Characterization of DAT missense mutations in novel mouse models

Abstract

The dopamine transporter (DAT) is responsible for dopamine (DA) clearance in the synaptic cleft, and thus crucial for maintaining DA homeostasis in the central nervous system (CNS). Mutations in the DAT coding gene have been associated with both movement and psychiatric disorders, such as parkinsonism and attention-deficit hyperactivity disorder (ADHD). However, the detailed contribution of DAT in human disease development is yet poorly understood. In the present study, we performed in vivo and ex vivo experiments to investigate the role of two DAT missense mutations, DAT I312F and DAT-D421N, that previously have been identified in a single patient diagnosed with a unique combination of both early onset parkinsonism and ADHD. Knock in mice homozygous for either DAT-I312F or DAT-D421N or compound heterozygous for the two mutations, corresponding to the patient genotype, were investigated. Striatal synaptosomes isolated from the three mice strains revealed dramatic reductions in DA uptake capacity for all three strains (Vmax: I312F = 61%, IFxDN = 24% and D421N = 10% of WT Vmax). Furthermore, confocal imaging of immunolabelled coronal brain slices displayed an apparent stronger immunosignal for striatal DAT in I312F KI mice, whereas the dopaminergic network in D421N KI mice showed reduced density, maybe reflecting neurodegeneration. Together these findings suggest that the two DAT mutations lead to altered DAT function and cause distinct DAergic dysregulation. Altered DA homeostasis was suggested by behavioral studies where the DAT-I312F KI mice demonstrated drastic increased locomotor activity both horizontally and vertically, however, without any signs of motor dysfunction. The results from my thesis strengthens the link of the two DAT missense mutations as risk factors for both ADHD and neurodegenerative early-onset parkinsonism and contribute to the insight of DAergic associated pathologies.

Popular Abstract

Characterization of new mouse models with mutation in the dopamine transporter gene

Dysregulations of dopamine signaling in the brain are believed to be the underlying cause of several neurological and psychiatric disorders. Yet it remains unclear how the exact mechanisms of dopamine dysregulation contributes to the development of these diseases. Many studies focus on the implication of mutations or other factors that can negatively affect the dopamine signaling, to increase the understanding of the development of human diseases and potentially lead to better clinical treatments in the future.

Dopamine is a neurotransmitter in the central nervous system that is important for regulation of physiological functions, such as movement control, attention, and reward mechanism. Upon activation, dopamine is released from a dopamine neuron into the synaptic cleft and binds to receptors located on other neurons, which then activates next neuron. To stop the dopamine signaling, dopamine is transported back into the dopamine neuron by a protein called dopamine transporter (DAT). This process is crucial for maintaining normal dopamine concentrations in the brain. Interestingly, mutations in different genes that result in disrupted dopamine signaling are associated to both psychiatric and neurological disorders.

DAT mutation in human disease
Recent studies have identified several mutations in the DAT gene in patients diagnosed with ADHD, bipolar disorder, autism and movement disorders. One patient diagnosed with the unique combination of both ADHD and early-onset parkinsonism was recently identified with two mutations in the DAT gene; DAT-I312F and DAT-D421N. To study the potential role of these mutations in disease development, studies in cell cultures were carried out. These studies revealed that both mutations lead to severe consequences of the transporter function. In my master project, I have further investigated the impact of the I312F and D421N mutations in living animals. Three mouse strains were therefore generated; two strains carried either I312F or D421N mutations and the third carried both mutations, to mimic the combination of mutations found in the patient. My results show that the transport activity mediated by DAT was remarkably reduced in all three mice strains, especially D421N mice (10% of control). Furthermore, microscopy imaging displayed elevated signal intensity of DAT in I312F mice and a reduction in the number of dopaminergic neurons in D421N, most likely due to neurodegeneration. Lastly, in behavioral studies on I312F mice, we saw a strong hyperactivity, which is one of the most characteristic symptoms of human ADHD. Together, my experiments indicate that there is a linkage between the I312F mutation and ADHD symptoms as well as between the D421N mutation and the neurodegenerative progression. Both these conditions are found in the patient, suggesting that these mouse models could possibly serve as valuable tools to expand the insight into how DAT dysfunction might contribute to dopamine-associated diseases.

Master’s Degree Project in Molecular Biology 60 credits, 2017
Department of Biology, Lund University

Advisor: Ulrik Gether and Freja Herborg Hansen
Department of Neuroscience, University of Copenhagen

• author: Berlin, Frida
• supervisor: Ulrik Gether
• organization: Degree Projects in Molecular Biology
• course: MOBT01 20162
• year: 2017
• type: H2 - Master's Degree (Two Years)
• subject: Biology and Life Sciences
• language: English
• id: 8927074
• date added to LUP: 2017-10-10 11:59:27
• date last changed: 2017-10-10 11:59:27