Leader: Luigi Bubacco (UNIPD); Other collaborator(s):
We plan to study the role of protein dyshomeostasis in neurodegenerative diseases. The experimental approaches exploited will span from the biophysics of protein aggregation to the cellular and in vivo analyses of the processes that govern protein clearance and mitochondrial removal in neurons and microglia.
Brief description of the activities and of the intermediate results: This project is aimed to integrate proteomic, interactomics, and bioinformatics approaches to identify novel players involved in PD-related neuroinflammation and understand how aging impacts their function. The first aim of this project is to understand which protein interacts with LRRK2 within microglia in basal conditions and in inflammatory conditions associated to protein dishomeostasis (WP1). To this aim, BV2 microglia cell line cultures have been established in our lab. As a strategy for profiling LRRK2 interactors, we will rely on Proximity labeling, a technique that enables to capture both transient and constitutive interactions and has been already successfully employed to profile LRRK2 interactors in HEK cells. Proximity labelling involves the conjugation of a protein of interest with the ascorbate peroxidase 2 (APEX2), which upon biotin phenol administration biotinylates surrounding proteins in a 20 nm radius. The second aim of the project is to investigate how the expression of LRRK2 substrates and interactors changes along chronological aging (WP2). This is of interest as LRRK2 pathogenic mutations display an age-dependent increase in penetrance. To achieve this goal, a bioinformatic analysis has been carried out using publicly available LRRK2 interactomics data and healthy aging transcriptomic data. Interactomics data of human and mouse LRRK2 has been downloaded from the IntAct database and processed through R for subsequent analysis, currently ongoing.
The third and final aim of this project is to understand how aging, LRRK2 mutations, and systemic inflammation synergize to promote neurodegeneration in PD (WP3). To this aim, we use the LRRK2-G2019S transgenic mice, treated with chronic low-dose intraperitoneal LPS. These mice fit perfectly with the scope of the project, as they display age-dependent neurodegeneration and neuroinflammation.
BV2 cells have been confirmed to respond to both LPS and IFN-γ using IL-1β ELISA assay and qPCR, respectively. Transfection of BV2 cells with LRRK2-APEX2 constructs was carried out with the Glial-Mag transfection reagent. Despite successful transfection, APEX2-mediated proximity biotinylation did not work in BV2 cells. However, the APEX2 reaction was carried out in HEK293 cells, suggesting a cell-type specific problem with microglial cells. As an alternative strategy to profile LRRK2 interactors in microglia, we employed GFP-tagged nanobodies targeted against LRRK2, which can be co-immunoprecipitated using GFP-trap, a resin composed of GFP-targeted nanobodies. Despite confirming the colocalization between LRRK2-targeted nanobodies and LRRK2, co-immunoprecipitation was unsuccessful in BV2 cells. Currently, we are testing the APEX2 system in TLR-4-expressing HEK293 cells, which respond to LPS and may be useful in identifying immune-specific interactors of LRRK2, although a subsequent validation will be needed.
In parallel, we utilized A549 cells, which express high levels of endogenous LRRK2, and confirmed existing literature demonstrating their robust response to IFN-γ stimulation. Notably, LRRK2 activity toward its substrate, RAB10, is significantly elevated upon IFN-γ exposure. This model system provides a robust platform for studying how LRRK2 protein and phospho-protein networks are rewired during inflammatory responses, with the added advantage of using cells with endogenous LRRK2 expression (work ongoing).
For Aim 2, we anticipate completing the analysis by early 2025. This will involve integrating the LRRK2-interactome data with aging- and PD-related gene networks, as well as the protein/phospho-protein interactors identified under inflammatory conditions from Aim 1. The top-ranked candidates from this combined dataset will then be validated in young and aged brain tissues from BAC-G2019S mice (Aim 3) through histological and biochemical assessments. These mice are currently aging, and all necessary ethical approvals have been obtained from the Ministry of Health (authorization no. 1005/2024-PR, response to protocol D2784.195).
Coming soon