PREP co-expression promotes formation of soluble aSyn dimers in N2A cells.
A, schematic presentation of GLuc fragment-tagged aSyn reporter constructs used in this study. B, the effect of co-expression of PREP or PREP(S554A) with aSyn was studied by PCA. aSyn-GLuc1 and aSyn-GLuc2 were kept constant (31.25 ng of plasmid DNA per well each), whereas increasing amounts of PREP or PREP(S554A) plasmid were transfected (12.5, 37.5, and 62.5 ng). The total amount of plasmid was adjusted to 125 ng per well with mock plasmid when needed. Both PREP and catalytically inactive PREP(S554A) significantly increased the dimerization of aSyn with increasing levels of expression (one-way ANOVA; ***, p < 0.001 PREP versus control). The average values are displayed as percent change as compared with control (cells expressing aSyn-GLuc1 and aSyn-GLuc2 only; mean ± S.E.; n = 4 independent experiments). C, the effect of KYP-2047 on aSyn dimerization in PREP-expressing cells was studied by PCA. Cells transfected with aSyn-GLuc1 and aSyn-GLuc2 (31.25 ng each) and PREP or PREP(S554A) (37.5 ng) were treated with at 1, 5, or 10 μm KYP-2047 for 4 h. Both 5 and 10 μm doses of KYP-2047 significantly decreased aSyn dimerization when incubated with active PREP (one-way ANOVA; *, p < 0.05 5 μm KYP-2047 versus control; **, p < 0.01 10 μm KYP-2047 versus control), but KYP-2047 did not show any effect on aSyn dimerization in PREP(S554A)-expressing cells. DMSO was used as vehicle control. The average values are displayed as percent change as compared with control (mean ± S.E.; n = 3 independent experiments). D, the effect of KYP-2047 on aSyn dimerization without co-expression of PREP was studied by PCA. Cells transfected with aSyn-GLuc1 and aSyn-GLuc2 (31.25 ng each) were treated with 10 μm KYP-2047 for 4 h. No change in aSyn dimerization was observed. DMSO was used as vehicle control. The average values are displayed as percent change as compared with control (mean ± S.E.; n = 4 independent experiments). E, solubility of aSyn reporter proteins with co-expression of PREP or PREP (S554A) and KYP-2047 treatment was studied by cellular fractionation and Western blot. Cells transfected with aSyn-GLuc1 and aSyn-GLuc2 (0.75 μg each), PREP, or PREP(S554A) plasmids were transfected at 1.5 μg. 5 μm KYP was added for 24 h. No shift of aSyn reporters from Triton-soluble (S) to insoluble fractions (I) was observed. Blots were stained with antibodies to PREP, HA tag (aSyn-GLuc1/2), and GAPDH as a loading control.