Nuclear alpha-synuclein is present in the human brain and is modified in dementia with Lewy bodies

Dementia with Lewy bodies (DLB) is pathologically defined by the cytoplasmic accumulation of alpha-synuclein (aSyn) within neurons in the brain. Predominately pre-synaptic, aSyn has been reported in various subcellular compartments in experimental models. Indeed, nuclear alpha-synuclein (aSynNuc) is evident in many models, the dysregulation of which is associated with altered DNA integrity, transcription and nuclear homeostasis. However, the presence of aSynNuc in human brain cells remains controversial, yet the determination of human brain aSynNuc and its pathological modification is essential for understanding synucleinopathies. Here, using a multi-disciplinary approach employing immunohistochemistry, immunoblot, and mass-spectrometry (MS), we confirm aSynNuc in post-mortem brain tissue obtained from DLB and control cases. Highly dependent on antigen retrieval methods, in optimal conditions, intra-nuclear pan and phospho-S129 positive aSyn puncta were observed in cortical neurons and non-neuronal cells in fixed brain sections and in isolated nuclear preparations in all cases examined. Furthermore, an increase in nuclear phospho-S129 positive aSyn immunoreactivity was apparent in DLB cases compared to controls, in both neuronal and non-neuronal cell types. Our initial histological investigations identified that aSynNuc is affected by epitope unmasking methods but present under optimal conditions, and this presence was confirmed by isolation of nuclei and a combined approach of immunoblotting and mass spectrometry, where aSynNuc was approximately tenfold less abundant in the nucleus than cytoplasm. Notably, direct comparison of DLB cases to aged controls identified increased pS129 and higher molecular weight species in the nuclei of DLB cases, suggesting putative pathogenic modifications to aSynNuc in DLB. In summary, using multiple approaches we provide several lines of evidence supporting the presence of aSynNuc in autoptic human brain tissue and, notably, that it is subject to putative pathogenic modifications in DLB that may contribute to the disease phenotype

Non-Invasive Mouse Models of Post-Traumatic Osteoarthritis

SummaryAnimal models of osteoarthritis (OA) are essential tools for investigating the development of the disease on a more rapid timeline than human OA. Mice are particularly useful due to the plethora of genetically modified or inbred mouse strains available. The majority of available mouse models of OA use a joint injury or other acute insult to initiate joint degeneration, representing post-traumatic osteoarthritis (PTOA). However, no consensus exists on which injury methods are most translatable to human OA. Currently, surgical injury methods are most commonly used for studies of OA in mice; however, these methods may have confounding effects due to the surgical/invasive injury procedure itself, rather than the targeted joint injury. Non-invasive injury methods avoid this complication by mechanically inducing a joint injury externally, without breaking the skin or disrupting the joint. In this regard, non-invasive injury models may be crucial for investigating early adaptive processes initiated at the time of injury, and may be more representative of human OA in which injury is induced mechanically. A small number of non-invasive mouse models of PTOA have been described within the last few years, including intra-articular fracture of tibial subchondral bone, cyclic tibial compression loading of articular cartilage, and anterior cruciate ligament (ACL) rupture via tibial compression overload. This review describes the methods used to induce joint injury in each of these non-invasive models, and presents the findings of studies utilizing these models. Altogether, these non-invasive mouse models represent a unique and important spectrum of animal models for studying different aspects of PTOA

Microstructural analysis of collagen and elastin fibres in the kangaroo articular cartilage reveals a structural divergence depending on its local mechanical environment

