Proteoglycan Metabolism Research Articles

Genetic architecture and polygenic risk score prediction of degenerative suspensory ligament desmitis (DSLD) in the Peruvian Horse.

Introduction: Spontaneous rupture of tendons and ligaments is common in several species including humans. In horses, degenerative suspensory ligament desmitis (DSLD) is an important acquired idiopathic disease of a major energy-storing tendon-like structure. DSLD risk is increased in several breeds, including the Peruvian Horse. Affected horses have often been used for breeding before the disease is apparent. Breed predisposition suggests a substantial genetic contribution, but heritability and genetic architecture of DSLD have not been determined. Methods: To identify genomic regions associated with DSLD, we recruited a reference population of 183 Peruvian Horses, phenotyped as DSLD cases or controls, and undertook a genome-wide association study (GWAS), a regional window variance analysis using local genomic partitioning, a signatures of selection (SOS) analysis, and polygenic risk score (PRS) prediction of DSLD risk. We also estimated trait heritability from pedigrees. Results: Heritability was estimated in a population of 1,927 Peruvian horses at 0.22 ± 0.08. After establishing a permutation-based threshold for genome-wide significance, 151 DSLD risk single nucleotide polymorphisms (SNPs) were identified by GWAS. Multiple regions of enriched local heritability were identified across the genome, with strong enrichment signals on chromosomes 1, 2, 6, 10, 13, 16, 18, 22, and the X chromosome. With SOS analysis, there were 66 genes with a selection signature in DSLD cases that was not present in the control group that included the TGFB3 gene. Pathways enriched in DSLD cases included proteoglycan metabolism, extracellular matrix homeostasis, and signal transduction pathways that included the hedgehog signaling pathway. The best PRS predictive performance was obtained when we fitted 1% of top SNPs using a Bayesian Ridge Regression model which achieved the highest mean of R2 on both the probit and logit liability scales, indicating a strong predictive performance. Discussion: We conclude that within-breed GWAS of DSLD in the Peruvian Horse has further confirmed that moderate heritability and a polygenic architecture underlies the trait and identified multiple DSLD SNP associations in novel tendinopathy candidate genes influencing disease risk. Pathways enriched with DSLD risk variants include ones that influence glycosaminoglycan metabolism, extracellular matrix homeostasis, signal transduction pathways.

Abstract PR009: A new invasive zebrafish model of ewing sarcoma reveals EWSR1-FLI1-driven dysregulation of heparan sulfate proteoglycan metabolism and ERK signaling in developing tumors

Abstract Ewing sarcoma is a malignant tumor of bones or soft tissues with an extremely poor prognosis for patients with metastatic or relapsed disease. Ewing sarcoma is associated with the appearance of a chimeric fusion oncogene, most frequently EWSR1-FLI1 (EF1). The interaction of Ewing sarcoma cells with the tumor microenvironment is essential for tumor growth, angiogenesis, and metastasis. The tumor microenvironment includes surrounding blood vessels, immune cells, signaling molecules, and extracellular matrix. The dependence of a tumor cell on its microenvironment opens new possibilities to target not just cancer cells but also the tumor microenvironment itself. In the absence of a representative in vivo model, the role of tumor cell/microenvironmental interactions in Ewing sarcoma initiation and progression is not well-understood. To address these concerns, we developed a new in vivo genetic model of Ewing sarcoma by integrating the human EWSR1-FLI1 fusion oncogene into the zebrafish genome. Our system allows broad mosaic Cre-inducible expression of human EF1 in zebrafish, which causes rapid onset of Ewing sarcoma at high penetrance. This invasive model allows studying the behavior of GFP-labeled cancer cells during tumor initiation and progression, in a complex developmental context which is not currently possible in mammals. We found that observed tumors express canonical EF1 target genes and stain for known Ewing sarcoma markers including CD99. Proteomic analysis revealed that EF1 expression affects the expression of enzymes involved in heparan sulfate proteoglycan (HSPG) metabolism. Such dysregulation of proteoglycan metabolism is associated with activation of ERK1/2 signaling pathway in tumor cells resulting in increased tumor cell proliferation and survival. Proteoglycans are key regulators of signaling of cell surface receptors, playing an essential role in cell-to-cell communication. Targeting heparan sulfate proteoglycans with the specific heparan sulfate antagonist Surfen reduces ERK1/2 signaling and decreases tumorigenicity of Ewing sarcoma cells in vitro and in vivo. Taken together, our new zebrafish model will open new possibilities to study EWSR1-FLI- driven tumorigenesis, as well as mechanisms of tumor development and progression. Our results highlight the important role of the extracellular matrix in Ewing sarcoma tumor growth and the potential of agents targeting proteoglycan metabolism as novel therapies for this disease. Citation Format: Elena Vasileva, Mikako Warren, Timothy J. Triche, James F. Amatrud. A new invasive zebrafish model of ewing sarcoma reveals EWSR1-FLI1-driven dysregulation of heparan sulfate proteoglycan metabolism and ERK signaling in developing tumors [abstract]. In: Proceedings of the AACR Special Conference: Sarcomas; 2022 May 9-12; Montreal, QC, Canada. Philadelphia (PA): AACR; Clin Cancer Res 2022;28(18_Suppl):Abstract nr PR009.

