polyglutamine expansion causes neurodegeneration by altering the neuronal differentiation program

Polyglutamine expansion causesneurodegeneration by altering the neuronaldifferentiation program

Gretta Abou-Sleymane1,4, Frederic Chalmel3,{,{, Dominique Helmlinger1,{,}, Aurelie Lardenois3,

Christelle Thibault1, Chantal Weber1,4, Karine Merienne1,4, Jean-Louis Mandel1,4, Olivier Poch3,

Didier Devys1,2,4 and Yvon Trottier1,4,*

1Department of Molecular Pathology, 2Department of Transcription and 3Department of Structural Genomic and

Biology, Institut de Genetique et Biologie Moleculaire et Cellulaire (IGBMC), CNRS/INSERM/ULP, BP10142, 67404

Illkirch Cedex, CU de Strasbourg, France and 4Chaire de Genetique Humaine, College de France, France

Received October 10, 2005; Revised and Accepted January 11, 2006

Huntington’s disease (HD) and spinocerebellar ataxia type 7 (SCA7) belong to a group of inherited neuro-degenerative diseases caused by polyglutamine (polyQ) expansion in corresponding proteins.Transcriptional alteration is a unifying feature of polyQ disorders; however, the relationship betweenpolyQ-induced gene expression deregulation and degenerative processes remains unclear. R6/2 and R7Emouse models of HD and SCA7, respectively, present a comparable retinal degeneration characterized byprogressive reduction of electroretinograph activity and important morphological changes of rod photo-receptors. The retina, which is a simple central nervous system tissue, allows correlating functional, morpho-logical and molecular defects. Taking advantage of comparing polyQ-induced degeneration in two retinamodels, we combined gene expression profiling and molecular biology techniques to decipher the molecularpathways underlying polyQ expansion toxicity. We show that R7E and R6/2 retinal phenotype strongly cor-relates with loss of expression of a large cohort of genes specifically involved in phototransduction functionand morphogenesis of differentiated rod photoreceptors. Accordingly, three key transcription factors (Nrl,Crx and Nr2e3) controlling rod differentiation genes, hence expression of photoreceptor specific traits, aredown-regulated. Interestingly, other transcription factors known to cause inhibitory effects on photoreceptordifferentiation when mis-expressed, such as Stat3, are aberrantly re-activated. Thus, our results suggest thatindependently from the protein context, polyQ expansion overrides the control of neuronal differentiationand maintenance, thereby causing dysfunction and degeneration.

INTRODUCTION

Huntington’s disease (HD) and spinocerebellar ataxia type 7(SCA7) are inherited neurodegenerative diseases that belongto polyglutamine (polyQ) disorders, which also include spino-bulbar muscular atrophy, dentatorubro-pallidoluysian atrophyand the SCA1, SCA2, SCA3, SCA6 and SCA17 (1). Thecausal mutation is a CAG repeat expansion in the corresponding

genes encoding an expanded polyQ tract in the proteins. PolyQexpansions confer a gain of toxic properties to mutant proteins,which display aberrant interactions with normal proteinpartners and accumulate into neurons to form intranuclearinclusions (NIs), a microscopic hallmark of these diseases (2).

Although polyQ disorders share common genetic features,they generate distinct pattern of neuronal degenerationdespite overlapping protein expression areas (1). For instance,

# The Author 2006. Published by Oxford University Press. All rights reserved.For Permissions, please email: [email protected]

{These two authors contributed equally.

‡Present address: Division of Bioinformatics and Division of Biochemistry, Swiss Institute of Bioinformatics, Biozentrum, University of Basel,

CH-4056 Basel, Switzerland.}Present address: Department of Genetics, Harvard Medical School, Boston, MA 02115, USA.

*To whom correspondence should be addressed. Tel: þ33 388653412; Fax: þ33 388653246; Email: [email protected]

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the primary target in HD is the striatum, whereas SCA7 causesdegeneration of the cerebellum and brain stem and is theonly polyQ disorder affecting the retina. The mechanism under-lying selective neurodegeneration is unknown. When com-pared with adult onset cases, juvenile patients who carry verylarge expansions often present a more severe phenotype result-ing from broader brain degeneration, suggesting that neuronspresent different sensitivity depending on polyQ expansionlength. In mouse models for polyQ disorders, expression oftruncated mutant proteins bearing the polyQ expansion ismore harmful than full-length proteins and can cause toxicityin neuronal cell types normally spared by the disease process,supporting the idea that polyQ expansion itself is toxic, whilethe protein context modulates the toxicity and contributes tothe selectivity of neuronal degeneration (3).

Studies of mouse models for polyQ diseases revealed thatmutant proteins induce neuronal dysfunction prior to causingcell death. Several non-exclusive mechanisms underlyingpolyQ toxicity have been proposed, such as impairment ofprotein folding and degradation, calcium homeostasis,axonal transport or synaptic transmission (2). Many of thesedefects are known to trigger and maintain neuronal stress con-ditions. Cumulative evidences indicate that deregulation ofgene expression also occurs during polyQ pathogenesis(4,5). This might be the result of sequestration of transcrip-tional regulators into polyQ aggregates or of aberrant inter-actions between mutant proteins and nuclear factorsregulating gene expression. For instance, mutant huntingtin(htt), the protein involved in HD, interacts with and impairsthe function of various transcriptional activators such asSP1, TAF4 and CBP, which may contribute to neuronaldegeneration in HD (5). Interestingly, ATXN7, the SCA7gene product, was shown to be a subunit of the TFTC/STAGA transcription co-activator complex (6). It was recentlyproposed that mutant ATXN7 impairs the histone acetyltrans-ferase activity of the complex (7,8). Gene deregulationmight also result from a programmed cellular response topolyQ-induced stress (9).

Other evidences of gene deregulation are provided by DNAmicroarray analyses comparing gene expression profiles ofpolyQ disorder models versus control. These studies revealedthat polyQ toxicity affects expression of genes involved in mul-tiple cellular functions such as neuronal signaling, calciumregulation, stress and inflammation (4,10,11). Although theseexpression changes could have serious consequences forneuronal function, how they relate to the disease processremains unclear (4). Correlations between phenotypic featuresof mouse models and gene deregulation were difficult toestablish. These issues are crucial to dissociate primary fromsecondary effects of polyQ toxicity and to draw a compre-hensive scheme of the pathomechanism involved in polyQdiseases.

