Ganglioside GQ1b ameliorates cognitive impairments in an Alzheimer's disease mouse model, and causes reduction of amyloid precursor protein.

Brain-derived neurotrophic factor (BDNF) plays crucial roles in memory impairments including Alzheimer's disease (AD). Previous studies have reported that tetrasialoganglioside GQ1b is involved in long-term potentiation and cognitive functions as well as BDNF expression. However, in vitro and in vivo functions of GQ1b against AD has not investigated yet. Consequently, treatment of oligomeric Aß followed by GQ1b significantly restores Aß1-42-induced cell death through BDNF up-regulation in primary cortical neurons. Bilateral infusion of GQ1b into the hippocampus ameliorates cognitive deficits in the triple-transgenic AD mouse model (3xTg-AD). GQ1b-infused 3xTg-AD mice had substantially increased BDNF levels compared with artificial cerebrospinal fluid (aCSF)-treated 3xTg-AD mice. Interestingly, we also found that GQ1b administration into hippocampus of 3xTg-AD mice reduces Aß plaque deposition and tau phosphorylation, which correlate with APP protein reduction and phospho-GSK3ß level increase, respectively. These findings demonstrate that the tetrasialoganglioside GQ1b may contribute to a potential strategy of AD treatment.

A novel brain-derived neurotrophic factor-modulating peptide attenuates Aß1-42-induced neurotoxicity in vitro.

Brain-derived neurotrophic factor (BDNF) is a member of the neurotrophin family, which plays important roles in learning and memory formation and in protecting neurons from diverse neurotoxic insults, such as amyloid-beta (Aß). Since BDNF expression is decreased in patients with Alzheimer's disease, various strategies have attempted to increase BDNF levels. In a previous study, we screened and identified a novel BDNF-modulating peptide (consisting of methionine-valine-glycine, named Neuropep-1) by a positional scanning-synthetic peptide combinatorial library (PS-SPCL). Neuropep-1 exhibited neuroprotective effects against in vitro and in vivo Alzheimer's disease models. Based on the previous PS-SPCL data, we modified the amino acid sequence of Neuropep-1 in this study to identify a more potent novel BDNF-modulating peptide. By replacing the valine in the second position with aspartic acid, the resulting Neuropep-4 was found to be highly effective in inducing BDNF expression even at concentrations of 1pM in the SH-SY5Y cell line and rat primary cortical neurons. In addition, among the tested peptides, Neuropep-4 provided neurons with the strongest protection against oligomeric and/or fibrillar Aß1-42-induced cell death through BDNF upregulation. These results suggest the potential of Neuropep-4 as a therapeutic candidate for treating neurodegenerative diseases, such as AD.

The ganglioside GQ1b regulates BDNF expression via the NMDA receptor signaling pathway.

Gangliosides are sialic acid-containing glycosphingolipids which play a role in neuronal functions. Among the gangliosides, tetrasialoganglioside GQ1b shows neurotrophic factor-like actions, such as increasing neurite outgrowth, cell proliferation, and long-term potentiation. In addition, we recently reported that GQ1b improves spatial learning and memory performance in naïve rats. However, it is still unknown how GQ1b exerts its diverse neuronal functions. Thus, we hypothesized that GQ1b might influence synaptic activity by regulating brain-derived neurotrophic factor (BDNF) expression, which is an important protein for synaptic plasticity and cognition. Interestingly, GQ1b treatment increased BDNF expression in GQ1b-null SH-SY5Y cell lines and rat primary cortical neurons. Additionally, we confirmed whether the observed effects were due to GQ1b or due to a ganglioside with fewer sialic acid molecules (GT1b and GD1b) created by the sialidases present on the plasma membranes, by directly applying GT1b and GD1b or GQ1b co-treated with a sialidase inhibitor. Treatment with GT1b or GD1b had no effect on BDNF expression, whereas co-treatment with a sialidase inhibitor and GQ1b significantly increased BDNF levels. Moreover, GQ1b restored the decreased BDNF expression induced by the ganglioside synthesis inhibitor, D-PDMP, in rat primary cortical neurons. GQ1b treatment significantly increased BDNF levels, whereas pretreatment with the N-methyl-d-aspartate (NMDA) receptor antagonist D-AP5 blocked the effects of GQ1b on BDNF expression, suggesting that GQ1b regulates BDNF expression via the NMDA receptor signaling. Finally, we performed an intracerebroventricular GQ1b injection, which resulted in increased prefrontal and hippocampal BDNF expression in vivo. These findings demonstrate, for the first time, that tetrasialoganglioside GQ1b regulates BDNF expression in vitro and in vivo.

