Autophagic Signaling Research Articles

Fetal e-cigarette exposure programs a neonatal brain hypoxic-ischemic sensitive phenotype via altering DNA methylation patterns and autophagy signaling pathway.

Maternal e-cigarette (e-cig) exposure is a pressing perinatal health concern. Emerging evidence reveals its potential adverse impacts on brain development in offspring, yet the underlying mechanisms are poorly understood. The present study tested the hypothesis that fetal e-cig exposure induces an aberrant DNA methylation profile in the developing brain, leading to alteration of autophagic flux signaling and programming of a sensitive phenotype to neonatal hypoxic-ischemic encephalopathy (HIE). Pregnant rats were exposed to chronic intermittent e-cig aerosol. Neonates were examined at the age of 9 days old. Maternal e-cig exposure decreased the body weight and brain weight but enhanced the brain-to-body weight ratio in the neonates. E-cig exposure induced a gender-dependent increase in hypoxic-ischemia-induced brain injury in male neonates associated with enhanced reactive oxygen species (ROS) activity. It differentially altered DNA methyltransferase expression and enhanced both global DNA methylation levels and specific CpG methylation at the autophagy-related gene 5 (ATG5) promoter. In addition, maternal e-cig exposure caused downregulations of ATG5, microtubule-associated protein 1 light chain 3β, and sirtuin 1 expression in neonatal brains. Of importance, knockdown of ATG5 in neonatal pups exaggerated neonatal HIE. In conclusion, the present study reveals that maternal e-cig exposure downregulates autophagy-related gene expression via DNA hypermethylation, leading to programming of a hypoxic-ischemic sensitive phenotype in the neonatal brain.

Rimonabant ameliorates hepatic ischemia/reperfusion injury in rats: Involvement of autophagy via modulating ERK- and PI3K/AKT-mTOR pathways

Hepatic ischemia/reperfusion (HIR), which can result in severe liver injury and dysfunction, is usually associated with autophagy and endocannabinoid system derangements. Whether or not the modulation of the autophagic response following HIR injury is involved in the hepatoprotective effect of the cannabinoid receptor 1(CB1R) antagonist rimonabant remains elusive and is the aim of the current study. Rats pre-treated with rimonabant (3 mg/kg) or vehicle underwent 30 min hepatic ischemia followed by 6 hrs. reperfusion. Liver injury was evaluated by serum ALT, AST, bilirubin (total and direct levels) and histopathological examination. The inflammatory, profibrotic and oxidative responses were investigated by assessing hepatic tumor necrosis factor α (TNFα), nuclear factor kappa B (NF-κB), transforming growth factor (TGF-β), lipid peroxidation and reduced glutathione. The hepatic levels of CB1R and autophagic markers p62, Beclin-1, and LC3 as well as the autophagic signaling inhibitors ERK1/2, PI3K, Akt and mTOR were also determined. Rimonabant significantly attenuated HIR-induced increases in hepatic injury, inflammation, profibrotic responses and oxidative stress and improved the associated pathological features. Rimonabant modulated the expression of p62, Beclin-1, and LC3, down-regulated CB1R, and dcreased pERK1/2, PI3K, Akt, and mTOR activities. The current study suggests that rimonabant can protect the liver from IR injury at least in part by inducing autophagy, probably by modulating ERK- and/or PI3K/AKT-mTOR signaling.

Targeting Autophagy with Natural Compounds in Cancer: A Renewed Perspective from Molecular Mechanisms to Targeted Therapy.

Natural products are well-characterized to have pharmacological or biological activities that can be of therapeutic benefits for cancer therapy, which also provide an important source of inspiration for discovery of potential novel small-molecule drugs. In the past three decades, accumulating evidence has revealed that natural products can modulate a series of key autophagic signaling pathways and display therapeutic effects in different types of human cancers. In this review, we focus on summarizing some representative natural active compounds, mainly including curcumin, resveratrol, paclitaxel, Bufalin, and Ursolic acid that may ultimately trigger cancer cell death through the regulation of some key autophagic signaling pathways, such as RAS-RAF-MEK-ERK, PI3K-AKT-mTOR, AMPK, ULK1, Beclin-1, Atg5 and p53. Taken together, these inspiring findings would shed light on exploiting more natural compounds as candidate small-molecule drugs, by targeting the crucial pathways of autophagy for the future cancer therapy.

