Fig. 1

A53T SynGFP mice overexpress alpha-synuclein and the transgene localizes to neurons. a A53T SynGFP mice were generated by cloning the human SNCA gene, containing a point mutation at nt157 G > A causing an Ala53Thr change, into the MoPrp.Xho vector putting it under control of the murine prion promotor. SynGFP expression levels were determined by Western blot of brain lysate in WT and A53T (High) mice. Original data from Western blot also shows additional mouse lines that were not used in this study, including the A53T SynGFP (Low) line and the E46K SynGFP (High and Low) line. This blot was stained with antibodies to alpha-synuclein and GAPDH (loading control). Differences in apparent sizes of WT and mutant SynGFP proteins are due to the different linker regions used between alpha-synuclein and GFP. b Quantification of Western blot. SynGFP expression levels determined by Western blot of brain lysate indicate an overexpression of A53T SynGFP in this mouse line (High) of ~ 8-fold compared to a previously characterized human wild-type alpha-synuclein-GFP mouse line (WT SynGFP). Group data shows a significant difference in the normalized protein expression in the different transgenic mouse lines (one-way ANOVA (interaction F(4,5) = 570.1), p < 0.0001; Tukey’s multiple comparisons test, A53T (High) protein expression is greater than all other mouse lines: p < 0.0001 for all 4 comparisons, A53T (Low) protein expression is greater than E46K (Low) and WT: p = 0.0003 and p < 0.0001 respectively, E46K (High) protein expression is greater than E46K (Low) and WT: p = 0.0002 and p < 0.0001 respectively, and E46K (Low) protein expression is greater than WT: p = 0.007; A53T (Low) protein expression is not significantly different from E46K (High): p = 0.9047; N = 2 animals per line. c–e In A53T SynGFP transgenic animals, protein expression of the transgene was present in punctate staining in the neuropil and in relatively homogenous staining in a subset of cell bodies. Characterization of the cell types expressing SynGFP was determined using immunohistochemistry (IHC) and demonstrated that SynGFP is detectable in a subset of NeuN-positive cells (arrows), but rarely found in GFAP-positive or Iba1-positive cells. Arrow heads show SynGFP-negative cells. Scale bar equals 10 µm. f The majority of A53T SynGFP positive cells were NeuN-positive (99.7%), while a very small fraction of A53T SynGFP positive cells were GFAP-positive (0.3%), and none (0.0%) were Iba1 positive (N = 815 cells). g Of the total NeuN-positive cells, 55.1% were GFP positive, indicating expression of the A53T SynGFP transgene in a large fraction of this cell type population. 10.3% of GFAP-positive cells were GFP positive, and no Iba1-positive cells were GFP positive. Based on this characterization, expression of the A53T SynGFP transgene is significantly increased in neuronal cells, rather than non-neuronal cells, in these animals (Chi-square (2) = 106.4, p < 0.0001, N = 1553 cells)