Anti-Semaphorin 4D Rescues Motor, Cognitive, and Respiratory Phenotypes in a Rett Syndrome Mouse Model
, Elizabeth E. Evans 3, Vikas Mishra 3, Leslie Balch 3, Allison Eberhardt 3, Maurice Zauderer 3,† and Wendy A. Gold 1,2,4,5,*,†Round 1
Reviewer 1 Report
The current study reports the beneficial effects of anti- SEMA4D Ab in preventing and improving the symptoms and deficits in a trans- genic mouse model of Rett syndrome. The presented data suggest that anti- SEMA4D Ab immunotherapy is a potentially promising therapeutic strategy to improve phenotype, coordination, cognition, locomotion and respiration in Rett syndrome.
Specific comments:
- Anti-Semaphorin 4D antibody rescues motor, cognitive and respiratory phenotypes in a Rett syndrome mouse model
- Materials & Methods section. Animal model. The information on hemizygous Mecp2T158A/y mutant mice is missing.
- Missing important information about the Rett syndrome-associated mutation being positioned in the X-linked gene (encoding methyl-CpG binding protein 2). Only 10% of Rett syndrome patients carry a T158M mutation whereas a T158A mutation is far rarer.
- Unclear why electroencephalography was not performed on these mice.
- The following statement needs clarification: A patent application for the USE OF SEMAPHORIN-4D BINDING MOLECULES FOR THE TREATMENT OF RETT SYNDROME has been lodged. Lodged where?
- Unclear why in figure legends the mice are named the Rett-syndrome mice instead of a proper strain nomenclature especially considering the fact that both, transgenic and knock-in mice were used in this study.
- Lines 210-211. References 22-25?
- Lines 204-215. It has not been proven in this study that anti-Sema4D Ab penetrate BBB because its level was not measured in brain or its ability to cross BBB was not tested in vitro using brain endothelial cell layers. In general, Abs poorly cross BBB. Was a receptor-mediated transcytosis involved in anti-Sema4D Ab’s BBB crossing as endothelial cells? Endothelial cells express Plexin B1. Could the observed effect be mediated by BBB cell activation, increased permeability, and/or mediator’s secretion? Or it is the opposite effect where microglia activation leads to the BBB disruption? How anti-Sema4D Ab affect Plexin B1 expression in brain tissues?
- Figure 4. Panel A shows the lack of Sema4D expression in mouse brain. The reviewer does not see the WT mice in both pre-symptomatic and symptomatic cohorts in this Figure. Interesting that Sema4D is still highly expressed in brains of anti-Sema4D Ab treated mice.
Author Response
The current study reports the beneficial effects of anti- SEMA4D Ab in preventing and improving the symptoms and deficits in a trans- genic mouse model of Rett syndrome. The presented data suggest that anti- SEMA4D Ab immunotherapy is a potentially promising therapeutic strategy to improve phenotype, coordination, cognition, locomotion and respiration in Rett syndrome.
Specific comments:
1. Anti-Semaphorin 4D antibody rescues motor, cognitive and respiratory phenotypes in a Rett syndrome mouse model
No action required
2. Materials & Methods section. Animal model. The information on hemizygous Mecp2T158A/y mutant mice is missing.
We have expanded on the model in this section.
3. Missing important information about the Rett syndrome-associated mutation being positioned in the X-linked gene (encoding methyl-CpG binding protein 2). Only 10% of Rett syndrome patients carry a T158M mutation whereas a T158A mutation is far rarer.
This has been explained in the materials and methods section
4. Unclear why electroencephalography was not performed on these mice.
This is a valid question. Unfortunately, our laboratory does not have this equipment or the expertise to perform EEGs. In addition, due to the continuous monitoring of the mice throughout the trial, we were unable to send these mice to any collaborating laboratories. However, despite this, we believe that the accumulative power of the behaviour tests performed in this study strongly supports the efficacy of anti-SEMA4D treatment.
5. The following statement needs clarification: A patent application for the USE OF SEMAPHORIN-4D BINDING MOLECULES FOR THE TREATMENT OF RETT SYNDROME has been lodged. Lodged where?
The patent applications were filed in USPTO (United States Patent and Trademark Office) and PCT (Patent Cooperation Treaty).
6. Unclear why in figure legends the mice are named the Rett-syndrome mice instead of a proper strain nomenclature especially considering the fact that both, transgenic and knock-in mice were used in this study.
This has been updated in the legend and the rest of the manuscript.
7. Lines 210-211. References 22-25?
This has been corrected.
8. Lines 204-215. It has not been proven in this study that anti-Sema4D Ab penetrate BBB because its level was not measured in brain or its ability to cross BBB was not tested in vitro using brain endothelial cell layers. In general, Abs poorly cross BBB. Was a receptor-mediated transcytosis involved in anti-Sema4D Ab’s BBB crossing as endothelial cells? Endothelial cells express Plexin B1. Could the observed effect be mediated by BBB cell activation, increased permeability, and/or mediator’s secretion? Or it is the opposite effect where microglia activation leads to the BBB disruption? How anti-Sema4D Ab affect Plexin B1 expression in brain tissues?
The reviewer is correct that the BBB is relatively efficient at blocking antibody penetration. But it is not perfect. The references provided demonstrate that 0.1-0.3% of circulating antibody will penetrate into the brain, irrespective of its target. The proposed mechanism by which Abs cross the BBB are discussed in Wang et al. We were unable to effectively measure drug in the brain in this study due to difficulty in obtaining CSF from mice, however, in other studies (including a clinical study in Huntington’s Disease, manuscript in preparation), we have detected drug in the CSF at the expected levels.
