Expression Of Chondroitin Sulfate Proteoglycans Research Articles

CD200+ and CD200− macrophages accumulated in ischemic lesions of rat brain: The two populations cannot be classified as either M1 or M2 macrophages

Two types of macrophages in lesion core of rat stroke model were identified according to NG2 chondroitin sulfate proteoglycan (NG2) and CD200 expression. NG2(+) macrophages were CD200(-), and vice versa. NG2(-) macrophages expressed two splice variants of CD200 that are CD200L and CD200S. CD200(+) macrophages expressed CD8, CD68, CD163, CCL2, inducible nitric oxide synthase, interleukin-1β, Toll-like receptor 4 and transforming growth factor β, whilst NG2(+) cells expressed a costimulatory factor CD86. Both cell types expressed insulin-like growth factor 1 and CD200R. These results demonstrate that the two macrophage types cannot be classified as either M1 or M2.

NG2+ progenitors derived from embryonic stem cells penetrate glial scar and promote axonal outgrowth into white matter after spinal cord injury.

The glial scar resulting from spinal cord injury is rich in chondroitin sulfate proteoglycan (CSPG), a formidable barrier to axonal regeneration. We explored the possibility of breaching that barrier by first examining the scar in a functional in vitro model. We found that embryonic stem cell-derived neural lineage cells (ESNLCs) with prominent expression of nerve glial antigen 2 (NG2) survived, passed through an increasingly inhibitory gradient of CSPG, and expressed matrix metalloproteinase 9 (MMP-9) at the appropriate stage of their development. Outgrowth of axons from ESNLCs followed because the migrating cells sculpted pathways in which CSPG was degraded. The degradative mechanism involved MMP-9 but not MMP-2. To confirm these results in vivo, we transplanted ESNLCs directly into the cavity of a contused spinal cord 9 days after injury. A week later, ESNLCs survived and were expressing both NG2 and MMP-9. Their axons had grown through long distances (>10 mm), although they preferred to traverse white rather than gray matter. These data are consistent with the concept that expression of inhibitory CSPG within the injury scar is an important impediment to regeneration but that NG2+ progenitors derived from ESNLCs can modify the microenvironment to allow axons to grow through the barrier. This beneficial action may be partly due to developmental expression of MMP-9. We conclude that it might eventually be possible to encourage axonal regeneration in the human spinal cord by transplanting ESNLCs or other cells that express NG2.

Neuroprotective Effect of Simvastatin via Inducing the Autophagy on Spinal Cord Injury in the Rat Model.

Simvastatin, an inhibitor of 3-hydroxy-3-methylglutaryl-coenzyme A reductase, is invariably used to treat cardiovascular diseases. Simvastatin has been recently demonstrated to have a neuroprotective effect in nervous system diseases. The present study aimed to further verify the neuroprotection and molecular mechanism of simvastatin on rats after spinal cord injury (SCI). The expression of Beclin-1 and LC3-B was evidently enhanced at postoperation days 3 and 5, respectively. However, the reduction of the mTOR protein and ribosomal protein S6 kinase p70 subtype (p70S6K) phosphorylation level occurred at the same time after SCI. Simvastatin significantly increased the expression of brain-derived neurotrophic factor (BDNF) and glial cell line-derived neurotrophic factor (GDNF). Meanwhile, immunofluorescence results indicated that the expression of chondroitin sulfate proteoglycan (CSPG) and caspase-3 protein was obviously reduced by simvastatin. Furthermore, Nissl staining and Basso, Beattie, and Bresnahan (BBB) scores showed that the quantity and function of motor neurons were visibly preserved by simvastatin after SCI. The findings of this study showed that simvastatin induced autophagy by inhibiting the mTOR signaling pathway and contributed to neuroprotection after SCI.

