Another significant finding in our study
Another significant finding in our study was the demonstration that the axon-protective action associated with CK2 inhibition correlated with preservation of mitochondrial structure and function in Gap19 weight (Fig. 11). Because of the consistent protection conferred by CK2 inhibition during OGD, improved axon function after injury may be a result of attenuated oxidative injury during the spatiotemporal progression of WM injury. The main evidence supporting this concept is that preservation of axonal mitochondrial integrity correlated with axon function recovery. We previously reported that loss of CFP (+) fluorescence in Thy-1 mito-CFP mice is an indicator of decreased ATP production (Baltan et al., 2011a; Murphy et al., 2013) and subsequently interventions that conserved mitochondrial integrity restored ATP production. CDK5 is suggested to have a direct impact on mitochondrial dynamics and function by increasing the generation of reactive oxygen species and phosphorylation of the mitochondrial fission protein Drp-1, resulting in mitochondrial defects (Cherubini et al., 2015; Jahani-Asl et al., 2015; Klinman & Holzbaur, 2015; Morel et al., 2010; Park et al., 2015; Sun et al., 2008) and acting as a downstream signaling pathway upon CK2 activation. On the other hand, CDK5 inhibition following the ischemic period failed to exert protection to axon function, suggesting that CDK5 signaling during ischemia, but not during the recovery phase, is important to alleviate oxidative injury. The evidence that inhibition of active AKT confers post-ischemic protection to axon function suggests a novel effect of PTEN/AKT pathway activation in mediating mitochondrial injury via regulation of GSK3β in ischemic WM. AKT has been reported to upregulate GLT-1 in astrocytes (Ji et al., 2013; Li et al., 2006; Zhang et al., 2013), which would contribute to increased glutamate release during OGD and ATP depletion, as well as enhanced excitotoxicity (Baltan et al., 2008a). Consistent with this, AKT inhibition with MK-2206 or ARQ-092 promoted axon function recovery when applied before injury. Note that post-ischemic injury requires blockade by AQ-092, which is a specific blocker for the active form of AKT (Lapierre et al., 2016; Yu et al., 2015), suggesting that AKT mediates ischemic WM injury once activated. Therefore, it is necessary to target this active form of AKT to promote recovery. Moreover, a member of the AKT/GSK3ß signaling pathway, the GSK3β isoform, is proposed as an important therapeutic target for cerebral ischemia (Cowper-Smith et al., 2008; Koh et al., 2008) and an intriguing relationship between GSK3β and mitochondria is emerging such that GSK3β inhibition reduces the generation of mitochondrial reactive oxygen species and neuronal damage (Valerio et al., 2011). GSK3β has been reported to play a dual role in myocardial ischemia, where the kinase is activated during ischemia and inhibited during reperfusion, and inhibition of GSK3β provided protection against ischemic injury (Zhai et al., 2011). We observed a similar increase in GSK3βS9 phosphorylation following reperfusion in WM indicative of inactivation of the kinase. CK2 inhibition during ischemia attenuated the inactivation of GSK3β during reperfusion and improved axon function recovery. Our results suggest that CK2 inhibition protects axonal structural integrity and function by either directly maintaining axonal metabolism and/or indirectly by preserving oligodendrocyte health. In addition, future studies will be important to verify whether GSK3β is a universal target that protects both gray and WM against stroke. It is intriguing that inhibition of the CDK5 and AKT pathways provides similar functional protection to aging axons. The impact of aging on WM is of general interest because the global effects of aging on myelinated nerve fibers are more intricate and extensive than those in cortical gray matter. Aging axons are larger, have thicker myelin, and are characterized by longer and thicker mitochondria that correlate with lower ATP levels and increased generation of nitric oxide, protein nitration, and lipid peroxidation (Stahon et al., 2016). Moreover, disruption in Ca2+ homeostasis and defective unfolded protein responses in aging axons are common (Stahon et al., 2016). Consequently, excitotoxic and oxidative injury dominate ischemic injury mechanisms in aging WM and interventions that protect young WM become ineffective or impede recovery in aging WM. Because ARQ-092 provides post-ischemic protection to aging axon function, even to MONs obtained from old mice (20-months-old), we propose that AKT is a common molecular target to protect WM function independent of age.