Article_M2

RESEARCH ARTICLE
Opposite Phenotypes of Muscle Strength and
Locomotor Function in Mouse Models of
Partial Trisomy and Monosomy 21 for the
Proximal Hspa13-App Region
Véronique Brault1,2,3,4
, Arnaud Duchon1,2,3,4
, Caroline Romestaing5
, Ignasi Sahun6
,
Stéphanie Pothion7
, Mona Karout1,2,3,4
, Christelle Borel8
, Doulaye Dembele1,2,3,4
, Jean-
Charles Bizot9
, Nadia Messaddeq1,2,3,4
, Andrew J. Sharp10
, Damien Roussel5
, Stylianos
E Antonarakis8,11
, Mara Dierssen6
, Yann Hérault1,2,3,4,12
*
1 Institut de Génétique et de Biologie Moléculaire et Cellulaire, Illkirch, France, 2 Centre National de la
Recherche Scientifique, UMR7104, Illkirch, France, 3 Institut National de la Santé et de la Recherche
Médicale, U964, Illkirch, France, 4 Université de Strasbourg, Illkirch, France, 5 LEHNA, CNRS UMR502,
Université de Lyon, Villeurbanne, France, 6 Genes and Disease Program, Center for Genomic Regulation,
Barcelona, Spain, and CIBER de Enfermedades Raras (CIBERER), Barcelona, Spain, 7 Transgenese et
Archivage Animaux Modèles, TAAM, CNRS, UPS44, Orléans, France, 8 Department of Genetic Medicine
and Development, University of Geneva Medical School, Geneva, Switzerland, 9 Key-Obs S.A., Allée du
Titane, Orléans, France, 10 Department of Genetics and Genomic Sciences, Mount Sinai School of
Medicine, New York, New York, United States of America, 11 iGE3 Institute of Genetics and Genomics of
Geneva, Geneva, Switzerland, 12 Institut Clinique de la Souris, PHENOMIN, GIE CERBM, Illkirch, France
* herault@igbmc.fr
Abstract
The trisomy of human chromosome 21 (Hsa21), which causes Down syndrome (DS), is the
most common viable human aneuploidy. In contrast to trisomy, the complete monosomy
(M21) of Hsa21 is lethal, and only partial monosomy or mosaic monosomy of Hsa21 is
seen. Both conditions lead to variable physiological abnormalities with constant intellectual
disability, locomotor deficits, and altered muscle tone. To search for dosage-sensitive
genes involved in DS and M21 phenotypes, we created two new mouse models: the
Ts3Yah carrying a tandem duplication and the Ms3Yah carrying a deletion of the Hspa13-
App interval syntenic with 21q11.2-q21.3. Here we report that the trisomy and the monoso-
my of this region alter locomotion, muscle strength, mass, and energetic balance. The
expression profiling of skeletal muscles revealed global changes in the regulation of genes
implicated in energetic metabolism, mitochondrial activity, and biogenesis. These genes
are downregulated in Ts3Yah mice and upregulated in Ms3Yah mice. The shift in skeletal
muscle metabolism correlates with a change in mitochondrial proliferation without an alter-
ation in the respiratory function. However, the reactive oxygen species (ROS) production
from mitochondrial complex I decreased in Ms3Yah mice, while the membrane permeability
of Ts3Yah mitochondria slightly increased. Thus, we demonstrated how the Hspa13-App in-
terval controls metabolic and mitochondrial phenotypes in muscles certainly as a conse-
quence of change in dose of Gabpa, Nrip1, and Atp5j. Our results indicate that the copy
PLOS Genetics | DOI:10.1371/journal.pgen.1005062 March 24, 2015 1 / 27
a11111
OPEN ACCESS
Citation: Brault V, Duchon A, Romestaing C, Sahun
I, Pothion S, Karout M, et al. (2015) Opposite
Phenotypes of Muscle Strength and Locomotor
Function in Mouse Models of Partial Trisomy and
Monosomy 21 for the Proximal Hspa13-App Region.
PLoS Genet 11(3): e1005062. doi:10.1371/journal.
pgen.1005062
Editor: Gregory S. Barsh, Stanford University School
of Medicine, United States
Received: July 12, 2014
Accepted: February 9, 2015
Published: March 24, 2015
Copyright: © 2015 Brault et al. This is an open
access article distributed under the terms of the
Creative Commons Attribution License, which permits
unrestricted use, distribution, and reproduction in any
medium, provided the original author and source are
credited.
Data Availability Statement: Data from the
expression study are under the GEO accession
number (GSE58463).
Funding: The project was supported by the French
National Centre for Scientific Research (CNRS), the
French National Institute of Health and Medical
Research (INSERM), the University of Strasbourg
and the “Centre Europeen de Recherche en
Biomedecine”, the “Fondation Jerome Lejeune”, and
the European commission (AnEUploidy project
LSHG-CT-2006-037627 to YH, MD and SEA). The

number variation in the Hspa13-App region has a peripheral impact on locomotor activity by
altering muscle function.
Author Summary
Down syndrome is the most common genetic cause of intellectual disabilities, and marked
hypotonia is among the constant diagnostic traits. Here we observe the opposite changes
in locomotion, muscle strength, and energetic balance in new mouse models of DS and
M21 for the Hspa13-App proximal region of human chromosome 21. The differential ex-
pression analysis revealed downregulation of skeletal muscle genes controlling energetic
metabolism, mitochondrial activity, and biogenesis in Ts3Yah, while upregulation of simi-
lar set of genes was found in Ms3Yah mice. This phenomenon correlates with the changes
in mitochondrial proliferation with increased membrane permeability of Ts3Yah mito-
chondria and decreased mitochondrial ROS production in Ms3Yah mice. Our results
demonstrate the opposite phenotypic effect of trisomy and monosomy of the Hspa13-App
syntenic region of human chromosome 21, highlighting new physiological mechanisms
for hypotonia in DS individuals.
Introduction
DS and M21 are complex genetic conditions that arise from an altered dosage of genes on
human chromosome 21 (Hsa21). Full M21 is rare and generally not compatible with life, with
only a few cases reported in literature, and the oldest case survived for only a few days after
birth [1–5]. The remaining M21 cases are partial deletion or mosaics. The phenotypes of M21
are variable among individuals and depend on the specific deleted region of Hsa21. Some pa-
tients have only mild to moderate intellectual disabilities [6, 7], while others are more severely
affected and present multiple dysmorphic craniofacial, skeletal, and cardiac features, as well as
muscular, ocular, pulmonary, renal, and genitourinary abnormalities [8–12].
DS, with an incidence of one in 700 live births [13], is still the most common viable cause of
chromosomal aneuploidy in humans and the most frequent genetic cause of intellectual dis-
ability. Besides the characteristic of facial dysmorphology, hypotonia is one of the major traits
observable at birth in this disorder. More than 80 features occur with various degrees of expres-
sion and frequency in DS, and some have been associated with regions of Hsa21 [12, 14]. How-
ever, the causes of delayed motor performance affecting both gross and fine motor skills, weak
muscle strength, and exercise performance are not known. Motor impairments are prominent
throughout life and may contribute to the delay in cognitive skills [15, 16]. Poor locomotor
skills have been attributed to impaired coordinated input due to cerebellar dysfunction [17, 18]
but might have a more complex origin combining deficit in balance and postural control, stiff-
ness, reduced muscular strength, and hypotonia.
Hypotonia is a state of low muscular tonicity due to decreased springlike properties of the
striated muscle fibres, which suffer from a lack of energy to keep working at a normal level for
a long time and a slower speed of response. Hypotonia may also be accompanied by reduced
muscular strength. It can be caused by abnormal muscle function, abnormal neurological
input, or metabolic diseases. Impaired exercise endurance is observed in DS individuals and
could arise from endocrinal or metabolic perturbations [19, 20]. However, little is known about
the origin of hypotonia in DS.
Copy Number Variation Modifying Muscle Function
laboratory of SEA was supported by grants from The
Swiss National Science Foundation (144082), the EU
AnEUploidy project (LSHG-CT-2006-037627), and
the ERC (249968). This study been supported by
French state funds through the “Agence Nationale de
la Recherche” under the frame programme
Investissements d’Avenir labelled ANR-10-IDEX-
0002-02, ANR-10-LABX-0030-INRT, ANR-10-INBS-
07 PHENOMIN. The funders had no role in study
design, data collection and analysis, decision to
publish, or preparation of the manuscript.
Competing Interests: The authors have declared
that no competing interests exist.

