Detection of Dietary Chalcone and Flavonoid Metabolites in Mice Using UPLC-MS/MS and Their Modulatory Effects on Amyloid ß Aggregation.

Amyloid ß-protein (Aß42) aggregates have been demonstrated to induce cognitive decline and neurodegeneration in Alzheimer's disease (AD). Thus, functional food ingredients that inhibit Aß42 aggregation are valuable for AD prevention. Although several food ingredients have been studied for their anti-aggregation activity, information on their bioavailability in the brain, incorporated forms, and relevance to AD etiology is limited. Here, we first detected the sulfate- and glucuronic-acid-conjugated forms of green perilla-derived chalcone (1) and taxifolin (2), which inhibit Aß42 aggregation, in the brain, small intestine, and plasma of mice (1 and 2 were administered orally) using ultra-performance liquid chromatography-tandem mass spectrometry. We observed that the conjugated metabolites (sulfate (4) and glucuronide (5)) of 1 prevented the fibrillization and oligomerization of Aß42. These findings imply that the conjugated metabolites of 1 can prove beneficial for AD treatment.

Lysine-targeting inhibition of amyloid ß oligomerization by a green perilla-derived metastable chalcone in vitro and in vivo.

Oligomers of amyloid ß (Aß) represent an early aggregative form that causes neurotoxicity in the pathogenesis of Alzheimer's disease (AD). Thus, preventing Aß aggregation is important for preventing AD. Despite intensive studies on dietary compounds with anti-aggregation properties, some identified compounds are susceptible to autoxidation and/or hydration upon incubation in water, leaving unanswered issues regarding which active structures in metastable compounds are actually responsible for the inhibition of Aß aggregation. In this study, we observed the site-specific inhibition of 42-mer Aß (Aß42) oligomerization by the green perilla-derived chalcone 2',3'-dihydroxy-4',6'-dimethoxychalcone (DDC), which was converted to its decomposed flavonoids (dDDC, 1-3) via nucleophilic aromatic substitution with water molecules. DDC suppressed Aß42 fibrillization and slowed the transformation of the ß-sheet structure, which is rich in Aß42 aggregates. To validate the contribution of dDDC to the inhibitory effects of DDC on Aß42 aggregation, we synthesized 1-3 and identified 3, a catechol-type flavonoid, as one of the active forms of DDC. 1H-15N SOFAST-HMQC NMR revealed that 1-3 as well as DDC could interact with residues between His13 and Leu17, which were near the intermolecular ß-sheet (Gln15-Ala21). The nucleation in Aß42 aggregates involves the rate-limiting formation of low-molecular-weight oligomers. The formation of a Schiff base with dDDC at Lys16 and Lys28 in the dimer through autoxidation of dDDC was associated with the suppression of Aß42 nucleation. Of note, in two AD mouse models using immunoaffinity purification-mass spectrometry, adduct formation between dDDC and brain Aß was observed in a similar manner as reported in vitro. The present findings unraveled the lysine-targeting inhibitory mechanism of metastable dietary ingredients regarding Aß oligomerization.

Interactions of amyloid coaggregates with biomolecules and its relevance to neurodegeneration.

The aggregation of amyloidogenic proteins is a pathological hallmark of various neurodegenerative diseases, including Alzheimer's disease, Parkinson's disease, and amyotrophic lateral sclerosis. In these diseases, oligomeric intermediates or toxic aggregates of amyloids cause neuronal damage and degeneration. Despite the substantial effort made over recent decades to implement therapeutic interventions, these neurodegenerative diseases are not yet understood at the molecular level. In many cases, multiple disease-causing amyloids overlap in a sole pathological feature or a sole disease-causing amyloid represents multiple pathological features. Various amyloid pathologies can coexist in the same brain with or without clinical presentation and may even occur in individuals without disease. From sparse data, speculation has arisen regarding the coaggregation of amyloids with disparate amyloid species and other biomolecules, which are the same characteristics that make diagnostics and drug development challenging. However, advances in research related to biomolecular condensates and structural analysis have been used to overcome some of these challenges. Considering the development of these resources and techniques, herein we review the cross-seeding of amyloidosis, for example, involving the amyloids amyloid ß, tau, α-synuclein, and human islet amyloid polypeptide, and their cross-inhibition by transthyretin and BRICHOS. The interplay of nucleic acid-binding proteins, such as prions, TAR DNA-binding protein 43, fused in sarcoma/translated in liposarcoma, and fragile X mental retardation polyglycine, with nucleic acids in the pathology of neurodegeneration are also described, and we thereby highlight the potential clinical applications in central nervous system therapy.

