Dyrk1a is expressed in Camk2a-positive cells and its full inactivation in the glutamatergic neurons induces brain defects

Dyrk1a is ubiquitously expressed in different neuronal cell populations of the brain but with regional differences: the protein level being higher in the olfactory bulb, cerebellar cortex, cortical structures and granular and pyramidal cell layers of the hippocampus [35]. We checked DYRK1A expression in adult glutamatergic neurons, by co-immunohistochemical localisation with an antibody against CAMK2A and DYRK1A. In the wild-type adult mouse, both proteins were found in pyramidal and granular neurons of the hippocampus and dentate gyrus and in neurons of the cortex (S1 Fig).

We wanted to better understand the function of DYRK1A in adult glutamatergic neurons, circumventing the effect of Dyrk1a on early brain development. For this, we inactivated both copies of Dyrk1a using a conditional approach, to generate a full knock-out in those neurons. A floxed Dyrk1a allele (Dyrk1a^cKO allele) was designed such that exon 7 that codes for the serine/threonine protein kinase active site signature domain was flanked by two loxP sites (Fig 1A). We used the Tg(Camk2aCre)4Gsc transgene [33] to generate the Dyrk1a^Camk2aCre allele (shortened as Dyrk1a^C). This transgene starts to express the Cre recombinase at three weeks of age in the cortex and hippocampus, when most neural circuits are already formed, as described previously [33,36,37]. Our laboratory has previously shown that Cre deletion occurs in around 60% of the Camk2Cre-positive cells in the cortex and in around 80% of the Camk2Cre-positive cells of the hippocampus [38]. We checked the ability of the Cre to recombine the Dyrk1a floxed allele in Dyrk1a^{Camk2aCre/Camk2aCre} (recombination of both Dyrk1a^cKO alleles with the Cre recombinase; noted here Dyrk1a^C/C) mice. The generation of the deleted allele was detected by PCR analysis exclusively in brain areas where Camk2a is expressed (Fig 1B). Quantification of Dyrk1a mRNA in different brain regions confirmed that Dyrk1a is expressed at different relative levels in brain subregions (Fig 1C). Nevertheless, decrease of the Dyrk1a transcripts was found in the hippocampus, cortex and thalamus/hypophysis but not in the cerebellum of Dyrk1a^C/C mice (Fig 1C). Loss of the DYRK1A protein was confirmed in the hippocampus by Western blot analysis (Fig 1D) and immunohistology (Fig 1E). This reduction was more evident within the pyramidal cell layers of the CA1 and CA3 composed mostly of glutamatergic neurons.

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Fig 1. Generation of mice deficient for Dyrk1a in the glutamatergic neurons.

(A) Targeting strategy for conditional inactivation of Dyrk1a. Exon 7 containing the serine/threonine protein kinase active site was flanked with loxP sites (red arrowheads) in two steps: a targeted allele was first generated by homologous recombination in ES cells, then in vivo expression of the Flp recombinase resulted in recombinaison of the FRT sites (green arrowheads) and removal of the selection cassette (white box) generating the conditional allele (cKO). The knock-out allele (KO) was observed in the brain of Dyrk1a^C/C. Arrows represent primers for PCR genotyping. (B) Genomic DNA was isolated from different organs from a Dyrk1a^C/C mouse and genotyped for the presence of the knock-out allele with primers Lf and Er, giving a 232 bp PCR product for the KO allele. (C) Ratio of relative mRNA of Dyrk1a in different brain structures in Dyrk1a^C/C and disomic control mice. (D) Autoradiographic image and quantification of immunoblots of DYRK1A protein in the hippocampus of Dyrk1a^C/C mice relative to control mice. Band intensities were estimated using ImageJ and normalized against the loading control GAPDH. Data are presented as point plots with mean ± SD with unpaired Student’s t-test, *p<0.05, **p<0.01, ***p<0.001 (n = 5 ctrl and 4 Dyrk1a^C/C hippocampus for mRNA analysis and n = 3 per genotype for protein analysis). (E) DYRK1A immunohistochemistry of coronal brain sections around Bregma -1.5 mm (Paxinos adult mouse brain atlas, Franklin and Paxinos, 1997) at the level of the hippocampus and of the cortex from a control and a Dyrk1a^C/C mouse (2-month-old males). Expression of DYRK1A was detected in the cytoplasm of the neurons of the control (Ctrl) and a net decrease of expression was observed in Dyrk1a^C/C neurons as visible in the high-magnification images of the cortex (1), Cornus Ammonis 1(CA1, 2), Cornus Ammonis 3(CA3, 3) and Dentate Gyrus (DG, 4) (white arrows point at neurons with little or no DYRK1A signal). DYRK1A signal is in grey while the blue color corresponds to DAPI in the representative image of the analyzed brain region. DAPI signal was removed from high-magnification images to better visualize DYRK1A signal and nuclei hence appear as black dots on those images.

https://doi.org/10.1371/journal.pgen.1009777.g001

We analysed the implication of DYRK1A in glutamatergic neurons by looking at brain morphology and cognitive phenotypes. Brain weight was significantly decreased in Dyrk1a^C/C mice compared to control mice (90% of the control weight; Fig 2A). We selected sections at Bregma -1.5 to have the hippocampus and the cortex for morphometric analysis, as those two structures are known to be impacted in by Dyrk1a gene dosage change [31,39] (Fig 2B). Contouring of the brain unravelled reduced surface area of the total brain surface in Dyrk1a^C/C mice (~88% of control; Fig 2C). The area of the hippocampus including the cornus ammonis fields (CA1, CA2 and CA3) and dentate gyrus (DG) did not significantly differ between the two genotypes (Fig 2D). We measured the thickness of the oriens layer at the CA1, CA2 and CA3 levels, of the pyramidal layer at the CA1 level, of the radiatum layer, of the CA1 and DG molecular layers and of the granular layer of the DG and did not find any difference between control and Dyrk1a^C/C mice (S2A Fig). Within the hippocampus, the dorsal CA1 region has a central role in episodic-like memory formation and was shown to consolidate memory during object novelty discrimination [40–42]. We counted cells within a specific frame in the middle of the CA1, but found no difference in cell density (S2B Fig). Dyrk1a was reported to increase cell density in layer VI of the somatosensory cortex [39]. Specific decrease in cortical thickness was observed at the level of the dorsal motor cortex (~78% of controls, Fig 2E) and of the somatosensory cortex (~76% of controls, Fig 2E) whereas decrease in thickness at the more ventral auditory cortex level was not significant (Fig 2E). Measurements of the thickness of different layers in the somatosensory cortex (Fig 2F) indicate decrease in the thickness of molecular layer I, external granular and pyramidal layers II/III, internal pyramidal layer V and internal polymorphic layer VI (Fig 2G). Only the internal granular layer IV was found unchanged (Fig 2G). To investigate how change in cellularity might relate to cortical thickness, we counted the number of cells present in SSC layers II-III, V and VI. We found that cellular density in layers II-III, V and VI was increased by about 30% in Dyrk1a^C/C mice (Fig 3D). We confirmed that neuronal density was indeed increased in the SSC of Dyrk1a^C/C mice by labelling neurons with the NeuN marker and counting NeuN-positive cells (Fig 3B and 3E). We used S100b immunostaining to visualize astrocytes in the cortex to see if decreased cortical size could be due to decrease number of this cell population (Fig 3C and 3F). As for the total cell population and the neuronal population, we found the same number of S100b-positive cells in both genotypes resulting in an increased density in the cortex of Dyrk1a^C/C mice (Fig 3F).

