Brown and Beige (or brite) adipose tissues burn lipid by converting chemical energy into heat, and have been considered as a new therapeutic target to counteract obesity. The regulation of thermogenic function in adipose tissue at the transcriptional level has been extensively studied in the past several years. However, the upstream signaling pathways that control the transcriptional machinery of thermogenic genes and effector mechanisms in brown or beige adipocytes remain largely unknown. Our recent study demonstrated that overactivation of mTOR Complex 1 (mTORC1) signaling is associated with impaired thermogenic function in vivo (Liu et al., 2014, Cell Metabolism). Consistent with this, our preliminary data showed that inactivation of mTORC1 by adipose-specific ablation of raptor, a key component of mTORC1, up-regulated the expression of thermogenic genes in brown adipose tissue (BAT) and inguinal white adipose tissue (iWAT or beige fat). In addition, we also found that the autophagy, a pathway downstream of mTORC1 is activated by cold stress and a β3-adrenoceptor agonist in vivo and in cells, and inhibition of autophagy diminishes β3-adrenoceptor agonist- induced UCP1 expression in primary adipocytes. In support of this, inhibition of autophagy by adipose-specific deletion of autophagy protein 7 (ATG7) suppresses basal and cold-induced energy expenditure, lipolysis and UCP1 expression in iWAT in vivo. Based on these findings, we hypothesize that autophagy plays a critical role in regulating the browning of white adipose tissue and mediates the beneficial effect of mTORC1 inhibition on thermogenesis in human brown adipocytes. We will first determine whether and how inhibiting mTORC1 and autophagy alters beige adipocyte differentiation and thermogenesis via cell-autonomous mechanisms. We will then investigate whether autophagy is an essential effector downstream of mTORC1 in regulating thermogenesis in WAT using adipose-specific autophagy-related protein 7 (ATG7)/raptor double KO mice. Lastly, we will delineate the role of mTORC1 and autophagy in regulating thermogenesis in primary human brown adipocytes. This study will lead to the identification of the mTORC1/autophagy pathway as a critical regulator of beige adipocyte differentiation and recruitment in response to various environmental factors. In addition, elucidation of the underlying signaling mechanisms involved in the browning of white fat may reveal promising new anti-obesity drug targets and lead to novel therapeutic approaches for obesity-associated metabolic diseases.

The apically located inter-cellular tight junctions (TJ) within the intestinal epithelium act as a paracellular barrier and prevent permeation of noxious luminal antigens. Loss of intestinal TJ barrier function is a key pathogenic factor in intestinal disorders and inflammatory bowel disease (IBD). Emerging evidence shows that defects in autophagy play an important role in the susceptibility, etiology, and progression of IBD. Although clinical data and animal studies show a direct link between defective intestinal TJ barrier and intestinal inflammation in IBD patients and animal models of IBD, the role of autophagy in the regulation of intestinal epithelial TJ barrier remains unknown. Our preliminary studies indicated that autophagy plays a key role in the enhancement of intestinal TJ barrier. Specifically, autophagy reduces paracellular TJ permeability by degradation of the pore forming tight junction protein claudin-2 and increasing protein levels of barrier protective transmembrane TJ protein occludin. Induction of autophagy causes a selective increase in lysosomal targeting of claudin-2 from the membrane and causes an increase in membrane retention of occludin. Thus, our central hypothesis is that autophagy selectively modulates TJ membrane protein composition to induce an enhancement of the intestinal TJ barrier. The overall specific aims of this application are: Specific Aim 1. To delineate the intracellular vesicular trafficking mechanisms in autophagy-induced enhancement of intestinal epithelial TJ barrier. Specific Aim 2. To elucidate the role of intracellular signaling in autophagy regulation of intestinal TJ barrier. Specific Aim 3. To delineate the role of autophagy in intestinal TJ barrier function and inflammation in animal models of IBD. This proposal will provide novel insights into the crucial role that autophagy plays in the homeostasis of intestinal barrier and bridge the gap in scientific knowledge that will be important for therapeutic efforts against IBD.

Although ignored until recently for not complying with the central dogma of molecular biology, non-coding RNAs (ncRNAs) are emerging as important novel regulators of diverge cellular processes. The capacity of ncRNAs to engage in “molecular multitasking” positions them to link multiple genetic risk factors for polygenic human genetic disorders, such as psychiatric diseases, into functional networks. Circular RNAs (circRNAs) are a novel category of non-coding RNAs that are derived from the back-splicing and covalent joining of exons and introns of protein-coding genes, yet lack the capacity to become translated into protein. Recent studies have suggested that circRNAs are enriched in the brain, are developmentally regulated, and are abundant in dendrites and synapses. Despite this, nothing is known about the function of circRNAs in the mammalian brain and their potential involvement in autophagy and neuropsychiatric disease. Autophagy is a coordinated lysosomal process for the degradation and recycling of cellular components, organelles, and protein aggregates that has been heavily implicated in the pathophysiology of neurodegenerative disorders. However, recent data suggest that numerous autophagy-associated genes display reduced expression in postmortem brains of subjects with psychiatric disorders and that neurons utilize autophagy during normal neuronal development and function. Despite the above, the importance of autophagy in psychiatric disease pathogenesis and pathophysiology has not been fully elucidated. Our proposed research aims in examining the role of altered in psychiatric disorders circRNAs that are either derived from autophagy-related genes or are upstream regulators of autophagy gene expression in neuronal autophagy and development and function. I specifically propose the following 3 aims. Aim 1: Test the hypothesis that alterations in psychiatric disease-associated circRNAs dysregulate linear gene expression related to autophagy and neuronal development and function. Aim 2: Test the hypothesis that manipulation of psychiatric disease-related circRNAs can alter autophagy and neuronal development and function. Aim 3: Test the hypothesis that treatment with autophagy-inducing drugs can rescue circRNA-mediated alterations in neuronal development and function. Taken together our proposed research will elucidate the mechanisms that underlie the effects of autophagy-associated circRNAs on neuronal autophagy and function, while examining in parallel their relevance for psychiatric disease therapeutics.

