Upregulation of Connexin 43 Expression Via C -Jun N-Terminal Kinase Signaling in Prion Disease

Abstract

Prion infection leads to neuronal cell death, glial cell activation, and the accumulation of misfolded prion proteins. However, the altered cellular environments in animals with prion diseases are poorly understood. In the central nervous system, cells connect the cytoplasm of adjacent cells via connexin (Cx)-assembled gap junction channels to allow the direct exchange of small molecules, including ions, neurotransmitters, and signaling molecules, which regulate the activities of the connected cells. Here, we investigate the role of Cx43 in the pathogenesis of prion diseases. Upregulated Cx43 expression, which was dependent on c-Jun N-Terminal Kinase (JNK)/c-Jun signaling cascades, was found in prion-affected brain tissues and hippocampal neuronal cells. Scrapie infection-induced Cx43 formed aggregated plaques within the cytoplasmic compartments at the cell-cell interfaces. The ethidium bromide (EtBr) uptake assay and scrape-loading dye transfer assay demonstrated that increased Cx43 has functional consequences for the activity of Cx43 hemichannels. Interestingly, blockade of PrP^Sc accumulation reduced Cx43 expression through the inhibition of JNK signaling, indicating that PrP^Sc accumulation may be directly involved in JNK activation-mediated Cx43 upregulation. Overall, our findings describe a scrapie infection-mediated novel regulatory signaling pathway of Cx43 expression and may suggest a role for Cx43 in the pathogenesis of prion diseases.

Keywords

Connexin 43 gap junction JNK prion protein scrapie

INTRODUCTION

Gap junctions are a complex of membrane channels that allow the diffusion of small molecules (<1.5 kDa) between adjacent cells to form a functional syncytium. Gap junctions are composed of two hemichannels (connexons) formed by hexamers of connexins (Cxs) [1]. Of the Cx genes that code for gap junction proteins, Cx43 is the most ubiquitous; it is abundantly expressed in the brain and highly expressed during embryonic development [2, 3]. In the central nervous system (CNS), Cx43 is widely expressed in neurons, astrocytes, and oligodendrocytes [4 –6], and various signal molecules, including ATP, glutamate, and Ca^2 + ions, diffuse through functional Cx43 hemichannels [7 –11], thereby modulating crucial CNS processes [12].

Cx43 has contributed to neuronal death in vitro [11, 13], in a rat cortical ablation model [14], and in ischemic brain injury [15]. Cx43 plays a protective role against oxidative stress-induced cell death [16, 17]; deletion or blockade of this protein prevents chronic neuropathic pain following spinal cord injury [18, 19] and fetal ischemia [20]. The deletion of Cx43 or expression of a truncated form increases the vulnerability to stroke [21, 22]. In addition, Cx43 is involved in neuronal differentiation [23 –25], cellular mortality [26 –29], hippocampal proliferation, and the survival of newborn cells [30].

Moreover, the role of Cx43 has been implicated in various pathological conditions of the brain. Increased Cx43 levels have been identified within amyloid plaques in the brains of Alzheimer’s disease patients and in an animal model of the disease [31, 32], as well as in the anterior horns of mSOD1-Tg mice, which represent an amyotrophic lateral sclerosis mouse model [33], the MPTP-lesioned striatum of a Parkinson’s disease model [34], and the hippocampal regions of patients with mesial temporal lobe epilepsy [35]. In contrast, the loss of Cx43 expression is found in the actively demyelinating lesions of multiple sclerosis and in the active perivascular lesions of neuromyelitis optica [36]. Therefore, Cx43 is associated with the pathophysiology of various tissue conditions. However, to the best of our knowledge, there are no data available regarding the role of Cx43 in prion diseases.

