Sex-specific gene regulation of neuronal chloride co-transporter, kcc2

神经元氯协同转运蛋白 kcc2 的性别特异性基因调控

• 批准号: 7841928
• 负责人: WOLFGANG B. LIEDTKE
• 金额: $ 19.5万
• 依托单位: DUKE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-05-15 至 2012-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7841928
• 关键词: Absence Epilepsy; Address; Affect; Automobile Driving; Binding; Binding Sites; Biological Assay; Brain; Carrier Proteins; Chemicals; Chloride Ion; Chlorides; Chronic; Clinical; Coumestrol; DNA Binding; DNA Markers; DNA Sequence; Development; Dieldrin; Disease; Down-Regulation; Electroporation; Embryo; Employee Strikes; Endocrinology; Environment; Epilepsy; Estradiol; Estrogen Antagonists; Estrogen Receptors; Estrogens; Female; Figs - dietary; GABA-A Receptor; Gene Expression; Gene Expression Regulation; Gene Targeting; Generalized seizures; Genes; Genetic; Genetic Transcription; Genotype; Glycine; Glycine Receptors; Image; Individual; Knockout Mice; Light; Link; Luciferases; Measurement; Measures; Mediating; Mediator of activation protein; Methodology; Methods; Modeling; Molecular; Movement; Mus; Mutate; Neuronal Injury; Neurons; Neurosciences; Neurotransmitters; Nucleic Acid Regulatory Sequences; Outcome Measure; Pain; Pattern; Perinatal; Pesticides; Phenotype; Physiological; Physiology; Phytoestrogens; Plant Roots; Plasmids; Plastics; Play; Potassium Chloride; Pregnancy; Prevalence; Protein Isoforms; Public Health; Rattus; Refractory; Regulation; Reporter; Reporter Genes; Response Elements; Role; Severities; Site; Staging; Stream; Syndrome; Testing; Therapeutic; Time; Transcription Repressor/Corepressor; Transcriptional Regulation; Universities; Woman; Xeno; Xenobiotics; Y Chromosome; base; bisphenol A; chronic pain; clomeleon; dimorphism; environmental toxicology; gamma-Aminobutyric Acid; male; men; mimetics; neuronal excitability; neuropsychiatry; novel; offspring; painful neuropathy; promoter; protein expression; public health relevance; pup; ratiometric; research study; response; sex; sexual dimorphism; symporter; tissue culture; transmission process

DESCRIPTION (provided by applicant): Chronic pathological pain and certain epileptic syndromes are neuropsychiatric disorders that share an increased female prevalence and refractoriness to treatment. The latter feature is considered to be linked to pathologically increased neuronal excitability caused by increased neuronal chloride (Cl-), which in turn is rooted in down-regulation of the dominant neuronal Cl--transporter, KCC2, which extrudes Cl-. Here we propose experiments to elucidate sex-specific regulation of the kcc2 gene by estrogens, based on a hypothesis that neuronal Cl- is dysregulated in response to neuronal injury in a sexually dimorphic manner, with the consequence of rendering women more susceptible to the above diseases. We have obtained exciting preliminary results (1) showing that kcc2 transcription is regulated by the repressor REST/NRSF which binds to a novel RE1/NRSE DNA binding site in kcc2 regulatory regions, (2) demonstrating this regulation to underlie the early developmental transformation of GABAergic transmission from excitatory to inhibitory, (3) developing a novel method to culture cortical primary neurons from individual rat E17 embryos which are being sex-typed by X-and Y-chromosome specific DNA markers. The latter method, straightforward yet possibly a groundbreaking novelty, permits strictly separate female vs. male primary cortical neuronal culture. We intend to elaborate molecular mechanisms how neuronal Cl- and KCC2 are regulated sex-specifically by exposing male vs. female neurons to 17-¿-estradiol and xenobiotic estrogen-mimetics. For this, we will electroporate kcc2 reporter gene constructs, wildtype and mutated for binding sites, driving a secreted luciferase reporter, which will facilitate establishment of a time-course of kcc2 transcription. For direct determination of Cl-, the fluorescent Cl--indicator clomeleon will be co-transfected. Cultures will be exposed to physiologically relevant concentrations of estradiol and practically relevant concentrations of xeno-estrogens (coumestrol, bisphenol-A, dieldrin). Use of the latter compounds will allow us to address modulation of estrogen responses by these ubiquitous compounds. Any sex-specific regulation will be confirmed in primary cultures derived from gene-targeted mice (estrogen-receptor (ER)-a, -¿ and non-classical-ER-knockin). These experiments will be conducted in a highly collaborative environment at Duke University, involving molecular and physiology neuroscience labs, in addition molecular endocrinology and environmental toxicology input. Results can be expected to shed new light on a fundamental matter, neuronal Cl--regulation, which very likely has sex-specific regulation as a basis for increased female prevalence in therapy-refractory neuropsychiatric diseases. PUBLIC HEALTH RELEVANCE: Neuronal chloride dictates nerve cells' excitability, and is reduced in chronic pathological pain as well as in certain forms of epilepsy, diseases characterized by therapeutic refractoriness and strong female preponderance. Experiments are described that will elucidate the regulation of the dominant electroneutral chloride transporter of mature neurons, KCC2. Estrogen and xenobiotic estrogen-mimetics will be used for stimulation of primary cortical neurons in culture, which will be maintained strictly separate for male vs. female, based on a novel methodology platform described here. Neurons derived from late-pregnancy embryos of rats and mice, the latter genetically encoded to lack functional estrogen-receptors, will be subjected to assays probing function and regulation of the kcc2 gene, namely reporter gene assays and measurement of neuronal chloride.

