Potential treatment for OCD by exploiting the alternative splicing of a glutamate transporter




Saathvika Rajamani

Introduction

Obsessive Compulsive Disorder (OCD) is a chronic anxiety disorder. The number of affected people outranks those affected by other mental illnesses such as Schizophrenia and Autism. Twin studies and family based studies of OCD affected individuals identified polymorphisms in a particular region in chromosome 9, which was further narrowed down to the region that coded for SLC1A1 gene.
This gene, Solute Carrier 1 Family 1, encodes a protein called Excitatory Amino Acid Carrier 1 (EAAC1) which is a glutamate transporter. This glutamate transporter carries glutamate across the plasma membrane of the cell. It is an antiporter that imports one molecule of glutamate, a hydrogen ion and three sodium ions and exports one potassium ion.

SLC1A1 gene is expressed predominantly in the brain and the kidney. Disorders that arise due to the mutations in this gene also include Schizophrenia and Autism. Three alternative transcripts have been identified for this gene using bioinformatics tools. They have two promoters, one and two, out of which the latter is an internal promoter. The splice variants under the control of promoter one have either exon 2 or 11 missing.

Glutamate is a primary neurotransmitter. High levels of glutamate can induce neurotoxicity and has been linked to various neurodegenerative disorders. The primary transcript encodes for EAAC1 which mediates efficient glutamate transport. The splice variants II and III encode for a protein that interferes with the formation of the functional EAAC transporter. This leads to a decrease in glutamate transport and an overall increase of glutamate, which further leads to neurotoxicity.
Glutamate levels were found to be comparatively higher in patients suffering from OCD when compared to unaffected individuals upon analyzing the cerebrospinal fluids of both.
The purpose of this experiment was to identify compounds that would alter the splicing of SLC1A1 transcripts.


Materials and Methods

• Human Embryonic Kidney 293 cells or HEK-293 cells were treated with compounds and the RNA was extracted.
• Primer pairs were designed with primers in exon 1 and 8.
• Reverse Transcriptase PCR was done with these primers on RNA isolated from both untreated and treated samples.
• RT-PCR products were visualized using gel electrophoresis with 1% agarose gel.
• The amplified products were purified and sequenced.
• The sequences were analyzed using NCBI-BLAST for confirmation.


Results

RT-PCR was performed on the extracted RNA samples with primers in exons 1 and 8. The bands of sizes 846 bp and 777 bp were observed.

The RNA sample treated with compound 135 showed two distinct RT-PCR products upon visualizing in the gel after electrophoresis.

The ratio of the two bands was distinctly different, with the primary transcript expressed less and the splice variant expressed more when compared with the other samples.

Upon gel purification and sequencing, the 846bp transcript was identified as the primary transcript that did not miss an exon while the 777bp was identified as the splice variant II which was missing exon 2.


Discussion

In this project, the treatment of the HEK 293 cells with certain compounds induced alternative splicing of the transcript. When the RNA from the cells treated with compound 135 were extracted and amplified, two distinct RT-PCR products were observed. Upon sequencing the products were identified as the primary transcript that did not lack an exon, and the splice variant which lacked exon 2. This splice variant codes for a protein that blocks the formation of the functional glutamate transporter, a trimer, whereas the primary transcript codes for the functional glutamate transporter itself.

Compound 135 induced higher expression of the splice variant. This would lead to lower formation of functional glutamate transporters and result in less efficient glutamate transport into the cells. This compound should thus be avoided in the diets of the affected individuals.

Overall, this project has shown that compounds have the ability to alternatively splice SLC1A1 transcript. More compounds would be used in the treatments and protein based assays would also be carried out in the future for further analysis.


References

1. Arnold, Paul Daniel, et al. "Glutamate transporter gene SLC1A1 associated with obsessive-compulsive disorder." Archives of general psychiatry 63.7 (2006): 769-776.
2. Dickel, Diane E., et al. "Association testing of the positional and functional candidate gene SLC1A1/EAAC1 in early-onset obsessive-compulsive disorder." Archives of general psychiatry 63.7 (2006): 778-785.
3. Shugart, Y. Y., et al. "A family‐based association study of the glutamate transporter gene SLC1A1 in obsessive–compulsive disorder in 378 families." American Journal of Medical Genetics Part B: Neuropsychiatric Genetics 150.6 (2009): 886-892.
4. Grewer, C., Gameiro, A., & Rauen, T. (2014). SLC1 Glutamate Transporters. Pflugers Archiv : European Journal of Physiology, 466(1), 3–24. http://doi.org/10.1007/s00424-013-1397-7

Figures


Figure 1- Excitatory Amino Acid Carrier 1


Figure 2-The splice variants of SLC1A1 gene.


Figure 3-Agarose gel electrophoresis image of RT-PCR products of treated and untreated RNA samples with SLC1A1 primer pairs for exons 1-8


Figure 4-Diagrammatic representation of the full-length transcript and the splice variant that is missing exon 2. It also shows the locations of the primers.


Obsessive Compulsive Disorder (OCD) is a chronic anxiety disorder that causes the affected individuals to have recurring thoughts and subsequent repetitive behavior. Obsessions could lead to new compulsions and vice versa. OCD affected individuals have high levels of glutamate neurotransmitter in their cerebrospinal fluids. Genome scan on diseased individuals led to the identification of several polymorphisms in a region in Chromosome 9. The region was later found to correspond to that of gene SLC1A1. This gene codes for a glutamate transporter that transports glutamate into the cells, primarily the neurons. Alternative splicing of this gene having 12 exons results in the production of two unique splice variants that missed either exon 2 or exon 11. These alternative transcripts negatively regulate the primary transcript regulated glutamate transport. Upon treatment with several compounds, the splicing was shown to vary, proving that splicing can be modified based on treatments. Treatment with compound 135 especially showed an increase in splice variant production that missed exon 2. This compound might not be advisable for OCD-affected individuals.

Full Paper

Acknowledgments

I would like to thank Dr. Berish Rubin for guidance and providing me with the opportunity to work on this project. I would like to thank Catharina Grubaugh and Anthony Evans for their help and patience throughout this project.


This document was last modified 05/17/2017.
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