Transmissible spongiform encephalopathies or prion diseases are infectious and fatal neurodegenerative diseases. A mutation and an abnormal structure of the prion protein (PrP^C) are associated with a protease-resistant and infectious form (PrP^Sc), which is considered a causative factor for prion diseases. Neuropathologically, prion diseases are characterized by a long incubation period of many months to several decades, neuronal vacuolation (spongiosis), neuronal cell death, and glial cell activation (microgliosis and astrocytosis) [37]. These changes lead to the release of inflammatory molecules, such as proinflammatory cytokines, reactive oxygen species, proteases, and complement proteins that induce neuronal damage and remove the damaged cells [37].

The aim of the present study was to determine the specific role of Cx43 in prion pathogenesis using cellular and mouse models of prion disease. In this study, we report for the first time that Cx43 expression is increased via the c-Jun N-Terminal Kinase (JNK) signaling pathway in both in vitro and in vivo models of prion disease.

MATERIALS AND METHODS

Animals and scrapie strains

C57BL/6J mice and golden Syrian hamsters (4 to 6 weeks of age) were purchased from Central Lab Animal, Inc. (Seoul, Republic of Korea). The original stocks of the ME7, 22L, 139A, and 263K scrapie strains were kindly provided by Dr. Alan Dickinson of the Agriculture and Food Research Council and Medical Research Council Institute (Neuropathogenesis Unit, Edinburgh, UK). For scrapie infection, the mice were intracerebrally inoculated with 30 μl of 1% w/v brain homogenates of the ME7, 22L, and 139A inocula (for mice) or 50 μl of 1% w/v hamster brain homogenate of the 263K inoculum (for hamsters) in phosphate-buffered saline (PBS, pH 7.4) using a stereotaxic apparatus (Stoelting, Wood Dale, IL, USA). The control mice received 30∼50 μl of 1% w/v normal brain homogenate. The scrapie-infected and uninfected mice were sacrificed at 10–150 days post-inoculation (dpi) or at the terminal stage (160 dpi for ME7, 140 dpi for 22L, and 170 dpi for 139A) when the mice displayed typical clinical signs of the disease. All experiments were performed in accordance with Korean laws and with the approval of the Hallym Medical Center Institutional Animal Care and Use Committee (HMC2011-0-0115-07).

Subcellular fractionation

Mouse brain tissues were lysed in cold hypotonic buffer (10 mM Tris-HCl, pH 7.4; 1 mM DTT; 5 mM MgCl₂; 10 mM KCl; 10 mM NaF; and 1 mM Na₃VO₄) with a protease inhibitor cocktail tablet (Roche, Indianapolis, IN, USA) by passing through a 23-gauge syringe needle 10 times. The lysates were centrifuged at 500 g for 10 min to remove the nuclei and unbroken cells. The post-nuclear supernatants were subsequently centrifuged at 100,000 g for 1 h at 4°C to separate the membrane pellet from the cytosolic fraction. The membrane pellets were washed with ice-cold PBS and resuspended in modified RIPA buffer (50 mM Tris-HCl, pH 7.4; 150 mM NaCl; 2 mM EDTA; 1% Triton X-100; 1% Nonidet P (NP)-40; 0.25% sodium deoxycholate; 1 mM Na₃VO₄; and 10 mM NaF) with a protease inhibitor cocktail tablet by rocking for 1 h at 4°C, followed by centrifugation at 20,000 g for 10 min at 4°C. The supernatant contained the solubilized membrane proteins.

