Gβ2 mimics activation kinetic slowing of CaV2.2 channels by noradrenaline in rat sympathetic neurons

Several neurotransmitters and hormones acting through G protein-coupled receptors elicit a voltage-dependent regulation of Ca_V2.2 channels, having profound effects on cell function and the organism. It has been hypothesized that protein–protein interactions define specificity in signal transduction. Yet it is unknown how the molecular interactions in an intracellular signaling cascade determine the specificity of the voltage-dependent regulation induced by a specific neurotransmitter. It has been suspected that specific effector regions on the Gβ subunits of the G proteins are responsible for voltage-dependent regulation. The present study examines whether a neurotransmitter’s specificity can be revealed by simple ion-current kinetic analysis likely resulting from interactions between Gβ subunits and the channel-molecule. Noradrenaline is a neurotransmitter that induces voltage-dependent regulation. By using biochemical and patch-clamp methods in rat sympathetic neurons we examined calcium current modulation induced by each of the five Gβ subunits and found that Gβ₂ mimics activation kinetic slowing of Ca_V2.2 channels by noradrenaline. Furthermore, overexpression of the Gβ₂ isoform reproduces the effect of noradrenaline in the willing–reluctant model. These results advance our understanding on the mechanisms by which signals conveying from a variety of membrane receptors are able to display precise homeostatic responses.     

Protective effects of EphB2 on Aβ1–42 oligomer-induced neurotoxicity and synaptic NMDA receptor signaling in hippocampal neurons

Alzheimer’s disease (AD) is a neurodegenerative disorder characterized pathologically by the abnormal deposition of extracellular amyloid-β (Aβ) oligomers. However, the nature and precise mechanism of the toxicity of Aβ oligomers are not clearly understood. Aβ oligomers have been previously shown to cause a major loss of EphB2, a member of the EphB family of receptor tyrosine kinases. To determine the effect of EphB2 on Aβ oligomer-induced neurotoxicity and the underlying molecular mechanisms, we examined the EphB2 gene in cultured hippocampal neurons. Using a cellular model of AD, Aβ_1–42 oligomers were confirmed to induce neurotoxicity in a time-dependent manner and result in a major decrease of EphB2. EphB2 overexpression could prevent the neurotoxicity of hippocampal neurons from exposure to Aβ_1–42 oligomers for 1 h. Further analysis revealed that EphB2 overexpression increased synaptic NR1 and NR2B expression in Aβ_1–42 oligomer-treated neurons. Moreover, EphB2 overexpression prevented Aβ_1–42 oligomer-induced downregulation of dephosphorylated p38 MAPK and phosphorylated CREB. Together, these results suggest that EphB2 is a factor which protects hippocampal neurons against the toxicity of Aβ_1–42 oligomers, and we infer that the protection of EphB2 is achieved by increasing the synaptic NMDA receptor level and downstream p38 MAPK and CREB signaling in hippocampal neurons. This study provides new molecular insights into the neuroprotective effect of EphB2 and highlights its potential therapeutic role in the management of AD.     

Docosahexaenoic acid withstands the Aβ25-35-induced neurotoxicity in SH-SY5Y cells

Background

Docosahexaenoic acid (DHA, C22:6, n-3) ameliorates the memory-related learning deficits of Alzheimer's disease (AD), which is characterized by fibrillar amyloid deposits in the affected brains. Here, we have investigated whether DHA-induced inhibition of Amyloid β-peptide_25-35 (Aβ_25-35) fibrillation limits or deteriorates the toxicity of the human neuroblastoma cells (SH-SY5Y).

Experimental methods

In vitro fibrillation of Aβ_25-35 was performed in the absence or presence of DHA. Afterwards, SH-SY5Y cells were incubated with Aβ_25-35 in absence or presence 20 μM DHA to evaluate its effect on the Aβ_25-35-induced neurotoxicity by MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide)]-redox and TUNEL (TdT-mediated dUTP-biotin nick end-labeling) assay and immunohistochemistry. The level of Aβ_25-35-induced lipid peroxide (LPO) was determined in the absence or presence of oligomer-specific antibody. Fatty acid profile was estimated by gas chromatography.

Results

DHA significantly reduced the Aβ_25-35 in vitro fibrillation, as indicated by fluorospectroscopy and transmission electron microscopy. Aβ_25-35 decreased the MTT-redox activity and increased the apoptotic damage and levels of LPO when compared with those of the controls. However, when the SH-SY5Y cells were treated with Aβ_25-35 in the presence of DHA, MTT redox potential significantly increased and the levels LPO decreased, suggesting an inhibition of the Aβ_25-35-induced neurotoxity. DHA improved the Aβ_25-35 induced DNA damage and axodendritic loss, with a concomitant increase in the cellular level of DHA, suggesting DHA protects the cell from neurotoxic degeneration.