Activity-differential search for amyloid-ß aggregation inhibitors using LC-MS combined with principal component analysis.

Aggregation of amyloid ß42 (Aß42) is one of the hallmarks of Alzheimer's disease (AD). Inhibition of Aß42 aggregation is thus a promising approach for AD therapy. Kampo medicine has been widely used to combat dementias such as AD. Crude drug known as Shoyaku is an ingredient of Kampo that could have potential as a natural source of novel drugs. However, given that a mixture of compounds, rather than singular compounds, could contribute to the biological functions of crude drug, there are very limited studies on the structure and mechanism of each constituent in crude drug which may have anti-Aß42 aggregation properties. Herein we provide an efficient method, using LC-MS combined with principal component analysis (PCA), to search for activity-dependent compounds that inhibit Aß42 aggregation from 46 crude drug extracts originating from 18 plants. Only 5 extracts (Kakou, Kayou, Gusetsu, Rensu, and Renbou) from lotus demonstrated differentially inhibitory activities depending on the part of the plant from which they are derived (e.g. petiole, leaf, root node, stamen, and receptacle, respectively). To compare the anti-aggregative properties of compounds of active crude drug with those of inactive crude drug, these extracts were subjected to LC-MS measurement, followed by PCA. From 12 candidate compounds identified from the analysis, glucuronized and glucosidized quercetin, as well as 6 flavonoids (datiscetin, kaempferol, morin, robinetin, quercetin, and myricitrin), including catechol or flatness moiety suppressed Aß42 aggregation, whereas curcumol, a sesquiterpene, did not. In conclusion, this study offers a new activity-differential methodology to identify bioactive natural products in crude drugs that inhibit Aß42 aggregation and that could be applied to future AD therapies.

Synthetic Biology-Based Discovery of Diterpenoid Pyrones from the Genome of Eupenicillium shearii.

Diterpenoid pyrones are a type of mainly fungal meroterpenoid metabolite consisting of a diterpene connected to a pyrone, some of which show potent bioactivity. Through genome mining and heterologous expression, nine new diterpenoid pyrones, shearones A-I (1-9), were discovered from the fungus Eupenicillium shearii IFM 42152, and their biosynthetic enzyme activities were revealed. Some of these heterologously biosynthesized diterpenoid pyrones exhibited moderate antiaggregative ability against amyloid ß42 in vitro.

Aptamers targeting amyloidogenic proteins and their emerging role in neurodegenerative diseases.

Aptamers are oligonucleotides selected from large pools of random sequences based on their affinity for bioactive molecules and are used in similar ways to antibodies. Aptamers provide several advantages over antibodies, including their small size, facile, large-scale chemical synthesis, high stability, and low immunogenicity. Amyloidogenic proteins, whose aggregation is relevant to neurodegenerative diseases, such as Alzheimer's, Parkinson's, and prion diseases, are among the most challenging targets for aptamer development due to their conformational instability and heterogeneity, the same characteristics that make drug development against amyloidogenic proteins difficult. Recently, chemical tethering of aptagens (equivalent to antigens) and advances in high-throughput sequencing-based analysis have been used to overcome some of these challenges. In addition, internalization technologies using fusion to cellular receptors and extracellular vesicles have facilitated central nervous system (CNS) aptamer delivery. In view of the development of these techniques and resources, here we review antiamyloid aptamers, highlighting preclinical application to CNS therapy.

A screen for sleep and starvation resistance identifies a wake-promoting role for the auxiliary channel unc79.

