Loss Of Atg7 Research Articles

Loss of Atg5 in Sertoli cells enhances the susceptibility of cadmium-impaired testicular spermatogenesis in mice.

Cadmium (Cd), one of the most common contaminants in diet and drinking water, impairs testicular germ cell development and spermatogenesis. Autophagy is essential for maintaining Sertoli cell function and Sertoli-germ cell communication. However, the role of Sertoli cell autophagy in Cd-caused spermatogenesis disorder remains unclear. Here, the mice of autophagy-related gene 5 (Atg5) knockouts in Sertoli cells were used to investigate the effect of autophagy deficiency on Cd-impaired spermatogenesis and its underlying mechanisms. Results showed that Sertoli cell-specific knockout of Atg5 exacerbated Cd-reduced sperm count and MVH (a specific marker for testicular germ cells) level in mice. Additionally, Sertoli cell Atg5 deficiency reduced the number of spermatocytes and decreased the level of meiosis-related proteins (SYCP3 and STRA8) in Cd-treated mouse testes. Loss of Atg5 in Sertoli cell exacerbated Cd-reduced the level of retinoic acid (RA) and retinal dehydrogenase (ALDH1A1 and ALDH1A) in mouse testes. Meanwhile, we found that the level of transcription factor WT1 was significantly downregulated in Atg5-/- plus Cd-treated testes. Further experiments showed that Wt1 overexpression restored Cd-decreased the levels of ALDH1A1 in Sertoli cells. Collectively, the above data suggest that knockout of Atg5 in Sertoli cell enhances the susceptibility of Cd-impaired testicular spermatogenesis. These findings provide new insights into autophagy of Sertoli cell preventing environmental toxicants-impaired testicular spermatogenesis.

Autophagy prevents early proinflammatory responses and neutrophil recruitment during Mycobacterium tuberculosis infection without affecting pathogen burden in macrophages.

The immune response to Mycobacterium tuberculosis infection determines tuberculosis disease outcomes, yet we have an incomplete understanding of what immune factors contribute to a protective immune response. Neutrophilic inflammation has been associated with poor disease prognosis in humans and in animal models during M. tuberculosis infection and, therefore, must be tightly regulated. ATG5 is an essential autophagy protein that is required in innate immune cells to control neutrophil-dominated inflammation and promote survival during M. tuberculosis infection; however, the mechanistic basis for how ATG5 regulates neutrophil recruitment is unknown. To interrogate what innate immune cells require ATG5 to control neutrophil recruitment during M. tuberculosis infection, we used different mouse strains that conditionally delete Atg5 in specific cell types. We found that ATG5 is required in CD11c+ cells (lung macrophages and dendritic cells) to control the production of proinflammatory cytokines and chemokines during M. tuberculosis infection, which would otherwise promote neutrophil recruitment. This role for ATG5 is autophagy dependent, but independent of mitophagy, LC3-associated phagocytosis, and inflammasome activation, which are the most well-characterized ways that autophagy proteins regulate inflammation. In addition to the increased proinflammatory cytokine production from macrophages during M. tuberculosis infection, loss of ATG5 in innate immune cells also results in an early induction of TH17 responses. Despite prior published in vitro cell culture experiments supporting a role for autophagy in controlling M. tuberculosis replication in macrophages, the effects of autophagy on inflammatory responses occur without changes in M. tuberculosis burden in macrophages. These findings reveal new roles for autophagy proteins in lung resident macrophages and dendritic cells that are required to suppress inflammatory responses that are associated with poor control of M. tuberculosis infection.

ATG5 provides host protection acting as a switch in the atg8ylation cascade between autophagy and secretion.

ATG5 is a part of the E3 ligase directing lipidation of ATG8 proteins, a process central to membrane atg8ylation and canonical autophagy. Loss of Atg5 in myeloid cells causes early mortality in murine models of tuberculosis. This invivo phenotype is specific to ATG5. Here, we show using human cell lines that absence of ATG5, but not of other ATGs directing canonical autophagy, promotes lysosomal exocytosis and secretion of extracellular vesicles and, in murine Atg5fl/fl LysM-Cre neutrophils, their excessive degranulation. This is due to lysosomal disrepair in ATG5 knockout cells and the sequestration by an alternative conjugation complex, ATG12-ATG3, of ESCRT protein ALIX, which acts in membrane repair and exosome secretion. These findings reveal a previously undescribed function of ATG5 in its host-protective role in murine experimental models of tuberculosis and emphasize the significance of the branching aspects of the atg8ylation conjugation cascade beyond the canonical autophagy.

Loss of ATG5 in KRT14+ cells leads to accumulated functional impairments of salivary glands via pyroptosis.

