ABSTRACT
Warburg micro syndrome (WMS) is a hereditary autosomal neuromuscular disorder in humans caused by mutations in Rab18, Rab3GAP1, or Rab3GAP2 genes. Rab3GAP1/2 forms a heterodimeric complex, which acts as a guanosine nucleotide exchange factor and activates Rab18. Although the genetic causes of WMS are known, it is still unclear whether loss of the Rab3GAP-Rab18 module affects neuronal or muscle cell physiology or both, and how. In this work, we characterize a Rab3GAP2 mutant Drosophila line to establish a novel animal model for WMS. Similarly to symptoms of WMS, loss of Rab3GAP2 leads to highly decreased motility in Drosophila that becomes more serious with age. We demonstrate that these mutant flies are defective for autophagic degradation in multiple tissues including fat cells and muscles. Loss of Rab3GAP-Rab18 module members leads to perturbed autolysosome morphology due to destabilization of Rab7-positive autophagosomal and late endosomal compartments and perturbation of lysosomal biosynthetic transport. Importantly, overexpression of UVRAG or loss of Atg14, two alternative subunits of the Vps34/PI3K (vacuole protein sorting 34/phosphatidylinositol 3-kinase) complexes in fat cells, mimics the autophagic phenotype of Rab3GAP-Rab18 module loss. We find that GTP-bound Rab18 binds to Atg6/Beclin1, a permanent subunit of Vps34 complexes. Finally, we show that Rab3GAP2 and Rab18 are present on autophagosomal and autolysosomal membranes and colocalize with Vps34 Complex I subunits. Our data suggest that the Rab3GAP-Rab18 module regulates autolysosomal maturation through its interaction with the Vps34 Complex I, and perturbed autophagy due to loss of the Rab3GAP-Rab18 module may contribute to the development of WMS.
Subject(s)
Abnormalities, Multiple/genetics , Cataract/congenital , Class III Phosphatidylinositol 3-Kinases/genetics , Cornea/abnormalities , Drosophila Proteins/genetics , Hypogonadism/genetics , Intellectual Disability/genetics , Lysosomes/metabolism , Microcephaly/genetics , Optic Atrophy/genetics , rab GTP-Binding Proteins/genetics , rab3 GTP-Binding Proteins/genetics , Abnormalities, Multiple/metabolism , Abnormalities, Multiple/pathology , Adaptor Proteins, Vesicular Transport/genetics , Adaptor Proteins, Vesicular Transport/metabolism , Adipocytes/metabolism , Adipocytes/pathology , Animals , Autophagy/genetics , Autophagy-Related Proteins/genetics , Autophagy-Related Proteins/metabolism , Beclin-1/genetics , Beclin-1/metabolism , Cataract/genetics , Cataract/metabolism , Cataract/pathology , Class III Phosphatidylinositol 3-Kinases/deficiency , Cornea/metabolism , Cornea/pathology , Disease Models, Animal , Drosophila Proteins/deficiency , Drosophila Proteins/metabolism , Drosophila melanogaster , Gene Expression Regulation , Humans , Hypogonadism/metabolism , Hypogonadism/pathology , Intellectual Disability/metabolism , Intellectual Disability/pathology , Lysosomes/pathology , Microcephaly/metabolism , Microcephaly/pathology , Muscles/metabolism , Muscles/pathology , Neurons/metabolism , Neurons/pathology , Optic Atrophy/metabolism , Optic Atrophy/pathology , Protein Binding , Sequence Homology, Amino Acid , Signal Transduction , Tumor Suppressor Proteins/genetics , Tumor Suppressor Proteins/metabolism , rab GTP-Binding Proteins/deficiency , rab GTP-Binding Proteins/metabolism , rab3 GTP-Binding Proteins/deficiency , rab7 GTP-Binding ProteinsABSTRACT