Mouse models provide a powerful tool to study the physiological, cellular, and molecular as-
pects of human diseases. Moreover, the possibility to manipulate large genomic regions in the
mouse genome has offered a chance to develop mouse models of aneuploidies and investigate
the relationship between phenotype and genotype. Hsa21 genes are found in three syntenic re-
gions localised on mouse chromosomes 16 (Mmu16, 23.3 Mb, 115 Hsa21 orthologous genes),
17 (Mmu17, 1.1 Mb, 19 Hsa21 orthologous genes), and 10 (Mmu10, 2.3 Mb, 41 Hsa21 ortholo-
gous genes) (http://www.ensembl.org).
Several mouse models have been generated over the years that display many features of DS
[21–25], but locomotor activity has not previously been fully investigated in these models.
There is little in literature about the locomotor phenotype of the trisomic models for genes lo-
cated on the Mmu16. Moreover, conflicting results have been reported on the motor abilities of
Ts65Dn mice that are trisomic for 102 orthologous of Hsa21 genes (http://www.ensembl.org)
located between Mprl39 and Zfp295 [26, 27]. Additionally, this model is trisomic for 50 genes
and 10 pseudogenes present in the centromeric part of Mmu17 that is irrelevant to DS [28].
Studies from Costa and collaborators reported lower maximal treadmill running speeds, defi-
cits in balance and motor coordination assessed by the rotarod test, and reduction in grip force
[29, 30], whereas Hyde and collaborators found no impairment in motor learning in Ts65Dn
mice [31, 32]. Several analyses have described abnormal electrical and biochemical properties
in motor neurons and muscle cells both in human and DS mouse models [33–35]. The analysis
of the distal part of the Hsa21 did not reveal locomotor deficit in a mouse model trisomic for
the Abcg1-U2af1 segment on Mmu17 [25] and demonstrated that the Cstb-Prmt2 segment is
also not necessary, as going back to two copies of this region in the Tc1 transchromosomic
model does not rescue the locomotor deficit observed in this model (although five genes in this
region were not tested here as they are not trisomic in Tc1) [36].
To assess the contribution of the DS phenotype associated with the 9.4 Mb Hspa13-App
Hsa21 syntenic region on Mmu16 that is not trisomic in the widely used Ts65Dn model, we
generated through chromosome engineering a trisomic mouse model (Ts3Yah) and the corre-
sponding monosomic model (Ms3Yah) for this region. Those mice were evaluated for different
behavioural phenotypes associated with DS and displayed opposite locomotor phenotypes.
While Ts3Yah mice have impaired locomotion that is associated with increased grip strength
and muscle mass, the opposite phenotype was observed in Ms3Yah animals. The transcription
profile analysis of skeletal muscles revealed coordinate changes in muscle metabolic activity
and mitochondrial biogenesis. This finding was confirmed by alteration in the oxidative capaci-
ty and mitochondrial content of the skeletal muscles. This study unravels new insight into the
metabolic changes that result in defects of skeletal muscle strength and performance that are
associated with Hsa21 aneuploidy and suggest new candidate genes involved in DS and M21.
Results
Generating models of aneuploidy for the Hspa13-App region on mouse
chromosome 16
Chromosomal engineering based on the MICER [37] and long-range Cre/loxP-mediated re-
combination [38] were used in embryonic stem (ES) cells to produce the segmental duplication
(Dp[16Hspa13-App]2Yah; MGI5519056) and the corresponding deletion (Del[16Hspa13-App]
3Yah; MGI5519049) of the Hspa13-App 9.4 Mb interval located on Mmu16, as described in
Materials and Methods and Fig. 1. This mouse syntenic region contains 15 Hsa21 orthologous
genes (Fig. 1A, S1 Table). As a result of the insertion points of the targeting vectors, the Hspa13
gene at the proximal end of the 9.5 Mb region was in two functional copies in the duplication
and lost in the deletion, whereas the App gene at the distal end was only in one functional copy

Fig 1. Generating a 9.2 Mb deletion and reciprocal tandem duplication between the Hspa13 and App loci. (A) The 9.2 Mb targeted region defined by
the Hspa13 and App genes as shown from a capture derived from the UCSC genome browser (http://genome.ucsc.edu/). Black headband: chromosomes
bands; blue headband: contigs; red rectangle boxes: protein coding genes. (B) The targeting vectors containing a loxP site (green arrow), a selectable
antibiotic resistance gene (puro or neo), and a part of the Hprt gene (3’ or 5’ Hprt, red arrows) were integrated successively in the Hspa13 locus
(Hspa13tm1Yah) and in the App gene (Apptm1Yah). The start and end positions of the genomic homologous regions present in the 5’ and 3’-Hprt vectors are

in the duplication and lost in the deletion. The derived aneuploid mouse models corresponding
to the tandem duplication and the reciprocal deletion were respectively named Dp(16)
(Hspa13-App)2Yah for the trisomy (abbreviated as Ts3Yah) and Del(16)(Hspa13-App)3Yah
for the monosomy (abbreviated as Ms3Yah). The two models were validated by the Southern
blot analysis (Fig. 1B), fluorescent in situ hybridisation (FISH) analysis (Fig. 1E), and array-
based comparative genomic hybridisation (Fig. 1F) as described in Materials and Methods.
Ts3Yah and Ms3Yah mice are viable and fertile, with normal life spans, and exhibit no obvious
gross anatomical anomalies on the C57BL/6J genetic background. Ts3Yah show no weight
difference (weight at 10 weeks, n = 12 male mice per genotype: control 26.4±0.9g vs Ts3Yah
26.5±0.7g, Student’s t-test p = 0.97). While their weight is not different at birth from their litter-
mates (weight taken at J2, n = 9 male mice per genotype: control 1.8g±0.1g vs Ms3Yah
1.9g±0.1 g, Student’s t-test p = 0.78), adult Ms3Yah mice weight approximately 10% less than
their wild-type littermates (weight at 10 weeks, n = 10 male mice per genotype: control
28.6±0.6 g vs Ms3Yah 24.6g±1.1g, Student’s t-test p = 0.005).
Locomotor performances are oppositely affected in mice trisomic and
monosomic for the Hspa13-App region
Motor function was assessed first using two rotarod motor performance tests: one with five-
minute sessions of incremental fixed speeds and the second consisting of two five-minute trials
with accelerating speed. Ts3Yah mice showed performances that were significantly lower than
their diploid littermates in both tests at age 8 to 12 months, and Ms3Yah mice performed sig-
nificantly lower than their diploid littermates at the same age as well (Fig. 2A). Looking at both
Ts3Yah and Ms3Yah mice, the weight of the animal was not correlated with its performance,
excluding that the increased performance in Ms3Yah animals was due to mice being leaner.
The effects of the trisomy and monosomy on rotarod test performance were however not ob-
served in the young mice aged 3 months (S1A Fig). In order to assess muscle strength, the max-
imal grip force was measured and normalised to the body weight of the animal. Muscle
strength was increased in Ts3Yah and decreased in Ms3Yah mice compared to diploid litter-
mates at 3 to 4 months and 8 to 12 months of age (Fig. 2B, at 8 to 12 months of age). Muscle en-
durance capacity was tested by having the mice run on a treadmill and measuring the maximal
distance travelled. Ts3Yah mice were only able to run about half of the distance covered by dip-
loid control mice, indicating that they were more susceptible to fatigue (Fig. 2C), whereas
Ms3Yah mice showed performances comparable to diploid controls (S1B Fig). Spontaneous lo-
comotor activity and habituation were measured in an open field by recording the distance
from mouse genome assembly GRm38. The Stchtm1Yah allele was checked by Southern blot analysis. The digestion of DNA with SacI (S) restriction
enzyme and hybridisation with external probes A and B gives additional fragments of 19.7 kb and 14.3 kb, respectively, for the Stchtm1Yah allele compared
to the wild-type (wt) allele of 17.2 kb. The hybridisation of the same DNA digest with the Gap1 probe gives two additional fragments of 19.7 and 14.3 kb for the
Hspa13tm1Yah. (C) The integration of the 3’Hprt vector was confirmed in the same manner by digestion with the PsiI (P) enzyme and hybridisation with
probes C (wt, 11.5 kb; Apptm1Yah allele, 10.9 kb), D (wt, 11.5 kb; Apptm1Yah allele, 9.9 kb), and Gap2 (wt, 11.5 kb; Apptm1Yah allele, 10.9 and 9.9 kb). (D)
After Cre-mediated recombination, the deletion (Ms3Yah allele) and duplication (Ts3Yah allele) were identified by NdeI (N) digestion, and a probe was made
on the ampicillin gene (Ampi) present in both targeting vectors that recognises recombinant clones with the Ms3Yah (23.2 kb) and Ts3Yah (18.2 kb) alleles
from nonrecombinant ones that still carry the Hspa13tm1Yah (26 kb) and Apptm1Yah (15.4 kb) alleles. Puro: puromycin, neo: neomycin. (E) Interphase FISH
analysis with BAC probes that map in the region of the deletion or duplication (BAC bMQ-34K13; red) and outside (BAC bMQ-381L17; green). The wild-type
(diploid) showed two red and two green adjacent signals. On the other hand, nuclei from Ms3Yah showed two green and only one red signal due to the
deletion of the Hspa13-App region, and nuclei from Ts3Yah showed two green and three red signals. (F) The CGH profile of Mmu16 was established using
NimbleGen mouse HD2 whole genome CGH oligonucleotide arrays comprising 2,100,000 isothermal probes 50–75 bp length, with a median spacing of 1.1
kb throughout the genome (UCSC NCBI37/mm9/July 2007 assembly) enabling high-resolution CNV detection. It confirmed the loss of one copy of the
Hspa13-App fragment in Ms3Yah mice and the presence of an additional copy of the same fragment in Ts3Yah animals. Plotted are log 2 transformed
hybridisation ratios of Ms3Yah and Ts3Yah versus diploid mouse DNA.
doi:10.1371/journal.pgen.1005062.g001