Macroautophagy/autophagy is critically involved in the process of salivary gland (SG) diseases such as xerostomia, which has a serious impact on quality of life. KRT14+ progenitor cells are found to be the main progenitors for maintaining the ductal homeostasis of the submandibular SGs. In this study, we investigated the role of ATG5 in SG KRT14+ cells in mice and humans. Human labial salivary glands (LSG) from primary Sjogren's syndrome (pSS) and non-pSS patients (normal), and submandibular glands (SMG) from Atg5flox/flox ; Krt14-Cre (cKO) mice were used. ATG5+ KRT14+ and p62+ KRT14+ cells were detected by immunofluorescence staining in LSG. TUNEL, immunofluorescence, immunohistochemistry, and western blot were performed to detect cell death in SMG. Saliva was collected in 12-week-old (12 W) and 32-week-old (32 W) mice, then the concentration of calcium and buffering capacity were detected to analyze the function of SG. We found that LSG from pSS patients showed increased p62 and decreased ATG5 in KRT14+ cells. We further revealed that in 32 W, (1) the function of salivary glands was significantly impaired in cKO mice, (2) cell death increased in cKO mice, but cl-Caspase 3 was not significantly changed, and (3) cleaved gasdermin D increased and was highly expressed in KRT14+ cells of cKO mice. After applying a pyroptosis inhibitor to 32 W mice, the reduced saliva flow rate was rescued. In addition, pyroptosis was also found in KRT14+ cells of pSS patients. Collectively, our results indicate that Atg5 deficiency would induce pyroptosis in mice SG, which could lead to functional impairments of SG.

Activation of Autophagic Flux Maintains Mitochondrial Homeostasis during Cardiac Ischemia/Reperfusion Injury.

Reperfusion injury after extended ischemia accounts for approximately 50% of myocardial infarct size, and there is no standard therapy. HDAC inhibition reduces infarct size and enhances cardiomyocyte autophagy and PGC1α-mediated mitochondrial biogenesis when administered at the time of reperfusion. Furthermore, a specific autophagy-inducing peptide, Tat-Beclin 1 (TB), reduces infarct size when administered at the time of reperfusion. However, since SAHA affects multiple pathways in addition to inducing autophagy, whether autophagic flux induced by TB maintains mitochondrial homeostasis during ischemia/reperfusion (I/R) injury is unknown. We tested whether the augmentation of autophagic flux by TB has cardioprotection by preserving mitochondrial homeostasis both in vitro and in vivo. Wild-type mice were randomized into two groups: Tat-Scrambled (TS) peptide as the control and TB as the experimental group. Mice were subjected to I/R surgery (45 min coronary ligation, 24 h reperfusion). Autophagic flux, mitochondrial DNA (mtDNA), mitochondrial morphology, and mitochondrial dynamic genes were assayed. Cultured neonatal rat ventricular myocytes (NRVMs) were treated with a simulated I/R injury to verify cardiomyocyte specificity. The essential autophagy gene, ATG7, conditional cardiomyocyte-specific knockout (ATG7 cKO) mice, and isolated adult mouse ventricular myocytes (AMVMs) were used to evaluate the dependency of autophagy in adult cardiomyocytes. In NRVMs subjected to I/R, TB increased autophagic flux, mtDNA content, mitochondrial function, reduced reactive oxygen species (ROS), and mtDNA damage. Similarly, in the infarct border zone of the mouse heart, TB induced autophagy, increased mitochondrial size and mtDNA content, and promoted the expression of PGC1α and mitochondrial dynamic genes. Conversely, loss of ATG7 in AMVMs and in the myocardium of ATG7 cKO mice abolished the beneficial effects of TB on mitochondrial homeostasis. Thus, autophagic flux is a sufficient and essential process to mitigate myocardial reperfusion injury by maintaining mitochondrial homeostasis and partly by inducing PGC1α-mediated mitochondrial biogenesis.

Investigating immunoregulatory effects of myeloid cell autophagy in acute and chronic inflammation.

Studies have highlighted a critical role for autophagy in the regulation of multiple cytokines. Autophagy inhibits the release of interleukin (IL)‐1 family cytokines, including IL‐1α, IL‐1β and IL‐18, by myeloid cells. This, in turn, impacts the release of other cytokines by myeloid cells, as well as other cells of the immune system, including IL‐22, IL‐23, IL‐17 and interferon‐γ. Here, we assessed the impact of genetic depletion of the autophagy gene Atg7 in myeloid cells on acute and chronic inflammation. In a model of acute lipopolysaccharide‐induced endotoxemia, loss of autophagy in myeloid cells resulted in increased release of proinflammatory cytokines, both locally and systemically. By contrast, loss of Atg7 in myeloid cells in the Lyn −/− model of lupus‐like autoimmunity resulted in reduced systemic release of IL‐6 and IL‐10, with no effects on other cytokines observed. In addition, Lyn −/− mice with autophagy‐deficient myeloid cells showed reduced expression of autoantibodies relevant to systemic lupus erythematosus, including anti‐histone and anti‐Smith protein. In vitro, loss of autophagy, through pharmacological inhibition or small interfering RNA against Becn1, inhibited IL‐10 release by human and mouse myeloid cells. This effect was evident at the level of Il10 messenger RNA expression. Our data highlight potentially important differences in the role of myeloid cell autophagy in acute and chronic inflammation and demonstrate a direct role for autophagy in the production and release of IL‐10 by macrophages.

