Monoclonal antibodies against nitrated nerve growth factor reveal an oxidation-dependent pathogenic hallmark in ALS

Abstract

Nerve growth factor (NGF) is a member of the neurotrophin family, essential for neuronal survival and phenotypic maintenance. However, in vitro, its function can be disrupted by oxidative posttranslational modifications such as tyrosine nitration. Nitrated NGF (NO 2 NGF) has been shown to have a gain-of-toxic, pro-apoptotic, activity in motoneuron cultures. Herein, we report the generation and characterization of monoclonal antibodies (mAbs) that specifically recognize NO 2 NGF to unravel its formation in vivo. Using hybridoma technology, we produced mAbs with high affinity and selectivity for NO 2 NGF, as demonstrated immunochemically and by surface plasmon resonance. The antibodies elicit neutralizing capacity to NO 2 NGF in neuronal cells. Nitrated Tyr52 within the NGF 48–58 sequence was identified as the primary antigenic determinant by crystallographic analysis of antibody:peptide complexes at atomic resolution, peptide-based epitope mapping and molecular dynamics simulations, whereas local sequence NGF motifs around the nitrated tyrosine are important for protein specificity. The antibodies revealed NO 2 NGF accumulation in amyotrophic lateral sclerosis (ALS) rodent models and human subjects. Indeed, immunofluorescence showed selective accumulation of NO 2 NGF in spinal cord regions undergoing motor neuron degeneration, as well as in sciatic nerves and neuromuscular junctions. Our findings establish NGF nitration as an oxidative hallmark in ALS and demonstrate that monoclonal antibodies targeting this chemical modification are powerful tools for both mechanistic studies and biomarkers development. This work proposes a link between neurotrophin nitration and neurodegenerative disease progression and opens avenues for therapeutic exploration along the peroxynitrite-tyrosine nitration pathway.