Autophagic Flux In Response Research Articles

MLKL Protects Pulmonary Endothelial Cells in Acute Lung Injury.

The role of autophagy in pulmonary microvascular endothelial cells (PMVECs) is controversial in LPS-induced acute lung injury (ALI). Mixed lineage kinase domain-like pseudokinase (MLKL) has recently been reported to maintain cell survival by facilitating autophagic flux in response to starvation rather than its well-recognized role in necroptosis. Using a mouse PMVEC and LPS-induced ALI model, we showed that in PMVECs, MLKL was phosphorylated (p-MLKL) and autophagic flux was accelerated at the early stage of LPS stimulation (1-3 h), manifested by increases in concentrations of lipidated MAP1LC3B/LC3B (microtubule-associated protein 1 light chain 3 β; LC3-II), decreases in concentrations of SQSTM1/p62 (sequestosome 1), and fusion of the autophagosome and lysosome by pHluorin-mKate2-human LC3 assay, which were all reversed by either MLKL inhibitor or siRNA MLKL. In mice, the inhibition of MLKL increased vascular permeability and aggravated mouse ALI upon 3-hour LPS stimulation. The p-MLKL induced by short-term LPS formed multimers to facilitate the closure of the phagophore by HaloTag-LC3 autophagosome completion assay. The charged multivesicular body protein 2A (CHMP2A) is essential in the process of phagophore closure into the nascent autophagosome. In agreement with the p-MLKL change, CHMP2A concentrations markedly increased during 1-3-hour LPS stimulation. CHMP2A knockdown blocked autophagic flux upon LPS stimulation, whereas CHMP2A overexpression boosted autophagic flux and attenuated mouse ALI even in the presence of MLKL inhibitor. We propose that the activated MLKL induced by short-term LPS facilitates autophagic flux by accelerating the closure of the phagophore via CHMP2A, thus protecting PMVECs and alleviating LPS-induced ALI.

Abstract PR03: Cooperative anti-tumor effects of combined inhibition of KRASG12C plus autophagy in preclinical models of KRASG12C-driven lung cancer

Abstract Approximately 25% of lung cancers are driven by mutationally-activated KRAS. A major obstacle in treating lung cancer is resistance to current therapeutic treatments. The recent FDA approval of sotorasib, a direct pharmacological inhibitor of KRASG12C, marks a critical milestone in the treatment of this important subset of lung cancer. However, the twin problems of primary or acquired resistance to direct pharmacological inhibition of KRASG12C remain major obstacles in sustaining the deepest and most durable patient responses. In response to cellular stresses such as RAS pathway-targeted therapeutics, RAS-mutated cancer cells have been demonstrated to increase autophagy, an intracellular recycling pathway. Moreover, combined inhibition of RAS pathway signaling plus autophagy had potent anti-tumor effects in preclinical models of melanoma or pancreatic cancer. However, the mechanism(s) underlying treatment-induced autophagy is not well understood. Here, we demonstrate that KRASG12C-driven lung cancer cells increase autophagic flux in response to treatment with KRASG12C inhibitors. Moreover, combined treatment of such cells with a specific, selective and potent inhibitor of the ULK1/2 protein kinases, master regulators of autophagy, led to superior anti-tumor effects in human cell line xenograft models. Previous work to understand why inhibiting autophagy sensitizes KRAS mutant cancer cells to targeted therapy has been confounded by the use of non-specific lysosomal inhibitors such as hydroxychloroquine. Using selective autophagy inhibitors, such as ULK1/2i, provides a unique opportunity to investigate the precise mechanism as to why inhibition of autophagy sensitizes cells to KRASG12C inhibition. Additionally, about 30% of lung cancer patients with KRAS mutations also have deletions or inactivating mutations in LKB1, a protein involved in regulation of nutrient sensing and autophagy. Patients with KRAS-mutated lung cancer whose tumors also lack LKB1 expression are characterized by aggressive behavior and resistance to standard treatment. In other KRAS-driven cancers, the LKB1>AMPK>ULK1 signaling axis is a proposed mechanism as to how autophagic flux increases following KRAS pathway inhibition. However, our preliminary data suggests that LKB1 is dispensable for sotorasib-induced autophagy in KRASG12C-driven lung cancer cells. To this end, KRASG12C-driven lung cancer cell lines with loss of LKB1 expression significantly increased autophagic flux after treatment with either a KRASG12C inhibitor or inhibitors of RAF>MEK>ERK signaling suggesting that the mechanism(s) of autophagy induction in KRASG12C-driven may be different to those detected in other KRAS mutated cancers. Consequently, we have generated new genetically engineered mouse models of KRASG12C-driven lung cancer in which LKB1 is silenced to further investigate the role of LKB1 in the autophagy response of KRASG12C-driven lung cancers to pathway-targeted blockade of oncogenic KRAS signaling. Citation Format: Phaedra C. Ghazi, Conan Kinsey, Madhumita Bogdan, Bryan D. Smith, Daniel L. Flynn, Martin McMahon. Cooperative anti-tumor effects of combined inhibition of KRASG12C plus autophagy in preclinical models of KRASG12C-driven lung cancer [abstract]. In: Proceedings of the AACR Special Conference: Targeting RAS; 2023 Mar 5-8; Philadelphia, PA. Philadelphia (PA): AACR; Mol Cancer Res 2023;21(5_Suppl):Abstract nr PR03.