The retina provides several advantages over other CNSregions to study the mechanism of neuronal degener-ation. Indeed, many of the molecular events regulating retinadevelopment, differentiation and function are known and cor-relations between functional, morphological and moleculardefects underlying the retinopathy can often be made(12,13). The retina is composed of six neuronal cell typesspatially subdivided into laminated layers. Photoreceptors,

which constitute 70% of retinal neurons, comprise 97% ofrods and 3% of cones in mouse retina. Differentiated photo-receptors display a characteristic morphology that definesthe retinal outer nuclear and segment layers composed,respectively, of their small cell bodies, surrounding verycompact nuclei, and of their protuberant cytoplasm.

The SCA7 retinal dystrophy was reproduced in one knockinand two transgenic mouse models (14–16). Despite differentlevel and cell type specificity of mutated gene expression,these models display similar phenotypes characterized byintranuclear accumulation of mutant ATXN7 correlatingwith progressive electroretinogram (ERG) dysfunction andshortening of segment layers. Retinal dysfunction occursprior to loss of photoreceptors. Opposite to SCA7 knockinmice that die from severe neurological phenotype at 14–19weeks of age (16), SCA7 transgenic mice R7E, whichexpress the mutant ATXN7 harboring 90Q in rod photo-receptors, have a normal life span (14). Strikingly, analysisof aged R7E revealed that rod ERG activity decreases toflat response despite limited loss of rod cells (17). Instead,the ERG defect appeared to result from morphologicalchange of rods, which entirely lose their segments (17).Reduction of ERG activity is also accompanied by down-regulation of the rhodopsin (Rho) gene expression, suggestingthat early transcriptional impairment underlies R7E roddysfunction (17).

Interestingly, the HD mouse model, R6/2, also developsa progressive retinal degeneration comparable to the R7Eretinopathy (18). This transgenic model ubiquitously expressesthe mutated HD gene exon-1, which encodes the N-terminal90 amino acids of htt protein with 150 glutamines (represent-ing only 3% of htt) (19). R6/2 has a broader spectrum of braindegeneration than typically seen in HD. Similitudes of R7Eand R6/2 phenotypes demonstrate that, independently of theprotein context, polyQ expansion is sufficient to triggerretinal degeneration in mouse.

In the present study, we aimed at elucidating the molecularand cellular events underlying retinal degeneration in R7Eand R6/2 mice. To this end, we performed gene expression pro-filing analysis of R7E and R6/2 retina. To correlate gene dereg-ulation along the progression of R7E retinal degeneration, weexamined R7E and the control R7N mice at onset and moderatestage of pathology. We also compared gene expression changesin R7E and R6/2 retina to highlight common deregulatedmolecular pathways, which have a high probability of beingrelevant to polyQ-induced photoreceptor degeneration. Ourstudy reveals a strong correlation between the progressive dys-function and morphological change of photoreceptors and lossof expression of mature rod genes in both R7E and R6/2 retina.More interestingly, we provide evidence that polyQ expansion,regardless of the protein context, compromises the geneticprogram maintaining photoreceptor differentiation, hence theexpression of photoreceptor specific traits.

RESULTS

To get insight into molecular and cellular pathways involvedin polyQ-induced retinal degeneration, we used genome-wide oligonucleotide microarrays (MOE430A Affymetrix) to

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characterize gene expression profiles in retina of R7E versusR7N mice [which express wild-type (wt) ATXN7 with 10Q]and of R6/2 versus wt littermates. Retina RNA was preparedfrom four to six animals from each mouse line. Each RNAsample was independently hybridized on a different DNAmicroarray. Hybridization of control RNA samples (R7N orwt) indicated that nearly 50% of the array probe sets (about11 500 out of 22 690) display a positive signal based onabsent/present detection calls (MAS analysis). To select thedifferentially expressed genes, three consecutive filters wereapplied as detailed in Materials and Methods. A list of allderegulated transcripts identified in this study is available asSupplementary Material, Table S1.

Gene expression changes during SCA7 retinopathyprogression

We first examined gene expression profiles in R7E comparedwith R7N at 3 and 9 weeks of age, which correspond to onsetand moderate stages of disease, respectively (14,17). Until 3weeks of age, R7E retina develops normally and displays noobvious phenotype. At 3 weeks, NIs are detected in somerod photoreceptors and the first functional abnormalitiesdetected by scotopic ERG occur between 3 and 4 weeks ofage. At 9 weeks, moderate stage is characterized by the pre-sence of NIs in most rod cells and a marked reduction ofERG response. Nine-week-old retina also displays morpho-logical alterations of rod photoreceptors characterized byloss of segments and enlarged nuclei with atypical decon-densed chromatin (17,60). However, there is no significantneuronal loss at this stage. Later on, R7E retinopathyworsens towards flattening of ERG recording, complete lossof segment layers and thinning of the outer nuclear layer.

Comparison of gene expression of R7E versus R7N atretinopathy onset showed that the level of 106 transcriptswas changed by 1.5-fold or more. Of these, 42% were over-expressed and 58% under-expressed in R7E versus R7N. Atmoderate stage of disease, a substantially higher number oftranscripts (486) showed a change in expression level with aconserved ratio between over- (38%) and under-expressed(62%) transcripts. Venn diagram (Fig. 1A) intersecting geneexpression changes at onset and moderate disease progressionshows that 50% of differentially expressed transcripts at 3weeks of age were specific to the onset of disease. The other50% of early altered transcripts also displayed expressionchanges at moderate stage, the majority of which beingunder-expressed (73%).

Gene expression changes primarily due topolyQ expansion

Retinas of 9-week-old R7E and R6/2 mice display very similarretinopathy according to their rod ERG dysfunction andmorphological alterations, which result in outer nuclear layerdisorganization and segment layers reduction (18). R6/2ERG dysfunction also involves cones and light-inducedpost-synaptic neurons, consistent with the ubiquitousexpression of mutant htt-exon-1. We compared geneexpression changes in 9-week-old R6/2 versus R7E retina,assuming that common changes are likely to concern rod

photoreceptor expressed genes and to be primarily due topolyQ expansion independent of protein context. To allowcomparison of these two models of different genetic back-ground, we first identified modulated transcripts in R6/2versus wt littermates. Out of 311 differentially representedtranscripts, 74% were under-expressed and 26% over-expressed. Intersecting differentially expressed transcripts ofR6/2 and R7E revealed 113 common deregulated transcripts,most of which (87 transcripts) were under-expressed(Fig. 1B). Of these 113 transcripts, 24 were altered atdisease onset in R7E retina.

