Necdin shapes serotonergic development and SERT activity modulating breathing in a mouse model for Prader-Willi syndrome

Essential revisions:

1) The histological analysis does not indicate enough specifics on how quantification was performed, notably it does not appear that stereological methods were utilized. In the absence of such methods there are significant concerns for bias.

Effectively, we didn’t explain our method of quantification in the text. Now, we introduced this explanation in the Material and methods in the Immunohistochemistry and quantification part as following:

“Brainstem structures were sampled by selecting the raphe obscurus area and counting was performed on 3 sagittal sections of 100µm/animals which represent the entire PET1-YFP positive cell population of the raphe obscurus (ROb/B2) and pallidus (RPa/B1), both nuclei being difficult to separate. For each section, a Z-stack composed of 10 confocal images (8 µm focal spacing) was acquired. For quantification, stereological method has been applied on each Z-stack image using the eCELLence software developed by Glance Vision Technologies (Italy). The total cell number/ per animal was obtained by summing the sub-total of cells counted for the 3 Z-stacks.”

2) The authors focus on the serotonin system and the effect of modulating the 5-HT system genetically or pharmacologically. Previous studies have shown that other excitatory neuromodulators (Substance P and TRH) likewise partially rescue the respiratory deficit in brainstem-spinal cord preparations from Ndn-KO mouse pups (Pagliardini Advances in Experimental Medicine and Biology 2008). This raises the possibility that the serotonergic deficit is only one of many deficits in this genetic mutant. This point should be addressed and weighed in light of previous publications.

This point is extremely relevant. The reasons why, in regards to the respiratory distress, we studied the 5-HT neurons and system are: 1) previously we showed that Necdin is not expressed in the NKR1 positive neurons of the preBötzinger complex ((Zanella et al., 2008b); Figure S3), excluding a cell autonomous alteration of those cells; 2) Necdin is expressed in medullary 5HT neurons and Necdin deficiency in medullary preparations of Ndn-KO neonates alters the serotonergic modulation of the inspiratory rhythm generator (Zanella et al., 2008b), 3) Abnormal projections of 5-HT fibers toward both the rostral CNS and the RVLM were previously reported in Ndn-KO (Lee et al., 2005; Pagliardini et al., 2005). In addition, differences in the morphology of 5-HT vesicles from medullary fibers in sections performed at the level of the pre-Bötzinger complex were observed (Pagliardini et al., 2005; Zanella et al., 2008b). 4) Substance P, TRH and 5-HT are excitatory neuromodulators acting in part on the preBötzinger Complex to increase the frequency and stabilize inspiratory burst generation (Teran et al., 2015); both Substance P and TRH increase the frequency of inspiratory bursts and decrease the incidence of irregularly rhythm in brainstem-spinal cord preparations from Ndn-KO mouse pups (Pagliardini et al., 2005; Pagliardini et al., 2008). However, most substance P and TRH inputs to the preBötzinger complex come from the medullary 5-HT neurons (Hodges et al., 2008b; Holtman et al., 1994; Kachidian et al., 1991; Ptak et al., 2009).

While we agree with the reviewers that the 5-HT system is not the only one affected in Ndn-KO mice, the specific point raised here actually further emphasizes the critical role of 5-HT neuron alterations in driving respiratory rhythm perturbation in Ndn-KO mice. Furthermore our results also show that increase of extracellular 5-HT level alone is sufficient to restore eupneic breathing.

Consequently, in order to clarify this point, in the Introduction of our article, we included:

“Furthermore, it was previously shown that, in perinatal Ndn-KO mice in vitro, an irregular inspiratory rhythm and apneas resulted from an alteration in the regulation of the Inspiratory Rhythm Generator (IRG) (Ren et al., 2003; Zanella et al., 2008). 5-HT application, as well as other neuromodulators such as substance P and thyrotropin-releasing hormone (TRH), stabilized the inspiratory rhythm and reduced the incidence of apneas (Pagliardini et al., 2005; Zanella et al., 2008). Interestingly, 5-HT neurons co-release substance P and TRH, and represent the main source of these peptides to the IRG (Hodges et al., 2008; Holtman et al., 1994; Kachidian et al., 1991; Ptak et al., 2009). An anatomical study of the developing Ndn-KO medulla reported alterations in the majority of axonal tracts within the medulla and in particular an abnormal morphology and orientation of 5-HT and Substance P axonal fibers (Pagliardini et al., 2005; Pagliardini et al., 2008).”

