Regional age-related changes in neuronal nitric oxide synthase (nNOS), messenger RNA levels and activity in SAMP8 brain

Abstract

Background: Nitric oxide (NO) is a multifunctional molecule synthesized by three isozymes of the NO synthase (NOSs) acting as a messenger/modulator and/or a potential neurotoxin. In rodents, the role of NOSs in sleep processes and throughout aging is now well established. For example, sleep parameters are highly deteriorated in senescence accelerated-prone 8 (SAMP8) mice, a useful animal model to study aging or age-associated disorders, while the inducible form of NOS (iNOS) is down-regulated within the cortex and the sleep-structures of the brainstem. Evidence is now increasing for a role of iNOS and resulting oxidative stress but not for the constitutive expressed isozyme (nNOS). To better understand the role of nNOS in the behavioural impairments observed in SAMP8 versus SAMR1 (control) animals, we evaluated age-related variations occurring in the nNOS expression and activity and nitrites/nitrates (NOx-) levels, in three brain areas (n = 7 animals in each group). Calibrated reverse transcriptase (RT) and real-time polymerase chain reaction (PCR) and biochemical procedures were used.

Results: We found that the levels of nNOS mRNA decreased in the cortex and the hippocampus of 8- vs 2-month-old animals followed by an increase in 12-vs 8-month-old animals in both strains. In the brainstem, levels of nNOS mRNA decreased in an age-dependent manner in SAMP8, but not in SAMR1. Regional age-related changes were also observed in nNOS activity. Moreover, nNOS activity in hippocampus was found lower in 8-month-old SAMP8 than in SAMR1, while in the cortex and the brainstem, nNOS activities increased at 8 months and afterward decreased with age in SAMP8 and SAMR1. NOx- levels showed profiles similar to nNOS activities in the cortex and the brainstem but were undetectable in the hippocampus of SAMP8 and SAMR1. Finally, NOx- levels were higher in the cortex of 8 month-old SAMP8 than in age-matched SAMR1.

Conclusion: Concomitant variations occurring in NO levels derived from nNOS and iNOS at an early age constitute a major factor of risk for sleep and/or memory impairments in SAMP8.

Figure 1

Biochemical characterization of nNOS during aging in the cortex, the hippocampus and the brainstem of SAMR1 and SAMP8. nNOS mRNA levels in arbitrary units (A.U.) (A), nNOS enzymatic activity in pmol of citrulline per minute per mg of protein (B) and Nitrites and nitrates (NOx) levels in nmol per mg of tissue (C) were detected in SAMR1 (solid line and open box) and SAMP8 (dotted line and dark box). Data are mean ± S.E.M) (n = 7 mice in each group). Statistics: significantly different from 2 months *P < 0.05, ** P < 0.01; or from 8 months ^aP < 0.05, ^bP < 0.01; significantly different from strain ^cP < 0.01.