The Slit3 gene is related to the preference of nicotine in people


Researchers at Queen Mary University of London have shown that zebrafish can provide genetic clues to smoking, a complex human behavior.

By studying genetically-altered zebrafish they were able to pinpoint a human gene, Slit3, involved in nicotine addiction and also discover the ways in which it may act.

While zebrafish have been used extensively in genetic research, they’ve been used only in developmental models, such as identifying genes associated with disease, rather than to predict genes involved in a complex cognitive behaviour such as smoking.

Although smoking has long been known to have a genetic element, relatively little has been known about the genes involved since it has been difficult to identify them from human studies alone.

In a study published in eLife journal, the researchers tested families of genetically altered zebrafish for nicotine preference.

When one family showed a much stronger nicotine preference compared to the others, the researchers identified all the mutations in the family, eventually narrowing down to a mutation in the Slit3 gene linked to the behaviour.

To see if the same gene affected nicotine preference in people, the researchers looked for association between variants in the human Slit3 gene and smoking behaviour, such as decreased or increased desire to smoke and how easy it was to quit, in groups of people in the UK and Finland.

They found 3 variants in the human Slit3 gene that were significantly linked to smoking activity.

To then learn more about how the Slit3 gene might be working, the researchers tested both mutant and wild type fish for sensitivity to a dopaminergic drug.

To then learn more about how the Slit3 gene might be working, the researchers tested both mutant and wild type fish for sensitivity to a dopaminergic drug.

In humans this drug affects the startle reflex – our physical reaction to a sudden loud noise – that is linked to addictions, including nicotine addiction. When tested with the startle reaction, the mutant fish showed decreased sensitivity to the drug.

After testing various different receptors that might be involved in the reduced drug sensitivity, the researchers found that only one receptor was implicated – the serotonin receptor 5HT 1AA.

Caroline Brennan, Professor of Molecular Genetics at Queen Mary University of London, led the research.

She explained: “This gives us a hypothesis for how the Slit3 gene works in humans. It is somehow altering the level of serotonin receptors present; and the differences in the levels are presumably then influencing sensitivity to nicotine addiction.”

Professor Brennan added: “As well as finding out more about the genes involved in nicotine addiction, most importantly, we’ve found an easier way of finding these genes in the future.

Although zebrafish are a ‘lower’ organism, they have a similar genetic structure to humans and share 70% of genes with us. 84% of genes known to be associated with human disease have a zebrafish counterpart; and while there has been scepticism regarding their usefulness in terms of human cognition, we have shown that they can give insight into the genetics of that as well.”

Tobacco smoking is the leading preventable cause of death worldwide placing a heavy social and financial burden on society (13). It is well established that aspects of smoking behaviour have a strong genetic component (47).

However, identifying causal genetic factors and exploring the mechanisms by which they act is challenging in human studies: the field has been characterized by small effect sizes and lack of replication such that there are remarkably few genes and loci that can be confidently linked to smoking.

The strongest evidence for causal effects is for functional variants in CHRNA5 (8) and CYP2A6 (4), affecting amount smoked and nicotine metabolism, respectively. Recent large studies have identified numerous new association loci, but their significance is yet to be biologically characterised (67).

As approaches to identify genetic risk are difficult in humans, research has been facilitated by studies in animal models, with a focus on genomic analysis of inbred and selectively-bred, naturally occurring genetic strains (9).

This type of study produces quantitative trait loci (QTL) maps of multiple loci, each with a small impact on the phenotype. However, as with human studies, it is inherently difficult to identify relevant genes from QTL maps, as the overall phenotype cannot be predicted by individual genotypes.

Mutagenesis studies in animal model systems can overcome these limitations: e.g. N-ethyl-N-nitrosourea (ENU) mutagenesis introduces thousands of point mutations into the genome with the potential to generate much stronger phenotypes than those occurring in a natural population thereby facilitating identification of causal mutations.

Examination of phenotypic variation in ENU mutagenized model species could be applied to identify novel, naturally occurring variants influencing human addictive behaviour by identifying key genes and pathways affecting conserved behavioural phenotypes.