travelled by the mouse during the first 15 minutes and the last 15 minutes of a 30 minute ses-
sion. No difference was found in Ts3Yah mice (S1C Fig), while Ms3Yah mice had a slight over-
all increase in the distance travelled and did not reduce their exploratory activity as much as
the controls animals during the last fifteen minutes of the session, suggesting a slight hyperac-
tivity (Fig. 2D). The mice were tested for hind limb coordination by walking across a notched
bar. No significant difference was observed for the percentage of errors in hind limb coordina-
tion in Ts3Yah mice (S1D Fig), while Ms3Yah mice made significantly less errors than their
control littermates (Fig. 2E).
Copy number variation of the Hspa13-App region triggers opposite
expression changes of pathways involved in mitochondrial function and
biogenesis in the muscle
Muscle strength and locomotion variation suggest that the observed motor phenotypes at least
partly result from a peripheral muscular phenotype. Hence, we decided to focus our attention
on the skeletal muscles. To identify molecular changes in the skeletal muscles of Ts3Yah and
Ms3Yah mice, we studied the transcriptional profiles of gastrocnemius muscles isolated from
Fig 2. Evaluation of motor skills. (A) The motor skill performances of Ts3Yah mice, Ms3Yah mice, and their diploid controls (n = 10–15 per genotype) were
analysed using two rotarod tests. The first test consisted of four consecutive trials of 5 minutes at constant speed and reported as the mean ± sem of the time
that mice remained on the rod (repeated ANOVA ‘genotype,’ ‘speed’: Ts3Yah vs diploid: F[1, 69] = 10.042 p = 0.004; Ms3Yah vs diploid: F[1, 87] = 8.809
p = 0.006). This test was followed by another test involving two trials at accelerating speed (4–40 rpm) for 5 minutes and the calculated mean rotational
velocity at the time of falling for the two trials and each group. (B) Grip force, immediately prior to the animal releasing its grasp from the grid, was measured in
grams and normalised to the weight of the animal. (C) The endurance of Ts3Yah and Ms3Yah mice to exercise was measured as the maximal running
distance travelled on a treadmill until exhaustion. (D) Spontaneous locomotor activity and habituation were assessed in an open field by measuring the
distance travelled during the first 15 minutes and the last 15 minutes of a 30-minute session. (E) Motor coordination skills were tested with a notched bar by
looking at the percentage of errors made by the mouse with its hind paws when crossing the notched bar. Data are presented as mean ± sem and analysed
with Student’s t-test, *p<0.05, **p<0.01, ***p<0.001.

adult mice. From the 45,281 probe sets sampled on the microarray, 43,058 and 43,632 were ex-
pressed in the Ts3Yah and the Ms3Yah, respectively. There were 2,302 (corresponding to 1,614
genes) and 2,246 (corresponding to 1,631 genes) probes differentially expressed in Ts3Yah and
Ms3Yah muscles, respectively, compared to their euploid littermate controls, with a significant
p-value <0.05 and resampling p-values of 0.081 (Ts3Yah) and 0.073 (Ms3Yah) (see Materials
and Methods for details of the methods used). About half of them (1,129 and 1,169, respective-
ly) were upregulated and half of them (1,173 and 1,077, respectively) downregulated. Fold
changes (FC) observed varied between 0.5 and 2.1 for both Ts3Yah and Ms3Yah, with only
about 5% of the deregulated genes having a FC>1.2 or <0.8 (Fig. 3A), indicating that the triso-
my and the monosomy of the Hspa13-App region do not have a dramatic effect on gene expres-
sion. The final lists of the most deregulated probes (FC>|1.2|) for Ts3Yah and Ms3Yah are
Fig 3. Gene expression profile of skeletal muscles from Ts3Yah and Ms3Yah transgenic mice. (A) Distribution of the deregulated genes depending on
their fold change. (B) Clustering derived from statistically deregulated genes (Student’s t-test, p<0.05) with FC >|1.2| in Ts3Yah compared to their diploid
control littermates and in Ms3Yah compared to their diploid control littermates. The red and green colours indicate relative expression levels (red: increased
expression; green: decreased expression). (C) Quantitative PCR analysis of genes within the Hspa13-App trisomic or monosomic region (aneuploid),
bordering the Hspa13-App region and selected for their significant deregulation within the transcriptome. Data are presented as mean ± sem and analysed
with Student’s t-test, ~p<0.1, *p<0.05, **p<0.01.

presented along with their annotation, FC, and p-value in Supplementary Tables (S2–S3 Ta-
bles), respectively, and the clustering of those deregulated genes are presented in Fig. 3B. Three
genes from the Hspa13-App region (Chodl, Atp5j, and D16Ertd472e [C21orf91 in human])
were among the most deregulated in Ts3Yah muscles. In addition, several protein kinases (Ttn,
Nmrk2, Csnk2a1-rs3, Pctk1, Pacsin3, Prkaa2 Raf1, Cdc2l2, Smg1, Hk2, Ckmt2, Dapk2, Mpp3,
and Atp1b1), three members of translation initiation factors (Eif2c2, Eif4e3, and Eif2b5), and a
binding protein of a translation initiation factor (Eif4ebp2) were overexpressed, and a few mito-
chondrial-located proteins and enzymes were underexpressed (Atp5a1, Ckmt2, Mgst1, Stard7,
Abhd10, Cyb5b, Pdpr, Hk2, Tmem65, Tomm22, Hsd3b2, and Tmem160). In Ms3Yah muscles,
only 26 genes were downregulated at FC<0.8, and six of them (App, Atp5j, Chodl,
D16Ertd472e, Usp25, and Jam2) corresponded to genes mapping within the Hspa13-App
monosomic segment. Interestingly, among the 84 genes upregulated in Ms3Yah muscles, 29
were mitochondrial related (Hadhb, Alas1, Ndufa8, Brp17, Aifm1, Acadl, Gbas, Mfn2, Acaa2,
As3mt, Mrpl47, Adh1, Tmem143, Akr1b10, Slc25a11, Pptc7, Cyc1, Uqcc, LOC433224, Slc25a20,
Mpc2, Rmnd1, Coq5, Slc25a3, Slc25a4, Idh3g, Hmgcl, Akr1b10, Coq6, Ppif, and Ndufaf4). By
comparing the lists of the most deregulated probes in Ts3Yah and Ms3Yah muscles, we found
eight different genes displaying opposite deregulation in the two conditions: Chodl, Atp5j, and
D16Ertd472e in the Hspa13-App region and Wfs1, Ffar3, mt-Nd4l, Gpihbp1, and Hist1h4h lo-
cated elsewhere (see genes in bold in S2–S3 Tables).
We selected the genes located in the Hspa13-App interval and at its borders for the analysis
of their expression profile by quantitative PCR analysis (QRT-PCR) (Fig. 3C). Only three
genes within the Hspa13-App interval, namely, Ncam2, Samsn1, and Prss7 (also known as
Tmprss15), were not detected by QRT-PCR analysis in the striated muscle as reported in the lit-
erature. Nevertheless, most of the genes present within this interval and were not detected by
the microarray experiment were found expressed in skeletal muscles by QRT-PCR analysis.
The genes present in three copies in Ts3Yah muscles were upregulated at around 1.5 fold, at
the exception of Cxadr and Chodl. As expected, the expression of App that is not duplicated in
the Ts3Yah chromosome was unchanged in Ts3Yah muscle. In Ms3Yah animals, all the mono-
somic genes were globally 0.5 fold underexpressed compared to their disomic controls. In addi-
tion, we measured the expression levels of five genes at the border of the rearrangement,
namely, Adamts1, Adamts5, Usp16, Cct8, and Bach1 (Fig. 3C). No significant change of expres-
sion was observed for border genes present in two copies in the genome of Ts3Yah and
Ms3Yah mice. Finally, genes found differentially expressed in Ts3Yah and Ms3Yah were tested
by QRT-PCR. Most of the genes were validated and found to be significantly deregulated in
Ms3Yah muscle as in the microarray analysis, although deregulation was only observed for
Wnt4 in Ts3Yah animals (Fig. 3C).
We further analysed our microarray data with the software tool ‘Gene Set Enrichment Anal-
ysis’ (GSEA; http://www.broad.mit.edu/gsea) [39, 40]. This analysis helps find correlation of
gene expression. The genes belonging to a defined pathway are ranked together according to
their change in expression in Ts3Yah and Ms3Yah muscles compared to their diploid controls
and calculating a maximum enrichment score (ES) for each gene set. More than 20 informative
gene sets were significantly downregulated in Ts3Yah and upregulated in Ms3Yah muscles
compared to diploid muscles (S4–S5 Tables), whereas no category was found for upregulated
in Ts3Yah or downregulated in Ms3Yah muscles. Most of the deregulated gene sets were asso-
ciated with mitochondrial energy metabolism (electron transport chain and oxidative phos-
phorylation or ‘VOXPHOS’), fatty acid metabolism (fatty acid degradation), and carbohydrate
metabolism (TCA cycle, glycolysis and gluconeogenesis, and butanoate metabolism). About
half of the genes assigned to mitochondrial gene sets overlapped with those categories. The
complementary GSEA ([40]) revealed a major overlap in the leading edge subset of genes

