Stefania Ceruti

Astrocytes in the damaged brain: molecular and cellular insights into their reactive response and healing potential

Long considered merely a trophic and mechanical support to neurons, astrocytes have progressively taken the center stage as their ability to react to acute and chronic neurodegenerative situations became increasingly clear. Reactive astrogliosis starts when trigger molecules produced at the injury site drive astrocytes to leave their quiescent state and become activated. Distinctive morphological and biochemical features characterize this process (cell hypertrophy, upregulation of intermediate filaments, and increased cell proliferation). Moreover, reactive astrocytes migrate towards the injured area to constitute the glial scar, and release factors mediating the tissue inflammatory response and remodeling after lesion. A novel view of astrogliosis derives from the finding that subsets of reactive astrocytes can recapitulate stem cell/progenitor features after damage, fostering the concept of astroglia as a promising target for reparative therapies. But which biochemical/signaling pathways modulate astrogliosis with respect to both the time after injury and the type of damage? Are reactive astrocytes overall beneficial or detrimental for neuroprotection and tissue regeneration? This debate has been animating this research field for several years now, and an integrated view on the results obtained and the possible future perspectives is needed. With this Commentary article we have attempted to answer the above-mentioned questions by reviewing the current knowledge on the molecular mechanisms controlling and sustaining the reaction of astroglia to injury and its stem cell-like properties. Moreover, the cellular/molecular mechanisms supporting the detrimental or beneficial features of astrogliosis have been scrutinized to gain insights on possible pharmacological approaches to enhance astrocyte neuroprotective activities.

Adenosine Receptors and Neurological Disease: Neuroprotection and Neurodegeneration

Adenosine receptors modulate neuronal and synaptic function in a range of ways that may make them relevant to the occurrence, development and treatment of brain ischemic damage and degenerative disorders. A(1) adenosine receptors tend to suppress neural activity by a predominantly presynaptic action, while A(2A) adenosine receptors are more likely to promote transmitter release and postsynaptic depolarization. A variety of interactions have also been described in which adenosine A(1) or A(2) adenosine receptors can modify cellular responses to conventional neurotransmitters or receptor agonists such as glutamate, NMDA, nitric oxide and P2 purine receptors. Part of the role of adenosine receptors seems to be in the regulation of inflammatory processes that often occur in the aftermath of a major insult or disease process. All of the adenosine receptors can modulate the release of cytokines such as interleukins and tumor necrosis factor-alpha from immune-competent leukocytes and glia. When examined directly as modifiers of brain damage, A(1) adenosine receptor (AR) agonists, A(2A)AR agonists and antagonists, as well as A(3)AR antagonists, can protect against a range of insults, both in vitro and in vivo. Intriguingly, acute and chronic treatments with these ligands can often produce diametrically opposite effects on damage outcome, probably resulting from adaptational changes in receptor number or properties. In some cases molecular approaches have identified the involvement of ERK and GSK-3beta pathways in the protection from damage. Much evidence argues for a role of adenosine receptors in neurological disease. Receptor densities are altered in patients with Alzheimers disease, while many studies have demonstrated effects of adenosine and its antagonists on synaptic plasticity in vitro, or on learning adequacy in vivo. The combined effects of adenosine on neuronal viability and inflammatory processes have also led to considerations of their roles in Lesch-Nyhan syndrome, Creutzfeldt-Jakob disease, Huntingtons disease and multiple sclerosis, as well as the brain damage associated with stroke. In addition to the potential pathological relevance of adenosine receptors, there are earnest attempts in progress to generate ligands that will target adenosine receptors as therapeutic agents to treat some of these disorders.

Blockade of A2A adenosine receptors prevents basic fibroblast growth factor‐induced reactive astrogliosis in rat striatal primary astrocytes

Previous literature data show that blockade of A2A adenosine receptors via selective antagonists induces protection in various models of neurodegenerative diseases. The mechanisms underlying this effect are still largely unknown. Since it is known that excessive reactive astrogliosis is a factor contributing to cell death in diseases characterized by neurodegenerative events, the present study has been aimed at determining whether selective A2A receptor antagonists can counteract the formation of reactive astrocytes induced in vitro by basic fibroblast growth factor (bFGF), a typical trigger of this reaction. Exposure of primary rat striatal astrocytes to the selective A2A antagonist SCH58261 resulted in concentration‐dependent abolition of bFGF induction of astrogliosis in vitro. This effect could also be reproduced with the chemically unrelated A2A antagonist KW‐6002. The direct activation of A2A adenosine receptors by selective receptor agonists was not sufficient per se to induce astrogliosis, suggesting that the A2A receptor needs to act in concert with other bFGF‐induced genes to trigger the formation of reactive astrocytes. These results provide a mechanism at the basis of the neuroprotection induced by A2A receptor antagonists in models of brain damage and highlight this adenosine receptor subtype as a novel target for the pharmacological modulation of the gliotic reaction.

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.

