Synthia H. Sun

Expanded-Polyglutamine Huntingtin Protein Suppresses the Secretion and Production of a Chemokine (CCL5/RANTES) by Astrocytes

Huntingtons disease (HD) is a hereditary neurological disease caused by expended CAG repeats in the HD gene, which codes for a protein called Huntingtin (Htt). The resultant mutant Huntingtin (mHtt) forms aggregates in neurons and causes neuronal dysfunction. In astrocytes, the largest population of brain cells, mHtt also exists. We report herein that astrocyte-conditioned medium (ACM) collected from astrocytes of R6/2 mice (a mouse model of HD) caused primary cortical neurons to grow less-mature neurites, migrate more slowly, and exhibit lower calcium influx after depolarization than those maintained in wild-type (WT) ACM. Using a cytokine antibody array and ELISA assays, we demonstrated that the amount of a chemokine [chemokine (C-C motif) ligand 5 (CCL5)/regulated on activation normal T cell expressed and secreted (RANTES)] released by R6/2 astrocytes was much less than that by WT astrocytes. When cortical neurons were treated with the indicated ACM, supplementation with recombinant CCL5/RANTES ameliorated the neuronal deficiency caused by HD-ACM, whereas removing CCL5/RANTES from WT-ACM using an anti-CCL5/RANTES antibody mimicked the effects evoked by HD-ACM. Quantitative PCR and promoter analyses demonstrated that mHtt hindered the activation of the CCL5/RANTES promoter by reducing the availability of nuclear factor κB-p65 and, hence, reduced the transcript level of CCL5/RANTES. Moreover, ELISA assays and immunocytochemical staining revealed that mHtt retained the residual CCL5/RANTES inside R6/2 astrocytes. In line with the above findings, elevated cytosolic CCL5/RANTES levels were also observed in the brains of two mouse models of HD [R6/2 and Hdh(CAG)150] and human HD patients. These findings suggest that mHtt hinders one major trophic function of astrocytes which might contribute to the neuronal dysfunction of HD.

ATP-stimulated Ca2+ influx and phospholipase D activities of a rat brain-derived type-2 astrocyte cell line, RBA-2, are mediated through P2X7 receptors

Abstract: This study characterizes and examines the P2 receptor‐mediated signal transduction pathway of a rat brain‐derived type 2 astrocyte cell line, RBA‐2. ATP induced Ca2+ influx and activated phospholipase D (PLD). The ATP‐stimulated Ca2+ influx was inhibited by pretreating cells with P2 receptor antagonist, pyridoxal‐phosphate‐6‐azophenyl‐2′,4′‐disulfonic acid (PPADS), in a concentration‐dependent manner. The agonist 2′‐ and 3′‐O‐(4‐benzoylbenzoyl)adenosine 5′‐triphosphate (BzATP) stimulated the largest increases in intracellular Ca2+ concentrations ([Ca2+]i); ATP, 2‐methylthioadenosine triphosphate tetrasodium, and ATPγS were much less effective, whereas UTP, ADP, α,β‐methylene‐ATP, and β,γ‐methylene‐ATP were ineffective. Furthermore, removal of extracellular Mg2+ enhanced the ATP‐ and BzATP‐stimulated increases in [Ca2+]i. BzATP stimulated PLD in a concentration‐ and time‐dependent manner that could be abolished by removal of extracellular Ca2+ and was inhibited by suramin, PPADS, and oxidized ATP. In addition, PLD activities were activated by the Ca2+ mobilization agent, ionomycin, in an extracellular Ca2+ concentration‐dependent manner. Both staurosporine and prolonged phorbol ester treatment inhibited BzATP‐stimulated PLD activity. Taken together, these data indicate that activation of the P2X7 receptors induces Ca2+ influx and stimulates a Ca2+‐dependent PLD in RBA‐2 astrocytes. Furthermore, protein kinase C regulates this PLD.

