Fabio Bianco

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.

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.

A role for P2X7 in microglial proliferation

Microglia, glial cells with an immunocompetent role in the CNS, react to stimuli from the surrounding environment with alterations of their phenotypic response. Amongst other activating signals, the endotoxin lipopolysaccharide (LPS) is widely used as a tool to mimic bacterial infection in the CNS. LPS‐activated microglia undergo dramatic changes in cell morphology/activity; in particular, they stop proliferating and differentiate from resting to effector cells. Activated microglia also show modifications of purinoreceptor signalling with a significant decrease in P2X7 expression. In this study, we demonstrate that the down‐regulation of the P2X7 receptor in activated microglia may play an important role in the antiproliferative effect of LPS. Indeed, chronic blockade of the P2X7 receptor by antagonists (oxidized ATP, KN62 and Brilliant Blue G), or treatment with the ATP‐hydrolase apyrase, severely decreases microglial proliferation, down‐regulation of P2X7 receptor expression by small RNA interference (siRNA) decreases cell proliferation, and the proliferation of P2X7‐deficient N9 clones and primary microglia, in which P2X7 expression is down‐regulated by siRNA, is unaffected by either LPS or P2X7 antagonists. Furthermore, flow cytometric analysis indicates that exposure to oxidized ATP or treatment with LPS reversibly decreases cell cycle progression, without increasing the percentage of apoptotic cells. Overall, our data show that the P2X7 receptor plays an important role in controlling microglial proliferation by supporting cell cycle progression.

Microglial microvesicle secretion and intercellular signaling.

Microvesicles (MVs) are released from almost all cell brain types into the microenvironment and are emerging as a novel way of cell-to-cell communication. This review focuses on MVs discharged by microglial cells, the brain resident myeloid cells, which comprise ∼10–12% of brain population. We summarize first evidence indicating that MV shedding is a process activated by the ATP receptor P2X7 and that shed MVs represent a secretory pathway for the inflammatory cytokine IL-β. We then discuss subsequent findings which clarify how IL-1 β can be locally processed and released from MVs into the extracellular environment. In addition, we describe the current understanding about the mechanism of P2X7-dependent MV formation and membrane abscission, which, by involving sphingomyelinase activity and ceramide formation, may share similarities with exosome biogenesis. Finally we report our recent results which show that microglia-derived MVs can stimulate neuronal activity and participate to the propagation of inflammatory signals, and suggest new areas for future investigation.

The surface-exposed chaperone, Hsp60, is an agonist of the microglial TREM2 receptor

Triggering receptor expressed in myeloid (TREM) cells 2, a receptor expressed by myeloid cells, osteoclasts and microglia, is known to play a protective role in bones and brain. Mutations of the receptor (or of its coupling protein, DAP12) sustain in fact a genetic disease affecting the two organs, the polycystic lipomembraneous osteodysplasia with sclerosing leukoencephalopathy (PLOSL or Nasu‐Hakola disease). So far, specific agonist(s) of TREM2 have not been identified and its (their) transduction mechanisms are largely unknown. Heat shock protein 60 (Hsp60) is a mitochondrial chaperone that can also be harboured at the cell surface. By using constructs including the extracellular domain of TREM2 and the Fc domain of IgGs we have identified Hsp60 as the only TREM2‐binding protein exposed at the surface of neuroblastoma N2A cells and astrocytes, and lacking in U373 astrocytoma. Treatment with Hsp60 was found to stimulate the best known TREM2‐dependent process, phagocytosis, however, only in the microglial N9 cells rich in the receptor. Upon TREM2 down‐regulation, the Hsp60‐induced stimulation of N9 phagocytosis was greatly attenuated. Hsp60 is also released by many cell types, segregated within exosomes or shedding vesicles which might then undergo dissolution. However, the affinity of its binding (Kd = 3.8 μM) might be too low for the soluble chaperone released from the vesicles to the extracellular space to induce a significant activation of TREM2. It might in contrast be appropriate for the binding of TREM2 to Hsp60 exposed at the surface of cells closely interacting with microglia. The ensuing stimulation of phagocytosis could play protective effects on the brain.

Different properties of P2X7 receptor in hippocampal and cortical astrocytes

P2X7 receptor is a ligand-gated ion channel, which can induce the opening of large membrane pores. Here, we provide evidence that the receptor induces pore formation in astrocytes cultured from cortex, but not from the hippocampus. Furthermore, P2X7 receptor activation promptly induces p38 mitogen-activated protein kinase (MAPK) phosphorylation in cortical but not in hippocampal astrocytes. Given the role of p38 MAPK activation in pore opening, these data suggest that defective coupling of the receptor to the enzyme could occur in hippocampal cultures. The different capabilities of the receptor to open membrane pores cause relevant functional consequences. Upon pore formation, caspase-1 is activated and pro-IL1-β is cleaved and released extracellularly. The receptor stimulation does not result in interleukin-1beta secretion from hippocampal astrocytes, although the pro-cytokine is present in the cytosol of lipopolysaccharide-primed cultures. These results open the possibility that activation of P2X7 receptors differently influences the neuroinflammatory processes in distinct brain regions.