Objective: To assess the microstructure of the collagen and elastin fibres in articular cartilage under different natural mechanical loading conditions and determine the relationship between the microstructure of collagen and its mechanical environment. Method: Articular cartilage specimens were collected from the load bearing regions of the medial femoral condyle and the medial distal humerus of adult kangaroos. The microstructure of collagen and elastin fibres of these specimens was studied using laser scanning confocal microscopy (LSCM) and the orientation and texture features of the collagen were analysed using ImageJ. Results: A zonal arrangement of collagen was found in kangaroo articular cartilage: the collagen fibres aligned parallel to the surface in the superficial zone and ran perpendicular in the deep zone. Compared with the distal humerus, the collagen in the femoral condyle was less isotropic and more clearly oriented, especially in the superficial and deep zones. The collagen in the femoral condyle was highly heterogeneous, less linear and more complex. Elastin fibres were found mainly in the superficial zone of the articular cartilage of both femoral condyle and distal humerus. Conclusions: The present study demonstrates that the collagen structure and texture of kangaroo articular cartilage is joint-dependent. This finding emphasizes the effects of loading on collagen development and suggests that articular cartilage with high biochemical and biomechanical qualities could be achieved by optimizing joint loading, which may benefit cartilage tissue engineering and prevention of joint injury. The existence of elastin fibres in articular cartilage could have important functional implications

Therapeutic value of graded aerobic exercise training in rheumatoid arthritis

Women with rheumatoid arthritis performed 1 of 3 low intensity aerobic exercise protocols (15, 25, and 35 minutes) 3 times per week for 12 weeks. A nontraining group served as controls. All exercise groups improved their aerobic capacity, exercise time, and joint counts. Subjects described improvement in activities of daily living and reduced joint pain and fatigue. Exercise duration up to 35 minutes can be therapeutic, and as little as 15 minutes of exercise 3 times/week is sufficient to improve aerobic capacity in rheumatoid arthritis patients with severe limitations.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/37771/1/1780280106_ftp.pd

Time evolution of in vivo articular cartilage repair induced by bone marrow stimulation and scaffold implantation in rabbits

