An early-life microbiota metabolite protects against obesity by regulating intestinal lipid metabolism.

The mechanisms by which the early-life microbiota protects against environmental factors that promote childhood obesity remain largely unknown. Using a mouse model in which young mice are simultaneously exposed to antibiotics and a high-fat (HF) diet, we show that Lactobacillus species, predominant members of the small intestine (SI) microbiota, regulate intestinal epithelial cells (IECs) to limit diet-induced obesity during early life. A Lactobacillus-derived metabolite, phenyllactic acid (PLA), protects against metabolic dysfunction caused by early-life exposure to antibiotics and a HF diet by increasing the abundance of peroxisome proliferator-activated receptor γ (PPAR-γ) in SI IECs. Therefore, PLA is a microbiota-derived metabolite that activates protective pathways in the small intestinal epithelium to regulate intestinal lipid metabolism and prevent antibiotic-associated obesity during early life.

Hyperglycemia Increases Severity of Staphylococcus aureus Osteomyelitis and Influences Bacterial Genes Required for Survival in Bone.

Hyperglycemia, or elevated blood glucose, renders individuals more prone to developing severe Staphylococcus aureus infections. S. aureus is the most common etiological agent of musculoskeletal infection, which is a common manifestation of disease in hyperglycemic patients. However, the mechanisms by which S. aureus causes severe musculoskeletal infection during hyperglycemia are incompletely characterized. To examine the influence of hyperglycemia on S. aureus virulence during invasive infection, we used a murine model of osteomyelitis and induced hyperglycemia with streptozotocin. We discovered that hyperglycemic mice exhibited increased bacterial burdens in bone and enhanced dissemination compared to control mice. Furthermore, infected hyperglycemic mice sustained increased bone destruction relative to euglycemic controls, suggesting that hyperglycemia exacerbates infection-associated bone loss. To identify genes contributing to S. aureus pathogenesis during osteomyelitis in hyperglycemic animals relative to euglycemic controls, we used transposon sequencing (TnSeq). We identified 71 genes uniquely essential for S. aureus survival in osteomyelitis in hyperglycemic mice and another 61 mutants with compromised fitness. Among the genes essential for S. aureus survival in hyperglycemic mice was the gene encoding superoxide dismutase A (sodA), one of two S. aureus superoxide dismutases involved in detoxifying reactive oxygen species (ROS). We determined that a sodA mutant exhibits attenuated survival in vitro in high glucose and in vivo during osteomyelitis in hyperglycemic mice. SodA therefore plays an important role during growth in high glucose and promotes S. aureus survival in bone. Collectively, these studies demonstrate that hyperglycemia increases the severity of osteomyelitis and identify genes contributing to S. aureus survival during hyperglycemic infection.

Inhibition of nucleotide biosynthesis disrupts lipid accumulation and adipogenesis.

Energy balance and nutrient availability are key determinants of cellular decisions to remain quiescent, proliferate, or differentiate into a mature cell. After assessing its environmental state, the cell must rewire its metabolism to support distinct cellular outcomes. Mechanistically, how metabolites regulate cell fate decisions is poorly understood. We used adipogenesis as our model system to ascertain the role of metabolism in differentiation. We isolated adipose tissue stromal vascular fraction cells and profiled metabolites before and after adipogenic differentiation to identify metabolic signatures associated with these distinct cellular states. We found that differentiation alters nucleotide accumulation. Furthermore, inhibition of nucleotide biosynthesis prevented lipid storage within adipocytes and downregulated the expression of lipogenic factors. In contrast to proliferating cells, in which mechanistic target of rapamycin complex 1 is activated by purine accumulation, mechanistic target of rapamycin complex 1 signaling was unaffected by purine levels in differentiating adipocytes. Rather, our data indicated that purines regulate transcriptional activators of adipogenesis, peroxisome proliferator-activated receptor γ and CCAAT/enhancer-binding protein α, to promote differentiation. Although de novo nucleotide biosynthesis has mainly been studied in proliferation, our study points to its requirement in adipocyte differentiation.

