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The blood-brain barrier (BBB) is a formidable obstacle to the delivery of therapeutics to the brain. Antibodies that bind transferrin receptor (TfR), which is enriched in brain endothelial cells, have been shown to cross the BBB and are being developed as fusion proteins to deliver therapeutic cargos to brain targets. Various antibodies have been developed for this purpose and their in vivo evaluation demonstrated that either low affinity or monovalent receptor binding re-directs their transcellular trafficking away from lysosomal degradation and toward improved exocytosis on the abluminal side of the BBB. However, these studies have been performed with antibodies that recognize different TfR epitopes and have different binding characteristics, preventing inter-study comparisons. In this study, the efficiency of transcytosis in vitro and intracellular trafficking in endosomal compartments were evaluated in an in vitro BBB model for affinity variants (K d from 5 to174 nM) of the rat TfR-binding antibody, OX26. Distribution in subcellular fractions of the rat brain endothelial cells was determined using both targeted quantitative proteomics-selected reaction monitoring and fluorescent imaging with markers of early- and late endosomes. The OX26 variants with affinities of 76 and 108 nM showed improved trancytosis (P app values) across the in vitro BBB model compared with a 5 nM OX26. Although ~40% of the 5 nM OX26 and ~35% of TfR co-localized with late-endosome/lysosome compartment, 76 and 108 nM affinity variants showed lower amounts in lysosomes and a predominant co-localization with early endosome markers. The study links bivalent TfR antibody affinity to mechanisms of sorting and trafficking away from late endosomes and lysosomes, resulting in improvement in their transcytosis efficiency., Open Practices: Open Science: This manuscript was awarded with the Open Materials Badge. For more information see: https://cos.io/our-services/open-science-badges/ Cover Image for this issue: doi: 10.1111/jnc.14193., (© 2018 The Authors. Journal of Neurochemistry published by John Wiley & Sons Ltd on behalf of International Society for Neurochemistry.)

Glaucoma, a neurodegenerative disease, is characterized by a progressive loss of retinal ganglion cells (rgc). Up- and down-regulated autoantibody immunoreactivities in glaucoma patients have been demonstrated. Previous studies showed protective effects of down-regulated antibodies [gamma (γ)-synuclein and glial fibrillary acidic protein [GFAP]) on neuroretinal cells. The aim of this study was to test these protective antibody effects on rgc in an organ culture model and to get a better understanding of cell-cell interactions of the retina in the context of the protective effect. We used an adolescent retinal organ culture (pig) with an incubation time of up to 4 days. Retinal explants were incubated with different antibodies for 24 h (anti-GFAP, anti-γ-synuclein and anti-myoglobin antibody as a control). Brn3a and TUNEL staining were performed. We also conducted glutamine synthetase staining and quantification of the retinal explants. Mass spectrometry analyses were performed as well as protein analyses via microarray. We detected a continuous decrease of rgc/mm in the retinal explants throughout the 4 days of incubation with increased TUNEL rgc staining. Immunohistochemical analyses showed a protective effect of anti-γ-synuclein (increased rgc/mm of 41%) and anti-GFAP antibodies (increased rgc/mm of 37%). Mass spectrometric, microarray and immunohistochemical analyses demonstrated Müller cell involvement and decreased endoplasmic reticulum stress response in the antibody-treated retinae. We could detect that the tested antibodies have a protective effect on rgc which seems to be the result of reduced stress levels in the retina as well as a shift of glutamine synthetase localization in the endfeet of the Müller cells towards the inner retinal layer. Loss of retinal ganglion cells (rgc) in glaucoma leads to blindness. Several antibodies are down-regulated in glaucoma patients. Our aim was to test if these antibodies have a protective effect of rgc in a retinal organ culture. This could be shown with an increase of rgc numbers. This effect results through reduced stress levels and the shift of glutamine synthetase localization., (© 2016 International Society for Neurochemistry.)

The co-involvement of tumour necrosis factor-α (TNF-α) and interleukin-6 (IL-6) during blood-brain barrier (BBB) injury has been reported in various models of neuroinflammation, although the precise functional interplay between these archetypal proinflammatory cytokines remains largely undefined within this context. In the current paper, we tested the hypothesis that TNF-α-mediated BBB disruption is measurably attributable in-part to induction of microvascular endothelial IL-6 production. In initial experiments, we observed that treatment of human brain microvascular endothelial cells (HBMvECs) with TNF-α (0-100 ng/mL, 0-24 h) robustly elicited both time- and dose-dependent induction of IL-6 expression and release, as well as expression of the IL-6 family receptor, GP130. Further experiments demonstrated that the TNF-α-dependent generation of reactive oxygen species, down-regulation of adherens/tight junction proteins, and concomitant elevation of HBMvEC permeability, were all significantly attenuated by blockade of IL-6 signalling using either an anti-IL-6 neutralizing antibody or an IL-6 siRNA. Based on these observations, we conclude that TNF-α treatment of HBMvECs in vitro activates IL-6 production and signalling, events that were shown to synergize with TNF-α actions to elicit HBMvEC permeabilization. These novel findings offer a constructive insight into the specific contribution of downstream cytokine induction to the injurious actions of TNF-α at the BBB microvascular endothelium interface. The co-involvement of tumour necrosis factor-α (TNF-α) and interleukin-6 (IL-6) during blood-brain barrier (BBB) injury has been widely reported. Using human brain microvascular endothelial cells (HBMvEC), we show that TNF-α-mediated BBB disruption is measurably attributable in-part to induction of endothelial IL-6 production and signalling. We demonstrate that the TNF-α-dependent generation of reactive oxygen species (ROS), down-regulation of interendothelial junctions, and concomitant elevation of HBMvEC permeability, could be significantly attenuated by using either an IL-6 neutralizing antibody or an IL-6-specific siRNA. These findings provide insight into the complex nature of proinflammatory cytokine injury at the BBB microvascular endothelium interface., (© 2015 International Society for Neurochemistry.)

Low-density lipoprotein receptors (LDLRs) mediate the uptake of lipoprotein particles into cells, as studied mainly in peripheral tissues. Here, we show that nerve growth factor (NGF) increases LDLR levels in PC6.3 cells and in cultured septal neurons from embryonic rat brain. Study of the mechanisms showed that NGF enhanced transcription of the LDLR gene, acting mainly via Tropomyosin receptor kinase A receptors. Simvastatin, a cholesterol-lowering drug, also increased the LDLR expression in PC6.3 cells. In addition, pro-NGF and pro-brain-derived neurotrophic factor, acting via the p75 neurotrophin receptor (p75NTR) also increased LDLRs. We further observed that Myosin Regulatory Light Chain-Interacting Protein/Inducible Degrader of the LDLR (Mylip/Idol) was down-regulated by pro-NGF, whereas the other LDLR regulator, proprotein convertase subtilisin kexin 9 (PCSK9) was not significantly changed. On the functional side, NGF and pro-NGF increased lipoprotein uptake by neuronal cells as shown using diacetyl-labeled LDL. The addition of serum-derived lipoprotein particles in conjunction with NGF or simvastatin enhanced neurite outgrowth. Collectively, these results show that NGF and simvastatin are able to stimulate lipoprotein uptake by neurons with a positive effect on neurite outgrowth. Increases in LDLRs and lipoprotein particles in neurons could play a functional role during brain development, in neuroregeneration and after brain injuries. Nerve growth factor (NGF) and pro-NGF induce the expression of low-density lipoprotein receptors (LDLRs) in neuronal cells leading to increased LDLR levels. Pro-NGF also down-regulated myosin regulatory light chain-interacting protein/inducible degrader of the LDLR (Mylip/Idol) that is involved in the degradation of LDLRs. NGF acts mainly via Tropomyosin receptor kinase A (TrkA) receptors, whereas pro-NGF stimulates p75 neurotrophin receptor (p75NTR). Elevated LDLRs upon NGF and pro-NGF treatments enhanced lipoprotein uptake by neurons. Addition of LDL particles further led to the stimulation of neurite outgrowth in PC6.3 cells after NGF or simvastatin treatments, suggesting a stimulatory role of lipoproteins on neuronal differentiation. In contrast, pro-NGF had no effect on neurite outgrowth either in the absence or presence of LDL particles. The precise mechanisms by which increased lipoproteins uptake can affect neurite outgrowth warrant further studies., (© 2015 International Society for Neurochemistry.)