(genes in the gene set that contribute most to the ES) between Ts3Yah and Ms3Yah enriched
gene sets (see S4–S5 Tables for leading edge genes in each gene set) (see among the top-ranked
genes sets, one called ‘PGC,’ for peroxisome proliferator-activated receptor-gamma coactiva-
tor) and corresponding to a set of genes involved in oxidative phosphorylation and whose ex-
pression is coordinately decreased in human diabetic muscles and activated upon expression of
Pgc-1α (Ppargc-1α) in C2C12 cells [41]. Leading edge genes from this set show a 25% to 50%
overlap with leading edge genes from genes sets implicated in energy and metabolic pathways
identified in our analysis. Fig. 4 shows GSEA-scoring plots and their corresponding heat maps
for ‘PGC’ and the four gene sets, including ‘electron transport chain,’ ‘TCA cycle,’ ‘fatty acid
degradation,’ and ‘glycolysis/gluconeogenesis,’ that show the most overlap with gene set ‘PGC.’
Fig 4. Pathways related to energy metabolism are underexpressed in Ts3Yah skeletal muscles and overexpressed in Ms3Yah skeletal muscles.
GSEA-scoring plots of gene expression profile in gastrocnemius muscles from Ts3Yah (A) and Ms3Yah (B) mice. Four plots of representative gene sets
relevant to metabolic pathways that were significantly enriched and the ‘PGC’ gene set corresponding to genes involved in OXPHOS that were activated by
PGC1-α are shown. The top part of each plot shows the running enrichment score for the gene set as the analysis goes down the ranked list. The middle
portion shows the position of members of the gene set in the ranked list of genes. The lower portion plots the value of the ranking metric of the genes in the
expression data set. Below the enrichment plots, the corresponding heat maps show the leading edge subsets of genes that contribute most to the
enrichment score. The red and blue colours indicate relative expression levels (red: increased expression; blue: decreased expression).

Together, these findings suggest a shift in energy metabolism to a more oxidative state with in-
creased mitochondrial biogenesis and/or activity in Ms3Yah skeletal muscles and an opposite
suppression of the oxidative state in Ts3Yah muscles that might be triggered by signaling path-
way(s) involving transcriptional coactivators of the PGC family or other cooperating partners
involved in this regulatory pathway.
The Hspa13-App region is implicated in the regulation of skeletal muscle
oxidative capacity
Changes in locomotor capacity, muscle strength, and deregulation of the transcriptional con-
trol of energy metabolism led us to explore the muscle properties in Ts3Yah and Ms3Yah ani-
mals. We found an increased muscle mass in Ts3Yah animals and decreased muscle mass in
Ms3Yah animals (Fig. 5A) that could account for the change in muscle strength observed in
the animals. Gastrocnemius muscles are composed predominantly of type II glycolytic fibres
and are poor in oxidative fast-twitch fibres that contain high amounts of mitochondria. We es-
timated the mitochondrial content of the gastrocnemius muscle by quantifying mtDNA and
comparing the copy number of mtCOX2 normalised by nuclear DNA in transgenic animals
versus their diploid control littermates. We observed a significant increase in mtDNA content
in Ms3Yah muscles and close to significant decrease in Ts3Yah muscles (Student’s t-test
Ts3Yah vs diploid, p = 0.06; Fig. 5B).
Mitochondrial activity was assessed by staining histological muscles sections for the pres-
ence of the mitochondrial enzyme succinate dehydrogenase (SDH). In tibialis anterioris (TA),
a skeletal muscle rich in glycolytic fibres, the SDH-stained fibres versus nonstained fibres were
counted. Ms3Yah muscles revealed significant increase in SDH-stained fibres compared to dip-
loid and Ts3Yah muscles (Fig. 5C, top), but no change in the percentage of slow-twitch oxida-
tive type I myofibers using MyHCI staining was observed (Fig. 5D, top). In the soleus, mainly
composed of oxidative fibres, differences between highly oxidative and less oxidative fibres
were determined by scoring darkly SDH-stained fibres from lightly stained fibres. A significant
increase in the number of oxidative fibres was found in Ms3Yah compared to diploid and
Ts3Yah muscles (Fig. 5C, bottom). An increased proportion of type I fibres was found in
Ms3Yah compared to both diploid and Ts3Yah muscles. A decreased proportion of type I myo-
fibers was also found in Ts3Yah soleus compared to diploid (Fig. 5D, bottom). Hence, we
found that monosomy increased the oxidative state of the muscle while trisomy decreased it.
We analysed the TA in electron microscopy. In skeletal muscle fibres, mitochondria are usually
localised between myofibrils, aligned parallel to the myofibrils, and in pairs at the Z-disc of the
sarcomere and packed strands underneath the sarcolemma. In Ts3Yah TA, some fibres (esti-
mated <10%) were found having few small-sized mitochondria compared to normal fibres
(Fig. 5E). In contrast, we could find areas with increased mitochondrial mass present between
adjacent fibres and containing few degenerative mitochondria in Ms3Yah muscles (Fig. 5E).
Overall, these results indicate increased muscle oxidative capacity in Ms3Yah muscles and de-
creased oxidative capacity in Ts3Yah muscles due to change in mitochondrial content.
Analysis of mitochondrial function in skeletal muscles
To determine if mitochondrial function is altered in the skeletal muscles of Ts3Yah and
Ms3Yah mice, we assessed mitochondrial functional capacity in isolated equal amounts of mi-
tochondrial preparations from hind limb muscles. We measured mitochondrial respiration
using pyruvate/malate (PM), whose breakdown products generate NADH and lead to the re-
duction of mitochondrial complex I and the citric acid intermediate succinate (S), which do-
nates electrons to FAD-containing complex II of the electron transport chain. While Ts3Yah

mitochondrial respiration rates during state 3 respiration with ADP was comparable to the re-
spiratory rates of diploid mitochondria (S2A Fig), they were significantly higher compared to
controls in both states 2 (basal respiration in the presence of substrate alone) (ANOVA ‘geno-
type,’ ‘substrate’; F[1, 13] = 3.674, p = 0.078) (Fig. 6A) and 4o (respiration in presence of oligo-
mycin, an inhibitor of ATPase) (ANOVA ‘genotype,’ ‘substrate’; F[1, 13] = 56.681, p = 0.026)
(Fig. 6B). The fact that the respiratory flux is increased in both states 2 and 4o in Ts3Yah mito-
chondria suggests an increase in the membrane permeability. The respiratory control ratio,
Fig 5. Histological analyses of skeletal muscles. (A) Mass of gastrocnemius muscles reported to body weight of the mice (n = 10–15 per group). (B)
Quantification of the mitochondrial copy number in the gastrocnemius muscles of Ts3Yah and Ms3Yah mice relative to their diploid controls. The levels of
mtDNA were normalised to levels of nucleus-encoded DNA (n = 5 per group). (C) Transverse sections of TA and soleus muscle were stained for SDH activity
and SDH+ fibres counted in the entire sections. Results are given as percentages of stained fibres (n = 5–7 per group). (D) Changes in the proportion of type I
slow-twitch fibres in TA and soleus of Ts3Yah and Ms3Yah mice using MyHC immunohistological staining (n = 5–7 per group). Data are presented as
mean ± sem. Student’s t-test (A and B) and ANOVA with all pairwise multiple comparison procedures (Holm-Sidak method) (C and D), ~p<0.1, *p<0.05,
**p<0.01, ***p<0.001. (E) Transmission electron microscope (TEM) analysis of TA muscles. The arrows are pointing at degenerative mitochondria. The
cropped image is shown with 2.5x magnification of the original image. M: mitochondrion. Scale bars are 2 μm for the two first panels and 6 μm for the last.

