MicroRNA-mediated silencing of the Kv4.2 complex in epilepsy

 描述（由申请人提供）：超过三分之一的癫痫患者在目前可用的治疗方法下并非无癫痫。难治性癫痫的原因是多方面的;然而，大脑中神经元兴奋性和同步性升高和不受控制是基本属性。因此，利用大脑的内在机制来控制兴奋性和抑制同步性的疗法具有很大的前景，但关键取决于对这些过程的更好理解。拟议研究对这一挑战的贡献将是评估microRNA诱导的瞬时失活A型钾电流沉默，这是大脑控制神经元兴奋性的固有机制，可能是癫痫的治疗靶点。A型电流是大脑兴奋性和同步性的关键门控器，A型电流介导通道亚单位的缺陷增加了癫痫发作和癫痫的易感性。因此，操纵这些电流可以治疗癫痫，但目前没有特定的A型通道调节药物可用。在脑中，A型电流主要由钾通道Kv4.2及其辅助亚基（Kv4.2复合物）介导。Kv4.2复合物的蛋白质水平在癫痫的啮齿动物模型中降低，表明Kv4.2复合物的下调是癫痫的病理机制。如果详细了解，导致Kv4.2复合物下调的机制可以作为操纵A型电流的替代药物靶点。该项目的中心假设是，癫痫中Kv4.2和A型电流的下调是由microRNA介导的Kv4.2复合物沉默引起的，这有助于神经元的过度兴奋性和同步性，因此可能是治疗靶点。这一假设得到了强有力的初步数据的支持，这些数据表明，抑制Kv4.2靶向microRNA降低了小鼠中诱发癫痫持续状态的严重程度，降低了培养的野生型神经元中红藻氨酸诱导的兴奋性毒性，但没有Kv4.2 KO神经元，并防止了红藻氨酸诱导的Kv4.2在体外下调。将追求三个目标。目的1将检查在获得性癫痫的小鼠模型中抑制Kv4.2靶向microRNA是否防止癫痫发作后Kv4.2复合物下调并降低癫痫发作频率和严重程度。目的2通过分析靶向Kv4.2复合体的机制和定量比较候选microRNA对Kv4.2复合体表达和功能的调控效果，筛选出最有效的靶向Kv4.2的microRNA。目的3将通过将分析扩展到两种遗传性难治性癫痫小鼠模型，神经元特异性Pten缺失小鼠和Cntnap 2 KO小鼠，来测试这些发现的生理相关性。与Cntnap 2相反，Pten缺失导致Kv4.2降低，这使得能够评估增加的microRNA诱导的Kv4.2沉默作为具有和不具有可检测的Kv4.2缺陷的癫痫的病理机制和治疗靶点。该方法具有创新性，因为它将操纵A型电流表达式来修改函数。这项研究有望推进有关大脑如何调节兴奋性的知识，最终可能导致治疗难治性癫痫的新策略。

 DESCRIPTION (provided by applicant): More than one third of individuals with epilepsy are not seizure-free with currently available treatments. Causes of intractable epilepsy are manifold; yet, elevated and uncontrolled neuronal excitability and synchronicity in the brain are fundamental attributes. Therapies that exploit the brain's intrinsic mechanisms to control excitabil- ity and suppress synchronicity thus hold great promise, but critically depend on a better understanding of these processes. The contribution of the proposed research to this challenge will be to assess microRNA-induced silencing of transient inactivating A-type potassium currents as an inherent mechanism of the brain that con- trols neuronal excitability and could be a therapeutic target for epilepsy. A-type currents are crucial gate keep- ers of excitability and synchronicity in the brain, and defects in A-type current-mediating channel subunits in- crease the susceptibility to seizures and epilepsy. Manipulating these currents could thus be therapeutic in epi- lepsy, but there are currently no specific A-type channel-modulating drugs available. In the brain, A-type cur- rents are mainly mediated by the potassium channel Kv4.2 and its auxiliary subunits (Kv4.2 complex). Protein levels of the Kv4.2 complex are decreased in rodent models of epilepsy, suggesting that downregulation of the Kv4.2 complex is a pathological mechanism in epilepsy. If understood in detail, the mechanisms that lead to this downregulation of the Kv4.2 complex could thus serve as an alternative drug target to manipulate A-type currents. The central hypothesis of this project is that downregulation of Kv4.2 and A-type currents in epilepsy is caused by microRNA-mediated silencing of the Kv4.2 complex, which contributes to neuronal hyperexcitabil- ity and -synchronicity and could thus be a therapeutic target. This hypothesis is supported by strong pilot data showing that inhibition of a Kv4.2-targeting microRNA decreases severity of provoked status epilepticus in mice, reduces kainic acid-induced excitotoxicity in cultured wild type, but not Kv4.2 KO neurons and prevents kainic acid-induced downregulation of Kv4.2 in vitro. Three aims will be pursued. Aim 1 will examine if inhibition of Kv4.2-targeting microRNAs prevents Kv4.2 complex downregulation following seizures and reduces seizure frequency and severity in a mouse model of acquired epilepsy. Aim 2 will identify the most potent Kv4.2- targeting microRNAs by analyzing the mechanisms and quantitatively comparing the efficacy of candidate mi- croRNAs to regulate Kv4.2 complex expression and function. Aim 3 will test the physiological relevance of these findings by expanding the analyses to two mouse models of genetic intractable epilepsy, neuron-specific Pten deletion mice and Cntnap2 KO mice. In contrast to Cntnap2, Pten deletion leads to reduced Kv4.2, ena- bling assessment of increased microRNA-induced Kv4.2 silencing as pathological mechanism and therapeutic target in epilepsy with and without detectable Kv4.2 defects. The approach is innovative, because it will manipulate A-type current expression to modify function. The research is expected to advance knowledge about how the brain regulates excitability, which ultimately could lead to new strategies to treat intractable epilepsy.