GNS561 Exhibits Potent Antiviral Activity against SARS-CoV-2 through Autophagy Inhibition

Since December 2019, SARS-CoV-2 has spread quickly worldwide, leading to more than 280 million confirmed cases, including over 5,000,000 deaths. Interestingly, coronaviruses were found to subvert and hijack autophagic process to allow their viral replication. Autophagy-modulating compounds thus rapidly emerged as an attractive strategy to fight SARS-CoV-2 infection, including the well-known chloroquine (CQ). Here, we investigated the antiviral activity and associated mechanism of GNS561/Ezurpimtrostat, a small lysosomotropic molecule inhibitor of late-stage autophagy. Interestingly, GNS561 exhibited antiviral activity of 6–40 nM depending on the viral strain considered, currently positioning it as the most powerful molecule investigated in SARS-CoV-2 infection. We then showed that GNS561 was located in lysosome-associated-membrane-protein-2-positive (LAMP2-positive) lysosomes, together with SARS-CoV-2. Moreover, GNS561 increased LC3-II spot size and caused the accumulation of autophagic vacuoles and the presence of multilamellar bodies, suggesting that GNS561 disrupted the autophagy mechanism. To confirm our findings, we used the K18-hACE2 mouse model and highlighted that GNS561 treatment led to a decline in SARS-CoV-2 virions in the lungs associated with a disruption of the autophagy pathway. Overall, our study highlights GNS561 as a powerful drug in the treatment of SARS-CoV-2 infection and supports the hypothesis that autophagy blockers could be an alternative strategy for COVID-19.

Role of cardiolipins, mitochondria, and autophagy in the differentiation process activated by all-trans retinoic acid in acute promyelocytic leukemia

The role played by lipids in the process of granulocytic differentiation activated by all-trans retinoic acid (ATRA) in Acute-Promyelocytic-Leukemia (APL) blasts is unknown. The process of granulocytic differentiation activated by ATRA in APL blasts is recapitulated in the NB4 cell-line, which is characterized by expression of the pathogenic PML-RARα fusion protein. In the present study, we used the NB4 model to define the effects exerted by ATRA on lipid homeostasis. Using a high-throughput lipidomic approach, we demonstrate that exposure of the APL-derived NB4 cell-line to ATRA causes an early reduction in the amounts of cardiolipins, a major lipid component of the mitochondrial membranes. The decrease in the levels of cardiolipins results in a concomitant inhibition of mitochondrial activity. These ATRA-dependent effects are causally involved in the granulocytic maturation process. In fact, the ATRA-induced decrease of cardiolipins and the concomitant dysfunction of mitochondria precede the differentiation of retinoid-sensitive NB4 cells and the two phenomena are not observed in the retinoid-resistant NB4.306 counterparts. In addition, ethanolamine induced rescue of the mitochondrial dysfunction activated by cardiolipin deficiency inhibits ATRA-dependent granulocytic differentiation and induction of the associated autophagic process. The RNA-seq studies performed in parental NB4 cells and a NB4-derived cell population, characterized by silencing of the autophagy mediator, ATG5, provide insights into the mechanisms underlying the differentiating action of ATRA. The results indicate that ATRA causes a significant down-regulation of CRLS1 (Cardiolipin-synthase-1) and LPCAT1 (Lysophosphatidylcholine-Acyltransferase-1) mRNAs which code for two enzymes catalyzing the last steps of cardiolipin synthesis. ATRA-dependent down-regulation of CRLS1 and LPCAT1 mRNAs is functionally relevant, as it is accompanied by a significant decrease in the amounts of the corresponding proteins. Furthermore, the decrease in CRLS1 and LPCAT1 levels requires activation of the autophagic process, as down-regulation of the two proteins is blocked in ATG5-silenced NB4-shATG5 cells.

Effect of Exercise Training On Autophagic Process in White Adipose Tissue of High Fat Diet-Induced Obese Mice

