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Overexpression of Neuroglobin Promotes Energy Metabolism and Autophagy Induction in Human Neuroblastoma SH-SY5Y Cells.

ABSTRACT: Neuroglobin (NGB) is an O₂-binding globin mainly expressed in the central and peripheral nervous systems and cerebrospinal fluid. Previously, it was demonstrated that NGB overexpression protects cells from hypoxia-induced death. To investigate processes promoted by NGB overexpression, we used a cellular model of neuroblastoma stably overexpressing an NGB-FLAG construct. We used a proteomic approach to identify the specific profile following NGB overexpression. To evaluate the role of NGB overexpression in increasing energetic metabolism, we measured oxygen consumption rate (OCR) and the extracellular acidification rate through Seahorse XF technology. The effect on autophagy induction was evaluated by analyzing SQSTM1/p62 and LC3-II expression. Proteomic analysis revealed several differentially regulated proteins, involved in oxidative phosphorylation and integral mitochondrial proteins linked to energy metabolism. The analysis of mitochondrial metabolism demonstrated that NGB overexpression increases mitochondrial ATP production. Indeed, NGB overexpression enhances bioenergetic metabolism, increasing OCR and oxygen consumption. Analysis of autophagy induction revealed an increase of LC3-II together with a significant decrease of SQSTM1/p62, and NGB-LC3-II association during autophagosome formation. These results highlight the active participation of NGB in several cellular processes that can be upregulated in response to NGB overexpression, playing a role in the adaptive response to stress in neuroblastoma cells.

SUBMITTER: Manganelli V

PROVIDER: S-EPMC8699457 | biostudies-literature |

REPOSITORIES: biostudies-literature

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The proteome of neuroglobin-overexpressing SH-SY5Y neuroblastoma cells

Project description:Neuroglobin (NGB) is an oxygen-binding globin expressed in vertebrates mainly in the central and peripheral nervous system, but also in extra-neuronal cells. NGB is involved in oxygen transport, storage, and homeostasis. The role hypothesized for this globin is in fact related to neuroprotection against oxidative stress. Also, NGB is functionally and physically associated to mitochondria, possibly playing a role in ATP production and regulation. NGB actually exerts its function upon up-regulation. Thus, NGB-overexpressing SH-SY5Y neuroblastoma cells (NGB-FLAG) were analyzed by shotgun label-free quantitative proteomics in comparison with control cells (CTRL).

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Overexpression of miR-128 specifically inhibits the truncated isoform of NTRK3 and upregulates BCL2 in SH-SY5Y neuroblastoma cells.

Project description:BACKGROUND: Neurotrophins and their receptors are key molecules in the regulation of neuronal differentiation and survival. They mediate the survival of neurons during development and adulthood and are implicated in synaptic plasticity. The human neurotrophin-3 receptor gene NTRK3 yields two major isoforms, a full-length kinase-active form and a truncated non-catalytic form, which activates a specific pathway affecting membrane remodeling and cytoskeletal reorganization. The two variants present non-overlapping 3'UTRs, indicating that they might be differentially regulated at the post-transcriptional level. Here, we provide evidence that the two isoforms of NTRK3 are targeted by different sets of microRNAs, small non-coding RNAs that play an important regulatory role in the nervous system. RESULTS: We identify one microRNA (miR-151-3p) that represses the full-length isoform of NTRK3 and four microRNAs (miR-128, miR-485-3p, miR-765 and miR-768-5p) that repress the truncated isoform. In particular, we show that the overexpression of miR-128 - a brain enriched miRNA - causes morphological changes in SH-SY5Y neuroblastoma cells similar to those observed using an siRNA specifically directed against truncated NTRK3, as well as a significant increase in cell number. Accordingly, transcriptome analysis of cells transfected with miR-128 revealed an alteration of the expression of genes implicated in cytoskeletal organization as well as genes involved in apoptosis, cell survival and proliferation, including the anti-apoptotic factor BCL2. CONCLUSIONS: Our results show that the regulation of NTRK3 by microRNAs is isoform-specific and suggest that neurotrophin-mediated processes are strongly linked to microRNA-dependent mechanisms. In addition, these findings open new perspectives for the study of the physiological role of miR-128 and its possible involvement in cell death/survival processes.

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Mechanical cues protect against silica nanoparticle exposure in SH-SY5Y neuroblastoma.

Project description:The increasing appearance of engineered nanomaterials in broad biomedical and industrial sectors poses an escalating health concern from unintended exposure with unknown consequences. Routine in vitro assessments of nanomaterial toxicity are a vital component to addressing these mounting health concerns; however, despite the known role of cell-cell and cell-matrix contacts in governing cell survival, these physical interactions are generally ignored. Herein, we demonstrate that exposure to amorphous silica particles destabilizes mitochondrial membrane potential, stimulates reactive oxygen species (ROS) production and promotes cytotoxicity in SH-SY5Y human neuroblastoma through mechanisms that are potently matrix dependent, with SH-SY5Y cells plated on the softest matrix displaying a near complete recovery in viability compared to dose-matched cells plated on tissue-culture plastic. Cells on the softest matrix (3 kPa) further displayed a 50% reduction in ROS production and preserved mitochondrial membrane potential. The actin cytoskeleton is mechanosensitive and closely related to ROS production. SH-SY5Y cells exposed to a 100 μg/mL dose of 50 nm silica particles displayed distinct cytoskeletal aberrations and a 70% increase in cell stiffness. Overall, this study establishes that the mechanical environment can significantly impact silica nanoparticle toxicity in SH-SY5Y cells. The mechanobiochemical mechanisms behind this regulation, which are initiated at the cell-matrix interface to adjust cytoskeletal structure and intracellular tension, demand specific attention for a comprehensive understanding of nanotoxicity.

| S-EPMC7877221 | biostudies-literature

Metabolomic Responses to Manganese Dose in SH-SY5Y Human Neuroblastoma Cells.

Project description:Manganese (Mn)-associated neurotoxicity has been well recognized. However, Mn is also an essential nutrient to maintain physiological function. Our previous study of human neuroblastoma SH-SY5Y cells showed that Mn treatment comparable to physiological and toxicological concentrations in human brain resulted in different mitochondrial responses, yet cellular metabolic responses associated with such different outcomes remain uncharacterized. Herein, SH-SY5Y cells were examined for metabolic responses discriminated by physiological and toxicological levels of Mn using high-resolution metabolomics (HRM). Before performing HRM, we examined Mn dose (from 0 to100 μM) and time effects on cell death. Although we did not observe any immediate cell death after 5 h exposure to any of the Mn concentrations assessed (0-100 μM), cell loss was present after a 24-h recovery period in cultures treated with Mn ≥ 50 μM. Exposure to Mn for 5 h resulted in a wide range of changes in cellular metabolism including amino acids (AA), neurotransmitters, energy, and fatty acids metabolism. Adaptive responses at 10 μM showed increases in neuroprotective AA metabolites (creatine, phosphocreatine, phosphoserine). A 5-h exposure to 100 µM Mn, a time before any cell death occurred, resulted in decreases in energy and fatty acid metabolites (hexose-1,6 bisphosphate, acyl carnitines). The results show that adjustments in AA metabolism occur in response to Mn that does not cause cell death while disruption in energy and fatty acid metabolism occur in response to Mn that results in subsequent cell death. The present study establishes utility for metabolomics analyses to discriminate adaptive and toxic molecular responses in a human in vitro cellular model that could be exploited in evaluation of Mn toxicity.

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