# Compromised retinoic acid receptor beta expression accelerates the onset of motor, cellular and molecular abnormalities in a mouse model of Huntington's disease

Nicolas Zinter, Tao Ye, Hanna Semaan, Valerie Fraulob, Damien Plassard, Wojciech Krezel

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Nicolas Zinter, Tao Ye, Hanna Semaan, Valerie Fraulob, Damien Plassard, et al.. Compromised retinoic acid receptor beta expression accelerates the onset of motor, cellular and molecular abnormalities in a mouse model of Huntington's disease. Neurobiology of Disease, 2025, 212, pp.106943. 10.1016/j.nbd.2025.106943. hal-05168939

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# Compromised retinoic acid receptor beta expression accelerates the onset of motor, cellular and molecular abnormalities in a mouse model of Huntington's disease

Nicolas Zinter ID, Tao Ye, Hanna Semaan, Valérie Fraulob, Damien Plassard, Wojciech Krezel*

Institut de Génétique et de Biologie Moléculaire et Cellulaire, INSERM U1258, CNRS UMR 7104, Université de Strasbourg, Illkirch, France.

<figure><img src="image_3.png" /></figure>

## ARTICLE INFO

**Keywords:**

Huntington disease

Nuclear receptors - Retinoic Acid Receptor Beta

Disease progression

Motor behavior - Cell identity

Transcriptomics

## ABSTRACT

The mechanisms underlying detrimental effects of mutant Huntingtin on striatal dysfunction in Huntington's disease (HD) are not well understood. Although retinoic acid receptor beta (RARβ) emerged recently as one of the top regulators of transcriptionally downregulated genes in the striatum of HD patients and mouse models, its involvement in disease progression remains elusive. Here we challenged functional relevance of RARβ dysregulation in HD onset and progression. Using a series of genetic mouse models, we investigated whether genetically reduced *Rarβ* expression synergizes with disease- causing mutant huntingtin (mHTT) fragment in R6/1 mice to accelerate HD-like behavioral, cellular and molecular striatal deregulations. We report that genetically compromised *Rarβ* signaling accelerates onset of motor abnormalities in the R6/1 HD mouse model. Transcriptional profiling revealed that downregulation of *Rarβ* expression in *Rarβ<sup>+/+</sup>*; R6/1 mice also accelerates transcriptional signature of disease progression and aging by emergence of a cluster of upregulated genes related to cell-cycle, stem cell maintenance and telencephalon development, contributing thereby to degradation of striatal cell-identity. Reactivation of proliferative activity in the neurogenic niche and development-related transcriptional programs in the striatum prompt an attempt of lineage infidelity in HD striatum which may lead as a consequence to disease-driving energy crisis, as suggested by downregulation of oxidative phosphorylation genes, a well-accepted correlate of HD physiopathology, and a metabolic condition required for maintenance of proliferative activity and differentiation but not compatible with high energetic demand of differentiated and active neurons. Overall, our data indicate that RARβ delays disease progression, perhaps by delaying aging process.

## 1. Background

Huntington's disease (HD) is an autosomal dominant neurodegenerative disease characterized by progressive motor and cognitive symptoms. The age of onset of motor symptoms is highly variable, but starts, on average, in the early 40s and progresses until the death of patients, often occurring 10-20 years after (Ross et al., 2014). Impairment of the striatum, and in particular the selective degeneration of medium spiny neurons (MSNs), is the hallmark of this disease (Vonsattel and DiFiglia, 1998). Although these changes are key components of HD

physiopathology, a number of additional correlates were also associated with disease progression, including increased proliferation and neurogenesis in the neurogenic niche of the ventricular and subventricular zone (SVZ), but meaning of this correlate remains not well understood (Curtis et al., 2003; Curtis et al., 2005; Gil et al., 2005).

HD is caused by abnormal CAG repeat expansions in the first exon of the Huntingtin (HTT) gene. The length of the resulting polyglutamine repeat correlates negatively with the age of onset of the disease. Several murine models of HD have been generated, including R6/1 transgenic mice carrying the first exon of human mutant Huntingtin (mHTT)

Abbreviations: CPU, Caudate Putamen Nucleus; Drd1, Dopaminergic Receptor D1; Drd2, Dopaminergic Receptor D2; HD, Huntington Disease; HRP, Horseradish Peroxidase; HTT, Huntingtin; M, Module; mHTT, *Mutant Huntingtin*; MSNs, Medium Spiny Neurons; NAc, Accumbens Nucleus; PBS, Phosphate-buffered Saline; PFA, Paraformaldehyde; RARβ, Retinoic Acid Receptor Beta; RT, Room Temperature; snMSNs, Striatonigral Medium Spiny Neurons; spMSNs, Striatopallidal Medium Spiny Neurons; SSC, Standard Saline Citrate; SVZ, subventricular Zone; TEA, Triethanolamine.

* Corresponding author at: Institut de Génétique et de Biologie Moléculaire et Cellulaire (IGBMC), Inserm U1258; CNRS UMR 7104, Université de Strasbourg, 1 rue L. Fries, Illkirch 67404, France.

E-mail address: krezel@igbmc.fr (W. Krezel).
(Mangiarini et al., 1996), as well as an allelic series of mice with knock-
in (KI) insertions of human *mHTT* exon 1 with different lengths of CAG
(Q) repeats ranging from Q20, corresponding to a non-toxic form of the
CAG stretch, up to Q175 corresponding to the toxic form (Pouladi et al.,
2013). Like in human, these mouse models develop progressive motor
and cognitive dysfunction with different age of onset, depending on the
model and the length of CAG repeats (Nithianantharajah et al., 2008;
Menalled et al., 2012; Smith et al., 2014; Langfelder et al., 2016).
Transcriptional changes appeared to be much more sensitive markers of
disease progression than behavior as they were observed at pre-
symptomaptic age in Q140 and Q175 *mHTT* KI mice and were also
present in Q80, Q92, and Q111 models which do not display any overt
phenotype (Langfelder et al., 2016). An information emerging from the
transcriptomic analyses of diverse mouse models was down-regulation
of cellular identity genes in HD striatum (Langfelder et al., 2016;
Vashishtha et al., 2013; Achour et al., 2015; Lee et al., 2020; Malaiya
et al., 2021). Although less well studied, some reports also revealed an
upregulation of developmental genes (Langfelder et al., 2016; Le Gras
et al., 2017). These and other molecular signatures of disease progres-
sion may provide insight into HD physiopathology, which is clearly
complex. Specifically, using weighted gene co-expression network
analysis (WGCNA), one study allowed to define 37 modules (M1-37) of
striatal genes which were co-expressed at 2, 6, and 10 months of age in
the allelic series of Q20-Q175 KI models (Langfelder et al., 2016).
Although some of these modules co-varied in age- and CAG repeat-
dependent manner, the identification of the most relevant one(s) for
understanding mechanisms of the disease remains a challenge.

Retinoic acid receptor beta (RARβ), a ligand-dependent transcription factor mediating retinoic acid (the major active form of vitamin A) signaling emerged recently as a possible major regulator of gene networks affected in HD (Lee et al., 2020; Ciancia et al., 2022; Niewiadomska-Cimicka et al., 2017). Reduced RARβ expression observed consistently in the striatum of HD patients and animal models (Niewiadomska-Cimicka et al., 2017; Hodges et al., 2006; Luthi-Carter et al., 2000) could contribute to the physiopathology of HD. In line with this possibility, RARβ loss of function in *RARβ*⁻/⁻ mice led to HD-like motor deficits most probably through mitochondrial-dependent dysfunctions (Ciancia et al., 2022). RARβ deficiency may directly affect striatonigral and striatopallidal projection MSNs (called hereafter snMSNs and spMSNs), the main cell types expressing RARβ in mouse and human striatum (Lee et al., 2020; Ciancia et al., 2022). spMSN function and survival appeared particularly dependent on RARβ activity. Accordingly, loss of dopamine D2 receptor (*Drd2*)-expressing spMSN was observed in *RARβ*⁻/⁻ striatum, whereas somatic inactivation of *RARβ* in postmitotic *Drd2*⁺ neurons or in the whole striatum recapitulated both behavioral and spMSN deficits displayed by *RARβ*⁻/⁻ mice (Ciancia et al., 2022).

Despite these correlative observations, the role of compromised
RARβ signaling in HD is not clear. By investigating animal behavior, we
report here that genetically compromised *RARβ* signaling accelerates
onset of HD-like motor abnormalities in R6/1 transgenic mice. Tran-
scriptional profiling reveals that compromising *RARβ* signaling in R6/1
mice accelerates the transcriptional signature of disease progression by
emergence of upregulated clusters of genes related to cell-cycle, stem
cell maintenance and telencephalon development, with concomitant
downregulation of striatal cell-identity genes. The reactivation of cell
division and developmental programs, further documented here by
increased proliferative activity of the neurogenic niche in the striatum,
could prompt an attempt of lineage infidelity which may lead to disease-
driving energy crisis in striatal neurons, as suggested by transcriptional
changes coherent with reduction of oxidative phosphorylation, required
for maintenance of proliferative and developmental activities but un-
sustainable for high energy demand of differentiated and active neurons.
Upregulation of developmental genes with concomitant loss of cell
identity is one of the key features of the aging process (Yang et al.,
2023a), suggesting that compromised *RARβ* signaling accelerates this

process in HD.

## 2. Methods

2.1. *Animals*

Hemizygous R6/1 mice (Mangiarini et al., 1996) were maintained on the CBA genetic background for more than 10 generations, whereas mice carrying a null mutation for *RARβ* (*RARβ*⁻/⁻) were generated from heterozygous crosses as previously described (Ghyselinck et al., 1997) and were maintained on mixed genetic background of about 50% C57BL/6J and 50% 129/SV for more than 10 generations. Both lines were crossed to generate the first generation of R6/1<sup>Tg/0</sup>; *RARβ*⁺/⁻ mice and their littermate controls, which were used for behavioral tests and subsequent molecular analyses. For all experiments, mice were housed (3–4/cage) with water and food available *ad libitum* and 12 h/12 h light/dark cycle with the beginning of the light phase at 7 am. Mice were sacrificed 2 days after the last behavioral experiment for further cellular and molecular analyses. All experiments were carried out in accordance with the Directive of the European Parliament: 2010/63/EU and in compliance with the guidelines of CNRS and French ministry of Agriculture and Food (decree 87848).

