Michela Matteoli

Astrocyte-Derived ATP Induces Vesicle Shedding and IL-1β Release from Microglia

ATP has been indicated as a primary factor in microglial response to brain injury and inflammation. By acting on different purinergic receptors 2, ATP is known to induce chemotaxis and stimulate the release of several cytokines from these cells. The activation of purinergic receptors 2 in microglia can be triggered either by ATP deriving from dying cells, at sites of brain injury or by ATP released from astrocytes, in the absence of cell damage. By the use of a biochemical approach integrated with video microscopy experiments, we investigated the functional consequences triggered in microglia by ATP released from mechanically stimulated astrocytes, in mixed glial cocultures. Astrocyte-derived ATP induced in nearby microglia the formation and the shedding of membrane vesicles. Vesicle formation was inhibited by the ATP-degrading enzyme apyrase or by P2X7R antagonists. Isolation of shed vesicles, followed by IL-1β evaluation by a specific ELISA revealed the presence of the cytokine inside the vesicular organelles and its subsequent efflux into the extracellular medium. IL-1β efflux from shed vesicles was enhanced by ATP stimulation and inhibited by pretreatment with the P2X7 antagonist oxidized ATP, thus indicating a crucial involvement of the pore-forming P2X7R in the release of the cytokine. Our data identify astrocyte-derived ATP as the endogenous factor responsible for microvesicle shedding in microglia and reveal the mechanisms by which astrocyte-derived ATP triggers IL-1β release from these cells.

Hardwiring the Brain: Endocannabinoids Shape Neuronal Connectivity

The roles of endocannabinoid signaling during central nervous system development are unknown. We report that CB1 cannabinoid receptors (CB1Rs) are enriched in the axonal growth cones of γ-aminobutyric acid–containing (GABAergic) interneurons in the rodent cortex during late gestation. Endocannabinoids trigger CB1R internalization and elimination from filopodia and induce chemorepulsion and collapse of axonal growth cones of these GABAergic interneurons by activating RhoA. Similarly, endocannabinoids diminish the galvanotropism of Xenopus laevis spinal neurons. These findings, together with the impaired target selection of cortical GABAergic interneurons lacking CB1Rs, identify endocannabinoids as axon guidance cues and demonstrate that endocannabinoid signaling regulates synaptogenesis and target selection in vivo.

Vesicular transmitter release from astrocytes

Astrocytes can release a variety of transmitters, including glutamate and ATP, in response to stimuli that induce increases in intracellular Ca2+ levels. This release occurs via a regulated, exocytotic pathway. As evidence of this, astrocytes express protein components of the vesicular secretory apparatus, including synaptobrevin 2, syntaxin, and SNAP‐23. Additionally, astrocytes possess vesicular organelles, the essential morphological elements required for regulated Ca2+‐dependent transmitter release. The location of specific exocytotic sites on these cells, however, remains to be unequivocally determined.

Acid sphingomyelinase activity triggers microparticle release from glial cells.

We have earlier shown that microglia, the immune cells of the CNS, release microparticles from cell plasma membrane after ATP stimulation. These vesicles contain and release IL‐1β, a crucial cytokine in CNS inflammatory events. In this study, we show that microparticles are also released by astrocytes and we get insights into the mechanism of their shedding. We show that, on activation of the ATP receptor P2X7, microparticle shedding is associated with rapid activation of acid sphingomyelinase, which moves to plasma membrane outer leaflet. ATP‐induced shedding and IL‐1β release are markedly reduced by the inhibition of acid sphingomyelinase, and completely blocked in glial cultures from acid sphingomyelinase knockout mice. We also show that p38 MAPK cascade is relevant for the whole process, as specific kinase inhibitors strongly reduce acid sphingomyelinase activation, microparticle shedding and IL‐1β release. Our results represent the first demonstration that activation of acid sphingomyelinase is necessary and sufficient for microparticle release from glial cells and define key molecular effectors of microparticle formation and IL‐1β release, thus, opening new strategies for the treatment of neuroinflammatory diseases.

Nucleotide-mediated calcium signaling in rat cortical astrocytes: Role of P2X and P2Y receptors

