Formation Of Lysosomes Research Articles

Irisin suppresses PDGF-BB-induced proliferation of vascular smooth muscle cells in vitro by activating AMPK/mTOR-mediated autophagy.

Restenosis is a pivotal factor that restricts the efficacy of coronary artery bypass grafting. Inhibition of vascular smooth muscle cells (VSMCs) proliferation can improve intimal hyperplasia and lumen stenosis. Irisin, a polypeptide secreted by muscle cells, has been demonstrated to have a protective role in various cardiovascular diseases. However, the effect and mechanism of irisin on VSMCs proliferation and phenotype switching remain unclear. Cell proliferation ability was assessed using the methylthiazolyldiphenyl-tetrazolium bromide (MTT) assay and 5-ethynyl-2'-deoxyuridine (EdU) incorporation. Cell cycle analysis was performed using flow cytometry, while expression levels of contractile and synthesis-related proteins were determined through RT-qPCR and Western blot. The VSMCs were infected with an adenovirus carrying GFP-LC3, and the proportion of cells showing positive expression was assessed. Additionally, the formation of autophagic lysosomes in cells was observed through transmission electron microscopy. In this study, we have demonstrated the inhibitory effects of irisin on the proliferation and phenotypic transition of platelet-derived growth factor-BB (PDGF-BB)-induced VSMCs. More importantly, we have discovered that irisin can activate the AMP-activated protein kinase/mammalian target of rapamycin (AMPK/mTOR) signaling pathway to mediate autophagy in PDGF-BB-induced VSMCs. The inhibitory effect of irisin on PDGF-BB-induced VSMCs proliferation was significantly attenuated by the AMPK inhibitor, Compound C. Conversely the mTOR inhibitor, rapamycin further enhanced the inhibitory effect of irisin on PDGF-BB induced VSMCs proliferation. In conclusion, our findings suggest that irisin effectively suppresses the aberrant proliferation of VSMCs following PDGF-BB stimulation by modulating autophagy levels through the AMPK/mTOR signaling pathway.

Immune System Responses against Mycobacterium tuberculosis and its Mechanisms to Escape from the Immune System

Background: Mycobacterium tuberculosis (MTB) employs a variety of strategies to evade the host immune response, enabling its persistence and the development of tuberculosis. These evasion tactics involve thwarting lysosome formation, manipulating intracellular pH, and disrupting apoptosis and autophagy processes within host cells. Specifically, MTB interferes with lysosome acidification by modulating calcium ions (Ca2+), iron ions, and hydrogen ions (H+ ), creating an optimal environment for its survival within host cells. Furthermore, MTB inhibits host cell apoptosis and autophagy, critical defense mechanisms against intracellular pathogens. Understanding these immunological escape mechanisms is paramount for developing effective tuberculosis therapies. Future research should focus on targeting MTB evasion strategies to pave the way for innovative tuberculosis treatments. Conclusion: There is still a long road ahead of us to understand the immunological escape mechanism used by MTB. Over the past 50 years, numerous studies have looked into the immune response and the pathogenic processes of MTB.

Lysosomal Storage Diseases: Natural Products Inducing Autophagy

Background: The link between autophagy and lysosomal function has been wellrecognised in recent decades; defective autophagy and lysosomal function lead to various disorders, notably Lysosomal Storage Disorders (LSDs). The malfunction of multiple mechanistic pathways influences the contribution of LSDs. Different ways are employed in such situations, but one novel approach could resolve the problem by inducing the autophagic pathway, which aids in maintaining proper autophagy and lysosomal degradation function. Methods: Autophagic Inducer functions on the activation of Transcriptional factor EB (TFEB) and its mechanism; mTOR Complex Inhibition dependently or independently may repair the malfunction of the entire mechanism. Finding a potential autophagic inducer is still a work in progress, but targeting TFEB and mTOR could redefine LSD treatment. The development of experimentally available TFEB modulators could enhance autophagic flux promote lysosomal function and increase lysosomal biogenesis and can be a promising technique for treating illnesses caused by ALP dysfunction, such as lysosomal storage disorder. Results: MTORC1 suppression causes TFEB to be transported to the nucleus and transcription of multiple genes involved in the formation of autophagosomes and lysosomes, indicating that MTORC1 has positive effects in treating lysosomal storage diseases such as Pompe disease, Batton disease, Fabry disease, etc. thus modulating autophagy attenuates the above condition. Conclusion: This review comprises autophagy and lysosome association, and their malfunction leads to various lysosomal diseases. Several natural products are also discussed, which can be possible treatment options.