During neuronal differentiation, axonal elongation is regulated by both external and intrinsic stimuli, including neurotropic factors, cytoskeleton dynamics, second messengers such as cyclic adenosine monophosphate (cAMP), and neuronal excitability. Chloride intracellular channel 1 (CLIC1) is a cytoplasmic hydrophilic protein that, upon stimulation, dimerizes and translocates to the plasma membrane, where it contributes to increase the membrane chloride conductance. Here, we investigated the expression of CLIC1 in primary hippocampal neurons and retinal ganglion cells (RGCs) and examined how the functional expression of CLIC1 specifically modulates neurite outgrowth of neonatal murine RGCs. Using a combination of electrophysiology and immunohistochemistry, we found that CLIC1 is expressed in hippocampal neurons and RGCs and that the chloride current mediated by CLIC1 is required for maintaining growth cone morphology and sustaining cAMP-stimulated neurite elongation in dissociated immunopurified RGCs. In cultured RGCs, inhibition of CLIC1 ionic current through the pharmacological blocker IAA94 or a specific anti-CLIC1 antibody directed against its extracellular domain prevents the neurite outgrowth induced by cAMP. CLIC1-mediated chloride current, which results from an increased open probability of the channel, is detected only when cAMP is elevated. Inhibition of protein kinase A prevents such current. These results indicate that CLIC1 functional expression is regulated by cAMP via protein kinase A and is required for neurite outgrowth modulation during neuronal differentiation. Using a combination of electrophysiology and immunohistochemistry, we found that the chloride intracellular channel 1 (CLIC1) protein modulates the speed of neurite growth. The chloride current mediated by CLIC1 is essential for maintaining growth cone morphology and is required for sustaining cAMP-stimulated neurite elongation in dissociated immunopurified neurons. The presence of either the CLIC1 current blocker IAA94 or the anti-CLIC1 antibody inhibits neurite growth of Retina Ganglion Cells cultured in the presence of 10 micromolar forskolin for 24 h., (© 2014 International Society for Neurochemistry.)

GPR37, also known as parkin-associated endothelin-like receptor (Pael-R), is an orphan G protein-coupled receptor (GPCR) that aggregates intracellularly in a juvenile form of Parkinson's disease. However, little is known about the structure or function of this receptor. Here, in order to better understand the functioning of this receptor, we focused on the GPR37 C-terminal tail, in particular on a cystein-enriched region. Thus, we aimed to reveal the role of these residues on receptor plasma membrane expression and function, and also whether the presence of this cysteine-rich domain is linked to the previously described receptor-mediated cytotoxicity. Interestingly, while the deletion of six cysteine residues within this region did not affect receptor internalization it promoted GPR37 plasma membrane expression and signaling. Furthermore, the removal of the C-terminal cysteine-rich domain protected against GPR37-mediated apoptosis and cell death. Overall, we identified a GPR37 domain, namely the C-terminal tail cysteine-rich domain, which played a critical role in receptor cell surface expression, function and GPR37-mediated cytotoxicity. These results might contribute to better comprehend the pathophysiology (i.e. in Parkinson's disease) of this rather unknown member of the GPCR family., (© 2013 International Society for Neurochemistry.)

Acute inflammation is a prominent feature of central nervous system (CNS) insult and is detrimental to the CNS tissue. Although this reaction spontaneously diminishes within a short period of time, the mechanism underlying this inflammatory resolution remains largely unknown. In this study, we demonstrated that an initial infiltration of Ly6C(+) Ly6G(-) immature monocyte fraction exhibited the same characteristics as myeloid-derived suppressor cells (MDSCs), and played a critical role in the resolution of acute inflammation and in the subsequent tissue repair by using mice spinal cord injury (SCI) model. Complete depletion of Ly6C(+) Ly6G(-) fraction prior to injury by anti-Gr-1 antibody (clone: RB6-8C5) treatment significantly exacerbated tissue edema, vessel permeability, and hemorrhage, causing impaired neurological outcomes. Functional recovery was barely impaired when infiltration was allowed for the initial 24 h after injury, suggesting that MDSC infiltration at an early phase is critical to improve the neurological outcome. Moreover, intraspinal transplantation of ex vivo-generated MDSCs at sites of SCI significantly reduced inflammation and promoted tissue regeneration, resulting in better functional recovery. Our findings reveal the crucial role of an Ly6C(+) Ly6G(-) fraction as MDSCs in regulating inflammation and tissue repair after SCI, and also suggests an MDSC-based strategy that can be applied to acute inflammatory diseases., (© 2012 International Society for Neurochemistry.)

The association of gangliosides with specific proteins in the central nervous system was examined by coimmunoprecipitation with an anti-ganglioside antibody. The monoclonal antibody to the ganglioside GD3 (R24) immunoprecipitated the Csk (C-terminal src kinase)-binding protein (Cbp). Sucrose density gradient analysis showed that Cbp of rat cerebellum was detected in detergent-resistant membrane (DRM) raft fractions. R24 treatment of the rat primary cerebellar cultures induced Lyn activation and tyrosine phosphorylation of Cbp. Treatment with anti-ganglioside GD1b antibody also induced tyrosine phosphorylation. Furthermore, over-expressions of Lyn and Cbp in Chinese hamster ovary (CHO) cells resulted in tyrosine 314 phosphorylation of Cbp, which indicates that Cbp is a substrate for Lyn. Immunoblotting analysis showed that the active form of Lyn and the Tyr314-phosphorylated form of Cbp were highly accumulated in the DRM raft fraction prepared from the developing cerebellum compared with the DRM raft fraction of the adult one. In addition, Lyn and the Tyr314-phosphorylated Cbp were highly concentrated in the growth cone fraction prepared from the developing cerebellum. Immunoelectron microscopy showed that Cbp and GAP-43, a growth cone marker, are localized in the same vesicles of the growth cone fraction. These results suggest that Cbp functionally associates with gangliosides on growth cone rafts in developing cerebella., (© 2012 International Society for Neurochemistry.)

The nerve growth factor (NGF) precursor, proNGF, is implicated in various neuropathological states. ProNGF signals apoptosis by forming a complex with the receptors p75 and sortilin, however, it can also induce neurite growth, proposed to be mediated by the receptor of mature NGF, tyrosine kinase receptor A (TrkA). The way in which these dual effects occur in adult neurons is unclear. We investigated the neurotrophic effects of proNGF on peptidergic sensory neurons isolated from adult mouse dorsal root ganglia and found that proNGF stimulated neurite extension and branching, requiring p75, sortilin and TrkA. Neurite growth rarely occurred in sortilin-expressing neurons but was commonly observed in TrkA-positive, sortilin-negative neurons that associated closely with sortilin-positive glia. ProNGF was unable to induce local trophic effects at growth cones where sortilin-positive glia was absent. We propose that in adult sensory neurons the neurotrophic response to proNGF is mediated by NGF and TrkA, and that peri-somatic glia may participate in sortilin- and p-75 dependent cleavage of proNGF. The potential ability of local glial cells to provide a targeted supply of NGF may provide an important way to promote trophic (rather than apoptotic) outcomes under conditions where regeneration or sprouting is required., (© 2012 The Authors. Journal of Neurochemistry © 2012 International Society for Neurochemistry.)

Nogo-66 is a 66-amino-acid-residue extracellular domain of Nogo-A, which plays a key role in inhibition neurite outgrowth of central nervous system through binding to the Nogo-66 receptor (NgR) expressed on the neuron. Recent studies have confirmed that NgR is also expressed on the surface of macrophages/microglia in multiple sclerosis, but its biological effects remain unknown. In the present study, our results demonstrated that Nogo-66 triggered microglia anti-adhesion and inhibited their migration in vitro, which was mediated by NgR. We also assessed the roles of small GTP (glycosyl phosphatidylinositol)-binding proteins of the Rho family as the downstream signal transducers on the microglia adhesion and mobility induced by Nogo-66. The results showed that Nogo-66 activated RhoA and reduced the activity of Cdc42 in the meanwhile, which further triggered the anti-adhesion and migration inhibition effects to microglia. Nogo-66 inhibited microglia polarization and membrane protrusion formation, thus might eventually contribute to the decreasing capability of cell mobility. Taken together, the Nogo-66/NgR pathway may modulate neuroinflammation via mediating microglia adhesion and migration in addition to its role in neurons. Better understanding the relationship between Nogo-66/NgR and neuroinflammation may help targeting NgR for treating central nervous system diseases related with inflammation., (© 2011 The Authors. Journal of Neurochemistry © 2011 International Society for Neurochemistry.)

Parkinson's disease is a chronic neurodegenerative movement disorder characterized by the loss of dopaminergic neurons in the substantia nigra pars compacta. New therapeutic approaches aiming at delaying or reversing the neurodegenerative process are under active investigations. In this work, we found that harpagoside, an iridoid purified from the Chinese medicinal herb Scrophularia ningpoensis, could not only prevent but also rescue the dopaminergic neurodegeneration in MPTP/MPP(+) intoxication with promising efficacy. Firstly, in cultured mesencephalic neurons, harpagoside significantly attenuated the loss of TH-positive neuron numbers and the shortening of axonal length. Secondly, in a chronic MPTP mouse model, harpagoside dose-dependently improved the loco-motor ability (rotarod test), increased the TH-positive neuron numbers in the substantia nigra pars compacta (unbiased stereological counting) and increased the striatal DAT density ((125) I-FP-CIT autoradiography). Thirdly, harpagoside markedly elevated the GDNF mRNA and GDNF protein levels in MPTP/MPP(+) lesioned models. However, the protecting effect of harpagoside on the dopaminergic degeneration disappeared when the intrinsic GDNF action was blocked by either the Ret inhibitor PP1 or the neutralizing anti-GDNF antibody. Taken together, we conclude that harpagoside attenuates the dopaminergic neurodegeneration and movement disorder mainly through elevating glial cell line-derived neurotrophic factor., (© 2011 The Authors. Journal of Neurochemistry © 2011 International Society for Neurochemistry.)

