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

Point mutations in the gene encoding the human dopamine transporter (hDAT, SLC6A3) cause a syndrome of infantile/juvenile dystonia and parkinsonism. To unravel the molecular mechanism underlying these disorders and investigate possible pharmacological therapies, here we examined 13 disease-causing DAT mutants that were retained in the endoplasmic reticulum when heterologously expressed in HEK293 cells. In three of these mutants, i.e. hDAT-V158F, hDAT-G327R, and hDAT-L368Q, the folding deficit was remedied with the pharmacochaperone noribogaine or the heat shock protein 70 (HSP70) inhibitor pifithrin-µ such that endoplasmic reticulum export of and radioligand binding and substrate uptake by these DAT mutants were restored. In Drosophila melanogaster, DAT deficiency results in reduced sleep. We therefore exploited the power of targeted transgene expression of mutant hDAT in Drosophila to explore whether these hDAT mutants could also be pharmacologically rescued in an intact organism. Noribogaine or pifithrin-µ treatment supported hDAT delivery to the presynaptic terminals of dopaminergic neurons and restored sleep to normal length in DAT-deficient (fumin) Drosophila lines expressing hDAT-V158F or hDAT-G327R. In contrast, expression of hDAT-L368Q in the Drosophila DAT mutant background caused developmental lethality, indicating a toxic action not remedied by pharmacochaperoning. Our observations identified those mutations most likely amenable to pharmacological rescue in the affected children. In addition, our findings also highlight the challenges of translating insights from pharmacochaperoning in cell culture to the clinical situation. Because of the evolutionary conservation in dopaminergic neurotransmission between Drosophila and people, pharmacochaperoning of DAT in D. melanogaster may allow us to bridge that gap.

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

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.

Relax, Cool Down and Scaffold: How to Restore Surface Expression of Folding-Deficient Mutant GPCRs and SLC6 Transporters.

Many diseases arise from mutations, which impair protein folding. The study of folding-deficient variants of G protein-coupled receptors and solute carrier 6 (SLC6) transporters has shed light on the folding trajectory, how it is monitored and how misfolding can be remedied. Reducing the temperature lowers the energy barrier between folding intermediates and thereby eliminates stalling along the folding trajectory. For obvious reasons, cooling down is not a therapeutic option. One approach to rescue misfolded variants is to use membrane-permeable orthosteric ligands. Antagonists of GPCRs are-in many instances-effective pharmacochaperones: they restore cell surface expression provided that they enter cells and bind to folding intermediates. Pharmacochaperoning of SLC6 transporters is less readily achieved because the ionic conditions in the endoplasmic reticulum (ER) are not conducive to binding of typical inhibitors. The second approach is to target the heat-shock protein (HSP) relay, which monitors the folding trajectory on the cytosolic side. Importantly, orthosteric ligands and HSP-inhibitors are not mutually exclusive. In fact, pharmacochaperones and HSP-inhibitors can act in an additive or synergistic manner. This was exemplified by rescuing disease-causing, folding-deficient variants of the human dopamine transporters with the HSP70 inhibitor pifithrin-µ and the pharmacochaperone noribogaine in Drosophila melanogaster.