TARDBP/TDP-43 regulates autophagy in both MTORC1-dependent and MTORC1-independent manners

In a recent paper we addressed the mechanism by which defective autophagy contributes to TARDBP/TDP-43-mediated neurodegenerative disorders. We demonstrated that TARDBP regulates MTORC1-TFEB signaling by targeting RPTOR/raptor, a key component and an adaptor protein of MTORC1. Loss of TARDBP decreased the mRNA stability of RPTOR and this regulation in turn enhanced autophagosomal and lysosomal biogenesis in an MTORC1-dependent manner. Meanwhile, loss of TARDBP could also impair autophagosome-lysosome fusion in an MTORC1-independent manner. Importantly, we found that modulation of MTOR activity by treatment with rapamycin and phosphatidic acid had strong effects on the neurodegenerative phenotypes of TBPH (Drosophila TARDBP)-depleted flies. Taken together, our data reveal that multiple dysfunctions in the autophagic process contribute to TARDBP-linked neurodegeneration and may help to identify potential therapeutic targets in the future.     

The Multifunctional Drosophila melanogaster V-ATPase Is Encoded by a Multigene Family

In animals, V-ATPases are believed to play roles in the plasma membrane,as well as endomembrane. To understand these different functions, it isnecessary to adopt a genetic approach in a physiologically tractable modelorganism. For this purpose, Drosophila melanogaster is ideal,because of the powerful genetics associated with the organism and because ofthe unusually informative epithelial phenotype provided by the Malpighiantubule. Recently, the first animal knockouts of a V-ATPase weredescribed in Drosophila. The resulting phenotypes have generalutility for our understanding of V-ATPase function and suggest a screen fornovel subunits and associated proteins. Genome project resources haveaccelerated our knowledge of the V-ATPase gene family size and the newDrosophila genes vhaSFD, vha100-1, vha100-2, vha100-3, vha16-2,vha16-3, vha16-4, vhaPPA1, vhaPPA2, vhaM9.7.1, and vhaM9.7.2are described. The Drosophila V-ATPase model is thus well-suited toboth forward and reverse genetic analysis of this complex multifunctionalenzyme.     

Pharmacological inhibition of lysosomes activates the MTORC1 signaling pathway in chondrocytes in an autophagy-independent manner

Mechanistic target of rapamycin (serine/threonine kinase) complex 1 (MTORC1) is a protein-signaling complex at the fulcrum of anabolic and catabolic processes, which acts depending on wide-ranging environmental cues. It is generally accepted that lysosomes facilitate MTORC1 activation by generating an internal pool of amino acids. Amino acids activate MTORC1 by stimulating its translocation to the lysosomal membrane where it forms a super-complex involving the lysosomal-membrane-bound vacuolar-type H⁺-ATPase (v-ATPase) proton pump. This translocation and MTORC1 activation require functional lysosomes. Here we found that, in contrast to this well-accepted concept, in epiphyseal chondrocytes inhibition of lysosomal activity by v-ATPase inhibitors bafilomycin A₁ or concanamycin A potently activated MTORC1 signaling. The activity of MTORC1 was visualized by phosphorylated forms of RPS6 (ribosomal protein S6) and EIF4EBP1, 2 well-known downstream targets of MTORC1. Maximal RPS6 phosphorylation was observed at 48-h treatment and reached as high as a 12-fold increase (p < 0.018). This activation of MTORC1 was further confirmed in bone organ culture and promoted potent stimulation of longitudinal growth (p < 0.001). Importantly, the same effect was observed in ATG5 (autophagy-related 5)-deficient bones suggesting a macroautophagy-independent mechanism of MTORC1 inhibition by lysosomes. Thus, our data show that in epiphyseal chondrocytes lysosomes inhibit MTORC1 in a macroautophagy-independent manner and this inhibition likely depends on v-ATPase activity.     

