The role of Miro at endoplasmic reticulum-mitochondria contacts in health and disease

内质网-线粒体接触（ermc）在一系列生理过程中发挥关键作用，包括钙稳态、自噬、细胞凋亡和脂质代谢（Shirokova et al., 2020）。在肌萎缩性侧索硬化症、阿尔茨海默病和帕金森病（PD）等疾病中，已经观察到这些细胞器间接触的功能和超微结构失调（De Mario et al., 2017）。事实上，据推测ermc的功能障碍是神经退行性疾病致病终点的主要早期贡献者。尽管有这些观察结果，我们对ermc调控机制的理解仍然有限，特别是在神经元中。这一知识可以为疾病功能障碍提供清晰的认识，并有助于确定新的治疗靶点。线粒体Rho GTPase （Miro）家族是线粒体外膜蛋白，定位于ermc。已知Miro与ermc中积累的几种蛋白质相互作用，如丝分裂蛋白（Misko等人，2010），果蝇和酵母的同源物都与ermc中的调节作用有关（Lee等人，2016；Kornmann等人，2011）。此外，Miro敲除和pd相关的Miro - 1突变体改变了ERMC超微结构，同时失调了人类细胞系中的钙稳态（Modi等人，2019；Berenguer-Escuder等人，2019）。虽然有越来越多的证据表明，有一种调节机制控制着接触者及其组成部分的数量，可能是由Miro支持的，但针对接触者的类型以及Miro调节它们的机制仍不清楚。因此，我们的目标是利用共聚焦、超分辨率和电子显微镜来探索Miro在原代培养神经元和离体脑组织中ERMC调节中的作用。此外，我们将应用分子和细胞生物学技术来探索Miro功能障碍对特定蛋白质接触的功能和结构影响。我们的研究将利用实验室中已有的小鼠Miro1/2敲除系（López-Doménech等人，2018）和miro1构建体，包括pd相关突变体和信号突变体。我们希望进一步了解ermc的调控过程及其在神经退行性过程中的失调。

Endoplasmic reticulum-mitochondria contacts (ERMCs) play a key role in a range of physiological processes including calcium homeostasis, autophagy, apoptosis, and lipid metabolism (Shirokova et al., 2020). The dysregulation of both the function and ultrastructure of these inter-organellar contacts has been observed in diseases such as Amyotrophic lateral sclerosis, Alzheimer's, and Parkinson's disease (PD) (De Mario et al., 2017). Indeed, it is postulated that the malfunction of ERMCs is a major early contributor to the pathogenic end-point in neurodegenerative disease. Despite these observations, our understanding of the mechanisms underpinning the regulation of ERMCs is still limited, particularly in neurons. This knowledge could provide clarity to dysfunction in disease and help identify new targets for therapeutics. The Mitochondrial Rho GTPase (Miro) family are outer mitochondrial membrane proteins that localize at ERMCs. Miro is known to interact with several proteins which accumulate at ERMCs, such as the Mitofusins (Misko et al.,2010), and both the Drosophila and Yeast homologs are associated with regulatory roles at ERMCs (Lee et al., 2016; Kornmann et al., 2011). Moreover, Miro knock-out and PD-related Miro1-mutants altered ERMC ultrastructure whilst simultaneously dysregulating calcium homeostasis in human cell lines (Modi et al., 2019; Berenguer-Escuder et al., 2019). While there is growing evidence of a regulatory mechanism controlling the number of contacts and their components, potentially underpinned by Miro, the types of contacts targeted and the mechanism by which Miro regulates them remains unclear. Thus, we aim to utilise confocal, super resolution, and electron microscopy to explore the role of Miro in ERMC regulation in primary cultured neurons and ex vivo brain tissue. Moreover, we will apply molecular and cell biology techniques to explore the functional and structural implications of Miro dysfunction on specific protein contacts. Our investigation will utilise pre-existing in-lab mouse Miro1/2 knock-out lines (López-Doménech et al., 2018) and Miro1-constructs, including PD-related mutants and signaling mutants. We hope to provide further insight into the regulatory processes at ERMCs and their dysregulation in the neurodegenerative process.