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The current PhD thesis focuses on the role of sphingosine-1-phophate receptors (S1PRs) and their modulators in mice central nervous system (CNS). Beside the well-recognized immunomodulatory effect of the compounds, new insights are now suggesting that these molecules may directly interact with S1PRs located on CNS cells. In this context, the project aims at i) investigating the presence and the activity of S1PRs in mouse cortical synaptosomes (isolated nerve endings) and gliosomes (astrocytic processes) in both control and EAE mice ii) studying the effect of siponimod (BAF312) on oligodendroglia cells differentiation. While the first part of the project was carried out at the University of Genoa (Italy), experiments on oligodendroglial cells were performed at the University of Hasselt (Belgium). Firstly, we performed Western blot and confocal analyses which confirmed the existence of S1P1R and S1P3R in both glutamatergic cortical synaptosomes and gliosomes. Then, we performed functional studies (superfusion experiments) to investigate the effect of S1PRs activation in controlling glutamate transmission. By using different ligands (the broad-spectrum agonist sphingosine-1-phosphate (S1P), the S1P1R agonist CS-2100 and the selective S1P3R agonist CYM-5541) we demonstrated that presynaptic S1P1Rs are able to inhibit the 12 mM KCl-evoked release of preloaded [3H]D-aspartate (an analogue of glutamate) in cortical synaptosomes, while the activation of presynaptic S1P3Rs significantly enhances it. In contrast, the S1P1R agonist was ineffective in cortical gliosomes while CYM-5541 inhibited the 15 mM KCl-evoked [3H]D-aspartate exocytosis from cortical gliosomes. The opposite outcomes can be explained by the coupling of the receptors to different types of G-protein which, when activated, can generate opposite intracellular signals. The hypothesis is further support by the evidence that S1P failed to affect the [3H]D-aspartate release in both cortical preparations. The concomitant activation of both S1P1R and S1P3R avoid the possibility to observe a clear effect in glutamate transmission in presence of this agonist. Then, we investigated the expression of both receptors in cortical synaptosomes and gliosomes from experimental autoimmune encephalomyelitis (EAE). In particular, S1P1R and S1P3R are significantly increased in cortical synaptosomal lysates from EAE mice at the acute stage of disease, but unchanged in the gliosomal preparation. Interestingly, oral therapeutic administration of fingolimod (FTY720; 0.03 mg/Kg/day for 14 days) recovered the receptor densities to almost physiological levels and also restored their release-regulating activity. Surprisingly, biotinylation studies performed in synaptosomes and gliosomes from healthy mice suggested that the receptors are not expressed in the outer layer on the cellular membrane, since the S1P1R and S1P3R immunopositivity was absent in the biotinylated samples from both preparations. This evidence suggests that S1P1R and S1P3R may be preferentially located intracellularly or in the inner side of the plasma membrane. Similarly, biotinylation studies performed on control and EAE mice unveiled no changes in the membrane expression of the receptors.The results obtained in this first part improve the knowledge on the existence and the release-regulating activity of S1PRs in components of the tripartite synapsis (namely the presynaptic and the astrocytic one). All experiments were performed on cortical samples and not on the spinal cord, where inflammatory infiltration is known to be much higher in EAE animal model. The choice of this specific area for our investigation allowed to study the effects of FTY720 beyond its immunomodulatory activity, adding new insights on the effect of this S1PRs modulator on glutamatergic transmission. The second part of the project focused on the effect of another S1PRs modulator (siponimod, BAF312) on OPC in vitro differentiation. Despite very preliminary, the results suggested that treatment with BAF312 (10 nM) seems to induce a partial OPC differentiation as we can observed by the outcomes emerged from the immunostaining images as well as by the qPCR analysis. In parallel, at the same concentration, the treatment with BAF312 seems to enhance both TrkB mRNA expression and p-TrkB protein density. As an increased release of neurotrophic factors has been observed in literature following FTY720 administration, the increased expression and activation of TrkB receptors could represent an indirect way in which the drug can exert its neuroprotective effect. Further experiments will be conducted to confirm the preliminary results and study the effective role of BAF312 in these cells.

