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CK2 inhibition protects white matter from ischemic injury
Accepted Manuscript Title: CK2 Inhibition Protects White Matter from Ischemic Injury Authors: Selva Baltan, Chinthasagar Bastian, John Quinn, Danielle Aquila, Andrew McCray, Sylvain Brunet PII: DOI: Reference:
S0304-3940(18)30554-8 https://doi.org/10.1016/j.neulet.2018.08.021 NSL 33756
To appear in:
Neuroscience Letters
Received date: Revised date: Accepted date:
26-6-2018 13-8-2018 14-8-2018
Please cite this article as: Baltan S, Bastian C, Quinn J, Aquila D, McCray A, Brunet S, CK2 Inhibition Protects White Matter from Ischemic Injury, Neuroscience Letters (2018), https://doi.org/10.1016/j.neulet.2018.08.021 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
CK2 Inhibition Protects White Matter from Ischemic Injury Selva Baltan, Chinthasagar Bastian, John Quinn, Danielle Aquila, Andrew McCray, and Sylvain
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Brunet* Department of Neurosciences, Cleveland Clinic Foundation, Cleveland, Ohio 44195.
Correspondence should be addressed to:
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Sylvain Brunet, PhD Department of Neurosciences Lerner Research Institute Cleveland Clinic Foundation 9500 Euclid Avenue/NC30 Cleveland, OH 44195 USA Tel: (216) 408-9586 Fax: (216) 444-7927
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email: [email protected] Number of figures = 3
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Number of tables = 0
Number of words: Abstract (182), Review (2827),
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Running Title: CK2 Inhibition Protects White Matter from Ischemic Injury.
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Acknowledgements: This work was supported by grants from NIA (AG033720) to S.B and NINDS (NS094881) to S.B and S.B.
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HighlightsWM components express abundant levels of the CK2α subunit.
CK2 signaling differentially regulates the CDK5 and AKT/GSK3β signaling pathways to mediate WM ischemic injury.
Inhibition of CK2 and the active conformation of AKT confers post-ischemic protection to young, aging, and old WM, providing a common therapeutic target independent of age. Neuronal responses to CK2 signaling during ischemia vary, depending upon the
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experimental model used. Abstract
Strokes occur predominantly in the elderly and white matter (WM) is injured in most strokes, contributing to the disability associated with clinical deficits. Casein kinase 2 (CK2) is expressed
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in neuronal cells and was reported to be neuroprotective during cerebral ischemia. Recently, we
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reported that CK2 is abundantly expressed by glial cells and myelin. However, in contrast to its
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role in cerebral (gray matter) ischemia, CK2 activation during ischemia mediated WM injury via
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the CDK5 and AKT/GSK3β signaling pathways1. Subsequently, CK2 inhibition using the small molecule inhibitor CX-4945 correlated with preservation of oligodendrocytes as well as
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conservation of axon structure and axonal mitochondria, leading to improved functional recovery.
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Notably, CK2 inhibition promoted WM function when applied before or after ischemic injury by differentially regulating the CDK5 and AKT/GSK3β pathways. Specifically, blockade of the active
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conformation of AKT conferred post-ischemic protection to young, aging, and old WM, suggesting a common therapeutic target across age groups. CK2 inhibitors are currently being used in clinical
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trials for cancer patients; therefore, it is important to consider the potential benefits of CK2
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inhibitors during an ischemic attack. We have recently identified that casein kinase 2 (CK2) inhibition protects white matter (WM) from ischemic injury. We focused our research on CK2 because upregulation of CK2 activity is associated with many diseases, including ischemic injury6–8. This review highlights and contrasts the role of CK2 in WM and gray matter (GM) portions of the brain following ischemia together with
their molecular mechanisms. Finally, we incorporate findings from cancer research to explore whether some of those identified mechanisms of CK2 activity can be extrapolated to stroke research.
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CK2 is not a conventional protein kinase (PK). CK2 is composed of two catalytic -subunits ( and ’) and two regulatory β-subunits. First, despite the initial lack of substrates following its discovery, CK2 was subsequently shown to phosphorylate numerous substrates, including other PKs, thus acting as a “master regulator”11. Second, unlike other PKs, the catalytic activity of CK2 is not regulated by second messengers or phosphorylation11 and it was found to be constitutively However,
CK2
was
recently
proposed
to
be
activated
via
a
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active.
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polymerization/depolymerization mechanism13 together with reactive oxygen species (ROS) 16,17.