Purpose: Tissue engineering techniques were used to study cartilage repair over a 12-month period in a rabbit model. Methods: A full-depth chondral defect along with subchondral bone injury were originated in the knee joint, where a biostable porous scaffold was implanted, synthesized of poly(ethyl acrylate-co-hydroxyethyl acrylate) copolymer. Morphological evolution of cartilage repair was studied 1 and 2 weeks, and 1, 3, and 12 months after implantation by histological techniques. The 3-month group was chosen to compare cartilage repair to an additional group where scaffolds were preseeded with allogeneic chondrocytes before implantation, and also to controls, who underwent the same surgery procedure, with no scaffold implantation. Results: Neotissue growth was first observed in the deepest scaffold pores 1 week after implantation, which spread thereafter; 3 months later scaffold pores were filled mostly with cartilaginous tissue in superficial and middle zones, and with bone tissue adjacent to subchondral bone. Simultaneously, native chondrocytes at the edges of the defect started to proliferate 1 week after implantation; within a month those edges had grown centripetally and seemed to embed the scaffold, and after 3 months, hyaline-like cartilage was observed on the condylar surface. Preseeded scaffolds slightly improved tissue growth, although the quality of repair tissue was similar to non-preseeded scaffolds. Controls showed that fibrous cartilage was mainly filling the repair area 3 months after surgery. In the 12-month group, articular cartilage resembled the untreated surface. Conclusions: Scaffolds guided cartilaginous tissue growth in vivo, suggesting their importance in stress transmission to the cells for cartilage repair.This study was supported by the Spanish Ministry of Science and Innovation through MAT2010-21611-C03-00 project (including the FEDER financial support), by Conselleria de Educacion (Generalitat Valenciana, Spain) PROMETEO/2011/084 grant, and by CIBER-BBN en Bioingenieria, Biomateriales y Nanomedicina. The work of JLGR was partially supported by funds from the Generalitat Valenciana, ACOMP/2012/075 project. CIBER-BBN is an initiative funded by the VI National R&D&i Plan 2008-2011, Iniciativa Ingenio 2010, Consolider Program, CIBER Actions and financed by the - Instituto de Salud Carlos III with assistance from the European Regional Development Fund.Sancho-Tello Valls, M.; Forriol, F.; Gastaldi, P.; Ruiz Sauri, A.; Martín De Llano, JJ.; Novella-Maestre, E.; Antolinos Turpín, CM.... (2015). Time evolution of in vivo articular cartilage repair induced by bone marrow stimulation and scaffold implantation in rabbits. International Journal of Artificial Organs. 38(4):210-223. https://doi.org/10.5301/ijao.5000404S210223384Becerra, J., Andrades, J. A., Guerado, E., Zamora-Navas, P., López-Puertas, J. M., & Reddi, A. H. (2010). Articular Cartilage: Structure and Regeneration. Tissue Engineering Part B: Reviews, 16(6), 617-627. doi:10.1089/ten.teb.2010.0191Nelson, L., Fairclough, J., & Archer, C. (2009). Use of stem cells in the biological repair of articular cartilage. Expert Opinion on Biological Therapy, 10(1), 43-55. doi:10.1517/14712590903321470MAINIL-VARLET, P., AIGNER, T., BRITTBERG, M., BULLOUGH, P., HOLLANDER, A., HUNZIKER, E., … STAUFFER, E. (2003). HISTOLOGICAL ASSESSMENT OF CARTILAGE REPAIR. The Journal of Bone and Joint Surgery-American Volume, 85, 45-57. doi:10.2106/00004623-200300002-00007Hunziker, E. B., Kapfinger, E., & Geiss, J. (2007). The structural architecture of adult mammalian articular cartilage evolves by a synchronized process of tissue resorption and neoformation during postnatal development. Osteoarthritis and Cartilage, 15(4), 403-413. doi:10.1016/j.joca.2006.09.010Onyekwelu, I., Goldring, M. B., & Hidaka, C. (2009). Chondrogenesis, joint formation, and articular cartilage regeneration. Journal of Cellular Biochemistry, 107(3), 383-392. doi:10.1002/jcb.22149Ahmed, T. A. E., & Hincke, M. T. (2010). Strategies for Articular Cartilage Lesion Repair and Functional Restoration. Tissue Engineering Part B: Reviews, 16(3), 305-329. doi:10.1089/ten.teb.2009.0590Hangody, L., Kish, G., Kárpáti, Z., Udvarhelyi, I., Szigeti, I., & Bély, M. (1998). Mosaicplasty for the Treatment of Articular Cartilage Defects: Application in Clinical Practice. Orthopedics, 21(7), 751-756. doi:10.3928/0147-7447-19980701-04Steinwachs, M. R., Guggi, T., & Kreuz, P. C. (2008). Marrow stimulation techniques. Injury, 39(1), 