Mapping methionine-driven metabolic reprogramming in ALS via bioorthogonal D2O-SRS and 2PEF imaging

Amyotrophic lateral sclerosis (ALS) is a motor neuron disease characterized by progressive paralysis and eventual death due to the degeneration of both upper and lower motor neurons. Recent research suggests that overproduction of reactive oxygen species (ROS) in malfunctioning mitochondria coupled with weakened antioxidant defenses, may drive disease progression. Lmethionine (Met) is known to support cellular metabolism and activate endogenous antioxidant enzymes. We hypothesized that supplementation with methionine could enhance antioxidant defenses in an in vitro ALS model and help control disease progression. In this study, we employed an optical imaging strategy combining bioorthogonal deuterium oxide (D₂O)-probed stimulated Raman scattering (DO-SRS) with two-photon excitation fluorescence (2PEF) microscopy to directly observe how a methionine-enriched diet affects oxidative stress and cellular metabolism in neurodegenerative cells. Our results indicate that excess methionine promotes the synthesis of lipids and unsaturated lipid membranes while reducing protein synthesis and oxidative stress. Additionally, this regulation increased both the size and abundance of lipid droplets while reducing stress granule volume, suggesting a shift toward lipid-based stress adaptation These findings suggest that methionine supplementation may protect against oxidative imbalance and support cellular repair in cellular neurodegenerative disease models.