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 의학박사 학위논문

Bank Vole 프리온 단백 발현 형질 전환 마우스의

신경세포에서 다양한 프리온 감염 세포주 확립

Establishment of advanced in vitro model from Bank Vole

transgenic mice for susceptibility to various prion strains

Choi, Hong Seok (최 홍 석)

의학과 (Department of Medical Science)

미생물학 전공 (Major in microbiology)

한림대학교 대학원
Graduate School, Hallym University

2016년도
 김용선 교수지도

의학 박사 학위논문

최홍석의 박사 학위논문을 합격으로 판정함.

2016 년 12 월

심사위원장 권 형 주 ________________

심사위원 최 경 찬 ________________

심사위원 박 재 봉 ________________

심사위원 김 재 일 ________________

심사위원 김 용 선 ________________
 CONTENTS

I . Chapter 1

Dysfunction of Mitochondrial Dynamics in the Brains of Scrapie-Infected Mice.....1

1.1 Intoduction..................................................................................................... 3

1.2 Materials and methods..........................................................................................5

1.3 Result....................................................................................................................8

1.4 Discussion...........................................................................................................11

1.5 References..........................................................................................................19

II . Chapter 2

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

Prion Disease..............................................................................................................23

2.1 Intoduction..................................................................................................... 25

2.2 Materials and methods........................................................................................27

2.3 Result..................................................................................................................34

2.4 Discussion...........................................................................................................39

2.5 References..........................................................................................................53
III . Chapter 3

Establishment of Advanced In Vitro Model from Bank Vole Transgenic Mice for

Susceptibility to Various Prion Strains..................................................................62

3.1 Intoduction.....................................................................................................63

3.2 Materials and methods........................................................................................66

3.3 Result..................................................................................................................70

3.4 Discussion...........................................................................................................73

3.5 References..........................................................................................................81

3.6 Abstract...............................................................................................................85

3.7 Abstract in korea.................................................................................................86

IV. Appendix...................................................................................................................88
 LIST OF FIGURES

Figure 1.1. Deposition of PK-resistant prion isoforms in ME7-infected mice.................15

Figure 1.2. Differential expression of mitochondrial fusion and fission proteins in

hippocampi of ME7-infected mice...................................................................................16

Figure 1.3. Subcellular localization of mitochondrial fusion and fission proteins in

hippocampi of ME7-infected mice...................................................................................17

Figure 1.4. Electron microscopic analysis of the mitochondria in hippocampal neurons

of ME7-infected mice……………………….................….............................................18

Figure 2.1. Increased expression of Cx43 protein and mRNA in the brains of scrapie

infected mice....................................................................................................................43

Figure 2.2. Immunohistochemical staining of Cx43 in brain sections from control and

scrapie-infected mice.......................................................................................................45

Figure 2.3. Increased Cx43 protein in membrane fractions of scrapie infected

brains………………………………………………………………………………….46

Figure 2.4. Subcellular distribution of Cx43 and PrP in hippocampal cell lines with or

without scrapie infection..................................................................................................47

Figure 2.5. JNK activation-mediated Cx43 upregulation in scrapie-infected cells and

mic………………………………………………………………………………….......48

Figure 2.6. Increased EtBr uptake in scrapie-infected cells.............................................49

Figure 2.7. Increased scrape-loading dye transfer in scrapie-infected cells.....................50

Figure 2.8. Attenuation of JNK signaling-mediated Cx43 upregulation by inhibiting the

accumulation of PrPSc......................................................................................................52

Figure 3.1. Cell-type characterization and expressions of the PrP in the BV neuronal cell
lines………………………………………......................................................................76

Figure 3.2. Analysis of PK and TL-resistant PrPSc in the BV neuronal cell lines infected

with different prion strains.............................................................................................78

Figure 3.3. Accumulation of PK-resistant prion isoforms in ZW-22L and BV-22L

scrapie-infected ICR mice………………………………………...............................79

LIST OF TABLES

Table 1. Accumulation of PK-resistant prion isoforms in ZW-22L and BV-22L scrapie-

infected ICR mice………………………………………………………………………79

 I. Chapter 1

Dysfunction of mitochondrial dynamics in the brains of scrapie-

infected mice

Abstract

Mitochondrial dysfunction is a common and prominent feature of many

neurodegenerative diseases, including prion diseases; it is induced by oxidative stress in

scrapie-infected animal models. In previous studies, we found swelling and dysfunction

of mitochondria in the brains of scrapie-infected mice compared to brains of controls, but

the mechanisms underlying mitochondrial dysfunction remain unclear. To examine

whether the dysregulation of mitochondrial proteins is related to the mitochondrial

dysfunction associated with prion disease, we investigated the expression patterns of

mitochondrial fusion and fission proteins in the brains of ME7 prion-infected mice.

Immunoblot analysis revealed that Mfn1 was up-regulated in both whole brain and

specific brain regions, including the cerebral cortex and hippocampus, of ME7-infected

mice compared to controls. Additionally, expression levels of Fis1 and Mfn2 were

elevated in the hippocampus and the striatum, respectively, of the ME7-infected brain. In

contrast, Dlp1 expression was significantly reduced in the hippocampus in the ME7-

infected brain, particularly in the cytosolic fraction. Finally, we observed abnormal

mitochondrial enlargement and histopathological change in the hippocampus of the ME7-

infected brain. These observations suggest that the mitochondrial dysfunction, which is

1
presumably caused by the dysregulation of mitochondrial fusion and fission proteins, may

contribute to the neuropathological changes associated with prion disease.

Key words : Prion disease, Mitochondrial fusion, Fission, Scrapie, Mitochondrial

dysfunction

2
1.1 Introduction

Prion diseases, also known as transmissible spongiform encephalopathies (TSEs), are

fatal neurodegenerative disorders that affect both humans and animals [1, 2]. Scrapie is a

prototypical prion disease that affects sheep and goats. Clinically, scrapie is characterized

by a long latent period, progressive ataxia, tremor, wasting and ultimately death [3]. Many

scrapie strains have been isolated from sheep and goats and used to examine not only the

range of various pathogenesis pathways but also the neuropathological mechanisms

induced by prion disease [4]. Typical features of the disease include the formation of

spongiform vacuoles and astrocytosis, the formation of amyloid plaques in some cases

and neuronal loss in the brain [5, 6]. A key event in prion disease is the conformational

misfolding of the endogenously expressed cellular prion protein (PrPC) into the scrapie

form of the pathogenic prion protein (PrPSc) [5].

A number of recent studies have demonstrated that mitochondria are dynamic organelles

that continually undergo fission and fusion with one another [7–9]. Mitochondria can

change in number and morphology within a cell during development, throughout the cell

cycle and when challenged with various cytotoxic stimuli [9]. In mammals, the key

molecules involved in mitochondrial fission are dynamin-like protein 1 (Dlp1, also

referred to as Drp1) and fission 1 (Fis1). The process opposing fission, i.e., mitochondrial

fusion, is controlled in mammalian cells by mitofusin 1 (Mfn1), mitofusin 2 (Mfn2) and

optic atrophy 1 (Opa1) [7]. The sizes, shapes and interconnectivities of mitochondria are

determined by their fusion and fission [9]. It has been suggested that the mitochondrial

defects associated with Alzheimer’s disease (AD), Parkinson’s disease (PD) and

Huntington’s disease (HD) may result, at least in part, from a disruption of the fusion and

3
fission mechanisms of mitochondria [8, 9]. A recent study reported that the expression of

Dlp1 is decreased and that mitochondria are abnormally elongated in the fibroblasts of

AD patients and in neuronal cell lines overexpressing amyloid precursor protein (APP)

[10]. The study suggests that APP causes an imbalance between mitochondrial fusion and

fission that results in an abnormal distribution of mitochondria, which in turn contributes

to mitochondrial and neuronal dysfunction. Several observations suggest a link between

mitochondrial dysfunction and PD. Pink1 and parkin, which are PD-related genes,

promote mitochondrial fission and inhibit mitochondrial fusion in Drosophila [11].

Additionally, HD research has focused on mitochondrial dysfunction. A mouse model of

HD exhibits early defects in respiration and ATP production [12]. Moreover, mutant

huntingtin seems to disrupt mitochondrial Ca2+ buffering [13] and to cause mitochondrial

ultrastructural changes in the lymphoblasts of HD patients [14]. Previous studies reported

that dysfunction and enlargement of the mitochondria occur by oxidative stress in animal

models of prion disease [15, 16]. However, the underlying mechanism responsible for

this mitochondrial dysfunction remains unclear.

In the present study, we investigated the mitochondrial fusion and fission proteins that

may be involved in the mitochondrial dysfunction observed in prion disease. We show

that mitochondrial fusion and fission proteins are differentially modulated in the terminal

stage of an experimental mouse model of prion disease and that this modulation may

contribute to the morphological damage and reduction in number of mitochondria in

infected neuronal cells.

4
1.2 Materials and methods

1) Antibodies

The following monoclonal and polyclonal antibodies were used mouse monoclonal

anti-PrP (3F10) [17], mouse monoclonal anti-COX IV (Abcam), goat polyclonal anti-

enolase (Santa Cruz), mouse monoclonal to β-actin (Sigma–Aldrich), mouse

monoclonal anti-Opa1 and mouse monoclonal anti-Dlp1 (BD Transduction

Laboratories), mouse monoclonal anti-Mfn2, chicken polyclonal anti-Mfn1 (Novus

Biologicals) and rabbit polyclonal anti-Fis1 (Santa Cruz).

2) Animals and scrapie strains

Six-week-old C57BL/6 mice were obtained from the Central Laboratory Animal

(Republic of Korea) and divided into two groups: one group was infected with the ME7

scrapie strain, and the other included age-matched controls. The ME7 scrapie strain was

kindly provided by Dr. Alan Dickinson (Neuropathogenesis Unit, Edinburgh, UK). This

scrapie strain was maintained by serial intracerebral passages of brain homogenate from

a terminally affected mouse. The mice were intracerebrally inoculated with 30 μl of 1%

(w/v) brain homogenate in 0.01 M phosphate-buffered saline (PBS, pH 7.4) from either

a normal brain or an ME7-infected C57BL/6 mouse brain at the terminal stage of the

disease. When the clinical signs of prion disease were evident in the terminal stage (160

days post inoculation, dpi), the mice were sacrificed.

3) Western blot analysis

Whole brains or hippocampal regions were homogenized gently in 10-fold greater

5
volumes (w/v) of 50 mM Tris–HCl (pH 7.4) containing 150 mM NaCl, 1 mM EDTA,

0.25% Na-deoxycholate, 1% NP-40 and protease inhibitor cocktails (Roche). The

protein concentration was determined using the BCA assay (Thermo Scientific). The

homogenates were treated with Proteinase K (PK) at a concentration of 50 μg/ml. Equal

amounts of protein (10–50 lg in all assays) were separated by SDS–PAGE using 10%,

12% or 15% acrylamide gels and then transferred to nitrocellulose membranes (Thermo

Scientific). After blocking with 5% skim milk for 1 h, the membranes were incubated

with the individual antibodies overnight at 4 °C and then incubated with horseradish

peroxidase-conjugated secondary antibody in Tris-buffered saline with 0.05% Tween-20

(TBST) containing 5% skim milk for 1 h at room temperature. The blots were visualized

with the SuperSignal West Pico Chemiluminescent Substrate (Thermo Scientific). The

expression levels of each protein were quantified using ImageJ software (NIH).

4) Transmission electron microscopy (TEM)

The animals were perfused with 0.1 M PBS (pH 7.4) containing 4% paraformaldehyde

and 2.5% glutaraldehyde under deep anesthesia with 16.5% urethane. The brains were

removed and fixed in the 0.1 M PBS fixative that was used for perfusion. The bilateral

hippocampal regions were trimmed into small pieces immediately after their surgical

removal and kept in the fixative for 2 h at 4 °C. Post-fixation was performed in 0.1 M

PBS with 1% osmium tetroxide followed by dehydration through a graded ethanol series

and embedding in Epon 812. Ultra-thin sections (75 nm) prepared by using an

ultramicrotome (RMC MTXL) were stained with uranyl acetate and lead citrate and were

subsequently observed with a transmission electron microscope (JEM-1011, JEOL). The

numbers of total and damaged mitochondria were counted in the hippocampal neurons

6
in the control and ME7-infected brains and then calculated based on fifteen arbitrarily

selected hippocampal neurons. The sizes of the mitochondria were measured under high

magnification (x50,000) using TEM (iTEM, Olympus Soft Imaging Solutions, GmbH),

and the values were calculated as the means ± SDs of the lengths and widths of thirty

arbitrarily selected mitochondria from each group. Statistical analyses of the numbers

and sizes of the mitochondria were performed using Jandel SigmaStat software (V 3.5).