## 2.2. *Behavioral phenotyping*

All behavioral analyses were carried out using 8 week-old males. The
pipeline consisted of the following tests, performed as listed with one
test on each consecutive day.

2.2.1. *Open field*

Novelty-induced locomotion was tested in open field arenas measuring 40 x 40 cm and equipped with infrared beams for automatic detection and monitoring of animal movement (Bioseb, France). Mice were individually placed in the periphery of the open field arena and allowed to explore the apparatus freely for 30 min. The distance traveled, the number of rears and time spent in the central and peripheral regions were recorded over the test session. The test was performed in a room homogeneously illuminated at 150 Lux.

2.2.2. *Rotarod*

An accelerating (4–40 rpm in 5 min) cylinder, rotarod (Bioseb, France), was used for analyses of motor coordination. For each animal, a complete test session consisted of three trials separated by 15 min intervals of recovery. The latency time to fall from the rotarod was recorded as an index of motor coordination (the shorter latency to fall, the worse coordination performance). Before the first trial, mice were allowed to stay on the rotarod for 30 s for habituation period before starting the acceleration phase.

2.2.3. *Spontaneous locomotion in actimetric cages*

Spontaneous locomotor activity was measured in actimetric cages (Immetronic, Pessac, France) during 32 h (11 am until 7 pm the next day). Briefly, each mouse was placed in an individual cage equipped with infra-red photo beam cells that measure horizontal movements. During the experiment mice had free access to food and water and followed habitual for them dark/light cycle with day starting at 7 am and finishing at 7 pm.

2.3. *In situ hybridization and cell counts*

Brain cryosectioning (14 µm) was performed to collect striatal
samples spanning bregma 1.3 – 0.6 mm, and on the day of analysis slides
were thawed and dried for 15 min at room temperature (RT), post-fixed
with 4% paraformaldehyde (PFA) dissolved in phosphate-buffered sa-
line (PBS), acetylated with 0.1 M triethanolamine (TEA), rinsed with
standard saline citrate (SSC), dehydrated by an increasing ethanol
gradient, air-dried and hybridized at 65°C overnight with digoxigenin-labeled *Drd2* RNA probe (1681 bp fragment of 5' region of *Drd2* cDNA). After hybridization the sections were rinsed, blocked and incubated for 1 h at RT with an anti-digoxigenin, alkaline phosphatase-conjugated antibody (Roche). The sections were subsequently rinsed and incubated in the dark with a NBT/BCIP solution (Nitroblue tetrazolium chloride/ 5-bromo-4-chloro-3-indyl-phosphate; Roche) until the staining appeared (overnight or up to 48 h). If the staining reaction was longer than overnight, the NBT/BCIP solution was changed after 24 h. Imaging was performed using a Nanozoomer digital scanner (Hamamatsu Photonics, France) and cells were counted manually by two independent experimenters using the ImageJ software.

## 2.4. Western blotting

Striatum samples from 8 week-old mice were homogenized manually in RIPA lysis buffer (Tris HCl 50 mM, NaCl 30 mM, EDTA 2 mM, Triton 0.01X) supplemented with a protease inhibitor cocktail (#11873580001, Roche Diagnostic). Samples were then sonicated (2 × 3 s) and left 30 min on ice. Following a 30 min centrifugation, protein concentration was quantified using Bradford assay (#5000006, Biorad) and 10 µg of protein extracts were separated by SDS-PAGE, and electro-transferred (400 mA during 1 h) onto 0.2 µm nitrocellulose membrane (#A29612452, Amersham). Immunodetection was carried out using rabbit anti-RARβ, diluted at 1:1000 (Rochette-Egly et al., 1992); mouse anti-GAPDH, 1:5000 (CAB932Hu22, USCN) and horseradish peroxidase (HRP)-conjugated secondary antibodies (Goat anti-mouse, 1:5000, G-21040, Invitrogen; Goat anti-rabbit, 1:5000, #111 035 44, Jackson Immunoresearch). Enzymatic detection was carried out using Super Signal West Femto Maximum Sensitivity Substrate (#34095, Thermo scientific). GAPDH was used as a loading control when quantifying RARβ expression.

## 2.5. Immunohistochemistry

Immunohistochemistry were performed on 14 µm brain cryosections that were thawed and dried for 10 min at room temperature. Sections were then fixed with paraformaldehyde 4% for 15 min and washed 3 times in PBS. Antigen retrieval was then carried out for 20 min at 90°C in 0.01M citrate buffer, pH 6. Slides were cooled down for 1 h at room temperature under agitation and washed 3 times in PBS during 10 min, and permeabilized and blocked in a solution of PBS + Triton 0.1% + FCS 10% for 1 h at room temperature. Incubation with primary antibodies (Table S1) was done overnight at +4°C, then sections were washed 3 times 10 min in PBS and incubated with secondary antibodies (Table S1) for 1 h at room temperature.

## 2.6. Transcriptomic analysis

NAc samples from n=3 animals/genotype were punched on thick cryosections (300 µm) and RNA was extracted using the RNeasy MicroPlus kit from Qiagen following the manufacturer's protocol. RNA-Seq libraries were generated from 200 ng total RNA using TruSeq Stranded mRNA Library Prep Kit and TruSeq RNA Single Indexes kits A and B (Illumina, San Diego, CA), according to the manufacturer's instructions. Briefly, following purification with poly-T oligonucleotide-attached magnetic beads, the mRNA was fragmented using divalent cations at 94°C for 2 minutes. The cleaved RNA fragments were copied into first strand cDNA using reverse transcriptase and random primers. Strand specificity was achieved by replacing dTTP with dUTP during 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, the products were purified and enriched with 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. Excess PCR primers were removed by purification using AMPure XP

beads (Beckman-Coulter, Villepinte, France) and the final cDNA libraries were checked for quality and quantified using capillary electrophoresis. Out of initial n=3 samples/genotype one control R6/1<sup>0/0</sup>; *Rarβ<sup>+/+</sup>* sample was not used due to poor quality. Libraries were then single-read sequenced with a length of 50 bp on an Illumina HiSeq 4000 sequencer. Image analysis and base calling were carried out using RTA v.2.7.7 and bcl2fastq v.2.17.1.14. Reads were mapped onto the mm10 assembly of *Mus musculus* genome using TopHat v.2.0.14 (Kim et al., 2013) and Bowtie 2 v.2.1.0 (Langmead and Salzberg, 2012). Gene expression was quantified from uniquely aligned reads using HTSeq-count v.0.6.1 (Anders et al., 2015) with annotations from Ensembl release 90 and intersection-nonempty mode. Only non-ambiguously assigned reads were retained for further analyses. Comparisons of interest have been performed using R 3.5.1 with DESeq2 version 1.22.1 (Love et al., 2014). More precisely, read counts were normalized from the estimated size factors using the median-of-ratios method and, for comparisons between each group, a Wald test was used to estimate the P-values. Sequencing data are available under GSE270980 accession number in Gene Expression Omnibus platform.

To check the existence of an interaction between mHTT and *Rarβ<sup>+/-</sup>* as an aggravating factor, a Likelihood-Ratio Test was used with DESeq2. Unwanted variation was identified with sva v.3.30.1 (Leek, 2014) and taken into account in the statistical model. P-values were then adjusted for multiple testing with the IHW method (Ignatiadis et al., 2016).

The enrichment analyses of deregulated genes in the present study with reference to the gene modules identified by Langfelder et al. (Langfelder et al., 2016) was performed using the ORA methods of EGSEA R package (1.26.0; (Alhamdoosh et al., 2017)). For this purpose, the differentially expressed genes (p<0.05) were converted to NCBI Entrez gene id with Ensembl biomart release 90. A custom gene set collection was created from the modules downloaded from (Langfelder et al., 2016) using buildCustom Idx of EGSEA. Over-representation analysis of each deregulated gene list was performed using egsea.ora function.

The enrichment analyses were additionally conducted using the GSEA method implemented in GSEA software version 4.3.2 (Subramanian et al., 2005; Mootha et al., 2003). The identical custom gene set collection and the ontology gene sets of MSigDB were employed. Genes were preranked using the formula sign(logFC) * -log10 (pval) following the DESeq2 analysis.

## 2.7. Quantitative PCR

The RNA was prepared from striatal preparations of newborn mice using RNAeasy micro kit (Qiagen, France). cDNA was synthesized using QuantiTect Reverse Transcription Kit (Qiagen, France), according to manufacturer's recommendations and was preceded by genomic DNA elimination. cDNA was synthesized Gene-specific primers for *Rarβ* detection were designed using Primer3 software (http://biotools.umassmed.edu/bioapps/primer3): forward primer: GCAGTTCTTATCACGATGGC, reverse primer: GATCCTCGATTTCTACACCG. Reaction was performed in duplicates in LightCycler 480 (Roche, France) using DNA SYBR Green I Master Kit (Roche, Diagnostics) with cDNA and gene-specific primers (5 pmol), following the manufacturer's instructions.

