Identifying dominant-negative actions of a dopamine transporter variant in patients with parkinsonism and neuropsychiatric disease
Freja Herborg, Kathrine L. Jensen, Sasha Tolstoy, Natascha V. Arends, Leonie P. Posselt, Aparna Shekar, Jenny I. Aguilar, Viktor K. Lund, Kevin Erreger, Mattias Rickhag, Matthew D. Lycas, Markus N. Lonsdale, Troels Rahbek-Clemmensen, Andreas T. Sørensen, Amy H. Newman, Annemette Løkkegaard, Ole Kjærulff, Thomas Werge, for the iPSYCH researchers, Lisbeth B. Møller, Heinrich J.G. Matthies, Aurelio Galli, Lena E. Hjermind, Ulrik Gether
Freja Herborg, Kathrine L. Jensen, Sasha Tolstoy, Natascha V. Arends, Leonie P. Posselt, Aparna Shekar, Jenny I. Aguilar, Viktor K. Lund, Kevin Erreger, Mattias Rickhag, Matthew D. Lycas, Markus N. Lonsdale, Troels Rahbek-Clemmensen, Andreas T. Sørensen, Amy H. Newman, Annemette Løkkegaard, Ole Kjærulff, Thomas Werge, for the iPSYCH researchers, Lisbeth B. Møller, Heinrich J.G. Matthies, Aurelio Galli, Lena E. Hjermind, Ulrik Gether
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Research Article Cell biology Neuroscience

Identifying dominant-negative actions of a dopamine transporter variant in patients with parkinsonism and neuropsychiatric disease

Abstract

Dysfunctional dopaminergic neurotransmission is central to movement disorders and mental diseases. The dopamine transporter (DAT) regulates extracellular dopamine levels, but the genetic and mechanistic link between DAT function and dopamine-related pathologies is not clear. Particularly, the pathophysiological significance of monoallelic missense mutations in DAT is unknown. Here, we use clinical information, neuroimaging, and large-scale exome-sequencing data to uncover the occurrence and phenotypic spectrum of a DAT coding variant, DAT-K619N, which localizes to the critical C-terminal PSD-95/Discs-large/ZO-1 homology–binding motif of human DAT (hDAT). We identified the rare but recurrent hDAT-K619N variant in exome-sequenced samples of patients with neuropsychiatric diseases and a patient with early-onset neurodegenerative parkinsonism and comorbid neuropsychiatric disease. In cell cultures, hDAT-K619N displayed reduced uptake capacity, decreased surface expression, and accelerated turnover. Unilateral expression in mouse nigrostriatal neurons revealed differential effects of hDAT-K619N and hDAT-WT on dopamine-directed behaviors, and hDAT-K619N expression in Drosophila led to impairments in dopamine transmission with accompanying hyperlocomotion and age-dependent disturbances of the negative geotactic response. Moreover, cellular studies and viral expression of hDAT-K619N in mice demonstrated a dominant-negative effect of the hDAT-K619N mutant. Summarized, our results suggest that hDAT-K619N can effectuate dopamine dysfunction of pathological relevance in a dominant-negative manner.

Authors

Freja Herborg, Kathrine L. Jensen, Sasha Tolstoy, Natascha V. Arends, Leonie P. Posselt, Aparna Shekar, Jenny I. Aguilar, Viktor K. Lund, Kevin Erreger, Mattias Rickhag, Matthew D. Lycas, Markus N. Lonsdale, Troels Rahbek-Clemmensen, Andreas T. Sørensen, Amy H. Newman, Annemette Løkkegaard, Ole Kjærulff, Thomas Werge, for the iPSYCH researchers, Lisbeth B. Møller, Heinrich J.G. Matthies, Aurelio Galli, Lena E. Hjermind, Ulrik Gether

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Figure 2

DAT-K619N displays functional impairments and reduced surface expression in vitro.

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DAT-K619N displays functional impairments and reduced surface expression...
(A–C) Evaluation of DAT-K619N functions and surface expression in transiently transfected HEK293 cells. (A) Functional comparison of DAT-K619N to WT using [3H]-dopamine (DA) uptake. Uptake curves (left) are average curves of 6 experiments each performed in triplicate and normalized to the fitted maximal uptake capacity (Vmax) of DAT-WT. The DAT-K619N variant demonstrated reduced Vmax (right bar diagram) compared with DAT-WT (P < 0.01, 1-sample 2-tailed t test, n = 6) with no accompanying change in Km (Km =1.4 ± 0.3 μM for K619N vs. 1.7 ± 0.4 μM for DAT-WT, P > 0.05 Student’s t test). (B) Amphetamine-induced amperometric currents with representative traces of amperometric currents (left) and quantification of the amphetamine-induced peak currents relative to DAT-WT (right). DA-release through DAT-K619N was impaired compared with DAT-WT (P < 0.05, 1-sample 2-tailed t test, n = 9 for WT and n = 7 for DAT-K619N). (C) Surface biotinylation of transiently transfected HEK293 cells. The amount of DAT-K619N relative to DAT-WT was decreased in the surface and the total protein fractions (P < 0.05, 1-sample 2-tailed t test, n = 4). (D) Confocal live imaging of surface-expressed WT and DAT-K619N in HEK239 cells by labeling with the fluorescent cocaine analog, JHC 1-64 (20 nM). Mean intensity of the JCH 1-64 signal was reduced for DAT-K619N relative to DAT-WT. Images were acquired from 4 independent transfections, and intensities were normalized to WT mean intensity for each imaging session (P < 0.05, 1-sample 2-tailed t test, n = 21 images per group). Data are mean ± SEM. *P < 0.05, **P < 0.01.