Plasma microglial-derived extracellular vesicles are increased in frail patients with Mild Cognitive Impairment and exert a neurotoxic effect.

Extracellular vesicles (EVs) are mediators of cellular communication that can be released by almost all cell types in both physiological and pathological conditions and are present in most biological fluids. Such characteristics make them attractive in the research of biomarkers for age-related pathological conditions. Based on this, the aim of the present study was to examine the changes in EV concentration and size in the context of frailty, a geriatric syndrome associated with a progressive physical and cognitive decline. Specifically, total EVs and neural and microglial-derived EVs (NDVs and MDVs respectively) were investigated in plasma of frail and non-frail controls (CTRL), mild cognitive impairment (MCI) subjects, and in Alzheimer's disease (AD) patients. Results provided evidence that AD patients displayed diminished NDV concentration (3.61 × 109 ± 1.92 × 109 vs 7.16 × 109 ± 4.3 × 109 particles/ml) and showed high diagnostic performance. They are able to discriminate between AD and CTRL with an area under the curve of 0.80, a sensitivity of 78.95% and a specificity of 85.7%, considering the cut-off of 5.27 × 109 particles/ml. Importantly, we also found that MDV concentration was increased in frail MCI patients compared to CTRL (5.89 × 109 ± 3.98 × 109 vs 3.16 × 109 ± 3.04 × 109 particles/ml, P < 0.05) and showed high neurotoxic effect on neurons. MDV concentration discriminate frail MCI vs non-frail CTRL (AUC = 0.76) with a sensitivity of 80% and a specificity of 70%, considering the cut-off of 2.69 × 109 particles/ml. Altogether, these results demonstrated an alteration in NDV and MDV release during cognitive decline, providing important insight into the role of EVs in frailty status.

Testing Aß toxicity on primary CNS cultures using drug-screening microfluidic chips.

Open microscale cultures of primary central nervous system (CNS) cells have been implemented in microfluidic chips that can expose the cells to physiological fluidic shear stress conditions. Cells in the chips were exposed to differently aggregated forms of beta-amyloid (Aß), i.e. conditions mimicking an Alzheimer's Disease environment, and treated with CNS drugs in order to assess the contribution of glial cells during pharmacological treatments. FTY720, a drug approved for the treatment of Multiple Sclerosis, was found to play a marked neuroprotective role in neuronal cultures as well as in microglia-enriched neuronal cultures, preventing neurodegeneration after cell exposure to neurotoxic oligomers of Aß.

Microglia convert aggregated amyloid-ß into neurotoxic forms through the shedding of microvesicles.

Alzheimer's disease (AD) is characterized by extracellular amyloid-ß (Aß) deposition, which activates microglia, induces neuroinflammation and drives neurodegeneration. Recent evidence indicates that soluble pre-fibrillar Aß species, rather than insoluble fibrils, are the most toxic forms of Aß. Preventing soluble Aß formation represents, therefore, a major goal in AD. We investigated whether microvesicles (MVs) released extracellularly by reactive microglia may contribute to AD degeneration. We found that production of myeloid MVs, likely of microglial origin, is strikingly high in AD patients and in subjects with mild cognitive impairment and that AD MVs are toxic for cultured neurons. The mechanism responsible for MV neurotoxicity was defined in vitro using MVs produced by primary microglia. We demonstrated that neurotoxicity of MVs results from (i) the capability of MV lipids to promote formation of soluble Aß species from extracellular insoluble aggregates and (ii) from the presence of neurotoxic Aß forms trafficked to MVs after Aß internalization into microglia. MV neurotoxicity was neutralized by the Aß-interacting protein PrP and anti-Aß antibodies, which prevented binding to neurons of neurotoxic soluble Aß species. This study identifies microglia-derived MVs as a novel mechanism by which microglia participate in AD degeneration, and suggest new therapeutic strategies for the treatment of the disease.

Analysis of SNAP-25 immunoreactivity in hippocampal inhibitory neurons during development in culture and in situ.