Loss of Vhl alters trabecular bone loss during S. aureus osteomyelitis in a cell-specific manner.

Osteomyelitis, or bone infection, is a major complication of accidental trauma or surgical procedures involving the musculoskeletal system. Staphylococcus aureus is the most frequently isolated pathogen in osteomyelitis and triggers significant bone loss. Hypoxia-inducible factor (HIF) signaling has been implicated in antibacterial immune responses as well as bone development and repair. In this study, the impact of bone cell HIF signaling on antibacterial responses and pathologic changes in bone architecture was explored using genetic models with knockout of either Hif1a or a negative regulator of HIF-1α, Vhl. Deletion of Hif1a in osteoblast-lineage cells via Osx-Cre (Hif1aΔOB ) had no impact on bacterial clearance or pathologic changes in bone architecture in a model of post-traumatic osteomyelitis. Knockout of Vhl in osteoblast-lineage cells via Osx-Cre (VhlΔOB ) caused expected increases in trabecular bone volume per total volume (BV/TV) at baseline and, intriguingly, did not exhibit an infection-mediated decline in trabecular BV/TV, unlike control mice. Despite this phenotype, bacterial burdens were not affected by loss of Vhl. In vitro studies demonstrated that transcriptional regulation of the osteoclastogenic cytokine receptor activator of NF-κB ligand (RANKL) and its inhibitor osteoprotegerin (OPG) is altered in osteoblast-lineage cells with knockout of Vhl. After observing no impact on bacterial clearance with osteoblast-lineage conditional knockouts, a LysM-Cre model was used to generate Hif1aΔMyeloid and VhlΔMyeloid mouse models to explore the impact of myeloid cell HIF signaling. In both Hif1aΔMyeloid and VhlΔMyeloid models, bacterial clearance was not impacted. Moreover, minimal impacts on bone architecture were observed. Thus, skeletal HIF signaling was not found to impact bacterial clearance in our mouse model of post-traumatic osteomyelitis, but Vhl deletion in the osteoblast lineage was found to limit infection-mediated trabecular bone loss, possibly via altered regulation of RANKL-OPG gene transcription.

Context-Dependent Roles for Toll-Like Receptors 2 and 9 in the Pathogenesis of Staphylococcus aureus Osteomyelitis.

Staphylococcus aureus is the major causative agent of bacterial osteomyelitis, an invasive infection of bone. Inflammation generated by the immune response to S. aureus contributes to bone damage by altering bone homeostasis. Increases in the differentiation of monocyte lineage cells into bone-resorbing osteoclasts (osteoclastogenesis) promote bone loss in the setting of osteomyelitis. In this study, we sought to define the role of Toll-like receptor (TLR) signaling in the pathogenesis of S. aureus osteomyelitis. We hypothesized that S. aureus-sensing TLRs 2 and 9, both of which are known to alter osteoclastogenesis in vitro, promote pathological changes to bone, including increased osteoclast abundance, bone loss, and altered callus formation during osteomyelitis. Stimulation of osteoclast precursors with S. aureus supernatant increased osteoclastogenesis in a TLR2-dependent, but not a TLR9-dependent, manner. However, in vivo studies using a posttraumatic murine model of osteomyelitis revealed that TLR2-null mice experienced similar bone damage and increased osteoclastogenesis compared to wild type (WT) mice. Therefore, we tested the hypothesis that compensation between TLR2 and TLR9 contributes to osteomyelitis pathogenesis. We found that mice deficient in both TLR2 and TLR9 (Tlr2/9-/-) have decreased trabecular bone loss in response to infection compared to WT mice. However, osteoclastogenesis is comparable between WT and Tlr2/9-/- mice, suggesting that alternative mechanisms enhance osteoclastogenesis in vivo during osteomyelitis. Indeed, we discovered that osteoclast precursors intracellularly infected with S. aureus undergo significantly increased osteoclast formation, even in the absence of TLR2 and TLR9. These results suggest that TLR2 and TLR9 have context-dependent roles in the alteration of bone homeostasis during osteomyelitis.