Disruption of ATG9A‐dependent basal autophagy causes an accumulation of ubiquitin‐rich condensates which act as a platform for inflammatory signaling

Excessive inflammation underlies many human diseases, such as neurodegeneration, autoimmune disorders, cancer, and COVID‐19. Thus, cellular mechanisms that control inflammation are of high therapeutic interest. Autophagy has been implicated in the suppression of inflammation, yet mechanistic links between autophagy and inflammation are not completely understood. Previous work demonstrated that loss of ATG9A, but not ATG5 or ATG7, increased inflammatory signaling through the STING‐IRF3 cascade, suggesting that perhaps an autophagy‐independent function of ATG9A regulates inflammation.1 Here, we show that loss of the essential basal autophagy regulators ATG9A and ATG101 sensitizes cells to dsDNA‐induced IRF3 activation. Importantly, this effect was not observed with the loss of ATG5 or ATG7, suggesting that the canonical LC3‐lipidation autophagy machinery is not required for suppression of IRF3 and inflammatory signaling.2 In an effort to understand the role of ATG9A and ATG101 in suppressing inflammatory signaling, we found that loss of ATG9A and ATG101, but not ATG5 or ATG7, caused an accumulation of ubiquitin‐rich condensates. We also found that the accumulation of ubiquitin‐rich condensates coincided with an overactivation of the ubiquitin‐sensing, IRF3‐targeted kinase TBK1. Importantly, we found that inhibiting the accumulation of ubiquitin‐rich condensates via knock‐out of p62/SQSTM1 abrogated the increased inflammatory signaling caused by loss of ATG9A. Together, our data suggest a model in which the loss of basal autophagy machinery causes an accumulation of ubiquitin‐rich condensates, which, in turn, act as a platform for the aberrant activation of TBK1‐mediated inflammatory signaling. We propose that these data have important implications for diseases of protein aggregation wherein similar ubiquitin‐rich condensates form and aberrant inflammatory signaling plays a pathological role.References(1) Saitoh, T.; Fujita, N.; Hayashi, T.; Takahara, K.; Satoh, T.; Lee, H.; Matsunaga, K.; Kageyama, S.; Omori, H.; Noda, T.; Yamamoto, N.; Kawai, T.; Ishii, K.; Takeuchi, O.; Yoshimori, T.; Akira, S. Atg9a Controls DsDNA‐Driven Dynamic Translocation of STING and the Innate Immune Response. PNAS 2009, 106 (49). https://doi.org/10.1073/pnas.0911267106.(2) Kannangara, A. R.; Poole, D. M.; McEwan, C. M.; Youngs, J. C.; Weerasekara, V. K.; Thornock, A. M.; Lazaro, M. T.; Balasooriya, E. R.; Oh, L. M.; Soderblom, E. J.; Lee, J. J.; Simmons, D. L.; Andersen, J. L. BioID Reveals an ATG9A Interaction with ATG13‐ATG101 in the Degradation of P62/SQSTM1‐ubiquitin Clusters. EMBO reports 2021, 22 (10). https://doi.org/10.15252/embr.202051136.

Autophagy deficiency promotes lung metastasis of prostate cancer via stabilization of TWIST1.

The role of autophagy in prostate cancer metastasis remains controversial, and the effects of the autophagy-related gene ATG5 on prostate cancer metastasis are poorly understood. This study aims to explore the effects of ATG5 on prostate cancer metastasis and its molecular mechanism. The metastatic characteristics of LNCaP and DU145 cells were assessed by NOD/SCID mouse experiments, western blot, transwell assay, and wound-healing assay. Double membrane autophagic vesicle observation and the adenovirus-expressing mCherry-GFP-LC3B fusion protein were used to assess the autophagic characteristics of LNCaP and DU145 cells. The role of p62 in the accumulation of TWIST1 was confirmed by western blot under different conditions. The lentivirus particles of shATG5, NOD/SCID mice experiments, western blot, transwell assay, and wound-healing assay were used to confirm the role of ATG5 in TWIST1 accumulation and prostate cancer cell metastasis. We identified a stabilizing effect of p62 on TWIST1 in the autophagic regulation of EMT and prostate cancer metastasis. The loss of ATG5 in DU145 cells resulted in autophagy deficiency and p62 accumulation, which stabilized TWIST1 and increased the TWIST1 level in prostate cancer cells, and eventually promoted EMT and metastasis. In comparison, LNCaP cells with regular ATG5 expression and autophagy status retained remarkable epithelial cell characteristics and had limited metastatic characteristics. Similar results were also found in wild-type LNCaP cells and LNCaP cells with stable ATG5 interference. Our research revealed ATG5-mediated autophagy as a key mechanism that controls the metastasis of prostate cancer by regulating p62 abundance and TWIST1 stabilization.

