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.
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