Brevican isoforms associate with neural membranes

Brevican is a neural-specific proteoglycan of the brain extracellular matrix, which is particularly abundant in the terminally differentiated CNS. It is expressed by neuronal and glial cells, and as a component of the perineuronal nets it decorates the surface of large neuronal somata and primary dendrites. One brevican isoform harbors a glycosylphosphatidylinositol anchor attachment site and, as shown by ethanolamine incorporation studies, is indeed glypiated in stably transfected HEK293 cells as well as in oligodendrocyte precursor Oli-neu cells. The major isoform is secreted into the extracellular space, although a significant amount appears to be tightly attached to the cell membrane, as it floats up in sucrose gradients. Flotation is sensitive to detergent treatment. Brevican is most prominent in the microsomal, light membrane and synaptosomal fractions of rat brain membrane preparations. The association with the particulate fraction is in part sensitive to chondroitinase ABC and phosphatidylinositol-specific phospholipase C treatment. Furthermore, brevican staining on the surface of hippocampal neurons in culture is diminished after hyaluronidase or chondroitinase ABC treatment. Taken together, this could provide a mechanism by which perineuronal nets are anchored on neuronal surfaces.     

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.     

Neurocan is dispensable for brain development

Neurocan is a component of the extracellular matrix in brain. Due to its inhibition of neuronal adhesion and outgrowth in vitro and its expression pattern in vivo it was suggested to play an important role in axon guidance and neurite growth. To study the role of neurocan in brain development we generated neurocan-deficient mice by targeted disruption of the neurocan gene. These mice are viable and fertile and have no obvious deficits in reproduction and general performance. Brain anatomy, morphology, and ultrastructure are similar to those of wild-type mice. Perineuronal nets surrounding neurons appear largely normal. Mild deficits in synaptic plasticity may exist, as maintenance of late-phase hippocampal long-term potentiation is reduced. These data indicate that neurocan has either a redundant or a more subtle function in the development of the brain.     

Altered production and proteolytic processing of brevican by transforming growth factor beta in cultured astrocytes

Brevican, a proteoglycan of the lectican family, inhibits neurite outgrowth and may also stabilize synapses. Little is known about its expression or function in vitro. This study seeks to determine whether a brevican-containing matrix is present in neural cultures, and if so, how the production of brevican may be modulated. To accomplish this, the content of brevican and its proteolytic fragments were measured in primary cultures of neurons, astrocytes and microglia after treatment with cytokines. These experiments revealed that astrocytes and neurons express several isoforms of brevican, whereas microglia do not produce this proteoglycan. Cleavage fragments of brevican were found primarily in neuronal and astrocyte culture medium. ADAMTS4 (a disintegrin and metalloproteinase with thrombospondin motifs), a protease that selectively cleaves lecticans, was detected in cultures of neurons, astrocytes and microglia. When astrocytes were challenged with various cytokines, it was found that treatment with transforming growth factor beta (TGFbeta) resulted in a marked increase in intact brevican in the culture medium that was accompanied by a trend for a decrease in ADAMTS-generated fragments of brevican and apparent ADAMTS activity. Thus, TGFbeta may play a role in neuronal plasticity through its regulation of brevican and the activity of the ADAMTSs.     

Reelin and ApoE receptors cooperate to enhance hippocampal synaptic plasticity and learning

