Oligodendrocyte-lineage cell exocytosis and L-type prostaglandin D synthase promote oligodendrocyte development and myelination
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.
The manuscript will be of interest to glial and myelin disease researchers. The well-designed combination of in vitro and in vivo approaches uncovers a potential mechanism of autocrine/paracrine signaling in oligodendrocyte maturation which provides an exciting avenue for future investigation.https://doi.org/10.7554/eLife.77441.sa0
In the developing central nervous system (CNS), oligodendrocyte precursor cells (OPCs) differentiate into oligodendrocytes (Bergles and Richardson, 2016; Hill et al., 2014; Kang et al., 2010), which form myelin sheaths around axons. Myelin is essential for the propagation of action potentials and for the metabolism and health of axons (Fünfschilling et al., 2012; Larson et al., 2018; Mukherjee et al., 2020; Saab et al., 2016; Schirmer et al., 2018; Simons and Nave, 2016). When oligodendrocytes and myelin are damaged in demyelinating diseases such as multiple sclerosis (MS) and leukodystrophy, sensory, motor, and cognitive deficits can ensue (Gruchot et al., 2019; Lubetzki et al., 2020; Stadelmann et al., 2019). In a broader range of neurological disorders involving neuronal loss, such as brain/spinal cord injury and stroke, the growth and myelination of new axons are necessary for neural repair (Wang et al., 2020). Thus, understanding oligodendrocyte development and myelination is critical for developing treatments for a broad range of neurological disorders.
Over the past several decades, researchers have made great progress in elucidating the cell-intrinsic regulation of oligodendrocyte development and myelination (e.g. transcription factors, epigenetic mechanisms, and cell death pathways) (Aggarwal et al., 2013; Bergles and Richardson, 2016; Budde et al., 2010; Dugas et al., 2010; Elbaz and Popko, 2019; Elbaz et al., 2018; Emery and Lu, 2015; Emery et al., 2009; Fedder-Semmes and Appel, 2021; Foerster et al., 2020; Harrington et al., 2010; Herbert and Monk, 2017; Howng et al., 2010; Koenning et al., 2012; Mitew et al., 2018; Nawaz et al., 2015; Snaidero et al., 2017; Sun et al., 2018; Wang et al., 2017; Xu et al., 2020; Zhao et al., 2018; Zuchero et al., 2015), as well as the cell-extrinsic regulation by other cell types (e.g. neurons (Gibson et al., 2014; Hines et al., 2015; Mayoral et al., 2018; Osso et al., 2021; Redmond et al., 2016; Wake et al., 2011), microglia/macrophages (Butovsky et al., 2006; Sherafat et al., 2021), and lymphocytes Dombrowski et al., 2017). However, it remains unclear whether interactions among oligodendrocyte-lineage cells (OPCs and oligodendrocytes) affect oligodendrocyte development and myelination.
One of the most abundant transcripts encoding secreted proteins in oligodendrocyte-lineage cells is Ptgds, encoding lipocalin-type prostaglandin D synthase (L-PGDS; Zhang et al., 2014; Zhang et al., 2016). Oligodendrocytes and meningeal cells are major sources of L-PGDS in the CNS (Urade et al., 1993; Urade, 2021; Zhang et al., 2014; Zhang et al., 2016). L-PGDS has two functions: as an enzyme and as a carrier (Urade and Hayaishi, 2000, Urade, 2021). As an enzyme, L-PGDS converts prostaglandin H2 to prostaglandin D2 (PGD2) (Urade et al., 1985). PGD2 regulates sleep, pain, and allergic reactions (Eguchi et al., 1999; Satoh et al., 2006; Urade and Hayaishi, 2011). L-PGDS also binds and transports lipophilic molecules such as thyroid hormone, retinoic acid, and amyloid-β (Urade and Hayaishi, 2000) and promotes Schwann cell myelination in the peripheral nervous system (Trimarco et al., 2014). Yet, its function in the development of the CNS is unknown.
To determine whether cell-cell interactions within the oligodendrocyte lineage regulate oligodendrocyte development, we blocked VAMP1/2/3-dependent exocytosis from oligodendrocyte-lineage cells in vivo and found impairment in oligodendrocyte development, myelination, and motor behavior in mice. Similarly, exocytosis-deficient OPCs exhibited impaired development in vitro. Adding oligodendrocyte-lineage cell-secreted molecules promoted oligodendrocyte development. These results suggest that an autocrine/paracrine loop promotes oligodendrocyte development and myelination. We assessed L-PGDS as a candidate autocrine/paracrine signal and further discovered that oligodendrocyte development and myelination were impaired in L-PGDS-knockout mice. Moreover, overexpression of the gene encoding L-PGDS partially restored the myelination defect of exocytosis-deficient mice. These results reveal a new autocrine/paracrine loop model for the regulation of oligodendrocyte development in which VAMP1/2/3-dependent exocytosis from oligodendrocyte-linage cells and secreted L-PGDS promote oligodendrocyte development and myelination.
Expression of botulinum toxin B in oligodendrocyte-lineage cells in vivo
If oligodendrocyte-lineage cells use autocrine/paracrine mechanisms to promote development and myelination, one would predict that (1) blocking secretion from oligodendrocyte-lineage cells would impair oligodendrocyte development and myelination and, in turn, that (2) adding oligodendrocyte-lineage cell-secreted molecules might promote oligodendrocyte development. Membrane fusion relies on soluble N-ethylmaleimide-sensitive fusion protein attachment protein receptors (SNARE) family proteins located on vesicles (v-SNAREs) and target membranes (t-SNAREs). The binding of v-SNAREs and t-SNARES form intertwined α-helical bundles that generate force for membrane fusion (Pobbati et al., 2006). VAMP1/2/3 are v-SNAREs that drive the fusion of vesicles with the plasma membrane to mediate exocytosis (Chen and Scheller, 2001). We found that oligodendrocyte-lineage cells express high levels of VAMP2 and VAMP3 and low levels of VAMP1 in vivo (Figure 1A–C; Zhang et al., 2014; Zhang et al., 2016), consistent with previous reports in vitro (Feldmann et al., 2009; Feldmann et al., 2011; Madison et al., 1999). We found that VAMP2+ and VAMP3+ puncta are distributed throughout cultured OPCs, including.
In the soma and processes (Figure 1—figure supplement 1). Botulinum toxin B specifically cleaves VAMP1/2/3 (Yamamoto et al., 2012), but not VAMP4, 5, 7, or 8 (Yamamoto et al., 2012), and inhibits the release of vesicles containing proteins (Somm et al., 2012) as well as small molecules such as neural transmitters (Poulain et al., 1988). Of note, botulinum toxin B does not cleave VAMP proteins that are involved in the vesicular transport between the trans-Golgi network, endosomes, and lysosomes (Antonin et al., 2000; Hoai et al., 2007; Pols et al., 2013). Similarly, botulinum toxins do not affect ion channel- or membrane transporter-mediated release of small molecules.
To block VAMP1/2/3-dependent exocytosis from oligodendrocyte-lineage cells, we crossed Pdgfra-CreER transgenic mice, which express Cre recombinase in OPCs (PDGFRα+Olig2+; Kang et al., 2010), with loxP-stop-loxP-botulinum toxin B light chain-IRES-green fluorescent protein (GFP) (inducible botulinum toxin B, or ibot) transgenic mice (Slezak et al., 2012), allowing expression of botulinum toxin B-light chain in OPCs and their progeny. The light chain contains the catalytically active domain of the toxin but lacks the heavy chain, which allows cell entry (Montal, 2010), thus confining toxin expression to the targeted cell type. Therefore, the ibot transgenic mice allow for the inhibition of VAMP1/2/3-dependent exocytosis in a cell-type-specific and temporally controlled manner (Slezak et al., 2012).
In our study, we used double-transgenic mice hemizygous for both Cre and ibot and referred to them as the PD:ibot mice thereafter. To validate our model and test its recombination efficiency, we injected 0.1 mg of 4-hydroxytamoxifen in each PD:ibot mouse daily for 2 days between postnatal days 2–4 (P2-4) and examined GFP expression at P8 and P30. We assessed whether GFP expression is restricted to oligodendrocyte-lineage cells (specificity) and what proportion of oligodendrocyte-lineage cells express GFP (efficiency/coverage). At P8, when the vast majority of oligodendrocyte-lineage cells are undifferentiated OPCs, we detected specific expression of GFP in oligodendrocyte-lineage cells (Figure 1D–F). GFP was efficiently expressed by oligodendrocyte-lineage cells throughout the brain, including the cerebral cortex grey matter, corpus callosum, and striatum (Figure 1G). At P30, when substantial numbers of OPCs have differentiated (PDGFRα–Olig2+), we observed a similarly high specificity of GFP expression in oligodendrocyte-lineage cells (Figure 1—figure supplement 2). These observations are consistent with previous reports on the specificity and efficiency of the Pdgfra-CreER transgenic line (Kang et al., 2010). As controls, we used wildtype mice, mice with only the Cre transgene or only the ibot transgene subjected to the same tamoxifen injection scheme. In all control conditions, we detected very little GFP expression (Figure 1—figure supplement 3).
To directly assess the expression of botulinum toxin B-light chain and the cleavage of the VAMP proteins in oligodendrocyte-lineage cells from PD:ibot mice, we purified OPCs from PD:ibot and control mice by immunopanning and allowed them to differentiate into oligodendrocytes in culture. We performed western blot analysis of the cultures and detected botulinum toxin B-light chain in PD:ibot but not in control cells (Figure 1H). Furthermore, the levels of full-length VAMP2 and VAMP3 proteins were lower in PD:ibot cells compared with control cells (Figure 1I, J, L and M). Based on these observations, we conclude that the botulinum toxin-GFP transgene is specifically and efficiently expressed by oligodendrocyte-lineage cells in PD:ibot mice.