During central nervous system (CNS) development, individual oligodendrocytes myelinate multiple axons, thus requiring the outgrowth and extensive branching of oligodendroglial processes. Laminin (Lm)-deficient mice have a lower percentage of myelinated axons, which may indicate a defect in the ability to properly extend and branch processes. It remains unclear, however, to what extent extracellular matrix (ECM) receptors contribute to oligodendroglial process remodeling itself. In the current study, we report that the ECM receptor dystroglycan is necessary for Lm enhancement of filopodial formation, process outgrowth, and process branching in differentiating oligodendroglia. During early oligodendroglial differentiation, the disruption of dystroglycan-Lm interactions, via blocking antibodies or dystroglycan small interfering RNA (siRNA), resulted in decreased filopodial number and length, decreased process length, and decreased numbers of primary and secondary processes. Later in oligodendrocyte differentiation, dystroglycan-deficient cells developed fewer branches, thus producing less complex networks of processes as determined by Sholl analysis. In newly differentiating oligodendroglia, dystroglycan was localized in filopodial tips, whereas, in more mature oligodendrocytes, dystroglycan was enriched in focal adhesion kinase (FAK)-positive focal adhesion structures. These results suggest that dystroglycan-Lm interactions influence oligodendroglial process dynamics and therefore may regulate the myelination capacity of individual oligodendroglia., (© 2011 The Authors. Journal of Neurochemistry © 2011 International Society for Neurochemistry.)

Deposition of amyloid β protein (Aβ) in the brain is the hallmark of Alzheimer's disease (AD) pathogenesis. Beta-site amyloid precursor protein (APP) cleaving enzyme 1 (BACE1) is the β-secretase in vivo essential for generation of Aβ. Previously we demonstrated that BACE1 is ubiquitinated and the degradation of BACE1 is mediated by the ubiquitin-proteasome pathway (UPP). However the mechanism underlying regulation of BACE1 degradation by UPP remains elusive. Ubiquitin carboxyl-terminal hydrolase L1 (UCHL1) is a deubiquitinating enzyme highly specific to neuron, catalyzing the hydrolysis of ubiquitin conjugates from ubiquitinated substrates. UCHL1 regulates ubiquitin-dependent protein degradation. However, whether UCHL1 is particularly involved in the proteasomal degradation of BACE1 and what is the role of UCHL1 in AD pathogenesis remain elusive. To investigate the effect of UCHL1 on BACE1 degradation, HUCH cells, a UCHL1 stably over-expressed HEK293 cell line, was established. We found that inhibition of UCHL1 significantly increased BACE1 protein level in a time-dependent manner. Half life of BACE1 was reduced in HUCH cells compared with HEK. Over-expression of UCHL1 decreased APP C-terminal fragment C99 and Aβ levels in HUCH cells. Moreover, disruption of Uchl1 gene significantly elevated levels of endogenous BACE1, C99 and Aβ in the Uchl1-null gad mice. These results demonstrated that UCHL1 accelerates BACE1 degradation and affects APP processing and Aβ production. This study suggests that potentiation of UCHL1 might be able to reduce the level of BACE1 and Aβ in brain, which makes it a novel target for AD drug development., (© 2011 The Authors. Journal of Neurochemistry © 2011 International Society for Neurochemistry.)

Tissue plasminogen activator (tPA) is necessary for hippocampal long-term potentiation. Synaptically released zinc also contributes to long-term potentiation, especially in the hippocampal CA3 region. Using cortical cultures, we examined whether zinc increased the concentration and/or activity of tPA. Two hours after a 10-min exposure to 300 μM zinc, expression of tPA and its substrate, plasminogen, were significantly increased, as was the proteolytic activity of tPA. In contrast, increasing extracellular or intracellular calcium levels did not affect the expression or secretion of tPA. Changing zinc influx or chelating intracellular zinc also failed to alter tPA/plasminogen induction by zinc, indicating that zinc acts extracellularly. Zinc-mediated extracellular activation of matrix metalloproteinase (MMP) underlies the up-regulation of brain-derived neurotrophic factor (BDNF) and tropomyosin receptor kinase (Trk) signaling. Consistent with these findings, co-treatment with a neutralizing antibody against BDNF or specific inhibitors of MMPs or Trk largely reversed tPA/plasminogen induction by zinc. Treatment of cortical cultures with p-aminophenylmercuric acetate, an MMP activator, MMP-2, or BDNF alone induced tPA/plasminogen expression. BDNF mRNA and protein expression was also increased by zinc and mediated by MMPs. Thus, an extracellular zinc-dependent, MMP- and BDNF-mediated synaptic mechanism may regulate the levels and activity of tPA., (© 2011 The Authors. Journal of Neurochemistry © 2011 International Society for Neurochemistry.)

Alpha-synuclein (SNCA) is a predominantly neuronal protein involved in the control of neurotransmitter release. The levels of SNCA expression are closely linked to the pathogenesis of Parkinson's disease; however, the biochemical pathways and transcriptional elements that control SNCA expression are not well understood. We previously used the model system of neurotrophin-mediated PC12 cell neuronal differentiation to examine these phenomena. Although these studies were informative, they were limited to the use of a cell line; therefore, in the current work, we have turned our attention to cultured primary rat cortical neurons. In these cultures, SNCA expression increased with time in culture, as the neurons mature. Luciferase assays based on transient transfections of fusion constructs encoding components of the transcriptional control region of SNCA identified various promoter areas that have a positive or negative effect on SNCA transcription. Intron 1, previously identified by us as an important regulatory region in the PC12 cell model, cooperates with regions 5' to exon 1 to mediate gene transcription. Using selective pharmacological tools, we find that tyrosine kinase receptors and the phosphatidyl-inositol 3 kinase signaling pathway are involved in mediating these effects. The exogenous application of the neurotrophin brain-derived neurotrophic factor (BDNF) is sufficient on its own to promote the transcriptional activation of SNCA through this pathway, but a neutralizing antibody against BDNF failed to affect SNCA transcription in maturing cultures, suggesting that BDNF is not the main factor involved in maturation-induced SNCA transcription in this model. Further in vivo studies are needed to establish the role of neurotrophin signaling in the control of SNCA transcription., (© 2011 The Authors. Journal of Neurochemistry © 2011 International Society for Neurochemistry.)

Sustained neuronal activity induces synaptic remodeling, in part, by altering gene expression. We have used a major climbing fiber pathway onto cerebellar Purkinje cells to investigate the effects of sustained climbing fiber-evoked glutamatergic synaptic transmission on transcription, expression and phosphorylation of proteins related to the regulation of inhibitory GABA(A) receptor function. Binocular horizontal optokinetic stimulation was used to modulate climbing fiber signals to Purkinje cells in the flocculus and nodulus of rabbits and mice. Purkinje cells in the flocculus and nodulus ipsilateral to the eye stimulated in the Posterior→Anterior direction received increased climbing fiber activity. Purkinje cells in flocculus and nodulus ipsilateral to the eye stimulated in the Anterior→Posterior direction received decreased climbing fiber activity. We identified changes in levels of gene transcripts in floccular and nodular Purkinje cells with the technique of differential display RT-PCR. Increased climbing fiber input reduced transcript levels and expression of GABA receptor-associated protein (GABARAP). Using a protein 'pull down' technique, we showed that GABARAP interacts with serine phosphorylated GABA(A)γ2, gephyrin and β-tubulin. Serine de-phosphorylation of GABA(A)γ2 reduced association of GABARAP with GABA(A)γ2. Climbing fiber activity did not influence the expression of GABA(A)γ2. Rather, it decreased its serine phosphorylation. Climbing fiber discharge decreased both expression of GABARAP and serine phosphorylation of GABA(A)γ2. Consequently, climbing fiber activity may reduce the surface expression of GABA(A) receptors in Purkinje cells rendering Purkinje cells less susceptible to interneuronal GABAergic inhibition., (© 2011 The Authors. Journal of Neurochemistry © 2011 International Society for Neurochemistry.)

Cold pre-conditioning reduces subsequent brain injury in small animals but the underlying mechanisms remain undefined. As hypothermia triggers systemic macrophage tumor necrosis factor alpha (TNF-α) production and other neural pre-conditioning stimuli depend on this cytokine, we reasoned that microglia and TNF-α would be similarly involved with cold pre-conditioning neuroprotection. Also, as slice cultures closely approximate their in vivo counterpart and include quiescent microglia, we used rat hippocampal slice cultures to confirm this hypothesis. Furthermore, inflammatory cytokine gene screening with subsequent PCR and immunostaining confirmation of targeted mRNA and related protein changes showed that cold pre-conditioning triggered a significant rise in TNF-α that localized to microglia and a significant rise in interleukin (IL)-11 that localized mainly to hippocampal pyramidal neurons and, more rarely, astrocytes. Importantly, co-stimulation with cold and IL-11, an anti-inflammatory cytokine that inhibits TNF-α expression, abrogated the otherwise evident protection. Instead, cold pre-conditioning coupled with blockade of IL-11 signaling further enhanced neuroprotection from that seen with cold pre-conditioning alone. Thus, physiological activation of brain pro-inflammatory cytokine signaling, and its amplification by inhibition of coincident anti-inflammatory cytokine signaling, may be opportune targets for the development of novel therapeutics that can mimic the protection seen in cold pre-conditioning., (© 2010 The Authors. Journal of Neurochemistry © 2010 International Society for Neurochemistry.)

