HCN Channel Trafficking in Epilepsy

描述(申请人提供)：颞叶癫痫(TLE)是难治性癫痫发作的常见原因。TLE癫痫发作倾向的增加是由神经元兴奋性异常引起的。在TLE中表现兴奋性变化的候选通道之一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，Ih对膜电位动态平衡和神经元兴奋性至关重要。在海马锥体神经元中，h通道亚单位hcn1和hcn2在远端比近端的树突显著丰富，这一现象对神经元的兴奋性至关重要；树突ih的上调降低了兴奋性，而减少则增加了兴奋性。在TLE大鼠模型中，我们发现在癫痫持续状态后24小时，即自发发作(潜伏期)之前的时间点，兴奋性降低，而Ih和远端HCN1的浓集增加。相反，在自发性癫痫发作后，我们发现兴奋性增加，伴随着ih的减少和hcn1从远端树突到胞体的重新定位。有趣的是，在自发性癫痫发作后，与H通道结合蛋白的相互作用显著减少。此外，在切片培养中，我们发现HCN1远端树突状细胞的定位是以活性依赖的方式控制的，需要激活N-甲基-D-天冬氨酸受体(NMDAR)和钙调蛋白依赖的蛋白激酶II(CaMKII)活性。我们推测，TLE早期h通道远端丰富的增强是对兴奋性增加的稳态反应，而h通道在癫痫发生过程中从远端树突到胞体的重新定位是一个异常过程，有助于兴奋性的增加和自发癫痫的发展。我们假设：1)h通道定位的控制调节TLE的兴奋性，2)从内嗅皮层到CA1树突的活动通过NMDAR介导的蛋白激酶激活和HCN亚单位磷酸化来控制h通道定位，3)HCN亚单位的异常磷酸化扰乱h通道的运输，推翻正常的动态平衡的h通道定位控制，导致TLE自发性癫痫的发生。我们将利用生理学、细胞生物学和生化技术来解决以下特定目标：1)TLE远端树突状细胞h通道是否在自发癫痫发作后潜伏期增加和减少；2)从内嗅皮层到CA1的临时氨基输入、NMDAR激活、CaMKII活性和HCN亚基的磷酸化控制h通道的定位；3)HCN亚单位磷酸化异常阻止了对运输和靶向至关重要的相互作用，并在慢性TLE中错误定位h通道。公共卫生相关性：在公共卫生方面，尽管有许多新的内科和外科治疗，难治性癫痫发作仍然是导致颞叶癫痫(TLE)患者残疾的一个重要原因。这项工作的最终目的是为了更好地了解控制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一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.