A cholinergic neuroskeletal interface promotes bone formation during postnatal growth and exercise

Abstract

The autonomic nervous system is a master regulator of homeostatic processes and stress responses. Sympathetic noradrenergic nerve fibers decrease bone mass, but the role of cholinergic signaling in bone has remained largely unknown. Here, we describe that early postnatally, a subset of sympathetic nerve fibers undergoes an interleukin-6 (IL-6)-induced cholinergic switch upon contacting the bone. A neurotrophic dependency mediated through GDNF-family receptor-α2 (GFRα2) and its ligand, neurturin (NRTN), is established between sympathetic cholinergic fibers and bone-embedded osteocytes, which require cholinergic innervation for their survival and connectivity. Bone-lining osteoprogenitors amplify and propagate cholinergic signals in the bone marrow (BM). Moderate exercise augments trabecular bone partly through an IL-6-dependent expansion of sympathetic cholinergic nerve fibers. Consequently, loss of cholinergic skeletal innervation reduces osteocyte survival and function, causing osteopenia and impaired skeletal adaptation to moderate exercise. These results uncover a cholinergic neuro-osteocyte interface that regulates skeletogenesis and skeletal turnover through bone-anabolic effects.

Keywords: anabolic; autonomic; bone; cholinergic; development; exercise; neuroskeletal; osteocyte; skeletal; sympathetic.

Graphical abstract

Figure 1

Characterization of the cholinergic system in bone (A) Immunofluorescence of pan-neural TUJ1 in Nes-GFP femur. Insets show bone (A′) and growth plate (Aʺ) areas. Scale bars, 500 μm (A) and 100 μm (Aʹ and Aʺ). (B and C) Immunofluorescence of osteolineage markers in WT long bones with high-magnification insets. (D) Frequency of TUJ1⁺ cells among Nestin-GFP⁺ and osteolineage cells expressing RUNX2 or osteolectin. (E and F) Genetic tracing of cholinergic cells in (E) cortical bone and (F) growth plate of ChAT-IRES-cre;Ai35D mice. Scale bars, 200 μm. See also Figure S1C. (G and H) Immunofluorescence of VAChT⁺ cholinergic nerve fibers in (G) periosteum and (H) cortical bone in WT femur. See also Figures S1E–S1G. (I and J) Immunofluorescence of VIP⁺ or VAChT⁺ cholinergic nerve fibers in cortical bone of (I) a Nes-GFP mouse, or (J) WT or GFRα2 KO mice. (K and L) Area covered by (K) VAChT⁺ or VIP⁺ cholinergic nerve fibers in cortical bone or (L) VAChT⁺ cells near the growth plate of WT or GFRα2 KO mice. (M) Immunofluorescence of VAChT⁺ cells in the growth plate of WT or GFRα2 KO tibias. Scale bars, 200 μm. (N) Summary of changes in pan-neural and cholinergic markers in GFRα2 KO mice. N.A., not assessed. (B, C, and G–J) Scale bars, 100 μm. (D, K, and L) Data are mean ± SEM; ^∗∗p < 0.01, unpaired two-tailed t test. (A–I) Arrowheads depict nerve fiber staining and arrows depict non-neural staining. (A–J and M) Nuclei were counterstained with DAPI (blue). EMCN, endomucin.

Figure 2

Interleukin-6 induces a cholinergic switch in sympathetic neurons (A) Schematic of neonatal sympathectomy and analysis at adulthood. (B and C) Immunofluorescence of (B) GFRα2⁺ or (C) VAChT⁺ cholinergic nerve fibers in skulls (B) and the cortical bone (C) of adult mice subjected to neonatal chemical sympathectomy (6-OHDA) or saline treatment, with quantification of cholinergic nerve fibers. See also Figures S2A–S2C. (D) Immunofluorescence of VAChT in genetically traced sympathetic nerve fibers from TH-cre;Ai14D bones. (E) Schematic of superior cervical ganglion (SCG) isolation and culture. (F and G) qRT-PCR analysis of (F) cholinergic and (G) noradrenergic gene expression from WT SCG cultures. (H and I) Immunofluorescence of (H) noradrenergic (TH) and cholinergic (GFRα2) markers in 14-day cultured WT SCGs and (I) quantification. (J) Heatmap depicting fold change in mRNA expression of cholinergic and noradrenergic genes from day 14 SCG cultures relative to day 7 SCG cultures (n= 3–5). See also Figures S3A–S3G. (C, D, and H) Nuclei were counterstained with DAPI. (B–D and H) Scale bars, 100 μm. (B, C, F, G, and I) Data are mean ± SEM. ^∗p < 0.05, ^∗∗p < 0.01, and ^∗∗∗p < 0.001; ANOVA and pairwise comparisons.