GM-CSF reduces expression of chondroitin sulfate proteoglycan (CSPG) core proteins in TGF-β-treated primary astrocytes

GM-CSF plays a role in the nervous system, particularly in cases of injury. A therapeutic effect of GM-CSF has been reported in rat models of various central nervous system injuries. We previously showed that GM-CSF could enhance long-term recovery in a rat spinal cord injury model, inhibiting glial scar formation and increasing the integrity of axonal structure. Here, we investigated molecular the mechanism(s) by which GM-CSF suppressed glial scar formation in an in vitro system using primary astrocytes treated with TGF-β. GM-CSF repressed the expression of chondroitin sulfate proteoglycan (CSPG) core proteins in astrocytes treated with TGF-β. GM-CSF also inhibited the TGF-β-induced Rho-ROCK pathway, which is important in CSPG expression. Finally, the inhibitory effect of GM-CSF was blocked by a JAK inhibitor. These results may provide the basis for GM-CSF’s effects in glial scar inhibition and ultimately for its therapeutic effect on neural cell injuries. [BMB Reports 2014; 47(12): 679-684]

Transforming growth factor β-induced expression of chondroitin sulfate proteoglycans is mediated through non-Smad signaling pathways

The expression of chondroitin sulfate proteoglycans (CSPGs) by reactive astrocytes is a major factor contributing to glial scarring and regenerative failure after spinal cord injury, but the molecular mechanisms underlying CSPG expression remain largely undefined. One contributing factor is transforming growth factor β (TGFβ), which is upregulated after injury and has been shown to induce expression of CSPGs in vitro. TGFβ typically mediates its effects through the Smad2/3 signaling pathway, and it has been suggested that this pathway is responsible for CSPG expression. However, there is evidence that TGFβ can also activate non-Smad signaling pathways. In this study, we report that TGFβ-induced expression of three different CSPGs – neurocan, brevican, and aggrecan – is mediated through non-Smad signaling pathways. We observed significant increases in TGFβ-induced expression of neurocan, brevican, and aggrecan following siRNA knockdown of Smad2 or Smad4, which indicates that Smad signaling is not required for the expression of these CSPGs. In addition, we show that neurocan, aggrecan, and brevican levels are significantly reduced when TGFβ is administered in the presence of either the PI3K inhibitor LY294002 or the mTOR inhibitor rapamycin, but not the MEK1/2 inhibitor U0126. This suggests that TGFβ mediates this effect through non-Smad-dependent activation of the PI3K–Akt–mTOR signaling pathway, and targeting this pathway may therefore be an effective means of reducing CSPG expression in the injured CNS.

ME-11 * REDUCTION OF NFkB IN THE IN THE TUMOR MICROENVIRONMENT ALTERS THE NEURAL EXTRACELLULAR MATRIX AND REDUCES GLIOBLASTOMA GROWTH AND DISPERSION

Identifying major support mechanisms in the neural microenvironment could have significant impact against growth and invasion of malignant gliomas. The NFkB pathway is a major signaling hub that drives tumor progression and at the same time regulates the inflammatory responses of normal tissues. Here we show that animals with a stunted NFkB response exhibit reduced reactivity against intracranial gliomas, which surprisingly reduces tumor growth and invasion. NFkB-deficient mice (RelA + /- TNFR1-/-) were injected intracranially with mouse GL261 glioblastoma cells to analyze tumor phenotype. Histological and MRI analyses showed significantly smaller tumors in RelA + /- mice compared to control littermates. Interestingly, immunohistochemical studies indicated that tumors in RelA + /- mice had vascularization and infiltration of microglia/macrophages comparable to controls, but significant reduction of M2 microglia/macrophage polarization and GFAP-positive astroglia. The lower reactivity of RelA + /- astroglia was also evident following intracranial implantation of highly invasive mouse glioblastoma cells (Mut4; GFAP-Cre:Nf1loxP/+ Trp53-/+ PTENloxP/+), suggesting that the astrocyte compartment played a major role in tumor growth or invasion. To test this hypothesis we prepared cultures of brain slices, where astrocytes and their secreted ECM are the major components challenging glioma cells. Interestingly, glioblastoma cells were significantly less invasive in brain slices from NFkB-deficient mice compared to wild-type tissue. Further analysis of RelA-/–astrocytes demonstrated reduced migration towards glioblastoma cells, increased expression of neural chondroitin sulfate proteoglycans and reduced expression of the metalloproteases that cleave these inhibitory ECM proteins, lending support to the reduced tumor progression observed in vivo. Taken together, these results strongly suggest that activation of NFkB in the neural microenvironment underlies the reactive glial response that promotes tumor growth and invasion. In particular, our results highlight astroglial NFkB signaling as a driver of glioma malignancy that should be considered for long-term targeted strategies.

