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Prostaglandin D2 synthase/GPR44: a signaling axis in PNS myelination

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Abstract

Neuregulin 1 type III is processed following regulated intramembrane proteolysis, which allows communication from the plasma membrane to the nucleus. We found that the intracellular domain of neuregulin 1 type III upregulated the prostaglandin D2 synthase (L-pgds, also known as Ptgds) gene, which, together with the G protein–coupled receptor Gpr44, forms a previously unknown pathway in PNS myelination. Neuronal L-PGDS is secreted and produces the PGD2 prostanoid, a ligand of Gpr44. We found that mice lacking L-PGDS were hypomyelinated. Consistent with this, specific inhibition of L-PGDS activity impaired in vitro myelination and caused myelin damage. Furthermore, in vivo ablation and in vitro knockdown of glial Gpr44 impaired myelination. Finally, we identified Nfatc4, a key transcription factor for myelination, as one of the downstream effectors of PGD2 activity in Schwann cells. Thus, L-PGDS and Gpr44 are previously unknown components of an axo-glial interaction that controls PNS myelination and possibly myelin maintenance.

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Figure 1: NRG1 type III undergoes a regulated intramembrane proteolysis cleavage that is Schwann cell dependent.
Figure 2: L-pgds is the most upregulated gene in neurons infected with NRG1 ICD.
Figure 3: L-PGDS is secreted and enzymatically active.
Figure 4: L-pgds−/− are hypomyelinated.
Figure 5: H-pgds and L-pgds are required for myelin formation and maintenance.
Figure 6: L-PGDS enzymatic activity is important for PNS myelination and maintenance.
Figure 7: Gpr44 promotes myelination.
Figure 8: PGD2 induced Nfatc4 dephosphorylation in primary Schwann cells.

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Change history

  • 17 November 2014

    In the version of this article initially published online, a P value was incorrect on p. 5, first full paragraph. It read “We found similar alterations in 6-month-old sciatic nerves of L-pgds−/− mice, although the difference was not significant (P = 0.649; Supplementary Table 2).” The correct P value is 0.0649. Also, the first Results subheading read “NRG1 type III is cleaved and activates L-PGDS by γ-secretase”; it should have read “NRG1 type III is cleaved by γ-secretase and activates L-PGDS.” The errors have been corrected for the print, PDF and HTML versions of this article.

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Acknowledgements

We thank V. Marzano and A. Urbani for shotgun mass spectrometry analyses, F. Clarelli for heat map and statistical analyses, G. Fitzgerald (University of Pennsylvania) for providing Gpr44−/− mice, P. Podini for excellent technical assistance with electron microscopy analyses, and P. Del Boccio for LC-MS/MS analyses. We are grateful to M. Buono, S. Previtali and A. Bolino for critical reading of the manuscript and suggestions, and L. Massimino for artwork. Part of this work was carried out in ALEMBIC, an advanced microscopy laboratory at the San Raffaele Scientific Institute. M.G.F. conducted this study in partial fulfillment for her Ph.D. in Molecular and Cellular Neuroscience, San Raffaele University. This study was supported by the Italian Minister of Health (award number GR08-35, C.T.), the European Marie Curie Reintegration Grant (award number IRG 239430-2008, C.T.) and the Agence Nationale pour la Recherche (ANR blanc programme, B.B.B.). C.T. is also supported by Italian Fondazione Italiana Sclerosi Multipla.

Author information

Authors and Affiliations

Authors

Contributions

A.T. designed and conducted the majority of the experiments. M.G.F., V.A. and A.L. contributed to in vitro and biochemical studies. P.B. and F.M.B. performed expression studies. G.D. and A.Q. performed morphological and ultrastructural analyses. Y.U. and B.B.-B. provided transgenic lines and provided input. D.P. and P.S. performed the MS/MS-LC analyses. C.T. designed the experimental plan, supervised the project and wrote the manuscript.

Corresponding author

Correspondence to Carla Taveggia.

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Competing interests

C.T. and A.T. submitted a patent on July 1 2014 (PCT/EP2014/063995) based on the work described in this paper.

Integrated supplementary information

Supplementary Figure 1 Differentially expressed genes in the microarray analyses.

a) Heat map diagram showing the expression levels of the differentially expressed genes from the comparison between NRG1 ICD infected DRG neurons and not infected (Limma moderated t–test P = <0.001; fold change cut off > 1.5 or < 0.66 df=6). N = 4 different biological replicates in each experimental condition. b) Venn diagram showing the number of differentially expressed genes in the comparisons tested in the microarray analyses (P = <0.01; fold change cut–off: 2). N = 4 different biological replicates in each experimental condition. c) RT–PCR on mRNA prepared from DRG neurons not infected, infected with a lentivirus expressing NRG1 ICD or EGFP as control. L–PGDS is upregulated only in neurons overexpressing NRG1 ICD. N = 3 different independent mRNA preparations and analyses.

