Huntington's 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 B-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.
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 Ca 2ϩ influx and activated phospholipase D (PLD). The ATP-stimulated Ca 2ϩ influx was inhibited by pretreating cells with P2 receptor antagonist, pyridoxalphosphate-6-azophenyl-2Ј,4Ј-disulfonic acid (PPADS), in a concentration-dependent manner. The agonist 2Ј-and 3Ј-O-(4-benzoylbenzoyl)adenosine 5Ј-triphosphate (BzATP) stimulated the largest increases in intracellular Ca 2ϩ concentrations ([Ca 2ϩ ] i ); ATP, 2-methylthioadenosine triphosphate tetrasodium, and ATP␥S were much less effective, whereas UTP, ADP, ␣,-methylene-ATP, and ,␥-methylene-ATP were ineffective. Furthermore, removal of extracellular Mg 2ϩ enhanced the ATP-and BzATP-stimulated increases in [Ca 2ϩ ] i . BzATP stimulated PLD in a concentration-and time-dependent manner that could be abolished by removal of extracellular Ca 2ϩ and was inhibited by suramin, PPADS, and oxidized ATP. In addition, PLD activities were activated by the Ca 2ϩ mobilization agent, ionomycin, in an extracellular Ca 2ϩ concentration-dependent manner. Both staurosporine and prolonged phorbol ester treatment inhibited BzATP-stimulated PLD activity. Taken together, these data indicate that activation of the P2X 7 receptors induces Ca 2ϩ influx and stimulates a Ca 2ϩ -dependent PLD in RBA-2 astrocytes. Furthermore, protein kinase C regulates this PLD. Key Words: ATP-2Ј-and 3Ј-O-(4-benzoylbenzoyl)adenosine 5Ј-triphosphate -Ca 2ϩ influx-Phospholipase D -P2X 7 receptor-Type-2 astrocytes.
ATP is an important signaling molecule in the nervous system and it's signaling is mediated through the metabotropic P2Y and ionotropic P2X receptors. ATP is known to stimulate Ca(2+) 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 [(3)H]GABA from RBA-2 cells is mediated through the P2X(7) receptors. ATP and the ATP analogue 3'-O-(4-benoylbenoyl)-adenosine-5'-triphosphate (BzATP) both stimulated [(3)H]GABA release in a concentration dependent manner, while the nonselective P2 receptor antagonist pyridoxalphosphate-6-azophenyl-2',4'-disulfonic acid (PPADS), the P2X(7)-sensitive antagonist oxidized ATP (oATP), and high extracellular Mg(2+) all inhibited the ATP-stimulated [(3)H]GABA release. The ATP-stimulated [(3)H]GABA release was not affected neither by removing extracellular Na(+) nor by changes in the intracellular or extracellular Ca(2+) concentration. The GABA transporter inhibitors nipecotic acid and beta-alanine also had no effect. The ATP-stimulated [(3)H]GABA release was blocked, however, when media Cl(-) was replaced with gluconate and when extracellular HCO(3)(-) 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 [(3)H]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 [(3)H]GABA release. We concluded that in RBA-2 cells, ATP stimulates [(3)H]GABA release through the P2X(7) receptors via a Cl(-)/HCO(3)(-)-dependent mechanism that is regulated by PKC, PKA, MEK/ERK, and PLD.
Recent evidence suggests that the P2X 7 receptor may play a role in the pathophysiology of preclinical models of pain and inflammation. Therefore, pharmacological agents that target this receptor may potentially have clinical utility as antiinflammatory and analgesic therapy. We investigated and characterized the previously reported P2X 7 antagonist N-(adamantan-1-ylmethyl)-5-[(3R-amino-pyrrolidin-1-yl)methyl]-2-chloro-benzamide, hydrochloride salt (AACBA; GSK314181A). In vitro, AACBA was a relatively potent inhibitor of both human P2X 7 -mediated calcium flux and quinolinium,4-[(3-methyl-2(3H)-benzoxazolylidene)methyl]-1-[3-(triemethylammonio)propyl]-diiodide (YO-PRO-1) uptake assays, with IC 50 values of approximately 18 and 85 nM, respectively. Compared with the human receptor, AACBA was less potent at the rat P2X 7 receptor, with IC 50 values of 29 and 980 nM in the calcium flux and YO-PRO-1 assays, respectively. In acute in vivo models of pain and inflammation, AACBA dose-dependently reduced lipopolysaccharideinduced plasma interleukin-6 release and prevented or reversed carrageenan-induced paw edema and mechanical hypersensitivity. In chronic in vivo models of pain and inflammation, AACBA produced a prophylactic, but not therapeutic-like, prevention of the clinical signs and histopathological damage of collageninduced arthritis. Finally, AACBA could not reverse L 5 spinal nerve ligation-induced tactile allodynia when given therapeutically. Consistent with previous literature, these results suggest that P2X 7 receptors do play a role in animal models of pain and inflammation. Further study of P2X 7 antagonists both in preclinical and clinical studies will help elucidate the role of the P2X 7 receptor in pain and inflammatory mechanisms and may help identify potential clinical benefits of such molecules.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.
hi@scite.ai
10624 S. Eastern Ave., Ste. A-614
Henderson, NV 89052, USA
Copyright © 2024 scite LLC. All rights reserved.
Made with 💙 for researchers
Part of the Research Solutions Family.