Alzheimer's disease (AD) is characterized by progressive dementia with senile plaques (amyloid-β peptide, Aβ) and neurofibrillary tangles (hyperphosphorylated tau, p-tau). Current drugs only temporarily relieve symptoms. Therefore, preventative food components are of interest. Epidemiological studies suggest a polyphenol-rich diet reduces AD risk, with rosmarinic acid (RA), a phenylpropanoid polyphenol from Lamiaceae herbs, showing promise due to its Aβ aggregation inhibition *in vitro*. Previous research showed RA inhibited Aβ oligomerization in Tg2576 mice, but its effects on tau phosphorylation and cognitive dysfunction remained unclear. Phosphorylated tau is neurotoxic, inducing neuronal death and contributing to the major neuropathological lesions of AD. While RA has shown promise in suppressing stress-induced tau aggregation, the molecular mechanism and its impact on tau pathology *in vivo* at physiologically relevant concentrations remained unclear. This study aimed to characterize the mechanism of RA's preventative effect in AD using 3xTg-AD mice, which exhibit both Aβ and p-tau overexpression, by assessing pathology and memory before the onset of AD pathology. The study employed behavioral tests (minimizing influence on subsequent analyses), immunohistochemistry to visualize early pathological changes, and transcriptome analysis of the hippocampus to comprehensively examine RA's effects.

Several studies support the potential of polyphenols, particularly rosmarinic acid (RA), in preventing Alzheimer's disease. Epidemiological studies have linked polyphenol-rich diets to a reduced risk of AD. *In vitro* studies demonstrated RA's potent ability to inhibit amyloid-β (Aβ) aggregation and alleviate synaptic toxicity by directly binding to Aβ's β-sheet structure. Previous *in vivo* studies using Tg2576 mice showed that RA supplementation inhibited Aβ oligomerization, but the impact on tau phosphorylation and cognitive function remained elusive. Other studies have explored RA's effects on drug-induced cognitive deficits and intraventricular Aβ injection models. While some research indicated RA's ability to suppress stress-induced tau aggregation, the detailed molecular mechanism and the effect of RA intake on tau pathology in AD models were not fully understood. Many *in vitro* studies used higher RA concentrations than those expected in the brain, raising questions about the relevance of *in vitro* mechanisms to *in vivo* effects.

This study used 6-8 week old male 3xTg-AD mice, divided into two groups: a control group fed a normal AIN-93G diet, and an RA group fed the same diet supplemented with 0.5% RA. Body weight and food consumption were monitored bi-weekly. After 8 months, mice were sacrificed, and blood and organs collected. Behavioral tests (Y-maze for spatial working memory and novel object recognition test for object recognition memory) were conducted at 10 months of age. Immunohistochemistry was performed to assess Aβ and p-tau accumulation in brain sections. Transcriptome analysis (DNA microarray) of the hippocampus was conducted on RNA from four mice per group. Quantitative RT-PCR analyzed the expression of *Jnk* family members and *Dusp1* in hippocampus and cerebral cortex. An enzyme-linked immunosorbent assay (ELISA) measured JNK3 protein levels in the hippocampus. Statistical analysis employed Student's t-test, with significance set at p<0.05.

RA supplementation improved spatial working memory and object recognition memory in 3xTg-AD mice, as evidenced by increased alternation ratio in the Y-maze test and higher discrimination index in the novel object recognition test. Immunohistochemical analysis revealed that RA significantly suppressed Aβ accumulation in cortical regions near the amygdala and p-tau accumulation in the hippocampal CA1 region, a key area for memory. Transcriptome analysis identified 549 differentially expressed genes (DEGs) in the hippocampus of the RA group. Gene ontology (GO) analysis showed enrichment in terms related to nervous system development, memory, and neurotransmission. KEGG pathway analysis revealed downregulation of the JNK signaling pathway, specifically JNK3, a key kinase involved in tau phosphorylation. This downregulation was further confirmed by qRT-PCR and ELISA, showing reduced expression of JNK3 mRNA and protein in the hippocampus. Immunohistochemistry confirmed decreased p-JNK and p-c-Jun (a JNK substrate) in the hippocampal CA1 region. RA also exhibited a systemic anti-inflammatory effect, reducing the expression levels of inflammatory mediators (IL-1β, TNF-α, CCL5, CXCL13, HMGB1) in both the brain (hippocampus, cerebral cortex) and peripheral tissues (spleen, intestine). The expression of TLR2 and TLR4, involved in neuroinflammation, was also reduced.

This study demonstrates that RA intake effectively inhibited the progression of AD pathology and improved cognitive function in 3xTg-AD mice. The improvement in cognitive performance correlated with the reduction in both Aβ and p-tau accumulation, particularly in the hippocampal CA1 region crucial for memory. The observed downregulation of the JNK signaling pathway, especially JNK3, provides a potential mechanistic explanation for the beneficial effects of RA. JNK3 is a key kinase involved in tau phosphorylation, and its inhibition likely contributes to the reduced p-tau levels. The systemic anti-inflammatory effect of RA further supports this mechanism, as inflammation is known to activate the JNK pathway. The observed reduction in peripheral inflammation might also contribute to the overall neuroprotective effect by reducing the inflammatory signals reaching the brain. Future research should investigate the precise pharmacokinetics of RA and its metabolites to determine whether peripheral anti-inflammatory effects precede those in the brain or if low concentrations directly impact the brain.

This study provides strong evidence that RA supplementation effectively mitigates Alzheimer's disease pathology and improves cognitive function in a mouse model. The mechanism likely involves the downregulation of the JNK signaling pathway, particularly JNK3, leading to reduced tau phosphorylation and a systemic anti-inflammatory effect. These findings support the potential of RA as a preventative or therapeutic agent for AD. Further research should focus on clarifying the detailed pharmacokinetic aspects of RA and its metabolites and exploring potential synergistic effects with other therapeutic approaches.

The study used a mouse model, which may not perfectly reflect the complexity of human AD. The sample size, while adequate for statistical analysis, could be increased for greater power. While the study shows a correlation between RA treatment and JNK pathway downregulation, further studies are needed to conclusively establish a causal relationship. The study focused on male mice, and further investigation is needed to determine if the effects of RA are similar in females.