S100B is a soluble protein secreted by astrocytes that exerts pro-survival or pro-apoptotic effects depending on the concentration reached in the extracellular millieu. The S100B receptor termed RAGE (for receptor for advanced end glycation products) is highly expressed in the developing brain but is undetectable in normal adult brain. In this study, we show that RAGE expression is induced in cortical neurons of the ischemic penumbra. Increased RAGE expression was also observed in primary cortical neurons exposed to excitotoxic glutamate (EG). S100B exerts effects on survival pathways and neurite extension when the cortical neurons have been previously exposed to EG and these S100B effects were prevented by anti-RAGE blocking antibodies. Furthermore, nuclear factor kappa B (NF-κB) is activated by S100B in a dose- and RAGE-dependent manner and neuronal death induced by NF-κB inhibition was prevented by S100B that restored NF-κB activation levels. Together, these findings suggest that excitotoxic damage can induce RAGE expression in neurons from ischemic penumbra and demonstrate that cortical neurons respond to S100B through engagement of RAGE followed by activation of NF-κB signaling. In addition, basal NF-κB activity in neurons is crucial to modulate the extent of pro-survival or pro-death S100B effects., (© 2011 The Authors. Journal of Neurochemistry © 2011 International Society for Neurochemistry.)

We report a novel mechanism for dopamine D(1) receptor (D(1) R)-mediated extracellular signal-regulated kinases (Erk) activation in rat striatum. Erk signaling depends on phosphorylation and dephosphorylation events mediated by specific kinases and phosphatases. The tyrosine phosphatase Shp-2, that is required for Erk activation by tyrosine kinase receptors, has been recently shown to regulate signaling downstream of few G protein-coupled receptors. We show that the D(1) R interacts with Shp-2, that D(1) R stimulation results in Shp-2 tyrosine phosphorylation and activation in primary striatal neuronal cultures and that D(1) R/Shp-2 interaction is required for transmitting D(1) R-dependent signaling to Erk1/2 activation. D(1) R-mediated Erk1/2 phosphorylation in cultured striatal neurons is in fact abolished by over-expression of the inactive Shp-2(C/S) mutant and by small interfering RNA-induced Shp-2 silencing. Moreover, by using selective inhibitors we show that both D(1) R-induced Shp-2 activation and Erk1/2 phosphorylation are dependent on the cyclic AMP/protein kinase A pathway and require Src. These results, which were substantiated also in transfected human embryonic kidney 293 cells, provide a novel mechanism by which to converge D(1) R signaling to the Erk pathway and suggest that Shp-2 or the D(1) R/Shp-2 interface could represent a potential drug target for disorders of dopamine transmission involving malfunctioning of D(1) R signaling., (© 2011 The Authors. Journal of Neurochemistry © 2011 International Society for Neurochemistry.)

In addition to supporting rapid nerve conduction, myelination nurtures and stabilizes axons and protects them from acute toxic insults. One myelin molecule that protects and sustains axons is myelin-associated glycoprotein (MAG). MAG is expressed on the innermost wrap of myelin, apposed to the axon surface, where it interacts with axonal receptors that reside in lateral membrane domains including gangliosides, the glycosylphosphatidylinositol-anchored Nogo receptors, and β1-integrin. We report here that MAG protection extends beyond the axon to the neurons from which those axons emanate, protecting them from excitotoxicity. Compared to wild type mice, Mag-null mice displayed markedly increased seizure activity in response to intraperitoneal injection of kainic acid, an excitotoxic glutamate receptor agonist. Mag-null mice also had larger lesion volumes in response to intrastriatal injection of the excitotoxin NMDA. Prior injection of a soluble form of MAG partially protected Mag-null mice from NMDA-induced lesions. Hippocampal neurons plated on proteins extracted from wild-type rat or mouse myelin were resistant to kainic acid-induced excitotoxicity, whereas neurons plated on proteins from Mag-null myelin were not. Protection was reversed by anti-MAG antibody and replicated by addition of soluble MAG. MAG-mediated protection from excitotoxicity was dependent on Nogo receptors and β1-integrin. We conclude that MAG engages membrane-domain resident neuronal receptors to protect neurons from excitotoxicity, and that soluble MAG mitigates excitotoxic damage in vivo., (© 2011 The Authors. Journal of Neurochemistry © 2011 International Society for Neurochemistry.)

The cellular prion protein (PrP(C)) is a cell-surface glycoprotein mainly expressed in the CNS. The structural conversion of PrP(C) generates the prion, the infectious agent causing transmissible spongiform encephalopathies, which are rare and fatal diseases affecting animals and humans. Despite decades of intensive research, the mechanism of prion-associated neurodegeneration and the physiologic role of PrP(C) are still obscure. Recent evidence, however, supports the hypothesis that PrP(C) may be involved in the control of Ca(2+) homeostasis. Given the universal significance of Ca(2+) as an intracellular messenger for both the life and death of cells, this possibility may help explain the complex, often controversial, dataset accumulated on PrP(C) physiology, and the events leading to prion-associated neuronal demise. In this study, we have compared local Ca(2+) movements in cerebellar granule neurons (CGN) derived from wild-type (WT), or PrP-knockout (KO), mice, by means of the Ca(2+)-sensitive photo-probe, aequorin, genetically targeted to specific intracellular domains and delivered to CGN by lentiviral vectors. The use of an aequorin that localizes to the cytosolic domains proximal to the plasma membrane has allowed us to demonstrate that there was a dramatic increase of store-operated Ca(2+) entry in PrP-KO CGN compared to WT neurons. Notably, this phenotype was rescued upon restoring PrP(C) expression. The Ca(2+)-phenotype of PrP-KO neurons can in part be explained by the lower expression of two major Ca(2+)-extruding proteins, namely the plasma membrane and the sarco-endoplasmic reticulum Ca(2+)-ATPases. The lower sarco-endoplasmic reticulum Ca(2+)-ATPase content may also contribute to explain why PrP-KO CGN accumulated less Ca(2+) in the endoplasmic reticulum than the WT counterpart., (© 2011 The Authors. Journal of Neurochemistry © 2011 International Society for Neurochemistry.)

We have previously shown that the multi-functional phosphoprotein osteopontin (OPN) is present in the substantia nigra (SN) and that its mRNA and protein expression are up-regulated following toxic insult. We now report the effects of the arginine-glycine-aspartic acid (RGD)-containing peptide fragment of OPN and OPN inactivation on the survival of tyrosine hydroxylase (TH) positive neurones in primary rat ventral mesencephalic (VM) cultures and in SN in the rat. Treatment of VM cultures with the fragment of OPN containing the RGD integrin binding domain did not decrease TH positive cell number, but instead the peptide fragment protected against cell loss induced by both MPP(+) and lipopolysaccharide (LPS). Incorporation of an OPN antibody into VM cultures caused a concentration-dependent loss of TH positive neurones. The OPN antibody also exacerbated MPP(+) - and LPS-induced cell loss at all concentrations tested. In the rat, administration of the RGD-containing peptide fragment of OPN protected TH positive neurones against a mechanically-induced lesion and against 6-hydroxydopamine- and LPS-induced cell loss. The protection against 6-hydroxydopamine toxicity was confirmed in a separate study using stereological analysis. By contrast, stereotaxic injection of the OPN antibody into the SN resulted in a loss of TH positive cells. These results suggest that OPN may be necessary for the survival of TH positive cells in SN but through the RGD-containing peptide fragment may also have neuroprotective properties relevant to Parkinson's disease., (© 2010 The Authors. Journal Compilation © 2010 International Society for Neurochemistry.)

Neural stem cells (NSCs) proliferate and generate new neurons in the adult brain. A carbohydrate-binding protein (lectin), Galectin-1, is expressed in the NSCs in the subependymal zone (SEZ) of the adult mouse brain. The infusion and knockout of Galectin-1 in the SEZ results in an increase and decrease, respectively, of NSCs and subsequently born progenitor cells. The molecular mechanism of this effect, however, has been unknown. Previous studies outside the brain suggest that Galectin-1 binds to a carbohydrate structure of beta1 Integrin and modulates cell adhesion. Here, we studied the functional interaction between Galectin-1 and beta1 Integrin in the adult mouse SEZ. Beta1 Integrin was purified from adult SEZ tissue by binding to a Galectin-1 affinity column, and this binding depended on Galectin-1's carbohydrate-binding activity. In adult brain sections, Galectin-1-binding activity was detected on beta1 Integrin-expressing cells in the SEZ. Furthermore, in the adult SEZ, the simultaneous infusion of a beta1 Integrin-neutralizing antibody with Galectin-1 protein reversed the increasing effect of Galectin-1 on the number of adult neural progenitor cells (NPCs). Finally, intact beta1 Integrin was required for Galectin-1's function in NPC adhesion in vitro. These results suggest that the interaction between beta1 Integrin and Galectin-1 plays an important role in regulating the number of adult NPCs through mechanisms including cell adhesion.

