Psychomotor impairments and therapeutic implications revealed by a mutation associated with infantile Parkinsonism-Dystonia

Abstract

Parkinson disease (PD) is a progressive, neurodegenerative disorder affecting over 6.1 million people worldwide. Although the cause of PD remains unclear, studies of highly penetrant mutations identified in early-onset familial parkinsonism have contributed to our understanding of the molecular mechanisms underlying disease pathology. Dopamine (DA) transporter (DAT) deficiency syndrome (DTDS) is a distinct type of infantile parkinsonism-dystonia that shares key clinical features with PD, including motor deficits (progressive bradykinesia, tremor, hypomimia) and altered DA neurotransmission. Here, we define structural, functional, and behavioral consequences of a Cys substitution at R445 in human DAT (hDAT R445C), identified in a patient with DTDS. We found that this R445 substitution disrupts a phylogenetically conserved intracellular (IC) network of interactions that compromise the hDAT IC gate. This is demonstrated by both Rosetta molecular modeling and fine-grained simulations using hDAT R445C, as well as EPR analysis and X-ray crystallography of the bacterial homolog leucine transporter. Notably, the disruption of this IC network of interactions supported a channel-like intermediate of hDAT and compromised hDAT function. We demonstrate that Drosophila melanogaster expressing hDAT R445C show impaired hDAT activity, which is associated with DA dysfunction in isolated brains and with abnormal behaviors monitored at high-speed time resolution. We show that hDAT R445C Drosophila exhibit motor deficits, lack of motor coordination (i.e. flight coordination) and phenotypic heterogeneity in these behaviors that is typically associated with DTDS and PD. These behaviors are linked with altered dopaminergic signaling stemming from loss of DA neurons and decreased DA availability. We rescued flight coordination with chloroquine, a lysosomal inhibitor that enhanced DAT expression in a heterologous expression system. Together, these studies shed some light on how a DTDS-linked DAT mutation underlies DA dysfunction and, possibly, clinical phenotypes shared by DTDS and PD.

Bile Acids Gate Dopamine Transporter Mediated Currents

“…These conformers occasionally gave rise to simultaneous opening of both the EC and IC gates such that formation of an intermittent water channel was detected. Notably, the sodium permeation pathway (Aguilar et al, 2021) coincides with that of water channeling (Cheng et al, 2018;Cheng and Bahar, 2019). This path, observed in silico, may also be associated with DATmediated ion fluxes or leak currents (Ingram et al, 2002;Erreger et al, 2008).…”

“…Recently, molecular modeling found that the infantile Parkinsonism-Dystonia associated substitution, R445C in hDAT, disrupted a phylogenetically conserved intracellular network of interactions and promoted a channel-like intermediate of hDAT. These rearrangements lead to the permeation of Na + from both the EC and IC solutions (Aguilar et al, 2021). Interestingly, docking simulations suggested that OCA could also bind near H444/R445 to stabilize the IF state or act as an anion-lipid (Figure 6A for OCA(−) in the IFo state).…”

Molecular and Clinical Repercussions of GABA Transporter 1 Variants Gone Amiss: Links to Epilepsy and Developmental Spectrum Disorders

“…It recently proved to have great translational potential in the case of folding-impaired DAT variants (Mazhar Asjad et al, 2017). We, and others, have examined the trafficking and activity of dopamine transporter deficiency syndrome (DTDS)-linked mutants in Drosophila ( Kasture et al, 2016 ; Mazhar Asjad et al, 2017; Aguilar et al, 2021 ). Drug screens carried out in Drosophila were led by data from in silico and in vitro experiments, and have also been validated in induced pluripotent stem cells (iPSCs) obtained from DTDS patients ( Ng et al, 2021 ).…”

The Use of Drosophila to Understand Psychostimulant Responses

“…Experiments in cell culture show that many psychiatric disorder-associated DAT variants exhibit heterogenous molecular phenotypes, including differences in DA uptake kinetics, reverse transport, and altered binding to psychostimulant drugs [ 125 ]. Drosophila is an efficient model for studying the effects of human DAT mutants in vivo and have shown that DAT mutations associated with early-onset Parkinson’s [ 126 ] as well as ASD [ 107 ] lead to impairments in motor behavior. Drosophila is a feasible system for performing similar experiments to unravel the behavioral and molecular nature of DAT variants associated with ADHD, offering a practical model to identify the molecular mechanisms involved in response to psychostimulant drugs.…”