GSK3-CRMP2 signaling mediates axonal regeneration induced by Pten knockout

Knockout of phosphatase and tensin homolog (PTEN−/−) is neuroprotective and promotes axon regeneration in mature neurons. Elevation of mTOR activity in injured neurons has been proposed as the primary underlying mechanism. Here we demonstrate that PTEN−/− also abrogates the inhibitory activity of GSK3 on collapsin response mediator protein 2 (CRMP2) in retinal ganglion cell (RGC) axons. Moreover, maintenance of GSK3 activity in Gsk3S/A knockin mice significantly compromised PTEN−/−-mediated optic nerve regeneration as well as the activity of CRMP2, and to a lesser extent, mTOR. These GSK3S/A mediated negative effects on regeneration were rescued by viral expression of constitutively active CRMP2T/A, despite decreased mTOR activation. Gsk3S/A knockin or CRMP2 inhibition also decreased PTEN−/− mediated neurite growth of RGCs in culture and disinhibition towards CNS myelin. Thus, the GSK3/CRMP2 pathway is essential for PTEN−/− mediated axon regeneration. These new mechanistic insights may help to find novel strategies to promote axon regeneration.

The manuscript by Leibinger et al. investigates the signaling principles relevant to PTEN-loss mediated growth and regeneration responses using the optic nerve as model system. Work by others identified (a while back) robust installment of axon growth following a crush injury to the optic nerve in PTEN deficient retinal ganglion cells. This response was shown to involve PI3K/PTEN dependent regulation of downstream mTOR function. On the other hand, this current group previously demonstrated that axon regeneration in the same injury model can be modulated by the PI3K/PTEN and Akt regulated kinase GSK3. Here, the group uses a number of different experimental system and combinational approaches to analyze the possibility that GSK3 (as well as CRMP2) is involved in mediating PTEN-loss induced axon regeneration responses. The main conclusion is that, indeed, GSK3/CRMP2 is essential for PTEN-/mediated axon regeneration. The authors employ a number of epstasis experiments to detail the signalling relationship. For example, they show that maintenance of GSK3 activity in mice with overactivated GSK3 compromised PTEN-loss mediated optic nerve regeneration. One of the major findings of this study is that GSK3 in itself is able to activation mTOR function in crush injury models, indicating that one could bypass PI3K/PTEN to module this pathway. The work is well executed and results are convincing. However, the paper is difficult to read, not alone because of the difficult signaling relationships tested. The paper is an obvious progression from previous publications, and therefore deserves publication. I only have few comments on how to improve the work.
It would be of interest to see if the increase in GSK3 phosphorylation in control animals following ONC dependent on Rapamycin? A further readout for PTEN-loss is the increase in neuronal soma size. The data indicate that there might be a dichotomy on this PI3K/PTEN dependent function, which would be of great interest to the community. The authors should analyze cell size of RGC in the presence/absence of PTEN with or without constitutive activity of GSK3alpha/beta (see Figure 4). The discussion is stating the obvious that is already introduced/mentioned in the introduction/result sections. It would be of great interest to incorporate further topics, such as the potential involvement of PTEN dependent, but PI3K independent functions. Do the authors believe that all regenerative growth that can be installed through PTEN-loss involve its lipid phosphatase (i.e. is all PTEN-loss induced growth dependent on mTOR and GSK3)? Or is it conceivable that PTEN dependent, but PI3K independent function could regulate axon regeneration…. Or that PTEN feedback regulation by GSK3 (PTEN T366) is involved in axon regeneration? I believe the authors could expand discussing the implications of their findings further….
Reviewer #2 (Remarks to the Author): Phosphatase and tensin homolog (PTEN) has been identified as a prominent intrinsic inhibitor of CNS axon regeneration. PTEN knockout mediates axon regeneration in CNS through the PI3K/AKT/TSC signaling pathway and mTOR is a key downstream signal of PI3K/AKT in regulating the process of cellular growth. However, The precise mechanisms by which PTEN/mTOR controls axon regeneration remain to be elucidated. in this study, Leibinger et al. utilized different genetic mouse models to investigate the underlying mechanism of knockout of (PTEN) promoting axon regeneration after optic nerve crush injury. Their study shows that PTEN knockout significantly increased inhibitory phosphorylation of both GSK3α and GSK3β and reduced CRMP2 phosphorylation. The inactivation of GSK3 and the downstream release of axonal CRMP2 from inhibition is an essential part of the mechanism of the axon growth promoting effect of PTEN knockout. PTEN knockout induces inhibitory GSK3 phosphorylation is mTOR independent. Although this hypothesis needs to be further confirmed in other more common CNS injury model such as spinal cord injury, targeting GSK3/CRMP2 pathway could be a new strategy to promote axon regeneration in CNS in order to avoid the potential risk of cancer triggering by direct inhibition of PTEN. Authors applied βIII-tubulin as a marker for RGC in their histology study and RGC survival quantification. βIII-tubulinis a general neuronal marker but not a specific RGC marker. RBPMS and/or Brn3a should be the selective markers of ganglion cells in the mammalian retina. Type error in Line 610. I should be H instead.
Reviewer #3 (Remarks to the Author): The manuscript by Leibinger et al. reported a detailed signaling pathway downstream of Pten knockout in promoting optic nerve regeneration. Specifically, the study showed that downstream of Pten inhibition, GSK3 inactivation and the subsequent activation of CRMP2 were crucial for optic nerve regeneration. In particular, direct activation of CRMP2 was able to fully rescue Pten-deletion induced optic nerve regeneration without GSK3 phosphorylation and mTor activation. Overall, the results presented in the study were largely nice and clear. The major strength of the study is the identification of CRMP2 as an important downstream mediator of Pten-GSK3 pathway to regulate optic nerve regeneration. The major weakness was the lack of overall novelty. Addressing the following comments with either new experiments or text revising would greatly strengthen the manuscript. Figure 2, all the results were based on western blot of the whole retina tissue, in which proteins from RGCs were only a very small percentage of the total retina proteins. Thus, the results were not convincing enough. The authors should at least mention the potential weakness. Figure 3, the results were very similar to a previous published study, in which active Akt-induced optic nerve regeneration was impaired by GSK3b-S9A or GSK3a-S21A mutants. In Figure 3A, B, the axon lengths in many conditions were shorter than 4um, probably less than the cell diameter. It is difficult to accurately quantify axon length with such short axons.

