Indiana University-Purdue University Indianapolis (IUPUI) / Down syndrome (DS) is caused by three copies of human chromosome 21 (Hsa21) and results in phenotypes including intellectual disability. Ts65Dn mice, the most extensively studied DS model, have three copies of ~50% of the genes on Hsa21 and display many phenotypes associated with DS, including cognitive deficits. DYRK1A is a dosage-sensitive gene found in three copies in humans with Trisomy 21 and in Ts65Dn mice and is involved in CNS development. Overexpression of DYRK1A is hypothesized to cause many of the cognitive and developmental deficits observed in DS and has been touted as a target for drug development in DS. Definitive evidence that excessive expression/activity of Dyrk1a directly contributes to specific phenotypes in DS mouse models is limited, and there is no direct evidence that verified pharmacological inhibition of Dyrk1a in vivo causes enduring improvement in DS cognitive phenotypes. In part, this reflects the remarkably limited knowledge of the temporal regulation of Dyrk1a expression and activity in different brain regions across development in DS mouse models. To establish the therapeutic potential of Dyrk1a inhibitors, the first aim of this study was to determine when and in what brain regions excessive Dyrk1a is evident and to identify developmental periods when elevated expression of Dyrk1a may be contributing to enduring aberrant functional development. This aim provided systematic quantification of Dyrk1a protein level at key postnatal (P) ages (P12, P15, P18, P24 P30, P42) in Ts65Dn mice, at ages of translational relevance to clinical applications in humans (birth, early adolescence, late adolescence, young adult). Western blot analysis showed that significant elevation of Dyrk1a with the largest effect sizes occurred in trisomic mice on P15. The second aim of this study was to test whether treating Ts65Dn with a novel Dyrk1a inhibitor (CX-4945) during the time of Dyrk1a elevation would rescue the behavioral and structural abnormalities observed. From P14-P18, Ts65Dn and euploid mice were treated with 75mg/kg CX-4945 or DMSO (vehicle) and tested on a homing task and locomotor activity in a novel arena on P17-P18, counterbalanced for order. At the cessation of treatment, hippocampal cell proliferation was assessed. While there was a lack of statistically significant improvements with CX-4945 treatment, there were modest effect sizes. In addition, several of the behavioral studies were significantly underpowered, making it difficult to conclusively ascertain the efficacy of CX-4945 on specific phenotypes. Nevertheless, this study demonstrates that Dyrk1a is dynamically expressed across development in mice, and suggests that consideration of the spatial and temporal expression of Dyrk1a may well be critical in the development of therapeutics for DS.

Indiana University-Purdue University Indianapolis (IUPUI) / Down syndrome (DS) is a genetic condition caused by the triplication of human chromosome 21 and presents with many phenotypes including decreased brain size, hypocellularity in the brain, and assorted ocular phenotypes. Some of the ocular phenotypes seen are increased risk of cataracts, accommodation difficulties, increased risk of refractive errors, and increased retinal thickness. The Ts65Dn mouse model of DS is a classically used mouse model as it presents a number of phenotypes also seen in those with DS. Some of these phenotypes include decreased brain volume, abnormal synaptic plasticity, and ocular phenotypes. These ocular phenotypes include decreased visual acuity, cataracts, and increased retinal thickness. The Ts65Dn mouse model is trisomic for Dyrk1a, a gene of interest in DS research. We hypothesize that there will be a genotypic and sex effect of retinal thickness and retinal ganglion cell (RGC) count at postnatal day 15 in the Ts65Dn mouse model of DS. Retinal slices were taken from male and female trisomic and euploid Ts65Dn mice at P15 and fluorescently labeled for RGCs and bipolar cells via immunohistochemistry. The retinas were measured for total retinal thickness and RNA-binding protein (RBPMS) positive cells in the RGC layer were counted. There was no genotypic or sex effect when comparing retinal thickness in trisomic mice as compared to euploid mice. There was a genotypic effect of RBPMS positive cell count in which the trisomic mice had a higher number of RBPMS positive cells than euploid mice. Increased retinal thickness along with increased RGC number have both been implicated with decreased apoptosis in the retina. In the Ts65Dn mouse model along with in individuals with DS, this could be due to an increase in DYRK1A protein levels reducing apoptosis. In future studies, determining DYRK1A’s influence in retinal thickness and RGC number could result in a treatment for overactive DYRK1A that could normalize retinal thickness and RGC number in those with DS.

