Cspg4high microglia contribute to microgliosis during neurodegeneration.

Microglia play a critical role in the pathogenic process of neurodegenerative diseases, such as Parkinson's disease (PD) and Alzheimer's disease (AD). Upon pathological stimulation, microglia are converted from a surveillant to an overactivated phenotype. However, the molecular characters of proliferating microglia and their contributions to the pathogenesis of neurodegeneration remain unclear. Here, we identify chondroitin sulfate proteoglycan 4 (Cspg4, also known as neural/glial antigen 2)-expressing microglia as a specific subset of microglia with proliferative capability during neurodegeneration. We found that the percentage of Cspg4+ microglia was increased in mouse models of PD. The transcriptomic analysis of Cspg4+ microglia revealed that the subcluster Cspg4high microglia displayed a unique transcriptomic signature, which was characterized by the enrichment of orthologous cell cycle genes and a lower expression of genes responsible for neuroinflammation and phagocytosis. Their gene signatures were also distinct from that of known disease-associated microglia. The proliferation of quiescent Cspg4high microglia was evoked by pathological α-synuclein. Following the transplantation in the adult brain with the depletion of endogenous microglia, Cspg4high microglia grafts showed higher survival rates than their Cspg4- counterparts. Consistently, Cspg4high microglia were detected in the brain of AD patients and displayed the expansion in animal models of AD. These findings suggest that Cspg4high microglia are one of the origins of microgliosis during neurodegeneration and may open up a avenue for the treatment of neurodegenerative diseases.

MiR126-targeted-nanoparticles combined with PI3K/AKT inhibitor as a new strategy to overcome melanoma resistance.

Metastatic melanoma poses significant challenges as a highly lethal disease. Despite the success of molecular targeting using BRAFV600E inhibitors (BRAFis) and immunotherapy, the emergence of early recurrence remains an issue and there is the need for novel therapeutic approaches. This study aimed at creating a targeted delivery system for the oncosuppressor microRNA 126 (miR126) and testing its effectiveness in combination with a phosphatidylinositol 3-kinase (PI3K)/ protein kinase B (AKT) inhibitor for treating metastatic melanoma resistant to BRAFis. To achieve this, we synthesized chitosan nanoparticles containing a chemically modified miR126 sequence. These nanoparticles were further functionalized with an antibody specific to the chondroitin sulfate proteoglycan 4 (CSPG4) melanoma marker. After evaluation in vitro, the efficacy of this treatment was evaluated through an in vivo experiment using mice bearing resistant human melanoma. The co-administration of miR126 and the PI3K/AKT inhibitor in these experiments significantly reduced tumor growth and inhibited the formation of liver and lung metastases. These results provide evidence for a strategy to target an oncosuppressive nucleic acid sequence to tumor cells while simultaneously protecting it from plasma degradation. The system described in this study exhibits encouraging potential for the effective treatment of therapy-resistant metastatic melanoma while also presenting a prospective approach for other forms of cancer.

Metabolic disorders exacerbate the formation of glial scar after stroke.

Metabolic disorders are risk factors for stroke exacerbating subsequent complications. Rapidly after brain injury, a glial scar forms, preventing excessive inflammation and limiting axonal regeneration. Despite the growing interest in wound healing following brain injury, the formation of a glial scar in the context of metabolic disorders is poorly documented. In this study, we used db/db mice to investigate the impact of metabolic perturbations on brain repair mechanisms, with a focus on glial scarring. First, we confirmed the development of obesity, poor glucose regulation, hyperglycaemia and liver steatosis in these mice. Then, we observed that 3 days after a 30-min middle cerebral artery occlusion (MCAO), db/db mice had larger infarct area compared with their control counterparts. We next investigated reactive gliosis and glial scar formation in db/+ and db/db mice. We demonstrated that astrogliosis and microgliosis were exacerbated 3 days after stroke in db/db mice. Furthermore, we also showed that the synthesis of extracellular matrix (ECM) proteins (i.e., chondroitin sulphate proteoglycan, collagen IV and tenascin C) was increased in db/db mice. Consequently, we demonstrated for the first time that metabolic disorders impair reactive gliosis post-stroke and increase ECM deposition. Given that the damage size is known to influence glial scar, this study now raises the question of the direct impact of hyperglycaemia/obesity on reactive gliosis and glia scar. It paves the way to promote the development of new therapies targeting glial scar formation to improve functional recovery after stroke in the context of metabolic disorders.

