General phenotypic features of SCA7 mice carrying 140 CAG repeats.
Previous SCA7266Q/5Q mice are severely affected and die at about 16 weeks of age (26). SCA7140Q/5Q heterozygous mice have a longer 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 significantly 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 SCA7140Q/5Q and SCA7140Q/140Q males were significantly 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). Further characterization was mainly performed on SCA7140Q/5Q mice, as they represent the genetic status of most SCA7 patients.
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 (Additional file 3; Figure S1). From about 40 weeks of age, SCA7140Q/5Q mice showed already severe motor deficit and were easily distinguishable from control 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 sacrificed at this stage.
Degeneration of photoreceptors, pigmented epithelium and Bruch’s membrane.
To determine the onset and progression of SCA7140Q/5Q mouse retinopathy, 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 (Fig. 2a-c and Additional file 3; Figure S2). 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 significant reduction from 23 weeks onward (Additional file 3; Figure S2).
ERG studies in SCA7 patients showed that cone dysfunction precedes rod dysfunction (3). We evaluated cone function of WT and SCA7140Q/5Q mice by measuring the cone-derived b-wave amplitudes of photopic (single flashes ERG of 1, 3 and 10 Cd.s.m-2) and flicker responses (5 frequencies between 2 and 25 Hz) at 5, 10, 15 and 23 weeks. At 5 weeks, b-wave amplitudes of both photopic and flicker responses of SCA7140Q/5Q mice were similar to WT littermates. However, at 10 weeks, the b-wave amplitudes of photopic and flicker responses of SCA7140Q/5Q mice significantly decreased relative to WT mice, between 32% and 36% for photopic flashes and between 37% and 45% depending on flicker frequency (Fig. 2d-e and Additional file 4: Table S3). The loss of b-wave amplitudes of photopic responses and flicker further worsened with age, with reductions between 81% and 100% at 23 weeks.
The green and blue opsin (OPN1MW, OPN1SW, respectively) of cones and the rhodopsin (RHO) of rods are light-sensitive pigments that initiate vision and represent major components of photoreceptor outer segments. RT-qPCR showed that expression of these genes progressively decreased with age in SCA7140Q/5Q mice (Fig. 2f). Notably, the level of Opn1mw and Opn1sw RNAs already showed about 65% (p < 0.01) and 90% (p < 0.01) reduction, respectively, in 10-week-old mutant mice, when cone-derived ERG responses began to decrease. The decrease of cone opsin RNAs was earlier and more drastic than for the Rho RNA, consistent with the primary alteration of cone function in SCA7 patients. Therefore, the loss of cone ERG of SCA7140Q/5Q mice closely correlates with the loss of cone opsins expression, and both occur before a significant shortening of outer segments detected by OCT.
Histology and electron microscopy indicated that photoreceptors had almost completely lost their outer segments at 44 weeks in SCA7140Q/5Q mice and at 20 weeks in SCA7140Q/140Q mice, while the thickness of inner segments showed only a slight reduction in both SCA7 genotypes, compared to WT retina (Fig. 2g and Additional file 3: Figure S2i-k). Additional degenerative signs of SCA7 photoreceptors included disorganization of the remnant outer segments, swollen mitochondria and vesicular membrane accumulation in inner segments, several dark stained inner segments and photoreceptor nuclei (Fig. 2h-k), and ectopic localization of photoreceptor nuclei in the subretinal space (data not shown). Interestingly, we found striking anomalies of the retinal pigmented epithelium (RPE) and Bruch’s membrane of SCA7140Q/5Q mice never reported previously to our knowledge. Firstly, the thickness of RPE was clearly increased in SCA7 mice as compared to controls (Fig. 2l). Secondly, on electron microscopy, while the normal RPE (Fig. 2m) contains translucent infolding membranes in contact all along the basement membrane, SCA7 RPE showed discontinuous and opaque infolding membranes, with homogenous deposits between the plasma membrane and the basement membrane (Fig. 2n, o). In addition, the Bruch’s membrane of SCA7 retina showed enlargement and disorganization, as compared to WT in which the Bruch’s membrane was thin and regular (Fig. 2m-o).
Deficits in motor activity and coordination.