NT-12 * IMPACT OF HUMANIZED CHONDROITINASE ABC ON OV THERAPY FOR INTRACRANIAL TUMORS

Breast cancer is a leading cause of cancer death and source of brain metastases (BM) among women in the US. With a dismal two year survival of 2%, novel treatment modalities are essential. Chondroitin sulfate proteoglycan (CSPG) expression has been directly associated with metastatic potential in breast cancer. The Chondroitinase ABC (Chase) enzyme is able to digest CSPGs, thereby disrupting critical extracellular matrix interactions. We recently generated an oncolytic virus expressing Chase which resulted in some efficacy in mice bearing intracranial gliomas. Here, we developed a second generation Chase mutant (ChaseM) vector, which results in optimal enzymatic expression and function in mammalian cells. ChaseM decreases neurosphere formation in vitro, akin to the phenotype observed with pharmacologic blockade of CSPG assembly. In nude mice bearing glioma xenografts, OV-ChaseM led to a significant increase in median survival over control virus treated animals (p = 0.005). CSPGs in the CNS are formed as an integral part of the glial scar and implicated in the sequestration of inflammation at the site of insult. Thus, we hypothesize that the dissolution of CSPGs by OV-ChaseM will facilitate the influx of immune cells into the tumor stroma, alleviate microglia suppression and elevate T-cell responses following viral therapy, resulting in long term responses. We generated a syngeneic model of BM to test the ability of OV-ChaseM to facilitate these effects. Intracranial implantation of DB7 breast cancer cells results in 100% tumor development and pathology akin to human BM disease. In this model, a single dose of OV-ChaseM resulted in a significant survival advantage compared to controls (p = 0.02). Ongoing studies are evaluating the contribution of host anti-tumor immune activation towards this survival efficacy. With this data, we aim to broaden the scope of treatment for brain cancer patients and provide therapeutic evidence for regiments to be implemented in the clinic.

Effects of granulocyte colony-stimulating factor and granulocyte-macrophage colony-stimulating factor on glial scar formation after spinal cord injury in rats.

This study investigated the effects of granulocyte colony-stimulating factor (G-CSF) on glial scar formation after spinal cord injury (SCI) in rats and compared the therapeutic effects between G-CSF and granulocytemacrophage colony-stimulating factor (GM-CSF) to evaluate G-CSF as a potential substitute for GM-CSF in clinical application. Rats were randomly assigned to 1 of 4 groups: a sham-operated group (Group 1), an SCI group without treatment (Group 2), an SCI group treated with G-CSF (Group 3), and an SCI group treated with GM-CSF (Group 4). G-CSF and GM-CSF were administered via intraperitoneal injection immediately after SCI. The effects of G-CSF and GM-CSF on functional recovery, glial scar formation, and axonal regeneration were evaluated and compared. The rats in Groups 3 and 4 showed better functional recovery and more decreased cavity sizes than those in Group 2 (p < 0.05). Both G-CSF and GM-CSF suppressed intensive expression of glial fibrillary acidic protein around the cavity at 4 weeks and reduced the expression of chondroitin sulfate proteoglycans (p < 0.05). Also, early administration of G-CSF and GM-CSF protected axon fibers from destructive injury and facilitated axonal regeneration. There were no significant differences in comparisons of functional recovery, glial scar formation, and axonal regeneration between G-CSF and GM-CSF. G-CSF suppressed glial scar formation after SCI in rats, possibly by restricting the expression of glial fibrillary acidic protein and chondroitin sulfate proteoglycans, which might facilitate functional recovery from SCI. GM-CSF and G-CSF had similar effects on glial scar formation and functional recovery after SCI, suggesting that G-CSF can potentially be substituted for GM-CSF in the treatment of SCI.