Hair Loss and Telogen Effluvium Related to COVID-19: The Potential Implication of Adipose-Derived Mesenchymal Stem Cells and Platelet-Rich Plasma as Regenerative Strategies.

The diffusion of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) inducing coronavirus disease 2019 (COVID-19) has increased the incidence of several dermatological disorders, including hair loss (HL). This article aims to review the literature regarding the incidence of HL and telogen effluvium (TE) in COVID-19 patients and critically appraise the available evidence regarding the role of regenerative strategies like Platelet-Rich Plasma (PRP) and Human Follicle Stem Cells (HFSCs). A literature review regarding the correlation of HL and TE in COVID-19 patients analyzing the biomolecular pathway involved and the role of regenerative strategies was performed using PubMed, MEDLINE, Embase, PreMEDLINE, Scopus, and the Cochrane databases. Observational studies revealed an escalated incidence of pattern HL and TE in COVID-19 patients. Psychological stress, systemic inflammation, and oxidative stress are potential culprits. Proinflammatory cytokines and stress hormones negatively affect the normal metabolism of proteoglycans. Reduced anagenic expression of proteoglycans is a potential mediating mechanism that connects HL to COVID-19. Currently, only one study has been published on PRP against HL in COVID-19 patients. Further controlled trials are required to confirm PRP and HFSCs efficacy in COVID-19 patients.

Abstract 1636: A new invasive zebrafish model of Ewing sarcoma reveals EWSR1-FLI1-driven dysregulation of heparan sulfate proteoglycan metabolism in developing tumors

Abstract Ewing sarcoma (ES) is a malignant bone and soft-tissue tumor with an extremely poor prognosis for patients with metastatic or relapsed disease. ES is characterized by the appearance of a chimeric fusion oncogene, most frequently EWSR1-FLI1 (EF1). In the absence of a representative in vivo model the role of tumor cell/microenvironmental interactions in tumor initiation and progression remains a subject of constant debate. To address these concerns, we developed an innovative Cre-inducible model of ES in zebrafish which allows broad mosaic expression of human EF1 in zebrafish, which causes rapid onset of ES at high penetrance. This invasive model allows study of the behavior of GFP-labeled cancer cells during tumor initiation and progression, in a complex developmental context which is not currently possible in mammals. We found that observed tumors express canonical EF1 target genes and stain for known ES markers including CD99. Proteomic analysis revealed that EF1 expression affects heparan sulfate proteoglycan (HSPG) metabolism and activates HSPG-mediated ERK signaling in EF1 expressing cells, promoting cancer cell survival and proliferation. Proteoglycans are key regulators of signaling of cell surface receptors, playing an essential role in cell-to-cell communication. Targeting heparan sulfate proteoglycans with the specific heparan sulfate antagonist Surfen reduces ERK1/2 signaling and decreases tumorigenicity of ES cells in vitro and in vivo. These results highlight the important role of the extracellular matrix in ES tumor growth and the potential of agents targeting proteoglycan metabolism as novel therapies for this disease. Citation Format: Elena Vasileva, Mikako Warren, Timothy Triche, James Amatruda. A new invasive zebrafish model of Ewing sarcoma reveals EWSR1-FLI1-driven dysregulation of heparan sulfate proteoglycan metabolism in developing tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1636.

Dysregulated heparan sulfate proteoglycan metabolism promotes Ewing sarcoma tumor growth.

