Upregulation Of Autophagy Research Articles

Synergistic Targeting of BTK and E-Selectin/CXCR4 in the Microenvironment of Mantle Cell Lymphomas

Synergistic Targeting of BTK and E-Selectin/CXCR4 in the Microenvironment of Mantle Cell Lymphomas

ATG5 Dependent Autophagy in Graft-Versus-Host Disease Target Organs Mitigates Disease Severity Post Allogeneic Bone Marrow Transplant

In immune mediated diseases such as graft-versus-host disease (GVHD), research has focused almost entirely on hematopoietic cells. It has recently been posited that mechanisms within target tissues also play a significant role in regulating the severity of disease. Autophagy protects cells from a variety of stressors and is known to affect immune cell responses after allogeneic bone marrow transplant (allo-BMT). However, a definitive and exclusive role of target tissue pathways on mitigating GVHD has heretofore not been demonstrated. Because GVHD target organs are under stress from alloreactive T cells, we sought to determine the role of target tissue autophagy in mitigating the severity of GVHD. We hypothesized that the stress from alloreactive T cells would result in upregulation of target tissue autophagy. To test this in vivo, we utilized LC3-GFP-RFP reporter mice as transplant recipients in the well-characterized MHC disparate GVHD murine model BALB/c→B6. In these reporter mice, autophagosomes are marked with a dual GFP-RFP tag, and can be assessed for in vivo induction of autophagy by confocal microscopy through quantification of red punctal abundance. The recipient B6 reporter mice were lethally irradiated with split doses of 5Gy and transplanted with 5x106 BM and 3x106 splenic T-cells from either syngeneic B6 (syn) or allogeneic BALB/c (allo) donors. We assessed for induction of autophagy in GVHD target organs (gut, liver) and non-target organs (kidney, heart) and observed that autophagy was induced to a greater extent in target organs. Because the primary driver of morbidity and mortality after allo-BMT is gastrointestinal (GI) GVHD, we next hypothesized that autophagy protects the GI epithelium from alloimmune damage. To determine the effects of autophagy exclusively in the GI tract, we generated mice that lack a macroautophagy protein, ATG5, in the GI epithelium only (Atg5fl/flVil1-Cre) and utilized them as recipients in BALB/c→B6 allo-BMT. As hypothesized, survival of allo Atg5fl/flVil1-Cre mice was significantly reduced (P<0.0001) compared to littermate controls. Importantly, all syn Atg5 KO mice survived the entire period of observation, demonstrating that protection from conditioning regimen toxicity did not require ATG5-dependent autophagy in the gut. We harvested both GVHD target and non-target organs and performed histopathological analysis at day 7 post-transplant and found that histological GVHD severity in the gut was significantly higher in allo Atg5fl/flVil1-Cre mice compared to Cre-null WT littermate controls (P=0.0063). All other organs were unaffected by loss of ATG5 in the GI epithelium, and we observed no difference between WT and Atg5 KO syn recipients in any organ. We harvested splenocytes and tissue-resident lymphocytes from the gut and found similar numbers of donor CD4 and CD8 T cells, activation markers CD69 and CD62L, and IFNγ and TNFα positive cells. Furthermore, similar levels of the proinflammatory serum cytokines IFNγ, TNFα, and IL-6 were observed in the sera of WT and Atg5 KO mice. We next determined whether autophagy is critical for protection in other GVHD target organs after allo-BMT. To this end, we generated Atg5fl/flAlb-Cre mice, which lack Atg5 expression in hepatocytes. Using a similar model of acute GVHD as above, we observed dramatically shorter survival in the Atg5fl/flAlb-Cre allo recipients (P=0.025) along with an increase in the hepatocyte damage markers bilirubin (P=0.0001), alanine aminotransferase (P=0.036), and alkaline phosphatase (P=0.0002) in Atg5 KO allo recipients. As before, there were similar numbers of splenic and liver-resident donor T cells, activation markers, and proinflammatory cytokines. To determine if autophagy is exclusively protective in GVHD target organs, we next generated Atg5fl/flMyh6-Cre mice, which lack Atg5 expression in cardiac myocytes, and Atg5fl/flAlb-Cre mice, which lack Atg5 expression in podocytes, as a control for lack of ATG5 in non-GVHD target organs. Using a similar GVHD model as above, we monitored these mice and littermate controls for survival and severity of GVHD symptoms. In contrast to our findings in the liver and gut, we observed similar survival (P=0.17, Myh6; P=0.49, Nphs2) and GVHD severity between allo recipients. Collectively, our results demonstrated that autophagy is a tissue-intrinsic protective response that regulates disease severity in GVHD target organs. Disclosures No relevant conflicts of interest to declare.

