Single-Nucleus Phosphatome Rewiring and Phosphoproteomics Reveal PFKFB2 as an LRRK2-Linked Metabolic Node in Human Parkinsonian Substantia Nigra

Aberrant signalling by leucine-rich repeat kinase 2 (LRRK2) is a major driver of Parkinson’s disease (PD) biology, linking Rab phosphorylation to vesicular trafficking, endolysosomal dysfunction and immune-cell regulation. However, how LRRK2-linked trafficking programs intersect with metabolic and mitochondrial remodeling within vulnerable human substantia nigra cell states remains poorly defined. Here, phosphatome-wide systems analysis, single-nucleus transcriptomics, structural modelling and deep-phosphoproteomic validation were integrated to identify the glycolytic regulator PFKFB2 as a phosphorylation-regulated metabolic node in the Parkinsonian substantia nigra. Sample-level pseudobulk profiling of the GSE184950 human substantia nigra single-nucleus RNA-seq dataset revealed distributed phosphatome remodeling across the PD spectrum, with PFKFB2 emerging as a transcriptionally reduced and dynamically rewired systems hub. Full-atlas analysis across 390,360 nuclei showed that this tissue-level decrease resolved into disease-stage-specific cellular redistribution: PFKFB2 was reduced across oligodendrocyte-lineage and neuronal compartments in PD, whereas Parkinson’s disease dementia (PDD) showed microglial induction alongside astrocytic, astrocyte/glial-intermediate and neuronal loss. Stratification by PFKFB2 expression revealed marked transcriptional polarity, with PFKFB2-positive cellular states enriched for PD-associated genes, LRRK2–Rab trafficking, lysosomal/autophagy and glycolytic programs, while PFKFB2-negative states retained stronger mitochondrial/OXPHOS-associated signatures. Mechanistically, sequence-based phosphosite prioritization and AlphaFold-guided peptide docking identified the flexible, putatively disordered C-terminal regulatory tail of PFKFB2 as a kinase-accessible region, prioritizing the conserved Ser483 locus as the strongest LRRK2-compatible structural candidate. Independent phosphoproteomic interrogation of human and mouse LRRK2 perturbation datasets further supported the orthologous PFKFB2/Pfkfb2 Ser483/Ser486 region as a conserved LRRK2-responsive phosphosite candidate, including inhibitor-sensitive reduction and inhibitor-resistant retention under A2016T LRRK2 conditions. Together, these findings position the PFKFB2 signalling axis as a cell-state-resolved metabolic bridge linking LRRK2–Rab trafficking programs with the mitochondrial–endolysosomal disease architecture of the Parkinsonian substantia nigra.