Two apolipoprotein E (apoE) receptors, the very low density lipoprotein (VLDL) receptor and apoE receptor 2 (apoER2), are also receptors for Reelin, a signaling protein that regulates neuronal migration during brain development. In the adult brain, Reelin is expressed by GABA-ergic interneurons, suggesting a potential function as a modulator of neurotransmission. ApoE receptors have been indirectly implicated in memory and neurodegenerative disorders because their ligand, apoE, is genetically associated with Alzheimer disease. We have used knockout mice to investigate the role of Reelin and its receptors in cognition and synaptic plasticity. Mice lacking either the VLDL receptor or the apoER2 show contextual fear conditioning deficits. VLDL receptor-deficient mice also have a moderate defect in long term potentiation (LTP), and apoER2 knockouts have a pronounced one. The perfusion of mouse hippocampal slices with Reelin has no effect on baseline synaptic transmission but significantly enhances LTP in area CA1. This Reelin-dependent augmentation of LTP is abolished in VLDL receptor and apoER2 knockout mice. Our results reveal a role for Reelin in controlling synaptic plasticity in the adult brain and suggest that both of its receptors are necessary for Reelin-dependent enhancement of synaptic transmission in the hippocampus. Thus, the impairment of apoE receptor-dependent neuromodulation may contribute to cognitive impairment and synaptic loss in Alzheimer disease.     

Pathway-specific alteration of synaptic plasticity in Tg2576 mice

Various animal models of Alzheimer disease (AD) are characterized by deficits in spatial memory that are causally related to altered synaptic function and impairment of long-term potentiation (LTP) in the hippocampus. In Tg2576 AD mice, we compared LTP in 2 major hippocampal pathways, Schaffer collateral (SC) and mossy fiber (MF) pathways. Whereas LTP was completely abolished in the SC pathway of Tg2576 mice, we found no decrease in LTP induced by stimulation of the MF pathway. In fact, we found that in the MF pathway, LTP was slightly, but significantly, enhanced compared with that in the MF pathway of WT littermates. This pathway-specific impairment of LTP is not attributable to alterations in transmitter release, as indicated by an unaltered paired-pulse ratio. These results suggest that the spatial memory deficits normally seen in AD models arise primarily from LTP impairment at the SC pathway.     

cDNA cloning, chromosomal localization, and expression analysis of human BEHAB/brevican, a brain specific proteoglycan regulated during cortical development and in glioma

BEHAB (Brain Enriched HyAluronan Binding)/brevican, a brain-specific member of the lectican family of chondroitin sulfate proteoglycans (CSPGs), may play a role in both brain development and human glioma. BEHAB/brevican has been cloned from bovine, mouse and rat. Two isoforms have been reported: a full-length isoform that is secreted into the extracellular matrix (ECM) and a shorter isoform with a sequence that predicts a glycophosphatidylinositol (GPI) anchor. Here, we report the characterization of BEHAB/brevican isoforms in human brain. First, BEHAB/brevican maps to human chromosome 1q31. Second, we report the sequence of both isoforms of human BEHAB/brevican. The deduced protein sequence of full-length, secreted human BEHAB/brevican is 89.7, 83.3 and 83.2% identical to bovine, mouse and rat homologues, respectively. Third, by RNase protection analysis (RPA) we show the developmental regulation of BEHAB/brevican isoforms in normal human cortex. The secreted isoform is highly expressed from birth through 8years of age and is downregulated by 20years of age to low levels that are maintained in the normal adult cortex. The GPI isoform is expressed at uniformly low levels throughout development. Fourth, we confirm and extend previous studies from our laboratory, here demonstrating the upregulation of BEHAB/brevican mRNA in human glioma quantitatively. RPA analysis shows that both isoforms are upregulated in glioma, showing an approximately sevenfold increase in expression over normal levels. In contrast to the developmental regulation of BEHAB/brevican, where only the secreted isoform is regulated, both isoforms are increased in parallel in human glioma. The distinct patterns of regulation of expression of the two isoforms suggest distinct mechanisms of regulation of BEHAB/brevican during development and in glioma.     

