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Research Report

Role of GFAP in morphological retention and distribution ofreactive astrocytes induced by scrapie encephalopathy in mice

Hiroshi Gomia,c,⁎, Takashi Yokoyamab, Shigeyoshi Itoharac

aLaboratory of Molecular Endocrinology and Metabolism, Institute for Molecular and Cellular Regulation, Gunma University,3-39-15 Showa-machi, Maebashi, Gunma 371-8512, JapanbPrion Disease Research Center, National Institute of Animal Health, 3-1-5 Kannondai, Tsukuba, Ibaraki 305-0856, JapancLaboratory for Behavioral Genetics, Brain Science Institute, Riken, 2-1 Hirosawa, Wako, Saitama 351-0198, Japan

A R T I C L E I N F O

⁎ Corresponding author. Fax: +81 27 220 8877.E-mail address: hgomi@showa.gunma-u.a

0006-8993/$ – see front matter © 2009 Elsevidoi:10.1016/j.brainres.2009.11.025

A B S T R A C T

Article history:Accepted 11 November 2009Available online 18 November 2009

We have previously demonstrated that mutant mice bearing astrocytes deficient in glialfibrillary acidic protein (GFAP) exhibited typical spongiform degeneration and prion plaquedeposition. However, it remains to be determined whether there are astrocyte-specificalterations in the reactive response of astrocytes. Herein,we analyzedmorphological featuresof Gfap−/− reactive astrocytes. Light microscopic morphometry of mutant reactive astrocytesrevealed reduced outlined cell area andshorter distances amongexpanded cell space butwithlarger nuclei. Electronmicroscopy revealedmutant cells containing very few and sparse glialfilaments as well as abnormal cytoarchitecture of reactive astrocytic processes. Furthermore,paired cell formation appeared frequently. The results suggest that GFAP is necessary formorphological retention and distribution of reactive astrocytes during prion disease, and thatthere is a GFAP-dependent function of glial filaments in reactive astrocytes.

© 2009 Elsevier B.V. All rights reserved.

Keywords:Glial fibrillary acidic proteinScrapieReactive astrocytesCell morphologyNeurodegeneration

1. Introduction

Astrocytes show a remarkable cellular response to a widerange of neuropathological conditions of the CNS. Hypertro-phic swelling and reactive astrocytosis are the most commonresponsive features of astrocytes. Enhanced expression andprominent accumulation of astrocytic intermediate filaments(IFs) is an unambiguous morphological indicator of reactiveastrocytosis. Glial fibrillary acidic protein (GFAP) is a major IFprotein expressed in reactive astrocytes. Vimentin is alsoknown to be expressed in reactive astrocytes (Schiffer et al.,1986) and forms an IF network with GFAP and/or nestin that isanother IF protein that is usually expressed in neuroepithelialstem cells (Lin et al., 1995; Galou et al., 1996; Eliasson et al.,1999).

c.jp (H. Gomi).

er B.V. All rights reserved

Genetic deprivation of astrocytic IF proteins in mice hasprovided insights into the roles of astrocytes and reactiveastrocytosis in both physiological and pathological aspects(Gomi et al., 1995; Pekny et al., 1995; McCall et al., 1996; Galouet al., 1996; Shibuki et al., 1996; Colucci-Guyon et al., 1999;Pekny et al., 1999; Menet et al., 2001). Within a single astrocyte,it is thought that mutualistic interaction of IF protein isimportant for the formation of functional astrocytic IFs.Studies of GFAP- and/or vimentin-deficient astrocytes haveelucidated the partnership of astrocytic IFs in resting andreactive astrocytes. In vimentin-deficient astrocytes, GFAPformed astrocytic IFs in vitro and in vivo, and these filamentsdisplayed a more densely packed aberrant organization thanwild-type astrocytes (Galou et al., 1996; Eliasson et al., 1999).The results suggest that vimentin, even at a low level of

.

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expression, is necessary for normal IF formation in astrocytes.On the other hand, in GFAP-deficient resting astrocytes,vimentin did not form astrocytic IFs or did so with very lowefficiency in vivo (Pekny et al., 1995; McCall et al., 1996). Bycontrast, in GFAP-deficient reactive astrocytes, vimentinformed IFs together with re-expressed nestin, but the bundledstructure of filaments in these astrocytes was much sparserthan that in wild-type reactive astrocytes (Eliasson et al.,1999). These experimental data also suggest that nestin doesnot form astrocytic IFs in direct partnership with GFAP inreactive astrocytes. Consequently, GFAP- and vimentin-defi-cient reactive astrocytes showed complete absence of astro-cytic IFs (Eliasson et al., 1999).

