Figure 7. Effects of transglutaminase inhibition on neurite extension in SH-SY5Y cells.

To determine functions of polyamination and MT stability in neuronal development, we differentiated SH-SY5Y neuroblastoma in the presence of IR072 (fig S5), an irreversible transglutaminase inhibitor. A) Control SH-SY5Y cells were differentiated, fixed and stained with beta III tubulin antibody. Normal neurite extension and neuron-like phenotypes were observed. B) SH-SY5Y cells differentiated in the presence of the IR072 showed significant inhibition of neurite outgrowth and a phenotype more like undifferentiated SH-SY5Y cells. C) Length distribution of neurites for control SH-SY5Y cells showed an average of 76μm with some neurites >150μm. D) Length distribution of neurites for differentiated SH-SY5Y cells treated with IR072 showed a significant shift towards shorter neurites with an average length of 29μm with few neurites >90μm. The difference in mean neurite length for control and IR072 treated cells was statistically significant (P<0.00001). This suggests that transglutaminase activity is required for efficient elongation of neurites during differentiation of SH-SY5Y cells. E) Cold/Ca²⁺ fractionations were done on SH-SY5Y cells under 3 conditions (undifferentiated, differentiated, differentiated with IR072). Western blots with DM1A antibody (α-tubulin) showed that: 1) in undifferentiated cells with low transglutaminase activity and TG2 protein levels, <10% of total tubulin was cold stable, with no cold/Ca²⁺ stable tubulin. 2) In fully differentiated cells where both transglutaminase activity and TG2 proteins level peaked, cold stable tubulin level increased significantly to >40% of total tubulin and cold/Ca²⁺stable tubulin was >20% of total tubulin); 3) In differentiated cells treated with IR072 where transglutaminase activity was significantly reduced, cold/Ca² stable tubulin was dramatically lowered <10%. This suggests a strong correlation between transglutaminase activity and microtubule stability, both of which may contribute to neurite development.