The Ewing sarcoma family of tumors is a group of malignant small round blue cell tumors (SRBCTs) that affect children, adolescents, and young adults. The tumors are characterized by reciprocal chromosomal translocations that generate chimeric fusion oncogenes, the most common of which is EWSR1-FLI1. Survival is extremely poor for patients with metastatic or relapsed disease, and no molecularly targeted therapy for this disease currently exists. The absence of a reliable genetic animal model of Ewing sarcoma has impaired investigation of tumor cell/microenvironmental interactions in vivo. We have developed a new genetic model of Ewing sarcoma based on Cre-inducible expression of human EWSR1-FLI1 in wild-type zebrafish, which causes rapid onset of SRBCTs at high penetrance. The tumors express canonical EWSR1-FLI1 target genes and stain for known Ewing sarcoma markers including CD99. Growth of tumors is associated with activation of the MAPK/ERK pathway, which we link to dysregulated extracellular matrix metabolism in general and heparan sulfate proteoglycan catabolism in particular. Targeting heparan sulfate proteoglycans with the specific heparan sulfate antagonist Surfen reduces ERK1/2 signaling and decreases tumorigenicity of Ewing sarcoma cells in vitro and in vivo. These results highlight the important role of the extracellular matrix in Ewing sarcoma tumor growth and the potential of agents targeting proteoglycan metabolism as novel therapies for this disease.

Damage to the connective tissue system in patients with post-traumatic osteomyelitis

In 63 patients with suppurative bone process the activity of N-acetyl-fi-D-glucosaminidaze lysosomal enzyme in urine as well as oxyproline and hexuronic acids excretion were evaluated. Significant changes in metabolism of collagen and proteoglycans of osseous organic matrix were detected and it was confirmed by high excretion of their structural components. The more active and vaster the suppurative process was the more quantitative excretion of collagen and glu- cosaminoglycans and higher enzyme activity were. The suggestion was formulated that high excretion of N-acetyl-fl-D-glucosaminidaze in urine noted in the patients with suppurative inflammatory complications was the indicator of pathologic changes in tubular apparatus of kidney. The detection of excretion of the connective tissue metabolites (oxyproline and hexuronic acids) could be used for the diagnostic and prognostic evaluation of the disease severity and for the choice of method for the treatment of suppurative process.

Integration of a miniaturized DMMB assay with high-throughput screening for identifying regulators of proteoglycan metabolism

Defective biosynthesis or function of proteoglycans causes pathological conditions in a variety of tissue systems. Osteoarthritis (OA) is a prevalent degenerative joint disorder characterized by progressive cartilage destruction caused by imbalanced proteoglycan synthesis and degradation. Identifying agents that regulate proteoglycan metabolism may benefit the development of OA-modifying therapeutics. High-throughput screening (HTS) of chemical libraries has paved the way for achieving this goal. However, the implementation and adaptation of HTS assays based on proteoglycan measurement remain underexploited. Using primary porcine chondrocytes as a model, we report a miniaturized dimethyl-methylene blue (DMMB) assay, which is commonly used to quantitatively evaluate sulfated glycosaminoglycan (GAG) content, with an optimized detection range and reproducibility and its integration with HTS. Treatment with TGF-β1 and IL1-α, known as positive and negative proteoglycan regulators, respectively, supported the assay specificity. A pre-test of chemical screening of 960 compounds identified both stimulators (4.48%) and inhibitors (6.04%) of GAG production. Fluorophore-assisted carbohydrate electrophoresis validated the activity of selected hits on chondroitin sulfate expression in an alginate culture system. Our findings support the implementation of this simple colorimetric assay in HTS to discover modifiers of OA or other diseases related to dysregulated proteoglycan metabolism.

Inherited Proteoglycan Biosynthesis Defects—Current Laboratory Tools and Bikunin as a Promising Blood Biomarker

Proteoglycans consist of proteins linked to sulfated glycosaminoglycan chains. They constitute a family of macromolecules mainly involved in the architecture of organs and tissues as major components of extracellular matrices. Some proteoglycans also act as signaling molecules involved in inflammatory response as well as cell proliferation, adhesion, and differentiation. Inborn errors of proteoglycan metabolism are a group of orphan diseases with severe and irreversible skeletal abnormalities associated with multiorgan impairments. Identifying the gene variants that cause these pathologies proves to be difficult because of unspecific clinical symptoms, hardly accessible functional laboratory tests, and a lack of convenient blood biomarkers. In this review, we summarize the molecular pathways of proteoglycan biosynthesis, the associated inherited syndromes, and the related biochemical screening techniques, and we focus especially on a circulating proteoglycan called bikunin and on its potential as a new biomarker of these diseases.

