Expression Of Chondroitin Sulfate Proteoglycans Research Articles

Chondroitin sulfate proteoglycans mRNA expression and degradation in the zebra finch following traumatic brain injury

Traumatic brain injury (TBI) is one of the leading causes of fatality and disability worldwide. From minutes to months following damage, injury can result in a complex pathophysiology that can lead to temporary or permanent deficits including an array of neurodegenerative symptoms. These changes can include behavioral dysregulation, memory dysfunctions, and mood changes including depression. The nature and severity of impairments resulting from TBIs vary widely given the range of injury type, location, and extent of brain tissue involved. In response to the injury, the brain induces structural and functional changes to promote repair and minimize injury size. Despite its high prevalence, effective treatment strategies for TBI are limited. PNNs are part of the neuronal extracellular matrix (ECM) that mediate synaptic stabilization in the adult brain and thus neuroplasticity. They are associated mostly with inhibitory GABAergic interneurons and are thought to be responsible for maintaining the excitatory/inhibitory balance of the brain. The major structural components of PNNs include multiple chondroitin sulfate proteoglycans (CSPGs) as well as other structural proteins. Here we examine the effects of injury on CSPG expression, specifically around the changes in the side change moieties. To investigate CSPG expression following injury, adult male and female zebra finches received either a bilateral penetrating, or no injury and qPCR analysis and immunohistochemistry for components of the CSPGs were examined at 1- or 7-days post-injury. Next, to determine if CSPGs and thus PNNs should be a target for therapeutic intervention, CSPG side chains were degraded at the time of injury with chondroitinase ABC (ChABC) CSPGs moieties were examined. Additionally, GABA receptor mRNA and aromatase mRNA expression was quantified following CSPG degradation as they have been implicated in neuronal survival and neurogenesis. Our data indicate the CSPG moieties change following injury, potentially allowing for a brief period of synaptic reorganization, and that treatments that target CSPG side chains are successful in further targeting this brief critical period by decreasing GABA mRNA receptor expression, but also decreasing aromatase expression.

Extracellular matrix abnormalities in the hippocampus of subjects with substance use disorder

Contextual triggers are significant factors contributing to relapse in substance use disorders (SUD). Emerging evidence points to a critical role of extracellular matrix (ECM) molecules as mediators of reward memories. Chondroitin sulfate proteoglycans (CSPGs) are a subset of ECM molecules that form perineuronal nets (PNN) around inhibitory neurons. PNNs restrict synaptic connections and help maintain synapses. Rodent models suggest that modulation of PNNs may strengthen contextual reward memories in SUD. However, there is currently a lack of information regarding PNNs in the hippocampus of people with SUD as well as how comorbidity with major depressive disorder (MDD) may affect PNNs. We used postmortem hippocampal tissues from cohorts of human and nonhuman primates with or without chronic alcohol use to test the hypothesis that PNNs are increased in subjects with SUD. We used histochemical labeling and quantitative microscopy to examine PNNs, and qRT-PCR to examine gene expression for ECM molecules, synaptic markers and related markers. We identified increased densities of PNNs and CSPG-labeled glial cells in SUD, coinciding with decreased expression of the ECM protease matrix metalloproteinase 9 (Mmp9), and increased expression for the excitatory synaptic marker vesicle associated membrane protein 2 (Vamp2). Similar increases in PNNs were observed in monkeys with chronic alcohol self-administration. Subjects with MDD displayed changes opposite to SUD, and subjects with SUD and comorbid MDD had minimal changes in any of the outcome measures examined. Our findings demonstrate that PNNs are increased in SUD, possibly contributing to stabilizing contextual reward memories as suggested by preclinical studies. Our results also point to a previously unsuspected role for CSPG expression in glial cells in SUD. Evidence for increased hippocampal PNNs in SUD suggests that targeting PNNs to weaken contextual reward memories is a promising therapeutic approach for SUD, however comorbidity with MDD is a significant consideration.

Hindbrain boundaries as niches of neural progenitor and stem cells regulated by the extracellular matrix proteoglycan chondroitin sulphate.

