Β-amyloid Fibril Formation Research Articles

Effects of Melissa officinalis Extract Containing Rosmarinic Acid on Cognition in Older Adults Without Dementia: A Randomized Controlled Trial.

Previous in vitro and in vivo studies on Alzheimer's disease (AD) models have reported that rosmarinic acid (RA) can inhibit the formation of amyloid-β fibrils as well as the oligomerization and deposition of amyloid-β protein. Melissa officinalis (M. officinalis) extract containing 500 mg of RA is tolerable and safe in healthy individuals and patients with mild AD dementia. This randomized placebo-controlled double-blind trial aimed to assess the effects of M. officinalis extract on cognition in older adults without dementia. This study included individuals who were diagnosed with subjective or mild cognitive impairment (n = 323). The trial involved M. officinalis extract supplementation (500 mg of RA per day) period of 96 weeks followed by a washout period of 24 weeks. The primary endpoint was the Alzheimer's Disease Assessment Scale-cognitive subscale score, and the secondary endpoints were other cognitive measure results as well as safety and tolerability. There were no significant differences in cognitive measures between the placebo and M. officinalis groups from baseline to 96 weeks. However, based on the analysis of Clinical Dementia Rating Sum of Boxes scores in participants without hypertension, the score was found to be increased by 0.006 and decreased by 0.085 in the M. officinalis and placebo groups, respectively; this difference was statistically significant (p = 0.036). Furthermore, there were no differences in vital signs, physical and neurological measures, or hippocampal volume between the two groups. These results indicate that M. officinalis extract may help prevent cognitive decline in older adults without hypertension.

Dracaena cochinchinensis stemwood extracts inhibit amyloid-β fibril formation and promote neuronal cell differentiation.

Alzheimer’s disease (AD) is a progressive neurodegenerative disorder characterized by the deposition of amyloid plaques in the brain. The prevention of amyloid-β (Aβ)-induced neuronal toxicity is considered a major target for drug development for AD treatment. Dracaena cochinchinensis (Lour.) S.C. Chen, a Thai folk medicine named “Chan-Daeng,” is a member of the Asparagaceae family. The stemwood of D. cochinchinensis has been traditionally used for its antipyretic, pain relief, and anti-inflammatory effects. The aim of the present study was to determine the pharmacological activities of ethanol and water extracts of D. cochinchinensis stemwood in blocking the Aβ fibril formation, preventing Aβ-mediated cell toxicity, and promoting neuronal differentiation in cultured PC12 cells. The herbal extracts of D. cochinchinensis stemwood prevented the formation of Aβ fibrils and disassembled the aggregated Aβ in a dose-dependent manner. Additionally, they prevented Aβ fibril-mediated cell death. The synergy of the herbal extract with a low dose of the nerve growth factor showed an increase in the protein expression of neurofilaments, that is, NF68, NF160, and NF200. These findings suggest that the extracts of D. cochinchinensis stemwood may be used for AD treatment by targeting Aβ fibril formation and inducing neuron regeneration.

Antidiabetic, Antiglycation, and Antioxidant Activities of Ethanolic Seed Extract of Passiflora edulis and Piceatannol In Vitro.

