Investigating cellular dysfunction related to lysosomal impairment in CLN2 disease
Lead Research Organisation:
Royal Veterinary College
Department Name: Comparative Biomedical Sciences CBS
Abstract
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.
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.
People |
ORCID iD |
Claire Russell (Primary Supervisor) | |
Lisa Kiani (Student) |
Studentship Projects
Project Reference | Relationship | Related To | Start | End | Student Name |
---|---|---|---|---|---|
BB/M009513/1 | 01/10/2015 | 31/03/2024 | |||
1964932 | Studentship | BB/M009513/1 | 06/11/2017 | 29/01/2022 | Lisa Kiani |
Description | Characterisation of cellular phenotypes in a zebrafish model of lysosomal dysfunction, allowing investigation of the underlying mechanisms. Development of imaging-based high-throughput screening using transgenic zebrafish. |
Exploitation Route | Publication of our findings may provide more knowledge of the mechanisms involved in lysosome dysfunction and related diseases. Our high-throughput imaging assays provide the opportunity to screen for therapeutics in an animal model of CLN2 disease. |
Sectors | Healthcare,Pharmaceuticals and Medical Biotechnology,Other |
Description | CoB Practical Course Grant |
Amount | £1,000 (GBP) |
Organisation | Company of Biologists |
Sector | Charity/Non Profit |
Country | United Kingdom |
Start | 06/2018 |
End | 06/2018 |
Description | EuFishBioMed Short term fellowship / travel grant |
Amount | € 375 (EUR) |
Organisation | EuFishBioMed |
Sector | Private |
Country | Germany |
Start | 09/2019 |
End | 09/2019 |
Description | LIDo DCDF fellowship |
Amount | £24,192 (GBP) |
Organisation | Biotechnology and Biological Sciences Research Council (BBSRC) |
Sector | Public |
Country | United Kingdom |
Start | 05/2022 |
End | 11/2022 |
Description | Marine Biological Laboratory Scholarship |
Amount | $3,090 (USD) |
Organisation | Marine Biological Laboratory |
Sector | Academic/University |
Country | United States |
Start | 08/2018 |
End | 08/2018 |
Title | Transgenic zebrafish |
Description | Several transgenic zebrafish lines created to measure lysosome biology and autophagy in live zebrafish larvae. |
Type Of Material | Model of mechanisms or symptoms - non-mammalian in vivo |
Year Produced | 2018 |
Provided To Others? | No |
Impact | Allowed us to characterise disease phenotypes in our model. |
Description | Dr Philipp Gut, Nestle Research Centre |
Organisation | Nestlé (Global) |
Department | Nestlé Institute of Health Sciences |
Country | Switzerland |
Sector | Private |
PI Contribution | My PhD is co-supervised by Dr Gut as part of the LIDo iCase scheme, including a mandatory placement which I completed in 2019. |
Collaborator Contribution | My PhD project has been a shared effort between us and our collaborators, including the development of transgenic zebrafish lines and the use of these in investigating CLN2 disease. |
Impact | Transgenic zebrafish lines to measure lysosomal biology and autophagy in our CLN2 disease model. |
Start Year | 2017 |