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Identifier 000397236
Title Study of the neuroprotective and neurogenic actions of synthetic micromolecules in animal models of neurodegeneration
Alternative Title Μελέτη των νευροπροστατευτικών -νευροαναγεννητικών δράσεων νέων συνθετικών μικρομορίων σε ζωικά μοντέλα νευροεκφύλισης
Author Ευσταθόπουλος, Πασχάλης
Thesis advisor Gravanis, Achilleas
Select a value Charalampopoulos, Ioannis
Vekrellis, Konstantinos
Abstract The adult mammalian brain possesses only a limited capacity for regeneration under pathological conditions that cause neuronal cell loss. However, the identification of endogenous adult neural stem cells (aNSCs) that can proliferate and differentiate into functional neurons, unveil new intriguing possibilities towards restoration of brain function in case of a disease. aNSCs that lie mainly in the area of the dentate gyrus (DG) and in the subventricular zone (SVZ) are generated continuously and after they differentiate they get integrated to the existing circuit. These cells are responsive to intrinsic as well as external stimuli that affect their proliferation, their survival and differentiation. Such molecules are neurotrophins such as Nerve growth factor (NGF) and Brain derived neurotrophic factor (BDNF), as well as other growth factors such as hormones, which are also responsible for the patterning of nervous system during development. Increasing neurogenesis with genetic or pharmacological manipulation improves performance on various cognitive tasks while it has been recently shown that there are specific types of functions of the DG that require the integration of new neurons such as pattern separation. Neurogenesis has been implicated in the pathophysiology of many neurodegenerative and neuropsychiatric diseases. Alterations in neurogenesis have been observed in various animal models of Alzheimer’s disease (AD) and have been linked to accumulation of aβ amyloid and the development of neuroinflammation. On the other hand stimulating adult hippocampal neurogenesis in these models either by means of environmental enrichment, physical exercise or with pharmacological ways reduces amyloid pathology and improves cognitive function. Finally, adult neurogenesis is dramatically reduced during ageing and pharmacological agents that succeed to restore this decline were also effective to alleviate age-related memory deficits. Nevertheless, the clinical use of many molecules that have been shown to possess neurogenic properties is often problematic either because of their peptidic nature and their inability to cross the blood brain barrier or because they accompanied by off target adverse effects. For this reason, modern pharmacology is seeking of small lipophilic molecules, which can mimic the actions of endogenous growth factors and show good availability to the CNS as well as a good safety profile. In this study I have tested the neurogenic potential of two small molecules that belong to two different classes. One of them is fingolimod that has been approved for the treatment of multiple sclerosis based on its ability to inhibit the egress of lymphocytes from lymphoid nodes, thus preventing its migration to the site of inflammation. It acts through activation of 5 subtypes of S1P receptors that belong to GPCR superfamily but also as an intracellular messenger itself by regulating the expression of many genes such as BDNF and other growth factors. However, S1P is also synthesized de novo in the CNS and S1P receptors are abundantly expressed in various regions and by a plethora of cell types in the CNS and the therapeutic effects of fingolimod have been tested for a wide range of CNS disorders, from neurodegenerative to neuropsychiatric conditions. Intrigued by the role of S1P during development but also in the maintenance of an intact nervous system I sought to determine if there are any effects of the -already in clinical use- agonist of S1PRs, fingolimod in regulation of adult neurogenesis, which may contribute to its beneficial actions. My results show that fingolimod can induce the proliferation and survival of adult hippocampal progenitors in vivo and in vitro through activation of MAPK pathway and induction of BDNF while it affects behavior that have been associated with the functional role of adult neurogenesis. The other molecule that it was tested for its ability to induce adult neurogenesis was a synthetic analogue of the endogenous neurosteroid Dehydroepiandrosterone (DHEA), called BNN27. DHEA is synthesized in the periphery from the adrenal glands, but also is synthesized de novo and secreted locally in the CNS in high concentration. DHEA has been shown to promote neuronal survival in various models of neurodegeneration both in vivo and in vitro through divergent mechanisms and recent data from our laboratory support the