Chondroitin Sulfate Proteoglycan Research Articles

Chondroitin sulfate proteoglycans prevent immune cell phenotypic conversion and inflammation resolution via TLR4 in rodent models of spinal cord injury

Chondroitin sulfate proteoglycans (CSPGs) act as potent inhibitors of axonal growth and neuroplasticity after spinal cord injury (SCI). Here we reveal that CSPGs also play a critical role in preventing inflammation resolution by blocking the conversion of pro-inflammatory immune cells to a pro-repair phenotype in rodent models of SCI. We demonstrate that enzymatic digestion of CSPG glycosaminoglycans enhances immune cell clearance and reduces pro-inflammatory protein and gene expression profiles at key resolution time points. Analysis of phenotypically distinct immune cell clusters revealed CSPG-mediated modulation of macrophage and microglial subtypes which, together with T lymphocyte infiltration and composition changes, suggests a role for CSPGs in modulating both innate and adaptive immune responses after SCI. Mechanistically, CSPG activation of a pro-inflammatory phenotype in pro-repair immune cells was found to be TLR4-dependent, identifying TLR4 signalling as a key driver of CSPG-mediated immune modulation. These findings establish CSPGs as critical mediators of inflammation resolution failure after SCI in rodents, which leads to prolonged inflammatory pathology and irreversible tissue destruction.

Chondroitin sulfate proteoglycan 4,6 sulfation regulates sympathetic nerve regeneration after myocardial infarction.

Sympathetic denervation of the heart following ischemia/reperfusion induced myocardial infarction (MI) is sustained by chondroitin sulfate proteoglycans (CSPGs) in the cardiac scar. Denervation predicts risk of sudden cardiac death in humans. Blocking CSPG signaling restores sympathetic axon outgrowth into the cardiac scar, decreasing arrhythmia susceptibility. Axon growth inhibition by CSPGs can depend on the sulfation status of the glycosaminoglycan (CS-GAG) side chains. Tandem sulfation of CS-GAGs at the 4th (4S) and 6th (6S) positions of n-acetyl-galactosamine inhibits outgrowth in several types of central neurons, but we don't know if sulfation is similarly critical during peripheral nerve regeneration. We asked if CSPG sulfation prevented sympathetic axon outgrowth after MI. Reducing 4S with the 4-sulfatase enzyme Arylsulfatase-B (ARSB) enhanced outgrowth of dissociated rat sympathetic neurons over CSPGs. Likewise, reducing 4S with ARSB restored axon outgrowth from mouse sympathetic ganglia co-cultured with cardiac scar tissue. We quantified enzymes responsible for adding and removing sulfation, and found that CHST15 (4S dependent 6-sulfotransferase) was upregulated, and ARSB was downregulated after MI. This suggests a mechanism for production and maintenance of sulfated CSPGs in the cardiac scar. We decreased 4S,6S CS-GAGs in vivo by transient siRNA knockdown of Chst15 after MI, and found that reducing 4S,6S restored tyrosine hydroxylase (TH) positive sympathetic nerve fibers in the cardiac scar. Reinnervation reduced isoproterenol induced arrhythmias. Our results suggest that modulating CSPG-sulfation after MI may be a therapeutic target to promote sympathetic nerve regeneration in the cardiac scar and reduce post-MI cardiac arrhythmias.

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.