Characterization of the signalling pathways involved in ATP and basic fibroblast growth factor‐induced astrogliosis

1 A brief challenge of rat astrocytes with either α,β‐methyleneATP (α,β‐meATP) or basic fibroblast growth factor (bFGF) resulted, three days later, in morphological differentiation of cells, as shown by marked elongation of astrocytic processes. The P2 receptor antagonist suramin prevented α,β‐meATP‐ but not bFGF‐induced astrocytic elongation. Similar effects on astrocytic elongation were also observed with ATP and other P2 receptor agonists (β,γmeATP, ADPβS, 2meSATP and, to a lesser extent, UTP). 2 Pertussis toxin completely abolished α,β‐meATP‐ but not bFGF‐induced effects. No effects were exerted by α,β‐meATP on cyclic AMP production; similarly, neomycin had no effects on elongation of processes induced by the purine analogue, suggesting that adenylyl cyclase and phospholipase C are probably not involved in α,β‐meATP‐induced effects (see also the accompanying paper by Centemeri et al., 1997 ). The tyrosine‐kinase inhibitor genistein greatly reduced bFGF‐ but not α,β‐meATP‐induced astrocytic elongation. 3 Challenge of cultures with α,β‐meATP rapidly and concentration‐dependently increased [3H]‐arachidonic acid (AA) release from cells, suggesting that activation of phospholipase A2 (PLA2) may be involved in the long‐term functional effects evoked by purine analogues. Consistently, exogenously added AA markedly elongated astrocytic processes. Moreover, various PLA2 inhibitors (e.g. mepacrine and dexamethasone) prevented both the early α,β‐meATP‐induced [3H]‐AA release and/or the associated long‐term morphological changes, without affecting the astrocytic elongation induced by bFGF. Finally, the protein kinase C (PKC) inhibitor H7 fully abolished α,β‐meATP‐ but not bFGF‐induced effects. 4 Both α,β‐meATP and bFGF rapidly and transiently induced the nuclear accumulation of Fos and Jun. Both c‐fos and c‐jun induction by the purine analogue could be fully prevented by pretreatment with suramin. In contrast, the effects of bFGF were unaffected by this P2 receptor antagonist. 5 It was concluded that α,β‐meATP‐ and bFGF‐morphological differentiation of astrocytes occurs via independent transductional pathways. For the purine analogue, signalling involves a Gi/Go protein‐coupled P2Y‐receptor which may be linked to activation of PLA2 (involvement of an arachidonate‐sensitive PKC is speculated); for bFGF, a tyrosine kinase receptor is involved. Both pathways merge on some common intracellular target, as suggested by induction of primary response genes, which in turn may regulate late response genes mediating long‐term phenotypic changes of astroglial cells. 6 These findings implicate P2 receptors as novel targets for the pharmacological regulation of reactive astrogliosis, which has intriguing implications in nervous system diseases characterized by degenerative events.

Modulation of Apoptosis by Adenosine in the Central Nervous System: a Possible Role for the A3 Receptor

A great body of evidence has been accumulating in the last 20 years supporting a role for adenosine as a neurotransmitter and neuromodulator in the central nervous system.1 In brain, adenosine acts as a potent depressant of excitatory neurotransmission and is colocalized (either as adenosine per se or as its precursor molecule, adenosine triphosphate (ATP)) with “classic” excitatory transmitters in many presynaptic terminals, whence it is released during physiological neurotransmission. It is now well established that the multiple effects of this nucleoside are mediated by activation of specific cell surface receptors, which, based on biochemical, pharmacological and molecular cloning studies, have been classified into four subtypes, denoted as A1, A2A, A2B and A3.2 All the adenosine receptors are members of the guanine nucleotide-binding protein (G protein)-coupled receptor family and possess seven transmembrane helical regions.3 The functional roles of some of the adenosine receptor subtypes (e.g., the A1 and A2A receptors) are relatively well established (see below), whereas the role(s) of the A2B and the recently cloned A3 receptors are still largely unknown.

Calcitonin Gene-Related Peptide-Mediated Enhancement of Purinergic Neuron/Glia Communication by the Algogenic Factor Bradykinin in Mouse Trigeminal Ganglia from Wild-Type and R192Q Cav2.1 Knock-In Mice: Implications for Basic Mechanisms of Migraine Pain