OBJECTIVE: Class III phosphoinositide 3-kinase, also known as VPS34 (vacuolar protein sorting 34), is a highly conserved enzyme regulating important cellular functions such as NADPH oxidase (NOX) assembly, membrane trafficking, and autophagy. Although VPS34 is expressed in platelets, its involvement in platelet activation remains unclear. Herein, we investigated the role of VPS34 in platelet activation and thrombus formation using VPS34 knockout mice. APPROACH AND RESULTS: Platelet-specific VPS34-deficient mice were generated and characterized. VPS34 deficiency in platelets did not influence tail bleeding time. In a ferric chloride-induced mesenteric arteriolar thrombosis model, VPS34-/- mice exhibited a prolonged vessel occlusion time compared with wild-type mice (42.05±4.09 versus 18.30±2.47 minutes). In an in vitro microfluidic whole-blood perfusion assay, thrombus formation on collagen under arterial shear was significantly reduced for VPS34-/- platelets. VPS34-/- platelets displayed an impaired aggregation and dense granule secretion in response to low doses of collagen or thrombin. VPS34 deficiency delayed clot retraction but did not influence platelet spreading on fibrinogen. We also demonstrated that VPS34 deficiency altered the basal level of autophagy in resting platelets and hampered NOX assembly and mTOR (mammalian target of rapamycin) signaling during platelet activation. Importantly, we identified the NOX-dependent reactive oxygen species generation as the major downstream effector of VPS34, which in turn can mediate platelet activation. In addition, by using a specific inhibitor 3-methyladenine, VPS34 was found to operate through a similar NOX-dependent mechanism to promote human platelet activation. CONCLUSIONS: Platelet VPS34 is critical for thrombosis but dispensable for hemostasis. VPS34 regulates platelet activation by influencing NOX assembly.
Subject(s)
Blood Coagulation , Blood Platelets/enzymology , Class III Phosphatidylinositol 3-Kinases/blood , NADPH Oxidases/blood , Phosphatidylinositol Phosphates/blood , Platelet Activation , Thrombosis/enzymology , Adult , Animals , Autophagy , Chlorides , Class III Phosphatidylinositol 3-Kinases/deficiency , Class III Phosphatidylinositol 3-Kinases/genetics , Collagen/blood , Disease Models, Animal , Female , Ferric Compounds , Genotype , Humans , Male , Mice, Inbred C57BL , Mice, Knockout , Phenotype , Platelet Aggregation , Reactive Oxygen Species/blood , Signal Transduction , TOR Serine-Threonine Kinases/blood , Thrombin/metabolism , Thrombosis/blood , Thrombosis/chemically induced , Thrombosis/genetics , Time Factors , Young AdultABSTRACT
The PI 3-kinase Vps34 (Pik3c3) synthesizes phosphatidylinositol 3-phosphate (PI3P), a lipid critical for both endosomal membrane traffic and macroautophagy. Human genetics have implicated PI3P dysregulation, and endosomal trafficking in general, as a recurring cause of demyelinating Charcot-Marie-Tooth (CMT) peripheral neuropathy. Here, we investigated the role of Vps34, and PI3P, in mouse Schwann cells by selectively deleting Vps34 in this cell type. Vps34-Schwann cell knockout (Vps34SCKO ) mice show severe hypomyelination in peripheral nerves. Vps34-/- Schwann cells interact abnormally with axons, and there is a delay in radial sorting, a process by which large axons are selected for myelination. Upon reaching the promyelinating stage, Vps34-/- Schwann cells are significantly impaired in the elaboration of myelin. Nerves from Vps34SCKO mice contain elevated levels of the LC3 and p62 proteins, indicating impaired autophagy. However, in the light of recent demonstrations that autophagy is dispensable for myelination, it is unlikely that hypomyelination in Vps34SCKO mice is caused by impaired autophagy. Endosomal trafficking is also disturbed in Vps34-/- Schwann cells. We investigated the activation of the ErbB2/3 receptor tyrosine kinases in Vps34SCKO nerves, as these proteins, which play essential roles in Schwann cell myelination, are known to traffic through endosomes. In Vps34SCKO nerves, ErbB3 was hyperphosphorylated on a tyrosine known to be phosphorylated in response to neuregulin 1 exposure. ErbB2 protein levels were also decreased during myelination. Our findings suggest that the loss of Vps34 alters the trafficking of ErbB2/3 through endosomes. Abnormal ErbB2/3 signaling to downstream targets may contribute to the hypomyelination observed in Vps34SCKO mice.
