Astrocytic Ceruloplasmin Deficiency Triggers Iron Toxicity and Neurodegeneration in a LRRK2 Parkinson’s Tri-Culture Model

Astrocytes and microglia carrying the LRRK2-G2019S mutation contribute to non-cell- autonomous dopaminergic neuron (DAn) degeneration in Parkinson’s disease (PD), but the mechanisms underlying their interplay remain unclear. Here, we developed a novel induced pluripotent stem cell (iPSC)-derived tri-culture system comprising healthy DAn and either LRRK2-mutant or isogenic control iPSC-derived astrocytes and microglia. Using integrated functional assays and transcriptomic profiling, we found that mutant astrocytes adopt a hyperreactive state, driving microglial activation and subsequent DAn degeneration. Mechanistically, we identified a selective downregulation of ceruloplasmin (CP), a copper-dependent ferroxidase, in mutant astrocytes, leading to disrupted iron homeostasis with accumulation of Fe ²⁺ and ROS. This iron dysregulation mediated both microglial reactivity and neurodegeneration. Notably, pharmacological restoration of CP re-established iron homeostasis, reduced microglial activation, and protected DAn from degeneration. Our findings uncover a novel astrocyte-microglia-neuron axis driving PD pathogenesis and showcase the power of our unique stem cell tri-culture platform for dissecting disease mechanisms and discovering therapeutic targets.