The role of lysosomal ion homeostasis and lysosomal proteases in healthy ageing and neurodegeneration

The lysosome is a multifaceted organelle involved in several cellular mechanisms. Its acidic lumen is crucial for the activation of proteases involved in the breakdown and recycling of macromolecules. Changes in lysosomal pH and protease activity have previously been reported in cellular models of familial Alzheimer disease (FAD), however, measurement of these changes has been problematic within the field, with drastic variations depending on the probes used. We have performed a comparison of current lysosomal pH measurement techniques to identify the best probes and preferred method for accurate pH measurements. We have utilized these probes to accurately measure and confirm the presence of significantly increased lysosomal pH in patient FAD cells with mutations in PSEN1, the gene encoding presenilin 1 (PSEN1). Resultant changes in cathepsin protease levels, which are optimally active at an acidic pH, are also evident in these cells as well as hyperfunctioning of the TRPML1 channel. Here, we highlight the use of a compound which enhances vATPase function, aiding in the acidification of the lysosomes, in order to rectify the pH defect and resultant changes in cathepsin activity in PSEN1 cells. With previous findings of decreased acidity in ageing yeast vacuoles which function similarly to lysosomes, these methods have been applied to measure significant increase in lysosomal pH in passage aged human cells as well as in older patient derived cells from a range of ages (6-96). These changes in pH correlate to decreased Cathepsin D activity which has previously been shown to cause downstream cellular dysfunction and contribute to the build-up of neurotoxic protein aggregate hallmarks of Alzheimer disease (AD). Further investigation in hippocampal sections taken from WT and PDAPP mice aged 3 and 15 months as well as in hippocampal sections of the ageing human brain(0.3-104 years old) shows decreased Cathepsin D staining suggestive of increased lysosomal pH. Here, we provide the first report of changes in lysosomal pH during healthy ageing in humans, providing a possible link between the pathogenesis of FAD and late onset sporadic AD. CLN10 is a childhood neurodegenerative lysosomal storage disease caused by mutation in cathepsin D. We have performed high throughput screening of an FDA approved drug library for potential modulators of cathepsin D activity. This screening resulted in numerous hit iii compounds, 2 of which were validated as increasing cathepsin D activity in patient derived CLN10 cells. These compounds have the potential to be developed for the treatment of CLN10 disease, as well as the further potential use for targeting cathepsin D activity for the treatment of FAD. Together, data in this thesis support the emerging importance of the role of lysosomes in AD as well as in healthy ageing with particular emphasis of the importance of lysosomal pH and cathepsin activity, with the latter also linking to CLN10 disease. We believe that lysosomal pH and cathepsin D modulation provide promising therapeutic targets for the treatment of age associated neurodegeneration, with the scope to also investigate further the mechanisms behind healthy ageing