Determination of the clinical relevance of Parkinson disease-associated intronic enhancer of the alpha-synuclein gene, in a novel mouse deletion model

在新型小鼠缺失模型中确定帕金森病相关的α-突触核蛋白基因内含子增强子的临床相关性

• 批准号: 10665271
• 负责人: Hanseok Ko
• 金额: $ 24.56万
• 依托单位: JOHNS HOPKINS UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2023
• 资助国家: 美国
• 起止时间: 2023-04-15 至 2025-03-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10665271
• 关键词: Age; Age Months; Automobile Driving; Behavioral; Biological; Biological Assay; Cells; Chromatin; Cis-Acting Sequence; Clinical; Data; Disease; Disease Progression; Engineering; Enhancers; Equilibrium; Exhibits; Exposure to; Fluorescent in Situ Hybridization; Functional disorder; Gene Amplification; Genes; Genetic; Genetic Transcription; Genetic study; Genotype; Heterozygote; Histologic; Homozygote; Human; Immunohistochemistry; Inflammation; Injections; Introns; Knockout Mice; Lewy Bodies; Midbrain structure; Modeling; Motor; Mus; Mutate; Mutation; Nerve Degeneration; Neurons; Onset of illness; Orthologous Gene; Oxidopamine; Parkinson Disease; Parkinsonian Disorders; Pathologic; Pathology; Phenotype; Population; RNA Splicing; Reporter; Risk; Risk Factors; Risk Reduction; Role; SNCA gene; Severities; Severity of illness; Smell Perception; Symptoms; Techniques; Testing; Tetanus Helper Peptide; Tetracyclines; Titrations; Transcript; Transcriptional Regulation; Transgenic Mice; Untranslated RNA; Variant; Viral Vector; Work; alpha synuclein; behavioral phenotyping; clinically relevant; disease phenotype; disorder risk; dopaminergic neuron; glial activation; molecular phenotype; mouse model; mutant; neuron loss; neurotoxic; novel; novel therapeutics; olfactory bulb; phenotypic data; pre-formed fibril; promoter; protein misfolding; risk variant; single molecule; sporadic Parkinson' s Disease; therapeutic development; Age; Age Months; Automobile Driving; Behavioral; Biological; Biological Assay; Cells; Chromatin; Cis-Acting Sequence; Clinical; Data; Disease; Disease Progression; Engineering; Enhancers; Equilibrium; Exhibits; Exposure to; Fluorescent in Situ Hybridization; Functional disorder; Gene Amplification; Genes; Genetic; Genetic Transcription; Genetic study; Genotype; Heterozygote; Histologic; Homozygote; Human; Immunohistochemistry; Inflammation; Injections; Introns; Knockout Mice; Lewy Bodies; Midbrain structure; Modeling; Motor; Mus; Mutate; Mutation; Nerve Degeneration; Neurons; Onset of illness; Orthologous Gene; Oxidopamine; Parkinson Disease; Parkinsonian Disorders; Pathologic; Pathology; Phenotype; Population; RNA Splicing; Reporter; Risk; Risk Factors; Risk Reduction; Role; SNCA gene; Severities; Severity of illness; Smell Perception; Symptoms; Techniques; Testing; Tetanus Helper Peptide; Tetracyclines; Titrations; Transcript; Transcriptional Regulation; Transgenic Mice; Untranslated RNA; Variant; Viral Vector; Work; alpha synuclein; behavioral phenotyping; clinically relevant; disease phenotype; disorder risk; dopaminergic neuron; glial activation; molecular phenotype; mouse model; mutant; neuron loss; neurotoxic; novel; novel therapeutics; olfactory bulb; phenotypic data; pre-formed fibril; promoter; protein misfolding; risk variant; single molecule; sporadic Parkinson' s Disease; therapeutic development