The interplay between neural progenitors and stem cells (NPSCs), and their extracellular matrix (ECM) is a crucial regulatory mechanism that determines their behavior. Nonetheless, how the ECM dictates the state of NPSCs remains elusive. The hindbrain is valuable to examine this relationship, as cells in the ventricular surface of hindbrain boundaries (HBs), which arise between any two neighboring rhombomeres, express the NPSC marker Sox2, while being surrounded with the membrane-bound ECM molecule chondroitin sulphate proteoglycan (CSPG), in chick and mouse embryos. CSPG expression was used to isolate HB Sox2+ cells for RNA-sequencing, revealing their distinguished molecular properties as typical NPSCs, which express known and newly identified genes relating to stem cells, cancer, the matrisome and cell cycle. In contrast, the CSPG- non-HB cells, displayed clear neural-differentiation transcriptome. To address whether CSPG is significant for hindbrain development, its expression was manipulated in vivo and in vitro. CSPG manipulations shifted the stem versus differentiation state of HB cells, evident by their behavior and altered gene expression. These results provide further understanding of the uniqueness of hindbrain boundaries as repetitive pools of NPSCs in-between the rapidly growing rhombomeres, which rely on their microenvironment to maintain their undifferentiated state during development.

Photobiomodulation inhibits the expression of chondroitin sulfate proteoglycans after spinal cord injury via the Sox9 pathway.

Both glial cells and glia scar greatly affect the development of spinal cord injury and have become hot spots in research on spinal cord injury treatment. The cellular deposition of dense extracellular matrix proteins such as chondroitin sulfate proteoglycans inside and around the glial scar is known to affect axonal growth and be a major obstacle to autogenous repair. These proteins are thus candidate targets for spinal cord injury therapy. Our previous studies demonstrated that 810 nm photobiomodulation inhibited the formation of chondroitin sulfate proteoglycans after spinal cord injury and greatly improved motor function in model animals. However, the specific mechanism and potential targets involved remain to be clarified. In this study, to investigate the therapeutic effect of photobiomodulation, we established a mouse model of spinal cord injury by T9 clamping and irradiated the injury site at a power density of 50 mW/cm2 for 50 minutes once a day for 7 consecutive days. We found that photobiomodulation greatly restored motor function in mice and downregulated chondroitin sulfate proteoglycan expression in the injured spinal cord. Bioinformatics analysis revealed that photobiomodulation inhibited the expression of proteoglycan-related genes induced by spinal cord injury, and versican, a type of proteoglycan, was one of the most markedly changed molecules. Immunofluorescence staining showed that after spinal cord injury, versican was present in astrocytes in spinal cord tissue. The expression of versican in primary astrocytes cultured in vitro increased after inflammation induction, whereas photobiomodulation inhibited the expression of versican. Furthermore, we found that the increased levels of p-Smad3, p-P38 and p-Erk in inflammatory astrocytes were reduced after photobiomodulation treatment and after delivery of inhibitors including FR 180204, (E)-SIS3, and SB 202190. This suggests that Smad3/Sox9 and MAPK/Sox9 pathways may be involved in the effects of photobiomodulation. In summary, our findings show that photobiomodulation modulates the expression of chondroitin sulfate proteoglycans, and versican is one of the key target molecules of photobiomodulation. MAPK/Sox9 and Smad3/Sox9 pathways may play a role in the effects of photobiomodulation on chondroitin sulfate proteoglycan accumulation after spinal cord injury.

MicroRNA-143 targets SIRT2 to mediate the histone acetylation of PLAUR and modulates functions of astrocytes in spinal cord injury

This study aimed to explore effects of microRNA (miR)-143 on the proliferation, apoptosis, and cytokine secretion in astrocytes after spinal cord injury (SCI). After gain- and loss-of-function assays and transforming growth factor (TGF)-β stimulation in astrocytes, the cell viability, proliferation, and apoptosis were examined. The expression of miR-143, SIRT2, and PLAUR and levels of astrocyte-related glial fibrillary acidic protein (GFAP), Vimentin, chondroitin sulfate proteoglycan (CSPG), and connective tissue growth factor (CTGF) were also measured. The binding relationship between miR-143 and SIRT2 was assessed, as well as the correlation of PLAUR with SIRT2. In established SCI rat models, the locomotion function and astrocyte hyperplasia were detected. The TGF-β stimulation decreased miR-143 but increased SIRT2 expression in astrocytes. Mechanistically, miR-143 negatively targeted SIRT2 and SIRT2 down-regulation inhibited the H3K27 deacetylation of PLAUR promoter to increase PLAUR expression. miR-143 up-regulation inhibited TGF-β stimulated-proliferation, promoted cell apoptosis, and reduced GFAP, Vimentin, CSPG, and CTGF expression in astrocytes, which was counterweighed by SIRT2 overexpression. SIRT2 silencing reduced the proliferation and GFAP, Vimentin, CSPG, and CTGF expression while augmenting the apoptosis in TGF-β stimulated astrocytes, which was abrogated by PLAUR silencing. The injection of miR-143 agomir improved the locomotion function and reduced the astrocyte hyperplasia in SCI rats, which was reversed by silencing PLAUR. miR-143 targeted SIRT2 to affect PLAUR expression via the regulation of histone acetylation, which repressed the astrocyte activation in vivo and in vitro to improve the locomotion function in SCI rats.

