title: The phosphorylation status and cytoskeletal remodeling of striatal astrocytes treated with quinolinic acid.
abstract: 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 100 µM QUIN, cells were exposed to (32)P-orthophosphate and/or protein kinase A (PKA), protein kinase dependent of Ca(2+)/calmodulin II (PKCaMII) or protein kinase C (PKC) inhibitors, H89 (20 μM), KN93 (10 μM) 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 (50 µM DL-AP5 and CNQX, respectively) glutamate receptors as well as to metabotropic glutamate receptor (mGLUR; 50 µM MCPG), mGLUR1 (100 µM MPEP) and mGLUR5 (10 µM 4C3HPG) prevented the hyperphosphorylation provoked by QUIN. Also, intra and extracellular Ca(2+) quelators (1mM EGTA; 10 µM BAPTA-AM, respectively) prevented QUIN-mediated effect, while Ca(2+) influx through voltage-dependent Ca(2+) channel type L (L-VDCC) (blocker: 10 µM 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.
authors.affiliation: Departamento de Bioquímica, Instituto de Ciências Básicas da Saúde, Universidade Federal do Rio Grande do Sul, Porto Alegre, RS 90035-003, Brazil.
authors.forename: Paula
authors.initials: P
authors.lastname: Pierozan
authors.affiliation: Departamento de Bioquímica, Instituto de Ciências Básicas da Saúde, Universidade Federal do Rio Grande do Sul, Porto Alegre, RS 90035-003, Brazil.
authors.forename: Fernanda
authors.initials: F
authors.lastname: Ferreira
authors.affiliation: Departamento de Bioquímica, Instituto de Ciências Básicas da Saúde, Universidade Federal do Rio Grande do Sul, Porto Alegre, RS 90035-003, Brazil.
authors.forename: Bárbara
authors.initials: B
authors.lastname: Ortiz de Lima
authors.affiliation: Departamento de Bioquímica, Instituto de Ciências Básicas da Saúde, Universidade Federal do Rio Grande do Sul, Porto Alegre, RS 90035-003, Brazil.
authors.forename: Carolina
authors.initials: C
authors.lastname: Gonçalves Fernandes
authors.affiliation: Departamento de Farmacologia, Universidade Federal de São Paulo (UNIFESP/EPM), São Paulo, SP, Brazil.
authors.forename: Priscila
authors.initials: P
authors.lastname: Totarelli Monteforte
authors.affiliation: Departamento de Farmacologia, Universidade Federal de São Paulo (UNIFESP/EPM), São Paulo, SP, Brazil.
authors.forename: Natalia
authors.initials: N
authors.lastname: de Castro Medaglia
authors.affiliation: Departamento de Farmacologia, Universidade Federal de São Paulo (UNIFESP/EPM), São Paulo, SP, Brazil.
authors.forename: Claudia
authors.initials: C
authors.lastname: Bincoletto
authors.affiliation: Departamento de Farmacologia, Universidade Federal de São Paulo (UNIFESP/EPM), São Paulo, SP, Brazil.
authors.forename: Soraya
authors.initials: S
authors.lastname: Soubhi Smaili
authors.affiliation: Departamento de Bioquímica, Instituto de Ciências Básicas da Saúde, Universidade Federal do Rio Grande do Sul, Porto Alegre, RS 90035-003, Brazil. Electronic address: rpureur@ufrgs.br.
authors.forename: Regina
authors.initials: R
authors.lastname: Pessoa-Pureur
mesh_terms: D008841:Actin Cytoskeleton / Q000187:drug effects* / Q000378:metabolism; D000818:Animals; D017209:Apoptosis / Q000187:drug effects; D001253:Astrocytes / Q000166:cytology* / Q000187:drug effects / Q000378:metabolism; D015153:Blotting, Western; D002118:Calcium / Q000378:metabolism; D049109:Cell Proliferation / Q000187:drug effects; D002478:Cells, Cultured; D003342:Corpus Striatum / Q000166:cytology* / Q000187:drug effects / Q000378:metabolism; D005260:Female; D005904:Glial Fibrillary Acidic Protein / Q000378:metabolism; D005971:Glutamates / Q000378:metabolism; D007124:Immunoenzyme Techniques; D010766:Phosphorylation / Q000187:drug effects; D011247:Pregnancy; D017378:Quinolinic Acid / Q000494:pharmacology*; D051381:Rats; D017208:Rats, Wistar; D014746:Vimentin / Q000378:metabolism
chemical_list: D005904:Glial Fibrillary Acidic Protein; D005971:Glutamates; D014746:Vimentin; D017378:Quinolinic Acid; D002118:Calcium
keywords: Astrocyte, cell signaling; Cytoskeleton remodelling; GFAP phosphorylation; Quinolinic acid