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Prenatal Treatment of Down Syndrome to Improve Brain Development and Neurocognition

唐氏综合症的产前治疗可改善大脑发育和神经认知

基本信息

批准号：
9589746
负责人：
Diana Bianchi
金额：
$ 117.42万
依托单位：
NATIONAL HUMAN GENOME RESEARCH INSTITUTE
依托单位国家：
美国
项目类别：
财政年份：
资助国家：
美国
起止时间：
至
项目状态：
未结题

来源：
https://reporter.nih.gov/project-details/9589746
关键词：
Adult Affect Age Amniotic Fluid Animals Apigenin Apoptosis Behavior Behavior assessment Behavioral Biological Sciences Birth Boston Brain Brain region CASP3 gene Cell Culture Techniques Cell Cycle Cell Density Cell Line Cell physiology Cells Cellular biology Cerebellum Cerebral cortex Child Chromosomes, Human, Pair 21 Clinical Cognition Communication Complex Computer software DNA Data Databases Defect Development Diagnosis Discrimination Down Syndrome Equipment FDA approved Fetus Future Gene Expression Genes Genetic Genotype Gestational Age Goals Growth Hippocampus (Brain)Histology Hospitals Human Image Individual Infant Inflammation Intellectual functioning disability Laboratories Lead Learning Life Liquid substance Longevity Manuscripts Maps Medical center Memory Microscope Mitochondria Modeling Molecular Molecular Analysis Mothers Mus National Human Genome Research Institute Neurocognition Neuronal Differentiation Neurons Newborn Infant Oxidative Stress Pathway interactions Pharmaceutical Preparations Pharmacology Pharmacotherapy Phenotype Postdoctoral Fellow Preclinical Drug Evaluation Pregnancy Pregnant Women Prenatal Diagnosis Process Production Proteins Publications RNA Reagent Research Assistant Research Personnel Rodent Scientist Second Pregnancy Trimester Signal Pathway Somatosensory Cortex Staining method Stains Synaptic plasticity System Testing The Jackson Laboratory Therapeutic Therapeutic Intervention Thick Tissues Transcript Translating Ultrasonics Visual Woman Work base behavioral study biobank brain abnormalities brain tissue cell type cognitive function cryostat drug candidate drug testing experimental study fetal frontal lobe human data imaging system improved in utero in vitro testing in vivo laboratory equipment lateral ventricle learning extinction live cell imaging material transfer agreement mitochondrial dysfunction morphometry mouse Trisomy 16 mouse Ts65Dn mouse model myelination neurogenesis neuroinflammation novel personalized medicine postnatal preclinical trial prenatal prenatal testing prenatal therapy programs pup response screening sex synaptogenesis touchscreen transcriptome treatment effect vocalization

项目摘要

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 (https://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. We established our laboratory at the National Human Genome Research Institute (NHGRI) on January 9th, 2017, and during the past eight months we achieved the following objectives: Objective 1: Laboratory Set-Up We equipped 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 Lafayette operant learning system (to analyze complex cognitive function associated with specific brain regions). Objective 2: Hiring staff Dr. Faycal Guedj and research assistant Ashley Siegel moved with Dr. Bianchi from Tufts Medical Center (Boston) to establish the new laboratory. Dr. Guedj was appointed as a Staff Scientist and Ms. Siegel was hired as an Animal Biologist. In February 2017, Mrs. Nicole Reed joined the laboratory as a Laboratory Manager. Mr. Jason Swinderman was appointed as a Post-Baccalaureate fellow in March 2017. We are in the process of hiring post-doctoral fellows with expertise in human cell culture, drug screening and rodent behavior and histology. Objective 3: Establish a Human Amniocyte Biobank for Drug Testing Following Material Transfer Agreements between NHGRI and Tufts Medical Center (Boston, MA) and between NHGRI and Women and Infants Hospital (Providence, RI), we received discarded anonymized amniocytes obtained for clinical indications and established a biobank. The cells have either normal chromosomes or trisomy 21. We are currently analyzing phenotypic variability between different individual cell lines and are testing the hypothesis that individualized therapeutic interventions (personalized medicine) may be better than one therapeutic molecule for all. 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, we purchased the three most commonly used mouse models of Down syndrome, including Ts1Cje, Ts65Dn and Dp (16)1Yey strains from the Jackson Laboratory. We had previously used these models in our former laboratory at Tufts Medical Center. Preliminary evidence suggests that there are significant phenotypic differences between these three models. For example, Dp (16)1Yey has no observable prenatal brain abnormalities. Yet, this mouse develops behavioral abnormalities at about two weeks of postnatal age. We bred animals for future molecular analyses of dysregulated genes and pathways that result in the cellular and behavioral deficits observed in Down syndrome. We also analyzed the morphometric and cellular changes in the adult brains of these three strains. Our studies focused on the cerebral cortex, hippocampus and cerebellum, three regions that have been shown to be severely affected in humans with Down syndrome. We observed a significant decrease in the thickness of layer IV of the somatosensory cortex in Ts65Dn mice and an increased volume of the lateral ventricles in the Dp (16)1Yey mice. No changes in brain morphometry was observed in the Ts1Cje mouse model. Analysis of cerebellar morphometry and cell densities in the three target brain regions are ongoing. In parallel with the molecular and cellular experiments, we analyzed Ts65Dn pup vocalization after separation from their mothers. We also demonstrated a significant delay in vocalization in the trisomic pups during the first week of life. Objective 6: Publications In response to a review of a submitted manuscript, we are finalizing quantitative PCR experiments to validate gene expression data. We are also in the process of finalizing two other manuscripts for submission. The first one describes the preclinical trial data that we obtained using the first potential treatment, apigenin, in human amniocytes derived from fetuses with DS and in the Ts1Cje mouse model. The second one describes the results obtained using the touch screen operant learning in the Ts1Cje, Ts65Dn and Dp (16)1Yey mouse models. In this manuscript, we have used the visual discrimination task to test hippocampal learning and Extinction task to test frontal cortex-dependent learning inhibition.

唐氏综合症，也被称为21三体，是导致智力残疾的最常见遗传原因，每700名新生儿中就有1名患有唐氏综合症。在之前的工作中，我们使用了一种基于多种人类细胞类型和组织以及来自三种不同DS小鼠模型（Dp (16)1Yey， Ts65Dn和Ts1Cje）的胎儿脑组织的基因表达数据的综合和寿命方法来识别一致失调的信号通路和细胞过程。其中包括细胞周期缺陷、炎症、氧化应激和线粒体功能障碍等。我们还使用Connectivity Map数据库（https://www.broadinstitute.org/connectivity-map-cmap）来识别fda批准的分子，这些分子预计可以抵消这些通路异常，并且可以在体外使用人类细胞和在体内使用小鼠模型进行测试（Guedj et al, 2016 PMID 27586445）。我们确定了17个分子，预计可以挽救人类和小鼠DS的转录组变化（Guedj et al. 2016 pmiid 27586445）。