defined as the respiration in state 3 (maximal ADP-stimulated respiration) divided by that in
state 4o, remained unchanged, showing intact mitochondria (S2B Fig). The activity of cyto-
chrome oxidase (COX), the terminal enzyme in the mitochondrial electron transport chain,
was investigated by isolating its respiratory activity using antimycin and was not changed in
Ts3Yah mitochondria (S2C Fig). To further explore proton leak in Ts3Yah mitochondria, we
used the batch of mitochondria subjected to respiration with succinate and measured the Δψm
in parallel with respiration in the absence of net ATP synthesis (presence of oligomycin) to cal-
culate the basal proton conductance of the membrane [42]. A voltage plot for the conductance
over a range of potentials was generated by starting in state 4o and progressively limiting elec-
tron transport by titrating malonate and simultaneously determining respiration and Δψm. The
curve of the relationship between oxygen consumption and membrane potential in Ts3Yah mi-
tochondria was shifted to the left compared to diploid controls indicating that the rate of oxy-
gen consumed to counteract proton leak was slightly increased (Fig. 6C). In addition, reporting
the kinetic response of substrate oxidation activity to mitochondrial potential during states 2,
3, and 4o revealed increased respiratory activity in Ts3Yah compared to diploid controls as
seen by the shift of the slope to the right (Fig. 6D). Finally, in non-phosphorylating conditions
(state 4o), the mitochondrial potential in Ts3Yah mitochondria (186.7±1.9 mV) was the same
as in diploid controls (185.0±2.2 mV), indicating that the higher rate of oxygen consumption
Fig 6. Evaluation of mitochondrial function. Mitochondria were isolated from hind limb muscle to test their respiration capacities. (A) Yield of mitochondrial
protein per gram of muscle obtained from the mitochondrial extraction. (B–D) The rates of oxygen consumptions were measured in isolated muscle
mitochondria in the presence of pyruvate/malate (PM) or succinate (S) as described in Materials and Methods. (B) State 2, basal was measured in the
presence of substrates only. (C) State 3, ADP-stimulated respiration was measured with the addition of 500 μM ADP. (D) State 4o, after addition of
oligomycin. (E) COX activity was given by measuring mitochondrial oxygen consumption during PM respiration with the addition of 10 mM antimycin, 2 mM
ascorbate, and 0.5 mM TMPD. (F) Basal proton leak was visualised by plotting the rate of oxygen consumed against mitochondrial membrane potential.
(G) Kinetic response of substrate oxidation activity to membrane potential at states 3, 2, and 4o in Ts3Yah and diploid isolated mitochondria. Data are
presented as mean ± sem (n = 5–6 per genotype) with Student’s t-test (A and G) and two-way ANOVA with all pairwise multiple comparison procedures
(Holm-Sidak method) (D), *p<0.05, **p<0.01.

observed in Ts3Yah was not due to an increase in oxidation of succinate. Together, those re-
sults suggest a slight change in the inner membrane proton conductance in Ts3Yah mitochon-
dria. The mitochondrial respiration and COX activity in Ms3Yah mitochondria were similar to
that of diploid controls (S2D–S2G Fig). However, the mitochondrial protein yield calculated
from the reported amount of mitochondria extracted to the weight of muscle used for the ex-
traction was significantly increased (37%; Student’s t-test, p = 0.024; Fig. 6E), suggesting that
the increased oxidative capacity observed in Ms3Yah muscle is due solely to increased mito-
chondrial proliferation and not to increased mitochondrial respiration.
The kinetic analysis between oxygen consumption and membrane potential in Ms3Yah mi-
tochondria was similar to that of diploid controls indicating a similar basal proton leak, as ex-
pected by the absolute respiratory rates observed in Ms3Yah mitochondria. However, although
the basal proton motive force values were not significantly different between genotypes
(Ms3Yah: 182 ± 3 mV; diploid control: 191±4 mV), the chemical component of the proton mo-
tive force ΔpH was significantly lower (~30%; t-test, p = 0.001) in Ms3Yah mice (14±1 mV)
than in diploid controls (21±1 mV) (Fig. 6F). We also observed a 26% decrease in Ms3Yah mi-
tochondrial ROS production (Fig. 6G, decrease in H2O2 release as a marker of ROS produc-
tion) under basal respiration with S, which corresponds to the respiratory state that produces
most ROS (ANOVA ‘genotype,’ ‘substrate’; F[1,31] = 6.068, p = 0.02, and post hoc tests with all
pairwise multiple comparison procedure using Holm-Sidak method: Ms3Yah vs diploid with
succinate as substrate p = 0.001). ROS production from S comes from a reverse electron flux
from complex II to complex I (NAD) that produces superoxide at a very high rate and is highly
dependent on the pH gradient (ΔpH) across the mitochondrial inner membrane [43]. Overall,
our analysis revealed that there is a propensity of increased membrane permeability in Ts3Yah
mitochondria and of decreased ROS production by Ms3Yah mitochondria that is due to a de-
crease of pH across the mitochondrial inner membrane.
Discussion
The phenotypic characterisation of two new mouse models trisomic and monosomic for the
9.4 Mb Mmu16 Hspa13-App region syntenic to the centromeric 21q11.2-q11.3 region revealed
the contribution of gene dosage to locomotion, muscle strength, and mass and energy balance.
The locomotor deficits observed in DS were attributed to impaired coordination input due to
cerebellar or more general central dysfunction. Structural anomalies have been observed in the
cerebellum of DS patients [44–46], but the presence of other motor-related phenotypes such as
increased time for feedback adjustments in grip force, higher static grip forces, and difficulties to
maintain constant muscle power suggests a more complex origin [47, 48]. Studies on motor con-
trol have described specific preferential muscle coactivation patterns in people who have DS dur-
ing voluntary movements or postural adjustments. This coactivation of the agonist-antagonist
muscle pair might produce less resistance to motion and might contribute to hypotonia [49, 50].
The locomotor deficit in Ts3Yah is accompanied by increased grip strength but decreased
muscle endurance. Functional characterisation of the soleus muscle of Ts65Dn mice revealed
that the muscle weakness observed in vivo was not the result of a deficit in force-generating ca-
pacity of the muscle in the basal state but was rather due to post-fatigue muscle weakness [51].
Hence, hypotonia and motor deficits observed in DS might be associated more with the fatiga-
bility of the muscle due to a lower energetic state. Indeed, hypotonia corresponds to a state of
low muscular tonicity as the consequence of lack of energy to keep working at a normal level
for a long time (and of a slower speed of response [52, 53]. Lower exercise endurance capacity
with diminished basal metabolism and aerobic capacity has been observed in DS persons
[54–56]. Accordingly, walking anomalies observed in DS children are associated with

