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

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.SIGNIFICANCE STATEMENT 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.

Preserving Mitochondrial Structure and Motility Promotes Recovery of White Matter After Ischemia.

Stroke significantly affects white matter in the brain by impairing axon function, which results in clinical deficits. Axonal mitochondria are highly dynamic and are transported via microtubules in the anterograde or retrograde direction, depending upon axonal energy demands. Recently, we reported that mitochondrial division inhibitor 1 (Mdivi-1) promotes axon function recovery by preventing mitochondrial fission only when applied during ischemia. Application of Mdivi-1 after injury failed to protect axon function. Interestingly, L-NIO, which is a NOS3 inhibitor, confers post-ischemic protection to axon function by attenuating mitochondrial fission and preserving mitochondrial motility via conserving levels of the microtubular adaptor protein Miro-2. We propose that preventing mitochondrial fission protects axon function during injury, but that restoration of mitochondrial motility is more important to promote axon function recovery after injury. Thus, Miro-2 may be a therapeutic molecular target for recovery following a stroke.

Mitochondrial dynamics and preconditioning in white matter.

Mechanisms of ischemic preconditioning have been extensively studied in gray matter. However, an ischemic episode affects both the gray matter (GM) and white matter (WM) portions of the brain. Inhibition of mitochondrial fission is one of the mechanisms of preconditioning neuronal cell bodies against ischemia. Although axons are anatomical extensions of neuronal cell bodies, injury mechanisms differ between GM and WM. Indeed, axonal dysfunction is responsible for much of the disability associated with clinical deficits observed after stroke; however, the signaling process underlying preconditioning remains unexplored in axons. Using mouse optic nerve, which is a pure isolated WM tract, we show that mitochondria in myelinated axons undergo rapid and profuse fission during oxygen glucose deprivation (OGD) that is mediated by translocation of cytoplasmic Dynamin Related Protein-1 (Drp-1) to mitochondria. OGD-induced mitochondrial fission correlates with reduced mitochondrial motility and loss of axon function. Mitochondrial fragmentation and loss of motility become permanent during the recovery period. Inhibiting mitochondrial fission by administering mitochondrial division inhibitor-1 (Mdivi-1) during OGD preserves mitochondrial shape and motility and promotes axon function recovery. In contrast, preconditioning WM by applying Mdivi-1 only before OGD fails to conserve mitochondrial shape or motility and fails to benefit axon function. Our findings suggest that inhibition of mitochondrial fission during ischemia promotes axon function recovery, but is not sufficient to precondition WM against ischemia. These results raise caution in that approaches to preconditioning neuronal cell bodies may not successfully translate into functional improvement following ischemia.