Ciprofloxacin reduces tenocyte viability and proteoglycan synthesis in short-term explant cultures of equine tendon.

Fluoroquinolones are an effective, broad-spectrum antibiotic used to treat an array of bacterial infections. However, they are associated with an increased risk of tendinopathy and tendon rupture even after discontinuation of treatment. This condition is known as fluoroquinolone-associated tendinopathy, the underlying mechanisms of which are poorly understood. While many factors may be involved in the pathophysiology of tendinopathies in general, changes in tenocyte metabolism and viability, as well as alteration of proteoglycan metabolism are prominent findings in the scientific literature. This study investigated the effects of ciprofloxacin, a common fluoroquinolone, on cell viability, proteoglycan synthesis, and proteoglycan mRNA expression in equine superficial digital flexor tendon explants after 96 h treatment with between 1–300 µg/mL ciprofloxacin, and again after 8 days discontinuation of treatment. Ciprofloxacin caused significant reductions in cell viability by between 25–33% at all dosages except 10 µg/mL, and viability decreased further after 8 days discontinuation of treatment. Proteoglycan synthesis significantly decreased by approximately 50% in explants treated with 100 µg/mL and 300 µg/mL, however this effect reversed after 8 days in the absence of treatment. No significant mRNA expression changes were observed after the treatment period with the exception of versican which was down-regulated at the highest concentration of ciprofloxacin. After the recovery period, aggrecan, biglycan and versican genes were all significantly downregulated in explants initially treated with 1–100 µg/mL. Results from this study corroborate previously reported findings of reduced cell viability and proteoglycan synthesis in a whole tissue explant model and provide further insight into the mechanisms underlying fluoroquinolone-associated tendinopathy and rupture. This study further demonstrates that certain ciprofloxacin induced cellular changes are not rapidly reversed upon cessation of treatment which is a novel finding in the literature.

COVID-19 Can Exacerbate Pattern Hair Loss and Trigger Telogen Effluvium - The Role of Proteoglycan Replacement Therapy with Nourkrin® in Clinical Treatment of COVID-19 Associated Hair Loss

The unfolding coronavirus disease 2019 (COVID-19) has increased the incidence of several dermatological disorders, including diffuse hair loss. Research revealed an escalated incidence of pattern hair loss (PHL) and telogen effluvium (TE) in COVID-19 patients. Psychological stress, systemic inflammation and oxidative stress are potential culprits. Reduced anagenic expression of proteoglycans is a potential mediating mechanism that connects hair loss to critical health conditions such as COVID-19. Proinflammatory cytokines and stress hormones negatively affect the normal metabolism of proteoglycans, which are known regulators of the hair growth cycle.

Порушення метаболізму протеогліканів і колагену у нирках за цукрового діабету: клініко-патогенетичні механізми та лабораторні маркери

