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Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease with no effective cure. Astrocytes display a toxic phenotype in ALS and contribute to motoneuron (MN) degeneration. Modulating astrocytes’ neurotoxicity can reduce MN death. Our previous studies showed the beneficial effect of mesenchymal stem cell (MSC) administration in SOD1G93A ALS mice, but the mechanisms are still unclear. We postulated that the effects could be mediated by extracellular vesicles (EVs) secreted by MSCs. We investigated, by immunohistochemical, molecular, and in vitro functional analyses, the activity of MSC-derived EVs on the pathological phenotype and neurotoxicity of astrocytes isolated from the spinal cord of symptomatic SOD1G93A mice and human astrocytes (iAstrocytes) differentiated from inducible neural progenitor cells (iNPCs) of ALS patients. In vitro EV exposure rescued mouse and human ALS astrocytes’ neurotoxicity towards MNs. EVs significantly dampened the pathological phenotype and neuroinflammation in SOD1G93A astrocytes. In iAstrocytes, exposure to EVs increased the antioxidant factor Nrf2 and reduced reactive oxygen species. We previously found nine miRNAs upregulated in MSC-derived EVs. Here, the transfection of SOD1G93A astrocytes with single miRNA mimics reduced astrocytes’ activation and the expression of neuroinflammatory factors. Moreover, miR-466q and miR-467f mimics downregulate Mapk11, while miR-466m-5p and miR-466i-3p mimics promote the nuclear translocation of Nrf2. In iAstrocytes, transfection with miR-29b-3p mimic upregulated NQO1 antioxidant activity and reduced neurotoxicity towards MNs. MSC-derived EVs modulate astrocytes’ reactive phenotype and neurotoxicity through anti-inflammatory and antioxidant-shuttled miRNAs, thus representing a therapeutic strategy in ALS.

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.

Antibodies recognizing the amino-terminal domain of receptor subunit proteins modify the receptor efficiency to controlling transmitter release in isolated nerve endings (e.g., synaptosomes) indirectly confirming their presence in these particles but also allowing to speculate on their subunit composition. Western blot analysis and confocal microscopy unveiled the presence of the GluA1, GluA2, GluA3, and GluA4 receptor subunits in cortical synaptosomes. Functional studies confirmed the presence of presynaptic release-regulating AMPA autoreceptors in these terminals, whose activation releases [3H]D-aspartate ([3H]D-Asp, here used as a marker of glutamate) in a NBQX-dependent manner. The AMPA autoreceptors traffic in a constitutive manner, since entrapping synaptosomes with the pep2-SVKI peptide (which interferes with the GluA2-GRIP1/PICK1 interaction) amplified the AMPA-evoked releasing activity, while the inactive pep2-SVKE peptide was devoid of activity. Incubation of synaptosomes with antibodies recognizing the NH2 terminus of the GluA2 and the GluA3 subunits increased, although to a different extent, the GluA2 and 3 densities in synaptosomal membranes, also amplifying the AMPA-evoked glutamate release in a NBQX-dependent fashion. We then analyzed the releasing activity of complement (1:300) from both treated and untreated synaptosomes and found that the complement-induced overflow occurred in a DL-t-BOA-sensitive, NBQX-insensitive fashion. We hypothesized that anti-GluA/GluA complexes in neuronal membranes could trigger the classic pathway of activation of the complement, modifying its releasing activity. Accordingly, the complement-evoked release of [3H]D-Asp from antiGluA2 and anti-GluA3 antibody treated synaptosomes was significantly increased when compared to untreated terminals and facilitation was prevented by omitting the C1q component of the immunocomplex. Antibodies recognizing the NH2 terminus of the GluA1 or the GluA4 subunits failed to affect both the AMPA and the complement-evoked tritium overflow. Our results suggest the presence of GluA2/GluA3-containing release-regulating AMPA autoreceptors in cortical synaptosomes. Incubation of synaptosomes with commercial anti-GluA2 or anti-GluA3 antibodies amplifies the AMPA-evoked exocytosis of glutamate through a complement-independent pathway, involving an excessive insertion of AMPA autoreceptors in plasma membranes but also affects the complement-dependent releasing activity, by promoting the classic pathway of activation of the immunocomplex. Both events could be relevant to the development of autoimmune diseases typified by an overproduction of anti-GluA subunits.

Human bone marrow-derived mesenchymal stem cells (hBM-MSCs) are considered a great promise in the repair and regeneration of bone. Considerable efforts have been oriented towards uncovering the best strategy to promote stem cells osteogenic differentiation. In previous studies, hBM-MSCs exposed to physical stimuli such as pulsed electromagnetic fields (PEMFs) or directly seeded on nanostructured titanium surfaces ($TiO_{2}$) were shown to improve their differentiation to osteoblasts in osteogenic condition. In the present study, the effect of a daily PEMF-exposure on osteogenic differentiation of hBM-MSCs seeded onto nanostructured $TiO_{2}$ (with clusters under 100 nm of dimension) was investigated. $TiO_{2}$-seeded cells were exposed to PEMF (magnetic field intensity: 2 mT; intensity of induced electric field: 5 mV; frequency: 75 Hz) and examined in terms of cell physiology modifications and osteogenic differentiation. Results showed that PEMF exposure affected $TiO_{2}$-seeded cells osteogenesis by interfering with selective calcium-related osteogenic pathways, and greatly enhanced hBM-MSCs osteogenic features such as the expression of early/late osteogenic genes and protein production (e.g., ALP, COL-I, osteocalcin and osteopontin) and ALP activity. Finally, PEMF-treated cells resulted to secrete into conditioned media higher amounts of BMP-2, DCN and COL-I than untreated cell cultures. These findings confirm once more the osteoinductive potential of PEMF, suggesting that its combination with $TiO_{2}$ nanostructured surface might be a great option in bone tissue engineering applications.

Presynaptic mGlu2/3 autoreceptors exist in rat spinal cord nerve terminals as suggested by the finding that LY379268 inhibited the 15 mM KCl-evoked release of [H-3]D-aspartate ([H-3]D-Asp) in a LY341495-sensitive manner. Spinal cord glutamatergic nerve terminals also possess presynaptic release regulating 5-HT2A heteroreceptors. Actually, the 15 mM KCl-evoked [H-3]D-Asp exocytosis from spinal cord synaptosomes was reduced by the 5-HT2A agonist (+/-)DOI, an effect reversed by the 5-HT2A antagonists MDL11,939, MDL100907, ketanserin and trazodone (TZD). We investigated whether mGlu2/3 and 5-HT2A receptors colocalize and cross-talk in these terminals and if 5-HT2A ligands modulate the mGlu2/3-mediated control of glutamate exocytosis. Western blot analysis and confocal microscopy highlighted the presence of mGlu2/3 and 5-HT2A receptor proteins in spinal cord VGLUT1 positive synaptosomes, where mGlu2/3 and 5-HT2A receptor immunoreactivities largely colocalize. Furthermore, mGlu2/3 immunoprecipitates from spinal cord synaptosomes were also 5-HT2A immunopositive. Interestingly, the 100 pM LY379268-induced reduction of the 15 mM MCI-evoked [H-3]D-Asp overflow as well as its inhibition by 100 nM (+/-)DOI became undetectable when the two agonists were concomitantly added. Conversely, 5-HT2A antagonists (MDL11,939, MDL100907, ketanserin and TZD) reinforced the release-regulating activity of mGlu2/3 autoreceptors. Increased expression of mGlu2/3 receptor proteins in synaptosomal plasmamembranes paralleled the gain of function of the mGlu2/3 autoreceptors elicited by 5-HT2A antagonists. Based on these results, we propose that in spinal cord glutamatergic terminals i) mGlu2/3 and 5-HT2A receptors colocalize and interact one each other in an antagonist-like manner, ii) 5-HT2A antagonists are indirect positive allosteric modulator of mGlu2/3 autoreceptors controlling glutamate exocytosis. (C) 2018 Elsevier Ltd. All rights reserved.

