Chondroitin Sulfate Proteoglycan Research Articles

Chondroitin sulfate proteoglycan 4: An attractive target for antibody-based immunotherapy.

Multifunctional molecules involved in tumor progression and metastasis have been identified as valuable targets for immunotherapy. Among these, chondroitin sulfate proteoglycan 4 (CSPG4), a significant tumor cell membrane-bound proteoglycan, has emerged as a promising target, especially in light of advances in chimeric antigen receptor (CAR) T-cell therapy. The profound bioactivity of CSPG4 and its role in pivotal processes such as tumor proliferation, migration, and neoangiogenesis underline its therapeutic potential. We reviewed the molecular intricacies of CSPG4, its functional attributes within tumor cells, and the latest clinical-translational advances targeting it. Strategies such as blocking monoclonal antibodies, conjugate therapies, bispecific antibodies, small-molecule inhibitors, CAR T-cell therapies, trispecific killer engagers, and ribonucleic acid vaccines against CSPG4 were assessed. CSPG4 overexpression in diverse tumors and its correlation with adverse prognostic outcomes emphasize its significance in cancer biology. These findings suggest that targeting CSPG4 offers a promising avenue for future cancer therapy, with potential synergistic effects when combined with existing treatments.

Multifunctional Hierarchical Nanoplatform with Anisotropic Bimodal Mesopores for Effective Neural Circuit Reconstruction after Spinal Cord Injury.

A persistent inflammatory response, intrinsic limitations in axonal regenerative capacity, and widespread presence of extrinsic axonal inhibitors impede the restoration of motor function after a spinal cord injury (SCI). A versatile treatment platform is urgently needed to address diverse clinical manifestations of SCI. Herein, we present a multifunctional nanoplatform with anisotropic bimodal mesopores for effective neural circuit reconstruction after SCI. The hierarchical nanoplatform features of a Janus structure consist of dual compartments of hydrophilic mesoporous silica (mSiO2) and hydrophobic periodic mesoporous organosilica (PMO), each possessing distinct pore sizes of 12 and 3 nm, respectively. Unlike traditional hierarchical mesoporous nanomaterials with dual-mesopores interlaced with each other, the two sets of mesopores in this Janus nanoplatform are spatially independent and possess completely distinct chemical properties. The Janus mesopores facilitate controllable codelivery of dual drugs with distinct properties: the hydrophilic macromolecular enoxaparin (ENO) and the hydrophobic small molecular paclitaxel (PTX). Anchoring with CeO2, the resulting mSiO2&PMO-CeO2-PTX&ENO nanoformulation not only effectively alleviates ROS-induced neuronal apoptosis but also enhances microtubule stability to promote intrinsic axonal regeneration and facilitates axonal extension by diminishing the inhibitory effect of extracellular chondroitin sulfate proteoglycans. We believe that this functional dual-mesoporous nanoplatform holds significant potential for combination therapy in treating severe multifaceted diseases.

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.

A Novel Network Pharmacology Strategy Based on the Universal Effectiveness-Common Mechanism of Medical Herbs Uncovers Therapeutic Targets in Traumatic Brain Injury.

Many herbs can promote neurological recovery following traumatic brain injury (TBI). There must lie a shared mechanism behind the common effectiveness. We aimed to explore the key therapeutic targets for TBI based on the common effectiveness of the medicinal plants. The TBI-effective herbs were retrieved from the literature as imputes of network pharmacology. Then, the active ingredients in at least two herbs were screened out as common components. The hub targets of all active compounds were identified through Cytohubba. Next, AutoDock vina was used to rank the common compound-hub target interactions by molecular docking. A highly scored compound-target pair was selected for in vivo validation. We enrolled sixteen TBI-effective medicinal herbs and screened out twenty-one common compounds, such as luteolin. Ten hub targets were recognized according to the topology of the protein-protein interaction network of targets, including epidermal growth factor receptor (EGFR). Molecular docking analysis suggested that luteolin could bind strongly to the active pocket of EGFR. Administration of luteolin or the selective EGFR inhibitor AZD3759 to TBI mice promoted the recovery of body weight and neurological function, reduced astrocyte activation and EGFR expression, decreased chondroitin sulfate proteoglycans deposition, and upregulated GAP43 levels in the cortex. The effects were similar to those when treated with the selective EGFR inhibitor. The common effectiveness-based, common target screening strategy suggests that inhibition of EGFR can be an effective therapy for TBI. This strategy can be applied to discover core targets and therapeutic compounds in other diseases.

