Enhanced accumulation of Kir4.1 protein, but not mRNA, in a murine model of cuprizone-induced demyelination

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0006-8993/$ - see frohttp://dx.doi.org/10

Abbreviations: A

GFAP, glial fibrilla

OPC, oligodendroc

NMO, neuromyelitnCorresponding autE-mail address: m

Please cite this acuprizone-induce

Research Report

Enhanced accumulation of Kir4.1 protein,but not mRNA, in a murine modelof cuprizone-induced demyelination

Mitsunari Nakajimaa,n, Takuya Kawamuraa, Ryuji Tokuia, Kohei Furutaa,Mami Suginoa, Masayuki Nakanishib, Satoshi Okuyamaa,Yoshiko Furukawaa

aDepartment of Pharmaceutical Pharmacology, School of Clinical Pharmacy, College of Pharmaceutical Sciences,Matsuyama University, 4-2 Bunkyo-cho, Matsuyama 790-8578, Ehime, JapanbDepartment of Biochemistry, School of Clinical Pharmacy, College of Pharmaceutical Sciences, Matsuyama University,4-2 Bunkyo-cho, Matsuyama 790-8578, Ehime, Japan

a r t i c l e i n f o

Article history:

Accepted 18 September 2013

Two channel proteins, inwardly rectifying potassium channel 4.1 (Kir4.1) and water channel

aquaporin-4 (AQP4), were recently identified as targets of an autoantibody response in

Keywords:

Multiple sclerosis

Neuromyelitis optica

Aquaporin-4

Astroglia

Autoantibody

nt matter & 2013 Elsevie.1016/j.brainres.2013.09.02

PP, Alzheimer's precurs

ry acidic protein; Iba-1,

yte precursor cells; Kir4.

is optica; PBS(� ), magnhor. Fax: þ81 89 926 [email protected]

rticle as: Nakajima, M.,d demyelination. Brain

a b s t r a c t

patients with multiple sclerosis and neuromyelitis optica, respectively. In the present study,

we examined the expression patterns of Kir4.1 and AQP4 in a mouse model of demyelination

induced by cuprizone, a copper chelator. Demyelination was confirmed by immunohisto-

chemistry using an anti-proteolipid protein antibody in various brain regions, including the

corpus callosum, of cuprizone-fed mice. Activation of microglial and astroglial cells was also

confirmed by immunohistochemistry, using an anti-ionized calcium binding adapter molecule

and a glial fibrillary acidic protein antibody. Western blot analysis revealed the induction of

Kir4.1 protein, but not AQP4, in the cortex of cuprizone-fed mice. Immunohistochemical

analysis confirmed the Kir4.1 protein induction in microvessels of the cerebral cortex. Real-

time polymerase chain reaction analysis revealed that mRNA levels of Kir4.1 and AQP4 in the

cortex did not change during cuprizone administration. These findings suggest that enhanced

accumulation of Kir4.1 protein in the brain with an inflammatory condition facilitates the

autoantibody formation against Kir4.1 in patients with multiple sclerosis.

& 2013 Elsevier B.V. All rights reserved.

r B.V. All rights reserved.4

or protein; AQP4, aquaporin-4; CC, corpus callosum; CNS, central nervous system;

ionized calcium binding adapter molecule; olig2, oligodendrocyte transcription factor 2;

1, inwardly rectifying potassium channel 4.1; MS, multiple sclerosis;

esium and calcium-free phosphate buffered saline; PLP, proteolipid protein.-u.ac.jp (M. Nakajima).

et al., Enhanced accumulation of Kir4.1 protein, but not mRNA, in a murine model ofResearch (2013), http://dx.doi.org/10.1016/j.brainres.2013.09.024

dx.doi.org/10.1016/j.brainres.2013.09.024



mailto:[email protected]





Fig. 1 – Changes in the body weight of cuprizone-fed mice.Body weight changes in male mice (A) and female mice(B). Mice were fed a diet containing 0.2% cuprizone from 3 to 8weeks of age, followed by a normal diet for 2 weeks.A reduction in body weight was observed at 4, 6, and 8 weeksof age in cuprizone-fed mice of both sexes. Mice with reducedbody weight recovered from the effect of cuprizone at 8–10weeks of age. The inter-group differences between the control(Con) and cuprizone treated (Cup) mice were significant at 4, 6,and 8 weeks of age in both sexes. *: po0.01.