Background and Purpose We recently proposed the existence of mGlu3-preferring autoreceptors in spinal cord terminals and of mGlu2-preferring autoreceptors in cortical terminals. This study aims to verify our previous conclusions and to extend their pharmacological characterization. Experimental Approach We studied the effect of LY566332, an mGlu2 receptors positive allosteric modulator (PAM) and of LY2389575, a selective mGlu3 receptor negative allosteric (NAM) modulator, on the mGlu2/3 agonist LY379268-mediated inhibition of glutamate exocytosis [measured as KCl-evoked release of preloaded [3H]-D-aspartate ([3H]-D-Asp)]. The mGlu2 PAM BINA and the mGlu3 NAM ML337, as well as selective antibodies recognizing the N-terminal of the receptor proteins, were used to confirm the pharmacological characterization of the native receptors. Key Results Cortical synaptosomes possess LY566332-sensitive autoreceptors that are slightly, although significantly, susceptible to LY2389575. Differently, LY566332-insensitive and LY2389575-sensitive autoreceptors exist in spinal cord terminals. BINA and ML337 mimicked LY566332 and LY2389575, respectively, in controlling LY379268-mediated inhibition of glutamate exocytosis from both cortical and spinal cord synaptosomes. Incubation of cortical synaptosomes with anti-mGlu2 antibody prevented the LY379268-induced inhibition of glutamate exocytosis, and this response was also partially reduced by the anti-mGlu3 antibody. Incubation of spinal cord synaptosomes with the anti-mGlu3 antibody nulled the LY379268-mediated reduction of glutamate exocytosis from these terminals, while the anti-mGlu2 antibody was inactive. Western blot analysis and confocal microscopy were largely consistent with these functional observations. Conclusions We confirm that mGlu3-preferring autoreceptors exist in spinal cord terminals. Differently, cortical glutamatergic terminals possess mGlu2/ mGlu3 heterodimers, whose inhibitory effect is largely mediated by the mGlu2 receptors.

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disorder due to loss of upper and lower motor neurons (MNs). The mechanisms of neuronal death are largely unknown, thus prejudicing the successful pharmacological treatment. One major cause for MN degeneration in ALS is represented by glutamate(Glu)-mediated excitotoxicity. We have previously reported that activation of Group I metabotropic Glu receptors (mGluR1 and mGluR5) at glutamatergic spinal cord nerve terminals produces abnormal Glu release in the widely studied SOD1G93A mouse model of ALS. We also demonstrated that halving mGluR1 expression in the SOD1G93A mouse had a positive impact on survival, disease onset, disease progression, and on a number of cellular and biochemical readouts of ALS. We generated here SOD1G93A mice with reduced expression of mGluR5 (SOD1G93AGrm5-/+) by crossing the SOD1G93A mutant mouse with the mGluR5 heterozigous Grm5-/+ mouse. SOD1G93AGrm5-/+ mice showed prolonged survival probability and delayed pathology onset. These effects were associated to enhanced number of preserved MNs, decreased astrocyte and microglia activation, reduced cytosolic free Ca2+ concentration, and regularization of abnormal Glu release in the spinal cord of SOD1G93AGrm5-/+ mice. Unexpectedly, only male SOD1G93AGrm5-/+ mice showed improved motor skills during disease progression vs. SOD1G93A mice, while SOD1G93AGrm5-/+ females did not. These results demonstrate that a lower constitutive level of mGluR5 has a significant positive impact in mice with ALS and support the idea that blocking Group I mGluRs may represent a potentially effective pharmacological approach to the disease.

Glutamate (Glu)-mediated excitotoxicity is a major cause of amyotrophic lateral sclerosis (ALS) and our previous work highlighted that abnormal Glu release may represent a leading mechanism for excessive synaptic Glu. We demonstrated that group I metabotropic Glu receptors (mGluR1, mGluR5) produced abnormal Glu release in SOD1G93A mouse spinal cord at a late disease stage (120 days). Here, we studied this phenomenon in pre-symptomatic (30 and 60 days) and early-symptomatic (90 days) SOD1G93A mice. The mGluR1/5 agonist (S)-3,5-Dihydroxyphenylglycine (3,5-DHPG) concentration dependently stimulated the release of [3H]d-Aspartate ([3H]d-Asp), which was comparable in 30- and 60-day-old wild type mice and SOD1G93A mice. At variance, [3H]d-Asp release was significantly augmented in 90-day-old SOD1G93A mice and both mGluR1 and mGluR5 were involved. The 3,5-DHPG-induced [3H]d-Asp release was exocytotic, being of vesicular origin and mediated by intra-terminal Ca2+ release. mGluR1 and mGluR5 expression was increased in Glu spinal cord axon terminals of 90-day-old SOD1G93A mice, but not in the whole axon terminal population. Interestingly, mGluR1 and mGluR5 were significantly augmented in total spinal cord tissue already at 60 days. Thus, function and expression of group I mGluRs are enhanced in the early-symptomatic SOD1G93A mouse spinal cord, possibly participating in excessive Glu transmission and supporting their implication in ALS. Please define all abbreviations the first time they appear in the abstract, the main text, and the first figure or table caption.

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 SOD1

Mouse hippocampal glutamatergic nerve endings express presynaptic release-regulating NMDA autoreceptors (NMDARs). The presence of GluN1, GluN2A, GluN2B, and GluN3A subunits in hippocampal vesicular glutamate transporter type 1-positive synaptosomes was confirmed with confocal microscopy. GluN2C, GluN2D, and GluN3B immunopositivity was scarcely present. Incubation of synaptosomes with the anti-GluN1, the anti-GluN2A, the anti-GluN2B, or the anti-GluN3A antibody prevented the 30 mu M NMDA/1 mu M glycine-evoked [H-3]d-aspartate ([H-3]d-ASP) release. The NMDA/glycine-evoked [H-3]d-ASP release was reduced by increasing the external protons, consistent with the participation of GluN1 subunits lacking the N1 cassette to the receptor assembly. The result also excludes the involvement of GluN1/GluN3A dimers into the NMDA-evoked overflow. Complement (1:300) released [H-3]d-ASP in a dizocilpine-sensitive manner, suggesting the participation of a NMDAR-mediated component in the releasing activity. Accordingly, the complement-evoked glutamate overflow was reduced in anti-GluN-treated synaptosomes when compared to the control. We speculated that incubation with antibodies had favored the internalization of NMDA receptors. Indeed, a significant reduction of the GluN1 and GluN2B proteins in the plasma membranes of anti-GluN1 or anti-GluN2B antibody-treated synaptosomes emerged in biotinylation studies. Altogether, our findings confirm the existence of presynaptic GluN3A-containing release-regulating NMDARs in mouse hippocampal glutamatergic nerve endings. Furthermore, they unveil presynaptic alteration of the GluN subunit insertion in synaptosomal plasma membranes elicited by anti-GluN antibodies that might be relevant to the central alterations occurring in patients suffering from autoimmune anti-NMDA diseases.

Background and purpose Presynaptic, release-regulating metabotropic glutamate 2 and 3 (mGlu2/3) autoreceptors exist in the CNS. They represent suitable targets for therapeutic approaches to central diseases that are typified by hyperglutamatergicity. The availability of specific ligands able to differentiate between mGlu2 and mGlu3 subunits allows us to further characterize these autoreceptors. In this study we investigated the pharmacological profile of mGlu2/3 receptors in selected CNS regions and evaluated their functions in mice with experimental autoimmune encephalomyelitis (EAE). Experimental approach The comparative analysis of presynaptic mGlu2/3 autoreceptors was performed by determining the effect of selective mGlu2/3 receptor agonist(s) and antagonist(s) on the release of [(3)H]-D-aspartate from cortical and spinal cord synaptosomes in superfusion. In EAE mice, mGlu2/3 autoreceptor-mediated release functions were investigated and effects of in vivo LY379268 administration on impaired glutamate release examined ex vivo. Key results Western blot analysis and confocal microscopy confirmed the presence of presynaptic mGlu2/3 receptor proteins. Cortical synaptosomes possessed LY541850-sensitive, NAAG-insensitive autoreceptors having low affinity for LY379268, while LY541850-insensitive, NAAG-sensitive autoreceptors with high affinity for LY379268 existed in spinal cord terminals. In EAE mice, mGlu2/3 autoreceptors completely lost their inhibitory activity in cortical, but not in spinal cord synaptosomes. In vivo LY379268 administration restored the glutamate exocytosis capability in spinal cord but not in cortical terminals in EAE mice. Conclusions and implications We propose the existence of mGlu2-preferring and mGlu3-preferring autoreceptors in mouse cortex and spinal cord respectively. The mGlu3 -preferring autoreceptors could represent a target for new pharmacological approaches for treating demyelinating diseases.

