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

Abstract Aberrant signaling by leucine-rich repeat kinase 2 (LRRK2) drives Parkinson’s disease (PD) biology, linking Rab phosphorylation to endolysosomal dysfunction. However, how LRRK2-linked trafficking intersects with metabolic remodeling within vulnerable human substantia nigra cell states remains poorly defined. Here, phosphatome-wide systems analysis, single-nucleus transcriptomics, structural modeling, and independent phosphoproteomic evidence were integrated to identify the glycolytic regulator PFKFB2 as a phosphorylation-associated metabolic node. Sample-level pseudobulk profiling of a human substantia nigra single-nucleus RNA-seq data set revealed distributed phosphatome remodeling across the PD spectrum, prioritizing PFKFB2 as a transcriptionally reduced, 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 and neuronal loss. Stratification by PFKFB2 expression revealed marked transcriptional polarity: PFKFB2-positive cellular states were enriched for LRRK2–Rab trafficking and lysosomal programs, whereas PFKFB2-negative states retained mitochondrial/OXPHOS signatures; paired donor-pseudobulk analysis confirmed that the principal PFKFB2-associated PDD microglial program was reproducible across donors. Structurally, AlphaFold-guided peptide docking identified the flexible 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 data sets supported the orthologous PFKFB2/Pfkfb2 Ser483/Ser486 region as an LRRK2-responsive phosphosite candidate, exhibiting inhibitor-sensitive reduction and inhibitor-resistant retention under A2016T LRRK2 conditions. Together, these findings position the PFKFB2 signaling axis as a cell-state-resolved metabolic interface linking LRRK2–Rab trafficking with the mitochondrial–endolysosomal disease architecture of the Parkinsonian substantia nigra.