CSPG4P12 polymorphism served as a susceptibility marker for esophageal cancer in Chinese population.

BACKGROUND: Chondroitin sulfate proteoglycan 4 pseudogene 12 (CSPG4P12) has been implicated in the pathogenesis of various cancers. This study aimed to evaluate the association of the CSPG4P12 polymorphism with esophageal squamous cell carcinoma (ESCA) risk and to explore the biological impact of CSPG4P12 expression on ESCA cell behavior. METHODS: A case-control study was conducted involving 480 ESCA patients and 480 healthy controls to assess the association between the rs8040855 polymorphism and ESCA risk. The CSPG4P12 rs8040855 genotype was identified using the TaqMan-MGB probe method. Logistic regression model was used to evaluate the association of CSPG4P12 SNP with the risk of ESCA by calculating the odds ratios (OR) and 95% confidence intervals (95%CI ). The effects of CSPG4P12 overexpression on cell proliferation, migration, and invasion were examined in ESCA cell lines. Co-expressed genes were identified via the CBioportal database, with pathway enrichment analyzed using SangerBox. The binding score of CSPG4P12 to P53 was calculated using RNA protein interaction prediction (RPISeq). Additionally, Western Blot analysis was performed to investigate the impact of CSPG4P12 overexpression on the P53/PI3K/AKT signaling pathway. RESULTS: The presence of at least one rs8040855 G allele was associated with a reduced susceptibility to ESCA compared to the CC genotype (OR = 0.51, 95%CI = 0.28-0.93, P = 0.03). Stratification analysis revealed that the CSPG4P12 rs8040855 C allele significantly decreased the risk of ESCA among younger individuals (≤ 57 years) and non-drinkers (OR = 0.31, 95%CI = 0.12-0.77, P = 0.01; OR = 0.42, 95%CI=0.20-0.87, P = 0.02, respectively). CSPG4P12 expression was found to be downregulated in ESCA tissues compared to adjacent normal tissues. Overexpression of CSPG4P12 in ESCA cells inhibited their proliferation, migration, and invasion capabilities. Furthermore, Western Blot analysis indicated that CSPG4P12 overexpression led to a reduction in PI3K and p-AKT protein expression levels. P53 silencing rescues the inhibitory effect of CSPG4P12 on p-AKT. CONCLUSION: The CSPG4P12 rs8040855 variant is associated with reduced ESCA risk and the overexpression of CSPG4P12 inhibited the migration and invasion of ESCA cells by P53/PI3K/AKT pathway. These findings suggest that CSPG4P12 may serve as a novel biomarker for ESCA susceptibility and a potential target for therapeutic intervention.

A chimeric human/dog-DNA vaccine against CSPG4 induces immunity with therapeutic potential in comparative preclinical models of osteosarcoma.

The high mortality rate of osteosarcoma (OSA) patients highlights the requirement of alternative strategies. The young age of patients, as well as the rarity and aggressiveness of the disease, limits opportunities for the robust testing of novel therapies, suggesting the need for valuable preclinical systems. Having previously shown the overexpression of the chondroitin sulfate proteoglycan (CSPG)4 in OSA, herein the functional consequences of its downmodulation in human OSA cells were evaluated in vitro, with a significant impairment of cell proliferation, migration, and osteosphere generation. The potential of a chimeric human/dog (HuDo)-CSPG4 DNA vaccine was explored in translational comparative OSA models, including human xenograft mouse models and canine patients affected by spontaneous OSA. The adoptive transfer of HuDo-CSPG4 vaccine-induced CD8+ T cells and sera in immunodeficient human OSA-bearing mice delayed tumor growth and metastasis development. HuDo-CSPG4 vaccination resulted safe and effective in inducing anti-CSPG4 immunity in OSA-affected dogs, which displayed prolonged survival as compared to controls. Finally, HuDo-CSPG4 was also able to induce a cytotoxic response in a human surrogate setting in vitro. On the basis of these results and the high predictive value of spontaneous OSA in dogs, this study paves the way for a possible translation of this approach to humans.