Abstract 1379: Studying the autophagic flux in response to hypoxia in BAP1-mutated pleural mesothelioma cells linked to asbestos exposure

Abstract Introduction. Malignant mesothelioma is a rare cancer linked to asbestos exposure. It comes from the oncogenic transformation of mesothelioma cells, a protective membrane that covers several internal cavities including the pleura, peritoneum and pericardium. Malignant pleural mesothelioma (MPM) accounts for more than 90% of cases. The average survival of these patients is approximately 8 to 12 months, due to the resistance of tumors towards treatments. A well-known hallmark in cancer is tumor suppression gene inactivation. BAP1 is the most mutated gene in pleural mesothelioma (25-60%). BAP1 has a deubiquitinase activity involved in chromatin modifications, homologous recombination, DNA damage and programmed cell death. Moreover, hypoxia is an important microenvironment component within tumors, and it is demonstrated in patients with MPM. The bio-persistent localization of asbestos in mesothelioma cells has been shown to induce chronic inflammation that activates autophagic flux to survive. Autophagy is induced by several types of cellular stresses, including hypoxia. This suggests that MPM cells are dependent on autophagic flux when subjected to stress to ensure their continuity and proliferation. Hypothesis/Objectives. The hypothesis of this project is that MPM has vulnerabilities in the autophagy/lysosome process, which can thus be targeted by chemical inhibitors to improve treatment response and efficacy. The specific objectives are to i) examine autophagy flux in MPM cells with BAP1 mutations, ii) demonstrate autophagy modulation in MPM cells towards the hypoxic response, and iii) assess the potential for autophagy and lysosome inhibitors in MP cells with mutations on BAP1 or towards the hypoxic response. Methods/Results. To achieve this, molecular biology approaches are used to reintroduce (Gateway system), or repress (CRISRP/Cas9) the expression of BAP1 in Mero-25 and Mero-41 cells, respectively. Models were confirmed by western blot. The hypoxic chamber is used to manipulate cells in 1% O2, in which we confirmed the activation of autophagy in response to hypoxia in a kinetic timeframe of 24, 48 and 72hr. Subsequently, the autophagic flux was studied in presence of Bafilomycin A1 indicating an activation of this cellular process. The potential and cytotoxicity of autophagy or lysosome inhibitors will be evaluated by XTT and clonogenic assays. Conclusion. Malignant pleural mesothelioma has an unmet need in novel therapeutic strategies. Our studies could contribute to improve anticancer treatments using combinatory approaches that modulate autophagy. Citation Format: Chloe Michaud, Sandra Turcotte. Studying the autophagic flux in response to hypoxia in BAP1-mutated pleural mesothelioma cells linked to asbestos exposure [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 1379.

Dysregulation of endoplasmic reticulum stress response in skin wounds in a streptozotocin-induced diabetes mouse model.