Development, Pathogenesis, and Regeneration of the Intervertebral Disc: Current and Future Insights Spanning Traditional to Omics Methods.

The intervertebral disc (IVD) is the fibrocartilaginous joint located between each vertebral body that confers flexibility and weight bearing capabilities to the spine. The IVD plays an important role in absorbing shock and stress applied to the spine, which helps to protect not only the vertebral bones, but also the brain and the rest of the central nervous system. Degeneration of the IVD is correlated with back pain, which can be debilitating and severely affects quality of life. Indeed, back pain results in substantial socioeconomic losses and healthcare costs globally each year, with about 85% of the world population experiencing back pain at some point in their lifetimes. Currently, therapeutic strategies for treating IVD degeneration are limited, and as such, there is great interest in advancing treatments for back pain. Ideally, treatments for back pain would restore native structure and thereby function to the degenerated IVD. However, the complex developmental origin and tissue composition of the IVD along with the avascular nature of the mature disc makes regeneration of the IVD a uniquely challenging task. Investigators across the field of IVD research have been working to elucidate the mechanisms behind the formation of this multifaceted structure, which may identify new therapeutic targets and inform development of novel regenerative strategies. This review summarizes current knowledge base on IVD development, degeneration, and regenerative strategies taken from traditional genetic approaches and omics studies and discusses the future landscape of investigations in IVD research and advancement of clinical therapies.

Effects of lithium administration on vertebral bone disease in mucopolysaccharidosis I dogs.

Mucopolysaccharidosis (MPS) I is a lysosomal storage disease characterized by deficient activity of the enzyme alpha-L-iduronidase, leading to abnormal accumulation of heparan and dermatan sulfate glycosaminoglycans in cells and tissues. Patients commonly exhibit progressive skeletal abnormalities, in part due to failures of endochondral ossification during postnatal growth. Previously, using the naturally-occurring canine model, we showed that bone and cartilage cells in MPS I exhibit elevated lysosomal storage from an early age and that animals subsequently exhibit significantly diminished vertebral trabecular bone formation. Wnts are critical regulators of endochondral ossification that depend on glycosaminoglycans for signaling. The objective of this study was to examine whether lithium, a glycogen synthase kinase-3 inhibitor and stimulator of Wnt/beta-catenin signaling, administered during postnatal growth could attenuate progression of vertebral trabecular bone disease in MPS I. MPS I dogs were treated orally with therapeutic levels of lithium carbonate from 14 days to 6 months-of-age. Untreated heterozygous and MPS I dogs served as controls. Serum was collected at 3 and 6 months for assessment of bone turnover markers. At the study end point, thoracic vertebrae were excised and assessed using microcomputed tomography and histology. Lithium-treated animals exhibited significantly improved trabecular spacing, number and connectivity density, and serum bone-specific alkaline phosphatase levels compared to untreated animals. Growth plates from lithium-treated animals exhibited increased numbers of hypertrophic chondrocytes relative to both untreated MPS I and heterozygous animals. These findings suggest that bone and cartilage cells in MPS I are still capable of responding to exogenous osteogenic signals even in the presence of significant lysosomal storage, and that targeted osteogenic therapies may represent a promising approach for attenuating bone disease progression in MPS I.

Progression of vertebral bone disease in mucopolysaccharidosis VII dogs from birth to skeletal maturity.

