Neurobiology of Disease 33 (2009) 260–273

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Neurobiology of Disease

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Mutant γPKC found in spinocerebellar ataxia type 14 induces aggregate-independentmaldevelopment of dendrites in primary cultured Purkinje cells

Takahiro Seki a, Takayuki Shimahara a, Kazuhiro Yamamoto a, Nana Abe a, Taku Amano a, Naoko Adachi b,Hideyuki Takahashi b, Kaori Kashiwagi b, Naoaki Saito b, Norio Sakai a,⁎a Department of Molecular and Pharmacological Neuroscience, Graduate School of Biomedical Sciences, Hiroshima University, 1-2-3 Kasumi, Minami-ku, Hiroshima 734-8551, Japanb Laboratory of Molecular Pharmacology, Biosignal Research Center, Kobe University, Kobe 657-8501, Japan

⁎ Corresponding author. Fax: +81 82 257 5144.E-mail address: nsakai@hiroshima-u.ac.jp (N. Sakai).Available online on ScienceDirect (www.scienced

0969-9961/$ – see front matter © 2008 Elsevier Inc. Aldoi:10.1016/j.nbd.2008.10.013

a b s t r a c t

a r t i c l e i n f o

Article history:

Missense mutations in prot Received 22 September 2008Accepted 18 October 2008Available online 8 November 2008

Keywords:γPKCSpinocerebellar ataxia type 14 (SCA14)Missense mutationAggregate formationPurkinje cellsDendritesTranslocationFRAPOligomer formation

ein kinase Cγ (γPKC) gene have been found in spinocerebellar ataxia type 14(SCA14), an autosomal dominant neurodegenerative disease. We previously demonstrated that mutant γPKCfound in SCA14 is susceptible to aggregation and induces apoptosis in cultured cell lines. In the present study,we investigated whether mutant γPKC formed aggregates and howmutant γPKC affects the morphology andsurvival of cerebellar Purkinje cells (PCs), which are degenerated in SCA14 patients. Adenovirus-transfectedprimary cultured PCs expressing mutant γPKC-GFP also had aggregates and underwent apoptosis. Long-termtime-lapse observation revealed that PCs have a potential to eliminate aggregates of mutant γPKC-GFP.Mutant γPKC-GFP disturbed the development of PC dendrites and reduced synapse formation, regardless ofthe presence or absence of its aggregates. In PCs without aggregates, mutant γPKC-GFP formed solubleoligomers, resulting in reduced mobility and attenuated translocation of mutant γPKC-GFP upon stimulation.These molecular properties of mutant γPKC might affect the dendritic morphology in PCs, and be involved inthe pathogenesis of SCA14.

© 2008 Elsevier Inc. All rights reserved.

Introduction

Autosomal dominant spinocerebellar ataxias (SCAs) are a hetero-geneous group of neurological disorders clinically characterized byprogressive ataxia due to cerebellar dysfunction. SCAs are classifiedinto at least 28 types by chromosomal loci of the causal genes (Scholset al., 2004; Cagnoli et al., 2006; Duenas et al., 2006). CAG trinu-cleotide repeat expansions in coding regions were first identified asresponsible mutations in several SCAs (SCA1, 2, 3, 6, 7, 17 anddentatorubral pallidoluysian atrophy (DRPLA)) (Martin, 1999; Everettand Wood, 2004). Missense and deletion mutations in responsiblegenes were later found in other SCAs (SCA5, 11, 13, 14, 15 and 27)(Duenas et al., 2006; Houlden et al., 2007; van de Leemput et al., 2007).Among these, SCA14 is caused by missense mutations in the PRKCGgene encoding protein kinase Cγ (γPKC), first identified by Chen et al.in 2003 (Chen et al., 2003). To date, 23 mutations have been identifiedin different SCA14 families, including a 2-amino acid-deletion mutant(ΔK100-H101) (van de Warrenburg et al., 2003; Yabe et al., 2003;Stevanin et al., 2004; Alonso et al., 2005; Chen et al., 2005; Klebe et al.,2005, 2007; Verbeek et al., 2005a,b; Dalski et al., 2006; Hiramoto et al.,2006; Vlak et al., 2006; Nolte et al., 2007; Wieczorek et al., 2007).

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PKC is a family of serine/threonine kinases that play importantroles in various cellular functions. Among PKC subtypes, γPKC isspecifically present in the central nervous system and is especiallyabundant in cerebellar Purkinje cells (PCs) (Saito et al., 1988). γPKCknockout mice showed mildly impaired motor coordination andincomplete developmental elimination of synapses between PCs andclimbing fibers (Chen et al., 1995; Kano et al., 1995). However, theseataxic symptoms in γPKC knockout mice were not prominentcompared with those in SCA14 patients. Morphologies of thecerebellum and cerebellar PCs in γPKC knockout mice were alsoindistinguishable from those in wild type mice (Kano et al., 1995),while prominent cerebellar atrophy and degeneration of PCs wereobserved in SCA14 patients (Yamashita et al., 2000). Moreover, SCA14is inherited in an autosomal dominant fashion, raising the possibilitythat gain of toxic function of mutant γPKC, not loss of γPKC function,underlies the pathogenic mechanism of SCA14.

We previously demonstrated that 7 mutant γPKCs tended to formaggregates in culture cells (Seki et al., 2005b) and that this aggregateformation caused apoptotic cell death via impairment of the ubiquitinproteasome system and induction of ER stress (Seki et al., 2007).Similar aggregate formation of mutant or misfolded proteins isfrequently observed in various neurodegenerative diseases (Tayloret al., 2002; Ross and Poirier, 2004), suggesting that aggregateformation is involved in the common pathogenesis of neurodege-nerative diseases, including SCA14. In the present study, to elucidate

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the pathogenesis of SCA14, we expressed mutant γPKC-GFP inprimary cultured PCs as a cellular model of SCA14. We found thataggregates of mutant γPKC are formed in PCs and triggers PCapoptosis. However, PCs had a protective potential to eliminate thetoxic aggregates of mutant protein. Regardless of the presence orabsence of aggregation, mutant γPKC impaired dendritic developmentand synapse formation of PCs. These phenomena might be induced bythe reducedmobility and the attenuated translocation of oligomerizedmutant γPKC.

Materials and methods

Materials

SUMITOMO Nerve-Cell Culture System (Neuron culture mediumand Dissociation solutions) was obtained from Sumitomo Bakelite(Tokyo, Japan). Anti-calbindin D28K mouse monoclonal antibody wasfrom Swant (Bellinzona, Switzerland). Anti-GluRδ1/2 rabbit polyclonalantibody and anti-amyloid oligomer rabbit polyclonal antibody (A11)were from Millipore (Billerica, MA, USA). Anti-ubiquitin mousemonoclonal antibody and anti-γPKC rabbit polyclonal antibody werefromSanta Cruz Biotechnology (Santa Cruz, CA, USA). Anti-proteasome20S rabbit polyclonal antibody was from Biomol International(Plymouth Meeting, PA, USA). Anti-activated caspase-3 antibody wasfrom cell signaling technology (Boston, MA, USA). Alexa Fluora 546(Alexa546)-conjugated goat anti-rabbit and mouse antibodies,normal goat serum (NGS) and Hank's balanced salt solution werefrom Invitrogen (Carlsbad, CA, USA). Poly-L-ornithine (0.01%) andpoly-L-lysine (0.01%) solutions, 3,3′,5′-triiodo-L-thyronine (T3) so-dium salt, Hoechst 33342 and Dulbecco's modified Eagle's medium(DMEM) were from Sigma-Aldrich (Saint Louis, MO, USA). Glass-bottomed culture dishes (35 mm diameter) were from MatTek(Ashland, MA, USA). Glass-bottomed culture dishes with grids(35 mm diameter) were from Matsunami Glass (Kishiwada, Japan).

Cerebellar dissociated culture from mouse embryos

E18 embryos were removed from pregnant ICR mice anesthetizedwith diethyleter. Their cerebelli were dissected and kept in ice-coldHank's balanced salt solution. Cerebellar cells were dissociated byusing dissociation solutions of SUMITOMO Nerve-Cell Culture Systemaccording to the manufacturer's protocol. Dissociated cerebellar cellswere suspended in a neuron culture medium of SUMITOMO Nerve-Cell Culture System, plated at 2×105 cells/100 μl on the center of a35-mm diameter glass-bottomed culture dish coated with polyL-ornithine and poly L-lysine, and cultured in a humidified atmospherecontaining 5% CO2 at 37 °C. After the cells had attached to the bottom(1–2 h later), 1 ml of culture medium was added to each dish. Theculture medium was supplemented with 100 pM T3. Cells werecultured for 21–28 days in vitro (DIV). Half of the medium waschanged every 3–4 days.

Construction of adenoviral vectors to express γPKC-GFP by using atetracycline (Tet)-regulated system

To express WT and mutant γPKC-GFP specifically in PCs, weconstructed two types of adenoviral vectors to regulate the expressionof γPKC-GFP by a Tet-regulated system in promoter- and Tet-dependent manners (Fig. 1A). The first vector, Ad-L7-tTA, encodestetracycline transactivator (tTA) cDNA under the control of the L7promoter, which induces gene expression in a PC-specific manner. Thesecond vector, Ad-TetOP-γPKC-GFP, encodes WT or mutant γPKC-GFPcDNA under the control of the TetOp minimal promoter, which istransactivated by tTA.

