Sphingosine-1-phosphate signaling regulates the ability of Müller glia to become neurogenic, proliferating progenitor-like cells

Reviewer #1 (Public review):

Summary:

This study shows that the pro-inflammatory S1P signaling regulates the responses of muller glial cells to damage. The authors describe the expression of S1P signaling components. Using agonist and antagonist of the pathways they also investigate their effect on the de-differentiation and proliferation of Muller glial cells in damaged retina of postnatal chicks. They show that S1PR1 is highly expressed in resting MG and non-neurogenic MGPCs. This receptor suppresses the proliferation and neuronal activity promotes MGPC cell cycle re-entry and enhanced the number of regenerated amacrine-like cells after retinal damage. The formation of MGPCs in damaged retinas is impaired in the absence of microglial cells. This study further shows that ablation of microglial cells from the retina increases the expression of S1P-related genes in MG, whereas inhibition of S1PR1 and SPHK1 partially rescues the formation of MGPCs in damaged retinas depleted of microglia. The studies also show that expression of S1P-related genes is conserved in fish and human retinas.

Strengths:

This is well-conducted study, with convincing images and statistically relevant data

Weaknesses:

However, given that S1P is upstream N 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)

Reviewer #2 (Public review):

Summary:

Sphingosine-1-phosphate (S1P) metabolic and signaling genes are expressed highly in retinal Müller glia (MG) cells. This study tested how S1P signaling regulates glial phenotype, dedifferentiation of, reprogramming into proliferating MG-derived progenitor cells (MGPCs), and neuronal differentiation of the progeny of MGPCs using in vivo chick retina. Major techniques used are Sc-RNASeq and immunohistochemistry to determine the gene expression and proliferation of MG cells that co-label with signaling antibodies or mRNA FISH following treating the in vivo eyes with various S1P signaling antagonists, agonists, and signal modulators. The major conclusions drawn are supported by the results presented. However, the methodology they have used to modulate the S1P pathway using various chemical drugs raises questions about the outcomes and whether those are the real effects of S1P receptor modulation or S1P synthesis inhibition.

Strengths:

- Use of elaborated single-cell RNAseq expression data.
- Use of FISH for S1P receptors and kinase as a good quality antibody is not available.
- Use of EdU assay in combination with IHC
- Comparison with human and Zebrafish Sc-RNA data

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?

Author response:

Reviewer #1:

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). 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 to better highlight these important distinctions between NFκB- and S1P-signaling.

Reviewer #2:

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 µl of liquid vitreous, 800 µl 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, parsed with S1P receptor expression patterns, together with measurements of retinal levels of S1P following exposure to drugs targeting enzymes that catalyze synthesis or degradation of S1P, as described above.