Neuropep-1 ameliorates learning and memory deficits in an Alzheimer's disease mouse model, increases brain-derived neurotrophic factor expression in the brain, and causes reduction of amyloid beta plaques.

Alzheimer's disease (AD) is a neurodegenerative disease characterized by amyloid beta (Aß) deposits, hyperphosphorylated tau deposition, and cognitive dysfunction. Abnormalities in the expression of brain-derived neurotrophic factor (BDNF), which plays an important role in learning and memory formation, have been reported in the brains of AD patients. A BDNF modulating peptide (Neuropep-1) was previously identified by positional-scanning synthetic peptide combinatorial library. Here we examine the neuroprotective effects of Neuropep-1 on several in vitro neurotoxic insults, and triple-transgenic AD mouse model (3xTg-AD). Neuropep-1 protects cultured neurons against oligomeric Aß1-42, 1-methyl-4-phenylpyridinium, and glutamate-induced neuronal cell death. Neuropep-1 injection also significantly rescues the spatial learning and memory deficits of 3xTg-AD mice compared with vehicle-treated control group. Neuropep-1 treatment markedly increases hippocampal and cortical BDNF levels. Furthermore, we found that Neuropep-1-injected 3xTg-AD mice exhibit dramatically reduced Aß plaque deposition and Aß levels without affecting tau pathology. Neuropep-1 treatment does not alter the expression or activity of full-length amyloid precursor protein, α-, ß-, or γ-secretase, but levels of insulin degrading enzyme, an Aß degrading enzyme, were increased. These findings suggest Neuropep-1 may be a therapeutic candidate for the treatment of AD.

Mecamylamine attenuates dexamethasone-induced anxiety-like behavior in association with brain derived neurotrophic factor upregulation in rat brains.

Mecamylamine (MEC), which was initially developed as a ganglionic blocker for the treatment of hypertension has been investigated as a potent antagonist for most types of nicotinic acetylcholine receptors (nAChRs). Most studies of MEC have focused on its inhibitory effects for nAChRs; however its biological uses have recently been expanded to the treatment of psychological disorders accompanying anxiety-related symptoms. Although MEC shows obvious anxiolytic action, there is no clear evidence on its function. In this study, we investigated whether MEC affects brain derived neurotrophic factor (BDNF) expression in vitro and in vivo. MEC increased BDNF expression in differentiated SH-SY5Y cells and the cerebral cortex region of rat brains. To determine if the anxiolytic effect of MEC is associated with BDNF upregulation, the elevated plus maze (EPM) task was conducted in a dexamethasone (DEX)-induced anxiety model. MEC reduced DEX-induced anxiety-like behavior, and increased BDNF expression in the cerebral cortex of rats. These results suggest that the anxiolytic effect of MEC in EPM might be associated with BDNF upregulation in the cerebral cortex region of rats. The therapeutic efficacy of MEC for anxiety might be partly dependent on BDNF modulation.

A novel trimeric peptide, Neuropep-1-stimulating brain-derived neurotrophic factor expression in rat brain improves spatial learning and memory as measured by the Y-maze and Morris water maze.

Abundant studies have shown possible links between low levels of brain-derived neurotrophic factor (BDNF) and neurological diseases such as Alzheimer's disease, Parkinson's disease, and depression, as well as stress and anxiety; therefore, BDNF could be a therapeutic target for neurological disorders. In the present study, a positional scanning-synthetic peptide combinatorial library was utilized to identify a peptide modulator of BDNF expression in the hippocampal neuronal cell line, H19-7. A novel tripeptide (Neuropep-1) induced a significant increase of BDNF mRNA and protein levels in H19-7 cells. Pre-treatment of TrkB inhibitor (K252a) did not block Neuropep-1-induced BDNF up-regulation. These results indicate that Neuropep-1 may up-regulate BDNF expression that might be independent of the TrkB receptor pathway. Tail vein injection of Neuropep-1 significantly up-regulated BDNF expression, TrkB phosphorylation, and its downstream signals including activation of Akt, ERK, and cAMP response element binding in the rat hippocampus. To evaluate improvement of spatial learning and memory (SLM) by Neuropep-1-induced BDNF up-regulation, the Y-maze and Morris water maze tests were performed. These results showed Neuropep-1 injection improved SLM performance with increase of BDNF and TrkB expression, activation of TrkB downstream signals in rat hippocampus compared with the control group. However, phosphorylation levels of TrkB were not changed when it was normalized to the level of TrkB expression. The difference on TrkB phosphorylation in Neuropep-1-injected rats may be affected by behavioral tests. These results suggest that Neuropep-1 may improve SLM via activation of the BDNF/TrkB signaling pathway in the rat hippocampus. Therefore, our findings represent that Neuropep-1 might be a potential candidate for treatment of learning and memory disorders as well as neurological diseases involving the abnormal expression of BDNF.

