Roberta Brambilla

Inhibition of astroglial nuclear factor κB reduces inflammation and improves functional recovery after spinal cord injury

In the central nervous system (CNS), the transcription factor nuclear factor (NF)-κB is a key regulator of inflammation and secondary injury processes. After trauma or disease, the expression of NF-κB–dependent genes is highly activated, leading to both protective and detrimental effects on CNS recovery. We demonstrate that selective inactivation of astroglial NF-κB in transgenic mice expressing a dominant negative (dn) form of the inhibitor of κBα under the control of an astrocyte-specific promoter (glial fibrillary acidic protein [GFAP]–dn mice) leads to a dramatic improvement in functional recovery 8 wk after contusive spinal cord injury (SCI). Histologically, GFAP mice exhibit reduced lesion volume and substantially increased white matter preservation. In parallel, they show reduced expression of proinflammatory chemokines and cytokines, such as CXCL10, CCL2, and transforming growth factor–β2, and of chondroitin sulfate proteoglycans participating in the formation of the glial scar. We conclude that selective inhibition of NF-κB signaling in astrocytes results in protective effects after SCI and propose the NF-κB pathway as a possible new target for the development of therapeutic strategies for the treatment of SCI.

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.

Transgenic Inhibition of Astroglial NF-κB Improves Functional Outcome in Experimental Autoimmune Encephalomyelitis by Suppressing Chronic Central Nervous System Inflammation

In the CNS, the transcription factor NF-κB is a key regulator of inflammation and secondary injury processes. Following trauma or disease, the expression of NF-κB-dependent genes is activated, leading to both protective and detrimental effects. In this study, we show that transgenic inactivation of astroglial NF-κB (glial fibrillary acidic protein-IκBα-dominant-negative mice) resulted in reduced disease severity and improved functional recovery following experimental autoimmune encephalomyelitis. At the chronic stage of the disease, transgenic mice exhibited an overall higher presence of leukocytes in spinal cord and brain, and a markedly higher percentage of CD8+CD122+ T regulatory cells compared with wild type, which correlated with the timing of clinical recovery. We also observed that expression of proinflammatory genes in both spinal cord and cerebellum was delayed and reduced, whereas the loss of neuronal-specific molecules essential for synaptic transmission was limited compared with wild-type mice. Furthermore, death of retinal ganglion cells in affected retinas was almost abolished, suggesting the activation of neuroprotective mechanisms. Our data indicate that inhibiting NF-κB in astrocytes results in neuroprotective effects following experimental autoimmune encephalomyelitis, directly implicating astrocytes in the pathophysiology of this disease.

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.

Inhibition of Gap-Junctional Communication Induces the Trans-differentiation of Osteoblasts to an Adipocytic Phenotype in Vitro

Osteoblasts and adipocytes are thought to differentiate from a common stromal progenitor cell. These two phenotypically mature cell types show a high degree of plasticity, which can be observed when cells are grown under specific culture conditions. Gap junctions are abundant among osteoblastic cellsin vivo and in vitro, whereas they are down-regulated during adipogenesis. Gap junctional communication (GJC) modulates the expression of genes associated with the mature osteoblastic phenotype. Inhibition of GJC utilizing 18-α-glycyrrhetinic acid (AGRA) blocks the maturation of pre-osteoblastic cells in vitro. Moreover, cytoplasmic lipid droplets are detectable at the end of the culture period, suggesting that GJC inhibition may favor an adipocytic phenotype. We used several human osteoblastic cell lines, as well as bone-derived primary osteoblastic cells, to show that confluent cultures of human osteoblastic cells grown under osteogenic conditions developed an adipocytic phenotype after 3 days of complete inhibition of GJC using AGRA or oleamide, two dissimilar nontoxic reversible inhibitors. Development of an adipogenic phenotype was confirmed by the accumulation of triglyceride droplets and the increase in mRNA expression of the adipocytic markers peroxisome proliferator-activated receptor γ2 and lipoprotein lipase. Glycyrrhizic acid, a noninhibitory AGRA analog, or α-bromopalmitate, a nondegradable fatty acid, had no effect. Modulation of skeletal GJC may represent a new pharmacological target by which inhibition of marrow adipogenesis can take place with the parallel enhancement of osteoblastogenesis, thus providing a novel therapeutic approach to the treatment of human age-related osteopenic diseases and postmenopausal osteoporosis.

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.