LKB1/p53/TIGAR/autophagy-dependent VEGF expression contributes to PM2.5-induced pulmonary inflammatory responses

One of the health hazards of PM2.5 exposure is to induce pulmonary inflammatory responses. In our previous study, we demonstrated that exposing both the immortalized and primary human bronchial epithelial cells to PM2.5 results in a significant upregulation of VEGF production, a typical signaling event to trigger chronic airway inflammation. Further investigations showed that PM2.5 exposure strongly induces ATR/CHK1/p53 cascade activation, leading to the induction of DRAM1-dependent autophagy to mediate VEGF expression by activating Src/STAT3 pathway. In the current study, we further revealed that TIGAR was another transcriptional target of p53 to trigger autophagy and VEGF upregulation in Beas-2B cells after PM2.5 exposure. Furthermore, LKB1, but not ATR and CHK1, played a critical role in mediating p53/TIGAR/autophagy/VEGF pathway activation also by linking to Src/STAT3 signaling cascade. Therefore, on combination of the previous report, we have identified both ATR/CHK1/p53/DRAM1- and LKB1/p53/TIGAR- dependent autophagy in mediating VEGF production in the bronchial epithelial cells under PM2.5 exposure. Moreover, the in vivo study further confirmed VEGF induction in the airway potentially contributed to the inflammatory responses in the pulmonary vascular endothelium of PM2.5-treated rats. Therefore, blocking VEGF expression or autophagy induction might be the valuable strategies to alleviating PM2.5-induced respiratory injuries.

A New Possible Mechanism by Which Punicalagin Protects against Liver Injury Induced by Type 2 Diabetes Mellitus: Upregulation of Autophagy via the Akt/FoxO3a Signaling Pathway.

The aim of this study was to investigate the protective effect of punicalagin (PU), which is a main component of pomegranate polyphenols, against liver injury induced by Type 2 diabetes mellitus (T2DM) and to explore the molecular mechanism based on autophagy in vivo and in vitro. In T2DM mice, we found that PU significantly improved liver histology, reversed serum biochemical abnormalities, and increased the autophagosome number in the liver. In HepG2 cells cultured in a high-glucose environment, PU upregulated the glucose uptake level. Both in vivo and in vitro, PU upregulated the expression of autophagy-related proteins, such as LC3b and p62, and reduced the phosphorylated Akt/total Akt and phosphorylated FoxO3a/total FoxO3a protein ratios, and these effects were enhanced by LY294002 (a PI3K/Akt inhibitor). In summary, our current findings suggest that PU protects against liver injury induced by T2DM by restoring autophagy through the Akt/FoxO3a signaling pathway.

Molecular evolution of autophagy rate-limiting factor LAMP2 in placental mammals

Autophagy is the cellular process of removal of misfolded or damaged macromolecules and organelles. Experimental studies have demonstrated autophagy as a major mechanism of lifespan extension in long-lived mammals such as bats and mole rat rodents. Moreover, the role of this biological process has been well documented in protection against age-associated diseases and viral infection. However, studies on the molecular adaptive changes of autophagy factors during evolution are scarce. Here, we conducted a bioinformatics study of the molecular evolution of the Lysosomal Associated Membrane Protein 2 (LAMP2), as a rate-limiting factor in the lysosomal degradation stage of autophagy (the communal step of two of autophagy types: macroautophagy and chaperone-mediated). Analyzing LAMP2 across placental mammals, our phylogenetic-based maximum likelihood analyses indicate that the majority of the coding sites undergo purifying selection. However, around 27% of sites display a relaxation of purifying constraints (average ω = 0.42128), among which, 14 particular sites undergo positive selection (ω > 1). These sites are mostly located in the first luminal domain of LAMP2 (N-domain), with a hotspot region in the 135–144 codons interval. Therefore, the N-domain may account for the functional diversity and regulation of LAMP2. In addition, the identified positive selection sites could act as key regulatory sites in the LAMP2 function. On the other hand, testing the rate of evolution in LAMP2 along different clades of placental mammals revealed a relatively relaxed evolution in LAMP2 along megabats’ clade. It is not clear yet whether an expedited evolution of LAMP2 in megabats has contributed to their reported up-regulation of autophagy. Finally, our data indicate positive selection sites along the ancestral branch of the clades of rodents, mouse-related rodents, and mole-rats; and suggest the potentially important regulatory role of these sites in LAMP2. Identifying the residues under positive selection, our findings pave the way for future experimental investigations to define how these selective substitutions have functionally affected autophagy.