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Fig 2. Consequence of Dyrk1a inactivation in glutamatergic neurons on brain morphology.

(A) Brain weight from male and female mice aged 3 months old (n = 7–9 per genotype). (B) Representative coronal sections of control (Ctrl) (left) and Dyrk1a^C/C (right) brains at Bregma -1.5 stained with cresyl violet and luxol blue that were used for measurements (Magnification 20X). (C) Dot plots of total brain area measurements (red line around the brain in B). (D) Dot plots of hippocampal areas (red area around hipp in B). (E) Measurements of the thickness of the cortex at the 3 levels represented by red lines in figure B. (F) Representative cresyl violet and luxol blue stained coronal sections of somatosensory cortex layers in control (Ctrl) and Dyrk1a^C/C brains at Bregma -1.5. (G) Measurements of the thickness of the different layers presented in F. Data are presented as point plots with mean ± SD with unpaired Student’s t-test, *p<0.05, **p<0.01, ***p<0.001 (n = 3 females per genotype). AUD: auditory cortex, SSC: somatosensory cortex, DMC: dorso motor cortex, Hipp: hippocampus.

https://doi.org/10.1371/journal.pgen.1009777.g002

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Fig 3. Analysis of cell density in the SSC of Dyrk1a^C/C mice.

Representative cresyl violet and luxol blue stained (A), NeuN-labeled (B) and S100b-labeled (C) coronal sections at Bregma -1.5 with the counting frames. (D-F) Relative density of cells counted within a frame of 0.1 cm width at the level of the SSC. Data are presented as point plots with mean ± SD with unpaired Student’s t-test, *p<0.05, **p<0.01, ***p<0.001 (n = 3 females aged 3 months per genotype).

https://doi.org/10.1371/journal.pgen.1009777.g003

Full Dyrk1a inactivation in the glutamatergic neurons impacts general behaviour and cognition

To analyse mouse behaviour in Dyrk1a^C/C mice, we first focused our attention on locomotor activity and exploratory activity. Measurement of horizontal, or vertical, locomotor activity during circadian cycle did not differ in Dyrk1a^C/C mice compared to control mice (S3A and S3B Fig). The analysis of exploratory behaviour in a novel environment (open field (OF) test) indicated normal locomotor activity for the Dyrk1a^C/C mice (S3C Fig) but their exploratory pattern was altered as they spent significantly more time in the centre of the OF (Fig 4A), suggesting a decreased anxiety. This phenotype was confirmed in the elevated plus maze with Dyrk1a^C/C mice spending significantly more time in the open arms than control mice (Fig 4B). In this test, Dyrk1a^C/C mice were also more active, visiting more arms than the control mice (Fig 4C). The locomotor performance was assessed in the rotarod task. Dyrk1a^C/C mice exhibited slightly better performance in this test than their control littermates, with an increase in latency to fall, indicating that motor balance is not affected in those mice (Fig 4D).

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Fig 4. Impact of Dyrk1a inactivation in glutamatergic neurons on general behavior, locomotor activity and cognition.

(A) The exploratory behavior of a new environment was analyzed by the percentage of time spent in the center of an open field over 30 min of test. Dyrk1a^C/C mice spent more time in the center of the arena, suggesting that they are less anxious. (B) Confirmation of the phenotype in a new group of mice using the Elevated Plus Maze test (EPM) with Dyrk1a^C/C mice showing a higher percentage of time spent in the open arms of the maze. (C) Mouse activity, measured by the number of entries in both open and closed arms, was increased in Dyrk1a^C/C animals. (D) Evaluation of the locomotor performance on the rotarod during consecutive trials with increased rotational speeds. The latency is the mean of 3 independent trials. Dyrk1a^C/C mice showed increased performance. (E) In the NOR test, the percentage of time spent exploring the familiar (FO) and the new (NO) objects show that control mice spend significantly more time on the NO while Dyrk1a^C/C mice do not make any difference between the two objects. (F) Exploration times of the two identical objects during the presentation phase of the NOR show that Dyrk1a^C/C mice tend to have an increased exploration time. (G) Total exploratory time of the two objects during the test indicates that the absence of object discrimination of the Dyrk1a^C/C mice is not due to a lack of interest of the objects. (H) Percentage of freezing time during the habituation phase (before the foot shock; basal level of activity) (Habituation; Mann-Whitney rank sum test, p = 0.71) and during the 6 min of contextual exposure 24 hours later indicate a deficit of contextual learning in Dyrk1a^C/C mice. (I-J) Pain sensitivity was evaluated by measuring the mouse latency to elicit a response to pain when put on a plate (52°C). In this test, Dyrk1a^C/C and Dyrk1a^+/- mice had a lower threshold than their control littermates. (K) Interest in social interaction was measured by the time spent sniffing the cage containing a congener (C1) during the Crawley test. This time was reduced for Dyrk1a^C/C mice compared to control mice (unpaired t-test p = 0.01) while the time spent exploring the empty cage (EC) was the same between the two groups (unpaired t-test p = 0.23). Data are presented as point plots with mean ± SD. Statistical analyses were done with unpaired Student’s t-test or Mann-Whitney rank sum test if normality test failed, except E: paired T-test FO vs NO and one sample T-test vs 50% mean (in red: ***p<0.001); A, D, H, I and K: tests were done on young males (1.5–3.5 months old depending on the test with animals aged ± 3 weeks), n = 8–10 per genotype,. B-C (EPM) and E-G (NOR) were done with another batch of males a 4 months of age, n = 18 and 16 controls for each test respectively and 13 Dyrk1a^C/C(2 animals were removed from the NOR test because they did not reach the minimum exploration time during the presentation session); *p<0.05, **p<0.01, ***p<0.001.

