Leucine-rich repeat kinase 2 (LRRK2) mutations are among the most significant genetic risk factors for developing late onset Parkinson’s disease (PD). To understand whether a therapeutic can modulate LRRK2 levels as a potential disease modifying strategy, it is important to have methods in place to measure the protein with high sensitivity and specificity. To date, LRRK2 measurements in cerebrospinal fluid (CSF) have used extracellular vesicle enrichment via differential ultracentrifugation and western blot detection. Our goal was to develop a methodology which could be deployed in a clinical trial, therefore throughput, robustness and sensitivity were critical. To this end, we developed a Stable Isotope Standard Capture by Anti-peptide Antibody (SISCAPA) assay which is capable of detecting LRRK2 from 1 ml of human CSF. The assay uses a commercially available LRRK2 monoclonal antibody (N241A/34) and does not require extracellular vesicle enrichment steps. The assay includes stable isotope peptide addition which allows for absolute quantitation of LRRK2 protein. We determined that the assay performed adequately for CSF measurements and that blood contamination from traumatic lumbar puncture does not pose a serious analytical challenge. We then applied this technique to 106 CSF samples from the MJFF LRRK2 Cohort Consortium which includes healthy controls, sporadic PD patients and LRRK2 mutation carriers with and without PD. Of the 105 samples that had detectable LRRK2 signal, we found that the PD group with the G2019S LRRK2 mutation had significantly higher CSF LRRK2 levels compared to all other groups. We also found that CSF LRRK2 increased with the age of the participant. Taken together, this work represents a step forward in our ability to measure LRRK2 in a challenging matrix like CSF which has implications for current and future LRRK2 therapeutic clinical trials.

中文翻译：

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人类脑脊液中 LRRK2 的定量测量表明 G2019S 患者的水平升高

富含亮氨酸的重复激酶 2 (LRRK2) 突变是发展迟发性帕金森病 (PD) 的最重要的遗传风险因素之一。要了解治疗剂是否可以调节 LRRK2 水平作为潜在的疾病改善策略，重要的是要有方法来以高灵敏度和特异性测量蛋白质。迄今为止，脑脊液 (CSF) 中的 LRRK2 测量已通过差异超速离心和蛋白质印迹检测使用细胞外囊泡富集。我们的目标是开发一种可以在临床试验中部署的方法，因此通量、稳健性和灵敏度至关重要。为此，我们开发了一种稳定同位素标准捕获抗肽抗体 (SISCAPA) 测定法，该测定法能够检测 1 ml 人脑脊液中的 LRRK2。该检测使用市售的 LRRK2 单克隆抗体 (N241A/34)，不需要细胞外囊泡富集步骤。该测定包括稳定同位素肽添加，允许对 LRRK2 蛋白进行绝对定量。我们确定该测定对 CSF 测量进行了充分的分析，并且外伤性腰椎穿刺造成的血液污染不会构成严重的分析挑战。然后，我们将该技术应用于来自 MJFF LRRK2 队列联盟的 106 份 CSF 样本，其中包括健康对照、散发性 PD 患者和有和没有 PD 的 LRRK2 突变携带者。在具有可检测 LRRK2 信号的 105 个样本中，我们发现具有 G2019S LRRK2 突变的 PD 组与所有其他组相比具有显着更高的 CSF LRRK2 水平。我们还发现 CSF LRRK2 随着参与者年龄的增长而增加。总之，这项工作代表了我们在具有挑战性的矩阵（如 CSF）中测量 LRRK2 的能力向前迈进了一步，这对当前和未来的 LRRK2 治疗临床试验具有影响。