Supplementary Figure 2 L–PGDS protein is not retained in DRG neurons.

a) Representative western blotting analyses of DRG neurons lysates not infected or infected with NRG1 ICD or L–PGDS as control. Only in L–PGDS overexpressing cultures it is possible to detect L–PGDS protein. Actin serves as a loading control. N = 3 different independent experiments. b) Representative western blotting analyses of DRG conditioned media from not infected or L–PGDS infected neurons. 14 days after infection, cultures were grown in the presence of neurobasal media for additional 48 hours after which the media was tested for L–PGDS expression. L–PGDS is released in the conditioned media. N = 3 different independent experiments. For uncropped pictures of western blots, see Supplementary Figure 10. c) Ponceau loading control of the Western blot showed in Figure 3a. N = 3 different independent experiments. d) Ponceau loading control of the Western blot showed in Figure 3b. N = 3 different independent experiments. e) LC–MS/MS analyses show that PGD2 is not present in conditioned media of DRG neurons overexpressing EGFP. Chromatograms are representative of three different experiments. N = 3 conditioned media preparations.

Supplementary Figure 3 H–PGDS–/– are normally myelinated.

a) Representative immunofluorescence of two months old s–type, L–PGDS–/– and H–PGDS–/– sciatic nerve cross sections stained for H–PGDS (rhodamine). H–PDGS is expressed in wild–type and in L–PGDS–/– in similar amounts. Sections were also stained for neurofilament (fluorescein) and nuclei (DAPI). Lack of signal in H–PGDS–/– confirmed antibody specificity. N = 3 independent experiments. Bar: 50 μm. b–d) Morphological analyses of 1 month wild–type and H–PGDS–/– sciatic nerves. b) Electron micrographs and c) g–ratio analyses confirmed normal myelination in H–PGDS–/–. g–ratio as a function of axon diameter is similar in wild–type (red line) and H–PGDS–/– (black line) (t–test analysis; P = 0.4261; t=0.7964, df=662). The graph represents the g–ratio obtained from more than 250 myelinated axons. d) Distribution of myelinated fibers is similar in H–PGDS–/– and wild–type 1 month sciatic nerves. Fisher’s exact test; P = 0.4449 (total vs 1–2 μm), P = 0.5546 (total vs 2–3 μm), P = 0.2521 (total vs 3–4 μm), P = 0.1694 (total vs 4–5 μm), P = 1 (total vs 5–6 μm). Over 80 fibers for each genotype were counted. N = 3 mice per genotype. Bar: 2 μm. e–g) Morphological analyses of 6 month wild–type and H–PGDS–/– sciatic nerves. e) Electron micrographs and f) g–ratio analyses confirmed normal myelination in H–PGDS–/–. g–ratio as a function of axon diameter is similar in wild–type (red line) and H–PGDS–/– (black line) (t–test analysis; P = 0.6071; t=0.5725, df=388). The graph represents the g–ratio obtained from more than 150 myelinated axons. g) Distribution of myelinated fibers is similar in H–PGDS–/– and wild–type 6 month sciatic nerves. Fisher’s exact test; P = 0.2325 (total vs 1–2 μm), P = 0.8385 (total vs 2–3 μm), P = 0.811 (total vs 3–4 μm), P = 0.1056 (total vs 4–5 μm), P = 0.4431 (total vs 5–6 μm), P = 1 (total vs 6–7 μm), P = 0.2243 (total vs 7–8 μm), P = 1 (total vs >8 μm). Over 60 fibers for each genotype were counted. N = 3 mice per genotype. Bar: 2 μm.

Supplementary Figure 4 In vitro H–PGDS–/–;L–PGDS–/– neurons are hypomyelinated.

a–b) Representative electron microscopy analyses of wild–type (a) and H–PGDS–/–;L–PGDS–/– (b) mouse DRG neurons seeded with rat wild–type Schwann cells and maintained in myelinating conditions for 9 days. Null cocultures are hypomyelinated, and when formed myelin is aberrant (arrow). They also present several naked axons (asterisks), unlike control cultures. N = 9 different independent coculture experiments. Bar: 1 μm c) High power images showing the significant difference in myelin sheath thickness in null cocultures as compared to that of wild–type axons of similar caliber. N = 9 different independent coculture experiments. Bar: 500 nm.