Validation of gene expression data

Expression of the Rho gene was previously shown to be drasti-cally reduced in R7E and R6/2 retina (17). QuantitativeRT–PCR (Q-PCR) in 3.5- and 9-week-old R7E measured,respectively, a 3-fold and 10-fold decrease in mRNA level.Similarly, in 10-week-old R6/2, Rho gene expression wasdecreased by 4.9-fold. Our microarray data corroboratethese expression changes, showing reduction of Rho expressionby 1.6- and 2.3-fold, respectively, at onset and moderate stagein R7E and by 2.0-fold in R6/2. To further test the reliability ofmicroarray data, expression profiles of a subset of 15 geneswere examined by Q-PCR (Table 1). These genes were selectedbecause they displayed different expression profiles and broadvariation spectra ranging from the minimal 1.5 cut-off valueand up to 26-fold variation. Many of them are discussedfurther. For all genes tested, Q-PCR data confirmed thealtered mRNA expression and changed orientation detectedby microarray analysis, even for variations near the 1.5-foldcut-off value. Expression changes measured by Q-PCR were

Figure 1. Summary of gene expression changes in R7E and R6/2 retina. (A)Venn diagram showing mRNA expression changes in R7E versus R7N atonset (R7E 3w) and moderate stage (R7E 9w) of retinopathy. (B) Venndiagram intersection showing retina mRNA changes primarily due to polyQexpansion toxicity. mRNA expression changes in R6/2 versus wt (R6/2 9w)were compared with changes in R7E versus R7N (R7E 9w) at a time whenR7E and R6/2 retinopathies are similar. In brackets, numbers and arrowsindicate the repartition of over- and under-expressed transcripts.

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in general more important, indicating that the microarray dataunder-estimated the level at which genes were deregulated.Q-PCR performed on several genes at onset and moderatedisease stages in R7E mice demonstrated that sensitivity ofmicroarray was sufficient to detect progressive decrease orincrease of gene expression (e.g. Rho, Hes5, etc.) that corre-lated with aggravation of phenotype. Consequently, we areconfident that transcriptional alterations identified in our micro-array analysis are likely to reflect not only changes in trend butalso progressive modifications in gene expression occurringduring disease aggravation in mutant mice retina.

Deregulated genes gather into specific functional pathwaysover time

Given the large number of transcripts showing altered level ofexpression in R7E and R6/2 retina, we used a systematicapproach to interpret the biological significance of thesegene deregulations. First, we re-annotated each transcript byanalyzing the corresponding Affymetrix probe sets sequenceswith RetScope platform (Chalmel F. and Poch O. unpublisheddata). Briefly, RetScope, based on the analysis of the BLAST(20) homology searches in the GenBank (21), UniProt (22)and Human genome (23) public databases, associates withhigh confidence each probe set sequence with existingfull-length transcript and protein accession numbers. Then,GOAnno (24) was used to predict Gene Ontology (25)annotations for each identified protein. Finally, we identifiedover-represented GO Biological Process (GOBP) terms bycalculating a z-score using the whole human proteome asreference (26). We reasoned that genes over-represented in agiven GOBP term have a higher probability of being relevantto polyQ-induced retinopathy. Among the 727 deregulated

transcripts (Supplementary Material, Table S1), 541 wereassigned to GOBP terms, and 50% of them gather in enrichedGOBP terms presenting a z-score of 3 or greater (correspond-ing to a probability of less than 0.00135).

Disease onset in R7E was associated with under-expressedgenes significantly enriched in signal transduction, cell com-munication and most importantly visual perception GOBPterms (Fig. 2). The number and enrichment of under-expressedgenes of these three functional categories increased at moder-ate stage of R7E phenotype. In contrast to signal transductionand cell communication, visual perception represents a levelof higher specificity with respect to the GO annotationsystem. Enrichment of down-regulated genes involved invisual perception makes perfect sense with the early and pro-gressive ERG defect in R7E retinopathy and by itself validatesthe method that we used to identify deregulated pathways.

The moderate stage of R7E phenotype was also character-ized by enrichment of down-regulated genes involved indevelopment, morphogenesis and organogenesis as well asup- or down-regulated genes associated with cell growth,maintenance and organization. Expression alteration ofgenes belonging to these categories might account for theimportant morphological changes (e.g. loss of segments andenlarged nuclei) of rod photoreceptors seen at 9 weeks.Down-regulation of genes involved in regulation of celldeath was also over-represented, although rod photoreceptordeath is observed at later stages only.

Strikingly, altered genes in R6/2 were significantly enrichedin the same functional categories as in R7E at 9 weeks. This con-cerned genes involved in visual perception, signal transduction,cell communication, as well as in development, morphogenesis,organogenesis and cell growth/maintenance (Fig. 2). Theseresults highlight at the molecular level the similitude of

Table 1. Real-time Q-PCR validation of microarray expression data

Gene name R7E (onset) R7E (moderate) R6/2 (moderate)

AFC Q-PCR AFC Q-PCR AFC Q-PCR

Rhodopsin (Rho) 21.6 23.0 22.3 210 22.0 24.9Rod transducin alpha subunit (Gnat1) (21.2) 21.5 25.0a (21.2)Rod phosphodiesterase 6 beta (Pde6b) (21.4) 22.2 25.5 NS 22.5Blue cone opsin (Bcp) NS 21.9 22.1 226.0 2430.0Cone transducin alpha subunit (Gnat2) NS þ1.5 þ2.1 þ2.0 22.6 25.8Cone arrestin (Arr3) NS (21.4) 21.5 219.0 295.0Rhodopsin kinase (Rhok) NS 2 3.5 2 3.6 2 2.1Rod outer segment membrane protein

1 (Rom)(21.4) 22.5 24.6 21.6

Cone-rod homeobox containing gene (Crx) (21.4) 21.6 21.5 (21.4) 21.9Neural retina leucine zipper protein (Nrl) (21.4) 22.2 23.0 21.5Nuclear receptor (Nr2e3) NS 22.1 25.0 NSX box binding protein (Xbp1) NS þ1.8 þ2.0 NSMethyl binding domain 4 protein (Mbd4) þ2.2 þ2.0 þ1.9 þ1.9 NSHairy and enhancer of split 5 (Hes5) NS þ2.4 þ2.4 NSProtein lnhibitor of activated STAT3 (Pias3) NS (21.3) 21.4 21.5 21.7

For each gene are shown the AFC and real-time Q-PCR fold change between mutant and control mice. The genes tested displayed significant AFC(�1.5-fold change, P, 0.015) in at least one comparison group (R7E versus R7N or R6/2 versus wt); non-significant AFC (NS); AFC values inferiorto the 1.5 cut-off but presenting P, 0.015 (in brackets) are also indicated. (2) and (þ), respectively, indicate lower and higher expression in themutant versus control animals. Some of the listed genes were tested at the onset stage in the R6/2 mice. The real time Q-PCR fold changebetween mutant and control mice, for Rho, Bcp, Gnat2 and Arr3 were 21.6, 24.2, 21.7 and 23, respectively.aFold change was measured by densitometry analysis of northern blot.




