Single-cell resolution uncovers cell type-specific dysregulation in Parkin-deficient neuron-microglia co-cultures

ABSTRACT Background Mutations in the E3 ubiquitin ligase Parkin (encoded by PRKN ) are the most frequently known cause of recessively inherited Parkinson’s disease. In addition to the loss of dopaminergic neurons, microglial activation is another pathological feature observed in Parkinson’s disease. While postmortem brain samples show the end stage of the disease, neurons and glia derived from patients’ induced pluripotent stem cells (iPSCs) provide a model for detecting early pre-degenerative disease trajectories. However, mixed cell populations often confound these cultures, leading to heterogeneous disease phenotypes. Methods Here, we tease apart the cell type-specific phenotypes underlying Parkin-linked Parkinson’s disease by performing single-nucleus RNA sequencing in iPSC-derived co-cultures of dopaminergic neurons and microglia from PRKN mutation carriers and healthy controls. We validated our transcriptomic key findings through inflammatory cytokine profiling and live-cell calcium imaging. Results Single-nucleus RNA sequencing identified seven major cell types composed of neuronal, glial, and precursor cells, with dopaminergic neurons accounting for the largest cell population. Pathway analysis revealed cell type-specific dysregulated biological processes in Parkin-deficient cells, including gene expression differences in dopaminergic neurons that control mitophagy and dopamine homeostasis, whereas microglia showed changes in calcium homeostasis and inflammatory signaling. Functional analysis verified elevated secretion of monocyte chemotactic protein 1 in PRKN -mutant co-cultures compared with controls, linking Parkin deficiency to increased microglial chemotactic signaling. Furthermore, lower intracellular calcium levels and diminished calcium release following treatment confirmed impaired calcium homeostasis in PRKN -mutant microglia. Conclusions Profiling at the single-cell level resolved distinct cell subpopulations, enabling us to identify cell type-specific pathway disturbances underlying Parkin deficiency. This unique dataset provides a basis for understanding the impairment of individual cell types and the impact of cellular crosstalk in Parkinson’s disease pathology.