The results provide preliminary evidence that these anthocyanins can modulate the PI3K/Akt/GSK3β signaling pathway in a vertebrate model, but they do not establish a treatment for Alzheimer’s disease in people. The researchers induced selected Alzheimer’s-like features by exposing adult wild-type zebrafish to aluminum chloride in water for four consecutive days. On Day 4, fish also received an intraperitoneal aluminum chloride injection to reinforce model induction.

Donepezil, an acetylcholinesterase inhibitor used in Alzheimer’s treatment, served as the positive control. C3R, C3S, and C3X were each administered at 2 milligrams per kilogram by intragastric gavage once daily for five days, beginning with the first day of aluminum exposure. That common dose was selected from preliminary screening at 0.5, 1, and 2 milligrams per kilogram to standardize the comparison rather than to identify a separate maximum effective dose for each compound.

The principal experiment included 15 fish per group: untreated controls, aluminum-exposed model animals, donepezil controls, and one group for each cyanidin glycoside. Behavioral testing showed that the model reproduced several of the changes the investigators sought to study. In the novel object recognition test, aluminum-exposed fish spent less time exploring a novel object and showed less preference for it than untreated controls, indicating impaired recognition memory.

In the color-biased T-maze, model fish took longer to enter the rewarded green chamber and spent less time there after learning the task. All three cyanidin glycosides increased exploration of the novel object and improved T-maze performance relative to the model group. C3R and C3X also shortened the latency to enter the upper region of a novel tank, where exploration was reduced in the model group, indicating attenuation of anxiety-like behavior.

These memory effects were not attributable to a simple increase in general movement: total distance traveled and average velocity did not differ significantly between the model and treatment groups in the relevant novel object recognition and T-maze comparisons. Brain biochemical analysis indicated that the compounds partly reversed the cholinergic and oxidative abnormalities associated with aluminum exposure. C3R, C3S, and C3X shifted these measurements toward control values, as did donepezil.

The glycosides also increased superoxide dismutase activity and glutathione content and reduced malondialdehyde, which the investigators used as a measure of lipid peroxidation. Together, these observations are consistent with reduced oxidative stress and partial restoration of cholinergic function. The experiment did not establish whether changes in oxidative stress preceded the behavioral or cellular effects, and it did not exclude contributions from other mechanisms.

Histological and ultrastructural examinations supported the idea that the treatments protected brain tissue. Hematoxylin and eosin staining showed loose cellular organization, reduced neuronal density, and vascular congestion in the model group, whereas treated fish displayed more intact tissue and greater cellular density. Nissl staining revealed fewer and more disorganized neurons and fewer Nissl bodies after aluminum exposure.

Treatment with each glycoside or donepezil significantly increased the amount of Nissl-positive material. Transmission electron microscopy showed disrupted membrane boundaries, cytoplasmic vacuolization, cellular edema, swollen mitochondria, and fewer identifiable organelles in model animals. C3R produced the clearest preservation of membranes, mitochondria, and other organelles, while C3S and C3X also produced improvements that left some edema and vacuolization.

These images provide morphological evidence of neuronal protection, but they cannot determine whether mitochondrial fission, fusion, or caspase-dependent apoptosis was directly affected. Gene-expression measurements showed that aluminum exposure altered molecular markers associated with cholinergic function, the amyloid precursor protein family, neurotrophic signaling, inflammation, and PI3K/Akt/GSK3β signaling. The model brain had increased expression of ache, aplp2, il6, il1b, tnfa, and rela, along with reduced chata, bdnf, and ntrk2b.

Expression of pik3r1 and akt2 decreased, whereas gsk3ba increased. C3R, C3S, C3X, and donepezil moved these measures toward control levels, with significant reductions in the elevated inflammatory transcripts and restoration of pathway-related transcripts. Western blotting produced a parallel protein-level pattern: treated fish had lower acetylcholinesterase and higher choline acetyltransferase, while phosphorylation of Tau at Ser396 declined.

Immunofluorescence imaging likewise showed weaker acetylcholinesterase signals and stronger choline acetyltransferase signals after treatment, with C3R producing relatively pronounced recovery. Aluminum exposure reduced the phosphorylation of PI3K p85 at Tyr458 and Akt at Ser473 while increasing the ratio of phosphorylated to total GSK3β at Tyr216. C3R and C3X significantly reversed these alterations, and donepezil produced similar effects.