VT68.2: An Antibody to Chondroitin Sulfate Proteoglycan 4 (CSPG4) Displays Reactivity against a Tumor-Associated Carbohydrate Antigen.

The anti-CSPG4 monoclonal antibodies (mAbs) have shown anti-tumor activity and therapeutic potential for treating breast cancer. In addition, CSPG4 is a dominant tumor-associated antigen that is also involved in normal-tissue development in humans. Therefore, the potential for off-tumor activity remains a serious concern when targeting CSPG4 therapeutically. Previous work suggested that glycans contribute to the binding of specific anti-CSPG4 antibodies to tumor cells, but the specificity and importance of this contribution are unknown. In this study, the reactivity of anti-CSPG4 mAbs was characterized with a peptide mimetic of carbohydrate antigens expressed in breast cancer. ELISA, flow cytometry, and microarray assays were used to screen mAbs for their ability to bind to carbohydrate-mimicking peptides (CMPs), cancer cells, and glycans. The mAb VT68.2 displayed a distinctly strong binding to a CMP (P10s) and bound to triple-negative breast cancer cells. In addition, VT68.2 showed a higher affinity for N-linked glycans that contain terminal fucose and fucosylated lactosamines. The functional assays demonstrated that VT68.2 inhibited cancer cell migration. These results define the glycoform reactivity of an anti-CSPG4 antibody and may lead to the development of less toxic therapeutic approaches that target tumor-specific glyco-peptides.

Chondroitinase and Antidepressants Promote Plasticity by Releasing TRKB from Dephosphorylating Control of PTPσ in Parvalbumin Neurons.

Perineuronal nets (PNNs) are an extracellular matrix structure rich in chondroitin sulfate proteoglycans (CSPGs), which preferentially encase parvalbumin-containing (PV+) interneurons. PNNs restrict cortical network plasticity but the molecular mechanisms involved are unclear. We found that reactivation of ocular dominance plasticity in the adult visual cortex induced by chondroitinase ABC (chABC)-mediated PNN removal requires intact signaling by the neurotrophin receptor TRKB in PV+ neurons. Additionally, we demonstrate that chABC increases TRKB phosphorylation (pTRKB), while PNN component aggrecan attenuates brain-derived neurotrophic factor (BDNF)-induced pTRKB in cortical neurons in culture. We further found that protein tyrosine phosphatase σ (PTPσ, PTPRS), receptor for CSPGs, interacts with TRKB and restricts TRKB phosphorylation. PTPσ deletion increases phosphorylation of TRKB in vitro and in vivo in male and female mice, and juvenile-like plasticity is retained in the visual cortex of adult PTPσ-deficient mice (PTPσ+/-). The antidepressant drug fluoxetine, which is known to promote TRKB phosphorylation and reopen critical period-like plasticity in the adult brain, disrupts the interaction between TRKB and PTPσ by binding to the transmembrane domain of TRKB. We propose that both chABC and fluoxetine reopen critical period-like plasticity in the adult visual cortex by promoting TRKB signaling in PV+ neurons through inhibition of TRKB dephosphorylation by the PTPσ-CSPG complex.SIGNIFICANCE STATEMENT Critical period-like plasticity can be reactivated in the adult visual cortex through disruption of perineuronal nets (PNNs) by chondroitinase treatment, or by chronic antidepressant treatment. We now show that the effects of both chondroitinase and fluoxetine are mediated by the neurotrophin receptor TRKB in parvalbumin-containing (PV+) interneurons. We found that chondroitinase-induced visual cortical plasticity is dependent on TRKB in PV+ neurons. Protein tyrosine phosphatase σ (PTPσ, PTPRS), a receptor for PNNs, interacts with TRKB and inhibits its phosphorylation, and chondroitinase treatment or deletion of PTPσ increases TRKB phosphorylation. Antidepressant fluoxetine disrupts the interaction between TRKB and PTPσ, thereby increasing TRKB phosphorylation. Thus, juvenile-like plasticity induced by both chondroitinase and antidepressant treatment is mediated by TRKB activation in PV+ interneurons.

Loss of ABCA8B decreases myelination by reducing oligodendrocyte precursor cells in mice.

