Histone Modifications in a Mouse Model of Early Adversities and Panic Disorder: Role for Asic1 and Neurodevelopmental Genes

Davide Cittaro, Valentina Lampis, Alessandra Luchetti, Roberto Coccurello, Alessandro Guffanti, Armando Felsani, Anna Moles, Elia Stupka, Francesca R D' Amato, Marco Battaglia, Davide Cittaro, Valentina Lampis, Alessandra Luchetti, Roberto Coccurello, Alessandro Guffanti, Armando Felsani, Anna Moles, Elia Stupka, Francesca R D' Amato, Marco Battaglia

Abstract

Hyperventilation following transient, CO2-induced acidosis is ubiquitous in mammals and heritable. In humans, respiratory and emotional hypersensitivity to CO2 marks separation anxiety and panic disorders, and is enhanced by early-life adversities. Mice exposed to the repeated cross-fostering paradigm (RCF) of interference with maternal environment show heightened separation anxiety and hyperventilation to 6% CO2-enriched air. Gene-environment interactions affect CO2 hypersensitivity in both humans and mice. We therefore hypothesised that epigenetic modifications and increased expression of genes involved in pH-detection could explain these relationships. Medullae oblongata of RCF- and normally-reared female outbred mice were assessed by ChIP-seq for H3Ac, H3K4me3, H3K27me3 histone modifications, and by SAGE for differential gene expression. Integration of multiple experiments by network analysis revealed an active component of 148 genes pointing to the mTOR signalling pathway and nociception. Among these genes, Asic1 showed heightened mRNA expression, coherent with RCF-mice's respiratory hypersensitivity to CO2 and altered nociception. Functional enrichment and mRNA transcript analyses yielded a consistent picture of enhancement for several genes affecting chemoception, neurodevelopment, and emotionality. Particularly, results with Asic1 support recent human findings with panic and CO2 responses, and provide new perspectives on how early adversities and genes interplay to affect key components of panic and related disorders.

Figures

Figure 1
Figure 1
(A) Profile heatmap around TSS of RefSeq genes. Read counts were extracted for all ChIP-seq experiments within a region spanning ±5 kb around TSS. The gradient blue-to-red color indicates high-to-low counts in the corresponding region. (B) Clustered heatmap of ChIP-seq profiles. Correlations were evaluated over the genome-wide signal of all experiments, including genomic DNA, and shown as a diagonal matrix. Rows and columns are clustered according to inter-sample correlation. Color intensity is proportional to the correlation value reported in each cell. Markers for actively transcribed regions (H3K4me3 and H3Ac) cluster together in the upper left region. H3K27me3 clusters with input DNA, probably due to its broad enrichment profile.
Figure 2
Figure 2
(A) Active circuit of 145 genes identified by network analysis (see methods). Each node is colored by the fold change found by SAGE when comparing RCF vs. CT mice. (B) Functional analysis of the active circuit. Scores are combined for EnrichR analysis on Reactome Pathways and MGI Mouse Phenotype gene sets.
Figure 3. Asic1 gene Expression.
Figure 3. Asic1 gene Expression.
Histograms on the left represent mean ± SD levels of expression of Asic1 gene found by SAGE analysis in Set 2 MOs of RCF and CT mice, as described in the main text: mean log 2(E) ± SD Fold Change respectively: 6.88 ± 0.37 and 5.89 ± 0.17, p = 0.047. Histograms on the right represent mean ± SD levels of expression of Asic1 gene found by real time PCR in the MO of 5 RCF and 4 CT mice: mean ± SD of Delta Cycle Threshold (dCT) as referred to Atcb housekeeping gene, respectively: 9.52 ± 1.16 and 12.10 ± 1.38, Student t = 2.64, p = 0.033. Asterisks over the RCF bars denote significant difference in comparison to the CT group.
Figure 4. Respiratory responses.
Figure 4. Respiratory responses.
Mean ± SD values of the percentage of increment (delta) of tidal volume from baseline (air) to 6% CO2-enriched air, among 5 RCF female (26.88 ± 8.10) vs. 5 CT female (16.63 ± 4.12) adult (PND = 80–90) mice (Student t = 2.54, DF = 8, p = 0.03), and 5 RCF male (30.46 + 6.34) vs. 3 CT male (20.68 + 2.02) adult (PND = 80–90) mice (Student t = 2.53, DF = 6, p = 0.05). Asterisk indicates a significance of p ≤ 0.05 in the comparisons of RCF to CT animals divided by sex. The results of an ANOVA with females and males pooled together are reported in the main text.
Figure 5. Nociception.
Figure 5. Nociception.
Results of the formalin test performed in 6 RCF vs. 7 CT adult (PND = 80–90) male mice. Responses were recorded continuously for 40 minutes and calculated in blocks of consecutive 5 minute periods. The figure shows the time spent licking the injected paw (Mean ± SE in seconds) for each time interval; ‘*’ and ‘**’ indicate significance of respectively p 

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