Activation of P2X7 receptors induced [3H]GABA release from the RBA-2 type-2 astrocyte cell line through a Cl−/HCO3−-dependent mechanism

ATP is an important signaling molecule in the nervous system and its signaling is mediated through the metabotropic P2Y and ionotropic P2X receptors. ATP is known to stimulate Ca2+ influx and phospholipase D (PLD) activity in the type‐2 astrocyte cell line, RBA‐2; in this study, we show that the release of preloaded [3H]GABA from RBA‐2 cells is mediated through the P2X7 receptors. ATP and the ATP analogue 3′‐O‐(4‐benoylbenoyl)‐adenosine‐5′‐triphosphate (BzATP) both stimulated [3H]GABA release in a concentration dependent manner, while the nonselective P2 receptor antagonist pyridoxalphosphate‐6‐azophenyl‐2′,4′‐disulfonic acid (PPADS), the P2X7‐sensitive antagonist oxidized ATP (oATP), and high extracellular Mg2+ all inhibited the ATP‐stimulated [3H]GABA release. The ATP‐stimulated [3H]GABA release was not affected neither by removing extracellular Na+ nor by changes in the intracellular or extracellular Ca2+ concentration. The GABA transporter inhibitors nipecotic acid and β‐alanine also had no effect. The ATP‐stimulated [3H]GABA release was blocked, however, when media Cl− was replaced with gluconate and when extracellular HCO3− was removed. The Cl− channel/exchanger blockers 4,4′‐diisothiocyanatostilbene‐2′,2′‐disulfonic acid (DIDS) and 4‐acetamido‐4′‐ isothiocyanatostilbene‐2′,2′‐disulfonic acids (SITS), but not diphenylamine‐2‐carboxylic acid (DPC) and furosemide, blocked the ATP‐stimulated [3H]GABA release. The anionic selectivity of the process was F− > Cl− > Br− which is the same as that reported for volume‐sensitive Cl− conductance. Treating cells with phorbol‐12‐myristate 13‐acetate (PMA), forskolin, dibutyryl‐cAMP, PD98059, neomycin, and D609 all inhibited the ATP‐stimulated [3H]GABA release. We concluded that in RBA‐2 cells, ATP stimulates [3H]GABA release through the P2X7 receptors via a Cl−/HCO3−‐dependent mechanism that is regulated by PKC, PKA, MEK/ERK, and PLD. GLIA 37:8–18, 2002.

Activation of P2X7 purinoceptor-stimulated TGF-β1 mRNA expression involves PKC/MAPK signalling pathway in a rat brain-derived type-2 astrocyte cell line, RBA-2

The present study investigates the receptor and mechanisms involved in ATP-stimulated transforming growth factor-beta 1 (TGF-beta 1) mRNA expression of a type-2 astrocyte cell line, RBA-2. RT-PCR analysis revealed that RBA-2 type-2 astrocytes possess abundant P2X(4) and P2X(7) receptors. ATP and P2X(7) receptor-sensitive agonist, BzATP, both stimulated TGF-beta 1 mRNA expression in a time and dose-dependent manner. The stimulation required a minimum of 500 muM ATP; BzATP was much more potent that ATP, and P2X(7)-selective antagonist, oATP, inhibited the effects. In addition, ATP metabolites ADP, AMP and adenosine were ineffective in stimulation of TGF-beta 1 mRNA expression. Thus, the effect of ATP was mediated through the P2X(7) receptors. To investigate further the mechanisms by which the P2X(7) receptor mediated the TGF-beta 1 mRNA expression, the cells were treated with inhibitors for mitogen-activated kinase (MAPK) or protein kinase C (PKC), PD98059 or GF109203X, respectively. Both PD98059 and GF109203X inhibited the ATP-stimulated TGF-beta 1 mRNA expression. Furthermore, ATP and BzATP stimulated ERK1/2 activation and the activation was inhibited by PKC inhibitors, GF109203X and Gö6976. In conclusion, activation of P2X(7) receptors enhanced TGF-beta 1 mRNA expression and the effect involved PKC/MAPK signalling pathway in RBA-2 type-2 astrocytes.

Microglial phagocytosis attenuated by short-term exposure to exogenous ATP through P2X7 receptor action

Microglia, the CNS resident macrophages responsible for the clearance of degenerating cellular fragments, are essential to tissue remodeling and repair after CNS injury. ATP can be released in large amounts after CNS injury and may mediate microglial activity through the ionotropic P2X and the metabotropic P2Y receptors. This study indicates that exposure to a high concentration of ATP for 30 min rapidly induces changes of the microglial cytoskeleton, and significantly attenuates microglial phagocytosis. A pharmacological approach showed that ATP‐induced inhibition of microglial phagocytotic activity was due to P2X7R activation, rather than that of P2YR. Activation of P2X7R by its agonist, 2′‐3′‐O‐(4‐benzoyl)benzoyl‐ATP (BzATP), produced a Ca2+‐independent reduction in microglial phagocytotic activity. In addition, the knockdown of P2X7R expression by lentiviral‐mediated shRNA interference or the blockade of P2X7R activation by the specific antagonists, oxidized ATP (oxATP) and brilliant blue G, has efficiently restored the phagocytotic activity of ATP and BzATP‐treated microglia. Our results reveal that P2X7R activation may induce the formation of a Ca2+‐independent signaling complex, which results in the reduction of microglial phagocytosis. This suggests that exposure to ATP for a short‐term period may cause insufficient clearance of tissue debris by microglia through P2X7R activation after CNS injury, and that blockade of this receptor may preserve the phagocytosis of microglia and facilitate CNS tissue repair.