In response to acute injury, vimentin- and GFAP-deficientreactive astrocytes in mice showed impaired glial scarformation (Pekny et al., 1999), limited hypertrophy of cellprocesses (Wilhelmsson et al., 2004), and attenuated glialreaction to retinal detachment (Nakazawa et al., 2007). Whilethe complete absence of IFs in reactive astrocytes seems to bethe cause of various prominent impairments in cellularfunction, the GFAP or vimentin single IF protein-dependentreactive response of astrocytes has not been fully character-ized, especially in neurodegenerative diseases. We havepreviously demonstrated that GFAP-deficient mice exhibitedneuropathological changes typical of prion diseases wheninoculated with infectious scrapie prions (PrPSc; Gomi et al.,1995). In the present study, we examined GFAP-deficientreactive astrocytes and reassessed the detailed morphologicalproperties of mutant reactive astrocytes in scrapie ence-

Fig. 1 – Immunoblot analysis of cytoskeletal proteins. Cerebral tisand Gfap−/− (KO) mice at 2, 12, 20, and 23 weeks after intracerebra(Mo) subjected to SDS-PAGE (10 μl/lane), and reacted with monoanti-GFAP antibody recognizes the 50 kDa GFAP inWTmice but nanti-vimentin antibody recognizes the 57 kDa vimentin in both Wfurther cross-reacts with 50 kDa GFAP in WT mice (open arrowhreactions with monoclonal anti-α-tubulin and anti-β-actin antibarrowhead) and 43 kDa β-actin (lower arrowhead).

phalopathy. Morphometric and electron microscopic ana-lysis of reactive astrocytes demonstrated that GFAP playsa role in morphological retention and distribution of reactiveastrocytes.

2. Results

2.1. Immunoblot analysis of scrapie-infected brain

We previously reported that GFAP-deficient mice exhibitedneuropathological changes typical of prion diseases wheninoculated with infectious PrPSc (Gomi et al., 1995). For thedetection of PrPSc in the brain of infected mice and analysisof time-course changes in PrPSc deposition, PrPSc waspurified from brain extracts and immunoblotting wasperformed at 2, 12, and 20 weeks after inoculation usingrabbit polyclonal anti-PrP antiserum (Yokoyama et al., 1995).Amplified PrPSc in the brain of infected mice were detectableat 20 weeks after inoculation (Supplementary Fig. 1). Foranalysis of brain cytoskeletal protein, immunoblotting wasperformed on cerebral tissue homogenates (Fig. 1). In PrPSc-infected Gfap+/+ mice, a prominent increase in GFAPexpression was detected 20 weeks after infection alongwith an upregulation in vimentin expression. The expres-sion level of vimentin in Gfap−/− mice was not remarkablydifferent from that in Gfap+/+ mice. No significant changes inexpression levels of tubulin and actin cytoskeletons weredetected throughout the time-course of disease progression

sue homogenates (10%w/w)were prepared from Gfap+/+ (WT)l inoculation (wpi) of scrapie prion (Sc) and mock inoculationclonal antibodies against cytoskeletal proteins. Monoclonalot in KOmice (closed arrowhead in upper panels). MonoclonalT and KO mice (closed arrowhead in middle panels) and

ead). Lower panels show the immunolabeling of mixedodies. Each antibody detects the 56 kDa α-tubulin (upper

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in either mouse genotype. In agreement with vimentinimmunohistochemistry (Gomi et al., 1995), reactive changesof Gfap−/− astrocytes were confirmed by increased expressionof vimentin in immunoblot analysis.

2.2. Light microscopic and morphometric analysis ofreactive astrocytes

We performed a light microscopic analysis of cell shapewith toluidine blue (TB)-stained semi-thin sections preparedfrom plastic resin-embedded tissues. In optic nerve sec-tions, the overall degree of cytoplasmic swelling andhypertrophic thickening of processes in Gfap−/− reactiveastrocytes was less than that in Gfap+/+ reactive astrocytes(Figs. 2A and B). Similar morphology was observed inreactive astrocytes distributed in the cerebral cortex andhippocampus (Figs. 2C−F).

