Fig. 4

Age-dependent activation of the PINK1-parkin pathway in mitochondria from brains of APP-PSEN1-SREBF2 mice. Mitochondria-rich fractions were isolated from brain extracts of WT and the indicated transgenic mice at increasing ages. a and b Representative immunoblots showing the expression levels of PINK1, parkin and PARL in the mitochondria-rich fraction. PACT: C-terminal PARL fragment. c and d Phos-tag™ SDS-PAGE analysis of phospho-PINK (p-PINK1) in the mitochondria-rich fraction from WT and the indicated transgenic mice. As a control, samples were treated with 50 U of alkaline phosphatase (PP) for 1 h at 37 °C to inhibit phosphorylation. SREBF2 mice were treated with the autophagy inducer rapamycin (RAPA, 5 mg/kg, i.p.) for 24 h. PDH and TOMM20 were used to confirm equal loading. e Representative K63 polyubiquitin (K63-polyUb) western blot. K63-linkage specific polyubiquitin antibodies were used to detect K63 ubiquitination of proteins in the mitochondrial fractions. During mitochondria isolation, deubiquitination of the proteins was prevented by including 10 mM N-ethylmaleimide (NEM) in the isolation buffer. a, b, c, and e Densitometry of the bands representing specific protein immunoreactivity was assessed from samples grouped in the indicated range of age and values were normalized to the corresponding PDH or TOMM20 values. C-terminal PARL (PACT) fragment values were normalized to unprocessed PARL values (n = 3–4 per range of age and genotype). SF2: SREBF2 mice, A-P: APP-PSEN1 mice, A-P-SF2: APP-PSEN1-SREBF2 mice. See Supplementary Figure 18 for uncropped blots. f Hippocampal slices from 10-month-old WT and the indicated transgenic mice. Shown are confocal photomicrograph of K63-polyUb (green) and TOMM20 (red) immunoreactivity. To recover the mitochondrial GSH content, mice were treated with 1.25 mmol/kg of GSH ethyl ester (GSHee) every 12 h for 2 weeks. Nuclei were counterstained by DRAQ5 (blue). Scale bars: 25 μm. The Pearson’s correlation coefficient (PCC) was calculated from 3 random fields per condition. One-way ANOVA. *P < 0.05; **P < 0.01 (data are mean ± SD)