Down syndrome (DS) is a developmental disorder with structural, functional, molecular, and genetic alterations that show a dynamic onset and severity (Antonarakis et al., 2020; de Moraes et al., 2008; Grieco et al., 2015; Keeling et al., 1997; LaCombe and Roper, 2020; Llambrich et al., 2022a; McCarron et al., 2017; Steingass et al., 2011). The alterations associated with DS simultaneously affect multiple systems that are likely interrelated over development, with changes in one system modulating the others (Llambrich et al., 2022b). Many studies have provided evidence of these developmental alterations and have demonstrated the ability of dietary supplements such as epigallocatechin-3-gallate (EGCG) or green tea extracts enriched in EGCG (GTE-EGCG) to modulate these systems separately (Catuara-Solarz et al., 2016; de la Torre et al., 2016; Goodlett et al., 2020; Guedj et al., 2009; Llambrich et al., 2022b; Rondal, 2020; Stagni et al., 2015; Starbuck et al., 2021). However, a holistic evaluation of the simultaneous effects of trisomy and GTE-EGCG in those intertwined systems is missing. The variety in experimental setups and readouts obtained in different preclinical studies hinders the comparison and integration of results and, as a result, evidence is usually contradictory, reporting both positive and negative treatment effects. This lack of consistency can lead to biased interpretations about the etiology of the disorder and the potential effect of pharmacological treatments.

Much of this DS research was based on the Ts65Dn mouse model (Davisson et al., 1990; Reeves et al., 1995), as it was one of the first preclinical models available for DS and has been widely used for experimental testing. These mice carry a segment with approximately 120 genes homologous to Hsa21 (starting upstream of Mrpl39 to the telomeric end of Mmu16), translocated to a small centromeric part of Mmu17 (Duchon et al., 2011; Herault et al., 2017; Reinholdt et al., 2011). Ts65Dn mice are trisomic for about two-thirds of the genes orthologous to Hsa21, but also carry genes originating from the Mmu17 that are not related with DS, including about 46 protein-coding genes, 35 nonprotein-coding genes and 35 pseudogenes (Muñiz Moreno et al., 2020). These genetic alterations do not fully represent DS’s aneuploidy and other mouse and rat models have been developed recently that more faithfully represent the trisomic nature of DS (Kazuki et al., 2020; Kazuki et al., 2022). However, in this study, we used the Ts65Dn mouse model because it recapitulates the main skeletal, brain, cognitive, brain metabolite, and genetic alterations associated with DS (Blazek et al., 2011; Costa et al., 2010; Dierssen et al., 2002; Escorihuela et al., 1998; Gupta et al., 2016; Huang et al., 2000; Llambrich et al., 2022b; Même et al., 2014; Starbuck et al., 2021); and the effects of GTE-EGCG pharmacological modulation have been extensively evaluated using this mouse model (Catuara-Solarz et al., 2016; Goodlett et al., 2020; Jamal et al., 2022; Llambrich et al., 2022b; McElyea et al., 2016; Stagni et al., 2016; Starbuck et al., 2021; Stringer et al., 2017).

At the structural level, people with DS show skeletal and brain alterations that progress through ontogeny (Antonarakis et al., 2020; de Moraes et al., 2008; Fischer-Brandies et al., 1986; Keeling et al., 1997; LaCombe and Roper, 2020; Pearlson et al., 1998; Starbuck et al., 2021). Children with DS have a decreased buildup of bone mass and a low bone turnover rate, resulting in more osteoclast than osteoblast activity, smaller bone area, lower bone mineral density (BMD), and an increased risk of osteoporosis at adulthood (Carfì et al., 2017; Keeling et al., 1997; LaCombe and Roper, 2020). Individuals with DS also present midfacial hypoplasia and flattened nasal bridge along with skull malformations resulting in a shorter, wider, and rounder skull (Blazek et al., 2011; LaCombe and Roper, 2020; Richtsmeier et al., 2000; Suri et al., 2010; Thomas and Roper, 2021). Previous studies have shown the potential of GTE-EGCG to modulate craniofacial and postcranial morphology, as well as the microarchitecture and BMD of the long bones, showing positive, negative or no treatment effects (Abeysekera et al., 2016; Blazek et al., 2015a; Jamal et al., 2022; Llambrich et al., 2022a; McElyea et al., 2016; Starbuck et al., 2021; Stringer et al., 2017). We previously detected dose-, time-, and anatomical structure-dependent effects in a study using the same mouse model and the same treatment regime with two different GTE-EGCG doses (Llambrich et al., 2022a). A dose of 100 mg/kg/day of GTE-EGCG exacerbated facial dysmorphologies (Starbuck et al., 2021), modified the skeletal dysmorphologies associated with DS without rescuing the bones shape (Llambrich et al., 2022a), and altered the integration between the skull and the brain (Llambrich et al., 2022b). However, a lower dose of 30 mg/kg/day significantly reduced the facial dysmorphologies (Starbuck et al., 2021) but did not show additional rescuing effects in other skeletal traits (Llambrich et al., 2022a).

