Autophagic Status Research Articles

B cell function in patients with systemic lupus erythematosus is regulated by the upregulation of JunD

PurposeSystemic lupus erythematosus (SLE) is largely caused by B cell dysfunction. JunD is an activator protein 1 family protein that has been linked to the regulation of apoptotic and proliferative activities. However, the precise mechanism(s) by which JunD functions remains to be fully elucidated. Accordingly, this study aimed to clarify the functional importance of JUND gene expression in SLE, with further analyses of the functional role that JunD plays as a regulator of B cell proliferation and immune function. MethodsReverse transcriptase quantitative polymerase chain reaction techniques were used to analyze JunD expression in B cells of patients with SLE and healthy subjects. Cell Counting Kit-8 (CCK-8) assays and flow cytometry methods were used to characterise proliferative activity, cell cycle progression, and apoptosis of B cells in which JunD was either knocked down or overexpressed. The immune status and autophagic activity of these cells were assessed using Western immunoblotting and enzyme-linked immunosorbent assay (ELISA). Additionally, a JunD knockdown mouse model was established, and the functional role of B cell JunD expression in the pathogenesis of SLE was assessed using Western immunoblotting, ELISA, and haematoxylin and eosin staining. ResultsB cells from patients with SLE exhibited upregulation of JunD, with overexpression facilitating in vitro cellular proliferation and modulation of the immune and autophagic status of these B cells. JunD knockdown was also sufficient to modulate in vivo immune function and the autophagic status of B cells. ConclusionJunD was upregulated in the B cells of patients with SLE, where it regulates proliferation, autophagy, and immunity.

Circadian clock genes REV-ERBα regulates the secretion of IL-1β in deciduous tooth pulp stem cells by regulating autophagy in the process of physiological root resorption of deciduous teeth

Physiological root resorption is a common occurrence during the development of deciduous teeth in children. Previous research has shown that the regulation of the inflammatory microenvironment through autophagy in DDPSCs is a significant factor in this process. However, it remains unclear why there are variations in the autophagic status of DDPSCs at different stages of physiological root resorption. To address this gap in knowledge, this study examines the relationship between the circadian clock of DDPSCs, the autophagic status, and the periodicity of masticatory behavior. Samples were collected from deciduous teeth at various stages of physiological root resorption, and DDPSCs were isolated and cultured for analysis. The results indicate that the circadian rhythm of important autophagy genes, such as Beclin-1 and LC3, and the clock gene REV-ERBα in DDPSCs, disappears under mechanical stress. Additionally, the study found that REV-ERBα can regulate Beclin-1 and LC3. Evidence suggests that mechanical stress is a trigger for the regulation of autophagy via REV-ERBα. Overall, this study highlights the importance of mechanical stress in regulating autophagy of DDPSCs via REV-ERBα, which affects the formation of the inflammatory microenvironment and plays a critical role in physiological root resorption in deciduous teeth.

Diminished LC3 expression with unchanged Beclin 1 levels in right atrial appendage tissue of diabetic patients undergoing coronary artery bypass graft

Type 2 diabetes potentiates the risk of heart failure. A vital physiologic process, autophagy, may be impaired in the diabetic heart. The purpose of the present work was to explore the autophagic status in the human diabetic heart. Techniques like immunohistochemistry and western blotting were employed to examine the expression of some of the important proteins involved in autophagic machinery. Our brief study reports, for the first time, evidence of decreased cardiac autophagic levels in diabetic patients.

MiR-141-3p promotes hypoxia-induced autophagy in human placental trophoblast cells.