The TetOp promoter was digested with Xho I and Hind III frompTet-Splice (Invitrogen, Carlsbad, CA, USA) and subcloned into the Xho

I/Hind III site of pShuttle (Stratagene, La Jolla, CA, USA). The newplasmid was designated TetOp-pShuttle. WT and mutant γPKC-GFPcDNAs with the polyadenylation signal were amplified by polymerasechain reaction (PCR) using plasmids γPKC-GFP in pcDNA3 (Seki et al.,2005b) as templates. The sense and antisense primers used were5′-GAATTCGCCATGGCTGGTCTG-3′ and 5′-ATCCCCAGCATGCCTGC-TATT-3′, respectively. The PCR products were subcloned into thedownstream region of the TetOp promoter in TetOp-pShuttle vector,which was digested with Hind III and blunted by a DNA blunting kit(Takara Bio, Otsu, Japan). DsRed monomer cDNAwith polyadenylationsignal was digested with Age I and Mlu I from pDsRed monomer-C1(Clontech Mountain View, CA, USA), blunted and subcloned intoTetOp-pShuttle. The L7 promoter was a kind gift from Prof. Morgan(St. Jude Children's Research Hospital, Memphis, TN). In the presentstudy, a region of the L7 gene approximately 1.2 kb upstream from thetranscriptional initiation site was used as a promoter. tTA cDNA wasobtained from a pTet-tTAk plasmid (Invitrogen). Both the L7 promoterand tTA cDNA with polyadenylation signal were subcloned intopShuttle.

Ad-L7-tTA, Ad-TetOp-γPKC-GFP and Ad-TetOp-DsRed monomerwere constructed by using an AdEasy adenoviral vector system(Stratagene) according to the manufacturer's protocol. Briefly, theshuttle vector was recombinated with pAdEasy-1, which is anadenoviral backbone cosmid vector, in E. coli strain BJ5183. Therecombinant adenoviral genomewas digested from the cosmid vectorby Pac I and transfected into HEK293 cells, which stably express the E1gene and produce the E1 gene-deleted adenoviral vector. Proliferatedadenoviral vectors were extracted from HEK293 cells and concen-trated by cesium chloride ultracentrifugation.

Expression and live imaging of γPKC-GFP in PCs

OnDIV14orDIV21, cultured cellswere infectedwith two adenoviralvectors, Ad-L7-tTA (4×106 pfu/dish) and Ad-TetOp-γPKC-GFP (WT,S119P or G128D, 6×105 pfu/dish). After a further 7–14 days ofcultivation, γPKC-GFP fluorescence was detected at 488-nm argonlaser excitation using a 505–530-nm band-pass barrier filter with aconfocal scanning fluorescent microscope (LSM 510 META, Carl Zeiss,Esslingen, Germany). PCs were morphologically characterized byrelatively large somata and highly branched dendrites. γPKC-GFPfluorescence images of PCs were obtained as Z-stack images of γPKC-GFP fluorescence. The areas of γPKC-GFP-expressing PCs werecalculated from Z-stack projected images by Image-Pro Plus 5.1(Media Cybernetics, Bethesda, MD, USA).

When cells were plated on a glass-bottomed dish with a grid,γPKC-GFP fluorescence of PCs was observed daily from 4 to 7 daysafter the adenoviral infection on DIV22. After observation on thefirst day, cells were treated and cultured with or without tetracycline(1 μg/ml) to arrest the expression of γPKC-GFP by the Tet-regulatedsystem (Fig. 1A). The same γPKC-GFP-expressing PCs were observeddaily according to the grid on the culture dish.

To observe sequential changes in PCs expressing γPKC-GFP moreprecisely, long-tem time-lapse imaging was conducted by using anincubation imaging system, LCV100 (Olympus, Tokyo, Japan), underregulated conditions (5% CO2 at 37 °C). Sequential GFP fluorescenceimages of 15–20 different PCs per dish were obtained every 30 min for60 h from 4 days after the adenoviral infection on DIV21–22.

Observation of γPKC-GFP translocation, calcium imaging and fluorescentrecovery after photobleaching (FRAP) assay

On DIV21–28, the culture medium was replaced with 950 μl ofHEPES buffer (NaCl 165 mM, KCl 5 mM, CaCl2 1 mM, MgCl2 1 mM,HEPES 5 mM, glucose 10 mM, pH 7.4). Translocations of WT andmutant γPKC-GFP were triggered by a direct application of 50 μl ofHEPES buffer containing a high concentration of KCl (100 mM). γPKC-

Fig. 1. PC-selective expression of γPKC-GFP by adenoviral vectors utilizing tetracycline (Tet)-regulated gene expression system. (A) Construction of adenoviral vectors and schematicdiagram of Tet-regulated gene expression system. To express γPKC-GFP selectively in PCs, we infected two adenoviral vectors. The first vector, Ad-L7-tTA, was constructed to expresstetracycline transactivator (tTA) under the control of the L7 promoter, which induces gene expression in a PC-specific manner. The second vector, Ad-TetOp-γPKC-GFP, encodesWTormutant γPKC-GFP cDNA under the control of the TetOp minimal promoter, which is transactivated by tTA. By the co-infection of these two adenoviral vectors, tTA binds to the TetOppromoter and activates the transcription of γPKC-GFP, leading to the selective expression of γPKC-GFP in PCs. In the presence of tetracycline (Tet), γPKC-GFP expression is turned off,since Tet-bound tTA is not able to bind to and activate the TetOp promoter. (B) PC-selective expression ofWT-GFP in primary cultured cerebellar cells. Culture cells were infected withadenoviral vectors (Ad-L7-tTA and Ad-TetOp-WT-GFP) on DIV14, followed by fixation on DIV28 and immunostaining with anti-Calbindin D28K, a marker of cerebellar PCs, mousemonoclonal antibody (diluted 1:1000) and Alexa546-conjugated secondary antibody. Left and right images are representative of GFP fluorescence and calbindin staining (Alexa546fluorescence), respectively. Images were projected from Z-stack images obtained by using a confocal laser microscope. Bar=20 μm.

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GFP fluorescent images of PC dendrites were recorded every 0.5 s for5 min before and after the stimulation by using a confocal lasermicroscope.

For calcium imaging, calcium green-1 (CG1) AM (Invitrogen) wasused as a calcium indicator, as described previously (Seki et al.,2005b). Briefly, cells were loaded with 10 μM CG1 AM for 60 min atroom temperature before observation. The emissions of GFP and CG1were both excited with an argon laser at 488 nm and were separatedby emission fingerprinting (Carl Zeiss, LSM 510 META). GFP and CG1fluorescent images of PC dendrites were recorded every 1 s for 2 minbefore and after the high KCl stimulation. The fluorescent intensity ofCG1 was calculated from 3 different regions of interest (ROIs) in thecytoplasm of PC dendrites.

For the FRAP assay, γPKC-GFP fluorescent images in somata anddendrites of PCs were obtained by using a confocal laser microscopewith relatively low resolution to rapidly monitor the changes in GFPfluorescence. γPKC-GFP was photobleached by repetitive irradiationof a maximal excitation laser to small circular regions in somata anddendrites of PCs. Fluorescent images were recorded every 0.2 s for2 min before and after photobleaching. Changes in fluorescence of the

bleached area were normalized by the whole fluorescence in imagesand analyzed by GraphPad Prism (GraphPad Software Inc., San Diego,CA, USA).

In these experiments, we observed PCs without any aggregates ofγPKC-GFP. All experiments were performed at room temperature.

Immunostaining and nuclear staining

On DIV21–29, cells were fixed with 4% paraformaldehyde in PBSfor more than 30 min. After washing twice with PBS, the cells weretreated with PBS containing 0.3% Triton X-100 and 5% normalgoat serum (NGS) for 10 min at RT. The cells were then incubatedwith PBS-T (0.03% Triton X-100 in PBS) containing the primaryantibody and 5% NGS for N1 h at RT. After washing four times withPBS-T, the cells were incubated with PBS-T containing Alexa546-conjugated goat anti-mouse or rabbit IgG antibodies (diluted 1:500)and 5% NGS for N1 h at RT. After three washes, the fluorescence ofGFP and Alexa546 was observed with a confocal scanning fluore-scent microscope (LSM 510 META, Carl Zeiss) at 488-nm argonlaser excitation using a 505–530-nm band-pass barrier filter and at

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543-nm HeNe laser excitation using a 560-nm long-pass barrier filter,respectively.

Apoptotic cell death triggered by mutant γPKC-GFP was evaluatedby nuclear fluorescent staining with Hoechst 33342 (Seki et al., 2006).Hoechst 33342 staining was conducted concomitantly with Calbindinimmunostaining by adding Hoechst 33342 (0.5 μg/ml) to thesecondary antibody solution. The fluorescence of Hoechst 33342was monitored with a confocal laser scanning fluorescent microscopeat 364-nm ultraviolet laser excitation using a 385–470-nm band-passbarrier filter. Cells with condensed or fragmented nuclei wereconsidered to be apoptotic.

Results

PC-selective expression of γPKC-GFP in primary culture of mousecerebellar cells

To express γPKC-GFP selectively in PCs, we utilized the L7promoter and the tetracycline (Tet)-regulated gene expression system(Chen et al., 1998; Sakai et al., 2002) (Fig. 1A). Since a gene under theTetOp promoter is expressed only in the presence of tetracyclinetransactivator (tTA), the expression is regulated by the promoterupstream of tTA. Therefore, gene expression can be regulated in a cell-specific manner by the use of a cell-specific promoter. Furthermore,gene expression can be regulated in a time-specific manner since theexpression is arrested by the addition of Tet. To achieve PC-selectiveexpression of γPKC-GFP using this system, we constructed twoadenoviral vectors: Ad-L7-tTA, which expresses tTA under the controlof the L7 promoter, a promoter that specifically functions in PCs, andAd-TetOp-γPKC-GFP, which has γPKC-GFP (wild type (WT), S119P or

Fig. 2. Mutant γPKC-GFP tended to form aggregates in primary cultured PCs. (A) Representwithout (center) and with (right) aggregates. Images were projected from Z-stack images obvectors on DIV14 and observed on DIV28. Bar=20 μm. (B, C) Quantitative analyses of aggregatWTor mutant γPKC-GFP per experiment. Cells were infected with adenoviral vectors on DIV1PCs having aggregates of WT or mutant γPKC-GFP from 5 independent experiments. ⁎⁎⁎pb

G128D mutant) cDNA downstream of the TetOp promoter (Fig. 1A).We first attempted to express WT γPKC-GFP (WT-GFP) selective-ly in PCs in mouse cerebellar primary culture by co-infection withAd-L7-tTA and Ad-TetOp-WT-GFP. When adenoviral vectors wereinfected on DIV14, most WT-GFP-expressing cells had typical PC-likemorphologies, such as large somata and highly branched dendrites onDIV28, and were immunostained with an antibody against calbindinD28K, which is expressed specifically in PCs in the cerebellum(Fig. 1B). Similar expression patterns were found in primary culturedcells infected on DIV21 and observed on DIV28 (data not shown).

