DETERMINANTS OF VULNERABILITY TO EXCITOTOXIC DEATH

谷氨酸介导的兴奋性毒性损伤与缺血性神经元死亡和多种神经退行性疾病有关。 已知对兴奋性毒性损伤的敏感性在不同类型的CNS神经元之间变化，在发育过程中变化，并且受到各种神经营养因子的影响。 该提案将使用两个良好的特征模型系统，小脑颗粒细胞神经元和脊髓运动神经元的原代培养物，以确定是否以及在何种程度上改变谷氨酸受体的表达，亚基组成或表型特性的差异，我们已经发现这两种类型的神经元谷氨酸诱导的细胞死亡的敏感性。 该提案有三个具体目标。 1.）的人。成熟的小脑颗粒细胞对兴奋性毒性损伤有抵抗力。 未成熟颗粒细胞表达NMDA受体亚单位NR2B，而成熟颗粒细胞表达NR2C亚单位。 为了直接测试NR2表达的变化是否影响颗粒细胞对兴奋性毒性死亡的敏感性，我们将比较在NR2B或NR2C表达占主导地位的条件下培养的颗粒细胞的敏感性，并使用复制缺陷型病毒载体来改变这两个亚基的表达。 2.）的情况。我们已经发现，暴露于脑源性神经营养因子（BDNF）的脊髓运动神经元的纯化培养物，大大增加了这些细胞对兴奋性毒性死亡的脆弱性。 为了测试这种效应是否是谷氨酸受体（GluR）表型变化的结果，我们将使用电生理学方法比较BDNF存在或不存在下生长的运动神经元。 我们还将比较运动神经元的GluR表型从BDNF处理的文化，有或没有被暴露于谷氨酸。 3.）第三章BDNF对GluR表达和运动神经元对兴奋性毒性损伤的敏感性的影响将在来自转基因动物的脊髓的原代培养物中进行研究，其中运动神经元可以通过绿色荧光蛋白（GFP）的细胞类型特异性表达来鉴定。 我们将把这些结果与用GFP转基因动物和BDNF敲除小鼠杂交产生的突变小鼠的脊髓培养物的实验结果进行比较。

Glutamate-mediated excitotoxic injury has been implicated in ischemic neuronal death and a variety of neurodegenerative disorders. Sensitivity to excitotoxic injury is known to vary between different types of CNS neurons, to change during development, and to be influenced by various neurotrophic factors. This proposal will use two well-characterized model systems, primary cultures of cerebellar granule cell neurons and spinal motor neurons, to determine whether and to what degree alterations in the expression, subunit composition, or phenotypic properties of glutamate receptors contribute to differences we have found in the sensitivity of these two types of neurons to glutamate-induced cell death. The proposal has three specific aims. 1.) Mature cerebellar granule cells are resistant to excitotoxic injury. Immature granule cells express the NMDA- receptor subunit NR2B, whereas mature granule cells express the NR2C subunit. To test directly whether changes in NR2 expression influence the sensitivity of granule cells to excitotoxic death, we will compare the sensitivity of granule cells cultured under conditions where NR2B or NR2C expression predominates and use replication-deficient viral vectors to alter the expression of these two subunits. 2.) We have found that exposure of purified cultures of spinal motor neurons to brain-derived neurotrophic factor (BDNF) greatly increases the vulnerability of these cells to excitotoxic death. To test whether this effect is the result of changes in glutamate-receptor (GluR) phenotype, we Will use electrophysiological methods to compare motor neurons that have been grown in the presence or absense of BDNF. We will also compare the GluR phenotype of motor neurons from BDNF-treated cultures that have or have not been exposed to glutamate. 3.) The effect of BDNF on the GluR expression and sensitivity of motor neurons to excitotoxic injury will be studied in primary cultures of spinal cord from transgenic animals in which motor neurons can be identified by cell-type specific expression of the Green Fluorescent Protein (GFP). The results will be compared with those from experiments on spinal cord cultures from mutant mice we will generate by crossbreeding the GFP-transgenic animals with BDNF knockout mice.