Yuji Yoshiike

Potent anti-amyloidogenic and fibril-destabilizing effects of polyphenols in vitro: implications for the prevention and therapeutics of Alzheimer's disease.

Cerebral deposition of amyloid β‐peptide (Aβ) in the brain is an invariant feature of Alzheimers disease (AD). A consistent protective effect of wine consumption on AD has been documented by epidemiological studies. In the present study, we used fluorescence spectroscopy with thioflavin T and electron microscopy to examine the effects of wine‐related polyphenols (myricetin, morin, quercetin, kaempferol (+)‐catechin and (–)‐epicatechin) on the formation, extension, and destabilization of β‐amyloid fibrils (fAβ) at pH 7.5 at 37°C in vitro. All examined polyphenols dose‐dependently inhibited formation of fAβ from fresh Aβ(1–40) and Aβ(1–42), as well as their extension. Moreover, these polyphenols dose‐dependently destabilized preformed fAβs. The overall activity of the molecules examined was in the order of: myricetin = morin = quercetin > kaempferol > (+)‐catechin = (–)‐epicatechin. The effective concentrations (EC50) of myricetin, morin and quercetin for the formation, extension and destabilization of fAβs were in the order of 0.1–1 µm. In cell culture experiments, myricetin‐treated fAβ were suggested to be less toxic than intact fAβ, as demonstrated by 3‐[4,5‐dimethylthiazol‐2‐yl]‐2,5‐diphenyltetrazolium bromide assay. Although the mechanisms by which these polyphenols inhibit fAβ formation from Aβ, and destabilize pre‐formed fAβin vitro are still unclear, polyphenols could be a key molecule for the development of preventives and therapeutics for AD.

Formation of filamentous tau aggregations in transgenic mice expressing V337M human tau

Formation of neurofibrillary tangles (NFTs) is the most common feature in several neurodegenerative diseases, including Alzheimers disease (AD). Here we report the formation of filamentous tau aggregations having a beta-sheet structure in transgenic mice expressing mutant human tau. These mice contain a tau gene with a mutation of the frontotemporal dementia parkinsonism (FTDP-17) type, in which valine is substituted with methionine residue 337. The aggregation of tau in these transgenic mice satisfies all histological criteria used to identify NFTs common to human neurodegenerative diseases. These mice, therefore, provide a preclinical model for the testing of therapeutic drugs for the treatment of neurodegenerative disorders that exhibit NFTs.

Nordihydroguaiaretic acid potently breaks down pre-formed Alzheimer's β-amyloid fibrils in vitro

Inhibition of the accumulation of amyloid β‐peptide (Aβ) and the formation of β‐amyloid fibrils (fAβ) from Aβ, as well as the degradation of pre‐formed fAβ in the CNS would be attractive therapeutic objectives for the treatment of Alzheimers disease (AD). We previously reported that nordihydroguaiaretic acid (NDGA) inhibited fAβ formation from Aβ(1–40) and Aβ(1–42) dose‐dependently in the range of 10–30 µmin vitro. Utilizing fluorescence spectroscopic analysis with thioflavin T and electron microscopic study, we show here that NDGA dose‐dependently breaks down fAβ(1–40) and fAβ(1–42) within a few hours at pH 7.5 at 37°C. At 4 h, the fluorescence of fAβ(1–40) and fAβ(1–42) incubated with 50 µm NDGA was 5% and 10% of the initial fluorescence, respectively. The activity of NDGA to break down these fAβs was observed even at a low concentration of 0.1 µm. At 1 h, many short, sheared fibrils were observed in the mixture incubated with 50 µm NDGA, and at 4 h, the number of fibrils reduced markedly, and small amorphous aggregates were observed. We next compared the activity of NDGA to break down fAβ(1–40) and fAβ(1–42), with other molecules reported to inhibit fAβ formation from Aβ and/or to degrade pre‐formed fAβ both in vivo and in vitro. At a concentration of 50 µm, the overall activity of the molecules examined in this study was in the order of: NDGA >> rifampicin = tetracycline > poly(vinylsulfonic acid, sodium salt) = 1,3‐propanedisulfonic acid, disodium salt > β‐sheet breaker peptide (iAβ5). In cell culture experiments, fAβ disrupted by NDGA were less toxic than intact fAβ, as demonstrated by 3‐(4,5‐dimethylthiazol‐2‐yl)‐2,5‐diphenyltetrazolium bromide assay. Although the mechanisms by which NDGA inhibits fAβ formation from Aβ, as well as breaking down pre‐formed fAβin vitro, are still unclear, NDGA could be a key molecule for the development of therapeutics for AD.