26-31. doi:10.1016/j.injury.2008.01.042Brittberg, M., Lindahl, A., Nilsson, A., Ohlsson, C., Isaksson, O., & Peterson, L. (1994). Treatment of Deep Cartilage Defects in the Knee with Autologous Chondrocyte Transplantation. New England Journal of Medicine, 331(14), 889-895. doi:10.1056/nejm199410063311401Richter, W. (2009). Mesenchymal stem cells and cartilagein situregeneration. Journal of Internal Medicine, 266(4), 390-405. doi:10.1111/j.1365-2796.2009.02153.xBartlett, W., Skinner, J. A., Gooding, C. R., Carrington, R. W. J., Flanagan, A. M., Briggs, T. W. R., & Bentley, G. (2005). Autologous chondrocyte implantationversusmatrix-induced autologous chondrocyte implantation for osteochondral defects of the knee. The Journal of Bone and Joint Surgery. British volume, 87-B(5), 640-645. doi:10.1302/0301-620x.87b5.15905Little, C. J., Bawolin, N. K., & Chen, X. (2011). Mechanical Properties of Natural Cartilage and Tissue-Engineered Constructs. Tissue Engineering Part B: Reviews, 17(4), 213-227. doi:10.1089/ten.teb.2010.0572Vikingsson, L., Gallego Ferrer, G., Gómez-Tejedor, J. A., & Gómez Ribelles, J. L. (2014). An «in vitro» experimental model to predict the mechanical behavior of macroporous scaffolds implanted in articular cartilage. Journal of the Mechanical Behavior of Biomedical Materials, 32, 125-131. doi:10.1016/j.jmbbm.2013.12.024Weber, J. F., & Waldman, S. D. (2014). Calcium signaling as a novel method to optimize the biosynthetic response of chondrocytes to dynamic mechanical loading. Biomechanics and Modeling in Mechanobiology, 13(6), 1387-1397. doi:10.1007/s10237-014-0580-xMauck, R. L., Soltz, M. A., Wang, C. C. B., Wong, D. D., Chao, P.-H. G., Valhmu, W. B., … Ateshian, G. A. (2000). Functional Tissue Engineering of Articular Cartilage Through Dynamic Loading of Chondrocyte-Seeded Agarose Gels. Journal of Biomechanical Engineering, 122(3), 252-260. doi:10.1115/1.429656Palmoski, M. J., & Brandt, K. D. (1984). Effects of static and cyclic compressive loading on articular cartilage plugs in vitro. Arthritis & Rheumatism, 27(6), 675-681. doi:10.1002/art.1780270611Khoshgoftar, M., Ito, K., & van Donkelaar, C. C. (2014). The Influence of Cell-Matrix Attachment and Matrix Development on the Micromechanical Environment of the Chondrocyte in Tissue-Engineered Cartilage. Tissue Engineering Part A, 20(23-24), 3112-3121. doi:10.1089/ten.tea.2013.0676Agrawal, C. M., & Ray, R. B. (2001). Biodegradable polymeric scaffolds for musculoskeletal tissue engineering. Journal of Biomedical Materials Research, 55(2), 141-150. doi:10.1002/1097-4636(200105)55:23.0.co;2-jPérez Olmedilla, M., Garcia-Giralt, N., Pradas, M. M., Ruiz, P. B., Gómez Ribelles, J. L., Palou, E. C., & García, J. C. M. (2006). Response of human chondrocytes to a non-uniform distribution of hydrophilic domains on poly (ethyl acrylate-co-hydroxyethyl methacrylate) copolymers. Biomaterials, 27(7), 1003-1012. doi:10.1016/j.biomaterials.2005.07.030Horbett, T. A., & Schway, M. B. (1988). Correlations between mouse 3T3 cell spreading and serum fibronectin adsorption on glass and hydroxyethylmethacrylate-ethylmethacrylate copolymers. Journal of Biomedical Materials Research, 22(9), 763-793. doi:10.1002/jbm.820220903Kiremitçi, M., Peşmen, A., Pulat, M., & Gürhan, I. (1993). Relationship of Surface Characteristics to Cellular Attachment in PU and PHEMA. Journal of Biomaterials Applications, 7(3), 250-264. doi:10.1177/088532829300700304Lydon, M. ., Minett, T. ., & Tighe, B. . (1985). Cellular interactions with synthetic polymer surfaces in culture. Biomaterials, 6(6), 396-402. doi:10.1016/0142-9612(85)90100-0Campillo-Fernandez, A. J., Pastor, S., Abad-Collado, M., Bataille, L., Gomez-Ribelles, J. L., Meseguer-Dueñas, J. M., … Ruiz-Moreno, J. M. (2007). Future Design of a New Keratoprosthesis. Physical and Biological Analysis of Polymeric Substrates for Epithelial Cell Growth. Biomacromolecules, 8(8), 2429-2436. doi:10.1021/bm0703012Funayama, A., Niki, Y., Matsumoto, H., Maeno, S., Yatabe, T., Morioka, H., … Toyama, Y. (2008). Repair of full-thickness articular cartilage defects using injectable type II collagen gel embedded with cultured chondrocytes in a rabbit model. Journal of Orthopaedic Science, 13(3), 225-232. doi:10.1007/s00776-008-1220-zKitahara, S., Nakagawa, K., Sah, R. L., Wada, Y., Ogawa, T., Moriya, H., & Masuda, K. (2008). In Vivo Maturation of Scaffold-free Engineered Articular Cartilage on Hydroxyapatite. Tissue Engineering Part A, 14(11), 1905-1913. doi:10.1089/ten.tea.2006.0419Martinez-Diaz, S., Garcia-Giralt, N., Lebourg, M., Gómez-Tejedor, J.