Conclusion

DHA not only inhibits the in vitro fibrillation but also resists the Aβ_25-35-induced toxicity in the neuronal cells. This might be the basis of the DHA-induced amelioration of Aβ-induced neurodegeneration and related cognitive deficits.     

Thioredoxin-1 inhibits amyloid-β25–35-induced activation of NLRP1/caspase-1/GSDMD pyroptotic pathway in PC12 cells

Molecular Biology Reports - Alzheimer’s disease (AD), the most common neurodegenerative disease, is charactered by these accepted pathological features, such as β-amyloid (Aβ)...     

The protective effects of tanshinone IIA on neurotoxicity induced by β-amyloid protein through calpain and the p35/Cdk5 pathway in primary cortical neurons

The characteristic pathological change of Alzheimer's disease (AD) include deposits of β-amyloid protein (Aβ) in brain, neurofibrillary tangles (NFTs), as well as a few neuronal loss. Evidence shows that Aβ causes calcium influx and induces the cleavage of p35 into p25. Furthermore, the binding of p25 to cyclin-dependent kinase 5 (Cdk5) constitutively activates Cdk5. The p25/Cdk5 complex then hyperphosphorylates tau. Tanshinone IIA (tanIIA), a natural product extracted from Chinese herbal medicine Salvia miltiorrhiza BUNGE, has been reported to exert antioxidative activity. However, its neuroprotective activity remains unclear. The present study determined whether tanIIA protects neurons against Aβ(25-35)-induced cytotoxicity and detected the association of this protective effect with calpain and the p35/Cdk5 pathway. The results showed that tanIIA protected neurons against the neurotoxicity of Aβ(25-35), increased the viability of neurons, decreased expression of phosphorylated tau in neurons induced by Aβ(25-35), improved the impairment of the cell ultrastructure (such as nuclear condensation and fragmentation, and neurofibril collapse). Further more, we found that tanIIA maintained the normal expression of p35 on peripheral membranes, and decreased p25 expression in the cytoplasm. TanIIA also inhibited the translocation of Cdk5 from the nucleus into the cytoplasm of primary neurons induced by Aβ(25-35). These data suggested that tanIIA possessed neuroprotective action and the protection may involve in calpain and the p35/Cdk5 pathway.     

Structural Determination of Aβ25-35 Micelles by Molecular Dynamics Simulations

Amyloid-β (Aβ) peptides and other amyloidogenic proteins can form a wide range of soluble oligomers of varied morphologies at the early aggregation stage, and some of these oligomers are biologically relevant to the pathogenesis of Alzheimer's disease. Spherical micelle-like oligomers have been often observed for many different types of amyloids. Here, we report a hybrid computational approach to systematically construct, search, optimize, and rank soluble micelle-like Aβ_25-35 structures with different side-chain packings at the atomic level. Simulations reveal for the first time, to our knowledge, that two Aβ micelles with antiparallel peptide organization and distinct surface hydrophobicity display high structural stability. Stable micelles experience a slow secondary structural transition from turn to α-helix. Energetic analysis coupled with computational mutagenesis reveals that van der Waals and solvation energies play a more pronounced role in stabilizing the micelles, whereas the electrostatic energies present a stable but minor energetic contribution to peptide assemblies. Modeled Aβ micelles with shapes and dimensions similar to those of experimentally derived spherical structures also provide detailed information about the roles of structural dynamics and transition in the formation of amyloid fibrils. The strong binding affinity of our micelles to antibodies implies that micelles may be a biologically relevant species.     

β-amyloid-induced changes in calcium homeostasis in cultured hippocampal neurons of the rat

