Human Molecular Genetics, 2006, Vol. 15, No. 5
doi:10.1093/hmg/ddi483
Advance Access published on January 24, 2006
691–703
Polyglutamine expansion causes
neurodegeneration by altering the neuronal
differentiation program
Gretta Abou-Sleymane1,4, Frédéric Chalmel3,{,{, Dominique Helmlinger1,{,}, Aurélie Lardenois3,
Christelle Thibault1, Chantal Weber1,4, Karine Mérienne1,4, Jean-Louis Mandel1,4, Olivier Poch3,
Didier Devys1,2,4 and Yvon Trottier1,4,*
Department of Molecular Pathology, 2Department of Transcription and 3Department of Structural Genomic and
Biology, Institut de Génétique et Biologie Moléculaire et Cellulaire (IGBMC), CNRS/INSERM/ULP, BP10142, 67404
Illkirch Cédex, CU de Strasbourg, France and 4Chaire de Génétique Humaine, Collège 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 neurodegenerative diseases caused by polyglutamine (polyQ) expansion in corresponding proteins.
Transcriptional alteration is a unifying feature of polyQ disorders; however, the relationship between
polyQ-induced gene expression deregulation and degenerative processes remains unclear. R6/2 and R7E
mouse models of HD and SCA7, respectively, present a comparable retinal degeneration characterized by
progressive reduction of electroretinograph activity and important morphological changes of rod photoreceptors. The retina, which is a simple central nervous system tissue, allows correlating functional, morphological and molecular defects. Taking advantage of comparing polyQ-induced degeneration in two retina
models, we combined gene expression profiling and molecular biology techniques to decipher the molecular
pathways underlying polyQ expansion toxicity. We show that R7E and R6/2 retinal phenotype strongly correlates with loss of expression of a large cohort of genes specifically involved in phototransduction function
and morphogenesis of differentiated rod photoreceptors. Accordingly, three key transcription factors (Nrl,
Crx and Nr2e3) controlling rod differentiation genes, hence expression of photoreceptor specific traits, are
down-regulated. Interestingly, other transcription factors known to cause inhibitory effects on photoreceptor
differentiation when mis-expressed, such as Stat3, are aberrantly re-activated. Thus, our results suggest that
independently from the protein context, polyQ expansion overrides the control of neuronal differentiation
and maintenance, thereby causing dysfunction and degeneration.
INTRODUCTION
Huntington’s disease (HD) and spinocerebellar ataxia type 7
(SCA7) are inherited neurodegenerative diseases that belong
to polyglutamine (polyQ) disorders, which also include spinobulbar muscular atrophy, dentatorubro-pallidoluysian atrophy
and the SCA1, SCA2, SCA3, SCA6 and SCA17 (1). The
causal mutation is a CAG repeat expansion in the corresponding
genes encoding an expanded polyQ tract in the proteins. PolyQ
expansions confer a gain of toxic properties to mutant proteins,
which display aberrant interactions with normal protein
partners and accumulate into neurons to form intranuclear
inclusions (NIs), a microscopic hallmark of these diseases (2).
Although polyQ disorders share common genetic features,
they generate distinct pattern of neuronal degeneration
despite overlapping protein expression areas (1). For instance,
*To whom correspondence should be addressed. Tel: þ33 388653412; Fax: þ33 388653246; Email: yvon@igbmc.u-strasbg.fr
{
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.
# The Author 2006. Published by Oxford University Press. All rights reserved.
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Human Molecular Genetics, 2006, Vol. 15, No. 5
which constitute 70% of retinal neurons, comprise 97% of
rods and 3% of cones in mouse retina. Differentiated photoreceptors display a characteristic morphology that defines
the retinal outer nuclear and segment layers composed,
respectively, of their small cell bodies, surrounding very
compact nuclei, and of their protuberant cytoplasm.
The SCA7 retinal dystrophy was reproduced in one knockin
and two transgenic mouse models (14 –16). Despite different
level and cell type specificity of mutated gene expression,
these models display similar phenotypes characterized by
intranuclear accumulation of mutant ATXN7 correlating
with progressive electroretinogram (ERG) dysfunction and
shortening of segment layers. Retinal dysfunction occurs
prior to loss of photoreceptors. Opposite to SCA7 knockin
mice that die from severe neurological phenotype at 14 –19
weeks of age (16), SCA7 transgenic mice R7E, which
express the mutant ATXN7 harboring 90Q in rod photoreceptors, have a normal life span (14). Strikingly, analysis
of aged R7E revealed that rod ERG activity decreases to
flat response despite limited loss of rod cells (17). Instead,
the ERG defect appeared to result from morphological
change of rods, which entirely lose their segments (17).
Reduction of ERG activity is also accompanied by downregulation of the rhodopsin (Rho) gene expression, suggesting
that early transcriptional impairment underlies R7E rod
dysfunction (17).
Interestingly, the HD mouse model, R6/2, also develops
a progressive retinal degeneration comparable to the R7E
retinopathy (18). This transgenic model ubiquitously expresses
the mutated HD gene exon-1, which encodes the N-terminal
90 amino acids of htt protein with 150 glutamines (representing only 3% of htt) (19). R6/2 has a broader spectrum of brain
degeneration than typically seen in HD. Similitudes of R7E
and R6/2 phenotypes demonstrate that, independently of the
protein context, polyQ expansion is sufficient to trigger
retinal degeneration in mouse.
In the present study, we aimed at elucidating the molecular
and cellular events underlying retinal degeneration in R7E
and R6/2 mice. To this end, we performed gene expression profiling analysis of R7E and R6/2 retina. To correlate gene deregulation along the progression of R7E retinal degeneration, we
examined R7E and the control R7N mice at onset and moderate
stage of pathology. We also compared gene expression changes
in R7E and R6/2 retina to highlight common deregulated
molecular pathways, which have a high probability of being
relevant to polyQ-induced photoreceptor degeneration. Our
study reveals a strong correlation between the progressive dysfunction and morphological change of photoreceptors and loss
of 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 genetic
program maintaining photoreceptor differentiation, hence the
expression of photoreceptor specific traits.
