Targeting Mitochondrial Fission for Neuroprotection in Diabetic Neuropathy

项目总结/摘要 糖尿病周围神经病变（PDN）表现为慢性疼痛或感觉丧失， 糖尿病合并症影响至少一半的糖尿病患者人群。因为只有姑息治疗 尽管这些药物是可用的，但迫切需要预防或逆转“垂死”退化的疗法。 PDN中的外周轴突。最近的证据表明，糖尿病损害线粒体结构， 在感觉神经元中起作用。然而，其潜在机制尚不清楚。 线粒体的形状是由相反的裂变和融合事件控制的。线粒体突变 融合酶引起神经系统疾病，其表现与糖尿病患者的神经系统并发症相似， 患者具体来说，线粒体融合蛋白-2突变导致腓骨肌萎缩症2A型，即外周型腓骨肌萎缩症。 以原发性轴突变性为特征的神经病变，而Opa 1突变导致显性视神经病变 萎缩症是遗传性失明的最常见形式。 线粒体分裂酶动力蛋白相关蛋白1（Drp 1）通过以下物质的去磷酸化而被激活： 高度保守的抑制性PKA磷酸化位点。两种磷酸酶靶向该位点以促进 线粒体分裂，Ca 2+依赖性磷酸酶钙调神经磷酸酶和神经元特异性和线粒体- 含有Bβ2调节亚基（PP 2 A/Bβ2）的蛋白磷酸酶2A的局部亚型。我们产生 小鼠敲除（KO）Bβ2，发现神经元中线粒体延长，与缺失a Drp 1激活剂。Bβ2 KO导致缺血性卒中后梗死体积显著减少，表明 线粒体延长具有神经保护作用。相反，敲除A激酶促发蛋白1（AKAP 1）， 这种蛋白质将PKA募集到线粒体外膜，以维持Drp 1磷酸化， 抑制状态，导致线粒体断裂并加剧中风损伤。 初步证据支持Bβ2 KO小鼠对周围神经病变具有抗性， 1型和2型糖尿病模型，本发明的建议寻求Bβ2（和其它， 尚未发现的神经元特异性Drp 1激活剂）作为治疗PDN的药物靶标。我们进一步 我建议研究糖尿病如何导致感觉神经元的线粒体断裂，以及如何抑制 线粒体断裂保护糖尿病中的外周轴突。使用新的小鼠模型， 体内成像方法，我们将测试总体假设，即线粒体的失调， 裂变/融合平衡有助于糖尿病神经病变的发病机制，抑制Drp 1- 依赖性线粒体分裂通过改善线粒体代谢提供神经保护， 减少ROS，调节线粒体Ca 2+转运和增强感觉轴突的再生。 我们预计这些研究将阐明PDN病因，提出新的治疗策略，从而 有助于提高快速增长的糖尿病人群的生活质量。

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.