Post Ischemia Intermittent Hypoxia Induces Hippocampal Neurogenesis and Synaptic Alterations and Alleviates Long-Term Memory Impairment

Adult hippocampal neurogenesis is important for learning and memory, especially after a brain injury such as ischemia. Newborn hippocampal neurons contribute to memory performance by establishing functional synapses with target cells. This study demonstrated that the maturation of hippocampal neurons is enhanced by postischemia intermittent hypoxia (IH) intervention. The effects of IH intervention in cultured neurons were mediated by increased synaptogenesis, which was primarily regulated by brain-derived neurotrophic factor (BDNF)/PI3K/AKT. Hippocampal neo-neurons expressed BDNF and exhibited enhanced presynaptic function as indicated by increases in the pSynapsin expression, synaptophysin intensity, and postsynapse density following IH intervention after ischemia. Postischemia IH-induced hippocampal neo-neurons were affected by presynaptic activity, which reflected the dynamic plasticity of the glutamatergic receptors. These alterations were also associated with the alleviation of ischemia-induced long-term memory impairment. Our results suggest that postischemia IH intervention rescued ischemia-induced spatial learning and memory impairment by inducing hippocampal neurogenesis and functional synaptogenesis via BDNF expression.

Activation of P2X7 receptors decreases glutamate uptake and glutamine synthetase activity in RBA‐2 astrocytes via distinct mechanisms

Glutamate clearance by astrocytes is critical for controlling excitatory neurotransmission and ATP is an important mediator for neuron‐astrocyte interaction. However, the effect of ATP on glutamate clearance has never been examined. Here we report that treatment of RBA‐2 cells, a type‐2‐like astrocyte cell line, with ATP and the P2X7 receptor selective agonist 3′‐O‐(4‐benzoylbenzoyl) adenosine 5′‐triphosphate (BzATP) decreased the Na+‐dependent [3H]glutamate uptake within minutes. Mechanistic studies revealed that the decreases were augmented by removal of extracellular Mg2+ or Ca2+, and was restored by P2X7 selective antagonist , periodate‐oxidized 2′,3′‐dialdehyde ATP (oATP), indicating that the decreases were mediated through P2X7 receptors. Furthermore, stimulation of P2X7 receptors for 2 h inhibited both activity and protein expression of glutamine synthetase (GS), and oATP abolished the inhibition. In addition, removal of extracellular Ca2+ and inhibition of protein kinase C (PKC) restored the ATP‐decreased GS expression but failed to restore the P2X7‐decreased [3H]glutamate uptake. Therefore, P2X7‐mediated intracellular signals play a role in the down‐regulation of GS activity/expression. Activation of P2X7 receptors stimulated increases in intracellular Na+ concentration ([Na+]i) suggesting that the P2X7‐induced increases in [Na+]i may affect the local Na+ gradient and decrease the Na+‐dependent [3H]glutamate uptake. These findings demonstrate that the P2X7‐mediated decreases in glutamate uptake and glutamine synthesis were mediated through distinct mechanisms in these cells.

The P2X7 receptor-mediated phospholipase D activation is regulated by both PKC-dependent and PKC-independent pathways in a rat brain-derived Type-2 astrocyte cell line, RBA-2