A two-wave technique of calciometry with the use of a fluorescence dye, fura-2/AM, was applied for examination of the effect of a protein, β-amyloid (the main component of senile plaques in Alzheimer’s disease), on calcium homeostasis in cultured neurons of the rat hippocampus; β-amyloid was added to the culture medium. In most neurons, the effect of β-amyloid appeared as a more than twofold increase in the basic calcium concentration, as compared with the control (153.4 ± 11.5 and 71.7 ± 5.4 nM, respectively; P < 0.05). The characteristics of calcium transients induced by application of hyperpotassium solution also changed; the amplitude of these transients decreased, and the duration of a part corresponding to calcium release from the cell (rundown of the transient) increased. The mean amplitude of calcium transients under control conditions was 447.5 ± 20.1 nM, while after incubation in the presence of β-amyloid this index dropped to 278.4 ± 22.6 nM. Under control conditions, the decline phase of calcium transients lasted, on average, 100 ± 6 sec, while after incubation of hippocampal cell cultures in the presence of β-amyloid this phase lasted 250 ± 10 sec. Therefore, an excess of β-amyloid influences significantly calcium homeostasis in the nerve cells by disturbing functions of the calcium-controlling systems, such as voltage-operated calcium channels of the plasma membrane and calcium stores of the mitochondria and endoplasmic reticulum. Neirofiziologiya/Neurophysiology, Vol. 40, No. 1, pp. 9–12, January–February, 2008.     

Negative Cooperativity between α3β1and α2β1Integrins in Human Mammary Carcinoma MDA MB 231 Cells

The α₃β₁integrin has been implicated as a receptor for several matrix components, including collagen, fibronectin, and laminins. The function of α₃β₁seems to be very versatile involving cell adhesion to or migration on ECM, establishment of cell–cell contacts in aggregates, as well as linkage to intracellular tyrosine phosphorylation cascades. Here we report a strong induction of attachment of α₃β₁integrin expressing human breast carcinoma cell line MDA MB 231 to matrix proteins by two α₃integrin subunit function-blocking monoclonal antibodies (P1B5 and ASC-1). In contrast, stimulation of adhesion to ECM by inhibitory α₃integrin-specific antibodies was not observed in the α₃β₁integrin-expressing nonmalignant human mammary epithelial cell line MCF-10A or the human breast carcinoma cell line MDA MB 468 that expressed relatively low amounts of α₃β₁integrin at the cell surface. This increase was specific for collagens and not observed on fibronectin or laminin. Physiological concentrations of bivalent cations were not required. MAb P1B5 did not induce homotypic aggregation of MDA MB 231 cells. The P1B5-induced increase in cell attachment to collagens could be prevented but not reduced below control levels by blocking mAb to the α₂integrin subunit. Function blocking anti-α₅integrin subunit mAb was without effect while anti-β₁-mAb completely abolished adhesion. Our data indicate that negative cooperativity between integrins results in transdominant inhibition of α₂β₁function by α₃β₁in human MDA MB 231 but not MDA MB 468 tumor cells or nonmalignant MCF-10A cells.     

Calcium-sensing receptor antagonist (calcilytic) NPS 2143 specifically blocks the increased secretion of endogenous Aβ42 prompted by exogenous fibrillary or soluble Aβ25–35 in human cortical astrocytes and neurons—Therapeutic relevance to Alzheimer's disease

The “amyloid-β (Aβ) hypothesis” posits that accumulating Aβ peptides (Aβs) produced by neurons cause Alzheimer's disease (AD). However, the Aβs contribution by the more numerous astrocytes remains undetermined. Previously we showed that fibrillar (f)Aβ_25–35, an Aβ₄₂ proxy, evokes a surplus endogenous Aβ₄₂ production/accumulation in cortical adult human astrocytes. Here, by using immunocytochemistry, immunoblotting, enzymatic assays, and highly sensitive sandwich ELISA kits, we investigated the effects of fAβ_25–35 and soluble (s)Aβ_25–35 on Aβ₄₂ and Aβ₄₀ accumulation/secretion by human cortical astrocytes and HCN-1A neurons and, since the calcium-sensing receptor (CaSR) binds Aβs, their modulation by NPS 2143, a CaSR allosteric antagonist (calcilytic). The fAβ_25–35-exposed astrocytes and surviving neurons produced, accumulated, and secreted increased amounts of Aβ₄₂, while Aβ₄₀ also accrued but its secretion was unchanged. Accordingly, secreted Aβ₄₂/Aβ₄₀ ratio values rose for astrocytes and neurons. While slightly enhancing Aβ₄₀ secretion by fAβ_25–35-treated astrocytes, NPS 2143 specifically suppressed the fAβ_25–35-elicited surges of endogenous Aβ₄₂ secretion by astrocytes and neurons. Therefore, NPS 2143 addition always kept Aβ₄₂/Aβ₄₀ values to baseline or lower levels. Mechanistically, NPS 2143 decreased total CaSR protein complement, transiently raised proteasomal chymotrypsin activity, and blocked excess NO production without affecting the ongoing increases in BACE1/β-secretase and γ-secretase activity in fAβ_25–35-treated astrocytes. Compared to fAβ_25–35, sAβ_25–35 also stimulated Aβ₄₂ secretion by astrocytes and neurons and NPS 2143 specifically and wholly suppressed this effect. Therefore, since NPS 2143 thwarts any Aβ/CaSR-induced surplus secretion of endogenous Aβ₄₂ and hence further vicious cycles of Aβ self-induction/secretion/spreading, calcilytics might effectively prevent/stop the progression to full-blown AD.     