Preeclampsia (PE) is a pregnancy-specific disorder and a significant contributor to maternal, fetal and neonatal morbidity and mortality worldwide. Its pathogenesis is generally accepted as insufficient trophoblast invasion of the maternal endometrium and inadequate remodeling of the maternal spiral arteries. These impairments lead to elevated levels of hypoxia and oxidative stress. Autophagy has become a highly researched field in obstetrics, and this process may be essential for preimplantation development beyond the four- and eight-cell stages, and for blastocyst survival, extra-villous trophoblast functions, invasion and vascular remodeling. Several studies have shown that autophagy activation, shown by an increase in autophagy vacuoles or microtubule-associated protein 1A/1B-light chain 3 (LC3) dots, was more common in PE than in normal pregnancy. Thus, changes in autophagic status are seen in preeclamptic placentas. MicroRNA-141-3p (miR-141-3p), a multifunctional miRNA, is involved in a variety of physiological and pathological processes, including PE and autophagy. However, the influence of miR-141-3p on autophagy regulation in trophoblast cells has yet to be described. Therefore, the objective of our study was to investigate the role of miR-141-3p in autophagy induced by hypoxia in human placental trophoblast cells. Our results found that hypoxia induced autophagy in trophoblast cells and dramatically elevated the expression of miR-141-3p. Overexpression of miR-141-3p improved autophagic activity, whereas low expression of miR-141-3p inhibited autophagic activity. Therefore, our data demonstrated that miR-141-3p promoted hypoxia-induced autophagy in placental trophoblast cells, which may be related to the development of preeclampsia.

Impaired Autophagy Causes Severe Corneal Neovascularization.

To investigate the role of macrophage autophagy in the process of corneal neovascularization (CNV). In vivo, mice CNV was induced by alkali injury and compared with rapamycin-treated alkaline burn mice. Western blot was used to determine the autophagic status of the macrophages. We quantified the levels of macrophage polarization markers (CD86, INOS, CD163, CD206) by RT-qPCR and measured inflammatory factors through ELISA (IL-6 and TNF-α) in the early phase after injury. In vitro, the human umbilical vein endothelial cells (HUVECs) were co-cultured with macrophage-conditioned medium (MCM) induced by the THP-1 cell line to simulate the neovascular microenvironment. The vascularization capacity of HUVECs was examined using the CCK-8 assay kit, tube formation assay, and scratch wound-healing assay. In vivo, the mRNA expression of Beclin-1 and ATG5 was increased, together with the upregulation of M1 macrophage markers (CD86 and INOS) in corneas after early alkali injury. The area of CNV is effectively relieved in the rapamycin-treated mice. In vitro, upregulation of autophagy level by pretreatment with 3-methyladenine (3-MA) could increase the mRNA expression of the M1 markers. Macrophage-conditioned medium with impaired autophagy contains more IL-6 and TNF-α compared to the M1 macrophage-conditioned medium, promoting HUVEC proliferation, migration, and tube formation capacity. Enhancing the autophagy level with rapamycin (RAPA) could reverse this phenomenon. Impaired autophagy promoted macrophage polarization toward M1 type and increased the expression of IL-6 and TNF-α, which led to severe CNV. Using the autophagy activator (RAPA) could effectively alleviate CNV by promoting autophagy.

Intervention time decides the status of autophagy, NLRP3 activity and apoptosis in macrophages induced by ox‐LDL

BackgroundIt has been determined through extensive studies that autophagy, the Nucleotide-binding oligomerization domain-like receptor containing pyrin domain 3 (NLRP3) inflammasome and apoptotic responses in macrophages jointly contribute to atherogenesis and its development in the presence of lipid abnormalities. Few studies have investigated in full-scale if the intervention time for lipids abnormality or NLRP3 activation have a significant effect on autophagy, NLRP3 or the apoptotic status in macrophages.MethodsHuman THP-1 monocyte-derived macrophages were established by challenging THP-1 monocytes with 80 µg/ml oxidized low-density lipoprotein (ox-LDL) for specific durations. Foam cell formation was observed by Oil Red O (ORO) staining. Western blots were employed to determine protein expression. Transmission electron microscope (TEM) and immunofluorescence microscopy were applied to observe the autophagic status of cells. Cell apoptosis was evaluated by terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL).ResultsThe cells were treated with ox-LDL for 12 h and 36 h, which were considered to represent early and advanced stages of atherogenesis for this study. The results showed that inhibition of ox-LDL phagocytosis by cytochalasin D in the early stage improved autophagic status, reduced NLRP3 activation and the apoptotic response significantly. In contrast, cytochalasin D had little effect on blocking the detrimental effect of ox-LDL at the advanced stage. Moreover, the changes in autophagy, apoptosis and NLRP3 expression after treatment with small interfering (si) RNA targeting NLRP3 in the early and advanced stages of atherogenesis were consistent with the above data.ConclusionsInterventions against lipid disorders or inflammatory reactions in the early or advanced stages of atherogenesis may have different results depending on when they are applied during the process of atherosclerotic pathogenesis. These results may help improve therapeutic strategies for atherosclerosis prevention. Furthermore, a healthy lifestyle should still be recommended as the most important and inexpensive measure to prevent atherogenesis.