Ganglioside GQ1b improves spatial learning and memory of rats as measured by the Y-maze and the Morris water maze tests.

Gangliosides are major components of cell membranes and are particularly enriched in the mammalian brain where they represent the major lipid constituents of the neuronal cell surface. In the central nervous system, gangliosides have a close connection to many neurophysiological functions related to neurogenesis, proliferation, synaptogenesis, and synaptic transmission. The previously reported effect of the tetra-sialoganglioside GQ1b in hippocampal CA1 neurons of brain slices showed that GQ1b enhanced ATP-induced long-term potentiation (LTP). However, there has been no clear evidence of the effects of GQ1b on learning and memory as measured using behavioral test. In the present study, we performed the Y-maze and the Morris water maze (MWM) tests to reveal the effects of GQ1b on spatial learning and memory following intracerebroventricular (ICV) injection of GQ1b. GQ1b-treated rats showed highly increased performance on the Y-maze and the MWM tests without any significant alteration of basal locomotor activity. Therefore, our behavioral data strongly suggest that GQ1b improves spatial learning and memory in rats. Also, these data support the previous finding that GQ1b treatment in hippocampal CA1 neurons of rodent brain slices increased ATP-induced LTP.

Effects of treadmill exercise on hypoactivity of the hypothalamo-pituitary-adrenal axis induced by chronic administration of corticosterone in rats.

The stress response alters behavior, autonomic function and secretion of multiple hormones, including CRF, ACTH, and glucocorticoid, through the HPA axis. Consecutive stress exposures lead to HPA axis dysregulation such as hyperactivity in Alzheimer's disease and depression, and hypoactivity in post-traumatic stress disorder. In the present study, we established a model of hypoactivated HPA axis in rat through chronic administration of corticosterone (40mg/kg, s.c.) for 19 consecutive days. In this model, CRF mRNA expression in the hypothalamus and ACTH levels in serum were significantly decreased by chronic administration of corticosterone. In addition, the effect of treadmill exercise was investigated in our hypoactivated HPA axis rat model. Treadmill exercise recovered the dysregulated hypoactivity of the HPA axis induced by corticosterone administration for 19 days. The results of the present study suggest that treadmill exercise may aid recovery of hypoactivated HPA axis dysregulation in psychological diseases such as post-traumatic stress disorder.

Decreased hippocampal cholinergic neurostimulating peptide precursor protein associated with stress exposure in rat brain by proteomic analysis.

The stress response alters behavior, autonomic function, and secretion of multiple hormones, including corticotropin-releasing factor, adrenocorticotropin hormone, and cortisol, through the hypothalamic-pituitary-adrenal axis. Constitutive stress responses lead to a number of psychiatric disorders, including depression, posttraumatic stress disorder, Alzheimer's disease (AD), and other anxiety disorders through increased stress hormones and other unknown factors. Here, we performed a proteomic analysis of rat brain exposed to restraint stress compared with a nonstress group by using 2D-DIGE and MALDI-TOF analysis. Several proteins were identified by peptide mass fingerprint (PMF), including down-regulated hippocampal cholinergic neurostimulating peptide precursor protein (HCNP-pp). The current study demonstrates that HCNP-pp mRNA and protein expression are decreased in rat hippocampus after stress exposure. The level of HCNP-pp in H19-7, a rat hippocampal cell line, significantly decreases with dexamethasone treatment, a synthetic glucocorticoid. Thus, this finding suggests that HCNP-pp expression may decrease in response to stress exposure. Decreased HCNP-pp from stress exposure may result in lower levels of HCNP that might contribute to a loss of acetylcholine production.

