Dual targeting of hepatocyte DGAT2 and stellate cell FASN alleviates nonalcoholic steatohepatitis in mice.

Nonalcoholic steatohepatitis (NASH) is a malady of multiple cell types associated with hepatocyte triglyceride (TG) accumulation, macrophage inflammation, and stellate cell-induced fibrosis, with no approved therapeutics yet available. Here, we report that stellate cell fatty acid synthase (FASN) in de novo lipogenesis drives the autophagic flux that is required for stellate cell activation and fibrotic collagen production. Further, we employ a dual targeting approach to NASH that selectively depletes collagen through selective stellate cell knockout of FASN (using AAV9-LRAT Cre in FASNfl/fl mice), while lowering hepatocyte triglyceride by depleting DGAT2 with a GalNac-conjugated, fully chemically modified siRNA. DGAT2 silencing in hepatocytes alone or in combination with stellate cell FASNKO reduced liver TG accumulation in a choline-deficient NASH mouse model, while FASNKO in hepatocytes alone (using AAV8-TBG Cre in FASNfl/fl mice) did not. Neither hepatocyte DGAT2 silencing alone nor FASNKO in stellate cells alone decreased fibrosis (total collagen), while loss of both DGAT2 plus FASN caused a highly significant attenuation of NASH. These data establish proof of concept that dual targeting of DGAT2 plus FASN alleviates NASH progression in mice far greater than targeting either gene product alone.

De novo lipogenesis fuels adipocyte autophagosome and lysosome membrane dynamics.

Adipocytes robustly synthesize fatty acids (FA) from carbohydrate through the de novo lipogenesis (DNL) pathway, yet surprisingly DNL contributes little to their abundant triglyceride stored in lipid droplets. This conundrum raises the hypothesis that adipocyte DNL instead enables membrane expansions to occur in processes like autophagy, which requires an abundant supply of phospholipids. We report here that adipocyte Fasn deficiency in vitro and in vivo markedly impairs autophagy, evident by autophagosome accumulation and severely compromised degradation of the autophagic substrate p62. Our data indicate the impairment occurs at the level of autophagosome-lysosome fusion, and indeed, loss of Fasn decreases certain membrane phosphoinositides necessary for autophagosome and lysosome maturation and fusion. Autophagy dependence on FA produced by Fasn is not fully alleviated by exogenous FA in cultured adipocytes, and interestingly, imaging studies reveal that Fasn colocalizes with nascent autophagosomes. Together, our studies identify DNL as a critical source of FAs to fuel autophagosome and lysosome maturation and fusion in adipocytes.

Molecular Assessment of Epiretinal Membrane: Activated Microglia, Oxidative Stress and Inflammation.

Fibrocellular membrane or epiretinal membrane (ERM) forms on the surface of the inner limiting membrane (ILM) in the inner retina and alters the structure and function of the retina. ERM formation is frequently observed in ocular inflammatory conditions, such as proliferative diabetic retinopathy (PDR) and retinal detachment (RD). Although peeling of the ERM is used as a surgical intervention, it can inadvertently distort the retina. Our goal is to design alternative strategies to tackle ERMs. As a first step, we sought to determine the composition of the ERMs by identifying the constituent cell-types and gene expression signature in patient samples. Using ultrastructural microscopy and immunofluorescence analyses, we found activated microglia, astrocytes, and Müller glia in the ERMs from PDR and RD patients. Moreover, oxidative stress and inflammation associated gene expression was significantly higher in the RD and PDR membranes as compared to the macular hole samples, which are not associated with inflammation. We specifically detected differential expression of hypoxia inducible factor 1-α (HIF1-α), proinflammatory cytokines, and Notch, Wnt, and ERK signaling pathway-associated genes in the RD and PDR samples. Taken together, our results provide new information to potentially develop methods to tackle ERM formation.

Loss of Na(+)/K(+)-ATPase in Drosophila photoreceptors leads to blindness and age-dependent neurodegeneration.