Abstract 5979: Recombinant N-acetylgalactosamine-4-sulfatase (arylsulfatase B; ARSB) inhibits lung metastases in murine B16F10 syngeneic tumor model

Abstract Activity of 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) and dermatan sulfate, is reduced in several malignant cell lines and human tissues, including melanomas, mammary, prostate, and colon. Following tail vein injection of B16F10 melanoma cells in 12-week-old female C57BL/6J mice (n=11) and subsequent treatment by tail vein injection of bioactive, recombinant human ARSB (0.2 mg/kg), on days 2 and 7, development of metastatic lung lesions was markedly reduced. The average number of lesions declined from mean value of 114 (range: 10-278; n=5) to mean value of 2.5 (range: 0-9; n=6). These findings are consistent with our previous report of significant increase in survival and decrease in size of B16F10 subcutaneous melanomas in C57BL/6J mice treated with exogenous ARSB. The underlying mechanisms leading to inhibition of melanoma proliferation have profound impact on vital signaling pathways and transcriptional events. These pathways involve more or less binding with C4S when ARSB activity is modified. Notably, galectin-3 binding with C4S increases following exogenous ARSB, leading to decline in free nuclear galectin-3. This leads to reduced expression of the melanoma biomarker chondroitin sulfate proteoglycan CSPG)-4; also known as melanoma-associated chondroitin sulfate proteoglycan (MCSP). Expression of Programmed Death-Ligand 1 (PD-L1) is also reduced, declining from 1.34 ± 0.23 ng/mg protein (n=5) to 0.50 ± 0.05 ng/mg protein (n=6) following ARSB treatment. 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 contribute to decline in matrix metalloproteinases MMP2 and MMP9 and thereby restrain invasiveness and metastasis. Following treatment with exogenous ARSB, the serum MMP2 declined from 267 ± 38 (ng/ml; n=5) to 121 ± 16 ng/ml (n=6), consistent with the observed decline in melanoma lesions. The impact of ARSB on signaling pathways and transcriptional events demonstrates how modification of a critical sulfation and the resulting impact on binding with C4S influences vital cell processes and can suppress tumor development. 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. Exogenous ARSB may become an effective treatment for acquired deficiency of ARSB in melanoma. Citation Format: Joanne Kramer Tobacman, Sumit Bhattacharyya, Insug O-Sullivan. Recombinant N-acetylgalactosamine-4-sulfatase (arylsulfatase B; ARSB) inhibits lung metastases in murine B16F10 syngeneic tumor model [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5979.

Abstract 99: Versican regulates T cell abundance within the colorectal cancer microenvironment

