Characterization of miRNAs on neural development and plasticity

今年，我们研究了大鼠创伤性脑损伤(TBI)，以确定在抑制基因表达和调控生物过程(如细胞分化、增殖和凋亡)方面重要的miRNAs。脑外伤会导致暂时性或永久性的大脑结构和功能损伤，这是与损伤相关的死亡和残疾的主要原因。脑外伤的幸存者除了常见的情绪和行为问题外，还表现出神经精神异常，如认知缺陷，这些问题是导致脑外伤后残疾的主要原因。负责认知和情绪的海马体是脑部易受脑外伤影响的区域之一。脑损伤后海马区的病理改变是细胞丢失、神经回路紊乱、突触传递和可塑性受损，所有这些都会导致神经精神症状。脑外伤后海马区损伤和恢复的机制尚不清楚。然而，这些结构和功能的变化是由几个大脑区域的基因表达变化引起的，就像在人类和动物的脑损伤模型中所显示的那样。同样，基因表达变化的机制也不清楚。我们研究的目标是microRNAs，它对大脑的正常功能至关重要，它们的丢失会导致突触蛋白表达、突触传递、树突棘形态、学习和记忆的变化。先前的研究表明，脑外伤改变了海马区信使RNA的表达。MiRNA表达的改变更有可能影响其靶mRNA的表达，因此miRNAs可能在脑损伤中发挥调节基因表达的作用。由于颅脑损伤后神经元损伤和智能损害的分子和细胞机制尚不清楚，我们使用深度测序技术在大鼠控制的皮质撞击损伤模型(CCI)中描绘了脑损伤后24小时和7天时海马区miRNA转录组的变化。 我们还开发了一种生物信息学分析，称为miRNA-基因-GO浓缩，以计算检测CCI中miRNA表达变化的潜在影响。分析按以下三个一般步骤进行：1)通过TargetScan数据库将深度测序得到的大鼠miRNA与其小鼠同源基因进行匹配；2)编译通过计算预测的小鼠miRNA同源基因并评估其富集性的统计学意义；3)从基因本体论数据库中获得注释过度表达的基因的GO项，并对三组GO项(生物过程、分子功能和细胞成分)中的每一个进行富集化分析。GO浓缩分析表明，CCI后24小时和7天的miRNAs都对应于TBI的继发性损伤阶段(即组织学、生化、代谢和细胞变化)，发生在最初创伤后几分钟到几天甚至几个月。这些类型的细胞反应是启动继发性损伤的关键，并被认为影响与脑损伤相关的神经元的结构和功能损伤。这些发现与已知的创伤后病理改变是一致的。此外，还为CCI后的两个时间点确定了不同的GO术语。在24小时内，miRNAs被调节以抑制炎症反应中的白细胞死亡，防止应激诱导的蛋白质变性和聚集，并保护细胞免受能量耗竭。在7天的时间点，细胞切换到支持修复和结构重塑的细胞环境的miRNAs。 我们对CCI损伤对海马区miRNA转录组的影响以及损伤后海马区miRNAs潜在调控的细胞功能和生物学过程的研究发现，在CCI后的不同时间，不同组的miRNAs受到调节，提示多个miRNAs协同调节细胞通路，促进脑损伤的病理变化和治疗。这些不同的miRNAs在CCI后不同时间的表达谱可作为评估TBI进展的分子标记。生物信息学分析表明，miRNAs以CCI后特定时间的方式针对多个GO项，这表明在脑损伤的不同阶段，miRNAs共同作用于调节大脑中广泛的细胞功能。这些已确定的生物途径可能是开发新的脑损伤治疗方法的潜在靶点。 除了创伤性脑损伤，我们通过结合深度测序、生物信息学和实时成像来研究miRNAs在突触发育中的作用。通过对miRNA转录本的分析，鉴定了在脊椎形态发生过程中在海马神经元中差异表达的miRNA。生物信息学分析用于确定经鉴定的miRNAs统计上富含的信使核糖核酸靶标和细胞过程。通过海马神经元活体成像检测几个miRNAs的功能。我们的研究表明miRNAs对于脊柱的形态发生是必不可少的。我们还确定了这些miRNAs调控脊柱的靶基因，并证明了翻译依赖的肌动蛋白重排在脊柱重塑中发挥着重要作用。这些发现表明，通过影响不同靶点的表达，miRNAs协调了对突触发育至关重要的细胞过程网络。

This year we studied traumatic brain injury (TBI) in rat to identify miRNAs which are important in repressing gene expression and regulating biological processes, such as cell differentiation, proliferation and apoptosis. TBI leads to temporary or permanent structural and functional impairment of the brain which is a leading cause of injury-related death and disability. Survivors of TBI show neuropsychiatric abnormalities such as cognitive deficits in addition to emotional and behavioral problems which are common and contribute substantially to post-TBI disabilities. One of the brain regions vulnerable to TBI is the hippocampus which is responsible for cognition and emotion. Pathological changes observed in TBI-hippocampus are cell loss, disturbed neural circuits, impaired synaptic transmission and plasticity, all of which lead to neuropsychiatric symptoms. The mechanisms responsible for hippocampus damage and recovery post-TBI remain unclear. However, these structural and functional changes result from altered gene expression in several brain regions, as has been shown in human and animal models of TBI. And again, the mechanisms for gene expression changes are also unclear. The target of our study, microRNAs, are critical for proper brain functions and their loss causes changes in synaptic protein expression, synaptic transmission, dendritic spine morphology, learning and memory. Previous studies have shown that TBI alters mRNA (messenger RNA) expression in the hippocampus. Altered miRNA expression more than likely impacts their target mRNA expression, so it is possible that miRNAs play a role in regulating gene expression in TBI. Since molecular and cellular mechanisms subserving neuronal damage and impairment in TBI mental abilities are largely unknown, we used deep-sequencing to delineate miRNA transcriptome changes in