Promoting axon regeneration by inhibiting RNA N6-methyladenosine demethylase ALKBH5

  1. Dong Wang
  2. Tiemei Zheng
  3. Songlin Zhou
  4. Mingwen Liu
  5. Yaobo Liu
  6. Xiaosong Gu
  7. Susu Mao
  8. Bin Yu

  • Curated by eLife

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    This important study combines a screen of known N6-methyladenine (m6A)-dependent RNA modifying factors to identify ALKBH5 as critical in crush injury response. They demonstrate through gain and loss of function an effect on ALKBH5 m6A-dependent Lpin2 mRNA stability during injury-induced axon regeneration in both dorsal root ganglia nerve and optic nerve regeneration. The results provide new insight into the role of RNA modification on neural injury. However, the limitations of the experimental design on the conclusions drawn require additional consideration. With additional control experiments and further consideration of the limitations, the paper will provide a link between N6-methyladenine and neurotrauma.

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Abstract

A key limiting factor of successful axon regeneration is the intrinsic regenerative ability in both the peripheral nervous system (PNS) and central nervous system (CNS). Previous studies have identified intrinsic regenerative ability regulators that act on gene expression in injured neurons. However, it is less known whether RNA modifications play a role in this process. Here, we systematically screened the functions of all common m 6 A modification-related enzymes in axon regeneration and report ALKBH5, an evolutionarily conserved RNA m 6 A demethylase, as a regulator of axonal regeneration in rodents. In PNS, knockdown of ALKBH5 enhanced sensory axonal regeneration, whereas overexpressing ALKBH5 impaired axonal regeneration in an m 6 A-dependent manner. Mechanistically, ALKBH5 increased the stability of Lpin2 mRNA and thus limited regenerative growth associated lipid metabolism in dorsal root ganglion neurons. Moreover, in CNS, knockdown of ALKBH5 enhanced the survival and axonal regeneration of retinal ganglion cells after optic nerve injury. Together, our results suggest a novel mechanism regulating axon regeneration and point ALKBH5 as a potential target for promoting axon regeneration in both PNS and CNS.

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  1. Version published to 10.7554/elife.85309 on eLife
  2. eLife

    eLife assessment

    This important study combines a screen of known N6-methyladenine (m6A)-dependent RNA modifying factors to identify ALKBH5 as critical in crush injury response. They demonstrate through gain and loss of function an effect on ALKBH5 m6A-dependent Lpin2 mRNA stability during injury-induced axon regeneration in both dorsal root ganglia nerve and optic nerve regeneration. The results provide new insight into the role of RNA modification on neural injury. However, the limitations of the experimental design on the conclusions drawn require additional consideration. With additional control experiments and further consideration of the limitations, the paper will provide a link between N6-methyladenine and neurotrauma.

  3. eLife

    Reviewer #1 (Public Review):

    This manuscript described the role of ALKBH5, an evolutionarily conserved mRNA m6A demethylase as a key regulator of axon regeneration. The authors screened the function of m6A regulators during axon regeneration and found that ALKBH5 limits regenerative growth associated with DRG neurons, by enhancing the stability of Lpin2 mRNA via erasing a single m6A modification in the 3'UTR. The major strength of the manuscript is the convincing importance of ALKDH5 as an attenuator to initially suppress the axon regeneration in the CNS and in the PNS proven by in vivo model system. These findings further suggest the potential use of ALKDH5 inhibitors to enhance neural regeneration upon physical injury.

  4. eLife

    Reviewer #2 (Public Review):

    The authors wanted to determine if the mRNA modification m6A is involved in axonal regeneration pathways. They performed a small-scale siRNA screen targeting major components of this pathway to determine if not down if any of these genes would influence axonal regeneration. They identified ALKBH5, an m6A demethylating enzyme, as a gene that represses axonal regeneration after injury, and when knocked down, promotes axonal growth. They identify a putative mRNA target of ALKBH5, Lpin2, which they believe is demethylated by ALKBH5, resulting in higher levels of m6A on this transcript and thus greater mRNA degradation and reduced expression.

