Oligodendrocyte-lineage cell exocytosis and L-type prostaglandin D synthase promote oligodendrocyte development and myelination
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Evaluation Summary:
This manuscript uses a combination of in vitro and in vivo approaches and uncovers a potential mechanism of autocrine/paracrine signaling in oligodendrocyte maturation, which provides an exciting avenue for future investigation. In particular, the authors examined the role of oligodendroglial exocytosis, and specifically the role of L-type prostaglandin D synthase (LPGDS), in modulating oligodendrocyte differentiation and myelination. This work will be of interest to glial and myelin disease researchers.
(This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. Reviewer #1 and Reviewer #3 agreed to share their names with the authors.)
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Abstract
In the developing central nervous system, oligodendrocyte precursor cells (OPCs) differentiate into oligodendrocytes, which form myelin around axons. Oligodendrocytes and myelin are essential for the function of the central nervous system, as evidenced by the severe neurological symptoms that arise in demyelinating diseases such as multiple sclerosis and leukodystrophy. Although many cell-intrinsic mechanisms that regulate oligodendrocyte development and myelination have been reported, it remains unclear whether interactions among oligodendrocyte-lineage cells (OPCs and oligodendrocytes) affect oligodendrocyte development and myelination. Here, we show that blocking vesicle-associated membrane protein (VAMP) 1/2/3-dependent exocytosis from oligodendrocyte-lineage cells impairs oligodendrocyte development, myelination, and motor behavior in mice. Adding oligodendrocyte-lineage cell-secreted molecules to secretion-deficient OPC cultures partially restores the morphological maturation of oligodendrocytes. Moreover, we identified L-type prostaglandin D synthase as an oligodendrocyte-lineage cell-secreted protein that promotes oligodendrocyte development and myelination in vivo. These findings reveal a novel autocrine/paracrine loop model for the regulation of oligodendrocyte and myelin development.
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Author Response
Reviewer #1 (Public Review):
Pan et al. examined the role of oligodendroglial exocytosis, and specifically the role of L-type prostaglandin D synthase (LPGDS), in modulating oligodendrocyte differentiation and myelination. The topic of autocrine and paracrine signaling within the oligodendrocyte lineage is under-studied and the authors use a novel approach for oligodendrocyte precursor-specific inhibition of VAMP-mediated exocytosis using inducible expression of botulinum toxin with the PDGRFa-CreER transgenic mouse line (PD:ibot). Using a combination of in vitro culture systems and immunohistological analysis in vivo, the authors find ibot expression in OPCs leads to reduced oligodendrogenesis and myelination, leading to a behavioral deficit in rotarod performance. Additional transcriptomic analysis in PD:ibot mice …
Author Response
Reviewer #1 (Public Review):
Pan et al. examined the role of oligodendroglial exocytosis, and specifically the role of L-type prostaglandin D synthase (LPGDS), in modulating oligodendrocyte differentiation and myelination. The topic of autocrine and paracrine signaling within the oligodendrocyte lineage is under-studied and the authors use a novel approach for oligodendrocyte precursor-specific inhibition of VAMP-mediated exocytosis using inducible expression of botulinum toxin with the PDGRFa-CreER transgenic mouse line (PD:ibot). Using a combination of in vitro culture systems and immunohistological analysis in vivo, the authors find ibot expression in OPCs leads to reduced oligodendrogenesis and myelination, leading to a behavioral deficit in rotarod performance. Additional transcriptomic analysis in PD:ibot mice revealed Ptgds, the gene encoding LPGDS, was significantly overexpressed in both mature oligodendrocytes and OPCs. Further pharmacological experiments with cultured OPCs showed direct LPGDS inhibition led to a similar inhibition of oligodendrogenesis as PD:ibot mice. Together, this study reveals VAMP-mediated exocytosis in OPCs is required for normal oligodendrogenesis and identifies LPGDS as a new chemical regulator of oligodendrocyte myelination. These findings are strengthened by careful characterization of the PD:ibot mouse line and effective use of culture systems and pharmacology to uncover a cellular mechanism. Quantification is performed at several levels of resolution using immunohistochemistry, electron micrography, and protein/transcriptomic analyses and control experiments were largely carefully considered.
We thank the reviewer for recognizing the strength of our study.
