ABSTRACT
Sequestosome-1 (SQSTM1/p62) is involved in cellular processes such as autophagy and metabolic reprogramming. Mutations resulting in the loss of function of SQSTM1 lead to neurodegenerative diseases including frontotemporal dementia. The pathogenic mechanism that contributes to SQSTM1-related neurodegeneration has been linked to its role as an autophagy adaptor, but this is poorly understood, and its precise role in mitochondrial function and clearance remains to be clarified. Here, we assessed the importance of SQSTM1 in human induced pluripotent stem cell (iPSC)-derived cortical neurons through the knockout of SQSTM1. We show that SQSTM1 depletion causes altered mitochondrial gene expression and functionality, as well as autophagy flux, in iPSC-derived neurons. However, SQSTM1 is not essential for mitophagy despite having a significant impact on early PINK1-dependent mitophagy processes including PINK1 recruitment and phosphorylation of ubiquitin on depolarized mitochondria. These findings suggest that SQSTM1 is important for mitochondrial function rather than clearance.
Subject(s)
Cerebral Cortex/cytology , Mitochondria/metabolism , Neurons/metabolism , Sequestosome-1 Protein/metabolism , Cell Differentiation , Cell Respiration , Gene Expression Regulation , Humans , Induced Pluripotent Stem Cells/cytology , Induced Pluripotent Stem Cells/metabolism , Membrane Potential, Mitochondrial , Mitochondria/pathology , Mitophagy , Oxidative Phosphorylation , Protein Kinases/metabolism , Reproducibility of ResultsABSTRACT
Genetic and biochemical evidence points to an association between mitochondrial dysfunction and Parkinson's disease (PD). PD-associated mutations in several genes have been identified and include those encoding PTEN-induced putative kinase 1 (PINK1) and parkin. To identify genes, pathways, and pharmacological targets that modulate the clearance of damaged or old mitochondria (mitophagy), here we developed a high-content imaging-based assay of parkin recruitment to mitochondria and screened both a druggable genome-wide siRNA library and a small neuroactive compound library. We used a multiparameter principal component analysis and an unbiased parameter-agnostic machine-learning approach to analyze the siRNA-based screening data. The hits identified in this analysis included specific genes of the ubiquitin proteasome system, and inhibition of ubiquitin-conjugating enzyme 2 N (UBE2N) with a specific antagonist, Bay 11-7082, indicated that UBE2N modulates parkin recruitment and downstream events in the mitophagy pathway. Screening of the compound library identified kenpaullone, an inhibitor of cyclin-dependent kinases and glycogen synthase kinase 3, as a modulator of parkin recruitment. Validation studies revealed that kenpaullone augments the mitochondrial network and protects against the complex I inhibitor MPP+. Finally, we used a microfluidics platform to assess the timing of parkin recruitment to depolarized mitochondria and its modulation by kenpaullone in real time and with single-cell resolution. We demonstrate that the high-content imaging-based assay presented here is suitable for both genetic and pharmacological screening approaches, and we also provide evidence that pharmacological compounds modulate PINK1-dependent parkin recruitment.
Subject(s)
Mitochondria/metabolism , RNA, Small Interfering/metabolism , Small Molecule Libraries/metabolism , Ubiquitin-Protein Ligases/metabolism , Benzazepines/chemistry , Benzazepines/metabolism , Benzazepines/pharmacology , HeLa Cells , Humans , Hydrazones/chemistry , Hydrazones/metabolism , Hydrazones/pharmacology , Indoles/chemistry , Indoles/metabolism , Indoles/pharmacology , Membrane Potential, Mitochondrial/drug effects , Mitophagy/drug effects , Principal Component Analysis , Protein Kinases/chemistry , Protein Kinases/genetics , Protein Kinases/metabolism , RNA Interference , Small Molecule Libraries/chemistry , Small Molecule Libraries/pharmacology , Ubiquitin-Conjugating Enzymes/antagonists & inhibitors , Ubiquitin-Conjugating Enzymes/genetics , Ubiquitin-Conjugating Enzymes/metabolism , Ubiquitin-Protein Ligases/antagonists & inhibitors , Ubiquitin-Protein Ligases/geneticsABSTRACT
The discovery of mutations within genes associated with autosomal recessive Parkinson's disease allowed for the identification of PINK1/Parkin regulated mitophagy as an important pathway for the removal of damaged mitochondria. While recent studies suggest that AKT-dependent signalling regulates Parkin recruitment to depolarised mitochondria, little is known as to whether this can also regulate PINK1 mitochondrial accumulation and downstream mitophagy. Here, we demonstrate that inhibition of AKT signalling decreases endogenous PINK1 accumulation in response to mitochondria depolarisation, subsequent Parkin recruitment, phosphorylation of ubiquitin, and ultimately mitophagy. Conversely, we show that upon stimulation of AKT signalling via insulin, the mitophagy pathway is increased in SHSY5Y cells. These data suggest that AKT signalling is an upstream regulator of PINK1 accumulation on damaged mitochondria. Importantly, we show that the AKT pathway also regulates endogenous PINK1-dependent mitophagy in human iPSC-derived neurons.
