Changes in resting-state functional connectivity after stroke in a mouse brain lacking extracellular matrix components.

In the brain, focal ischemia results in a local region of cell death and disruption of both local and remote functional neuronal networks. Tissue reorganization following stroke can be limited by factors such as extracellular matrix (ECM) molecules that prevent neuronal growth and synaptic plasticity. The brain's ECM plays a crucial role in network formation, development, and regeneration of the central nervous system. Further, the ECM is essential for proper white matter tract development and for the formation of structures called perineuronal nets (PNNs). PNNs mainly surround parvalbumin/GABA inhibitory interneurons, of importance for processing sensory information. Previous studies have shown that downregulating PNNs after stroke reduces the neurite-inhibitory environment, reactivates plasticity, and promotes functional recovery. Resting-state functional connectivity (RS-FC) within and across hemispheres has been shown to correlate with behavioral recovery after stroke. However, the relationship between PNNs and RS-FC has not been examined. Here we studied a quadruple knock-out mouse (Q4) that lacks four ECM components: brevican, neurocan, tenascin-C and tenascin-R. We applied functional connectivity optical intrinsic signal (fcOIS) imaging in Q4 mice and wild-type (129S1 mice) before and 14 days after photothrombotic stroke (PT) to understand how the lack of crucial ECM components affects neuronal networks and functional recovery after stroke. Limb-placement ability was evaluated at 2, 7 and 14 days of recovery through the paw-placement test. Q4 mice exhibited significantly impaired homotopic RS-FC compared to wild-type mice, especially in the sensory and parietal regions. Changes in RS-FC were significantly correlated with the number of interhemispheric callosal crossings in those same regions. PT caused unilateral damage to the sensorimotor cortex and deficits of tactile-proprioceptive placing ability in contralesional fore- and hindlimbs, but the two experimental groups did not present significant differences in infarct size. Two weeks after PT, a general down-scaling of regional RS-FC as well as the number of regional functional connections was visible for all cortical regions and most notable in the somatosensory areas of both Q4 and wild-type mice. Q4 mice exhibited higher intrahemispheric RS-FC in contralesional sensory and motor cortices compared to control mice. We propose that the lack of growth inhibiting ECM components in the Q4 mice potentially worsen behavioral outcome in the early phase after stroke, but subsequently facilitates modulation of contralesional RS-FC which is relevant for recovery of sensory motor function. We conclude that Q4 mice represent a valuable model to study how the elimination of ECM genes compromises neuronal function and plasticity mechanisms after stroke.

ß-Sarcoglycan Deficiency Reduces Atherosclerotic Plaque Development in ApoE-Null Mice.

BACKGROUND: Smooth muscle cells are important for atherosclerotic plaque stability. Their proper ability to communicate with the extracellular matrix is crucial for maintaining the correct tissue integrity. In this study, we have investigated the role of ß-sarcoglycan within the matrix-binding dystrophin-glycoprotein complex in the development of atherosclerosis. RESULTS: Atherosclerotic plaque development was significantly reduced in ApoE-deficient mice lacking ß-sarcoglycan, and their plaques contained an increase in differentiated smooth muscle cells. ApoE-deficient mice lacking ß-sarcoglycan showed a reduction in ovarian adipose tissue and adipocyte size, while the total weight of the animals was not significantly different. Western blot analysis of adipose tissues showed a decreased activation of protein kinase B, while that of AMP-activated kinase was increased in mice lacking ß-sarcoglycan. Analysis of plasma in ß-sarcoglycan-deficient mice revealed reduced levels of leptin, adiponectin, insulin, cholesterol, and triglycerides but increased levels of IL-6, IL-17, and TNF-α. CONCLUSIONS: Our results indicate that the dystrophin-glycoprotein complex and ß-sarcoglycan can affect the atherosclerotic process. Furthermore, the results show the effects of ß-sarcoglycan deficiency on adipose tissue and lipid metabolism, which may also have contributed to the atherosclerotic plaque reduction.

Primary hippocampal neurons, which lack four crucial extracellular matrix molecules, display abnormalities of synaptic structure and function and severe deficits in perineuronal net formation.