Physiological stress promotes adaptive plasticity, but the mechanisms responsible for maladaptation in the presence of an excessive or dysregulated response still remain unknown. Exposure to an acute inescapable foot shock (FS) stress protocol can induce both rapid and sustained changes in synaptic function and neuroarchitecture, suggesting that the effects of a single stressful event are far from being simply acute. Evaluating short- and long-term modifications triggered by acute stress could be a useful tool to dissect adaptive and maladaptive components underlying stress reaction. On this basis, the aim of the present thesis was to study some early and delayed alterations caused by acute FS stress in neuronal presynaptic (synaptosomes) and astroglia peri-synaptic (gliosomes) compartments purified from prefrontal cortex (PFC) in vulnerable (VUL) and resilient (RES) rats. We first applied the sucrose test paradigm, to identify RES or VUL rats at different time points after FS stress, thus showing that the anhedonic behaviour started 6h after FS stress and lasted at least for 48h. FS stress was characterized by an immediate and transient rise of corticosterone levels in both VUL and RES rats respect to controls. 24h after FS stress, when no augmentation of corticosterone level was still detectable, the depolarization-evoked glutamate release was more pronounced in PFC gliosomes of VUL, but not of RES rats. No release changes were found immediately, 6h and 48h after FS stress. No modifications were detected also in basal glutamate release in PFC gliosomes of both VUL and RES rats at each time point investigated. When focusing on PFC synaptosomes, we observed that the depolarization-evoked glutamate release was significantly increased in both VUL and RES rats respect to controls 24h after FS stress, while the basal glutamate release was increased in VUL animals only. Studying the molecular mechanisms underlying these functional alterations, we demonstrated that the excessive depolarization-evoked glutamate release measured 24h after FS-stress in VUL PFC gliosomes was mediated by glutamate transporters operating in the reverse mode. We also found that synapsin I expression did not change in synaptic membranes purified from PFC synaptosomes, while synapsin I phosphorylation in Ser9 was significantly increased in both RES and VUL rats, thus paralleling the increased glutamate release in PFC synaptosomes. Moreover, mineral corticoid receptors expression was significantly increased in RES rat PFC nuclear fraction whereas that of glucocorticoid receptors was increased in VUL rats. The present data suggest that acute FS stress induces adaptive/maladaptive stress responses at the synapse level, affecting the neuronal presynaptic counterpart and the surrounding astroglia cells with the alteration of specific molecular mechanisms that might represent new potential pharmacological targets for future therapeutic interventions.

Amyotrophic lateral sclerosis is an adult-onset neurodegenerative disease that develops because of motor neuron death. Several mechanisms occur supporting neurodegeneration, including mitochondrial dysfunction. Recently, we demonstrated that the synaptosomes from the spinal cord of SOD1G93A mice, an in vitro model of presynapses, displayed impaired mitochondrial metabolism at early pre-symptomatic stages of the disease, whereas perisynaptic astrocyte particles, or gliosomes, were characterized by mild energy impairment only at symptomatic stages. This work aimed to understand whether mitochondrial impairment is a consequence of upstream metabolic damage. We analyzed the critical pathways involved in glucose catabolism at presynaptic and perisynaptic compartments. Spinal cord and motor cortex synaptosomes from SOD1G93A mice displayed high activity of hexokinase and phosphofructokinase, key glycolysis enzymes, and of citrate synthase and malate dehydrogenase, key Krebs cycle enzymes, but did not display high lactate dehydrogenase activity, the key enzyme in lactate fermentation. This enhancement was evident in the spinal cord from the early stages of the disease and in the motor cortex at only symptomatic stages. Conversely, an increase in glycolysis and lactate fermentation activity, but not Krebs cycle activity, was observed in gliosomes from the spinal cord and motor cortex of SOD1G93A mice although only at the symptomatic stages of the disease. The cited enzymatic activities were enhanced in spinal cord and motor cortex homogenates, paralleling the time-course of the effect observed in synaptosomes and gliosomes. The observed metabolic modifications might be considered an attempt to restore altered energetic balance and indicate that mitochondria represent the ultimate site of bioenergetic impairment.