Despite these strengths, there are some points that need to be further addressed. The interpretation of autocrine/paracrine signaling relies on a critical culture experiment in which PD:ibot OPCs were cultured in the presence of PD:ibot or control OPC well inserts. However, these results had a marginal effect size, raising questions as to the extent to which VAMP inhibition specifically had effects through the blockade of exocytosis (resulting in an autocrine/paracrine signaling deficit) or inhibited oligodendrogenesis in a cell-intrinsic mechanism (e.g. VAMP-dependent trafficking of critical myelination components, such as PLP (Feldmann et al., 2011)).
We agree with the reviewer that both cell autonomous and cell non-autonomous effects may contribute to the defect associated with VAMP inhibition. We performed additional experiments to investigate the contribution of cell non-autonomous mechanisms. We took advantage of the fact that all OPCs purified from PD:ibot mice were not botulinum-GFP-expressing (efficiency ~65% Figure 6B, page 24). The GFP- cells in PD:ibot OPC cultures did not express botulinum toxin and were competent in exocytosis. We compared the development of GFP- control cells in cultures generated from PD:ibot mice vs. control cells in cultures generated from control mice. Interestingly, we found that the percentages and sizes of lamellar cells in control cells in PD:ibot cultures were smaller than in control cells in control cultures (Figure 6C, D text page 25). Although both groups of cells were competent in exocytosis, they were surrounded by exocytosis-deficient vs. exocytosis-competent neighbor cells. The differences in the growth capacity of control cells in the presence of different neighbor cells reveal cell non-autonomous contributions of botulinum-expressing cells in oligodendrocyte development.
As described above under Essential Revisions 4), we performed additional experiments on the role of the secreted protein L-PGDS in oligodendrocyte development. We found that adding a protein that inactivates PGD2, HPGD extracellularly to oligodendrocyte cultures inhibited their development (Figure 7F, G, page 33). Adding L-PGDS protein extracellularly to PD:ibot oligodendrocyte cultures rescued their development defect (Figure 9A, B, page 33). Moreover, overexpressing Ptgds in PD:ibot mice partially rescued the myelination defect (Figure 9E-H, page 36). These observations further strengthened our conclusion that cell non-autonomous mechanisms contribute to the effect of botulinum toxin on oligodendrocyte and myelin development.
Nevertheless, these results do not rule out the cell autonomous effect of botulinum on oligodendrocyte development and, therefore, we included the potential contribution of both cell autonomous and cell non-autonomous mechanisms in the text.
Additionally, the authors claim the reduced number of oligodendrocytes in PD:ibot mice in vivo is not due to oligodendrocyte apoptosis and provide evidence by cleaved caspase-3 immunostaining of the cerebral cortex. While statistically not significant, the data is highly variable.
We thank the reviewer for pointing out the variability of the caspase-3 results. We performed a more thorough analysis of activated caspase-3 at multiple developmental stages. Again, we did not find any statistically significant difference in apoptosis between PD:ibot and control oligodendrocytes, OPCs, or cells of other lineages (Figure 3-figure supplement 1, text page 13).
If true, this would suggest oligodendrocyte differentiation is inhibited, which would coincide with a reduction of OPC proliferation. A complementary experiment comparing the rates of OPC proliferation between control and PD:ibot mice in vivo would provide further clarity on how oligodendrocyte density is being reduced.
We analyzed OPC proliferation in vivo by staining and quantifying Ki67+PDGFRa+ cells. Intriguingly, we found a modest increase in OPC proliferation in PD:ibot mice (Figure 3-figure supplement 3, text page 14).
The relevance of these myelination deficits is assessed with a rotarod assay, however, the mice used for these experiments are several times older (2-5 months) than those used for all other histological quantification (P8-P30). The large variance in results could be due to age-related differences in myelination, and it is unclear whether the deficits at early timepoints show a linear progression with age.
We thank the reviewer for the insightful comment. We have separately labeled data points from 2 months old and 5 months old mice (Figure 3Q-S, text page 17). With the data we have so far (n=20-27 per genotype), there isn’t a striking progression of phenotype with age. Future analysis at multiple time points may resolve any age-dependent changes in the phenotype.