RESULTS
To get insight into molecular and cellular pathways involved
in polyQ-induced retinal degeneration, we used genomewide oligonucleotide microarrays (MOE430A Affymetrix) to
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the primary target in HD is the striatum, whereas SCA7 causes
degeneration of the cerebellum and brain stem and is the
only polyQ disorder affecting the retina. The mechanism underlying selective neurodegeneration is unknown. When compared with adult onset cases, juvenile patients who carry very
large expansions often present a more severe phenotype resulting from broader brain degeneration, suggesting that neurons
present different sensitivity depending on polyQ expansion
length. In mouse models for polyQ disorders, expression of
truncated mutant proteins bearing the polyQ expansion is
more harmful than full-length proteins and can cause toxicity
in neuronal cell types normally spared by the disease process,
supporting the idea that polyQ expansion itself is toxic, while
the protein context modulates the toxicity and contributes to
the selectivity of neuronal degeneration (3).
Studies of mouse models for polyQ diseases revealed that
mutant proteins induce neuronal dysfunction prior to causing
cell death. Several non-exclusive mechanisms underlying
polyQ toxicity have been proposed, such as impairment of
protein folding and degradation, calcium homeostasis,
axonal transport or synaptic transmission (2). Many of these
defects are known to trigger and maintain neuronal stress conditions. Cumulative evidences indicate that deregulation of
gene expression also occurs during polyQ pathogenesis
(4,5). This might be the result of sequestration of transcriptional regulators into polyQ aggregates or of aberrant interactions between mutant proteins and nuclear factors
regulating gene expression. For instance, mutant huntingtin
(htt), the protein involved in HD, interacts with and impairs
the function of various transcriptional activators such as
SP1, TAF4 and CBP, which may contribute to neuronal
degeneration in HD (5). Interestingly, ATXN7, the SCA7
gene product, was shown to be a subunit of the TFTC/
STAGA transcription co-activator complex (6). It was recently
proposed that mutant ATXN7 impairs the histone acetyltransferase activity of the complex (7,8). Gene deregulation
might also result from a programmed cellular response to
polyQ-induced stress (9).
Other evidences of gene deregulation are provided by DNA
microarray analyses comparing gene expression profiles of
polyQ disorder models versus control. These studies revealed
that polyQ toxicity affects expression of genes involved in multiple cellular functions such as neuronal signaling, calcium
regulation, stress and inflammation (4,10,11). Although these
expression changes could have serious consequences for
neuronal function, how they relate to the disease process
remains unclear (4). Correlations between phenotypic features
of mouse models and gene deregulation were difficult to
establish. These issues are crucial to dissociate primary from
secondary effects of polyQ toxicity and to draw a comprehensive scheme of the pathomechanism involved in polyQ
diseases.
The retina provides several advantages over other CNS
regions to study the mechanism of neuronal degeneration. Indeed, many of the molecular events regulating retina
development, differentiation and function are known and correlations between functional, morphological and molecular
defects underlying the retinopathy can often be made
(12,13). The retina is composed of six neuronal cell types
spatially subdivided into laminated layers. Photoreceptors,
Human Molecular Genetics, 2006, Vol. 15, No. 5
693
characterize gene expression profiles in retina of R7E versus
R7N mice [which express wild-type (wt) ATXN7 with 10Q]
and of R6/2 versus wt littermates. Retina RNA was prepared
from four to six animals from each mouse line. Each RNA
sample was independently hybridized on a different DNA
microarray. Hybridization of control RNA samples (R7N or
wt) indicated that nearly 50% of the array probe sets (about
11 500 out of 22 690) display a positive signal based on
absent/present detection calls (MAS analysis). To select the
differentially expressed genes, three consecutive filters were
applied as detailed in Materials and Methods. A list of all
deregulated transcripts identified in this study is available as
Supplementary Material, Table S1.
We first examined gene expression profiles in R7E compared
with R7N at 3 and 9 weeks of age, which correspond to onset
and moderate stages of disease, respectively (14,17). Until 3
weeks of age, R7E retina develops normally and displays no
obvious phenotype. At 3 weeks, NIs are detected in some
rod photoreceptors and the first functional abnormalities
detected by scotopic ERG occur between 3 and 4 weeks of
age. At 9 weeks, moderate stage is characterized by the presence of NIs in most rod cells and a marked reduction of
ERG response. Nine-week-old retina also displays morphological alterations of rod photoreceptors characterized by
loss of segments and enlarged nuclei with atypical decondensed chromatin (17,60). However, there is no significant
neuronal loss at this stage. Later on, R7E retinopathy
worsens towards flattening of ERG recording, complete loss
of segment layers and thinning of the outer nuclear layer.
Comparison of gene expression of R7E versus R7N at
retinopathy onset showed that the level of 106 transcripts
was changed by 1.5-fold or more. Of these, 42% were overexpressed and 58% under-expressed in R7E versus R7N. At
moderate stage of disease, a substantially higher number of
transcripts (486) showed a change in expression level with a
conserved ratio between over- (38%) and under-expressed
(62%) transcripts. Venn diagram (Fig. 1A) intersecting gene
expression changes at onset and moderate disease progression
shows that 50% of differentially expressed transcripts at 3
weeks of age were specific to the onset of disease. The other
50% of early altered transcripts also displayed expression
changes at moderate stage, the majority of which being
under-expressed (73%).
Gene expression changes primarily due to
polyQ expansion
Retinas of 9-week-old R7E and R6/2 mice display very similar
retinopathy according to their rod ERG dysfunction and
morphological alterations, which result in outer nuclear layer
disorganization and segment layers reduction (18). R6/2
ERG dysfunction also involves cones and light-induced
post-synaptic neurons, consistent with the ubiquitous
expression of mutant htt-exon-1. We compared gene
expression changes in 9-week-old R6/2 versus R7E retina,
assuming that common changes are likely to concern rod
Figure 1. Summary of gene expression changes in R7E and R6/2 retina. (A)
Venn diagram showing mRNA expression changes in R7E versus R7N at
onset (R7E 3w) and moderate stage (R7E 9w) of retinopathy. (B) Venn
diagram intersection showing retina mRNA changes primarily due to polyQ
expansion 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 when
R7E and R6/2 retinopathies are similar. In brackets, numbers and arrows
indicate the repartition of over- and under-expressed transcripts.
photoreceptor expressed genes and to be primarily due to
polyQ expansion independent of protein context. To allow
comparison of these two models of different genetic background, we first identified modulated transcripts in R6/2
versus wt littermates. Out of 311 differentially represented
transcripts, 74% were under-expressed and 26% overexpressed. Intersecting differentially expressed transcripts of
R6/2 and R7E revealed 113 common deregulated transcripts,
most of which (87 transcripts) were under-expressed
(Fig. 1B). Of these 113 transcripts, 24 were altered at
disease onset in R7E retina.