<p dir="ltr">Down syndrome (DS) arises from the triplication of human chromosome 21 (Hsa21), leading to a spectrum of phenotypes characterized by neurodevelopmental and cognitive abnormalities. The Ts65Dn mouse model emulates DS by harboring three copies of genes found on Hsa21 resulting in trisomy 21 (Ts21)- like traits, including disruptions in neuronal pathways, delays in sensorimotor and behavior milestones, and deficits in learning and memory tasks. There is no cure for DS and available therapies primarily address symptoms stemming from Ts21-associated phenotypes. <i>DYRK1A</i>, a gene triplicated in Ts21, has a pivotal role in pathways of neurodevelopment and has been a focus of inhibition treatment research aimed at preempting abnormal brain phenotypes. This study aimed to find a point of substantial <i>Dyrk1a </i>expression dysregulation during a period of critical neonatal neurodevelopment and employ targeted pharmacological and genetic knockdown methods to alleviate the presence or severity of characteristically abnormal brain and behavior phenotypes. The hypothesis of this study was that administering a targeted intervention prior to a point of known overexpression in trisomic pups would ameliorate molecular, sensorimotor, and neurobehavioral deficits, redirecting growth trajectories of Ts65Dn neonatal pups towards more neurotypical outcomes. To test this hypothesis, the spatiotemporal pattern of DYRK1A expression was quantified during the first three weeks of neonatal development across the hippocampus, cerebral cortex, and cerebellum of the Ts65Dn mouse model and found to fluctuate according to the genotype, age, sex, and brain region of the subject. <i>Dyrk1a </i>protein and mRNA expression levels were delineated in trisomic animals by age, exploring the correlation between expression and age, sex, genotype, and brain region. Next a constitutive <i>Dyrk1a </i>knockdown model was integrated with the Ts65Dn model to investigate the impact of gene copy number reduction on protein and mRNA expression levels during phases of known DYRK1A dysregulation. On postnatal day 6, protein expression was rescued in all three brain regions of male animals but was rescued only in the cerebellum of females. There were no significant differences in mRNA transcript levels in either sex at this age. Finally, genetic elements were introduced into the Ts65Dn model to facilitate a spatiotemporally controlled functional reduction of <i>Dyrk1a</i> and discern how the timing of gene copy number reduction affects molecular and neurobehavioral development in a trisomic system. Results from these studies suggest that only functionally reducing <i>Dyrk1a </i>gene copy number on the day of birth is not sufficient to rescue the majority of deficits and delays present in the Ts65Dn mouse model of DS. These findings significantly enhance the understanding of trisomic <i>Dyrk1a </i>expression dynamics during neonatal development and shed light on tailored therapeutic approaches to modulate intrinsic DS characteristics based on age, sex, and phenotypic considerations.</p>

Motor deficits are a hallmark of Down syndrome (DS), yet little is known about their exact cause. Despite the rich understanding of the neurobiology of DS, there is still a lack of targetable mechanisms for early intervention aimed at alleviating motor changes in people with DS. Therefore, we utilized a mouse model of DS known as Ts65Dn to characterize for the first time the effects of trisomy 21 on spinal cord (SC) development. A central molecular player in SC patterning and cell-type specification, Oligodendrocyte transcription factor 2 (Olig2), is located on human chromosome 21 (Hsa21) and is triplicated in both people with DS and in Ts65Dn mice. To observe the effects of the supernumerary Olig2, we used immunohistochemistry to visualize the OLIG2-derived cellular populations (i.e., motor neurons (MNs) and oligodendrocytes (OLs)), as well as adjacent and interacting cell populations (i.e., ventral spinal interneurons (INs)). We limited our analyses to two embryonic ages—embryonic days (E) 12.5 and 14.5. Our results indicate that there is no overall change in the numbers of OLs at either E12.5 