Beccano-Kelly Dayne A, Kuhlmann Naila, Tatarnikov Igor, Volta Mattia, Munsie Lise N, Chou Patrick, Cao Li-Ping, Han Heather, Tapia Lucia, Farrer Matthew J, Milnerwood Austen J
Centre for Applied Neurogenetics, Medical Genetics, University of British Columbia Vancouver, BC, Canada ; Djavad Mowafaghian Centre for Brain Health, Faculty of Medicine, University of British Columbia Vancouver, BC, Canada.
Centre for Applied Neurogenetics, Medical Genetics, University of British Columbia Vancouver, BC, Canada ; Djavad Mowafaghian Centre for Brain Health, Faculty of Medicine, University of British Columbia Vancouver, BC, Canada ; Graduate Program in Neuroscience, University of British Columbia Vancouver, BC, Canada.
Front Cell Neurosci. 2014 Sep 26;8:301. doi: 10.3389/fncel.2014.00301. eCollection 2014.
Mutations in Leucine-Rich Repeat Kinase-2 (LRRK2) result in familial Parkinson's disease and the G2019S mutation alone accounts for up to 30% in some ethnicities. Despite this, the function of LRRK2 is largely undetermined although evidence suggests roles in phosphorylation, protein interactions, autophagy and endocytosis. Emerging reports link loss of LRRK2 to altered synaptic transmission, but the effects of the G2019S mutation upon synaptic release in mammalian neurons are unknown. To assess wild type and mutant LRRK2 in established neuronal networks, we conducted immunocytochemical, electrophysiological and biochemical characterization of >3 week old cortical cultures of LRRK2 knock-out, wild-type overexpressing and G2019S knock-in mice. Synaptic release and synapse numbers were grossly normal in LRRK2 knock-out cells, but discretely reduced glutamatergic activity and reduced synaptic protein levels were observed. Conversely, synapse density was modestly but significantly increased in wild-type LRRK2 overexpressing cultures although event frequency was not. In knock-in cultures, glutamate release was markedly elevated, in the absence of any change to synapse density, indicating that physiological levels of G2019S LRRK2 elevate probability of release. Several pre-synaptic regulatory proteins shown by others to interact with LRRK2 were expressed at normal levels in knock-in cultures; however, synapsin 1 phosphorylation was significantly reduced. Thus, perturbations to the pre-synaptic release machinery and elevated synaptic transmission are early neuronal effects of LRRK2 G2019S. Furthermore, the comparison of knock-in and overexpressing cultures suggests that one copy of the G2019S mutation has a more pronounced effect than an ~3-fold increase in LRRK2 protein. Mutant-induced increases in transmission may convey additional stressors to neuronal physiology that may eventually contribute to the pathogenesis of Parkinson's disease.
富含亮氨酸重复激酶2（LRRK2）的突变会导致家族性帕金森病，仅G2019S突变在某些种族中占比高达30%。尽管如此，LRRK2的功能在很大程度上仍未明确，不过有证据表明其在磷酸化、蛋白质相互作用、自噬和内吞作用中发挥作用。新出现的报道将LRRK2的缺失与突触传递改变联系起来，但G2019S突变对哺乳动物神经元突触释放的影响尚不清楚。为了评估既定神经网络中的野生型和突变型LRRK2，我们对LRRK2基因敲除、野生型过表达和G2019S基因敲入小鼠3周龄以上的皮质培养物进行了免疫细胞化学、电生理和生化特性分析。LRRK2基因敲除细胞中的突触释放和突触数量基本正常，但观察到谷氨酸能活性离散性降低且突触蛋白水平下降。相反，野生型LRRK2过表达培养物中的突触密度适度但显著增加，尽管事件频率没有变化。在基因敲入培养物中，谷氨酸释放明显升高，而突触密度没有任何变化，这表明生理水平的G2019S LRRK2会提高释放概率。其他研究表明与LRRK2相互作用的几种突触前调节蛋白在基因敲入培养物中的表达水平正常；然而，突触素1的磷酸化显著降低。因此，对突触前释放机制的干扰和突触传递增强是LRRK2 G2019S的早期神经元效应。此外，基因敲入和过表达培养物的比较表明，一份G2019S突变的作用比LRRK2蛋白增加约3倍更为显著。突变诱导的传递增加可能会给神经元生理带来额外的应激源，最终可能导致帕金森病的发病机制。
