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- Cesare Usai is Research Associate of the Institute of Biophysics (IBF), National Research Council, Italy. He was born... moreCesare Usai is Research Associate of the Institute of Biophysics (IBF), National Research Council, Italy. He was born in 1944 and received his degree in Physics at the University of Genoa, Italy, in 1970. PHILIPS fellowship in 1971 and Researcher at the IBF since 1973. Visiting scientist, Dept. of Biophysics and Medical Physics, University of California, Berkeley, in 1985-86. Senior Researcher and Leader of the division “Biomembranes” of the IBF since 1989. Assistant director of IBF from 1992 to 2003. Professor on contract of the University of Genoa, Faculty of Mathematical, Physical and Natural Sciences from 1993 to 2010. His experience in advanced fluorescence microscopy and spectroscopy techniques has been applied to different biological problems: neural degeneration (Amiotrophic Lateral Sclerosis, Charcot-Marie Syndrome)edit
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... more
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 ne...
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Mesenchymal stromal/stem cells (MSCs) are characterized by neuroprotective, immunomodulatory, and neuroregenerative properties, which support their therapeutic potential for inflammatory/neurodegenerative diseases, including multiple... more
Mesenchymal stromal/stem cells (MSCs) are characterized by neuroprotective, immunomodulatory, and neuroregenerative properties, which support their therapeutic potential for inflammatory/neurodegenerative diseases, including multiple sclerosis (MS) and amyotrophic lateral sclerosis (ALS). One mode of action through which MSCs exert their immunomodulatory effects is release of extracellular vesicles that carry proteins, mRNAs, and microRNAs (miRNAs), which, once transferred, modify the function of target cells. We identified nine miRNAs significantly dysregulated in IFN-γ-primed MSCs, but present at different levels in their derived small extracellular vesicles (s-EV). We show that miR-467f and miR-466q modulate the pro-inflammatory phenotype of activated N9 microglia cells and of primary microglia acutely isolated from late symptomatic SOD1G93A mice, a murine ALS model, by downregulating Tnf and Il1b expression. Further analysis of the mode of action of miR-467f and miR-466q indicat...
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Background Mesenchymal stromal/stem cells (MSCs) are characterized by neuroprotective, immunomodulatory, and neuroregenerative properties, which support their therapeutic potential for neurodegenerative diseases driven by... more
Background Mesenchymal stromal/stem cells (MSCs) are characterized by neuroprotective, immunomodulatory, and neuroregenerative properties, which support their therapeutic potential for neurodegenerative diseases driven by microglia-associated inflammation, such as amyotrophic lateral sclerosis (ALS). One mode of action through which MSCs exert their immunomodulatory effects is the release of extracellular vesicles, including exosomes, that carry proteins, mRNAs, and microRNAs (miRNAs), which, once transferred, modify the function of target cells. We have investigated the role of miRNAs present in exosomes derived from IFN-γ-primed mouse MSCs in the modulation of microglia activation, and analysed their effect on target genes and signaling pathways. Methods We compared miRNA expression in IFN-γ-primed vs unprimed mouse MSCs by microarray and measured the levels of relevant miRNAs in their respective exosomes through RT-PCR. To assess the effect of dysregulated MSC-derived miRNAs, we ...
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The effect of GABA on glutamate release from astrocytes has been studied in healthy mice and in a murine transgenic model of amyotrophic lateral sclerosis (ALS), using mouse spinal cord gliosomes labeled with [(3)H]d-aspartate... more
The effect of GABA on glutamate release from astrocytes has been studied in healthy mice and in a murine transgenic model of amyotrophic lateral sclerosis (ALS), using mouse spinal cord gliosomes labeled with [(3)H]d-aspartate ([(3)H]d-ASP). GABA concentration-dependently evoked the release of [(3)H]d-ASP. The effect of GABA was not mimicked by GABA(A) or GABA(B) receptor agonists or counteracted by antagonists, excluding receptor involvement. However, it was prevented by the GABA transport inhibitor N-(4,4-phenyl-3-butenyl)-nipecotic acid (SKF 89976A), suggesting participation of GABA transporters type 1 (GAT1) placed on glutamate-releasing astrocyte-derived gliosomes. Accordingly, GAT1 co-expressed with glutamate-aspartate transporter (GLAST) and glutamate transporter type 1 (GLT1) in the majority of glial particles. [(3)H]d-aspartate release was Ca(2+)-independent and not blocked by the glutamate uptake inhibitor dl-threo-b-benzyloxyaspartic acid (dl-TBOA); instead, it was abrogated by the anion channel blockers niflumic acid and 5-nitro-2-(3-phenylpropylamino) benzoic acid (NPPB). The GAT1-mediated release of [(3)H]d-ASP was significantly enhanced in spinal cord gliosomes from the mouse model of ALS. This excessive [(3)H]d-ASP release was very precocious, largely preceding the onset of the disease symptoms. These data indicate that GAT1, GLAST and GLT1 coexist on the same gliosome in mouse spinal cord and that activation of GAT1 transporters elicits glutamate release by anion channel opening. This phenomenon might have pathological relevance, because [(3)H]d-ASP release is enhanced in experimental ALS.