Abstract Background: Clinical benefit from immunotherapy is seen in a minority of patients with metastatic colorectal cancer (CRC). Identifying how the tumor microenvironment (TME) influences immune infiltration and treatment response is an unmet need. Versican (VCAN), a chondroitin sulfate proteoglycan, common in the CRC TME has immunoregulatory properties and can be cleaved releasing an immunostimulatory product, versikine (Vkine). Here we evaluate the impact of the abundance of VCAN and Vkine on CD8+ T cell infiltration in CRC. Methods: Samples include 306 primary colon tumors, an additional 176 primary CRCs with matched liver metastases (175 samples), and 328 normal samples (238 colon and 90 liver). Samples were stained via immunohistochemistry (IHC) for VCAN, Vkine, and CD8. CD8+ tumor infiltrating lymphocytes (TILs) were counted per high powered field (HPF, 400X magnification) and presented as averages. Intensity of VCAN and Vkine expression were scored from 0-3+. Samples designated a 0/1 were considered low accumulation and samples scored as a 2/3+ were designated high. Samples scored low for VCAN but high for Vkine were designated VCAN proteolytic predominant (VPP) and all other combinations considered VCAN proteolytic weak (VPW). To create an in vitro model, the mouse MC38 cell line was CRISPR engineered to over-express VCAN. Conditioned media was then concentrated and added to a collagen matrix in a transwell with activated T cells. The number of cells and their subtype that migrated through the matrix were evaluated with flow cytometry. Results: VCAN accumulation was highest in cancerous stroma with 58% of cancers being VCAN high and only 14% of adjacent normal tissues (p<0.001). Vkine accumulation was similar between groups, with 67% of tumors and 54% of adjacent normal samples scored as Vkine high. The VPP phenotype was least common in tumor tissues with only 27% of tumor samples designated VPP versus 43% of adjacent normal samples (p<0.001). VCAN high tumors had significantly fewer TILs with 4 TILs/HPF vs 13 in the VCAN low tumors (p<0.001). High levels of Vkine correlated with greater TIL abundance (Vkine high: 10 TILs/HPF, Vkine low: 5 TILs/HPF; p=0.02). Versican proteolysis had the strongest correlation with TIL abundance in cancers (VPP: 18 TILs/HPF, VPW: 5 TILS/HPF; p<0.001). In vitro, the addition of VCAN containing media into transwell matrices reduced both CD4+ and CD8+ T cell migration through the matrix (Control: 4277 CD8+ T cells, VCAN: 2603 CD8+ T cells; p<0.001 and Control: 7770 CD4+ T cells, VCAN: 5340 CD4+ T cells; p=0.007). Conclusions: VCAN accumulation in human CRCs is common and correlates with reduced TIL abundance, whereas VCAN proteolysis correlates with increased TILs. In vitro, VCAN demonstrated the ability to reduce T cell migration. Future studies will explore the mechanism by which VCAN abundance leads to decreased T cell infiltration and the importance of this as an immunotherapy biomarker. Citation Format: Sean Kraus, Philip Emmerich, Mitch Hayes, Kristina Matkowskyj, Wei Zhang, Cheri Pasch, Fotis Asimakopoulos, Dustin A. Deming. Versican regulates T cell abundance within the colorectal cancer microenvironment [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 99.

Abstract 5245: Anti-CSPG4 CAR-Macrophages synergize with anti-CD47 in melanoma cytotoxicity

Abstract While CAR T-cells have been a success in treating liquid tumors, these successes have not been replicated in solid tumors. There are several reasons for these challenges, including physical matrix barriers and increased tumor antigen heterogeneity in solid tumors. Macrophages are frequently found in melanoma tumors, even in those that have low T-cell infiltrate, suggesting macrophages are well equipped to penetrate the physical barriers surrounding solid tumors. Macrophages are also antigen-presenting cells, thus, if macrophages phagocytose the tumor, they will likely present additional antigens to T-cells, potentially enhancing the adaptive immune response in cancer treatment. Thus, we hypothesized that macrophages expressing chimeric antigen receptors (CAR-Macrophages or CAR-Ms) are able to access solid tumors, phagocytose cancer cells, and subsequently re-educate the tumor immune environment by directly polarizing tumor-associated macrophages and by presenting novel antigens to recruit and educate adaptive immune cells. To test these hypotheses, we generated CAR-Ms with primary human macrophages using an scFV specific to chondroitin sulfate proteoglycan 4 (CSPG4), a highly expressed antigen enriched and specific to melanoma tumors, and an intracellular activation domain to promote phagocytosis. We found that anti-CSPG4 CAR-Ms phagocytosed human melanoma cells at high efficiency, however, this phagocytosis did not result in high levels of melanoma cell cytotoxicity. To improve CAR-M-mediated melanoma cell death engulfment, we blocked the anti-CD47/SIRPα (“don’t eat me”) signaling axis with an anti-CD47 blocking antibody, which resulted in additive effects for phagocytosis, and a robust effect on the melanoma cell killing. Preliminary in vivo xenograft experiments suggest that anti-CSPG4 CAR-Ms alone delay melanoma growth. We are now testing whether combining anti-CD47 and CSPG4 CAR-Ms therapies in vivo can 1) further reduce primary tumor growth and 2) prevent recurrence following surgical resection by treating remnants of the tumor. Our work shows that CSPG4 CAR-M-mediated killing of CSPG4-positive cancer cells is significantly enhanced when in combination with anti-CD47 antibody treatment. Citation Format: Daniel Greiner, Qian Xue, Trinity Waddell, Minna Roh-Johnson. Anti-CSPG4 CAR-Macrophages synergize with anti-CD47 in melanoma cytotoxicity [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5245.

