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.