Subject(s)
Mitochondria/metabolism , Mitophagy , Neurons/metabolism , Parkinson Disease/metabolism , Protein Kinases/metabolism , Proto-Oncogene Proteins c-akt/physiology , Cell Line, Tumor , Humans , Induced Pluripotent Stem Cells , Neurons/cytology , Phosphorylation , Proto-Oncogene Proteins c-akt/genetics , Signal Transduction , Ubiquitin/metabolism , Ubiquitin-Protein Ligases/metabolismABSTRACT
Mutations that result in the defective trafficking of γ2 subunit containing GABAA receptors (γ2-GABAARs) are known to reduce synaptic inhibition. Whether perturbed clustering of non-mutated GABAARs similarly reduces synaptic inhibition in vivo is less clear. In this study we provide evidence that the loss of postsynaptic γ2-GABAARs upon postnatal ablation of gephyrin, the major scaffolding protein of inhibitory postsynapses, from mature principal neurons within the forebrain results in reduced induction of long-term potentiation (LTP) and impaired network excitability within the hippocampal dentate gyrus. The preferential reduction in not only synaptic γ2-GABAAR cluster number at dendritic sites but also the decrease in γ2-GABAAR density within individual clusters at dendritic inhibitory synapses suggests that distal synapses are more sensitive to the loss of gephyrin expression than proximal synapses. The fact that these mice display behavioural features of anxiety and epilepsy emphasises the importance of postsynaptic γ2-GABAAR clustering for synaptic inhibition.
Subject(s)
Carrier Proteins/genetics , Long-Term Potentiation , Membrane Proteins/genetics , Prosencephalon/metabolism , Receptors, GABA-A/metabolism , Synaptic Potentials , Animals , Carrier Proteins/metabolism , Cell Line , Dentate Gyrus/cytology , Dentate Gyrus/metabolism , Dentate Gyrus/physiology , Male , Membrane Proteins/metabolism , Mice , Mice, Inbred C57BL , Neurons/metabolism , Neurons/physiology , Prosencephalon/cytology , Prosencephalon/physiology , Receptors, GABA-A/genetics , Synapses/metabolism , Synapses/physiologyABSTRACT
Pathological studies on Parkinson's disease (PD) patients suggest that PD pathology progresses from the enteric nervous system (ENS) and the olfactory bulb into the central nervous system. We have previously shown that environmental toxins acting locally on the ENS mimic this PD-like pathology progression pattern in mice. Here, we show for the first time that the resection of the autonomic nerves stops this progression. Moreover, our results show that an environmental toxin (i.e. rotenone) promotes the release of alpha-synuclein by enteric neurons and that released enteric alpha-synuclein is up-taken by presynaptic sympathetic neurites and retrogradely transported to the soma, where it accumulates. These results strongly suggest that pesticides can initiate the progression of PD pathology and that this progression is based on the transneuronal and retrograde axonal transport of alpha-synuclein. If confirmed in patients, this study would have crucial implications in the strategies used to prevent and treat PD.