Characterisation of α3β1 and αvβ3 integrin N-oligosaccharides in metastatic melanoma WM9 and WM239 cell lines

It is well documented that glycan synthesis is altered in some pathological processes, including cancer. The most frequently observed alterations during tumourigenesis are extensive expression of β1,6-branched complex type N-glycans, the presence of poly-N-acetyllactosamine structures, and high sialylation of cell surface glycoproteins. This study investigated two integrins, α₃β₁ and α_vβ₃, whose expression is closely related to cancer progression. Their oligosaccharide structures in two metastatic melanoma cell lines (WM9, WM239) were analysed with the use of matrix-assisted laser desorption ionisation mass spectrometry. Both examined integrins possessed heavily sialylated and fucosylated glycans, with β1,6-branches and short polylactosamine chains. In WM9 cells, α₃β₁ integrin was more variously glycosylated than α_vβ₃; in WM239 cells the situation was the reverse. Functional studies (wound healing and ELISA integrin binding assays) revealed that the N-oligosaccharide component of the tested integrins influenced melanoma cell migration on vitronectin and α₃β₁ integrin binding to laminin-5. Additionally, more variously glycosylated integrins exerted a stronger influence on these parameters. To the best of our knowledge, this is the first report concerning structural characterisation of α_vβ₃ integrin glycans in melanoma or in any cancer cells.     

Binding of HIV-1 virions to α4β7 expressing cells and impact of antagonizing α4β7 on HIV-1 infection of primary CD4+ T cells

HIV-1 envelope glycoprotein is reported to interact with α4β7, an integrin mediating the homing of lymphocytes to gut-associated lymphoid tissue, but the significance of α4β7 in HIV-1 infection remains controversial. Here, using HIV-1 strain Ba L, the gp120 of which was previously shown to be capable of interacting with α4β7, we demonstrated that α4β7 can mediate the binding of whole HIV-1 virions to α4β7-expressing transfectants. We further constructed a cell line stably expressing α4β7 and confirmed the α4β7-mediated HIV-1 binding. In primary lymphocytes with activated α4β7 expression, we also observed significant virus binding which can be inhibited by an anti-α4β7 antibody. Moreover, we investigated the impact of antagonizing α4β7 on HIV-1 infection of primary CD4+ T cells. In α4β7-activated CD4+ T cells, both anti-α4β7 antibodies and introduction of shorthairpin RNAs specifically targeting α4β7 resulted in a decreased HIV-1 infection. Our findings indicate that α4β7 may serve as an attachment factor at least for some HIV-1 strains. The established approach provides a promising means for the investigation of other viral strains to understand the potential roles of α4β7 in HIV-1 infection.     

Substoichiometric inhibition of Aβ1-40 aggregation by a tandem Aβ40-1-Gly8-1-40 peptide

Aβ peptides aggregate to form insoluble and neurotoxic fibrils associated with Alzheimer’s disease. Inhibition of the aggregation has been the subject of numerous studies. Here we describe a novel, substoichiometric inhibitor of Aβ_1-40 fibrillization as a tandem dimeric construct consisting of Aβ_40-1 (reverse sequence) linked to Aβ_1-40 via an eight residue glycine linker. At molar ratios of the tandem peptide to Aβ_1-40 of 1:10 to 1:25 inhibition of fibrillization, as measured by ThioflavinT, was observed. We postulate that the tandem construct binds to a fibrillar intermediate but the reverse sequence delays or prevents further monomer association.     

A modified protocol to prepare seed-free starting solutions of amyloid-β (Aβ)1–40 and Aβ1–42 from the corresponding depsipeptides

Preparing reliable, seed-free stock solutions of the highly amyloidogenic peptides amyloid-β (Aβ) is difficult. Besides the formation of aggregates during synthesis and storage, dissolution of the peptide is a critical step because vortexing can induce aggregation. To overcome this, synthesis of the more water-soluble depsi-Aβ_1–42 peptide, from which the native sequence is easily obtained, has been suggested. We further refined this technique, including a cutoff filtration step and switching the depsipeptide in basic conditions, to stabilize the formed native peptide. The obtained solutions of native Aβ_1–40 and Aβ_1–42 peptides were homogeneous and aggregate free, as indicated by thioflavin T and circular dichroism analysis.     