The article considers the issue of disorders of connective tissue metabolism in diabetes mellitus. Glycosylation of structural components of connective tissue and glucose toxicity have been found to determine the pathogenesis of late complications of diabetes mellitus. The most common concept of the pathogenesis of diabetes is metabolic, according to which all variants of diabetes mellitus, including blood vessels, their basement membrane, are associated with primary disorders of lipid, glycoprotein, protein and carbohydrate metabolism due to complete or partial insufficiency. It has been found that the formation of interstitial fibrosis in the kidneys of patients with diabetes begins in the preclinical stages of diabetic nephropathy. The leading cause of interstitial fibrogenesis is hyperglycemia; exacerbate proteinuria fibrosis, activation of the renin-angiotensin system, chronic inflammation and the formation of myofibroblasts in the interstitium. According to the results of the study of aspects of early diagnosis of kidney damage in type 1 diabetes mellitus, it was found that the development of diabetic nephrosclerosis is characterized by qualitative and quantitative changes in collagen composition in the glomeruli and interstitium, rebalance between collagen synthesis and breakdown, glycosaminoglycans, increased synthesis of fibrogenic growth factors and oxidative modification of proteins. The formation of diabetic nephropathy in patients with diabetes is also characterized by the accumulation of collagen types IV and VI, the appearance of interstitial collagen types III and I in the glomeruli, as well as the accumulation of collagen of all types in tubulointerstitium. Quantitative and qualitative characteristics of sulfated glycosaminoglycans of urine in human diabetes indicated different degrees of development of diabetic nephropathy. Glycosaminoglycans hyperexcretion was observed in patients with diabetes mellitus without proteinuria. In patients with microalbuminuria, glycosaminoglycans hyperexcretion was even more pronounced. It was also found that in diabetes, the total excretion of sulfated glycosaminoglycans in the urine doubles. Conclusion. Thus, in diabetes mellitus, an important pathogenetic link in the violation of the morpho-functional state of the kidneys is the degradation of collagen and proteoglycans of the basement membranes of the glomeruli, as well as interstitial fibrosis. This is reflected in changes in urinary glycosaminoglycans excretion, in particular heparansulfate and chondroitin sulfate, which may serve as a marker of proteoglycan metabolism disorders in the kidneys. Patients with diabetes also have an increase in the urine of hydroxyproline, which indicates an increase in the intensity of collagen metabolism in patients

CRISPR screening of porcine sgRNA library identifies host factors associated with Japanese encephalitis virus replication

Japanese encephalitis virus (JEV) is a mosquito-borne zoonotic flavivirus that causes encephalitis and reproductive disorders in mammalian species. However, the host factors critical for its entry, replication, and assembly are poorly understood. Here, we design a porcine genome-scale CRISPR/Cas9 knockout (PigGeCKO) library containing 85,674 single guide RNAs targeting 17,743 protein-coding genes, 11,053 long ncRNAs, and 551 microRNAs. Subsequently, we use the PigGeCKO library to identify key host factors facilitating JEV infection in porcine cells. Several previously unreported genes required for JEV infection are highly enriched post-JEV selection. We conduct follow-up studies to verify the dependency of JEV on these genes, and identify functional contributions for six of the many candidate JEV-related host genes, including EMC3 and CALR. Additionally, we identify that four genes associated with heparan sulfate proteoglycans (HSPGs) metabolism, specifically those responsible for HSPGs sulfurylation, facilitate JEV entry into porcine cells. Thus, beyond our development of the largest CRISPR-based functional genomic screening platform for pig research to date, this study identifies multiple potentially vulnerable targets for the development of medical and breeding technologies to treat and prevent diseases caused by JEV.

Differential miRNA Expression: Signature for Glaucoma in Pseudoexfoliation.

PurposeTo investigate the microRNA (miRNA) profile in patients with different stages of pseudoexfoliation (PXF).MethodsPeripheral blood of patients with PXF (naïve to medical therapy and with no systemic disease/drugs) with ocular hypertension (OHT) and pseudoexfoliation glaucoma (PXG) was evaluated in triplicate for miRNA profiling using polymerase chain reaction (PCR) arrays. Those identified in the discovery stage were validated with evaluation of serum transforming growth factor-β1 (TGF-β1) levels by ELISA. The downstream targets of TGF-β1 and unfolded protein response (UPR) were analyzed using reverse transcription-quantitative polymerase chain reaction (RT-qPCR). Predicted targets of the identified miRNA and KEGG pathway analysis were done using miRbase and DIANA tools mirPathv3.1.ResultsWe found hsa-miR-122-5p, hsa-miR-124-3p and hsa-miR-424-5p to be upregulated in PXG targeting 3 specific pathways namely TGF-β1, fibrosis/ECM and proteoglycan metabolism with common effectors like SMAD/3/2. The unfolded protein response (UPR) genes were significantly downregulated in all stages of PXF suggesting this as the key mechanism for protein aggregates in PXF syndrome. Serum TGF-β1 was significantly upregulated as disease progressed to later stages in PXG. This elevation in advanced stages was associated with significantly differential expression of downstream pathways and fibrotic genes in OHT compared to PXG predominantly through the SMAD3, a canonical pathway marker.ConclusionCirculatory miRNA differentially regulating TGF-β1 and downstream targets including UPR genes may be the key mechanisms for glaucoma onset in PXF.

Integral Roles of Specific Proteoglycans in Hair Growth and Hair Loss: Mechanisms behind the Bioactivity of Proteoglycan Replacement Therapy with Nourkrin® with Marilex® in Pattern Hair Loss and Telogen Effluvium.

