Pharmacological Chaperones of the Dopamine Transporter Rescue Dopamine Transporter Deficiency Syndrome Mutations in Heterologous Cells

Abstract

A number of pathological conditions have been linked to mutations in the dopamine transporter gene, including hereditary dopamine transporter deficiency syndrome (DTDS). DTDS is a rare condition that is caused by autosomal recessive loss-offunction mutations in the dopamine transporter (DAT), which often affects transporter trafficking and folding. We examined the possibility of using pharmacological chaperones of DAT to rescue DTDS mutations. After screening a set of known DAT ligands for their ability to increase DAT surface expression, we found that bupropion and ibogaine increased DAT surface expression, whereas others, including cocaine and methylphenidate, had no effect. Bupropion and ibogaine increased wild type DAT protein levels and also promoted maturation of the endoplasmic reticulum (ER)-retained DAT mutant K590A. Rescue of K590A could be blocked by inhibiting ER to Golgi transport using brefeldin A. Furthermore, knockdown of coat protein complex II (COPII) component SEC24D, which is important in the ER export of wild type DAT, also blocked the rescue effects of bupropion and ibogaine. These data suggest that bupropion and ibogaine promote maturation of DAT by acting as pharmacological chaperones in the ER. Importantly, both drugs rescue DAT maturation and functional activity of the DTDS-associated mutations A314V and R445C. Together, these results are the first demonstration of pharmacological chaperoning of DAT and suggest this may be a viable approach to increase DAT levels in DTDS and other conditions associated with reduced DAT function. The dopamine transporter (DAT)4 is responsible for controlling levels of extracellular dopamine and maintaining dopamine stores by transporting dopamine back into neurons after release (1, 2). DAT is part of the Na ϩ /Cl Ϫ -dependent neurotransmitter symporter solute carrier 6 (SLC6) family, which also includes the serotonin, norepinephrine, taurine, and GABA transporters (GAT) (3). A variety of pathological conditions have been associated with mutations in both coding and non-coding regions of the DAT gene (SLC6A3), most of which result in reduced DAT function (4 -7). A recently discovered condition, hereditary DAT deficiency syndrome (DTDS) is caused by autosomal recessive loss-of-function mutations in DAT (6,8,9). The disorder is characterized by parkinsonismdystonia and elevated dopamine metabolites in the cerebrospinal fluid. To date, very few diagnosed DTDS patients have survived to adulthood; the majority of patients die in infancy or adolescence (8, 9). When expressed in heterologous cells, DTDS mutations prevent DAT protein maturation past the endoplasmic reticulum (ER) and result in reduction or elimination of dopamine uptake activity (9). ER retention is a common consequence of mutations in membrane proteins and does not necessarily reflect complete misfolding (10). However, the quality control in the ER is so stringent that mutated proteins that are otherwise partially or completely functional can be retained and degraded (11). Indeed, folding of eve...

Pharmacochaperoning in a Drosophila model system rescues human dopamine transporter variants associated with infantile/juvenile parkinsonism

“…In a recent study, four disease-causing hDAT mutants ( i.e. hDAT-L224P, -A314V, -R445C, and -P529L) were examined for their responsiveness to pharmacochaperoning; pretreatment of HEK293 cells with by ibogaine and bupropione restored surface expression of and substrate uptake by hDAT-A314V and of hDAT-R445C ( 45 ). We have not been able to recapitulate these findings; it is evident from Fig.…”

“…We do not consider it likely that this discrepancy can be attributed to our using noribogaine, because noribogaine is more potent than ibogaine on DAT ( 46 ). It appears more likely that the discrepancy can be accounted for by the different transfection regimen; we assessed pharmacochaperoning in transiently transfected cells, and Beerepoot et al ( 45 ) used stably transfected cells. Clonal selection may magnify a pharmacochaperoning effect, which escapes detection when assessed in the entire population.…”

Behavioral Effects of a Potential Novel TAAR1 Antagonist

“…Cells were passaged 24 h prior to transfection at 50% confluency (∼2 × 10 6 cells in a 10 cm plate). Transfections were carried out using the PEI method as described previously ( Ehrhardt et al, 2006 ; Lam et al, 2013 ; Beerepoot et al, 2016 ). PEI and plasmid DNA (3 μl:1 μg PEI:DNA ratio) were added into separate tubes (tube 1: PEI, tube 2: DNA) followed by 200 μL of DMEM into each tube, containing no supplements.…”

Big Lessons from Tiny Flies:<em> Drosophila Melanogaster </em>as a Model to Explore Dysfunction of Dopaminergic and Serotonergic Neurotransmitter Systems

“…Two DTDS-causing mutants (hDAT-V158F and hDAT-G327R) were amenable to functional rescue by noribogaine and pifithrin-µ in living flies: pharmacochaperoning restored axonal delivery of the otherwise ER-retained mutant protein and corrected the sleepless phenotype [61][62][63]. Another research group reported that some of the mutants are also responsive to pharmacological rescue by bupropion [64], although this finding has not been confirmed in behavioral assays in flies. Monoamine transporters have a rich pharmacology comprising atypical inhibitors and partial substrates, i.e.…”