5) Statistical analyses

The compared values were calculated as the mean ± SD of three brains from each group,

and statistical significance was determined using Student’s t-tests.

7
1.3 Result

1) Imbalanced expression of mitochondrial fusion and fission proteins in infected brains

First, we identified deposits of PK-resistant PrPSc in ME7-infected brains in the end

stage; the normal PrPC proteins were completely degraded by the PK treatment, as shown

in Fig. 1. To determine whether the mitochondrial fusion and fission proteins are affected

by prion infection, their expression patterns were investigated in whole brains of the

control and ME7-infected mice at the end stage of disease (160 dpi) (Fig. 2). Of the

mitochondrial fusion and fission proteins (fusion proteins: Opa1, Mfn1 and Mfn2; fission

proteins: Dlp1 and Fis1), Western blot analysis revealed that only Mfn1 was differentially

expressed in the infected whole brains compared to the age-matched control brains (Fig.

2A and B). It has previously been reported that the hippocampal regions are more severely

damaged than any other regions in brains infected with the ME7 scrapie strain [18]. Thus,

to compare the expression levels of the mitochondrial fusion and fission proteins in

various brain regions, we dissected both the control and ME7-infected brains into the

following regions: cerebral cortex, hippocampus, cerebellum, striatum and brainstem.

We found that the levels of Mfn1 and Fis1 were significantly increased in the

hippocampi of the infected brains, whereas the Dlp1 levels were significantly decreased

at the end stage (Fig. 2C and D). Additionally, the levels of Mfn1 and Mfn2 were

increased in the cerebral cortex and striatum, respectively, of infected brains (data not

shown). In the cerebellum and brain stem, none of the five mitochondrial fusion and

fission proteins was significantly differentially expressed between the control and

infected mice (data not shown). These results indicate that the differential regulation of

mitochondrial fusion and fission proteins may be involved in the mitochondrial

dysfunction of the ME7-infected brains, particularly in the hippocampus [16].

8
2) Analysis of fission proteins in the hippocampal cytosol of infected brain

To examine the localization of the mitochondrial fusion and fission proteins in the

organelles and the expression levels of these proteins in the cytosol and mitochondria,

mitochondrial and cytosolic fractions were isolated from the hippocampi of control and

ME7-infected mice; again the hippocampus was chosen because this region is known to

be severely damaged in ME7-infected brains [18]. Immunoblot analyses revealed that

Dlp1 levels in the mitochondrial fractions from the hippocampi were not altered by ME7

infection (Fig. 3A and B) but were significantly decreased in the cytosolic fractions of the

infected hippocampi; this finding is consistent with the decrease in Dlp1 levels in the

affected hippocampi that is illustrated in Fig. 2C and D. The concentration of Opa1, which

is primarily localized to the inner membrane of the mitochondria, was not altered in the

mitochondria or cytosol isolated from the hippocampi of the infected brains (Fig. 3C and

D).

3) Morphological changes in the mitochondria in the hippocampal region of infected brain

The modulation of mitochondrial dynamics due to fusion and fission protein

concentration probably contributed to alterations in mitochondrial shape [9]. Electron

microscopic analyses were performed to assess the relationship between mitochondrial

fusion and fission proteins and mitochondrial shapes in the hippocampal region. As

shown in Fig. 4, the mitochondria in the normal hippocampal neurons were present in a

variety of shapes, including rod, round and elliptical shapes. The cristae were normal in

appearance. In contrast, enlarged mitochondria with damaged cristae were clearly

observed in many of the hippocampal neurons in infected brains (Fig. 4A). The numbers

9
of total neuronal mitochondria were significantly decreased in the hippocampi of the

infected mice compared to those of controls (Fig. 4B). Additionally, the lengths and

widths of the mitochondria in the hippocampal neurons of infected brains were

significantly increased compared to those of the controls, which is consistent with the

observation of enlarged hippocampal mitochondria visualized in Fig. 4B. The

morphological changes seen in mitochondria may be involved in the mitochondrial

dysfunction in the hippocampi of ME7-infected mice.

10
1.4 Discussion

In this study, we demonstrated for the first time that the expression patterns of

mitochondrial fusion and fission proteins were altered in an experimental mouse model

of prion disease; i.e., ME7 scrapie-infected mice. Of the mitochondrial fusion and fission

proteins examined, the levels of Mfn1 were found to be significantly increased in whole

brains of the ME7-infected mice. Within the different dissected regions of the ME7-

infected brains, we found differences in the expression levels of various mitochondrial

fusion and fission proteins compared to controls. Particularly notable were the alterations

in the expression of Mfn1 in the cerebral cortex; Mfn1, Dlp1 and Fis1 in the hippocampus;

and Mfn2 in the striatum.

A recent study suggested that most neurodegenerative diseases may be associated with

the dysregulation of mitochondrial fusion and fission proteins [19]. Mitochondrial fusion

and fission are regulated by large dynamin-related GTPases. Mitochondrial fusion, which

is regulated by three large GTPases (Mfn1, Mfn2 and Opa1), involves the coordinated

fusion of both the outer and inner mitochondrial membranes [20]. The overexpression of

mitofusins causes the normally punctate mitochondria to become elongated, whereas the

repression of mitofusins causes the fragmentation of the mitochondrial network in tissue

culture cells [21]. Interestingly, we found increased level of Mfn1, which is a

transmembrane protein that localizes to the outer membranes of mitochondria, in the

homogenates of whole brain, hippocampus and cerebral cortex of the infected brain. In

addition the expression level of Mfn2 was increased in the striatum of the infected brain

(Data not shown). Although electron microscopy analyses did not reveal the detailed

membrane structures of the mitochondria in this study, initial outer membrane fusion may

11
have occurred in the brains of the infected mice due to increases in the Mfn1 and Mfn2

proteins. Moreover, although there may be distinct pathways or mechanisms for Mfn1

and Mfn2, a functional interplay between the two proteins may be involved in the control

of mitochondrial fusion [22].

Different strains of mouse-adapted scrapie have been reported to preferentially target

different specific brain regions; as noted previously, the ME7 strain is known to be

particularly associated with hippocampal damage [23]; therefore, we focused on the

hippocampal region of the ME7-infected brains. Our study demonstrated that ME7

infection led to significant increases in the levels of Mfn1 and Fis1 in the hippocampi of

the ME7-infected brains, whereas Dlp1 was significantly decreased. Dlp1 localizes in

several organelles, including the endoplasmic reticulum, microtubules and peroxisomes,

but primarily resides in the mitochondria and cytosol [24]. We isolated the cytosolic

fraction of the hippocampal region and investigated the changes in the expression of

mitochondrial fusion and fission proteins in both the purified mitochondria fraction and

the cytosol. Dlp1 levels were decreased in cytosolic fractions but not in mitochondria

fractions. These observations imply that imbalances in mitochondrial dynamics may

contribute to the enlargement and the degeneration of mitochondria that occurs in the

hippocampi of scrapie-infected mice.

Changes in the shape of the mitochondria have been observed in ME7 scrapie-infected

brain even in the preclinical stage of infection [25]. The mitochondrial enlargement

demonstrated during the end stage of prion disease in this study was also observed in a

previous study that demonstrated structural abnormalities of the mitochondria in the

neurons of the hippocampi and cerebral cortices of brains from scrapie-infected hamsters

[15]. Fragmentation and clustering of mitochondria in the soma have been observed in

12
the brains of patients with sporadic AD, and alterations in the expressions of several

fusion and fission proteins have been observed in these brains [26]. Thus, we sought to

determine the relationship between expression levels of the mitochondrial fusion and

fission proteins and the alterations in the shapes, numbers and sizes of the mitochondria

in a prion disease model. In the ME7 mouse model, we found that the total number of

neuronal mitochondria was reduced significantly and that a number of enlarged and

degenerated mitochondria appeared. These mitochondria had damaged cristae and matrix.

The relationship between the mitochondrial damage and the induction of

neurodegeneration and disease is uncertain. Clearly the fact that the number of

mitochondria was reduced combined with degeneration of the remaining mitochondria

would lead to a loss of neuronal function. It needs to be noted that the loss of mitochondria

could affect the quantity of proteins observed; this is particularly important in instances

where the level of protein decreased (e.g., Dlp1 in the cytosol). Furthermore, in those

instances where there was no change in protein levels (e.g., Opa1), the reduced number

of mitochondria could mask an increase.

Growing evidence suggests that the delicate balance between mitochondrial fission and

fusion is critical for mitochondrial functions, including energy production, Ca2+

signaling, reactive oxygen species (ROS) production, apoptosis and senescence [7,27].

Dysregulation of the mitochondrial fusion and fission proteins in neurons may be a

common pathway leading to the mitochondrial and neuronal dysfunctions that are critical

in the pathogenesis of prion disease. It remains unclear, however, how the alterations in

mitochondrial fusion and fission proteins that were observed in this study contribute to

neurodegeneration in prion diseases. Taken together, the findings of this study suggest

that the disruption of the balance of mitochondrial fusion and fission proteins, which may

13
be a common feature of prion diseases, is involved in the mitochondrial and neuronal

dysfunction in the brains of ME7-infected mice.

14
Fig. 1.1. Deposition of PK-resistant prion isoforms in ME7-infected mice. Whole

brains of control (n = 3) and infected mice (n = 3) in the end stage (160 dpi) were

homogenized and blotted with anti-PrP antibody (3F10). A portion of each sample was

treated with PK (PK 50 ug/ml). β-Actin was used as a loading control.

15
Fig. 1.2. Differential expression of mitochondrial fusion and fission proteins in

hippocampi of ME7-infected mice. The mitochondrial dynamic proteins of the whole

brains and hippocampal regions of the control (n = 3) and infected mice (n = 3) in the end

stage of disease were blotted. β-Actin was used as a loading control for A and C. The

intensities of the bands in panels A and C were measured and quantified (B and D). The

values are expressed as the mean ± SD (n = 3). CON: control (white bars); ME7: ME7-

infected (black bars). Statistically significant differences are indicated (*p < 0.05 and **p

< 0.01).

16
Fig. 1.3. Subcellular localization of mitochondrial fusion and fission proteins in

hippocampi of ME7-infected mice. Opa1 (fusion) and Dlp1 (fission) from the

mitochondria (A) and cytosolic (C) fractions of the hippocampi of the control (n = 3) and

ME7-infected mice (n = 3) at the end stage of the disease were blotted (160 dpi). β-Actin

was used as a loading control. The intensities of the bands in panels A and C were

measured and quantified (B and D). The values are expressed as the mean ± SD (n = 3).

CON: control (white bars); ME7: ME7-infected (black bars). Note that both of the Opa1

bands are primarily localized to the mitochondria of hippocampi in both the control and

infected specimens, whereas substantial amounts of Dlp1 were localized to the cytosol.

Statistically significant differences are indicated (*p < 0.05).

17
Fig. 1.4. Electron microscopic analysis of the mitochondria in hippocampal neurons

of ME7-infected mice. (A) Control mitochondria were elliptical in shape and exhibited

compact cristae, whereas the infected mitochondria were enlarged and exhibited partially

swollen cristae. Scale bar, 1 ㎛. (B) The total number of mitochondria in the hippocampal

neurons of the control and ME7-infected mice (the total number of mitochondria were

counted from 15 neuronal cells from each group). (C) The lengths (white bar) and widths

(black bar) of the mitochondria in the hippocampal neurons of the control and ME7-

infected mice (the lengths and widths of 30 mitochondria in each group were measured).

Statistically significant differences are indicated (*p < 0.01). CON: control; ME7: ME7-

infected.

18
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22
 II. Chapter 2

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 PrPSc accumulation reduced Cx43

expression through the inhibition of JNK signaling, indicating that PrPSc 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.

23
Keywords: Connexin 43, gap junction, JNK, prion protein, scrapie

24
2.1 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 Ca2+ 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,

25
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

(PrPC) are associated with a protease-resistant and infectious form (PrPSc), 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.

26
2.2 Materials and methods

1) 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).

2) Subcellular fractionation

Mouse brain tissues were lysed in cold hypotonic buffer (10mM Tris-HCl, pH 7.4; 1mM

DTT; 5mM MgCl2; 10mM KCl; 10mM NaF; and 1mM 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

27
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 (50mM Tris-HCl, pH 7.4; 150mM NaCl; 2mM EDTA; 1% Triton X-100; 1%

Nonidet P (NP)-40; 0.25% sodium deoxycholate; 1mM Na3VO4; and 10mM 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.

3) Western blot analysis

The mouse brains and cultured cells were lysed in modified RIPA buffer with 20mM

Tris-HCl (pH 7.5), 150mM NaCl, 1% Triton, 0.5% sodium deoxycholate, 0.1% sodium

dodecyl sulfate (SDS), 1% NP-40, 10mM NaF, 1mM Na3VO4, 1mM EDTA, 1mM EGTA

and 0.1Mphenylmethylsulfonyl 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

antiphospho- 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

28
appropriate secondary antibodies conjugated to horseradish peroxidase. To detect PrPSc,

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 ImageQuant™ LAS 4000 imager (GE Healthcare Life Sciences,

Piscataway, NJ, USA) using an enhanced chemiluminescence western blot detection

system (Amersham Biosciences, Piscataway, NJ, USA).

