Fragile X Messenger Ribonucleoprotein 1 (FMR1), a novel inhibitor of osteoblast/osteocyte differentiation, regulates bone formation, mass, and strength in young and aged male and female mice

Abstract

Fragile X Messenger Ribonucleoprotein 1 (FMR1) gene mutations lead to fragile X syndrome, cognitive disorders, and, in some individuals, scoliosis and craniofacial abnormalities. Four-month-old (mo) male mice with deletion of the FMR1 gene exhibit a mild increase in cortical and cancellous femoral bone mass. However, consequences of absence of FMR1 in bone of young/aged male/female mice and the cellular basis of the skeletal phenotype remain unknown. We found that absence of FMR1 results in improved bone properties with higher bone mineral density in both sexes and in 2- and 9-mo mice. The cancellous bone mass is higher only in females, whereas, cortical bone mass is higher in 2- and 9-mo males, but higher in 2- and lower in 9-mo female FMR1-knockout mice. Furthermore, male bones show higher biomechanical properties at 2mo, and females at both ages. Absence of FMR1 increases osteoblast/mineralization/bone formation and osteocyte dendricity/gene expression in vivo/ex vivo/in vitro, without affecting osteoclasts in vivo/ex vivo. Thus, FMR1 is a novel osteoblast/osteocyte differentiation inhibitor, and its absence leads to age-, site- and sex-dependent higher bone mass/strength.

References

[1] Christensen DL et al. Prevalence and charactersitics of autism spectrum disorder among children aged 8 years - autism and developmental disabilities monitoring network, 11 sites, United States, 2012. Surveill Summ., 2012, 65: 1-23

[2] Grigsby J. The fragile X mental retardation 1 gene (FMR1): historical perspective, phenotypes, mechanism, pathology, and epidemiology. Clin. Neuropsychol., 2016, 30: 815-833

[3] Coffee B et al. Incidence of fragile X syndrome by newborn screening for methylated FMR1 DNA. Am. J. Hum. Genet., 2009, 85: 503-514

[4] Kaufmann WE, Reiss AL. Molecular and cellular genetics of fragile X syndrome. Am. J. Med. Genet., 1999, 88: 11-24

[5] Bakker CE et al. Fmr1 knockout mice: a model to study fragile X mental retardation. The Dutch-Belgian fragile X consortium. Cell, 1994, 78: 23-33

[6] Bernardet M, Crusio WE. Fmr1 KO mice as a possible model of autistic features. Sci. World J., 2006, 6: 1164-1176

[7] Davids JR, Hagerman RJ, Eilert RE. Orthopaedic aspects of fragile-X syndrome. J. Bone Jt. Surg. Am., 1990, 72: 889-896

[8] Butler MG, Pratesi R, Watson MS, Breg WR, Singh DN. Anthropometric and craniofacial patterns in mentally retarded males with emphasis on the fragile X syndrome. Clin. Genet., 1993, 44: 129-138

[9] Heulens I et al. Craniofacial characteristics of fragile X syndrome in mouse and man. Eur. J. Hum. Genet, 2013, 21: 816-823

[10] Hjalgrim H, Fisher HB, Brondum-Nielsen K, Nolting D, Kjaer I. Aspects of skeletal development in fragile X syndrome fetuses. Am. J. Med. Genet, 2000, 95: 123-129

[11] NIH. Fragile X Syndrome, https://rarediseases.info.nih.gov/diseases/6464/fragile-x-syndrome (2021).

