In vitro antiaggregation and deaggregation potential of Rhizophora mucronata and its bioactive compound (+)- catechin against Alzheimer’s beta amyloid peptide (25–35)

Objective: Amyloid hypothesis states that endogenous β-amyloid peptides (Aβ), especially its aggregated oligomers and fibrils are the key pathogenic factors leading to Alzheimer’s disease (AD). Therefore, inhibition of Aβ fibrillation rather than blocking its production is considered promising therapeutic intervention. Hence, the present study was carried out to assess the effect of methanolic leaf extract of R. mucronata (MERM) and its bioactive compound catechin on in vitro fibrillation of Aβ (25–35).

Methodology: Antiaggregation and disaggregation effect by MERM and (+)- catechin against Aβ (25–35) were assessed in three different phases by thioflavin T (ThT) fluorescence assay and confocal microscopic analysis. The conformational changes in the aggregated Aβ fibrils in the presence and absence of MERM and catechin were analysed by Fourier transform infrared (FTIR), transmission electron microscopy (TEM) and CD spectroscopy.

Results: Results of ThT and confocal microscopic studies showed decrease in fluorescence intensity in MERM and catechin-treated groups illustrating that both MERM and catechin effectively inhibited fibril aggregation as well as destabilized preformed Aβ fibril. TEM revealed that MERM incubated samples were virtually devoid of structured fibrils but had an amorphous-like consistency, whereas the control contained structured fibrils of various width and length. FTIR analysis showed decrease in absorbance at 1630 cm^?1 (amide I region) in MERM-treated groups substantiating the results of ThT assay. Circular dichroism data indicate that catechin prevents the formation of β-structured aggregates of Aβ peptide.

Conclusion: Results suggest that MERM and catechin might have direct interaction with Aβ peptide preventing its fibrillation.     

Protective effect of colostrinin on neuroblastoma cell survival is due to reduced aggregation of beta-amyloid

Colostrinin (CLN), a mixture of proline-rich polypeptides, has shown a stabilizing effect on cognitive function in Alzheimer's patients measured by the Alzheimer's disease Assessment Scale-cognitive (ADAS-cog) and in Instrumental Activities of Daily Living (ILDL) in recently conducted clinical trials. The aim of this study was to elucidate a possible mode of action of CLN in the treatment of Alzheimer's disease. Here, we report that CLN prevents the aggregation of beta-amyloid peptide Abeta (1-40) in vitro. The impact of CLN on the fibril formation was monitored by optical and electron microscopy. The electron micrographs illustrate that, at 25 microM, Abeta (1-40) peptides formed fibrils after 24-48 h of incubation. The presence of 0.25 microM CLN completely abolished the fibril formation. Abeta (1-40) peptides grow into dense fibers when examined at the 20th day. In the presence of CLN, however, the fibrils are much shorter and less dense. Addition of CLN as late as the 17th day can still dissolves the preformed fibrils. These observations were compared to the effect of CLN on the neurotoxic activity of beta-amyloid peptides in the cell culture model (SHSY-5Y). The beta-amyloid peptides were pre-incubated with CLN at various times and used to treat SHSY-5Y neuroblastoma cells for up to 4 days. The cytotoxic effect was monitored by trypan blue exclusion. We demonstrated that 24-48 h treatment was the onset of toxicity of 10-50 microM of beta-amyloid peptides. Pre-incubation of 0.0025-0.25muM of CLN with 25 microM of beta-amyloid peptides leads to near-complete abolition of cytotoxicity. Low doses of CLN (2.5 nM) can attain cytotoxic protection levels similar to those of highest doses (0.25 microM). Thus, the time course for the appearance of beta-amyloid fibrils coincides with that for cytotoxicity, and that the reduction of fibrils of beta-amyloid peptides by CLN is concomitant with the reduction of the cytotoxic effects of beta-amyloid on SHSY-5Y neuroblastoma cells. Our studies suggest that the neuroprotective effects exerted by CLN are related to the reduction of beta-amyloid fibrils.     