MiR-140-3p regulates the osteogenic differentiation ability of bone marrow mesenchymal stem cells by targeting spred2-mediated autophagy.

Understanding the function and regulatory mechanism of miR-140-3p on the osteogenic differentiation of bone mesenchymal stem cells (BMSCs). Alizarin Red staining, Alkaline phosphatase (ALP) staining, and ALP activity were used to detect the ability osteogenic differentiation. miR-140-3p or Spred2 overexpression into BMSCs using lentiviral vectors and the result were analyzed by Reverse transcription quantitative polymerase chain reaction (RT-qPCR). The relation between miR-140-3p and Spred2 was examined by luciferase reporter assay. CCK8 assay was used to detect the proliferation of BMSCs. RT-qPCR and Western blot analysis were both used to detect altered gene and protein in osteogenic differentiation of BMSCs, respectively. The BMSCs which were induced for 21days were analyzed by Alizarin Red staining, (ALP) staining and ALP activity. RT-qPCR analysis showed that overexpressed miR-140-3p promotes osteogenic differentiation. Western blots results indicated that the overexpression of Spred2 suppressed miR-140-3p. Luciferase reporter assay indicated that Spred2 can integrate with miR-140-3p directly. Meanwhile, the protein level of ALP, OCN, and Runx2, the markers of chondrogenesis, was increased when miR-140-3p increased or Spred2 overexpressed in the osteoinductive medium applied to the BMSCs. Our study demonstrated the association between miR-140-3p and Spred2 in osteogenic differentiation of BMSCs for the first time. Furthermore, our detections also revealed that Spred2-induced autophagic signaling accelerates the progress of osteogenic differentiation ability of BMSCs.

The Role of Autophagy in Anti-Cancer and Health Promoting Effects of Cordycepin.

Cordycepin is an adenosine derivative isolated from Cordyceps sinensis, which has been used as an herbal complementary and alternative medicine with various biological activities. The general anti-cancer mechanisms of cordycepin are regulated by the adenosine A3 receptor, epidermal growth factor receptor (EGFR), mitogen-activated protein kinases (MAPKs), and glycogen synthase kinase (GSK)-3β, leading to cell cycle arrest or apoptosis. Notably, cordycepin also induces autophagy to trigger cell death, inhibits tumor metastasis, and modulates the immune system. Since the dysregulation of autophagy is associated with cancers and neuron, immune, and kidney diseases, cordycepin is considered an alternative treatment because of the involvement of cordycepin in autophagic signaling. However, the profound mechanism of autophagy induction by cordycepin has never been reviewed in detail. Therefore, in this article, we reviewed the anti-cancer and health-promoting effects of cordycepin in the neurons, kidneys, and the immune system through diverse mechanisms, including autophagy induction. We also suggest that formulation changes for cordycepin could enhance its bioactivity and bioavailability and lower its toxicity for future applications. A comprehensive understanding of the autophagy mechanism would provide novel mechanistic insight into the anti-cancer and health-promoting effects of cordycepin.

Oncolytic Virus-Induced Autophagy in Glioblastoma.

Simple SummaryGlioblastoma (GBM) is the most common and aggressive brain tumor with an incidence rate of nearly 3.19/100,000. Current therapeutic options fall short in improving the survival of patients with GBM. Various genetic and microenvironmental factors contribute to GBM progression and resistance to therapy. The development of gene therapies using self-replicating oncolytic viruses can advance GBM treatment. Due to GBM heterogeneity, oncolytic viruses have been genetically modified to improve the antiglioma effect in vitro and in vivo. Oncolytic viruses can activate autophagy signaling in GBM upon tumoral infection. Autophagy can be cytoprotective, whereby the GBM cells catabolize damaged organelles to accommodate to virus-induced stress, or cytotoxic, whereby it leads to the destruction of GBM cells. Understanding the molecular mechanisms that control oncolytic virus-induced autophagic signaling in GBM can fuel further development of novel and more effective genetic vectors.Autophagy is a catabolic process that allows cells to scavenge damaged organelles and produces energy to maintain cellular homeostasis. It is also an effective defense method for cells, which allows them to identify an internalized pathogen and destroy it through the fusion of the autophagosome and lysosomes. Recent reports have demonstrated that various chemotherapeutic agents and viral gene therapeutic vehicles provide therapeutic advantages for patients with glioblastoma as monotherapy or in combination with standards of care. Despite nonstop efforts to develop effective antiglioma therapeutics, tumor-induced autophagy in some studies manifests tumor resistance and glioma progression. Here, we explore the functional link between autophagy regulation mediated by oncolytic viruses and discuss how intracellular interactions control autophagic signaling in glioblastoma. Autophagy induced by oncolytic viruses plays a dual role in cell death and survival. On the one hand, autophagy stimulation has mostly led to an increase in cytotoxicity mediated by the oncolytic virus, but, on the other hand, autophagy is also activated as a cell defense mechanism against intracellular pathogens and modulates antiviral activity through the induction of ER stress and unfolded protein response (UPR) signaling. Despite the fact that the moment of switch between autophagic prosurvival and prodeath modes remains to be known, in the context of oncolytic virotherapy, cytotoxic autophagy is a crucial mechanism of cancer cell death.

