The Creatine Transporter Unfolded: A Knotty Premise in the Cerebral Creatine Deficiency Syndrome

Abstract

Creatine provides cells with high-energy phosphates for the rapid reconstitution of hydrolyzed adenosine triphosphate. The eponymous creatine transporter (CRT1/SLC6A8) belongs to a family of solute carrier 6 (SLC6) proteins. The key role of CRT1 is to translocate creatine across tissue barriers and into target cells, such as neurons and myocytes. Individuals harboring mutations in the coding sequence of the human CRT1 gene develop creatine transporter deficiency (CTD), one of the pivotal underlying causes of cerebral creatine deficiency syndrome. CTD encompasses an array of clinical manifestations, including severe intellectual disability, epilepsy, autism, development delay, and motor dysfunction. CTD is characterized by the absence of cerebral creatine, which implies an indispensable role for CRT1 in supplying the brain cells with creatine. CTD-associated variants dramatically reduce or abolish creatine transport activity by CRT1. Many of these are point mutations that are known to trigger folding defects, leading to the retention of encoded CRT1 proteins in the endoplasmic reticulum and precluding their delivery to the cell surface. Misfolding of several related SLC6 transporters also gives rise to detrimental pathologic conditions in people; e.g., mutations in the dopamine transporter induce infantile parkinsonism/dystonia, while mutations in the GABA transporter 1 cause treatment-resistant epilepsy. In some cases, folding defects are amenable to rescue by small molecules, known as pharmacological and chemical chaperones, which restore the cell surface expression and transport activity of the previously non-functional proteins. Insights from the recent molecular, animal and human case studies of CTD add toward our understanding of this complex disorder and reveal the wide-ranging effects elicited upon CRT1 dysfunction. This grants novel therapeutic prospects for the treatment of patients afflicted with CTD, e.g., modifying the creatine molecule to facilitate CRT1-independent entry into brain cells, or correcting folding-deficient and loss-of-function CTD variants using pharmacochaperones and/or allosteric modulators. The latter justifies a search for additional compounds with a capacity to correct mutation-specific defects.

Keywords: SLC6A8; creatine; creatine transporter 1; creatine transporter deficiency; intellectual disability; pharmacochaperoning; protein misfolding.

Figure 1

The multi-layered physiological roles of creatine transporter 1 (CRT1). The primary function of creatine transporter 1 (CRT1) is to refill cellular creatine reserves, to compensate for its steady degradation (1.7% daily), in end-consumer cells incapable of self-synthesizing sufficient amounts of creatine (i.e., neurons, myocytes, and cardiomyocytes). CRT1 orchestrates creatine flux throughout the body by tuning the amount of creatine extracted from the blood by individual organs. CRT1 plays a key role in providing the brain with creatine, essential to higher cognitive functions. It shifts creatine across tissue barriers like the kidney tubules, the blood-brain barrier (BBB), and the inner blood-retinal barrier. It thus mediates reabsorption of creatine from the primary urine and facilitates the maintenance of cerebral and retinal tissue energetics, respectively. At the placenta, CRT1 takes up creatine, crucial for embryofetal development. It also supports the functioning and integrity of the intestinal barrier, and is an immunoregulator in leukocytes and stabilizes erythrocyte plasma membranes. Cr, creatine; BBB, blood-brain barrier; BCSFB, blood-cerebrospinal fluid barrier; iBRB, inner blood-retinal barrier; GAA, guanidinoacetate; RBC, red blood cell; WBC, white blood cell.

Figure 2

Creatine transporter deficiency (CTD)-associated variants mapped onto a CRT1 topology. CRT1 adopts the common structural fold of SLC6 proteins with 12 transmembrane domains (TMs) and cytoplasmic N- and C-termini. Most of the reported sequence alterations in CTD cluster in the region encompassing TMDs 7 and 8. Only those variants with the ascertained clinical phenotypes are displayed on the topology. Sixteen mutations are known to be folding-deficient. Individual mutants vary in their response to the chemical chaperone 4-PBA.

Figure 3

A molecular view of CTD and the putative therapeutic approaches. CTD mutations trigger folding deficits or impair CRT1 substrate uptake activity (both resulting in the lack of functional CRT1 proteins at the cell surface; top right). Three approaches can be used to restore creatine supply in the cells: (1) conjugating creatine with salts, taken up by other transporters; and (2) creating lipophilic creatine analogs, to establish transporter-independent delivery of cyclocreatine, creatine benzyl, and fatty acid esters (top left), or (3) pharmacochaperoning to correct folding defects in CRT1 variants (recovering surface expression and creatine uptake activity of CTD mutant protein, top middle) by treatment with small molecules, that act either directly on the CRT1 itself (i.e., transporter ligands) or manipulate the cellular folding machinery (e.g., heat shock protein inhibitors and chemical chaperones).