ATP is the dominant messenger for astrocyte‐to‐astrocyte calcium‐mediated communication. Definition of the exact ATP/P2 receptors in astrocytes and of their coupling to intracellular calcium ([Ca2+]i) has important implications for brain physiology and pathology. We show that, with the only exception of the P2X6 receptor, primary rat cortical astrocytes express all cloned ligand‐gated P2X (i.e., P2X1–5 and P2X7) and G‐protein‐coupled P2Y receptors (i.e., P2Y1, P2Y2, P2Y4, P2Y6, and P2Y12). These cells also express the P2Y‐like UDP‐glucose receptor, which has been recently recognized as the P2Y14 receptor. Single‐cell image analysis showed that only some of these receptors are coupled to [Ca2+]i. While ATP induced rapid and transient [Ca2+]i increases (counteracted by the P2 antagonists suramin, pyridoxal‐phosphate‐6‐azophenyl‐2′‐4′‐disulfonic acid and oxidized ATP), the P2X1/P2X3 agonist αβmeATP produced no changes. Conversely, the P2X7 agonist BzATP markedly increased [Ca2+]i; the presence and function of the P2X7 receptor was also confirmed by the formation of the P2X7 pore. ADP and 2meSADP also produced [Ca2+]i increases antagonized by the P2Y1 antagonist MRS2179. Some cells also responded to UTP but not to UDP. Significant responses to sugar‐nucleotides were also detected, which represents the first functional response reported for the putative P2Y14 receptor in a native system. Based on agonist preference of known P2 receptors, we conclude that, in rat astrocytes, ATP‐induced calcium rises are at least mediated by P2X7 and P2Y1 receptors; additional receptors (i.e., P2X2, P2X4, P2X5, P2Y2, P2Y4, and P2Y14) may also contribute.

ATP Mediates Calcium Signaling Between Astrocytes and Microglial Cells: Modulation by IFN-γ

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-γ. 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 P2X7 purinergic receptor on microglial cells and greatly increased membrane permeability, eventually leading to microglial apoptosis. IFN-γ increased ATP release and potentiated the P2X7-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.

SNAP-25 Modulation of Calcium Dynamics Underlies Differences in GABAergic and Glutamatergic Responsiveness to Depolarization

SNAP-25 is a component of the SNARE complex implicated in synaptic vesicle exocytosis. In this study, we demonstrate that hippocampal GABAergic synapses, both in culture and in brain, lack SNAP-25 and are resistant to the action of botulinum toxins type A and E, which cleave this SNARE protein. Relative to glutamatergic neurons, which express SNAP-25, GABAergic cells were characterized by a higher calcium responsiveness to depolarization. Exogenous expression of SNAP-25 in GABAergic interneurons lowered calcium responsiveness, and SNAP-25 silencing in glutamatergic neurons increased calcium elevations evoked by depolarization. Expression of SNAP-25(1-197) but not of SNAP-25(1-180) inhibited calcium responsiveness, pointing to the involvement of the 180-197 residues in the observed function. These data indicate that SNAP-25 is crucial for the regulation of intracellular calcium dynamics and, possibly, of network excitability. SNAP-25 is therefore a multifunctional protein that participates in exocytotic function both at the mechanistic and at the regulatory level.

LRRK2 Controls Synaptic Vesicle Storage and Mobilization within the Recycling Pool

Mutations in leucine-rich repeat kinase 2 (LRRK2) are the single most common cause of inherited Parkinsons disease. Little is known about its involvement in the pathogenesis of Parkinsons disease mainly because of the lack of knowledge about the physiological role of LRRK2. To determine the function of LRRK2, we studied the impact of short hairpin RNA-mediated silencing of LRRK2 expression in cortical neurons. Paired recording indicated that LRRK2 silencing affects evoked postsynaptic currents. Furthermore, LRRK2 silencing induces at the presynaptic site a redistribution of vesicles within the bouton, altered recycling dynamics, and increased vesicle kinetics. Accordingly, LRRK2 protein is present in the synaptosomal compartment of cortical neurons in which it interacts with several proteins involved in vesicular recycling. Our results suggest that LRRK2 modulates synaptic vesicle trafficking and distribution in neurons and in consequence participates in regulating the dynamics between vesicle pools inside the presynaptic bouton.

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 Ca2+, a treatment that was found to produce a large and sustained increase in intracellular calcium [Ca2+] 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 [Ca2+] i increase upon specific stimuli is present in cultured hippocampal astrocytes.

A Common Exocytotic Mechanism Mediates Axonal and Dendritic Outgrowth

Outgrowth of the dendrites and the axon is the basis of the establishment of the neuronal shape, and it requires addition of new membrane to both growing processes. It is not yet clear whether one or two exocytotic pathways are responsible for the respective outgrowth of axons and dendrites. We have previously shown that tetanus neurotoxin-insensitive vesicle-associated membrane protein (TI-VAMP) defines a novel network of tubulovesicular structures present both at the leading edge of elongating dendrites and axons of immature hippocampal neurons developing in primary culture and that TI-VAMP is an essential protein for neurite outgrowth in PC12 cells. Here we show that the expression of the N-terminal domain of TI-VAMP inhibits the outgrowth of both dendrites and axons in neurons in primary culture. This effect is more prominent at the earliest stages of the development of neurons in vitro. Expression of the N-terminal domain deleted form of TI-VAMP has the opposite effect. This constitutively active form of TI-VAMP localizes as the endogenous protein, particularly concentrating at the leading edge of growing axons. Our results suggest that a common exocytotic mechanism that relies on TI-VAMP mediates both axonal and dendritic outgrowth in developing neurons.