Fig 2: Development of sensitive single-molecule array assays and standards for fluid measures of total LRRK2, total Rab10, and pT73-Rab10. (A) AlphaFold structure of Rab10 and (B) LRRK2. Specific epitopes selected for the quantification of total LRRK2, total Rab10, and pT73-Rab10. The assay for Rab10 and pT73-Rab10 share the same detector antibody on the C-terminus but differ in capture antibody epitopes. (C) Representative SDS-PAGE gels with Coomassie stain (blue) for the quantification of recombinant His-Rab10 (derived from E. coli cells) and FLAG-G2019S-LRRK2 (derived from HEK-293T cells). Recombinant bovine-serum albumin standards are shown. (D) Representative regression curves for the measurement of recombinant Rab10 and (E) recombinant LRRK2 in the assay buffer. Goodness-of-fit (r-squared), limit-of-detection (LOD), and lower-limit of quantification (LLOQ) are indicated and calculated from duplicate and triplicate values for each assay point. (F) Representative phos-tag immunoblot analysis showing the proportion of Rab10 protein phosphorylated in protein lysates. HEK-293T lysates were generated through transient transfection of plasmids expressing FLAG-R1441G-LRRK2 with human FLAG-Rab10. The ratio of pT73-Rab10 to total Rab is calculated from three independent preparations as 15.2% ± 0.9% SEM. Protein preparations analyzed by phos-tag analysis are utilized as standards for single-molecule arrays (SiMOA). (G) Representative regression curves for the analysis of LRRK2, (H) Rab10, and (I) pT73-Rab10, as measured in diluted (sample buffer) HEK-293T protein lysates. Prior to lysis, cells were treated with MLi2 (200 nM, 30 min, red dots and lines) or vehicle (blue dots and lines). Assay quality parameters (orange font) are indicated based on triplicate values. Error bars for experimental replicates are typically less than 5% and too short to be visualized graphically in most of the plots

Fig 3: Sepsis increases the extracellular ratio of pT73-Rab10 to total Rab10 in serum that primarily localizes to exosome-enriched serum fractions. (A) Microbe profile of cecal slurry generated from healthy CD-1 outbred male mice. Percentages of phylum-level taxonomy, identified by 16S rRNA amplicon sequencing, are depicted with the “Other” category including Actinobacteria, Cyanobacteria, and Verrocomicrobia. Slurries were injected intraperitoneally into a cohort of outbred male (n = 8) and female (n = 16) CD-1 mice, with serum procured from facial vein draws. At the 8 hr time point after injection, 4 female and 4 male mice were selected at random to measure serum (B) interferon-γ (IFNγ) and (C) TNF with ELISA analysis. (D) Before and after graphs show changes in serum LRRK2 levels, (E) total Rab10 and (F) the ratio of pT73-Rab10 to total Rab10 72 hrs post-injection. P values shown are from paired samples (before and after) t-tests. (G) Procedure for “Exo-Spin Serum mini columns’’ size-exclusion fractionation of 100 microliters of human serum spread into fractions 1–3. (H) Proteomic analysis of eluted fractions via mass spectrometry assessments of the relative abundance of unique peptides associated with characteristic serum exosome proteins CD9 (plasma-membrane protein enriched in exosomes and extracellular vesicles), myeloperoxidase (a soluble myeloid-cell produced enzyme enriched in exosomes and extracellular vesicles), and a small soluble cytokine HCC-1 (CCL14). (I) Column graphs show levels of LRRK2, (J) Rab10, (K) and pT73-Rab10 as measured in three different human biobanked serum samples (one male and one female with PD, and one healthy control). Green bars are measured from fraction one, yellow bars are fraction two, and purple are fraction three. Columns show group mean and error bars show SEM

Fig 4: The ratio of pT73-Rab10 to total Rab10 in serum is elevated in mouse models of late-onset PD. (A) Levels of total LRRK2, (B) total Rab10 and (C) the ratio of pT73-Rab10 to total Rab10 as measured by SiMOA in venous serum from VPS35WT/WT (2 males and 4 females) mice, VPS35WT/D620N (4 males and 4 females) mice, and VPS35D620N/D620N (6 males) mice. (D) Graphs show numbers of neutrophils (CD11b + Ly6G+), (E) classical monocytes (CD11b + Ly6G- Ly6C+), and (F) CD4 T-cells measured by flow cytometry in VPS35WT/WT and VPS35D620N/D620N serum samples. (G) Representative immunoblots and quantification of cultured bone marrow derived macrophages demonstrating elevated pT73-Rab10 to total Rab10 levels with mutant VPS35 expression. (H) Levels of total LRRK2, (I) total Rab10 and (J) the ratio of pT73-Rab10 to total Rab10 from Snca−/− (3 males and 6 females) mice, and PAC-SNCA/Snca−/− (5 males and 4 females) mice. (K) Graphs show numbers of neutrophils (CD11b + Ly6G+), (L) classical monocytes (CD11b + Ly6G- Ly6C+), and (M) CD4 T-cells measured by flow cytometry in Snca−/− and PAC-SNCA/Snca−/− mice blood. (N) Representative immunoblots and quantification of cultured bone marrow-derived macrophages demonstrating low but equivalent pT73-Rab10 to total Rab10 between Snca−/− and PAC-SNCA/Snca−/− mice. Mouse ages ranged from 3–6 months. Columns depict group mean, dots represent the mean values from an animal or independent experiment, and error bars show SEM. P values are from two-tailed t-tests or one-way ANOVA analysis with Tukey’s multiple comparison when three groups are tested

Fig 5: LRRK2 protein levels predict pT73-Rab10 levels that are elevated in PD cases with worse MDS-UPDRS scores. (A) Scatter plots show relationships between measured serum levels of total Rab10 and LRRK2, (B) pT73-Rab10 and LRRK2, and (C) the ratio of pT73-Rab10 to Rab10 and LRRK2. N = 522 cases and controls (see Table 1). R values show correlation coefficients and p values correspond to mean linear regression analysis of lines (red) with 95% confidence intervals shown (red dashed curves). (D) Box and whisker plot comparing the mean MDS-UPDRS part III scores in PD cases split to lower (< 0.5) and higher (> 0.5) ratios of pT73-Rab10/Rab10. P value indicated is from a Mann-Whitney U test for comparisons of groups with unequal sizes