RNA in extracellular vesicles during adolescence reveal immune, energetic and microbial imprints of early life adversity.

Exposure to early life adversity (ELA), including childhood maltreatment, is one of the most significant risk factors for the emergence of neuropsychiatric disorders in adolescence and adulthood. Despite this relationship being well established, the underlying mechanisms remain unclear. One way to achieve this understanding is to identify molecular pathways and processes that are perturbed as a consequence of childhood maltreatment. Ideally, these perturbations would be evident as changes in DNA, RNA or protein profiles in easily accessible biological samples collected in the shadow of childhood maltreatment. In this study, we isolated circulating extracellular vesicles (EVs) from plasma collected from adolescent rhesus macaques that had either experienced nurturing maternal care (CONT) or maternal maltreatment (MALT) in infancy. RNA sequencing of RNA in plasma EVs and gene enrichment analysis revealed that genes related to translation, ATP synthesis, mitochondrial function and immune response were downregulated in MALT samples, while genes involved in ion transport, metabolism and cell differentiation were upregulated. Interestingly, we found that a significant proportion of EV RNA aligned to the microbiome and that MALT altered the diversity of microbiome-associated RNA signatures found in EVs. Part of this altered diversity suggested differences in prevalence of bacterial species in CONT and MALT animals noted in the RNA signatures of the circulating EVs. Our findings provide evidence that immune function, cellular energetics and the microbiome may be important conduits via which infant maltreatment exerts effects on physiology and behavior in adolescence and adulthood. As a corollary, perturbations of RNA profiles related to immune function, cellular energetics and the microbiome may serve as biomarkers of responsiveness to ELA. Our results demonstrate that RNA profiles in EVs can serve as a powerful proxy to identify biological processes that might be perturbed by ELA and that may contribute to the etiology of neuropsychiatric disorders in the aftermath of ELA.

Estrogen-dependent association of HDAC4 with fear in female mice and women with PTSD.

Women are at increased risk of developing post-traumatic stress disorder (PTSD) following a traumatic event. Recent studies suggest that this may be mediated, in part, by circulating estrogen levels. This study evaluated the hypothesis that individual variation in response to estrogen levels contributes to fear regulation and PTSD risk in women. We evaluated DNA methylation from blood of female participants in the Grady Trauma Project and found that serum estradiol levels associates with DNA methylation across the genome. For genes expressed in blood, we examined the association between each CpG site and PTSD diagnosis using linear models that adjusted for cell proportions and age. After multiple test correction, PTSD associated with methylation of CpG sites in the HDAC4 gene, which encodes histone deacetylase 4, and is involved in long-term memory formation and behavior. DNA methylation of HDAC4 CpG sites were tagged by a nearby single-nucleotide polymorphism (rs7570903), which also associated with HDAC4 expression, fear-potentiated startle and resting-state functional connectivity of the amygdala in traumatized humans. Using auditory Pavlovian fear conditioning in a rodent model, we examined the regulation of Hdac4 in the amygdala of ovariectomized (OVX) female mice. Hdac4 messenger RNA levels were higher in the amygdala 2 h after tone-shock presentations, compared with OVX-homecage control females. In naturally cycling females, tone-shock presentations increased Hdac4 expression relative to homecage controls for metestrous (low estrogen) but not the proestrous (high estrogen) group. Together, these results support an estrogenic influence of HDAC4 regulation and expression that may contribute to PTSD in women.

Gephyrin plays a key role in BDNF-dependent regulation of amygdala surface GABAARs.

Brain-derived neurotrophic factor (BDNF) is critically involved in synaptic plasticity and neurotransmission. Our lab has previously found that BDNF activation of neurotrophic tyrosine kinase, receptor, type 2 (TrkB) is required for fear memory formation and that GABAA receptor (GABAAR) subunits and the GABAA clustering protein gephyrin are dynamically regulated during fear memory consolidation. We hypothesize that TrkB-dependent internalization of GABAARs may partially underlie a transient period of amygdala hyperactivation during fear memory consolidation. We have previously reported that BDNF modulates GABAAR α1 subunit sequestration in cultured hippocampal and amygdala neurons by differential phosphorylation pathways. At present, no studies have investigated the regulation of gephyrin and GABAAR α1 subunits following BDNF activation in the amygdala. In this study, we confirm the association of GABAAR α1 and γ2 subunits with gephyrin on mouse amygdala neurons by coimmunoprecipitation and immunocytochemistry. We then demonstrate that rapid BDNF treatment, as well as suppression of gephyrin protein levels on amygdala neurons, induced sequestration of surface α1 subunits. Further, we find that rapid exposure of BDNF to primary amygdala cultures produced decreases in gephyrin levels, whereas longer exposure resulted in an eventual increase. While total α1 subunit levels remained unchanged, gephyrin was downregulated in whole cell homogenates, but enhanced in complexes with GABAARs. Our data with anisomycin suggest that BDNF may rapidly induce gephyrin protein degradation, with subsequent gephyrin synthesis occurring. Together, these findings suggest that gephyrin may be a key factor in BDNF-dependent GABAAR regulation in the amygdala. This work may inform future studies aimed at elucidating the pathways connecting BDNF, GABAA systems, gephyrin, and their role in underlying amygdala-dependent learning.