Subject(s)
Brain/drug effects , Enteric Nervous System/drug effects , Insecticides/toxicity , Neurons/drug effects , Parkinson Disease, Secondary/chemically induced , Rotenone/toxicity , alpha-Synuclein/metabolism , Animals , Axonal Transport/drug effects , Brain/metabolism , Brain/pathology , Enteric Nervous System/metabolism , Enteric Nervous System/pathology , Humans , Mice , Neurons/metabolism , Parkinson Disease, Secondary/metabolism , Parkinson Disease, Secondary/pathology , Primary Cell Culture , Sympathectomy , VagotomySubject(s)
rab GTP-Binding Proteins/metabolism , Animals , Endocytosis , Endosomes/metabolism , Humans , RNA-Binding ProteinsABSTRACT
Inhibitory glycine receptors (GlyRs) are composed of homologous α- (α1-4) and ß-subunits. The ß-subunits (GlyRß) interact via their large cytosolic loops with the postsynaptic scaffolding protein gephyrin and are therefore considered essential for synaptic localization. In situ hybridization studies indicate a widespread distribution of GlyRß transcripts throughout the mammalian central nervous system (CNS), whereas GlyRα mRNAs and proteins display more restricted expression patterns. Here we report the generation of a monoclonal antibody that specifically recognizes rodent GlyRß (mAb-GlyRß) and does not exhibit crossreactivity with any of the GlyRα1-4 subunits. Immunostaining with this antibody revealed high densities of punctate GlyRß immunoreactivity at inhibitory synapses in mouse spinal cord, brainstem, midbrain, and olfactory bulb but not in the neocortex, cerebellum, or hippocampus. This contrasts the abundance of GlyRß transcripts in all major regions of the rodent brain and suggests that GlyRß protein levels are regulated posttranscriptionally. When mAb-GlyRß was used in double-labeling experiments with GlyRα1-, α2-, α3-, or α4-specific antibodies to examine the colocalization of GlyRß with these GlyR subunits in the mouse retina, >90% of the GlyRα1-3 clusters detected were found to be GlyRß-immunoreactive. A subset (about 50%) of the GlyRα4 puncta in the inner plexiform layer, however, was found to lack GlyRß and gephyrin immunostaining. These GlyRα4-only clusters were apposed to bassoon immunoreactivity and hence synaptically localized. Their existence points to a gephyrin-independent synaptic localization mechanism for a minor subset of GlyRs.
Subject(s)
Antibodies, Monoclonal , Brain Chemistry , Brain/metabolism , Receptors, Glycine/analysis , Spinal Cord/metabolism , Animals , Antibodies, Monoclonal/immunology , Antibody Specificity , Humans , Immunohistochemistry , Mice , Mice, Inbred C57BL , Microscopy, Confocal , Rats , Receptors, Glycine/biosynthesis , Spinal Cord/chemistry , Synapses/chemistry , Synapses/metabolismABSTRACT
The scaffolding protein gephyrin is essential for the clustering of glycine and GABA(A) receptors (GABA(A)Rs) at inhibitory synapses. Here, we provide evidence that the size of the postsynaptic gephyrin scaffold is controlled by dephosphorylation reactions. Treatment of cultured hippocampal neurons with the protein phosphatase inhibitors calyculin A and okadaic acid reduced the size of postsynaptic gephyrin clusters and increased cytoplasmic gephyrin staining. Protein phosphatase 1 (PP1) was found to colocalize with gephyrin at selected postsynaptic sites and to interact with gephyrin in transfected cells and brain extracts. Alanine or glutamate substitution of the two established serine/threonine phosphorylation sites in gephyrin failed to affect its clustering at inhibitory synapses and its ability to recruit gamma2 subunit containing GABA(A)Rs. Our data are consistent with the postsynaptic gephyrin scaffold acting as a platform for PP1, which regulates gephyrin cluster size by dephosphorylation of gephyrin- or cytoskeleton-associated proteins.
