Author response:
The following is the authors’ response to the original reviews.
Public Reviews:
Reviewer #1 (Public review):
Weaknesses:
However, given that S1P is upstream NF-κB signaling, it is unclear if it offers conceptual innovations as compared to previous studies from the same team (Palazzo et al. 2020; 2022, 2023)
We find distinct differences between the impacts of S1P- and NFkB-signaling on glial activation, neuronal differentiation of the progeny of MGPCs and neuronal survival in damaged retinas. In the current study we demonstrate that 2 consecutive daily intravitreal injections of S1P selectively activated mTor (pS6) and Jak/Stat3 (pStat3), but not MAPK (pERK1/2) signaling in Müller glia. Further, inhibition of S1P synthesis (SPHK1 inhibitor) decreased ATF3, mTor (pS6) and pSmad1/5/9 levels in activated Müller glia in damaged retinas. Inhibition of NFkB-signaling in damaged chick retinas did not impact the above-mentioned cell signaling pathways (Palazzo et al., 2020). Thus, S1P-signaling impacts cell signaling pathways in MG that are distinct from NFκB, but we cannot exclude the possibility of cross-talk between NFkB and these pathways. Further, inhibition of NFκB-signaling potently decreases numbers of dying cells and increases numbers of surviving ganglion cells (Palazzo et al 2020). Consistent with these findings, a TNF orthologue, which presumably activates NFκB-signaling, exacerbates cell death in damage retinas (Palazzo et al., 2020). By contrast, 5 different drugs targeting S1P-signaling had no effect on numbers of dying cells and only one S1PR1 inhibitor modestly decreased numbers of dying cells (current study). Although two different inhibitors of NFkB-signaling suppressed the proliferation of microglia in damaged retinas (Palazzo et al., 2020), all of the S1P-targeting drugs had no effect upon the proliferation of microglia (current study). In addition, inhibition of NFκB does not influence the neurogenic potential of MGPCs in damaged chick retinas (Palazzo et al., 2020), whereas inhibition of S1P receptors (S1PR1 and S1PR3) and inhibition of S1P synthesis (SPHK1) significantly increased the differentiation of amacrine-like neurons in damaged retinas (current study). Collectively, in comparison to the effects of pro-inflammatory cytokines and NFκB-signaling, our current findings indicate that S1P-signaling through S1PR1 and S1PR3 in Müller glia has distinct effects upon cell signaling pathways, neuronal regeneration and cell survival in damaged retinas. We will revise text in the Discussion (pages 33-34) to better highlight these important distinctions between NFκB- and S1P-signaling.
Reviewer #2 (Public review):
Weaknesses:
The methodology is not very clean. A number of drugs (inhibitors/ antagonists/agonists signal modulators) are used to modulate S1P expression or signaling in the retina without evidence that these drugs are reaching the target cells. No alternative evaluation if the drugs, in fact, are effective. The drug solubility in the vehicle and in the vitreous is not provided, and how did they decide on using a single dose of each drug to have the optimal expected effect on the S1P pathway?
Müller glia are the predominant retinal cell type that expresses S1P receptors. Consistent with these patterns of expression, we report Müller glia-specific effects of different agonists and antagonists that increase or decrease S1P-signaling. Since we compare cell-level changes within contralateral eyes wherein one retina is exposed to vehicle and the other is exposed to vehicle plus drug, it seems highly probable that the drugs are eliciting effects upon the Müller glia. It is possible, but very unlikely, that the responses we observed could have resulted from drugs acting on extra-retinal tissues, which might secondarily release factors that elicit cellular responses in Müller glia. However, this seems unlikely given the distinct patterns of expression for different S1P receptors in Müller glia, and the outcomes of inhibiting Sphk1 or S1P lyase on retinal levels of S1P.
For example, we provide evidence that S1PR1 and S1PR3 expression is predominant in Müller glia in the chick retina using single cell-RNA sequencing and fluorescence in situ hybridization (FISH). Thus, we expect that S1PR1/3-targeting small molecule inhibitors to directly act on Müller glia, which is consistent with our read-outs of cell signaling with injections of S1P in undamaged retinas. We show that SPHK1 and SGPL1, which encode the enzymes that synthesize or degrade S1P, are expressed by different retinal cell types, including the Müller glia. The efficacy of the drugs that target SPHK1 and SGPL1 was assessed by measuring levels of S1P in the retina. By using liquid chromatography and tandem mass spectroscopy (LC-MS/MS), we provide data that inhibition of S1P synthesis (inhibition of SPHK1) significantly decreased levels of S1P in normal retinas, whereas inhibition of S1P degradation (inhibition of SGPL1) increased levels of S1P in damaged retinas (Fig. 5). These data suggest that the SPHK1 inhibitor and the SGPL1 inhibitor specifically act at the intended target to influence retinal levels of S1P. Further, inhibition of SPHK1 (to decrease levels S1P) results in decreased levels of ATF3, pS6 (mTor) and pSMAD1/5/9 in Müller glia, consistent with the notion that reduced levels of S1P in the retina impacts signaling at Müller glia. Finally, we find similar cellular responses to chemically different agonists or antagonists, and we find opposite cellular responses to agonists and antagonists, which are expected to be complimentary if the drugs are specifically acting at the intended targets in the retina. We will revise the Discussion to better address caveats and concerns regarding the actions and specificity of different drugs within the retina following intravitreal delivery.
