p66Shc Inactivation Modifies RNS Production, Regulates Sirt3 Activity, and Improves Mitochondrial Homeostasis, Delaying the Aging Process in Mouse Brain
Effects of aging on mitochondrial content, biogenesis, and structure in p66Shc(−/−) mouse brain. (a) Quantification of mtDNA and nuclear DNA by qPCR. (b) The mtDNA/nDNA ratio was calculated from 3- and 24-month WT (grey) and p66Shc(−/−) (black) groups. The mRNA levels of PGC-1α were measured in TRIzol-treated brain extracts from all the studied groups; GADPH mRNA was used as standard (). (c) Mitochondrial morphology was evaluated using electron microscopy of fixed brain slices ( for each experimental group) (magnification:×20,000). (d) At least 300 tubular and fragmented mitochondria were counted per arbitrary area. The percentage distribution of tubular and fragmented brain mitochondria was determined in a minimum of 8–10 random fields at ×4400 magnification to ensure a representative area of analysis (). Mitochondria whose length was more than three times their width were considered tubular, while the remaining round mitochondria were considered fragmented. The analysis was performed by two different investigators in a blinded fashion. Values represent the ; A represents compared to the 3-month-old group, B represents between 24-month-old WT and p66Shc(−/−) groups, one-way analysis of variance (ANOVA) and Bonferroni post hoc test.