In DS, the craniofacial size and shape alterations are accompanied with structural brain alterations. People with DS show a reduced overall brain volume from birth, with disproportionately smaller hippocampus and cerebellum, and larger ventricles (Hamner et al., 2018; Movsas et al., 2016; Patkee et al., 2020; Pinter et al., 2001; Rodrigues et al., 2019; Smigielska-Kuzia et al., 2011). Evidence for the effects of GTE-EGCG on brain anatomy is limited. Our own previous study showed that administration of 100 mg/kg/day of GTE-EGCG altered the brain shape of Ts65Dn mice at adulthood (Llambrich et al., 2022b), while another study administering green tea polyphenols corresponding to 0.6–1 mg EGCG per day in YACtg152F7 mice showed reduced thalamic-hypothalamic volume and reduced overall brain weight and volume (Guedj et al., 2009).

The structural brain alterations in DS are associated with cognitive disabilities, causing functional disability (Guidi et al., 2011; Rodrigues et al., 2019; Stagni et al., 2016). People with DS show relative impairment in executive function, short-term memory, working memory, and explicit long-term memory (Grieco et al., 2015; Lott and Dierssen, 2010; McCarron et al., 2017; Real de Asua et al., 2015; Steingass et al., 2011; Zis and Strydom, 2018); together with a neurodevelopmental delay in the acquisition of both gross and fine motor skills during childhood (Beqaj et al., 2017; Ferreira-Vasques and Lamônica, 2015; Frank and Esbensen, 2015; Kim et al., 2017; Malak et al., 2015). Regarding cognition, the therapeutic results of green tea extracts are also contradictory. A study administering 60 mg/kg GTE-EGCG to 3- to 4-month-old mBACtgDyrk1a and Ts65Dn mice for 4 weeks rescued working memory (Souchet et al., 2015), and interventional studies in humans with DS have shown the ability of (GTE-)EGCG to improve memory recognition, working memory, inhibitory control, and adaptive behavior (de la Torre et al., 2016; De la Torre et al., 2014). However, other studies found no effect or negative effects of (GTE-)EGCG on cognition. In children aged 6–12 years, a randomized phase Ib clinical trial administering 10 mg/kg/day of EGCG for 6 months combined with a dietary supplement did not improve cognition and functionality (Cieuta-Walti et al., 2022). In mice, GTE-EGCG at a dose of 30 mg/kg/day had no effect on visuospatial learning and memory, and even resulted in reduced swimming speed in the Morris water maze and increased thigmotaxic behavior in 5- to 6-month-old Ts65Dn mice (Catuara-Solarz et al., 2015).

Several studies have shown that the cognitive functional alterations observed in DS are related with changes at a molecular level on the concentration of hippocampal metabolites, as people with DS show increased myo-inositol levels when compared to euploid population, and people with DS and dementia show reduced levels of N-acetylaspartate (NAA) when compared to people with DS without dementia (Beacher et al., 2005; Lamar et al., 2011). These findings warrant further investigation into the potential role of hippocampal metabolites in cognitive function, particularly given that few studies to date have investigated the effects of trisomy on the concentration of the main metabolites in the hippocampus of Ts65Dn mice (Huang et al., 2000; Même et al., 2014; Santin et al., 2014) and there is currently no evidence on the effects of (GTE-)EGCG treatment.

Finally, all these structural, functional, and molecular alterations are the result of a complex genetic imbalance involving the triplicated genes in chromosome 21 and the interactions of many genes across the genome (Olson et al., 2004). Indeed, skeletal malformations can be associated with the dysregulation of genes such as Regulator of Calcineurin 1 (RCAN1), Superoxide Dismutase 1 (SOD1), Engrailed Homeobox 2 (EN2), ETS Proto-Oncogene 2 (ETS2), Sonic Hedgehog (SHH), Dual-Specificity Tyrosine-(Y)-Phosphorylation Regulated Kinase 1 A (DYRK1A), SRY-Box Transcription Factor 9 (SOX9), and Orthodenticle Homeobox 2 (OTX2) (Arron et al., 2006; Billingsley et al., 2013; McElyea et al., 2016; Roper et al., 2009; Thomas and Roper, 2021; Weisfeld-Adams et al., 2016), while cognitive impairment and altered brain development may be associated with RCAN1, SOD1, Oligodendrocyte Transcription Factor 1 (OLIG1), Oligodendrocyte Transcription Factor2 (OLIG2), SIM BHLH Transcription Factor 2 (SIM2), Down Syndrome Cell Adhesion Molecule (DSCAM), DYRK1A, Down Syndrome Critical Region 1 (DSCR1), Synaptojanin 1 (SYNJ1) and Potassium Inwardly Rectifying Channel Subfamily J Member 6 (KCNJ6) (Chang et al., 2003; Hoeffer et al., 2007; Kazemi et al., 2016; Kleschevnikov et al., 2017; Lana-Elola et al., 2011; Stagni and Bartesaghi, 2022; Stagni et al., 2018). From these genes, DYRK1A, a gene involved in both skeletal and neuronal development that is overexpressed by trisomy of chromosome 21 (Blazek et al., 2015a; Dierssen and de Lagrán, 2006), has been proposed as a target gene for therapy (Atas-Ozcan et al., 2021; Becker et al., 2014; Dierssen and de Lagrán, 2006; García-Cerro et al., 2014; Jarhad et al., 2018; Ji et al., 2015; Lee et al., 2009; Rondal, 2020). The potential of EGCG to modulate gene expression in the Ts65Dn mouse model for DS has limited evidence, with one study administering 200 mg/kg EGCG on embryonic days 7 and 8 twice daily showing decreases in Protein patched homolog 1 (Ptch) and Ets2 RNA expression and significant increases in Rcan1 and Shh RNA expression in the first pharyngeal arch of Ts65Dn mice at embryonic day (E) 9.5 (McElyea et al., 2016).