Western blot analysis

The mouse brains and cultured cells were lysed in modified RIPA buffer with 20 mM Tris-HCl (pH 7.5), 150 mM NaCl, 1% Triton, 0.5% sodium deoxycholate, 0.1% sodium dodecyl sulfate (SDS), 1% NP-40, 10 mM NaF, 1 mM Na₃VO₄, 1 mM EDTA, 1 mM EGTA and 0.1 M phenylmethylsulfonyl fluoride (PMSF), as well as a protease inhibitor cocktail. The homogenates were centrifuged at 15,000 g at 4°C for 30 min, the supernatants were collected, and the protein concentrations were determined using a BCA protein assay kit (Pierce, Rockford, IL, USA). Equal protein amounts were separated by 10 or 12% SDS-PAGE and then transferred to PVDF membrane using an electrotransfer system (Bio-Rad, Hercules, CA, USA). To detect the target proteins, mouse monoclonal anti-PrP (3F10, 1:3,000) [38] rabbit polyclonal anti-Cx43 (1:1,000; Cell Signaling Technology, Beverly, MA, USA), mouse monoclonal anti-JNK (1:1,000; Santa Cruz Biotechnology, Santa Cruz, CA, USA), mouse monoclonal anti-phospho-JNK (1:1,000, Santa Cruz Biotechnology), rabbit polyclonal anti-c-Jun (1,000; Santa Cruz Biotechnology), rabbit polyclonal anti-phospho-c-Jun (1:1000; Cell Signaling Technology), and mouse monoclonal anti-β-actin (1:10,000; Sigma-Aldrich, Saint Louis, MO, USA) antibodies were used with the appropriate secondary antibodies conjugated to horseradish peroxidase. To detect PrP^Sc, 50 μg/ml or 20 μg/ml of proteinase K (PK) were treated for 30 min at 37°C in cell lysates or brain homogenates, respectively. The target signals were visualized by digital images captured with an ImageQuanttrademark LAS 4000 imager (GE Healthcare Life Sciences, Piscataway, NJ, USA) using an enhanced chemiluminescence western blot detection system (Amersham Biosciences, Piscataway, NJ, USA).

Maintenance and scrapie infection of cultured cell lines

Mouse hippocampal neuronal cell lines, including ZW13-2 (wild-type PrP) and Zpl2-4 (PrP knockout) cells, were previously established [39]. The cells were cultured in Dulbecco’s modified Eagle’s medium (Hyclone, Logan, UT, USA) with 10% fetal bovine serum (FBS, Hyclone), 100 units/ml penicillin, and 100 μg/ml streptomycin in a 37°C incubator with 5% CO₂. The ZW13-2 cells were persistently infected with the 22L and 139A scrapie strains as previously described [40]. The infected cells were maintained in Opti-MEM (Sigma-Aldrich) with 10% fetal calf serum (Hyclone) and sub-cultured every 3 days at a 1:2 split for the first 10 passages. The infected cells stably produced PrP^Sc for over 50 passages. For immunocytochemistry, dye uptake assay, and scrape-loading dye transfer assay, cells were seeded onto coverslips at 2 × 10⁴ cells per well in 24-well plates and allowed to adhere for 2 days.

Cell blot assay

Stable scrapie infection was confirmed after five passages as previously described [40]. Briefly, cells were grown to confluence on Thermanox plastic cover slips (Nalgene Nunc International, Rochester, NY, USA) and transferred to nitrocellulose membrane. After drying for 1 h at 37°C, the membrane was incubated in lysis buffer (50 mM Tris-HCl, pH 8.0; 150 mM NaCl; 0.5% sodium deoxycholate; 0.5% Triton X-100; 2 mM PMSF) containing PK (5 μg/ml) at 37°C for 10 min. The membrane was placed into 3 M guanidinium thiocyanate (Sigma-Aldrich), 10 mM Tris·HCl (pH 8.0) for 10 min followed by immunostaining with anti-PrP antibody 3F10(1:3,000).

Semi-quantitative RT-PCR

Total RNA was extracted from the brain samples using TRI reagent (Sigma-Aldrich) according to the manufacturer’s protocols. cDNA was synthesized using the Moloney murine leukemia virus reverse transcriptase (Promega, Madison, WI, USA). The primer sequences were as follows: connexin43 (127 bp), sense, 5′-CTGAGTGCGGTCTACACCTG-3′, anti-sense, 5′-GAGCGAGAGACACCAAGGAC-3′; β-actin (196 bp), sense, 5′-TGTGATGGACTCCGGTGACGG-3′, antisense, 5′-ACAGCTTCTCTTTGATGTCACGC-3′. The PCR products were separated by electrophoresis on a 1% agarose gel and visualized under UV light.