Autophagic state prospectively identifies facultative stem cells in the intestinal epithelium.

The intestinal epithelium exhibits a rapid and efficient regenerative response to injury. Emerging evidence supports a model where plasticity of differentiated cells, particularly those in the secretory lineages, contributes to epithelial regeneration upon ablation of injury-sensitive stem cells. However, such facultative stem cell activity is rare within secretory populations. Here, we ask whether specific functional properties predict facultative stem cell activity. We utilize in vivo labeling combined with ex vivo organoid formation assays to evaluate how cell age and autophagic state contribute to facultative stem cell activity within secretory lineages. Strikingly, we find that cell age (time elapsed since cell cycle exit) does not correlate with secretory cell plasticity. Instead, high autophagic vesicle content predicts plasticity and resistance to DNA damaging injury independently of cell lineage. Our findings indicate that autophagic status prior to injury serves as a lineage-agnostic marker for the prospective identification of facultative stem cells.

Increased Frequency of Mutations in the Gene Responsible for Familial Mediterranean Fever (MEFV) in a Cohort of Patients with Chronic Idiopathic Neutropenia

▪Introduction-Aim: Chronic idiopathic neutropenia (CIN) is a neutrophil disorder characterized by the prolonged and unexplained reduction in the number of peripheral blood (PB) absolute neutrophil counts (ANC). The underlying pathogenesis in CIN implicates the production of proinflammatory cytokines by activated lymphocytes and monocytes that induce excessive apoptotic death of the bone marrow (BM) granulocytic progenitor cells. Clonal hematopoiesis identified by next generation sequencing (NGS) of myeloid genes is found in 11% of CIN patients conferring an increased risk for MDS/AML transformation whereas the non-clonal patients display usually a benign course. The basis for the immune cell activation and proinflammatory cytokine production in CIN remains obscure. Based on previously reported data showing increased frequency of mutations of the MEFV gene encoding pyrin in patients with idiopathic inflammatory conditions other than typical Familial Mediterranean Fever (FMF), we sought to investigate the common MEFV mutations in a cohort of well characterized CIN patients.Patients-Methods: We have studied 50 patients fulfilling the previously reported diagnostic criteria of CIN (median ANC 1.5x10 9/L, range 0.2-1.7 x10 9/L), 44 females and 6 males with a median age of 56 years (range 25-87 years) and a long-follow-up (median 132 months, range 8-336 months) in the Department of Hematology of the University Hospital of Heraklion, Crete, Greece. Nonisotopic RNase cleavage assay (NIRCA) analysis was used as first screening method to detect MEFV exons 10 and 2 mutations in DNA extracted from PB or BM samples from CIN patients, confirmed by direct NGS analysis. These sequences contain the main disease-related mutations and polymorphisms.Results: Genetics alterations of MEFV were detected in 22 out of 50 CIN patients (44%). Pathogenic mutations (variants associated with typical or “atypical” FMF phenotype in Greek population) were identified in 10/50 CIN patients (20%). The 20% frequency of MEFV mutations in exon 10 and/or exon 2 in CIN patients is significantly higher compared to the carrier rate of common MEFV mutations in the healthy Greek population (0.7%) according to our previously reported data (P<0.0001, Fisher's exact test). NGS analysis confirmed the mutational pattern of NIRCA and specifically showed: (a) one patient with heterozygous I720M (ATC>ATG; Ile>Met), two patients with heterozygous A744S (GCC>TCC; Ala>Ser) and one with homozygosity, one patient with heterozygous M694V (ATG>GTG; Met>Val), one with heterozygous K695R (AAG>AGG; Lys>Arg) and one with heterozygous M680I (ATG>ATC; Met>Ile), all in exon 10, and (b) four patients with homozygous R202Q mutation in exon 2 (one patient with homozygous A744S co-mutation in exon 10) and two patients with R202Q heterozygosity combined with heterozygosity of I720M and A744S of exons 10, respectively. None of the patients displayed any symptoms/signs of FMF or other systemic inflammatory disease. No statistically significant differences were identified between MEFV mutated and non-mutated CIN patients in the severity of neutropenia or in lymphocyte, monocyte, hemoglobin and platelet counts. A significant difference was identified between the two patient groups in serum IgG (1440±264 vs 1133±245 mg/dl; P = 0.0023, Mann-Whitney test) but not IgA or IgM levels.Discussion: This study reports for the first time that 20% of unselected, consecutive patients with CIN carry mutations of the MEFV gene without clinical manifestations of FMF. Whether these patients represent atypical cases of FMF or the identified MEFV genetic alterations have a pathogenetic/modifying effect in the inflammatory responses associated with CIN is an open/novel field of research. As a first step we are currently investigating the neutrophil autophagic status, IL-1β production and the neutrophil extracellular trap (NET) formation in CIN patients with mutations in MEFV to clarify their potential effect in the immune deregulation known to characterize CIN. DisclosuresNo relevant conflicts of interest to declare.