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CK2 and WM Ischemia
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Transient CK2 inhibition is a novel and effective therapeutic strategy to protect WM against ischemic stroke. We recently reported that a brief application of the CK2 inhibitor CX-4945
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preserves mouse optic nerve (MON) axon function following ischemic injury (Figure 1A-C)1. This
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is the first report that a PK inhibitor protects young, aging, and old WM from ischemic injury (Figure 1D-F)1. Improving MON function following ischemia is an important endpoint, as axon
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injury is an independent risk factor and burden for adverse outcomes following a stroke, even in
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intravenous thrombolysis patients19. To investigate the mechanisms by which CK2 inhibition protects WM from ischemic injury, we
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investigated the impact of CX-4945 on the cellular components of MONs. WM is composed of astrocytes, oligodendrocytes, microglia, and axons20. First, we showed that the CK2α subunit is expressed in glial cell compartments. We evaluated the expression and localization of CK2 in MONs using immunohistochemistry and glial cell-specific antibodies in conjunction with confocal imaging to support a biological basis for CK2 inhibitor action in MONs. The expression of CK2α
subunit co-localized with GFAP (+) astrocyte nuclei and some processes and also with NF-200 (+) axons. Almost all Olig2 (+) oligodendrocytes were strongly immunoreactive for CK2α. Immunolabeling was also evident on PLP (+) myelin. This extensive expression of CK2α in glial cells in addition to axons implicate them as cellular targets of CX-49451. Expectedly, CK2
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inhibition protected axons and oligodendrocytes from ischemic injury. We assessed the impact of CK2 inhibition on nuclear morphology, oligodendrocytes, and the axon cytoskeleton, which are critical elements that show widespread injury after ischemia21,22. CK2 inhibition preserved APC (+) oligodendrocytes and SMI-31 (+) axonal labeling (Figure 2A). Whether CK2 inhibition directly or indirectly protects axon function and/or structure remains unresolved. We suggest that
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oligodendrocyte death predisposes axons to injury and improper conduction because
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oligodendrocytes not only provide the structural and electrical framework for fast and
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synchronized axonal conduction23, but they also support axons metabolically24. Therefore,
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oligodendrocyte death will lead to disruption of axonal myelin and nodes of Ranvier to alter axonal conduction, while interruption of metabolic support to the axon will increase the strain on already-
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decreased levels of ATP in the face of decreased oxygen levels during ischemic stress 25. Note
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that axons are independent of neuronal cell bodies in terms of energy supply and are completely dependent upon local energy production. We previously reported that in WM, post-injury
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protection correlated with conservation of mitochondrial integrity during ischemic injury41,42. Therefore, we postulated that the protective effects of CK2 inhibition in promoting axon function
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recovery after ischemia correlate with axonal mitochondrial preservation. Indeed, when we monitored mitochondrial fluorescence in MONs isolated from Thy-1 mito CFP mice28 during
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control conditions and during oxygen glucose deprivation (OGD) with or without CX-4945 treatment, we confirmed this hypothesis. Mitochondria in control MONs displayed short tubular morphology (Figure 2B, left panel). After OGD, there was a dramatic loss of mitochondrial CFP (+) fluorescence (Figure 2B, middle panel). The remaining mitochondria exhibited a small punctate morphology that is typical of ischemia-induced mitochondrial fission31. Pretreatment of
MONs with CX-4945 effectively attenuated the loss of mitochondrial fluorescence and preserved mitochondrial morphology (Figure 2B, right panel, and 2C). These findings confirmed and expanded our previous reports that approaches conferring post-ischemic protection to WM
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function correlate with preserved mitochondrial integrity. During ischemia, the impact of CK2 on mitochondria varies in different organs/tissues. For example, CK2 upregulation during ischemia leads to disrupted mitochondrial homeostasis and mediates cardiomyocyte ischemic injury31. Specifically, ischemia/reperfusion (I/R) progressively increases CK2 to curtail FUN14 domain protein 1 (FUNDC1)-dependent mitophagy via post-
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transcriptional inactivation of FUNDC1, thus impairing mitochondrial protective systems to amplify
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cardiomyocyte death signals. On the other hand, the impact of CK2 inhibition in a model of transient ischemia (middle cerebral artery occlusion (MCAO) model) suggested that NADPH
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oxidase isoform 2 (NOX2) activation releases ROS after CK2 inhibition, triggering the release of
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apoptogenic factors (AIF) from the mitochondria and inducing DNA damage after ischemic brain
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injury32. It is not clear why these different results have been observed; however, it may be that in
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WM and heart, the source of ROS is determined by mitochondria and that in GM it is dictated by NADPH oxidase regulating CK2 signaling differently.
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CK2 Signaling Pathways to Protect WM from Ischemic Injury
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CK2 activates CDK5 during ischemia and AKT/GSK3 throughout the ischemic and reperfusion periods. We have shown that inhibition of CDK5 with roscovitine protects WM from ischemic injury
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only when applied before OGD in young and aging WM33. This result suggests that CDK5 is activated during OGD only and that it is substantial such that inhibition during OGD leads to improved axon function. Interestingly, CDK5 inhibition with indolinone A and knocking out CDK5 in mice are protective in MCAO models34. In addition, in a MCAO model, post-ischemic application of a CDK5 inhibitory peptide, P5-TAT, decreased infarct volume35. These results suggest that
CDK5 inhibition could be a potential therapeutic target to protect both WM and GM when applied during an ischemic episode, independent of age. Systematically investigating the AKT signaling pathway showed that only the pan-AKT inhibitor
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MK-2206 was beneficial to axon function when applied before OGD in young and aging MONs1. These results raised a question as to why individual inhibition of the AKT or CDK5 pathways failed to exert post-ischemic protection, while CK2 inhibition was beneficial before or after injury. MK2206 targets the inactive conformation of AKT, where the PH domain engages the kinase domain, thus preventing phosphorylation and activation36,37. Therefore, we further tested the possibility that AKT inhibition after OGD required an inhibitor that targets the active conformation of AKT.
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For these experiments, we examined the effectiveness of a new selective allosteric pan-AKT
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inhibitor, ARQ-092, which targets the inactive and active conformations of AKT 38 with equal
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potency. When ARQ-092 was applied before OGD, we observed a level of protection similar to
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MK-2206. However, in contrast to MK-2206, ARQ-092 promoted axon function recovery when applied after the end of OGD. This protection was observed in both young and aging MONs. On
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the other hand, in GM, AKT signaling is associated with neuronal cell survival45 and
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oligodendrocyte progenitor cell (OPC) and oligodendrocyte survival42,43; therefore, for a therapeutic intervention to be successful, the reason for this difference needs to be determined.
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Note that these later conclusions were reached from experiments that inhibited AKT for a long
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duration (chronic inhibition). Overall, this difference may be related to the AKT isoform expressed in WM and other organs/tissues together with the duration of AKT inhibition. This possibility is
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currently being investigated. Overall, this evidence suggests that acute CDK5 and AKT inhibition are sufficient to protect axon function against ischemia when applied before injury, while inhibition of the active conformation of AKT is necessary to exert post-ischemic protection independent of age.
Activating GSK3 signaling to inhibit PK signaling during ischemia/reperfusion (I/R) is protective of WM ischemic injury. Glycogen synthase kinase (GSK3), which was the first substrate identified for AKT44, is inhibited by AKT phosphorylation at positions S9 and S2145. When GSK3 is active, it is known to promote the inactivation and degradation of its substrates45. GSK3 substrates also
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have to be phosphorylated at another site (i.e., “primed”) by another PK in order for GSK3 to phosphorylate them45. We have shown that CK2/AKT1/GSK3 signaling is present in HEK cells as well as in the oligodendrocyte cell line MO3.13. In addition, we have shown that the CK2/AKT/GSK3 axis is present in MONs. Specifically, OGD/reperfusion inactivates GSK3by
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increasing GSK3S9 phosphorylation, which is attenuated by CX-4945 (Figure 2D and E).