Immunohistochemistry

Immunohistochemical procedures were performed as previously described [41]. The sections were blocked with 10% normal donkey serum in Tris-buffered saline (50 mM Tris-HCl and 150 mM NaCl, pH 7.6) and then incubated overnight at 4°C with rabbit polyclonal anti-Cx43 antibody (1:100). After washing in PBS, the sections were first incubated with biotinylated donkey anti-rabbit IgG antibody (1:500; Vector Laboratories, Burlingame, CA, USA) for 1 h and then with avidin-biotin peroxidase complex (ABC Kit, Vector Laboratories). The sections were mounted in Permount (Thermo Fisher Scientific, Inc., Waltham, MA, USA). The control sections were stained without primaryantibody.

Immunocytochemistry

The cells were fixed with 4% paraformaldehyde in PBS for 15 min at room temperature and then treated with 0.1% Triton X-100 in PBS. After a brief wash with PBS, the cells were incubated with blocking buffer (5% normal goat serum and 0.1% Tween 20 in PBS) for 1 h, followed by incubation with rabbit polyclonal anti-Cx43 antibody (1:200, Cell Signaling Technology) and mouse monoclonal anti-PrP (3F10, 1:200) [38] overnight at 4°C. The cells were subsequently incubated with either Alexa Fluor 568 goat anti-rabbit IgG or Alexa Fluor 488 goat anti-mouse IgG antibodies (Invitrogen, Carlsbad, CA, USA) for 1 h. Control reactions omitting the primary antibodies resulted in no labeling with the secondary antibodies (data not shown). After rinsing with PBS, the cells were then mounted in 4,6-diamidino-2-phenylindole (DAPI)-containing Vectashield Mounting Medium (Vector Laboratories) to label nuclei and visualized using a confocal laser scanning microscope (LSM 700; Carl Zeiss, Oberkochen, Germany).

Dye uptake assay

To evaluate the functional Cx43 hemichannel, an uptake assay using the hemichannel-permeable reporter dye ethidium bromide (EtBr) was performed as previously described [42]. The cells were incubated with 5 μM EtBr in PBS in the presence or absence of a connexin hemichannel blocker LaCl₃ (500 μM) for 5 min at 37°C. For JNK inhibition, the cells were pretreated with the JNK inhibitor SP600125 (50 μM) for 6 h and then incubated with 5 μM EtBr for 5 min at 37°C. The cells were then washed with Hank’s balanced salt solution (HBSS), fixed with 4% paraformaldehyde in PBS for 15 min at room temperature and washed with HBSS. Dye uptake was examined with a confocal laser scanning microscope.

Scrape-loading dye transfer assay

To determine the functional Cx43 hemichannels, an uptake assay using the gap junction permeable reporter dye Lucifer yellow (LY) (Sigma-Aldrich) was performed as previously described [43]. The cells were incubated with 0.01% LY in the presence or absence of the Cx channel blocker lanthanum chloride (LaCl₃, 500 μM) for 5 min at 37°C. For JNK inhibition, the cells were pretreated with a JNK inhibitor SP600125 (50 μM) for 6 h and then incubated with 0.01% LY in PBS for 5 min at 37°C. The cells were washed with HBSS and fixed with 100% MeOH for 15 min at –20°C. The cells were observed under a confocal laser scanning microscope. The distances of LY diffusion after scrape loading were measured in at least twenty random areas from each sample, and the fluorescence intensity was assessed using Image J software and compared between the control and infected cells with or without each treatment.

Brilliant blue G (BBG) treatment

Uninfected and 22L scrapie-infected cells (1 × 10⁶ cells/100-mm dish) were treated or not treated with BBG (0.6–60 μM) for 3 days and then lysed. The expression levels of PrP^Sc, JNK, phospho-JNK, and Cx43 were determined by western blotting as previously described.

