Prenatal Treatment of Down Syndrome to Improve Brain Development and Neurocognition

National Human Genome Research Institute

Investigators

Linked publications, trials & patents

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 large 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). We also generated stable NPC lines expressing a fluorescent nuclear marker (NucLight Red) and the cell cycle FUCCI construct (Cell Cycle Green/Red) for use in live-cell imaging. To gain better insights into the molecular mechanisms underlying atypical brain development in fetuses with DS, we performed transcriptome analyses on fibroblasts, iPSCs, and neural progenitor cells. Pathway analyses showed dysregulation across the cell cycle, DNA damage/repair, inflammation, oxidative stress, mitochondrial dysfunction and oxidative phosphorylation. We compared array and single cell RNA sequencing and found a shared set of differentially expressed genes using the two different technologies. Live-cell imaging of NPCs was used to validate the dysregulated pathways. 2) Screening of the generated cell lines for therapeutic responses to several drug candidates identified using the Connectivity Map (CMap) database. To objectively select drug candidates that can rescue transcriptomic changes in DS, we used the Connectivity Map (CMap) database. We are using our novel transcriptome data from T21 and Eup NPCs to identify more drug candidates using the CMap and its newer version, the Library of Integrated Network-Based Cellular Signatures (LINCS). To date, we have screened 12 drug candidates identified with the CMap to evaluate their cytotoxicity and efficacy in improving neurogenesis and rescuing the redox imbalance in T21 NPCs. Analysis is currently ongoing, but some drugs have 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. In collaboration with Yann Heraults lab (IGBMC, Strasbourg, France), we rederived a novel mouse model Ts66Yah (on a F1 genetic background) that carries the same Mmu16 trisomy as the Ts65Dn without the non-orthologous Mmu17 trisomic genes. In the past year, we expanded our fetal phenotyping and natural history studies to include the Ts66Yah mouse model. Congenital anomalies were only seen in trisomic embryos in the Ts66Yah mouse model. The most common anomalies in Ts66Yah embryos are renal pelvis dilatation, congenital heart defects, and hepatic congestion/inflammation. No macroscopic anomalies were observed in the brains (absence of microcephaly), lungs or spleens of euploid or trisomic embryos. To extend our previous gene expression findings in E15.5 embryonic forebrain, we analyzed E18.5 embryonic forebrains and placentas from the Ts1Cje, Ts65Dn, Dp(16)1/Yey and Ts66Yah mouse models. Analysis of gene expression changes in the Ts66Yah mouse confirmed the exclusive trisomy of Mmu16 orthologous genes. We also confirmed major genomic differences between these four mouse models and demonstrated that the Ts66Yah model is the one that mimics closely the human karyotype (freely segregating chromosome) and genotype (overexpression of only Mmu16 orthologous genes). Little overlap was found in dysregulated genes in the brain and placenta across strains, however, we were able to identify and validate several diploid genes that were consistently dysregulated in all tissues and mouse models analyzed. Those genes were also shown to be dysregulated in human T21 NPCs. Their contributions to the cellular and cognitive DS phenotypes are under extensive investigation. Comparative Pathway and network analyses in the human derived iPSCs, NSCs and the four different mouse models are ongoing. To evaluate motor development and coordination, communication and learning/memory in the four mouse models of DS, we are using high throughput behavioral testing, including FOX scale, ultrasonic vocalization (USV), motor development and contextual olfactory memory for neonates; LABORAS home cage 24h behavioral monitoring, conventional rodent behavioral testing and translational touchscreen paradigms in adult mice and littermate controls (WT). During the past year we have completed our pilot touchscreen studies on Ts1Cje, Ds65Tn, and Dp(16)1/Yey mice and submitted them for publication. 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. Analyses of motor development and USVs in the Ts66Yah mouse model showed milder motor and communication deficits in trisomic pups when compared to their WT littermates and the Ts65Dn mouse model. These data corroborate the potential contribution of non-Mmu16 orthologous genes to the severity of the Ts65Dn behavioral phenotypic deficits. In adulthood, we also showed that Ts66Yah have milder behavioral deficits in the open field, rotarod, fear conditioning, novel object recognition and Morris water maze tests. We are actively back-crossing the Ts66Yah on a C57BL/6J background to investigate the potential contribution of the genetic background to the Ts66Yah phenotype and compare this model to the Dp(16)1/Yey and Ts1Cje mouse models (both maintained on a C57BL/6J genetic background). (4) Administration of promising candidate drugs identified in section (2) to the best mouse model of DS and evaluate its safety and efficacy. During the past year we reported promising therapeutic effects of apigenin in both human amniocytes from fetuses with T21 and in the Ts1Cje mouse model (Guedj et al, 2020). We demonstrated that the therapeutic effects of apigenin are pleiotropic, resulting in decreased oxidative stress, activation of pro-proliferative and pro-neurogenic genes (KI67, Nestin, SOX2 and PAX6), reduction of the pro-inflammatory cytokines INFG, IL1A and IL12P70 through the inhibition of NFB signaling, and increase of the anti-inflammatory cytokines IL10 and IL12P40. Further studies are needed to refine dosing, as well as to demonstrate therapeutic effects in a second mouse model of DS.