Molecular Determination of in vivo Cellular Calcium Signaling During Nerve Damage

描述（由申请人提供）：在再生医学的快速发展领域的一个关键成就将是能够促进损伤，疾病或显微手术切断神经元的修复。细胞内钙信号在神经元对创伤性损伤的反应中起着重要的作用，但这一信号通路的机制仍不清楚。在神经元损伤后，细胞质钙水平的升高可引发变性，但对于有效的修复和再生也是至关重要的。我们将 阐明在一个完整的生理背景下，钙信号在受损的神经元和调节它的分子组成部分的反应。我们的工作结合了先进的生物光子和遗传技术在线虫C。elegans创造了一个独特的强大的实验系统。结合先进的激光手术技术和延时荧光成像，我们可以选择性地损伤完整的成年C。elegans和光学测量整个神经元的细胞内钙信号和生长动力学。利用新兴的光遗传学光活化技术，我们可以刺激受损的神经元，提高细胞内钙信号，以促进再生。C. elegans使我们能够通过分析基因突变来确定这些细胞事件背后的特定分子成分的作用。这项工作的目的如下：第一，确定介导受损神经元中空间定位的细胞内钙动力学和再生反应的分子和细胞机制，第二，确定凋亡caspase被钙激活以在早期再生中发挥有益作用的新途径的分子细节，第三，通过人工操纵受损神经元中的钙生理学来刺激额外的再生。因此，我们的研究将详细了解受损神经元内的钙信号级联，这对神经治疗学的发展至关重要。许多研究表明，通过各种手段抑制钙作为一种神经保护措施。这项工作将定义新的分子靶点，并提出新的治疗策略，用于调节钙信号以刺激神经元再生，建立一个详细的框架来解释其作用。我们的研究结果将把这些可能性，以全面了解细胞的事件，控制 神经元再生

DESCRIPTION (provided by applicant): A critical accomplishment in the rapidly developing field of regenerative medicine will be the ability to foster repair of neurons severed by injury, disease, or microsurgery. Intracellular calcium signaling plays a fundamental role in the neuronal response to traumatic damage, yet the mechanisms that orchestrate this signaling pathway remain largely unknown. Following neuronal insult, elevation of cytoplasmic calcium levels can trigger degeneration but also is critical for efficient repair and regeneration. We will elucidate within an intact physiological context, the calcium signaling response in a damaged neuron and the molecular components that modulate it. Our work combines advanced biophotonic and genetic techniques within the nematode worm C. elegans to create a uniquely powerful experimental system. Combining advanced laser surgery techniques and time-lapse fluorescence imaging, we can selectively damage individual neurons within intact adult C. elegans and optically measure intracellular calcium signaling and outgrowth dynamics throughout the neuron. Employing the emerging technology of optogenetic photo- activation, we can stimulate the damaged neuron elevating cellular calcium signals to enhance regeneration. The genetic tractability of C. elegans allows us to pinpoint the roles of specific molecular components underlying these cellular events through analysis of genetic mutations. The aims of this work are the following: first, to determine the molecular and cellular mechanisms that mediate spatially localized intracellular calcium dynamics and regeneration response in a damaged neuron, second to define the molecular details of a novel pathway by which an apoptotic caspace is activated by calcium to perform a beneficial role in early regeneration, and third to stimulate additional regeneration by artificially manipulating calcium physiology in a damaged neuron. As such, our study will generate a detailed understanding of the calcium signaling cascade within a damaged neuron that is critical for the advancement of neurotherapeutics. Numerous studies have suggested the inhibition of calcium through various means as a neuroprotective measure. This work will define new molecular targets and suggests novel therapeutic strategies for the modulation of calcium signaling to stimulate neuronal regeneration, establishing a detailed framework in which to interpret their effects. Our findings will put these possibilities into a comprehensive understanding of the cellular events that control neuronal regeneration.