Alteration in chondroitin sulfate proteoglycan expression at the epicenter of spinal cord is associated with the loss of behavioral function in Tiptoe walking Yoshimura mice.

The objective of this study was to explore the correlation between the alteration in chondroitin sulfate proteoglycan (CSPG) expression at the epicenter of spinal cord and the loss of behavioral function in tiptoe walking Yoshimura mice. The tiptoe walking Yoshimura mice (twy) and Institute of Cancer Research (ICR) mice, aged 20 and 26weeks, were used in the present study. The behavior assessment, micro-computed tomography and immunofluorescent staining were performed. The compressed spinal cord was histologically analyzed. The results showed that the expression of CSPG was statistically higher at the compressed spinal cord for twy mice compared with that at the normal spinal cord for ICR mice. At the 26th week, a large ossification block at the posterior longitudinal ligament of C1-3 was obviously observed at the micro-CT image We observed the BMS Score was significantly correlated with the expression of glial fibrillary acidic protein, CSPG and hyaluronan (P<0.05). These findings suggest that compression injury induces the higher CSPG expression at the compressed spinal cord in the twy mice. Furthermore, the alteration in CSPG expression at the epicenter of spinal cord is associated with the loss of behavioral function in twy mice.

Beneficial effects of gfap/vimentin reactive astrocytes for axonal remodeling and motor behavioral recovery in mice after stroke.

The functional role of reactive astrocytes after stroke is controversial. To elucidate whether reactive astrocytes contribute to neurological recovery, we compared behavioral outcome, axonal remodeling of the corticospinal tract (CST), and the spatio-temporal change of chondroitin sulfate proteoglycan (CSPG) expression between wild-type (WT) and glial fibrillary acidic protein/vimentin double knockout (GFAP(-/-) Vim(-/-) ) mice subjected to Rose Bengal induced cerebral cortical photothrombotic stroke in the right forelimb motor area. A foot-fault test and a single pellet reaching test were performed prior to and on day 3 after stroke, and weekly thereafter to monitor functional deficit and recovery. Biotinylated dextran amine (BDA) was injected into the left motor cortex to anterogradely label the CST axons. Compared with WT mice, the motor functional recovery and BDA-positive CST axonal length in the denervated side of the cervical gray matter were significantly reduced in GFAP(-/-) Vim(-/-) mice (n = 10/group, P < 0.01). Immunohistological data showed that in GFAP(-/-) Vim(-/-) mice, in which astrocytic reactivity is attenuated, CSPG expression was significantly increased in the lesion remote areas in both hemispheres, but decreased in the ischemic lesion boundary zone, compared with WT mice (n = 12/group, P < 0.001). Our data suggest that attenuated astrocytic reactivity impairs or delays neurological recovery by reducing CST axonal remodeling in the denervated spinal cord. Thus, manipulation of astrocytic reactivity post stroke may represent a therapeutic target for neurorestorative strategies.

Targeted inhibition of KCa3.1 attenuates TGF‐β‐induced reactive astrogliosis through the Smad2/3 signaling pathway