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.

GSK-3β Is Required for Memory Reconsolidation in Adult Brain

Activation of GSK-3β is presumed to be involved in various neurodegenerative diseases, including Alzheimers disease (AD), which is characterized by memory disturbances during early stages of the disease. The normal function of GSK-3β in adult brain is not well understood. Here, we analyzed the ability of heterozygote GSK-3β knockout (GSK+/−) mice to form memories. In the Morris water maze (MWM), learning and memory performance of GSK+/− mice was no different from that of wild-type (WT) mice for the first 3 days of training. With continued learning on subsequent days, however, retrograde amnesia was induced in GSK+/− mice, suggesting that GSK+/− mice might be impaired in their ability to form long-term memories. In contextual fear conditioning (CFC), context memory was normally consolidated in GSK+/− mice, but once the original memory was reactivated, they showed reduced freezing, suggesting that GSK+/− mice had impaired memory reconsolidation. Biochemical analysis showed that GSK-3β was activated after memory reactivation in WT mice. Intraperitoneal injection of a GSK-3 inhibitor before memory reactivation impaired memory reconsolidation in WT mice. These results suggest that memory reconsolidation requires activation of GSK-3β in the adult brain.

Vitamin A exhibits potent antiamyloidogenic and fibril-destabilizing effects in vitro.

Cerebral deposition of amyloid beta-peptide (Abeta) in the brain is an invariant feature of Alzheimer disease (AD). Plasma or cerebrospinal fluid concentrations of antioxidant vitamins and carotenoids, such as vitamins A, C, E, and beta-carotene, have been reported to be lower in AD patients, and these vitamins clinically have been demonstrated to slow the progression of dementia. In this study, we used fluorescence spectroscopy with thioflavin T (ThT) and electron microscopy to examine the effects of vitamin A (retinol, retinal, and retinoic acid), beta-carotene, and vitamins B2, B6, C, and E on the formation, extension, and destabilization of beta-amyloid fibrils (fAbeta) in vitro. Among them, vitamin A and beta-carotene dose-dependently inhibited formation of fAbeta from fresh Abeta, as well as their extension. Moreover, they dose-dependently destabilized preformed fAbetas. The overall activity of the molecules examined was in the order of retinol = retinal > beta-carotene > retinoic acid. Although the exact mechanisms are still unclear, vitamins A and beta-carotene could be key molecules for the prevention and therapy of AD.

Hyperphosphorylated tau in parahippocampal cortex impairs place learning in aged mice expressing wild-type human tau

To investigate how tau affects neuronal function during neurofibrillary tangle (NFT) formation, we examined the behavior, neural activity, and neuropathology of mice expressing wild‐type human tau. Here, we demonstrate that aged (>20 months old) mice display impaired place learning and memory, even though they do not form NFTs or display neuronal loss. However, soluble hyperphosphorylated tau and synapse loss were found in the same regions. Mn‐enhanced MRI showed that the activity of the parahippocampal area is strongly correlated with the decline of memory as assessed by the Morris water maze. Taken together, the accumulation of hyperphosphorylated tau and synapse loss in aged mice, leading to inhibition of neural activity in parahippocampal areas, including the entorhinal cortex, may underlie place learning impairment. Thus, the accumulation of hyperphosphorylated tau that occurs before NFT formation in entorhinal cortex may contribute to the memory problems seen in Alzheimers disease (AD).