Mucopolysaccharidosis (MPS) VII is a lysosomal storage disorder characterized by deficient ß-glucuronidase activity, leading to accumulation of incompletely degraded heparan, dermatan and chondroitin sulfate glycosaminoglycans. Patients with MPS VII exhibit progressive spinal deformity, which decreases quality of life. Previously, we demonstrated that MPS VII dogs exhibit impaired initiation of secondary ossification in the vertebrae and long bones. The objective of this study was to build on these findings and comprehensively characterize how vertebral bone disease manifests progressively in MPS VII dogs throughout postnatal growth. Vertebrae were collected postmortem from MPS VII and healthy control dogs at seven ages ranging from 9 to 365 days. Microcomputed tomography and histology were used to characterize bone properties in primary and secondary ossification centers. Serum was analyzed for bone turnover biomarkers. Results demonstrated that not only was secondary ossification delayed in MPS VII vertebrae, but that it progressed aberrantly and was markedly diminished even at 365 days-of-age. Within primary ossification centers, bone volume fraction and bone mineral density were significantly lower in MPS VII at 180 and 365 days-of-age. MPS VII growth plates exhibited significantly lower proliferative and hypertrophic zone cellularity at 90 days-of-age, while serum bone-specific alkaline phosphatase (BAP) was significantly lower in MPS VII dogs at 180 days-of-age. Overall, these findings establish that vertebral bone formation is significantly diminished in MPS VII dogs in both primary and secondary ossification centers during postnatal growth.

Effects of nanocrystalline hydroxyapatite concentration and skeletal site on bone and cartilage formation in rats.

Most fractures heal by a combination of endochondral and intramembranous ossification dependent upon strain and vascularity at the fracture site. Many biomaterials-based bone regeneration strategies rely on the use of calcium phosphates such as nano-crystalline hydroxyapatite (nHA) to create bone-like scaffolds. In this study, nHA was dispersed in reactive polymers to form composite scaffolds that were evaluated both in vitro and in vivo. Matrix assays, immunofluorescent staining, and Western blots demonstrated that nHA influenced mineralization and subsequent osteogenesis in a dose-dependent manner in vitro. Furthermore, nHA dispersed in polymeric composites promoted osteogenesis by a similar mechanism as particulated nHA. Scaffolds were implanted into a 2-mm defect in the femoral diaphysis or metaphysis of Sprague-Dawley rats to evaluate new bone formation at 4 and 8 weeks. Two formulations were tested: a poly(thioketal urethane) scaffold without nHA (PTKUR) and a PTKUR scaffold augmented with 22 wt% nHA (22nHA). The scaffolds supported new bone formation in both anatomic sites. In the metaphysis, augmentation of scaffolds with nHA promoted an intramembranous healing response. Within the diaphysis, nHA inhibited endochondral ossification. Immunohistochemistry was performed on cryo-sections of the bone/scaffold interface in which CD146, CD31, Endomucin, CD68, and Myeloperoxidase were evaluated. No significant differences in the infiltrating cell populations were observed. These findings suggest that nHA dispersed in polymeric composites induces osteogenic differentiation of adherent endogenous cells, which has skeletal site-specific effects on fracture healing. STATEMENT OF SIGNIFICANCE: Understanding the mechanism by which synthetic scaffolds promote new bone formation in preclinical models is crucial for bone regeneration applications in the clinic where complex fracture cases are seen. In this study, we found that dispersion of nHA in polymeric scaffolds promoted in vitro osteogenesis in a dose-dependent manner through activation of the PiT1 receptor and subsequent downstream Erk1/2 signaling. While augmentation of polymeric scaffolds with nHA enhanced intramembranous ossification in metaphyseal defects, it inhibited endochondral ossification in diaphyseal defects. Thus, our findings provide new insights into designing synthetic bone grafts that complement the skeletal site-specific fracture healing response.

Hypoxic Preconditioning Enhances Bone Marrow-Derived Mesenchymal Stem Cell Survival in a Low Oxygen and Nutrient-Limited 3D Microenvironment.

