Cell type-specific functions of microRNA in epilepsy

摘要 在美国，至少有340万人患有癫痫。尽管新批准的抗癫痫发作迅速增加 在过去的几十年里，癫痫在所有病例中仍然有30%是难治性的。可选的和 因此，迫切需要概念上新颖的治疗策略。一组有希望的新疗法 靶标是microRNAs，一种抑制翻译并诱导其降解的非编码小RNA 靶向mRNAs。MicroRNA通常针对同一途径的几个组件，使它们变得强大 生物过程的调节者。虽然这在试图治疗癫痫等复杂疾病时是一个优势， 不利之处是副作用的可能性增加。这是一个主要障碍，降低了人们对 使用microRNA作为治疗脑部疾病的靶点。我们将使用特定于细胞类型的方法来解决此问题 检验microRNAs在控制大脑多个方面实现特异性的假设的策略 通过不同的细胞类型、脑回路和细胞类型特定的靶点来调节它们的不同角色来发挥作用， 这可以被用来优化治疗策略。大多数针对microRNAs的临床前研究 癫痫患者使用了细胞类型非特异性的反义寡核苷酸方法。这些研究 揭示了几个被证明对癫痫发作控制至关重要的微RNA也影响神经元 在健康条件下的形态，潜在地降低了它们作为治疗靶点的价值。建议数 研究将遵循概念上的新方法，通过使用microRNA海绵来克服这一问题。 MicroRNA海绵隔离并在功能上抑制microRNA，与反交配子相反，它可以 在特定细胞类型启动子的控制下表达，从而仅在选定的靶细胞中抑制microRNA 大脑中的种群。以两个癫痫相关的microRNA为例，我们将首先使用microRNA 神经元亚型和星形胶质细胞特异性启动子和腺相关基因联合调控下的海绵 病毒基因转移以测试microRNAs对癫痫敏感性和神经元形态的影响 根据大脑中的细胞类型进行区分(目标1)。提供对潜在机制和细胞的洞察 ，我们将把microRNA海绵与转基因小鼠结合起来， Cre依赖地表达表位标记的RNA诱导沉默复合体(RISC)，允许特定的 仅从表达病毒海绵的细胞中分离RISC相关的mRNA(目标2)。比较一下 转导细胞的RISC相关转录组，与microRNA特异性海绵相比，我们将 通过实验识别特定细胞类型的microRNA靶标。我们的战略将利用多元化 MicroRNAs调节数百个mRNAs的功能，同时通过操纵microRNAs使特异性最大化 在与目标表型最相关的细胞类型中。这项研究可能是迈向 开发安全有效的微核糖核酸靶向治疗癫痫。

SUMMARY At least 3.4 million people in the USA live with epilepsy. Despite a rapid increase in newly approved anti-seizure drugs in the last decades, epilepsy is still intractable in about thirty percent of all cases. Alternative and conceptually novel therapeutic strategies are therefore urgently needed. One group of promising novel treatment targets are microRNAs, small noncoding RNAs that suppress the translation and induce the degradation of their target mRNAs. MicroRNAs often target several components of the same pathway, making them powerful regulators of biological processes. While this is an advantage when trying to treat complex diseases like epilepsy, a disadvantage is the increased potential for side effects. This is a major obstacle reducing enthusiasm for the use of microRNA as treatment targets in brain disorders. We will address this issue by using cell type-specific strategies to test the hypothesis that microRNAs achieve specificity in controlling multiple aspects of brain function by mediating their diverse roles through different cell types, brain circuits, and cell type-specific targets, which could be leveraged to optimize treatment strategies. Most preclinical studies targeting microRNAs in epilepsy have used cell type-unspecific antagomir (antisense oligonucleotide) approaches. These studies revealed that several microRNAs shown to be crucial for seizure control in epilepsy also affect neuronal morphology under healthy conditions, potentially reducing their value as treatment targets. The proposed research will follow conceptually novel approaches to overcome this problem by using microRNA sponges. MicroRNA sponges sequester and functionally inhibit microRNAs and can, in contrast to antagomirs, be expressed under the control of cell type-specific promoters, thus inhibiting microRNAs only in select target cell populations in the brain. Taking two epilepsy-relevant microRNAs as examples, we will first use microRNA sponges under the control of neuron subtype- and astrocyte-specific promoters combined with adeno-associated viral gene transfer to test if the effects of the microRNAs on seizure susceptibility and neuronal morphology can be separated by cell types in the brain (aim 1). To provide insight into the underlying mechanisms and the cell type-specific mRNA targets of the microRNAs, we will combine the microRNA sponges with transgenic mice that Cre-dependently express epitope-tagged RNA-induced silencing complex (RISC) allowing for the specific isolation of RISC-associated mRNA from only those cells that express the viral sponge (aim 2). Comparing the RISC-associated transcriptome from cells transduced with scrambled versus microRNA-specific sponges we will experimentally identify cell type-specific microRNA targets. Our strategy will take advantage of the multiplex function of microRNAs regulating hundreds of mRNAs while maximizing specificity by manipulating microRNAs in the cell types that are the most relevant for the targeted phenotype. This research may be the first step towards the development of safe and effective microRNA-targeted therapies in epilepsy.