difficulties to maintain constant muscle power [57]. Interestingly, our Ts3Yah mouse model
shows locomotor deficits as a consequence of energy decrease and lack of endurance.
The contribution of skeletal muscles to DS locomotor phenotypes and their physiological
and biochemical characterisation have been poorly explored. A recent analysis of the soleus
muscle in Ts65Dn mice failed to highlight a clear change in mitochondrial content or in muscle
fibre type composition in the muscle although it identified alterations in pathways involved in
glucose and fat metabolism as well as ATP biosynthesis [51]. The examination of hormonal
and metabolic responses after a maximal effort in a treadmill test in DS and age-matched con-
trol people revealed that exercise limitation in DS people was caused by limited aerobic capaci-
ty [56]. This limitation was attributed to the disruption of the hormonal response (cortisol and
catecholamines) leading to an incapacity to control glycolysis, neoglucogenesis, and lipolysis
when muscle exercise is pursued [56]. Altered mitochondrial activity and increased oxidative
stress would play a similar role in muscular function as found in the central nervous system,
with DS accelerated aging and neurodegeneration [58–61].
Ms3Yah mitochondria revealed a decreased ROS production under respiration with succi-
nate that could be linked to a lower pH gradient (ΔpH) of the mitochondrial inner membrane,
whereas no change was observed for the membrane electrical potential (Δψ). ROS production
with succinate is mainly generated by reverse electron flux from complex II to complex I
[62–65] and is at least threefold more sensitive to the pH gradient (ΔpH) across the mitochon-
drial inner membrane than to the membrane potential (Δψ) [43]. PH gradient can be generated
by proton pumping during substrate oxidation or by the ATP synthase pumping protons into
the mitochondrial matrix during ATP synthesis. Interestingly, Atp5J, which encodes the F6
subunit of the mitochondrial ATP synthase [66], is found in the Hspa13-App region and shows
opposite deregulation in Ts3Yah and Ms3Yah mice. Change in the dosage of this subunit
might lead to a change in ATP synthase efficiency and thus to a change in pH gradient across
the mitochondrial inner membrane. Hence, transgenic mice overexpressing Atp5J have sus-
tained a decrease in intracellular pH in tissues leading to acidosis [67]. A deficit of mitochon-
drial ATP production due to alteration of the catalytic OXPHOS capacity and associated to a
compensatory enhancement of glycolysis and mitochondrial mass was seen in fibroblasts from
DS subjects [68]. Increased respiration during non-phosphorylating conditions, an indicator of
nonoptimal OXPHOS efficiency, was also observed in the same study and suggests a greater
proton leak across the mitochondrial inner membrane of DS fibroblasts. Interestingly, as seen
by Valenti and collaborators [68], we did not observe major changes in mitochondrial respira-
tory capacity in Ts3Yah and Ms3Yah mitochondria, but Ts3Yah mitochondria showed a slight
increase in respiration during non-phosphorylating conditions and a shift in the relationship
between oxygen consumption and mitochondrial membrane potential indicative of a greater
proton leak. This finding, together with the presence in the Hspa13-App region of the gene cod-
ing for a subunit of the ATP synthase, suggest that ATP synthesis might be affected in Ts3Yah
mitochondria. It would be interesting to further measure the ATPase activity as well as the
ratio of ATP synthesis over oxygen consumption in Ts3Yah mitochondria.
The transcriptome analysis of Ts3Yah and Ms3Yah skeletal muscles identifies the deregula-
tion of genes involved in energy metabolic pathways. These results are consistent with previous
findings in the hearts of DS fetuses and in DS amniocytes [69, 70]. Moreover, our analysis sug-
gests that Pgc-1α or members of the Pgc-1α pathway are important to the molecular changes
observed in Ts3Yah and Ms3Yah muscles. Consistent with this hypothesis, we increased ex-
pression of Pgc-1α in the transcriptome of Ms3Yah muscles (FC = 1.25, p = 0.0027) and in-
creased expression in Ms3Yah (FC = 1.18, p = 0.03) and decrease expression in Ts3Yah
(FC = 0.81, p = 0.01) muscles of ERRα (ESRRA), encoding the orphan nuclear receptor ERRα
(NR3B1) that is known to have co-expression with Pgc-1α in tissues with high energy demand

and whose transcriptional activity has been extensively linked to members of the PGC-1 family
of coactivators [71]. Pgc-1α is a transcriptional coactivator of steroid and nuclear receptors and
was found to play a central role as a modulator of oxidative metabolism in response to external
physiological stimuli by activating the expression of a wide array of genes involved in fuel in-
take, gluconeogenesis, and fatty acid oxidation [72]. Most importantly, Pgc-1α controls mito-
chondrial biogenesis through the regulation of the synthesis of mitochondrial proteins
involved not only in oxidative phosphorylation [73, 74] but also in the mitochondrial ribosom-
al machinery, protein transport, and dynamic [75]. Interestingly, the gene encoding the mito-
chondrial enzyme citrate synthase (Cs) needed for pyruvate-derived acetyl-CoA entry to the
TCA cycle and whose activity is a marker of increased mitochondrial biogenesis is found in the
leading edge genes of the mitochondrial gene set identified in the GSEA analysis. In addition,
genes coding for proteins involved in the transcription and translation of mitochondrial genes
(Tfam, Tufm, Tfb1m, Tfb2m, Lars2, Wars2, Mtif2, and Mtrf1) and for proteins participating in
mitochondrial protein import and translocation across the outer and inner mitochondrial
membrane (Mipep,Surf1, Tomm70a, Timm17b, Timm9, Timm44, and Tomm22) were found in
mitochondrial leading edge genes in either Ts3Yah or Ms3Yah or both (see leading edge gene
set ‘HUMAN_MITODB_6_2002,’ S4–S5 Tables). Hence, molecular changes observed in
Ts3Yah and Ms3Yah muscles point at gene(s) within the Hspa13-App region affecting the
Pgc1a coactivator network regulating energy metabolism and mitochondrial biogenesis.
Eight of the 15 genes conserved between human and mouse in the Hspa13-App region,
namely, Hspa13, Nrip1, Usp25, Btg3, D16Ertd472e, Mrpl39, Jam2, Atp5J, and Gabpa, were glob-
ally 0.5 fold downregulated in Ms3Yah muscles and 1.5 fold upregulated in Ts3Yah muscles
following gene dosage. Among those genes, two (Gabpa and Nrip1) are strong candidates for
the muscular phenotypes as they are known to be part of the network of proteins involved in
the transcriptional control of cellular energy homeostasis. Gabpa (GA-binding protein subunit
α) encodes the ETS DNA-binding subunit of the NRF2 (nuclear respiratory factor) transcrip-
tion factor that was found to interact with PGC-1α and ERRα to drive the expression of mito-
chondrial genes [76, 77]. Genetic disruption and knockdown of mouse Gabpa caused early
embryonic lethality [78, 79] with mitochondrial dysfunction and have been proposed as a pos-
sible explanation for embryonic loss. Yang and collaborators (2014) recently deleted Gabpa in
cultured primary mouse fibroblasts and found out that this resulted in a reduction of mito-
chondrial mass, ATP production, and mitochondrial methyltransferase Tfb1m, which is essen-
tial for mitochondrial protein translation and mitochondrial biogenesis [80]. However, the
Ms3Yah mice with only one copy of Gabpa have increased mitochondrial biogenesis, and the
Gabpα heterozygous knockout mice display no phenotype and express similar levels of GABPα
protein as the wild-type mice in their muscles [79]. Moreover, no increase in protein expression
of GABPα could be seen in human DS fibroblasts [81].
The receptor-interacting protein (RIP140 or NRIP1) identified as a corepressor of nuclear
receptors [82–84] was found to affect oxidative metabolism and mitochondrial biogenesis by
negatively controlling mitochondrial pathways regulated by PGC-1α [85]. Nrip1 null mice dis-
played an increase in the number of oxidative fibres as measured by SDH and MyHC isoforms
staining and an increase in the quantity of mitochondria, whereas overexpression of NRIP1 re-
duced oxidative activity and mitochondrial biogenesis in skeletal muscle [86]. In addition, the
absence of NRIP1 resulted in the upregulation of the same gene sets (oxidative phosphoryla-
tion, fatty acid oxidation, TCA cycle, and glycolysis) that we found deregulated in our models
[86]. A siRNA experiment to decrease NRIP1 expression in trisomic human fetal fibroblasts
enabled to recover normal levels of nuclear-encoded mitochondrial genes and normal mito-
chondrial function in those fibroblasts [87]. As such Nrip1 changes in copy number might ex-
plain most of the phenotypes observed in Ts3Yah and Ms3Yah mice, but we cannot exclude

the contribution of other genes from the interval. Indeed, the change in the steady-state level of
a subunit of mitochondrial ribosomal protein (MRP) encoded by the Mrpl39 gene within the
Hspa13-App region may also trigger perturbation in the translation of mitochondrial-encoded
proteins of the OXPHOS system, leading to variations of mitochondrial function observed in
Ts3Yah and Ms3Yah mice. Translational deficiencies due to mutations in genes encoding
MRPs have been described for four of the more than 70 MRPs, leading to growth retardation,
cardiomyopathy, hypotonia, and brain anomalies [88–91] but are expected to range from le-
thality to slightly impaired energy metabolic deficiencies and give rise to various tissue-specific
defects such as neuromuscular disorders or metabolic acidosis [92, 93]. In addition, deficit in
the OXPHOS machinery can trigger the mitochondrial membrane permeability transition
(MPT) leading to mitochondrial apoptosis or necrosis [94] as observed in Ms3Yah muscles.
DS and M21 are complex genetic diseases, and mouse models represent invaluable tools to
decipher Hsa21 dosage-sensitive genes and associated deregulated molecular pathways. Our
new models show that subtle deregulations contribute to the complexity of the DS-related pa-
thology and indicate the need to further study the contribution of different Hsa21 regions to
the modulation of DS. In particular, this study contributes to understanding the motor deficits
in people who have DS by providing new insight into the effect of trisomy on muscle dysfunc-
tion and proposing new deregulated pathways that might represent novel therapeutic targets
for DS therapy.
Materials and Methods
Ethics statement
All animals were treated in compliance with animal welfare policies from the French Ministry of
Agriculture (Law 87 848 and YH accreditation 45–31 and 67–369). Mice were bred on C57BL/6J
background for at least five generations prior to the functional analysis. The animals were bred
under SPF conditions and were treated in compliance with animal welfare policies. In all the ex-
periments, disomic littermates matched for age and gender were used as controls. A series of
behavioural experiments were conducted in order to evaluate motor and muscular functions. All
experimental procedures have been described previously in [36]. Behavioural protocols (rotarod,
grip strength, treadmill, notched bar, and open field) were approved by the local Ethics Commit-
tee for Animal Experimentation under the accreditation number (2012–069). For all these tests,
mice were kept in SPF conditions with free access to food and water. The light-dark cycle was
12:12, with lights on at 7:00 a.m. All the tests were done between 9:00 a.m. and 4:00 p.m.
After weaning, male mice were gathered by litters in the same cage. The different apparatus
used were placed in a dimly lit testing room (approximatively 60 lux). To produce experimental
groups, only animals coming from litters containing a minimum of two male pups were select-
ed. Groups of animals were transferred to the experimental room 30 minutes before each ex-
perimental test. No invasive procedure was used, and the mice were euthanized at the end of
the analysis.
Generating the Ts3Yah and Ms3Yah mouse strains
The Ms3Yah monosomic mice, Del(16Stch-App)3Yah, and the Ts3Yah trisomic mice, Dp
(16Stch-App)2Yah, were generated by chromosomal engineering in vivo as described in more
details in S1 Text. Briefly, Hspa13 and App targeting vectors containing loxP sites and isolated
from 5’Hprt and 3’Hprt libraries [95] were inserted by homologous recombination in HM1–1
Hprt-deficient ES cells in a trans configuration [96] (Fig. 1B–C). Transient expression of the
Cre recombinase generated clones with the tandem duplication and the deletion of the
Hspa13-App fragment (Fig. 1D) that were subsequently injected to C57BL/6J blastocysts to

