Changes in GABA Expression in Zebrafish with a Mutation in the slc6a8b Gene

Benjamin O’Connell, Dr. Susan Chapman, Joseph Sheheen

Clemson University

Abstract

The SLC6 transporter family facilitates the movement of small substrates across the blood brain barrier (BBB). Since the developing brain needs a vast range of different molecules in order to mature properly, it is critical for transporters to be fully functional. Thus, disorders involving SLC6 transporters have long been thought to be associated with developmental defects leading to autism spectrum disorders (ASDs). One specific member of the SLC6 family is the creatine transporter (CRT), which is encoded by the gene SLC6A8. Mutations in the SLC6A8 gene result in disruption of creatine transport across the BBB and into brain cells, resulting in intellectual disability and the onset of ASDs. One area of interest is how protein expression changes during embryonic development of patients with mutations to their SLC6A8 gene. In this project, GABA expression was studied using immunostaining techniques on wholemount and sections of slc6a8b knockout larval zebrafish. GABA was chosen due to its role in brain development. Because it starts DNA synthesis in neuronal progenitor cells, GABA is essential for signaling when and how an embryonic brain should grow. Zebrafish embryos act as excellent models of brain development and determining how GABA expression changes in slc6a8b knockout zebrafish will provide valuable insight into how GABA alters brain development in humans with the same mutation.

Introduction

Creatine is most widely known for its role in recycling adenosine diphosphate (ADP) to create adenosine triphosphate (ATP), the body’s standard “energy” molecule. This cycle is essential for maintaining a steady supply of ATP into areas of the body with high energy needs, such as the developing embryonic brain. Without creatine, and therefore without sufficient ATP, the brain cannot develop correctly. This is known as Cerebral Creatine Deficiency Syndrome (CCDS). CCDS typically results in the patient developing an autism spectrum disorder (ASD). One specific type of CCDS is caused by a mutation in the SLC6A8 gene, the gene that codes for the creatine transporter (CRT). Without a functioning transporter, creatine cannot enter the brain. Because this mutation is thought to be linked to the X chromosome in humans, it appears to be more prevalent in males, due to each male only having 1 X chromosome. Females, who have 2 X chromosomes, could still shows symptoms of CCDS. However, it is less common. In zebrafish, two copies of this gene are expressed, slc6a8a and slc6a8b. They are thought to work in tandem.
One of the aims of this project is to determine how CCDS impacts GABA expression and, subsequently, embryonic brain development. It is hypothesized that creatine is binding to the GABA A receptor in an antagonistic role [2]. This aids neuromodulation of GABA signaling. Without creatine to regulate GABA signaling, GABA will not be expressed properly. Because GABA is key in stimulating neuronal progenitor cell differentiation, the irregular GABA expression results in improper brain development.
In this project, slc6a8b mutants are compared to wild type zebrafish. The slc6a8b mutants are the offspring of slc6a8b knockout heterozygotes. While a method to determine the exact genotype of the test subjects has not yet been implemented, differences in GABA expression allow zebrafish that are homozygous or heterozygous for the knockout to be easily distinguished from wild type zebrafish.

Results

Conclusions

Wild type zebrafish and slc6a8b mutants clearly display differences in GABA expression. This is evident when comparing the two cryosections. The wild type zebrafish have much greater GABA expression in the region near ventral thalamus (VT) while the mutant zebrafish express GABA closer to the dorsal thalamus (DT) and posterior tuberculum. Finding these differences in GABA expression are essential in determining how developing embryonic brains are changed by a CCDS. Understanding the impact of the creatine deficiency will be invaluable in one day designing treatment or preventative measures for the condition.

Currently, methods are being discussed to genotype the slc6a8b mutants. This would improve the accuracy of the data as phenotypic results are currently being used to identify the fish. It is also planned to continue research into slc6a8a mutants as well.

References

Dodd, J. R., and Christie, D. L. (2007) Selective amino acid substitutions convert the creatine transporter to a γ-aminobutyric acid transporter. J. Biol. Chem282, 15528–15533.

Mueller, T., Vernier, P., Wullimann, MF. (2006) A phylotypic stage in vertebrate brain development : GABA cell patterns in zebrafish compared with mouse.. Journal of Comparative Neurology, Wiley, 494 (4), pp.620-634.

Materials and Methods

Embryos were stored in methanol at 4°C. To begin the preparation protocol, the embryos were washed 3 separate times for 5 minutes in 1x PBS. The embryos were then fixed in 4% PFA for one hour. Next, the embryos were placed in PBST (0.5% Triton X-100 + 95.5% 1x PBS) for 4, 5-minute rounds. Acetone on ice was then applied for 7 minutes to aid permeabilization. 4 more rounds of PBST were done to aid permeabilization. To help prevent nonspecific binding, the embryos were placed in a blocking solution (10% lamb serum + 90% PBST) for 2, 1-hour rounds. In order to reduce autofluorescence, a hydrogen peroxide solution (0.3% 30%-hydrogen peroxide + 97.7% blocking solution) was applied for 30 minutes. 2 more 5-minute washes in blocking solution were then done to remove the hydrogen peroxide. To apply the primary antibody, the embryos were placed in a solution of 1 μL rabbit anti-GABA antibody + 500 μL blocking solution overnight on a rocker at 4°C. To begin the second day of preparations, 4, 5-minute washes of PBST were applied on a nutator. The secondary antibody (in a solution of 1 μL alexa 488 nm goat anti-rabbit antibody + 200 μL blocking solution) was then applied on a nutator for 2 hours. From this point onward, the embryos were kept in the dark to preserve fluorescence. To rinse the embryos, 3 more, 5-minute washes in PBST were applied. The embryos were then stored in PBS until needed. Some embryos were visualized in wholemount under a confocal microscope.

The cryosectioned embryos were dehydrated in a series of sucrose solutions as follows: 5% sucrose for 30 minutes, 15% sucrose for 30 minutes, and 30% sucrose overnight. They were then placed in an embedding mold and covered in OCT for 30 minutes. The mold, still full of OCT, was then dipped in 2-methylbutane chilled by dry ice.  The molds were left in the 2-methylbutane for approximately 40 seconds until all the OCT was solidly frozen. The embryos were then transversely sectioned by a microtome in a cryostat at approximately -18°C. After the sections were added to glass slides, the OCT was dissolved away by a 15 second wash in PBS. Slowfade was then added before the sections were imaged under a fluorescent microscope.