NOS3 Inhibition Confers Post-Ischemic Protection to Young and Aging White Matter Integrity by Conserving Mitochondrial Dynamics and Miro-2 Levels

Abstract

White matter (WM) damage following a stroke underlies a majority of the neurological disability that is subsequently observed. Although ischemic injury mechanisms are age-dependent, conserving axonal mitochondria provides consistent post-ischemic protection to young and aging WM. Nitric oxide synthase (NOS) activation is a major cause of oxidative and mitochondrial injury in gray matter during ischemia; therefore, we used a pure WM tract, isolated male mouse optic nerve, to investigate whether NOS inhibition provides post-ischemic functional recovery by preserving mitochondria. We show that pan-NOS inhibition applied before oxygen-glucose deprivation (OGD) promotes functional recovery of young and aging axons and preserves WM cellular architecture. This protection correlates with reduced nitric oxide (NO) generation, restored glutathione production, preserved axonal mitochondria and oligodendrocytes, and preserved ATP levels. Pan-NOS inhibition provided post-ischemic protection to only young axons, whereas selective inhibition of NOS3 conferred post-ischemic protection to both young and aging axons. Concurrently, genetic deletion of NOS3 conferred long-lasting protection to young axons against ischemia. OGD upregulated NOS3 levels in astrocytes, and we show for the first time that inhibition of NOS3 generation in glial cells prevents axonal mitochondrial fission and restores mitochondrial motility to confer protection to axons by preserving Miro-2 levels. Interestingly, NOS1 inhibition exerted post-ischemic protection selectively to aging axons, which feature age-dependent mechanisms of oxidative injury in WM. Our study provides the first evidence that inhibition of glial NOS activity confers long-lasting benefits to WM function and structure and suggests caution in defining the role of NO in cerebral ischemia at vascular and cellular levels. White matter (WM) injury during stroke is manifested as the subsequent neurological disability in surviving patients. Aging primarily impacts CNS WM and mechanisms of ischemic WM injury change with age. Nitric oxide is involved in various mitochondrial functions and we propose that inhibition of glia-specific nitric oxide synthase (NOS) isoforms promotes axon function recovery by preserving mitochondrial structure, function, integrity, and motility. Using electrophysiology and three-dimensional electron microscopy, we show that NOS3 inhibition provides a common target to improve young and aging axon function, whereas NOS1 inhibition selectively protects aging axons when applied after injury. This study provides the first evidence that inhibition of glial cell NOS activity confers long-lasting benefits to WM structure and function.

Long-Term Results of Pharmacological Correction of Inos, Enos, Nnos Mrna Expression Disorders in Rat Hippocampus After Chronic Prenatal Hypoxia

“…Also, we found inhibition of eNOS mRNA expression in the hippocampus CA1 zone of 1.5-month-old rats undergoing CPH. This does not contradict the data of other researchers and the ideas about the cascade mechanism of ischemic neurodegeneration, which shows an increase in the expression of iNOS and nNOS [2,6,7,9,11,13,16]. The activity and expression of eNOS may vary with the time and severity of ischemic brain damage.…”

“…Expression of mRNA of the NOS endothelial isoform, according to the literature, demonstrates a contradictory nature. According to the numerous data, eNOS is activated at the beginning of hypoxic exposure, and at chronic oxygen deficiency its synthesis decreases [9,11,13]. Wang H. et al (2009) found that eNOS expression was reduced after OGD reperfusion injury.…”

“…Constitutive isoforms of NO synthase are constantly present in the corresponding cells, they are associated with their membrane proteins and have predominantly physiological significance, since the amount of nitric oxide formed by them is relatively small [12]. Neuronal NOS acts in the regulation of growth and differentiation of central nervous system (CNS) cells and, presumably, in their recovery after local ischemic brain damage [9,13]. Endothelial NOS plays a leading role in ensuring a constant baseline level of NO, which is associated with the implementation of local endothelial cytoprotection mechanisms and the maintenance of vascular homeostasis [3,9].…”

Pathophysiological Role of TRPM2 in Age-Related Cognitive Impairment in Mice

“…Memory function is one of the most vulnerable age-related characteristics (Larrabee and Crook, 1994). Previous reports show that aging results in damage to the white matter and hippocampus (Stahon et al, 2016;Bastian et al, 2018;Bishop and Yankner, 2010). However, it is not clear whether one is damaged to a greater or lesser extent than the other.…”

Common genetic variation indicates separate etiologies for periventricular and deep white matter hyperintensities

“…Some of these genes such as SH3PXD2A have been previously associated with total WMH and ischemic stroke 16,41 . The NOS3 gene is associated with coronary artery disease, migraine, vascular dysfunction, SVD, and ischemic stroke 29,43,51,52 .…”