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Therefore, GSK3 activation in WM following OGD/reperfusion is associated with protection from
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ischemic injury. This later result is indeed surprising since in most cells, including neurons and OPCs, GSK3 inactivation is associated with increased survival and prevention of apoptosis46,47.
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However, in liver cells, GSK3 deletion increases cell death and apoptosis48. Thus, an alteration
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in signaling may be distinctive in different cells, thus accounting for the variable outcomes on cell
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survival between GM and WM with ischemia. We therefore propose that the protective effect of CK2 inhibition is partly mediated by a reduction in the inhibitory effect of CK2/AKT on GSK3,
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thus resulting in its activation. Furthermore, activated GSK3 can then inactivate its substrates, which are themselves substrates and primed by other PKs activated by I/R, thus increasing the
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number of PKs whose signaling is inhibited. Therefore, this predisposes a state of energy
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conservation in WM when ATP is momentarily unavailable. CK2 Signaling During Ischemia in Brain GM In GM, CK2 signaling can be either protective or damaging. For example, in a model of transient forebrain ischemia, the preservation or activation of CK2 activity was proposed to be important for neuronal survival after cerebral ischemia8. Specifically, It was reported that the regions
vulnerable to ischemia showed a decrease in CK2 activity, whereas resistant regions were associated with an increase8. In contrast, in a model of global ischemia, an increase in CK2 activity in the dentate gyrus was reported50.
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Further investigation into the impact of transient cerebral ischemia and the role of CK2 signaling in a mouse model of ischemia (MCAO51) revealed that MCAO was associated with a decrease in CK2 (- and ’) levels and activity. On the other hand, the changes in CK2 activity following ischemia varied as a function of time such that the reduction in CK2 levels was attenuated at 1 h and 3 h, remained unchanged at 6 h and 12 h, and was ~60% below normal at 24 h and 48 h.
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Inhibition of CK2 with tetrabromocinnamic acid (TBCA) was associated with an increase in GM injury. And finally, using an MCAO model and the same experimental approach as Kim et al. 51,
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three other reports have concluded that CK2 signaling is protective of GM from ischemic injury52– 54
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. Overall these studies suggest that neuronal responses to CK2 signaling vary depending upon
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the experimental model used.
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Could the experimental design that prolongs CK2 inhibition alter the outcome in ischemic
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experiments55? It is possible that neuronal injury observed with TCBA (MCAO model) and siRNA (cortical neuronal culture model) was caused by the duration of the reduction in CK2 protein
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levels/activity, since decreased CK2 activity for an extended period of time mediates cell death in non-neuronal cells56. In addition, the CK2 inhibitor used may have been injurious because some
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CK2 inhibitors have been shown to generate ROS57. Future experiments using a MCAO model subjected to a shorter duration of CK2 signaling inhibition using a different CK2 inhibitor, such as
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CX-4945, need to be conducted in order to answer these questions. All of these experiments in GM matter injury models have identified an array of signaling mechanisms that are potentially important. First, Kim et al. proposed that CK2 acts as a negative modulator of NADPH oxidase55. Then Blanquet et al.50 found that increased CK2 correlated with
inhibition of the activity of MKK(3/6), p38, and deacetylases. Subsequent studies investigated the mechanisms by which CK2 inhibition of NOX2 led to neuronal survival using superoxide dismutase 1 (SOD1) transgenic mutant and gp91(Nox2) knock-out mice32. These results suggest that NOX2 activation releases ROS after CK2 inhibition, triggering the release of apoptogenic
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factors (AIF) from mitochondria and inducing DNA damage after ischemic brain injury. Overall, they concluded that there is a reciprocal relationship between ROS and CK2 protein levels. And finally, studies were performed to test the mechanism of compounds that showed neuroprotection in a MCAO model such as apelin-13, 5d, a novel analogue of the racemic 3-n-butylphthalide, and cilostazol52–54. The authors concluded that the neuroprotective effects observed with apelin-13
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and 5d may be partly mediated by an increase in CK2 activity, such that apelin-13 regulated CK2
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by activating the apelin receptor/Gi/Gq-CK2 and restoring p-eIF2α-ATF4-CHOP/GRP78 to
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attenuate neuronal apoptosis, and 5d decreased NADPH oxidase activity by positively regulating
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CK252,53. Experiments with cilostazol suggested that its protective mechanism was produced by the maxi-K channel opening coupled to upregulation of CK2 phosphorylation and downregulation
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of PTEN phosphorylation54. The exact mechanisms of how channel opening increases and how
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PTEN phosphorylation decreases CK2 have not been elucidated, nor has the impact of CK2 inhibitor been tested to abrogate the effect of cilostazol. The authors suggest that CK2 activation
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may serve as a therapeutic target for inhibiting neuronal cell apoptosis in ischemic cells and thus
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may offer a cell survival strategy. These proposed mechanisms of CK2 protection identified in GM
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and neuronal injury remain to be tested in WM.
A Lesson from Cancer Research Interestingly, cancer research has been instrumental in identifying novel therapeutic targets and mechanism(s) for cerebral ischemic injury. In cancer, CK2 activity has been shown to be upregulated such that cells become dependent upon high CK2 levels for their survival9. CK2 has
been shown to increase glucose metabolism in bladder cancer cells, thus contributing to the Warburg effect58. Could the protective effect of CK2 inhibition in WM be mediated by a metabolic switch during the ischemic period? For instance, Zhang et al.58 have shown that CK2 inhibition was associated with decreased levels of AKTS4 phosphorylation and decreased levels of
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glycolysis-related genes. Also, CK2 could regulate glycolysis by phosphorylating glycolytic enzymes59,60. Taken together, these results suggest that CK2 inhibition may suppress high glycolysis levels by reducing CK2 and AKT phosphorylation of enzymes/transporter in the glycolytic pathway. It is plausible that I/R leads to increased glycolysis, resulting in an increase in metabolic intermediates for the tricarboxylic acid cycle; however, with decreasing O2 levels, this
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would lead to increased lactate levels, decreased pH, and activation of the pentose phosphate
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pathway (PPP). Note that an increase in glycolysis in the face of compromised oxidative
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phosphorylation leads to increased lactate and an increase in PPP pathway activation, which is
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essential to generate the building blocks (lipids, nucleic acids) that are necessary to sustain the high mitotic rates of tumors61. Therefore, we hypothesize that during ischemia and early
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reperfusion when ATP is compromised25, it is more important to relieve the metabolic constraints
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than to generate the building blocks for repair. However, after this transient repression of CK2
(Figure 3).