Detection of surface asialoglycoprotein receptor expression in hepatic and extra-hepatic cells using a novel monoclonal antibody.

The asialoglycoprotein receptor (ASGPR) is a heterodimeric membrane protein which is involved in the internalization of desialylated glycoproteins and also in the binding and uptake of various pathogenic viruses. To facilitate the analysis of ASGPR expression, we generated a monoclonal antibody, termed ASSA-1, that is specific to the ASGPR H1 subunit based on ELISA and Western blots analysis. ASSA-1 also reacted to surface-displayed ASGPR in live cells thus enabling analysis of ASGPR expression by immunofluorescence flow cytometry, which we used to analyze established human liver cell lines previously confirmed to be positive for ASGPR mRNA expression. In agreement with previous reports, surface ASGPR was also detected in extra-hepatic cells and, surprisingly, even in human T cell lines, which was then further confirmed in activated, but not in resting, primary human peripheral blood lymphocytes. These observations suggest that ASGPR has a broad pattern of expression that even extends into cells from the immune system, which biological meanings still have to be analyzed. We expect that monoclonal antibody ASSA-1 will serve as a new powerful tool in analyzing the biological role of ASGPR in hepatic and extra-hepatic cells.

Structure-activity relationships of anti-HIV-1 peptides with disulfide linkage between D- and L-cysteine at positions i and i+3, respectively, derived from HIV-1 gp41 C-peptide.

The constrained a-helical structure of a C-peptide is useful for enhancing anti-HIV-1 activity. The i and i+3 positions in an a-helical structure are located close together, therefore D-Cys (dC) and L-Cys (C) were introduced at the positions, respectively, to make a dC-C disulfide bond in 28mer C-peptides. Accordingly, this study tested whether a dC-C disulfide bond would increase the a-helicity and anti-HIV-1 activity of peptides. A C-peptide can be divided into three domains, the N-terminal hydrophobic domain (HPD), middle interface domain (IFD), and C-terminal hydrogen domain (HGD), based on the binding property with an N-peptide. In general, the dC-C modifications in HPD enhanced the anti-HIV-1 activity, while those in IFD and HGD resulted in no or much less activity. The modified peptides with no activity clearly showed much less a-helicity than the native peptides, while those with higher activity showed an almost similar or slightly increased alpha-helicity. Therefore, the present results suggest that the introduction of a dC-C bridge in the N-terminal hydrophobic domain of a C-peptide may be useful for enhancing the anti-HIV-1 activity.

Stronger anti-HIV-1 activity of C-peptide derived from HIV-1 89.6 gp41 C-terminal heptad repeated sequence.

C34-LAI containing amino acids 118 to 151 of the HIV-1(LAI) gp41 ectodomain exhibits potent anti-HIV-1 activity. However, the N-terminal halves of C34 peptides vary more according to the HIV-1 strain than the C-terminal halves. Therefore, an analysis was conducted on the anti-HIV-1 activities of the C34 peptides derived from various HIV-1 strains. C34-89.6 exhibited the strongest anti-HIV-1 activity among the C34 peptides tested. Interestingly, its N-terminal half was more acidic than those of the other C34 peptides, whereas its C-terminal half was more basic. Since the C-peptides derived from the HIV-1(LAI) strain are used extensively, the anti-HIV-1 activities of these peptides were compared between the HIV-1 strains 89.6 and LAI. When using chimeric peptides, it was found that the C-terminal basic region of C34-89.6 was more critical than its N-terminal basic region. The anti-HIV-1 activity of T20-89.6 and C28-89.6 was also stronger than that of T20-LAI and C28-LAI, respectively. The anti-HIV-1 activity of C28-89.6 was weakened when the C-terminal basic residues were changed to the corresponding residues of C28-LAI. However, no conformational differences were found among the C28 peptides. Accordingly, these results imply that introducing the C-terminal basic residues of the HIV-1 89.6 C-peptide may be useful for developing potent anti-HIV-1 drugs.

Selective isolation and identification of HLA-DR-associated naturally processed and presented epitope peptides.