Indices of Defective Autophagy in Whole Muscle and Lysosome Enriched Fractions From Aged D2-mdx Mice.

Duchenne muscular dystrophy (DMD) is a fatal, progressive muscle disease caused by the absence of functional dystrophin protein. Previous studies in mdx mice, a common DMD model, identified impaired autophagy with lysosomal insufficiency and impaired autophagosomal degradation as consequences of dystrophin deficiency. Thus, we hypothesized that lysosomal abundance would be decreased and degradation of autophagosomes would be impaired in muscles of D2-mdx mice. To test this hypothesis, diaphragm and gastrocnemius muscles from 11 month-old D2-mdx and DBA/2J (healthy) mice were collected. Whole muscle protein from diaphragm and gastrocnemius muscles, and protein from a cytosolic fraction (CF) and a lysosome-enriched fraction (LEF) from gastrocnemius muscles, were isolated and used for western blotting. Initiation of autophagy was not robustly activated in whole muscle protein from diaphragm and gastrocnemius, however, autophagosome formation markers were elevated in dystrophic muscles. Autophagosome degradation was impaired in D2-mdx diaphragms but appeared to be maintained in gastrocnemius muscles. To better understand this muscle-specific distinction, we investigated autophagic signaling in CFs and LEFs from gastrocnemius muscles. Within the LEF we discovered that the degradation of autophagosomes was similar between groups. Further, our data suggest an expanded, though impaired, lysosomal pool in dystrophic muscle. Notably, these data indicate a degree of muscle specificity as well as model specificity with regard to autophagic dysfunction in dystrophic muscles. Stimulation of autophagy in dystrophic muscles may hold promise for DMD patients as a potential therapeutic, however, it will be critical to choose the appropriate model and muscles that most closely recapitulate findings from human patients to further develop these therapeutics.

Abstract 2421: PTCH1 C-terminal domain truncations in colorectal cancer increase mitogenic signalling and autophagy

