AAV1.NT-3 gene therapy in a CMT2D model: phenotypic improvements in GarsP278KY/+ mice

AUTHORS

Burcak Ozes, Kyle Moss, Morgan Myers, Alicia Ridgley, Lei Chen, Darren Murrey, Zarife Sahenk

ABSTRACT

Glycyl–tRNA synthetase mutations are associated to the Charcot–Marie–Tooth disease type-2D. The GarsP278KY/+ model for Charcot–Marie–Tooth disease type-2D is known best for its early onset severe neuropathic phenotype with findings including reduced axon size, slow conduction velocities and abnormal neuromuscular junction. Muscle involvement remains largely unexamined. We tested the efficacy of neurotrophin 3 gene transfer therapy in two Gars mutants with severe (GarsP278KY/+) and milder (GarsΔETAQ/+) phenotypes via intramuscular injection of adeno-associated virus setoype-1, triple tandem muscle creatine kinase promoter, neurotrophin 3 (AAV1.tMCK.NT-3) at 1 × 1011 vg dose. In the GarsP278KY/+ mice, the treatment efficacy was assessed at 12 weeks post-injection using rotarod test, electrophysiology and detailed quantitative histopathological studies of the peripheral nervous system including neuromuscular junction and muscle. Neurotrophin 3 gene transfer therapy in GarsP278KY/+ mice resulted in significant functional and electrophysiological improvements, supported with increases in myelin thickness and improvements in the denervated status of neuromuscular junctions as well as increases in muscle fibre size along with attenuation of myopathic changes. Improvements in the milder phenotype GarsΔETAQ/+ was less pronounced. Furthermore, oxidative enzyme histochemistry in muscles from Gars mutants revealed alterations in the content and distribution of oxidative enzymes with increased expression levels of Pgc1a. Cox1, Cox3 and Atp5d transcripts were significantly decreased suggesting that the muscle phenotype might be related to mitochondrial dysfunction. Neurotrophin 3 gene therapy attenuated these abnormalities in the muscle. This study shows that neurotrophin 3 gene transfer therapy has disease modifying effect in a mouse model for Charcot–Marie–Tooth disease type-2D, leading to meaningful improvements in peripheral nerve myelination and neuromuscular junction integrity as well as in a unique myopathic process, associated with mitochondria dysfunction, all in combination contributing to functional outcome. Based on the multiple biological effects of this versatile molecule, we predict neurotrophin 3 has the potential to be beneficial in other aminoacyl-tRNA synthetase-linked Charcot–Marie–Tooth disease subtypes.

Clinical Prediction of High-Turnover Bone Disease After Kidney Transplantation

AUTHORS

Satu M. Keronen, Leena A. L. Martola, Patrik Finne, Inari S. Burton, Xiaoyu F. Tong, Heikki P. Kröger & Eero O. Honkanen

ABSTRACT

Bone histomorphometric analysis is the most accurate method for the evaluation of bone turnover, but non-invasive tools are also required. We studied whether bone biomarkers can predict high bone turnover determined by bone histomorphometry after kidney transplantation. We retrospectively evaluated the results of bone biopsy specimens obtained from kidney transplant recipients due to the clinical suspicion of high bone turnover between 2000 and 2015. Bone biomarkers were acquired concurrently. Of 813 kidney transplant recipients, 154 (19%) biopsies were taken at a median of 28 (interquartile range, 18–70) months after engraftment. Of 114 patients included in the statistical analysis, 80 (70%) presented with high bone turnover. Normal or low bone turnover was detected in 34 patients (30%). For discriminating high bone turnover from non-high, alkaline phosphatase, parathyroid hormone, and ionized calcium had the areas under the receiver operating characteristic curve (AUCs) of 0.704, 0.661, and 0.619, respectively. The combination of these markers performed better with an AUC of 0.775. The positive predictive value for high turnover at a predicted probability cutoff of 90% was 95% while the negative predictive value was 35%. This study concurs with previous observations that hyperparathyroidism with or without hypercalcemia does not necessarily imply high bone turnover in kidney transplant recipients. The prediction of high bone turnover can be improved by considering alkaline phosphatase levels, as presented in the logistic regression model. If bone biopsy is not readily available, this model may serve as clinically available tool in recognizing high turnover after engraftment.

Bisphosphonate-enoxacin inhibit osteoclast formation and function by abrogating RANKL-induced JNK signalling pathways during osteoporosis treatment

AUTHORS

Qiang Xu, Ping Zhan, Xiaofeng Li, Fengbo Mo, Huaen Xu, Yuan Liu, Qi Lai, Bin Zhang,Min Dai, Xuqiang Liu

ABSTRACT

Osteoporosis is an age-related disease characterized by low mineral density, compromised bone strength and increased risk of fragility fracture. Most agents for treating osteoporosis focus primarily on anti-resorption by inhibiting osteoclast activity. Bisphosphonate (BP) is a potent anti-resorptive agent that has been used clinically for decades and is proven to be effective. However, BP has a variety of side effects and is far from being an ideal anti-osteoporosis agent. BP selectively binds to calcium crystals, which are subsequently taken up or released by osteoclasts. Based on the action of BP, we previously demonstrated the inhibitory effect of a novel bone-targeting BP derivative, bisphosphonate-enoxacin (BE). In the current study, we used bone marrow-derived osteoclast cultures to further assess the inhibitory effect of BE on osteoclastogenesis and employed reverse transcription PCR and real-time PCR to examine expression of osteoclast-specific genes. Additionally, we used bone resorption and F-actin immunofluorescence assays to evaluate the effect of BE on osteoclast function and investigated the potential mechanisms affecting osteoclast differentiation and function in vitro. Furthermore, an ovariectomized (OVX) rat model was established to evaluate the therapeutic effects of BE on preventing bone loss. Results showed that BE exerted potent inhibitory effects on osteoclast formation and bone resorption by specifically abrogating RANKL-induced JNK signalling, and that it preserved OVX rat bone mass in vivo without any notable side effects. Collectively, these results indicated that the BP derivative BE may have significant potential as a treatment for osteoporosis and other osteolytic diseases.

