DNA repair and alpha-synucleinopathy in Lewy body disorders

基因组是固有防御的最重要的决定因素，其完整性的丧失可能导致病理学的最终结果。 条件。细胞DNA修复系统已经进化来减轻这种风险，但在神经学方面知之甚少。 精神错乱。我们建议检查dna修复的变化是否改善了以边缘为主的路易体障碍的行为和组织学结果，这是一种神经疾病，在这种情况下，不可溶的α-突触核蛋白沉积填充了 与嗅觉、情感和记忆有关的大脑。氧化的、受损的DNA的修复通过以下方式促进 脱嘌呤/脱嘧啶核酸内切酶I(APE1)。多效性ape1基因既具有DNA修复功能，又具有氧化还原功能。 功能--前者在帕金森氏症患者的脑组织中受损。已知氧化的α-突触核蛋白进入 Ape1和α-突触核病之间的关系到目前为止还没有被仔细研究过。我们已经收集了试点数据来支持中心假设，即在边缘为主的路易体疾病的细胞和动物模型中，APE1缓和了暴露于预先形成的α-突触核蛋白纤维的病理后遗症。嗅球/前嗅核(OB/AON)复合体是路易体疾病中最早显示包涵体的脑区之一。我们的试验数据显示，在OB/AON中注入纤维后，雄性小鼠的反应是嗅觉丧失、边缘致密包涵体，并在6个月内APE1表达下降。相比之下，女性的反应是APE1在全球范围内增加，不会出现嗅觉缺陷，边缘内含物也较少。这些性别差异与男性患路易体疾病的风险是女性的2至3倍平行。我们最近的人类尸检工作类似地揭示了性别改变边缘路易体疾病对边缘脑区APE1表达的影响。新的实验数据还表明，shRNA介导的APE1基因敲除增加了初级神经元中的包涵体。在这里，我们建议进行进一步的机制研究，以检验ape1和α-突触核病之间的因果关系。 目的1.我们将验证慢病毒介导的APE1在原代神经元/星形胶质细胞和原代细胞中被击倒的假设 经α-突触核蛋白纤维处理的神经元将导致APE1表达和核酸内切酶活性的丧失，更大的DNA损伤，更多的蛋白质聚集和细胞丢失。我们将使用两个shRNA序列来解决这一假设，并确定过表达人WT APE1是否可以防止击倒表型，以确保shRNA特异性。 在AIM 2中，我们将检验APE1减轻α-突触核蛋白原纤维的行为和组织学影响的假设 雄性和雌性OB/AON大鼠输液。我们将比较人类野生型APE1过表达大鼠和转基因大鼠 过表达突变APE1并伴有DNA修复受损(D210A突变)或氧化还原能力丧失(C65A)的大鼠 突变)。认知、焦虑、嗅觉和运动行为将按月进行评估。在3个月、6个月和12个月时将测量APE1的表达和活性、DNA损伤标记物、蛋白病理性包涵体和细胞计数。 如果得到资助，这些研究将是第一次检查α-突触核病症和dna修复能力之间的关系，并表征边缘路易氏病变的性别差异。这些实验旨在支持或反对这样的观点，即dna修复机制应该被作为阻止α-突发性核素性疾病的合理目标。

The genome is the foremost determinant of inherent defenses, and loss of its integrity can culminate in pathological conditions. Cellular DNA repair systems have evolved to mitigate this risk, but are poorly understood in neurological disorders. We propose to examine if changes in DNA repair modify behavioral and histological outcomes in limbic-predominant Lewy body disorder, a neurological condition in which insoluble deposits of α-synuclein protein fill areas of the brain involved in olfaction, emotion, and memory. The repair of oxidized, damaged DNA is facilitated by apurinic/apyrimidinic endonuclease I (APE1). The pleiotropic APE1 gene has both a DNA repair function and a redox function—the former is impaired in brain tissue from Parkinson’s patients. It is known that oxidized α-synuclein enters the nucleus and elicits breaks in DNA, but the relationship between APE1 and α-synucleinopathy has not, until now, been scrutinized. We have collected pilot data to support the central hypothesis that APE1 tempers the pathological sequelae of exposure to preformed α-synuclein fibrils in cellular and animal models of limbic-predominant Lewy body disorders. The olfactory bulb/anterior olfactory nucleus (OB/AON) complex is one of the earliest brain regions to display inclusions in Lewy body disorders. Our pilot data reveal that male mice respond to fibril infusions in the OB/AON with loss of smell, dense limbic inclusions, and a decrease in APE1 expression within six months. In contrast, females respond with a global increase in APE1, do not develop smell deficits, and display fewer limbic inclusions. These sex differences parallel the 2 to 3-fold higher risk of Lewy body disorders in men compared to women. Our recent human postmortem work similarly reveals that gender modifies the impact of limbic Lewy body disease on APE1 expression in limbic brain regions. New pilot data also show that shRNA-mediated knockdown of APE1 increases inclusions in primary neurons. Here, we propose further mechanistic studies that will put the causality of the link between APE1 and α-synucleinopathy to the test. AIM 1. We will test the hypothesis that lentivirus-mediated APE1 knockdown in primary neuron/astrocyte and primary neuron cultures treated with α-synuclein fibrils will elicit loss of APE1 expression and endonuclease activity, greater DNA damage, and more protein aggregations and cell loss. We will employ two shRNA sequences to address this hypothesis and determine if overexpressing human WT APE1 prevents the knockdown phenotype, to ensure shRNA specificity. In AIM 2, we will test the hypothesis that APE1 mitigates the behavioral and histological impact of α-synuclein fibril infusions in the male and female rat OB/AON. We will contrast human wildtype APE1-overexpressing rats to transgenic rats that overexpress mutant APE1 with impaired DNA repair (D210A mutation) or loss of redox capacity (C65A mutation). Cognitive, anxiety, olfactory, and motor behavior will be assessed at monthly intervals. APE1 expression and activity, DNA damage markers, proteinopathic inclusions, and cell counts will be measured at 3, 6, and 12 months. If funded, these studies will be the first to examine the relationship between α-synucleinopathy and DNA repair capacity and to characterize sex differences in limbic Lewy pathology. The experiments are designed to either support or reject the notion that DNA reparatory mechanisms should be pursued as a rational target for stymieing α-synucleinopathic disease.