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. 2018 Aug 1;38(31):6921-6932.
doi: 10.1523/JNEUROSCI.1862-17.2018. Epub 2018 Jun 29.

Muscarinic Receptor M 3 R Signaling Prevents Efficient Remyelination by Human and Mouse Oligodendrocyte Progenitor Cells

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Free PMC article

Muscarinic Receptor M 3 R Signaling Prevents Efficient Remyelination by Human and Mouse Oligodendrocyte Progenitor Cells

R Ross Welliver et al. J Neurosci. .
Free PMC article

Abstract

Muscarinic receptor antagonists act as potent inducers of oligodendrocyte differentiation and accelerate remyelination. However, the use of muscarinic antagonists in the clinic is limited by poor understanding of the operant receptor subtype, and questions regarding possible species differences between rodents and humans. Based on high selective expression in human oligodendrocyte progenitor cells (OPCs), we hypothesized that M3R is the functionally relevant receptor. Lentiviral M3R knockdown in human primary CD140a/PDGFαR+ OPCs resulted in enhanced differentiation in vitro and substantially reduced the calcium response following muscarinic agonist treatment. Importantly, following transplantation in hypomyelinating shiverer/rag2 mice, M3R knockdown improved remyelination by human OPCs. Furthermore, conditional M3R ablation in adult NG2-expressing OPCs increased oligodendrocyte differentiation and led to improved spontaneous remyelination in mice. Together, we demonstrate that M3R receptor mediates muscarinic signaling in human OPCs that act to delay differentiation and remyelination, suggesting that M3 receptors are viable targets for human demyelinating disease.SIGNIFICANCE STATEMENT The identification of drug targets aimed at improving remyelination in patients with demyelination disease is a key step in development of effective regenerative therapies to treat diseases, such as multiple sclerosis. Muscarinic receptor antagonists have been identified as effective potentiators of remyelination, but the receptor subtypes that mediate these receptors are unclear. In this study, we show that genetic M3R ablation in both mouse and human cells results in improved remyelination and is mediated by acceleration of oligodendrocyte commitment from oligodendrocyte progenitor cells. Therefore, M3R represents an attractive target for induced remyelination in human disease.

Keywords: CHRM3; demyelination; human; lentivirus; remyelination; transplantation.

