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Dopamine transporter trafficking following methylphenidate (Ritalin) treatment in human dopaminergic neurons
The student will investigate the trafficking of the dopamine transporter between intracellular and synaptic localization in a human dopaminergic cell line via live cell imaging following pharmacological treatment.
Attention Deficit-Hyperactivity Disorder (ADHD) is one of the most frequent psychiatric disorders in children and adolescents, with up to 5% affected worldwide. Psychostimulant such as methylphenidate (MPH), are most effective treatment in ADHD, however, the molecular mechanism of action of MPH is still not fully elucidated. MPH is known to blocks the dopamine transporter (DAT) in the central nervous system, however, the effects on the dynamic DAT availability has not been assessed. For this purpose, human neuroblastoma dopaminergic cell line SH-SY5Y will be used following transfection of HaloTag® labelled DAT and MARCKS-GFP (membrane specific labelling). DAT trafficking between the cytoplasm and the membrane will be assessed using live cell imaging microscopy after treatment with different doses of MPH (range of 1-100nM and 1-100μM). Time-lapse imaging will be acquired following by data assessment using several software, including FIJI ImageJ (exporting and drift correction), Illastik (for machine learning strategy), CellPorfiler (image segmentation & quantification) and statistical software. The findings will elucidate whether MPH treatment alters DAT availability on the membrane and thus affects its functionality.
Attention Deficit-Hyperactivity Disorder (ADHD) is one of the most frequent psychiatric disorders in children and adolescents, with up to 5% affected worldwide. Psychostimulant such as methylphenidate (MPH), are most effective treatment in ADHD, however, the molecular mechanism of action of MPH is still not fully elucidated. MPH is known to blocks the dopamine transporter (DAT) in the central nervous system, however, the effects on the dynamic DAT availability has not been assessed. For this purpose, human neuroblastoma dopaminergic cell line SH-SY5Y will be used following transfection of HaloTag® labelled DAT and MARCKS-GFP (membrane specific labelling). DAT trafficking between the cytoplasm and the membrane will be assessed using live cell imaging microscopy after treatment with different doses of MPH (range of 1-100nM and 1-100μM). Time-lapse imaging will be acquired following by data assessment using several software, including FIJI ImageJ (exporting and drift correction), Illastik (for machine learning strategy), CellPorfiler (image segmentation & quantification) and statistical software. The findings will elucidate whether MPH treatment alters DAT availability on the membrane and thus affects its functionality.
The aim of this project is to investigate the effect of MPH on DAT trafficking and membrane availability.
The aim of this project is to investigate the effect of MPH on DAT trafficking and membrane availability.
Applicants should submit a CV, courses and grades taken during Bachelor and Master Studies, and a motivation letter. Any laboratory knowledge and experience should be listed. Student should be highly motivated to establish and learn new methods. Experience with cell culture will be an advance but it is not necessary.
Applicants should submit a CV, courses and grades taken during Bachelor and Master Studies, and a motivation letter. Any laboratory knowledge and experience should be listed. Student should be highly motivated to establish and learn new methods. Experience with cell culture will be an advance but it is not necessary.