Anna Niewiadomska-Cimicka

Frédéric Doussau

Jean-Baptiste Perot

Michel Roux

Celine Keime

Antoine Hache

Françoise Piguet

Ariana Novati

Chantal Weber

Binnaz Yalcin

Aurélie Eisenmann

Julien Flament

Keywords: Spinocerebellar ataxia type 7, Cell-type speci c gene deregulation, Purkinje cells, Polyglutamine expansion, Cerebellum, Peripheral neuropathy, Retinopathy

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Introduction

Spinocerebellar ataxia type 7 (SCA7) is an adult-onset neurodegenerative disorder caused by a CAG repeat expansion in the ATXN7 gene that is translated into an elongated polyglutamine (polyQ) tract in ATXN7 [1]. SCA7 expansions range from 37 to more than 200 repeats [2] and the length of the expanded repeats correlates inversely with the onset of symptoms and positively with disease severity and progression. Rare very large expansions ranging from 200-460 repeats cause infantile-onset SCA7, affect non-neural tissues and lead to early death [3][4][5][6][7]. ATXN7 is a subunit of Spt-Ada-Gcn5 Acetyltransferase (SAGA) [8], a multiprotein complex that regulates RNA polymerase II-dependent transcription through histone acetylation and deubiquitination activities.

The adult form of SCA7 is primarily characterized by the di culty to coordinate balance, gait, and speech. Other common clinical features include spastic ataxia gait, dysphagia, intentional tremors, slow eye movements, ophthalmoplegia, as well as pyramidal signs [9,[START_REF] David | Molecular and clinical correlations in autosomal dominant cerebellar ataxia with progressive macular dystrophy (SCA7)[END_REF]. The pathology arises primarily from progressive damage of the cerebellum and its associated tracts. Neuronal loss is observed in the Purkinje cell layer (PCL), the granule cell layer (GCL), the inferior olivary nuclei and the basis pontis associated with the atrophy of spinocerebellar and pyramidal tracts [9,[START_REF] Martin | Spinocerebellar ataxia type 7[END_REF][START_REF] Rub | Spinocerebellar ataxia type 7 (SCA7): widespread brain damage in an adult-onset patient with progressive visual impairments in comparison with an adult-onset patient without visual impairments[END_REF][START_REF] Horton | Schmahmann Spinocerebellar ataxia type 7: clinical course, phenotype-genotype correlations, and neuropathology[END_REF][START_REF] Martin | On an autosomal dominant form of retinal-cerebellar degeneration: an autopsy study of ve patients in one family[END_REF][START_REF] Martin | Spinocerebellar ataxia type 7 (SCA7) -correlations between phenotype and genotype in one large Belgian family[END_REF][16]. Alteration of other structures including the telencephalon, midbrain and medulla oblongata is observed and may contribute to additional symptoms [START_REF] David | Molecular and clinical correlations in autosomal dominant cerebellar ataxia with progressive macular dystrophy (SCA7)[END_REF][START_REF] Martin | Spinocerebellar ataxia type 7[END_REF][START_REF] Bang | Huh Pontine atrophy precedes cerebellar degeneration in spinocerebellar ataxia 7: MRI-based volumetric analysis[END_REF]. A peripheral neuropathy has been reported in some but not all neurophysiological studies [START_REF] Rub | Spinocerebellar ataxia type 7 (SCA7): widespread brain damage in an adult-onset patient with progressive visual impairments in comparison with an adult-onset patient without visual impairments[END_REF]18,[START_REF] Van De Warrenburg | Peripheral nerve involvement in spinocerebellar ataxias[END_REF]. A feature of SCA7, which differs from other inherited ataxias is the decrease of visual acuity due to loss of cone and rod photoreceptors [START_REF] David | Molecular and clinical correlations in autosomal dominant cerebellar ataxia with progressive macular dystrophy (SCA7)[END_REF][START_REF] Rub | Spinocerebellar ataxia type 7 (SCA7): widespread brain damage in an adult-onset patient with progressive visual impairments in comparison with an adult-onset patient without visual impairments[END_REF][START_REF] Bang | Huh Pontine atrophy precedes cerebellar degeneration in spinocerebellar ataxia 7: MRI-based volumetric analysis[END_REF][START_REF] Aleman | Jacobson Spinocerebellar ataxia type 7 (SCA7) shows a cone-rod dystrophy phenotype[END_REF].

The visual impairment in SCA7 mouse models revealed a progressive loss of photoreceptor-speci c gene expression, resulting in shortage of proteins necessary for renewal of outer segments, the highly dynamic photosensitive structure of terminally differentiated photoreceptors [2,[START_REF] Spada | Polyglutamine-expanded ataxin-7 antagonizes CRX function and induces cone-rod dystrophy in a mouse model of[END_REF][START_REF] Abou-Sleymane | et Polyglutamine expansion causes neurodegeneration by altering the neuronal differentiation program[END_REF][START_REF] Helmlinger | Glutamineexpanded ataxin-7 alters TFTC/STAGA recruitment and chromatin structure leading to photoreceptor dysfunction[END_REF][START_REF] Ye Mova | et Polyglutamine toxicity induces rod photoreceptor division, morphological transformation or death in Spinocerebellar ataxia 7 mouse retina[END_REF]. Gene downregulation is due to dysfunction of GCN5-dependent acetylation activity of SAGA leading to histone hypoacetylation on photoreceptor gene promoters [START_REF] Palhan | et Polyglutamine-expanded ataxin-7 inhibits STAGA histone acetyltransferase activity to produce retinal degeneration[END_REF], and to alterations of the transactivation activity of cell-type speci c transcription factors [START_REF] Spada | Polyglutamine-expanded ataxin-7 antagonizes CRX function and induces cone-rod dystrophy in a mouse model of[END_REF]26,[START_REF] Merienne | Preventing polyglutamine-induced activation of c-Jun delays neuronal dysfunction in a mouse model of SCA7 retinopathy[END_REF]. The results support the notion that ATXN7 mutation primarily alters the expression of photoreceptor identity genes and hence the maintenance of terminal neuronal differentiation [2]. Interestingly, zebra sh studies showed that Atxn7 is involved in the differentiation of photoreceptor and Purkinje cell (PC) during development [28,[START_REF] Yanicostas | Requirement for zebra sh ataxin-7 in differentiation of photoreceptors and cerebellar neurons[END_REF]. Whether PCs or other neurons also progressively lose their differentiation identity in SCA7 remains to be investigated.

In contrast to the retinopathy, the cellular and molecular mechanisms underlying SCA7 motor incoordination remain to be determined. Different transgenic mice showed different vulnerability of PCs to mutant ATXN7 (mATXN7) expression [START_REF] Yvert | Expanded polyglutamines induce neurodegeneration and trans-neuronal alterations in cerebellum and retina of SCA7 transgenic mice[END_REF][START_REF] Yvert | Mandel SCA7 mouse models show selective stabilization of mutant ataxin-7 and similar cellular responses in different neuronal cell types[END_REF][START_REF] Chou | Polyglutamine-expanded ataxin-7 causes cerebellar dysfunction by inducing transcriptional dysregulation[END_REF][START_REF] Custer | et Bergmann glia expression of polyglutamine-expanded ataxin-7 produces neurodegeneration by impairing glutamate transport[END_REF][START_REF] Mookerjee | Posttranslational modi cation of ataxin-7 at lysine 257 prevents autophagy-mediated turnover of an N-terminal caspase-7 cleavage fragment[END_REF][START_REF] Furrer | Reduction of mutant ataxin-7 expression restores motor function and prevents cerebellar synaptic reorganization in a conditional mouse model of SCA7[END_REF][36] and little overlap of cerebellar gene deregulation [START_REF] Chou | Polyglutamine-expanded ataxin-7 causes cerebellar dysfunction by inducing transcriptional dysregulation[END_REF][START_REF] Friedrich | et Comparative analyses of Purkinje cell gene expression pro les reveal shared molecular abnormalities in models of different polyglutamine diseases[END_REF]38]. Interestingly, transgenic mice overexpressing mATXN7 in the whole brain except PCs or speci cally in Bergmann glia developed motor dysfunction [START_REF] Custer | et Bergmann glia expression of polyglutamine-expanded ataxin-7 produces neurodegeneration by impairing glutamate transport[END_REF][START_REF] Mookerjee | Posttranslational modi cation of ataxin-7 at lysine 257 prevents autophagy-mediated turnover of an N-terminal caspase-7 cleavage fragment[END_REF], questioning the contribution of different cerebellar cell types to the pathology. However, the previous approaches have been insu cient to provide a deep understanding of the SCA7 cerebellar pathology at a single cell-type resolution.

Knock-in mouse models, in which a CAG repeat expansion is introduced in Atxn7 show proper temporal and spatial expression of ATXN7 splicing isoforms and thus provide a genetically and physiologically more representative disease model. SCA7266Q/5Q knock-in mice show motor de cit and shrinkage of PC body size, but present a severe infantile-like pathology and early death, which limit the characterization of the motor phenotype [START_REF] Yoo | SCA7 knockin mice model human SCA7 and reveal gradual accumulation of mutant ataxin-7 in neurons and abnormalities in shortterm plasticity[END_REF]. A cognate SCA7100Q/5Q knock-in line develops late-onset pathology (half-life of ~19 months) and did not go through deep characterization [START_REF] Chen | Gcn5 loss-of-function accelerates cerebellar and retinal degeneration in a SCA7 mouse model[END_REF]. Yet, the broad spectrum of SCA7 pathology has not been fully recapitulated in the current animal models. Furthermore, the contribution of SAGA dysfunction to cerebellar pathology is unclear as conditional deletion of GCN5 in mouse PCs led to very mild ataxia [START_REF] Chen | Gcn5 loss-of-function accelerates cerebellar and retinal degeneration in a SCA7 mouse model[END_REF].

