Neuroglobin (NGB) is a hexacoordinated hemeprotein mainly expressed in neurons. Following its upregulation and mitochondrial localization, NGB plays a pro-survival role against neuronal stress. Previously, we built a stable NGB-FLAG-overexpressing neuroblastoma cell line and showed that NGB promotes autophagy and localizes in autophagolysosomes. Here we studied the interactome of NGB-FLAG cells to identify novel autophagy-related NGB-binding partners and investigate how its upregulation could induce autophagy. LC3-II and p62 levels as well as mTORC1 activity were analyzed to evaluate autophagy in NGB-FLAG cells. NGB interactors were identified by affinity purification-mass spectrometry and protein-protein interaction network analysis and validated by immunoprecipitation. The increase of LC3-II and decrease of p62 in NGB-FLAG compared to control confirmed that NGB overexpression promotes autophagy. Interactome analysis identified the Regulatory associated protein of mTOR (RPTOR) as one of 134 putative NGB interactors, further validated by immunoprecipitation. NGB overexpression also determined a consistent increment of RPTOR phosphorylation at Ser792 which is required for mTORC1 inhibition, then confirmed by lower levels of phospho-mTOR and phospho-ULK1 in NGB-FLAG compared to control. Collectively, our data suggests that NGB is a positive regulator of autophagy. Through association with RPTOR, NGB may promote its activation and inhibit mTORC1 repressive activity on autophagy initiation.

Proteins tend to misfold upon stressful events that alter their homeostasis, potentially leading to protein aggregation. A tight regulation of synthesis, folding and degradation, defined as proteostasis network (PN), is required to ensure the functionality of the cell. PN is of utmost importance in post-mitotic cells such as neurons, where protein quality must be preserved for their entire lifetime. Most neurodegenerative disorders are associated with dysregulation of this network. Here, we describe the alteration in key components of the PN during chronic stress and link them with the increase in the amyloid burden and with the aggregation of the protein TDP-43, a major player in Amyotrophic Lateral Sclerosis and other neurodegenerative diseases. Neuroblastoma SH-SY5Y cells were treated with a panel of environmental stressors and analyzed after 24 h and 72 h. Treatments resulted in altered PN functionality, including proteasome impairment, halted protein synthesis, engulfed bulk and selective autophagy, in the absence of overt cell death. Thioflavin staining showed increased amyloid burden throughout treatments, associated with phosphorylated TDP-43 (pTDP-43). Biochemical analyses further revealed the cleavage and increased insolubility of pTDP-43. Our results suggest that TDP-43 is a central player during the integrated stress response to chr onic insults and that increased amyloid burden may reflect the global wellfare of a cellular system, pointing toward the alteration of the PN as the main drive for the onset of sporadic neurodegenerative disorders.

Background/Objectives: In this study, we investigated the effect on antioxidant defenses of a tomato extract obtained by supercritical CO₂ extraction (sCO₂TE), evaluating whether this green extraction method preserves biological activity compared to a conventional tomato extract (CTE) and focusing on superoxide dismutase (SOD) and glutathione peroxidase (GPx) regulation, Nuclear factor erythroid 2-related factor 2 (NRF2) activation, reactive oxygen species (ROS) and lipid peroxidation modulation. Methods: Human glioblastoma astrocytoma U-373 cells were pre-treated with sCO₂TE or conventional tomato extract (CTE) and subsequently exposed to sodium arsenite (AsNaO₂) to induce oxidative stress, or lipopolysaccharide (LPS) to trigger inflammatory signaling. Cell viability was assessed by Trypan Blue and MTT [3-(4,5-dimethylthiazolyl-2)-2,5-diphenyltetrazolium bromide]; cell toxicity by propidium iodide staining. Intracellular ROS and lipid peroxidation were measured by flow cytometry. Gene expression of NRF2, SOD1 and GPX1 was analyzed by qRT-PCR, NRF2 activation and modulation of ERK1/2 (Extracellular Signal-Regulated Kinase 1/2) and NF-κB (Nuclear Factor kappa-light-chain-enhancer of activated B cells) were evaluated by Western blot. Results: Pre-treatment with sCO₂TE significantly reduced AsNaO₂-induced ROS production and lipid peroxidation, showing a stronger effect compared to CTE. sCO₂TE enhanced the expression of NRF2 phosphorylation and its downstream targets SOD1 and GPX1, particularly under oxidative stress conditions. In addition, sCO₂TE attenuated LPS-induced phosphorylation of ERK1/2 and NF-κB p65, suggesting anti-inflammatory activity. Conclusions: These findings demonstrate that sCO₂TE preserves the antioxidant and anti-inflammatory properties of tomato-derived bioactives. The comparable efficacy of sCO₂TE and CTE supports the use of sCO₂ as a sustainable and solvent-free extraction method for the development of nutraceutical formulations targeting oxidative stress and neuroinflammation.