Tanshinones Inhibit Amyloid Aggregation by Amyloid-β Peptide,Disaggregate Amyloid Fibrils,and Protect Cultured Cells

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Neuronal fibrillogenesis: amyloid fibrils from primary neuronal cultures impair long-term memory in the crab Chasmagnathus

Amyloid beta protein (Abeta) fibrillogenesis is considered one of the crucial steps of Alzheimer's disease (AD) pathogenesis. The effect of endogenous neuronal amyloid fibrils on memory processes is unknown. To investigate this issue, we first characterised the Abeta fibrillar aggregates secreted by cerebellar granule cells and then we evaluated the effect of neuronal fibrils on an invertebrate model of memory. An increase of fibril formation, assessed by Thioflavin T (ThT) fluorescence, was observed in the conditioned medium of apoptotic neurons during 48 h of the apoptotic process. Moreover, the depolarisation-stimulated secretion of cerebellar granule cells contains monomers of endogenous Abeta, which undergo cell-free fibrillogenesis over several days of incubation. The pattern of single endogenous fibrils, examined by electron microscopy, was similar to that of synthetic Abeta while a tighter and more complex interfibrillar organization was observed in endogenous fibrils. The biological effect of neuronal fibrils was studied in a long-term memory (LTM) paradigm, namely the context-signal learning of the crab Chasmagnathus. Pre-training injection of neuronal fibril extract (protein concentration, 1 microg/ml) induced amnesia in a dose-dependent manner. On the contrary, no effect on retention was observed with the administration of two orders higher doses (100 microg/ml) of synthetic Abeta1-40. These results indicate that only naturally secreted fibrils, but not synthetic Abeta, clearly interfere with memory process.     

Amyloid β-Protein Aggregation Produces Highly Reproducible Kinetic Data and Occurs by a Two-Phase Process

Protein aggregation can lead to major disturbances of cellular processes and is associated with several diseases. We report kinetic and equilibrium data by ThT fluorescence and enzyme-linked immunosorbent assay of sufficient quality and reproducibility to form a basis for mechanistic understanding of amyloid β-peptide (Aβ) fibril formation. Starting from monomeric peptide in a pure buffer system without cosolvents, we find that the kinetics of Aβ aggregation vary strongly with peptide concentration in a highly predictable manner. The free Aβ concentration in equilibrium with fibrils was found to vary with total peptide concentration in a manner expected for a two-phase system. The free versus total Aβ concentration was linear up to ca. 0.2 μM, after which free Aβ decreased with total Aβ toward an asymptotic value. Our results imply that Aβ fibril formation arises from a sequence of events in a highly predictable manner.     

A natural scavenger of peroxynitrites, rosmarinic acid, protects against impairment of memory induced by Abeta(25-35)

Peroxynitrite (ONOO⁻)-mediated damage is regarded to be responsible for the cognitive dysfunction induced by amyloid beta protein (Aβ) in Alzheimer's disease (AD). In the present study, we examined the protective effects of rosmarinic acid (RA), a natural scavenger of ONOO⁻, on the memory impairment in a mouse model induced by acute i.c.v. injection of Aβ_25–35. Mice daily received i.p. several doses of RA after the injection of Aβ_25–35. RA prevented the memory impairments induced by Aβ_25–35 in the Y maze test and novel object recognition task. RA, at the effective lowest dose (0.25 mg/kg), prevented Aβ_25–35-induced nitration of proteins, an indirect indicator of ONOO⁻ damage, in the hippocampus. At this dose, RA also prevented nitration of proteins and impairment of recognition memory induced by ONOO⁻-i.c.v.-injection. Co-injection of the non-memory-impairing dose of ONOO⁻ with Aβ_25–35 blocked the protective effects of RA (0.25 mg/kg). These results demonstrated that the memory protective effects of RA in the neurotoxicity of Aβ_25–35 is due to its scavenging of ONOO⁻, and that daily consumption of RA may protect against memory impairments observed in AD.     

