Dynamin-related protein 1 and mitochondrial fission adapters regulate presynaptic function

该项目的长期目标是改善由于线粒体功能障碍而导致的神经传递缺陷， 作为阻止疾病发展到较晚退化阶段的一种方式，增加人口的健康寿命 越来越容易患上与年龄相关的神经疾病。动力蛋白相关蛋白1(Drp1)促进 线粒体分裂，已被确定为限制线粒体异常的治疗靶点 阿尔茨海默氏症和亨廷顿氏病的碎片化。该项目的目标是确定Drp1如何 与线粒体分裂适配器的相互作用会影响突触前终末功能。我们建议，有必要 因为高水平的线粒体呼吸支持突触传递使突触前终末成为 高细胞应激环境。受调控的线粒体分裂对细胞存活非常重要 细胞应激源，通过Drp1起作用，但在神经元突触前终末利用的适配器是 未知。在特定的目标1中，我们将研究线粒体分裂适配蛋白Mff和MFf的丢失 FIS1影响线粒体动态平衡和突触传递。在具体目标2中，我们将检查不同的 Drp1基因缺失时线粒体功能和超微结构参数的变化及挽救功能的尝试 通过将Drp1重新表达定位于线粒体外膜。表型差异将与 在目标1中，生成受调控的线粒体分裂对突触功能的影响的完整图景。 Drp1也可能促进突触质膜的断裂，但这一额外功能的影响 关于突触传递的问题还没有解决。在特定的目标3中，我们将测试Drp1有助于 突触小泡的回收和再循环，并确定膜相关的Drp1是否足以 促进SV恢复，恢复突触传递。在合作中，PI和另外两个世界级的 研究人员已经开发出新的方法来解剖DRP1的异构体特定角色(S)，使用 将小鼠的花萼作为模型系统举行。使用病毒介导的转基因的组合，高级 电生理学，以及高分辨率光学和电子显微镜，特定的Drp1亚型 支持线粒体分裂与突触传递和突触前SV提取将是系统性的 测试过。与传统的小突触不同，巨大的“花盏状”兴奋性突触的实验可达性 突触允许从突触前终末进行记录，从而允许操纵突触前[ATP]和 实时跟踪膜外/内吞作用。这种方法对于剖析能量支持是必要的。 突触线粒体在突触小泡再循环机制中的作用。本项目的成果 可用于通知、预测和测试常规谷氨酸能突触的功能和功能障碍 与疾病相关的神经退行性变首先出现。从这个项目产生的知识将确定可行的 在Drp1功能改变的情况下，恢复突触功能的干预途径可以利用 在治疗上减轻疾病相关的突触功能障碍和神经退行性变。

The long-term goal of this project is to ameliorate neurotransmission defects due to mitochondrial dysfunction, as a way to stop disease progression to later degenerative stages, increasing healthspan in populations increasingly subject to age-related neurological diseases. Dynamin-related protein 1 (DRP1) acts to promote mitochondrial fission and has been identified as a therapeutic target for limiting aberrant mitochondrial fragmentation in Alzheimer’s and Huntington’s disease. The goal of this project is to determine how DRP1 interaction with mitochondrial fission adapters impacts presynaptic terminal function. We propose that the need for high levels of mitochondrial respiration to support synaptic transmission makes the presynaptic terminal a high cellular stress environment. Regulated mitochondrial fission is important for cell survival in response to cellular stressors, acting through DRP1, but the adapters utilized at the neuronal presynaptic terminal are unknown. In Specific Aim 1, we will examine how loss of the mitochondrial fission adapter proteins MFF and FIS1 affect mitochondria homeostasis and synaptic transmission. In Specific Aim 2, we will examine distinct parameters of mitochondrial function and ultrastructure when DRP1 is eliminated, and attempt to rescue function by targeting DRP1 re-expression to mitochondrial outer membrane. Phenotypic differences will be corelated with those in Aim 1, to generate a complete picture of the effect of regulated mitochondrial fission on synaptic function. DRP1 may also facilitate scission of plasma membrane at the synapse, but the impact of this additional function on synaptic transmission is unresolved. In Specific Aim 3, we will test the hypothesis that DRP1 facilitates synaptic vesicle retrieval and recycling, and determine whether membrane-associated DRP1 is sufficient to facilitate SV retrieval, and restore synaptic transmission. In collaboration, the PI and two other world-class investigators have developed novel approaches to allow dissection of the isoform-specific role(s) of DRP1, using the mouse calyx of Held as a model system. Using a combination of viral-mediated transgenesis, advanced electrophysiology, and high-resolution light and electron microscopy, the ability of specific DRP1 isoforms to support mitochondrial fission versus synaptic transmission and presynaptic SV retrieval will be systematically tested. In contrast to small conventional synapses, experimental accessibility of giant ‘calyx-like’ excitatory synapses allow recordings from the presynaptic terminal, permitting manipulation of presynaptic [ATP] and tracking membrane exo/endocytosis in real time. This approach is necessary to dissect the energy-supporting roles of synaptic mitochondria from mechanisms underlying synaptic vesicle recycling. Results from this project can be used to inform, predict, and test function and dysfunction at conventional glutamatergic synapses where disease-relevant neurodegeneration first appears. Knowledge generated from this project will identify viable routes of intervention for restoring function to synapses where DRP1 function is altered, which can be leveraged therapeutically to alleviate disease-related synaptic dysfunction and neurodegeneration.