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KCC2 expression supersedes NKCC1 in mature fiber cells in mouse and rabbit lenses.

ABSTRACT: Na-K-Cl cotransporter 1 (NKCC1) and K-Cl cotransporter 2 (KCC2) have fundamental roles in neuron differentiation that are integrated with gamma-aminobutyric acid (GABA) and glutamate receptors, GABA synthesized by GAD25/65/67 encoded by GAD1/GAD2 genes, and GABA transporters (GATs). Cells in the eye lens express at least 13 GABA receptor subunits, ?-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) and N-methyl D-aspartate (NMDA) glutamate receptors, GAD1/GAD2, GAT1-4 and vGAT, and NKCC1. NKCC1:KCC2 ratios determine the switch in GABA actions from trophic/growth promoting early in development to their classic inhibitory roles in adult neurons. Lens epithelial cells cover the anterior surface and differentiate to elongated fiber cells in the lens interior with comparable morphology and sub-cellular structures as neurons. NKCC1 is expressed before KCC2 in neuron development and increases cell chloride, which stimulates differentiation and process formation. Subsequently, KCC2 increases and extrudes cell chloride linked with maturation. KCC2 has an additional structural moonlighting role interacting with F-actin scaffolding in dendritic spine morphogenesis. We examined KCC2 versus NKCC1 spatial expression in relation to fiber cell developmental status within the lens.Immunofluorescence and immunoblots were used to detect expression in mouse and rabbit lenses.NKCC1 was restricted to peripheral elongating lens fiber cells in young adult mouse and rabbit lenses. Lens KCC2 expression included the major KCC2b neuronal isoform and was detected in interior fiber cells with decreased NKCC1 expression and localized at the membranes. Lens expression of RE-1 silencing transcription factor (REST) regulated KCC2 is consistent with GAD1 and GAD2, several GABA and glutamate receptor subunits, miR-124, and other REST-regulated genes expressed in lenses.NKCC1 in peripheral elongating fiber cells is superseded by KCC2 expression in interior mature fiber cells that also express >20 additional integral GABA biology genes, AMPA/NMDA glutamate receptors, and an array of accessory proteins that together underlie morphogenesis in neurons. The present findings provide further evidence that this fundamental neuronal regulation is extensively conserved in lens and identify additional parallels in the morphogenetic programs that underlie lens fiber cell and neuronal differentiation and contribute to the development of visual acuity.

SUBMITTER: Frederikse PH

PROVIDER: S-EPMC4605749 | biostudies-literature | 2015

REPOSITORIES: biostudies-literature

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KCC2 expression supersedes NKCC1 in mature fiber cells in mouse and rabbit lenses.

Frederikse Peter H PH Kasinathan Chinnaswamy C

Molecular vision 20151013

<h4>Purpose</h4>Na-K-Cl cotransporter 1 (NKCC1) and K-Cl cotransporter 2 (KCC2) have fundamental roles in neuron differentiation that are integrated with gamma-aminobutyric acid (GABA) and glutamate receptors, GABA synthesized by GAD25/65/67 encoded by GAD1/GAD2 genes, and GABA transporters (GATs). Cells in the eye lens express at least 13 GABA receptor subunits, α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) and N-methyl D-aspartate (NMDA) glutamate receptors, GAD1/GAD2, GAT1-4 and ...[more]