Supplementary Figure 5 The sorting process is normal in H–PGDS–/–;L–PGDS–/– and saphenous nerves are hypomyelinated.

a) Morphological analyses of P2 wild–type and H–PGDS–/–;L–PGDS–/– sciatic nerves. H–PGDS–/–;L–PGDS–/– are significantly hypomyelinated, but not impaired in axonal sorting. Bottom panel shows a significantly hypomyelinated axon a feature common to many null fibers. N = 3 mice per genotype. Bar: 2 μm, top panels, 1 μm bottom panels. b–d) Morphological analyses of 8 month wild–type and H–PGDS–/–;L–PGDS–/– saphenous nerves. b) Electron micrographs and c) g–ratio analyses confirmed hypomyelination in H–PGDS–/–;L–PGDS–/– saphenous nerves. g–ratio as a function of axon diameter is similar in wild–type (red line) and H–PGDS–/–;L–PGDS–/– (black line) (t–test analysis; P = <0.0001; t=8.448, df=662). The graph represent the g–ratio obtained from more than 300 myelinated axons. d) Distribution of myelinated fibers is similar in H–PGDS–/–;L–PGDS–/– and wild–type saphenous nerves. Fisher’s exact test; P = 1 (total vs 1–2 μm), P = 0.7667 (total vs 2–3 μm), P = 0.9181 (total vs 3–4 μm), P = 0.6889 (total vs 4–5 μm), P = 0.6445 (total vs 5–6 μm), P = 0.4917 (total vs 6–7 μm). Over 100 fibers for each genotype were counted. N = 3 mice per genotype. Bar: 2 μm.

Supplementary Figure 6 The number of myelinated axons and Remak fibers are normal in H–PGDS–/–;L–PGDS–/–.

a) The total number of myelinated axons per area is similar in motor roots of 1 month old wild–type and H–PGDS–/–;L–PGDS–/–. Myelinated fibers were counted over the entire root (n=3). (t–test analysis; P = 0.0517; t=2.744, df=4). N = 3 mice per genotype. Error bars represent means ± s.e.m. b) The total number of myelinated axons per area is similar in sensory roots of 1 month old wild–type and H–PGDS–/–;L–PGDS–/– mice. Myelinated fibers were counted over the entire root (n=3) (t–test analysis; P = 0.051; t=2.756, df=4). N = 3 mice per genotype. Error bars represent means ± s.e.m. c) The total number of myelinated axons per area is similar in motor roots of 8 months old wild–type and H–PGDS–/–;L–PGDS–/– mice. Myelinated fibers were counted over the entire root (n=3) (t–test analysis; P = 0.9689; t=0.04154, df=4). N = 3 mice per genotype. Error bars represent means ± s.e.m. d) The total number of myelinated axons per area is similar in sensory roots of 8 months old wild–type and H–PGDS–/–;L–PGDS–/– mice. Myelinated fibers were counted over the entire root (n=3) (t–test analysis; P = 0.1571; t=1.739, df=4). N = 3 mice per genotype. Error bars represent means ± s.e.m. e) The total number of myelinated axons per area is similar in 9 months old sciatic nerves of wild–type and H–PGDS–/–;L–PGDS–/– mice. Myelinated fibers were counted over the entire sciatic nerve cross sections (t–test analysis; P = 0.6075; t=0.5566, df=4). N = 3 mice per genotype. Error bars represent means ± s.e.m. f–g) The number of axons per Remak bundle in wild–type and H–PGDS–/–;L–PGDS–/– sciatic nerves were binned into separate groups and are shown as a percentage of the total axons. Over 120 bundles for each genotype were counted. The number of axons/bundle is similar in 1 month (e) and 9 months old mice (f). Fisher’s exact test 1 month old mice; P = 0.5021 (total vs 1–5), P = 0.9063 (total vs 5–10), P = 0.6173 (total vs 11–15 μm), P = 0.2234 (total vs 16–20 μm), P = 1 (total vs 21–25 μm), P = 0.8052 (total vs 26–30 μm), P = 0.2923 (total vs 31–35 μm), P = 0.0513 (total vs 36–40 μm), P = 0.251 (total vs 41–45 μm). Fisher’s exact test 9 months old mice; P = 0.6537 (total vs 1–5), P = 0.2255 (total vs 5–10), P = 0.3149 (total vs 11–15 μm), P = 1 (total vs 16–20 μm), P = 0.5191 (total vs 21–25 μm), P = 0.3447 (total vs 26–30 μm), P = 1 (total vs 31–35 μm), P = 0.3539 (total vs 36–40 μm), P = 0.2685 (total vs 41–45 μm), P = 0.359 (total vs > 50). N = 3 mice per genotype.