The regulation of sleep and metabolism are highly interconnected, and dysregulation of sleep is linked to metabolic diseases that include obesity, diabetes, and heart disease. Furthermore, both acute and long-term changes in diet potently impact sleep duration and quality. To identify novel factors that modulate interactions between sleep and metabolic state, we performed a genetic screen for their roles in regulating sleep duration, starvation resistance, and starvation-dependent modulation of sleep. This screen identified a number of genes with potential roles in regulating sleep, metabolism, or both processes. One such gene encodes the auxiliary ion channel UNC79, which was implicated in both the regulation of sleep and starvation resistance. Genetic knockdown or mutation of unc79 results in flies with increased sleep duration, as well as increased starvation resistance. Previous findings have shown that unc79 is required in pacemaker for 24-hours circadian rhythms. Here, we find that unc79 functions in the mushroom body, but not pacemaker neurons, to regulate sleep duration and starvation resistance. Together, these findings reveal spatially localized separable functions of unc79 in the regulation of circadian behavior, sleep, and metabolic function.

Searching for Natural Products That Delay Nucleation Phase and Promote Elongation Phase of Amyloid ß42 toward Alzheimer's Disease Therapeutics.

Aggregation of amyloid ß42 (Aß42) is one of the hallmarks of Alzheimer's disease (AD). The mechanism of Aß42 aggregation mainly consists of two phases, nucleation and elongation (including plateau region as a saturation phase). During the nucleation phase, the monomer gradually forms toxic oligomers. During the elongation phase, each nucleus acts as a template and associates with monomers to initiate less toxic fibrillization. We previously proposed a method of classifying compounds into nine groups based on their ability to modulate the nucleation and/or elongation phases. An orcein derivative (O4), which is a phenoxazine dye isolated from the lichen Roccella tinctoria and containing a 2,5-cyclohexadienone moiety, was reported to convert oligomers into relatively inert fibrils, resulting in the reduction of the neurotoxicity of Aß42. Focusing on O4 in the pursuit of anti-AD drugs, we herein screened 480 natural products including NPDepo (RIKEN) for the compounds that delayed the nucleation phase and promoted the elongation phase. The signal intensities for Aß42 treated with each of the 15 compounds that met these criteria were lowered in dot blotting using antioligomer antibody, and the fibril formation of Aß42 in the presence of these compounds was observed in transmission electron microscopy. Among the 15 compounds, 12 compounds (80%) reduced the toxicity of Aß42 against mouse neuroblastoma Neuro-2a cells. Some of these anticytotoxic compounds contain 2-pyrone and 4-pyrone that interacted with Aß42, maybe by shifting the equilibrium of Aß from toxic oligomer into inert fibrils.

[Development of a Novel Alzheimer's Disease Model Based on the Theory of the Toxic-conformer of Amyloid ß].

Development of therapeutics for Alzheimer's disease (AD) is an urgent research task. Amyloid ß (Aß) is one of the causative proteins of AD. Irie et al. identified a toxic conformer among the various structures of 42-mer Aß (Aß42). This conformer, which possesses a turn structure at the positions Glu22-Asp23, exhibits rapid oligomerization and potent neurotoxicity. By the generation of conformationally-specific antibodies against this toxic conformer of Aß, elevation of the toxic conformer in the AD brain was strongly suggested. To investigate the pathogenic role of the toxic conformer in AD, passive immunization experiments against conventional AD model mice were conducted. Specific antibody administration improved the behavioral abnormalities observed in AD model mice without affecting senile plaque pathology. Next, knock-in mice exclusively producing the toxic conformer of Aß were generated. These mice exhibited cognitive dysfunction and oligomerization of Aß, which preceded the onset of the plaque deposition. Taken together, the toxic conformer of Aß is confirmed to be involved in the pathogenesis of AD, and our knock-in mice could be useful in analyzing the Aß oligomer-related pathology of AD.

Detection of Amyloid ß Oligomers with RNA Aptamers in AppNL-G-F/NL-G-F Mice: A Model of Arctic Alzheimer's Disease.