Validation of gene expression data
Expression of the Rho gene was previously shown to be drastically reduced in R7E and R6/2 retina (17). Quantitative
RT – 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 was
decreased by 4.9-fold. Our microarray data corroborate
these expression changes, showing reduction of Rho expression
by 1.6- and 2.3-fold, respectively, at onset and moderate stage
in R7E and by 2.0-fold in R6/2. To further test the reliability of
microarray data, expression profiles of a subset of 15 genes
were examined by Q-PCR (Table 1). These genes were selected
because they displayed different expression profiles and broad
variation spectra ranging from the minimal 1.5 cut-off value
and up to 26-fold variation. Many of them are discussed
further. For all genes tested, Q-PCR data confirmed the
altered mRNA expression and changed orientation detected
by microarray analysis, even for variations near the 1.5-fold
cut-off value. Expression changes measured by Q-PCR were
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Gene expression changes during SCA7 retinopathy
progression
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Human Molecular Genetics, 2006, Vol. 15, No. 5
Table 1. Real-time Q-PCR validation of microarray expression data
Gene name
R7E (moderate)
R6/2 (moderate)
AFC
Q-PCR
AFC
Q-PCR
AFC
21.6
(21.2)
(21.4)
NS
NS
NS
NS
(21.4)
23.0
22.3
21.5
22.2
21.9
þ2.1
(21.4)
2 3.5
22.5
210
25.0a
25.5
22.1
þ2.0
21.5
2 3.6
24.6
22.0
(21.2)
NS
226.0
22.6
219.0
2 2.1
21.6
21.6
22.2
22.1
þ1.8
þ1.9
þ2.4
(21.3)
21.5
23.0
25.0
þ2.0
þ1.9
þ2.4
21.4
(21.4)
21.5
NS
NS
NS
NS
21.5
(21.4)
(21.4)
NS
NS
þ2.2
NS
NS
þ1.5
þ2.0
Q-PCR
24.9
22.5
2430.0
25.8
295.0
21.9
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 inferior
to 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 the
mutant 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 change
between mutant and control mice, for Rho, Bcp, Gnat2 and Arr3 were 21.6, 24.2, 21.7 and 23, respectively.
a
Fold change was measured by densitometry analysis of northern blot.
in general more important, indicating that the microarray data
under-estimated the level at which genes were deregulated.
Q-PCR performed on several genes at onset and moderate
disease stages in R7E mice demonstrated that sensitivity of
microarray was sufficient to detect progressive decrease or
increase of gene expression (e.g. Rho, Hes5, etc.) that correlated with aggravation of phenotype. Consequently, we are
confident that transcriptional alterations identified in our microarray analysis are likely to reflect not only changes in trend but
also progressive modifications in gene expression occurring
during disease aggravation in mutant mice retina.
Deregulated genes gather into specific functional pathways
over time
Given the large number of transcripts showing altered level of
expression in R7E and R6/2 retina, we used a systematic
approach to interpret the biological significance of these
gene deregulations. First, we re-annotated each transcript by
analyzing the corresponding Affymetrix probe sets sequences
with RetScope platform (Chalmel F. and Poch O. unpublished
data). 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 with
high confidence each probe set sequence with existing
full-length transcript and protein accession numbers. Then,
GOAnno (24) was used to predict Gene Ontology (25)
annotations for each identified protein. Finally, we identified
over-represented GO Biological Process (GOBP) terms by
calculating a z-score using the whole human proteome as
reference (26). We reasoned that genes over-represented in a
given GOBP term have a higher probability of being relevant
to polyQ-induced retinopathy. Among the 727 deregulated
transcripts (Supplementary Material, Table S1), 541 were
assigned to GOBP terms, and 50% of them gather in enriched
GOBP terms presenting a z-score of 3 or greater (corresponding to a probability of less than 0.00135).
Disease onset in R7E was associated with under-expressed
genes significantly enriched in signal transduction, cell communication and most importantly visual perception GOBP
terms (Fig. 2). The number and enrichment of under-expressed
genes of these three functional categories increased at moderate stage of R7E phenotype. In contrast to signal transduction
and cell communication, visual perception represents a level
of higher specificity with respect to the GO annotation
system. Enrichment of down-regulated genes involved in
visual perception makes perfect sense with the early and progressive ERG defect in R7E retinopathy and by itself validates
the method that we used to identify deregulated pathways.
The moderate stage of R7E phenotype was also characterized by enrichment of down-regulated genes involved in
development, morphogenesis and organogenesis as well as
up- or down-regulated genes associated with cell growth,
maintenance and organization. Expression alteration of
genes belonging to these categories might account for the
important morphological changes (e.g. loss of segments and
enlarged nuclei) of rod photoreceptors seen at 9 weeks.
Down-regulation of genes involved in regulation of cell
death was also over-represented, although rod photoreceptor
death is observed at later stages only.
Strikingly, altered genes in R6/2 were significantly enriched
in the same functional categories as in R7E at 9 weeks. This concerned genes involved in visual perception, signal transduction,
cell communication, as well as in development, morphogenesis,
organogenesis and cell growth/maintenance (Fig. 2). These
results highlight at the molecular level the similitude of
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Rhodopsin (Rho)
Rod transducin alpha subunit (Gnat1)
Rod phosphodiesterase 6 beta (Pde6b)
Blue cone opsin (Bcp)
Cone transducin alpha subunit (Gnat2)
Cone arrestin (Arr3)
Rhodopsin kinase (Rhok)
Rod outer segment membrane protein
1 (Rom)
Cone-rod homeobox containing gene (Crx)
Neural retina leucine zipper protein (Nrl)
Nuclear receptor (Nr2e3)
X box binding protein (Xbp1)
Methyl binding domain 4 protein (Mbd4)
Hairy and enhancer of split 5 (Hes5)
Protein lnhibitor of activated STAT3 (Pias3)
R7E (onset)
Human Molecular Genetics, 2006, Vol. 15, No. 5
695
retinal degeneration and dysfunction in the two models. Expectedly, 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 dysfunction, consistent with the ubiquitous expression of mutated
htt-exon-1 in R6/2 retina.