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activity during I/R, it is important to release this inhibition so that CK2 can aid in the repair process
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Conclusion
In this review, we have discussed how inhibition of the CK2/CDK5 signaling axis protects WM
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only when inhibited before ischemia, while inhibition of the CK2/AKT/GSK3 signaling axis protects WM either before or after an ischemic injury in young, aging, and old WM (Figure 3). CK2 activation mediates WM ischemic injury in a differential spatiotemporal manner such that CDK5 signaling becomes important during ischemia, while AKT signaling emerges as the main pathway during the reperfusion period following ischemia. Consequently, interventions selectively
targeting the activated form of AKT confer post-ischemic functional recovery in young and aging WM. In contrast, ischemia-mediated regulation of CK2 activity impacts neuronal survival differently. The reasons for this discrepancy may stem from differences in the dominant CK2 substrates, the duration of the CK2 inhibition, the involvement of inflammation, and the choice of
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CK2 pharmacological inhibitor used in these studies. As the role of CK2 in the control of apoptosis in non-neuronal cells is well-established, we would advocate, based on our results in WM, that future studies use a selective CK2 inhibitor like CX-4945 at an optimal concentration and duration. Experiments using a short duration of treatment with CX-4945 to investigate neuronal survival in an in vivo model are also warranted. Until then, the effective use of CK2 signaling inhibition to
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protect the brain against ischemic injury will require the development of a targeted approach for
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WM in order to minimize damage to GM. It is essential to preserve, protect, and repair both
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neuronal and glial cells of the brain to improve functional recovery. Nevertheless, continued
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research into the role of CK2 signaling in WM may reveal more effective therapeutic targets for WM that may be useful for neurodegenerative diseases that primarily affect glial cells and myelin
injury.
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Figure Legends
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such as traumatic brain injury, multiple sclerosis, periventricular leukomalacia, and spinal cord
Figure 1. The CK2 small molecule inhibitor, CX-4945, promotes function recovery in young
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and aging axons following OGD. Time course shows minimal recovery following OGD (black; B, young; D, aging). Examples of CAP traces (A, young; and D, aging) taken during baseline (a),
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OGD (b), and recovery (c) for control and CX-4945 (5 μM) pre-treatment (red) and post-treatment (magenta). Arrows show sustained CAP at the end of OGD following CX-4945 in young and aging MONs. Pre-treatment with CX-4945 (red: A, B and C, young; and D, E and F, aging) applied before OGD promoted a consistent and sustained CAP area recovery, preserved CAP area during OGD, and improved axon function following recovery. CX-4945 applied after OGD (magenta: A,
B and C, young; and D, E and F, aging) promoted a consistent and sustained CAP area recovery. ** p < 0.01, and *** p < 0.001, one-way ANOVA with Newman-Keuls post hoc test.1 Figure 2. CK2 inhibition prevents OGD-induced axon injury, oligodendrocyte death, and
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mitochondrial disruption, and it also attenuates OGD-upregulated GSK3β phosphorylation in mouse optic nerve. A) OGD (1 h) caused widespread loss of SMI-31 (+) axons (green) and adenomatous polyposis coli (APC (+), oligodendrocytes, magenta), while CX-4945 (5 μM) prevented axonal injury and oligodendrocyte loss. Glial nuclei were labeled with Sytox (blue, white arrows). Note that the merged images in the right panels are enlarged areas indicated by the squares with dashed lines in the middle panels. Scale bar = 20 µm. B) Two-photon confocal
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images of axonal CFP (+) mitochondria in MONs from Thy-1 mito-CFP mice after control, OGD,
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and OGD + CX-4945. OGD attenuated CFP fluorescence; in addition, mitochondria became
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smaller and less numerous. CX-4945 pre-treatment preserved CFP (+) mitochondrial
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fluorescence and morphology. C) Histogram summarizing the data. Scale bar = 10 μm. *** p < 0.001, one-way ANOVA with Newman-Keuls post hoc test. D) and E) An increase in GSK3βS9
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was observed in MONs following OGD and reperfusion; however, this was attenuated by CX-
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4945 (5 µM) pre-treatment. *** p < 0.001, one-way ANOVA with Newman-Keuls post hoc test.1
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Figure 3. CK2 inhibition confers WM functional protection against ischemia by differentially regulating the CDK5 and AKT signaling pathways. A) Ischemic injury in young
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WM follows a sequential order, initiated by loss of ionic homeostasis leading to excitotoxicity and then merging into oxidative injury. Our findings suggest that ischemia in WM results in CK2
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activation. Functional protection was evident when CX-4945, a CK2 inhibitor, was applied before or after glutamate accumulation, which may suggest that CX-4945 could simultaneously target the excitotoxic and oxidative pathways. CK2 recruits CDK5 and AKT/GSK3β signaling to mediate WM ischemic injury in a spatiotemporal manner such that CDK5 signaling becomes important during ischemia, while AKT signaling emerges as the main pathway during the reperfusion period
following ischemia. B) A detailed molecular model of the regulation of CK2/CDK5 and CK2/AKT/GSK3 signaling pathways mediating WM injury with ischemia. Note that recruitment of the oxidative pathway is mediated by positive feedback from CK2/AKT/GSK3 signaling to increase glycolysis in the face of decreased O2, glucose, and ATP, leading to accentuated
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glycolytic metabolites and ROS levels. PPP, Pentose Phosphate Pathway; NADPH, Nicotinamide Adenine Dinucleotide Phosphate Hydrogen; GSH, Glutathione; ROS, Reactive Oxygen Species; CK2, Casein Kinase 2; CDK5, cell division PK 5; AKT, PKB; GSK3, Glycogen synthase kinase3 ; and I/R, Ischemia/Reperfusion.
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Figure 1 Brunet et al.