Increased expression of chondroitin sulfate proteoglycans in dentate gyrus and amygdala causes postinfectious seizures.

Alterations in the extracellular matrix are common in patients with epilepsy and animal models of epilepsy, yet whether they are the cause or consequence of seizures and epilepsy development is unknown. Using Theiler's murine encephalomyelitis virus (TMEV) infection-induced model of acquired epilepsy, we found de novo expression of chondroitin sulfate proteoglycans (CSPGs), a major extracellular matrix component, in dentate gyrus (DG) and amygdala exclusively in mice with acute seizures. Preventing the synthesis of CSPGs specifically in DG and amygdala by deletion of the major CSPG aggrecan reduced seizure burden. Patch-clamp recordings from dentate granule cells revealed enhanced intrinsic and synaptic excitability in seizing mice that was significantly ameliorated by aggrecan deletion. In situ experiments suggested that dentate granule cell hyperexcitability results from negatively charged CSPGs increasing stationary cations on the membrane, thereby depolarizing neurons, increasing their intrinsic and synaptic excitability. These results show increased expression of CSPGs in the DG and amygdala as one of the causal factors for TMEV-induced acute seizures. We also show identical changes in CSPGs in pilocarpine-induced epilepsy, suggesting that enhanced CSPGs in the DG and amygdala may be a common ictogenic factor and potential therapeutic target.

Abstract LB_C21: Exogenous N-acetylgalactosamine-4-sulfatase (ARSB)-mediated tumor suppression involves effects on chondroitin 4-sulfate binding with SHP-2 and galectin-3, MAPK signaling, and transcriptional events, including epigenetic regulation of PD-L1

Abstract In experiments in melanoma, prostate, colon, and mammary cells and tissues, the enzyme N-acetylgalactosamine-4-sulfatase (Arylsulfatase B: ARSB), which removes the 4-sulfate group at the non-reducing end of chondroitin 4-sulfate (C4S), acts as a tumor suppressor. Following treatment with recombinant human (rh) ARSB in previous experiments and in recent experiments in the B16F10 mouse model of malignant melanoma, specific molecular pathways have been identified that retard tumor progression. These pathways involve more or less binding with chondroitin 4-sulfate when ARSB activity is modified. Galectin-3 binding with C4S increases following exogenous ARSB, leading to lower nuclear galectin-3 and reduced expression of chondroitin sulfate proteoglycan (CSPG)4 and of carbohydrate sulfotransferase (CHST)15 in melanoma cells and tissues. Nuclear Sp1 also declines, inhibiting transcription of both CSPG4, also known as the melanoma-associated chondroitin sulfate proteoglycan (MCSP), and of CHST15, which is required for 6-sulfation of N-acetylgalactosamine 4-sulfate. These proteins are associated with more aggressive tumors, and decline in their expression is consistent with the tumor suppressive function of ARSB. Inverse to the effect on galectin-3, SHP2, the ubiquitous non-receptor tyrosine phosphatase (PTPN11), binds less to C4S following treatment with exogenous ARSB. The resulting activation of SHP2 impacts on MAPK signaling, reducing phospho-ERK1/2 and phospho(T180/Y182)-p38-MAPK. These effects lead to decline in expression of the matrix metalloproteinases MMP2 and MMP9 and thereby restrain invasiveness and metastasis. Treatment of cultured melanoma cells with siRNA for ARSB leads to opposite effects to those of exogenous ARSB, including epigenetic effects modifying Programmed Death-Ligand 1 (PD-L1) expression. Treatment with rhARSB significantly reduces PD-L1 protein and gene expression, in contrast to increases when ARSB is silenced. The effects of rhARSB on PD-L1 expression are attributable to declines in phospho-JNK and nuclear c-Jun when free galectin-3 is less and to associated increases in histone deacetylase (HDAC) 3 expression and activity. Consistent with these effects, the ARSB-silencing induced increase in PD-L1 is inhibited by peptide inhibitors of the JNK-c-Jun interaction and by HDAC3 inhibitor. ARSB silencing reduces PD-L1 promoter acetylation, as shown by chromatin immunoprecipitation with anti-acetylhistone H3 antibody. The impact of rhARSB on signaling pathways, transcriptional events, and epigenetic mechanisms demonstrates how a critical sulfation and the resulting modification of chondroitin 4-sulfate can influence vital cell processes and can suppress tumor development. Effects on subcutaneous and pulmonary melanomas demonstrate the impact of exogenous ARSB in melanoma and suggest future prospects as a therapeutic agent. Recombinant human ARSB is an approved agent for the treatment of Mucopolysaccharidosis VI and is used safely and effectively for this congenital deficiency of ARSB. Citation Format: Joanne K. Tobacman, Insug O-Sullivan, Sumit Bhattacharyya. Exogenous N-acetylgalactosamine-4-sulfatase (ARSB)-mediated tumor suppression involves effects on chondroitin 4-sulfate binding with SHP-2 and galectin-3, MAPK signaling, and transcriptional events, including epigenetic regulation of PD-L1 [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2023 Oct 11-15; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2023;22(12 Suppl):Abstract nr LB_C21.

