Jun-ichi Takasaki

Phenolic compounds prevent amyloid β-protein oligomerization and synaptic dysfunction by site-specific binding

Background: Epidemiological evidence suggests that consumption of phenolic compounds reduce the incidence of Alzheimer disease (AD). Results: Myricetin and rosmarinic acid reduced cellular and synaptic toxicities by inhibition of amyloid β-protein (Aβ) oligomerization. Myricetin promoted NMR changes of Aβ. Conclusion: Phenolic compounds are worthy therapeutic candidates for AD. Significance: Phenolic compounds blocked early assembly processes of Aβ through differently binding. Cerebral deposition of amyloid β protein (Aβ) is an invariant feature of Alzheimer disease (AD), and epidemiological evidence suggests that moderate consumption of foods enriched with phenolic compounds reduce the incidence of AD. We reported previously that the phenolic compounds myricetin (Myr) and rosmarinic acid (RA) inhibited Aβ aggregation in vitro and in vivo. To elucidate a mechanistic basis for these results, we analyzed the effects of five phenolic compounds in the Aβ aggregation process and in oligomer-induced synaptic toxicities. We now report that the phenolic compounds blocked Aβ oligomerization, and Myr promoted significant NMR chemical shift changes of monomeric Aβ. Both Myr and RA reduced cellular toxicity and synaptic dysfunction of the Aβ oligomers. These results suggest that Myr and RA may play key roles in blocking the toxicity and early assembly processes associated with Aβ through different binding.

Familial Parkinson disease mutations influence α-Synuclein assembly

Lewy bodies composed of aggregates of α-synuclein (αS) in the brain are the main histopathological features of Lewy body diseases (LBD) such as Parkinsons disease and dementia with Lewy bodies. Mutations such as E46K, A30P and A53T in the αS gene cause autosomal dominant LBD in a number of kindreds. Although these mutations accelerate fibril formation, their precise effects at early stages of the αS aggregation process remain unknown. To answer this question, we examined the aggregation including monomer conformational dynamics and oligomerization of the E46K, A30P, A53T and A30P/A53T mutations and wild type (WT) using thioflavin S assay, circular dichroism spectroscopy, photo-induced cross-linking of unmodified proteins, electron microscopy, and atomic force microscopy. Relative to WT αS, E46K αS accelerated the kinetics of the secondary structure change and oligomerization, whereas A30P αS decelerated them. These effects were reflected in changes in average oligomer size. The mutant oligomers of E46K αS functioned as fibril seeds significantly more efficiently than those of WT αS, whereas the mutant oligomers of A30P αS were less efficient. Our results that mutations of familial LBD had opposite effects at early stages of αS assembly may provide new insight into the molecular mechanisms of LBD.

Effect of melatonin on α-synuclein self-assembly and cytotoxicity

α-Synuclein (αS) assembly has been implicated as a critical step in the development of Lewy body diseases such as Parkinsons disease and dementia with Lewy bodies. Melatonin (Mel), a secretory product of the pineal gland, is known to have beneficial effects such as an antioxidant function and neuroprotection. To elucidate whether Mel has an antiassembly effect, here we used circular dichroism spectroscopy, photoinduced crosslinking of unmodified proteins, thioflavin S fluorescence, size exclusion chromatography, electron microscopy and atomic force microscopy to examine the effects of Mel on the αS assembly. We also examined the effects of Mel on αS-induced cytotoxicity by assaying 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide metabolism in αS-treated, primary neuronal cells. Initial studies revealed that Mel blocked αS fibril formation as well as destabilizing preformed αS fibrils. Subsequent evaluation of the assembly-stage specificity of the effect showed that Mel was able to inhibit protofibril formation, oligomerization, and secondary structure transitions. Importantly, Mel decreased αS-induced cytotoxicity. These data suggest a mechanism of action for Mel, inhibition of assembly of toxic polymers and protection of neurons from their effect.

Effects of sex hormones on Alzheimer's disease-associated β-amyloid oligomer formation in vitro.