Targeting adipocytic discoidin domain receptor 2 impedes fat gain while increasing bone mass

AUTHORS

Xiaoyu Yang, Jing Li, Liting Zhao, Yazhuo Chen, Zhijun Cui, Taotao Xu, Xu Li, Shufang Wu & Yan Zhang

ABSTRACT

Obesity is closely associated with low-bone-mass disorder. Discoidin domain receptor 2 (DDR2) plays essential roles in skeletal metabolism, and is probably involved in fat metabolism. To test the potential role of DDR2 in fat and fat-bone crosstalk, Ddr2 conditional knockout mice (Ddr2Adipo) were generated in which Ddr2 gene is exclusively deleted in adipocytes by Adipoq Cre. We found that Ddr2Adipo mice are protected from fat gain on high-fat diet, with significantly decreased adipocyte size. Ddr2Adipo mice exhibit significantly increased bone mass and mechanical properties, with enhanced osteoblastogenesis and osteoclastogenesis. Marrow adipocyte is diminished in the bone marrow of Ddr2Adipo mice, due to activation of lipolysis. Fatty acid in the bone marrow was reduced in Ddr2Adipo mice. RNA-Seq analysis identified adenylate cyclase 5 (Adcy5) as downstream molecule of Ddr2. Mechanically, adipocytic Ddr2 modulates Adcy5-cAMP-PKA signaling, and Ddr2 deficiency stimulates lipolysis and supplies fatty acid for oxidation in osteoblasts, leading to the enhanced osteoblast differentiation and bone mass. Treatment of Adcy5 specific inhibitor abolishes the increased bone mass gain in Ddr2Adipo mice. These observations establish, for the first time, that Ddr2 plays an essential role in the crosstalk between fat and bone. Targeting adipocytic Ddr2 may be a potential strategy for treating obesity and pathological bone loss simultaneously.

Correlated evolution of larval development, egg size and genome size across two genera of snapping shrimp

AUTHORS

Kristin M. Hultgren, Solomon T. C. Chak, Jeremy Bjelajac, Kenneth S. Macdonald

ABSTRACT

Across plants and animals, genome size is often correlated with life-history traits: large genomes are correlated with larger seeds, slower development, larger body size and slower cell division. Among decapod crustaceans, caridean shrimps are among the most variable both in terms of genome size variation and life-history characteristics such as larval development mode and egg size, but the extent to which these traits are associated in a phylogenetic context is largely unknown. In this study, we examine correlations among egg size, larval development and genome size in two different genera of snapping shrimp, Alpheus and Synalpheus, using phylogenetically informed analyses. In both Alpheus and Synalpheus, egg size is strongly linked to larval development mode: species with abbreviated development had significantly larger eggs than species with extended larval development. We produced the first comprehensive dataset of genome size in Alpheus (n = 37 species) and demonstrated that genome size was strongly and positively correlated with egg size in both Alpheus and Synalpheus. Correlated trait evolution analyses showed that in Alpheus, changes in genome size were clearly dependent on egg size. In Synalpheus, evolutionary path analyses suggest that changes in development mode (from extended to abbreviated) drove increases in egg volume; larger eggs, in turn, resulted in larger genomes. These data suggest that variation in reproductive traits may underpin the high degree of variation in genome size seen in a wide variety of caridean shrimp groups more generally.

Bio-clickable mussel-inspired peptides improve titanium-based material osseointegration synergistically with immunopolarization-regulation

AUTHORS

Jie Suna, Yingkang Huang, Huan Zhao, Junjie Niu, Xuwei Ling, Can Zhu, Lin Wang, Huilin Yang, Zhilu Yang, Guoqing Pan, Qin Shi

ABSTRACT

Upon the osteoporotic condition, sluggish osteogenesis, excessive bone resorption, and chronic inflammation make the osseointegration of bioinert titanium (Ti) implants with surrounding bone tissues difficult, often lead to prosthesis loosening, bone collapse, and implant failure. In this study, we firstly designed clickable mussel-inspired peptides (DOPA-N3) and grafted them onto the surfaces of Ti materials through robust catechol-TiO2 coordinative interactions. Then, two dibenzylcyclooctyne (DBCO)-capped bioactive peptides RGD and BMP-2 bioactive domain (BMP-2) were clicked onto the DOPA-N3-coated Ti material surfaces via bio-orthogonal reaction. We characterized the surface morphology and biocompatibility of the Ti substrates and optimized the osteogenic capacity of Ti surfaces through adjusting the ideal ratios of BMP-2/RGD at 3:1. In vitro, the dual-functionalized Ti substrates exhibited excellent promotion on adhesion and osteogenesis of mesenchymal stem cells (MSCs), and conspicuous immunopolarization-regulation to shift macrophages to alternative (M2) phenotypes and inhibit inflammation, as well as enhancement of osseointegration and mechanical stability in osteoporotic rats. In summary, our biomimetic surface modification strategy by bio-orthogonal reaction provided a convenient and feasible method to resolve the bioinertia and clinical complications of Ti-based implants, which was conducive to the long-term success of Ti implants, especially in the osteoporotic or inflammatory conditions.