Department of Molecular Pharmacology, Albert Einstein College of Medicine, 1300 Morris Park Avenue, Forchheimer Building, Room 209, Bronx, NY, 10461, USA.
Laboratorio de Aminoácidos Excitadores, Instituto Nacional de Neurología y Neurocirugía, 14269, Mexico City, Mexico.
Neurotox Res. 2020 Oct;38(3):751-764. doi: 10.1007/s12640-020-00262-5. Epub 2020 Jul 29.
In a previous study, we have shown that methylmercury (MeHg) exposure causes focal aggregation of intracellular transgenic mCherry protein in dendrites of cephalic (CEP) neurons in Caenorhabditis elegans (C. elegans). However, the underlying mechanism is unknown. We hypothesized that reduced cellular release of mCherry via extracellular vesicles by MeHg contributes to its accumulation and intracellular aggregation. Thus, we characterized vesicular structures in CEP dendrites, which were 1-3 μm in diameter and could readily bud off from the plasma membrane of the dendrites. Chronic treatment of C. elegans with MeHg (5 μM, 4-10 days) reduced the number of vesicles attached to CEP dendrites (attached vesicles) and vesicles unattached to CEP dendrites (unattached vesicles), as well as the presence of extracellular mCherry, supporting the hypothesis that release of mCherry by microvesicle formation is inhibited by MeHg. Leucine-rich repeat kinase 2 (LRRK2) has an important function in membrane biology. Further investigation showed that the effects of MeHg were modified by human LRRK2. In worms with the wild-type LRRK2, the vesicle numbers were significantly reduced by MeHg (0.5 and 5 μM). The effects of MeHg on the presence of extracellular mCherry and attached vesicles were modified by the human wild-type LRRK2. Independent of MeHg treatment, the G2019S mutant LRRK2 showed reduced number of unattached vesicles; however, the levels of extracellular mCherry were increased. Knockdown of C. elegans irk-1, the homolog of human LRRK2, reduced the number of attached vesicles, corroborating that LRRK2 plays an important role in the formation of microvesicles.
在之前的研究中，我们已经表明，甲基汞（MeHg）暴露会导致头（CEP）神经元树突内细胞内转基 因 mCherry 蛋白的局灶性聚集。然而，其潜在机制尚不清楚。我们假设 MeHg 通过细胞外囊泡减少 mCherry 的细胞释放，导致其积累和细胞内聚集。因此，我们对 CEP 树突中的囊泡结构进行了表征，这些囊泡的直径为 1-3μm，很容易从树突的质膜上出芽。慢性用 MeHg（5μM，4-10 天）处理秀丽隐杆线虫（C. elegans）减少了附着于 CEP 树突的囊泡（附着囊泡）和未附着于 CEP 树突的囊泡（未附着囊泡）的数量，以及细胞外 mCherry 的存在，支持了 MeHg 抑制通过微囊泡形成释放 mCherry 的假说。富含亮氨酸重复激酶 2（LRRK2）在膜生物学中具有重要功能。进一步的研究表明，MeHg 的作用被人类 LRRK2 修饰。在具有野生型 LRRK2 的蠕虫中，MeHg（0.5 和 5μM）显著减少了囊泡数量。MeHg 对细胞外 mCherry 和附着囊泡的影响被人类野生型 LRRK2 修饰。独立于 MeHg 处理，G2019S 突变 LRRK2 显示出未附着囊泡数量减少；然而，细胞外 mCherry 的水平增加。秀丽隐杆线虫 irk-1 的敲低，人类 LRRK2 的同源物，减少了附着囊泡的数量，证实了 LRRK2 在微囊泡形成中发挥重要作用。