We performed extensive and longitudinal motor performance analysis of SCA7140Q/5Q and WT male littermates using a variety of motor activity paradigms. The spontaneous activity measured with the open field showed that SCA7140Q/5Q mice become significantly hypoactive at 16 weeks, and hypoactivity worsened with age (Fig. 3a-d). Specifically, 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, 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 significant 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 significantly 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. On footprint, SCA7 and WT mice performed similarly in all gait parameters until 17 weeks. From 21 weeks, SCA7140Q/5Q mice made significantly shorter steps (stride length) with forelimbs and hindlimbs of each body side (Fig. 3j). Similarly, the stance width between forelimbs or between hindlimbs was consistently shorter in SCA7140Q/5Q mice from 21 weeks (Fig. 3k). Temporal gait analysis as recorded by Catwalk showed that the swing duration of right and left forelimbs were significantly affected in SCA7140Q/5Q mice from 25 weeks, while the swing duration of hindlimbs were not affected (Fig. 3l, m). Finally, the grip strength test showed that the muscle strength of SCA7140Q/5Q mice normalized to animal body weight 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. Mice from the second colony were tested using the standardized protocols established for the first colony (see Methods for further details). As in the first colony, SCA7140Q/5Q males gained less weight than WT littermates, reaching a plateau at 14 weeks (Additional file 3: Figure S3a). 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 first colony (Additional file 3: Figure S3b), indicating a significant environment effect regardless of the genotype. As for the first colony, open field 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 (Additional file 3: Figure S3c-f). In contrast to the first colony, the exploration of the open central area (Additional file 3: Figure S3g) suggested a tendency of SCA7 mice to anxiety behavior in the second environment. As for the first colony, SCA7140Q/5Q mice of the second colony took significantly longer time to cross the beam walking bar (Additional file 3: Figure S3h). However, accelerated rotarod, which had a smaller rod diameter than the previous apparatus used for the first 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 (Additional file 3: Figure S3i).
In summary, the hypoactivity of SCA7140Q/5Q mice revealed by open field tests appeared at an early time point (onset at 16 weeks), while defects in motor performances (as shown in rotarod of the first colony) and in specific proprioceptive abilities (as reflected 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.
Morphological and functional alterations of the peripheral nervous system.
To understand the cause of motor alterations in SCA7140Q/5Q mice, the activity of the peripheral nervous system was first assessed over time by measuring the sciatic nerve function by electromyography. At 15 weeks, median amplitudes of plantar M-wave 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 fibers and decreased number of small fibers (Fig. 4c). On electron microscopy (Fig. 4d-o), several structural alterations were observed in myelinated fibers of SCA7140Q/5Q mice, including myelin degeneration and infolding-like structures as well as axonal modifications characterized by inner swelling tongue and autophagy. Non-myelinated fibers 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 (28). 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 significant reduction of brain areas in the three coronal sections (from -6.7% to -8%) with both white and grey matter alterations (Additional file 3; Figure S4). 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 fimbria of the hippocampus.
We then aimed to capture SCA7 mouse brain alterations at earlier disease stage using high resolution MRI. MRI measurement clearly showed a global atrophy of the SCA7 mouse brains compared to WT at 25 weeks (p < 0.05) (Fig. 5a). Moreover, the decrease of whole brain volume of SCA7140Q/140Q mice was two times higher than SCA7140Q/5Q (p < 0.05), confirming the stronger severity of the disease in homozygous mice. Interestingly, our automated segmentation pipeline highlighted specific brain regions particularly atrophied in the mouse model such as the corpus callosum, and subparts of the hippocampus and cortex (Fig. 5b and Additional file 3: Figure S5). No significant 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 significantly 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).
Morphological and functional alterations of Purkinje cells.
Since PC are primary targets in SCA7, their morphology, connectivity and function were investigated. The area of PC soma was significantly 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 fibers (CF) of inferior olivary neurons. To this purpose, we quantified vGLUT2-immunolabeling of CF-PC contact points in the molecular cell layer using the image analysis Imaris software. The average density and average volume of vGLUT2 contact points were similar in WT and SCA7 mice (data not shown). However, extension of climbing fiber terminals along the PC dendritic tree was significantly 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 firing pattern even in the absence of synaptic inputs (38, 39). Any alteration in the precision of their pacemaking activity may be the result of PC cellular dysfunctions and may affect cerebellar functions (40). To investigate the correlation between PC function, morphological phenotypes and motor deficits, we assessed whether the intrinsic properties of PC spontaneous discharge were altered in SCA7140Q/5Q mice at 2 disease stages (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). 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 firing frequency (measurement of the mean ISI) and the CV2, which estimates the variability of the firing pattern between two consecutive ISIs (41, 42). At 22 weeks, no difference in the firing 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 firing rate in lobule VI (p = 0.004) (Fig. 6o). Our results show that intrinsic properties of PC excitability are altered in symptomatic SCA7 mice.
