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1.
Nat Commun ; 8(1): 740, 2017 09 29.
Article in English | MEDLINE | ID: mdl-28963530

ABSTRACT

Dopamine regulates reward, cognition, and locomotor functions. By mediating rapid reuptake of extracellular dopamine, the dopamine transporter is critical for spatiotemporal control of dopaminergic neurotransmission. Here, we use super-resolution imaging to show that the dopamine transporter is dynamically sequestrated into cholesterol-dependent nanodomains in the plasma membrane of presynaptic varicosities and neuronal projections of dopaminergic neurons. Stochastic optical reconstruction microscopy reveals irregular dopamine transporter nanodomains (∼70 nm mean diameter) that were highly sensitive to cholesterol depletion. Live photoactivated localization microscopy shows a similar dopamine transporter membrane organization in live heterologous cells. In neurons, dual-color dSTORM shows that tyrosine hydroxylase and vesicular monoamine transporter-2 are distinctively localized adjacent to, but not overlapping with, the dopamine transporter nanodomains. The molecular organization of the dopamine transporter in nanodomains is reversibly reduced by short-term activation of NMDA-type ionotropic glutamate receptors, implicating dopamine transporter nanodomain distribution as a potential mechanism to modulate dopaminergic neurotransmission in response to excitatory input.The dopamine transporter (DAT) has a crucial role in the regulation of neurotransmission. Here, the authors use super-resolution imaging to show that DAT clusters into cholesterol-dependent membrane regions that are reversibly regulated by ionotropic glutamate receptors activation.


Subject(s)
Cell Membrane/metabolism , Cholesterol/metabolism , Dopamine Plasma Membrane Transport Proteins/metabolism , Dopaminergic Neurons/metabolism , Presynaptic Terminals/metabolism , Animals , Cell Line , Cell Membrane/ultrastructure , Dopamine Plasma Membrane Transport Proteins/ultrastructure , Dopaminergic Neurons/ultrastructure , Mice , Microscopy , Neurons/metabolism , Neurons/ultrastructure , Presynaptic Terminals/ultrastructure , Receptors, Ionotropic Glutamate/metabolism , Synaptic Transmission , Tyrosine 3-Monooxygenase/metabolism , Vesicular Monoamine Transport Proteins/metabolism
2.
J Clin Invest ; 124(7): 3107-20, 2014 Jul.
Article in English | MEDLINE | ID: mdl-24911152

ABSTRACT

Parkinsonism and attention deficit hyperactivity disorder (ADHD) are widespread brain disorders that involve disturbances of dopaminergic signaling. The sodium-coupled dopamine transporter (DAT) controls dopamine homeostasis, but its contribution to disease remains poorly understood. Here, we analyzed a cohort of patients with atypical movement disorder and identified 2 DAT coding variants, DAT-Ile312Phe and a presumed de novo mutant DAT-Asp421Asn, in an adult male with early-onset parkinsonism and ADHD. According to DAT single-photon emission computed tomography (DAT-SPECT) scans and a fluoro-deoxy-glucose-PET/MRI (FDG-PET/MRI) scan, the patient suffered from progressive dopaminergic neurodegeneration. In heterologous cells, both DAT variants exhibited markedly reduced dopamine uptake capacity but preserved membrane targeting, consistent with impaired catalytic activity. Computational simulations and uptake experiments suggested that the disrupted function of the DAT-Asp421Asn mutant is the result of compromised sodium binding, in agreement with Asp421 coordinating sodium at the second sodium site. For DAT-Asp421Asn, substrate efflux experiments revealed a constitutive, anomalous efflux of dopamine, and electrophysiological analyses identified a large cation leak that might further perturb dopaminergic neurotransmission. Our results link specific DAT missense mutations to neurodegenerative early-onset parkinsonism. Moreover, the neuropsychiatric comorbidity provides additional support for the idea that DAT missense mutations are an ADHD risk factor and suggests that complex DAT genotype and phenotype correlations contribute to different dopaminergic pathologies.


Subject(s)
Attention Deficit Disorder with Hyperactivity/genetics , Attention Deficit Disorder with Hyperactivity/metabolism , Dopamine Plasma Membrane Transport Proteins/genetics , Dopamine Plasma Membrane Transport Proteins/metabolism , Mutant Proteins/genetics , Mutant Proteins/metabolism , Mutation, Missense , Parkinsonian Disorders/genetics , Parkinsonian Disorders/metabolism , Adult , Amino Acid Sequence , Amino Acid Substitution , Animals , Attention Deficit Disorder with Hyperactivity/complications , Brain/diagnostic imaging , Brain/metabolism , Cohort Studies , DNA Mutational Analysis , Dopamine/metabolism , Dopamine Plasma Membrane Transport Proteins/chemistry , Female , HEK293 Cells , Humans , Male , Models, Molecular , Molecular Sequence Data , Mutant Proteins/chemistry , Oocytes/metabolism , Parkinsonian Disorders/complications , Pedigree , Positron-Emission Tomography , Protein Conformation , Recombinant Proteins/chemistry , Recombinant Proteins/genetics , Recombinant Proteins/metabolism , Sequence Homology, Amino Acid , Sodium/metabolism , Tomography, Emission-Computed, Single-Photon , Xenopus
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