Project No.2248
Primary Supervisor
Dr J. Arjuna Ratnayaka – University of Southampton
Co-Supervisor(s)
Professor Louise C. Serpell – University of Sussex
Dr David Tumbarello – University of Southampton
Summary
Background: Lysosomal damage is a key feature of neurodegeneration, responsible for blinding diseases including age-related macular degeneration (AMD) and neuropathies like Alzheimer’s disease (AD){1}.
Toxic macromolecules, including self-assembling proteins amyloid-beta (Abeta), accumulate in lysosomes{2,3}. Lysosomes are also damaged by post-translational modification of certain cargos and their inefficient breakdown{4,5}. This project will investigate how lysosomes become damaged, how
lysosomal-deficiency can impair cell function, and whether boosting the production of new lysosomes can rescue cellular defects. Approaches: Two experts working on protein misfolding diseases of the retina and brain have come together to offer an exciting project, combining molecular-biology and optogenetic tools, live/3D-imaging with computing algorithms, to develop a novel pipeline for “lysosomal-fingerprinting”. Studies will utilize cell models of the retinal pigment epithelium (RPE) and retinal/CNS neurons in the supervisors’ laboratories. Baseline lysosome size/number and distribution in healthy cells will be quantified and compared with pathogenic conditions in AMD/AD. Ubiquitination and proteasome-activity will be evaluated alongside, as these can compete with degradation of lysosome-targeted cargos{1}. Fluorescently-labelled molecules (photoreceptor outer segments [OS], oxidatively modified-OS and Abeta, which are internalised by RPE cells and neurons), and genetic constructs targeting lysosomes will report their localisation, lysosomal enzyme activity and efficiency of cargo degradation. We have pioneered the use of computing algorithms in these studies{3-5} that will delineate between healthy/damaged lysosomes and those with/without cargos, which will be confirmed by 3D/electron microscopy. Live-cell imaging will report lysosomal mobility and availability. Other trafficking vesicles will also be assessed, as functional lysosomes must fuse with these to reform{1}. Finally, genetic manipulation will induce the biogenesis of new lysosomes to determine whether cellular defects can be rescued. Impact: Developing a pipeline to characterise lysosomal dysfunction at molecular level can unravel a major pathway of cellular damage linked with neurodegeneration, providing novel insights into maintaining healthy cells in old age.
1 Keeling, E., Lotery, A. J., Tumbarello, D. A. & Ratnayaka, J. A. Impaired Cargo Clearance in the Retinal Pigment Epithelium (RPE) Underlies Irreversible Blinding Diseases. Cells 7, doi:10.3390/cells7020016 (2018).
2 Marshall, K. E., Vadukul, D. M., Staras, K. & Serpell, L. C. Misfolded amyloid-beta-42 impairs the endosomal-lysosomal pathway. Cell Mol Life Sci, doi:10.1007/s00018-020-03464-4 (2020).
3 Lynn, S. A. et al. Oligomeric Aβ(1-42) Induces an AMD-Like Phenotype and Accumulates in Lysosomes to Impair RPE Function. Cells 10, doi:10.3390/cells10020413 (2021).
4 Keeling, E. et al. Oxidative Stress and Dysfunctional Intracellular Traffic Linked to an Unhealthy Diet Results in Impaired Cargo Transport in the Retinal Pigment Epithelium (RPE). Molecular nutrition & food research, e1800951, doi:10.1002/mnfr.201800951 (2019).
5 Keeling, E. et al. An In-Vitro Cell Model of Intracellular Protein Aggregation Provides Insights into RPE Stress Associated with Retinopathy. International journal of molecular sciences 21, doi:10.3390/ijms21186647 (2020).