Distinct Roles of CK2- and AKT-Mediated NF-κB Phosphorylations in Clasmatodendrosis (Autophagic Astroglial Death) within the Hippocampus of Chronic Epilepsy Rats

The downregulation of glutathione peroxidase-1 (GPx1) plays a role in clasmatodendrosis (an autophagic astroglial death) in the hippocampus of chronic epilepsy rats. Furthermore, N-acetylcysteine (NAC, a GSH precursor) restores GPx1 expression in clasmatodendritic astrocytes and alleviates this autophagic astroglial death, independent of nuclear factor erythroid-2-related factor 2 (Nrf2) activity. However, the regulatory signal pathways of these phenomena have not been fully explored. In the present study, NAC attenuated clasmatodendrosis by alleviating GPx1 downregulation, casein kinase 2 (CK2)-mediated nuclear factor-κB (NF-κB) serine (S) 529 and AKT-mediated NF-κB S536 phosphorylations. 2-[4,5,6,7-Tetrabromo-2-(dimethylamino)-1H-benzo[d]imidazole-1-yl]acetic acid (TMCB; a selective CK2 inhibitor) relieved clasmatodendritic degeneration and GPx1 downregulation concomitant with the decreased NF-κB S529 and AKT S473 phosphorylations. In contrast, AKT inhibition by 3-chloroacetyl-indole (3CAI) ameliorated clasmatodendrosis and NF-κB S536 phosphorylation, while it did not affect GPx1 downregulation and CK2 tyrosine (Y) 255 and NF-κB S529 phosphorylations. Therefore, these findings suggest that seizure-induced oxidative stress may diminish GPx1 expression by increasing CK2-mediated NF-κB S529 phosphorylation, which would subsequently enhance AKT-mediated NF-κB S536 phosphorylation leading to autophagic astroglial degeneration.


Experimental Animals and Chemicals
Male Sprague Dawley (SD) rats (200-250 g) were cared under controlled environmental conditions (23-25 • C, 12 h light/dark cycle) and freely accessed to water and conventional rat diets. All experimental protocols described below were approved by the Institutional Animal Care and Use Committee of Hallym University (Hallym 2021-3, approval date: 17 May 2021). All reagents were obtained from Sigma-Aldrich (St. Louis, MO, USA), except as noted.

NAC Treatment
Chronic epilepsy rats were given N-acetylcysteine (NAC, 70 mg/kg/day, i.p.) over a 7-day period [17]. Five hours after the last injection, the animals were used for experiments.

Western Blot
Under urethane anesthesia (1.5 g/kg, i.p.), rats were decapitated, and the hippocampus was rapidly dissected out and homogenized in lysis buffer containing protease inhibitor cocktail (Roche Applied Sciences, Branford, CT, USA) and phosphatase inhibitor cocktail (PhosSTOP ® , Roche Applied Science, Branford, CT, USA). The protein concentration was measured using a Micro BCA Protein Assay Kit (Pierce Chemical, Dallas, TX, USA). Thereafter, Western blotting was performed by the standard protocol (n = 7 rats in each group). After electrophoresis, proteins were transferred to polyvinylidene fluoride membranes that were subsequently incubated with a blocking solution followed by immunoblotting with the primary antibody (Table 1). For chemiluminescent detection and analysis, an Image-Quant LAS4000 system (GE Healthcare Korea, Seoul, South Korea) was used. The β-actin value was used for the normalization of each protein value. The phosphoprotein/total protein ratio was represented as the phosphorylation ratio [14,30,34].

Tissue Preparation and Immunohistochemistry
Animals were anesthetized with urethane anesthesia (1.5 g/kg, i.p.) and perfused with 4% paraformaldehyde in 0.1 M phosphate buffer (PB, pH 7.4) through the left ventricle followed by post-fixation in the same fixative overnight. After immersion with 30% sucrose overnight, brains were sectioned at 30 µm. Sections were blocked with 3% bovine serum albumin in PBS for 30 min, and later incubated overnight with mixtures of primary antibod-  (Table 1) in PBS containing 0.3% Triton X-100. After washing, tissues were reacted with Brilliant Violet-421, Cy2-or Cy3-fluorescent dye conjugated secondary antibodies (Jackson Immuno Research Laboratories, West Grove, PA, USA). The fluorescent intensity was quantified in the randomly selected 2-3 reactive astrocytes or clasmatodendritic astrocytes in the stratum radiatum of the CA1 region (n = 7 rats in each group) with AxioVision Rel. 4.8 (Carl Zeiss Korea, Seoul, Republic of Korea) and ImageJ software. For quantification of clasmatodendritc astrocytes, cell counts were conducted in areas of interest (1 × 10 4 µm 2 ) of 10 sections per each animal [14,34].