Role of tau in the polymerization of peptides from β-amyloid precursor protein

The composition of paired helical filaments (PHFs), the intracellular amyloid fibrils that accumulate in the brains of Alzheimer patients, is not completely known. We investigated whether synthetic peptides from β-amyloid precursor protein (APP) can form PHF-like fibrils. Two peptides formed fibrils morphologically similar to PHFs. The presence of tau protein, a known PHF component, greatly enhanced the numbers of fibrils formed from one peptide, from the C-terminus of APP, and became associated with the fibrils. A τ fragment corresponding to the tubulin-binding region was sufficient to induce fibril formation. Tau did not alter fibril formation by the other peptide, which was from the β/A4 region of APP. These results raise the possibility that a C-terminal fragment of APP, along with tau, may be involved in PHF formation. Thus the proteolytic processing of APP may generate fragments that contribute to both amyloids and both histopathologic lesions of Alzheimer's disease.     

Benfotiamine prevents increased β-amyloid production in HEK cells induced by high glucose

Objective To determine whether high glucose enhances β-amyloid (Aβ) production in HEK293 Swedish mutant (APPsw) cells with Aβ precursor protein (APP) overexpression, and whether under this condition benfotiamine reduces the increased Aβ production. Methods HEK293 APPsw cells were cultured with different concentrations of glucose for different times. TheAβ content in the supernatant was determined by ELISA. To investigate the mechanism by which benfotiamine reduced Aβ production, glycogen synthase kinase-3 (GSK-3) activity and expression were measured after the cells were cultured with 5.5 g/L glucose for 12 h. Results With 1.0, 3.0, 4.5, 5.5, 6.5, 7.5, 8.5, or 10.5 g/L glucose, Aβ production by HEK293 APPsw cells was highest in the presence of 5.5 g/L glucose for 6 and 12 h. The difference in Aβ content between 5.5 and 1.0 g/L was most marked after incubation for 12 h. Benfotiamine at 20 and 40 μg/mL significantly reduced Aβ production in cells incubated with 5.5 g/L glucose for 12 h. Moreover, 40 μg/mL benfotiamine significantly enhanced the ratio of phosphorylated GSK-3 to total GSK-3, together with consistent down-regulation of GSK-3 activity. Conclusion High glucose increases Aβ production by HEK293 APPsw cells while benfotiamine prevents this increase. This is correlated with the modulation of GSK-3 activity.     

Charge Dependent Retardation of Amyloid β Aggregation by Hydrophilic Proteins

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Intrinsic Structural Heterogeneity and Long-Term Maturation of Amyloid β Peptide Fibrils

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Abeta42 gene vaccine prevents Abeta42 deposition in brain of double transgenic mice

Aβ₄₂ peptide aggregation and deposition is an important component of the neuropathology of Alzheimer's disease (AD). Gene-gun mediated gene vaccination targeting Aβ₄₂ is a potential method to prevent and treat AD. APPswe/PS1ΔE9 transgenic (Tg) mice were immunized with an Aβ₄₂ gene construct delivered by the gene gun. The vaccinated mice developed Th2 antibodies (IgG1) against Aβ₄₂. The Aβ₄₂ levels in brain were decreased by 41% and increased in plasma 43% in the vaccinated compared with control mice as assessed by ELISA analysis. Aβ₄₂ plaque deposits in cerebral cortex and hippocampus were reduced by 51% and 52%, respectively, as shown by quantitative immunolabeling. Glial cell activation was also significantly attenuated in vaccinated compared with control mice. One rhesus monkey was vaccinated and developed anti-Aβ₄₂ antibody. These new findings advance significantly our knowledge that gene-gun mediated Aβ₄₂ gene immunization effectively induces a Th2 immune response and reduces the Aβ₄₂ levels in brain in APPswe/PS1ΔE9 mice. Aβ₄₂ gene vaccination may be safe and efficient immunotherapy for AD.     