Since a holistic evaluation of these systems is missing, we designed a longitudinal experimental setup to follow the simultaneous development of structural, functional, molecular, and genetic alterations in the Ts65Dn mouse model. In this study, we conducted a multi-modal in vivo imaging study using micro computed tomography (µCT), magnetic resonance imaging (MRI) and magnetic resonance spectroscopy (MRS) to investigate the integrated development of craniofacial shape, BMD, brain volumes, and hippocampal metabolites in wildtype and Ts65Dn mice. Additionally, we evaluated the changes in body weight and performed a battery of neurodevelopmental and adult cognitive tests to assess cognitive function from birth to adulthood throughout development. At endpoint, we also evaluated tibia microarchitecture from ex vivo µCT scans and used RNAseq to analyze cerebellar gene expression in the same mice at 8 months old (Figure 1). Furthermore, we evaluated the pleiotropic effects of prenatal chronic GTE-EGCG treatment and explored the effects of treatment discontinuation on these systems, providing the first controlled longitudinal study assessing the simultaneous effects of treatment across different systems.

Figure 1

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Our longitudinal experimental setup allowed us to investigate the pleiotropic effects of trisomy, GTE-EGCG treatment and treatment discontinuation at the structural, functional, molecular, and genetic levels in the Ts65Dn DS mouse model (Figure 1). We compared wildtype (WT) and Ts65Dn (TS) untreated mice to evaluate trisomy effects, and WT and TS treated mice to evaluate treatment effects.

Structural characterization

Structurally, we investigated the longitudinal effects of trisomy, treatment, and treatment discontinuation on body weight, skeletal system, and adult brain volume (Figure 2).

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Body weight

First, we investigated mouse body growth by monitoring body weight over development. TS untreated mice tended to present lower body weights than WT untreated mice over development from birth to adulthood, but these genotype differences did not reach significance at any developmental stage (Figure 2A; P_BW1=0.7630; P_BW2=0.3688; P_BW3=0.1094).

Chronic GTE-EGCG treatment, which started prenatally and was maintained until 5 months, significantly reduced the body growth of TS treated mice from PD1 to PD17 (Figure 2A at BW₁) in comparison with WT untreated (p<0.0001), WT treated (p<0.0001), and TS untreated mice (p<0.0001). At adulthood before treatment discontinuation (Figure 2A at BW₂), these differences were maintained, but only reached significance when compared to WT untreated (p=0.0322) and WT treated mice (p=0.0103). After three months of treatment discontinuation, both WT and TS treated mice showed a mild increase in body weight, making TS treated mice not significantly different from WT untreated (Figure 2A; P_BW3=0.9284), and causing WT treated mice to be significantly different from WT untreated (Figure 2A; P_BW3=0.0259).

Skeletal development

Since people with DS show craniofacial abnormalities, reduced BMD, and altered bone microarchitecture (Carfì et al., 2017; Kao et al., 1992; LaCombe and Roper, 2020; Thomas and Roper, 2021; Vicente et al., 2020), we investigated the effects of genotype, treatment, and treatment discontinuation in these systems by performing in vivo µCT scans throughout development and ex vivo µCT scans at endpoint as indicated in Figure 1.

Craniofacial morphology

We first performed geometric morphometric analyses on the craniofacial 3D models obtained from in vivo µCT scans to investigate craniofacial shape throughout development. The results confirmed our previous findings (Llambrich et al., 2022a; Llambrich et al., 2022b), showing that TS untreated mice presented a significantly different craniofacial shape than WT untreated mice at µCT₁ (p<0.0001) and µCT₂ (p=0.0011) (Supplementary file 2a). In this study, we further detected that the craniofacial differences induced by genotype persisted until adulthood (Figure 2B and P_µCT3=0.0009; Figure 2C and P_µCT4=0.0055).

Prenatal chronic GTE-EGCG treatment only showed a significant effect in the craniofacial shape at PD3, where both WT and TS treated mice were different from their untreated counterparts (P_WT=0.0418; P_TS=0.0005). However, the treatment never rescued the craniofacial shape in TS treated mice, as these mice were significantly different from WT untreated mice at all stages (Figure 2B and C; Supplementary file 2a). Discontinuing the treatment for three months did not induce any effects (Figure 2C), maintaining the craniofacial dysmorphologies already observed at µCT₃ (Figure 2B).