Abstract 13433: Enhancing Autophagic Flux Maintains Mitochondrial Homeostasis in Cardiac Ischemia/Reperfusion Injury

Introduction and Hypothesis: Histone deacetylase (HDAC) inhibition enhances cardiomyocyte autophagic activity, mitochondria biogenesis, and blunts ischemia/reperfusion (I/R) injury when given at the time of reperfusion. Similarly, augmentation of autophagic flux using a specific autophagy-inducing peptide, Tat-Beclin (TB), is cardioprotective by reducing infarct ~50% and improved systolic function. TB increases autophagic flux and reduced reactive oxygen species (ROS) in the infarct border zone, which is abolished in ATG7 KO mice. We will test whether TB cleans damaged mitochondria and induces mitochondrial biogenesis during cardiac I/R injury. Methods: 8-12-week-old, wild-type, C57BL6 mice were randomized into two groups: TB and Tat-Scrambled (TS) control peptide. Each group was subjected to I/R surgery (45min ischemia, 24h reperfusion). Mitochondrial abundance, quality, and dynamics were assayed. In addition, cultured adult mouse ventricular myocytes (AMVMs) from conditional cardiomyocyte ATG7 knockout (ATG7 KO) and control mice, and neonatal rat ventricular myocytes (NRVMs) treated with ATG7 and control siRNA were used to evaluate the role of autophagy. Results: TB increased mitochondria DNA (mtDNA) content in the border zone significantly. In NRVMs subjected to I/R, TB reduced cell death by ~40%, decreased ROS, and increased mtDNA content significantly by ~50%. TB also increased the intact mtDNA significantly. Moreover, TB promoted the expression of PGC1α (a major driver of mitochondrial biogenesis) both in the infarct border zone and NRVMs subjected to I/R by ~40%, and increased levels of mitochondrial dynamics gene transcripts Drp1, Fis1, and MFN1 / 2. Conversely, ATG7 knockdown in NRVMs and cardiomyocyte-specific ATG7 KO in myocardium abolished the beneficial effects of TB on mtDNA content. To test the changes in the myocardium are cardiomyocyte-specific, we verified the effects of TB and the loss of ATG7 in isolated AMVMs. Conclusions: Autophagic flux is an essential process to mitigate myocardial reperfusion injury acting, at least in part, by inducing PGC1α-mediated mitochondrial biogenesis. Augmentation of autophagic flux may emerge as a viable clinical therapy to reduce I/R injury in myocardial infarction.

Ulk1 Kinase Mediates Clearance of Free α-Globin in β-Thalassemia

Normal erythroid maturation is highly regulated to maximize the production of HbA heterotetramer (α2β2) and minimize the accumulation of potentially toxic free α- or β-globin subunits. In β-thalassemia, β-globin gene (HBB) mutations cause buildup of free α-globin, which forms intracellular precipitates that impair erythroid cell maturation and viability. Endogenous mechanisms exist to detoxify free α-globin, as evidenced by clinical observations that most individuals with HBB haploinsufficiency (β-thalassemia trait) are asymptomatic. Previously, we showed that free α-globin is eliminated in erythroid precursors by the ubiquitin-proteasome (UPS) system and by a lysosomal-dependent process presumed to be autophagy (Khandros et al., Blood 2012;119:5265). Our current study investigates the latter.Atg5 is required for basal autophagy and autophagosome formation via conjugation with Atg12 in most cell types. We investigated whether Atg5 played a selective role in eliminating free α-globin during unbalanced globin chain synthesis. β-thalassemic mice (HbbTh3/+) carrying a conditional Atg5 allele (Atg5fl) in which exon 3 is flanked by loxP sites were interbred with mice expressing erythroid-specific Cre recombinase (EpoR-Cre). Loss of Atg5 in β-thalassemic erythroid precursors inhibited autophagy, as evidenced by the accumulation of the markers p62/SQSTM1 and LC3. However, there was minimal effect on the β-thalassemic phenotype as measured by RBC count, Hb, hematocrit, reticulocyte count, and spleen weight. Moreover, loss of Atg5 did not alter the level of insoluble α-globin in β-thalassemic RBC fractions, as assessed by triton-acetic acid-urea (TAU) gel electrophoresis to resolve the α- and β-globin chains. Thus, Atg5, a key component of canonical autophagy, is not required for the elimination of free α-globin in β-thalassemia.The Ulk1 (unc-51−like kinase 1) protein kinase participates in canonical and alternative autophagy pathways and mediates mitophagy during erythropoiesis. To determine the role of Ulk1 in a-globin degradation, we introduced a null allele into HbbTh3/+ mice. Ulk1−/− HbbTh3/+ mice were born at normal Mendelian ratio, but exhibited a high rate of perinatal death. Only 20% of double-mutant mice survived for 30 days (n=5), compared to 70% of Ulk1+/+ HbbTh3/+ mice (n=13) (P <0.05). To examine the consequences of Ulk1 loss in β-thalassemic erythroblasts, we transplanted whole bone marrow cells from double mutants and controls (CD45.2) into lethally irradiated wild-type hosts (CD45.1; >87% engraftment after 30 days). Loss of Ulk1 exacerbated the β-thalassemic phenotype, as evidence by reduced Hb (10.85 ± 0.18 vs 10.21 ± 0.18 g/dL, n=14 vs 8; P <0.01), reduced RBC number (7.37 ± 0.13 vs 6.35 ± 0.09 × 106/mL; P <0.0001), increased reticulocyte count (17.99 ± 2.76 vs 39.41 ± 2.14%; P <0.0001), and increased spleen weight (0.24 ± 0.01 vs 0.34 ± 0.03 g; P <0.01). Moreover, insoluble α-globin was increased by approximately 2-fold in Ulk1−/− HbbTh3/+ reticulocytes, ascompared to Ulk1+/+ HbbTh3/+ cells (Figure). To assess the turnover of newly synthesized free α-globin, we pulse-labeled reticulocytes with 35S amino acids and chased with or without a proteasome inhibitor (MG132) or a lysosome inhibitor (chloroquine or bafilomycin A1). Analysis of control (Ulk1+/+ HbbTh3/+) reticulocytesshowed that newly synthesized insoluble free α-globin was cleared by separate proteasomal and lysosomal pathways. In marked contrast, the lysosomal-dependent α-globin degradation pathway was completely eliminated in Ulk1−/− HbbTh3/+ reticulocytes. Thus, we conclude that Ulk1 mediates the degradation of free α-globin in β-thalassemia.Our findings illustrate a new mechanism through which protein quality-control pathways can modulate the severity of β-thalassemia by eliminating unstable free α-globin, and they open up possibilities for new therapeutic approaches, as Ulk1 activity can be modulated pharmacologically. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Developmental Consequences of Defective ATG7-Mediated Autophagy in Humans

