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Perineuronal net (PNN) is a highly structured portion of the CNS extracellular matrix (ECM) regulating synaptic plasticity and a range of pathologic conditions including posttraumatic regeneration and epilepsy. Here we studied Wisteria floribunda agglutinin-stained histological sections to quantify the PNN size and enrichment of chondroitin sulfates in mouse brain and spinal cord. Somatosensory cortex sections were examined during the period of PNN establishment at postnatal days 14, 21 and 28. The single cell PNN size and the chondroitin sulfate intensity were quantified for all cortex layers and specifically for the cortical layer IV which has the highest density of PNN-positive neurons. We demonstrate that the chondroitin sulfate proteoglycan staining intensity is increased between P14 and P28 while the PNN size remains unchanged. We then addressed posttraumatic changes of the PNN expression in laminae 6 and 7 of cervical spinal cord following hemisection injury. We demonstrate increase of the chondroitin sulfate content at 1.6–1.8 mm rostrally from the injury site and increase of the density of PNN-bearing cells at 0.4–1.2 mm caudally from the injury site. We further demonstrate decrease of the single cell PNN area at 0.2 mm caudally from the injury site suggesting that the PNN ECM takes part in the posttraumatic tissue rearrangement in the spinal cord. Our results demonstrate new insights on the PNN structure dynamics in the developing and posttraumatic CNS.

The High Mobility Group Box 1 (HMGB1) is the most abundant nuclear nonhistone protein that is involved in transcription regulation. In addition, HMGB1 has previously been found as an extracellularly acting protein enhancing neurite outgrowth in cultured neurons. Although HMGB1 is widely expressed in the developing central nervous system of vertebrates and invertebrates, its function in the developing mouse brain is poorly understood. Here, we have analyzed developmental defects of the HMGB1 null mouse forebrain, and further examined our findings in ex vivo brain cell cultures. We find that HMGB1 is required for the proliferation and differentiation of neuronal stem cells/progenitor cells. Enhanced apoptosis is also found in the neuronal cells lacking HMGB1. Moreover, HMGB1 depletion disrupts Wnt/&beta, catenin signaling and the expression of transcription factors in the developing cortex, including Foxg1, Tbr2, Emx2, and Lhx6. Finally, HMGB1 null mice display aberrant expression of CXCL12/CXCR4 and reduced RAGE signaling. In conclusion, HMGB1 plays a critical role in mammalian neurogenesis and brain development.

A striking result from epidemiological studies show a correlation between low alcohol intake and lower incidence for ischemic stroke and severity of derived brain injury. Although reduced apoptosis and inflammation has been suggested to be involved, little is known about the mechanism mediating this effect in vivo. Increase in intracellular chloride concentration and derived depolarizing GABAAR-mediated transmission are common consequences following various brain injuries and are caused by the abnormal expression levels of the chloride cotransporters NKCC1 and KCC2. Downstream pro-apoptotic signaling through p75NTR may link GABAA depolarization with post-injury neuronal apoptosis. Here, we show that changes in GABAergic signaling, Cl− homeostasis, and expression of chloride cotransporters in the post-traumatic mouse brain can be significantly reduced by administration of 3% ethanol to the drinking water. Ethanol-induced upregulation of KCC2 has a positive impact on neuronal survival, preserving a large part of the cortical peri-infarct zone, as well as preventing the massive post-ischemic upregulation of the pro-apoptotic protein p75NTR. Importantly, intracortical multisite in vivo recordings showed that ethanol treatment could significantly ameliorate stroke-induced reduction in cortical activity. This surprising finding discloses a pathway triggered by low concentration of ethanol as a novel therapeutically relevant target.

The immune function of AMIGO2 is currently unknown. Here, we revealed novel roles of AMIGO2 in modulating T-cell functions and EAE using Amigo2-knockout (AMG2KO) mice. Amigo2 was abundantly expressed by murine T helper (Th) cells. Its deficiency impaired transplanted T-cell infiltration into the secondary lymphoid organs and dampened Th-cell activation, but promoted splenic Th-cell proliferation and abundancy therein. AMG2KO Th cells had respectively elevated T-bet in Th1- and GATA-3 in Th2-lineage during early Th-cell differentiation, accompanied with increased IFN-γ and IL-10 but decreased IL-17A production. AMG2KO mice exhibited ameliorated EAE, dampened spinal T-cell accumulation, decreased serum IL-17A levels and enhanced splenic IL-10 production. Adoptive transfer of encephalitogenic AMG2KO T cells induced milder EAE and dampened spinal Th-cell accumulation and Tnf expression. Mechanistically, Amigo2-overexpression in 293T cells dampened NF-kB transcriptional activity, while Amigo2-deficiency enhanced Akt but suppressed GSK-3β phosphorylation and promoted nuclear translocations of NF-kB and NFAT1 in Th-cells. Collectively, our data demonstrate that AMIGO2 is important in regulating T-cell functions and EAE, and may be harnessed as a potential therapeutic target for multiple sclerosis.

Perineuronal nets (PNN) ensheath GABAergic and glutamatergic synapses on neuronal cell surface in the central nervous system (CNS), have neuroprotective effect in animal models of Alzheimer disease and regulate synaptic plasticity during development and regeneration. Crucial insights were obtained recently concerning molecular composition and physiological importance of PNN but the microstructure of the network remains largely unstudied. Here we used histochemistry, fluorescent microscopy and quantitative image analysis to study the PNN structure in adult mouse and rat neurons from layers IV and VI of the somatosensory cortex. Vast majority of meshes have quadrangle, pentagon or hexagon shape with mean mesh area of 1.29µm(2) in mouse and 1.44µm(2) in rat neurons. We demonstrate two distinct patterns of chondroitin sulfate distribution within a single mesh - with uniform (nonpolar) and node-enriched (polar) distribution of the Wisteria floribunda agglutinin-positive signal. Vertices of the node-enriched pattern match better with local maxima of chondroitin sulfate density as compared to the uniform pattern. PNN is organized into clusters of meshes with distinct morphologies on the neuronal cell surface. Our findings suggest the role for the PNN microstructure in the synaptic transduction and plasticity.

