The contributions of excitation and contraction to muscle deterioration in a Drosophila model of CFL2 nemaline myopathy

项目总结 线状肌病(NM)是一种以肌肉无力为特征的骨骼肌疾病，发病率为1 在50,000个活产婴儿中。在组织病理学上，断裂的肌肉中有明显的肌动蛋白积聚。 到目前为止，已发现的致病突变是肌肉内肌动蛋白细丝结构的关键基因。 这些蛋白的改变如何导致NM病理的分子机制还不是很清楚。 Cofilin-2是一种受影响的肌动蛋白结合蛋白，对肌动蛋白细丝的切断起重要作用。此文件已归档 异构体是出生后和成熟骨骼肌中的主要形式，其功能已被重点研究。 肌节上的肌动蛋白是肌肉的主要收缩单位。Cofilin-2是已知的生物化学 比肌瘤肌动蛋白更容易与细胞质非肌瘤肌动蛋白结合，但这一特性的影响 关于NM级数的研究尚未见报道。果蝇肌肉特异性粘附素(DmCFL)基因敲除模型的建立 我们的实验室显示，在生长过程中，与肌节增加有关的进行性肌肉缺陷。我 分析了DmCFL基因敲除模型的肌肉强化准备产生的RNA测序数据 并发现与兴奋收缩偶联(ECC)相关的基因存在差异表达。ECC是 运动神经元发出的信号传递到肌肉的过程，最终导致 收缩。我的初步数据显示神经肌肉一侧的肌动蛋白结构紊乱 连接(NMJ)，这是肌肉从运动神经元接收信号的地方。基于文献和 这些初步发现，我假设非肌瘤肌动蛋白的cofilin调节对于 在肌肉恶化之前，ECC需要适当的NMJ和收缩机械结构。至 针对这一假设，我将使用DmCFL击倒模型来分析cofilin减少对 NMJ信号转导(目标1)和肌肉收缩(目标2)。前者将通过以下方式实现 利用分子、显微镜和电生理技术分析NMJ蛋白的变化 肌肉(目标1A)和运动神经元(目标1B)的定位、形态和功能。我会比较一下 从果蝇幼虫肌肉到cofilin-2基因敲除小鼠肌肉样本的形态发现。 将使用针对钙信号机制的荧光成像技术来评估收缩 (目标2 A)。使用一种改进的锻炼方法(目标2B)，我将发现锻炼强度如何影响 DmCFL基因敲除幼虫的收缩和表型进展。这些实验将共同提供 洞察NMJ的状态和COFILIN NM的收缩活动，同时利用简单性和高 果蝇的进化保护水平。在回答这个与临床疾病相关的问题时 这通常表现在生命的早期，我会进一步发展技术技能和科学推理 需要作为一名细胞和发育生物学家。这些研究将补充我的临床活动，因为我训练 成为一名对儿科感兴趣的全能内科科学家和独立研究员。

PROJECT SUMMARY Nemaline myopathy (NM) is a skeletal muscle disease hallmarked by muscle weakness with an incidence of 1 in 50,000 live births. On histopathology, there is an obvious presence of actin accumulations in disrupted muscle. The causative mutations identified thus far are in genes critical for actin filament structure within the muscle, yet the molecular mechanisms for how alteration of these proteins leads to NM pathology is not well understood. Cofilin-2, which is important for actin filament severing, is one such affected actin-binding protein. This cofilin isoform is the predominant form in postnatal and mature skeletal muscle; its function has mainly been studied with respect to actin at the sarcomere, the muscle’s main contractile unit. Cofilin-2 is known to biochemically bind more readily to cytoplasmic non-sarcomeric actin than sarcomeric actin, but the impact of this characteristic on NM progression has not been studied. A Drosophila model of muscle-specific cofilin (DmCFL) knockdown was shown by our lab to have progressive muscular defects linked to sarcomere addition during growth. I analyzed RNA sequencing data produced from muscle-enriched preparation of the DmCFL knockdown model and found that genes associated with excitation-contraction coupling (ECC) are differentially expressed. ECC is the process by which signals from the motor neuron are communicated to the muscle ultimately leading to contraction. My preliminary data show disordered actin organization at the muscle side of the neuromuscular junction (NMJ), which is where the muscle receives signals from the motor neuron. Based on the literature and these preliminary findings, I hypothesize that cofilin regulation of non-sarcomeric actin is critical for the proper NMJ and contraction machinery structure needed for ECC prior to muscle deterioration. To address this hypothesis, I will use the DmCFL knockdown model to analyze the impact of decreased cofilin on the NMJ signal transduction (Aim 1) and muscle contraction (Aim 2). The former will be accomplished by using molecular, microscopy, and electrophysiological techniques to analyze changes in NMJ protein localization, morphology, and function at the muscle (Aim 1A) and motor neuron (Aim 1B). I will compare the morphological findings from Drosophila larval muscle to those from cofilin-2 knockout mouse muscle samples. Contraction will be assessed using fluorescent imaging techniques targeted to the calcium signaling machinery (Aim 2A). Using a modified exercise approach (Aim 2B), I will discover how exercise intensity influences contraction and phenotype progression in DmCFL knockdown larva. These experiments will collectively provide insight into the status of the NMJ and contractile activity in cofilin NM while leveraging the simplicity yet high level of evolutionary conservation of Drosophila. In answering this question relevant to a clinical disease that typically manifests early in life, I will further develop the technical skills and scientific reasoning needed as a cell and developmental biologist. These studies will complement my clinical activities as I train to become a well-rounded physician-scientist and independent investigator interested in pediatrics.