Phosphoinositides (PIs) control fundamental cell processes, and inherited defects of PI kinases or phosphatases cause severe human diseases including Lowe syndrome due to mutations in OCRL that encodes a PI(4,5)P2 5-phosphatase. Here we unveil a lysosomal response to the arrival of autophagosomal cargo where OCRL plays a key role. We identify mitochondrial DNA and TLR9 as the cargo and the receptor that triggers and mediates, respectively, this response. This lysosome-cargo response is required to sustain the autophagic flux and involves a local increase in PI(4,5)P2 that is confined in space and time by OCRL. Depleting or inhibiting OCRL leads to an accumulation of lysosomal PI(4,5)P2, an inhibitor of the calcium channel mucolipin-1 that controls autophagosome-lysosome fusion. Hence, autophagosomes accumulate in OCRL-depleted cells and in the kidneys of Lowe syndrome patients. Importantly, boosting the activity of mucolipin-1 with selective agonists restores the autophagic flux in cells from Lowe syndrome patients.
Lowe syndrome and Dent-2 disease are caused by mutation of the inositol 5-phosphatase OCRL1. Despite our increased understanding of the cellular functions of OCRL1, the underlying basis for the renal tubulopathy seen in both human disorders, of which a hallmark is low molecular weight proteinuria, is currently unknown. Here, we show that deficiency in OCRL1 causes a defect in endocytosis in the zebrafish pronephric tubule, a model for the mammalian renal tubule. This coincides with a reduction in levels of the scavenger receptor megalin and its accumulation in endocytic compartments, consistent with reduced recycling within the endocytic pathway. We also observe reduced numbers of early endocytic compartments and enlarged vacuolar endosomes in the sub-apical region of pronephric cells. Cell polarity within the pronephric tubule is unaffected in mutant embryos. The OCRL1-deficient embryos exhibit a mild ciliogenesis defect, but this cannot account for the observed impairment of endocytosis. Catalytic activity of OCRL1 is required for renal tubular endocytosis and the endocytic defect can be rescued by suppression of PIP5K. These results indicate for the first time that OCRL1 is required for endocytic trafficking in vivo, and strongly support the hypothesis that endocytic defects are responsible for the renal tubulopathy in Lowe syndrome and Dent-2 disease. Moreover, our results reveal PIP5K as a potential therapeutic target for Lowe syndrome and Dent-2 disease.
The endocannabinoid system (eCBs), named after the plant Cannabis sativa, comprises cannabinoid receptors, endogenous ligands known as “endocannabinoids”, and enzymes involved in the biosynthesis and degradation of these ligands, as well as putative transporters for these ligands. ECBs proteins and small molecules have been detected in early embryonic stages of many vertebrate models. As a result, cannabinoid receptors and endogenous as well as exogenous cannabinoids influence development and behavior in many vertebrate species. Understanding the precise mechanisms of action for the eCBs will provide an invaluable guide towards elucidation of vertebrate development and will also help delineate how developmental exposure to marijuana might impact health and cognitive and executive functioning in adulthood. Here we review the developmental roles of the eCBs in vertebrates, focusing our attention on the zebrafish model. Since little is known regarding the eCBs in zebrafish, we provide new data on the expression profiles of eCBs genes during development and in adult tissue types of this model organism. We also highlight exciting areas for future investigations, including the synaptic regulation of eCBs, its role in reward and addiction, and in nervous system development and plasticity.
Highlights d The dopamine metabolite 5,6-dihydroxyindole (DHI) binds directly to Nurr1 d DHI forms a covalent adduct with Nurr1, reacting as the indolequinone with Cys566 d The Nurr1-metabolite structure reveals a previously unreported ligand-binding pocket d DHI stimulates the transcription of Nurr1 target genes underlying dopamine homeostasis
BackgroundAPOL1 is found in human kidney podocytes and endothelia. Variants G1 and G2 of the APOL1 gene account for the high frequency of nondiabetic CKD among African Americans. Proposed mechanisms of kidney podocyte cytotoxicity resulting from APOL1 variant overexpression implicate different subcellular compartments. It is unclear where endogenous podocyte APOL1 resides, because previous immunolocalization studies utilized overexpressed protein or commercially available antibodies that crossreact with APOL2. This study describes and distinguishes the locations of both APOLs.MethodsImmunohistochemistry, confocal and immunoelectron microscopy, and podocyte fractionation localized endogenous and transfected APOL1 using a large panel of novel APOL1-specific mouse and rabbit monoclonal antibodies.ResultsBoth endogenous podocyte and transfected APOL1 isoforms vA and vB1 (and a little of isoform vC) localize to the luminal face of the endoplasmic reticulum (ER) and to the cell surface, but not to mitochondria, endosomes, or lipid droplets. In contrast, APOL2, isoform vB3, and most vC of APOL1 localize to the cytoplasmic face of the ER and are consequently absent from the cell surface. APOL1 knockout podocytes do not stain for APOL1, attesting to the APOL1-specificity of the antibodies. Stable re-transfection of knockout podocytes with inducible APOL1-G0, -G1, and -G2 showed no differences in localization among variants.ConclusionsAPOL1 is found in the ER and plasma membrane, consistent with either the ER stress or surface cation channel models of APOL1-mediated cytotoxicity. The surface localization of APOL1 variants potentially opens new therapeutic targeting avenues.
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