4‐Phenylbutyrate rescues folding‐deficient creatine transporter‐1 variants linked to the creatine transporter deficiency syndrome

Abstract

Mutations in the coding sequence of the hCRT‐1 gene (SLC6A8) have been associated with the creatine transporter deficiency (CTD) syndrome. CTD encompasses a range of moderate to severe conditions, from epilepsy, mental retardation, autism, development delay, behavioural problems and motor dysfunction to gastrointestinal symptoms. Diseases arising from misfolding of other SLC6 transporters have been reported over recent years; e.g. folding‐deficient mutants of the dopamine transporter (hDAT) cause infantile/juvenile Parkinsonism dystonia in children, while orthostatic intolerance arises from misfolding of the human noradrenaline transporter (hNET). In hCRT‐1, dozens of naturally occurring variants have been linked to CTD. Remarkably, some of these mutations occur at highly conserved residues across the SLC6 family; e.g. a single point mutation inducing a sleepless phenotype in Drosophila DAT (dDAT‐G108Q), leads to severe mental retardation in children when mutated at the equivalent glycine residue in hCRT‐1 (hCRT‐1‐G132V). Similarly, the P554L mutation causes Parkinsonism/dystonia in hDAT, but triggers drug‐resistant epilepsy in children harbouring the same mutation in hCRT‐1. Here, we examined 16 clinically relevant hCRT‐1 variants to assess the molecular basis of CTD. Confocal microscopy imaging revealed that the YFP‐tagged mutant CRTs are trapped in the endoplasmic reticulum (ER), co‐localised with an ER‐resident chaperone calnexin, whereas the wild type hCRT‐1 reached the plasma membrane. The chemical chaperone 4‐phenylbutyrate (4‐PBA) proved to be effective in functionally rescuing several of the folding‐deficient CRT‐1 mutants. Radiolabeled creatine uptake assays were also verified by assessing the surface expression of the variant transporters by cell surface biotinylation and immunoblotting of detergent lysates (i.e. the mature fully‐glycosylated band appeared in response to positive chaperone action). In addition, the rescue action of 4‐PBA was also recapped in primary hippocampal neurons expressing the wild type transporter and CTD‐triggering mutations. Overall, this work is clinically relevant and ought to confer pristine therapeutic prospects awaited for by many CTD patients.Support or Funding InformationThis work was supported by the Austrian Science Fund (FWF) project no P31255‐B27 to Sonja Sucic.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.