74 Study of the Mechanism of Action of Chloroquine And Neuroinflammation in a Zebrafish Model of X-Linked Adrenoleukodystrophy

Faculty Mentor: Joshua Bonkowsky (Pediatrics, University of Utah)

X-linked Adrenoleukodystrophy (X-ALD) is neurodegenerative disease caused by mutations in the ABCD1 gene, which is believed to affect the myelin in the central nervous system. X-ALD is one of the most common leukodystrophies, however, there are no good experimental mice models for the disease. The Bonkowsky lab developed a zebrafish model for X-ALD, as zebrafish are an ideal model due to being relatively inexpensive, quick to mature, and good for high-throughput screening. Using this model chloroquine was identified as a promising drug treatment for X-ALD. However, the zebrafish model the Bonkowsky lab was using lost its decreased motor behavior phenotype in the abcd1 mutant fish likely due to SNPs, which also indicated that the Abcd1 gene began to be expressed in the mutant fish, which was not suitable for the mutant to be a proper model. Therefore, the first aim of this project was to evaluate and characterize several new ABCD1 zebrafish lines. The second aim of this project was to use one or more of these ABCD1 zebrafish lines to characterize the mechanism of action of chloroquine and to characterize its potential to control the inflammatory response. We evaluated three different fish lines (sa21198, sa34298, and a CRISPR knockout line) for a motor behavior phenotype at 5-, 6-, and 7-days post-fertilization as a preliminary way to select a good model. We did not observe a decreased motor behavior phenotype in any of the abcd1 mutant fish compared to wild-type fish for any of the three lines. We then evaluated the RNA expression levels of Abcd1 for wildtype and mutant fish of each line to ensure that even without a motor phenotype, the fish were still viable as a model. At this time work has not been done yet on characterizing the mechanism of action of chloroquine due to the time-consuming characterizing and figuring out how to genotype these three fish lines. However, going forward the sa21198 and the CRISPR KO line has been selected to work with due to ease of genotyping. The CRISPR KO line is also going to be used in the future for several different experiments.