Targeting Mitochondrial Fission for Neuroprotection in Diabetic Neuropathy

项目摘要/摘要 以慢性疼痛或感觉丧失为表现的外周糖尿病神经病变(PDN)是一种令人衰弱的疾病 糖尿病的共病，影响至少一半的糖尿病患者。因为只有姑息治疗 ，迫切需要预防或逆转脑白质瘤“死亡性”退化的治疗方法 PDN中的外周轴突。最近的证据表明，糖尿病会损害线粒体结构和 感觉神经元的功能。然而，潜在的机制尚不清楚。 线粒体的形状由相反的分裂和融合事件控制。线粒体的突变 融合酶引起的神经系统疾病类似于糖尿病患者的神经系统并发症 病人。具体地说，丝裂素-2突变导致2A型夏科-玛丽-图思病，一种外周血型 以原发轴突变性为特征的神经病，而OPA1基因突变导致显性视神经 萎缩是遗传性失明最常见的形式。 线粒体裂变酶Dynamin-Related Protein 1(Drp1)通过去磷酸化激活 一个高度保守的抑制性PKA磷酸化位点。两种磷酸酶以此为靶点促进 线粒体分裂，钙依赖的磷酸酶钙调神经磷酸酶和神经元特异性的线粒体- 含有Bβ2调节亚基(PP2A/Bβ2)的蛋白磷酸酶2A的定位异构体。我们生成了 小鼠敲除Bβ2(KO)，在神经元中发现延长的线粒体，与a DRP1激活剂。Bβ2 KO可显著缩小缺血性卒中后的脑梗塞体积，表明 线粒体的延长具有神经保护作用。相反，敲除A激酶锚定蛋白1(AKAP1)， 一种蛋白质，它将PKA招募到线粒体膜外，以维持DRp1的磷酸化和 抑制状态，导致线粒体碎裂，加重中风损伤。 初步证据表明，Bβ2 KO小鼠在两种情况下都对周围神经病变具有抵抗力 对于1型和2型糖尿病模型，本提案寻求Bβ2(和其他， 到目前为止，尚未发现神经元特异性DRp1激活剂)作为治疗PDN的药物靶点。我们进一步 建议研究糖尿病是如何导致感觉神经元线粒体断裂的，以及如何抑制 线粒体碎裂可保护糖尿病患者的外周轴突。使用新的鼠标型号和创新的 活体成像方法，我们将测试最重要的假设，线粒体失调 分裂/融合平衡参与了糖尿病神经病变的发病机制，而抑制DRp1- 依赖的线粒体分裂通过改善线粒体代谢来提供神经保护， 降低ROS，调节线粒体钙转运，促进感觉神经轴突再生。 我们期望这些研究将阐明PDN的病因，提出新的治疗策略，从而 帮助改善快速增长的糖尿病人口的生活质量。

Project Summary / Abstract Presenting with chronic pain or loss of sensation, peripheral diabetic neuropathy (PDN) is a debilitating comorbidity of diabetes that affects at least half the diabetic patient population. Since only palliative treatments are available, there is an urgent need for therapies that prevent or reverse the “dying back” degeneration of peripheral axons in PDN. Recent evidence suggests that diabetes compromises mitochondrial structure and function in sensory neurons. However, the underlying mechanisms are unknown. Mitochondrial shape is controlled by opposing fission and fusion events. Mutations in mitochondrial fusion enzymes cause neurological disorders that present similarly to neurological complications in diabetic patients. Specifically, mitofusin-2 mutations result in Charcot-Marie-Tooth disease type 2A, a peripheral neuropathy characterized by primary axon degeneration, while mutations in Opa1 cause dominant optic atrophy, the most common form of hereditary blindness. The mitochondrial fission enzyme dynamin-related protein 1 (Drp1) is activated by dephosphorylation of a highly conserved inhibitory PKA phosphorylation site. Two phosphatases target this site to promote mitochondrial fission, the Ca2+-dependent phosphatase calcineurin and a neuron-specific and mitochondria- localized isoform of protein phosphatase 2A containing the Bβ2 regulatory subunit (PP2A/Bβ2). We generated a mouse knock-out (KO) of Bβ2 and found elongated mitochondria in neurons, consistent with deletion of a Drp1 activator. Bβ2 KO results in a striking reduction in infarct volume following ischemic stroke, indicating that mitochondrial elongation is neuroprotective. Conversely, knocking out A Kinase Anchoring Protein 1 (AKAP1), the protein that recruits PKA to the outer mitochondrial membrane to maintain Drp1 in a phosphorylated and inhibited state, causes mitochondrial fragmentation and exacerbates stroke injury. Supported by preliminary evidence that Bβ2 KO mice are resistant to peripheral neuropathy in both type-1 and type-2 diabetes models, the present proposal seeks proof-of-concept evidence for Bβ2 (and other, as yet undiscovered, neuron-specific Drp1 activators) as a drug target for the treatment of PDN. We further propose to investigate how diabetes causes mitochondrial fragmentation in sensory neurons and how inhibiting mitochondrial fragmentation protects peripheral axons in diabetes. Using new mouse models and innovative in vivo imaging approaches, we will test the overarching hypothesis that dysregulation of the mitochondrial fission/fusion equilibrium contributes to the pathogenesis of diabetic neuropathy, and that inhibition of Drp1- dependent mitochondrial fission provides neuroprotection via improvement of mitochondrial metabolism, reduction of ROS, modulation of mitochondrial Ca2+ transport and enhanced regeneration of sensory axons. We anticipate that these studies will shed light on PDN etiology, suggest new therapeutic strategies, and thus help improve quality of life for a rapidly growing diabetic population.