Blocking VAMP1/2/3-dependent exocytosis from oligodendrocyte-lineage cells impairs oligodendrocyte development, myelination, and motor behavior
In PD:ibot mice, we found that the numbers of differentiated oligodendrocytes (CC1+) were reduced in the cerebral cortex at P11, 15, and 30 (Figure 2A and B), whereas the numbers of OPCs (PDGFRα+) did not change (Figure 2C, E, F and H). Olig2 labels both OPCs and differentiated oligodendrocytes, and the densities of Olig2+ cells in PD:ibot mice were also reduced, likely due to the reduction in differentiated oligodendrocytes (Figure 2C, D, F and G). At P8, the vast majority of PDGFRα-CreER-expressing cells are OPCs (Paukert et al., 2014). Therefore, it is more likely that blocking exocytosis from OPCs rather than oligodendrocytes affects oligodendrocyte development during the early postnatal period.
To determine the stage(s) of oligodendrocyte development affected by botulinum toxin, we performed RNAscope in situ hybridization in vivo using probes for Enpp6, a marker for pre-myelinating oligodendrocytes, and Mbp, a marker for oligodendrocytes. Interestingly, both markers showed a significant reduction in PD:ibot mice compared with controls, demonstrating that the oligodendrocyte development defect in PD:ibot mice manifest as early as the pre-myelinating stage (Figure 2I, J and K).
We next examined myelin development in PD:ibot mice and found that immunofluorescence of myelin basic protein (MBP), one of the main components of CNS myelin, is reduced in PD:ibot mice (Figure 3A–H). Moreover, many MBP+ ibot-GFP–expressing cells exhibit round cell morphology whereas MBP+GFP– control cells form elongated myelin internodes along axon tracks (Figure 3I). Transmission electron microscopy allows for the assessment of myelin structure at a high resolution. Thus, to further examine myelination in PD:ibot mice, we performed transmission electron microscopy imaging and found a reduction in the percentage of myelinated axons (Fig. 3 J, L) and reduced myelin thickness in PD:ibot mice (g-ratio: axon diameter divided by the diameter of axon +myelin; Figure 3K and M). The density and diameter of axons did not differ between PD:ibot and control mice (Figure 3N and O).
To determine whether the reduction of oligodendrocytes in PD:ibot mice is caused by cell death, we performed immunostaining with an antibody against cleaved caspase-3, which labels apoptotic cells. We observed no difference in the total apoptotic cells (cleaved caspase-3+), apoptotic OPCs (cleaved caspase-3+PDGFRα+), apoptotic oligodendrocyte-lineage cells (cleaved caspase-3+Olig2+), or apoptotic cells from other lineages (cleaved caspase-3+Olig2–) between PD:ibot and control mice in the cerebral cortex at P8, 15 and 30 (Figure 3—figure supplement 1).
To determine whether botulinum toxin-B-expressing cells contribute to the population of surviving differentiated oligodendrocytes, we examined the overlap between GFP+ botulinum-expressing cells and differentiated oligodendrocytes (Olig2+PDGFRα– cells and CC1+ cells) and found that botulinum-expressing cells can survive and become differentiated oligodendrocytes at P8 and P30 (Figure 3—figure supplement 2A). We next compared the ratio of CC1+ cells to Olig2+ cells in GFP+ cells in PD:ibot mice, GFP– cells in PD:ibot mice, and all cells (GFP–) in control cells and did not observe statistically significant difference between any of the groups (Figure 3—figure supplement 2B). To examine whether OPC proliferation is affected in PD:ibot mice, we quantified the percentage of Ki-67+ proliferating cells among PDGFRα+ OPCs and found an increase in OPC proliferation (Figure 3—figure supplement 3).
To investigate whether the expression of botulinum toxin B-light chain affects oligodendrocyte development and myelination in non-cell-type-specific manners, we blocked exocytosis from astrocytes or endothelial cells by crossing ibot transgenic mouse with Gfap-Cre (line 77.6) and Tek-Cre strains, respectively. We found astrocyte- and endothelial cell-specific expression of ibot-GFP in these mice but did not detect any obvious changes in oligodendrocyte density or myelin proteins. These observations suggest that botulinum toxin B-light chain peptides have specific effects on the targeted cell types. However, we could not rule out the possibility that embryonic and/or early postnatal compensation may mask the effect when a constitutive Cre is used.
To examine the functional consequences of blocking VAMP1/2/3-dependent exocytosis from oligodendrocyte-lineage cells, we assessed the motor behavior of PD:ibot and littermate control mice using the rotarod test. We placed mice on a gradually accelerating rotarod and recorded the time each mouse stayed on the rotarod. We found that PD:ibot mice stayed on the rotarod for significantly shorter amounts of time than littermate control mice on all 3 days of testing (Figure 3P-S). Therefore, blocking VAMP1/2/3-dependent exocytosis from oligodendrocyte-lineage cells led to deficits in neural circuit function.
PD:ibot mice exhibit changes in the transcriptomes of OPCs and oligodendrocytes
We next aimed to uncover the molecular changes in OPCs and oligodendrocytes in PD:ibot mice. We performed immunopanning to purify OPCs and oligodendrocytes from the brains of P17 PD:ibot and littermate control mice and performed RNA-sequencing (RNA-seq). We detected broad and robust gene expression changes in oligodendrocytes, and moderate changes in OPCs (Figure 4A and B, Supplementary files 1 and 2), demonstrating that VAMP1/2/3-dependent exocytosis from oligodendrocyte-lineage cells is critical for establishing and/or maintaining the normal molecular attributes of oligodendrocytes and OPCs. Notably, the expression of signature genes of differentiated oligodendrocytes such as Plp1, Mbp, Aspa, and Mobp was significantly reduced in oligodendrocytes purified from PD:ibot mice compared with controls (Figure 4E–H, Supplementary files 1 and 2). This result was not secondary to a reduction in oligodendrocyte density, as we loaded a similar amount of cDNA libraries from PD:ibot and control oligodendrocytes for sequencing, and processed all sequencing data with the same pipeline. Therefore, VAMP1/2/3-dependent exocytosis from oligodendrocyte-lineage cells is critical for the expression of mature oligodendrocyte genes. The expression of genes encoding oligodendrocyte-lineage cell marker proteins used for immunohistochemistry, Qk (encoding the Qk protein recognized by the CC1 antibody), Olig2, and PDGFRα did not change in PD:ibot mice (Figure 4—figure supplement 1). Therefore, our oligodendrocyte count is not confounded by changes in marker gene expression. We next performed gene ontology (GO) analysis to reveal the molecular pathways and cellular processes altered in each type of glial cell in PD:ibot mice (Figure 4I–L, Supplementary file 3). Genes associated with filopodium assembly, calcium ion transport, and plasma membrane raft assembly pathways were increased, whereas genes associated with lipid biosynthetic process, axon ensheathment, and myelination pathways were reduced in oligodendrocytes in PD:ibot mice. Genes associated with the trans-synaptic signaling, chemical synaptic transmission, and GPCR signaling pathways were increased in OPCs in PD:ibot mice. To assess whether oligodendrocyte-lineage cell exocytosis affects other glial cell types, such as astrocytes and microglia, we also purified these cells by immunopanning and performed RNA-seq. We observed moderate changes in astrocytes and microglia (Figure 4C and D). For example, genes associated with phagocytosis, such as Cd68 and C1qc, were increased in microglia from PD:ibot mice (Supplementary file 2), suggesting the importance of oligodendrocyte exocytosis in oligodendrocyte-microglial interactions.
Blocking VAMP1/2/3-dependent exocytosis from oligodendrocyte-lineage cells impairs oligodendrocyte development in vitro
VAMP1/2/3-dependent exocytosis from oligodendrocyte-lineage cells may directly affect oligodendrocyte development or change the attributes of other cell types, and, in turn, indirectly affect oligodendrocytes. For example, OPC-secreted molecules may affect axonal growth, and subsequently, axonal signals may affect oligodendrocytes indirectly. Therefore, we next employed purified OPC and oligodendrocyte cultures to determine whether exocytosis has direct roles in oligodendrocyte-lineage cells in the absence of other cell types.
We performed immunopanning to purify OPCs from P7 PD:ibot and control mice injected with 4-hydroxytamoxifen as described above. We cultured the OPCs for two days in the proliferation medium and then switched to the differentiation medium and cultured them for another seven days. To assess oligodendrocyte differentiation and maturation, we assessed the levels of MBP protein, a marker for differentiated oligodendrocytes, and detected lower MBP levels in PD:ibot cells compared with controls (Figure 5B and C). Similarly, Mbp mRNA levels were also lower as determined by quantitative real-time PCR (Figure 5D).
Additionally, we assessed the morphological maturation of oligodendrocytes in vitro (Figure 5E–H). OPCs are initially bipolar, and as they differentiate, they grow a few branches to become star-like. The cells next grow more branches to become arborized and then extend myelin-sheath-like flat membranous structures, acquiring a ‘lamellar’ morphology (Figure 5A; Zuchero et al., 2015) (also referred to as a ‘fried egg’ or ‘pancake’ morphology). We used the CellMask dye to analyze the morphological maturation of oligodendrocytes. At day 3 of differentiation, we found that a larger proportion of PD:ibot cells than control cells are at the early ‘star’, stage whereas a smaller proportion of PD:ibot cells than control cells have proceeded to the late ‘lamellar’ stage (Figure 5F). At day 7 of differentiation, more PD:ibot cells have proceeded from the ‘star’ to the ‘arborized’ stage compared with day 3, but the percentage of cells that have proceeded to the late ‘lamellar’ stage remains lower in PD:ibot than in control cultures (Figure 5E and G). We next quantified the size of lamellar cells, which have large sheaths of myelin-like membrane. Interestingly, we found that lamellar cells from PD:ibot mice are significantly smaller than those from control mice (Figure 5E and H). We also used a membrane-staining version of the CellMask dye at day 7 of differentiation and found similar results (Figure 5—figure supplement 1). Together, these observations suggest that VAMP1/2/3-dependent exocytosis is required for the morphological maturation of oligodendrocytes and that exocytosis has a direct effect on cells within the oligodendrocyte lineage to promote their development.
