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不是没有癫痫发作。顽固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的升高和失控是基本特征。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前景光明，但关键是取决于对这些过程的更好理解。这项拟议的研究对这一挑战的贡献将是评估microRNA诱导的瞬时失活A-型钾电流沉默作为大脑的一种内在机制，控制神经元的兴奋性，并可能成为癫痫的治疗靶点。A型电流是脑内兴奋性和同步性的重要门控器，A型电流介导通道亚单位的缺陷增加了癫痫和癫痫的易感性。因此，操纵这些电流可能对癫痫有治疗作用，但目前还没有特效的A型通道调节药物。在脑内，A型电流主要由钾通道Kv4.2及其辅助亚单位(Kv4.2复合体)介导。在癫痫的啮齿动物模型中，Kv4.2复合体的蛋白水平降低，提示Kv4.2复合体的下调是癫痫的一种病理机制。如果详细了解，导致Kv4.2复合体下调的机制可能因此成为操纵A型电流的替代药物靶点。这个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复合体的沉默引起的，这有助于神经元的高度兴奋性和同步性，因此可能成为治疗的靶点。这一假说得到了强有力的试点数据的支持，该数据表明，抑制Kv4.2靶向的microRNA可以降低小鼠诱发的癫痫状态的严重程度，减少红藻氨酸诱导的野生型兴奋性毒性，但不能抑制Kv4.2 KO神经元的兴奋毒性，并在体外阻止红藻氨酸诱导的Kv4.2下调。我们将追求三个目标。目的1将在获得性癫痫小鼠模型中研究抑制Kv4.2靶向microRNAs是否能阻止癫痫发作后Kv4.2复杂下调，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的microRNAs的作用机制，并定量比较候选microRNAs对Kv4.2复合体的表达和功能的调节作用，确定最有效的靶向Kv4.2的microRNAs。目的3将通过将分析扩展到两个遗传顽固性癫痫小鼠模型，神经元特异性Pten缺失小鼠和cntnap2KO小鼠，来检验这些发现的生理学相关性。与cntnap2相反，Pten缺失导致Kv4.2降低，使人们能够评估microRNA诱导的Kv4.2沉默增加是癫痫的病理机制和治疗靶点，无论是否存在Kv4.2缺陷。这种方法是创新的，因为它将操作A型电流表达式来修改函数。这项研究有望推进关于大脑如何调节兴奋性的知识，这最终可能导致治疗顽固性癫痫的新策略。

 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.