Background. Some studies have established a relationship between obesity and the autophagic process in adipose tissue. This study aimed to investigate the effect of exercise training on the autophagic process in white adipose tissue (WAT) of high fat diet-induced obese mice.Methods and Results. C57BL/6 mice were assigned into three groups included: 1) Control 2), High-Fat Diet-induced Obesity (HFD-Ob), and 3) High-Fat Diet with Exercise Training (HFD-Ex). The subjects of HFD-Ob were fed a high-fat diet for 14 weeks. The mice of HFD-Ex had eight weeks of endurance training on a treadmill in addition to having the HFD. The Real-Time–PCR and western blot methods were used to measure the mRNA and protein levels of markers of the autophagic process. HFD caused an upregulation in the factors of the autophagosome formation, including ATG5 and ATG7, LC3, and the exercise training could augment the upregulation. Further, the training program prevented the change in LAMP2 expression (a marker of autophagolysosome), which being reduced by HFD. The lysosomal clearance factors (CTSB and CTSL) were raised in HFD-Ob and differently changed in HFD-Ex.Conclusion. HFD-induced obesity promoted the early and last steps of autophagy whereas defected the intermediate-step of it. Interestingly, the exercise training enhanced the early phase of autophagy, which being increased by HFD. Further, the training program could modify the rising effect of HFD on the last step of autophagy. It seems that a part of the protective effect of exercise training on obesity-related complications may be mediated by modulating the autophagic process in white adipose tissue.

The Tubulin Code and Tubulin-Modifying Enzymes in Autophagy and Cancer

Microtubules are tubulin polymers that constitute the structure of eukaryotic cells. They control different cell functions that are often deregulated in cancer, such as cell shape, cell motility and the intracellular movement of organelles. Here, we focus on the crucial role of tubulin modifications in determining different cancer characteristics, including metastatic cell migration and therapy resistance. We also discuss the influence of microtubule modifications on the autophagic process—the cellular degradation pathway that influences cancer growth. We discuss findings showing that inducing microtubule modifications can be used as a means to kill cancer cells by inhibiting autophagy.

Rapamycin induces autophagy and increases heat tolerance in Drosophila melanogaster

Autophagy is a physiological process that facilitates the recycling of intracellular cytosolic components as a response to diverse stressful conditions. By increasing the turnover of damaged structures and clearance of long-lived and larger protein aggregates, the induction of autophagy increases tolerance to abiotic stress in a range of organisms. However, the contribution of this process to heat-tolerance of insect models remains poorly studied to date. Here, we report that rapamycin exposure in Drosophila melanogaster induces autophagy in flies, which in turn correlates with an increase in heat tolerance and quicker recovery from heat-coma. This confirms the potentially important role of the autophagic process in heat tolerance mechanisms in this organism, opening the path to further characterization of its relationship to thermal acclimation and molecular level processes related to stress.

Role of FGFR2c and Its PKCε Downstream Signaling in the Control of EMT and Autophagy in Pancreatic Ductal Adenocarcinoma Cells

Pancreatic ductal adenocarcinoma (PDAC) is a treatment-resistant malignancy characterized by a high malignant phenotype including acquired EMT signature and deregulated autophagy. Since we have previously described that the aberrant expression of the mesenchymal FGFR2c and the triggering of the downstream PKCε signaling are involved in epidermal carcinogenesis, the aim of this work has been to assess the contribution of these oncogenic events also in the pancreatic context. Biochemical, molecular and immunofluorescence approaches showed that FGFR2c expression impacts on PDAC cell responsiveness to FGF2 in terms of intracellular signaling activation, upregulation of EMT-related transcription factors and modulation of epithelial and mesenchymal markers compatible with the pathological EMT. Moreover, shut-off via specific protein depletion of PKCε signaling, activated by high expression of FGFR2c resulted in a reversion of EMT profile, as well as in a recovery of the autophagic process. The detailed biochemical analysis of the intracellular signaling indicated that PKCε, bypassing AKT and directly converging on ERK1/2, could be a signaling molecule downstream FGFR2c whose inhibition could be considered as possible effective therapeutic approach in counteracting aggressive phenotype in cancer.

Calcitriol confers neuroprotective effects in traumatic brain injury by activating Nrf2 signaling through an autophagy-mediated mechanism

Background The present study aimed to further explore the potential interaction between oxidative stress and autophagy in the progression of traumatic brain injury (TBI) and therapeutic mechanism of calcitriol, the active form of vitamin D (VitD). Methods Neuroprotective effects of calcitriol were examined following TBI. We further evaluated the impacts of TBI and calcitriol treatment on autophagic process and nuclear factor E2-related factor 2 (Nrf2) signaling. Results We found that treatment of calcitriol markedly ameliorated the neurological deficits and histopathological changes following TBI. The brain damage impaired autophagic flux and impeded Nrf2 signaling, the major regulator in antioxidant response, consequently leading to uncontrolled and excessive oxidative stress. Meanwhile, calcitriol promoted autophagic process and activated Nrf2 signaling as evidenced by the reduced Keap1 expression and enhanced Nrf2 translocation, thereby mitigating TBI-induced oxidative damage. In support, we further found that chloroquine (CQ) treatment abrogated calcitriol-induced autophagy and compromised Nrf2 activation with increased Keap1 accumulation and reduced expression of Nrf2-targeted genes. Additionally, both CQ treatment and Nrf2 genetic knockout abolished the protective effects of calcitriol against both TBI-induced neurological deficits and neuronal apoptosis. Conclusions Therefore, our work demonstrated a neuroprotective role of calcitriol in TBI by triggering Nrf2 activation, which might be mediated by autophagy.