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retinal degeneration and dysfunction in the two models. Expect-edly, a cohort of genes was enriched in categories unique to R6/2. Under-expressed genes involved in synaptic transmission,transport and cell adhesion are likely to underlie pan-retina dys-function, consistent with the ubiquitous expression of mutatedhtt-exon-1 in R6/2 retina.

Down-regulation of genes essential for photoreceptor lighttransduction

The most compelling finding of the above analysis is the pre-eminent deregulation of genes associated with visual functionin R7E and R6/2 retina, which occurs in the absence of overtcell death. In 9-week-old R7E, this concerned 27 genes, mostof which were under-expressed (Table 2). Of these, eightgenes displayed expression changes already at the onset ofR7E retinopathy. A first group of 14 R7E deregulated genesare directly involved in phototransduction cascade of rodphotoreceptors: Rho, transducin subunits (Gngt1, Gnb1,Gnat1 and Gnb3), cGMP phosphodiesterase subunits (Pde6aand Pde6b), genes involved in ion channel structure and regu-lation (Cnga, Guca1b and Slc24a1) and in other phototrans-duction functions (Sag, Rhok, Rdh12, Rcv and Rbp). Asecond group of genes are implicated in morphogenesis ofphotoreceptor segments (Rom1 and Rds). A third group iscomposed of genes having diverse but essential functions inphotoreceptor, because they cause retinopathy when mutatedin human and mouse (Rp1, Tulp1, Impdh1 or Rs1h) orbecause their orthologs are involved in retinal degenerationin flies (Pitpnm1 and Ppef2).

In R6/2, six rod phototransduction genes (Rho, Gngt1,Gnb1, Sag, Rhok and Rdh12) were as well down-regulated.Moreover, in R6/2, six cone-specific genes involved invision (Bcp, Gcp, Gnat2, Pde6c, Pde6h and Arr3) were

under-expressed, consistent with the ubiquitous expressionof the mutant htt-exon-1 transgene. It is noteworthy that theexpression of three cone-specific genes (Bcp and Gnat2 andPde6c) and the retinal pigmentary epithelium (RPE)-specificgene Myo7A were also altered in R7E. Up-regulation ofMyo7A, which is involved in phagocytosis of photoreceptorouter segment disks by the RPE, suggests an increasedphagocytic activity of these cells in response to structuraldisorganization of the R7E photoreceptor segment layers.

As shown in Table 1, Q-PCR analysis validated the dereg-ulation of eight tested genes (Rho, Bcp, RhoK, Rom1, Gnat1,Gnat2, Arr3 and Pde6b) in R7E and R6/2 retina. Furthermore,Q-PCR analysis of some of these genes at onset and moderatedisease stages revealed that deregulation progresses alongphenotype aggravation in both R7E and R6/2 retina. Together,these data indicate that the abnormal ERGs and structural dis-organization of segment layers in R7E and R6/2 retina resultfrom a very specific down-regulation of genes involved infunction and morphogenesis of photoreceptors.

Altered expression of genes controlling photoreceptordifferentiation

To specify the unique properties of photoreceptor cells, thedevelopmental pathway in vertebrate retina is regulated by aseries of transcription factors acting before and during theterminal differentiation of photoreceptors (12,13). Enrichmentof a large cohort of deregulated genes involved in visual per-ception as well as in development, morphogenesis, organo-genesis and cell growth, maintenance and organizationsuggested that the genetic controls maintaining photoreceptordifferentiation are impaired in R7E and R6/2 retina. Amongthe deregulated transcripts belonging to these pathways, wefound a number of transcription factors controlling various

Figure 2. Enriched GOBP terms corresponding to R7E and R6/2 deregulated genes. Under-expressed (A) and over-expressed (B) genes in retina of R7E onsetand moderate and R6/2 moderate. Only GOBP terms that present a z-score value �3 and include at least six genes for R7E onset and 10 genes for R7E and R6/2moderate are shown. Number of genes/GOBP term is indicated at the right side of each bar. Basic level GOBP terms containing more than 5000 human proteinsin GO hierarchy are not represented. Black bars represent GOBP terms common to R7E and R6/2, whereas gray bars are GOBP terms specific to one mutantmouse.




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aspects of neuronal cell fate specification and differentiation(Table 3).

Under-expressed transcription factor genes. Among thisgroup, Crx, Nrl and Nr2e3 present a special interest, as theyare key regulators of the terminal differentiation and main-tenance of rod photoreceptors (27–29). These genes areexpressed in immature rod cells and interplay together toensure the proper expression of rod- and repression of cone-specific genes during rod terminal differentiation. Crx is alsorequired for terminal differentiation of cones (27). Twoother genes, ErrB and Mef2C, are markers of differentiationfate, as their expression increases in maturing photoreceptors(30,31). Mef2C is also known to be involved in neuronaldifferentiation process. For these five genes, the expressionlevel in R7E retina progressively decreased from 1.4 to 2.9-fold between onset and moderate stage of pathology. Althoughthe 1.4-fold variation seen at onset is inferior to the 1.5-foldused as a confident cut-off for our microarray analysis, thedata are consistent with the progressive aggravation of theR7E phenotype. In R6/2 retina, Nrl, Errb and Mef2cexpressions were also significantly decreased, and Crx wasreduced by 1.4-fold. We performed Q-PCR analysis of Crx,

Nrl and Nr2e3 at R7E moderate stage and of Crx in R6/2and confirmed their reduced level of expression (Table 1).

Reduced expression of the late differentiation regulatorsCrx, Nrl and Nr2e3 is expected to cause deregulation of alarge subset of their target genes in both mouse models. Toevaluate the extent to which dysfunction of these transcriptionfactors contributes to polyQ-induced retinopathy, we com-pared our data sets with recent expression profiling studies,which revealed the transcriptional networks regulated by Nrland Crx in mouse (32–35). Using Affymetrix microarrayanalysis, Yoshida et al. (32) identified 164 differentiallyexpressed genes in Nrl2/2 versus wt mature retina, whichare likely candidate Nrl-regulated genes. We found 15 Nrl-regulated genes presenting altered expression level at onsetand 45 at moderate stage of R7E retinopathy (SupplementaryMaterial, Table S2), an increasing number inversely correlat-ing with the progressive down-regulation of Nrl gene alongdisease phenotype (Table 3). Similarly, 24 Nrl-regulatedgenes were also altered in R6/2 retina. Many of the Nrl-regulated genes showing expression changes in R7E andR6/2 retina function in phototransduction cascade. Expressionprofiling data also revealed that Nr2e3 and Mef2c are regu-lated by Nrl (32,33). Nr2e3 inhibits expression of cone-specific genes in rod cells. Evidence for Nr2e3 dysfunction

