Leucine-rich repeat kinase 2 impairs the release sites of Parkinson's disease vulnerable dopamine axons

The end-stage pathology of Parkinson’s disease (PD) involves the loss of dopamine-producing neurons in the substantia nigra compacta, but synaptic deregulation of these neurons begins much earlier in the disease process. Understanding the mechanisms of axonal deficits may provide opportunities for early therapeutic intervention, yet they remain largely unknown. Within the substantia nigra compacta, different dopamine neuron subpopulations exhibit variable vulnerability patterns in PD, complicating our effort to understand the mechanisms of synaptic dysfunction and vulnerability. Here, we utilized intersectional genetic mouse models to uncover cell-intrinsic mechanisms of synaptic perturbations in vulnerable dopamine neurons, with a focus on the LRRK2 kinase— an important protein closely linked to PD. A combination of immunofluorescence and advanced proximity labeling methods revealed higher LRRK2 expression in the most vulnerable dopamine neurons. Further analysis using high-resolution imaging showed that mutant LRRK2 disrupts the release sites of the vulnerable dopamine axons in mice, which correlates with decreased dopamine release in the striatum as reported for these mice. Proteomic and biochemical approaches demonstrate that pathogenic mutant LRRK2 increases the phosphorylation of Rab3 proteins, that are crucial for neurotransmitter release. This alters Rab3 interactions with their effector proteins, impacting their synaptic functions as relevant for neurotransmitter release. Overall, our findings emphasize the cell-autonomous dysfunctions of LRRK2 mutations in the neuronal cell types most impacted in the disease, providing a framework for therapeutic strategies to address early nigrostriatal synaptic deficits in PD.帕金森病（Parkinson’s disease, PD）的终末期病理表现为黑质致密部（substantia nigra compacta）内多巴胺能神经元的丢失，但此类神经元的突触失调在疾病进程中出现得更早。阐明轴突缺陷的潜在机制或可为早期治疗干预提供契机，但该机制目前仍在很大程度上未被阐明。在黑质致密部中，不同的多巴胺能神经元亚群在PD中表现出各异的易感性模式，这加大了我们理解突触功能障碍与神经元易感性机制的难度。 本研究利用交叉遗传小鼠模型（intersectional genetic mouse models），探究易感性多巴胺能神经元中突触扰动的细胞内在机制，重点关注富亮氨酸重复激酶2（LRRK2 kinase）——一种与PD密切相关的关键蛋白。结合免疫荧光（immunofluorescence）与先进的邻近标记（proximity labeling）技术，研究人员发现易感性最高的多巴胺能神经元中LRRK2的表达水平显著更高。 通过高分辨率成像（high-resolution imaging）开展的进一步分析显示，突变型LRRK2会破坏小鼠体内易感性多巴胺能轴突的递质释放位点，这与此前报道的此类小鼠纹状体（striatum）内多巴胺释放减少的现象相一致。蛋白质组学（proteomic）与生物化学（biochemical）实验证实，致病性突变型LRRK2会增强Rab3蛋白（Rab3 proteins）的磷酸化水平——这类蛋白对神经递质释放至关重要。该改变会扰乱Rab3与其效应蛋白（effector proteins）的相互作用，进而影响其参与神经递质释放的突触功能。 总体而言，本研究结果凸显了LRRK2突变在PD受累最严重的神经元类型中的细胞自主性功能异常，为针对PD早期黑质纹状体突触缺陷的治疗策略提供了理论框架。