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女性患病率和治疗难治性增加的神经精神疾病。后一个特征被认为与神经元氯化物（Cl-）增加引起的病理性神经元兴奋性增加有关，而神经元氯化物（Cl-）增加又根源于主要神经元Cl-转运体KCC 2（其挤出Cl-）的下调。在这里，我们提出的实验来阐明性别特异性调节的kcc 2基因的雌激素，基于一个假设，即神经元Cl-是失调的神经元损伤的性二型的方式，使妇女更容易患上上述疾病的后果。我们已经获得了令人兴奋的初步结果：（1）表明kcc 2转录受阻遏物REST/NRSF的调控，该阻遏物与kcc 2调控区的一个新的RE 1/NRSE DNA结合位点结合，（2）证明这种调控是GABA能传递从兴奋性到抑制性的早期发育转变的基础，（3）建立了一种新的方法，从E17胚胎中培养皮层原代神经元，并通过X和Y染色体特异性DNA标记进行性别分型。后一种方法，简单，但可能是一个突破性的新奇，允许严格分开的女性与男性的初级皮层神经元文化。我们打算详细阐述神经元Cl-和KCC 2是如何通过将雄性与雌性神经元暴露于17-<$-雌二醇和异源性雌激素模拟物来进行性别特异性调节的分子机制。为此，我们将电穿孔kcc 2报告基因构建体，野生型和突变的结合位点，驱动分泌的荧光素酶报告基因，这将有助于建立kcc 2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。将培养物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。将在源自基因靶向小鼠的原代培养物中确认任何性别特异性调节（雌激素受体（ER）-α、-β和非经典-ER-敲入）。这些实验将在杜克大学的高度协作环境中进行，涉及分子和生理学神经科学实验室，以及分子内分泌学和环境毒理学输入。结果可以预期揭示一个基本的问题，神经元Cl-调节，这很可能有性别特异性调节的基础上增加女性患病率的治疗难治性神经精神疾病的新的光。 公共卫生关系：神经元氯化物决定神经细胞的兴奋性，并且在慢性病理性疼痛以及某些形式的癫痫中减少，这些疾病的特征是治疗无效和女性优势。 实验描述，将阐明成熟神经元，KCC 2的占主导地位的电中性氯转运的调节。雌激素和异源性雌激素模拟物将用于刺激培养物中的原代皮质神经元，基于本文所述的新方法平台，其将严格区分雄性与雌性。来自大鼠和小鼠妊娠晚期胚胎的神经元，后者遗传编码为缺乏功能性雌激素受体，将进行检测探测kcc 2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