Figure 7

Proteolytic processing of p75NTR in the cerebral cortex of EPM2A -/- mice at different ages. A) Proteolytic processing of p75NTR (15 days of age). Representative western blot developed against cytoplasmic domain of p75NTR (ICD). The blot revealed fragments at 24 kDa consistent with the p75NTR-CTF, and a fragment at 19 kDa consistent with p75NTR-ICD, which was also weakly observed in the wild-type. We also detected an increase of p75NTR and ICD-p75 cleavage (*P < 0.05). Comparable amounts of samples are loaded, however α-actin was also used as a loading control. B) Quantification of the ratio ICD-p75NTR/ECD-p75NTR (15 days of age). C) Confocal images of ICD-p75NTR in GAD67 positive neurons D) There is not translocation of ICD units to nucleus in the cerebral cortex of mice with 15 days of age. We previously separated cytoplasm and nuclear fractions and we observed that there is not translocation of p75NTR-ICD and Notch-ICD to the nucleus. Representative blot of ICD units are shown (n = 5). As a loading control of cytoplasm fraction we used actin and as a loading control of nuclear fraction was used NeuN. E) Changes of soluble proteins in the cerebral cortex (n = 5). Low speed centrifugation of tissue extracts was used to isolate soluble fraction, which was analyzed by western blotting. These representative blots revealed that the main changes between wild-type and EPM2A-/- mice were noticed at 15 days-old and at 1 month-old. There was a significant decrease in the proteins implicated in neuroprotection and neurorepair: TTR, ApoJ, BDNF, NGF, VEGF; and there was a significant increase in proteins implicated with neuropathological situations: β-Amyloid 1-40 and 1-42 and MMP9. Results are mean ± SEM *p < 0.05, **p < 0.01 wild-type mice vs EPM2A -/- mice (ANOVA followed by Student’s t test).