or E14.5. However, there tend to be more OL-fated cells within the pMN domain, where they originate, and migrating cells tend to be clustered closer to the pMN domain at E12.5. IN populations show some changes in Ts65Dn mice at E12.5, with both total and abventricular PAX6+ cell numbers and abventricular NKX2.2+ cell numbers increased in Ts65Dn embryos compared to euploid mice. However, at E14.5 the number of NKX2.2+ cells is unchanged. No difference in the NKX6.1+ population was seen at either time-point. In contrast, there are significant changes in the MN population at both E12.5 and E14.5. Specifically, at E12.5, the total ISL1+ MN population is significantly increased and shows altered regional distribution in the ventral horn of Ts65Dn SCs. Conversely, the Ts65Dn spinal MN population is normalized to euploid levels at E14.5. Overall, our results suggest that neurogenesis, gliogenesis, and cell-type specification of OLIG2-lineage cells are altered in the developing SC of Ts65Dn mice. Thus, this work identifies a novel target for future therapeutic interventions aimed at ameliorating motor changes in DS.

<p>The relationship between gene copy number and protein expression levels has not thoroughly been examined in humans or mouse models of Down syndrome (DS) in relationship to developmental changes in the trisomic brain. Found on human chromosome 21 (Hsa21) and triplicated in DS, Dual-specificity tyrosine-phosphorylated regulated kinase 1A (<i>DYRK1A)</i> has been linked in DS to neurological deficits by restricting cell growth and proliferation. Little information exists regarding DYRK1A during perinatal development and how its expression may lead to cognitive deficits, and none exists that explores the gene-to-protein relationship during these critical time periods. This study aims to 1) Quantify variable DYRK1A expression across development as a function of age, sex, and brain region in trisomic Ts65Dn mice compared to euploid counterparts and 2) establish that the spatiotemporal pattern of developmental DYRK1A in the brain is not influenced solely by gene copy number, and that reduction of <i>Dyrk1a</i> in euploid and trisomic mice does not result in a corresponding global reduction of DYRK1A expression. DYRK1A was quantified in three areas of the postnatal brain at seven ages using the Ts65Dn mouse, the most studied model of DS, and found that trisomic expression is significantly increased on postnatal day ([P]6), declining by the third week to near euploid levels. We also uncovered a sexual dimorphic expression of DYRK1A when comparing animals of different sexes within the same genotype. Data from <i>Dyrk1a</i> knockdown mice indicated that reducing only <i>Dyrk1a</i> in euploid and in otherwise trisomic animals yields highly variable levels of DYRK1A, dependent on sex and tissue type, supporting the non-intuitive relationship between gene dosage and protein expression. These data emphasize the need to understand the age-dependent regulation of antecedent conditions that are causing changes in <i>Dyrk1a</i> expression in the brain.</p>

<p>Down syndrome (DS) is a genetic condition caused by the triplication of human chromosome  21 and presents with many phenotypes including decreased brain size, hypocellularity in the brain,  and assorted ocular phenotypes. Some of the ocular phenotypes seen are increased risk of cataracts,  accommodation difficulties, increased risk of refractive errors, and increased retinal thickness. The  Ts65Dn mouse model of DS is a classically used mouse model as it presents a number of  phenotypes also seen in those with DS. Some of these phenotypes include decreased brain volume,  abnormal synaptic plasticity, and ocular phenotypes. These ocular phenotypes include decreased  visual acuity, cataracts, and increased retinal thickness. The Ts65Dn mouse model is trisomic for <em>Dyrk1a</em>, a gene of interest in DS research. We hypothesize that there will be a genotypic and sex effect of retinal thickness and retinal ganglion cell (RGC) count at postnatal day 15 in the Ts65Dn  mouse model of DS. Retinal slices were taken from male and female trisomic and euploid Ts65Dn  mice at P15 and fluorescently labeled