Background: Heparin and heparin-related sulphated carbohydrates inhibit ligand binding of the receptor for advanced glycation end products (RAGE). Here, we have studied the ability of heparin to inhibit homophilic interactions of RAGE in living cells and studied how heparin related structures interfere with RAGE&ndash, ligand interactions. Methods: Homophilic interactions of RAGE were studied with bead aggregation and living cell protein-fragment complementation assays. Ligand binding was analyzed with microwell binding and chromatographic assays. Cell surface advanced glycation end product binding to RAGE was studied using PC3 cell adhesion assay. Results: Homophilic binding of RAGE was mediated by V1- and modulated by C2-domain in bead aggregation assay. Dimerisation of RAGE on the living cell surface was inhibited by heparin. Sulphated K5 carbohydrate fragments inhibited RAGE binding to amyloid &beta, peptide and HMGB1. The inhibition was dependent on the level of sulfation and the length of the carbohydrate backbone. &alpha, d-Glucopyranosiduronic acid (glycyrrhizin) inhibited RAGE binding to advanced glycation end products in PC3 cell adhesion and protein binding assays. Further, glycyrrhizin inhibited HMGB1 and HMGB1 A-box binding to heparin. Conclusions: Our results show that K5 polysaccharides and glycyrrhizin are promising candidates for RAGE targeting drug development.

Promoting adult hippocampal neurogenesis is expected to induce neuroplastic changes that improve mood and alleviate anxiety. However, the underlying mechanisms remain largely unknown and the hypothesis itself is controversial. Here we show that mice lacking Jnk1, or c-Jun N-terminal kinase (JNK) inhibitor-treated mice, display increased neurogenesis in adult hippocampus characterized by enhanced cell proliferation and survival, and increased maturation in the ventral region. Correspondingly, anxiety behaviour is reduced in a battery of tests, except when neurogenesis is prevented by AraC treatment. Using engineered retroviruses, we show that exclusive inhibition of JNK in adult-born granule cells alleviates anxiety and reduces depressive-like behaviour. These data validate the neurogenesis hypothesis of anxiety. Moreover, they establish a causal role for JNK in the hippocampal neurogenic niche and anxiety behaviour, and advocate targeting of JNK as an avenue for novel therapies against affective disorders.

International audience; Chondroitin sulfate (CS) glycosaminoglycans inhibit regeneration in the adult central nervous system (CNS). We report here that HB-GAM (heparin-binding growth-associated molecule; also known as pleiotrophin), a CS-binding protein expressed at high levels in the developing CNS, reverses the role of the CS chains in neurite growth of CNS neurons in vitro from inhibition to activation. The CS-bound HB-GAM promotes neurite growth through binding to the cell surface proteoglycan glypican-2; furthermore, HB-GAM abrogates the CS ligand binding to the inhibitory receptor PTPσ (protein tyrosine phosphatase sigma). Our in vivo studies using two-photon imaging of CNS injuries support the in vitro studies and show that HB-GAM increases dendrite regeneration in the adult cerebral cortex and axonal regeneration in the adult spinal cord. Our findings may enable the development of novel therapies for CNS injuries. Chondroitinase ABC (ChABC) treatment has been extensively used to demonstrate that the CS side chains of CSPGs are potent inhibitors of regeneration and plasticity in the extracellular matrix (ECM) of brain and spinal cord 1–7. We have sought for less invasive ways to enhance CNS regeneration and plasticity, which might be useful for the development of therapies for CNS traumas. To this end, we considered endogenously occurring molecules that might modulate functions of the CNS matrix in the juvenile brain which displays high plasticity in comparison to the adult brain. HB-GAM/pleiotrophin was initially isolated as a heparin-binding neurite outgrowth-promoting factor for central neurons 8,9. Its expression peaks during the first 3–4 weeks of postnatal development in rat brain 10 corresponding to heightened plasticity of the juvenile brain 11. The expression level at this stage is very high, up to 10–15 μ g/g of wet tissue weight 8 , which might be sufficient to modulate matrix structures 12 that typically inhibit neural plasticity and regeneration. HB-GAM is secreted from neurons and glial cells upon cleav-age of a classic-type secretion signal 10 , binds CS side chains of CSPGs with nanomolar K d values 12–14 , and lines nearly all fiber tracts of the early postnatal rat brain 15. Furthermore, the growth factor Midkine, which displays homology with HB-GAM, was recently reported to partially overcome CSPG inhibition of neurite extension 16. Taken together, these studies suggest HB-GAM as a candidate molecule to modify interactions of CNS neu-rons with inhibitory ECM structures such as CSPGs. However, the possibility that HB-GAM could overcome or even reverse the inhibitory effects of the ECM on growth and regeneration of neurites has not previously been explored. Results HB-GAM promotes neurite outgrowth on CSPG substrates. We first studied the effect of HB-GAM on neurite growth from primary CNS neurons plated on aggrecan, a major CSPG in the CNS 17. As expected, the aggrecan matrix effectively prevented neurite outgrowth from the CNS neurons (Fig. 1a–c). Coating of HB-GAM together with aggrecan overcame the inhibitory effect (Fig. 1c,d). Moreover, delayed addition of HB-GAM to culture media promoted neurite extension from the primary CNS neurons already inhibited by aggrecan (Supplementary Fig. S1).