Fig. 2 – Reactive astrocytes in several regions of the brain. Gfap+/+

(590 nm thick) from the optic nerve (A and B), cerebral cortex (C anDistributed reactive astrocytes are indicated by arrowheads. Bar

Wethenevaluated themorphologyof reactiveastrocytes bymorphometry. Protoplasmic reactive astrocytes in hippocam-pus and white matter fibrous reactive astrocytes in the corpuscallosum were assessed using several parameters, includingoutlined cell area, nuclear size, vascular endfoot area, longestdistance among expanded cell space(e.g., distance betweenextended processes and expanded cytosolic edges), and thenumber of accumulated lipofuscin granules (Fig. 3). In both celltypes, cell area and longest distance among the outlined areawere significantly reduced in Gfap−/− reactive astrocytes (cellareas: 10% reduction in protoplasmic and 16% reduction infibrous astrocytes; the longest distance: 11% reduction inprotoplasmic and 15% reduction in fibrous astrocytes) whencompared with that in Gfap+/+ reactive astrocytes. By con-trast, nucleus sizes were significantly enlarged in Gfap−/−

cells (nucleus sizes: 14% increase in protoplasmic and 41%increase in fibrous astrocytes). No significant difference was

(A, C, and E) and Gfap−/− (B, D, and F) mice. Semi-thin sectionsd D), and hippocampus (E and F) were stained with 0.25% TB.s, 20 μm (A and B) and 50 μm (C−F).

Fig. 3 – Morphometric analysis of resting and reactive astrocytes. Morphological shapes of representative reactive astrocytes ofhippocampalprotoplasmic (AandB) andwhitematter (corpus callosum) fibrous reactiveastrocytes (CandD) fromGfap+/+ (AandC)or Gfap−/− (B and D)mice (arrowheads). Scattered vacuoles in neuropil or intralamellar and intra-astrocytic vacuoles are indicatedby *. The inset shows lipofuscin granules (Lp). Bars, 20 μm. Resting and reactive astrocytes were analyzed using severalparameters: outlined cell area, nuclear size, longest distance among expanded cell space, number of lipofuscin granules ofhippocampal protoplasmic astrocytes, and fibrous astrocytes in the corpus callosum (E) from Gfap+/+ (WT) and Gfap−/− (KO) mice.*P<0.015, **P<0.005, ***P<0.0005.

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detected in the number of cytosolic lipofuscin granules. Thesize of swollen astrocytic foot processes that surround bloodvessels (perivascular endfoot) was measured for hippocam-pal blood vessels that had diameters smaller than 30 μm(Supplementary Fig. 2). Although, Gfap−/− cells had a slighttendency to have an increased endfoot size, the differencebetween genotypes was insignificant. Morphometry ofresting astrocytes in tissues from naïve animals did not

show any significant differences in parameters analyzedbetween Gfap+/+ and Gfap−/− cells (Fig. 3E).

2.3. Electron microscopic analysis of reactive astrocytes

In Gfap+/+ mice, ultrastructural examination showed accumu-lation of glial filaments in the cytoplasm and processes ofhippocampal protoplasmic (Figs 4A and B) and white matter

Fig. 4 – Electron micrographs of protoplasmic reactive astrocytes. Typical shapes of protoplasmic reactive astrocytes in thehippocampus from Gfap+/+ (A) and Gfap−/−(C) mice. Fine structures of processes with higher magnification indicated by opensquares in (A) and (C) are shown in (B) and (D), respectively. In Gfap+/+ reactive astrocytes, the cytoplasm and processes are filledwith canonical glial filament (Gf) bundles. However, in Gfap−/− reactive astrocytes, IFs are very sparse (closed arrowheads) andthe cytoplasm and processes show clearing matrix (**), clustered glycogen granules (Gg), and microtubules (open arrowheads).Atypical constrictions of expanding processes are also shown (arrows). Morphology of shaft edges of reactive astrocyticprocesses fromGfap+/+ (E) andGfap−/− (F)mice. Constricted structure of themiddle regions of processes (arrows) shown in Gfap−/−

reactive astrocytes (G). Abbreviations: An, astrocytic nuclei; Ap, astrocytic processes; De, dendrites; V, blood vessels; En,perivascular endfeet of astrocytic processes; *, vacuoles; At, axon terminals; m,mitochondrion; Te, terminal edges of processes.Bars, 10 μm (A and C), 500 nm (B and D), and 2 μm (E to G).

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fibrous reactive astrocytes (Figs. 5A and B). Filament structuresalso accumulated in the vascular endfeet of reactive astro-cytes (Figs. 6A, B). In contrast to Gfap+/+ reactive astrocytes,clearingmatrix of the cytoplasm and processes that containedglycogen granules were observed in Gfap−/− reactive astrocytes(Figs. 5C, D and 6C, D). Also observed were microtubules thatstood out as cytosolic substrates (Figs. 4D, 5D, and 6D).Although there were very few and sparse glial filaments inGfap−/− reactive astrocytes (Figs. 4D and 5), a marked cytoarch-itectural change in the shape of Gfap−/− reactive astrocyticprocesses was observed. Shaft edges of processes of Gfap+/+

hippocampal protoplasmic reactive astrocytes showed athinly sharpened morphology (Fig. 4E). By contrast, Gfap−/−

processes frequently showed clubbing (Fig. 4F). Furthermore,the middle regions of processes showed unnatural constric-tions (Figs. 4C, G).