BackgroundAutophagy is the major intracellular degradation route in mammalian cells. Systemic ablation of core autophagy-related (ATG) genes in mice leads to embryonic or perinatal lethality, and conditional models show neurodegeneration. Impaired autophagy has been associated with a range of complex human diseases, yet congenital autophagy disorders are rare.MethodsWe performed a genetic, clinical, and neuroimaging analysis involving five families. Mechanistic investigations were conducted with the use of patient-derived fibroblasts, skeletal muscle–biopsy specimens, mouse embryonic fibroblasts, and yeast.ResultsWe found deleterious, recessive variants in human ATG7, a core autophagy-related gene encoding a protein that is indispensable to classical degradative autophagy. Twelve patients from five families with distinct ATG7 variants had complex neurodevelopmental disorders with brain, muscle, and endocrine involvement. Patients had abnormalities of the cerebellum and corpus callosum and various degrees of facial dysmorphism. These patients have survived with impaired autophagic flux arising from a diminishment or absence of ATG7 protein. Although autophagic sequestration was markedly reduced, evidence of basal autophagy was readily identified in fibroblasts and skeletal muscle with loss of ATG7. Complementation of different model systems by deleterious ATG7 variants resulted in poor or absent autophagic function as compared with the reintroduction of wild-type ATG7.ConclusionsWe identified several patients with a neurodevelopmental disorder who have survived with a severe loss or complete absence of ATG7, an essential effector enzyme for autophagy without a known functional paralogue. (Funded by the Wellcome Centre for Mitochondrial Research and others.)

Essential Role of CRIM1 on Endometrial Receptivity in Goat.

In domestic ruminants, endometrial receptivity is related to successful pregnancy and economic efficiency. Despite several molecules having been reported in the past regarding endometrial receptivity regulation, much regarding the mechanism of endometrial receptivity regulation remains unknown due to the complex nature of the trait. In this work, we demonstrated that the cysteine-rich transmembrane bone morphogenetic protein (BMP) regulator 1 (CRIM1) served as a novel regulator in the regulation of goat endometrial receptivity in vitro. Our results showed that hormones and IFN-τ increased the expression of CRIM1 in goat endometrial epithelial cells (EECs). Knockdown of CRIM1 via specific shRNA hindered cell proliferation, cell adhesion and prostaglandins (PGs) secretion and thus derailed normal endometrial receptivity. We further confirmed that receptivity defect phenotypes due to CRIM1 interference were restored by ATG7 overexpression in EECs while a loss of ATG7 further impaired receptivity phenotypes. Moreover, our results showed that changing the expression of ATG7 affected the reactive oxygen species (ROS) production. Moreover, mR-143-5p was shown to be a potential upstream factor of CRIM1-regulated endometrial receptivity in EECs. Overall, these results suggest that CRIM1, as the downstream target of miR-143-5p, has effects on ATG7-dependent autophagy, regulating cell proliferation, cell adhesion and PG secretion, and provides a new target for the diagnosis and treatment of early pregnancy failure and for improving the success rates of artificial reproduction.