The lncRNA PVT1 regulates autophagy in regulatory T cells to suppress heart transplant rejection in mice by targeting miR-146a

Regulatory T cells (Tregs) are indispensable for the maintenance of immune tolerance. The purpose of this study was to investigate the effect of the interaction of the lncRNA PVT1 and miR-146a on Treg autophagy and reveal the mechanism to alleviate transplant rejection. PVT1 and miR-146a expression levels were analyzed by qRT-PCR. Bioinformatic analysis and methylation profiling were used to determine the relationship between PVT1 and miR-146a. Altered autophagic status in Tregs was detected by western blotting. The effect of autophagy on Treg function was assessed in cell coculture in vitro and in animal models. Our results showed that PVT1 expression was reduced in Tregs during rejection and negatively correlated with miR-146a expression. Higher PVT1 expression was associated with higher autophagy in Tregs. Further, highly autophagic Tregs had stronger inhibitory effects on CD4+ T cells in vitro, prolonged allograft survival and alleviated rejection in vivo. Mechanistic studies showed that overexpression of PVT1 enhanced TNF receptor-associated factor (TRAF) 6 expression by directly targeting miR-146a. MiR-146a overexpression reversed PVT1-induced Treg autophagy and inhibited PVT1-induced TRAF6 expression. The present study shows a novel regulatory pathway of the autophagy program that comprises PVT1, miR-146a, and TRAF6. Our findings may provide potential targets and new therapeutic strategies for transplant rejection.

In vivo autophagy quantification: Measuring LC3 and P62 puncta in 3D image system from zebrafish larvae.

Autophagy is a central pathway in maintaining cellular homeostasis through the recycling of damaged proteins and organelles. Detection of LC3 protein levels by immunofluorescence or western blot analysis is one of the most common ways to measure autophagy. For quantitative autophagy analysis, LC3 western blot analysis is commonly used, whereas immunostaining is used for qualitative autophagy analysis. However, zebrafish larvae have a lot of proteases that rapidly degrade LC3 protein in samples. P62 is another autophagy marker that bind to damaged proteins and can reflects autophagic status. This study demonstrates a fast and accurate way to quantify autophagy from LC3 and/or P62 immunostaining images. We used a three-dimensional analysis of whole-mount LC3 immunostaining images of zebrafish larvae. Counting LC3 and P62 punctate by two dimensions can be used as a qualitative method for the analysis of autophagy. However, here we demonstrate that 3D image analysis can be used as a quantitative, rapid tool for monitoring autophagy in zebrafish larvae and avoiding drawbacks of LC3 western blot analysis.

