1. Mouse models of telomerase dysfuction as a models for age-related osteoporosis Tracy Brennan Aug 21, 2012

2. Purpose Determine if the accelerated aging mice present an age-dependent bone phenotype that recapitulates the characteristics observed in human senile osteoporosis.

3. Indices of Senile Osteoporosis Decreased trabecular bone and trabecular thickness Decreased cortical bone Increased bone porosity Decreased bone formation rate Decreased number of osteoblasts Decreased number of osteoclasts Increased number of adipocytes Jee WSS and Yao W J Musculoskel Neuron Interact 2001; Chan and Duque Gerontology 2002; Clarke, et al. Journal of Clinical Endocrinology and Metabolism 1996; Diab, et al. Bone 2006 http://parathyroid.com

4. MicroCT Analysis: Trabecular bone (Young) WTWrn -/- Terc -/- Wrn -/- Terc -/- N=13N=24N=14N=13

5. MicroCT Analysis: Trabecular bone (OLD) WTWrn -/- Terc -/- Wrn -/- Terc -/- N=14N=17N=14N=9

6. MicroCT Analysis: Cortical bone (Young) WTWrn -/- Terc -/- Wrn -/- Terc -/- N=13N=24N=14N=13

7. MicroCT Analysis: Cortical bone (OLD) WTWrn -/- Terc -/- Wrn -/- Terc -/- N=14N=17N=14N=9

8. Number of Pores 0 100 200 300 400 500 600 700 800 900 Young WTWrn-/-Terc-/-Wrn-/- Terc-/- Number of Pores 0 50 100 150 200 250 300 350 400 Old Total Pore Volume 0 5000 10000 15000 20000 25000 30000 35000 40000 45000 Young Total Pore Volume 0 2000 4000 6000 8000 10000 12000 Old WTWrn-/-Terc-/-Wrn-/- Terc-/- WTWrn-/-Terc-/-Wrn-/- Terc-/- WTWrn-/-Terc-/-Wrn-/- Terc-/- MicroCT Analysis: Porosity

9. Osteoblast Number 0 50 100 150 200 250 300 350 400 450 Young WTWrn-/-Terc-/-Wrn-/- Terc-/- 0 50 100 150 200 250 300 Old WTWrn-/- Osteoblast Number 0 20 40 60 80 100 120 140 Young Osteoclast Numbers 0 10 20 30 40 50 60 70 80 Old WTWrn-/-Terc-/-Wrn-/- Terc-/- WTWrn-/- Wrn-/- Terc-/- Osteoclast Numbers

10. Bone Lining Cells 0 50 100 150 200 250 Young WTWrn-/-Terc-/-Wrn-/- Terc-/- Bone Lining Cells 84 86 88 90 92 94 96 98 100 102 Old WTWrn-/-

11. Bone formation rate in old mice

12. Senile osteoporosis is recognized as a geriatric syndrome with a particular pathophysiology. It has been pointed out that senile osteoporosis is the product of a skeleton in an advanced stage of life with multiple mechanisms in the development stages of the disease that interact together and the product is an osteoporotic bone. While mechanisms of postmenopausal osteoporosis have been studied using animal models, clinical epidemiology and drug testing in human subjects; the mechanism of senile osteoporosis has not been well characterized, mainly because of a lack of experimental models. Senile osteoporosis is a complex disease that requires models that more closely resembles to human condition. Senile Osteoporosis

13. Current Models of Osteoporosis Ovariectomy/Gonadomectomy – Sex steroid deficiency substantially reduces bone mass and changes bone architecture and turnover, but does not cause spontaneous factures. – These cause very rapid bone changes that may not be accurately reflecting the affects of true aging on bone. (Wronksi, et al. Calcif Tissue Int 1988; Wronksi, et al. Calcif Tissue Int 1989; Wronksi, et al. Calcif Tissue Int 1992; Kalu, DN Bone Miner 1991; Seedor, et al. J Bone Miner Res 1991; Black, et al. Calcif Tissue Int 1989) Immobilization (Lee, et al. J Histotech 1997; Lee, WSS and Ma, YF Morphologie 1999) Treatment with particular factors (ie glucocorticoid) (Weinstein, et al. J Clin Invest 1998; Mangolagas, et al. J Bone Miner Res 1999) SAMP6 – the first rodent model of senile osteoporosis with spontaneous fractures at older age (Matsushita, et al Am J Pathol 1986) – Because these differences are already apparent at low maturity, low bone mass seems to result primarily from a deficiency in bone mass acquisition during growth and not from excessive bone loss during aging. (Matsushita, et al Am J Pathol 1986) Most “osteoporotic” animal models are selected on the basis of low bone mass and/or density, but do not experience spontaneous fracture.

14. Aging Models Werner syndrome (WS): – The autosomal recessive disorder is caused by a mutation in the WRN gene, which encodes a DNA helicase of the RecQ family (which are highly conserved throughout evolution and important for maintanence of genomic stability). (Yu, et al Nature 1990; Suzuki, et al Nucl. Acids Res. 1997) – Patients are normal until the second decade of life, when they develop many of the pathologies that phenocopy human aging, including alopecia, scleroderma-like skin pathology, accelerated atherosclerosis, ischemic heart disease, osteoporosis, biolateral ocular catracts, type II diabetes mellitusm and hypogonadism. (Epstein, et al Medicine 1966; Martin and Oshima Nature 2000) Dyskeratosis Congenita (DC): – The autosomal dominant form of DC is caused by a mutation in the TERC gene, which encodes the RNA component of the telomerase complex. (Vulliamy, et al Nature 2001) – Patients have abnormal skin pigmentation, nail dystrophy, mucosal leukoplakia, and increased cancer risk (most often succumbing to bone marrow failure by the fourth decade of life). (Dokal Lancet. 2001)

15. Aging Models Wrn -/- mice do not exhibit any premature aging phenotypes. (Lombard, et al Mol. Cell Biol., 2000) Terc -/- mice lack detectable telomerase activity and are viable for the six generations analyzed. Early generations of Terc -/- mice show little change in telomere length and no premature aging phenotypes. But in subsequent generations (5 to 6), the telomeres shrink and mice show a number of premature aging phenotypes, including gray hair, increased incidence of spontaneous malignancies, and decreased life-span. (Sci. Aging Knowl. Environ., 2003) Wrn -/- Terc -/- mice have a low bone mass phenotype shown by DXA on 14.7 month old mice, and that age-related osteoporosis is the result of impaired osteoblast differentiation. (Pignolo, et al Aging Cell 2008)

16. Osteoporosis As the US population becomes increasingly older, understanding and minimizing aging-related bone loss is increasingly becoming a national priority. Osteoporosis, clinically defined as bone fracture related to decreased bone density, is one of the most physically debilitating conditions associated with aging. In older people, the fractures (especially hip fractures) – Almost always result in hospitalization – 20% fatal – ~50% produce permanent disabilities World Health Organization