Synaptosomal associated protein of 25 kDa (SNAP-25) is a component of the soluble N-ethylmaleimide-sensitive fusion protein (NSF) attachment protein receptor (SNARE) complex which plays a central role in synaptic vesicle exocytosis. We have previously demonstrated that adult rat hippocampal GABAergic synapses, both in culture and in brain, are virtually devoid of SNAP-25 immunoreactivity and are less sensitive to the action of botulinum toxin type A, which cleaves this SNARE protein [Neuron 41 (2004) 599]. In the present study, we extend our findings to the adult mouse hippocampus and we also provide demonstration that hippocampal inhibitory synapses lacking SNAP-25 labeling belong to parvalbumin-, calretinin- and cholecystokinin-positive interneurons. A partial colocalization between SNAP-25 and glutamic acid decarboxylase is instead detectable in developing mouse hippocampus at P0 and, at a lesser extent, at P5. In rat embryonic hippocampal cultures at early developmental stages, SNAP-25 immunoreactivity is detectable in a percentage of GABAergic neurons, which progressively reduces with time in culture. Consistent with the presence of the substrate, botulinum toxin type A is partially effective in inhibiting synaptic vesicle recycling in immature GABAergic neurons. Since SNAP-25, beside its role as a SNARE protein, is involved in additional processes, such as neurite outgrowth and regulation of calcium dynamics, the presence of higher levels of the protein at specific stages of neuronal differentiation may have implications for the construction and for the functional properties of brain circuits.

Evidence of a role for cyclic ADP-ribose in calcium signalling and neurotransmitter release in cultured astrocytes.

Astrocytes possess different, efficient ways to generate complex changes in intracellular calcium concentrations, which allow them to communicate with each other and to interact with adjacent neuronal cells. Here we show that cultured hippocampal astrocytes coexpress the ectoenzyme CD38, directly involved in the metabolism of the calcium mobilizer cyclic ADP-ribose, and the NAD+ transporter connexin 43. We also demonstrate that hippocampal astrocytes can release NAD+ and respond to extracellular NAD+ or cyclic ADP-ribose with intracellular calcium increases, suggesting the existence of an autocrine cyclic ADP-ribose-mediated signalling. Cyclic ADP-ribose-induced calcium changes are in turn responsible for an increased glutamate and GABA release, this effect being completely inhibited by the cyclic ADP-ribose specific antagonist 8-NH2-cADPR. Furthermore, addition of NAD+ to astrocyte-neuron co-cultures results in a delayed intracellular calcium transient in neuronal cells, which is strongly but not completely inhibited by glutamate receptor blockers. These data indicate that an astrocyte-to-neuron calcium signalling can be triggered by the CD38/cADPR system, which, through the activation of intracellular calcium responses in astrocytes, is in turn responsible for the increased release of neuromodulators from glial cells.

Chronic blockade of glutamate receptors enhances presynaptic release and downregulates the interaction between synaptophysin-synaptobrevin-vesicle-associated membrane protein 2.

During development of neuronal circuits, presynaptic and postsynaptic functions are adjusted in concert, to optimize interneuronal signaling. We have investigated whether activation of glutamate receptors affects presynaptic function during synapse formation, when constitutive synaptic vesicle recycling is downregulated. Using primary cultures of hippocampal neurons as a model system, we have found that chronic exposure to both NMDA and non-NMDA glutamate receptor blockers during synaptogenesis produces an increase in miniature EPSC (mEPSC) frequency, with no significant changes in mEPSC amplitude or in the number of synapses. Enhanced synaptic vesicle recycling, selectively in glutamatergic nerve terminals, was confirmed by the increased uptake of antibodies directed against the lumenal domain of synaptotagmin. No increased uptake was detected in neuronal cultures grown in the chronic presence of TTX, speaking against an indirect effect caused by decreased electrical activity. Enhanced mEPSC frequency correlated with a reduction of synaptophysin-synaptobrevin-vesicle-associated membrane protein 2 (VAMP2) complexes detectable by immunoprecipitation. Intracellular perfusion with a peptide that inhibits the binding of synaptophysin to synaptobrevin-VAMP2 induced a remarkable increase of mEPSC frequency in control but not in glutamate receptor blocker-treated neurons. These findings suggest that activation of glutamate receptors plays a role in the downregulation of the basal rate of synaptic vesicle recycling that accompanies synapse formation. They also suggest that one of the mechanisms through which this downregulation is achieved is an increased interaction of synaptophysin with synaptobrevin-VAMP2.

ATP mediates calcium signaling between astrocytes and microglial cells: modulation by IFN-gamma.