Carbohydrate-protein interactions between HNK-1-reactive sulfoglucuronyl glycolipids and the proteoglycan lectin domain mediate neuronal cell adhesion and neurite outgrowth

Lecticans, a family of chondroitin sulfate proteoglycans, represent the largest group of proteoglycans expressed in the nervous system. We previously showed that the C-type lectin domains of lecticans bind two classes of sulfated cell surface glycolipids, sulfatides and HNK-1-reactive sulfoglucuronylglycolipids (SGGLs). In this paper, we demonstrate that the interaction between the lectin domain of brevican, a nervous system-specific lectican, and cell surface SGGLs acts as a novel cell recognition system that promotes neuronal adhesion and neurite outgrowth. The Ig chimera of the brevican lectin domain bind to the surface of SGGL-expressing rat hippocampal neurons. The substrate of the brevican chimera promotes adhesion and neurite outgrowth of hippocampal neurons. The authentic, full-length brevican also promotes neuronal cell adhesion and neurite outgrowth. These activities of brevican substrates are neutralized by preincubation of cells with HNK-1 monoclonal antibodies and by pretreatment of the brevican substrates with purified SGGLs. Brevican and HNK-1 carbohydrates are coexpressed in specific layers of the developing hippocampus where axons from entorhinal neurons elongate. Our observations suggest that cell surface SGGLs and extracellular lecticans comprise a novel cell-substrate recognition system operating in the developing nervous system.     

The overexpression of RBM3 alleviates TBI‐induced behaviour impairment and AD‐like tauopathy in mice

The therapeutic hypothermia is an effective tool for TBI‐associated brain impairment, but its side effects limit in clinical routine use. Hypothermia up‐regulates RNA‐binding motif protein 3 (RBM3), which is verified to protect synaptic plasticity. Here, we found that cognitive and LTP deficits, loss of spines, AD‐like tau pathologies are displayed one month after TBI in mice. In contrast, the deficits of LTP and cognitive, loss of spines and tau abnormal phosphorylation at several sites are obviously reversed in TBI mice combined with hypothermia pre‐treatment (HT). But, the neuroprotective role of HT disappears in TBI mouse models under condition of blocking RBM3 expression with RBM3 shRNA. In other hand, overexpressing RBM3 by AAV‐RBM3 plasmid can mimic HT‐like neuroprotection against TBI‐induced chronic brain injuries, such as improving LTP and cognitive, loss of spines and tau hyperphosphorylation in TBI mouse models. Taken together, hypothermia pre‐treatment reverses TBI‐induced chronic AD‐like pathology and behaviour deficits in RBM3 expression dependent manner, RBM3 may be a potential target for neurodegeneration diseases including Alzheimer disease.     

β-adducin (Add2) KO mice show synaptic plasticity, motor coordination and behavioral deficits accompanied by changes in the expression and phosphorylation levels of the α- and γ-adducin subunits

Adducins are a family of proteins found in cytoskeleton junctional complexes, which bind and regulate actin filaments and actin-spectrin complexes. In brain, adducin is expressed at high levels and is identified as a constituent of synaptic structures, such as dendritic spines and growth cones of neurons. Adducin-induced changes in dendritic spines are involved in activity-dependent synaptic plasticity processes associated with learning and memory, but the mechanisms underlying these functions remain to be elucidated. Here, β-adducin knockout (KO) mice were used to obtain a deeper insight into the role of adducin in these processes. We showed that β-adducin KO mice showed behavioral, motor coordination and learning deficits together with an altered expression and/or phosphorylation levels of α-adducin and γ-adducin. We found that β-adducin KO mice exhibited deficits in learning and motor performances associated with an impairment of long-term potentiation (LTP) and long-term depression (LTD) in the hippocampus. These effects were accompanied by a decrease in phosphorylation of adducin, a reduction in α-adducin expression levels and upregulation of γ-adducin in hippocampus, cerebellum and neocortex of mutant mice. In addition, we found that the mRNA encoding β-adducin is also located in dendrites, where it may participate in the fine modulation of LTP and LTD. These results strongly suggest coordinated expression and phosphorylation of adducin subunits as a key mechanism underlying synaptic plasticity, motor coordination performance and learning behaviors.     