2.4. High frequency appearance of paired reactiveastrocytes in Gfap−/− mice

We found the frequent appearance of a tandem nucleararrangement of reactive astrocytes in the TB-stained sectionsobtained from Gfap−/− mice (Figs. 7A and B). The percentemergence of these cell types to the total analyzed cells was26% (vs. 5% in Gfap+/+ mice) in hippocampal protoplasmicastrocytes and 15% (vs. 4% in Gfap+/+ mice) in white matterfibrous astrocytes (Fig. 7C). To determine whether restingGfap−/− astrocytes, like reactive Gfap−/− astrocytes, exhibit anincrease in paired nuclei formation, we examined tissuesfrom naïve animals. The percent emergence of paired nucleiin resting Gfap−/− astrocytes was 7% (vs. 3% in Gfap+/+ mice) inprotoplasmic astrocytes and 6% (vs. 5% in Gfap+/+ mice) infibrous astrocytes. To clarify whether the paired arrangement

Fig. 5 – Electron micrographs of fibrous reactive astrocytes. Morphological shapes of white matter reactive astrocytes in thecorpus callosum of Gfap+/+ (A) and Gfap−/− (C) mice. Fine structures of the cytoplasm with a higher magnification indicated byopen squares in (A) and (C) are shown in (B) and (D), respectively. In Gfap+/+ reactive astrocytes, the cytoplasm and processescontain IF bundles (Gf). IFs are very sparse (closed arrowheads) and seem to run in the same direction as microtubules (openarrowheads) in Gfap−/− reactive astrocytes (E). Abbreviations: An, astrocytic nuclei; On, oligodendrocytic nuclei; Gg, glycogengranules; Ax, axons; m, mitochondrion; *, vacuoles in intralamellar, periaxonal, or extramyelinic spaces; **, clearing matrix.Bars, 10 μm (A and C), 500 nm (B and D), and 1 μm (E).

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of nuclei is related to nuclear deformation (karyolobism) or amultinucleated change, we serially analyzed semi-thin sec-tions. In most cells, no nuclear fusion of paired nuclei (i.e., twoindependent nuclei) was confirmed; however, there was aminority of U- or J-shaped deformed polymorphic nuclei(Supplementary Figs. 3 and 4). In the former case, paired nucleiappeared to be separated by a set of probable apposing plasmamembranes by electron microscopy (Figs. 7D, E, and inset in E).Thus, it can be interpreted that paired cells appear to adjoin.Such a cell arrangement was distributed in several regions,including the cerebral cortex, hippocampus, and corpuscallosum, suggesting an astrocytic subtype-nonspecific orga-nization. Although this morphological feature seems torepresent histological evidence of recent cellular division ofreactive astrocytes (Norenberg et al., 1972; Pittella andBrasileiro-Filho, 1987), none of the karyomitotic chromosomal

appearances rarely observed in anoxic encephalopathy (Die-mer and Kliken, 1976) were discernible in samples from thisneuropathological stage of prion disease.

2.5. Morphological imaging of reactive astrocytes inGfap−/− mice

In Fig. 8, we demonstrate a possiblemorphological delineationof astrocytic IF-poor reactive astrocytes induced by scrapie-induced neurodegenerative diseases. In normal reactiveastrocytes filled with rich glial filaments, processes projectingfrom the swollen cell body expand sharply and strongly.However, in reactive astrocytes with poor glial filaments,morphological abnormality is exhibited by distorted shape ofcell body and processes. Processes on shaft edges wereclubbed. Generally, there is a smaller outlined cell area and

Fig. 6 – Electron micrographs of vascular endfeet of reactive astrocytes. Enlarged vascular endfeet of Gfap+/+ (A) and Gfap−/− (C)mice. Open squares in (A) and (C) are shown in (B) and (D) at higher magnification, respectively. Glial filaments accumulate inGfap+/+ endfeet; but it is difficult to identify IF structure in Gfap−/− endfeet. Abbreviations: V, lumina of blood vessels; En,perivascular endfeet of astrocytic processes; Ap, astrocytic processes; De, dendrites; Gf, glial filament bundles; **, clearingmatrix; Gg, glycogen granules; At, axon terminals; m, mitochondrion; Bars, 1 μm.