Shinbaro2 enhances axonal extension beyond the glial scar for functional recovery in rats with contusive spinal cord injury

Spinal cord injury (SCI) is a devastating event that often results in the inflammatory condition of glial scar tissue formation, impeding neural regeneration and recovery. Reducing the inflammatory response and inhibiting glial formation are promising strategies for improving SCI outcomes. Here, we introduce a new role for Shinbaro2 (Sh2), known for its anti-inflammatory and pain-reducing effects, in ameliorating glial scars formed in the damaged spinal cord and promoting axon growth after SCI. Sh2 was applied at various concentrations to cultivate primary spinal cord neurons. Concentrations of 1 and 2 mg/mL effectively enhanced cell viability and axonal outgrowth in spinal cord neurons subjected to hydrogen peroxide or laceration injury. Sh2 helped reduce neuroinflammation by increasing anti-inflammatory M2 macrophages (arginase 1) and decreasing inflammatory cells, ultimately reducing lesion size. In scar formation, Sh2 inhibited the expression of β-catenin and nestin in reactive astrocytes in the injured spinal cord. Moreover, Sh2 suppressed the expression of chondroitin sulfate proteoglycans and SOX9, which are involved in scar formation. Furthermore, Sh2 promoted the sprouting of serotonergic axons and the growth of neurofibrillary tangles, enhancing motor function recovery in SCI. These findings highlight the potential of Sh2 as an SCI therapeutic intervention, offering hope for neural and functional restoration in individuals with this debilitating condition.

Exogenous recombinant N-acetylgalactosamine-4-sulfatase (Arylsulfatase B; ARSB) inhibits progression of B16F10 cutaneous melanomas and modulates cell signaling

In the syngeneic, subcutaneous B16F10 mouse model of malignant melanoma, treatment with exogenous ARSB markedly reduced tumor size and extended survival. In vivo experiments showed that local treatment with exogenous N-acetylgalactosamine-4-sulfatase (Arylsulfatase B; ARSB) led to reduced tumor growth over time (p < 0.0001) and improved the probability of survival up to 21 days (p = 0.0391). Tumor tissue from the treated mice had lower chondroitin 4-sulfate (C4S) content and lower sulfotransferase activity. The free galectin-3 declined, and the SHP2 activity increased, due to altered binding with chondroitin 4-sulfate. These changes induced effects on transcription, which were mediated by Sp1, phospho-ERK1/2, and phospho-p38 MAPK. Reduced mRNA expression of chondroitin sulfate proteoglycan 4 (CSPG4), carbohydrate sulfotransferase 15 (N-acetylgalactosamine 4-sulfate 6-O-sulfotransferase), and matrix metalloproteinases 2 and 9 resulted. Experiments in the human melanoma cell line A375 demonstrated similar responses to exogenous ARSB as in the tumors, and inverse effects followed ARSB siRNA. ARSB, which removes the 4-sulfate group at the non-reducing end of C4S, acts as a tumor suppressor, and treatment with exogenous ARSB impacts on vital cell signaling and reduces the expression of critical genes associated with melanoma progression.

