Prenatal Treatment of Down Syndrome to Improve Brain Development and Neurocognition

National Human Genome Research Institute

Investigators

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Abstract

During the past year, we achieved the following objectives for each of our goals: 1) Deep molecular and cellular phenotyping of iPSCs and iPSC-derived neural progenitor cells (NPCs) from individuals with T21 and age and sex matched euploid individuals. We generated a collection of induced pluripotent stem cells (iPSCs) from individuals with trisomy 21 (T21) and age/sex matched euploid controls (Eup) and further differentiated them to neural progenitor cells (NPC). NPCs were used to generate stable cell lines expressing a fluorescent nuclear marker (NucLight Red) for use in live-cell imaging and drug screening. To gain better insights into the molecular mechanisms underlying atypical brain development in fetuses with DS, we continued to perform transcriptome analyses on fibroblasts, and both transcriptome and proteome analyses on iPSCs and NPCs. Pathway analyses showed dysregulation across the cell cycle, DNA damage/repair, inflammation, oxidative stress, mitochondrial dysfunction, and oxidative phosphorylation. We compared array, single cell RNA sequencing, and proteomics data and found a shared set of differentially expressed genes using the multiple technologies. Live-cell imaging assays of NPCs were used to validate the dysregulated pathways. Mitochondrial function was examined by measuring oxygen consumption and respiratory capacity. Assays to measure increased oxidative stress and antioxidant capacity are ongoing. Studies examining the altered cell type ratios in differentiation of T21 versus Eup NPCs to neurons and glia are ongoing. 2) Screening of the generated cell lines for therapeutic responses to several drug candidates identified using the Connectivity Map (CMap) database. We used the Connectivity Map (CMap) database to objectively select drug candidates that can rescue transcriptomic changes in Down syndrome. Transcriptome data from T21 and Eup NPCs allows us to identify additional drug candidates using the CMap and the Library of Integrated Network-Based Cellular Signatures (LINCS). We selected compounds to review for toxicity and teratogenicity using LINCS neuron and NPC specific signatures. To date, we have screened 12 drug candidates identified with the CMap and 12 identified with LINCS and evaluated each drugs cytotoxicity and efficacy in improving proliferation in T21 NPCs. A subset of drugs has been selected for further evaluation, and others have been eliminated from consideration due to toxicity. The goal of these studies is to identify several drug candidates that can be used in preclinical safety and efficacy studies using the best mouse model that is selected after the studies below are completed. 3) Deep molecular, cellular and behavioral phenotyping of several mouse models of DS to determine the one that most closely mimics the humans DS phenotype with an emphasis on the embryo and placenta. During this past year, we were able to continue and finalize the deep phenotyping across the lifespan of the Dp(16)1/Yey, Ts65Dn, Ts66Yah and Ts1Cje mouse models of Down syndrome (DS). These strains harbor a trisomy of overlapping mouse chromosome 16 (Mmu16) regions that are orthologous to human chromosome 21 (Hsa21). These models are all cytogenetically different from each other. As a result of their karyotypic differences, the Dp(16)1/Yey, Ts65Dn, Ts66Yah and Ts1Cje mouse models all exhibit distinct prenatal gene expression and postnatal phenotypes, however, they share several dysregulated pathways that could be targeted in future prenatal therapies, including neuroinflammation, interferon signaling, oxidative stress response and sirtuin pathway. This past year we focused on the novel Ts66Yah model that carries the identical segmental Mmu16 trisomy as Ts65Dn, but in which the Mmu17 non-orthologous region was removed using CRISPR/Cas9 technology. Our findings demonstrated that the Ts66Yah mouse model closely recapitulates the human DS karyotype even though it carries only 44% of Hsa21 orthologous genes. We demonstrated that the Ts65Dn mouse exhibits a more severe phenotype throughout the lifespan compared to the Ts66Yah mouse. Several Mmu17 non-orthologous genes were uniquely overexpressed in Ts65Dn embryonic forebrain; this produced major differences in dysregulated genes and pathways between the two strains. Despite these genome-wide differences, the primary Mmu16 trisomy effects were highly conserved in both models, resulting in several common dysregulated disomic genes and pathways. During the neonatal period, delays in motor development, communication and olfactory spatial memory were observed in both Ts66Yah and Ts65Dn pups, but were more pronounced in Ts65Dn. Adult Ts66Yah mice showed working memory deficits and sex-specific effects in exploratory behavior and spatial hippocampal memory, while long-term memory was preserved. Like the neonates, adult Ts66Yah mice exhibited fewer and milder behavioral deficits when compared to Ts65Dn mice. Our findings suggest that trisomy of the non-orthologous Mmu17 genes significantly contributes to the phenotype of the Ts65Dn model and may explain why preclinical trials that have primarily used this model have unsuccessfully translated to human therapies. We also established several translational outcome measures throughout the lifespan to evaluate motor development, communication, and learning/memory in mouse models of DS. Using these outcome measures, we showed that differences in karyotype and prenatal gene expression profiles were associated with distinct behavioral phenotypes the Dp(16)1/Yey, Ts65Dn, Ts66Yah and Ts1Cje mouse models. These outcome measures will be used to evaluate the effects of prenatal therapy in future studies. During the past year we completed our pilot touchscreen studies on Ts1Cje, Ts65Dn, and Dp(16)1/Yey mice and submitted them for publication. The results are currently under revision. Humans with DS exhibit learning deficits in the Cambridge Neuropsychological Test Automated Battery (CANTAB). We translated the CANTAB Visual Distinction (VD) and Extinction tasks using rodent touchscreen behavioral testing to investigate learning and inhibitory control. Dp(16)1/Yey, and Ts1Cje models did not demonstrate motivation or learning deficits during early pre-training, however, Ts1Cje mice showed a significant learning delay after the introduction of the incorrect stimulus (late pre-training), suggesting prefrontal cortex defects in this model. Both Dp(16)1/Yey and Ts1Cje mice display learning deficits in VD but these deficits were more pronounced in the Dp(16)1/Yey mouse model. Both mouse models also exhibit compulsive behavior and abnormal cortical inhibitory control during Extinction compared to WT. Ts65Dn mice outperformed WT in pre-training stages, largely by initiating a significantly higher number of trials due to their hyperactive behavior. Both Ts65Dn and WT showed poor performance during late pre-training and VD. These studies demonstrate significant learning deficits and compulsive behavior in the Ts1Cje and Dp(16)1/Yey mouse models of DS. They also demonstrate that the mouse genetic background (B6 vs. F1 hybrid) and the absence of hyperactive behavior are key determinants of successful learning in touchscreen behavioral testing. (4) Administration of promising candidate drugs identified in section (2) to the best mouse model of DS and evaluate its safety and efficacy. This will begin once the previous goals are accomplished.