Abstract The canonical Hedgehog (Hh) signaling pathway, driving activation of SMO and GLI1, has been implicated in colorectal cancer (CRC) stemness and resistance to chemotherapy. We recently reported that the C-terminal domain (CTD) of the Hh receptor PTCH1 limits autophagic flux and ERK signaling by non-canonical mechanisms (independently of SMO/GLI1). Given that those novel functions are implicated in tumorigenesis, we explored the existence of PTCH1 CTD mutations in cancer specimens. Analysis of the TCGA Cancer Atlas database and the Genomics England 100,000 Genomes Project revealed relatively frequent somatic mutations in the CTD of PTCH1 in CRC: S1203(fs) and R1308(fs), which result in premature truncations of the CTD in about 4% of cases. PTCH1 CTD mutations were strongly associated with BRAF mutations (61% samples) and with right-sided disease (10/10 of samples with sidedness information). Co-immunoprecipitation analysis of truncated PTCH1(S1203*) and PTCH1(R1308*) showed loss of interaction with ATG101 and GRB2, required for regulation of autophagic flux and ERK signaling, respectively. To determine the pathogenic role of those mutations, we engineered SW620 metastatic CRC cell lines using CRISPR/Cas9 to create two clones with indel mutations between S1203 and R1308. Truncation of the CTD increased basal ERK and AKT phosphorylation levels, proliferation, invasion and migration compared to isogenic cells expressing wild type PTCH1. The mutant clones also showed persistent proliferation in the presence of glycolysis inhibitors and of the autophagy inducer rapamycin. Moreover, both clones displayed an enhanced autophagic flux by analysis of the LC3BI/II and p62 markers in the presence and absence of Bafilomycin A1. RNA-seq analysis revealed that the CTD truncation resulted in increase in GO terms of metabolic pathways, pathways in cancer, mTOR signaling pathway, PI3K-AKT pathway, MAPK signaling pathway and regulation of actin cytoskeleton, among others. The clones morphology changed together with a remarkable cadherin switch and loss of vimentin at RNA and protein level, suggestive of mesenchymal-epithelial transition, a process necessary for establishment of metastasis following cancer cell spread. Interestingly, one clone showed enhanced apoptosis in response to FOLFOX (5-fluorouracil/lecovorin/oxaliplatin), the most effective chemotherapy for metastatic CRC, as determined by cleaved PARP and Caspase 7 activity, compared to the parental cells. These findings reveal important tumor suppressor functions of the CTD of PTCH1 acting to suppress autophagy and mitogenic signaling, which are dysregulated by CTD mutations in a subset of colon cancer patients. Our results provide mechanistic insights of the effect of PTCH1 mutations in CRC and identify potential therapeutic vulnerabilities. Citation Format: Begoña Caballero-Ruiz, Cintli C. Morales-Alcala, Henry M. Wood, Gianluca Canettieri, Natalia A. Riobo-Del Galdo. PTCH1 C-terminal domain truncations in colorectal cancer increase mitogenic signalling and autophagy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2421.

Whole Cigarette Smoke Condensates Induce Accumulation of Amyloid Beta Precursor Protein with Oxidative Stress in Murine Astrocytes.

Although cigarette smoking has been postulated to be a potential risk factor for Alzheimer’s disease (AD), the toxic mechanism is still unclear. Additionally, astrocytes have been identified as a potential target, given they play multiple roles in maintaining normal brain function. In this study, we explored the toxic mechanism of whole cigarette smoke condensates (WCSC) using murine astrocytes. Cell proliferation, the percentage of cells in the G2/M phase, and LDH concentrations in the cell supernatants were all reduced in WCSC-treated cells. In addition, oxidative stress was induced, together with shortening of processes, structural damage of organelles, disturbances in mitochondrial function, blockage of autophagic signals, accumulation of amyloid β precursor protein, and loss of chemotactic functions. Based on these results, we hypothesize that dysfunction of astrocytes may contribute to the occurrence of cigarette-smoking-induced AD.

Docosahexaenoic acid promotes the formation of autophagosomes in MCF-7 breast cancer cells through oxidative stress-induced growth inhibitor 1 mediated activation of AMPK/mTOR pathway

Docosahexaenoic acid (DHA) is known to regulate autophagy in cancer cells. We explored whether oxidative stress-induced growth inhibitor 1 (OSGIN1) is involved in the regulation of autophagy by DHA in breast cancer cells and the possible mechanisms involved. DHA upregulated the levels of OSGIN1, LC3-II and SQSTM1/p62. By contrast, DHA dose-dependently decreased the levels of mTOR and p-mTORS2448 expression. Using GFP/RFP-LC3 fluorescence staining, we showed that cells treated with DHA showed a dose-dependent response in autophagic signals. OSGIN1 Overexpression mimicked DHA treatment in that LC3-II and GFP/RFP-LC3 signals as well as the expression of p-AMPKαT172 and p-RaptorS792 were significantly increased, whereas mTOR, p-mTORS2448, and p-ULK1S757 expression were decreased. With knockdown of OSGIN1 expression, these outcomes were reversed. Moreover, OSGIN1 overexpression transiently elevated the accumulation of OSGIN1 and reactive oxygen species (ROS) in the mitochondrial fraction and subsequently increased p-AMPKαT172 and p-RaptorS792 expression. Upon pretreatment with Mito-TEMPO, a scavenger of mitochondrial ROS, these outcomes were reversed. Taken together, these results suggest that DHA can transiently elevate the generation of ROS in mitochondria and promote autophagosome formation through activation of the p-AMPKαT172/p-Raptor S792 and inactivation of the p-mTORS2448/p-ULK1Ser757 signaling pathways, and these effects depend on OSGIN1 protein in MCF-7 cells.