Follicular proteoglycans are key players with structural, functional, and regulatory roles in the growth and cycling behaviour of the hair follicles. The expression pattern of specific proteoglycans is strongly correlated with follicular phase transitions, which further affirms their functional involvement. Research shows that bioactive proteoglycans, e.g., versican and decorin, can actively trigger follicular phase shift by their anagen-inducing, anagen-maintaining, and immunoregulatory properties. This emerging insight has led to the recognition of “dysregulated proteoglycan metabolism” as a plausible causal or mediating pathology in hair growth disorders in both men and women. In support of this, declined expression of proteoglycans has been reported in cases of anagen shortening and follicular miniaturisation. To facilitate scientific communication, we propose designating this pathology “follicular hypoglycania (FHG),” which results from an impaired ability of follicular cells to replenish and maintain a minimum relative concentration of key proteoglycans during anagen. Lasting FHG may advance to structural decay, called proteoglycan follicular atrophy (PFA). This process is suggested to be an integral pathogenetic factor in pattern hair loss (PHL) and telogen effluvium (TE). To address FHG and PFA, a proteoglycan replacement therapy (PRT) program using oral administration of a marine-derived extract (Nourkrin® with Marilex®, produced by Pharma Medico Aps, Aarhus, Denmark) containing specific proteoglycans has been developed. In clinical studies, this treatment significantly reduced hair fall, promoted hair growth, and improved quality of life in patients with male- and female-pattern hair loss. Accordingly, PRT (using Nourkrin® with Marilex®) can be recommended as an add-on treatment or monotherapy in patients with PHL and TE.

Prognostic value and clinical laboratory assessment of connective tissue exchange disorders in the course of children’s traumatic brain injury

The author of this paper has carried out clinical laboratory assessment of connective tissue disorders with the purpose of the prognostic value of the course of closed craniocerebral injury of children with different severity. 115 children with closed craniocerebral injury with different severity in the age from 3 to 17 were examined. There were 90 boys and 25 girls among them. The average age of children was (13.5 ± 4.5) years. The distribution of children into groups was carried out according to the clinical forms of traumatic brain injury. The author has accomplished analysis of certain phenotypological signs among patients of clinical groups. The analysis has demonstrated that the most significant phenotypic markers of patients were: postural disorder (anterior curvature / lateral curvature), hypermobility of the joints, platypodia and myopia. Biochemical research methods were used to assess the condition of the connective tissue. The content of connective tissue markers has been analyzed (glucosaminoglycans in the blood serum and daily excretion of oxyproline in the urine). The author has studied biochemical changes on the basis of a comparative analysis of undifferentiated connective tissue disease (UCTD) phenotypic mar kers among the patients of the clinical groups, which demonstrate that the patients had a decrease of general glucosaminoglycans in the blood serum in the presence of UCTD markers, and their redistribution towards a decrease or increase in the I fraction against the background of a significant decrease in the III fraction and an increase in the uronic acids excretion. An increase in oxyproline was observed due to an increase in collagen catabolism. The changes in the metabolism of proteoglycans and collagen, established during the analysis of the results of biochemical studies, indicate instability and insufficiency of connective tissue structures of the patients’ body with various signs of dysplasia. Key words: children, traumatic brain injury, connective tissue, phenotypic marker, glucosaminoglycans, oxyproline

Targeting soluble tumor necrosis factor as a potential intervention to lower risk for late-onset Alzheimer\u2019s disease associated with obesity, metabolic syndrome, and type 2 diabetes

