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.

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.

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.

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.

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.

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.

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.

Mechanistic analyses of the suppression of amyloid ß42 aggregation by apomorphine.

(R)-Apomorphine (1) has the potential to reduce the accumulation of amyloid ß-protein (Aß42), a causative agent of Alzheimer's disease (AD). Although the inhibition of Aß42 aggregation by 1 is ascribable to the antioxidative effect of its phenol moiety, its inhibitory mechanism at the molecular level remains to be fully elucidated. LC-MS and UV analyses revealed that 1 is autoxidized during incubation to produce an unstable o-quinone form (2), which formed a Michael adduct with Lys 16 and 28 of Aß42. A further autoxidized form of 1 (3) with o-quinone and phenanthrene moieties suppressed Aß42 aggregation comparable to 1, whereas treating 1 with a reductant, tris(2-carboxyethyl)phosphine diminished its inhibitory activity. 1H-15N SOFAST-HMQC NMR studies suggested that 1 interacts with Arg5, His13,14, Gln15, and Lys16 of the Aß42 monomer. These regions form intermolecular ß-sheets in Aß42 aggregates. Since 3 did not perturb the chemical shift of monomeric Aß42, we performed aggregation experiments using 1,1,1,3,3,3-hexafluoro-2-propanol-treated Aß42 to investigate whether 3 associates with Aß42 oligomers. Compounds 1 and 3 delayed the onset of the oligomer-driven nucleation phase. Despite their cytotoxicity, they did not exacerbate Aß42-mediated neurotoxicity in SH-SY5Y neuroblastoma cells. These results demonstrate that extension of the conjugated system in 1 by autoxidation can promote its planarity, which is required for intercalation into the ß-sheet of Aß42 nuclei, thereby suppressing further aggregation.

Synthesized Aß42 Caused Intracellular Oxidative Damage, Leading to Cell Death, via Lysosome Rupture.

Neuronal cellular accumulation of amyloid beta peptide (Aß) has been implicated in the pathogenesis of Alzheimer's disease (AD). Intracellular accumulation of Aß42, a toxic form of Aß, was observed as an early event in AD patients. However, its contribution and the cellular mechanism of cell death remained unclear. We herein revealed the mechanism by which Aß42 incorporated into cells leads to cell death by using chemically synthesized Aß42 variants. The Aß42 variant Aß42 (E22P) which has an increased tendency to oligomerize, accumulated in lysosomes at an earlier stage than wild-type Aß42, leading to higher ROS production and lysosomal membrane oxidation, and resulting in cell death. On the other hand, Aß42 (E22V), which is incapable of oligomerization, did not accumulate in cells or affect the cell viability. Moreover, intracellular localization of EGFP-Galectin-3, a ß-galactoside binding lectin, showed that accumulation of oligomerized Aß42 in lysosomes caused lysosomal membrane permeabilization (LMP). Overexpression of lysosome-localized LAMP1-fused peroxiredoxin 1 and treatment with U18866A, an inhibitor of cholesterol export from lysosomes that causes an increase in lysosomal membrane stability, attenuated Aß42-mediated LMP and cell death. Our findings show that lysosomal ROS generation by toxic conformer of Aß led to cell death via LMP, and suggest that these events are potential targets for AD prevention.Key words: Amyloid-beta (Aß), Cell death, Lysosome, Lysosomal membrane permeabilization, Reactive oxygen species (ROS).

Inhibitory Activities of Antioxidant Flavonoids from Tamarix gallica on Amyloid Aggregation Related to Alzheimer's and Type 2 Diabetes Diseases.

The prevention of amyloid aggregation is promising for the treatment of age-related diseases such as Alzheimer's (AD) and type 2 diabetes (T2D). Ten antioxidant flavonoids isolated from the medicinal halophyte Tamarix gallica were tested for their amyloid aggregation inhibition potential. Glucuronosylated flavonoids show relatively strong inhibitory activity of Amyloid ß (Aß) and human islet amyloid polypeptide (hIAPP) aggregation compared to their aglycone analogs. Structure-activity relationship of the flavonoids suggests that the catechol moiety is important for amyloid aggregation inhibition, while the methylation of the carboxyl group in the glucuronide moiety and of the hydroxyl group in the aglycone flavonoids decreased it.