Down-regulation of genes essential for photoreceptor light
transduction
The most compelling finding of the above analysis is the preeminent deregulation of genes associated with visual function
in R7E and R6/2 retina, which occurs in the absence of overt
cell death. In 9-week-old R7E, this concerned 27 genes, most
of which were under-expressed (Table 2). Of these, eight
genes displayed expression changes already at the onset of
R7E retinopathy. A first group of 14 R7E deregulated genes
are directly involved in phototransduction cascade of rod
photoreceptors: Rho, transducin subunits (Gngt1, Gnb1,
Gnat1 and Gnb3), cGMP phosphodiesterase subunits (Pde6a
and Pde6b), genes involved in ion channel structure and regulation (Cnga, Guca1b and Slc24a1) and in other phototransduction functions (Sag, Rhok, Rdh12, Rcv and Rbp). A
second group of genes are implicated in morphogenesis of
photoreceptor segments (Rom1 and Rds). A third group is
composed of genes having diverse but essential functions in
photoreceptor, because they cause retinopathy when mutated
in human and mouse (Rp1, Tulp1, Impdh1 or Rs1h) or
because their orthologs are involved in retinal degeneration
in 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 in
vision (Bcp, Gcp, Gnat2, Pde6c, Pde6h and Arr3) were
under-expressed, consistent with the ubiquitous expression
of the mutant htt-exon-1 transgene. It is noteworthy that the
expression of three cone-specific genes (Bcp and Gnat2 and
Pde6c) and the retinal pigmentary epithelium (RPE)-specific
gene Myo7A were also altered in R7E. Up-regulation of
Myo7A, which is involved in phagocytosis of photoreceptor
outer segment disks by the RPE, suggests an increased
phagocytic activity of these cells in response to structural
disorganization of the R7E photoreceptor segment layers.
As shown in Table 1, Q-PCR analysis validated the deregulation 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 moderate
disease stages revealed that deregulation progresses along
phenotype aggravation in both R7E and R6/2 retina. Together,
these data indicate that the abnormal ERGs and structural disorganization of segment layers in R7E and R6/2 retina result
from a very specific down-regulation of genes involved in
function and morphogenesis of photoreceptors.
Altered expression of genes controlling photoreceptor
differentiation
To specify the unique properties of photoreceptor cells, the
developmental pathway in vertebrate retina is regulated by a
series of transcription factors acting before and during the
terminal differentiation of photoreceptors (12,13). Enrichment
of a large cohort of deregulated genes involved in visual perception as well as in development, morphogenesis, organogenesis and cell growth, maintenance and organization
suggested that the genetic controls maintaining photoreceptor
differentiation are impaired in R7E and R6/2 retina. Among
the deregulated transcripts belonging to these pathways, we
found a number of transcription factors controlling various
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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 onset
and 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 R6/2
moderate 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 proteins
in 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 mutant
mouse.
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Human Molecular Genetics, 2006, Vol. 15, No. 5
Table 2. Deregulation of genes essential for photoreceptor function
Gene name
Opsins
Transducins
Phosphodiesterases
Arrestins
Kinase
Segment morphogenesis
Others
Rhodopsin
Blue cone opsin
Green cone opsina
Transducin gamma 1 subunit
Transducin beta 1 subunit
Rod transducin alpha subunita
Transducin beta 3 subunit
Cone transducin alpha subunita
Transducin beta 4 subunit
Phosphodiesterase 6aa
Phosphodiesterase 6ba
Phosphodiesterase 6c
Phosphodiesterase 6ha
Cyclic nucleotide gated channel alpha 1’
Guanylate cyclase activator 1Ba
Solute carrier family 24, member 1
Retinal S-Antigena
Cone arrestin
Rhodopsin kinasea
Rod outer segment membrane protein 1a
Retinal degeneration, slowa
Retinol deshydrogenase 12a
Recoverina
Retinitis pigmentosa 1a
Tubby-like protein 1a
Inosine 5’-phosphate deshydrogenase 1a
Phosphatidylinositol membrane associated 1a
Protein phosphatase, EF hand calcium-binding
domain 2a
Retinoschisis 1 homologa
Retinol-binding protein
Myosine VIIAa
Symbol
R7E (onset)
R7E (moderate)
R6/2 (moderate)
Rho
Bcp
Gcp
Gngt1
Gnb1
Gnat1
Gnb3
Gnat2
Gnb4
Pde6a
Pde6b
Pde6c
Pde6h
Cnga1
Guca1b
Slc24a1
Sag
Arr3
Rhok
Rom1
Rds
Rdh12
Rcv
Rp1
Tulp1
Impdh1
Pitpnm1
Ppef 2
21.6
22.3
21.9
21.7
21.8
22.2
23.2
21.5
þ1.7
þ2.1
þ1.6
22.1
22.2
þ1.5
22.0
226.0
246.4
21.5
21.9
Rs1h
Rbp
Myo7A
þ1.6
21.5
22.2
22.3
22.0
21.8
23.5
22.5
22.7
21.9
21.5
22.1
22.6
21.6
22.7
21.5
219.0
22.1
21.6
22.8
21.6
22.2
21.7
21.5
21.6
21.9
22.0
22.5
þ1.9
þ2.1
21.6
The AFC of the differentially expressed genes are represented. (2) and (þ) indicate, respectively, lower and higher expression in the mutant versus
control.
a
Genes that cause retina disorders when mutated in human or other organisms.
aspects of neuronal cell fate specification and differentiation
(Table 3).
Under-expressed transcription factor genes. Among this
group, Crx, Nrl and Nr2e3 present a special interest, as they
are key regulators of the terminal differentiation and maintenance of rod photoreceptors (27 –29). These genes are
expressed in immature rod cells and interplay together to
ensure the proper expression of rod- and repression of conespecific genes during rod terminal differentiation. Crx is also
required for terminal differentiation of cones (27). Two
other genes, ErrB and Mef2C, are markers of differentiation
fate, as their expression increases in maturing photoreceptors
(30,31). Mef2C is also known to be involved in neuronal
differentiation process. For these five genes, the expression
level in R7E retina progressively decreased from 1.4 to 2.9fold between onset and moderate stage of pathology. Although
the 1.4-fold variation seen at onset is inferior to the 1.5-fold
used as a confident cut-off for our microarray analysis, the
data are consistent with the progressive aggravation of the
R7E phenotype. In R6/2 retina, Nrl, Errb and Mef2c
expressions were also significantly decreased, and Crx was
reduced by 1.4-fold. We performed Q-PCR analysis of Crx,
Nrl and Nr2e3 at R7E moderate stage and of Crx in R6/2
and confirmed their reduced level of expression (Table 1).