The myelin sheath, which is wrapped around axons, is a lipid-enriched structure produced by mature oligodendrocytes. Disruption of the myelin sheath is observed in several neurological diseases, such as multiple sclerosis. A crucial component of myelin is sphingomyelin, levels of which can be increased by ABCA8, a member of the ATP-binding cassette transporter family. ABCA8 is highly expressed in the cerebellum, specifically in oligodendroglia. However, whether ABCA8 plays a role in myelination and mechanisms that would underlie this role remain unknown. Here, we found that the absence of Abca8b, a mouse ortholog of ABCA8, led to decreased numbers of cerebellar oligodendrocyte precursor cells (OPCs) and mature oligodendrocytes in mice. We show that in oligodendrocytes, ABCA8 interacts with chondroitin sulfate proteoglycan 4 (CSPG4), a molecule essential for OPC proliferation, migration, and myelination. In the absence of Abca8b, localization of CSPG4 to the plasma membrane was decreased, contributing to reduced cerebellar CSPG4 expression. Cerebellar CSPG4+ OPCs were also diminished, leading to decreased mature myelinating oligodendrocyte numbers and cerebellar myelination levels in Abca8b-/- mice. In addition, electron microscopy analyses showed that the number of nonmyelinated cerebellar axons was increased, whereas cerebellar myelin thickness (g-ratio), myelin sheath periodicity, and axonal diameter were all decreased, indicative of disordered myelin ultrastructure. In line with disrupted cerebellar myelination, Abca8b-/- mice showed lower cerebellar conduction velocity and disturbed locomotion. In summary, ABCA8 modulates cerebellar myelination, in part through functional regulation of the ABCA8-interacting protein CSPG4. Our findings suggest that ABCA8 disruption may contribute to the pathophysiology of myelin disorders.

CSPG4 expression in soft tissue sarcomas is associated with poor prognosis and low cytotoxic immune response.

BACKGROUND: Soft tissue sarcomas (STS) are heterogeneous and pro-metastatic tumors. Identification of accurate prognostic factors and novel therapeutic targets are crucial. CSPG4 is a cell surface proteoglycan with oncogenic functions. It recently emerged as a potential target for immunotherapy, including cell therapy based on CSPG4-specific chimeric antigen receptor (CAR)-redirected cytokine-induced killer lymphocytes (CSPG4-CAR.CIKs) in STS. However, expression of CSPG4 is poorly known in STS so far. METHODS: We analyzed CSPG4 gene expression in 1378 localized STS clinical samples, and searched for correlations with clinicopathological data, including disease-free survival (DFS), and with tumor immune features. RESULTS: CSPG4 expression was heterogeneous across samples. High expression was associated with younger patients' age, more frequent undifferentiated pleomorphic sarcoma and myxofibrosarcoma pathological subtypes, more frequent internal trunk tumor site, and more CINSARC high-risk samples. No correlation existed with pathological tumor size and grade, and tumor depth. Patients with high CSPG4 expression displayed 49% (95% CI 42-57) 5-year DFS versus 61% (95% CI 56-68) in patients with low expression (p = 3.17E-03), representing a 49% increased risk of event in the "CSPG4-high" group (HR = 1.49, 95% CI 1.14-1.94). This unfavorable prognostic value persisted in multivariate analysis, independently from other variables. There were significant differences in immune variables between "CSPG4-high" and "CSPG4-low" tumors. The "CSPG4-low" tumors displayed profiles suggesting higher anti-tumor cytotoxic immune response and higher potential vulnerability to immune checkpoint inhibitors (ICI). By contrast, the "CSPG4-high" tumors displayed profiles implying an immune-excluded tumor microenvironment, potentially induced by hypoxia, resulting from an immature chaotic microvasculature, and/or the presence of contractile myofibroblasts. CONCLUSIONS: Patients with "CSPG4-high" STS, theoretically candidate for CAR.CIKs, display shorter DFS and an immune environment unfavorable to vulnerability to CAR.CIKs, which could be improved by combining anti-angiogenic drugs able to normalize the tumor vasculature. By contrast, "CSPG4-low" STS are better candidates for immune therapy involving ICI.

LAR inhibitory peptide promotes recovery of diaphragm function and multiple forms of respiratory neural circuit plasticity after cervical spinal cord injury.