The current dogma in neural regeneration research implies that chondroitin sulfate proteoglycans (CSPGs) inhibit plasticity and regeneration in the adult central nervous system (CNS). We argue that the role of the CSPGs can be reversed from inhibition to activation by developmentally expressed CSPG-binding factors. Heparin-binding growth-associated molecule (HB-GAM; also designated as pleiotrophin) has been studied as a candidate molecule that might modulate the role of CSPG matrices in plasticity and regeneration. Studies in vitro show that in the presence of soluble HB-GAM chondroitin sulfate (CS) chains of CSPGs display an enhancing effect on neurite outgrowth. Based on the in vitro studies, we suggest a model according to which the HB-GAM/CS complex binds to the neuron surface receptor glypican-2, which induces neurite growth. Furthermore, HB-GAM masks the CS binding sites of the neurite outgrowth inhibiting receptor protein tyrosine phosphatase sigma (PTPσ), which may contribute to the HB-GAM-induced regenerative effect. In vivo studies using two-photon imaging after local HB-GAM injection into prick-injury of the cerebral cortex reveal regeneration of dendrites that has not been previously demonstrated after injuries of the mammalian nervous system. In the spinal cord, two-photon imaging displays HB-GAM-induced axonal regeneration. Studies on the HB-GAM/CS mechanism in vitro and in vivo are expected to pave the way for drug development for injuries of brain and spinal cord.

Background: Social deficit is one of the core symptoms of neuropsychiatric diseases, in which immune genes play an important role. Although a few immune genes have been shown to regulate social and emotional behaviors, how immune gene network(s) may jointly regulate sociability has not been investigated so far. Methods: To decipher the potential immune-mediated mechanisms underlying social behavior, we first studied the brain microarray data of eight inbred mouse strains with known variations in social behavior and retrieved the differentially expressed immune genes. We then made a protein-protein interaction analysis of them to find the major networks and explored the potential association of these genes with the behavior and brain morphology in the mouse phenome database. To validate the expression and function of the candidate immune genes, we selected the C57BL/6 J and DBA/2 J strains among the eight inbred strains, compared their social behaviors in resident-intruder and 3-chambered social tests and the mRNA levels of these genes, and analyzed the correlations of these genes with the social behaviors. Results: A group of immune genes were differentially expressed in the brains of these mouse strains. The representative C57BL/6 J and DBA/2 J strains displayed significant differences in social behaviors, DBA/2 J mice being less active in social dominance and social interaction than C57BL/6 J mice. The mRNA levels of H2-d1 in the prefrontal cortex, hippocampus, and hypothalamus and C1qb in the hippocampus of the DBA/2 J strain were significantly down-regulated as compared to those in the C57BL/6 J strain. In contrast, Polr3b in the hippocampus and Tnfsf13b in the prefrontal cortex of the DBA/2 J strain were up-regulated. Furthermore, C1qb, Cx3cl1, H2-d1, H2-k1, Polr3b, and Tnfsf13b were predicted to be associated with various behavioral and brain morphological features across the eight inbred strains. Importantly, the C1qb mRNA level was confirmed to be significantly correlated with the sociability in DBA/2 J but not in C57BL/6 J mice. Conclusions: Our study provided evidence on the association of immune gene network(s) with the brain development and behavior in animals and revealed neurobiological functions of novel brain immune genes that may contribute to social deficiency in animal models of neuropsychiatric disorders.

High mobility group box 1 (HMGB1, also called amphoterin) facilitates neurite outgrowth in early development, yet can exacerbate pathology and inhibit regeneration by inducing adverse neuroinflammation when released from dying cells, suggesting that HMGB1 plays a critical, yet undefined role in neuroregeneration. We explored whether HMGB1 contributes to recovery after complete spinal cord transection in adult zebrafish. Quantitative PCR and in situ hybridization revealed that HMGB1 mRNA levels decreased between 12 h to 11 days after spinal cord injury (SCI), then returned to basal levels by 21 days. Western blot and immunohistological analyses indicated that the time course of HMGB1 protein expression after SCI parallels that of mRNA. Immunofluorescence staining revealed that HMGB1 translocates from nuclei into the cytoplasm of spinal motoneurons at 4 and 12 h (acute stage) following SCI, then accumulates in the nuclei of motoneurons during the ensuing chronic stage (after 6 days following SCI). Immunohistology of transgenic zebrafish, expressing green fluorescent protein in blood vessels, showed enhanced HMGB1 expression in blood vessels in the vicinity of motoneurons. Application of anti-sense HMGB1 morpholinos inhibited locomotor recovery by 34 % and decreased axonal regeneration by 34 % compared to fish treated with a control morpholino. The present study shows that HMGB1 expression increases in both endothelial cells and motoneurons, suggesting that HMGB1 promotes recovery from SCI not only through enhancing neuroregeneration, but also by increasing angiogenesis. The inflammatory effects of HMGB1 are minimized through the decrease in HMGB1 expression during the acute stage.

Syndecan-3 (N-syndecan) is a transmembrane heparan sulfate proteoglycan abundantly expressed in developing brain. In addition to acting as a coreceptor, syndecan-3 acts as a signaling receptor upon binding of its ligand HB-GAM (heparin-binding growth-associated molecule; pleiotrophin), which activates the cortactin-src kinase signaling pathway. This leads to rapid neurite extension in neuronal cells, which makes syndecan-3 as an interesting transmembrane receptor in neuronal development and regeneration. However, little is known about the signaling mechanism of syndecan-3. Here we have analyzed formation of ligand-N-syndecan signaling complexes at the cell surface using fluorescence resonance energy transfer (FRET) and bioluminescence resonance energy transfer (BRET). We show that ligand binding leads to dimerization of syndecan-3 at the cell surface. The dimerized syndecan-3 colocalizes with actin in the filopodia of cells. Several amino acid residues (K383, G392 and G396) in the transmembrane domain are shown to be important for the ligand-induced dimerization, whereas the cytosolic domain is not required for the dimerization.