Statistical analysis

The data are expressed as the mean±SEM. Significant differences between the experimental groups were evaluated using one-way analysis of variance (ANOVA). Statistical significance was defined as ^*p < 0.05, ^**p < 0.01, and ^***p < 0.001.

RESULTS

Upregulation of Cx43 expression in scrapie-infected mouse brains

We first investigated the expression levels of the Cx43 protein in the brain tissues of control mice and mice infected with one of three scrapie strains (ME7, 22L, or 139A). These strains possess distinct incubation periods and neuropathological features [44]. In the whole brain and dissected brain tissues, including the cerebral cortex, hippocampus, striatum, cerebellum, and brain stem, we demonstrated that Cx43 protein Fig. 1A,B) and mRNA (Fig. 1C) were highly expressed in animals with scrapie infection compared with the controls. These results suggest that both the mRNA and protein levels of Cx43 are upregulated after scrapie infection, and the upregulated protein levels of Cx43 in most brain regions are a result of increased gene expression in scrapie-infected mice.

To further determine whether the increased Cx43 protein expression is correlated with disease progression, we performed western blotting using whole brains obtained at 50, 100, and 150 dpi for the ME7 strain (for mice) and at 10, 30, 60, and 90 dpi for the 263K strain (for hamsters) (Fig. 1D). No difference between the control and scrapie-infected brains was identified at 50 dpi in ME7 mice or in 263K hamsters at 30 dpi. However, Cx43 protein expression tended to increase relative to the stage of scrapie development and was significantly increased at 90 dpi (for hamsters) and 150 dpi (for mice). These time points were correlated with marked accumulations of PrP^Sc, indicating that Cx43 protein increased at the end stage of scrapie infection.

To confirm the increase in Cx43 after scrapie infection, we subsequently performed immunohistochemical staining of Cx43 in various brain regions of the control and scrapie-infected mice. Cx43 was prominent in most brain regions, including the cerebral cortex, hippocampus, striatum, cerebellum, and brain stem, at the end stage of scrapie infection compared with levels in the controls (Fig. 2). A marked increase in Cx43 immunoreactivity was seen in the hippocampus and the Purkinje layer of the cerebellum in the scrapie-infected mice. In addition, Cx43, which showed a diffuse staining pattern, localized to regions of cell-cell contact or blood vessels (arrows in Fig. 2). Overall, these findings suggest that scrapie infection increases Cx43 expression in the brain.

Increased Cx43 in the membrane fraction from the brains of scrapie-infected mice

Because Cx43 is an integral plasma membrane protein, we subsequently examined whether the increased Cx43 is related to membrane localization using cytosolic and membrane fractions prepared from control brains and from ME7, 22L, and 139A scrapie-infected brains as described in the Materials and Methods. Calnexin and enolase were used as markers of the membrane and cytosolic fractions, respectively. As expected, the Cx43 protein was preferentially detected in the membrane fraction of all scrapie-infected brains compared with levels in the controls (Fig. 3), which is consistent with the immunoblot analysis data (Fig. 1). These data suggest that increases in membrane-associated Cx43 protein result from scrapie-induced pathological conditions.