Accumulation of mutant ATXN7 in retina and cerebellum.
Studies of SCA7 cellular and animal models showed that mATXN7 tends to misfold and form protein aggregates (reviewed in (16)). Immunofluorescence analysis of control adult retina and cerebellum showed that WT ATXN7 is barely detectable (Fig. 7a and b, left panels). In contrast, the immunolabeling progressively increased with age in SCA7 mouse tissues, indicating that mATXN7 accumulates over time. This resulted in different labeling profiles depending of the neuronal population. In SCA7 retina at 12 weeks, mATXN7 aggregates were readily detected in many photoreceptor nuclei and in some neuronal nuclei of ganglion and inner nuclear layers (Fig. 7a). mATXN7 aggregates became widespread in retinal neuron nuclei at later stages. In SCA7 cerebellum, PC showed the earliest and most important mATXN7 accumulation that progressively increased between 12 and 19 weeks and progressively led to the formation of large nuclear aggregates at later stages (Fig. 7b). mATXN7 nuclear aggregates were also observed in the MCL and granule cell layer (GCL). All cerebellar lobules showed the accumulation of mATXN7 in PC nuclei (Fig. 7c).
Western blot analysis showed the expression of WT ATXN7 as a 110 kilodalton (kDa) band in control retina and cerebellar samples (Fig. 7d). In SCA7 samples, the mutant form of ATXN7 migrated at ~140 kDa. Importantly, mATXN7 was also detected as high molecular weight smear from 5-8 weeks of age, indicating that mATXN7 accumulates as insoluble oligomeric forms in retinal and cerebellar tissues, much before the formation of visible nuclear aggregates as detected by immunolabeling of tissues.
Cerebellar gene expression changes in SCA7 mice.
To gain molecular insight into the cerebellar dysfunctions of SCA7140Q/5Q mice, gene expression profiles of cerebellar samples of symptomatic SCA7 mice and their WT littermates were analyzed by RNA-seq. Analysis of differentially expressed genes indicated that 406 genes are downregulated in SCA7 cerebella and 270 genes are upregulated (p < 0.05) (Fig. 8a). Ingenuity Pathway Analysis (IPA) (36) of the 676 differentially expressed genes revealed that the most significantly affected functions (p ≤ 0.0002) concern major neuronal signaling pathways involving glutamate, G-protein coupled receptors, calcium, CREB and cAMP, and LTD (Fig. 8b and Additional file 5: Table S4), most of which have previously been implicated in the pathogenesis of other ataxias (40).
IPA also pointed out the impairment of interferon signaling pathway (Fig. 8b). 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 (Additional file 6: Table S5). Moreover, functional annotations of the 270 upregulated genes using STRING (35) further highlighted a strong enrichment (false discovery rate (FDR) <2*10-6) of type 1 interferon pathway (Additional file 3: Figure S6). The interferon regulator factor 7 gene (Irf7), which is the second most significantly upregulated protein-coding gene (2.7 fold; p = 2.28*10-10) in SCA7 mouse cerebella, composed with high confidence (0.700) a protein-protein association network of 19 direct edges that includes signal transducer and activator of transcription 1 (Stat1), which further interconnects with the proto-oncogene Jun. Together, Irf7, Stat1 and Jun constituted the center a large network of 189 edges involving multiple upregulated genes coding for proteins involved in immune system response (Additional file 3: Figure S6), further emphasizing the important activation of interferon signaling pathway in SCA7 cerebella. Chort et al. (43) 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-enriched genes.