The objective of this work was to investigate the antidiabetic, antiglycation, and antioxidant potentials of ethanolic extract of seeds of Brazilian Passiflora edulis fruits (PESE), a major by-product of the juice industry, and piceatannol (PIC), one of the main phytochemicals of PESE. PESE, PIC, and acarbose (ACB) exhibited IC50 for alpha-amylase, 32.1 ± 2.7, 85.4 ± 0.7, and 0.4 ± 0.1 µg/mL, respectively, and IC50 for alpha-glucosidase, 76.2 ± 1.9, 20.4 ± 7.6, and 252 ± 4.5 µg/mL, respectively. The IC50 of PESE, PIC, and sitagliptin (STG) for dipeptidyl-peptidase-4 (DPP-4) was 71.1 ± 2.6, 1137 ± 120, and 0.005 ± 0.001 µg/mL, respectively. PESE and PIC inhibited the formation of advanced glycation end-products (AGE) with IC50 of 366 ± 1.9 and 360 ± 9.1 µg/mL for the initial stage and 51.5 ± 1.4 and 67.4 ± 4.6 µg/mL for the intermediate stage of glycation, respectively. Additionally, PESE and PIC inhibited the formation of β-amyloid fibrils in vitro up to 100%. IC50 values for 1,1-diphenyl-2-picrylhydrazyl radical (DPPH•) scavenging activity of PESE and PIC were 20.4 ± 2.1, and 6.3 ± 1.3 µg/mL, respectively. IC50 values for scavenging hypochlorous acid (HOCl) were similar in PESE, PIC, and quercetin (QCT) with values of 1.7 ± 0.3, 1.2 ± 0.5, and 1.9 ± 0.3 µg/mL, respectively. PESE had no cytotoxicity to the human normal bronchial epithelial (BEAS-2B), and alpha mouse liver (AML-12) cells up to 100 and 50 µg/mL, respectively. However, 10 µg/mL of the extract was cytotoxic to non-malignant breast epithelial cells (MCF-10A). PESE and PIC were found to be capable of protecting cultured human cells from the oxidative stress caused by the carcinogen NNKOAc at 100 µM. The in vitro evidence of the inhibition of alpha-amylase, alpha-glucosidase, and DPP-4 enzymes as well as antioxidant and antiglycation activities, warrants further investigation of the antidiabetic potential of P. edulis seeds and PIC.

Clusterin in Alzheimer's disease: An amyloidogenic inhibitor of amyloid formation?

Clusterin is a heterodimeric glycoprotein (α- and β-chain), which has been described as an extracellular molecular chaperone. In humans, clusterin is an amyloid-associated protein, co-localizing with fibrillar deposits in several amyloidoses, including Alzheimer's disease. To clarify its potential implication in amyloid formation, we located aggregation-prone regions within the sequence of clusterin α-chain, via computational methods. We had peptide-analogues, which correspond to each of these regions, chemically synthesized and experimentally demonstrated that all of them can form amyloid-like fibrils. We also provide evidence that the same peptide-analogues can inhibit amyloid-β fibril formation, potentially making them appropriate drug candidates for Alzheimer's disease. At the same time, our findings hint that the respective aggregation-prone clusterin regions may be implicated in the molecular mechanism in which clusterin inhibits amyloid formation. Furthermore, we suggest that molecular chaperones with amyloidogenic properties might have a role in the regulation of amyloid formation, essentially acting as functional amyloids.

Deletion of Specific Conserved Motifs from the N-Terminal Domain of αB-Crystallin Results in the Activation of Chaperone Functions.

Smaller oligomeric chaperones of α-crystallins (αA- and αB-) have received increasing attention due to their improved therapeutic potential in preventing protein aggregating diseases. Our previous study suggested that deleting 54–61 residues from the N-terminal domain (NTD) of αB-crystallin (αBΔ54–61) decreases the oligomer size and increases the chaperone function. Several studies have also suggested that NTD plays a significant role in protein oligomerization and chaperone function. The current study was undertaken to assess the effect of deleting conserved 21–28 residues from the activated αBΔ54–61 (to get αBΔ21–28, Δ54–61) on the structure–function of recombinant αBΔ21–28, Δ54–61. The αBΔ21–28, Δ54–61 mutant shows an 80% reduction in oligomer size and 3- to 25-fold increases in chaperone activity against model substrates when compared to αB-WT. Additionally, the αB∆21–28, ∆54–61 was found to prevent β-amyloid (Aβ1–42) fibril formation in vitro and suppressed Aβ1–42-induced cytotoxicity in ARPE-19 cells in a more effective manner than seen with αB-WT or αB∆54–61. Cytotoxicity and reactive oxygen species (ROS) detection studies with sodium iodate (SI) showed that the double mutant protein has higher anti-apoptotic and anti-oxidative activities than the wild-type or αB∆54–61 in oxidatively stressed cells. Our study shows that the residues 21–28 and 54–61 in αB-crystallin contribute to the oligomerization and modulate chaperone function. The deletion of conserved 21–28 residues further potentiates the activated αBΔ54–61. We propose that increased substrate affinity, altered subunit structure, and assembly leading to smaller oligomers could be the causative factors for the increased chaperone activity of αBΔ21–28, Δ54–61.

Membrane Interactions Accelerate the Self-Aggregation of Huntingtin Exon 1 Fragments in a Polyglutamine Length-Dependent Manner.

