Cyclophilin D Knock-Out Mice Show Enhanced Resistance to Osteoporosis and to Metabolic Changes Observed in Aging Bone

<div><p>Pathogenic factors associated with aging, such as oxidative stress and hormone depletion converge on mitochondria and impair their function via opening of the mitochondrial permeability transition pore (MPTP). The MPTP is a large non-selective pore regulated by cyclophilin D (CypD) that disrupts mitochondrial membrane integrity. MPTP involvement has been firmly established in degenerative processes in heart, brain, and muscle. Bone has high energy demands and is therefore expected to be highly sensitive to mitochondrial dysfunction. Despite this, the role of mitochondria and the MPTP in bone maintenance and bone pathology has not been elucidated. Our goal was to determine whether mitochondria are impaired in aging bone and to see if protecting mitochondria from MPTP opening via CypD deletion protects against bone loss. We found that bone mass, strength, and formation progressively decline over the course of 18 months in C57BL/6J mice. Using metabolomics and electron microscopy, we determined that oxidative metabolism is impaired in aging bone leading to a glycolytic shift, imbalance in nucleotides, and decreased NAD⁺/NADH ratio. Mitochondria in osteocytes appear swollen which is a major marker of MPTP opening. CypD deletion by CypD knockout mouse model (CypD KO) protects against bone loss in 13- and 18-month-old mice and prevents decline in bone formation and mitochondrial changes observed in wild type C57BL/6J mice. Together, these data demonstrate that mitochondria are impaired in aging bone and that CypD deletion protects against this impairment to prevent bone loss. This implicates CypD-regulated MPTP and mitochondrial dysfunction in the impairment of bone cells and in aging-related bone loss. Our findings suggest mitochondrial metabolism as a new target for bone therapeutics and inhibition of CypD as a novel strategy against bone loss.</p></div

CypD deletion protects against the decline in bone architecture, strength, and formation in 13-mo-old mice.

<p>A) Histological evaluation of mouse bones, Trabecular Bone to Total Area ratios; B) Biomechanical torsion test of mouse femurs; C) Bone formation assay. Quantitative analysis of Mineral Apposition Rate (MAR), Mineralizing Surface/Bone Surface (MS/BS) and Bone Formation Rate (BFR). Scale bar is 5 μm; D) Bone resorption assay. TRAP-positive osteoclast surface relative to bone surface (Oc.S/B.S.) and CTX-I bone resorption/OC activity marker measured using ELISA. Data are Means ± SD (n = 5). *, <i>p</i><0.05 vs 3 mo as determined with <i>t-</i>test.</p

CypD deletion protects against metabolic changes in bone samples from 13-mo-old mice.

<p>Metabolomic analysis of mouse bones using MS-LC. Data are Means ± SD (n = 3). *, <i>p</i><0.05 as determined with <i>t-</i>test.</p

Decline in bone architecture, strength, and formation in 13-mo-old C57BL/6J mice.

<p>A) Histological evaluation of mouse bones. Sections stained with ABH/OG were analyzed using histomorphometry and OsteoMeasure software and trabecular Bone to Total Area ratios were calculated; B) Biomechanical torsion test of mouse femurs; C) Bone formation assay. Shown are representative sections and quantitative analyses of mouse bones labeled with Alizarin Red and Calcein for dynamic Mineral Apposition Rate (MAR), Mineralizing Surface/Bone Surface (MS/BS) and Bone Formation Rate (BFR) assay. Scale bar is 5 μm; D) TRAP-positive osteoclast surface relative to bone surface (Oc.S/B.S.) and CTX-I bone resorption/OC activity marker measured using ELISA. Data are Means ± SD (n = 5). *, <i>p</i><0.05 vs 3 mo as determined with <i>t-</i>test.</p

Metabolic changes in bone samples from 13-mo-old C57BL/6J mice.

<p>Small metabolites were extracted from bone shafts of tibia and femurs and analyzed using metabolomic LC-MS. Metabolites are grouped into appropriate metabolic pathways. Data are Means ± SD (n = 3). *, <i>p</i><0.05 vs 3 mo as determined with <i>t</i>-test.</p

CypD deletion protects against osteocyte mitochondrial swelling in bone samples from 13-mo-old mice.

<p>A) Electron micrographs of osteocytes (OTs) in mouse tibia. Arrowheads indicate mitochondria. Boxed mitochondria are enlarged and shown in the insets; B) Quantitative analysis of mitochondrial morphology. Fifteen cells per sample and 3 mice per group were blindly analyzed by three independent scorers and the number of mitochondria in a swollen conformation was determined. Data are Means ± SD. *, <i>p</i><0.05 as determined with <i>t</i>-test.</p

CypD KO mice do not show bone loss at 13 and 18 months of age.

<p>Representative microCT images of mouse tibiae, femurs, and L3 from 3-, 13-, and 18- mo-old CypD KO mice and graphs showing quantitative volumetric analysis of microCT data. Data are Means ± SD (n = 5–15). *, <i>p</i><0.05 vs 3 mo as determined with ANOVA.</p