Chondroitin sulfate proteoglycans (CSPGs), up-regulated in and around the lesion after traumatic spinal cord injury (SCI), are key extracellular matrix inhibitory molecules that limit axon growth and consequent recovery of function. CSPG-mediated inhibition occurs via interactions with axonal receptors, including leukocyte common antigen- related (LAR) phosphatase. We tested the effects of a novel LAR inhibitory peptide in rats after hemisection at cervical level 2, a SCI model in which bulbospinal inspiratory neural circuitry originating in the medullary rostral ventral respiratory group (rVRG) becomes disconnected from phrenic motor neuron (PhMN) targets in cervical spinal cord, resulting in persistent partial-to-complete diaphragm paralysis. LAR peptide was delivered by a soaked gelfoam, which was placed directly over the injury site immediately after C2 hemisection and replaced at 1 week post-injury. Axotomized rVRG axons originating in ipsilateral medulla or spared rVRG fibers originating in contralateral medulla were separately assessed by anterograde tracing via AAV2-mCherry injection into rVRG. At 8 weeks post-hemisection, LAR peptide significantly improved ipsilateral hemidiaphragm function, as assessed in vivo with electromyography recordings. LAR peptide promoted robust regeneration of ipsilateral-originating rVRG axons into and through the lesion site and into intact caudal spinal cord to reach PhMNs located at C3-C5 levels. Furthermore, regenerating rVRG axons re-established putative monosynaptic connections with their PhMNs targets. In addition, LAR peptide stimulated robust sprouting of both modulatory serotonergic axons and contralateral-originating rVRG fibers within the PhMN pool ipsilateral/caudal to the hemisection. Our study demonstrates that targeting LAR-based axon growth inhibition promotes multiple forms of respiratory neural circuit plasticity and provides a new peptide-based therapeutic strategy to ameliorate the devastating respiratory consequences of SCI.

Increasing toll-like receptor 2 on astrocytes induced by Schwann cell-derived exosomes promotes recovery by inhibiting CSPGs deposition after spinal cord injury.

BACKGROUND: Traumatic spinal cord injury (SCI) is a severely disabling disease that leads to loss of sensation, motor, and autonomic function. As exosomes have great potential in diagnosis, prognosis, and treatment of SCI because of their ability to easily cross the blood-brain barrier, the function of Schwann cell-derived exosomes (SCDEs) is still largely unknown. METHODS: A T10 spinal cord contusion was established in adult female mice. SCDEs were injected into the tail veins of mice three times a week for 4 weeks after the induction of SCI, and the control group was injected with PBS. High-resolution transmission electron microscope and western blot were used to characterize the SCDEs. Toll-like receptor 2 (TLR2) expression on astrocytes, chondroitin sulfate proteoglycans (CSPGs) deposition and neurological function recovery were measured in the spinal cord tissues of each group by immunofluorescence staining of TLR2, GFAP, CS56, 5-HT, and ß-III-tublin, respectively. TLR2f/f mice were crossed to the GFAP-Cre strain to generate astrocyte specific TLR2 knockout mice (TLR2-/-). Finally, western blot analysis was used to determine the expression of signaling proteins and IKKß inhibitor SC-514 was used to validate the involved signaling pathway. RESULTS: Here, we found that TLR2 increased significantly on astrocytes post-SCI. SCDEs treatment can promote functional recovery and induce the expression of TLR2 on astrocytes accompanied with decreased CSPGs deposition. The specific knockout of TLR2 on astrocytes abolished the decreasing CSPGs deposition and neurological functional recovery post-SCI. In addition, the signaling pathway of NF-κB/PI3K involved in the TLR2 activation was validated by western blot. Furthermore, IKKß inhibitor SC-514 was also used to validate this signaling pathway. CONCLUSION: Thus, our results uncovered that SCDEs can promote functional recovery of mice post-SCI by decreasing the CSPGs deposition via increasing the TLR2 expression on astrocytes through NF-κB/PI3K signaling pathway.

The glycosyltransferase EXTL2 promotes proteoglycan deposition and injurious neuroinflammation following demyelination.

