Blockade of 67-kDa Laminin Receptor Facilitates AQP4 Down-Regulation and BBB Disruption via ERK1/2-and p38 MAPK-Mediated PI3K/AKT Activations

Recently, we have reported that dysfunctions of 67-kDa laminin receptor (67LR) induced by status epilepticus (SE, a prolonged seizure activity) and 67LR neutralization are involved in vasogenic edema formation, accompanied by the reduced aquaporin 4 (AQP4, an astroglial specific water channel) expression in the rat piriform cortex (PC). In the present study, we found that the blockade of 67LR activated p38 mitogen-activated protein kinase (p38 MAPK) and extracellular signal-regulated kinase 1/2 (ERK1/2) signaling pathways, which enhanced phosphatidylinositol 3 kinase (PI3K)/AKT phosphorylations in endothelial cells and astrocytes, respectively. 67LR-p38 MAPK-PI3K-AKT activation in endothelial cells increased vascular permeability. In contrast, 67LR-ERK1/2-PI3K-AKT signaling pathways in astrocytes regulated astroglial viability and AQP4 expression. These findings indicate that PI3K/AKT may integrate p38 MAPK and ERK1/2 signaling pathways to regulate AQP4 expression when 67LR functionality is reduced. Thus, we suggest that 67LR-p38 MAPK/ERK1/2-PI3K-AKT-AQP4 signaling cascades may mediate serum extravasation and AQP4 expression in astroglio-vascular systems, which is one of the considerable therapeutic targets for vasogenic edema in various neurological diseases.


Introduction
Status epilepticus (SE, a prolonged seizure activity) is one of the neurologic emergencies that lead to death or permanent neurologic injury. As SE causes 3-5% of symptomatic epilepsy (~35% of epileptic syndromes), it is a high-risk factor for developing acquired epilepsy [1]. SE results in neuronal damage and astroglial death, which trigger long-term and profound alterations in the neuronal network that leads to the development of temporal lobe epilepsy (TLE) [2][3][4][5]. Furthermore, SE leads to the leakage of blood serum components into the parenchyma (referred to as vasogenic edema) across the blood-brain barrier (BBB) leading to the impaired astrocyte function and the altered potassium homeostasis. This vasogenic edema formation evokes neuroinflammation and paroxysmal neuronal discharge, which play an important role in epileptogenesis [6,7].
The BBB maintains the brain microenvironment to ensure proper nervous system functions by segregating the systemic environment. The BBB is formed by the endothelial cells lining the blood vessels, astrocytic endfeet surrounding the blood vessels and pericytes embedded in the basement membranes between the endothelial cells and the astrocytes. Astrocytes envelop >99% of the BBB endothelium and play an important role in inducing and maintaining BBB. Thus, BBB disruption

Experimental Animals and Chemicals
Adult male Sprague-Dawley (SD) rats (7 weeks old) were used in the present study. Animals were housed in an acclimatized room (temperature, 22 ± 2 °C; humidity, 55 ± 5%; a 12-h light/dark cycle). The animals had access to water and food ad libitum. All efforts were made to reduce the number of animals and to minimize their suffering. Animal procedures were approved by the Institutional Animal Care and Use Committee of Hallym University (Chuncheon, South Korea, Hallym 2017- 54, 19th February 2017 and Hallym 2018-2, 26th April 2018). All reagents were obtained from Sigma-Aldrich (St. Louis, MO, USA), except as noted.

Experimental Animals and Chemicals
Adult male Sprague-Dawley (SD) rats (7 weeks old) were used in the present study. Animals were housed in an acclimatized room (temperature, 22 ± 2 • C; humidity, 55 ± 5%; a 12-h light/dark cycle). The animals had access to water and food ad libitum. All efforts were made to reduce the number of animals and to minimize their suffering. Animal procedures were approved by the Institutional Animal Care and Use Committee of Hallym University (Chuncheon, South Korea, Hallym 2017- 54, 19th February 2017 and Hallym 2018-2, 26th April 2018). All reagents were obtained from Sigma-Aldrich (St. Louis, MO, USA), except as noted.

SE Induction
To induce SE, animals were pretreated with an intraperitoneal injection of LiCl (127 mg/kg, i.p.) and atropine methylbromide (5 mg/kg, i.p.) 24 h and 20 min before pilocarpine (30 mg/kg, i.p.) treatment, Cells 2020, 9, 1670 4 of 19 respectively. Control animals received an equal volume of normal saline instead of pilocarpine. Two hours after SE, animals received diazepam (Valium; Roche, Neuilly sur-Seine, France; 10 mg/kg, i.p.) to terminate SE. Three days after SE, animals were used for immunohistochemistry and Western blot.