Data Analysis
The Mann-Whitney test was applied to analyze statistical significance of data obtained from two groups. The Kruskal-Wallis test with Dunn-Bonferroni post hoc test was used for the comparison of data obtained four groups. The Spearman test was applied to identify the relationship between two variables. A p-value less than 0.05 was considered significant.

Figure 3. Effects of NAC on GPx1 expression and AKT S473 phosphorylation in CA1 astrocytes.
Compared to control rats, AKT S473 phosphorylation is enhanced in clasmatodendritic (vacuolized) CA1 astrocytes (Clas, arrows) more than reactive CA1 astrocytes (Reac), which is attenuated by NAC treatment. (A) Representative photos of GPx1 expression, AKT S473 signal and their intensities. Bar = 25 µm. (B) Quantification of AKT S473 intensity in CA1 astrocytes (* ,# p < 0.05 vs. vehicle and reactive astrocytes, respectively, n = 20 cells in 7 rats, respectively; Kruskal-Wallis test with Dunn-Bonferroni post hoc test). (C) Linear regression analysis between GPx1 and AKT S473 intensities in reactive and clasmatodendritic CA1 astrocytes of chronic epilepsy rats (n = 80 cells in 14 rats; Spearman test). ed by NAC treatment. (A) Representative photos of GPx1 expression, AKT S473 signal and their intensities. Bar = 25 μm. (B) Quantification of AKT S473 intensity in CA1 astrocytes (* ,# p < 0.05 vs. vehicle and reactive astrocytes, respectively, n = 20 cells in 7 rats, respectively; Kruskal-Wallis test with Dunn-Bonferroni post hoc test). (C) Linear regression analysis between GPx1 and AKT S473 intensities in reactive and clasmatodendritic CA1 astrocytes of chronic epilepsy rats (n = 80 cells in 14 rats; Spearman test).  Compatible with immunofluorescent studies, Western blot data also revealed that NAC augmented AKT S473 and NF-κB S536 phosphorylation levels, as compared to the vehicle ( Figures 5A-C and S2). These findings indicate that AKT-mediated NF-κB S536 phosphorylation may participate in clasmatodendritic degeneration, and that NAC may ameliorate clasmatodendrosis by inhibiting this pathway as well as CK2-mediated NF-κB S529 phosphorylation. and reactive astrocytes, respectively, n = 20 cells in 7 rats, respectively; Kruskal-Wallis test with Dunn-Bonferroni post hoc test). (C) Linear regression analysis between GPx1 and NF-κB S536 intensities in reactive and clasmatodendritic CA1 astrocytes of chronic epilepsy rats (n = 80 cells in 14 rats; Spearman test).
Compatible with immunofluorescent studies, Western blot data also revealed that NAC augmented AKT S473 and NF-κB S536 phosphorylation levels, as compared to the vehicle ( Figures 5A-C and S2). These findings indicate that AKT-mediated NF-κB S536 phosphorylation may participate in clasmatodendritic degeneration, and that NAC may ameliorate clasmatodendrosis by inhibiting this pathway as well as CK2-mediated NF-κB S529 phosphorylation.  (Figures 3 and 4), NAC diminishes AKT S473 and NF-κB S536 phosphorylation levels, as compared to the vehicle (Veh). (A) Representative Western blot of AKT, AKT S473, NF-κB and NF-κB S536 levels. (B,C) Quantification of AKT S473 and NF-κB S536 phosphorylation levels based on Western blot data (* ,# p < 0.05 vs. control animals and vehicle-treated epilepsy rats, respectively, n = 7 rats, respectively; Kruskal-Wallis test with Dunn-Bonferroni post hoc test).
Antioxidants 2023, 12, x FOR PEER REVIEW 12 of 20 the vehicle (Figures 8A-E and S3). These findings indicate that CK2-mediated NF-κB S529 phosphorylation may diminish GPx1 expression during clasmatodendrosis, and that AKT-mediated NF-κB S536 phosphorylation may be a consequence of GPx1 downregulation induced by this pathway.