Reactive astrogliosis, characterized by cellular hypertrophy and various alterations in gene expression and proliferative phenotypes, is considered to contribute to brain injuries and diseases as diverse as trauma, neurodegeneration, and ischemia. KCa3.1 (intermediate-conductance calcium-activated potassium channel), a potassium channel protein, has been reported to be up-regulated in reactive astrocytes after spinal cord injury in vivo. However, little is known regarding the exact role of KCa3.1 in reactive astrogliosis. To elucidate the role of KCa3.1 in regulating reactive astrogliosis, we investigated the effects of either blocking or knockout of KCa3.1 channels on the production of astrogliosis and astrocytic proliferation in response to transforming growth factor (TGF)-β in primary cultures of mouse astrocytes. We found that TGF-β increased KCa3.1 protein expression in astrocytes, with a concomitant marked increase in the expression of reactive astrogliosis, including glial fibrillary acidic protein and chondroitin sulfate proteoglycans. These changes were significantly attenuated by the KCa3.1 inhibitor 1-((2-chlorophenyl) (diphenyl)methyl)-1H-pyrazole (TRAM-34). Similarly, the increase in glial fibrillary acidic protein and chondroitin sulfate proteoglycans in response to TGF-β was attenuated in KCa3.1(-/-) astrocytes. TRAM-34 also suppressed astrocytic proliferation. In addition, the TGF-β-induced phosphorylation of Smad2 and Smad3 proteins was reduced with either inhibition of KCa3.1 with TRAM-34 or in KCa3.1(-/-) astrocytes. These findings highlight a novel role for the KCa3.1 channel in reactive astrogliosis phenotypic modulation and provide a potential target for therapeutic intervention for brain injuries. Reactive astrogliosis is characterized by the expression of glial fibrillary acidic protein and chondroitin sulfate proteoglycans. We demonstrate that either pharmacological blockade or knockout of KCa3.1 channels reduces reactive gliosis in cultured astrocytes caused by TGF-β, and also reduces TGF-β-induced phosphorylation of Smad2/3.

Analysis of Human Embryonic Stem Cells with Regulatable Expression of the Cell Adhesion Molecule L1 in Regeneration after Spinal Cord Injury

Cell replacement therapy is one potential avenue for central nervous system (CNS) repair. However, transplanted stem cells may not contribute to long-term recovery of the damaged CNS unless they are engineered for functional advantage. To fine tune regenerative capabilities, we developed a human neural cell line expressing L1, a regeneration-conducive adhesion molecule, under the control of a doxycycline regulatable Tet-off promoter. Controlled expression of L1 is desired because overexpression after regenerative events may lead to adverse consequences. The regulated system was tested in several cell lines, where doxycycline completely eliminated green fluorescent protein or L1 expression by 3-5 days in vitro. Increased colony formation as well as decreased proliferation were observed in H9NSCs without doxycycline (hL1-on). To test the role of L1 in vivo after acute compression spinal cord injury of immunosuppressed mice, quantum dot labeled hL1-on or hL1-off cells were injected at three sites: lesion; proximal; and caudal. Mice transplanted with hL1-on cells showed a better Basso Mouse Scale score, when compared to those with hL1-off cells. As compared to the hL1-off versus hL1-on cell transplanted mice 6 weeks post-transplantation, expression levels of L1, migration of transplanted cells, and immunoreactivity for tyrosine hydroxylase were higher, whereas expression of chondroitin sulfate proteoglycans was lower. Results indicate that L1 expression is regulatable in human stem cells by doxycycline in a nonviral engineering approach. Regulatable expression in a prospective nonleaky Tet-off system could hold promise for therapy, based on the multifunctional roles of L1, including neuronal migration and survival, neuritogenesis, myelination, and synaptic plasticity.

Neuronal and Astroglial TGFβ-Smad3 Signaling Pathways Differentially Regulate Dendrite Growth and Synaptogenesis

