Advances in Hyaluronan Biology: Signaling, Regulation, and Disease Mechanisms

Hyaluronan is an extracellular glycosaminoglycan polymer consisting of linear disaccharide units containing alternating glucuronate and N-acetylglucosamine.Many cell types make hyaluronan, which unlike most other macromolecules is assembled at the plasmamembrane and concurrently translocated through the hyaluronan synthase enzyme. The normal function of large hyaluronan polymers (\u3e1MDa) in tissue cushioning, hydration, and lubrication is well established. The aberrant accumulation and degradation of hyaluronan and the receptor-mediated signaling of smaller hyaluronan fragments have also been extensively implicated in a variety of pathological states including inflammation and cancer. More recently, the discovery that hyaluronan can either be a structural matrix component or appear as smaller processed polymers and oligomers that differentially engage a diverse range of signaling receptors has created an exciting paradigm shift and reenergized hyaluronan research in a broad range of fields. In this special issue, eight review articles focus on summarizing the latest contributions to understanding hyaluronan synthesis and catabolism and the regulation of hyaluronan functions. Seven novel primary research articles also investigate multiple roles of hyaluronan in disease progression and targeting

An organotypic slice culture model of chronic white matter injury with maturation arrest of oligodendrocyte progenitors

<p>Abstract</p> <p>Background</p> <p>CNS myelination disturbances commonly occur in chronic white matter lesions in neurodevelopmental and adult neurological disorders. Recent studies support that myelination failure can involve a disrupted cellular repair mechanism where oligodendrocyte (OL) progenitor cells (OPCs) proliferate in lesions with diffuse astrogliosis, but fail to fully differentiate to mature myelinating OLs. There are no <it>in vitro </it>models that reproduce these features of myelination failure.</p> <p>Results</p> <p>Forebrain coronal slices from postnatal day (P) 0.5/1 rat pups were cultured for 1, 5, or 9 days <it>in vitro </it>(DIV). Slices rapidly exhibited diffuse astrogliosis and accumulation of the extracellular matrix glycosaminoglycan hyaluronan (HA), an inhibitor of OPC differentiation and re-myelination. At 1 DIV ~1.5% of Olig2⁺OLs displayed caspase-3 activation, which increased to ~11.5% by 9 DIV. At 1 DIV the density of PDGFRα⁺and PDGFRα⁺/Ki67⁺OPCs were significantly elevated compared to 0 DIV (<it>P </it>< 0.01). Despite this proliferative response, at 9 DIV ~60% of white matter OLs were late progenitors (preOLs), compared to ~7% in the postnatal day 10 rat (<it>P </it>< 0.0001), consistent with preOL maturation arrest. Addition of HA to slices significantly decreased the density of MBP⁺OLs at 9 DIV compared to controls (217 ± 16 <it>vs. </it>328 ± 17 cells/mm², respectively; <it>P </it>= 0.0003), supporting an inhibitory role of HA in OL lineage progression in chronic lesions.</p> <p>Conclusions</p> <p>Diffuse white matter astrogliosis and early OPC proliferation with impaired OL maturation were reproduced in this model of myelination failure. This system may be used to define mechanisms of OPC maturation arrest and myelination failure related to astrogliosis and HA accumulation.</p

Hyaluronan Synthesis, Catabolism, and Signaling in Neurodegenerative Diseases

The glycosaminoglycan hyaluronan (HA), a component of the extracellular matrix, has been implicated in regulating neural differentiation, survival, proliferation, migration, and cell signaling in the mammalian central nervous system (CNS). HA is found throughout the CNS as a constituent of proteoglycans, especially within perineuronal nets that have been implicated in regulating neuronal activity. HA is also found in the white matter where it is diffusely distributed around astrocytes and oligodendrocytes. Insults to the CNS lead to long-term elevation of HA within damaged tissues, which is linked at least in part to increased transcription of HA synthases. HA accumulation is often accompanied by elevated expression of at least some transmembrane HA receptors including CD44. Hyaluronidases that digest high molecular weight HA into smaller fragments are also elevated following CNS insults and can generate HA digestion products that have unique biological activities. A number of studies, for example, suggest that both the removal of high molecular weight HA and the accumulation of hyaluronidase-generated HA digestion products can impact CNS injuries through mechanisms that include the regulation of progenitor cell differentiation and proliferation. These studies, reviewed here, suggest that targeting HA synthesis, catabolism, and signaling are all potential strategies to promote CNS repair