Figures

Figure 1.
Figure 1.
M3R KD induces human CD140a/PDGFαR+ OPC differentiation in vitro. A, Western blot confirmation of M3R KD by LV shRNAi in CHO-M3R cells. B–D, Fetal PDGFαR/CD140a+ hOPCs were infected with M3R (A, B) or control shRNAi LV, selected with puromycin and switched to mitogen-free media to encourage oligodendrocyte differentiation. Oligodendrocyte differentiation, determined by the proportion and morphological complexity of O4+ oligodendrocytes (green) and OLIG2+ cells (red), was increased in M3R KD hOPCs (B–D, quantified in E, mean ± SEM). MBP qRT-PCR, normalized to control OPCs in PDGF-AA, showed increase myelin protein gene expression following M3R KD (F, mean ± SEM). *p < 0.05 versus Ctrl (two-way ANOVA followed by Bonferroni post hoc test). ***p < 0.001 versus Ctrl (two-way ANOVA followed by Bonferroni post hoc test). There were 3 fetal samples (18- to 20-week gestational age). Scale bar, 20 μm.
Figure 2.
Figure 2.
M3R modulates muscarinic-evoked Ca2+ response in hOPCs. Fetal CD140a+ hOPCs were cultured and infected with intracellular Ca2+ reporter GCaMP6s and M3R or control shRNAi LV. Time-lapse microscopy of Ca2+ response following Oxo-M treatment was recorded and analyzed. A, Pseudo-color representation of Ca2+ response from control (top) and M3R KD (bottom) hOPCs. B, Normalized intensity plots for control (top) and M3R (bottom) KD hOPCs. C, Dose–response curves of percentage [Ca2+]i responding cells following Oxo-M addition. Per-cell quantification of average Ca2+ peak amplitude (D), total number of Ca2+ spikes (E), and area under the curve (F) in Ca2+-responsive hOPCs. M3R KD (red) resulted in a highly significant decrease in Ca2+ response at higher Oxo-M doses. *p < 0.05 (two-way ANOVA with Tukey's HSD post test). **p < 0.01 (two-way ANOVA with Tukey's HSD post test). ***p < 0.001 (two-way ANOVA with Tukey's HSD post test). There were two fetal preparations (n > 30 cells per condition/dose, mean ± SEM). Scale bar, 25 μm.
Figure 3.
Figure 3.
Long-term LV KD of M3R promotes remyelination by transplanted hOPCs. Shiverer/rag2 pups were transplanted with hOPCs following LV infection and selection. At 12 weeks after implantation, human cells were stained for hNA (red) and MBP (green) (A). Immunostaining for differentiated oligodendrocytes (CC1, green) showed that M3R KD increased the resultant density of human hNA+CC1+ oligodendrocytes (arrows) in the corpus callosum following engraftment (B, quantified in C). Analysis of MBP synthesis revealed that, compared with controls, M3R KD cells produced substantially more MBP-stained fibers per transplanted cell in the corpus callosum (D, quantified in E). Within myelinated regions, M3R KD led to an increased number of MBP (green) ensheathed axons (neurofilament, red) compared with controls (F, quantified in G). Dotted lines indicate outline of the corpus callosum. *p < 0.05 (t test). n = 3 animals per group, mean ± SEM shown. Scale bars: A, 500 μm; B, 20 μm; D, 10 μm; F, 20 μm.
Figure 4.
Figure 4.
Systemic solifenacin treatment enhances OPC differentiation following focal demyelination of mouse spinal cord. Young adult (8- to 10-week-old) mouse spinal cords were lesioned via direct injection of lysolecithin into dorsal white matter. Animals received daily subcutaneous administration of either isotonic saline (top row) or 10 mg/kg solifenacin (bottom row) from day of lesion until death at 14 dpl. In situ hybridization for Plp1 (A, quantified in F) and CC1 immunostaining (C, quantified in H) revealed increased density of oligodendrocytes in solifenacin animals relative to controls (quantified in F and H, respectively) with no observable effect on overall density of oligodendrocyte lineage cells (Olig2 in B, quantified in G), microglial response (Iba1, D), or astrogliosis (Gfap, E). *p < 0.05 (Student's t test). n = 3 per group, mean ± SEM shown. Scale bars: A, B, 40 μm; C–E, 100 μm.
Figure 5.
Figure 5.
Conditional M3R knock-out in OPCs enhances their differentiation following focal demyelination of mouse spinal cords. Genetic ablation of M3R in NG2+ OPCs was induced in NG2CreERT2:Rosa-YFP:M3Rfl/fl young adult (8- to 10-week-old) mice via daily intraperitoneal injection of 200 mg/kg tamoxifen. Mice received either tamoxifen or canola oil (vehicle) for 5 d, and their spinal cords were lesioned via direct injection of lysolecithin 1 week after the final day of intraperitoneal injections. Animals were killed at 14 dpl, and spinal cord tissue was collected for histological analysis. In situ hybridization for Plp1 revealed increased oligodendrocyte density in M3R icKO animals relative to WT controls (A, quantified in B). We observed a significantly greater proportion of oligodendrocyte lineage cells that were oligodendrocytes in M3R icKO animals relative to controls (immunostaining for CC1 in E, quantified in F). M3R deletion did not influence the overall density of oligodendrocyte lineage cells (Olig2 in C, quantified in D). OPC-specific conditional M3R knock-out had no observable effect on microglial response (Iba1, G) or astrogliosis (Gfap, H). *p < 0.05 (Student's t test). n = 4 or 5 per group, mean ± SEM shown. Scale bar, 200 μm.
Figure 6.
Figure 6.
Conditional ablation of M3R in OPCs accelerates remyelination. OPC-specific M3R knock-out was induced in adult NG2CreERT2:Rosa-YFP:M3Rfl/fl mice by daily intraperitoneal administration of 200 mg/kg tamoxifen or vehicle for 5 d. Animals were lesioned 1 week after the last day of injection. Animals were killed at 14 dpl and spinal cord tissue processed into resin for electron microscopy. Analysis revealed that M3R icKO in OPCs significantly increased the proportion of oligodendrocyte (OL) remyelinated axons in lesions compared with WT animals (A, quantified in B, mean ± SEM). C, Quantification of myelin sheath thickness in WT (green) and M3R iCKO (red) at 14 dpl by g-ratio analysis. SC, Schwann cell-remyelinated fiber. **p < 0.01 (Student's t test). n = 3 or 4 animals, ≥300 axons per animal. Scale bar, 2 μm.

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