# 3. Results

## 3.1. Compromised *RARβ* exacerbates motor deficits in a mouse model of HD

To gain insight into the role of RARβ in HD, we investigated whether compromising *Rarβ* expression in a HD mouse model might accelerate the onset of HD-like symptoms. To this end, we generated R6/1 mice carrying only one copy of functional *Rarβ* and monitored their motor behaviors at 8 weeks, which is pre-symptomatic in R6/1 model, as a readout of disease symptoms. We found that 8 week-old R6/1<sup>Tg/0</sup>;
Rarβ⁺/⁻ mice displayed significant ~50% deficit in motor coordination in the rotarod test, which was not observed in mice carrying only the human mHTT transgene (R6/1Tg/0; Rarβ⁺/⁺) or heterozygous for the Rarβ null mutation (R6/10/0; Rarβ⁺/⁻) (Fig. 1A). Accordingly, two-way ANOVA analyses revealed a significant interaction between mHTT transgene and Rarβ heterozygosity (F(1,50)=4.2, p<0.05) for this performance, whereas post-hoc analyses supported significant reduction of latency to fall from accelerated rotarod in R6/1Tg/0; Rarβ⁺/⁻ mice as compared to wild-type (R6/10/0; Rarβ⁺/⁺) and R6/10/0; Rarβ⁺/⁻ or R6/1Tg/0; Rarβ⁺/⁺ littermates. Such locomotor coordination deficit did not affect overall novelty-induced locomotion and rearing behaviors in the open field test, as these performances were not altered in any group of mice and attained on average ~100 meters of distance and ~170 rears during 30 minutes of the test (Fig. S1A, S1B). These measures were not biased by abnormal anxiety states as the percentage of time spent in the center of the open arena during the first 5 minutes of the test was on average ~7% and not different between any of the tested groups (Fig. S1C). Importantly, R6/1Tg/0; Rarβ⁺/⁺ mice displayed spontaneous hyperlocomotion in actimetric cages during the active, dark phase of the light/dark cycle and Rarβ heterozygosity exacerbated this hyperactivity (see R6/1Tg/0; Rarβ⁺/⁻ mice), without any effect on locomotion in transgene-negative R6/10/0; Rarβ⁺/⁻ mice (Fig. 1B, C). This is supported by significant R6/1 x Rarβ interaction (F(1,52)=14.2, p<0.001, two-way ANOVA) and respective post-hoc analyses.

## 3.2. Compromised RARβ signaling in R6/1 mice does not induce loss of spMSNs

Since studies of Rarβ knockout mice revealed a role of RARβ in post-natal protection of about 20% of Drd2⁺ spMSNs from cell death (Ciancia et al., 2022), we hypothesized that compromised RARβ expression in HD striatum may contribute to, and possibly explain the high sensitivity of Drd2⁺ spMSNs to die over the course of the disease. In our genetic model, by inactivating one Rarβ allele in R6/1Tg/0; Rarβ⁺/⁻ mice, we might expect to observe Drd2⁺ spMSN cell death. To address this possibility, we first investigated whether Rarβ heterozygosity indeed reduces RARβ protein levels. Our western blot analyses indicated that in the caudate putamen (CPu) of 8 week-old R6/10/0; Rarβ⁺/⁻ mice, RARβ level was reduced to ~15 ± 4% of the level observed in wild-type controls and it did not decrease any further in compound R6/1Tg/0;

Rarβ⁺/⁻ mice, remaining at 16 ± 5% (Fig. 2A, B). RARβ protein level was also significantly reduced in transgenic, R6/1Tg/0; Rarβ⁺/⁺ mice but to lower extent (50 ± 5% of wild-type level). This is supported by the main effect of Rarβ and R6/1 genotypes (F (1, 8) = 54.8, p<0.001 and F(1, 8) = 10, p=0.01, respectively), and the significant interaction between the two genotypes (F(1, 8) = 12.1, p<0.01). The effect of Rarβ heterozygosity and R6/1 transgene on RARβ expression was synergistic in the nucleus accumbens shell (NAc), as supported by main effect of each genotype (F(1, 8) = 14.9 for R6/1, p<0.01 and F (1, 8) = 69.1, p<0.001 for RARβ) and significant reduction of RARβ in R6/1Tg/0; Rarβ⁺/⁻ as compared to R6/10/0; Rarβ⁺/⁺ or R6/1Tg/0; Rarβ⁺/⁺ NAc (Fig. 2B). To gain insight into early changes of Rarβ expression in HD relevant mouse model we also quantified Rarβ mRNA by qPCR in the striatum of newborn R6/1Tg/0 and their littermate R6/10/0 control mice, but did not observe any significant difference (Fig. S2A).

We then quantified the number of Drd2⁺ spMSNs using in situ hybridization to detect corresponding cells in the striatum. Though one might have expected a reduced density of Drd2⁺ cells per square millimeter analyzed in our disease model, we instead observed an increase both in CPu and NAc, which was associated exclusively with the presence of the mHTT transgene as supported by main effect of R6/1 genotype as the only significant difference in two-way ANOVA analyses (F (1, 16) = 10.62, p<0.01 for CPu and F (1, 16) = 77, p=0.01 for NAc), even though follow-up post-hoc analyses did not support specific difference between any individual group (Fig. S2B-D). This result could reflect a decrease of striatal volume due to mHTT-induced atrophy reported previously in several HD mouse models (Gil-Mohapel et al., 2011). To consider this possibility, we scored striatal area at each bregma level used for analyses (Fig. 2C). Reduction of striatal size was evident from the main effect of mHTT transgene (F (1,16)= 5.71, p<0.05), supporting an atrophy induced by the presence of the mHTT transgene, but it was not aggravated by monoallelic inactivation of Rarβ. Normalization of cell counts with respect to the surface change led to the conclusion of an absence of significant change in the number of Drd2⁺ cells in both CPu and NAc in any of the genotypes, as compared with the wild-type group of 8 weeks-old mice (Fig. 2D, 2E, S2C, S2D).

<figure><img src="image_4.png" /><figcaption>**Fig. 1.** Compromised RARβ signaling in the R6/1 mouse model exacerbates motor deficits linked to HD. (A) Motor coordination evaluated in accelerated rotarod as mean latency to fall from the rotating cylinder. (B) Spontaneous locomotor activity measured in actimetric cages over 32 h. (C) Spontaneous locomotor activity scores (number of beam breaks) over the dark phase of the light/dark cycle in actimetric cages measured between 7 pm and 7 am. Statistical differences were calculated using Bonferroni multiple comparisons as post-hoc follow-up of ANOVA analyses : *, p < 0.05; **, p < 0.01; ***, p < 0.001. Error bars represent SEM.</figcaption></figure>
<figure><img src="image_5.png" /><figcaption>**Fig. 2.** Compromised RARβ signaling in R6/1 mice does not induce loss of spMSNs. (A) Western blot detection of RARβ and GAPDH in CPu and in NAc. (B) Quantification data of RARβ expression in allelic series of mouse lines. (C) Measure of striatal area for each genotype was reported as percent of mean striatal area in the wild type (R6/1<sup>0/0</sup>; *RARβ<sup>+/-</sup>*) group. (D) Numbers of Drd2<sup>+</sup> MSNs cell were normalized with respect to striatal area changes for each genotype and reported in CPu and (E) in NAc. Statistical differences were calculated using Bonferroni multiple comparisons as post-hoc follow-up of ANOVA analyses : *, p < 0.05; **, p < 0.01; ***, p < 0.001. Error bars represent SEM.</figcaption></figure>

**3.3. Transcriptomic analyses reveal synergistic dysregulation of cell cycle and neurodevelopmental gene sets associated with HD progression**

In addition to hypothesis-driven analyses of Drd2<sup>+</sup> cell numbers, we used transcriptomic profiling as an unbiased approach to determine the contribution of reduced RARβ expression in HD-related changes. We decided to focus such analyses on the NAc region, as RARβ down-regulation was exacerbated and almost absent in R6/1<sup>Tg/0</sup>; *RARβ<sup>+/-</sup>* mice, but also because this striatal region displays somewhat higher RARβ expression comparing to dorsal or posterior striatum and was the most affected at transcriptional level by RARβ loss-of-function in *RARβ<sup>-/-</sup>* (Niewiadomska-Cimicka et al., 2017; Liao et al., 2005). Although transcriptomic data of individual samples did not clearly cluster according to genotype documenting some biological and/or technical variability (Fig. S3A), we found that, at 8 weeks of age, R6/1<sup>Tg/0</sup>; *RARβ<sup>+/-</sup>* mice (wild-type for the *RARβ* locus) displayed marked transcriptional changes of 341 downregulated and 81 upregulated genes in comparison to wild-type littermates (at cutoff of p<0.05; Fig. 3A and Table S2). These, and an additional pool of genes were affected by inactivation of one copy of

RARβ in R6/1<sup>Tg/0</sup>; *RARβ<sup>+/-</sup>* mice, which displayed 370 downregulated and 134 upregulated genes when compared to the wild-type condition, and 233 downregulated and 232 upregulated genes in comparison to R6/1<sup>Tg/0</sup>; *RARβ<sup>+/-</sup>* mice (Fig. 3A, Table S2). Thus, whereas some of the gene expression changes observed in mice carrying only mHTT transgene (R6/1<sup>Tg/0</sup>; *RARβ<sup>+/-</sup>* ) could already reflect reduced RARβ expression clearly present at 8 weeks of age (Fig. 2), an early reduction of RARβ signaling (germline inactivation of one allele) further revealed contribution of this receptor to progression of disease-associated changes. Specifically, RARβ-dependent changes in the R6/1 background were not merely an additive effect of *RARβ* heterozygosity and mHTT presence as there were only 12 down and 9 upregulated genes in R6/1<sup>0/0</sup>; *RARβ<sup>+/-</sup>* as compared to wild-type R6/1<sup>0/0</sup>; *RARβ<sup>+/-</sup>* striatum (Fig. 3A, Table S2).

