Exploiting zebrafish genetics to identify genes affecting addiction-related phenotypes.

Exploiting zebrafish genetics to identify genes affecting addiction-related phenotypes
The cost of substance abuse in the US alone is estimated at $166 billion annually in preventable health
care costs, with tobacco ($130 billion) and alcohol ($25 billion) abuse contributing to the overwhelming
majority of this expense. The World Health Organization declared tobacco-related disease a global
epidemic, predicted to cause an estimated 8 million annual deaths worldwide by 2030, if unabated.
There is a vital need for increased understanding of the genetic and cell biological factors influencing
vulnerability to addiction including smoking to help identify individuals at risk and to inform treatment
strategies. As approaches to identify genetic risk are difficult in humans, research on genetic risk can be
facilitated by the use of animal models. Zebrafish are an established developmental and behavioral
genetic model species that show conserved responses to common drugs of abuse as well as behaviors
that model core phenotypes predictive of vulnerability to addiction. We shall identify genes affecting core
phenotypes associated with addiction by screening lines of ethyl nitrosurea (ENU)-mutagenised
zebrafish for sensitivity to nicotine reward and impulsivity. We test the hypothesis that genes that show
persistent adaptive changes in expression following chronic nicotine exposure are also addiction
vulnerability genes.
Our specific aims are:
1) To isolate 25 heritable loci affecting a) nicotine reward and b) impulsivity.
i) We will use behavioral assays in adult ENU-mutagenized zebrafish to identify families
showing altered nicotine reward and impulse control. We have developed a scalable, fully
automated behavioral assay system for this purpose. We will use analysis of microarray
or RNAseq data to identify genes that show persistent changes in expression following
chronic nicotine exposure as candidates for genes affecting nicotine addiction
vulnerability. We will pre-load the screen with families carrying mutations in these
candidate genes.
ii) We shall use exome sequencing to map the mutations in at least 10 families. The use of
ENU- mutagenized lines facilitates identification of the causal mutations (by generation of
strong phenotypes and/or by the ENU mutations acting as markers).
2) To identify the cell biological processes underlying the observed behavioral phenotypes.
i) We shall use additional (predicted functional) ENU mutations or targeted knockouts of the
identified genes to confirm genotype-phenotype associations.
ii) We shall perform RNA sequencing to gain insight into possible effects on gene
expression and cellular pathways affected.
iii) We shall perform developmental analyses to test the hypothesis that behavioral effects
are mediated by effects on neuronal patterning or neural circuit formation at
developmental timepoints.
The expected outcomes from this study are identification of novel genetic factors influencing core
behaviors predictive of vulnerability to drug addiction and increased understanding of the cellular
processes affected. These findings will enable future human genetic variation studies in these identified
genes, with the potential to develop more personalized therapies. Results from this work will have a
positive impact on individualized treatment efforts for tobacco dependence and addiction as a whole.

Grant Number: 5U01DA044400-05
NIH Institute/Center: NIH
Principal Investigator: Caroline Brennan