Published evidence has linked glutamate with the pathogenesis of Alzheimer's disease (AD) and the up-regulation of a variety of chemokines, including monocyte chemotactic protein-1 (MCP-1)/chemokine ligand 2, with AD-associated pathological changes. In this study, we assessed the potential molecular basis for the role of glutamate in hippocampal inflammation by determining its effects on MCP-1 induction. We also attempted to identify the mechanism by which resveratrol (trans-3,5,4'-trihydroxystilbene), a polyphenolic phytostilbene, modulates the expression of MCP-1 in the glutamate-stimulated hippocampus. An ex vivo study using rat hippocampal slices demonstrated a time- and dose-dependent increase in MCP-1 release from glutamate-exposed hippocampus. This increase was accompanied by enhanced MCP-1 gene expression via the activation of the MEK/extracellular signal-regulated kinase (ERK) pathway and interleukin-1beta (IL-1beta) expression. The inhibition of the MEK/ERK pathway with SL327, which is capable of crossing the blood-brain barrier, nearly abolished the observed glutamate-induced effects. Furthermore, anti-IL-1beta antibodies suppressed the glutamate-induced expression of MCP-1 mRNA and protein, whereas an isotype-matched antibody exerted only minimal effects. It is worthy of note that resveratrol, to a similar degree as SL327, down-regulated glutamate-induced IL-1beta expression and reduced the expression of MCP-1 mRNA and protein release via the inactivation of ERK1/2. These results indicate that the activation of the MEK/ERK pathway and the consequent IL-1beta expression are essential for glutamate-stimulated MCP-1 production in the hippocampus. Additionally, our data reveal an anti-inflammatory mechanism of resveratrol involving the inactivation of the ERK1/2 pathway in the hippocampus, which is linked principally to AD-associated cognitive dysfunction.

The objective of our study was to determine granulocyte-macrophage colony-stimulating factor (GM-CSF) activity in the brain following GM-CSF induction. We injected recombinant mouse GM-CSF into the brains of 8-month-old C57BL6 mice via intracerebroventricular injections and studied the activities of microglia, astrocytes, and neurons. We also sought to determine whether an anti-GM-CSF antibody could suppress endogenous microglial activity in the C57BL6 mice and could also suppress microglial activity induced by the recombinant mouse GM-CSF in another group of C57BL6 mice. Using quantitative real-time RT-PCR, we assessed microglial, astrocytic, and neuronal activity by measuring mRNA expression of pro-inflammatory cytokines, GFAP, and the neuronal marker NeuN in the cerebral cortex tissues from C57BL6 mice. We performed immunoblotting and immunohistochemistry of activated microglia in different regions of the brains from control (phosphate-buffered saline-injected C57BL6 mice) and experimental mice (recombinant GM-CSF-injected C57BL6 mice, GM-CSF antibody-injected C57BL6 mice, and recombinant mouse GM-CSF plus anti-GM-CSF antibody-injected C57BL6 mice). We found increased mRNA expression of CD40 (9.75-fold), tumor necrosis factor-alpha (2.1-fold), CD45 (1.73-fold), and CD11c (1.70-fold) in the cerebral cortex of C57BL6 mice that were induced with recombinant GM-CSF, compared with control mice. Further, the anti-GM-CSF antibody suppressed microglia in mice that were induced with recombinant GM-CSF. Our immunoblotting and immunohistochemistry findings of GM-CSF-associated cytokines in C57BL6 mice induced with recombinant GM-CSF, in C57BL6 mice injected with the anti-GM-CSF antibody, and in C57BL6 mice injected with recombinant mouse GM-CSF plus anti-GM-CSF antibody concurred with our real-time RT-PCR findings. These findings suggest that GM-CSF is critical for microglial activation and that anti-GM-CSF antibody suppresses microglial activity in the CNS. The findings from this study may have implications for anti-inflammatory effects of Alzheimer's disease and experimental autoimmune encephalomyelitis mice (a multiple sclerosis mouse model).

Brain-derived neurotrophic factor (BDNF) is involved in many aspects of neuronal biology and hippocampal physiology. Status epilepticus (SE) is a condition in which prolonged seizures lead to neuronal degeneration. SE-induced in rodents serves as a model of Temporal Lobe Epilepsy with hippocampal sclerosis, the most frequent epilepsy in humans. We have recently described a strong correlation between TrkB decrease and p75ntr increase with neuronal degeneration (Neuroscience 154:978, 2008). In this report, we report that local, acute intra-hippocampal infusion of function-blocking antibodies against BDNF prevented both early TrkB down-regulation and neuronal degeneration after SE. Conversely, the infusion of recombinant human BDNF protein after SE greatly increased neuronal degeneration. The inhibition of BDNF mRNA translation by the infusion of antisense oligonucleotides induced a rapid decrease of BDNF protein levels, and a delayed increase. If seizures were induced at the time endogenous BDNF was decreased, SE-induced neuronal damage was prevented. On the other hand, if seizures were induced at the time endogenous BDNF was increased, SE-induced neuronal damage was exacerbated. These results indicate that under a pathological condition BDNF exacerbates neuronal injury.

Previous reports indicated the presence of both gangliosides and sialidase in the nuclear envelope (NE) of primary neurons and the NG108-15 neural cell line. GM1, one of the major gangliosides of this membrane, was shown to be tightly associated with a sodium-calcium exchanger in the inner membrane of the NE and to potentiate exchanger activity. GD1a was the other major ganglioside detected in the NE and, like GM1, occurs in both inner and outer membranes. A subsequent report indicated the presence of sialidase activity in the NE without specification as to which of the two membranes express it. The present study was undertaken to determine the nature and locus of this activity within the NE of two cell lines: NG108-15 and SH-SY5Y. Western blot analysis of the separated membranes revealed occurrence of Neu3 in the inner membrane and Neu1 in the outer membrane of the NE. Moreover, sialidase activity at both sites was shown capable of catalyzing conversion of endogenous GD1a to GM1.

Metabotropic glutamate (mGlu) receptors mediate in part the CNS effects of glutamate. These receptors interact with a large array of intracellular proteins in which the final role is to regulate receptor function. Here, using co-immunoprecipitation and pull-down experiments we showed a close and specific interaction between mGlu(5) receptor and NECAB2 in both transfected human embryonic kidney cells and rat hippocampus. Interestingly, in pull-down experiments increasing concentrations of calcium drastically reduced the ability of these two proteins to interact, suggesting that NECAB2 binds to mGlu(5) receptor in a calcium-regulated manner. Immunoelectron microscopy detection of NECAB2 and mGlu(5) receptor in the rat hippocampal formation indicated that both proteins are codistributed in the same subcellular compartment of pyramidal cells. In addition, the NECAB2/mGlu(5) receptor interaction regulated mGlu(5b)-mediated activation of both inositol phosphate accumulation and the extracellular signal-regulated kinase/mitogen-activated protein kinase pathway. Overall, these findings indicate that NECAB2 by its physical interaction with mGlu(5b) receptor modulates receptor function.

Oxidative stress and down-regulated trophic factors are involved in the pathogenesis of nigrostriatal dopamine(DA)rgic neurodegeneration in Parkinson's disease. Fibroblast growth factor 9 (FGF9) is a survival factor for various cell types; however, the effect of FGF9 on DA neurons has not been studied. The antioxidant melatonin protects DA neurons against neurotoxicity. We used MPP(+) to induce neuron death in vivo and in vitro and investigated the involvement of FGF9 in MPP(+) intoxication and melatonin protection. We found that MPP(+) in a dose- and time-dependent manner inhibited FGF9 mRNA and protein expression, and caused death in primary cortical neurons. Treating neurons in the substantia nigra and mesencephalic cell cultures with FGF9 protein inhibited the MPP(+)-induced cell death of DA neurons. Melatonin co-treatment attenuated MPP(+)-induced FGF9 down-regulation and DA neuronal apoptosis in vivo and in vitro. Co-treating DA neurons with melatonin and FGF9-neutralizing antibody prevented the protective effect of melatonin. In the absence of MPP(+), the treatment of FGF9-neutralizing antibody-induced DA neuronal apoptosis whereas FGF9 protein reduced it indicating that endogenous FGF9 is a survival factor for DA neurons. We conclude that MPP(+) down-regulates FGF9 expression to cause DA neuron death and that the prevention of FGF9 down-regulation is involved in melatonin-provided neuroprotection.