The curcumin analog (PAC) suppressed cell survival and induced apoptosis and autophagy in oral cancer cells

PAC (3,5-Bis (4-hydroxy-3-methoxybenzylidene)-N-methyl-4-piperidone), a novel bioactive curcumin analog, has been reported to have anticancer properties against various tumors. However, the anti-cancer effects of PAC on oral cavity squamous cell carcinoma were not studied yet. Our aim is to investigate the anti-oral cancer properties of PAC in vitro, and determine the molecular mechanisms underlying these effects. Viability assays including MTT and LDH were conducted to measure cell proliferation. Flow cytometry-based cytotoxicity assay was performed to detect autophagic cell death and oxidative stress markers. Western blotting was used for measuring protein expression/activation in apoptotic, autophagic and pro-carcinogenic cellular signaling pathways. We demonstrated that PAC preferentially and, in a dose, -dependent way kills oral cancer cells, but was not toxic to normal human gingival cells. PAC destabilizes cell-cycle distributions, inhibits the expression of oncogenes (cyclin D1) and that of cyclin-dependent kinase inhibitor (p21WAF1) is upregulated, increases the expression of p53 gene, and inhibits epithelial-mesenchymal transition markers in oral cancer cells. The PAC effect involve various signaling pathways including NF-κB, MAPK, Wnt, caspase-3/9 and PARP1. Finally, PAC demonstrated ability to induce autophagy, decrease production of reactive oxygen species, increase intracellular glutathione (GSH) activity, and reduce mitochondrial membrane potential in oral cancer cells. In conclusion, PAC inhibits the proliferation and increases the apoptosis and autophagy and oxidative stress of oral cancer cells. These effects involve ERK1/2, p38/JNK, NF-κB and Wnt cellular signaling pathways. Overall, our study suggests the potential use of PAC to treat oral cancer.

Lifelong Aerobic Exercise Alleviates Sarcopenia by Activating Autophagy and Inhibiting Protein Degradation via the AMPK/PGC-1α Signaling Pathway.

Sarcopenia is an aging-induced syndrome characterized by a progressive reduction of skeletal muscle mass and strength. Increasing evidence has attested that appropriate and scientific exercise could induce autophagy or optimize the functional status of autophagy, which plays a critical role in senescent muscular dystrophy. As a publicly recognized strategy for extending lifespan and improving the health of the elderly, the underlying mechanisms of lifelong regular aerobic exercise for the prevention of sarcopenia have not been fully elucidated. To explore the role of lifelong aerobic exercise in the beneficial regulation of autophagic signaling pathways in senescent skeletal muscle, the natural aging mice were used as the sarcopenia model and subjected to lifelong treadmill running to evaluate corresponding parameters related to skeletal muscle atrophy and autophagic signaling pathways. Compared with the young control mice, the aged mice showed a significant reduction in skeletal muscle mass, gastrocnemius muscle weight/body weight (GMW/BW) ratio, and cross-sectional areas (CSA) of skeletal muscle fibers (p < 0.01). In contrast, lifelong aerobic exercise effectively rescued these reduced biomarkers associated with muscle atrophy. Moreover, as shown in the activated AMPK/PGC-1α signaling pathway, lifelong aerobic exercise successfully prevented the aging-induced impairment of the ubiquitin-proteasome system (UPS), excessive apoptosis, defective autophagy, and mitochondrial dysfunction. The exercise-induced autophagy suppressed the key regulatory components of the UPS, inhibited excessive apoptosis, and optimized mitochondrial quality control, thereby preventing and delaying aging-induced skeletal muscle atrophy.

