Gintonin Binds to Reduced LPA4 Receptor Subtype in Human Cortical Neurons in Alzheimer’s Disease Brains
, Inyeong Park 2, Sung-Hee Hwang 4, Yeshin Kim 5, Jae-Won Jang 5, Hyung-Seok Kim 6, Seong-Min Choi 7, Sang Jin Kim 8,9, Hwa Jin Cho 10, Ik-Hyun Cho 11, Jong-Hoon Kim 12, Do-Geun Kim 2,* and Seung-Yeol Nah 3,*
Round 1
Reviewer 1 Report
Comments and Suggestions for AuthorsThe study by Kim et al reports that in the human brain, gintonin (GT) binds preferentially to cortical neurons expressing LPAR4 receptor subtype and that this receptor is reduced in Alzheimer´s disease patients. Although the study could be relevant to the field, additional experiments are necessary to strengthen the main conclusions of the manuscript, as indicated below.
Figure 1. The cellular expression pattern of NeuN and GT immunostainings look very similar, which is surprising as NeuN is a nuclear antigen. Please, explain this. In addition, the Hoechst or DAPI fluorescent staining should be presented. Furthermore, the Methods should contain a brief description of how the colocalized area is calculated.
The apparent reduction of NeuN-expressing neurons and increase in activated astrocytes and microglia in AD brains should be supported with quantitative data.
Figure 2. Please, note that the % of colocalized areas of GT and LPAR5 and GT and LPAR6 do not fit well with the images presented. Are the images representative?
Discussed. The new data should be appropriately interpreted and discussed.
Author Response
Reviewe#1
The study by Kim et al reports that in the human brain, gintonin (GT) binds preferentially to cortical neurons expressing LPAR4 receptor subtype and that this receptor is reduced in Alzheimer´s disease patients. Although the study could be relevant to the field, additional experiments are necessary to strengthen the main conclusions of the manuscript, as indicated below.
Comment #1
Figure 1. The cellular expression pattern of NeuN and GT immunostainings look very similar, which is surprising as NeuN is a nuclear antigen. Please, explain this. In addition, the Hoechst or DAPI fluorescent staining should be presented. Furthermore, the Methods should contain a brief description of how the colocalized area is calculated.
The apparent reduction of NeuN-expressing neurons and increase in activated astrocytes and microglia in AD brains should be supported with quantitative data.
Author’s response:
NeuN is primarily expressed in the nuclei of neurons but is also known to exhibit some expression in the cytoplasm (Nassauw, Wu et al. 2005, Dredge and Jensen 2011, Lin, Wang et al. 2016) as shown in below example. When we compared with DAPI staining, NeuN signals extend beyond the DAPI defined nuclear region. This indicates that if gintonin shows colocalization with NeuN, it implies that GT might also reside within the cytoplasmic environment. In addition, Methods for calculation of colocalization was described in line 137-144.
The arrows indicate the regions corresponding to DAPI and NeuN signals, revealing that the NeuN signal extends over a broader area compared to the DAPI signal. This observation indirectly suggests that the NeuN protein is localized not only in the nucleus but also within the cytoplasm of the cells.
We also added additional data in Figure 1(C, F and I) with apparent reduction of NeuN-expressing neurons and increase in activated astrocytes and microglia in AD brains in line 172-183.
Comment #2
Figure 2. Please, note that the % of colocalized areas of GT and LPAR5 and GT and LPAR6 do not fit well with the images presented. Are the images representative?
Author’s response:
According to Reviewer#1 comments, we added each % co-localization of gintonin with LPA4, LPA5 and LPA6, respectively, line 206-209.
Comment #3
Discussed. The new data should be appropriately interpreted and discussed.
Author’s response:
We added new data in line 172-183 and line 206-209 and discussed them in line 258-260 and line 277-279.
Reference
Dredge, B. K. and K. B. Jensen (2011). "NeuN/Rbfox3 Nuclear and Cytoplasmic Isoforms Differentially Regulate Alternative Splicing and Nonsense-Mediated Decay of Rbfox2." PLOS ONE 6(6): e21585.
