THE PHOSPHORYLATION STATE OF THE MICROTUBULE-ASSOCIATED PROTEIN TAU AS AFFECTED BY GLUTAMATE, COLCHICINE AND BETA-AMYLOID IN PRIMARY RAT CORTICAL NEURONAL CULTURES

The effects of the excitatory amino acid glutamate, the microtubule de stabilizing agent colchicine, and beta(25-35)-amyloid peptide on the p hosphorylation state of tau were studied in rat cortical neurons in pr imary culture. Using immunocytochemistry and Western-blot analysis, we demonstrated that a proportion of tau in these cultures is normally h ighly phosphorylated, but mast of this tau fraction is dephosphorylate d after treatment of the cultures with glutamate or colchicine, but no t with beta-amyloid; the glutamate- and colchicine-induced changes in tau phosphorylation commenced before cell death, as assessed by releas e of lactate dehydrogenase, Dephosphorylation of tau was readily revea led by using the monoclonal antibodies Tau.1 and AT8, which have phosp hate-sensitive epitopes that both centre around serine-199 and -202 (n umbering of the largest tau isoform). On Western blots and by immunocy tochemistry, AT8 labelling strongly decreased after glutamate and colc hicine treatments, whereas Tau.1 staining was more intense. Neurofilam ent monoclonal antibodies, including RT97, 8D8, SMI31 and SMI310, all additionally known to recognize tau in a phosphorylation-dependent man ner, also demonstrated that glutamate and colchicine treatments of the cultures induced a dephosphorylation of tau. We also showed immunocyt ochemically that there is an increase in tau immunoreactivity in neuro nal perikarya in response to glutamate and colchicine treatment, and t his occurs concomitantly with the dephosphorylation of tau. Treatment of the primary rat cortical neuronal cultures with beta(25-35)-amyloid peptide, under conditions which induce neuronal degeneration, did not induce a change in tau phosphorylation, and failed to act synergistic ally with glutamate to produce an increase in dephosphorylation of tau over that produced by glutamate treatment alone. These findings demon strate that glutamate and colchicine induce tau dephosphorylation, as opposed to increased tau phosphorylation, which would be more indicati ve of Alzheimer-type neurodegeneration.