Subject(s)
Axons/enzymology , Class III Phosphatidylinositol 3-Kinases/deficiency , Neuronal Outgrowth/physiology , Receptor, ErbB-2/metabolism , Receptor, ErbB-3/metabolism , Schwann Cells/enzymology , Animals , Autophagy/physiology , Axons/pathology , Cell Proliferation/physiology , Class III Phosphatidylinositol 3-Kinases/genetics , Endosomes/enzymology , Endosomes/pathology , Female , Male , Mice, 129 Strain , Mice, Inbred C57BL , Mice, Knockout , Motor Activity/physiology , Peripheral Nerves/enzymology , Peripheral Nerves/growth & development , Peripheral Nerves/pathology , Phosphorylation , Schwann Cells/pathology , Sciatic Nerve/enzymology , Sciatic Nerve/growth & development , Sciatic Nerve/pathology , Signal TransductionABSTRACT
Phosphoinositides play important roles in numerous intracellular membrane pathways. Little is known about the regulation or function of these lipids in rod photoreceptor cells, which have highly active membrane dynamics. Using new assays with femtomole sensitivity, we determined that whereas levels of phosphatidylinositol-3,4-bisphosphate and phosphatidylinositol-3,4,5-trisphosphate were below detection limits, phosphatidylinositol-3-phosphate (PI(3)P) levels in rod inner/outer segments increased more than 30-fold after light exposure. This increase was blocked in a rod-specific knockout of the PI-3 kinase Vps34, resulting in failure of endosomal and autophagy-related membranes to fuse with lysosomes, and accumulation of abnormal membrane structures. At early ages, rods displayed normal morphology, rhodopsin trafficking, and light responses, but underwent progressive neurodegeneration with eventual loss of both rods and cones by twelve weeks. The degeneration is considerably faster than in rod knockouts of autophagy genes, indicating defects in endosome recycling or other PI(3)P-dependent membrane trafficking pathways are also essential for rod survival.
Subject(s)
Class III Phosphatidylinositol 3-Kinases/genetics , Phosphatidylinositol Phosphates/metabolism , Retinal Cone Photoreceptor Cells/metabolism , Retinal Degeneration/genetics , Retinal Rod Photoreceptor Cells/metabolism , Rhodopsin/genetics , Animals , Autophagy/genetics , Autophagy-Related Proteins/deficiency , Autophagy-Related Proteins/genetics , Cell Survival , Class III Phosphatidylinositol 3-Kinases/deficiency , Endosomes/metabolism , Gene Expression Regulation , Light , Light Signal Transduction , Lysosomes/metabolism , Membrane Fusion , Mice , Mice, Inbred C57BL , Mice, Knockout , Retinal Cone Photoreceptor Cells/pathology , Retinal Degeneration/metabolism , Retinal Degeneration/pathology , Retinal Rod Photoreceptor Cells/pathology , Rhodopsin/metabolismABSTRACT
Phosphatidylinositol phosphates are key regulators of vesicle identity, formation and trafficking. In mammalian cells, the evolutionarily conserved class III PtdIns 3-kinase PIK3C3/VPS34 is part of a large multiprotein complex that catalyzes the localized phosphorylation of phosphatidylinositol to phosphatidylinositol-3-phosphate (PtdIns3P). We demonstrate that PIK3C3 has a key function in vesicular trafficking, endocytosis and autophagosome-autolysosome formation in the highly specialized glomerular podocytes.