The epigenetic effects of amyloid-β1-40 on global DNA and neprilysin genes in murine cerebral endothelial cells

Amyloid-β (Aβ) is the core component of senile plaques, which are the pathological markers for Alzheimer’s disease and cerebral amyloid angiopathy. DNA methylation/demethylation plays a crucial role in gene regulation and could also be responsible for presentation of senescence. Oxidative stress, which may be induced by Aβ, is thought to be an important contributor of DNA hyper-methylation; however, contradicting this is the fact that global DNA hypo-methylation has been found in aging brains. It therefore remains largely unknown as to whether Aβ does in fact cause DNA methylation/demethylation. Neprilysin (NEP) is one of the enzymes responsible for Aβ degradation, with its expression decreasing in both Alzheimer and aging brains. Using high-performance liquid chromatography (HPLC), we explore whether Aβ is responsible for alteration of the global DNA methylation status on a murine cerebral endothelial cells model, and also use methylation-specific PCR (MSPCR) to examine whether DNA methylation status is altered on the NEP promoter region. We find that Aβ reduces global DNA methylation whilst increasing NEP DNA methylation and further suppressing the NEP expression in mRNA and protein levels. Our results support that Aβ induces epigenetic effects, implying that DNA methylation may be part of a vicious cycle involving the reduction in NEP expression along with a resultant increase in Aβ accumulation, and that Aβ may induce global DNA hypo-methylation.     

Prevention of pathological change and cognitive degeneration of Tg2576 mice by inoculating Aβ1–15 vaccine

This study aims to discuss the effect of preventing pathological changes and cognitive degeneration of Tg2576 mice by inoculating the subunit fragment of Aβ vaccine. Thirty-two Tg2576 mice were randomly divided into four groups, each having eight mice: Group I, the control group, inoculated with adjuvants; Group II, the Aβ42 group, inoculated with Aβ^₄₂ vaccine; Group III, the Aβ_1―15 group, inoculated with Aβ_1―15 vaccine; and Group IV, the Aβ_36―42 group, inoculated with Aβ_36―42 vaccine. The titer of the serum anti-body against Aβ₄₂ (Group II) was significantly higher than that of the control group (Group I), and a low level of antibodies could be detected in the brain homogenate in the three vaccine-inoculated groups. Morris water maze test showed that the Aβ42 group, Aβ_1―15 group and Aβ_36―42 group were obviously im-proved compared with the control group. The cultured splenocytes sampled from each group were induced by Con A or their respective antigens, and the cell proliferation of the three vaccine-inoculated groups was significantly higher than that of the control group. In the Aβ₄₂ group, IL2 and IFN-γ were relatively low and IL4 and IL10 were relatively high. By contrast, IL4 and IL10 were much higher in the Aβ_1―15 group and IL2 and IFN-γ were much higher in the Aβ_36―42 group. The immunohistochemical test showed a large number of senile plaques in the brain cortex and hippocampus of the mice in the con-trol group, no senile plaque in the brain of the Aβ_1―15 group and Aβ₄₂ group mice, and a small number of senile plaques in the brain of the Aβ_36―42 group mice. The results suggest that the subunit fragment of Aβ_1―15 vaccine could prevent not only cognitive and behavioral degeneration but also Aβ deposition and formation of senile plaques in Tg2576 mice.     

Brusatol inhibits amyloid-β-induced neurotoxicity in U-251 cells via regulating the Nrf2/HO-1 pathway

Amyloid-β (Aβ) has been reported to cause oxidative damage of neurons leading to neurotoxicity in a variety of diseases and cancers. As an anticancer drug, brusatol (BR) has been shown to have potent cytotoxic effects on various cancer cell lines. In this study, the effect and mechanism of BR on Aβ-induced neurotoxicity was investigated in U-251 glioma cells. Using the MTT assay, the results suggest that BR ameliorated cell injury induced by Aβ in U-251 cells. After running Hoechst and Western blot assays, BR prevented cell apoptosis induced by Aβ in U-251 cells. In addition, BR inhibited the increased reactive oxygen species and mitochondrial membrane potential levels induced by Aβ in U-251 cells using the DCFH-DA and Rh123 method. Furthermore, BR induced the Nrf2/HO-1 pathway by inhibiting the PI3K/AKT/mTOR pathway to inhibit neurotoxicity elicited by Aβ. These results suggest that brustasol is a valuable potential antitumor drug available for chemotherapy.