Research Interests: Biology, Neurochemistry, Calcium, Medicine, Transgenic Mice, and 15 moreAmyotrophic Lateral Sclerosis, Humans, Mice, Animals, Spinal Cord, Astrocyte, Superoxide Dismutase, Mouse Model, Chelating Agents, Neurosciences, Glutamic Acid, Gene Expression Regulation, Glutamate Receptor, Gliosis, and Glutamate release
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ABSTRACT 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... more
ABSTRACT 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. HIGHLIGHTSThe presence of nanoscale TiO2 surface features enhances hBM‐MSC osteogenesis.Ca2+ homeostasis changes are involved in surface‐driven hBM‐MSC lineage commitment.2D and 3D morphological parameters provide insights into hBM‐MSC adhesion phase.
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Research Interests: Spinal Cord and Receptor
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Research Interests: Neuroscience, Chemistry, Medicine, Baclofen, Exocytosis, and 3 moreAgonist, Glutamate Receptor, and Gabaergic
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... more
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, ...
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The ecto-enzyme CD38 is gaining momentum as a novel therapeutic target for patients with hematological malignancies, with several anti-CD38 monoclonal antibodies in clinical trials with promising results. In chronic lymphocytic leukemia... more
The ecto-enzyme CD38 is gaining momentum as a novel therapeutic target for patients with hematological malignancies, with several anti-CD38 monoclonal antibodies in clinical trials with promising results. In chronic lymphocytic leukemia (CLL) CD38 is a marker of unfavorable prognosis and a central factor in the pathogenetic network underlying the disease: activation of CD38 regulates genetic pathways involved in proliferation and movement. Here we show that CD38 is enzymatically active in primary CLL cells and that its forced expression increases disease aggressiveness in a xenograft model. The effect is completely lost when using an enzyme-deficient version of CD38 with a single amino-acid mutation. Through the enzymatic conversion of NAD into ADPR (ADP-ribose) and cADPR (cyclic ADP-ribose), CD38 increases cytoplasmic Ca(2+) concentrations, positively influencing proliferation and signaling mediated via chemokine receptors or integrins. Consistently, inhibition of the enzymatic act...
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Connexin 43 (Cx43) hexameric hemichannels, recently demonstrated to mediate NAD(+) transport, functionally interact in the plasma membrane of several cells with the ectoenzyme CD38 that converts NAD(+) to the universal calcium mobilizer... more
Connexin 43 (Cx43) hexameric hemichannels, recently demonstrated to mediate NAD(+) transport, functionally interact in the plasma membrane of several cells with the ectoenzyme CD38 that converts NAD(+) to the universal calcium mobilizer cyclic ADP-ribose (cADPR). Here we demonstrate that functional uncoupling between CD38 and Cx43 in CD38-transfected 3T3 murine fibroblasts is paralleled by decreased [Ca(2+)](i) levels as a result of reduced intracellular conversion of NAD(+) to cADPR. A sharp inverse correlation emerged between [Ca(2+)](i) levels and NAD(+) transport (measured as influx into cells and as efflux therefrom), both in the CD38(+) cells (high [Ca(2+)](i), low transport) and in the CD38(-) fibroblasts (low [Ca(2+)](i), high transport). These differences were correlated with distinctive extents of Cx43 phosphorylation in the two cell populations, a lower phosphorylation with high NAD(+) transport (CD38(-) cells) and vice versa (CD38(+) cells). Conversion of NAD(+)-permeabl...
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The presynaptic α‐adrenoceptors located on the terminals of the cholinergic nerves of the guinea‐pig myenteric plexus have been characterized according to their sensitivities to α‐adrenoceptor agonists and antagonists. Electrical... more
The presynaptic α‐adrenoceptors located on the terminals of the cholinergic nerves of the guinea‐pig myenteric plexus have been characterized according to their sensitivities to α‐adrenoceptor agonists and antagonists. Electrical stimulation of the cholinergic nerves supplying the longitudinal muscle of the guinea‐pig ileum caused a twitch response. Clonidine caused a concentration‐dependent inhibition of the twitch response; the maximum inhibition obtained was 80 to 95% of the twitch response. Oxymetazoline and xylazine were qualitatively similar to clonidine but were about 5 times less potent. Phenylephrine and methoxamine also inhibited the twitch response but were at least 10,000 times less potent than clonidine. The twitch‐inhibitory effects of clonidine, oxymetazoline and xylazine, but not those of phenylephrine or methoxamine, were reversed by piperoxan (0.3 to 1.0 μg/ml). Lysergic acid diethylamide (LSD) inhibited the twitch response, but also increased the basal tone of the...