C3S increased PI3K Tyr458 phosphorylation and reduced GSK3β Tyr216 and Tau Ser396 phosphorylation, but it did not significantly restore Akt Ser473 phosphorylation, indicating a partial pathway response. The interpretation requires care because Akt canonically influences GSK3β through inhibitory phosphorylation at Ser9 rather than Tyr216, and the study did not directly measure GSK3β kinase activity. The measurements therefore indicate coordinated pathway-associated regulation rather than proof that Akt directly controls GSK3β phosphorylation at Tyr216.

A separate experiment tested the functional involvement of PI3K signaling by administering the PI3K inhibitor LY294002 two hours before the cyanidin glycosides. Blocking PI3K attenuated the treatment-related increases in choline acetyltransferase, phosphorylated PI3K, and phosphorylated Akt. At the same time, it increased acetylcholinesterase, phosphorylated GSK3β, and phosphorylated Tau at Ser396.

The inhibitor result supports participation of PI3K/Akt signaling in the biochemical effects observed in the model, but it does not identify the direct molecular targets of the glycosides. Total PI3K, Akt, and GSK3β abundance did not change significantly among the relevant groups. Molecular docking was used to explore possible structural differences among the compounds.

C3R received the most favorable predicted scores for human PI3Kα and AKT1, followed by C3X and C3S for PI3Kα, while C3S ranked ahead of C3X for AKT1. These calculations are hypotheses about possible accommodation at the proteins, not evidence of direct binding or functional regulation, and they cannot fully account for C3R’s stronger biological effects in the experiment. Among the three compounds, C3R emerged as the most consistently effective candidate under the shared dose and short treatment schedule.

Its advantages appeared across behavioral testing, biochemical markers, histological and ultrastructural observations, cholinergic protein measurements, and PI3K/Akt/GSK3β-associated changes. The comparison does not show that C3R’s rutinoside-linked structure caused its stronger performance; the source presents the distinct sugar groups as a possible basis for further investigation rather than a demonstrated explanation. The results instead identify C3R as a priority for systematic follow-up.

Controlled gavage also ensured that the fish received measured compound doses, but it did not reproduce the gastrointestinal conditions, food matrix, and exposure patterns associated with eating cyanidin-rich foods. Translation of the findings remains limited in several ways. The model lasted only five treatment days and reproduced selected features such as cognitive impairment, oxidative stress, cholinergic dysfunction, inflammation, and increased Tau Ser396 phosphorylation.

It did not reproduce the chronic, age-dependent development of Alzheimer’s disease. The experiment also did not determine whether behavioral benefits persist after treatment ends, whether general antioxidant effects or interactions with aluminum contributed to the phenotype, or how the compounds behave after longer exposure. The gastrointestinal stability, bioavailability, metabolism, pharmacokinetics, and brain exposure of C3R, C3S, and C3X were not established, and species-specific differences limit direct extrapolation from zebrafish to mammals.

The researchers identified mammalian work, including tests in APP/PS1 mice, as a necessary next step for evaluating pharmacokinetic properties, blood–brain barrier penetration, efficacy, and direct targets. For now, the study offers a controlled in vivo screening result with broad but preliminary implications. Food-derived anthocyanins can produce measurable behavioral, biochemical, histological, ultrastructural, and molecular effects in an adult vertebrate exposed to a toxin-induced Alzheimer’s-like phenotype.

C3R performed best overall among the three equal-dose treatments, and the inhibitor experiment linked part of its biochemical profile to PI3K/Akt signaling. However, the work does not show that dietary supplementation could treat or prevent Alzheimer’s disease in humans.

Subject of Research: Neuroprotective effects of three food-derived cyanidin glycosides in an aluminum-induced Alzheimer’s-like adult zebrafish model

Article Title: Neuroprotective Effects of Food‐Derived Cyanidin Glycosides in an Alzheimer's Disease‐Like Zebrafish Model Through Modulation of PI3K/Akt/GSK3β Signaling

Article References: Wu, Y., Tie, F., Gao, Y., Li, J., Wang, R., Hu, N., Dong, Q., & Wang, H. (2026). Neuroprotective Effects of Food‐Derived Cyanidin Glycosides in an Alzheimer's Disease‐Like Zebrafish Model Through Modulation of PI3K/Akt/GSK3β Signaling. Food Science & Nutrition, 14(10), Article e72486. https://doi.org/10.1002/fsn3.72486

Image Credits: AI Generated

DOI: 10.1002/fsn3.72486

Keywords: cyanidin glycosides, cyanidin-3-O-rutinoside, zebrafish model, Alzheimer’s-like pathology, PI3K/Akt/GSK3β, cholinergic function, oxidative stress, Tau phosphorylation, anthocyanins, neuroprotection, Neuroprotective, Effects