Bone mineral density

The BMD of the humerus was estimated throughout development from the in vivo µCT scans. Although we observed that TS untreated mice tended to present lower BMD than WT untreated mice over development and adulthood, the differences did not reach significance at any developmental stage (Figure 2D, Supplementary file 2b).

Prenatal GTE-EGCG chronic treatment significantly modified the developmental trajectory of humerus BMD in WT and TS treated mice (P_WT <0.0001; P_TS=0.0023), but never rescued the trisomic phenotype, as TS treated mice remained different from WT untreated (p<0.0001). At PD3, WT and TS treated mice showed similar levels of BMD as compared to untreated mice (Figure 2D at µCT₁) but tended to show lower BMD at PD29 (Figure 2D at µCT₂). This tendency for reduced BMD was rescued at adulthood (Figure 2D at µCT₃), but after 3 months of treatment discontinuation, both WT and TS treated groups showed a decline in BMD (Figure 2D at µCT₄), which caused WT and TS treated mice to be different than WT untreated control mice (P_WT=0.0001; P_TS=0.0002), suggesting a rebound effect following treatment retrieval.

Tibia development and microarchitecture

Finally, we complemented our structural skeletal analysis by evaluating the length and microarchitecture of the tibia from ex vivo µCT scans at endpoint, as indicated in Figure 1.

To obtain a global view of the genotype and treatment effects, we performed a linear discriminant analysis (LDA) combining the results of all tibia microarchitecture tests into a single analysis that maximized the differences among groups of mice (Figure 2E). The LDA showed limited overlap between groups, as mice separated by treatment along the first axis, and by genotype along the second axis. These results suggested genotype differences and treatment effects that did not rescue the trisomic phenotype (Figure 2E). The multivariate pairwise comparisons after one-way PERMANOVA using Mahalanobis distances detected significant differences between all groups of mice except between WT untreated and TS untreated mice (Supplementary file 2c), confirming the treatment effects in both genotypes.

To explore the overall differences detected by the LDA in finer detail, we grouped the results of the tibia microarchitecture tests according to the structural domain they evaluated (cortical bone strength, cortical bone size, and trabecular bone), and repeated the multivariate pairwise comparisons (Supplementary file 2c). Even though we did not detect any significant difference between WT untreated and TS untreated mice in any structural domain (Supplementary file 2c), TS untreated mice tended to show specific reductions in cortical thickness, polar moment of inertia, cross-sectional area, and bone area (Figure 2—figure supplement 1 C,E,F,G); and the univariate pairwise comparison tests revealed significant differences in the tibia length (Figure 2—figure supplement 1A; p=0.0170) and periosteal perimeter (Figure 2—figure supplement 1I; p=0.0154).

After GTE-EGCG chronic treatment and discontinuation, WT treated mice were not significantly different than WT untreated mice for any specific structural domain (Supplementary file 2c), but the univariate pairwise tests indicated significantly reduced tibia length (Figure 2—figure supplement 1A; p=0.0006) and cortical porosity (Figure 2—figure supplement 1D; p=0.0120), together with increased cortical BMD (Figure 2—figure supplement 1B; p=0.0120). TS treated mice were significantly different than TS untreated mice for the cortical bone strength and cortical bone size domains (Supplementary file 2c), showing significantly more mineralized (Figure 2—figure supplement 1B; p=0.0039), thicker (Figure 2—figure supplement 1C; p=0.0400), and less porous (Figure 2—figure supplement 1D; p=0.0005) cortical bone. However, they appeared different from WT untreated mice in both cortical domains (Supplementary file 2c) as well as in the cortical BMD (Figure 2—figure supplement 1B; p=0.0097) and cortical porosity tests (Figure 2—figure supplement 1D; p=0.0107). These results could be explained either by adverse effects of the treatment during development that were persistent after discontinuation, or by a negative effect following treatment withdrawal.

Functional characterization

After evaluating the effects of trisomy and chronic prenatal GTE-EGCG treatment and its discontinuation at a structural level, we investigated its simultaneous effects at a functional level. We performed a battery of cognitive tests to evaluate early neurodevelopment and adult cognition before and after treatment discontinuation (Figure 3), at the timepoints specified in Figure 1.

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Neurodevelopment

We first evaluated early brain function and neurodevelopment over the first weeks after birth by performing a battery of neurodevelopmental tests from PD1 until PD18 (Figure 3A). The LDA with the results from all neurodevelopmental tests combined showed that the treatment was the main differentiating factor among groups of mice, as Axis 1 separated treated and untreated mice (Figure 3A). Despite some overlap, Axis 2 separated WT and TS mice. These results suggested mild genotype differences and treatment effects that did not rescue the trisomic phenotype (Figure 3A). The multivariate pairwise comparisons after one-way PERMANOVA using Mahalanobis distances (Supplementary file 2f) detected significant differences between TS untreated and WT untreated mice (p=0.0001), and between TS treated mice and both TS untreated (p=0.0001) and WT untreated mice (p=0.0001), confirming these findings.

