Abstract
Ankyrin repeat and kinase domain containing 1 (ANKK1) is a member of the receptor-interacting protein serine/threonine kinase family, known to be involved in cell proliferation, differentiation and activation of transcription factors. Genetic variation within the ANKK1 locus is suggested to play a role in vulnerability to addictions. However, ANKK1 mechanism of action is still poorly understood. It has been suggested that ANKK1 may affect the development and/or functioning of dopaminergic pathways. To test this hypothesis, we generated a CRISPR-Cas9 loss of function ankk1 zebrafish line causing a 27 bp insertion that disrupts the ankk1 sequence introducing an early stop codon. We found that ankk1 transcript levels were significantly lower in ankk1 mutant (ankk127ins) fish compared to their wild type (ankk1+/+) siblings. In ankk1+/+ adult zebrafish brain, ankk1 protein was detected in isocortex, hippocampus, basolateral amygdala, mesencephalon, and cerebellum, resembling the mammalian distribution pattern. In contrast, ankk1 protein was reduced in the brain of ankk127ins/27ins fish. Quantitative polymerase chain reaction analysis revealed an increase in expression of drd2b mRNA in ankk127ins at both larval and adult stages. In ankk1+/+ adult zebrafish brain, drd2 protein was detected in cerebral cortex, cerebellum, hippocampus, and caudate homolog regions, resembling the pattern in humans. In contrast, drd2 expression was reduced in cortical regions of ankk127ins/27ins being predominantly found in the hindbrain. No differences in the number of cell bodies or axonal projections detected by anti-tyrosine hydroxylase immunostaining on 3 days post fertilization (dpf) larvae were found. Behavioral analysis revealed altered sensitivity to effects of both amisulpride and apomorphine on locomotion and startle habituation, consistent with a broad loss of both pre and post synaptic receptors. Ankk127ins mutants showed reduced sensitivity to the effect of the selective dopamine receptor antagonist amisulpride on locomotor responses to acoustic startle and were differentially sensitive to the effects of the non-selective dopamine agonist apomorphine on both locomotion and habituation. Taken together, our findings strengthen the hypothesis of a functional relationship between ANKK1 and DRD2, supporting a role for ANKK1 in the maintenance and/or functioning of dopaminergic pathways. Further work is needed to disentangle ANKK1’s role at different developmental stages.
Highlights
Addiction or substance use disorder (SUD) is a complex condition characterized by the uncontrolled use of drugs despite harmful and adverse consequences
We found that drd2b mRNA was upregulated in ankk127ins/27ins whole brain samples at larval and adult stages, but no differences in the number of dopaminergic neurons nor in axon pathfinding were detected in 3 dpf larvae
We exemplify how zebrafish can be exploited as a suitable model to study the development and functioning of the vertebrate nervous system, and to interrogate the functional impact of genetic variation relevant for human disease, including psychiatric disease and substance use disorder. This is the first study aiming at the characterization, at both behavioral and molecular levels, of a loss of function line for ankk1 and its effect on the development and functioning of the dopaminergic system
Summary
Addiction or substance use disorder (SUD) is a complex condition characterized by the uncontrolled use of drugs despite harmful and adverse consequences Environmental factors such as early life trauma, altered family structure, social pressure, and isolation during childhood increase the risk of developing SUDs (Wong et al, 2013; Baarendse et al, 2014; Koeneke et al, 2020), genetic factors contribute to the liability of the disorder, with heritability estimates ranging from 40 to 60% (see Prom-Wormley et al, 2017 and Lopez-Leon et al, 2021, for reviews). ANKK1 maps to chromosome 11q22-q23 (chr11: 11,338,038–113,400,418; GRCh38/hg38) in a 512 kb gene cluster that includes the neural cell adhesion molecule 1 (NCAM1), tetratricopeptide repeat domain 12 (TTC12) and dopamine receptor 2 (DRD2) genes (Neville et al, 2004; Mota et al, 2012). In the adult mouse brain, ANKK1 protein is expressed in neural stem cells, in post-mitotic
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