Reviewer #3 (Public Review):
The authors pose an important question of whether oligodendrocyte lineage cells have an autocrine/paracrine signaling loop that contributes to their differentiation and myelination. While prior studies have demonstrated oligodendrocyte lineage cells have cell-intrinsic pathways that impact differentiation and myelination, there isn't a strong precedent for oligodendrocytes to promote their own differentiation via autocrine/paracrine mechanisms. The notion that oligodendrocyte lineage cells promote their own differentiation in an autocrine/paracrine manner is an intriguing one that adds a new layer to our understanding of how oligodendrocyte maturation is controlled. I anticipate this paper will prompt a new direction of future investigations to uncover the extent of oligodendrocyte autocrine/paracrine signaling.
To test the possible role of oligodendrocyte-secreted molecules on oligodendrocyte development, Pan et al. utilized a mouse model where the release of a subset of secretory vesicles (specifically VAMP1/2/3-dependent vesicles) is blocked. Blocking this vesicular release prevented or delayed the differentiation of oligodendrocytes in vivo and in vitro. Further, the authors identified changes to the mRNA and secreted protein levels of prostaglandin D2 synthase (L-PGDS). Prior RNA sequencing and snRNA sequencing datasets of the oligodendrocyte lineage have identified Ptgds as a highly abundant mRNA transcript in oligodendrocyte lineage cells, particularly mature oligodendrocytes. Ptgds encodes L-PGDS, which has an unknown role in oligodendrocyte function. L-PGDS has been shown to regulate Schwann cell myelin formation in the peripheral nervous system, prompting the question of whether this protein acts similarly in the central nervous system. The paper has a clear set of well-rounded experiments, with a few remaining points that would strengthen the conclusions:
We thank the reviewer for the positive comments on our study.
One of the foundational conclusions of the study is that VAMP1/2/3-dependent exocytosis is critical to oligodendrocyte maturation, by using a PDGFRa-CreER mouse line combined with iBot mice that express botulinum toxin in Cre-expressing cells (abbreviated as PD:iBot). Prior work has demonstrated in vitro that oligodendrocyte morphological maturation, myelin gene expression and myelin protein transport can all be impacted by the loss of VAMPs, including VAMP3. This paper establishes the importance of these SNARE proteins in the oligodendrocyte lineage in vivo: the number of mature (CC1+) oligodendrocytes and myelin basic protein staining is substantially reduced in PD:iBot mice.
- The data in Figure 3M suggests that PD:iBot oligodendrocytes (GFP+) are lacking MBP+ sheaths and that any myelin formed is by the smaller percent of oligodendrocytes that do not express botulinum (GFP- cells). Furthermore, the efficiency of iBot expression (as evaluated by GFP+ cells) shows that 80% of OPCs and just 60% of oligodendrocyte lineage cells express GFP at P8 and supplementary data shows just 30% of oligodendrocyte lineage cells express GFP at P30. This raises the question of whether PD:iBot cells are unable to differentiate and die. While the authors show no change in caspase-dependent apoptosis in PD:iBot cells in vivo and in vitro, the data still suggests that blocking VAMP-dependent exocytosis itself slows or prevents the progression to a fully myelinating oligodendrocyte in vivo rather than the putative autocrine/paracrine signals are required for OPC differentiation. Confirming whether botulinum-expressing cells also contribute to the population of surviving, differentiated oligodendrocytes in vivo to strengthen the conclusions that autocrine/paracrine secreted molecules contribute to the oligodendrocyte maturation in vivo.
We thank the reviewers for raising a key point in characterizing the consequence of botulinum toxin expression in oligodendrocyte-lineage cells. We analyzed the overlap between GFP+ botulinum-expressing cells and the population of differentiated oligodendrocytes (Olig2+PDGFRa-CC1+ cells) and found that botulinum-expressing cells can survive and become differentiated oligodendrocytes (Figure 3-figure supplement 2, text page 14). Additionally, we performed a more thorough analysis of activated caspase-3+ apoptotic cells than was included in first submission and did not detect statistically significant differences between PD:ibot and control mice (Figure 3-figure supplement 1, text page 13).
- The paper has complementary in vitro data to pinpoint a mechanism that results in hindered oligodendrocyte maturation. The authors conduct a well-designed set of in vitro co-culture experiments in Fig4 K-M that led them to conclude oligodendrocyte morphology is impacted by secreted molecules from other oligodendrocytes.