V-ATPase and osmotic imbalances activate endolysosomal LC3 lipidation

Recently a noncanonical activity of autophagy proteins has been discovered that targets lipidation of microtubule-associated protein 1 light chain 3 (LC3) onto macroendocytic vacuoles, including macropinosomes, phagosomes, and entotic vacuoles. While this pathway is distinct from canonical autophagy, the mechanism of how these nonautophagic membranes are targeted for LC3 lipidation remains unclear. Here we present evidence that this pathway requires activity of the vacuolar-type H⁺-ATPase (V-ATPase) and is induced by osmotic imbalances within endolysosomal compartments. LC3 lipidation by this mechanism is induced by treatment of cells with the lysosomotropic agent chloroquine, and through exposure to the Heliobacter pylori pore-forming toxin VacA. These data add novel mechanistic insights into the regulation of noncanonical LC3 lipidation and its associated processes, including LC3-associated phagocytosis (LAP), and demonstrate that the widely and therapeutically used drug chloroquine, which is conventionally used to inhibit autophagy flux, is an inducer of LC3 lipidation.     

A SNX10/V-ATPase pathway regulates ciliogenesis in vitro and in vivo

Sorting nexins (SNXs) are phosphoinositide-binding proteins implicated in the sorting of various membrane proteins in vitro, but the in vivo functions of them remain largely unknown. We reported previously that SNX10 is a unique member of the SNX family genes in that it has vacuolation activity in cells. We investigate the biological function of SNX10 by loss-of-function assay in this study and demonstrate that SNX10 is required for the formation of primary cilia in cultured cells. In zebrafish, SNX10 is involved in ciliogenesis in the Kupffer's vesicle and essential for left-right patterning of visceral organs. Mechanistically, SNX10 interacts with V-ATPase complex and targets it to the centrosome where ciliogenesis is initiated. Like SNX10, V-ATPase regulates ciliogenesis in vitro and in vivo and does so synergistically with SNX10. We further discover that SNX10 and V-ATPase regulate the ciliary trafficking of Rab8a, which is a critical regulator of ciliary membrane extension. These results identify an SNX10/V-ATPase-regulated vesicular trafficking pathway that is crucial for ciliogenesis, and reveal that SNX10/V-ATPase, through the regulation of cilia formation in various organs, play an essential role during early embryonic development.     

Dual suppressive effect of MTORC1 on autophagy

The lysosome is a key subcellular organelle that receives and degrades macromolecules from endocytic, secretory and autophagic pathways. Lysosomal function is thus critical for an efficient autophagic process. However, the molecular mechanisms mediating lysosomal function upon autophagic induction are largely unknown. Our laboratory recently discovered that upon autophagy activation, the lysosome is activated, and this functional activation is dependent on MTORC1 suppression, suggesting that MTORC1 exerts a suppressive effect on lysosomal function. Therefore, data from our study demonstrate that MTORC1 exerts a dual inhibitory effect on autophagy, blocking autophagy not only at the initiation stage via suppression of the ULK1 complex, but also at the degradation stage via inhibition of lysosomal function. We think that understanding the negative regulatory effect of MTORC1 on lysosomal function expands the functional scope of MTORC1 in autophagy regulation, and offers new clues for developing novel interventional strategies in autophagy- and lysosome-related diseases.     

Sustained activation of autophagy suppresses adipocyte maturation via a lipolysis-dependent mechanism

ABSTRACT

Dysregulation of macroautophagy/autophagy is implicated in obesity and insulin resistance. However, it remains poorly defined how autophagy regulates adipocyte development. Using adipose-specific rptor/raptor knockout (KO), atg7 KO and atg7 rptor double-KO mice, we show that inhibiting MTORC1 by RPTOR deficiency led to autophagic sequestration of lipid droplets, formation of LD-containing lysosomes, and elevation of basal and isoproterenol-induced lipolysis in vivo and in primary adipocytes. Despite normal differentiation at an early phase, progressive degradation and shrinkage of cellular LDs and downregulation of adipogenic markers PPARG and PLIN1 occurred in terminal differentiation of rptor KO adipocytes, which was rescued by inhibiting lipolysis or lysosome. In contrast, inactivating autophagy by depletion of ATG7 protected adipocytes against RPTOR deficiency-induced formation of LD-containing lysosomes, LD degradation, and downregulation of adipogenic markers in vitro. Ultimately, atg7 rptor double-KO mice displayed decreased lipolysis, restored adipose tissue development, and upregulated thermogenic gene expression in brown and inguinal adipose tissue compared to RPTOR-deficient mice in vivo. Collectively, our study demonstrates that autophagy plays an important role in regulating adipocyte maturation via a lipophagy and lipolysis-dependent mechanism.     

