Creatine deficiency syndrome (CDS) is caused by mutation of the X-linked creatine transporter, SLC6A8. Symptoms include intellectual disability, epilepsy, delayed speech/dysphasia, autistic behavioral disorders, delayed motor skills (walking, sitting), slow growth and individuals tire easily. Less often there are abnormal heart rhythms, microcephaly, midfacial hypoplasia and self-mutilation. The onset of symptoms occurs between 3-36 months of age, indicating a window of opportunity for maximal treatment benefit. CDS is the second most common X-linked intellectual disability after Fragile-X syndrome, estimated at 1% of all intellectual disabilities of unknown etiology, which approximates to at least 3.5 million males globally. As genetic testing improves the number of confirmed cases will likely rise significantly as under diagnosis in males is addressed. Currently, no treatment modality exists as creatine cannot cross the blood brain barrier or enter brain cells without a functioning transporter, making creatine supplementation ineffective. Importantly, it appears that brain cells do not have the ability to synthesis creatine, unlike the liver, pancreas and kidney. Creatine is an essential biochemical for ATP metabolism, acting as a phosphate sink in the form of phosphocreatine in brain tissues with fluctuating and dynamic energy demands. To rapidly test the creatine analogs we have synthesized, to identify candidate targets, there is a need for high throughput screening tools. Testing in whole animals is critical to demonstrate that analogs can pass through the blood brain barrier and enter complex neuronal tissues. Zebrafish are genetically tractable, available in large numbers and develop quickly external to the mother. Importantly, they have phenotypic and genetic similarities to humans, with similar organ systems to mammals and sharing significant genetic sequence identity to humans makes screening in zebrafish attractive. ZIRC has recently started to release the first of four KO zebrafish lines, one with an SLC6A8 null mutation and three KO lines of the paralog (currently designated as si:ch211-117c9.5, or SLC6A8b), which has 69% protein similarity to SLC6A8a (99% similarity of transmembrane domains). The purpose of this proposal is to further develop and validate these KO lines, cross breeding to incorporate all four SLC6A8 mutant alleles, and test the ability of our analogs to enter brain tissue in the double null mutants. To do this we propose the following three aims:
Aim 1 : Validate and further develop SLC6A8 KO lines, Aim 2: Screen for creatine analogs that can passively transport into brain tissue without the need for a functional creatine transporter, Aim 3: Analyze the morphology of GABAergic regions to identify changes in SLC6A8 embryos.
Creatine transporter deficiency syndrome affects at least 3.5 million people, resulting in X-linked intellectual disability and autism spectrum disorders. SLC6A8 codes for the creatine transporter, without which creatine cannot pass the blood-brain barrier, nor enter neuronal cells, thus hindering ATP metabolism in areas of the brain with fluctuating and dynamic energy needs. We will validate several SLC6A8 knockout fish lines, produced by ZIRC, as a tool for high throughput screening of creatine analogs for their ability to passively transport into the brain, to rapidly identify potential therapeutic candidates.
