Age-related changes in mitochondrial respiration and oxidative damage in the cerebral cortex of the Fischer 344 rat

Abstract

This study probed possible age-related changes in mitochondrial bioenergetics in naïve Fischer 344 rats. Synaptic and extrasynaptic mitochondria were isolated from the cortex of one hemisphere of young (3-5 months), middle (12-14 months), or aged (22-24 months) rats. Respiration parameters were obtained using a Clarke-type electrode. Aged rats displayed no significant alterations in respiration, indicating mitochondria must be more resilient to the aging process than previously thought. Synaptic mitochondria displayed lower respiration capacities than the extrasynaptic fraction. Aged F344 rats appear capable of normal electron transport chain function without declines in ability to produce ATP. Markers of cortical oxidative damage (3-nitrotyrosine [3-NT], 4-hydroxynonenal [4-HNE], and protein carbonyls [PC]) were collected from the post-mitochondrial supernatant (PMS) from the contralateral hemisphere, and from mitochondrial samples following respiration analysis. Age-related increases in PC and 3-NT levels were found in synaptic mitochondria, whereas significant extrasynaptic elevations were only found in middle aged rats. These findings support an age-related increase in oxidative damage in the cortex, while proposing the two fractions of mitochondria are differentially affected by the aging process. Levels of oxidative damage that accumulates in the cortex with age does not appear to significantly impair cortical mitochondrial respiration of F344 rats.

Fig. 1

Typical oxygen utilization traces of synaptic and extrasynaptic mitochondria isolated from a naïve cortex of an aged animal. state I: no substrates for respiration added; no oxygen utilization apparent. state II: addition of P/M; basal rate of respiration. state III: two separate additions of ADP; each addition connects the ETC with oxidative phosphorylation; high level of oxygen utilization indicated ADP being converted to ATP. state IV: addition of oligomycin; electrons are blocked from returning into the matrix through the ATP synthase, the ETC slows only to maintain mitochondrial membrane potential that is lost through inner membrane into the matrix, and oxygen utilization is greatly reduced. state V: addition of FCCP; results in an uncoupling of the ETC to ATP synthesis, represents maximum rate of respiration since no bottle-necking occurs at ATP synthase - protons are allowed to rush back into the matrix. Rotenone is then added to shut down complex 1-driven respiration. state V (succinate): addition of succinate; maximum rate of respiration via complex 2 due to presence of FCCP still in the system.

Fig. 2

Overall oxygen utilization rate (nmol 0₂/min/mg). The overall oxygen utilization rate was not significantly different in either mitochondrial fraction as a function of age. Bars represent group means ± SD; numbers inside the bars represent sample size.

Fig. 3

Actual states of respiration for synaptic and extrasynaptic fractions. a. state II – P/M b. state III – ADP c. state IV – Oligomycin d. state V – FCCP e. state V – Succinate. f. respiratory control ratio (RCR) – which is an index of how coupled respiration is to ATP production and is calculated by dividing state III by state IV. Bars represent group means ± SD; numbers inside the bars represent sample size.

Fig. 4

Markers of oxidative damage are expressed as a percent of values observed in young animals. Relative optical densities of protein carbonyls (PC) were determined in the a. post mitochondrial supernatant (PMS) and in b. purified mitochondrial fractions (synaptic and extrasynaptic). Relative levels of 4-hydroxynonenal (4-HNE)-protein adducts, the most prevalent toxic peroxidation product formed during oxidant stress, were assessed in the c. PMS and d. both mitochondrial fractions. Relative levels of nitrotyrosine (3-NT), a biomarker of reactive nitrogen species formation, were measured in the e. PMS and f. both mitochondrial fractions. Bars represent group means ± SD; numbers inside bars represent the sample size. * p < 0.05; ** p < 0.01; ***p < 0.001 compared to young. # p < 0.01 compared to middle aged rats.

Fig. 5

The relationship between the levels of oxidative damage (PC, 4-HNE, and 3-NT) and the RCR values in a. synaptic mitochondria and c. extrasynaptic mitochondria. The relationship between the levels of oxidative damage and the overall oxygen utilization rates in b. synaptic mitochondria and d. extrasynaptic mitochondria. Symbols represent individual values. * p < 0.05, ** 0.01, *** 0.005.