Background

To help shed light on impaired neuronal signaling in obesity, we are exploring the contribution of the dynamic epitranscriptome to nutrient response signaling using our published human induced pluripotent stem cell-derived neuronal cultures which model those of the arcuate nucleus.

Methods

Focusing on purified NPY/AGRP orexigenic neurons, we have shown that our in vitro cultures respond to hormonal influences such as adiponectin and leptin through activation/inactivation and up-/down-regulation of the neuropeptides NPY and AGRP, respectively. Additionally, as in fasted mice, FTO expression is decreased by low-glucose conditions (LG). N6-methyladenosine (m6A) RNA modifications were assessed in these neurons under conditions of fasting/low glucose, AICAR activation, and hormonal treatment with the following methods: immunofluorescence using αm6A (Synaptic Systems) with RNAse treated cells as control for specificity, colorimetric assay, and meRIPseq. Functional enrichment of m6A gene sets were tested with DAVID.

Results

We show that m6A-modified RNA is present in the soma of these hypothalamic neurons, along dendrites and at the synapse where it may play a role in signaling and plasticity. Activation of the neurons with either AICAR treatment or 2hr LG results in definitive increase in overall m6A levels as measured by ICC and colorimetric assay. Leptin treatment of activated neurons reset the m6A levels. meRIPseq showed significant enrichment in peaks for the DRACH motif and motifs for zinc fingers known to regulate extracellular matrix remodeling at synapses. Neuronal response to LG caused significant changes at 5603 m6A peaks in 2855 unique genes of which 933 are localized outside of the soma. These 933 genes were significantly enriched in the processes of synapse assembly and cell-cell signaling and the mouse phenotype abnormal CNS synaptic transmission (q Bonf=5E-18;5E-8;3E-8).

Conclusions

This epitranscriptomic code may mediate signaling to maintain energy balance by these key neurons.