Essential Tremor: Gene expression profiling in cerebellar Purkinje cells

描述（由申请人提供）：特发性震颤（ET）是最常见的神经系统疾病之一，发生在4%的40岁及以上的成年人中，甚至更常见于高龄（即，70岁时7-8%，90岁以上时>20%）。尽管其患病率高，ET的潜在发病机制知之甚少，因此，目前的治疗是经验性的，疗效差。人类死后研究是目前推进ET潜在病理生理机制研究的最有力途径，因为ET的基因尚未被鉴定，并且目前不存在转基因小鼠来提供动物模型。我们的小组一直在进行系统的死后研究，这揭示了ET病例大脑中可识别的结构变化。我们已经证明，绝大多数ET病例（>90%）的神经病理学结果局限于小脑本身，特别是浦肯野细胞（PC），它提供了小脑皮质的整个神经元输出。在ET脑中，受损的PC轴突增加了6至7倍，被鉴定为PC轴突近端部分的圆形轴突（即，“鱼雷”）。与此PC轴突损伤密切相关的是PC数量减少约30-40%和异位（移位）PC数量增加。我们正在进行的病理学研究进一步表明，鱼雷很可能只是一个先进的PC轴突损伤的标志。早期的变化现在在ET脑中变得明显，包括近端PC轴突的小到中等大小的增厚的分级增加（即，推测的前体-鱼雷）和PC复发性轴突侧支形成的增加，伴随着PC轴突的皮质内段的发芽沿着增加。这些研究暗示PC变性是ET疾病过程的核心特征，与缓慢进行性轴突损伤和皮质内小脑连接的显著重塑相关。虽然这些尸检研究已经将我们对疾病发病机制的理解推进到了细胞水平，但现在是时候进行分子理解了。在这个建议中，我们将解决是否可以识别分子改变小脑PC从ET患者与神经正常对照。我们将通过微阵列筛选从ET与对照尸检脑的PC分离的RNA进行基因表达分析。我们将采用激光捕获显微切割专门针对PC，从而促进与ET相关的细胞特异性变化的精确评估。ET脑将限于尸检时不存在其他神经退行性病变的脑。对所得表达数据的分析将鉴定在ET与对照PC中差异表达和/或生物学分组的基因和/或分子途径。将通过定量真实的时间PCR验证基因表达的变化。这项研究将是第一个启动ET研究的分子表达方法，并将为提出ET的分子因果机制提供基础，这将是未来研究的重点。

DESCRIPTION (provided by applicant): Essential tremor (ET) is among the most common neurological diseases, occurring in 4% of adults age 40 and older, and even more frequently in advanced age (i.e., 7-8% by age 70 and >20% by age 90+). Despite its high prevalence, the underlying pathogenesis of ET is poorly understood, and, as a consequence, current treatments are empiric and have poor efficacy. Human postmortem studies are currently the most robust avenue for advancing the study of the underlying pathophysiological mechanisms of ET, as genes have yet to be identified for ET, and no transgenic mice currently exist to provide an animal model. Our group has been conducting systematic postmortem studies, which have revealed identifiable structural changes in the brains of ET cases. We have demonstrated that neuropathologic findings in the vast majority of ET cases (>90%) localize to the cerebellum itself and, in particular, to the Purkinje cells (PC), which provide the entire neuronal output from cerebellar cortex. In ET brains, there is a 6- to 7-fold increase in damaged PC axons, identified as rounded swellings of the proximal portion of the PC axon (i.e., "torpedoes"). Strongly correlating with this PC axonal damage is an approximate 30-40% reduction in the number of PCs and an increase in the number of heterotopic (displaced) PCs. Our ongoing pathologic studies further indicates that the torpedo is likely only a marker of advanced PC axonal damage. Earlier changes are now becoming evident in ET brains, including a graded increase in small to intermediate-sized thickenings of the proximal PC axon (i.e., presumed precursor- torpedoes) and increases in PC recurrent axon collateral formation with increased sprouting along the intracortical segment of PC axons. These studies implicate PC degeneration as a core feature of the disease process in ET, associated with slowly progressive axonal damage and significant remodeling of intracortical cerebellar connectivity. While these postmortem studies have advanced our understanding of disease pathogenesis to a cellular level, it is time to now proceed to a molecular understanding. In this proposal, we will address whether molecular alterations can be identified in cerebellar PCs from ET patients vs. neurologically normal controls. We will perform gene expression analyses by microarray screening on RNA isolated from PCs of ET versus control autopsy brains. We will employ laser-capture microdissection to specifically target PCs, thereby facilitating a precise evaluation of cell-specific changes associated with ET. ET brains will be limited to those with an absence of other neurodegenerative pathologies at autopsy. Analysis of the resultant expression data will identify genes and/or molecular pathways that are differentially expressed and/or biologically grouped in ET versus control PCs. Changes in gene expression will be validated by quantitative real time PCR. This study will be the first to initiate a molecular expression approach in ET research, and will provide a basis for proposing molecular causal mechanisms for ET that will be the focus of future investigations.