Functional annotations of differentially expressed genes (DEGs) were made using gene set enrichment analyses (GSEA). As expected, mHTT alone in R6/1<sup>Tg/0</sup>; *RARβ<sup>+/-</sup>* mice affected some of the key biological functions associated with HD including synaptic transmission, mono-amine / dopaminergic signaling, mitochondrial components and activity (Table S3 and Fig. S3B). Although many similar functions were also
A

-Log₁₀-adjusted p-value)

<figure><img src="image_6.png" /></figure>

B

<figure><img src="image_7.png" /></figure>

C

<figure><img src="image_8.png" /></figure>

D

<figure><img src="image_9.png" /></figure>

E

DOWNREGULATED GENES

<figure><img src="image_10.png" /></figure>

UPREGULATED GENES

<figure><img src="image_11.png" /></figure>

R6/1Tg/0; *Rarβ+/-* vs R6/1Tg/0; *Rarβ+/*

F

<figure><img src="image_12.png" /></figure>

(caption on next page)
**Fig. 3.** Comparison of DEGs associated with compromised RARβ signaling in R6/1 mice. (A) Volcano plots illustrating significantly upregulated and downregulated genes and their numbers for specific comparisons. Statistical significance (grey line) was fixed at 1.3 for -log10(adjust. p-value), corresponding to p<0.05 (adjusted p-value). (B-C) Dot-plots illustrating gene set enrichment of DEGs (R6/1<sup>Tg/0</sup>; *Rarβ<sup>+/-</sup>* vs R6/1<sup>Tg/0</sup>; *Rarβ<sup>+/+</sup>* comparison) in modules identified by Langfelder and colleagues (see Supplementary Table 3 and 4 in (Langfelder et al., 2016) for module genes or annotations) including in particular M2 (GPCR signaling describing many cell-identity genes), M16 (developmental programs), M22 (mitochondrial functions, energy production). (B) Congruence of gene expression upregulation due to deletion of one copy of *Rarβ* in R6/1 mice with gene upregulation in specific modules was illustrated by red dots (log2FC > 0) whereas blue dots (downregulated genes; log2FC < 0) illustrate opposite changes. (C) Congruence of gene expression downregulation due to deletion of one copy of *Rarβ* in R6/1 mice with gene downregulation in specific modules was illustrated by blue dots whereas red dots (upregulated genes) illustrate opposite changes. Dot size represent statistical significance of such changes. Modules for which at least one genotype comparison displayed significant changes were shown. (D) Gene expression changes synergistically affected by R6/1 and *Rarβ<sup>+/-</sup>* in R6/1<sup>Tg/0</sup>; *Rarβ<sup>+/-</sup>* striatum were determined by Likelihood-Ratio test and analyzed for their enrichment in gene modules associated with HD progression. (E) Venny plot representing R6/1<sup>Tg/0</sup>; *Rarβ<sup>+/-</sup>* specific DEGs related to module M16 in comparison to M16 downregulated (left panel) and upregulated (right panel) genes in Q175 mice across different ages. (F) Direction of transcriptional changes of DEGs in the striatum of aging, 30-week-old R6/1 mice compared to age-matched WT mice (AC; Achour et al. 2015) and 8 week-old R6/1<sup>Tg/0</sup>; *Rarβ<sup>+/-</sup>* mice compared to R6/1<sup>Tg/0</sup>; *Rarβ<sup>+/+</sup>* in the present study (ZI).

affected in compound R6/1<sup>Tg/0</sup>; *Rarβ<sup>+/-</sup>* mice, the direction of changes was not always congruent. For example, gene sets associated with control of animal behavior, monoamine / catecholamine transport and signaling were similarly downregulated. On the other hand, mitochondrial complex and ribosome functions were upregulated in presence of mHTT in R6/1<sup>Tg/0</sup>; *Rarβ<sup>+/+</sup>* mice, but downregulated upon concomitant *Rarβ* deletion in R6/1<sup>Tg/0</sup>; *Rarβ<sup>+/-</sup>* mice. Reduced synaptic plasticity, a functional annotation of R6/1<sup>Tg/0</sup>; *Rarβ<sup>+/+</sup>* DEGs, was not among the top affected functions in R6/1<sup>Tg/0</sup>; *Rarβ<sup>+/-</sup>* mice. Inversely, an upregulation of genes related to telencephalon development, cell-cycle transition or stem-cell maintenance, increased cilium, axoneme assembly, was scored in R6/1<sup>Tg/0</sup>; *Rarβ<sup>+/-</sup>* mice, but was not observed in R6/1<sup>Tg/0</sup>; *Rarβ<sup>+/+</sup>* mice (Table S3 and Fig. S3B). Finally, although we did not identify any specific functional cluster of DEGs related to retinoic acid metabolism, we observed a decrease of *Aldh1a1* expression, known for its role in striatal retinoic acid synthesis(McCaffery and Drager, 1994), specifically in R6/1<sup>Tg/0</sup>; *Rarβ<sup>+/-</sup>* mice reflecting thereby a synergistic effect of mHTT expression and *Rarβ* heterozygosity supported by likelihood-ratio analyses (Table S2). In addition, concomitant increase of *Rdh12* and decrease of *Dhrs3* expression were associated with expression of mHTT suggesting increased production of retinaldehyde in both R6/1<sup>Tg/0</sup>; *Rarβ<sup>+/-</sup>* and R6/1<sup>Tg/0</sup>; *Rarβ<sup>+/+</sup>* striatum possibly contributing to neurotoxic, atrophy driving factors in these mice.

To identify transcriptional changes associated with HD onset and/or progression, we compared our data with those reported by Langfelder et al. (Langfelder et al., 2016) in relation with Huntingtin CAG length- and age-dependent disease progression in the murine allelic series. Using pre-ranked enriched GSEA (EGSEA), as expected, we found that differentially expressed genes (DEGs) in R6/1<sup>Tg/0</sup>; *Rarβ<sup>+/+</sup>* mice (wild-type for *Rarβ*) were the strongest enriched in M2, the module (module's designation according to Langfelder et al.) harboring downregulated striatal cell identity genes along with other functional gene sets and identified as having the strongest association with CAG length and best preserved in human HD datasets (Fig. 3B-C, see M2 gene list in Table S4). Significant enrichments were also observed for M11, the module with high numbers of glial genes (see M11 gene list in Table S4), M43 and M9, associated with mitochondrial functions, and M7 with cell death genes. Comparison of R6/1<sup>Tg/0</sup>; *Rarβ<sup>+/-</sup>* with R6/1<sup>0/0</sup>; *Rarβ<sup>+/+</sup>* mice revealed similar and frequently enhanced profiles of enrichment with emergence of genes belonging to M39 related to stress response and DNA damage repair (Fig. 3B, C, Table S4). The genetic interaction between compromised *Rarβ* expression and mHTT transgene was, however, the best visible through comparison of DEGs in R6/1<sup>Tg/0</sup>; *Rarβ<sup>+/-</sup>* and R6/1<sup>Tg/0</sup>; *Rarβ<sup>+/+</sup>* mice (Fig. 3B, C, Table S4). The major transcriptional changes associated with inactivation of one *Rarβ* allele in R6/1 mice were observed in modules ordered according to their significance from 22 through 16, 5, 2, 30, 11, 24, 25, 9, 42 to 4, out of which modules M2, 11, 25 and 9 were reported as highly correlated with disease progression by Langfelder et al. Surprisingly, some of such transcriptional changes showed opposite trend with respect to that observed within specific modules. For example, some M2 relevant genes were upregulated in R6/1<sup>Tg/0</sup>; *Rarβ<sup>+/-</sup>* when compared to R6/1<sup>Tg/0</sup>; *Rarβ<sup>+/+</sup>* whereas

this module represents genes downregulated in HD and in R6/1<sup>Tg/0</sup>; *Rarβ<sup>+/+</sup>* as compared to R6/1<sup>0/0</sup>; *Rarβ<sup>+/-</sup>* striatum (Fig. 3C, Table S4). When inspecting functional annotations of this group of genes, we observed that most of them were associated with neurodevelopment including a subset of 192 genes downregulated in R6/1<sup>Tg/0</sup>; *Rarβ<sup>+/+</sup>* striatum and frequently upregulated upon additional deletion of one *Rarβ* allele (R6/1<sup>Tg/0</sup>; *Rarβ<sup>+/-</sup>* vs R6/1<sup>Tg/0</sup>; *Rarβ<sup>+/+</sup>* or a subset of 99 genes deregulated exclusively in R6/1<sup>Tg/0</sup>; *Rarβ<sup>+/-</sup>* striatum (R6/1<sup>Tg/0</sup>; *Rarβ<sup>+/-</sup>* vs R6/1<sup>0/0</sup>; *Rarβ<sup>+/+</sup>*) (Fig. S3C, Table S4). The remaining group of 72 genes was common to all three comparisons and included striatal and dopamine signaling related genes which expression remained globally downregulated without any specific selectivity for snMSNs or spMSNs expressing respectively dopamine D1 or D2 receptors (Table S4). Likewise, M22 related genes (strongly enriched in mitochondrial genes) were downregulated upon deletion of one *Rarβ* allele in R6/1 mice (R6/1<sup>Tg/0</sup>; *Rarβ<sup>+/-</sup>* vs R6/1<sup>Tg/0</sup>; *Rarβ<sup>+/+</sup>*) in contrast to their upregulation in R6/1<sup>Tg/0</sup>; *Rarβ<sup>+/-</sup>* mice carrying mHTT transgene alone (Fig. 3B, C, Table S4). Such opposition in gene expression changes may reflect temporal dynamics of M22 DEGs which in Q175 mouse model of HD also displayed increased number of downregulated genes specifically at the early symptomatic age of 6 months (44 down- and 101 up-regulated genes), but not at pre-symptomatic age of 2 months (0 down- and 8 up-regulated) or advanced symptomatic age of 10 months (3 down- and 161-upregulated genes) (Langfelder et al., 2016). Importantly, almost all genes deregulated in R6/1 mice upon deletion of *Rarβ* (R6/1<sup>Tg/0</sup>; *Rarβ<sup>+/-</sup>* vs R6/1<sup>Tg/0</sup>; *Rarβ<sup>+/+</sup>* comparison) displayed congruent changes with those observed in striatum of early symptomatic. 6-month-old Q175 mice, including marked downregulation of mitochondrial genes from module M22, and upregulation of cell cycle and development-related genes in M16 and M2 (see above), and supported by EGSEA results (Fig. 3B, C, see corresponding gene lists in Table S4). In particular, M16 and M20 (cell cycle and development) modules emerged as synergistically dysregulated by mHTT transgene and *Rarβ* heterozygosity, as indicated by likelihood-ratio analyses with M16 module genes displaying particularly high statistical significance and gene number (Fig. 3D). In addition, we took advantage of the recent study of epigenetic aging in HD mouse models by Brulé et al. We found that upregulated R6/1<sup>Tg/0</sup>; *Rarβ<sup>+/-</sup>* vs R6/1<sup>Tg/0</sup>; *Rarβ<sup>+/+</sup>* genes were strongly enriched in all four modules (called hereafter Brulé M1-M4) identified in this study by euchromatin score as positively associated with aging (Brulé M1-M4) or presence of HD-causing mutation (Brulé M2, Brulé M4) (Fig. S3F and Table S4). Globally, these data and together with an early emergence of behavioral phenotype in R6/1<sup>Tg/0</sup>; *Rarβ<sup>+/-</sup>* suggests that such R6/1<sup>Tg/0</sup>; *Rarβ<sup>+/-</sup>* vs R6/1<sup>Tg/0</sup>; *Rarβ<sup>+/+</sup>* specific transcriptional changes are perhaps the most relevant for disease progression.

