Dottorato in MEDICINA SPERIMENTALE

Titolo della tesi: Investigation of the neuroprotective mechanisms of Neuroglobin in human neuroblastoma SH-SY5Y cells

For a long time, Hb and Mb had been considered the only globin types present in vertebrates. In 2000, through database mining of genome-sequencing data a third vertebrate globin member was identified in human and mouse brain, hence named neuroglobin (Ngb). Numerous in vitro and in vivo evidence proved that Ngb upregulation plays a pro-survival role against neuronal damage, whereas decreased Ngb levels exacerbate brain injury. It has been reported to inhibit the intrinsic apoptotic pathway by multiple mechanisms as well as to act as ROS and RNS scavenger, thereby reducing oxidative stress. Currently, although our understanding of Ngb cytoprotective mechanisms has been extensively increased, it remains largely limited to its ability to inhibit cell death upon neurotoxic stress. Thereby, the aim of this project is to investigate whether Ngb may promote neuronal survival by modulating additional cellular processes beyond its anti-apoptotic role. Proteome analysis of our established human Ngb-overexpressing neuroblastoma SH-SY5Y (NGB-FLAG) cell line revealed an increased expression of both mitochondrial structural proteins and oxidative phosphorylation enzymes along with elevated levels of transmembrane lysosomal proteins. Based on these findings, we first investigated whether Ngb overexpression influenced mitochondrial function. Cell energy metabolism analysis, performed using Seahorse technology, showed an increment in both mitochondrial respiration and ATP production. The concomitant increase in mtDNA copy number and NRF1 mRNA levels overall suggested that Ngb overexpression may promote the maintenance of a large pool of mitochondria with an increased respiration rate. Next, through proteomics and protein-protein interaction analysis along with co-immunoprecipitation experiments, we demonstrated that overexpressed Ngb associates with LC3 on autolysosomes, and also that, via Raptor activation, it may reduce mTORC1-mediated inhibitory signaling on ULK1 complex, thereby promoting autophagy. In parallel, we investigated the neuroprotective effect of Ngb overexpression in three-dimensional cell cultures, which were obtained by hanging drop method and then treated with the mitochondrial toxin 1-methyl-4-phenylpyridinium ion (MPP+). Overall, Ngb overexpression was shown to reduce MPP+-induced oxidative stress and apoptotic cell death, as well as to prevent amyloid protein accumulation. Conclusively, our findings confirm that Ngb overexpression enhances mitochondrial metabolic activity and, for the first time ever, demonstrate that Ngb actively promotes autophagy. Furthermore, Ngb-overexpressing spheroids appear to provide a valuable tool for further investigations of neuroprotective activity of this globin. Functional data on oxidative stress and cell death experimentally confirmed their reliability, while the observation of reduced amyloid protein accumulation may lay the foundations for studying the effects of Ngb upregulation on more specific neurodegenerative processes.