-A., Vila, G., Caceres, E., … Monllau, J. C. (2010). In Vivo Evaluation of 3-Dimensional Polycaprolactone Scaffolds for Cartilage Repair in Rabbits. The American Journal of Sports Medicine, 38(3), 509-519. doi:10.1177/0363546509352448Wang, Y., Bian, Y.-Z., Wu, Q., & Chen, G.-Q. (2008). Evaluation of three-dimensional scaffolds prepared from poly(3-hydroxybutyrate-co-3-hydroxyhexanoate) for growth of allogeneic chondrocytes for cartilage repair in rabbits. Biomaterials, 29(19), 2858-2868. doi:10.1016/j.biomaterials.2008.03.021Alió del Barrio, J. L., Chiesa, M., Gallego Ferrer, G., Garagorri, N., Briz, N., Fernandez-Delgado, J., … De Miguel, M. P. (2014). Biointegration of corneal macroporous membranes based on poly(ethyl acrylate) copolymers in an experimental animal model. Journal of Biomedical Materials Research Part A, 103(3), 1106-1118. doi:10.1002/jbm.a.35249Diego, R. B., Olmedilla, M. P., Aroca, A. S., Ribelles, J. L. G., Pradas, M. M., Ferrer, G. G., & Sánchez, M. S. (2005). Acrylic scaffolds with interconnected spherical pores and controlled hydrophilicity for tissue engineering. Journal of Materials Science: Materials in Medicine, 16(8), 693-698. doi:10.1007/s10856-005-2604-7Serrano Aroca, A., Campillo Fernández, A. J., Gómez Ribelles, J. L., Monleón Pradas, M., Gallego Ferrer, G., & Pissis, P. (2004). Porous poly(2-hydroxyethyl acrylate) hydrogels prepared by radical polymerisation with methanol as diluent. Polymer, 45(26), 8949-8955. doi:10.1016/j.polymer.2004.10.033Diani, J., Fayolle, B., & Gilormini, P. (2009). A review on the Mullins effect. European Polymer Journal, 45(3), 601-612. doi:10.1016/j.eurpolymj.2008.11.017Mullins, L. (1969). Softening of Rubber by Deformation. Rubber Chemistry and Technology, 42(1), 339-362. doi:10.5254/1.3539210Jurvelin, J. S., Buschmann, M. D., & Hunziker, E. B. (2003). Mechanical anisotropy of the human knee articular cartilage in compression. Proceedings of the Institution of Mechanical Engineers, Part H: Journal of Engineering in Medicine, 217(3), 215-219. doi:10.1243/095441103765212712Shapiro, F., Koide, S., & Glimcher, M. J. (1993). Cell origin and differentiation in the repair of full-thickness defects of articular cartilage. The Journal of Bone & Joint Surgery, 75(4), 532-553. doi:10.2106/00004623-199304000-00009SELLERS, R. S., ZHANG, R., GLASSON, S. S., KIM, H. D., PELUSO, D., D’AUGUSTA, D. A., … MORRIS, E. A. (2000). Repair of Articular Cartilage Defects One Year After Treatment with Recombinant Human Bone Morphogenetic Protein-2 (rhBMP-2)*. The Journal of Bone and Joint Surgery-American Volume, 82(2), 151-160. doi:10.2106/00004623-200002000-00001Hunziker, E. B., Michel, M., & Studer, D. (1997). Ultrastructure of adult human articular cartilage matrix after cryotechnical processing. Microscopy Research and Technique, 37(4), 271-284. doi:10.1002/(sici)1097-0029(19970515)37:43.0.co;2-oAppelman, T. P., Mizrahi, J., Elisseeff, J. H., & Seliktar, D. (2009). The differential effect of scaffold composition and architecture on chondrocyte response to mechanical stimulation. Biomaterials, 30(4), 518-525. doi:10.1016/j.biomaterials.2008.09.063Chung, C., & Burdick, J. A. (2008). Engineering cartilage tissue. Advanced Drug Delivery Reviews, 60(2), 243-262. doi:10.1016/j.addr.2007.08.027HUNZIKER, E. B., & ROSENBERG, L. C. (1996). Repair of Partial-Thickness Defects in Articular Cartilage. The Journal of Bone & Joint Surgery, 78(5), 721-33. doi:10.2106/00004623-199605000-00012Schulze-Tanzil, G. (2009). Activation and dedifferentiation of chondrocytes: Implications in cartilage injury and repair. Annals of Anatomy - Anatomischer Anzeiger, 191(4), 325-338. doi:10.1016/j.aanat.2009.05.003Umlauf, D., Frank, S., Pap, T., & Bertrand, J. (2010). Cartilage biology, pathology, and repair. Cellular and Molecular Life Sciences, 67(24), 4197-4211. doi:10.1007/s00018-010-0498-0Karystinou, A., Dell’Accio, F., Kurth, T. B. A., Wackerhage, H., Khan, I. M., Archer, C. W., … De Bari, C. (2009). Distinct mesenchymal progenitor cell subsets in the adult human synovium. Rheumatology, 48(9), 1057-1064. doi:10.1093/rheumatology/kep192Sakaguchi, Y., Sekiya, I., Yagishita, K., & Muneta, T. (2005). Comparison of human stem cells derived from various mesenchymal tissues: Superiority of synovium as a cell source. Arthritis & Rheumatism, 52(8), 2521-2529. doi:10.1002/art.21212Schaefer, D., Martin, I., Jundt, G., Seidel, J., Heberer, M., Grodzinsky, A., … Freed, L. E. (2002). Tissue-engineered composites for the repair of large osteochondral defects. Arthritis & Rheumatism, 46(9), 2524-2534. doi:10.1002/art.1049