Versican promotes T helper 17 cytotoxic inflammation and impedes oligodendrocyte precursor cell remyelination

Remyelination failure in multiple sclerosis (MS) contributes to progression of disability. The deficient repair results from neuroinflammation and deposition of inhibitors including chondroitin sulfate proteoglycans (CSPGs). Which CSPG member is repair-inhibitory or alters local inflammation to exacerbate injury is unknown. Here, we correlate high versican-V1 expression in MS lesions with deficient premyelinating oligodendrocytes, and highlight its selective upregulation amongst CSPG members in experimental autoimmune encephalomyelitis (EAE) lesions modeling MS. In culture, purified versican-V1 inhibits oligodendrocyte precursor cells (OPCs) and promotes T helper 17 (Th17) polarization. Versican-V1-exposed Th17 cells are particularly toxic to OPCs. In NG2CreER:MAPTmGFP mice illuminating newly formed GFP+ oligodendrocytes/myelin, difluorosamine (peracetylated,4,4-difluoro-N-acetylglucosamine) treatment from peak EAE reduces lesional versican-V1 and Th17 frequency, while enhancing GFP+ profiles. We suggest that lesion-elevated versican-V1 directly impedes OPCs while it indirectly inhibits remyelination through elevating local Th17 cytotoxic neuroinflammation. We propose CSPG-lowering drugs as potential dual pronged repair and immunomodulatory therapeutics for MS.

FC017: Adaptive Remodelling of Mesangial Extracellular Matrix Proteoglycan Composition during IGA Nephropathy

Abstract BACKGROUND AND AIMS IgA nephropathy (IgAN) is characterized by the deposition of galactose deficient IgA (gd-IgA1) containing immune complexes in the mesangium leading to activation of the mesangial cells and local inflammation. Hallmarks of the disease are increased by mesangial proliferation and matrix expansion. The exact composition of the mesangial matrix is not known and nor is the full role of the expansion in disease progression. One important group of proteins found in extracellular matrixes is proteoglycans (PGs). PGs are composed of a core protein to which one or several negatively charged glycosaminoglycan (GAG) side chains are attached. Both the core protein and GAGs determine the properties of PGs and changes in the amount and structure of PGs could influence disease progression. In this study, we aimed to elucidate changes in the PG expression in the glomerulus in IgAN as well as how gd-IgA1 affects the mesangial cells’ PG expression. METHOD Using previously published glomerular transcriptomic data from patients with IgAN compared to healthy controls we investigated PG expression as well as changes in expression on a glomerular level [1]. Secondly, we investigated how gd-IgA1 affects the PG production of the cells in vitro using data from a previously published proteomic data set [1] as well as new in vivo experiments using real-time PCR as well as immunohistochemistry. To further explore the effects on PGs in IgAN, we employed a novel glycoproteomic approach that provides the combined characterization of GAG linkage regions and core protein identities in complex sample matrices. RESULTS We found 31 PGs to be expressed in the human glomeruli and of those 6 PGs were found to be significantly regulated in patients with IgAN. SDC1 (syndecan-1), was downregulated, while MXRA5 (matrix-remodeling associated 5), CD44 (CD44), TNC (tenascin C), FMOD (fibromodulin) and PRELP (PRELP) were up-regulated. The proteomic data set on mesangial cells treated with gd-IgA1 revealed that mesangial cells in vitro expressed 19 PGs and of those 10 were significantly regulated. Chondroitin sulfate proteoglycan 4, glypican-1, agrin, aggrecan, versican, lumican, protein AMBP, tenascin, collagen alpha-1(VII) chain and laminin subunit alpha-4. All of the significantly regulated PGs had an increased expression, except aggrecan, which was down-regulated. An interesting finding from the glycoproteomic experiments was the identification of collagen VII as a novel PG carrying chondroitin GAG in mesangial cells. In addition, immunohistochemistry revealed that gd-IgA1 treatment of mesangial cells does not lead to a total increase in GAGs, but rather to a switch from heparan sulfate to chondroitin sulfate GAGs. CONCLUSION In conclusion, this study gives a comprehensive view of the PGs expressed by mesangial cells and their alterations in response to gd-IgA1 as part of the expansion of the mesangium in IgAN. The switch of heparan sulfate GAGs to chondroitin sulfate GAGs is an interesting finding but the role of this switch needs further investigation.