Mouse cortical GABAergic synaptosomes possess presynaptic inhibitory GABA(B) autoreceptors. Accordingly, (+/-) baclofen (3 m mu M) inhibits in a CGP53423-sensitive manner the 12 mM KCl-evoked release of preloaded [H-3]GABA. Differently, the existence of presynaptic release-regulating metabotropic glutamate type 1 (mGlu1) heteroreceptors in these terminals is still matter of discussion, although confocal microscopy unveiled the existence of mGlu1 alpha with GABA(B1) or GABA(B2) proteins in cortical VGAT-positive synaptosomes. The group I mGlu agonist 3,5-DHPG failed to modify on its own the 12 mM KCl-evoked [H-3]GABA exocytosis from cortical nerve endings, but, when added concomitantly to the GABA(B) agonist, it significantly reduced the 3 m mu M (+/-)baclofen-induced inhibition of [H-3]GABA exocytosis. Conversely, the mGlu1 antagonist LY367385 (0.03-1 m mu M), inactive on its own on GABA exocytosis, amplified the 3 m mu M (+/-)baclofen-induced inhibition of [H-3]GABA overflow. The (+/-) baclofen-induced inhibition of [H-3] GABA exocytosis was more pronounced in cortical synaptosomes from Grm1(crv4/crv4) mice, which bear a spontaneous mutation of the Grm1 gene leading to the functional inactivation of the mGlu1 receptor. Inasmuch, the expression of GABA(B2) receptor protein in cortical synaptosomal lysates from Grm1(crv4/crv4) mice was increased when compared to controls. Altogether, these observations seem best interpreted by assuming that mGlu1 coexist with GABA(B) receptors in GABAergic cortical synaptosomes, where they control GABA receptors in an antagonist-like manner. We then asked whether the mGlu1-mediated control of GABA(B) receptors is restricted to GABAergic terminals, or if it occurs also in other subpopulations of nerve endings. Release-regulating GABAB receptors also exist in glutamatergic nerve endings. (+/-)baclofen (1 m mu M) diminished the 12 mM KCl-evoked [H-3]D-aspartate overflow. Also in these terminals, the concomitant presence of 1 mu m M LY367385, inactive on its own, significantly amplified the inhibitory effect exerted by (+/-)baclofen on [H-3]D-aspartate exocytosis. Confocal microscopy confirmed the colocalization of mGlu1 with GABA(B1) and GABA(B2) labeling in vesicular glutamate type1 transporter-positive particles. Our results support the conclusion that mGlu1 receptors modulate in an antagonist-like manner presynaptic release-regulating GABA(B) receptors. This receptor-receptor interaction could be neuroprotective in central disease typified by hyperglutamatergicity.

Mouse cortical GABAergic synaptosomes possess presynaptic inhibitory GABAB autoreceptors. Accordingly, (±)baclofen (3 μM) inhibits in a CGP53423-sensitive manner the 12 mM KCl-evoked release of preloaded [3H]GABA. Differently, the existence of presynaptic release-regulating metabotropic glutamate type 1 (mGlu1) heteroreceptors in these terminals is still matter of discussion, although confocal microscopy unveiled the existence of mGlu1α with GABAB1 or GABAB2 proteins in cortical VGAT-positive synaptosomes. The group I mGlu agonist 3,5-DHPG failed to modify on its own the 12 mM KCl-evoked [3H]GABA exocytosis from cortical nerve endings, but, when added concomitantly to the GABAB agonist, it significantly reduced the 3 μM (±)baclofen-induced inhibition of [3H]GABA exocytosis. Conversely, the mGlu1 antagonist LY367385 (0.03–1 μM), inactive on its own on GABA exocytosis, amplified the 3 μM (±)baclofen-induced inhibition of [3H]GABA overflow. The ( ± )baclofen-induced inhibition of [3H]GABA exocytosis was more pronounced in cortical synaptosomes from Grm1crv4/crv4 mice, which bear a spontaneous mutation of the Grm1 gene leading to the functional inactivation of the mGlu1 receptor. Inasmuch, the expression of GABAB2 receptor protein in cortical synaptosomal lysates from Grm1crv4/crv4 mice was increased when compared to controls. Altogether, these observations seem best interpreted by assuming that mGlu1 coexist with GABAB receptors in GABAergic cortical synaptosomes, where they control GABA receptors in an antagonist-like manner. We then asked whether the mGlu1-mediated control of GABAB receptors is restricted to GABAergic terminals, or if it occurs also in other subpopulations of nerve endings. Release-regulating GABAB receptors also exist in glutamatergic nerve endings. (±)baclofen (1 μM) diminished the 12 mM KCl-evoked [3H]D-aspartate overflow. Also in these terminals, the concomitant presence of 1 μM LY367385, inactive on its own, significantly amplified the inhibitory effect exerted by (±)baclofen on [3H]D-aspartate exocytosis. Confocal microscopy confirmed the colocalization of mGlu1 with GABAB1 and GABAB2 labeling in vesicular glutamate type1 transporter-positive particles. Our results support the conclusion that mGlu1 receptors modulate in an antagonist-like manner presynaptic release-regulating GABAB receptors. This receptor–receptor interaction could be neuroprotective in central disease typified by hyperglutamatergicity.

Presynaptic mGlu2/3 autoreceptors exist in rat spinal cord nerve terminals as suggested by the finding that LY379268 inhibited the 15 mM KCl-evoked release of [

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disorder due to loss of upper and lower motor neurons (MNs). The mechanisms of neuronal death are largely unknown, thus prejudicing the successful pharmacological treatment. One major cause for MN degeneration in ALS is represented by glutamate(Glu)-mediated excitotoxicity. We have previously reported that activation of Group I metabotropic Glu receptors (mGluR1 and mGluR5) at glutamatergic spinal cord nerve terminals produces abnormal Glu release in the widely studied SOD1

Mesenchymal stem cells from human bone marrow (hBM-MSC) are widely utilized for clinical applications involving bone healing. In this context, their use has been often optimized in association to variously designed titanium substrates, being this material of great use in orthopaedic implants. According to recent findings, the ability of hBM-MSC to differentiate towards a specific lineage is not only driven by biochemical signals, but physical stimuli, such as rigidity or roughness of the substrate, can also support a commitment towards osteogenic differentiation. Moreover, the presence of features with defined dimensional scales, in particular nanometer-size, also proved to elicit specific biological effects. Here we evaluated the effectiveness of a nano-patterned titanium surface in sustaining hBM-MSC adhesion, growth and differentiation by means of a panel of biophysical tools: morphometry, electrophysiology, intracellular calcium measurements and immunocytochemistry. The results substantiate the idea that this micro-textured titanium dioxide is a good surface for growth and differentiation of hBM-MSC and it exhibits a stimulating action mainly in the initial period of differentiation. Moreover, the basal concentration of free cytosolic Calcium [Ca2+]i is confirmed to be a good hallmark of the hBM-MSC maturation stage. The study could provide relevant hints to help improving the biocompatibility and osteointegration potential of clinical titanium implants.

