Figure 1: Properties of limbic α-synucleinopathy in humans, rats, and mice.

Sprague-Dawley outbred rats were bilaterally infused with preformed fibrils (n = 20; 15 μg in 3 μL) or an equivalent volume of phosphate-buffered saline (PBS; n = 12) in the OB/AON at 4 months of age (a-b). Four months later, hemibrains were immersion-fixed in formalin, sectioned in the sagittal plane, and immunostained with clone EP1536Y against pSer129 α-synuclein via TSA amplification as in [4]. Arrow in a points to tissue disturbance along the needle track, stained with the pan-nuclear Hoechst reagent. Panel b was imaged at 5-micron resolution on an Odyssey M (extended in Fig. S1). The other hemisphere was reserved for biochemical assays (f-h). Infusion sites in additional animals are included in Fig. S1. Insoluble fractions were extracted from postmortem limbic tissues of humans with (n = 13) or without (n = 13) diagnoses of Lewy body disorders (c-e), or from limbic tissues of rats and mice infused in the OB/AON with PBS (control) or preformed fibrils (f-k). For panels i-k, outbred mice were bilaterally infused in the OB/AON with preformed fibrils (5 μg in 1 μL; n = 10) or an equivalent volume of PBS (n = 10) at 9–11 months of age. Six weeks post-infusion, amygdala and piriform cortical tissues were collapsed for ultracentrifugation and detergent-based fractionation. Recombinant α-synuclein monomers were loaded onto the last lane in panel i as a negative control for the phospho-specific EP1536Y antibody. LBD = Lewy body disorder; A.U. = arbitrary units. Triton X-100 = Tx. Each subject is illustrated as a colored dot as the statistical unit. Two-tailed, multiplicity-adjusted p values per two-way ANOVA/Bonferroni are shown, and factor effects are listed. For details on methods and subject numbers, please consult the Supplemental Files. Gaussian/heteroscedastic raw data that failed assumptions of parametric testing are also in the Supplemental Files.