Localization of LRRK2 to membranous and vesicular structures in mammalian brain
Localization of LRRK2 to membranous and vesicular structures in mammalian brain
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DOI：
10.1002/ana.21019
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发表时间：
2006-11-01
影响因子：
11.2
通讯作者：
Dawson, Valina L.
中科院分区：
文献类型：
--
作者：
Biskup, Saskia;Moore, Darren J.;Dawson, Valina L.
目的：PARK 8基因负责晚发型常染色体显性帕金森氏病编码一个未知生物学功能的大型新蛋白质，称为富含亮氨酸重复激酶2（LRRK 2）。方法：用多克隆抗体分别对LRRK 2的氨基端和羧基端进行标记，检测LRRK 2的生化、亚细胞和免疫组织化学分布。结果：LRRK 2在大鼠脑中的表达量约为280 kDa。亚细胞分级显示LRRK 2存在于大鼠脑的微粒体、突触囊泡富集和突触体胞质组分以及线粒体外膜中。大鼠和人脑组织和原代大鼠皮质神经元，LRRK 2特异性抗体的免疫组织化学分析显示，广泛的神经元特异性标记仅定位于胞体，树突和轴突内的点状结构。原代皮层神经元的共聚焦共定位分析显示LRRK 2免疫反应性与线粒体和溶酶体特异性标记物部分但显著重叠。此外，在啮齿动物基底神经节的超微结构分析检测LRRK 2免疫反应与膜和囊泡细胞内结构，包括溶酶体，内涵体，运输囊泡，和线粒体。LRRK 2与多种膜和囊泡结构、膜结合细胞器、和微管表明LRRK 2对脂质或脂质相关蛋白的亲和力，并可能表明在生物发生和/或代谢中的潜在作用。或调节哺乳动物脑内的泡状和膜状细胞内结构。
Objective: The PARK8 gene responsible for late-onset autosomal dominant Parkinson's disease encodes a large novel protein of unknown biological function termed leucine-rich repeat kinase 2 (LRRK2). The studies herein explore the localization of LRRK2 in the mammalian brain.Methods: Polyclonal antibodies generated against the amino or carboxy termini of LRRK2 were used to examine the biochemical, subcellular, and immunohistochemical distribution of LRRK2.Results: LRRK2 is detected in rat brain as an approximate 280kDa protein by Western blot analysis. Subcellular fractionation demonstrates the presence of LRRK2 in microsomal, synaptic vesicle-enriched and synaptosomal cytosolic fractions from rat brain, as well as the mitochondrial outer membrane. Immunohistochemical analysis of rat and human brain tissue and primary rat cortical neurons, with LRRK2-specific antibodies, shows widespread neuronal-specific labeling localized exclusively to punctate structures within perikarya, dendrites, and axons. Confocal colocalization analysis of primary cortical neurons shows partial yet significant overlap of LRRK2 immunoreactivity with markers specific for mitochondria and lysosomes. Furthermore, ultrastructural analysis in rodent basal ganglia detects LRRK2 immunoreactivity associated with membranous and vesicular intracellular structures, including lysosomes, endosomes, transport vesicles, and mitochondria.Interpretation: The association of LRRK2 with a variety of membrane and vesicular structures, membrane-bound organelles, and microtubules suggests an affinity of LRRK2 for lipids or lipid-associated proteins and may suggest a potential role in the biogenesis and/or regulation of vesicular and membranous intracellular structures within the mammalian brain.