BS-512-01 PHARMACOLOGICAL SYMPATHETIC REINNERVATION AFTER MYOCARDIAL INFARCTION PREVENTS ARRHYTHMIAS IN THE MOUSE HEART

Sympathetic control of the heart plays a key role in modulating cardiac function and electrophysiology. Sympathetic hypo-innervation following myocardial infarction (MI) increases risk for development of ventricular arrhythmias. We have previously shown that chondroitin sulfate proteoglycans (CSPGs) play a role in post-MI hypo-innervation by signaling through the neuronal protein tyrosine phosphatase receptor σ (PTPσ), and that genetic deletion of PTPσ from the sympathetic neurons of mice restores innervation and prevents arrhythmias post-MI.

Antigen mimicry as an effective strategy to induce CSPG4-targeted immunity in dogs with oral melanoma: a veterinary trial

BackgroundMelanoma is the most lethal form of skin cancer in humans. Conventional therapies have limited efficacy, and overall response is still unsatisfactory considering that immune checkpoint inhibitors induce lasting clinical...

PGE receptor EP4 signaling increased hyaluronan‐vesican complex in abdominal aortic aneurysm

Chronic inflammation and dysregulation of extracellular matrices are hallmarks of abdominal aortic aneurysm (AAA) pathophysiology. We have demonstrated that PGE receptor EP4 signaling in vascular smooth muscle cells (VSMCs) has a pivotal role in AAA formation via immune infiltrates and impairment of elastic fiber repair. However, EP4 signaling‐mediated changes in extracellular matrices have not been fully understood, and expressions of the chondroitin sulfate proteoglycan versican are controversial. Versican has multiple binding partners (e.g., hyaluronan and fibrillin‐1) and modulates VSMC phenotypes. We, therefore, aimed to clarify an expression change in versican in AAA.We administrated angiotensin‐II (AngII) to VSMC‐specific EP4 overexpression mice (EP4‐Tg) to induce AAA. The expression level of versican V0/V1 was gradually increased in the tunica media of EP4‐Tg mice 4 days after AngII administration, but not in Non‐Tg mice. This increase in versican V0/V1 became prominent 14 days after AngII administration in EP4‐Tg mice. Protein expression of versican V0/V1 was increased in the tunica media of human AAA tissues. EP4 stimulation in VSMCs derived from EP4‐Tg mice increased levels of versican mRNAs (20.0±1.4‐fold, n=5, p<0.01) and versican V0/V1 proteins (4.1±0.7‐fold, n=5, p<0.01), while administration of EP1/3 or EP2 agonist did not. The EP4‐mediated increase in versican mRNAs was completely suppressed by Actinomycin D. Versican V0/V1 are cleaved by a disintegrin and metalloproteinase with thrombospondin motifs (ADAMTS‐1) and ADAMTS‐5. The expression levels of ADAMTS‐1, ADAMTS‐5, and cleaved versican protein expressions were not changed in EP4‐Tg VSMCs. The expression level of Hyaluronan synthase1 (Has1) mRNA was also increased in EP4‐Tg VSMCs (66.9±10.1‐fold, n=5, p<0.01). Western blotting revealed that versican V0/V1 proteins of approximately 200‐400 kDa in size in the supernatants of EP4‐Tg VSMCs stimulated with an EP4 agonist were increased by hyaluronan digestion with hyaluronidase (6.3±1.5‐fold, n=6, p<0.01). Transmission electron microscopic analysis demonstrated that elastin contractile units between elastic laminae and VSMCs were disrupted in EP4‐Tg mice administrated with 10 days of AngII, but not in Non‐Tg mice.These data suggested that EP4 signaling in VSMCs increased hyaluronan‐versican V0/V1 complex. Versican V0/V1 proteins were increased in the early stage of a mouse AAA model. It has been suggested that versican has inhibitory roles in elastic fiber formation. EP4‐mediated hyaluronan‐versican V0/V1 complex may exacerbate AAA via disruption of elastin contractile units.

