Nuzzo, Maria Teresa
Role of estrogen-induced neuroglobin expression in neurodegeneration and neuroprotection [Tesi di dottorato]
Università degli studi Roma Tre, 2016-02-15

Since its discovery, Neuroglobin (Ngb) has received great attention by the scientific community for its peculiar functions. Even though it is a member of the globin family, Ngb displays a low degree of homology with other globins including Myoglobin (Mb) and Hemoglobin (Hb), only sharing with them the quaternary structure and the capability to bind to oxygen and other gaseous ligands. On the other hand, Ngb is a hexa-coordinated globin, whereas the other members of the globin family show a penta-coordination, index of a highest reactivity. Furthermore Ngb exhibits characteristic enzymatic properties. Several works focused on the definition of Ngb functions, starting from its localization inside the cell. Although the molecular mechanism(s) involved is(are) yet matter of debate, as a whole Ngb is considered a protein whose over-expression results protective in the nervous system against a panel of pathologic conditions, including neurodegenerative diseases. Recently our laboratory have reported that 17?-estradiol (E2), the most active between estrogens, is an endogenous modulator of Ngb. In fact, E2 is able to promote in several cells of the nervous system (i.e., hypothalamic neurons, astrocytes, human neuroblastoma cell lines), via ER?, Ngb over-expression and binding to mitochondrial cytochrome c, a crucial event against oxidative stress-induced apoptosis. E2 is a steroid hormone with pleiotropic effects on the entire organism playing roles that encompass from the control of reproductive functions to advanced cerebral functions. Estrogen receptors (ERs) are ligand activated transcription factors, so operating genomically, but also involved in rapid mechanisms, activated when they are localized at the plasma membrane. There are two estrogen receptors subtypes (i.e., ER? and ER?), responsible for opposing functions and displaying a different expression pattern among tissues. To further complicate the picture, E2 finely tunes their expression, so modulating its effects in target cells. As an example, E2 promotes the cell proliferation via ER?, whereas ER? activities are responsible for the apoptotic cell death. On the other hand, in the nervous system, both ERs activation is required for neuron survival. Thus, both receptor subtypes mediates the E2 neuroprotective and neurotrophic effects, working against a series of brain damages, as well as responsible for sexual differences in the onset of neurodegenerative disorders and the gravity of their symptoms. Previous data obtained in our laboratory sustain the idea that one of the mechanisms by which E2 acts as a neuroprotective hormone is the promotion of high Ngb level into mitochondria which, in turn, is fundamental for Ngb anti-apoptotic activity. In this thesis this idea has been further enlarged by characterizing E2-induced Ngb over-expression in mitochondria both in neuroprotection and in neurodegeneration. This information will be useful to better define the signaling pathways at the root of E2-induced Ngb over-expression that can be used to discover novel molecules able to maintain Ngb up-regulation avoiding the side effects linked to the E2 treatment. In particular, our project has been mainly focused in discovery the protein that could realize Ngb transport to mitochondria upon E2 stimulus. For the first time it has been ascertained that E2 enhances Huntingtin (Htt) expression both in vitro (human neuroblastoma cells SK-N-BE) and in vivo (striatal and hippocampal rat tissues), thanks to rapid (i.e., Akt activation) and long-term (transcription) ER?-dependent pathways. Htt is a ubiquitous protein with several functions vital for the cell survival attributed to, and so also considered a neuroprotectant protein. Htt involvement in anti-apoptotic E2/Ngb process has been verified by several steps: (i) E2 requires Htt to promote Ngb expression; (ii) Htt and Ngb interact, and this binding is strongly induced by E2; (iii) Htt realizes E2-induced Ngb transport from nucleus to mitochondria upon oxidative stress conditions; (iv) Htt is important for E2-induced and Ngb mediated cellular protection against apoptosis. These results allowed to define a novel neuroprotective axis (i.e., E2/Htt/Ngb), active under oxidative stress conditions. In order to verify if this axis is active even under neurodegenerative conditions its existence in Huntington’s Disease (HD) models has been tested. HD is a neurodegenerative dominant autosomal pathology caused by a mutation in the Htt codifying gene. This mutation determines the development of an oversized glutamine tract (PolyQ) in the N-terminal region of Htt, so losing its physiological activities and also acquiring toxicity, hence resulting in the neurodegeneration for striatal neurons at the first, and other brain regions with the progression of pathology. Estrogenic induction of Htt expression levels is active only for the wild type form (wt) of the protein, whereas E2 effects were completely impaired in the hippocampus of 13 weeks old R6/2 transgenic mice and in the murine cell lines expressing mHtt (STHdhQ111 striatal cell line). Additionally, wtHtt is required for E2 promotion of its interaction with Ngb and for Ngb migration to mitochondria. As a consequence, the presence of mHtt also impairs the anti-apoptotic effect usually guaranteed by E2/Htt/Ngb axis, clearly inoperative under neurodegenerative conditions. These results show that only the wt form of Htt takes part of the cellular defensive system elicited by E2, whereas upon mutation Htt loses these beneficial functions. Thus, the discovery of drugs able to increase Ngb levels in mitochondria, mimicking E2 effects, could be promising for the development of treatment of neurodegenerative diseases. This substance could so guarantee neuronal survival against neurodegenerative processes. The screening involved compounds known from literature as Ngb inductors (i.e., valproic acid and cinnamic acid), natural estrogen-mimetic compounds (i.e., Resveratrol and Naringenin), the selective ER? ligand dyarilpropionitrile (DPN), the brain neurotrophic factor BDNF and the environmental pollutant Bisphenol A (BPA). Both Naringenin and DPN (specific ligands of ER?) satisfy the research requests. Previous studies from literature pointed the therapeutically potential of ERs ligands, further supporting the results here reported indicating that DPN, selectively activating ER?, enhances Ngb expression levels, without exerting any parallel influence on Htt levels, which requires ER? activation. This induction is sufficient for the protection against programmed cell death upon oxidative stress, as Ngb over-expression is preserved. The Ngb anti-apoptotic function has been attributed to various functions Ngb potentially exerts in different cellular sites. This is the reason why it has been evaluated Ngb localization consequent to DPN administration. Results show that DPN, like E2, carries Ngb to mitochondria without any Htt involvement, unlike E2. As a whole, these data confirm the pivotal role played by Ngb in neuroprotective effects of E2, highlighting the importance of Ngb over-expression. In addition, the principal results obtained during this three year project were: (i) the discovery of a novel neuroprotective axis (i.e., E2/Htt/Ngb), active under oxidative stress conditions; (ii) the loss of this beneficial axis activities during neurodegeneration; (iii) the description of novel beneficial functions for Htt protein which are loss upon its mutation; (iv) the discovery that the activation of Htt/Ngb pathway is shared by several substances with neuroprotective effects; (v) the discovery that ERs activate diverse and parallel pathways to guarantee neuroprotection; and (vi) the identification of a compound that preserves E2/Ngb anti-apoptotic results (i.e., DPN) specifically activating only ER? pathway. As a whole, these data elucidate the role of E2-induced neuroglobin expression in neurodegeneration and neuroprotection opening a new avenues in the field of neurodegeneration treatment with the discovery of DPN protective action.

diritti: info:eu-repo/semantics/openAccess
Marino, Maria

Tesi di dottorato. | Lingua: en. | Paese: | BID: TD18017412