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Ischemic stroke, a leading cause of disability and mortality, initiates a complex damage cascade within the neurovascular unit (NVU), leading to blood-brain barrier (BBB) disruption and neuroinflammation that severely exacerbates secondary injury. Neuroglobin (Ngb), an endogenous protein induced by brain injury, represents a high-potential neuroprotective target. While the precise mechanisms underlying its protective action remain incompletely elucidated, substantial evidence points to its multifaceted ability to mitigate ischemic damage. To fully unlock this potential, a fundamental understanding of how neurons, astrocytes, microglia, and pericytes, coordinate their function in response to stress, and specifically identifying the role Ngb plays within this integrated cellular network, is required. This review examines the post-stroke interplay among these cells, analyzing current knowledge about how Ngb modulates the collective inflammatory response by suppressing pro-inflammatory pathways and fostering a neuroprotective environment. Furthermore, Ngb’s upregulation in glial cells and pericytes promotes direct neuronal repair mechanisms, such as neurite outgrowth and axonal regeneration, while supporting neuronal survival and BBB integrity. Importantly, evidence suggests that Ngb’s efficacy is most pronounced when its intracellular concentration exceeds the levels achieved through physiological upregulation. In this regard, we integrate broad preclinical evidence with specific insights from nanoparticle-mediated delivery systems that enable effective Ngb transport to NVU cells. These synthesized findings demonstrate beneficial outcomes in stroke models, driven by the modulation of mitochondrial dynamics, cytoskeletal remodeling, and synaptic regeneration pathways. Collectively, the literature indicates that targeted therapeutic Ngb may enhancement strategies effectively complement endogenous levels to orchestrate protective responses across the NVU. Nonetheless, a detailed investigation into the therapeutic utility of Ngb is still required to fully translate encouraging preclinical findings into successful clinical application for improving stroke outcomes. Graphical Abstract Neuroglobin (Ngb) is an endogenous protein protecting the Neurovascular Unit (NVU) after stroke. This review integrates evidence showing Ngb’s action in glial cells, pericytes, and neurons to suppress inflammation and enhance tissue repair. Systemic Ngb-nanoparticles complement endogenous levels, leading to improved neurological outcomes and underscoring Ngb’s high therapeutic potential

Neuroglobin is an endogenous neuroprotective protein. We determined the safety of direct delivery of Neuroglobin in the rat retina and its effects on retinal inflammatory chemokines and microglial during transient hypoxia. Exogenous Neuroglobin protein was delivered to one eye and a sham injection to the contralateral eye of six rats intravitreally. Fundus photography, Optical Coherence Topography, electroretinogram, histology and Neuroglobin, chemokines level were determined on days 7 and 30. Another 12 rats were subjected to transient hypoxia to assess the effect of Neuroglobin in hypoxia exposed retina by immunohistochemistry, retinal Neuroglobin concentration and inflammatory chemokines. Intravitreal injection of Neuroglobin did not incite morphology or functional changes in the retina. Retinal Neuroglobin protein was reduced by 30% at day 7 post hypoxia. It was restored to normoxic control levels with intravitreal exogenous Neuroglobin injections and sustained up to 30 days. IL-6, TNFα, IL-1B, RANTES, MCP-1 and VEGF were significantly decreased in Neuroglobin treated hypoxic retinae compared to non-treated hypoxic controls. This was associated with decreased microglial activation in the retina. Our findings provide proof of concept suggesting intravitreal Neuroglobin injection is non-toxic to the retina and can achieve the functional level to abrogate microglial and inflammatory chemokines responses during transient hypoxia.

