Cortés Adriana, Phung Toan K, de Mena Lorena, Garrido Alicia, Infante Jon, Ruíz-Martínez Javier, Galmés-Ordinas Miquel À, Glendinning Sophie, Pérez Jesica, Roig Ana, Soto Marta, Cosgaya Marina, Ravasi Valeria, Fernández Manel, Rubiano-Castro Alejandro, Díaz Ramón, Hernández-Eguiazu Haizea, Sánchez-Quintana Coro, Vinagre-Aragón Ana, Mondragón Elisabet, Croitoru Ioana, Rivera-Sánchez María, Corrales-Pardo Andrea, Sierra María, Tolosa Eduardo, Malagelada Cristina, Nirujogi Raja S, Fernández-Irigoyen Joaquín, Santamaría Enrique, Alessi Dario R, Martí María J, Ezquerra Mario, Fernández-Santiago Rubén
Proteored-ISCIII, Proteomics Platform, Clinical Neuroproteomics Unit, Navarrabiomed, Departamento de Salud, UPNA, IdiSNA, Pamplona ES 31008, Spain.
Medical Research Council Protein Phosphorylation and Ubiquitylation Unit, University of Dundee, Dundee DD1 5EH, UK.
Brain. 2025 Jun 3;148(6):2075-2092. doi: 10.1093/brain/awae404.
Leucine-rich repeat kinase 2 (LRRK2) inhibition is a promising disease-modifying therapy for LRRK2-associated Parkinson's disease (L2PD) and idiopathic PD. However, pharmacodynamic readouts and progression biomarkers for clinical trials aiming for disease modification are insufficient, given that no endogenous marker reflecting enhanced kinase activity of the most common LRRK2 G2019S mutation has yet been reported in L2PD patients. Using phospho-/proteomic analyses, we assessed the impact of LRRK2-activating mutations in peripheral blood mononuclear cells from an LRRK2 clinical cohort from Spain (n = 174). The study groups encompassed G2019S L2PD patients (n = 37), non-manifesting LRRK2 mutation carriers of G2019S (here termed G2019S L2NMCs) (n = 27), R1441G L2PD patients (n = 14), R1441G L2NMCs (n = 11), idiopathic PD patients (n = 40) and healthy controls (n = 45). We identified 207 differentially regulated proteins in G2019S L2PD compared with controls (39 upregulated and 168 downregulated) and 67 in G2019S L2NMCs (10 upregulated and 57 downregulated). G2019S downregulated proteins affected the endolysosomal pathway, proteostasis and mitochondria, e.g. ATIC, RAB9A or LAMP1. At the phospho-proteome level, we observed increases in endogenous phosphorylation levels of pSer106 RAB12 in G2019S carriers, which were validated by immunoblotting after 1 year of follow-up (n = 48). Freshly collected peripheral blood mononuclear cells from three G2019S L2PD, one R1441G L2PD, one idiopathic PD and five controls (n = 10) showed strong diminishment of pSer106 RAB12 phosphorylation levels after in vitro administration of the MLi-2 LRRK2 inhibitor. Using machine learning, we identified an 18-feature G2019S phospho-/protein signature discriminating G2019S L2PD, L2NMCs and controls with 96% accuracy that was correlated with disease severity, i.e. UPDRS-III motor scoring. Using easily accessible peripheral blood mononuclear cells from a LRRK2 clinical cohort, we identified elevated levels of pSer106 RAB12 as an endogenous biomarker of G2019S carriers. Our data suggest that monitoring pSer106 RAB12 phosphorylation could be a relevant biomarker for tracking LRRK2 activation, particularly in G2019S carriers. Future work might determine whether pSer106 RAB12 could help with patient enrichment and monitoring drug efficacy in LRRK2 clinical trials.
富含亮氨酸重复激酶2（LRRK2）抑制是一种很有前景的针对LRRK2相关帕金森病（L2PD）和特发性帕金森病的疾病修饰疗法。然而，鉴于在L2PD患者中尚未报道反映最常见LRRK2 G2019S突变增强激酶活性的内源性标志物，用于旨在进行疾病修饰的临床试验的药效学生物标志物和疾病进展生物标志物并不充分。通过磷酸化蛋白质组分析，我们评估了来自西班牙的LRRK2临床队列（n = 174）外周血单个核细胞中LRRK2激活突变的影响。研究组包括G2019S L2PD患者（n = 37）、G2019S的未表现出症状的LRRK2突变携带者（此处称为G2019S L2NMCs）（n = 27）、R1441G L2PD患者（n = 14）、R1441G L2NMCs（n = 11）、特发性帕金森病患者（n = 40）和健康对照（n = 45）。与对照相比，我们在G2019S L2PD中鉴定出207种差异调节蛋白（39种上调和168种下调），在G2019S L2NMCs中鉴定出67种（10种上调和57种下调）。G2019S下调的蛋白影响内溶酶体途径、蛋白质稳态和线粒体，例如ATIC、RAB9A或LAMP1。在磷酸化蛋白质组水平，我们观察到G2019S携带者中pSer106 RAB12的内源性磷酸化水平升高，在随访1年后通过免疫印迹得到验证（n = 48）。来自3例G2019S L2PD、1例R1441G L2PD、1例特发性帕金森病患者和5例对照（n = 10）的新鲜采集的外周血单个核细胞在体外给予MLi - 2 LRRK2抑制剂后，pSer106 RAB12磷酸化水平显著降低。通过机器学习，我们鉴定出一种18特征的G2019S磷酸化/蛋白质特征，以96%的准确率区分G2019S L2PD、L2NMCs和对照，该特征与疾病严重程度相关，即统一帕金森病评定量表第三部分（UPDRS - III）运动评分。通过使用来自LRRK2临床队列的易于获取的外周血单个核细胞，我们鉴定出pSer106 RAB12水平升高是G2019S携带者的一种内源性生物标志物。我们的数据表明，监测pSer106 RAB12磷酸化可能是追踪LRRK2激活的相关生物标志物，特别是在G2019S携带者中。未来的工作可能会确定pSer106 RAB12是否有助于LRRK2临床试验中的患者富集和监测药物疗效。