Isoangustone A induces autophagic cell death in colorectal cancer cells by activating AMPK signaling

Phytochemicals, especially flavonoids, have been widely investigated for their diversified pharmacological activities including anticancer activities. Previously we identified isoangustone A from licorice-derived compounds as a potent inducer of cell death. In the present study, the exact mechanism by which isoangustone A induced cell death was further investigated, with autophagy as an indispensible part of this process. Isoangustone A treatment activated autophagic signaling and induced a complete autophagic flux in colorectal cancer cells. Knockdown of ATG5 or pre-treatment with autophagy inhibitors significantly reversed isoangustone A-induced apoptotic signaling and loss of cell viability, suggesting autophagy plays an important role in isoangustone A-induced cell death. Isoangustone A inhibited Akt/mTOR signaling, and overexpressing of a constitutively activated Akt mildly suppressed isoangustone A-induced cell death. More importantly, isoangustone A inhibited cellular ATP level and activated AMPK, and pre-treatment with AMPK inhibitor or overexpression of dominant negative AMPKα2 significantly reversed isoangustone A-induced autophagy and cell death. Further study shows isoangustone A dose-dependently inhibited mitochondrial respiration, which could be responsible for isoangustone A-induced activation of AMPK. Finally, isoangustone A at a dosage of 10 mg/kg potently activated AMPK and autophagic signaling in and inhibited the growth of SW480 human colorectal xenograft in vivo. Taken together, induction of autophagy through activation of AMPK is an important mechanism by which isoangustone A inhibits tumor growth, and isoangustone A deserves further investigation as a promising anti-cancer agent.

Ablation of optineurin impairs metabotropic glutamate receptor 5 signaling in mouse hippocampus

Optineurin (OPTN) is a ubiquitously expressed autophagy adaptor that forms a functional interactome with Group I metabotropic glutamate receptors (mGluR1 and 5), members of the Gαq/11protein receptor family. OPTN attenuates mGluR1 coupling to phospholipase Cβ (PLCβ) and subsequent inositol trisphosphate (IP3) formation in response to agonist stimulation. We have also demonstrated that pathological mGluR5 signaling impairs autophagic machinery via GSK3β/ZBTB16 pathway in both Alzheimer's and Huntington's disease mouse models. In this study, we investigated whether mGluR5 signaling is regulated in an OPTN-dependent manner. We knocked out Optn gene in STHdhQ7/Q7 striatal cells using CRISPR/Cas9 approach and employed OPTN null mice to probe the impact of OPTN loss on mGluR5 canonical and autophagic signaling. We show that OPTN deletion enhances Ca2+ mobilization following mGluR5 activation in STHdhQ7/Q7 cells and impairs mGluR5-mediated ERK1/2 phosphorylation in both STHdhQ7/Q7 cells and acute hippocampal slices. In addition, OPTN loss enhanced basal GSK3β activity and increased the breakdown of ZBTB16 in STHdhQ7/Q7 cells, and abolished mGluR5-mediated inactivation of GSK3β/ZBTB16 pathway in hippocampal slices. OPTN deletion also increased the expression levels of autophagy adaptor P62 in hippocampal slices, an effect that was reversed by mGluR5 antagonism. Overall, the data suggest that mGluR5 and OPTN crosstalk is essential for mGluR5 signaling and therefore can potentially contribute to the pathophysiology of neurodegenerative diseases.

MTORC1 hyperactivation in skeletal muscle produces early‐onset myopathy associated with autophagy dysregulation

By upregulating protein anabolism, mTORC1 maintains muscle mass and growth. However, mTORC1 upregulation is also known to promote muscle aging and produce age-associated myopathy. Whether mTORC1 activation is sufficient to produce myopathy or indirectly promotes it by accelerating tissue aging is elusive. Here we examined the effect of muscular mTORC1 hyperactivation, produced by concomitant deletion of Tsc1 and Depdc5 (MCK-TD). MCK-TD mice produced severe myopathy, associated with loss of skeletal muscle mass and force, as well as cardiac muscle hypertrophy and bradypnea. The pathologies were manifested at eight weeks of age, leading to a highly penetrant fatality at around twelve weeks of age. Transcriptome analysis indicates that both proteasomal and autophagic pathways were highly upregulated in MCK-TD skeletal muscle, in addition to inflammation, oxidative stress, and DNA damage signaling pathways. Upon mTORC1 hyperactivation, autophagosome levels were unexpectedly increased with AMPK and ULK1 upregulation. Despite the strong upregulation of autolysosomal capacities, autophagosomal-lysosomal fusion was not efficient, leading to prominent accumulation of undegraded autophagosomes and autophagy substrates. Administration of a superoxide scavenger tempol normalized most of these molecular pathologies and subsequently restored muscle histology and force generation. These results collectively indicate that mTORC1 hyperactivation is sufficient to produce early-onset myopathy in mice. Mitochondrial oxidative stress is likely the major output of how mTORC1 hyperactivation can acutely damage the muscle tissue and dysregulate autophagic and proteasomal signaling pathways.