SCA7 is part of a group comprising 5 other SCAs (SCA1, SCA2, SCA3, SCA6 and SCA17) that are also caused by polyQ expansion in different gene products. PolyQ SCAs cause damage to the cerebellum, often with high vulnerability of PCs [START_REF] Klockgether | Update on degenerative ataxias[END_REF]. Despite shared genetic, clinical, and neuropathological features, more is needed to identify points of intersection and divergence of the pathophysiology of polyQ SCAs [START_REF] Niewiadomska-Cimicka | Trottier Gene Deregulation and Underlying Mechanisms in Spinocerebellar Ataxias With Polyglutamine Expansion[END_REF]. While SCA proteins do not share any domain and have different cellular functions, changes in gene expression are central features in most polyQ SCAs. Therefore, the comparison of differentially expressed genes should provide insight into converging disease mechanisms.

To get insight into the mechanisms underlying motor incoordination and cerebellar degeneration, we used a new SCA7 knock-in mice line carrying 140 CAG repeats. A comprehensive and longitudinal characterization of this model using a battery of analyses (motor and behavioral tests, retina imaging, MRI, electrophysiology, neuropathology) indicates that SCA7140Q/5Q mice remarkably recapitulate the major clinical features observed in patients, including cerebellar damage, speci c cerebral atrophy, peripheral nerves pathology and photoreceptor dystrophy, which account for progressive impairment of behavior, motor and vision functions. Using this relevant model, we developed a methodology to analyze cell-type speci c gene expression in the cerebellum and determined the morphological, electrophysiological and gene expression features of the most vulnerable neurons, the PCs. Our study further show that both SAGA-dependent epigenetic marks -histone H3 acetylation and H2B deubiquitination -are altered and correlate with gene deregulation. Finally, in contrast to other cerebellar cell types, PCs show a profound downregulation of neuronal identity genes, which are also downregulated in other polyQ SCAs. Thus, our study uncovers a signature of PC identity genes highly vulnerable to polyQ expansion mutation, revealing a converging pathomechanisms in polyQ SCAs.

Methods

Mouse information.

The SCA7 knock-in mouse line, which is derived from the SCA7 mouse model [START_REF] Yoo | SCA7 knockin mice model human SCA7 and reveal gradual accumulation of mutant ataxin-7 in neurons and abnormalities in shortterm plasticity[END_REF] by CAG repeat contraction, harbors an expansion ranging from 140-150 CAG repeats in exon1 of the Atxn7 locus. Mice are kept on C57Bl/6J background and bred in the Mouse Clinical Institute (Illkirch, France) with a 12 h light/dark cycle and free access to food and water. Genotyping was performed by PCR as described previously [START_REF] Yoo | SCA7 knockin mice model human SCA7 and reveal gradual accumulation of mutant ataxin-7 in neurons and abnormalities in shortterm plasticity[END_REF]. Wild type (WT) and SCA7 males and females were analyzed, unless indicated.

Behavioral and motor performance analysis.

To ensure the replicability of SCA7 mouse phenotype, two mouse colonies were raised in the husbandry of different research centers and were analyzed using standardized protocols by independent experimenters and instruments. The experiments were all performed during the light phase of a 12h light/dark cycle. Footprint. After coating the front-and hind feet with 2 different colors of non-toxic inks, mice walked through a tunnel (length: 50 cm, width: 9 cm, height: 6 cm) covered with paper. The following ve footprint patterns were manually measured: matching between paws, stride length, stance width, distance between fore-and hindlimbs.

Grip strength test. This test measures the maximal muscle strength (g) using an isometric dynamometer connected to a grid (Bioseb). Mice were held by the tail and allowed to grip the metal grid with the 4 paws, then they were pulled backwards until they released it. Each mouse was submitted to 3 consecutive trials and the maximal strength developed by the mouse before releasing the grid was recorded. The average value of the three trials adjusted to body weight was used as index of the muscle strength.

CatWalk. Gait analyses were performed using CatWalk XT 8.1 (Noldus Information Technology, Wageningen, The Netherlands). The CatWalk apparatus consists of a closed corridor resting on a glass plate. The walkway (20 cm) is illuminated with red light from the ceiling and green light from the edges of the plate (green intensity threshold: 0.30; camera gain: 20). When an animal enters the corridor, the light re ected by the paws is detected by a camera placed 25 cm below the walkway. Each mouse was placed on the walkway at one extremity and allowed to move in the corridor in both directions. Only runs lasting a maximum of 10 seconds with 60% maximal speed variation were counted. Five successful runs per animal were recorded. The swing (s) de ned as the duration of no contact of a paw with the glass plate was analyzed separately for fore-and hindlimbs and averaged between left and right sides.

Optical Coherence Tomography and electroretinography.

Optical Coherence Tomography (OCT). Pupils were dilated with a bilateral topical application of atropine (1%, Virbac, France). Animals were then anesthetized using iso urane (4% for induction, 2% R2200 spectral domain OCT (Bioptigen, North Carolina, USA). Cornea was kept moisturized with a thin layer of ocular gel (Ocry-gel, TVM, France) applied with surgical eye spears. For each eye, a rectangular scan was obtained, corresponding to a region of 1.4 x 1.4 mm centered on the optic nerve. The thicknesses of the whole retina (including the pigmentary epithelium), the segments, the outer nuclear layer, the inner nuclear layer and the inner plexiform layer for a given eye were calculated as the average of 2 measures performed manually at 0.5 mm from the optic nerve center using the Bioptigen InVivoVue Neuroanatomical study.

Neuroanatomical study was carried out using 10 mice at 33 weeks (n=5 per group) and 12 mice at 50 weeks (n=6 per group). Brain samples were xed in 4% buffered formalin for 48 hours. Seventy-eight brain parameters, made of area and length measurements, were taken blind to the genotype across three coronal sections as described [START_REF] Mikhaleva | Yalcin Histomorphological Phenotyping of the Adult Mouse Brain[END_REF]. Data were analyzed using a t-test with equal variances to determine whether a brain region is associated with neuroanatomical defect or not.

Magnetic resonance imaging (MRI).

After paraformaldehyde (PFA) perfusion of 25-week-old mice, brains were removed and stained for one week in a solution of Gadolinium (Dotarem©, Guerbet, France) in PBS at 2.5 mM. This protocol enhances the signal-and contrast-to-noise ratios on MR images of xed brains [START_REF] Dhenain | Volk Passive staining: a novel ex vivo MRI protocol to detect amyloid deposits in mouse models of Alzheimer's disease[END_REF], Ex vivo MRI experiments were performed on a horizontal 11.7 T Bruker scanner (Bruker, Ettlingen, Germany). A quadrature cryoprobe (Bruker, Ettlingen, Germany) was used for radiofrequency transmission and reception. High-resolution anatomical images were acquired using Echo Planar Imaging sequence (Resolution= 100x100x200 µm , TR= 3000 ms, TE= 28 ms). In order to preserve integrity to avoid deformations, brains were kept inside skulls for ex vivo MRI experiments. Anatomical images were coregistered in the same space to create a study template. Transformations to match the study template to an atlas composed of a high-resolution template and a map of region's labels adapted from the Allen mouse brain Atlas [START_REF] Lein | Genome-wide atlas of gene expression in the adult mouse brain[END_REF] were calculated. Finally, transformations to match the study template and labels to each individual anatomic image were calculated. The automated segmentation pipeline was performed using an in-house python library (Sammba-MRI, https://github.com/sammba-mri/sammbamri).

Immuno uorescence.

Cryosections. Mice were transcardialy perfused with 1xPBS followed by 4% PFA/1xPBS Floating cerebellar sections were processed as described above with the following modi cations.

Sections were incubated in Eppendorf tubes for 3 days at 4°C with primary antibodies in 300 μl of blocking buffer. Secondary antibodies were applied for 2h at RT. Sections were positioned on slides directly before mounting.

Retina images were acquired on a Leica DM 4000B with a Cool SNAP HQ2 camera with 40x objective with zoom factor 2. z-slices with 0.2 µm z-step using 6.5 µm x 6.5 µm pixel size.

Images were saved in tiff format. Subsequently, RGB channels were split in Fiji. Channel with vGLUT2 acquisition («.ims» format) was exported to Imaris v 9.3.1 (Bitplane AG) for 3D reconstruction. The region of interest, namely the molecular cell layer, was selected and 3D volumes were created with the surface module of Imaris. Next, vGLUT2 was analyzed only on this region with the Imaris spot module.

The detection of objects in 3-dimensional space was based on both intensity and size (with the estimated XY diameter of 1µm). Only spots ranging from 0.52 to 30 µm were selected to eliminate

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background. Density and volume measurements of spots were analyzed in Excel and GraphPad Prism 6. The extension of vGLUT2-positive climbing ber terminals along the dendrites of PC was measured using Fiji. Namely, the ratio of the extension of the 3D reconstructed vGLUT2 spots in the molecular cell layer over the total length of the molecular cell layer was calculated for the central part of lobule VI and for the side part of lobule X.

Histology and electron microscopy analysis.

Eyes were prepared for histology and electron microscopy as in [START_REF] Ye Mova | et Polyglutamine toxicity induces rod photoreceptor division, morphological transformation or death in Spinocerebellar ataxia 7 mouse retina[END_REF]. Semi-thin (2 μm) sagittal sections were cut with an ultramicrotome (Leica Ultracut UCT) and stained with toluidine blue, and histologically analyzed by light microscopy. For sciatic nerve preparation, animals were perfused with 5 mL of PBS and 10 mL 4% PFA in saline, and tissues were xed in 2.5% PFA/2.5% glutaraldehyde in cacodylate buffer. Ultra-thin (70 nm) sections were cut and contrasted with uranyl acetate and lead citrate and examined at 70kv with a Morgagni 268D electron microscope. Images were captured digitally by Mega View III camera (Soft Imaging System) and contrast was adjusted for display purposes.

Western blot analysis.