In Vivo Autophagy Up-Regulation of Small Intestine Enterocytes in Chinese Soft-Shelled Turtles during Hibernation.

Many studies have focused on how autophagy plays an important role in intestinal homeostasis under pathological conditions. However, its role in the intestine during hibernation remains unclear. In the current study, we characterized in vivo up-regulation of autophagy in enterocytes of the small intestine of Chinese soft-shelled turtles during hibernation. Autophagy-specific markers were used to confirm the existence of autophagy in enterocytes through immunohistochemistry (IHC), immunofluorescence (IF), and immunoblotting. IHC staining indicated strong, positive immunoreactivity of the autophagy-related gene (ATG7), microtubule-associated protein light chain (LC3), and lysosomal-associated membrane protein 1 (LAMP1) within the mucosal surface during hibernation and poor expression during nonhibernation. IF staining results showed the opposite tendency for ATG7, LC3, and sequestosome 1 (p62). During hibernation ATG7 and LC3 showed strong, positive immunosignaling within the mucosal surface, while p62 showed strong, positive immunosignaling during nonhibernation. Similar findings were confirmed by immunoblotting. Moreover, the ultrastructural components of autophagy in enterocytes were revealed by transmission electron microscopy (TEM). During hibernation, the cumulative formation of phagophores and autophagosomes were closely associated with well-developed rough endoplasmic reticulum in enterocytes. These autophagosomes overlapped with lysosomes, multivesicular bodies, and degraded mitochondria to facilitate the formation of autophagolysosome, amphisomes, and mitophagy in enterocytes. Immunoblotting showed the expression level of PTEN-induced kinase 1 (PINK1), and adenosine monophosphate-activated protein kinase (AMPK) was enhanced during hibernation. Furthermore, the exosome secretion pathway of early–late endosomes and multivesicular bodies were closely linked with autophagosomes in enterocytes during hibernation. These findings suggest that the entrance into hibernation is a main challenge for reptiles to maintain homeostasis and cellular quality control in the intestine.

Glucocorticoids preserve the t-tubular system in ventricular cardiomyocytes by upregulation of autophagic flux.

A major contributor to contractile dysfunction in heart failure is remodelling and loss of the cardiomyocyte transverse tubular system (t-system), but underlying mechanisms and signalling pathways remain elusive. It has been shown that dexamethasone promotes t-tubule development in stem cell-derived cardiomyocytes and that cardiomyocyte-specific glucocorticoid receptor (GR) knockout (GRKO) leads to heart failure. Here, we studied if the t-system is altered in GRKO hearts and if GR signalling is required for t-system preservation in adult cardiomyocytes. Confocal and 3D STED microscopy of myocardium from cardiomyocyte-specific GRKO mice revealed decreased t-system density and increased distances between ryanodine receptors (RyR) and L-type Ca2+ channels (LTCC). Because t-system remodelling and heart failure are intertwined, we investigated the underlying mechanisms in vitro. Ventricular cardiomyocytes from failing human and healthy adult rat hearts cultured in the absence of glucocorticoids (CTRL) showed distinctively lower t-system density than cells treated with dexamethasone (EC50 1.1nM) or corticosterone. The GR antagonist mifepristone abrogated the effect of dexamethasone. Dexamethasone improved RyR-LTCC coupling and synchrony of intracellular Ca2+ release, but did not alter expression levels of t-system-associated proteins junctophilin-2 (JPH2), bridging integrator-1 (BIN1) or caveolin-3 (CAV3). Rather, dexamethasone upregulated LC3B and increased autophagic flux. The broad-spectrum protein kinase inhibitor staurosporine prevented dexamethasone-induced upregulation of autophagy and t-system preservation, and autophagy inhibitors bafilomycin A and chloroquine accelerated t-system loss. Conversely, induction of autophagy by rapamycin or amino acid starvation preserved the t-system. These findings suggest that GR signalling and autophagy are critically involved in t-system preservation and remodelling in the heart.