The accumulation of aggregated protein is a typical hallmark of many human neurodegenerative disorders, including polyglutamine-related diseases such as chorea Huntington. Misfolding of the amyloidogenic proteins gives rise to self-assembled complexes and fibres. The huntingtin protein is characterised by a segment of consecutive glutamines which, when exceeding ~ 37 residues, results in the occurrence of the disease. Furthermore, it has also been demonstrated that the 17-residue amino-terminal domain of the protein (htt17), located upstream of this polyglutamine tract, strongly correlates with aggregate formation and pathology. Here, we demonstrate that membrane interactions strongly accelerate the oligomerisation and β-amyloid fibril formation of htt17-polyglutamine segments. By using a combination of biophysical approaches, the kinetics of fibre formation is investigated and found to be strongly dependent on the presence of lipids, the length of the polyQ expansion, and the polypeptide-to-lipid ratio. Finally, the implications for therapeutic approaches are discussed.

Kinetics of human butyrylcholinesterase inhibition by 1,9-dimethyl-methylene blue

Alzheimer’s disease (AD) is an irreversible and progressive neurodegenerative disorder, characterized by β-amyloid plaques, neurofibrillary tangles and loss of cholinergic neurons. Butyrylcholinesterase (BChE) inhibition is one of the most critical strategy for the treatment of AD since BChE causes inactivation of neurotransmitter acetylcholine and has positive effects on promoting the formation of β-amyloid fibrils. Our previous studies showed that various phenothiazine-derived compounds such as thionine and toluidine blue O (TBO) cause a potent inhibition of human cholinesterases. TBO was also found to reduce amyloid precursor protein processing in-vitro and in-vivo models of AD. In this study, it was aimed to determine the inhibitory effect of 1,9-dimethyl-methylene blue (DMMB), a phenothiazine-derived compound, on human plasma BChE and explore its inhibitory mechanism. The inhibition of human BChE was assessed by the colorimetric method of Ellman using butyrylthiocholine as substrate and 0-0.375 μM of DMMB. The kinetic findings showed that DMMB acts as a linear mixed-type inhibitor of human BChE with Ki value of 23 ± 0.004 nM and α= 3.6 ± 1.6. It was concluded that DMMB, which is a potent inhibitor effective at nM level may be helpful in designing new cholinesterase inhibitors for the treatment of AD.

Cytosolic Free N-Glycans Are Retro-Transported Into the Endoplasmic Reticulum in Plant Cells.

During endoplasmic reticulum (ER)-associated degradation, free N-glycans (FNGs) are produced from misfolded nascent glycoproteins via the combination of the cytosolic peptide N-glycanase (cPNGase) and endo-β-N-acetylglucosaminidase (ENGase) in the plant cytosol. The resulting high-mannose type (HMT)-FNGs, which carry one GlcNAc residue at the reducing end (GN1-FNGs), are ubiquitously found in developing plant cells. In a previous study, we found that HMT-FNGs assisted in protein folding and inhibited β-amyloid fibril formation, suggesting a possible biofunction of FNGs involved in the protein folding system. However, whether these HMT-FNGs occur in the ER, an organelle involved in protein folding, remained unclear. On the contrary, we also reported the presence of plant complex type (PCT)-GN1-FNGs, which carry the Lewisa epitope at the non-reducing end, indicating that these FNGs had been fully processed in the Golgi apparatus. Since plant ENGase was active toward HMT-N-glycans but not PCT-N-glycans that carry β1-2xylosyl and/or α1-3 fucosyl residue(s), these PCT-GN1-FNGs did not appear to be produced from fully processed glycoproteins that harbored PCT-N-glycans via ENGase activity. Interestingly, PCT-GN1-FNGs were found in the extracellular space, suggesting that HMT-GN1-FNGs formed in the cytosol might be transported back to the ER and processed in the Golgi apparatus through the protein secretion pathway. As the first step in elucidating the production mechanism of PCT-GN1-FNGs, we analyzed the structures of free oligosaccharides in plant microsomes and proved that HMT-FNGs (Man9-7GlcNAc1 and Man9-8GlcNAc2) could be found in microsomes, which almost consist of the ER compartments.