Defective autophagy in Sf1 neurons perturbs the metabolic response to fasting and causes mitochondrial dysfunction

ObjectiveThe ventromedial nucleus of the hypothalamus (VMH) is a critical component of the forebrain pathways that regulate energy homeostasis. It also plays an important role in the metabolic response to fasting. However, the mechanisms contributing to these physiological processes remain elusive. Autophagy is an evolutionarily conserved mechanism that maintains cellular homeostasis by turning over cellular components and providing nutrients to the cells during starvation. Here, we investigated the importance of the autophagy-related gene Atg7 in Sf1-expressing neurons of the VMH in control and fasted conditions. MethodsWe generated Sf1-Cre; Atg7loxP/loxP mice and examined their metabolic and cellular response to fasting. ResultsFasting induces autophagy in the VMH, and mice lacking Atg7 in Sf1-expressing neurons display altered leptin sensitivity and impaired energy expenditure regulation in response to fasting. Moreover, loss of Atg7 in Sf1 neurons causes alterations in the central response to fasting. Furthermore, alterations in mitochondria morphology and activity are observed in mutant mice. ConclusionTogether, these data show that autophagy is nutritionally regulated in VMH neurons and that VMH autophagy participates in the control of energy homeostasis during fasting.

Loss of Atg7 causes chaotic nucleosome assembly of mouse bone marrow CD11b+Ly6G- myeloid cells.

Atg7, a critical component of autophagy machinery, is essential for counteracting hematopoietic aging. However, the non-autophagic role of Atg7 on hematopoietic cells remains fundamentally unclear. In this study, we found that loss of Atg7, but not Atg5, another autophagy-essential gene, in the hematopoietic system reduces CD11b myeloid cellularity including CD11b+Ly6G+ and CD11b+Ly6G- populations in mouse bone marrow. Surprisingly, Atg7 deletion causes abnormally accumulated histone H3.1 to be overwhelmingly trapped in the cytoplasm in the CD11b+Ly6G-, but not the CD11b+Ly6G+ compartment. RNA profiling revealed extensively chaotic expression of the genes required in nucleosome assembly. Functional assays further indicated upregulated aging markers in the CD11b+Ly6G- population. Therefore, our study suggests that Atg7 is essential for maintaining proper nucleosome assembly and limiting aging in the bone marrow CD11b+Ly6G- population.

Loss of Atg7 in Endothelial Cells Enhanced Cutaneous Wound Healing in a Mouse Model

BackgroundEmerging evidence has linked autophagy to skin wound healing; however, the underlying cellular and molecular mechanisms remain poorly understood. The present study was designed to determine the role of autophagy in endothelial cell (EC)-mediated skin wound healing in mice. MethodsAutophagy-related gene (Atg7) in mouse ECs was inactivated by the Cre-loxP system under the control of an EC-specific VE-Cadherin (Cdh5) promoter (Atg7EC−/− mice). Full-thickness skin wounds were created on the dorsum of wild-type (WT), Cdh5-Cre+, floxed Atg7 (Atg7F/F), and Atg7EC−/− mice. Autophagic activity was determined by autophagic flux assay in the primary culture of ECs isolated from these mice. The wound re-epithelialization and angiogenesis was examined by histological analyses. The angiogenic activity of ECs was evaluated by tube formation assay in vitro. EC proliferation was examined by a cell count CCK-8 kit. EC-originated intercellular communication with dermal fibroblasts and keratinocytes was assessed by measuring the effect of EC conditional medium on the growth of keratinocytes and fibroblasts. The levels of VEGF, EGF, bFGF in EC conditional medium were measured by ELISA. ResultsAutophagy deficiency in ECs markedly enhanced the re-epithelialization and the wound closure during skin wound healing. However, it has minimal impact on angiogenesis in the wounded skin. Notably, autophagy deficiency in ECs did not affect their proliferation and migration or angiogenic activity per se but enhanced the EC conditional medium-induced proliferation and migration of keratinocytes and fibroblasts. ConclusionsThese results demonstrate for the first time an inhibitory role of autophagy in the EC-originated paracrine regulation of skin wound healing.

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

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

Inactivation of MTOR promotes autophagy-mediated epithelial injury in particulate matter-induced airway inflammation