https://doi.org/10.1371/journal.pgen.1009777.g004

Impact of the loss of Dyrk1a in glutamatergic neurons on cognition was evaluated using different memory tests. Working memory was assessed by recording spontaneous alternation in the Y-maze. The percentage of alternation between the three arms was similar between Dyrk1a^C/C and control mice (S3D Fig) indicating a normal working memory in both genotypes. In this test, the number of visited arms during the 5 min session was not significantly different in the Dyrk1a^C/C mice compared to the controls, although those mice showed more variability (S3E Fig). Long-term explicit memory requiring the hippocampus and related medial and temporal lobe structures was tested with the novel object recognition test (NOR) with 24 hours delay. Although Dyrk1a^C/C mice showed as much interest exploring the objects during the presentation session (Fig 4E) and during the discrimination session (Fig 4F) compared to control animals, they did not make any difference between the two objects during the retention trial (Fig 4G) by contrast to control mice who spent significantly more time on the novel object compared to the familiar one. Thus, the NOR test unravelled a deficit in long-term explicit memory in the Dyrk1a^C/C mice. Then, we tested associative learning using the contextual fear-conditioning test. During the habituation, mice showed the same basal level of freezing whatever their genotypes were (Fig 4H, Habituation). However, Dyrk1a^C/C mice showed significantly less freezing than control mice during contextual discrimination, indicating poorer performance in contextual learning (Fig 4H, Context). During the cued learning, Dyrk1a^C/C mice responded like control mice to the conditioned stimulus, indicating normal cued fear (S3F Fig). As decreased freezing could be due to a deficit in pain sensitivity rather than a deficit in memory, we tested the mice in the hot plate test. Dyrk1a^C/C mice had a decreased latency to elicit a first response to noxious thermal stimulus, suggesting that they were more sensitive to pain than control mice (Fig 4I). As pain sensitivity was never tested in Dyrk1a knock-out heterozygous mice (shortened as Dyrk1a^+/-), we also tested those mice in the hot plate. We also found that those mice are more sensitive to pain (Fig 4J).

As in Human DYRK1A heterozygous mutations lead to autistic behaviour in MRD7, mouse sociability was investigated in this full inactivation of Dyrk1a in the glutamatergic neurons. We presented an empty cage and a cage containing a congener to the tested mouse and measured the time spent by the tested mouse to sniff either cage. Both Dyrk1a^C/C and control mice showed social preference as they spent significantly more time sniffing the cage containing the congener than the empty cage (S3G Fig, Social preference). However, the total amount of time spent with their congener was decreased in Dyrk1a^C/C mice compared to control mice whereas the time spent exploring the empty cage did not differ (Fig 4K). Preference for social novelty was tested by placing a new congener in the empty cage. Both genotypes spent significantly more time sniffing the cage containing the new congener compared to the cage with the familiar one (>60% of the time allocated for the new congener) (S3H Fig, Social novelty preference). There was also no significant difference in the total time control and transgenic animals spent sniffing both congeners (S3I Fig, Social contact).

Finally, as DYRK1A haploinsufficiency in human is causing epilepsy, we challenged the homozygous inactivation in Dyrk1a^C/C and control mice with two different doses of the seizure-provoking agent pentylenetetrazol (PTZ) and the occurrence of myoclonic, clonic and tonic seizures was scored. At both 30 mg/kg (S3J Fig) and 50 mg/kg (S3K Fig), Dyrk1a^C/C susceptibility to seizure was like control mice. Altogether, those results indicate that Dyrk1a full inactivation in glutamatergic neurons does not increase susceptibility to PTZ-induced seizure.

Hence, Dyrk1a inactivation in glutamatergic neurons only impacts specific cognitive function such as explicit long-term memory, contextual fear memory and exploratory behavior while having no impact in others such as working memory, social behaviour and epileptic susceptibility.

Dyrk1a inactivation in the glutamatergic neurons lowers expression of genes involved in neurotransmission in the hippocampus, while enhancing expression of genes implicated in the regulation of transcription

The hippocampus is a key structure in memory formation. Long-term object recognition memory analysed in the NOR test was shown to require interaction between the hippocampus and the perirhinal cortex [43–45] while contextual fear memory involves a neural circuit including the hippocampus, amygdala and medial prefrontal cortex [46]. Although we could not detect any morphological defect in the hippocampus of Dyrk1a^C/C mice, those mice are defective in both long-term recognition and contextual fear memories. To unravel the potential molecular mechanisms underlying the learning defects of Dyrk1a^C/C mice, we performed genome-wide transcriptional profiling (RNA-seq) of Dyrk1a^C/C and control mice in the hippocampus at postnatal day 30, as the brain is fully developed and mature at this stage. Analysis of the RNA-seq exon reads was performed using hypergeometric test and Bonferroni correction (DEseq algorithm, P<0.025) as previously published [38,47,48] and identified 297 up-regulated and 257 down-regulated genes in Dyrk1a^C/C compared with controls (S1 Table). To determine the putative cell types associated with the deregulated genes, we compared the sets of up- and down-regulated genes with the markers of hippocampal cell types obtained from single cell RNA-seq (see Materials and Methods). As a result, up-regulated genes were enriched in oligodendrocyte-expressed genes (hypergeometric test, bonferroni corrected, P<3.4E-13) whereas down-regulated genes were enriched in neuronal markers (hypergeometric test, bonferroni corrected, pyramidal markers P<2.3E-4, interneuronal markers P<8.3E-3) (S2 Table). This decrease in neuronal markers expression is not reflected by a decrease in neuronal cells in the hippocampus as we did not observe a decrease in the thickness of the pyramidal cell layers or the DG granular cell layer, and did not find a deficit in cell density within the CA1 of the Dyrk1a^C/C hippocampus (S2 Fig). Next, we performed GO enrichment analyses of the lists of up- and down-regulated genes with using a Benjamini cut-off of P < 0.05 (Table 1). The strongest enrichments for up-regulated genes were related to transcriptional regulation and DNA methylation. This category of genes did not have any overlap with the oligodendrocyte overexpressed genes at the exception of the SRY-related HMG-box transcription factor Sox8 and we did not find any enriched specific function for the list of the oligodendrocyte markers that are up-regulated in Dyrk1a^C/C hippocampi. We counted the number of Olig2+ cells in the corpus-callosum and found no difference between Dyrk1a^C/C and control mice, suggesting that increased oligodendrocyte markers is not due to an increased number of oligodendrocytes (S4 Fig). Using the list of up-regulated genes that are oligodendrocyte-specific, we searched if this list was enriched for specific biological processes with FunRich (http://funrich.org) and found that eight of the twenty-seven listed genes were implicated in cell communication and signal transduction, suggesting that Dyrk1a inactivation in glutamatergic neurons might have a secondary impact on communication between those neurons and oligodendrocytes. Interestingly, among up-regulated genes, we found Nr4a1 (Nurr77), Arc (Arg3.1), Npas4, Fos (cFos), Egr1 (Zif268) and Fosb, six immediate-early genes (IEGs) encoding proteins involved in transduction signals that are induced in response to a wide variety of cellular stimuli and that are implicated in neuronal plasticity. Looking at known late response genes known to be activated by NPAS4 [49] in glutamatergic neurons, only three out of thirty-four (10%) of them were significantly deregulated in the hippocampus of Dyrk1a^C/C compared to control mice (S4 Table), with Fam198b being up-regulated and Csrnp1and Slc2a1 being down-regulated. Among target genes of NPAS4 shared between excitatory and inhibitory cells, four out of twelve (~30%) that we looked at were found deregulated in the hippocampus of Dyrk1a^C/C compared to control mice (Lmo2 and Fosl2, up-regulated; Mylk and Nptx2, down-regulated). Down-regulated genes found in the hippocampus transcriptome of Dyrk1a^C/C mice were associated with presynaptic vesicle exocytosis, regulation of neurotransmitter levels and neuron projection, and pointed at a perturbation of chemical synaptic transmission via the deregulation of proteins involved in synaptic vesicle exocytosis. Particularly, genes coding for proteins of the SNARE complex (Snap25, Stx1a, Napa and Napb), regulating its activity (Doc2b, Snph) or implicated in vesicular synaptic cycle (Anxa7, Amph, Syn2, Syngr1) were found downregulated in the hippocampus of Dyrk1a^C/C mice. This complex is known to mediate synaptic vesicle docking and fusion with the presynaptic membrane during neuromediator release. The SNARE complex was recently found, also with NPAS4, as a common pathway misregulated in models of DS overexpressing DYRK1A [50].