The activity of Na(+)/K(+)-ATPase establishes transmembrane ion gradients and is essential to cell function and survival. Either dysregulation or deficiency of neuronal Na(+)/K(+)-ATPase has been implicated in the pathogenesis of many neurodegenerative disorders such as Alzheimer's disease, Parkinson's disease and rapid-onset dystonia Parkinsonism. However, genetic evidence that directly links neuronal Na(+)/K(+)-ATPase deficiency to in vivo neurodegeneration has been lacking. In this study, we use Drosophila photoreceptors to investigate the cell-autonomous effects of neuronal Na(+)/K(+) ATPase. Loss of ATPα, an α subunit of Na(+)/K(+)-ATPase, in photoreceptors through UAS/Gal4-mediated RNAi eliminated the light-triggered depolarization of the photoreceptors, rendering the fly virtually blind in behavioral assays. Intracellular recordings indicated that ATPα knockdown photoreceptors were already depolarized in the dark, which was due to a loss of intracellular K(+). Importantly, ATPα knockdown resulted in the degeneration of photoreceptors in older flies. This degeneration was independent of light and showed characteristics of apoptotic/hybrid cell death as observed via electron microscopy analysis. Loss of Nrv3, a Na(+)/K(+)-ATPase ß subunit, partially reproduced the signaling and degenerative defects observed in ATPα knockdown flies. Thus, the loss of Na(+)/K(+)-ATPase not only eradicates visual function but also causes age-dependent degeneration in photoreceptors, confirming the link between neuronal Na(+)/K(+) ATPase deficiency and in vivo neurodegeneration. This work also establishes Drosophila photoreceptors as a genetic model for studying the cell-autonomous mechanisms underlying neuronal Na(+)/K(+) ATPase deficiency-mediated neurodegeneration.

Long-distance mechanism of neurotransmitter recycling mediated by glial network facilitates visual function in Drosophila.

Neurons rely on glia to recycle neurotransmitters such as glutamate and histamine for sustained signaling. Both mammalian and insect glia form intercellular gap-junction networks, but their functional significance underlying neurotransmitter recycling is unknown. Using the Drosophila visual system as a genetic model, here we show that a multicellular glial network transports neurotransmitter metabolites between perisynaptic glia and neuronal cell bodies to mediate long-distance recycling of neurotransmitter. In the first visual neuropil (lamina), which contains a multilayer glial network, photoreceptor axons release histamine to hyperpolarize secondary sensory neurons. Subsequently, the released histamine is taken up by perisynaptic epithelial glia and converted into inactive carcinine through conjugation with ß-alanine for transport. In contrast to a previous assumption that epithelial glia deliver carcinine directly back to photoreceptor axons for histamine regeneration within the lamina, we detected both carcinine and ß-alanine in the fly retina, where they are found in photoreceptor cell bodies and surrounding pigment glial cells. Downregulating Inx2 gap junctions within the laminar glial network causes ß-alanine accumulation in retinal pigment cells and impairs carcinine synthesis, leading to reduced histamine levels and photoreceptor synaptic vesicles. Consequently, visual transmission is impaired and the fly is less responsive in a visual alert analysis compared with wild type. Our results suggest that a gap junction-dependent laminar and retinal glial network transports histamine metabolites between perisynaptic glia and photoreceptor cell bodies to mediate a novel, long-distance mechanism of neurotransmitter recycling, highlighting the importance of glial networks in the regulation of neuronal functions.

A Drosophila metallophosphoesterase mediates deglycosylation of rhodopsin.

Oligosaccharide chains of newly synthesized membrane receptors are trimmed and modified to optimize their trafficking and/or signalling before delivery to the cell surface. For most membrane receptors, the functional significance of oligosaccharide chain modification is unknown. During the maturation of Rh1 rhodopsin, a Drosophila light receptor, the oligosaccharide chain is trimmed extensively. Neither the functional significance of this modification nor the enzymes mediating this process are known. Here, we identify a dmppe (Drosophila metallophosphoesterase) mutant with incomplete deglycosylation of Rh1, and show that the retained oligosaccharide chain does not affect Rh1 localization or signalling. The incomplete deglycosylation, however, renders Rh1 more sensitive to endocytic degradation, and causes morphological and functional defects in photoreceptors of aged dmppe flies. We further demonstrate that the dMPPE protein functions as an Mn(2+)/Zn(2+)-dependent phosphoesterase and mediates in vivo dephosphorylation of α-Man-II. Most importantly, the dephosphorylated α-Man-II is required for the removal of the Rh1 oligosaccharide chain. These observations suggest that the glycosylation status of membrane proteins is controlled through phosphorylation/dephosphorylation, and that MPPE acts as the phosphoesterase in this regulation.

Mutation of a TADR protein leads to rhodopsin and Gq-dependent retinal degeneration in Drosophila.