To address the role of the transforming growth factor beta (TGFβ)-Smad3 signaling pathway in dendrite growth and associated synaptogenesis, we used small inhibitory RNA to knockdown the Smad3 gene in either cultured neurons and or primary astrocytes. We found that TGFβ1 treatment of primary neurons increased dendrite extensions and the number of synapsin-1-positive synapses. When Smad3 was knockdown in primary neurons, dendrite growth was inhibited and the number of synapsin-1-positive synapses reduced even with TGFβ1 treatment. When astrocyte-conditioned medium (ACM), collected from TGFβ1-treated astrocytes (TGFβ1-stimulated ACM), was added to cultured neurons, dendritic growth was inhibited and the number of synapsin-1-positive puncta reduced. When TGFβ1-stimulated ACM was collected from astrocytes with Smad3 knocked down, this conditioned media promoted the growth of dendrites and the number of synapsin-1-positive puncta in cultured neurons. We further found that TGFβ1 signaling through Smad3 increased the expression of chondroitin sulfate proteoglycans, neurocan, and phosphacan in ACM. Application of chondroitinase ABC to the TGFβ1-stimulated ACM reversed its inhibitory effects on the dendrite growth and the number of synapsin-1-positive puncta. On the other hand, we found that TGFβ1 treatment caused a facilitation of Smad3 phosphorylation and translocation to the nucleus induced by status epilepticus (SE) in wild-type (Smad3(+/+)) mice, and this treatment also caused a promotion of γ-aminobutyric acid-ergic synaptogenesis impaired by SE in Smad3(+/+) as well as in Smad3(-/-) mice, but more dramatic promotion in Smad3(+/+) mice. Thus, we provide evidence for the first time that TGFβ-Smad3 signaling pathways within neuron and astrocyte differentially regulate dendrite growth and synaptogenesis, and this pathway may be involved in the pathogenesis of some central nervous system diseases, such as epilepsy.

Chondroitin sulfate proteoglycans in the nervous system: inhibitors to repair.

Chondroitin sulfate proteoglycans (CSPGs) are widely expressed in the normal central nervous system, serving as guidance cues during development and modulating synaptic connections in the adult. With injury or disease, an increase in CSPG expression is commonly observed close to lesioned areas. However, these CSPG deposits form a substantial barrier to regeneration and are largely responsible for the inability to repair damage in the brain and spinal cord. This review discusses the role of CSPGs as inhibitors, the role of inflammation in stimulating CSPG expression near site of injury, and therapeutic strategies for overcoming the inhibitory effects of CSPGs and creating an environment conducive to nerve regeneration.

Targeting inhibitory chondroitin sulphate proteoglycans to promote plasticity after injury.

Chondroitin sulphate proteoglycans (CSPGs) are one of the major families of inhibitory extracellular matrix molecules in the central nervous system. The expression of various CSPGs is strong during early nervous system development; however, it is downregulated during maturation and up-regulated again after nervous system injury. In vivo injection of an enzyme called chondroitinase ABC, which removes the inhibitory chondroitin sulphate chains on the CSPGs, in the injured area promotes both the regeneration and plasticity of the neurons. Here, we describe the method of in vivo injection of the chondroitinase ABC into the cortex of adult rat brain and the histochemical method to assess the successfulness of the digestion.

Immunoablation of cells expressing the NG2 chondroitin sulphate proteoglycan

Expression of the transmembrane NG2 chondroitin sulphate proteoglycan (CSPG) defines a distinct population of NG2-glia. NG2-glia serve as a regenerative pool of oligodendrocyte progenitor cells in the adult central nervous system (CNS), which is important for demyelinating diseases such as multiple sclerosis, and are a major component of the glial scar that inhibits axon regeneration after CNS injury. In addition, NG2-glia form unique neuron–glial synapses with unresolved functions. However, to date it has proven difficult to study the importance of NG2-glia in any of these functions using conventional transgenic NG2 ‘knockout' mice. To overcome this, we aimed to determine whether NG2-glia can be targeted using an immunotoxin approach. We demonstrate that incubation in primary anti-NG2 antibody in combination with secondary saporin-conjugated antibody selectively kills NG2-expressing cells in vitro. In addition, we provide evidence that the same protocol induces the loss of NG2-glia without affecting astrocyte or neuronal numbers in cerebellar brain slices from postnatal mice. This study shows that targeting the NG2 CSPG with immunotoxins is an effective and selective means for killing NG2-glia, which has important implications for studying the functions of these enigmatic cells both in the normal CNS, and in demyelination and degeneration.