BACKGROUND: Chondroitin sulfate proteoglycans (CSPGs) are potent inhibitors of axonal regrowth and remyelination. More recently, they have also been highlighted as a modulator of macrophage infiltration into the central nervous system in experimental autoimmune encephalomyelitis, an inflammatory model of multiple sclerosis. METHODS: We interrogated results from single nucleotide polymorphisms (SNPs) lying in or close to genes regulating CSPG metabolism in the summary results from two publicly available systematic studies of multiple sclerosis (MS) genetics. A demyelinating injury model in the spinal cord of exostosin-like 2 deficient  (EXTL2-/-) mice was used to investigate the effects of dysregulation of EXTL2 on remyelination. Cell cultures of bone marrow-derived macrophages and primary oligodendrocyte precursor cells and neurons were supplemented with purified CSPGs or conditioned media to assess potential mechanisms of action. RESULTS: The strongest evidence for genetic association was seen for SNPs mapping to the region containing the glycosyltransferase exostosin-like 2 (EXTL2), an enzyme that normally suppresses CSPG biosynthesis. Six of these SNPs showed genome-wide significant evidence for association in one of the studies with concordant and nominally significant effects in the second study. We then went on to show that a demyelinating injury to the spinal cord of EXTL2-/- mice resulted in excessive deposition of CSPGs in the lesion area. EXTL2-/- mice had exacerbated axonal damage and myelin disruption relative to wild-type mice, and increased representation of microglia/macrophages within lesions. In tissue culture, activated bone marrow-derived macrophages from EXTL2-/- mice overproduce tumor necrosis factor α (TNFα) and matrix metalloproteinases (MMPs). CONCLUSIONS: These results emphasize CSPGs as a prominent modulator of neuroinflammation and they highlight CSPGs accumulating in lesions in promoting axonal injury.

Modulating proteoglycan receptor PTPσ using intracellular sigma peptide improves remyelination and functional recovery in mice with demyelinated optic chiasm.

Multiple sclerosis (MS) is an autoimmune disease characterized by myelin and axonal damage in the central nervous system (CNS). Glial scar which is a hallmark of MS contains repair inhibitory molecules including chondroitin sulfate proteoglycans (CSPGs). CSPGs inhibit repair of damaged area through various receptors including protein tyrosine phosphatase sigma (PTPσ). In the current study we use intracellular sigma peptide (ISP), an inhibitor of PTPσ signaling, in LPC-induced focal demyelination of mouse optic chiasm. ISP treatment resulted in decreased demyelination, reduced astrogliosis, and increased newly generated oligodendrocytes which subsequently led to enhanced remyelination. Analyzing of electrophysiological (as performed by visual evoked potential recording) and behavioral (performed by visual cliff test) outcomes showed that ISP-treatment improved the integrity of optic pathway as well as the visual acuity. When ISP was administrated only during the repair phase, histological, electrophysiological and behavioral studies showed its regenerative effect. Our results demonstrated the possibility of using ISP as a new strategy to inhibit PTPσ for myelin protection, myelin repair in demyelinated axons, and functional neural pathway conductivity restoration in patients suffering from MS.

Modulation of Receptor Protein Tyrosine Phosphatase Sigma Increases Chondroitin Sulfate Proteoglycan Degradation through Cathepsin B Secretion to Enhance Axon Outgrowth.

Severed axon tips reform growth cones following spinal cord injury that fail to regenerate, in part, because they become embedded within an inhibitory extracellular matrix. Chondroitin sulfate proteoglycans (CSPGs) are the major axon inhibitory matrix component that is increased within the lesion scar and in perineuronal nets around deafferented neurons. We have recently developed a novel peptide modulator (intracellular sigma peptide) of the cognate receptor of CSPGs, protein tyrosine phosphatase σ (RPTPσ), which has been shown to markedly improve sensorimotor function, micturition, and coordinated locomotor behavior in spinal cord contused rats. However, the mechanism(s) underlying how modulation of RPTPσ mediates axon outgrowth through inhibitory CSPGs remain unclear. Here, we describe how intracellular sigma peptide modulation of RPTPσ induces enhanced protease Cathepsin B activity. Using DRG neurons from female Sprague Dawley rats cultured on an aggrecan/laminin spot assay and a combination of biochemical techniques, we provide evidence suggesting that modulation of RPTPσ regulates secretion of proteases that, in turn, relieves CSPG inhibition through its digestion to allow axon migration though proteoglycan barriers. Understanding the mechanisms underlying RPTPσ modulation elucidates how axon regeneration is impaired by proteoglycans but can then be facilitated following injury.SIGNIFICANCE STATEMENT Following spinal cord injury, chondroitin sulfate proteoglycans (CSPGs) upregulate and potently inhibit axon regeneration and functional recovery. Protein tyrosine phosphatase σ (RPTPσ) has been identified as a critical cognate receptor of CSPGs. We have previously characterized a synthetic peptide (intracellular sigma peptide) that targets the regulatory intracellular domain of the receptor to allow axons to regenerate despite the presence of CSPGs. Here, we have found that one important mechanism by which peptide modulation of the receptor enhances axon outgrowth is through secretion of a protease, Cathepsin B, which enables digestion of CSPGs. This work links protease secretion to the CSPG receptor RPTPσ for the first time with implications for understanding the molecular mechanisms underlying neural regeneration and plasticity.