[12] Leboucher A et al. Fmr1-deficiency impacts body composition, skeleton, and bone microstructure in a mouse model of fragile X syndrome. Front. Endocrinol. (Lausanne), 2019, 10: 678

[13] Michaelsen-Preusse K, Feuge J, Korte M. Imbalance of synaptic actin dynamics as a key to fragile X syndrome? J. Physiol., 2018, 596: 2773-2782

[14] Booker SA, Kind PC. Mechanisms regulating input-output function and plasticity of neurons in the absence of FMRP. Brain Res. Bull, 2021, 175: 69-80

[15] Wang JS, Wein MN. Pathways controlling formation and maintenance of the osteocyte dendrite network. Curr. Osteoporos. Rep., 2022, 20: 493-504

[16] JAX® Mice, C. R. S. Aged C57BL/6J mice for research studies: Considerations, applications and best practices. (2017). https://resources.jax.org/white-papers/whitepaper-aged-b6

[17] Woo S, Rooser J, Dusevich V, Kalajzic I, Bonewald LF. Cell line IDG-SW3 replicates osteoblast-to-late-osteocyte differentiation in vitro and accelerates bone formation in vivo. J. Bone Miner. Res., 2011, 26: 2634-2646

[18] Davis HM et al. Cx43 overexpression in osteocytes prevents osteocyte apoptosis and preserves cortical bone quality in aging mice. JBMR Plus, 2018, 2: 206-216

[19] Davis HM et al. Disruption of the Cx43/miR21 pathway leads to osteocyte apoptosis and increased osteoclastogenesis with aging. Aging Cell, 2017, 16: 551-563

[20] Plotkin LI, Bellido T. Osteocytic signalling pathways as therapeutic targets for bone fragility. Nat. Rev. Endocrinol, 2016, 12: 593-605

[21] Kato Y, Windle JJ, Koop BA, Mundy GR, Bonewald LF. Establishment of an osteocyte-like cell line, MLO-Y4. J. Bone Miner. Res, 1997, 12: 2014-2023

[22] Zhang C, Bakker AD, Klein-Nulend J, Bravenboer N. Studies on osteocytes in their 3D native matrix versus 2D in vitro models. Curr. Osteoporos. Rep., 2019, 17: 207-216

[23] Dallas SL, Prideaux M, Bonewald LF. The osteocyte: an endocrine cell… and more. Endocr. Rev., 2013, 34: 658-690

[24] Qin L, Liu W, Cao H, Xiao G. Molecular mechanosensors in osteocytes. Bone Res., 2020, 8: 23

[25] Wang Y, McNamara LM, Schaffler MB, Weinbaum S. A model for the role of integrins in flow induced mechanotransduction in osteocytes. Proc. Natl. Acad. Sci. U. S. A., 2007, 104: 15941-15946

[26] Kidd SA et al. Fragile X syndrome: a review of associated medical problems. Pediatrics, 2014, 134: 995-1005

[27] Nolan SO et al. Deletion of Fmr1 results in sex-specific changes in behavior. Brain Behav., 2017, 7: e00800

[28] Nolan SO, Hodges SL, Lugo JN. High-throughput analysis of vocalizations reveals sex-specific changes in Fmr1 mutant pups. Genes Brain Behav., 2020, 19: e12611

[29] Reynolds CD, Nolan SO, Jefferson T, Lugo JN. Sex-specific and genotype-specific differences in vocalization development in FMR1 knockout mice. Neuroreport, 2016, 27: 1331-1335

[30] Sharma A et al. Sexing bones: improving transparency of sex reporting to address bias within preclinical studies. J. Bone Miner Res., 2023, 38: 5-13

[31] Steppe L, Bulow J, Tuckermann J, Ignatius A, Haffner-Luntzer M. Correction: Steppe et al. Bone mass and osteoblast activity are sex-dependent in mice lacking the estrogen receptor alpha in chondrocytes and osteoblast progenitor cells. Int. J. Mol. Sci. 2022, 23, 2902. Int. J. Mol. Sci., 2022, 23: 6020

[32] Essex AL et al. TREM2 R47H variant causes distinct age- and sex-dependent musculoskeletal alterations in mice. J. Bone Miner. Res., 2022, 37: 1366-1381

[33] Deosthale P et al. Sex-specific differences in direct osteoclastic versus indirect osteoblastic effects underlay the low bone mass of Pannexin1 deletion in TRAP-expressing cells in mice. Bone Rep., 2021, 16: 101164