Calcium-mediated intercellular communication is a mechanism by which astrocytes communicate with each other and modulate the activity of adjacent cells, including neurons and oligodendrocytes. We have investigated whether microglia, the immune effector cells involved in several diseases of the CNS, are actively involved in this communication network. To address this issue, we analyzed calcium dynamics in fura-2-loaded cocultures of astrocytes and microglia under physiological conditions and in the presence of the inflammatory cytokine IFN-gamma. The intracellular calcium increases in astrocytes, occurring spontaneously or as a result of mechanical or bradykinin stimulation, induced the release of ATP, which, in turn, was responsible for triggering a delayed calcium response in microglial cells. Repeated stimulations of microglial cells by astrocyte-released ATP activated P2X(7) purinergic receptor on microglial cells and greatly increased membrane permeability, eventually leading to microglial apoptosis. IFN-gamma increased ATP release and potentiated the P2X(7)-mediated cytolytic effect. This is the first study showing that ATP mediates a form of calcium signaling between astrocytes and microglia. This mechanism of intercellular communication may be involved in controlling the number and function of microglial cells under pathophysiologic CNS conditions.

Different localizations and functions of L-type and N-type calcium channels during development of hippocampal neurons.

Using immunocytochemical assays and patch-clamp and calcium-imaging recordings, we demonstrate that L-type and N-type calcium channels have distinct patterns of expression and distribution and play different functional roles during hippocampal neuron differentiation. L-type channels, which support the depolarization-induced calcium influx in neurons from the very early developmental stages, are functionally restricted to the somatodendritic compartment throughout neuronal development and play a crucial role in supporting neurite outgrowth at early developmental stages. N-type channels, which start contributing at later neuronal differentiation stages (3-4 DIV), are also functionally expressed in the axons of immature neurons. At this developmental stage preceding synaptogenesis, N-type (but not L-type) channels are involved in controlling synaptic vesicle recycling. It is only at later developmental stages (10-12 DIV), when the neurons have established a clear axodendritic polarity and form synaptic contacts, that N-type channels are progressively excluded from the axon. Electrophysiological recordings of single neurons growing in microislands revealed that synaptic maturation coincides with a progressive increase in N-type channels in the somatodendritic region and a progressive decrease in the N-type channels supporting glutamate release from the presynaptic terminal. These results indicate that L-type and N-type calcium channels undergo dynamic, developmentally regulated rearrangements in regional distribution and function and also suggest that different mechanisms may be involved in the sorting and/or stabilization of these two types of channels in different plasma membrane domains during neuronal differentiation.

Synaptogenesis in hippocampal cultures.

Hippocampal cultures offer unique advantages for the study of neuronal development and synaptogenesis. Studies performed on this model enabled dissection of the temporal sequence of events which lead to the differentiation of pre- and postsynaptic compartments.

Tetanus toxin blocks the exocytosis of synaptic vesicles clustered at synapses but not of synaptic vesicles in isolated axons.

Recycling synaptic vesicles are already present in isolated axons of developing neurons (Matteoli et al., Zakharenko et al., 1999). This vesicle recycling is distinct from the vesicular traffic implicated in axon outgrowth. Formation of synaptic contacts coincides with a clustering of synaptic vesicles at the contact site and with a downregulation of their basal rate of exo-endocytosis (Kraszewski et al, 1995; Coco et al., 1998) We report here that tetanus toxin-mediated cleavage of synaptobrevin/vesicle-associated membrane protein (VAMP2), previously shown not to affect axon outgrowth, also does not inhibit synaptic vesicle exocytosis in isolated axons, despite its potent blocking effect on their exocytosis at synapses. This differential effect of tetanus toxin could be seen even on different branches of a same neuron. In contrast, botulinum toxins A and E [which cleave synaptosome-associated protein of 25 kDa. (SNAP-25)] and F (which cleaves synaptobrevin/VAMP1 and 2) blocked synaptic vesicle exocytosis both in isolated axons and at synapses, strongly suggesting that this process is dependent on "classical" synaptic SNAP receptor (SNARE) complexes both before and after synaptogenesis. A tetanus toxin-resistant form of synaptic vesicle recycling, which proceeds in the absence of external stimuli and is sensitive to botulinum toxin F, E, and A, persists at mature synapses. These data suggest the involvement of a tetanus toxin-resistant, but botulinum F-sensitive, isoform of synaptobrevin/VAMP in synaptic vesicle exocytosis before synapse formation and the partial persistence of this form of exocytosis at mature synaptic contacts.

Astrocytes are required for the oscillatory activity in cultured hippocampal neurons.

Synchronous oscillations of intracellular calcium concentration ([Ca2+]i) and of membrane potential occurred in a limited population of glutamatergic hippocampal neurons grown in primary cultures. The oscillatory activity occurred in synaptically connected cells only when they were in the presence of astrocytes. Microcultures containing only one or a few neurons also displayed oscillatory activity, provided that glial cells participated in the network. The glutamate-transporter inhibitors L-trans-pyrrolidine-2, 4-dicarboxylic acid (PDC) and dihydrokainate, which produce an accumulation of glutamate in the synaptic microenvironment, impaired the oscillatory activity. Moreover, in neurons not spontaneously oscillating, though in the presence of astrocytes, oscillations were induced by exogenous L-glutamate, but not by the stereoisomer D-glutamate, which is not taken up by glutamate transporters. These data demonstrate that astrocytes are essential for neuronal oscillatory activity and provide evidence that removal of glutamate from the synaptic environment is one of the major mechanisms by which glial cells allow the repetitive excitation of the postsynaptic cell.

