Abstract
As a highly prevalent neurodevelopmental disorder, attention-deficit hyperactivity disorder (ADHD) mostly affects children and adolescents across the globe, and it is regarded as a public health concern. The etiology of ADHD is multifactorial, with the interaction between multiple genetic and environmental factors potentially leading to a wide variety of clinical outcomes in ADHD patients. Methylphenidate (MPH) is one of the treatments of choice for ADHD, given that it seems to ameliorate symptoms and normalize brain maturational delays that are often found in ADHD patients. One of its most well-known therapeutic mechanisms of action refers to the blockage of dopamine transporters (DAT). However, previous controversial findings indicate that this drug might target alternative cellular components. Genetic evidence indicated that Wnt signaling is involved in the pathophysiology of ADHD, as this pathway plays important functions in neurodevelopment and maintaining the homeostasis of the central nervous system. Concomitantly, cellular and molecular studies have shown that MPH might be able to modulate this pathway while inducing neuronal differentiation in neural cell lines. Based on this context, the main goal of this thesis was to investigate the involvement of the Wnt pathway in the treatment of patient-specific neural cell lines with MPH. To do so, we generated induced pluripotent stem cells (iPSCs) from 5 ADHD patients and 5 matching controls. Subsequently, neural stem cells (NSCs) and forebrain cortical neurons (FCNs) were derived from biologically characterized iPSCs. Subsequently, a proteomic analysis was conducted on these three developmental stages to examine the relationship between Wnt and ADHD. The results showed that levels of active β-catenin were higher in ADHD NSCs, while no disparities were seen between ADHD and control iPSCs or FCNs. To further validate our hypothesis that Wnt activity is more active in ADHD NSCs, we conducted a reporter assay to functionally evaluate Wnt activity in this cell type. The examination of EC50 and IC50 values following the administration of increasing doses of Wnt3a and DKK1 (an agonist and an antagonist of Wnt signaling, respectively) revealed that the ADHD group does indeed have excessive activation of this pathway. To test the effects of MPH treatment in this scenario, these cells were subjected to MPH treatment at 10 nM and 100 nM. As a result, acute MPH treatment activated this pathway only in ADHD NSCs and specifically at the therapeutic dose of 10 nM. Chronic exposure to this dose resulted in upregulated β-catenin expression in ADHD NSCs, indicating that MPH effects in the Wnt pathway are long-lasting. Growth rates from iPSCs and NSCs were also compared between groups through two different methods: xCELLigence and WST-1 assays. Both techniques showed that ADHD NSCs proliferate significantly less than controls, whereas no differences were observed for the early developmental stage of iPSCs. Furthermore, a concentration of MPH 10 nM somewhat enhanced the growth of ADHD NSCs. However, this effect was mitigated when Wnt activity was blocked beforehand via DKK1 treatment. This demonstrates that MPH regulates the proliferation of ADHD NSCs in a Wnt-dependent manner. Moreover, we found links across genetic, clinical, and cellular parameters, demonstrating the exceptional specificity of our findings and the possibility for our cell lines to be translated into practical applications. Overall, these findings reinforce that the Wnt pathway can be implicated in ADHD-related phenotypes and might be a potential therapeutic target for MPH. Moreover, they open doors for future and deeper investigation in ADHD research in order to elucidate novel preventive measures and therapeutic targets.
Yde Ohki, Cristine Marie