Aggregate formation of WT and mutant γPKC-GFP in primarycultured PCs

We examined whether mutant γPKC-GFP forms aggregates inprimary cultured PCs. Among 23 mutants found from SCA14 families,two mutant (S119P and G128D) γPKCs, which have been demon-strated to form aggregates with higher frequency (Seki et al., 2005b),were selected in the present study. By infecting adenoviral vectors onDIV14 or DIV21, WT-GFP and two mutant γPKC-GFPs (S119P-GFP andG128D-GFP) were expressed in primary cultured PCs, and GFP fluore-scence in PCs was observed on DIV28. Although aggregates were notfound in most PCs expressing WT-GFP, cytoplasmic aggregates werefrequently observed in PCs expressing S119P-GFP (Fig. 2A) or G128D-GFP (data not shown). Although several PCs had aggregates of mutantγPKC-GFP in both somata and dendrites as shown in Fig. 2A, theseaggregates were preferably formed in somata of PCs Approximately60% of S119P-GFP-expressing PCs had aggregates (59.7±3.6% of PCsinfected on DIV14, n=5, and 65.0±6.2% of PCs infected on DIV21, n=5)and approximately 50% of G128D-GFP-expressing PCs had aggregates

ative images of GFP fluorescence in living PCs expressing WT-GFP (left) and S119P-GFPtained by using a confocal laser microscope. These cells were infected with adenovirale formation ofWTandmutant γPKC-GFPs in PCs. We evaluated 100–120 PCs expressing4 (B) or DIV21 (C) and observed on DIV28. Data represent the mean percentage±SEM of0.001 vs WT (unpaired t-test).

Fig. 3. Long-term observation of mutant γPKC aggregate production and effects of cessation of mutant γPKC expression on its preformed aggregates. (A) Time-lapse observation ofGFP fluorescence in living PCs expressing S119P-GFP. Living PCs infected on DIV22 were observed from DIV26 every 30 min for 60 h. The upper images represent PCs with aggregatesof S119P-GFP that continuously enlarged. Themiddle images represent PCs that died soon after the aggregates were formed. The lower images represent PC inwhich aggregates weretransiently formed and disappeared. The time point and scale bar (20 μm) are shown on each image. (B, C) Daily observation of living PCs having aggregates of S119P-GFP by usingglass-bottomed dish with a grid. Images represent PCs without (B) and with (C) Tet (1 μg/ml) treatment on day 0 (DIV26, before treatment), day 1 (DIV27, 1 day after treatment) andday 3 (DIV29, 3 days after treatment). Images were projected from Z-stack images obtained by using a confocal laser microscope. Bar=20 μm.

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(44.1±6.9% of PCs infected on DIV14, n=5, and 48.9±5.0% of PCsinfected on DIV21, n=5), which were significantly greater than thoseof WT-GFP-expressing PCs (1.5±0.5% of PCs infected on DIV14, n=5,and 3.6±1.0% of PCs infected on DIV21, n=5) (Figs. 2B, C). These resultsindicate that mutant γPKC-GFP forms cytoplasmic aggregates inprimary cultured PCs. To exclude the possibility that excessiveexpression of mutant γPKC-GFP by adenoviral infection resulted inaggregate formation, we compared the level of exogenous γPKC-GFPwith that of endogenous γPKC. Primary cultured PCs were immunos-tained with anti γPKC antibody that detects both endogenous andexogenous γPKC. Sufficient staining was seen in PCs non-expressingmutant γPKC-GFP, compared with PCs expressing mutant γPKC-GFP,regardless of the presence of aggregation (Supplemental Fig. 1),suggesting that the expression level of exogenous γPKC-GFP in PCswere smaller than that of endogenous γPKC.

Next, we attempted to elucidate processes by which aggregates ofmutant γPKC-GFP were formed by long-term time-lapse imaging ofliving PCs. Four days after infection, sequential GFP fluorescence of PCsexpressing S119P-GFP was observed every 10 min for 60 h. During the60-h observation period, aggregates of S119P-GFP were frequentlyformed in PCs without aggregates at the beginning of the observation(in 46 out of 55 observed PCs). Three patterns of behavior wereobserved in PCs with aggregates (Fig. 3A). In the most frequentlyobserved pattern (20 out of 46 observed PCs), small aggregates in thePC soma were continuously enlarged and occupied almost the entiresoma (Fig. 3A upper panels, Supplemental movie 1). These PCs werestill alive with branched dendrites in spite of the presence of largeaggregates. In the second pattern (15 out of 46 observed PCs),aggregates in PC soma were also enlarged briefly after the start ofaggregate formation and this was followed by cell death with roundlyshaped and shedding dendrites (Fig. 3A middle panels, Supplementalmovie 2). Condensed nuclear staining was observed in these PCs,indicating that apoptosis occurred (data not shown). In the thirdpattern (11 out of 46 observed PCs), aggregates in the soma wereenlarged to some extent, but gradually disappeared (Fig. 3A lowerpanels, Supplemental movie 3). The disappearance of aggregates wasalso observed in the experiments using glass-bottomed dishes withgrids, in which the same PCs expressing S119P-GFP were observeddaily from 4 to 7 days after adenoviral infection on DIV22. Aggregatesof S119P-GFP, which were seen on the first observation day (DIV26),had spontaneously disappeared on DIV29 in a few PCs (3 out of 43observed PCs, Table 1). These results suggest that aggregates ofmutant γPKC-GFP in PCs are formed in a reversible manner and thatPCs have the ability to eliminate aggregates of mutant γPKC-GFP.

To further confirm the ability of PCs to eliminate aggregates, weinvestigated how the arrest of mutant γPKC expression affects on the

Table 1Aggregate formation and apoptosis of PCs expressing S119P-GFP

PCs on DIV26 PCs on DIV29

With aggregates Apoptosis

Without aggregates 62.9% 12.9%(39/62) (8/62)

With aggregates 90.6%a 40.6%a

(29/32) (13/32)With aggregates+Tet (1 μg/ml) 17.6%b, c 17.6%e

(6/34) (6/34)

From DIV26 to DIV29, we daily observed the same PCs without and with aggregates of S119with tetracycline (1 μg/ml) immediately after the observation on DIV26. After observation onwith nuclear staining with Hoechst 33342 to detect apoptotic PCs. We counted the numimmunoreactivity in the same PCs observed on DIV26. The table shows the percentages of inthe three left columns indicate the numbers of corresponding cells per observed cells. Parentcells.

a pb0.01 vs cells without aggregates on DIV26.b pb0.001 vs cells without aggregates on DIV26.c pb0.001 vs cells with aggregates on DIV26.d pb0.05 vs cells without aggregates on DIV26.e pb0.05 vs cells with aggregates on DIV26 (chi-square test).

preformed aggregates, since the addition of tetracycline (Tet) turns offthe gene expression in the Tet-regulated system (Fig. 1A). The samePCs expressing S119P-GFP were observed daily by using a glass-bottomed dish with a grid. After observation on the first day (day 0 inFigs. 3B, C), cells were treated with or without Tet (1 μg/ml) andcultured further 3 days. In most PCs not treated with Tet, aggregates ofS119P-GFP on day 0 were enlarged on the next day, occasionallyfollowed by cell death (Fig. 3B). On the other hand, in most Tet-treatedPCs, large aggregates observed on day 0 had become dot-likeaggregates in somata and dendrites on the next day, followed bycomplete disappearance of aggregates on day 3 (Fig. 3C, Table 1).Furthermore, in PCs without aggregates, the expression of mutantγPKC-GFP was almost completely lost 1 or 2 days after Tet treatment(data not shown). These results indicate that the ability of PCs toeliminate aggregates appeared more prominently after the cessationof mutant γPKC-GFP expression.