UNC13B regulates the sensitivity of Wilms' tumor cells to doxorubicin by modulating lysosomes.

Wilms' tumor is a malignant neoplasm where current medical advancements have significantly improved survival rates; however, challenges persist such as the resistance of the tumor to chemotherapy drugs like doxorubicin. This necessitates higher dosages, leading to decreased sensitivity. However, using high doses of doxorubicin can have late effects on the heart. Unc-13 homolog B (UNC13B) may be involved in the drug resistance in several tumors, yet its role in modulating drug sensitivity in Wilms' tumor remains unexplored. UNC13B levels were quantified using reverse transcription-qPCR and Western blotting. The half-maximal inhibitory concentration for doxorubicin, vincristine, and actinomycin-D was determined using CCK-8 assays. Cell cycle and apoptosis were analyzed using flow cytometry, and lysosomal changes were observed using Lyso-Tracker staining. The present study initially evaluated UNC13B expression levels in the Wilms' tumor 17.94 cell line. Additionally, through short hairpin RNA-mediated knockdown, changes in doxorubicin sensitivity in 17.94 Wilms' tumor cells were assessed. Concurrently, preliminary investigations into the role of UNC13B in regulating lysosomes was performed, revealing a significant positive association between UNC13B levels and lysosome formation in the 17.94 cell line. Lysosomes likely serve a role in the sensitivity of Wilms' tumor cell lines to drugs. Elevated UNC13B expression was observed in the 17.94 Wilms' tumor cell line compared to normal kidney cells. UNC13B knockdown also resulted in increased apoptosis levels upon doxorubicin treatment. Immunofluorescence revealed UNC13B localization within cellular vesicles, and its knockdown significantly decreased lysosome levels. Overall, the findings of the present study demonstrate that UNC13B regulates the sensitivity of the Wilms' tumor 17.94 cell line to doxorubicin by modulating lysosome formation within cells. The results suggest that UNC13B is likely an enriched target involved in lysosomal regulation in certain tumors, offering a new approach for optimizing chemotherapy in Wilms' tumor and other cancers with high UNC13B expression.

Lipopolysaccharide-Induced Lysosomal Cell Death Through Reactive Oxygen Species in Rat Liver Cell Clone 9.

In sepsis, bacterial components, particularly lipopolysaccharide (LPS), trigger organ injuries such as liver dysfunction. Although sepsis induces hepatocyte damage, the mechanisms underlying sepsis-related hepatic failure remain unclear. In this study, we demonstrated that the LPS-treated rat hepatocyte cell line Clone 9 not only induced reactive oxygen species (ROS) generation and apoptosis but also increased the expression of the autophagy marker proteins LC3-II and p62, and decreased the expression of intact Lamp2A, a lysosomal membrane protein. Additionally, LPS increased lysosomal membrane permeability and galectin-3 puncta formation, and promoted lysosomal alkalization in Clone 9 cells. Pharmacological inhibition of caspase-8 and cathepsin D (CTSD) suppressed the activation of caspase-3 and rescued the viability of LPS-treated Clone 9 cells. Furthermore, LPS induced CTSD release associated with lysosomal leakage and contributed to caspase-8 activation. Pretreatment with the antioxidant N-acetylcysteine (NAC) not only diminished ROS generation and increased the cell survival rate, but also decreased the expression of activated caspase-8 and caspase-3 and increased the protein level of Lamp2A in LPS-treated Clone 9 cells. These results demonstrate that LPS-induced ROS causes lysosomal membrane permeabilization and lysosomal cell death, which may play a crucial role in hepatic failure in sepsis. Our results may facilitate the development of new strategies for sepsis management.

Involvement of the choroid plexus in Alzheimer’s disease pathophysiology: findings from mouse and human proteomic studies