Abstract 5530: Metastasis founder cells activate immunsuppression early in human metastatic colonization

Abstract Metastatic dissemination occurs very early in melanoma, often before diagnosis, rendering prevention of metastasis formation an important therapeutic goal. However, the earliest steps of lethal metastasis in patients are incompletely understood. Therefore, in this study we searched for the earliest detectable disseminated cancer cells (DCC) in sentinel lymph node biopsies of 492 stage I-III melanoma patients. We used micromanipulator-assisted isolation and single-cell transcriptome analysis of these extremely rare DCC and identified melanoma -associated chondroitin sulfate proteoglycan (MCSP) expressing melanoma cells as strong candidates for metastasis founder cells in lymph nodes. Based on a median follow-up time of 6 years, their detection was the strongest predictor of systemic metastasis and death upon multivariable analysis. Single-cell transcriptome analysis revealed that melanoma DCC were exposed to CD8 T cell attack during the transition from single cells to metastasis-initiating clusters, activated the extracellular vesicular exosomal pathway, and expressed the immunomodulatory proteins CD155 and CD276, but rarely PD-L1. CD155- and CD276-positive extracellular vesicles from patient-derived DCC models showed an immunosuppressive activity on CD8 T cells. In conclusion, our data suggest that either direct targeting of metastasis-founder cell with MCSP or their immune escape mechanisms may be key to curing early-stage melanoma. Citation Format: Severin Guetter, Courtney Koenig, Huiqin Koerkel-Qu, Aleksandra Markiewicz, Sebastian Scheitler, Marie Katzer, Mark Berneburg, Philipp Renner, Beatrix Cucuruz, Leonhard Guttenberger, Veronika Naimer, Kathrin Weidele, Steffi Treitschke, Christian Werno, Hanna Jaser, Tonia Bargmann, Armin Braun, Florian Weber, Katharina Limm, Reinhard Rachel, Felix Baumann, Lisa Schmidleithner, Kathrin Schambeck, Anja Ulmer, Sebastian Haferkamp, Christoph Klein, Melanie Werner-Klein. Metastasis founder cells activate immunsuppression early in human metastatic colonization [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5530.

Assembly of neuron- and radial glial-cell-derived extracellular matrix molecules promotes radial migration of developing cortical neurons

Radial neuronal migration is a key neurodevelopmental event for proper cortical laminar organization. The multipolar-to-bipolar transition, a critical step in establishing neuronal polarity during radial migration, occurs in the subplate/intermediate zone (SP/IZ), a distinct region of the embryonic cerebral cortex. It has been known that the extracellular matrix (ECM) molecules are enriched in the SP/IZ. However, the molecular constitution and functions of the ECM formed in this region remain poorly understood. Here, we identified neurocan (NCAN) as a major chondroitin sulfate proteoglycan in the mouse SP/IZ. NCAN binds to both radial glial-cell-derived tenascin-C (TNC) and hyaluronan (HA), a large linear polysaccharide, forming a ternary complex of NCAN, TNC, and HA in the SP/IZ. Developing cortical neurons make contact with the ternary complex during migration. The enzymatic or genetic disruption of the ternary complex impairs radial migration by suppressing the multipolar-to-bipolar transition. Furthermore, both TNC and NCAN promoted the morphological maturation of cortical neurons in vitro. The present results provide evidence for the cooperative role of neuron- and radial glial-cell-derived ECM molecules in cortical development.

