Vps35 p. D620N causes Lrrk2 kinase hyperactivity, chronic microglial activation and inflammation
Isaac Bul Deng1, Mengfui Bu1,2, Jordan Follett1
1Department of Neurology, McKnight Brain Institute, University of Florida, Gainesville, Florida, USA.
Abstract:
Pathogenic variants in leucine-rich repeat kinase 2 (LRRK2), vacuolar protein sorting 35 (VPS35), and RAB32 cause dominantly inherited parkinsonism, indistinguishable from idiopathic late-onset Parkinson's disease (PD). All three causes constitutively activate LRRK2 kinase activity to augment immune responses, enhancing immunity to fight pathogens, but similar mechanisms in the brain increase the vulnerability of dopaminergic neurons to degeneration. Although VPS35 p.D620N possess the highest constitutive increase in LRRK2 kinase activity among known variants in LRRK2 or RAB32, its effects on the immune system remain poorly understood. LRRK2 and Rab32 are highly expressed in myeloid cells including microglia; thus we examined the transcriptomic and functional consequences of Vps35 p.D620N in knock-in mice (VKI). Microglia were isolated from brains of six-month-old VKI mice and were analyzed via single-cell RNA sequencing. Differential gene expression highlighted pathways involved in antimicrobial humoral immune response, lysosomal stress sensing, and phagocytosis. Notably, genes of S100 family proteins, along with lipocalin 2 (Lcn2), were significantly upregulated, and those measures were complimented by immunohistochemistry and quantitative PCR. In contrast, pathways involved in synaptic transmission, neuronal development, and homeostatic immune signaling were downregulated. Peripheral stimulation with lipopolysaccharide amplified microglial activation and phagocytic markers in wildtype mice, and VKI mice also display enhanced morphological activation and increased synaptic engulfment. Collectively, Vps35 p.D620N drives a chronic pro-inflammatory microglial phenotype characterized by heightened innate immune signaling, lysosomal stress, and enhanced phagocytic activity. VKI microglia are sensitized to peripheral immune challenges and may promote synaptic remodeling and neurodegenerative vulnerability in PD. These results provide mechanistic insight into how retromer dysfunction and LRRK2 kinase hyperactivity intersect with microglial biology to influence PD pathogenesis.
Insights
The VPS35 p.D620N variant in Parkinson's disease (PD) causes chronic inflammation in microglia, a key brain immune cell. This heightened immune response and cellular stress may increase neurodegeneration risk in PD.
Area of Science:
- Neuroscience
- Immunology
- Genetics
Background:
- Pathogenic variants in LRRK2, VPS35, and RAB32 cause inherited parkinsonism, activating LRRK2 kinase and immune responses.
- VPS35 p.D620N shows the highest LRRK2 kinase activity increase, but its immune effects are unclear.
- LRRK2 and Rab32 are highly expressed in microglia, suggesting a role in neuroinflammation.
Purpose of the Study:
- To investigate the transcriptomic and functional consequences of the VPS35 p.D620N variant in microglia.
- To understand how retromer dysfunction and LRRK2 hyperactivity influence Parkinson's disease pathogenesis.
Main Methods:
- Single-cell RNA sequencing of microglia from VKI mice carrying the VPS35 p.D620N variant.
- Differential gene expression analysis to identify affected pathways.
- Immunohistochemistry and quantitative PCR to validate findings.
- Functional assays including peripheral lipopolysaccharide stimulation.
Main Results:
- VKI microglia exhibit upregulated genes in antimicrobial immunity, lysosomal stress, and phagocytosis (e.g., S100 proteins, Lcn2).
- Downregulation of pathways related to synaptic transmission and homeostatic immune signaling observed.
- VKI microglia show enhanced morphological activation and synaptic engulfment, especially after peripheral immune challenge.
Conclusions:
- VPS35 p.D620N induces a chronic pro-inflammatory microglial phenotype with heightened innate immunity and lysosomal stress.
- VKI microglia are sensitized to immune challenges, potentially promoting synaptic remodeling and neurodegenerative vulnerability in PD.
- Mechanistic insights into the intersection of retromer dysfunction, LRRK2 hyperactivity, and microglial biology in PD pathogenesis.