2a) The key experiment is the transwell co-culture experiment with control and iBot cells, which suggests that blocking secretion itself has the predominant impact on cell morphology: by eye, both group3 and 4 show the largest reduction in lamellar area and the difference between group 3 and 4 is slight. At day 3 of culture (Fig 4E), the authors show the clearest effect as a reduction in cells with lamellar morphology. The quantification of the lamellar cell area is less obvious than the % of cells with arborized vs lamellar shape, as seen in Figures E & F. I would recommend that the authors show representative images of these observations and quantification of morphologies for the transwell experiments. The impact of secreted factors may be clearer with this measure.
We added representative images (Figure 6G). We quantified both the % and size of lamellar cells. The size of lamellar cells is significantly higher in group 4 than in group 3. Although the % of lamellar cells is numerically higher in group 4 than in group 3, the difference is not statistically significant. To further assess whether cell non-autonomous mechanisms contribute to the oligodendrocyte development defect in PD:ibot mice, we performed additional analysis in culture. We took advantage of the fact that all OPCs purified from PD:ibot mice were not botulinum-GFP-expressing (efficiency ~65% Figure 6B). The GFP- cells in PD:ibot OPC cultures did not express botulinum toxin and were competent in exocytosis. We compared the development of GFP- control cells in cultures generated from PD:ibot mice vs. control cells in cultures generated from control mice. Interestingly, we found that the percentages and sizes of lamellar cells in control cells in PD:ibot cultures is smaller than in control cells in control cultures (Figure 6C, D, text page 25). Although both groups of cells were competent in exocytosis, they were surrounded by exocytosis-deficient vs. exocytosis-competent neighbor cells. The differences in the growth capacity of control cells in the presence of different neighbor cells reveal cell non-autonomous contributions of botulinum-expressing cells in oligodendrocyte development.
2b) On a related note, the cell morphology data is dependent on MBP staining. The authors show that MBP protein is reduced in cells from iBot mice. Since MBP+ cell area/arborized or lamellar structure is being quantified, there remains the possibility that the cells could display a more complex morphology (lamellar) that may be missed by only staining for MBP. The authors use a CellMask dye to show cellular morphology, which is a great idea. The authors state that it labels the plasma membrane; however, the methods (and images) indicate that a cytoplasmic CellMask was used (cat.no. H32720 labels nuclei and cytoplasm, not membranes). These conclusions about cell morphology vs simply MBP expression would be strengthened by an alternative membrane label (e.g., a CellMask plasma membrane dye).
We thank the reviewers for the insightful suggestion. We used the membrane version of CellMask and repeated the transwell co-culture experiment. The new results are consistent with the results based on MBP (Figure 6-figure supplement 1, text page 23). In addition, we used the membrane version of CellMask for all the new cell culture experiments (L-PGDS rescue, HPGD etc.)
- The authors sought to identify what secreted factors may be affected by blocking VAMP1/2/3-dependent exocytosis. Pan et al. opted for a strategy of examining transcriptional changes, asserting that important genes may be upregulated in response to compensate for blocked secretion. While this is an indirect way to identify secreted candidates, the authors found a fortuitous result that Ptgds was substantially increased in the PD:iBot oligodendrocyte cells. To confirm that L-PGDS secretion is reduced from iBot cells, the authors show Western blots. By eye the change in L-PGDS is variable, however, the authors conduct several experiments with an inhibitor and product of L-PGDS that nonetheless indicate L-PGDS activity can contribute to the morphological maturation of oligodendrocytes. A caveat is that the AT-56 inhibitor reduces MBP+ cells, and the quantification of morphology is dependent on MBP staining (again, see my note in 2b about the CellMask dye). A report on differentiation (% MBP+ cells) may be a more accurate reflection of the result.
We repeated the AT-56 experiment using the membrane version of CellMask and again found that AT-56 inhibits oligodendrocyte maturation (Figure 7-figure supplement 2, text page 33).
The key, compelling experiment demonstrating the role of prostaglandin D2 is the authors' rescue experiment in Fig 4G.
As described above under Essential Revisions 4), we performed additional rescue experiments on the role of L-PGDS in oligodendrocyte development. We found that adding L-PGDS protein extracellularly to PD:ibot oligodendrocyte cultures rescued their development defect (Figure 9A, B, page 34). Moreover, overexpressing Ptgds in PD:ibot mice partially rescued the myelination defect (Figure 9E-H, page 36).