Assembly of neuron- and radial glial-cell-derived extracellular matrix molecules promotes radial migration of developing cortical neurons.

Radial neuronal migration is a key neurodevelopmental event for proper cortical laminar organization. The multipolar-to-bipolar transition, a critical step in establishing neuronal polarity during radial migration, occurs in the subplate/intermediate zone (SP/IZ), a distinct region of the embryonic cerebral cortex. It has been known that the extracellular matrix (ECM) molecules are enriched in the SP/IZ. However, the molecular constitution and functions of the ECM formed in this region remain poorly understood. Here, we identified neurocan (NCAN) as a major chondroitin sulfate proteoglycan in the mouse SP/IZ. NCAN binds to both radial glial-cell-derived tenascin-C (TNC) and hyaluronan (HA), a large linear polysaccharide, forming a ternary complex of NCAN, TNC, and HA in the SP/IZ. Developing cortical neurons make contact with the ternary complex during migration. The enzymatic or genetic disruption of the ternary complex impairs radial migration by suppressing the multipolar-to-bipolar transition. Furthermore, both TNC and NCAN promoted the morphological maturation of cortical neurons in vitro. The present results provide evidence for the cooperative role of neuron- and radial glial-cell-derived ECM molecules in cortical development.

CSPG4-dependent cytotoxicity for C. difficile TcdB is influenced by extracellular calcium and chondroitin sulfate.

Clostridioides difficile is a leading cause of antibiotic-associated gastrointestinal illness, and many disease pathologies are caused by the toxin TcdB. TcdB engages multiple cell surface receptors, with receptor tropisms differing among the variants of the toxin. Chondroitin sulfate proteoglycan 4 (CSPG4) is a critical receptor for multiple forms of TcdB, and insights into TcdB-CSPG4 interactions are applicable to many disease-causing strains of C. difficile. CSPG4 is modified by chondroitin sulfate (CS) and contains laminin-G repeats stabilized by Ca2+, yet the relative contributions of CS and Ca2+ to TcdB cytotoxicity have not been determined. This study demonstrates distinct roles in TcdB cell binding and cell entry for Ca2+ and CS, respectively. These effects are specific to CSPG4 and contribute to the activities of a prominent isoform of TcdB that utilizes this receptor. These findings advance an understanding of factors contributing to TcdB's mechanism of action and contribution to C. difficile disease.

Reprogramming of astrocytes to neuronal-like cells in spinal cord injury: a systematic review.

A Systematic Review OBJECTIVES: To determine the therapeutic efficacy of in vivo reprogramming of astrocytes into neuronal-like cells in animal models of spinal cord injury (SCI). PRISMA 2020 guidelines were utilized, and search engines Medline, Web of Science, Scopus, and Embase until June 2023 were used. Studies that examined the effects of converting astrocytes into neuron-like cells with any vector in all animal models were included, while conversion from other cells except for spinal astrocytes, chemical mechanisms to provide SCI models, brain injury population, and conversion without in-vivo experience were excluded. The risk of bias was calculated independently. 5302 manuscripts were initially identified and after eligibility assessment, 43 studies were included for full-text analysis. After final analysis, 13 manuscripts were included. All were graded as high-quality assessments. The transduction factors Sox2, Oct4, Klf4, fibroblast growth factor 4 (Fgf4) antibody, neurogenic differentiation 1 (Neurod1), zinc finger protein 521 (Zfp521), ginsenoside Rg1, and small molecules (LDN193189, CHIR99021, and DAPT) could effectively reprogramme astrocytes into neuron-like cells. The process was enhanced by p21-p53, or Notch signaling knockout, valproic acid, or chondroitin sulfate proteoglycan inhibitors. The type of mature neurons was both excitatory and inhibitory. Astrocyte reprogramming to neuronal-like cells in an animal model after SCI appears promising. The molecular and functional improvements after astrocyte reprogramming were demonstrated in vivo, and further investigation is required in this field.