Activation of CD4 helper T-cells is mediated by the presentation of antigenic peptides in context of self-MHC class II molecules. So far, the rules after which antigen-presenting cells (APC) select a particular epitope within a given protein antigen have been not fully elucidated. Nevertheless, immunoaffinity purification of APC-derived MHC class II molecules and the subsequent elutions of their with associated naturally processed and presented peptide epitopes (NPPE) have helped tremendously in understanding the nature of this rather complex process. In the present study, a novel approach for identifying such NPPEs is introduced, which is based on the culture of APCs in a completely protein-free medium during the antigen presenting process. These APCs do still express a high level of MHC class II as determined by HLA-DR cell surface staining, but the repertoire of the associated NPPEs is drastically reduced when compared to peptides eluted from cells maintained under normal culture condition. Actually, reverse phase-high pressure liquid chromatography (RP-HPLC) revealed that the entire NPPE repertoire consisted of less than ten major peaks, which is more than a 100-fold reduction of background peptide peaks as seen in cells from serum-containing culture conditions. Feeding APCs with exogenous antigens further confirmed the advantage of this novel system. While exogenous antigen-derived peptide peaks in an NPPE-eluate from RP-HPLC are hardly to detect by conventional procedures, the very low background of serum- and protein-free cultured APCs immensely facilitated this process, providing an improved tool for the identification and characterization of NPPEs.

alpha-Melanocyte-stimulating hormone inhibits lipopolysaccharide-induced tumor necrosis factor-alpha production in leukocytes by modulating protein kinase A, p38 kinase, and nuclear factor kappa B signaling pathways.

The neuropeptide alpha-melanocyte-stimulating hormone (alpha-MSH) inhibits inflammation by down-regulating the expression of proinflammatory cytokines such as tumor necrosis factor-alpha (TNF-alpha) in leukocytes via stimulation of alpha-MSH cell surface receptors. However, the signaling mechanism of alpha-MSH action has not yet been clearly elucidated. Here, we have investigated signaling pathways by which alpha-MSH inhibits lipopolysaccharide (LPS)-induced TNF-alpha production in leukocytes such as THP-1 cells. We focused on the possible roles of protein kinase A (PKA), p38 kinase, and nuclear factor kappa B (NF kappa B) signaling. In THP-1 cells, LPS is known to activate p38 kinase, which in turn activates NF kappa B to induce TNF-alpha production. We found that pretreatment of cells with alpha-MSH blocked LPS-induced p38 kinase and NF kappa B activation as well as TNF-alpha production. This response was proportional to alpha-MSH receptor expression levels, and addition of an alpha-MSH receptor antagonist abolished the inhibitory effects. In addition, alpha-MSH treatment activated PKA, and PKA inhibition abrogated the inhibitory effects of alpha-MSH on p38 kinase activation, NF kappa B activation, and TNF-alpha production. Taken together, our results indicate that stimulation of PKA by alpha-MSH causes inhibition of LPS-induced activation of p38 kinase and NF kappa B to block TNF-alpha production.

A vector system for introducing foreign HIV-1 env genes and pseudotyping of MuLV particles with the recombinant HIV-1 envelope proteins for anti-HIV-1 assay.

We attempted to incorporate the HIV-1 envelope proteins derived from various HIV-1 strains into MuLV particles for developing a rapid and safe anti-HIV-1 screening system. In a previous study, only HIV-1 envelope protein lacking cytoplasmic 144 amino acids has been reported to be able to incorporate into MuLV particles. We designed and constructed a vector, pcKCX, expressing the envelope glycoprotein with cytoplasmic truncation by introducing the partial foreign HIV-1 env gene corresponding to the ectodomain of its envelope protein. Three HIV-1 env genes of AD8, BaL or 89.6 strains were cloned, and the HIV-1/MuLV pseudotypes were generated in the transfected TELCeB6 cells with all the cloned plasmids. The pseudotypes displayed host specificity depending on their original HIV-1 strains and their infection to the target cells was inhibited by treatment of a potent anti-HIV-1 peptide C34. A stable cell clone against the HIV-1(BaL) strain was found to express the R5 tropic envelope glycoprotein on the cell surface and to produce continuously HIV-1(BaL)/MuLV pseudotypes. These results suggested that the vector system is useful for cloning of various foreign HIV-1 env genes and the recombinant envelope glycoproteins effectively incorporate into MuLV particles. The HIV-1/MuLV pseudotypes may be useful for anti-HIV-1 assay.