RNA aptamers have garnered attention for diagnostic applications due to their ability to recognize diverse targets. Oligomers of 42-mer amyloid ß-protein (Aß42), whose accumulation is relevant to the pathology of Alzheimer's disease (AD), are among the most difficult molecules for aptamer recognition because they are prone to aggregate in heterogeneous forms. In addition to designing haptens for in vitro selection of aptamers, the difficulties involved in determining their effect on Aß42 oligomerization impede aptamer research. We previously developed three RNA aptamers (E22P-AbD4, -AbD31, and -AbD43) with high affinity for protofibrils (PFs) derived from a toxic Aß42 dimer. Notably, these aptamers recognized diffuse staining, which likely originated from PFs or higher-order oligomers with curvilinear structures in a knock-in AppNL-G-F/NL-G-F mouse, carrying the Arctic mutation that preferentially induced the formation of PFs, in addition to a PS2Tg2576 mouse. To determine which oligomeric sizes were mainly altered by the aptamer, ion mobility-mass spectrometry (IM-MS) was carried out. One aptamer, E22P-AbD43, formed adducts with the Aß42 monomer and dimer, leading to suppression of further oligomerization. These findings support the utility of these aptamers as diagnostics for AD.

Synthetic and Biophysical Studies on the Toxic Conformer in Amyloid ß with the E22Δ Mutation in Alzheimer Pathology.

The toxic conformer of the 40- or 42-mer-amyloid ß-proteins (Aß) (Aß40, Aß42) with a turn at positions 22 and 23 plays a role in oligomer formation, leading to neurotoxicity as part of the pathogenesis of Alzheimer's disease (AD). A deletion mutant at Glu22 (E22Δ) of Aß, known as an Osaka mutation, accelerates oligomerization. Although E22Δ-Aß has not been found to be toxic to cultured neuronal cells and is instead synaptotoxic in long-term potentiation, there is no information on the toxic conformer of E22Δ-Aß in AD. The site-directed spin labeling study of E22Δ-Aß40 by continuous wave-electron spin resonance (CW-ESR) spectroscopy in part showed the spatial proximity between positions 10 and 35, which are characteristic of the toxic conformation of Aß, indicating the existence of a toxic conformer of Aß with the E22Δ mutation. To obtain structural insight, E22Δ-Aß42 substitutes with proline (F20P, A21P, D23P, and V24P), in which proline is known as a turn inducer but is a ß-sheet breaker, were synthesized. An enzyme immunoassay using the 24B3 antibody recognizing toxic conformer of Aß was carried out. 24B3 reacted with these substitutes of E22Δ-Aß42 as well as E22Δ-Aß42 in a similar manner to WT-Aß42. Notably, only A21P-E22Δ-Aß42 exhibited strong neurotoxicity in rat primary neurons after 8 days of incubation, with potent high-order oligomerization compared with E22Δ-Aß42. These results suggest that E22Δ-Aß42 could enhance neurotoxicity by generating a toxic oligomer conformation with a turn near position 21.

Synthetic biology based construction of biological activity-related library of fungal decalin-containing diterpenoid pyrones.

A synthetic biology method based on heterologous biosynthesis coupled with genome mining is a promising approach for increasing the opportunities to rationally access natural product with novel structures and biological activities through total biosynthesis and combinatorial biosynthesis. Here, we demonstrate the advantage of the synthetic biology method to explore biological activity-related chemical space through the comprehensive heterologous biosynthesis of fungal decalin-containing diterpenoid pyrones (DDPs). Genome mining reveals putative DDP biosynthetic gene clusters distributed in five fungal genera. In addition, we design extended DDP pathways by combinatorial biosynthesis. In total, ten DDP pathways, including five native pathways, four extended pathways and one shunt pathway, are heterologously reconstituted in a genetically tractable heterologous host, Aspergillus oryzae, resulting in the production of 22 DDPs, including 15 new analogues. We also demonstrate the advantage of expanding the diversity of DDPs to probe various bioactive molecules through a wide range of biological evaluations.