Human neuroglobin (Ngb) is a globin featuring a disulfide bond (Cys46–Cys55) and a redox-active cysteine residue (Cys120) and plays a dual role in cellular stress responses. In this study, we investigated how wild-type (WT) Ngb and its two mutants, C120S Ngb, in which Cys120 is replaced by serine, and A15C Ngb, which contains an engineered Cys15–Cys120 disulfide bridge, modulate oxidative stress in triple-negative breast cancer (MDAMB231) and hormone receptor-positive breast cancer (MCF-7) cells. In both cell lines, WT Ngb enhanced cell survival under H2O2-induced oxidative stress by scavenging reactive oxygen species (ROS) through oxidation of Cys120. In contrast, the C120S and A15C mutants lost this protective capacity and instead promoted apoptosis. Mass spectrometry analysis confirmed the oxidation of Cys120 to sulfenic acid in WT Ngb, whereas both mutants exhibited impaired redox activity, leading to elevated ROS levels, lipid peroxidation, and activation of caspase-9/3. AO/EB staining further revealed that WT Ngb attenuated DNA damage, while the mutants exacerbated apoptosis in both MDAMB231 and MCF-7 cells. These results demonstrate that Cys120 acts as a critical redox switch, dictating whether Ngb exerts cytoprotective or pro-apoptotic effects across different breast cancer cell types. Our findings suggest that WT Ngb may help protect normal tissues during cancer therapy, whereas engineered Ngb mutants could be used to selectively sensitize both triple-negative and hormone receptor-positive breast cancer cells to oxidative damage, offering a novel redox-targeted therapeutic strategy.

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.

Neuroglobin (Ngb) is predominantly expressed in neurons of the central and peripheral nervous systems and it clearly seems to be involved in neuroprotection. Engineering Ngb to observe structural and dynamic alterations associated with perturbation in ligand binding might reveal important structural determinants, and could shed light on key features related to its mechanism of action. Our results highlight the relevance of the CD loop and of Phe106 as distal and proximal controls involved in ligand binding in murine neuroglobin. We observed the effects of individual and combined mutations of the CD loop and Phe106 that conferred to Ngb higher CO binding velocities, which we correlate with the following structural observations: the mutant F106A shows, upon CO binding, a reduced heme sliding hindrance, with the heme present in a peculiar double conformation, whereas in the CD loop mutant “Gly-loop”, the original network of interactions between the loop and the heme was abolished, enhancing binding via facilitated gating out of the distal His64. Finally, the double mutant, combining both mutations, showed a synergistic effect on CO binding rates. Resonance Raman spectroscopy and MD simulations support our findings on structural dynamics and heme interactions in wild type and mutated Ngbs.

Retinal neurodegeneration affects an increasing number of people worldwide causing vision impairments and blindness, reducing quality of life, and generating a great economic challenge. Due to the complexity of the tissue, and the diversity of retinal neurodegenerative diseases in terms of etiology and clinical presentation, so far, there are no cures and only a few early pathological markers have been identified. Increasing efforts have been made to identify and potentiate endogenous protective mechanisms or to abolish detrimental stress responses to preserve retinal structure and function. The discovering of the intracellular monomeric globin neuroglobin (NGB), found at high concentration in the retina, has opened new possibilities for the treatment of retinal disease. Indeed, the NGB capability to reversibly bind oxygen and its neuroprotective function against several types of insults including oxidative stress, ischemia, and neurodegenerative conditions have raised the interest in the possible role of the globin as oxygen supplier in the retina and as a target for retinal neurodegeneration. Here, we provide the undercurrent knowledge on NGB distribution in retinal layers and the evidence about the connection between NGB level modulation and the functional outcome in terms of retinal neuroprotection to provide a novel therapeutic/preventive target for visual pathway degenerative disease.

Previous studies have indicated that paracrine factors (conditioned medium) increase wound closure and reduce reactive oxygen species in a traumatic brain injury in vitro model. Although the beneficial effects of conditioned medium from human adipose tissue-derived mesenchymal stem cells (hMSCA-CM) have been previously suggested for various neurological diseases, their actions on astrocytic cells are not well understood. In this study, we have explored the effect of hMSCA-CM on human astrocyte model (T98G cells) subjected to scratch assay. Our results indicated that hMSCA-CM improved cell viability, reduced nuclear fragmentation, attenuated the production of reactive oxygen species, and preserved mitochondrial membrane potential and ultrastructural parameters. In addition, hMSCA-CM upregulated neuroglobin in T98G cells and the genetic silencing of this protein prevented the protective action of hMSCA-CM on damaged cells, suggesting that neuroglobin is mediating, at least in part, the protective effect of hMSCA-CM. Overall, this evidence suggests that the use of hMSCA-CM is a promising therapeutic strategy for the protection of astrocytic cells in central nervous system (CNS) pathologies.