Impact of MeCP2 Overexpression on Myelination Defects Found in Pitt‐Hopkins Syndrome Model Mice

IntroductionStudies examining neurodevelopmental disorders (NDDs) have emphasized a relationship between Pitt Hopkins syndrome (PTHS), which is caused by mutations in the Transcription Factor 4(TCF4) gene, and myelination defects that suggest novel molecular phenotypes in NDDs (Phan et al., 2020). Known phenotypic overlap between PTHS and Rett syndrome (RTT) suggests that there are possible common signaling pathways between the two NDDs. We have found that increasing expression of Methyl‐CpG‐Binding Protein 2 (MeCP2), the causative protein in most cases of RTT, decreases hyperactivity and corrects impairments in learning and memory in a mouse model of PTHS (Tcf4+/‐).AimThe current study sought to understand if the positive effects of MeCP2 overexpression on behavioral phenotypes in the Tcf4+/‐ model were due to a reversal of myelination deficits.Methods/ResultsWe generated four distinct animal genotypes (WT, MECP2Tg1/o, Tcf4+/‐, MECP2Tg1/o; Tcf4+/‐) and performed RNA‐sequencing from the hippocampus and striatum followed by quantitative RT‐PCR, revealing that myelin‐associated genes are differentially expressed in MECP2Tg1/o; Tcf4+/‐ mice in both brain regions. However, Western blotting revealed decreased expression for the myelinating oligodendrocytes (OLs) protein Myelin Oligodendrocyte Glycoprotein (MOG) in mice of both the Tcf4+/‐ and MECP2Tg1/o; Tcf4+/‐ genotypes without changes in the OL precursor cell marker Chondroitin Sulfate Proteoglycan (NG2). We are currently quantifying potential changes in OL morphology and number (mature and immature cells), and myelin synthesis via immunohistochemistry of brain sections.ConclusionsAltogether, our preliminary results indicate that behavioral phenotypic rescue in MECP2Tg1/o; Tcf4+/‐ mice may not result from a correction of myelination deficits characteristic of Tcf4+/‐animals.

Regulation of perineuronal net components in the synaptic bouton vicinity on lumbar α-motoneurons in the rat after spinalization and locomotor training: New insights from spatio-temporal changes in gene, protein expression and WFA labeling

Chondroitin sulfate proteoglycans (CSPGs) consist of core proteins and glycosaminoglycan side chains. Tenascins, and hyaluronan and proteoglycan link protein 1 (HAPLN), link CSPGs with a hyaluronan backbone to constitute perineuronal nets (PNNs), which ensheath preferentially highly active neurons to maintain architecture and stabilize synapses, but restrict repair plasticity. Spinal cord injury increases CSPG core protein levels in the lesion proximity, limiting permissiveness of the extracellular milieu for fiber regrowth, however regulation of PNNs structure in the vicinity of distant α-motoneurons (MNs) in the course of degeneration and reorganization of their inputs requires research. Here, we examined early and late changes in CSPGs, HAPLN1, tenascin-R, and glial activation along the spinal cord in male rats with complete spinal cord transection (Th10), and their impact on PNNs ensheathing lumbar MNs innervating ankle extensor and flexor muscles, which are in different loading states in paraplegic rats. We show that (1) distance from the lesion site and time after injury (2–5 weeks) differentiate degree of changes in transcription rates (measured with RT-qPCR) of PNNs proteins with increased CSPGs and decreased HAPLN1 transcripts, suggesting long-term PNN destabilization in majority of spinal segments, (2) in lumbar segments PNN composition is not MN-class (extensor vs flexor) specific, both showing early decrease and late upregulation of Wisteria floribunda agglutinin (WFA) labeling in vicinity of synaptic boutons on MNs, (3) long-term locomotor training tends to reduce WFA(+) PNNs, but not their protein components (immunofluorescence measurements) around MNs. Our results suggest that training-induced regulation may target glycan structures of CSPGs.

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.