Inactivation of astroglial NF‐κB promotes survival of retinal neurons following ischemic injury

Reactive astrocytes have been implicated in neuronal loss following ischemic stroke. However, the molecular mechanisms associated with this process are yet to be fully elucidated. In this work, we tested the hypothesis that astroglial NF‐κB, a key regulator of inflammatory responses, is a contributor to neuronal death following ischemic injury. We compared neuronal survival in the ganglion cell layer (GCL) after retinal ischemia‐reperfusion in wild‐type (WT) and in GFAP‐IκBα‐dn transgenic mice, where the NF‐κB classical pathway is suppressed specifically in astrocytes. The GFAP‐IκBα‐dn mice showed significantly increased survival of neurons in the GCL following ischemic injury as compared with WT littermates. Neuroprotection was associated with significantly reduced expression of pro‐inflammatory genes, encoding Tnf‐α, Ccl2 (Mcp1), Cxcl10 (IP10), Icam1, Vcam1, several subunits of NADPH oxidase and NO‐synthase in the retinas of GFAP‐IκBα‐dn mice. These data suggest that certain NF‐κB‐regulated pro‐inflammatory and redox‐active pathways are central to glial neurotoxicity induced by ischemic injury. The inhibition of these pathways in astrocytes may represent a feasible neuroprotective strategy for retinal ischemia and stroke.

Transgenic Inhibition of Glial NF-kappa B Reduces Pain Behavior and Inflammation after Peripheral Nerve Injury

&NA; The transcription factor nuclear factor kappa B (NF‐&kgr;B) is a key regulator of inflammatory processes in reactive glial cells. We utilized a transgenic mouse model (GFAP‐I&kgr;B&agr;‐dn) where the classical NF‐&kgr;B pathway is inactivated by overexpression of a dominant negative (dn) form of the inhibitor of kappa B (I&kgr;B&agr;) in glial fibrillary acidic protein (GFAP)‐expressing cells, which include astrocytes, Schwann cells, and satellite cells of the dorsal root ganglion (DRG) and sought to determine whether glial NF‐&kgr;B inhibition leads to a reduction in pain behavior and inflammation following chronic constriction injury (CCI) of the sciatic nerve. As expected, following CCI nuclear translocation, and hence activation, of NF‐&kgr;B was detected only in the sciatic nerve of wild type (WT) mice, and not in GFAP‐I&kgr;B&agr;‐dn mice, while upregulation of GFAP was observed in the sciatic nerve and DRGs of both WT and GFAP‐I&kgr;B&agr;‐dn mice, indicative of glial activation. Following CCI, mechanical and thermal hyperalgesia were reduced in GFAP‐I&kgr;B&agr;‐dn mice compared to those in WT, as well as gene and protein expression of CCL2, CCR2 and CXCL10 in the sciatic nerve. Additionally, gene expression of TNF, CCL2, and CCR2 was reduced in the DRGs of transgenic mice compared to those of WT after CCI. We can therefore conclude that transgenic inhibition of NF‐&kgr;B in GFAP‐expressing glial cells attenuated pain and inflammation after peripheral nerve injury. These findings suggest that targeting the inflammatory response in Schwann cells and satellite cells may be important in treating neuropathic pain.

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.

Induction of COX-2 and reactive gliosis by P2Y receptors in rat cortical astrocytes is dependent on ERK1/2 but independent of calcium signalling.

The present study has been aimed at characterizing the ATP/P2 receptor (and transductional pathways) responsible for the morphological changes induced in vitro by αβmethyleneATP on rat astrocytes obtained from cerebral cortex, a brain area highly involved in neurodegenerative diseases. Exposure of cells to this purine analogue resulted in elongation of cellular processes, an event reproducing in vitro a major hallmark of in vivo reactive gliosis. αβmethyleneATP‐induced gliosis was prevented by the P2X/P2Y blocker pyridoxalphosphate‐6‐azophenyl‐2′‐4′‐disulfonic acid, but not by the selective P2X antagonist 2′,3′‐O‐(2,4,6‐trinitrophenyl)‐ATP, ruling out a role for ligand‐gated P2X receptors. Conversely, the Gi/Go protein inactivator pertussis toxin completely prevented αβmethyleneATP‐induced effects. No effects were induced by αβmethyleneATP on intracellular calcium concentrations. RT‐PCR and western blot analysis showed that αβmethyleneATP–induced gliosis involves up‐regulation of cyclooxygenase‐2 (but not lipooxygenase). Also this effect was fully prevented by pyridoxalphosphate‐6‐azophenyl‐2′‐4′‐disulfonic acid. Experiments with inhibitors of mitogen‐activated protein kinases (MAPK) suggest that extracellular signal regulated protein kinases (ERK)1/2 mediate both cyclooxygenase‐2 induction and the associated in vitro gliosis. These findings suggest that purine‐induced gliosis involves the activation of a calcium‐independent G‐protein‐coupled P2Y receptor linked to ERK1/2 and cyclooxygenase‐2. Based on the involvement of cyclooxygenase‐2 and inflammation in neurodegenerative diseases, these findings open up new avenues in the identification of novel biological targets for the pharmacological manipulation of neurodegeneration.