Maternal obesity modulates sexually dimorphic epigenetic regulation and expression of leptin receptor in offspring hippocampus.

Maternal obesity during pregnancy is associated with a greater risk for obesity and neurodevelopmental deficits in offspring. This developmental programming of disease is proposed to involve neuroendocrine, inflammatory, and epigenetic factors during gestation that disrupt normal fetal brain development. The hormones leptin and insulin are each intrinsically linked to metabolism, inflammation, and neurodevelopment, which led us to hypothesise that maternal obesity may disrupt leptin or insulin receptor signalling in the developing brain of offspring. Using a C57BL/6 mouse model of high fat diet-induced maternal obesity (mHFD), we performed qPCR to examine leptin receptor (Lepr) and insulin receptor (Insr) gene expression in gestational day (GD) 17.5 fetal brain. We found a significant effect of maternal diet and offspring sex on Lepr regulation in the developing hippocampus, with increased Lepr expression in female mHFD offspring (p < 0.05) compared to controls. Maternal diet did not alter hippocampal Insr in the fetal brain, or Lepr or Insr in prefrontal cortex, amygdala, or hypothalamus of female or male offspring. Chromatin immunoprecipitation revealed decreased binding of histones possessing the repressive histone mark H3K9me3 at the Lepr promoter (p < 0.05) in hippocampus of female mHFD offspring compared to controls, but not in males. Sex-specific deregulation of Lepr could be reproduced in vitro by exposing female hippocampal neurons to the obesity related proinflammatory cytokine IL-6, but not IL-17a or IFNG. Our findings indicate that the obesity-related proinflammatory cytokine IL-6 during pregnancy leads to sexually dimorphic changes in the modifications of histones binding at the Lepr gene promoter, and concomitant changes to Lepr transcription in the developing hippocampus. This suggests that exposure of the fetus to metabolic inflammatory molecules can impact epigenetic regulation of gene expression in the developing hippocampus.

Obesity during pregnancy in the mouse alters the Netrin-1 responsiveness of foetal arcuate nucleus neuropeptide Y neurones.

When individuals undergo gestation in an obese dam, they are at increased risk for impairments in the ability of the brain to regulate body weight. In rodents, gestation in an obese dam leads to a number of changes to the development of the hypothalamic neurones that regulate body weight, including reduced neuronal connectivity at birth. In the present study, we aimed to clarify how this neural circuitry develops normally, as well as to explore the mechanism underpinning the deficiency in connectivity seen in foetuses developing in obese dams. First, we developed an in vitro model for observing and manipulating the axonal growth of foetal arcuate nucleus (ARN) neuropeptide (NPY) neurones. We then used this model to test 2 hypotheses: (i) ARN NPY neurones respond to Netrin-1, one of a small number of axon growth and guidance factors that regulate neural circuit formation throughout the developing brain; and (ii) Netrin-1 responsiveness would be lost upon exposure to the inflammatory cytokine interleukin (IL)-6, which is elevated in foetuses developing in obese dams. We observed that ARN NPY neurones responded to Netrin-1 with a significant expansion of their growth cones, comprising the terminal apparatus that neurones use to navigate. Unexpectedly, we found further that NPY neurones from obese pregnancies had a reduced responsiveness to Netrin-1, raising the possibility that ARN NPY neurones from foetuses developing in obese dams were phenotypically different from normal NPY neurones. Finally, we observed that IL-6 treatment of normal NPY neurones in vitro led to a reduced growth cone responsiveness to Netrin-1, essentially causing them to behave similarly to NPY neurones from obese pregnancies. These results support the hypothesis that IL-6 can disrupt the normal process of axon growth from NPY neurones, and suggest one possible mechanism for how the body weight regulating circuitry fails to develop properly in the offspring of obese dams.

A novel role for the DNA repair gene Rad51 in Netrin-1 signalling.

Mutations in RAD51 have recently been linked to human Congenital Mirror Movements (CMM), a developmental disorder of the motor system. The only gene previously linked to CMM encodes the Netrin-1 receptor DCC, which is important for formation of corticospinal and callosal axon tracts. Thus, we hypothesised that Rad51 has a novel role in Netrin-1-mediated axon development. In mouse primary motor cortex neurons, Rad51 protein was redistributed distally down the axon in response to Netrin-1, further suggesting a functional link between the two. We next manipulated Rad51 expression, and assessed Netrin-1 responsiveness. Rad51 siRNA knockdown exaggerated Netrin-1-mediated neurite branching and filopodia formation. RAD51 overexpression inhibited these responses, whereas overexpression of the CMM-linked R250Q mutation, a predicted loss-of-function, had no effect. Thus, Rad51 appears to negatively regulate Netrin-1 signalling. Finally, we examined whether Rad51 might operate by modulating the expression of the Unc5 family, known negative regulators of Netrin-1-responsiveness. Unc5b and Unc5c transcripts were downregulated in response to Rad51 knockdown, and upregulated with RAD51 overexpression, but not R250Q. Thus, Rad51 negatively regulates Netrin-1 signalling, at least in part, by modulating the expression of Unc5s. Imbalance of positive and negative influences is likely to lead to aberrant motor system development resulting in CMMs.