Neuroprotective Effects of Two N-Benzylamides in Models of Oxidative Stress, Neurite Outgrowth and Neurodegeneration.

Oxidative stress is a central pathological mechanism in neurodegenerative diseases (NDDs), contributing to mitochondrial dysfunction, impaired proteostasis, and progressive neuronal loss. The sigma-1 receptor (S1R) is an endoplasmic reticulum (ER)-resident chaperone that coordinates cellular stress responses and has emerged as a promising therapeutic target for neuroprotection. In this study, we investigated the neuroprotective properties and mechanisms of the structurally related N-benzylamides, N-benzylcinnamide (NBCA) and N-benzylbenzamide (NBBA) using complementary in vitro, in vivo and in silico approaches. Neuroprotective activity was initially evaluated using glutamate-induced oxidative injury in HT22 hippocampal neurones and H2O2-induced oxidative stress in SH-SY5Y neuroblastoma cells. Both compounds significantly attenuated oxidative injury, although NBCA consistently showed greater efficacy and was therefore selected for detailed mechanistic investigation. NBCA reduced intracellular reactive oxygen species (ROS) production and lipid peroxidation while restoring endogenous antioxidant enzyme activities, mitochondrial membrane potential, ATP production, and cholinergic homeostasis. Immunofluorescence analysis showed that NBCA modulated nuclear factor erythroid 2-related factor 2 (NRF2) and S1R immunoreactivity after oxidative stress. NBCA also promoted neurite outgrowth in wild-type (WT) Neuro-2a cells, whereas these effects were absent in S1R knockout cells, supporting the involvement of S1R-associated signalling. Molecular docking predicted favourable interactions between NBCA and the stress-response proteins S1R, BiP and TMEM97, suggesting modulation of interconnected cellular stress-response networks. In Caenorhabditis elegans, NBCA improved resistance to oxidative stress, preserved learning and memory, delayed amyloid-β-induced paralysis, and reduced amyloid deposition in transgenic Alzheimer's disease (AD) models. Collectively, these findings demonstrate that NBCA exerts neuroprotective effects across multiple experimental models by preserving redox homeostasis, mitochondrial function, and neuronal integrity. The convergence of pharmacological, genetic, imaging, computational, and whole-organism evidence supports the involvement of S1R-associated signalling in NBCA's biological activity. Together, these findings demonstrate that modulation of S1R-associated cellular stress-response networks is a promising strategy for preserving neuronal function during oxidative stress and neurodegeneration and identify NBCA as a valuable lead compound for future mechanistic and therapeutic investigation.