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Palmitic acid (PA) is significantly increased in the hypothalamus of mice, when fed chronically with a high-fat diet (HFD). PA impairs insulin signaling in hypothalamic neurons, by a mechanism dependent on autophagy, a process of lysosomal-mediated degradation of cytoplasmic material. In addition, previous work shows a crosstalk between autophagy and the primary cilium (hereafter cilium), an antenna-like structure on the cell surface that acts as a signaling platform for the cell. Ciliopathies, human diseases characterized by cilia dysfunction, manifest, type 2 diabetes, among other features, suggesting a role of the cilium in insulin signaling. Cilium depletion in hypothalamic pro-opiomelanocortin (POMC) neurons triggers obesity and insulin resistance in mice, the same phenotype as mice deficient in autophagy in POMC neurons. Here we investigated the effect of chronic consumption of HFD on cilia; and our results indicate that chronic feeding with HFD reduces the percentage of cilia in hypothalamic POMC neurons. This effect may be due to an increased amount of PA, as treatment with this saturated fatty acid in vitro reduces the percentage of ciliated cells and cilia length in hypothalamic neurons. Importantly, the same effect of cilia depletion was obtained following chemical and genetic inhibition of autophagy, indicating autophagy is required for ciliogenesis. We further demonstrate a role for the cilium in insulin sensitivity, as cilium loss in hypothalamic neuronal cells disrupts insulin signaling and insulin-dependent glucose uptake, an effect that correlates with the ciliary localization of the insulin receptor (IR). Consistently, increased percentage of ciliated hypothalamic neuronal cells promotes insulin signaling, even when cells are exposed to PA. Altogether, our results indicate that, in hypothalamic neurons, impairment of autophagy, either by PA exposure, chemical or genetic manipulation, cause cilia loss that impairs insulin sensitivity.

Macroautophagy/autophagy is an intracellular process involved in the breakdown of macromolecules and organelles. Recent studies have shown that PKD2/PC2/TRPP2 (polycystin 2, transient receptor potential cation channel), a nonselective cation channel permeable to Ca2+ that belongs to the family of transient receptor potential channels, is required for autophagy in multiple cell types by a mechanism that remains unclear. Here, we report that PKD2 forms a protein complex with BECN1 (beclin 1), a key protein required for the formation of autophagic vacuoles, by acting as a scaffold that interacts with several co-modulators via its coiled-coil domain (CCD). Our data identified a physical and functional interaction between PKD2 and BECN1, which depends on one out of two CCD domains (CC1), located in the carboxy-terminal tail of PKD2. In addition, depletion of intracellular Ca2+ with BAPTA-AM not only blunted starvation-induced autophagy but also disrupted the PKD2-BECN1 complex. Consistently, PKD2 overexpression triggered autophagy by increasing its interaction with BECN1, while overexpression of PKD2D509V, a Ca2+ channel activity-deficient mutant, did not induce autophagy and manifested diminished interaction with BECN1. Our findings show that the PKD2-BECN1 complex is required for the induction of autophagy, and its formation depends on the presence of the CC1 domain of PKD2 and on intracellular Ca2+ mobilization by PKD2. These results provide new insights regarding the molecular mechanisms by which PKD2 controls autophagy.Abbreviations: ADPKD: autosomal dominant polycystic kidney disease; ATG: autophagy-related; ATG14/ATG14L: autophagy related 14; Baf A1: bafilomycin A1; BCL2/Bcl-2: BCL2 apoptosis regulator; BCL2L1/BCL-XL: BCL2 like 1; BECN1: beclin 1; CCD: coiled-coil domain; EBSS: Earle's balanced salt solution; ER: endoplasmic reticulum; GAPDH: glyceraldehyde-3-phosphate dehydrogenase; GFP: green fluorescent protein; GOLGA2/GM130: golgin A2; GST: glutathione s-transferase; LAMP1: lysosomal associated membrane protein 1; MAP1LC3/LC3: microtubule associated protein 1 light chain 3; MTORC1: mechanistic target of rapamycin kinase complex 1; NBR1: NBR1 autophagy cargo receptor; PIK3C3/VPS34: phosphatidylinositol 3-kinase catalytic subunit type 3; PKD2/PC2: polycystin 2, transient receptor potential cation channel; RTN4/NOGO: reticulon 4; RUBCN/RUBICON: rubicon autophagy regulator; SQSTM1/p62: sequestosome 1; UVRAG: UV radiation resistance associated; WIPI2: WD repeat domain, phosphoinositide interacting 2. ispartof: AUTOPHAGY vol:17 issue:7 pages:1714-1728 ispartof: location:United States status: published

Polycystin-2 (PC2) is a calcium channel that can be found in the endoplasmic reticulum, the plasmatic membrane, and the primary cilium. The structure of PC2 is characterized by a highly ordered C-terminal tail with an EF-motif (calcium-binding domain) and a canonical coiled-coil domain (CCD; interaction domain), and its activity is regulated by interacting partners and post-translational modifications. Calcium mobilization into the cytosol by PC2 has been mainly associated with cell growth and differentiation, and therefore mutations or dysfunction of PC2 lead to renal and cardiac consequences. Interestingly, PC2-related pathologies are usually treated with rapamycin, an autophagy stimulator. Autophagy is an intracellular degradation process where recycling material is sequestered into autophagosomes and then hydrolyzed by fusion with a lysosome. Interestingly, several studies have provided evidence that PC2 may be required for autophagy, suggesting that PC2 maintains a physiologic catabolic state.

