ABSTRACT
Since their discovery decades ago, the primary physiological and pathological effects of potassium channels have been attributed to their ion conductance, which sets membrane potential and repolarizes action potentials. For example, Kv3 family channels regulate neurotransmitter release by repolarizing action potentials. Here we report a surprising but crucial function independent of potassium conductance: by organizing the F-actin cytoskeleton in mouse nerve terminals, the Kv3.3 protein facilitates slow endocytosis, rapid endocytosis, vesicle mobilization to the readily releasable pool, and recovery of synaptic depression during repetitive firing. A channel mutation that causes spinocerebellar ataxia inhibits endocytosis, vesicle mobilization, and synaptic transmission during repetitive firing by disrupting the ability of the channel to nucleate F-actin. These results unmask novel functions of potassium channels in endocytosis and vesicle mobilization crucial for sustaining synaptic transmission during repetitive firing. Potassium channel mutations that impair these "non-conducting" functions may thus contribute to generation of diverse neurological disorders.
Subject(s)
Endocytosis/physiology , Shaw Potassium Channels/metabolism , Synaptic Transmission/physiology , Synaptic Vesicles/metabolism , Actins/metabolism , Animals , CHO Cells , Cricetulus , Mice , Mutation , Presynaptic Terminals/metabolism , Shaw Potassium Channels/geneticsABSTRACT
Peroxisome proliferator-activated receptor-γ (PPARγ), is a transcription factor that governs pathways, such as lipid metabolism and immune response, that have been implicated in the etiology of LOAD. Previously, we established HepG2-derived cell-lines with stable knockdown of PPARγ gene, and showed an increase in mRNA levels of genes mapped in the APOE linkage disequilibrium (LD) region on chromosome 19q13.32, with the greatest effect observed for APOE-mRNA. Here, we extended the analysis using our PPARγ knockdown model system and investigated the broader effect on expression changes of genes implicated in LOAD via genome wide association studies (GWAS). We applied the nCounter gene expression assay (NanoString) using a panel of twenty-four LOAD-associated genes inferred by proximity to the top significantly associated SNPs. Two independent PPARγ knockdown cell-lines showed changes in mRNA levels of a total of seven genes compared to a control HepG2 cell-line; six of which, ABCA7, APOE, CASS4, CELF1, PTK2B, and ZCWPW1, were upregulated and one, DSG2, was downregulated upon PPARγ knockdown. Our results propose that PPARγ may act as a master regulator of the transcription of several genes involved in LOAD pathogenesis. Our study provided the premise for further analyses including a larger set of genes positioned within a wider range of linkage disequilibrium (LD) regions tagged by all LOAD significantly associated SNPs.
Subject(s)
Alzheimer Disease/genetics , Gene Expression Regulation , Genetic Predisposition to Disease , PPAR gamma/genetics , ATP-Binding Cassette Transporters/genetics , ATP-Binding Cassette Transporters/metabolism , Adaptor Proteins, Signal Transducing/genetics , Adaptor Proteins, Signal Transducing/metabolism , Age of Onset , Alzheimer Disease/metabolism , Alzheimer Disease/physiopathology , Apolipoproteins E/genetics , Apolipoproteins E/metabolism , CELF1 Protein/genetics , CELF1 Protein/metabolism , Desmoglein 2/genetics , Desmoglein 2/metabolism , Focal Adhesion Kinase 2/genetics , Focal Adhesion Kinase 2/metabolism , Gene Expression Profiling , Gene Knockdown Techniques , Genome-Wide Association Study , Hep G2 Cells , Humans , Linkage Disequilibrium , Models, Biological , PPAR gamma/antagonists & inhibitors , PPAR gamma/deficiency , Polymorphism, Single Nucleotide , RNA, Small Interfering/genetics , RNA, Small Interfering/metabolism , Signal TransductionABSTRACT
Chromosome 19q13.32 is a gene rich region, and has been implicated in multiple human phenotypes in adulthood including lipids traits, Alzheimer's disease, and longevity. Peroxisome Proliferator Activated Receptor Gamma (PPARγ) is a ligand-activated nuclear transcription factor that plays a role in human complex traits that are also genetically associated with the chromosome 19q13.32 region. Here, we study the effects of PPARγ on the regional expression regulation of the genes clustered within chromosome 19q13.32, specifically TOMM40, APOE, and APOC1, applying two complementary approaches. Using the short hairpin RNA (shRNA) method in the HepG2 cell-line we knocked down PPARγ expression and measured the effect on mRNA expression. We discovered PPARγ knock down increased the levels of TOMM40-, APOE-, and APOC1-mRNAs, with the highest increase in expression observed for APOE-mRNA. To complement the PPARγ knockdown findings we also examined the effects of low doses of PPARγ agonists (nM range) on mRNA expression of these genes. Low (nM) concentrations of pioglitazone (Pio) decreased transcription of TOMM40, APOE, and APOC1 genes, with the lowest mRNA levels for each gene observed at 1.5nM. Similar to the effect of PPARγ knockdown, the strongest response to pioglitazone was also observed for APOE-mRNA, and rosiglitazone (Rosi), another PPARγ agonist, produced results that were consistent with these. In conclusion, our results further established a role for PPARγ in regional transcriptional regulation of chr19q13.32, underpinning the association between PPARγ, the chr19q13.32 genes cluster, and human complex traits and disease.
