Abstract

Lysosomal dysfunction is well known to cause Lysosomal Storage Disorders, the common cause of childhood neurodegeneration. However, more recently lysosomal dysfunction has also been implicated in diseases of aging, such as Alzheimer and Parkinson diseases. It is thought this is due to lysosomal deacidification, which disrupts lysosomal degradative processes, causing accumulation of autophagic cargo in lysosomes. This is something neurons are more prone to due to them being postmitotic.The vacuolar-ATPase (vATPase), is an ATP dependent proton pump which maintains lysosomal pH. Dysfunction prevents protons from entering the lysosome, causing a rise in lysosomal pH. The V0a subunit has 4 isoforms that are targeted to different membranes in the cell/body. The V0a1 isoform localises in the presynaptic nerve terminal, to synaptic vesicles. There is both genetic and functional data linking disrupted vATPase function to Alzheimer disease (AD). Mutations in the V0a3 subunit cause early onset AD, and Presenilin 1 familial Alzheimer disease (AD) causes a defect in vATPase processing that is central to pathogenesis. Boosting vATPase function is therefore a potential therapeutic strategy for AD. However, there are currently no good chemical starting points to achieve this increased activity.To facilitate the identification of novel activators, we have developed a robust high content imaging for lysosomal pH. This assay uses cells treated with a low concentration of the vATPase inhibitor bafilomycin A1 to mimic the lysosomal deacidification seen in AD, and pH-insensitive and sensitive fluorophore-conjugated dextrans, endocytosed to the lysosome, as markers for the lysosome and its pH. We have validated the assay using known inhibitors and activators of the vATPase, and now intend to use it in a high throughput screen to identify molecules that can restore the pH of deacidified lysosomes