In a recent study published in Molecular Psychiatry, researchers from China examined the effect of two drugs used for the treatment of attention-deficit/hyperactivity disorder (ADHD)— methylphenidate (MPH) and atomoxetine (ATX)— on the Drosophila melanogaster brain at single-cell resolution.
They found that glial and neuronal cells responded to the drugs and showed distinct transcriptional changes. Further, the study provides a list of target candidate genes to support drug repurposing for ADHD in the future.
Study: The effects of methylphenidate and atomoxetine on Drosophila brain at single-cell resolution and potential drug repurposing for ADHD treatment. Image Credit: AtlasStudio/Shutterstock.com
Background
ADHD is a neurodevelopmental disorder with a complex pathogenesis and etiology, affecting 7.2% of the world’s population and 6.2% of China’s. Various pharmacological stimulants (such as MPH) and non-stimulants (such as ATX) are used to treat the symptoms and cognitive dysfunction in ADHD, acting mostly by regulating inter-synaptic neurotransmitter levels.
However, evidence suggests that other potential targets may also play a role in these drugs' observed clinical and experimental effects, emphasizing the need for in-depth investigation of their underlying mechanisms.
Understanding how MPH and ATX regulate various cell types and the associated genes in humans is challenging, given the lack of access to human brain samples. Therefore, Drosophila is leveraged as a model organism with its evolutionarily conserved genes, central nervous system, and diverse cell types.
As developing new drugs is a lengthy and expensive process, multiple studies have explored drug repurposing and identifying new gene targets for neurological diseases.
In the present study, researchers analyzed Drosophila's behavior and single-cell level gene expression in response to treatment with MPH and ATX and further explored the implications for ADHD treatment.
About the study
To assess the effect of the drugs on behavior, starved, male, wild-type Drosophila flies were treated with optimized doses of ATX (1.5 mg/ml, n = 24), MPH (0.25 mg/ml, n = 24), or a control (5% sucrose and yeast solution, n = 24) for 24 hours.
The locomotory activity of flies was video-recorded and analyzed using the in-house EasyFlyTracker software, and short-term distances were measured. Following behavior analysis, the flies showing increased locomotory activity after drug treatment were dissected, and six samples were isolated from 20 brains per treatment.
Single-cell ribonucleic acid sequencing (scRNA-SEQ) libraries were prepared and analyzed using bioinformatics tools such as Basecall software, FlyBase, Cell Ranger, Seurat, FinaAllMarker, Metascape, and FlyPhoneDB for sequence alignment, cell-type clustering, marker identification, differential gene expression analysis, and cell-cell communication analysis.
Primary clusters were re-clustered to identify subclusters, including glial cells and monoaminergic neurons.
New potential drug targets were identified using the druggable genome database to explore the opportunities for drug repurposing and target identification.
A drug set enrichment analysis was performed to verify the relevance of the repurposing targets identified in the study to ADHD treatment. Access to this information was provided by constructing a website.
Results and discussion
In the behavioral analysis, the flies showed increased locomotory activity (hyperactivity-like behavior) in response to MPH or ATX compared to the control. Corroborating with previous literature, the flies were shown to travel a significantly greater distance in 10 minutes post-exposure to MPH or ATX.
In the scRNASEQ analysis, 28 gene clusters were identified at low resolution, and glial cells and neurons were distinguished using canonical markers and genes. Detailed annotations included monoaminergic neurons, mushroom body Kenyon cells, ellipsoid body cells, optic lobe cells, projection neurons, unannotated clusters, and glial cells.
The researchers used dopaminergic neurons (Monoamines, C20) as targets for drug-related analysis. 694 drug-responsive differentially expressed genes (DEGs) for MPH and 248 for ATX across all clusters.
The top 20 pathways associated were identified, and 230 genes were found to be shared between the two groups. The paths were mostly related to neurotransmitter regulation, indicating the role of an imbalance of neurotransmitters in ADHD.
In addition to dopamine-related genes, MPH and ATX were also shown to inhibit other receptor genes. Interestingly, MPH induced a broader range of cell-type responses compared to ATX. Findings from pathway analysis highlighted diverse responses in distinct cell types, emphasizing the importance of precise treatment.
While GABAergic (pertaining to gamma amino-butyric acid) and monoaminergic neurons were significantly affected by MPH and ATX, the overall cell-type proportions remained relatively stable.
Four glial subtypes with diverse functions were identified. Ensheathing and astrocyte-like glial cells were significantly involved in drug response and associated pathways.
Further, the researchers demonstrated the link between candidate ADHD genes, FDA-approved ADHD drug targets, and their homologs in Drosophila, as drugs backed by genetic evidence are more likely to be approved.
ADHDrug (http://adhdrug.cibr.ac.cn/), the web tool developed in the present study, allows the retrieval of all the provided drug and target information supported by drug enrichment analysis.
Conclusion
Although the study's approach cannot be applied clinically, the findings provide a rapid, cost-effective pipeline for repurposing drugs for ADHD by successfully exploring potential targets and compiling a candidate list.
In the future, studies incorporating disease models for ADHD while considering gender-based and developmental effects could help improve our understanding of drug targets against ADHD, opening new avenues for potential therapy for the disorder.
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