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1. Introduction

In the last decade, two disease-specific autoantibodies wereidentified in the sera of patients with neuromyelitis optica(NMO) and multiple sclerosis (MS), which are chronic inflam-matory, demyelinating disorders of the central nervous sys-tem (CNS). The first autoantibody discovered in humans withNMO acts against the water channel aquaporin-4 (AQP4)(Lennon et al., 2004, 2005), which exerts pathogenic effectsin vivo (Bradl et al., 2009). Historically, NMO was classified asa subtype of MS, but the discovery of the autoantibodyagainst AQP4 now allows for discrimination between NMOand MS (Roemer et al., 2007). The second autoantibodydiscovered acts against the inwardly rectifying potassiumchannel 4.1 (Kir4.1). The Kir4.1 autoantibody also has patho-genic effects in vivo (Srivastava et al., 2012). The antigens ofthe two autoantibodies colocalize along the plasma mem-branes of astrocyte endfeet (Masaki et al., 2010), and couldmediate the coupling between water homeostasis and gluta-mate buffering actions in the CNS (Wen et al., 1999).

Mice fed the copper chelator cuprizone (bis-cyclohexaneoxaldihydrazone) develop reproducible demyelination in theCNS. This model is useful for investigating the underlyingpathologic processes of demyelinating disorders such as NMOand MS (Kipp et al., 2009; Gudi et al., 2009). Both demyelina-tion and remyelination mechanisms can be investigatedusing this animal model. Cuprizone feeding reduces themyelin sheath and activates microglial and astroglial cells.Expression of autoantigens such as AQP4 and Kir4.1, how-ever, has not been analyzed in this model.

In the present study, we induced demyelination in thebrains of mice by feeding them cuprizone, and investigatedthe demyelinating process by focusing on the expression ofAQP4 and Kir4.1.

2. Results

2.1. Cuprizone feeding led to reduced body weight,demyelination, and hydrocephalus

To assess the effect of cuprizone treatment on the myelinsheath, we used young male and female mice. Demyelinationwas induced by feeding 3-weeks old male and female mice adiet containing 0.2% cuprizone for 5 weeks. The administrationof cuprizone reduced the viability of male mice (control: 11/11;cuprizone treatment: 36/41), while female mice tolerated thecuprizone toxin (control: 15/15; cuprizone treatment: 31/31).Fig. 1 shows the change in body weight of mice duringcuprizone treatment and the post-treatment period. A reduc-tion in body weight was observed in both male and femalemice during cuprizone treatment and a quick recovery fol-lowed after 2 weeks. The body weight reduction was greater inmales than in females (compare A and B in Fig. 1).

Myelination was determined by immunohistochemistryfor proteolipid protein (PLP), a marker of mature oligoden-drocytes (Pott et al., 2009). Fig. 2 clearly demonstrates thereduction of the myelin sheath in almost all areas in thebrain of both male and female cuprizone-treated mice,

Please cite this article as: Nakajima, M., et al., Enhanced accumucuprizone-induced demyelination. Brain Research (2013), http://

although the degree of demyelination was low in someregions such as the brain stem, the medulla of the cerebel-lum, and the fimbria of the hippocampus. Hydrocephalus wasalso observed in cuprizone-treated mice, while we observedno apparent atrophic change in the cortex, hippocampus, orstriatum (Fig. 2 B and D). Dilation of the ventricles did notshow clear reduction in the post cuprizone-treatment period(data not shown).

Fig. 3 shows the quantitative change in the myelin sheath inthe corpus callosum (CC) and the cerebral cortex of cuprizone-fed mice. The reduction of PLP-immunoreactivity in the CC andthe cerebral cortex was confirmed in bothmale and female mice.During the recovery period, PLP-immunoreactivity returned tocontrol levels in males. The degree of recovery was lower infemales (Fig. 3 Cupþ2w).

The phenotypes of reduced viability and body weight losswere greater in males than females, while trouble in recoveryfrom demyelination was greater in females than males.