We recently proposed the existence of mGluWe studied the effect of LY566332, an mGluCortical synaptosomes possess LY566332-sensitive autoreceptors that are slightly, although significantly, susceptible to LY2389575. In contrast, LY566332-insensitive and LY2389575-sensitive autoreceptors are present in spinal cord terminals. BINA and ML337 mimicked LY566332 and LY2389575, respectively, in controlling LY379268-mediated inhibition of glutamate exocytosis from both cortical and spinal cord synaptosomes. Incubation of cortical synaptosomes with anti-mGluWe confirmed that mGlu

Glycine GlyT2 transporters are localized on glycine-storing nerve endings. Their main function is to accumulate glycine to replenish synaptic vesicles. Glycine was reported to be costored with γ-aminobutyric acid (GABA) in cerebellar interneurons that may coexpress glycine and GABA transporters, and this is confirmed here by confocal microscopy analysis showing coexpression of GAT1 and GlyT2 transporters on microtubule-associated protein-2-positive synaptosomes. It was found that GABA uptake elicited glycine release from cerebellar nerve endings by various mechanisms. We investigated whether and by what mechanisms activation of glycine transporters could mediate release of GABA. Nerve endings purified from cerebellum were prelabeled with [3H]GABA and exposed to glycine. Glycine stimulated [3H]GABA release in a concentration-dependent manner. The glycine effect was insensitive to strychnine or to 5,7-dichlorokynurenate but it was abolished when GlyT2 transporters were blocked. About 20% of the evoked release was dependent on external Ca2+ entered by reversal of plasmalemmal Na+/Ca2+exchangers. A significant portion of the GlyT2-mediated release of [3H]GABA (about 50% of the external Ca2+-independent release) occurred by reversal of GABA GAT1 transporters. Na+ ions, reaching the cytosol during glycine uptake through GlyT2, activated mitochondrial Na+/Ca2+ exchangers, causing an increase in cytosolic Ca2+, which in turn triggered a Ca2+-induced Ca2+ release process at inositoltrisphosphate receptors. Finally, the increased availability of Ca2+ in the cytosol allowed the opening of anion channels permeable to GABA. In conclusion, GlyT2 transporters not only take up glycine to replenish synaptic vesicles but can also mediate release of GABA by reversal of GAT1 and permeation through anion channels. © 2013 Wiley Periodicals, Inc.

Vascular remodelling plays a central role in asthma and chronic obstructive pulmonary disease (COPD). Bradykinin (BK) is a vasoactive proinflammatory peptide mediating acute responses in asthma. We investigated the role of angiogenic factors in relation to BK receptors in asthma and COPD.Bronchial biopsies from 33 patients with COPD, 24 old (≥50 years) patients with (≥50 years) asthma, 18 old control smokers, 11 old control non-smokers, 15 young (≤40yrs) patients with (≤40 years) asthma and 10 young control non-smokers were immunostained for CD31, vascular endothelial growth factor-A (VEGF-A), angiogenin and BK receptors (B2R and B1R). Fibroblast and endothelial co-localisation of relevant molecules were performed by immunofluorescence. BK-induced VEGF-A and angiogenin release was studied (ELISA) in bronchial fibroblasts from subjects with asthma and COPD.In bronchial lamina propria of old patients with asthma, CD31 and VEGF-A(+) cell numbers were higher than old control non-smokers (p0.05). Angiogenin(+), B2R(+) and B1R(+) cell numbers in old patients with asthma were higher than in old control non-smokers, control smokers and patients with COPD (p0.01). Angiogenin(+) cell numbers were higher in patients with COPD than both old control groups (p0.05). In all patients with asthma the number of B2R(+) cells was positively related to the numbers of B1R(+) (rs=0.43), angiogenin(+) (rs=0.42) and CD31 cells (rs=0.46) (p0.01). Angiogenin(+) cell numbers were negatively related to forced expiratory volume in 1 s (rs=-0.415, p=0.008). Double immunofluorescence revealed that CD31 cells of capillary vessels coexpressed B2R and that fibroblasts coexpressed B2R, VEGF-A and angiogenin. BK (10(-6)M) induced significant angiogenin release in fibroblasts from asthma and to a lesser extent in COPD.Unlike COPD, this study suggests the involvement of BK receptors in bronchial vascular remodelling in asthma.

Glutamate-mediated excitotoxicity plays a major role in ALS and reduced astrocytic glutamate transport was suggested as a cause. Based on previous work we have proposed that abnormal release may represent another source of excessive glutamate. In this line, here we studied the modulation of glutamate release in ALS by Group I metabotropic glutamate (mGlu) receptors, that comprise mGlu1 and mGlu5 members. Synaptosomes from the lumbar spinal cord of SOD1/G93A mice, a widely used murine model for human ALS, and controls were used in release, confocal or electron microscopy and Western blot experiments. Concentrations of the mGlu1/5 receptor agonist 3,5-DHPG >0.3 μM stimulated the release of [(3)H]d- aspartate, used to label the releasing pools of glutamate, both in control and SOD1/G93A mice. At variance, ≤0.3 μM 3,5-DHPG increased [(3)H]d-aspartate release in SOD1/G93A mice only. Experiments with selective antagonists indicated the involvement of both mGlu1 and mGlu5 receptors, mGlu5 being preferentially involved in the high potency effects of 3,5-DHPG. High 3,5-DHPG concentrations increased IP3 formation in both mouse strains, whereas low 3,5-DHPG did it in SOD1/G93A mice only. Release experiments confirmed that 3,5-DHPG elicited [(3)H]d-aspartate exocytosis involving intra-terminal Ca(2+) release through IP3-sensitive channels. Confocal microscopy indicated the co-existence of both receptors presynaptically in the same glutamatergic nerve terminal in SOD1/G93A mice. To conclude, activation of mGlu1/5 receptors produced abnormal glutamate release in SOD1/G93A mice, suggesting that these receptors are implicated in ALS and that selective antagonists may be predicted for new therapeutic approaches. This article is part of a Special Issue entitled 'Metabotropic Glutamate Receptors'.

Abscisic acid (ABA), a long known phytohormone, has been recently demonstrated to be present also in humans, where it targets cells of the innate immune response, mesenchymal and hemopoietic stem cells and cells involved in the regulation of systemic glucose homeostasis. LANCL2, a peripheral membrane protein, is the mammalian ABA receptor. We show that N-terminal glycine myristoylation causes LANCL2 localization to the plasmamembrane and to cytoplasmic membrane vesicles, where it interacts with the α subunit of a Gi protein and starts the ABA signaling pathway via activation of adenylate cyclase. Demyristoylation of LANCL2 by chemical or genetic means triggers its nuclear translocation. Nuclear enrichment of native LANCL2 is also induced by ABA treatment. Therefore human LANCL2 is a non-transmembrane G protein-coupled receptor susceptible to hormone-induced nuclear translocation.

Isomorphic mutation of the SBDS gene causes Shwachman-Diamond syndrome (SDS). SDS is a rare genetic bone marrow failure and cancer predisposition syndrome. SDS cells have ribosome biogenesis and their protein synthesis altered, which are two high-energy consuming cellular processes. The reported changes in reactive oxygen species production, endoplasmic reticulum stress response and reduced mitochondrial functionality suggest an energy production defect in SDS cells. In our work, we have demonstrated that SDS cells display a Complex IV activity impairment, which causes an oxidative phosphorylation metabolism defect, with a consequent decrease in ATP production. These data were confirmed by an increased glycolytic rate, which compensated for the energetic stress. Moreover, the signalling pathways involved in glycolysis activation also appeared more activated; i.e. we reported AMP-activated protein kinase hyper-phosphorylation. Notably, we also observed an increase in a mammalian target of rapamycin phosphorylation and high intracellular calcium concentration levels ([Ca2+]i), which probably represent new biochemical equilibrium modulation in SDS cells. Finally, the SDS cell response to leucine (Leu) was investigated, suggesting its possible use as a therapeutic adjuvant to be tested in clinical trials.