The proteoglycan brevican binds to fibronectin after proteolytic cleavage and promotes glioma cell motility

The adult neural parenchyma contains a distinctive extracellular matrix that acts as a barrier to cell and neurite motility. Nonneural tumors that metastasize to the central nervous system almost never infiltrate it and instead displace the neural tissue as they grow. In contrast, invasive gliomas disrupt the extracellular matrix and disperse within the neural tissue. A major inhibitory component of the neural matrix is the lectican family of chondroitin sulfate proteoglycans, of which brevican is the most abundant member in the adult brain. Interestingly, brevican is also highly up-regulated in gliomas and promotes glioma dispersion by unknown mechanisms. Here we show that brevican secreted by glioma cells enhances cell adhesion and motility only after proteolytic cleavage. At the molecular level, brevican promotes epidermal growth factor receptor activation, increases the expression of cell adhesion molecules, and promotes the secretion of fibronectin and accumulation of fibronectin microfibrils on the cell surface. Moreover, the N-terminal cleavage product of brevican, but not the full-length protein, associates with fibronectin in cultured cells and in surgical samples of glioma. Taken together, our results provide the first evidence of the cellular and molecular mechanisms that may underlie the motility-promoting role of brevican in primary brain tumors. In addition, these results underscore the important functional implications of brevican processing in glioma progression.     

Hyaluronan-based extracellular matrix under conditions of homeostatic plasticity

Neuronal networks are balanced by mechanisms of homeostatic plasticity, which adjusts synaptic strength via molecular and morphological changes in the pre- and post-synapse. Here, we wondered whether the hyaluronic acid-based extracellular matrix (ECM) of the brain is involved in mechanisms of homeostatic plasticity. We hypothesized that the ECM, being rich in chondroitin sulfate proteoglycans such as brevican, which are suggested to stabilize synapses by their inhibitory effect on structural plasticity, must be remodelled to allow for structural and molecular changes during conditions of homeostatic plasticity. We found a high abundance of cleaved brevican fragments throughout the hippocampus and cortex and in neuronal cultures, with the strongest labelling in perineuronal nets on parvalbumin-positive interneurons. Using an antibody specific for a brevican fragment cleaved by the matrix metalloprotease ADAMTS4, we identified the enzyme as the main brevican-processing protease. Interestingly, we found ADAMTS4 largely associated with synapses. After inducing homeostatic plasticity in neuronal cell cultures by prolonged network inactivation, we found increased brevican processing at inhibitory as well as excitatory synapses, which is in line with the ADAMTS4 subcellular localization. Thus, the ECM is remodelled in conditions of homeostatic plasticity, which may liberate synapses to allow for a higher degree of structural plasticity.     

Brevican is degraded by matrix metalloproteinases and aggrecanase-1 (ADAMTS4) at different sites

Brevican is a member of the lectican family of chondroitin sulfate proteoglycans that is predominantly expressed in the central nervous system. The susceptibility of brevican to digestion by matrix metalloproteinases (MMP-1, -2, -3, -7, -8, -9, -10, and -13 and membrane type 1 and 3 MMPs) and aggrecanase-1 (ADAMTS4) was examined. MMP-1, -2, -3, -7, -8, -10, and -13 degraded brevican into a few fragments with similar molecular masses, whereas the degradation products of aggrecanase-1 had apparently different sizes. NH(2)-terminal sequence analyses of the digestion fragments revealed that cleavages of the brevican core protein by these metalloproteinases occurred commonly within the central non-homologous domain. MMP-1, -2, -3, -7, -8, -10, and -13 preferentially attacked the Ala(360)-Phe(361) bond, whereas aggrecanase-1 cleaved the Glu(395)-Ser(396) bond, which are similar to the cleavage sites observed with cartilage proteoglycan (aggrecan) for the MMPs and aggrecanase-1, respectively. These data demonstrate that MMP-1, -2, -3, -7, -8, -10, and -13 and aggrecanase-1 digest brevican in a similar pattern to aggrecan and suggest that they may be responsible for the physiological turnover and pathological degradation of brevican.     