Cerebellum and retina were dissected and snap frozen in liquid nitrogen. Their whole cell extracts were obtained by lysis and sonication of the tissue in, respectively, 500 µl and 150 µl of the buffer containing 50mM Hepes-KOH (pH 7.5), 140mM NaCl, 1mM EDTA, 0.1% Na-deoxycholate, 1% SDS, 1% Triton-X100, 1x Protease Inhibitor Cocktail (Roche), 20mM N-ethylmaleide, 20mM nicotinamide. The last two compounds were not used for ATXN7 detection. Protein concentrations were measured using Bradford protein assay. 10 µg (histone analysis) and 30 µg (ATXN7 analysis) of total proteins were used for electrophoresis. Electrophoresis was run in 4-12% Bis-Tris Protein Gels in NuPAGE MOPS SDS Running Buffer (ThermoFisher) for histone analysis and in NuPAGE™ 3-8% Tris-Acetate Protein Gels in NuPAGE™ Tris-Acetate SDS Running Buffer (ThermoFisher) for ATXN7 detection.

Following electrophoresis, proteins were transferred on nitrocellulose membrane for 1.5 h at 200 mA or overnight at 30mA. Antibodies were diluted in 1x PBSTM (1xPBS with 0.1% Tween, and 5% non-fat milk). The following primary antibodies were applied overnight at 4°C: anti-ATXN7, 1:1 000 (ThermoFisher, PA1-749); anti-H2B, 1:10 000 (IGBMC clone H2-2A8); anti-H2Bub 1:2 000 (MediMabs, MM-0029); anti-H3K9ac 1:2 000 (Abcam ab4441); anti-GAPDH 1:10 000 (Merck Millipore, MAB374); anti-TUBB 1:2 000 (IGBMC, 2A2). Secondary antibodies: Peroxidase A niPure F(ab') Fragment Goat Anti-Rabbit IgG (H+L) (GARPO; 1:10 000, Jackson ImmunoResearch Lab.) and Peroxidase A niPure Goat Anti-Mouse IgG (H+L) (GAMPO; 1:10 000, Jackson ImmunoResearch Lab.) were applied in 1x

PBSTM for 2h at RT, washed three times in 1xPBST, and proteins were detected using SuperSignal

West Pico PLUS Chemiluminescent kit (ThermoFisher). Chemiluminescent signal was acquired on Amersham Imager 600. Images were analyzed using Fiji.

RNA-seq library preparation and sequencing. Analysis of public datasets.

For SCA1 dataset (GEO: GSE75778), FASTQ les were retrieved from Sequence Read Archive using fastq-dump (SRA Toolkit v2.8.0, BioProject PRJNA305316, samples: GSM1967518-GSM1967520; GSM1967524-GSM1967529; GSM1967533 -GSM1967538; GSM1967542 -GSM1967544), and only the rst 50 bases of read 1 sequences were retained for further analysis. For SCA2 dataset (Array Express: E-MTAB-6293), FASTQ les were retrieved from Sequence Read Archive using fastq-dump (SRA Toolkit v2.8.0, BioProject PRJEB24319, samples ERR2237861-ERR2237880, ERR2237882 -ERR2237893). Reads were then mapped onto the mm10 assembly of mouse genome using Tophat v2.0.14 [46] with bowtie v2.1.0 aligner. Gene expression was quanti ed using HTSeq v0.6.1 [START_REF] Anders | HTSeq--a Python framework to work with high-throughput sequencing data[END_REF] and gene annotations from Ensembl release 90. Statistical analysis was performed using R and DESeq2 v1.18. 1 [48].

Microarray probe sets of Doyle et al. [START_REF] Doyle | Application of a translational pro ling approach for the comparative analysis of CNS cell types[END_REF] were converted into gene names according to mm10 annotation using the Affymetrix NetAffx Annotation File (Mouse430_2 Annotations, Release 36). Next, the cell-type subdivided datasets were curated to indicate the genes present uniquely in one cell-type dataset and in more than 1 cell-type datasets. The obtained gene lists were used to study cell-type distribution of transcriptional changes in SCA7.

Quantitative RT-PCR.

Total RNA samples were extracted from 1 cerebellum/sample or 2 retinas/animal/sample using TriReagent (MRC) following the manufacturer's instruction. 500ng of puri ed RNA was then reverse transcribed using QuantiTect® Reverse Transcription kit (Qiagen) according to the manufacturer's protocol. The real-time qPCR (RT-qPCR) reactions were performed on obtained cDNAs (1/20 dilution) using QuantiTect SYBR Green PCR Master Mix (Qiagen), and speci c primers (Table S1) with a Light Cycler 480 (Roche). Gene expressions were normalized based on the expression of Hprt or 36B4 (Rplp0).

Statistical analysis. ERG studies in SCA7 patients showed that cone dysfunction precedes rod dysfunction [START_REF] Bang | Huh Pontine atrophy precedes cerebellar degeneration in spinocerebellar ataxia 7: MRI-based volumetric analysis[END_REF]. We evaluated De cits in motor activity and coordination.

We performed longitudinal motor performance analysis of SCA7140Q/5Q and WT male littermates using a variety of motor activity paradigms. Morphological and functional alterations of the peripheral nervous system.

A peripheral neuropathy was reported in some but not all neurophysiological studies in patients [START_REF] Rub | Spinocerebellar ataxia type 7 (SCA7): widespread brain damage in an adult-onset patient with progressive visual impairments in comparison with an adult-onset patient without visual impairments[END_REF]18,[START_REF] Van De Warrenburg | Peripheral nerve involvement in spinocerebellar ataxias[END_REF] and has never been explored in mouse models. To understand the cause of motor alterations in SCA7140Q/5Q mice, the activity of the peripheral nervous system was rst assessed over time by measuring the sciatic nerve function by electromyography. At 15 weeks, median amplitudes of plantar Mwave and H-wave of SCA7140Q/5Q mice were similar to WT littermates (Fig 4a-b). However, both plantar M-wave and H-wave amplitudes were reduced by 21% (p < 0.05) and by 33% (p < 0.05), respectively, at 32 weeks in SCA7 mice. At 43 weeks, the plantar M-wave amplitude was reduced by 31% (p < 0.05), while the H-wave was almost absent (95%, p < 0.0001). To correlate these functional abnormalities with structural status, sciatic nerve sections were analyzed by histology and electron microscopy. Compared to WT mice, cross section of sciatic nerve of SCA7 mice showed clear alterations of myelinated bers and decreased number of small bers (Fig 4c). On electron microscopy (Fig 4d-o), several structural alterations were observed in myelinated bers of SCA7140Q/5Q mice, including myelin degeneration and infolding-like structures as well as axonal modi cations characterized by inner swelling tongue and autophagy. Nonmyelinated bers displayed abnormal structures of Remak bundles. In conclusion, motor components of the sciatic nerve morphology and activity are affected in SCA7140Q/5Q mice.

Distinct morphological alterations in SCA7 mouse brain.

Brains of SCA7 patients show prominent neurodegeneration in the cerebellum and atrophy of other brain regions. To establish a systematic survey of brain damage of SCA7 mice, the morphology of three different coronal brain sections of SCA7140Q/5Q and WT mice was studied using neuroanatomical measurement of 78 anatomy parameters across 20 distinct brain subregions [START_REF] Mikhaleva | Yalcin Histomorphological Phenotyping of the Adult Mouse Brain[END_REF]. At 33 weeks, there was no major surface difference between SCA7140Q/5Q mice and WT littermates. However, end-stage SCA7140Q/5Q mice (50 weeks) showed signi cant reduction of brain areas in the three coronal sections (from -6.7% to -8%) with both white and grey matter alterations (S4 Fig) . Atrophy of grey matter structures included the somatosensory cortex, the pons, while affected white matter regions comprised the corpus callosum, the anterior commissure, the internal capsule and the mbria of the hippocampus. We then aimed to capture SCA7 mouse brain alterations at earlier disease stage using high resolution MRI. MRI Morphological and functional alterations of Purkinje cells.

Since PCs are highly vulnerable neurons in inherited ataxias, their morphology, connectivity and function were investigated. The area of PC soma was signi cantly smaller in lobule VI (p < 0.002) of 34-week SCA7140Q/5Q compared to age-matched WT mice, while in lobules IX and X PC soma did not differ (Fig 6a-e). In contrast, PC circularity index was affected in lobules IX and X (p < 0.006 and p < 0.0005, respectively), but not in lobule VI (Fig 6f-h). We then analyzed synapses integrity between PC and climbing bers (CF) of inferior olivary neurons. To this purpose, we quanti ed vGLUT2-immunolabeling of CF-PC contact points in the MCL. The average density and average volume of vGLUT2 contact points were similar in WT and SCA7 mice (data not shown). However, extension of climbing ber terminals along the PC dendritic tree was signi cantly reduced in lobule VI and X by 15.8% (p=0.029) and 23% (p=0.034), respectively, in SCA7140Q/5Q compared to WT mice (Fig 6i ,j). Moreover, co-immunolabeling of vGLUT2 and CALB1 revealed frequent aggregation of vGLUT2 contact points along large dendritic arborization of PC in SCA7 cerebellum (Fig 6k). Therefore, despite different morphological alterations of PC soma of lobule VI and X, synaptic contacts between PC and CF were affected in both lobules.

Intrinsic membrane properties of PC determine their spontaneous ring pattern even in the absence of synaptic inputs [START_REF] Gruol | Morphological and physiological differentiation of Purkinje neurons in cultures of rat[END_REF][START_REF] Hausser | Tonic synaptic inhibition modulates neuronal output pattern and spatiotemporal synaptic[END_REF]. Any alteration in the precision of their pacemaking activity may be the result of PC cellular dysfunctions and may affect cerebellar functions [START_REF] Hoxha | Purkinje Cell Signaling De cits in Animal Models of Front Synaptic[END_REF]. To investigate the correlation between PC function, morphological phenotypes and motor de cits, we assessed whether the intrinsic properties of PC spontaneous discharge were altered in SCA7140Q/5Q mice at 22 and 38 weeks. We recorded spontaneous activity of PC located in lobule VI and IX/X in WT and SCA7 littermates using juxtacellular recordings of PC in acute cerebellar slices at a near physiological temperature (32°C) (Fig 6l Accumulation of mutant ATXN7 in retina and cerebellum.