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Table 1. Gene ontology enrichment derived from up- and down-regulated genes in the Dyrk1a^C/C hippocampus.

https://doi.org/10.1371/journal.pgen.1009777.t001

Behavioural defects are induced in Dyrk1a^C/+ mice while partial rescue of memory alterations is observed in Dp1Yey/Dyrk1a^C/+ mice

We investigated the respective consequences of Dyrk1a dosage in glutamatergic neurons on the cognitive phenotypes observed respectively in MRD7 and DS mouse models. We analysed mice heterozygous for the Dyrk1a knockout allele in glutamatergic neurons to investigate the implication of this gene in the cognitive phenotypes of MRD7. We also performed a rescue experiment consisting on the return to two copies of Dyrk1a in the glutamatergic neurons of Dp1Yey trisomic mice. For this, we compared animals carrying Dyrk1a^Camk2aCre/+ (noted Dyrk1a^C/+), Dp1Yey, and Dp1Yey/Dyrk1a^Camk2aCre/+ (noted Dp1Yey/Dyrk1a^C/+), with Dyrk1a^cKO/+ as controls in behavioural tests. Mouse locomotor behaviour was tested in the open-field (OF). Dyrk1a^C/+ mice did not show any significant difference in locomotor activity compared to controls. Surprisingly, whereas in our conditions Dp1Yey mice travelled the same total distance in the OF as control mice, Dp1Yey/Dyrk1a^C/+ mice travelled significantly more distance (Fig 5A), suggesting that those mice are hyperactive. The percentage of time spent by the mice in the centre of the open field arena did not differ between genotypes (Fig 5B), suggesting normal anxiety-related behaviour. Mice heterozygous for Dyrk1a in glutamatergic neurons presented the same behaviour as control mice, indicating that removing only one copy of Dyrk1a is not enough to trigger decreased anxiety, as observed in the complete knockout of Dyrk1a in glutamatergic neurons (Figs 5B vs 4A). Analysis of working memory was done using the Y maze test. All the four groups of mice visited the same number of arms during the test, suggesting a normal locomotor activity (S5A Fig). On the other hand, Dp1Yey mice showed lower percentage of spontaneous alternation as compared to control mice (Fig 5C), confirming the phenotype already observed in previous studies [50,51]. This decreased performance was not restored by Dyrk1a normalization in glutamatergic neurons (Fig 5C). Haploinsufficiency of Dyrk1a in those neurons, like the inactivation of the two copies of Dyrk1a, did not trigger any change in working memory (Fig 5C). We therefore also tested Dyrk1a^+/- mice in the same test. Dyrk1a^+/- animals showed the same activity (number of visited arms; S5B Fig) and the same level of alternation as their wild-type littermates, indicating a normal working memory (S5C Fig).

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Fig 5. Consequence of the normalization of Dyrk1a in glutamatergic neurons of Dp1Yey mice on animal cognition.

(A) The total distance travelled in the open field during a 30 min session is significantly increased in the Dp1Yey/Dyrk1a^C/+ mice compared to the control mice (Kruskal-Wallis One Way Analysis of Variance on Ranks, p = 0.009 with Dunn’s post hoc multiple comparison procedures versus control, Dp1Yey/Dyrk1^C//+ vs control, p = 0.007). (B) Percentage time spent in the center of the OF does not vary between genotypes (One way ANOVA, F(3, 40) = 2.76, p = 0.054). (C) Percentage of spontaneous alternation of the mice during a 5 min session in a Y-maze. Lower percentage of alternation was found in Dp1Yey mice and in Dp1Yey/Dyrk1a^C/+ indicating a deficit in working memory in Dp1Yey mice that is not rescued in Dp1Yey/Dyrk1a^C/+ mice (One way ANOVA, Holm-Sidak method for multiple comparisons versus control group, F(3,49) = 4.3, p = 0.009, Dp1Yey vs control q = 2.48, p = 0.03, Dp1Yey/Dyrk1a^C/+ q = 3.24, p = 0.006). (D-E) Novel object recognition was assessed with 24 hours time laps. (D) Time spent exploring the two identical objects during the first object presentation session was decreased in Dp1Yey/Dyrk1a^C/+ mice (Kruskal-Wallis One Way Analysis of Variance on Ranks, p = 0.02 with Dunn’s post hoc multiple comparison procedures versus control, Dp1Yey/Dyrk1a^C//+ vs control, p = 0.02). (E) When introducing the novel object during the retention period, both control and Dp1Yey/Dyrk1a^C/+ lines spent significantly more time exploring the novel object than the familiar one (Paired t-test novel object vs familiar object, ctrl p = 0.003, Dp1Yey/Dyrk1a^C/+ p = 0.02), whereas Dp1Yey and Dyrk1a^C/+ mice did not (Paired t-test novel object vs familiar object, Dp1Yey p = 0.57, Dyrk1a^C/+ p = 0.56), revealing a significant deficit in memory for both Dp1Yey and Dyrk1a^C/+ mice which is rescued in Dp1Yey/Dyrk1a^C/+ mice. (F) Mice were tested for social novelty preference. All the genotypes but Dp1Yey spent significantly more time sniffing the new congener (Paired t-test congener vs empty cage, ctrl p = 0.004, Dp1Yey p = 0.13, Dp1Yey/Dyrk1a^C/+ p = 0.002, Dyrk1a^C/+ p = 0.002). Data are presented as point plots with mean ± SD. (n = 8–15 per genotype, *p<0.05, **p<0.01). Males (in blue) and females (in red) are pooled in the same graph as the statistical analyses did not reveal significant effect of sex.

