Pharmacochaperoning of the dopamine transporter

The dopamine transporter (DAT) is a neurotransmitter transporter (NTT) belonging to the solute carrier 6 (SLC6) gene family. Its main physiological role is to retrieve the previously released dopamine from the synaptic cleft, where it operates in a relay with the vesicular monoamine transporter (vMAT) to refill synaptic vesicles. It is known that point mutations in DAT (slc6A3 gene) occur in infantile dystonia/parkinsonism. The mutations are thought to impair DAT folding and thus cause retention of misfolded DATs in the endoplasmic reticulum (ER). The main aims of this project are: (1) to examine whether some of these naturally occurring DAT variants can be functionally rescued, by employing different compounds or ligands (chemical- and pharmacochaperones) that interact with the transporter and boost its delivery to its site of action at the cell surface, and (2) to extend the studies to DATs close relative, the noradrenaline transporter (NET), a variant of which causes orthostatic intolerance due to intracellular retention. My research plan is contingent on a model where SCL6 transporters first associate with luminal proteinaceous chaperones (e.g. calnexin) and cytosolic heat shock proteins (HSPs); upon release of luminal chaperones, they form oligomers, adopt their final conformation and release HSPs from their C-termini. This in turn renders the ER export motif located on transporter C-terminal domains accessible to coatomer-II/COPII machinery, such that ER export can occur. The following evidence supports this model: (i) SLC6 transporters must oligomerise to exit the ER, (ii) the ER export motif (the binding site for SEC24, i.e. the cargo binding component of the COPII complex) resides in the C-terminus of NTTs (e.g. GAT-1/SERT/NET/DAT), (iii) many SERT C-terminal mutations, the SEC24 interaction site included, give rise to protein misfolding. Transporters are synthesised in the ER, but prior to reaching their target membrane, they must pass stringent ER quality control mechanisms to prevent misfolded proteins from reaching the cell surface. The notion that the C-terminus is initially occupied by a HSP relay conforms well to the finding that 4-phenylbuytrate (4-PBA), which affects HSP expression, markedly enhances SERT surface expression. Besides, non-specific chemical chaperones (e.g. 4-PBA and dimethylsulfoxide) couldsuccessfully rescueER-trappedSERT mutants. Pharmacochaperoning, however, shows impeccable specificity (e.g. only ibogaine could rescue several misfolded SERTs). Inhibiting HSPs and/or applying pharmacological and chemical chaperones to ER-retained DAT and NET variants may salvage their expression and activity at the cell surface. Evidently, this research is therapeutically relevant because it poses novel potential in the treatment of neuropsychiatric disorders resulting from defective folding of monoamine transporter proteins.

The most significant outcome of this project has been unravelling the molecular basis of a clinical syndrome known as infantile/juvenile dystonia and parkinsonism. Single point mutations in the gene encoding the human dopamine transporter (hDAT, SLC6A3) are known to be linked to this disease. The principal physiological role of hDAT is to mediate the reuptake of the neurotransmitter dopamine from the synaptic cleft, thus achieving rapid termination of neurotransmission. Dysfunction of the transporter hence leads to a number of dopamine-related disorders, ranging from the attention deficit hyperactivity disorder and depression to movement disorders and/or parkinsonism. To unravel the mechanism underlying the latter condition, and to explore the potential pharmacological therapies, we investigated thirteen disease-associated hDAT mutants that occur in children afflicted with this severe disorder. When expressed heterologously in HEK293 cells, all of the mutants displayed impaired cellular localisation. They were all retained in the endoplasmic reticulum compartment of the cells, rather than at the plasma membrane, as is the case with the wild type hDAT protein. We found that all mutants were trapped inside the cells, due to protein folding defects. In three of the mutants, i.e. hDAT- V158F, hDAT-G327R, and hDAT-L368Q, the folding deficit could be remedied by treatment with the pharmacochaperone noribogaine or the heat shock protein 70 (HSP70) inhibitor pifithrin-, such that endoplasmic reticulum export of and radioligand binding and substrate uptake by these DAT mutants were all restored. In the fruit fly Drosophila melanogaster, DAT function deficiency results in reduced sleep duration. Hence, we utilised the power of targeted transgene expression of mutant hDATs in the fly system, to study whether these disease-linked hDAT variants could also be pharmacologically rescued in an intact living organism. Noribogaine or pifithrin- treatment instigated the trafficking and delivery of hDAT to the presynaptic terminals of dopaminergic neurons and restored sleep in DAT-deficient (fumin) Drosophila lines expressing hDAT- V158F or hDAT-G327R to normal length. On the other hand, expression of hDAT-L368Q in the Drosophila DAT mutant background caused developmental lethality, likely due to a toxic effect that could not be remedied by pharmacochaperoning. In summary, our observations identified mutations amenable to pharmacological rescue in the affected children. In addition, our findings highlight the challenges of translating insights from pharmacochaperoning in the cell culture systems to the ultimate clinical setting. Because of the evolutionary conservation in dopaminergic neurotransmission between flies and people, studies like this one may allow us to bridge that gap.