Prostatic acid phosphatase (PAP), the first diagnostic marker and present therapeutic target for prostate cancer, modulates nociception at the dorsal root ganglia (DRG), but its function in the central nervous system has remained unknown. We studied expression and function of TMPAP (the transmembrane isoform of PAP) in the brain by utilizing mice deficient in TMPAP (PAP-/- mice). Here we report that TMPAP is expressed in a subpopulation of cerebral GABAergic neurons, and mice deficient in TMPAP show multiple behavioral and neurochemical features linked to hyperdopaminergic dysregulation and altered GABAergic transmission. In addition to increased anxiety, disturbed prepulse inhibition, increased synthesis of striatal dopamine, and augmented response to amphetamine, PAP-deficient mice have enlarged lateral ventricles, reduced diazepam-induced loss of righting reflex, and increased GABAergic tone in the hippocampus. TMPAP in the mouse brain is localized presynaptically, and colocalized with SNARE-associated protein snapin, a protein involved in synaptic vesicle docking and fusion, and PAP-deficient mice display altered subcellular distribution of snapin. We have previously shown TMPAP to reside in prostatic exosomes and we propose that TMPAP is involved in the control of GABAergic tone in the brain also through exocytosis, and that PAP deficiency produces a distinct neurological phenotype.

Mitochondria play a variety of functional roles in cortical neurons, from metabolic support and neuroprotection to the release of cytokines that trigger apoptosis. In dendrites, mitochondrial structure is closely linked to their function, and fragmentation (fission) of the normally elongated mitochondria indicates loss of their function under pathological conditions, such as stroke and brain trauma. Usingin vivotwo-photon microscopy in mouse brain, we quantified mitochondrial fragmentation in a full spectrum of cortical injuries, ranging from severe to mild. Severe global ischemic injury was induced by bilateral common carotid artery occlusion, whereas severe focal stroke injury was induced by Rose Bengal photosensitization. The moderate and mild traumatic injury was inflicted by focal laser lesion and by mild photo-damage, respectively. Dendritic and mitochondrial structural changes were tracked longitudinally using transgenic mice expressing fluorescent proteins localized either in cytosol or in mitochondrial matrix. In response to severe injury, mitochondrial fragmentation developed in parallel with dendritic damage signified by dendritic beading. Reconstruction from serial section electron microscopy confirmed mitochondrial fragmentation. Unlike dendritic beading, fragmentation spread beyond the injury core in focal stroke and focal laser lesion models. In moderate and mild injury, mitochondrial fragmentation was reversible with full recovery of structural integrity after 1–2 weeks. The transient fragmentation observed in the mild photo-damage model was associated with changes in dendritic spine density without any signs of dendritic damage. Our findings indicate that alterations in neuronal mitochondria structure are very sensitive to the tissue damage and can be reversible in ischemic and traumatic injuries.SIGNIFICANCE STATEMENTDuring ischemic stroke or brain trauma, mitochondria can either protect neurons by supplying ATP and adsorbing excessive Ca2+, or kill neurons by releasing proapoptotic factors. Mitochondrial function is tightly linked to their morphology: healthy mitochondria are thin and long; dysfunctional mitochondria are thick (swollen) and short (fragmented). To date, fragmentation of mitochondria was studied either in dissociated cultured neurons or in brain slices, but not in the intact living brain. Using real-timein vivotwo-photon microscopy, we quantified mitochondrial fragmentation during acute pathological conditions that mimic severe, moderate, and mild brain injury. We demonstrated that alterations in neuronal mitochondria structural integrity can be reversible in traumatic and ischemic injuries, highlighting mitochondria as a potential target for therapeutic interventions.

Background The components of nucleosomes, which contain DNA and histones, are released into the circulation from damaged cells and can promote inflammation. We studied whether the on-admission levels of circulating nucleosomes predict the development of severe acute pancreatitis (AP), in particular among the patients who present without clinical signs of organ dysfunction. Methods This is a prospective study of 74 AP patients admitted to Helsinki University Hospital from 2003 to 2007. Twenty-three patients had mild, 27 moderately severe, and 24 severe AP as defined by the revised Atlanta criteria. 14/24 severe AP patients had no sign of organ dysfunction on admission (modified marshall score

Kv2.1 is a potassium channel α-subunit abundantly expressed throughout the brain. It is a main component of delayed rectifier current (I(K)) in several neuronal types and a regulator of excitability during high-frequency firing. Here we identify AMIGO (amphoterin-induced gene and ORF), a neuronal adhesion protein with leucine-rich repeat and immunoglobin domains, as an integral part of the Kv2.1 channel complex. AMIGO shows extensive spatial and temporal colocalization and association with Kv2.1 in the mouse brain. The colocalization of AMIGO and Kv2.1 is retained even during stimulus-induced changes in Kv2.1 localization. AMIGO increases Kv2.1 conductance in a voltage-dependent manner in HEK cells. Accordingly, inhibition of endogenous AMIGO suppresses neuronal I(K) at negative membrane voltages. In conclusion, our data indicate AMIGO as a function-modulating auxiliary subunit for Kv2.1 and thus provide new insights into regulation of neuronal excitability.

AMIGO-1 is the parent member of a novel family of three cell surface leucine-rich repeat (LRR) proteins. Its expression is induced by the binding of HMGB1 ( h igh- m obility g roup b ox 1 protein) to RAGE ( r eceptor for a dvanced g lycation e nd products) on neurons. Binding of HMGB1 to RAGE is known to have a direct effect on cellular growth regulation and mobility, and AMIGO-1 directly supports growth of neuronal processes and fasciculation of neurites. In addition, the second member of the AMIGO-family, AMIGO-2, has been implicated in adhesion of tumor cells in adenocarcinoma and survival of neurons. We have determined the crystal structure of AMIGO-1 at 2.0 A resolution, which reveals a typical cell surface LRR domain arrangement with N- and C-terminal capping domains with disulfide bridges, followed by a C2-type Ig domain. AMIGO-1 is a dimer, with the LRR regions forming the dimer interface, and sequence conservation analysis and static light-scattering measurements suggest that all three AMIGO family proteins form similar dimers. Based on the AMIGO-1 structure, we have also modeled AMIGO-2 and present small-angle X-ray scattering data on AMIGO-2 and AMIGO-3. Our mutagenesis studies show that AMIGO-1 dimerization is necessary for proper cell surface expression and thus probably for proper or stable folding in the endoplastic reticulum and for the function of the protein. Based on the data presented earlier, we also suggest that dimerization through the LRR–LRR interface is likely to be involved in cell–cell adhesion by AMIGO-1, while extensive glycosylation may have a role.