hippocampus at 24hr and day 7 post-TBI in the rat controlled cortical impact injury model (CCI). We also developed a bioinformatics analysis, called miRNA-gene-GO Enrichment, to computationally examine the potential effects of miRNA expression changes in CCI. The analysis was performed in these three general steps: 1) match rat miRNAs resulting from deep-sequencing to their mouse orthologs via the TargetScan database, 2) compile computationally predicted target genes of the mouse miRNA orthologs and estimate their statistical significance of enrichment, and 3) obtain the GO terms that annotate the over-represented genes from the Gene Ontology database and perform enrichment analysis on each of three sets of GO terms (biological process, molecular function, and cellular component). The GO enrichment analysis revealed that both the 24hr and 7 day post-CCI miRNAs correspond to the secondary damage stage (i.e. histological, biochemical, metabolic and cellular changes) of TBI, which occurs minutes to days or even months after the initial trauma. These types of cellular responses are essential to the initiation of secondary damage, and are thought to impact TBI-related structural and functional impairments of neurons. These findings are consistent with known post-trauma pathological changes. In addition, distinct GO terms are identified for the two post-CCI time points. At 24 hours the miRNAs are modulated to inhibit leukocyte cell death for the inflammatory response, prevent stress-induced protein denaturation and aggregation, and protect cells from energy exhaustion. At the 7 day time point, cells switch to miRNAs that support a cellular environment for repair and structural remodeling. Our study of the effect of CCI injury on the miRNA transcriptome in the hippocampus and identification of cellular functions and biological processes potentially regulated by miRNAs in the damaged hippocampus found distinct sets of miRNAs regulated at different post-CCI times, and suggest that multiple miRNAs cooperatively regulate cellular pathways for the pathological changes and management of brain injury. These distinct miRNAs expression profiles at different post-CCI times may be used as molecular markers to assess TBI progression. The bioinformatics analysis showed the miRNAs target to multiple GO terms in a post-CCI time specific manner which indicated that miRNAs act together to regulate a broad range of cellular functions in the brain at different stages of TBI. These identified biological pathways may be potential targets for development of new TBI treatments. In addition to traumatic brain injury, we investigated the role of miRNAs in synapse development by combining deep-sequencing, bioinformatics and live imaging. miRNAs differentially expressed in hippocampal neurons during spine morphogensis were identified by profiling miRNA transcriptomes. Bioinformatics analysis was conducted to determine mRNA targets and cellular processes statistically enriched by the identified miRNAs. The functions of several miRNAs were examined by live imaging of hippocampal neurons. Our study shows that miRNAs are essential for spine morphogenesis. We also identified the target genes through which these miRNAs regulate spines, and demonstrated that translation-dependent actin rearrangement plays important roles in spine remodeling. These findings reveal that by impinging on the expression of diverse targets, miRNAs orchestrate networks of cellular processes essential for synapse development.