Alzheimer's-afflicted neurons contain phosphorylated forms of tau that are not present in healthy adults, these can be recognized with great specificity by monoclonal antibodies such as paired helical filament-1 (PHF-1) [Greenberg S. G. and Davies P. (1990)Proc. natn. Acad. Sci. U.S.A.87, 5827-5831; Greenberg S. G.et al. (1992)J. biol. Chem.267, 564-569]. The PHF-1 phospho-tau epitope is also present in immature neurons undergoing axodendritic differentiation [Pope W. B.et al. (1993)Expl Neurol.120, 106-113]. Analogous to its presence in immature neurons, we report here that the PHF-1 tau epitope spontaneously occurs in the human neuroblastoma cell line SHSY5Y, where its level can be regulated by differentiation and by molecules found in the extracellular matrix. Confocal immunofluorescence studies showed PHF-1 epitope to be constitutively expressed in the somatic cytoplasm as well as in short neurites typical of undifferentiated SHSY5Y cells. Induction of differentiation with retinoic acid produced cells with a neuronal morphology and a redistribution of the expression of PHF-1 tau in the long neurites. Protracted exposure to retinoic acid decreased the levels of PHF-1 immunofluorescence without a loss of neurites, similar to the developmental down-regulation seenin situ. The effects of retinoic acid on PHF-1 immunofluorescence were modifiable by fibronectin, which can be released by some neuroblastoma cell lines [Ciccarone V.et al. (1989)Cancer Res.49, 219-225; Yoshihara T.et al. (1992)Int. J. Cancer51, 620-626.]. Exogenous human fibronectin caused a marked up-regulation of PHF-1 immunofluorescence. Quantitative analysis of 15 multicellular areas, from six different cultures, per experimental condition showed a 16-fold increase compared to untreated controls. Up-regulation by fibronectin was also evident in undifferentiated cells. Cell counts indicated no proliferative effects of the fibronectin under the conditions used. Laminin also caused an increase of PHF-1 tau in retinoic acid-treated cells. Data obtained from immunoblots verified the results observed with immunofluorescence. The data show that the PHF-1 tau epitope is spontaneously expressed by non-degenerating human neuroblastoma cells, down-regulated by cellular differentiation, induced by retinoic acid and up-regulated by the extracellular matrix components fibronectin and laminin. One explanation of the data is that fibronectin maintains a population of SHSY5Y cells in a biochemical state of differentiation in which PHF-1 tau is expressed. This effect occurs depsite the presence of morphological changes accompanying long-term retinoic acid-induced differentiation. This study shows that molecules of the extracellular matrix can regulate the phosphorylation state of tau, increasing expression of an epitope previously linked specifically to axon formation in developing neurons and to Alzheimer's neurodegeneration in the adult. We hypothesize, therefore, that extracellular matrix molecules, in their known ability to influence axon outgrowth, may do so via a mechanism associated with tau phosphorylation, and that matrix-related mechanisms could play a role in generating aberrant phospho-tau epitopes in Alzheimer's disease.
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