Amyloid-β depresses excitatory cholinergic synaptic transmission in Drosophila

Objective Decline, disruption, or alterations of nicotinic cholinergic mechanisms contribute to cognitive dysfunctions like Alzheimer’s disease (AD). Although amyloid-β (Aβ) aggregation is a pathological hallmark of AD, the mechanisms by which Aβ peptides modulate cholinergic synaptic transmission and memory loss remain obscure. This study was aimed to investigate the potential synaptic modulation by Aβ of the cholinergic synapses between olfactory receptor neurons and projection neurons (PNs) in the olfactory lobe of the fruit fly. Methods Cholinergic spontaneous and miniature excitatory postsynaptic current (mEPSC) were recorded with whole-cell patch clamp from PNs in Drosophila AD models expressing Aβ40, Aβ42, or Aβ42Arc peptides in neural tissue. Results In fly pupae (2 days before eclosion), overexpression of Aβ42 or Aβ42Arc, but not Aβ40, led to a significant decrease of mEPSC frequency, while overexpression of Aβ40, Aβ42, or Aβ42Arc had no significant effect on mEPSC amplitude. In contrast, Pavlovian olfactory associative learning and lifespan assays showed that both short-term memory and lifespan were decreased in the Drosophila models expressing Aβ40, Aβ42, or Aβ42Arc. Conclusion Both electrophysiological and behavioral results showed an effect of Aβ peptide on cholinergic synaptic transmission and suggest a possible mechanism by which Aβ peptides cause cholinergic neuron degeneration and the consequent memory loss.     

Modulation of Alzheimer's beta-amyloid neurotoxicity by site-directed single-chain antibody

A single-chain antibody was constructed from variable regions of heavy and light genes of the parental anti-beta-amyloid peptide IgM 508 antibody. This antibody exhibits anti-aggregating properties, leading to disaggregation of Alzheimer beta-amyloid (betaA) fibrils and prevents its toxic effect on cultured PC-12 cells. Sequencing of the small antibody, namely 508 (Fv), revealed that the V(L) domain contained a cysteine residue in the complementary determining region (CDR)3 (residue 96) which affects its solubility and stability. The cysteine codon was replaced using SOE PCR, and one of the mutants obtained, namely 508F(Fv) (containing phenylalanine instead of cysteine), showed an increased storage stability and higher affinity compared to the wild type. Antibody 508F(Fv) prevents the neurotoxicity of betaA (90% cell viability) and disrupts the fibril structure of beta-amyloid (62% decrease in ThT fluorescence). The ability of antibody 508F(Fv) to dissolve already-formed betaA fibrils makes it a good candidate for intracellular expression and modulation of APP processing as the first step towards the production of therapeutic protection molecules for Alzheimer's disease treatment.     

Melatonin Potentiates the Neuroprotective Properties of Resveratrol Against Beta-Amyloid-Induced Neurodegeneration by Modulating AMP-Activated Protein Kinase Pathways

Background and Purpose

Recent studies have demonstrated that resveratrol (RSV) reduces the incidence of age-related macular degeneration, Alzheimer''s disease (AD), and stroke, while melatonin (MEL) supplementation reduces the progression of the cognitive impairment in AD patients. The purpose of this investigation was to assess whether the co-administration of MEL and RSV exerts synergistic effects on their neuroprotective properties against β-amyloid (Aβ)-induced neuronal death.

Methods

The neuroprotective effects of co-treatment with MEL and RSV on Aβ1-42-induced cell death, was measured by MTT reduction assay. Aβ1-42 caused an increase in intracellular levels of reactive oxygen species (ROS), as assessed by H₂-DCF-DA dye, and a reduction of total glutathione (GSH) levels and mitochondrial membrane potential, as assessed using monochlorobimane and rhodamine 123 fluorescence, respectively. Western blotting was used to investigate the intracellular signaling mechanism involved in these synergic effects.

Results

We treated a murine HT22 hippocampal cell line with MEL or RSV alone or with both simultaneously. MEL and RSV alone significantly attenuated ROS production, mitochondrial membrane-potential disruption and the neurotoxicity induced by Aβ1-42. They also restored the Aβ1-42-induced depletion of GSH, back to within its normal range and prevented the Aβ1-42-induced activation of glycogen synthase kinase 3β (GSK3β). However, co-treatment with MEL and RSV did not exert any significant synergistic effects on either the recovery of the Aβ1-42-induced depletion of GSH or on the inhibition of Aβ1-42-induced GSK3β activation. Aβ1-42 treatment increased AMP-activated protein kinase (AMPK) activity, which is associated with subsequent neuronal death. We demonstrated that MEL and RSV treatment inhibited the phosphorylation of AMPK.