https://doi.org/10.1371/journal.pgen.1009777.g005

We further tested the mice in the NOR test for long term reference memory. Both control and Dp1Yey/Dyrk1a^C/+ mice showed a significant preferential exploration of the novel object during the retention trial (Fig 5E) whereas Dp1Yey and Dyrk1a^C/+ mice spent the same time on the two objects (Fig 5E). The deficit of novel object exploration during the retention phase in Dp1Yey and Dyrk1a^C/+ mice was not due to a lack of familiar object exploration during the presentation phase as both genotypes showed similar exploration times than control mice (Fig 5D). Only Dp1Yey/Dyrk1a^C/+ showed a slight decrease in object exploration compared to control mice during the presentation phase mice (Fig 5D), but this did not impair their retention capacity during the test phase. Hence, the deficit in object recognition in Dp1Yey mice could be rescued by normalization of Dyrk1a copy number in glutamatergic neurons and is also generated by the absence of one copy of the gene in the same neuronal cell line. In the fear conditioning test, all genotypes showed more freezing during the context phase after conditioning than during the habituation phase and no difference was observed between genotypes in the context response (S5D Fig). In the sociability 3-chambers test, all the four groups of mice showed preference for the cage containing the mouse rather than the empty cage (S5E Fig). No difference was found between the four groups in the total amount of time spent sniffing the cage containing the congener (S5F Fig). Hence, by contrast to Dyrk1a^C/C mice, Dyrk1a^C/+ mice do not present decreased social exploratory behaviour. Dp1Yey mice did not spend significantly more time with the novel mouse compared to the familiar one, indicating no preference for social novelty (Fig 5F). This phenotype was rescued by returning to two copies of Dyrk1a in glutamatergic neurons (Fig 5F). Dyrk1a^C/+ mice also showed preference for social novelty (Fig 5F).

Hence, both increase in Dyrk1a copy number in glutamatergic neurons of trisomic mice and haploinsufficiency of Dyrk1a in glutamatergic neurons impact explicit memory supporting a key role of Dyrk1a in glutamatergic function as a modulator of explicit memory, but other functions require normalization of Dyrk1a in other cell types to be restored.

Proteomic analysis suggests an impact of Dyrk1a gene dosage on synaptic activity

To examine the contribution of DYRK1A in molecular pathways linked to the cognitive phenotypes associated to T21 in the glutamatergic neurons, we performed proteomic profiling of the hippocampus of control, Dp1Yey, Dp1Yey/Dyrk1a^C/+ and Dyrk1a^C/+ mice. We identified 63 proteins that were up-regulated and 16 that were down-regulated in the hippocampi of Dp1Yey mice compared with controls. Among those, 40 of the up-regulated and 12 of the down-regulated proteins were back to control levels in Dp1Yey/Dyrk1a^C/+ hippocampi, while one up-regulated protein in Dp1Yey was down-regulated in Dp1Yey/Dyrk1a^C/+ and 4 down-regulated proteins in Dp1Yey were up-regulated in Dp1Yey/Dyrk1a^C/+ mice. We found 51 up-regulated and 7 down-regulated proteins in the hippocampus of Dyrk1a^C/+ mice. Eleven of those proteins (CAMK2A, ATP6V1C1, DPP3, ERGIC1, GPM6A, CENPV, RPS28, AGAP2, SNX6, ABCA1, BRK1) were also deregulated in Dp1Yey and back to normal level in Dp1Yey/Dyrk1a^C/+, suggesting that they are impacted by Dyrk1a copy number in glutamatergic neurons (Fig 6A and S5 Table). We performed GO enrichment analysis on the list of deregulated proteins using the ToppCluster website, selecting a Bonferroni cut-off of P<0.05. Enrichment analysis indicates that pathways and GO components that are mostly affected by Dyrk1a gene dosage are synaptic, dendritic and axonal components (Fig 6B and 6C; S6 Table). Normalization of Dyrk1a copy number in the glutamatergic neurons did not rescue specific pathways but had a more global effect with 50 to 80% of the proteins present in each Dp1Yey enriched GO returning to normal amount in Dp1Yey/Dyrk1a^C/+ mice (Fig 6B). Interestingly, decreased Dyrk1a gene dosage was found to impact pre-synaptic proteins as observed in the transcriptome of Dyrk1a^C/C hippocampi, whereas increased Dyrk1a gene dosage was associated with the post-synapse and growth cone (Fig 6C). Proteins enriched in the hippocampus of Dyrk1a^C/+ mice were linked to translational activity whereas increased Dyrk1a gene dosage was associated to ATPase activity (Fig 6C).

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Fig 6.

Proteomic analysis (A) Venn diagram showing the numbers of deregulated proteins in the different mouse models. The numbers of proteins shown for the Dp1Yey/Dyrk1a^C/+ model (in dark blue) correspond to proteins that were deregulated in the Dp1Yey and back to normal levels in this model (proteins that are regulated by Dyrk1a in the trisomy). Proteins deregulated by both Dyrk1a up and down-regulation (common to Dp1Yey, Dp1Yey/Dyrk1a^C/+ and Dyrk1a^C/+) are listed in the grey shaded box. (B) Radar plots of GO terms that are mostly enriched in the Dp1Yey model (in red with scale bar corresponding to the log of the p-value) and of the proportion of the proteins found deregulated in Dp1Yey which amount is normalized by the return in 2 copies in the Dp1Yey/Dyrk1a^C/+ model (in blue with scale bar corresponding to the % of Dp1Yey deregulated protein back to normal levels). (C) Visual representation of the GO enrichments for the deregulated proteins in Dp1Yey and Dyrk1a^C/+ hippocampi with connection between common terms. The different categories of GO are represented by different colors: pink for Cellular components”, blue for “Molecular functions”, green for “Pathways”, orange for “Biological processes” and grey for “Phenotypes”. The list of proteins in the box corresponds to proteins present in the common deregulated GO terms (in red deregulated proteins in Dp1Yey and in green proteins deregulated in Dyrk1a^C/+). (D) Western blots of DYRK1A, NR2B, PSD95, SYNGAP and CAMK2 proteins following IPs of wild-type mice brain extracts. We found in NR2B, PSD95 and CAMK2 in the IPs of DYRK1A. We also detected DYRK1A in the IPs of NR2B, PSD95 and SYNGAP.

