Fig. 5

Nilotinib ameliorates Vps13a^−/− mouse red cell features and its passage into brain across the BBB prevents Lyn activation and improves autophagy. a Left panel. Quantitation of acanthocytes by brightfield microscopic analysis on Vps13a^−/− and Vps13a^−/− mice treated with nilotinib (25 mg/kg/d for 6 weeks). Data from 50 visual fields was collected by two blinded researchers. Results are means ± SEM n = 6; *P < 0.05 versus WT; °P < 0.05 versus vehicle treated Vps13a^−/− by 2-way ANOVA with Bonferroni correction for multiple comparison. Right panel. Red cell distribution histograms generated for red blood cell volume (RBC Volume) and cell hemoglobin concentration (RBC-HC) of RBCs from wild-type (WT) control, Vps13a^−/− mice treated with nilotinib (25 mg/kg/d for 6 weeks). One experiment representative of six others with similar result is shown. The blue circle indicates the presence of a subpopulation of dense red cells, containing acanthocytes as described in human patients (see also Lupo et al. [42]). b Total Lyn was immunoprecipitated from red cell cytosol fractions of Vps13a^−/− mice treated with vehicle or with nilotinib (25 mg/kg/d for 6 weeks (6Ws)) and detected with antibody against active Lyn (phospho-Lyn 396) or antibody against total Lyn (Wb: Western-blot). The experiment shown is representative of 6 experiments. IgG is used as loading control as catalase in whole cell lysate (WCL). Lower panel. Densitometric analysis of the immunoblots; means ± SEM (n = 6; P < 0.05 vs. WT by t-test). c Quantification of nilotinib in isolated basal ganglia from wild-type (WT) and Vps13a^−/− mice treated either with vehicle or nilotinib. Data are means ± SD (n = 6; °P < 0.05 vs. vehicle treated Vps13a^−/− by 2-way ANOVA for multiple comparison). d Total Lyn was immunoprecipitated from basal ganglia of Vps13a^−/− mice treated with vehicle or with nilotinib (25 mg/kg/d for 6 weeks (6Ws)). The experiment shown is representative of 6 experiments, each from an individual Vps13a^−/− mouse and each with similar results. IgG and catalase are used as loading control. WCL: whole cell lysate. Lower panel. Densitometric analysis of the immunoblots; means ± SEM (n = 6; °P < 0.05 vs. WT by t-test). e Total Lyn was immunoprecipitated from basal ganglia of wild-type and Vps13a^−/− mice treated with vehicle or with nilotinib (25 mg/kg/d for 6 months (6Mo), 12 months old mice) and detected with antibody against active Lyn (phospho-Lyn 396) or antibody against total Lyn (Wb: Western-blot). The experiment shown is representative of 6 experiments, each from an individual Vps13a^−/− mouse and each with similar results. IgG is shown as loading control as well as GAPDH in whole cell lysate (WCL). Lower panel. Densitometric analysis of the immunoblots; means ± SEM (n = 6; *P < 0.05 vs. WT; °P < 0.05 vs. vehicle treated Vps13a^−/− by 2-way ANOVA with Bonferroni correction for multiple comparison). f Western blot (Wb) analysis of Ulk1 (Atg1), Beclin-1, Vps34, Rab5, p62, phospho-tau At8, and At180 and total tau in isolated basal ganglia from 18 months old wild-type, and Vps13a^−/− mice treated with either vehicle or nilotinib (25 mg/kg/d for 6 months (6Mo)). GAPDH was used as loading control (See Additional file 1: Fig. 14S for data on nilotinib treated 12 months-old mice). Right panel. Densitometric analyses of the immunoblot bands similar to those shown are presented at right. Data are means ± SEM (n = 6; *P < 0.05 vs. WT; °P < 0.05 vs. vehicle treated Vps13a^−/− by 2-way ANOVA with Bonferroni correction for multiple comparison). g Western blot (Wb) analysis of γ-Synuclein and Synaptotagmin in isolated basal ganglia from 12 months old wild-type, and Vps13a^−/− mice treated with either vehicle or nilotinib (25 mg/kg/d for 3 months (3Mo)) and 18 months old wild-type, and Vps13a^−/− mice treated with either vehicle or nilotinib (25 mg/kg/d for 6 months (6Mo)). GAPDH was used as loading control. Lower panel. Densitometric analyses of the immunoblot bands similar to those shown are presented. Data are means ± SEM (n = 6; *P < 0.05 vs. WT; °P < 0.05 vs. vehicle treated Vps13a^−/− by 2-way ANOVA with Bonferroni correction for multiple comparison)