The microtubule associated protein Tau (MAPT) is a neuronal specific protein and plays important roles in regulating microtubule stabilities and neuronal polarization. Alternative RNA splicing of the single Tau gene produces six different isoforms in the central nerve system, which differ by the absence or presence of one or two acidic insertions (called 0N, 1N or 2N) in the amino-terminus and three or four microtubule binding repeats (called 3R and 4R) in the carboxyl-terminal region. In cultured rat hippocampal neurons, the development-dependent alterations in the cellular distributions of 3R-and 4R-Tau suggest that they regulate the microtubule cytoskeleton of growing neurons in a spatially and temporally specific and yet differential manner. Moreover, earlier studies in our laboratory also suggest that 3R-and 4R-Tau isoforms interact with microtubules of different dynamic states in axonal growth cone. Therefore, in order to investigate the different effects of 3R- and 4R-Tau proteins on microtubule stabilities during neuronal development, I have constructed two plasmids respectively consisting of the 3R and 4R domains of rat Tau with the C-termini tagged by DsRed (called 3RDsRed and 4RDsRed respectively). The 3RDsRed and 4RDsRed constructs are transfected into cultured hippocampal neurons, and the transfected neurons are subjected to fluorescence immunostaining to study the effects of over-expressing 3R- and 4R-regions of Tau on structural polarization and neurite growth. The results reveal that the over-expression of neither 3R- nor 4R-region affects neuronal polarity. Nevertheless, over-expressing 4R region of Tau protein impedes neurite growth, while 3R region does not overtly affect neurite growth. Combined with previous studies, our results suggest that 3R and 4R Tau isoforms display distinct roles on stabilizing microtubule and thus participate in the regulation of neurite growth during the development of rat hippocampal neurons in culture.

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