Growing evidence indicates that Wingless-type (Wnt) signaling plays an important role in the maturation of the central nervous system. We report here that Wingless-type family member 5A (Wnt-5a) is expressed early in development and stimulates dendrite spine morphogenesis, inducing de novo formation of spines and increasing the size of the preexisting ones in hippocampal neurons. Wnt-5a increased intracellular calcium concentration in dendritic processes and the amplitude of NMDA spontaneous miniature currents. Acute application of Wnt-5a increased the amplitude of field excitatory postsynaptic potentials (fEPSP) in hippocampal slices, an effect that was prevented by calciumchannel blockers. The physiological relevance of our findings is supported by studies showing that Wnt scavengers decreased spine density, miniature excitatory postsynaptic currents, and fEPSP amplitude. We conclude that Wnt-5a stimulates different aspects of synaptic differentiation and plasticity in the mammalian central nervous system.T he Wingless-type (Wnt) signaling pathway modulates several developmental processes, and it is activated by the interaction of the Wnt ligand with members of the Frizzled (Fz) family of seven transmembrane cell-surface receptors (1). It has been reported that Wnt signaling plays a key role in diverse aspects of neuronal development and connectivity (2), regulating axon guidance and remodeling (3), dendrite development (4), synapse formation, (5) and synaptic plasticity (6, 7). Several components of the Wnt pathway are localized at adult synapses, indicating that the molecular machinery required to transduce Wnt signaling is structurally localized at central synapses (8). Different pathways have been described downstream of Fz receptors: the canonical Wnt/β-catenin pathway and the noncanonical ones which involve intracellular signaling by Ca 2+ (the Wnt/Ca 2+ pathway) and the JNK cascade (the Wnt/JNK pathway) (9, 10). Different canonical Wnt ligands have been shown to modulate the presynaptic region. Wnt-7a increases the clustering of synapsin 1 in cerebellar neurons (3) and regulates the trafficking of the α 7 nicotinic acetylcholine receptor to presynaptic terminals in hippocampal neurons (11). In addition, double-mutant mice lacking Wnt-7a and Dishevelled 1 show impaired neurotransmitter release at existing synapses, suggesting a role for Wnt signaling in synaptic transmission (5). Wnt-7a and Wnt-3a were shown to induce the recycling and exocytosis of synaptic vesicles in mature hippocampal neurons and to enhance synaptic transmission in adult hippocampal slices (12). Wnt7a/b levels also were increased in CA3 pyramidal neurons by an enriched environment in which the increase in synapse number at the hippocampal stratum lucidum was shown to be mediated by Wnt signaling (13). Wnt-3a is able to modulate presynaptic differentiation (14,15), and it is released from synapses by an activity-dependent mechanism that facilitates postsynaptic long-term potentiation (6).Recent studies indicated that a different Wnt l...

The mechanisms that induce Alzheimer's disease (AD) are largely unknown thereby deterring the development of disease-modifying therapies. One working hypothesis of AD is that Aβ excess disrupts membranes causing pore formation leading to alterations in ionic homeostasis. However, it is largely unknown if this also occurs in native brain neuronal membranes. Here we show that similar to other pore forming toxins, Aβ induces perforation of neuronal membranes causing an increase in membrane conductance, intracellular calcium and ethidium bromide influx. These data reveal that the target of Aβ is not another membrane protein, but that Aβ itself is the cellular target thereby explaining the failure of current therapies to interfere with the course of AD. We propose that this novel effect of Aβ could be useful for the discovery of anti AD drugs capable of blocking these “Aβ perforates”. In addition, we demonstrate that peptides that block Aβ neurotoxicity also slow or prevent the membrane-perforating action of Aβ.

Alzheimer disease is a progressive neurodegenerative brain disorder that leads to major debilitating cognitive deficits. It is believed that the alterations capable of causing brain circuitry dysfunctions have a slow onset and that the full blown disease may take several years to develop. Therefore, it is important to understand the early, asymptomatic, and possible reversible states of the disease with the aim of proposing preventive and disease-modifying therapeutic strategies. It is largely unknown how amyloid ␤-peptide (A␤), a principal agent in Alzheimer disease, affects synapses in brain neurons. In this study, we found that similar to other pore-forming neurotoxins, A␤ induced a rapid increase in intracellular calcium and miniature currents, indicating an enhancement in vesicular transmitter release. Significantly, blockade of these effects by low extracellular calcium and a peptide known to act as an inhibitor of the A␤-induced pore prevented the delayed failure, indicating that A␤ blocks neurotransmission by causing vesicular depletion. This new mechanism for A␤ synaptic toxicity should provide an alternative pathway to search for small molecules that can antagonize these effects of A␤.