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shorter process length above a certain diameter (such as thatpossessed by shaft edges), but larger nuclei than those innormal reactive astrocytes. Interestingly, the paired cellformation appears frequently.

3. Discussion

In prion diseases, extensive reactive astrocytosis accompa-nied by an increase in GFAP expression is a typical neuro-pathological finding. To evaluate the role of GFAP in reactiveastrocytosis in an experimental murine model of priondisease, we and other groups infected Gfap−/−mice with PrPSc

(Gomi et al., 1995; Tatzelt et al., 1996). Our previous histopath-ological data alluded to a morphological abnormality inreactive astrocytes of Gfap−/− mice as indicated by a changein immunohistochemical staining of marker proteins vimen-tin and S-100 (Gomi et al., 1995). Therefore, in the presentstudy, we analyzed morphological features of GFAP-deficientastrocytes in detail.

By light microscopic morphometry in TB-stained astro-cytes, we observed reduced outlined cell area and shorterdistances among expanded cell space, but larger nuclei inswollen Gfap−/− reactive astrocytes. Since there were nosignificant changes in the morphology of resting astrocytes,the differences could be attributed to a reduced hypertrophicchange in Gfap−/− reactive astrocytes. However, a technicaldifficulty in measuring thinner processes by our methodprevented the actual determination of process length and cellsize. Wilhelmsson et al. (2004) have also reported shorterGfap−/− reactive astrocytic processes when visualized by

immunohistochemistry using the astrocytic marker, gluta-mine synthase; but they found that extended processesoccupied the same spacial neuropil volume as wild-typeastrocytes when visualized using fluorescent dye injection.These results indicated that the substantial process length isnot different between Gfap−/− and wild-type astrocytes, butGfap−/− astrocytes are presumably thinner and more difficultto visualize. Therefore, our results from TB-stained semi-thinsections can be interpreted to indicate that there is probably ashorter process length above a certain diameter (e.g., shownby shaft edges of processes in the present study) in Gfap−/−

reactive astrocytes. Notably, Anderová et al. (2001) previouslyshowed that Gfap−/− astrocytes in the spinal cord have reducedprocess length as visualized using fluorescent dye injection.Complete absence of astrocytic IFs in GFAP- and vimentin-deficient mice (Gfap−/− Vim−/−) induced fewer processes inreactive retinal Müller cells (Kinouchi et al., 2003) and smallerirregularly-shaped endfeet of reactive Müller cells (Lundkvistet al., 2004). Furthermore, Gfap−/− Vim−/−reactive astrocytesprevented hypertrophy of astrocytic processes depicted by ashorter and irregular shape in the entorhinal cortex lesionmodel (Wilhelmsson et al., 2004); cultured Gfap−/− Vim−/−

primary astrocytes had smaller cell sizes and exhibitedfewer and shorter processes (Lepekhin et al., 2001). Thus,these results revealed a novel role of astrocytic IFs indetermining astrocyte morphology (Pekny and Nilsson, 2005).

Electron microscopy showed mutant cells containing veryfew and sparse glial filaments, as well as abnormally shapedprocesses. Consistent with immunohistochemical detectionof vimentin in Gfap−/− reactive astrocytes (Gomi et al., 1995), anupregulation of vimentin expression in cerebral tissue was

Fig. 7 – Frequently appearing paired reactive astrocytes in Gfap−/− mice. The representative TB-stained semi-thin sections ofhippocampal protoplasmic (A) and white matter fibrous (B) reactive astrocytes (arrows). The percent appearance of tandemnuclear arrangement in resting and reactive astrocytes from Gfap+/+ (WT) and Gfap−/− (KO) mice. (C) Electron micrographs ofpaired protoplasmic (D) and fibrous (E) reactive astrocytes. Arrowheads indicate probable plasma membranes separating twoastrocytic cell bodies. Inset in (E) shows a high-magnification image of intervening membranes that extend to outer plasmamembranes. Abbreviations: An, astrocytic nuclei; V, blood vessels; *, extracellular space. Bars, 10 μm (A and B), 2 μm (D and E),and 200 μm (inset in E).

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demonstrated by immunoblot analysis. It has been reportedthat scrapie-infected mice exhibited increased expression ofnestin in reactive astrocytes (Jin et al., 2004). Therefore, our

Fig. 8 – Morphological image of reactive astrocytes. In normal reaprocesses from the swollen cell body expand radially with a lengpoor glial filaments, the cell body is slightly distorted in shape wiIn addition, paired cell formation is frequently seen in cells with

data suggest that vimentin forms glial filamentswith resumednestin in Gfap−/− reactive astrocytes in persistent neurodegen-erative diseases, as observed in cases of acute injury

ctive astrocytes (rAs) filledwith rich glial filaments, extendingth-thickness balance. However, in reactive astrocytes withth irregularly constricted processes and clubbing shaft edges.poor glial filaments.

