Muscarinic Receptor M₃R Signaling Prevents Efficient Remyelination by Human and Mouse Oligodendrocyte Progenitor Cells

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.

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.

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.

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 NG2CreER^T2:Rosa-YFP:M₃R^fl/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.