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Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease characterized by progressive degeneration of motor neurons (MNs). Astrocytes display a toxic phenotype in ALS, which results in MN damage. Glutamate (Glu)-mediated... more
Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease characterized by progressive degeneration of motor neurons (MNs). Astrocytes display a toxic phenotype in ALS, which results in MN damage. Glutamate (Glu)-mediated excitotoxicity and group I metabotropic glutamate receptors (mGluRs) play a pathological role in the disease progression. We previously demonstrated that in vivo genetic ablation or pharmacological modulation of mGluR5 reduced astrocyte activation and MN death, prolonged survival and ameliorated the clinical progression in the SOD1G93A mouse model of ALS. This study aimed to investigate in vitro the effects of mGluR5 downregulation on the reactive spinal cord astrocytes cultured from adult late symptomatic SOD1G93A mice. We observed that mGluR5 downregulation in SOD1G93A astrocytes diminished the cytosolic Ca2+ overload under resting conditions and after mGluR5 simulation and reduced the expression of the reactive glial markers GFAP, S100β and viment...
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Isomorphic mutation of SBDS gene is the cause of Shwachman-Diamond syndrome (SDS). SDS is a rare genetic bone marrow failure and cancer predisposition syndrome. SDS cells have altered ribosome biogenesis and protein synthesis, two... more
Isomorphic mutation of SBDS gene is the cause of Shwachman-Diamond syndrome (SDS). SDS is a rare genetic bone marrow failure and cancer predisposition syndrome. SDS cells have altered ribosome biogenesis and protein synthesis, two high-energy consuming cellular processes. The reported increment in reactive oxygen species production, endoplasmic reticulum stress response and reduced mitochondrial functionality suggest a defect in the energy production in SDS cells. In this study, we analyzed the energetic metabolism in SDS cells and find that the oxygen consumption is impaired when it is induced by pyruvate/malate or succinate. This induces poor ATP production and AMP accumulation with a consequent alteration in the ATP/AMP ratio. Also respiratory chain activity was impaired because of faulty function of the complex IV; this defect is not dependent from impaired protein synthesis despite ribosome biogenesis and transduction defects in SDS. In fact, COX5A and Cox2, two subunits of Com...
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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... more
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 (TiO2) 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 TiO2 (with clusters under 100 nm of dimension) was investigated. TiO2-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 TiO2-seeded cells osteogenesis by ...
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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... more
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 ...
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Research Interests: Signal Transduction, Cell line, Brain, G protein-coupled receptors, Female, and 14 moreAnimals, Microglia, NF-kappa B, Nicotinic Acetylcholine Receptors, Clinical Sciences, SYNAPSES, Experimental Autoimmune Encephalomyelitis, Sirtuin, Neurosciences, Glutamic Acid, Neuroprotective Agents, Excitatory Postsynaptic Potentials, Tissue culture techniques, and G Protein Coupled Receptors
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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... more
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/glycin...
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In this study we report for the first time the localization of a photoreceptor pigment in the cilia of the colored heterotrich ciliates Blepharisma japonicum red and blue form, Fabrea salina, and Stentor coeruleus, as result of a confocal... more
In this study we report for the first time the localization of a photoreceptor pigment in the cilia of the colored heterotrich ciliates Blepharisma japonicum red and blue form, Fabrea salina, and Stentor coeruleus, as result of a confocal microscopy investigation. Optical sectioning confocal microscopy has been used for studying the spatial distribution of the pigment in the cell body, surprisingly showing that, besides its expected presence in the cortical region immediately below the cell membrane, it is located in the cilia too. In order to ascertain possible differences in the pigment fluorescence properties along the cell body, we have measured emission spectra from different parts of it (anterior, posterior, and cilia). Our results clearly indicate that in all cases the spectra are the same, within experimental errors. Finally, we have evaluated the pigment relative fluorescence efficiency of these ciliates. In an ordered scale from lower to greater efficiency, we have S. coeruleus, B. japonicum blue, B. japonicum red, and F. salina. The possible implications of our findings for the process of photosensory transduction are discussed.
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Research Interests: Neuroscience, Biophysics, Chemistry, Neuroethology, Biology, and 15 moreMedicine, Comparative Physiology, Anisotropy, Biological Sciences, Calcium channels, Natural Product, Nickel, Cadmium, Analysis of Variance, Model System, Membrane Fluidity, Paramecium, Calcium Channel Blockers, Cell Membrane, and Medical and Health Sciences
GABAB receptors modulate swimming behavior in Paramecium by inhibiting dihydropyridine-sensitive Ca2+ channels via G-proteins. Prolonged occupancy of GABAB receptors by baclofen results in a decrease in GABAB receptor functions. Since... more
GABAB receptors modulate swimming behavior in Paramecium by inhibiting dihydropyridine-sensitive Ca2+ channels via G-proteins. Prolonged occupancy of GABAB receptors by baclofen results in a decrease in GABAB receptor functions. Since changes in the number of cell-surface GABAA receptors have been postulated to be of importance in modulating inhibitory synaptic transmission in neurons, we have studied the cell-surface expression and maintenance of GABAB receptors in P. primaurelia. In this study, we use immunostaining in electron and confocal microscopy to demonstrate that constitutive internalization of GABAB receptors in P. primaurelia is mediated by clathrin-dependent and -independent endocytosis. Indeed, GABAB receptors colocalize with the adaptin complex AP2, which is implicated in the selective recruitment of integral membrane proteins to clathrin-coated vesicles, and with caveolin 1, which is associated with uncoated membrane invaginations. Furthermore, when endocytosis is bl...