THE GLUTAMATE TRANSPORTER EAAT4 IN PUKINJE CELL BIOLOGY

谷氨酸运输对于神经递质谷氨酸的突触失活是必不可少的。在星形胶质细胞和神经元中都发现了一个谷氨酸转运体家族。这些转运蛋白在神经系统疾病的病理生理学中发挥重要作用，特别是ALS和阿尔茨海默病。EAAT4是一种选择性定位于小脑浦肯野细胞的谷氨酸转运体，具有配体门控氯离子通道的特性。它在小脑生理学中的作用以及在共济失调的可能发病机制中的作用尚不清楚。在这项提案中，提出了在浦肯野细胞生物学中研究EAAT4的实验，并对EAAT4功能障碍可能导致的疾病产生了更大的影响。首先，将开发一种建立器官型小脑培养物的技术。这些培养物将保持其体内结构，并表达小脑中存在的谷氨酸转运体，包括EAAT4。这个系统将允许操纵条件和评估影响谷氨酸转运体的因素。其次，将研究EAAT4分子敲除导致的表型以及伴随这种蛋白质表达减少而导致的小脑的神经解剖学和神经生理学变化。最后，我将研究EAAT4功能障碍是否导致SCA1转基因小鼠的浦肯野细胞死亡；SCA1转基因小鼠是人类神经退行性疾病脊髓小脑性共济失调的模型。

Glutamate transport is essential for the synaptic inactivation of the neurotransmitter glutamate. A family of glutamate transporters has been identified in both astroglia and neurons. These transporters play important roles in the pathophysiology of neurologic disorders, notably ALS and Alzheimers disease. EAAT4 is a glutamate transporter selectively localized to cerebellar Purkinje cells and has the properties of a ligand-gated chloride ion channel. Its role in cerebellar physiology and in the possible pathogenesis of ataxic disorders is unknown. In this proposal, experiments are presented to study EAAT4 in Purkinje cell biology with larger implications for disorders to which EAAT4 dysfunction may contribute. First, a technique for the establishment of organotypic cerebellar cultures will be developed. These cultures will maintain their in vivo architecture and express the glutamate transporters, including EAAT4, that are present in the cerebellum. This system will allow the ability to manipulate conditions and assess factors that affect this glutamate transporter. Second, the phenotype that results from molecular knockdown of EAAT4 will be studied along with the neuroanatomical and neurophysiological changes in the cerebellum that accompany this reduction in protein expression. Finally, I will study whether EAAT4 dysfunction contributes to Purkinje cell death in the SCA1 transgenic mouse; a model for the human neurodegenerative disorder spinocerebellar ataxia.