for RGCs and bipolar cells via immunohistochemistry. The  retinas were measured for total retinal thickness and RNA-binding protein (RBPMS) positive cells in the RGC layer were counted. There was no genotypic or sex effect when comparing retinal  thickness in trisomic mice as compared to euploid mice. There was a genotypic effect of RBPMS  positive cell count in which the trisomic mice had a higher number of RBPMS positive cells than  euploid mice. Increased retinal thickness along with increased RGC number have both been  implicated with decreased apoptosis in the retina. In the Ts65Dn mouse model along with in  individuals with DS, this could be due to an increase in DYRK1A protein levels reducing apoptosis.  In future studies, determining DYRK1A’s influence in retinal thickness and RGC number could  result in a treatment for overactive <em>DYRK1A</em> that could normalize retinal thickness and RGC  number in those with DS.</p>

Indiana University-Purdue University Indianapolis (IUPUI) / Down syndrome (DS) is caused by three copies of human chromosome 21 (Hsa21) and results in an array of phenotypes including intellectual disability. Ts65Dn mice, the most extensively studied DS model, have three copies of ~50% of the genes on Hsa21 and display many phenotypes associated with DS, including cognitive deficits. DYRK1A is found in three copies in humans with Trisomy 21 and in Ts65Dn mice, and is involved in a number of critical pathways including CNS development and osteoclastogenesis. Epigallocatechin-3-gallate (EGCG), the main polyphenol in green tea, inhibits Dyrk1a activity. We have shown that a three-week EGCG treatment (~10mg/kg/day) during adolescence normalizes skeletal abnormalities in Ts65Dn mice, yet the same dose did not rescue deficits in the Morris water maze spatial learning task (MWM) or novel object recognition (NOR). Others have reported that An EGCG dose of 2-3 mg per day (90mg/ml) improved hippocampal-dependent task deficits in Ts65Dn mice. The current study investigated deficits in a radial arm maze pattern separation task in Ts65Dn mice. Pattern separation requires differentiation between similar memories acquired during learning episodes; distinguishing between these similar memories is thought to depend on distinctive encoding in the hippocampus. Pattern separation has been linked to functional activity of newly generated granule cells in the dentate gyrus. Recent studies in Ts65Dn mice have reported significant reductions in adult hippocampal neurogenesis, and after EGCG treatment, enhanced hippocampal neurogenesis. Thus, it was hypothesized that Ts65Dn mice would be impaired in the pattern separation task, and that EGCG would alleviate the pattern separation deficits seen in trisomic mice, in association with increased adult hippocampal neurogenesis. At weaning, Ts65Dn mice and euploid littermates were randomly assigned to the water control, or EGCG [0.4 mg/mL], with both treatments yielding average daily intakes of ~50 mg/kg/day. Beginning on postnatal day 75, all mice were trained on a radial arm maze-delayed non-matching-to-place pattern separation task. Euploid mice performed significantly better over training than Ts65Dn mice, including better performance at each of the three separations. EGCG did not significantly alleviate the pattern separation deficits in Ts65Dn mice. After the behavioral testing commenced, animals were given ad libitum food access for five days, received a 100mg/kg injection of BrdU, and were perfused two hours later. Coronal sections through the dorsal hippocampus were processed for BrdU labeling, and cells were manually counted throughout the subgranular zone of the dentate gyrus. The euploid controls had significantly more BrdU labeled cells than Ts65Dn mice, however, EGCG does not appear to increase proliferation of the hippocampal neuroprogenitor cells. This is the first report of deficits in Ts65Dn mice on a pattern separation task. To the extent that pattern separation depends on the functional involvement of newly generated neurons in an adult dentate gyrus, this approach in Ts65Dn mice may help identify more targeted pharmacotherapies for cognitive deficits in individuals with DS.