Figure 3

Interleukin-6 promotes postnatal development of skeletal sympathetic cholinergic innervation (A and B) Proximity ligation assay of IL-6 in BM section from postnatal day-3 developing femur. Scale bars, 100 μm (A) and 50 μm (B). (C) Schematic of in vivo IL-6 blockade. (D–F) Immunofluorescence of (D) VAChT⁺, or (E) GFRα2⁺ cholinergic nerve fibers (green) in (D) femoral cortical bone and (E) skulls from 6.5-week-old mice following IL-6 blockade, with (F) quantification of fiber area. Dashed lines indicate bone surface. (G–I) Immunofluorescence of (G) VAChT⁺ or (H) GFRα2⁺ cholinergic nerve fibers in (G) femoral cortical bone and (H) skulls from male IL-6 KO mice, with (I) quantification of fiber area. Dashed lines indicate cranial sutures. (D, E, G, and H) Scale bars, 100 μm. Nuclei were counterstained with DAPI (blue). (F and I) Data are mean ± SEM, ^∗p < 0.05, ^∗∗p < 0.01, unpaired two-tailed t test.

Figure 4

Osteolineage cells contribute to the non-neuronal cholinergic system (A–E) Immunofluorescence of cholinergic bone-lining cells near the growth plate (gp) in (A–C) ChAT-IRES-cre;Ai35D and (D and E) ChAT-IRES-cre;Ai14D mice with co-staining of osteolineage markers. Arrows depict co-localization in high-magnification insets (Bʹ–Eʺ). 2HG, 2^nd harmonic generation imaging of collagen. See also Figures S4A and S4B. (F) Quantification of co-localization of ChAT-labeled cells. (G and H) Immunofluorescence of cholinergic bone-lining cells near the (G) growth plate and (H) endosteal regions of ChAT-IRES-cre;Ai14D;Nes-GFP tibias. Arrows depict nerve fibers. Arrowheads depict osteolineage cells. See also Figures S4C and S4D. (A–E) Scale bars, 100 μm (A and B) and 200 μm (G and H). (I) Flow cytometry gating strategy for analysis of CD31⁻CD45⁻Ter119⁻PDGFRα⁺Sca1⁻ (PαS⁻) cells, CD31⁻CD45⁻Ter119⁻PDGFRα⁺Sca1⁺ (PαS⁺) cells, CD31⁻CD45⁻Ter119⁻PDGFRα⁻CD51⁺Sca1⁺ (OPS⁺) cells, and CD31⁻CD45⁻Ter119⁻PDGFRα⁻CD51⁺Sca1⁻ (OPS⁻) cells. (J) Frequency of ChAT-IRES-cre-traced osteolineage cells in endosteal or central BM. See also Figures S4E and S4F. (K) Acetylcholine content in osteolineage cells from WT and Nes-GFP mice. (L) Acetylcholine content in primary WT osteoblasts (OB) and osteocytes (OC) from digested WT bone fragments. (M) Schematic of CD51⁺ osteolineage cell isolation and culture. (N and O) Acetylcholine content (N) and qRT-PCR analysis (O) of cultured CD51⁺ osteolineage cells. (P) Acetylcholine content from endosteal and central BM serum of mice lacking an α7 nicotinic receptor in LepR-cre targeted niche cells 1 month after bone marrow transplantation. (F and J–P) Data are mean ± SEM, ^∗p < 0.05, ^∗∗p < 0.01, ^∗∗∗p < 0.001, and ^∗∗∗∗p < 0.0001; unpaired two-tailed t test (F and J–O) or ANOVA and pairwise comparisons (P).

