The impact of aging on CNS white matter (WM) is of general interest because the global effects of aging on myelinated nerve fibers are more complex and profound than those in cortical gray matter. It is important to distinguish between axonal changes created by normal aging and those caused by neurodegenerative diseases, including multiple sclerosis, stroke, glaucoma, Alzheimer's disease, and traumatic brain injury. Using three-dimensional electron microscopy, we show that in mouse optic nerve, which is a pure and fully myelinated WM tract, aging axons are larger, have thicker myelin, and are characterized by longer and thicker mitochondria, which are associated with altered levels of mitochondrial shaping proteins. These structural alterations in aging mitochondria correlate with lower ATP levels and increased generation of nitric oxide, protein nitration, and lipid peroxidation. Moreover, mitochondria-smooth endoplasmic reticulum interactions are compromised due to decreased associations and decreased levels of calnexin and calreticulin, suggesting a disruption in Ca 2ϩ homeostasis and defective unfolded protein responses in aging axons. Despite these age-related modifications, axon function is sustained in aging WM, which suggests that age-dependent changes do not lead to irreversible functional decline under normal conditions, as is observed in neurodegenerative diseases.

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.

White matter (WM) is frequently affected by stroke. Aging WM recovers less following stroke compared to younger WM. Free radicals accumulate with aging resulting in increased oxidative stress. Mitochondria/endoplasmic reticulum (ER) interactions are crucial for protection from oxidative stress. We hypothesized that aging WM is more susceptible to oxidative stress damage mediated by mitochondrial/ER dysfunction, leading to decreased ATP synthesis and impaired Ca2+ homeostasis. Mouse optic nerves (MON) obtained from C57BL/6J and Thy-1 CFP(+) mice at 1 (young) or 12 months (aging) of age were used. Axon function was quantified by recording evoked compound action potentials. Mitochondrial/ER structure and interactions were determined by 3D-electron microscopy, Western blotting, ATP assays, and CFP (+) fluorescent mitochondrial imaging. Oxidative stress was assessed by NOS and glutathione assays and by Western blotting for oxidative stress markers, 3-NT and 4-HNE. Oxygen-glucose deprivation (OGD) for 60 min resulted in decreased axon function recovery of aging WM. Aging WM showed elevated NOS activity and increased levels of by-products of lipid peroxidation (4-HNE) and protein nitration (3-NT), indicating aggravated oxidative stress. Structurally, aging axons were larger, with thicker myelin, and were characterized by longer and thicker mitochondria due to altered levels of mitochondrial shaping proteins. This was further confirmed by 3D-EM and CFP (+) imaging and may underlie the decreased ATP levels detected in aging WM. Moreover, mitochondrial-ER interactions were compromised due to decreased association between the organelles and due to decrease in levels of mitochondrial trafficking protein miro-2, which suggests defective Ca2+ homeostasis in aging axons. Calnexin, an ER stress response chaperone protein, was decreased under baseline conditions in aging axons, but did not show upregulation following OGD when compared to young axons, suggesting a defect in unfolded protein responses. We conclude that aging WM is increasingly vulnerable to stroke because of inherent structural changes in axons and mitochondria/ER interactions, leading to depletion of ATP and defective Ca2+ dynamics, resulting in increased oxidative stress.