Exploring the Bone Proteome to Help Explain Altered Bone Remodeling and Preservation of Bone Architecture and Strength in Hibernating Marmots

Periods of physical inactivity increase bone resorption and cause bone loss and increased fracture risk. However, hibernating bears, marmots, and woodchucks maintain bone structure and strength, despite being physically inactive for prolonged periods annually. We tested the hypothesis that bone turnover rates would decrease and bone structural and mechanical properties would be preserved in hibernating marmots (Marmota flaviventris). Femurs and tibias were collected from marmots during hibernation and in the summer following hibernation. Bone remodeling was significantly altered in cortical and trabecular bone during hibernation with suppressed formation and no change in resorption, unlike the increased bone resorption that occurs during disuse in humans and other animals. Trabecular bone architecture and cortical bone geometrical and mechanical properties were not different between hibernating and active marmots, but bone marrow adiposity was significantly greater in hibernators. Of the 506 proteins identified in marmot bone, 40 were significantly different in abundance between active and hibernating marmots. Monoaglycerol lipase, which plays an important role in fatty acid metabolism and the endocannabinoid system, was 98-fold higher in hibernating marmots compared with summer marmots and may play a role in regulating the changes in bone and fat metabolism that occur during hibernation

Nanoscale Subsynaptic Domains Underlie the Organization of the Inhibitory Synapse

Summary: Inhibitory synapses mediate the majority of synaptic inhibition in the brain, thereby controlling neuronal excitability, firing, and plasticity. Although essential for neuronal function, the central question of how these synapses are organized at the subsynaptic level remains unanswered. Here, we use three-dimensional (3D) super-resolution microscopy to image key components of the inhibitory postsynaptic domain and presynaptic terminal, revealing that inhibitory synapses are organized into nanoscale subsynaptic domains (SSDs) of the gephyrin scaffold, GABAARs and the active-zone protein Rab3-interacting molecule (RIM). Gephyrin SSDs cluster GABAAR SSDs, demonstrating nanoscale architectural interdependence between scaffold and receptor. GABAAR SSDs strongly associate with active-zone RIM SSDs, indicating an important role for GABAAR nanoscale organization near sites of GABA release. Finally, we find that in response to elevated activity, synapse growth is mediated by an increase in the number of postsynaptic SSDs, suggesting a modular mechanism for increasing inhibitory synaptic strength. : Crosby et al. reveal that inhibitory synapses are composed of nanoscale subsynaptic domains of GABAA receptors, the inhibitory scaffold gephyrin and the active-zone protein RIM. During plasticity, additional subsynaptic domains are recruited to the synapse, suggesting a mechanism for activity-dependent synaptic growth. Keywords: GABAA receptor, gephyrin, RIM, VGAT, super-resolution nanoscopy, inhibitory synapse, homeostatic plasticity, structured illumination microscop

Combination Therapy with Zoledronic Acid and Parathyroid Hormone Improves Bone Architecture and Strength following a Clinically-Relevant Dose of Stereotactic Radiation Therapy for the Local Treatment of Canine Osteosarcoma in Athymic Rats.

Clinical studies using definitive-intent stereotactic radiation therapy (SRT) for the local treatment of canine osteosarcoma (OSA) have shown canine patients achieving similar median survival times as the current standard of care (amputation and adjuvant chemotherapy). Despite this, there remains an unacceptable high risk of pathologic fracture following radiation treatment. Zoledronic acid (ZA) and parathyroid hormone (PTH) are therapeutic candidates for decreasing this fracture risk post-irradiation. Due to differing mechanisms, we hypothesized that the combined treatment with ZA and PTH would significantly improve bone healing more than ZA or PTH treatment alone. Using an orthotopic model of canine osteosarcoma in athymic rats, we evaluated bone healing following clinically-relevant doses of radiation therapy (12 Gy x 3 fractions, 36 Gy total). Groups included 36 Gy SRT only, 36 Gy SRT plus ZA, 36 Gy SRT plus ZA and PTH, 36 Gy SRT plus PTH, and 36 Gy SRT plus localized PTH treatment. Our study showed significant increases in bone volume and increased polar moments of inertia (in the distal femoral metaphysis) 8 weeks after radiation in the combined (ZA/PTH) treatment group as compared to radiation treatment alone. Histomorphometric analysis revealed evidence of active mineralization at the study endpoint as well as successful tumor-cell kill across all treatment groups. This work provides further evidence for the expanding potential indications for ZA and PTH therapy, including post-irradiated bone disease due to osteosarcoma

Representative fluorescent micrograph.

<p>Image illustrates single (line with one arrowhead) and double-labeled surfaces (line with two arrowheads) in trabecular bone.</p

Radiograph severity scoring on a scale from 0–5.

<p>A. 0 –No evidence of tumor-associated osteolysis. B. 1 –Mild trabecular lysis apparent (≤ 50% of the diameter of the femur affected. C. 2 –Moderate trabecular lysis apparent (> 50% of the femur’s diameter affected). D. 3 –Severe osteolysis evident with 1 cortex involved. E. 4 –Severe osteolysis evident with both cortices affected. F. 5 –Fracture.</p

Evidence of previous osteosarcoma.

<p><b>(H&E)</b> a) Distal metaphysis exhibiting trabecular destruction, necrosis, and replacement fibrosis throughout regions of previous tumor burden. The growth plate (asterisk), metaphysis (dagger), and epiphysis (double dagger) are shown for reference. (20x) Note the metaphyseal tumor does not cross the growth plate to the epiphysis, where normal trabecular architecture is evident. b) Loosely organized fibrous connective tissue and necrosis in tumor-burdened areas. (100x).</p

Active Osteosarcoma at the study endpoint.

<p>a) Rat. Distal Metaphysis of Femur. (H&E. 200x). Replacing normal trabecular architecture are poorly differentiated neoplastic cells that are polygonal to spindle shaped with lightly eosinophilic cytoplasm. b) Rat. Distal Metaphysis of Femur. (H&E. 400x). Neoplastic cells are irregularly polygonal with indistinct cell borders and moderate amounts of cytoplasm. Nuclei contain multiple, distinct nucleoli and a mitotic figure is present (asterisk).</p

Fluorescent micrograph.

<p>Areas of dystrophic mineralization confounded quantification of single-labeled surfaces. As an alternative variable, digital images of the region of interest were used to quantify the total number of positive florescent pixels. There were no significant differences between treatment groups.</p

Effects of radiation and adjunct therapy on treated bone.

<p>Represented are bone volume (a), and polar moment of inertia (b) in the distal metaphysis of tumor-burdened femora. Data represent mean (+/- SE) values.</p

Assessment of bone resorption over the region of interest.

<p>(a) There was no difference (p = 0.0627) between groups in osteoclast surface. (b) There was no difference (p = 0.1108) between groups in osteoclast number. (c) TRAP surface showed a statistically significant difference (p = 0.0152) between groups, however, pairwise comparisons (Dunn’s test) was not able to detect which groups.</p