The extracellular matrix (ECM) of the brain plays crucial roles during the development, maturation, and regeneration of the CNS. In a subpopulation of neurons, the ECM condenses to superstructures called perineuronal nets (PNNs) that surround synapses. Camillo Golgi described PNNs a century ago, yet their biological functions remain elusive. Here, we studied a mouse mutant that lacks four ECM components highly enriched in the developing brain: the glycoproteins tenascin-C and tenascin-R and the chondroitin sulfate proteoglycans brevican and neurocan. Primary embryonic hippocampal neurons and astrocytes were cultivated using a cell insert system that allows for co-culture of distinct cell populations in the absence of direct membrane contacts. The wild-type and knock-out cells were combined in the four possible permutations. Using this approach, neurons cultivated in the presence of mutant astrocytes displayed a transient increase of synapses after 2 weeks. However, after a period of 3 weeks or longer, synapse formation and stabilization were compromised when either neuron or astrocyte cell populations or both were of mutant origin. The development of PNN structures was observed, but their size was substantially reduced on knock-out neurons. The synaptic activity of both wild-type and knock-out neurons was monitored using whole-cell patch clamping. The salient observation was a reduced frequency of IPSCs and EPSCs, whereas the amplitudes were not modified. Remarkably, the knock-out neuron phenotypes could not be rescued by wild-type astrocytes. We conclude that the elimination of four ECM genes compromises neuronal function.

Fibromodulin deficiency reduces low-density lipoprotein accumulation in atherosclerotic plaques in apolipoprotein E-null mice.

OBJECTIVE: The aim of this study was to analyze how an altered collagen structure affects development of atherosclerotic plaques. METHODS AND RESULTS: Fibromodulin-null mice develop an abnormal collagen fibril structure. In apolipoprotein E (ApoE)-null and ApoE/fibromodulin-null mice, a shear stress-modifying carotid artery cast induced formation of atherosclerotic plaques of different phenotypes; inflammatory in low-shear stress regions and fibrous in oscillatory shear stress regions. Electron microscopy showed that collagen fibrils were thicker and more heterogeneous in oscillatory shear stress lesions from ApoE/fibromodulin-null mice. Low-shear stress lesions were smaller in ApoE/fibromodulin-null mice and contained less lipids. Total plaque burden in aortas stained en face with Oil Red O, as well as lipid accumulation in aortic root lesions, was also decreased in ApoE/fibromodulin-null mice. In addition, lipid accumulation in RAW264.7 macrophages cultured on fibromodulin-deficient extracellular matrix was decreased, whereas levels of interleukin-6 and -10 were increased. Our results show that an abnormal plaque collagen fibril structure can influence atherosclerotic plaque development. CONCLUSIONS: The present findings suggest a more complex role for collagen in plaque stability than previously anticipated, in that it may promote lipid-accumulation and inflammation at the same time as it provides mechanical stability.

Tracking the distribution of persistent and mobile wastewater-derived substances in the southern and central North Sea using anthropogenic gadolinium from MRI contrast agents as a far-field tracer.

The use of the rare earth element gadolinium (Gd) in contrast agents for magnetic resonance imaging has led to a significant (micro-)contamination of riverine and coastal environments in many parts of the world. This study comprises a detailed investigation on the rare earth elements and yttrium inventory of the North Sea and also reports data for the major tributaries Thames, Rhine, Ems, Weser and Elbe. We show that large parts of the southern North Sea, including the Wadden Sea UNESCO Natural World Heritage site, are (micro)contaminated with Gd from Gd-based contrast agents (GBCA). Their dispersion reveals their estuarine input and allows to effectively track water masses and currents. The chemical persistence and conservative behavior of GBCA, coupled with the low detection limits of state-of-the-art analytical methods, makes the anthropogenic Gd a sensitive screening proxy for monitoring similarly stable, but potentially hazardous, persistent chemical/pharmaceutical substances in natural waters.

The lecticans of mammalian brain perineural net are O-mannosylated.

O-Mannosylation is an important protein modification in brain. During the last years, a few mammalian proteins have been identified as targets of the protein-O-mannosyltransferases 1 and 2. However, these still cannot explain the high content of O-mannosyl glycans in brain and the strong brain involvement of congenital muscular dystrophies caused by POMT mutations (Walker-Warburg syndrome, dystroglycanopathies). By fractionating and analyzing the glycoproteome of mouse and calf brain lysates, we could show that proteins of the perineural net, the lecticans, are O-mannosylated, indicating that major components of neuronal extracellular matrix are O-mannosylated in mammalian brain. This finding corresponds with the high content of O-mannosyl glycans in brain as well as with the brain involvement of dystroglycanopathies. In contrast, the lectican neurocan is not O-mannosylated when recombinantly expressed in EBNA-293 cells, revealing the possibility of different control mechanisms for the initiation of O-mannosylation in different cell types.