4) 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 PrPSc 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.

5) 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

29
drying for 1 h at 37°C, the membrane was incubated in lysis buffer (50mM Tris-HCl, pH

8.0; 150mM NaCl; 0.5% sodium deoxycholate; 0.5% Triton X-100; 2mM PMSF)

containing PK (5 ㎍/ml) at 37°C for 10 min. The membrane was placed into 3 M

guanidinium thiocyanate (Sigma-Aldrich), 10mM Tris·HCl (pH 8.0) for 10 min followed

by immunostaining with anti-PrP antibody 3F10 (1:3,000).

6) 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′, antisense, 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.

7) Immunohistochemistry

Immunohistochemical procedures were performed as previously described [41]. The

sections were blocked with 10% normal donkey serum in Tris-buffered saline (50mM

Tris-HCl and 150mM 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,

30
Inc., Waltham, MA, USA). The control sections were stained without primary antibody.

8) 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).

9) 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

31
room temperature and washed with HBSS. Dye uptake was examined with a confocal

laser scanning microscope.

10) 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.

11) 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 PrPSc,

JNK, phospho-JNK, and Cx43 were determined by western blotting as previously

described.

12) Statistical analysis

The data are expressed as the mean±SEM. Significant differences between the

32
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.

33
2.3 Results

1) 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 PrPSc, 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

34
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.

2) 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.

3) 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

35
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

PrPSc 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.

4) 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 PrPSc (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

36
and infected cell lines were treated with various concentrations of an ATP-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.

5) 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

37
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 hemichannels and GJIC and likely occurs via JNK

activation.

6) Suppression of Cx43 expression with decreased JNK activity by BBG-mediated

blockade of prion conversion

To further investigate whether Cx43 induction is regulated by PrPSc accumulation, we

conducted a blocking experiment of PrPSc accumulation using BBG, which has anti-prion

activity through the inhibition of PrPSc 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, PrPSc, and p-JNK were

subsequently measured. As shown in Fig. 8 BBG treatment efficiently inhibited PrPSc

accumulation in a dosedependent manner. In correlation with the decrease in PrPSc, 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 PrPSc accumulation may be responsible

for JNK signaling-mediated Cx43 upregulation.

38
2.4 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 PrPSc 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,

13, 15, 18, 20, 33, 35, 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 Ca2+, 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

39
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, 60, 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

proinflammatory 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

40
as stroke, Alzheimer’s disease, Parkinson’s disease, and Huntington’s disease [57], it

remains a matter of debate whether Cx43 is neuroprotective or deleterious.

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 remains unresolved. 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 PrPSc appears

41
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, PrPSc 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 PrPSc (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.

42
Fig. 2.1 Increased expression of Cx43 protein and mRNA in the brains of scrapie

infected mice. A) Cx43 protein expression in whole brain and in dissected brains of the

control and ME7 (160 dpi), 22 L (140 dpi), and 139A (170 dpi) scrapie-infected mice was

evaluated by western blot with anti-Cx43 antibody. β-actin was used as a loading control.

B) The intensities of the Cx43 bands in panel A were measured and quantified. The values

are expressed as the mean±SEM (n = 3). C) In whole brains, the Cx43 mRNA levels were

43
analyzed by RT-PCR with three individuals per group. The error bars represent the SEM.

D) Cx43 protein expression was analyzed in the whole brains of the control and scrapie-

infected mice by western blot at the indicated time points after inoculation. β-actin was

used as a loading control, and PrPSc was detected using anti-PrP antibody after PK (50

μg/ml) treatment. Each experiment was repeated at least three times, and similar results

were obtained. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001.

44
Fig. 2.2 Immunohistochemical staining of Cx43 in brain sections from control and

scrapie-infected mice. Cx43 protein was detected in various brain sections obtained from

the control and ME7, 22 L and 139A scrapie-infected mice at 150 days post-inoculation.

Arrows, Cx43-positive cell-cell cotact or blood vessels. Scale bar, 25 μm.

45
Fig. 2.3 Increased Cx43 protein in membrane fractions of scrapie infected brains.

Whole brains were lysed, separated into cytosol and membrane fractions, and subjected

to western blot analysis as described in the Materials and Methods. Each fraction of the

brain homogenates was evaluated using anti-calnexin as a membrane marker and anti-

enolase as a cytosolic marker.

46
Fig. 2.4 Subcellular distribution of Cx43 and PrP in hippocampal cell lines with or

without scrapie infection. Double immunofluorescence staining was carried using anti-

Cx43 (red) and anti-PrP (green) in the PrP knockout (Zpl2-4) and wild-type (ZW13-2)

hippocampal neuronal cell lines with or without the 22 L or 139A scrapie infection. DAPI

was used for nuclear staining. Note that Cx43 was mainly localized in plaque formations

at the cell-cell contact areas (merged and bright-field images). Arrows indicate parts of

the gap junctions. Scale bars, 20μm.

47
Fig. 2.5 JNK activation-mediated Cx43 upregulation in scrapie-infected cells and

mice. A) Increased Cx43 expression and JNK activation in control and scrapie-infected

ZW13-2 cells (left panels) and mouse brains (right panels). To detect PrPSc, cell lysates

and brain homogenates were treated for 30 min at 37°C with PK at 50 μg/ml or 20 μg/ml,

respectively. B, C) JNK inhibition attenuates Cx43 expression in a dose- (B) and time-

dependent (C) manner. Control, 22L-, and 139A-infected ZW13-2 cells were treated with

0, 25, 50, and 100 μM of the JNK inhibitor SP600125 for 6 h (B) or with 50 μM

SP600125 for the time indicated (C). Each protein was analyzed by western blot with the

indicated antibodies.

48
Fig. 2.6 Increased EtBr uptake in scrapie-infected cells. An EtBr uptake assay was

performed using control or scrapie-infected ZW13-2 cells. Cells were pretreated with 500

μM LaCl3 for 5 min (middle panels) or 50 μM of the JNK inhibitor SP600125 for 6 h

(bottom panels) and then incubated with 5 μM EtBr for 5 min at 37°C; the fluorescent

signal was then analyzed using a confocal microscope.

49
50
Fig. 2.7 Increased scrape-loading dye transfer in scrapie-infected cells. Control or

scrapie-infected ZW13-2 cells were incubated with 0.1% Lucifer yellow (LY) for 5 min

at 37°C (top panels) or with 500 μM LaCl3 treatment (middle panels). For JNK inhibition,

the cells were pretreated with the JNK inhibitor SP600125 (50 μM) for 6 h prior to LY

treatment (bottom panels). LY fluorescence was determined using a confocal microscope

(A) and mean length of LY-filled area (the distance from the center to the edge, n = 20)

was represented by bars (B). The arrow indicates scrape; lines drawn in upper panels

indicate the distance for dye transfer. ∗∗∗p < 0.001. Scale bar, 50 μm.

51
Fig. 2.8 Attenuation of JNK signaling-mediated Cx43 upregulation by inhibiting the

accumulation of PrPSc. Control and 22 L scrapie-infected ZW13-2 cells were treated

with various concentrations of Brilliant blue G (BBG; 0-60 μM) for 3 days, and the

expression levels of Cx43, phosphorylated JNK, and PrPSc were analyzed by western blot

with the indicated antibodies. To detect PrPSc, the total cell lysates were treated with 50

μg/ml of PK for 30 min at 37°C. β-actin was used as a loading control.

52
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61
 III. Chapter 3

Establishment of Advanced In Vitro Model from Bank Vole

Transgenic Mice for Susceptibility to Various Prion Strains

Abstract

Prions are infectious agents that cause a devastating neurodegenerative disease in both
humans and animals. Unlike other rodents, bank vole (Myodes glareolus, BV) is
susceptible to prions from a diverse range of species, including humans. There are two
lines of BV, one homozygous for methionine (BV109M) and the other for isoleucine at
codon 109 (BV109I) of prion protein (PrP). Here, we established neuronal cell lines from
embryonic brain of BV109I mouse that was immortalized by an introduction of plasmid
DNA encoding for SV40-T antigen. BV109I mice develop age-dependent signs of
spontaneous neurologic illness. The established BV cell lines infected with 22L scrapie
strain (BV-22L) showed a replication and an accumulation of disease-associated forms
of the PrP. BV-22L infected neuronal cells were then intracerebrally injected into ICR
mice showing clinical symptoms of disease after 147 days post-inoculation. We also
confirmed that various prion strains, including ME7, 263K, CWD and 22L, were
successfully infected into BV neuronal cells. The newly established BV cell models may
enhance our understanding on the cellular mechanisms of prion replication and can be a
useful tool for the study of the pathogenic mechanisms of prion disease.

Key words: bank vole, neuronal cell lines, prion disease, scrapie

62
3.1. Introduction

Prions are infectious agents that cause a devastating neurodegenerative disease in both

humans and animals. The prion-associated diseases including transmissible spongiform

encephalopathies (TSE) or CJD in human were characterized as “infectivity” which are

distinguish from other neurodegenerative disease such as Alzheimer’s and Parkinson’s

disease; this property seems to reside, at least in part, in an aberrant scrapie form of prion

protein (PrPSc) of a constitutively expressed cellular prion protein (PrPC) [1]. It has been

suggested that the conformer protein, PrPC interacted with abnormal prion protein, PrPSc

and then generated newly formed PrPSc through undefined process, including post-

translational modification, combination with factor X, and mutations and so on (Prusiner,

1998). PrPC is constitutively expressed in most cells, including neuronal cells, in particular

with highest expression level in subset of neurons of central nervous system (CNS). Despite

the distribution of PrPC in various tissues, there are still many arguing on the cellular

functions of PrPC [2, 3, 4].

Scrapie is one of the prion diseases developed in sheep and goat, and its clinical

symptoms characterized by a long latent period, progressive ataxia, tremor, wasting and

death [5]. Due to the property of long latent period, scrapie is regarded as a slow infectious

disease. It is successfully transmitted to several species of rodents, including mice,

hamsters and rats. Many kinds of scrapie strains were isolated and used to study

pathogenesis of prion diseases [6, 7]. The typical features of the prion diseases are

histopathological spongiform degeneration in CNS, reactive gliosis or astrocytosis and

neuronal loss observed in some cases, and formation of amyloid plaques. It has been

reported that a common cause of prion diseases including scrapie is the conformational

63
misfolding of the endogenously expressed prion protein [8]. A predominant α-helical-rich

prion protein PrPC have changed into a disease-associated β-sheet-rich conformation in

terms PrPSc [9], but the mechanism is still unknown. PrPSc has two different biochemical

features compared to PrPC which is ability to formation of amyloid fibrils and relatively

resistant to proteolysis with proteinase K (PK). These features are used to diagnostically

distinguish PrPSc from PrPC. The mice presented clinical signs of scrapie agent has shown

the microglial activation in the regions of brain showing vacuolation, PrP deposition and

neuronal apoptosis. [10, 11]. Under the same condition for proteolysis with PK, PrPSc

produces a smaller protease-resistant molecule of 142 amino acids whereas PrPC is

completely hydrolyzed [12]. Prion rods formed by limited proteolysis, and detergent

extraction are indistinguishable from the filaments that aggregate to form PrP amyloid

plaques in the CNS [13].

Study for transmission barrier needs to elucidate the basis of prion replication and

acquiring knowledge to decipher the risk of interspecies transmission [14]. For the study

of transmission activity between species barrier, the animal models that are susceptible to

various prion strains are quite available. Recently bank voles (Myodes glareouls, BV)

were introduced as an animal model for the research of prion diseases. This animal model

has an important difference compared to a model of mouse and Syrian hamster. A

transgenic (Tg) mice expressing BVPrP showed a high susceptibility to prion strains

originated from various species, including sheep, goats and humans [15]. In addition, BV

mice shows a relatively short incubation period compared to a conventional prion-animal

model [16]. Two lines of BV mouse that is homozygous phenotype with either methionine

or isoleucine at codon 109 on BV Prnp and in termed as Tg mouse BV109M and BV109I

were investigated, respectively [17]. BV109M was shown to be susceptible to sporadic

64
and genetic Creutzfeldt-Jakob disease (CJD) [18], sheep scrapie [19], mouse- and

hamster-adapted scrapie strains [20], cattle and sheep BSE [18] and atypical BSE [14].

The studies with BV109I was revealed that the susceptibility of PrP109I phenotype is

similar to the methionine-carrying line, although the differences depends on the specific

prion strains [21].