Glycine receptors (GlyRs), together with GABA(A) and nicotinic acetylcholine (ACh) receptors, form part of the ligand-activated ion channel superfamily and regulate the excitability of the mammalian brain stem and spinal cord. Here we report that the ability of the neurotransmitter glycine to gate recombinant and native ionotropic GlyRs is modulated by the G protein betagamma dimer (Gbetagamma). We found that the amplitude of the glycine-activated Cl- current was enhanced after application of purified Gbetagamma or after activation of a G protein-coupled receptor. Overexpression of three distinct G protein alpha subunits (Galpha), as well as the Gbetagamma scavenger peptide ct-GRK2, significantly blunted the effect of G protein activation. Single-channel recordings from isolated membrane patches showed that Gbetagamma increased the GlyR open probability (nP(o)). Our results indicate that this interaction of Gbetagamma with GlyRs regulates both motor and sensory functions in the central nervous system.

Abstract-D-Glucose infusion and gestational diabetes induce vasodilatation in humans and increase L-arginine Key Words: humans Ⅲ endothelium Ⅲ glucose Ⅲ arginine Ⅲ nitric oxide T he cationic amino acid L-arginine is the substrate for nitric oxide (NO) synthesis via endothelial NO synthase (eNOS) 1 and is taken up primarily by the Na ϩ -independent high-affinity (K m Ϸ100 to 400 mol/L) systems y ϩ /CAT-1 and y ϩ /CAT-2B (where CAT indicates cationic amino acid transporter) in human umbilical vein endothelial cells (HUVECs). 2,3 L-Arginine transport and NO synthesis (Larginine/NO pathway) are increased in HUVECs from patients with gestational diabetes. 2 Interestingly, long-term incubation (24 hours) of HUVECs from normal pregnancies with elevated D-glucose mimics the effect of gestational diabetes on the L-arginine/NO pathway. 4 In addition, elevated D-glucose for 24 hours 4,5 or 5 days 6 increases eNOS gene expression. A recent report shows that D-glucose infusion induces vasodilatation in humans, 7 and in animal models, an elevation of plasma D-glucose results in rapid (seconds to minutes) vasodilatation. 8 -10 Therefore, rapid fluctuations in the D-glucose level are crucial in maintaining human fetal endothelial function. [2][3][4][5]11 D-Glucose activates protein kinase C (PKC), an enzyme involved with long-term stimulation of the L-arginine/NO pathway, 5,12-14 and (within 1 hour) p42 and p44 mitogen-activated protein (MAP) kinases (p42/44 mapk ). 5,14,15 p42/44 mapk activation may itself be dependent on PKC activation and NO synthesis. 5,14 However, the effect of short-term incubation with elevated D-glucose on the endothelial L-arginine/NO pathway has not been investigated. 4,11,16,17 The present study shows that a 2-minute incubation with 25 mmol/L D-glucose increases L-arginine transport and NO synthesis in HUVECs. The underlying cellular mechanisms involve phosphorylation of eNOS at Ser 1177 via phosphatidylinositol 3-kinase (PI3-k) and activation of eNOS and p42/ p44 mapk by D-glucose. 18 Materials and Methods Cell CultureHuman umbilical vein endothelium was isolated (collagenase digestion 0.25 mg/mL) and cultured (37°C, 5% CO 2 , confluent passage 2) in medium 199 containing 5 mmol/L D-glucose, 10% newborn calf serum, 10% fetal calf serum, 3.2 mmol/L L-glutamine, 100 mol/L L-arginine, and 100 U/mL penicillin-streptomycin (primary culture medium). [2][3][4] Before an experiment (24 hours), the incubation medium was changed to serum-free medium 199.

