Aging‐induced Mitochondrial Dysfunction in the Mouse Brain Microvessels: Effect of Neuronal Nitric Oxide Synthase Inhibition
Authors: Sure Venkata NLR. , Sakamuri Siva SVP. , Sperling Jared A. , Merdzo Ivan , Mahalingam Padmini S. , Evans Wesley R. , Katakam Prasad VG. 
Published: June 2021

Aging‐induced cerebrovascular changes contribute to the increased risk of stroke and neurodegenerative diseases. Notably, aging‐induced impairments of cerebral microcirculation have been implicated in cognitive dysfunction. Mitochondrial dysfunction plays an important role in the age‐related cerebrovascular changes; however, mitochondrial respiratory dysfunction has never been studied in the microvasculature. Similarly, neuronal nitric oxide synthase (nNOS) is known to play a critical role in various cells of cerebrovasculature, but its effects on mitochondrial respiration in the young and aged brain microvessels have never been examined previously. Here, we studied the mitochondrial respiration and the responses to nNOS inhibition in the in the aged‐microvessels from mouse brain.

Methods

We isolated the microvessels from the saline‐perfused brains from young (3 months of age) and aged mice (18 months of age) and collected microvessels by a combination of gradient centrifugation and filtration (40μm – 300μm). Isolated microvessels were treated with saline or nNOS inhibitor, ARL‐17477 (1μM, ARL) and plated into the wells of Seahorse XFe24 microculture plate. Oxygen consumption rate (OCR) was measured at the basal level and after the injection of oligomycin, FCCP and antimycin/Rotenone. Basal and maximal respiration were measured along with ATP production, proton leak, and maximal respiration. We also measured the reactive oxygen species (ROS) levels by electron spin resonance spectrometry (ESR).

Results

Aged‐microvessels showed 40% decrease in the basal respiration, whereas maximal respiration is decreased by 49% when compared with the microvessels from the young mouse brain. ATP production, proton leak, and non‐mitochondrial respiration were decreased by 36%, 40%, and 72% respectively in the aged‐microvessels when compared to the young vessels. Aged‐microvessels also showed increased ROS levels by 38.6%. ARL treatment increased the maximal respiration by 21.2% (101.8±6.4 vs 84.0±8.9 picomoles of O2/min/μg, p<0.05) and ATP production by 19% (52.3±4.4 vs 43.9±3.6 picomoles of O2/min/μg) in mouse brain microvessels. Basal respiration, proton leak, and non‐mitochondrial respiration were not altered by ARL treatment in the microvessels in young mice. In contrast, none of the respiratory parameters were affected by ARL in the aged‐microvessels. Interestingly, nNOS inhibition significantly decreased ROS levels in microvessels from both young and old mice compared to the respective untreated microvessels (p<0.05). Notably, ROS levels following nNOS inhibition was significantly greater in microvessels from young than aged mice (p<0.05) indicating a reduction of nNOS function with aging.

Conclusions

Our study showed that mitochondrial respiratory parameters were compromised in the aged‐microvessels from the mouse brain, indicating a possible role of mitochondrial dysfunction in the aging‐associated cerebral microvascular impairments. In addition, nNOS appears to negatively regulate mitochondrial respiration in the microvasculature of the young mouse brain but the nNOS effect on mitochondrial respiration was lost in aged brain microvessels

Support or Funding Information

American Heart Association (PVG: 14SDG20490359 and VNS: 16PRE31450006), and National Institute of Health: (PVK: R01NS094834)

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.