Inhibition of Sphingosine Kinase 1 Reduces Sphingosine-1-Phosphate and Exacerbates Amyloid-Beta-Induced Neuronal Cell Death in Mixed-Glial-Cell Culture
Round 1
Reviewer 1 Report
Comments and Suggestions for AuthorsThe research article investigated the potential role of impaired sphingosine-1-phosphate (S1P) metabolism on the aetiology of Alzheimer's disease. The intracellular levels of S1P in microglia and astrocyte cells were decreased by the researchers using the SK1 pharmacological inhibitor PF-543. After that, the authors investigated nitric oxide levels, ROS production, glutamate uptake, amyloid-beta (Aß) uptake, etc., to explore the molecular mechanism of S1P-induced AD. The authors have discovered that abnormal S1P metabolism may contribute to the aetiology of AD by preventing glial functions, including Aß clearance, which exacerbates neuroinflammation and damages neurons.
The experiments are performed adequately, and the paper is well-written. However, the following points need to be addressed by the authors in the manuscript:
1. A graphical abstract or summary figure could be included by the authors to enhance the clarity of research findings and conclusions.
2. Why did the authors prefer dot blot over Western blot to check the expression levels in Fig. 1?
3. An explanation/hypothesis is required for the no change in S1P level after PF-543 treatment in Fig. 1 results.
4. Fig. 3a, b, and c (and subsequent figures) show an extra DMSO control group. The rationale behind taking these extra groups must be mentioned in the methods.
5. Authors have performed ROS generation experiments in primary microglia but not in other experiments like Nitrite production, SK1 level, etc. Either explain the omission of those experiments or add this result from primary microglia in supplementary results.
6. An explanation for a reduced level of glutamate uptake by PF-543 (as well as +LPS) must be provided in the discussion, as this result is totally unexpected compared to the other results of PF-543 treatment.
Author Response
Please see the attachment.
Author Response File: 
Author Response.pdf
Reviewer 2 Report
Comments and Suggestions for AuthorsDear Authors
This is a vey well organizaed manuscript but are necessary to solve these minnor suggestions before the formal acceptation.The discussion is well elaborated. The conclusion should replace in line 602 the word ..... are accompanied in AD pathology by A beta neurotoxicity since your findings are associated to Abeta neurotoxicity (neuron and glia cocultures: astrocytes and microglia). .
Minnor comments
Abstract
Line 1-11. The begining of abstract is obvious until ¨Abstract: Alzheimer’s disease (AD) is a major neurodegenerative disease characterized by two pathological 9features, senile plaques and neurofibrillary tangles. Although the onset mechanism of AD remains unclear, 10cumulative evidence suggest that glial cells such as astrocytes and microglia can contribute to the pathogenesis of AD.
I would suggest to remove this part of reduce this obvious content
These authors evaluated Sphingolisps in A-beta treated neurons following the signaling scheme according to postmorten published studies in AD patients.
The authors indicate that to elucidate the role of S1P in AD pathology, we investigated the effect of atered S1P metabolism induced by SK1 inhibition on glial cell functions such as nitric oxide (NO) and ROS production. However this is not totalluy true. It is more appropiate to talk about Amyloid beta neurotoxicity that Alzheimer disease. In fact, they don`t use a transgenic model of AD for the study of this aim. Please, it is more appropiate the term neurotoxicity than Alzheimer disease model.
Methods
The line preparation of rat primary microglia was carried out as previously described [16] PLease,l describe this method in detail. Thanks¡
They also prepared neuron/glia mixed culture was prepared as described previously from hippocampus of 20-day-old Wistar rat embryos by the similar manner as described above for the astrocytes culture [17]
However, the most affected area in AD is the prefrontal cortex
Line 163. Please, explain why these Cells were treated with LPS (10 ng/mL) with PF-543 (10 or 20 μM) for 24 h.
Line 171. In addition, explain why filuted protein samples were subjected to dot blotting. 2 μL sample (1 μg protein)was blotted on nitrocellulose membrane and air-dried for 1 h. In fact, this concentration seem to be very low for soluble markers for inmunoblot. Shall you explain the reason?
Please, indicate the nm selected for detecting 2.7. Cell Viability Assay
Intracellular ROS generation was measured using the cell permeable fluorescent dye 196
H2DCFDA, as described previously [17]. Please, describe this method in detail.
What does mean ¨Cultured astrocytes were replated on 6 cm dish (Thermo Fisher Scientific Inc.) and stabilized for 24 h?
Line 282. Please, give more details about the preparation of this part. ¨2.12. Preparation of ABeta 1-42 Solution. Human Abeta 1-42 was dissolved in DMSO to 10 mM as a stock solution and kept at -80°C before using. A-beta 1-42 stock solution was diluted with culture medium to 10 μM and pre-incubated for 7 days at 37°C to form aggregate, then used for experiments, usually at 100 2 nM Abeta 1-42 monomer concentration¨
Line 256. The Neuron-phagocytosed microglia were counted as cells co-expressing Iba-1 and beta-3-tubulin and Iba-1 positive cells containing β3-tubulin positive fragments, and calculated as a percentage of the total number of microglia. However, this stmimation stimated the total nuumber of microglia but no those microglial involved cells in phagocytosis.
Aβ uptake microglia were evaluated by double immunostaining for Aβ and Iba-1. Aβ uptake microglia were counted as cells co-expressing Aβ and Iba-1, and calculated as a percentage of the total number of microglia. This calculation is correct but phagocytosis of microglia should be evaluated by a marker that demonstrate the existence of this mechanism.