BackgroundInsulin impairment and inflammation are two features common to type 2 diabetes and Alzheimer’s disease; however, the molecular and signaling interactions underlying this relationship are not well understood. Mounting evidence point to the associations between the disruption of metabolite processing in insulin impairment and neurodegenerative conditions such as Alzheimer’s. Although the brain depends partially on metabolites processed in the periphery, to date, little is known about how soluble tumor necrosis factor signaling (solTNF) impacts integrated peripheral immune and metabolic feedback signals in states of energy overload and insulin insensitivity.MethodsC57Bl/6J mice were fed a high-fat high-carbohydrate diet (HFHC) for 14 weeks. The brain-permeant biologic XPro1595® was used to block solTNF-dependent pathways. Metabolic and immune alterations were evaluated in the gut, liver, and brain. Behavioral tests were performed. Untargeted metabolomics was carried out in the plasma and liver.ResultsHFHC diet promotes central insulin impairment and dysregulation of immune-modulatory gene expressed in the brain. Alteration of metabolites associated with type 2 diabetes and Alzheimer’s such as butanoate, glutamate, biopterin, branched-chain amino acids, purines, and proteoglycan metabolism was observed in HFHC-fed mice. solTNF inhibition ameliorates hepatic metabolic disturbances and hepatic and intestinal lipocalin-2 levels, and decreases insulin impairment in the brain and behavioral deficits associated with HFHC diet.ConclusionsOur novel findings suggest that HFHC diet impacts central insulin signaling and immune-metabolic interactions in a solTNF-dependent manner to increase the risk for neurodegenerative conditions. Our novel findings indicate that selective solTNF neutralization can ameliorate peripheral and central diet-induced insulin impairment and identify lipocalin-2 as a potential target for therapeutic intervention to target inflammation and insulin disturbances in obesogenic environments. Collectively, our findings identify solTNF as a potential target for therapeutic intervention in inflammatory states and insulin disturbances in obesogenic environments to lower risk for AD.

Molecular properties underlying regional vulnerability to Alzheimer's disease pathology.

Amyloid deposition and neurofibrillary degeneration in Alzheimer's disease specifically affect discrete neuronal systems, but the underlying mechanisms that render some brain regions more vulnerable to Alzheimer's disease pathology than others remain largely unknown. Here we studied molecular properties underlying these distinct regional vulnerabilities by analysing Alzheimer's disease-typical neuroimaging patterns of amyloid deposition and neurodegeneration in relation to regional gene expression profiles of the human brain. Graded patterns of brain-wide vulnerability to amyloid deposition and neurodegeneration in Alzheimer's disease were estimated by contrasting multimodal amyloid-sensitive PET and structural MRI data between patients with Alzheimer's disease dementia (n = 76) and healthy controls (n = 126) enrolled in the Alzheimer's Disease Neuroimaging Initiative (ADNI). Regional gene expression profiles were derived from brain-wide microarray measurements provided by the Allen brain atlas of the adult human brain transcriptome. In a hypothesis-driven analysis focusing on the genes coding for the amyloid precursor (APP) and tau proteins (MAPT), regional expression levels of APP were positively correlated with the severity of regional amyloid deposition (r = 0.44, P = 0.009), but not neurodegeneration (r = 0.01, P = 0.96), whereas the opposite pattern was observed for MAPT (neurodegeneration: r = 0.46, P = 0.006; amyloid: r = 0.08, P = 0.65). Using explorative gene set enrichment analysis, amyloid-vulnerable regions were found to be characterized by relatively low expression levels of gene sets implicated in protein synthesis and mitochondrial respiration. By contrast, neurodegeneration-vulnerable regions were characterized by relatively high expression levels of gene sets broadly implicated in neural plasticity, with biological functions ranging from neurite outgrowth and synaptic contact over intracellular signalling cascades to proteoglycan metabolism. At the individual gene level this data-driven analysis further corroborated the association between neurodegeneration and MAPT expression, and additionally identified associations with known tau kinases (CDK5, MAPK1/ERK2) alongside components of their intracellular (Ras-ERK) activation pathways. Sensitivity analyses showed that these pathology-specific imaging-genetic associations were largely robust against changes in some of the methodological parameters, including variation in the brain donor sample used for estimating regional gene expression profiles, and local variations in the Alzheimer's disease-typical imaging patterns when these were derived from an independent patient cohort (BioFINDER study). These findings highlight that the regionally selective vulnerability to Alzheimer's disease pathology relates to specific molecular-functional properties of the affected neural systems, and that the implicated biochemical pathways largely differ for amyloid accumulation versus neurodegeneration. The data provide novel insights into the complex pathophysiological mechanisms of Alzheimer's disease and point to pathology-specific treatment targets that warrant further exploration in independent studies.