NG2 Molecule Expression in Acute Lymphoblastic Leukemia B Cells: A Flow-Cytometric Marker for the Rapid Identification of KMT2A Gene Rearrangements.

B-lineage acute lymphoblastic leukemias (B-ALL) harboring rearrangements of the histone lysine [K]-Methyltransferase 2A (KMT2A) gene on chromosome 11q23 (KMT2A-r) represent a category with dismal prognosis. The prompt identification of these cases represents an urgent clinical need. Considering the correlation between rat neuron glial-antigen 2 (NG2) chondroitin-sulfate-proteoglycan molecule expression and KMT2A-r, we aimed to identify an optimized cytofluorimetric diagnostic panel to predict the presence of KMT2A-r. We evaluated 88 NG2+ B-ALL cases identified with an NG2 positivity threshold >10% from a cohort of 1382 newly diagnosed B-ALLs referred to the Division of Hematology of 'Sapienza' University of Rome. Eighty-five of 88 (96.6%) NG2+ B-ALLs harbored KMT2A-r and were mainly pro-B ALL (77/85; 91%). Only 2 B-ALLs with KMT2A-r showed NG2 expression below 10%, probably due to the steroid therapy administered prior to cytofluorimetric analysis.Compared to KMT2A-r-cases, KMT2A r+ B-ALLs showed a higher blast percentage, significantly higher mean fluorescence intensity (MFI) of CD45, CD38, and CD58, and significantly lower MFI of CD34, CD22, TdT, and CD123.The study confirmed differences in CD45, CD34, CD22, and TdT MFI within the same immunologic EGIL group (European Group for the immunological classification of leukemias), indicating no influence of the B-ALLs EGIL subtype on the KMT2A-r+ B-ALLs immunophenotype. Our data demonstrate the association between NG2 and KMT2A-r in B-ALLs identify a distinctive immunophenotypic pattern, useful for rapid identification in diagnostic routines of these subtypes of B-ALLs with a poor prognosis that benefits from a specific therapeutic approach.

Chemo-enzymatic synthesis of tetrasaccharide linker peptides to study the divergent step in glycosaminoglycan biosynthesis.

Glycosaminoglycans are extended linear polysaccharides present on cell surfaces and within the extracellular matrix that play crucial roles in various biological processes. Two prominent glycosaminoglycans, heparan sulfate and chondroitin sulfate, are covalently linked to proteoglycan core proteins through a common tetrasaccharide linker comprising glucuronic acid, galactose, galactose, and xylose moities. This tetrasaccharide linker is meticulously assembled step by step by four Golgi-localized glycosyltransferases. The addition of the fifth sugar moiety, either N-acetylglucosamine or N-acetylgalactosamine, initiates further chain elongation, resulting in the formation of heparan sulfate or chondroitin sulfate, respectively. Despite the fundamental significance of this step in glycosaminoglycan biosynthesis, its regulatory mechanisms have remained elusive. In this study, we detail the expression and purification of the four linker-synthesizing glycosyltransferases and their utilization in the production of fluorescent peptides carrying the native tetrasaccharide linker. We generated five tetrasaccharide peptides, mimicking the core proteins of either heparan sulfate or chondroitin sulfate proteoglycans. These peptides were readily accepted as substrates by the EXTL3 enzyme, which adds an N-acetylglucosamine moiety, thereby initiating heparan sulfate biosynthesis. Importantly, EXTL3 showed a preference towards peptides mimicking the core proteins of heparan sulfate proteoglycans over the ones from chondroitin sulfate proteoglycans. This suggests that EXTL3 could play a role in the decision-making step during glycosaminoglycan biosynthesis. The innovative strategy for chemo-enzymatic synthesis of fluorescent-labeled linker-peptides promises to be instrumental in advancing future investigations into the initial steps and the divergent step of glycosaminoglycan biosynthesis.