idea that some of these effects are mediated by activation of NGF receptors (namely TrkA and p75NTR). However, the therapeutic use of DHEA is compromised because it widely affects the endocrine system by its own or after conversion to other steroid hormones with possible tumorogenic action. In particular breast, endometrial, or prostate cancers have been associated with disturbances on estrogens and androgen levels. Moreover the exact actions of DHEA cannot be distinguished from its metabolites. BNN27, has been developed by modifications of DHEA structure in order to sustain its neuroprotective properties and avoid its further metabolism to androgens and estrogen. In this way, BNN27 lacks of the possible tumorogenic potential of the parent molecule, DHEA. In order to test whether BNN27 could reproduce the anti-apoptotic effects of DHEA mediated by activation of NGF receptors’ initiated signaling, we used cultures of sympathetic neurons that are known to be dependent on NGF for their survival as well as NGF null mice embryos that exhibit massive cell death in the DRGs during the embryonic development. BNN27 was found efficient to reduce apoptosis due to NGF deprivation in both cases. Based on the neuroprotective actions of BNN27 I further sought to investigate if this molecule could support neurogenesis in the adult rodent hippocampus. BNN27, was not effective in inducing either proliferation or survival of neural stem cells in the area in WT mice nor did it reversed neurogenic deficits in old animals. However, long-term administration of BNN27 ameliorated the neurogenic and cholinergic deficits in an animal model of amyloidosis, the 5XFAD mice, while it also reduced the amyloid burden. One the other part of this study I investigated the effects of microconically patterned silicon substrates on the behavior of peripheral nervous system neurons and glial cells. Directed axonal outgrowth is necessary for nerve regeneration after an injury but also in a broad range of applications in neuroscience such as construction of microfluidic devices or neuronal interfaces. Among the factors that control the orientation of a regrowing axon are cellular cues and substrate topography. Based on their design, different topographies that have been tried so far to manipulate cell growth, migration, and differentiation of various cell types could be described by the following geometries: continuous and discontinuous. These geometries could be further classified based on the directionality as anisotropic, in which cues are provided along a single axis or isotropic topographies which are uniform in all directions, providing cues along multiple axesExamples of continuous topographies are photolithographically fabricated grooved silicon substrates or electrospun polymer fibers at parallel or random orientation, while discontinuous geometries include silicon or gold pillars or posts. Recently the bioengineering lab in IESL in FORTH developed and characterized micropatterned silicon culture substrates fabricated with the use ultra-fast pulsed laser structuring. Upon increasing laser fluence surface roughness is also increased and acquires anisotropic geometrical characteristics. More specifically as we pass from flat silicon to surfaces that display a higher degree of roughness, substrates are comprised of microcones with elliptical shape and specific orientation. Surface roughness and wettablity of the micropatterned Si substrates, influence fibroblast adhesion as well as differentiation capacity of PC12 cells as response to NGF. At the framework of our collaboration we aim to investigate the effects of substrate of topography in cell outgrowth and morphology of PNS populations.We showed that cultured Schwann cells and sympathetic neurons of SCGs migrate or grow their axons along the major axis of microcones in parallel alignement. Moreover when Schwann cells are present they seem to drive the growing axons as it was revealed in coculture studies of the populations examined as well as in whole DRG explant cultures a classic model to study neurite outgrowth and Schwann cell migration. Our study demonstrates for first time that a discontinuous topography could drive the directional outgrowth of neurons and glial cells of the peripheral nervous system if it contains at least a feature of anisotropy which is the elliptical shape of the microcones. This distinct inherent property of our microstructures combined with the conductance of the material, provides a useful system to explore and control neuronal functions and subsequent network characteristics.
Language English
Subject Fingolimod
Neurogenesis
ΒΝΝ27
Νευρογένεση
Φινγκολιμόδη
Issue date 2015-12-18
Collection   School/Department--School of Medicine--Department of Medicine--Doctoral theses
  Type of Work--Doctoral theses
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