Antidementia medication acetylcholinesterase inhibitors have therapeutic benefits on osteoporotic bone by attenuating osteoclastogenesis and bone resorption

Abstract

This study was designed to determine whether the use of acetylcholinesterase inhibitors (AChEIs), a group of drugs that stimulate acetylcholine receptors and are used to treat Alzheimer's disease (AD), is associated with osteoporosis protection and inhibition of osteoclast differentiation and function. Firstly, we examined the effects of AChEIs on RANKL-induced osteoclast differentiation and function with osteoclastogenesis and bone resorption assays. Next, we investigated the impacts of AChEIs on RANKL-induced nuclear factor κB and NFATc1 activation and expression of osteoclast marker proteins CA-2, CTSK and NFATc1, and dissected the MAPK signaling in osteoclasts in vitro by using luciferase assay and Western blot. Finally, we assessed the in vivo efficacy of AChEIs using an ovariectomy-induced osteoporosis mouse model, which was analyzed using microcomputed tomography, in vivo osteoclast and osteoblast parameters were assessed using histomorphometry. We found that Donepezil and Rivastigmine inhibited RANKL-induced osteoclastogenesis and impaired osteoclastic bone resorption. Moreover, AChEIs reduced the RANKL-induced transcription of Nfatc1, and expression of osteoclast marker genes to varying degrees (mainly Donepezil and Rivastigmine but not Galantamine). Furthermore, AChEIs variably inhibited RANKL-induced MAPK signaling accompanied by downregulation of AChE transcription. Finally, AChEIs protected against OVX-induced bone loss mainly by inhibiting osteoclast activity. Taken together, AChEIs (mainly Donepezil and Rivastigmine) exerted a positive effect on bone protection by inhibiting osteoclast function through MAPK and NFATc1 signaling pathways through downregulating AChE. Our findings have important clinical implications that elderly patients with dementia who are at risk of developing osteoporosis may potentially benefit from therapy with the AChEI drugs. Our study may influence drug choice in those patients with both AD and osteoporosis.

Keywords: acetylcholinesterase inhibitors; drug choice; osteoclast; osteoporosis.

Figure 1

Donepezil and Rivastigmine inhibited osteoclast formation, whereas Galantamine showed no effect. (a) RANKL‐induced osteoclastogenesis using BMMs extracted from wild‐type mouse treated with Galantamine (G), Donepezil (D) and Rivastigmine (R) at various concentrations (0.1/0.5/1.0/5.0 μM). Light microscope images depicting the dose‐dependent effect of AChEIs on RANKL‐induced osteoclast formation at ×100 magnification and the representative images were shown to indicate the level of TRAP staining. (b) The representative images of positive and negative control. (c) Qualification of the number of TRAP⁺ MNCs, counted as osteoclasts (n = 3). *p < 0.05, **p < 0.01 relative to vehicle control, RANKL‐treated cultures. All the experiments were repeated three times and the results are presented as mean ± SD. AChEIs, acetylcholinesterase inhibitors; BMM, bone marrow macrophage.

Figure 2

Donepezil and Rivastigmine attenuated RANKL‐induced bone resorption by osteoclasts, whereas Galantamine showed no effect. (a−h) Representative images of TRAP stained (a−d) and scanning electron microscopy of resorption pits (e−h) formed by bone marrow‐derived osteoclasts in bone slices treated with RANKL only (a, e), RANKL+Galantamine (b, f), RANKL + Donepezil (c, g) and RANKL+Rivastigmine (d, h) (mag = 100× for A‐D and mag = ×400 for e−h). (I) Quantification of TRAP positive multinucleated cells per bone slice. (j) Quantification of relative pit area per osteoclast. Experiments were carried out in triplicate and results are presented as mean ± SD. *p < 0.05 versus control group with RANKL treatment only.