Kon-tiki enhances PS2 integrin adhesion and localizes its ligand, Thrombospondin, in the myotendinous junction.

Cell-extracellular-matrix adhesion is mediated by cell receptors, mainly integrins and transmembrane proteoglycans, which can functionally interact. How these receptors are regulated and coordinated is largely unknown. We show that the conserved transmembrane Drosophila proteoglycan Kon-tiki (Kon, also known as Perdido) interacts with the αPS2ßPS integrin (αPS2 is encoded by inflated and ßPS by myospheroid) to mediate muscle-tendon adhesion. kon and inflated double mutant embryos show a synergistic increase in muscle detachment. Furthermore, Kon modulates αPS2ßPS signaling at the muscle attachment, since phosphorylated Fak is reduced in kon mutants. This reduction in integrin signaling can be rescued by the expression of a truncated Kon protein containing its transmembrane and extracellular domains, suggesting that these domains are sufficient to mediate this signaling. We show that these domains are sufficient to properly localize the αPS2ßPS ligand, Thrombospondin, to the muscle attachment, and to partially rescue Kon-dependent muscle-tendon adhesion. We propose that Kon can engage in a protein complex with αPS2ßPS and enhance integrin-mediated signaling and adhesion by recruiting its ligand, which would increase integrin-binding affinity to the extracellular matrix, resulting in the consolidation of the myotendinous junction.

Naturally occurring cancers in pet dogs as pre-clinical models for cancer immunotherapy.

Despite the significant progress in tumor prevention, early detection, diagnosis and treatment made over recent decades, cancer is still an enormous public health challenge all around the world, with the number of people affected increasing every year. A great deal of effort is therefore being devoted to the search for novel safe, effective and economically sustainable treatments for the growing population of neoplastic patients. One main obstacle to this process is the extremely low percentage of therapeutic approaches that, after successfully passing pre-clinical testing, actually demonstrate activity when finally tested in humans. This disappointing and expensive failure rate is partly due to the pre-clinical murine models used for in vivo testing, which cannot faithfully recapitulate the multifaceted nature and evolution of human malignancies. These features are better mirrored in natural disease models, i.e., companion animals affected by cancers. Herein, we discuss the relevance of spontaneous canine tumors for the evaluation of the safety and anti-tumor activity of novel therapeutic strategies before in-human trials, and present our experience in the development of a vaccine that targets chondroitin sulphate proteoglycan (CSPG)4 as an example of these comparative oncology studies.

Cytoskeletal Signal-Regulated Oligodendrocyte Myelination and Remyelination.

Myelination and remyelination in the central nervous system (CNS) are essential for rapid conduction of action potentials and for appropriate neuronal communications supporting higher brain functions. Myelination is dependent on developmental stage and is controlled by neuronal axons-oligodendrocyte (OL) signaling. Numerous studies of the initial myelination and remyelination stages in the CNS have demonstrated several key cytoskeletal signals in axons and OLs. In this review, we focus on cytoskeletal signal-regulated OL myelination and remyelination, with particular attention to neuronal Notch proteins, bidirectional Eph/ephrin signaling, OL integrin and cadherin superfamily proteins, OL actin rearrangement, and OL tyrosine kinase Fyn substrate proteins during the initial myelination and remyelination stages in the CNS.

Furanodienone overcomes temozolomide resistance in glioblastoma through the downregulation of CSPG4-Akt-ERK signalling by inhibiting EGR1-dependent transcription.