Syndecan-3 may act alone or as a coreceptor with RET to promote cell spreading, neurite outgrowth, and migration of cortical neurons by GNDF, NRTN, and ARTN., Glial cell line–derived neurotrophic factor (GDNF) family ligands (GFLs) are potent survival factors for dopaminergic neurons and motoneurons with therapeutic potential for Parkinson’s disease. Soluble GFLs bind to a ligand-specific glycosylphosphatidylinositol-anchored coreceptor (GDNF family receptor α) and signal through the receptor tyrosine kinase RET. In this paper, we show that all immobilized matrix-bound GFLs, except persephin, use a fundamentally different receptor. They interact with syndecan-3, a transmembrane heparan sulfate (HS) proteoglycan, by binding to its HS chains with high affinity. GFL–syndecan-3 interaction mediates both cell spreading and neurite outgrowth with the involvement of Src kinase activation. GDNF promotes migration of cortical neurons in a syndecan-3–dependent manner, and in agreement, mice lacking syndecan-3 or GDNF have a reduced number of cortical γ-aminobutyric acid–releasing neurons, suggesting a central role for the two molecules in cortical development. Collectively, syndecan-3 may directly transduce GFL signals or serve as a coreceptor, presenting GFLs to the signaling receptor RET.

Catechol-O-methyltransferase (COMT) has soluble (S-COMT) and membrane bound (MB-COMT) isoforms. Our aims were to assess the behavioral phenotype of S-COMT mutant mice and to clarify the role of MB-COMT in dopamine metabolism in different brain areas.Behavioral phenotype of the S-COMT mutant mice was assessed using a test battery designed to describe anxiety phenotype, spontaneous locomotor activity, sensorymotor gating, social behavior, and pain sensitivity. Microdialysis was used to explore the effect of S-COMT deficiency on extracellular dopamine under an L: -dopa load (carbidopa /L: -dopa 30/10 mg/kg i.p.).In behavioral tests, mature adult S-COMT mutants that only possessed MB-COMT exhibited enhanced acoustic startle without alterations in sensorimotor gating. They also showed barbering of vibrissae and nonaggressive social dominance, suggesting a change in their social interactions. In addition, S-COMT deficiency slightly and sex-dependently affected spinal pain reflex and the effect of morphine on hot-plate latency. In microdialysis studies under L: -dopa load, S-COMT mutants of both sexes had higher accumbal dopamine levels, but male S-COMT mutant mice showed paradoxically lower prefrontal cortical dopamine concentrations than wild-type animals. S-COMT deficiency induced the accumulation of 3,4-dihydroxyphenylacetic acid in all brain areas, which was accentuated after L: -dopa loading. The lack of S-COMT decreased extracellular homovanillic acid levels. However, after L: -dopa loading, homovanillic acid concentrations in the prefrontal cortex of S-COMT mutants were similar to those of wild-type mice.A lack of S-COMT has a notable, albeit small, brain-area and sex-dependent effect on the O-methylation of dopamine and 3,4-dihydroxyphenylacetic acid in the mouse brain. It also induces subtle changes in mouse social interaction behaviors and nociception.

Extracellularly occurring HMGB1, either released during cell injury or actively secreted from cells, has profound effects on behaviour of a wide variety of cell types. Extracellular HMGB1 regulates migratory responses of many cell types, including neuron and growth cone migration, invasive migration of tumour cells, and migration of endothelial and immune cells. RAGE (Receptor for Advanced Glycation End Products) plays a key role as a cell surface receptor in most, if not all HMGB1-dependent migration mechanisms. HMGB1 binds to the distal immunoglobulin-like domain of RAGE, activating a signalling pathway that ends up in modulation of the cytoskeleton for regulation of cell motility. In addition to RAGE, proteoglycans and sulfated carbohydrate epitopes of glycolipids and glycoproteins may play a role as cell surface binding sites of HMGB1, affecting migratory behaviour of cells. In addition to physiological and pathophysiological cell migration control, HMGB1 has been widely studied as a molecule linking tissue injury to inflammatory mechanisms. HMGB1 by itself has little if any proinflammatory activity but it appears to activate innate immunity mechanisms as a complex with DNA, lipids and/or proinflammatory cytokines. The inflammation-inducing activity of HMGB1/DNA complexes may depend on both RAGE and Toll-like receptors of the immune cell surface. In addition to the receptors activating innate immunity, receptors downregulating inflammation upon HMGB1 release have been recently found, and include thrombomodulin and the CD-24/Siglec pathway.

OBJECTIVES: To study in patients with acute pancreatitis (AP) the plasma soluble form of the receptor for advanced glycation end products (sRAGE) and high-mobility group box chromosomal protein 1 (HMGB1) levels, followed-up for 12 days after hospitalization, in relation to the occurrence of organ failure and mortality. METHODS: Thirty-eight patients with severe AP and organ failure (grade 2). A control group (127 patients) consisted of 38 patients with severe AP without organ failure (grade 1) and 89 patients with mild AP (grade 0). Plasma samples for determination of HMGB1 and sRAGE levels were collected on admission and on days 1 and 2, days 3 and 4, and days 7 and 12 after admission. RESULTS: The median of the highest sRAGE levels was higher in grade 2 patients (472 pg/mL; interquartile range [IQR], 259-912) than in grade 0 plus grade 1 patients (349 pg/mL; IQR, 209-544; P = 0.024). Among the patients with detectable HMGB1, the median of the highest HMGB1 levels was 117 ng/mL (IQR, 56-212; n = 24) in grade 2 patients and 87 ng/mL (IQR, 54-161; n = 62) in grade 0 plus grade 1 patients (P = 0.310). CONCLUSIONS: We demonstrate that sRAGE level, but not HMGB1 level, is significantly higher in AP patients who develop organ failure than in AP patients without organ failure who recover.

