermore, Pns11 potentially blocks autophagosome degradation by directly targeting and mediating the reduced expression of N-glycosylated Lamp1 on lysosomal membranes. Taken together, these results highlight how RGDV remodels autophagosomes to benefit viral propagation in its insect vector.

MP20-02 PROTECTIVE ROLE OF METFORMIN IN ACUTE RENAL ISCHEMIC REPERFUSION INJURY

You have accessJournal of UrologyCME1 May 2022MP20-02 PROTECTIVE ROLE OF METFORMIN IN ACUTE RENAL ISCHEMIC REPERFUSION INJURY Junrong Zou, Xiaofeng Zou, Guoxi Zhang, Kecheng Lou, Shangzhi Feng, Guancheng Xiao, and Hailan He Junrong ZouJunrong Zou More articles by this author , Xiaofeng ZouXiaofeng Zou More articles by this author , Guoxi ZhangGuoxi Zhang More articles by this author , Kecheng LouKecheng Lou More articles by this author , Shangzhi FengShangzhi Feng More articles by this author , Guancheng XiaoGuancheng Xiao More articles by this author , and Hailan HeHailan He More articles by this author View All Author Informationhttps://doi.org/10.1097/JU.0000000000002553.02AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookLinked InTwitterEmail Abstract INTRODUCTION AND OBJECTIVE: To figure out the role of metformin in acute renal ischemic reperfusion (IR) injury. METHODS: HK-2 cell was treated with, PBS, metformin, 3MA and rapamycin. Western-blot was used to observe cell autophagy and apoptosis level. 15 C57 mice were divided randomly into Control group, IR group, IR+ 3-MA group, IR+ rapamycin group, IR+ metformin group, with 3 mice in each group. In this research, the IR mouse model was established through clamping the bilateral renal artery and vein for 20min. The above mice were treated with PBS, metformin (200mg/kg), 3-MA (30mg/kg) and rapamycin (1mg/kg) by intraperitoneal injection respectively. All the mice were sacrificed after anesthesia 24h later, serum, the kidney was obtained for sequent experiments. The kidney was divided into two parts, one of which was fixed by 2.5% glutaraldehyde for autophagosome and apoptosis body observation under electron microscope, the other part was fixed by 10% formalin for the later histological experiments, including IHC, TUNEL staining and H&E.This study was approved by the Hospital Ethics Committee. RESULTS: Metformin inhibits autophagy and apoptosis level in HK-2 cell. In the IR model, metformin showed a more effective protective role compared to 3-MA and control group. After metformin treatment, IHC staining showed less LC3B and cleaved-caspase3 expression, and LAMP2 (lysosome) expression was inhibited. Metformin decreased serum creatinine and β2-microglobulin. TUNEL staining showed apoptosis level decreased after metformin treatment. We also observed decreased autophagosome and apoptosis body in metformin group through electron microscope scan. In addition, H&E staining showed kidney necrosis was inhibited after metformin treatment. CONCLUSIONS: Altogether, in this research, we established IR mouse model of kidney stone. We firstly found metformin inhibits autophagy and apoptosis in renal cell, which indicated metformin plays a protective role of metformin in acute renal ischemic reperfusion injury. Source of Funding: National Natural Science Foundation of China (81860456, 81760462) © 2022 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 207Issue Supplement 5May 2022Page: e317 Advertisement Copyright & Permissions© 2022 by American Urological Association Education and Research, Inc.MetricsAuthor Information Junrong Zou More articles by this author Xiaofeng Zou More articles by this author Guoxi Zhang More articles by this author Kecheng Lou More articles by this author Shangzhi Feng More articles by this author Guancheng Xiao More articles by this author Hailan He More articles by this author Expand All Advertisement PDF DownloadLoading ...

Chaperone-mediated autophagy plays an important role in regulating retinal progenitor cell homeostasis

PurposeTo explore the function and regulatory mechanism of IFITM3 in mouse neural retinal progenitor cells (mNRPCs), which was found to be very important not only in the development of the retina in embryos but also in NRPCs after birth.MethodsPublished single-cell sequencing data were used to analyze IFITM3 expression in mNRPCs. RNA interference was used to knock down the expression of IFITM3. CCK-8 assays were used to analyze cell viability. RNA-seq was used to assess mRNA expression, as confirmed by real-time quantitative PCR, and immunofluorescence assays and western blots were used to validate the levels of relative proteins, and autophagy flux assay. Lysosomal trackers were used to track the organelle changes.ResultsThe results of single-cell sequencing data showed that IFITM3 is highly expressed in the embryo, and after birth, RNA-seq showed high IFITM3 expression in mNRPCs. Proliferation and cell viability were greatly reduced after IFITM3 was knocked down. The cell membrane system and lysosomes were dramatically changed, and lysosomes were activated and evidently agglomerated in RAMP-treated cells. The expression of LAMP1 was significantly increased with lysosome agglomeration after treatment with rapamycin (RAMP). Further detection showed that SQSTM1/P62, HSC70 and LAMP-2A were upregulated, while no significant difference in LC3A/B expression was observed; no autophagic flux was generated.ConclusionIFITM3 regulates mNRPC viability and proliferation mainly through chaperone-mediated autophagy (CMA) but not macroautophagy (MA). IFITM3 plays a significant role in maintaining the homeostasis of progenitor cell self-renewal by sustaining low-level activation of CMA to eliminate deleterious factors in cells.