Lin, Y.-S., H.-Y. Wang, D.-F. Huang, P.-F. Hsieh, M.-Y. Lin, C.-H. Chou, I. J. Wu, G.-J. Huang, S. S.-F. Gau and H.-S. Huang (2016). "Neuronal Splicing Regulator RBFOX3 (NeuN) Regulates Adult Hippocampal Neurogenesis and Synaptogenesis." PLOS ONE 11(10): e0164164.
Nassauw, L. V., M. Wu, F. D. Jonge, D. Adriaensen and J.-P. Timmermans (2005). "Cytoplasmic, but not nuclear, expression of the neuronal nuclei (NeuN) antibody is an exclusive feature of Dogiel type II neurons in the guinea-pig gastrointestinal tract." Histochemistry and Cell Biology 124(5): 369-377.
Author Response File: 
Author Response.pdf
Reviewer 2 Report
Comments and Suggestions for AuthorsThe authors used brain tissue taken from a brain bank of both non Alzheimer's and Alzheimer's affected samples.
They constructed fluorescent gintonin (an active component of ginseng) and found in double labelling experiments that it binds to neurons, not glial cells in the human cortex within these brain samples. They further found which of the LPA receptor subtypes it binds to: subtype 4, and they show that this receptor's expression goes down in Alzheimer's disease. The fluorescence experiments were complemented with Western blot experiments. This is a well designed study and the findings are very valuable in my opinion, as the study gives a clue to a possible future drug target. The manuscript is well written, the figures are comprehensible, so I think it is almost ready for publishing. Before publishing, however, I suggest changing the second sentence of the abstract: this sentence is unclear. Please state whether ginseng has been scientifically proven to keep brain healthy or whether this idea is part of traditional medicine.
Comments on the Quality of English LanguageThe English Language is not wrong but I would look closely at the English once more - it can be noticed that this paper was not written by native speakers and there are some sentences that are unclear.
Author Response
Reviewer #2
The authors used brain tissue taken from a brain bank of both non-Alzheimer's and Alzheimer's affected samples.
Comment #1
They constructed fluorescent gintonin (an active component of ginseng) and found in double labelling experiments that it binds to neurons, not glial cells in the human cortex within these brain samples. They further found which of the LPA receptor subtypes it binds to: subtype 4, and they show that this receptor's expression goes down in Alzheimer's disease. The fluorescence experiments were complemented with Western blot experiments. This is a well designed study and the findings are very valuable in my opinion, as the study gives a clue to a possible future drug target. The manuscript is well written, the figures are comprehensible, so I think it is almost ready for publishing. Before publishing, however, I suggest changing the second sentence of the abstract: this sentence is unclear. Please state whether ginseng has been scientifically proven to keep brain healthy or whether this idea is part of traditional medicine.
Author’s response:
We edited the sentence again according to Reviewer#2 comment.
Comment #2
The English Language is not wrong but I would look closely at the English once more - it can be noticed that this paper was not written by native speakers and there are some sentences that are unclear.
Author’s response:
We edited the revised article by native English editor.
Author Response File: Author Response.pdf
Round 2
Reviewer 1 Report
Comments and Suggestions for AuthorsThe authors indicate that NeuN exhibits some expression in the cytoplasm. However, the expression patterns of NeuN and GT immunostainings look very similar. Thus, single anti-NeuN immunostaining should be performed to show convincingly that the pattern presented in the figure is specific.
Author Response
Comment #1
The authors indicate that NeuN exhibits some expression in the cytoplasm. However, the
expression patterns of NeuN and GT immunostainings look very similar. Thus, single anti
NeuN immunostaining should be performed to show convincingly that the pattern presented in
the figure is specific.
Author’s response:
We conducted single anti-NeuN immunostaining to validate the specificity of the observed
pattern further. The experimental results are attached herewith for review and consideration.
Author Response File: Author Response.pdf
Round 3
Reviewer 1 Report
Comments and Suggestions for AuthorsThe manuscript has been improved after revision.
