Analysis of skeleton in a mouse model of Rett syndrome

Q Science (General);QM Human anatomy;R Medicine (General);RD Surgery;RJ Pediatrics

Kamal, Bushra ; Cobb, Stuart

University:University of Glasgow

Department:Institute of Neuroscience and Psychology

关键词: Rett syndrome, anatomy, bone, MECP2, biomechanical properties, trabecular bone, cortical bone, collagen;   

Others  :  http://theses.gla.ac.uk/6092/1/2015Kamal%20PhD.pdf

来源: University of Glasgow

【 摘 要 】

Rett Syndrome (RTT) is an X-linked genetic disorder and a major cause of intellectual disability in girls. Mutations in the methyl-CpG binding protein 2 (MECP2) gene, are the primary cause of the disorder. Despite the dominant neurological phenotypes that characterise RTT, MECP2 is expressed ubiquitously throughout the body and a number of peripheral phenotypes such as growth retardation (reduced height and weight), skeletal deformities (scoliosis/kyphosis), reduced bone mass and low energy fractures are also common yet under-reported clinical features of the disorder. In order to explore whether MeCP2 protein deficiency results in altered structural and functional properties of bone and to test the potential reversibility of any such defects, I have conducted series of histological, imaging and biomechanical tests of bone using an accurate genetic (functional knockout) mouse model of RTT. Initial experiments using a GFP reporter mouse line demonstrated the presence of MeCP2 in bone cells and the effective silencing on the gene in functional knockout mice. Different aspects of the study were conducted in different types of bone tissues that were especially suited for individual assays. For instance, biomechanical three point bending tests were conducted in long bone (femur) whilst trabecular geometry measures were measured in spinal vertebrae.Both hemizygous Mecp2stop/y male mice in which Mecp2 is silenced in all cells and female Mecp2stop/+ mice in which Mecp2 is silenced in ~50% of cells as a consequence of random X-chromosome inactivation (XCI), revealed, lighter and smaller long bones and significant reductions in cortical bone mechanical properties (~ 39.5% reduction in stiffness, 31% reduction in ultimate load and 37% reduction in Young’s modulus respectively in Mecp2stop/y male mice; %) and material properties (microhardess reduced 12.3% in Mecp2stop/y male mice and 14% inMecp2stop/+ female mice) as compared to age wild type control mice. Micro structural analysis conducted using µCT also revealed a significant reduction in cortical (54% reduction in cortical thickness, 30% in bone volume, 20% in total area, and 38% in marrow area) and trabecular (~30% in trabecular thickness) bone parameters as compared to age matched wild-type controls MeCP2-deficent mice. Histological analysis using Sirius red staining as a marker of collagen revealed a ~25% reduction in collagen content in MeCP2 deficient mice as compared to age matched wild type controls. In experiments designed to establish the potential for reversal of MeCP2-related deficits, unsilencing of Mecp2 in adult mice by tamoxifen-induced and cre-mediated excision of a stop cassette located at the endogenous Mecp2 locus (male; Mecp2stop/y, CreER and female; Mecp2+/stop, CreER), resulted in a restoration of biomechanical properties towards the wild-type levels. Specifically, Male Mecp2stop/y, CreER mice displayed improvement in mechanical properties (stiffness 40%, ultimate load 10%, young’s modulus 61% and micro hardness 12%) and structural bone parameter (trabecular thickness 80%) as compared to Mecp2stop/y male mice. Female Mecp2+/stop, CreER, displayed a significant improvement (19%) in microhardess measures as compared to Mecp2 deficient mice. Overall, the results of my studies show that MeCP2-deficiency results in overt, but potentially reversible, alterations in the biomechanical integrity of bone and highlights the importance of targeting skeletal phenotypes in considering the development of pharmacological and gene-based therapies for Rett Syndrome