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

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，女性患病率和治疗难治性增加。后一种特征被认为与神经元氯离子（Cl-）增加引起的神经元兴奋性病理增加有关，而神经元氯离子（Cl-）的增加又源于主要的神经元Cl-转运体KCC2的下调，KCC2可以挤出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，基于神经元Cl-在神经损伤反应中以两性二态方式失调，导致女性更容易患上述疾病的假设。我们已经获得了令人兴奋的初步结果(1)表明kcc2转录受到抑制因子REST/NRSF的调控，该抑制因子与kcc2调控区域的一个新的RE1/NRSE DNA结合位点结合；(2)表明这种调控是gaba能传递从兴奋性到抑制性的早期发育转变的基础。(3)利用x染色体和y染色体特异性DNA标记进行性别分型的大鼠E17胚胎个体皮层原代神经元的培养。后一种方法，直截了当，但可能是突破性的新奇，允许严格区分雌性和雄性初级皮层神经元培养。我们打算通过将雄性和雌性神经元暴露于17-雌二醇和异种雌激素模拟物中，详细阐述神经元Cl-和KCC2是如何被性别特异性调节的分子机制。为此，我们将电穿孔kcc2报告基因结构，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kcc2转录的时间过程。为了直接测定Cl-，荧光Cl-指示剂clomeleon将被共转染。培养物将暴露于生理上相关浓度的雌二醇和实际相关浓度的异雌激素（库雌醇、双酚a、狄氏剂）。后一种化合物的使用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。任何性别特异性调节将在基因靶向小鼠（雌激素受体(ER)-a， -¿和非经典ER敲除蛋白）的原代培养中得到证实。这些实验将在杜克大学一个高度协作的环境中进行，包括分子和生理神经科学实验室，以及分子内分泌学和环境毒理学的投入。研究结果有望揭示一个基本问题，即神经元Cl-调节，它很可能具有性别特异性调节，是治疗难治性神经精神疾病中女性患病率增加的基础。公共卫生相关性：神经元氯化物支配神经细胞的兴奋性，在慢性病理性疼痛和某些形式的癫痫（以治疗难治性和女性优势为特征的疾病）中降低。实验将阐明成熟神经元中主要的电中性氯离子转运体KCC2的调控。雌激素和异种雌激素模拟物将用于刺激培养中的初级皮质神经元，根据这里描述的新方法平台，将严格区分雄性和雌性。来自大鼠和小鼠妊娠晚期胚胎的神经元，后者的基因编码缺乏功能性雌激素受体，将被用于检测kcc2基因的功能和调控，即报告基因检测和神经元氯化物的测量。

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.