BackgroundStructural and functional changes of the choroid plexus (ChP) have been reported in Alzheimer’s disease (AD). Nonetheless, the role of the ChP in the pathogenesis of AD remains largely unknown. We aim to unravel the relation between ChP functioning and core AD pathogenesis using a unique proteomic approach in mice and humans.MethodsWe used an APP knock-in mouse model, APPNL-G-F, exhibiting amyloid pathology, to study the association between AD brain pathology and protein changes in mouse ChP tissue and CSF using liquid chromatography mass spectrometry. Mouse proteomes were investigated at the age of 7 weeks (n = 5) and 40 weeks (n = 5). Results were compared with previously published human AD CSF proteomic data (n = 496) to identify key proteins and pathways associated with ChP changes in AD.ResultsChP tissue proteome was dysregulated in APPNL-G-F mice relative to wild-type mice at both 7 and 40 weeks. At both ages, ChP tissue proteomic changes were associated with epithelial cells, mitochondria, protein modification, extracellular matrix and lipids. Nonetheless, some ChP tissue proteomic changes were different across the disease trajectory; pathways related to lysosomal function, endocytosis, protein formation, actin and complement were uniquely dysregulated at 7 weeks, while pathways associated with nervous system, immune system, protein degradation and vascular system were uniquely dysregulated at 40 weeks. CSF proteomics in both mice and humans showed similar ChP-related dysregulated pathways.ConclusionsTogether, our findings support the hypothesis of ChP dysfunction in AD. These ChP changes were related to amyloid pathology. Therefore, the ChP could become a novel promising therapeutic target for AD.

The Therapeutic Effects of Bromelain against Colorectal Cancer: A Systematic Review

Background: Colorectal cancer (CRC) is one of the most prevalent cancers worldwide. Objective: Considering the side effects of chemotherapy treatments, we reviewed the anti-cancer effects and mechanisms of bromelain on colon cancer cells in this study. Methods: The PRISMA guidelines were followed in the design of this systematic review. Various databases, including PubMed, Web of Science, Cochrane Library, and Scopus, were thoroughly searched. Finally, 14 articles were retrieved after considering the study's inclusion and exclusion criteria. The desired data were extracted, entered into an Excel file, and the study results were reviewed. Results: According to the included studies, bromelain can significantly reduce the survival and death of cloned cancer cells through different mechanisms. These mechanisms include impeding tumor growth and metastasis by reducing mucins production/secretion and increasing/reducing reactive oxygen species (ROS) production. Moreover, bromelain induces apoptosis via reduced expression of Bcl-2, extracellular signal-related kinase (ERK), Akt, activation caspase system (caspase-3, 7, 8, and 9), and extranuclear p53. Ferroptosis was another mechanism of causing cell death. In addition, bromelain activates the autophagy pathway, lysosome formation, and deregulation of other autophagyrelated proteins. Conclusion: Bromelain effectively inhibits colon cancer cells' growth, proliferation, and metastasis and reduces their survival by different mechanisms. Therefore, after examining clinical studies, it can be used as an effective drug for treating CRC.

Lysosomal Peptide Self-Assembly to Control Cell Behavior.

Lysosomes are membrane-enclosed organelles that play key roles in degrading and recycling cellular debris, cellular signaling, and energy metabolism processes. Confinement of amphiphilic peptides in the lysosome to construct functional nanostructures through noncovalent interactions is an emerging approach to tune the homeostasis of lysosome. After briefly introducing the importance of lysosome and its functions, we discuss the advantages of lysosomal nanostructure formation for disease therapy. We next discuss the strategy for triggering the self-assembly of peptides in the lysosome, followed by a concise outlook of the future perspective about this emerging research direction.

Macrophage ATG16L1 expression suppresses metabolic dysfunction-associated steatohepatitis progression by promoting lipophagy.

Metabolic dysfunction-associated steatohepatitis (MASH) is an unmet clinical challenge due to the rapid increased occurrence but lacking approved drugs. Autophagy-related protein 16-like 1 (ATG16L1) plays an important role in the process of autophagy, which is indispensable for proper biogenesis of the autophagosome, but its role in modulating macrophage-related inflammation and metabolism during MASH has not been documented. Here, we aimed to elucidate the role of ATG16L1 in the progression of MASH. Expression analysis was performed with liver samples from human and mice. MASH models were induced in myeloid-specific Atg16l1-deficient and myeloid-specific Atg16l1-overexpressed mice by high-fat and high-cholesterol diet or methionine- and choline-deficient diet to explore the function and mechanism of macrophage ATG16L1 in MASH. Macrophage-specific Atg16l1 knockout exacerbated MASH and inhibited energy expenditure, whereas macrophage-specific Atg16l1 transgenic overexpression attenuated MASH and promotes energy expenditure. Mechanistically, Atg16l1 knockout inhibited macrophage lipophagy, thereby suppressing macrophage β-oxidation and decreasing the production of 4-hydroxynonenal, which further inhibited stimulator of interferon genes(STING) carbonylation. STING palmitoylation was enhanced, STING trafficking from the endoplasmic reticulum to the Golgi was promoted, and downstream STING signaling was activated, promoting proinflammatory and profibrotic cytokines secretion, resulting in hepatic steatosis and hepatic stellate cells activation. Moreover, Atg16l1-deficiency enhanced macrophage phagosome ability but inhibited lysosome formation, engulfing mtDNA released by pyroptotic hepatocytes. Increased mtDNA promoted cGAS/STING signaling activation. Moreover, pharmacological promotion of ATG16L1 substantially blocked MASH progression. ATG16L1 suppresses MASH progression by maintaining macrophage lipophagy, restraining liver inflammation, and may be a promising therapeutic target for MASH management.

