Lysosomes are highly acidic organelles that integrate important cellular processes in all cell types. There is a preponderance of risk genes for neurological disorders associated with lysosome dysfunction. In the lysosome, lumenal Ca2+ is critical to function, and defects in every known lysosomal Ca2+ release channel leads to a distinct neurological disorder. Yet, dysregulated lysosomal Ca2+ can also arise due to defective import. While much more known of mechanisms that release lysosomal Ca2+, there is a paucity of information on the pathophysiology of Ca2+ import. Notably, the only known lysosomal Ca2+ importer in animals, the P-type ATPase ATP13A2, was recently discovered by my laboratory using newly developed reporter technology for lysosomal Ca2+ imaging. This importer was previously identified as a major risk gene for Parkinson's disease. Thus, a structural level understanding of how by ATP13A2 imports Ca2+ into the lysosome is highly significant. The premise of this proposal is that ATP13A2 function is mechanistically similar to that of SERCA but with lower affinity and/or efficiency of Ca2+ transport. This premise is based on unpublished data from my laboratory using homology modeling, which predicts very high similarity between ATP13A2 and SERCA. SERCA (Sarco/Endoplasmic Reticulum Ca2+ ATPase) is one of the best studied P2-type ATPases. In contrast, ATP13A2 is a P5-type ATPase, an ATPase sub-class yet to be mechanistically characterized. The steps outlined in this proposal will identify and study the molecular mechanism of how ATP13A2 drives lysosomal Ca2+ import by mapping lysosomal Ca2+ dynamics in real-time in live mammalian cells. We plan to create and characterize a photostable lysosomal Ca2+ reporter and develop an assay to map the kinetics of lysosomal Ca2+ import in situ in live cells. Preliminary data shows that we have identified the relevant molecular components to make this photostable organellar Ca2+ reporter. Further, an initial bioinformatics analysis and homology modeling has revealed a remarkable similarly between ATP13A2 and SERCA. This will allow us to pinpoint residues important to Ca2+ import by a P5-type ATPase. By expressing various ATP13A2 mutants and using real-time Ca2+ mapping, we shall be able to identify residues critical to the function of ATP13A2. Successful completion of this research will identify how a major risk gene for Parkinson's disease imports lysosomal Ca2+ and elucidate the first structure-activity relationship in P5-type ATPases. Also, by providing the first practical technology to quantitatively map lysosomal Ca2+ fluxes in live cells (in real-time), we will be in a position to study new lysosomal Ca2+ importers and existing lysosome Ca2+ release channels connected to various neurodegenerative diseases.
Many neurodegenerative diseases arise due to lysosome dysfunction in the different cell-types of the brain as a result of lysosomal Ca2+ dysregulation. A major obstacle to leveraging lysosomes as a therapeutic target stems from lack of knowledge of lysosomal Ca2+ import mechanisms. Our research seeks to provide the first insights into lysosomal Ca2+ import at the molecular level.