Table 2. Deregulation of genes essential for photoreceptor function

Gene name Symbol R7E (onset) R7E (moderate) R6/2 (moderate)

Opsins Rhodopsina Rho 21.6 22.3 22.0Blue cone opsin Bcp 21.9 226.0Green cone opsina Gcp 246.4

Transducins Transducin gamma 1 subunit Gngt1 21.7 22.2 21.5Transducin beta 1 subunit Gnb1 21.8 23.2 21.9Rod transducin alpha subunita Gnat1 21.5Transducin beta 3 subunit Gnb3 þ1.6 þ1.7Cone transducin alpha subunita Gnat2 þ2.1 22.6Transducin beta 4 subunit Gnb4 þ1.6

Phosphodiesterases Phosphodiesterase 6aa Pde6a 22.1Phosphodiesterase 6ba Pde6b 22.2Phosphodiesterase 6c Pde6c þ1.5 21.6Phosphodiesterase 6ha Pde6h 22.7

Channel structure and regulation Cyclic nucleotide gated channel alpha 1’ Cnga1 22.2Guanylate cyclase activator 1Ba Guca1b 21.5 22.3Solute carrier family 24, member 1 Slc24a1 22.0

Arrestins Retinal S-Antigena Sag 21.8 21.5Cone arrestin Arr3 219.0

Kinase Rhodopsin kinasea Rhok 23.5 22.1Segment morphogenesis Rod outer segment membrane protein 1a Rom1 22.5 21.6

Retinal degeneration, slowa Rds 22.7Others Retinol deshydrogenase 12a Rdh12 21.9 22.8 22.0

Recoverina Rcv 21.6Retinitis pigmentosa 1a Rp1 21.5 22.2Tubby-like protein 1a Tulp1 21.7Inosine 5’-phosphate deshydrogenase 1a Impdh1 21.5Phosphatidylinositol membrane associated 1a Pitpnm1 21.6Protein phosphatase, EF hand calcium-binding

domain 2aPpef2 21.9

Retinoschisis 1 homologa Rs1h 22.1 22.5 21.6Retinol-binding protein Rbp þ1.9Myosine VIIAa Myo7A þ2.1

The AFC of the differentially expressed genes are represented. (2) and (þ) indicate, respectively, lower and higher expression in the mutant versuscontrol.aGenes that cause retina disorders when mutated in human or other organisms.




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in R7E retina is suggested by up-regulation of cone-specificgenes (Gnat2 and Pde6c) (Table 2). As for Nrl, we comparedour data set with candidate Crx-regulated genes revealedby SAGE analysis of Crx2/2 versus wt P10 retina (34,35).Again, the number of Crx-regulated genes showing altera-tion in R7E increased from onset (9 genes) to moderatestage (16 genes) in correlation with the progressive down-regulation of Crx gene (Table 3 and Supplementary Material,Table S3). Similarly, nine Crx-regulated genes were alteredin R6/2. In addition, change trends of genes deregulated inR7E and R6/2 were generally in agreement with the expectedtranscriptional consequence of Crx and Nrl inactivation.Together, these results indicate that reduced level ofCrx, Nrl and Nr2e3 expression in R7E and R6/2 retina isin part responsible for the loss of expression of many roddifferentiation genes.

Other transcription factors under-expressed in R7E andR6/2 retina might also contribute to the loss of differentiatedstate of retinal neurons (Table 3). Pax6, Mab21l1 and Lhx2genes are essential for early eye development and remainexpressed in adult retina (36). Nurr codes for a master regula-tor of neuronal differentiation and maintenance in other brainregions (37) and might display a similar role in the retina.

Over-expressed transcription factor genes. Three R7E over-expressed genes, Optx2, Hes5 and Stat3, display crucial func-tions in the retinal development (Table 3). Optx2 is requiredfor early eye development (38), whereas the Notch effectorHes5 is expressed in retinal progenitors and modulates glialcell fate specification (39). Stat3 is thought to play a keyrole in retinal development by maintaining retinal precursorsin an undifferentiated state (40). Interestingly, persistentOptx2, Hes5 or Stat3 expression in developing retina causedinhibitory effects on photoreceptor differentiation (38,39,41).Our microarray data indicated that Hes5 transcript was notdetected (‘absent call’) in control retina, consistent withprevious in situ hybridization study on mature retina (39).Q-PCR analysis confirmed that Hes5 was re-expressed inR7E (Table 1). Immunofluorescence analysis of wt retina

revealed a faint Hes5 staining restricted to ganglion cell andinner nuclear layers (Fig. 3) and no staining in photoreceptorlayer. In R7E retina, anti-Hes5 antibody stained rod photo-receptors containing NIs, but not those in which NIs wereabsent (presumably due to the absence of transgene expression)(Fig. 3). Our data suggest that Hes5 gene was aberrantlyre-expressed in R7E photoreceptors.

We also confirmed by western blot analysis that increasedStat3 gene expression correlated with increased level ofStat3 protein and its phosphorylated (tyrosine 727) form in9-week-old R7E and R6/2 (Fig. 4A). Interestingly, phos-phorylation of Stat3 was detected as early as 3 weeks ofage, preceding the increase in Stat3 protein, and was sustainedover time in R7E retina, whereas it remained undetected inR7N controls. Another study found that phosphorylatedStat3 precedes the increase of Stat3 protein level, suggestingthat Stat3 regulates its own promoter (42). We also foundthat phospho-Stat3 was enriched in the nuclear fractionwhen compared with the cytoplasmic one in the R7E(Fig. 4B), consistent with the known process of Stat3activation.

Stat proteins are terminal transcription factors of a well-documented signaling pathway, which involves external cyto-kine stimuli, membrane receptors, Janus tyrosine kinases (Jak)and several negative regulators such as protein inhibitor ofactivated Stat (Pias) and suppressor of cytokine signaling(Socs) (43). Affymetrix microarray MOE430A contained probesets to assess expression of several of these genes controllingStat3 activation. We found that only Pias3 gene displayedaltered expression in both R7E and R6/2 retina when com-pared with their controls (Table1). Decreased expression ofPias3, which inhibits Stat3 binding to the DNA regulatoryelements, is consistent with Stat3 protein activation. Weconfirmed by Q-PCR analysis that Pias3 gene expressionwas decreased in both R7E and R6/2 retina.

Altogether, these results indicate that transcription factorsassociated to early steps of retinal development and able tocause negative effect on photoreceptor differentiation areaberrantly over-expressed in polyQ retina.