An RNA aptamer with potent affinity for a toxic dimer of amyloid ß42 has potential utility for histochemical studies of Alzheimer's disease.

Oligomers of ß-amyloid 42 (Aß42), rather than fibrils, drive the pathogenesis of Alzheimer's disease (AD). In particular, toxic oligomeric species called protofibrils (PFs) have attracted significant attention. Herein, we report RNA aptamers with higher affinity toward PFs derived from a toxic Aß42 dimer than toward fibrils produced from WT Aß42 or from a toxic, conformationally constrained Aß42 variant, E22P-Aß42. We obtained these RNA aptamers by using the preincubated dimer model of E22P-Aß42, which dimerized via a linker located at Val-40, as the target of in vitro selection. This dimer formed PFs during incubation. Several physicochemical characteristics of an identified aptamer, E22P-AbD43, suggested that preferential affinity of this aptamer toward PFs is due to its higher affinity for the toxic dimer unit (KD = 20 ± 6.0 nm) of Aß42 than for less-toxic Aß40 aggregates. Comparison of CD data from the full-length and random regions of E22P-AbD43 suggested that the preferential binding of E22P-AbD43 toward the dimer might be related to the formation of a G-quadruplex structure. E22P-AbD43 significantly inhibited the nucleation phase of the dimer and its associated neurotoxicity in SH-SY5Y human neuroblastoma cells. Of note, E22P-AbD43 also significantly protected against the neurotoxicity of WT Aß42 and E22P-Aß42. Furthermore, in an AD mouse model, E22P-AbD43 preferentially recognized diffuse aggregates, which likely originated from PFs or higher-order oligomers with curvilinear structures, compared with senile plaques formed from fibrils. We conclude that the E22P-AbD43 aptamer is a promising research and diagnostic tool for further studies of AD etiology.

Evaluation of Toxic Amyloid 42 Oligomers in Rat Primary Cerebral Cortex Cells and Human iPS-derived Neurons Treated with 10-Me-Aplog-1, a New PKC Activator.

Amyloid ß42 (Aß42), a causative agent of Alzheimer's disease (AD), is derived extracellularly from Aß precursor protein (APP) following the latter's cleavage by ß-secretase, but not α-secretase. Protein kinase Cα (PKCα) activation is known to increase α-secretase activity, thereby suppressing Aß production. Since Aß42 oligomer formation causes potent neurotoxicity, APP modulation by PKC ligands is a promising strategy for AD treatment. Although bryostatin-1 (bryo-1) is a leading compound for this strategy, its limited natural availability and the difficulty of its total synthesis impedes further research. To address this limitation, Irie and colleagues have developed a new PKC activator with few side effects, 10-Me-Aplog-1, (1), which decreased Aß42 in the conditioned medium of rat primary cerebral cortex cells. These results are associated with increased α-secretase but not PKCε-dependent Aß-degrading enzyme. The amount of neuronal embryonic lethal abnormal vision (nELAV), a known ß-secretase stabilizer, was reduced by treatment with 1. Notably, 1 prevented the formation of intracellular toxic oligomers. Furthermore, 1 suppressed toxic oligomerization within human iPS-derived neurons such as bryo-1. Given that 1 was not neurotoxic toward either cell line, these findings suggest that 1 is a potential drug lead for AD therapy.

Insulin deficiency promotes formation of toxic amyloid-ß42 conformer co-aggregating with hyper-phosphorylated tau oligomer in an Alzheimer's disease model.