In
3. In Figure 4, It showed that activation of GSK3a/b had no negative effects on RGC survival promoted by Pten knockout after nerve crush. This result suggests that GSK3 activation have no negative effect on Pten knockout-induced Akt-mTor activation. In Figure 5B, C, Pten knockout-induced mTor activation (increased level of pS6) was reduced by GSK3 activation (SA mutants). In Figure 5I, J double knocking out GSK3a/b increased RGCs survival by activation of mTor. Taken together, it seems that GSK3 activation reduces Pten knockout-induced mTor activation but has no negative effect on neuronal survival, whereas GSK3 knockout activates mTor and promotes neuronal survival. It seems confusing regarding how Akt-mTor activity regulates neuronal survival. A better explanation would be very helpful.
Reviewer #1: I only have few comments on how to improve the work.
It would be of interest to see if the increase in GSK3 phosphorylation in control animals following ONC dependent on Rapamycin?
Response: We followed this suggestion and performed these additional experiments.
Respective data are implemented as suppl. Fig. 1. A-D. The mTOR inhibition (rapamycin treatment) did expectedly not affect the ONC-induced phosphorylation of GSK3α or GSK3β.
Reviewer #1: A further readout for PTEN-loss is the increase in neuronal soma size. The data indicate that there might be a dichotomy on this PI3K/PTEN dependent function, which would be of great interest to the community. The authors should analyze cell size of RGC in the presence/absence of PTEN with or without the constitutive activity of GSK3alpha/beta (see Figure 4). Response: We also followed this suggestion and data are presented as suppl. Fig. 2.
The RGC size was significantly increased after PTEN -/-(A, B), and reduced again by constitutively active GSK3(α/β) S/A . In contrast, the single knockin (GSK3β S/A ) showed no effect, suggesting that the full activity of both GSK3 isoforms is required to abrogate the PTEN -/effect. We also investigated the effect of GSK3 knockouts on the soma sizes with and without ONC (C, D). Consistently, the average soma sizes increased only after a total GSK3 knockout, whereas every single knockout alone showed no effect (C, D).
Reviewer #1: The discussion is stating the obvious that is already introduced/mentioned in the introduction/result sections. It would be of great interest to incorporate further topics, such as the potential involvement of PTEN dependent, but PI3K independent functions. Do the authors believe that all regenerative growth that can be installed through PTEN-loss involve its lipid phosphatase (i.e. is all PTEN-loss induced growth dependent on mTOR and GSK3)? Or is it conceivable that PTEN dependent, but PI3K independent function could regulate axon regeneration…. Or that PTEN feedback regulation by GSK3 (PTEN T366) is involved in axon regeneration?