Within the trigeminal ganglion, crosstalk between neurons and satellite glial cells (SGCs) contributes to neuronal sensitization and transduction of painful stimuli, including migraine pain, at least partly through activation of purinergic receptor mechanisms. We previously showed that the algogenic mediator bradykinin (BK) potentiates purinergic P2Y receptors on SGCs in primary trigeminal cultures. Our present study investigated the molecular basis of this effect in wild-type (WT) mice and CaV2.1 α1 R192Q mutant knock-in (KI) mice expressing a human mutation causing familial hemiplegic migraine type 1. Single-cell calcium imaging of WT cultures revealed functional BK receptors in neurons only, suggesting a paracrine action by BK to release a soluble mediator responsible for its effects on SGCs. We identified this mediator as the neuropeptide calcitonin gene-related peptide (CGRP), whose levels were markedly increased by BK, while the CGRP antagonist CGRP8-37 and the anti-migraine drug sumatriptan inhibited BK actions. Unlike CGRP, BK was ineffective in neuron-free SGC cultures, confirming the CGRP neuronal source. P2Y receptor potentiation induced by CGRP in SGCs was mediated via activation of the extracellular signal-regulated kinase 1/2 pathways, and after exposure to CGRP, a significant release of several cytokines was detected. Interestingly, both basal and BK-stimulated CGRP release was higher in KI mouse cultures, where BK significantly upregulated the number of SGCs showing functional UTP-sensitive P2Y receptors. Our findings suggest that P2Y receptors on glial cells might be considered as novel players in the cellular processes underlying migraine pathophysiology and might represent new targets for the development of innovative therapeutic agents against migraine pain.

Cyclo-oxygenase-2 mediates P2Y receptor-induced reactive astrogliosis

Excessive cyclo‐oxygenase‐2 (COX‐2) induction may play a role in chronic neurological diseases characterized by inflammation and astrogliosis. We have previously identified an astroglial receptor for extracellular nucleotides, a P2Y receptor, whose stimulation leads to arachidonic acid (AA) release, followed, 3 days later, by morphological changes resembling reactive astrogliosis. Since COX‐2 may be upregulated by AA metabolites, we assessed a possible role for COX‐2 in P2Y receptor‐mediated astrogliosis. A brief challenge of rat astrocytes with the ATP analogue α,β‐methylene ATP (α,βmeATP) resulted, 24 h later, in significantly increased COX‐2 expression. The selective COX‐2 inhibitor NS‐398 completely abolished α,βmeATP‐induced astrocytic activation. Constitutive astroglial COX‐1 or COX‐2 did not play any role in purine‐induced reactive astrogliosis. PGE2, a main metabolite of COX‐2, also induced astrocytic activation. These data suggest that a P2Y receptor mediates reactive astrogliosis via induction of COX‐2. Antagonists selective for this receptor may counteract excessive COX‐2 activation in both acute and chronic neurological diseases.

Roles of P2 receptors in glial cells: focus on astrocytes.

Central nervous system glial cells release and respond to nucleotides under both physiological and pathological conditions, suggesting that these molecules play key roles in both normal brain function and in repair after damage. In particular, ATP released from astrocytes activates P2 receptors on astrocytes and other brain cells, allowing a form of homotypic and heterotypic signalling, which also involves microglia, neurons and oligodendrocytes. Multiple P2X and P2Y receptors are expressed by both astrocytes and microglia; however, these receptors are differentially recruited by nucleotides, depending upon specific pathophysiological conditions, and also mediate the long-term trophic changes of these cells during inflammatory gliosis. In astrocytes, P2-receptor-induced gliosis occurs via activation of the extracellular-regulated kinases (ERK) and protein kinase B/Akt pathways and involves induction of inflammatory and anti-inflammatory genes, cyclins, adhesion and antiapoptotic molecules. While astrocytic P2Y1 and P2Y2,4 are primarily involved in short-term calcium-dependent signalling, multiple P2 receptor subtypes seem to cooperate to astrocytic long-term changes. Conversely, in microglia, exposure to inflammatory and immunological stimuli results in differential functional changes of distinct P2 receptors, suggesting highly specific roles in acquisition of the activated phenotype. We believe that nucleotide-induced activation of astrocytes and microglia may originally start as a defence mechanism to protect neurons from cytotoxic and ischaemic insults; dysregulation of this process in chronic inflammatory diseases eventually results in neuronal cell damage and loss. On this basis, full elucidation of the specific roles of P2 receptors in these cells may help exploit the beneficial neuroprotective features of activated glia while attenuating their harmful properties and thus provide the basis for novel neuroprotective strategies that specifically target the purinergic system.

EFFECTS OF ATP ANALOGUES AND BASIC FIBROBLAST GROWTH FACTOR ON ASTROGLIAL CELL DIFFERENTIATION IN PRIMARY CULTURES OF RAT STRIATUM

We have used primary cultures of rat striatum to study the effects of ATP analogues on the elongation of astrocytic processes, a parameter of astroglial cell differentiation. Parallel studies were performed with basic fibroblast growth factor, a known regulator of astroglial cell function. After three days in culture, both the growth factor and αβ‐methylene‐ATP induced dramatic increases in the mean length of astrocytic processes/cell. For both agents, effects were dose‐dependent. The effect of αβ‐methylene‐ATP was antagonized by the trypanoside suramin and mimicked by 2‐methyl‐thio‐ATP, suggesting the involvement of a suramin‐sensitive P2‐purinoceptor. Neither an additive nor a synergistic effect between αβ‐methylene‐ATP and basic fibroblast growth factor on the elongation of processes was detected in cultures exposed to both agents. Indeed, an inhibition with respect to the effects induced by either agent alone was recorded, suggesting that the growth factor and the purine analogue can modulate astrocytic differentiation by activation of common intracellular pathways.