In this study we investigated whether BVPrP-expressing neuronal cell lines (BV cells)

are susceptible to a wide range of prion sources. Taking advantage of prion strains adapted

BV cell lines, this study aimed to investigate which prion strains are actually associated

with distinctive PrPSc types using this biochemical phenotypes. Firstly we established the

neuronal cell line derived from BV109I mouse and then assessed the susceptibility of the

cells to 22L scrapie strains. We examined the characterization of 22L strains-adapted BV

neuronal cells, resulting in observed high localization titers of PrPSc accumulation in

cytoplasm.

These wild-type BV, BV-/- and 22L-adapted BV neuronal cell lines will be useful

experimental systems to reveal the physiological functions of both PrPC and PrPSc, and to

investigate the conformational change of prion isoforms and the pathological mechanisms

in prion diseases.

65
3.2. Materials and Methods

1) Animals and prion strains

The prion strains, 22L, ME7, and 263K were kindly provided by Alan Dickinson

(Neuropathogenesis Unit, UK): 22L prion strain was maintained by serial intracerebral

passage of brain homogenate from terminally affected mice. Six-week-old male ICR mice

were obtained from the Central Lab. Animal Inc. (Republic of Korea) and the inbred ICR

mice divided into age-matched controls and experimental group infected with 22L prion

strain. CWD prion strain was passaged to the Tg PrPElk mice [22]. (Jeon et al., 2013).

Mice were inoculated intracerebrally with 30 μl of 1 % (w/v) brain homogenates in 0.01

M phosphate-buffered saline (pH 7.4) from either normal brain or prion-infected ICR

mouse brain at the terminal stages of the disease. Animals were sacrificed when clinical

signs of the disease were manifested and then the brain of both control and prion-infected

mice was harvested for protein analysis and histological pathology of prion disease. All

animal experiment has been conducted in accordance with the guidelines laid down by

the Hallym Medical Center for the Institutional Animal Care and Use Committee.

2) Establishment of neuronal cell lines from BV109I mice

Primary neuronal cells were isolated from 14-days-old embryos of BVPrP-expressing

Tg mice (BV109I). This BV109I mice produced from PrP knockout mouse were kindly

provided by Dr. Joaquin Castilla (Proteomics Unit, Spain). The cells were cultured on

poly-L-lysine (P-L-L) (Sigma-Aldrich, USA) coated dishes with culture media DMEM

(Hyclone, USA) containing 20% FBS, 100 unit/ml of penicillin and 100 unit/ml of

streptomycin (Gibco BRL, USA) at 37℃, 5% CO2. After one week, medium was replaced

66
by selective medium (DMEM with 0.25 mg/ml G418, Hyclone), and changed in every 3

days. When a single colony formed, it was transferred to 24-well plate dish and examined.

At last the cells from a single colony were transferred to 6-well dishes and maintained in

culture medium, DMEM with 10% FBS and 1% antibiotics.

3) Scrapie-infected cell lines

The cells including BV and mouse hippocampal neuronal ZW13-2 (PrP wild-type) [23]

were cultured in DMEM with 10% FBS (Hyclone), 100 units/ml of penicillin and 100

µg/ml of streptomycin in incubator. The BV cells were persistently infected with 22L

prion strain as previously described [24]. The infected cells maintained in Opti-MEM

(Gibco BRL) with 10% FCS (Hyclone). Stable scrapie infection was confirmed after 10

passages by a cell blot assay as previously described [24]. The infected cells stably

produced PrPSc over 50 passages.

4) Reverse transcription-polymerase chain reaction (RT-PCR)

To assess the expression levels of SV40 and PrP, total RNA was extracted using Trizol

reagent (Invitrogen), and synthesized cDNA by AMV transcriptase (Promega, USA). To

determine the integration of SV40 large T antigen, genomic DNA was extracted from the

primary cultured cells using DNA extraction kit (Qiagen, USA) according to the

Manufacture’s instruction. PCR was performed with the following primers (Bioneer,

Republic of Korea).

PrP,

sense: 5’ ATGGCGAACCTTGGCTACTGG-3’

antisense: 5’-CCCTCATCCCACGATCAGGAAGATG-3’

67
 SV40 large T antigen,

sense: 5’-TGAGGCTACTGCTGACTCTCAACA-3’

antisense: 5’-GCATGACTCAAAAAACTTAGCAATTCTG-3’

GAPDH,

sense: 5’-TGGTATCGTGGAAGGACTCATGAC-3’

antisense: 5’-ATGCCAGTGAGCTTCCCGTTCAGC-3’ (reference 2004??).

The obtained PCR products were examined by electrophoresis with 1 % TBE-agarose gel.

5) Western blot analysis.

The mouse brains and neuronal cells were lysed in 10 volumes of RIPA buffer (Thermo

Fisher Scientific, USA) containing protease inhibitor cocktails (Roche, Germany). The

brain homogenates and cell lysates were centrifuged at 10,000 g for 15 min at 4℃ and

then the supernatant were taken. The protein concentration was determined by BCA assay

(Pierce, USA). To detect PrPSc, 5 to 50 μg/ml of proteinase K (PK) and 25 to 100 μg/ml

of thermolysin (TL) was treated for 30 min at 37°C in brain homogenates and cell lysates.

Equal amounts of proteins were boiled with 5X SDS Gel-loading buffer [100mM Tris-

HCl (pH 6.8), 4% SDS, 0.2% bromophenol blue, 20% glycerol, and 10% -

mercaptoethanol] for 10 min, separated by 15% SDS-gel. The separated proteins were

transferred to nitrocellulose membrane (Amersham, USA) at 260 mA for 1.5 hr. After

blocking with 5% skim milk in TBST containing 0.05% for 1 hr, the membranes were

incubated with either one of following antibodies at 4°C for overnight : mouse anti-PrP

antibody (3F10; 1:3,000) [25], mouse monoclonal anti-NeuN antibody (1:5,000,

Chemicon, USA) as a neuronal marker and mouse monoclonal anti-GFAP (1:3,000,

68
Chemicon) as an astrocyte marker. Incubation with appropriate secondary antibodies

conjugated with horseradish peroxidase (Sigma-Aldrich) carried out for 1 hr at room

temperature. The membranes were visualized by chemiluminescence substrate as

described in Manufacture’s instruction. (Amersham). The target signals were visualized

by digital images and captured with an ImageQuant™ LAS 4000 imager (GE Healthcare

Life Sciences, USA) using an enhanced chemiluminescence Western blot detection

system (Amersham). Western blot analysis (n=3 for each group) was performed at least

three times.

69
3.3. Result

1) Neuronal cell lines established from BV109I mice

Primary neuronal cells were isolated from the cortical region of 14-days-old embryos of

BV109I mice and transfected with retroviruses expressing SV40 large-T-antigen to be

immortalized. The cells were cultured in selective media until the establishment of cell

lines as described in Materials and Methods, and four distinct neuronal cell lines were

established: BV5, BV6, BV7, and BV8. As shown in Fig. 1A, all of cell lines were

characterized by RT-PCR with a SV40 large T antigen-specific primers showing similar

expression levels. To further confirm the cell type of each cell lines, Western blot and

immunofluorescence analyses were carried out using different cell markers. In both

experiments, four cells were positively reacted with a neuronal cell marker, NeuN, but

not with an astroglial cell marker, GFAP. Characterization of the cell type yielded similar

outcome in both Western blot (Fig 1B) and immunofluorescence analysis (data not

shown). For the positive controls, brain tissues of BV109I mice and ZW13-2 cell line

were examined.

To examine the expression levels of BVPrP mRNA and protein in both BV neuronal cell

lines and the brain of BV109I mice, we performed RT-PCR and Western blot analysis,

respectively. The expression of prnp gene was detected in BV5 and BV 6 cell lines, but

not in BV 7 and BV 8 (Fig. 1C). In consistent with the mRNA expression level of BVPrP,

the BVPrP protein was strongly detected as three typical glycoforms in BV5 and BV6

cell lines, however BV7 and BV8 cell lines showed no detectable BVPrP. Taken together,

these newly established cells originated from BV109I mice were characterized as

neuronal cell lines, which can be used as a model of BVPrP-expressing (BV5 and BV6)

70
and BVPrP-deficient (BV7 and BV8) neuronal cells.

2) BV neuronal cells are susceptible to infection with different prion strains

Unlike other rodents, BVs are highly susceptible to prions from many different species,

suggesting that BVs do not impose a species barrier, which normally restricts the

transmission of prions from one species to another [26]. We intracerebrally inoculated

various prion strains into mouse or hamster to use as prion inoculums, which lastly were

used to establish prion-infected BV neuronal cell lines. When the established BV neuronal

cells expresses PrPC, we assessed their susceptibility to various prion infection. As shown

in Fig. 2, the PK-resistant PrP were detected up to 30 passages in various prion-infected

BV cells, and 22L-infected BV6 cells (Bv-22L) consistently produced stronger

expression of PrPSc than ME7-, 263K- or CWD-infected BV cells (BV-ME7, BV-263K,

or BV-CWD, respectively) (Fig 2A). The levels of PrPSc in BV-22L cells were compared

with the PrPSc in the brain of 22L-infected ICR mice (Fig. 2B). The detected PrP protein

by anti-PrP antibody (3F10) has shown the typical migration pattern of PrPC towards

PrPSc under the PK treatment in different prion-infected BV neuronal cells. We next

confirmed the BV cells-adapted prion strains by the detection of thermolysin (TL)-

resistant PrPSc. As shown in Fig. 2C, the TL-resistant prion isoforms were detected in the

brain of 22L-infected ICR mouse and BV cells following TL treatment, whereas normal

prion isoforms in the BV6 cell line inoculated with the wild-type ICR mouse brain

homogenates was completely degraded by TL treatment. Taken together, our data indicate

that BV neuronal cells are a new, highly susceptible model of prion strain infection.

71
3) Transmission of BV-22L neuronal cells to ICR mice.

Intracerebral inoculation of ICR mice with cell lysates from 22L-infected ZW or BV

neuronal cells resulted in prion disease with clearly evident, progressive clinical signs. In

this studies on two independent cell-passaged scrapie strains, ZW-22L and BV-22L,

showed a different survival rates at first passage, however the survival rate became the

similar in second passage with a scrapie strain (Table 1). The incubation period at first

passage in mice was 149.2 ± 6.14 days (ZW-22L) and 185.3 ± 5.76 days (BV-22L)

decreasing to 145.5 ± 2.7 days (ZW-22L) and 147.8 ± 4 days (BV-22L), respectively, at

the second passages.

To further determine the accumulation of PK-resistant PrPSc in the brains of ZW-22L

and BV-22L inoculated mice at the end stage of the disease, we performed

immunoblotting using whole brains obtained from control and ZW-22L or BV-22L

inoculated mice. As shown in Fig. 3, we found no difference the accumulation of PK-

resistant PrPSc between the brains of ZW-22L and BV-22L inoculated mice (Fig. 3A)

although the total PrP proteins were slightly increased in infected brains. The PrPC in

control mice was completely degraded by the PK treatment at the end stage. Consistent

with these results, the levels and the rate of PK-resistant PrPSc accumulation in the brain

homogenates of second passaged ZW-22L and BV-22L mice were very similar. Taken

together, our data demonstrated that BV neuronal cells are a new, highly susceptible

model of various prion strains infection.

72
1.4. Discussion

In this study we hypothesized that PrPC protein expressing in BV in termed BVPrP mice

may improves the sensitivity of cells to various prion agents. To address this, we

established the neuronal cell lines expressing BVPrP and subjected the cells to in vivo

transmission of mouse-adapted prion strains. It is first time to establish the neuronal cells

originated from BV Tg mice. The characteristics of BV cells infected with 22L scrapie

were similar to scrapie-infected ZW13-2 cells which showed a high level of PK-resistant

PrPSc and the ability to induce scrapie in mice upon intracerebral inoculation. In vivo and

in vitro transmission studies with transgenic mice have clearly demonstrated an inverse

correlation between the expression level of PrPC and the incubation time of experimental

prion diseases.

It has been reported that several cell systems permissive to the replication of different

mouse-adapted prion strains have been developed [27]. They could be a suitable tool for

defining the mechanisms underlying prion-induced brain damage; their simplicity, in fact,

allows analyzing the events during prion pathogenesis better than in vivo models. Most

PrPC expressing cell lines are refractory to mammalian prion infection with unknown

reasons [28]. Prion strains have an exquisite host cell tropism in vivo [29] as well as in

tissue cultured cells [30].

Prions that are fully infectious between individuals of the identical mammalian or yeast

species may transmit poorly or not at all across species, which is called the species barrier.

It has been widely accepted that the species barrier may be largely due to the difference

of the sequence and conformation of PrP protein [31]. This difference from the variants

of PrP sequence has shown in the studies using Tg mice [32]. The characterization of Tg

73
mice expressing wild-type BVPrP showed remarkable finding (Watts et al., 2012).

