Mapping the Impact ofDUSP6in an ADHD iPSC derived-dopaminergic neuronal model

Abstract

Although the gene DUSP6 has been implicated as a risk gene for ADHD in recent GWAS studies, its functional role in the aetiology of the condition remains poorly understood. DUSP6 is reported to regulate dopaminergic neurotransmission by decreasing available synaptic dopamine, suggesting a potential mechanism by which DUSP6 may confer risk to ADHD. In this study, we employed CRISPR-Cas9 to knockout DUSP6 in induced pluripotent stem cells (iPSCs) derived from an individual with ADHD. These isogenic engineered cells were then differentiated into highly homogeneous dopaminergic neurons, including both heterozygous (Het) and homozygous (Hom) knockout lines, along with parental control lines, to assess changes in dopaminergic neurotransmission at both cellular and transcriptomic levels. Enzyme Linked Immunosorbent Assay (ELISA) analyses showed a dose-dependent trend of increasing dopamine levels, at extracellular level, in the knockout lines. RNA sequencing further supported this finding, showing that downregulation of DUSP6 lead to the upregulation of differentially expressed genes (DEGs) associated with the regulation of dopamine secretion and synaptic functions critical for dopamine signalling. These DEGs appear to encode components of the synaptic machinery essential for effective dopamine transmission or act as regulators of dopaminergic neurotransmission. Additionally, RNA sequencing uncovered other potential biological mechanisms through which DUSP6 may contribute to increase the risk for ADHD, including effects on neurogenesis, extracellular matrix-associated processes, lipid metabolism, and sex-specific gene expression. Furthermore, we identified overlaps between DUSP6 knockout DEGs and those associated with other neuropsychiatric disorders, including major depression (MDD), bipolar disorder (BD), and schizophrenia (SCZ), suggesting shared genetic pathways potentially influenced by DUSP6. Together, this study provides deeper insights into the molecular underpinnings of the role played by DUSP6 in the genetic aetiology of ADHD and its broader implications across related neuropsychiatric conditions.