3) The study would have much higher impact if the truly novel findings were disentangled from replication of previous results, although this replication is also important to report. The authors fail to place their experiments in the context of previously published work, in some cases contradicting their own previous studies (e.g. number of 5_HT neurons, (Zanella, 2008)) but confirming that of others (Pagliardini, 2005) with no reference to either study. This will put undue strain on readers to identify which are new observations and which are repeats of previously published observations.

Because we submitted the paper as a Short Report which has space limitation, we have not placed our experiments and results in the precise context of previously published work, including authors’ publications. Now, we introduced in our revised manuscript (see point 2) pioneer studies conducted in the Necdin mutant mouse that highlighted respiratory deficits and dysfunction of neuromodulation such as the 5-HT system (mainly by R. Wevrick’s and Muscatelli’s teams); however we knew neither the cause nor the consequences of this dysfunction. In our current study, our aim was to investigate the development and functional maturation of the 5-HT system since we first showed that Necdin is expressed in post-mitotic 5-HT precursors (in this paper) and throughout embryonic and postnatal development of 5-HT neurons. To conduct a rationale and complete investigation, we had to replicate experiments in the mouse model in order to make a complete story on deciphering causal link between 5-HT alteration and respiratory deficits in the same mouse model. Indeed some 5-HT linked alterations were previously described in different Ndn-KO models (different KO constructs, different genetic background, homozygous versus heterozygous mutants) and by different teams.

In this article we studied homozygous mutants (-/-) named here Ndn-KO. Necdin is an imprinted gene paternally expressed only, the maternal allele being silent. Previously we have shown that Necdin mice deleted for the paternal allele only (Ndn +m/-p) presented a milder phenotype than the Ndn-/- (deleted for both paternal and maternal alleles) due to a stochastic and faint expression of the maternal allele (Rieusset et al., 2013). This unexpected expression was the cause of a high variability in the severity of the phenotype that might result in not significant differences compared with the wild-type mice. To avoid this variability, we chose here to study the -/- Ndn mice.

To clarify this point we propose to add in the text, introduction of the Results/Discussion part:

“Necdin is an imprinted gene, paternally expressed only. In order to avoid a variability in our results due to a stochastic and faint expression of the maternal allele (Rieusset et al., 2013), here, we chose to work on Ndn-/- mice (named here Ndn-KO) instead of Ndn +m/-p mice as it has been done previously (Zanella et al., 2008).”

Importantly, the experiments we made in our first publication (Zanella et al., 2008) were performed on Ndn+m/-p mice named in the article Ndn-KO mice. Then in the review written by Zanella (Zanella et al., 2008b) based on the study published in Journal of Neurosciences, the Ndn-KO were cited as Ndn -/- instead of Ndn+m/-p; this was an error in the review.

If we consider the experiments performed on 5-HT neurons/system “which are repeats of previously published observations”, they are restricted to:

1) In the first paragraph entitled “Lack of Necdin affects the development and function of 5HT neurons”:

– Necdin expression in 5-HT neurons by immunohistochemistry (using a specific antibody validated on Ndn-/- brain sections) was already reported in Zanella et al. (Zanella et al., 2008). However here we precisely defined the extent of expression in the 5-HT neurons of the different raphe nuclei throughout embryonic and post-natal stages, showing for the first time that Necdin is expressed in 5HT post-mitotic precursors Figure 1—figure supplement 1A-I.