Different from mice, BVs are highly susceptible to a wide range of prion strains isolated

from numerous species, including humans (Agrimi et al., 2008). Tg mice expressing the

109I polymorphic variant of BVPrP develop a spontaneous neurodegenerative disease

that recapitulates all of the neuropathological hallmarks of prion disease [26]. This model

was used as the shortest incubation time model for prion diseases because of its

outstanding susceptibility to propagate most of the existing prion strains from different

species. In a previous study, we established PrP hippocampal neuronal cell lines including

ZW 13-2 and showed very limited data that ZW 13-2 cell lines could be infected with the

22L and 139A strains of scrapie [23, 24]. Moreover, since only mouse prions were

produced in the cell lines by inoculating mouse prions, we demonstrated that prions could

be replicated and their biological characteristics were retained in the scrapie-infected

neuronal cell lines. This study showed the prion adaptation of the neuronal cell line

derived from the embryonic brain of BV109I mouse. We found that the scrapie-infected

cell line produced three major protease-resistant and PrP-immunoreactive prion proteins

with apparent molecular masses ranging. It showed that brain homogenate from 22L-

infected mice contained similar levels of total PrP and PK-resistant PrPSc. BV-22L

infected neuronal cells were intracerebrally injected into ICR mice, resulting in develop

the clinical symptoms of disease on incubation period averaged at 147 days dpi. The

incubation period was different between first and second passage in case of BV-22L

scrapie infected ICR mice, which presented on average of 185 days (BV-22L) at first

passage and of 147 days (BV-22L) at second passage, resulting in getting shorter

approximately 30 days in second passage than first passage.

Other alternatives of animal bioassay include both cell-based systems and cell-free

74
methodologies. Although cell lines are not generally suitable for prion infection or

replication, various cell lines, most notably N2a cells, have been used to study prions in

vitro. The cell line used for prion infection is not permissive for the infection of all prion

strains, i.e. N2a cell is resistant to strain ME7, 22A, and 301V, but susceptible to RML

and 22L [31].

In a current study, the various prion strains, including ME7, 263K, CWD, and 22L, were

successfully infected into BV neuronal cells and confirmed the PK-resistant PrPSc by

Western blot analysis. These results are consistent with the results described in Prusiner

et al. [31]. However, PrPSc produced in ME7, 263K, or CWD-infected BV neuronal cells

disappeared after over 50 passages although we determined that PrPSc was detectable in

up to 30 passages. There are many assumptions; the formation of PrP Sc in acute

conversion kills the host cells and the difference in PrP allotype between the host and

inoculum [20] and so on.

It is clear that different prion strains-adapted BV cells, which can be a target for prion

infection in vivo, are established in this study for the first time. The establishment of BV

cell models may enhance our understanding on the cellular mechanisms of prion

replication. From a viewpoint of the applied research and public health, prion-adapted

BV109I cell system will be a very useful model for the screening of prion types and

therapeutic compounds, and for the development of new diagnostic marker that are

urgently needed.

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Figure 3.1. Cell-type characterization and expressions of the PrP in the BV neuronal

cell lines. (A) Expression levels of SV40 large T antigen mRNA were determined by RT-

PCR analysis. Tails from PrP-expressing wild-type or PrP-knockout mice were used as a

negative control for SV40 T. GAPDH was used as a loading control. (B) The BV5, BV6,

BV7 and BV8 cell lines were positive to NeuN which did react with protein lysates of

ZW13-2 neuronal cells as a positive control. In contrast, GFAP (a ~50 kDa protein band,

an astrocyte marker) was detected only in the brain homogenate of BV109I mouse. (C)

mRNA expressions of PrP (789 bp) was checked. Genomic DNA from the tails of PrP-

expressing BV109I or PrP-knockout mouse were used as a positive or negative control,

76
respectively. BV5 and BV6; Expression of BV109I neuronal cell lines. BV7 and BV8;

PrP knockout neuronal cell lines. (D) The expression of PrPC protein in BV neuronal cell

lines were analyzed by immunoblotting with anti-PrP antibody (3F10).

77
Figure 3.2. Analysis of PK and TL-resistant PrPSc in the BV neuronal cell lines

infected with different prion strains. Various prion-infected cells were used in the

Western blot. (A) and (B) The PK-digested PrPSc isoforms were detected using anti-3F10

PrP antibody. A portion of each sample was treated with PK (PK 5 or 10 μg/ml). (C) 22L-

infected mouse and BV6 cells following digestion with thermolysin (TL, 25 through 100

μg/ml).

78
Table 1. Transmission of 22L scrapie-infected BV neuronal cell lines to ICR mice

Figure 3.3. Accumulation of PK-resistant prion isoforms in ZW-22L and BV-22L

scrapie-infected ICR mice. PK-resistant PrPSc isoforms were identified in the brains of

ICR mice that were intracerebrally inoculated with ZW-22L or BV-22L-infected cell

lysates. (A) Whole brains of control (n = 4), first-infected mice (n = 4) and (B) second-

infected mice (n = 3) at the end stage were homogenized and immunoblotted with anti-

79
3F10 PrP antibody. To detect PK-resistant PrPSc, the total cell lysates were treated with

50 ug/ml of PK for 30 min at 37°C.

80
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84
 Establishment of Advanced In Vitro Model from Bank Vole Transgenic
Mice for Susceptibility to Various Prion Strains

2016. 88 page
Doctoral Degree
Hong-Seok Choi
Department of Microbiology
Advisor Prof. Yong-Sun Kim

ABSTRACT

Prions are infectious agents that cause a devastating neurodegenerative disease in both
humans and animals. Unlike other rodents, bank vole (Myodes glareolus, BV) is
susceptible to prions from a diverse range of species, including humans. There are two
lines of BVs, one homozygous for methionine (BV109M) and the other for isoleucine at
codon 109 (BV109I) of prion protein (PrP). Here, we established neuronal cell lines from
embryonic brain of BV109I mouse that was immortalized by an introduction of plasmid
DNA encoding for SV40-T antigen. Bv109I mice develop age-dependent signs of
spontaneous neurologic illness. The established BV cell lines infected with 22L scrapie
strain (BV-22L) showed a replication and an accumulation of disease-associated forms
of the PrP. BV-22L infected neuronal cells were then intracerebrally inoculated into ICR
mice showing clinical symptoms of disease after ~147 days post-inoculation. We also
confirmed that various prion strains, including ME7, 263K, CWD and 22L, were
successfully infected into BV neuronal cells. The newly established BV cell models may
enhance our understanding on the cellular mechanisms of prion replication and can be a
useful tool for the study of the pathogenic mechanisms of prion diseases.

Key words : bank vole, neuronal cell lines, prion disease, scrapie

85
 Bank Vole 프리온 단백 발현 형질 전환 마우스의 신경세포에서 다양한

프리온 감염 세포주 확립

2016. 88p.

박사학위논문

최홍석

의학과 미생물학 전공

지도교수: 김용선

국문초록

프리온 질환은 사람과 동물 모두에서 발생하는 감염성 신경질환이다. 다른 설치류와는 달리

bank vole (Myodes glareolus, BV)은 인간을 포함한 다양한 종의 프리온에 감염될 수 있다.

프리온 질병의 발생에서 신경세포는 중요한 역할을 한다. 본 연구에서는 BV 유전자 이식

마우스의 배아뇌조직으로부터 해마부위 신경세포를 분리하고 SV large T antigen 감염에

의한 세포 불멸화 과정을 통해 신경세포주를 확립하였다. BV 신경세포주는 스크래피

스트레인 (22L)을 감염시켰으며, 50번 이상의 계대 배양한 이 세포주에서 지속적으로

비정상적인 프리온 단백의 복제나 축적을 확인하였으며 단백질 분해효소 K에 저항성을

가지는 비정상적인 프리온 단백의 축적을 확인하였다. 또한 22L 스크래피에 감염된 BV

신경세포주를 마우스 뇌에 주입한 결과 평균 147일 후에 임상증상을 확인하였다. BV

신경세포주는 스크래피 뿐만 아니라 다른 여러 종의 프리온 스트레인에 감염되었다. 따라서

본 연구에서 처음으로 확립된 BV 신경세포주는 다양한 프리온 스트레인에 감염될 수 있으며

프리온 질환의 발병원인을 연구하는 유용한 도구로 이용될 수 있을 것이다.

핵심어: 프리온 질환, Bank Vole 형질전환 마우스, 스크래피, 신경세포주

86
 Establishment of Advanced In Vitro Model from Bank Vole

Transgenic Mice for Susceptibility to Various Prion Strains

The undersigned certify that they have read, and recommended to the Graduate

school for acceptance, a thesis entitled “Establishment of Advanced In Vitro Model from

Bank Vole Transgenic Mice for Susceptibility to Various Prion Strains” submitted by

Hong-Seok Choi in partial fulfillment or the requirements for the degree of Ph.D. in

Medical Science.

Dr. Hyung-Joo Kwon

Department of Microbiology

___________________

Dr. Kyung-Chan Choi

Department of Pathology

___________________

Dr. Jae-Bong Park

Department of Biochemistry

___________________

Dr. Jae-Il Kim

Department of Food Science & Nutrition

___________________

Dr. Yong-Sun Kim

Department of Microbiology

___________________

December, 2016

87
 IV. Appendix

1. Choi HS, Choi YG, Shin HY, Oh JM, Park JH, Kim JI, Carp RI, Choi EK, Kim YS,

Dysfunction of mitochondrial dynamics in the brains of scrapie-infected mice. Biochem

Biophys Res Commun, 448(2):157-62, 2014

2. Lee GH*, Jang B*, Choi HS*, Kim HJ, Park JH1, Jeon YC, Carp RI, Kim YS, Choi

EK, Upregulation of Connexin 43 Expression Via C-Jun N-Terminal Kinase Signaling in

Prion Disease. J Alzheimers Dis, 49(4):1005-19, 2015.

* These authors contributed equally to this work.

88
 Biochemical and Biophysical Research Communications 448 (2014) 157–162

Contents lists available at ScienceDirect

Biochemical and Biophysical Research Communications

journal homepage: www.elsevier.com/locate/ybbrc

Dysfunction of mitochondrial dynamics in the brains of scrapie-infected

mice
Hong-Seok Choi a,b, Yeong-Gon Choi b, Hae-Young Shin b, Jae-Min Oh b, Jeong-Ho Park a,b,
Jae-Il Kim c, Richard I. Carp d, Eun-Kyoung Choi b,⇑, Yong-Sun Kim a,b,⇑
a
Department of Microbiology, College of Medicine, Hallym University, 1 Okcheon-dong, Chuncheon, Gangwon-do 200-702, Republic of Korea
b
Ilsong Institute of Life Science, Hallym University, 1605-4 Gwanyang-dong, Dongan-gu, Anyang, Gyeonggi-do 431-060, Republic of Korea
c
Department of Food Science and Nutrition, Pukyong National University, 599-1 Daeyeon-3-dong, Nam-gu, Busan 608-737, Republic of Korea
d
New York State Institute for Basic Research in Developmental Disabilities, 1050 Forest Hill Road, Staten Island, NY 10314, USA

a r t i c l e i n f o a b s t r a c t

Article history: Mitochondrial dysfunction is a common and prominent feature of many neurodegenerative diseases,
Received 7 April 2014 including prion diseases; it is induced by oxidative stress in scrapie-infected animal models. In previous
Available online 19 April 2014 studies, we found swelling and dysfunction of mitochondria in the brains of scrapie-infected mice com-
pared to brains of controls, but the mechanisms underlying mitochondrial dysfunction remain unclear. To
Keywords: examine whether the dysregulation of mitochondrial proteins is related to the mitochondrial dysfunction
Prion disease associated with prion disease, we investigated the expression patterns of mitochondrial fusion and fission
Mitochondrial fusion
proteins in the brains of ME7 prion-infected mice. Immunoblot analysis revealed that Mfn1 was up-reg-
Fission
Scrapie
ulated in both whole brain and specific brain regions, including the cerebral cortex and hippocampus, of
Mitochondrial dysfunction ME7-infected mice compared to controls. Additionally, expression levels of Fis1 and Mfn2 were elevated
in the hippocampus and the striatum, respectively, of the ME7-infected brain. In contrast, Dlp1 expres-
sion was significantly reduced in the hippocampus in the ME7-infected brain, particularly in the cytosolic
fraction. Finally, we observed abnormal mitochondrial enlargement and histopathological change in the
hippocampus of the ME7-infected brain. These observations suggest that the mitochondrial dysfunction,
which is presumably caused by the dysregulation of mitochondrial fusion and fission proteins, may con-
tribute to the neuropathological changes associated with prion disease.
Ó 2014 Elsevier Inc. All rights reserved.

