Cell\u2013cell coupling and DNA methylation abnormal phenotypes in the after-hours mice

Federico Tinarelli,Edoardo Balzani,Valter Tucci,Ilaria Colombi,Gavin Kelsey,Elena Ivanova,Erica Barini,Michela Chiappalone,Laura Gasparini,Celina Garcia Garcia

doi:10.1186/s13072-020-00373-5

Abstract

BackgroundDNA methylation has emerged as an important epigenetic regulator of brain processes, including circadian rhythms. However, how DNA methylation intervenes between environmental signals, such as light entrainment, and the transcriptional and translational molecular mechanisms of the cellular clock is currently unknown. Here, we studied the after-hours mice, which have a point mutation in the Fbxl3 gene and a lengthened circadian period.MethodsIn this study, we used a combination of in vivo, ex vivo and in vitro approaches. We measured retinal responses in Afh animals and we have run reduced representation bisulphite sequencing (RRBS), pyrosequencing and gene expression analysis in a variety of brain tissues ex vivo. In vitro, we used primary neuronal cultures combined to micro electrode array (MEA) technology and gene expression.ResultsWe observed functional impairments in mutant neuronal networks, and a reduction in the retinal responses to light-dependent stimuli. We detected abnormalities in the expression of photoreceptive melanopsin (OPN4). Furthermore, we identified alterations in the DNA methylation pathways throughout the retinohypothalamic tract terminals and links between the transcription factor Rev-Erbα and Fbxl3.ConclusionsThe results of this study, primarily represent a contribution towards an understanding of electrophysiological and molecular phenotypic responses to external stimuli in the Afh model. Moreover, as DNA methylation has recently emerged as a new regulator of neuronal networks with important consequences for circadian behaviour, we discuss the impact of the Afh mutation on the epigenetic landscape of circadian biology.

Highlights

Gavin Kelsey and Valter Tucci jointly directed this work.The regulation of the 24-h circadian rhythms of many physiological processes, such as sleep–wake cycles, behavioural responses, and endocrine modifications, depends on the entrainment of the internal clock with environmental signals, the most important of which is light [1]
We investigated whether Fbxl3 mutation affects neuronal network communication basic processes
We monitored the electrophysiological activity of in vitro neuronal networks for 27 consecutive hours using microelectrode arrays (MEAs; Fig. 1a)

Summary

Introduction

Gavin Kelsey and Valter Tucci jointly directed this work.The regulation of the 24-h circadian rhythms of many physiological processes, such as sleep–wake cycles, behavioural responses, and endocrine modifications, depends on the entrainment of the internal clock with environmental signals, the most important of which is light [1]. The internal clock is implemented by interlocked transcriptional and translational molecular mechanisms and is In mammals, such as humans and mice, non-visual light signals detected by the retina activate molecular events along the retinohypothalamic tract (RHT) that convey the external environmental information to the SCN. The transfer of information between the environment and the central internal clock is accomplished by the photopigment melanopsin, encoded by the gene Opn, at the intrinsically photosensitive retinal ganglion cells (ipRGCs) of the retina. The activation of this molecular system leads the main neuronal response to Tinarelli et al Epigenetics & Chromatin (2021) 14:1 light-entraining processes [4]. We studied the after-hours mice, which have a point mutation in the Fbxl gene and a lengthened circadian period

Methods

Results

Conclusion