Dietary carotenoids related to risk of incident Alzheimer dementia (AD) and brain AD neuropathology: a community-based cohort of older adults

Studies have reported a protective relation to cognitive decline with long-term intake of total and individual dietary carotenoids. However, the underlying mechanisms have not yet been clearly established in humans. To evaluate the prospective association between intakes of total and individual carotenoids and risk of incident Alzheimer dementia (AD) and explore the underlying neuropathological basis. Among 927 participants from the Rush Memory and Aging Project who were free from AD at baseline and were followed up for a mean of 7 y, we estimated HRs for AD using Cox proportional hazards models by intakes of energy-adjusted carotenoids. Brain AD neuropathology was assessed in postmortem brain autopsies among 508 deceased participants. We used linear regression to assess the association of carotenoid intake with AD-related neuropathology. Higher intake of total carotenoids was associated with substantially lower hazard of AD after controlling for age, sex, education, ApoE-ε4, participation in cognitively stimulating activities, and physical activity level. Comparing the top and bottom quintiles (median intake: 24.8 compared with 6.7 mg/d) of total carotenoids, the multivariate HR (95% CI) was 0.52 (0.33, 0.81), P-trend<0.01. A similar association was observed for lutein-zeaxanthin, a weaker linear inverse association was observed for β-carotene, and a marginally significant linear inverse association was found for β-cryptoxanthin. Among the deceased participants, consumers of higher total carotenoids (top compared with bottom tertile, 18.2 compared with 8.2 mg/d) had less global AD pathology (b: -0.10; SE=0.04; P-trend=0.01). For individual carotenoids, lutein-zeaxanthin and lycopene were inversely associated with brain global pathology, whereas lutein-zeaxanthin showed additional inverse associations with AD diagnostic score, neuritic plaque severity, and neurofibrillary tangle density and severity. Our findings support a beneficial role of total carotenoid consumption, in particular lutein/zeaxanthin, on AD incidence that may be related to the inhibition of brain β-amyloid deposition and fibril formation.

K2S2O8-mediated coupling of 6-amino-7-aminomethyl-thiazolino-pyridones with aldehydes to construct amyloid affecting pyrimidine-fused thiazolino-2-pyridones.

We herein present the synthesis of diversely functionalized pyrimidine fused thiazolino-2-pyridones via K2S2O8-mediated oxidative coupling of 6-amino-7-(aminomethyl)-thiazolino-2-pyridones with aldehydes. The developed protocol is mild, has wide substrate scope, and does not require transition metal catalyst or base. Some of the synthesized compounds have an ability to inhibit the formation of Amyloid-β fibrils associated with Alzheimer's disease, while others bind to mature amyloid-β and α-synuclein fibrils.

Modulation of β-Amyloid Fibril Formation in Alzheimer's Disease by Microglia and Infection.

Amyloid plaques are a pathological hallmark of Alzheimer’s disease. The major component of these plaques are highly ordered amyloid fibrils formed by amyloid-β (Aβ) peptides. However, whilst Aβ amyloid fibril assembly has been subjected to detailed and extensive analysis in vitro, these studies may not reproduce how Aβ fibrils assemble in the brain. This is because the brain represents a highly complex and dynamic environment, and in Alzheimer’s disease multiple cofactors may affect the assembly of Aβ fibrils. Moreover, in vivo amyloid plaque formation will reflect the balance between the assembly of Aβ fibrils and their degradation. This review explores the roles of microglia as cofactors in Aβ aggregation and in the clearance of amyloid deposits. In addition, we discuss how infection may be an additional cofactor in Aβ fibril assembly by virtue of the antimicrobial properties of Aβ peptides. Crucially, by understanding the roles of microglia and infection in Aβ amyloid fibril assembly it may be possible to identify new therapeutic targets for Alzheimer’s disease.

Shexiang Baoxin Pill, a Traditional Chinese Herbal Formula, Rescues the Cognitive Impairments in APP/PS1 Transgenic Mice.

