Deletion Of Autophagy-related Gene Research Articles

Influenza A virus infection‐induced macroautophagy facilitates MHC class II‐restricted endogenous presentation of an immunodominant viral epitope

CD4+ T cells recognize peptides presented by major histocompatibility complex class II molecules (MHC-II). These peptides are generally derived from exogenous antigens. Macroautophagy has been reported to promote endogenous antigen presentation in viral infections. However, whether influenza A virus (IAV) infection-induced macroautophagy also leads to endogenous antigen presentation through MHC-II is still debated. In this study, we show that IAV infection leads to endogenous presentation of an immunodominant viral epitope NP311-325 by MHC-II to CD4+ T cells. Mechanistically, such MHC-II-restricted endogenous IAV antigen presentation requires denovo protein synthesis as it is inhibited by the protein synthesis inhibitor cycloheximide, and a functional ER-Golgi network as it is totally blocked by Brefeldin A. These results indicate that MHC-II-restricted endogenous IAV antigen presentation is dependent on denovo antigen and/or MHC-II synthesis, and transportation through the ER-Golgi network. Furthermore, such endogenous IAV antigen presentation by MHC-II is enhanced by TAP deficiency, indicating some antigenic peptides are of cytosolic origin. Most importantly, the bulk of such MHC-II-restricted endogenous IAV antigen presentation is blocked by autophagy inhibitors (3-MA and E64d) and deletion of autophagy-related genes, such as Beclin1 and Atg7. We have further demonstrated that in dendritic cells, IAV infection prevents autophagosome-lysosome fusion and promotes autophagosome fusion with MHC class II compartment (MIIC), which likely promotes endogenous IAV antigen presentation by MHC-II. Our results provide strong evidence that IAV infection-induced autophagosome formation facilitates endogenous IAV antigen presentation by MHC-II to CD4+ T cells. The implication for influenza vaccine design is discussed.

Deletion of autophagy-related gene 7 in dopaminergic neurons prevents their loss induced by MPTP

Parkinson’s disease (PD) is a neurodegenerative disease caused by a gradual loss of midbrain dopaminergic (mDA) neurons in the substantia nigra pars compacta (SNpc) during aging. 1-Methyl-4-phenyl-1, 2, 3, 6-tetrahydropyridine (MPTP) is one of the neurotoxins used widely to induce PD-like symptoms in PD animal models, including rodents and non-human primates. It has been reported that deletion of autophagy-related gene 7 (Atg7) in the brain results in a reduction of mDA neurons in adulthood. In this study, we used tyrosine hydroxylase (TH)-Cre mice to generate conditional knockout (CKO) mice with the specific deletion of Atg7 in mDA neurons. Consistent with previous reports, adult Atg7 CKO mice contained fewer TH-positive mDA neurons compared with wild-type (WT) controls. TH-expressing neurons containing puncta-like structures with p62 and ubiquitin immunoreactivity were observed in the midbrain of Atg7 CKO mice but were not detected in control mice. However, MPTP-induced loss of mDA neurons was not observed in Atg7 CKO mice. Our results indicate that Atg7-involved autophagy is required not only for the survival of mDA neurons in the mouse brain, but also for MPTP-induced mDA neuron degeneration.

A mouse model to study the role of autophagy in obesity induced placental dysfunction

