Introduction
Parkinson’s disease (PD) is a progressive neurodegenerative disorder characterised by motor and non-motor symptoms, including cognitive impairment.1 Pathologically, PD is defined by dopaminergic neuron loss and accumulation of α-synuclein (α-Syn) aggregates.1 Aging is the greatest risk factor for PD, and cellular senescence—an irreversible cell-cycle arrest—is a key hallmark of aging tissues. Senescent cells adopt a senescence-associated secretory phenotype (SASP), releasing pro-inflammatory factors that promote chronic neuroinflammation.2 Increasing evidence suggests that pathological protein aggregation induces senescence in multiple brain cell types, including microglia.3 As the resident immune cells of the central nervous system, microglia play a central role in maintaining brain homeostasis, but disease-associated microglial states can exacerbate inflammation.4 Leucine-rich repeat kinase 2 (LRRK2), a kinase strongly implicated in both familial and sporadic PD, is highly expressed in microglia and regulates inflammatory signalling.5 Previous studies demonstrate that LRRK2 can induce neuronal senescence via p53–p21 signalling and promote α-Syn aggregation6, while inhibition of LRRK2 ameliorates neuroinflammation and cytotoxicity.7 However, the role of LRRK2 hyperactivity in α-Syn–driven microglial senescence remains unclear.
Aims/Objectives
This project aims to determine whether α-Syn promotes microglial senescence through increased LRRK2 expression and activity in PD. The objectives are to (i) map the spatial relationship between LRRK2 expression, senescence markers and α-Syn pathology in human PD brain tissue, (ii) investigate how α-Syn drives senescence in iPSC-derived microglia carrying the common LRRK2 mutation, G2019S, and (iii) assess whether inhibiting LRRK2 activity can attenuate α-Syn–driven senescence in iPSC-derived microglia.
Methods and Research Plan
Post-mortem human brain tissue from PD cases across Braak stages (Lewy body pathology), Parkinson’s disease dementia (PDD), dementia with Lewy bodies (DLB) and age and sex-matched controls (male, n = 12 and female, n = 3 for each group) will be analysed using imaging mass cytometry (IMC). The prefrontal cortex, a region affected by α-Syn pathology in cognitively impaired PD cases, will be examined. This approach can quantify senescent microglial populations and assess their spatial relationship to α-Syn pathology.
Complementary in-vitro studies will be using human iPSC-derived microglia carrying the LRRK2 G2019S mutation along with heterozygous and homozygous knockouts (KO). iPSC-derived microglia will be exposed to α-Syn pre-formed fibrils to assess LRRK2 activity and senescence induction. To directly test the contribution of LRRK2 activity, iPSC-derived microglia will be treated with LRRK2 kinase inhibitors.
Results
Meta-analysis of published single-nucleus RNA-sequencing datasets (n = 11) indicates increased expression of canonical senescence gene sets in microglia from PD, PDD and DLB compared with healthy controls (meta logFC = 0.5 and p value = 1e-04). Immunofluorescence staining on fixed-formalin parafilm-embedded (FFPE) tissue from PDD patients show presence of senescence markers, LRRK2 and pathology markers. iPSC cells containing the LRRK2 G2019S mutation, heterozygous and homozygous knockouts have been validated and characterised for differentiation into iPSC-derived microglia.
Conclusion
This work aims to define how α-Syn pathology mediated LRRK2 activity drives microglial senescence in PD. By combining spatial analysis of human tissue with in-vitro iPSC-derived microglial models, this project offering mechanistic insight into senescence-associated pathology in microglia in PD.