2.2. Glial response to cuprizone treatment

We examined the glial response to cuprizone treatment usingimmunohistochemistry for glial cell markers. Cuprizone feedingdid not affect the number of oligodendrocyte transcriptionfactor 2 (olig2)-positive oligodendrocyte precursor cells (OPC)in the CC during cuprizone-treatment period, although thenumber of OPC significantly increased in males, but not in

lation of Kir4.1 protein, but not mRNA, in a murine model ofdx.doi.org/10.1016/j.brainres.2013.09.024




Fig. 2 – Demyelination patterns in cuprizone-fed mice. Overview of sagittal brain sections stained immunohistochemicallywith an anti-PLP antibody. (A) and (C) are brain sections from 8-week-old control male and female mice, respectively. (B) and(D) are brain sections from cuprizone-treated male and female mice, respectively. Reduced expression of a matureoligodendrocyte marker (PLP) and enlarged ventricles were observed in mice fed cuprizone for 5 weeks. Almost all PLPdisappeared from the brains of mice treated with cuprizone, except for some regions such as the brain stem (*), the medulla ofcerebellum (thin arrows), and the fimbria of the hippocampus (arrows) in B and D. Scale bar¼1 mm.

Fig. 3 – Changes in PLP immunoreactivity in the brains during cuprizone treatment. (A) and (B) show the changes in PLPimmunoreactivity in the corpus callosum (CC) and cerebral cortex (Cx), respectively, of male control mice (Con), cuprizone-treated mice (Cup), and cuprizone-treated mice with a 2-week recovery period (Cupþ2w). (C) and (D) show the results forfemale mice. PLP immunoreactivity in the CC and Cx in 6 sections from 3 male or female mice were measured and areexpressed in each column. In the cuprizone-treated groups (Cup), PLP immunoreactivity was significantly reduced in the CCand Cx in both sexes. The reduced PLP immunoreactivity recovered from the effect of cuprizone at 8–10 weeks of age(Cupþ2w), and the recovery was more rapid in male mice. *: po0.05.

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females, during the recovery period (Fig. 4, Cupþ2w). Thisincrease in the OPC number in males was consistent with thelarge increase in the PLP-immunoreactivity in males duringthe recovery period (Fig. 3A and B, Cupþ2w).


The number of ionized calcium binding adapter molecule(Iba-1)-positive macrophages/microglial cells increased in theCC of both male and female mice during the cuprizonetreatment and during the recovery period (Fig. 5).





Fig. 4 – Changes in olig2-immunoreactivity in the brains during cuprizone treatment. (A) and (C) micrographs of sagittalsections stained with anti-olig2 antibody. (a) and (d) are from control mice (Con), (b) and (e) are from cuprizone-treated mice(Cup), (c) and (f) are from cuprizone-treated mice with a 2-week recovery period (Cupþ2w). (a)–(c) are from the corpus callosum(CC), (d)–(f) are from the cerebral cortex (Cx). (B) olig2-positive cells in the CC in 5-6 sections from 3 male mice were countedand are expressed in each column. (D) olig2-positive cells from female mice were counted and are expressed. Note theincrease of olig2-positive cells in the cuprizone-treated mice with a 2-week recovery period (Cupþ2w) in (B). *: po0.01. Scalebar in A and C¼50 μm.

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Cuprizone feeding increased glial fibrillary acidic protein(GFAP)-immunoreactivity in the CC (Fig. 6). The degree ofincreased GFAP-immunoreactivity was greater in females.

GFAP-immunoreactivity also increased in the cerebralcortex in response to cuprizone treatment, while immunor-eactivity for the olig2 OPC marker or the Iba-1 macrophage/microglia marker in the cerebral cortex did not change(Figs. 4–6A and C).

2.3. Levels of Kir4.1 protein were increased duringcuprizone treatment

Although glial responses to the cuprizone treatment wereclearly detected in the CC based on olig2, Iba-1, and GFAP(Figs. 4–6), an astroglial GFAP-response was also observed inthe cerebral cortex (Fig. 6). Astrocytes are closely related tocerebral blood vessels as well as neuronal synapses, andAQP4 and Kir4.1 proteins colocalize along the plasma mem-branes of astrocyte endfeet (Masaki et al., 2010; Iadecola andNedergaard, 2007). In fact, AQP4 and Kir4.1 proteins did notshow strong immunoreactivity for each antibody in the CC ofcontrol and cuprizone-treated brains (data not shown). Thus,we focused on the cerebral microvasculature for the analysisof AQP4 and Kir4.1 protein expression.