Increased Cx43 expression in scrapie-infected hippocampal cell lines

Gap junctions are complexes of intercellular channels between adjacent cells [1]. We subsequently examined whether a scrapie infection-induced increase in Cx43 expression is associated with gap junction plaque formation in hippocampal neuronal cell lines, as previously established [39]. Although Cx43 is a primary gap junction protein in astrocytes, we demonstrated that the ZW13-2 and Zpl2-4 hippocampal neuronal cell lines endogenously express Cx43. Thus, these cell lines were used as an in vitro model of prion replication after infection with either of two mouse-derived scrapie strains (22L or 139A). As shown in Fig. 4, Cx43 (red) was detected in both ZW13-2 and Zpl2-4 cells, and intense expression of Cx43 was mainly localized within plaques at the cell-cell contact areas. In the presence of PrP (ZW13-2), Cx43 staining consisted of sparse but large puncta distinctly separated from the nucleus (blue). Interestingly, Cx43 was clearly detected with more aggregates between neighboring scrapie-infected ZW13-2 cells but not between Zpl2-4 cells or uninfected cells. Uptake of infection was confirmed by the PK-resistant PrP^Sc levels in cultures after multiple passages (Supplementary Figure 1). These results demonstrated that pathogenic PrP or scrapie infection induce the upregulation of Cx43 expression and the formation of gap junction-like plaques in neuronal cells.

Association of JNK activation and Cx43 upregulation in scrapie-infected cells and mice

JNK is a stress-activated kinase reported to be important in intracellular signaling pathways that regulate Cx43 expression [45 –47]. To determine whether JNK signaling is involved in the induction of Cx43, we examined the activation of JNK and its downstream molecule c-Jun in scrapie-infected neuronal cells and mice. Scrapie infection was confirmed by the detection of PK-resistant PrP^Sc (Fig. 5A, second panels). Interestingly, Cx43 was upregulated and associated with JNK activation, which was demonstrated by increased phospho-JNK (P-JNK) in scrapie-infected neuronal cells and in infected brains (Fig. 5A). The levels of P-c-Jun were also significantly increased in association with JNK activation; however, the total levels of JNK and c-Jun were not altered. To further elucidate the functional roles of JNK in the induction of Cx43 by scrapie infection, control and infected cell lines were treated with various concentrations of anATP-competitive inhibitor of JNK, SP600125 (0–100 μM), for 6 h. As shown in Fig. 5B and C, increased Cx43 and P-JNK/P-c-Jun by scrapie infection were effectively downregulated by SP600125 treatment in a dose- and time-dependent manner. These findings suggest that JNK-c-Jun signaling is essential for the Cx43 overexpression induced by scrapie infection.

Increased dye uptake by scrapie-induced Cx43 expression

The uptake of the fluorescent dye EtBr has been used as an indicator of hemichannel opening [42, 48]. To determine whether Cx43 upregulation by scrapie infection affects the opening of Cx43 hemichannels, we performed EtBr uptake assays in scrapie-infected and un-infected ZW13-2 neuronal cells. As shown in Fig. 6 enhanced EtBr uptake was observed in both the 22L and 139A scrapie-infected cells compared with the control cells. The scrapie infection-enhanced EtBr uptake was inhibited in the presence of LaCl₃ (500 μM; a known connexin hemichannel blocker) and the JNK inhibitor SP600125, indicating that scrapie infection-mediated upregulation of Cx43 controls the functional state of hemichannels.

The scrape-loading dye transfer technique is used to determine the intrinsic gap junction intercellular communication (GJIC) [43, 49]. Next, scrapie-infected and un-infected ZW13-2 neuronal cells were subjected to a scrape-loading dye transfer assay in the absence or presence of LaCl₃ and a JNK inhibitor as described in the Materials and Methods. As shown in Fig. 7A (top panels), gap junction-mediated intercellular transfer of LY was increased in the 22L and 139A scrapie-infected cells compared with the control cells. This increase was abolished by either hemichannel inhibition (LaCl₃) or JNK inhibition (SP600125) (Fig. 7A, middle and bottom panels, respectively). Hemichannel inhibition almost blocked GJIC in either control or infected cells (Fig. 7A middle panels), whereas JNK inhibition maintained basal levels of GJIC in control and infected cells (Fig. 7A ottom panels) without a significant difference between control and infected cells. The distances of LY diffusion were measured and compared with those in the controls and infected cells with or without treatments (Fig. 7B). These results indicate that the upregulation of Cx43 expression by scrapie infection represents the efficient communication capacity of Cx43\enlargethispage 4pt hemichannels and GJIC and likely occurs via JNK activation.

