Using zebrafish disease model in molecular research on neurodevelopmental disorder generation

Abstract

In this thesis, zebrafish were used as a model organism to study two different projects: issues stemming from the dopaminergic system during the neurodevelopmental period, specifically ADHD, and a Parkinson’s disease model representing dopaminergic system disorders in old age. This approach allowed for an investigation of the lifelong effects of disorders caused by dopaminergic system dysfunction. Attention-deficit/hyperactivity disorder (ADHD) is a prevalent condition characterized by persistent inattention, hyperactivity, and impulsivity, often leading to significant impairments in daily functioning. Numerous medications have been developed to manage ADHD, with methylphenidate (commonly known as Ritalin or Concerta) being the most widely used active compound. While methylphenidate effectively improves attention, focus, and emotional regulation, its long-term use can lead to behavioral issues such as increased depression and anxiety, as well as physical side effects like sleep disturbances and decreased sensitivity to rewards. These side effects are more challenging to compensate for in adults compared to children, which may leave permanent effects. This study aims to investigate the long-term effects of methylphenidate use in adult women, with a focus on its role in sleep disorders, circadian rhythm disruption, and the potential implications for pregnancy, specifically on the susceptibility of offspring to accelerated brain aging. Our hypothesis is that methylphenidate use during pregnancy may contribute to changes in offspring telomere length and gene methylation patterns associated with brain aging, thereby increasing their vulnerability to neurodegeneration. To evaluate this, telomere length and methylation analyses were conducted on genes linked to brain aging in second-generation offspring. Additionally, our hypothesis is that therapeutic interventions, such as melatonin for regulating sleep disturbances and oxytocin as an alternative to mitigate methylphenidate’s side effects, may have protective effects. Zebrafish were used as the model organism in this study due to their high genetic similarity to humans and their ease of egg production, enabling multi-generational studies. As a result of this study, it was determined that neuroinflammation caused by circadian rhythm disruption and maternal stress was reduced through melatonin & methylphenidate and oxytocin & methylphenidate combinational treatments. In the offspring of the next generation, it was observed that the telomere length inherited at birth was shorter when treated with methylphenidate only. In the second research project of this thesis, the origins of Parkinson’s disease from two distinct locations—the brain and the gut—were investigated. Parkin-son’s disease is a neurodegenerative disorder characterized by the loss or reduction of dopaminergic neurons in the central nervous system. It is associated with aggregation of alpha-synuclein fibrils and resulting in motor function impairments. In the previous phase of this research, transgenic zebrafish models of Parkinson’s disease were developed by integrating human alpha-synuclein gene into the zebrafish genome at the single-cell stage. In this phase of thesis, six-month-old transgenic zebrafish were used to test the hypothesis that Parkinson’s disease can originate from either the brain or the gut. Alpha-synuclein protein was injected into the brain and gut, and its migration between these two organs was analyzed. The migration of alpha-synuclein fibrils was validated using immuno-histochemistry techniques. Behavioral changes and motor function impairments were assessed using novel tank tests, swim endurance tests, and hyposmia tests. As a result of the study, it was validated that alpha-synuclein fibrils injected into the brain-to-gut and gut-to-brain migrate at different speeds.