- Although it's not a direct demonstration that L-PDGS secretion from oligodendrocytes is the key factor, the global L-PDGS knockout mice phenocopy many of the observations of the PD:iBot mice. This is a nice set of observations consistent with the author's hypothesis that L-PDGS impacts oligodendrocyte maturation. Future work should pinpoint whether oligodendrocyte-derived L-PDGS is critical.
We agree with the reviewer that pinpointing whether oligodendrocyte-derived L-PGDS promotes oligodendrocyte development and myelination is an interesting direction to pursue in future work. We are breeding L-PGDS conditional knockout mice to address this question and may report the results in a separate paper in the future.
Minor points:
- The authors demonstrate that PD:iBot expresses botulinum and loses VAMP2 protein levels in oligodendrocyte lineage cells, but there is no demonstration of whether VAMP3 is expressed or similarly affected. Prior work has demonstrated in vitro that oligodendrocytes express both VAMP2 and VAMP3 (VAMP1 not detected). This would more clearly demonstrate which VAMP-mediated vesicular transport is blocked for the effects observed.
We agree with the reviewer and examined VAMP3 levels with Western blot. We found diminished levels of VAMP3 in oligodendrocyte-lineage cells from PD:ibot mice (Figure 1 J, M, text page 10).
- It is satisfying to observe a behavioral effect in the PD:iBot mice. I would advise caution in interpreting any direct link between oligodendrocytes maturation and the rotarod behavioral difference at this time. Blocking secretion from PDGFRa-Cre expressing cells may have many indirect effects (beyond myelination) in both the CNS and other cell types that can express PDGFRa and VAMPs1/2/3. I was pleased that the authors did not conclude any direct links at this time.
We agree with the reviewer.
Overall, the authors had a well-rounded manuscript with clearly described and thoughtful experiments. The data support the conclusion that VAMP-mediated exocytosis is critical for oligodendrocyte maturation. The evidence that reduced L-PDGS secretion from the oligodendrocytes can explain the effects of the iBot mice is not as clear cut, but their data does demonstrate that L-PDGS is an important molecule for the differentiation of oligodendrocytes. This work will lead a new direction for future studies to investigate autocrine/paracrine signaling in oligodendrocyte maturation.
We thank the reviewer for the positive comments on our manuscript. As detailed in Essential Revisions 4), we now provide additional evidence on the potential contribution of L-PGDS in the oligodendrocyte development defect in PD:ibot mice.
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Evaluation Summary:
This manuscript uses a combination of in vitro and in vivo approaches and uncovers a potential mechanism of autocrine/paracrine signaling in oligodendrocyte maturation, which provides an exciting avenue for future investigation. In particular, the authors examined the role of oligodendroglial exocytosis, and specifically the role of L-type prostaglandin D synthase (LPGDS), in modulating oligodendrocyte differentiation and myelination. This work will be of interest to glial and myelin disease researchers.
(This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. Reviewer #1 and Reviewer #3 agreed to share their names with the authors.)
