Neurocentre Magendie, INSERM U1215
Heike Blockus, Sebastian V. Rolotti, Miklos Szoboszlay, et al.
Amr Abouelezz, Holly Stefen, Mikael Segerstråle, et al.
Answers to the questions from the authors:
– The inhibition of Tpm3.1 leads to a decrease of AnkG and also promotes the disruption of the somatodendritic vesicles filter at the AIS. It is known that this last effect is also triggered the invalidation of AnkG. Is it possible that Tpm3.1 is promoting such an effect by disrupting the actin and decreasing AnkG by direct interaction? Do you plan to run interaction experiments between Tpm3.1 and AnkG / other AIS components, considering also that some of them show periodic patterns within the AIS, like Tpm3.1?
This is a crucial point. We are very interested in elucidating the exact mechanism through which Tpm3.1 contributes to the stability of the structure. As you mentioned, disrupting AnkG accumulation (for example through knocking it down) leads to the loss of AIS structure and function. The question is whether the effects we see on AIS structure and function upon Tpm3.1 inhibition are a result of the loss of AnkG, or is the loss of AnkG itself one of the effects of Tpm3.1 inhibition, which acts through a different mechanism. According to the current literature and our unpublished results, Tpm3.1 would be situated around 100 nm away from AnkG. Therefore, direct interaction between Tpm3.1/actin rings and AnkG is not plausible. However, it is possible that other components of the AIS lattice, such as betaIV-spectrin, are acting as a link that mediates this interaction. We are actively looking into this!
– You show that Tpm3.1 is expressed in the mature AIS, do you know if it is also true for the stages where the AIS is developing? In this case, would it be possible to downregulate Tpm3.1 at DIV0 with and assess the consequences on AIS formation?
The literature suggests that Tpm3.1 localizes to the axons of developing neurons early during development. Hannan et al. (Mol. Cell. Neurosci., 1995) described the segregation of Tpm3.1 as the “earliest known marker for neuronal polarity”. We did not test the effects of downregulating Tpm3.1 at the early stages. In the experiments we conducted with Tpm3 KO mice, we introduced Cre at DIV0 in cultures. However, the experiments were run at DIV10 and even at DIV10, neurons had some residual Tpm3.1 left (which contributes to the milder effects of KO compared to acute inhibition). It would be interesting to test the effect of direct acute inhibition at the early stages and seeing if Tpm3.1 affects AIS formation or if it is only required for AIS maintenance.