We will evaluate the biological necessity of an intronic enhancer of SNCA and determine its relevance to Parkinson disease (PD). SNCA is frequently mutated in familial PD; it encodes alpha-synuclein (α-syn), the primary constituent of Lewy bodies (LB). LB formation/accumulation is a pathological hallmark of PD and is driven by protein misfolding promoted by SNCA structural mutation, gene amplification, or genetic/environmental insults that elevate α-syn levels. Noncoding variants predicted to impact SNCA transcriptional regulatory control are also risk factors for sporadic PD. We recently identified PD-associated variants in SNCA intron 4. These variants lie within a dopaminergic (DA) neuron open chromatin region (OCR) that we have shown interacts with the SNCA promoter and is a cis-regulatory enhancer in catecholaminergic neurons. We predict that this sequence, and the variants therein, impact SNCA transcriptional control and modulate PD risk. Hypothesis - Deletion of the Snca enhancer in mice will reduce Snca transcription in PD-relevant cells with potential impact on their viability/distribution, and on motor/non-motor PD phenotypes (Aim 1); deletion will reduce DA neuron vulnerability to PD-relevant insults and ameliorate the onset and severity of disease (Aim2). Several highly effective strategies exist to elicit Parkinsonian pathology in mice. Tetracycline (tet)-dependent expression of PD mutant and wild-type (WT) forms of SNCA in DA neurons result in marked and progressive loss of ventral midbrain DA neuron populations, consistent with PD pathology. Likewise, intrastriatal injection of α-syn pre-formed fibrils (PFF) also result in a mouse model that exhibits progressive DA neuronal loss. Consistent with the known role of Snca levels impacting PD risk and progression, α-syn deficient mice are protected from PFF-induced neurodegeneration and from tet-dependent expression (via viral vector) of the PD missense mutant (hA53T) or WT SNCA. α-syn null mice are also protected from the neurotoxic effects of other PD promoting (and Snca-elevating) insults, like MPTP, 6-OHDA, and LPS. We will test whether cell-dependent titration of Snca levels, similarly, ameliorates risk and progression in a new mouse model. We have engineered mouse lines lacking this enhancer (Snca Enhdel) and provide preliminary evidence that this sequence impacts Snca transcription. Using established techniques, we propose to assay the effect of Snca Enhdel on prodromal, behavioral, and motor phenotypes (Aim 1a), as well as on PD-relevant neuron viability/distribution and microglial activation in the midbrain and olfactory bulb (Aim 1b) via immunohistochemistry and single molecule fluorescence in situ hybridization. Similarly, we will determine whether Snca Enhdel reduces onset and progression of prodromal/motor/non-motor disease phenotypes (Aim 2a) and impacts DA neuron vulnerability (Aim 2b) when exposed to PD-relevant insults (intrastriatal injection of SNCA PFF and adenoviral-delivery of SNCA hA53T). We will establish the extent to which modulating Snca transcription impacts risk of PD relevant pathology in mice, and its potential as a novel therapeutic avenue.

我们将评估SNCA中内含子增强子的必要生物学，并确定其与 帕金森病（PD）。 SNCA经常在家族性PD中突变；它编码α-突触核蛋白（α-Syn）， 主要构成路易尸体（LB）。 LB形成/积累是PD的病理标志，是 由SNCA结构突变，基因扩增或遗传/环境促进的蛋白质错误折叠驱动 侮辱升高α-syn水平。预测会影响SNCA转录调节控制的非编码变体 也是零星PD的危险因素。我们最近确定了SNCA内含子4中的PD相关变体。 我们显示的变体位于多巴胺能（DA）神经元开放染色质区域（OCR）中 SNCA启动子是儿茶酚胺能神经元中的顺式调节增强子。我们预测这一点 序列以及其中的变体会影响SNCA转录控制并调节PD风险。 假设 - 小鼠中SNCA增强子的缺失将减少与PD相关细胞中的SNCA转录 对其生存力/分布以及运动/非运动PD表型的潜在影响（AIM 1）；删除会 减少DA神经元对PD相关的损伤的脆弱性并改善疾病的发作和严重程度（AIM2）。 有几种高效的策略来引起小鼠帕金森病病理学。四环素（TET）依赖性 DA神经元中SNCA的PD突变体和野生型（WT）形式的表达导致明显的渐进性 腹脑中脑DA神经元群体的丧失与PD病理一致。同样，纹状体注射 α-Syn预形成原纤维（PFF）也导致了表现出进行性DA神经元损失的小鼠模型。 与影响PD风险和进展的SNCA水平的已知作用一致，α-Syn缺乏小鼠是 免受PFF诱导的神经退行性的保护，并免受PD的TET依赖性表达（通过病毒载体） 错义突变体（HA53T）或WT SNCA。 α-Syn Null小鼠也受到其他保护的保护 PD促进（和SNCA升高）侮辱，例如MPTP，6-OHDA和LPS。我们将测试是否依赖细胞 同样，SNCA水平的滴定可以缓解新的小鼠模型中的风险和进展。 我们已经设计了鼠标线，缺乏此增强器（SNCA增强），并提供了初步证据表明 此序列影响SNCA转录。使用既定技术，我们建议维护SNCA的效果 增强前序，行为和运动表型（AIM 1A）以及PD相关神经元 中脑和嗅球中的生存力/分布和小胶质细胞激活（AIM 1B）通过 免疫组织化学和单分子荧光原位杂交。同样，我们将确定 SNCA是否可以增强前驱/运动/非运动疾病表型的发作和进展（AIM 2A）并影响DA神经元脆弱性（AIM 2B）时暴露于PD相关感染（纹状体内注射 SNCA pff和snca ha53t的腺病毒递送）。我们将确定调节SNCA的程度 转录会影响小鼠中PD相关病理的风险及其作为一种新型治疗途径的潜力。