There is increasing evidence for the neuronal coexistence of classical transmitters. Implications in favor of cotransmission have often been represented by the identification, in the same neuron, of the putative cotransmitters, their synthetic enzymes and/or their vesicular transporters. In contrast, coexpression of neurotransmitter transporters on the plasma membrane of the same nerve terminal, although a potentially important indication for cotransmission, has received poor attention. We here used preparations of isolated nerve endings to functionally identify transporters coexpressed on the plasma membrane of the same terminal, in order to verify if such transporter coexpression indeed exists in neuronal systems in which cotransmission has already been established or reasonably suspected through other technical approaches. We could observe that functional transporters for glycine and glutamate are coexpressed on nerve terminals in the cerebellum; transporters for dopamine and GABA coexist on striatal terminals; transporters for glycine and GABA, previously found to coexist as cotransmission markers on nerve terminals of spinal cord and cerebellum, are not coexpressed in neocortex and hippocampus, where cotransmission has not been proposed to occur; transporters for GABA, glycine and glutamate are colocalized on nerve terminals of the spinal cord. Confocal microscopy experiments were performed to substantiate functional data, highlighting the presence of the co-existing transporters under study on MAP-2 positive synaptosomes. It is concluded that investigating the colocalization of functional neurotransmitter transporters on the plasma membrane of nerve terminals can provide useful information on the possibility of cotransmission.

Glutamate-mediated excitotoxicity plays a major role in the degeneration of motor neurons in amyotrophic lateral sclerosis and reduced astrocytary glutamate transport, which in turn increases the synaptic availability of the amino acid neurotransmitter, was suggested as a cause. Alternatively, here we report our studies on the exocytotic release of glutamate as a possible source of excessive glutamate transmission. The basal glutamate efflux from spinal cord nerve terminals of mice-expressing human soluble superoxide dismutase (SOD1) with the G93A mutation [SOD1/G93A(+)], a transgenic model of amyotrophic lateral sclerosis, was elevated when compared with transgenic mice expressing the wild-type human SOD1 or to non-transgenic controls. Exposure to 15 mM KCl or 0.3 μM ionomycin provoked Ca(2+)-dependent glutamate release that was dramatically increased in late symptomatic and in pre-symptomatic SOD1/G93A(+) mice. Increased Ca(2+) levels were detected in SOD1/G93A(+) mouse spinal cord nerve terminals, accompanied by increased activation of Ca(2+)/calmodulin-dependent kinase II and increased phosphorylation of synapsin I. In line with these findings, release experiments suggested that the glutamate release augmentation involves the readily releasable pool of vesicles and a greater capability of these vesicles to fuse upon stimulation in SOD1/G93A(+) mice.

Bradykinin (BK) induces fibroblast contraction but the structural changes and intracellular mechanisms involved have not been completely explored. We stimulated HFL-1 fibroblasts with BK to assess: 1) fibroblast contractility; 2) the role of alpha-smooth muscle actin (SMA) in contraction by small interfering RNA (siRNA); 3) alpha-SMA protein expression; 4) alpha-SMA and F-actin structure; 5) intracellular calcium concentration; and 6) phosphorylated myosin light-chain (pMLC) and MLC kinase (MLCK) expression. BK triggered concentration- and time-dependent fibroblast gel contraction in conjunction with alpha-SMA over expression, but not in alpha-SMA-siRNA-treated cells. BK also increased alpha-SMA(+) and F-actin(+) cell number and stress fibre polymerisation (detectable at 5-60 min). These BK-induced changes were associated with an increase in intracellular calcium concentration, which peaked within 15 s, and activation of pMLC, which was detectable at 5-60 min. No MLCK content modification was observed. The different manifestations of the BK-induced fibroblast activation were downregulated at different levels (25-100%) by HOE140, a specific BK B2 receptor (B2R) antagonist and by the Ca(2+) chelator, EGTA. Thus, BK-induced fibroblast contraction, associated with differentiation into alpha-SMA(+) myofibroblasts, is mediated through the activation of the B2R and involves the Ca(2+)/calmodulin pMLC-dependent pathway.

BACKGROUND: Increased numbers of activated neutrophils have been reported in the bronchial mucosa of patients with stable chronic obstructive pulmonary disease (COPD), particularly in severe disease. OBJECTIVES: To investigate the expression of neutrophilic chemokines and adhesion molecules in bronchial biopsies from patients with stable COPD of different severity (GOLD stages I-IV) compared with age-matched control subjects, smokers with normal lung function and never smokers. METHODS: The expression of CCL5, CXCL1, 5, 6, 7 and 8, CXCR1, CXCR2, CD11b and CD44 was measured in the bronchial mucosa using immunohistochemistry, confocal immunofluorescence, real-time quantitative polymerase chain reaction (RT-QPCR) and Western blotting (WB). RESULTS: The numbers of CCL5+ epithelial cells and CCL5+ and CXCL7+ immunostained cells were increased in the bronchial submucosa of patients with stable severe COPD compared with control never smokers and smokers with normal lung function. This was also confirmed at the level of mRNA expression. The numbers of CCL5+ cells in the submucosa of patients with COPD were 2-15 times higher than any other chemokines. There was no correlation between the number of these cells and the number of neutrophils in the bronchial submucosa. Compared with control smokers, the percentage of neutrophils co-expressing CD11b and CD44 receptors was significantly increased in the submucosa of patients with COPD. CONCLUSION: The increased expression of CCL5 and CXCL7 in the bronchial mucosa of patients with stable COPD, together with an increased expression of extracellular matrix-binding receptors on neutrophils, may be involved in the pathogenesis of COPD.

Charcot-Marie-Tooth (CMT) is the most frequent inherited neuromuscular disorder, affecting 1 person in 2500. CMT1A, the most common form of CMT, is usually caused by a duplication of chromosome 17p11.2, containing the PMP22 (peripheral myelin protein-22) gene; overexpression of PMP22 in Schwann cells (SC) is believed to cause demyelination, although the underlying pathogenetic mechanisms remain unclear. Here we report an abnormally high basal concentration of intracellular calcium ([Ca(2+)](i)) in SC from CMT1A rats. By the use of specific pharmacological inhibitors and through down-regulation of expression by small interfering RNA, we demonstrate that the high [Ca(2+)](i) is caused by a PMP22-related overexpression of the P2X7 purinoceptor/channel leading to influx of extracellular Ca(2+) into CMT1A SC. Correction of the altered [Ca(2+)](i) in CMT1A SC by small interfering RNA or with pharmacological inhibitors of P2X7 restores functional parameters of SC (migration and release of ciliary neurotrophic factor), which are typically defective in CMT1A SC. More significantly, stable down-regulation of the expression of P2X7 restores myelination in co-cultures of CMT1A SC with dorsal root ganglion sensory neurons. These results establish a pathogenetic link between high [Ca(2+)](i) and impaired SC function in CMT1A and identify overexpression of P2X7 as the molecular mechanism underlying both abnormalities. The development of P2X7 inhibitors is expected to provide a new therapeutic strategy for treatment of CMT1A neuropathy.

Abscisic acid (ABA) is a phytohormone recently identified as a new endogenous pro-inflammatory hormone in human granulocytes. Here we report the functional activation of human monocytes and vascular smooth muscle cells by ABA. Incubation of monocytes with ABA evokes an intracellular Ca2+ rise through the second messenger cyclic ADP-ribose, leading to NF-kappaB activation and consequent increase of cyclooxygenase-2 expression and prostaglandin E2 production and enhanced release of MCP-1 (monocyte chemoattractant protein-1) and of metalloprotease-9, all events reportedly involved in atherogenesis. Moreover, monocytes release ABA when exposed to thrombin-activated platelets, a condition occurring at the injured vascular endothelium; monocyte-derived ABA behaves as an autocrine and paracrine pro-inflammatory hormone-stimulating monocyte migration and MCP-1 release, as well as vascular smooth muscle cells migration and proliferation. These results, and the presence of ABA in human arterial plaques at a 10-fold higher concentration compared with normal arterial tissue, identify ABA as a new signal molecule involved in the development of atherosclerosis and suggest a possible new target for anti-atherosclerotic therapy.