Dysfunction in key cellular organelles has been linked to diabetic complications. This study intended to investigate the alterations in the unfolded protein response (UPR), autophagy, and mitochondrial function, which are part of the endoplasmic reticulum (ER) stress response, in wound healing (WH) under diabetes conditions. WH mouse models were used to evaluate the UPR, autophagy, mitochondrial fusion, fission, and biogenesis as well as mitophagy in the skin of control and diabetic mice at baseline and 10 days after wounding. The autophagic flux in response to high-glucose conditions was also evaluated in keratinocyte and fibroblast cell cultures. WH was impaired in the diabetic mouse model, and we found that the UPR and autophagy pathways were activated in skin wounds of control mice and in the non-wounded skin of diabetic mice. Moreover, high-glucose conditions induced autophagy in the keratinocyte and fibroblast cell cultures. However, mitophagy did not change in the skin of diabetic mice or the wounded skin. In addition, mitochondrial fusion was activated in control but not in the skin wounds of diabetic mice, while mitochondrial biogenesis is downregulated in the skin of diabetic mice. In conclusion, the activation of the UPR, autophagy, and mitochondrial remodeling are crucial for a proper WH. These results suggest that the increase in ER stress and autophagy in the skin of diabetic mice at baseline significantly escalated to pathological levels after wounding, contributing to impaired WH in diabetes.

Wheat Germ Spermidine and Clove Eugenol in Combination Stimulate Autophagy In Vitro Showing Potential in Supporting the Immune System against Viral Infections.

Impaired autophagy, responsible for increased inflammation, constitutes a risk factor for the more severe COVID-19 outcomes. Spermidine (SPD) is a known autophagy modulator and supplementation for COVID-19 risk groups (including the elderly) is recommended. However, information on the modulatory effects of eugenol (EUG) is scarce. Therefore, the effects of SPD and EUG, both singularly and in combination, on autophagy were investigated using different cell lines (HBEpiC, SHSY5Y, HUVEC, Caco-2, L929 and U937). SPD (0.3 mM), EUG (0.2 mM) and 0.3 mM SPD + 0.2 mM EUG, significantly increased autophagy using the hallmark measure of LC3-II protein accumulation in the cell lines without cytotoxic effects. Using Caco-2 cells as a model, several crucial autophagy proteins were upregulated at all stages of autophagic flux in response to the treatments. This effect was verified by the activation/differentiation and migration of U937 monocytes in a three-dimensional reconstituted intestinal model (Caco-2, L929 and U937 cells). Comparable benefits of SPD, EUG and SPD + EUG in inducing autophagy were shown by the protection of Caco-2 and L929 cells against lipopolysaccharide-induced inflammation. SPD + EUG is an innovative dual therapy capable of stimulating autophagy and reducing inflammation in vitro and could show promise for COVID-19 risk groups.

Detection of Autophagy-Related Gene Expression by Conjunctival Impression Cytology in Age-Related Macular Degeneration.

Purpose: To investigate the association of autophagy-related gene expression with age-related macular degeneration (AMD). Methods: Patients with AMD were recruited for analysis by conjunctival impression cytology. mRNA was assessed by real-time polymerase chain reaction (RT-PCR) to evaluate whether the expression of 26 autophagy-related genes (ATGs) was correlated with AMD. Further studies on cell viability and autophagic flux in response to oxidative stress by H2O2 were performed in human retinal pigment epithelial (RPE) cell lines based on the results of impression cytology. Results: Both the neovascular AMD (nAMD) and polypoidal choroidal vasculopathy (PCV) groups had significantly higher mRNA levels of gamma-aminobutyric acid receptor-associated protein-like 1 (GABARAPL1) and microtubule-associated proteins 1A/1B light chain 3B (MAP1LC3B) than the control group, but there was no significant difference between these two groups. Age difference existed only in the AMD group. GABARAPL1 and MAP1LC3B mRNA expression increased significantly after acute oxidative stress in adult retinal pigment epithelial (ARPE-19) cells. Cell viability significantly increased and decreased in the cells harboring GABARAPL1 expression vector and silenced with siRNA against GABARAPL1, respectively, during short-term oxidative stress, whereas viability increased in the GABARAPL1-silenced cells after long-term oxidative stress. Silencing GABARAPL1 itself caused a reduction in autophagic flux under both short and long-term oxidative stress. Conclusion: Our study showed the possibility of assessing autophagy-related gene expression by conjunctival impression cytology. GABARAPL1 was significantly higher in AMD. Although an in vitro study showed an initial protective effect of autophagy, a cell viability study revealed the possibility of a harmful effect after long-term oxidative injury. The underlying mechanism or critical factors require further investigation.

