Role of IFN-γ stimulation in the regulation of the Parkinson’s Disease-associated kinase LRRK2

Parkinson's disease (PD) is a multifactorial neurodegenerative disorder in which both central and peripheral inflammation contribute to pathogenesis. Mutations in LRRK2 (leucine-rich repeat kinase 2) are the most common cause of monogenic PD, and its kinase activity, reported by phosphorylation of a subset of Rab GTPases, including Rab10 at Thr73, is a promising therapeutic target. However, LRRK2 inhibitors, while reducing mutant LRRK2-mediated cell toxicity and lysosomal stress, cause pulmonary side effects in type II pneumocytes, supporting the importance of studying its physiological activity in relevant cell types. The pro-inflammatory cytokine IFN-γ upregulates LRRK2 expression and activity in immune cells, but whether IFN-γ engages the LRRK2–Rab10 pathway in type II pneumocytes remains unclear. Here we used A549 cells, a human type II pneumocyte model with high endogenous LRRK2 expression, to dissect the IFN-γ–LRRK2 axis by western blotting and confocal immunocytochemistry. We found that IFN-γ activated the JAK-STAT pathway, with a transient pSTAT1 (Tyr701) peak at 15 minutes and total STAT1 accumulation at 24 hours, independently of LRRK2 kinase activity, as shown by insensitivity to the LRRK2 inhibitor MLi-2. IFN-γ increased Rab10 phosphorylation at Thr73 after 24 hours, but not acutely, without altering LRRK2 protein or mRNA levels, indicating activation of the LRRK2 pool rather than transcriptional induction, in contrast to what has been described in myeloid cells and microglia. The pRab10 increase was abolished by MLi-2, confirming its dependence on LRRK2 kinase activity. Unexpectedly, across three immunostaining protocols, the anti-prab10 antibody used in this study revealed an IFN-γ-induced nuclear signal that did not co-localize with LAMP1 or pericentrin. Critically, a MLi-2 sensitive nuclear signal was also detected on Rab10 KO cells demonstrating that the nuclear ICC signal cannot be unambiguously attributed to pRab10. The identity of this epitope remains to be established, for example by immunoprecipitation coupled to mass spectrometry. These data validate A549 cells as a peripheral model of IFN-γ-driven LRRK2 pathway activation, reveal a delayed, expression-independent engagement of LRRK2 kinase activity, and uncover a specificity caveat for pRab10 immunostaining that is relevant to the interpretation of LRRK2 biomarker studies. The experimental work presented in this thesis was carried out at the Laboratory of Brain Physiopathology at the Department of Biology, University of Padova.