Increased LAMP1 Expression Enhances SARS-CoV-1 and SARS-CoV-2 Production in Vero-Derived Transgenic Cell Lines

Coronaviridae is a family of single-stranded RNA (ssRNA) viruses that can cause diseases with high mortality rates. SARS-CoV-1 and MERS-CoV appeared in 2002-2003 and 2012, respectively. A novel coronavirus, SARS-CoV-2, emerged in 2019 in Wuhan (China) and has caused more than 5 million deaths in worldwide. The entry of SARS-CoV-1 into the cell is due to the interaction of the viral spike (S) protein and the cell protein, angiotensin-converting enzyme 2 (ACE2). After infection, virus assembly occurs in Golgi apparatus-derived vesicles during exocytosis. One of the possible participants in this process is LAMP1 protein. We established transgenic Vero cell lines with increased expression of human LAMP1 gene and evaluated SARS-CoV-1 and SARS-CoV-2 production. An increase in the production of both viruses in LAMP1-expressing cells when compared with Vero cells was observed, especially in the presence of trypsin during infection. From these results it can be assumed that LAMP1 promotes SARS-CoV-1 and SARS-CoV-2 production due to enhanced exocytosis.

Autophagy Contributes to the Rapamycin-Induced Improvement of Otitis Media.

Otitis media (OM) is a pervasive disease that involves hearing loss and severe complications. In our previous study, we successfully established a mouse model of human OM using Tlr2tm1Kir (TLR2–/–) mice with middle ear (ME) inoculation of streptococcal peptidoglycan-polysaccharide (PGPS). In this study, we found that hearing loss and OM infections in OM mice were significantly alleviated after treatment with rapamycin (RPM), a widely used mechanistic target of RPM complex 1 (mTORC1) inhibitor and autophagy inducer. First of all, we tested the activity of mTORC1 by evaluating p-S6, Raptor, and mTOR protein expression. The data suggested that the protein expression level of p-S6, Raptor and mTOR are decreased in TLR2–/– mice after the injection of PGPS. Furthermore, our data showed that both the autophagosome protein LC3-II, Beclin-1, ATG7, and autophagy substrate protein p62 accumulated at higher levels in mice with OM than in OM-negative mice. The expression of lysosomal-associated proteins LAMP1, Cathepsin B, and Cathepsin D increased in the OM mice compared with OM-negative mice. Rab7 and Syntaxin 17, which is necessary for the fusion of autophagosomes with lysosomes, are reduced in the OM mice. In addition, data also described that the protein expression level of p-S6, mTOR and Raptor are lower than PGPS group after RPM treatment. The accumulation of LC3-II, Beclin-1, and ATG7 are decreased, and the expression of Rab7 and Syntaxin 17 are increased significantly after RPM treatment. Our results suggest that autophagy impairment is involved in PGPS-induced OM and that RPM improves OM at least partly by relieving autophagy impairment. Modulating autophagic activity by RPM may be a possible effective treatment strategy for OM.

Ivermectin induces mitophagy in H9c2 cells via activation of the PINK1/Parkin pathway

Abstract Ivermectin (IVM), a remarkable broad-spectrum anthelmintic and insecticides, which also contributes to clinical application. However, IVM induction of cytotoxicity through mitophagy has not been consistently proven in vitro. Here we investigate the cytotoxic effects of ivermectin in mammalian nontarget cells. We have used cck-8 assay to evaluate the cell viability. The Fluorescence labeling was detected the occurrence of autophagy and mitophagy. The ATP bioluminescence detection kit was used to detect changes in intracellular ATP levels. In addition, the western blot assay was applied to reflect the expression of autophagy-related and mitophagy- related proteins. Cellular calcium concentration analysis was captured with a flow cytometer. We also used western blot to reveal expression of the lysosomal membrane protein Lamp2. The expression of lysosomal cathepsin mRNA was evaluated by RT-PCR. The cell viability was significantly diminished in H9c2 cells and the number of autophagosomes increases in a dose-dependent way, at the same time, LC3-II / I ratio was increased. We also found that the H9c2 cellular ATP level was decreased and observed the mPTP opening. The co-localization of mitochondria and lysosomes was detected, also, we found the concentration of Ca2+, the expression of Lamp 2 and mRNA expression levels of Cathepsin B and Cathepsin L significantly increased in a dose-dependent way in H9c2 cells. Finally, the results of expression of PINK1 and Parkin protein increased simultaneously in H9c2 cells. IVM induced mitophagy in H9c2 cells via PINK1/Parkin signaling.

Determining Activation Status of Natural Killer Cells Following Stimulation via Cytokines and Surface Receptors.

Stimulation of Natural Killer (NK) cells with cytokines, target cell interaction, or antibody mediatedactivation of receptors on the NK cell surface enables the dissection of specific signaling intermediates in different activation pathways. NK cell activation status is commonly measured by production of interferon gamma (IFNγ) and expression of the degranulation marker LAMP-1 (CD107a). Cytotoxic potency can also be assessed by the production of perforin, granzymes, and tumor necrosis factor alpha (TNFα). NK cell receptor mediated activation by antibodies requires crosslinking of the receptor-specific antibodies; thus, in vitro activation assays are performed by binding antibodies to cell culture plates. All parameters can be measured by flow cytometry.