OBJECTIVE: Skeletal tissues such as intervertebral disc and articular cartilage possess limited innate potential to regenerate, in part due to their avascularity and low cell density. Despite recent advances in mesenchymal stem cell (MSC)-based disc and cartilage regeneration, key challenges remain, including the sensitivity of these cells to in vivo microenvironmental stress such as low oxygen and limited nutrition. The objective of this study was to investigate whether preconditioning with hypoxia and/or transforming growth factor-ß 3 (TGF-ß3) can enhance MSC survival and extracellular matrix production in a low oxygen and nutrient-limited microenvironment. DESIGN: MSCs from multiple bovine donors were preconditioned in monolayer in normoxia or hypoxia, with or without TGF-ß3, and the global effects on gene expression were examined using microarrays. Subsequently, the effects of preconditioning on MSC survival and extracellular matrix production were examined using low oxygen and nutrient-limited pellet culture experiments. RESULTS: Hypoxic preconditioning resulted in upregulation of genes associated with growth, cell-cell signaling, metabolism, and cell stress response pathways, and significantly enhanced MSC survival for all donors in low oxygen and nutrient-limited pellet culture. In contrast, TGF-ß3 preconditioning diminished survival. The nature and magnitude of the effects of preconditioning with either hypoxia or TGF-ß3 on glycosaminoglycan production were donor dependent. CONCLUSIONS: These results strongly support the use of hypoxic preconditioning to improve postimplantation MSC survival in avascular tissues such as disc and cartilage.

Dysregulated BMP signaling through ACVR1 impairs digit joint development in fibrodysplasia ossificans progressiva (FOP).

The development of joints in the mammalian skeleton depends on the precise regulation of multiple interacting signaling pathways including the bone morphogenetic protein (BMP) pathway, a key regulator of joint development, digit patterning, skeletal growth, and chondrogenesis. Mutations in the BMP receptor ACVR1 cause the rare genetic disease fibrodysplasia ossificans progressiva (FOP) in which extensive and progressive extra-skeletal bone forms in soft connective tissues after birth. These mutations, which enhance BMP-pSmad1/5 pathway activity to induce ectopic bone, also affect skeletal development. FOP can be diagnosed at birth by symmetric, characteristic malformations of the great toes (first digits) that are associated with decreased joint mobility, shortened digit length, and absent, fused, and/or malformed phalanges. To elucidate the role of ACVR1-mediated BMP signaling in digit skeletal development, we used an Acvr1R206H/+;Prrx1-Cre knock-in mouse model that mimics the first digit phenotype of human FOP. We have determined that the effects of increased Acvr1-mediated signaling by the Acvr1R206H mutation are not limited to the first digit but alter BMP signaling, Gdf5+ joint progenitor cell localization, and joint development in a manner that differently affects individual digits during embryogenesis. The Acvr1R206H mutation leads to delayed and disrupted joint specification and cleavage in the digits and alters the development of cartilage and endochondral ossification at sites of joint morphogenesis. These findings demonstrate an important role for ACVR1-mediated BMP signaling in the regulation of joint and skeletal formation, show a direct link between failure to restrict BMP signaling in the digit joint interzone and failure of joint cleavage at the presumptive interzone, and implicate impaired, digit-specific joint development as the proximal cause of digit malformation in FOP.

Molecular profiling of failed endochondral ossification in mucopolysaccharidosis VII.

Mucopolysaccharidosis (MPS) VII is a lysosomal storage disorder characterized by deficient activity of ß-glucuronidase, leading to progressive accumulation of incompletely degraded heparan, dermatan, and chondroitin sulfate glycosaminoglycans (GAGs). Patients with MPS VII exhibit progressive skeletal deformity including kyphoscoliosis and joint dysplasia, which decrease quality of life and increase mortality. Previously, using the naturally-occurring canine model, we demonstrated that one of the earliest skeletal abnormalities to manifest in MPS VII is failed initiation of secondary ossification in vertebrae and long bones at the requisite postnatal developmental stage. The objective of this study was to obtain global insights into the molecular mechanisms underlying this failed initiation of secondary ossification. Epiphyseal tissue was isolated postmortem from the vertebrae of control and MPS VII-affected dogs at 9 and 14 days-of-age (n = 5 for each group). Differences in global gene expression across this developmental window for both cohorts were measured using whole-transcriptome sequencing (RNA-Seq). Principal Component Analysis revealed clustering of samples within each group, indicating clear effects of both age and disease state. At 9 days-of-age, 1375 genes were significantly differentially expressed between MPS VII and control, and by 14 days-of-age, this increased to 4719 genes. A targeted analysis focused on signaling pathways important in the regulation of endochondral ossification was performed, and a subset of gene expression differences were validated using qPCR. Osteoactivin (GPNMB) was the top upregulated gene in MPS VII at both ages. In control samples, temporal changes in gene expression from 9 to 14 days-of-age were consistent with chondrocyte maturation, cartilage resorption, and osteogenesis. In MPS VII samples, however, elements of key osteogenic pathways such as Wnt/ß-catenin and BMP signaling were not upregulated during this same developmental window suggesting that important bone formation pathways are not activated. In conclusion, this study represents an important step towards identifying therapeutic targets and biomarkers for bone disease in MPS VII patients during postnatal growth.

