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。TLE中癫痫发作倾向增加是由异常神经元兴奋性引起的。在TLE中表现兴奋性变化的一个候选者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，其对于膜电位稳态和神经元兴奋性至关重要。在海马锥体神经元中，h通道亚基HCN 1和HCN 2在远侧树突中比近侧树突显著富集，这是神经元兴奋性的关键现象;树突Ih的上调降低兴奋性，而减少增加兴奋性。在TLE大鼠模型中，我们发现在癫痫持续状态后24小时（自发性癫痫发作（潜伏期）之前的时间点），HCN 1的兴奋性降低，Ih和远端富集增强。相反，在自发性癫痫发作后，我们发现兴奋性增加加上减少Ih和HCN 1从远端树突到索马的再定位。有趣的是，与h通道结合蛋白的相互作用在自发性癫痫发作后显著减少。此外，在切片培养中，我们发现HCN 1远端树突定位以活性依赖性方式控制，需要激活N-甲基-D-天冬氨酸受体（NMDAR）和钙调蛋白依赖性蛋白激酶II（CaMK II）活性。我们的理由是，早期增强的h通道远端富集TLE是一个稳态反应增加的活动，降低兴奋性，而h通道重新定位从远端树突的索马在海马癫痫发生代表一个异常的过程，有助于增加兴奋性和自发性癫痫发作的发展。我们假设：1）h通道定位的控制调节TLE中的兴奋性，2）从内嗅皮层到CA 1树突的输入的活性通过NMDAR介导的蛋白激酶活化和HCN亚基磷酸化来控制h通道定位，和3）HCN亚基的异常磷酸化破坏h通道运输并超越h通道定位的正常稳态、活性依赖性控制，导致TLE中自发性癫痫发作。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）远端树突h通道是否在TLE中在潜伏期期间增加和在自发性癫痫发作后减少，2）从内嗅皮层到CA 1的时间氨输入、NMDAR激活、CaMK II活性和HCN亚基的磷酸化控制h通道定位，和3）异常的HCN亚基磷酸化阻止了对运输和靶向重要的相互作用，并使慢性TLE中的h通道错误定位。 公共卫生相关性：在公共卫生方面，尽管有许多新的医疗和手术治疗，但难治性癫痫发作仍然是颞叶癫痫患者残疾的重要原因。这项工作的最终目标是更好地了解控制TLE中自发性和难治性癫痫发作的分子因素，并希望确定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.