Epigenetic consequences of early-life caregiver maltreatment in rat medial prefrontal cortex neurons

Roth, Tania L.Negative experiences with a caregiver during infancy can result in long-lasting changes in brain function and behavior. Victims of child abuse have increased incidences of cognitive abnormalities and psychiatric disorders correlating to aberrant neural development and function, but underlying mechanisms are not well-understood. It is our central hypothesis that brain and behavior changes conferred by early child adversity occur through epigenetic changes such as DNA methylation. DNA methylation is an epigenetic modification that alters gene transcription without changing the DNA sequence. Using a rodent model of early-life caregiver maltreatment (involving exposure to an adverse caregiving environment from postnatal days [PN]1-7), we have previously demonstrated abnormal methylation of DNA associated with the Brain-derived neurotrophic factor (Bdnf) gene in the medial prefrontal cortex (mPFC) of developing and adult male and female rats. Bdnf is crucial for brain development and neuroplasticity, and aberrant Bdnf regulation has been implicated in several cognitive abnormalities and psychiatric disorders linked to early-life adversity. We have also found that early-life maltreatment alters mRNA levels of proteins known to regulate DNA methylation (i.e. DNMTs, MeCP2) patterns in the mPFC. Thus, this dissertation extends these foundational studies by 1) better characterizing Bdnf DNA methylation across multiple time points during development in the healthy and stressed rodent mPFC and 2) by characterizing Bdnf DNA methylation in specific cell populations within the mPFC. The first aim of this dissertation was to use a developmental approach to compare Bdnf DNA methylation patterns of male and female rats receiving nurturing care or maltreatment in infancy. We have previously published work that demonstrates altered Bdnf DNA methylation in the adolescent and adult rodent mPFC after exposure to early-life caregiver maltreatment. In this prior work, however, the methods used (methyl-specific PCR) did not allow us to establish a trajectory of methylation across normal or abnormal (maltreatment) development. Here, we used bisulfite sequencing PCR (BSP) to measure percent methylation of individual CG sites at Bdnf exon IV in the mPFC of rats at three developmental time points: PN8 (infancy), PN30 (adolescence), and PN90 (adulthood). Results demonstrated that males maintained a stable level of methylation across the lifespan, regardless of quality of care. Conversely, females showed a drastic increase in Bdnf IV methylation during adulthood if they experienced early-life maltreatment, while control groups maintained a low level of methylation across all developmental time-points. The second aim of this dissertation was to use fluorescence-activated cell sorting (FACS) to compare Bdnf DNA methylation changes in neurons and non-neurons of the mPFC in adult rats exposed to maltreatment or nurturing care during infancy. In the PFC, one third of the cells are glia (while the other two thirds are neurons), consisting of astrocytes, oligodendrocytes, and microglia. Thus far, work in our lab has used punches of frozen tissue and extracted DNA from the homogenate, which consists of both neurons and glia. Here, we extracted nuclei from the mPFC of adult animals that had experienced our maltreatment paradigm and used fluorescence-activated cell sorting (FACS) to isolate neurons before performing BSP to identify methylation levels. Our data indicate that early-life stress-induced DNA methylation changes in Bdnf exon IV are present in neurons but not non-neurons. Specifically, females that experienced early-life maltreatment exhibited increased methylation compared to controls, while no changes were present in male Bdnf methylation. These changes mirror our previous findings in the whole mPFC (all cell types) and demonstrate the specificity of our previous findings to neurons. The studies in this dissertation have, for the first time, established cell-type specific changes in DNA methylation in response to maltreatment and characterized the trajectory of Bdnf methylation in the healthy and stressed mPFC. These findings help to advance our understanding of the impact of early-life stress on DNA methylation patterns in the mPFC. While further studies are needed to investigate behavioral trajectories of early-life stressed animals and characterize functional outcomes of our methylation findings in the mPFC, these studies provide empirical support for the ability of maltreatment to produce changes in DNA methylation within the mPFC and support the notion that brain and behavior changes conferred by early child adversity can occur through changes in DNA methylation.University of Delaware, Department of Psychological and Brain SciencesPh.D