Reduced expression of the late differentiation regulators
Crx, Nrl and Nr2e3 is expected to cause deregulation of a
large subset of their target genes in both mouse models. To
evaluate the extent to which dysfunction of these transcription
factors contributes to polyQ-induced retinopathy, we compared our data sets with recent expression profiling studies,
which revealed the transcriptional networks regulated by Nrl
and Crx in mouse (32 –35). Using Affymetrix microarray
analysis, Yoshida et al. (32) identified 164 differentially
expressed genes in Nrl2/2 versus wt mature retina, which
are likely candidate Nrl-regulated genes. We found 15 Nrlregulated genes presenting altered expression level at onset
and 45 at moderate stage of R7E retinopathy (Supplementary
Material, Table S2), an increasing number inversely correlating with the progressive down-regulation of Nrl gene along
disease phenotype (Table 3). Similarly, 24 Nrl-regulated
genes were also altered in R6/2 retina. Many of the Nrlregulated genes showing expression changes in R7E and
R6/2 retina function in phototransduction cascade. Expression
profiling data also revealed that Nr2e3 and Mef2c are regulated by Nrl (32,33). Nr2e3 inhibits expression of conespecific genes in rod cells. Evidence for Nr2e3 dysfunction
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Human Molecular Genetics, 2006, Vol. 15, No. 5
697
Table 3. Deregulation of transcription factors implicated in retinal development
R7E
(onset)
R7E
(moderate)
R612
(moderate)
Function
Reference
Cone-rod homeobox containing gene (Crx)
Neural retina leucine zipper protein (Nrl)
Nuclear receptor (Nr2e3)
Estrogen-related receptor beta (ErrB)
Myocyte enhancer factor 2C (Mef2c)
Paired box gene 6 (Pax6)
Mab-21-like 1 (Mab21l1)
LIM homeobox protein 2 (Lhx2)
Nuclear receptor (Nurr)
Optic homeobox 2 (Optx2)
Signal transducer and activator
of transcription 3 (Stat3)
Hairy and enhancer of split 5 (Hes5)
(21.4)
(21.4)
NS
(21.4)
21.5
NS
(21.4)
21.5
NS
NS
NS
21.6
22.2
22.1
22.9
22.9
NS
21.8
NS
21.6
þ1.5
þ1.7
(21.4)
21.5
NS
21.8
22.2
21.8
NS
NS
NS
NS
þ2.1
Differentiation and maintenance of cones and rods
Differentiation and maintenance of rods
Differentiation and maintenance of rods
Expression increases in differentiating photoreceptors
Expression increases in differentiating retina
Early eye development
Early eye development
Early eye development
Differentiation and maintenance of neurons
Early eye development
Retinal progenitor proliferation
(27)
(28)
(29)
(30)
(31)
(36)
(36)
(36)
(37)
(38)
(40)
þ2.4
NS
Retinal gliogenesis, inhibits neurogenesis
(39)
NS
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-off
but presenting P , 0.015 are also indicated in brackets. NS, non-significant.
in R7E retina is suggested by up-regulation of cone-specific
genes (Gnat2 and Pde6c) (Table 2). As for Nrl, we compared
our data set with candidate Crx-regulated genes revealed
by SAGE analysis of Crx2/2 versus wt P10 retina (34,35).
Again, the number of Crx-regulated genes showing alteration in R7E increased from onset (9 genes) to moderate
stage (16 genes) in correlation with the progressive downregulation of Crx gene (Table 3 and Supplementary Material,
Table S3). Similarly, nine Crx-regulated genes were altered
in R6/2. In addition, change trends of genes deregulated in
R7E and R6/2 were generally in agreement with the expected
transcriptional consequence of Crx and Nrl inactivation.
Together, these results indicate that reduced level of
Crx, Nrl and Nr2e3 expression in R7E and R6/2 retina is
in part responsible for the loss of expression of many rod
differentiation genes.
Other transcription factors under-expressed in R7E and
R6/2 retina might also contribute to the loss of differentiated
state of retinal neurons (Table 3). Pax6, Mab21l1 and Lhx2
genes are essential for early eye development and remain
expressed in adult retina (36). Nurr codes for a master regulator of neuronal differentiation and maintenance in other brain
regions (37) and might display a similar role in the retina.
Over-expressed transcription factor genes. Three R7E overexpressed genes, Optx2, Hes5 and Stat3, display crucial functions in the retinal development (Table 3). Optx2 is required
for early eye development (38), whereas the Notch effector
Hes5 is expressed in retinal progenitors and modulates glial
cell fate specification (39). Stat3 is thought to play a key
role in retinal development by maintaining retinal precursors
in an undifferentiated state (40). Interestingly, persistent
Optx2, Hes5 or Stat3 expression in developing retina caused
inhibitory effects on photoreceptor differentiation (38,39,41).
Our microarray data indicated that Hes5 transcript was not
detected (‘absent call’) in control retina, consistent with
previous in situ hybridization study on mature retina (39).
Q-PCR analysis confirmed that Hes5 was re-expressed in
R7E (Table 1). Immunofluorescence analysis of wt retina
revealed a faint Hes5 staining restricted to ganglion cell and
inner nuclear layers (Fig. 3) and no staining in photoreceptor
layer. In R7E retina, anti-Hes5 antibody stained rod photoreceptors containing NIs, but not those in which NIs were
absent (presumably due to the absence of transgene expression)
(Fig. 3). Our data suggest that Hes5 gene was aberrantly
re-expressed in R7E photoreceptors.