– Alteration of 5HT projections have been observed by Pagliardini et al. (2008). This study is now cited in the Introduction. Here we observed a similar alteration illustrated in Figure 1—figure supplement 1J. We modified the text as following:

“While 5-HT neuronal somas from rostral midbrain project theirs axons to the mesencephalon at E12.5, in Ndn-KO embryos, we observed a decrease in those ascending 5-HT projections (Figure 1—figure supplement 1J) as previously described (Pagliardini et al., 2008).”

– The counting of 5-HT neurons in Ndn-KO pups (Figure 1 B-C).

Concerning the number of 5-HT neurons, to explain why “in some cases contradicting their own previous studies (e.g. number of 5-HT neurons, (Zanella, 2008) but confirming that of others (Pagliardini, 2005) with no reference to either study”. As explained above, Ndn+m/-p presented a milder phenotype than the Ndn-/- (deleted for both paternal and maternal alleles) due to a stochastic and faint expression of the maternal allele (Rieusset et al., 2013). In particular in this previous article (Rieusset et al., 2013), we showed that the number of 5HT neurons in the Ndn+m/-p is not significantly different compared to wild-type, as we already observed in the publication of Zanella et al., 2008. However, in Ndn-/- mice we observed a marked reduced number of 5-HT neurons (Rieusset et al., 2013). Here, by counting the Pet1eYFP+ neurons in Ndn-/- we confirmed the results and variability described in Rieusset et al., 2013. We clarified this point in the text as following:

“At E16.5, we observed a lack of organization of the 5-HT cytoarchitecture in Ndn-KO with misplaced 5-HT neurons leading in neonates to a significant ~30% reduction in the total number of 5HT-expressing neurons as quantified in the B1-B2 caudal Raphe nuclei (Figure 1 B-C); we already observed such a significant reduction in Ndn-/- mutants but not in Ndn+m/-p mutants (Rieusset et al., 2013).”

We modified the conclusion of this paragraph as following:

“Overall, our results showthat Necdin is critical for the establishment and functional features of 5-HT neurons during development.”

2) In the second paragraph entitled “Lack of Necdin increases the expression and activity of serotonin transporter SERT”:

– Neurite abnormalities with the presence of abnormal 5-HT vesicles/varicosities were observed in 5HT fibers (Pagliardini, 2005 and Zanella, 2008). In fact, we couldn’t prove that those abnormal 5-HT vesicles are either varicosities defined as “swellings that release their neurotransmitters at neuroeffector junctions” or spheroids defined as “dystrophic degenerative structures“. That is the reason why we named them here enLarged Punctiform Axonal (LPA) 5-HT staining. Furthermore, for the first time, we quantified them in different brain regions and in different genetic mutant mice (Ndn KO, Sert KO, Ndn/Sert DKO). We modified the text as following:

“An alteration of 5-HT metabolism was already observed in Ndn-KO mice (Zanella et al., 2008), along with abnormal 5-HT fibers with “large varicosities “(Pagliardini et al., 2005; Zanella et al., 2008). Using immunohistochemistry (IHC), we quantified these enlarged punctiform axonal (LPA) 5-HT staining, showing a significant increase in different Ndn-KO brain regions compared with WT (Figure 2A-B).”

3) In the third paragraph entitled “Genetic ablation or pharmacological inhibition of SERT uptake restores normal breathing in Ndn-KO mice”.

– We reported in a previous publication that in wild type mice: “Fluoxetine Treatment Abolishes the in vitro Respiratory Response to Acidosis in Neonatal Mice” (Voituron et al., 2010). Here, since we investigated the effect of fluoxetine in Ndn-KO, the experiments in control animals was mandatory.

We modified the text as following:

“Looking at the chemosensitive response to central acidosis in en bloc brainstem-spinal cord, we found that fluoxetine treatment abolished the capacity of WT to respond to acidosis as we previously observed (Voituron et al., 2010) (Figure 4—figure supplement 2A-D).”

All the other experiments of this paper “are new published observations”.