Astrogliosis and associated CSPG upregulation adversely affect dendritogenesis, spinogenesis and synaptic activity in the cerebellum of a double-hit rat model of protein malnutrition (PMN) and lipopolysaccharide (LPS) induced bacterial infection

The extracellular matrix (ECM) plays a vital role in growth, guidance and survival of neurons in the central nervous system (CNS). The chondroitin sulphate proteoglycans (CSPGs) are a type of ECM proteins that are crucial for CNS homeostasis. The major goal of this study was to uncover the effects of astroglial activation and associated intensified expression of CSPGs on dendritogenesis, spinogenesis as well as on synaptic activity in cerebellum following protein malnutrition (PMN) and lipopolysaccharide (LPS) induced bacterial infection. Female Wistar albino rats (3 months old) were switched to control (20% protein) or low protein (LP, 8% protein) diet for 15 days followed by breeding. A set of pups born to control/LP mothers and maintained on respective diets throughout the experimental period constituted the control and LP groups, while a separate set of both control and LP group pups exposed to bacterial infection by a single intraperitoneal injection of LPS (0.3 mg/ kg body weight) on postnatal day-9 (P-9) constituted control+LPS and LP+LPS groups respectively. The consequences of astrogliosis induced CSPG upregulation on cerebellar cytoarchitecture and synaptic activity were studied using standard immunohistochemical and histological tools on P-21 and 6 months of age. The results revealed reactive astrogliosis and associated CSPG upregulation in a double-hit model of PMN and LPS induced bacterial infection resulted in disrupted dendritogenesis, reduced postsynaptic density protein (PSD-95) levels and a deleterious impact on normal spine growth. Such alterations frequently have the potential to cause synaptic dysregulation and inhibition of plasticity both during development as well as adulthood. At the light of our results, we can envision that upregulation of CSPGs in PMN and LPS co-challenged individuals might emerge as an important modulator of brain circuitry and a major causative factor for many neurological disorders.

Discovery of Hippo signaling as a regulator of CSPG4 expression and as a therapeutic target for Clostridioides difficile disease.

The signaling pathways and networks regulating expression of chondroitin sulfate proteoglycan 4 (CSPG4), a cancer-related protein that serves as a receptor for Clostridiodes difficile TcdB, are poorly defined. In this study, TcdB-resistant/CSPG4-negative HeLa cells were generated by exposure to increasing concentrations of the toxin. The cells that emerged (HeLa R5) lost expression of CSPG4 mRNA and were resistant to binding by TcdB. mRNA expression profiles paired with integrated pathway analysis correlated changes in the Hippo and estrogen signaling pathways with a CSPG4 decrease in HeLa R5 cells. Both signaling pathways altered CSPG4 expression when modulated chemically or through CRISPR-mediated deletion of key transcriptional regulators in the Hippo pathway. Based on the in vitro findings, we predicted and experimentally confirmed that a Hippo pathway inactivating drug (XMU-MP-1) provides protection from C. difficile disease in a mouse model. These results provide insights into key regulators of CSPG4 expression and identify a therapeutic for C. difficile disease.

Glial scar survives until the chronic phase by recruiting scar-forming astrocytes after spinal cord injury

Spinal cord injury (SCI) causes reactive astrogliosis, the sequential phenotypic change of astrocytes in which naïve astrocytes (NAs) transform into reactive astrocytes (RAs) and subsequently become scar-forming astrocytes (SAs), resulting in glial scar formation around the lesion site and thereby limiting axonal regeneration and motor/sensory functional recovery. Inhibiting the transformation of RAs into SAs in the acute phase attenuates the reactive astrogliosis and promotes regeneration. However, whether or not SAs once formed can revert to RAs or SAs is unclear. We performed selective isolation of astrocytes from glial scars at different time points for a gene expression analysis and found that the expression of Sox9, an important transcriptional factor for glial cell differentiation, was significantly increased in chronic phase astrocytes (CAs) compared to SAs in the sub-acute phase. Furthermore, CAs showed a significantly lower expression of chondroitin sulfate proteoglycan (CSPG)-related genes than SAs. These results indicated that SAs changed their phenotypes according to the surrounding environment of the injured spinal cord over time. Even though the integrin-N-cadherin pathway is critical for glial scar formation, collagen-I-grown scar-forming astrocytes (Col-I-SAs) did not change their phenotype after depleting the effect of integrin or N-cadherin. In addition, we found that Col-I-SAs transplanted into a naïve spinal cord formed glial scar again by maintaining a high expression of genes involved in the integrin-N-cadherin pathway and a low expression of CSPG-related genes. Interestingly, the transplanted Col-I-SAs changed NAs into SAs, and anti-β1-integrin antibody blocked the recruitment of SAs while reducing the volume of glial scar in the chronic phase. Our findings indicate that while the characteristics of glial scars change over time after SCI, SAs have a cell-autonomous function to form and maintain a glial scar, highlighting the basic mechanism underlying the persistence of glial scars after central nervous system injury until the chronic phase, which may be a therapeutic target.