Neuronal ceroid lipofuscinosis related ER membrane protein CLN8 regulates PP2A activity and ceramide levels.

The neuronal ceroid lipofuscinoses (NCLs) are a group of inherited neurodegenerative lysosomal storage disorders. CLN8 deficiency causes a subtype of NCL, referred to as CLN8 disease. CLN8 is an ER resident protein with unknown function; however, a role in ceramide metabolism has been suggested. In this report, we identified PP2A and its biological inhibitor I2PP2A as interacting proteins of CLN8. PP2A is one of the major serine/threonine phosphatases in cells and governs a wide range of signaling pathways by dephosphorylating critical signaling molecules. We showed that the phosphorylation levels of several substrates of PP2A, namely Akt, S6 kinase, and GSK3ß, were decreased in CLN8 disease patient fibroblasts. This reduction can be reversed by inhibiting PP2A phosphatase activity with cantharidin, suggesting a higher PP2A activity in CLN8-deficient cells. Since ceramides are known to bind and influence the activity of PP2A and I2PP2A, we further examined whether ceramide levels in the CLN8-deficient cells were changed. Interestingly, the ceramide levels were reduced by 60% in CLN8 disease patient cells compared to controls. Furthermore, we observed that the conversion of ER-localized NBD-C6-ceramide to glucosylceramide and sphingomyelin in the Golgi apparatus was not affected in CLN8-deficient cells, indicating transport of ceramides from ER to the Golgi apparatus was normal. A model of how CLN8 along with ceramides affects I2PP2A and PP2A binding and activities is proposed.

Quantitative MRI correlates with histological grade in a percutaneous needle injury mouse model of disc degeneration.

Low back pain due to disc degeneration is a major cause of morbidity and health care expenditures worldwide. While stem cell-based therapies hold promise for disc regeneration, there is an urgent need to develop improved in vivo animal models to further develop and validate these potential treatments. The objectives of this study were to characterize a percutaneous needle injury model of intervertebral disc degeneration in the mouse caudal spine, and compare two non-invasive quantitative imaging techniques, microcomputed tomography and magnetic resonance imaging (MRI), as effective measures of disc degeneration in this model. Percutaneous needle injury of mouse caudal discs was undertaken using different needle sizes and injury types (unilateral or bilateral annulus fibrosus (AF) puncture). Mice were euthanized 4 weeks post-injury, and MRI and microcomputed tomography were used to determine T2 relaxation time of the NP and disc height index, respectively. Disc condition was then further assessed using semi-quantitative histological grading. Bilateral AF puncture with either 27 or 29G needles resulted in significantly lower T2 relaxation times compared to uninjured controls, while disc height index was not significantly affected by any injury type. There was a strong, inverse linear relationship between histological grade and NP T2 relaxation time. In this study, we demonstrated that quantitative MRI can detect disc degeneration in the mouse caudal spine 4 weeks following percutaneous needle injury, and may therefore serve as a surrogate for histology in longitudinal studies of both disc degeneration and cell-based therapies for disc regeneration using this model. © 2018 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 36:2771-2779, 2018.

RNA sequencing identifies gene regulatory networks controlling extracellular matrix synthesis in intervertebral disk tissues.