Specific compositions of amyloid-β peptides as the determinant of toxic β-aggregation

Alzheimers disease (AD) may be caused by toxic aggregates formed from amyloid-β (Aβ) peptides. By using Thioflavin T, a dye that specifically binds to β-sheet structures, we found that highly toxic forms of Aβ-aggregates were formed at the initial stage of fibrillogenesis, which is consistent with recent reports on Aβ oligomers. Formation of such aggregates depends on factors that affect both nucleation and elongation. As reported previously, addition of Aβ42 systematically accelerated the nucleation of Aβ40, most likely because of the extra hydrophobic residues at the C terminus of Aβ42. At Aβ42-increased specific ratio (Aβ40: Aβ42 = 10: 1), on the other hand, not only accelerated nucleation but also induced elongation were observed, suggesting pathogenesis of early-onset AD. Because a larger proportion of Aβ40 than Aβ42 was still required for this phenomenon, we assumed that elongation does not depend only on hydrophobic interactions. Without any change in the C-terminal hydrophobic nature, elongation was effectively induced by mixing wild type Aβ40 with Italian variant Aβ40 (E22K) or Dutch variant (E22Q). We suggest that Aβ peptides in specific compositions that balance hydrophilic and hydrophobic interactions promote the formation of toxic β-aggregates. These results may introduce a new therapeutic approach through the disruption of this balance.

Molecular chaperone-mediated tau protein metabolism counteracts the formation of granular tau oligomers in human brain.

Intracellular accumulation of filamentous tau proteins is a defining feature of neurodegenerative diseases termed tauopathies. The pathogenesis of tauopathies remains largely unknown. Molecular chaperones such as heat shock proteins (HSPs), however, have been implicated in tauopathies as well as in other neurodegenerative diseases characterized by the accumulation of insoluble protein aggregates. To search for in vivo evidence of chaperone‐related tau protein metabolism, we analyzed human brains with varying degrees of neurofibrillary tangle (NFT) pathology, as defined by Braak NFT staging. Quantitative analysis of soluble protein levels revealed significant positive correlations between tau and Hsp90, Hsp40, Hsp27, α‐crystallin, and CHIP. An inverse correlation was observed between the levels of HSPs in each specimen and the levels of granular tau oligomers, the latter of which were isolated from brain as intermediates of tau filaments. We speculate that HSPs function as regulators of soluble tau protein levels, and, once the capacity of this chaperone system is saturated, granular tau oligomers form virtually unabated. This is expressed pathologically as an early sign of NFT formation. The molecular basis of chaperone‐mediated protection against neurodegeneration might lead to the development of therapeutics for tauopathies.

GABAA Receptor-Mediated Acceleration of Aging-Associated Memory Decline in APP/PS1 Mice and Its Pharmacological Treatment by Picrotoxin

Advanced age and mutations in the genes encoding amyloid precursor protein (APP) and presenilin (PS1) are two serious risk factors for Alzheimers disease (AD). Finding common pathogenic changes originating from these risks may lead to a new therapeutic strategy. We observed a decline in memory performance and reduction in hippocampal long-term potentiation (LTP) in both mature adult (9–15 months) transgenic APP/PS1 mice and old (19–25 months) non-transgenic (nonTg) mice. By contrast, in the presence of bicuculline, a GABAA receptor antagonist, LTP in adult APP/PS1 mice and old nonTg mice was larger than that in adult nonTg mice. The increased LTP levels in bicuculline-treated slices suggested that GABAA receptor-mediated inhibition in adult APP/PS1 and old nonTg mice was upregulated. Assuming that enhanced inhibition of LTP mediates memory decline in APP/PS1 mice, we rescued memory deficits in adult APP/PS1 mice by treating them with another GABAA receptor antagonist, picrotoxin (PTX), at a non-epileptic dose for 10 days. Among the saline vehicle-treated groups, substantially higher levels of synaptic proteins such as GABAA receptor α1 subunit, PSD95, and NR2B were observed in APP/PS1 mice than in nonTg control mice. This difference was insignificant among PTX-treated groups, suggesting that memory decline in APP/PS1 mice may result from changes in synaptic protein levels through homeostatic mechanisms. Several independent studies reported previously in aged rodents both an increased level of GABAA receptor α1 subunit and improvement of cognitive functions by long term GABAA receptor antagonist treatment. Therefore, reduced LTP linked to enhanced GABAA receptor-mediated inhibition may be triggered by aging and may be accelerated by familial AD-linked gene products like Aβ and mutant PS1, leading to cognitive decline that is pharmacologically treatable at least at this stage of disease progression in mice.