4) The abnormality of serotonergic function in the respiratory system in the hindbrain of Ndn-null mice has been studied by several groups over the last decade, compromising the novelty of the study. For example, the authors neglect to reference key publications: the papers from the Yoshikawa group about the discovery of mouse necdin, their necdin knockout mouse and work on neuronal abnormalities consequent to lack of necdin; the papers from the Wevrick group on the discovery of human necdin, the disruption of serotonergic axonal projections in necdin null mice, their discovery that serotonergic neurons in the hindbrain of necdin-null mice have abnormal cytoarchitecture and abnormal varicosities; the migration defect in necdin-deficient neurons; and the author's own work showing 5-HT abnormalities by HPLC (Zanella Advances in Experimental Medicine and Biology 2008) and the effect of fluoxetine in WT mice (Voituron, 2010). The authors should re-write these parts of the manuscript to better summarize key previous work, and put their new findings in this context.

We agree with this comment. This point is somehow linked to point 2 and 3 and the modifications made to answer those both points should also be considered in point 4. Indeed, we tried to introduce all the key previous work in different parts of the text: Introduction, Results and Discussion. All the studies and publications cited by the reviewers have been now introduced. The publication (book review) of Zanella et al. in Advances in Experimental Medicine and Biology 2008 has been replaced by the original study: Zanella et al., 2008.

[Editors' note: what now follows is the author responses after they submitted for further consideration.]

[…] Reviewer #2:

The authors have responded to the original review primarily by adding a sentence at the end of each section describing how their work repeats and extends, to a greater or lesser extent, work previously done by others.

We actually modified considerably all parts of the manuscript. We introduced and discussed previous literature, in order to better contextualize our results. In the Introduction, we extended the description of breathing deficits in PWS patients, and the previous results obtained on Ndn-KO mice linked to the breathing deficits. We also extended the conclusion, starting by summarizing the previous results published on Necdin, 5-HT and breathing in order to discriminate our novel results from those previous results. We explained how and when Fluoxetine might be used in PWS patients to alleviate their breathing deficits. We also modified the Material and methods section providing more explanations on the use of Malgel2-/- mice (see next paragraph). In the present version, we have also included 25 new references (compared to the initial version). In the previous version, there were some novel results described in the figure legends but not in the main text. In the present version, we thus transferred this information from the figure legends to the result part.

Furthermore, they state that the work is all novel because they now use homozygous null mice rather than paternally inherited heterozygous mice.

We never made such a claim and we apologize for this misunderstanding: the novelty aspect of our study is discussed compared to the literature in the Results/Discussion and conclusion sections, and never refers to the use of heterozygous vs. homozygous mice. The use of homozygous mice allowed us to reduce the variability in the severity of their phenotype (apnea). Indeed, we previously observed that heterozygous mice show an important phenotypic variability, ranging from strong to light phenotypic alteration, due to a stochastic expression of the maternal allele (Rieusset et al., 2013). Here, the use of homozygous mice is justified scientifically and ethically, as it allowed us to get significant results using a smaller number of mice, since their phenotype (apnea in particular) was of greater homogeneity. We explained why we used homozygous rather than heterozygous mice in the “Material and Methods” part, which is the most appropriated section, avoiding any misunderstanding concerning the novelty of our results (such as considering that the novelty is the use of Ndn-/- mice).

It remains unclear which experiments provide truly novel data and which a confirmation of their work and that of others. Once the previously demonstrated findings are subtracted from the results, the novel results may become apparent.

In order to clarify this point, we made a table listing all figures (see table below) indicating which precisely are the repeated and novel experiments, the references of the repeated experiments and the justification to repeat them. Less than 10% of experiments have been replicated, consequently more than 90% are novel.

The manuscript can then be properly assessed for its suitability for publication, if the truly novel results are of sufficient interest. In summary, the authors need to rewrite the entire manuscript to describe what is already known about the serotonergic system in necdin +/- and necdin KO mice, then state their novel results.