Adrenal chromaffin cells synthesize and secrete catecholamines and neuropeptides that may regulate hormonal and paracrine signaling in stress and also during inflammation. The aim of our work was to study the role of the cytokine interleukin-1beta (IL-1beta) on catecholamine release and synthesis from primary cell cultures of human adrenal chromaffin cells. The effect of IL-1beta on neuropeptide Y (NPY) release and the intracellular pathways involved in catecholamine release evoked by IL-1beta and NPY were also investigated. We observed that IL-1beta increases the release of NPY, norepinephrine (NE), and epinephrine (EP) from human chromaffin cells. Moreover, the immunoneutralization of released NPY inhibits catecholamine release evoked by IL-1beta. Moreover, IL-1beta regulates catecholamine synthesis as the inhibition of tyrosine hydroxylase decreases IL-1beta-evoked catecholamine release and the cytokine induces tyrosine hydroxylase Ser40 phosphorylation. Moreover, IL-1beta induces catecholamine release by a mitogen-activated protein kinase (MAPK)-dependent mechanism, and by nitric oxide synthase activation. Furthermore, MAPK, protein kinase C (PKC), protein kinase A (PKA), and nitric oxide (NO) production are involved in catecholamine release evoked by NPY. Using human chromaffin cells, our data suggest that IL-1beta, NPY, and nitric oxide (NO) may contribute to a regulatory loop between the immune and the adrenal systems, and this is relevant in pathological conditions such as infection, trauma, stress, or in hypertension.

This study was undertaken to elucidate the molecular mechanisms by which lithium regulates the development of spinal cord-derived neural progenitor cells (NPCs) in vitro and after transplanted in vivo. Our results show that lithium at the therapeutic concentration significantly increases the proliferation and neuronal differentiation of NPCs in vitro. Specific ELISAs, western blotting, and quantitative real-time RT-PCR assays demonstrate that lithium treatment significantly elevates the expression and production of brain-derived neurotrophic factor (BDNF) by NPCs in culture. Application of a BDNF neutralizing antibody in culture leads to a marked reduction in the neurogenesis of lithium-treated NPCs to the control level. However, it shows no effects on the proliferation of lithium-treated NPCs. These findings suggest that the BDNF pathway is possibly involved in the supportive role of lithium in inducing NPC neurogenesis but not proliferation. This study also provides evidence that lithium is able to elevate the neuronal generation and BDNF production of NPCs after transplantation into the adult rat ventral horn with motoneuron degeneration because of spinal root avulsion, which highlights the therapeutic potential of lithium in cell replacement strategies for spinal cord injury because of its ability to promote neuronal differentiation and BDNF production of grafted NPCs in the injured spinal cord.

CD47 or integrin-associated protein promotes cell death in blood and tumor cells. Recently, CD47 signaling has been identified in neurons as well. In this study, we investigated the role of CD47 in neuronal cell death. Exposure of primary mouse cortical neurons to the CD47 ligand thrombospondin-1 or the specific CD47-activating peptide 4N1K induced cell death. Activation of CD47 elevated levels of active caspase 3 and increased the generation of reactive oxygen species (ROS) in a time-dependent manner. Both ROS scavengers and caspase inhibitors attenuated cell death. But ROS scavenging did not reduce the activation of caspase 3, and combination treatments with a caspase inhibitor plus free radical scavenger did not yield additive protection. Taken together, these data suggest that parallel and redundant pathways of oxidative stress and caspase-mediated cell death are involved. We conclude that CD47 mediates neuronal cell death through caspase-dependent and caspase-independent pathways.

The microtubule associated protein tau is a major component of neurofibrillary tangles in Alzheimer disease brain, however the neuropathological processes behind the formation of neurofibrillary tangles are still unclear. Previously, 14-3-3 proteins were reported to bind with tau. 14-3-3 Proteins usually bind their targets through specific serine/threonine -phosphorylated motifs. Therefore, the interaction of tau with 14-3-3 mediated by phosphorylation was investigated. In this study, we show that the phosphorylation of tau by either protein kinase A (PKA) or protein kinase B (PKB) enhances the binding of tau with 14-3-3 in vitro. The affinity between tau and 14-3-3 is increased 12- to 14-fold by phosphorylation as determined by real time surface plasmon resonance studies. Mutational analyses revealed that Ser214 is critical for the phosphorylation-mediated interaction of tau with 14-3-3. Finally, in vitro aggregation assays demonstrated that phosphorylation by PKA/PKB inhibits the formation of aggregates/filaments of tau induced by 14-3-3. As the phosphorylation at Ser214 is up-regulated in fetal brain, tau's interaction with 14-3-3 may have a significant role in the organization of the microtubule cytoskeleton in development. Also as the phosphorylation at Ser214 is up-regulated in Alzheimer's disease brain, tau's interaction with 14-3-3 might be involved in the pathology of this disease.

Oligodendrocytes stain more strongly for iron than any other cell in the CNS, and they require iron for the production of myelin. For most cell types transferrin is the major iron delivery protein, yet neither transferrin receptor protein nor mRNA are detectable in mature oligodendrocytes. Thus an alternative iron delivery mechanism must exist. Given the significant long term consequences of developmental iron deficiency and the iron requirements for normal myelination, identification of the iron delivery mechanism for oligodendrocytes is important. Previously we have reported that oligodendrocytes bind H-ferritin and that H-ferritin binds to white matter tracts in vivo. Recently, T cell immunoglobulin and mucin domain-containing protein-2 (Tim-2) was shown to bind and internalize H-ferritin. In the present study we show that Tim-2 is expressed on oligodendrocytes both in vivo and in vitro. Further, the onset of saturable H-ferritin binding in CG4 oligodendrocyte cell line is accompanied by Tim-2 expression. Application of a blocking antibody to the extracellular domain of Tim-2 significantly reduces H-ferritin binding to the differentiated CG4 cells and primary oligodendrocytes. Tim-2 expression on CG4 cells is responsive to iron; decreasing with iron loading and increasing with iron chelation. Taken together, these data provide compelling evidence that Tim-2 is the H-ferritin receptor on oligodendrocytes suggesting it is the primary mechanism for iron acquisition by these cells.

Peripheral nerve lesion leads to the production of interleukin 6 (IL-6)-related neuropoietic cytokines involved in nerve protection and regeneration. This family of cytokines mainly signal through the signal transducer and activator of transcription (STAT) pathway that is locally activated in injured nerves. IL-6 is also involved in pain that frequently arises from peripheral nerve lesion. We investigated the possible activation of this major IL-6 signaling system in the spinal cord after peripheral nerve injury and its role in neuropathic pain. Ligation of L5-L6 spinal nerves (SNL) evoked an accumulation of active, phosphorylated form of STAT3 in microglial cells of dorsal spinal cord mostly in projection areas of injured nerves. SNL resulted also in a massive induction of IL-6 mRNA expression in dorsal root ganglia and increased concentration of IL-6 in dorsal spinal cord. Intrathecal injection of anti-rat IL-6 antibodies prevented the SNL-induced accumulation of phospho-STAT3 in the spinal cord. STAT3 pathway blockade with Janus kinase 2 inhibitor AG490 attenuated both mechanical allodynia and thermal hyperalgesia in SNL rats. These data show that in response to SNL injury Janus kinase/STAT3 system is activated mainly through IL-6 signaling in spinal microglia and that this transduction pathway participates in development of pain associated with nerve alteration.

We provide evidence that the sphingolipid ceramide, in addition to its pro-apoptotic function, regulates neural progenitor (NP) motility in vitro and brain development in vivo. Ceramide (N-palmitoyl d-erythro sphingosine and N-oleoyl d-erythro sphingosine) and the ceramide analog N-oleoyl serinol (S18) stimulate migration of NPs in scratch (wounding) migration assays. Sphingolipid depletion by inhibition of de novo ceramide biosynthesis, or ceramide inactivation using an anti-ceramide antibody, obliterates NP motility, which is restored by ceramide or S18. These results suggest that ceramide is crucial for NP motility. Wounding of the NP monolayer activates neutral sphingomyelinase indicating that ceramide is generated from sphingomyelin. In membrane processes, ceramide is co-distributed with its binding partner atypical protein kinase C zeta/lambda (aPKC), and Cdc42, alpha/beta-tubulin, and beta-catenin, three proteins involved in aPKC-dependent regulation of cell polarity and motility. Sphingolipid depletion by myriocin prevents membrane translocation of aPKC and Cdc42, which is restored by ceramide or S18. These results suggest that ceramide-mediated membrane association of aPKC/Cdc42 is important for NP motility. In vivo, sphingolipid depletion leads to ectopic localization of mitotic or post-mitotic neural cells in the embryonic brain, while S18 restores the normal brain organization. In summary, our study provides novel evidence that ceramide is critical for NP motility and polarity in vitro and in vivo.