Cardiac Shock Wave Therapy Alleviates Hypoxia/Reoxygenation-Induced Myocardial Necroptosis by Modulating Autophagy.

Regulated necrosis (necroptosis) is crucially involved in cardiac ischaemia-reperfusion injury (MIRI). The aim of our study is to investigate whether shock wave therapy (SWT) is capable of exerting protective effects by inhibiting necroptosis during myocardial ischaemia-reperfusion (I/R) injury and the possible role of autophagy in this process. We established a hypoxia/reoxygenation (H/R) model in vitro using HL-1 cells to simulate MIRI. MTS assays and LDH cytotoxicity assay were performed to measure cell viability and cell damage. Annexin V/PI staining was used to determine apoptosis and necrosis. Western blotting was performed to assess the changes in cell signaling pathways associated with autophagy, necroptosis, and apoptosis. Reactive oxygen species (ROS) production was detected using DHE staining. Autophagosome generation and degradation (autophagic flux) were analysed using GFP and RFP tandemly tagged LC3 (tfLC3). HL-1 cells were then transfected with p62/SQSTM1 siRNA in order to analyse its role in cardioprotection. Our results revealed that SWT increased cell viability in the H/R model and decreased receptor-interacting serine/threonine-protein kinase 1 (RIPK1) and RIPK3 expression. ROS production was also inhibited by SWT. Moreover, SWT decreased Beclin1 expression and the ratio of LC3-II/LC3-I following H/R. Simultaneously, in the tfLC3 assay, the SWT provoked a decrease in the cumulative autophagosome abundance. siRNA-mediated knockdown of p62 attenuated H/R-induced necroptosis, and SWT did not exert additive effects. Taken together, SWT ameliorated H/R injury by inhibiting necroptosis. SWT also relieved the blockade of autophagic flux in response to H/R injury. The restoration of autophagic flux by SWT might contribute to its cardioprotective effect on necroptosis following H/R injury.

Autophagy induced by Helicobacter pylori infection is necessary for gastric cancer stem cell emergence

The main cause of gastric cancer is the infection by the bacterium Helicobacter pylori which induces a chronic inflammation and an epithelial-to-mesenchymal transition (EMT) leading to the emergence of cells with cancer stem cell (CSC) properties. However, the underlying mechanisms have not been fully characterized. Moreover, H. pylori modulates the host cell autophagic process, but a few studies have investigated the role of this process in tumoral transformation. The aim of this study was to determine whether H. pylori-induced autophagy has a role in CSC emergence. Autophagic flux in response to H. pylori infection was characterized in AGS cell line expressing the tandem-tagged mCherry-GFP-LC3 protein and using a ratiometric flow cytometry analysis. Then, AGS and MKN45 cell lines were treated with bafilomycin or chloroquine, two pharmaceutical well-known inhibitors of autophagy, and different EMT and CSC characteristics were analyzed. First, a co-expression of the gastric CSC marker CD44 and the autophagic marker LC3 in mice and human stomach tissues infected with H.pylori was observed. Then, we demonstrated in vitro that H.pylori was able to activate the autophagy process with a reduced autophagic flux. Finally, infected cells were treated with autophagy inhibitors, which reduced (i) appearance of mesenchymal phenotypes and migration ability related to EMT and (ii) CD44 expression as well as tumorsphere formation capacities reflecting CSC properties. In conclusion, all these data show that H.pylori-induced autophagy is implicated in gastric CSC emergence and could represent an interesting therapeutic target.

The Lysosomal Membrane Protein Lamp2 Alleviates Lysosomal Cell Death by Promoting Autophagic Flux in Ischemic Cardiomyocytes.