Degenerative disk disease of the spine is a major cause of back pain and disability. Optimization of regenerative medical therapies for degenerative disk disease requires a deep mechanistic understanding of the factors controlling the structural integrity of spinal tissues. In this investigation, we sought to identify candidate regulatory genes controlling extracellular matrix synthesis in spinal tissues. To achieve this goal we performed high throughput next generation RNA sequencing on 39 annulus fibrosus and 21 nucleus pulposus human tissue samples. Specimens were collected from patients undergoing surgical discectomy for the treatment of degenerative disk disease. Our studies identified associations between extracellular matrix genes, growth factors, and other important regulatory molecules. The fibrous matrix characteristic of annulus fibrosus was associated with expression of the growth factors platelet derived growth factor beta (PDGFB), vascular endothelial growth factor C (VEGFC), and fibroblast growth factor 9 (FGF9). Additionally we observed high expression of multiple signaling proteins involved in the NOTCH and WNT signaling cascades. Nucleus pulposus extracellular matrix related genes were associated with the expression of numerous diffusible growth factors largely associated with the transforming growth signaling cascade, including transforming factor alpha (TGFA), inhibin alpha (INHA), inhibin beta A (INHBA), bone morphogenetic proteins (BMP2, BMP6), and others. CLINICAL SIGNIFICANCE: this investigation provides important data on extracellular matrix gene regulatory networks in disk tissues. This information can be used to optimize pharmacologic, stem cell, and tissue engineering strategies for regeneration of the intervertebral disk and the treatment of back pain. © 2017 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 36:1356-1369, 2018.

Whole Transcriptome Analysis of Notochord-Derived Cells during Embryonic Formation of the Nucleus Pulposus.

Recapitulation of developmental signals represents a promising strategy for treating intervertebral disc degeneration. During development, embryonic notochord-derived cells (NDCs) are the direct progenitors of cells that populate the adult nucleus pulposus (NP) and are an important source of secreted signaling molecules. The objective of this study was to define global gene expression profiles of NDCs at key stages of embryonic disc formation. NDCs were isolated from Shh-cre;ROSA:YFP mice at embryonic day 12.5 and postnatal day 0, representing opposite ends of the notochord to NP transformation. Differences in global mRNA abundance across this developmental window were established using RNA-Seq. Protein expression of selected molecules was confirmed using immunohistochemistry. Principal component analysis revealed clustering of gene expression at each developmental stage with more than 5000 genes significantly differentially expressed between E12.5 and P0. There was significantly lower mRNA abundance of sonic hedgehog pathway elements at P0 vs E12.5, while abundance of elements of the transforming growth factor-beta and insulin-like growth factors pathways, and extracellular matrix components including collagen 6 and aggrecan, were significantly higher at P0. This study represents the first transcriptome-wide analysis of embryonic NDCs. Results suggest signaling and biosynthesis of NDCs change dramatically as a function of developmental stage.

Pathogenesis and treatment of spine disease in the mucopolysaccharidoses.

The mucopolysaccharidoses (MPS) are a family of lysosomal storage disorders characterized by deficient activity of enzymes that degrade glycosaminoglycans (GAGs). Skeletal disease is common in MPS patients, with the severity varying both within and between subtypes. Within the spectrum of skeletal disease, spinal manifestations are particularly prevalent. Developmental and degenerative abnormalities affecting the substructures of the spine can result in compression of the spinal cord and associated neural elements. Resulting neurological complications, including pain and paralysis, significantly reduce patient quality of life and life expectancy. Systemic therapies for MPS, such as hematopoietic stem cell transplantation and enzyme replacement therapy, have shown limited efficacy for improving spinal manifestations in patients and animal models. Therefore, there is a pressing need for new therapeutic approaches that specifically target this debilitating aspect of the disease. In this review, we examine how pathological abnormalities affecting the key substructures of the spine - the discs, vertebrae, odontoid process and dura - contribute to the progression of spinal deformity and symptomatic compression of neural elements. Specifically, we review current understanding of the underlying pathophysiology of spine disease in MPS, how the tissues of the spine respond to current clinical and experimental treatments, and discuss future strategies for improving the efficacy of these treatments.

Delayed hypertrophic differentiation of epiphyseal chondrocytes contributes to failed secondary ossification in mucopolysaccharidosis VII dogs.

