Down syndrome, also known as trisomy 21, is the most common genetic cause of intellectual disability, affecting 1 in 700 newborns. In prior work, we used an integrated and lifespan approach based on gene expression data from multiple human cell types and tissues and fetal brain tissue from three different mouse models of DS (Dp (16)1Yey, Ts65Dn and Ts1Cje) to identify consistently dysregulated signaling pathways and cellular processes. These included cell cycle defects, inflammation, oxidative stress and mitochondrial dysfunction, among others. We also used the Connectivity Map database (www.broadinstitute.org/connectivity-map-cmap) to identify FDA-approved molecules that are predicted to counteract these pathway abnormalities and can be tested in vitro using human cells and in vivo using mouse models (Guedj et al, 2016 PMID 27586445). We identified 17 molecules predicted to rescue transcriptome changes in both humans and mice with DS (Guedj et al. 2016 PMID 27586445). We hypothesize that giving safe medications to pregnant women who are carrying fetuses diagnosed with trisomy 21 will reduce oxidative stress and inflammation, promote the production of new fetal nerve cells, and lead to improvement in brain growth, all of which will ultimately improve brain function, learning and memory after birth. During the past year we achieved the following objectives using both human cells and mouse models of Down syndrome. Objective 1: Laboratory Set-Up We continued to equip our new laboratory with general laboratory equipment, but also purchased some specific state-of-the-art equipment that will enable us to screen and test for prenatal therapies more effectively. To screen drugs in human cells from individuals with Down syndrome: In addition to general cell culture equipment, we acquired the Essen Bioscience IncuCyte live cell imaging system and the Beckman Coulter Biomek i5 liquid handler to be able to plate the cells, dispense the reagents/drugs, analyze image different aspects of cell biology (cell cycle, oxidative stress, mitochondria) and prepare DNA, RNA and proteins for downstream analyses. To perform molecular, cellular and behavioral phenotyping of mouse models of Down syndrome we purchased the Applied Biosystems QuantStudio 7 quantitative PCR machine to perform genotyping and analyze gene expression changes in both 96 and 384-well formats. We also purchased a cryostat and a Zeiss microscope equipped with the Stereo Investigator software to prepare tissue cryosections for staining and analyzing brain sections, respectively. Finally, to study behavioral endpoints in the mouse that can be translated to the behavioral assessment of infants and children with Down syndrome, we acquired the Metris BV Smart Chamber ultrasonic vocalization system (to analyze pup-mother communication) and the Lafayette operant learning system (to analyze complex cognitive function associated with specific brain regions). Objective 2: Achieving Full Staffing of Laboratory Team Due to the hiring limitations at NIH last year we were unable to hire new staff between January 2017 and January 2018. Once the limitations were eased, we were able to hire a post-doctoral fellow, Sarah Lee, PhD., and a new research associate, Sabina Khantsis. Our former post-baccalaureate student, Jason Swinderman, was accepted into many medical school programs. He ultimately chose to matriculate at the University of California San Francisco School of Medicine. In August our new post-bac, Monica Duran Martinez, joined us from the University of Puerto Rico. Objective 3: Establish a Human iPSC Biobank for Drug Testing Laboratory staff have undergone training in transformation of iPSCs to neurons. We have purchased cells from Coriell that have either normal chromosomes or trisomy 21. The transformed neurons will be used to evaluate effects of different therapies. Objective 4: Live Cell Imaging Our previous gene expression studies in cells from humans with DS have shown delayed cell cycles, increased oxidative stress and abnormal mitochondrial function. To analyze these abnormalities in living cells and investigate the effects of candidate drug treatments, we tested many live cell imaging compatible reagents, including NucLight, H2B-GFP and Fucci (cell cycle), Cytotox and Caspase 3/7 (apoptosis), Cell Rox, CM-H2DCFDA, Grx1-GFP and Orp1-GFP (oxidative stress) and MitoSox and MitoTracker (mitochondrial function). We defined the optimal concentrations for each reagent and will use them to investigate baseline differences between cells derived from humans with DS and euploid controls. These reagents will also be used to analyze the effects of treatment with several candidate drugs. Objective 5: Analysis of molecular, cellular and behavioral phenotypes in the Ts1Cje, Ts65Dn and Dp (16)1Yey Mouse Models of Down Syndrome To identify the best mouse model(s) for prenatal treatment, for the past 3 years we have been studying the three most commonly used mouse models of Down syndrome, including Ts1Cje, Ts65Dn and Dp (16)1Yey strains from the Jackson Laboratory. A comprehensive comparison of the brain anatomy, histology, gene expression, and behavior in these models, at 3 different points in the lifespan, was published this past year in Disease Models and Mechanisms. Objective 6: Publications in preparation The results of our first preclinical trial of treatment with the flavone, apigenin, on human amniocytes with trisomy 21 and in the Ts1Cje mouse model is expected to be submitted for publication soon. The results showed a reduction in oxidative stress, and some functional improvement of behavior in the Ts1Cje mouse.

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2
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2018
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Human Genome Research
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Aziz, Nadine M; Guedj, Faycal; Pennings, Jeroen L A et al. (2018) Lifespan analysis of brain development, gene expression and behavioral phenotypes in the Ts1Cje, Ts65Dn and Dp(16)1/Yey mouse models of Down syndrome. Dis Model Mech 11:
de Wert, Guido; Dondorp, Wybo; Bianchi, Diana W (2017) Fetal therapy for Down syndrome: an ethical exploration. Prenat Diagn 37:222-228
Ferr├ęs, Millie A; Bianchi, Diana W; Siegel, Ashley E et al. (2016) Perinatal Natural History of the Ts1Cje Mouse Model of Down Syndrome: Growth Restriction, Early Mortality, Heart Defects, and Delayed Development. PLoS One 11:e0168009