Lysosomal membrane permeabilization (LMP) has recently been recognized as an important cell death pathway in various cell types. However, studies regarding the correlation between LMP and cardiomyocyte death are scarce. Lysosomal membrane-associated protein 2 (Lamp2) is an important component of lysosomal membranes and is involved in both autophagy and LMP. In the present study, we found that the protein content of Lamp2 gradually decreased in response to oxygen, glucose and serum deprivation (OGD) treatment in vitro. To further elucidate its role in ischemic cardiomyocytes, particularly with respect to autophagy and LMP, we infected cardiomyocytes with adenovirus carrying full-length Lamp2 to restore its protein level in cells. We found that OGD treatment resulted in the occurrence of LMP and a decline in the viability of cardiomyocytes, which were remarkably reversed by Lamp2 restoration. Exogenous expression of Lamp2 also significantly alleviated the autophagic flux blockade induced by OGD treatment by promoting the trafficking of cathepsin B (Cat B) and cathepsin D (Cat D). Through drug intervention and gene regulation to alleviate and exacerbate autophagic flux blockade respectively, we found that impaired autophagic flux in response to ischemic injury contributed to the occurrence of LMP in cardiomyocytes. In conclusion, our present data suggest that Lamp2 overexpression can improve autophagic flux blockade probably by promoting the trafficking of cathepsins and consequently conferring cardiomyocyte resistance against lysosomal cell death (LCD) that is induced by ischemic injury. These results may indicate a new therapeutic target for ischemic heart damage.

PTBP3 promotes malignancy and hypoxia-induced chemoresistance in pancreatic cancer cells by ATG12 up-regulation.

Pancreatic ductal adenocarcinoma (PDAC) tumours exhibit a high level of heterogeneity which is associated with hypoxia and strong resistance to chemotherapy. The RNA splicing protein polypyrimidine tract‐binding protein 3 (PTBP3) regulates hypoxic gene expression by selectively binding to hypoxia‐regulated transcripts. We have investigated the role of PTBP3 in tumour development and chemotherapeutic resistance in human PDAC tissues and pancreatic cancer cells. In addition, we determined the sensitivity of cancer cells to gemcitabine with differential levels of PTBP3 and whether autophagy and hypoxia affect gemcitabine resistance in vitro. PTBP3 expression was higher in human pancreatic cancer than in paired adjacent tissues. PTBP3 overexpression promoted PDAC proliferation in vitro and tumour growth in vivo, whereas PTBP3 depletion had opposing effects. Hypoxia significantly increased the expression of PTBP3 in pancreatic cancer cells in vitro. Under hypoxic conditions, cells were more resistance to gemcitabine. Knockdown of PTBP3 results in decreased resistance to gemcitabine, which was attributed to attenuated autophagy. We propose that PTBP3 binds to multiple sites in the 3′‐UTR of ATG12 resulting in overexpression. PTBP3 increases cancer cell proliferation and autophagic flux in response to hypoxic stress, which contributes to gemcitabine resistance.

Autophagy mediates temporary reprogramming and dedifferentiation in plant somatic cells.

Somatic cells acclimate to changes in the environment by temporary reprogramming. Much has been learned about transcription factors that induce these cell-state switches in both plants and animals, but how cells rapidly modulate their proteome remains elusive. Here, we show rapid induction of autophagy during temporary reprogramming in plants triggered by phytohormones, immune, and danger signals. Quantitative proteomics following sequential reprogramming revealed that autophagy is required for timely decay of previous cellular states and for tweaking the proteome to acclimate to the new conditions. Signatures of previous cellular programs thus persist in autophagy-deficient cells, affecting cellular decision-making. Concordantly, autophagy-deficient cells fail to acclimatize to dynamic climate changes. Similarly, they have defects in dedifferentiating into pluripotent stem cells, and redifferentiation during organogenesis. These observations indicate that autophagy mediates cell-state switches that underlie somatic cell reprogramming in plants and possibly other organisms, and thereby promotes phenotypic plasticity.

Disruptions of Autophagy in the Rat Retina with Age During the Development of Age-Related-Macular-Degeneration-like Retinopathy.