The relative ability of cell autonomous HIV-1 restriction factors to interfere with the viral life cycle contributes to a host’s level of susceptibility to infection. Pharmacological enhancement of restriction factor efficacy would be a novel approach to treating HIV infection. However, the mechanistic basis for HIV blockage by restriction factors is not completely understood hampering efforts to employ restriction factor-based host directed therapies. The tripartite motif (TRIM) family of proteins consists of more than 70 members in humans, several of which have been identified as antiviral restriction factors. In this role, TRIMs can diminish viral replication directly by interfering with the viral life cycle or indirectly by fine tuning cellular innate immune responses. TRIM family member TRIM5α accomplishes both of these: first, it prevents retroviral infection of cells by a hitherto unexplained mechanism. Second, TRIM5α also acts as a pattern recognition receptor, promoting the establishment of an antiviral cellular state via the activation of inflammatory signaling pathways upon retroviral recognition. Although TRIMs appear to employ multiple approaches in antiretroviral defense, one strikingly common feature among the TRIM family is that many if not all TRIMs are involved in the regulation and execution of autophagy. In addition to its role as a known defense mechanism against intracellular pathogens (including HIV-1), autophagy is also increasingly recognized as a means of reducing or fine tuning inflammation. Here, we propose to test the hypothesis that autophagy underlies TRIM action in protecting cells against HIV-1 infection and in modulating the TRIM-dependent inflammatory signaling. The studies proposed here have several overarching goals. First, they seek to improve our understanding of the molecular mechanism whereby rhesus TRIM5α both regulates autophagy and directs the autophagic degradation of incoming HIV-1 capsids (Aim 1). Second, they will determine if modulations of the autophagy pathway affect TRIM5α-dependent activation of pro-inflammatory signaling upon lentiviral infection. Finally, they will address whether human TRIMs other than TRIM5α that restrict HIV also employ autophagy in their antiviral actions (Aim 2). We have assembled a team of autophagy and HIV experts to address these questions. Our studies have the potential to uncover the mode of action of several known antiretroviral proteins and lay the groundwork for our understanding of how TRIMs as a family can both positively and negatively affect inflammation. We expect these studies to show that autophagy is a unifying aspect of diverse TRIM actions in HIV defense. Since autophagy can be pharmacologically manipulated, our findings may indicate that modulations of autophagy could be a therapeutic approach to dealing with TRIM-related diseases including HIV/AIDS. Our expertise in TRIMs and autophagy, along with the financial and institutional support to be provided should the COBRE application be funded will ensure successful completion of these aims.

The function of T helper (TH) cells, the central organizers of adaptive immunity, is specified by the effector cytokines they produce. Regulation of TH cell cytokine secretion is not well understood and represents an important gap in our knowledge. Our recent data indicate that membrane- associated nucleic acid binding protein (Mnab, encoded by Rc3h2) is required for TH cell effector cytokine secretion. Mnab shares with its paralog Roquin (encoded by Rc3h1) a highly conserved N-terminus but possesses a unique hydrophobic C-terminus. Roquin is important for control of follicular helper T (Tfh) cell development through repression of Icos mRNA. Recently, Mnab was shown to play a redundant role with Roquin in Tfh cell development via repression of Icos and Ox40 mRNAs. Whether Mnab also targets other mRNAs and regulates function of other TH lineages is unclear. Our preliminary studies demonstrated that a Mnab deficiency led to profound defects in effector cytokine production in TH1, TH2 and TH17 cells, which differs from its known function, suggesting an important role of the distinct C-terminus of Mnab. Based on our preliminary data, we formulate a novel hypothesis: Mnab targets mRNAs encoding proteins in the stress response-autophagy pathway, a common pathway that is critical in TH cell effector cytokine production. The overall specific aims of this project are: Aim 1. Determine the role of Mnab and UPR-autophagy in TH cell function. Aim 2. Delineate the molecular mechanism whereby Mnab controls mRNA stability. Aim 3. Determine the role of Mnab in TH cell function in vivo using disease models. By addressing our hypothesis, these studies will reveal a novel post-transcription control mechanism of TH cell effector function. Manipulation of the corresponding pathways may be of therapeutic benefit in human disease, such as autoimmune and inflammatory disorders.