Figure 3

The effect of AChEI on RANKL‐stimulated NFAT and NF‐κB activity, and downstream protein expression. (a−f) BMM cells stably transfected with an NFAT (a−c) and NF‐κB (d−f) transcriptional luciferase reporter construct, were pretreated with Galantamine (a, d), Donepezil (b, e) and Rivastigmine (c, f) for 1 h and then exposed to RANKL for 6 h and 24 h for NF‐κB and NFAT respectively. (g−i) BMM cells were pretreated with 10 μM Galantamine (g), Donepezil (h) and Rivastigmine (i) for 1 h before RANKL (100 ng/mL) stimulation for 0, 1, 3, 5, and 7 days. Representative blots of cell lysates for NFATc1, Carbonic Anhydrase 2 (CA2) and Cathepsin‐K (CTSK) were presented. *p < 0.05, **p < 0.01 compared to control group. BMM, bone marrow macrophage; NF‐κB, nuclear factor κB.

Figure 4

AChEI inhibited the RANKL‐induced MAPK signaling pathway. Representative Western blot images of p‐P38, P38, p‐JNK, JNK, p‐ERK, ERK, and GAPDH at 0, 10, 20, 30, and 60 min stimulated by RANKL (50 ng/mL) with or without Galantamine (a), Donepezil (b) and Rivastigmine (c). The relative ratio of phosphorylated proteins and unphosphorylated proteins were quantitatively determined. The expression of all the proteins mentioned above was determined relative to GAPDH expression. Significant differences between the treatment and control groups are indicated as *p < 0.05 and **p < 0.01.

Figure 5

The mRNA expression of AChE increased during osteoclastogenesis but significantly decreased in AChEIs‐treated BMMs. The primers for qRT‐PCR were presented in the Table (a) with GAPDH as the reference. *p < 0.05, **p < 0.01 compared with the 0 h (b) or RANKL only (c) control group. BMM, bone marrow macrophage.

Figure 6

Micro‐CT analysis of hind limbs revealing AChEI inhibits OVX‐induced bone loss in vivo. (a, b) Representative 3D reconstructions of trabecular bone, cortical bone and bone parameters assessed by micro‐CT in distal femur (a, A′−J′) and proximal tibia (b, A’−J’) in an age‐matched five groups of mice, respectively. Trabecular bone parameters (a, B’−E’, and b, B’−E’) are shown as trabecular bone volume fraction (BV/TV, %; a, B’ and b, B’), trabecular number (Tb.N, 1/mm; a, C’ and b, C’), trabecular thickness (Tb.Th, mm; a, D’ and b, D’) and trabecular separation (Tb.Sp, mm; a, E’ and  E’). Cortical bone parameters (a, G’‐J’ and b, G’−J’) are shown as total cortical area (Tt.Ar, mm²; a, G’ and b, G’), cortical bone area (Ct.Ar, mm²; a, H’ and b, H’), cortical area fraction (Ct.Ar/Tt. Ar, %; a, I’ and b, I’) and cortical thickness (Ct.Th, μm; a, J’ and b, J’). Data are presented as mean ± SD. OVX Ovariectomy, Gal Galantamine, Don Donepezil, Riv Rivastigmine. *p < 0.05, **p < 0.01 compared with OVX control group.

Figure 7

Donepezil and Rivastigmine protected against OVX‐induced bone loss via inhibiting osteoclast activity in vivo. (a, b) Representative low‐power images of H&E (a) and TRAP (b) stained femur sections. A higher magnification micrograph of the area within a square in B was presented below each, and yellow arrows in B indicate TRAP‐stained osteoclasts within the femur. The red bar and black bar represent 200 and 100 μm, respectively. (c) Quantitative histomorphometric analysis of bone parameters: Number of osteoclasts per bone perimeter (N.Oc/B. Pm, mm⁻¹), Osteoclast surface relative to bone surface (Oc.S/BS, %), Number of osteoblasts per bone perimeter (N.Ob/B.Pm, mm⁻¹), and Osteoblast surface relative to bone surface (Ob.S/BS, %). n = 7 mice per group. Bar charts represent mean ± SD. *p < 0.05 compared with the OVX control group.

Figure 8

A schematic diagram illustrating the suppression of AChEIs on RANKL‐induced MAPK and NFATc1 activation during osteoclastogenesis, thus increasing bone mineral density.