AQP4-immunoreactivity was detected along the microvesselsin the cerebral cortex of control mice. Cuprizone treatment did


not change the amount of AQP4-immunoreactivity in eithermale or female mice (Fig. 7A–F). Immunoreactivity to Kir4.1was light along the microvessels in the cerebral cortex of controlmice. Cuprizone treatment increased Kir4.1-immunoreactivityalong the microvessels in both male and female mice (Fig. 7G–L).Western blot analysis revealed increased levels of GFAP in thecerebral cortex during cuprizone treatment and during therecovery period. While cuprizone treatment did not affect thelevels of AQP4, it increased the levels of Kir4.1 in the cerebralcortex in both male and female mice (Fig. 8).

2.4. Levels of Kir4.1 mRNA were sustained duringcuprizone treatment

Real-time PCR analysis of the cerebral cortex revealedincreased levels of GFAP mRNA in the cuprizone-treated mice(Fig. 9). Kir4.1 mRNA expression and the AQP4 mRNA expres-sion did not change in either male or female mice (Fig. 9).

3. Discussion

The present study revealed that Kir4.1 protein levels wereincreased in the brains of cuprizone-fed mice. The channelprotein Kir4.1 is one of the most important targets of theautoantibody response in patients with MS (Srivastava et al.,





Fig. 5 – Changes in Iba-1-immunoreactivity in the brains during cuprizone treatment. (A) and (C) micrographs of sagittalsections stained with an anti-Iba-1 antibody. (a) and (d) are from control mice (Con), (b) and (e) are from cuprizone-treated mice(Cup), (c) and (f) are from cuprizone-treated mice with a 2-week recovery period (Cupþ2w). (a)–(c) are from the corpus callosum(CC), (d)–(f) are from the cerebral cortex (Cx). (B) Iba-1-positive cells in the CC in 6 sections from 3 male mice were counted andare expressed in each column. (D) Iba-1-positive cells from female mice were counted and are expressed. Note the increase ofIba-1-positive cells in the cuprizone-treated mice (Cup) and the cuprizone-treated mice with a 2-week recovery period(Cupþ2w) in (B) and (D). *: po0.01. Scale bar in A and C¼50 μm.

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2012). Therefore, the discovery of increased Kir4.1 levelsenhances the value of this cuprizone-treatment model. Thefinding that the Kir4.1 protein is targeted by the immunesystem is very interesting, but there is no sign of an adaptiveimmune response in the lesions observed in the cuprizonemodel (Acs and Kalman, 2012). The major contribution of themodel, therefore, is the finding that the Kir4.1 proteinaccumulates in inflammatory conditions. A chronic inflam-matory condition is one of the most prominent featuresobserved in the CNS of patients with MS (Gasperini et al.,2013).

Real-time PCR analysis revealed that the Kir4.1 proteinaccumulates without an increased transcription in thebrains of cuprizone-treated mice. mRNA levels of Kir4.1 didnot increase during cuprizone treatment, suggesting thatthe Kir4.1 protein increases via mechanisms other than anincreased transcription. A strong candidate for this mechanismis the failure of the Kir4.1 protein degradation system. Studiesof Kir4.1 protein degradation have not yet been performed. Theanalysis of protein degradation systems such as ubiquitin/proteasome or autophagy might elucidate the mechanisms ofKir4.1 protein accumulation and present a new target for MStherapies.

Hydrocephalus was first noted in the cuprizone model in1970 (Kesterson and Carlton, 1970), and the present findingsconfirmed this pathology (Fig. 2). An imbalance of AQP4 andKir4.1 might lead to a failure of water homeostasis, and could


be how hydrocephalus is induced in the cuprizone model.Blood–brain barrier permeability has been intensively inves-tigated in this model and it has been established that theblood–brain barrier remains intact (Sansom et al., 1973;Kondo et al., 1987; Bakker and Ludwin, 1987). AQP4 andKir4.1 colocalize along the microvessels in the brain (Fig. 7;Masaki et al., 2010) and mutually mediate water homeostasis(Wen et al., 1999). An increased accumulation of the Kir4.1protein might lead to a failure of water homeostasis in thebrains of cuprizone-fed mice.