Aggregates of mutant γPKC-GFP induced apoptotic cell death in PCs

We next examined whether mutant γPKC-GFP induced apoptoticcell death in primary cultured PCs. Apoptotic PCs were assessed bynuclear chromatin condensation (Seki et al., 2006). Fig. 4A showsfluorescent images of S119P-GFP-expressing PCs stained with anti-calbindin antibody and with Hoechst 33342 for nuclear staining.Apoptotic cells with chromatin condensation (arrow) were frequentlyobserved in PCs having aggregates of S119P-GFP, while PCs withoutaggregates had normally stained nuclei (arrowhead). These apoptoticPCs with aggregates were immunostained with anti-activatedcaspase-3 antibody (Supplemental Fig. 2). Furthermore, apoptoticPCs were characterized by reduced or dot-like immunostaining withanti-calbindin antibody (Fig. 4A, arrow). In both cells infected onDIV14 and those infected on DIV21, the percentages of apoptotic PCswere significantly increased in PCs expressing mutant γPKC-GFP,compared with that in PCs expressing WT-GFP (Figs. 4B, C). Allapoptotic PCs had aggregates of mutant γPKC-GFP, indicating thatthese aggregates were cytotoxic to primary cultured PCs, similarly toour previous findings in CHO cells (Seki et al., 2007). The importanceof aggregate formation of mutant γPKC-GFP in PC apoptosis wasfurther confirmed by daily observation of the same PCs using glass-bottomed dishes with grids. PCs expressing S119P-GFP were observedon DIV26, 4 days after adenoviral infection on DIV22, and they werefixed on DIV29 and stained with anti-calbindin antibody and Hoechst33342. In PCs without aggregates on DIV26, all apoptotic PCs on DIV29had aggregates of S119P-GFP formed during 3-day cultivation fromDIV26 to DIV29 (Table 1). Moreover, PCs with aggregates on DIV26were more frequently apoptotic on DIV29 than those without

Surviving PCs with reduced calbindin With aggregates/apoptosis

12.9% 100%(8/62) (8/8)15.6% 100%(5/32) (13/13)0%d, e 100%(0/34) (6/6)

P-GFP cultured on a glass-bottomed dish with grid. Cells indicated as +Tet were treatedDIV29, cells were fixed and immunostained with anti-calbindin antibody concomitantlyber of PCs with aggregates, apoptotic PCs and surviving PCs with reduced calbindindividually categorized cells in the observed cells. Parentheses under the percentages inheses in the right column indicate the number of cells with aggregates per the apoptotic

Fig. 4. Apoptotic cell death in PCs expressing WT and mutant γPKC-GFP. (A) Representative GFP fluorescence (left), calbindin immunostaining (center) and nuclear staining (right)images of PCs expressing S119P-GFP. Cells were infected on DIV21, fixed on DIV28 and immunostained with anti-calbindin antibody, concomitantly with nuclear staining withHoechst 33342 (0.5 μg/ml). Cells with fragmented or condensed nuclei were regarded as apoptotic cells (arrow). All apoptotic PCs had aggregates of mutant γPKC-GFP and were lessstained with anti-calbindin antibody. Arrowhead indicates a surviving PC without aggregates of S119P-GFP and with normal nucleus. Bar=10 μm. (B, C) Quantitative analysis ofapoptotic cell death in PCs expressing WT and mutant γPKC-GFP. Cells were infected with adenoviral vectors on DIV14 (B) or DIV21 (C) and fixed on DIV28. We evaluated100–120 calbindin-positive PCs expressing γPKC-GFP per experiment. Data represent the mean percentage±SEM of apoptotic PCs from 4–5 independent experiments. ⁎⁎pb0.01 and⁎⁎⁎pb0.001 vs WT (unpaired t-test).

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aggregates. In these PCs, several surviving PCs on DIV29 withaggregates had reduced immunostainingwith anti-calbindin antibody(Table 1), indicating the possibility that reduction in calbindin mightprecede apoptosis. On the other hand, Tet-induced arrest of S119P-GFPexpression significantly rescued PCs with aggregates on DIV26 fromapoptosis (Table 1). In addition, all apoptotic PCs in Tet-treated cellshad aggregates on DIV29. These cells were not rescued by theclearance of mutant γPKC-GFP because the apoptotic process wouldhave already overtaken the executive threshold of the apoptosistriggered by aggregate accumulation. Furthermore, aggregates ofmutant γPKC-GFP in PC somata were colocalized with ubiquitin and

Table 2Effects of mutant γPKC-GFP on the morphology of PCs

DIV14-infected cells

WT-GFP S119P-GFP G

Aggregation − − + −Number of observed cells 31 29 29 28

Area (μm2) 5445±227 3826±208c 3455±186c 3829±2Dendritic area (μm2) 5060±219 3404±200c 3023±174c 3449±2Somatic area (μm2) 385±17 422±20 432±21 380±1

Maximal dendrite width (μm) 7.17±0.26 6.47±0.25 6.40±0.28 6.42±0Length of the longest dendrite (μm) 131.6±4.0 118.3±3.5a 115.3±4.4b 124.2±3Branch numbers of the longest dendrites 13.3±0.6 11.4±0.5a 11.2±0.6a 11.9±0

Cell area, dendritic area, somatic area, maximal dendrite width, lengths of the longest denγPKC-GFP were measured from images of GFP fluorescence. PCs with aggregates and those wrespectively, in the upper line of the table). Data represent means±SEM of 19–31 PCs infecte

a pb0.05.b pb0.01.c pb0.001 vs WT-GFP (unpaired t-test).

proteasome 20S immunoreactivities (Supplemental Fig. 3), similarlyto our previous findings in CHO and SH-SY5Y cells (Seki et al., 2007).These results indicate that aggregate formation of mutant γPKC-GFPhas an important role in triggering apoptosis of primary cultured PCs,probably by affecting UPS in PCs.

Morphological characteristics of PCs expressing WT and mutantγPKC-GFP

We then focused on the morphology of PCs expressing WT andmutant γPKC-GFP, especially on their dendrites. As shown in Fig. 2A

DIV21-infected cells

128D-GFP WT-GFP S119P-GFP G128D-GFP

+ − − + − +28 30 29 30 20 19

13c 3288±226c 4818±156 3829±182c 3716±151c 3841±200c 3917±242b

01c 2891±213c 4480±152 3436±182c 3345±145c 3483±185c 3505±234c

8 397±21 338±11 392±18a 370±18 358±27 412±25b

.30 6.36±0.26a 6.22±0.24 5.30±0.26a 5.41±0.26a 5.38±0.29a 5.66±0.24

.8 118.3±5.0a 109.3±2.7 97.0±4.0a 95.2±3.9b 96.9±3.9a 97.5±4.4a

.6 10.6±0.6b 12.4±0.6 9.8±0.5b 10.7±0.5a 9.6±0.6b 9.9±0.6b

drites and branch numbers of the longest dendrites of PCs expressing WT and mutantithout aggregates of mutant γPKC-GFP were separately analyzed (indicated by + and −,d on DIV14 and DIV21.

Fig. 5. Effects of mutant γPKC-GFP on dendritic spine density of PCs. (A) Representative GFP fluorescence (left), GluRδ2 immunostaining (center) and merged (right) images of PCsexpressing WT-GFP (upper) and S119P-GFP (lower), infected on DIV14. Images were projected from Z-stack images obtained by using a confocal laser microscope. Cells were fixed onDIV28 and immunostained with anti GluRδ1/2 rabbit polyclonal antibody (diluted 1:200) and with Alexa546-conjugated secondary antibody. Since GluRδ1 is not expressed in thecerebellum, anti-GluRδ1/2 antibody solely detectsGluRδ2 in cerebellar primaryculture. Bar=5 μm. (B)Quantitativeanalysis of dendritic spine densities of PCs expressingWTandmutantγPKC-GFP, infected on DIV14. In these experiments, we selectively observed PCs without any aggregates of mutant γPKC-GFP. Dots that were positive in both GFP fluorescence andGluRδ2 immunostaining were regarded as dendritic spines. Each value is the mean±SEM of 9–10 PCs (numbers in the parenthesis). ⁎⁎pb0.01 and ⁎⁎⁎pb0.001 vsWT (unpaired t-test).

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left, PCs expressingWT-GFP had highly branched dendrites, while PCsexpressing S119P-GFP had leaner dendrites with reduced branches.These tendencies were observed in both PCs without and withaggregates of G128D-GFP (data not shown). To analyze thesephenomena quantitatively, the area of whole PCs including dendritesand somata, maximal dendritic width, number of branches and lengthof the longest dendrites of PCs were measured by using fluorescenceγPKC-GFP as an index showing cell morphology (Table 2). In PCsexpressing mutant γPKC-GFP, cells with aggregates and cells withoutaggregates were analyzed separately. It is possible that γPKC-GFP,especially in PCs having aggregates of mutant γPKC-GFP, is notdistributed the edge of the dendrites of PCs. To exclude thepossibility, we coexpressed mutant γPKC-GFP with DsRed monomer.We confirmed that the distribution of γPKC-GFP with aggregates inPCs was similar to the distribution of coexpressed DsRed monomer(Supplemental Fig. 4), suggesting that the distribution of γPKC-GFPreached to the edge of PC dendrites and reflected the morphology ofPCs. Quantitative analysis of the areas of PCs was performed on

DIV28 for both PCs infected on DIV14 and those infected on DIV21.The areas of PCs expressing WT-GFP (Table 2) were comparable tothose of PCs expressing GFP (5706±230 μm2 in PCs infected onDIV14 (n=14), 5174±217 μm2 in PCs infected on DIV21 (n=23)),suggesting that overexpression of WT-GFP did not affect PC cellarea. The areas of PCs expressing mutant γPKC-GFP, especially thoseof dendrites, were significantly reduced compared with those of PCsexpressing WT-GFP (Table 2). Other parameters (maximal width,number of branches and length of the longest dendrites) were alsosignificantly reduced or tended to be reduced compared with thosein WT-GFP expressing PCs (Table 2). Intriguingly, there was nosignificant difference in any of the parameters between PCs withaggregates and those without aggregates of mutant γPKC-GFP(Table 2), indicating that mutant γPKC-GFP induces maldevelop-ment of dendrites in primary cultured PCs regardless of thepresence or absence of its aggregates.

Furthermore, we found that many PCs expressing mutant γPKC-GFP had two or more dendritic shafts from somata (Fig. 2A center and

268 T. Seki et al. / Neurobiology of Disease 33 (2009) 260–273

right), while many PCs expressing WT-GFP had a single dendrite shaft(Fig. 2A left). The number of dendritic shafts is known to be changedwith maturation of PCs (Boukhtouche et al., 2006). In the prematurestage, PCs have multiple perisomatic protrusions with a few branches,

followed by the maturation of PCs with a single and highly-brancheddendritic tree. Therefore, it is possible that mutant γPKC-GFPhampered the maturation of PCs. To examine this possibility, wecounted the number of morphologically mature PCs, which had a

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single dendrite from the soma, in cells shown in Table 2. As shown inSupplemental Table 1, the number of mature PCs was significantlyreduced or tended to be reduced in PCs expressing mutant γPKC-GFPcompared with those expressing WT-GFP. Interestingly, somatic areasof immature PCs tended to be increased compared with those ofmature PCs (Supplemental Table 1), which may be the reasonwhy thesomatic areas in PCs expressing mutant γPKC-GFP was increased(Table 2). In contrast, the dendritic areas in mature PCs and immaturePCs were similar (Supplemental Table 1).

