Split-GFP tools to investigate the subcellular dynamics of the Parkinson’s disease kinase LRRK2

Parkinson’s disease (PD) is a neurodegenerative movement disorder for which no cure is available. Over the past 20 years, genetics provided a step-change opportunity to unravel the molecular basis of PD by identifying disease-causing mutations and risk-alleles. Among all the genetic contributors of PD, mutations in the LRRK2 gene, encoding a complex GTPase-kinase, represent the most common cause of disease, making LRRK2 a pursued target in quest of disease-modifying therapies. To date, an impressive number of LRRK2 inhibitors have been developed. However, without the understanding of the role covered by LRRK2 in the physiology of neuronal and non-neuronal cells, it is difficult to predict whether a chronic regime of LRRK2 inhibition is a safe therapeutic option. One approach to understand the function of a protein is to investigate its subcellular localization and how it changes upon perturbation of the system. In this project, two different tools based on Split-GFP technology were used to create a system to study the subcellular localization of LRRK2: LRRK2 in fusion with N-terminal β11 strand, that represents a direct way to see how LRRK2 interacts with different compartments where reconstitution of the GFP signal would occur, and SPLICS probes, which allow to study contacts between organelles and how LRRK2 affects them in different conditions. The first tool, β11-LRRK2 construct, did not exhibit the expected fluorescent signal, therefore the development of a tandem-β11 LRRK2, that could overcome this limitation, is in progress. On the other hand, SPLICS probes revealed that the G2019S mutation increase the number of contact sites between lysosomes and mitochondria.