Investigating cellular dysfunction related to lysosomal impairment in CLN2 disease

The lysosome is the centre of cellular waste degradation and recycling, as well as an important
metabolic signaling hub. Both functions have a major influence over cellular homeostasis.
Studying lysosomal storage disorders (LSDs) provides valuable insights into functions of the
lysosome and the widespread cellular impact during lysosomal impairment. This study focuses
on lysosomal impairment in CLN2 disease, an LSD caused by recessively inherited
dysfunction of lysosomal serine protease Tripeptidyl Peptidase 1 (TPP1). Predominantly using
a zebrafish model of CLN2 disease (tpp1-/-), the aim of this project was to investigate the
cellular dysfunction related to lysosomal impairment as a consequence of Tpp1 deficiency.
I present novel transgenic zebrafish lines with autophagosome and lysosome markers, LC3 and
Lamp1 respectively, tagged by ZsGreen fluorescent protein. Using these lines, I was able to
reliably quantify lysosomal number, size and morphology, as well as autophagic flux in vivo.
This dataset highlights severe lysosomal abnormality in tpp1-/- mutant zebrafish, alongside
blocked autophagic flux and alterations in autophagosome position. The impact of lysosomal
impairment on cellular homeostasis in tpp1-/- mutant zebrafish is evidenced by hyperactivity of
a key metabolic regulator, mammalian target of rapamycin complex 1 (mTORC1) and a
number 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 of
pregnenolone, a compound previously identified to reduce seizures in the tpp1-/- mutant
zebrafish. I went on to explore the potential mechanisms of pregnenolone and the insights it
provides into disease pathogenesis, with a particular focus on lysosomal biology, autophagy
and cholesterol metabolism.