Sympathetic nerve innervation and metabolism in ischemic myocardium in response to remote ischemic perconditioning

Sympathetic nerve denervation after myocardial infarction (MI) predicts risk of sudden cardiac death. Therefore, therapeutic approaches limit infarct size, improving adverse remodeling and restores sympathetic innervation have a great clinical potential. Remote ischemic perconditioning (RIPerc) could markedly attenuate MI-reperfusion (MIR) injury. In this study, we aimed to assess its effects on cardiac sympathetic innervation and metabolism. Transient myocardial ischemia is induced by ligature of the left anterior descending coronary artery (LAD) in male Sprague–Dawley rats, and in vivo cardiac 2-[18F]FDG and [11C]mHED PET scans were performed at 14–15 days after ischemia. RIPerc was induced by three cycles of 5-min-long unilateral hind limb ischemia and intermittent 5 min of reperfusion during LAD occlusion period. The PET quantitative parameters were quantified in parametric polar maps. This standardized format facilitates the regional radioactive quantification in deficit regions to remote areas. The ex vivo radionuclide distribution was additionally identified using autoradiography. Myocardial neuron density (tyrosine hydroxylase positive staining) and chondroitin sulfate proteoglycans (CSPG, inhibiting neuron regeneration) expression were assessed by immunohistochemistry. There was no significant difference in the mean hypometabolism 2-[18F]FDG uptake ratio (44.6 ± 4.8% vs. 45.4 ± 4.4%) between MIR rats and MIR + RIPerc rats (P > 0.05). However, the mean [11C]mHED nervous activity of denervated myocardium was significantly elevated in MIR + RIPerc rats compared to the MIR rats (35.9 ± 7.1% vs. 28.9 ± 2.3%, P < 0.05), coupled with reduced denervated myocardium area (19.5 ± 5.3% vs. 27.8 ± 6.6%, P < 0.05), which were associated with preserved left-ventricular systolic function, a less reduction in neuron density, and a significant reduction in CSPG and CD68 expression in the myocardium. RIPerc presented a positive effect on cardiac sympathetic-nerve innervation following ischemia, but showed no significant effect on myocardial metabolism.

The Composition and Cellular Sources of CSPGs in the Glial Scar After Spinal Cord Injury in the Lamprey.

Axon regrowth after spinal cord injury (SCI) is inhibited by several types of inhibitory extracellular molecules in the central nervous system (CNS), including chondroitin sulfate proteoglycans (CSPGs), which also are components of perineuronal nets (PNNs). The axons of lampreys regenerate following SCI, even though their spinal cords contain CSPGs, and their neurons are enwrapped by PNNs. Previously, we showed that by 2 weeks after spinal cord transection in the lamprey, expression of CSPGs increased in the lesion site, and thereafter, decreased to pre-injury levels by 10 weeks. Enzymatic digestion of CSPGs in the lesion site with chondroitinase ABC (ChABC) enhanced axonal regeneration after SCI and reduced retrograde neuronal death. Lecticans (aggrecan, versican, neurocan, and brevican) are the major CSPG family in the CNS. Previously, we cloned a cDNA fragment that lies in the most conserved link-domain of the lamprey lecticans and found that lectican mRNAs are expressed widely in lamprey glia and neurons. Because of the lack of strict one-to-one orthology with the jawed vertebrate lecticans, the four lamprey lecticans were named simply A, B, C, and D. Using probes that distinguish these four lecticans, we now show that they all are expressed in glia and neurons but at different levels. Expression levels are relatively high in embryonic and early larval stages, gradually decrease, and are upregulated again in adults. Reductions of lecticans B and D are greater than those of A and C. Levels of mRNAs for lecticans B and D increased dramatically after SCI. Lectican D remained upregulated for at least 10 weeks. Multiple cells, including glia, neurons, ependymal cells and microglia/macrophages, expressed lectican mRNAs in the peripheral zone and lesion center after SCI. Thus, as in mammals, lamprey lecticans may be involved in axon guidance and neuroplasticity early in development. Moreover, neurons, glia, ependymal cells, and microglia/macrophages, are responsible for the increase in CSPGs during the formation of the glial scar after SCI.