Previous studies showed that mATXN7 tends to misfold and form intracellular protein aggregates (reviewed in [2]). To determine the dynamic of mATXN7 aggregation in SCA7 mice we performed immuno uorescence and western blot analysis of retina and cerebellar sections. In WT retina and cerebellum, ATXN7 immunolabeling is barely detectable (S6a,b Fig) . In contrast, the immunolabeling progressively increased with age in SCA7 mouse tissues, indicating that mATXN7 accumulates over time. This resulted in different labeling pro les depending of the neuronal population. mATXN7 aggregates were readily detected in many photoreceptor nuclei at 12 weeks, became widespread in all retinal neuron nuclei at later stages. In SCA7 cerebellum, PCs showed the earliest mATXN7 accumulation that progressively increased between 12 and 19 weeks, leading to the formation of large nuclear aggregates at later stages. mATXN7 nuclear aggregates were also observed in the MCL and GCL. All cerebellar lobules showed the accumulation of mATXN7 in PC nuclei (S6c Fig).

Western blot analysis showed the expression of normal ATXN7 as a 110 kilodalton (kDa) band in control retina and cerebellar samples (S6d Fig) . In SCA7 samples, the mutant form of ATXN7 migrated at ~140 kDa. From 5-8 weeks of age, mATXN7 was also detected as high molecular weight smear in these tissues, indicating that mATXN7 accumulates as insoluble oligomeric forms, much before the formation of visible nuclear aggregates as detected by immunolabeling of tissues.

Cerebellar gene expression changes in SCA7 mice. in the pathogenesis of other ataxias [START_REF] Hoxha | Purkinje Cell Signaling De cits in Animal Models of Front Synaptic[END_REF].

IPA also pointed out the impairment of interferon signaling pathway (Fig 7b). Interestingly, among the 10 most predicted upstream regulators (p ≤ 7*10-9) of the differentially expressed genes, 8 are implicated in the modulation of interferons signaling pathways (Table S5). Moreover, functional annotations of the 270 upregulated genes using STRING [START_REF] Szklarczyk | STRING v10: protein-protein interaction networks, integrated over the tree of life[END_REF] Chort et al. [START_REF] Chort | Interferon beta induces clearance of mutant ataxin 7 and improves locomotion in SCA7 knock-in mice[END_REF] previously reported the increased level of interferon-beta and its receptor in the cerebellum of the SCA7266Q/5Q model. Our transcriptome data provide additional evidence of the activation of a regulatory network involving interferon signaling.

Downregulation of a large subset of Purkinje cell identity genes.

To gain insight into the cell type-speci c distribution of differentially expressed genes in SCA7 cerebella, we took advantage of the publicly available actively translated mRNA pro les of all the major cerebellar cell types [START_REF] Doyle | Application of a translational pro ling approach for the comparative analysis of CNS cell types[END_REF]. To compare with our SCA7 RNA-seq dataset, the published datasets [START_REF] Doyle | Application of a translational pro ling approach for the comparative analysis of CNS cell types[END_REF] were reannotated using Affymetrix microarray probesets annotations on mm10 genome assembly. This reannotation led to a total of 9671 actively translated transcripts in the different cell types of WT cerebellum (S8 Fig ). Taking the cellular distribution into account, we were able to determine the cell-type speci city of 470 differentially expressed genes of SCA7 cerebellum and found that 240 of them were assigned to a single cerebellar cell type and 230 were present in more than one cell type (Table S7). The analysis revealed that all the cerebellar cell types presented differentially expressed genes (Fig 7c,d), indicating that SCA7 pathology is widespread across cerebellar cell types.

However, despite the rarity of PC in the cerebellum estimated to 0.1% of cerebellar cells, 23% (106 out of 470) of SCA7 deregulated genes were assigned to PC. In contrast, 15% of SCA7 deregulated genes were assigned to granule cells, while this cell type represents 90% of cerebellar cells. Importantly, while granule cell genes were both up-and downregulated, almost all PC genes (104 out of 106 genes) were downregulated (Fig 7d), indicating that repression of PC genes is a major component of SCA7 cerebellar pathology. Another publicly available datasets from Kratz et al.

[58] allowed us to identify 5 additional PC genes that were downregulated in SCA7 mouse cerebella (Table S7), resulting in a total 109 downregulated PC genes.

To ascertain the speci city of these 109 genes in PC, their pattern of expression was analyzed using in situ hybridization datasets reported in The Brain Transcriptome Database (BrainTx; CDT) [START_REF] Sato | Cerebellar development transcriptome database (CDT-DB): pro ling of spatio-temporal gene expression during the postnatal development of mouse cerebellum[END_REF] and in the Allen Brain Atlas [START_REF] Lein | Genome-wide atlas of gene expression in the adult mouse brain[END_REF]. We established three categories of genes according to the level of enriched expression in PC, as speci cally expressed in PC compared to other cerebellar cells, enriched in PC or nonenriched in PC (e.g. equally present in other cell types). The analysis showed that 83 genes had speci c enrichment in PC, and are thus considered as PC identity genes that showed reduced expression in SCA7 mice. The remaining genes were expressed in wider range of cerebellar cell types or were not present in in situ hybridization datasets and were then excluded from further analysis (Table S8).

RT-qPCR was used to study the time course of repression of a subset of PC identity genes in SCA7140Q/5Q cerebella. The Grid2, Calb1, Fam21 and Rora genes showed decreased expression in SCA7 mice from 27 weeks, coinciding with the onset of motor alterations (S9 Fig) . Interestingly, the 3 PC genes Pcp4, Fam107b and Rgs8 showed downregulation already at 12 weeks, a pre-symptomatic stage of the disease, suggesting a strong implication in PC pathology of SCA7 mice.

A signature of downregulated PC identity genes in SCA1, SCA2 and SCA7.

To further explore whether PC genes downregulated in SCA7 have pathological relevance in cerebellar ataxia, we looked at publicly available transcriptomic datasets of SCA1 and SCA2 mouse models in which the mutant genes were speci cally expressed in PC and caused spontaneous ring dysfunction [60,61]. In these 2 models, expression level of a subset genes was also shown to be altered compared to their respective WT littermates [60, 61]. Before proceeding with the comparative analysis, we have reanalyzed the RNA-seq raw data of SCA1 mouse cerebella at 5, 12 and 28 weeks and SCA2 cerebella at 6 weeks with the same software which was used for SCA7. We then compared the differentially expressed genes in the cerebellum of the three SCA models. The analysis revealed that 136 genes common to the 3 datasets were downregulated and only 5 were upregulated (Fig 7e,f). Interestingly, out of the 136 downregulated genes, 67 were PC identity genes including 10 genes previously associated with genetic ataxias (Table 1 and Table S9). These 67 genes were downregulated already at 5 weeks in SCA1 and at 6 weeks in SCA2 mice, which are disease onset stages of these models [61, 62], and constitute a signature of early pathological processes of PC. The comparison performed with the SCA1 dataset at later disease stages revealed 13 additional downregulated PC identity genes in common with SCA2 and SCA7 mice (Table 1), suggesting that these 13 genes were associated with the progression of PC pathology.

The biological signi cance of the early downregulation of the 67 PC identity genes was researched using 3 analyses. First, the KEGG pathway analysis indicated that this set was enriched for genes involved in LTD (FDR 0.0036), cGMP-PKG signaling pathway (FDR 0.0068) and phosphatidylinositol signaling system (FDR 0.0069) (Fig 7g). Second, the STRING analysis [START_REF] Szklarczyk | STRING v10: protein-protein interaction networks, integrated over the tree of life[END_REF] was used to search for functional The network also showed central positions to the regulator of G-protein signaling 8 (RGS8), phospholipase C beta 3 (PLCB3) and cGMP-dependent kinase PRKG1, the latter two are related to LTD and synapse functions of PC [START_REF] Hoxha | Purkinje Cell Signaling De cits in Animal Models of Front Synaptic[END_REF]63]. Finally, our list of 67 PC identity genes was compared to a published list of genes composing the SCA1 Magenta gene module [61]. This module was established using the WGCNA (Weighted Gene Coexpression Network Analysis) of SCA1 cerebellar transcriptomes and reported to be enriched in PC genes involved in LTD and glutamate signaling and to be of high relevance for SCA1 pathogenesis [61]. We found that 48 out of 67 PC-enriched genes were present in the Magenta module. Furthermore, two major hub genes described in the module, namely Fam107b and Rgs8, were within the top strongest downregulated genes in SCA1, SCA2 and SCA7 models, and were already downregulated at 12 weeks in SCA7 mice, hence before onset of motor symptoms (S9 Fig).

Altogether, these analyses suggest that the dysfunction of synaptic LTD of PC constitutes a common pathway in the early pathogenesis of SCA1, SCA2 and SCA7.

Finally, to identify mechanisms which could account for the downregulation of the 67 PC identity genes, we rst looked at RORa (RAR-related orphan receptor alpha), a key transcription factor involved in PC differentiation and survival [64] and showing downregulation in SCA1, SCA2 and SCA7 mice (Table 1).

Among the 67 PC genes, 19 genes were known RORa-target genes [64, 65] (Table 1), suggesting that RORa dysfunction accounts for PC genes deregulation. We then performed in silico analysis of 2-kb promoter sequence of the 67 PC genes to search for de novo motifs and transcription factors binding sites using MEME, TOMTOM, AME or FIMO [66]. The analysis did not reveal major enrichment of other transcription factor binding motifs in the promoter of the 67 PC identity genes affected, compared to control sequences (e.g. shu ed promoter sequences or promoter sequences of 67 PC-enriched non-deregulated genes) (data not shown). Deregulation of gene expression may depend on alteration of diverse processes including chromatin structure, nucleosomal occupancy and histone modi cations, which play important roles in transcriptional regulation. Given the presence of ATXN7 in the histone-modifying SAGA complex, we determined whether the bulk level of histone marks regulated by SAGA were altered in SCA7 cerebellum. The level of H2Bub was signi cantly increased in SCA7140Q/5Q cerebellum compared to WT, while the level of H3K9ac showed no signi cant difference (Fig 8a,b). However, a signi cant decrease of H3K9ac was observed in SCA7140Q/140Q homozygous mice, which were more severely affected that the SCA7 heterozygous mice (S10 Fig) . Together, this suggests that mutant ATXN7 differentially alters the histone-modifying activities of SAGA complex along the progression of cerebellar pathology and could impact on transcription of cerebellar genes.