The folding of amyloid β-protein (Aβ) into oligomeric, protofibrillar, and fibrillar assemblies is hypothesized to be the key pathogenic event in Alzheimers disease (AD), with oligomeric assemblies thought to be the most neurotoxic. Inhibitors of oligomer formation, therefore, could be valuable therapeutics for patients with AD. Epidemiological studies have indicated that estrogen therapy reduces the risk of developing AD in women. Here, we examined the effects of estrogen (estrone (E1), estradiol (E2), and estriol (E3)) and related sexual steroids (androstenedione (AND) and testosterone (TES)) on the in vitro oligomer formation of Aβ(1-40) and Aβ(1-42) using a method of photo-induced cross-linking of unmodified proteins (PICUP) and electron microscopic studies. Estrogens (E1, E2, and E3) inhibited low-order Aβ oligomer formation, and among them, E3 had the strongest in vitro activity. Estrogen could be a potential therapeutic agent to prevent or delay AD progression, and further understanding of the fact that these very similar molecules have different anti-oligomeric effects would contribute to the development of new agents.

Vitamin A has Anti-Oligomerization Effects on Amyloid-β In Vitro

Inhibition of amyloid-β (Aβ) aggregation is an attractive therapeutic strategy for treatment of Alzheimers disease (AD). We previously reported that vitamin A and β-carotene inhibit fibrillation of Aβ40 and Aβ42 (Ono et al, 2004, Exp Neurol). In this study, we firstly examined the effects of vitamin A (retinoic acid, retinol, and retinal), β-carotene, vitamin B2, vitamin B6, vitamin C, vitamin E, coenzyme Q10, and α-lipoic acid on oligomerization of Aβ40 and Aβ42 in vitro; vitamin A and β-carotene dose-dependently inhibited oligomerization of Aβ40 and Aβ42. Furthermore, retinoic acid decreased cellular toxicity by inhibition of Aβ42 oligomerization. Second, we analyzed how vitamin A inhibits Aβ aggregation by using fluorescence spectroscopy and thioflavin T assay with two Aβ fragments, Aβ1-16 and Aβ25-35. A fluorescence peak of retinoic acid was greatly restrained in the presence of Aβ25-35, and retinoic acid inhibited aggregation of Aβ25-35, but not of Aβ1-16, which suggest the specific binding of retinoic acid to the C-terminal portion of Aβ. Thus, vitamin A and β-carotene might be key molecules for prevention of AD.

Anti-amyloidogenic effects of soybean isoflavones in vitro: Fluorescence spectroscopy demonstrating direct binding to Aβ monomers, oligomers and fibrils

Alzheimers disease is characterized by the presence of extracellular deposits of amyloid, primarily composed of the amyloid β-protein (Aβ). A growing body of evidence indicates that oligomeric forms of Aβ play a critical role in disease causation. Soybean isoflavones are flavonoids with an isoflavone backbone. Isoflavones have been reported to protect against Aβ-induced neurotoxicity in cultured cell systems, the molecular mechanisms remain unclear. Our previous studies demonstrated that red wine-related flavonoids with a flavone backbone are able to inhibit Aβ assembly and destabilize preformed Aβ aggregates. Here, we show that isoflavones, especially glycitein and genistein, have anti-fibrillization, anti-oligomerization and fibril-destabilizing effects on Aβ(1-40) and Aβ(1-42)in vitro at physiological pH and temperature, by using nucleation-dependent polymerization monitored by thioflavin T fluorescence, atomic force microscopy, electron microscopy, and photo-induced cross-linking of unmodified proteins followed by SDS-PAGE. Our three-dimensional fluorescence spectroscopic analyses demonstrated that glycitein interacted with Aβ monomers, oligomers and fibrils, indicating specific binding of glycitein to these Aβ species. Glycitein also interacted with different Aβ fragments (Aβ(1-42), Aβ(1-40), Aβ(1-16) and Aβ(25-35)), exhibiting the highest fluorescence enhancement with Aβ(25-35). We speculated that glyciteins anti-amyloidogenic properties are specifically mediated by its binding to Aβ monomers, oligomers and fibrils. Isoflavones may hold promise as a treatment option for preventative strategies targeting amyloid formation in Alzheimers disease.

Effects of antiparkinsonian agents on β-amyloid and α-synuclein oligomer formation in vitro.

