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Background and Purpose: To deepen our knowledge of the role of complement in synaptic impairment in experimental autoimmune encephalomyelitis (EAE) mice, we investigated the distribution of C1q and C3 proteins and the role of complement as a promoter of glutamate release in purified nerve endings (synaptosomes) and astrocytic processes (gliosomes) isolated from the cortex of EAE mice at the acute stage of the disease (21 ± 1 day post‐immunization). Experimental Approach: EAE cortical synaptosomes and gliosomes were analysed for glutamate release efficiency (measured as release of preloaded [3H]D‐aspartate ([3H]D‐ASP)), C1q and C3 protein density, and for viability and ongoing apoptosis. Key Results: In healthy mice, complement releases [3H]D‐ASP from gliosomes more efficiently than from synaptosomes. The releasing activity occurs in a dilution‐dependent manner and involves the reversal of the excitatory amino acid transporters (EAATs). In EAE mice, the complement‐induced releasing activity is significantly reduced in cortical synaptosomes but amplified in cortical gliosomes. These adaptations are paralleled by decreased density of the EAAT2 protein in synaptosomes and increased EAAT1 staining in gliosomes. Concomitantly, PSD95, GFAP, and CD11b, but not SNAP25, proteins are overexpressed in the cortex of the EAE mice. Similarly, C1q and C3 protein immunostaining is increased in EAE cortical synaptosomes and gliosomes, although signs of ongoing apoptosis or altered viability are not detectable. Conclusion and Implications: Our results unveil a new noncanonical role of complement in the CNS of EAE mice relevant to disease progression and central synaptopathy that suggests new therapeutic targets for the management of MS. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

J. Neurochem. (2012) 121, 428-437. Abstract The impact of Regulated upon Activation Normal T cells Expressed and Secreted (RANTES) on the release of pre-loaded [3H] d-aspartate ([3H] d-ASP) from mouse spinal cord synaptosomes was investigated. RANTES (0.01-1 nM) failed to affect the spontaneous release, but facilitated the 15 mM K+-evoked overflow of [3H] d-ASP. Incubation of synaptosomes with antibodies raised against the chemokine receptor (CCR)1 and CCR5 proteins prevented RANTES-induced facilitation of glutamate exocytosis, whereas anti-CCR3 antibody was inefficacious. Accordingly, BX513 and d-Ala-peptide T-amide (DAPTA) CCR1 and CCR5 antagonists, respectively, prevented RANTES-induced effect, whereas the CCR3 antagonist SB 328437 was inactive. To compare these findings to previous results, we quantified the effects of CCR antagonists on the RANTES-induced modifications of the spontaneous and the K+-evoked [3H] d-ASP release in the mouse cortex. Here, CCR1 and CCR5, but not CCR3, antagonists prevented the RANTES-mediated [3H] d-ASP release, whereas RANTES-induced inhibition of the 12 mM K+-evoked [3H] d-ASP exocytosis was also antagonized by SB 328437. Facilitation of glutamate exocytosis in spinal cord relied on PLC-dependent mobilization of Ca2+ from IP3-sensitive stores; adenylyl cyclase was not involved. CCR1, CCR3 and CCR5 receptor proteins were present in spinal cord synaptosomal and gliosomal lysates, although RANTES-induced changes to glutamate release could not be observed in gliosomes. Our results confirm the role of RANTES as modulator of glutamate transmission. [ABSTRACT FROM AUTHOR]

J. Neurochem. (2010) 115, 343-352. Abstract We have investigated the spontaneous and the depolarisation-induced release of [3H] d-aspartate ([3H] d-ASP), a non-metabolisable analogue of glutamate, in spinal cord slices, synaptosomes and gliosomes from mice with experimental autoimmune encephalomyelitis (EAE) at 13, 21 and 55 days post-immunisation (d.p.i.), representing onset, peak and chronic phases of the pathology. At 13 and 21 d.p.i., the KCl-evoked, calcium-dependent overflow of [3H] d-ASP in spinal cord slices was significantly lower (30-40%), whereas at 55 d.p.i. it was significantly higher (30%), than that elicited in matched controls. When the release was measured from spinal cord synaptosomes and gliosomes in superfusion, a different picture emerged. The spontaneous and the KCl(15 mM)-induced release of [3H] d-ASP were significantly increased both in synaptosomes (17% and 45%, respectively) and gliosomes (26% and 25%, respectively) at 21, but not at 13, d.p.i. At 55 d.p.i., the KCl-induced [3H] d-ASP release was significantly increased (40%) only in synaptosomes. Finally, uptake of [3H] d-ASP was markedly (50-60%) increased in spinal cord synaptosomes, but not in gliosomes, obtained from EAE mice at 21 d.p.i., whereas no differences could be detected at 13 d.p.i. Our data indicate that glutamatergic neurotransmission is altered in the spinal cord of EAE mice. [ABSTRACT FROM AUTHOR]

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

The multifunctional protein high mobility group box 1 (HMGB1) is expressed in hippocampus and cerebellum of adult mouse brain. Our aim was to determine whether HMGB1 affects glutamatergic transmission by monitoring neurotransmitter release from glial (gliosomes) and neuronal (synaptosomes) re-sealed subcellular particles isolated from cerebellum and hippocampus. HMGB1 induced release of the glutamate analogue [3H]d-aspartate form gliosomes in a concentration-dependent manner, whereas nerve terminals were insensitive to the protein. The HMGB1-evoked release of [3H]d-aspartate was independent of modifications of cytosolic Ca2+ , but it was blocked bydl-threo-β-benzyloxyaspartate (dl-TBOA), an inhibitor of glutamate transporters. HMGB1 also stimulated the release of endogenous glutamate in a Ca2+-independent anddl-TBOA-sensitive manner. These findings suggest the involvement of carrier-mediated release. Moreover, dihydrokainic acid, a selective inhibitor of glutamate transporter 1 (GLT1), does not block the effect of HMGB1, indicating a role for the glial glutamate-aspartate transporter (GLAST) subtype in this response. We also demonstrate that HMGB1/glial particles association is promoted by Ca2+. Furthermore, although HMGB1 can physically interact with GLAST and the receptor for advanced glycation end products (RAGE), only its binding with RAGE is promoted by Ca2+. These results suggest that the HMGB1 cytokine could act as a modulator of glutamate homeostasis in adult mammal brain. [ABSTRACT FROM AUTHOR]

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