Beclin-1/LC3-II dependent macroautophagy was uninfluenced in ischemia-challenged vascular endothelial cells

Autophagy has been extensively studied and occurs in many biological settings. However, a question remains as to whether ischemia enhances Beclin-1/LC3-II-dependent macroautophagy in vascular endothelial cells, as has been previously thought. Furthermore, the effect of the level of autophagy on cell or skin flap survival still requires elucidation. We created a lethal ischemia model in human umbilical vascular endothelial cells (HUVECs), performed quantitative proteomics and bioinformatics analyses, and verified the autophagic status and effect both in vitro and in vivo. The significantly upregulated proteins encoded by autophagy-related genes (ATGs) included ATG2A, ATG3, ATG4B, ATG5, ATG7, ATG9A, ATG12, ATG16, and ATG101. The significantly enhanced lysosomal proteins were cathepsin B, cathepsin D, lysosome-associated membrane protein 1 (LAMP1), and LAMP2. However, the differentially expressed proteins excluded Beclin-1, microtubule-associated protein light chain 3 (LC3)-I, and LC3-II. Western blot analyses verified that the protein expression levels of Beclin-1, LC3-I, and LC3-II were neither upregulated nor downregulated in ischemia-challenged HUVECs. The autophagic status was not enhanced by rapamycin in ischemic HUVECs but appeared to be inhibited by chloroquine. Our in vivo study on rats showed that a downregulation in autophagic status jeopardized skin flap survival. In conclusion, Ischemia neither enhanced nor inhibited Beclin-1/LC3-II-dependent canonical macroautophagy both in vitro and in vivo, in contradiction to previous studies. An appropriate autophagic homeostasis can minimize cell or skin flap damage.

Bone marrow-derived mesenchymal stem cells modulate autophagy in RAW264.7 macrophages via the phosphoinositide 3-kinase/protein kinase B/heme oxygenase-1 signaling pathway under oxygen-glucose deprivation/restoration conditions.

BackgroundAutophagy of alveolar macrophages is a crucial process in ischemia/reperfusion injury-induced acute lung injury (ALI). Bone marrow-derived mesenchymal stem cells (BM-MSCs) are multipotent cells with the potential for repairing injured sites and regulating autophagy. This study was to investigate the influence of BM-MSCs on autophagy of macrophages in the oxygen-glucose deprivation/restoration (OGD/R) microenvironment and to explore the potential mechanism.MethodsWe established a co-culture system of macrophages (RAW264.7) with BM-MSCs under OGD/R conditions in vitro. RAW264.7 cells were transfected with recombinant adenovirus (Ad-mCherry-GFP-LC3B) and autophagic status of RAW264.7 cells was observed under a fluorescence microscope. Autophagy-related proteins light chain 3 (LC3)-I, LC3-II, and p62 in RAW264.7 cells were detected by Western blotting. We used microarray expression analysis to identify the differently expressed genes between OGD/R treated macrophages and macrophages co-culture with BM-MSCs. We investigated the gene heme oxygenase-1 (HO-1), which is downstream of the phosphoinositide 3-kinase/protein kinase B (PI3K/Akt) signaling pathway.ResultsThe ratio of LC3-II/LC3-I of OGD/R treated RAW264.7 cells was increased (1.27 ± 0.20 vs. 0.44 ± 0.08, t = 6.67, P < 0.05), while the expression of p62 was decreased (0.77 ± 0.04 vs. 0.95 ± 0.10, t = 2.90, P < 0.05), and PI3K (0.40 ± 0.06 vs. 0.63 ± 0.10, t = 3.42, P < 0.05) and p-Akt/Akt ratio was also decreased (0.39 ± 0.02 vs. 0.58 ± 0.03, t = 9.13, P < 0.05). BM-MSCs reduced the LC3-II/LC3-I ratio of OGD/R treated RAW264.7 cells (0.68 ± 0.14 vs. 1.27 ± 0.20, t = 4.12, P < 0.05), up-regulated p62 expression (1.10 ± 0.20 vs. 0.77 ± 0.04, t = 2.80, P < 0.05), and up-regulated PI3K (0.54 ± 0.05 vs. 0.40 ± 0.06, t = 3.11, P < 0.05) and p-Akt/Akt ratios (0.52 ± 0.05 vs. 0.39 ± 0.02, t = 9.13, P < 0.05). A whole-genome microarray assay screened the differentially expressed gene HO-1, which is downstream of the PI3K/Akt signaling pathway, and the alteration of HO-1 mRNA and protein expression was consistent with the data on PI3K/Akt pathway.ConclusionsOur results suggest the existence of the PI3K/Akt/HO-1 signaling pathway in RAW264.7 cells under OGD/R circumstances in vitro, revealing the mechanism underlying BM-MSC-mediated regulation of autophagy and enriching the understanding of potential therapeutic targets for the treatment of ALI.