描述（由申请人提供）：慢性病理性疼痛和某些癫痫综合征是神经精神疾病，其女性患病率较高且难以治疗。后一个特征被认为与神经元氯 (Cl-) 增加引起的病理性神经元兴奋性增加有关，而神经元氯 (Cl-) 增加又源于主要神经元 Cl- 转运蛋白 KCC2 的下调，KCC2 会排出 Cl-。在这里，我们提出实验来阐明雌激素对 kcc2 基因的性别特异性调节，基于这样的假设：神经元 Cl- 以性别二态性方式响应神经元损伤而失调，结果使女性更容易患上述疾病。我们获得了令人兴奋的初步结果（1）表明 kcc2 转录受到阻遏蛋白 REST/NRSF 的调节，该阻遏蛋白 REST/NRSF 与 kcc2 调节区域中的新 RE1/NRSE DNA 结合位点结合，（2）证明这种调节是 GABA 能传递从兴奋性到抑制性的早期发育转变的基础，（3）开发了一种培养来自个体大鼠 E17 的皮质原代神经元的新方法 通过 X 和 Y 染色体特异性 DNA 标记进行性别分型的胚胎。后一种方法简单但可能具有突破性的新颖性，允许严格分开女性和男性原代皮质神经元培养。我们打算通过将男性和女性神经元暴露于 17-β-雌二醇和异生雌激素模拟物来详细阐述神经元 Cl- 和 KCC2 如何进行性别特异性调节的分子机制。为此，我们将对 kcc2 报告基因构建体（野生型和结合位点突变）进行电穿孔，驱动分泌型荧光素酶报告基因，这将有助于建立 kcc2 转录的时间过程。为了直接测定 Cl-，将共转染荧光 Cl--指示剂 clomeleon。培养物将暴露于生理相关浓度的雌二醇和实际相关浓度的异种雌激素（香豆雌酚、双酚A、狄氏剂）。使用后一种化合物将使我们能够解决这些普遍存在的化合物对雌激素反应的调节问题。任何性别特异性调节都将在来自基因靶向小鼠（雌激素受体（ER）-a、-¿和非经典-ER-敲入）的原代培养物中得到证实。这些实验将在杜克大学高度协作的环境中进行，涉及分子和生理学神经科学实验室，此外还有分子内分泌学和环境毒理学输入。预计结果将为神经元氯离子调节这一基本问题提供新的线索，这种调节很可能具有性别特异性调节作为女性难治性神经精神疾病患病率增加的基础。 公共健康相关性：神经元氯决定神经细胞的兴奋性，在慢性病理性疼痛以及某些形式的癫痫（以治疗难治性和女性占主导地位的疾病为特征的疾病）中神经元氯会降低。 实验将阐明成熟神经元的主要电中性氯离子转运蛋白 KCC2 的调节。雌激素和异生雌激素模拟物将用于刺激培养物中的初级皮质神经元，基于此处描述的新颖方法平台，男性与女性将严格分开。来自大鼠和小鼠妊娠晚期胚胎的神经元（后者基因编码缺乏功能性雌激素受体）将接受检测 kcc2 基因的功能和调节，即报告基因检测和神经元氯化物的测量。

项目成果

Highly conductive carbon nanotube matrix accelerates developmental chloride extrusion in central nervous system neurons by increased expression of chloride transporter KCC2.

• DOI: 10.1002/smll.201201994
• 发表时间: 2013-04-08
• 期刊: SMALL
• 影响因子: 13.3
• 作者: Liedtke, Wolfgang;Yeo, Michele;Zhang, Hongbo;Wang, Yiding;Gignac, Michelle;Miller, Sara;Berglund, Ken;Liu, Jie
• 通讯作者: Liu, Jie