The aim of this study was to characterize the regulatory mechanisms of the P2X(7) receptor (P2X(7)R)-mediated phospholipase D (PLD) activation in a rat brain-derived Type-2 astrocyte cell line, RBA-2. A time course study revealed that activation of P2X(7)R resulted in a choline and not phosphorylcholine formation, suggesting that activation of P2X(7)R is associated with the phosphatidylcholine-PLD (PC-PLD) in these cells. GF 109203X, a selective protein kinase C (PKC) inhibitor, partially inhibited the P2X(7)R-mediated PLD activation, while blocking the phorbol 12-myristate 13-acetate (PMA)-stimulated PLD activity. In addition, PMA synergistically activated the P2X(7)R-mediated PLD activity. Furthermore, genistein, a tyrosine kinase inhibitor, blocked the P2X(7)R-activated PLD, while KN62, a Ca(2+)/calmodulin-dependent protein kinase II (CaMKII) inhibitor, was less effective, whereas the mitogen-activated protein kinase (MAPK) inhibitor PD98059 was ineffective. No additive inhibitory effects were found by simultaneous treatment of GF 109203X and KN62 on P2X(7)R-activated PLD. Taken together, these results demonstrate that both PKC-dependent and PKC-independent signaling pathways are involved in the regulation of P2X(7)R-mediated PLD activation. Additionally, CaMKII may participate in the PKC-dependent pathway, and tyrosine kinase may play a pivotal role on both PKC-dependent and PKC-independent pathways in the P2X(7)R-mediated PLD activation in RBA-2 cells.

P2X7R‐mediated Ca2+‐independent d‐serine release via pannexin‐1 of the P2X7R‐pannexin‐1 complex in astrocytes

d‐serine is a coagonist of N‐methyl‐d‐aspartate (NMDA) subtype of glutamate receptor and plays a role in regulating activity‐dependent synaptic plasticity. In this study, we examined the mechanism by which extracellular ATP triggers the release of d‐serine from astrocytes and discovered a novel Ca2+‐independent release mechanism mediated by P2X7 receptors (P2X7R). Using [3H] d‐serine, which was loaded into astrocytes via the neutral amino acid transporter 2 (ASCT2), we observed that ATP and a potent P2X7R agonist, 2′(3′)‐O‐(4‐benzoylbenzoyl)adenosine‐5′‐triphosphate (BzATP), stimulated [3H]D‐serine release and that were abolished by P2X7R selective antagonists and by shRNAs, whereas enhanced by removal of intracellular or extracellular Ca2+. The P2X7R‐mediated d‐serine release was inhibited by pannexin‐1 antagonists, such as carbenoxolone (CBX), probenecid (PBN), and 10Panx‐1 peptide, and shRNAs, and stimulation of P2X7R induced P2X7R‐pannexin‐1 complex formation. Simply incubating astrocytes in Ca2+/Mg2+‐free buffer also induced the complex formation, and that enhanced basal d‐serine release through pannexin‐1. The P2X7R‐mediated d‐serine release assayed in Ca2+/Mg2+‐free buffer was enhanced as well, and that was inhibited by CBX. Treating astrocytes with general protein kinase C (PKC) inhibitors, such as chelerythrine, GF109203X, and staurosporine, but not Ca2+‐dependent PKC inhibitor, Gö6976, inhibited the P2X7R‐mediated d‐serine release. Thus, we conclude that in astrocytes, P2X7R‐pannexin‐1 complex formation is crucial for P2X7R‐mediated d‐serine release through pannexin‐1 hemichannel. The release is Ca2+‐independent and regulates by a Ca2+‐independent PKC. The activated P2X7R per se is also functioned as a permeation channel to release d‐serine in part. This P2X7R‐mediated d‐serine release represents an important mechanism for activity‐dependent neuron‐glia interaction. GLIA 2015;63:877–893

Roles of P2X7 Receptor in Glial and Neuroblastoma Cells: The Therapeutic Potential of P2X7 Receptor Antagonists

Recently, one of the P2 purinergic receptors, the P2X7 receptor, has been extensively studied in nervous system and important functions have been revealed in both astrocytes and microglia. Stimulation of the receptors induces a sustained and nondesensitized increase in intracellular Ca2+ concentration ([Ca2+]i). In astrocytes purinergic receptors primarily regulate neurotransmission by inducing gliotransmitters release whereas in microglia the receptors stimulate the processing and release of proinflammation cytokines such as interleukin-1 and are thereby involved in inflammation and neurodegeneration. Thus, P2X7 receptors are considered not only to exert physiological functions but also mediate cell death. P2X7 receptors have also been identified in various cancer cells and in neuroblastoma cells. In these cells, the P2X7 receptor-mediated sustained Ca2+ signal is important in maintaining cellular viability and growth. Accordingly, these findings not only lead to a better understanding of roles of the receptor but also prompt the development of more potent, selective and safer P2X7 selective antagonists. These emerging antagonists bring new hope in the treatment of inflammatory-induced neurodegenerative diseases as well as neuroblastoma.