Neuroglobin (Ngb) is a nerve hemeprotein that acts as a neuroprotectant against multiple brain injuries, including oxidative stress and β-amyloid toxicity. Besides inhibiting apoptosis and scavenging ROS/RNS, Ngb overexpression promotes autophagy, a highly conserved catabolic process essential for maintaining cellular homeostasis under both basal and stress conditions. Although Ngb upregulation has proven effective in animal models of neurological diseases, its effects have not yet been studied in three-dimensional (3D) cell cultures, which better mimic in vivo tissue architecture than monolayers. Therefore, in the present work we developed a simple yet reliable 3D model of Ngb overexpression using human SH-SY5Y neuroblastoma cells and examined its response to 1-methyl-4-phenylpyridinium (MPP+), a well-established in vitro model of Parkinson's disease (PD)-related neurodegeneration. Control and Ngb-overexpressing spheroids were generated using the hanging drop method. Ngb overexpression decreased MPP + -induced ROS production and cell death, thus validating our 3D model. Additionally, biochemical fractionation and Thioflavin-S staining showed that Ngb upregulation reduces MPP + -induced impairment of autophagy and amyloid accumulation, key pathological features of neurodegeneration. Overall, our findings emphasize the importance of 3D cultures for studying Ngb-mediated neuroprotection and suggest that Ngb upregulation may prevent the disruption of neuronal proteostasis under neurotoxic stress.