Engineering injured spinal cord with bone marrow-derived stem cells and hydrogel-based matrices: a glance at the state of the art.

Concerning the broad topic of neural tissue engineering, we present a review relating to the state of the art in spinal cord injury repair strategies. Particular attention is given to spinal cord damage causes and effects, in neural and mesenchymal stem cell therapeutic approaches, in the use of hydrogels as cell carriers and in the mathematical modeling of involved phenomena. High importance is given to multidisciplinary strategies applied to spinal cord repair, since new research frontiers are believed to be now on 3D gel/cells and neuroprotective agent constructs for neural reconstruction purposes.

Hydrogel for cell housing in the brain and in the spinal cord

Purpose Neurons in the adult mammalian central nervous system do not proliferate or renew themselves and consequently strong interest in cell replacement therapies to repair brain and spinal cord damages has emerged in the last decade. Methods An injectable resorbable hydrogel with a controlled nanostructure, specifically designed for neural cell housing, was developed together with a new protocol for building three-dimensional (3D) biohybrid cell/hydrogel constructs: cells are housed within the polymeric matrix which is directly built with a specific cell culture media. This matrix was tested with standard glial populations, primary astrocytes and mesenchymal stem cells. Results Physico-chemical characterization of the hydrogel matrix confirmed a 2- week (+ 2 days) stability before massive degradation; mean mesh size of about 5 nm and thixotropic behavior with transition yield stress at 60+5 Pa. Cell survival within the hydrogel resulted in about 55±5% (minimum value) survivals, data also confirmed by optical assessments. Cell viability also remained high after extraction from the gel, indicating survival to inclusion latency period. Conclusions Since the intimate structure of the gel mimics extracellular matrix cells as would be expected to be found in an in vivo context, this polymeric formulation is a promising base for building 3D constructs for neural cell housing, in which cells are embedded and kept alive directly from the time of polycondensation.

The Expression of GHS-R in Primary Neurons Is Dependent upon Maturation Stage and Regional Localization

Ghrelin is a hormone with a crucial role in the regulation of appetite, regulation of inflammation, glucose metabolism and cell proliferation. In the brain ghrelin neurons are located in the cortex (sensorimotor area, cingular gyrus), and the fibres of ghrelin neurons in hypothalamus project directly to the dorsal vagal complex (DVC). Ghrelin binds the growth hormone secretagogue receptor (GHS-R) a G-protein-coupled receptor with a widespread tissue distribution, indeed these receptors are localized both in nonnervous, organs/tissues (i.e. adipose tissue, myocardium, adrenals, gonads, lung, liver, arteries, stomach, pancreas, thyroid, and kidney) as well as in central nervous system (CNS) and higher levels of expression in the pituitary gland and the hypothalamus and lower levels of expression in other organs, including brain. A GHS-R specific monoclonal antibody has been developed and characterized and through it we demonstrate that GHS-R is expressed in primary neurons and that its expression is dependent upon their developmental stage and shows differences according to the brain region involved, with a more pronounced expression in hippocampal rather than cortical neurons. A characterization of GHS-R within the central nervous system is of extreme importance in order to gain insights on its role in the modulation of neurodegenerative events such as Alzheimer’s disease.

A microfluidic device for depositing and addressing two cell populations with intercellular population communication capability

We present a method for depositing cells in the microchambers of a sealed microfluidic device and establishing flow across the chambers independently and serially. The device comprises a transparent poly(dimethylsiloxane) (PDMS) microfluidic network (MFN) having 2 cell chambers with a volume of 0.49 µL, 6 microchannels for servicing the chambers, and 1 microchannel linking both chambers. The MFN is sealed with a Si chip having 6 vias and ports that can be left open or connected to high-precision pumps. Liquids are drawn through each chamber in parallel or sequentially at flow rates from 0.1 to 10 µL min−1. Plugs of liquid as small as 0.5 µL can be passed in one chamber within 5 s to 5 min. Plugs of liquid can also be introduced into a chamber for residence times of up to 30 min. By injecting different liquids into 3 ports, 3 adjacent laminar streams of liquid can be drawn inside one chamber with lateral concentration gradients between the streams ranging from 20 to 500 µm. The flexibility of this device for depositing cells and exposing them to liquids in parallel or serially is illustrated by depositing two types of cells, murine N9 microglia and human SH-S5Y5 neuroblastoma. Microfluidic communication between the chambers is illustrated by stimulating N9 microglia using ATP to induce these cells to release plasma membrane vesicles. The vesicles are drawn through the second chamber containing neuroblastoma and collected in a port of the device for off-chip analysis using confocal fluorescence microscopy. Cells in the MFN can also be fixed using a solution of formaldehyde for further analysis after disassembly of the MFN and Si lid. This microfluidic device offers a simple, flexible, and powerful method for depositing two cell populations in separate chambers and may help investigating pathways between the cells populations.