s / Placenta 35 (2014) A1eA112 A21 mediated via its receptors, and suggest that this phenomenon may play a role in situations of dysregulation of the vasoactive systems. P1.33. A MOUSE MODEL TO STUDY THE ROLE OF AUTOPHAGY IN OBESITY INDUCED PLACENTAL DYSFUNCTION Alina Maloyan, Sribalasubashini Muralimanoharan, Leslie Myatt University of Texas Health Science Center, San Antonio, Texas, USA Objectives: Maternal obesity leads to placental dysfunction and programs the offspring for metabolic disease. We have shown activation of oxidative/ nitrative stress, increase in p53 and mitochondrial dysfunction In placentas of obese women. Autophagy ensures cellular survival by sequestration of damaged organelles and proteins in autophagosomes, followed by degradation and recycling through the lysosomal machinery. We found disruption of autophagosome maturation in placentas of obese women leading to defective autophagy. To investigate the role of autophagy in placental function, a conditional knockout mouse model was generated with placenta-specific deletion of autophagy-related gene 7 (atg7). We have previously reported that deletion of ATG7 in placenta programmed the offspring to obesity and hyperglycemia. Here we hypothesized that ATG7 deficiency alters placental oxidative stress, cell death and mitochondrial function. Methods: Placentas from ATG7 knockout (KO, n1⁄45 dams) and wild type (WT, n1⁄45 dams) mice were collected at C-section on day 17.5 of gestation. Markers of oxidative stress, mitochondrial metabolism and cell death were measured by Western Blot. Result: When compared to WT mice, the KO mice showed a significant reduction in protein expression of subunits of mitochondrial complexes II, III and V as well as in expression of antioxidants SOD2 and catalase (p<0.05) indicating mitochondrial dysfunction and a decrease in oxidative stress defences. In the KO mouse placenta there was a significant increase in expression of both PPARg, a key regulator of mitochondrial metabolism in obesity (KO, 1.5±0.1 vs. WT, 0.9±0.0) and of p53 [KO, 1.3±0.1 vs. WT, 0.7±0.1). Apoptotic cell death was reduced in KO placentas as indicated by a 40% reduction in expression of both p53 upregulated modulator of apoptosis (PUMA) and of activated caspase-3 (p<0.05). Conclusion: Disruption of placental autophagic flux in the murine placenta leads to placental dysfunction, and fetal programming, similar to that in pregnancies with maternal obesity. P1.34-N. UPREGULATION OF PLACENTAL AUTOPHAGY IN A RAT MODEL OF DEXAMETHASONE-INDUCED FETAL GROWTH RESTRICTION Aisha Rasool, Ainslie Garrod, Elizabeth Cowley, Susan Greenwood, Elizabeth Cottrell University of Manchester, Manchester, UK Objectives: Exposure to stressors during pregnancy (e.g. poor nutrition or psychological stress) is associated with reduced fetal and placental growth. In animal models, the reduction in placental weight often precedes fetal growth restriction (FGR), suggesting placental adaptations support fetal growth in the face of an adverse environment. Autophagy, a conserved cellular process of ‘self-eating’ may be one mechanism by which this adaptation occurs, mobilizing placental energy stores to maintain fetal growth, at the expense of placental growth. Glucocorticoids (GCs) play an integral role in the response to in utero stressors, and in addition can stimulate autophagy in a number of non-placental cell types. Here, we sought to investigate the role of GCs in stimulating placental autophagy, using an established rat model of dexamethasone (DEX)-induced FGR. Methods: Pregnant rats were treated with dexamethasone acetate (DEX; 1ug/mL) or vehicle (0.1% ethanol) in drinking water from gestational day (G)13-G22. Fetal and placental weights were measured at G16, G19 and G22, and expression of autophagic genes assessed in placental tissue by qPCR. Result: Maternal DEX attenuated gestational weight gain (p<0.001), without affecting food intake. Placental weight was reduced by maternal DEX (by 14%, 28% and 34% compared with controls at G16, G19 and G22 respectively; p<0.001). Fetal weight was reduced to a lesser degree, at G19 and G22 (by 17.5% and 32.1%, respectively; p<0.05), indicating a degree of fetal protection from the growth-restricting effects of DEX. In the placenta, REDD1 and AMBRA1 gene expression were significantly increased by maternal DEX (p<0.01 for both), consistent with autophagic induction in this tissue. Conclusions: The current data suggest a role for glucocorticoids in the regulation of placental autophagy. Induction of autophagy in the placenta may underlie in part the ability of the placenta to support fetal growth in the face of increased stress or nutrient restriction. P1.35-N. SUPPLEMENTATION WITH INORGANIC NITRATE DURING PREGNANCY IMPROVES MATERNAL UTERINE ARTERY FUNCTION AND PLACENTAL EFFICIENCY IN MICE Elizabeth Cottrell , Ainslie Garrod , Mark Wareing , Mark Dilworth , Sarah Finn-Sell , SusanGreenwood , Philip Baker , Colin Sibley a University of Manchester, Manchester, UK; University of Auckland, Auckland, New