Date of Award
Bachelor of Science
gene, protein, mutant, phenotype, antibody
Infantile Neuronal Ceroid Lipofuscinosis (INCL) is caused by a mutation of the gene CLN1, which encodes palmitoyl protein thioesterase 1 (PPT1). PPT1 is a lysosomal enzyme that cleaves the thioester bond connecting long chain fatty acids of palmitoylated proteins. Without PPT1, palmitoylated proteins are not degraded properly and this causes unwanted build up in neurons leading to fatality. Recently, it has been thought that a mutation in a similar enzyme, palmitoyl-protein thioesterase 2 (PPT2), another lysosomal hydrolase, may also be responsible for INCL by causing a similar effect of Ppt1 deficiency. A bioinformatics analysis in this study showed similarities between human PPT1/PPT2 and Drosophila Ppt1/Ppt2. Therefore, using Drosophila melanogaster as a model, the role of Ppt1 and Ppt2 can be elucidated using the molecular genetic technique RNA interference (RNAi). RNAi targets the mRNA for destruction thus creating a mutant phenotype. In this study, knock-down of Ppt1 and Ppt2 mRNA expression was achieved using the Gal4-AUS system. Transgenic Ppt1-UAS and Ppt2-UAS fly lines were crossed into various Gal4 driver lines specific for different subsets of embryonic CNS neurons. Embryos from these crosses were immunohistochemically stained with FAS II antibody and stage 16 embryos were examined for neural defects. Preliminary results indicate that Ppt1 and Ppt2 RNAi knock-down disrupted nervous system development in Drosophila. Furthermore, mutant embryos were allowed to progress to a larval state where motor studies showed differences in locomotor ability. These results provide insights into the mechanism of Ppt1 and Ppt2 in causing INCL pathology.
O'Hern, Patrick J., "From Gene to Behavior: Investigation of Ppt1 and Ppt2 RNAi induced knock-down during Drosophila neurogenesis" (2011). Honors Theses. 1039.