They used 30 μM PF, which decreased cell viability in BV-2 cells and 10 μM is sufficiently effective for NO production in microglia (Suppl. Fig. S1a). Please, addd these supplementary result to the mean manuscript since it is important to known the rational use of these concentraitons
Line 293. In addition, include experiements from supplementary data that showed that 10 μM PF of astrocytes treatment had no effect on NO production; However, 20 μsignificantly increased it (Suppl. Fig. S2a). Please, add these findings to the mean part of results in the manuscript.
The figure 1 A shows do blot for SP-1 almost without signal. It is difficult to understand these ratios when the staining is almost null, except for LPS treatment. Explain the do blot thecnic also in more detail.
Line 329. Your findings clearly demonstrated that co-treatment of PF-543 significantly increased NO production in LPS-treated . However, what does mechanisms can explain the absence of effects by PF-543 treatment on NO production in your model?
Line 345-. We also found opposite results in experiment using astrocytes since PF-543 treatment remarkably increased both NO production and iNOS protein expression in LPS-stimulated astrocytes (Figs. 3d and e).Shall you explain these discreprances depeding of cells are astrocytes or mixed cutlures? What does mechanisms can explain these differences in terms of NO production?
Why does you include DMSO treated group in experiment-3 for mixed cultures but no with astrocytes?
Line 362. They further ¨elucidated the effect of PF-543 on other inflammatory indicator, generation of intracellular ROS¨. Please, take into account that ROS production and inflammation are different aspects. ROS levels are not representative of inflamamtion (in general) althogh free radical production can be exacerbated by inflammatory mediators.ç
Line 406. They also evalauted whether decrease in S1P content by PF-543 affect glutamate uptake by astrocyte. For the enalysis of this aim, cultured astrocyte was stimulated with LPS in the presence or absence PF-543 for 24 h, and glutamate uptake was determined by measuring intracellular [3H]-conjugated glutamate levels. Although the addition of PF-543 or LPS significantly decreased astrocytic glutamate uptake, co-treatment of PF-543 with LPS further reduced it compared to each alone (Fig. 6). These results suggest that decreased cellular S1P induced by PF-543 in astrocytes may abbreviate synaptic glutamate clearance. Shall you explain a possible differential contribution of astrocyte glutamate transportes in glutamate reaptake in astrocytes?
Line 414. Primary astrocytes were treated with 10 ng/mL LPS in the presence or absence of 20 μM PF-543 for 24 h. Glutamate uptake was assessedusing [3H]-conjugated glutamate and liquid scintillation counter. Explain the exactly time of PF administration after LPS treatments in these experiments.
Line 420. The experiment in figure x indicate that decrease in cellular S1P content (by PF-543) impairs clearance of A by microglia, which may accelerate A accumulation. However, why you exactly selected 7 days after treament for evaluating this effect?
Line 451. The figure 8 showed interested data on the 3.5. Decrease in S1P content Exacerbated Neuronal Damage and Increased MicroglialPhagocytosis of Neurons in A-Treated Neuron/Glia Mixed Culture. However, the PF group alone was not included in this design. Shall you explain the reason?
Although results of neuronal loss seems to be clear, the staining for Beta 3 tubuline and merge (color combine) staining are saturated. Please, put better photos in this figure-5. In addition, the Hoersch staining can be used for the evaluation of apoptotic markers but there are also markers of neurodegeneration (Fluoro Jade staining, etc).
Line 471. Collectively, these experiments demonstrated that decrease in glial S1P aggravates Abeta-induced neuronal loss by enhanced microglial phagocytosis of cell debris. However, we have not evaluated if microglial-phagocytic markers are overexpressed in this mixed culture despite their NO overproduction. In addition, we don´t treat these mixture neuron/microglia cocultures with LPS in order to see if phagocytosis is impaired by PF trreatment? Shall you explain these commment?
Line 482. The figure 9 showed neuron/glia mixed culture treated with pre-aggregated Ab1eta (100 nM at monomer concentration) with or without 20 μM PF-543 for 48 h. However, the experiment did not show a PF treated group alone.
How exactly the neuron-microglia phagocytoses have been evaluated in both experiments? This is not clear for me since we don`t have a phagocytoses marker and Iba-1 staining is a general markers of microglial cells.
Line 489.- 3.6. Decrease in S1P content Reduced mRNA Expression of PPAR-γ and CD36, Molecules
Associated with Aβ Uptake in BV-2 Microglia
Why PPAR gamma has been evaluated here? Shall you explain it?
Figure-10. The effect of PF-543 on mRNA expression of PPAR-γ and CD36 in BV-2 microglia. Cells were treated with pre-aggregated A1 beta (1 μM at monomer concentration) with or without 20 μM PF-543 for 24 h. However, why some spingolip marker were not tested in your model? I understand the inclussion of CD36 or PPAR gamma evaluation but is there any connexion between CD36 signalling and Abeta neurotoxicity in showed data in figures 8 or 9. So, why these authors have not evalauted CD36 or phagocytic markers in these experiments (Figures 8 and 9)? Shall you explain it?
Line 601. The discussion is well elaborate but, in my modest opinion, the conclusion can not indicate that This study demonstrates that the decrease in intracellular S1P inhibited microglial 602 A beta uptake and increased neuronal damage, those are accompanied in AD pathology¨. I would suggest replace in AD pathology by A beta neurotoxicity since your findings have not been obtained in a rodent model of Alzheimer disease .
This is a vey well organizaed manuscript but are necessary to solve these minnor suggestions before the formal acceptation.
Thanks¡
Author Response
Please see the attachment.
Author Response File: Author Response.pdf
Round 2
Reviewer 1 Report
Comments and Suggestions for AuthorsThe revised manuscript contains all the answers to my queries. Therefore, the manuscript can be accepted.