Drug-induced reinforcement of behaviour, that reflects the hedonic value of drugs of abuse including nicotine, is highly conserved in both mammalian and non-mammalian species (1013).

Conditioned place preference (CPP), where drug exposure is paired with specific environmental cues, is commonly used as a measure of drug-induced reward or reinforcement (14).

ENU Mutagenesis screens for cocaine or amphetamine-induced CPP have been undertaken in zebrafish (915), however, despite successful isolation of lines with altered reinforcement responses to these drugs, the causal mutations have not been identified and the predictive validity of these screens for human behaviour has not been established.

Larval locomotor response to nicotine has also been used to explore nicotine response genetics (16) but the relevance of larval locomotion to addiction is somewhat less clear.

Here, we conducted a forward genetic screen of families of ENU-mutagenized zebrafish for nicotine-induced CPP. Zebrafish express homologues of all 16 members of the nicotinic acetylcholine receptor family present in mammals (1719) with similar binding characteristics (2021).

However, as a result of a local gene duplication event in the ray fish lineage and a teleost genome tetraploidation event zebrafish have duplicate copies of nicotinic receptor, α2, α4, α7, α9, α10, β1, β3 and β5. In addition, zebrafish have additional receptors (α8 and α11) that have been lost in humans (19).

Zebrafish show robust CPP to nicotine (2124). Nicotinic receptor partial agonists, that modulate striatal dopamine release in response to nicotine in mammalian systems, also inhibit nicotine-induced CPP in zebrafish (21).

Further, on prolonged exposure to nicotine or ethanol, adult zebrafish show conserved adaptive changes in gene expression and develop dependence-related behaviours, such as persistent drug seeking despite adverse stimuli or reinstatement of drug seeking following periods of abstinence (24).

These data demonstrate the existence of a conserved nicotine-responsive reward pathway and support the suitability of zebrafish to examine the genetic and molecular mechanisms underlying behavioural responses to nicotine.

To evaluate the use of a behavioural CPP screen in zebrafish to predict loci affecting human smoking behaviour we initially assessed

1) the ability of varenicline and bupropion, pharmacological agents used to treat human nicotine addiction, to reduce zebrafish nicotine-induced place preference and

2) the heritability of nicotine responses in ENU-mutagenized fish. We then screened 30 families of ENU-mutagenized fish to identify families with increased/decreased CPP for nicotine.

For two families with altered CPP response, the phenotype was confirmed following independent replication with a larger number of fish. Exome sequence information was used to generate a list of coding, loss of function candidate mutations affecting the phenotype.

One family with a mutation co-segregating with increased nicotine CPP was selected for further study. Firstly, the effect of the identified gene on nicotine-induced CPP was confirmed using an independent line carrying a similar predicted loss of function mutation in the same gene.

We then characterized the mutation using gene expression analysis, immunohistochemical analysis of neuronal pathways and behavioural responses to acoustic startle; a response known to be modulated by serotonergic and dopaminergic signalling and, in humans, associated with vulnerability to addiction (2527).

Finally, we used focused SNP analysis of human cohorts to assess the predictive validity of findings in fish for human smoking behaviour.

In agreement with previous studies zebrafish showed a robust CPP to nicotine. Nicotine-induced CPP was abolished by varenicline and bupropion and found to be heritable in fish.

The screening of ENU mutagenized families identified mutations in the slit3 gene influencing sensitivity to rewarding effects of nicotine. Slit3 mutant larvae and adult fish showed reduced behavioural sensitivity to amisulpride and larvae showed increased ht1aa receptor expression. No effect on neuronal pathfinding was detected.

Analysis of the SLIT3 locus in two independent human cohorts identified two genetic markers that predict level of cigarette consumption and likelihood of cessation. This proof of principle study demonstrates that screening of zebrafish is able to predict loci affecting complex human behavioural phenotypes and suggests a role for SLIT3 signalling in the development of dopaminergic and serotonergic pathways affecting behaviours associated with nicotine sensitivity.

Queen Mary University London


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