When the neurodevelopmental tests were grouped into functional domains (developmental landmarks, neuromotor tests and reflexes) (Supplementary file 2f), the multivariate pairwise comparisons only indicated significant differences between WT untreated and TS untreated mice for the reflexes (p=0.0423). TS untreated mice tended to be delayed in most individual tests (Figure 3—figure supplement 1), and the univariate pairwise comparison tests confirmed significant delays in the acquisition rate of the vertical climbing (Figure 3—figure supplement 1E; p=0.0256) and tactile response (Figure 3—figure supplement 1I; p=0.0064), together with the average day of acquisition of the surface righting response (Figure 3—figure supplement 1C; p=0.0320). However, we also observed that TS untreated mice tended to be anticipated in the vibrissae placing, blast response, and Preyer reflex (Figure 3—figure supplement 1 J,M,N), even though these results did not reach significance.

Prenatal chronic treatment with GTE-EGCG caused TS treated mice to be significantly different than TS untreated mice for the domains neuromotor tests and reflexes, and WT treated mice to be different from WT untreated mice for the domain reflexes (Supplementary file 2f). The univariate tests indicated that treatment significantly modulated the average day of acquisition and acquisition rate of multiple neurodevelopmental tests in both WT and TS treated mice, causing either a delay or an anticipation in most tests (Figure 3—figure supplement 1). However, the treatment never rescued the trisomic phenotype, as TS treated mice remained significantly different than WT untreated mice for the three functional domains and most of the individual tests (Supplementary file 2f) (Figure 3—figure supplement 1).

Finally, we investigated the mothers’ behavior during 24 hr at PD8 to evaluate maternal care and observed that, despite being trisomic, all Ts65Dn mothers performed similarly and there was no large intra-group variation for any of the behaviors analyzed, confirming that maternal care did not influence the results of the neurodevelopmental tests (Figure 3—figure supplement 2). Furthermore, no significant differences were detected between treated and untreated mothers in any readout (Figure 3—figure supplement 2), confirming that the GTE-EGCG treatment did not modulate maternal care.

Adult cognition

Then, we evaluated adult cognitive performance by means of open field (OF), elevated plus maze (EPM), sociability/preference for social novelty (SPSN), novel object recognition (NOR) and passive avoidance (PA) testing at two timepoints, Cog.₁ and Cog.₂, before and after treatment discontinuation (Figure 3B and C), as specified in Figure 1.

The LDA with the results from all tests combined showed minor overlap between groups at both Cog.₁ and Cog.₂ (Figure 3B and C), with WT untreated mice occupying the negative extreme of Axis 1, WT treated and TS untreated mice presenting an intermediate position, and TS treated mice falling on the positive extreme of Axis 1, opposite to WT untreated mice (Figure 3B and C). These results suggested genotype differences between WT and TS untreated mice that were confirmed as significant by the multivariate pairwise comparisons after one-way PERMANOVA at Cog.₁ (Supplementary file 2g; p=0.0125) but not at Cog.₂ (Supplementary file 2h; p=0.4596).

When the cognitive tests were grouped into cognitive domains (anxiety, arousal, and memory), the multivariate pairwise comparisons only detected a significant difference between WT untreated and TS untreated mice for the arousal tests at Cog.₁ (p=0.0028) (Supplementary file 2g and h). Indeed, TS untreated mice showed a tendency to cover longer distances at higher speed in all arousal tests when compared with WT untreated mice (Figure 3—figure supplement 3 E-J). These differences reached significance for the distance and speed during OF, as well as for the distance and speed during NOR at Cog.₁ (Figure 3—figure supplement 3 E,F,I,J, top). TS untreated mice also tended to show increased exploratory and risk-taking behavior, as they usually presented a tendency of increased time spent in the center of the arena during habituation for NOR at Cog.₁ (Figure 3—figure supplement 3C, top), increased percentage of time spent in the open arm during EPM at Cog.₁ (Figure 3—figure supplement 3D, top), increased time in the center during OF at Cog.₂ (Figure 3—figure supplement 3A, bottom), and reduced time to enter the dark chamber during the training session of PA at Cog.₁ (Figure 3—figure supplement 3M, top), although these differences did not reach statistical significance. Finally, TS untreated mice showed impaired memory robustness and formation at the latest stage, with significantly less preference for the novel object during NOR when compared with WT untreated mice (Figure 3—figure supplement 3K, bottom; p=0.0257), and a tendency for reduced latency to enter the dark chamber during training and testing sessions for PA at Cog.₂ (Figure 3—figure supplement 3 M,N, bottom).

