Disruption of Excitable Axonal Domains by Glucose Metabolite Methylglyoxal

项目摘要/摘要 有髓轴突兴奋域的改变，特别是轴突起始段(AIS)和 兰维尔结节是包括糖尿病在内的各种神经退行性疾病的关键病理生理学机制。 AIS长度的缩短已被证明降低了神经元的兴奋性，并与认知能力有关 2型糖尿病和阿尔茨海默病的损害。然而，细胞和分子机制的研究进展 这些结构域在疾病条件下是如何改变的，人们仍然知之甚少。这一关键的知识鸿沟 限制了场操纵AIS和节点进行治疗的能力。目前的建议旨在澄清 神经系统病理生理学的这一重要方面。此应用程序的总体目标是识别 AIS和结节破坏过程中的关键分子环节。以前的研究和初步数据 现已确定葡萄糖的高活性副产物甲基乙二醛(MG)的含量升高 新陈代谢，作为AIS和结节破坏的潜在中介。这些数据也支持钙痛， 细胞内依赖钙的半胱氨酸蛋白酶参与了这一过程。中心假设是 乙二醛通过激活钙蛋白来破坏AIS和结蛋白复合体并抑制神经系统 功能。我们将通过三个具体目标来检验这一假设。目的1：检验MG减少的假说 具有新型清除肽的水平将改善db/db小鼠的AIS缩短和认知障碍， 一种已建立的2型糖尿病模型。目的2：检验MG升高导致AIS/Node的假说 改变，降低神经网络活性(Aim 2A，体外；小鼠皮质神经元培养和多电极 阵列)和认知损害(目标2B，活体内；全身给药MG或乙二醛酶抑制剂1， 一种对野生型小鼠的MG解毒的酶)。目的3：验证钙调神经痛调节效应的假设 MG对AIS/节点结构、神经网络活性的影响(Aim 3A，体外；药理Calain抑制)，以及 认知功能(AIM 3B，C，体内；钙调蛋白的基因操作，一种特定的内源性抑制物 肌腱痛)。AIM 3B将评估钙蛋白酶过度激活和MG增加的联合影响 基因敲除小鼠；Aim 3C将评估过度表达小鼠的MG和Calain抑制增加 卡巴斯丁。这一应用在概念上是创新的，因为我们提出提升MG的关键目标是 活体神经元内AIS和Ranvier结节的结构。多电极阵列的创新使用将 确定MG和AIS缩短增加对神经网络功能的影响。拟议的研究 意义重大，因为完成AIMS将使MG和CalPain成为翻译的潜在目标 旨在治疗认知障碍的研究--例如在AIM 1中测试的新型MG清道夫 2型糖尿病的损害。这些结果也有可能影响多种神经退行性变 例如阿尔茨海默氏症等疾病，从而最终对该领域产生持续和强大的影响。

Project summary/abstract Alterations in the excitable domains of myelinated axons, specifically the axon initial segment (AIS) and the nodes of Ranvier, are key pathophysiologies in various neurodegenerative conditions, including diabetes. Shortening of AIS length has been shown to lower neuronal excitability, and is also implicated in cognitive impairment in type 2 diabetes and Alzheimer’s disease. However, the cellular and molecular mechanisms of how these domains are altered in disease conditions remain poorly understood. This critical gap in knowledge limits the field’s ability to manipulate the AIS and nodes for treatment. The current proposal seeks to elucidate this important aspect of nervous system pathophysiology. The overall objective of this application is to identify a critical molecular link in the process of AIS and nodal disruption. The prior studies and preliminary data provided here have identified elevations in methylglyoxal (MG), a highly reactive byproduct of glucose metabolism, as a potential mediator for AIS and nodal disruption. These data also support that calpains, calcium-dependent intracellular cysteine proteases, are involved in this process. The central hypothesis is that methylglyoxal disrupts AIS and nodal protein complexes via calpain activation and inhibits nervous system function. We will test this hypothesis via three Specific Aims. Aim 1: Test the hypothesis that reduction of MG levels with novel scavenging peptides will ameliorate AIS shortening and cognitive impairment in db/db mice, an established model for type 2 diabetes. Aim 2: Test the hypothesis that elevated MG causes AIS/node changes, reduced neural network activity (Aim 2A, in vitro; mouse cortical neuron culture and multi-electrode arrays), and cognitive impairment (Aim 2B, in vivo; systemic administration of MG or inhibitor of glyoxalase 1, an enzyme that detoxifies MG, in wild-type mice). Aim 3: Test the hypothesis that calpains mediate the effects of MG on AIS/node structures, neural network activity (Aim 3A, in vitro; pharmacological calpain inhibition), and cognitive function (Aim 3B, C, in vivo; genetic manipulation of calpastatin, a specific endogenous inhibitor of calpains). Aim 3B will assess combined effects of increased MG and calpain over-activation in calpastatin knockout mice; and Aim 3C will assess increased MG and calpain inhibition in mice over-expressing calpastatin. This application is conceptually innovative, as we propose that the key targets of elevated MG are the structures of the AIS and nodes of Ranvier in live neurons. Innovative use of multi-electrode arrays will determine the effects of increased MG and AIS shortening on neural network function. The proposed research is significant, because completion of the aims will validate MG and calpains as potential targets for translational research aimed at treatments – such as the novel MG scavengers tested in Aim 1 – for comorbid cognitive impairment in type 2 diabetes. These results also have potential to impact a wide variety of neurodegenerative conditions, such as Alzheimer’s, thus ultimately providing a sustained and powerful influence on the field.