Synaptic function of Chromosome 21- encoded microRNAs

描述(申请人提供)：适当和功能性突触连接的形成和维持是一个高度调节的过程，调节不当会导致认知障碍。鉴于关于非编码RNA调节基因表达的新信息，突触功能可能至少部分受到非编码RNA的调节，包括microRNAs(MiRNAs)。一些miRNAs参与了棘突发生、树突分枝和突触发生。因此，可以想象，miRNAs在大脑中的过度或不足表达可能会导致突触功能障碍，从而导致神经或神经精神障碍。人类21号染色体(HSA21)编码5个已知的miRNAs，21三体(TS21，即唐氏综合症)是最常见的智力残疾遗传形式。因此，TS21为研究miRNA过表达对突触的形成和功能的影响提供了一个独特的模型。这个项目的目标是阐明 通过检验HSA21 miRNAs过度表达导致突触传递功能障碍从而导致TS21患者认知功能障碍的假设，发现HSA21 miRNAs在TS21患者产生的突触功能障碍中的作用可能与TS21患者的认知障碍有关。此外，有初步证据表明，这些miRNAs可能通过依赖于甲基CpG结合蛋白2(MeCP2)的途径影响突触的完整性。利用创新的诱导多能干细胞(IPS)和诱导神经元(IN)细胞技术，可以研究HSA21miRNAs对人类神经元突触的影响。在对照INS中过表达HSA21miRNAs后，将通过形态和功能分析，包括电生理学和钙成像来评估突触功能。MeCP2将通过双荧光素酶分析被确认为这些miRNAs的靶标，其在miRNA介导的突触修饰中的作用将通过敲除和拯救来测试。此外，还将使用患者特有的INS来阐明HSA21miRNAs的过度表达是否会导致TS21中的突触缺陷。通过定量聚合酶链式反应验证HSA21 miRNAs在TS21 INS中的表达水平，并将其与MeCP2的表达水平(通过Western Blot测定)相关联后，我们将对突触的形态和功能进行表征，以便与对照INS进行比较。然后，我们将建立HSA21 miRNA过度表达与突触缺陷之间的因果关系，使用强硬的诱骗来对抗miRNAs，并在细胞内“拯救”TS21-的突触功能，以及MeCP2的表达水平。这项研究具有创新性，因为我们将使用跨学科的分析方法，结合最新发展的细胞和iPS细胞技术来研究HSA21miRNAs在神经系统中的功能，这将拓宽我们对其生物学功能的认识，并为TS21的治疗提供机制和分子基础的洞察。

DESCRIPTION (provided by applicant): The formation and maintenance of appropriate and functional synaptic connections is a highly regulated process, with misregulation resulting in disordered cognition. Given the emerging information about gene expression regulation by non-coding RNAs, synaptic functions are likely to be regulated at least in part by non- coding RNAs, including microRNAs (miRNAs). Several miRNAs have been implicated in spinogenesis, dendritic arborization, and synaptogenesis. Thus, over- or under-expression of miRNAs in the brain could conceivably contribute to synaptic dysfunction resulting in neurological or neuropsychiatric disorders. Human chromosome 21 (HSA21) codes for 5 known miRNAs, and Trisomy 21 (TS21, i.e. Down syndrome) is the most common genetic form of intellectual disability. Thus, TS21 provides a unique model to study the effect of miRNA overexpression on the formation and functionality of synapses. The objective of this project is to elucidate the role of HSA21 miRNAs in synaptic dysfunction in human neurons generated from TS21 patients, which may be implicated in the cognitive disability in TS21 by testing the hypothesis that overexpression of HSA21 miRNAs leads to dysfunction in synaptic transmission that causes cognitive impairment in TS21 patients. Furthermore, there is preliminary evidence that suggests these miRNAs may affect synaptic integrity via a methyl CpG binding protein 2 (MeCP2) dependent pathway. Utilizing the innovative induced pluripotent stem (iPS) cell and induced neuronal (iN) cell technologies, it is possible to study the effects of HSA21 miRNAs on the synapses of human neurons. Following overexpression of HSA21 miRNAs in control iNs, synaptic function will be assessed by morphological and functional analyses, including electrophysiology and Calcium imaging. MeCP2 will be confirmed as a target of these miRNAs by dual luciferase assay, and its role in the miRNA-mediated modification of synapses will be tested via knockdown and rescue. Furthermore, patient-specific iNs will be used to elucidate whether the overexpression of HSA21 miRNAs causes synaptic defects in TS21. After verifying the expression levels of HSA21 miRNAs in TS21 iNs via qPCR and correlating them with the level of MeCP2 (determined by Western Blot), we will morphologically and functionally characterize the synapse for comparison with control iNs. We will then establish a cause-effect relationship between HSA21 miRNA overexpression and synaptic defects using Tough Decoys to antagonize the miRNAs and "rescue" the synaptic function of TS21-iN cells, as well as the expression level of MeCP2. The proposed research is innovative, because we will use interdisciplinary analytical methodologies and combine the newly developed iN cell and iPS cell technologies to examine the functions of HSA21 miRNAs in the nervous system, which will broaden our knowledge of their biological functions, as well as provide insight into mechanistic and molecular bases for the treatment of TS21.