BackgroundShexiang Baoxin Pill (SBP), a formulated traditional Chinese medicine (TCM), has been widely used to treat cardiovascular diseases for years. This herbal mixture has been shown to promote differentiation of cultured neuronal cells. Here, we aimed to investigate the effects of SBP in attenuating cognitive impairment in APP/PS1 transgenic mice.MethodsEthanol and water extracts of SBP, denoted as SBPEtOH and SBPwater, were standardized and applied onto cultured rat pheochromocytoma PC12 cells. The potential effect of SBPEtOH extract in attenuating the cognitive impairments in APP/PS1 transgenic mice was shown by following lines of evidence: (i) inhibition of Aβ fibril formation, (ii) suppression of secretions of cytokines, and (iii) improvement of behavioral tests by Morris water maze.ResultsSBPwater and SBPEtOH inhibited the formation of β-amyloid fibrils and protected the Aβ-induced cytotoxicity in cultured PC12 cells. In APP/PS1 transgenic mice, the treatment of SBPEtOH inhibited expressions of NO, NOS, AChE, as well as aggregation of Aβ. Besides, the levels of pro-inflammatory cytokines were suppressed by SBP treatment in the transgenic mice. Importantly, the behavioral tests by Morris Water maze indicated that SBP attenuated cognitive impairments in APP/PS1 transgenic mice.ConclusionThe current result has supported the notion that SPB might ameliorate the cognitive impairment through multiple targets, suggesting that SBP could be considered as a promising anti-AD agent.

In Vitro Anti-cholinesterase Activity and Mass spectrometric Analysis of Curculigo orchioides Gaertn., Rhizome Extract

Curculigo orchioides Gaertn, is commonly known as golden grass. Acetylcholinesterase is an enzyme involved in non-cholinergic and cholinergic activity in both the central and peripheral nervous systems, most of the AChE is found in a tetrameric form bound to neuronal cell membranes. The AChE induces amyloid-β fibril formation and forms very toxic AChE-Aβ complexes in the brain. The study aims to determine the anticholinesterase activity and chemical composition analysed by GC-MS, LC-MS, FT-IR, and HPTLC analysis in the methanolic extract of C. orchioides Gaertn rhizome. The in vitro anti-acetylcholinesterase activity was performed by bio-autograph and spectrophotometry. The methanolic extract of rhizome has better inhibition of the AChE enzyme in vitro experiments. These results might be useful in explaining the effect of the methanolic extract of the rhizome as an anti-acetylcholinesterase agent for the management of AD.

Melatonin Receptor as a Drug Target for Neuroprotection.

Melatonin, a neurohormone secreted from the pineal gland, circulates throughout the body and then mediates several physiological functions. The pharmacological effects of melatonin can be mediated through its direct antioxidant activity and receptor-dependent signaling. This article will mainly review receptor-dependent signaling. Human melatonin receptors include melatonin receptor type 1 (MT1) and melatonin receptor type 2 (MT2), which are widely distributed throughout the brain. Several lines of evidence have revealed the involvement of the melatonergic system in different neurodegenerative diseases. Alzheimer's disease pathology negatively affects the melatonergic system. Melatonin effectively inhibits β-amyloid (Aβ) synthesis and fibril formation. These effects are reversed by pharmacological melatonin receptor blockade. Reductions in MT1 and MT2 expression in the amygdala and substantia nigra pars compacta have been reported in Parkinson's disease patients. The protective roles of melatonin against ischemic insults via its receptors have also been demonstrated. Melatonin has been reported to enhance neurogenesis through MT2 activation in cerebral ischemic/reperfusion mice. The neurogenic effects of melatonin on mesenchymal stem cells are particularly mediated through MT2. Understanding the roles of melatonin receptors in neuroprotection against diseases may lead to the development of specific analogs with specificity and potency greater than those of the original compound. These successfully developed compounds may serve as candidate preventive and disease-modifying agents in the future.

Phenolic composition, antioxidant activity, anticholinesterase potential and modulatory effects of aqueous extracts of some seaweeds on β-amyloid aggregation and disaggregation