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experiments (Lin et al., 1995; Eliasson et al., 1999; Pekney et al.,1999). However, the amount of IFs is insufficient for morpho-logical retention in Gfap−/− reactive astrocytes. Under physio-logical conditions, Gfap−/− astrocytes in the spinal cord, asvisualized using fluorescent dye injection, exhibited shorterand thinner processes than Gfap+/+ astrocytes (Anderová et al.,2001). Therefore, it is presumed that a primary morphologicalabnormality of Gfap−/− astrocytes in the basal phase isinherited in the reactive phase induced by scrapie encepha-lopathy. Nevertheless, the accumulation of sufficient glialfilaments is not essential for hypertrophic cell swelling in thereactive phase, even though there was a morphologicalabnormality in Gfap−/− reactive astrocytes. In this context, aseries of studies on aquaporin-4, a glial membrane waterchannel, suggest that water movement into reactive astro-cytes would be involved in edema-associated swelling ofreactive astrocytes in prion diseases (Vizuete et al., 1999;Manley et al., 2000; Rodríguez et al., 2006).

Enlarged nuclei of Gfap−/− reactive astrocytes appear to be aunique phenotype. The role of IFs in nucleus localization andshape control has recently been noted as one of the importantfunctions of IFs. It is suggested that an IF network with plectin,a cytoskeletal linker protein, joins the nuclear membranemediated byKASH and SUNnuclearmembrane integral proteinfamilies (Wilhelmsen et al., 2005; Starr, 2007). Absence of theeffect of IFs on nuclear localization and shape was observed inIF-free cells such as vimentin, desmin, and ketatin (Minin andMoldaver, 2008). Therefore, loss of GFAP might be responsiblefor the phenotypic enlargement of nuclei in Gfap−/− reactiveastrocytes, because nuclear hypertrophy is one of the commonreactive responses observed in reactive astrocytes.

Another morphological feature of Gfap−/− reactive astro-cytes in cases of scrapie encephalopathy is the frequentappearance of paired cell formation. Several aspects such aschanges in cell proliferation and migration of reactive astro-cytes can be examined. Paired cell formation of astrocytes isprominently observed in hepatic encephalopathy and hepa-tocirrhosis, and it represents a proliferative feature of reactiveastrocytes (Norenberg et al., 1972; Pittella, 1981; Brumback andLapham, 1989). In hepatic encephalopathy, paired cell forma-tion is assumed to a result of newly formed daughter cells thathave yet not migrated away from the parent cell. In brains ofhumans with prion diseases and scrapie-infected animals,expression of proliferating cell nuclear antigen (also known ascyclin) has been demonstrated in reactive astrocytes (Biernatet al., 1995; Ye et al., 1998). Neurotoxic PrP peptides (PrP106–126 or PrP90–231) have also elicited astrocyte proliferation(Florio et al., 1996; Thellung et al., 2007). Consistently, anincreased cell labeling with 5-bromo-2′-deoxyuridine (BrdU)has been reported in scrapie-infected mice brains (Na et al.,2009). A portion of BrdU-positive cells was depicted with apaired nuclear arrangement that was co-labeled with orwithout neuronal markers. The incorporation of BrdU incells during scrapie infection correlated well with the expres-sion of resumed nestin that has often been associated withreactive astrocytosis (Jin et al., 2004). Therefore, the paired cellarrangement observed in the present study may also reflectaspects of neurogenesis and astrogenesis (Larsson et al., 2004;Widestrand et al., 2007). A possible interpretation of prolifer-ative changes in Gfap−/− reactive astrocytes may be associated

with more facilitative cell division. In the process of cellduplication, GFAP must be processed for structural modifica-tions, such as destruction/assembly of filaments, through thephosphorylation or dephosphorylation of its head domain(Inagaki et al., 1994; Takemura et al., 2002a, 2002b). Suchregulation of GFAP contributes to extensive remodeling offrameworks in mitotic astrocytic IFs. Without GFAP, anabbreviated structural change in IFs might trigger a facilitatedcell division. Furthermore, Lepekhin et al. (2001) demonstratedthat cultured Gfap−/− astrocytes exhibited a defect in cellmigration in vitro. If cell migration is disturbed in Gfap−/−

reactive astrocytes in vivo, the incidence of non-migrated cellsafter duplication will increase. Although recent reports havedemonstrated the possible involvement of some signalingpathways or autocrine/paracrine secretory functions in astro-cyte migration using an in vitro scratch wound model (Lim etal., 2007; Borán and García, 2007; Lee et al., 2009), it remainsunknown how IFs function in cell migration.

