Contribution of microglia to the pathogenic mechanisms behind LRRK2-associated Parkinson's disease

Microglia are immune cells of the brain playing critical roles during the inflammatory response. Among the genes mutated in familial Parkinson’s disease, those encoding LRRK2 and α-synuclein have been associated with neuroinflammatory processes. Previous work showed that LRRK2 is a positive regulator of inflammation, while aggregated α-synuclein released by dying neurons can activate microglia triggering the neuroinflammatory process. The main goal of this project was to gain novel insights into the contribution of microglial cells to the pathogenic mechanisms of LRRK2-associated Parkinson’s disease. To this aim, we initially performed RNA-sequencing (RNA-seq) of Lrrk2 wild-type (WT) and knockout (KO) primary microglia treated with α-synuclein pre-formed fibrils (PFFs) or lipopolysaccharide (LPS). We found that LPS and α-synuclein triggered in part overlapping but also different responses, while loss of Lrrk2 had a subtle effect in attenuating the pro-inflammatory response. Moreover, treatment with α-synuclein PFFs caused a significant induction of the antioxidant superoxide dismutase-2 (SOD2) enzyme in WT cells, with the effect attenuated in Lrrk2 KO microglia cells. Since loss of Lrrk2 in primary microglia revealed only small differences in gene expression both under resting or pro-inflammatory conditions, we next moved to an experimental condition that more closely resembles the physiological situation, e.g. acutely isolated microglia cells from LPS-injected adult mouse brains. Using single cell RNA sequencing (scRNA-seq), we confirmed a subset of the genes nominated in primary culture experiments, including IL-1, SOD2 and TXNIP as differentially expressed upon inflammatory stimulation with LPS. However, the overall effect on gene transcription due to loss of Lrrk2 remained subtle. To further explore the impact of LRRK2 on microglia function, we performed ex vivo phagocytic assays. Lrrk2 KO microglia displayed the highest phagocytic activity compared to microglia isolated from WT and pathogenic mutants G2019S and R1441C mice upon treatment with α-synuclein PFFs, suggesting that LRRK2 negatively regulates phagocytosis or delays lysosomal degradation. Additionally, we performed intrastriatal brain injections with lipopolysaccharide (LPS)-inflammatory agent or with PBS as a control, that demonstrated a significant morphological changes in microglial shape (increase in circularity and soma area) in LPS-stimulated animals compare to PBS-treated and a modest differences between LRRK2 genotypes. Additionally, the branch complexity was reduced in LPS-injected animals with stronger effect in Lrrk2-KO and R1441C genotypes and a less response in G2019S animals. Next, we evaluated LRRK2 mRNA and protein expression at the single cell level. We found that LRRK2 has a low and sparse pattern of expression in unstimulated resident microglia. However, upon stimulation with LPS, the proportion of microglia cells expressing LRRK2 increased, while LRRK2 levels remained unchanged per cell. This is, to date, the first attempt to analyze LRRK2 protein expression at the single cell level in brain cells. In summary, the present work provides new insights into the biology of LRRK2 in the brain resident microglia and shows that inflammatory stimulations with α-synuclein PFFs cause a significant induction of pro-inflammatory and anti-oxidant responses, which are attenuated in LRRK2-KO microglia.