Table 3. Deregulation of transcription factors implicated in retinal development

Gene symbol R7E(onset)

R7E(moderate)

R612(moderate)

Function Reference

Cone-rod homeobox containing gene (Crx) (21.4) 21.6 (21.4) Differentiation and maintenance of cones and rods (27)Neural retina leucine zipper protein (Nrl) (21.4) 22.2 21.5 Differentiation and maintenance of rods (28)Nuclear receptor (Nr2e3) NS 22.1 NS Differentiation and maintenance of rods (29)Estrogen-related receptor beta (ErrB) (21.4) 22.9 21.8 Expression increases in differentiating photoreceptors (30)Myocyte enhancer factor 2C (Mef2c) 21.5 22.9 22.2 Expression increases in differentiating retina (31)Paired box gene 6 (Pax6) NS NS 21.8 Early eye development (36)Mab-21-like 1 (Mab21l1) (21.4) 21.8 NS Early eye development (36)LIM homeobox protein 2 (Lhx2) 21.5 NS NS Early eye development (36)Nuclear receptor (Nurr) NS 21.6 NS Differentiation and maintenance of neurons (37)Optic homeobox 2 (Optx2) NS þ1.5 NS Early eye development (38)Signal transducer and activator

of transcription 3 (Stat3)NS þ1.7 þ2.1 Retinal progenitor proliferation (40)

Hairy and enhancer of split 5 (Hes5) NS þ2.4 NS Retinal gliogenesis, inhibits neurogenesis (39)

The AFC is shown and (2) and (þ) indicate, respectively, lower and higher expression in the mutant versus control mice. Values inferior to 1.5 cut-offbut presenting P, 0.015 are also indicated in brackets. NS, non-significant.




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DISCUSSION

Mature rod photoreceptors are issued from sequential devel-opment of retinal progenitor cells (RPC) during vertebrateretinogenesis. In the initial phase, a pool of dividing RPCbecomes post-mitotic precursors committed to rod fate.Then, following a period of specification, immature rodsterminally differentiate into mature rods, which expressspecific proteins essential to establish a characteristic cellshape and to execute light transduction. Commitment and

differentiation of rod photoreceptors require transcriptionalprograms that are being uncovered (12,13). In the presentstudy, we have used gene expression profiling to build a com-prehensive scheme of the pathomechanism involved in twomouse models of rod photoreceptor degeneration inducedby different polyQ expansion proteins. Our data show thatpolyQ expansion, regardless of the protein context, causes aprogressive loss of expression of mature rod genes by com-promising the genetic programs involved in the maintenanceof rod differentiation state, resulting in rod dysfunction anddegeneration.

Correlating rod dysfunction and morphological changewith polyQ-induced transcriptional alterations

In SCA7 R7E mouse model, rod-specific expression ofATXN7 with expanded polyQ causes a progressive roddegeneration timely defined by molecular (NI formation),functional (scotopic ERG loss), histological (segment layerreduction) and cell shape anomalies, occurring prior overtcell death (17). Using DNA microarrays and Q-PCR, weshow that R7E rod phenotype was attributable to reducedexpression of a large subset of genes specifically involved inphototransduction and segment morphogenesis of maturerods. Previous studies (15,16) reported similar findings inother SCA7 mouse models by analyzing a restrained numberof selected photoreceptor genes. Our genomewide analysisgives now an exhaustive view over time of the loss ofexpression of genes essential to mature rod features. Likewise,expression of mutant htt-exon-1 in R6/2 mouse retina, whichcauses a retinopathy comparable to R7E (18), alters the regu-lation of a similar subset of mature rod genes. Mutanthtt-exon-1, which is expressed in cones as well and causesphotopic ERG dysfunction of R6/2 retina, also leads toreduced expression of cone phototransduction genes. Down-regulation of mature photoreceptor genes is progressive and

Figure 3. Aberrant photoreceptor expression of Hes5 in R7E retina. Hes5 staining is observed in ganglion cells (GC) and inner nuclear layer (INL) of wt and R7Eretina, but not in wt photoreceptors (ONL) or in R7E photoreceptors that do not contain NIs presumably due to the absence of transgene expression (arrowhead).In contrast, R7E photoreceptors containing NIs displayed Hes5 staining. The animals are 13 weeks of age. Scale bar: 50 mm.

Figure 4. Aberrant activation of Stat3. (A) Stat3 expression is increased inR7E retina when compared with controls at different stages of the pathology.Phospho-Stat3, the activated form of Stat3, is detected as early as 3 weeks ofage (arrow). This activation persists and increases at later stages. Stat3 andphospho-Stat3 are also increased in R6/2 when compared with control’sretina. Anti-TFIIFalpha antibody is used to control the loaded proteinamounts. (B) Phospho-Stat3 is enriched in the nuclear fraction comparedwith the cytoplasmic one in the R7E mice.




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affects an increasing number of genes along phenotype aggra-vation in R7E and R6/2. Together, these results indicate thatphotoreceptors expressing polyQ expansion proteins progress-ively loose their differentiation features due to transcriptionalalterations of mature photoreceptor genes. Apart from genesdirectly implicated in rod maturation state, a large number ofgenes involved in development, morphogenesis, cell growth,maintenance and organization are also significantly deregu-lated in 9-week-old retina, a disease time point whensegment layer is severely reduced in both polyQ modelsand R7E rod nuclei lose their chromatin organization (60).This indicates that important reprogramming of geneexpression accompanied reshaping of rod cells to neuronsbeing non-functional and showing immature traits.

Down-regulation of rod differentiation program

Consistent with the loss of rod photoreceptor differentiationfeatures, three key transcription factors (Crx, Nrl and Nr2e3)that control terminal differentiation and maintenance ofmature rod are down-regulated in R7E and R6/2 retina.Mice lacking the homeobox Crx develop precursor neuronscommitted to rod fate, but fail to develop proper outer seg-ments and to express phototransduction proteins (27,34).The bZIP Nrl is essential for expression of rod-specific andrepression of cone-specific genes in rods. Nrl and Crx proteinscan interact to exhibit transcriptional synergy in gene acti-vation. Inactivation of Nrl in mice results in a complete lossof rods at the expense of supernumerary S-cones (28). OneNrl repression mechanism of cone genes proceeds via induc-tion of the orphan nuclear receptor Nr2e3 expression. Rd7mutant mouse, which carries a spontaneous Nr2e3 deletion,develops aberrant hybrid cone–rod photoreceptors (29,44).The reduced Crx and Nrl expressions have a direct impacton R7E and R6/2 photoreceptor expression profiles, as alarge number of Crx- and Nrl-target genes show deregulationin polyQ retina models, even though our microarray analysesare performed at early stages (onset and moderate phenotype)of retinopathy. Strikingly, the number of Crx- and Nrl-targetgenes deregulated in R7E retina increases from onset tomoderate stage, inversely correlating with the progressivedown-regulation of Crx and Nrl genes along phenotype aggra-vation. Moreover, Nr2e3 dysfunction in R7E retina issuggested by increased level of cone-specific gene expression.Thus, we conclude that progressive loss of Crx, Nr2e3 and Nrlfunctions in R7E and R6/2 retina results in the failure ofrod photoreceptors to maintain the expression of mature rodgenes.