Knockdown of miR-146a in regulatory T cells suppresses heart transplantation rejection in mice by increasing autophagy

Clinical trials of regulatory T cells (Tregs) have shown that adoptive transfer of Tregs has great promise for the treatment of rejection. However, strategies to improve Treg function are needed in order to enhance their efficacy and reduce the number of Tregs required for adoptive transfer. Autophagy is a process for degrading intracellular components, and it mediates cell death, lymphocyte homeostasis, and Treg function. Studies have shown that the survival and function of Tregs with disrupted autophagy are defective. We found that the autophagic status of Tregs was compromised during acute rejection, allowing us to enhance Treg autophagy by regulating microRNA-146a (miR-146a), which is highly expressed in Tregs and is implicated in their function and metabolism. MiR-146a antagomir-mediated miR-146a knockdown promoted Treg autophagy, as evaluated by Western blot analysis. Further, we evaluated whether altering autophagy affects Treg function in both an in vitro cell coculture model and a heart transplantation model in mice. An increase in autophagy enhanced the inhibitory effects of Tregs on CD4+ T cells and dendritic cells (DCs) in vivo and in vitro. In addition, adoptive transfer of highly autophagic Tregs treated with miR-146a antagomir significantly alleviated rejection. Collectively, these data provide a new method that uses miR-146a knockdown to increase Treg efficacy by increasing autophagy.

Axonal Protection by Nicotinamide Riboside via SIRT1-Autophagy Pathway in TNF-Induced Optic Nerve Degeneration

Nicotinamide adenine dinucleotide (NAD+) synthesis pathway has been involved in many biological functions. Nicotinamide riboside (NR) is widely used as an NAD+ precursor and known to increase NAD+ level in several tissues. The present study aimed to examine the effect of NR on tumor necrosis factor (TNF)-induced optic nerve degeneration and to investigate whether it alters SIRT1 expression and autophagic status in optic nerve. We also examined the localization of nicotinamide riboside kinase 1 (NRK1), which is a downstream enzyme for NR biosynthesis pathway in retina and optic nerve. Intravitreal injection of TNF or TNF plus NR was performed on rats. The p62 and LC3-II protein levels were examined to evaluate autophagic flux in optic nerve. Immunohistochemical analysis was performed to localize NRK1 expression. Morphometric analysis showed substantial axonal protection by NR against TNF-induced axon loss. TNF-induced increment of p62 protein level was significantly inhibited by NR administration. NR administration alone significantly increased the LC3-II levels and reduced p62 levels compared with the basal levels, and upregulated SIRT1 levels in optic nerve. Immunohistochemical analysis showed that NRK1 exists in retinal ganglion cells (RGCs) and nerve fibers in retina and optic nerve. NR administration apparently upregulated NRK1 levels in the TNF-treated eyes as well as the control eyes. Pre-injection of an SIRT1 inhibitor resulted in a significant increase of p62 levels in the NR plus TNF treatment group, implicating that SIRT1 regulates autophagy status. In conclusion, NRK1 exists in RGCs and optic nerve axons. NR exerted protection against axon loss induced by TNF with possible involvement of upregulated NRK1 and SIRT1-autophagy pathway.