Structural insights into mechanisms for inhibiting amyloid ß42 aggregation by non-catechol-type flavonoids.

The prevention of 42-mer amyloid ß-protein (Aß42) aggregation is promising for the treatment of Alzheimer's disease. We previously described the site-specific inhibitory mechanism for Aß42 aggregation by a catechol-type flavonoid, (+)-taxifolin, targeting Lys16,28 after its autoxidation. In contrast, non-catechol-type flavonoids (morin, datiscetin, and kaempferol) inhibited Aß42 aggregation without targeting Lys16,28 with almost similar potencies to that of (+)-taxifolin. We herein provided structural insights into their mechanisms for inhibiting Aß42 aggregation. Physicochemical analyses revealed that their inhibition did not require autoxidation. The (1)H-(15)N SOFAST-HMQC NMR of Aß42 in the presence of morin and datiscetin revealed the significant perturbation of chemical shifts of His13,14 and Gln15, which were close to the intermolecular ß-sheet region, Gln15-Ala21. His13,14 also played a role in radical formation at Tyr10, thereby inducing the oxidation of Met35, which has been implicated in Aß42 aggregation. These results suggest the direct interaction of morin and datiscetin with the Aß42 monomer. Although only kaempferol was oxidatively-degraded during incubation, its degradation products as well as kaempferol itself suppressed Aß42 aggregation. However, neither kaempferol nor its decomposed products perturbed the chemical shifts of the Aß42 monomer. Aggregation experiments using 1,1,1,3,3,3-hexafluoro-2-propanol-treated Aß42 demonstrated that kaempferol and its degradation products inhibited the elongation rather than nucleation phase, implying that they interacted with small aggregates of Aß42, but not with the monomer. In contrast, morin and datiscetin inhibited both phases. The position and number of hydroxyl groups on the B-ring of non-catechol-type flavonoids could be important for their inhibitory potencies and mechanisms against Aß42 aggregation.

Semisynthesis and Structure-Activity Studies of Uncarinic Acid C Isolated from Uncaria rhynchophylla as a Specific Inhibitor of the Nucleation Phase in Amyloid ß42 Aggregation.

Oligomers of the 42-mer amyloid-ß protein (Aß42), rather than fibrils, cause synaptic dysfunction in the pathology of Alzheimer's disease (AD). The nucleation phase in a nucleation-dependent aggregation model of Aß42 is related to the formation of oligomers. Uncaria rhynchophylla is one component of "Yokukansan", a Kampo medicine, which is widely used for treating AD symptoms. Previously, an extract of U. rhynchophylla was found to reduce the aggregation of Aß42, but its active principles have yet to be identified. In the present work, uncarinic acid C (3) was identified as an inhibitor of Aß42 aggregation that is present in U. rhynchophylla. Moreover, compound 3 acted as a specific inhibitor of the nucleation phase of Aß42 aggregation. Compound 3 was synthesized from saponin A (10), an abundant byproduct of rutin purified from Uncaria elliptica. Comprehensive structure-activity studies on 3 suggest that both a C-27 ferulate and a C-28 carboxylic acid group are required for its inhibitory activity. These findings may aid the development of oligomer-specific inhibitors for AD therapy.

Synthesis and characterization of the amyloid ß40 dimer model with a linker at position 30 adjacent to the intermolecular ß-sheet region.

