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).
During this quarter, the research efforts focused on the refinement of the experimental model for proximity labeling, the expansion of the bioinformatic analysis pipeline, and significant international collaborations that strengthened the bioinformatic component of the project.
A review chapter titled “Involvement of microglial aging in neurodegenerative diseases: a focus on Parkinson’s disease” was submitted to Elsevier. This chapter provides a comprehensive overview of the current knowledge surrounding the interplay between microglial aging and disease-driving mechanisms in Parkinson’s disease (PD). The manuscript is currently under revision.
As part of WP1, a pilot experiment was conducted using TLR4-expressing HEK293 cells, successfully transfected with both LRRK2-APEX2 and NES-APEX2 constructs via Lipofectamine 2000. The APEX2-mediated biotinylation reaction, performed following the protocol described in Bonet-Ponce et al. (2020), was successful and validated by streptavidin-HRP immunoblotting. These findings indicate that TLR4-HEK293 cells represent a promising platform for studying LRRK2 interactors in the context of immune activation. However, due to limited time to complete the remaining steps of the experimental pipeline, we have prioritized Task 2 (bioinformatic analysis).
In this context, two international collaborations significantly advanced the bioinformatic component of the project. First, during a visiting period at the University College London School of Pharmacy, under the supervision of Claudia Manzoni, we conducted differential expression analysis (DEA) on a previously cleared dataset of whole blood transcriptomics from PD patients and healthy controls. The analysis revealed an age-associated downregulation of adaptive immune pathways and upregulation of innate immune signatures, consistent with the concept of “inflammaging.” Notably, the LRRK2 interactors RAB38 and ABLIM1 showed differential expression between young and aged PD patients, but not in healthy individuals.
Second, during a visiting period at the Yusuf Hamied Department of Chemistry, University of Cambridge (Michele Vendruscolo Laboratory), under the supervision of James Tompkins,
we analyzed a single-nucleus RNA-seq (snRNA-seq) dataset of prefrontal cortex samples from healthy individuals across different age groups. DEA indicated a selective increase in LRRK2 expression in microglia and oligodendrocyte precursor cells (OPCs) with aging, primarily driven by an increased proportion of LRRK2-expressing cells. Moreover, significant transcriptomic changes were exclusive to microglia, where 13 LRRK2 interactors were differentially expressed across age groups.
A high-dimensional weighted gene co-expression network analysis (hdWGCNA) was performed to explore microglial heterogeneity. This analysis identified 15 microglial subclusters, with two of them, associated with older individuals, exhibiting the highest levels of LRRK2 expression. These findings point toward specific microglial subpopulations that may be particularly relevant in aging-associated neuroinflammation.
Overall, the results generated in this period consolidate the rationale for leveraging transcriptomic data to pinpoint aging- and inflammation-associated changes in LRRK2 signaling. The integration and prioritization of candidate interactors are ongoing and will guide validation in brain tissues from G2019S transgenic mice (WP3), which continue to age under controlled conditions. All necessary ethical authorizations remain valid (Ministry of Health, authorization no. 1005/2024-PR, protocol D2784.195).
Coming soon