Proteoglycan abnormalities in olfactory epithelium tissue from subjects diagnosed with schizophrenia

Emerging evidence points to proteoglycan abnormalities in the pathophysiology of schizophrenia (SZ). In particular, markedly abnormal expression of chondroitin sulfate proteoglycans (CSPGs), key components of the extracellular matrix, was observed in the medial temporal lobe. CSPG functions, including regulation of neuronal differentiation and migration, are highly relevant to the pathophysiology of SZ. CSPGs may exert similar functions in the olfactory epithelium (OE), a continuously regenerating neural tissue that shows cell and molecular abnormalities in SZ. We tested the hypothesis that CSPG expression in OE may be altered in SZ. CSPG-positive cells in postmortem OE from non-psychiatric control (n=9) and SZ (n=10) subjects were counted using computer-assisted light microscopy. ‘Cytoplasmic’ CSPG (c-CSPG) labeling was detected in sustentacular cells and some olfactory receptor neurons (c-CSPG+ORNs), while ‘pericellular’ CSPG (p-CSPG) labeling was found in basal cells and some ORNs (p-CSPG+ORNs). Dual labeling for CSPG and markers for mature and immature ORNs suggests that c-CSPG+ORNs correspond to mature ORNs, and p-CSPG+ORNs to immature ORNs. Previous studies in the same cohort demonstrated that densities of mature ORNs were unaltered (Arnold et al., 2001). In the present study, numerical densities of c-CSPG+ORNs were significantly decreased in SZ (p<0.025; 99.32% decrease), suggesting a reduction of CSPG expression in mature ORNs. Previous studies showed a striking increase in the ratios of immature neurons with respect to basal cells. In this study, we find that the ratio of p-CSPG+ORNs/CSPG+basal cells was significantly increased (p=0.03) in SZ, while numerical density changes of p-CSPG+ORNs (110.71% increase) or CSPG+basal cells (53.71% decrease), did not reach statistical significance. Together, these results indicate that CSPG abnormalities are present in the OE of SZ and specifically point to a reduction of CSPG expression in mature ORNs in SZ. Given the role CSPGs play in OE cell differentiation and axon guidance, we suggest that altered CSPG expression may contribute to ORN lineage dysregulation, and olfactory identification abnormalities, observed in SZ.

Abstract 4058: CSPG4 mediates melanoma cell survival in response to BRAFV600E inhibition.

Abstract Resistance to chemotherapy remains a major cause of malignant melanoma treatment failure. While recent clinical trials have shown significant patient response to chemotherapeutics targeting BRAFV600E, e.g. dabrafenib, tumors eventually become resistant and disease recurs. Development of additional novel therapies is therefore critical to better manage melanoma patients. Analysis of patient-derived melanoma lesions has revealed robust expression of the cell adhesion protein chondroitin sulfate proteoglycan 4 (CSPG4). While not prognostic due to its expression in premalignant nevi, CSPG4 has been revealed to promote melanoma progression by enhancing cell growth, migration and invasion in vitro and in vivo. Previous studies have shown CSPG4 to enhance activation of ERK1/2, PI3K and FAK, suggesting that CSPG4 likely contributes to oncogenic growth and survival through activation of a number of parallel signaling cascades. CSPG4 therefore represents a potential avenue for melanoma cell survival in response to chemotherapy. In support of this hypothesis, melanoma cells expressing endogenous or exogenous CSPG4 are more resistant to cell death induced by dabrafenib than cells lacking endogenous expression of CSPG4. Likewise, suppression of CSPG4 via siRNA reduced melanoma cell survival in response to dabrafenib. Melanoma cells expressing CSPG4 show enhanced growth in soft agar versus cells lacking CSPG4 expression, even in the presence of BRAFV600E inhibition. To determine the mechanism of CSPG4-driven resistance to BRAFV600E inhibition, activation of a number of oncogenic growth and survival pathways was interrogated. While ERK1/2 phosphorylation in the presence of BRAFV600E inhibition is unaffected by CSPG4, AKT phosphorylation is enhanced by expression of CSPG4. Moreover, this CSPG4-driven AKT activation in response to dabrafenib is dependent on PI3K, as treatment of melanoma cells with the PI3K inhibitor LY294002 eliminated increased AKT phosphorylation by CSPG4 in response to dabrafenib. CSPG4 enhances Src-FAK complex assembly, membrane lipid subdomain localization, and activation in melanoma cells through syntenin-1-dependent scaffolding. A mutant of CSPG4 engineered to eliminate lipid microdomain localization of CSPG4, termed CSPG4C2230A, was unable to recapitulate this phenotype.CSPG4C2230A also failed to protect melanoma cells from cytotoxicity or promote growth in soft agar in the presence of BRAFV600E inhibition. Altogether, these data indicate that CSPG4 confers protection against cytotoxicity induced by BRAFV600E inhibition by organizing progression-associated signalosomes involved in AKT activation, making CSPG4 an attractive target for the development of adjuvant therapies. Ongoing studies focus on determining the mechanism of CSPG4-mediated AKT activation and on developing CSPG4-targeting melanoma treatments for use in preclinical models. Citation Format: Leah E. Colvin Wanshura, Jianbo Yang, Matthew A. Price, Jennifer H. Carlson, Arkadiusz Z. Dudek, James B. McCarthy. CSPG4 mediates melanoma cell survival in response to BRAFV600E inhibition. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4058. doi:10.1158/1538-7445.AM2013-4058