Pyrroloquinoline quinone protected autophagy-dependent apoptosis induced by mono(2-ethylhexyl) phthalate in INS-1 cells

Mono(2-ethylhexyl) phthalate (MEHP) is the main metabolite of di(2-ethylhexyl) phthalate (DEHP) in organisms and is commonly used as a plasticizer. Exposure to DEHP impairs the function of islet beta cells (INS-1 cells), which is related to insulin resistance and type 2 diabetes. At present, some research data have also confirmed that MEHP has a certain damage effect on INS-1 cells. In our experiment, we found that MEHP would lead to the increase of reactive oxygen species (ROS) and the upregulation of autophagy. And downregulated ROS production by N-acetyl-L-cysteine could also reduce autophagy. In addition, MEHP-induced lysosomal membrane permeability (LMP) subsequently released cathepsin D. Additionally, MEHP induced the collapse of mitochondrial transmembrane potential and release of cytochrome c. Addition of autophagy inhibitor 3-methyladenine relieved MEHP-induced apoptosis as assessed by the expression of cleaved caspase 3, cleaved caspase 9, and terminal deoxynucleotidyl transferase dUTP nick end labeling assay, indicating that MEHP-induced apoptosis was autophagy dependent. Cathepsin D inhibitor, pepstatin A, suppressed MEHP-induced mitochondria release of cytochrome c and apoptosis as well. Meanwhile, pyrroloquinoline quinone (PQQ), a new B vitamin, improved the above phenomenon. Taken together, our results indicate that MEHP induces autophagy-dependent apoptosis in INS-1 cells by lysosomal-mitochondrial axis. PQQ improved this process by downregulating ROS and provided a degree of protection. Our study provides a new perspective for MEHP on the cytotoxic mechanism and PQQ protection in INS-1 cells.

Dimethylfumarate Inhibits Colorectal Carcinoma Cell Proliferation: Evidence for Cell Cycle Arrest, Apoptosis and Autophagy.

Recent studies have proven that Dimethylfumarate (DMF) has a marked anti-proliferative impact on diverse cancer entities e.g., on malignant melanoma. To explore its anti-tumorigenic potential, we examined the effects of DMF on human colon carcinoma cell lines and the underlying mechanisms of action. Human colon cancer cell line HT-29 and human colorectal carcinoma cell line T84 were treated with or without DMF. Effects of DMF on proliferation, cell cycle progression, and apoptosis were analyzed mainly by Bromodeoxyuridine (BrdU)- and Lactatdehydrogenase (LDH)-assays, caspase activation, flowcytometry, immunofluorescence, and immunoblotting. In addition, combinational treatments with radiation and chemotherapy were performed. DMF inhibits cell proliferation in both cell lines. It was shown that DMF induces a cell cycle arrest in G0/G1 phase, which is accompanied by upregulation of p21 and downregulation of cyclin D1 and Cyclin dependent kinase (CDK)4. Furthermore, upregulation of autophagy associated proteins suggests that autophagy is involved. In addition, the activation of apoptotic markers provides evidence that apoptosis is involved. Our results show that DMF supports the action of oxaliplatin in a synergetic manner and failed synergy with radiation. We demonstrated that DMF has distinct anti-tumorigenic, cell dependent effects on colon cancer cells by arresting cell cycle in G0/G1 phase as well as activating both the autophagic and apoptotic pathways and synergizes with chemotherapy.

EGFR-mediated autophagy in tumourigenesis and therapeutic resistance.

The epidermal growth factor receptor (EGFR) is a receptor tyrosine kinase (RTK) that links extracellular signals to the control of cell survival, growth, proliferation and differentiation. Due to its frequent overexpression and hyperactivation, EGFR has been a therapeutic target for human malignancies. Unfortunately, the specialized inhibitors of EGFR or EGFR-mediated pathways have not yet achieved the desired clinical effects. Therefore, it is necessary to elucidate the EGFR-mediated molecular basis of tumourigenesis, development and therapeutic resistance and to identify potential therapeutic targets. Interestingly, emerging research has indicated that autophagy is closely related to tumourigenesis, tumour progression and chemoresistance. Both autophagy upregulation and downregulation have been observed in cancers, suggesting its dual oncogenic and tumour suppressor properties during malignant transformation. Importantly, EGFR has been demonstrated to be a critical determinant of whether autophagy has a cytoprotective or cytotoxic effect. Therefore, here, we mainly focus on the function of EGFR in autophagy, especially the potential mechanism. The EGFR-mediated pathways or proteins involved in autophagy regulation include (1) the EGFR-mTOR pathway; (2) the EGFR-RAS pathway; (3) EGFR-Beclin1; (4) the EGFR-STAT3 pathway and (5) EGFR-LAPTM4B (oncoprotein lysosomal-associated transmembrane protein 4B). In addition, we also describe the role of EGFR-mediated autophagy in chemoresistance and tumour therapy. We attempt to summarized the mechanism by which EGFR-mediated signalling pathways participate in regulating autophagy and to investigate how to use the existing knowledge to identify potential cancer therapeutic targets.