Subject(s)
Carrier Proteins/metabolism , Membrane Proteins/metabolism , Protein Phosphatase 1/metabolism , Synapses/metabolism , Animals , Carrier Proteins/genetics , Cells, Cultured , Cytoskeleton/metabolism , Cytoskeleton/ultrastructure , Enzyme Inhibitors/pharmacology , Hippocampus/cytology , Humans , Marine Toxins , Membrane Proteins/genetics , Neurons/drug effects , Neurons/metabolism , Neurons/ultrastructure , Okadaic Acid/pharmacology , Oxazoles/pharmacology , Protein Phosphatase 1/antagonists & inhibitors , Rats , Receptors, GABA/metabolism , Recombinant Fusion Proteins/genetics , Recombinant Fusion Proteins/metabolism , Synapses/drug effectsABSTRACT
Renal reabsorption of inorganic phosphate (P(i)) is mainly mediated by the Na(+)-dependent P(i)-cotransporter NaPi-IIa that is expressed in the brush-border membrane (BBM) of renal proximal tubules. Regulation and apical expression of NaPi-IIa are known to depend on a network of interacting proteins. Most of the interacting partners identified so far associate with the COOH-terminal PDZ-binding motif (TRL) of NaPi-IIa. In this study GABA(A) receptor-associated protein (GABARAP) was identified as a novel interacting partner of NaPi-IIa applying a membrane yeast-two-hybrid system (MYTH 2.0) to screen a mouse kidney library with the TRL-truncated cotransporter as bait. GABARAP mRNA and protein are present in renal tubules, and the interaction of NaPi-IIa and GABARAP was confirmed by using glutathione S-transferase pulldowns from BBM and coimmunoprecipitations from transfected HEK293 cells. Amino acids 36-68 of GABARAP were identified as the determinant for the described interaction. The in vivo effects of this interaction were studied in a murine model. GABARAP(-/-) mice have reduced urinary excretion of P(i), higher Na(+)-dependent (32)P(i) uptake in BBM vesicles, and increased expression of NaPi-IIa in renal BBM compared with GABARAP(+/+) mice. The expression of Na(+)/H(+) exchanger regulatory factor (NHERF)1, an important scaffold for the apical expression of NaPi-IIa, is also increased in GABARAP(-/-) mice. The absence of GABARAP does not interfere with the regulation of the cotransporter by either parathyroid hormone or acute changes of dietary P(i) content.
Subject(s)
Cytoskeletal Proteins/genetics , Cytoskeletal Proteins/metabolism , Kidney Tubules, Proximal/physiology , Membrane Proteins/genetics , Membrane Proteins/metabolism , Sodium-Phosphate Cotransporter Proteins, Type IIa/genetics , Sodium-Phosphate Cotransporter Proteins, Type IIa/metabolism , Animals , Apoptosis Regulatory Proteins , Cell Line , Embryonic Stem Cells/physiology , Endocytosis/drug effects , Endocytosis/physiology , Epithelial Cells/physiology , Gene Expression/physiology , Gene Library , Homeostasis/physiology , Humans , Kidney/cytology , Kidney Tubules, Proximal/cytology , Mice , Mice, Mutant Strains , Microtubule-Associated Proteins , Microvilli/physiology , Parathyroid Hormone/pharmacology , Phosphates/metabolism , Phosphates/pharmacology , Phosphoproteins/genetics , Phosphoproteins/metabolism , Phosphorus, Dietary/pharmacology , RNA, Messenger/metabolism , Sodium-Hydrogen Exchangers/genetics , Sodium-Hydrogen Exchangers/metabolismABSTRACT
Collybistin (Cb) is a brain-specific guanine nucleotide exchange factor that has been implicated in plasma membrane targeting of the postsynaptic scaffolding protein gephyrin found at glycinergic and GABAergic synapses. Here we show that Cb-deficient mice display a region-specific loss of postsynaptic gephyrin and GABA(A) receptor clusters in the hippocampus and the basolateral amygdala. Cb deficiency is accompanied by significant changes in hippocampal synaptic plasticity, due to reduced dendritic GABAergic inhibition. Long-term potentiation is enhanced, and long-term depression reduced, in Cb-deficient hippocampal slices. Consistent with the anatomical and electrophysiological findings, the animals show increased levels of anxiety and impaired spatial learning. Together, our data indicate that Cb is essential for gephyrin-dependent clustering of a specific set of GABA(A) receptors, but not required for glycine receptor postsynaptic localization.
Subject(s)
Carrier Proteins/physiology , Guanine Nucleotide Exchange Factors/physiology , Hippocampus/physiology , Membrane Proteins/physiology , Neuronal Plasticity , Receptors, GABA-A/physiology , Synapses/physiology , Synaptic Transmission , Amygdala/physiology , Animals , Guanine Nucleotide Exchange Factors/genetics , Long-Term Potentiation , Long-Term Synaptic Depression , Maze Learning , Mice , Mice, Knockout , Motor Activity , Patch-Clamp Techniques , Presynaptic Terminals/physiology , Receptors, Glycine/metabolism , Rho Guanine Nucleotide Exchange FactorsABSTRACT
The role the cytoskeleton plays in generating and/or maintaining gephyrin-dependent receptor clusters at inhibitory synapses is poorly understood. Here, the effects of actin cytoskeleton disruption were investigated in eGFP-gephyrin-transfected cells and hippocampal neurons. While gephyrin was not associated with microfilaments in transfected cells, it colocalized with G-actin and cytochalasin-D-induced F-actin patches. The linker region between the MoeA and MogA homology domains of gephyrin was required for colocalization with F-actin patches and for the binding of gephyrin to ena/VASP, an actin anti-capping factor that, in vitro, caused gephyrin binding to polymerized actin. In hippocampal neurons, treatment with cytochalasin D resulted in the redistribution of the neuronal ena/VASP homologue Mena into actin patches and, at early stages of development, a reduction in the number of gephyrin clusters. Our data suggest that Mena binding to F-actin allows for gephyrin recruitment to the leading edge of uncapped actin filaments.