Western Blot
Western blot was performed by the standard protocol. Briefly, sample proteins (10 µg) were separated on a Bis-Tris sodium dodecyl sulfate-poly-acrylamide electrophoresis gel (SDS-PAGE). Separated proteins then were transferred to polyvinylidene fluoride membranes. The membranes were incubated with a relatively specific primary antibody ( Table 1). The ECL Kit (GE Healthcare Korea, Seoul, South Korea) was used to detect signals. The bands were detected and quantified on ImageQuant LAS4000 system (GE Healthcare Korea, Seoul, South Korea). β-Actin antibody was used as a loading control for the quantitative analysis of relative expression levels of proteins. The ratio of phosphoprotein to total protein was described as the phosphorylation ratio. Thereafter, the density value of each sample obtained from 67LR IgG-infused animals (67LR IgG) and SE-induced animals (SE) was compared to that obtained from control IgG-infused animals (Cont IgG) and non-SE-induced animals (Control), respectively.

Immunohistochemistry
Standard procedures for immunohistochemistry were used to detect serum extravasation. Briefly, free-floating sections were washed 3 times in PBS (0.1 M, pH 7.3). Next, to inactivate the endogenous peroxidase, sections were incubated in 3% H 2 O 2 and 10% methanol in PBS (0.1 M) for 20 min at room temperature. Later, sections were incubated in biotinylated rat IgG and ABC complex (Vector, #PK-6100, Burlingame, CA, USA, diluted 1:200). Tissue sections were developed in 3,3 -diaminobenzidine in 0.1 M Tris buffer and mounted on gelatin-coated slides. Some sections were incubated with a cocktail solution containing the primary antibodies (Table 1) in PBS containing 0.3% Triton X-100 overnight at room temperature. Thereafter, sections were visualized with appropriate Cy2-and Cy3-conjugated secondary antibodies. GFAP and SMI-71 were used for the markers of astrocytes and endothelial cells, respectively. To establish the specificity of the immunostaining, a negative control test was carried out with pre-immune serum instead of the primary antibody. No immunoreactivity was observed for the negative control in any structures. All experimental procedures in this study were performed under the same conditions and in parallel. Immunoreaction was observed using an Axio Scope microscope (Carl Zeiss Korea, Seoul, Korea).

Measurements of Serum Extravasation and the Volume of GFAP-Deleted Lesion
Serum extravasation was measured, as previously described [14,17,18,31]. Aforementioned, sections were incubated in biotinylated rat IgG and ABC complex (Vector, #PK-6100, Burlingame, CA, USA, diluted 1:200). Tissue sections were developed in 3,3 -diaminobenzidine in 0.1 M Tris buffer and mounted on gelatin-coated slides. Thereafter, sections (10 sections per each animal) were captured, and areas of interest were selected. Then, the measurement of serum extravasation was performed on 5× images using AxioVision Rel. 4.8 software. Serum extravasation (rat IgG immunodensity) measurements were represented as the number of a 256 grayscale. Intensity values were corrected by subtracting the average values of background noise (mean background intensity) obtained from five image inputs. Intensity of each section was standardized by setting the threshold level (mean background intensity obtained from five image inputs). Manipulation of the images was restricted to threshold and brightness adjustments to the whole image. The volume of GFAP-deleted lesion in the PC was measured by AxioVision Rel. 4.8 software and estimated by the modified Cavalieri method: Cells 2020, 9, 1670 6 of 19 V = Σarea × section thickness (30 µm) × 1/the fraction of the sections (1/6). The volumes are reported in mm 3 .

Statistical Analysis
The animal number (n) of each experimental group used for the evaluation was seven. The data obtained from each animal (different samples from the same experiment) were analyzed. Quantitative data were expressed as mean ± standard error of the mean. After evaluating the values on normality using Shapiro-Wilk W-test, data are analyzed by one-way ANOVA followed by Newman-Keuls post-hoc test. p < 0.05 was considered to be statistically different.

The Effects of SE on p-p38 MAPK, pAKT, and pERK1/2 Levels in Endothelial Cells and Astrocytes
Next, we explored whether SE influences p38 MAPK, AKT, and ERK1/2 phosphorylations in cellular specific manners. Following SE, p-p38 MAPK signal was preferentially upregulated in endothelial cells ( Figure 6). Furthermore, pERK1/2 signals were enhanced in remaining (surviving) astrocytes ( Figure 6), although its total level was reduced on Western blot ( Figure 5A,B). pAKT signal was increased in both astrocytes and endothelial cells ( Figure 6). SB202190 and U0126 abrogated the up-regulated pAKT level in endothelial cells and astrocytes, respectively (Figure 7). 3CAI abolished it in both cells following SE (Figure 7). Thus, our findings suggest that SE-induced activations of 67LR-p38 MAPK-PI3K and 67LR-ERK1/2-PI3K signaling pathways may increase AKT activity in endothelial cells and astrocytes, respectively.