Objective. To assess whether autoantibodies against ribosomal P (anti-P), which are possibly pathogenic in neuropsychiatric systemic lupus erythematosus (NPSLE), alter glutamatergic synaptic transmission and to what extent the cross-reacting neuronal surface P antigen (NSPA) is involved.Methods. We analyzed glutamatergic transmission and long-term potentiation (LTP) mediated by AMPA receptor (AMPAR) and N-methyl-D-aspartate receptor (NMDAR) by field excitatory postsynaptic potential (EPSP) at the CA3-CA1 synapse. AMPAR activation by patch-clamp recordings in primary ventral spinal cord neurons was analyzed. In primary hippocampal neurons, NSPA distribution was assessed by double immunofluorescence, and intracellular calcium changes were evaluated using Fura-2 AM. NSPA-LacZ reporter-knockin mice expressing a truncated NSPA were used to assess NSPA expression pattern and function in the brain using b-galactosidase staining and comparative electrophysiology, calcium responses, and water maze memory tests.Results. NSPA was expressed in the brain in hippocampal CA1, dentate gyrus and ventral, but not dorsal, CA3 regions, encompassing postsynaptic regions and partial colocalization with NMDAR. Notably, NSPA-LacZ reporter-knockin mice showed impaired memory, and decreased NMDAR activity and LTP, with neurons insensitive to anti-P autoantibodies. Anti-P autoantibodies enhanced CA1 postsynaptic transmission, increasing AMPAR and NMDAR activity and leading to LTP abrogation after prolonged (20-minute) incubation.Conclusion. Our findings indicate that the neuronal cell surface target of anti-P, NSPA, is involved in glutamatergic synaptic transmission and plasticity related to memory in the hippocampus, and mediates the deleterious effects of anti-P on these processes. Cognitive impairment, as well as other diffuse NPSLE manifestations, may develop when anti-P autoantibodies have access to brain regions coexpressing NSPA, AMPAR, and NMDAR.Neuropsychiatric systemic lupus erythematosus (NPSLE) syndromes include diffuse brain dysfunctions without overt brain inflammation, which makes

A role for Wnt signal transduction in the development and maintenance of brain structures is widely acknowledged. Recent studies have suggested that Wnt signaling may be essential for synaptic plasticity and neurotransmission. However, the direct effect of a Wnt protein on synaptic transmission had not been demonstrated. Here we show that nanomolar concentrations of purified Wnt3a protein rapidly increase the frequency of miniature excitatory synaptic currents in embryonic rat hippocampal neurons through a mechanism involving a fast influx of calcium from the extracellular space, induction of post-translational modifications on the machinery involved in vesicle exocytosis in the presynaptic terminal leading to spontaneous Ca 2؉ transients. Our results identify the Wnt3a protein and a member of its complex receptor at the membrane, the low density lipoprotein receptor-related protein 6 (LRP6) coreceptor, as key molecules in neurotransmission modulation and suggest crosstalk between canonical and Wnt/Ca 2؉ signaling in central neurons.

Alzheimer's disease (AD) is a neurodegenerative disorder characterized by a progressive deterioration of cognitive abilities, amyloid-β peptide (Aβ) accumulation and synaptic alterations. Previous studies indicated that hyperforin, a component of the St John's Wort, prevents Aβ neurotoxicity and some behavioral impairments in a rat model of AD. In this study we examined the ability of tetrahydrohyperforin (IDN5607), a stable hyperforin derivative, to prevent the cognitive deficit and synaptic impairment in an in vivo model of AD. In double transgenic APPswe/PSEN1ΔE9 mice, IDN5706 improves memory and prevents the impairment of synaptic plasticity in a dose-dependent manner, inducing a recovery of long-term potentiation. In agreement with these findings, IDN5706 prevented the decrease in synaptic proteins in hippocampus and cortex. In addition, decreased levels of tau hyperphosphorylation, astrogliosis, and total fibrillar and oligomeric forms of Aβ were determined in double transgenic mice treated with IDN5706. In cultured cells, IDN5706 decreased the proteolytic processing of the amyloid precursor protein that leads to Aβ peptide generation. These findings indicate that IDN5706 ameliorates AD neuropathology and could be considered of therapeutic relevance in AD treatment.