Cell non-autonomous effect of botulinum toxin-B in oligodendrocyte development in vitro
To examine whether cell non-autonomous effect contributes to the oligodendrocyte development defect associated with botulinum toxin-B expression, we compared the growth of wild-type cells in cultures containing vs. not containing botulinum-expressing cells. We took advantage of the fact that all OPCs purified from PD:ibot mice were not botulinum-GFP-expressing (efficiency ~65%, Figure 6A and B). 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 the sizes of lamellar cells in control cells from PD:ibot cultures were smaller than in control cells from control cultures (Figure 6C and D). Although both groups of cells were competent in exocytosis, they were surrounded by exocytosis-deficient vs. exocytosis-competent neighbor cells. The difference in the growth capacity of control cells in the presence of different neighbor cells reveals cell non-autonomous contributions of botulinum-expressing cells in oligodendrocyte development.
Oligodendrocyte-lineage cell-secreted molecules partially restore oligodendrocyte morphological maturation in secretion-deficient cells
Cell non-autonomous effects on oligodendrocyte development may be mediated by contact-dependent mechanisms or secreted molecules. To distinguish between these possibilities, we next assessed whether adding oligodendrocyte-lineage cell-secreted molecules could restore differentiation in VAMP1/2/3-dependent exocytosis-deficient OPCs. We prepared co-cultures of OPCs separated by inserts with 1 μm-diameter pores to allow for the diffusion of secreted molecules (Figure 6E). We plated PD:ibot and control cells on inserts and on the bottom of culture wells in four combinations: (1) control-inserts-control-wells; (2) PD:ibot-inserts-control-wells; (3) PD:ibot-inserts-PD:ibot-wells; and (4) control-inserts-PD:ibot-wells (Figure 6F) and examined oligodendrocyte morphological differentiation on the bottom of culture wells by quantifying lamellar cells as described above. We performed one-way ANOVA with a multiple comparison test comparing every group to every other group. Comparing group 3 vs. group 4, we found that adding secreted molecules from control cells on inserts partially rescued the size of lamellar cells of PD:ibot cells on the bottom of culture wells (Figure 6G and H). We used both a cytosol-staining version (Figure 6G) and a membrane-staining version (Figure 6—figure supplement 1) of the CellMask dye and found similar results. These observations lend further support to the hypothesis that oligodendrocyte-lineage cell-secreted molecules promote oligodendrocyte development.
Blocking L-PGDS leads to oligodendrocyte development defects in vitro
We next sought to uncover the identity of the secreted molecules that promote oligodendrocyte and myelin development. We mined our oligodendrocyte and OPC RNA-seq dataset to identify highly expressed genes encoding secreted proteins (Zhang et al., 2014; Zhang et al., 2016). After testing a few candidate genes using in vitro OPC cultures, we focused on Ptgds, one of the most abundant genes encoding a secreted protein expressed by oligodendrocyte-lineage cells in both humans and mice (Zhang et al., 2014; Zhang et al., 2016). Its expression increases during development (Kang et al., 2011) as oligodendrocyte development and myelination occur. Interestingly, Ptgds is important for Schwann cell myelination in the peripheral nervous system (Trimarco et al., 2014). Yet, its function in the development of the CNS is unknown. Ptgds encodes the L-PGDS protein, which converts prostaglandin H2 to prostaglandin D2 (PGD2) (Urade and Hayaishi, 2000). We performed western blot analyses to assess L-PGDS secretion by botulinum toxin B-expressing OPCs/oligodendrocytes in culture. We detected an increase in intracellular L- PGDS in OPC/oligodendrocyte cultures from PD:ibot mice compared with controls (Figure 7A). Secreted L-PGDS, however, is lower in PD:ibot compared with control cultures (Figure 7A–C), suggesting that botulinum toxin B inhibits L-PGDS secretion. L-PGDS secretion is not completely eliminated, most likely because not all cells in the culture express botulinum toxin (efficiency:~65%, Figure 6B) and wild-type cells may compensate by increasing secretion when extracellular L-PGDS levels are low.
To determine the role of L-PGDS in oligodendrocyte development and CNS myelination, we first assessed oligodendrocyte development in vitro in the presence of AT-56, a specific L-PGDS inhibitor (Irikura et al., 2009). We found that AT-56 inhibits wild-type oligodendrocyte development in a dose-dependent manner in vitro (Figure 7D and E), suggesting a requirement of L-PGDS in oligodendrocyte development, without affecting their survival (Figure 7—figure supplement 1). Using cytoplasm-staining and membrane-staining versions of CellMask dyes, we observed similar results (Figure 7—figure supplement 2).
L-PGDS synthesizes PGD2, whereas another enzyme, 15-hydroxyprostaglandin dehydrogenase (HPGD) inactivates PGD2 (Conner et al., 2001). To assess the involvement of PGD2 in oligodendrocyte development, we added HPGD to OPC cultures from control mice. Interestingly, we found that HPGD reduced the percentage of oligodendrocytes with mature lamellar morphology (Figure 7F and G), lending further support to the potential role of the L-PGDS/PGD2 pathway in oligodendrocyte development.
L-PGDS is required for oligodendrocyte development in vivo
Having discovered the role of L-PGDS in oligodendrocyte development in vitro, we next assessed whether L-PGDS regulates oligodendrocyte development in vivo. We examined oligodendrocytes in L-PGDS global knockout mice and found a significant decrease in CC1+ oligodendrocytes in the corpus callosum and cerebral cortex of L-PGDS-knockout mice at P9 (Figure 8A and B). The density of OPCs (PDGFRα+) and cleaved caspase-3 positive cells and the intensity of SMI-32, a marker for damaged axons, and Iba1, a marker for microglial reactivity, did not differ between L-PGDS-knockout and control mice (Figure 8C–K).
L-PGDS and PGD2 restore the development of secretion-deficient oligodendrocytes
Next, we determined whether L-PGDS is sufficient to rescue the maturation defect of PD:ibot cells in vitro. Indeed, the exogenous addition of recombinant L-PGDS protein partially rescued the percentage of cells with lamellar morphology from PD:ibot mice (Figure 9A and B), further supporting the role of L-PGDS in oligodendrocyte development. To assess the role of PGD2, the synthesis product of the L-PGDS enzyme, in oligodendrocyte development, we added PGD2 to cultures from PD:ibot mice and observed a partial rescue of the oligodendrocyte maturation defect of PD:ibot cells (Figure 9C and D). Although other cell types could mediate the effects of systemic L-PGDS knockout, the observation that L-PGDS and PGD2 rescue the morphological maturation of oligodendrocytes in purified cultures in vitro supports a direct role of PGD2 in oligodendrocyte-lineage cell development.
Overexpression of L-PGDS partially rescues the myelin defect in PD:ibot mice in vivo
To assess the effect of L-PGDS on myelin development in vivo, we bred the L-PGDS overexpressing transgenic mouse strain (Ptgds-TG, line B7) (Pinzar et al., 2000) with PD:ibot mice to obtain triple transgenic mice (Pdgfra-CreER; lox-stop-lox-botulinum toxin-B light chain-GFP, Ptgds-TG). Compared with PD:ibot mice, the triple transgenic mice exhibited enhanced myelination (Figure 9E–H), providing further evidence of the role of L-PGDS in oligodendrocyte and myelin development in vivo. Interestingly, we found that Ptgds is the most highly upregulated gene in OPCs and oligodendrocytes from PD:ibot mice compared with control mice based on RNA-seq (Figure 4A and B). Moreover, Hpgd, which inactivates PGD2, is the most robustly downregulated gene in microglia from PD:ibot mice compared with control mice (Figure 4D). These intriguing observations may reflect homeostatic mechanisms that maintain L-PGDS and PGD2 levels. When L-PGDS secretion is blocked by botulinum toxin, extracellular PGD2 levels decrease, which induces an increase in Ptgds mRNA to promote PGD2 production and a decrease in Hpgd mRNA to reduce PGD2 inactivation.
Given the enriched expression of L-PGDS by oligodendrocyte-lineage cells (Zhang et al., 2014; Zhang et al., 2016) and its localization in the extracellular space (Figure 7A and C; Hoffmann et al., 1993), our results indicate that L-PGDS is an oligodendrocyte-lineage cell-secreted autocrine/paracrine molecule that promotes oligodendrocyte development and myelination. Our results are consistent with the following model: OPCs secrete autocrine/paracrine signals such as L-PGDS to promote oligodendrocyte development and myelination. When VAMP1/2/3-dependent exocytosis is blocked, L-PGDS secretion is defective, leading to defective oligodendrocyte development and myelination. Overexpression of Ptgds (L-PGDS) in exocytosis-deficient PD:ibot mice restores L-PGDS levels and partially rescues myelination defect. Our discovery of the role of exocytosis and L-PGDS in oligodendrocytes provides insight into the mechanisms regulating oligodendrocyte development and myelination and reveals novel molecular targets for future efforts aimed toward enhancing myelination for neural repair.
In this study, we showed that oligodendrocyte-lineage cell-secreted molecules promote oligodendrocyte development and myelination in an autocrine/paracrine manner. We identified L-PGDS as one such secreted molecule, thus revealing a novel cellular mechanism regulating oligodendrocyte development.
Previously, the roles of VAMP3 and related pathways in myelin protein delivery and oligodendrocyte morphogenesis have been investigated largely in vitro using cultures of an OPC-like cell line (Oli-Neu cells) or primary oligodendrocytes. For example, VAMP3 and VAMP7 knockdown inhibits the transport of a myelin protein, Proteolipid Protein1 in vitro (Feldmann et al., 2011). Tetanus toxin, which cleaves VAMP1/2/3, inhibits oligodendrocyte branching in vitro (Sloane and Vartanian, 2007). Syntaxin4, a potential binding partner of VAMP3, is required for the transcription of MBP in oligodendrocytes in vitro (Bijlard et al., 2015). Our study established a requirement of VAMP1/2/3-dependent exocytosis in oligodendrocyte development, myelination, and motor behavior in vivo and identified L-PGDS as an oligodendrocyte-lineage cell-secreted protein that promotes oligodendrocyte development and myelination.
VAMP1/2/3-dependent exocytosis is not the only pathway employed by oligodendrocyte-lineage cells to release molecules that mediate cell-cell interactions. For example, oligodendrocytes release exosome-like vesicles that inhibit the growth of myelin-like membranes in vitro (Bakhti et al., 2011). Tetanus toxin cleaves VAMP1/2/3 but does not affect exosome release (Fader et al., 2009). Therefore, the role of VAMP1/2/3-dependent exocytosis in promoting myelination and the effect of exosome-like vesicles in inhibiting myelination are likely parallel pathways independent of each other. In future studies, it could be interesting to determine the signals that regulate VAMP1/2/3-dependent exocytosis and VAMP1/2/3-independent exosome release during development and disease in vivo and thus define how these two seemingly opposing effects are coordinated to shape precise and dynamic myelination.