Indeed, 90% of the results presented in this article are novel results that have never been published in neither heterozygous nor homozygous Necdin-KO mice. In the new version, novel results are clearly stated in the Abstract and in the conclusion (“Here, we showed…healthy mice.”). In the “Results and Discussion” part we also present our results with respect to the previous ones.

Overall, we hope that the readers will see the novelty of our results, listed below:

– The expression of Necdin in early post-mitotic precursors (expressing Pet1) followed by an expression in all 5-HT neurons;

– The misplacement and disorganization of 5-HT raphe nuclei in Ndn-KO mice. The decrease in the number of 5-HT neurons in the B1-B2 nuclei, a result not previously observed in our Ndn+m/-p mice but observed in Ndn-/- mice and we explained why (see table);

– An in vivo alteration in cell migration of 5-HT precursors;

– An alteration of firing properties of 5-HT neurons recorded on brain slices of mutant;

– The quantification of enlarged Punctiform Axonal (LPA) 5-HT stainings in different brain regions and in different genetic backgrounds (WT, Ndn-KO, Slc6a4 and Ndn/Slc6a4-DKO) showing the lack of Sert expression is sufficient to suppress the 5-HT LPA stainings;

– The overexpression of Sert resulting from a post-transcriptional or post-traductional regulation and resulting in an increase activity of SERT transport and uptake (indeed the experiment using ASP+ allows the visualization and quantification of ASP+ uptake through SERT (Lau et al., 2015));

– The genetic ablation of Slc6a4 (encoding SERT) in Ndn-KO (Ndn/Slc6a4-DKO) leading to a rescue of the phenotype (normalization of apnea and loss of accumulation of 5-HT in LPAs);

– The in vivo positive effect of early Fluoxetine treatment in Ndn-KO mice allowing apnea to be normalized;

– The in vivo adverse effect of early Fluoxetine treatment in WT mice, inducing apnea. We are not aware of any published or unpublished similar results and so considered that they are novel.

The manuscript also has many awkward phrases that make it difficult to read.

We are really sorry for the grammatical errors (this article was written by French scientists). The new manuscript has been read and corrected by Pr. Keith Dudley (a native English scientist) and Dr Simon McMullan (Australian Senior scientist).

In the Abstract alone, they need to edit, for example:

“Necdin-deficient mouse is the best relevant model” – awkwardly phrased.

Now replaced by: “The Necdin-deficient ouse is the only model…”.

“Increase of 5-HT reuptake activity of the Serotonin Transporter (SERT)” – I don't think that was demonstrated by the experiments shown as they measured SERT transport in Figure 2, not reuptake.

We agree that the reuptake is not appropriated. However uptake is the appropriate word since we measure in Figure 2E-H (and Figure 2—figure supplement 2) the SERT uptake activity using ASP+ as fluorescent substrate and we measured the kinetic parameters of Asp+ uptake in primary cultures of raphe neurons. Indeed those experiments measure SERT transport activity and the uptake activity (Lau et al., 2015).

“Or fluoxetine treatment, a SERT inhibitor reuptake” – what is a SERT inhibitor reuptake?

This was a big mistake that we didn’t see. Of course it has been corrected by: a 5-HT reuptake inhibitor.

“Is sufficient to cause respiratory deficit in Necdin-KO pups” – this is vague.

This has been replaced by: is sufficient to cause the apneas in Necdin-KO pups.

“SERT inhibition is a therapeutic perspective for PW patients” – what does therapeutic perspective mean, and why PW and not PWS is defined earlier in the same paragraph?

This has been replaced by:and that Fluoxetine opens a novel perspective to improve ventilator control in patients with PWS.

The rest of the manuscript has similar issues with awkwardly phrased and ungrammatical English that makes it difficult to interpret.

We have modified the Abstract, and the text has been proofread and corrected by Pr. Keith Dudley (a native English speaking scientist) and Dr. Simon McMullan (Australian Senior scientist), as mentioned above.