Investigating the effect of pressure sheds light on the dynamics and plasticity of proteins, intrinsically correlated to functional efficiency. Here we detail the structural response to pressure of neuroglobin (Ngb), a hexacoordinate globin likely to be involved in neuroprotection. In murine Ngb, reversible coordination is achieved by repositioning the heme more deeply into a large internal cavity, the “heme sliding mechanism”. Combining high pressure crystallography and coarse-grain simulations on wild type Ngb as well as two mutants, one (V101F) with unaffected and another (F106W) with decreased affinity for CO, we show that Ngb hinges around a rigid mechanical nucleus of five hydrophobic residues (V68, I72, V109, L113, Y137) during its conformational transition induced by gaseous ligand, that the intrinsic flexibility of the F-G loop appears essential to drive the heme sliding mechanism, and that residue Val 101 may act as a sensor of the interaction disruption between the heme and the distal histidine.

Astrocytes are specialized cells capable of regulating inflammatory responses in neurodegenerative diseases or traumatic brain injury. In addition to playing an important role in neuroinflammation, these cells regulate essential functions for the preservation of brain tissue. Therefore, the search for therapeutic alternatives to preserve these cells and maintain their functions contributes in some way to counteract the progress of the injury and maintain neuronal survival in various brain pathologies. Among these strategies, the conditioned medium from human adipose-derived mesenchymal stem cells (CM-hMSCA) has been reported with a potential beneficial effect against several neuropathologies. In this study, we evaluated the potential effect of CM-hMSCA in a model of human astrocytes (T98G cells) subjected to scratch injury. Our findings demonstrated that CM-hMSCA regulates the cytokines IL-2, IL-6, IL-8, IL-10, GM-CSF, and TNF-α, downregulates calcium at the cytoplasmic level, and regulates mitochondrial dynamics and the respiratory chain. These actions are accompanied by modulation of the expression of different proteins involved in signaling pathways such as AKT/pAKT and ERK1/2/pERK, and may mediate the localization of neuroglobin (Ngb) at the cellular level. We also confirmed that Ngb mediated the protective effects of CM-hMSCA through regulation of proteins involved in survival pathways and oxidative stress. In conclusion, regulation of brain inflammation combined with the recovery of fundamental cellular aspects in the face of injury makes CM-hMSCA a promising candidate for the protection of astrocytes in brain pathologies.

Most neurodegenerative diseases such as Alzheimer's disease are proteinopathies linked to the toxicity of amyloid oligomers. Treatments to delay or cure these diseases are lacking. Using budding yeast, we report that the natural lipid tripentadecanoin induces expression of the nitric oxide oxidoreductase Yhb1 to prevent the formation of protein aggregates during aging and extends replicative lifespan. In mammals, tripentadecanoin induces expression of the Yhb1 orthologue, neuroglobin, to protect neurons against amyloid toxicity. Tripentadecanoin also rescues photoreceptors in a mouse model of retinal degeneration and retinal ganglion cells in a Rhesus monkey model of optic atrophy. Together, we propose that tripentadecanoin affects p‐bodies to induce neuroglobin expression and offers a potential treatment for proteinopathies and retinal neurodegeneration. The natural lipid tripentadecanoin induces the expression of Yhb1 in budding yeast and its orthologue Neuroglobin in mammals. Yhb1 induction prevents the formation of age‐induced protein aggregates and delays aging in yeast. Neuroglobin induction prevents the toxicity of diverse amyloid oligomers in neurons and chemically or genetically induced retinal degeneration in animals.