The real cause of AD is still unclear. Aβ containing 40 or 42 amino acids is the main component of extracellular senile plaques, which are the most important marker of AD pathology. There is compelling evidence supporting the crucial role of amyloid in the pathophysiology of AD, from genetic, in vitro, and in vivo studies9. Transgenic animals, like APPswe/PSEN1dE9 (APP/PS1) mice, reveal early and progressive accumulation of amyloid and develop memory decline, similar to symptoms in humans, from about 3 months old22. The amyloid PET scan has become the most important biomarker tool for diagnosing AD. However, the burden of amyloid in the brain is not perfectly correlated with the severity of AD, and almost all clinical trials of amyloid-cleaning therapy have failed7,14. Most studies showed that although the amyloid burden was reduced, dementic symptoms continued to progress23. The amyloid hypothesis was recently challenged24. To test the amyloid hypothesis, treatment in many ongoing trials was moved to the early or asymptomatic phase, to try and prevent mental decline25. In this study, we tested the effects of PAW on transgenic APP/PS1 mice. Based on prior research, significant amyloid accumulation appeared at 3 months of age, behavior and memory declines occurred a few months later, and abnormal amyloid PET results were found after 9 months of age in APP/PS1 animals26. Therefore, AD mice were given PAW from 5 months old, and memory surveys with the NOR test were conducted every 3 months. Results showed that memory continuously and obviously declined in AD mice without PAW, but memory decline was slower in AD mice treated with PAW. The memory performance became significantly different at 6 months after treatment (Fig. 1). Amyloid PET scans showed that the amyloid burden was higher in AD mice without PAW treatment, especially in the cortex, hippocampus, and hypothalamus (Figs 2 and 3). These findings support the close relationships of amyloid burdens in the cortex and hippocampus with memory declines. Amyloid-reduction therapy should potentially be effective. Results also implied that treatment to reduce the amyloid burden might begin in the early stage, before or soon after the accelerating accumulation of amyloid. PAW has been tested in a few diseases, and its antioxidative and anti-inflammatory effects were documented19,20. The mechanisms of its reduction of the amyloid burden by PAW could be in multiple manners. The antioxidative and anti-inflammatory actions could suppress APP, β-secretase, and γ-secretase gene expressions and reduce the Aβ formation27,28,29. Inhibition of polymerization from monomeric or oilgomeric Aβ is also a possible mechanism30. In our previous report, the amount of small water clusters created in PAW showed a slightly negative charged20. These small water clusters might prevent polymerization of Aβ in APP/PS1 mice. Enhancement of expressions of amyloid-degradation enzymes, including neprilysin, is another possible mechanism31. In this study, oligomeric Aβ protein levels were measured, but they showed no change with PAW treatment in APP/PS1 mice (Fig. 6). The synthesis of the Aβ protein is regulated by neprilysin, PSEN1, BACE1, and the APP. Levels of these proteins also did not change in our present data. We suggest that the effect of PAW on reducing the formation of senile plaques did not occur in the synthesis of the Aβ protein, but in preventing aggregations of Aβ proteins. Further studies on the reduction mechanism of PAW are needed.

The tau protein which is also the another main component of pathological biomarker, intracellular neurofibrillary tangles, is considered to be another target for the diagnosis and treatment of AD14. Interactions between amyloid and tau are well studied22. Amyloid enhances tau phosphorylation, and amyloid alters tau splicing32,33. In this study, p-tau accumulated in the hippocampus of APP/PS1 mice with a high amyloid burden along with aging. APP/PS1 mice given PAW showed reduced p-tau deposition in the hippocampus compared to APP/PS1 mice without PAW, and such deposition is considered the beginning site of AD pathology, by comparison of APP/PS1 mice without PAW (Fig. 4). These findings suggest that PAW can reduce p-tau accumulation, which might be attributed to reduced amyloid plaque formation in APP/PS1 mice. However, the detailed mechanisms still need further investigation.

Inflammation occurs in the pathologically vulnerable region of AD brains and contributes to AD pathogenesis. Interleukin (IL)-1 and IL-6 are immunoregulatory cytokines which are overexpressed in the AD brains34. Reduction of inflammation in AD brain might help to delay the progression of AD. In our present data, we analyzed the inflammation by measuring protein levels of IL-1β and IL-6 using an ELISA assay. Although the protein levels of IL-1β and IL-6 were not significantly reduced in PAW-treated APP/PS1 mice, decreased values were observed (Fig. S4). The number of mice used for the analysis of inflammation was not sufficient to show a significant difference. Further experiments are needed to elucidate the reduction effects on inflammation in APP/PS1 mice.

This study showed that PAW reduced the amyloid and p-tau burden in APP/PS1 mice. Although the precise molecular mechanisms are still unclear, our data might provide a new potential strategy for preventing of AD. Although there might be multiple mechanisms, aggregation or polymerization of amyloid, p-tau accumulation and anti-inflammation could all be involved. In conclusion, PAW confers effects of reducing amyloid and p-tau accumulation, borderline significant anti-inflammation and decelerating memory declines. These novel findings indicate the potential use of PAW in the therapy of early AD.