Amyloid fibrils in senile plaque mainly consist of the 40-mer and 42-mer amyloid ß-proteins (Aß40 and Aß42). Although Aß42 plays more important role in the pathogenesis of Alzheimer's disease (AD), Aß40 could be involved in the progression of AD pathology because of its large amount. Recent studies revealed that variable sizes of Aß oligomers contributed to the neuronal death and cognitive dysfunction. However, how large oligomeric species are responsible for AD pathogenesis remains unclear. We previously proposed a toxic dimer model of Aß with turn structure at positions 22 and 23 using solid-state NMR and systematic proline replacement. Based on this model, we herein show the synthesis and biological activities of an E22P-Aß40 dimer at position 30, which was connected to l,l-2,6-diaminopimeric acid. The E22P-Aß40 dimer formed stable 6∼8-mer oligomers without amyloid fibrils, but was not neurotoxic on human neuroblastoma cells. On the other hand, E22P-Aß40 generated high molecular-weight oligomers into fibrils, and showed the neurotoxicity. These results suggest that such kind of Aß40 dimer with a parallel ß-sheet might not be pathological.

Site-specific inhibitory mechanism for amyloid ß42 aggregation by catechol-type flavonoids targeting the Lys residues.

The aggregation of the 42-residue amyloid ß-protein (Aß42) is involved in the pathogenesis of Alzheimer disease (AD). Numerous flavonoids exhibit inhibitory activity against Aß42 aggregation, but their mechanism remains unclear in the molecular level. Here we propose the site-specific inhibitory mechanism of (+)-taxifolin, a catechol-type flavonoid, whose 3',4'-dihydroxyl groups of the B-ring plays a critical role. Addition of sodium periodate, an oxidant, strengthened suppression of Aß42 aggregation by (+)-taxifolin, whereas no inhibition was observed under anaerobic conditions, suggesting the inhibition to be associated with the oxidation to form o-quinone. Because formation of the Aß42-taxifolin adduct was suggested by mass spectrometry, Aß42 mutants substituted at Arg(5), Lys(16), and/or Lys(28) with norleucine (Nle) were prepared to identify the residues involved in the conjugate formation. (+)-Taxifolin did not suppress the aggregation of Aß42 mutants at Lys(16) and/or Lys(28) except for the mutant at Arg(5). In addition, the aggregation of Aß42 was inhibited by other catechol-type flavonoids, whereas that of K16Nle-Aß42 was not. In contrast, some non-catechol-type flavonoids suppressed the aggregation of K16Nle-Aß42 as well as Aß42. Furthermore, interaction of (+)-taxifolin with the ß-sheet region in Aß42 was not observed using solid-state NMR unlike curcumin of the non-catechol-type. These results demonstrate that catechol-type flavonoids could specifically suppress Aß42 aggregation by targeting Lys residues. Although the anti-AD activity of flavonoids has been ascribed to their antioxidative activity, the mechanism that the o-quinone reacts with Lys residues of Aß42 might be more intrinsic. The Lys residues could be targets for Alzheimer disease therapy.

Altered regulation of sleep and feeding contributes to starvation resistance in Drosophila melanogaster.

Animals respond to changes in food availability by adjusting sleep and foraging strategies to optimize their fitness. Wild populations of the fruit fly, Drosophila melanogaster, display highly variable levels of starvation resistance that are dependent on geographic location, food availability and evolutionary history. How behaviors that include sleep and feeding vary in Drosophila with increased starvation resistance is unclear. We have generated starvation-resistant flies through experimental evolution to investigate the relationship between foraging behaviors and starvation resistance. Outbred populations of D. melanogaster were selected for starvation resistance over 60 generations. This selection process resulted in flies with a threefold increase in total lipids that survive up to 18 days without food. We tested starvation-selected (S) flies for sleep and feeding behaviors to determine the effect that selection for starvation resistance has had on foraging behavior. Flies from three replicated starvation-selected populations displayed a dramatic reduction in feeding and prolonged sleep duration compared to fed control (F) populations, suggesting that modified sleep and feeding may contribute to starvation resistance. A prolonged larval developmental period contributes to the elevated energy stores present in starvation-selected flies. By preventing S larvae from feeding longer than F larvae, we were able to reduce energy stores in adult S flies to the levels seen in adult F flies, thus allowing us to control for energy storage levels. However, the reduction of energy stores in S flies fails to generate normal sleep and feeding behavior seen in F flies with similar energy stores. These findings suggest that the behavioral changes observed in S flies are due to genetic regulation of behavior rather than elevated lipid levels. Testing S-F hybrid individuals for both feeding and sleep revealed a lack of correlation between food consumption and sleep duration, indicating further independence in genetic factors underlying the sleep and feeding changes observed in S flies. Taken together, these findings provide evidence that starvation selection results in prolonged sleep and reduced feeding through a mechanism that is independent of elevated energy stores. These findings suggest that changes in both metabolic function and behavior contribute to the increase in starvation resistance seen in flies selected for starvation resistance.