Glial cell line-derived neurotrophic factor (GDNF) protects and repairs dopamine neurons. It binds to GDNF family receptor alpha1 (GFRalpha1) and activates receptor tyrosine kinase. Heparan sulphate proteoglycans (HSPGs) also participate in the signalling of GDNF, though binding to HS may hinder the diffusion of infused GDNF. We assessed the importance of heparin-binding determinants in the neuroprotective effects of GDNF in the 6-OHDA rat model of Parkinson's disease. We utilized a truncated, non-heparin-binding Delta38N-GDNF or combined wtGDNF with heparin-binding growth-associated molecule (HB-GAM, pleiotrophin). Tissue diffusion of wtGDNF+/-HB-GAM and Delta38N-GDNF was also compared. A protective effect against ipsilateral d-amphetamine-induced turning was seen with 10 microg wtGDNF, 17 microg HB-GAM+10 microg wtGDNF or 10 microg Delta38N-GDNF at 8 weeks post lesion. This effect was most pronounced with wtGDNF alone. HB-GAM (17 or 50 microg) also reduced rotational behaviour, but did not protect dopaminergic cells. Otherwise, the survival of TH-positive cells in the substantia nigra correlated with the behavioural data. Although Delta38N-GDNF was more widely distributed than wtGDNF (irrespective of its origin), stable in a brain extract, and potent in mitogen-activated kinase assay, it was inferior in vivo. The results imply that GDNF binding to HSs is needed for the optimum neuroprotective effect.

article i nfo HB-GAM (also known as pleiotrophin) is a cell matrix-associated protein that is highly expressed in bone. It affects osteoblast function, and might therefore play a role in bone development and remodeling. We aimed to investigate the role of HB-GAM in bone in vivo and in vitro. The bones of HB-GAM deficient mice with an inbred mouse background were studied by histological, histomorphometrical, radiological, biomechanical and μ-CT analyses and the effect of immobilization was evaluated. HB-GAM localization in vivo was studied. MLO-Y4 osteocytes were subjected to fluid shear stress in vitro, and gene and protein expression were studied by subtractive hybridization, quantitative PCR and Western blot. Human osteoclasts were cultured in the presence of rhHB-GAM and their formation and resorption activities were assayed. In agreement with previous reports, the skeletal structure of the HB-GAM knockout mice developed normally. However, a growth retardation of the weight-bearing bones was observed by 2 months of age, suggesting a link to physical activity. Adult HB-GAM deficient mice were characterized by low bone formation and osteopenia, as well as resistance to immobilization-dependent bone remodeling. HB-GAM was localized around osteocytes and their processes in vivo and furthermore, osteocytic HB-GAM expression was upregulated by mechanical loading in vitro. HB-GAM did not affect on human osteoclast formation or resorption in vitro. Taken together, our results suggest that HB-GAM is an osteocyte-derived factor that could participate in mediating the osteogenic effects of mechanical loading on bone.

The central nervous system (CNS) has traditionally been considered to be immunologically privileged, but over the years there has been a re-evaluation of this dogma. To date, studies have tended to focus on the immune functions of glial cells, whereas the roles of neurons have been regarded as passive and their immune-regulatory properties have been less examined. However, recent findings indicate that CNS neurons actively participate in immune regulation by controlling their glial cell counterparts and infiltrated T cells. Here, we describe the immune-regulatory roles of CNS neurons by both contact-dependent and contact-independent mechanisms. In addition, we specifically deal with the immune functions of neuronal cell adhesion molecules, many of which are key modulators of neuronal synaptic formation and plasticity.

High mobility group box 1 protein (HMGB1), a cytokine actively secreted by phagocytes and passively released from necrotic cells, is an inflammatory mediator in experimental hepatic ischemia/reperfusion injury. We characterized its expression in human liver transplantation. In 20 patients, in addition to systemic samples, blood was drawn from portal and hepatic veins during and after reperfusion to assess changes within the graft. Plasma HMGB1, tumor necrosis factor alpha (TNF-alpha), and interleukin-6 (IL-6) levels were measured, and HMGB1 immunohistochemistry was performed on biopsies taken before and after reperfusion. Plasma HMGB1 was undetectable before reperfusion, and levels in systemic circulation peaked after graft reperfusion. At portal declamping, HMGB1 levels were substantially higher in the caval effluent [188 (80-371) ng/mL] than in portal venous blood [0 (0-3) ng/mL, P < 0.001]. HMGB1 release from the graft continued thereafter. HMGB1 levels were not related to TNF-alpha or IL-6 levels. HMGB1 expression was up-regulated in biopsies taken after reperfusion (P = 0.020), with intense hepatocyte and weak neutrophil staining. HMGB1 levels in hepatic venous blood correlated with graft steatosis (r = 0.497, P = 0.03) and peak postoperative alanine aminotransferase levels (r = 0.588, P = 0.008). Our results indicate that HMGB1 originates from the graft and is a marker of hepatocellular injury in human liver transplantation.

Intercellular adhesion molecules (ICAMs) bind to leukocyte β2 integrins, which, among other functions, provide costimulatory signals for T-cell activation. ICAM-5 (telencephalin) is expressed in the somadendritic region of neurons of the mammalian brain. The receptor for ICAM-5 is the integrin LFA-1, a major leukocyte integ-rin expressed in lymphocytes and microglia. In conditions of brain ischemia, epilepsy, and encephalitis, the soluble form of ICAM-5 (sICAM-5) has been detected in physiologic fluids. Here, we report that sICAM-5 attenuates the T-cell receptor-mediated activation of T cells as demonstrated by the decreased expression of the activation markers CD69, CD40L, and CD25 (IL-2R). This effect is most clearly seen in CD45ROLow (naive), and not in CD45ROHigh (memory) T cells, and is most effective early in priming, but not in the presence of strong costimulatory signals. Furthermore, sICAM-5 promotes the mRNA expression of the cytokines TGF-β1 and IFN-γ, but not TNF. The formation of sICAM-5 is promoted by activated T cells through the cleavage of ICAM-5 from neurons. This suggests that ICAM-5 is involved in immune privilege of the brain and acts as an anti-inflammatory agent.

