Cell type-specific functions of microRNA in epilepsy

microRNA 在癫痫中的细胞类型特异性功能

• 批准号: 10427844
• 负责人: Christina Gross
• 金额: $ 23.85万
• 依托单位: CINCINNATI CHILDRENS HOSP MED CTR
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-02-15 至 2024-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10427844
• 关键词: 3-Dimensional; Address; Affect; 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 (Brain); Individual; Lead; Mediating; Messenger RNA; MicroRNAs; Molecular; Morphology; Mus; Neurons; Pathogenesis; Pathway interactions; Persons; Pharmaceutical Preparations; Pharmacology; 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; base; cell type; cellular transduction; excitatory neuron; high resolution imaging; inhibitor; insight; mouse model; negative affect; neglect; neuronal circuitry; novel; novel strategies; novel therapeutic intervention; preclinical study; prevent; promoter; reconstruction; side effect; targeted treatment; therapeutic target; transcriptome; transcriptome sequencing; treatment optimization; treatment strategy

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 万人患有癫痫症。尽管新批准的抗癫痫药物迅速增加 在过去的几十年里，癫痫症在大约百分之三十的病例中仍然是难治性的。另类和 因此，迫切需要概念上新颖的治疗策略。一组有前途的新疗法 靶标是 microRNA，即抑制翻译并诱导其降解的小非编码 RNA。 目标 mRNA。 MicroRNA 通常针对同一途径的多个组件，这使其功能强大 生物过程的调节者。虽然这在尝试治疗癫痫等复杂疾病时是一个优势， 缺点是副作用的可能性增加。这是降低人们参与热情的一个主要障碍。 使用 microRNA 作为脑部疾病的治疗靶点。我们将通过使用细胞类型特异性来解决这个问题 检验 microRNA 在控制大脑多个方面实现特异性这一假设的策略 通过不同的细胞类型、脑回路和细胞类型特异性目标介导其不同的作用来发挥功能， 可以利用它来优化治疗策略。大多数针对 microRNA 的临床前研究 癫痫症已使用细胞类型非特异性 antagomir（反义寡核苷酸）方法。这些研究 研究表明，几种对癫痫发作控制至关重要的 microRNA 也会影响神经元 健康条件下的形态，可能会降低其作为治疗目标的价值。拟议的 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 antagomir 相比，可以 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 大脑中的人群。以两种与癫痫相关的microRNA为例，我们首先使用microRNA 神经元亚型和星形胶质细胞特异性启动子与腺相关启动子结合控制下的海绵 病毒基因转移以测试 microRNA 对癫痫易感性和神经元形态的影响是否可以 按大脑中的细胞类型分开（目标 1）。提供对潜在机制和细胞的深入了解 microRNA 的类型特异性 mRNA 目标，我们将 microRNA 海绵与转基因小鼠结合起​​来， Cre 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细胞中分离 RISC 相关 mRNA（目标 2）。比较 使用乱序海绵与 microRNA 特异性海绵转导的细胞中的 RISC 相关转录组，我们将 通过实验鉴定细胞类型特异性的 microRNA 靶标。我们的策略将利用多路复用的优势 microRNA 的功能调节数百个 mRNA，同时通过操纵 microRNA 最大化特异性 与目标表型最相关的细胞类型。这项研究可能是迈向的第一步 开发安全有效的 microRNA 靶向疗法治疗癫痫。