Astrocytes specifically remove surface-adsorbed fibrinogen and locally express chondroitin sulfate proteoglycans

Surface-adsorbed fibrinogen (FBG) was recognized by adhering astrocytes, and was removed from the substrates in vitro by a two-phase removal process. The cells removed adsorbed FBG from binary proteins’ surface patterns (FBG+laminin, or FBG+albumin) while leaving the other protein behind. Astrocytes preferentially expressed chondroitin sulfate proteoglycan (CSPG) at the loci of fibrinogen stimuli; however, no differences in overall CSPG production as a function of FBG surface coverage were identified. Removal of FBG by astrocytes was also found to be independent of transforming growth factor type β (TGF-β) receptor based signaling as cells maintained CSPG production in the presence of TGF-β receptor kinase inhibitor, SB 431542. The inhibitor decreased CSPG expression, but did not abolish it entirely. Because blood contact and subsequent FBG adsorption are unavoidable in neural implantations, the results indicate that implant-adsorbed FBG may contribute to reactive astrogliosis around the implant as astrocytes specifically recognize adsorbed FBG.

Conditional Sox9 ablation reduces chondroitin sulfate proteoglycan levels and improves motor function following spinal cord injury

Chondroitin sulfate proteoglycans (CSPGs) found in perineuronal nets and in the glial scar after spinal cord injury have been shown to inhibit axonal growth and plasticity. Since we have previously identified SOX9 as a transcription factor that upregulates the expression of a battery of genes associated with glial scar formation in primary astrocyte cultures, we predicted that conditional Sox9 ablation would result in reduced CSPG expression after spinal cord injury and that this would lead to increased neuroplasticity and improved locomotor recovery. Control and Sox9 conditional knock-out mice were subject to a 70 kdyne contusion spinal cord injury at thoracic level 9. One week after injury, Sox9 conditional knock-out mice expressed reduced levels of CSPG biosynthetic enzymes (Xt-1 and C4st), CSPG core proteins (brevican, neurocan, and aggrecan), collagens 2a1 and 4a1, and Gfap, a marker of astrocyte activation, in the injured spinal cord compared with controls. These changes in gene expression were accompanied by improved hind limb function and locomotor recovery as evaluated by the Basso Mouse Scale (BMS) and rodent activity boxes. Histological assessments confirmed reduced CSPG deposition and collagenous scarring at the lesion of Sox9 conditional knock-out mice, and demonstrated increased neurofilament-positive fibers in the lesion penumbra and increased serotonin immunoreactivity caudal to the site of injury. These results suggest that SOX9 inhibition is a potential strategy for the treatment of SCI.