Cell type-specific functions of microRNA in epilepsy

总结 在美国，至少有340万人患有癫痫。尽管新批准的抗癫痫药物迅速增加， 在过去的几十年里，癫痫在大约30%的病例中仍然是难以治愈的。替代和 因此，迫切需要概念新颖的治疗策略。一组有前途的新疗法 靶点是microRNA，即小的非编码RNA，其抑制翻译并诱导其降解。 靶向mRNA。microRNA通常针对同一途径的几个组件，使其功能强大 生物过程的调节器。虽然这在试图治疗癫痫等复杂疾病时是一个优势， 缺点是增加了副作用的可能性。这是一个主要的障碍，降低了人们的热情， microRNA作为脑疾病治疗靶点的用途。我们将通过使用特定于细胞类型的 测试microRNA在控制大脑多个方面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异性靶点介导其不同的作用来发挥作用， 这可以用来优化治疗策略。大多数临床前研究靶向microRNA， 癫痫已经使用了细胞类型非特异性的反义寡核苷酸（反义寡核苷酸）方法。这些研究 揭示了几种对癫痫发作控制至关重要的microRNA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。拟议 研究将遵循概念上新颖的方法，通过使用microRNA海绵来克服这个问题。 microRNA海绵隔离并功能性抑制microRNA，与微RNA海绵相反， 在细胞类型特异性启动子的控制下表达，从而仅在选择的靶细胞中抑制microRNA 大脑中的人口。以两种癫痫相关microRNA为例，我们将首先使用microRNA 海绵在神经元亚型和星形胶质细胞特异性启动子的控制下， 病毒基因转移来测试microRNA对癫痫易感性和神经元形态的影响是否可以 根据大脑中的细胞类型进行分离（目标1）。为了深入了解潜在的机制和细胞 我们将联合收割机与转基因小鼠结合， 依赖性表达表位标记的RNA诱导沉默复合物（RISC），允许特异性 仅从表达病毒海绵的那些细胞中分离RISC相关mRNA（目的2）。比较 RISC相关的转录组从细胞转导与乱序与microRNA特异性海绵，我们将 实验上鉴定细胞类型特异性microRNA靶。我们的策略是利用 microRNA的功能调节数百种mRNA，同时通过操纵microRNA来最大化特异性 在与靶向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microRNA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.