Mucopolysaccharidosis (MPS) VII is a lysosomal storage disorder characterized by deficient ß-glucuronidase activity, which leads to the accumulation of incompletely degraded glycosaminoglycans (GAGs). MPS VII patients present with severe skeletal abnormalities, which are particularly prevalent in the spine. Incomplete cartilage-to-bone conversion in MPS VII vertebrae during postnatal development is associated with progressive spinal deformity and spinal cord compression. The objectives of this study were to determine the earliest postnatal developmental stage at which vertebral bone disease manifests in MPS VII and to identify the underlying cellular basis of impaired cartilage-to-bone conversion, using the naturally-occurring canine model. Control and MPS VII dogs were euthanized at 9 and 14 days-of-age, and vertebral secondary ossification centers analyzed using micro-computed tomography, histology, qPCR, and protein immunoblotting. Imaging studies and mRNA analysis of bone formation markers established that secondary ossification commences between 9 and 14 days in control animals, but not in MPS VII animals. mRNA analysis of differentiation markers revealed that MPS VII epiphyseal chondrocytes are unable to successfully transition from proliferation to hypertrophy during this critical developmental window. Immunoblotting demonstrated abnormal persistence of Sox9 protein in MPS VII cells between 9 and 14 days-of-age, and biochemical assays revealed abnormally high intra and extracellular GAG content in MPS VII epiphyseal cartilage at as early as 9 days-of-age. In contrast, assessment of vertebral growth plates and primary ossification centers revealed no significant abnormalities at either age. The results of this study establish that failed vertebral bone formation in MPS VII can be traced to the failure of epiphyseal chondrocytes to undergo hypertrophic differentiation at the appropriate developmental stage, and suggest that aberrant processing of Sox9 protein may contribute to this cellular dysfunction. These results also highlight the importance of early diagnosis and therapeutic intervention to prevent the progression of debilitating skeletal disease in MPS patients.

The role of N-glycosylation in folding, trafficking, and functionality of lysosomal protein CLN5.

CLN5 is a soluble lysosomal protein with unknown function. Mutations in CLN5 lead to neuronal ceroid lipofuscinosis, a group of inherited neurodegenerative disorders that mainly affect children. CLN5 has eight potential N-glycosylation sites based on the Asn-X-Thr/Ser consensus sequence. Through site-directed mutagenesis of individual asparagine residues to glutamine on each of the N-glycosylation consensus sites, we showed that all eight putative N-glycosylation sites are utilized in vivo. Additionally, localization studies showed that the lack of N-glycosylation on certain sites (N179, N252, N304, or N320) caused CLN5 retention in the endoplasmic reticulum, indicating that glycosylation is important for protein folding. Interestingly, one particular mutant, N401Q, is mislocalized to the Golgi, suggesting that N401 is not important for protein folding but essential for CLN5 trafficking to the lysosome. Finally, we analyzed several patient mutations in which N-glycosylation is affected. The N192S patient mutant is localized to the lysosome, indicating that this mutant has a functional defect in the lysosome. Our results suggest that there are functional differences in various N-glycosylation sites of CLN5 which affect folding, trafficking, and lysosomal function of CLN5.

Directed evolution of a small-molecule-triggered intein with improved splicing properties in mammalian cells.

Laboratory-created small-molecule-dependent inteins enable protein structure and function to be controlled posttranslationally in living cells. Previously we evolved inteins that splice efficiently in Saccharomyces cerevisiae only in the presence of the cell-permeable small molecule 4-hydroxytamoxifen (4-HT). In mammalian cells, however, these inteins exhibited lower splicing efficiencies and slower splicing in the presence of 4-HT, as well as higher background splicing in the absence of 4-HT. Here we further evolved ligand-dependent inteins in yeast at 30°C and 37°C. The resulting second-generation evolved inteins exhibit substantially improved splicing yields and kinetics. The improvements carried over to mammalian cells, in which the newly evolved inteins spliced with substantially greater (∼2- to 8-fold) efficiency while maintaining low background splicing levels. These second-generation inteins augment the promise of ligand-dependent protein splicing for probing protein function in mammalian cells.