Assessing stimulation-dependent changes in LRRK2 and GCase expression/activity and convergence at the lysosome in cryopreserved monocytes.

Abstract

Both leucine-rich repeat kinase 2 (LRRK2) and glucocerebrosidase (GCase) are promising targets for the treatment of Parkinson's disease (PD). Evidence suggests that both proteins are involved in similar biological pathways, including lysosomal function. However, studies to date have largely investigated the enzymes in isolation and any relationship between LRRK2 and GCase remains unclear. Recent data suggest that it is the response to immunological challenges that is altered in PD and that circulating baseline cytokine levels are not useful diagnostic or predictive biomarkers in PD. Taken together, this project aims to optimize a blood collection and cryopreservation protocol to facilitate the collection and study of monocytes from PD patients and healthy controls, with a particular focus on measuring stimulation-dependent changes in LRRK2 and GCase protein activity levels and lysosomal function. Human PBMCs were isolated from whole blood and plated fresh or post-cryopreservation. PBMCs were treated with MLi2 or CBE +/- IFN-γ. Treatment effects on cathepsin activity, LRRK2 and GCase enzymatic activity, and antigen presentation were assessed in monocytes via flow cytometry. Media was collected RESULT: We first assessed the effects of cryopreservation on our read-outs. Despite cryopreservation having a small effect at baseline, both fresh and cryopreserved monocytes gated from total PBMCs exhibited comparable changes in response to IFN-γ. We observed that LRRK2 kinase inhibition decreases cathepsin activity in monocytes in a stimulation-dependent manner. This was coupled with increased LRRK2 expression and alterations in immune cell activation with LRRK2 kinase inhibition. Similarly, an increase in GCase activity levels were observed in monocytes upon IFN-γ treatment. These results show that cryopreservation of PBMCs doesn't affect our ability to measure stimulation-dependent changes in both LRRK2 and GCase expression/activity levels. These assays are therefore to be repeated in idiopathic PD PBMCs. Furthermore, these results suggest a fundamental role of LRRK2, specifically LRRK2 kinase activity, in lysosomal function in immune cells. Given the parallel increases in GCase activity, future research aims to investigate how these two enzymes interact and converge on biological pathways regulating inflammation and the lysosome in patient monocytes.