A regulated secretory pathway in cultured hippocampal astrocytes.

Glial cells have been reported to express molecules originally discovered in neuronal and neuroendocrine cells, such as neuropeptides, neuropeptide processing enzymes, and ionic channels. To verify whether astrocytes may have regulated secretory vesicles, the primary cultures prepared from hippocampi of embryonic and neonatal rats were used to investigate the subcellular localization and secretory pathway followed by secretogranin II, a well known marker for dense-core granules. By indirect immunofluorescence, SgII was detected in a large number of cultured hippocampal astrocytes. Immunoreactivity for the granin was detected in the Golgi complex and in a population of dense-core vesicles stored in the cells. Subcellular fractionation experiments revealed that SgII was stored in a vesicle population with a density identical to that of the dense-core secretory granules present in rat pheochromocytoma cells. In line with these data, biochemical results indicated that 40-50% of secretogranin II synthesized during 18-h labeling was retained intracellularly over a 4-h chase period and released after treatment with different secretagogues. The most effective stimulus appeared to be phorbol ester in combination with ionomycin in the presence of extracellular Ca(2+), a treatment that was found to produce a large and sustained increase in intracellular calcium [Ca(2+)](i) transients. Our findings indicate that a regulated secretory pathway characterized by (i) the expression and stimulated exocytosis of a typical marker for regulated secretory granules, (ii) the presence of dense-core vesicles, and (iii) the ability to undergo [Ca(2+)](i) increase upon specific stimuli is present in cultured hippocampal astrocytes.

Internalization and proteolytic action of botulinum toxins in CNS neurons and astrocytes.

Tetanus and botulinum toxins bind and are internalized at the neuromuscular junction. Botulinum neurotoxins (BoNTs) enter the cytosol at the motor nerve terminal; tetanus neurotoxin (TeNT) proceeds retroaxonally inside the motor axon to reach the spinal cord inhibitory interneurons. Although the major target of BoNTs is the peripheral cholinergic terminals, CNS neurons are susceptible to intoxication as well. We investigated the route of entry and the proteolytic activity of BoNT/B and BoNT/F in cultured hippocampal neurons and astrocytes. We show that, differently from TeNT, which enters hippocampal neurons via the process of synaptic vesicle (SV) recycling, BoNTs are internalized and cleave the substrate synaptobrevin/VAMP2 via a process independent of synaptic activity. Labeling of living neurons with Texas Red-conjugated BoNTs and fluoresceinated dextran revealed that these toxins enter hippocampal neurons via endocytic processes not mediated by SV recycling. Botulinum toxins also exploit endocytosis to enter cultured astrocytes, where they partially cleave cellubrevin, a ubiquitous synaptobrevin/VAMP isoform. These results indicate that, in spite of their closely related protein structure, TeNT and BoNTs use different routes to penetrate hippocampal neurons. These findings bear important implications for the identification of the protein receptors of clostridial toxins.

The role of glial cells in synaptic function.

Glial cells represent the most abundant cell population in the central nervous system and for years they have been thought to provide just structural and trophic support to neurons. Recently, several studies were performed, leading to the identification of an active interaction between glia and neurons. This paper focuses on the role played by glial cells at the level of the synapse, reviewing recent data defining how glia is determinant in synaptogenesis, in the modulation of fully working synaptic contacts and in synaptic plasticity.

Synaptic and intrinsic mechanisms shape synchronous oscillations in hippocampal neurons in culture.

We have detected spontaneous, synchronous calcium oscillations, associated with variations in membrane potential, in hippocampal neurons maintained in primary culture. The oscillatory activity is synaptically driven, as it is blocked by tetrodotoxin, by the glutamate receptor antagonist 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX) and by toxins inhibiting neurotransmitter release from presynaptic nerve endings. Neuronal oscillations do not require for their expression the presence of a polyneuronal network and are not primarily influenced by the gamma-aminobutyric acid (GABA(A)) receptor antagonist picrotoxin, suggesting that they entirely rely on glutamatergic neurotransmission. Synaptic and intrinsic conductances shape the synchronized oscillations in hippocampal neurons. The concomitant activation of N-methyl-D-aspartate (NMDA) receptors and voltage-activated L-type calcium channels allows calcium entering from the extracellular medium and sustaining the long depolarization, which shapes every single calcium wave.