Amyotrophic lateral sclerosis (ALS) is an adult-onset fatal neurodegenerative disease characterized by muscle wasting, weakness, and spasticity due to a progressive degeneration of cortical, brainstem, and spinal motor neurons. The etiopathological causes are still largely obscure, although astrocytes definitely play a role in neuronal damage. Several mechanisms have been proposed to concur to neurodegeneration in ALS, including mitochondrial dysfunction. We have previously shown profound modifications of glutamate release and presynaptic plasticity in the spinal cord of the SOD1G93A mouse model of ALS. In this work, we characterized, for the first time, the aerobic metabolism in two specific compartments actively involved in neurotransmission (i.e. the presynaptic district, using purified synaptosomes, and the perisynaptic astrocyte processes, using purified gliosomes) in SOD1G93A mice at different stages of the disease. ATP/AMP ratio was lower in synaptosomes isolated from the spinal cord, but not from other brain areas, of SOD1G93A vs. control mice. The energy impairment was linked to altered oxidative phosphorylation (OxPhos) and increment of lipid peroxidation. These metabolic dysfunctions were present during disease progression, starting at the very pre-symptomatic stages, and did not depend on a different number of mitochondria or a different expression of OxPhos proteins. Conversely, gliosomes showed a reduction of the ATP/AMP ratio only at the late stages of the disease and an increment of oxidative stress also in the absence of a significant decrement in OxPhos activity. Data suggest that the presynaptic neuronal moiety plays a pivotal role for synaptic energy metabolism dysfunctions in ALS. Changes in the perisynaptic compartment seem subordinated to neuronal damage.

Glutamate release induced by mild depolarization was studied in astroglial preparations from the adult rat cerebral cortex, that is acutely isolated glial sub-cellular particles (gliosomes), cultured adult or neonatal astrocytes, and neuron-conditioned astrocytes. K+ (15, 35 mmol/L), 4-aminopyridine (0.1, 1 mmol/L) or veratrine (1, 10 micromol/L) increased endogenous glutamate or [3H]D-aspartate release from gliosomes. Neurotransmitter release was partly dependent on external Ca2+, suggesting the involvement of exocytotic-like processes, and partly because of the reversal of glutamate transporters. K+ increased gliosomal membrane potential, cytosolic Ca2+ concentration [Ca2+]i, and vesicle fusion rate. Ca2+ entry into gliosomes and glutamate release were independent from voltage-sensitive Ca2+ channel opening; they were instead abolished by 2-[2-[4-(4-nitrobenzyloxy)phenyl]ethyl]isothiurea (KB-R7943), suggesting a role for the Na+/Ca2+ exchanger working in reverse mode. K+ (15, 35 mmol/L) elicited increase of [Ca2+]i and Ca2+-dependent endogenous glutamate release in adult, not in neonatal, astrocytes in culture. Glutamate release was even more marked in in vitro neuron-conditioned adult astrocytes. As seen for gliosomes, K+-induced Ca2+ influx and glutamate release were abolished by KB-R7943 also in cultured adult astrocytes. To conclude, depolarization triggers in vitro glutamate exocytosis from in situ matured adult astrocytes; an aptitude grounding on Ca2+ influx driven by the Na+/Ca2+ exchanger working in the reverse mode.

The Bergmann glia is equipped with Ca2+-permeable AMPA receptors for glutamate, indispensable for structural and functional relations between the Bergmann glia and parallel/climbing fibers-Purkinje cell synapses. To better understand roles for the Bergmann AMPA receptors, herein we investigate on gliotransmitter release and Ca2+ signals in isolated Bergmann glia processes obtained from adult rat cerebellum. We found that: 1) the rat cerebellar purified astrocyte processes (gliosomes) expressed astrocytic and Bergmann markers and exhibited negligible contamination by nerve terminals, microglia, or oligodendrocytes; 2) activation of Ca2+-permeable AMPA receptors caused Ca2+ signals in the processes, and the release of glutamate from the processes; 3) effectiveness of rose bengal, trypan blue or bafilomycin A1, indicated that activation of the AMPA receptors evoked vesicular glutamate release. Cerebellar purified nerve terminals appeared devoid of glutamate-releasing Ca2+-permeable AMPA receptors, indicating that neuronal contamination may not be the source of the signals detected. Ultrastructural analysis indicated the presence of vesicles in the cytoplasm of the processes; confocal imaging confirmed the presence of vesicular glutamate transporters in Bergmann glia processes. We conclude that: a vesicular mechanism for release of the gliotransmitter glutamate is present in mature Bergmann processes; entry of Ca2+ through the AMPA receptors located on Bergmann processes is coupled with vesicular glutamate release. The findings would add a new role for a well-known Bergmann target for glutamate (the Ca2+-permeable AMPA receptors) and a new actor (the gliotransmitter glutamate) at the cerebellar excitatory synapses onto Purkinje cells.