Age-related macular degeneration (AMD) is one of the main causes of vision impairment in the elderly. Autophagy is the process of delivery of cytoplasmic components into lysosomes for cleavage; its age-related malfunction may contribute to AMD. Here we showed that the development of AMD-like retinopathy in OXYS rats is accompanied by retinal transcriptome changes affecting genes involved in autophagy. These genes are associated with kinase activity, immune processes, and FoxO, mTOR, PI3K-AKT, MAPK, AMPK, and neurotrophin pathways at preclinical and manifestation stages, as well as vesicle transport and processes in lysosomes at the progression stage. We demonstrated a reduced response to autophagy modulation (inhibition or induction) in the OXYS retina at age 16 months: expression of genes Atg5, Atg7, Becn1, Nbr1, Map1lc3b, p62, and Gabarapl1 differed between OXYS and Wistar (control) rats. The impaired reactivity of autophagy was confirmed by a decreased number of autophagosomes under the conditions of blocked autophagosome–lysosomal fusion according to immunohistochemical analysis and transmission electron microscopy. Thus, the development of AMD signs occurs against the background of changes in the expression of autophagy-related genes and a decrease in autophagy reactivity: the ability to enhance autophagic flux in response to stress.

Frontline Science: Autophagy is a cell autonomous effector mechanism mediated by NLRP3 to control Trypanosoma cruzi infection.

Autophagy and inflammasome activation are cell-autonomous and cross-regulated processes involved in host resistance against infections. Our group previously described that NLRP3 inflammasome is required for the control of Trypanosoma cruzi, the causative agent of Chagas disease. However, the involvement of autophagy in this process was unclear. Here, we demonstrated that T. cruzi was able to induce an increase in LC3-II expression as well as autophagosome and autolysosome formation in peritoneal macrophages (PMs) from C57BL/6 wild-type mice. Moreover, the pharmacologic inhibition of autophagic machinery impaired the ability of PMs to control T. cruzi replication. Importantly, NLRP3 was required for the induction of a regular autophagic flux in response to T. cruzi, an effect mediated by its participation in the autolysosomes formation. Together, these results indicate autophagy as an effector mechanism mediated by NLRP3 to control T. cruzi infection.

Quantifying autophagic flux in kidney tissue using structured illumination microscopy.

Kidney disease is estimated to affect 15% of the world's population. Autophagy is a key homeostatic pathway in eukaryotic cells, which has been linked to numerous pathological states. In the kidney, autophagy has been shown to modulate both acute and chronic injuries. Despite the importance of autophagy in kidney disease, few techniques to precisely monitor autophagic flux in kidney tissue are available. Here we describe an improved technique to quantify autophagic flux using an RFP-GFP-LC3 reporter mouse and super-resolution microscopy. Using structured illumination microscopy, we can resolve individual autophagosomes within kidney tubular cells. We describe the preparation of slides, staining, imaging and data processing. 3D surface rendering is utilized to categorize and quantify autophagosomes by number, size, fluorescence and autophagic flux in response to ischemia.

A Potent and Selective ULK1 Inhibitor Suppresses Autophagy and Sensitizes Cancer Cells to Nutrient Stress

SummaryIn response to stress, cancer cells generate nutrients and energy through a cellular recycling process called autophagy, which can promote survival and tumor progression. Accordingly, autophagy inhibition has emerged as a potential cancer treatment strategy. Inhibitors targeting ULK1, an essential and early autophagy regulator, have provided proof of concept for targeting this kinase to inhibit autophagy; however, these are limited individually in their potency, selectivity, or cellular activity. In this study, we report two small molecule ULK1 inhibitors, ULK-100 and ULK-101, and establish superior potency and selectivity over a noteworthy published inhibitor. Moreover, we show that ULK-101 suppresses autophagy induction and autophagic flux in response to different stimuli. Finally, we use ULK-101 to demonstrate that ULK1 inhibition sensitizes KRAS mutant lung cancer cells to nutrient stress. ULK-101 represents a powerful molecular tool to study the role of autophagy in cancer cells and to evaluate the therapeutic potential of autophagy inhibition.