The role of labile iron on brain proteostasis; could it be an early event of neurodegenerative disease?

Iron deposits in the brain are a natural consequence of aging. Iron accumulation, especially in the form of labile iron, can trigger a cascade of adverse effects, eventually leading to neurodegeneration and cognitive decline. Aging also increases the dysfunction of cellular proteostasis. The question of whether iron alters proteostasis is now being pondered. Herein, we investigated the effect of ferric citrate, considered as labile iron, on various aspects of proteostasis of neuronal cell lines, and also established an animal model having a labile iron diet in order to evaluate proteostasis alteration in the brain along with behavioral effects. According to an in vitro study, labile iron was found to activate lysosome formation but inhibits lysosomal clearance function. Furthermore, the presence of labile iron can alter autophagic flux and can also induce the accumulation of protein aggregates. RNA-sequencing analysis further reveals the upregulation of various terms related to proteostasis along with neurodegenerative disease-related terms. According to an in vivo study, a labile iron-rich diet does not induce iron overload conditions and was not detrimental to the behavior of male Wistar rats. However, an iron-rich diet can promote iron accumulation in a region-dependent manner. By staining for autophagic markers and misfolding proteins in the cerebral cortex and hippocampus, an iron-rich diet was actually found to alter autophagy and induce an accumulation of misfolding proteins. These findings emphasize the importance of labile iron on brain cell proteostasis, which could be implicated in developing of neurological diseases.

Staphylococcus aureus promotes its intracellular survival by inhibiting Rab11-Rab11FIP4-mediated vesicle trafficking

Mastitis in dairy cows is mainly caused by bacteria, in which Staphylococcus aureus appears frequently. Epithelial cells, as a major physical barrier of mammary gland, play an important role in preventing mastitis in dairy cows. Our previous study reported that Rab11fip4 (an effector of Rab11) was significantly changed in response to stimulation by S. aureus. So, in this study, the role of Rab11A in phagocytosis of bovine mammary epithelial cells (MAC-T) against S. aureus was evaluated. First, changes of Rab11A and Rab11fip4 were analyzed in response to S. aureus by immunofluorescence and western blotting. Subsequently, the effects of Rab11A and Rab11fip4 on proliferation of S. aureus, as well as formation and function of late endosomes (LEs) and lysosomes (LYSs) were investigated. The results showed that, after infection, Rab11A and Rab11fip4 were recruited to phagosomes containing S. aureus. Rab11A promoted bacterial clearance and rescues the destruction of LEs and LYSs by S. aureus, whereas Rab11fip4 did the opposite. These findings provide new insights into phagocytosis and control of S. aureus in host cells, thus lay the foundation to elucidate the pathogenesis of S. aureus in bovine mastitis.

Gallic acid promotes macrophage phagosome acidification and phagolysosome formation by activating NLRP3/mTOR signaling pathway

IntroductionGallic acid (GA) has a good therapeutic effect in bacteriological inhibition and plays a variety of functions in maintaining the stability of the immune system. The aim of the present study was to investigate the effect of GA on the bactericidal activity of macrophages against Vibrio vulnificus (Vv). MethodsA cell counting kit-8 (CCK-8) assay was carried out to test the cytotoxicity of GA on J774A.1 cells. Concentration of proinflammatory cytokines in J774A.1 cells were evaluated by ELISA. The internalization and degradation of Vv in the phagosomes were observed by transmission electron microscopy (TEM). The phagosome acidification and phagolysosome formation were detected to evaluate the bacteria-clearing function of J774A.1 cells. The bactericidal activity of GA in vivo was also investigated by collecting the survival time of Vv infected mice and observing the inflammatory infiltration of organs. ResultsOur results demonstrated that GA at 50 μM significantly inhibited the proinflammatory cytokines levels, promoted phagosome acidification and phagolysosome formation in J774A.1 cells with Vv infection. This may be related to the activation of NLRP3/mTOR signaling pathway. Additionally, GA treatment improves the survival and bactericidal activity of mice infected with Vv. ConclusionsIn summary, GA exerts bactericidal activity against Vv infection by regulating the formation and acidification of phagocytic lysosomes in macrophages.

