Using forward and reverse genetic approaches to identify genes and cell biological processes underlying vulnerability to addiction

Addiction including smoking is the leading preventable cause of death and disability in the UK (Ekpu and Brown, 2015). Twin studies show significant genetic contribution addiction including smoking behaviour and response to treatment (e.g. Agrawal, 2012). Greater understanding of genetic variants influencing addiction related behaviour and the cellular processes affected may lead to the development of new therapies and identification of vulnerable individuals, providing opportunities for preventative interventions.
This proposal will identify new gene variants linked to addiction-related behaviour and treatment outcome and the cellular processes affected by; analysing lines of mutagenised zebrafish that show altered addiction-related behaviour; analysing developmental and behavioural impact of specific targeted mutations and; by interrogation of human SNP data. Zebrafish are an ideal, cost effective model system in which to conduct these studies as large numbers of mutant lines that can be screened for behavioural phenotypes exist and, once a candidate allele is identified, the transparent nature of embryonic and larval forms makes them superbly suited for neurodevelopmental analysis to determine the cellular processes affected. Further they are vertebrate so findings in zebrafish are of translational relevance.

Research plans:

Approach 1: Characterisation of mutant zebrafish lines showing altered behavioural phenotypes associated with drug addiction: nicotine preference, pre-pulse inhibition and impulse control.

As the result of a recent MRC CASE studentship and BBSRC on going project grant, we have isolated 9 lines of ENU-mutagenised zebrafish that show differential nicotine seeking behaviour, pre-pulse inhibition and/or impulse control. We have identified 2 of the causal ENU mutations and using focused SNP analysis in human cohorts we have identified 2 novel SNPs in one of the homologous human genes that predict smoking behaviour. These data strengthen the hypothesis that the genes are causally linked to addiction related phenotypes and demonstrate the translational validity of using zebrafish to explore the genetics of human nicotine-seeking and addiction.

Here we will use developmental and pharmacological analyses to characterize the cellular processes by which the identified mutations influence addiction-related behaviours and genotype (using exome sequencing or site specific pcr) the remaining lines in collaboration with Elisabeth Busch-Nentwich from the Sanger Institute.

Approach 2. Targeted zebrafish gene mutations.

Through collaboration with Elisabeth Busch-Nentwich we will generate targeted mutations using gene editing technology to test the hypothesis that variants identified in human association studies are causally related to drug seeking behaviour and explore the cellular processes affected. In particular, recent human studies highlight the acetylcholine receptor gene cluster in nicotine dependence but more detailed localisation of the responsible genes is not possible because of strong linkage disequilibrium (The Tobacco Genetics Consortium 2010). The fish model offers the opportunity to explore effects of individual genes: we have obtained lines of fish carrying loss of function mutations within the dopamine receptor 2, ankk1, dopamine transporter, and nicotinic receptor alpha5, and alpha9 genes. Gene editing techniques can be used to analyse the effect of other specific variants associated with human addiction-related behaviour.

Approach 3. Analysis of identified genes in human studies.

Through collaboration with human geneticists (R.Walton, R.Keers, QMUL) we will take genes identified in zebrafish experiments and identify functional human SNPs from public databases and/or re-sequencing and explore the relationship to treatment response cohorts of human subjects and in Finnish twin studies in collaboration with Jaakko Kaprio, Helsinki, seeking to identify markers which could increase the predictive value of current diagnostics.

These studies will increase understanding of the genetic factors that influence vulnerability to addiction and the cell biological mechanisms by which they act and will, ultimately, lead to the development of novel therapeutics and a consequent impact on public health: ‘Each discovery of a biologically relevant locus is a potential first step in a translational journey’ (Hirschhorn, 2009).

Training opportunities:

Zebrafish behavioural analysis (adults and larvae) and developmental biology (IHC, ISH, confocal analysis).
Training in gene editing (CRISPR) providing a unique opportunity to train the student in cutting egde techniques in a model species at the forefront of behavioural genetics research.
In addition the student will be trained in human genome analysis (SNP analysis) of large data sets.

Anticipated start date: 2 October 2017.