Examination of gene expression using translating ribosome affinity purification (TRAP) in genetic models of Parkinson's disease

帕金森病（PD）是第二常见的神经退行性疾病，大约1%的60岁以上的人受其影响。针对PD病理的早期阶段和预防或延迟进展，需要开发疾病改善治疗。这一发展需要在神经退行性变之前和随后的神经退行性变相关的早期细胞机制的特征。PD涉及投射到背纹状体的多巴胺能神经元的选择性死亡。投射到腹侧纹状体的多巴胺能神经元基本上幸免。造成这种敏感性差异的细胞和遗传过程尚不清楚。目前的假设认为，在背侧纹状体和腹侧纹状体之间，DA神经元在轴突水平上的基因表达调控存在差异。本项目将利用转基因PD啮齿动物模型比较背侧纹状体和腹侧纹状体多巴胺能神经元的基因表达。我们将使用一种新开发的方法（'TRAP'）从多巴胺能神经元中选择性地纯化翻译核糖体。TRAP涉及增强的绿色荧光蛋白（eGFP）标记核糖体蛋白L10a的表达。eGFP-L10a标签翻译核糖体，通过免疫沉淀纯化。eGFP-L10a将在多巴胺转运子启动子的控制下选择性地在多巴胺能神经元中表达。与其他转录组学方法相比，这将提供更准确的表达读数。本项目旨在证明多巴胺能神经元轴突水平是否存在局部mRNA翻译，背侧纹状体和腹侧纹状体之间的表达特征是否不同，PD遗传模型中这种表达是否改变，以及这种表达是否随着年龄的增长而改变。研究基因表达的结果可用于指导多巴胺能神经元功能的功能研究。该项目的结果也将为TRAP基因表达谱的应用提供一个神经学上的先例，以探索其他神经退行性疾病的起源，包括阿尔茨海默病、肌萎缩侧索硬化症和亨廷顿病。

Parkinson's disease (PD) is the second most common neurodegenerative condition, affecting approximately 1% of individuals over the age of 60. Development of disease-modifying treatments is required to target early stages of PD pathology and prevent or delay progression. This development requires characterisation of the early cellular mechanisms that precede and are associated with subsequent neurodegeneration.PD involves the selective death of dopaminergic neurons projecting to the dorsal striatum. Dopaminergic neurons projecting to the ventral striatum are largely spared. The cellular and genetic processes responsible for this differential sensitivity are not understood. The current hypothesis proposes that there is a differential regulation of gene expression at the axon level in DA neurons between dorsal and ventral striata.This project will use transgenic rodent models of PD to compare gene expression between dopaminergic neurons of the dorsal and ventral striatum. We will use a newly developed method ('TRAP') to purify translating ribosomes selectively from dopaminergic neurons. TRAP involves expression of enhanced green fluorescent protein (eGFP) tagged ribosomal protein L10a. eGFP-L10a tags translating ribosomes, enabling purification by immunoprecipitation. eGFP-L10a will be selectively expressed in dopaminergic neurons by placing it under the control of a dopamine transporter promoter. This will give a more accurate readout of expression compared to other transcriptomic methods.The project aims to demonstrate whether there is local mRNA translation at the axon level of dopaminergic neurons, whether the signature of expression differs between dorsal and ventral striata, whether this expression is altered in genetic models of PD and whether this changes with aging. Results from investigating gene expression can then be used to guide functional studies of dopaminergic neuron function. The results derived from this project will also provide a neurological precedent for application of TRAP gene expression profiling to explore origins of other neurodegenerative conditions, including Alzheimer's disease, amyotrophic lateral sclerosis and Huntington's disease.