МОРФОЛОГИЧЕСКИЕ И УЛЬТРАСТРУКТУРНЫЕ ИЗМЕНЕНИЯ ТКАНИ ПЕЧЕНИ ПРИ БИЛИАРНОЙ ОККЛЮЗИИ РАЗЛИЧНОЙ ДЛИТЕЛЬНОСТИ

An analysis of the morphological state of the liver was carried out in 160 patients with obstructive jaundice of non-tumor etiology who received treatment in the Novgorod Regional Clinical Hospital and Central City Clinical Hospital. The results of liver biopsy during surgery were studied. Pronounced structural and functional changes in liver tissue were revealed, which were further aggravated due to an increase in the duration of occlusion. In cases with long-term chronic jaundice, after decompression of the bile ducts, sclerotic changes, histiolymphocytic infiltration and proliferating biliary epithelium, fibrosis and foci of hepatocyte necrosis remain in the portal tracts. Ultrastructure analysis revealed changes in the cell membrane, cytoplasmic structures, destruction of mitochondrial membranes, formation of secondary lysosomes, disruption of the integrity of the nuclear membrane and signs of karyolysis. The study of puncture and surgical biopsies for obstructive jaundice makes it possible to establish the form and severity of cholestasis and, along with other diagnostic indicators, affects disease prognosis and the tactics of surgical treatment. Taking the different timing and severity of cholestasis into account, it is necessary to use therapeutic technologies in advance that lead to an improvement in liver morphology.

Inhibition of Amino Acids Influx into Proximal Tubular Cells Improves Lysosome Function in Diabetes.

Key Points Collectrin serves as a chaperone for the trafficking of neutral amino acid (AA) transporters in the apical membranes of proximal tubular cells (PTCs). Cltrn knockout reduced AAs influx into PTCs, inactivated mTOR, activated transcription factor EB, improved lysosome function, and ameliorated vacuolar formation of PTCs in diabetic mice treated with streptozotocin and high-fat diet.The inhibition of neutral AA transporter, such as B0AT1 (SLC6A19), and transcription factor EB activator is a new therapeutic strategy against diabetic kidney disease. Background Inhibition of glucose influx into proximal tubular cells (PTCs) by sodium–glucose cotransporter 2 inhibitors revealed prominent therapeutic effects on diabetic kidney disease. Collectrin (CLTRN) serves as a chaperone for the trafficking of neutral amino acid (AA) transporters in the apical membranes of PTCs. We investigated the beneficial effects of reduced influx of AAs into PTCs in diabetes and obesity model of Cltrn−/y mice. Methods Cltrn+/y and Cltrn−/y mice at age 5 weeks were assigned to standard diet and streptozotocin and high-fat diet (STZ-HFD)–treated groups. Results At age 22–23 weeks, body weight and HbA1c levels significantly increased in STZ-HFD-Cltrn+/y compared with standard diet-Cltrn+/y; however, they were not altered in STZ-HFD-Cltrn−/y compared with STZ-HFD-Cltrn+/y. At age 20 weeks, urinary albumin creatinine ratio was significantly reduced in STZ-HFD-Cltrn−/y compared with STZ-HFD-Cltrn+/y. Under the treatments with STZ and HFD, the Cltrn gene deficiency caused significant increase in urinary concentration of AAs such as Gln, His, Gly, Thr, Tyr, Val, Trp, Phe, Ile, Leu, and Pro. In PTCs in STZ-HFD-Cltrn+/y, the enlarged lysosomes with diameter of 10 μm or more were associated with reduced autolysosomes, and the formation of giant lysosomes was prominently suppressed in STZ-HFD-Cltrn−/y. Phospho-mTOR and inactive form of phospho-transcription factor EB were reduced in STZ-HFD-Cltrn−/y compared with STZ-HFD-Cltrn+/y. Conclusions The reduction of AAs influx into PTCs inactivated mTOR, activated transcription factor EB, improved lysosome function, and ameliorated vacuolar formation of PTCs in STZ-HFD-Cltrn−/y mice.