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S0304-3940(18)30554-8 https://doi.org/10.1016/j.neulet.2018.08.021 NSL 33756
To appear in:
Neuroscience Letters
Received date: Revised date: Accepted date:
26-6-2018 13-8-2018 14-8-2018
Please cite this article as: Baltan S, Bastian C, Quinn J, Aquila D, McCray A, Brunet S, CK2 Inhibition Protects White Matter from Ischemic Injury, Neuroscience Letters (2018), https://doi.org/10.1016/j.neulet.2018.08.021 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
CK2 Inhibition Protects White Matter from Ischemic Injury Selva Baltan, Chinthasagar Bastian, John Quinn, Danielle Aquila, Andrew McCray, and Sylvain
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Brunet* Department of Neurosciences, Cleveland Clinic Foundation, Cleveland, Ohio 44195.
Correspondence should be addressed to:
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Sylvain Brunet, PhD Department of Neurosciences Lerner Research Institute Cleveland Clinic Foundation 9500 Euclid Avenue/NC30 Cleveland, OH 44195 USA Tel: (216) 408-9586 Fax: (216) 444-7927
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email: [email protected] Number of figures = 3
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Number of tables = 0
Number of words: Abstract (182), Review (2827),
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Running Title: CK2 Inhibition Protects White Matter from Ischemic Injury.
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Acknowledgements: This work was supported by grants from NIA (AG033720) to S.B and NINDS (NS094881) to S.B and S.B.
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HighlightsWM components express abundant levels of the CK2α subunit.
CK2 signaling differentially regulates the CDK5 and AKT/GSK3β signaling pathways to mediate WM ischemic injury.
Inhibition of CK2 and the active conformation of AKT confers post-ischemic protection to young, aging, and old WM, providing a common therapeutic target independent of age. Neuronal responses to CK2 signaling during ischemia vary, depending upon the
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experimental model used. Abstract
Strokes occur predominantly in the elderly and white matter (WM) is injured in most strokes, contributing to the disability associated with clinical deficits. Casein kinase 2 (CK2) is expressed
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in neuronal cells and was reported to be neuroprotective during cerebral ischemia. Recently, we
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reported that CK2 is abundantly expressed by glial cells and myelin. However, in contrast to its
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role in cerebral (gray matter) ischemia, CK2 activation during ischemia mediated WM injury via
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the CDK5 and AKT/GSK3β signaling pathways1. Subsequently, CK2 inhibition using the small molecule inhibitor CX-4945 correlated with preservation of oligodendrocytes as well as
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conservation of axon structure and axonal mitochondria, leading to improved functional recovery.
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Notably, CK2 inhibition promoted WM function when applied before or after ischemic injury by differentially regulating the CDK5 and AKT/GSK3β pathways. Specifically, blockade of the active
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conformation of AKT conferred post-ischemic protection to young, aging, and old WM, suggesting a common therapeutic target across age groups. CK2 inhibitors are currently being used in clinical
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trials for cancer patients; therefore, it is important to consider the potential benefits of CK2
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inhibitors during an ischemic attack. We have recently identified that casein kinase 2 (CK2) inhibition protects white matter (WM) from ischemic injury. We focused our research on CK2 because upregulation of CK2 activity is associated with many diseases, including ischemic injury6–8. This review highlights and contrasts the role of CK2 in WM and gray matter (GM) portions of the brain following ischemia together with
their molecular mechanisms. Finally, we incorporate findings from cancer research to explore whether some of those identified mechanisms of CK2 activity can be extrapolated to stroke research.
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CK2 is not a conventional protein kinase (PK). CK2 is composed of two catalytic -subunits ( and ’) and two regulatory β-subunits. First, despite the initial lack of substrates following its discovery, CK2 was subsequently shown to phosphorylate numerous substrates, including other PKs, thus acting as a “master regulator”11. Second, unlike other PKs, the catalytic activity of CK2 is not regulated by second messengers or phosphorylation11 and it was found to be constitutively However,
CK2
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recently
proposed
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active.
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polymerization/depolymerization mechanism13 together with reactive oxygen species (ROS) 16,17.
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CK2 and WM Ischemia
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Transient CK2 inhibition is a novel and effective therapeutic strategy to protect WM against ischemic stroke. We recently reported that a brief application of the CK2 inhibitor CX-4945
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preserves mouse optic nerve (MON) axon function following ischemic injury (Figure 1A-C)1. This
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is the first report that a PK inhibitor protects young, aging, and old WM from ischemic injury (Figure 1D-F)1. Improving MON function following ischemia is an important endpoint, as axon
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injury is an independent risk factor and burden for adverse outcomes following a stroke, even in
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intravenous thrombolysis patients19. To investigate the mechanisms by which CK2 inhibition protects WM from ischemic injury, we
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investigated the impact of CX-4945 on the cellular components of MONs. WM is composed of astrocytes, oligodendrocytes, microglia, and axons20. First, we showed that the CK2α subunit is expressed in glial cell compartments. We evaluated the expression and localization of CK2 in MONs using immunohistochemistry and glial cell-specific antibodies in conjunction with confocal imaging to support a biological basis for CK2 inhibitor action in MONs. The expression of CK2α
subunit co-localized with GFAP (+) astrocyte nuclei and some processes and also with NF-200 (+) axons. Almost all Olig2 (+) oligodendrocytes were strongly immunoreactive for CK2α. Immunolabeling was also evident on PLP (+) myelin. This extensive expression of CK2α in glial cells in addition to axons implicate them as cellular targets of CX-49451. Expectedly, CK2
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inhibition protected axons and oligodendrocytes from ischemic injury. We assessed the impact of CK2 inhibition on nuclear morphology, oligodendrocytes, and the axon cytoskeleton, which are critical elements that show widespread injury after ischemia21,22. CK2 inhibition preserved APC (+) oligodendrocytes and SMI-31 (+) axonal labeling (Figure 2A). Whether CK2 inhibition directly or indirectly protects axon function and/or structure remains unresolved. We suggest that
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oligodendrocyte death predisposes axons to injury and improper conduction because
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oligodendrocytes not only provide the structural and electrical framework for fast and
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synchronized axonal conduction23, but they also support axons metabolically24. Therefore,
