Expression of mutant alpha-synuclein modulates microglial phenotype in vitro.
Expression of mutant alpha-synuclein modulates microglial phenotype in vitro.
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DOI：
10.1186/1742-2094-8-44
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发表时间：
2011-05-09
影响因子：
9.3
通讯作者：
Combs CK
中科院分区：
文献类型：
--
作者：
Rojanathammanee L;Murphy EJ;Combs CK
反应性小胶质细胞增多是帕金森病(PD)脑的组织学特征，与沉积的α-突触核蛋白水平呈正相关。这表明小胶质细胞介导的炎症事件可能在疾病的病理生理学中起作用。编码α-突触核蛋白的基因突变导致家族性帕金森病。根据我们先前的发现，α-突触核蛋白的表达调节小胶质细胞的表型，我们假设该蛋白的突变形式的表达可能有助于帕金森病脑内反应性小胶质细胞的特征。为了研究野生型和突变型α突变体对小胶质细胞表型的影响，将野生型(WT)和突变型α突变型突变体(A30P和A53T)瞬时导入小鼠小胶质细胞系BV2，表达人野生型(WT)和突变型突变体(A30P和A53T)蛋白。通过Western印迹分析、酶联免疫吸附试验、吞噬试验和神经毒性试验检测小胶质细胞表型的变化。正如预期的那样，α-突触核蛋白的过度表达在转基因细胞中诱导了一种反应性表型。α-突触核蛋白的表达增加了环氧合酶-2的蛋白水平。转染组细胞分泌促炎细胞因子肿瘤坏死因子-α和白介素6，并增加一氧化氮的产生。转基因细胞的吞噬能力也受损，与溶酶体相关膜蛋白1(LAMP-1)的蛋白水平降低有关。尽管细胞因子分泌增加，但在神经元-小胶质细胞共培养中，转基因细胞并没有表现出比对照未转基因BV2细胞更强的神经毒性能力。这些数据表明，α-突触核蛋白的过度表达导致小胶质细胞进入一种以花生四烯酸代谢酶水平升高为特征的反应表型，细胞因子和反应性氮物种的分泌都叠加在吞噬能力受损的基础上。
Increased reactive microglia are a histological characteristic of Parkinson's disease (PD) brains, positively correlating with levels of deposited α-synuclein protein. This suggests that microglial-mediated inflammatory events may contribute to disease pathophysiology. Mutations in the gene coding for α-synuclein lead to a familial form of PD. Based upon our prior findings that α-synuclein expression regulates microglial phenotype we hypothesized that expression of mutant forms of the protein may contribute to the reactive microgliosis characteristic of PD brains. To quantify the effects of wild type and mutant α-synuclein over-expression on microglial phenotype a murine microglial cell line, BV2, was transiently transfected to express human wild type (WT), and mutant α-synuclein (A30P and A53T) proteins. Transfected cells were used to assess changes in microglia phenotype via Western blot analysis, ELISA, phagocytosis, and neurotoxicity assays. As expected, over-expression of α-synuclein induced a reactive phenotype in the transfected cells. Expression of α-synuclein increased protein levels of cycloxygenase-2 (Cox-2). Transfected cells demonstrated increased secretion of the proinflammatory cytokines, tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6), as well as increased nitric oxide production. Transfected cells also had impaired phagocytic ability correlating with decreased protein levels of lysosomal-associated membrane protein 1 (LAMP-1). In spite of the increased cytokine secretion profile, the transfected cells did not exhibit increased neurotoxic ability above control non-transfected BV2 cells in neuron-microglia co-cultures. These data demonstrated that over-expression of α-synuclein drives microglial cells into a form of reactive phenotype characterized by elevated levels of arachidonic acid metabolizing enzymes, cytokine secretion, and reactive nitrogen species secretion all superimposed upon impaired phagocytic potential.
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