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.

Epidermal Growth Factor Suppresses the Development of GABAergic Neurons Via the Modulation of Perineuronal Net Formation in the Neocortex of Developing Rodent Brains.

Previously, we reported that epidermal growth factor (EGF) suppresses GABAergic neuronal development in the rodent cortex. Parvalbumin-positive GABAergic neurons (PV neurons) have a unique extracellular structure, perineuronal nets (PNNs). PNNs are formed during the development of PV neurons and are mainly formed from chondroitin sulfate (CS) proteoglycans (CSPGs). We examined the effect of EGF on CSPG production and PNN formation as a potential molecular mechanism for the inhibition of inhibiting GABAergic neuronal development by EGF. In EGF-overexpressing transgenic (EGF-Tg) mice, the number of PNN-positive PV neurons was decreased in the cortex compared with that in wild-type mice, as in our previous report. The amount of CS and neurocan was also lower in the cortex of EGF-Tg mice, with a similar decrease observed in EGF-treated cultured cortical neurons. PD153035, an EGF receptor (ErbB1) kinase inhibitor, prevented those mentioned above excess EGF-induced reduction in PNN. We explored the molecular mechanism underlying the effect of EGF on PNNs using fluorescent substrates for matrix metalloproteinases (MMPs) and a disintegrin and metalloproteinases (ADAMs). EGF increased the enzyme activity of MMPs and ADAMs in cultured neurons. These enzyme activities were also increased in the EGF-Tg mice cortex. GM6001, a broad inhibitor of MMPs and ADAMs, also blocked EGF-induced PNN reductions. Therefore, EGF/EGF receptor signals may regulate PNN formation in the developing cortex.

Molecular profiling of the hippocampus of children with autism spectrum disorder.

Several lines of evidence point to a key role of the hippocampus in Autism Spectrum Disorders (ASD). Altered hippocampal volume and deficits in memory for person and emotion related stimuli have been reported, along with enhanced ability for declarative memories. Mouse models have demonstrated a critical role of the hippocampus in social memory dysfunction, associated with ASD, together with decreased synaptic plasticity. Chondroitin sulfate proteoglycans (CSPGs), a family of extracellular matrix molecules, represent a potential key link between neurodevelopment, synaptic plasticity, and immune system signaling. There is a lack of information regarding the molecular pathology of the hippocampus in ASD. We conducted RNAseq profiling on postmortem human brain samples containing the hippocampus from male children with ASD (n = 7) and normal male children (3-14 yrs old), (n = 6) from the NIH NeuroBioBank. Gene expression profiling analysis implicated molecular pathways involved in extracellular matrix organization, neurodevelopment, synaptic regulation, and immune system signaling. qRT-PCR and Western blotting were used to confirm several of the top markers identified. The CSPG protein BCAN was examined with multiplex immunofluorescence to analyze cell-type specific expression of BCAN and astrocyte morphology. We observed decreased expression of synaptic proteins PSD95 (p < 0.02) and SYN1 (p < 0.02), increased expression of the extracellular matrix (ECM) protease MMP9 (p < 0.03), and decreased expression of MEF2C (p < 0.03). We also observed increased BCAN expression with astrocytes in children with ASD, together with altered astrocyte morphology. Our results point to alterations in immune system signaling, glia cell differentiation, and synaptic signaling in the hippocampus of children with ASD, together with alterations in extracellular matrix molecules. Furthermore, our results demonstrate altered expression of genes implicated in genetic studies of ASD including SYN1 and MEF2C.

