Fig. 4

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