The toxic conformer of amyloid ß-protein (Aß) ending at 42 (Aß42), which contains a unique turn conformation at amino acid residue positions 22 and 23 and tends to form oligomers that are neurotoxic, was reported to play a critical role in the pathomechanisms of Alzheimer's disease (AD), in which diabetes mellitus (DM)-like mechanisms are also suggested to be operative. It remains to be established whether the attenuation of insulin signaling is involved in an increase of toxic Aß42 conformer levels. The present study investigated the association between impaired insulin metabolism and formation of toxic Aß42 conformers in the brains of an AD mouse model. In particular, we studied whether insulin deficiency or resistance affected the formation of toxic Aß42 conformers in vivo. We induced insulin deficiency and resistance in 3xTg-AD mice, a mouse AD model harboring two familial AD-mutant APP (KM670/671NL) and PS1 (M146 V) genes and a mutant TAU (P301L) gene, by streptozotocin (STZ) injection and a high fructose diet (HFuD), respectively. Cognitive impairment was significantly worsened by STZ injection but not by HFuD. Dot blot analysis revealed significant increases in total Aß42 levels and the ratio of toxic Aß42 conformer/total Aß42 in STZ-treated mice compared with control and HFuD-fed mice. Immunostaining showed the accumulation of toxic Aß42 conformers and hyper-phosphorylated tau protein (p-tau), which was more prominent in the cortical and hippocampal neurons of STZ-treated mice compared with HFuD-fed and control mice. HFuD-fed mice showed only a mild-to-moderate increase of these proteins compared with controls. Toxic Aß42 conformers were co-localized with p-tau oligomers (Pearson's correlation coefficient = 0.62) in the hippocampus, indicating their co-aggregation. Toxic Aß42 conformer levels were inversely correlated with pancreatic insulin secretion capacity as shown by fasting immunoreactive insulin levels in STZ-treated mice (correlation coefficient = -0.5879, p = .04441), but not HFuD-fed mice, suggesting a decrease in serum insulin levels correlates with toxic Aß42 conformer formation. Levels of p-Akt and phosphorylated glycogen synthase kinase-3ß measured by a homogeneous time-resolved fluorescence assay were significantly lower in STZ-treated mice than in HFuD-fed mice, suggesting a greater inhibition of brain insulin signaling by STZ than HFuD, although both levels were significantly decreased in these groups compared with controls. Iba1-positive and NOS2-positive areas in the cortex and hippocampus were significantly increased in STZ-treated mice and to a lesser extent in HFuD-fed mice compared with controls. These findings suggest that insulin deficiency rather than insulin resistance and the resultant impairment of brain insulin signaling facilitates the formation of toxic Aß42 conformer and its co-aggregation with p-tau oligomers, and that insulin deficiency is an important pathogenic factor in the progression of AD.

Synthetic and biochemical studies on the effect of persulfidation on disulfide dimer models of amyloid ß42 at position 35 in Alzheimer's etiology.

Protein persulfidation plays a role in redox signaling as an anti-oxidant. Dimers of amyloid ß42 (Aß42), which induces oxidative stress-associated neurotoxicity as a causative agent of Alzheimer's disease (AD), are minimum units of oligomers in AD pathology. Met35 can be susceptible to persulfidation through its substitution to homoCys residue under the condition of oxidative stress. In order to verify whether persulfidation has an effect in AD, herein we report a chemical approach by synthesizing disulfide dimers of Aß42 and their evaluation of biochemical properties. A homoCys-disulfide dimer model at position 35 of Aß42 formed a partial ß-sheet structure, but its neurotoxicity was much weaker than that of the corresponding monomer. In contrast, the congener with an alkyl linker generated ß-sheet-rich 8-16-mer oligomers with potent neurotoxicity. The length of protofibrils generated from the homoCys-disulfide dimer model was shorter than that of its congener with an alkyl linker. Therefore, the current data do not support the involvement of Aß42 persulfidation in Alzheimer's disease.

Three Structural Features of Functional Food Components and Herbal Medicine with Amyloid ß42 Anti-Aggregation Properties.

