Novel non-stimulants rescue hyperactive phenotype in an adgrl3.1 mutant zebrafish model of ADHD

Abstract

ADHD is a highly prevalent neurodevelopmental disorder. The first-line therapeutic for ADHD, methylphenidate, can cause serious side effects including weight loss, insomnia, and hypertension. Therefore, the development of non-stimulant-based therapeutics has been prioritized. However, many of these also cause other effects, most notably somnolence. Here, we have used a uniquely powerful genetic model and unbiased drug screen to identify novel ADHD non-stimulant therapeutics. We first found that adgrl3.1 null (adgrl3.1−/−) zebrafish larvae showed a robust hyperactive phenotype. Although the hyperactivity was rescued by three ADHD non-stimulant therapeutics, all interfered significantly with sleep. Second, we used wild-type zebrafish larvae to characterize a simple behavioral phenotype generated by atomoxetine and screened the 1200 compound Prestwick Chemical Library® for a matching behavioral profile resulting in 67 hits. These hits were re-assayed in the adgrl3.1−/−. Using the previously identified non-stimulants as a positive control, we identified four compounds that matched the effect of atomoxetine: aceclofenac, amlodipine, doxazosin, and moxonidine. We additionally demonstrated cognitive effects of moxonidine in mice using a T-maze spontaneous alternation task. Moxonidine, has high affinity for imidazoline 1 receptors. We, therefore, assayed a pure imidazoline 1 agonist, LNP599, which generated an effect closely matching other non-stimulant ADHD therapeutics suggesting a role for this receptor system in ADHD. In summary, we introduce a genetic model of ADHD in zebrafish and identify five putative therapeutics. The findings offer a novel tool for understanding the neural circuits of ADHD, suggest a novel mechanism for its etiology, and identify novel therapeutics.

A Unified Approach to Investigating 4 dpf Zebrafish Larval Behaviour through a Standardised Light/Dark Assay

“…We also validated using an established transgenic line, adgrl3.1 -/-, which was employed because previous studies had shown robust hyperactivity compared to WT (Sveinsdóttir et al, 2023). We aimed to demonstrate the ability of our assay to distinctly establish specific drug effects by demonstrating the common adverse effect, sedation, in the WT ATO exposed larvae (Bastiaens, 2007).…”

adgrl3.1-deficient zebrafish show noradrenaline-mediated externalizing behaviors, and altered expression of externalizing disorder-candidate genes, suggesting functional targets for treatment

“…The fish used in this study were homozygous adgrl3.1 mutant zebrafish ( adgrl3.1-/- ) created by CRISPR-Cas9 genome engineering, as previously described 29 , and age-matched adgrl3.1 +/+ controls. Adult fish were housed in an aquarium facility at the University of Portsmouth (UK), and kept on a 14:10 light: dark cycle (28°C, pH~8).…”

“…For example, Adgrl3 -/- rats and mice, and both adgrl3.1 antisense morpholino oligonucleotide (MO) knock-down and adgrl3.1 -/- knock-out zebrafish larvae display persistent hyperactivity 24, 27 which is rescued by both stimulant and non-stimulant ADHD treatments (e.g. the dopamine (DAT) reuptake inhibitor methylphenidate 28 and the noradrenaline (NET) reuptake inhibitor atomoxetine 29 28 24 .…”

“…In addition, extensive previous research has demonstrated that atomoxetine significantly reduces impulse control deficits in rodents 31 , humans 32 and zebrafish 33 . In terms of hyperactivity, we recently carried out a screen of adgrl3.1 -/- zebrafish larvae and found several drugs rescue hyperactivity, including aceclofenac, amlodipine, doxazosin, and moxonidine 29 . Although the molecular mechanisms of action by which this gene impacts externalizing behaviors is not clear, collectively, these data suggest that ADGRL3 is functionally involved with two core externalising symptoms (hyperactivity and reduced impulse-control) representing an ideal tool for identifying targets for the development of novel therapeutics.…”

adgrl3.1-deficient zebrafish show noradrenaline-mediated externalizing behaviors, and altered expression of externalizing disorder-candidate genes, suggesting functional targets for treatment

“…Generation of adglr3.1 −/− sh The sh used in this study were homozygous adgrl3.1 mutant zebra sh (adgrl3.1-/-) created by CRISPR-Cas9 genome engineering, as previously described 29 , and age-matched adgrl3.1+/+ controls. Adult sh were housed in an aquarium facility at the University of Portsmouth (UK), and kept on a 14:10 light: dark cycle (28°C, pH ~ 8).…”