Biomolecular Dynamics, Katholieke Universiteit Leuven, Leuven, Belgium.
Mol Cell Neurosci. 2011 Feb;46(2):516-26. doi: 10.1016/j.mcn.2010.12.004. Epub 2010 Dec 9.
Parkinson's and Parkinson's-plus diseases are associated with abnormal, aggregated forms of the protein, α-synuclein. We have investigated the effects of calcium on α-synuclein aggregation in vitro and in vivo. We treated monomeric α-synuclein with calcium in vitro and used fluorescence imaging, fluorescence correlation and scanning electron microscopy to investigate protein aggregation. Incubation of fluorescent-labelled monomeric α-synuclein (24h) at low concentration (10 μM) with calcium resulted in surface aggregates (1.5±0.7 μm(2)) detected by fluorescence microscopy saturating at a half-maximum calcium concentration of 80 μM, whilst incubations without calcium showed few protein aggregates. Scanning electron microscopy revealed that α-synuclein surface plaques (0.5-1 μm) form in the presence of calcium and comprise 10-20 nm globular particles. Incubation of α-synuclein at high concentration (75 μM; 6h) resulted in soluble oligomeric aggregates detected by fluorescence correlation spectroscopy in a calcium dependent process, saturating at a half maximum calcium concentration of 180 μM. In cell culture experiments, we used thapsigargin or calcium ionophore A23187 to induce transient increases of intracellular free calcium in human 1321N1 cells expressing an α-synuclein-GFP construct and observed calcium flux and α-synuclein aggregation by fluorescence microscopy. The cell culture data shows that a transient increase in intracellular free calcium significantly increased the proportion of cells bearing cytoplasmic α-synuclein aggregates 6 and 12h post-treatment (P, 0.01). Our data indicates that calcium accelerates α-synuclein aggregation on surfaces, in free solution and in cultured cells and suggests that surface adsorption may play an important role in the calcium-dependent aggregation mechanism.
帕金森病和帕金森病综合征与蛋白质α-突触核蛋白的异常聚集形式有关。我们研究了钙对α-突触核蛋白在体外和体内聚集的影响。我们在体外用钙处理单体α-突触核蛋白，并使用荧光成像、荧光相关和扫描电子显微镜来研究蛋白质聚集。用荧光标记的单体α-突触核蛋白（孵育 24 小时）在低浓度（10 μM）下与钙孵育，导致通过荧光显微镜检测到表面聚集物（1.5±0.7 μm²），半最大钙浓度为 80 μM 时达到饱和，而没有钙孵育的情况下，很少有蛋白质聚集。扫描电子显微镜显示，α-突触核蛋白表面斑块（0.5-1 μm）在钙存在的情况下形成，包含 10-20 nm 的球形颗粒。在高浓度（75 μM；孵育 6 小时）下孵育α-突触核蛋白会导致通过荧光相关光谱检测到可溶性寡聚体聚集物，这是一个依赖于钙的过程，半最大钙浓度为 180 μM 时达到饱和。在细胞培养实验中，我们使用他莫昔芬或钙离子载体 A23187 诱导表达α-突触核蛋白-GFP 构建体的人 1321N1 细胞内细胞内游离钙的短暂增加，并通过荧光显微镜观察钙流和α-突触核蛋白聚集。细胞培养数据表明，细胞内游离钙的短暂增加显著增加了处理后 6 和 12 小时携带细胞质α-突触核蛋白聚集物的细胞比例（P，0.01）。我们的数据表明，钙加速了α-突触核蛋白在表面、游离溶液和培养细胞中的聚集，并表明表面吸附可能在钙依赖性聚集机制中起重要作用。
