Initial evidence linking synaptic superoxide production with poor short-term memory in aged mice

Abstract

Unregulated production of reactive oxygen species (ROS) is a marker of cellular and organismal aging linked to cognitive decline in humans and rodents. The sources of elevated ROS contributing to cognitive decline are unknown. Because NADPH oxidase (Nox) inhibition may prevent memory decline with age, we hypothesized that Nox and not mitochondrial sources of synaptic ROS production are linked to individual variance in cognitive performance in aged mice. Young (8 months) and aged (26 months) mice were tested in the novel object recognition task (NORT). Mitochondrial and Nox ROS production was assayed in isolated synaptosomes using spin trapping electron paramagnetic resonance (EPR) spectroscopy. Aged mice exhibited variance in NORT performance, with some performing similar to young mice while others exhibited poorer short-term memory. EPR studies indicated that Nox rather than mitochondria was the major ROS source at the synapse, and Nox-induced but not mitochondrial-induced ROS levels correlated with NORT performance in aged mice. Our findings support the hypothesis that variance in Nox-specific synaptic ROS production may predict short-term memory deficits with age.

Figure 1

Mice exhibit variance in age-related novel object recognition performance. One-trial novel object recognition was assessed across young (8 mo) and aged (26 mo) male C57BL6N mice. (A) Percentage time exploring the novel object compared to total object exploration during choice phase. Data are presented as mean ± SEM, **p<0.001, main effect of age. (B) Individual variance in novel object recognition across aged and young mice. N=13-15/group. Dashed line depicts the cutoff criterion (2 standard deviations below mean performance in young mice) for grouping aged mice into “impaired” and “unimpaired” categories. The mice assigned for EPR spin trapping analysis are depicted using open symbols.

Figure 2

EPR spin trapping spectroscopy elucidates that Nox is a major source of superoxide in synaptosomes with minor contribution from synaptosomal mitochondria in young and old mice brains. (A) Representative EPR spectra recorded over ~ 6 minutes from mixing of 5 mM NADPH with 0.2–0.5 mg synaptosomal protein isolated from brain of age-impaired (gray lines) or age-unimpaired (black lines) mice at 37°C with 70 mM DEPMPO. In the incubations, 10 mM DETC was included to inhibit the SOD enzyme resulting in enhanced, and hence, quantifiable EPR signals. (B) Observed superoxide bursts upon adding NADPH in all groups (solid lines) and the effect of inclusion of the Nox2 inhibitor apocynin (300 µM final concentration, dashed lines), n=6, 5, and 4 for young, age-impaired, and age-unimpaired groups, respectively. See methods for details. Data are mean ± SEM. (C) Representative EPR spectra of superoxide production from metabolically active synaptosomal mitochondria in the same preparations under the same conditions except that Nox substrate is replaced by mitochondria ones. State 3 respiration was initiated by the addition of 10 mM malate + 10 mM pyruvate. Addition of ADP was not necessary to initiate state 3 oxygen consumption indicating that synaptosomes contain a pool of the ATPase substrate. The mitochondrial superoxide signals were quantified in panel (D). (E) Novel object recognition performance correlates with the yield Nox-produced superoxide at the synapses in old but not in young brains. Area under superoxide bursts’ EPR signals over the first 6 minutes from activity initiations were collected and normalized by the total synaptosomal protein contents for each group. Linear regression analysis is only applied on the old groups and the solid line represents the least-square best fit for the 9 data points obtained for the two old groups. Statistically significant linear negative correlation was obtained (R = −0.772, SD = 8.64, p = 0.01) with the general trend of higher superoxide yields in old brains from animals with lower recognition performance. Young animals however performed consistently better in the behavioral test independently of the superoxide levels in their synaptosomes.