Autoimmune Mechanisms of Diabetic Neuropathy

糖尿病神经病变的病理生理学机制知之甚少。我们之前报道过，从 2型糖尿病合并神经病变患者体外培养诱导的程序性细胞死亡 人神经母细胞瘤细胞通过自身抗体介导的途径激活caspase- 依赖性细胞凋亡。最近的研究支持，caspase不依赖的途径也可以促进 PCD。这一过程可能涉及自噬的激活，自噬是一种隔离蛋白质和细胞器的途径 在自噬体内对细胞压力做出反应。我们提出了一种新的假设，即自身抗体 存在于伴有神经病变的2型糖尿病患者的血清中，从而激活自噬，caspase- 神经元中的依赖和非依赖的PCD通过Fas依赖的途径。Fas(CD95)是 细胞膜结合死亡受体家族。我们的初步数据支持激动剂 自身抗体结合并激活Fas受体。我们将监测自身抗体的发展。 并将它们的存在与早期自主神经和周围神经病变的既定标记物相关联 以及后来的2型糖尿病患者队列。我们将解剖诱导的途径(S) 自噬和PCD。我们认为自噬是清除损伤的早期细胞保护反应。 线粒体依次进展为caspase依赖和非依赖的PCD，并减少 三磷酸腺苷水平。我们推测，自体抗体诱导的自噬将涉及掺入LC3n，a 自噬小体的特异性标志物，以及PI-3激酶的激活(III类)。培养的SH-SY5Y(人 神经母细胞瘤细胞)将暴露于来自以下来源的补体灭活血清：1.2型糖尿病 记录有糖尿病神经病变的患者，2.年龄和性别匹配的2型糖尿病患者 神经病变的证据和3.健康、年龄和性别匹配的对照组。平行的动物研究将是 检查初级神经元(结状神经节、背根)是否原位激活自噬 神经节和肌间神经丛)取自雌性Zucker糖尿病肥胖大鼠，是一种诱导型 2糖尿病和链脲佐菌素诱导的糖尿病大鼠，这是一个经过验证的糖尿病神经病变模型。我们会 同时评估有神经病变的糖尿病大鼠血清是否诱导自噬和caspase依赖。 培养的大鼠结状神经节、背根神经节和肌间神经元的和/或非依赖性PCD与血清的比较 从没有神经病变的糖尿病大鼠和瘦的、非糖尿病的对照组中，并检查这些的时间进程 事件。我们假设线粒体功能障碍和三磷酸腺苷水平降低将发挥关键作用。 在caspase依赖和非依赖PCD的顺序激活中。与公共卫生的相关性：这些 研究将导致对自身免疫机制基础的新见解，自噬和 糖尿病神经病变病理生理学中的程序性细胞死亡。 。

The pathophysiology of diabetic neuropathy is poorly understood. We reported previously that sera from patients with type 2 diabetes mellitus with neuropathy induced programmed cell death (PCD) in cultured human neuroblastoma cells via an autoantibody-mediated pathway involving activation of caspase- dependent apoptosis. Recent studies support that a caspase-independent pathway can also contribute to PCD. This process may involve activation of autophagy, a pathway that sequesters proteins and organelles in autophagosomes in response to cellular stress. We propose the novel hypothesis that autoantibodies present in the sera of type 2 diabetic patients with neuropathy sequentially activate autophagy, caspase- dependent and -independent PCD in neurons via a Fas-dependent pathway. Fas (CD95) is a member of the cell membrane-bound death receptor family. Our preliminary data support the hypothesis that agonist autoantibodies bind and activate the Fas receptor. We will monitor the development of autoantibody(-ies) and correlate their presence with established markers of autonomic and peripheral neuropathy in an early and later cohort of patients with type 2 diabetes mellitus. We will dissect the pathway(s) that induce autophagy and PCD. We propose that autophagy is an early cytoprotective response to remove injured mitochondria that progresses sequentially to caspase-dependent and -independent PCD with decrease in ATP levels. We hypothesize that autoantibody-induction of autophagy will involve incorporation of LC3n, a specific marker for autophagosomes, and activation of PI-3 kinase (class III). Cultured SH-SY5Y (human neuroblastoma cells) will be exposed to complement-inactivated sera obtained from: 1. type 2 diabetic patients with documented diabetic neuropathy, 2. Age- and gender- matched type 2 diabetic patients without evidence of neuropathy and 3. Healthy, age- and gender-matched controls. Parallel animal studies will be performed examining whether autophagy is activated in situ in primary neurons (nodose ganglia, dorsal root ganglia and myenteric plexus) obtained from the female Zucker Diabetic Fatty rat, an inducible model of type 2 diabetes and the streptozotocin-induced diabetic rat, a well validated model of diabetic neuropathy. We will also assess whether sera from diabetic rats with neuropathy induce autophagy and caspase-dependent and/or -independent PCD in cultured rat nodose ganglia, DRG and myenteric neurons compared to sera from diabetic rats without neuropathy and lean, non-diabetic controls, and examine the time-course for these events. We hypothesize that mitochondrial dysfunction and decreased levels of ATP will play a pivotal role in sequential activation of caspase-dependent and -independent PCD. Relevance to public health: These studies will lead to novel insights regarding the mechanistic basis of autoimmunity, autophagy and programmed cell death in the pathophysiology of diabetic neuropathy. .