Subject(s)
Autophagy , Class III Phosphatidylinositol 3-Kinases/metabolism , Endocytosis , Lysosomes/metabolism , Phagosomes/metabolism , Podocytes/cytology , Podocytes/enzymology , Animals , Class III Phosphatidylinositol 3-Kinases/deficiency , Drosophila melanogaster/cytology , Humans , Mice , Models, BiologicalABSTRACT
The molecular mechanisms that maintain podocytes and consequently, the integrity of the glomerular filtration barrier are incompletely understood. Here, we show that the class III phosphoinositide 3-kinase vacuolar protein sorting 34 (Vps34) plays a central role in modulating endocytic pathways, maintaining podocyte homeostasis. In mice, podocyte-specific conditional knockout of Vps34 led to early proteinuria, glomerular scarring, and death within 3-9 weeks of age. Vps34-deficient podocytes exhibited substantial vacuolization and foot process effacement. Although the formation of autophagosomes and autophagic flux were impaired, comparisons between podocyte-specific Vps34-deficient mice, autophagy-deficient mice, and doubly deficient mice suggested that defective autophagy was not primarily responsible for the severe phenotype caused by the loss of Vps34. In fact, Rab5-positive endosomal compartments, endocytosis, and fluid-phase uptake were severely disrupted in Vps34-deficient podocytes. Vps34 deficiency in nephrocytes, the podocyte-like cells of Drosophila melanogaster, resulted in a block between Rab5- and Rab7-positive endosomal compartments. In summary, these data identify Vps34 as a major regulator of endolysosomal pathways in podocytes and underline the fundamental roles of endocytosis and fluid-phase uptake for the maintenance of the glomerular filtration barrier.
Subject(s)
Class III Phosphatidylinositol 3-Kinases/physiology , Endocytosis , Homeostasis , Podocytes/physiology , Animals , Autophagy , Class III Phosphatidylinositol 3-Kinases/deficiency , Drosophila melanogaster , Endosomes/metabolism , Kidney Glomerulus/pathology , Mice , Mice, Inbred C57BL , Proteinuria/etiology , SclerosisABSTRACT
PIK3C3/Vps34 is the class III PtdIns3K that is evolutionarily conserved from yeast to mammals. Its central role in mammalian autophagy has been suggested through the use of pharmacological inhibitors and the study of its binding partners. However, the precise role of PIK3C3 in mammals is not clear. Using mouse strains that allow tissue-specific deletion of PIK3C3, we have described an essential role of PIK3C3 in regulating autophagy, and liver and heart function.
Subject(s)
Autophagy , Class III Phosphatidylinositol 3-Kinases/metabolism , Liver/enzymology , Mammals/metabolism , Myocardium/enzymology , Animals , Class III Phosphatidylinositol 3-Kinases/deficiency , Fibroblasts/enzymology , Gene Deletion , Humans , Liver/pathology , Mice , Myocardium/pathology , Phagosomes/metabolism , Phagosomes/ultrastructure , T-Lymphocytes/enzymologyABSTRACT
A critical regulator of autophagy is the Class III PI3K Vps34 (also called PIK3C3). Although Vps34 is known to play an essential role in autophagy in yeast, its role in mammals remains elusive. To elucidate the physiological function of Vps34 and to determine its precise role in autophagy, we have generated Vps34(f/f) mice, in which expression of Cre recombinase results in a deletion of exon 4 of Vps34 and a frame shift causing a deletion of 755 of the 887 amino acids of Vps34. Acute ablation of Vps34 in MEFs upon adenoviral Cre infection results in a diminishment of localized generation of phosphatidylinositol 3-phosphate and blockade of both endocytic and autophagic degradation. Starvation-induced autophagosome formation is blocked in both Vps34-null MEFs and liver. Liver-specific Albumin-Cre;Vps34(f/f) mice developed hepatomegaly and hepatic steatosis, and impaired protein turnover. Ablation of Vps34 in the heart of muscle creatine kinase-Cre;Vps34(f/f) mice led to cardiomegaly and decreased contractility. In addition, while amino acid-stimulated mTOR activation was suppressed in the absence of Vps34, the steady-state level of mTOR signaling was not affected in Vps34-null MEFs, liver, or cardiomyocytes. Taken together, our results indicate that Vps34 plays an essential role in regulating functional autophagy and is indispensable for normal liver and heart function.