We also confirmed by western blot analysis that increased
Stat3 gene expression correlated with increased level of
Stat3 protein and its phosphorylated (tyrosine 727) form in
9-week-old R7E and R6/2 (Fig. 4A). Interestingly, phosphorylation of Stat3 was detected as early as 3 weeks of
age, preceding the increase in Stat3 protein, and was sustained
over time in R7E retina, whereas it remained undetected in
R7N controls. Another study found that phosphorylated
Stat3 precedes the increase of Stat3 protein level, suggesting
that Stat3 regulates its own promoter (42). We also found
that phospho-Stat3 was enriched in the nuclear fraction
when compared with the cytoplasmic one in the R7E
(Fig. 4B), consistent with the known process of Stat3
activation.
Stat proteins are terminal transcription factors of a welldocumented signaling pathway, which involves external cytokine stimuli, membrane receptors, Janus tyrosine kinases (Jak)
and several negative regulators such as protein inhibitor of
activated Stat (Pias) and suppressor of cytokine signaling
(Socs) (43). Affymetrix microarray MOE430A contained probe
sets to assess expression of several of these genes controlling
Stat3 activation. We found that only Pias3 gene displayed
altered expression in both R7E and R6/2 retina when compared with their controls (Table1). Decreased expression of
Pias3, which inhibits Stat3 binding to the DNA regulatory
elements, is consistent with Stat3 protein activation. We
confirmed by Q-PCR analysis that Pias3 gene expression
was decreased in both R7E and R6/2 retina.
Altogether, these results indicate that transcription factors
associated to early steps of retinal development and able to
cause negative effect on photoreceptor differentiation are
aberrantly over-expressed in polyQ retina.
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Gene symbol
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Human Molecular Genetics, 2006, Vol. 15, No. 5
differentiation of rod photoreceptors require transcriptional
programs that are being uncovered (12,13). In the present
study, we have used gene expression profiling to build a comprehensive scheme of the pathomechanism involved in two
mouse models of rod photoreceptor degeneration induced
by different polyQ expansion proteins. Our data show that
polyQ expansion, regardless of the protein context, causes a
progressive loss of expression of mature rod genes by compromising the genetic programs involved in the maintenance
of rod differentiation state, resulting in rod dysfunction and
degeneration.
Correlating rod dysfunction and morphological change
with polyQ-induced transcriptional alterations
Figure 4. Aberrant activation of Stat3. (A) Stat3 expression is increased in
R7E 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 of
age (arrow). This activation persists and increases at later stages. Stat3 and
phospho-Stat3 are also increased in R6/2 when compared with control’s
retina. Anti-TFIIFalpha antibody is used to control the loaded protein
amounts. (B) Phospho-Stat3 is enriched in the nuclear fraction compared
with the cytoplasmic one in the R7E mice.
DISCUSSION
Mature rod photoreceptors are issued from sequential development of retinal progenitor cells (RPC) during vertebrate
retinogenesis. In the initial phase, a pool of dividing RPC
becomes post-mitotic precursors committed to rod fate.
Then, following a period of specification, immature rods
terminally differentiate into mature rods, which express
specific proteins essential to establish a characteristic cell
shape and to execute light transduction. Commitment and
In SCA7 R7E mouse model, rod-specific expression of
ATXN7 with expanded polyQ causes a progressive rod
degeneration timely defined by molecular (NI formation),
functional (scotopic ERG loss), histological (segment layer
reduction) and cell shape anomalies, occurring prior overt
cell death (17). Using DNA microarrays and Q-PCR, we
show that R7E rod phenotype was attributable to reduced
expression of a large subset of genes specifically involved in
phototransduction and segment morphogenesis of mature
rods. Previous studies (15,16) reported similar findings in
other SCA7 mouse models by analyzing a restrained number
of selected photoreceptor genes. Our genomewide analysis
gives now an exhaustive view over time of the loss of
expression of genes essential to mature rod features. Likewise,
expression of mutant htt-exon-1 in R6/2 mouse retina, which
causes a retinopathy comparable to R7E (18), alters the regulation of a similar subset of mature rod genes. Mutant
htt-exon-1, which is expressed in cones as well and causes
photopic ERG dysfunction of R6/2 retina, also leads to
reduced expression of cone phototransduction genes. Downregulation of mature photoreceptor genes is progressive and
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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 R7E
retina, 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.
Human Molecular Genetics, 2006, Vol. 15, No. 5
affects an increasing number of genes along phenotype aggravation in R7E and R6/2. Together, these results indicate that
photoreceptors expressing polyQ expansion proteins progressively loose their differentiation features due to transcriptional
alterations of mature photoreceptor genes. Apart from genes
directly implicated in rod maturation state, a large number of
genes involved in development, morphogenesis, cell growth,
maintenance and organization are also significantly deregulated in 9-week-old retina, a disease time point when
segment layer is severely reduced in both polyQ models
and R7E rod nuclei lose their chromatin organization (60).
This indicates that important reprogramming of gene
expression accompanied reshaping of rod cells to neurons
being non-functional and showing immature traits.
Consistent with the loss of rod photoreceptor differentiation
features, three key transcription factors (Crx, Nrl and Nr2e3)
that control terminal differentiation and maintenance of
mature rod are down-regulated in R7E and R6/2 retina.
Mice lacking the homeobox Crx develop precursor neurons
committed to rod fate, but fail to develop proper outer segments and to express phototransduction proteins (27,34).
The bZIP Nrl is essential for expression of rod-specific and
repression of cone-specific genes in rods. Nrl and Crx proteins
can interact to exhibit transcriptional synergy in gene activation. Inactivation of Nrl in mice results in a complete loss
of rods at the expense of supernumerary S-cones (28). One
Nrl repression mechanism of cone genes proceeds via induction of the orphan nuclear receptor Nr2e3 expression. Rd7
mutant mouse, which carries a spontaneous Nr2e3 deletion,
develops aberrant hybrid cone – rod photoreceptors (29,44).
The reduced Crx and Nrl expressions have a direct impact
on R7E and R6/2 photoreceptor expression profiles, as a
large number of Crx- and Nrl-target genes show deregulation
in polyQ retina models, even though our microarray analyses
are performed at early stages (onset and moderate phenotype)
of retinopathy. Strikingly, the number of Crx- and Nrl-target
genes deregulated in R7E retina increases from onset to
moderate stage, inversely correlating with the progressive
down-regulation of Crx and Nrl genes along phenotype aggravation. Moreover, Nr2e3 dysfunction in R7E retina is
suggested by increased level of cone-specific gene expression.