1. Introduction
Prion diseases, also known as transmissible spongiform enceph-
alopathies (TSEs), are fatal neurodegenerative disorders that affect
both humans and animals [1,2]. Scrapie is a prototypical prion dis-
ease that affects sheep and goats. Clinically, scrapie is character-
ized by a long latent period, progressive ataxia, tremor, wasting
and ultimately death [3]. Many scrapie strains have been isolated
from sheep and goats and used to examine not only the range of
various pathogenesis pathways but also the neuropathological
mechanisms induced by prion disease [4]. Typical features of the
disease include the formation of spongiform vacuoles and astrocy-
tosis, the formation of amyloid plaques in some cases and neuronal
loss in the brain [5,6]. A key event in prion disease is the conforma-
tional misfolding of the endogenously expressed cellular prion
⇑ Corresponding authors.
E-mail addresses: ekchoi@hallym.ac.kr (E.-K. Choi), yskim@hallym.ac.kr
(Y.-S. Kim).
protein (PrPC) into the scrapie form of the pathogenic prion protein
(PrPSc) [5].
A number of recent studies have demonstrated that mitochon-
dria are dynamic organelles that continually undergo fission and
fusion with one another [7–9]. Mitochondria can change in number
and morphology within a cell during development, throughout the
cell cycle and when challenged with various cytotoxic stimuli [9].
In mammals, the key molecules involved in mitochondrial fission
are dynamin-like protein 1 (Dlp1, also referred to as Drp1) and fis-
sion 1 (Fis1). The process opposing fission, i.e., mitochondrial
fusion, is controlled in mammalian cells by mitofusin 1 (Mfn1),
mitofusin 2 (Mfn2) and optic atrophy 1 (Opa1) [7]. The sizes,
shapes and interconnectivities of mitochondria are determined
by their fusion and fission [9]. It has been suggested that the mito-
chondrial defects associated with Alzheimer’s disease (AD), Parkin-
son’s disease (PD) and Huntington’s disease (HD) may result, at
least in part, from a disruption of the fusion and fission mecha-
nisms of mitochondria [8,9]. A recent study reported that the
expression of Dlp1 is decreased and that mitochondria are

http://dx.doi.org/10.1016/j.bbrc.2014.04.069
0006-291X/Ó 2014 Elsevier Inc. All rights reserved.
158 H.-S. Choi et al. / Biochemical and Biophysical Research Communications 448 (2014) 157–162
abnormally elongated in the fibroblasts of AD patients and in neu-
ronal cell lines overexpressing amyloid precursor protein (APP)
[10]. The study suggests that APP causes an imbalance between
mitochondrial fusion and fission that results in an abnormal distri-
bution of mitochondria, which in turn contributes to mitochondrial
and neuronal dysfunction. Several observations suggest a link
between mitochondrial dysfunction and PD. Pink1 and parkin,
which are PD-related genes, promote mitochondrial fission and
inhibit mitochondrial fusion in Drosophila [11]. Additionally, HD
research has focused on mitochondrial dysfunction. A mouse
model of HD exhibits early defects in respiration and ATP produc-
tion [12]. Moreover, mutant huntingtin seems to disrupt mito-
chondrial Ca2+ buffering [13] and to cause mitochondrial
ultrastructural changes in the lymphoblasts of HD patients [14].
Previous studies reported that dysfunction and enlargement of
the mitochondria occur by oxidative stress in animal models of
prion disease [15,16]. However, the underlying mechanism respon-
sible for this mitochondrial dysfunction remains unclear.
In the present study, we investigated the mitochondrial fusion
and fission proteins that may be involved in the mitochondrial dys-
function observed in prion disease. We show that mitochondrial
fusion and fission proteins are differentially modulated in the ter-
minal stage of an experimental mouse model of prion disease and
that this modulation may contribute to the morphological damage
and reduction in number of mitochondria in infected neuronal
cells.
2. Materials and methods
2.1. Antibodies
The following monoclonal and polyclonal antibodies were used:
mouse monoclonal anti-PrP (3F10) [17], mouse monoclonal anti-
COX IV (Abcam), goat polyclonal anti-enolase (Santa Cruz), mouse
monoclonal to b-actin (Sigma–Aldrich), mouse monoclonal
anti-Opa1 and mouse monoclonal anti-Dlp1 (BD Transduction
Laboratories), mouse monoclonal anti-Mfn2, chicken polyclonal
anti-Mfn1 (Novus Biologicals) and rabbit polyclonal anti-Fis1
(Santa Cruz).
2.2. Animals and scrapie strains
Six-week-old C57BL/6 mice were obtained from the Central
Laboratory Animal (Republic of Korea) and divided into two
groups: one group was infected with the ME7 scrapie strain, and
the other included age-matched controls. The ME7 scrapie strain
was kindly provided by Dr. Alan Dickinson (Neuropathogenesis
Unit, Edinburgh, UK). This scrapie strain was maintained by serial
intracerebral passages of brain homogenate from a terminally
affected mouse. The mice were intracerebrally inoculated with
30 ll of 1% (w/v) brain homogenate in 0.01 M phosphate-buffered
saline (PBS, pH 7.4) from either a normal brain or an ME7-infected
C57BL/6 mouse brain at the terminal stage of the disease. When
the clinical signs of prion disease were evident in the terminal
stage (160 days postinoculation, dpi), the mice were sacrificed.
2.3. Western blot analysis
Whole brains or hippocampal regions were homogenized gently
in 10-fold greater volumes (w/v) of 50 mM Tris–HCl (pH 7.4)
containing 150 mM NaCl, 1 mM EDTA, 0.25% Na-deoxycholate, 1%
NP-40 and protease inhibitor cocktails (Roche). The protein
concentration was determined using the BCA assay (Thermo
Scientific). The homogenates were treated with Proteinase K (PK)
at a concentration of 50 lg/ml. Equal amounts of protein
(10–50 lg in all assays) were separated by SDS–PAGE using 10%,
12% or 15% acrylamide gels and then transferred to nitrocellulose
membranes (Thermo Scientific). After blocking with 5% skim milk
for 1 h, the membranes were incubated with the individual antibod-
ies overnight at 4 °C and then incubated with horseradish peroxi-
dase-conjugated secondary antibody in Tris-buffered saline with
0.05% Tween-20 (TBST) containing 5% skim milk for 1 h at room tem-
perature. The blots were visualized with the SuperSignal West Pico
Chemiluminescent Substrate (Thermo Scientific). The expression
levels of each protein were quantified using ImageJ software (NIH).
2.4. Transmission electron microscopy (TEM)
The animals were perfused with 0.1 M PBS (pH 7.4) containing
4% paraformaldehyde and 2.5% glutaraldehyde under deep anes-
thesia with 16.5% urethane. The brains were removed and fixed
in the 0.1 M PBS fixative that was used for perfusion. The bilateral
hippocampal regions were trimmed into small pieces immediately
after their surgical removal and kept in the fixative for 2 h at 4 °C.
Post-fixation was performed in 0.1 M PBS with 1% osmium tetrox-
ide followed by dehydration through a graded ethanol series and
embedding in Epon 812. Ultra-thin sections (75 nm) prepared by
using an ultramicrotome (RMC MTXL) were stained with uranyl
acetate and lead citrate and were subsequently observed with a
transmission electron microscope (JEM-1011, JEOL). The numbers
of total and damaged mitochondria were counted in the hippocam-
pal neurons in the control and ME7-infected brains and then calcu-
lated based on fifteen arbitrarily selected hippocampal neurons.
The sizes of the mitochondria were measured under high magnifi-
cation (50,000) using TEM (iTEM, Olympus Soft Imaging
Solutions, GmbH), and the values were calculated as the
means ± SDs of the lengths and widths of thirty arbitrarily selected
mitochondria from each group. Statistical analyses of the numbers
and sizes of the mitochondria were performed using Jandel
SigmaStat software (V 3.5).
2.5. Statistical analyses
The compared values were calculated as the mean ± SD of three
brains from each group, and statistical significance was deter-
mined using Student’s t-tests.
3. Results
3.1. Imbalanced expression of mitochondrial fusion and fission
proteins in infected brains
First, we identified deposits of PK-resistant PrPSc in ME7-infected
brains in the end stage; the normal PrPC proteins were completely
degraded by the PK treatment, as shown in Fig. 1. To determine
whether the mitochondrial fusion and fission proteins are affected
by prion infection, their expression patterns were investigated in
whole brains of the control and ME7-infected mice at the end stage
of disease (160 dpi) (Fig. 2). Of the mitochondrial fusion and fission
proteins (fusion proteins: Opa1, Mfn1 and Mfn2; fission proteins:
Dlp1 and Fis1), Western blot analysis revealed that only Mfn1 was
differentially expressed in the infected whole brains compared to
the age-matched control brains (Fig. 2A and B). It has previously been
reported that the hippocampal regions are more severely damaged
than any other regions in brains infected with the ME7 scrapie strain
[18]. Thus, to compare the expression levels of the mitochondrial
fusion and fission proteins in various brain regions, we dissected
both the control and ME7-infected brains into the following regions:
cerebral cortex, hippocampus, cerebellum, striatum and brainstem.
We found that the levels of Mfn1 and Fis1 were significantly
 H.-S. Choi et al. / Biochemical and Biophysical Research Communications 448 (2014) 157–162 159

3.2. Analysis of fission proteins in the hippocampal cytosol of infected

brain

To examine the localization of the mitochondrial fusion and fis-

Fig. 1. Deposition of PK-resistant prion isoforms in ME7-infected mice. Whole
brains of control (n = 3) and infected mice (n = 3) in the end stage (160 dpi) were
homogenized and blotted with anti-PrP antibody (3F10). A portion of each sample
was treated with PK (PK 50 lg/ml). b-Actin was used as a loading control.
increased in the hippocampi of the infected brains, whereas the Dlp1
levels were significantly decreased at the end stage (Fig. 2C and D).
Additionally, the levels of Mfn1 and Mfn2 were increased in the cere-
bral cortex and striatum, respectively, of infected brains (data not
shown). In the cerebellum and brain stem, none of the five mitochon-
drial fusion and fission proteins was significantly differentially
expressed between the control and infected mice (data not shown).
These results indicate that the differential regulation of mitochon-
drial fusion and fission proteins may be involved in the mitochon-
drial dysfunction of the ME7-infected brains, particularly in the
hippocampus [16].
sion proteins in the organelles and the expression levels of these
proteins in the cytosol and mitochondria, mitochondrial and cyto-
solic fractions were isolated from the hippocampi of control and
ME7-infected mice; again the hippocampus was chosen because
this region is known to be severely damaged in ME7-infected
brains [18]. Immunoblot analyses revealed that Dlp1 levels in the
mitochondrial fractions from the hippocampi were not altered by
ME7 infection (Fig. 3A and B) but were significantly decreased in
the cytosolic fractions of the infected hippocampi; this finding is
consistent with the decrease in Dlp1 levels in the affected hippo-
campi that is illustrated in Fig. 2C and D. The concentration of
Opa1, which is primarily localized to the inner membrane of the
mitochondria, was not altered in the mitochondria or cytosol iso-
lated from the hippocampi of the infected brains (Fig. 3C and D).
3.3. Morphological changes in the mitochondria in the hippocampal
region of infected brain
The modulation of mitochondrial dynamics due to fusion and
fission protein concentration probably contributed to alterations
in mitochondrial shape [9]. Electron microscopic analyses were
performed to assess the relationship between mitochondrial fusion
and fission proteins and mitochondrial shapes in the hippocampal

Fig. 2. Differential expression of mitochondrial fusion and fission proteins in hippocampi of ME7-infected mice. The mitochondrial dynamic proteins of the whole brains and
hippocampal regions of the control (n = 3) and infected mice (n = 3) in the end stage of disease were blotted. b-Actin was used as a loading control for A and C. The intensities
of the bands in panels A and C were measured and quantified (B and D). The values are expressed as the mean ± SD (n = 3). CON: control (white bars); ME7: ME7-infected
(black bars). Statistically significant differences are indicated (⁄p < 0.05 and ⁄⁄p < 0.01).
160 H.-S. Choi et al. / Biochemical and Biophysical Research Communications 448 (2014) 157–162

Fig. 3. Subcellular localization of mitochondrial fusion and fission proteins in hippocampi of ME7-infected mice. Opa1 (fusion) and Dlp1 (fission) from the mitochondria (A)
and cytosolic (C) fractions of the hippocampi of the control (n = 3) and ME7-infected mice (n = 3) at the end stage of the disease were blotted (160 dpi). b-Actin was used as a
loading control. The intensities of the bands in panels A and C were measured and quantified (B and D). The values are expressed as the mean ± SD (n = 3). CON: control (white
bars); ME7: ME7-infected (black bars). Note that both of the Opa1 bands are primarily localized to the mitochondria of hippocampi in both the control and infected
specimens, whereas substantial amounts of Dlp1 were localized to the cytosol. Statistically significant differences are indicated (⁄p < 0.05).