Discussion

SCA7 is a devastating neurological disease affecting primarily the cerebellum and retina, and no treatment can currently prevent or slow down the disease process. Accurate models of the disease are required as con dent tools for a better understanding of SCA7 pathogenesis and for developing therapeutic strategies. In this study, we used a new and highly relevant mouse model to determine the molecular and cellular basis of the cerebellar pathology of SCA7. By combing cerebellar gene pro ling and single-cell-type resolution as well as morphological and functional studies, PCs came out as the most affected cerebellar cell type and showed profound loss of expression of neuronal identity genes, critical for their spontaneous ring and synaptic functions. These observations suggest disease mechanisms primarily altering the maintenance of PC differentiation, and are consistent with similar pathological features described for SCA7 mouse photoreceptors in this and earlier studies. Like in SCA7 mice, PC identity genes were also affected in pre-symptomatic SCA1 and SCA2 mice, indicating convergence of pathomechanisms underlying PC vulnerability in these polyQ SCAs.

Retinal, PNS and CNS features of SCA7 mice.

Unlike previous mouse models, the SCA7140Q/5Q line combines most cardinal disease features observed in adult SCA7 patients, including retinal, cerebral, cerebellar and peripheral nerve pathologies.

Symptoms develop and progress in a suitable time window for preclinical trials and can be quantitatively evaluated using a series of robust motor, functional, histological and molecular parameters (S11 Fig) . In contrast to the severely affected mouse line, SCA7140Q/5Q mice present no complication related to husbandry, as male fertility and offspring delivery are normal.

The SCA7140Q/5Q line recapitulates photoreceptor degenerative features as observed in earlier mouse models [2,[START_REF] Spada | Polyglutamine-expanded ataxin-7 antagonizes CRX function and induces cone-rod dystrophy in a mouse model of[END_REF][START_REF] Abou-Sleymane | et Polyglutamine expansion causes neurodegeneration by altering the neuronal differentiation program[END_REF][START_REF] Yvert | Expanded polyglutamines induce neurodegeneration and trans-neuronal alterations in cerebellum and retina of SCA7 transgenic mice[END_REF][START_REF] Yoo | SCA7 knockin mice model human SCA7 and reveal gradual accumulation of mutant ataxin-7 in neurons and abnormalities in shortterm plasticity[END_REF]. In normal retinal, photoreceptor function is maintained by sustained expression of photoreceptor identity genes essential to the daily renewal of outer segments, a structure entirely lled with discs of folded double membranes where phototransduction takes place. In SCA7140Q/5Q retina, photoreceptor dysfunction correlates with the progressive reduced expression of photoreceptor identity genes such as opsin genes and the disappearance of outer segments as shown by OCT and electron microscopy. In zebra sh, knock-down of atxn7 impedes the formation of photoreceptor outer segments during development of the eye [28,[START_REF] Yanicostas | Requirement for zebra sh ataxin-7 in differentiation of photoreceptors and cerebellar neurons[END_REF]. Together, this suggests that mATXN7 toxicity alters a normal function of ATXN7 in the maintenance of outer segments, a cell-type speci c structure characteristic of photoreceptor terminal differentiation [67]. SCA7140Q/5Q shows alterations in the RPE and Bruch's membrane, also reported in patients [START_REF] Rub | Spinocerebellar ataxia type 7 (SCA7): widespread brain damage in an adult-onset patient with progressive visual impairments in comparison with an adult-onset patient without visual impairments[END_REF]. These observations warrant further investigation as the interaction between photoreceptor and RPE is essential for proper visual function. Interestingly, the homogenous deposits observed between RPE plasma membrane and basement membrane of SCA7140Q/5Q mice, resemble sub-RPE deposits reported in Malattia Leventinese mouse model [68], a dominantly inherited macular degenerative disease. These mice bears a mutation in Efemp1, a proposed interactor of ATXN7 [69].

Peripheral pathologies account for the neurological phenotype in SCA7140Q/5Q mice. Electromyograph studies revealed that motor peripheral activities are reduced at 32 weeks, due to lesions of myelin and myelinated bers of sciatic nerve. The sensory component might also be affected as non-myelinated bers also show alterations. SCA7140Q/5Q mice ndings parallel the most recent study of SCA7 patients, who showed abnormal motor and sensory nerve conductions [18], and complement previous neuropathological evidence of degenerating signs in the dorsal root and anterior horn of patients' spinal cord [START_REF] Rub | Spinocerebellar ataxia type 7 (SCA7): widespread brain damage in an adult-onset patient with progressive visual impairments in comparison with an adult-onset patient without visual impairments[END_REF][START_REF] Martin | On an autosomal dominant form of retinal-cerebellar degeneration: an autopsy study of ve patients in one family[END_REF].

Post mortem brains showed atrophy and neuronal loss in diverse brain regions, which could account for a variety of neurological symptoms present in adult-onset SCA7 patients [START_REF] Martin | Spinocerebellar ataxia type 7[END_REF][START_REF] Rub | Spinocerebellar ataxia type 7 (SCA7): widespread brain damage in an adult-onset patient with progressive visual impairments in comparison with an adult-onset patient without visual impairments[END_REF]. SCA7140Q/5Q mice also present signi cant decreased volume of selected brain regions at 24 weeks. Interestingly, MRI analysis shows atrophy of the corpus callosum, hippocampus and cortical regions, which parallels observations of human SCA7 [START_REF] Rub | Spinocerebellar ataxia type 7 (SCA7): widespread brain damage in an adult-onset patient with progressive visual impairments in comparison with an adult-onset patient without visual impairments[END_REF][START_REF] Adanyeguh | Autosomal dominant cerebellar ataxias: Imaging biomarkers with high effect[END_REF]. In particular, recent studies showed altered functional connectivity between patients' cerebellum and hippocampus or parahippocampal areas that may account for speci c memory dysfunctions [START_REF] Martin | Spinocerebellar ataxia type 7 (SCA7) -correlations between phenotype and genotype in one large Belgian family[END_REF][START_REF] Hernandez-Castillo | Disruption of visual and motor connectivity in spinocerebellar ataxia type 7[END_REF][START_REF] Hernandez-Castillo | Speci c cerebellar and cortical degeneration correlates with ataxia severity in spinocerebellar ataxia type 7[END_REF][START_REF] Chirino | Motor and cognitive impairments in spinocerebellar ataxia type 7 and its correlations with cortical volumes[END_REF]. The hippocampus is a brain region where mutant ATXN7 rapidly accumulates in SCA7140Q/5Q (data not shown) and in SCA7266Q/5Q mice [START_REF] Yoo | SCA7 knockin mice model human SCA7 and reveal gradual accumulation of mutant ataxin-7 in neurons and abnormalities in shortterm plasticity[END_REF]. In the latter model, mATXN7 accumulation was correlated to speci c alteration of the short-term synaptic plasticity of hippocampal neurons [START_REF] Yoo | SCA7 knockin mice model human SCA7 and reveal gradual accumulation of mutant ataxin-7 in neurons and abnormalities in shortterm plasticity[END_REF]. The cerebellar cortex showed no signi cant atrophy, which can be due to strong variability of volume measurement by MRI in this structure. Indeed, in our ex vivo imaging setup, the cerebellum was peripheral in the eld of view, where the magnetic eld was less homogeneous and coil sensitivity was lower. Direct morphometric analysis of SCA7 cerebellar sections showed that the vermis width is slightly but signi cantly smaller before the onset of the motor symptoms. At end-disease stage (52 weeks), SCA7140Q/5Q brain showed additional atrophy of the pons and somatosensory cortex as well as the anterior commissure, the internal capsule and the mbria of the hippocampus. Overall, brain alterations in the SCA7140Q/5Q mice recapitulate many characteristics of human SCA7.

PC pathology of SCA7 mice.

Previous analysis of the cerebellar pathology in SCA7 lacked single-cell resolution of functional and molecular alterations. Our study of SCA7140Q/5Q mice indicates that PCs show morphological alterations of soma and synapses, impairment of spontaneous ring, and early and progressive repression of PC genes involved in ring and synaptic functions.

It is well known that precision of PC pacemaking activity is essential for normal cerebellar functions and any impairment of PC intrinsic excitability leads to ataxic behaviors [START_REF] Hoxha | Purkinje Cell Signaling De cits in Animal Models of Front Synaptic[END_REF]. In several rodent models of ataxia, PCs are characterized by a lower rate of spontaneous ring compared to WT and/or by an irregular ring pattern [START_REF] Hoxha | Purkinje Cell Signaling De cits in Animal Models of Front Synaptic[END_REF]62,[START_REF] Dorgans | Characterization of the dominant inheritance mechanism of Episodic Ataxia type[END_REF][START_REF] Stahl | Flocculus Purkinje cell signals in mouse Cacna1a calcium channel mutants of escalating severity: an investigation of the role of ring irregularity in ataxia[END_REF][76]. Here, we show that PCs in SCA7 mice re with a higher regularity in lobules VI and IX/X and a lower frequency in lobule VI, compared to WT littermates. Properties of PC pacemaking activity are shaped by a complex interplay between Ca2+, Na+ and K+ conductance [START_REF] Raman | Ionic currents underlying spontaneous action potentials in isolated cerebellar Purkinje neurons[END_REF] and mutations or deletions of genes encoding for these ion channels are causal factors of many types of ataxia [START_REF] Hoxha | Purkinje Cell Signaling De cits in Animal Models of Front Synaptic[END_REF]. In SCA7 mice, expression of several PC genes associated with potassium channels is downregulated (Table 1) and may explain the alterations of PC pacemaking activity [78]. This hypothesis is reinforced by the fact that in SCA7 mice PCs exhibit a higher regularity of their ring pattern (low CV2), a phenotype already observed in ataxic waddles mice [START_REF] White | et Pathogenesis of severe ataxia and tremor without the typical signs of neurodegeneration[END_REF] Previous whole cerebellum gene pro ling in polyQ SCAs models were limited to provide single-cell resolution of gene deregulation, which is constraining given the cellular heterogeneity of the cerebellum and the low percentage of PC population. Our study determined cerebellar cell-type speci c gene deregulation in SCA7 mouse cerebellum. PCs differ from other cerebellar cell types by the high frequency of deregulated cell identity genes and their consistent downregulation. Many of these downregulated genes are essential for major functions of PCs -spontaneous ring and synaptic functions involving glutamate signaling and LTD -and are mutated in other genetic ataxias (Table 1).