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oligodendrocyte death will lead to disruption of axonal myelin and nodes of Ranvier to alter axonal conduction, while interruption of metabolic support to the axon will increase the strain on already-
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decreased levels of ATP in the face of decreased oxygen levels during ischemic stress 25. Note
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that axons are independent of neuronal cell bodies in terms of energy supply and are completely dependent upon local energy production. We previously reported that in WM, post-injury
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protection correlated with conservation of mitochondrial integrity during ischemic injury41,42. Therefore, we postulated that the protective effects of CK2 inhibition in promoting axon function
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recovery after ischemia correlate with axonal mitochondrial preservation. Indeed, when we monitored mitochondrial fluorescence in MONs isolated from Thy-1 mito CFP mice28 during
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control conditions and during oxygen glucose deprivation (OGD) with or without CX-4945 treatment, we confirmed this hypothesis. Mitochondria in control MONs displayed short tubular morphology (Figure 2B, left panel). After OGD, there was a dramatic loss of mitochondrial CFP (+) fluorescence (Figure 2B, middle panel). The remaining mitochondria exhibited a small punctate morphology that is typical of ischemia-induced mitochondrial fission31. Pretreatment of
MONs with CX-4945 effectively attenuated the loss of mitochondrial fluorescence and preserved mitochondrial morphology (Figure 2B, right panel, and 2C). These findings confirmed and expanded our previous reports that approaches conferring post-ischemic protection to WM
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function correlate with preserved mitochondrial integrity. During ischemia, the impact of CK2 on mitochondria varies in different organs/tissues. For example, CK2 upregulation during ischemia leads to disrupted mitochondrial homeostasis and mediates cardiomyocyte ischemic injury31. Specifically, ischemia/reperfusion (I/R) progressively increases CK2 to curtail FUN14 domain protein 1 (FUNDC1)-dependent mitophagy via post-
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transcriptional inactivation of FUNDC1, thus impairing mitochondrial protective systems to amplify
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cardiomyocyte death signals. On the other hand, the impact of CK2 inhibition in a model of transient ischemia (middle cerebral artery occlusion (MCAO) model) suggested that NADPH
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oxidase isoform 2 (NOX2) activation releases ROS after CK2 inhibition, triggering the release of
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apoptogenic factors (AIF) from the mitochondria and inducing DNA damage after ischemic brain
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injury32. It is not clear why these different results have been observed; however, it may be that in
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WM and heart, the source of ROS is determined by mitochondria and that in GM it is dictated by NADPH oxidase regulating CK2 signaling differently.
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CK2 Signaling Pathways to Protect WM from Ischemic Injury
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CK2 activates CDK5 during ischemia and AKT/GSK3 throughout the ischemic and reperfusion periods. We have shown that inhibition of CDK5 with roscovitine protects WM from ischemic injury
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only when applied before OGD in young and aging WM33. This result suggests that CDK5 is activated during OGD only and that it is substantial such that inhibition during OGD leads to improved axon function. Interestingly, CDK5 inhibition with indolinone A and knocking out CDK5 in mice are protective in MCAO models34. In addition, in a MCAO model, post-ischemic application of a CDK5 inhibitory peptide, P5-TAT, decreased infarct volume35. These results suggest that
CDK5 inhibition could be a potential therapeutic target to protect both WM and GM when applied during an ischemic episode, independent of age. Systematically investigating the AKT signaling pathway showed that only the pan-AKT inhibitor
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MK-2206 was beneficial to axon function when applied before OGD in young and aging MONs1. These results raised a question as to why individual inhibition of the AKT or CDK5 pathways failed to exert post-ischemic protection, while CK2 inhibition was beneficial before or after injury. MK2206 targets the inactive conformation of AKT, where the PH domain engages the kinase domain, thus preventing phosphorylation and activation36,37. Therefore, we further tested the possibility that AKT inhibition after OGD required an inhibitor that targets the active conformation of AKT.
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For these experiments, we examined the effectiveness of a new selective allosteric pan-AKT
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inhibitor, ARQ-092, which targets the inactive and active conformations of AKT 38 with equal
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potency. When ARQ-092 was applied before OGD, we observed a level of protection similar to
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MK-2206. However, in contrast to MK-2206, ARQ-092 promoted axon function recovery when applied after the end of OGD. This protection was observed in both young and aging MONs. On
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the other hand, in GM, AKT signaling is associated with neuronal cell survival45 and
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oligodendrocyte progenitor cell (OPC) and oligodendrocyte survival42,43; therefore, for a therapeutic intervention to be successful, the reason for this difference needs to be determined.
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Note that these later conclusions were reached from experiments that inhibited AKT for a long
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duration (chronic inhibition). Overall, this difference may be related to the AKT isoform expressed in WM and other organs/tissues together with the duration of AKT inhibition. This possibility is
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currently being investigated. Overall, this evidence suggests that acute CDK5 and AKT inhibition are sufficient to protect axon function against ischemia when applied before injury, while inhibition of the active conformation of AKT is necessary to exert post-ischemic protection independent of age.
Activating GSK3 signaling to inhibit PK signaling during ischemia/reperfusion (I/R) is protective of WM ischemic injury. Glycogen synthase kinase (GSK3), which was the first substrate identified for AKT44, is inhibited by AKT phosphorylation at positions S9 and S2145. When GSK3 is active, it is known to promote the inactivation and degradation of its substrates45. GSK3 substrates also
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have to be phosphorylated at another site (i.e., “primed”) by another PK in order for GSK3 to phosphorylate them45. We have shown that CK2/AKT1/GSK3 signaling is present in HEK cells as well as in the oligodendrocyte cell line MO3.13. In addition, we have shown that the CK2/AKT/GSK3 axis is present in MONs. Specifically, OGD/reperfusion inactivates GSK3by
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increasing GSK3S9 phosphorylation, which is attenuated by CX-4945 (Figure 2D and E).
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Therefore, GSK3 activation in WM following OGD/reperfusion is associated with protection from
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ischemic injury. This later result is indeed surprising since in most cells, including neurons and OPCs, GSK3 inactivation is associated with increased survival and prevention of apoptosis46,47.