Emerging role of neuregulin-1beta1 in pathogenesis and progression of multiple sclerosis

Emerging role of neuregulin-1beta1 in pathogenesis and progression of multiple sclerosis

Low-Molecular Weight Protamine Overcomes Chondroitin Sulfate Inhibition of Neural Regeneration.

Protamine is an arginine-rich peptide that replaces histones in the DNA-protein complex during spermatogenesis. Protamine is clinically used in cardiopulmonary bypass surgery to neutralize the effects of heparin that is required during the treatment. Here we demonstrate that protamine and its 14–22 amino acid long fragments overcome the neurite outgrowth inhibition by chondroitin sulfate proteoglycans (CSPGs) that are generally regarded as major inhibitors of regenerative neurite growth after injuries of the adult central nervous system (CNS). Since the full-length protamine was found to have toxic effects on neuronal cells we used the in vitro neurite outgrowth assay to select a protamine fragment that retains the activity to overcome the neurite outgrowth inhibition on CSPG substrate and ended up in the 14 amino acid fragment, low-molecular weight protamine (LMWP). In contrast to the full-length protamine, LMWP displays very low or no toxicity in our assays in vitro and in vivo. We therefore started studies on LMWP as a possible drug lead in treatment of CNS injuries, such as the spinal cord injury (SCI). LMWP mimicks HB-GAM (heparin-binding growth-associated molecule; pleiotrophin) in that it overcomes the CSPG inhibition on neurite outgrowth in primary CNS neurons in vitro and inhibits binding of protein tyrosine phosphatase (PTP) sigma, an inhibitory receptor in neurite outgrowth, to its CSPG ligand. Furthermore, the chondroitin sulfate (CS) chains of the cell matrix even enhance the LMWP-induced neurite outgrowth on CSPG substrate. In vivo studies using the hemisection and hemicontusion SCI models in mice at the cervical level C5 revealed that LMWP enhances recovery when administered through intracerebroventricular or systemic route. We suggest that LMWP is a promising drug lead to develop therapies for CNS injuries.

Activation of A2B adenosine receptor protects against demyelination in a mouse model of schizophrenia.

The purpose of the present study was to explore the effects of A2B adenosine receptor (A2BAR) on learning, memory and demyelination in a dizocilpine maleate (MK-801)-induced mouse model of schizophrenia (SCZ). BAY 60-6583, an agonist of A2BAR, or PSB 603, an antagonist of A2BAR, was used to treat SCZ in this model. The Morris Water Maze (MWM) was utilized to determine changes in cognitive function. Moreover, western blotting, immunohistochemistry and immunofluorescence were conducted to investigate the myelination and oligodendrocyte (OL) alterations at differentiation and maturation stages. The MWM results showed that learning and memory were impaired in SCZ mice, while subsequent treatment with BAY 60-6583 alleviated these impairments. In addition, western blot analysis revealed that myelin basic protein (MBP) and chondroitin sulphate proteoglycan 4 (NG2) expression levels were significantly decreased in MK-801-induced mice, while the expression of G protein-coupled receptor 17 (GPR17) was increased. Additionally, the number of anti-adenomatous polyposis coli clone CC-1/OL transcription factor 2 (CC-1+/Olig2+) cells was also decreased. Notably, BAY 60-6583 administration could reverse these changes, resulting in a significant increase in MBP and NG2 protein expression, and in the number of CC-1+/Olig2+ cells, while GPR17 protein expression levels were decreased. The present study indicated that the selective activation of A2BAR using BAY 60-6583 could improve the impaired learning and memory of SCZ mice, as well as protect the myelin sheath from degeneration by regulating the survival and maturation of OLs.