Autophagy plays a critical role in Klotho gene deficiency-induced arterial stiffening and hypertension.

Klotho is an anti-aging gene that shortens the life span when disrupted and extends the lifespan when overexpressed. This study investigated whether autophagy plays a role in Klotho gene deficiency-induced arterial stiffening and hypertension. Klotho mutant heterozygous (KL+/-) mice and age- and sex-matched wild-type (WT) mice were used. Arteries were examined for autophagy using Western blot assays. Pulse wave velocity (PWV), a direct measure of arterial stiffness, and blood pressure (BP) increased significantly in KL (+/-) mice. The autophagy level, as measured by LC3-II expression and autophagy flux, increased in aortas of KL (+/-) mice, indicating that Klotho gene deficiency upregulated autophagy. Chloroquine diminished Klotho gene deficiency-induced increases in PWV and BP and eliminated the upregulation of autophagic flux in KL (+/-) mice. Klotho gene deficiency-induced arterial stiffness was accompanied by upregulation of MMP9, TGFβ-1, TGFβ-3, RUNX2, and ALP, but these changes were effectively mitigated by chloroquine. Chloroquine also halted an increase in scleraxis expression in aortas of Klotho (+/-) mice. In cultured mouse aortic smooth muscle cells, Klotho gene deficiency increased autophagy, leading to upregulation of scleraxis, a key transcription factor of collagen synthesis. Klotho gene deficiency failed to upregulate scleraxis expression when autophagy was inhibited, suggesting that autophagy is a critical mediator of Klotho gene deficiency-induced upregulation of scleraxis. Suppression of enhanced autophagy by chloroquine lessens Klotho gene deficiency-induced arterial stiffening and hypertension by stopping upregulation of MMP9 and scleraxis. The enhanced autophagic activity plays a crucial role in Klotho gene deficiency-induced arterial stiffening and hypertension. KEY MESSAGES: Klotho gene deficiency upregulates autophagy. Upregulation of autophagy plays a role in the pathogenesis of arterial stiffening. Autophagy regulates MMP9 activity and scleraxis expression.

BIRC5/Survivin is a novel ATG12–ATG5 conjugate interactor and an autophagy-induced DNA damage suppressor in human cancer and mouse embryonic fibroblast cells

ABSTRACT BIRC5/Survivin is known as a dual cellular functions protein that directly regulates both apoptosis and mitosis in embryonic cells during embryogenesis and in cancer cells during tumorigenesis and tumor metastasis. However, BIRC5 has seldom been demonstrated as a direct macroautophagy/autophagy regulator in cells. ATG7 expression and ATG12–ATG5-ATG16L1 complex formation are crucial for the phagophore elongation during autophagy in mammalian cells. In this study, we observed that the protein expression levels of BIRC5 and ATG7 were inversely correlated, whereas the expression levels of BIRC5 and SQSTM1/p62 were positively correlated in normal breast tissues and tumor tissues. Mechanistically, we found that BIRC5 negatively modulates the protein stability of ATG7 and physically binds to the ATG12–ATG5 conjugate, preventing the formation of the ATG12–ATG5-ATG16L1 protein complex in human cancer (MDA-MB-231, MCF7, and A549) and mouse embryonic fibroblast (MEF) cells. We also observed a concurrent physical dissociation between BIRC5 and ATG12–ATG5 (but not CASP3/caspase-3) and upregulation of autophagy in MDA-MB-231 and A549 cells under serum-deprived conditions. Importantly, despite the fact that upregulation of autophagy is widely thought to promote DNA repair in cells under genotoxic stress, we found that BIRC5 maintains DNA integrity through autophagy negative-modulations in both human cancer and MEF cells under non-stressed conditions. In conclusion, our study reveals a novel role of BIRC5 in cancer cells as a direct regulator of autophagy. BIRC5 may act as a “bridging molecule”, which regulates the interplay between mitosis, apoptosis, and autophagy in embryonic and cancer cells. Abbreviations ACTA1: actin; ATG: autophagy related; BIRC: baculoviral inhibitor of apoptosis repeat-containing; BAF: bafilomycin A1; CQ: chloroquine; CASP3: caspase 3; HSPB1/Hsp27: heat shock protein family B (small) member 1/heat shock protein 27; IAPs: inhibitors of apoptosis proteins; IP: immunoprecipitation; MAP1LC3/LC3: microtubule associated protein 1 light chain 3; PLA: proximity ligation assay; SQSTM1/p62: sequestosome 1; siRNA: small interfering RNA

Aggregable Nanoparticles-Enabled Chemotherapy and Autophagy Inhibition Combined with Anti-PD-L1 Antibody for Improved Glioma Treatment.