Tangzhiqing decoction attenuates cognitive dysfunction of mice with type 2 diabetes by regulating AMPK/mTOR autophagy signaling pathway

Ethnopharmacological relevanceTangzhiqing decoction (TZQD) is an effective prescription developed by Jiangsu Province Hospital of Chinese Medicine for the treatment of diabetes mellitus (DM) and its complications, which has a clear cerebral protective effect on mice with diabetic cognitive dysfunction, but its specific mechanism has not been well elucidated. Aims of the studyThis study aims to verify the protection of TZQD on cognitive function in mice with type 2 diabetes mellitus (T2DM) and explore the possible underlying mechanisms. Materials and methodsSix active ingredients in TZQD were detected using high-performance liquid chromatography analysis. In vivo experiments, the protection of TZQD on cognitive function and hippocampal neurons in type 2 diabetes mice was verified to obtain the optimal intervention dose of TZQD. TZQD and 3-methyladenine (3 MA) respectively or jointly intervened in mice with T2DM for 12 weeks, followed by detecting the cognitive difference, hippocampus cornu ammonis 1 (CA1) region injury, and hippocampal neuronal apoptosis in each group. Simultaneously, the investigation of autophagosome formation and organelle impairment in hippocampal neurons, along with the examination of AMPK/mTOR pathway proteins and autophagy-related proteins, was conducted to elucidate the potential mechanisms, through which TZQD modulates autophagy and enhances cognitive function. In vitro experiments, TZQD-containing serum and AMPK inhibitor Compound C (CC) were used to intervene in mouse hippocampal neuron HT22 cells under high glucose environment, further clarifying the regulatory role of TZQD on the AMPK/mTOR pathway and its impact on HT22 cell apoptosis and autophagy. ResultsIn vivo experiment results showed that TZQD had an obvious hypoglycemic effect. Different doses of TZQD could improve cognitive function and hippocampus damage in diabetes mice, with the middle dose of TZQD showing the best effect. TZQD increased the swimming speed of diabetes mice, improved their spatial recognition and memory ability, and reduced hippocampal neuronal apoptosis, Nissl body injury, and p-tau217 protein deposition. In addition, through transmission electron microscopy (TEM), immunofluorescence, and Western blot (WB) detection, TZQD significantly improved the organelle damage of hippocampal neurons in diabetes mice, promoted the formation of autophagy lysosomes, increased the expression of autophagy-related proteins like Beclin 1, LC3II/LC3I, LAMP1, and LAMP2, reduced the level of P62 and promoted autophagy flow, which, however, were all significantly weakened by 3 MA. Meanwhile, TZQD regulated the expressions of AMPK/mTOR pathway proteins. In vitro experimental study results showed that TZQD can regulate the expression ratio of p-AMPK/AMPK alpha 1 and p-mTOR/mTOR in HT22 cells under high glucose conditions and improved the morphology and vitality of HT22 cells. By employing techniques such as monodansylcadaverine (MDC) staining, Lysosomal red fluorescent probe staining, and Annexin V-FITC/PI double staining, the investigation revealed that TZQD administration resulted in enhanced autophagosome formation, preservation of a lysosomal acidic milieu, and consequent mitigation of HT22 cell apoptosis under high glucose conditions. ConclusionsTZQD can regulate the AMPK/mTOR pathway to activate autophagy to attenuate hippocampal neuronal apoptosis, thereby protecting cognitive function in diabetic mice.

ITGB3 Promotes the Progression of JAK2V617F-Positive Myeloproliferative Neoplams By Regulating Calreticulin to Inhibit Cellular Autophagy