A comprehensive atlas of Aggrecan, Versican, Neurocan and Phosphacan expression across time in wildtype retina and in retinal degeneration

As photoreceptor cells die during retinal degeneration, the surrounding microenvironment undergoes significant changes that are increasingly recognized to play a prominent role in determining the efficacy of therapeutic interventions. Chondroitin Sulphate Proteoglycans (CSPGs) are a major component of the extracellular matrix that have been shown to inhibit neuronal regrowth and regeneration in the brain and spinal cord, but comparatively little is known about their expression in retinal degeneration. Here we provide a comprehensive atlas of the expression patterns of four individual CSPGs in three models of inherited retinal degeneration and wildtype mice. In wildtype mice, Aggrecan presented a biphasic expression, while Neurocan and Phosphacan expression declined dramatically with time and Versican expression remained broadly constant. In degeneration, Aggrecan expression increased markedly in Aipl1-/- and Pde6brd1/rd1, while Versican showed regional increases in the periphery of Rho-/- mice. Conversely, Neurocan and Phosphacan broadly decrease with time in all models. Our data reveal significant heterogeneity in the expression of individual CSPGs. Moreover, there are striking differences in the expression patterns of specific CSPGs in the diseased retina, compared with those reported following injury elsewhere in the CNS. Better understanding of the distinct distributions of individual CSPGs will contribute to creating more permissive microenvironments for neuro-regeneration and repair.

MTORC1 is a key regulator that mediates OGD- and TGFβ1-induced myofibroblast transformation and chondroitin-4-sulfate expression in cardiac fibroblasts.

Ischemia-reperfusion infarct-derived chondroitin sulfate proteoglycans (CSPGs) are important for sustaining denervation of the infarct. Sympathetic denervation within the heart after myocardial infarction (MI) predicts the probability of a higher risk for serious ventricular arrhythmias. Chondroitin-4-sulfate (C4S) is the predominant chondroitin sulfate component in the heart. However, the mechanisms that induce CSPG expression in fibroblasts following MI remain to be elucidated. The present study found that oxygen-glucose deprivation (OGD) and TGFβ1 stimulation induced myofibroblast transformation and C4S synthesis in vitro by using reverse transcription-quantitative PCR, western blotting and immunofluorescence. MTT assay was used to detect cell viability following OGD or OGD + TGF lotreatment. Using the PI3K inhibitor ZSTK474, the Akt inhibitor MK2206, or the mTOR inhibitor AZD8055, it was observed that OGD and TGFβ1 stimulation induced myofibroblast transformation and that C4S synthesis was mTOR-dependent, whereas the upstream canonical PI3K/Akt axis was dispensable by using western blotting and immunofluorescence. siRNA knockdown of Smad3, Raptor, or Rictor, indicated that mTORC1 was critical for promoting OGD- and TGFβ1-induced myofibroblast transformation and C4S synthesis by using western blotting and immunofluorescence. This response, may be mediated via cooperation between canonical Smad3 and mTORC1 signaling. These data suggested that inhibiting myofibroblast transformation may reduce C4S synthesis. Target mTORC1 may provide additional insight into the regeneration of sympathetic nerves and the reduction of fibrosis after MI at the cellular level. These findings may contribute to the understanding of the mechanism by which C4S overproduction in the hearts of patients with MI is associated with myocardial fibrosis.

Tenascins Interfere With Remyelination in an Ex Vivo Cerebellar Explant Model of Demyelination

Oligodendrocytes form myelin membranes and thereby secure the insulation of axons and the rapid conduction of action potentials. Diseases such as multiple sclerosis highlight the importance of this glial cell population for brain function. In the adult brain, efficient remyelination following the damage to oligodendrocytes is compromised. Myelination is characterized by proliferation, migration, and proper integration of oligodendrocyte precursor cells (OPCs). These processes are among others controlled by proteins of the extracellular matrix (ECM). As a prominent representative ECM molecule, tenascin-C (Tnc) exerts an inhibitory effect on the migration and differentiation of OPCs. The structurally similar paralogue tenascin-R (Tnr) is known to promote the differentiation of oligodendrocytes. The model of lysolecithin-induced demyelination of cerebellar slice cultures represents an important tool for the analysis of the remyelination process. Ex vivo cerebellar explant cultures of Tnc −/− and Tnr −/− mouse lines displayed enhanced remyelination by forming thicker myelin membranes upon exposure to lysolecithin. The inhibitory effect of tenascins on remyelination could be confirmed when demyelinated wildtype control cultures were exposed to purified Tnc or Tnr protein. In that approach, the remyelination efficiency decreased in a dose-dependent manner with increasing concentrations of ECM molecules added. In order to examine potential roles in a complex in vivo environment, we successfully established cuprizone-based acute demyelination to analyze the remyelination behavior after cuprizone withdrawal in SV129, Tnc −/− , and Tnr −/− mice. In addition, we documented by immunohistochemistry in the cuprizone model the expression of chondroitin sulfate proteoglycans that are inhibitory for the differentiation of OPCs. In conclusion, inhibitory properties of Tnc and Tnr for myelin membrane formation could be demonstrated by using an ex vivo approach.

