Department of Pharmacology, Physiology, & Therapeutics, University of North Dakota School of Medicine and Health Sciences, 504 Hamline Street, Neuroscience Building, Grand Forks, ND 58203, USA.
J Neuroinflammation. 2011 May 9;8:44. doi: 10.1186/1742-2094-8-44.
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.
帕金森病 (PD) 大脑中的反应性小胶质细胞增多是其组织学特征，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导致疾病的病理生理学。α-突触核蛋白基因编码突变导致家族性 PD。基于我们之前发现的α-突触核蛋白表达调节小胶质细胞表型的研究结果，我们假设该蛋白的突变形式的表达可能导致 PD 大脑中反应性小胶质细胞增多的特征。
为了量化野生型和突变型α-突触核蛋白过表达对小胶质细胞表型的影响，我们瞬时转染小鼠小胶质细胞系 BV2 表达人野生型 (WT) 和突变型α-突触核蛋白 (A30P 和 A53T) 蛋白。通过 Western blot 分析、ELISA、吞噬作用和神经毒性测定来评估转染细胞中小胶质细胞表型的变化。
正如预期的那样，α-突触核蛋白过表达诱导转染细胞呈现反应性表型。α-突触核蛋白的表达增加了环氧化酶-2 (Cox-2) 的蛋白水平。转染细胞表现出促炎细胞因子肿瘤坏死因子-α (TNF-α) 和白细胞介素-6 (IL-6) 的分泌增加，以及一氧化氮产生增加。转染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。尽管细胞因子分泌谱增加，但在神经元-小胶质细胞共培养物中，与对照未转染的 BV2 细胞相比，转染细胞并未表现出增加的神经毒性能力。
这些数据表明，α-突触核蛋白的过表达使小胶质细胞呈现出一种反应性表型，其特征是花生四烯酸代谢酶水平升高、细胞因子分泌和活性氮物种分泌增加，同时吞噬能力受损。
