Biology subjects

Mabry, S. J.

Publications and source records attributed to Mabry, S. J..

2 recordsLinked to original sources

bioRxiv2022

Syntaxin1 Ser14 Phosphorylation is Required for Non-Vesicular Dopamine Release

Amphetamine (AMPH), a psychostimulant commonly prescribed for the treatment of neuropsychiatric and neurological disorders, has a high liability for abuse. The abuse and psychomotor stimulant properties of AMPH are primarily associated with its ability to increase dopamine (DA) neurotransmission. This increase is mediated, in large part, by non-vesicular DA release (DA efflux). DA efflux is the result of reversal of the DA transporter (DAT) promoted by AMPH. Syntaxin 1 (Stx1) is a SNARE protein that plays a pivotal role in vesicular release. Previously, we have shown that Stx1 also interacts with the distal DAT N-terminus, an event promoted by AMPH. Stx1 is phosphorylated at Ser14 by casein kinase II (CK2). Using Drosophila Melanogaster as an animal model, we show that this phosphorylation event is critical for non-vesicular DA release and regulates the expression of AMPH preference as well as the ability of AMPH to promote mating drive. We also show that reverse transport of DA mediated by DAT underlies these complex behaviors promoted by AMPH. Our molecular dynamics (MD) simulations of the phosphorylated DAT/Stx1 complex demonstrate that the phosphorylation state of these proteins plays a key role in allowing DAT to dwell in an efflux-willing state. This state also supports constitutive DA efflux (CDE), an event that occurs in the absence of AMPH. The DAT-Stx1 phosphorylated complex is characterized by the breakdown of two key salt bridges in DAT, K66-D345 and E428-R445, which are critical for the formation of the intracellular (IC) gate and for transport function. The breaking of these salt bridges leads to an opening and hydration of the DAT intracellular vestibule, allowing DA to bind from the cytosol, a mechanism that we hypothesize leads to CDE. We further determine the importance of Stx1 phosphorylation in CDE by pharmacologically inhibiting CK2 with CX-4945, a molecule currently in phase II clinical trials for cancer treatment. CX-4945 treatment prevented the expression of CDE in isolated Drosophila Melanogaster brains as well as behaviors associated with CDE. Thus, our results suggest that Stx1 phosphorylation is a possible pharmacological target for the treatment of AMPH abuse.

neuroscience↗

bioRxiv2021

Psychomotor impairments and therapeutic implications revealed by a mutation linked with Infantile Parkinsonism-Dystonia

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 through enhanced DAT surface expression via the lysosomal inhibitor chloroquine. Together, these studies shed light on how a DTDS-linked DAT mutation underlies DA dysfunction and, more broadly, the clinical phenotypes shared by DTDS and PD.

neuroscience↗