Discussion
Astroglial activation generates H2O2 that evokes an imbalance of redox homeostasis in the brain [42]. Therefore, the defense system removing H2O2 is essential for astroglial viability. GPx1 plays an important role in GSH-mediated H2O2 elimination [22,23]. Indeed, GPx expression is increased in glial cells around surviving neurons [43] and GPx1 inhibits the ROS-mediated AKT activation [32,33]. In the present study, GPx1 was upregulated in reactive CA1 astrocytes, suggesting that increased GPx1 expression in reactive astrocytes may be an adaptive response against oxidative stress. However, GPx1 expression was significantly diminished in clasmatodendritic CA1 astrocytes concomitant with increased NF-κB S529 phosphorylation, which was recovered by NAC. NF-κB signaling pathway activates autophagy after heat shock [35]. Indeed, NF-κB S529, but not S276 and S311, phosphorylation is involved in clasmatodendritic astrocytes [2]. Since NAC acts as a direct ROS scavenger per se as well as a GSH precursor leading to increased GPx1/2 expression [44][45][46], our findings suggest that the antioxidant properties of NAC may improve GPx1 downregulation in clasmatodendritic astrocytes by inhibiting NF-κB S529 phosphorylation.
S529 phosphorylation increases NF-κB-mediated nuclear transcriptional activity, which is regulated by CK2 [36]. CK2 is a highly conserved and constitutively active serine/threonine kinase that promotes cell viability, proliferation and differentiation [47,48]. CK2 activity is enhanced by phosphorylation of Y255 and T360/S362 sites, which are

Discussion
Astroglial activation generates H 2 O 2 that evokes an imbalance of redox homeostasis in the brain [42]. Therefore, the defense system removing H 2 O 2 is essential for astroglial viability. GPx1 plays an important role in GSH-mediated H 2 O 2 elimination [22,23]. Indeed, GPx expression is increased in glial cells around surviving neurons [43] and GPx1 inhibits the ROS-mediated AKT activation [32,33]. In the present study, GPx1 was upregulated in reactive CA1 astrocytes, suggesting that increased GPx1 expression in reactive astrocytes may be an adaptive response against oxidative stress. However, GPx1 expression was significantly diminished in clasmatodendritic CA1 astrocytes concomitant with increased NF-κB S529 phosphorylation, which was recovered by NAC. NF-κB signaling pathway activates autophagy after heat shock [35]. Indeed, NF-κB S529, but not S276 and S311, phosphorylation is involved in clasmatodendritic astrocytes [2]. Since NAC acts as a direct ROS scavenger per se as well as a GSH precursor leading to increased GPx1/2 expression [44][45][46], our findings suggest that the antioxidant properties of NAC may improve GPx1 downregulation in clasmatodendritic astrocytes by inhibiting NF-κB S529 phosphorylation.
The present data show AKT S473 hyperphosphorylation in clasmatodendritic CA1 astrocytes exhibiting low GPx1 intensity. Oxidative stress triggers AKT activation [57], which inhibits ROS-induced GPx1 upregulation [58]. Therefore, the present data are simply interpreted as that AKT may be one of the upstream molecules to suppress GPx1 expression in clasmatodendritic astrocytes. In the present study, however, AKT inhibition by 3CAI did not improve GPx1 downregulation in clasmatodendritic astrocytes, although it attenuated clasmatodendrosis. Therefore, our findings indicate that AKT S473 hyperphosphorylation may not be relevant to reduced GPx1 expression during clasmatodendritic degeneration.
On the other hand, CK2 also activates AKT by phosphorylation at S129 site [59][60][61]. In addition, the present study reveals that both CK2 inhibition by TMCB and AKT inhibition by 3CAI attenuated clasmatodendritic degeneration. Considering these, it is plausible that CK2-mediated AKT S129 phosphorylation would also elicit clasmatodendrosis by NF-κB S536 phosphorylation. However, CK2-mediated AKT S129 phosphorylation is necessary for the cell viability in HEK-293T cells [59]. Indeed, CX-4945 (a CK2 inhibitor) exerts strong anti-proliferative activity by blocking AKT S129 phosphorylation in cancer cells [60,61]. Therefore, it is likely that CK2-mediated AKT S129 phosphorylation may not be involved in clasmatodendritic degeneration or astroglial viability in the epileptic hippocampus.
Astrocytes contribute to the slow afterhyperpolarizing potential (sAHP), which is a major intrinsic mechanism of neuronal inhibition and its termination [66]. 4,5,6,7-Tetrabromotriazole (TBB, a CK2 inhibitor) augments sAHP [67]. Since the inhibition of clasmatodendrosis shortens seizure duration in chronic epilepsy rats [14], the present data provide evidence that clasmatodendrosis may be an epiphenomenon maintaining prolonged seizure duration in the epileptic hippocampus.