Cell membranes isolated from nervous tissue can be easily injected into Xenopus oocytes, thereby effectively "microtransplanting" functional neurotransmitter receptors. This technique therefore allows a direct functional characterization of the original membrane receptor/ion channel proteins and the associated molecules while still embedded in their natural lipid environment. Cell membranes will contain components from different types of cells, i.e. neurons and glial cells, expressing their own receptors, with possibly different properties. To study the receptor properties of a single cell type, we injected oocytes with membranes isolated only from glia (gliosomes) of adult mouse neocortex and we focused our work on GABA(A) receptors incorporated in the oocyte cell membrane. We found that GABA(A)-activated currents allowed a good biophysical and pharmacological characterization of glial GABA(A) receptors. Therefore, the microtransplantation of gliosomes into oocytes can represent a good model to study the electrical and pharmacological properties of adult glial cells under different physiological and pathological conditions. Moreover, since gliosomes can be isolated from frozen tissues, this approach can be extended to post-mortem human tissues.

Zn2+ and Cu2+ released from synaptic terminals, have modulatory roles in regulating neuronal excitability. Synaptic neuronal GABAARS are relatively Zn2+-insensitive, while the extrasynaptic receptors have a higher sensitivity to block. Cu2+ is thought to block GABAARS through the same mechanism as Zn2+. So far, there is no information about Zn2+ and Cu2+ effects on glial GABAARS. Here, we compared the effects of these cations on neuronal and glial GABAARS expressed by oocytes injected with synaptosomes and gliosomes. We found that while the percentage of zinc-sensitive receptors was similar in both cases, the percentage of zinc-insensitive receptors was significantly different. Indeed, at 200 μM Zn2+ the mean percentage (± S.E.) of GABA remaining current in synaptosome-injected oocytes was 37.4 ± 1.1 % (n = 11) and in gliosome-injected oocytes was 32.6 ± 1.6 % (n = 9). The EC50 for GABA significantly changed from control only for neuronal receptors (EC50 140 ± 10 μM in control vs 290 ± 50 μM in zinc, n=6). Preliminary results of Cu2+ effects also showed a significant difference between neuronal and glial GABAARS: at 1 μM Cu2+, the percentages of remaining GABA-currents were 46.3 ± 2 % (n = 11) and 39.1 ± 1.1 (n = 10). At 100 μM Cu2+, the values were 18.4 ± 1.40 % (n = 13) and 13.75 ± 1.5 (n = 10). These findings indicate that neuronal and glial GABA currents are affected in a different way by the two cations, suggesting a different modulatory effect at the synaptic level.

Glial subcellular re-sealed particles (referred to as gliosomes here) were purified from rat cerebral cortex and investigated for their ability to release glutamate. Confocal microscopy showed that the glia-specific proteins glial fibrillary acidic protein (GFAP) and S-100, but not the neuronal proteins 95-kDa postsynaptic density protein (PSD-95), microtubule-associated protein 2 (MAP-2) and beta-tubulin III, were enriched in purified gliosomes. Furthermore, gliosomes exhibited labelling neither for integrin-alphaM nor for myelin basic protein, which are specific for microglia and oligodendrocytes respectively. The Ca2+ ionophore ionomycin (0.1-5 microm) efficiently stimulated the release of tritium from gliosomes pre-labelled with [3H]d-aspartate and of endogenous glutamate in a Ca(2+)-dependent and bafilomycin A1-sensitive manner, suggesting the involvement of an exocytotic process. Accordingly, ionomycin was found to induce a Ca(2+)-dependent increase in the vesicular fusion rate, when exocytosis was monitored with acridine orange. ATP stimulated [3H]d-aspartate release in a concentration- (0.1-3 mm) and Ca(2+)-dependent manner. The gliosomal fraction contained proteins of the exocytotic machinery [syntaxin-1, vesicular-associated membrane protein type 2 (VAMP-2), 23-kDa synaptosome-associated protein (SNAP-23) and 25-kDa synaptosome-associated protein (SNAP-25)] co-existing with GFAP immunoreactivity. Moreover, GFAP or VAMP-2 co-expressed with the vesicular glutamate transporter type 1. Consistent with ultrastructural analysis, several approximately 30-nm non-clustered vesicles were present in the gliosome cytoplasm. It is concluded that gliosomes purified from adult brain contain glutamate-accumulating vesicles and can release the amino acid by a process resembling neuronal exocytosis.