The Role of Coactivator‐associated Arginine Methyltransferase 1 (CARM1) During Sarcopenia

CARM1 is emerging as an important regulator of skeletal muscle plasticity. We recently reported reduced muscle mass, along with aberrant autophagic and atrophic signaling in CARM1 skeletal muscle-specific knockout (mKO) mice. Notably, CARM1 interacts with forkhead box O1 (FOXO1), a master transcriptional regulator of the atrophy program, and knockout of CARM1 in skeletal muscle results in elevated FOXO1 protein content. Although CARM1 appears to be indispensable for preserving muscle mass, the impact of CARM1 deletion during the sarcopenia of aging remains to be elucidated. The purpose of this study was to more comprehensively understand the function of CARM1 during the progressive loss of skeletal muscle mass and strength with age. Survival analysis revealed that mKO mice have a significantly shorter lifespan compared to their wild type (WT) littermates. For instance, the survival rate of mKO and WT animals at 22 months is 17% and 33%, respectively. In order to assess functional and physiological adaptations to aging, grip strength and tibialis anterior (TA) muscle weights were assessed in 3 month (young) and 22 month (old) animals. We detected main effects (p < 0.05) of aging and genotype on forelimb grip strength. In particular, grip strength was significantly lower by 24% in old mKO versus old WT mice. Main effects (p < 0.05) of aging and genotype were also observed for TA muscle weight normalized to body weight. Relative to old WT mice, TA muscle weight was significantly lower by 7% in old mKO animals. We also employed Western blot analyses to assess FOXO1Ser256 activation status (phosphorylated/total), as well as the mammalian target of rapamycin complex 1 (mTORC1)Ser2448, a key regulator of muscle protein synthesis. The phosphorylation status of FOXO1Ser256 was lower (p = 0.07) in old mKO versus old WT mice, suggesting that CARM1 deletion leads to greater FOXO1 nuclear entry and transcription of atrogenes with aging. Compared to old WT animals, phosphorylation levels of mTORC1Ser2448 were also lower (p < 0.05) in old mKO mice, suggesting that CARM1 is important for driving muscle protein synthesis throughout the lifespan. Collectively, these results highlight the importance of CARM1 for combating the progressive and generalized loss of skeletal muscle mass and strength that occurs with age. Manipulating the expression and/or activity of CARM1 in skeletal muscle may offer a viable therapeutic strategy for mitigating sarcopenia.

G protein β5-ATM complexes drive acetaminophen-induced hepatotoxicity

Excessive ingestion of the common analgesic acetaminophen (APAP) leads to severe hepatotoxicity. Here we identify G protein β5 (Gβ5), elevated in livers from APAP overdose patients, as a critical regulator of cell death pathways and autophagic signaling in APAP-exposed liver. Liver-specific knockdown of Gβ5 in mice protected the liver from APAP-dependent fibrosis, cell loss, oxidative stress, and inflammation following either acute or chronic APAP administration. Conversely, overexpression of Gβ5 in liver was sufficient to drive hepatocyte dysfunction and loss. In hepatocytes, Gβ5 depletion ameliorated mitochondrial dysfunction, allowed for maintenance of ATP generation and mitigated APAP-induced cell death. Further, Gβ5 knockdown also reversed impacts of APAP on kinase cascades (e.g. ATM/AMPK) signaling to mammalian target of rapamycin (mTOR), a master regulator of autophagy and, as a result, interrupted autophagic flux. Though canonically relegated to nuclear DNA repair pathways, ATM also functions in the cytoplasm to control cell death and autophagy. Indeed, we now show that Gβ5 forms a direct, stable complex with the FAT domain of ATM, important for autophosphorylation-dependent kinase activation. These data provide a viable explanation for these novel, G protein-independent actions of Gβ5 in liver. Thus, Gβ5 sits at a critical nexus in multiple pathological sequelae driving APAP-dependent liver damage.