We observed a decrease in the percentage of PDGFRa+ OPCs expressing botulinum toxin-B from P8 to P30 in PD:ibot mice (Figure 1G and Figure 1—figure supplement 2C). Since OPC proliferation is increased and the overall OPC densities do not change in PD:ibot mice, a decrease in the percentage of OPCs expressing the toxin is consistent with the death of some toxin-expressing OPCs. Although our cleaved caspase-3 immunohistochemistry results did not show a difference in OPC survival between PD:ibot and control cells (Figure 3—figure supplement 1), we cannot exclude the possibilities that OPC die through non-apoptotic mechanisms or that microglia clear dead cells too rapidly for accurate counting.
OPCs are present throughout the CNS in adults (Hughes et al., 2013; Kang et al., 2010), even in demyelinated lesions in patients with multiple sclerosis (Franklin, 2002). Therefore, inducing oligodendrocyte and new myelin formation is an attractive strategy for treating demyelinating diseases. However, remyelination therapy has not been successful so far (Franklin, 2002), underscoring the need for a more complete understanding of the mechanisms regulating oligodendrocyte and myelin development. Our discovery of the role of L-PGDS in oligodendrocyte development and myelination adds to the knowledge of the molecular regulation of myelination. Interestingly, mixed results have been reported on the level of the Ptgds gene and the L-PGDS protein in multiple sclerosis patients and mouse models (Jäkel et al., 2019; Kagitani-Shimono et al., 2006; Penkert et al., 2021). During remyelination in mice, PGD2 levels increase (Penkert et al., 2021). Future studies should clarify the involvement of L-PGDS in multiple sclerosis and its therapeutic potential in promoting remyelination.
A recent study shows that the gene encoding L-PGDS, Ptgds, marks a subpopulation of OPCs more resilient to spinal cord injury than other OPCs (Floriddia et al., 2020). Thus, the function of L-PGDS in OPC heterogeneity and their responses to injury and other neurological disorders will be interesting to explore in the future.
The product of the L-PGDS enzyme, PGD2, binds and activates two G-protein-coupled receptors, DP1 and DP2 (Gpr44) (Narumiya and Furuyashiki, 2011). In addition, PGD2 undergoes non-enzymatic conversion to 15d-PGJ2, which activates the peroxisome proliferator-activated receptor-γ (Scher and Pillinger, 2005). Future studies should aim to identify the receptor(s) that mediates the effect of L-PGDS on oligodendrocyte development and myelination, as well as the downstream signaling pathways.
OPCs and oligodendrocytes express numerous genes encoding secreted proteins (Zhang et al., 2014). Although we identified the role of L-PGDS in oligodendrocyte development, our results do not rule out contributions from other secreted molecules. Our RNA-seq dataset provides a roadmap for future investigation of the roles of additional oligodendrocyte-lineage cell-secreted molecules in the brain.
Blocking exocytosis with botulinum toxin B may reduce the delivery of proteins and lipids to the plasma membrane, therefore causing cell-autonomous effects on oligodendrocyte development in addition to blocking secretion. Both cell-autonomous and cell-non-autonomous mechanisms may be involved in the effect of blocking exocytosis on oligodendrocyte development (Fekete et al., 2022; Lam et al., 2022). Our transwell rescue experiment and the comparisons between control cells in PD:ibot culture and control cells in control culture (Figure 6) support the importance of secreted molecules but do not rule out cell-autonomous mechanisms.
Two recent publications from the Zuchero and Nishiyama groups also utilized the ibot mice to investigate the role of VAMP2/3-mediated exocytosis in oligodendrocyte development and myelination (Fekete et al., 2022; Lam et al., 2022). Together with data presented here, the three complementary studies utilized different Cre lines to express botulinum toxin-B in oligodendrocyte lineage cells (NG2-Cre in Fekete et al., CNP-Cre in Lam et al., and PDGFRa-CreER in this study). Interestingly, all three ibot mouse strains exhibit myelination defects in the central nervous system (the spinal cord in Fekete et al., the brain in this study, and the brain and the spinal cord in Lam et al.). These consistent results from three mouse strains obtained by independent groups demonstrate an important role of VAMP2/3-mediated exocytosis in myelin development. VAMP2/3-mediated exocytosis may affect membrane expansion, secretion, and signaling. To provide mechanistic insight into the function of VAMP2/3 in myelin development, these three studies focused on different aspects of VAMP2/3 function. In NG2:ibot mice, Fekete et al. observed elevated levels of Fyn kinase, which is a signaling molecule implicated in regulating oligodendrocyte maturation and myelination. Using CNP:ibot mice, Lam et al. demonstrated the role of VAMP2/3-dependent exocytosis in membrane expansion and insertion of plasma membrane proteins during oligodendrocyte maturation, and further performed elegant imaging studies to capture exocytosis events of VAMP2/3-containing vesicles in oligodendrocytes in vitro and in vivo. In this study, we demonstrated the cell-non-autonomous function of VAMP2/3-dependent exocytosis in oligodendrocyte development and identified a novel role of a secreted molecule, L-PGDS, in oligodendrocyte development. Because VAMP2/3-mediated exocytosis may contribute to the membrane insertion and secretion of a variety of molecules, which may in turn activate multiple intracellular signaling pathways, it is not surprising that multiple mechanisms mediate the effect of VAMP2/3 in oligodendrocyte development and myelination. These three complementary studies provide key insight toward a comprehensive understanding of the role of VAMP2/3 in myelination.
Although all three ibot mouse strains exhibit myelin defects, other phenotypes differ among the strains. For example, the ibot mice in Fekete et al. and this study have reduced oligodendrocyte densities, whereas Lam et al. did not observe an overt loss of oligodendrocytes. We used the CC1 antibody to detect differentiated oligodendrocytes and Fekete et al. used CC1 (i.e. QKI7) and several additional markers to validate a reduction in mature oligodendrocytes (ASPA, GST-p, Nkx2.2). Differences in the timing of botulinum toxin-B expression driven by the Cre lines may account for the divergent results. Fekete et al. used NG2-Cre and this study used PDGFRa-CreER, both Cre lines drive toxin expression in OPCs, whereas Lam et al. used CNP-Cre that predominantly drives toxin expression in oligodendrocytes. The intriguing divergent phenotypes suggest the possibility of a stage-dependent requirement of VAMP2/3-mediated exocytosis in the development of oligodendrocyte-lineage cells, which may be investigated in the future to clarify the complex roles of VAMP2/3 in oligodendrocyte development and myelination. Together, the three complementary studies revealed the importance of VAMP2/3-mediated exocytosis in myelination through a combination of cell-autonomous and cell-non-autonomous mechanisms.
Materials and methods
Lead contact and materials availability
Further information and requests for resources and reagents should be directed to and will be fulfilled by the Lead Contact, Ye Zhang (firstname.lastname@example.org). This study did not generate new unique reagents.
All animal experimental procedures (protocols: #R-16–079 and #R-16–080) were approved by the Chancellor’s Animal Research Committee at the University of California, Los Angeles, and conducted in compliance with national and state laws and policies. All the mice were group-housed in standard cages (maximum 5 mice per cage). Rooms were maintained on a 12 hr light/dark cycle. Pdgfra-CreER (Jax #018280), ibot (Jax #018056), Gfap-Cre (Jax #024098), and Tek-Cre (Jax, #004128) mouse strains were obtained from Jackson Laboratories. L-PGDS global knockout (Ptgds–/–) mouse strain was originally from Urade, cryopreserved by Garret FitzGerald (Urade and Hayaishi, 2000). L-PGDS overexpressing transgenic mouse strain (Ptgds-TG, line B7) was originally from Urade, cryopreserved by JCRB Laboratory Animal Resource Bank (Pinzar et al., 2000). All PD:ibot and control mice, including wildtype, Cre-only and ibot-only mice used were congenic. We used PD:ibot and littermate control mice raised under the same maternal care in the same cage to minimize environmental confounding factors.
OPC purification and culture
Whole brains excluding the olfactory bulbs and the cerebellum from one pup at postnatal day 7 to day 8 were used to make each batch of OPC culture. OPCs were purified using an immunopanning method described before (Emery and Dugas, 2013). Briefly, the brains were digested into single-cell suspensions using papain. Microglia and differentiated oligodendrocytes were depleted using anti-CD45 antibody- (BD Pharmingen, cat #550539) and GalC hybridoma-coated panning plates, respectively. OPCs were then collected using an O4 hybridoma-coated panning plate. For most culture experiments, cells were plated on 24-well plates at a density of 30,000 per well. For comparison of OPC differentiation at different densities, OPCs were plated at densities of 5000 per well and 40,000 per well. For all experiments, OPCs were first kept in proliferation medium containing growth factors PDGF (10 ng/ml, Peprotech, cat #100–13 A), CNTF (10 ng/ml, Peprotech, cat #450–13), and NT-3 (1 ng/ml, Peprotech, cat #450–03) for 2–3 days, and then switched to differentiation medium containing thyroid hormone (40 ng/ml, Sigma, cat #T6397-100MG) but without PDGF or NT-3 for seven days to differentiate them into oligodendrocytes as previously described (Emery and Dugas, 2013). Half of the culture media was replaced with fresh media every other day. All the cells were maintained in a humidified 37°C incubator with 10% CO2. Cells from both female and male mice were used. For coculture experiments with inserts, OPCs were purified from PD:ibot and littermate control mice as described above. A total of 100,000 cells per well were plated on inserts with 1 μm diameter pores (VWR, cat #62406–173), and the inserts were placed on top of wells with cells plated at 30,000 cells per well density on 24-well culture plates. 200 μl medium was added per insert and 500 μl medium was added per well under the inserts.
