Effects of microtubule stabilization and destabilization on tau immunoreactivity in cultured hippocampal neurons.
Tau immunoreactivity is altered in neurofibrillary tangles (NFT) and degenerating neurites in Alzheimer's disease (AD). In addition, cytoskeletal proteins including tau are excessively phosphorylated in AD. Previous data indicated that calcium influx can cause antigenic changes in tau in cultured rat hippocampal and human cortical neurons similar to those seen in NFT. The present study used cultured hippocampal neurons to test the hypothesis that disruption of microtubules is a key event leading to altered antigenic properties of tau that result from calcium influx. As previously reported, we found that glutamate (100-500 microM) and calcium ionophore A23187 (0.5-1 microM) elevated intraneuronal calcium levels and caused a reduction in microtubules, a marked increase in staining with Alz-50 and 5E2, and a decrease in tau-1 immunoreactivity. The microtubule-disrupting agent colchicine (1 microM) caused increased immunoreactivity of neurons towards tau antibodies Alz-50 and 5E2, and these effects of colchicine occurred in the absence of an increase in intracellular calcium levels. The microtubule-stabilizing drug taxol (100 nM) reduced neuronal immunoreactivity towards Alz-50 and 5E2 in untreated cultures and in cultures exposed to glutamate or A23187. Western blot analysis indicated that A23187 caused a reduction in tau levels which was partially prevented by taxol, suggesting that tau associated with microtubules is less susceptible to calcium-mediated degradation. Acid phosphatase treatment increased neuronal immunoreactivity towards tau-1 and reduced immunoreactivity towards Alz-50. The calcium-induced alterations in tau immunoreactivity were, and the colchicine-induced alterations were not, affected by acid phosphatase treatment. Taken together, the data indicate that microtubule depolymerization can cause antigenic changes in tau similar to those seen in NFT independently of an increase in intraneuronal calcium levels. Stabilization of microtubules prevented the antigenic changes in tau suggesting that microtubules affect the availability and/or properties of epitopes on tau that are recognized by antibodies that stain NFT.
Sanders-Brown Research Center on Aging, University of Kentucky, Lexington 40536-0230.
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Mattson
D000470:Alkaloids / Q000494:pharmacology*; D000818:Animals; D000001:Calcimycin / Q000494:pharmacology; D002118:Calcium / Q000378:metabolism*; D002478:Cells, Cultured; D003078:Colchicine / Q000494:pharmacology*; D004622:Embryo, Mammalian; D016257:Fura-2; D005971:Glutamates / Q000494:pharmacology; D018698:Glutamic Acid; D006624:Hippocampus / Q000378:metabolism / Q000648:ultrastructure*; D007150:Immunohistochemistry; D007700:Kinetics; D008870:Microtubules / Q000187:drug effects / Q000502:physiology / Q000648:ultrastructure*; D009474:Neurons / Q000187:drug effects / Q000378:metabolism / Q000648:ultrastructure*; D017239:Paclitaxel; D011712:Pyramidal Tracts / Q000187:drug effects / Q000378:metabolism / Q000648:ultrastructure; D051381:Rats; D016875:tau Proteins / Q000032:analysis / Q000378:metabolism*
D000470:Alkaloids; D005971:Glutamates; D016875:tau Proteins; D000001:Calcimycin; D018698:Glutamic Acid; D017239:Paclitaxel; D003078:Colchicine; D002118:Calcium; D016257:Fura-2