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.

Enzyme-immobilized nanoclay hydrogel simultaneously reduces inflammation and scar deposition to treat spinal cord injury

Spinal cord injury (SCI) is a severely disabling pathological condition and always results in sensory and motor functions loss. After SCI, inflammation-mediated secondary tissue damage and subsequent formation of neuroglial scar suppress neuron survival and regeneration, therefore resulting in severe neurofunctional dysfunction. Although nanoclay biomaterials demonstrated useful immunomodulation feature in non-neuronal tissue, it is unclear if they can inhibit inflammation in SCI lesions and promote neurofunctional recovery. In this study, we immobilized chondroitinase ABC enzyme (ChABC), which can degrade chondroitin sulfate proteoglycans (CSPGs) of neuroglial scar, inside nanoclay (Laponite®, Lap) hydrogel through electrostatic interaction. The presented ChABC-immobilized Lap hydrogel (ChABC@Lap) exhibited shear-thinning properties and excellent injectability, allowing to locally deliver it into injured area through a non-invasive manner. Cell biological experiments and SCI rat model research revealed that both pure Lap (without ChABC) and ChABC@Lap hydrogels significantly reduced pro-inflammatory macrophage marker (iNOS) intensity. Importantly, compared with Lap hydrogel, ChABC@Lap hydrogel significantly decreased GFAP and CS-56 marked scar deposition, increased Tuj-1+ neurons regeneration, and promoted NF200+ neurons survival, thereby improving SCI rat of electrophysiological characteristics and motor function. In summary, the designed enzyme-immobilized nanoclay hydrogel offers a promising strategy for SCI therapy through modulating simultaneously inflammation and glial scar deposition.

Temporal and differential proteomic profile of molecular mediators associated with chronic and acute wound healing.

The underlying pathophysiology of nonhealing chronic wounds is poorly understood due to the changes occurring at the gene level and the complexity arising in their proteomic profile. Here, we elucidated the temporal and differential profile of the normal and diabetic wound-healing mediators along with their interactions and associated pathways. Skin tissues corresponding to normal and diabetic wounds were isolated at Days 0, 3, 6, and 9 representing different healing phases. Temporal gene expression was analyzed by quantitative real-timePCR. Concurrently, differential protein patterns in the wound tissues were identified by Nano LC-ESI-TOF mass spectrometry and later confirmed by Western blot analysis. Gene ontology annotation, protein-protein interaction, and protein pathway analysis were performed using DAVID, PANTHER, and STRING bioinformatics resources. Uniquely identified proteins (complement C3, amyloid beta precursor protein, and cytoplasmic linker associated protein 2) in the diabetic wound tissue implied that these proteins are involved in the pathogenesis of diabetic wound. They exhibit enhanced catalytic activity, trigger pathways linked with inflammation, and negatively regulate wound healing. However, in the normal wound tissue, axin 1, chondroitin sulfate proteoglycan 4, and sphingosine-1-phosphate receptor were identified, which are involved in proliferation, angiogenesis, and remodeling. Our findings demonstrate the correlation between elevated gene expression of tumor necrosis factor-α, interleukin(IL)-1β, and identified mediators: aryl hydrocarbon receptor nuclear translocator, 5'-aminolevulinate synthase 2, and CXC-family, that inflicted an inflammatory response by activating downstream MAPK, JAK-STAT, and NF-κB pathways. Similarly, in normal wound tissue, the upregulated IL-4 and hepatocyte growth factor levels in conjunction with the identified proteins, serine/threonine-protein kinase mTOR and peroxisome proliferator-activated receptor gamma, played a significant role in the cellular response to platelet-derived growth factor stimulus, dermal epithelialization, and cell proliferation, processes associated with the repair mechanism. Furthermore, Western blot analysis indicated elevated levels of inflammatory markers and reduced levels of proliferative and angiogenic factors in the diabetic wound.