Identifying dominant-negative actions of a dopamine transporter variant in patients with parkinsonism and neuropsychiatric disease
Freja Herborg, … , Lena E. Hjermind, Ulrik Gether
Freja Herborg, … , Lena E. Hjermind, Ulrik Gether
Published August 10, 2021
Citation Information: JCI Insight. 2021;6(18):e151496. https://doi.org/10.1172/jci.insight.151496.
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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 6

DAT-K619N exerts a dominant-negative effect on DAT-WT.

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DAT-K619N exerts a dominant-negative effect on DAT-WT. (A) Evaluation of...
(A) Evaluation of dominant-negative effects of DAT-K619N in vitro. DA uptake was measured in HEK293 cells, cotransfected with equal amounts (1.5 μg) of DAT-K619N and DAT-WT, and compared with cells transfected only with DAT-WT (1.5 μg + 1.5 μg empty vector), DAT-K619N (1.5 μg + 1.5 μg empty vector), or with 3 μg of DAT-WT as a control. Each experiment was performed in triplicate and normalized to Vmax of DAT-WT (1.5 μg + 1.5 μg empty vector). The Vmax of DAT-K619N/DAT-WT cotransfected cells (1.5 μg + 1.5 μg) was reduced relative to DAT-WT (1.5 μg + 1.5 μg empty vector) to a level similar to DAT-K619N alone (*P < 0.016, 1-sample 2-tailed t test, Bonferroni-adjusted significance level [= 0.05/3] n = 4–7) indicating dominant-negative actions of hDAT-K619N. (B) Evaluation of dominant-negative effects in vivo performed by overexpressing HA-hDAT-WT or HA-hDAT-K619N selectively in dopaminergic neurons, using bilateral midbrain AAV injections in TH-Cre mice, and performing [3H]-DA uptake on striatal synaptosomes. Injections with AAV encoding mCherry was used for comparison with endogenous DA uptake levels. Uptake curves are average curves of 4 experiments each performed in triplicate. Quantification of Vmax normalized to mCherry showed HA-hDAT-K619N but not HA-hDAT-WT reduced DA uptake below endogenous uptake capacity (*P < 0.005, 1-sample 2-tailed t test, Bonferroni-adjusted significance level: **P < 0.01/2; n = 4. (C) Western blot analysis of striatal synaptosomal preparations. Representative blots for total DAT, HA-DAT, and TH are shown. Specific detection of HA-hDAT-WT and HA-hDAT-K619N using anti-HA antibody confirmed both constructs were trafficked to striatal terminals. (D–F) Quantification of relative expression levels revealed a reduction in both DAT (E) and TH (F) in HA-hDAT-K619N–injected mice (*P < 0.025, 1-sample 2-tailed t test, Bonferroni-adjusted significance level [= 0.05/2], n = 4); HA-DAT levels did not differ significantly between HA-DAT-WT and HA-DAT-K619N–injected mice (P = 0.21, 1-sample 2-tailed t test, n = 4).