To understand whether synergistic effect of R6/1<sup>Tg/0</sup> and *Rarβ<sup>+/-</sup>* on gene expression changes reflects acceleration of disease progression, we asked whether synergistically affected DEGs related to module M16 in 8-weeks old R6/1<sup>Tg/0</sup>; *Rarβ<sup>+/-</sup>* vs R6/1<sup>Tg/0</sup>; *Rarβ<sup>+/+</sup>* mice may correspond to changes characteristic of mouse HD models at fully symptomatic age. Thus, when comparing to age-dependent transcriptional deregulation in
the Q175 KI mouse model (2, 6 and 10-months old) reported by Langfelder and colleagues, we observed that 25% of such changes were found in symptomatic, 6 month-old Q175 mice but were absent from pre-symptomatic, 2 month-old, or advanced stage 10 month-old Q175 mice (Fig. 3E). Specifically, upregulation of e.g. Ascl1, Ckap2l, Dlx2, Foxm1, Lmnb1, Rrm2, Rsph4a, Sox4, Ttc21a, Vwa5b1 in 6 month-old Q175 and R6/1Tg/0; Rarβ+/- striatum points to reactivation of transcriptional programs supporting cells stemness, differentiation and migration and indicates that early compromised Rarβ expression in R6/1Tg/0; Rarβ+/- mice accelerates onset of some HD-like transcriptional changes typical for the “pivotal age” of transition from non-symptomatic to symptomatic phase at 6 months. Comparison of all DEGs common between R6/1Tg/0; Rarβ+/- vs R6/1Tg/0; Rarβ+/- and Q175 striatum at different ages revealed a high number (59.5% of all DEGs from R6/1Tg/0; Rarβ+/- vs R6/1Tg/0; Rarβ+/- comparison) and congruence of such transcriptional changes especially evident for the group of 6 month old Q175 mice (50%), but also 10 month old Q175 mice (40%) (Fig. S3F-H, Table S5). This is further supported by significant overlap of congruent changes between DEGs from R6/1Tg/0; Rarβ+/- vs R6/1Tg/0; Rarβ+/- mice at 8 weeks and DEGs reported by Achour et al. (Achour et al., 2015) in 30 week-old R6/1Tg/0 striatum (Fig. 3F), corresponding respectively to 8% of all DEGs in Achour et al. study and 10% in R6/1Tg/0; Rarβ+/- vs R6/1Tg/0; Rarβ+/- comparison. Finally, R6/1Tg/0; Rarβ+/- vs R6/1Tg/0; Rarβ+/- transcriptional changes overlapping with fully symptomatic R6/1Tg/0 or Q175 mice are directly relevant for striatal/forebrain development, cell cycle control or stemness and include for example

Ebf1, Dlx2, Ascl1, Arx, Foxm1, Zic1, Gli3, Ntrk2, Sox4, Sox8, Sox11 whereas other genes like Wdr6, Bahcc1, Pcdhgb1, Mex3a, Ckap2l, Fhl2 are involved in control of cell proliferation in neural or extra-neural tissues (see Table S5 for full list of overlapping gene changes).

### 3.4. Compromised RARβ signaling drives reactivation of cell-cycle programs and lineage infidelity in HD mice

As supported by comparisons of individual compound mutant mice but also Likelihood-Ratio Test of synergy between mHTT transgene and Rarβ+/-, an upregulation of genes associated with cell-cycle regulation was the most consistent change induced by Rarβ heterozygosity in R6/1 mice. Furthermore, the direction of changes in this cluster correlated with those observed in DEGs of M16 and M20 modules by Langfelder and colleagues. We hypothesized that such changes in the NAC may reflect an overall reactivation of cell-cycle/division transcriptional programs, which should be best visible in the ventricular and sub-ventricular zone (collectively referred to as SVZ hereafter) (Fig. 4A), an adult neurogenic niche harboring actively dividing cell. Analyses of Ki67+ proliferating cells in the SVZ region revealed that mHTT differently affected the number of proliferating cells depending on the presence of Rarβ heterozygosity as supported by significant R6/1 x Rarβ interaction (F(1, 20) = 34.9, p<0.001) (Fig. 4B, C, Fig. S4A). Accordingly, whereas no significant changes in number of Ki67+ cells were observed in R6/1Tg/0; Rarβ+/- and R6/1Tg/0; Rarβ+/-, their number was significantly increased in R6/1Tg/0; Rarβ+/- SVZ. As revealed by analyses

<figure><img src="image_13.png" /><figcaption>**Fig. 4.** Compromised Rarβ signaling drives reactivation of cell-cycle programs and lineage infidelity in HD mice. (A) Cartoon depicting the ventricular / sub-ventricular zone (called throughout SVZ, red line) in a mouse coronal brain section. (B) Examples of immunofluorescent identification of proliferating Ki67+ and PH3+ cells in R6/1Tg/0; Rarβ+/- SVZ (see Fig. S4 for all reaming genotypes). Blue arrowheads point to a double positive cell. The letter V indicates the ventricular side. (C) Quantification of Ki67+ cells (% of cells). (D) Quantification of PH3+ cells among Ki67+ cells in SVZ (% of cells). (E) Examples of immunofluorescence identifying GFAP+, SOX2+, Nestin+ neural stem cells (NSCs) in R6/1Tg/0; Rarβ+/- SVZ (see Fig. S4 for all reaming genotypes). Blue arrowheads point to a triple positive cell. The letter V indicates the ventricular side. (F) NSC quantification (% of cells). Statistical differences were calculated using Bonferroni multiple comparisons as post-hoc follow-up of ANOVA analyses: *, p < 0.05; **, p < 0.01; ***, p < 0.001. Error bars represent SEM.</figcaption></figure>
of phospho-histone 3 co-immunostaining (PH3+), the percentage of cells in S-phase was similar among all genotypes and ranged at about 5.5-8% of Ki67+ cells (Fig. 4D). We therefore asked whether changes in the number of proliferating cells may result from covariation of the reservoir of neuroblasts / neural stem cells (called hereafter collectively NSCs). The number of GFAP+; SOX2+; Nestin+ triple positive NSCs was not affected in R6/1<sup>Tg/0</sup>; *Rarβ<sup>+/-</sup>* mice (Fig. 4E, F, Fig. S4B). However, a significant increase of NSCs correlated with higher cell proliferation index was noted in R6/1<sup>Tg/0</sup>; *Rarβ<sup>+/-</sup>* SVZ. This is supported by significant interaction between R6/1<sup>Tg/0</sup> and *Rarβ<sup>+/-</sup>* interaction (F(1,20) = 5.119, p=0.0349 and group effects for both R6/1 (F (1,20) = 16.99, p=0.0005) and *Rarβ* (F (1,20) = 13.41, p=0.0015; Fig. 4F, Fig. S4) and suggests that the increased proliferation rate observed in these mice results from an increased pool of ependymal NSCs. Since increased proliferative activity was previously observed in HD patients and mouse models, we speculated that absence of such changes in 2-month-old R6/1<sup>Tg/0</sup>; *Rarβ<sup>+/-</sup>* mice may reflect early phase of physiopathology and may increase with age. In line with this possibility an increase of proliferation index and NSC cells was observed in 14-weeks old R6/1<sup>Tg/0</sup>; *Rarβ<sup>+/-</sup>* mice (Fig. S4C-F).