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Reviewer #1 (Public Review):
Pan et al. examined the role of oligodendroglial exocytosis, and specifically the role of L-type prostaglandin D synthase (LPGDS), in modulating oligodendrocyte differentiation and myelination. The topic of autocrine and paracrine signaling within the oligodendrocyte lineage is under-studied and the authors use a novel approach for oligodendrocyte precursor-specific inhibition of VAMP-mediated exocytosis using inducible expression of botulinum toxin with the PDGRFa-CreER transgenic mouse line (PD:ibot). Using a combination of in vitro culture systems and immunohistological analysis in vivo, the authors find ibot expression in OPCs leads to reduced oligodendrogenesis and myelination, leading to a behavioral deficit in rotarod performance. Additional transcriptomic analysis in PD:ibot mice revealed Ptgds, the …
Reviewer #1 (Public Review):
Pan et al. examined the role of oligodendroglial exocytosis, and specifically the role of L-type prostaglandin D synthase (LPGDS), in modulating oligodendrocyte differentiation and myelination. The topic of autocrine and paracrine signaling within the oligodendrocyte lineage is under-studied and the authors use a novel approach for oligodendrocyte precursor-specific inhibition of VAMP-mediated exocytosis using inducible expression of botulinum toxin with the PDGRFa-CreER transgenic mouse line (PD:ibot). Using a combination of in vitro culture systems and immunohistological analysis in vivo, the authors find ibot expression in OPCs leads to reduced oligodendrogenesis and myelination, leading to a behavioral deficit in rotarod performance. Additional transcriptomic analysis in PD:ibot mice revealed Ptgds, the gene encoding LPGDS, was significantly overexpressed in both mature oligodendrocytes and OPCs. Further pharmacological experiments with cultured OPCs showed direct LPGDS inhibition led to a similar inhibition of oligodendrogenesis as PD:ibot mice. Together, this study reveals VAMP-mediated exocytosis in OPCs is required for normal oligodendrogenesis and identifies LPGDS as a new chemical regulator of oligodendrocyte myelination. These findings are strengthened by careful characterization of the PD:ibot mouse line and effective use of culture systems and pharmacology to uncover a cellular mechanism. Quantification is performed at several levels of resolution using immunohistochemistry, electron micrography, and protein/transcriptomic analyses and control experiments were largely carefully considered.
Despite these strengths, there are some points that need to be further addressed. The interpretation of autocrine/paracrine signaling relies on a critical culture experiment in which PD:ibot OPCs were cultured in the presence of PD:ibot or control OPC well inserts. However, these results had a marginal effect size, raising questions as to the extent to which VAMP inhibition specifically had effects through the blockade of exocytosis (resulting in an autocrine/paracrine signaling deficit) or inhibited oligodendrogenesis in a cell-intrinsic mechanism (e.g. VAMP-dependent trafficking of critical myelination components, such as PLP (Feldmann et al., 2011)). Additionally, the authors claim the reduced number of oligodendrocytes in PD:ibot mice in vivo is not due to oligodendrocyte apoptosis and provide evidence by cleaved caspase-3 immunostaining of the cerebral cortex. While statistically not significant, the data is highly variable. If true, this would suggest oligodendrocyte differentiation is inhibited, which would coincide with a reduction of OPC proliferation. A complementary experiment comparing the rates of OPC proliferation between control and PD:ibot mice in vivo would provide further clarity on how oligodendrocyte density is being reduced. The relevance of these myelination deficits is assessed with a rotarod assay, however, the mice used for these experiments are several times older (2-5 months) than those used for all other histological quantification (P8-P30). The large variance in results could be due to age-related differences in myelination, and it is unclear whether the deficits at early timepoints show a linear progression with age.
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Reviewer #2 (Public Review):
The report employs an oligodendrocyte botulinum B toxin transgenic mouse line by which they are able to perturb vesicular release in a cell-specific manner. Using this mouse line the authors contend that disrupting VAMP in this ibot mouse line is evidence for vesicular release from oligodendrocytes and extracellular release of signaling factors that contribute to the maturation of oligodendrocytes.
Using a transcriptomic approach the authors determined that expression of Prostaglandin-Endoperoxide Synthase 1 was elevated in OPCs and a pharmacological inhibitor results in a concentration-dependent reduction in OL maturation. Lastly, the authors determined that a global L-PGDS knockout mouse phenocopies the ibot mouse with respect to reduced developmental myelination.
These data build upon prior work …
Reviewer #2 (Public Review):
The report employs an oligodendrocyte botulinum B toxin transgenic mouse line by which they are able to perturb vesicular release in a cell-specific manner. Using this mouse line the authors contend that disrupting VAMP in this ibot mouse line is evidence for vesicular release from oligodendrocytes and extracellular release of signaling factors that contribute to the maturation of oligodendrocytes.
Using a transcriptomic approach the authors determined that expression of Prostaglandin-Endoperoxide Synthase 1 was elevated in OPCs and a pharmacological inhibitor results in a concentration-dependent reduction in OL maturation. Lastly, the authors determined that a global L-PGDS knockout mouse phenocopies the ibot mouse with respect to reduced developmental myelination.
These data build upon prior work identifying elevated expression of L-PGDS in OPCs. These data also provide preliminary evidence for the extracellular release of signals from OPCs which may then, in an autocrine or local paracrine manner, impact the potential for the maturation of this cell type.