In addition to the maldevelopment of dendrites, spine-likeprotrusions in dendrites seemed to be reduced in PCs expressingmutant γPKC-GFP without aggregates (Fig. 2A). To elucidate the effectof mutant γPKC-GFP on spine formation, spines of PC dendrites werevisualized by immunostaining of GluRδ2, which is a δ glutamatereceptor subtype specifically localized at the dendritic spines of PCswith parallel fibers (Landsend et al., 1997). GluRδ2was stained as a dotin PC dendrites, colocalized with γPKC-GFP-positive spine-likeprotrusions (Fig. 5A), indicating that these protrusions were parallelfiber-PC spines. The density of GluRδ2-positive spineswas significantlyreduced in mutant γPKC-GFP-expressing PC dendrites withoutaggregates compared with that in WT-GFP-expressing PC dendrites(Figs. 5A, B). These results suggest that mutant γPKC-GFP reducedGluRδ2-positive spines in PC dendrites, independently of aggregateformation.

Reduced translocation and mobility of mutant γPKC-GFP in primarycultured PCs

γPKC is known to be translocated from the cytosol to plasmamembrane and activated by various stimulations, including receptoractivation, increase in intracellular Ca2+ concentration and produc-tion of various lipids (Sakai et al., 1997; Shirai et al., 1998; Seki et al.,2005a). We examined the translocation of WT and mutant γPKC-GFP in PC dendrites by high potassium-induced depolarization. Inthis experiment, we observed PCs without aggregates of WT andmutant γPKC-GFP. Translocation was induced by bath application of1/20 volume of 100 mM KCl into the HEPES buffer containing 5 mMKCl. A large portion of the WT-GFP in PC dendrites was rapidlytranslocated from the cytosol to the plasma membrane by high KClstimulation, followed by returned to the cytosol within 2 min (Fig.6A upper panels, Supplemental movie 4). Although G128D-GFP inPC dendrites was also rapidly and transiently translocated to theplasma membrane, only a slight portion of G128D-GFP wastranslocated and most of the G128D-GFP remained in the cytosol(Fig. 6A lower panels, Supplemental movie 6). Similar phenomenawere also observed in PCs expressing S119P-GFP (Supplementalmovie 5). The reduction of cytosolic γPKC-GFP was measured todetermine the ‘translocation amplitude’ of WT and mutant γPKC-GFP. Figs. 6B and C show temporal changes in the cytosolicfluorescence (black line) and the fluorescence ratio (membrane/cytosol, red line) of WT-GFP and G128D-GFP, respectively, in Fig. 6A.Translocation reached a peak at the time when the fluorescenceratio reached maximum (dotted line). We quantified the transloca-tion amplitude of γPKC-GFP by the reduction of cytosolic fluores-cence at the translocation peak. The reduction of cytosolic mutant

Fig. 6. Translocation of WT andmutant γPKC-GFP in PC dendrites induced by high KCl stimul(upper panels) and G128D-GFPmutant (lower panels) before (Pre) and 10 s, 2 min and 10minon DIV28. WT-GFP was rapidly and prominently translocated from the cytosol to plasma methis translocation was subtle and the majority of cytosolic G128D-GFP was retained in thefluorescent ratio (membrane/cytosol, red line) of WT-GFP (B) and G128D-GFP (C) in PC dendrGFP at the translocation peak, when the fluorescence ratio reachedmaximum, as the index foofWTandmutant γPKC-GFP in PC dendrites. Data represent means±SEM of the reduction inγPKC-GFP were significantly less than those of WT. ⁎pb0.05 vs WT (unpaired t-test). (E) TemG128D-GFP (blue) in response to high KCl stimulation. Data represent mean percentage ofG128D-GFP: n=7).

γPKC-GFP was significantly smaller than that of WT-GFP (Fig. 6D).These results suggest that the translocation ability of mutant γPKC-GFP is attenuated in PC dendrites.

It is possible that this attenuated translocation of mutant γPKC-GFP is due to impairment in the regulation of intracellular Ca2+

concentration ([Ca2+]i) in PCs expressing mutant γPKC-GFP. Toexamine this possibility, changes in [Ca2+]i in response to high KClstimulation were measured in PC dendrites expressing WT andmutant γPKC-GFP by using calcium green-1 (CG1) as an intracellularCa2+ indicator. As shown in Fig. 6E, [Ca2+]i in PC dendrites was elevatedimmediately after the stimulation, followed by a gradual decline to thebasal level. These changes in [Ca2+]i were similar in PCs expressingWT-GFP, S119P-GFP and G128D-GFP, indicating that the attenuatedtranslocation of mutant γPKC-GFP is not caused by the dysregulationof [Ca2+]i.

To elucidate the molecular basis of the attenuated translocationof mutant γPKC-GFP, the mobilities of WT and mutant γPKC-GFPwere examined by fluorescence recovery after photobleaching(FRAP) analysis (Meyvis et al., 1999). In this experiment, weobserved PCs without aggregates of WT and mutant γPKC-GFP.After photobleaching by irradiation of a strong excitation laser inthe WT-GFP-expressing PC soma, the fluorescence of WT-GFP waslost at the photobleached area (red dot circle) immediately afterphotobleaching (Fig. 7A). However, the fluorescence at the photo-bleached area was almost completely recovered 4 s after photo-bleaching by the rapid influx of surrounding unbleached WT-GFP(Fig. 7A upper panels, Supplemental movie 7). In contrast, thebleached area of the PC soma expressing S119P-GFP was stillreduced 4 s after photobleaching and the recovery of fluorescencewas retarded (Fig. 7A lower panels, Supplemental movie 8). Similarphenomena were also observed in PCs expressing G128D-GFP(Supplemental movie 9). Fig. 7B shows temporal changes in thefluorescence of WT and mutant γPKC-GFP in the PC somata. Whilethe fluorescence of WT-GFP (black line) rapidly recovered afterphotobleaching, the fluorescence recovery of S119P-GFP (red line)and G128D-GFP (blue line) was markedly delayed. Half times offluorescence recovery in PC somata expressing S119P-GFP andG128D-GFP were significantly longer than that of PC somataexpressing WT-GFP (Fig. 7C). Similar results were obtained byFRAP analyses in PC dendrites (Figs. 7D, E). Furthermore, nativePAGE experiments revealed that most mutant γPKC-GFP formedhigh molecular weight species, while WT-GFP mainly existed asmonomer or dimer in SH-SY5Y cells (Supplemental Fig. 5). Theseresults suggest that the mobility of mutant γPKC-GFP is markedlyreduced in PC somata and dendrites by the formation of highmolecular weight species, leading to its attenuated translocationupon high KCl stimulation.

It is plausible that this reduced mobility of mutant γPKC-GFP iscaused by oligomer formation. To address the issue, primarycultured PCs were immunostained with anti-amyloid oligomerantibody (A11). The A11 antibody is known to recognize thecommon structure of soluble amyloid oligomers derived fromvarious aggregate-prone proteins, including amyloid β protein, α-synuclein and expanded polyglutamine repeat (Kayed et al., 2003;Nagai et al., 2007). PCs expressing WT-GFP were only weakly

ation. (A) Representative images of GFP fluorescence in PC dendrites expressingWT-GFPafter high KCl (100mMKCl, 50 μl) stimulation. PCs were infected on DIV21 and observedmbrane. Although G128D-GFP was also rapidly translocated to the plasma membrane,cytosol. Bar=5 μm. (B, C) Temporal changes in fluorescent intensity (black line) and

ites shown in A. We evaluated the reduction in fluorescence intensity of cytosolic γPKC-r translocation amplitude (B and C). (D) Quantitative analyses of translocation intensitiesfluorescent intensity at the translocation peak (n=8). Translocation intensities of mutantporal changes in [Ca2+]i of PC dendrites expressing WT-GFP (black), S119P-GFP (red) andthe fluorescence intensity of CG1 before stimulation (WT-GFP: n=8, S119P-GFP: n=10,

Fig. 7. FRAP analyses of WTandmutant γPKC-GFP in PCs. (A) Representative images of GFP fluorescence in PC somata expressingWT-GFP (upper) and S119P-GFP (lower) before (Pre)and 0.2, 2 and 20 s after photobleaching. Bleached areas were shown as red dot circles in images before photobleaching. Cells were infected on DIV21 and observed on DIV28.Bar=2 μm. (B–E) Temporal changes in fluorescent intensities (B, D) and half times of fluorescent recoveries (C, E) in bleached areas in PC somata (B, C) and dendrites (D, E) expressingWTandmutant γPKC-GFP. In (B and D) black, red and blue lines represent average fluorescent intensities of WT-GFP, S119P-GFP and G128D-GFP, respectively. In (C and E) half time ofrecovery was calculated by fitting the changes in fluorescent intensities after photobleaching to single exponential functions. Data represent means±SEM of 7–13 PCs. ⁎pb0.05,⁎⁎pb0.01 and ⁎⁎⁎pb0.001 vs WT (unpaired t-test).