region. As shown in Fig. 4, the mitochondria in the normal hippo-
campal neurons were present in a variety of shapes, including rod,
round and elliptical shapes. The cristae were normal in appearance.
In contrast, enlarged mitochondria with damaged cristae were
clearly observed in many of the hippocampal neurons in infected
brains (Fig. 4A). The numbers of total neuronal mitochondria were
significantly decreased in the hippocampi of the infected mice
compared to those of controls (Fig. 4B). Additionally, the lengths
and widths of the mitochondria in the hippocampal neurons of
infected brains were significantly increased compared to those of
the controls, which is consistent with the observation of enlarged
hippocampal mitochondria visualized in Fig. 4B. The morphologi-
cal changes seen in mitochondria may be involved in the mito-
chondrial dysfunction in the hippocampi of ME7-infected mice.
4. Discussion
In this study, we demonstrated for the first time that the
expression patterns of mitochondrial fusion and fission proteins
were altered in an experimental mouse model of prion disease;
i.e., ME7 scrapie-infected mice. Of the mitochondrial fusion and fis-
sion proteins examined, the levels of Mfn1 were found to be signif-
icantly increased in whole brains of the ME7-infected mice. Within
the different dissected regions of the ME7-infected brains, we
found differences in the expression levels of various mitochondrial
fusion and fission proteins compared to controls. Particularly nota-
ble were the alterations in the expression of Mfn1 in the cerebral
cortex; Mfn1, Dlp1 and Fis1 in the hippocampus; and Mfn2 in
the striatum.
A recent study suggested that most neurodegenerative diseases
may be associated with the dysregulation of mitochondrial fusion
and fission proteins [19]. Mitochondrial fusion and fission are reg-
ulated by large dynamin-related GTPases. Mitochondrial fusion,
which is regulated by three large GTPases (Mfn1, Mfn2 and
Opa1), involves the coordinated fusion of both the outer and inner
mitochondrial membranes [20]. The overexpression of mitofusins
causes the normally punctate mitochondria to become elongated,
whereas the repression of mitofusins causes the fragmentation of
the mitochondrial network in tissue culture cells [21]. Interest-
ingly, we found increased level of Mfn1, which is a transmembrane
protein that localizes to the outer membranes of mitochondria, in
the homogenates of whole brain, hippocampus and cerebral cortex
of the infected brain. In addition the expression level of Mfn2 was
increased in the striatum of the infected brain (Data not shown).
Although electron microscopy analyses did not reveal the detailed
membrane structures of the mitochondria in this study, initial
outer membrane fusion may have occurred in the brains of the
infected mice due to increases in the Mfn1 and Mfn2 proteins.
Moreover, although there may be distinct pathways or mecha-
nisms for Mfn1 and Mfn2, a functional interplay between the
two proteins may be involved in the control of mitochondrial
fusion [22].
Different strains of mouse-adapted scrapie have been reported
to preferentially target different specific brain regions; as noted
previously, the ME7 strain is known to be particularly associated
 H.-S. Choi et al. / Biochemical and Biophysical Research Communications 448 (2014) 157–162 161

Fig. 4. Electron microscopic analysis of the mitochondria in hippocampal neurons of ME7-infected mice. (A) Control mitochondria were elliptical in shape and exhibited
compact cristae, whereas the infected mitochondria were enlarged and exhibited partially swollen cristae. Scale bar, 1 lm. (B) The total number of mitochondria in the
hippocampal neurons of the control and ME7-infected mice (the total number of mitochondria were counted from 15 neuronal cells from each group). (C) The lengths (white
bar) and widths (black bar) of the mitochondria in the hippocampal neurons of the control and ME7-infected mice (the lengths and widths of 30 mitochondria in each group
were measured). Statistically significant differences are indicated (⁄p < 0.01). CON: control; ME7: ME7-infected.

with hippocampal damage [23]; therefore, we focused on the hip-
pocampal region of the ME7-infected brains. Our study demon-
strated that ME7 infection led to significant increases in the
levels of Mfn1 and Fis1 in the hippocampi of the ME7-infected
brains, whereas Dlp1 was significantly decreased. Dlp1 localizes
in several organelles, including the endoplasmic reticulum, micro-
tubules and peroxisomes, but primarily resides in the mitochon-
dria and cytosol [24]. We isolated the cytosolic fraction of the
hippocampal region and investigated the changes in the expression
of mitochondrial fusion and fission proteins in both the purified
mitochondria fraction and the cytosol. Dlp1 levels were decreased
in cytosolic fractions but not in mitochondria fractions. These
observations imply that imbalances in mitochondrial dynamics
may contribute to the enlargement and the degeneration of mito-
chondria that occurs in the hippocampi of scrapie-infected mice.
Changes in the shape of the mitochondria have been observed
in ME7 scrapie-infected brain even in the preclinical stage of infec-
tion [25]. The mitochondrial enlargement demonstrated during the
end stage of prion disease in this study was also observed in a pre-
vious study that demonstrated structural abnormalities of the
mitochondria in the neurons of the hippocampi and cerebral corti-
ces of brains from scrapie-infected hamsters [15]. Fragmentation
and clustering of mitochondria in the soma have been observed
in the brains of patients with sporadic AD, and alterations in the
expressions of several fusion and fission proteins have been
observed in these brains [26]. Thus, we sought to determine the
relationship between expression levels of the mitochondrial fusion
and fission proteins and the alterations in the shapes, numbers and
sizes of the mitochondria in a prion disease model. In the ME7
mouse model, we found that the total number of neuronal mito-
chondria was reduced significantly and that a number of enlarged
and degenerated mitochondria appeared. These mitochondria had
damaged cristae and matrix. The relationship between the mito-
chondrial damage and the induction of neurodegeneration and dis-
ease is uncertain. Clearly the fact that the number of mitochondria
was reduced combined with degeneration of the remaining mito-
chondria would lead to a loss of neuronal function. It needs to be
noted that the loss of mitochondria could affect the quantity of
proteins observed; this is particularly important in instances
where the level of protein decreased (e.g., Dlp1 in the cytosol). Fur-
thermore, in those instances where there was no change in protein
levels (e.g., Opa1), the reduced number of mitochondria could
mask an increase.
Growing evidence suggests that the delicate balance between
mitochondrial fission and fusion is critical for mitochondrial func-
tions, including energy production, Ca2+ signaling, reactive oxygen
species (ROS) production, apoptosis and senescence [7,27]. Dysreg-
ulation of the mitochondrial fusion and fission proteins in neurons
may be a common pathway leading to the mitochondrial and neu-
ronal dysfunctions that are critical in the pathogenesis of prion dis-
ease. It remains unclear, however, how the alterations in
mitochondrial fusion and fission proteins that were observed in
162 H.-S. Choi et al. / Biochemical and Biophysical Research Communications 448 (2014) 157–162
this study contribute to neurodegeneration in prion diseases.
Taken together, the findings of this study suggest that the disrup-
tion of the balance of mitochondrial fusion and fission proteins,
which may be a common feature of prion diseases, is involved in
the mitochondrial and neuronal dysfunction in the brains of
ME7-infected mice.
Acknowledgments
This research was supported by Hallym University Research
Fund, 2012 (HRF-201210-000), the National Research Foundation
of Korea Grant funded by the Korean Government (NRF-2011-
619-E0001) and by the framework of international cooperation
program managed by the National Research Foundation of Korea
(NRF-2011-616-H00002).
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DOI 10.3233/JAD-150283
IOS Press

Upregulation of Connexin 43 Expression

Via C-Jun N-Terminal Kinase Signaling
in Prion Disease
Geon-Hwi Leea,b,1 , Byungki Janga,1 , Hong-Seok Choia,c,1 , Hee-Jun Kima , Jeong-Ho Parka,c ,
Yong-Chul Jeona , Richard I. Carpd , Yong-Sun Kima,c and Eun-Kyoung Choia,b,∗
a IlsongInstitute of Life Science, Hallym University, Anyang, Gyeonggi-do, Republic of Korea
b Department of Biomedical Gerontology, Graduate School of Hallym University, Chuncheon, Gangwon-do,
Republic of Korea
c Department of Microbiology, College of Medicine, Hallym University, Chuncheon, Gangwon-do, Republic of Korea
d New York State Institute for Basic Research in Developmental Disabilities, Staten Island, NY, USA

Accepted 24 September 2015

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 PrPSc accumulation reduced Cx43 expression
through the inhibition of JNK signaling, indicating that PrPSc 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 Cx43 is the most ubiquitous; it is abundantly expressed

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,
1 These authors contributed equally to this work.
∗ Correspondence
to: Eun-Kyoung Choi, Ilsong Institute of Life
Science and Department of Biomedical Gerontology, Hallym Uni-
versity, 15 Gwanpyeong-ro, 170beon-gil, Dongan-gu, Anyang,
Gyeonggi-do, 431-815, Republic of Korea. Tel.: +82 31 380 1893;
Fax: +82 31 388 3427; E-mail: ekchoi@hallym.ac.kr.
in the brain and highly expressed during embryonic
development [2, 3]. In the central nervous system
(CNS), Cx43 is widely expressed in neurons, astro-
cytes, and oligodendrocytes [4–6], and various signal
molecules, including ATP, glutamate, and Ca2+ 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 protec-
tive role against oxidative stress-induced cell death
[16, 17]; deletion or blockade of this protein prevents
chronic neuropathic pain following spinal cord injury

ISSN 1387-2877/16/$35.00 © 2016 – IOS Press and the authors. All rights reserved
1006 G.-H. Lee et al. / Scrapie Infection-Mediated Cx43 Upregulation
[18, 19] and fetal ischemia [20]. The deletion of Cx43
or expression of a truncated form increases the vulner-
ability 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 rep-
resent 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 neuromyeli-
tis optica [36]. Therefore, Cx43 is associated with the
pathophysiology of various tissue conditions. How-
ever, 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 neurodegenera-
tive diseases. A mutation and an abnormal structure
of the prion protein (PrPC ) are associated with a
protease-resistant and infectious form (PrPSc ), which is
considered a causative factor for prion diseases. Neu-
ropathologically, 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 cel-
lular 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) sig-
naling 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 origi-
nal 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 (Neuropathogene-
sis Unit, Edinburgh, UK). For scrapie infection, the
mice were intracerebrally inoculated with 30 ␮l of
1% w/v brain homogenates of the ME7, 22L, and
139A inocula (for mice) or 50 ␮l of 1% w/v ham-
ster 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 ␮l 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 dis-
ease. All experiments were performed in accordance
with Korean laws and with the approval of the Hal-
lym 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 Na3 VO4 )
with a protease inhibitor cocktail tablet (Roche, Indi-
anapolis, 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 Na3 VO4 ; 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 solubi-
lized 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 deoxy-
cholate, 0.1% sodium dodecyl sulfate (SDS), 1%
NP-40, 10 mM NaF, 1 mM Na3 VO4 , 1 mM EDTA,
 G.-H. Lee et al. / Scrapie Infection-Mediated Cx43 Upregulation
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-␤-actin (1:10,000; Sigma-
Aldrich, Saint Louis, MO, USA) antibodies were used
with the appropriate secondary antibodies conjugated
to horseradish peroxidase. To detect PrPSc , 50 ␮g/ml
or 20 ␮g/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 dig-
ital images captured with an ImageQuant™ LAS 4000
imager (GE Healthcare Life Sciences, Piscataway, NJ,
USA) using an enhanced chemiluminescence western
blot detection system (Amersham Biosciences, Piscat-
away, 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 knock-
out) 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 ␮g/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 PrPSc for over 50 passages. For immuno-
cytochemistry, 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.
1007
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% Tri-
ton X-100; 2 mM PMSF) containing PK (5 ␮g/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 sam-
ples using TRI reagent (Sigma-Aldrich) according to
the manufacturer’s protocols. cDNA was synthesized
using the Moloney murine leukemia virus reverse tran-
scriptase (Promega, Madison, WI, USA). The primer
sequences were as follows: connexin43 (127 bp),
sense, 5 -CTGAGTGCGGTCTACACCTG-3 , anti-
sense, 5 -GAGCGAGAGACACCAAGGAC-3 ; ␤-
actin (196 bp), sense, 5 -TGTGATGGACTCCGGT
GACGG-3 , antisense, 5 -ACAGCTTCTCTTTGATG
TCACGC-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 Laborato-
ries). The sections were mounted in Permount
(Thermo Fisher Scientific, Inc., Waltham, MA, USA).
The control sections were stained without primary
antibody.
1008 G.-H. Lee et al. / Scrapie Infection-Mediated Cx43 Upregulation
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 Technol-
ogy) 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 antibod-
ies (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 incu-
bated with 5 ␮M EtBr in PBS in the presence or
absence of a connexin hemichannel blocker LaCl3
(500 ␮M) for 5 min at 37◦ C. For JNK inhibition, the
cells were pretreated with the JNK inhibitor SP600125
(50 ␮M) for 6 h and then incubated with 5 ␮M 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 tempera-
ture 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 per-
formed 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 ␮M) for 5 min at 37◦ C. For JNK inhibition, the
cells were pretreated with a JNK inhibitor SP600125
(50 ␮M) 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 dif-
fusion after scrape loading were measured in at least
twenty random areas from each sample, and the fluo-
rescence 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 ␮M) for 3 days and then lysed. The
expression levels of PrPSc , JNK, phospho-JNK, and
Cx43 were determined by western blotting as previ-
ously described.
Statistical analysis
The data are expressed as the mean ± SEM. Sig-
nificant 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 incuba-
tion 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 pro-
gression, 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
 G.-H. Lee et al. / Scrapie Infection-Mediated Cx43 Upregulation 1009