Context: Seaweeds contain bioactive compounds with different biological activities. They are used as functional ingredients for the development of therapeutic agents to combat degenerative diseases.Objective: This study investigated the phenolic composition, antioxidant activity, cholinesterase inhibitory and anti-amyloidogenic activities of aqueous extracts of Gracilaria beckeri (J.Agardh) Papenfuss (Gracilariaceae) (RED-AQ), Ecklonia maxima (Osbeck) Papenfuss (Lessoniaceae) (ECK-AQ), Ulva rigida (C.Agardh) Linnaeus (Ulvaceae) (URL-AQ) and Gelidium pristoides (Turner) Kützing (Gelidiaceae) (GEL-AQ).Materials and methods: Phenolic composition of the seaweed extracts was determined using liquid chromatography mass spectrometry. Radical scavenging and metal chelating activities were assessed in vitro. The effect of the extracts (21–84 µg/mL) on acetylcholinesterase and butyrylcholinesterase activities were also investigated using an in vitro colorimetric assay. Transmission electron microscope and thioflavin-T fluorescence assay were used to examine the anti-amyloidogenic activities of the extracts.Results: Phloroglucinol, catechin, epicatechin 3-glucoside were identified in the extracts. ECK-AQ (IC50=30.42 and 280.47 µg/mL) exhibited the highest OH• scavenging and metal chelating activities, while RED-AQ (41.23 and 334.45 µg/mL) exhibited the lowest. Similarly, ECK-AQ (IC50 = 49.41 and 52.11 µg/mL) exhibited higher inhibitory effects on acetylcholinesterase and butyrylcholinesterase activities, while RED-AQ (64.56 and 63.03 µg/mL) showed the least activities. Rapid formation of β-amyloid (Aβ1-42) fibrils and aggregates was observed in electron micrographs of the control after 72 and 96 h. The reduction of Aβ1-42 aggregates occurred after co-treatment with the seaweed extracts.Discussion and conclusion: ECK-AQ, GEL-AQ, URL-AQ and RED-AQ may possess neuroprotective potential and could be explored for the management of Alzheimer’s disease.

Relevance of N-terminal residues for amyloid-β binding to platelet integrin αIIbβ3, integrin outside-in signaling and amyloid-β fibril formation

A pathological hallmark of Alzheimer's disease (AD) is the aggregation of amyloid-β peptides (Aβ) into fibrils, leading to deposits in cerebral parenchyma and vessels known as cerebral amyloid angiopathy (CAA). Platelets are major players of hemostasis but are also implicated in AD. Recently we provided strong evidence for a direct contribution of platelets to AD pathology. We found that monomeric Aβ40 binds through its RHDS sequence to integrin αIIbβ3, and promotes the formation of fibrillar Aβ aggregates by the secretion of adenosine diphosphate (ADP) and the chaperone protein clusterin (CLU) from platelets. Here we investigated the molecular mechanisms of Aβ binding to integrin αIIbβ3 by using Aβ11 and Aβ16 peptides. These peptides include the RHDS binding motif important for integrin binding but lack the central hydrophobic core and the C-terminal sequence of Aβ. We observed platelet adhesion to truncated N-terminal Aβ11 and Aβ16 peptides that was not mediated by integrin αIIbβ3. Thus, no integrin outside-in signaling and reduced CLU release was detected. Accordingly, platelet mediated Aβ fibril formation was not observed. Taken together, the RHDS motif of Aβ is not sufficient for Aβ binding to platelet integrin αIIbβ3 and platelet mediated Aβ fibril formation but requires other recognition or binding motifs important for platelet mediated processes in CAA. Thus, increased understanding of the molecular mechanisms of Aβ binding to platelet integrin αIIbβ3 is important to understand the role of platelets in amyloid pathology.

Strategy to discover full-length amyloid-beta peptide ligands using high-efficiency microarray technology.

Although the formation of β-amyloid (Aβ) fibrils in neuronal tissues is a hallmark of Alzheimer disease (AD), small-sized Aβ oligomers rather than mature fibrils have been identified as the most neurotoxic species. Therefore, the design of new inhibitors, able to prevent the aggregation of Aβ, is believed to be a promising therapeutic approach to AD. Unfortunately, the short-lived intermediate structures that occur in a solution along the Aβ aggregation pathway escape conventional experimental investigations and there is urgent need of new tools aimed at the discovery of agents targeting monomeric Aβ and blocking the early steps of amyloid aggregation. Here, we show the combination of high-efficiency slides (HESs) with peptide microarrays as a promising tool for identifying small peptides that bind Aβ monomers. To this aim, HESs with two immobilized reference peptides, (i.e., KLVFF and Semax) with opposite behavior, were investigated for binding to fluorescently labeled Aβ peptide. Transmission electron microscopy was used to demonstrate Aβ fibrillar aggregates missing. The use of HESs was critical to ensure convenient output of the fluorescent microarrays. The resulting sensitivity, as well as the low sample consumption and the high potential for miniaturization, suggests that the proposed combination of peptide microarrays and highly efficient slides would be a very effective technology for molecule profiling in AD drug discovery.

Cell-penetrating Chaperone Peptide Prevents Protein Aggregation And Protects Against Cell Apoptosis.