Polycystin-2 (PC2) is a calcium channel that can be found in the endoplasmic reticulum, the plasmatic membrane, and the primary cilium. The structure of PC2 is characterized by a highly ordered C-terminal tail with an EF-motif (calcium-binding domain) and a canonical coiled-coil domain (CCD; interaction domain), and its activity is regulated by interacting partners and post-translational modifications. Calcium mobilization into the cytosol by PC2 has been mainly associated with cell growth and differentiation, and therefore mutations or dysfunction of PC2 lead to renal and cardiac consequences. Interestingly, PC2-related pathologies are usually treated with rapamycin, an autophagy stimulator. Autophagy is an intracellular degradation process where recycling material is sequestered into autophagosomes and then hydrolyzed by fusion with a lysosome. Interestingly, several studies have provided evidence that PC2 may be required for autophagy, suggesting that PC2 maintains a physiologic catabolic state.

// Daniel Pena-Oyarzun 1,2,* , Rodrigo Troncoso 1,3,* , Catalina Kretschmar 1,4 , Cecilia Hernando 1,4 , Mauricio Budini 4 , Eugenia Morselli 5 , Sergio Lavandero 1,2,6 and Alfredo Criollo 1,4 1 Advanced Center for Chronic Diseases, Facultad Ciencias Quimicas y Farmaceuticas & Facultad Medicina, Universidad de Chile, Santiago, Chile 2 Center for Molecular Studies of the Cell, Facultad de Medicina, Universidad de Chile, Santiago, Chile 3 Instituto de Nutricion y Tecnologia de los Alimentos, Universidad de Chile, Santiago, Chile 4 Instituto de Investigacion en Ciencias Odontologicas, Facultad de Odontologia, Universidad de Chile, Santiago, Chile 5 Departamento de Fisiologia, Facultad de Ciencias Biologicas, Pontificia Universidad Catolica de Chile, Santiago, Chile 6 Department of Internal Medicine (Cardiology Division), University of Texas Southwestern Medical Center, Dallas, TX, USA * These authors have contributed equally to this work Correspondence to: Alfredo Criollo, email: // Sergio Lavandero, email: // Keywords : hyperosmotic stress, polycystin-2, mTOR, Autophagy Received : May 10, 2017 Accepted : June 21, 2017 Published : July 05, 2017 Abstract Various intracellular mechanisms are activated in response to stress, leading to adaptation or death. Autophagy, an intracellular process that promotes lysosomal degradation of proteins, is an adaptive response to several types of stress. Osmotic stress occurs under both physiological and pathological conditions, provoking mechanical stress and activating various osmoadaptive mechanisms. Polycystin-2 (PC2), a membrane protein of the polycystin family, is a mechanical sensor capable of activating the cell signaling pathways required for cell adaptation and survival. Here we show that hyperosmotic stress provoked by treatment with hyperosmolar concentrations of sorbitol or mannitol induces autophagy in HeLa and HCT116 cell lines. In addition, we show that mTOR and AMPK, two stress sensor proteins involved modulating autophagy, are downregulated and upregulated, respectively, when cells are subjected to hyperosmotic stress. Finally, our findings show that PC2 is required to promote hyperosmotic stress-induced autophagy. Downregulation of PC2 prevents inhibition of hyperosmotic stress-induced mTOR pathway activation. In conclusion, our data provide new insight into the role of PC2 as a mechanosensor that modulates autophagy under hyperosmotic stress conditions.

Muscle atrophy involves a massive catabolism of intracellular components leading to a significant reduction in cellular and tissue volume. In this regard, autophagy, an intracellular mechanism that degrades proteins and organelles, has been implicated with muscle breakdown. Recently, it has shown that polycystin-2 (PC2), a membrane protein that belongs to the transient receptor potential (TRP) family, is required for the maintenance of cellular proteostasis, by regulating autophagy in several cell types. The role of PC2 in the control of atrophy and autophagy in skeletal muscle remains unknown. Here, we show that PC2 is required for the induction of atrophy in C2C12 myotubes caused by nutrient deprivation or rapamycin exposure. Consistently, overexpression of PC2 induces atrophy in C2C12 myotubes as indicated by decreasing of the myogenic proteins myogenin and caveolin-3. In addition, we show that inhibition of mTORC1, by starvation or rapamycin is inhibited in cells when PC2 is silenced. Importantly, even if PC2 regulates mTORC1, our results show that the regulation of atrophy by PC2 is independent of autophagy. This study provides novel evidence regarding the role of PC2 in skeletal muscle cell atrophy.