Suppression of Cx43 expression with decreased JNK activity by BBG-mediated blockade of prion conversion

To further investigate whether Cx43 induction is regulated by PrP^Sc accumulation, we conducted a blocking experiment of PrP^Sc accumulation using BBG, which has anti-prion activity through the inhibition of PrP^Sc accumulation [50]. Un-infected and 22L scrapie-infected ZW13-2 neuronal cells were incubated with various concentrations of BBG (0–60 μM) for 3 days, and the expression levels of Cx43, PrP^Sc, and p-JNK were subsequently measured. As shown in Fig. 8 BBG treatment efficiently inhibited PrP^Sc accumulation in a dose-dependent manner. In correlation with the decrease in PrP^Sc, the expression levels of Cx43 and p-JNK were gradually reduced following BBG treatment of 22L scrapie-infected cells. In contrast, no changes were observed in the uninfected cells treated with BBG. These results suggest that PrP^Sc accumulationmay be responsible for JNK signaling-mediated Cx43 upregulation.

DISCUSSION

In the present study, we demonstrated that the upregulation of Cx43 expression, which controls functional properties of hemichannels, is mediated by JNK activation and correlated with PrP^Sc accumulation in both in vivo and in vitro systems of prion diseases. These findings represent the first demonstration of the link between the Cx43 protein and prion pathogenesis.

Cx43 hemichannels contribute to cell death and are responsible for tissue damage [11 , 51]. Several signaling molecules are linked to brain inflammation and unwarranted cell death. It is conceivable that the excessive diffusion of neurotransmitters or ions, such as glutamate, ATP, and Ca^2 +, from damaged cells to adjacent normal cells via Cx43-promoted gap junctions may accelerate this pathophysiological process [8 –10]. Astrocytes and microglia release glutamate via gap junctions [8 , 52–54], which then induces neuronal cell death [54, 55].

Under various pathological conditions, astrocytes become reactive, triggering a long-lasting process that comprises complex phenotypic changes [56]. Astrocyte activation, in which there is both hypertrophy and proliferation, involves changes in the cellular phenotype and in the expression of transporters, receptors and ion channels, which can be deleterious for neurons and thus lead to neuronal cell death [57]. These changes can explain how connexins can undergo both up- and downregulation depending on the type of brain pathology, nature of the injury, time scale and distance from the lesioned area [58, 59].

Continuous astrocyte-neuron interactions are required to maintain Cx30 expression and increased Cx43 levels in astrocytes [60]. The expression level of Cx43 and its hemichannel activity were significantly increased in scrapie-infected brains and in cultured hippocampal cells, even though the neuronal loss associated with prion diseases reduces the level of Cx43 in astrocytes associated with the lost neurons. In our study, it is difficult to identify which cell type(s) is mainly stained for Cx43. However, it is possible that astroglial Cx43 upregulation may be caused by scrapie infection-mediated pathological changes since reactive astrocytes are one of the major pathological changes associated with prion diseases. Using previously established hippocampal neuronal cell lines [39], increased Cx43 was observed following inoculation with two independent scrapie strains. Although Cx43 is primarily expressed in astrocytes [4 , 61] and astrocyte gap junctions coupled with CNS cells [59], these neuronal cell lines are a useful in vitro system to study the functional role of Cx43 in prion diseases because they express endogenous Cx43. In addition, our data suggest that scrapie infection-enhanced Cx43 proteins may consistently exacerbate the pathogenesis of prion diseases despite the limited involvement of functional Cx43 expression in the glial cells from this study.

