Please use this identifier to cite or link to this item: http://hdl.handle.net/2307/4585
Title: Neuroglobin : molecular,cellular and biomedical aspects
Other Titles: Neuroglobina: aspetti cellulari, molecolari e biomedici
Authors: De Marinis, Elisabetta
metadata.dc.contributor.advisor: Ascenzi, Paolo
Keywords: neuroglobin
receptor
estroben
Issue Date: 12-Dec-2011
Publisher: Università degli studi Roma Tre
Abstract: Although globins are among the best-investigated vertebrate proteins, no other distinct types of globins have been identified so far in this taxon. In 2000, it was identified a third globin type in humans and rodents. This protein was predominantly expressed in the brain, and therefore they have called it neuroglobin (Ngb). The discovery of Ngb aroused a great interest among scientific community inducing to consider heme-globins not only as mere O2 storage/delivery proteins. Ngb is a highly conserved protein, with an evolutionary rate that is about threefold slower than that of myoglobin and hemoglobin. Thus, Ngb has remained largely unchanged during evolution, pointing to an important role of this protein. In particular, an important role in neuroprotection has been addressed to Ngb, especially against ischemia and oxidative stress-related neurodegenerative diseases, but many divergences between in vivo and in vitro experimental approaches still render unclear the biological role of this novel globin. Several mechanisms underlying Ngb neuroprotective effects have been proposed. Indeed, Ngb has been hypothesized: (i) to act as an O2 buffer, (ii) to facilitate O2 diffusion to the mitochondria, (iii) to catalyze the formation and the decomposition of reactive nitrogen and/or oxygen species, and (iv) to be part of intracellular signaling pathways by inhibiting the dissociation of GDP from Gα proteins and triggering the release of the Gβγ complex, and by reducing cytochrome c. Although it is unlikely that Ngb has so many distinct roles, there is no doubt that Ngb displays a protective function(s) in the brain. The emerging neuroprotective role of Ngb arises the challenge to investigate the mechanisms able to modulate its expression. Indeed, a significant contribution to highlight the role played by Ngb in neuroprotection could derive from the identification of Ngb endogenous modulator(s) (e.g., neuroactive hormones and neurotransmitters), but, as far as we know, no Ngb involvement in the hormone signal transduction pathways has been identified yet. Thus, aim of this project is to approach to the knowledge of Ngb physiological role (i) identifying Ngb endogenous modulator(s), (ii) identifying the molecular mechanisms responsible of Ngb expression and induction, and (iii) the role played by Ngb in neuroprotective signaling pathways. In the first part of the project was evaluated if sex steroid hormones may act as endogenous modulators of Ngb levels in SK-N-BE human neuroblastoma cell line and in mouse primary hippocampal neurons. The reported results indicate that physiological concentration of the estrogen 17β-estradiol (E2), but not androgens, acts as endogenous modulator of Ngb in both cell models. This effect is mediated by estrogen receptor β (ERβ) via genomic and extranuclear signals involving p38/MAPK pathway. In the second part, the involvement of Ngb in the neuroprotective effects of E2 against H2O2-induced toxicity has been investigated in SK-N-BE cells. Indeed, E2 exerts a protective effect against the H2O2-induced injury, and requires ERβ. E2 pretreatment impairs H2O2-induced caspase-3 and PARP activation, enhancing cell viability. However, in Ngb-silenced SK-N-BE cells E2 was unable to counteract the H2O2-induced decrease in cell number and the activation of the pro-apoptotic cascade suggesting that Ngb can be regarded as part of signals activated by E2 to exert neuroprotective effects, definitely validating the role played by Ngb as an anti-apoptotic neuroprotective globin. Thus, it has been clarified the Ngb sub-cellular localization to understand how this novel globin can intercept the apoptotic pathway. Therefore, in SK-N-BE cells has been demonstrated that Ngb is expressed in the nucleus, mitochondria and is scattered in the cytoplasm. E2 reallocates Ngb mainly at mitochondria strengthened the hypothesis that Ngb directly interferes with the intrinsic pathway of apoptosis, being mitochondria just the starting site of this process. Indeed, it has been assessed that Ngb co-immunoprecipitates with cytochrome c in mitochondrial fraction and this association is enhanced pretreating SK-N-BE cells with E2, suggesting that E2-induced reallocation of Ngb facilitates Ngb-cytochrome c interaction. Remarkably, E2 pretreatment before the addition of H2O2 strongly enhances Ngb co-immunoprecipitation with cytochrome c. This E2 effect is stronger during oxidative stress condition rather than in basal condition and requires ERβ activity. Thus, the mechanism underlying Ngb protection against H2O2 stress is the interception of the intrinsic pathway of apoptosis interfering directly with cytochrome c release. In the light of Ngb neuroprotective potential, linked with E2-mediated signals, the third part of the project was aimed to characterize the E2-mediated regulation of Ngb levels in astrocytes, where E2 exerts a well known anti-inflammatory effect. In mouse primary cortical astrocytes E2 affects Ngb expression at physiological concentration. The effect of E2 on Ngb levels specifically requires ERβ, confirming also in astrocytes the direct involvement of ERβ in Ngb modulation, as already reported for human neuroblastoma SK-N-BE and mouse hippocampal neurons. Although it has been established that Ngb is an E2-inducible protein and that, from a functional point of view, the E2-mediated Ngb upregulation allows to promote the E2-induced outcomes, also a putative role of Ngb as a compensatory protein responding to challenging stimuli, must be considered. The finding that lipopolysaccharide (LPS) is able to increase Ngb protein levels, although with a lesser degree compared with E2, further provides an additional contribution to understand the role of Ngb also as offsetting protein. Interestingly, although both E2 and LPS are able to increase Ngb protein levels, a negative cross-talk between ERs and LPS-induced signal (i.e., NFκB) seems to be present. In fact, ERα-activated signals (which are not involved in E2-mediated Ngb upregulation) block LPS-mediated Ngb increase, whereas on the other hand, LPS impairs the ERβ-induced upregulation of Ngb protein levels. Therefore, the co-activation of ERα and ERβ is pivotal to regulate Ngb expression in presence of LPS-activated signals (i.e., NFκB). Despite LPS, via NFκB, is able to increase Ngb levels, the role of this globin is not addressed to promote LPS effects, as observed for E2. Indeed, Ngb seems to be pivotal to mediate the E2 anti-inflammatory effects (i.e., inhibition of IL-6 and IP-10 synthesis), since Ngb knocking down prevents the protective effect of E2. As a whole, the well known neuroprotective effects elicited by E2 may, at least in part, be explained by an enhanced Ngb expression in neurons and astrocytes. The principal role played by Ngb in the brain could be the reduction of neuronal death by resetting the trigger level of apoptosis and inhibition of pro-inflammatory molecules expression, leading to the onset of physiological response to stress. E2 acts to accelerate Ngb neuroprotective effect rapidly enhancing its protein levels in both neurons and astrocytes. In addition, the possibility that other hormones and neurotransmitters may upregulate Ngb levels in brain a potential new opportunity for the development of neuroprotective strategies and drugs against stroke damage, inflammation, neurodegenerative diseases (e.g., Alzheimer’s and Parkinson’s disease), excitotoxicity, and injuries related to oxygen or glucose deprivation.
URI: http://hdl.handle.net/2307/4585
Access Rights: info:eu-repo/semantics/openAccess
Appears in Collections:X_Dipartimento di Biologia
T - Tesi di dottorato

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