Donepezil increased collagen expression in ovine osteoporotic mesenchymal stem cells

Introduction:

Acetylcholine (ACh) and its receptors, transporters, synthesis and degradation enzymes were found in bone. The neuronal as well as non-neuronal cholinergic system is discussed to be involved in bone mass accrual, bone composition, and regulation of proliferation and differentiation of osteoblasts and its progenitors, mesenchymal stem cells (MSC). Recently, a retrospective cohort study reported that patients with hip fractures that additionally suffer from Alzheimer's disease (AD) had improved bone quality, better fracture healing, and less healing complication if they were treated with Donepezil which is used in AD therapy [1]. Donepezil inhibits the ACh degradation molecule acetylcholinesterase (AChE) selectively and reversibly. A case-control study showed a reduced fracture risk in AD patients who used the AChE inhibitor (AChEI) Donepezil [2]. Thus, we investigated the application of Donepezil on ovine MSC of an osteoporosis model regarding their proliferation rate and gene-expression.

Methods:

MSC of 20 female bone adult merino sheep were isolated, characterized and divided into 4 groups: a) untreated control group (C), b) sheep with bilateral ovariectomy (OVX), c) sheep with OVX and Vitamin D and calcium malnutrition (OD), and d) sheep with OVX, malnutrition and application of 320 mg i.m. of the corticosteroid Methylprednisolone (ODS). Eight months after OVX a bone biopsy of the iliac crest was performed, MSC isolated and cultured. MSC were treated with 15.4 nM Donepezil for 12 hours and AChE activity (AChE Assay kit, abcam, Cambridge, UK), proliferation rate (colorimetric BrdU assay, Roche, Basel, Switzerland), mRNA expression, matrix deposition by Picro-Sirius Red staining (Abcam, Cambridge, UK) were analyzed.

Results:

Application of Donepezil resulted in a significant decrease of AChE activity (p < 0.001). Inhibition of AChE did not lead to a significant increase in proliferation (BrdU assay, p = 0.063). On mRNA level no reduction of AChE and also butyrylcholinesterase was observed after application of Donepezil. Expression of the osteogenic markers osteocalcin was not regulated by Donepezil while the mRNA concentration of collagen 1α1 was increased in Donepezil treated MSC (p = 0.033) as well as the collagen deposition determined by Picro-Sirius Red staining.

Discussion:

In conclusion, AChE inhibition via Donepezil resulted in an increased synthesis of osteoid which consists mainly of collagen 1α1. Thus, we suppose that increased ACh levels through AChEI do not support MSC proliferation but synthesis of bone matrix. References: [1] Eimar H et al. 2013J Musculoskelet Neuronal Interact 13(4):454 – 463. [2] Tamimi I et al. 2012 JBMR 27(7):1518 – 1527. The study was supported by DFG (SFB/TRR 79 project B7 and T1).