Expression of insulin signalling components in the sensory epithelium of the human saccule.

Several studies have demonstrated a link between diabetes and the dysfunction of the inner ear. Few studies, however, have reported the signalling mechanisms involved in metabolic control in human inner ear cells. Knowledge of the expression and role of the insulin receptor and downstream signalling components in the inner ear is sparce. Our immunohistochemistry approach has shown that the insulin receptor, insulin receptor substrate 1 (IRS1), protein kinase B (PKB) and insulin-sensitive glucose transporter (GLUT4) are expressed in the sensory epithelium of the human saccule, which also exhibits expression of a calcium-sensitive cAMP/cGMP phosphodiesterase 1C (PDE1C) and the vasopressin type 2 receptor. IRS1 and PDE1C are selectively expressed in sensory epithelial hair cells, whereas the other components are expressed in sensory epithelial supporting cells or in both cell types, as judged from co-expression or non-co-expression with glial fibrillary acidic protein, a marker for supporting cells. Furthermore, IRS1 appears to be localized in association with sensory nerves, whereas GLUT4 is expressed in the peri-nuclear area of stromal cells, as is the case for aquaporin 2. Thus, the insulin receptor, insulin signalling components and selected cAMP signalling components are expressed in the human saccule. In addition to well-known mechanisms of diabetes complications, such as neuropathy and vascular lesions, the expression of these proteins in the saccule could have a role in the observed link between diabetes and balance/hearing disorders.

Mouse development is not obviously affected by the absence of dermatan sulfate epimerase 2 in spite of a modified brain dermatan sulfate composition.

Dermatan sulfate epimerase 2 (DS-epi2), together with its homolog DS-epi1, transform glucuronic acid into iduronic acid in DS polysaccharide chains. Iduronic acid gives DS increased chain flexibility and promotes protein binding. DS-epi2 is ubiquitously expressed and is the predominant epimerase in the brain. Here, we report the generation and initial characterization of DS-epi2 null mice. DS-epi2-deficient mice showed no anatomical, histological or morphological abnormalities. The body weights and lengths of mutated and wild-type littermates were indistinguishable. They were fertile and had a normal lifespan. Chondroitin sulfate (CS)/DS isolated from the newborn mutated mouse brains had a 38% reduction in iduronic acid compared with wild-type littermates, and compositional analysis revealed a decrease in 4-O-sulfate and an increase in 6-O-sulfate containing structures. Despite the reduction in iduronic acid, the adult DS-epi2-/- brain showed normal extracellular matrix features by immunohistological stainings. We conclude that DS-epi1 compensates in vivo for the loss of DS-epi2. These results extend previous findings of the functional redundancy of brain extracellular matrix components.

Circulating Hepatocyte Growth Factor Reflects Activation of Vascular Repair in Response to Stress.

Hepatocyte growth factor (HGF) is released by stressed human vascular cells and promotes vascular cell repair responses in both autocrine and paracrine ways. Subjects with a low capacity to express HGF in response to systemic stress have an increased cardiovascular risk. Human atherosclerotic plaques with a low content of HGF have a more unstable phenotype. The present study shows that subjects with a low ability to express HGF in response to metabolic stress have an increased risk to suffer myocardial infarction and stroke.

Sialoadhesin-positive macrophages bind regulatory T cells, negatively controlling their expansion and autoimmune disease progression.

An important regulatory suppressive function in autoimmune and other inflammatory processes has been ascribed to CD4(+)Foxp3(+) regulatory T cells (Tregs), which requires direct cell-cell communication between Tregs, effector T cells, and APCs. However, the molecular basis for these interactions has not yet been clarified. We show here that sialoadhesin (Sn), the prototype of the siglec family of sialic acid-binding transmembrane proteins, expressed by resident and activated tissue-infiltrating macrophages, directly binds to Tregs, negatively regulating their expansion in an animal model of multiple sclerosis (MS), experimental autoimmune encephalomyelitis (EAE). In this model, macrophages infiltrate the CNS exhibiting tissue-destructing and demyelinating activity, leading to MS-like symptoms. We show here that severity of EAE symptoms is reduced in Sn knockout (KO) mice compared with wild-type littermates due to an up-regulation of CD4(+)Foxp3(+) Treg lymphocytes. Through the use of a Sn fusion protein, Tregs were shown to express substantial amounts of Sn ligand on their cell surface, and direct interaction of Sn(+) macrophages with Tregs specifically inhibited Treg but not effector T lymphocyte proliferation. Conversely, blocking of Sn on macrophages by Sn-specific Abs resulted in elevated proliferation of Tregs. Data indicate that Sn(+) macrophages regulate Treg homeostasis which subsequently influences EAE progression. We propose a new direct cell-cell interaction-based mechanism regulating the expansion of the Tregs during the immune response, representing a "dialogue" between Sn(+) macrophages and Sn-accessible sialic acid residues on Treg lymphocytes.

