Novel actions of neurosteroids on GABA (A) receptor trafficking

描述（由申请人提供）：紧张性抑制通过特定的神经元群体的持续活动来决定神经元的兴奋性和神经元回路的活动。 高亲和力突触外γ-氨基丁酸A受体（GABAAR），其是神经类固醇的主要靶标。神经甾体被广泛接受为GABA能抑制的内源性调节剂。神经类固醇水平的波动被认为在癫痫中起着关键作用，也与自闭症、焦虑、抑郁、经前综合征和精神分裂症有关。然而，到目前为止，在理解神经类固醇水平的波动如何改变突触外GABAAR亚单位表达水平方面存在根本性的差距，这是发生在大量神经精神疾病中的GABA能抑制变化的重要因素。我们的长期目标是充分了解神经类固醇影响突触外GABAARs表达水平的机制。在这个提议中，我们将讨论神经甾体在蛋白激酶C（PKC）依赖的含α4亚基的GABAAR磷酸化中的作用。我们将确定这如何影响介导紧张性抑制的GABAAR亚型的细胞表面积累。我们的中心假设是，神经类固醇调节由α4β3和δ亚基组装的GABAAR的磷酸化，其介导齿状回和丘脑中的大部分紧张性抑制。神经类固醇特异性增强α4亚基中丝氨酸443（S443）的PKC依赖性磷酸化。这种增加的磷酸化导致由α4β3和δ亚基组成的受体插入质膜的增强，这些受体起源于分泌途径。受体细胞表面稳定性的这些增强是导致强直性抑制功效持续增加的原因。在强有力的初步数据的指导下，将通过追求三个具体目标来检验这一假设：（1）确定PKC依赖性磷酸化在调节介导紧张性抑制的GABAAR的特异性细胞表面积累中的作用（2）可视化神经类固醇介导的GABAAR的细胞表面稳定性和膜运输的变化和（3）确定神经类固醇对GABAAR的作用。依赖于GABAAR活性的磷酸化和紧张性抑制的功效。总之，这一建议将证明一个新的和意想不到的机制，神经甾体发挥持久和持续的变化，紧张性抑制的疗效。了解神经类固醇介导的GABAARs磷酸化和细胞表面表达的变化有可能转化为更好地理解神经精神疾病。此外，这些信息可能导致开发更有效的治疗焦虑症、抑郁症、经前综合症、精神分裂症和药物滥用的方法。

DESCRIPTION (provided by applicant): Tonic inhibition determines the excitability of neurons and the activity of neuronal circuits through the persistent activity of specialized populations of high affinity extrasynaptic γ-aminobutyric acid A receptors (GABAARs), that are the principle targets of neurosteroids. Neurosteroids are widely accepted as endogenous regulators of GABAergic inhibition. Fluctuations in the levels of neurosteroids are accepted to play a critical role in epilepsy, and are also relevant to autism, anxiety, depression, premenstrual syndrome and schizophrenia. However, to date, there is a fundamental gap in understanding how fluctuations in the levels of neurosteroids change extrasynaptic GABAAR subunit expression levels, which are a significant factor in the changes in GABAergic inhibition that occur in a plethora of neuropsychiatric disorders. Our long-term goal is to fully understand the mechanism by which neurosteroids influence the expression levels of extrasynaptic GABAARs. In this proposal we will address the role that neurosteroids play in protein kinase C (PKC)-dependent phosphorylation of α4 subunit-containing GABAARs. We will determine how this influences the cell surface accumulation of the GABAAR subtypes that mediate tonic inhibition. Our central hypothesis is that neurosteroids modulate the phosphorylation of GABAARs assembled from α4β3 and δ subunits, which mediate the majority of tonic inhibition in the dentate gyrus and thalamus. Neurosteroids specifically potentiate PKC-dependent phosphorylation of serine 443 (S443) in the α4 subunit. This increased phosphorylation leads to enhanced insertion of receptors composed of α4β3 and δ subunits into the plasma membrane that originate within the secretory pathway. These enhancements of receptor cell surface stability are responsible for sustained increases in the efficacy of tonic inhibition. Guided by strong preliminary data, this hypothesis will be tested by pursuing three specific aims: (1) Determining the role of PKC-dependent phosphorylation in modulating the specific cell surface accumulation of GABAARs that mediate tonic inhibition (2) Visualizing neurosteroid- mediated changes in cell surface stability and membrane trafficking of GABAARs and (3) Ascertaining the effects of neurosteroid-dependent phosphorylation on the activity of GABAARs and the efficacy of tonic inhibition. In summary this proposal will demonstrate a new and unexpected mechanism by which neurosteroids exert persistent and sustained changes in the efficacy of tonic inhibition. Understanding neurosteroid-mediated changes in phosphorylation and cell surface expression of GABAARs has the potential to translate into better understanding of neuropsychiatric disorders. Moreover such information is likely to lead to the development of more efficacious treatments for anxiety, depression, premenstrual syndrome, schizophrenia and substance abuse.