The primary molecules for mediating the innate immune response are the Toll-like family of receptors (TLRs). Recent work has established that amyloid-beta (Abeta) fibrils, the primary components of senile plaques in Alzheimer's disease (AD), can interact with the TLR2/4 accessory protein CD14. Using antibody neutralization assays and tumor necrosis factor alpha release in the human monocytic THP-1 cell line, we determined that both TLR2 and TLR4 mediated an inflammatory response to aggregated Abeta(1-42). This was in contrast to exclusive TLR ligands lipopolysaccharide (LPS) (TLR4) and tripalmitoyl cysteinyl seryl tetralysine (Pam(3)CSK(4)) (TLR2). Atomic force microscopy imaging showed a fibrillar morphology for the proinflammatory Abeta(1-42) species. Pre-treatment of the cells with 10 microg/mL of a TLR2-specific antibody blocked approximately 50% of the cell response to fibrillar Abeta(1-42), completely blocked the Pam(3)CSK(4) response, and had no effect on the LPS-induced response. A TLR4-specific antibody (10 microg/mL) blocked approximately 35% of the cell response to fibrillar Abeta(1-42), completely blocked the LPS response, and had no effect on the Pam(3)CSK(4) response. Polymyxin B abolished the LPS response with no effect on Abeta(1-42) ruling out bacterial contamination of the Abeta samples. Combination antibody pre-treatments indicated that neutralization of TLR2, TLR4, and CD14 together was much more effective at blocking the Abeta(1-42) response than the antibodies used alone. These data demonstrate that fibrillar Abeta(1-42) can trigger the innate immune response and that both TLR2 and TLR4 mediate Abeta-induced tumor necrosis factor alpha production in a human monocytic cell line.

Neuroglycan C (NGC) is a transmembrane-type of chondroitin sulfate proteoglycan with an epidermal growth factor (EGF)-like module that is exclusively expressed in the CNS. Because ectodomain shedding is a common processing step for many transmembrane proteins, we examined whether NGC was subjected to proteolytic cleavage. Western blotting demonstrated the occurrence of a soluble form of NGC with a 75 kDa core glycoprotein in the soluble fraction of the young rat cerebrum. In contrast, full-length NGC with a 120 kDa core glycoprotein and its cytoplasmic fragment with a molecular size of 35 kDa could be detected in the membrane fraction. The soluble form of NGC was also detectable in culture media of fetal rat neurons, and the full-length form existed in cell layers. The amount of the soluble form in culture media was decreased by adding a physiological protease inhibitor such as a tissue inhibitor of metalloproteinase (TIMP)-2 or TIMP-3, but not by adding TIMP-1. Both EGF-like and neurite outgrowth-promoting activity of the NGC ectodomain may be regulated by this proteolytic processing.

As the first immune cells to infiltrate the nervous system after traumatic PNS and CNS injury, neutrophils (polymorphonuclear leukocytes, PMNs) may promote injury by releasing toxic soluble factors that may affect neuronal survival. Direct neurotoxicity of matrix metalloproteinases (MMPs), reactive oxygen species (ROS), and cytokines released by PMNs was investigated by culturing dorsal root ganglion (DRG) cells with PMN-conditioned media containing MMP inhibitor (GM6001), ROS scavengers, or tumor necrosis factor alphaR (TNF-alphaR) neutralizing antibody. Although DRGs exposed to PMN-conditioned media had 53% fewer surviving neurons than controls, neuronal cell loss was prevented by GM6001 (20 micromol/L), catalase (1000 U/mL), or TNF-alphaR neutralizing antibody (1.5 microg/mL), elevating survival to 77%, 94%, and 95%, respectively. In accordance with protection by GM6001, conditioned media collected from MMP-9 null PMNs was less neurotoxic than that collected from wild-type PMNs. Additionally, MMP inhibition reduced PMN-derived ROS; removal of ROS reduced PMN-derived MMP-9 activity; and TNF-alpha inhibition reduced both PMN-derived MMP-9 activity and ROS in PMN cultures. Our data provide the first direct evidence that PMN-driven neurotoxicity is dependent on MMPs, ROS, and TNF-alpha, and that these factors may regulate PMN release of these soluble factors or interact with one another to mediate PMN-driven neurotoxicity.

S100B protein is found in brain, has been used as a marker for brain injury and is neurotrophic. Using a well-characterized in vitro model of brain cell trauma, we have previously shown that strain injury causes S100B release from neonatal rat neuronal plus glial cultures and that exogenous S100B reduces delayed post-traumatic neuronal damage even when given at 6 or 24 h post-trauma. The purpose of the current studies was to measure post-traumatic S100B release by specific brain cell types and to examine the effect of an antibody to S100 on post-traumatic delayed (48 h) neuronal injury and the protective effect of exogenous S100B. Neonatal rat cortical cells grown on a deformable elastic membrane were subjected to a strain (stretch) injury produced by a 50 ms displacement of the membrane. S100B was measured with an ELISA kit. Trauma released S100B from pure cultures of astrocytes, microglia, and neurons. Anti-S100 reduced released S100B to below detectable levels, increased delayed neuronal injury in traumatized cells and negated the protective effect of exogenous S100B on injured cells. Heat denatured anti-S100 did not exacerbate injury. These studies provide further evidence for a protective role for S100B following neuronal trauma.

Interleukin (IL)-6 is a pro-inflammatory cytokine now widely recognized to contribute to the molecular events that follow CNS injury. Little is known, however, about its action on axonal sprouting and regeneration in the brain. We addressed this issue using the model of transection of Schaffer collaterals in mice organotypic hippocampal slice cultures. Transection of slice cultures was associated with a marked release of IL-6 that could be neutralized by an IL-6 blocking antibody. We monitored functional recovery across the lesion by recording synaptic responses using a multi-electrode array. We found that application of IL-6 antibodies to the cultures after lesioning significantly reduced functional recovery across the lesion. Furthermore, the level of expression of the 43-kDa growth-associated protein (GAP-43) was lower in slices treated with the IL-6 neutralizing antibody than in those treated with a control IgG. Conversely, addition of exogenous IL-6 to the culture medium resulted in a dose-dependent enhancement of functional recovery across the lesion and a higher level of expression of GAP-43. Co-culture of CA3 hemi-slices from thy1-YFP mice with CA1 hemi-slices from wild-type animals confirmed that IL-6-treated co-cultures exhibited an increased number of growing fluorescent fibres across the lesion site. Taken together these data indicate that IL-6 plays an important role in CNS repair mechanisms by promoting regrowth and axon regeneration.

Astroglial activation constitutes a dominant response to all types of injuries of the CNS. Despite the ubiquitous nature of this cellular reaction to neural injury, a little is known concerning the signaling mechanisms that initiate it. Recently, we demonstrated that astrocytic hypertrophy and enhanced expression of glial fibrillary acidic protein resulting from toxicant-induced neurodegeneration are linked to activation of the janus kinase (JAK)-signal transducer and activator of transcription-3 (STAT3) pathway. These observations implicate ligands at the gp130 receptor as potential upstream effectors of astrogliosis. Here we used the brain slice preparation to examine potential activators of the JAK-STAT3 pathway. Following incubation of freshly cut striatal slices in phosphate-free oxygenated buffer for up to 75 min, we found that slicing the striatum itself was a sufficient stimulus to initiate a rapid activation of the JAK-STAT3 pathway as assessed with immunoblots of pSTAT3((tyr705)) using phospho-state specific antibodies. The mRNA for the gp130 cytokines, leukemia inhibitory factor, interleukin-6 and oncostatin M or the beta-chemokine, monocyte chemoattractive protein (CCl2) also were up-regulated in the slice. Moreover, we could enhance the activation of STAT3((tyr705)) by adding exogenous cytokines to the slice and we could inhibit phosphorylation of STAT3((tyr705)) by addition of tyrosine kinase inhibitors (Lav A and AG490) or neutralizing antibodies directed against leukemia inhibitory factor or oncostatin M. These data suggest that STAT3 activation is an early event in slice-induced glial activation and establishes the brain slice preparation method as a reliable model to examine the signaling mechanisms that underlie glial activation.

Cystatin C (CysC) is an endogenous cysteine proteases inhibitor produced by mature astrocytes in the adult brain. Previously we isolated CysC as a factor activating the glial fibrillary acidic protein (GFAP) promoter, and showed that CysC is expressed in astrocyte progenitors during development. Here we show that protease inhibitor activity increased daily in conditioned medium, and that this activity was mainly a result of CysC released from primary cultured cells. Human CysC added to the culture medium of primary brain cells increased the number of GFAP-positive and nestin-positive cells. Human CysC also increased the number of neurospheres formed from embryonic brain, and thus it increases the number of neural stem/precursor cells in a manner similar to glycosylated rat CysC. The addition of a neutralizing antibody, on the other hand, greatly decreased the number of GFAP and glutamate aspartate transporter (GLAST)-positive astrocytes. This decrease was reversed by the addition of CysC but not by another cysteine protease inhibitor. Thus, the promotion of astrocyte development by CysC appears to be independent of its protease inhibitor activity. The antibody increased the number of oligodendrocytes and their precursors. Therefore, CysC modifies glial development in addition to its activity against neural stem/precursor cells.

N-Acetylaspartylglutamate (NAAG) is a neuropeptide found in high concentrations in the brain. Using whole-cell recordings of CA1 pyramidal neurons in acute hippocampal slices, we found that either (i) the application of exogenous NAAG or (ii) an increase of endogenous extracellular NAAG, caused by the inhibition of its catabolic enzyme glutamate carboxypeptidase II (GCP II), resulted in a significant reduction in the amplitude of the isolated NMDA receptor (NMDAR) component of the evoked excitatory postsynaptic current (EPSC). Conversely, reduction of endogenous extracellular NAAG caused by either (i) perfusion with a soluble form of pure human GCP II or (ii) affinity purified antibodies against NAAG, enhanced the amplitude of the isolated NMDAR current. Bath application of GCP II inhibitor induced a progressive loss of spontaneous NMDAR miniatures. Furthermore, NAAG blocked the induction of long-term potentiation at Schaffer collateral axons-CA1 pyramidal neuron synapses. All together, these results suggest that NAAG acts as an endogenous modulator of NMDARs in the CA1 area of the hippocampus.