Sengottuvel et al., 2011). Moreover, BRN3a is only a suitable marker for non-injured
RGCs. It is therefore not applicable for quantification of RGC survival after optic nerve crush since the expression of this protein is downregulated after axotomy (see our data presented in Fig. 1, below).

To demonstrate the complete overlap of RBPMS and βIII-tubulin staining and the downregulation of BRN3a in axotomized RGCs to this reviewer we provide data below
showing retinal flat mounts from either untreated wt mice (con) or animals 21 days after optic nerve crush (ONC) (Fig. 1, below).  Reviewer #2: Type error in Line 610. I should be H instead.

Response: We corrected this typo.
Reviewer #3: Addressing the following comments with either new experiments or text revising would greatly strengthen the manuscript.
In Figure 2, all the results were based on western blot of the whole retina tissue, in which proteins from RGCs were only a very small percentage of the total retina proteins. Thus, the results were not convincing enough. The authors should at least mention the potential weakness.

vs. PTEN +/+ retinae by Western blot analysis and immunohistochemistry and thereby verify that WB analysis of retinal lysates can be used for reliable measurements in
RGCs. We, therefore, feel that adding more immunohistochemical data into Fig. 2 would not add any benefit to the reader and would be rather redundant. We, therefore, would prefer to leave it as it is.
Reviewer #3: In Figure 3, the results were very similar to a previously published study, in which active Akt-induced optic nerve regeneration was impaired by GSK3b-S9A or GSK3a-S21A mutants. In Figure 3A, B, the axon lengths in many conditions were shorter than 4um, probably less than the cell diameter. It is difficult to accurately quantify axon length with such short axons. activation have no negative effect on Pten knockout-induced Akt-mTor activation. In Figure   5B, C, Pten knockout-induced mTor activation (increased level of pS6) was reduced by GSK3 activation (SA mutants). In Figure 5I, J double knocking out GSK3a/b increased RGCs survival by activation of mTor. Taken together, it seems that GSK3 activation reduces Pten knockout-induced mTor activation but has no negative effect on neuronal survival, whereas GSK3 knockout activates mTor and promotes neuronal survival. It seems confusing regarding how Akt-mTor activity regulates neuronal survival. A better explanation would be very helpful.

Response: This comment appears to be based on a misunderstanding because
Response: This is a valuable point. We think that mTOR activity must be above a certain threshold to increase neuronal survival and soma size. This is consistent with our observation that only PTEN -/and GSK3(α/β) -/-, which both enhanced pS6 levels to a significantly stronger extent compared to every single knockout alone (GSK3α or GSK3β), were neuroprotective.
As shown by the current study, PTEN -/activates mTOR via two routes: Indirectly via

GSK3 inhibition and directly via AKT/TSC/Rheb. Under conditions of constitutive active GSK3 (SA mutants) mTOR activity is slightly reduced in PTEN -/-RGCs.
However, the remaining mTOR activity seems to be still sufficiently high to confer neuroprotection. Moreover, according to our newly generated data (suppl. Fig. 2 To address this point we included the following paragraph into the discussion:

"The observation that GSK3β -/failed to rescue axotomized RGCs from degeneration and did not increase soma size, while both PTEN -/and the GSK3(α/β) -/induced higher pS6 levels, soma sizes and were neuroprotective suggests that a mTOR activity only above a certain level affects neuroprotection and soma size. Consistently, only the double knockin GSK3(α/β) S/A , but not the single GSK3β S/A knockin compromised PTEN -/induced increase in the soma size, which was previously described as a consequence of S6k1 activity downstream of mTOR (Yang et al., 2014). In addition, the higher pS6 levels and soma sizes observed after PTEN -/compared to GSK3(α/β) -/also correlated with a stronger neuroprotection and may be due to the additional mTOR activation via the PI3K/AKT/Rheb axis."
Reviewer #3: In Fig. 9, the overexpression of active CRMP2 mutant was able to fully restore Pten knockout-induced optic nerve regeneration impaired by GSK3b activation. Because GSK3b activation blocked mTor activation, it seemed that mutant CRMP2 promoted optic nerve regeneration with impaired mTor activation.

2017), it is quite possible that the remaining mTOR activity is not required for the CRMP2 T/A effect on regeneration."
Reviewer #3: Moreover, if CRMP2 activation could fully rescue axon growth impaired by GSK3-SA mutants, is CRMP2 the only downstream target of GSK3 to regulate axon regeneration?