Biology subjects

MacKenzie, A.

Publications and source records attributed to MacKenzie, A..

2 recordsLinked to original sources

bioRxiv2019

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

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 that was consistent with these previous studies. 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 evidence of reduced anxiety-like behaviour in male GAL5.1KO animals mirroring that seen in humans. 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.

genetics↗

bioRxiv2019

Genetic, epigenetic and pharmacological influences modulating tissue specific regulation of the cannabinoid receptor -1 (CB1) gene; implications for cannabinoid pharmacogenetics.

Cannabinoid receptor-1 (CB1) represents a potential drug target against conditions that include obesity and substance abuse. However, drug trials targeting CB1 (encoded by the CNR1 gene) have been compromised by differences in patient response. Towards addressing the hypothesis that genetic and epigenetic changes within the regulatory regions controlling CNR1 expression contribute to these differences, we isolated the human CNR1 promotor (CNR1prom) and demonstrate its activity in primary cells and transgenic mice. We also provide evidence of CNR1prom in CB1 autoregulation and its repression by DNA-methylation. We further characterised a conserved regulatory sequence (ECR1) in CNR1 intron 2 that contained a polymorphism in linkage disequilibrium with disease associated SNPs. Deletion of ECR1 from mice using CRISPR genome editing significantly reduced CNR1 expression in the hippocampus. These mice also displayed reduced ethanol intake and hypothermia response to CB1 agonism. Moreover, human specific C-allele variants of ECR1 (ECR1(C)) drove higher levels of CNR1prom activity in hippocampal cells than did the ancestral T-allele. We further demonstrate a role for the AP-1 transcription factor in driving higher ECR1(C) activity. In the context of the known roles of CB1 the current study suggests a mechanism through which ECR1(C) may be neuroprotective in the hippocampus against stress. The cell-specific approaches used in our study to determine the functional effects of genetic and epigenetic changes on the activity of tissue-specific regulatory elements at the CNR1 locus represent an important step in gaining a mechanistic understanding of cannabinoid pharmacogenetics.

genetics↗