Versican V2 assembles the extracellular matrix surrounding the nodes of ranvier in the CNS.

The CNS-restricted versican splice-variant V2 is a large chondroitin sulfate proteoglycan incorporated in the extracellular matrix surrounding myelinated fibers and particularly accumulating at nodes of Ranvier. In vitro, it is a potent inhibitor of axonal growth and therefore considered to participate in the reduction of structural plasticity connected to myelination. To study the role of versican V2 during postnatal development, we designed a novel isoform-specific gene inactivation approach circumventing early embryonic lethality of the complete knock-out and preventing compensation by the remaining versican splice variants. These mice are viable and fertile; however, they display major molecular alterations at the nodes of Ranvier. While the clustering of nodal sodium channels and paranodal structures appear in versican V2-deficient mice unaffected, the formation of the extracellular matrix surrounding the nodes is largely impaired. The conjoint loss of tenascin-R and phosphacan from the perinodal matrix provide strong evidence that versican V2, possibly controlled by a nodal receptor, organizes the extracellular matrix assembly in vivo.

Evidence for a protective role of placental growth factor in cardiovascular disease.

Placental growth factor (PlGF) is a mitogen for endothelial cells, but it can also act as a proinflammatory cytokine. Because it promotes early stages of plaque formation in experimental models of atherosclerosis and was implicated in epidemiological associations with risk of cardiovascular disease (CVD), PlGF has been attributed a pro-atherogenic role. Here, we investigated whether PlGF has a protective role in CVD and whether elevated PlGF reflects activation of repair processes in response to vascular stress. In a population cohort of 4742 individuals with 20 years of follow-up, high baseline plasma PlGF was associated with increased risk of cardiovascular death, myocardial infarction, and stroke, but these associations were lost or weakened when adjusting for cardiovascular risk factors known to cause vascular stress. Exposure of cultured endothelial cells to high glucose, oxidized low-density lipoprotein (LDL) or an inducer of apoptosis enhanced the release of PlGF. Smooth muscle cells and endothelial cells treated with PlGF small interference RNA demonstrated that autocrine PlGF stimulation plays an important role in vascular repair responses. High expression of PlGF in human carotid plaques removed at surgery was associated with a more stable plaque phenotype and a lower risk of future cardiovascular events. When adjusting associations of PlGF with cardiovascular risk in the population cohort for plasma soluble tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) receptor-2, a biomarker of cellular stress, a high PlGF/TRAIL receptor-2 ratio was associated with a lower risk. Our findings provide evidence for a protective role of PlGF in CVD.

Brevican distinctively assembles extracellular components at the large diameter nodes of Ranvier in the CNS.

Brevican is known to be an abundant extracellular matrix component in the adult brain and a structural constituent of perineuronal nets. We herein show that brevican, tenascin-R (TN-R) and phosphacan are present at the nodes of Ranvier on myelinated axons with a particularly large diameter in the central nervous system. A brevican deficiency resulted in a reorganization of the nodal matrices, which was characterized by the shift of TN-R, and concomitantly phosphacan, from an axonal diameter-dependent association with nodes to an axonal diameter independent association. Supported by the co-immunoprecipitation results, these observations indicate that the presence of TN-R and phosphacan at nodes is normally brevican-dependent, while in the absence of brevican these molecules can also be recruited by versican V2. The versican V2 and Bral1 distribution was not affected, thus indicating a brevican-independent role of these two molecules for establishing hyaluronan-binding matrices at the nodes. Our results revealed that brevican plays a crucial role in determining the specialization of the hyaluronan-binding nodal matrix assemblies in large diameter nodes.

Uranium in stream and mineral water of the Federal Republic of Germany.

