br Materials and methods br Results br Discussion

Materials and methods
Results
Discussion One novel finding in our study is that CK2 inhibition preserved axon function and structure in WM against ischemia. Consistent with these findings, oligodendrocytes, astrocytes, myelin, and calcitonin gene related peptide were found to express CK2α. The robust expression pattern of CK2α in glial cells and components is intriguing and suggests an extensive kinase regulation of WM structure and function (Brechet et al., 2008; Moreno et al., 1999; Rosenberger et al., 2016; Yoshimura & Rasband, 2014) and a significant role during ischemia (Kim et al., 2009; Shichinohe et al., 2015). These results are consistent with previous reports that CK2 levels and activity are increased in cerebral ischemia (Hu & Wieloch, 1993; Ka et al., 2015; Kim et al., 1999), as well as in kidneys exposed to ischemia (Ka et al., 2015). The evidence that CK2α is expressed in immature oligodendrocytes (Huillard et al., 2010b) combined with our results suggest that CK2α is an important molecular target in WM for development and acute injury in experimental models (Wang et al., 2015). Furthermore, because CK2α was shown to be expressed in primary human astrocytes (Rosenberger et al., 2016), CK2 inhibition may have important clinical implications. Future experiments will investigate whether glial cell-specific CK2 inhibition can confer protection to WM integrity. However, in contrast, CK2 inhibition was not protective of neurons in an in vivo model of transient focal ischemia (Kim et al., 2009). There may be several reasons to explain the lack of protection to neurons against ischemia. First, we used CX-4945, which is a specific small molecule inhibitor that crosses the BBB to block CK2α (Zheng et al., 2013), as opposed to inhibitor TBCA (Kim et al., 2009), which was used in that study and could inhibit other protein kinases (Pagano et al., 2004; Pagano et al., 2007). Second, some CK2 inhibitors, even those closely related chemically, have been reported to generate reactive oxygen species (Schneider et al., 2009). Third, strains of mice may differ in their sensitivity to ischemia or drugs (C57BL/6J versus CD1 mice (Kim et al., 2009; Sheldon et al., 1998)). Finally, the role of CK2 in neurons and in myelinated axons and glia may be different during an ischemic episode. This provides further evidence that mechanisms of ischemic injury are not the same in gray and WM portions of the brain, and therefore that assessing therapeutic interventions in both neuronal cell bodies and in axons is essential in order to achieve global protection and restoration of function in the brain after stroke. A provocative aspect of our study was the demonstrated efficacy of CK2 inhibition by CX-4945 when administered after an ischemic episode. Ischemic injury in young WM follows a sequential order initiated by loss of ionic homeostasis leading to excitotoxicity and then merging into oxidative injury (Fig. 11); ionic deregulation directly impairs axon excitability, function, and structure due to toxic accumulation of Na+ and Ca2+ (Fern et al., 1995; Stys et al., 1990; Underhill & Goldberg, 2007; Wolf et al., 2001), whereas excessive glutamate accumulation overactivates AMPA/Kainate receptors, causing oligodendrocyte death and myelin disruption. Finally, the oxidative pathway attacks WM constituents via formation of reactive oxygen species (Back et al., 2005; Juurlink, 1997; Oka et al., 1993). Because functional protection was evident when CX-4945 was applied before or after glutamate accumulation, this finding may suggest that CX-4945 simultaneously targets the excitotoxic and oxidative pathways and may have multiple distinct sites of action: one related to glutamate accumulation and the other involving a post-excitotoxic mechanism, as we have previously shown (Baltan et al., 2011a). This suggestion is supported by the observation that axon function was preserved during OGD and recovery was 50% higher with pre-injury CK2 inhibitor application compared to post-injury application. Axon function solely relies on local ATP production to maintain excitability via regulation of Na+-K+ ATPase activity. OGD causes a prominent reduction in ATP levels and loss of CFP (+) mitochondria, while CK2 inhibition resulted in sustained CFP (+) mitochondria (Baltan, 2012a; Baltan et al., 2011a; Baltan et al., 2013; Murphy et al., 2013). Because CK2 is abundantly expressed by astrocytes, it is plausible that Na+-dependent glutamate release is modified, secondary to preservation of ATP levels and Na+ levels (Baltan, 2014b), thus leading to reduced excitotoxic injury to oligodendrocytes. Alternatively, a reduction in AMPA/Kainate receptor signaling on oligodendrocytes may lead to reduced Ca2+ and Na+ entry, thus ameliorating injury (Baltan, 2009; Baltan, 2015). Furthermore, CK2 phosphorylation of AMPA receptor GluA1 subunit regulates its membrane expression (Lussier et al., 2014) and regulation of oligodendrocyte NMDA receptor subunits may alter oligodendrocyte sensitivity to glutamate (Baltan, 2015; Spitzer et al., 2016). Regulation of oligodendrocyte NMDA receptors modulates metabolic support and interactions between oligodendrocytes and axons to impact functional recovery (Saab et al., 2016). Furthermore, CK2 was shown to phosphorylate the NMDA receptor subunit GluN2B, leading to its internalization and replacement with GluN2A-containing NMDA receptors in postsynaptic densities to regulate synaptic activity in neurons (Sanz-Clemente et al., 2010; Sanz-Clemente et al., 2013). Further experiments are currently underway to assess these possibilities. CK2 inhibition promoted axon function recovery, prevented oligodendrocyte death and axonal damage, and preserved axonal mitochondrial integrity against ischemia. Although the effectiveness of CDK5 and AKT inhibition remains to be quantified based on axonal recovery, we propose WM integrity is equally protected as CK2 inhibition since these are the downstream signaling pathways activated via CK2.