The influx of the toxic cation Cd2+ was studied in fura 2-loaded rat cerebellar granule neurons. In cells depolarized with Ca2(+)-free, high-KCI solutions, the fluorescence emission ratio (R) increased in the presence of 100 microM Cd2(+). This increase was fully reversed by the Cd2+ chelator tetrakis(2-pyridylmethyl)ethylenediamine, indicating a cadmium influx into the cell. The rate of increase, dR/dt, was greatly reduced (67+/-5%) by 1 microM nimodipine and enhanced by 1 microM Bay K 8644. Concurrent application of nimodipine and omega-agatoxin IVA (200 nM) blocked Cd2+ permeation almost completely (88+/-5%), whereas omega-conotoxin MVIIC (2 microM) reduced dR/dt by 24+/-8%. These results indicate a primary role of voltage-dependent calcium channels in Cd2+ permeation. Stimulation with glutamate or NMDA and glycine also caused a rise of R in external Cd2+. Simultaneous application of nimodipine and omega-agatoxin IVA moderately reduced dR/dt (25+/-3%). NMDA-driven Cd2(+) entry was almost completely prevented by 1 mM Mg2+, 50 microM memantine, and 10 microM 5,7-dichlorokynurenic acid, suggesting a major contribution of NMDA-gated channels in glutamate-stimulated Cd2+ influx. Moreover, perfusion with alpha-amino-3-hydroxy-5-methylisoxazole-4-propionate caused a slow increase of R. These results suggest that Cd2+ permeates the cell membrane mainly through the same pathways of Ca2+ influx.

It is widely accepted that glycine transporters of the GLYT1 type are situated on astrocytes whereas GLYT2 are present on glycinergic neuronal terminals where they mediate glycine uptake. We here used purified preparations of mouse spinal cord nerve terminals (synaptosomes) and of astrocyte-derived subcellular particles (gliosomes) to characterize functionally and morphologically the glial versus neuronal distribution of GLYT1 and GLYT2. Both gliosomes and synaptosomes accumulated [3H]GABA through GAT1 transporters and, when exposed to glycine in superfusion conditions, they released the radioactive amino acid not in a receptor-dependent manner, but as a consequence of glycine penetration through selective transporters. The glycine-evoked release of [3H]GABA was exocytotic from synaptosomes but GAT1 carrier-mediated from gliosomes. Based on the sensitivity of the glycine effects to selective GLYT1 and GLYT2 blockers, the two transporters contributed equally to evoke [3H]GABA release from GABAergic synaptosomes; even more surprising, the 'neuronal' GLYT2 contributed more efficiently than the 'glial' GLYT1 to mediate the glycine effect in [3H]GABA releasing gliosomes. These functional results were largely confirmed by confocal microscopy analysis showing co-expression of GAT1 and GLYT2 in GFAP-positive gliosomes and of GAT1 and GLYT1 in MAP2-positive synaptosomes. To conclude, functional GLYT1 are present on neuronal axon terminals and functional GLYT2 are expressed on astrocytes, indicating not complete selectivity of glycine transporters in their glial versus neuronal localization in the spinal cord.

The effect of euplotin C -- a lipophilic bioactive metabolite produced by the ciliate Euplotes crassus -- on the kinetics of both phagocytosis of latex particles and fluid-phase uptake of dextran, was studied in the single-cell ciliate Paramecium primaurelia. The inhibition of food vacuole formation was concentration- and time-dependent (p

Abscisic acid (ABA) is a phytohormone involved in fundamental physiological processes of higher plants, such as response to abiotic stress (temperature, light, drought), regulation of seed dormancy and germination, and control of stomatal closure. Here, we provide evidence that ABA stimulates several functional activities [phagocytosis, reactive oxygen species and nitric oxide (NO) production, and chemotaxis] of human granulocytes through a signaling pathway sequentially involving a pertussis toxin (PTX)-sensitive G protein/receptor complex, protein kinase A activation, ADP-ribosyl cyclase phosphorylation, and consequent cyclic-ADP-ribose overproduction, leading to an increase of the intracellular Ca 2+ concentration. The increase of free intracellular ABA and its release by activated human granulocytes indicate that ABA should be considered as a new pro-inflammatory cytokine in humans. This discovery is an intriguing example of conservation of a hormone and its signaling pathway from plants to humans and provides insight into the molecular mechanisms of granulocyte activation, possibly leading to the development of new antiinflammatory drugs.

Exposure to Pulsed Electromagnetic Field (PEMF) has been shown to affect proliferation and differentiation of human mesenchymal stem cells derived from bone marrow stroma (BM-hMSC). These cells offer considerable promise in the field of regenerative medicine, but their clinical application is hampered by major limitations such as poor availability and the time required to differentiate up to a stage suitable for implantation. For this reason, several research efforts are focusing on identifying strategies to speed up the differentiation process. In this work we investigated the in vitro effect of PEMF on Ca2+-related mechanisms promoting the osteogenic differentiation of BM-hMSC. Cells were daily exposed to PEMF while subjected to osteogenic differentiation and various Ca2+-related mechanisms were monitored using multiple approaches for identifying functional and structural modifications related to this process. The results indicate that PEMF exposure promotes chemically induced osteogenesis by mechanisms that mainly interfere with some of the calcium-related osteogenic pathways, such as permeation and regulation of cytosolic concentration, leaving others, such as extracellular deposition, unaffected. The PEMF effect is primarily associated to early enhancement of intracellular calcium concentration, which is proposed here as a reliable hallmark of the osteogenic developmental stage.

Dimethyl fumarate (DMF), recently approved as an oral immunomodulatory treatment for relapsing-remitting multiple sclerosis (MS), metabolizes to monomethyl fumarate (MMF) which crosses the blood–brain barrier and has demonstrated neuroprotective effects in experimental studies. We postulated that MMF exerts neuroprotective effects through modulation of microglia activation, a critical component of the neuroinflammatory cascade that occurs in neurodegenerative diseases such as MS. To ascertain our hypothesis and define the mechanistic pathways involved in the modulating effect of fumarates, we used real-time PCR and biochemical assays to assess changes in the molecular and functional phenotype of microglia, quantitative Western blotting to monitor activation of postulated pathway components, and ex vivo whole-cell patch clamp recording of excitatory post-synaptic currents in corticostriatal slices from mice with experimental autoimmune encephalomyelitis (EAE), a model for MS, to study synaptic transmission. We show that exposure to MMF switches the molecular and functional phenotype of activated microglia from classically activated, pro-inflammatory type to alternatively activated, neuroprotective one, through activation of the hydroxycarboxylic acid receptor 2 (HCAR2). We validate a downstream pathway mediated through the AMPK–Sirt1 axis resulting in deacetylation, and thereby inhibition, of NF-κB and, consequently, of secretion of pro-inflammatory molecules. We demonstrate through ex vivo monitoring of spontaneous glutamate-mediated excitatory post-synaptic currents of single neurons in corticostriatal slices from EAE mice that the neuroprotective effect of DMF was exerted on neurons at pre-synaptic terminals by modulating glutamate release. By exposing control slices to untreated and MMF-treated activated microglia, we confirm the modulating effect of MMF on microglia function and, thereby, its indirect neuroprotective effect at post-synaptic level. These findings, whereby DMF-induced activation of a new HCAR2-dependent pathway on microglia leads to the modulation of neuroinflammation and restores synaptic alterations occurring in EAE, represent a possible novel mechanism of action for DMF in MS. Electronic supplementary material The online version of this article (doi:10.1007/s00401-015-1422-3) contains supplementary material, which is available to authorized users.

