Mitochondrial Calcium and Neuronal Health

摘要 长期以来，人们一直认为钙(Ca~(2+))信号通过钙单一转运体传递到线粒体 神经功能的中枢。包括钙离子在内的单转运蛋白分子组成的发现 感觉调节器MICU1，紧随其后的是许多MICU1功能丧失患者的识别 进行性神经功能障碍，以运动和学习障碍为主，在 我们和其他人创造的老鼠模型。新的证据表明，急性MICU1蛋白降解 YME1L在缺氧时也会发生，这表明线粒体钙稳态的失调可能 在脑缺血/中风中也起作用。然而，与MICU1缺乏相关的神经元发病机制， 更广泛地说，单转运体调控在健康神经元中的作用仍然知之甚少。MICU1形成二聚体 与其并列的MICU2和MICU3，以及它自己。MICU2也被认为与人类神经系统有关 MICU3在成年神经元中很少，而MICU3在成年大脑中丰富。我们的中心假设 MICU1/2/3对线粒体钙摄取的控制是协调线粒体功能的关键 突触活动。这种控制还可以防止线粒体钙超载和氧化剂失调，从而导致 神经应激。此外，我们假设依赖于MICU的神经元把关的动态调整： 发育--在生命早期从主要由MICU1-MICU2转变为MICU1-MICU3二聚体--以及 低氧应激，通过MICU1的特定蛋白降解，具有潜在的致病后果。为了测试 这些想法，我们将使用神经元特异性的MICU1基因敲除小鼠，我们发现这种小鼠表现出神经变性 特点是线粒体钙超载，线粒体和神经元超微结构改变，运动和 认知障碍。我们还产生了MICU2和MICU3缺失的小鼠，并获得了MICU1 和MICU2患者来源的细胞。我们已经建立了先进的功能成像和大体积3D 超微结构能力。因此，我们可以很好地研究与脑出血相关的神经元发病机制。 每个或多个MICU亚型丢失。在目标1中，我们将测试这样的假设，即神经元源自MICU1， MICU2或MICU3缺陷小鼠以及MICU1和MICU2缺陷的患者成纤维细胞来源的神经元具有明显的 钙信号、线粒体动力学和突触小泡释放的损伤。在目标2中，我们将测试 神经元MICU1/2/3缺失促进线粒体氧化剂的产生和氧化剂介导的细胞损伤。这个 结果将有助于确定氧化剂是否与MICU相关的神经元/脑损伤有关。在目标3中，我们将测试 假设不同MICU亚型的相对丰度在神经元发育过程中是动态的， 每种异构体的丢失都会特别影响神经解剖和大脑功能，主要是运动区。 和海马体。在目标4中，我们将测试缺血再灌注是否诱导YME1L介导的MICU1的周转 以促进线粒体钙超载损伤，并使单一转运体对抑制剂敏感。这些研究是 预计将确定MICU1丢失是否是脑缺氧的致病成分和治疗修改因子。

ABSTRACT Propagation of calcium (Ca2+) signals to the mitochondria through the Ca2+ uniporter has long been considered central to neuronal function. The discovery of the molecular components of the uniporter including the Ca2+ sensing regulator MICU1, was followed by the identification of many MICU1 loss-of-function patients who display progressive neurological disorder, dominated by motor and learning impairments, which were recapitulated in mouse models created by us and others. Emerging evidence indicates that acute MICU1 proteolytic degradation by YME1L also occurs during hypoxia, suggesting that dysregulation of mitochondrial Ca2+ homeostasis may also play a role in ischemia/stroke. However, the neuronal pathogenesis associated with MICU1 deficiency and, more broadly, the role of uniporter regulation in healthy neurons, remain poorly understood. MICU1 forms dimers with its paralogs, MICU2 and MICU3, and with itself. MICU2 has also been linked to human neurological impairments but is scarce in adult neurons, whereas MICU3 is abundant in adult brain. Our central hypothesis is that control of mitochondrial Ca2+ uptake by MICU1/2/3 is essential for coordinating mitochondrial function with synaptic activity. This control also prevents mitochondrial Ca2+ overload and oxidant dysregulation that lead to neuronal stress. Further, we postulate dynamic tuning of neuronal MICU-dependent gatekeeping: during development—by a switch from primarily MICU1-MICU2 to MICU1-MICU3 dimers during early life—and under hypoxic stress, by specific proteolytic degradation of MICU1, with potentially pathogenic consequences. To test these ideas, we will use a neuron-specific MICU1 knockout mouse which we found to display neurodegeneration characterized by mitochondrial Ca2+ overload, altered mitochondrial and neuronal ultrastructure, and motor and cognitive impairments. We have also generated mice with MICU2 and MICU3 loss and have obtained MICU1 and MICU2 patient-derived cells. We have set up advanced functional imaging and large volume 3D ultrastructure capacities. Thus, we are well positioned to study the neuronal pathogenesis associated with the loss of each or multiple MICU isoforms. In Aim#1 we will test the hypothesis that neurons derived from MICU1, MICU2 or MICU3-deficient mice and MICU1 and MICU2-deficient patient fibroblast-derived neurons have distinct impairments in Ca2+ signaling, mitochondrial dynamics and synaptic vesicle release. In Aim#2 we will test if neuronal MICU1/2/3 loss promotes mitochondrial oxidant production and oxidant-mediated cell injury. The results will help to decide if oxidants are relevant to MICU-linked neuronal/brain injury. In Aim#3 we will test the hypothesis that the relative abundance of the different MICU isoforms is dynamic in neurons during development, and the loss of each isoform specifically affects neuroanatomy and brain function primarily in the motor areas and the hippocampus. In Aim#4 we will test if ischemia-reperfusion induces YME1L-mediated turnover of MICU1 to promote mitochondrial Ca2+ overload injury and to sensitize the uniporter to inhibitors. These studies are expected to decide if MICU1 loss is a pathogenic component and therapy-modifying factor in brain hypoxia.