The immediate early gene pip92 is rapidly and transiently induced by serum, basic fibroblast growth factor (bFGF), nerve growth factor (NGF) and phobol ester, as well as various toxic stimuli. Rho GTPases, such as RhoA, Rac1 and Cdc42, have been implicated in both cytoskeletal rearrangement and cell cycle control. Rac1 and Cdc42 induce neurite outgrowth in many types of neuronal cells. A downstream effector of both Rac1 and Cdc42, p21-activated kinase (Pak1), is highly enriched in neurons. In the present study, we examined the signal transduction pathways involved in pip92 induction, focusing on the involvement of Rho family guanosine 5'-triphosphate (GTP)ases. We also examined the functional role of pip92 expression during FGF-induced neuronal differentiation in embryonic hippocampal cells. Significant and robust activation of c-Jun N-terminal Kinase (JNK), Rac1 and extracellular signal-regulated kinase (ERK) appeared to be important for pip92 induction in response to bFGF. Transient transfection of kinase-inactive MEKK7 or chemical inhibitors of JNK significantly decreased the activation of Rac1 by FGF. However, blockade of Rac1 did not affect JNK activity. Moreover, a MEK-ERK blockade did not affect Rac1 activity. Activation of JNK and Rac1 induced Pak1 activity, which could then phosphorylate and activate transcription factor Elk1. Stimulation of Pak1-dependent Elk1 was required for the bFGF-induced activation of pip92. Suppression of endogenous pip92 expression by siRNA significantly enhanced bFGF-induced neurite outgrowth, while the ectopic expression of pip92 suppressed the neurite extension. Taken together, these data suggest that neurogenic growth factor-induced expression of pip92 is critical for the regulation of neuronal differentiation, occurring through the subsequent activation of Rac1, JNK, Pak1 and Elk1.

Recent evidence has demonstrated that hyaluronan synthase 2 mRNA is up-regulated after brain ischemia. After a cerebral ischemic event, microglia and macrophages are the major inflammatory cells and are activated by hyaluronan (HA). However, it is unclear how these cells compare with regard to HA responsiveness. We show here that peritoneal macrophages and RAW 264.7 macrophages produced more than five- and 10-fold more tumor necrosis factor-alpha (TNF-alpha) than primary microglia and BV-2 microglia, respectively. Antibody blockade study showed that CD44, Toll-like receptor-4 receptor and the receptor for HA-mediated motility were responsible for HA-induced TNF-alpha release. Furthermore, HA induced higher levels of phosphorylated MAPK in RAW 264.7 cells when compared with BV-2 cells. HA-mediated TNF-alpha production required p38 MAPK, extracellular-regulated kinase and c-Jun N-terminal kinase phosphorylation in both cell types. The levels of HA-induced TNF-alpha mRNA expression in BV-2 cells were only twofold lower compared with RAW 264.7 cells, suggesting that a translational event is involved in the differential production of TNF-alpha. Western blot analysis revealed that HA treatment resulted in more rapid phosphorylation of eukaryotic initiation factor 4E-binding protein 1 (4E-BP1) and more effective dissociation of 4E-BP1 from eukaryotic initiation factor 4E in RAW 264.7 cells than in BV-2 cells. Additionally, HA-induced phosphorylation of 4E-BP1 was dependent on MAPK signaling, indicating that RAW 264.7 cells exhibited higher levels of hyperphosphorylated 4E-BP1 possibly due to the overactivation of MAPK. The results suggest that resident microglia and blood-derived monocytes/macrophages exhibit differential sensitivities in response to extracellular mediators after brain ischemia.

Beta amyloid peptide-containing neuritic plaques are a defining feature of Alzheimer's disease pathology. Beta amyloid are 38-43 residue peptides derived by proteolytic cleavage of amyloid precursor protein. Although much attention has focused on the proteolytic events leading to beta amyloid generation, the function of amyloid precursor protein remains poorly described. Previously, we reported that amyloid precursor protein functions as a pro-inflammatory receptor on monocytic lineage cells and defined a role for amyloid precursor protein in adhesion by demonstrating that beta(1) integrin-mediated pro-inflammatory activation of monocytes is amyloid precursor protein dependent. We demonstrated that antibody-induced cross-linking of amyloid precursor protein in human THP-1 monocytes and primary mouse microglia stimulates a tyrosine kinase-based pro-inflammatory signaling response leading to acquisition of a reactive phenotype. Here, we have identified pro-inflammatory mediators released upon amyloid precursor protein-dependent activation of monocytes and microglia. We show that amyloid precursor protein cross-linking stimulated tyrosine kinase-dependent increases in pro-inflammatory cytokine release and a tyrosine kinase-independent increase in beta amyloid 1-42 generation. These data provide much needed insight into the function of amyloid precursor protein and provide potential therapeutic targets to limit inflammatory changes associated with the progression of Alzheimer's disease.

Neuronal and glial cells organizing the central nervous system (CNS) are generated from common neural precursor cells (NPCs) during neural development. However, the expression of cell-surface glycoconjugates that are crucial for determining the properties and biological function of these cells at different stages of development has not been clearly defined. In this study, we investigated the expression of several stage-specific glycoconjugate antigens, including several b-series gangliosides GD3, 9-O-acetyl GD3, GT1b and GQ1b, stage-specific embryonic antigen-1 (SSEA-1) and HNK-1, in mouse embryonic NPCs employing immunocytochemistry and flow cytometry. In addition, several of these antigens were positively identified by chemical means for the first time. We further showed that the SSEA-1 immunoreactivity was contributed by both glycoprotein and glycolipid antigens, and that of HNK-1 was contributed only by glycoproteins. Functionally, SSEA-1 may participate in migration of the cells from neurospheres in an NPC cell culture system, and the effect could be repressed by anti-SSEA-1 antibody. Based on this observation, we identified beta1 integrin as one of the SSEA-1 carrier glycoproteins. Our data thus provide insights into the functional role of certain glycoconjugate antigens in NPCs during neural development.

Activator protein 1 (AP-1) has been reported to regulate the gene expression in a wide variety of cellular processes in response to stimuli. In this study, we investigated the DNA-protein binding activities and promoter activity in the N-methyl-D-aspartate R2B (NR2B) gene AP-1 site in normal and ethanol-treated cultured neurons. The identity of the AP-1 site as the functional binding factor is suggested by the specific binding of nuclear extract derived from cultured cortical neurons to the labeled probes and the specific antibody-induced supershift. Mutations in the core sequence resulted in a significantly reduced promoter activity and the ability to compete for the binding. Moreover, treatment of the cultured neuron with 75 mm ethanol for 5 days caused a significant increase in the AP-1 binding activity and promoter activity. The AP-1 DNA-binding complex in control and ethanol-treated nuclear extract was composed of c-Fos, FosB, c-Jun, JunD, and phosphorylated CREB (p-CREB). Western blot analysis showed that p-CREB and FosB significantly increased, whereas c-Jun decreased. The DNA affinity precipitation assay indicated that FosB, p-CREB, and c-Jun increased in the AP-1 complex following ethanol treatment. These results suggest that AP-1 is an active regulator of the NR2B transcription and ethanol-induced changes may result at multiple levels in the regulation including AP-1 proteins expression, CREB phosphorylation and perhaps reorganization of dimmers.

Astrocytes become activated in response to brain injury, as characterized by increased expression of glial fibrillary acidic protein (GFAP) and increased rates of cell migration and proliferation. Damage to brain cells causes the release of cytoplasmic nucleotides, such as ATP and uridine 5'-triphosphate (UTP), ligands for P2 nucleotide receptors. Results in this study with primary rat astrocytes indicate that activation of a G protein-coupled P2Y(2) receptor for ATP and UTP increases GFAP expression and both chemotactic and chemokinetic cell migration. UTP-induced astrocyte migration was inhibited by silencing of P2Y(2) nucleotide receptor (P2Y(2)R) expression with siRNA of P2Y(2)R (P2Y(2)R siRNA). UTP also increased the expression in astrocytes of alpha(V)beta(3/5) integrins that are known to interact directly with the P2Y(2)R to modulate its function. Anti-alpha(V) integrin antibodies prevented UTP-stimulated astrocyte migration, suggesting that P2Y(2)R/alpha(V) interactions mediate the activation of astrocytes by UTP. P2Y(2)R-mediated astrocyte migration required the activation of the phosphatidylinositol-3-kinase (PI3-K)/protein kinase B (Akt) and the mitogen-activated protein kinase/extracellular signal-regulated kinase (MEK/ERK) signaling pathways, responses that also were inhibited by anti-alpha(V) integrin antibody. These results suggest that P2Y(2)Rs and their associated signaling pathways may be important factors regulating astrogliosis in brain disorders.