Many of the newly discovered therapeutic peptides and molecules are limited by their inability to cross the cell membrane. In the present study we employed a cell penetrating peptide (CPP), VPTLK, derived from Ku70 protein, to facilitate the entry of a mini-chaperone across the cell membrane. Our previous studies suggest that the mini-chaperone peptide representing the chaperone site in αA-crystallin, which can inhibit protein aggregation associated with proteopathies, has therapeutic potential. We have prepared a synthetic mini-chaperone by fusing the VPTLK sequence to N-terminus of mini-chaperone (FVIFLDVKHFSPEDLTVKGRD) to get VPTLKFVIFLDVKHFSPEDLTVKGRD peptide, which we call "CPPGRD." The amino acids, GRD, were added to increase the solubility of the peptide. The chaperone-like function of CPPGRD was measured using unfolding conditions for alcohol dehydrogenase and α-lactalbumin. The anti-apoptotic action of the peptide chaperone was evaluated using H2O2-induced Cos-7 and ARPE-19 cell apoptosis assays. The results show that the CPPGRD has both chaperone function and anti-apoptotic activity. Additionally, the CPPGRD was found to prevent β-amyloid fibril formation and suppress β-amyloid toxicity. The present study demonstrates that the CPPGRD protects unfolding proteins from aggregation and prevents cellular apoptosis. Therefore, the CPPGRD is a mini-chaperone with potential to become a therapeutic agent for protein aggregation diseases.

Receptor-mediated toxicity of human amylin fragment aggregated by short- and long-term incubations with copper ions.

Human amylin (hA1-37) is a polypeptide hormone secreted in conjunction with insulin from the pancreatic β-cells involved in the pathogenesis of type 2 diabetes mellitus (T2DM). The shorter fragment hA17-29 than full-length peptide is capable to form amyloids "in vitro". Here, we monitored the time course of hA17-29 β-amyloid fibril and oligomer formation [without and with copper(II)], cellular toxicity of different amyloid aggregates, and involvement of specific receptors (receptor for advanced glycation end-products, RAGE; low-affinity nerve growth factor receptor, p75-NGFR) in aggregate toxicity. Fibril and oligomer formation of hA17-29 incubated at 37°C for 0, 48, and 120h, without or with copper(II), were measured by the thioflavin T fluorescence assay and ELISA, respectively. Toxicity of hA17-29 aggregates and effects of anti-RAGE and anti-p75-NGFR antibodies were evaluated on neuroblastoma SH-SY5Y viability. Fluorescence assay of hA17-29 indicates an initial slow rate of soluble fibril formation (48h), followed by a slower rate of insoluble aggregate formation (120h). The highest quantity of oligomers was recorded when hA17-29 was pre-aggregated for 48h in the presence of copper(II) showing also the maximal cell toxicity (-44% of cell viability, p<0.01 compared to controls). Anti-RAGE or anti-p75-NGFR antibodies almost abolished cell toxicity of hA17-29 aggregates. These results indicate that copper(II) influences the aggregation process and hA17-29 toxicities are especially attributable to oligomeric aggregates. hA17-29 aggregate toxicity seems to be mediated by RAGE and p75-NGFR receptors which might be potential targets for new drugs in T2DM treatment.

370 - Inhibition of Amyloid-β Fibril Formation and Its Cytotoxicity by Antioxidant-Conjugated Amyloid-β C-Terminal Peptides

Amyloid-β (Aβ) deposition and oxidative stress observed in the brains of patients with Alzheimer’s disease (AD) are important targets for therapeutic intervention. In this study, we focused on the critical role of the C-terminal region of Aβ in the self-aggregation process and oxidative injury in the brains of AD patients. Two series of novel multifunctional antioxidants (TxAβ x-42 and CAAβ x-42 ) were designed by conjugating antioxidants (Trolox (Tx) or Caffeic acid (CA)) to C-terminal motifs of Aβ peptide, Aβ x-42 (x = 34, 36, 38, 40). Among the designed compounds, TxAβ 36-42 and CAAβ 38-42 showed significant anti-aggregation activity and a powerful capacity for scavenging intracellular ROS, resulting in protection of neuronal cells from Aβ 1-42 -induced neurotoxicity. These results suggest that antioxidants containing the C-terminal motif of Aβ 1-42 could become promising therapeutic agents for AD.