According to previous findings, the increased production of reactive oxygen species and pro-inflammatory cytokines, including IL-1α, IL-1β, and TNF-α, in the brains of scrapie-infected mice can lead astrocytes to become reactive [62]. These astrocytes can be well-coupled via gap junctions, leading to increases in hemichannel activity because of the increased Cx43 mRNA and protein levels in scrapie-infected brains. Consistent with this phenomenon, Cx43 was associated with gap junctional plaques via distribution to neighboring cells after scrapie infection (Fig. 4). Although increases in Cx43 and gap junctional communication and their effects on neuronal death and inflammatory responses have been linked to chronic and progressive neurodegenerative diseases such as stroke, Alzheimer’s disease, Parkinson’s disease, and Huntington’s disease [57], it remains a matter of debate whether Cx43 is neuroprotective ordeleterious.

Accumulating evidence has indicated that connexins act as phosphoproteins via a shift in their electrophoretic mobility or direct incorporation of ³²P [63]. The JNK signaling cascade is an important intracellular signaling pathway that regulates Cx43 [47]. Our data demonstrated that upregulated Cx43 was a result of JNK activation and was blocked by SP600125, a potent inhibitor of JNK. In this study, the assays used to measure Cx43 predominately identified a single band, and we were unable to distinguish the phosphorylation status of Cx43. Because Cx43 can be phosphorylated by various kinases, the activation of a specific kinase may not correlate with Cx43 phosphorylation in our models. A recent study has suggested that astroglial Cx43 plays a protective role in oxidative stress-induced cell death, which depends on the phosphorylation state of Cx43 [17]. In addition, the opening of hemichannels formed by pannexin 1, the mammalian ortholog of the invertebrate gap junction protein innexin, has also been implicated in neuronal death after ischemia [64]. Moreover, during the astrogliosis response observed 24 h after reperfusion, de novo synthesis of pannexin 2 occurs in hippocampal rat astrocytes [65]. Thus, the functional role of Cx43 and pannexin in the pathogenesis of prion diseases remainsunresolved.

In addition, previous reports have demonstrated that JNK signaling activation is involved in the pathogenesis of scrapie-infected hamster brains [66] and JNK signaling cascades are necessary for Cx43 expression [45 –47]. JNK regulates downstream gene expression through the activation or inactivation of various transcriptional factors, such as c-Jun, c-fos, and SP1 [67]. We also demonstrated that the activated JNK/c-Jun cascade is responsible for Cx43 expression in both in vitro and in vivo models of prion disease. Surprisingly, the accumulation of pathogenic PrP^Sc appears to occur upstream of JNK activation, which is then followed by increased Cx43 expression. Although PrP did not co-localize in Cx43 plaque formation, scrapie infection increased Cx43 aggregates at the cell-cell contact areas. Therefore, PrP^Sc accumulation may be responsible for the upregulation of Cx43 expression and gap junction formation.

Overall, for the first time, these results demonstrated that scrapie infection activates JNK signaling cascades to enhance Cx43 and gap junctions in neuronal cells and in the brain. Pathogenic PrP^Sc (or scrapie)-mediated upregulation of Cx43 through JNK activation may exacerbate prion pathology, including neuronal death. Although blocking gap junctions, which inhibit the diffusion of neurotoxic molecules, has been proposed as a candidate therapy for various neurodegenerative diseases, it can also limit essential physiological signals. Thus, this strategy has both neurotoxic and neuroprotective effects on disease progression. A previous report demonstrated that a JNK inhibitor reduced PrP106-126 peptide-induced neuronal apoptosis [68]. Therefore, we suggest that blocking the gap junctions (functional Cx hemichannels) expressed by neurons, astrocytes, and microglia in the region of prion propagation might represent a novel strategy to reduce disease phenotypes in prion diseases.

Footnotes

ACKNOWLEDGMENTS

We thank Dr. Joy J. Goto (California State University, Fresno, USA) for critical reading of this paper and helpful discussions. This work was supported by the National Research Foundation of Korea (NRF) grant funded by the Korean Government (NRF-2013R1A1A2007071) and by the Hallym University Specialization Fund (HRF-S-41).

Authors’ disclosures available online ().

Appendix

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