Regarding treatment effects, the results from the LDA at Cog.₁ suggested that the prenatal chronic treatment induced changes in both WT and TS treated mice (Figure 3B) that exacerbated the differences in TS treated mice and induced cognitive changes in WT treated mice that resulted in a cognitive performance similar to TS untreated mice (Figure 3B). Indeed, WT treated mice were significantly different than WT untreated mice when all cognitive tests were considered together as well as for the anxiety and arousal domains (Supplementary file 2g). In the case of TS treated mice, the multivariate pairwise comparisons revealed significant differences with WT untreated mice when considering all groups together as well as for the anxiety and arousal domains (Supplementary file 2g), confirming the lack of rescuing treatment effects. Both WT and TS treated mice showed significantly increased time in the center during NOR, and increased time in the open arm during EPM at Cog.₁ when compared with WT untreated mice (Figure 3—figure supplement 3 C,D, top), indicating increased exploratory and risk-taking behavior. Similarly, both WT and TS treated mice showed increased locomotor activity, as they covered significantly more distance at more speed than WT untreated mice for the distance and speed during OF, as well as the distance and speed during NOR at Cog.₁ (Figure 3—figure supplement 3 E,F,I,J, top). However, in the memory tests (Figure 3—figure supplement 3 K-N), the pairwise comparison tests revealed only a significant difference between TS untreated and TS treated mice for the preference for subject 2 (S2) at Cog.₁, where treated mice had greater preference for the novel stranger (Figure 3—figure supplement 3L, top; p=0.0137).

After treatment discontinuation at Cog.₂, the LDA (Figure 3C) presented a similar pattern as the LDA at Cog.₁ (Figure 3B), suggesting that discontinuing the treatment for one month did not substantially change the cognitive patterns induced after chronic GTE-EGCG treatment. However, new significant differences emerged between WT untreated and both WT treated and TS treated mice when evaluating the memory domain (Supplementary file 2h), suggesting that the treatment discontinuation altered the memory of both WT and TS mice. Furthermore, TS treated mice remained significantly different than WT untreated mice for all tests combined and all cognitive domains (Supplementary file 2h), confirming that the effects of treatment discontinuation did not rescue the trisomic cognitive phenotype. Indeed, significant differences between WT untreated and TS treated mice remained for the percentage of time spent in the open arm during EPM at Cog.₂ (Figure 3—figure supplement 3D, bottom; p=0.0476), and new significant differences emerged between WT untreated mice and both WT treated and TS treated mice for the time spent in the center and in the periphery during OF (Figure 3—figure supplement 3A, B, bottom), indicating that treated mice still presented a more explorative and risk-taking behavior. A similar trend was observed for the arousal tests, as even though no significant differences were detected between TS treated and TS untreated mice (Figure 3—figure supplement 3E-J, bottom), both treated groups showed hyperactivity as the univariate pairwise comparison tests confirmed significant differences between TS treated mice and WT untreated mice for all arousal tests (Figure 3—figure supplement 3E-J, bottom); and significant differences were detected between WT treated and WT untreated mice for the distance and speed during SPSN and the distance and speed during NOR (Figure 3—figure supplement 3G-J, bottom). In the memory tests, TS treated mice were significantly different than TS untreated (p=0.0046) but not than WT untreated mice for the preference for the novel object during NOR (Figure 3—figure supplement 3K, bottom; p=0.3566), which could suggest a beneficial treatment effect. Contrary to the other behavioral tests, the results for PA testing at Cog.₂ are influenced by the testing at Cog.₁ as mice may present a robust contextual fear memory trace created during the testing at Cog.₁, preventing them from entering the dark chamber even in the training phase of the test at Cog.₂. Indeed, WT untreated mice did not enter the dark box during training at Cog.₂, while both WT and TS treated mice showed a reduced tendency to enter the dark chamber which reached significance when comparing TS treated mice with WT untreated mice (Figure 3—figure supplement 3M, bottom; p<0.0001), indicating less robust contextual memory retention. After re-exposure to the fear conditioning in the training phase of PA at Cog.₂, all WT mice avoided the dark chamber while a few TS treated mice still showed a reduced tendency to enter the dark chamber (Figure 3—figure supplement 3N, bottom), reflecting altered memory robustness.

Molecular and genetic characterization

Finally, to complement the structural and functional evaluation and to obtain a complete holistic characterization of the alterations associated with DS, we performed in vivo MRS and ex vivo RNAseq at the timepoints specified in Figure 1 to investigate changes in hippocampal region metabolite concentration and cerebellar gene expression respectively (Figure 4).

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Hippocampal region metabolites

We performed in vivo MRS on a voxel placed in the hippocampal region at two timepoints, MRS₁ and MRS₂, as specified in Figure 1.

We first analyzed global differences in the metabolite spectra by performing an LDA containing the relative integrals of 10 spectral regions between 0.0 ppm and 4.3 ppm (Figure 4, Figure 4—figure supplement 1 and Supplementary file 1b). The LDA at MRS₁ separated WT untreated and TS untreated mice across Axis 1 (Figure 4A), and the multivariate pairwise comparisons after one-way PERMANOVA using Mahalanobis distances confirmed significant genotype differences (Supplementary file 2i; p=0.0136). After treatment discontinuation at MRS₂, the LDA showed milder genotype separation (Figure 4B) and the multivariate pairwise comparisons did not detect any significant difference between WT untreated and TS untreated mice (Supplementary file 2j; p=0.5790), confirming these findings.

