Polymerization of monomeric amyloid-β peptides (Aβ) into soluble oligomers and insoluble fibrils is one of the major pathways triggering the pathogenesis of Alzheimer's disease (AD). Using small molecules to prevent the polymerization of Aβ peptides can, therefore, be an effective therapeutic strategy for AD. In this study, we investigate the effects of mono- and biflavonoids in Aβ42-induced toxicity and fibrillogenesis and find that the biflavonoid taiwaniaflavone (TF) effectively and specifically inhibits Aβ toxicity and fibrillogenesis. Compared to TF, the monoflavonoid apigenin (AP) is less effective and less specific. Our data show that differential effects of the mono- and biflavonoids in Aβ fibrillogenesis correlate with their varying cytoprotective efficacies. We also find that other biflavonoids, namely, 2',8''-biapigenin, amentoflavone, and sumaflavone, can also effectively inhibit Aβ toxicity and fibrillogenesis, implying that the participation of two monoflavonoids in a single biflavonoid molecule enhances their activity. Biflavonoids, while strongly inhibiting Aβ fibrillogenesis, accumulate nontoxic Aβ oligomeric structures, suggesting that these are off-pathway oligomers. Moreover, TF abrogates the toxicity of preformed Aβ oligomers and fibrils, indicating that TF and other biflavonoids may also reduce the toxicity of toxic Aβ species. Altogether, our data clearly show that biflavonoids, possibly because of the possession of two Aβ binders separated by an appropriate size linker, are likely to be promising therapeutics for suppressing Aβ toxicity.

Reticulon 3 (RTN3) has previously been shown to interact with BACE1 and negatively regulate BACE1 activity. To what extent RTN3 deficiency affects BACE1 activity is an intriguing question. In this study, we aimed to address this by generating RTN3-null mice. Mice with complete deficiency of RTN3 grow normally and have no obviously discernible phenotypes. Morphological analyses of RTN3-null mice showed no significant alterations in cellular structure, although RTN3 is recognized as a protein contributing to the shaping of tubular endoplasmic reticulum. Biochemical analysis revealed that RTN3 deficiency increased protein levels of BACE1. This elevation of BACE1 levels correlated with enhanced processing of amyloid precursor protein at the ␤-secretase site. We also demonstrated that RTN3 deficiency in Alzheimer's mouse models facilitates amyloid deposition, further supporting an in vivo role of RTN3 in the regulation of BACE1 activity. Since it has been shown that RTN3 monomer is reduced in brains of Alzheimer's patients, our results suggest that long-lasting reduction of RTN3 levels has adverse effects on BACE1 activity and may contribute to Alzheimer's pathogenesis.

Alzheimer's disease (AD) is characterized by the presence of neuritic plaques in which dystrophic neurites (DNs) are typical constituents. We recently showed that DNs labeled by antibodies to the tubular endoplasmic reticulum (ER) protein reticulon-3 (RTN3) are enriched with clustered tubular ER. However, multi-vesicle bodies are also found in DNs, suggesting that different populations of DNs exist in brains of AD patients. To understand how different DNs evolve to surround core amyloid plaques, we monitored the growth of DNs in AD mouse brains (5xFAD and APP/PS1ΔE9 mice) by multiple approaches, including 2-dimensional and 3-dimesnional (3D) electron microscopy (EM). We discovered that a pre-autophagosome protein ATG9A was enriched in DNs when a plaque was just beginning to develop. ATG9A-positive DNs were often closer to the core amyloid plaque, while RTN3-immunoreactive DNs were mostly located in the outer layers of ATG9A-positive DNs. Proteins such as RAB7 and LC3 appeared in DNs at later stages during plaque growth, likely accumulated as a part of large autophagy vesicles, and were distributed relatively furthest from the core amyloid plaque. Reconstructing the 3D structure of different morphologies of DNs revealed that DNs in AD mouse brains were constituted in three layers that are distinct by enriching different types of vesicles, as validated by immune-EM methods. Collectively, our results provide the first evidence that DNs evolve from dysfunctions of preautophagosomes, tubular ER, mature autophagosomes, and the ubiquitin proteasome system during plaque growth.

BackgroundAggregation of soluble, monomeric β- amyloid (Aβ) to oligomeric and then insoluble fibrillar Aβ is a key pathogenic feature in development of Alzheimer’s disease (AD). Increasing evidence suggests that toxicity is linked to diffusible Aβ oligomers, rather than to insoluble fibrils. The use of naturally occurring small molecules for inhibition of Aβ aggregation has recently attracted significant interest for development of effective therapeutic strategies against the disease. A natural polyphenolic flavone, Kaempferol-3-O-rhamnoside (K-3-rh), was utilized to investigate its effects on aggregation and cytotoxic effects of Aβ42 peptide. Several biochemical techniques were used to determine the conformational changes and cytotoxic effect of the peptide in the presence and absence of K-3-rh.ResultsK-3-rh showed a dose-dependent effect against Aβ42 mediated cytotoxicity. Anti-amyloidogenic properties of K-3-rh were found to be efficient in inhibiting fibrilogenesis and secondary structural transformation of the peptide. The consequence of these inhibitions was the accumulation of oligomeric structural species. The accumulated aggregates were smaller, soluble, non-β-sheet and non-toxic aggregates, compared to preformed toxic Aβ oligomers. K-3-rh was also found to have the remodeling properties of preformed soluble oligomers and fibrils. Both of these conformers were found to remodel into non-toxic aggregates. The results showed that K-3-rh interacts with different Aβ conformers, which affects fibril formation, oligomeric maturation and fibrillar stabilization.ConclusionK-3-rh is an efficient molecule to hinder the self assembly and to abrogate the cytotoxic effects of Aβ42 peptide. Hence, K-3-rh and small molecules with similar structure might be considered for therapeutic development against AD.