Drugs and treatment
4-Hydroxy-tamoxifen stock solutions were made by dissolving 4-hydroxy-tamoxifen (Sigma, H7904) into pure ethanol at 10 mg/ml. The stock solutions were stored at –80°C until use. On the day of injection, an aliquot of 4-hydroxy-tamoxifen stock solution (100 μl) was thawed and mixed with 500 μl sunflower oil by vortexing for 5 min. Ethanol in the solution was vacuum evaporated in a desiccator (VWR, 89054–050) for an hour. 0.1 mg 4-hydroxy-tamoxifen was injected into each mouse subcutaneously daily for 2 days at P2 and P4. An L-PGDS inhibitor, AT-56 (Cayman Chemicals, cat #13160), and prostaglandin D2 (Cayman Chemicals, cat #12010) were dissolved in dimethyl sulfoxide (DMSO). To inhibit L-PGDS activity in vitro, AT-56 was added to the oligodendrocyte culture medium at 1 µM and 5 µM every other day. HPGD protein (R&D system, cat#5660-DH-010) was added to the oligodendrocyte culture medium at 3 µM and 6 µM daily. L-PGDS protein (Cayman Chemicals, cat#10006788) was added to the oligodendrocyte culture at 20 µM daily. For prostaglandin D2 treatment, prostaglandin D2 was added to the oligodendrocyte culture medium at 1 µM and 2 µM every 12 hr. An equal amount of DMSO was added to the control wells. Because a metabolite of prostaglandin D2, 15-d-prostaglandin J2, induces cell death, which can be prevented by N-acetyl cysteine (Lee et al., 2008), we included 1 mM N-acetyl cysteine, which is shown to improve cell survival, in the culture media of prostaglandin D2-treated and control cells.
Purification of microglia, oligodendrocytes, oligodendrocyte precursor cells, and astrocytes
Whole brains excluding the olfactory bulbs and the cerebellum from P17 PD:ibot and control littermates were used for the purification of microglia, oligodendrocytes, oligodendrocyte precursor cells, and astrocytes. A single-cell suspension was prepared as described above. Cells were incubated for 30 min on an anti-CD45 antibody (BD Pharmingen, cat#550539, 1.25 µg/ml)-coated panning plate to harvest microglia, followed by two sequential CD45-coated panning plates to deplete remaining microglia. Cell suspension was then incubated for 30 min on a GalC hybridoma-coated panning plate to collect differentiated oligodendrocytes, followed by two more GalC hybridoma-coated plates to deplete any remaining differentiated oligodendrocytes. The remaining cells were incubated for 30 min on an O4 hybridoma-coated panning plate to collect oligodendrocyte precursor cells, followed by two O4 hybridoma-coated panning plates to deplete remaining OPCs. The cell suspension was then incubated with an anti-HepaCAM antibody (R&D Systems, cat# MAB4108)-coated panning plate to collect astrocytes.
Total RNA was extracted using the miRNeasy Mini kit (Qiagen cat #217004). The concentrations and integrities of the RNA were measured using TapeStation (Agilent) and Qubit. Sixty ng total RNA from each sample was used for library preparation. cDNA was generated using the Nugen Ovation V2 kit (Nugen) and fragmented using the Covaris sonicator. Sequencing libraries were prepared using the Next Ultra RNA Library Prep kit (New England Biolabs) with 12 cycles of PCR amplification. An Illumina HiSeq 4000 sequencer was used to sequence these libraries and each sample had an average of 19.1 ± 2.9 million 50 bp single-end reads.
RNA-seq data analysis
STAR package was used to map reads to mouse genome mm10 and HTSEQ was used to obtain raw counts from sequencing reads. EdgeR-Limma-Voom packages in R were used to calculate Reads per Kilobase per Million Mapped Reads (RPKM) values from raw counts. DESeq2 package was used to analyze differential gene expression.
Immunohistochemistry and immunocytochemistry
Mice were anesthetized with isoflurane and transcardially perfused with phosphate-buffered saline (PBS) followed by 4% paraformaldehyde (PFA). Brains were removed and post-fixed in 4% PFA at 4 °C overnight. Brains were washed with PBS and cryoprotected in 30% sucrose at 4 °C for 2 days before embedding in optimal cutting temperature compound (Fisher, cat #23-730-571) and stored at –80 °C. Brains were sectioned using a cryostat (Leica) into 30-μm-thick sections and floating sections were blocked and permeabilized in 5% donkey serum with 0.3% Tween-20 in PBS and then stained with primary antibodies against GFP (Aves Labs, Inc, cat #GFP-1020, dilution 1:500), PDGFRα (R&D Systems, cat #AF1062, dilution 1:500), Olig2 (Millipore, cat #211F1.1, dilution 1:500), CC1 (Millipore, cat #OP80, dilution 1:500), MBP (Abcam, cat #ab7349, dilution 1:500), Iba1 (FUJIFILM Wako, cat# 019–19741, dilution 1:1000), SMI-32 (BioLegend, cat# 801702, dilution 1:500), and cleaved caspase-3 (Cell Signaling, cat #9661 S, dilution 1:500) at 4 °C overnight. Heat-induced epitope retrieval is performed for sections stained with SMI-32. All other staining did not require antigen retrieval. Sections were washed three times with PBS and incubated with fluorescent secondary antibodies (Invitrogen) at 4 °C overnight. Sections were mounted onto Superfrost Plus micro slides (Fisher, cat #12-550-15) and covered with mounting medium (Fisher, cat #H1400NB) and glass coverslips. Slides were imaged with a Zeiss Apotome epifluorescence microscope. Adjacent regions are tiled using Zeiss tiling function. Tiling borders in some representative images are caused by differing background intensities where different tiles are stitched together and are removed using ‘Substract Background’ function in Fiji with the setting (rolling ball radius = 50 pixels).
A large area of cerebral cortex, including motor cortex and somatosensory cortex, from bregma 1 mm to –2 mm was used for the following quantification: MBP coverage in the cortex, the density of oligodendrocyte-lineage marker genes (CC1, Olig2, PDGFRα, Enpp6, and Mbp), and the density of apoptotic cells (cleaved caspase-3+).
For immunocytochemistry of cultured cells, cells were fixed with 4% PFA and 0.3% Tween-20 in PBS. After blocking in 5% donkey serum, cells were then stained with the primary antibodies described above, VAMP2 (Synaptic Systems, cat #104 211, dilution 1:100), and VAMP3 (Synaptic Systems, cat #104 103, dilution 1:500) at 4 °C overnight. After three washes in PBS, cells were stained with secondary antibodies and the cytoplasm-staining CellMask Blue (Invitrogen, cat #H32720, 1:1,000) at 4 °C overnight. To stain the cells with the membrane-staining CellMask (Invitrogen, cat#C37608 Dilution 1:1000), we added the dye into the culture medium and incubate it with the cells for 13 min at 37 °C. After staining, cells were washed once with PBS and fixed with 4% PFA for 15 min at room temperature. Cells were washed three times with PBS before covered with mounting medium (Fisher, cat #H1400NB). Slides were imaged with a Zeiss Apotome epifluorescence microscope.
Fluorescence microscopy images were cropped, and brightness contrast was adjusted with identical settings across genotype, treatment, and control groups using Photoshop and ImageJ. All the images were randomly renamed using the following website (https://www.random.org/) and quantified with the experimenter blinded to the genotype and treatment condition of the samples. Cells with MBP+ membrane spreading out were identified as lamellar cells. Illustrations were made with Biorender.
RNAscope in situ hybridization
P15 mice were transcardially perfused with PBS followed by 4% PFA. Brains were removed and postfixed in 4% PFA for an additional 2 hr at room temperature and then overnight at 4 °C. Tissues were then dehydrated in 30% sucrose, embedded in OCT compound, and cut into 20- to 30-μm-thick sections. RNAscope Multiplex Fluorescent Reagent Kit v2 (ACDBio, cat# 323100) was used per manufacturer’s protocol. Probes used in this paper were purchased from ACDBio for mouse Enpp6 (cat#511021-C2) and Mbp (cat# 451491). Slides were imaged with a Zeiss Apotome epifluorescence microscope at equal power and exposure across all samples stained with the same set of probes.
Transmission electron microscopy
Brain specimens for transmission electron microscopy were prepared as described before (Salazar et al., 2018). Mice were anesthetized using isoflurane and transcardially perfused with 0.1 M phosphate buffer (PB) followed by 4% PFA with 2.5% glutaraldehyde in 0.1 M PB buffer. Brains were removed and post-fixed in 4% PFA with 2.5% glutaraldehyde in 0.1 M PB for another two days. Brains were sliced with Young Mouse Brain Slicer Matrix (Zivic Instruments, cat #BSMYS001-1) and a small piece of the corpus callosum was isolated from brain sections at 0–1 mm anterior to Bregma. After washing, samples were then post-fixed in 1% osmium tetroxide in 0.1 M PB (pH 7.4) and dehydrated through a graded series of ethanol concentrations. After infiltration with Eponate 12 resin, samples were embedded in fresh Eponate 12 resin and polymerized at 60 °C for 48 hr. Ultrathin sections of 70 nm thickness were prepared and placed on formvar/carbon-coated copper grids and stained with uranyl acetate and lead citrate. Grids were examined using a JEOL 100 CX transmission electron microscope at 60 kV and images were captured by an AMT digital camera (Advanced Microscopy Techniques Corporation, model XR611) by the Electron Microscopy Core Facility, UCLA Brain Research Institute. Before analysis, TEM images were blinded as described above. An axon that is encircled by 2 or more compacted layers of electron-dense lines is defined as a myelinated axon. g-ratio was determined by dividing the mean diameter of the area inside myelin by the mean diameter of the same axon with myelin. Approximately 100 myelinated axons from each group were analyzed for g-ratio analysis. For measuring the percentage of myelinated axons, approximately 900 axons from three mice were used for each group. More than 1300 axons from four mice from each group were used to measure axon density and approximately 500 axons from four mice from each group were used for axon diameter measurement.
RNA extraction and qPCR
100 k Day 7 differentiated oligodendrocytes were lysed with the Qiazol lysis buffer and RNA was extracted using the PureLink RNA mini kit (Invitrogen 12183018 A) following the manufacturer’s protocol. cDNA was generated using the SuperScript III First-Strand Synthesis SuperMix (Invitrogen 18080400). The primer for Mbp was designed using the primerblast tool from NCBI. Primerblast was used to validate the specificity of primers. The sequences for Mbp and Gapdh primers were provided in the key resource table. PowerUp SYBR Green Master Mix (Applied Biosystems A25742) and a QuantStudio 3 Real-Time PCR System (Thermo fisher, cat# A28567) were used for qRT-PCR reaction.