PMID: 26539026

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Project description:ContextDisturbances in markers of cortical γ-aminobutyric acid neurotransmission are a common finding in schizophrenia. The nature of γ-aminobutyric acid neurotransmission (hyperpolarizing or depolarizing) depends on the local intracellular chloride concentration. In the central nervous system, the intracellular chloride level is determined by the activity of 2 cation-chloride transporters, NKCC1 and KCC2. The activities of these transporters are in turn regulated by a network of serine-threonine kinases that includes OXSR1, STK39, and the WNK kinases WNK1, WNK3, and WNK4.ObjectiveTo compare the levels of NKCC1, KCC2, OXSR1, STK39, WNK1, WNK3, and WNK4 transcripts in prefrontal cortex area 9 between subjects with schizophrenia and healthy comparison subjects.DesignReal-time quantitative polymerase chain reaction technique was used to measure transcript levels in the prefrontal cortex.SettingHuman brain specimens were obtained from autopsies conducted at the Allegheny County Medical Examiner's Office, Pittsburgh, Pennsylvania.ParticipantsPostmortem brain specimens from 42 subjects with schizophrenia and 42 matched healthy comparison subjects. Brain specimens from 18 macaque monkeys exposed to haloperidol, olanzapine, or sham long-term.Main outcome measuresRelative expression levels for NKCC1, KCC2, OXSR1, STK39, WNK1, WNK3, and WNK4 transcripts compared with the mean expression level of 3 housekeeping transcripts.ResultsOXSR1 and WNK3 transcripts were substantially overexpressed in subjects with schizophrenia relative to comparison subjects. In contrast, NKCC1, KCC2, STK39, WNK1, and WNK4 transcript levels did not differ between subject groups. OXSR1 and WNK3 transcript expression levels were not changed in antipsychotic-exposed monkeys and were not affected by potential confounding factors in the subjects with schizophrenia.ConclusionIn schizophrenia, increased expression levels, and possibly increased kinase activities, of OXSR1 and WNK3 may shift the balance of chloride transport by NKCC1 and KCC2 and alter the nature of γ-aminobutyric acid neurotransmission in the prefrontal cortex.

Project description:The ocular lens contains only two cell types: epithelial cells and fiber cells. The epithelial cells lining the anterior hemisphere have the capacity to continuously proliferate and differentiate into lens fiber cells that make up the large proportion of the lens mass. To understand the transcriptional changes that take place during the differentiation process, high-throughput RNA-Seq of newborn mouse lens epithelial cells and lens fiber cells was conducted to comprehensively compare the transcriptomes of these two cell types.RNA from three biologic replicate samples of epithelial and fiber cells from newborn FVB/N mouse lenses was isolated and sequenced to yield more than 24 million reads per sample. Sequence reads that passed quality filtering were mapped to the reference genome using Genomic Short-read Nucleotide Alignment Program (GSNAP). Transcript abundance and differential gene expression were estimated using the Cufflinks and DESeq packages, respectively. Gene Ontology enrichment was analyzed using GOseq. RNA-Seq results were compared with previously published microarray data. The differential expression of several biologically important genes was confirmed using reverse transcription (RT)-quantitative PCR (qPCR).Here, we present the first application of RNA-Seq to understand the transcriptional changes underlying the differentiation of epithelial cells into fiber cells in the newborn mouse lens. In total, 6,022 protein-coding genes exhibited differential expression between lens epithelial cells and lens fiber cells. To our knowledge, this is the first study identifying the expression of 254 long intergenic non-coding RNAs (lincRNAs) in the lens, of which 86 lincRNAs displayed differential expression between the two cell types. We found that RNA-Seq identified more differentially expressed genes and correlated with RT-qPCR quantification better than previously published microarray data. Gene Ontology analysis showed that genes upregulated in the epithelial cells were enriched for extracellular matrix production, cell division, migration, protein kinase activity, growth factor binding, and calcium ion binding. Genes upregulated in the fiber cells were enriched for proteosome complexes, unfolded protein responses, phosphatase activity, and ubiquitin binding. Differentially expressed genes involved in several important signaling pathways, lens structural components, organelle loss, and denucleation were also highlighted to provide insights into lens development and lens fiber differentiation.RNA-Seq analysis provided a comprehensive view of the relative abundance and differential expression of protein-coding and non-coding transcripts from lens epithelial cells and lens fiber cells. This information provides a valuable resource for studying lens development, nuclear degradation, and organelle loss during fiber differentiation, and associated diseases.

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KCC2 expression supersedes NKCC1 in mature fiber cells in mouse and rabbit lenses.

Publications

KCC2 expression supersedes NKCC1 in mature fiber cells in mouse and rabbit lenses.

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