Male mice exhibited more serious phenotypes than femalemice after cuprizone treatment, such as increased mortalityand decreased body weight (Fig. 1). The finding that malemice are more sensitive to the cuprizone toxin than femalemice is consistent with previous reports (Acs and Kalman,2012). In contrast, the pathologic damage in female mice suchas sustained reduced PLP-immunoreactivity and lower olig2-induction following cuprizone feeding were unexpected(Figs. 3 and 4). These findings contrast with a previous reportthat the patterns of demyelination and remyelination werecomparable between C57BL/6J male and female mice (Tayloret al., 2009). These inconsistencies between the two experi-mental results might be due to a strain differences: the micein the present study were C57BL/6J and 129 hybrids. Never-theless, the findings of the present study suggest that themurine model of cuprizone-induced demyelination could beuseful for investigating the pathogenesis of MS.





Fig. 6 – Changes in GFAP-immunoreactivity in the brains during cuprizone treatment. (A) and (C) micrographs of sagittalsections stained with anti-GFAP antibody. (a) and (d) are from control mice (Con), (b) and (e) are from cuprizone-treated mice(Cup), (c) and (f) are from cuprizone-treated mice with a 2-week recovery period (Cupþ2w). (a)–(c) are from the corpus callosum(CC), (d)–(f) are from the cerebral cortex (Cx). (B) GFAP-immunoreactivity in the CC in 6 sections from 3 male mice wasmeasured and is expressed in each column. (D) GFAP immunoreactivity from female mice was measured and is expressed.Note the increase in GFAP immunoreactivity in the cuprizone-treated mice (Cup) and the cuprizone-treated mice with a2-week recovery period (Cupþ2w) in (B) and (D). *: po0.01. Scale bar in A and C¼50 μm.

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While demyelination was observed during cuprizone treat-ment, the number of olig2-positive OPC was not clearly reducedin the CC during the treatment period, but rather increasedduring the recovery period (Figs. 2–4). These results might bedue to the apoptosis of mature myelin-consisting oligodendro-cytes in response to cuprizone toxin and the tolerance of olig2-positive OPCs to the toxin and their proliferation after stress, orto the dedifferentiation and proliferation of the remainingmature oligodendrocytes (Matsushima and Morell, 2001; Kippet al., 2009).

Whether autoantibodies against Kir4.1 are generated is aninteresting issue and should be examined in a future study.The possibility of anti-Kir4.1 antibody production in mice fedcuprizone must be carefully considered, however, becausethere is HLA-allele dependency in the pathogenesis of MS,and therefore possible MHC-allele dependency on the anti-body production in these mice.

4. Experimental procedures

4.1. Animals

Mice were maintained under a controlled temperature andphotoperiod (23 1C, 12-h light and 12-h dark) with ad libitumfood and water. All experimental procedures followedthe Guideline for Animal Experimentation prepared by theAnimal Care and Use Committee of Matsuyama University.


C57BL/6J and 129 hybrid mice were used. In the present study,we used totally 95 mice. Demyelination was induced byfeeding 3-week old male and female mice a diet containing0.2% cuprizone (bis-cyclohexanone oxaldihydrazone, Sigma-Aldrich #C9012, St. Louis, MO) mixed into ground standardrodent chow (CRF-1, Oriental yeast, Tokyo, Japan) for 5 weeks,followed by feeding a normal pellet diet for 2 weeks.