HMGB1/Amphoterin is a ubiquitous, highly conserved DNA-binding protein that can be also released to the extracellular space by various cell types. Extracellular HMGB1 regulates migratory responses of several cell types through binding to RAGE that communicates with the cytoskeleton to regulate cell motility. HMGB1-induced cell signalling has been associated with mechanisms of several diseases, including cancer, sepsis, rheumatoid arthritis, stroke and atherosclerosis. This article reviews the evidence linking the functional roles of HMGB1 to RAGE signalling. Furthermore, we discuss the molecular and cellular mechanisms that may explain the roles of HMGB1/RAGE in diverse disease processes.

Matrix metalloproteinase (MMP)-2 and -9 are pivotal in remodeling many tissues. However, their functions and candidate substrates for brain development are poorly characterized. Intercellular adhesion molecule-5 (ICAM-5; Telencephalin) is a neuronal adhesion molecule that regulates dendritic elongation and spine maturation. We find that ICAM-5 is cleaved from hippocampal neurons when the cells are treated with N-methyl-d-aspartic acid (NMDA) or α-amino-3-hydroxy-5-methylisoxazole-propionic acid (AMPA). The cleavage is blocked by MMP-2 and -9 inhibitors and small interfering RNAs. Newborn MMP-2– and MMP-9–deficient mice brains contain more full-length ICAM-5 than wild-type mice. NMDA receptor activation disrupts the actin cytoskeletal association of ICAM-5, which promotes its cleavage. ICAM-5 is mainly located in dendritic filopodia and immature thin spines. MMP inhibitors block the NMDA-induced cleavage of ICAM-5 more efficiently in dendritic shafts than in thin spines. ICAM-5 deficiency causes retraction of thin spine heads in response to NMDA stimulation. Soluble ICAM-5 promotes elongation of dendritic filopodia from wild-type neurons, but not from ICAM-5–deficient neurons. Thus, MMPs are important for ICAM-5–mediated dendritic spine development.

HMGB4 is a new member in the family of HMGB proteins that has been characterized in sperm cells, but little is known about its functions in somatic cells. Here we show that HMGB4 and the highly similar rat Transition Protein 4 (HMGB4L1) are expressed in neuronal cells. Both proteins had slow mobility in nucleus of living NIH-3T3 cells. They interacted with histones and their differential expression in transformed cells of the nervous system altered the post-translational modification statuses of histones in vitro. Overexpression of HMGB4 in HEK 293T cells made cells more susceptible to cell death induced by topoisomerase inhibitors in an oncology drug screening array and altered variant composition of histone H3. HMGB4 regulated over 800 genes in HEK 293T cells with a p-value ≤0.013 (n = 3) in a microarray analysis and displayed strongest association with adhesion and histone H2A –processes. In neuronal and transformed cells HMGB4 regulated the expression of an oligodendrocyte marker gene PPP1R14a and other neuronal differentiation marker genes. In conclusion, our data suggests that HMGB4 is a factor that regulates chromatin and expression of neuronal differentiation markers.

High mobility group (HMG) proteins concentrate in the nucleus, interacting with chromatin. Amphoterin is an HMG protein (HMGB1) that has been shown to have extranuclear functions and can be secreted from some cell types. Exogenous amphoterin can increase neurite growth, suggesting that the secreted protein may have growth promoting activities in neurons. Consistent with this, we show that depletion of amphoterin mRNA from cultured adult rat DRG neurons attenuates neurite outgrowth, pointing to autocrine or paracrine mechanisms for its growth-promoting effects. The mRNA encoding amphoterin localizes to axonal processes and we showed recently that its 3′-UTR is sufficient for axonal localization of heterologous transcripts (Donnelly et al., 2013). Here, we show that amphoterin mRNA is transported constitutively into axons of adult DRG neurons. A preconditioning nerve injury increases the levels of amphoterin protein in axons without a corresponding increase in amphoterin mRNA in the axons. A 60 nucleotide region of the amphoterin mRNA 3′-UTR is necessary and sufficient for its localization into axons of cultured sensory neurons. Amphoterin mRNA 3′-UTR is also sufficient for axonal localization in distal axons of DRG neuronsin vivo. Overexpression of axonally targeted amphoterin mRNA increases axon outgrowth in cultured sensory neurons, but axon growth is not affected when the overexpressed mRNA is restricted to the cell body.

N-syndecan (syndecan-3) is a transmembrane proteoglycan that is abundantly expressed in the major axonal pathways and in the migratory routes of the developing brain. When ligated by heparin-binding (HB) growth-associated molecule (GAM; pleiotrophin), N-syndecan mediates cortactin–Src kinase-dependent neurite outgrowth. However, the functional role of N-syndecan in brain development remains unexplored. In this study, we show that N-syndecan deficiency perturbs the laminar structure of the cerebral cortex as a result of impaired radial migration. In addition, neural migration in the rostral migratory stream is impaired in the N-syndecan–null mice. We suggest that the migration defect depends on impaired HB-GAM–induced Src kinase activation and haptotactic migration. Furthermore, we show that N-syndecan interacts with EGF receptor (EGFR) at the plasma membrane and is required in EGFR-induced neuronal migration.