We will provide the drug solubility specifications and estimates of the initial maximum dose per eye for each drug. For chick eyes between P7 and P14, these estimates will assume a volume of about 100 ul of liquid vitreous, 800 ul gel vitreous and an average eye weight of 0.9 grams. We will revise Table 1 (pharmacological compounds) with ranges of reported in vivo ED50’s (mg/kg) for drugs and we will list the calculated initial maximum dose (mg/kg equivalent) per eye. Doses were chosen based on estimates of the initial maximum ocular dose that were within the range of reported ED50’s. However, as is the case for any in vivo model system, it is difficult to predict rates of drug diffusion out of the vitreous, how quickly the drugs are cleared from the entire eye, how much of the compound enters the retina, and how quickly the drug is cleared from the retina. Accordingly, we assessed drug specificity and sites of activation by relying upon readouts of cell signaling pathways that are parsed with patterns of expression of different S1P receptors and measurements of retinal levels of S1P following exposure to drugs targeted enzymes that synthesize or degrade S1P, as described above.
Reviewer #1 (Recommendations for the authors):
I am wondering if Muller glia can be considered as fully differentiated at early postnatal stages as those used in this study. Is this mechanism operative in adult retinas? Could the authors perform studies in older animals, just to have the proof of principle that the proposed mechanism is retained.
Chickens are considered to be adult at about 4 months of age, when the females start laying eggs. Unfortunately, housing, maintenance, handling and experimentation on large adult chickens has proven to be challenging. Nevertheless, there is evidence that Muller glia reprogramming remains robust in mature chick retinas from the P1 through P30, but the zones of proliferation shift away from central retina and become increasingly confined to the retinal periphery (Fischer, 2005). MG “maturation” appears to occur in a central-to-peripheral gradient, much like the process of embryonic retinal differentiation, but a zone of regeneration-competent MG remains in the periphery during adolescent development (Fischer, 2005).
We have defined central vs peripheral retina in the Methods.
To partially address this question, we have generated a new supplemental Figure 6 showing (i) SPHK1 fluorescent in-situ labeling of central and peripheral regions at P10, and (ii) analysis of EdU+Sox2+ MGPCs in central versus regions treated with NMDA +/-S1PR1 inhibitor or NMDA+/- SPHK1 inhibitor. We find that patterns of S1PR1 transcription in the central region are similar to the peripheral region (not shown), and S1PR1 inhibition modestly increased numbers of MGPCs in central regions. Unlike the peripheral regions of retina, SPHK1 FISH signal in the central region remains low at 48 hours post-injury (supplemental Fig. 6). Additionally, we found that the SPHK1 inhibitor had no effect on numbers of proliferating MGPCs in the central regions of retina, whereas SPHK1 inhibitors stimulated proliferation of MGPCs in the periphery (Fig. 4). It is likely that mature MG in central retinal regions are not responsive to SPHK1 inhibition due to low levels of expression.
We have previously shown that Notch-related genes show unique patterns of expression in the central and peripheral retinas, and expression levels significantly change at P0, P7, and P21 (Ghai et al, 2010). We found that Notch inhibition reduced cell death and numbers of MGPCs in central regions but not peripheral regions. Recent sc-RNA sequencing analysis of murine macula and peripheral retinal regions has revealed interesting differences in NFKBIA/Z and NFIA expression, possibly indicating a difference in the early inflammatory transcriptional response to retinal damage (Zhang et al, 2024 biorxiv). We believe that spatial sequencing of peripheral “immature” and central “mature” chick Muller glia will be a useful tool in the future to reveal key differences in signaling pathway-related gene expression which confer a competence for regeneration in the periphery.
We have added text to the Results (pages 20-21) and Discussion (page 32) to address the S1P-signaling in central (mature MG) vs peripheral (immature MG) regions of the retina.
Minor points.
The abstract is difficult to follow and consists of a list of what activates or represses the formation of MGPC. Please rewrite the abstract to integrate information and provide a clearer message. Also, please include the species of study in the abstract and mention it again at the beginning of the results, at least.
We have rewritten the abstract to simplify and clarify our main points (p 2).
