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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]

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

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

Amyloid-β protein precursor (AβPP) is a large transmembrane protein highly expressed in the central nervous system and cleavage of it can produce amyloid-β peptides (Aβ) involved in synaptic dysfunction and loss associated with cognitive impairment in Alzheimer's disease (AD). Surprisingly, little is known about the synaptic and sub-synaptic distribution of AβPP in different types of nerve terminals. We used total, synaptic, sub-synaptic, and astrocytic membrane preparations obtained from the hippocampus of adult rats to define the localization of AβPP, using two different antibodies against different AβPP epitopes. Western blot analysis revealed that AβPP was not significantly enriched in synaptosomal as compared to total membranes. Within synapses, AβPP immunoreactivity was more abundant in pre- (60 ± 4%) than post- (30 ± 5%) or extra-synaptic fractions (10 ± 2%). Immunocytochemical analysis of purified nerve terminals indicated that AβPP was more frequently associated with glutamatergic (present in 31 ± 4% of glutamatergic terminals) rather than with GABAergic (16 ± 3%) or cholinergic terminals (4 ± 1%, n = 4). We also observed a general lack of co-localization of AβPP and GFAP immunoreactivities in the hippocampus of sections of adult rat brain, albeit we could detect the presence of AβPP in gliosomes (vesicular specializations of astrocytic membranes), suggesting that AβPP has a heterogeneous localization restricted to certain regions of astrocytes. These results provide the first direct demonstration that AβPP is mostly distributed among glutamatergic rather than GABAergic or cholinergic terminals of the adult rat hippocampus, in remarkable agreement with the particular susceptibility to dysfunction and degeneration of glutamatergic synapses in early AD. [ABSTRACT FROM AUTHOR]

Alzheimer's disease (AD) is characterized by a progressive cognitive impairment tightly correlated with the accumulation of amyloid-β (Aβ) peptides (mainly Aβ1-42). There is a precocious disruption of glutamatergic synapses in AD, in line with an ability of Aβ to decrease astrocytic glutamate uptake. Accumulating evidence indicates that caffeine prevents the burden of AD, likely through the antagonism of A2A receptors (A2AR) which attenuates Aβ-induced memory impairment and synaptotoxicity. Since A2AR also modulate astrocytic glutamate uptake, we now tested if A2AR blockade could prevent the decrease of astrocytic glutamate uptake caused by Aβ. In cultured astrocytes, Aβ1-42 (1 μM for 24 hours) triggered an astrogliosis typified by an increased density of GFAP, which was mimicked by the A2AR agonist, CGS 26180 (30 nM), and prevented by the A2AR antagonist, SCH 58261 (100 nM). Aβ1-42 also decreased D-aspartate uptake by 28 ± 4%, an effect abrogated upon genetic inactivation or pharmacological blockade of A2AR. In accordance with the long term control of glutamate transporter expression by A2AR, Aβ1-42 enhanced the expression and density of astrocytic A2AR and decreased GLAST and GLT-I expression in astrocytes from wild type, but not from A2AR knockout mice. This impact of Aβ1-42 on glutamate transporters and uptake, dependent on A2AR function, was also confirmed in an ex vivo astrocyte preparation (gliosomes) from rats intracerebroventricularly (icv) injected with Aβ1-42. These results provide the first demonstration for a direct key role of astrocytic A2AR in the ability of Aβ-induced impairment of glutamate uptake, which may underlie glutamatergic synaptic dysfunction and excitotoxicity in AD. [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]

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

The endocannabinoid system and endocannabinoid receptor-driven modulation of glutamate release were studied in rat brain cortex astroglial gliosomes. These preparations contained the endocannabinoids N-arachidonoylethanolamine (anandamide) and 2-arachidonoylglycerol, as well their major biosynthetic ( N-acyl-phosphatidylethanolamines-hydrolyzing-phospholipase D and diacylglycerol-lipase) and catabolic (fatty acid amide-hydrolase and monoacylglycerol-lipase) enzymes. Gliosomes expressed type-1 (CB1R), type-2 (CB2R) cannabinoid, and type-1 vanilloid (TRPV1) receptors, as ascertained by Western blotting and confocal microscopy. Methanandamide, a stable analogue of anandamide acting as CB1R, CB2R, and TRPV1 agonist, stimulated or inhibited the depolarization-evoked gliosomal [H] d-aspartate release, at lower and higher concentrations, respectively. Experiments with ACEA (arachidonyl-2′-chloroethylamide), JWH133 ((6aR,10aR)-3-(1,1-dimethylbutyl)-6a,7,10,10a-tetrahydro-6,6,9-trimethyl-6H-dibenzo[b,d]-pyran) and capsaicin, selective agonists at CB1R, CB2R and TRPV1, respectively, demonstrated that potentiation of [H] d-aspartate release was due to CB1R while inhibition to CB2R and TRPV1 engagement. These findings were confirmed by using selective receptor antagonists. Furthermore, CB1R activation caused increase of intracellular IP3 and Ca concentration, suggesting an involvement of phospholipase C. [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]

Abstract: 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. [Copyright &y& Elsevier]

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]

Abstract: The presence of nicotinic receptors on astrocytes in human and rat brain has been previously demonstrated however their possible functional role is still poorly understood. In this study we investigated on the presence of nicotinic receptors on gliosomes, purified from mouse cortex, and on their role in eliciting glutamate release. Epibatidine significantly increased basal release of [3H]d-aspartate and of endogenous glutamate from mouse gliosomes but not from synaptosomes. This effect was prevented by methyllycaconitine, α-bungarotoxin and mecamylamine but not by dihydro-β-erythroidine. Epibatidine provoked also a significant increase of calcium concentration in gliosomes but not in synaptosomes; the increase in [Ca2+] i induced by epibatidine and KCl in gliosomes was very similar to each other. The present results indicate that α7 nicotinic receptors exist on mouse cortical glial particles and stimulate glutamate release. [Copyright &y& Elsevier]

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]

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 G93A 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 SOD1 G93A 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 SOD1 G93A 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., (© 2019 International Society for Neurochemistry.)