4.2. Immunohistochemistry

Mice were perfused with 4% paraformaldehyde in magne-sium and calcium-free phosphate buffered saline (PBS[� ]).Brains were post-fixed with the 4% paraformaldehyde solu-tion for 2 days, and rinsed with PBS(� ). After serial immer-sion in 10%, 20%, and 30% sucrose in PBS(� ), the brains wereembedded in OCT compound (Sakura Finetechnical, Tokyo,Japan) and sectioned at a thickness of 30-μm using a cryostat.Sections were stored at �80 1C until use. Sections werethawed and re-fixed with 4% paraformaldehyde solution for30 min. Endogenous peroxidase activity was blocked byincubation with 3% hydrogen peroxide in PBS(� ) for 5 min.Sections were incubated overnight with primary antibodies.Immunoreactivity was detected using the EnVisionþsystemHRP Rabbit (Dako/Japan #K4003, Tokyo, Japan) for rabbitantibodies or M.O.M. Kit (Vector, Burlingame, CA) for mouseantibodies, and diaminobenzidine. Primary antibodies used inthe immunohistochemistry were as follows: rabbit anti-Kir4.1





Fig. 7 – Immunohistochemistry of the cerebral cortex of cuprizone-fed mice with antibodies against AQP4 or Kir4.1. (A)–(F) aremicrographs of sagittal sections stained with an anti-AQP4 antibody. (G)–(L) are with an anti-Kir4.1 antibody. (A)–(C) and (G)–(I)are from male mice. (D)–(F) and (J)–(L) are from female mice. (A), (D), (G) and (J) are from control mice (Con); (B), (E), (H), and (K)are from cuprizone-treated mice (Cup); (C), (F), (I), and (L) are from cuprizone-treated mice with a 2-week recovery period(Cupþ2w). Note the clear capillary wall staining in sections immunostained with an anti-AQP4 or Kir4.1 antibody. Whilecuprizone treatment did not affect AQP4-immunoreactivity (A-F), it induced Kir4.1-immunoreactivity along the microvessels(H and K).

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(1/200, Alomone Labs #APC-035, Jerusalem, Israel), rabbit anti-AQP4 (1/10,000, Millipore #AB3594, Temecula, CA), rabbit anti-GFAP (1/10, Dako/Japan #N1506), rabbit anti-Iba-1 (1/250, Wako#019-19741, Osaka, Japan), mouse monoclonal anti-olig2 (1/100, Millipore #MABN50), and mouse monoclonal anti-PLP (1/200, Serotec #MCA839G, Toronto, Ontario, Canada) antibodies.The sections were immunostained for PLP, Kir4.1, or AQP4 bymounting them onto slide glasses after blocking the endo-genous peroxidase activity, followed by immersion in xylenes,rehydration in PBS(-), and incubation with primary antibodies.The immunohistochemistry procedures were validated usinga negative control in which the primary antibodies wereomitted. The densities of the anti-PLP and GFAP antibodystains were measured with Image J, an image processing andanalysis program, on photographed sections (Dr. WayneRasband, NIH, Bethesda, MD). For quantification of olig2 andIba-1 immunoreactivity, immunohistochemistry-positive cellswere manually counted. Two sections (0.038mm2/section) permouse brain were analyzed for each antibody. Data obtainedfrom 3 brains for each column are presented as the fold-change of control mean7SEM. In each experiment, data fromthe control brains were standardized. When multiple controlslides were stained in an experiment, mean values of thecontrol slides were used for standardization of the experi-mental data. Therefore, control data have error bars in somecases, and in other cases lack error bars.


4.3. Western blot analysis

The cerebral cortices of mice were homogenized with a glassDounce homogenizer in RIPA buffer containing 50 mMTris–HCl (pH 8.0), 150 mM NaCl, 0.1% sodium dodecyl sulfate,1% NP40, 0.5% NaDOC, and a complete protease inhibitorcocktail (Boehringer Mannheim GmbH, Mannheim, Ger-many), and centrifuged for 30 min at 20,000g. After sodiumdodecyl sulfate-polyacrylamide gel electrophoresis (12.5%polyacrylamide for GFAP and AQP4; 7.5% for Kir4.1), theextracted proteins in the supernatant (20 μg for GFAP; 60 μgfor AQP4 and Kir4.1) were transferred to a polyvinylidenedifluoride membrane (BioRad Laboratories, Hercules, CA). Theblots were probed with the following antibodies, which weredetected using the ECL Plus Western Blotting DetectionSystem (GE Healthcare UK Limited, Little Chalfont Buckin-ghamshire, UK). Blots probed with rabbit anti-GFAP antibody(1/100, Dako/Japan #N1506) were reprobed with horseradishperoxidase-conjugated anti-GAPDH antibody (1/70000, Sigma#G9295). Blots probed with rabbit anti-AQP4 antibody(1/2000, Santa Cruz Biotechnology #sc-20812, Dallas, TX) werereprobed with rabbit anti-actin antibody (1/1000, Sigma#A2066), and blots probed with rabbit anti-Kir4.1 antibody(1/2000, Alomone Labs) were reprobed with rabbit anti-Alz-heimer's precursor protein (APP) antibody (1/2000, Invitrogen#51-2700, Camarillo, CA). GAPDH, actin, and APP were used as