Supplementary Figure 7 AT–56 effects on Schwann cells and neuronal survival, axon–Schwann cell interaction and myelination.

a) Graph showing the dose dependent effects of AT–56 treatment on Schwann cell survival, determined as percentage of Caspase 3+ cells over the total number of cells (3 coverslip/condition; 3 different experiments). 20 μM AT–56 does not alter Schwann cells survival. Error bars represent means ± s.e.m. (t–test analysis: untreated – 20 μM P = 0.1138; t=1.807, df=4; untreated – 50 μM P < 0.0001; t=7.028, df=7). N = 3 different experiments. b) Graph showing the dose dependent effects of AT–56 treatment on myelinating Schwann cell–DRG neuronal coculture, determined as number of MBP+ segments per field (3 coverslip/condition; 3 different experiments). Increased concentration of AT–56 impairs myelination. Error bars represent means ± s.e.m. (t–test analysis: untreated – 1 μM P = 0.3866; t=0.8806, df=26; untreated – 10 μM *** P <0.0001; t=5.651, df=31; untreated – 25 μM *** P <0.0001; t=6.736, df=22). N = 3 different experiments. c) Graph showing the dose dependent effects of AT–56 treatment on Schwann cell–DRG neuronal coculture survival. We counted the effects of AT–56 on Schwann cells and express it as percentage of Caspase 3+ Schwann cells over the total number of cells (3 coverslip/condition; 3 different experiments). 25 μM AT–56 does not alter Schwann cells survival. (t–test analysis: untreated – 25 μM p = 0.3874; t=0.9318 df=6). Error bars represent means ± s.e.m. N = 3 different experiments. d) Organotypic mouse DRG explants were maintained in the presence of 25 μM AT–56 for 7 days then stained for S100 (rhodamine) and β–tubulin (fluorescein). AT–56 treatment does not impair axon – Schwann cell association. N = 3 different coculture experiments. Bar: 50 μm.

Supplementary Figure 8 PGD2 receptors expression and their role in myelination.

a) qRT–PCR analyses in mRNA prepared from not infected or NRG1 ICD infected DRG neurons, primary rat Schwann cells and skeletal muscle, which serves as a control. Ptgdr expression is comparable among all samples. Expression levels were normalized to Gapdh levels. Data were analyzed with the CFX Manager SoftwareTM from Biorad on three mRNA different preparations. Error bars represent means ± s.d. (One–way ANOVA; F=10.77; P = 0.0648 SC – Not Inf; P = 0.2853 SC – ICD and P = 0.04 Not Inf – ICD). N = 3 different independent mRNA preparations and analyses. b) Cocultures of mouse organotypic Schwann cells DRG neurons infected with Gpr44 specific shRNAs and scramble (shscr) lentiviruses were maintained in myelinating conditions for 7 d, fixed and stained for MBP (rhodamine) and neurofilament (fluorescein). Less myelin segments are evident in shGpr44–infected cultures. Shown images are representative of three different independent experiments. N = 3 different coculture experiments. Bar: 100 μm.

Supplementary Figure 9 PGD2 does not modulate AKT and MAPK pathways.

PGD2 does not activate AKT–1 and MAPK pathways. Rat primary Schwann were grown to confluence and then starved for 16 hours. Schwann cells were then treated with 2.5 ng/ml NRG1β1, 10 nM Cyclosporin A, 100 nM EtOH, 100 nM PGD2, 2.5 μM forskolin, 100 nM PGD2 together with 2.5 μM forskolin. Schwann cells lysates were tested by western blotting analyses for (a) AKT–1 phosphorylation (ser 473) and total AKT–1 levels and (b) p44/p42 phosphorylation and total p44/p42 levels. Shown images are representative of three different independent experiments. N = 3 different independent experiments. For uncropped pictures of western blots, see Supplementary Figure 10. c) Model of L–PGDS/GPR44 activity. NRG1 ICD, upon generation, translocates into the nucleus to specifically activate L–PGDS mRNA expression. L–PGDS protein is then released in the extracellular milieu where is enzymatically active. PGD2 binds to and activates glial Gpr44 resulting in dephosphorylation and nuclear translocation of Nfatc4 to promote myelination.

Supplementary Figure 10 Uncropped pictures of the Western Blots shown in the manuscript.

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Supplementary Figures 1–10 and Supplementary Tables 2 and 3 (PDF 10177 kb)

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Supplementary Table 1: List of genes upregulated in the Illumina analyses.

List of genes whose mRNA were upregulated with a FC >2.0 and a P = <0.01 in DRG ICD infected neurons versus not infected, corrected for genes upregulated in EGFP infected neurons. N = 4 different independent RNA preparations and analyses. (XLS 34 kb)

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Trimarco, A., Forese, M., Alfieri, V. et al. Prostaglandin D2 synthase/GPR44: a signaling axis in PNS myelination. Nat Neurosci 17, 1682–1692 (2014). https://doi.org/10.1038/nn.3857

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