Subject(s)
Autophagy , Class III Phosphatidylinositol 3-Kinases/metabolism , Liver/enzymology , Liver/pathology , Myocardium/enzymology , Myocardium/pathology , Amino Acids/metabolism , Animals , Class III Phosphatidylinositol 3-Kinases/deficiency , Electrocardiography , Embryo, Mammalian/cytology , Enzyme Activation , Fibroblasts/enzymology , Fibroblasts/pathology , Gene Deletion , Liver/physiopathology , Liver/ultrastructure , Mice , Mice, Knockout , Phagosomes/metabolism , Phagosomes/pathology , Phagosomes/ultrastructure , Phosphatidylinositol Phosphates/metabolism , Signal Transduction , TOR Serine-Threonine Kinases/metabolismABSTRACT
The Pik3c3 gene encodes an 887 amino acid lipid kinase, phosphoinositide-3-kinase class 3 (PIK3C3). PIK3C3 is known to regulate various intracellular membrane trafficking events. However, little is known about its functions during early embryogenesis in mammals. To investigate the function of PIK3C3 in vivo, we generated Pik3c3 null mice. We show here that Pik3c3 heterozygous are normal and fertile. In contrast, Pik3c3 homozygous mutants are embryonic lethal and die between E7.5 and E8.5 of embryogenesis. Mutant embryos are poorly developed with no evidence of mesoderm formation, and suffer from severely reduced cell proliferations. Cell proliferation defect is also evident in vitro, where mutant blastocysts in culture fail to give rise to typical colonies formed by inner cell mass. Electron microscopic analysis revealed that epiblast cells in mutant embryos appear normal, whereas the visceral endoderm cells contain larger vesicles inside the lipid droplets. Finally, we provide evidence that mTOR signaling is drastically reduced in Pik3c3 null embryos, which could be a major contributor to the observed proliferation and embryogenesis defects.
Subject(s)
Cell Proliferation , Class III Phosphatidylinositol 3-Kinases/deficiency , Class III Phosphatidylinositol 3-Kinases/physiology , Embryonic Development , Animals , Blastocyst/pathology , Embryo, Mammalian , Endoderm , Mesoderm , Mice , Mice, Knockout , TOR Serine-Threonine Kinases/metabolismABSTRACT
The lipid kinase PIK3C3 (also known as VPS34) regulates multiple aspects of endo-membrane trafficking processes. PIK3C3 is widely expressed by neurons in the CNS, and its catalytic product PI3P is enriched in dendritic spines. Here we generated a line of conditional mutant mouse in which Pik3c3 is specifically deleted in hippocampal and in small subsets of cortical pyramidal neurons using the CaMKII-Cre transgene. We found that Pik3c3-deficiency initially causes loss of dendritic spines accompanied with reactive gliosis, which is followed by progressive neuronal degeneration over a period of several months. Layers III and IV cortical neurons are more susceptible to Pik3c3-deletion than hippocampal neurons. Furthermore, in aged conditional Pik3c3 mutant animals, there are extensive gliosis and severe secondary loss of wild type neurons. Our analyses show that Pik3c3 is essential for CNS neuronal homeostasis and Pik3c3flox/flox; CaMKII-Cre mouse is a useful model for studying pathological changes in progressive forebrain neurodegeneration.