Single Cell Ras-GTP Analysis Reveals Altered Ras Activity in a Subpopulation of Neurofibroma Schwann Cells but Not Fibroblasts

Neurofibromatosis type 1 (NF1) is a common genetic disorder characterized by multiple neurofibromas, peripheral nerve tumors containing mainly Schwann cells and fibroblasts. The NF1 gene encodes neurofibromin, a tumor suppressor postulated to function in part as a Ras GTPase-activating protein. The roles of different cell types and of elevated Ras-GTP in neurofibroma formation are unclear. To determine which neurofibroma cell type has altered Ras-GTP regulation, we developed an immunocytochemical assay for active, GTP-bound Ras. In NIH 3T3 cells, the assay detected overexpressed, constitutively activated K-, N-, and Ha-Ras and insulin-induced endogenous Ras-GTP. In dissociated neurofibroma cells from NF1 patients, Ras-GTP was elevated in Schwann cells but not fibroblasts. Twelve to 62% of tumor Schwann cells showed elevated Ras-GTP, unexpectedly revealing neurofibroma Schwann cell heterogeneity. Increased basal Ras-GTP did not correlate with increased cell proliferation. Normal human Schwann cells, however, did not demonstrate elevated basal Ras activity. Furthermore, compared with cells from wild type littermates, Ras-GTP was elevated in all mouse Nf1−/− Schwann cells but never in Nf1−/− mouse fibroblasts. Our results indicate that Ras activity is detectably increased in only some neurofibroma Schwann cells and suggest that neurofibromin is not an essential regulator of Ras activity in fibroblasts

Advances in Hyaluronan Biology: Signaling, Regulation, and Disease Mechanisms

Hyaluronan is an extracellular glycosaminoglycan polymer consisting of linear disaccharide units containing alternating glucuronate and N-acetylglucosamine.Many cell types make hyaluronan, which unlike most other macromolecules is assembled at the plasmamembrane and concurrently translocated through the hyaluronan synthase enzyme. The normal function of large hyaluronan polymers (\u3e1MDa) in tissue cushioning, hydration, and lubrication is well established. The aberrant accumulation and degradation of hyaluronan and the receptor-mediated signaling of smaller hyaluronan fragments have also been extensively implicated in a variety of pathological states including inflammation and cancer. More recently, the discovery that hyaluronan can either be a structural matrix component or appear as smaller processed polymers and oligomers that differentially engage a diverse range of signaling receptors has created an exciting paradigm shift and reenergized hyaluronan research in a broad range of fields. In this special issue, eight review articles focus on summarizing the latest contributions to understanding hyaluronan synthesis and catabolism and the regulation of hyaluronan functions. Seven novel primary research articles also investigate multiple roles of hyaluronan in disease progression and targeting

The BRG-1 Subunit of the SWI/SNF Complex Regulates CD44 Expression

Aberrant regulation of CD44, a transmembrane glycoprotein, has been implicated in the growth and metastasis of numerous tumors. Although both CD44 overexpression and loss have been implicated in tumor progression, the mechanism of CD44 down-regulation in these tumor types is not known. By immunoblot and reverse transcription-polymerase chain reaction analysis we determined that a cervical carcinoma cell line, C33A, lacks CD44 expression. To determine how CD44 is down-regulated in C33A cells, we utilized cell fusions of C33A cells with a CD44-expressing cell line (SAOS-2). We found that SAOS-2 fusion restored CD44 expression in C33A cells, suggesting that a trans-acting factor present in SAOS-2 cells promotes CD44 production. C33A cells are BRG-1-deficient, and we found that CD44 was absent in another BRG-1-deficient tumor cell line, indicating that loss of BRG-1 may be a general mechanism by which cells lose CD44. Reintroduction of BRG-1 into these cells restored CD44 expression. Furthermore, disruption of BRG-1 function through the use of dominant-negative BRG-1 demonstrated the requirement of BRG-1 in CD44 regulation. Finally, we show that Cyclin E overexpression resulted in the attenuation of CD44 stimulation, which is consistent with previous observations that Cyclin E can abrogate BRG-1 action. Taken together, these results suggest that BRG-1 is a critical regulator of CD44 expression, thus implicating SWI/SNF components in the regulation of cellular adhesion and metastasis