Glucosamine reduces the inhibition of proteoglycan metabolism caused by local anaesthetic solution in human articular cartilage: an in vitro study

BackgroundWe assessed whether local anaesthetics caused inhibition of proteoglycan metabolism in human articular cartilage and whether the addition of Glucosamine sulphate could prevent or allow recovery from this adverse effect on articular cartilage metabolism.MethodsCartilage explants obtained from 13 femoral heads from fracture neck of femur patients (average age 80 years, 10 female) were exposed to either 1% Lidocaine, 2% Lidocaine, 0.25% Bupivacaine, 0.5% Bupivacaine, 0.5% Levo-bupivacaine or a control solution (M199 culture medium). Glucosamine-6-Sulphate was added during or 1 h after exposure to 0.5% Bupivacaine to assess its protective and reparative effects. After exposure, the explants were incubated in culture medium containing radio labelled 35-sulphate and uptake was measured after 16 h to give an assessment of proteoglycan metabolism.ResultsThe reduction in 35-S uptake compared to control was 65% for 1% Lidocaine (p < 0.001), 79% for 2% Lidocaine (p < 0.001), 61% for 0.25% Bupivacaine (p < 0.001), 85% for 0.5% Bupivacaine (p < 0.001) and 77% for 0.5% Levobupivacaine (p < 0.001). Glucosamine was able to protect the articular cartilage by reducing the inhibition of proteoglycan metabolism of 0.5% Bupivacaine from 85 to 30% (p < 0.001). When added after 0.5% Bupivacaine exposure, Glucosamine allowed some recovery with inhibition of metabolism to 70% (p = 0.004).ConclusionOur results showed that all local anaesthetic solutions inhibited proteoglycan metabolism in articular cartilage and the addition of Glucosamine was able to reduce the inhibition of metabolism caused by 0.5% Bupivacaine. Intra-articular injection of local anaesthetics requires careful consideration of risks and benefits.

The microstructure and micromechanics of the tendon-bone insertion.

The exceptional mechanical properties of the load-bearing connection of tendon to bone rely on an intricate interplay of its biomolecular composition, microstructure and micromechanics. Here we identify that the Achilles tendon-bone insertion is characterized by an interface region of ∼500 μm with a distinct fibre organization and biomolecular composition. Within this region, we identify a heterogeneous mechanical response by micromechanical testing coupled with multiscale confocal microscopy. This leads to localized strains that can be larger than the remotely applied strain. The subset of fibres that sustain the majority of loading in the interface area changes with the angle of force application. Proteomic analysis detects enrichment of 22 proteins in the interfacial region that are predominantly involved in cartilage and skeletal development as well as proteoglycan metabolism. The presented mechanisms mark a guideline for further biomimetic strategies to rationally design hard-soft interfaces.

Comparative and Evolutionary Studies of Vertebrate Extracellular Sulfatase Genes and Proteins: SULF1 and SULF2

Extracellular sulfatases (SULF1; SULF2) (EC: 3.1.6.-) are members of the sulfatase enzyme family which exhibit endoglucosamine-6-sulfatase activity and carry out essential roles in proteoglycan metabolism. These enzymes regulate a number of critical signalling pathways and the sulfation state of glycoaminoglycans in the extracellular space. SULF1 and SULF2 amino acid sequences and structures and SULF-like gene locations were examined using bioinformatic data from several genome projects. Sequence alignments and conserved secondary structures and key amino acid residues and domains were studied. Comparative genomic analyses were conducted using the UC Santa Cruz Genome Browser. Phylogeny studies investigated the evolutionary relationships of these genes and proteins. Human and other vertebrate SULF1 and SULF2 sequences were conserved, including signal peptides, metal (Ca2+) and substrate binding sequences, active site residues and N-glycosylation sites (sulfatase domain); and a C-terminal positively charged hydrophilic domain. Predicted 2D structures were identified for the sulfatase domain of vertebrate SULF1 and SULF2 using a bacterial phosphatase structure (PDB:4UPK). Vertebrate SULF1 and SULF2 genes usually contained 18/19 or 20 coding exons, respectively. Transcription factor binding sites and miR-binding sites were identified within the human SULF1 and SULF2 gene promoters and 3’-UTR regions, respectively. The Estrogen Receptor Gene (ESR1) was identified in the SULF2 promoter which may contribute to the higher expression level for this gene in female reproductive tissues. SULF1 and SULF2 genes and proteins were present in all vertebrate genomes examined. Phylogenetic analyses suggested that an ancestral invertebrate SUL1 gene underwent a gene duplication event to form two separate lines of vertebrate gene evolution: SULF1 and SULF2.