Thus, we conclude that progressive loss of Crx, Nr2e3 and Nrl
functions in R7E and R6/2 retina results in the failure of
rod photoreceptors to maintain the expression of mature rod
genes.
La Spada et al. (15) reported that mutant ATXN7 can interfere with CRX and NRL transactivation function in vitro.
On the basis of direct interaction between ATXN7 and CRX
and on the cone and rod CRX expression, the authors
favored a model in which interference with transactivation
function of CRX, rather than NRL, would be the major
defect leading to cone and rod dystrophy in SCA7. Our
result suggests another mechanism whereby mutant ATXN7
and mutant htt-exon-1 cause Crx, Nrl and Nr2e3 dysfunction
by repressing their expression.
We also noted the reduced expression of other transcription
factors (Table 3), which are involved either in early eye
development (Pax6, Mab2l1 and Lhx2) or in neuronal differentiation (Mef 2c, Errbeta; and Nurr). Although the role of
these factors in mature retina is currently unknown, their
deregulation might also contribute to the RE7 and R6/2
retinal phenotype. Whatever is their contribution, downregulation of these factors reveals that polyQ expansion
toxicity has a broad effect on the transcriptional program of
mature retina.
Aberrant activation of transcription factors that inhibit
photoreceptor differentiation
Another important finding of our study is the increased level
of Optx2, Hes5 and Stat3 expression in polyQ retina models.
It has already been shown using several experimental conditions that persistent expression of these transcription
factors in developing vertebrate retina causes inhibitory
effect on rod photoreceptor differentiation (38,39,41). It is
notable that Stat3 protein is activated in the two polyQ
models, and in the case of R7E, activation is triggered at
onset and sustained along the pathogenic process. During
retinal development, Stat3 inactivation is required for rod
photoreceptor precursors to differentiate into mature rod and
for Crx and Rho gene expression (40,41). The cytokines
CNTF and LIF, which activate Stat3 signaling pathway, also
prevent photoreceptor differentiation in rodents (45,46). In
addition, LIF effect on photoreceptor differentiation is due
to inhibition of Crx and Nrl expression (46). The consequence
of activating Stat3 in adult retina is currently unknown.
However, in preclinical studies on animal models for photoreceptor degeneration, prolonged CNTF treatment increased
photoreceptor survival without rescuing their function
(47,48). These rod photoreceptors displayed enlarged nuclei
with less compacted chromatin, similar to photoreceptor
nuclei of aged R7E mice (60). On the basis of the current
knowledge of CNTF, LIF and Stat3 effects on photoreceptor
differentiation, aberrant Stat3 activation in R7E and R6/2
retina might be one of the early events leading to loss of rod
differentiation by causing repression of Crx and Nrl gene
expression.
Our study also points out on several pathways that could
contribute to Stat3 activation. Pias3 gene expression is
reduced in both R7E and R6/2 retina. Down-regulation of
Pias3 protein, which interacts with and inhibits DNAbinding activity of Stat3, could strengthen Stat3 activation
and its transcriptional activity. In Drosophila, proper
expression level of both Pias and Stat ortholog genes are
crucial for eye development (49). Interestingly, deletion of
dpias inhibits retinal cell differentiation. Besides the canonical
cytokine/receptor/Jak pathway, other routes of Stat activation
have been identified. A recent study shed light on a cross-talk
between Notch –Hes and Jak – Stat signaling pathways via
direct interaction of Hes1 or Hes5 with Jak2 and Stat3,
which promotes Stat3 phosphorylation and activation. Consequently, aberrant Hes5 expression in R7E retina may
contribute to the sustained Stat3 activation. Moreover, Stat
proteins can also be activated by diverse cellular stresses
(50). For instance, oxidative stress, which is associated with
polyQ-induced toxicity in several model systems, can activate
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Down-regulation of rod differentiation program
699
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Human Molecular Genetics, 2006, Vol. 15, No. 5
Stat3 pathway (51). We previously showed that neuronal
stress involving the activation of JNK/Jun/AP1 pathway
occurred in R7E and R6/2 mouse retina (9). Our microarray
data also reveal over-expression of regulators of oxidative
and endoplasmic reticulum stress response, such as Bach2
and Xbp1 genes, respectively (Table 1 and Supplementary
Material, Table S1), warranting further investigation of the
role of stress in the loss of neuronal differentiation induced
by polyQ expansion.
PolyQ toxicity and loss of neuronal identity
Animals
R7E and R7N transgenic lines were maintained on the inbred
C57BL/6 background (14). R6/2 line, purchased from the
Jackson Laboratories (Bar Harbor, ME, USA) was originally
on C57BL/6:CBA/J background. To avoid the rd1 mutation
carried by CBA/J inbred strain, R6/2 mice were backcrossed
on C57BL/6 background. The mice used in this study were
75% C57BL/6 and 25% CBAJ. Genotyping of R7N, R7E,
R6/2 and rd1 mutations was performed by PCR on tail DNA
according to the protocols previously described (17). The
experiments were performed in accordance with the National
Institutes of Health Guide for Care and use of Laboratory
Animals.
RNA isolation
Both retinas from one mouse were isolated, pooled, immediately frozen in liquid nitrogen and stored at 2808C. Retinas
were homogenized with an ultraturax homogenizer and total
RNA was extracted using Qiagen columns according to manufacturer’s instructions (Qiagen RNeasy kit). RNA quantity
and quality were analyzed using 260/280 nm absorbance
ratio and Agilent apparatus.
Affymetrix gene profiling and data analysis
For the microarray experiments, we analyzed five R7E versus
five R7N mice at 3 weeks of age, six R7E versus six R7N mice
at 9 weeks of age as well as four R6/2 versus four wt mice at 9
weeks of age. Biotinylated cRNA were prepared according to
the standard Affymetrix protocol (GeneChip Expression
Analysis Technical Manual, 2001, 701151 Rev1, Mat No.