VPS39-deficiency observed in type 2 diabetes impairs muscle stem cell differentiation via altered autophagy and epigenetics

Insulin resistance and lower muscle quality (strength divided by mass) are hallmarks of type 2 diabetes (T2D). Here, we explore whether alterations in muscle stem cells (myoblasts) from individuals with T2D contribute to these phenotypes. We identify VPS39 as an important regulator of myoblast differentiation and muscle glucose uptake, and VPS39 is downregulated in myoblasts and myotubes from individuals with T2D. We discover a pathway connecting VPS39-deficiency in human myoblasts to impaired autophagy, abnormal epigenetic reprogramming, dysregulation of myogenic regulators, and perturbed differentiation. VPS39 knockdown in human myoblasts has profound effects on autophagic flux, insulin signaling, epigenetic enzymes, DNA methylation and expression of myogenic regulators, and gene sets related to the cell cycle, muscle structure and apoptosis. These data mimic what is observed in myoblasts from individuals with T2D. Furthermore, the muscle of Vps39+/− mice display reduced glucose uptake and altered expression of genes regulating autophagy, epigenetic programming, and myogenesis. Overall, VPS39-deficiency contributes to impaired muscle differentiation and reduced glucose uptake. VPS39 thereby offers a therapeutic target for T2D.

Knock-down of LRP/LR influences signalling pathways in late-stage colorectal carcinoma cells

BackgroundThe 37 kDa/67 kDa laminin receptor (LRP/LR) is involved in several tumourigenic-promoting processes including cellular viability maintenance and apoptotic evasion. Thus, the aim of this study was to assess the molecular mechanism of LRP/LR on apoptotic pathways in late stage (DLD-1) colorectal cancer cells upon siRNA-mediated down-regulation of LRP/LR.MethodssiRNAs were used to down-regulate the expression of LRP/LR in DLD-1 cells which was assessed using western blotting and qPCR. To evaluate the mechanistic role of LRP/LR, proteomic analysis of pathways involved in proliferation and apoptosis were investigated. The data from the study was analysed using a one-way ANOVA, followed by a two-tailed student’s t-test with a confidence interval of 95%.ResultsHere we show that knock-down of LRP/LR led to significant changes in the proteome of DLD-1 cells, exposing new roles of the protein. Moreover, analysis showed that LRP/LR may alter components of the MAPK, p53-apoptotic and autophagic signalling pathways to aid colorectal cancer cells in continuous growth and survival. Knock-down of LRP/LR also resulted in significant decreases in telomerase activity and telomerase-related proteins in the DLD-1 cells.ConclusionsThese findings show that LRP/LR is critically implicated in apoptosis and cell viability maintenance and suggest that siRNA-mediated knock-down of LRP/LR may be a possible therapeutic strategy for the treatment of colorectal cancer.

PRIMA‑1met induces autophagy in colorectal cancer cells through upregulation of the mTOR/AMPK‑ULK1‑Vps34 signaling cascade.

p53-reactivation and induction of massive apoptosis-1, APR-017 methylated (PRIMA-1met; APR246) targets mutant p53 to restore its wild-type structure and function. It was previously demonstrated that PRIMA-1met effectively inhibited the growth of colorectal cancer (CRC) cells in a p53-independent manner, and distinctly induced apoptosis by upregulating Noxa in p53-mutant cell lines. The present study including experiments of western blotting, acridine orange staining and transmission electron microscopy revealed that PRIMA-1met induced autophagy in CRC cells independently of p53 status. Importantly, PRIMA-1met not only promoted autophagic vesicle (AV) formation and AV-lysosome fusion, but also increased lysosomal degradation. Furthermore, Cell Counting Kit-8 assay, colony formation assay and small interfering RNA transfection were performed to investigate the underling mechanisms. The study indicated that activation of the mTOR/AMPK-ULK1-Vps34 autophagic signaling cascade was key for PRIMA-1met-induced autophagy. Additionally, autophagy served a crucial role in the inhibitory effect of PRIMA-1met in cells harboring wild-type p53, which was closely associated with the increased expression of Noxa. Taken together, the results determined the effect of PRIMA-1met on autophagy, and further revealed mechanistic insights into different CRC cell lines. It was concluded that PRIMA-1met-based therapy may be an effective strategy for CRC treatment.