Disrupted mitochondrial function in the Opa3L122P mouse model for Costeff Syndrome impairs skeletal integrity

Human Molecular Genetics, 2016, Vol. 25, No. 12 2404–2416 doi: 10.1093/hmg/ddw107 Advance Access Publication Date: 22 April 2016 Original Article ORIGINAL ARTICLE Disrupted mitochondrial function in the Opa3L122P mouse model for Costeff Syndrome impairs skeletal integrity 1 School of Biosciences, Cardiff University, Cardiff CF10 3AX, UK, 2School of Optometry and Vision Sciences, Cardiff University, Cardiff CF24 4LU, UK, 3Caerleon Comprehensive School, Caerleon, Newport NP18 1NF, UK, 4 Department of Craniofacial Development and Stem Cell Biology, King’s College London, Guy’s Hospital, London SE1 9RT, UK, 5School of Engineering, Cardiff University, The Parade, Cardiff CF24 3AA, UK, 6Institute of Molecular and Experimental Medicine, School of Medicine, Cardiff University, Heath Park, Cardiff CF14 4XN, UK and 7Cardiff Eye Unit, University Hospital of Wales, Heath Park, Cardiff CF14 4XW, UK *To whom correspondence should be addressed: Tel: þ44 (0)2920 874977; Fax: þ44 (0)2920 876328; E-mail: Abstract Mitochondrial dysfunction connects metabolic disturbance with numerous pathologies, but the significance of mitochondrial activity in bone remains unclear. We have, therefore, characterized the skeletal phenotype in the Opa3L122P mouse model for Costeff syndrome, in which a missense mutation of the mitochondrial membrane protein, Opa3, impairs mitochondrial activity resulting in visual and metabolic dysfunction. Although widely expressed in the developing normal mouse head, Opa3 expression was restricted after E14.5 to the retina, brain, teeth and mandibular bone. Opa3 was also expressed in adult tibiae, including at the trabecular surfaces and in cortical osteocytes, epiphyseal chondrocytes, marrow adipocytes and mesenchymal stem cell rosettes. Opa3L122P mice displayed craniofacial abnormalities, including undergrowth of the lower mandible, accompanied in some individuals by cranial asymmetry and incisor malocclusion. Opa3L122P mice showed an 8fold elevation in tibial marrow adiposity, due largely to increased adipogenesis. In addition, femoral length and cortical diameter and wall thickness were reduced, the weakening of the calcified tissue and the geometric component of strength reducing overall cortical strength in Opa3L122P mice by 65%. In lumbar vertebrae reduced vertebral body area and wall thickness were accompanied by a proportionate reduction in marrow adiposity. Although the total biomechanical strength of lumbar vertebrae was reduced by 35%, the strength of the calcified tissue (rmax) was proportionate to a 38% increase in trabecular number. Thus, mitochondrial function is important for the development and maintenance of skeletal integrity, impaired bone growth and strength, particularly in limb bones, representing a significant new feature of the Costeff syndrome phenotype. Received: February 15, 2016. Revised: March 29, 2016. Accepted: March 30, 2016 C The Author 2016. Published by Oxford University Press. V This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted reuse, distribution, and reproduction in any medium, provided the original work is properly cited. 2404 Alice E. Navein1, Esther J. Cooke1, Jennifer R. Davies2, Terence G. Smith2, Lois H. M. Wells1,3, Atsushi Ohazama4, Christopher Healy4, Paul T. Sharpe4, Sam L. Evans5, Bronwen A. J. Evans6, Marcela Votruba2,7 and Timothy Wells1,* Human Molecular Genetics, 2016, Vol. 25, No. 12 Introduction integrity, uncovering a striking novel aspect of the Costeff syndrome phenotype. Results Skeletal Opa3 expression Opa3 was expressed ubiquitously in the developing head of WT mice until E13.5 (Fig. 1A and B), but from E14.5 expression of Opa3 became more restricted, being seen in the developing submandibular salivary glands at E14.5 (Fig. 1Ci), the sensory layer of the retina and in hair follicles (Fig. 1Cvii and Di) and the developing brain and mandibular bone, but not in Meckel’s cartilages (Fig. 1Cii and iii), or in developing skull bone (Fig. 1Dii). Expression was also observed in developing cap stage tooth germs at E14.5 (Fig. 1Civ–vi), whilst in the molars, Opa3 was strongly expressed in odontoblasts while it was weakly expressed in ameloblasts at E18.5 (Fig. 1Diii). In the incisors the immature odontoblasts showed strong Opa3 expression whereas Opa3 was weakly expressed in mature odontoblasts at E18.5 (Fig. 1Div–vii). In contrast, strong Opa3 expression was observed in ameloblasts in both anterior and posterior regions of incisors at E18.5 (Fig. 1Div–vii). Immunohistochemistry (IHC) was used to show that the most pronounced Opa3 expression in adult tibiae was observed in the marrow cavity (Fig. 2A). Discrete expression was observed in cells on the endosteal and trabecular surfaces (Fig. 2C) and on the surfaces of the cortical periosteum (Fig. 2E) and synovial cartilage (Fig. 2G). In addition, Opa 3 immunoreactivity was seen in cortical osteocytes (Fig. 2E) articular chondrocytes (Fig. 2G) and in the germinal, proliferative and hypertrophic chondrocytes of the proximal epiphyseal plate (Fig 2I and K). In the marrow cavity, Opa 3 immunoreactivity was observed around the unilocular lipid droplets of the adipocytes (Fig. 2I and K) and in distinct rosettes of mesenchymal stem cells in the mid-diaphyseal marrow (Fig. 2M). Skull bone structure in Costeff syndrome mice No abnormalities were observed in heads of E18.5 Opa3L122P mice (data not shown), but micro-computed tomography (m-CT) analysis revealed an impairment in postnatal skull development. Overall skull length was reduced by 10% in Costeff syndrome mice at P14 (Table 1, Fig. 3; P < 0.001), which appears to be accompanied in adult mice by a shortening of the snout (Fig. 3). An 8% reduction in nasal height in Costeff syndrome mice at P14 (Table 1 and Fig. 3; P < 0.05) was accompanied by reductions of 11% and 14% in the height and length of the lower mandible (Table 1 and Fig. 3; P < 0.01 and P < 0.001 respectively). Individual Costeff syndrome mice displayed varying degrees of cranial asymmetry, which was particularly observable in the shape of the occipital bone and accompanied by misalignment of the lower mandible and incisors (Table 1 and Fig. 3; P < 0.05). At P14 mean incisor length in Costeff syndrome mice was 86% of that in WT littermates, but this was only significantly different in the lower incisors (Table 1 and Fig. 3; P < 0.05). However, malocclusion in adult Opa3L122P mice (Fig. 3) was accompanied in some individuals by incisor elongation. Marrow adiposity in Costeff syndrome mice Adiposity in the tibial marrow cavity of Opa3L122P mice was increased 8-fold (Fig. 4A; P < 0.05), due largely to a 6-fold elevation The mitochondrion represents a sub-cellular powerstation, utilizing protons from dietary carbohydrates and fat to generate heat and adenosine triphosphate. This function is particularly vital in tissu (...truncated)