We purified OPCs from PD:ibot and control mice by immunopanning and cultured them in proliferation medium for 2–3 days and differentiation medium for 7 days as described above. To collect secreted samples, culture media were mixed with ethylenediaminetetraacetic acid (EDTA)-free protease inhibitor cocktail (Sigma, cat #4693159001) at a 6:1 ratio and centrifuged at 1000×g for 10 min to remove dead cells and debris. To collect whole-cell lysates, cells were washed with PBS, lysed with radioimmunoprecipitation assay buffer containing EDTA-free protease inhibitor cocktail, and centrifuged at 12,000×g for 10 min to remove cell debris.
All samples were mixed with sodium dodecyl sulfate (SDS) sample buffer (Fisher, cat # AAJ60660AC) and 2-mercaptoethanol before boiling for 5 min. Samples were separated by SDS-polyacrylamide gel electrophoresis, followed by transferring to polyvinylidene difluoride membranes via wet transfer at 300 mA for 1.5 hr. Membranes were blocked with clear milk blocking buffer (Fisher, cat #PI37587) for 1 hr at room temperature and incubated with primary antibodies against L-PGDS (Santa Cruz Biotechnology, cat #sc-390717, dilution 1:1000), GAPDH (Sigma, cat #CB1001, dilution 1:5000), BoNT-B Light Chain (R&D Systems, cat #AF5420-SP, dilution 1:1000), VAMP2 (Synaptic Systems, cat #104 211, dilution 1:1000), VAMP3 (Novus Biological, cat # NB300-510-0.025mg, dilution 1:1000) and MBP (Abcam, cat #ab7349, dilution 1:1000) at 4 °C overnight. Membranes were washed with tris-buffered saline with Tween 20 (TBST) three times and incubated with horseradish peroxidase-conjugated secondary antibodies (Mouse, Cell Signaling, cat #7076 S; Rabbit, Cell Signaling, cat #7074 S; Rat, Cell Signaling, cat #7077 S; Sheep, Thermo Fisher, cat #A16041) or Donkey anti-Mouse IgG (H+L) Highly Cross-Adsorbed Secondary Antibody, Alexa Fluor Plus 647 (Fisher, cat # PIA32787,1:1000) (only for GAPDH in Figure 5B) for 1 hr at room temperature. After three washes in TBST buffer, SuperSignal West Femto Maximum Sensitivity Substrate (Fisher, cat #PI34095) was added to the membranes, and the signal was visualized using a ChemiDoc MP Imaging system (BIO-RAD).
Mice were familiarized with being picked up and handled by the experimenter daily for three days before the test to reduce stress. Mice were also habituated to the rotarod testing room for 15 min prior to all testing. Both male and female adult mice (2–5 months old) were used in the rotarod test. Mice were given three trials per day for three consecutive days (5–60 rpm over 5 min, with approximately 30 min between successive trials). The latency to fall was measured and the experimenter was blinded to the genotype of the mice during the test.
Quantification and statistical analysis
The numbers of animals and replicates are described in the figures and figure legends. The RNA-seq data were analyzed using the DESeq2 package. Adjusted p-values smaller than 0.05 were considered significant. For all non-RNA-seq data, analyses were conducted using Prism 8 software (Graphpad). The normality of data was tested by the Shapiro-Wilke test. For data with a normal distribution, Welch’s t-test was used for two-group comparisons and one-way ANOVA was used for multi-group comparisons. An estimate of variation in each group is indicated by the standard error of the mean (S.E.M.). * p<0.05, ** p<0.01, *** <0.001. An appropriate sample size was determined when the study was being designed based on published studies with similar approaches and focus as our study. A biological replicate is defined as one mouse. Different culture wells from the same mouse or different images taken from the brains or cell cultures from the same mouse are defined as technical replicates. All statistical tests were performed with each biological replicate/mouse as an independent observation. The number of times each experiment was performed is indicated in figure legends. No data were excluded from the analyses. Mice and cell cultures were randomly assigned to treatment and control groups. Imaging analyses and behavior tests were conducted when the experimenter was blinded to the genotypes or treatment conditions.
This study did not generate new codes.
We deposited all RNA-seq data to the Gene Expression Omnibus under accession number GSE168569.
NCBI Gene Expression OmnibusID GSE168569. Oligodendrocyte-lineage cell exocytosis and L-type prostaglandin D synthase promote oligodendrocyte development and myelination.
Inhibition of myelin membrane sheath formation by oligodendrocyte-derived exosome-like vesiclesThe Journal of Biological Chemistry 286:787–796.https://doi.org/10.1074/jbc.M110.190009
Oligodendrocyte development and plasticityCold Spring Harbor Perspectives in Biology 8:a020453.https://doi.org/10.1101/cshperspect.a020453
Control of oligodendroglial cell number by the mir-17-92 clusterDevelopment 137:2127–2132.https://doi.org/10.1242/dev.050633
Induction and blockage of oligodendrogenesis by differently activated microglia in an animal model of multiple sclerosisThe Journal of Clinical Investigation 116:905–915.https://doi.org/10.1172/JCI26836
Snare-Mediated membrane fusionNature Reviews. Molecular Cell Biology 2:98–106.https://doi.org/10.1038/35052017
STAR: ultrafast universal RNA-seq alignerBioinformatics 29:15–21.https://doi.org/10.1093/bioinformatics/bts635
Regulatory T cells promote myelin regeneration in the central nervous systemNature Neuroscience 20:674–680.https://doi.org/10.1038/nn.4528
Molecular control of oligodendrocyte developmentTrends in Neurosciences 42:263–277.https://doi.org/10.1016/j.tins.2019.01.002
Purification of oligodendrocyte lineage cells from mouse cortices by immunopanningCold Spring Harbor Protocols 2013:854–868.https://doi.org/10.1101/pdb.prot073973
Transcriptional and epigenetic regulation of oligodendrocyte development and myelination in the central nervous systemCold Spring Harbor Perspectives in Biology 7:a020461.https://doi.org/10.1101/cshperspect.a020461
Ti-vamp/vamp7 and VAMP3/cellubrevin: two v-SNARE proteins involved in specific steps of the autophagy/multivesicular body pathwaysBiochimica et Biophysica Acta - Molecular Cell Research 1793:1901–1916.https://doi.org/10.1016/j.bbamcr.2009.09.011
The Akt-mTOR pathway drives myelin sheath growth by regulating cap-dependent translationThe Journal of Neuroscience 41:8532–8544.https://doi.org/10.1523/JNEUROSCI.0783-21.2021
Comprehensive analysis of expression, subcellular localization, and cognate pairing of SNARE proteins in oligodendrocytesJournal of Neuroscience Research 87:1760–1772.https://doi.org/10.1002/jnr.22020
Transport of the major myelin proteolipid protein is directed by VAMP3 and VAMP7The Journal of Neuroscience 31:5659–5672.https://doi.org/10.1523/JNEUROSCI.6638-10.2011
Why does remyelination fail in multiple sclerosis?Nature Reviews. Neuroscience 3:705–714.https://doi.org/10.1038/nrn917
Oligodendrocyte PTEN is required for myelin and axonal integrity, not remyelinationAnnals of Neurology 68:703–716.https://doi.org/10.1002/ana.22090
Advances in myelinating glial cell developmentCurrent Opinion in Neurobiology 42:53–60.https://doi.org/10.1016/j.conb.2016.11.003
Neuronal activity biases axon selection for myelination in vivoNature Neuroscience 18:683–689.https://doi.org/10.1038/nn.3992
Vamp4 cycles from the cell surface to the trans-golgi network via sorting and recycling endosomesJournal of Cell Science 120:1028–1041.https://doi.org/10.1242/jcs.03387
ZFP191 is required by oligodendrocytes for CNS myelinationGenes & Development 24:301–311.https://doi.org/10.1101/gad.1864510
Biochemical, functional, and pharmacological characterization of AT-56, an orally active and selective inhibitor of lipocalin-type prostaglandin D synthaseThe Journal of Biological Chemistry 284:7623–7630.https://doi.org/10.1074/jbc.M808593200
Myelin gene regulatory factor is required for maintenance of myelin and mature oligodendrocyte identity in the adult CNSThe Journal of Neuroscience 32:12528–12542.https://doi.org/10.1523/JNEUROSCI.1069-12.2012
RNA-seq analysis is easy as 1-2-3 with limma, glimma and edgerF1000Research 5:ISCB Comm J-1408.https://doi.org/10.12688/f1000research.9005.3
Remyelination in multiple sclerosis: from basic science to clinical translationThe Lancet. Neurology 19:678–688.https://doi.org/10.1016/S1474-4422(20)30140-X
Botulinum neurotoxin: a marvel of protein designAnnual Review of Biochemistry 79:591–617.https://doi.org/10.1146/annurev.biochem.051908.125345
15D-pgj2: the anti-inflammatory prostaglandin?Clinical Immunology 114:100–109.https://doi.org/10.1016/j.clim.2004.09.008
NIH image to imagej: 25 years of image analysisNature Methods 9:671–675.https://doi.org/10.1038/nmeth.2089
Oligodendrocytes: myelination and axonal supportCold Spring Harbor Perspectives in Biology 8:a020479.https://doi.org/10.1101/cshperspect.a020479
Myosin Va controls oligodendrocyte morphogenesis and myelinationThe Journal of Neuroscience 27:11366–11375.https://doi.org/10.1523/JNEUROSCI.2326-07.2007
A botulinum toxin-derived targeted secretion inhibitor downregulates the GH/IGF1 axisThe Journal of Clinical Investigation 122:3295–3306.https://doi.org/10.1172/JCI63232
Myelin in the central nervous system: structure, function, and pathologyPhysiological Reviews 99:1381–1431.https://doi.org/10.1152/physrev.00031.2018
Prostaglandin D2 synthase/GPR44: a signaling axis in PNS myelinationNature Neuroscience 17:1682–1692.https://doi.org/10.1038/nn.3857
Purification and characterization of rat brain prostaglandin D synthetaseThe Journal of Biological Chemistry 260:12410–12415.
