Phosphoproteomics Profiling Reveals Key Proteins Involved in Neuroinflammation and Impaired Axon Guidance Induced by Bilirubin Deficiency

Hypobilirubinemia, characterized by low bilirubin levels, is increasingly recognized as a pathological condition linked to various neurodevelopmental and neurodegenerative diseases. However, the neuropathological mechanisms of hypobilirubinemia remain unclear. In this study, we aimed to elucidate the potential molecular mechanism of neuronal structural and functional damage through integrated phosphoproteomics and proteomics analysis. Proteomic analysis of brain tissues from Biliverdin reductase-A knockout (Blvra^-/-) mice and wild-type (WT) mice identified 133 differentially expressed proteins. Proteins with decreased abundance were enriched in axonogenesis, while proteins showing increased abundance were primarily involved in the PI3K-Akt signaling pathway, including LAMA4 and ITGA1. Phosphoproteomic analysis revealed 390 proteins with decreased phosphorylation at 542 sites, while 82 proteins had increased phosphorylation at 96 sites. Proteins associated with phosphosites showing decreased phosphorylation were enriched in neurogenesis and the axon guidance pathway, while those associated with increased phosphorylation sites were linked to neuronal apoptosis. Western blotting validation confirmed the modulation of key proteins, such as DPYSL2, MAPK8, and PRKCD, within the protein-protein interaction network of differentially expressed phosphorylated proteins, which have previously been implicated in neuronal development and degeneration. Golgi staining further revealed reduced number of dendritic intersections, branch points, shortened neurite length, and decreased dendritic complexity in Blvra^-/- mice compared to WT mice. These results indicated that low bilirubin levels disrupt brain protein phosphorylation regulatory networks, which may drive neuroinflammation, promote neuronal apoptosis, and impair neuronal growth, potentially contributing to neurodevelopmental and neurodegenerative diseases.