Fig. 2.

Lentiviral-based overexpression of MUT ATXN7 induces cell loss and degeneration in the mouse cerebellum. a–e Calbindin immunostaining revealed loss of Purkinje cells (PC) in mice injected with an LV-MUT-ATXN7, 2 and 12 weeks post-injection (D and E); in mice injected with PBS or an LV-WT-ATXN7 no cell loss was observed at either 2 weeks or 12 weeks post-injection (A, B and C). f Quantification: in transduced regions, the number of calbindin-positive PCs decreased ~19 %, 2 weeks post-injection and ~64 % 12 weeks post-injection in MUT ATXN7 transduced regions, whereas injection with PBS or overexpression of WT ATXN7 did not affect calbindin-positive PCs. Values are expressed as mean ± standard deviation (SD). g and h Laser confocal microscopy showing the preservation of calbindin-positive PCs (red) in regions transduced with WT ATXN7 displaying diffuse immunoreactivity (green) (arrow in G); PCs (red) were depleted in areas transduced with LV-MUT ATXN7, attested by the presence of ATXN7-positive aggregates (green). i–m Cresyl violet staining showed preservation of PCs and GCL in mice injected with PBS or LV-WT-ATXN7, whereas loss of PCs and shrinkage of the GCL (L and M) were observed in mice injected with MUT ATXN7, at 2 and 12 weeks post-injection. n–o Quantification of GCL and ML thickness: overexpression of MUT ATXN7 induced a shrinkage of the GCL (~31 %) (M and N) and ML (~33 %) (M and O) near the injection site, 12 weeks post-injection. PBS injection or overexpression of WT ATXN7 did not affect PCs, the ML or the GCL thickness (I, J, K, N and O). Statistical analyses of calbindin immunostaining and cresyl violet staining were performed by one-way ANOVA followed by a post-hoc Fisher’s test. Values are expressed as mean ± standard deviation (SD). p–q Neurotracer staining is decreased in PCs and GCL (Q) in mice injected with LV-MUT-ATXN7 compared to mice injected with LV-WT-ATXN7 (P). r–s Fluoro-Jade B staining shows increased number of degenerating neurons in mice injected with LV-MUT-ATXN7 (S) compared to mice overexpressing WT ATXN7 (R). t–u The number of caspase-3-positive cells increased in cerebellar regions injected with LV-MUT-ATXN7 (U). Bars: A–E: 100 μm; G and H: 100 μm; I–M: 50 μm; P–Q: 20 μm; R–S: 50 μm; T–U: 20 μm