The Drosophila photoreceptor is a model system for genetic study of retinal degeneration. Many gene mutations cause fly photoreceptor degeneration, either because of excessive stimulation of the visual transduction (phototransduction) cascade, or through apoptotic pathways that in many cases involve a visual arrestin Arr2. Here we report a gene named tadr (for torn and diminished rhabdomeres), which, when mutated, leads to photoreceptor degeneration through a different mechanism. Degeneration in the tadr mutant is characterized by shrunk and disrupted rhabdomeres, the light sensory organelles of photoreceptor. The TADR protein interacted in vitro with the major light receptor Rh1 rhodopsin, and genetic reduction of the Rh1 level suppressed the tadr mutation-caused degeneration, suggesting the degeneration is Rh1-dependent. Nonetheless, removal of phospholipase C (PLC), a key enzyme in phototransduction, and that of Arr2 failed to inhibit rhabdomeral degeneration in the tadr mutant background. Biochemical analyses revealed that, in the tadr mutant, the G(q) protein of Rh1 is defective in dissociation from the membrane during light stimulation. Importantly, reduction of G(q) level by introducing a hypomorphic allele of G(alphaq) gene greatly inhibited the tadr degeneration phenotype. These results may suggest that loss of a potential TADR-Rh1 interaction leads to an abnormality in the G(q) signaling, which in turn triggers rhabdomeral degeneration independent of the PLC phototransduction cascade. We propose that TADR-like proteins may also protect photoreceptors from degeneration in mammals including humans.

Prolonged G(q) activity triggers fly rhodopsin endocytosis and degradation, and reduces photoreceptor sensitivity.

Rapid deactivation of the Drosophila light receptor rhodopsin, through a visual arrestin Arr2 and a pathway that involves a transcription factor dCAMTA, is required for timely termination of light responses in the photoreceptor neuron. Here we report that this process is also critical for maintenance of the photoreceptor sensitivity. In both dCAMTA- and arr2-mutant flies, the endocytosis of the major rhodopsin Rh1 was dramatically increased, which was mediated by a G(q) protein that signals downstream of rhodopsin in the visual transduction pathway. Consequently, the Rh1 level was downregulated and the photoreceptor became less sensitive to light. Remarkably, the G(q)-stimulated Rh1 endocytosis does not require phospholipase C, a known effector of G(q), but depends on a tetraspanin protein. Our work has identified an arrestin-independent endocytic pathway of G protein-coupled receptor in the fly. This pathway may also function in mammals and mediate an early feedback regulation of receptor signaling.

A potential dimerization region of dCAMTA is critical for termination of fly visual response.

CAMTAs are a group of Ca(2+)/calmodulin binding transcription activators that are implicated in brain tumor suppression, cardiac hypertrophy, and plant sensory responses. The sole fly CAMTA, dCAMTA, stimulates expression of an F-box gene, dFbxl4, to potentiate rhodopsin deactivation, which enables rapid termination of fly visual responses. Here we report that a dCAMTA fragment associated with a full-length protein in co-transfected human embryonic kidney 293 cells. The interaction site was mapped to a region within the DNA-binding CG-1 domain. With this potential dimerization site mutated, the full-length dCAMTA had defective nuclear localization. In transgenic flies, this mutant dCAMTA variant failed to stimulate expression of dFbxl4 and rescue the slow termination of light response phenotype of a dCAMTA null mutant fly. Our data suggest that dCAMTA may function as a dimer during fly visual regulation and that the CG-1 domain may mediate dimerization of CAMTA transcription factors.

The fly CAMTA transcription factor potentiates deactivation of rhodopsin, a G protein-coupled light receptor.

Control of membrane-receptor activity is required not only for the accuracy of sensory responses, but also to protect cells from excitotoxicity. Here we report the isolation of two noncomplementary fly mutants with slow termination of photoresponses. Genetic and electrophysiological analyses of the mutants revealed a defect in the deactivation of rhodopsin, a visual G protein-coupled receptor (GPCR). The mutant gene was identified as the calmodulin-binding transcription activator (dCAMTA). The known rhodopsin regulator Arr2 does not mediate this visual function of dCAMTA. A genome-wide screen identified five dCAMTA target genes. Of these, overexpression of the F box gene dFbxl4 rescued the mutant phenotypes. We further showed that dCAMTA is stimulated in vivo through interaction with the Ca(2+) sensor calmodulin. Our data suggest that calmodulin/CAMTA/Fbxl4 may mediate a long-term feedback regulation of the activity of Ca(2+)-stimulating GPCRs, which could prevent cell damage due to extra Ca(2+) influx.