Dual-functional hydrogel system for spinal cord regeneration with sustained release of arylsulfatase B alleviates fibrotic microenvironment and promotes axonal regeneration

Traumatic damage to the spinal cord does not spontaneously heal, often leading to permanent tissue defects. We have shown that injection of imidazole-poly(organophosphazene) hydrogel (I-5) bridges cystic cavities with the newly assembled fibronectin-rich extracellular matrix (ECM). The hydrogel-created ECM contains chondroitin sulfate proteoglycans (CSPGs), collagenous fibrils together with perivascular fibroblasts, and various fibrotic proteins, all of which could hinder axonal growth in the matrix. In an in vitro fibrotic scar model, fibroblasts exhibited enhanced sensitivity to TGF-β1 when grown on CSPGs. To alleviate the fibrotic microenvironment, the I-5 hydrogel was equipped with an additional function by making a complex with ARSB, a human enzyme degrading CSPGs, via hydrophobic interaction. Delivery of the I-5/ARSB complex significantly diminished the fibrotic ECM components. The complex promoted serotonergic axonal growth into the hydrogel-induced matrix and enhanced serotonergic innervation of the lumbar motor neurons. Regeneration of the propriospinal axons deep into the matrix and to the lumbar spinal cord was robustly increased accompanied by improved locomotor recovery. Therefore, our dual-functional system upgraded the functionality of the hydrogel for spinal cord regeneration by creating ECM to bridge tissue defects and concurrently facilitating axonal connections through the newly assembled ECM.

Roles of Chondroitin Sulfate Proteoglycans as Regulators of Skeletal Development.

The extracellular matrix (ECM) is critically important for most cellular processes including differentiation, morphogenesis, growth, survival and regeneration. The interplay between cells and the ECM often involves bidirectional signaling between ECM components and small molecules, i.e., growth factors, morphogens, hormones, etc., that regulate critical life processes. The ECM provides biochemical and contextual information by binding, storing, and releasing the bioactive signaling molecules, and/or mechanical information that signals from the cell membrane integrins through the cytoskeleton to the nucleus, thereby influencing cell phenotypes. Using these dynamic, reciprocal processes, cells can also remodel and reshape the ECM by degrading and re-assembling it, thereby sculpting their environments. In this review, we summarize the role of chondroitin sulfate proteoglycans as regulators of cell and tissue development using the skeletal growth plate model, with an emphasis on use of naturally occurring, or created mutants to decipher the role of proteoglycan components in signaling paradigms.

Aggrecan and versican: two brothers close or apart.

Aggrecan (Acan) and versican (Vcan) are large chondroitin sulfate proteoglycans of the extracellular matrix. They share the same structural domains at both N- and C-termini. The N-terminal G1 domain binds hyaluronan (HA), forms an HA-rich matrix, and regulates HA-mediated signaling. The C-terminal G3 domain binds other extracellular matrix molecules and forms a supramolecular structure that stores transforming growth factor β (TGFβ) and bone morphogenetic proteins (BMPs) and regulates their signaling. EGF-like motifs in the G3 domain may directly act like an EGF ligand. Both Acan and Vcan are present in cartilage, intervertebral disc, brain, heart, and aorta. Their localizations are essentially reciprocal. This review describes their structural domains, expression patterns and functions, and regulation of their expression.

Update on Perineuronal Net Staining With Wisteria floribunda Agglutinin (WFA).