## 4. Discussion

Reduced expression of *Rarβ* and dysregulation of a large number of its transcriptional target genes observed in the striatum of HD patients and animal models suggested that *Rarβ* may play a critical role in progression of HD-related transcriptional changes, thereby driving disease progression (Lee et al., 2020; Niewiadomska-Cimicka et al., 2017; Luthi-Carter et al., 2000). Although this possibility could be supported by the observation of a neuroprotective activity of RARβ for Drd2+ neuron functions and survival (Ciancia et al., 2022), the contribution of compromised RARβ signaling to HD physiopathology has so far been mostly correlative. By investigating animal behavior, we found here that compromised RARβ signaling is sufficient to precipitate HD-like motor abnormalities in the R6/1 mouse model of HD at 8 weeks of age, which corresponds to motor pre-symptomatic period in this model (Nithianantharajah et al., 2008; Naver et al., 2003). At this age, R6/1 mice lacking one functional copy of *Rarβ* displayed significant coordination deficits and spontaneous hyperlocomotion, whereas R6/1<sup>Tg/0</sup>; *Rarβ<sup>+/-</sup>* littermates displayed only weak spontaneous hyperlocomotion. Such precocious coordination deficits and exacerbation of hyperlocomotion indicate that *Rarβ* acts as a disease modifier gene preventing disease progression. The critical time window of RARβ protective activity is not clear, but may take place in early postnatal life or during adolescence. This is suggested by our observation that expression of RARβ in the striatum mice carrying mHTT transgene alone drops sometime between birth and 8 weeks of age. Accordingly, whereas we did not observe any difference in *Rarβ* expression in the striatum of newborn R6/1<sup>Tg/0</sup>; *Rarβ<sup>+/-</sup>* mice, at 8 weeks of age reduction of RARβ level in the R6/1<sup>Tg/0</sup>; *Rarβ<sup>+/-</sup>* CPu was strong and not significantly different from that observed in R6/1<sup>Tg/0</sup>; *Rarβ<sup>+/-</sup>* mice, whereas in NAc, the mHTT and *Rarβ* heterozygosity displayed some additive or synergistic effect leading to highly significant reduction of RARβ expression in R6/1<sup>Tg/0</sup>; *Rarβ<sup>+/-</sup>* mice. Likewise, significant HD-like transcriptional changes were present already at 8 weeks of age in the NAc of R6/1<sup>Tg/0</sup>; *Rarβ<sup>+/-</sup>* mice carrying the R6/1 transgene alone. The decrease of expression of RARβ may reflect sequestration of this protein by mHTT aggregates reported earlier, and leading ultimately to downregulation of *Rarβ* expression in the striatum due to its positive transcriptional auto-regulation (Niewiadomska-Cimicka et al., 2017). Such strong decrease of RARβ expression in the NAc may also partially reflect a synergy between mHTT and *Rarβ* heterozygosity in lowering *Aldh1a1* expression in R6/1<sup>Tg/0</sup>; *Rarβ<sup>+/-</sup>* NAc. Indeed, *Aldh1a1* is the key retinoic acid synthesizing enzyme in the striatum (McCaffery and Drager, 1994) and its low expression may compromise retinoic acid synthesis and ligand-dependent activity of RARβ including control of its own expression.

Further studies will be required to determine the mechanism and exact time course of reduction in RARβ expression and its contribution to the neurodegenerative process in early stages of postnatal life. A large array of transcriptional changes has been reported in HD patients and animal models with the most dramatic ones observed in the striatum, the primary site of degeneration (Achour et al., 2015; Hodges et al., 2006; Luthi-Carter et al., 2000; Benn et al., 2005; Francelle et al., 2017; Kuhn et al., 2007; Labadorf and Myers, 2015; Lee et al., 2022; Seredenina and Luthi-Carter, 2012; Handsaker et al., 2025). The understanding of biologically meaningful changes was much advanced by a recent study that reported Huntingtin CAG length- and age-dependent gene co-expression networks (Langfelder et al., 2016). Among 54 expression modules defined by these authors, 37 (M1-37) corresponded to striatal genes, out of which many co-varied in age- and CAG repeat-dependent manner (see Supplementary Table 3 in (Langfelder et al., 2016) for module genes and Supplementary Table 4 in (Langfelder et al., 2016) for their functional annotations). Through transcriptomic profiling of striatal tissue, we found that 8 week-old R6/1 transgenic mice display a significant HD-related profile of gene expression changes including downregulation of genes related to cell-identity, monoaminergic and synaptic signaling, out of which many were also reported for older R6/1 mice (Achour et al., 2015; Alcalá-Vida et al., 2022). These genes belong to the M2 module identified by Langfelder et al. as the strongest associated with CAG length and best preserved in human HD datasets. Many of the DEGs observed in R6/1 mice carrying mHTT transgene alone may reflect significant decrease of *Rarβ* expression and activity which we observed at 2 months of age in R6/1<sup>Tg/0</sup>; *Rarβ<sup>+/-</sup>* mice. Importantly, *Rarβ* expression was not affected in the striatum of newborn R6/1 mice, suggesting postnatal onset of such decrease. Thus, constitutive deletion of one functional copy of *Rarβ* in R6/1<sup>Tg/0</sup>; *Rarβ<sup>+/-</sup>* mice compromises RARβ signaling early during brain development and broadens the profile of HD-like gene deregulations observed in R6/1 mice consistent with acceleration of disease progression. Accordingly, around 60% of gene expression changes associated with inactivation of one *Rarβ* allele on R6/1 genetic background (R6/1<sup>Tg/0</sup>; *Rarβ<sup>+/-</sup>* vs R6/1<sup>Tg/0</sup>; *Rarβ<sup>+/-</sup>*) in 8 weeks-old mice overlapped and were congruent with DEGs in the striatum of early symptomatic 6-month-old and fully symptomatic 10-month-old Q175 mice. Likewise, similar overlap was observed with fully symptomatic 30 weeks-old R6/1 mice (Fig. 3H; Fig. S3, Table S5). Of particular interest was the emergence of an upregulated cluster of genes related to cell-cycle, stem cell maintenance and telencephalon development which usually appears at later stages of disease progression, or during aging (Yang et al., 2023a; Yang et al., 2023b; Brulé et al., 2025). We therefore suggest that in HD, the MSNs lose their identity in two distinct steps which can be dissociated in time and possibly at mechanistic level: (i) decreased expression of genes which are typically expressed in MSNs, e.g. as observed for Drd1 and Drd2 in both, R6/1<sup>Tg/0</sup>; *Rarβ<sup>+/-</sup>* and R6/1<sup>Tg/0</sup>; *Rarβ<sup>+/-</sup>* young mice, and therefore less influenced by deletion of one copy of Rarb in R6/1 mice (ii) later acquisition of novel transcriptional signatures, those of developmentally immature/active cells evident only in young R6/1<sup>Tg/0</sup>; *Rarβ<sup>+/-</sup>* or aged R6/1<sup>Tg/0</sup>; *Rarβ<sup>+/-</sup>* mice. We also demonstrated that acquisition of developmental identity is functionally relevant as the number of Ki67+ proliferating cells in the SVZ of R6/1<sup>Tg/0</sup>; *Rarβ<sup>+/-</sup>* mice was significantly increased, reflecting an increased number of GFAP+; SOX2+; Nestin+ triple positive NSCs and neuroblasts in the ependymal and SVZ layers. Whereas such an increase was evident in the R6/1<sup>Tg/0</sup>; *Rarβ<sup>+/-</sup>* adult neurogenic niche, where proliferation is naturally occurring, no evident proliferating cells were observed in other parts of the striatum including the CPu and the NAc, the region used for transcriptional profiling. Several lines of evidence suggest, however, that increased expression of those genes may be of relevance for the progression of HD. Increased mitotic activity was reproducibly observed in HD post-mortem studies (Curtis et al., 2003; Curtis et al., 2005; Pelegrí et al., 2008), although it was rarely studied and found only weakly increased or unchanged in mouse models of HD (Gil et al., 2005; Gil-Mohapel et al., 2011; Jin et al., 2005; Lázic
et al., 2004; Lazic et al., 2006). A change found consistently between HD patients and mouse models, was upregulation of striatal genes related to cell division and brain development (Langfelder et al., 2016; Brulé et al., 2025). Using Likelihood-Ratio analyses, we found that R6/1<sup>Tg/0</sup> synergized with *Rarβ<sup>+/-</sup>* in upregulation of those genes as compared to mice carrying only the mHTT transgene (Fig. 3D). These genes were synergistically enriched in modules M16 and M20, but also corresponded to a subset of M2-relevant genes upregulated upon deletion of *Rarβ* allele in R6/1 mice; the two latter modules positively correlating with CAG length in the study by Langfelder et al. (Langfelder et al., 2016). Interestingly, more than 50% of R6/1<sup>Tg/0</sup>; *Rarβ<sup>+/-</sup>* DEGs enriched in module 16 were almost exclusively associated with the onset of symptoms in Q175 KI mice at 6 months of age, as compared to pre-symptomatic 2 month-old or advanced stage 10 month-old Q175 KI mice (Fig. 3G and ref. (Langfelder et al., 2016)), suggesting potential importance of corresponding deregulations in the control of the onset of the disease. Depression of cell-cycle and developmental genes could possibly be considered as lineage infidelity and part of the putative regenerative process as in case of wound repair (Ge et al., 2017), or reflect corrosion of epigenetic landscape during aging (Yang et al., 2023a; Yang et al., 2023b; Brulé et al., 2025). Such developmental reactivation may have dramatic consequences for disease progression potentially by impacting mitochondrial activity. Specifically, neuronal functions critically depend on energy produced during oxidative phosphorylation (OXPHOS), the metabolic mode which is not compatible with cell-cycle and developmental processes which are supported by the glycolytic cycle, much less efficient in producing energy (Gascón et al., 2016; Tsogtbaatar et al., 2020). Accordingly, concomitant to upregulation of genes related to cell cycle, stem cell maintenance and development, we found that R6/1<sup>Tg/0</sup>; *Rarβ<sup>+/-</sup>* mice also display strong downregulation of genes involved in OXPHOS, ATP biosynthesis, or ATP-dependent processes like ribosomal functions. We therefore propose that the role of RARβ is to delay HD progression by preventing lineage infidelity attempt and acquisition of immature/developmental cell identity in disease conditions. The resulting downregulation of OXPHOS and ATP production required for developmental programs may lead in turn to energy crisis unsustainable for neuronal functions driving thereby neurodegeneration and onset of behavioral deficits. The relevance of this process expands perhaps beyond HD and may also concern “physiological” aging, as the expression of developmental genes contributing to loss of cell identify is one of the key features of aging (Yang et al., 2023a; Yang et al., 2023b). This possibility is particularly intriguing as it points to RARβ as an aging-delaying factor and its compromised signaling as an aging accelerator in HD. Accordingly, we observed high enrichment of R6/1<sup>Tg/0</sup>; *Rarβ<sup>+/-</sup>* vs R6/1<sup>Tg/0</sup>; *Rarβ<sup>+/-</sup>* DEGs (upregulated genes) in 4 modules of actively transcribed genes (high euchromatin score) identified by Brulé and colleagues as positively associated with aging and/or HD-causing (Brulé et al., 2025).