Coordinate control of axon defasciculation and myelination by laminin-2 and -8

Schwann cells form basal laminae (BLs) containing laminin-2 (Ln-2; heterotrimer α2β1γ1) and Ln-8 (α4β1γ1). Loss of Ln-2 in humans and mice carrying α2-chain mutations prevents developing Schwann cells from fully defasciculating axons, resulting in partial amyelination. The principal pathogenic mechanism is thought to derive from structural defects in Schwann cell BLs, which Ln-2 scaffolds. However, we found loss of Ln-8 caused partial amyelination in mice without affecting BL structure or Ln-2 levels. Combined Ln-2/Ln-8 deficiency caused nearly complete amyelination, revealing Ln-2 and -8 together have a dominant role in defasciculation, and that Ln-8 promotes myelination without BLs. Transgenic Ln-10 (α5β1γ1) expression also promoted myelination without BL formation. Rather than BL structure, we found Ln-2 and -8 were specifically required for the increased perinatal Schwann cell proliferation that attends myelination. Purified Ln-2 and -8 directly enhanced in vitro Schwann cell proliferation in collaboration with autocrine factors, suggesting Lns control the onset of myelination by modulating responses to mitogens in vivo

Conditional müller cell ablation causes independent neuronal and vascular pathologies in a novel transgenic model

Müller cells are the major glia of the retina that serve numerous functions essential to retinal homeostasis, yet the contribution of Müller glial dysfunction to retinal diseases remains largely unknown. We have developed a transgenic model using a portion of the regulatory region of the retinaldehyde binding protein 1 gene for conditional Müller cell ablation and the consequences of primary Müller cell dysfunction have been studied in adult mice. We found that selective ablation of Müller cells led to photoreceptor apoptosis, vascular telangiectasis, blood-retinal barrier breakdown and, later, intraretinal neovascularization. These changes were accompanied by impaired retinal function and an imbalance between vascular endothelial growth factor-A (VEGF-A) and pigment epithelium-derived factor. Intravitreal injection of ciliary neurotrophic factor inhibited photoreceptor injury but had no effect on the vasculopathy. Conversely, inhibition of VEGF-A activity attenuated vascular leak but did not protect photoreceptors. Our findings show that Müller glial deficiency may be an important upstream cause of retinal neuronal and vascular pathologies in retinal diseases. Combined neuropro-tective and anti-angiogenic therapies may be required to treat Müller cell deficiency in retinal diseases and in other parts of the CNS associated with glial dysfunction

A TLR/AKT/FoxO3 immune tolerance–like pathway disrupts the repair capacity of oligodendrocyte progenitors

Cerebral white matter injury (WMI) persistently disrupts myelin regeneration by oligodendrocyte progenitor cells (OPCs). We identified a specific bioactive hyaluronan fragment (bHAf) that downregulates myelin gene expression and chronically blocks OPC maturation and myelination via a tolerance-like mechanism that dysregulates pro-myelination signaling via AKT. Desensitization of AKT occurs via TLR4 but not TLR2 or CD44. OPC differentiation was selectively blocked by bHAf in a maturation-dependent fashion at the late OPC (preOL) stage by a noncanonical TLR4/TRIF pathway that induced persistent activation of the FoxO3 transcription factor downstream of AKT. Activated FoxO3 selectively localized to oligodendrocyte lineage cells in white matter lesions from human preterm neonates and adults with multiple sclerosis. FoxO3 constraint of OPC maturation was bHAf dependent, and involved interactions at the FoxO3 and MBP promoters with the chromatin remodeling factor Brg1 and the transcription factor Olig2, which regulate OPC differentiation. WMI has adapted an immune tolerance–like mechanism whereby persistent engagement of TLR4 by bHAf promotes an OPC niche at the expense of myelination by engaging a FoxO3 signaling pathway that chronically constrains OPC differentiation