Subject(s)
Actins/metabolism , Carrier Proteins/metabolism , Cell Adhesion Molecules/metabolism , Cytoskeleton/metabolism , Membrane Proteins/metabolism , Microfilament Proteins/metabolism , Phosphoproteins/metabolism , Actin Cytoskeleton/metabolism , Actins/genetics , Animals , Antineoplastic Agents/pharmacology , Bridged Bicyclo Compounds, Heterocyclic/pharmacology , Carrier Proteins/genetics , Cell Adhesion Molecules/genetics , Cells, Cultured , Cytochalasin D/pharmacology , Cytoskeleton/drug effects , Depsipeptides/pharmacology , Hippocampus/cytology , Humans , Membrane Proteins/genetics , Microfilament Proteins/genetics , Neurons/cytology , Neurons/metabolism , Nucleic Acid Synthesis Inhibitors/pharmacology , Phosphoproteins/genetics , Protein Structure, Tertiary , Rats , Recombinant Fusion Proteins/genetics , Recombinant Fusion Proteins/metabolism , Synapses/metabolism , Synapses/ultrastructure , Thiazoles/pharmacology , ThiazolidinesABSTRACT
GABA(A) receptors (GABA(A)Rs) containing the gamma2 subunit are thought to require the interacting protein GABARAP (GABA(A)R associated protein) for trafficking to the neuronal plasma membrane. In order to assess whether GABARAP is required for GABA(A) receptor accumulation at synaptic sites, we analysed a GABARAP knockout mouse. GABARAP deficient mice are phenotypically normal and do not show up-regulation of other GABARAP homologues. Also, the total number of GABA(A)Rs, as assessed by benzodiazepine binding, is unaffected by the loss of GABARAP. Immunocytochemistry of cortical sections showed no differences in the expression and punctate distribution of the gamma2 subunit and the receptor anchoring protein gephyrin between GABARAP deficient and wild-type mice. Thus, GABARAP is not essential for trafficking gamma2 subunit containing GABA(A)Rs to the neuronal plasma membrane or targeting them to inhibitory synapses.
Subject(s)
Cytoskeletal Proteins/physiology , Membrane Proteins/physiology , Receptors, GABA-A/physiology , Synapses/physiology , Animals , Antibodies, Blocking/pharmacology , Antibodies, Monoclonal/pharmacology , Apoptosis Regulatory Proteins , Carrier Proteins/pharmacology , Cloning, Molecular , Cytoskeletal Proteins/antagonists & inhibitors , Cytoskeletal Proteins/genetics , Electrophoresis, Polyacrylamide Gel , Flunitrazepam , GABA Modulators , Genotype , Immunoblotting , Immunohistochemistry , Membrane Proteins/antagonists & inhibitors , Membrane Proteins/genetics , Membrane Proteins/pharmacology , Mice , Mice, Knockout , Microscopy, Confocal , Microtubule-Associated Proteins , Receptors, GABA-A/genetics , TransfectionABSTRACT
Gephyrin is a bi-functional modular protein involved in molybdenum cofactor biosynthesis and in postsynaptic clustering of inhibitory glycine receptors (GlyRs). Here, we show that full-length gephyrin is a trimer and that its proteolysis in vitro causes the spontaneous dimerization of its C-terminal region (gephyrin-E), which binds a GlyR beta-subunit-derived peptide with high and low affinity. The crystal structure of the tetra-domain gephyrin-E in complex with the beta-peptide bound to domain IV indicates how membrane-embedded GlyRs may interact with subsynaptic gephyrin. In vitro, trimeric full-length gephyrin forms a network upon lowering the pH, and this process can be reversed to produce stable full-length dimeric gephyrin. Our data suggest a mechanism by which induced conformational transitions of trimeric gephyrin may generate a reversible postsynaptic scaffold for GlyR recruitment, which allows for dynamic receptor movement in and out of postsynaptic GlyR clusters, and thus for synaptic plasticity.