When evaluating the concentrations of specific main metabolites like N-acetylaspartate (NAA), creatine +phosphocreatine (Cr +PCr), choline +phosphocholine + glycerophosphocoline (tCho), myo-inositol (myo-Inos) and taurine (Tau) (Figure 4—figure supplement 1), TS untreated mice tended to show lower levels of NAA and tCho at MRS₁ when compared with WT untreated mice (Figure 4—figure supplement 1 A,C), but the univariate pairwise comparison tests only revealed a significant increase in the concentration of Cr +PCr (Figure 4—figure supplement 1B, top; p=0.0235). At MRS₂, the same pattern was observed but the significant differences between WT untreated and TS untreated mice disappeared (Figure 4—figure supplement 1, bottom).

Regarding treatment effects, the LDA at MRS₁ separated treated and untreated mice across Axis 2 (Figure 4A), suggesting treatment effects that did not rescue the trisomic phenotype. The multivariate pairwise comparisons after one-way PERMANOVA (Supplementary file 2i) showed significant differences between TS treated and both WT untreated (p=0.0496) and TS untreated mice (p=0.0361), confirming these findings. When evaluating the specific concentrations of NAA, Cr +PCr, tCho, myo-Inos and Tau, both WT treated and TS treated mice tended to show increased levels of Cr +PCr, which reached significance when comparing TS treated mice with WT untreated mice (Figure 4—figure supplement 1B, top; p=0.0317). Conversely, both WT and TS treated mice tended to show reduced levels of myo-inositol and tCho (Figure 4—figure supplement 1D, top), causing TS treated mice to have significantly reduced tCho when compared with WT untreated mice (Figure 4—figure supplement 1C, top; p=0.0296).

After treatment discontinuation at MRS₂, the LDA showed milder treatment separation across Axis 2 (Figure 4B) and the multivariate pairwise comparisons did not detect any significant difference between TS untreated and TS treated mice (Supplementary file 2j; p=0.2111). However, the differences between TS treated and WT untreated mice remained significant (p=0.0347), indicating that discontinuing the treatment for 3 months did not rescue the trisomic phenotype. When evaluating the specific concentrations of NAA, Cr +PCr, tCho, myo-Inos and Tau, the same pattern was observed and the significant differences between TS treated and WT untreated mice disappeared (Figure 4—figure supplement 1, bottom). However, significant differences emerged between WT untreated mice and WT treated mice for the concentration of tCho and myo-inositol (Figure 4—figure supplement 1 C,D, bottom; P_tCho=0.0479, P_myo-inositol=0.0261).

Gene expression

Finally, since people with DS show impaired motor skills (Quinzi et al., 2022), we investigated the effects of genotype and chronic treatment after three months of treatment discontinuation in the cerebellar gene expression by performing RNAseq at endpoint (8 M).

We first performed a PCA using the normalized expression of the 125 genes that are triplicated in the Ts65Dn mouse model and included in our dataset. The PCA showed major overlap between all groups, even though TS treated mice showed a larger range of variation (Figure 4C). This result suggested that the differences in gene expression of the triplicated genes were minimal and that GTE-EGCG prenatal chronic treatment and three months of discontinuation did not induce major permanent effects when considering all triplicated genes. The multivariate pairwise comparison tests after one-way PERMANOVA using Euclidean distances did not detect any significant difference between groups, confirming these findings (Supplementary file 2k).

Finally, we analyzed the differentially expressed genes (DEGs) between each pair of experimental groups across the entire transcriptome (Supplementary file 3). When analyzing the entire transcriptome, we only detected 24 DEGs between WT untreated and TS untreated mice even when applying a loose p-adjusted value of 0.1, indicating small differences in cerebellar gene expression (Supplementary file 3). Interestingly, we detected 12 DEGs between WT untreated and TS treated mice, and only 9 of these genes were also found to be differentially expressed between WT untreated and TS untreated mice, which could indicate mild treatment effects in TS mice (Supplementary file 3). However, we did not detect any DEGs between TS untreated and TS treated mice, and only five DEGs were detected between WT untreated and WT treated mice, indicating that either the prenatal chronic GTE-EGCG treatment did not largely modulate gene expression or that the treatment had transient effects that were mostly not detected after three months of treatment discontinuation and were not rescuing the trisomic phenotype (Supplementary file 3).

We investigated for the first time the simultaneous pleiotropic effects of trisomy and a chronic prenatal and postnatal treatment with GTE-EGCG on several organ systems and levels throughout development in the Ts65Dn mouse model for DS. Our longitudinal and holistic approach overcomes a main limitation of previous research, in which systems were analyzed independently at single time points using different experimental setups and delivered disparate and contradictory results that could not be generalized. Here, we followed the simultaneous development of structural, functional, molecular, and genetic systems on Ts65Dn mice and provided comparable results of genotype and prenatal chronic GTE-EGCG treatment effects before and after treatment discontinuation.

The data supporting the findings of this study are available in Dryad at https://doi.org/10.5061/dryad.1rn8pk11r.