The Effects of SE on p-p38 MAPK, pAKT, and pERK1/2 Levels in Endothelial Cells and Astrocytes
Next, we explored whether SE influences p38 MAPK, AKT, and ERK1/2 phosphorylations in cellular specific manners. Following SE, p-p38 MAPK signal was preferentially upregulated in endothelial cells ( Figure 6). Furthermore, pERK1/2 signals were enhanced in remaining (surviving) astrocytes ( Figure 6), although its total level was reduced on Western blot ( Figure 5A,B). pAKT signal was increased in both astrocytes and endothelial cells ( Figure 6). SB202190 and U0126 abrogated the up-regulated pAKT level in endothelial cells and astrocytes, respectively (Figure 7). 3CAI abolished it in both cells following SE (Figure 7). Thus, our findings suggest that SE-induced activations of 67LR-p38 MAPK-PI3K and 67LR-ERK1/2-PI3K signaling pathways may increase AKT activity in endothelial cells and astrocytes, respectively.

Discussion
The major findings in the present study are that the blockade of 67LR functions leads to serum extravasation and the reduced AQP4 expression via two different PI3K/AKT-mediated pathways: Figure 7. The effects of kinase inhibitors on localizations of pAKT expression in the PC following SE. Compared to the vehicle, SB202190 and U0126 reduce pAKT signal in endothelial cells and astrocytes, respectively. 3CAI diminishes it in both endothelial cells and astrocytes. Cells 2020, 9,1670 14 of 20 ERK1/2, our findings suggest that AKT activation may down-regulate AQP4 expression in astrocytes following SE and 67LR neutralization. Givant-Horwitz et al. [32] reported that 67LR is involved in laminin-mediated p38 MAPK activation, which participates in SE-induced vasogenic edema via the increased PI3K/AKT-mediated eNOS expression [14,16]. Furthermore, 67LR neutralization activates p38 MAPK-mediated VEGF expression, which also increases vascular permeability [17]. Indeed, SB202190 alleviates serum extravasation induced by SE and 67LR IgG infusion, while it does not affect AQP4 expression [17,18]. Consistent with these reports, the present study reveals that 67LR neutralization and SE increased p38 MAPK-mediated PI3K/AKT phosphorylations. Unlike the case of ERK1/2 phosphorylation, however, the up-regulated p-p38 MAPK level was restricted to endothelial cells showing the decreased SMI-71 expression. Furthermore, SB202190 and 3CAI mitigated serum extravasation and astroglial loss, accompanied by the decreased pAKT level in endothelial cells, but not astrocytes, following SE. Since SE-induced vasogenic edema results in extensive astroglial loss that subsequently aggravates serum extravasation due to the impairment of AQP4-dependent water elimination [12], these findings indicate that 67LR-p38 MAPK-PI3K/AKT signaling pathway may increase vascular permeability due to BBB disruption, which subsequently evokes astroglial loss. Therefore, our