[34] Davis HM et al. Osteocytic miR21 deficiency improves bone strength independent of sex despite having sex divergent effects on osteocyte viability and bone turnover. FEBS J., 2020, 287: 941-963

[35] Aguilar-Perez A et al. Age- and sex-dependent role of osteocytic pannexin1 on bone and muscle mass and strength. Sci. Rep., 2019, 9

[36] Pacheco-Costa R et al. Connexin37 deficiency alters organic bone matrix, cortical bone geometry, and increases Wnt/beta-catenin signaling. Bone, 2017, 97: 105-113

[37] Pacheco-Costa R et al. Defective cancellous bone structure and abnormal response to PTH in cortical bone of mice lacking Cx43 cytoplasmic C-terminus domain. Bone, 2015, 81: 632-643

[38] Moorer MC et al. Defective signaling, osteoblastogenesis, and bone remodeling in a mouse model of connexin43 C-terminal truncation. J. Cell Sci., 2017, 130: 531-540

[39] Leboucher A et al. The translational regulator FMRP controls lipid and glucose metabolism in mice and humans. Mol. Metab., 2019, 21: 22-35

[40] Youlten SE et al. Osteocyte transcriptome mapping identifies a molecular landscape controlling skeletal homeostasis and susceptibility to skeletal disease. Nat. Commun., 2021, 12

[41] Tabula Muris C et al. Single-cell transcriptomics of 20 mouse organs creates a Tabula Muris. Nature, 2018, 562: 367-372

[42] Robling AG, Bonewald LF. The osteocyte: new insights. Annu. Rev. Physiol., 2020, 82: 485-506

[43] Tiede-Lewis LM et al. Degeneration of the osteocyte network in the C57BL/6 mouse model of aging. Aging (Albany. NY), 2017, 9: 2190-2208

[44] Bouxsein ML et al. Guidelines for assessment of bone microstructure in rodents using micro-computed tomography. J. Bone Miner. Res., 2010, 25: 1468-1486

[45] Wallace JM, Golcuk K, Morris MD, Kohn DH. Inbred strain-specific effects of exercise in wild type and biglycan deficient mice. Ann. Biomed. Eng., 2010, 38: 1607-1617

[46] Jepsen KJ, Silva MJ, Vashishth D, Guo XE, van der Meulen MC. Establishing biomechanical mechanisms in mouse models: practical guidelines for systematically evaluating phenotypic changes in the diaphyses of long bones. J. Bone Miner. Res., 2015, 30: 951-966

[47] Dempster DW et al. Standardized nomenclature, symbols, and units for bone histomorphometry: a 2012 update of the report of the ASBMR Histomorphometry Nomenclature Committee. J. Bone Miner. Res., 2013, 28: 2-17

[48] Davis HM et al. Short-term pharmacologic RAGE inhibition differentially affects bone and skeletal muscle in middle-aged mice. Bone, 2019, 124: 89-102

[49] Stern AR et al. Isolation and culture of primary osteocytes from the long bones of skeletally mature and aged mice. Biotechniques, 2012, 52: 361-373

[50] Ayoubi M et al. 3D interrelationship between osteocyte network and forming mineral during human bone remodeling. Adv. Healthc. Mater., 2021, 10: e2100113

[51] Weiner S, Raguin E, Shahar R. High resolution 3D structures of mineralized tissues in health and disease. Nat. Rev. Endocrinol., 2021, 17: 307-316

[52] Losel PD et al. Introducing Biomedisa as an open-source online platform for biomedical image segmentation. Nat. Commun., 2020, 11

Funding

U.S. Department of Health & Human Services | National Institutes of Health (NIH)(R01-AR053643)

U.S. Department of Veterans Affairs (Department of Veterans Affairs)(I01BX005154)

U.S. Department of Health & Human Services | NIH | Office of Extramural Research, National Institutes of Health (OER)(R01-AG067997)