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CRISPR disruption and UK Biobank analysis of a highly conserved polymorphic enhancer suggests a role in male anxiety and ethanol intake

Abstract

Excessive alcohol intake is associated with 5.9% of global deaths. However, this figure is especially acute in men such that 7.6% of deaths can be attributed to alcohol intake. Previous studies identified a significant interaction between genotypes of the galanin (GAL) gene with anxiety and alcohol abuse in different male populations but were unable to define a mechanism. To address these issues the current study analysed the human UK Biobank cohort and identified a significant interaction (n = 115,865; p = 0.0007) between allelic variation (GG or CA genotypes) in the highly conserved human GAL5.1 enhancer, alcohol intake (AUDIT questionnaire scores) and anxiety in men. Critically, disruption of GAL5.1 in mice using CRISPR genome editing significantly reduced GAL expression in the amygdala and hypothalamus whilst producing a corresponding reduction in ethanol intake in KO mice. Intriguingly, we also found the evidence of reduced anxiety-like behaviour in male GAL5.1KO animals mirroring that seen in humans from our UK Biobank studies. Using bioinformatic analysis and co-transfection studies we further identified the EGR1 transcription factor, that is co-expressed with GAL in amygdala and hypothalamus, as being important in the protein kinase C (PKC) supported activity of the GG genotype of GAL5.1 but less so in the CA genotype. Our unique study uses a novel combination of human association analysis, CRISPR genome editing in mice, animal behavioural analysis and cell culture studies to identify a highly conserved regulatory mechanism linking anxiety and alcohol intake that might contribute to increased susceptibility to anxiety and alcohol abuse in men.

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Fig. 1: GAL5.1 is active in Gal expressing cells of the hypothlamus and amygdala and can be rapidly and accurately disrupted using CRISPR genome editing.
Fig. 2: GAL5.1 specifically regulates expression of the Gal gene in the hypothalamus and amygdala but not flanking genes.
Fig. 3: GAL5.1 disruption decreases ethanol intake and modulates anxiety-like behaviour in the novelty suppressed feeding test.
Fig. 4: mGAL5.1 deletion affects sex-specific aspects of anxiety-like behaviour in the open field test and the elevated zero maze.
Fig. 5: EGR1 interaction and modulation of the PKC response varies with GAL5.1 genotype.

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Acknowledgements

We would like to thank Wenlong Huang, Giuseppe D’Agostino and the staff of the University of Aberdeen Medical Research Facility for their help and advice with the animal tests. AMcE was funded by BBSRC project grant (BB/N017544/1) and EH was funded by Medical Research Scotland (PhD-719-2013). PB and DW are funded by the Scottish Government Rural and Environment Science and Analytical Services Division to the Rowett Institute. Association studies were conducted using the UK Biobank Resource: application number 4844 and was supported by a Wellcome Trust Strategic Award ‘Stratifying Resilience and Depression Longitudinally’ (STRADL) (Reference 104036/Z/14/Z). Dedicated to the memory of my dear brother Angus MacKenzie (1963–2018).

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McEwan, A.R., Davidson, C., Hay, E. et al. CRISPR disruption and UK Biobank analysis of a highly conserved polymorphic enhancer suggests a role in male anxiety and ethanol intake. Mol Psychiatry 26, 2263–2276 (2021). https://doi.org/10.1038/s41380-020-0707-7

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