https://doi.org/10.1371/journal.pgen.1009777.g006

Interaction of DYRK1A with post-synaptic proteins

Behavioural and proteomic analyses suggest a direct impact of DYRK1A at the glutamatergic synapse. Previous work from our laboratory already demonstrated a role of DYRK1A at the presynapse by showing interaction of DYRK1A with SYN1, a neuronal phosphoprotein associating with the cytoplasmic surface of the presynaptic vesicles and tethering them to the actin cytoskeleton [52,53], and with CAMK2 that was previously shown to phosphorylate SYN1 leading to the release of the vesicle pool [54–56]. Moreover, we also found that SYN1 was phosphorylated by DYRK1A on its S551 residue in vitro and in vivo, highlighting the role of DYRK1A in SYN1-dependent presynaptic vesicle trafficking [56]. CAMK2, deregulated in our proteomic analysis, is also present in the glutamatergic postsynapse and has a major role in the molecular cascade leading to LTP [57]. To investigate a potential role of DYRK1A at the postsynapse, we looked at DYRK1A protein interaction with CAMK2 and key proteins of the postsynaptic density complex (PSD), GLUN2B (NR2B), PSD95 and SYNGAP. We carried co-immunoprecipitation (co-IP) experiments with adult mouse brain lysates using antibodies against DYRK1A and these proteins and using GAPDH as a negative control. We found CAMK2A, NR2B and PSD95 present in the immunoprecipitates (IPs) of DYRK1A, while DYRK1A was found in the IPs of NR2B, PSD95 and SYNGAP (Fig 6D), showing that these proteins interact together.

Ethic statement

Animal research was performed in agreement with the EC directive 2010/63/UE86/609/CEE and in compliance with the animal welfare policies of the French Ministry of Agriculture (law 87 848). Behavioural experiments were approved by the local animal care, use and ethic committee of the IGBMC (Com’Eth, no.17, APAFIS 2012–069). The PTZ-induced seizures protocol received the accreditation number APAFIS#6321.

Mouse lines

The Dp(16Lipi-Zbtb21)1Yey (Dp1Yey) line was created by Yu and collaborators [96] and bears a 22.6 Mb segmental duplication of the Lipi-Zfp295 fragment of murine chromosome 16 syntenic to Hsa21 [79,96]. The transgenic Tg(Camk2-Cre)4Gsc line [33,97] expressing the Cre recombinase under the control of the Camk2a promoter was used to inactivate the targeted conditional knockout allele in glutamatergic neurons of the cortex and hippocampus after birth. The Dyrk1^tm1.ICS conditional knockout (noted Dyrk1a^cKO) was generated at the PHENOMIN- ICS (Institut Clinique de la Souris; Illkirch, France; www.phenomin.fr) in the frame of the Gencodys consortium (http://www.gencodys.eu/). The targeting vector was constructed as follows. A 1096 bps fragment encompassing exon 7 (ENSMUSE00001246185) was amplified by PCR (from BAC RP23-115D20 genomic DNA) and subcloned in an MCI proprietary vector. This MCI vector contains a LoxP site as well as a floxed and flipped Neomycin resistance cassette. A 3.8 kb fragment corresponding to the 3’ homology arm and 4.1 kb fragment corresponding to the 5’ homology arms were amplified by PCR and subcloned in step1 plasmid to generate the final targeting construct. The linearized construct was electroporated in C57BL/6N (B6N) mouse embryonic stem (ES) cells. After selection, targeted clones were identified by PCR using external primers and further confirmed by Southern blot with a Neo probe (5’ and 3’ digests) as well as a 5’ external probe. Two positive ES clones were injected into BALB/cN blastocysts. Resulting male chimeras were bred with Flp deleter females previously backcrossed in a C57BL/6N [98] (PMID: 10835623). Germline transmission of the conditional allele was obtained (Fig 1B). The Flp transgene was segregated by a further breeding step. Dyrk1a constitutive heterozygous knockout (Dyrk1a^+/-) mice were generated by mating Dyrk1a^cKO/+ animals with the Hprt1^tm1(Cre)Mnn line (http://jaxmice.jax.org/strain/004302.html) to obtain a germline deletion of Dyrk1a. As Dyrk1a^+/- animals could not be generated on a pure background due to lethality, Hprt1^tm1(Cre)Mnn females and Dyrk1a^cKO/+ males were first crossed with the C₃H/HeH line (C3B line; congenic line for the BALB/c allele at the Pde6b locus [99] to generate a F1 B6C3B mixed background before being mated together. Dyrk1a^+/- were kept on this mixed background by crossing Dyrk1a^+/- with wild-type F1 B6C3B animals. Validation of the Dyrk1a^+/- model is presented in S6 Fig.

Animals for behavioural analysis were obtained by first mating Tg(Camk2-Cre)4Gsc males with Dyrk1a^cKO/cKO females to generate Dyrk1a^C/+ animals. Those animals were tested for the absence of the Dyrk1a KO allele by genotyping in order to avoid to use animals that could have Dyrk1a germline recombination. Dyrk1a^C/+ males were further mated with Dyrk1a^cKO/cKO females to obtain Dyrk1a^C/C animals as well as Dyrk1a^cKO/+ and Dyrk1a^cKO/cKO that were used as controls (S10 Table). Dyrk1a^C/+ females obtained during the first round of mating were further mated with Dp1Yey males in order to produce mice: Dp1Yey (trisomic, for the Lipi-Zfp295 fragment containing the Dyrk1a gene), Dp1Yey/Dyrk1a^C/+ (trisomic for the Lipi-Zfp295 fragment but containing only two copies of Dyrk1a in glutamatergic neurons), Dyrk1a^C/+ (containing only one copy of Dyrk1a in the glutamatergic neurons) and Dyrk1a^C/C (knocked out for Dyrk1a in glutamatergic neurons). Wild-type, Dyrk1a^cKO/+ and Dyrk1a^cKO/cKO mice were used as disomic controls (S7 Table).

For the genotyping of the mice and identification of the Dyrk1a knockout allele in the brain, genomic DNA was isolated from tail and different organ biopsies using the NaCl precipitation technique. 50–100 ng of genomic DNA was used for PCR. Primers used for the identification of each allele and size of PCR products are described in Fig 1 and S8 Table. Details on the genotyping protocol used here are published [100].

The mice were housed in groups (2–4 per cage) and were maintained under specific pathogen-free (SPF) conditions and were treated in compliance with the animal welfare policies of the French Ministry of Agriculture 133 (law 87 848).

Mouse RT droplet digital PCR (ddPCR)

Total RNA was extracted from frozen brain tissues of young adult mice (males and females, 3 months old) (cerebellum, cortex, striatum, hippocampus and thalamus/hypothalamus) of five wt and four Dyrk1a^C/C mice as described in Lindner et al. 2020 [101]. For ddPCR, all primers were designed and synthesized as described in Lindner et al. 2020 [101] excepting Universal Probe Library probe used to Dyrk1a mRNA which is provided by Roche. Dyrk1a and Hprt primers and probes sequences are given in S8 Table. RNA reverse transcription, droplet generation, PCR amplification, droplets quantification and analysis are also described in Lindner et al 2020 [101]. We presented the results as a ratio of the mean of Dyrk1a RNA transcript in Dyrk1a^C/C tissue normalized to the mean of Dyrk1a RNA transcript in wt tissue. Experiments were performed following dMIQE guidelines for reporting ddPCR experiments (S9 Table) [101,102].