V-ATPase and osmotic imbalances activate endolysosomal LC3 lipidation

Recently a noncanonical activity of autophagy proteins has been discovered that targets lipidation of microtubule-associated protein 1 light chain 3 (LC3) onto macroendocytic vacuoles, including macropinosomes, phagosomes, and entotic vacuoles. While this pathway is distinct from canonical autophagy, the mechanism of how these nonautophagic membranes are targeted for LC3 lipidation remains unclear. Here we present evidence that this pathway requires activity of the vacuolar-type H⁺-ATPase (V-ATPase) and is induced by osmotic imbalances within endolysosomal compartments. LC3 lipidation by this mechanism is induced by treatment of cells with the lysosomotropic agent chloroquine, and through exposure to the Heliobacter pylori pore-forming toxin VacA. These data add novel mechanistic insights into the regulation of noncanonical LC3 lipidation and its associated processes, including LC3-associated phagocytosis (LAP), and demonstrate that the widely and therapeutically used drug chloroquine, which is conventionally used to inhibit autophagy flux, is an inducer of LC3 lipidation.     

Emerging role of podocyte autophagy in the progression of diabetic nephropathy

Glomerular podocytes are pivotal in maintaining glomerular filtration barrier function. As severe podocyte injury results in proteinuria in patients with diabetic nephropathy, determining the pathogenesis of podocyte injury may contribute to the development of new treatments. We recently showed that autophagy is involved in the pathogenesis of diabetes-related podocyte injury. Insufficient podocyte autophagy and podocyte loss are observed in diabetic patients with massive proteinuria. Podocyte loss and massive proteinuria occur in high-fat diet-induced diabetic mice with podocyte-specific autophagy deficiency, with podocytes of these mice and of diabetic rats having huge damaged lysosomes. Sera from diabetic patients and from rodents with massive proteinuria cause autophagy insufficiency, resulting in lysosome dysfunction and apoptosis of cultured podocytes. These findings suggest the importance of autophagy in maintaining lysosome homeostasis in podocytes under diabetic conditions. Impaired autophagy may be involved in the pathogenesis of podocyte loss, leading to massive proteinuria in diabetic nephropathy.     

TPT1 (tumor protein,translationally-controlled 1) negatively regulates autophagy through the BECN1 interactome and an MTORC1-mediated pathway

TPT1/TCTP (tumor protein, translationally-controlled 1) is highly expressed in tumor cells, known to participate in various cellular activities including protein synthesis, growth and cell survival. In addition, TPT1 was identified as a direct target of the tumor suppressor TP53/p53 although little is known about the mechanism underlying the anti-survival function of TPT1. Here, we describe a role of TPT1 in the regulation of the MTORC1 pathway through modulating the molecular machinery of macroautophagy/autophagy. TPT1 inhibition induced cellular autophagy via the MTORC1 and AMPK pathways, which are inhibited and activated, respectively, during treatment with the MTOR inhibitor rapamycin. We also found that the depletion of TPT1 potentiated rapamycin-induced autophagy by synergizing with MTORC1 inhibition. We further demonstrated that TPT1 knockdown altered the BECN1 interactome, a representative MTOR-independent pathway, to stimulate autophagosome formation, via downregulating BCL2 expression through activating MAPK8/JNK1, and thereby enhancing BECN1-phosphatidylinositol 3-kinase (PtdIns3K)-UVRAG complex formation. Furthermore, reduced TPT1 promoted autophagic flux by modulating not only early steps of autophagy but also autophagosome maturation. Consistent with in vitro findings, in vivo organ analysis using Tpt1 heterozygote knockout mice showed that autophagy is enhanced because of haploinsufficient TPT1 expression. Overall, our study demonstrated the novel role of TPT1 as a negative regulator of autophagy that may have potential use in manipulating various diseases associated with autophagic dysfunction.     