Thrombospondin-related anonymous protein, TRAP, has a critical role in the hepatocyte invasion step of Plasmodium sporozoites, the transmissible form of the parasite causing malaria. The extracellular domains of this sporozoite surface protein interact with hepatocyte surface receptors whereas its intracellular domain acts as a link to the sporozoite actomyosin motor system. Liver heparan sulfate proteoglycans have been identified as potential ligands for TRAP. Proteoglycan binding has been associated with the A- and TSR domains of TRAP. We present the solution NMR structure of the TSR domain of TRAP and a chemical shift mapping study of its heparin binding epitope. The domain has an elongated structure stabilized by an array of tryptophan and arginine residues as well as disulfide bonds. The fold is very similar to those of thrombospondin type-1 (TSP-1) and F-spondin TSRs. The heparin binding site of TRAP-TSR is located in the N-terminal half of the structure, the layered side chains forming an integral part of the site. The smallest heparin fragment capable of binding to TRAP-TSR is a tetrasaccharide.

F-spondin is a protein mainly associated with neuronal development. It attaches to the extracellular matrix and acts in the axon guidance of the developing nervous system. F-spondin consists of eight domains, six of which are TSR domains. The TSR domain family binds a wide range of targets. Here we present the NMR solution structures of TSR1 and TSR4. TSR domains have an unusual fold that is characterized by a long, nonglobular shape, consisting of two beta-strands and one irregular extended strand. Three disulfide bridges and stack of alternating tryptophan and arginine side-chains stabilize the structure. TSR1 and TSR4 structures are similar to each other and to the previously determined TSR domain X-ray structures from another protein, TSP, although TSR4 exhibits a mobile loop not seen in other structures.

Heparin-binding growth-associated molecule is a developmentally regulated extracellular matrix protein promoting neurite outgrowth, axonal guidance and synaptogenesis. In the hippocampus, heparin-binding growth-associated molecule is expressed in an activity-dependent manner, and has been shown to suppress long-term potentiation of glutamatergic synapses in the area CA1, but the mechanisms underlying this action are unknown. One of the mechanisms by which extracellular matrix proteins might modulate fast synaptic transmission is by altering GABAergic function. Therefore, we have studied the properties of GABAA receptor-mediated inhibition in hippocampus of mutant mice overexpressing heparin-binding growth-associated molecule (heparin-binding growth-associated molecule transgenics). Under control conditions the wild-type mice have much higher level of long-term potentiation than the transgenics. However, in the absence of the GABAA receptor-mediated-inhibition a similar level of long-term potentiation is seen in both strains. In field potential recordings blockade of GABAA receptors by picrotoxin resulted in more accentuated increase in the CA1 population spike in the transgenics than in the wild-type animals. Whole-cell patch-clamp recordings revealed that when compared with the wild-type animals the transgenic mice had higher frequency of spontaneous inhibitory postsynaptic currents in CA1 pyramidal neurons. However, the frequency of action potential-independent miniature inhibitory postsynaptic currents was similar in both strains. Further, the transgenics had reduced paired-pulse depression of inhibitory postsynaptic currents, which was insensitive to the blockade of GABAB receptors in contrast to wild-type mice. The results demonstrate that the mice overexpressing heparin-binding growth-associated molecule have accentuated hippocampal GABAA receptor-mediated inhibition, which in turn may explain the lowered predisposition of glutamatergic synapses to undergo plastic changes in these animals. Thus, our findings suggest a mechanism by which heparin-binding growth-associated molecule can regulate synaptic plasticity.

HB-GAM (heparin-binding growth-associated molecule, also designated as pleiotrophin) and midkine form a two-member family of extracellular matrix proteins that bind tightly to sulfated carbohydrate structures such as heparan sulfate. These proteins are used by developing neurons as extracellular cues in axonal growth and guidance. HB-GAM was recently reported to enhance differentiation of neural stem cells. Based on the solution structure of HB-GAM, we have recently shown that HB-GAM consists of two beta-sheet domains flanked by flexible lysine-rich N- and C-terminal tails with no apparent structure. These domains are homologous to thrombospondin type I repeats present in numerous extracellular proteins that interact with the cell surface. Our findings showed that the two beta-sheet domains fold independently. We showed that the domains (but not the lysine-rich tails) in HB-GAM are required and sufficient for interaction with hippocampal neurons. The individual domains bind heparan sulfate weakly and fail to produce significant biological effects in neurite outgrowth and long term potentiation assays. The amino acids in the linker region joining the two domains may be replaced with glycines with no effect on protein function. These results suggest a co-operative action of the two beta-sheet domains in the biologically relevant interaction with neuron surface heparan sulfate.

Hyperpolarizing fast inhibitory neurotransmission by gamma-aminobutyric acid and glycine requires an efficient chloride extrusion mechanism in postsynaptic neurons. A major effector of this task in adult animals is the potassium-chloride co-transporter KCC2 that is selectively and abundantly expressed postsynaptically in most CNS neurons. Yet, the role of KCC2 in adult brain at the systems level is poorly known. Here, we characterize the behaviour of mice doubly heterozygous for KCC2 null and hypomorphic alleles that retain 15-20% of normal KCC2 protein levels in the brain. These hypomorphic KCC2-deficient mice were viable and fertile but weighed 15-20% less than wild-type littermates at 2 weeks old and thereafter. The mice displayed increased anxiety-like behaviour in several tests including elevated plus-maze and were more susceptible to pentylenetetrazole-induced seizures. Moreover, the mice were impaired in water maze learning and showed reduced sensitivity to tactile and noxious thermal stimuli in von Frey hairs, hot plate and tail flick tests. In contrast, the mice showed normal spontaneous locomotor activity in open field and Y-maze tests, and intact motor coordination in rotarod and beam tests. The results suggest that requirements for KCC2-dependent fast hyperpolarizing inhibition may differ among various functional systems of the CNS. As shunting inhibition is expected to be intact in KCC2-deficient neurons, these mice may provide a useful tool to study the specific functions and relative importance of hyperpolarizing fast synaptic inhibition in adult CNS that may have implications for human neuropsychiatric disorders, such as epilepsy, pain and anxiety.