Selective Autophagy as a Potential Therapeutic Target in Age-Associated Pathologies

Progressive accumulation of damaged cellular constituents contributes to age-related diseases. Autophagy is the main catabolic process, which recycles cellular material in a multitude of tissues and organs. Autophagy is activated upon nutrient deprivation, and oncogenic, heat or oxidative stress-induced stimuli to selectively degrade cell constituents and compartments. Specificity and accuracy of the autophagic process is maintained via the precision of interaction of autophagy receptors or adaptors and substrates by the intricate, stepwise orchestration of specialized integrating stimuli. Polymorphisms in genes regulating selective autophagy have been linked to aging and age-associated disorders. The involvement of autophagy perturbations in aging and disease indicates that pharmacological agents balancing autophagic flux may be beneficial, in these contexts. Here, we introduce the modes and mechanisms of selective autophagy, and survey recent experimental evidence of dysfunctional autophagy triggering severe pathology. We further highlight identified pharmacological targets that hold potential for developing therapeutic interventions to alleviate cellular autophagic cargo burden and associated pathologies.

Vitamin D confers neuroprotective effects in traumatic brain injury by activating Nrf2 signaling through an autophagy-mediated mechanism

Background: Traumatic brain injury (TBI) initiates an oxidative cascade that contributes to the delayed progressive damage, whereas autophagy is critical in maintaining homeostasis during stressful challenge. We previously demonstrated that vitamin D (VitD) shows strong neuroprotective and anti-oxidative properties in the animal models of TBI. Therefore, the present study aimed to further explore the potential interrelationship between oxidative stress and autophagy in the progression of TBI and therapeutic mechanism of VitD. Methods: Neuroprotective effects of calcitriol, the active form of VitD, were examined following TBI. We further evaluated the impacts of TBI and VitD treatment on autophagic process and nuclear factor E2-related factor 2 (Nrf2) signaling. To confirm the mechanism, chloroquine (CQ) treatment and Nrf2 −/− mice were used to block autophagy and Nrf2 pathway, respectively. Results: We found that treatment of calcitriol markedly ameliorated the neurological deficits and histopathological changes following TBI. The brain damage impaired autophagic flux and impeded Nrf2 signaling, the major regulator in antioxidant response, consequently leading to uncontrolled and excessive oxidative stress. Meanwhile, calcitriol promoted autophagic process and activated Nrf2 signaling as evidenced by the reduced Keap1 expression and enhanced Nrf2 translocation, thereby mitigating TBI-induced oxidative damage. To further confirm whether autophagy was responsible for Keap1 degradation and Nrf2 activation, the lysosomal inhibitor, CQ, was used to block autophagy. Our data suggested that CQ treatment abrogated calcitriol-induced autophagy and compromised Nrf2 activation with increased Keap1 accumulation and reduced expression of Nrf2-targeted genes. Additionally, both CQ treatment and Nrf2 genetic knockout abolished the protective effects of VitD against both TBI-induced neurological deficits and neuronal apoptosis. Conclusions: Therefore, our work demonstrated a neuroprotective role of VitD in TBI by triggering Nrf2 activation, which might be mediated by autophagy.

Epigenetic Regulation of Autophagy Beyond the Cytoplasm: A Review

Autophagy is a highly conserved catabolic process induced under various stress conditions to protect the cell from harm and allow survival in the face of nutrient- or energy-deficient states. Regulation of autophagy is complex, as cells need to adapt to a continuously changing microenvironment. It is well recognized that the AMPK and mTOR signaling pathways are the main regulators of autophagy. However, various other signaling pathways have also been described to regulate the autophagic process. A better understanding of these complex autophagy regulatory mechanisms will allow the discovery of new potential therapeutic targets. Here, we present a brief overview of autophagy and its regulatory pathways with emphasis on the epigenetic control mechanisms.