Cerebral protein kinase C and its mRNA level in apolipoprotein E-deficient mice

It is known that protein kinase C (PKC) activity may be one of the fundamental cellular changes associated with memory function. Apolipoprotein E (apoE) deficiency causes cholinergic deficits and memory impairment. ApoE-deficient mouse has been employed as a serviceable model for studying the relation between apoE and the memory deficit induced by cholinergic impairment. Brain-fatty acid binding protein (b-FABP) might be functional during development of the nervous system. Peroxisome proliferator-activated receptor (PPAR) is involved in the early change in lipid metabolism. We investigated the alterations not only in cerebral PKC activity, but also in the gene expressions of PKC-beta, brain-FABP and PPAR-alpha in apoE-deficient mice. The results showed that there was a lower cerebral membrane-bound PKC activity in the apoE-deficient mice than in its wild type strain (C57BL/6). But there were no significant differences in cytosolic PKC activity. PKC-beta, b-FABP and PPAR-alpha mRNA expressions in cerebrum were lowered in apoE-deficient mice. These findings may be involved in the dysfunction of the brain neurotransmission system in apoE-deficient mouse. Alternatively, these results also suggest that cerebral apoE plays an important role in brain PKC activation by maintaining an appropriate expression of b-FABP and PPAR-alpha mRNAs.     

Phosphorylation of the AMPA receptor GluR1 subunit is required for synaptic plasticity and retention of spatial memory

Plasticity of the nervous system is dependent on mechanisms that regulate the strength of synaptic transmission. Excitatory synapses in the brain undergo long-term potentiation (LTP) and long-term depression (LTD), cellular models of learning and memory. Protein phosphorylation is required for the induction of many forms of synaptic plasticity, including LTP and LTD. However, the critical kinase substrates that mediate plasticity have not been identified. We previously reported that phosphorylation of the GluR1 subunit of AMPA receptors, which mediate rapid excitatory transmission in the brain, is modulated during LTP and LTD. To test if GluR1 phosphorylation is necessary for plasticity and learning and memory, we generated mice with knockin mutations in the GluR1 phosphorylation sites. The phosphomutant mice show deficits in LTD and LTP and have memory defects in spatial learning tasks. These results demonstrate that phosphorylation of GluR1 is critical for LTD and LTP expression and the retention of memories.     

Disruption of MET Receptor Tyrosine Kinase,an Autism Risk Factor,Impairs Developmental Synaptic Plasticity in the Hippocampus

As more genes conferring risks to neurodevelopmental disorders are identified, translating these genetic risk factors into biological mechanisms that impact the trajectory of the developing brain is a critical next step. Here, we report that disrupted signaling mediated MET receptor tyrosine kinase (RTK), an established risk factor for autism spectrum disorders, in the developing hippocampus glutamatergic circuit leads to profound deficits in neural development, synaptic transmission, and plasticity. In cultured hippocampus slices prepared from neonatal mice, pharmacological inhibition of MET kinase activity suppresses dendritic arborization and disrupts normal dendritic spine development. In addition, single‐neuron knockdown (RNAi) or overexpression of Met in the developing hippocampal CA1 neurons leads to alterations, opposite in nature, in basal synaptic transmission and long‐term plasticity. In forebrain‐specific Met conditional knockout mice (Met^fx/fx;emx1^cre), an enhanced long‐term potentiation (LTP) and long‐term depression (LTD) were observed at early developmental stages (P12–14) at the Schaffer collateral to CA1 synapses compared with wild‐type littermates. In contrast, LTP and LTD were markedly reduced at young adult stage (P56–70) during which wild‐type mice show robust LTP and LTD. The altered trajectory of synaptic plasticity revealed by this study indicate that temporally regulated MET signaling as an intrinsic, cell autonomous, and pleiotropic mechanism not only critical for neuronal growth and functional maturation, but also for the timing of synaptic plasticity during forebrain glutamatergic circuits development.