Autophagic Flux Is Regulated by Interaction Between the C-terminal Domain of PATCHED1 and ATG101

The Hedgehog (Hh) receptor Patched1 (PTCH1) is a well-known tumor suppressor that in its active form represses Smoothened (SMO) activity, inhibits proliferation, and induces apoptosis. The cytoplasmic C-terminal domain (CTD) regulates PTCH1 turnover and nucleates a proapoptotic complex. In this study, it was mechanistically determined that Autophagy-related 101 (ATG101), essential for mammalian autophagy, physically interacts with the CTD of PTCH1 and connects it to the ULK complex, which stimulates the autophagy machinery in response to changes in nutrient availability. This interaction results in a blockade of basal autophagic flux and accumulation of autophagosomes with undegraded cargo. Remarkably, this function of PTCH1 is independent of its repressive activity on SMO, as shown in SMO-deficient cells or in the presence of a SMO inhibitor, but is opposed by Sonic Hedgehog (SHH). These findings reveal a novel noncanonical function of PTCH1 that limits autophagy, mediated by ATG101, which could have therapeutic implications in Hh-dependent cancers.Implications: Loss-of-function of the tumor suppressor Patched1 might promote cancer cell fitness by increasing autophagic flux in response to metabolic or environmental stresses. Mol Cancer Res; 16(5); 909-19. ©2018 AACR.

Ryanodine Receptors in Autophagy: Implications for Neurodegenerative Diseases?

Intracellular Ca2+ signaling is important in the regulation of several cellular processes including autophagy. The endoplasmic reticulum (ER) is the main and largest intracellular Ca2+ store. At the ER two protein families of Ca2+ release channels, inositol 1,4,5-trisphosphate receptors (IP3Rs) and ryanodine receptors (RyRs), are expressed. Several studies have reported roles in the regulation of autophagy for the ubiquitously expressed IP3R. For instance, IP3R-mediated Ca2+ release supresses basal autophagic flux by promoting mitochondrial metabolism, while also promoting the rapid initial increase in autophagic flux in response to nutrient starvation. Insights into the contribution of RyRs in autophagy have been lagging significantly compared to the advances made for IP3Rs. This is rather surprising considering that RyRs are predominantly expressed in long-lived cells with specialized metabolic needs, such as neurons and muscle cells, in which autophagy plays important roles. In this review article, recent studies revealing roles for RyRs in the regulation of autophagy will be discussed. Several RyR-interacting proteins that have been established to modulate both RyR function and autophagy will also be highlighted. Finally, the involvement of RyRs in neurodegenerative diseases will be addressed. Inhibition of RyR channels has not only been shown to be beneficial for treating several of these diseases but also regulates autophagy.

Impaired autophagic flux is associated with the severity of trauma and the role of A2AR in brain cells after traumatic brain injury

Recent studies have shown that after traumatic brain injury (TBI), the number of autophagosomes is markedly increased in brain cells surrounding the wound; however, whether autophagy is enhanced or suppressed by TBI remains controversial. In our study, we used a controlled cortical impact system to establish models of mild, moderate and severe TBI. In the mild TBI model, the levels of autophagy-related protein 6 (Beclin1) and autophagy-related protein 12 (ATG12)-autophagy-related protein 5 (ATG5) conjugates were increased, indicating the enhanced initiation of autophagy. Furthermore, the level of the autophagic substrate sequestosome 1 (SQSTM1) was decreased in the ipsilateral cortex. This result, together with the results observed in tandem mRFP-GFP-LC3 adeno-associated virus (AAV)-infected mice, indicates that autophagosome clearance was also increased after mild TBI. Conversely, following moderate and severe TBI, there was no change in the initiation of autophagy, and autophagosome accumulation was observed. Next, we used chloroquine (CQ) to artificially impair autophagic flux in the injured cortex of the mild TBI model and found that the severity of trauma was obviously exacerbated. In addition, autophagic flux and trauma severity were significantly improved in adenosine A2A receptor (A2AR) knockout (KO) mice subjected to moderate TBI. Thus, A2AR may be involved in regulating the impairment of autophagic flux in response to brain injury. Our findings suggest that whether autophagy is increased after TBI is associated with whether autophagic flux is impaired, and the impairment of autophagic flux exacerbates the severity of trauma. Furthermore, A2AR may be a target for alleviating the impairment in autophagic flux after TBI.

IP3 Receptor-Mediated Calcium Signaling and Its Role in Autophagy in Cancer.

