The phosphorylation status and cytoskeletal remodeling of striatal astrocytes treated with quinolinic acid

Quinolinic acid (QUIN) is a glutamate agonist which markedly enhances the vulnerability of neural cells to excitotoxicity. QUIN is produced from the amino acid tryptophan through the kynurenine pathway (KP). Dysregulation of this pathway is associated with neurodegenerative conditions. In this study we treated striatal astrocytes in culture with QUIN and assayed the endogenous phosphorylating system associated with glial fibrillary acidic protein (GFAP) and vimentin as well as cytoskeletal remodeling. After 24h incubation with 100µM QUIN, cells were exposed to 32P-orthophosphate and/or protein kinase A (PKA), protein kinase dependent of Ca2+/calmodulin II (PKCaMII) or protein kinase C (PKC) inhibitors, H89 (20μM), KN93 (10μM) and staurosporin (10nM), respectively. Results showed that hyperphosphorylation was abrogated by PKA and PKC inhibitors but not by the PKCaMII inhibitor. The specific antagonists to ionotropic NMDA and non-NMDA (50µM DL-AP5 and CNQX, respectively) glutamate receptors as well as to metabotropic glutamate receptor (mGLUR; 50µM MCPG), mGLUR1 (100µM MPEP) and mGLUR5 (10µM 4C3HPG) prevented the hyperphosphorylation provoked by QUIN. Also, intra and extracellular Ca2+ quelators (1mM EGTA; 10µM BAPTA-AM, respectively) prevented QUIN-mediated effect, while Ca2+ influx through voltage-dependent Ca2+ channel type L (L-VDCC) (blocker: 10µM verapamil) is not implicated in this effect. Morphological analysis showed dramatically altered actin cytoskeleton with concomitant change of morphology to fusiform and/or flattened cells with retracted cytoplasm and disruption of the GFAP meshwork, supporting misregulation of actin cytoskeleton. Both hyperphosphorylation and cytoskeletal remodeling were reversed 24h after QUIN removal. Astrocytes are highly plastic cells and the vulnerability of astrocyte cytoskeleton may have important implications for understanding the neurotoxicity of QUIN in neurodegenerative disorders. •Quinolinic acid (QUIN) induces hypersphorylation of cytoskeletal proteins in striatal astrocytes.•Glutamate, Ca2+, PKA and PKC are implicated in the aberrantly phosphorylated GFAP and vimentin.•QUIN induces reorganization of actin and GFAP cytoskeleton.•Hyperphosphorylation and cytoskeletal remodeling are reversed after QUIN removal.•Disruption of cytoskeleton is a cytotoxic action of QUIN in striatal astrocytes.