Hing, B. | bioRxiv Science

bioRxiv2021

An ancient polymorphic regulatory region within the BDNF gene associated with obesity modulates anxiety-like behaviour in mice and humans.

Obesity and anxiety are morbidities notable for their increased impact on society during the recent COVID-19 pandemic. Understanding the mechanisms governing susceptibility to these conditions will increase quality of life and our resilience to future pandemics. In the current study we explored the function of a highly conserved regulatory region (BE5.1) within the BDNF gene that harbours a polymorphism strongly associated with obesity (rs10767664; p=4.69x10-26). Analysis in primary cells suggested that the major T-allele of BE5.1 was an enhancer whereas the obesity associated A-allele was not. However, CRISPR/CAS9 deletion of BE5.1 from the mouse genome (BE5.1KO) produced no significant effect on the expression of BDNF transcripts in the hypothalamus, no change in weight gain after 28 days and only a marginally significant increase in food intake. Nevertheless, transcripts were significantly increased in the amygdala of female mice and elevated zero maze and marble burying tests demonstrated a significant increase in anxiety-like behaviour that could be reversed by diazepam. Consistent with these observations, human GWAS cohort analysis demonstrated a significant association between rs10767664 and anxiousness in human populations. Intriguingly, interrogation of the human GTEx eQTL database demonstrated no effect on BDNF mRNA levels associated with rs10767664 but a highly significant effect on BDNF-antisense (BDNF-AS) gene expression and splicing suggesting a possible mechanism. We discuss our findings within the context of the known function and regulation of BDNF in obesity and anxiety whilst exploring the validity of interrogating GWAS data using comparative genomics and functional analysis using CRISPR genome editing in mice.

genetics↗

bioRxiv2020

PACAP induces light aversion in mice by an inheritable mechanism independent of CGRP

The neuropeptides CGRP and PACAP have emerged as mediators of migraine, yet the potential overlap of their mechanisms remains unknown. Infusion of PACAP, like CGRP, can cause migraine in people, and both peptides share similar vasodilatory and nociceptive functions. In this study, we have used light aversion in mice as a surrogate for migraine-like photophobia to compare CGRP and PACAP and ask whether CGRP or PACAP actions were dependent on each other. Similar to CGRP, PACAP induced light aversion in outbred CD-1 mice. The light aversion was accompanied by increased resting in the dark, but not anxiety in a light-independent open field assay. Unexpectedly, about a third of the CD-1 mice did not respond to PACAP, which was not seen with CGRP. The responder and nonresponder phenotypes were stable, inheritable, and not sex-linked, although there was generally a trend for greater responses among male mice. RNA-seq analysis of trigeminal ganglia yielded hieriechial clustering of responder and nonresponder mice and revealed a number of candidate genes, including greater expression of pituitary hormones and receptors in a subset of responder mice. Importantly, an anti-PACAP monoclonal antibody could block PACAP-induced light aversion but not CGRP-induced light aversion. Conversely, an anti-CGRP antibody could not block PACAP-induced light aversion. Thus, we propose that CGRP and PACAP act by independent convergent pathways that cause a migraine-like symptom in mice. SignificanceThe relationship between the neuropeptides CGRP and PACAP in migraine is relevant given that both peptides can induce migraine in people, yet to date only drugs that target CGRP are available. Using an outbred strain of mice, we were able to show that most, but not all, mice respond to PACAP in a preclinical photophobia assay. Our finding that CGRP and PACAP monoclonal antibodies do not cross-inhibit the other peptide indicates that CGRP and PACAP actions are independent and suggests that PACAP-targeted drugs may be effective in patients who do not respond to CGRP-based therapeutics.

neuroscience↗