Identifying ROCK2 as an intervention target for bilirubin encephalopathy

Neonatal bilirubin encephalopathy is primarily characterized by central auditory dysfunction and cognitive impairments. However, the precise molecular mechanisms underlying bilirubin-induced neurotoxicity remain poorly understood, hindering the development of effective therapeutic strategies. Using kinase activity prediction tools based on quantitative phosphoproteomics, we identified ROCK2 (Rho-associated protein kinase 2) as a critical kinase regulating the phosphorylation of proteins associated with bilirubin exposure. Molecular docking and MicroScale Thermophoresis assays revealed a strong binding affinity between bilirubin and ROCK2. Interestingly, bilirubin increased ROCK2 protein expression without affecting its mRNA levels, and cycloheximide chase assays revealed enhanced ROCK2 stability, implicating post-translational regulation. While bilirubin did not directly activate ROCK2 kinase activity in vitro, it elevated phosphorylation of its substrate LIMK1, suggesting that ROCK2 accumulation amplifies downstream signaling. In primary rat neurons, ROCK2 inhibitors (Belumosudil and Y-27632) ameliorated bilirubin-induced loss of mitochondrial membrane potential and neuronal cell death. Furthermore, using ROCK2 inhibitors and Rock2^+/- mice, we demonstrated that modulation of ROCK2 significantly alleviated bilirubin-induced auditory deficits and cognitive impairments. These behavioral improvements were linked to restored excitatory synaptic transmission in the cochlear nucleus and reduced dendritic damage in hippocampal neurons. These findings position ROCK2 as a promising molecular target for therapeutic intervention in bilirubin encephalopathy.