Conclusions

Together, our results suggest that co-administration of MEL and RSV acts as an effective treatment for AD by attenuating Aβ1-42-induced oxidative stress and the AMPK-dependent pathway.     

Morphological Development of β(1-40) Amyloid Fibrils

The Alzheimer's disease-related peptide β(1-40) amyloid self-associates to form fibrils exhibiting a morphology characteristic of amyloidogenic proteins. The mechanism of this fibrillization process has yet to be fully elucidated. In this study we have immobilized the β(1-40) amyloid to flat gold surfaces using thiol-based self-assembled monolayers. Atomic force microscopy reveals the presence of spherical units of β(1-40) amyloid immediately following the initiation of fibrillization. Short fibrillar structures, termed nascent fibrils, which appear to be formed by the association of these units are also present at this time point. At later time points extended, branching networks of fibrils are observed. Some fibrils exhibit a more beaded appearance and greater axial periodicity than others. No nascent fibrils are seen to be present. We believe that these data identify an early fibril structure which could act as an intermediate in β-amyloid fibrillization. The oligomeric units of which these nascent fibrils are comprised are also determined.     

(−)Nicotine inhibits the activations of phospholipases A2 and D by amyloid β peptide

It has been established that amyloid beta peptide (AβP) activates phospholipase A2, phospholipase C and phospholipase D of LA-N-2 cells and other cell types. Nicotine in addition to being a cholinergic agonist, may be neuroprotective. We have investigated the ability of (−)nicotine to blunt the phospholipase activations by AβP in LA-N-2 cells. (−)Nicotine inhibits the AβP activation of phospholipase A2, with an IC₅₀ of 76 μM and of phospholipase D with an IC₅₀ of 252 μM. (−)Nicotine did not blunt the AβP activation of phospholipase C. These inhibitions of AβP activations were not observed with (+)nicotine or cotinine. The (−)nicotine inhibition of AβP activation of these two phospholipases was unaffected by hexamethonium and D-tubocurarine. There was no inhibition of the phospholipase A2 activity present in homogenates of LA-N-2 cells. Exposure of LA-N-2 cells to (−)nicotine for 2 h resulted in the blockade of phospholipase A2 activation by kainate and AβP but did not affect the ability of quisqualate and AβP to activate phospholipase D. These data suggest that if the nicotine inhibition of AβP activations is receptor occupancy mediated then it is by an atypical receptor type.     

Anti-amyloid beta activity of metallothionein-III is different from its neuronal growth inhibitory activity: structure-activity studies

Recent studies have shown that metallothionein-III (MT-III), but not MT-I or -II, antagonizes both the neurotrophic and neurotoxic effects of amyloid β peptides (Aβs). Further, its anti-Aβ-toxicity effect was attributed to the fact that it inhibits the formation of fibrillar Aβ. MT-III alone also affects neuron survival in culture—promoting at low but inhibiting at high concentrations. To characterize these biological activities of MT-III in relation to its neuronal growth inhibitory activity discovered by Uchida et al. [Neuron 7 (1991) 337–347], we here studied effects of the P7S/P9A double mutant, and the N- and C-terminal domains of MT-III on primary cultures of rat embryonic cortical neurons in the presence and absence of Aβ. Results show that (i) only the wild-type MT-III inhibited the formation of SDS-resistant Aβ aggregates and protected cortical neurons from the toxic effect of Aβ, and (ii) both the wild type and the N-terminal domain of MT-III promote neuron survival at low concentrations but inhibited it at high concentrations. On the basis of these findings, we conclude that the anti-Aβ activity of MT-III is different from its neuronal growth inhibitory activity and suggest that the increased trophic activity of AD brain extracts could be attributed to its low MT-III content.     