*Drd2*+ spMSNs are the most prone to neurodegeneration in HD, and RARβ activity was shown previously to play a role in their protection against cell death otherwise observed in *Rarβ*<sup>+/-</sup> mice, but also in wild-type mice treated with 3NP, a mitochondrial toxin (Ciancia et al., 2022). Despite evident behavioral deficits in R6/1<sup>Tg/0</sup>; *Rarβ*<sup>+/-</sup> mice and downregulation of a large set of genes required for mitochondrial functions, we did not observe any decrease in the number of *Drd2*+ cells in the NAc or CPu of young R6/1<sup>Tg/0</sup>; *Rarβ*<sup>+/-</sup> animals. These data further support the notion that RARβ neuroprotective role goes beyond prevention of cell death, but is more essential for spMSNs functions.

## 5. Conclusion

We report in this study that compromising RARβ expression in the R6/1 transgenic mouse model of HD accelerates onset of HD-like motor abnormalities, including motor coordination deficits. Moreover, using unbiased transcriptional analyses, we shown that decreased RARβ signaling contributes to the progression of disease-related

transcriptional changes in R6/1 mice by reactivation of developmental programs accelerating loss of cell identity but also mitochondrial dysfunction. Such effects were synergistic, indicating that RARβ is essential for repression of developmental programs, cell-identity maintenance and for slowing disease progression in HD. Although approximate as calculation, we estimate that compromised RARβ signaling accelerates the disease progression by at least 2-fold. This is supported by two main observations: (i) at 2 months of age R6/1<sup>Tg/0</sup>; *Rarβ<sup>+/-</sup>* mice display behavioral phenotype usually evident at 4-6 months of age in R6/1<sup>Tg/0</sup> and 6-10 months old Q175 mice; (ii) inactivation of one copy of *Rarβ* in R6/1 mice led to gene expression changes (R6/1<sup>Tg/0</sup>; *Rarβ<sup>+/-</sup>* vs R6/1<sup>Tg/0</sup>; *Rarβ<sup>+/-</sup>* DEGs) which at 2-months of age were congruent with those observed in 6-month old R6/1<sup>Tg/0</sup> mice or 6-months or 10-months old Q175 symptomatic but not 2-month old Q175 pre-symptomatic mice.

## Ethics approval and consent to participate

All experiments were carried out in accordance with the Directive of the European Parliament: 2010/63/EU and in compliance with the guidelines of CNRS and French ministry of Agriculture and Food (decree 87848).

## Consent for publication

Not applicable.

## Funding

This work was supported by an ERA-Net (RAinRARE) grant, and by the institutional grants IdEx Unistra (ANR-10-IDEX-0002), and SFRI-STRAT'US project (ANR-20-SFRI-0012) and EUR IMCBio (ANR-17-EURE-0023) under the framework of the France 2030 Program

## CRediT authorship contribution statement

**Nicolas Zinter:** Writing – review & editing, Writing – original draft, Visualization, Validation, Investigation, Formal analysis. **Tao Ye:** Writing – original draft, Formal analysis. **Hanna Semaan:** Visualization, Validation, Investigation, Formal analysis. **Valérie Fraulob:** Investigation. **Damien Plassard:** Writing – original draft, Formal analysis. **Wojciech Krezel:** Writing – review & editing, Writing – original draft, Supervision, Project administration, Funding acquisition, Data curation, Conceptualization.

## Declaration of competing interest

The authors declare no conflict of interest

## Data availability

Data will be made available on request.

## Acknowledgments

We thank Mr. Valentin Fraxial for assistance in behavioral analyses, Dr. Gill Bates for sharing the R6/1 mouse model of HD and the Mouse Clinical Institute animal facility for excellent animal care. We thank Dr. Pascal Dollé for discussions and critical reading of the manuscript. Sequencing was performed by the GenomEast platform, a member of the ‘France Génomique’ consortium (ANR-10-INBS-0009). We also acknowledge that graphical abstract was created using Biorender (individual license attributed to WK).
# Appendix A. Supplementary data

Supplementary data to this article can be found online at https://doi.org/10.1016/j.nbd.2025.106943.

## References

Achour, M., Le Gras, S., Keime, C., Parmentier, F., Lejeune, F.X., Boutillier, A.L., Néri, C., Davidson, I., Merienne, K., 2015. Neuronal identity genes regulated by super-enhancers are preferentially down-regulated in the striatum of Huntington's disease mice. Hum. Mol. Genet. 24 (12), 3481-3496.

Alcalà-Vida, R., Lotz, C., Brulé, B., Seguin, J., Decraene, C., Awada, A., Bombardier, A., Cosquer, B., Pereira de Vasconcelos, A., Brouillet, E., et al., 2022. Altered activity-regulated H3K9 acetylation at TGF-beta signaling genes during egocentric memory in Huntington's disease. Prog Neurobiol. 219, 102363.

Alhamdoosh, M., Law, C.W., Tian, L., Sheridan, J.M., Ng, M., Ritchie, M.E., 2017. Easy and efficient ensemble gene set testing with EGSEA. F1000Res 6, 2010.

Anders, S., Pyl, P.T., Huber, W., 2015. HTSeq-a Python framework to work with high-throughput sequencing data. Bioinformatics (Oxford, England) 31 (2), 166-169.

Benn, C.L., Lances, C., Li, H., Strand, A.D., Woodman, B., Sathasivam, K., Li, S.-H., Ghazi-Noori, S., Hockly, E., Faruque, S.M.N.N., et al., 2005. Contribution of nuclear and extranuclear polyQ to neurological phenotypes in mouse models of Huntington's disease. Hum. Mol. Genet. 14 (20), 3065-3078.

Brulé, B., Alcalà-Vida, R., Penaud, N., Scuto, J., Mounier, C., Seguin, J., Khodaverdian, S. V., Cosquer, B., Birmelé, E., Le Gras, S., et al., 2025. Accelerated epigenetic aging in Huntington's disease involves polycomb repressive complex 1. Nat. Commun. 16 (1), 1550.

Ciancia, M., Rataj-Baniowska, M., Zinter, N., Baldassarro, V.A., Fraulob, V., Charles, A.L., Alvarez, R., Muramatsu, S.I., de Lera, A.R., Geny, B., et al., 2022. Retinoic acid receptor beta protects striatopallidal medium spiny neurons from mitochondrial dysfunction and neurodegeneration. Prog. Neurobiol. 102246.

Curtis, M.A., Penney, E.B., Pearson, A.G., van Roon-Mom, W.M., Butterworth, N.J., Dragunow, M., Connor, B., Faull, R.L., 2003. Increased cell proliferation and neurogenesis in the adult human Huntington's disease brain. Proc. Natl. Acad. Sci. U S A 100 (15), 9023-9027.

Curtis, M.A., Penney, E.B., Pearson, M., Dragunow, M., Connor, B., Faull, R.L.M., 2005. The distribution of progenitor cells in the subependymal layer of the lateral ventricle in the normal and Huntington's disease brain. Neuroscience 132 (3), 777-788.

Fricelle, L., Lotz, C., Outeiro, T., Brouillet, E., Merienne, K., 2017. Contribution of Neuroepigenetics to Huntington's Disease. Front. Hum. Neurosci. 11, 17.

Gascón, S., Murenu, E., Masserdotti, G., Ortega, F., Russo, G.L., Petrik, D., Deshpande, A., Heinrich, C., Karow, M., Robertson, S.P., et al., 2016. Identification and successful negotiation of a metabolic checkpoint in direct neuronal reprogramming. Cell Stem Cell 18 (3), 396-409.

Ge, Y., Gomez, N.C., Adam, R.C., Nikolova, M., Yang, H., Verma, A., Lu, C.P.-J., Polak, L., Yuan, S., Elemento, O., et al., 2017. Stem cell lineage infidelity drives wound repair and cancer. Cell 169 (4), 636-650.e614.

Ghyselinck, N.B., Dupé, V., Dierich, A., Messadde, N., Garnier, J.M., Rochette-Egly, C., Chambon, P., Mark, M., 1997. Role of the retinoic acid receptor beta (RARbeta) during mouse development. Int. J. Dev. Biol. 41 (3), 425-447.

Gil, J.M.A.C., Mohapel, P., Araújo, I.M., Popovic, N., Li, Y.-Y., Brundin, P., Petersén, Å., 2005. Reduced hippocampal neurogenesis in R6/2 transgenic Huntington's disease mice. Neurobiol. Dis. 20 (3), 744-751.

Gil-Mohapel, J., Simpson, J.M., Ghilan, M., Christie, B.R., 2011. Neurogenesis in Huntington's disease: can studying adult neurogenesis lead to the development of new therapeutic strategies? Brain Res. 1406, 84-105.

Handsaker, R.E., Kashin, S., Reed, N.M., Tan, S., Lee, W.S., McDonald, T.M., Morris, K., Kamitaki, N., Mullally, C.D., Morakabati, N.R., et al., 2025. Long somatic DNA-repeat expansion drives neurodegeneration in Huntington's disease. Cell 188 (3), 623-639.e619.

Hodges, A., Strand, A.D., Aragaki, A.K., Kuhn, A., Sengstag, T., Hughes, G., Elliston, L.A., Hartog, C., Goldstein, D.R., Thu, D., et al., 2006. Regional and cellular gene expression changes in human Huntington's disease brain. Hum. Mol. Genet. 15 (6), 965-977.

Ignatiadis, N., Klaus, B., Zaugg, J.B., Huber, W., 2016. Data-driven hypothesis weighting increases detection power in genome-scale multiple testing. Nature Methods 13 (7), 577-580.

Jin, K., LaFevre-Bernt, M., Sun, Y., Chen, S., Gafni, J., Crippen, D., Logvinova, A., Ross, C. A., Greenberg, D.A., Ellerby, L.M., 2005. FGF-2 promotes neurogenesis and neuroprotection and prolongs survival in a transgenic mouse model of Huntington's disease. Proc. Natl. Acad. Sci. USA 102 (50), 18189-18194.

Kim, D., Pertea, G., Trapnell, C., Pimentel, H., Kelley, R., Salzberg, S.L., 2013. TopHat2: accurate alignment of transcriptomes in the presence of insertions, deletions and gene fusions. Genome Biol. 14 (4), R36.

