DIABETIC ENTERIC NEUROPATHY

超出所提供的空间。糖尿病性神经病变的病理生理机制知之甚少。我们先前报道了2型糖尿病伴神经病变患者的血清通过自身抗体介导的途径诱导培养的人神经母细胞瘤细胞的程序性细胞死亡（PCD），该途径涉及半胱天冬酶依赖性凋亡的激活。最近的研究表明，caspase独立途径也可以促进PCD。这一过程可能涉及自噬的激活，自噬是一种响应细胞应激而将蛋白质和细胞器隔离在自噬体中的途径。我们提出了一个新的假说，即2型糖尿病患者血清中存在的自身抗体通过Fas依赖性途径依次激活神经元中的自噬、半胱天冬酶依赖性和非依赖性PCD。Fas（CD 95）是细胞膜结合死亡受体家族的成员。我们的初步数据支持激动剂自身抗体结合并激活Fas受体的假设。我们将监测自身抗体的发展，并将其存在与2型糖尿病患者早期和晚期的自主神经和周围神经病变的既定标志物相关联。我们将剖析诱导自噬和PCD的途径。我们认为，自噬是一种早期的细胞保护反应，以消除受损的线粒体，顺序进展到caspase依赖性和非依赖性的PCD与ATP水平的降低。我们假设自噬的自身抗体诱导将涉及自噬体的特异性标志物LC 3 n的掺入和PI-3激酶（III类）的激活。将培养的SH-SY 5 Y（人神经母细胞瘤细胞）暴露于从以下获得的补体灭活血清：1. 2型糖尿病患者有记录的糖尿病神经病变，2。年龄和性别匹配的2型糖尿病患者无神经病变的证据和3.健康、年龄和性别匹配的对照组。将进行平行动物研究，检查从雌性Zucker糖尿病肥胖大鼠（2型糖尿病的诱导模型）和链脲佐菌素诱导的糖尿病大鼠（糖尿病神经病变的充分验证模型）获得的原代神经元（结状神经节、背根神经节和肌间神经丛）中是否原位激活自噬。我们还将评估与来自无神经病变的糖尿病大鼠和瘦的非糖尿病对照的血清相比，来自具有神经病变的糖尿病大鼠的血清是否在培养的大鼠结状神经节、DRG和肌间神经元中诱导自噬和半胱天冬酶依赖性和/或非依赖性PCD，并检查这些事件的时间过程。我们推测，线粒体功能障碍和ATP水平下降将发挥关键作用，在连续激活半胱天冬酶依赖和非依赖PCD。与公共卫生的相关性：这些研究将为糖尿病神经病变的病理生理学中自身免疫、自噬和程序性细胞死亡的机制基础提供新的见解。.性能现场=

EXCEED THE SPACE PROVIDED. 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. . PERFORMANCE SITE ========================================Section End===========================================