Inhibition of microtubule detyrosination by parthenolide facilitates functional CNS axon regeneration

Public Review:

The primary goal of this paper is to examine microtubule detyrosination as a potential therapeutic target for axon regeneration. Using dimethylamino-parthenolide (DMAPT), this study extensively examines mechanistic links between microtubule detyrosination, hyper-interleukin-6 (hIL-6), and PTEN in neurite outgrowth in retinal ganglion cells in vitro. These findings provide convincing evidence that parthenolide has a synergistic effect on hIL-6- and PTEN-related mechanisms of neurite outgrowth in vitro. The potential efficacy of systemic DMAPT treatment to promote axon
regeneration in mouse models of optic nerve crush and spinal cord injury was also examined.

Strengths:

1. The examination of synergistic activities between parthenolide, hyperIL-6, and PTEN knockout is leveraged not only for potential therapeutic value, but also to validate and delineate mechanism of action.

2. The in vitro studies utilize a multi-level approach that combines cell biology and biochemistry approaches to dissect the mechanistic link from parthenolide to microtubule dynamics.

3. The studies provide a basis for others to test the role of DMAPT in other settings, particularly in the context of other effective pro-regenerative approaches.

Weaknesses:

1. In vivo studies are limited to select outcomes of recovery and do not validate or address mechanism of action in vivo.

2. Known activities of DMAPT beyond microtubule detyrosination, such as oxidative stress, mitochondrial function and NFkB inhibition, are not considered in experimental examinations or in the interpretation of findings.

Author Response:

Weaknesses:

1. In vivo studies are limited to select outcomes of recovery and do not validate or address mechanism of action in vivo.

2. Known activities of DMAPT beyond microtubule detyrosination, such as oxidative stress, mitochondrial function and NFkB inhibition, are not considered in experimental examinations or in the interpretation of findings.

Response: Our research indicates that parthenolide exhibits a regenerative effect within a nanomolar range and with a bell-shaped concentration-response curve in culture. Moreover, we demonstrate a close correlation between the inhibition of detyrosinated microtubules and regeneration and consider the effects of hIL-6 or PTEN-KO on detyrosination in mouse and human RGCs. Therefore, we offer a coherent and satisfactory mechanistic explanation for the effects of parthenolide. We, therefore, feel the request to experimentally explore additional, somewhat speculative possibilities is not reasonable or helpful, and this issue should not be considered as a weakness.

Moreover, to the best of our knowledge, no evidence suggests profound antioxidative effects of DMAPT or parthenolide within these low-concentration ranges and that these would affect axon regeneration. Antioxidative effects may also not explain the observed bell-shaped curve. Furthermore, we have already considered the effect of NFkappaB in our previous work (Gobrecht et al., 2016) and shown that NFkappaB remains unaffected by low concentrations of parthenolide. Hence, conducting additional experiments addressing oxidative stress or other speculative causes will not strengthen our findings and do not justify the additional sacrifice of animal lives. Nevertheless, we will consider discussing these points in a revised version.