The impact of synaptic vesicle endo-exocytosis on the trafficking of nerve terminal heterotransporters was studied by monitoring membrane expression and function of the GABA transporter-1 (GAT-1) and of type-1/2 glycine (Gly) transporters (GlyT-1/2) at spinal cord glutamatergic synaptic boutons. Experiments were performed by inducing exocytosis in wild-type (WT) mice, in amphiphysin-I knockout (Amph-I KO) mice, which show impaired endocytosis, or in mice expressing high copy number of mutant human SOD1 with a Gly93Ala substitution (SOD1(G93A)), a model of human amyotrophic lateral sclerosis showing constitutively excessive Glu exocytosis. Exposure of spinal cord synaptosomes from WT mice to a 35mM KCl pulse increased the expression of GAT-1 at glutamatergic synaptosomal membranes and enhanced the GAT-1 heterotransporter-induced [(3)H]d-aspartate ([(3)H]d-Asp) release. Similar results were obtained in the case of GlyT-1/2 heterotransporters. Preventing depolarization-induced exocytosis normalized the excessive GAT-1 and GlyT-1/2 heterotransporter-induced [(3)H]d-Asp release in WT mice. Impaired endocytosis in Amph-I KO mice increased GAT-1 membrane expression and [(3)H]GABA uptake in spinal cord synaptosomes. Also the GAT-1 heterotransporter-evoked release of [(3)H]d-Asp was augmented in Amph-I KO mice. The constitutively excessive Glu exocytosis in SOD1(G93A) mice resulted in augmented GAT-1 expression at glutamatergic synaptosomal membranes and GAT-1 or GlyT-1/2 heterotransporter-mediated [(3)H]d-Asp release. Thus, endo-exocytosis regulates the trafficking of GAT-1 and GlyT-1/2 heterotransporters sited at spinal cord glutamatergic nerve terminals. As a consequence, it can be hypothesized that the excessive GAT-1 and GlyT-1/2 heterotransporter-mediated Glu release, in the spinal cord of SOD1(G93A) mice, is due to the heterotransporter over-expression at the nerve terminal membrane, promoted by the excessive Glu exocytosis.

Lipopolysaccharide, the main component of the cell wall of Gram-negative bacteria, is known to activate microglial cells following its interaction with the CD14/Toll-like receptor complex (TLR-4). The activation pathway triggered by lipopolysaccharide in microglia involves enhanced basal levels of intracellular calcium ([Ca2+]i) and terminates with increased generation of cytokines/chemokines and nitric oxide. Here we demonstrate that in lipopolysaccharide-stimulated murine N9 microglial cells, cyclic ADP-ribose, a universal and potent Ca2+ mobiliser generated from NAD+ by ADP-ribosyl cyclases (ADPRC), behaves as a second messenger in the cell activation pathway. Lipopolysaccharide induced phosphorylation, mediated by multiple protein kinases, of the mammalian ADPRC CD38, which resulted in significantly enhanced ADPRC activity and in a 1.7-fold increase in the concentration of intracellular cyclic ADP-ribose. This event was paralleled by doubling of the basal [Ca2+]i levels, which was largely prevented by the cyclic ADP-ribose antagonists 8-Br-cyclic ADP-ribose and ryanodine (by 75% and 88%, respectively). Both antagonists inhibited, although incompletely, functional events downstream of the lipopolysaccharide-induced microglia-activating pathway, i.e. expression of inducible nitric oxide synthase, overproduction and release of nitric oxide and of tumor necrosis factor alpha. The identification of cyclic ADP-ribose as a key signal metabolite in the complex cascade of events triggered by lipopolysaccharide and eventually leading to enhanced generation of pro-inflammatory molecules may suggest a new therapeutic target for treatment of neurodegenerative diseases related to microglia activation.

The effects of gamma-aminobutyric acid (GABA) on the release of glutamate from mouse spinal cord nerve endings have been studied using superfused synaptosomes. GABA elicited a concentration-dependent release of [3H]D-aspartate ([3H]D-ASP; EC50= 3.76 microM). Neither muscimol nor (-)baclofen mimicked GABA, excluding receptor involvement. The GABA-evoked release was strictly Na+ dependent and was prevented by the GABA transporter inhibitor SKF89976A, suggesting involvement of GAT-1 transporters located on glutamatergic nerve terminals. GABA also potentiated the spontaneous release of endogenous glutamate; an effect sensitive to SKF89976A and low-Na+-containing medium. Confocal microscopy shows that the GABA transporter GAT-1 is coexpressed with the vesicular glutamate transporter vGLUT-1 and with the plasma membrane glutamate transporter EAAT2 in a substantial portion of synaptosomal particles. The GABA effect was external Ca2+ independent and was not decreased when cytosolic Ca2+ ions were chelated by BAPTA. The glutamate transporter blocker DL-TBOA or dihydrokainate inhibited in part (approximately 35%) the GABA (10 microM)-evoked [3H]D-ASP release; this release was strongly reduced by the anion channel blockers niflumic acid and NPPB. GABA, up to 30 microM, was unable to augment significantly the basal release of [3H]glycine from spinal cord synaptosomes, indicating selectivity for glutamatergic transmission. It is concluded that GABA GAT-1 transporters and glutamate transporters coexist on the same spinal cord glutamatergic terminals. Activation of these GABA transporters elicits release of glutamate partially by reversal of glutamate transporters present on glutamatergic terminals and largely through anion channels.

The effect of euplotin C—a lipophilic toxic metabolite produced by the protist ciliate Euplotes crassus—on the swimming behavior was studied in a single-celled system represented by the ciliate Paramecium primaurelia. When in P. primaurelia internal Ca2+ concentration coupled to membrane depolarization increases, a reversal in the direction of ciliary beating and consequently in swimming direction occurs. The ciliary reversal is correlated to Ca2+ influx amount. In this study, evidence was given that continuous ciliary reversal (CCR) duration, induced by high external KCl concentrations, is longer in euplotin C-treated cells than in control cells. To test the hypothesis that euplotin C increases CCR duration by modulating a specific subtype of Ca2+ channel, selective Ca2+ channel blockers were used. Blocking L-type channels by nimodipine and verapamil, N- and Q-type channels by Ω-conotoxins, fractions GVIA and MVIIC, significantly reduced the CCR duration evoked by membrane depolarization, suggesting an involvement of these channels in ciliary reversal in Paramecium. The effect of euplotin C on CCR duration persisted when Ω-conotoxin GVIA or Ω-conotoxin MVIIC were applied, conversely, it disappeared when L-type channel blockers were used. The magnitude of the block by nimodipine and verapamil in the presence of euplotin C was comparable with that observed in the absence of euplotin C, suggesting that the Ca2+ channels modulated by euplotin C were dihydropyridine-sensible calcium channels similar to L-type channels found in mammalian cells.

The number of neurotransmitter receptors on the plasma membrane is regulated by the traffic of intracellular vesicles. Golgi-derived vesicles provide newly synthesized receptors to the cell surface, whereas clathrin-coated vesicles are the initial vehicles for sequestration of surface receptors, which are ultimately degraded or recycled. We have previously shown that GABAB receptors display a punctuate vesicular pattern dispersed on the cell surface and throughout the cytoplasm and are internalized via clathrin-dependent and -independent endocytosis. Here we have studied constitutive GABAB receptor trafficking after internalization in Paramecium primaurelia by confocal laser scanning microscopy and multiple immunofluorescence analysis. After internalization, receptors are targeted to the early endosomes characterized by the molecular markers EEA1 and rab5. Some of these receptors, destined for recycling back to the plasma membrane, traffic from the early endosomes to the endosomal recycling compartment that is characterized by the presence of rab4-immunoreactivity (IR). Receptors that are destined for degradation exit the endosomal pathway at the early endosomes and traffic to the late endosome-lysosome pathway. In fact, some of the GABAB-positive compartments were identified as lysosomal structures by double staining with the lysosomal marker LAMP-1. GABAB vesicle structures also colocalize with TGN38-IR and rab11-IR. TGN38 and rab11 are proteins found in association with post-Golgi and recycling endosomes, respectively.