Western blot analysis

Twenty-five micrograms of total protein extracts from hippocampi (young adult males and females 3 months old, n = 3 per genotype) were electrophoretically separated in SDS-polyacrylamide gels (10%) and transferred to a nitrocellulose membrane (100V, 2h at room temperature). Non-specific binding sites were blocked with 5% skimmed milk in Tween20 0.1% Tris buffer saline 1h at room temperature. Immunostaining was carried out with a mouse monoclonal anti-Dyrk1a (Abnova, H00001859-M01) and an anti-Gapdh antibodies (ThermoFisher, MA5-15738), followed by secondary anti-mouse IgG conjugated with horseradish peroxidase (DAKO). The immunoreactions were visualized by ECL chemiluminescence system (Amersham) with the Amersham Imager 600. Semi-quantitative analysis was performed using ImageJ software (W. Rasband, NIH; http://rsb.info.nih.gov/ij/).

Immunohistological analysis

Young adult female mice (3 months old) for immunohistochemistry and young adult males (2-month-old) for immunofluorescence were deeply anesthetized with sodium pentobarbital and perfused intracardially with 30 ml PBS followed by 30 ml 4% paraformaldehyde in PBS. Brains were removed from the skull and immersed in the same fixative overnight. After rinsing with PBS, the brains were transferred into 70% ethanol until paraffin inclusion. For inclusion, brains were dehydrated and embedded in paraffin. Serial 10 μm sections were made with a microtome.

Brain sections were stained using the myelin-specific dye luxol fast blue and the Nissl staining cresyl violet. Briefly, brain sections were deparaffinised, rehydrated and incubated in 0.1% luxol fast blue (95% alcohol and 0.5% acetic acid) solution at room temperature overnight. After rinsing excess stain with 95% ethanol and deionized water, the slides were placed in 0.05% lithium carbonate solution for 10 seconds followed by 70% ethanol for 5 seconds. They were then rinsed in deionized water until the colourless grey matter contrasted with the blue-green white matter. Sections were then stained in 0.1% cresyl violet acetate solution for 5 minutes at 56°C in a water bath, rinsed in deionized water and quickly in 100% ethanol. Sections were dried, cleared and mounted.

Immunohistology was performed using a standard protocol. After deparaffinization, rehydration and antigen retrieval (10 mM citric acid, 0.05% Tween 20, pH6.0) for 45 min in a 94°C water bath, sections were incubated in a blocking solution (0.05% Tween20, 5% Horse serum) for 1 hour at room temperature. Sections were then incubated at 4°C overnight with the primary antibodies (Mouse anti-DYRK1A: Abnova, Cat. N. H00001859-M01; Rabbit anti-CAMK2A: Molecular probes, PA5-14315). After washing, sections were incubated with anti-Mouse and anti-Rabbit Alexa fluorescent 546 or 488 secondary antibodies for detection (Abcam, Paris). Control immunostainings were also performed with secondary antibodies only. The sections were mounted with Mowiol mounting medium (0.1M Tris (pH8.5, 25% glycerol, 10% w/v Mowiol 4–88 (Citifluor)) containing DAPI (5 μg/ml) and images were acquired using Hamamatsu Nanozoomer 2.0 (Hamamatsu, Hamamatsu City, Japan) and a Leica Upright fluorescent microscope (Leica Microsystems, Heidelberg).

Immunohistochemistry was performed using a standard protocol. Briefly, antigen retrieval was performed by heating the slides in Tris/EDTA buffer (10 mM Tris Base, 1 mM EDTA, 0.05% Tween 20, pH 9.0) or citrate buffer 1 mM, pH6 for 45 min in a 94°C water bath. Then, the sections were quenched in 0.3% oxygen peroxide solution for 20 min and blocked with 10% normal horse serum and 0.1% Triton X-100 in 1× PBS for 1 h at room temperature. The sections were incubated overnight at 4°C with primary antibodies (rabbit anti-Olig2, 1:500, Santa Cruz sc-48817; Rabbit anti NeuN, 1:500 Sigma, ZRB377; Rabbit anti-S100b, 1:1000, Sigma HPA015768). which was detected by incubating the sections with secondary biotinylated antibodies (Life Technologies, France) for 2 h at room temperature and then with an avidin-biotin complex at 37°C for 30 min. Dark coloration was developed with diaminobenzidine tetrahydrochloride and the sections were mounted with aqueous mounting medium (Agilent, Les Ulis, France).

Morphometric analysis and cell counting

Morphometric analysis was performed on three Dyrk1a^C/C and three control adult females mice (3 months old) based on the standard operating procedures for morphological phenotyping of the mouse brain using basic histology [103]. Coronal sections for morphometric analysis and cell counting were carefully selected using specific anatomical landmarks that enables to precisely select slides that are at the same antero-posterior level (sections showing upper and lower arms of the dentate gyrus of the same length). Surface and cortical thickness measurements as well as cell counting were conducted on scanned images using Hamamatsu Nanozoomer 2.0 from luxol fast blue/cresyl violet-stained and NeuN-labeled and S100b-labeled sections around Bregma -1.5 mm (Paxinos adult mouse brain atlas, Franklin and Paxinos, 1997). TIFF files were opened in ImageJ with the following settings: 9 decimal places (using the panel Analyze/Set Measurements) and “cm” as unit length (using Analyze/Set Scale). The polygone selection tool was used to measure area and the straight-line tool was selected to measure length. The thickness of the different cortical layers (layer I to layer VI) were estimated in the somatosensory cortex based on the shape and density of the neurons on these different layers. Cell count performed in the somatosensory cortex was done within a counting frame of 0.1 cm. Cell count performed in the CA1 was done within a counting frame of 0.04 cm width). Olig2+-positive cells within the corpus callosum were counted by measuring a distance of 1 mm from the midline of the brain and selecting the corpus callosum area underneath. Cell count was done manually. It should be noted that cell counts were not acquired using randomly sampled unbiased stereological standards and therefore cannot necessarily be extrapolated to the entire cortex and hippocampus.

RNA-seq libraries and analysis

Total RNA was Trizol-extracted from 2 wild-type and 2 Dyrk1a^C/C frozen P30 young adult male hippocampi, stage at which the brain is fully developed. RNA was treated with DNase (Qiagen) and purified on the RNeasy MinElute Cleanup Kit (Qiagen). 2 μg of total RNA were treated with the Ribo-Zero rRNA Removal Kit (Human/Mouse/Rat; Illumina). Depleted RNA was precipitated 1h at -80°C in three volumes of ethanol plus 1 μg of glycogen. RNA was then washed and resuspended in 36 μl of RNAse free water. RNA fragmentation buffer (NEBNext, New England Biolabs, Evry, France) was added to the solution and the RNA was fragmented by incubation at 95°C for 3 min. cDNA first strand synthesis was performed with random hexamer primers and cDNA second strand synthesis was performed with dUTPs, to ensure strand specificity. The RNA-seq library was synthetized with KAPA Hyper prep kit (Kapa Biosystems, Wilmington, MA, USA): a treatment with USER enzyme (NEB, M5505L) was added to digest the unspecific strand.