Metabolic signature of articular cartilage following mechanical injury: An integrated transcriptomics and metabolomics analysis

Mechanical injury to the articular cartilage is a key risk factor in joint damage and predisposition to osteoarthritis. Integrative multi-omics approaches provide a valuable tool to understand tissue behavior in response to mechanical injury insult and help to identify key pathways linking injury to tissue damage. Global or untargeted metabolomics provides a comprehensive characterization of the metabolite content of biological samples. In this study, we aimed to identify the metabolic signature of cartilage tissue post injury. We employed an integrative analysis of transcriptomics and global metabolomics of murine epiphyseal hip cartilage before and after injury. Transcriptomics analysis showed a significant enrichment of gene sets involved in regulation of metabolic processes including carbon metabolism, biosynthesis of amino acids, and steroid biosynthesis. Integrative analysis of enriched genes with putatively identified metabolite features post injury showed a significant enrichment for carbohydrate metabolism (glycolysis, galactose, and glycosylate metabolism and pentose phosphate pathway) and amino acid metabolism (arginine biosynthesis and tyrosine, glycine, serine, threonine, and arginine and proline metabolism). We then performed a cross analysis of global metabolomics profiles of murine and porcine ex vivo cartilage injury models. The top commonly modulated metabolic pathways post injury included arginine and proline metabolism, arginine biosynthesis, glycolysis/gluconeogenesis, and vitamin B6 metabolic pathways. These results highlight the significant modulation of metabolic responses following mechanical injury to articular cartilage. Further investigation of these pathways would provide new insights into the role of the early metabolic state of articular cartilage post injury in promoting tissue damage and its link to disease progression of osteoarthritis