The aggregation of β‐amyloid protein (Aβ) and α‐synuclein (αS) are hypothesized to be the key pathogenic event in Alzheimers disease (AD) and Lewy body diseases (LBD), with oligomeric assemblies thought to be the most neurotoxic. Inhibitors of oligomer formation, therefore, could be valuable therapeutics for patients with AD and LBD. Here, we examined the effects of antiparkinsonian agents (dopamine, levodopa, trihexyphenidyl, selegiline, zonisamide, bromocriptine, peroxide, ropinirole, pramipexole, and entacapone) on the in vitro oligomer formation of Aβ40, Aβ42, and αS using a method of photo‐induced cross‐linking of unmodified proteins (PICUP), electron microscopy, and atomic force microscopy. The antiparkinsonian agents except for trihexyphenidyl inhibited both Aβ and αS oligomer formations, and, among them, dopamine, levodopa, pramipexole, and entacapone had the stronger in vitro activity. Circular dichroism and thioflavin T(S) assays showed that secondary structures of Aβ and αS assemblies inhibited by antiparkinsonian agents were statistical coil state and that their seeding activities had disappeared. The antiparkinsonian agents could be potential therapeutic agents to prevent or delay AD and LBD progression.

Phenolic compounds prevent beta-amyloid-protein oligomerization and synaptic dysfunction by site-specific binding

P2-048 PHENOLIC COMPOUNDS PREVENT BETAAMYLOID-PROTEIN OLIGOMERIZATION AND SYNAPTIC DYSFUNCTION BY SITE-SPECIFIC BINDING Kenjiro Ono, Lei Li, Yusaku Takamura, Yuji Yoshiike, Tokuhei Ikeda, Junichi Takasaki, Hisao Nishijo, Akihiko Takashima, David Teplow, Michael Zagorski, Masahito Yamada, Kanazawa University, Kanazawa, Japan; Case Western Reserve University, Cleveland, Ohio, United States; University of Toyama, Toyama, Japan; National Center for Geriatrics and Gerontology, Obu, Aichi, Japan; Kanazawa University, Kanazawa, Ishikawa, Japan; Kanazawa University, Kanazawa, Japan; UCLA, Los Angeles, California, United States. Contact e-mail: onoken@med. kanazawa-u.ac.jp

Vitamin A has anti-oligomerization effects on Aβ in vitro

Inhibition of amyloid- (A) aggregation is an attractive therapeutic strategy for treatment of Alzheimers disease (AD). We previously reported that vitamin A and -carotene inhibit fibrillation of A40 and A42 (Ono et al, 2004, Exp Neurol). In this study, we firstly examined the effects of vitamin A (retinoic acid, retinol, and retinal), -carotene, vitamin B2, vitamin B6, vitamin C, vitamin E, coenzyme Q10, and -lipoic acid on oligomerization of A40 and A42 in vitro; vitamin A and -carotene dose-dependently inhibited oligomerization of A40 and A42. Furthermore, retinoic acid decreased cellular toxicity by inhibition of A42 oligomerization. Second, we analyzed how vitamin A inhibits A aggregation by using fluorescence spectroscopy and thioflavin T assay with two A fragments, A1-16 and A25-35. A fluorescence peak of retinoic acid was greatly restrained in the presence of A25-35, and retinoic acid inhibited aggregation of A25-35, but not of A1-16, which suggest the specific binding of retinoic acid to the C-terminal portion of A. Thus, vitamin A and -carotene might be key molecules for prevention of AD.

Melatonin inhibits α-Synuclein Self-assembly

Background: a-Synuclein (aS) assembly has been implicated as a critical step in the development of Lewy body diseases such as Parkinson’s disease. Melatonin (Mel), a secretory product of the pineal gland, is known to have beneficial effects such as an antioxidant function and neuroprotection. Recently, Mel has been shown by immunostaining to inhibit aS assembly. Methods: To elucidate whether Mel has an anti-assembly effect, here we used circular dichroism spectroscopy (CD), photo-induced cross-linking of unmodified proteins (PICUP), thioflavin S fluorescence (ThS), size exclusion chromatography (SEC), electron microscopy (EM) and atomic force microscopy (AFM) to examine the effects of Mel on the assembly of aS. We also examined the effects ofMel on aS-induced cytotoxicity by assaying 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) metabolism in aS-treated, primary neuronal cells. Results: Initial studies revealed that Mel blocked aS fibril formation as well as destabilizing preformed aS fibrils. Subsequent evaluation of the assembly-stage specificity of the effect showed that Mel was able to inhibit protofibril formation, pre-protofibrillar oligomerization, and initial coil-a-helix/ß-sheet secondary structure transitions. Importantly, Mel had protective effects in assays of cytotoxicity in which Mel was mixed with aS prior to peptide assembly. Conclusions: These data suggest a mechanism of action for Mel, inhibition of assembly of toxic polymers and protection of neurons from their effect.