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However, in liver cells, GSK3 deletion increases cell death and apoptosis48. Thus, an alteration
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in signaling may be distinctive in different cells, thus accounting for the variable outcomes on cell
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survival between GM and WM with ischemia. We therefore propose that the protective effect of CK2 inhibition is partly mediated by a reduction in the inhibitory effect of CK2/AKT on GSK3,
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thus resulting in its activation. Furthermore, activated GSK3 can then inactivate its substrates, which are themselves substrates and primed by other PKs activated by I/R, thus increasing the
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number of PKs whose signaling is inhibited. Therefore, this predisposes a state of energy
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conservation in WM when ATP is momentarily unavailable. CK2 Signaling During Ischemia in Brain GM In GM, CK2 signaling can be either protective or damaging. For example, in a model of transient forebrain ischemia, the preservation or activation of CK2 activity was proposed to be important for neuronal survival after cerebral ischemia8. Specifically, It was reported that the regions
vulnerable to ischemia showed a decrease in CK2 activity, whereas resistant regions were associated with an increase8. In contrast, in a model of global ischemia, an increase in CK2 activity in the dentate gyrus was reported50.
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Further investigation into the impact of transient cerebral ischemia and the role of CK2 signaling in a mouse model of ischemia (MCAO51) revealed that MCAO was associated with a decrease in CK2 (- and ’) levels and activity. On the other hand, the changes in CK2 activity following ischemia varied as a function of time such that the reduction in CK2 levels was attenuated at 1 h and 3 h, remained unchanged at 6 h and 12 h, and was ~60% below normal at 24 h and 48 h.
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Inhibition of CK2 with tetrabromocinnamic acid (TBCA) was associated with an increase in GM injury. And finally, using an MCAO model and the same experimental approach as Kim et al. 51,
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three other reports have concluded that CK2 signaling is protective of GM from ischemic injury52– 54
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. Overall these studies suggest that neuronal responses to CK2 signaling vary depending upon
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the experimental model used.
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Could the experimental design that prolongs CK2 inhibition alter the outcome in ischemic
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experiments55? It is possible that neuronal injury observed with TCBA (MCAO model) and siRNA (cortical neuronal culture model) was caused by the duration of the reduction in CK2 protein
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levels/activity, since decreased CK2 activity for an extended period of time mediates cell death in non-neuronal cells56. In addition, the CK2 inhibitor used may have been injurious because some
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CK2 inhibitors have been shown to generate ROS57. Future experiments using a MCAO model subjected to a shorter duration of CK2 signaling inhibition using a different CK2 inhibitor, such as
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CX-4945, need to be conducted in order to answer these questions. All of these experiments in GM matter injury models have identified an array of signaling mechanisms that are potentially important. First, Kim et al. proposed that CK2 acts as a negative modulator of NADPH oxidase55. Then Blanquet et al.50 found that increased CK2 correlated with
inhibition of the activity of MKK(3/6), p38, and deacetylases. Subsequent studies investigated the mechanisms by which CK2 inhibition of NOX2 led to neuronal survival using superoxide dismutase 1 (SOD1) transgenic mutant and gp91(Nox2) knock-out mice32. These results suggest that NOX2 activation releases ROS after CK2 inhibition, triggering the release of apoptogenic
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factors (AIF) from mitochondria and inducing DNA damage after ischemic brain injury. Overall, they concluded that there is a reciprocal relationship between ROS and CK2 protein levels. And finally, studies were performed to test the mechanism of compounds that showed neuroprotection in a MCAO model such as apelin-13, 5d, a novel analogue of the racemic 3-n-butylphthalide, and cilostazol52–54. The authors concluded that the neuroprotective effects observed with apelin-13
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and 5d may be partly mediated by an increase in CK2 activity, such that apelin-13 regulated CK2
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by activating the apelin receptor/Gi/Gq-CK2 and restoring p-eIF2α-ATF4-CHOP/GRP78 to
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attenuate neuronal apoptosis, and 5d decreased NADPH oxidase activity by positively regulating
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CK252,53. Experiments with cilostazol suggested that its protective mechanism was produced by the maxi-K channel opening coupled to upregulation of CK2 phosphorylation and downregulation
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of PTEN phosphorylation54. The exact mechanisms of how channel opening increases and how
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PTEN phosphorylation decreases CK2 have not been elucidated, nor has the impact of CK2 inhibitor been tested to abrogate the effect of cilostazol. The authors suggest that CK2 activation
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may serve as a therapeutic target for inhibiting neuronal cell apoptosis in ischemic cells and thus
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may offer a cell survival strategy. These proposed mechanisms of CK2 protection identified in GM
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and neuronal injury remain to be tested in WM.
A Lesson from Cancer Research Interestingly, cancer research has been instrumental in identifying novel therapeutic targets and mechanism(s) for cerebral ischemic injury. In cancer, CK2 activity has been shown to be upregulated such that cells become dependent upon high CK2 levels for their survival9. CK2 has
been shown to increase glucose metabolism in bladder cancer cells, thus contributing to the Warburg effect58. Could the protective effect of CK2 inhibition in WM be mediated by a metabolic switch during the ischemic period? For instance, Zhang et al.58 have shown that CK2 inhibition was associated with decreased levels of AKTS4 phosphorylation and decreased levels of
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glycolysis-related genes. Also, CK2 could regulate glycolysis by phosphorylating glycolytic enzymes59,60. Taken together, these results suggest that CK2 inhibition may suppress high glycolysis levels by reducing CK2 and AKT phosphorylation of enzymes/transporter in the glycolytic pathway. It is plausible that I/R leads to increased glycolysis, resulting in an increase in metabolic intermediates for the tricarboxylic acid cycle; however, with decreasing O2 levels, this
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would lead to increased lactate levels, decreased pH, and activation of the pentose phosphate
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pathway (PPP). Note that an increase in glycolysis in the face of compromised oxidative
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phosphorylation leads to increased lactate and an increase in PPP pathway activation, which is
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essential to generate the building blocks (lipids, nucleic acids) that are necessary to sustain the high mitotic rates of tumors61. Therefore, we hypothesize that during ischemia and early
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reperfusion when ATP is compromised25, it is more important to relieve the metabolic constraints
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than to generate the building blocks for repair. However, after this transient repression of CK2
(Figure 3).