Figure 5

GFRα2 loss causes osteopenia and osteocyte degeneration (A and B) Quantitative μCT analysis of 3D cortical (A) and trabecular (B) bone parameters in WT or GFRα2 KO female tibias: tissue volume (TV), bone volume (BV), cortical bone volume fraction (Ct.BV/TV), cortical thickness (Ct.Th), trabecular bone volume fraction (Tb.BV/TV), trabecular thickness (Tb.Th), trabecular separation (Tb.Sp), and trabecular number (Tb.N). See also Figures S5A and S5B. (C) 3D rendering of proximal mid-tibial diaphysis from WT or GFRα2 KO female mice. Scale bars, 1 mm. (D) Three-point bend testing of tibias from WT or GFRα2 KO female mice. See also Figure S5E. (E and F) Immunofluorescence of trabecular bone from WT or GFRα2 KO female mice injected with calcein and xylenol orange (E) with quantification of bone formation rate (BFR) and mineral apposition rate (MAR) (F). Scale bars, 50 μm. See also Figure S5F. (G) Quantification of tartrate-resistant acid phosphatase (TRAP⁺) multinucleated giant cells (MGCs) from WT or GFRα2 KO BM sections. See also Figure S5G. (H–K) Phalloidin staining (H and J, green) and quantification (I and K) of osteocytes embedded in (H and I) cortical bone or (J and K) trabecular bone from WT or GFRα2 KO mice. Nuclei were counterstained with DAPI (blue). Scale bars, 50 μm. (L) Transmission electron micrographs of osteocytes (left) and surrounding collagen matrix (right) from WT or GFRα2 KO humeri. Arrowheads depict osteocyte cell processes. Scale bars, 500 nm. See also Figures S6C and S6D. (A, B, D, F, G, I, and K) Data are mean ± SEM, ^∗p < 0.05, ^∗∗p < 0.01, ^∗∗∗p < 0.001, unpaired two-tailed t test.

Figure 6

GFRα2 signaling maintains osteocyte connectivity and survival (A) Schematic of primary calvarial osteoblast (pOB) isolation and differentiation into osteocyte-like cells (OLCs). (B) Quantitative analysis of SYTOX⁺ dead cells in d21 WT or GFRα2 KO OLCs. (C) Number of osteocytes quantified from low-magnification phalloidin-stained cortical bone sections. See also Figure S6A. (D and E) Heatmap depicting mRNA expression from (D) calvarial pOBs and differentiated OLCs (n = 3–5) or (E) primary osteoblasts (OB) and osteocytes (OC) from digested WT or GFRα2 KO femur fragments (n = 6). (F–H) Analysis of dynamic histomorphometry (F, femurs), three-point bend (G, tibias), and trabecular thickness (Tb.Th, tibias) in male WT or GFRα2 KO mice subjected to treadmill exercise (5× per week) with s.c. treatment of Scl-Ab r13c7 (1 per week) for 5 weeks. (I and J) Cell numbers (I) and frequency of apoptotic (J) pOBs after 4-day treatment with GDNF-family ligands and soluble receptors. (K) Proximity ligation assay of neurturin in BM section from WT femur, with co-staining for VAChT⁺ cholinergic nerve fibers. (L–Q) Fluorescence (L and O, green) and quantification of phalloidin⁺ (M and P) osteocytes (N and Q) from adult WT mice subjected to neonatal chemical sympathectomy (6-OHDA; L–N), or WT or neurturin (Nrtn) KO mice (O–Q). (K, L, and O) Scale bars, 100 μm. Nuclei were counterstained with DAPI (blue). (B, C, F–J, M, N, P, and Q) Data are mean ± SEM, ^∗p < 0.05, ^∗∗p < 0.01, ^∗∗∗p < 0.001, unpaired two-tailed t test.

Figure 7

Moderate exercise increases bone cholinergic innervation through interleukin-6 (A) Schematic of moderate exercise protocol and analysis. (B–J) Characterization of cholinergic cells in sedentary or exercised (B, C, I, and J) WT mice or (D–H) ChAT-IRES-cre;Ai14D;Nes-GFP mice treated with IL-6 inhibitors or control IgG. (B and C) Immunofluorescence (B) and quantification (C) of GFRα2⁺ cholinergic fibers in skulls. Tyrosine hydroxylase (TH)⁺ noradrenergic fibers are also shown. (D and E) Immunofluorescence (D) and quantification (E) of genetically marked cholinergic nerve fibers (arrowheads). Arrow depicts ChAT⁺ osteocyte. (F–H) Immunofluorescence (F) and flow cytometric quantification (G and H) of genetically traced cholinergic osteolineage cells including (G) CD31⁻CD45⁻Ter119⁻PDGFRα⁻CD51⁺Sca1⁺ OPS+ cells and (H) CD31⁻CD45⁻Ter119⁻PDGFRα⁻CD51⁺Sca1⁻ OPS− cells. (B, D, and F) Nuclei were counterstained with DAPI (blue). Scale bars, 100 μm. (I and J) Acetylcholine content in (I) OPS⁻ cells and (J) osteocytes. (K and L) Quantitative μCT analysis of (K) cortical thickness (Ct.Th) and (L) trabecular thickness (Tb.Th) in sedentary or exercised male control WT mice, WT mice treated with IL-6 inhibitors, GFRα2 KO mice, or IL-6 KO mice. (C, E, and G–L) Data are mean ± SEM, ^∗p < 0.05, ^∗∗p < 0.01, ^∗∗∗p < 0.001, unpaired two-tailed t test.