Aggregation of amyloid ß42 (Aß42) is one of the hallmarks of Alzheimer's disease (AD). There are numerous naturally occurring products that suppress the aggregation of Aß42, but the underlying mechanisms remain to be elucidated. Based on NMR and MS spectroscopic analysis, we propose three structural characteristics found in natural products required for the suppressive activity against Aß42 aggregation (i.e., oligomerization by targeting specific amino acid residues on this protein). These characteristics include (1) catechol-type flavonoids that can form Michael adducts with the side chains of Lys16 and 28 in monomeric Aß42 through flavonoid autoxidation; (2) non-catechol-type flavonoids with planarity due to α,ß-unsaturated carbonyl groups that can interact with the intermolecular ß-sheet region in Aß42 aggregates, especially aromatic rings such as those of Phe19 and 20; and (3) carboxy acid derivatives with triterpenoid or anthraquinoid that can generate a salt bridge with basic amino acid residues such as Lys16 and 28 in the Aß42 dimer or trimer. Here, we summarize the recent body of knowledge concerning amyloidogenic inhibitors, particularly in functional food components and Kampo medicine, and discuss their application in the treatment and prevention of AD.

An App knock-in mouse inducing the formation of a toxic conformer of Aß as a model for evaluating only oligomer-induced cognitive decline in Alzheimer's disease.

Irie and colleagues identified a "toxic conformer", which possesses a turn structure at positions 22-23, among various conformations of Aß and have been reporting its potent oligomeric capacity and neurotoxicity. This toxic conformer was detected in the brains of AD patients and AD model mice (Tg2576 line), and passive immunization targeting this conformer ameliorated the cognitive dysfunction in an AD model. In this study, we developed a novel AD mouse model (AppNL-P-F/NL-P-F) with Swedish mutation (NL), Iberian mutation (F), and mutation (P) overproducing E22P-Aß, a mimic of the toxic conformer, utilizing the knock-in technique that well recapitulates the Aß pathology of AD patients in mice and avoids the artificial phenotype observed in transgenic-type model mice. We confirmed that AppNL-P-F/NL-P-F mice produce Aß by ELISA and accumulate senile plaques by immunohistochemistry at eight months of age. In WB, we observed a potential trimer band and high molecular-weight oligomer bands without a monomeric band in the TBS-soluble fraction of AppNL-P-F/NL-P-F mice at six months of age. In the novel object recognition test, cognitive impairment was observed at six months of age in these mice. These findings suggest that the toxic conformer of Aß induces cognitive dysfunction mediated by its oligomer formation in this mouse brain. AppNL-P-F/NL-P-F mice may be a useful model for evaluating Aß oligomer-induced cognitive impairment in AD and will aid in exploring therapeutic targets for AD pathology.

Synthesis and biochemical characterization of quasi-stable trimer models of full-length amyloid ß40 with a toxic conformation.

Here, we report the first synthesis of quasi-stable trimer models of full-length Aß40 with a toxic conformation using a 1,3,5-phenyltris-l-alanyl linker at position 34, 36, or 38. The only trimer to exhibit weak neurotoxicity against SH-SY5Y cells was the one which was linked at position 38. This suggests that such a propeller-type trimer model is not prone to forming oligomers with potent neurotoxicity, which is in contrast with its corresponding dimer model.

Amyloid ß toxic conformer has dynamic localization in the human inferior parietal cortex in absence of amyloid plaques.

Amyloid ß (Aß) plays a critical role in the pathogenesis of Alzheimer's disease. Nevertheless, its distribution and clearance before Aß plaque formation needs to be elucidated. Using an optimized immunofluorescent staining method, we examined the distribution of Aß in the post-mortem parietal cortex of 35 subjects, 30 to 65 years of age, APOE ε3/ε3, without AD lesions. We used 11A1, an antibody against an Aß conformer which forms neurotoxic oligomers. 11A1 immunoreactivity (IR) was present in cortical neurons, pericapillary spaces, astrocytes and the extracellular compartment at 30 years of age. The percentage of neurons with 11A1 IR did not change with age, but the number and percentage of astrocytes with 11A1 IR gradually increased. Notably, the percentage of pericapillary spaces labeled with 11A1 IR declined significantly in the 5th decade of the life, at the same time that 11A1 IR increased in the extracellular space. Our findings indicate that the Aß toxic conformer is normally present in various cell types and brain parenchyma, and appears to be constitutively produced, degraded, and cleared from the inferior parietal cortex. The decrease in pericapillary Aß and the concomitant increase of extracellular Aß may reflect an age-associated impairment in Aß clearance from the brain.