Impaired osteoclast homeostasis in the cystatin B-deficient mouse model of progressive myoclonus epilepsy

Otto Manninen,Tero Puolakkainen,Jemina Lehto,Elina Harittu,Aki Kallonen,Marko Peura,Tiina Laitala-Leinonen,Outi Kopra,Riku Kiviranta,Anna-Elina Lehesjoki

doi:10.1016/j.bonr.2015.10.002

Abstract

Progressive myoclonus epilepsy of Unverricht–Lundborg type (EPM1) is an autosomal recessively inherited disorder characterized by incapacitating stimulus-sensitive myoclonus and tonic-clonic epileptic seizures with onset at the age of 6 to 16years. EPM1 patients also exhibit a range of skeletal changes, e.g., thickened frontal cranial bone, arachnodactyly and scoliosis. Mutations in the gene encoding cystatin B (CSTB) underlie EPM1. CSTB is an inhibitor of cysteine cathepsins, including cathepsin K, a key enzyme in bone resorption by osteoclasts. CSTB has previously been shown to protect osteoclasts from experimentally induced apoptosis and to modulate bone resorption in vitro. Nevertheless, its physiological function in bone and the cause of the bone changes in patients remain unknown. Here we used the CSTB-deficient mouse (Cstb−/−) model of EPM1 to evaluate the contribution of defective CSTB protein function on bone pathology and osteoclast differentiation and function. Micro-computed tomography of hind limbs revealed thicker trabeculae and elevated bone mineral density in the trabecular bone of Cstb−/− mice. Histology from Cstb−/− mouse bones showed lower osteoclast count and thinner growth plates in long bones. Bone marrow-derived osteoclast cultures revealed lower osteoclast number and size in the Cstb−/− group. Cstb−/− osteoclasts formed less and smaller resorption pits in an in vitro assay. This impaired resorptive capacity was likely due to a decrease in osteoclast numbers and size. These data imply that the skeletal changes in Cstb−/− mice and in EPM1 patients are a result of CSTB deficiency leading to impaired osteoclast formation and consequently compromised resorptive capacity. These results suggest that the role of CSTB in osteoclast homeostasis and modulation of bone metabolism extends beyond cathepsin K regulation.

Highlights

Progressive myoclonus epilepsy of Unverricht–Lundborg type (EPM1, OMIM 254800, progressive myoclonus epilepsy type 1) is an autosomal recessively inherited neurodegenerative disorder with onset at the age of 6–16 years
Quantitative real-time PCR Total RNA was isolated from osteoclasts (Cstb−/− mice n = 3; control mice, n = 3, all male, cells pooled) cultured for 6 days on RANKL using Qiagen RNAeasy kit (Qiagen, Heiden, Germany) following the instructions of the manufacturer
Qiagen RT2 Profiler PCR Array osteogenesis kit (Qiagen, Hilden, Germany) was used to assess gene expression differences in Cstb−/− osteoclasts compared to controls (136 ng of RNA used for both groups)

Summary

Introduction

Progressive myoclonus epilepsy of Unverricht–Lundborg type (EPM1, OMIM 254800, progressive myoclonus epilepsy type 1) is an autosomal recessively inherited neurodegenerative disorder with onset at the age of 6–16 years. In addition to the neurological features, EPM1 patients have heterogeneous bone findings (Korja et al, 2007, Suoranta et al, 2012, Danner et al, 2013). They exhibit diffuse thickening of cranial bones in head magnetic resonance imaging (Suoranta et al, 2012). The majority of EPM1 patients show other skeletal changes, most commonly arachnodactyly, scoliosis, enlarged sinuses, and accessory ossicles of the foot (Suoranta et al, 2012), the etiology of which is currently unknown

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Conclusion