Fig. 4.
MTSS1 is an ATXN2 translation target. (A, Left) Western blot of whole-cerebellum lysate from 24-wk-old mice shows 90% reduction (arrow) in the band that corresponds MTSS1 in ATXN2Q127 mice, while calbindin (CALB1) was reduced 50%. Actin is included as a loading control. (Right) Quantitation of Western blot results. *P < 0.01, **P < 0.001; Student’s t test. (B, Left) Western blots for active SFK-Y416 phosphorylation and total Src in cerebellar lysate from 24-wk-old Atxn2Q127 mice show an eightfold increase in SFK-Y416 abundance. Tubulin was used as a loading control. (Right) Quantitation of Western blot results. (C) Percent histograms of Purkinje neuron mean firing frequencies (Left), examples of extracellular recordings of 1-s duration of a spontaneously spiking Purkinje neuron in the respective condition (Center), and histograms of interspike intervals calculated for the 2-min recording periods of the same neuron for ATXN2Q127 and ATXN2Q127+dasatinib. (D) Mean firing rates. **P = 3.77E-8; one-way ANOVA and Tukey post hoc test. (E) Western blot for Atxn2 in cerebellar lysate from 4-wk-old MIMEX15 cerebellum and age-matched controls with tubulin as a loading control. (F) RNA-immunoprecipitation in HEK-293 cells for Flag-ATXN2Q22 and Flag-ATXN2Q108 shows enrichment for MTSS1 but not GAPDH mRNA. Error bars indicate SD. (G) Polyribosome fractionation in 293T cells transfected with the MTSS1 UTR reporter and pcDNA, ATXN2Q22, ATXN2Q108, or ATXN2Q22+ATXN2Q108. The green line indicates UV 254-nm absorbance (nucleic acids) with 40S, 60S, 80S, and polyribosome peaks labeled. (H) Remaining Purkinje neurons in human SCA2 cerebellum (ATXN2Q22/Q41) show reduced MTSS1 staining compared with an age-matched control (ATXN2Q22/Q22).