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stained with anti-amyloid oligomer antibody (Fig. 8, upper panels),while PCs expressing G128D-GFP (Fig. 8, lower panels) and S119P-GFP (data not shown) were prominently stained, especially in the

dendritic shafts. As aggregates of mutant γPKC-GFP were notdetected (Supplemental Fig. 6), this antibody specifically recognizesoligomer formation of mutant γPKC, indicating that mutant γPKC

Fig. 8. Oligomer formation of mutant γPKC-GFP in PCs. (A) Representative GFP fluorescence (left), amyloid oligomer immunostaining (center) and merged (right) images of PCsexpressingWT-GFP (upper) and G128D-GFP (lower), infected on DIV21. Imageswere projected from Z-stack images obtained by using a confocal laser microscope. Cells were fixed onDIV28 and immunostained with anti amyloid oligomer rabbit polyclonal antibody (diluted 1:1000) and with Alexa546-conjugated secondary antibody. Bar=20 μm.

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more preferably forms soluble oligomers with amyloid structurethan WT γPKC.

Discussion

In the present study, we demonstrated that aggregates of mutantγPKC were also formed in primary cultured PCs transfected byadenoviral vectors (Fig. 2) and that these aggregates were ubiquitin-positive and colocalized with proteasome (Supplemental Fig. 3). Wealso revealed that apoptotic cell death preferably occurred in PCshaving aggregates of mutant γPKC (Fig. 4). Colocalization ofaggregates with proteasome has been previously reported in PCs ofmodel mice of other SCAs caused by polyglutamine proteins(Cummings et al., 1998; Yvert et al., 2000). These findings indicatethat there is a common pathogenic mechanism among various SCAs,which might be impairment of the ubiquitin–proteasome system andinduction of ER stress by the aggregate formation of mutant proteins.Similar aggregate formation is commonly observed in variousneurodegenerative disorders (Taylor et al., 2002; Ross and Poirier,2004). It has been contended that aggregates of mutant proteins areformed as a result of a cellular protective mechanism against the toxicmonomer or oligomer of mutant proteins (Agorogiannis et al., 2004;Ardley et al., 2005). In the present study, apoptosis was frequentlyobserved in PCs having aggregates of mutant γPKC but not in PCswithout aggregates (Fig. 4, Table 1), suggesting that aggregates ofmutant γPKC are an inducer for apoptosis of primary cultured PCs. Incontrast to our findings, no aggregates of γPKC were found incerebellar PCs of autopsy brain tissue of a SCA14 patient with H101Ymutation (Chen et al., 2003), indicating that non-aggregated γPKCexerts toxic effect on PCs. In the present study, we also revealed thatmutant γPKC induces aggregate-independent maldevelopment ofdendrites and reduction in dendritic spines in PCs (Table 2, Fig. 5).Further experiments were necessary to elucidate precise roles ofaggregated γPKC in pathogenesis of SCA14.

In the present study, long-term time-lapse imaging and dailyobservation of the same PCs revealed that aggregate formation ofmutant γPKC was reversible and was not the critical point forinduction of apoptosis (Fig. 3, Table 1). It has been reported that

various pathologies of neurodegenerative disorders could be reversedafter the onset of disease by cessation of mutant protein expression inconditional mice models of polyglutamine diseases (Yamamoto et al.,2000; Li et al., 2007). These findings indicate the possibility thatreduction in the expression level of mutant proteinsmight be effectivein the treatment of neurodegenerative diseases even after the onset ofdisease. Also, loss of mutant γPKC aggregates was observed in severalPCs continuously expressing mutant γPKC (Fig. 3A, Table 1, Supple-mental movie 3), suggesting that PCs have a potential to eliminateaggregates.

Apoptotic PCs showed reduced immunoreactivity with anti-calbindin antibody (Fig. 4A). A similar tendency was found inapoptotic PCs expressing WT γPKC (data not shown). A similarobservation was reported in autopsies of patients with familial orsporadic spinocerebellar degeneration (Ishikawa et al., 1995). Reduc-tion in calbindin immunoreactivity was also observed in surviving PCswith aggregates of mutant γPKC (Table 1), indicating that its reductionproceeds or triggers apoptosis of PCs. Calbindin is thought to beimportant for maintaining the intracellular calcium homeostasis(Roberts, 1993) and for protecting neurons from calcium-mediatedneurotoxicity (Mattson et al., 1991). Therefore, reduced calbindinexpression might increase the vulnerability to neurotoxicity and becommonly involved in the neurodegeneration of PCs in spinoce-rebellar degenerations.

PCs expressing mutant γPKC showed morphological changes,maldevelopment of dendrites with reduced branches (Table 2),regardless of the presence or absence of aggregates. This resultsuggests that these morphological changes are induced by alteredproperties of mutant γPKC other than aggregate formation. Activa-tion of PKC, especially γPKC, has been reported to negatively regulatethe development of PC dendrites in rat cerebellar slice cultures(Metzger and Kapfhammer, 2000; Schrenk et al., 2002). Therefore,changes in kinase activity by the mutation might affect the dendriticdevelopment of PCs expressing mutant γPKC. Indeed, Verbeek et al.reported that mutations (G118D and C150F) increased the kinaseactivity of γPKC (Verbeek et al., 2005a). We have also found that thebasal kinase activity was elevated in most mutant γPKCs, whileseveral mutations decreased or did not alter the kinase activity of

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γPKC (Adachi et al., 2008). Since basal activities of mutant γPKCexamined in the present study (S119P and G128D) were alsoincreased, these increased activities of mutant γPKC might triggerthe maldevelopment of PC dendrites. However, Lin et al. reportedthat mutant (H101Y, S119P and G128D) γPKC expression reduced thekinase activity of endogenous γPKC, leading to vulnerability of theexpressed cells to oxidative stress (Lin et al., 2007). Verbeek et al. alsoreported that mutant (G118D, V138E, C142S) γPKC showed reducedphorbol ester-induced kinase activation at plasma membrane(Verbeek et al., 2008). At present, we cannot determine whetherthe change in kinase activity of mutant γPKC affects the dendriticdevelopment of primary cultured PCs.

Loss of dendritic spines has been demonstrated to be involved inthe early phase of various neurodegenerative disorders (Selkoe, 2002;Spires et al., 2004; Fuhrmann et al., 2007). The present study alsodemonstrated that GluRδ2-positive spines were reduced in dendritesof PCs expressing mutant γPKC (Figs. 5A, B). GluRδ2 is predominantlyexpressed in cerebellar PCs at the postsynaptic sites with parallel fiber(PF) from cerebellar granule cells (Landsend et al., 1997), whichoccupy over 90% of all dendritic synapses of PCs (Sotelo, 2004).Maldevelopment of PC dendrites might decrease the densities ofdendritic spines by reducing opportunity to encounter the terminalfrom granule cells in cerebellar primary culture. In addition, GluRδ2-mediated synapse formation between PFs and PCs has been reportedto be essential for the dendritic development of PCs (Kurihara et al.,1997; Sotelo, 2004), suggesting that the loss of PF-PC synapses mightfurther exacerbate maldevelopment of PC dendrites by mutant γPKC.

We demonstrated attenuated translocation ability of mutantγPKC in PC dendrites by high KCl stimulation (Fig. 6, Supplementalmovie 4–6). The translocation of γPKC has an important role in signaltransduction along PC dendrites, since γPKC translocation triggered bytrans-synaptic stimulus was propagated along the dendrites of PCs inγPKC-GFP transgenic mice (Sakai et al., 2004). The attenuatedtranslocation ability of mutant γPKC would affect the signaltransduction from dendrites to somata of PCs. This impaired signaltransduction might trigger the maldevelopment of dendrites and thedecrease in dendritic spines in PCs expressing mutant γPKC.Furthermore, Ca2+ imaging experiments revealed that the high KCl-induced [Ca2+]i elevation in PC dendrites was not affected by theexpression of mutant γPKC (Fig. 6E), suggesting that the attenuatedtranslocation of mutant γPKC is not due to the dysregulation of [Ca2+]i.Therefore, we focused on the mobility of mutant γPKC in PCs. FRAPanalyses revealed that mobility of soluble mutant γPKC was reducedin PC somata and dendrites (Fig. 7, Supplemental movie 6–9),indicating that the insufficient translocation of mutant γPKC is dueto reduced mobility by its oligomer formation (Fig. 8, SupplementalFig. 5). Oligomers with amyloid structure of mutant proteins arefrequently formed before aggregate formation in various neurode-generative diseases (Glabe, 2006). Recent evidences have revealedthat soluble oligomers aremore toxic than visible aggregates (Arrasateet al., 2004; Li et al., 2007). Based on these findings, it is possible thatsoluble oligomers of mutant γPKC might cause aberrant morphologyof PC dendrites.

This attenuated translocation of mutant γPKC is opposite to arecent report by Verbeek et al. (2008), showing that mutant γPKC wasrapidly and strongly translocated to plasma membrane in HeLa cells.They emphasized that accelerated translocation was caused byconformational alteration of mutant γPKC. We also observed thatmutant γPKC was normally translocated tomembrane by the receptorstimulation in CHO (Seki et al., 2005b) and SH-SY5Y cells (data notshown). We do not have an answer that can clearly account for thiscontradiction, however, one possible explanation is the difference inthe experimental conditions, especially used cells. We also observedthat the reduction of mobility was much milder when the mutantγPKC was expressed in cell lines, such as SH-SY5Y cell than in PCs. Inthis regard, the mobility of mutant γPKCmaymore prominently affect

in PC cells than cell lines and overcome the effect of its conformationalalteration, leading to attenuated translocation in PCs.

Since SCA14 is inherited in an autosomal dominant manner, it isconsidered that SCA14 is caused by gain of toxic function of mutantγPKC. However, the findings, the attenuated translocation of mutantγPKC, propose the possibility that loss of function of mutant γPKC isrelated to the pathogenesis of SCA14. While γPKC knockout miceshows only slight ataxic phenotype and normal cerebellar morpho-logy, other PKC subtype might compensate lost functions of γPKC. Incase of SCA14, it is possible that endogenously expressed wild typeγPKC and other PKC subtypes are incorporated into soluble oligomersof mutant γPKC, leading to totally loss of PKC function in PCs affectedwith SCA14. Our preliminary experiments revealed that mutant γPKCforms soluble oligomer with wild type γPKC and other PKC subtypes(data not shown). Further studies are necessary to elucidate therelationship of mutant γPKC to functions of endogenous γPKC andother PKC subtype.