The libraries were pooled (4/lane) on an Illumina HiSeq. 2000. Libraries were sequenced (50 cycles, single-end) yielding on average 40 million mapped reads. RNA-Seq libraries were mapped with GSNAP (version 2015-06-23) against mm9 mouse RefSeq annotations updated to the 28/7/2015.

DESeq 2 (v1.14) was used to perform statistical comparisons. All the enrichment analysis were made from standard hypergeometric tests with benjamini or bonferroni correction. The markers of hippocampal cell types were obtained from [104] and the common background genes were evaluated prior to the enrichment (hypergeometric test). GO annotations were updated to 25/6/2015.

Proteomic analysis

Fourty micrograms of total protein extracts from hippocampus of 4 months old males coming from cohorts that underwent behavioural analysis (4 controls, 5 Dp1Yey, 4 Dp1Yey; Dyrk1a^C/+ and 5 Dyrk1a^C/+) were used for the preparation. Samples were precipitated, reduced, alkylated and digested with LysC and trypsin at 37°C overnight. 10 μg of each sample were then labeled with TMT isobaric tags, pooled, desalted on a C18 spin-column and dried on a speed-vacuum before nanoLC-MS/MS analysis. Samples were separated on a C18 Accucore nano-column (75 μm ID x50 cm, 2.6 μm, 150 Å, Thermo Fisher Scientific) coupled in line with an Orbitrap ELITE mass spectrometer (Thermo Scientific, San Jose, California). Samples were analyzed in a Top15 HCD (High Collision Dissociation) mass spectrometry on 8h gradient. Data were processed by database searching using SequestHT (Thermo Fisher Scientific) with Proteome Discoverer 1.4 software (Thermo Fisher Scientific) against a mouse Swissprot database (release 2015–03). Peptides were filtered at 5% false discovery rate (FDR) and one peptide in rank 1. Protein quantitation (ratio of the intensity of the fragmented tag in sample “x” to the intensity of the fragmented tag in one control (disomic) sample used as the reference) was performed with reporter ions quantifier node in Proteome Discoverer 1.4 software with integration tolerance of 20 ppm, and the purity correction factor were applied according to the manufacturer’s instructions. A scaling factor normalization method was used in order to make sample ratios comparable. Ratios were normalized by calculating the mean of all the peptide ratios in one sample, calculating a scaling factor (sf = mean [ratio control ref]/mean [ratio sample x]) for each sample and multiplying each ratio by the sf. Data were filtered with the following criteria: minimum number of peptide ratios used to calculate the protein ratio equal to 2; variability of the peptide ratios <20%; ratio of Dp1Yey and Dyrk1a^C/+ samples compared to mean of disomic controls, x>1.2 or x<0.8 and ratio of Dp1Yey;Dyrk1a^C/+ samples compared to mean of disomic controls 0.8<x<1.2 among proteins that were selected as deregulated in Dp1Yey. GO enrichment was calculated in the ToppCluster website (https://toppcluster.cchmc.org/), looking at enrichment within the following features: Molecular functions, Biological processes, Cellular components, Phenotypes and Pathways, and using a Bonferroni correction cut-off of P<0.05. The results of the enrichments can be found in the S6 Table.

Co-immunoprecipitation

Immunoprecipitations were performed on fresh half brains of 3-month-old wild-type male mice. Brains were dissected and lysed in 1.2 ml RIPA lysis buffer (Santa-Cruz Biotechnology, France) using Precellys homogenizer tubes (Bertin Instruments, Montigny-le-Bretonneux, France). After centrifugation at 2800 g for 2×15 s, 1 ml brain extract was incubated with 2 μg of antibody of interest at 4°C for 1 h under gentle rotation. An aliquot of the remaining supernatant was kept for further immunoblotting as homogenate control. Then, 20 μl protein G agarose beads, previously washed three times with bead buffer, were added to the mix and gently rotated at 4°C for 30 min. After a 1 min spin at 10,000 g and removal of the supernatant, the pelleted immune complexes were washed three times with bead buffer before WB analysis with appropriate antibodies directed against DYRK1A (H00001859 M01, Interchim; 1:1000), NMDAR2B (Abcam, #ab65783), PSD95 (ab18258, Abcam, France; 1:1000), CAMK2A (PA5-14315, Thermo Fisher Scientific; 1:1000), SYNGAP (sc-8572, Santa Cruz biotechnologies; 1:5000) and GAPDH (MA5-15738, Thermo Fisher Scientific; 1:3000). Immunoblots were revealed with Clarity Western ECL Substrate (Bio-Rad).

Mouse behavioural analysis

A series of behavioural experiments were conducted in adult mice. Behavioural analyses of Dyrk1a^C/C animals were performed with males only. Both males and females were used for the behavioural analysis of the Dyrk1a rescue experiment in Dp1Yey glutamatergic neurons. Due to the difficulty to obtain Dp1Yey; Dyrk1a^C/+ mice (three alleles combination and subfertility of the Dp1Yey line with only around 30% transmission of the Dp1Yey allele) and according to the fact that no sex effect was observed in behavior tests done another trisomic model [105], both males and females were pooled for statistical analysis (males and females are represented with different colours on the graphs). Number of animals and age of the animals for each test are given in S10 Table. Protocols for the different tests are described in S1 Materials and Methods. For all these tests, mice were kept in ventilated cages with free access to food and water. The light cycle was controlled as 12 h light and 12 h dark (lights on at 7:00 AM) and the tests were conducted between 8:00 AM and 4:00 PM. Animals were transferred to the experimental room 30 min before each experimental test. Behavioural experimenters were blinded as to the genetic status of the animals. All the standard operating procedures for breeding and behavioural phenotyping have been already described [47,106,107] and are detailed in S1 Materials and Methods.

Statistical analysis

Statistical analyses were performed using SigmaPlot software. For histological assessments and behavioral tests comparing Dyrk1a^{Camk2aCre/Camk2aCre} animals to controls, statistical analyses were performed using unpaired t-test when appropriate or the non-parametric Mann-Whitney rank sum test unless otherwise stated in the text. For the four groups analyses (Dp1Yey, Dp1Yey, Dp1Yey; Dyrk1a^C/+, Dp1Yey, Dyrk1a^C/+and controls) a two-way ANOVA did not reveal a significant effect of sex and no interaction with the genotype. Therefore, the sex factor was dropped from the model and a one-way ANOVA and post hoc Tukey’s multiple comparison test were used to analyse differences between the four genotype groups.