All evidence taken together, we conclude that GFAP, amajor IF protein of astrocytes, functions in both physiologicaland pathological conditions as a key player in cytoarchitec-tural changes in reactive astrocytes and qualitative celldistribution in reactive astrocytosis that occurs in priondisease induced neurodegeneration. The present study givesus a neuropathological understanding of GFAP-specific func-tions and the meaning of astrocytic IF accumulation inreactive astrocytes. Our findings would also be of furtherinterest in their application to other neuropathologies andreactive gliosis models that support similar observations.

4. Experimental procedures

4.1. Mice and inoculation of PrPSc

The GFAP-deficient mice inoculated with PrPSc used in thisstudy have been previously described (Gomi et al., 1995). Micewere examined biochemically 2, 12, 20, and 23 weeks afterinoculation, and samples for morphological analysis wereprepared from mice 23 weeks after inoculation. All animalexperiments have been performed according to the guidelinesof the Animal Care, Regulation of Infectious Agents andExperimental Committee, National Institute of Animal Health.

4.2. Immunoblot analysis

Cerebral tissue homogenates (10% w/w) were prepared withhomogenate buffer containing 50 mM phosphate buffer (pH7.3), 2 mM EDTA, 2 mM EGTA, 20 μg/ml phenylmethylsulfonylfluoride, and 20 μg/ml tosyl phenylalanyl chloromethylketone. For cytoskeletal protein analysis, tissue homogenateswere lysed with an equal volume of 2× SDS sample buffercontaining 0.25 M Tris–HCl (pH 6.8), 2% SDS, 50% glycerol, and10% 2-mercaptoethanol. Purification of PrPSc was carried outas described by Gomi et al. (1995). Cerebral tissue homoge-nates and purified PrPSc were subjected to SDS-PAGE (10% or12.5% polyacrylamide gel). Separated proteins were trans-ferred to an Immobilon-P transfer membrane (Millipore) andblocked with 5% skim milk in 20 mM Tris buffered saline (pH7.3) supplemented with 0.05% Tween 20 (TBS-T) for 1 h.

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Blotting membranes were reacted with primary antibodies for1 h at room temperature. Anti-GFAP (1:100, GF 12.24; ProgenBiotechnic), anti-vimentin (1:100, VIM 3B4; Cymbus BioscienceLimited), anti-α-tubulin (1:300, T-9026; Sigma), and anti-β-actin (1:200, MAB 1501; Chemicon) monoclonal antibodieswere used for cytoskeletal protein analysis. Analysis of PrPSc

was performed using rabbit polyclonal anti-PrP antiserum(Yokoyama et al., 1995). After washing three times with TBS-Tfor 15 min at room temperature, membranes were reactedwith peroxidase-conjugated rat anti-mouse IgG (1:3000;Zymed Laboratories Inc.) or with goat anti-rabbit IgG (1:5000;Dako Inc.) (secondary antibodies) for 1 h at room temperature.After the secondary antibody reaction, membranes werewashed three times with TBS-T and signal detection wasaccomplished using enhanced chemiluminescent Westernblotting detection reagents (Amersham Biosciences).

4.3. Light and electron microscopy

For morphological studies of reactive astrocytes, tissuesamples were prepared from scrapie-infected mice (twowild-type and two GFAP-deficient). For analysis of restingastrocytes, tissue sampleswere prepared fromnon-infected 8-week-old mice (two wild-type and two GFAP-deficient).Animalswere anesthetized deeply by intraperitoneal injectionof sodium pentobarbital (Nembtal, 0.1 mg /g body weight).After intracardiac rinse perfusionwith 10ml of saline solution,brains were fixed by perfusion with 100 ml of 2% paraformal-dehyde and 2.5% glutaraldehyde in 0.1 M phosphate buffer (pH7.3) at 4 °C. Brain tissues were dissected into small blocks(∼3 mm thick) and postfixed in the same fixative overnight at4 °C. Tissue blocks were coronally sliced (100 μm thick) on amicroslicer (DTK-100, Dosaka) and postfixed with 2% osmiumtetroxide in 0.1 M phosphate buffer at 4 °C for 2 h. Tissue sliceswere dehydrated, infiltrated, and embedded in plastic resin.Semi-thin (590 nm thick) sections were cut with an UltratrimDiatome (Nisshin EM) on an Ultracut E ultramicrotome(Reichert-Jung) and stained with 0.25% TB (Waldeck Gmbh &Co. KG, Chroma). Stained sections were viewed with amicroscope (BX50, Olympus). Ultrathin (90 nm thick) sectionswere cut with an Ultra 45° Diatome (Nisshin EM) on anultramicrotome, stainedwith 4% uranyl acetate and thenwith0.4% lead citrate, and subjected to electron microscopicanalysis in JEM 1010 (JEOL Company) at an acceleration voltageof 80 kV. Electron microscopic pictures (from magnification×3000 to ×250,000) were scanned at a resolution of 720 dpi.