generate chimeras. Chimeras were mated with C57Bl6/J animals and pups carrying either the
Mmu16 with the deletion or the Mmu16 with the duplication that were identified by Southern
blot analysis. Those animals were subsequently bred with C57BL/6J mice to obtain the
Ms3Yah and Ts3Yah lines.
Behavioural analyses
Groups (n = 10–15) of transgenic and diploid littermate males with matched age and genetic
background (B6 from N5 backcross level) were used for the different behavioural tests, follow-
ing the recommendations from the standard operating procedures developed by the Eumor-
phia network (http://www.eumorphia.org). A series of behavioural experiments were
conducted in order to evaluate the motor conditions in mice with group of 10 to 15 males per
genotype. For all these tests, mice were kept in SPF conditions with free access to food and
water. Several tests were carried out, including open-field, rotarod, notched bar,and grip
strength at ages 3 to 4 months and rotarod, grip strength, treadmill endurance, and notched
bar at ages 8 to 12 months. All procedures, except for the treadmill exercise, are presented in
S1 text. The treadmill system consists of a belt, which is enclosed in a Plexiglas chamber, and a
stimulus device with a metal shock grid attached to the rear of the belt. The speed and slope
of the belt are electronically adjusted. The animals were acclimatised with a 20-minute run at
25 cm/s and a 5-degree incline the day before the running test. For the actual test, the experi-
ment was started at 25 cm/s and a 5-degree incline. Every 20 minutes, a progressive increase of
5 cm/s in speed is applied. The distance run and the time spent running were recorded, and a
mouse was considered exhausted and removed from the experiment if it received approximate-
ly 100 shocks (1 mA) in a period of 5 minutes or if it was spending more than 20 seconds on
the shock grid. The duration of running and the total distance covered evaluate the perfor-
mance of the mice.
Gene expression analysis
RNA extraction. Total RNA was extracted from frozen gastrocnemius muscles from
Ms3Yah and Ts3Yah and their respective control diploid littermates (n = 6) using TRIzol Re-
agent (Invitrogen) followed by RNase-Free DNaseI (Qiagen) treatment.
Microarray hybridisation and data analysis. Biotinylated cDNA were prepared from total
RNAs and hybridised to Illumina’s MouseWG-6 v2.0 Expression BeadChips. Raw data was nor-
malised by the quantile method [97], and values were log transformed. A principal component
analysis was done using the GeneSpring software (Agilent Technologies) and showed that one
diploid sample corresponding to the Ts3Yah control group had a completely different profile.
This sample was excluded from the analysis. The data were analysed separately for Ms3Yah and
Ts3Yah, comparing each set to its own littermate diploid group. The genes expressed above the
background level were determined by selecting the probe sets having a signal value above the
35th percentile of all expression values in at least one array. This corresponded to a threshold
value above 90 in raw data (6.49 in log scale). The genes differentially expressed were selected by
statistical analysis (Student’s t-test p<0.05 between transgenic mice and their respective diploid
littermates). Verification was done to ensure that selected probes have acceptable false discovery
rate (<10%, [98]), and resampling analysis was used to make 1,000 permutations of the samples
for each probe in order to calculate the false discovery rate p-value (Zoe software; http://www-
microarrays.u-strasbg.fr/base.php?page = analysisExpressionFilterZoeE.php). FC was calculated
as a ratio of averages from transgenic and diploid signals with values <1 representing probes
that are underexpressed in the transgenic (Ts3Yah or Ms3Yah) compared to diploid controls

and values >1 representing probes that are overexpressed in the transgenic compared to the
diploid controls.
Hierarchical clustering was carried out on significantly deregulated genes (p<0.05) with
Cluster 3.0 software [99] using Euclidian distances to calculate the distances between the genes
and between the samples. The calculated distances were then clustered by complete linkage
clustering. The red-to-green colour scale represents the mean-adjusted expression values,
where red corresponds to higher expression and green to lower expression.
Gene Set Enrichment Analysis (GSEA) was used to test sets of related genes that might be
coordinately deregulated in Ts3Yah and Ms3Yah gastrocnemius muscles [40, 41]. The whole
transcriptome output lists of Ts3Yah and Ms3Yah differentially expressed genes, together with
their diploid controls, were submitted to the GSEA tool, and the functional gene sets contain-
ing curated biological pathways from the MSigDB C2 curated database were tested [40]. A
number of 1,000 permutations were specified as recommended in order to assess the statistical
significance of the enrichment score (ES). The GSEA analysis report highlights enrichment
gene sets with an FDR<5%. All data are accessible under the GEO accession number
(GSE58463) [NCBI tracking system #17057227].
QRT-PCR
cDNA synthesis was performed using the SuperScript III First-Strand Synthesis SuperMix for
QRT-PCR (Invitrogen). A series of primer pairs (available upon request) were designed to
span intron-exon junctions. Efficiencies of the TaqMan assays were checked using a cDNA di-
lution series from the extracts of gastrocnemius muscle samples. The QPCR was performed
with 300 nM of each primer and 100 nM of HPLC purified FAM-TAMRA-labelled double-dye
TaqMan probes in a final reaction of 15 μl with a standard amplification procedure. Normali-
sation was performed by carrying out in parallel the amplification of five housekeeping genes
(Actb, Pgk1, Hprt1, Ppia, and Gnas) and by using the GeNorm procedure [100] in order to cor-
rect the variations of the amount of source RNA in the starting material. All the tested samples
were performed in triplicate, and the results were reported as the mean ± sem.
mtDNA quantification by quantitative real-time PCR
DNA was isolated from muscle tissue, and quantitative PCR using the TaqMan technology was
performed in triplicate to determine the relative quantity of the mitochondrial DNA marker
cyclooxygenase 2 (Cox2) and the genomic DNA marker myxovirus resistance 1 (Mx1) gene.
mtCOX2 and Mx1 primers were purchased from Sigma-Aldrich and TaqMan MGB probes for
mtCOX2 and Mx1 from Applied Biosystem. PCR conditions were as follows: (1) 50°C for 2
minutes, (2) 95°C for 10 minutes, (3) 95°C for 15 seconds, and (4) 60°C for 1 minute (steps 3
and 4 were repeated 50 times). The results were calculated from the difference in threshold
cycle (ΔCT) values for mtDNA and nuclear-specific amplification. Data were expressed as
mtDNA in transgenic mice relative to diploid control groups.
SDH and immunofluorescence staining on frozen muscle sections
Tibialis anterioris muscles and soleus tissues were collected from 5 to 7 mice aged 4 to 6
months per genotype and immediately frozen in 5 minutes and 3 minutes, respectively, in iso-
pentane cooled in liquid nitrogen (-190°C). A total of 10 μm thick serial sections were obtained
using a cryostat (Leica CM3050) at -25°C. The sections were processed for succinate dehydro-
genase (SDH) staining (S1 Text). Alternatively, type I fibres in tibialis and soleus were charac-
terised by MyHC-I immunostaining using the primary monoclonal mouse antibody against
type I (M844, Sigma) as described in S1 Text. Quantification was done by counting muscle