The concentration of uranium was determined in 944 samples from stream water by the inductively coupled plasma quadrupole mass spectrometry (ICP-QMS) method and represented on a color-shaded contour map. Uranium concentrations in surface water were determined to be between 0.007 µg/l and 43.7 µg/l with median of 0.33 µg/l. The regional distribution of uranium is influenced primarily by lithological and anthropogenic factors. In Mecklenburg, northern Brandenburg, and eastern Schleswig-Holstein, elevated uranium concentrations coincide with the extent of the last Weichselian ice sheet. The maximum concentrations are observed in the surface waters of the old mining districts in the western part of the Ore Mountains and in eastern Thuringia. Elevated concentrations are found in areas of agriculturally used loess soils. These concentrations correlate with the use of phosphate fertilizers. There is a zone of elevated concentrations up to 10.0 µg U/l in the Keuper Sandstone area south of the Thuringian Forest and from northwest of Stuttgart as far as Coburg. The distribution of elevated values in mineral water shows a clear correlation with the elevated values in surface water and the geology of those locations. Bunter and Keuper strata are the most important uranium source.

Distinctive peri-luminal presence of agrin in murine and human carotid atherosclerotic plaques.

The clinical consequences of arterial atherosclerotic lesions depend, apart from their size, on their composition of cellular and extracellular components. While an intact endothelium at the interface of atherosclerotic plaques towards the blood can prevent its erosion, underlying smooth muscle cells within the plaque can reduce the risk of plaque ruptures, due to the deposition of stabilizing extracellular matrix. Basement membranes underlay and support the function of endothelial cells, and embed smooth muscle cells in the media, the source of most smooth muscle cells within atherosclerotic plaques. In the present study mouse atherosclerotic plaques were comparatively analyzed for the basement membrane components laminin, type IV collagen, perlecan, and agrin. Distinct agrin immunofluorescence was found in the peri-luminal area in mouse carotid atherosclerotic plaques. Agrin was also clearly present in the media, with a significant increase in regions directly associated with plaque tissue. In addition, ten human endarterectomy specimens were investigated for this heparan sulfate proteoglycan. No statistically significant differences in agrin immunofluorescence were noticed between five specimens from symptomatic and five from asymptomatic patients. In all these plaques agrin was present in a distinctive manner in a narrow zone partially or almost completely surrounding the lumen. Additionally it was also present around the small lumina of the CD31-positive neovessels. The presence of agrin at locations with particular importance for the growth and stability of atherosclerotic plaques renders this molecule strategically positioned to influence plaque development and vulnerability.

Hyaluronan is organized into fiber-like structures along migratory pathways in the developing mouse cerebellum.

Hyaluronan is a free glycosaminoglycan which is abundant in the extracellular matrix of the developing brain. Although not covalently linked to any protein it can act as a backbone molecule forming aggregates with chondroitin sulfate proteoglycans of the lectican family and link proteins. Using neurocan-GFP as a direct histochemical probe we analyzed the distribution and organization of hyaluronan in the developing mouse cerebellum, and related its fine structure to cell types of specified developmental stages. We observed a high affinity of this probe to fiber-like structures in the prospective white matter which are preferentially oriented parallel to the cerebellar cortex during postnatal development suggesting a specially organized form of hyaluronan. In other layers of the cerebellar cortex, the hyaluronan organization seemed to be more diffuse. During the second postnatal week, the overall staining intensity of hyaluronan in the white matter declined but fiber-like structures were still present at the adult stage. This type of hyaluronan organization is different from perineuronal nets e.g. found in deep cerebellar nuclei. Double staining experiments with cell type specific markers indicated that these fiber-like structures are predominantly situated in regions where motile cells such as Pax2-positive inhibitory interneuron precursors and MBP-positive oligodendroglial cells are located. In contrast, more stationary cells such as mature granule cells and Purkinje cells are associated with lower levels of hyaluronan in their environment. Thus, hyaluronan-rich fibers are concentrated at sites where specific neural precursor cell types migrate, and the anisotropic orientation of these fibers suggests that they may support guided neural migration during brain development.

Brevican-deficient mice display impaired hippocampal CA1 long-term potentiation but show no obvious deficits in learning and memory.

Brevican is a brain-specific proteoglycan which is found in specialized extracellular matrix structures called perineuronal nets. Brevican increases the invasiveness of glioma cells in vivo and has been suggested to play a role in central nervous system fiber tract development. To study the role of brevican in the development and function of the brain, we generated mice lacking a functional brevican gene. These mice are viable and fertile and have a normal life span. Brain anatomy was normal, although alterations in the expression of neurocan were detected. Perineuronal nets formed but appeared to be less prominent in mutant than in wild-type mice. Brevican-deficient mice showed significant deficits in the maintenance of hippocampal long-term potentiation (LTP). However, no obvious impairment of excitatory and inhibitory synaptic transmission was found, suggesting a complex cause for the LTP defect. Detailed behavioral analysis revealed no statistically significant deficits in learning and memory. These data indicate that brevican is not crucial for brain development but has restricted structural and functional roles.