Acknowledgments

This study was supported by a Grant-in-Aid for Scientific Researchfrom the Ministry of Education, Sports and Culture and by grants fromTakeda Science Foundation, the Uehara Memorial Foundation, theNaito Foundation, Suzuken Memorial Foundation, the Tokyo Bioche-mical Research Foundation and the Japanese Smoking ResearchAssociation. Thisworkwas carried out using equipment at the AnalysisCenter of Life Science, Hiroshima University and the Research Centerfor Molecular Medicine, Faculty of Medicine, Hiroshima University.

Appendix A. Supplementary data

Supplementary data associated with this article can be found, inthe online version, at doi:10.1016/j.nbd.2008.10.013.

References

Adachi, N., Kobayashi, T., Takahashi, H., Kawasaki, T., Shirai, Y., Ueyama, T., Matsuda, T.,Seki, T., Sakai, N., Saito, N., 2008. Enzymological analysis of mutant protein kinaseC{gamma} causing spinocerebellar ataxia type 14 and dysfunction in Ca2+homeostasis. J. Biol. Chem. 283, 19854–19863.

Agorogiannis, E.I., Agorogiannis, G.I., Papadimitriou, A., Hadjigeorgiou, G.M., 2004.Protein misfolding in neurodegenerative diseases. Neuropathol. Appl. Neurobiol.30, 215–224.

Alonso, I., Costa, C., Gomes, A., Ferro, A., Seixas, A.I., Silva, S., Cruz, V.T., Coutinho, P.,Sequeiros, J., Silveira, I., 2005. A novel H101Q mutation causes PKCgamma loss inspinocerebellar ataxia type 14. J. Hum. Genet. 50, 523–529.

Ardley, H.C., Hung, C.C., Robinson, P.A., 2005. The aggravating role of the ubiquitin-proteasome system in neurodegeneration. FEBS Lett. 579, 571–576.

Arrasate, M., Mitra, S., Schweitzer, E.S., Segal, M.R., Finkbeiner, S., 2004. Inclusion bodyformation reduces levels of mutant huntingtin and the risk of neuronal death.Nature 431, 805–810.

Boukhtouche, F., Janmaat, S., Vodjdani, G., Gautheron, V., Mallet, J., Dusart, I., Mariani, J.,2006. Retinoid-related orphan receptor alpha controls the early steps of Purkinjecell dendritic differentiation. J. Neurosci. 26, 1531–1538.

Cagnoli, C., Mariotti, C., Taroni, F., Seri, M., Brussino, A., Michielotto, C., Grisoli, M., DiBella, D., Migone, N., Gellera, C., Di Donato, S., Brusco, A., 2006. SCA28, a novel formof autosomal dominant cerebellar ataxia on chromosome 18p11.22-q11.2. Brain129, 235–242.

Chen, C., Kano,M., Abeliovich, A., Chen, L., Bao, S., Kim, J.J., Hashimoto, K., Thompson, R.F.,Tonegawa, S.,1995. Impairedmotor coordination correlateswith persistentmultipleclimbing fiber innervation in PKC gamma mutant mice. Cell 83, 1233–1242.

Chen, D.H., Brkanac, Z., Verlinde, C.L., Tan, X.J., Bylenok, L., Nochlin, D., Matsushita, M.,Lipe, H., Wolff, J., Fernandez, M., Cimino, P.J., Bird, T.D., Raskind, W.H., 2003.Missense mutations in the regulatory domain of PKC gamma: a newmechanism fordominant nonepisodic cerebellar ataxia. Am. J. Hum. Genet. 72, 839–849.

Chen, D.H., Cimino, P.J., Ranum, L.P., Zoghbi, H.Y., Yabe, I., Schut, L., Margolis, R.L., Lipe,H.P., Feleke, A., Matsushita, M., Wolff, J., Morgan, C., Lau, D., Fernandez, M., Sasaki,H., Raskind, W.H., Bird, T.D., 2005. The clinical and genetic spectrum ofspinocerebellar ataxia 14. Neurology 64, 1258–1260.

Chen, J., Kelz, M.B., Zeng, G., Sakai, N., Steffen, C., Shockett, P.E., Picciotto, M.R., Duman,R.S., Nestler, E.J., 1998. Transgenic animals with inducible, targeted gene expressionin brain. Mol. Pharmacol. 54, 495–503.

Cummings, C.J., Mancini, M.A., Antalffy, B., DeFranco, D.B., Orr, H.T., Zoghbi, H.Y., 1998.Chaperone suppression of aggregation and altered subcellular proteasomelocalization imply protein misfolding in SCA1. Nat. Genet. 19, 148–154.

273T. Seki et al. / Neurobiology of Disease 33 (2009) 260–273

Dalski, A., Mitulla, B., Burk, K., Schattenfroh, C., Schwinger, E., Zuhlke, C., 2006. Mutationof the highly conserved cysteine residue 131 of the SCA14 associated PRKCG gene ina family with slow progressive cerebellar ataxia. J. Neurol. 253, 1111–1112.

Duenas, A.M., Goold, R., Giunti, P., 2006. Molecular pathogenesis of spinocerebellarataxias. Brain 129, 1357–1370.

Everett, C.M., Wood, N.W., 2004. Trinucleotide repeats and neurodegenerative disease.Brain 127, 2385–2405.

Fuhrmann, M., Mitteregger, G., Kretzschmar, H., Herms, J., 2007. Dendritic pathology inprion disease starts at the synaptic spine. J. Neurosci. 27, 6224–6233.

Glabe, C.G., 2006. Common mechanisms of amyloid oligomer pathogenesis indegenerative disease. Neurobiol. Aging 27, 570–575.

Hiramoto, K., Kawakami, H., Inoue, K., Seki, T., Maruyama, H., Morino, H., Matsumoto,M., Kurisu, K., Sakai, N., 2006. Identification of a new family of spinocerebellarataxia type 14 in the Japanese spinocerebellar ataxia population by the screening ofPRKCG exon 4. Mov. Disord. 21, 1355–1360.

Houlden, H., Johnson, J., Gardner-Thorpe, C., Lashley, T., Hernandez, D., Worth, P.,Singleton, A.B., Hilton, D.A., Holton, J., Revesz, T., Davis, M.B., Giunti, P., Wood, N.W.,2007. Mutations in TTBK2, encoding a kinase implicated in tau phosphorylation,segregate with spinocerebellar ataxia type 11. Nat. Genet. 39, 1434–1436.

Ishikawa, K., Mizusawa, H., Fujita, T., Ohkoshi, N., Doi, M., Komatsuzaki, Y., Iwamoto, H.,Ogata, T., Shoji, S., 1995. Calbindin-D 28 k immunoreactivity in the cerebellum ofspinocerebellar degeneration. J. Neurol. Sci. 129, 179–185.

Kano, M., Hashimoto, K., Chen, C., Abeliovich, A., Aiba, A., Kurihara, H., Watanabe, M.,Inoue, Y., Tonegawa, S., 1995. Impaired synapse elimination during cerebellardevelopment in PKC gamma mutant mice. Cell 83, 1223–1231.

Kayed, R., Head, E., Thompson, J.L., McIntire, T.M., Milton, S.C., Cotman, C.W., Glabe, C.G.,2003. Common structure of soluble amyloid oligomers implies common mecha-nism of pathogenesis. Science 300, 486–489.

Klebe, S., Durr, A., Rentschler, A., Hahn-Barma, V., Abele, M., Bouslam, N., Schols, L.,Jedynak, P., Forlani, S., Denis, E., Dussert, C., Agid, Y., Bauer, P., Globas, C., Wullner, U.,Brice, A., Riess, O., Stevanin, G., 2005. New mutations in protein kinase Cgammaassociated with spinocerebellar ataxia type 14. Ann. Neurol. 58, 720–729.

Klebe, S., Faivre, L., Forlani, S., Dussert, C., Tourbah, A., Brice, A., Stevanin, G., Durr, A.,2007. Another mutation in cysteine 131 in protein kinase C gamma as a cause ofspinocerebellar ataxia type 14. Arch. Neurol. 64, 913–914.

Kurihara, H., Hashimoto, K., Kano, M., Takayama, C., Sakimura, K., Mishina, M., Inoue, Y.,Watanabe, M., 1997. Impaired parallel fiber→Purkinje cell synapse stabilizationduring cerebellar development of mutant mice lacking the glutamate receptordelta2 subunit. J. Neurosci. 17, 9613–9623.

Landsend, A.S., Amiry-Moghaddam, M., Matsubara, A., Bergersen, L., Usami, S.,Wenthold, R.J., Ottersen, O.P., 1997. Differential localization of delta glutamatereceptors in the rat cerebellum: coexpressionwith AMPA receptors in parallel fiber-spine synapses and absence from climbing fiber-spine synapses. J. Neurosci. 17,834–842.

Li, M., Chevalier-Larsen, E.S., Merry, D.E., Diamond, M.I., 2007. Soluble androgenreceptor oligomers underlie pathology in a mouse model of spinobulbar muscularatrophy. J. Biol. Chem. 282, 3157–3164.

Lin, D., Shanks, D., Prakash, O., Takemoto, D.J., 2007. Protein kinase C gamma mutationsin the C1B domain cause caspase-3-linked apoptosis in lens epithelial cells throughgap junctions. Exp. Eye Res. 85, 113–122.

Martin, J.B., 1999. Molecular basis of the neurodegenerative disorders. N. Engl. J. Med.340, 1970–1980.