    This study has major weaknesses. The ALKBH5 knockout mouse is not used. Thus the experiment relies on the selectivity of the siRNA. Many experiments relied on the single siRNA. The knockdown efficiency was relatively poor, with only a small change in ALKBH5 protein levels. The authors never assess whether m6A levels are indeed affected by ALKBH5 depletion using their approach. The results are therefore unconvincing because of not using the appropriate mouse model. Additionally, the authors' attempt to identify a target of ALKBH5 was not done using the appropriate approach, which would involve globally profiling m6A levels in control and ALKBH5 knockout conditions. Since they did not do global profiling of m6A, the authors cannot report how the exact stoichiometry of m6A sites in Lpin2 is affected (and if other mRNAs are affected which might be the true targets of ALKBH5). Attempts by other investigators to identify bona fide targets of ALKBH5 have been difficult, and the authors did not do the appropriate unbiased transcriptome-wide screen but instead used generic gene expression approaches to come to their target. It is not clear if they have a direct or indirect target of ALKBH5 based on the presented data.

    Overall, the authors have not achieved their aims and the results do not support the overall conclusions. However, some studies related to Lpin2 overexpression and not down suggest that this gene indeed can influence axonal regeneration in some way. But whether it is a direct target of ALKBH5 and whether ALKBH5 indeed has any role in axonal regeneration is still not clear.

  5. eLife

    Reviewer #3 (Public Review):

    This is an elegant article that shows the reciprocal actions of Alkbh5 gene modulation and of a putative target, Lpin2. The work focuses on the DRG; it is important to note that the group also examines a retinal ganglion model where changes in Alkbh5 are not as prominent, and perhaps because of that the resulting effects of Alkbh5 modulation are not as pronounced. Thus, the effects of a broadly acting agent such as Alkbh5 might differ depending on the exact biology of the model. This doesn't diminish the finding at all, but the group nicely shows that the efficacy of Alkbh5 gene modulation might vary among different models.

    A strength of the manuscript is the paper's use of reciprocal experimental designs to demonstrate effects. For instance, in Fig. 3 the authors show that Alkbh5 knockdown (KD) improves axonal regeneration and in Fig. 4 they show that Alkbh5 KD increases levels of methylated LPIN2, a transcript implicated in promoting axonal regeneration. Conversely, Figs. 5 and 6 also show that WT Alkbh5 over-expression inhibits axonal regeneration while an inactive Alkbh5 mutant has no effect. Alkbh5 KD is expected to increase levels of its target transcripts, and indeed, the group focuses on LPIN2 and shows that Alkbh5 KD increases LPIN2 adenosine methylation which is associated with a reciprocal decrease in total LPIN2 transcript (because m6A promotes degradation of the labeled transcripts). In Fig. 7 they go on to look at a specific target site of m6A on LPIN2. The hypothesis is that methylation of the target m6A site on LPIN2 will lead to a reduction in transcript levels; they show that mutating this target adenosine prevents the resulting effects of Alkbh5, implicating adenosine methylation in the modulation of LPIN2 levels. Finally, they show that over-expressing LPIN2 inhibits axonal regeneration while inhibiting LPIN2 increases regeneration. Thus, the experimental design includes many levels of reciprocal actions that are observed examining regeneration, adenosine methylation, and LPIN2 transcript levels. Taken together this approach is very convincing. The last figure examines the extent to which the KD approach extends to other models; the authors show that Alkbh5 is less active in a retinal ganglion model. The limited efficacy in the retinal ganglion is disappointing but serves to highlight the strength of the actions in the DRG model and provide a warning that the actions of Alkbh5 might vary significantly depending on the particular pathophysiology to which its gene modulation is being applied.

    The manuscript does have some weaknesses, but the weaknesses are modest and do not change the overall interpretations of the manuscript. For instance, interpreting the quantitative efficacy in Fig. 1 and 2 depends on knowing the efficiency of uptake of the RNAi for Alkbh5 and subsequent virally transduce shAlkbh5, however, the authors do not show this efficiency. But such weaknesses are quite minor and do not change any of the conclusions.

  6. Version published to 10.1101/2022.12.21.521436v1 on bioRxiv