In contrast with our understanding the gene regulatory programs that specify different neuron types during development, the programs that maintain cell-type identities in adults only begin to be delineated [89]. One emerging concept is that selective transcription factors that initially specify the acquisition of neuron-type identity continue to function later to maintain identity of postmitotic neuronal type [START_REF] Deneris | Maintenance of postmitotic neuronal cell identity[END_REF].

Dysfunction of such key transcription factors is proposed to account for neurodegenerative disorders, such as Parkinson's disease [START_REF] Decressac | Bjorklund alpha-Synuclein-induced down-regulation of Nurr1 disrupts GDNF signaling in nigral dopamine neurons[END_REF]. In the cerebellum, RORa is highly expressed in fetal PC, is essential for their maturation [START_REF] Boukhtouche | RORalpha, a pivotal nuclear receptor for Purkinje neuron survival and differentiation: from development to ageing[END_REF] and is required to maintain adult PC characteristics [START_REF] Chen | Mature Purkinje cells require the retinoic acid-related orphan receptor-alpha (RORalpha) to maintain climbing ber mono-innervation and other adult characteristics[END_REF]. In our study, we show that RORa expression is decreased in the cerebellum of SCA1, SCA2 and SCA7, and that many downregulated PC identity genes are indeed target of RORa, suggesting that the transcriptional program of PC maintenance involving RORa is disrupted in these polyQ SCAs. Consistently, previous studies showed that mATXN7 alters the transactivation activity of RORa in cultured cells [START_REF] Strom | A role for both wild-type and expanded ataxin-7 in transcriptional regulation[END_REF], and that ATXN1 and TIP60 form a complex to control RORa activity [START_REF] Serra | RORalpha-mediated Purkinje cell development determines disease severity in adult SCA1 mice[END_REF]. Furthermore, postnatal loss of Rora expression worsened neurodegeneration of PCs in SCA1 mice [START_REF] Serra | RORalpha-mediated Purkinje cell development determines disease severity in adult SCA1 mice[END_REF], while increasing RORa level (through partial loss of TIP60) delayed cerebellar pathology [96].

Other mechanisms might also be involved in the loss of PC identity gene expression. Recent studies established that epigenetic regulation at super-enhancers is involved in the expression of cell identity genes [START_REF] Hnisz | Super-enhancers in the control of cell identity and disease[END_REF]98]. In Huntington's disease, it was found that neuronal identity genes regulated by superenhancers are preferentially downregulated in the disease striatum [START_REF] Achour | Neuronal identity genes regulated by super-enhancers are preferentially down-regulated in the striatum of Huntington's disease mice[END_REF]. Our study revealed that mATXN7 alters the level of both epigenetic marks (H3K9ac and H2Bub) dependent of SAGA function in SCA7 mouse cerebellum. It was previously shown that components of SAGA can be sequestered in mATXN7 aggregates [100]. mATXN7 aggregates also sequestered CBP [START_REF] Yvert | Mandel SCA7 mouse models show selective stabilization of mutant ataxin-7 and similar cellular responses in different neuronal cell types[END_REF], which is responsible for H3K27 acetylation, a mark of active enhancers. Therefore, further genome-wide investigation of SAGA-and CBPdependent epigenetic marks might provide insight into mechanisms leading to loss neuron identity gene expression in SCA7.

Supplementary Files

This is a list of supplementary les associated with this preprint. Click to download.

AllsupplFiguresS1S11.pdf

TableS1Niewiadomskaetal.pdf

  eld test. The open elds were placed in a room homogeneously illuminated at 150 lux. Mice of the rst colony were tested in automated open elds (44.3 x 44.3 x 16.8 cm, Panlab, Barcelona, Spain), virtually divided into central and peripheral regions. Each mouse was placed in the periphery of the open eld and allowed to explore freely for 30 min, with the experimenter out of the animal's sight. The distance traveled, the number of rears, average speeds and resting time were automatically recorded and re ect the general locomotor or motor activity phenotype. The number of entries and the time spent in the central and peripheral regions were used as index of emotionality/anxiety. Mice of the second colony were tested in an open eld (46 x 46 cm). Each mouse was tracked over 30 minutes with EthoVision XT 11 and 12 (Noldus Information Technology, The Netherlands) and automatically analyzed for the same parameters, except for the number of rears that were manually scored.Rotarod. To assess the ability to maintain balance, mice of the rst colony were tested on a rotating rod of 5 cm in diameter covered by grey rubber foam (Bioseb, Chaville, France). Mice were given three testing trials during which the rotation speed accelerated from 4 to 40 rpm in 5 min. Trials were separated by a minimum of 5-10 min interval. The average latency before falling from the rod was used as index of motor coordination performance. Mice of the second colony were assessed using an apparatus from TSE Systems (Bad Homburg, Germany) with 3.5 cm rod diameter without rubber foam, using the same parameters as for the rst colony. The oor of the rotarod was covered with soft material to avoid animal discomfort when falling. Light intensity was set to about 115 lux.Notched bar. The test was used to test hindlimb coordination. On the rst day, mice were trained on a 2 cm-wide and 110 cm long natural wooden notched bar made of 26 platforms of 2 cm long spaced by 27 gaps of 2 cm and connected to the goal box (12 x12 x 14 cm), under 115 lux. The second day mice were tested in the same conditions and time to cross the notched bar was measured. A back paw going through a gap was considered as an error.Beam walking test. The test consisted of a 110 cm long wooden beam (2 cm diameter), positioned 50 cm above the oor. One extremity of the beam was used as starting position while the opposite extremity was connected to a goal box (of 12 x12 x 14 cm for the rst colony, and 8.5 x 8.5 x 12 cm for the second colony). The oor of the goal boxes was covered with clean sawdust. The oor under the beam was covered with soft material. During the whole protocol, the light intensity in proximity of the beam was maintained at about 115 Lux. Each mouse was rst habituated to the goal box for 1 minute before three training and three testing trials. On the rst training trial, mice were placed on the beam near and facing the goal box and allowed to access it. On the second and third training trials, mice were placed further away (33-and 66-cm, respectively) facing the box and were allowed to reach and enter the box. On the testing sections, mice were placed at the beam extremity (100 cm, starting position), facing the box and were allowed to reach the goal box. After each training and testing trial, mice were left for 15 seconds in the goal box. The latency to reach the box (cut off 60 seconds) and the number of errors (slips) while crossing the beam were recorded.

  5 and processed with Fiji. Cerebellum images were acquired either on a confocal microscope (SP8, Leica) with 40x/1.3 oil objective with a zoom factor 1 or on a Spinning disc with Leica DMI 8 inverted microscope equipped with Hamamatsu Orca ash 4.0 Camera 5 (Gataca Sytem) connected with Metamorph software (Molecular Devices). For the acquisitions, HC PL APO 40x/1.3 oil objective and three diode lasers (405, 488, and 561 nm) were used. Image brightness and contrast were equivalently adjusted in Fiji for display purposes when necessary. PC i d i l it t PC size and circularity measurements. Sagittal oating sections (50 µm) of central vermis were immunostained with anti-Calbindin1 antibody and acquired using confocal SP8 (Leica) microscope. The obtained z-stacks were subdivided into 3 groups of 15 slices using Fiji. After background subtraction the PC layer was outlined manually in the vicinity of PC somas. Next, a customized Fiji macro allowing an automatic detection and measurement the PC area and circularity was applied. The macro was kindly provided by Stephane Collins (Inserm UMR1231 GAD, Dijon, France). Manuel correction was performed dendritic parts were identi ed as PC. Edge PC were excluded from measurements. Morphometry of cerebellar vermis sections. Sagittal 20 µm sections of central vermis were immunostained with anti-CALB1 antibody and counterstained with DAPI. Sections were scanned with Digital Slide Scanner Nanozoomer 2.0 HT (Hamamatsu) using 40x magni cation. Images of the cerebellum were visualized using NDPI viewer and exported with 5x magni cation into tiff format. Length and width of cerebellar sections as well as the thickness of the molecular cell layer were measured using Fiji software. vGLUT2 density, volume and extension in the molecular cell layer. Sagittal oating sections (50 µm) of central vermis were immunostained with anti-Calbindin1 and anti-vGLUT2 antibodies, counterstained with DAPI. Sections were acquired using YoKogawa CSU W1 Spinning disc with Leica DMI 8 inverted microscope and Hamamatsu Orca ash 4.0 Camera (Gataca Sytem) and a driver by the Metamorph software (Molecular Devices). For the acquisitions, HC PL APO 40x/1.3 oil objective and three diode lasers (405, 488, and 561 nm) were used. Stacks consisted of 225

  photopic (single ashes ERG of 1, 3 and 10 Cd.s.m-2) and icker responses (5 frequencies between 2 and 25 Hz) over time. At 5 weeks, b-wave amplitudes of both photopic and icker responses of SCA7140Q/5Q mice were similar to WT littermates. However, at 10 weeks, the b-wave amplitudes of photopic and icker responses of SCA7140Q/5Q mice signi cantly decreased relative to WT mice, between 32% and 36% for photopic ashes and between 37% and 45% depending on icker frequency (Fig 2d-e and S3 Table). The loss of b-wave amplitudes of photopic responses and icker further worsened with age, with reductions between 81% and 100% at 23 weeks.

  measurement clearly showed a global atrophy of the SCA7 mouse brains compared to WT at 25 weeks (p < 0.05) (Fig5a). Moreover, the decrease of whole brain volume of SCA7140Q/140Q mice was two times higher than SCA7140Q/5Q (p < 0.05), con rming the stronger severity of the disease in homozygous mice. Interestingly, our automated segmentation pipeline highlighted speci c brain regions particularly atrophied in the mouse model such as the corpus callosum, and subparts of the hippocampus and cortex (Fig 5b and S5 Fig). No signi cant atrophy of the cerebellar cortex was measured in SCA7 mice, which can be due to strong variability of volume measurement in this structure. To further correlate cerebellar neuropathology and motor alterations, morphometric parameters were measured on sagittal sections of the central vermis at different disease stages (Fig 5c-g). The vermis length (axis from lobule V to IX) of SCA7140Q/5Q mice was smaller to WT at 34 weeks (Fig 5c). Interestingly, the vermis width (axis from lobule VI to X) was signi cantly shorter in SCA7140Q/5Q mice at 12 weeks already and this difference remained at later stages but did not worsen with the motor phenotype (Fig 5d). Strikingly, there was no major or progressive alteration of the molecular cell layer (MCL) thickness in any of the lobules analyzed (Fig 5f, g and data not shown).

  partnerships and interactions between proteins encoded by the 67 PC genes. It revealed a network of 45 edges (p value = 1.2e-13) (Fig 7g) interconnecting a large number of proteins encoded by genes showing mutations in human or mouse model ataxias, such as Grid2, Calb1, Car8, Trpc3, Kcnma1, Inpp5a, Inpp4a.

