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2013/2014 It is well established that in healthy brain, the interplay between neurons and glia play a very important role in the maintenance of a correct physiological activity. Recent studies have shown the existence of an active involvement of glia in both the formation and function of the synapse (Araque et al. 1999; Parpura et al. 2012). These findings have led to reconsider the role of glia also in the case of pathological situations. Indeed, beyond its fundamental implication in healthy brain, glia has been found to play a critical role in several neurological disorders. It is now clear (Nguyen at al. 2012, Maezawa and Jin, 2010) that glial cells are involved not only in neurodegenerative diseases such as Alzheimer's disease (Angelova PR at al. 2014), but also in diseases characterized by intellectual disability. In this regard, recent evidence has shown the involvement of glial cells in Rett Syndrome (Ballas et al., 2009, Maezawa et al., 2009, Derecki et al., 2012 Okabe 2012). The purpose of my thesis is to investigate the role of different populations of astrocytes in the development and survival of neurons using in vitro models of both neurodegenerative and neurodevelopmental disorders. To this aim, in the first part of my project I participated in the setting up of a microfluidic system, an experimental model aim at studying the interconnections between different brain cells, focusing my attention on the contribution of glial cells derived from different brain areas in these pathological states. Secondly, through the use of the microfluidic system, we have shown that astrocytes derived from distinct brain areas have different effects on neurons if exposed to several harmful stimuli. Specifically I have evaluated both the development and viability of hippocampal neurons co-cultured with either cortical or hippocampal astrocytes in a neuroinflammatory context represented by the stimulation of beta amyloid peptide (Aß) together with several proinflammatory cytokines. Taking advantage of the microfluidic system, we showed that hippocampal and cortical astrocytes exposed to these stimuli were able to significantly increase neuronal cell death. Interestingly, hippocampal astrocytes induced an elevated neuronal cell death if compared to cortical astrocytes. (Bianco F, N Tonna, Lovchik RD, Mastrangelo R, Morini R, Ruiz A, E Delamarche, Matteoli M. Anal. Chem. 2012). To assess whether neuronal death was due to soluble factors released by astrocytes, we monitored the neuronal calcium responsiveness upon the exposure of astrocytes to Aß and IL 1ß. We found that both cortical and hippocampal astrocytes elicited a specific calcium transient in neurons upon stimulation, however, the calcium transients were higher in neurons exposed to stimulated hippocampal rather than cortical astrocytes. Such calcium transient were blocked by the specific NMDA receptor antagonist APV, thus highlighting that the calciumtransients were mediated by the activation of these receptors following the release of astrocytic glutamate. More importantly, hippocampal astrocytes exposed to neuroinflammatory environment induced higher cell death and neuronal calcium transient compared to cortical astrocytes (Bianco F, N Tonna, Lovchik RD, Mastrangelo R, Morini R, Ruiz A, E Delamarche, Matteoli M. Anal. Chem. 2012) We also used the microfluidic system to perform a pharmacological study in an experimental model of Alzheimer's disease. In particular, we evaluated the effectiveness of the drug FTY720 (Fingolimod, normally used for the treatment of multiple sclerosis) to prevent cell death in neurons cultured alone or co-cultured with microglia upon the stimulation with either fibrillar or oligomeric Aß form. The results showed that the drug prevented cell death in neurons exposed to Aß oligomers, both in the presence and absence of microglia. (Ruiz A, Joshi P, R Mastrangelo, Francolini M, Verderio C, Matteoli M Lab Chip. 2014). In the second part of my PhD thesis, I have focused my attention on the role of different populations of astrocytes in supporting neuronal development in a model of Rett Syndrome. Observations made by Prof. Tongiorgi's lab, showed a different neuronal atrophy at the level of cortex and hippocampus in mouse model of Rett Syndrome, thus prompting us to investigate the effects of glial cells derived from these two different brain areas on neurons. Neurons were grown in conditioned medium obtained from hippocampal or cortical astrocytes established from WT or KO mouse, and the growth in terms of neurites length was evaluated. The morphological analysis showed that neurons grown in conditioned medium derived from hippocampal astrocytes displayed longer neuritic processes than those grown in cortical astrocytes-derived medium. In contrast, the growth of hippocampal neurons in conditioned medium from wt cortical astrocytes, hippocampal and cortical astrocytes Rett not significantly different compared to neurons cultured alone. This suggests that hippocampal astrocytes have a higher trophic effect on neuronal development than cortical astrocytes. In light of these results, we can assume that the atrophy observed in cortical region of Rett Syndrome mouse model may be due to a lower trophic capability of cortical astrocytes compared to hippocampal in supporting neuronal development. Taken together these data suggest that astrocytic populations belonging to distinct brain areas have different capability in supporting neuronal growth, thus opening the possibility that region-specific atrophy observed in RETT mouse model may stem from a different release of trophic factors from glial cells. XXVII Ciclo 1984 |