The protective role of metformin in autophagic status in peripheral blood mononuclear cells of type 2 diabetic patients.

Autophagy plays an important role in the pathophysiology of type 2 diabetes (T2D). Metformin is the most common antidiabetic drug. The main objective of this study was to explore the molecular mechanism of metformin in starvation-induced autophagy in peripheral blood mononuclear cells (PBMCs) of type 2 diabetic patients. PBMCs were isolated from 10 diabetic patients and 7 non-diabetic healthy volunteers. The autophagic puncta and markers were measured with the help of monodansylcadaverine staining and western blot. Additionally, transmission electron microscopy was also performed. No significant changes were observed in the initial autophagy marker protein levels in PBMCs of T2D after metformin treatment though diabetic PBMCs showed a high level of phospho-mammalian target of rapamycin, p62 and reduced expression of phospho-AMP-activated protein kinase and lysosomal membrane-associated protein 2, indicating a defect in autophagy. Also, induction of autophagy by tunicamycin resulted in apoptosis in diabetic PBMCs as observed by caspase-3 cleavage and reduced expression of Bcl2. Inhibition of autophagy by bafilomycin rendered consistent expression of p62 indicating a defect in the final process of autophagy. Further, electron microscopic studies also confirmed massive vacuole overload and a sign of apoptotic cell death in PBMCs of diabetic patients, whereas metformin treatment reduced the number of autophagic vacuoles perhaps by lysosomal fusion. Thus, our results indicate that defective autophagy in T2D is associated with the fusion process of lysosomes which could be overcome by metformin.

Axonal protection by a small molecule SIRT1 activator, SRT2104, with alteration of autophagy in TNF-induced optic nerve degeneration.

To examine the effects of SRT2104, an SIRT1 activator, in optic nerve degeneration induced by TNF and to investigate whether it affects the autophagic status after induction of axonal degeneration. Experimental. Adult male Wistar rats received intravitreal injection of TNF alone, concomitant injection of SRT2104 and TNF, or injection of SRT2104 alone. The autophagic status in the optic nerve was evaluated to examine p62 and LC3-II expression by immunoblot analysis. The effect of SRT2104 on TNF-induced axon loss was determined by counting the number of axons. Intravitreal injection of SRT2104 showed a modest protective tendency in the 2-pmol-treated groups against TNF-induced axon loss, although the tendency was not significant on quantitative analysis. However, significant protective effects were found in the 20- or 200-pmol-treated groups. Injection of SRT2104 alone significantly decreased the p62 levels and increased the LC3-II levels as compared with the basal levels. Similarly, concomitant injection of SRT2104 and TNF significantly decreased the p62 levels and increased the LC3-II levels as compared with the TNF-treated group. Upregulation of SIRT1 expression was observed in the optic nerve after SRT2104 treatment. The SIRT1 activator SRT2104 exerts axonal protection in TNF-induced optic nerve degeneration. This effect may be associated with upregulated autophagic status in the optic nerve.

T-Type Calcium Channels as Potential Therapeutic Targets in Vemurafenib-Resistant BRAFV600E Melanoma

Melanoma is a malignant neoplasia that is highly resistant to chemotherapy and radiotherapy and is associated with poor prognosis in advanced stage. Targeting melanoma that harbors the common BRAFV600E mutation with kinase inhibitors, such as vemurafenib, reduces tumor burden, but these tumors frequently acquire resistance to these drugs. We previously proposed that T-type calcium channel (TTCC) expression may serve as a biomarker for melanoma progression and prognosis, and we showed that TTCC blockers reduce migration and invasion rates because of autophagy blockade only in BRAFV600E-mutant melanoma cells. Here, we demonstrated that high expression of the TTCC Cav3.1 isoform is related to autophagic status in vemurafenib-resistant BRAFV600E-mutant melanoma cells and human biopsies, and in silico analysis revealed an enrichment of Cav3.1 expression in post-treatment melanomas. We also demonstrated that the TTCC blocker mibefradil induces apoptosis and impairs migration and invasion via inhibition of autophagy in resistant melanoma cells and mouse xenograft models. Moreover, we identified an association between PTEN status and Cav3.1 expression in these cells as a marker of sensitivity to combination therapy in resistant cells. Together, our results suggest that TTCC blockers offer a potential targeted therapy in resistant BRAFV600E-mutant melanoma and a therapeutic strategy to reduce progression toward BRAF inhibitor resistance.