Inhibition of central axon regeneration: perspective from chondroitin sulfate proteoglycans in lamprey spinal cord injury.

Inhibition of central axon regeneration: perspective from chondroitin sulfate proteoglycans in lamprey spinal cord injury.

Raman spectroscopic investigation on the biomedical evolution of spinal cord injury and the therapeutic outcomes of its low-level laser therapy

The mechanism of low-level laser therapy (LLLT) of spinal cord injury (SCI) is currently unclear, and clinical application of LLLT treatments lack standardized protocols, since the treatment outcomes depends on various light parameters such as wavelength, fluence intensity, polarization and irradiation duration. In this work, Raman spectroscopy, was adopted to investigate the pathological process of SCI and monitor SCI-LLLT treatment so that accurate guidance on the appropriate selection of irradiation parameters may be achieved by an nonlabelled and nondesctructive intraoperative molecular diagnosis method. In vitro Raman spectroscopy combined with multivariate analysis technology was adopted to investigate the biochemical variations in spinal cord tissue during the progression of SCI and LLLT treatment. Besides, a discriminant model based on the principal component analysis-linear discriminant analysis algorithm (PCA-LDA) was established to distinguish the spectra of spinal tissue at pathological stages. The results of spectral analysis indicated that a series of biochemical events had occurred following SCI, such as chondroitin sulfate proteoglycan (CSPG) aggregation, vascular rupture, demyelination, and cell necrosis. It also demonstrated that LLLT could promote tissue recovery after injury by inhibition of CSPG expression and an increase in the concentration of myelin in tissues. In addition, the discriminant model achieved an accuracy of 96.25 % and 98.13 % for the SCI and SCI-LLLT groups, respectively, which could be used for predicting LLLT treatment outcomes during clinical applications. The study provides support for the development of intraoperative real-time in vivo Raman diagnostic methods to monitor the course of SCI-LLLT, allowing the selection of appropriate laser parameters and therapeutic strategies for clinical treatments.

CSPGs promote the migration of meningeal fibroblasts via p38 mitogen-activated protein kinase signaling pathway under OGD conditions

AimsUsually glial scar that occurs after central nervous system injury has significantly affected the local neural microenvironment. Meningeal fibroblasts play an essential role in the formation of the glial scar. However, how and why meningeal fibroblasts migrate to lesion sites is still unclear. Main methodsAstrocytes were subjected to oxygen-glucose deprivation/reperfusion (OGD/R) injury. And then, we measured the glial fibrillary acidic protein(GFAP) and chondroitin sulfate proteoglycans (CSPGs) expression of reactive astrocytes by western blot and quantitative polymerase chain reaction (qPCR) after they were co-cultured with meningeal fibroblasts. Following, we clarified the possibility that CSPGs induce the migration of meningeal fibroblasts to glial scar by transwell migration assay and the activation of the p38 MAPK signaling pathway during the migration by western blot. Key findingsWe found that co-cultured meningeal fibroblasts could alleviate the significantly increased expression of GFAP and CSPGs in the activation of reactive astrocytes induced by OGD/R. Additionally, CSPGs secreted by reactive astrocytes could induce the migration of meningeal fibroblasts and the expression of phospho-p38 in meningeal fibroblasts when meningeal fibroblasts were co-cultured with supernatant of reactive astrocytes. What's more, we could observe a noticeable increase in CSPGs that chondroitinase ABC could reverse their functions. Moreover, phospho-p38 could cause the expression of phospho-cofilin and the migration of CSPGs-induced meningeal fibroblasts. SignificanceOur study provides reliable evidence for explaining scar formation mechanisms and further studying to improve regeneration after an injury to the central nervous system.