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.