Examination of potential mechanisms of amyloid-induced defects in neuronal transport

Microtubule-based neuronal transport pathways are impaired during the progression of Alzheimer's disease and other neurodegenerative conditions. However, mechanisms leading to defects in transport remain to be determined. We quantified morphological changes in neuronal cells following treatment with fibrils and unaggregated peptides of beta-amyloid (Aβ). Aβ fibrils induce axonal and dendritic swellings indicative of impaired transport. In contrast, Aβ peptides induce a necrotic phenotype in both neurons and non-neuronal cells. We tested several popular hypotheses by which aggregated Aβ could disrupt transport. Using fluorescent polystyrene beads, we developed experimental models of physical blockage and localized release of reactive oxygen species (ROS) that reliably induce swellings. Like the beads, Aβ fibrils localize in close proximity to swellings; however, fibril internalization is not required for disrupting transport. ROS and membrane permeability are also unlikely to be responsible for fibril-mediated toxicity. Collectively, our results indicate that multiple initiating factors converge upon pathways of defective transport.     

Fibrillar Alzheimer’s amyloid peptide A β1–42stimulates low density lipoprotein binding and cell association, free radical production and cell cytotoxicity in PC12 cells

Amyloid forming peptides are known to disturb vital cellular functions and induce cell death. However, the mechanisms by which fibrillogenic peptides induce cytotoxic effects in various cells has not been established. In this study the effects on low density lipoprotein binding and uptake of fibrils of the Alzheimer’s amyloid β-peptide (Aβ_1–42), which is known to play a central role in the pathogenesis of Alzheimer’s disease, were investigated in pheochromocytoma PC12 cells. Fibrillar Aβ_1–42at μmol concentrations increased low-density lipoprotein (LDL) binding and cell asociation by 460% and 200% respectively, and LDL degradation by about 62%. Approximately 49% and 34% of Aβ fibril stimulated LDL cell association and degradation was inhibited by anti-LDL receptor antibodies. The soluble form of Aβ had no effect on any of these measures of LDL metabolism. The observed increased glutathione reductase activity, DNA fragmentation (TUNEL assay) and decreased DNA synthesis ([³H] thymidine incorporation assay) in cells treated with Aβ_1–42fibrils alone or together with LDL relative to controls, suggests that the interaction of fibrils with LDL receptors might be one possible pathway which contributes to fibril cytotoxicity.     

A new DNA vaccine fused with the C3d-p28 induces a Th2 immune response against amyloid-beta

To enhance anti-amyloid-beta(Aβ) antibody generation and induce a Th2 immune response,we constructed a new DNA vaccine p(Aβ3-10)10-C3d-p28.3 encoding ten repeats of Aβ3-10 and three copies of C3d-p28 as a molecular adjuvant.In this study,we administered this adjuvant intramuscularly to female C57BL/6J mice at 8-10 weeks of age.Enzyme linked immunosorbent assay was used to detect the titer of serum anti-Aβ antibody,isotypes,and cytokines in splenic T cells.A 3(4,5-cimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide assay was used to detect the proliferation rate of splenic T cells.Brain sections from a 12-month-old APP/PS1 transgenic mouse were used for detecting the binding capacities of anti-Aβ antibodies to Aβ plaques.The p(Aβ3-10)10-C3d-p28.3 vaccine induced high titers of anti-amyloid-β antibodies,which bound to Aβ plaques in APP/PS1 transgenic mouse brain tissue,demonstrating that the vaccine is effective against plaques in a mouse model of Alzheimer’s disease.Moreover,the vaccine elicited a predominantly IgG1 humoral response and low levels of interferon-γ in ex vivo cultured splenocytes,indicating that the vaccine could shift the cellular immune response towards a Th2 phenotype.This indicated that the vaccine did not elicit a detrimental immune response and had a favorable safety profile.Our results indicate that the p(Aβ3-10)10-C3d-p28.3 vaccine is a promising immunotherapeutic option for Aβ vaccination in Alzheimer’s disease.