Figures

  Figures

Figure 1 Lifespan

 1 Figure 1

Figure 2 Photoreceptor

 2 Figure 2

Figure 4 Peripheral

 4 Figure 4

Figure 6

 6 Figure 6

Figure 7

 7 Figure 7

Figure 8

 8 Figure 8

  Statistical analysis was performed using R and DESeq2 v1.6.3 Bioconductor library[48]. Gene functional annotations were performed using Ingenuity Pathway Analysis (IPA) and String v10.5 software[START_REF] Szklarczyk | STRING v10: protein-protein interaction networks, integrated over the tree of life[END_REF][START_REF] Kramer | Causal analysis approaches in Ingenuity Pathway Analysis[END_REF].

	Illumina Hiseq 4000 sequencer as Single-Read 50 base reads following Illumina's instructions. Image
	analysis and base calling were performed using RTA 2.7.3 and bcl2fastq 2.17.1.14. Adapter dimer reads
	were removed using DimerRemover (https://sourceforge.net/projects/dimerremover/)
	RNA-seq data analysis.

RNA-Seq libraries were prepared from 300 ng of total RNA using TruSeq Stranded mRNA Library Prep Kit and TruSeq RNA Single Indexes kits A and B (Illumina, San Diego, CA), according to manufacturer's instructions. Brie y, following puri cation with poly-T oligo attached magnetic beads, mRNA were fragmented using divalent cations at 94°C for 2 minutes. RNA fragments were reverse transcribed into rst strand cDNA using random primers. Strand speci city was achieved by replacing dTTP with dUTP for the second strand cDNA synthesis using DNA Polymerase I and RNase H. Following addition of a single 'A' base and subsequent ligation of the adapter on double stranded cDNA fragments, products were puri ed and ampli ed by PCR (30 sec at 98°C; [10 sec at 98°C, 30 sec at 60°C, 30 sec at 72°C] x 12 cycles; 5 min at 72°C) to create the cDNA library. Surplus PCR primers and adapter dimers were removed by puri cation using AMPure XP beads (Beckman-Coulter, Villepinte, France) and the nal cDNA libraries were checked for quality and quanti ed using capillary electrophoresis. Libraries were sequenced on Image analysis and base calling were performed using RTA 2.7.3 and bcl2fastq 2.17.1.14. Reads were mapped onto the mm10 assembly of mouse genome using Tophat v2.0.14

[46]

with bowtie v2.1.0 aligner. Gene expression was quanti ed using HTSeq v0.6.1

[START_REF] Anders | HTSeq--a Python framework to work with high-throughput sequencing data[END_REF]

and gene annotations from Ensembl release 86.

  SEM)] can be found in gure legends. Data were analyzed using GraphPad Prism 7 or 8 or Stat view. Differences between means were assessed with unpaired Student's t test, one-way analysis of variance (ANOVA) or ordinary two-way ANOVA or two-way ANOVA with repeated measurement, followed by post-hoc testing of pairwise comparisons among genotypes, (Bonferroni correction), as indicated. Differences between median were analyzed using Mann-Whitney test. Survival of mice with different genotype and sex was plotted using the Kaplan-Meier method and statistical comparisons were performed using the Log Mantel-Cox test. Further information are indicated in gure legends and in TableS2. Signi cance was established at p < 0.05. Data are expressed as mean ± SEM unless indicated. studies of SCA7 mouse retinopathy mainly focused on photoreceptor pathology. To get a broader insight into the SCA7140Q/5Q mouse retinopathy, longitudinal analysis was made by correlating non-invasive OCT and ERG, and histological and molecular analysis. The OCT measurement of 8 different morphological parameters indicated that the overall structure of SCA7140Q/5Q retina was normal at 9 weeks (Fig2a-cand S2 Fig). However, from 15 weeks, photoreceptor segment layers progressively got thinner with age as compared to WT littermates (Fig 2b). The same was observed for the outer nuclear layer (ONL) but occurred later (23 weeks) and not as severely (Fig 2c). There was no clear scalable alteration of thickness of the inner nuclear layer (INL), the inner plexiform layer (IPL) or any other eye structures in SCA7 mice, except for the anterior chamber that showed a signi cant reduction from 23 weeks onward (S2 Fig).

	SCA7140Q/5Q and SCA7140Q/140Q males
	were signi cantly shorter by 6.6% (p < 0.01) and 23% (p < 0.0001), respectively, and those of adult
	SCA7140Q/5Q and SCA7140Q/140Q females by 6.5% (p < 0.05) and 21% (p < 0.0001), respectively (Fig
	1c). SCA7140Q/5Q mice, which represent the genetic status of most SCA7 patients, were used for a
	detailed phenotypic characterization.
	Examination of general physical appearance and body shape of SCA7140Q/5Q mice did not reveal gross
	morphological abnormalities at 28 weeks of age. The proportion of lean, fat and bone tissues was similar
	to their WT littermates, when normalizing to body weight (S1 Fig). From about 40 weeks of age,
	SCA7140Q/5Q mice showed already severe motor de cit and were easily distinguishable from control

Information on number (n) values and what n represents (e.g., animals, biological replicates) and de nition of center and dispersion and precision measures [e.g., mean, median, standard error of the mean (

Results

General phenotypic features of SCA7 mice carrying 140 CAG repeats. SCA7140Q/5Q heterozygous mice have a median lifespan of 54 weeks for both males and females (Fig

1a

). SCA7140Q/140Q homozygous mice have a much shorter lifespan than heterozygotes. Age-related body weight increase of SCA7140Q/5Q and SCA7140Q/140Q mice signi cantly differed from their WT littermates (males F(11,220)=105, p < 0.0001; females F(11,242)=36,1, p < 0.0001). SCA7140Q/5Q females and males gained weight at the same rate as WT mice up to 12 and 14 weeks, respectively, and then plateaued until 24 weeks with an underweight of 18% for females and 20% for males compared to WT of the same sex and age (Fig

1b

). Subsequently, the weight of SCA7140Q/5Q decreased, while remaining stable for WT mice. The body weight of SCA7140Q/140Q mice plateaued earlier at 10 weeks for males and 12 weeks for females, and strongly decreased after 20 weeks. Compared to age-matched WT male and female littermates, the body lengths of adult animals due to their smaller body size and hypoactivity, and some with a hunched posture (kyphosis) and tremors. Because of the poor health status and following recommendation of our animal health regulator, SCA7140Q/5Q mice were generally sacri ced at this stage.

Degeneration of photoreceptors, pigmented epithelium and Bruch's membrane.

Previous

  The spontaneous activity measured with the open eld showed that SCA7140Q/5Q mice become signi cantly hypoactive at 16 weeks, and hypoactivity worsened with age (Fig3a-d). Speci cally, they travelled shorter distances in the arena, ran slower, rested longer times, and made less rears than WT littermates. However, SCA7140Q/5Q mice did not show anxiety behavior, On footprint, SCA7 and WT mice performed similarly in all gait parameters until 17 weeks. From 21 weeks, SCA7140Q/5Q mice made signi cantly shorter steps (stride length) with forelimbs and hindlimbs of each body side (Fig3j). Similarly, the stance width between forelimbs or between hindlimbs was consistently shorter in SCA7140Q/5Q mice from 21 weeks (Fig3k). Temporal gait analysis as recorded by Catwalk showed that the swing duration of right and left forelimbs were signi cantly affected in SCA7140Q/5Q mice from 25 weeks, while the swing duration of hindlimbs were not affected (Fig3l,m). Finally, the grip strength test showed that the muscle strength of SCA7140Q/5Q mice normalized to animal body weight Mice from the second colony were tested using the standardized protocols established for the rst colony (see Methods for further details). As in the rst colony, SCA7140Q/5Q males gained less weight than WT littermates, reaching a plateau at 14 weeks (S3a Fig). However, the average weights of adult SCA7140Q/5Q and WT males from the second colony were 6.5% (p < 0.0001) lighter than the mice of the rst colony (S3b Fig), indicating a signi cant environment effect regardless of the genotype. As for the rst colony, open eld analysis revealed the hypoactivity of SCA7140Q/5Q mice from 16 weeks of age, with a decreased total distance travelled in the arena, a decreased average speed, an increased resting time and a decreased number of rears (S3c-f Fig). In contrast to the rst colony, the exploration of the open central area (S3g Fig) suggested a tendency of SCA7 mice to anxiety behavior in the second environment. As for the rst colony, SCA7140Q/5Q mice of the second colony took signi cantly longer time to cross the beam walking bar (S3h Fig). However, accelerated rotarod, which had a smaller rod diameter than the previous apparatus used for the rst colony, led to short latency to fall even for WT at baseline and hence did not reveal any difference in the latency between the two genotypes (S3i Fig).

since they explored the central area similarly to WT mice (Fig

3e

).