Glioma treatment using targeted chemotherapy is still far from satisfactory due to not only the limited accumulation but also the multiple survival mechanisms of glioma cells, including up-regulation of both autophagy and programmed cell death ligand 1 (PD-L1) expression. Herein, we proposed a combined therapeutic regimen based on functional gold nanoparticles (AuNPs)-enabled chemotherapy, autophagy inhibition, and blockade of PD-L1 immune checkpoint. Specifically, the legumain-responsive AuNPs (D&H-A-A&C) could passively target the glioma site and form in situ aggregates in response to legumain, leading to enhanced accumulation of doxorubicin (DOX) and hydroxychloroquine (HCQ) at the glioma site. HCQ could inhibit the DOX-induced cytoprotective autophagy and thus resensitize glioma cells to DOX. Parallelly, inhibiting autophagy could also inhibit the formation of autophagy-related vasculogenic mimicry (VM) by glioma stem cells. In vivo studies demonstrated that D&H-A-A&C possessed promising antiglioma effect. Moreover, cotreatment with anti-PD-L1 antibody was able to neutralize immunosuppressed glioma microenvironment and thus unleash antiglioma immune response. In vivo studies showed D&H-A-A&C plus anti-PD-L1 antibody could further enhance antiglioma effect and efficiently prevent recurrence. The effectiveness of this strategy presents a potential avenue to develop a more effective and more personalized combination therapeutic regimen for glioma patients.

Stromal cells downregulate miR-23a-5p to activate protective autophagy in acute myeloid leukemia

Complex molecular cross talk between stromal cells and the leukemic cells in bone marrow is known to contribute significantly towards drug-resistance. Here, we have identified the molecular events that lead to stromal cells mediated therapy-resistance in acute myeloid leukemia (AML). Our work demonstrates that stromal cells downregulate miR-23a-5p levels in leukemic cells to protect them from the chemotherapy induced apoptosis. Downregulation of miR-23a-5p in leukemic cells leads to upregulation of protective autophagy by targeting TLR2 expression. Further, autophagy inhibitors when used as adjuvants along with conventional drugs can improve drug sensitivity in vitro as well in vivo in a mouse model of leukemia. Our work also demonstrates that this mechanism of bone marrow stromal cell mediated regulation of miR-23a-5p levels and subsequent molecular events are relevant predominantly in myeloid leukemia. Our results illustrate the critical and dynamic role of the bone marrow microenvironment in modulating miRNA expression in leukemic cells which could contribute significantly to drug resistance and subsequent relapse, possibly through persistence of minimal residual disease in this environment.

Glioblastoma ablates pericytes antitumor immune function through aberrant up-regulation of chaperone-mediated autophagy.

The contractile perivascular cells, pericytes (PC), are hijacked by glioblastoma (GB) to facilitate tumor progression. PC's protumorigenic function requires direct interaction with tumor cells and contributes to the establishment of immunotolerance to tumor growth. Cancer cells up-regulate their own chaperone-mediated autophagy (CMA), a process that delivers selective cytosolic proteins to lysosomes for degradation, with pro-oncogenic effects. However, the possible impact that cancer cells may have on CMA of surrounding host cells has not been explored. We analyzed the contribution of CMA to the GB-induced changes in PC biology. We have found that CMA is markedly up-regulated in PC in response to the oxidative burst that follows PC-GB cell interaction. Genetic manipulations to block the GB-induced up-regulation of CMA in PC allows them to maintain their proinflammatory function and to support the induction of effective antitumor T cell responses required for GB clearance. GB-induced up-regulation of CMA activity in PC is essential for their effective interaction with GB cells that help tumor growth. We show that CMA inhibition in PC promotes GB cell death and the release of high immunogenic levels of granulocyte-macrophage colony stimulating factor (GM-CSF), through deregulation of the expression of cell-to-cell interaction proteins and protein secretion. A GB mouse model grafted in vivo with CMA-defective PC shows reduced GB proliferation and effective immune response compared to mice grafted with control PC. Our findings identify abnormal up-regulation of CMA as a mechanism by which GB cells elicit the immunosuppressive function of PC and stabilize GB-PC interactions necessary for tumor cell survival.