ABSTRACT Particulate matter (PM) is able to induce airway epithelial injury, while the detailed mechanisms remain unclear. Here we demonstrated that PM exposure inactivated MTOR (mechanistic target of rapamycin kinase), enhanced macroautophagy/autophagy, and impaired lysosomal activity in HBE (human bronchial epithelial) cells and in mouse airway epithelium. Genetic or pharmaceutical inhibition of MTOR significantly enhanced, while inhibition of autophagy attenuated, PM-induced IL6 expression in HBE cells. Consistently, club-cell-specific deletion of Mtor aggravated, whereas loss of Atg5 in bronchial epithelium reduced, PM-induced airway inflammation. Interestingly, the augmented inflammatory responses caused by MTOR deficiency were markedly attenuated by blockage of downstream autophagy both in vitro and in vivo. Mechanistically, the dysregulation of MTOR-autophagy signaling was partially dependent on activation of upstream TSC2, and interacted with the TLR4-MYD88 to orchestrate the downstream NFKB activity and to regulate the production of inflammatory cytokines in airway epithelium. Moreover, inhibition of autophagy reduced the expression of EPS15 and the subsequent endocytosis of PM. Taken together, the present study provides a mechanistic explanation for how airway epithelium localized MTOR-autophagy axis regulates PM-induced airway injury, suggesting that activation of MTOR and/or suppression of autophagy in local airway might be effective therapeutic strategies for PM-related airway disorders. Abbreviations ACTB: actin beta; AKT: AKT serine/threonine kinase; ALI: air liquid interface; AP2: adaptor related protein complex 2; ATG: autophagy related; BALF: bronchoalveolar lavage fluid; COPD: chronic obstructive pulmonary disease; CXCL: C-X-C motif chemokine ligand; DOX: doxycycline; EGF: epidermal growth factor; EGFR: epidermal growth factor receptor; EPS15: epidermal growth factor receptor pathway substrate 15; HBE: human bronchial epithelial; H&E: hematoxylin & eosin; IKK: IKB kinase; IL: interleukin; LAMP2: lysosomal-associated membrane protein 2; LPS: lipopolysaccharide; MAP1LC3B/LC3B: microtubule-associated protein 1 light chain 3 beta; MTEC: mouse tracheal epithelial cells; MTOR: mechanistic target of rapamycin kinase; MYD88: MYD88 innate immune signal transduction adaptor; NFKB: nuclear factor of kappa B; NFKBIA: NFKB inhibitor alpha; PM: particulate matter; PtdIns3K: phosphatidylinositol 3-kinase; Rapa: rapamycin; RELA: RELA proto-oncogene, NFKB subunit; SCGB1A1: secretoglobin family 1A member 1; siRNA: small interfering RNAs; SQSTM1: sequestosome 1; TEM: transmission electronic microscopy; TLR4: toll like receptor 4; TSC2: TSC complex subunit 2.

The autophagy machinery restrains iNKT cell activation through CD1D1 internalization

ABSTRACTInvariant natural killer T (iNKT) cells are innate T cells with powerful immune regulatory functions that recognize glycolipid antigens presented by the CD1D protein. While iNKT cell-activating glycolipids are currently being explored for their efficacy to improve immunotherapy against infectious diseases and cancer, little is known about the mechanisms that control CD1D antigen presentation and iNKT cell activation in vivo. CD1D molecules survey endocytic pathways to bind lipid antigens in MHC class II-containing compartments (MIICs) before recycling to the plasma membrane. Autophagosomes intersect with MIICs and autophagy-related proteins are known to support antigen loading for increased CD4+ T cell immunity. Here, we report that mice with dendritic cell (DC)-specific deletion of the essential autophagy gene Atg5 showed better CD1D1-restricted glycolipid presentation in vivo. These effects led to enhanced iNKT cell cytokine production upon antigen recognition and lower bacterial loads during Sphingomonas paucimobilis infection. Enhanced iNKT cell activation was independent of receptor-mediated glycolipid uptake or costimulatory signals. Instead, loss of Atg5 in DCs impaired clathrin-dependent internalization of CD1D1 molecules via the adaptor protein complex 2 (AP2) and, thus, increased surface expression of stimulatory CD1D1-glycolipid complexes. These findings indicate that the autophagic machinery assists in the recruitment of AP2 to CD1D1 molecules resulting in attenuated iNKT cell activation, in contrast to the supporting role of macroautophagy in CD4+ T cell stimulation.

