Apigenin as a Candidate Prenatal Treatment for Trisomy 21: Effects in Human Amniocytes and the Ts1Cje Mouse Model [Mouse]

Down syndrome (DS), a genetic condition leading to intellectual disability, is characterized by triplication of human chromosome 21. Neuropathological hallmarks of DS include abnormal central nervous system development that manifests during gestation and extends throughout life. As a result, newborns and adults with DS exhibit cognitive and motor deficits and fail to meet typical developmental and lack independent life skills. In the last two decades, a number of preclinical treatment studies showed beneficial effects in the Ts65Dn mouse model. As of summer 2020, 13 pharmacological interventions have been tested with little evidence of success in humans with DS. Potential reasons for this failure may be related to the fact that these therapeutic interventions were carried out too late, and not during the prenatal and early postnatal critical periods for brain development. To date, no prenatal treatment studies have been reported in pregnant women carrying fetuses with T21. A limited number of prenatal treatment studies using fluoxetine, maternal choline supplementation and the neuroprotective peptides NAP and SAL have been described using the Ts65Dn mouse model of DS. In our previous studies, we integrated gene expression data from nine different cellular and tissue sources in both humans with DS and mouse models to identify common dysregulated signaling pathways and cellular processes. We demonstrated that pathway abnormalities associated with DS were the result of gene-dosage specific effects and the consequence of a global stress response with activation of compensatory mechanisms. To counteract these genome-wide abnormalities, we used the Connectivity Map database (www.broadinstitute.org/CMap) to discover molecules that could be repurposed to rescue the transcriptome and promote more typical brain development in individuals with DS. One of the molecules that had the most consistent negative scores (hence, negating the dysregulated gene expression signatures in DS) across tissues and species was apigenin (4’, 5, 7-trihydroxyflavone). Here, we hypothesized that prenatal treatment with apigenin would partly rescue the global gene expression dysregulation to improve neurogenesis and postnatal cognitive outcomes in DS. In the current study, we used a human in vitro cell model (amniocytes derived from live fetuses with Trisomy 21 and euploid fetuses) and the Ts1Cje mouse model of DS to evaluate the effects of apigenin as a potential therpay for prenatal brain defects and postnatal cognitive outcome in DS. We demonstrated that apigenin is a safe treatment that can rescue oxidative stress and total antioxidant capacity imbalance in human amniocytes from fetuses with T21. It also improved several postnatal behavioral deficits in the Ts1Cje mouse model. We also showed that apigenin achieves its therapeutic action by triggering the expression of neurogenic genes, suppressing inflammation via inhibiting NFκB and by reducing the production of pro-inflammatory cytokines, while promoting the production of anti-inflammatory cytokines, angiogenic and neurotrophic factors. For human studies, gene expression analysis was performed on treated and untreated amniocytes from fetuses with Trisomy 21 (T21) and age and sex matched euploid fetuses using the GeneChip® Human Transcriptome HT 2.0 array (Affymetrix, Santa Clara, CA). only one apigenin dose (2 µM) was evaluated. Twenty-eight arrays were used (seven T21 and seven euploid for both controls and treated). For mouse studies, total RNA was isolated from the developing forebrain using the RNA II kits following the manufacturer’s instructions (Macherey-Nagel, Bethlehem, PA). RNA was processed and hybridized on the GeneChip® Mouse Gene ST 1.0 array. Twenty arrays were used (5 WTPow, 5 Ts1CjePow, 5 WTApig, 5 Ts1CjeApig; 3 males and 2 females/group); each array corresponded to labeled cDNA from one sample. Analyses were performed using unpaired t-tests. Data were analyzed using a repeated-measures ANOVA that included genotype, treatment, and sample pairings between T21 and control samples. Probe sets for which either genotype or treatment was significant (at p<0.001 and a Benjamini-Hochberg False Discovery Rate (BH-FDR) of 10%) were considered as differentially expressed genes (DEX). Pathway analyses were carried out using the Database for Annotation, Visualization and Integrated Discovery (DAVID) and Ingenuity Pathway Analysis (IPA) on the top 1% up-/down-regulated genes (hereafter referred to as Marginally Expressed (MEX) Genes), and Gene Set Enrichment Analysis (GSEA) on the whole transcriptome.