Biochemical, structural, genetic, physiological, and pathophysiological features of lipocalin-type prostaglandin D synthaseBiochimica et Biophysica Acta 1482:259–271.https://doi.org/10.1016/s0167-4838(00)00161-8
Prostaglandin D2 and sleep/wake regulationSleep Medicine Reviews 15:411–418.https://doi.org/10.1016/j.smrv.2011.08.003
Specificity of botulinum protease for human VAMP family proteinsMicrobiology and Immunology 56:245–253.https://doi.org/10.1111/j.1348-0421.2012.00434.x
An RNA-sequencing transcriptome and splicing database of glia, neurons, and vascular cells of the cerebral cortexThe Journal of Neuroscience 34:11929–11947.https://doi.org/10.1523/JNEUROSCI.1860-14.2014
Cns myelin wrapping is driven by actin disassemblyDevelopmental Cell 34:152–167.https://doi.org/10.1016/j.devcel.2015.06.011
(1) It is unclear from the data presented whether PD:iBot cells are unable to differentiate and are therefore more likely to die. Experiments to test whether or not botulinum-expressing cells contribute to the population of surviving, differentiated oligodendrocytes are needed.
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 12). 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 12).
(2) The manuscript's key experiment is the transwell co-culture study. First, raw data should be shown for this experiment.
We included raw data for this experiment (Figure 6G, text page 23)
Second, appropriate statistical analyses should be performed.
As Reviewer#2 suggested, we performed one-way ANOVA comparing every group to every other group with multiple comparison tests (Figure 6E-H text page 23).
Third, a non-MBP-reliant membrane marker should be employed for analysis.
We thank the reviewers for the insightful suggestion. We used the membrane version of CellMask suggested by Reviewer#3 and repeated the transwell co-culture experiment. The results are consistent with the results based on MBP (Figure 6—figure supplement 1, text page 24). In addition, we used the membrane version of CellMask for all the new cell culture experiments described below (L-PGDS rescue, HPGD etc.).
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 are not botulinum-GFP-expressing (efficiency ~65% Figure 6B, text page 21). The GFP- cells in PD:ibot OPC cultures do not express botulinum toxin and are 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 21). Although both groups of cells are 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.
(3) Vamp3 levels should be examined in addition to Vamp2; otherwise, the authors cannot conclude Vamp1/2/3-dependent exocytosis is involved in the phenotypes observed.
We agree with the reviewers and examined Vamp3 levels with Western blot. We found diminished levels of Vamp3 in oligodendrocyte-lineage cells from PD:ibot mice (Figure 1J, M, text page 9).
(4) The data are convincing that botulinum-expressing oligodendrocytes do not mature properly. Further, the data support a role for L-PGDS in oligodendrocyte maturation. However, the link between these two points is currently weak, and there is a concern about correlation vs. causation in the interpretation of the data. Experiments to more conclusively show causation are required.
We agree with the reviewers that the potential link between the role of L-PGDS and the development defect of botulinum-expressing oligodendrocytes is a key question to investigate. We started preparing mouse strains for investigating this question years ago and are excited to report our new data in this revised manuscript. Our collaborators Yoshihiro Urade and Ko Fujimori’s groups in Japan generated L-PGDS overexpressing transgenic mouse strains (Ptgds-TG). Despite challenges in shipping live mice overseas exacerbated by the COVID-19 pandemic, we were able to cryopreserve the sperms of the TG mice, ship the sperm, and recover live mice in the U.S. We then generated triple transgenic mice (PDGFRα-creER; flox-stop-flox-botulinum toxin B-GFP; Ptgds-TG). Interestingly, we found that Ptgds-TG partially rescued the myelination defect of PD:ibot mice (Figure 9E-H, text page 29).
We further assessed whether L-PGDS can rescue the oligodendrocyte development defect of botulinum-expressing cells in purified OPC cultures in vitro to avoid the confounding effect of L-PGDS on other cell types in vivo. We found that recombinant L-PGDS protein rescued the PD:ibot oligodendrocyte development defect in vitro (Figure 9A, B, text page 27).
Thanks to an excellent suggestion by Reviewer#1, we also added HPGD, a protein that inactivates PGD2, to OPC cultures and found that HPGD inhibits oligodendrocyte maturation (Figure 7F, G, text page 26).
Together, these results strengthen the link between the role of L-PGDS and the development defect of botulinum-expressing oligodendrocytes.
Reviewer #1 (Recommendations for the authors):
– For in vivo experiments examining oligodendrocyte lineage cells, the authors note that mice with only the Cre transgene or only the ibot transgene were subjected to tamoxifen. Experimental panels note "Controls." It is unclear if all these mice are congenic, which should be noted in the methods either way.
All mice used were congenic. We used PD:ibot and control littermates raised under the same maternal care in the same cage for all experiments to minimize environmental confounding factors. We added this information in the methods (text page 42).
It is also unclear if the different control genotypes were pooled for the quantifications. This should also be clarified in the methods.
We noted in the figure legends whether Cre-only or ibot-only controls were used for each experiment.
Finally, the authors note "very little GFP expression" in controls. This seems critical data to show and quantify.
We quantified the data and included a new figure (Figure 1—figure supplement 3).
– Figure 1B has a typo in the y axis label "ExpressionI".
– In Figure 1I, only VAMP2 expression is analyzed by Western blot. As VAMP3 is implicated in PLP trafficking and may have cell-autonomous effects in oligodendrocyte differentiation/myelination, and also has differentiation-dependent changes in gene expression from OPC to NFO/MO (Figure 1C), showing the levels of VAMP3 in PD:ibot OPCs would provide better mechanistic understanding at the effects of ibot expression.
We analyzed VAMP3 levels and found a reduction in PD:ibot cells (Figure 1J, M, text page 9).
– It is unclear what region of cerebral cortex was used for quantification throughout the manuscript. Was the same region used for all experiments? These details should be noted in the methods.
We included the information in the method (text page 47).
– In Figure 3N, the representative EM images used for quantification are too small and could stand to be enlarged to better appreciate the differences.
We enlarged the EM images.
It is unclear what criteria were used to determine myelinated axons and how g ratio was quantified. These details should be provided in the methods section.
We added the information in the Methods section (text page 49).
– Add best-fit lines to Figure 3Q.
– Figure 3R-U:
– Mice used for behavioral testing are 2-5x older than all mice used for cellular quantification (P8/P17/P30 vs P60-150).
– Between P8 and P30, the relative reduction in oligos is progressive. A great deal of myelin is added between 2 months and 5 months. Which data points came from which age groups? Are there age-related differences driving these results?
We have separately labeled data points from 2 months old and 5 months old mice (Figure 3Q-S). 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.
– Comparison of botox effects in OPCs vs astrocytes/endothelial cells uses different methods of expression (i.e. inducible for OPCs with CreER and constitutive with Cre for astrocytes/endothelial cells). It should at least be noted that embryonic/early postnatal compensation could partially account for the lack of effect (e.g. secreted factors/interactions from both cell types could play a role in oligodendrocyte lineage development in vivo). Additionally, repeated tamoxifen injection is known to influence oligodendrogenesis. If gfap/tie2-Cre mice were not also treated with tamoxifen, this could serve as a potential confound and should be stated as such.
We agree with the reviewer and added this point (page 15). We injected the same amount of tamoxifen to the littermates (control) of PD:ibot mice. Therefore, the developmental defect in PD:ibot mice is unlikely due to tamoxifen treatment.
– Given how critical the well-insertion experiments are in Figure 4K-M to the claims in this study, I would suggest splitting these panels and making a new figure and adding example images used for quantification from these cultures.
We made a new figure reporting the insert experiment results and added example images (Figure 6E-H).
As an alternative experiment, the authors could consider co-culturing Hpgd-overexpressing microglia along with control OPCs.
We thank the reviewer for a great idea. Inspired by the reviewer’s suggestion, we designed a simplified version of the suggested experiment. We added HPGD protein to the culture and found that HPGD inhibited oligodendrocyte maturation (Figure 7F, G, text page 26). This experiment provides additional evidence for the role of PGD2 in oligodendrocyte development.
– In Figure 5, expression of mature oligodendrocyte markers is reduced in purified oligodendrocytes using equivalent amounts of cDNA to compensate for presumably lower numbers of oligodendrocytes in the PD:ibot mice. CC1 and olig2 were used to count cell numbers by immunohistochemistry. Do these genes also show reduced expression in the RNA sequenced oligodendrocytes? Could it be that the number of oligodendrocytes counted is underestimated due to the reduced expression of selected markers used for immunostaining?
Qk (its encoded protein is labeled by the CC1 antibody) and Olig2 mRNA levels did not show significant differences between PD:ibot and control purified oligodendrocytes. We added these result (Figure 4—figure supplement 1, text page 17).
– For Figure 7, gene name (Ptgds-/-) should be used in place of LPGDS-/-.
Corrected (new Figure 8).
– In the Discussion (line 622), the word "strongly" is a bit of an overstatement given the small effect size in the referenced experiment.
We removed this word.
Reviewer #2 (Recommendations for the authors):
There were a number of concerns in the article in its present form.
1. The notion of VAMP-dependent vesicular release from oligodendrocyte progenitor cells (OPCs) was interesting but warrants additional validation. Within neurons, there are definable sites of vesicular release and it would be particularly relevant to determine where within the OPC the tSNARE is expressed morphologically.
We agree with the reviewer that the subcellular distribution of VAMP-associate vesicles is an important question to study. We stained for VAMP2 and VAMP3 proteins in cultured OPCs and found distributed VAMP2/3 throughout the cell, including in the soma and the processes (Figure 1 —figure supplement 1, text page 5).
2. Figure 1 data show relative VAMP1, 2 and 3 expression but these data require some context to appreciate the relative abundance of these genes in relation to neurons.
We now included data on Vamp1, 2, and 3 expression in neurons, astrocytes, microglia, and endothelial cells in addition to OPCs and oligodendrocytes (Figure 1A-C). These comparisons show that Vamp3 expression is higher in OPCs and oligodendrocytes than in neurons and that Vamp2 expression in OPCs and oligodendrocytes is about 1/3 the level in neurons.
