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    • Apart from a few mutations which

    2021-11-18

    Apart from a few mutations, which affect intracellular and extracellular loops of hCRT-1, the mutations are primarily found in the hydrophobic core of the transporter; in fact, TMDs 7 and 8 appear to be a hot spot for mutations (Freissmuth et al., 2018). Several mutations are found at positions, which are conserved in SLC6 transporters, e.g. a single point mutation in TM3, which induces a sleepless phenotype in the Drosophila dopamine (dDAT-G108Q) (Kasture et al., 2016), leads to severe mental retardation in children when mutated at the equivalent glycine residue in hCRT-1 (hCRT-1-G132V) (Lion-François et al., 2006). Similarly, the P554L mutation causes Parkinsonism/dystonia in hDAT and triggers drug-resistant epilepsy in children harbouring the same mutation in CRT-1. Currently, there is no effective treatment for patients suffering from creatine transporter deficiency: the current standard therapy relies on supplementation of creatine monohydrate, L-arginine and glycine, but a long-term study of children only showed that short term benefits did not translate into a sustained improvement (van de Kamp et al., 2012); the symptoms substantially deteriorated in those patients who survived into adulthood. This unmet medical need justifies the search for effective therapeutic strategies in the treatment of creatine transporter deficiency. In the present study, we explored the hypothesis that many of the mutations, which trigger creatine transporter deficiency, result in misfolding of hCRT-1 and that this folding deficiency can be corrected by pharmacological means.
    Material and methods
    Results
    Discussion To the best of our knowledge we are the first to report that 4-PBA can effectively rescue the activity of several pathological hCRT-1 variants linked to CTD. 4-PBA is an approved drug, which is clinically used in the chronic management of urea Dexlansoprazole disorders in children, i.e. diseases caused by genetic defects in several enzymes involved in the urea cycle. Deficiency of these enzymes (i.e. carbamoyl phosphate synthetase I, ornithine transcarbamylase, argininosuccinate synthetase, argininosuccinate lyase, arginase I, citrin and ornithine translocase) leads to nitrogen waste build up in the blood plasma, forming ammonia glutamine. 4-PBA acts as a nitrogen-scavenger and assists kidneys in excreting excess ammonia from the body. Besides, 4-PBA is used for sickle cell disease due to its capacity to activate β-globin transcription (Dover et al., 1994; Qi et al., 2004) and is also under clinical investigation for use in cancer and Huntington's disease (Kolb et al., 2015). Moreover, the merits of 4-PBA in the therapy of cystic fibrosis are well renowned. Treatment with 4-PBA induces CFTR channel function in ΔF508-CFTR-expressing cystic fibrosis airway epithelial cells (Rubenstein and Zeitlin, 2000). This is achieved by 4-PBA stabilising the misfolded ΔF508-CFTR variant in the ER and allowing it to traffic to the cell membrane, though the mechanistic details underlying this effect happen to be more complex than initially presumed. We also observed that 4-PBA increased both, the ER-resident core glycosylated and the mature glycosylated species of wild type hCRT1 and of the responsive CTD mutants. This increase in total transporter levels does not per se argue against a chaperoning action of 4-PBA: the transporter must be first stabilised in the ER and subsequently exported. This is actually also seen with receptor ligands, which act as pharmacochaperone: in fact, in response to antagonists, a folding-deficient mutant of the A1-adenosine receptors is stabilised and first accumulates in the ER, because it escapes ER-associated degradation (Kusek et al., 2015). Regrettably none of the misfolded DAT variants linked to infantile/juvenile parkinsonism and dystonia (Kurian et al., 2009; Ng et al., 2014) responded to 4-PBA treatment (Asjad et al., 2017). Surprisingly, the closely related serotonin transporter (SERT), showed marked increases in surface expression upon exposure to low mM concentrations of 4-PBA (Fujiwara et al., 2013). We further substantiated this finding for folding-deficient versions of SERT (El-Kasaby et al., 2014; Koban et al., 2015), which responded to 4-PBA treatment (El-Kasaby, Sucic and Freissmuth, unpublished data).