Functional Rescue of a Misfolded Drosophila melanogaster Dopamine Transporter Mutant Associated with a Sleepless Phenotype by Pharmacological Chaperones

Abstract

Folding-defective mutants of the human dopamine transporter (DAT) cause a syndrome of infantile dystonia/parkinsonism. Here, we provide a proof-of-principle that the folding deficit is amenable to correction in vivo by two means, the cognate DAT ligand noribogaine and the HSP70 inhibitor, pifithrin-μ. We examined the Drosophila melanogaster (d) mutant dDAT-G108Q, which leads to a sleepless phenotype in flies harboring this mutation. Molecular dynamics simulations suggested an unstable structure of dDAT-G108Q consistent with a folding defect. This conjecture was verified; heterologously expressed dDAT-G108Q and the human (h) equivalent hDAT-G140Q were retained in the endoplasmic reticulum in a complex with endogenous folding sensors (calnexin and HSP70-1A). Incubation of the cells with noribogaine (a DAT ligand selective for the inward-facing state) and/or pifithrin-μ (an HSP70 inhibitor) restored folding of, and hence dopamine transport by, dDAT-G108Q and hDAT-G140Q. The mutated versions of DAT were confined to the cell bodies of the dopaminergic neurons in the fly brain and failed to reach the axonal compartments. Axonal delivery was restored, and sleep time was increased to normal length (from 300 to 1000 min/day) if the dDAT-G108Q-expressing flies were treated with noribogaine and/or pifithrin-μ. Rescuing misfolded versions of DAT by pharmacochaperoning is of therapeutic interest; it may provide opportunities to remedy disorders arising from folding-defective mutants of human DAT and of other related SLC6 transporters.

Modulation of social space by dopamine in Drosophila melanogaster, but no effect on the avoidance of the Drosophila stress odorant

“…Furthermore, flies might rely on sexually dimorphic dopaminergic neurons to generate proper social spacing, as shown previously for stress response [27]. Finally, we found no effect on social spacing of loss of function of VMAT in dopaminergic cells in the evening, which supports previous reports of the role of dopamine and VMAT in sleep and arousal, although no change in activity patterns themselves has been reported [28][29][30][31][32].…”

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

“…Trapping of proteins in the ER is indicative of their misfolding; the defective folding can be remedied by various chemical and pharmacological chaperones. We opted for the ibogaine metabolite noribogaine (12-hydroxyibogamine), because for SERT mutants it is a more effective pharmacochaperone than the parent compound ibogaine ( 14 ) and because noribogaine rescued hDAT-G140Q and dDAT-G108Q ( 20 ). In addition, we also tested pifithrin-μ, a cell-permeable small molecule inhibitor of HSP70 ( 21 ) and the HSP90 inhibitor 17-DMAG, because these compounds are predicted to relax control imposed by the heat shock protein relay ( 8 ), which operates on the C terminus ( 14 ).…”

“…Inhibitors bind to the outward-facing conformation, which is contingent on the presence of high Na + concentrations ( 24 ). Hence, binding of the radiolabeled inhibitor [ 3 H]CFT to intact cells only detects transporters at the cell surface ( 20 ). Binding of [ 3 H]CFT was increased in cells expressing DAT-V158F, DAT-G327R, and DAT-L368Q, if they had been pretreated with noribogaine or pifithrin-μ ( Fig.…”

“…The chaperone/COPII-exchange model predicts that the stalled folding intermediates can be assisted for ER export by manipulating the chaperone or folding sensor machinery ( 8 ); these predictions have been verified ( 14 , 15 , 19 ). More recently we employed pharmacochaperoning to rescue a misfolded mutant of drosophila DAT, dDAT-G108Q ( 20 ). In the present work, we combined a cellular analysis of disease-causing human DAT mutants with their expression in Drosophila melanogaster .…”

Sequence determinants of the Caenhorhabditis elegans dopamine transporter dictating in vivo axonal export and synaptic localization

“…Further progress in this area will likely require higher resolution evaluation of SEC24D:DAT complexes. Further progress in this area will likely require higher resolution evaluation of SEC24D:DAT complexes, and ultimately may provide new opportunities to remedy the disrupted trafficking that is evident with disease-associated DAT mutations (Kasture et al, 2016). As this work proceeds, the opportunities of the C. elegans model for assessment of DAT trafficking to identified, mature DA synapses in vivo may prove highly advantageous.…”