2. Figure 1 Panel D requires an indication as to where the subsequent higher magnification images are taken.
It would also be warranted to define the relative efficiencies in multiple locales, including gray matter and white matter ROIs.
We now added the quantification of efficiencies in cerebral cortex-grey mater, corpus callosum-white matter, and striatum (Figure 1G, text page 8).
3. The analysis of cell death at P30 was limited in its scope as cell death may not be limited to the oligodendrocyte lineage cells and no differences at one time point are inconclusive.
We performed a more thorough analysis of activated caspase-3 at multiple developmental stages (P8, P15, and P30) in oligodendrocytes, OPCs, and cells of other lineages. There is no significant difference between PD:ibot and control mice in any of these comparisons (Figure 3—figure supplement 1 text page 12).
4. The analysis of myelination by IHC was also considered insufficient to support the authors' conclusions that the hypomyelination phenotype is truly hypomyelination. Neurodegeneration would result in perturbed myelination during development. Analysis of axons and axon counts in ROI where hypomyelination is suggested should be performed using electron microscopy.
We performed analyses of axon densities and axon diameter in the corpus callosum region using electron microscopy. We did not detect any statistically significant differences between PD:ibot and control mice (Figure 3N, O, text page 12).
5. In many instances the data are not analyzed statistically or incompletely. For example, Figure 3Q lacks linear regression analyses and comparisons (3Q should also declare the number of myelinated axons analyzed as well).
We added linear regression and the number of axons analyzed in the figure legend (text page 14) and method (text page 49).
Figure 4M requires ANOVA for comparison across all treatment groups.
We indeed performed ANOVA for comparison across all treatment groups (i.e. we compared every group to every other group instead of comparing every group to one control group) with multiple comparison tests. We apologize for the lack of clarity on this point in the original manuscript. We edited the figure legend to clarify this point (New Figure 6H, text page 23).
6. The analysis of secreted factors depicted in Figure 4K-M is also poorly explained as it is described it is unclear why treatment groups 2 and 4 should differ given they both have PD:ibot and control with a permeable membrane between them.
We thank the reviewer for raising a great point. We think this result suggests that cell autonomous effect of blocking exocytosis may also contribute to the oligodendrocyte development defect in PD:ibot mice. We included the possibly contributions from both cell-autonomous and cell-non-autonomous mechanisms in Discussion (text page 35).
7. All analyses of OPC-OL differentiation based on morphology are considered suggestive but lack rigor as they are liable to subjective characterization. Additional validation using stage-specific markers is needed to support their conclusions.
We thank the reviewer for a great suggestion. We performed additional in vivo and in vitro experiments to characterize OPC differentiation with oligodendrocyte markers. We perform RNAscope experiments in vivo using probes for Enpp6, a marker for pre-myelinating oligodendrocytes, and Mbp, a marker for oligodendrocytes. Interestingly, both markers showed a significant reduction in PD:ibot mice compared with controls, suggesting that the oligodendrocyte development defect in PD:ibot mice manifest as early as the pre-myelinating stage (Figure 2I-K, text page 11). We performed quantitative real-time PCR and Western blot experiments to assess the mRNA and protein levels of MBP to assess OPC differentiation independent of morphology in vitro. We found that both MBP mRNA and protein levels are lower in PD:ibot OPC/oligodendrocyte cultures than in control cultures (Figure 5C, D, text page 19).
8. It is unclear why the authors chose to use RNAseq to evaluate secreted factors. Proteomic analysis of conditioned media would have been more appropriate and unbiased.
We agree with the reviewer that proteomics analysis of conditioned media is a more appropriate method for evaluating OPC/oligodendrocyte-secreted factors. We are performing proteomic experiments and will report the results in a separate paper in the future. We initially chose to perform RNAseq to characterize molecular changes in OPCs and oligodendrocytes in PD:ibot mice. We noticed intriguing changes of Ptgds and other candidate genes encoding secreted proteins. We therefore decided to assess the function of these candidate genes in oligodendrocyte development and found an interesting role of Ptgds.
9. The central hypothesis of the model put forth by the authors is that expression of Ptgds is a signal to promote OPC differentiation yet it is expressed across all stages of maturation in this cell lineage. Moreover, the authors contend that ptgds is expression is lower in OPCs in multiple sclerosis, while this is not entirely true since during the progressive phase of disease it is actually upregulated (PMID: 16409554). Hence, the overall premise of the correlation of expression with disease is not supported by the existing data.
We edited the text to reflect the complexity of Ptgds expression in multiple sclerosis based on existing literature (text page 34). In the future, comprehensive evaluation of Ptgds mRNA, L-PGDS protein, and PGD2 levels in different phases of multiple sclerosis (MS) will clarify potential involvement of L-PGDS in MS and the therapeutic potential of this pathway in MS.
10. Do global L-PGDS mice have a neurodegenerative phenotype?
We stained for an axon damage marker, SMI-32, and did not detect any difference between Ptgds knockout and littermate control mice (Figure 8H, I, text page 27). We also stained for a microglia marker, Iba1, to assess glial reactivity and did not detect any difference between Ptgds knockout and littermate control mice (Figure 8J, K, text page 27).
Reviewer #3 (Recommendations for the authors):
(1) In order to see that the impact on differentiation and myelination in vivo is plausibly due to autocrine/paracrine-acting secreted factors, rather than a consequence of blocking of secretion itself, it is important to show whether GFP+/ibot cells differentiate and survive in vivo. The authors should show quantification of the fraction of oligodendrocytes with MBP+ sheaths that are GFP+ or GFP- (this can be done in the cortex where myelin is sparse) or CC1+ cells if needed.
We analyzed the overlap between GFP+ botulinum-expressing cells and the population of differentiated oligodendrocytes (Olig2+PDGFRa-CC1+) and found that botulinum-expressing cells can survive and become differentiated oligodendrocytes (Figure 3—figure supplement 2, text page 12).
(2 and 3) The authors should confirm with an MBP staining-independent membrane label (not cytoplasm) the conclusions on lamellar cell morphology. Otherwise, the fraction of MBP+ cells may be a more accurate reflection of the data.
Completed as suggested, described above.
I'm not sure whether this is an artifact introduced in the creation of the pdf (why I've included this as a private recommendation), but there are some issues with the images in Figures 2 and 7. Several images have clear boundaries with differing background intensities within the single image. This makes the images appear as they have been pasted together from separate images and/or brightness/contrast was not uniformly treated across the entire image. How were the original images modified? Are these stitched images with different acquisition settings?
Specifically, in 2F: The first two Olig2 images have a clear boundary in the images. In the PDGFRa image on the right-hand side, there is an apparent "box" in the lower-left (boundary and different background intensities).
Specifically in 7: A and C have variations in the background intensities in tile-like patterns within the images.
If the high-resolution versions of these images lack this issue, please ignore! If not, I ask the authors please address this.
We thank the reviewer for pointing this out. We typically image many fields of view using our Zeiss Apotome widefield fluorescent microscope and stitch the tiled images together into a larger image using an automatic stitching function in the Zeiss Zen software. For some reason, the background fluorescence intensity is not even across each tile, and we often see clear boundaries with differing background intensities where different tiles are stitched together. We confirm that we only stitched images from adjacent fields of view together and we always uniformly adjusted brightness and contrast across the entire image. We have included images without clear boundaries in this revised manuscript.https://doi.org/10.7554/eLife.77441.sa2
Article and author information
UCLA Brain Research Institute (Knaub Postdoctoral Fellowship)
- Lin Pan
National Institute of Neurological Disorders and Stroke (R00NS089780)
- Ye Zhang
National Institute on Aging (R03AG065772)
- Ye Zhang
National Institute of Child Health and Human Development (P50HD103557)
- Ye Zhang
National Center for Advancing Translational Science UCLA CTSI Grant (UL1TR001881)
- Ye Zhang
W. M. Keck Foundation (W. M. Keck Foundation junior faculty award)
- Ye Zhang
UCLA Eli and Edythe Broad Center of Regenerative Medicine and Stem Cell Research (Innovation Award)
- Ye Zhang
Wendy Ablon Foundation (Ablon Scholar Award)
- Ye Zhang
National Institute of Neurological Disorders and Stroke (R01NS109025)
- Ye Zhang
Friends of the Semel Institute for Neuroscience & Human Behavior (Friends scholar award)
- Ye Zhang
The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.
We thank Akiko Nishiyama, J Bradley Zuchero, Hui Zong, and Mable Lam for their advice and editing of our manuscript. We thank Qingyun Li, Richard Breyer, Henry Lin, and Ginger Milne for their advice. We thank Garret FitzGerald for reagents. We thank the Eli and Edythe Broad Center of Regenerative Medicine and Stem Cell Research, UCLA BioSequencing Core Facility for their services, Mahnaz Akhavan and Suhua Feng for their technical support. This work is supported by the Knaub Postdoctoral Fellowship to LP, the National Institute of Neurological Disorders and Stroke of the National Institute of Health (NIH) R00NS089780, R01NS109025, R01NS099102 (CT), the National Institute of Aging of the NIH R03AG065772, the National Institute of Child Health and Human Development P50HD103557, the National Center for Advancing Translational Science UCLA CTSI Grant UL1TR001881, the W M Keck Foundation Junior Faculty Award, the UCLA Eli and Edythe Broad Center of Regenerative Medicine and Stem Cell Research Innovation Award, the Ablon Faculty Scholar Award, and the Friends of the Semel Institute for Neuroscience & Human Behavior Friends Scholar Award to YZ.
All animal experimental procedures (protocols: #R-16-079 and #R-16-080) were approved by the Chancellor's Animal Research Committee at the University of California, Los Angeles, and conducted in compliance with national and state laws and policies.
- Claude Desplan, New York University, United States
- Kelly Monk, Vollum Institute, Oregon Health & Science University, United States
- Received: January 29, 2022
- Preprint posted: February 14, 2022 (view preprint)
- Accepted: February 12, 2023
- Accepted Manuscript published: February 13, 2023 (version 1)
- Version of Record published: February 22, 2023 (version 2)
© 2023, Pan et al.
This article is distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use and redistribution provided that the original author and source are credited.
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