LRRK2 and WAVE2 regulate microglial‐transition through distinct morphological phenotypes to induce neurotoxicity in a novel two‐hit in vitro model of neurodegeneration

Abstract

We report a novel in vitro classification system that tracks microglial activation state and their potential neurotoxicity. Mixed live‐cell imaging was used to characterize transition through distinct morphological phenotypes, production of reactive oxygen species (ROS), formation of reactive microglial aggregates, and subsequent cytokine production. Transwell cultures were used to determine microglial migration (control and lipopolysaccharide (LPS) treated) to glutamate pre‐stressed or healthy neurons. This two‐hit paradigm was developed to model the vast evidence that neurodegenerative conditions, like Parkinson's disease (PD), may stem from the collective impact of multiple environmental stressors. We found that healthy neurons were resistant to microglial‐mediated inflammation, whereas glutamate pre‐stressed neurons were highly susceptible and in fact, appeared to recruit microglia. The LPS treated microglia progressed through distinct morphological states and expressed high levels of ROS and formed large cellular aggregates. Recent evidence implicates leucine‐rich repeat kinase 2 (LRRK2) as an important player in the microglial inflammatory state, as well as in the genesis of PD. We found that inhibition of the LRRK2 signaling pathway using the kinase inhibitor cis‐2,6‐dimethyl‐4‐(6‐(5‐(1‐methylcyclopropoxy)‐1H‐indazol‐3‐yl)pyrimidin‐4‐yl)morpholine (MLi2) or inhibition of the actin regulatory protein, Wiskott‐Aldrich syndrome family Verprolin‐homologous Protein‐2 (WAVE2), stunted microglial activation and prevented neurotoxicity. Furthermore, inhibition of LRRK2 kinase activity reduced pro‐inflammatory chemokines including MIP‐2, CRG‐2, and RANTES. These data together support the notion that LRRK2 and WAVE2 are important mediators of cytokine production and cytoskeletal rearrangement necessary for microglial‐induced neurotoxicity. Furthermore, our model demonstrated unique microglial phenotypic changes that might be mechanistically important for better understanding neuron‐microglial crosstalk.

LRRK2 G2019S promotes astrocytic inflammation induced by oligomeric α-synuclein through NF-κB pathway

“…Previous studies have shown that LRRK2 mutations could promote the progression of PD and aggravate environmental toxin-induced inflammation. 19 , 20 , 21 Meanwhile, knockout of LRRK2 gene prevents the neurotoxic effects and neuroinflammatory reactions caused by paraquat. 22 Although these studies have revealed the improving effect of LRRK2 G2019S mutation in neurotoxin-induced neuroinflammation, it is still difficult to speculate how LRRK2 is involved in the inflammatory processes.…”

Synergistic photobiomodulation with 808-nm and 1064-nm lasers to reduce the β-amyloid neurotoxicity in the in vitro Alzheimer's disease models

“…The transwell insert system is widely used in studying the crosstalk activities of two different kinds of cells, wherein the influence of one cell line on another kind of cell can be avoided during the cell treatment process. For example, the transwell system, expressing the LPS-treated microglia, was utilized by Fenner et al ( 2021 ) to demonstrate the inflammatory effects of healthy neurons. Song et al ( 2012 ) also cultured microglia and neuroblastoma in the transwell system.…”

Orchestration of synaptic functions by WAVE regulatory complex-mediated actin reorganization

“…LRRK2 phosphorylates WAVE2, stabilizes its levels and modulates the dynamics of WAVE2-mediated phagocytic activity of macrophage cells 141 . Several subsequent reports appear to support the association of WAVE2 with PD 142 , 143 , although the mechanistic basis of WAVE2 action in the pathogenesis of PD is still unclear.…”