Discussion
The major findings in the present study are that the blockade of 67LR functions leads to serum extravasation and the reduced AQP4 expression via two different PI3K/AKT-mediated pathways: p38 MAPK-and ERK1/2-mediated PI3K/AKT activation in endothelial cells and astrocytes, respectively ( Figure 9).
Cells 2020, 9,1670 15 of 20 findings suggest that AKT may be one of the important molecules regulating astroglio-endothelial interactions during vasogenic edema formation. Figure 9. Scheme of role of 67-kDa LR in vasogenic edema formation and AQP4 expression. Blockade of 67LR induced by its neutralization and SE increases p38 MAPK and ERK1/2 phosphorylations in endothelial cells and astrocytes, respectively. Subsequently, both kinases activate PI3K/AKT signaling pathway. Increased AKT activity enhances endothelial permeability and triggers astroglial loss. However, AKT activation reduces AQP4 expression in astrocytes, which worsens vasogenic edema.
In the present study, 67LR neutralization increased total ERK1/2 phosphorylation. Furthermore, U0126 abrogated serum extravasation and the down-regulation of AQP4 expression induced by 67LR neutralization. In contrast, SE decreased total ERK1/2 phosphorylation, and U0126 deteriorated vasogenic edema and AQP4 down-regulation following SE. Thus, it would contradict the role of 67LR dysfunction in ERK1/2-mediated AQP4 regulation. Unlike 67LR IgG infusion, SE leads to acute and devastating astroglial degenerations that are characterized by a pattern of selective vulnerability [12,27,[44][45][46][47]. Thus, it is likely that the SE-induced astroglial damage may decrease the total ERK1/2 phosphorylation and AQP4 expression. However, immunohistochemical study revealed that pERK1/2 signals were enhanced in remaining (surviving) astrocytes following SE, although total ERK1/2 phosphorylation was reduced on Western blot. Therefore, our findings indicate that SE- Figure 9. Scheme of role of 67-kDa LR in vasogenic edema formation and AQP4 expression. Blockade of 67LR induced by its neutralization and SE increases p38 MAPK and ERK1/2 phosphorylations in endothelial cells and astrocytes, respectively. Subsequently, both kinases activate PI3K/AKT signaling pathway. Increased AKT activity enhances endothelial permeability and triggers astroglial loss. However, AKT activation reduces AQP4 expression in astrocytes, which worsens vasogenic edema.
Givant-Horwitz et al. [32] reported that 67LR is involved in laminin-mediated p38 MAPK activation, which participates in SE-induced vasogenic edema via the increased PI3K/AKT-mediated eNOS expression [14,16]. Furthermore, 67LR neutralization activates p38 MAPK-mediated VEGF expression, which also increases vascular permeability [17]. Indeed, SB202190 alleviates serum extravasation induced by SE and 67LR IgG infusion, while it does not affect AQP4 expression [17,18]. Consistent with these reports, the present study reveals that 67LR neutralization and SE increased p38 MAPK-mediated PI3K/AKT phosphorylations. Unlike the case of ERK1/2 phosphorylation, however, the up-regulated p-p38 MAPK level was restricted to endothelial cells showing the decreased SMI-71 expression. Furthermore, SB202190 and 3CAI mitigated serum extravasation and astroglial loss, accompanied by the decreased pAKT level in endothelial cells, but not astrocytes, following SE. Since SE-induced vasogenic edema results in extensive astroglial loss that subsequently aggravates serum extravasation due to the impairment of AQP4-dependent water elimination [12], these findings indicate that 67LR-p38 MAPK-PI3K/AKT signaling pathway may increase vascular permeability due to BBB disruption, which subsequently evokes astroglial loss. Therefore, our findings suggest that AKT may be one of the important molecules regulating astroglio-endothelial interactions during vasogenic edema formation.
On the other hand, some AQP4 isoforms are reported: AQP4a (M1), AQP4b, AQP4c (M23), AQP4d, AQP4e, and AQP4f in the brain [56][57][58][59]. Among them, AQP4a, AQP4c, and AQP4e are water-permeable and sensitive to changes in extracellular osmolality [60][61][62], which alternatively splice into AQP4b, AQP4d, and AQP4f isoforms, respectively [63]. Furthermore, De Bellis et al. [64] have reported a newly characterized AQP4ex isoform that is generated by translational readthrough. AQP4ex contains a 29 amino acid C-terminal extension, which is involved in proper membrane localization of AQP4 and interaction with other intracellular proteins. As AQP4ex display a perivascular polarization and expression in dystrophin-dependent pools, it is necessary for anchoring of the perivascular AQP4. Indeed, the absence of AQP4ex isoform, AQP4 assemblies are mislocalized in the brain [64,65]. In the present study, we could not explore the alterations in AQP4 isoforms induced by 67LR neutralization and SE, since the commercial antibodies for AQP4 isoforms are unavailable. However, Cartagena et al. [66] have reported that in the penetrating ballistic-like brain injury (PBBI) model AQP4a (M1) decreases at 3 and 7days post-injury, while AQP4c (M23) levels were highly variable with no significant changes. AQP4ex also regulates water transport at the BBB level, and binds with human neuromyelitis optica autoantibody that is associated with autoimmune demyelinating diseases in the central nervous system [67]. In addition, the deletion of gap junction forming proteins connexin-43 (Cx43) and connexin-30 (Cx30) increases AQP4a (M1) and AQP4ex isoform levels, but reduces AQP4c (M23) isoform in astrocytes [68]. Considering these reports, further studies are needed to validate AQP4 isoforms affected by 67LR functions in astroglio-vascular interactions.

Conclusions
To the best of our knowledge, the present data reveal for the first time that 67LR affected the severity of vasogenic edema formation in two different signaling pathways. Blockade of 67LR functions activated p38 MAPK-PI3K/AKT axis, which increased vascular permeability. It also increased ERK1/2and PI3K-mediated AKT phosphorylation in astrocytes, which decreased AQP4 expression (Figure 9). Thus, our findings suggest that the regulation of 67-kDa LR expression/functions may be one of the considerable factors for medication of vasogenic edema formation and prevention of its complications.