Figure 9. Characterization of transglutaminase activity, TG2 protein and microtubule stability in postnatal mouse brain development.

To evaluate changes in transglutaminase activity and TG2 protein as neurons develop and mature, mouse brain was analyzed at 10d (pre myelination); 3wk (active myelination) and 3mo (mature adult neurons). A) Fluorescent intensity of MDC incorporated into casein shows significantly increased transglutaminase activity during postnatal brain development. During this interval, neurite outgrowth plays a minimal role, but consolidation of synaptic connections and myelination progresses toward adult levels. Changes in transglutaminase activity during this time may be related to axonal maturation, correlated with myelination, B) Quantitative analysis show that transglutaminase activity increased 1.5 and 3 fold in 3wk and 3mo brains respectively, as compared with 10d mouse brain. C) Consistent with changes in transglutaminase activity, TG2 protein levels in western blots increased. Kinesin (H2) immunoreactivity was a loading control. D) Similar changes in TG2 protein and activity levels occurred during postnatal mouse brain development. E) Alteration of MT stability during postnatal mouse brain development. Cold/Ca²⁺ fractionations were performed on mouse brain from the 3 age groups and analyzed by immunoblot with DM1A antibody, which recognizes α-tubulin. Cold stable and cold/Ca²⁺ stable tubulin levels are 45% and 25% of total tubulin at 10d, respectively. At 3wk, cold stable tubulin increased to 58% of total tubulin, of which cold/Ca²⁺ stable tubulin was 32%. At 3mo, cold stable tubulin increased to 64%, while cold/Ca²⁺ tubulin increased to 36%. Differences are statistically significant (** P< 0.001 between 10-day and 3-week groups, * P<0.05 between 3-week and 3-month groups) n = 6.