Deficits in KCC2 activity and the pathophysiology of Autism spectrum disorders

 描述(申请人提供)：自闭症谱系障碍(ASD)有共同的核心症状，包括焦虑增加，重复行为，社交能力降低。人们普遍认为，自闭症是由于兴奋性和抑制性GABA能神经传递之间的“平衡”存在细微差异。电子中和钾/氯共转运体2(KCC2，或SLC12A5)在出生后选择性地在中枢神经系统表达，使神经元能够维持较低的细胞内氯离子浓度，这是出生后发育抑制性神经传递的必要先决条件。与其在促进神经元抑制方面的作用一致，KCC2的表达缺陷在自闭症患者和多种自闭症动物模型中明显存在。KCC2活性分别通过丝氨酸940和苏氨酸残基906和1007的磷酸化而受到正向和负向调节。在这里，我们将直接测试这些调节过程是否影响GABA能抑制的出生后发展，以及ASD的病理生理学。为此，我们创建了小鼠，在小鼠中，通过将S940突变为丙氨酸(S940A)，磷酸化对KCC2活性的正向调制已被消融。T906/1007被带有非赖氨酸激酶(WNKS)磷酸化，我们还获得了WNK活性缺陷的品系小鼠。使用这些新试剂的初步研究使我们能够提出一个新的假设，并将在这里进行检验：KCC2的出生后激活是通过S940的相互磷酸化和T906/1007的去磷酸化来促进的。损害这些过程会降低GABA能的疗效，并直接导致ASD的病理生理学。我们将进行的实验来验证我们的假设，具体有以下几个目标。具体目的1.验证KCC2中S940的磷酸化是GABA能抑制的出生后发展的关键决定因素的假设。具体目的2.验证WNK依赖的KCC2磷酸化减缓超极化GABA能抑制出生后发展的假说。具体目的3.验证GABA能抑制作用减缓出生后发育会复制ASD核心行为缺陷的假说。总之，这些实验将为KCC2磷酸依赖调节缺陷如何促进ASD的病理生理提供关键的机械性见解。这些信息可能导致针对KCC2活性的更有效治疗的开发，以最终改善ASD的患者预后。这样的策略也可能与其他神经精神障碍相关，如癫痫、神经病理性疼痛和精神分裂症，在这些疾病中，KCC2活性缺陷被认为具有重要意义。

 DESCRIPTION (provided by applicant): Autism spectrum disorders (ASD) have common core symptoms which include increases in anxiety, and repetitive behaviors, with reduced sociability. It is widely believed that ASDs arise from subtle differences in the "equilibrium" between excitatory and inhibitory GABAergic neurotransmission. The electroneutral K+/Cl- co- transporter 2 (KCC2, or SLC12A5) is selectively expressed in the CNS after birth and allows neurons to maintain low intracellular Cl- concentrations an essential pre-requisite for the postnatal development of inhibitory neurotransmission. Consistent with its role in facilitating neuronal inhibition, deficits in KCC2 expression are evident in patients with ASDs and multiple autism animal models. KCC2 activity is subject to both positive and negative modulation via phosphorylation of serine's 940 and threonine residues 906 and 1007 respectively. Here, we will directly test if these regulatory processes influence the postnatal development of GABAergic inhibition, and the pathophysiology of ASDs. To do so we have created mice in which positive modulation of KCC2 activity by phosphorylation has been ablated via mutation of S940 to an alanine (S940A). T906/1007 are phosphorylated by with with-no-lysine kinases (WNKs), and we have also obtained line mouse lines with deficits in WNK activity. Preliminary studies using these new reagents have allowed us to formulate a novel hypothesis that will be tested here: The postnatal activation of KCC2 is facilitated by the reciprocal phosphorylation of S940 and dephosphorylation of T906/1007. Compromising these processes decreases the efficacy of GABAergic and directly contributes to the pathophysiology of ASDs. The experiments we will perform to test our hypothesis are detailed in the following aims. Specific Aim 1. To test the hypothesis that phosphorylation of S940 in KCC2 is a critical determinant for the postnatal development of GABAergic inhibition. Specific Aim 2. To test the hypothesis that WNK dependent phosphorylation of KCC2 slows the postnatal development of hyperpolarizing GABAergic inhibition. Specific Aim 3. To test the hypothesis that slowing the postnatal development of GABAergic inhibition reproduces the core behavioral deficits of ASDs. Collectively, these experiments will provide key mechanistic insights into how deficits in KCC2 phospho-dependent modulation contribute to the pathophysiology of ASD. This information may lead to the development of more effective therapies targeting KCC2 activity to ultimately improve patient outcomes for ASDs. Such strategies may also be relevant for other neuropsychiatric disorders, such as epilepsy, neuropathic pain, and schizophrenia in which deficits of KCC2 activity are believed to be of significance.