Neuron-glia interaction through Serotonin-BDNF-NGFR axis enables regenerative neurogenesis in Alzheimer's model of adult zebrafish brain
Authors: Sevgican Demir
Nambirajan Govindarajan
Weipang Chang
Mehmet Ilyas Cosacak
Kerstin Brandt
Stanislava Dimitrova Popova
Yixin Zhang
Prabesh Bhattarai
Konstantinos Ampatzis
Violeta Mashkaryan
Caghan Kizil
Journal: PLoS biology (2020)
Discipline: BIOLOGY & BIOCHEMISTRY
Keywords: Alzheimer's disease; neurogenesis; zebrafish; neural stem cells; interleukin-4
Affiliations: German Center for Neurodegenerative Diseases (DZNE) within Helmholtz Association, Dresden, Germany., CRTD-Center for Regenerative Therapies Technische Universität Dresden, Dresden, Germany., B CUBE, Center for Molecular Bioengineering, Technische Universität Dresden, Dresden, Germany., Karolinska Institutet, Neuroscience Department, Stockholm, Sweden.

Abstract:  It was recently suggested that supplying the brain with new neurons could counteract Alzheimer's disease (AD). This provocative idea requires further testing in experimental models in which the molecular basis of disease-induced neuronal regeneration could be investigated. We previously found that zebrafish stimulates neural stem cell (NSC) plasticity and neurogenesis in AD and could help to understand the mechanisms to be harnessed for developing new neurons in diseased mammalian brains. Here, by performing single-cell transcriptomics, we found that amyloid toxicity-induced interleukin-4 (IL4) promotes NSC proliferation and neurogenesis by suppressing the tryptophan metabolism and reducing the production of serotonin. NSC proliferation was suppressed by serotonin via down-regulation of brain-derived neurotrophic factor (BDNF)-expression in serotonin-responsive periventricular neurons. BDNF enhances NSC plasticity and neurogenesis via nerve growth factor receptor A (NGFRA)/ nuclear factor 'kappa-light-chain-enhancer' of activated B-cells (NFkB) signaling in zebrafish but not in rodents. Collectively, our results suggest a complex neuron-glia interaction that regulates regenerative neurogenesis after AD conditions in zebrafish. 

Retracted: No

PubPeer comments:
 This paper seems to contain some serious statistical flaws that invalidate several of their findings, also several major claims are insufficiently backed up by their data and their morpholino experiments are insufficiently controlled according to the standards in the field. The authors use multiple statistical comparisons of experimental conditions to derive their conclusions. Some examples are shown below. ![file] ![file] ![file] That is done in millions of papers, but needs proper statistical tests that are suited for multiple comparisons. The Material and Methods section of the paper states: All experiments were performed at least with **2 replicates**. For **comparison of 2 particular experimental groups, a two-tailed Student t test** was performed. Statistical analyses were performed using Microsoft Excel (Microsoft Corporation, Redmond, WA). p-values less than 0.05 were considered significant. Error bars in the figures indicate the standard deviation. Significance indicated by * (p 0.05), ** (p 0.01), and *** (p 0.001), n.s. (not significant, p 0.05). No sample set was excluded from the analyses unless the histological sections were damaged severely during the acquisition of the sections (constitutes less than 4% of all sections analysed). They use n=2 for statistical comparison (at least for one figure panel) and they use studentu00b4s t Test to do multiple comparisons? Students t-Test is designed for hypothesis testing using two groups. Performing multiple comparisons will increase its inherent uncertainty - known as alpha error - so that basic assumptions of the test are not met anymore. In other words: Multiple comparisons with students t-test are invalid. So are the claims that the authors of the paper derive from the data in panels 1F, 1G, 2H, 2O, 5B, S3E and S6H. According to the submitted raw data [Link] , some of the comparisons in **panel 2H, 2O, S3E, 5B and S6H are not significant with p 0.05 anymore, when analysed with a actual multiple comparison test**, such as one way ANOVA, follow by turkeyu00b4s post hoc test. **In Figure S6H, only 2 experimental animals are evaluated** per condition. The calculations for the t test were made using the count numbers of individual sections rather than their average within one experimental animal. Thus, major claims of the paper are not validly supported by the data, any more. But ignoring basic rules of statistics is not the only problem with this paper. Major claims of the paper are in my opinion not sufficiently supported by the data shown. For example the authors state: Here, by performing single-cell transcriptomics, we found that amyloid toxicity-induced interleukin-4 (IL4) promotes NSC proliferation and neurogenesis by suppressing the tryptophan metabolism and reducing the production of serotonin. Already this major claim is never fully proven, The authors do show that injection of Abeta42 peptide or Interleukin 4 (IL4) does reduce the intracellular staining of serotonin in the adult zebrafish telencephalon, but **the requirement of this serotonin reduction for the increase in neural stem cell proliferation after these injections is not proven**. In particular, while the authors combine injections of Abeta42 peptide or IL4 with injections of serotonin, which would constitute a solid rescue experiment, they never show a rescue, that is significant reduction of induced stem cell proliferation after Abeta42 peptide or IL4 injections by concomitant injections of serotonin. Next the authors claim that  NSC proliferation was suppressed by serotonin via down-regulation of brain-derived neurotrophic factor (BDNF)-expression in serotonin-responsive periventricular neurons. This is never convincingly shown. Instead, the authors suggest that BDNF is reduced upon serotonin injection and they show that injection of BDNF has the opposite effect of serotonin injection on neural stem cell proliferation. **This is correlation but not causal proof of the authoru00b4s model**, because there is no data to suggest that the reduction of BDNF after serotonin injection is functionally required for the reduction of neural stem cell proliferation after serotonin injection. Finally, the authors use morpholinos to knock down *ngfra* - a putative BDNF receptor. **These experiments lack elementary controls**, which render them inconclusive. First of all, no data is shown or cited that evaluates off-target effects of this morpholino or compares it to a *ngfra* mutant. Second, no data are shown or cited that support the biological activity of this morpholino. It is not known whether it reduces ngfra expression or direct biological function. The authors do show that a reduction of NfkB:egfp-positive proliferating stem cells, after ngfra morpholino transfection compared to control morpholino injection, but it is currently unclear whether this reporter reflects ngfra activity in the adult zebrafish brain. The sequence of the control morpholino is not given, precluding assessment of its suitability as mismatch control. This is against accepted standards in the field [Link] [Link] . Also the proposed rescue of BDNF-induced proliferation after transfection with the *ngfra* morpholino is not significant, when analyzed with appropriate statistical tests, such as one way ANOVA, followed by Turkeyu00b4s post hoc test, invalidating also the major claim of *ngfra*-dependance of the effect of BNDF on neural stem cell proliferation. It is my hope that the authors will take the opportunity to address the current concerns about the work. , 
 In line with Diodontus Minutus s comment, I cannot find details on the type of Morpholino used in this study. Did the authors use a standard Morpholino, a standard Morpholino in combination with endo-porter or a Vivo-Morpholino? The latter consists of a delivery moiety ( Vivo moiety ) that confers cell permeability. It is hard to believe (unless evidence is provided through control experiments) that a standard Morpholino (without endo-porter) injected into the ventricle is efficiently taken up by the target cells. For reasons of reproducibility it is important that the authors provide all essential details of methods used. ,