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Rescueing sleepless flies – Pharmacochaperoning of folding-deficient dopamine transporters, which give rise to infantile dystonia/Parkinsonism

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Freissmuth M., El- Kasaby A., Kasture A., Asjad H. M. M., Sucic S.

Acta Neurobiologiae Experimentalis

2017

tom 77

nr Suppl.1

Folding-defective mutants of the human dopamine transporter (DAT) cause a syndrome of infantile dystonia/Parkinsonism. 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: a mutation in the gene encoding dDAT was identified in the Zuker collection of Drosophila melanogaster, which leads to a sleepless phenotype in flies harboring the mutation dDAT‑G108Q. We examined the structure of dDAT‑G108Q by molecular dynamics simulations using the published crystal structure of dDAT as a starting point. These simulation provided evidence for structural instability of dDAT‑G108Q consistent with a folding defect. We verified this conjecture by visulaizing heterologously expressed dDAT‑G108Q and the human equivalent hDAT‑G140Q in the endoplasmic reticulum and by showing that it was found 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‑inhibtor) restored folding of, and hence dopamine transport by, dDAT‑G108Q and of 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 increased to normal length (from 300 to 1000 min/d), if 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, e.g. of the human creatine transporter-1, which gives rise to mental retardation when mutated at the equivalent glycine residue. FINANCIAL SUPPORT: SFB35-10 by the Austrian Science Fund/FWF.

Trafficking of SLC6A14 – An amino acid transporter B(0,+) to plasma membrane

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Kovalchuk V., Jurkiewicz D., Juraszek B., Samluk L., Sucic S., Freissmuth M., Nalecz K. A.

Acta Neurobiologiae Experimentalis

2017

tom 77

nr Suppl.1

INTRODUCTION: The solute carrier 6 (SLC6) family of genes codes transporters for neurotransmitters, amino acids, osmolytes and energy metabolites. In the brain SLC6 transporters play an important role in the reuptake of GABA, serotonin, dopamine, noradrenaline, glycine and proline. SLC6A14 is a Na/Cl-dependent amino acid transporter ATB(0,+) – specific towards neutral and cationic amino acids. It is present in astrocytes and in the blood-brain barrier. In the first step of trafficking to plasma membrane – ER exit, the neurotransmitter transporters interact with specific isoforms of SEC24 proteins, the components of Coatomer II (COPII). AIM(S): To verify, which proteins are involved in trafficking of ATB(0,+) to the cell surface from ER. METHOD(S): Trafficking of rat SLC6A14 to plasma membrane was studied in heterologous expression system. RESULTS: Immunofluorescence analysis showed that SLC6A14 appears in the plasma membrane after 48 h, with the majority of the transporter not leaving ER. This observation was confirmed by biotinylation of surface proteins and analysis of SLC6A14 glycosylation status in order to distinguish core glycosylation (taking place in ER) from full glycosylation (taking place in Golgi apparatus). Trafficking was attenuated after co‑transfection with the dominant negative mutants of Sar1 GTPase – the first protein involved in COPII formation. Further studies demonstrated that out of four SEC24 proteins, exclusively SEC24C co-localized and interacted with SLC6A14, a result confirmed in the proximity ligation assay. CONCLUSIONS: These observations confirm a hypothesis that lysine in position +2 towards an ER export signal is crucial forspecific interaction of SLC6 transporters with SEC24C. FINANCIAL SUPPORT: This work has been supported by the European Union’s Horizon 2020 research and innovation programme under the Marie Sklodowska-Curie grant agreement no 665735 (Bio4Med) and by the funding from Polish Ministry of Science and Higher Education within 2016–2020 funds for the implementation of international projects (agreement no 3548/H2020/COFUND2016/2).

Ograniczanie wyników

2 Acta Neurobiologiae Experimentalis

2 Freissmuth M.

2 Sucic S.

1 Asjad H. M. M.

1 El- Kasaby A.

1 Juraszek B.

1 Jurkiewicz D.

1 Kasture A.

1 Kovalchuk V.

1 Nalecz K. A.

1 Samluk L.

2 2017

Wyniki wyszukiwania

Rescueing sleepless flies – Pharmacochaperoning of folding-deficient dopamine transporters, which give rise to infantile dystonia/Parkinsonism

Trafficking of SLC6A14 – An amino acid transporter B(0,+) to plasma membrane