Mattson, M.P., Rychlik, B., Chu, C., Christakos, S., 1991. Evidence for calcium-reducingand excito-protective roles for the calcium-binding protein calbindin-D28 k incultured hippocampal neurons. Neuron 6, 41–51.

Metzger, F., Kapfhammer, J.P., 2000. Protein kinase C activity modulates dendriticdifferentiation of rat Purkinje cells in cerebellar slice cultures. Eur. J. Neurosci. 12,1993–2005.

Meyvis, T.K., De Smedt, S.C., Van Oostveldt, P., Demeester, J., 1999. Fluorescence recoveryafter photobleaching: a versatile tool for mobility and interaction measurements inpharmaceutical research. Pharm. Res. 16, 1153–1162.

Nagai, Y., Inui, T., Popiel, H.A., Fujikake, N., Hasegawa, K., Urade, Y., Goto, Y., Naiki, H.,Toda, T., 2007. A toxic monomeric conformer of the polyglutamine protein. Nat.Struct. Mol. Biol. 14, 32–340.

Nolte, D., Landendinger, M., Schmitt, E., Muller, U., 2007. Spinocerebellar ataxia 14:novel mutation in exon 2 of PRKCG in a German family. Mov. Disord. 22,265–267.

Roberts, W.M., 1993. Spatial calcium buffering in saccular hair cells. Nature 363, 74–76.Ross, C.A., Poirier, M.A., 2004. Protein aggregation and neurodegenerative disease. Nat.

Med. 10 (Suppl.), S10–S17.Saito, N., Kikkawa, U., Nishizuka, Y., Tanaka, C., 1988. Distribution of protein kinase C-like

immunoreactive neurons in rat brain. J. Neurosci. 8, 369–382.Sakai, N., Sasaki, K., Ikegaki, N., Shirai, Y., Ono, Y., Saito, N., 1997. Direct visualization of

the translocation of the gamma-subspecies of protein kinase C in living cells usingfusion proteins with green fluorescent protein. J. Cell Biol. 139, 1465–1476.

Sakai, N., Thome, J., Newton, S.S., Chen, J., Kelz, M.B., Steffen, C., Nestler, E.J., Duman, R.S.,2002. Inducible and brain region-specific CREB transgenic mice. Mol. Pharmacol. 61,1453–1464.

Sakai, N., Tsubokawa, H., Matsuzaki, M., Kajimoto, T., Takahashi, E., Ren, Y., Ohmori, S.,Shirai, Y., Matsubayashi, H., Chen, J., Duman, R.S., Kasai, H., Saito, N., 2004.Propagation of gammaPKC translocation along the dendrites of Purkinje cell ingammaPKC-GFP transgenic mice. Genes Cells 9, 945–957.

Schols, L., Bauer, P., Schmidt, T., Schulte, T., Riess, O., 2004. Autosomal dominantcerebellar ataxias: clinical features, genetics, and pathogenesis. Lancet Neurol. 3,291–304.

Schrenk, K., Kapfhammer, J.P., Metzger, F., 2002. Altered dendritic development ofcerebellar Purkinje cells in slice cultures from protein kinase Cgamma-deficientmice. Neuroscience 110, 675–689.

Seki, T., Matsubayashi, H., Amano, T., Shirai, Y., Saito, N., Sakai, N., 2005a. Phosphoryla-tion of PKC activation loop plays an important role in receptor-mediatedtranslocation of PKC. Genes Cells 10, 225–239.

Seki, T., Adachi, N., Ono, Y., Mochizuki, H., Hiramoto, K., Amano, T., Matsubayashi, H.,Matsumoto, M., Kawakami, H., Saito, N., Sakai, N., 2005b. Mutant protein kinaseCgamma found in spinocerebellar ataxia type 14 is susceptible to aggregation andcauses cell death. J. Biol. Chem. 280, 29096–29106.

Seki, T., Irie, N., Nakamura, K., Sakaue, H., Ogawa,W., Kasuga, M., Yamamoto, H., Ohmori,S., Saito, N., Sakai, N., 2006. Fused protein of deltaPKC activation loop and PDK1-interacting fragment (deltaAL-PIF) functions as a pseudosubstrate and an inhibitorymolecule for PDK1 when expressed in cells. Genes Cells 11, 1051–1070.

Seki, T., Takahashi, H., Adachi, N., Abe, N., Shimahara, T., Saito, N., Sakai, N., 2007.Aggregate formation of mutant protein kinase C gamma found in spinocerebellarataxia type 14 impairs ubiquitin-proteasome system and induces endoplasmicreticulum stress. Eur. J. Neurosci. 26, 3126–3140.

Selkoe, D.J., 2002. Alzheimer's disease is a synaptic failure. Science 298, 789–791.Shirai, Y., Kashiwagi, K., Yagi, K., Sakai, N., Saito, N., 1998. Distinct effects of fatty acids on

translocation of gamma- and epsilon- subspecies of protein kinase C. J. Cell Biol.143, 511–521.

Sotelo, C., 2004. Cellular and genetic regulation of the development of the cerebellarsystem. Prog. Neurobiol. 72, 295–339.

Spires, T.L., Grote, H.E., Garry, S., Cordery, P.M., Van Dellen, A., Blakemore, C., Hannan,A.J., 2004. Dendritic spine pathology and deficits in experience-dependentdendritic plasticity in R6/1 Huntington's disease transgenic mice. Eur. J. Neurosci.19, 2799–2807.

Stevanin, G.,Hahn,V., Lohmann,E., Bouslam,N.,Gouttard,M., Soumphonphakdy, C.,Welter,M.L., Ollagnon-Roman, E., Lemainque, A., Ruberg,M., Brice, A., Durr, A., 2004.Mutationin the catalytic domain of protein kinase C gamma and extension of the phenotypeassociated with spinocerebellar ataxia type 14. Arch. Neurol. 61, 1242–1248.

Taylor, J.P., Hardy, J., Fischbeck, K.H., 2002. Toxic proteins in neurodegenerative disease.Science 296, 1991–1995.

van de Leemput, J., Chandran, J., Knight, M.A., Holtzclaw, L.A., Scholz, S., Cookson, M.R.,Houlden, H., Gwinn-Hardy, K., Fung, H.C., Lin, X., Hernandez, D., Simon-Sanchez, J.,Wood, N.W., Giunti, P., Rafferty, I., Hardy, J., Storey, E., Gardner, R.J., Forrest, S.M.,Fisher, E.M., Russell, J.T., Cai, H., Singleton, A.B., 2007. Deletion at ITPR1 underliesataxia in mice and spinocerebellar ataxia 15 in humans. PLoS Genet. 3, e108.

van de Warrenburg, B.P., Verbeek, D.S., Piersma, S.J., Hennekam, F.A., Pearson, P.L.,Knoers, N.V., Kremer, H.P., Sinke, R.J., 2003. Identification of a novel SCA14mutationin a Dutch autosomal dominant cerebellar ataxia family. Neurology 61, 1760–1765.

Verbeek, D.S., Knight, M.A., Harmison, G.G., Fischbeck, K.H., Howell, B.W., 2005a. Proteinkinase C gammamutations in spinocerebellar ataxia 14 increase kinase activity andalter membrane targeting. Brain 128, 436–442.

Verbeek, D.S., Warrenburg, B.P., Hennekam, F.A., Dooijes, D., Ippel, P.F., Verschuuren-Bemelmans, C.C., Kremer, H.P., Sinke, R.J., 2005b. Gly118Asp is a SCA14 foundermutation in the Dutch ataxia population. Hum. Genet. 117, 88–91.

Verbeek, D.S., Goedhart, J., Bruinsma, L., Sinke, R.J., Reits, E.A., 2008. PKC{gamma}mutations in spinocerebellar ataxia type 14 affect C1 domain accessibility andkinase activity leading to aberrant MAPK signaling. J. Cell. Sci. 121, 2339–2349.

Vlak, M.H., Sinke, R.J., Rabelink, G.M., Kremer, B.P., van de Warrenburg, B.P., 2006. NovelPRKCG/SCA14 mutation in a Dutch spinocerebellar ataxia family: expanding thephenotype. Mov. Disord. 21, 1025–1028.

Wieczorek, S., Arning, L., Gizewski, E.R., Alheite, I., Timmann, D., 2007. Benign SCA14phenotype in a German patient associated with a missense mutation in exon 3 ofthe PRKCG gene. Mov. Disord. 22, 2135–2136.

Yabe, I., Sasaki, H., Chen, D.H., Raskind, W.H., Bird, T.D., Yamashita, I., Tsuji, S., Kikuchi, S.,Tashiro, K., 2003. Spinocerebellar ataxia type 14 caused by a mutation in proteinkinase C gamma. Arch. Neurol. 60, 1749–1751.

Yamamoto, A., Lucas, J.J., Hen, R., 2000. Reversal of neuropathology and motordysfunction in a conditional model of Huntington's disease. Cell 101, 57–66.

Yamashita, I., Sasaki, H., Yabe, I., Fukazawa, T., Nogoshi, S., Komeichi, K., Takada, A.,Shiraishi, K., Takiyama, Y., Nishizawa, M., Kaneko, J., Tanaka, H., Tsuji, S., Tashiro, K.,2000. A novel locus for dominant cerebellar ataxia (SCA14) maps to a 10.2-cMinterval flanked by D19S206 and D19S605 on chromosome 19q13.4-qter. Ann.Neurol. 48, 156–163.

Yvert, G., Lindenberg, K.S., Picaud, S., Landwehrmeyer, G.B., Sahel, J.A., Mandel, J.L., 2000.Expanded polyglutamines induce neurodegeneration and trans-neuronal altera-tions in cerebellum and retina of SCA7 transgenic mice. Hum. Mol. Genet. 9,2491–2506.