After a brief wash with PBS, the cells were blocked with 1% BSA/PBS for 1 h and then they were exposed to the primary antibodies diluted in 1% BSA/PBS for 1 h: an antibody against Smi-31 (1:1000 Sternberger) as an axonal marker and anti-β-III-tubulin (1:2000 Covance) as a neuronal marker. After several washes with PBS, the cells were permeabilized in 0.1% Triton X-100 for 15 min and they were then treated with 1 M glycine for 30 min to eliminate any autofluorescence. 2.3 ImmunofluorescenceĪfter 24 and 48 h in culture, the hippocampal cells were fixed for 20 min with warm 4% paraformaldehyde in PBS containing 4% sucrose. After 4 h, the medium was replaced by the maintenance medium: Neurobasal medium (Invitrogen) supplemented with 2% B-27, 1% N2, 0.5 mM Glutamine, 1 mM pyruvate, 1% penicillin/streptomycin. The cells recovered were plated on poly lysine-coated (1 mg/ml) coverslips at low density in 86% DMEM containing 10% horse serum, 0.5 mM Glutamine, 1% penicillin/streptomycin. Briefly, the hippocampus was dissected out from each pup and dissociated individually with the papain dissociation system (Worthington Biochemical Corp. Hippocampal cell cultures were prepared from 17-day-old mouse fetuses according to modified versions of established procedures. Four to five mice were housed per cage in a temperature controlled environment on a 12/12 h light-dark cycle, with food and water available ad libitum. The animals were bred at the Centro de Biología Molecular “Severo Ochoa” (Madrid, Spain) and they were maintained in accordance with the institutional guidelines. 3 month-old male mice were used in the experiments carried out here. Tau −/− mice were generated as described previously, crossing heterozygous ( Tau +/−) mice to obtain homozygous tau knockout mice ( Tau −/−) and control littermates ( Tau +/+). We observed differences in the level of smarce1, which codes for BAF-57, a protein involved in the repression of neuronal specific genes and whose expression is stimulated in tau deficient mice.
We confirmed that tau is indeed present in the nucleus of cells and hence, we tested the possible influence of the tau protein on gene expression showing that it can modify the expression of certain genes. Tau can also bind to nucleic acids and it has been observed in the nucleus of neuronal cells. Nevertheless, delayed axonogenesis was observed in tau deficient neurons, a feature that has yet to be fully explained. The mild phenotype of these mice could be due to functional redundancy between tau and other microtubule-associated proteins. Tau deficiency delays neurite extension in cultured neurons, although the tau deficient mice produced by gene-targeting are viable and they do not show important cytoskeletal abnormalities.
Tau is a microtubule associated protein that promotes tubulin assembly in vitro, as well as stabilizing assembled microtubules in cultured cells by suppressing microtubule dynamics.
0 kommentar(er)