Fig. 8 – Western blots of proteins from the cerebral cortex of cuprizone-fed mice using antibodies against GFAP, AQP4, andKir4.1. Representative images of GFAP (A), AQP4 (B), and Kir4.1 (C) Western blots from the cerebral cortex of cuprizone-fedmale mice are presented. Densitometry was used to quantify the relative expression levels of GFAP (D, G), AQP4 (E, H), andKir4.1 (F, I). GAPDH, actin, and APP were re-probed and used as internal control proteins for GFAP, AQP4, and Kir4.1,respectively. D–F are data from male mice, and G–I are from female mice. Note the increased density of the Kir4.1 proteinband in the cuprizone-treated mice (Cup) and the cuprizone-treated mice with a 2-week recovery period (Cupþ2w) in (F) and (I).*: po0.05.

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internal control proteins. Data obtained from 3 to 4 brainsfor each column are presented as the fold-change of controlmean7SEM.

4.4. Real-time polymerase chain reaction (PCR)

The cerebral cortices from mice were dissected, rapidlyfrozen in liquid nitrogen, and stored until use at �80 1C.Total RNA was isolated from the frozen tissues usingNucleoSpin RNA II (TaKaRa #740955, Kyoto, Japan). TheRNA was treated with rDNase on columns followingthe manufacturer's protocol. cDNA was synthesized usingSuperScript III First-Strand Synthesis SuperMix (Invitrogen#11752) for quantitative real time-PCR according to the


manufacturer's protocol. Reactions for quantitative TaqManPCR contained TaqMan Gene Expression Master Mix,TaqMan primer/probe sets (Applied Biosystems, Foster City,CA) and cDNA. A two-step PCR was performed using Chromo 4real-time PCR analysis system with Opticon Monitor Version3.1 (BioRad). Denaturation was at 95 1C for 15 s followed byanealing and extension at 60 1C for 60 s for 40 cycles, 50 1C for2 min, and 95 1C for a 10-min pre-treatment. All primer/probesets were purchased from Applied Biosystems: GFAP,Mm01253033_m1; Kir4.1 (Kcnj10), Mm00445028_m1; AQP4,Mm00802131_m1; GAPDH, Mm99999915_g1. GAPDH was usedas an internal control gene. Data obtained from 3 to 4 brainsfor each column are presented as the fold-change of controlmean7SEM.





Fig. 9 – Gene expression changes of GFAP, AQP4 and Kir4.1 in the brains during cuprizone treatment. Total mRNA wasisolated from control mice (Con) and cuprizone-treated mice (Cup). cDNA was synthesized and used for real-time PCR. A–C aredata from male mice and D–F are from female mice. Note that there were no changes in gene expression of Kir4.1 (C, F) orAQP4 (B, E), and this is in contrast to changes in GFAP (A, D) in the cuprizone-treated mice (Cup). *: po0.05. Bar¼SEM.

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4.5. Statistical analysis

The Mann–Whitney U test was used to analyze body weightchanges. Steel's test was used to analyze intergroup differ-ences in the immunohistochemistry findings. Dunnett's testwas used to analyze the intergroup differences in the Westernblot results. Student's t-test was used to analyze the inter-group differences in the quantitative real-time PCR results.A p value of less than 0.05 was considered statisticallysignificant. All data are presented as mean7SEM.

Author contributions

Mitsunari N. and Y.F. designed the research; T.K., R.T., K.F., M.S.,Masayuki N., and S.O. performed the research; Mitsunari N. andY.F. wrote the paper.

Acknowledgments

This work was supported by JSPS KAKENHI Grant number25461567.


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Enhanced accumulation of Kir4.1 protein, but not mRNA, in a murine model of cuprizone-induced demyelination