HCN Channel Trafficking in Epilepsy

描述（由申请人提供）：颞叶癫痫（TLE）是难治性癫痫发作的常见原因。癫痫发作倾向增加是由异常的神经元兴奋性引起的。TLE中表现出兴奋性变化的一个候选是超极化激活的环核苷酸门控通道（h通道）。H通道介导超极化激活电流，Ih，这是膜电位稳态和神经元兴奋性的关键。在海马锥体神经元中，h通道亚单位HCN1和HCN2在远端比近端树突显著富集，这一现象对神经元的兴奋性至关重要；树突Ih的上调会降低兴奋性，而降低则会增加兴奋性。在TLE大鼠模型中，我们发现在癫痫持续状态（自发性癫痫发作前的一个时间点）24小时后，兴奋性降低，Ih和远端HCN1的富集增强。相反，在自发性癫痫发作后，我们发现兴奋性增加，Ih降低，HCN1从远端树突重新定位到体细胞。有趣的是，自发性癫痫发作后，与h通道结合蛋白的相互作用显著减少。此外，在薄片培养中，我们发现HCN1远端树突定位以活性依赖的方式控制，需要激活n-甲基-d -天冬氨酸受体（NMDAR）和钙调素依赖性蛋白激酶II （CaMKII）活性。我们认为，颞叶颞叶中h通道远端富集的早期增强是一种对活动增加的稳态反应，从而降低了兴奋性，而海马癫痫发生过程中h通道从远端树突向体细胞的重新定位则是一种异常过程，有助于兴奋性增加和自发癫痫发作的发展。我们假设：1)h通道定位的控制调节TLE的兴奋性，2)从内皮层到CA1树突的输入活性通过nmdar介导的蛋白激酶激活和HCN亚基磷酸化控制h通道定位，3)HCN亚基的异常磷酸化破坏h通道运输并覆盖正常的稳态、活动依赖的h通道定位控制，导致TLE自发性癫痫发作。我们将利用生理学、细胞生物学和生物化学技术来解决以下具体目标：为了确定1)TLE中远端树突状h通道是否在潜伏期增加而在自发性癫痫发作后减少，2)从内皮层向CA1、NMDAR激活、CaMKII活性和HCN亚基磷酸化的颞氨输入控制h通道定位，以及3)HCN亚基异常磷酸化阻止了慢性TLE中h通道运输和靶向的重要相互作用和错误定位。公共卫生相关性：就公共卫生而言，尽管有许多新的医学和外科治疗方法，难治性癫痫发作仍然是颞叶癫痫（TLE）患者致残的一个重要原因。这项工作的最终目标是更好地了解控制TLE自发性和难治性癫痫发作的分子因素，希望找到预防和治疗这种常见癫痫原因的新靶点。

DESCRIPTION (provided by applicant): Temporal lobe epilepsy (TLE) is a common cause of refractory seizures. Increased seizure propensity in TLE is caused by abnormal neuronal excitability. One candidate for manifesting excitability changes in TLE is the hyperpolarization-activated cyclic nucleotide-gated channel (h channel). H channels mediate the hyperpolarization-activated current, Ih, which is critical for membrane potential homeostasis and neuronal excitability. In hippocampal pyramidal neurons, h channel subunits HCN1 and HCN2 are dramatically enriched in distal compared to proximal dendrites, a phenomenon critical for neuronal excitability; upregulation of dendritic Ih reduces excitability, whereas reduction increases excitability. In a rat model of TLE, we found that excitability was reduced and Ih and distal enrichment of HCN1 was enhanced 24h after status epilepticus, a time point before onset of spontaneous seizures (latency). In contrast, after onset of spontaneous seizures, we found increased excitability coupled with reduced Ih and relocalization of HCN1 from distal dendrites to soma. Interestingly, interaction with an h channel binding protein was dramatically reduced after onset of spontaneous seizures. Furthermore, in slice cultures we found that HCN1 distal dendritic localization is controlled in an activity-dependent manner, requiring activation of N-methyl-D- aspartate receptors (NMDAR) and calmodulin-dependent protein kinase II (CaMKII) activity. We reason that early enhancement of h channel distal enrichment in TLE is a homeostatic response to increased activity that reduces excitability, whereas h channel relocalization from distal dendrites to soma during hippocampal epileptogenesis represents an aberrant process that contributes to increased excitability and the development of spontaneous seizures. We hypothesize that: 1) control of h channel localization regulates excitability in TLE, 2) activity of inputs from entorhinal cortex to CA1 dendrites controls h channel localization through NMDAR-mediated activation of protein kinases and HCN subunit phosphorylation, and 3) Abnormal phosphorylation of HCN subunits disrupts h channel trafficking and overrides normal homeostatic, activity-dependent control of h channel localization, leading to onset of spontaneous seizures in TLE. We will utilize physiological, cell biological and biochemical techniques to address the following specific aims: To determine whether 1) distal dendritic h channels are increased during latency and reduced after onset of spontaneous seizures in TLE, 2) Temporoammonic inputs from entorhinal cortex to CA1, NMDAR activation, CaMKII activity, and phosphorylation of HCN subunits control h channel localization, and 3) abnormal HCN subunit phosphorylation prevents interactions important for trafficking and targeting and mislocalizes h channels in chronic TLE. PUBLIC HEALTH RELEVANCE: With respect to public health, despite numerous new medical and surgical treatments, refractory seizures remain a significant cause of disability in patients with temporal lobe epilepsy (TLE). The ultimate goal of this work is to gain a better understanding of molecular factors controlling the onset of spontaneous and refractory seizures in TLE, with the hope of identifying novel targets for the prevention and treatment of this common cause of epilepsy.