In progress

DBP3: Multiscale modeling of DAT function in dopamine neurons

PI:Alexander Sorkin, PhD, Richard B. Mellon Professor and Chair, Department of Cell Biology, School of Medicine, University of Pittsburgh

Collaborator(s): Gonzalo Torres

Funding Source(s): 5R01DA014204-14, Dopamine transporter regulation by endocytosis ; 4/1/01-4/30/17 (Sorkin); 7R01DA038598, Regulation of dopamine transporter function by G protein β−γ subunits (Torres); 9/15/14 - 6/30/19

 


How the DBP acts as a driver of test bed:

Super-resolution time-dependent image data on DA neurons (obtained by EM, immunogold labeling and fluorescence microscopy) using transgenic knock-in mouse model (with HA-tagged DAT) will drive the construction of a spatiotemporally realistic model and MCell simulations of DA reuptake and the effects of drugs/psychostimulants and DA reuptake (influx) or reverse transport (efflux) that will be validated against experiments.

 


FigVII.1Highlight:

The neurotransmitter dopamine (DA) and dopaminergic neurons are involved in behaviors critical for species’ survival including motivation, motor planning, sensorimotor integration, habit formation, reward prediction/valuation and neuroendocrine regulation, as well as in Parkinson’s disease, schizophrenia, ADHD and drug addiction. Dopamine transporters (DATs) are present exclusively in DA neurons, and their function is to terminate DA neurotransmission by removing extracellular (EC) DA. Because DAT functions at peri- and extra-synaptic areas of the neuronal plasma membrane, regulation of subcellular targeting and surface levels of DAT is critical for overall activity of DA systems. DAT is synthesized in the soma of DA neurons in the midbrain and transported long-distance to synaptic areas in axons into the striatum and frontal cortex. Within the synapse, DAT localization at the cell surface, and therefore its function, is regulated by endocytosis. The dynamics and molecular mechanisms controlling steady-state neuronal DAT localization and its basal trafficking and endocytosis are not well understood. Furthermore, DAT localization and function are regulated by the neuronal activity, various signaling receptors present in DA neurons and DAT inhibitors, such as amphetamine (AMPH) and cocaine. Overall, a significant limitation has been that most of our mechanistic understanding about DAT endocytosis is based on studies in model expression systems using non-neuronal cells. Generation of multiscale computational models is necessary to use these data to investigate the dynamics of endogenous DAT transport and trafficking within the DA neuron. Such a computational methodology would have the power to predict the effects of drugs of abuse on DAT function, and, ultimately, on the intensity and duration of the DA transmission in the brain.

The specific aims of this project are:

To use advanced image analysis methods to measure the rate parameters of diffusion-based and vesicle-mediated transport of DAT between somatodendritic and axonal compartments of the neuron using a new transgenic knock-in mouse, in which endogenous DAT is replaced by HA epitope-tagged DAT, and construct a particle model of DAT traffic to predict the relative contribution of these two modes of DAT motility to the equilibrium distribution of DAT in the neuron.

This project will use image analysis methods developed in TR&D3, and MCell-based models developed in TR&D2.

To use an HA-DAT experimental model to study endocytic cycling of DAT within axonal varicosities (presynaptic areas of dopaminergic neurons) and use the quantitative data generated to develop, validate and refine an MCell model of DAT transport and DA uptake.

We will use the MCell model to predict the effects of altered endocytosis and recycling of DAT on its activity.

To measure the effects of acute and long-term exposure to AMPHs on the trafficking of DAT in DA neurons and incorporate these data into the MCell4 endocytic cycling models from the previous aim to generate a model of “addicted” DA neuron.

We will collaborate with DBP1 and TR&D1 toward gaining insights into the molecular structure/dynamic basis of the change in DAT activity upon AMPH binding.

 


Publications resulting from this work:

    • Cheng MH, Block E, Hu F, Cobanoglu MC, Sorkin A, Bahar I (2015) Insights into the modulation of dopamine transporter function by amphetamine, orphenadrine, and cocaine binding Front Neurol. 6: 134 PMID: 26106364, PMC4460958