Kinetically stable alpha-synuclein oligomers (SPR-484) are stable after a freeze-thaw cycle and when incubated at 37 oC for 2 weeks. Tris-Gylcine Native PAGE migration of kinetically stable alpha-synuclein oligomers after a freeze-thaw cycle (A). Size-exclusion chromatography of SPR-484 after a freeze-thaw cycle (B) and 37oC incubation for 14 days (C). By peak area, approximately 90% of SPR-484 remains oligomeric after freeze-thaw and 37oC treatments. SEC was performed on Superdex 200 10/300 GL Increase column in phosphate buffer pH 7.4. Note: Monomeric alpha-synuclein is an intrinsically disordered 14 kDa protein. Due to its extended conformation in solution, migration of free monomeric alpha-synuclein is similar to that of a globular 60 kDa protein on Native PAGE and SEC.

Kinetically stable alpha-synuclein oligomers (SPR-484) are toxic to dopaminergic neurons and induce phosphorylation of alpha-synuclein Ser129, a pathology associated with Parkinson’s disease. Survival of rat primary dopaminergic neurons 11 days after treatment quantified by anti-MAP2 antibody and expressed as a percentage of control (A). Levels of alpha-synuclein pSer129 present in rat primary dopaminergic neurons 11 days after treatment quantified by ratio of anti-alpha-synuclein pSer129 antibody to anti-MAP2 antibodyexpressed as a percentage of control (B). Mean +/- s.e.m; ** p<0.01 stats vs control, one-way Anova followed by Dunnett’s test; § p<0.05, stats vs control, Student’s t-test. Data is representative of n=6 experimental repeats for each condition; # represents control, n.s. indicates not significant p>0.05.

Representative immunohistochemistry images of Parkinson’s-associated pSer129 pathology induced in rat primary dopaminergic cells by kinetically stable alpha-synuclein oligomers (SPR-484). Primary rat dopaminergic neurons 11 days after treatment with control PBS buffer (A). Primary rat dopaminergic neurons 11 days after treatment with 10 µg/mL SPR-484 (B). Nuclei appear blue (Hoechst), dopaminergic neurons appear red (MAP2) and pathology appears green (α-syn pSer129). Both cultures treated with chicken polyclonal anti-MAP-2 antibody, mouse monoclonal anti-αsyn pSer129-specific antibody, Alexa Fluor 488 goat anti-mouse IgG, Alexa Fluor 647 goat anti-chicken IgG, and fluorescent marker Hoechst in the same solution. White arrows emphasize several regions of strong pSer129 pathology. Scale bar represents 100 μm. Note: SPR-484 is generated recombinantly in E.coli and is non-phosphorylated prior to addition on neurons.