fibres using the ImageJ software (W. Rasband, NIH; http://rsb.info.nih.gov/ij/) on at least three
consecutive sections.
Electron microscopy
TA samples (n = 3–5) of 3-month and 12-month old mice were fixed in 2.5% glutaraldehyde
and 2.5% paraformaldehyde in cacodylate buffer (0.1 M, pH 7.4), postfixed in 1% osmium te-
troxide in 0.1M cacodylate buffer, and dehydrated through graded alcohol (50%, 70%, 90%,
and 100%) and propylene oxide. The samples were oriented longitudinally and embedded in
Epon 812. Semithin sections were cut at 2 μm, and ultrathin sections were cut at 70 nm and
contrasted with uranyl acetate and lead citrate and examined at 70 kv with a Morgagni 268D
electron microscope. The images were captured digitally by a MegaView III camera (Soft Imag-
ing System).
Analysis of mitochondrial function
Mitochondria were isolated from hind limb skeletal muscles from one or two adult mice per
mitochondrial preparation as described in S1 Text. The protein concentration of mitochondrial
suspensions was determined in duplicate by a biuret method with bovine serum albumin to
normalise the samples. Oxygen consumption was measured with a Clark oxygen electrode
(Rank Brothers Ltd) in a stirred and closed chamber as described in S1 text. The cytochrome-c
oxidase (COX) activity was determined by the mitochondrial respiration rate in the mitochon-
drial suspension, which includes 10 mM antimycin, 2 mM ascorbate, and 0.5 mM TMPD (N,
N,N’,N’-TetraMethyl-p-Phenylene-Diamine). Measurements of COX activity were done in du-
plicate on n = 5 per genotype.
Mitochondrial membrane potential was measured using an electrode sensitive to the lipo-
philic cation triphenylmethylphosphonium (TPMP+
). The protocol described in S1 Text allows
us to calculate the ΔpH value as the difference between membrane potential values in the pres-
ence (ΔpH + Δψ) and in the absence (Δψ) of nigericin.
Respiration and membrane potential were then progressively inhibited through successive
steady states induced by the addition of malonate. After each run, 2 μM FCCP was added to re-
lease TPMP+
back into the medium for baseline correction. Membrane potentials were calcu-
lated as described previously [101], assuming a TPMP+
binding correction of 0.35 mg protein/
μl for skeletal muscle mitochondria [102].
Radical oxygen species (ROS) production by isolated mitochondria was evaluated by measur-
ing the rate of H2O2 released using an SFM-25 fluorometer (Kontron) at excitation and emission
wavelengths of 560 nm and 584 nm, respectively. The respiratory buffer (1 ml) was supple-
mented with 5 U/ml horseradish peroxidase, 1 μM Amplex Red reagent, and mitochondria
(15–20 μg/ml). Respiratory substrates (5 mM succinate or 5 mM pyruvate/2.5 malate or 40 μM
palmitoyl-L-carnitine/2.5 malate) were added to start the reaction. The fluorescent signal was
calibrated using a standard curve obtained after successive addition of H2O2 (20 to 80 pmoles).
Statistics
Unless otherwise stated, statistical analyses were performed comparing two groups, the
Ts3Yah or Ms3Yah and their diploid control littermates, using Student’s t-test when appropri-
ate or the nonparametric Mann-Whitney rank sum test. For tests requiring serial recordings,
data were analysed by two-way ANOVA using the SigmaPlot software. Data are presented as
mean ± sem. Significant threshold was set for p<0.05.

Supplemental information
Supplemental information includes additional experimental procedures, two figures, and five
tables and can be found online.
Supporting Information
S1 Table. Orthologous genes on Mmu16 and Hsa21 Hspa13-App region.
(DOCX)
S2 Table. Ts3Yah vs diploid littermates. Genes whose expression is significantly altered in
Ts3Yah gastrocnemius muscles with FC>|1.2| (t-test p<0.05). Gene names from the
Hspa13-App region are in italics. Probes deregulated in Ms3Yah model are in bold.
(DOCX)
S3 Table. Ms3Yah vs diploid littermates. Genes whose expression is significantly altered in
Ms3Yah gastrocnemius muscles with FC>|1.2| (t-test p<0.05). Gene names from the
Hspa13-App region are in italics. Probes deregulated in Ms3Yah muscles are in bold.
(DOCX)
S4 Table. Gene set enrichment analysis in diploid vs Ts3Yah. List of significantly enriched
gene sets as indicated by false discovery rate (FDR) <5%. Genes sets with negative enrichment
score (gene sets that show enrichment at the bottom of the ranked list, corresponding to down-
regulated genes compared to diploid controls) are listed for the Ts3Yah line as there was no
gene set with FDR<5% in the list of gene sets with positive enrichment score. Set size: number
of genes in the gene set after filtering out genes not in the expression data set. ES: enrichment
score for the gene set, representing the degree to which the gene set is overrepresented at the
edges (top or bottom) of the ranked list in the expression dataset. NES: normalised enrichment
score, ES after normalisation across analysed gene sets. Nom p-value: nominal p value giving
the statistical significance of the enrichment score. FDR q-value: false discovery rate giving the
probability that NES represent a false positive.
(DOCX)
S5 Table. Gene set enrichment analysis in Ms3Yah vs diploid. List of significantly enriched
gene sets as indicated by false discovery rate (FDR) <5%. Gene sets with positive enrichment
score (gene sets that show enrichment at the top of the ranked list, corresponding to upregu-
lated genes compared to diploid controls) are given for the Ms3Yah line that had no gene set
with a significant negative enrichment score. Set size: number of genes in the gene set after fil-
tering out genes not in the expression data set. ES: enrichment score for the gene set represent-
ing the degree to which the gene set is overrepresented at the edges (top or bottom) of the
ranked list in the expression dataset. NES: normalised enrichment score, ES after normalisation
across analysed gene sets. Nom p-value: nominal p value giving the statistical significance of
the enrichment score. FDR q-value: false discovery rate giving the probability that NES repre-
sent a false positive.
(DOCX)
S1 Fig. Evaluation of motor skills. (A) Rotarod tests at incremental fixed speed (repeated
ANOVA ‘genotype,’ ‘speed’: Ts3Yah vs diploid: F[1, 69] = 2.395 p = 0.135; Ms3Yah vs diploid:
F[1, 90] = 2.607 p = 0.117) and at accelerating speed (two trials [4–40 rpm] in five minutes
with the mean rotational velocity at the time of falling calculated for the two trials). (B) Endur-
ance to exercise was measured as the maximal running distance travelled on a treadmill until
exhaustion. (C) Spontaneous locomotor activity and habituation were assessed in an open field
by measuring the distance travelled during the first 15 minutes and the last 15 minutes of a

30-minute session. (D) Motor coordination skills were tested with a notched bar by looking at
the percentage of errors made by the mouse with its hind paws when crossing the notched bar.
Data are presented as mean ± sem.
(TIF)
S2 Fig. Evaluation of mitochondrial function. Mitochondria were isolated from hind limb
muscle to test their respiration capacities. (A) State 3, ADP-stimulated respiration in Ts3Yah
mitochondria. (B) Respiratory control ratio (RCR) is the ratio of state 3 to state 4o. (C) COX
activity was monitored by measuring mitochondrial oxygen consumption during PM respira-
tion with the addition of 10 mM antimycin, 2 mM ascorbate, and 0.5 mM TMPD. Yield of mi-
tochondrial protein per gram of muscle is obtained from the mitochondrial extraction. (D–F)
Rates of oxygen consumption in Ms3Yah mitochondria measured in the presence of pyruvate/
malate (PM) or succinate (S). (D) State 2, basal was measured in the presence of substrates
only. (E) State 3, ADP-stimulated respiration (ADP) was measured with the addition of
500 μM ADP. (F) State 4o, after addition of oligomycin. (G) COX activity in Ms3Yah mito-
chondria. Data are presented as mean ± sem (n = 5–6 per genotype).
(TIF)
S1 Text. Supporting information about gene targeting and generation of chromosomal re-
arrangements in ES cells; Southern blot analysis; Fluorescent in situ hybridisation; Array-
based comparative genomic hybridisation; Open field; Rotarod test; Notched bar test; Grip
strength; SDH on muscle sections; Immunofluorescence staining (fibre typing); Isolation
of skeletal muscle mitochondria; Mitochondrial respiration and COX activity; Mitochon-
drial membrane potential.
(DOCX)
Acknowledgments
We thank Feijun Luo, Laurence Jeronne, Caroline Colombier, Francoise Levavasseur, and Val-
érie Nalesso for their participation in the creation of the Ts3Yah and Ms3Yah models. We are
also grateful to Ivana Prokic, Thanuja Gali Ramamoorthy, and Daniel Metzger for their help in
muscle histological analysis. We thank members of the research group, of the IGBMC laboratory,
of the ICS, and of the AnEUploidy consortium for their helpful comments (www.aneuploidy.
org). We are grateful as well to the animal caretakers of the ICS.
Author Contributions
Conceived and designed the experiments: VB YH. Performed the experiments: VB AD DR CR
IS SP MK CB NM AJS. Analyzed the data: VB DD YH. Contributed reagents/materials/analysis
tools: DD JCB AJS. Wrote the paper: VB DR SEA AJS MD YH.
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