Conclusions
The present study demonstrates for the first time that CK2-mediated NF-κB S529 phosphorylation evoked GPx1 downregulation in clasmatodendritic astrocytes, which subsequently led to AKT-mediated NF-κB S536 phosphorylation facilitating this autophagic astroglial degeneration ( Figure 12). Therefore, our findings suggest that GPx1 may integrate between CK2-and AKT-mediated signaling pathways during clasmatodendrosis induced by oxidative stress. Astrocytes contribute to the slow afterhyperpolarizing potential (sAHP), which is a major intrinsic mechanism of neuronal inhibition and its termination [66]. 4,5,6,7-Tetrabromotriazole (TBB, a CK2 inhibitor) augments sAHP [67]. Since the inhibition of clasmatodendrosis shortens seizure duration in chronic epilepsy rats [14], the present data provide evidence that clasmatodendrosis may be an epiphenomenon maintaining prolonged seizure duration in the epileptic hippocampus.

Conclusions
The present study demonstrates for the first time that CK2-mediated NF-κB S529 phosphorylation evoked GPx1 downregulation in clasmatodendritic astrocytes, which subsequently led to AKT-mediated NF-κB S536 phosphorylation facilitating this autophagic astroglial degeneration ( Figure 12). Therefore, our findings suggest that GPx1 may integrate between CK2-and AKT-mediated signaling pathways during clasmatodendrosis induced by oxidative stress. Figure 12. Schematic depiction representing the distinct role of NF-κB phosphorylation in clasmatodendritic CA1 astrocytes based on the present data and previous reports. Seizure activity decreases the GSH level and subsequently increases the ROS level. Aberrant CK2-mediated NF-κB S529 phosphorylation participates in GPx1 downregulation, which abolishes the GPx1-mediated inhibition of NF-κB S536 phosphorylation induced by AKT hyperactivation. In turn, the enhanced NF-κB S536 phosphorylation is involved in clasmatodendritic degeneration concomitant with AKT-mediated Bif-1 activation.
Supplementary Materials: The following supporting information can be downloaded at: www.mdpi.com/xxx/s1. Figure S1: Full-length images of Western blots in Figure 2A. Figure S2: Full-length images of Western blots in Figure 5A. Figure S3: Full-length images of Western blots in Figure 8A. Figure S4: Full-length images of Western blots in Figure 11A. Informed Consent Statement: Not applicable.
Data Availability Statement: Data sharing is not applicable to this article. Figure 12. Schematic depiction representing the distinct role of NF-κB phosphorylation in clasmatodendritic CA1 astrocytes based on the present data and previous reports. Seizure activity decreases the GSH level and subsequently increases the ROS level. Aberrant CK2-mediated NF-κB S529 phosphorylation participates in GPx1 downregulation, which abolishes the GPx1-mediated inhibition of NF-κB S536 phosphorylation induced by AKT hyperactivation. In turn, the enhanced NF-κB S536 phosphorylation is involved in clasmatodendritic degeneration concomitant with AKT-mediated Bif-1 activation.

Supplementary Materials:
The following supporting information can be downloaded at: https: //www.mdpi.com/article/10.3390/antiox12051020/s1. Figure S1: Full-length images of Western blots in Figure 2A. Figure S2: Full-length images of Western blots in Figure 5A. Figure S3: Full-length images of Western blots in Figure 8A. Figure S4: Full-length images of Western blots in Figure 11A.

Informed Consent Statement: Not applicable.
Data Availability Statement: Data sharing is not applicable to this article.

Conflicts of Interest:
The authors declare no conflict of interest.