The Impact of Autophagy on the Progression of the Myelodysplastic Syndromes

IntroductionAbout 75% of myelodysplastic syndromes (MDS) are lower risk categories with a paucity of definitive treatment and only supportive management as an option. MDS, defined by ineffective erythropoiesis produces anemia requiring frequent blood transfusions resulting in systemic iron overload. Refractory Anaemia with ring sideroblasts frequently characterised by mutations in the spliceosome machinery also contributes to cellular iron overload. Excess iron drives activation of the transcription factor NF-κB promoting pro-inflammatory cytokines production supporting tumour growth. Iron chelation is routinely used to treat transfusional iron overload. A number of observational studies have demonstrated that the iron chelator Deferasirox (DFX), improves haemoglobin levels in a subset of MDS patients (Messa, et al. 2008, Banerjee, et al. 2015). Iron metabolism is deregulated in cancer cells resulting in a net iron influx enhancing ROS production. Excess ROS promotes autophagy, a catabolic cellular recycling pathway clearing redundant and damaged organelles to sustain cellular metabolism. Autophagy is initiated by the Atg1-Atg13 protein complex and can be upregulated in cancer facilitating the propagation of the malignant clone. We have previously demonstrated that treatment with the iron chelator DFX, blocked growth of myeloid leukemia cell lines whilst sparing normal stem cells. Herein, we demonstrate that the growth inhibitory effects of DFX are mediated by its ability to inhibit autophagy. We therefore postulate that modulation of intracellular iron levels can be adopted as a viable tool to further elucidate the role of autophagy in this disease.AimsTo explore the impact of iron modulation on autophagy.To elucidate the role autophagy in myeloid cell growth and proliferation.MethodsThp-1 (myeloid cell line) expressing the autophagy reporter LC3-GFP was utilised. Autophagy results in LC3 recruitment to the autphagosomal membrane. These cells were treated with DFX in the presence/absence of chloroquine, a lysosomal enzyme inhibitor to visualise accumulated LC3-II. Cells were also treated with two other iron chelators, deferoxamine (DFO) and Dp44mT. Treated cells were then analysed using AMNIS Flow Imaging. Acute myeloid leukaemia cell lines were treated with DFX in the presence/absence of chloroquine and subjected to Western blotting using anti-LC3-II antibody. AML cell lines were also treated with 3-methyl adenine (3MA), an autophagy inhibitor. Doxycycline inducible CRISPR/Cas9 gene editing was used to delete essential autophagy genes, ATG 5 and 7 in Thp-1 cells and subjected to Western Blotting using ATG5/7 antibodies. Cell proliferation assays were done in the presence and absence of doxycycline. The impact of ATG5/7 deletion on LC3 was analysed using Western Blotting.ResultsCQ treatment of Thp-1-LC3-GFP cells demonstrated an increased number of LC3 specks, which were significantly reduced upon co-treatment with DFX. The total number of cells overall with LC3 specks was also reduced in the combination treatment of CQ and DFX. This was consistent with Western Blotting results that demonstrated lower LC3-II levels in this treatment. Cells treated with the other iron chelators, DFO and Dp44mT demonstrated no decrease in LC3 specks upon co-treatment with CQ and confirmed with Western Blotting demonstrating no change in LC3-II levels. Pharmacological inhibition of autophagy in AML cell lines using 3MA, showed dose dependent decrease in cell growth. ATG5 and ATG7 editing in Thp-1 cells using CRISPR/Cas9 resulted in significant impact on cell growth and proliferation. Loss of ATG5 and ATG7 in these cellswas confirmed using Western Blotting. LC3-II levels were significantly reduced in ATG5/7 deleted cells.ConclusionOur results demonstrate an essential role for the autophagy mediators ATG5/7 highlighting the importance of autophagy in promoting proliferation of malignant myeloid cells. The negative impact of DFX on cell growth and proliferation appears to be via its ability to inhibit autophagy. This is a unique property of DFX not observed with other iron chelators. Using DFX as a tool to modulate iron metabolism, these results have reinforced the role of deregulated iron metabolism in propagation of malignant haematopoietic cells. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Selective Deletion of ATG5 in Bone Marrow Leptin-Receptor-Expressing Perivascular Stromal Cells Causes Leukopenia

Bone marrow niche cells, specifically the Leptin-receptor-expressing perivascular stromal cells (LepR+), CXCL12-abundant reticular cells (CAR), and endothelial cells play an important role in the maintenance and self-renewal functions of hematopoietic stem cells (HSCs). This maintenance function provided by the niche cells is mediated by a number of secretory-related molecules such as stem cell factor (SCF), CXCL12 and Angiopoietin. In addition, functional autophagy within HSCs is known to play a critical role for maintaining HSC homeostasis, as the loss of ATG7 in HSCs leads to loss of normal HSC functions and severe myeloproliferation. However, it remains unclear as to how hematopoiesis will be affected if autophagy is selectively blocked in bone marrow niche cells. To investigate the effect of autophagy deficiency within bone marrow niche cells on hematopoiesis, we generated a knockout mouse model allowing for a selective ablation of an autophagy-essential protein, ATG5, in LepR+ perivascular stromal cells. We have confirmed that the deletion of Atg5 is indeed restricted to LepR+ cells, and that the loss of ATG5 is sufficient to block autophagic function, as evident through the accumulation of p62 protein. We found that the white blood cell count (WBC) is significantly reduced in 16-week old male ATG5 KO mice compare to wild type (wt) littermate control mice (p<0.05). Further, the myeloid population (CD11b+ Ly6G+) in both bone marrow and peripheral blood is significantly decreased in ATG5 KO mice (p value < 0.05, ATG KO vs. wt control). WBC count continues to decline in 24-week old ATG5 KO male mice (p=0.02) but not in female mice (p=0.58). Both LSK (Lin- Sca-1+ c-Kit+) and long term HSC (LT-HSCs, CD150+ CD48- LSK) populations are decreased in both 16-week and 24-week old male mice; however, this trend did not reach statistical significance (p>0.05). Although the absolute cell count of lymphocytes, including B and T cells (CD19+ and CD3+, respectively) in peripheral blood, is significantly dropped in ATG5 KO mice compared to wt controls, there is no significant change of mature B and T cells in bone marrow. These preliminary results suggest that deletion of a key autophagy regulator in LepR+ bone marrow niche cells will cause leukopenia. The underlying mechanism is currently under investigation and it will help us to better understand the role of autophagy in hematopoiesis within the bone marrow microenvironment. DisclosuresNo relevant conflicts of interest to declare.