9. Figure 4. Panel A shows the lack of Sema4D expression in mouse brain. The reviewer does not see the WT mice in both pre-symptomatic and symptomatic cohorts in this Figure. Interesting that Sema4D is still highly expressed in brains of anti-Sema4D Ab treated mice.
The representative images in figures 4-6 were age-matched, all were selected from brain tissue samples collected at study endpoint of WT and RTT mice in the 8-week cohort.
The antibody used for IHC detection of SEMA4D in brain tissue is polyclonal and it was confirmed that monoclonal anti-SEMA4D Mab67 used for in vivo treatment does not block detection with the IHC antibody. The reduced level of SEMA4D expression in these samples is therefore likely to be a true reflection of reduced SEMA4D levels following treatment. A possible explanation for reduced SEMA4D levels in mice treated with anti-SEMA4D antibody is antibody-induced internalization of the antigen-antibody complex, as described in Fisher et al.
Reviewer 2 Report
Thank you for allowing me to review the manuscript by Mao and colleagues “Anti-Semaphorin 4D rescues motor, cognitive and respiratory phenotypes in a Rett syndrome mouse model” an interesting study describing the use of anti-SEMA4D monoclonal antibody for rescuing some clinical features of Rett syndrome mouse model.
Major revisions
The authors describe interesting results for neurological symptoms of the Rett syndrome mouse model treated with anti-SEMA4D monoclonal antibody. They do not see any significant results on survival. Might they discuss this result?
The authors calculated the difference of coordination and cognition skills in wt (treated with isotype antibody), and Rett treated with anti-SEMA4D vs Rett (treated with isotype antibody). Have the authors calculated the difference between the wt and Rett treated with anti-SEMA4D? Can they add the eventual significance of wt vs Rett on anti-SEMA4D in the figure 1?
Line 276-279. “Anti-SEMA4D treatment reduced the mean SEMA4D 276 intensity in NeuN+ neuronal cells but remained elevated relative to wild-type in the cortex 277 and striatum in both pre-symptomatic and symptomatic cohorts (p < 0.05), as well as in 278 the pons in the symptomatic cohort (p < 0.01)”. These lines are not clear to me when I see Figure 4B to G. I can infer that also cerebellum SEMA4D in treated mice is increased in cerebellum vs wt with statistical significance.
The authors interestingly showed a significant improvement of anti-SEMA4D in the prevention and reversion of Rett syndrome neurological symptoms and signs. Presently, in translational research, the prevention of Rett syndrome neurological symptoms is still debating since the diagnosis is mostly achieved when neurological symptoms are already evident. Thus, I would suggest to emphasize a potential use of anti-SEMA4D in neurological phenotype reversion.
Minor revisions
Line 325 add Figure to the number 6.
In Figure 7 line 355 Rett syndrome is repeated twice.
Line 428 reference 38 is repeated twice.
Author Response
Thank you for allowing me to review the manuscript by Mao and colleagues “Anti-Semaphorin 4D rescues motor, cognitive and respiratory phenotypes in a Rett syndrome mouse model” an interesting study describing the use of anti-SEMA4D monoclonal antibody for rescuing some clinical features of Rett syndrome mouse model.
Major revisions
The authors describe interesting results for neurological symptoms of the Rett syndrome mouse model treated with anti-SEMA4D monoclonal antibody. They do not see any significant results on survival. Might they discuss this result?
This is a good observation and has been added to the discussion section.
The authors calculated the difference of coordination and cognition skills in wt (treated with isotype antibody), and Rett treated with anti-SEMA4D vs Rett (treated with isotype antibody). Have the authors calculated the difference between the wt and Rett treated with anti-SEMA4D? Can they add the eventual significance of wt vs Rett on anti-SEMA4D in the figure 1?
Our calculations, which are described in each figure legend included: Wildtype vs Rett treated with isotype control (* WT vs P), Wildtype vs Rett treated with anti-SEMA4D (~ WT vs T), and Rett treated with isotype control vs vs Rett treated with anti-SEMA4D ( ^ P vs T) using the symbols *, ~ and ^ which we placed at the top of the highest trace for clarity. We note that the figures invariably indicate that the difference between wt treated with isotype control and Rett treated with anti-SEMA4D is smaller than the difference between wt and Rett treated with isotype control. In many instances, the overlap in curves indicates that the difference cannot be significant. We think it would unnecessarily compromise the legibility of figures to add more information to this effect.
Line 276-279. “Anti-SEMA4D treatment reduced the mean SEMA4D 276 intensity in NeuN+ neuronal cells but remained elevated relative to wild-type in the cortex 277 and striatum in both pre-symptomatic and symptomatic cohorts (p < 0.05), as well as in 278 the pons in the symptomatic cohort (p < 0.01)”. These lines are not clear to me when I see Figure 4B to G. I can infer that also cerebellum SEMA4D in treated mice is increased in cerebellum vs wt with statistical significance.
This has been corrected in the manuscript:
The authors interestingly showed a significant improvement of anti-SEMA4D in the prevention and reversion of Rett syndrome neurological symptoms and signs. Presently, in translational research, the prevention of Rett syndrome neurological symptoms is still debating since the diagnosis is mostly achieved when neurological symptoms are already evident. Thus, I would suggest to emphasize a potential use of anti-SEMA4D in neurological phenotype reversion.
This has been included in our discussion:
Minor revisions
Line 325 add Figure to the number 6.
This is a reference and has been corrected.
In Figure 7 line 355 Rett syndrome is repeated twice.
This has been corrected
Line 428 reference 38 is repeated twice.
This has been corrected