O bjectives: Integrins are integral cell-surface proteins composed of an alpha chain and a beta chain, of which integrin beta 3 (ITGB3) was known to participate in cell adhesion as well as cell-surface mediated signaling and plays vital roles in different tumors. However, the role of ITGB3 in JAK2V617F-positive classical myeloproliferative neoplasms (cMPNs) remians unclear, and the aim of this study was to explore the effect of ITGB3 on cellular autophagy in JAK2V617F-positive cMPN cell line and its underlying mechanisms. Methods: ITGB3 expression in bone marrows from JAK2V617F-positive cMPN patients compared to normal controls were detected by quantitative reverse-transcription PCR (qPCR) and further validated by western blotting. Cell models of cMPNs were constructed using human HEL cell lines carrying JAK2V617F mutation and cytokine-dependent wild type BaF3 cells (Jak2 V617F BaF3 cells) with ectopic expression of Jak2 V617F seperately to perform phenotype and mechanism investigation. The putative targets of STAT5a on the promoters of ITGB3 were disclosed by public databases and a dual-luciferase reporter assay and chromatin immunoprecipitation assay (ChIP) was used to analyze the binding of STAT5a to ITGB3 promoter region. To observe the role of ITGB3 played in cMPN progression, stable-transfected HEL cell line with ITGB3 knockdown or overexpression were established by lentivirus packaging and cell transfection. Cell phenotype including cell proliferation, apoptosis and cell cycle distribution were detected by soft agar colony formation assay, BrdU staining, flow cytometry and Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assays. The variations of autophagic flux after ITGB3 dysregulation in HEL cells was analyzed using mRFP-GFP-LC3 adenovirus transfection and fluorescence microscopy, and LC3-II/I ratio as well as autophagy-related protein were detected by western blotting. Transmission electron microscopy was used to observe the formation of intracellular autophagosomes and autophagic lysosomes. RNA sequencing and GSEA analysis were applied to predict the downstream pathway and putative targets of ITGB3. Western blotting, immunofluorescence and qPCR were performed to detect the transcription and expression levels of genes. Results: 1.Western blotting and qRT-PCR detection showed increased expression of ITGB3 in bone marrow of JAK2V617F-positive cMPN patients compared to normal controls (A-B). Also, the evaluation of ITGB3 in a variety of myeloid tumor cell lines (HEL, HL60, K562, KG-1a) and wild-type BaF3 cells as well as Jak2 V617F BaF3 cells revealed that ITGB3 protein level is the highest in HEL (which harboring the JAK2V617F) and Jak2 V617F BaF3 cells (C-F). 2. Dual-luciferase reporter assay showed that STAT5a significantly induced the transcription activities of promoters of ITGB3 and inhibition of STAT5a reduced the transcription activities of promoters of ITGB3 (G-H). ITGB3 knockdown (I) in HEL cells reduced the proliferation capacity (J-L), promoted early apoptosis (M-O) and caused cell cycle arrest (P-Q). While ITGB3 overexpression in HEL cells (R) reduced the cell cycle arrest (S) and apoptosis (T-U). 3. Autophagic flux analysis showed that ITGB3 knockdown promoted the autophagic flux in HEL cells (V-X). Inhibition of ITGB3 increases the synthesis and degradation of autophagosomes and accelerates the process of cellular autophagy (Y-Z). Further identification of DEGs in ITGB3-dysregulated HEL cells (AA-AB) reveals that ITGB3 inhibits the expression of calreticulin (AC-AE), and ITGB3 might be involved in the regulation of autophagy in HEL cells through the regulation of calreticulin. Conclusions: 1. ITGB3 is significantly activated in HEL cell lines carrying the JAK2V617F mutation. 2. ITGB3 protein acts as a pro-oncogene in JAK2V617F-positive myeloproliferative neoplams progression by regulating calreticulin to inhibit autophagy, which deserves more in-depth study in JAK2V617F mutation-positive cMPNs.

Peroxisome Proliferator-Activated Receptor γ Regulates Lipid Metabolism in Sheep Trophoblast Cells through mTOR Pathway-Mediated Autophagy.

Peroxisome proliferator-activated receptor gamma (PPARγ) is a key nuclear receptor transcription factor that is highly expressed in trophoblastic cells during embryonic attachment and is accompanied by rapid cell proliferation and increased lipid accumulation. We previously showed that the autophagy pathway is activated in cells after activation of PPARγ, accompanied by increased lipid accumulation. In this study, we used PPARγ agonist rosiglitazone and inhibitor GW9662, as well as autophagy activator rapamycin and inhibitor 3-methyladenine, to unravel the probable mechanism of PPARγ engaged in lipid metabolism in sheep trophoblast cells (STCs). After 12 h, 24 h, and 48 h of drug treatment, the levels of autophagy-related proteins were detected by Western blot, the triglyceride content and MDA level of cells were detected by colorimetry, and the lipid droplets and lysosomes were localized by immunofluorescence. We found that PPARγ inhibited the activity of mammalian target of rapamycin (mTOR) pathway in STCs for a certain period of time, promoted the increase of autophagy and lysosome formation, and enhanced the accumulation of lipid droplets and triglycerides. Compared with cells whose PPARγ function is activated, blocking autophagy before activating PPARγ will hinder lipid accumulation in STCs. Pretreatment of cells with rapamycin promoted autophagy with results similar to rosiglitazone treatment, while inhibition of autophagy with 3-methyladenine reduced lysosome and lipid accumulation. Based on these observations, we conclude that PPARγ can induce autophagy by blocking the mTOR pathway, thereby promoting the accumulation of lipid droplets and lysosomal degradation, providing an energy basis for the rapid proliferation of trophoblast cells during embryo implantation. In brief, this study partially revealed the molecular regulatory mechanism of PPARγ, mTOR pathway, and autophagy on trophoblast cell lipid metabolism, which provides a theoretical basis for further exploring the functional regulatory network of trophoblast cells during the attachment of sheep embryos.

