Role of poly(ADP-ribosyl)ation in diabetic neuropathy

描述(申请人提供)：高血糖引起的氧化应激影响多种信号转导和代谢途径。其中之一是羟基和超氧阴离子自由基以及过氧亚硝酸根刺激的DNA单链断裂引起的聚腺苷二磷酸核糖基化。这一设想的总体假设是，内皮细胞和雪旺细胞多(ADP-核糖)合成酶(PARS)的激活与NAD耗竭、能量衰竭和潜在的氧化应激加剧有关，导致神经血管功能障碍、神经传导障碍、神经营养支持受损和周围糖尿病神经病变(PDN)的形态变化。其理论基础是：1)氧化应激在PDN的神经血流量(NBF)、传导和神经生长因子(NGF)缺陷中起关键作用；2)聚(ADP-核糖基)在与氧化应激相关的许多病理(如心血管和神经退行性疾病、炎症)中的自由基损伤中起关键作用；3)内皮细胞和雪旺细胞中存在PARs；4)短期(内皮细胞)和长期(雪旺细胞)暴露于高血糖后，DNA单链断裂；5)短期糖尿病患者内皮细胞中PARS激活的证据，以及最近证明其在糖尿病引起的内皮功能障碍中的关键作用，以及6)申请人的初步数据，证明了两种特殊的结构不同的PARS抑制剂3-氨基苯甲酰胺(3-AB)和1，5-异喹啉二醇(ISO)纠正了糖尿病引起的运动和感觉神经传导速度(MNCV和SNCV)和能量衰竭。实验方法将包括在体内评估PARS抑制剂对早期糖尿病肾病的NBF、传导、生物能量学、氧化应激和神经生长因子(NGF)丰度的影响，以及在这些措施的基础上加上对晚期PDN大鼠模型的神经形态测量，比较PARS缺陷(+/-或-/-)小鼠和野生型PDN，以及PARS在高血糖诱导血管神经细胞和内皮细胞凋亡中的作用。其具体目的是1)评估3-AB、ISO和PJ34(一种新的有效的PARS抑制剂)对早期糖尿病肾病(6周糖尿病大鼠)NBF、MNCV、SNCV、生物能量学、氧化应激和NGF水平的影响；2)评估PJ34对上述指标和晚期糖尿病大鼠(8周糖尿病大鼠)的形态计量学指标的影响；3)比较PARS缺陷(+/-或-/-小鼠)和野生型PDN的作用；以及4)评估3-AB、ISO和PJ34对高血糖诱导血管神经细胞和内皮细胞凋亡的作用。这些研究与PDN的发病机制直接相关，并可能为开发PARS抑制剂预防和治疗PDN提供理论依据。

DESCRIPTION (provided by applicant):Hyperglycemia-induced oxidative stress affects multiple signal transduction and metabolic pathways. One of them is poly(ADP-ribosyl)ation induced by hydroxyl and superoxide radicals and peroxynitrite- stimulated DNA single-strand breakage. The OVERALL HYPOTHESIS of this proposal is that activation of endothelial and Schwann cell poly(ADP-ribose)synthetase (PARS), associated with NAD depletion, energy failure and, potentially, exacerbation of oxidative stress, contributes to neurovascular dysfunction, nerve conduction deficits, impaired neurotrophic support and morphological changes of peripheral diabetic neuropathy (PDN). The RATIONALE is based on the: 1) key role of oxidative stress in nerve blood flow (NBF), conduction and nerve growth factor (NGF) deficits of PDN; 2) key role of poly(ADP-ribosyl)ation in free radical damage in numerous pathologies (e.g. cardiovascular and neurodegenerative diseases, inflammation) associated with oxidative stress; 3) presence of PARS in endothelial and Schwann cells; 4)presence of DNA single-strand breaks after a short (endothelial cells) and prolonged (Schwann cells) exposure to hyperglycemia; 5) evidence of PARS activation in endothelial cells in short-term diabetes and recent demonstration of its key role in diabetes-induced endothelial dysfunction, and 6) applicant's preliminary data demonstrating correction of diabetes-induced motor and sensory nerve conduction velocities (MNCV and SNCV) and energy failure by two specific structurally diverse PARS inhibitors, 3-aminiobenzamide (3-AB) and 1,5-isoquinolinediol (ISO). The EXPERIMENTAL APPROACH will include in vivo evaluation of PARS inhibitors on NBF, conduction, bioenergetics, oxidative stress and nerve growth factor (NGF) abundance in early DN, and on these measures plus nerve morphometry in advanced PDN in the rat model, comparison of PDN in PARS-deficient (+/- or-/-) mice and the wild-type, and the studies of the role of PARS in hyperglycemia-induced apoptosis in vasa nervorum and endothelial cells. The SPECIFIC AIMS are 1) evaluate 3-AB, ISO and PJ34 (a novel potent PARS inhibitor) on NBF, MNCV, SNCV, bioenergetics, oxidative stress and NGF levels in early DN (rats with 6-wk diabetes); 2) evaluate PJ34 on these and morphometric indices in advanced DN (rats with 8-mo diabetes); 3) compare PDN in PARS-deficient (+/- or -/- mice) and the wild-type; and 4) evaluate 3-AB, ISO and PJ34 on hyperglycemia-induced apoptosis in vasa nervorum and endothelial cells. The studies are of direct relevance to the pathogenesis of PDN and may provide the rationale for development of PARS inhibitors for its prevention and treatment.