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Reviewer #3 (Public Review):
The authors pose an important question of whether oligodendrocyte lineage cells have an autocrine/paracrine signaling loop that contributes to their differentiation and myelination. While prior studies have demonstrated oligodendrocyte lineage cells have cell-intrinsic pathways that impact differentiation and myelination, there isn't a strong precedent for oligodendrocytes to promote their own differentiation via autocrine/paracrine mechanisms. The notion that oligodendrocyte lineage cells promote their own differentiation in an autocrine/paracrine manner is an intriguing one that adds a new layer to our understanding of how oligodendrocyte maturation is controlled. I anticipate this paper will prompt a new direction of future investigations to uncover the extent of oligodendrocyte autocrine/paracrine …
Reviewer #3 (Public Review):
The authors pose an important question of whether oligodendrocyte lineage cells have an autocrine/paracrine signaling loop that contributes to their differentiation and myelination. While prior studies have demonstrated oligodendrocyte lineage cells have cell-intrinsic pathways that impact differentiation and myelination, there isn't a strong precedent for oligodendrocytes to promote their own differentiation via autocrine/paracrine mechanisms. The notion that oligodendrocyte lineage cells promote their own differentiation in an autocrine/paracrine manner is an intriguing one that adds a new layer to our understanding of how oligodendrocyte maturation is controlled. I anticipate this paper will prompt a new direction of future investigations to uncover the extent of oligodendrocyte autocrine/paracrine signaling.
To test the possible role of oligodendrocyte-secreted molecules on oligodendrocyte development, Pan et al. utilized a mouse model where the release of a subset of secretory vesicles (specifically VAMP1/2/3-dependent vesicles) is blocked. Blocking this vesicular release prevented or delayed the differentiation of oligodendrocytes in vivo and in vitro. Further, the authors identified changes to the mRNA and secreted protein levels of prostaglandin D2 synthase (L-PGDS). Prior RNA sequencing and snRNA sequencing datasets of the oligodendrocyte lineage have identified Ptgds as a highly abundant mRNA transcript in oligodendrocyte lineage cells, particularly mature oligodendrocytes. Ptgds encodes L-PGDS, which has an unknown role in oligodendrocyte function. L-PGDS has been shown to regulate Schwann cell myelin formation in the peripheral nervous system, prompting the question of whether this protein acts similarly in the central nervous system. The paper has a clear set of well-rounded experiments, with a few remaining points that would strengthen the conclusions:
One of the foundational conclusions of the study is that VAMP1/2/3-dependent exocytosis is critical to oligodendrocyte maturation, by using a PDGFRa-CreER mouse line combined with iBot mice that express botulinum toxin in Cre-expressing cells (abbreviated as PD:iBot). Prior work has demonstrated in vitro that oligodendrocyte morphological maturation, myelin gene expression and myelin protein transport can all be impacted by the loss of VAMPs, including VAMP3. This paper establishes the importance of these SNARE proteins in the oligodendrocyte lineage in vivo: the number of mature (CC1+) oligodendrocytes and myelin basic protein staining is substantially reduced in PD:iBot mice.
The data in Figure 3M suggests that PD:iBot oligodendrocytes (GFP+) are lacking MBP+ sheaths and that any myelin formed is by the smaller percent of oligodendrocytes that do not express botulinum (GFP- cells). Furthermore, the efficiency of iBot expression (as evaluated by GFP+ cells) shows that 80% of OPCs and just 60% of oligodendrocyte lineage cells express GFP at P8 and supplementary data shows just 30% of oligodendrocyte lineage cells express GFP at P30. This raises the question of whether PD:iBot cells are unable to differentiate and die. While the authors show no change in caspase-dependent apoptosis in PD:iBot cells in vivo and in vitro, the data still suggests that blocking VAMP-dependent exocytosis itself slows or prevents the progression to a fully myelinating oligodendrocyte in vivo rather than the putative autocrine/paracrine signals are required for OPC differentiation. Confirming whether botulinum-expressing cells also contribute to the population of surviving, differentiated oligodendrocytes in vivo to strengthen the conclusions that autocrine/paracrine secreted molecules contribute to the oligodendrocyte maturation in vivo.