Chorionic Villus-Derived Mesenchymal Stem Cell-Mediated Autophagy Promotes Proliferation and Invasiveness of Trophoblasts Under Hypoxia by Activating the JAK2/STAT3 Signalling Pathway

Trophoblast dysfunction during pregnancy is fundamentally involved in preeclampsia. To understand how human chorionic villous mesenchymal stem cells (CV-MSCs) operate in regulation of trophoblast function, we treated trophoblasts with CV-MSC supernatant under hypoxic conditions. The treatment markedly enhanced proliferation and invasion ability and augmented autophagy. Transcriptome and pathway analyses of trophoblasts before and after treatment revealed JAK2/STAT3 signalling as an upstream regulator. We further found that STAT3 mRNA and protein levels increased during CV-MSC treatment. Consistent with these findings, inhibition of JAK2/STAT3 signalling reduced autophagy, survival and invasion of trophoblasts even in the presence of CV-MSCs, and blocking autophagy did not affect STAT3 activation in trophoblasts treated with CV-MSCs. Importantly, overexpression of STAT3 increased the levels of autophagy in trophoblasts; thus, it regulated positively autophagy in hypoxic trophoblasts. Human placental explants also proved our finding, in which STAT3 was activated and LC3B-II levels were increased by CV-MSC treatment. In summary, our data suggest that CV-MSC-dependent activation of JAK2/STAT3 signalling is a prerequisite for upregulation of autophagy in trophoblasts. Funding Statement: This study was supported by the Clinical Medicine+X Project of the Affiliated Hospital of Qingdao University. Declaration of Interests: The authors declare no potential conflicts of interest. Ethics Approval Statement: Placentas (n = 2) were obtained from full‑ term births after caesarean section with parental permission. In line with the ethical protocols of the Affiliated Hospital of Qingdao University, China, every procedure was conducted accordingly.

141. Autophagy induction contributes to the neuroprotective effect of intermittent fasting in experimental spinal cord injury

BACKGROUND CONTEXT Spinal cord injury (SCI) is one of the leading causes of neurological disability and death. So far, there is no satisfactory treatment for spinal cord injury due to its complex and ill-defined pathophysiology. Recently, autophagy has been implicated to be protective in acute spinal cord injury rat models. PURPOSE We aimed to investigate the therapeutic value of a dietary intervention, namely, intermittent fasting (IF), on neuronal survival after acute SCI and its underlying mechanism relating to autophagy regulation. STUDY DESIGN/SETTING Experimental spinal cord injury in rats. PATIENT SAMPLE One hundred rats. OUTCOME MEASURES Behavioral test, Western blot, immunofluorescence and TUNEL staining were applied in the current study. METHODS Male Sprague–Dawley rats were randomly divided into the following groups: ad libitum feeding (ALF) group, ALF+acute SCI group, IF group, IF+acute SCI group and ALF+ acute SCI+3-methyladenine (3-MA) group. RESULTS We found remarkable improvement in the behavioral test in the IF group. Western blot revealed marked decrease of cleaved caspase-3 level as well as bax/bcl-2 ratio in the IF group, suggesting an inhibition of intrinsic apoptosis pathway. immunofluorescence further revealed more NeuN-positive cells in the IF group, revealing that IF preserved more neurons after acute SCI. In addition, the expression of autophagy markers like LC3-II and beclin 1 were also increased in the IF group compared to the ad libitum feeding group that underwent acute SCI, while p62, the substrate protein of autophagy, presented considerable decrease, suggesting an up regulation of autophagy by IF. Blocking autophagy with 3-MA, a selective inhibitor of autophagy, reversed the down regulated apoptosis-related markers by IF, further validating that the neuroprotective effect of IF was associated with autophagy enhancement. CONCLUSIONS Taken together, the above findings suggest that IF exerts a neuroprotective effect in acute SCI via up regulating autophagy and dietary interventions could be promising for SCI treatment. FDA DEVICE/DRUG STATUS This abstract does not discuss or include any applicable devices or drugs.

MicroRNAs play an essential role in autophagy regulation in various disease phenotypes.

