BH4 Oxidation-Derived H₂O₂ Activates ERK1/2 Signaling via B-Raf in Rat Dorsal Root Ganglion Neurons

Elevated Tetrahydrobiopterin (BH4) levels are linked to various pain conditions. Pharmacological or genetic reduction of BH4 levels has analgesic effects in rodent models of neuropathic and inflammatory pain, but also affects neurological and cardiovascular functions. Little is known about the downstream mechanisms of BH4 that could be targeted to attenuate BH4-induced pain hypersensitivity without lowering BH4 levels. In this study, we exposed ex vivo-cultured rat dorsal root ganglion (DRG) neurons to BH4 and analyzed the activity of the sensory neuron-sensitizing kinase ERK1/2 via high-content imaging. We show that BH4 exposure leads to increased pERK1/2 levels in a dose- and time-dependent manner. Interestingly, we found that H₂O₂, as a by-product of BH4 oxidation and not BH4 itself, induces increased pERK1/2 levels via MEK1/2 and B-Raf (but not A-Raf or C-Raf) and that this can be blocked by pharmacological interference. In conclusion, elevated BH4 levels, as observed in various pain conditions, may drive sensory neuron sensitization via oxidation-derived H₂O₂ and the B-Raf-MEK1/2-ERK1/2 axis, which presents a novel pathway that could be targeted to attenuate BH4-induced pain hypersensitivity without the necessity to reduce BH4 levels.