As chemically specialized forms of the extracellular matrix in the central nervous system, polyanionic perineuronal nets (PNs) contain diverse constituents, including chondroitin sulfate proteoglycans (CSPGs), hyaluronic acid, and tenascins. They are detectable by various histological approaches such as colloidal iron binding and immunohistochemical staining to reveal, for instance, the CSPGs aggrecan, neurocan, phosphacan, and versican. Moreover, biotin, peroxidase, or fluorescein conjugates of the lectins Vicia villosa agglutinin and soybean agglutinin enable the visualization of PNs. At present, the N-acetylgalactosamine-binding Wisteria floribunda agglutinin (WFA) is the most widely applied marker for PNs. Therefore, this article is largely focused on methodological aspects of WFA staining. Notably, fluorescent WFA labeling allows, after its conversion into electron-dense adducts, electron microscopic analyses. Furthermore, the usefulness of WFA conjugates for the oftentimes neglected in vivo and in vitro labeling of PNs is emphasized. Subsequently, we discuss impaired WFA-staining sites after long-lasting experiments in vitro, especially in autoptic brain samples with long postmortem delay and partial enzymatic degradation, while immunolabeling of aggrecan and CSPG link proteins under such conditions has proven more robust. In some hippocampal regions from perfusion-fixed mice, more PNs are aggrecan immunoreactive than WFA positive, whereas the retrosplenial cortex displays many WFA-binding PNs devoid of visible aggrecan immunoreactivity. Additional multiple fluorescence labeling exemplarily revealed in ischemic tissue diminished staining of WFA-binding sites and aquaporin 4 and concomitantly upregulated immunolabeling of neurofilament, light chains, and collagen IV. Finally, we briefly discuss possible future staining approaches based on nanobodies to facilitate novel technologies revealing details of net morphology.

Water Treadmill Training Ameliorates Neurite Outgrowth Inhibition Associated with NGR/RhoA/ROCK by Inhibiting Astrocyte Activation following Spinal Cord Injury.

Spinal cord injury (SCI) often results in damage to or degeneration of axons. Crosstalk between astrocytes and neurons plays a pivotal role in neurite outgrowth following SCI. Rehabilitative training is a recognized method for the treatment of SCI, but the specific mechanism underlying its effect on axonal outgrowth in the central nervous system (CNS) has not yet been determined. A total of 190 adult male SD rats weighing 200–250 g were randomly divided into eight groups for use as animal models of SCI. Rats were subjected to water treadmill training (TT) for 7 or 14 d. The Basso-Beattie-Bresnahan (BBB) motor function scale, hematoxylin-eosin (HE) staining, Nissl staining, Western blotting, and immunofluorescence were used to measure tissue morphology and the degree of neurological deficit and to determine quantitative expression and accurate localization of the corresponding proteins. We found that TT decreased tissue structure damage and improved functional recovery. TT also promoted the regeneration of neurons and reduced SCI-induced apoptosis SCI around the lesion, as well as significantly increasing the expression of GAP43 and NF200 after SCI. In addition, TT significantly inhibited the injury-induced increase in the expression of proinflammatory factors. Moreover, TT reduced the activation of astrocytes and microglia, accompanied by the reduced expression of C3d and increased expression of S100A10. Finally, TT effectively reduced the level of chondroitin sulfate proteoglycan (CSPG) surrounding the lesion and inhibited the NGR/RhoA/ROCK signaling pathway in neurons after SCI. Overall, we found that TT played a novel role in recovery from SCI by promoting axonal outgrowth associated with NGR/RhoA/ROCK signaling by inhibiting astrocyte activation after SCI.

Structural dynamics of receptor recognition and pH-induced dissociation of full-length Clostridioides difficile Toxin B.

Clostridioides difficile secretes Toxin B (TcdB) as one of its major virulence factors, which binds to intestinal epithelial and subepithelial receptors, including frizzled proteins and chondroitin sulfate proteoglycan 4 (CSPG4). Here, we present cryo-EM structures of full-length TcdB in complex with the CSPG4 domain 1 fragment (D1401-560) at cytosolic pH and the cysteine-rich domain of frizzled-2 (CRD2) at both cytosolic and acidic pHs. CSPG4 specifically binds to the autoprocessing and delivery domains of TcdB via networks of salt bridges, hydrophobic and aromatic/proline interactions, which are disrupted upon acidification eventually leading to CSPG4 drastically dissociating from TcdB. In contrast, FZD2 moderately dissociates from TcdB under acidic pH, most likely due to its partial unfolding. These results reveal structural dynamics of TcdB during its preentry step upon endosomal acidification, which provide a basis for developing therapeutics against C. difficile infections.