Dexmedetomidine Protects Against Oxygen\u2013Glucose Deprivation-Induced Injury Through Inducing Astrocytes Autophagy via TSC2/mTOR Pathway

Although there is an increment in stroke burden in the world, stroke therapeutic strategies are still extremely limited to a minority of patients. We previously demonstrated that dexmedetomidine (DEX) protects against focal cerebral ischemia via inhibiting neurons autophagy. Nevertheless, the role of DEX in regulating astrocytes autophagic status in oxygen–glucose deprivation, a condition that mimics cerebral ischemia, is still unknown. In this study, we have shown that DEX and DEX + RAPA (autophagy inducer) increased viability and reduced apoptosis of primary astrocytes in oxygen–glucose deprivation (OGD) model compared with DEX + 3-methyladenine (3-MA) (autophagy inhibitor). DEX induced the expression of microtubule-associated protein 1 light chain 3 (LC3) and Beclin 1, while reduced the expression of p62 in primary cultured astrocytes through induction of autophagy. In addition, DEX enhanced the expression of tuberous sclerosis complex 2 (TSC2) in primary cultured astrocytes, while reduced the expression of mammalian target of rapamycin (mTOR). In conclusion, our study suggests that DEX exerts a neuroprotection against OGD-induced astrocytes injury via activation of astrocytes autophagy by regulating the TSC2/mTOR signaling pathway, which provides a new insight into the mechanisms of DEX treatment for acute ischemic injury.

4-Phenylbutyric acid and rapamycin improved diabetic status in high fat diet/streptozotocin-induced type 2 diabetes through activation of autophagy

An accumulating body of evidence supports the role of autophagy in the pathophysiology of T2DM. Also, abnormal endoplasmic reticulum (ER) stress response that has been implicated as a cause of insulin resistance (IR) could also be affected by the autophagic status in β-cells. The present study was designed to investigate whether autophagy is regulated in T2DM as well as to investigate the modulatory effect of the ER stress inhibitor 4-phenylbutyric acid (4-PBA) and the autophagy inducer rapamycin (Rapa) on the autophagic and diabetic status using type 2 diabetic animal model with IR. Treatment of diabetic rats with either 4-PBA or Rapa improved significantly the states of hyperglycaemia and dyslipidaemia, increased the antioxidant capacity, reduced the levels of lipid peroxidation and ER stress and increased the autophagic flux. The obtained improvements were attributed mainly to the induction of autophagy with subsequent regulation of ER stress-oxidative activation and prevention of β-cell apoptosis.

Heterogeneity of autophagic status in pancreatic β cells under metabolic stress

Autophagy in β cells has been demonstrated to play a pivotal role in cellular homeostasis and the progression of glucose intolerance. Although autophagic activity is affected by metabolic stress both in vivo and in vitro, it remains unclear as to what extent the autophagic status in each β cell is different from its neighboring cells.To address this question, GFP-LC3 reporter mice, which can visualize the autophagic status of each β cell as green-fluorescent puncta, were crossed with obese diabetic db/db mice. Imaging of green-fluorescent puncta in the islets of GFP-LC3 mice revealed that β cells are a heterogeneous population, as the density of GFP-LC3 puncta in each cell was variable. Furthermore, the variability was greater in GFP-LC3; db/db mice than in non-diabetic GFP-LC3; db/+ mice. Furthermore, when GFP-LC3 mice were treated with a low dose of S961, which antagonizes insulin signaling without inducing overt hyperglycemia, the number of β cells with a high density of GFP puncta was increased, suggesting that insulin resistance affects autophagic status independently of glucose profiles. These results suggest that pancreatic β cells under metabolic stress are heterogeneous regarding their autophagic status, which provides insights into the cellular dynamics of each β cell rather than the whole β-cell population.