α-Synuclein's (α-syn) aggregation properties enabled the development of seed amplification assays (SAA), accurately distinguishing between idiopathic Parkinson's disease (PD) patients and healthy controls in cerebrospinal fluid (CSF) analyses.1 Among autosomal dominant PD in European ancestry populations, LRRK2 pathogenic variants are the most frequent (~2.6%), whereas VPS35-linked PD occurs in <0.06% of PD patients.2 LRRK2 and VPS35 are key regulators of intracellular transport.3 LRRK2-linked PD exhibits heterogeneous α-syn pathology as Lewy body pathology and CSF-SAAs are positive only in approximately two-thirds of patients.1, 4 Postmortem data from the single reported VPS35-linked PD patient revealed no typical Lewy body pathology.4 Different variants in the LRRK2 gene affect kinase activity to varying degrees, and the rare PD-causing variant Y1699C is associated with extensive kinase activation in vitro.5 Of note, the only known pathogenic VPS35 D620N variant also increases LRRK2 kinase activity.6 No prior SAA studies exist for the LRRK2 Y1699C and the VPS35 D620N variants. We performed CSF-SAA (Supplementary Methods) in 3 PD patients carrying the VPS35 D620N variant and 2 LRRK2 Y1699C variant carriers (1 affected, 1 unaffected) (Supplementary Clinical Information, Fig. S1). Strikingly, neither the affected VPS35 D620N-linked PD patients nor the LRRK2 Y1699C variant carriers were CSF-SAA positive (Table 1), even though the investigated LRRK2 family members carried an additional GBA1 E365K variant. Our results in LRRK2 Y1699C variant carriers align with the reported atypical postmortem findings, and both were normosmic, in keeping with the established association between preserved olfaction and SAA negativity in LRRK2-linked PD.1 By contrast, 2 of 3 VPS35-linked PD patients were anosmic yet showed negative SAA results (Table 1), potentially indicating that anosmia in VPS35-linked PD may not be associated with α-syn pathology. Together, SAA negativity in VPS35 D620N and LRRK2 Y1699C variant carriers may suggest that further mechanisms beyond the shared autophagic-lysosomal pathway potentially influence α-syn aggregation detectable by SAA. Within the framework of the recently proposed attempts to biologically define PD, particularly VPS35-linked PD would not fulfill the criteria for neuronal α-syn disease defined by the NSD-ISS (Neuronal α-Synuclein Disease Integrated Staging System) classification according to our data.7 To complement SAA analyses, we quantified in vivo LRRK2 kinase activity in neutrophils (Supplementary Methods, Table S1), revealing significant increases in VPS35 D620N (about threefold, n = 6) and LRRK2 Y1699C variant carriers (about eightfold, n = 3) compared to healthy controls (Fig. 1; Fig. S2). Thus, our findings suggest that LRRK2 kinase hyperactivation does not necessarily determine SAA positivity. Particularly, we confirmed that the LRRK2 Y1699C variant elevates kinase activity in vivo, extending previous in vitro observations.5 In summary, our study underscores the potential of SAAs to disentangle the mechanisms underlying rare monogenic forms of PD. The apparent dissociation between pronounced LRRK2 kinase activation and α-syn deposition may have key implications for patient stratification and the design of disease-modifying trials. Due to the rarity of these genetic PD subtypes, further research will require multicenter efforts to assemble larger samples and clarify the underlying pathophysiology. (1) Research Project: A. Conception, B. Organization, C. Execution; (2) Statistical Analysis: A. Design, B. Execution, C. Review and Critique; (3) Manuscript Preparation: A. Writing of the First Draft, B. Review and Critique. L.S.: 1A, 1B, 1C, 2A, 2B, 3A. N.P.: 1B, 1C, 2C, 3B. L.M.L.: 1C, 2C, 3B. T.K.: 1C, 2C, 3B. N.B.: 1C, 2C, 3B. L.S.-G.: 1B, 1C, 2C, 3B. M.M.: 1C, 2C, 3B. A.Z.: 1B, 1C, 2C, 3B. C.B.: 1C, 2C, 3B. J.-M.G.: 1B, 1C, 2C, 3B. N.G.: 1C, 2C, 3B. S.P.: 1A, 1B, 2C, 3B. P.S.: 1B, 1C, 2C, 3B. P.B.: 1B, 1C, 2C, 3B. W.V.: 1B, 1C, 2C, 3B. E.S.: 1A, 1B, 1C, 2C, 3B. C.B.: 1A, 1B, 1C, 2C, 3B. C.K.: 1A, 1B, 1C, 2C, 3B. M.B.: 1A, 1B, 1C, 2A, 2B, 3A. The contributions of the NIH author(s) were made as part of their official duties as NIH federal employees, are in compliance with agency policy requirements, and are considered Works of the United States Government. However, the findings and conclusions presented in this paper are those of the author(s) and do not necessarily reflect the views of the NIH or the U.S. Department of Health and Human Services. We thank the participants who donated CSF and blood for the study. T.K. would like to acknowledge support from the Global Parkinson's Genetics Program (GP2) funded by the Aligning Science Across Parkinson's (ASAP) initiative and implemented by the MJFF. Open Access funding enabled and organized by Projekt DEAL. Author disclosures are available in the Supporting Information. The data that support the findings of this study are available from the corresponding author upon reasonable request. Data S1: Comprises Supplementary Methods, Supplementary Clinical Information, Table S1 (Overview on additional individuals included in LRRK2 kinase activity analyses), Figure S1 (Pedigrees of Family 1 and 3), Figure S2 (Representative immunoblots demonstrating LRRK2 dependent Rab10Thr73phosphorylation in patient- and control-derived clinical samples), and Supplementary References. Please note: The publisher is not responsible for the content or functionality of any supporting information supplied by the authors. Any queries (other than missing content) should be directed to the corresponding author for the article.
Santinelli et al. (Mon,) studied this question.