When comparing the mean latency to fall from the accelerated rotarod, SCA7140Q/5Q mice fell earlier than WT mice with statistically signi cant differences from the age of 24 weeks (Fig

3f

). On the notched bar test, SCA7140Q/5Q mice performed similarly to WT littermates up to 17 weeks (Fig

3g, h

). From 20 weeks, performance worsened relative to age-matched WT mice for the time required and the number of mistakes done when crossing the bar. For beam walking test, SCA7140Q/5Q mice took longer time to cross the bar from 20 weeks (Fig

3i

), while the number of mistakes was not signi cantly different between genotype (data not shown).

The forward gait patterns were quantitatively assessed for spatial and temporal gait parameters, both for the fore-and hind-limbs, using respectively footprint and Catwalk, an automated gait analysis system. was similar to WT mice (Fig

3n

).

Replicability of mouse phenotyping across laboratories has important implications in fundamental

research and preclinical studies and is often hampered by methodological issues. To assess the replicability of SCA7 mouse phenotype, a second SCA7140Q/5Q mouse colony was raised in the husbandry of a different research center and was analyzed by independent experimenters and instruments.

In summary, the hypoactivity of SCA7140Q/5Q mice revealed by open eld tests appeared at an early time point (onset at 16 weeks), while defects in motor performances (as shown in rotarod of the rst colony) and in speci c proprioceptive abilities (as re ected by gait alteration in notched bar, beam walking, footprint and catwalk) appeared later between 20-25 weeks, depending of the test. Except for rotarod, the onset and progression of motor phenotypes of SCA7140Q/5Q were replicable in two different research centers.

  ). To focus on PC intrinsic excitability and to rule out network contributions, excitatory and inhibitory synaptic transmissions were pharmacologically blocked. PC discharges were evaluated using the mean ring frequency (measurement of the mean ISI) and the CV2, which estimates the variability of the ring pattern between two consecutive ISIs[START_REF] Arancillo | Sillitoe In vivo analysis of Purkinje cell ring properties during postnatal mouse development[END_REF] 56]. At 22 weeks, no difference in the ring properties of PCs located in lobule VI was detected between WT and SCA7 mice. In contrast, in symptomatic SCA7 mice of 38 weeks, PC exhibited a higher regularity of discharge in both lobules VI (p < 0.001) and IX/X (p < 0.001)

(Fig

6m,n

) and a decrease in the mean ring rate in lobule VI (p = 0.004) (Fig

6o

). Our results show that intrinsic properties of PC excitability are altered in symptomatic SCA7 mice.

  To gain molecular insight into the cerebellar dysfunctions of SCA7140Q/5Q mice, gene expression pro les of cerebellar samples of symptomatic SCA7 mice and their WT littermates were analyzed by RNAseq. Analysis of differentially expressed genes indicated that 406 genes are downregulated in SCA7 cerebella and 270 genes are upregulated (p < 0.05) (Fig7a). Ingenuity Pathway Analysis (IPA)[START_REF] Kramer | Causal analysis approaches in Ingenuity Pathway Analysis[END_REF] of the 676 differentially expressed genes revealed that the most signi cantly affected functions (p ≤ 0.0002) concern major neuronal signaling pathways involving glutamate, G-protein coupled receptors, calcium, CREB and cAMP, and LTD (Fig7b and S4 Table), most of which have previously been implicated

  . Interestingly, in waddles mice, mutation in Car8 gene leads to low CV2 of PC ring and marked ataxia syndrome in the absence of major morphological alteration of MCL, as in SCA7 mouse cerebellum. Car8 gene is one of the 83 PC genes downregulated in SCA7 mice. Interestingly, in waddles mice, the low CV2 of PC activity has been restored to regular values by chlorzoxazone, a pharmacological agent increasing the probability of opening of Ca2+-dependent K+ channels[START_REF] White | et Pathogenesis of severe ataxia and tremor without the typical signs of neurodegeneration[END_REF].The impairment of PC excitability in SCA7140Q/5Q mice is different in lobule VI and in lobule IX/X, since slowing-down of the ring frequency is only observed in lobule VI. We also observed different the cerebellar cortex[85], suggesting that PCs located in Zebrin II positive (ZII+) stripes are particularly vulnerable in SCA1, SCA2 and SCA7. The dysfunction of ZII+ PC subclass in our SCA7140Q/5Q mice is further supported by the decreased expression of the Zebrin II gene itself (or Aldolase C, Aldoc) and by ring impairment of PCs located in lobule VI and IX/X of the vermis, which are enriched in ZII+ PCs.

	Loss of PC identity gene expression.
	Synaptic functions integrating afferent signals that arrive from CF, parallel bers (PF) or interneurons are
	essential to control the output of PCs, and mutations affecting the function of synaptic circuits lead to
	ataxia [54]. SCA7140Q/5Q dysfunction at CF-PC synapses is revealed by the reduced vGLUT2
	immunolabeling in the MCL, whereas downregulation of PC genes encoding post-synaptic membrane-
	associated proteins involved in glutamate signaling and LTD, such as GLURδ2 (coded by Grid2), GRID2ip,
	GABBR1 and TRPC3, indicates functional alteration of PF-PC synapses [82-85].
	There is strong interest in identifying converging pathways of PC pathology across SCAs to improve our
	understanding of polyQ pathogenesis and to identify common potential targets for therapeutic
	intervention [86]. We identi ed 67 PC identity genes downregulated in SCA7 as well as in early
	symptomatic SCA1 and SCA2 mice [61, 62] (Table 1), suggesting that they are primary targets of the
	pathogenesis in the three SCAs. Downregulation of these PC genes points out toward alteration of LTD,

morphological alteration of PCs in lobule VI (smaller soma) and in lobule IX/X (altered circularity) that may be linked to their excitability impairment. In WT mice, intrinsic excitability is heterogeneous among PCs [80] and our recordings did show differences in the ring frequency between PCs located in lobule VI and in lobules IX/X. Based on the topographical organization of the olivo-cortico-cerebellar network [81], lobule-speci c differences in PC ring rate between WT and SCA7 may suggest that defects are associated to speci c cerebellar tasks. cGMP-PKG signaling and phosphatidylinositol signaling pathways, with the involvement of two hub proteins, PLCB3 and PRKGI. Interestingly, PRKG1 phosphorylates PLCB3 as well as IP3R, another downregulated PC genes [87]. Deletion of Prkg1 in PC leads to a severe decrease of LTD in PC synapses [63] and mutant mice exhibit an impaired adaptation of the vestibulo-ocular re ex, a function altered in several SCAs [88]. It is noteworthy that Plcb3 expression is restricted to the Zebrin II positive parasagittal stripes of

Acknowledgments.

thank Dr Huda Zoghbi for providing the SCA7 knock-in mice, the Mouse Clinical Institute of Illkirch for mouse phenotyping support, the GenomEast Platform at IGBMC, a member of the 'France Genomique' consortium (ANR-10-INBS-0009) for processing Illumina libraries, Stephan Collins for macro allowing PC morphological analysis, Filiz Kavla, Raphaël Bour and Christel Wagner for technical assistance, Tao Ye for fruitful discussion regarding motif enrichments, Doulaye Dembele for suggestions regarding statistical analysis. Special thanks to Nathalie Daigle and Hélène Puccio for critical reading of the manuscript and for fruitful discussion.

Funding. This Project has received funding from the European Union Joint Programme-Neurodegenerative Disease Research (JPND) Project ModelPolyQ (Grant Agreement No. 643417) jointly funded by national funding organisations: ANR-15-JPWG-0008-03 to Y.T. and BMBF 01ED1606 to H.H.P.N.; from the E-Rare Research Programme which is a transnational R&D programme jointly funded by national funding organisations within the framework of the ERA-NET E-Rare 3 to Y.T, H.H.P.N and E.B.; from the French foundation Connaître les Syndromes Cérébelleux (CSC) to Y.T.; from ANR ANR-17-RAR3-0008-04_ACT to Y.T.; from ANR-CE37-2015 (CeMod) and Fondation pour la Recherche Médicale (Equipes FRM DEQ20140329514) to P.I.; from ANR-10-LABX-0030-INRT under the frame programme Investissements d'Avenir labelled ANR-10-IDEX-0002-02 (to IGBMC). The 11.7T MRI scanner was funded by a grant from NeurATRIS: A Translational Research Infrastructure for Biotherapies in Neurosciences ("Investissements d'Avenir", ANR-11-INBS-0011).

Availability of data and materials

The RNA-seq data have been submitted to the NCBI Gene Expression Omnibus (GEO) (http://www.ncbi.nlm.nih.gov/geo/) under GSE138527 (Link https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE138527 Token for reviewers: slknackkxdwfjaj). Other datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request.

SCA7 mouse cerebellar pathology reveals preferential downregulation of key Purkinje cellidentity genes and shared disease signature with SCA1 and SCA2.

List Of Abbreviations

Spinocerebellar ataxia type 7 (SCA7); Purkinje cells (PC); polyglutamine (polyQ); mutant ATXN7 (mATXN7); Spt-Ada-Gcn5 Acetyltransferase (SAGA); long-term depression (LTD); optical coherence tomography (OCT); electroretinography activity (ERG); outer nuclear layer (ONL); inner nuclear layer (INL); inner plexiform layer (IPL); green cone opsin (OPN1MW); blue cone opsin (OPN1SW); rhodopsin (RHO); Quantitative RT-PCR (RT-qPCR); retinal pigmented epithelium (RPE); magnetic resonance imaging (MRI); false discovery rate (FDR); wild type (WT); heterozygous (HTZ); homozygous (HMZ); males (M); females

Consent for publication.

Not applicable.

Con ict of interest statement.

The authors declare no con ict of interest.

Author's contributions. ANC, EB, JF, PI, HHHN and YT conceived the project. Researchers that performed experiments and/or analyzed data: mouse behaviors (ANC, HM, AN, CW, AK, HHHN and AE), retina analyses (MJR), electromyography (FP), morphometry (ANC and BY), electrophysiology of Purkinje cells (FD and PI), MRI (JBP, JF and EB), tissue analysis and imaging (ANC, NM, MFC, AH and EG), molecular analysis (ANC, CK and AH). ANC and YT supervised the project and wrote the rst draft of the manuscript. All authors read, edited and approved the manuscript.