La Spada et al. (15) reported that mutant ATXN7 can inter-fere with CRX and NRL transactivation function in vitro.On the basis of direct interaction between ATXN7 and CRXand on the cone and rod CRX expression, the authorsfavored a model in which interference with transactivationfunction of CRX, rather than NRL, would be the majordefect leading to cone and rod dystrophy in SCA7. Ourresult suggests another mechanism whereby mutant ATXN7and mutant htt-exon-1 cause Crx, Nrl and Nr2e3 dysfunctionby repressing their expression.

We also noted the reduced expression of other transcriptionfactors (Table 3), which are involved either in early eye

development (Pax6, Mab2l1 and Lhx2) or in neuronal differ-entiation (Mef2c, Errbeta; and Nurr). Although the role ofthese factors in mature retina is currently unknown, theirderegulation might also contribute to the RE7 and R6/2retinal phenotype. Whatever is their contribution, down-regulation of these factors reveals that polyQ expansiontoxicity has a broad effect on the transcriptional program ofmature retina.

Aberrant activation of transcription factors that inhibitphotoreceptor differentiation

Another important finding of our study is the increased levelof Optx2, Hes5 and Stat3 expression in polyQ retina models.It has already been shown using several experimental con-ditions that persistent expression of these transcriptionfactors in developing vertebrate retina causes inhibitoryeffect on rod photoreceptor differentiation (38,39,41). It isnotable that Stat3 protein is activated in the two polyQmodels, and in the case of R7E, activation is triggered atonset and sustained along the pathogenic process. Duringretinal development, Stat3 inactivation is required for rodphotoreceptor precursors to differentiate into mature rod andfor Crx and Rho gene expression (40,41). The cytokinesCNTF and LIF, which activate Stat3 signaling pathway, alsoprevent photoreceptor differentiation in rodents (45,46). Inaddition, LIF effect on photoreceptor differentiation is dueto inhibition of Crx and Nrl expression (46). The consequenceof activating Stat3 in adult retina is currently unknown.However, in preclinical studies on animal models for photo-receptor degeneration, prolonged CNTF treatment increasedphotoreceptor survival without rescuing their function(47,48). These rod photoreceptors displayed enlarged nucleiwith less compacted chromatin, similar to photoreceptornuclei of aged R7E mice (60). On the basis of the currentknowledge of CNTF, LIF and Stat3 effects on photoreceptordifferentiation, aberrant Stat3 activation in R7E and R6/2retina might be one of the early events leading to loss of roddifferentiation by causing repression of Crx and Nrl geneexpression.

Our study also points out on several pathways that couldcontribute to Stat3 activation. Pias3 gene expression isreduced in both R7E and R6/2 retina. Down-regulation ofPias3 protein, which interacts with and inhibits DNA-binding activity of Stat3, could strengthen Stat3 activationand its transcriptional activity. In Drosophila, properexpression level of both Pias and Stat ortholog genes arecrucial for eye development (49). Interestingly, deletion ofdpias inhibits retinal cell differentiation. Besides the canonicalcytokine/receptor/Jak pathway, other routes of Stat activationhave been identified. A recent study shed light on a cross-talkbetween Notch–Hes and Jak–Stat signaling pathways viadirect interaction of Hes1 or Hes5 with Jak2 and Stat3,which promotes Stat3 phosphorylation and activation. Con-sequently, aberrant Hes5 expression in R7E retina maycontribute to the sustained Stat3 activation. Moreover, Statproteins can also be activated by diverse cellular stresses(50). For instance, oxidative stress, which is associated withpolyQ-induced toxicity in several model systems, can activate




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Stat3 pathway (51). We previously showed that neuronalstress involving the activation of JNK/Jun/AP1 pathwayoccurred in R7E and R6/2 mouse retina (9). Our microarraydata also reveal over-expression of regulators of oxidativeand endoplasmic reticulum stress response, such as Bach2and Xbp1 genes, respectively (Table 1 and SupplementaryMaterial, Table S1), warranting further investigation of therole of stress in the loss of neuronal differentiation inducedby polyQ expansion.

PolyQ toxicity and loss of neuronal identity

The detailed mechanisms whereby polyQ expansion causesloss of neuronal differentiation remain to be characterized.However, it is worth to note that deregulation of the geneticprogram maintaining photoreceptor differentiation by polyQexpansion seems unique, because such gene deregulationspecificities were not reported in transcriptome analyses ofother retinal degenerations associated with rd1, Rho or Rp1mutations (52,53) (Chalmel, F. and Poch, O., manuscript inpreparation). Our conclusions that polyQ induces loss ofneuronal differentiation are based on the study of a highlyorganized and well-characterized neuronal tissue, the retina.However, failure of neurons to maintain their differentiatedstate might be a common feature to polyQ disorders. Earlystudies on gene deregulation occurring in HD showed thatgenes encoding proteins essential for striatal neuron functionssuch as neurotransmitters, receptors and other neuronal signal-ing proteins were down-regulated (10,54). Although themechanism remains to be identified, recent studies showedthat one critical regulator of neuronal terminal differentiation,neuron-restrictive silencer factor (NRSF), was altered in HD.Indeed, NRSF protein was abnormally localized in the neur-onal nucleus in HD, resulting in repression of NRSF-targetgenes (55). Another study showed that polyQ expansioncaused up-regulation of NRSF gene expression and preventedneuronal differentiation of embryonic stem cells (56). Over-expression of NRSF was shown to inhibit neurite outgrowth(57). Morphological abnormalities of dendrites and defectsof neurite outgrowth were reported in HD and in in vitroand in vivo HD models (58,59). In regard to our findings inR7E and R6/2 retina, it is thus conceivable that mutant httcompromises neuronal identity of striatal neurons by deregu-lating the genetic program controlling their differentiation.

One puzzling aspect of polyQ disorders is that long-termneuronal dysfunction precedes neuronal cell loss. Little isknown about this gradual degenerative process or on themechanism of neuronal death. Our finding suggests that dys-function occurred because neurons loose their neuronal fea-tures. PolyQ expansion might directly affect the key proteinsorchestrating the program of neuronal differentiation. Alterna-tively, in response to permanent stress caused by polyQ expan-sion, vulnerable neurons might slowly progress to animmature state. By being in conflict with the mature environ-ment of the nervous system, these immature neurons might notreceive stimuli promoting their survival. Studies on howpolyQ expansion causes loss of neuronal differentiation andhow neurons loosing neuronal traits are condemn to deathwill thus be of major relevance for the development of thera-peutic approaches in polyQ disorders.

