"Water, water. Everywhere. Nor any drop to drink.” These lines, from Samuel Taylor Coleridge’s “Rime of the Ancient Mariner,” refer to a group of stranded and thirsty mariners surrounded by salt water, none of which is drinkable. This situation is analogous to the inability of people with X-linked creatine deficiency to shuttle creatine into the brain’s cells, where it plays a critical role in the brain’s energy needs. No matter how much creatine you give to people with this disorder, they can’t use it because their creatine transporters (CRTs) are defective. A treatment may be in sight. A research team led by Joseph Clark, Ph.D., professor at the University of Cincinnati and the Cincinnati Children’s Hospital Medical Center, Cincinnati, Ohio, recently constructed a mouse model of X-linked creatine deficiency and demonstrated that cyclocreatine, a creatine analog, enters this mouse’s brain cells and corrects the cognitive deficits characteristic of the human disease (Kurosawa et al. 2012). Clark’s findings are an important breakthrough in developing a therapy for human X-linked creatine deficiency.
The creatine/phosphocreatine/creatine kinase system
Creatine is a naturally occurring nitrogenous organic acid in vertebrates. It is actively transported into brain cells by the CRT, which is encoded by the sodium- and chloride-dependent creatine transporter 1 (SLC6A8) gene. Once in the brain cells, creatine is phosphorylated to phosphocreatine by creatine kinase (CK). This creatine/phosphocreatine/CK system maintains the brain’s ATP homeostasis, which affects neurotransmitter release, membrane potential, Ca2+ homeostasis and/or ion gradients.
There are three creatine deficiency syndromes. Two are very rare, and only a few hundred people have been diagnosed worldwide. They are caused by defects in the genes that regulate creatine synthesis and are treatable with creatine supplementation. The third syndrome, X-linked creatine deficiency (or creatine deficiency syndrome, X-Linked, OMIM ID: 300352), is more common. In fact, it is the second-most common cause of X-linked mental retardation and affects approximately 42,000 Americans and 1 million people worldwide. However, it is uncommon enough to be classified as a rare disease by NIH’s Office of Rare Diseases Research. Unfortunately, it is not treatable with creatine supplementation because the CRT is defective.
The symptoms of CRT deficiency include cognitive dysfunction, poor language skills, and autism spectrum disorders. Because the SLC6A8 gene is on the X chromosome, the effects of CRT deficiency are more severe in males.
Constructing the Slc6a8–/y mouse
Clark and his colleagues wanted to design a treatment to correct the cognitive deficits of CRT deficiency. No suitable animal model was available. Although a whole body Slc6a8 knockout mouse had been constructed (Skelton et al. 2011), its severe phenotype does not model the human condition. What was needed was a mouse in which the Slc6a8 gene is knocked out only in the cortex and hippocampus – the brain regions mostly responsible for cognitive and memory functions.
Clark and his colleagues constructed the brain-specific Slc6a8–/y mouse by innovatively using some of our Cre-lox and FLP-FRT mice. To develop a brain-specific Slc6a8–/y knockout mouse, Clark and his team first produced cre-conditional Slc6a8fl/fl mice. These mice harbor loxP sites that flank exons 2 through 4 of the Slc6a8 gene and a positive selection FRT-flanked neomycin phosphotransferase gene (neo) cassette. They subsequently removed the cassette by breeding Slc6a8fl/fl mice to the germ-line Flp deleter strain B6.129S4-Gt(ROSA)26Sortm1(FLP1)Dym/RainJ (009086). They then generated brain-specific Slc6a8–/y mice and Slc6a8fl/y littermate controls by mating Slc6a8fl/fl females to male B6.Cg-Tg(Camk2a-cre)T29-1Stl/J (005359) mice. These mice express Cre recombinase driven by the mouse calcium/calmodulin-dependent protein kinase II alpha (CamkIIalpha) promoter in the brain, particularly in the hippocampus and cortex, both of which control the cognitive functions affected by CRT deficiency.
Clark and his colleagues demonstrated that, like humans with X-linked creatine deficiency, Slc6a8–/y mice have very low creatine levels in the brain but normal creatine levels in the heart, skeletal muscle, and serum. Also, like humans with the disorder, they have cognitive deficits and normal musculoskeletal control.
Cyclocreatine normalizes cognitive functions in Slc6a8–/y mice
Clark and his team treated adult Slc6a8–/y mice with the creatine analog cyclocreatine (1-carboxymethyl-2-iminoimidazolidine). They chose this compound for several reasons: its kinetic properties are similar to creatine’s; like creatine, it is phosphorylated and dephosphorylated by mitochondrial and cytosolic CK; it passively diffuses across the blood-brain barrier and into brain cells without needing the CRT; and, having been investigated as a possible chemotherapeutic, its toxicology is known. Clark and his team hypothesized that cyclocreatine would enter the brain cells of Slc6a8–/y mice and improve their cognitive functions. Their findings are summarized below:
- The body morphometry and overall health of Slc6a8–/y mice, either before or after cyclocreatine, creatine or placebo treatments, are normal or near normal.
- After cyclocreatine treatment, the levels of cyclocreatine and cyclocreatine phosphate in Slc6a8–/y mouse brains increase substantially, suggesting that, once in the brain cells, cyclocreatine is phosphorylated and substitutes for creatine in the creatine/phosphocreatine/CK system.
- Cyclocreatine normalizes spatial learning and memory, novel object recognition, and the discriminating abilities of Slc6a8–/y mice.
In summary, Clark and his colleagues constructed a novel brain-specific Slc6a8–/y mouse model that mimics human X-linked creatine deficiency. They demonstrated that cyclocreatine, a creatine analog, does not need the CRT to enter the brain cells of this mouse and that it substitutes for creatine in the creatine/phosphocreatine/CK system. Importantly, cyclocreatine normalizes the cognitive deficits in adults of this mouse without affecting overall health and muscle function. Clark and his colleagues suggest that cyclocreatine is the ideal drug for treating human X-linked creatine deficiency because 1) its toxicology is known; 2) it is the most kinetically similar creatine analog known; and 3) its phosphorylation by mitochondrial creatine kinase mitigates creatine kinase crystallization and concomitant pathologies. Although further studies must be performed before cyclocreatine is approved for human use, the findings by Clark and his colleagues are an important step in developing a treatment for human X-linked creatine deficiency.