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

描述(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，女性患病率增加，难以治疗。后者被认为与神经元氯化物(Cl-)增加引起的神经元兴奋性的病理性增加有关，而这反过来又根源于主要的神经元Cl-转运体KCC2的下调，KCC2分泌出Cl-。在这里，我们建议通过实验来阐明雌激素对KCC2基因的性别特异性调节，该实验基于一个假设，即神经元Cl-以性别二态的方式对神经元损伤做出反应，结果是使女性更容易患上上述疾病。我们已经获得了令人振奋的初步结果：(1)表明KCC2的转录受抑制子REST/NRSF调控，该抑制子与KCC2调节区中一个新的Re1/NRSE DNA结合位点相结合；(2)证明这一调控是GABA能传递从兴奋到抑制的早期发育转变的基础；(3)开发了一种新的方法来培养单个大鼠E17胚胎的皮质原代神经元，这些神经元正在通过X和Y染色体特异性DNA标记进行性别鉴定。后一种方法简单明了，但可能是一项开创性的创新，允许将女性和男性的原代皮质神经元培养严格分开。我们打算阐述神经元Cl-和KCC2如何调节性别的分子机制--特别是通过将男性神经元与女性神经元暴露于17-雌二醇和异种雌激素模拟物。为此，我们将电穿孔KCC2报告基因的构建，野生型和突变的结合部位，驱动一个分泌型荧光素酶报告基因，这将有助于建立KCC2转录的时程。为了直接测定氯离子，荧光氯离子指示剂Clomeleon将被共转染。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(香豆素、双酚A、狄氏剂)。使用后一种化合物将使我们能够解决这些无处不在的化合物对雌激素反应的调节。任何性别特异性的调控都将在来自基因靶向小鼠的原代培养中得到证实(雌激素受体(ER)-α，-β和非经典的ER-敲门蛋白)。这些实验将在杜克大学高度协作的环境中进行，包括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投入。这一结果有望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，是女性在难治性神经精神疾病中患病率增加的基础。与公共卫生相关：神经细胞氯化物决定神经细胞的兴奋性，在慢性病理性疼痛和某些形式的癫痫中减少，这些疾病的特点是治疗难治性和强烈的女性优势。描述的实验将阐明成熟神经元的主要电子中频氯转运体KCC2的调节。雌激素和异种雌激素类似物将用于刺激培养中的原代皮质神经元，根据这里描述的一种新的方法学平台，这将严格区分男性和女性。从大鼠和小鼠的妊娠晚期胚胎中获得的神经元，在基因上编码为缺乏功能性雌激素受体，将接受KCC2基因的探测功能和调节的分析，即报告基因分析和神经元氯的测定。

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.