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Author details

1. Sergi Llambrich

   Biomedical MRI, Department of Imaging and Pathology, KU Leuven, Leuven, Belgium

   Contribution

   Conceptualization, Data curation, Formal analysis, Investigation, Visualization, Writing – original draft, Writing – review and editing

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0000-0001-9980-0208

2. Birger Tielemans

   Biomedical MRI, Department of Imaging and Pathology, KU Leuven, Leuven, Belgium

   Contribution

   Investigation

   Competing interests

   No competing interests declared

3. Ellen Saliën

   Biomedical MRI, Department of Imaging and Pathology, KU Leuven, Leuven, Belgium

   Contribution

   Formal analysis, Investigation

   Competing interests

   No competing interests declared

4. Marta Atzori

   Department of Human Genetics, KU Leuven, Leuven, Belgium

   Contribution

   Formal analysis

   Competing interests

   No competing interests declared

5. Kaat Wouters

   Laboratory of Biological Psychology, KU Leuven, Leuven, Belgium

   Contribution

   Formal analysis

   Competing interests

   No competing interests declared

6. Vicky Van Bulck

   Laboratory of Biological Psychology, KU Leuven, Leuven, Belgium

   Contribution

   Formal analysis

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0000-0002-9355-0567

7. Mark Platt

   Centre for Preclinical Imaging, Department of Molecular and Clinical Cancer Medicine, University of Liverpool, Liverpool, United Kingdom

   Contribution

   Formal analysis

   Competing interests

   No competing interests declared

8. Laure Vanherp

   Biomedical MRI, Department of Imaging and Pathology, KU Leuven, Leuven, Belgium

   Contribution

   Formal analysis

   Competing interests

   No competing interests declared

9. Nuria Gallego Fernandez

   Departament de Biologia Evolutiva, Ecologia i Ciències Ambientals (BEECA), Facultat de Biologia, Universitat de Barcelona, Barcelona, Spain

   Contribution

   Formal analysis

   Competing interests

   No competing interests declared

10. Laura Grau de la Fuente

    Departament de Biologia Evolutiva, Ecologia i Ciències Ambientals (BEECA), Facultat de Biologia, Universitat de Barcelona, Barcelona, Spain

    Contribution

    Formal analysis

    Competing interests

    No competing interests declared

11. Harish Poptani

    Centre for Preclinical Imaging, Department of Molecular and Clinical Cancer Medicine, University of Liverpool, Liverpool, United Kingdom

    Contribution

    Resources

    Competing interests

    No competing interests declared

12. Lieve Verlinden

    Clinical and Experimental Endocrinology, KU Leuven, Leuven, Belgium

    Contribution

    Methodology

    Competing interests

    No competing interests declared

13. Uwe Himmelreich

    Biomedical MRI, Department of Imaging and Pathology, KU Leuven, Leuven, Belgium

    Contribution

    Resources, Formal analysis, Methodology

    Competing interests

    No competing interests declared

    "This ORCID iD identifies the author of this article:" 0000-0002-2060-8895

14. Anca Croitor

    Biomedical MRI, Department of Imaging and Pathology, KU Leuven, Leuven, Belgium

    Contribution

    Formal analysis

    Competing interests

    No competing interests declared

15. Catia Attanasio

    Department of Human Genetics, KU Leuven, Leuven, Belgium

    Contribution

    Resources, Supervision

    Competing interests

    No competing interests declared

    "This ORCID iD identifies the author of this article:" 0000-0001-8077-5719

16. Zsuzsanna Callaerts-Vegh

    Laboratory of Biological Psychology, KU Leuven, Leuven, Belgium

    Contribution

    Resources, Supervision

    Competing interests

    No competing interests declared

    "This ORCID iD identifies the author of this article:" 0000-0001-9091-2078

17. Willy Gsell

    Biomedical MRI, Department of Imaging and Pathology, KU Leuven, Leuven, Belgium

    Contribution

    Methodology

    Competing interests

    No competing interests declared

    "This ORCID iD identifies the author of this article:" 0000-0001-7334-6107

18. Neus Martínez-Abadías

    Departament de Biologia Evolutiva, Ecologia i Ciències Ambientals (BEECA), Facultat de Biologia, Universitat de Barcelona, Barcelona, Spain

    Contribution

    Conceptualization, Resources, Data curation, Supervision, Funding acquisition, Visualization, Writing – original draft, Writing – review and editing

    Contributed equally with

    Greetje Vande Velde

    For correspondence

    neusmartinez@ub.edu

    Competing interests

    No competing interests declared

    "This ORCID iD identifies the author of this article:" 0000-0003-3061-2123

19. Greetje Vande Velde

    Biomedical MRI, Department of Imaging and Pathology, KU Leuven, Leuven, Belgium

    Contribution

    Conceptualization, Resources, Data curation, Supervision, Funding acquisition, Visualization, Methodology, Writing – original draft, Project administration, Writing – review and editing

    Contributed equally with

    Neus Martínez-Abadías

    For correspondence

    greetje.vandevelde@kuleuven.be

    Competing interests

    No competing interests declared

    "This ORCID iD identifies the author of this article:" 0000-0002-5633-3993

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