Hepatic peroxisomal β-oxidation suppresses lipophagy via RPTOR acetylation and MTOR activation

ABSTRACT

Hepatic lipid homeostasis is controlled by a coordinated regulation of various metabolic pathways involved in de novo synthesis, uptake, storage, and catabolism of lipids. Disruption of this balance could lead to hepatic steatosis. Peroxisomes play an essential role in lipid metabolism, yet their importance is often overlooked. In a recent study, we demonstrated a role for hepatic peroxisomal β-oxidation in autophagic degradation of lipid droplets. ACOX1 (acyl-Coenzyme A oxidase 1, palmitoyl), the rate-limiting enzyme of peroxisomal β-oxidation, increases with fasting or high-fat diet (HFD). Liver-specific acox1 knockout (acox1-LKO) protects mice from hepatic steatosis induced by starvation or HFD via induction of lipophagy. Mechanistically, we showed that hepatic ACOX1 deficiency decreases the total cytosolic acetyl-CoA levels, which leads to reduced acetylation of RPTOR/RAPTOR, a component of MTORC1, which is a key regulator of macroautophagy/autophagy. These results identify peroxisome-derived acetyl-CoA as a critical metabolic regulator of autophagy that controls hepatic lipid homeostasis.     

Immunohistochemical identification of a vacuolar proton pump (V-ATPase) in bone-resorbing cells of an advanced teleost species, Oreochromis niloticus

Poly-and monoclonal antibodies, raised against mammalian membrane-bound proton pump (V-ATPase) were applied to the bone-resorbing cells of Oreochromis niloticus to clarify if osteoclasts of an advanced teleost species display V-ATPase, a key enzyme in the process of bone resorption. All antibodies labelled cells at known sites of bone resorption, the endosteal bone surfaces surrounding the tooth anlagen. The best results were achieved with a monoclonal antibody (E11). Although the majority of labelled cells were flat and mononucleated, the occurrence of V-ATPase in these cells indicates that they function as active bone-resorbing cells. The monoclonal antibody E11 was also applied successfully to monocytes, cells that are believed to be related most closely to osteoclasts. The assignment of V-ATPase to boneresorbing cells of O. niloticus was confirmed by application of the additional osteoclast markers, tartrate-resistant acid phosphatase (TRAP) and tartrate-resistant ATPase (TraATPase). Co-expression of V-ATPase, TRAP and TraATPase in fish osteoclasts is demonstrated for the first time.     

A negative feedback regulation of MTORC1 activity by the lysosomal Ca2+ channel MCOLN1 (mucolipin 1) using a CALM (calmodulin)-dependent mechanism

Macroautophagy/autophagy is an evolutionarily conserved pathway that is required for cellular homeostasis, growth and survival. The lysosome plays an essential role in autophagy regulation. For example, the activity of MTORC1, a master regulator of autophagy, is regulated by nutrients within the lysosome. Starvation inhibits MTORC1 causing autophagy induction. Given that MTORC1 is critical for protein synthesis and cellular homeostasis, a feedback regulatory mechanism must exist to restore MTORC1 during starvation. However, the molecular mechanism underlying this feedback regulation is unclear. In this study, we report that starvation activates the lysosomal Ca²⁺ release channel MCOLN1 (mucolipin 1) by relieving MTORC1's inhibition of the channel. Activated MCOLN1 in turn facilitates MTORC1 activity that requires CALM (calmodulin). Moreover, both MCOLN1 and CALM are necessary for MTORC1 reactivation during prolonged starvation. Our data suggest that lysosomal Ca²⁺ signaling is an essential component of the canonical MTORC1-dependent autophagy pathway and MCOLN1 provides a negative feedback regulation of MTORC1 to prevent excessive loss of MTORC1 function during starvation. The feedback regulation may be important for maintaining cellular homeostasis during starvation, as well as many other stressful or disease conditions.