Sexually dimorphic miR-497 regulates alpha-synuclein and alpha-synucleinopathy

描述（由申请人提供）：路易体痴呆（DLB）是一种毁灭性的神经退行性疾病。皮层路易体病理（Cortical Lewy body pathology， LBP）是DLB的病理底物，在大多数痴呆患者的尸检系列中，有10%-30%的大脑存在这种病理底物。在分子水平上，LBP由α -突触核蛋白（α - sn）蛋白的异常沉积组成，有证据表明，在某些DLB病例中，α - sn基因剂量增加。导致腰痛的生化途径尚不清楚，我们的总体目标是帮助填补这一知识空白。与女性相比，男性发生尸检证实的皮质性腰痛的风险明显增加（约三倍）；这是在许多尸检系列中重复的观察结果。我们的总体假设是，一种特定的microRNA （miRNA; miR-497）有助于两性二态DLB的发病机制。在初步研究中，我们观察到：miR-497在人脑和大鼠原代培养的神经元中以两性二态表达；miR-497在患有LBP的人脑和LBP易感脑区富集；培养细胞中miR-497的表达改变了α - sn和DJ-1/PARK7的表达。我们的实验室在LBP和mirna方面都有专业知识，这使我们能够验证miR- 497调节α - sn表达并可能以两性二态方式影响LBP的假设。特异性目的1：验证miR-497在人脑中的性别二态表达，并在患有LBP的人脑中失调的假设。拟议的研究将代表miRNA分析的最新技术，使用miRNA微阵列来测试miRNA表达与LBP和性别地位之间的关系。初步数据表明，在患有LBP的大脑和易感LBP的人类大脑区域中，有性二态的miR-497有失调的系统性趋势。特异性目的2：验证性别二态的miR-497调控α - sn表达的假设。初步数据表明miR-497调控α - sn。将用表达miR-497和其他对照mirna的慢病毒载体转导原代大鼠神经元，以验证miR-497调节α - sn的假设。在这些细胞中的进一步实验将探究miR- 497对α - sn的调控是直接的还是通过对PARK7/DJ-1基因的中间调控。在用miR-497和对照mirna进行病毒转导后，我们将确定培养的原代神经元中miR-497靶点的完整列表。这些机械实验将有助于我们的最终目标，治疗这种目前无法治愈的疾病。公共卫生意义：了解性别二态性miR-497如何改变α - sn表达以诱导性别二态性病理，将是神经化学领域一个重要的新范式，对诊断和治疗策略具有临床/转化意义。原代细胞培养系统将为测试降低驱动α - sn表达的miR-497二态表达的新方法提供实验范例。

DESCRIPTION (provided by applicant): Dementia with Lewy bodies (DLB) is a devastating neurodegenerative disease. Cortical Lewy body pathology (LBP), which is the pathological substrate of DLB, is observed in 10%-30% of brains in most autopsy series of dementia patients. At the molecular level, LBP consists of aberrant deposits of alpha-synuclein (alpha-SN) protein and there is evidence that increased alpha-SN gene dosage is seen in some cases of DLB. The biochemical pathways that contribute to LBP are poorly understood and our overall goal is to help fill this knowledge gap. Men have strongly (~three-fold) increased risk for developing autopsy-confirmed cortical LBP compared to women; this is an observation replicated in many autopsy series. Our overarching hypothesis is that a particular microRNA (miRNA; miR-497) contributes to sexually-dimorphic DLB pathogenesis. In preliminary studies we made the following observations: miR-497 is expressed sexually dimorphically in human brain and in rat primary cultured neurons; miR-497 is enriched in human brains with LBP and in LBP- vulnerable brain areas; and, miR-497 expression in cultured cells alters alpha-SN and DJ-1/PARK7 expression. Our lab has expertise in both LBP and miRNAs which enable us to test the hypothesis that miR- 497 regulates alpha-SN expression and may influence LBP in a sexually dimorphic manner. Specific Aim 1: Test the hypothesis that miR-497 is sexually dimorphically expressed in human brain, and dysregulated in human brains with LBP. Proposed studies will represent the state of the art in miRNA profiling using miRNA microarrays to test for associations between miRNA expression and both LBP and gender status. Preliminary data indicate a systematic tendency for sexually dimorphic miR-497 to be dysregulated in brains with LBP and in human brain areas vulnerable to LBP. Specific Aim 2: Test the hypothesis that sexually dimorphic miR-497 regulates alpha-SN expression. Preliminary data indicate that miR-497 regulates alpha-SN. Primary rat neurons will be transduced with lentiviral vectors expressing miR-497 versus other control miRNAs to test the hypothesis that miR-497 regulates alpha-SN. Further experiments in these cells will query whether the regulation of alpha-SN by miR- 497 is direct or via an intermediate regulation of PARK7/DJ-1 gene. Following viral transduction with miR-497 and control miRNAs, we will determine the full list of miR-497 targets in cultured primary neurons. These mechanistic experiments will aid our ultimate goal of therapies to combat this currently untreatable disease. Public health significance: Understanding how sexually dimorphic miR-497 alters alpha-SN expression to induce sex-dimorphic pathology would be a major novel paradigm in neurochemistry with clinical/translational significance for both diagnostic and therapeutic strategies. The primary cel culture system will offer an experimental paradigm for testing novel methods of attenuating the dimorphic expression of miR-497 that drives alpha-SN expression.