Kuhn, A., Goldstein, D.R., Hodges, A., Strand, A.D., Sengstag, T., Kooperberg, C., Becanovic, K., Pouladi, M.A., Sathasivam, K., Cha, J.-H.J., et al., 2007. Mutant huntingtin's effects on striatal gene expression in mice recapitulate changes observed in human Huntington's disease brain and do not differ with mutant huntingtin length or wild-type huntingtin dosage. Hum. Mol. Genet. 16 (15), 1845-1861.

Labadorf, A.T., Myers, R.H., 2015. Evidence of extensive alternative splicing in post mortem human brain HTT transcription by mRNA sequencing. PLoS One 10 (10), e0141298.

Langfelder, P., Cantle, J.P., Chatzopoulou, D., Wang, N., Gao, F., Al-Ramahi, I., Lu, X.H., Ramos, E.M., El-Zein, K., Zhao, Y., et al., 2016. Integrated genomics and proteomics define huntingtin CAG length-dependent networks in mice. Nat. Neurosci. 19 (4), 623-633.

Langmead, B., Salzberg, S.L., 2012. Fast gapped-read alignment with Bowtie 2. Nature Methods 9 (4), 357-359.

Lazic, S.E., Grote, H., Armstrong, R.J.E., Blakemore, C., Hannan, A.J., van Dellen, A., Barker, R.A., 2004. Decreased hippocampal cell proliferation in R6/1 Huntington's mice. Neuroreport 15 (5), 811-813.

Lazic, S.E., Grote, H.E., Blakemore, C., Hannan, A.J., van Dellen, A., Phillips, W., Barker, R.A., 2006. Neurogenesis in the R6/1 transgenic mouse model of Huntington's disease: effects of environmental enrichment. Eur. J. Neurosci. 23 (7), 1829-1838.

Le Gras, S., Keime, C., Anthony, A., Lotz, C., De Longprez, L., Brouillet, E., Cassel, J.C., Boutillier, A.L., Merienne, K., 2017. Altered enhancer transcription underlies Huntington's disease striatal transcriptional signature. Sci. Rep. 7, 42875.

Lee, H., Fenster, R.J., Pineda, S.S., Gibbs, W.S., Mohammadi, S., Davila-Velderrain, J., Garcia, F.J., Therrien, M., Novis, H.S., Gao, F., et al., 2020. Cell type-specific transcriptomics reveals that mutant huntingtin leads to mitochondrial rna release and neuronal innate immune activation. Neuron 107 (5), 891-908.e898.

Lee, J.-M., Huang, Y., Orth, M., Gillis, T., Siciliano, J., Hong, E., Mysore, J.S., Lucente, D., Wheeler, V.C., Seong, I.S., et al., 2022. Genetic modifiers of Huntington disease differentially influence motor and cognitive domains. Am. J. Hum. Genet. 109 (5), 885-899.

Leek, J.T., 2014. svaseq: removing batch effects and other unwanted noise from sequencing data. Nucleic Acids Res. 42 (21), e161.

Liao, W.L., Tsai, H.C., Wu, C.Y., Liu, F.C., 2005. Differential expression of RARbeta isoforms in the mouse striatum during development: a gradient of RARbeta2 expression along the rostrocaudal axis. Dev. Dyn. 233 (2), 584-594.

Love, M.I., Huber, W., Anders, S., 2014. Moderated estimation of fold change and dispersion for RNA-seq data with DESeq2. Genome Biol. 15 (12), 550.

Luthi-Carter, R., Strand, A., Peters, N.L., Solano, S.M., Hollingsworth, Z.R., Menon, A.S., Frey, A.S., Spektor, B.S., Penney, E.B., Schilling, G., et al., 2000. Decreased expression of striatal signaling genes in a mouse model of Huntington's disease. Hum. Mol. Genet. 9 (9), 1259-1271.

Malaiya, S., Cortes-Gutierrez, M., Herb, B.R., Coffey, S.R., Legg, S.R.W., Cantle, J.P., Colantoniu, C., Carroll, J.B., Ament, S.A., 2021. Single-nucleus RNA-seq reveals dysregulation of striatal cell identity due to huntington's disease mutations. J. Neurosci. 41 (25), 5534-5552.

Mangiarini, L., Sathasivam, K., Seller, M., Cozens, B., Harper, A., Hetherington, C., Lawton, M., Trottier, Y., Lehrach, H., Davies, S.W., et al., 1996. Exon 1 of the HD gene with an expanded CAG repeat is sufficient to cause a progressive neurological phenotype in transgenic mice. Cell 87 (3), 493-506.

McCaffrey, P., Drager, U.C., 1994. High levels of a retinoic acid-generating dehydrogenase in the meso-telencephalic dopamine system. Proc. Natl. Acad. Sci. U S A 91 (16), 7772-7776.

Menalled, L.B., Kudwa, A.E., Miller, S., Fitzpatrick, J., Watson-Johnson, J., Keating, N., Ruiz, M., Mushlin, R., Alosio, W., McConnell, K., et al., 2012. Comprehensive behavioral and molecular characterization of a new knock-in mouse model of Huntington's disease: zQ175. PLoS One 7 (12), e49838.

Mootha, V.K., Lindgren, C.M., Eriksson, K.F., Subramanian, A., Sihag, S., Lehar, J., Puigserver, P., Carlsson, E., Ridderstråle, M., Laurila, E., et al., 2003. PGC-1alpha-responsive genes involved in oxidative phosphorylation are coordinately downregulated in human diabetes. Nat. Genet. 34 (2), 267-273.

Naver, B., Stub, C., Møller, M., Fenger, K., Hansen, A.K., Hasholt, L., Sørensen, S.A., 2003. Molecular and behavioral analysis of the r6/1 huntington's disease transgenic mouse. Neuroscience 122 (4), 1049-1057.

Niewiadomska-Cimicka, A., Krzyzosiak, A., Ye, T., Podlesny-drabiniok, A., Dembele, D., Dolle, P., Krezel, W., 2017. Genome-wide analysis of rabeta transcriptional targets in mouse striatum links retinoic acid signaling with huntington's disease and other neurodegenerative disorders. Mol. Neurobiol. 54 (5), 3859-3878.

Nithianantharajah, J., Barkus, C., Murphy, M., Hannan, A.J., 2008. Gene-environment interactions modulating cognitive function and molecular correlates of synaptic plasticity in Huntington's disease transgenic mice. Neurobiol. Dis. 29 (3), 490-504.

Pelegri, C., Duran-Vilaregut, J., del Valle, J., Crespo-Biel, N., Ferrer, I., Pallas, M., Camins, A., Vilaplana, J., 2008. Cell cycle activation in striatal neurons from Huntington's disease patients and rats treated with 3-nitropropionic acid. Int. J. Develop. Neurosci. 26 (7), 665-671.

Pouladi, M.A., Morton, A.J., Hayden, M.R., 2013. Choosing an animal model for the study of Huntington's disease. Nat. Rev. Neurosci. 14 (10), 708-721.

Rochette-Egly, C., Gaub, M.P., Lutz, Y., Ali, S., Scheuer, I., Chambon, P., 1992. Retinoic acid receptor-beta: immunodetection and phosphorylation on tyrosine residues. Mol. Endocrinol. 6 (12), 2197-2209.

Ross, C.A., Aylward, E.H., Wild, E.J., Langbehn, D.R., Long, J.D., Warner, J.H., Scahill, R. I., Leavitt, B.R., Stout, J.C., Paulsen, J.S., et al., 2014. Huntington disease: natural history, biomarkers and prospects for therapeutics. Nat. Rev. Neurol. 10 (4), 204-216.

Seredenina, T., Luthi-Carter, R., 2012. What have we learned from gene expression profiles in Huntington's disease? Neurobiol. Dis. 45 (1), 83-98.

Smith, G.A., Rocha, E.M., McLean, J.R., Hayes, M.A., Izen, S.C., Isaacson, O., Hallett, P.J., 2014. Progressive axonal transport and synaptic protein changes correlate with behavioral and neuropathological abnormalities in the heterozygous Q175 KI mouse model of Huntington's disease. Hum. Mol. Genet. 23 (17), 4510-4527.

Subramanian, A., Tamayo, P., Mootha, V.K., Mukherjee, S., Ebert, B.L., Gillette, M.A., Paulovich, A., Pomeroy, S.L., Golub, T.R., Lander, E.S., et al., 2005. Gene set
enrichment analysis: a knowledge-based approach for interpreting genome-wide expression profiles. Proc. Natl. Acad. Sci. USA 102 (43), 15545-15550.

Tsogtbaatar, E., Landin, C., Minter-Dykhouse, K., Folmes, C.D.L., 2020. Energy metabolism regulates stem cell pluripotency. Front. Cell Dev. Biol. 8, 87.

Vashishtha, M., Ng, C.W., Yildirim, F., Gipson, T.A., Kratter, I.H., Bodai, L., Song, W., Lau, A., Labadorf, A., Vogel-Ciernia, A., et al., 2013. Targeting H3K4 trimethylation in Huntington disease. Proc. Natl. Acad. Sci. U S A 110 (32), E3027-E3036.

Vonsattel, J.P., DiFiglia, M., 1998. Huntington disease. J. Neuropathol. Exp. Neurol. 57 (5), 369-384.

Yang, J.H., Hayano, M., Griffin, P.T., Amorim, J.A., Bonkowski, M.S., Apostolides, J.K., Salfati, E.L., Blanchette, M., Munding, E.M., Bhakta, M., et al., 2023a. Loss of epigenetic information as a cause of mammalian aging. Cell 186 (2), 305-326.e327.

Yang, N., Occean, J.R., Melters, D.P., Shi, C., Wang, L., Stransky, S., Doyle, M.E., Cui, C. Y., Delannoy, M., Fan, J., et al., 2023b. A hyper-quietescent chromatin state formed during aging is reversed by regeneration. Mol. Cell 83 (10), 1659-1676.e1611.