1020407, 03/2002; Affymetrix). Ten micrograms of fragmented cRNA were hybridized for 16 h at 458C on murine MOE
430A GeneChips. These GeneChips contain 22 600 probe
sets, about 15 000 of them are against well-annotated fulllength genes, whereas the remaining probe sets are against
gene clusters containing only EST sequences and some gene
clusters with non-EST sequences. GeneChips were washed
and stained in the Affymetrix Fluidics Station 400 and
further scanned using the Hewlett-Packard GeneArray
Scanner G2500A. Initial data preparation was performed by
Affymetrix MicroArray Suite Version 5.0 (MAS5) using Affymetrix default analysis settings and global scaling as normalization method. The trimmed mean target intensity of each
chip was arbitrarily set to 100. Absolute analyses generate a
signal value for each probe set and a detection call of
absent, present or marginal. To select differentially expressed
genes, three consecutive filters were applied for each of the
three comparison groups (3-week-old R7E versus R7N,
9-week-old R7E versus R7N and 9-week-old R6/2 versus
wt). First, we performed a Mann – Whitney statistical test
and considered as significant the genes with a P-value less
than 0.015. We then picked the genes presenting an Affymetrix fold change (AFC) 1.5. Finally, we selected genes
called ‘present’ in at least (n 2 1) mice in a group of n
mutant mice for the up-regulated genes, or n control mice
for the down-regulated genes.
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The detailed mechanisms whereby polyQ expansion causes
loss of neuronal differentiation remain to be characterized.
However, it is worth to note that deregulation of the genetic
program maintaining photoreceptor differentiation by polyQ
expansion seems unique, because such gene deregulation
specificities were not reported in transcriptome analyses of
other retinal degenerations associated with rd1, Rho or Rp1
mutations (52,53) (Chalmel, F. and Poch, O., manuscript in
preparation). Our conclusions that polyQ induces loss of
neuronal differentiation are based on the study of a highly
organized and well-characterized neuronal tissue, the retina.
However, failure of neurons to maintain their differentiated
state might be a common feature to polyQ disorders. Early
studies on gene deregulation occurring in HD showed that
genes encoding proteins essential for striatal neuron functions
such as neurotransmitters, receptors and other neuronal signaling proteins were down-regulated (10,54). Although the
mechanism remains to be identified, recent studies showed
that one critical regulator of neuronal terminal differentiation,
neuron-restrictive silencer factor (NRSF), was altered in HD.
Indeed, NRSF protein was abnormally localized in the neuronal nucleus in HD, resulting in repression of NRSF-target
genes (55). Another study showed that polyQ expansion
caused up-regulation of NRSF gene expression and prevented
neuronal differentiation of embryonic stem cells (56). Overexpression of NRSF was shown to inhibit neurite outgrowth
(57). Morphological abnormalities of dendrites and defects
of neurite outgrowth were reported in HD and in in vitro
and in vivo HD models (58,59). In regard to our findings in
R7E and R6/2 retina, it is thus conceivable that mutant htt
compromises neuronal identity of striatal neurons by deregulating the genetic program controlling their differentiation.
One puzzling aspect of polyQ disorders is that long-term
neuronal dysfunction precedes neuronal cell loss. Little is
known about this gradual degenerative process or on the
mechanism of neuronal death. Our finding suggests that dysfunction occurred because neurons loose their neuronal features. PolyQ expansion might directly affect the key proteins
orchestrating the program of neuronal differentiation. Alternatively, in response to permanent stress caused by polyQ expansion, vulnerable neurons might slowly progress to an
immature state. By being in conflict with the mature environment of the nervous system, these immature neurons might not
receive stimuli promoting their survival. Studies on how
polyQ expansion causes loss of neuronal differentiation and
how neurons loosing neuronal traits are condemn to death
will thus be of major relevance for the development of therapeutic approaches in polyQ disorders.
MATERIALS AND METHODS
Human Molecular Genetics, 2006, Vol. 15, No. 5
701
Q-PCR analysis
ACKNOWLEDGEMENTS
Total RNA (1 mg) was subjected to reverse transcription using
SuperScript Reverse Transcriptase (Invitrogen). For each
gene, primers were designed using Primer 3 software and
are available upon request. Real-time PCR was performed
with SYBRGreen using the Light Cycler apparatus. Specificity
of reactions was confirmed by melting curve analysis. Significant fold change was calculated based on the difference in the
calculated concentration between the transgenic and the
control mice after normalization to Ppia or Arbp as internal
controls. Three to six animals for each genotype, R7E, R6/2
and wt, were analysed.
We are grateful to Fabrice Klein, Léa Ben-Haı̈em, Myriam
Ravache and Stanislas DuManoir for helpful discussion and
Astrid Lunkes and Nathalie Daigle for critical reading of the
manuscript. We thank Doulay Dembele from the IGBMC
Affymetrix platform. This work was supported by funds
from INSERM, CNRS, Hôpital Universitaire de Strasbourg;
Collège de France (to J.L.M.); European Community
EUROSCA integrated project (LSHM-CT-2004-503304),
Hereditary Disease Foundation and French Ministry of
Science (to D.D. and Y.T.).
Conflict of Interest statement. None declared.
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 protease
inhibitors. Triton was then added to a final concentration of
0.1% to whole retinal homogenates. Retinas were then incubated on ice for 15 min, sonicated and centrifuged for
15 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 monoclonal
Phospho-Stat3 (Tyr705) (58E12) (Cell signaling). They were
revealed with appropriate anti-mouse or anti-rabbit peroxidase-conjugated secondary antibodies and the chemiluminescent reaction (Pierce, Rockford, IL, USA).
Immunohistofluorescence
Enucleated eyes were dissected to remove lens and cornea
and fixed in fresh 4% paraformaldehyde for 2 h at 48C.
Fixed retinas were placed for 1 h in 30% sucrose and
frozen in Cryomatrix compound (Thermo Shandon). Cryostat
sections (10 mm) were mounted on SuperFrost/Plus slides (O.
Kindler, Freiburg, Germany). Sections were permeabilized
and 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 in
PBS, sections were incubated with primary antibodies
diluted in 3% normal goat serum, 0.5% BSA, 0.1%
Tween-20 and 1 PBS. Primary antibodies used were
rabbit Anti-Hes5 affinity purified polyclonal antibody
AB5708 (Chemicon International) and mouse anti-ATXN7
monoclonal antibody (2A10) (17). Sections were then
washed and incubated with secondary antibodies diluted in
the same solution as primary antibody. The secondary antibodies used were CY3- and Oregon green-conjugated goat
anti-mouse and anti-rabbit IgG at a dilution of 1:200.
Nuclei were counterstained with 0.5 mg/ml 4,6-diaminido-2phenylindole.
SUPPLEMENTARY MATERIAL
Supplementary Material is available at HMG Online.
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