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activity during I/R, it is important to release this inhibition so that CK2 can aid in the repair process
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Conclusion
In this review, we have discussed how inhibition of the CK2/CDK5 signaling axis protects WM
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only when inhibited before ischemia, while inhibition of the CK2/AKT/GSK3 signaling axis protects WM either before or after an ischemic injury in young, aging, and old WM (Figure 3). CK2 activation mediates WM ischemic injury in a differential spatiotemporal manner such that CDK5 signaling becomes important during ischemia, while AKT signaling emerges as the main pathway during the reperfusion period following ischemia. Consequently, interventions selectively
targeting the activated form of AKT confer post-ischemic functional recovery in young and aging WM. In contrast, ischemia-mediated regulation of CK2 activity impacts neuronal survival differently. The reasons for this discrepancy may stem from differences in the dominant CK2 substrates, the duration of the CK2 inhibition, the involvement of inflammation, and the choice of
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CK2 pharmacological inhibitor used in these studies. As the role of CK2 in the control of apoptosis in non-neuronal cells is well-established, we would advocate, based on our results in WM, that future studies use a selective CK2 inhibitor like CX-4945 at an optimal concentration and duration. Experiments using a short duration of treatment with CX-4945 to investigate neuronal survival in an in vivo model are also warranted. Until then, the effective use of CK2 signaling inhibition to
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protect the brain against ischemic injury will require the development of a targeted approach for
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WM in order to minimize damage to GM. It is essential to preserve, protect, and repair both
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neuronal and glial cells of the brain to improve functional recovery. Nevertheless, continued
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research into the role of CK2 signaling in WM may reveal more effective therapeutic targets for WM that may be useful for neurodegenerative diseases that primarily affect glial cells and myelin
injury.
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Figure Legends
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such as traumatic brain injury, multiple sclerosis, periventricular leukomalacia, and spinal cord
Figure 1. The CK2 small molecule inhibitor, CX-4945, promotes function recovery in young
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and aging axons following OGD. Time course shows minimal recovery following OGD (black; B, young; D, aging). Examples of CAP traces (A, young; and D, aging) taken during baseline (a),
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OGD (b), and recovery (c) for control and CX-4945 (5 μM) pre-treatment (red) and post-treatment (magenta). Arrows show sustained CAP at the end of OGD following CX-4945 in young and aging MONs. Pre-treatment with CX-4945 (red: A, B and C, young; and D, E and F, aging) applied before OGD promoted a consistent and sustained CAP area recovery, preserved CAP area during OGD, and improved axon function following recovery. CX-4945 applied after OGD (magenta: A,
B and C, young; and D, E and F, aging) promoted a consistent and sustained CAP area recovery. ** p < 0.01, and *** p < 0.001, one-way ANOVA with Newman-Keuls post hoc test.1 Figure 2. CK2 inhibition prevents OGD-induced axon injury, oligodendrocyte death, and
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mitochondrial disruption, and it also attenuates OGD-upregulated GSK3β phosphorylation in mouse optic nerve. A) OGD (1 h) caused widespread loss of SMI-31 (+) axons (green) and adenomatous polyposis coli (APC (+), oligodendrocytes, magenta), while CX-4945 (5 μM) prevented axonal injury and oligodendrocyte loss. Glial nuclei were labeled with Sytox (blue, white arrows). Note that the merged images in the right panels are enlarged areas indicated by the squares with dashed lines in the middle panels. Scale bar = 20 µm. B) Two-photon confocal
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images of axonal CFP (+) mitochondria in MONs from Thy-1 mito-CFP mice after control, OGD,
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and OGD + CX-4945. OGD attenuated CFP fluorescence; in addition, mitochondria became
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smaller and less numerous. CX-4945 pre-treatment preserved CFP (+) mitochondrial
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fluorescence and morphology. C) Histogram summarizing the data. Scale bar = 10 μm. *** p < 0.001, one-way ANOVA with Newman-Keuls post hoc test. D) and E) An increase in GSK3βS9
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was observed in MONs following OGD and reperfusion; however, this was attenuated by CX-
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4945 (5 µM) pre-treatment. *** p < 0.001, one-way ANOVA with Newman-Keuls post hoc test.1
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Figure 3. CK2 inhibition confers WM functional protection against ischemia by differentially regulating the CDK5 and AKT signaling pathways. A) Ischemic injury in young
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WM follows a sequential order, initiated by loss of ionic homeostasis leading to excitotoxicity and then merging into oxidative injury. Our findings suggest that ischemia in WM results in CK2
A
activation. Functional protection was evident when CX-4945, a CK2 inhibitor, was applied before or after glutamate accumulation, which may suggest that CX-4945 could simultaneously target the excitotoxic and oxidative pathways. CK2 recruits CDK5 and AKT/GSK3β signaling to mediate WM ischemic injury in a spatiotemporal manner such that CDK5 signaling becomes important during ischemia, while AKT signaling emerges as the main pathway during the reperfusion period
following ischemia. B) A detailed molecular model of the regulation of CK2/CDK5 and CK2/AKT/GSK3 signaling pathways mediating WM injury with ischemia. Note that recruitment of the oxidative pathway is mediated by positive feedback from CK2/AKT/GSK3 signaling to increase glycolysis in the face of decreased O2, glucose, and ATP, leading to accentuated
SC RI PT
glycolytic metabolites and ROS levels. PPP, Pentose Phosphate Pathway; NADPH, Nicotinamide Adenine Dinucleotide Phosphate Hydrogen; GSH, Glutathione; ROS, Reactive Oxygen Species; CK2, Casein Kinase 2; CDK5, cell division PK 5; AKT, PKB; GSK3, Glycogen synthase kinase3 ; and I/R, Ischemia/Reperfusion.
U
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Figure 1 Brunet et al.
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Figure 2 Brunet et al.
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Figure 3 Brunet et al.
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CK2 CDK5 AKT
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+ Feedback
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-Oligodendrocyte death -Axonal injury Oxidative mechanism Mitochondrial dysfunction Lipid peroxidation Protein oxidation DNA damage
Early I/R Later I/R