Fig. 1. Increased expression of Cx43 protein and mRNA in the brains of scrapie-infected mice. A) Cx43 protein expression in whole brain
and in dissected brains of the control and ME7 (160 dpi), 22 L (140 dpi), and 139A (170 dpi) scrapie-infected mice was evaluated by western
blot with anti-Cx43 antibody. ␤-actin was used as a loading control. B) The intensities of the Cx43 bands in panel A were measured and
quantified. The values are expressed as the mean ± SEM (n = 3). C) In whole brains, the Cx43 mRNA levels were analyzed by RT-PCR with
three individuals per group. The error bars represent the SEM. D) Cx43 protein expression was analyzed in the whole brains of the control and
scrapie-infected mice by western blot at the indicated time points after inoculation. ␤-actin was used as a loading control, and PrPSc was detected
using anti-PrP antibody after PK (50 ␮g/ml) treatment. Each experiment was repeated at least three times, and similar results were obtained.
∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001.

between the control and scrapie-infected brains was increase relative to the stage of scrapie development
identified at 50 dpi in ME7 mice or in 263K hamsters and was significantly increased at 90 dpi (for ham-
at 30 dpi. However, Cx43 protein expression tended to sters) and 150 dpi (for mice). These time points were
1010 G.-H. Lee et al. / Scrapie Infection-Mediated Cx43 Upregulation

Fig. 2. Immunohistochemical staining of Cx43 in brain sections from control and scrapie-infected mice. Cx43 protein was detected in various
brain sections obtained from the control and ME7, 22 L and 139A scrapie-infected mice at 150 days post-inoculation. Arrows, Cx43-positive
cell-cell cotact or blood vessels. Scale bar, 25 ␮m.

correlated with marked accumulations of PrPSc , indi-
cating that Cx43 protein increased at the end stage of
scrapie infection.
To confirm the increase in Cx43 after scrapie
infection, we subsequently performed immunohisto-
chemical staining of Cx43 in various brain regions
of the control and scrapie-infected mice. Cx43 was
prominent in most brain regions, including the cere-
bral cortex, hippocampus, striatum, cerebellum, and
brain stem, at the end stage of scrapie infection com-
pared 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 pro-
tein, we subsequently examined whether the increased
Cx43 is related to membrane localization using cytoso-
lic 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
 G.-H. Lee et al. / Scrapie Infection-Mediated Cx43 Upregulation
Fig. 3. Increased Cx43 protein in membrane fractions of scrapie-
infected brains. Whole brains were lysed, separated into cytosol
and membrane fractions, and subjected to western blot analysis
as described in the Materials and Methods. Each fraction of the
brain homogenates was evaluated using anti-calnexin as a membrane
marker and anti-enolase as a cytosolic marker.
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 chan-
nels 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 demon-
strated 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 sepa-
rated from the nucleus (blue). Interestingly, Cx43 was
clearly detected with more aggregates between neigh-
boring scrapie-infected ZW13-2 cells but not between
Zpl2-4 cells or uninfected cells. Uptake of infection
was confirmed by the PK-resistant PrPSc levels in
cultures after multiple passages (Supplementary Fig-
ure 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.
1011
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 reg-
ulate 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 detec-
tion of PK-resistant PrPSc (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 an
ATP-competitive inhibitor of JNK, SP600125
(0–100 ␮M), for 6 h. As shown in Fig. 5B and C,
increased Cx43 and P-JNK/P-c-Jun by scrapie infec-
tion 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 ␮M;
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 com-
munication (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
1012 G.-H. Lee et al. / Scrapie Infection-Mediated Cx43 Upregulation

Fig. 4. Subcellular distribution of Cx43 and PrP in hippocampal cell lines with or without scrapie infection. Double immunofluorescence
staining was carried using anti-Cx43 (red) and anti-PrP (green) in the PrP knockout (Zpl2-4) and wild-type (ZW13-2) hippocampal neuronal
cell lines with or without the 22 L or 139A scrapie infection. DAPI was used for nuclear staining. Note that Cx43 was mainly localized in plaque
formations at the cell-cell contact areas (merged and bright-field images). Arrows indicate parts of the gap junctions. Scale bars, 20 ␮m.

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 main-
tained basal levels of GJIC in control and infected cells
(Fig. 7A ottom panels) without a significant differ-
ence between control and infected cells. The distances
of LY diffusion were measured and compared with
those in the controls and infected cells with or with-
out treatments (Fig. 7B). These results indicate that
the upregulation of Cx43 expression by scrapie infec-
tion represents the efficient communication capacity
of Cx43 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 PrPSc accumulation, we conducted a
blocking experiment of PrPSc accumulation using
BBG, which has anti-prion activity through the
inhibition of PrPSc accumulation [50]. Un-infected
and 22L scrapie-infected ZW13-2 neuronal cells
were incubated with various concentrations of BBG
(0–60 ␮M) for 3 days, and the expression lev-
els of Cx43, PrPSc , and p-JNK were subsequently
measured. As shown in Fig. 8 BBG treatment
efficiently inhibited PrPSc accumulation in a dose-
dependent manner. In correlation with the decrease
in PrPSc , 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
 G.-H. Lee et al. / Scrapie Infection-Mediated Cx43 Upregulation 1013

Fig. 5. JNK activation-mediated Cx43 upregulation in scrapie-infected cells and mice. A) Increased Cx43 expression and JNK activation in
control and scrapie-infected ZW13-2 cells (left panels) and mouse brains (right panels). To detect PrPSc , cell lysates and brain homogenates
were treated for 30 min at 37◦ C with PK at 50 ␮g/ml or 20 ␮g/ml, respectively. B, C) JNK inhibition attenuates Cx43 expression in a dose-
(B) and time-dependent (C) manner. Control, 22L-, and 139A-infected ZW13-2 cells were treated with 0, 25, 50, and 100 ␮M of the JNK
inhibitor SP600125 for 6 h (B) or with 50 ␮M SP600125 for the time indicated (C). Each protein was analyzed by western blot with the indicated
antibodies.

BBG. These results suggest that PrPSc accumulation
may be responsible for JNK signaling-mediated Cx43
upregulation.
DISCUSSION
In the present study, we demonstrated that the upreg-
ulation of Cx43 expression, which controls functional
properties of hemichannels, is mediated by JNK acti-
vation and correlated with PrPSc 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, 13, 15, 18, 20, 33,
35, 51]. Several signaling molecules are linked to brain
inflammation and unwarranted cell death. It is conceiv-
able that the excessive diffusion of neurotransmitters
or ions, such as glutamate, ATP, and Ca2+ , from dam-
aged cells to adjacent normal cells via Cx43-promoted
gap junctions may accelerate this pathophysiological
process [8–10]. Astrocytes and microglia release gluta-
mate 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 hypertro-
phy and proliferation, involves changes in the cellular
phenotype and in the expression of transporters, recep-
tors 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
1014 G.-H. Lee et al. / Scrapie Infection-Mediated Cx43 Upregulation

Fig. 6. Increased EtBr uptake in scrapie-infected cells. An EtBr uptake assay was performed using control or scrapie-infected ZW13-2 cells.
Cells were pretreated with 500 ␮M LaCl3 for 5 min (middle panels) or 50 ␮M of the JNK inhibitor SP600125 for 6 h (bottom panels) and then
incubated with 5 ␮M EtBr for 5 min at 37◦ C; the fluorescent signal was then analyzed using a confocal microscope.

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, 60, 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-1␣, IL-1␤, and
TNF-␣, in the brains of scrapie-infected mice can
lead astrocytes to become reactive [62]. These astro-
cytes 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 infec-
 G.-H. Lee et al. / Scrapie Infection-Mediated Cx43 Upregulation 1015

Fig. 7. Increased scrape-loading dye transfer in scrapie-infected cells. Control or scrapie-infected ZW13-2 cells were incubated with 0.1%
Lucifer yellow (LY) for 5 min at 37◦ C (top panels) or with 500 ␮M LaCl3 treatment (middle panels). For JNK inhibition, the cells were
pretreated with the JNK inhibitor SP600125 (50 ␮M) for 6 h prior to LY treatment (bottom panels). LY fluorescence was determined using a
confocal microscope (A) and mean length of LY-filled area (the distance from the center to the edge, n = 20) was represented by bars (B). The
arrow indicates scrape; lines drawn in upper panels indicate the distance for dye transfer. ∗∗∗ p < 0.001. Scale bar, 50 ␮m.
1016 G.-H. Lee et al. / Scrapie Infection-Mediated Cx43 Upregulation
Fig. 8. Attenuation of JNK signaling-mediated Cx43 upregula-
tion by inhibiting the accumulation of PrPSc . Control and 22 L
scrapie-infected ZW13-2 cells were treated with various concen-
trations of Brilliant blue G (BBG; 0-60 ␮M) for 3 days, and
the expression levels of Cx43, phosphorylated JNK, and PrPSc
were analyzed by western blot with the indicated antibodies.
To detect PrPSc , the total cell lysates were treated with 50 ␮g/ml
of PK for 30 min at 37◦ C. ␤-actin was used as a loading
control.
tion (Fig. 4). Although increases in Cx43 and gap
junctional communication and their effects on neu-
ronal death and inflammatory responses have been
linked to chronic and progressive neurodegenerative
diseases such as stroke, Alzheimer’s disease, Parkin-
son’s disease, and Huntington’s disease [57], it remains
a matter of debate whether Cx43 is neuroprotective or
deleterious.
Accumulating evidence has indicated that connexins
act as phosphoproteins via a shift in their elec-
trophoretic mobility or direct incorporation of 32 P [63].
The JNK signaling cascade is an important intracellu-
lar 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 phos-
phorylation status of Cx43. Because Cx43 can be
phosphorylated by various kinases, the activation of a
specific kinase may not correlate with Cx43 phospho-
rylation 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 phos-
phorylation state of Cx43 [17]. In addition, the opening
of hemichannels formed by pannexin 1, the mam-
malian ortholog of the invertebrate gap junction protein
innexin, has also been implicated in neuronal death
after ischemia [64]. Moreover, during the astroglio-
sis 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 remains
unresolved.
In addition, previous reports have demonstrated that
JNK signaling activation is involved in the pathogen-
esis 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 tran-
scriptional factors, such as c-Jun, c-fos, and SP1 [67].
We also demonstrated that the activated JNK/c-Jun cas-
cade is responsible for Cx43 expression in both in vitro
and in vivo models of prion disease. Surprisingly, the
accumulation of pathogenic PrPSc 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 infec-
tion increased Cx43 aggregates at the cell-cell contact
areas. Therefore, PrPSc accumulation may be respon-
sible for the upregulation of Cx43 expression and gap
junction formation.
Overall, for the first time, these results demon-
strated that scrapie infection activates JNK signaling
cascades to enhance Cx43 and gap junctions in neu-
ronal cells and in the brain. Pathogenic PrPSc (or
scrapie)-mediated upregulation of Cx43 through JNK
activation may exacerbate prion pathology, includ-
ing neuronal death. Although blocking gap junctions,
which inhibit the diffusion of neurotoxic molecules,
has been proposed as a candidate therapy for var-
ious neurodegenerative diseases, it can also limit
essential physiological signals. Thus, this strategy has
both neurotoxic and neuroprotective effects on dis-
ease 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 hemichan-
nels) expressed by neurons, astrocytes, and microglia
in the region of prion propagation might represent a
novel strategy to reduce disease phenotypes in prion
diseases.
ACKNOWLEDGMENTS
We thank Dr. Joy J. Goto (California State Uni-
versity, Fresno, USA) for critical reading of this
paper and helpful discussions. This work was sup-
ported 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).
 G.-H. Lee et al. / Scrapie Infection-Mediated Cx43 Upregulation
Authors’ disclosures available online (http://j-alz.
com/manuscript-disclosures/15-0283r1).
SUPPLEMENTARY MATERIAL
The supplementary material is available in the elec-
tronic version of this article: http://dx.doi.org/10.3233/
JAD-150283.
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