Autophagy is a highly conserved catabolic process and fundamental biological process in eukaryotic cells. It recycles intracellular components to provide nutrients during starvation and maintains quality control of organelles and proteins. In addition, autophagy is a well‐organized homeostatic cellular process that is responsible for the removal of damaged organelles and intracellular pathogens. Moreover, it also modulates the innate and adaptive immune systems. Micro ribonucleic acids (microRNAs) are a mature class of post‐transcriptional modulators that are widely expressed in tissues and organs. And, it can suppress gene expression by targeting messenger RNAs for translational repression or, at a lesser extent, degradation. Research indicates that microRNAs regulate autophagy through different pathways, playing an essential role in the treatment of various diseases. It is an important regulator of fundamental cellular processes such as proliferation, autophagy, and cell apoptosis. In this review article, we first review the current knowledge of autophagy and the function of microRNAs. Then, we summarize the mechanism of autophagy and the signaling pathways related to autophagy by citing at least the main proteins involved in the different phases of the process. Second, we introduce other members of RNA and report some examples in various pathologies. Finally, we review the current literature regarding microRNA‐based therapies for cancer, atherosclerosis, cardiac disease, tuberculosis, and viral diseases. MicroRNAs can cause autophagy upregulation or downregulation by targeting genes or affecting autophagy‐related signaling pathways. Therefore, the microRNAs have a huge potential in autophagy regulation, and it is the function as diagnostic and prognostic markers.

Functional Transcriptome Analysis in ARSACS KO Cell Model Reveals a Role of Sacsin in Autophagy

Autosomal recessive spastic ataxia of Charlevoix-Saguenay (ARSACS) is a rare early-onset neurological disease caused by mutations in SACS, which encodes sacsin. The complex architecture of sacsin suggests that it could be a key player in cellular protein quality control system. Molecular chaperones that operate in protein folding/unfolding and assembly/disassembly patterns have been described as essential modulators of selectivity during the autophagy process. We performed RNA-sequencing analysis to generate a whole-genome molecular signature profile of sacsin knockout cells. Using data analysis of biological processes significantly disrupted due to loss of sacsin, we confirmed the presence of decreased mitochondrial function associated with increased oxidative stress, and also provided a demonstration of a defective autophagic pathway in sacsin-depleted cells. Western blotting assays revealed decreased expression of LC3 and increased levels of p62 even after treatment with the lysosomal inhibitor bafilomycin A1, indicating impairment of the autophagic flux. Moreover, we found reduced co-immunolocalization of the autophagosome marker LC3 with lysosomal and mitochondrial markers suggesting fusion inhibition of autophagic compartments and subsequent failed cargo degradation, in particular failed degradation of damaged mitochondria. Pharmacological up-regulation of autophagy restored correct autophagic flux in sacsin knockout cells. These results corroborate the hypothesis that sacsin may play a role in autophagy. Chemical manipulation of this pathway might represent a new target to alleviate clinical and pathological symptoms, delaying the processes of neurodegeneration in ARSACS.

TIGAR Attenuates High Glucose-Induced Neuronal Apoptosis via an Autophagy Pathway.

Hyperglycemia-induced neuronal apoptosis is one of the important reasons for diabetic neuropathy. Long-time exposure to high glucose accelerates many aberrant glucose metabolic pathways and eventually leads to neuronal injury. However, the underlying mechanisms of metabolic alterations remain unknown. TP53-inducible glycolysis and apoptosis regulator (TIGAR) is an endogenous inhibitor of glycolysis and increases the flux of pentose phosphate pathway (PPP) by regulating glucose 6-phosphate dehydrogenase (G6PD). TIGAR is highly expressed in neurons, but its role in hyperglycemia-induced neuronal injury is still unclear. In this study, we observed that TIGAR and G6PD are decreased in the hippocampus of streptozotocin (STZ)-induced diabetic mice. Correspondingly, in cultured primary neurons and Neuro-2a cell line, stimulation with high glucose induced significant neuronal apoptosis and down-regulation of TIGAR expression. Overexpression of TIGAR reduced the number of TUNEL-positive neurons and prevented the activation of Caspase-3 in cultured neurons. Furthermore, enhancing the expression of TIGAR rescued high glucose-induced autophagy impairment and the decrease of G6PD. Nitric oxide synthase 1 (NOS1), a negative regulator of autophagy, is also inhibited by overexpression of TIGAR. Inhibition of autophagy abolished the protective effect of TIGAR in neuronal apoptosis in Neuro-2a. Importantly, overexpression of TIGAR in the hippocampus ameliorated STZ-induced cognitive impairment in mice. Therefore, our data demonstrated that TIGAR may have an anti-apoptosis effect via up-regulation of autophagy in diabetic neuropathy.