To gain insight into the cell type-specific distribution of differentially expressed genes in SCA7 cerebella, we took advantage of the publicly available actively translated mRNA profiles of all the major cerebellar cell types (37). To compare with our SCA7 RNA-seq dataset, the published datasets (37) 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 (Additional file 3: Figure S7). 89% of them were present in the SCA7 dataset. Out of 9671 actively translated transcripts, we identified 5058 transcripts present in only one of the nine major cerebellar cell type datasets, including 801 enriched transcripts in PC, 2357 in granule cells, 156 in Golgi cells, 340 in stellate and basket cells, 144 in unipolar bruch cells, 563 in oligodendrocytes expressing Olig2, 133 in oligodendrocytes expressing Cmtm5, 279 in Bergmann glia and 285 in astrocytes (Additional file 3: Figure S7 and Additional file 7: Table S6). Taking the cellular distribution into account, we were able to determine the cell type enrichment of 470 differentially expressed genes of SCA7 cerebellum and found that 240 of them were enriched in a single cerebellar cell type and 230 were present in more than one cell type (Additional file 8: Table S7). The analysis revealed that all the cerebellar cell types presented differentially expressed genes (Fig. 8c, 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 enriched in PC. In contrast, 15% of SCA7 deregulated genes were enriched in granule cells, while this cell type represents 90% of cerebellar cells. Importantly, while granule cell-enriched genes were both up- and downregulated, almost all PC-enriched genes (104 out of 106 genes) were downregulated (Fig. 8d), indicating that repression of PC-enriched genes is a major component of SCA7 cerebellar pathology. Another publicly available datasets from Kratz et al. (44) allowed us to identify 5 additional PC-enriched genes that were downregulated in SCA7 mouse cerebella (Additional file 8: Table S7), resulting in a total 109 downregulated PC-enriched genes. To ascertain the enrichment of these 109 genes in PC, their level of expression was analyzed using in situ hybridization datasets reported in The Brain Transcriptome Database (BrainTx; CDT) (45) and in the Allen Brain Atlas (30). We established three categories of genes according to the level of enriched expression in PC, as specifically expressed or strongly enriched in PC compared to other cerebellar cells, enriched in PC or non-enriched in PC (e.g. equally present in other cell types). The analysis showed that 56 genes were specific or strongly enriched in PC, 27 genes had moderate enrichment in PC, while 14 other genes were expressed in wider range of cerebellar cell types and 12 genes were not present in in situ hybridization datasets (Additional file 9: Table S8). In fine, 83 genes had validated enriched expression in PC of WT mice and showed reduced expression in PC of SCA7 mice.
RT-qPCR was used to study the time course of repression of a subset of PC-enriched 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 (Additional file 3: Figure S8). Interestingly, the 3 PC-enriched 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-enriched genes in SCA1, SCA2 and SCA7.
To further explore whether PC-enriched 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 specifically expressed in PC and caused PC pathology (46, 47). In these 2 models, expression level of a subset genes was also shown to be altered compared to their respective WT littermates (46, 47). 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 datasets of differentially expressed genes in three SCA models. The analysis revealed that 136 genes common to the 3 datasets were downregulated and only 5 were upregulated (Fig. 8e and 8f). Interestingly, out of the 136 downregulated genes, 67 were PC-enriched including 10 genes associated with genetic ataxias (Table 1 and Additional file 10: 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 (47, 48), 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-enriched 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 significance of the early downregulation of the 67 PC-enriched 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. 8g). Second, the STRING analysis (35) was used to search for functional partnerships and interactions between proteins encoded by the 67 PC genes. It revealed a network of 45 edges (p value = 1.2e-13) (Fig. 8g) interconnecting a large number of proteins encoded by genes showing mutations in human or mouse model ataxias: Grid2, Calb1, Car8, Trpc3, Kcnma1, Inpp5a, Inpp4a. 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 (40, 49). Finally, our list of 67 PC-enriched genes was compared to a published list of genes composing the SCA1 Magenta gene module (47). 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 (47). 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 (Additional file 3: Figure S8). 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-enriched genes, we first looked at RORa (RAR-related orphan receptor alpha), a key transcription factor involved in PC differentiation and survival (50) and showing downregulation in SCA1, SCA2 and SCA7 mice (Table 1). Among the 67 PC-enriched genes, 19 genes were known RORa-target genes (50, 51) (Table 1), suggesting that RORa dysfunction partially accounts for PC genes deregulation. We then performed in silico analysis of 2-kb promoter sequence of the 67 PC-enriched genes to search for de novo motifs and transcription factors binding sites using MEME, TOMTOM, AME or FIMO (52). The analysis did not reveal major enrichment of transcription factor binding motifs in the promoter of the 67 PC-enriched genes affected, compared to control sequences (e.g. shuffled 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 modifications, 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 significantly increased in SCA7140Q/5Q cerebellum compared to WT, while the level of H3K9ac showed no significant difference (Fig. 9a, b). However, a significant decrease of H3K9ac was observed in SCA7140Q/140Q homozygous mice, which were more severely affected that the SCA7 heterozygous mice (Additional file 3: Figure S9). Together, this suggests that mutant ATXN7 differentially alters the histone-modifying activities of SAGA complex along the progression of cerebellar pathology and might impact on transcription of cerebellar genes.