Conformation-specific antibodies to target amyloid ß oligomers and their application to immunotherapy for Alzheimer's disease.

Amyloid ß-protein (Aß) oligomers, intermediates of Aß aggregation, cause cognitive impairment and synaptotoxicity in the pathogenesis of Alzheimer's disease (AD). Immunotherapy using anti-Aß antibody is one of the most promising approaches for AD treatment. However, most clinical trials using conventional sequence-specific antibodies have proceeded with difficulty. This is probably due to the unintended removal of the non-pathological monomer and fibrils of Aß as well as the pathological oligomers by these antibodies that recognize Aß sequence, which is not involved in synaptotoxicity. Several efforts have been made recently to develop conformation-specific antibodies that target the tertiary structure of Aß oligomers. Here, we review the recent findings of Aß oligomers and anti-Aß antibodies including our own, and discuss their potential as therapeutic and diagnostic tools.

Non-toxic conformer of amyloid ß may suppress amyloid ß-induced toxicity in rat primary neurons: implications for a novel therapeutic strategy for Alzheimer's disease.

The 42-mer amyloid ß-protein (Aß42) oligomers cause neurotoxicity and cognitive impairment in Alzheimer's disease (AD). We previously identified the toxic conformer of Aß42 with a turn at positions 22-23 ("toxic" turn) to form oligomers and to induce toxicity in rat primary neurons, along with the non-toxic conformer with a turn at positions 25-26. G25P-Aß42 and E22V-Aß42 are non-toxic mutants that disfavor the "toxic" turn. Here we hypothesize that these non-toxic mutants of Aß42 could suppress Aß42-induced neurotoxicity, and examined their effects on the neurotoxicity, aggregation, and levels of the toxic conformer, which was evaluated by dot blotting using a monoclonal antibody (11A1) against the toxic conformer. G25P-Aß42 and E22V-Aß42 suppressed the neurotoxicity and aggregation of Aß42 as well as the formation of the toxic conformer. The neurotoxicity induced by Aß42 was also significantly reduced by the treatment of 11A1, but not of Aß-sequence specific antibodies (6E10 and 4G8). Since recent studies indicate that Aß oligomers contain parallel ß-sheet, the present results suggest that the non-toxic mutants of Aß42 without the "toxic" turn could prevent the propagation process of the toxic conformer of Aß42 resulting in suppression of the formation of the toxic oligomers. This could be a promising strategy for AD therapeutics.

Structure-activity studies on the side chain of a simplified analog of aplysiatoxin (aplog-1) with anti-proliferative activity.

We have recently developed a simplified analog of aplysiatoxin (aplog-1) as an activator of protein kinase C (PKC) with anti-proliferative activity like bryostain 1. To identify sites in aplog-1 that could be readily modified to optimize therapeutic performance and to develop a molecular probe for examining the analog's mode of action, substituent effects on the phenol ring were systematically examined. Whereas hydrophilic acetamido derivatives were less active than aplog-1 in inhibiting cancer cell growth and binding to PKCδ, introduction of hydrophobic bromine and iodine atoms enhanced both biological activities. The anti-proliferative activity was found to correlate closely with molecular hydrophobicity, and maximal activity was observed at a logP value of 4.0-4.5. On the other hand, an induction test with Epstein-Barr virus early antigen demonstrated that these derivatives have less tumor-promoting activity in vitro than aplog-1 regardless of the hydrophobicity of their substituents. These results would facilitate rapid preparation of molecular probes to examine the mechanism of the unique biological activities of aplog-1.