Investigating cellular dysfunction related to lysosomal impairment in CLN2 disease

点击翻译按钮获取中文摘要

The lysosome is the centre of cellular waste degradation and recycling, as well as an importantmetabolic signaling hub. Both functions have a major influence over cellular homeostasis.Studying lysosomal storage disorders (LSDs) provides valuable insights into functions of thelysosome and the widespread cellular impact during lysosomal impairment. This study focuseson lysosomal impairment in CLN2 disease, an LSD caused by recessively inheriteddysfunction of lysosomal serine protease Tripeptidyl Peptidase 1 (TPP1). Predominantly usinga zebrafish model of CLN2 disease (tpp1-/-), the aim of this project was to investigate thecellular dysfunction related to lysosomal impairment as a consequence of Tpp1 deficiency.I present novel transgenic zebrafish lines with autophagosome and lysosome markers, LC3 andLamp1 respectively, tagged by ZsGreen fluorescent protein. Using these lines, I was able toreliably quantify lysosomal number, size and morphology, as well as autophagic flux in vivo.This dataset highlights severe lysosomal abnormality in tpp1-/- mutant zebrafish, alongsideblocked autophagic flux and alterations in autophagosome position. The impact of lysosomalimpairment on cellular homeostasis in tpp1-/- mutant zebrafish is evidenced by hyperactivity ofa key metabolic regulator, mammalian target of rapamycin complex 1 (mTORC1) and anumber of changes in gene expression detected by whole genome RNA Sequencing of tpp1-/-mutant zebrafish, alongside healthy siblings.Furthermore, I observed improvement in the lysosomal phenotype with treatment ofpregnenolone, a compound previously identified to reduce seizures in the tpp1-/- mutantzebrafish. I went on to explore the potential mechanisms of pregnenolone and the insights itprovides into disease pathogenesis, with a particular focus on lysosomal biology, autophagyand cholesterol metabolism.