Lines 65-69. The sentence is unclear, perhaps there are words either missing or in excess and there is a need to check the spelling.
We have simplified this sentence to improve clarity and referenced our recently published review to support.
Lines 112-113. Please explain why " retinas were treated with saline, NMDA, or 2 or 3 doses insulin+FGF2 and the combination of NMDA and insulin+FGF2". There is a reference but readers will appreciate understanding right away why.
We have added a sentence to clarify the purpose of comparing gene expression patterns in MG and MGPCs in NMDA-damaged retinas versus retinas treated with insulin+FGF2.
Lines 223-257. This list of experiments is difficult to follow and perhaps should be summarized better. Somehow lines 257-261 say it all.
We have revised this section to clarify differences in outcomes between S1PR1/3 activators and inhibitors. We also stated the enzymatic functions of SPHK1 and SGPL1 to improve clarity.
Lines 392-441. Comparative expression analysis should be summarized as the message is somehow simple but the description is rather lengthy.
We have revised our comparative expression analyses to be more concise.
Reviewer #2 (Recommendations for the authors):
(1) Only a single dose of the drugs (inhibitor/ antagonists/agonists signal modulators) is used for each drug, as shown in Table 1. How do they know this is an effective dose?
We estimated the appropriate dose based on the initial maximum dose, which we based on the reported ED50 values for each drug. We have revised Table 1 to include this information.
(2) Most of the drugs appeared to be hydrophobic, but except for sphingosine and S1P, all are described to be injected with sterile saline. They must provide solubility characteristics of these drugs in solvents. For example, FTY720 is not water-soluble, which raises the question of all of their drugs' solubility, bioavailability to the cells of interest, and their effectivity in signal transduction in the retinal cells.
Some S1P-targeting compounds were delivered in 20% DMSO in saline to support the solubility of the different lipophillic small molecule agonists/antagonists. We have added information to the Methods to describe the use of DMSO to solubilize these drugs (p 6) in Table 1 and p 5. We have also revised Table 1 with ranges of reported ED50’s (mg/kg) for all drugs and listed the calculated initial maximum dose (mg/kg) per eye.
(3) Drugs were delivered to the vitreous chamber, but there was no information on how they would cross the inner limiting membrane to affect or modulate S1P metabolism in retinal MG or to bind the S1P receptors on MG or other retinal cell types.
All selected compounds are small-molecule drugs, many of which are structural analogues of sphingosine or S1P. These drugs would be classified as BDDCS Class II drugs, meaning they have low solubility but high cell permeability. Thus, it is highly probable that they diffuse across the ILM to act on S1P receptors on MG, but it is also likely that their bioavailability is more limited, requiring a higher dose, repeated doses, and the use of solubilizing agents. We have clarified our use of DMSO to solubilize these drugs (p 6) according to vendor recommendations (p 5). This information has been added to the Methods.
(4) Gene expression is a very dynamic process; without providing more evidence that the expression changes are the direct effect of the drug treatment, the conclusions made based on the gene expression profiles are not strong. Additional points:
We do not make assertions that changes in scRNA-seq expression profiles are the direct result of S1P-targetting drugs. We report significant changes in cellular expression profiles following NMDA-induced retinal damage or ablation of microglia. We feel that new experiments to assess the gene expression profiles of retinal cells that are directly downstream of the different S1P-targetting drugs is better suited for future studies.
(5) Please add in the introduction that there is only one sphingosine kinase in chicken, as no SPHK2 is known to be present.
We have added additional information regarding the expression of SPHK1 and SPHK2 genes in the chick genome (p 4).
(6) Fig 1d and in many other UMAP clusters, the low expressing genes are barely visible (Ex. 1d, S1PR2, and S1PR3); please extract them in separate UMAP clusters and provide them in supplements.
We have revised supplemental Figure 1 to include separate panels for each of the S1P-related gene.
(7) The Figure References for SPHK1 (Fig. 2e), SGPL1 (Fig. 2e), ASAH1 (Fig. 2f), CERS6 (Fig. 2f), and CERS5 (Fig. 2f) in the line # 124- 132 should belong to Figure 1, not Figure 2.
We have corrected these figure references (p 14).
(8) The description of the expression of zebrafish genes does not match the figures. For example, 'Similarly, sphk1 was detected in very few cells in the retina (Fig. 10j). By comparison, sphk2 was detected in a few bipolar cells and rod photoreceptors (Fig. 10j). Similar to patterns of expression seen in chick and human retinas, sgpl1 was detected in microglia and a few cells scattered among the different clusters of inner retinal neurons and rod photoreceptors (Fig. 10j)', the expression of these genes are not in very few or few scattered cells rather in many cells.
We have revised these statements to improve clarity and more accurately describe the data in Figure 10 (p 28).
