Fig. 1

Summary of approach. A Depiction of specimen sampling for studies. Longitudinal image of the right hemisphere of the human brain with the approximate location of sampling sites shown (black lines). For BAR, a single coronal section through the Caudate Nucleus and Putamen (Forebrain) and a single transverse section through the midbrain were pooled and used. B Depiction of studies. The distribution of αsyn aggregates is distinct between the synucleinopathies PD/DLB and MSA. For PD/DLB, αsyn aggregates are observed prominently in neuronal cell bodies and projections, termed neuronal inclusions (NI). In contrast, in the MSA brain, αsyn aggregates occur prominently in glia, termed glial inclusions (GI). Biotinylation by antibody recognition (BAR) was used to identify and compare interactomes of total αsyn (BAR-MJFR1) and aggregated αsyn (BAR-PSER129) directly in the PD/DLB and MSA brain. MJFR1 antibody maps to an epitope of a.a. 118–123 of αsyn’s c-terminus and captures physiological monomeric forms and aggregates. PSER129 preferentially labels αsyn aggregates, especially in the postmortem brain where physiological PSER129 is scarce [8], and thus, BAR-PSER129 will capture aggregate interactions. LC–MS/MS identified BAR-labeled proteins and differential abundance analysis was used to determine interactors (i.e., proteins enriched over BAR-NEG) and disease-enriched proteins (PD/DLB vs. MSA). The resulting proteins were analyzed using a combination of approaches: overlap analysis, pathway enrichment mapping, and protein interaction network mapping. IHC staining for C PSER129 and D αsyn (i.e., MJFR1) in forebrain and midbrain sections. Sections were stained using nickel-DAB chromogen (black) and counterstained with methyl green (green). Whole-section scans and high-magnification images of select pathology-bearing regions are shown with red and blue boxes denoting the approximate area of the high-magnification image. Signal thresholding was applied to 20X images of PSER129-stained tissues, specifically in gray matter (GM, red box) and white matter (WM, blue box) in the forebrain and midbrain for quantification. Enlarged 20X images showing the application of thresholding E and the subsequent quantification of PSER129 immunoreactivity across all cases F. Scale bars: C, D = 2 mm and 25 µm; E = 2 mm and 20 µm. MSA, n = 5; PD/DLB, n = 10.(mean ±SEM  *Tukey’s multiple comparisons test p-adj. < 0.05)