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

Abstract

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 and structural analysis of rareSLC2A2variants associated with Fanconi‐Bickel syndrome and metabolic traits

“…Various mechanisms for improper sorting of mutant transporters in GLUTs and other SLC s have been described (El‐Kasaby, Koban, Sitte, Freissmuth, & Sucic, ; Koban et al, ; Skach, , Toye, ; Wieman et al, ). Generally, these mechanisms involve the retention (in ER or Golgi apparatus) and eventual destruction of aggregated or misfolded mutant protein or the misdirection of mutant protein to the incorrect membrane (Asjad et al, ; El‐Kasaby et al, ; Koban et al, ; Skach, ; Toye, ).…”

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

“…Both, siRNA-induced depletion of HSP70-1A and of HSP90β and their inhibition by small molecules restore cell surface expression of functional transporters in some but not all folding-deficient mutants [ 57 ]. Finally, pharmacochaperoning of folding-deficient mutants of SERT [ 57 ] and DAT [ 60 , 61 ] by noribogaine results in the release of the heat-shock protein relay.…”

“…This prediction has been verified in several instances: (i) as mentioned above inhibitors of HSP90 enhance surface expression of the A 2A -receptor [ 52 ] and of the V 2 -vasopressin receptor [ 103 ]. Similarly, ER export and cell surface expression of some folding-deficient SERT mutants [ 57 ] and disease-associated DAT mutants [ 61 ] can be restored by inhibitors of HSP90; (ii) The HSP70 inhibitor pifithrin-μ (2-phenylethynesulfonamide) is also effective at restoring the surface expression of several folding-deficient SERT [ 57 ] and DAT mutants [ 60 , 61 ]. Importantly, pifithrin-μ also rescues folding-defective DAT mutants in vivo: Drosophila melanogaster , which lack a functional DAT, are sleepless [ 104 ].…”

“…Importantly, pifithrin-μ also rescues folding-defective DAT mutants in vivo: Drosophila melanogaster , which lack a functional DAT, are sleepless [ 104 ]. Reduced sleep length is also seen true for flies harbouring folding-deficient DAT mutants [ 60 , 61 , 105 ]. Pifithrin-μ is as effective as the pharmacochaperone noribogaine in restoring sleep, if these flies are administered the drugs via their food [ 60 , 61 ].…”

“…Reduced sleep length is also seen true for flies harbouring folding-deficient DAT mutants [ 60 , 61 , 105 ]. Pifithrin-μ is as effective as the pharmacochaperone noribogaine in restoring sleep, if these flies are administered the drugs via their food [ 60 , 61 ]. Similarly, the increase in V 2 -receptor expression, which is induced by HSP90 inhibitors, is also seen in people: hyponatraemia is a side effect that was frequently observed in cancer patients undergoing clinical trials with various HSP90 inhibitors [ 103 ].…”