4.4. Morphometry

Digitalized images of resting and reactive astrocytes in TB-stained semi-thin sections were obtained with a charge-coupled device camera (DP-70, Olympus) using an objectivelens (×100; UplanApo, Olympus). Morphometric analysis wasperformed to measure the outlined cell area, nuclear size,vascular endfoot area, blood vessel area, and longest distanceamong expanded cell space using ImageJ 1.31 software (http://rsb.info.nih.gov.nih-image/). The surrounding area includingthe cell body with shaft edges of processes drawn by serialoutline, which was recognizable by equivalent TB-stainedcolor and brightness, was defined as the outlined cell area. The

perivascular endfoot area was measured in cross sections ofblood vessels (4–30 μm in diameter) whose contacted endfootdid not show a serial link to astrocytic cell body. Relativepercent of perivascular endfoot area vs. blood vessel area wascalculated by the following equation: percent of perivascularendfoot size=100 × [perivascular endfoot area (μm2)/bloodvessel area (μm2)]. Numbers of analyzed resting astrocyteswere 183 in Gfap+/+ and 178 in Gfap−/− protoplasmic astrocytes;107 in Gfap+/+ and 122 in Gfap−/− fibrous astrocytes. Numbers ofanalyzed reactive astrocytes were 214 in Gfap+/+ and 206 inGfap−/− protoplasmic astrocytes; 87 in Gfap+/+ and 108 in Gfap−/−

fibrous astrocytes. Analyzed cells were randomly selected fromsections of the hippocampus and corpus callosum.

4.5. Statistical analysis

Data are presented as mean±SEM. Differences betweenmeans were assessed by Student's two-tailed t test and wereconsidered significant if P values were lower than 0.015.

Acknowledgments

This work was supported by the Grants-in-Aid for scientificresearch from the Japan Society for the Promotion of Science(JSPS) and the contract research expenses from O'HARA.

Appendix A. Supplementary data

Supplementary Fig. 1. Accumulation of scrapie prions (PrPSc) inthe brain. At 2, 12, and 20weeks after intracerebral inoculation(wpi) of the Obihiro strain of PrPSc in Gfap+/+ (WT), Gfap+/−(HZ),and Gfap−/−(KO) mice. PrPSc was purified and subjected toimmunoblot analysis using anti-PrP antiserum. Normal brainhomogenates inoculated with mock infection were used asnegative controls and PrPSc purified from PrPSc-inoculated ICRmouse brain were used as positive controls (PC).

Supplementary Fig. 2. Morphometric analysis of vascularendfeet of reactive astrocytes. Enlarged vascular endfeet ofhippocampal astrocytes from Gfap+/+ (A) and Gfap−/−(B) mice(arrowheads). Various sizes of vacuoles (⁎) were observed inthe neuropil. Bar, 20 μm. Percent of perivascular endfoot sizevs. vascular diameter (4–30 μm of diameter range) of Gfap+/+

(WT) and Gfap−/−(KO) mice is shown in the histogram (C).Supplementary Fig. 3. Semi-thin serial sections of proto-

plasmic reactive astrocytes with tandem nuclei. TB-stainedsemi-thin sections (590 nm) of hippocampal protoplasmicastrocytes are serially arranged from top left (−10) to bottomright (+10). Nuclear fusion of tandem nuclei is not observed(closed and open arrowheads).

Supplementary Fig. 4. Semi-thin serial sections of fibrousreactive astrocytes with tandem nuclear arrangement. TB-stained semi-thin sections (590 nm) of fibrous astrocytes inthe corpus callosum are arranged serially from top left (−10) tobottom right (+22). Tandemnuclei of cells are not fused (closedand open arrowheads), but nuclear fusion which results in apolymorphic nucleus (U-shape) is observed in another cell(arrows).

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Note: The supplementary material accompanying thisarticle is available at (doi:10.1016/j.brainres.2009.11.025).

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