Calcium ions (Ca2+) play a complex role in orchestrating diverse cellular processes, including cell death and survival. To trigger signaling cascades, intracellular Ca2+ is shuffled between the cytoplasm and the major Ca2+ stores, the endoplasmic reticulum (ER), the mitochondria, and the lysosomes. A key role in the control of Ca2+ signals is attributed to the inositol 1,4,5-trisphosphate (IP3) receptors (IP3Rs), the main Ca2+-release channels in the ER. IP3Rs can transfer Ca2+ to the mitochondria, thereby not only stimulating core metabolic pathways but also increasing apoptosis sensitivity and inhibiting basal autophagy. On the other hand, IP3-induced Ca2+ release enhances autophagy flux by providing cytosolic Ca2+ required to execute autophagy upon various cellular stresses, including nutrient starvation, chemical mechanistic target of rapamycin inhibition, or drug treatment. Similarly, IP3Rs are able to amplify Ca2+ signals from the lysosomes and, therefore, impact autophagic flux in response to lysosomal channels activation. Furthermore, indirect modulation of Ca2+ release through IP3Rs may also be achieved by controlling the sarco/endoplasmic reticulum Ca2+ ATPases Ca2+ pumps of the ER. Considering the complex role of autophagy in cancer development and progression as well as in response to anticancer therapies, it becomes clear that it is important to fully understand the role of the IP3R and its cellular context in this disease. In cancer cells addicted to ER–mitochondrial Ca2+ fueling, IP3R inhibition leads to cancer cell death via mechanisms involving enhanced autophagy or mitotic catastrophe. Moreover, IP3Rs are the targets of several oncogenes and tumor suppressors and the functional loss of these genes, as occurring in many cancer types, can result in modified Ca2+ transport to the mitochondria and in modulation of the level of autophagic flux. Similarly, IP3R-mediated upregulation of autophagy can protect some cancer cells against natural killer cells-induced killing. The involvement of IP3Rs in the regulation of both autophagy and apoptosis, therefore, directly impact cancer cell biology and contribute to the molecular basis of tumor pathology.

Resveratrol-induced autophagy is dependent on IP3Rs and on cytosolic Ca2 +

Previous work revealed that intracellular Ca2+ signals and the inositol 1,4,5-trisphosphate (IP3) receptors (IP3R) are essential to increase autophagic flux in response to mTOR inhibition, induced by either nutrient starvation or rapamycin treatment. Here, we investigated whether autophagy induced by resveratrol, a polyphenolic phytochemical reported to trigger autophagy in a non-canonical way, also requires IP3Rs and Ca2+ signaling. Resveratrol augmented autophagic flux in a time-dependent manner in HeLa cells. Importantly, autophagy induced by resveratrol (80μM, 2h) was completely abolished in the presence of 10μM BAPTA-AM, an intracellular Ca2+-chelating agent. To elucidate the IP3R's role in this process, we employed the recently established HEK 3KO cells lacking all three IP3R isoforms. In contrast to the HEK293 wt cells and to HEK 3KO cells re-expressing IP3R1, autophagic responses in HEK 3KO cells exposed to resveratrol were severely impaired. These altered autophagic responses could not be attributed to alterations in the mTOR/p70S6K pathway, since resveratrol-induced inhibition of S6 phosphorylation was not abrogated by chelating cytosolic Ca2+ or by knocking out IP3Rs. Finally, we investigated whether resveratrol by itself induced Ca2+ release. In permeabilized HeLa cells, resveratrol neither affected the sarco- and endoplasmic reticulum Ca2+ ATPase (SERCA) activity nor the IP3-induced Ca2+ release nor the basal Ca2+ leak from the ER. Also, prolonged (4 h) treatment with 100μM resveratrol did not affect subsequent IP3-induced Ca2+ release. However, in intact HeLa cells, although resveratrol did not elicit cytosolic Ca2+ signals by itself, it acutely decreased the ER Ca2+-store content irrespective of the presence or absence of IP3Rs, leading to a dampened agonist-induced Ca2+ signaling. In conclusion, these results reveal that IP3Rs and cytosolic Ca2+ signaling are fundamentally important for driving autophagic flux, not only in response to mTOR inhibition but also in response to non-canonical autophagy inducers like resveratrol. This article is part of a Special Issue entitled: ECS Meeting edited by Claus Heizmann, Joachim Krebs and Jacques Haiech.