The presence of opioid, b-adrenergic and cholinergic receptors has been demonstrated in ciliated protozoa, but little is known about c-aminobutyric acid (GABA) receptors. In this study we have analyzed the distribution of GABAA-type receptor subunits in Paramecium. Confocal laser microscopy using antibodies specific for a1-, a2-, a3-, a6-, b2/3-, c2-, e-, k-, and h-subunits showed that most receptors are aggregated in clusters and are distributed both on cell surface and in the cytoplasm. The intensity of labelling of the a6-, b2/3- and c2-subunits was more intense than the a1-, e-, and h-subunits, suggesting that the former are present in higher concentrations than the latter. � 2004 Federation of European Microbiological Societies. Published by Elsevier B.V. All rights reserved.

Cyclic ADP-ribose (cADPR), a universal calcium mobilizer from intracellular stores, was recently demonstrated to stimulate proliferation of various cell types. The role of cADPR in a specific process of monocyte- and plasma-mediated activation of T-lymphocytes by lipopolysaccharide (LPS) was addressed using human mononuclear cells from peripheral blood (PBMCs). Incubation of PBMCs with 0.1 μg/ml of LPS for 24 h provided a doubling in the intracellular levels of cADPR as compared with unstimulated PBMCs. The cADPR increase was abolished either by prior removal of monocytes or by pre-incubating a whole PBMC population with a monoclonal antibody against the monocyte marker CD14. The increased concentrations of intracellular cADPR elicited by LPS stimulation were paralleled by significant increases in NAD + levels and in the activities of ectocellular and membrane-bound fractions of ADP-ribosyl cyclase/ cADPR hydrolase activities. A cytosolic ADP-ribosyl cyclase was also detectable in PBMCs and its activity was comparably enhanced by LPS stimulation. This soluble cyclase is distinguished from the membrane-bound cyclase by both substrate and inhibitor sensitivities. LPS-stimulated PBMCs showed 2-3-fold increases of intracellular calcium ([Ca2+]), and these changes were prevented completely by the cADPR antagonist 8-Br-cADPR and by ryanodine. Both compounds, and the cyclase inhibitor nicotinamide, significantly inhibited the T-lymphocyte proliferation induced by LPS in PBMCs. These results demonstrate that cADPR plays a role of second messenger in the adaptive immune recognition process of LPS-stimulated proliferation of PBMCs., link_to_subscribed_fulltext

Recently, the thermosensing pathway in sponges (Porifera) was elucidated. The thermosensor triggering this cascade is a heat-activated cation channel,with the phytohormone abscisic acid (ABA), cyclic ADP-ribose (cADPR) and calcium acting as intracellular messengers, similarly to the drought-stress signaling cascade in higher plants. Here, we investigated the functional effects downstream of the temperature-signaling pathway in Axinella polypoides (Porifera, Demonspongiae). Short-term stimulation followed by long-term depression of amino acid incorporation, oxygen consumption and water filtration were observed after exposure of the sponge to a brief heat stress or to micromolar ABA. These effects could be prevented by the targeted interruption of the signaling pathway either at the level of the cation channel thermosensor or at the level of the cADPR-induced intracellular calcium increase. Moreover, release of cyclase activity into the sea water and generation of extracellular cADPR were observed following brief heat stress. Intact sponge cells were sensitive to extracellular cADPR and addition of purified cyclase increased sponge respiration similarly to heat stress. This is the first observation of functional effects exerted on Metazoa by the phytohormone ABA: conservation of the ABA/cADPR stress-signaling cascade points to its early evolution in a common precursor of modern Metazoa and Metaphyta. The functional effects induced by extracellular cyclase/cADPR suggest an evolutionary origin of cADPR as an ancient stress hormone in Porifera.

Fanconi Anemia (FA) is a rare and complex inherited blood disorder associated with bone marrow failure and malignancies. Many alterations in FA physiology appear linked to red-ox unbalance including alterations in the morphology and structure of nuclei, intermediate filaments and mitochondria, defective respiration, reduced ATP production and altered ATP/AMP ratio. These defects are consistently associated with impaired oxygen metabolism indeed treatment with antioxidants N-acetylcysteine (NAC) and resveratrol (RV) does rescue FA physiology. Due to the importance of the intracellular calcium signaling and its key function in the control of intracellular functions we were interested to study calcium homeostasis in FA. We found that FANCA cells display a dramatically low intracellular calcium concentration ([Ca(2+)]i) in resting conditions. This condition affects cellular responses to stress. The flux of Ca(2+) mobilized by H2O2 from internal stores is significantly lower in FANCA cells in comparison to controls. The low basal [Ca(2+)]i in FANCA appears to be an actively maintained process controlled by a finely tuned interplay between different intracellular Ca(2+) stores. The defects associated with the altered Ca(2+) homeostasis appear consistently overlapping those related to the unbalanced oxidative metabolism in FA cells underlining a contiguity between oxidative stress and calcium homeostasis.

SummaryParamecium primaurelia is a unicellular eukaryote that moves in freshwater by ciliary beating and responds to environmental stimuli by altering motile behaviour. The movements of the cilia are controlled by the electrical changes of the cell membrane: when the intraciliary Ca2+ concentration associated with plasma membrane depolarization increases, the ciliary beating reverses its direction, and consequently the swimming direction changes. The ciliary reversal duration is correlated with the amount of Ca2+ influx. Here we evaluated the effects due to the activation or blockade of NMDA receptors on swimming behaviour in Paramecium. Paramecia normally swim forward drawing almost linear tracks. We observed that the simultaneous administration of NMDA and glycine induced a partial ciliary reversal (PaCR) leading to a continuous spiral-like swim. Furthermore, the duration of continuous ciliary reversal (CCR), triggered by high external KCl concentrations, was longer in NMDA/glycine treated cells. NMDA action required the presence of Ca2+, as the normal forward swimming was restored when the ion was omitted from the extracellular milieu. The PaCR and the enhancement of CCR duration significantly decreased when the antagonists of the glutamate site D-AP5 or CGS19755, the NMDA channel blocker MK-801, or the glycine site antagonist DCKA were added. The action of NMDA/glycine was also abolished by Zn2+ or ifenprodil, the GluN2A and the GluN2B NMDA-containing subunit blockers, respectively. Searches of the Paramecium genome database currently available indicate that the NMDA-like receptor with ligand binding characteristics of an NMDA receptor-like complex, purified from rat brain synaptic membranes and found in some metazoan genome, is also present in Paramecium. These results provide evidence that functional NMDA receptors similar to those typical of mammalian neuronal cells are present in the single-celled organism Paramecium and thus suggest that the glutamatergic NMDA system is a phylogenetically old behaviour-controlling mechanism.

Sponges (phylum Porifera) are the phylogenetically oldest metazoan animals, their evolution dating back to 600 million years ago. Here we demonstrate that sponges express ADP-ribosyl cyclase activity, which converts NAD + into cyclic ADP-ribose, a potent and universal intracellular Ca 2+ mobilizer. In Axinella polypoides (Demospongiae, Axinellidae), ADP-ribosyl cyclase was activated by temperature increases by means of an abscisic acid-induced, protein kinase A-dependent mechanism. The thermosensor triggering this signaling cascade was a heat-activated cation channel. Elucidation of the complete thermosensing pathway in sponges highlights a number of features conserved in higher organisms: ( i ) the cation channel thermoreceptor, sensitive to heat, mechanical stress, phosphorylation, and anesthetics, shares all of the functional characteristics of the mammalian heat-activated background K + channel responsible for central and peripheral thermosensing; ( ii ) involvement of the phytohormone abscisic acid and cyclic ADP-ribose as its second messenger is reminiscent of the drought stress signaling pathway in plants. These results suggest an ancient evolutionary origin of this stress-signaling cascade in a common precursor of modern Metazoa and Metaphyta.