Spatacsin regulates directionality of lysosome trafficking by promoting the degradation of its partner AP5Z1.

The endoplasmic reticulum (ER) forms contacts with the lysosomal compartment, regulating lysosome positioning and motility. The movements of lysosomes are controlled by the attachment of molecular motors to their surface. However, the molecular mechanisms by which ER controls lysosome dynamics are still elusive. Here, using mouse brain extracts and mouse embryonic fibroblasts, we demonstrate that spatacsin is an ER-resident protein regulating the formation of tubular lysosomes, which are highly dynamic. Screening for spatacsin partners required for tubular lysosome formation showed spatacsin to act by regulating protein degradation. We demonstrate that spatacsin promotes the degradation of its partner AP5Z1, which regulates the relative amount of spastizin and AP5Z1 at lysosomes. Spastizin and AP5Z1 contribute to regulate tubular lysosome formation, as well as their trafficking by interacting with anterograde and retrograde motor proteins, kinesin KIF13A and dynein/dynactin subunit p150Glued, respectively. Ultimately, investigations in polarized mouse cortical neurons in culture demonstrated that spatacsin-regulated degradation of AP5Z1 controls the directionality of lysosomes trafficking. Collectively, our results identify spatacsin as a protein regulating the directionality of lysosome trafficking.

ASO-mediated knockdown or kinase inhibition of G2019S-Lrrk2 modulates lysosomal tubule-associated antigen presentation in macrophages

Genetic variation around the LRRK2 gene affects risk for both familial and sporadic Parkinson's disease (PD). LRRK2 levels have become an appealing target for potential PD therapeutics with LRRK2 antisense oligonucleotides (ASOs) now moving toward clinical trials. However, LRRK2 has been suggested to play a fundamental role in peripheral immunity, and it is currently unknown if targeting increased LRRK2 levels in peripheral immune cells will be beneficial or deleterious. Here it was observed that G2019S macrophages exhibited increased stimulation-dependent lysosomal tubule formation (LTF) and MHC-II trafficking from the perinuclear lysosome to the plasma membrane in an mTOR-dependent manner with concomitant increases in pro-inflammatory cytokine release. Both ASO-mediated knockdown of mutant Lrrk2 and LRRK2 kinase inhibition ameliorated this phenotype and decreased these immune responses in control cells. Given the critical role of antigen presentation, lysosomal function, and cytokine release in macrophages, it is likely LRRK2-targeting therapies with systemic activity may have therapeutic value with regard to mutant LRRK2, but deleterious effects on the peripheral immune system, such as altered pathogen control in these cells, should be considered when reducing levels of non-mutant LRRK2.

Dissolving Fusion Oncoprotein Condensates to Reverse Aberrant Gene Expression.

In a recent study, Wang and colleagues reported that a significant fraction of cancer-associated fusion proteins display a common structural topology, including an N-terminal phase separation-prone region (PS) from one parent protein and a C-terminal DNA-binding domain (DBD) from the other. This is reminiscent of the structural topology of transcription factors and led to the hypothesis that the PS-DBD fusions form aberrant transcriptional condensates through phase separation, which was supported through transcriptomic data analysis and cellular condensate assays. The authors developed a high-throughput screen based upon time-lapse, high-content imaging to identify 114 compounds that dissolved condensates formed by a chromatin-dissociated mutant of FUS::ERG (FUS::ERGmut). One of these compounds, LY2835219, was shown to dissolve FUS::ERGmut condensates by promoting lysosome formation and was also active against condensates formed by other PS-DBD fusions, including EWS::FLI1. Finally, condensate dissolution by LY2835219 was shown to reverse aberrant gene expression driven by EWS::FLI1, although how this compound specifically marshals lysosomes to target some PS-DBD fusions and not other condensate-forming proteins remains elusive. This work not only highlights likely roles for aberrant condensate formation in the oncogenic function of PS-DBD fusions, but also provides proof of principle for mechanistically unbiased screening to identify compounds that modulate fusion protein-driven condensates and their oncogenic functions.