Regulation of fetal brain development in short versus long lived mice

Abstract

How fetal brain development is regulated in mice with reduced life span is a primary objective of this research. Three studies were performed to investigate the fetal brain development in short and lived mice strains. The scientific premise and background of brain development and aging are provided review of literature (Chapter 1). In the first study (Chapter 2), experiments were performed to test early-life origin of brain aging. Mouse epigenetic clock (epiclock) database which represents specific genomic sites that are methylated in an age-correlated manner was profiled in three life stages: fetal (gestation day 15), postnatal (day 5), and adult (week 70) brains of male and female C57BL/6J inbred mice. Data analysis showed that the female adult brain was epigenetically younger than the male adult brain even when the chronological age (time from birth) was the same (week 70). Specific methylations in the developing brain predictive of epigenetic differences in the aging brain between sexes were identified by predictive modeling by neural network. This study also showed that gene expression of epiclock genes were similar between placenta and fetal brain. However, genes unrelated to epiclock did not show this pattern. Whole-genome bisulfite sequencing identified sites that were methylated in a coordinated manner in the placenta and in a sex-specific manner in the fetal brain. These sites showed that methylation level in the epiclock genes and genes associated with gonadotropin-releasing hormone (GnRH) signaling pathway genes changes in a fetal-sex dependent manner both in the placenta and fetal brain. Furthermore, these methylations were maintained in the brain in the adult life stages. These findings suggested the fetal origin of sex differences in brain aging is epigenetically linked to the placenta. In the second study (Chapter 3), experiments were performed to test if reducing life span of mouse by ablating Caveolin 1 (Cav-1), a prolongevity gene that codes for an abundant structural protein of plasma membrane in endothelial cells, dysregulate brain development at the fetal stage. Further relevance of studying this specific gene is that mice lacking Cav-1 show neurodegeneration and multiple hallmarks of Alzheimer's disease (AD) at an early age. As a result, most Cav-1- null mice die within a year. Gene expression in bulk brain tissue as well as single cells were analyzed in the Cav-1-null fetal brain compared to the wildtype (WT). The results of this study showed that lack of Cav-1 leads to extensive dysregulation of genes of fetal brain at specific gestation time (day 15). Several epigenetic clock genes were differentially methylated in Cav-1-KO compared to WT mouse fetal brain. Single nuclei RNA sequencing identified specific glial and neuronal cells being dysregulated in the fetal brain due to the absence of Cav-1. In addition, methylation analysis was performed to investigate effect of Cav-1 on epiclock genes. Based on these results, a model was proposed for fetal links of Alzheimer's symptoms in mice lacking Cav-1. Lastly, in the third study (Chapter 4), experiments were performed to test if reduced lifespan in mice due to murine leukemia virus induced cancer influences fetal brain development. In this experiment, gene expression pattern of fetal brain and placenta of AKR/J mice, which mostly survive for a year due to onset of cancer, was compared with C57BL/6J mice to understand molecular and cellular links between aging and leukemia. The C57BL/6J has longer life span ([greater than] 2 years) and is refractory to AK virus that causes leukemia in AKR/J mice. The gene expression studies showed that genes related to aging and neurodegenerative diseases are differentially regulated in the fetal brain and placenta of AKR/J mice compared to that in C57BL/6J. Targeted methylation profiling of a total of 2,045 single bases of mouse genome, which are associated with mouse epigenetic clock data, showed that brain of AKR/J mice ages faster than C57BL/6J mice suggesting a link between leukemia and neuronal aging. By generating a F2 mapping population from AKR/J x C57BL/6J crosses, Bulk Segregant Analysis (BSA) was performed with the pooled DNA of F2 progenies by whole-genome sequencing to identify genetic variants associated with accelerated brain aging in AKR/J mice. Single-cell ATAC-seq (Assay for Transposase-Accessible Chromatin by sequencing) analysis further predicted that specific transcription factors are involved in the differential gene regulation of fetal brain in AKR/J mice compared to C57BL/6J mice. Together, the results of these study provide foundational knowledge to establish molecular and cellular links between reproduction and aging.