Viral reprogramming of neuronal metabolism captured by label-free imaging in a 3D human brain tissue model.

SIGNIFICANCE: Herpes simplex virus type 1 (HSV-1) is implicated in neurodegenerative risk, yet the dynamic metabolic consequences of infection in human neurons remain poorly defined. Understanding of such bioenergetic adaptations could guide the design of improved interventions. AIM: Our aim is to quantify HSV-1-induced metabolic reprogramming in a three-dimensional human neuronal tissue model using label-free two-photon metabolic imaging. APPROACH: Human-induced neural stem cells matured within silk-collagen scaffolds were infected with low-grade HSV-1 and monitored for 10 days. Two-photon excited fluorescence intensity and fluorescence lifetime imaging quantified the optical redox ratio [FAD/(NAD(P)H + FAD)], NAD(P)H bound fraction, and lipofuscin accumulation. Here, NAD(P)H denotes reduced nicotinamide adenine dinucleotide (phosphate), and FAD denotes flavin adenine dinucleotide. Lactate release and uptake assays complemented optical measurements. RESULTS: Infection induced an early hypermetabolic response characterized by increased glycolysis and oxidative phosphorylation, reflected by shifts in reduced nicotinamide adenine dinucleotide (phosphate) NAD(P)H lifetime components and elevated lactate production. Over time, neurons exhibited lactate reutilization supporting mitochondrial activity, alongside increased lipofuscin signal and altered redox metrics consistent with oxidative imbalance and mitochondrial dysfunction. These data support a model of lactate-associated metabolic adaptation during viral stress. CONCLUSIONS: Endogenous contrast two-photon imaging enables temporally resolved detection of infection-induced metabolic remodeling in human neural tissue models, highlighting optical metabolic imaging as a powerful tool for studying viral contributions to neurodegeneration.