Dystrophin deficiency reduces atherosclerotic plaque development in ApoE-null mice.

Dystrophin of the dystrophin-glycoprotein complex connects the actin cytoskeleton to basement membranes and loss of dystrophin results in Duchenne muscular dystrophy. We have previously shown injury-induced neointima formation of the carotid artery in mice with the mdx mutation (causing dystrophin deficiency) to be increased. To investigate the role of dystrophin in intimal recruitment of smooth muscle cells (SMCs) that maintains plaque stability in atherosclerosis we applied a shear stress-modifying cast around the carotid artery of apolipoprotein E (ApoE)-null mice with and without the mdx mutation. The cast induces formation of atherosclerotic plaques of inflammatory and SMC-rich/fibrous phenotypes in regions of low and oscillatory shear stress, respectively. Unexpectedly, presence of the mdx mutation markedly reduced the development of the inflammatory low shear stress plaques. Further characterization of the low shear stress plaques in ApoE-null mdx mice demonstrated reduced infiltration of CD3(+) T cells, less laminin and a higher SMC content. ApoE-null mdx mice were also found to have a reduced fraction of CD3(+) T cells in the spleen and lower levels of cytokines and monocytes in the circulation. The present study is the first to demonstrate a role for dystrophin in atherosclerosis and unexpectedly shows that this primarily involves immune cells.

IL-22 affects smooth muscle cell phenotype and plaque formation in apolipoprotein E knockout mice.

OBJECTIVE: IL-22 is a recently discovered cytokine that belongs to the family of IL-10 related cytokines. It is produced by activated T-cells and innate lymphoid cells and has been suggested to be involved in tissue repair. As both inflammation and repair play important roles in atherosclerosis we investigated if IL-22 deficiency influences the disease process in Apoe(-/-) mice. METHODS: We generated IL-22(-/-)Apoe(-/-) mice and fed them high-fat-diet for 14 weeks to characterize atherosclerosis development. RESULTS: IL-22(-/-)Apoe(-/-) mice exhibited reduced plaque size both in the aorta (p = 0.0036) and the aortic root compared (p = 0.0012) with Apoe(-/-) controls. Moreover, plaque collagen was reduced in IL-22(-/-)Apoe(-/-) mice (p = 0.02) and this was associated with an increased expression of smooth muscle cell (SMC)-α-actin (p = 0.04) and caldesmon (p = 0.016) in the underlying media. Carotid arteries from IL-22(-/-)Apoe(-/-) mice displayed increased expression of genes associated with a contractile SMC phenotype e.g. α-actin (p = 0.004) and caldesmon (p = 0.03). Arterial SMCs were shown to express the IL-22 receptor and in vitro exposure to IL-22 resulted in a down-regulation of alpha actin and caldesmon gene expression in these cells. CONCLUSION: Our observations demonstrate that IL-22 is involved in plaque formation and suggest that IL-22 released by immune cells is involved in activation of vascular repair by stimulating medial SMC dedifferentiation into a synthetic phenotype. This response contributes to plaque growth by enabling SMC migration into the intima but may also help to stabilize the plaque.

Cartilage link protein interacts with neurocan, which shows hyaluronan binding characteristics different from CD44 and TSG-6.

The interaction of neurocan with hyaluronan was qualitatively characterized with alkaline phosphatase fusion proteins secreted by mammalian cells. The wild type neurocan hyaluronan binding domain fused to alkaline phosphatase bound to immobilized hyaluronan under physiological as well as moderately hypertonic conditions, whereas its ability to bind to immobilized chondroitin sulfate dropped rapidly with increasing salt concentration. Strong hyaluronan binding ability was still evident when in both link modules within the hyaluronan binding domain a basic amino acid was mutated, which is well conserved among link modules of hyaluronan binding proteins. A strong enhancement of the binding of neurocan to immobilized hyaluronan was observed after preincubation of the immobilized hyaluronan with cartilage link protein. Moreover, this preincubation mediated also the binding of a fusion protein representing only the immunoglobulin module of neurocan linked to alkaline phosphatase, which showed no binding to immobilized hyaluronan alone. The interaction of the neurocan immunoglobulin module with link protein could also be shown by overlay blot analysis. These observations suggest that the hyaluronan binding characteristics of paired link modules are different from those of single link modules, and that the reported temporal co-expression of cartilage link protein and of neurocan in developing brain implicates the possibility of a cooperative function of these molecules.