Glioblastoma multiforme (GBM) is a highly aggressive type of brain tumour. Patients with GBM respond poorly to chemotherapy and have poor survival outcomes. Neuron-glial antigen 2 (NG2), also known as chondroitin sulphate proteoglycan 4 (CSPG4), has been shown to contribute to critical processes, such as cell survival, proliferation, and chemotherapy resistance, during glioma progression. In this study, we found that furanodienone (FUR), a diene-type sesquiterpene isolated from the rhizomes of Rhizoma curcumae, exhibited a potential cytotoxic effect on temozolomide (TMZ)-resistant GBM cells in vitro by inhibiting CSPG4 and related signalling pathways. Studies investigating the mechanism demonstrated that FUR suppressed CSPG4-Akt-ERK signalling, inflammatory responses, and cytokine levels but activated caspase-dependent pathways and mitochondrial dysfunction. Furthermore, an immunofluorescence assay and a dual-luciferase reporter assay revealed that inhibition of EGR1-mediated transcription might have contributed to the FUR-dependent blockade of CSPG4 signalling and glioma cell survival. These results established a link between FUR-induced CSPG4 inhibition and the suppression of EGR1-dependent transcription. Attenuation of ERK1/2 and cytokine signalling might have generated the EGR1-dependent negative feedback loop of the CSPG4 pathway during FUR-induced apoptosis. These findings suggested that FUR could be a therapeutic candidate for the treatment of malignant glioma via targeting CSPG4 signalling.

CSPG4 as Target for CAR-T-Cell Therapy of Various Tumor Entities-Merits and Challenges.

Targeting cancer cells using chimeric-antigen-receptor (CAR-)T cells has propelled adoptive T-cell therapy (ATT) to the next level. A plentitude of durable complete responses using CD19-specific CAR-T cells in patients suffering from various lymphoid malignancies resulted in the approval by the food and drug administration (FDA) of CD19-directed CAR-T cells for the treatment of acute lymphoblastic leukemia (ALL) and diffuse large B-cell lymphoma (DLBCL). A substantial portion of this success in hematological malignancies can be traced back to the beneficial properties of the target antigen CD19, which combines a universal presence on target cells with no detectable expression on indispensable host cells. Hence, to replicate response rates achieved in ALL and DLBCL in the realm of solid tumors, where ideal target antigens are scant and CAR-T cells are still lagging behind expectations, the quest for appropriate target antigens represents a crucial task to expedite the next steps in the evolution of CAR-T-cell therapy. In this review, we want to highlight the potential of chondroitin sulfate proteoglycan 4 (CSPG4) as a CAR-target antigen for a variety of different cancer entities. In particular, we discuss merits and challenges associated with CSPG4-CAR-T cells for the ATT of melanoma, leukemia, glioblastoma, and triple-negative breast cancer.

CSPG4-Specific CAR T Cells for High-Risk Childhood B Cell Precursor Leukemia.

The advent of CD19-specific chimeric antigen receptor (CAR) T cells has proven to be a powerful asset in the arsenal of cancer immunotherapy of acute lymphoblastic leukemia and certain B cell lymphomas. However, a sizable portion of patients treated with CD19-CAR T cells relapse with CD19-negative cancer cells, necessitating the quest for back-up antigens. Chondroitin sulfate proteoglycan 4 (CSPG4) expression has been reported on leukemic blasts bearing the ill-fated MLL 11q23 rearrangement. We aimed at exploring the use of CSPG4-specific CAR T cells against mixed-lineage leukemia (MLL)-rearranged leukemic blasts, using the precursor B cell leukemia cell line KOPN8 (MLL-MLLT1 translocation) as a model. First, we confirmed CSPG4 expression on KOPN8 cells. Bulk T cells electroporated with mRNA encoding a CSPG4-specific CAR upregulated activation markers and secreted the Th1 cytokines TNF and IFNγ in an antigen-specific manner upon co-culture with KOPN8 cells. More importantly, CSPG4-specific CAR T cells evinced specific degranulation towards KOPN8 cells and specifically lysed KOPN8 target cells in chromium lysis experiments. CSPG4 is a well-established CAR target in cutaneous melanoma. Here, we provide proof-of-principle data for the use of CSPG4-specific CAR T cells against MLL-translocated leukemias.