Calcium dependence of synaptic vesicle recycling before and after synaptogenesis.

Using an immunocytochemical assay to monitor synaptic vesicle exocytosis/endocytosis independently of neurotransmitter release, we have investigated some aspects of vesicle recycling in hippocampal neurons at different developmental stages. A calcium- and depolarization-dependent exocytotic/endocytotic recycling of synaptic vesicles was found to take place in neurons already before the formation of synaptic contacts. The analysis of synaptic vesicle recycling at different calcium concentrations revealed the presence of two release components: the first one activated by low calcium concentrations and sustaining vesicle recycling before synaptogenesis, and a second one activated by high calcium concentrations, which is specifically turned on after the establishment of synaptic contacts. These data suggest that formation of synapses correlates with the activation of a putative low-affinity calcium sensor, which allows synaptic vesicle exocytosis to be triggered and turned off over extremely short time scales, in response to large increases in the level of intracellular calcium.

Non-synaptic localization of the glutamate transporter EAAC1 in cultured hippocampal neurons.

It has been postulated for several years that the high affinity neuronal glutamate uptake system plays a role in clearing glutamate from the synaptic cleft. Four different glutamate transporter subtypes are now identified, the major neuronal one being EAAC1. To be a good candidate for the reuptake of glutamate at the synaptic cleft, EAAC1 should be properly located at synapses, either at pre- or postsynaptic sites. We have investigated the distribution of EAAC1 in primary cultures of hippocampal neurons, which represent an advantageous model for the study of synaptogenesis and synaptic specializations. We have demonstrated that EAAC1 immunoreactivity is segregated in the somatodendritic compartment of fully differentiated hippocampal neurons, where it is localized in the dendritic shaft and in the spine neck, outside the area facing the active zone. No co-localization of EAAC1 immunoreactivity with the stainings produced by typical presynaptic and postsynaptic markers was ever observed, indicating that EAAC1 is not to be considered a synaptic protein. Accordingly, the developmental pattern of expression of EAAC1 was found to be different from that of typical synaptic markers. Moreover, EAAC1 was expressed in the somatodendritic compartment of hippocampal neurons already at stages preceding the formation of synaptic contacts, and was also expressed in GABAergic interneurons with identical subcellular distribution. Taken together, these data rule against a possible role for EAAC1 in the clearance of glutamate from within the cleft and in the regulation of its time in the synapse. They suggest an unconventional non-synaptic function of this high-affinity glutamate carrier, not restricted to glutamatergic fibres.

Synaptic vesicle endocytosis mediates the entry of tetanus neurotoxin into hippocampal neurons.

Tetanus neurotoxin causes the spastic paralysis of tetanus by blocking neurotransmitter release at inhibitory synapses of the spinal cord. This is due to the penetration of the toxin inside the neuronal cytosol where it cleaves specifically VAMP/synaptobrevin, an essential component of the neuroexocytosis apparatus. Here we show that tetanus neurotoxin is internalized inside the lumen of small synaptic vesicles following the process of vesicle reuptake. Vesicle acidification is essential for the toxin translocation in the cytosol, which results in the proteolytic cleavage of VAMP/ synaptobrevin and block of exocytosis.

Native nicotinic acetylcholine receptors in human Imr32 neuroblastoma cells: functional, immunological and pharmacological properties.

IMR32 cells express two classes of surface nicotinic receptors: those labelled with high affinity by [125I]neuronal toxin, and those labelled by [125I]alpha-bungarotoxin. Whole-cell patch-clamp recordings indicate that both classes of receptor are able to elicit inward currents that are totally blocked by d-tubocurarine but only partially blocked by alpha-bungarotoxin. In IMR32 cells, nicotine induces an increase in the intracellular level of free Ca2+. This increase, which is also completely blocked by d-tubocurarine and only partially blocked by alpha-bungarotoxin and Cd2+, is due to extracellular calcium influx through both the nicotinic receptors and the voltage-activated Ca2+ channels. By using subunit-specific polyclonal antibodies, we have demonstrated that the alpha-bungarotoxin receptors contain the alpha 7 subunit, but none of the other subunits whose transcripts are present in IMR32 cells. The pharmacological profile of these human alpha 7-containing alpha-bungarotoxin receptors is similar to that observed in the native chick alpha 7 receptor, but there are also some species-specific differences.