Cell type-specific functions of microRNA in epilepsy

microRNA 在癫痫中的细胞类型特异性功能

基本信息

批准号：
10569048
负责人：
Christina Gross
金额：
$ 19.88万
依托单位：
CINCINNATI CHILDRENS HOSP MED CTR
依托单位国家：
美国
项目类别：
财政年份：
2022
资助国家：
美国
起止时间：
2022-02-15 至 2024-07-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10569048
关键词：
3-Dimensional Address Affect Anticonvulsants Antisense Oligonucleotides Astrocytes Biological Biological Assay Biological Process Biology Brain Brain Diseases Brain region Cells Complex Computer Analysis Dendritic Spines Development Disadvantaged Disease Enterobacteria phage P1 Cre recombinase Environment Epilepsy Epitopes Frequencies Gene Transfer Hippocampus Individual Mediating Messenger RNA MicroRNAs Molecular Morphology Mus Neurons Pathogenesis Pathway interactions Persons Pharmaceutical Preparations Phenotype Physiological Play Population Porifera Potassium Channel Predisposition Publishing Quantitative Reverse Transcriptase PCR RNA purification RNA-Induced Silencing Complex Reporter Reporting Research Risk Role Seizures Specificity Testing Tissues Transgenic Mice Translations Untranslated RNA Validation Vertebral column Viral Viral Genes Work cell type cellular transduction excitatory neuron high resolution imaging inhibitor insight mouse model negative affect neglect neuronal circuitry novel novel strategies novel therapeutic intervention pharmacologic preclinical study prevent promoter reconstruction side effect targeted treatment therapeutic target transcriptome transcriptome sequencing treatment optimization treatment strategy

项目摘要

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.

概括美国至少有340万人患有癫痫病。尽管新批准的反西兹迅速增加在过去的几十年中，在所有情况中约30％的情况下，癫痫仍然很难。替代方案和因此，迫切需要在概念上进行新颖的治疗策略。一组有前途的新颖治疗目标是microRNA，小型非编码RNA，可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成部分，使它们变得强大生物过程的调节剂。尽管试图像癫痫一样治疗复杂疾病时，这是一个优势，但缺点是副作用的潜力增加。这是减少对人的热情的主要障碍将microRNA用作脑部疾病的治疗靶标。我们将使用特定于小区类型来解决此问题测试microRNA在控制大脑多个方面具有特异性的假设的策略通过通过不同的细胞类型，脑电路和细胞类型特异性靶标介导其多种作用来发挥作用，可以利用它来优化治疗策略。大多数针对microRNA的临床前研究癫痫已经使用了细胞型无特异性antagomir（反义寡核苷酸）方法。这些研究揭示了几种对癫痫中癫痫控制至关重要的microRNA也会影响神经元在健康状况下形态，可能会降低其作为治疗靶标的价值。提议研究将遵循概念上新颖的方法来通过使用microRNA海绵来克服这一问题。 microRNA Sponges隔离并在功能上抑制microRNA，并且与Antagomirs相反在细胞类型特异性启动子的控制下表达，因此仅在选定的靶细胞中抑制microRNA 大脑中的种群。以两个与癫痫相关的microRNA作为示例，我们将首先使用microRNA 在神经元亚型和星形胶质细胞特异性启动子的控制下的海绵以及与腺相关相关的病毒基因转移以测试microRNA对癫痫敏感性和神经元形态的影响是否可以通过大脑中的细胞类型分离（AIM 1）。为了洞悉基本机制和细胞 microRNA的类型特异性mRNA靶标，我们将将microRNA海绵与转基因小鼠结合在一起 CRE依赖性表达表位标记的RNA诱导的沉默复合物（RISC），允许特定仅从那些表达病毒海绵的细胞中分离出RISC相关的mRNA（AIM 2）。比较来自用炒作与microRNA特异性海绵转导的细胞的RISC相关转录组，我们将实验性地识别细胞类型特异性的microRNA靶标。我们的策略将利用多重通过操纵microRNA的microRNA调节数百个mRNA的功能，同时最大程度地提高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的第一步在癫痫中开发安全有效的microRNA靶向疗法。