MATERIALS AND METHODS

Animals

R7E and R7N transgenic lines were maintained on the inbredC57BL/6 background (14). R6/2 line, purchased from theJackson Laboratories (Bar Harbor, ME, USA) was originallyon C57BL/6:CBA/J background. To avoid the rd1 mutationcarried by CBA/J inbred strain, R6/2 mice were backcrossedon C57BL/6 background. The mice used in this study were75% C57BL/6 and 25% CBAJ. Genotyping of R7N, R7E,R6/2 and rd1 mutations was performed by PCR on tail DNAaccording to the protocols previously described (17). Theexperiments were performed in accordance with the NationalInstitutes of Health Guide for Care and use of LaboratoryAnimals.

RNA isolation

Both retinas from one mouse were isolated, pooled, immedi-ately frozen in liquid nitrogen and stored at 2808C. Retinaswere homogenized with an ultraturax homogenizer and totalRNA was extracted using Qiagen columns according to manu-facturer’s instructions (Qiagen RNeasy kit). RNA quantityand quality were analyzed using 260/280 nm absorbanceratio and Agilent apparatus.

Affymetrix gene profiling and data analysis

For the microarray experiments, we analyzed five R7E versusfive R7N mice at 3 weeks of age, six R7E versus six R7N miceat 9 weeks of age as well as four R6/2 versus four wt mice at 9weeks of age. Biotinylated cRNA were prepared according tothe standard Affymetrix protocol (GeneChip ExpressionAnalysis Technical Manual, 2001, 701151 Rev1, Mat No.1020407, 03/2002; Affymetrix). Ten micrograms of fragmen-ted cRNA were hybridized for 16 h at 458C on murine MOE430A GeneChips. These GeneChips contain 22 600 probesets, about 15 000 of them are against well-annotated full-length genes, whereas the remaining probe sets are againstgene clusters containing only EST sequences and some geneclusters with non-EST sequences. GeneChips were washedand stained in the Affymetrix Fluidics Station 400 andfurther scanned using the Hewlett-Packard GeneArrayScanner G2500A. Initial data preparation was performed byAffymetrix MicroArray Suite Version 5.0 (MAS5) using Affy-metrix default analysis settings and global scaling as normal-ization method. The trimmed mean target intensity of eachchip was arbitrarily set to 100. Absolute analyses generate asignal value for each probe set and a detection call ofabsent, present or marginal. To select differentially expressedgenes, three consecutive filters were applied for each of thethree comparison groups (3-week-old R7E versus R7N,9-week-old R7E versus R7N and 9-week-old R6/2 versuswt). First, we performed a Mann–Whitney statistical testand considered as significant the genes with a P-value lessthan 0.015. We then picked the genes presenting an Affy-metrix fold change (AFC) �1.5. Finally, we selected genescalled ‘present’ in at least (n2 1) mice in a group of nmutant mice for the up-regulated genes, or n control micefor the down-regulated genes.




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Q-PCR analysis

Total RNA (1 mg) was subjected to reverse transcription usingSuperScript Reverse Transcriptase (Invitrogen). For eachgene, primers were designed using Primer 3 software andare available upon request. Real-time PCR was performedwith SYBRGreen using the Light Cycler apparatus. Specificityof reactions was confirmed by melting curve analysis. Signifi-cant fold change was calculated based on the difference in thecalculated concentration between the transgenic and thecontrol mice after normalization to Ppia or Arbp as internalcontrols. Three to six animals for each genotype, R7E, R6/2and wt, were analysed.

Western blotting

Retinas were homogenized in lysis buffer containing 50 mM

Tris–HCl pH 8.0, 10% glycerol, 5 mM EDTA, 400 mM KCl,1 mM phenylmethylsulfonyl fluoride and a cocktail of proteaseinhibitors. Triton was then added to a final concentration of0.1% to whole retinal homogenates. Retinas were then incu-bated on ice for 15 min, sonicated and centrifuged for15 min at 48C. Supernatants were analyzed on 10–12%SDS–PAGE gel.

Primary antibodies used were mouse monoclonal Stat3(F-2): sc-8019 (Santa Cruz, USA), rabbit monoclonalPhospho-Stat3 (Tyr705) (58E12) (Cell signaling). They wererevealed with appropriate anti-mouse or anti-rabbit peroxi-dase-conjugated secondary antibodies and the chemilumines-cent reaction (Pierce, Rockford, IL, USA).

Immunohistofluorescence

Enucleated eyes were dissected to remove lens and corneaand fixed in fresh 4% paraformaldehyde for 2 h at 48C.Fixed retinas were placed for 1 h in 30% sucrose andfrozen in Cryomatrix compound (Thermo Shandon). Cryostatsections (10 mm) were mounted on SuperFrost/Plus slides (O.Kindler, Freiburg, Germany). Sections were permeabilizedand blocked for 1 h with 10% normal goat serum, 0.5%bovine serum albumin (BSA), 0.1% Tween-20 and 1 �

phosphate-buffered saline (PBS). After a washing step inPBS, sections were incubated with primary antibodiesdiluted in 3% normal goat serum, 0.5% BSA, 0.1%Tween-20 and 1 � PBS. Primary antibodies used wererabbit Anti-Hes5 affinity purified polyclonal antibodyAB5708 (Chemicon International) and mouse anti-ATXN7monoclonal antibody (2A10) (17). Sections were thenwashed and incubated with secondary antibodies diluted inthe same solution as primary antibody. The secondary anti-bodies used were CY3- and Oregon green-conjugated goatanti-mouse and anti-rabbit IgG at a dilution of 1:200.Nuclei were counterstained with 0.5 mg/ml 4,6-diaminido-2-phenylindole.

SUPPLEMENTARY MATERIAL

Supplementary Material is available at HMG Online.

ACKNOWLEDGEMENTS

We are grateful to Fabrice Klein, Lea Ben-Haıem, MyriamRavache and Stanislas DuManoir for helpful discussion andAstrid Lunkes and Nathalie Daigle for critical reading of themanuscript. We thank Doulay Dembele from the IGBMCAffymetrix platform. This work was supported by fundsfrom INSERM, CNRS, Hopital Universitaire de Strasbourg;College de France (to J.L.M.); European CommunityEUROSCA integrated project (LSHM-CT-2004-503304),Hereditary Disease Foundation and French Ministry ofScience (to D.D. and Y.T.).

Conflict of Interest statement. None declared.

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polyglutamine expansion causes neurodegeneration by altering the neuronal differentiation program

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