The paper has complementary in vitro data to pinpoint a mechanism that results in hindered oligodendrocyte maturation. The authors conduct a well-designed set of in vitro co-culture experiments in Fig4 K-M that led them to conclude oligodendrocyte morphology is impacted by secreted molecules from other oligodendrocytes.
2a) The key experiment is the transwell co-culture experiment with control and iBot cells, which suggests that blocking secretion itself has the predominant impact on cell morphology: by eye, both group3 and 4 show the largest reduction in lamellar area and the difference between group 3 and 4 is slight. At day 3 of culture (Fig 4E), the authors show the clearest effect as a reduction in cells with lamellar morphology. The quantification of the lamellar cell area is less obvious than the % of cells with arborized vs lamellar shape, as seen in Figures E & F. I would recommend that the authors show representative images of these observations and quantification of morphologies for the transwell experiments. The impact of secreted factors may be clearer with this measure.
2b) On a related note, the cell morphology data is dependent on MBP staining. The authors show that MBP protein is reduced in cells from iBot mice. Since MBP+ cell area/arborized or lamellar structure is being quantified, there remains the possibility that the cells could display a more complex morphology (lamellar) that may be missed by only staining for MBP. The authors use a CellMask dye to show cellular morphology, which is a great idea. The authors state that it labels the plasma membrane; however, the methods (and images) indicate that a cytoplasmic CellMask was used (cat.no. H32720 labels nuclei and cytoplasm, not membranes). These conclusions about cell morphology vs simply MBP expression would be strengthened by an alternative membrane label (e.g., a CellMask plasma membrane dye).
The authors sought to identify what secreted factors may be affected by blocking VAMP1/2/3-dependent exocytosis. Pan et al. opted for a strategy of examining transcriptional changes, asserting that important genes may be upregulated in response to compensate for blocked secretion. While this is an indirect way to identify secreted candidates, the authors found a fortuitous result that Ptgds was substantially increased in the PD:iBot oligodendrocyte cells. To confirm that L-PGDS secretion is reduced from iBot cells, the authors show Western blots. By eye the change in L-PGDS is variable, however, the authors conduct several experiments with an inhibitor and product of L-PGDS that nonetheless indicate L-PGDS activity can contribute to the morphological maturation of oligodendrocytes. A caveat is that the AT-56 inhibitor reduces MBP+ cells, and the quantification of morphology is dependent on MBP staining (again, see my note in 2b about the CellMask dye). A report on differentiation (% MBP+ cells) may be a more accurate reflection of the result. The key, compelling experiment demonstrating the role of prostaglandin D2 is the authors' rescue experiment in Fig 4G.
Although it's not a direct demonstration that L-PDGS secretion from oligodendrocytes is the key factor, the global L-PDGS knockout mice phenocopy many of the observations of the PD:iBot mice. This is a nice set of observations consistent with the author's hypothesis that L-PDGS impacts oligodendrocyte maturation. Future work should pinpoint whether oligodendrocyte-derived L-PDGS is critical.
Minor points:
The authors demonstrate that PD:iBot expresses botulinum and loses VAMP2 protein levels in oligodendrocyte lineage cells, but there is no demonstration of whether VAMP3 is expressed or similarly affected. Prior work has demonstrated in vitro that oligodendrocytes express both VAMP2 and VAMP3 (VAMP1 not detected). This would more clearly demonstrate which VAMP-mediated vesicular transport is blocked for the effects observed.
It is satisfying to observe a behavioral effect in the PD:iBot mice. I would advise caution in interpreting any direct link between oligodendrocytes maturation and the rotarod behavioral difference at this time. Blocking secretion from PDGFRa-Cre expressing cells may have many indirect effects (beyond myelination) in both the CNS and other cell types that can express PDGFRa and VAMPs1/2/3. I was pleased that the authors did not conclude any direct links at this time.
Overall, the authors had a well-rounded manuscript with clearly described and thoughtful experiments. The data support the conclusion that VAMP-mediated exocytosis is critical for oligodendrocyte maturation. The evidence that reduced L-PDGS secretion from the oligodendrocytes can explain the effects of the iBot mice is not as clear cut, but their data does demonstrate that L-PDGS is an important molecule for the differentiation of oligodendrocytes. This work will lead a new direction for future studies to investigate autocrine/paracrine signaling in oligodendrocyte maturation.
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