PHARMACOLOGICAL RESCUE OF CREATINE TRANSPORTER-1 VARIANTS

The human creatine transporter 1 (hCRT-1) is a member of the solute carrier 6 (SLC6) protein family. Mutations in the coding sequence of hCRT-1 gene have been associated with the creatine transporter deficiency (CTD) syndrome. CTD encompasses a range of moderate to severe disorders, from epilepsy, mental retardation, autism,development delay (psychomotor retardation with independent walking and development delay), behavioural problems (hyperactivity, shyness, stereotypic-, aggressive- and self-injurious behaviour), motor dysfunction (stiff gait, mildly increased reflexes, coordination dysfunction and dystonia), to gastrointestinal symptoms (neonatal feeding difficulties, failure to thrive, vomiting, constipation, and gastric and duodenal ulcers). We propose that point mutations in hCRT-1 impair protein folding and consequently cause CTD. Many disorders arising from misfolding of other SLC6 transporters have been reported; e.g. mutations in the human dopamine transporter (DAT) cause infantile/juvenile Parkinsonism/dystonia. In hCRT-1 itself, dozens of naturally occurring variants identified to date are associated with CTD. Strikingly, there is a number of cases where mutations at equivalent and usually highly conserved residues in different SLC6 proteins lead to protein misfolding; e.g. a single point mutation causing a sleepless phenotype in Drosophila melanogaster DAT (dDAT-G108Q), exists at the equivalent position in hCRT-1 (hCRT-1-G132V) and leads to severe mental retardation in boys. Similar is the case of the P554L mutation: in hDAT, P554L leads to Parkinsonism, whereas in hCRT-1, it causes severe mental retardation and drug-resistant epilepsy. Our goal is to study the known clinically-relevant hCRT-1 variants and assess whether the phenotypes arise from defective folding of the hCRT-1 protein. Pharmacochaperoning was used to functionally rescue folding-deficient versions of DAT and the serotonin transporter (SERT). In fact, dDAT-G108Q was rescued both in vitro in HEK293 cells, and in vivo, in flies. Our preliminary data indicate that hCRT-1-G132V (mutation of glycine equivalent to G108 in dDAT) is fully retained in the endoplasmic reticulum (ER), in contrast to the wild type hCRT-1 which reaches the plasma membrane of HEK293 cells. We hence plan to elucidate the mechanistic details in the folding trajectory of wild type hCRT-1 to understand how misfolding occurs - and eventually whether and how folding (and transporter function) can be restored by pharmacological and/or chemical chaperones. These experiments may lead to the development of innovative therapeutic options for patients suffering from CTD.

Genetic mutations in the coding sequence of the human creatine transporter 1 (hCRT-1) have been directly linked to the syndrome of creatine transporter deficiency (CTD). CTD encompasses a range of moderate to severe disorders, including epilepsy, intellectual disability, autism, development delay (psychomotor retardation with independent walking and development delay), behavioural problems (hyperactivity, shyness, stereotypic-, aggressive- and self-injurious behaviour), motor dysfunction (stiff gait, mildly increased reflexes, coordination dysfunction and dystonia), and gastrointestinal symptoms (neonatal feeding difficulties, failure to thrive, vomiting, constipation, and gastric and duodenal ulcers). We discovered that the majority of CTD-associated mutations impaired folding of the hCRT-1 protein. We employed pharmacochaperoning to functionally rescue folding-deficient versions of hCRT-1, which were retained in the endoplasmic reticulum (ER) compartment, in contrast to the wild type transporter, which was correctly targeted to the plasma membranes of cells. Treatment with a renowned chemical chaperone 4-phenylbutyrate (4-PBA) efficiently restored the ER export, cell surface expression, as well as creatine uptake by many of the misfolded disease variants of hCRT-1. Our novel data provide a proof-of-principle that CTD mutants are amenable to rescue, and justify the search for additional small molecules with therapeutic potential in the treatment of many children afflicted with CTD.