Gut microbiota-dependent 24-hydroxycholesterol metabolism contributes to capsaicin-induced amelioration of Alzheimer’s disease-like pathology in mice

Domain: neuroscience
Published: 2026-02-03
Editorial outcome: major_revision

Abstract
Dietary capsaicin intake appears to affect the pathogenesis of Alzheimer’s disease (AD), while the underlying mechanisms remain unclear. Here, we found in human cohorts that moderate-to-high level of dietary capsaicin intake was associated with improved cognitive performance. Similarly, long-term oral capsaicin administration in male 5×FAD mice ameliorated AD-like pathologies and reshaped gut microbial composition. Gut microbiota transfer from capsaicin-treated mice produced similar effects of capsaicin intake. Moreover, capsaicin elevated the level of host 24(S)-hydroxycholesterol (24-HC), relating to the increase of gut Oscillibacter genus abundance. The 24-HC elevation enhanced microglial phagocytic activity in the brain, and inhibited proinflammatory factors production via liver x receptor β (LXRβ)-mediated transcriptional regulation. Finally, we observed elevation of 24-HC in plasma in AD patients with higher level of dietary capsaicin intake, which correlated with cognitive scores and plasma Aβ and p-tau biomarkers. These findings suggest the potential of capsaicin or capsaicin-rich diets in the prevention or treatment of AD and related diseases.

Introduction
Alzheimer’s disease (AD) is the most common cause of dementia in the elderly, with extracellular deposition of beta-amyloid (Aβ) and intraneuronal accumulation of hyperphosphorylated tau protein in the brain as two pathologic hallmarks1. There is no cure for AD to date2. In recent years, increasing attention has been paid to the influences of dietary pattern on AD onset and development3,4. Especially, emerging evidences have suggested a significant correlation of AD pathogenesis with dietary intake of capsaicin from chili peppers-rich foods5, which are prevalent in a number of regions worldwide including the Southwest China. Natural and synthetic capsaicin analogs showed many protective effects like antioxidation and anti-inflammation that potentially reduce the risk or degree of AD6. However, the exact protective effect and underlying mechanisms of capsaicin against AD are still elusive.

It has been recently unveiled that gut bacterial profile had strongly links to the development of AD7. Gut dysbiosis like imbalance between the Firmicutes and Bacteroidetes was widely recognized both in AD patients and animal models, which was even observed at the preclinical stage8,9. Transplantation of fecal microbiota from AD patients or transgenic mice both induced AD-like pathologies in microbiota-depleted healthy mice10–13, conversely, fecal microbiota transfer from wild-type mice and direct gavage of probiotics like Lactobacillus and Bifidobacterium both produced anti-AD outcomes14,15. Of note, capsaicin intake through diets appeared to be capable of reshaping the constitution and function of gut microbes16, but whether and how it contributes to the potential anti-AD effect of capsaicin are unknown.

The contribution of gut microbiota malfunction to AD pathogenesis is mainly mediated by the dysregulation of host metabolism. For example, gut dysbiosis in AD mice elevated levels of 12-hydroxy-heptadecatrienoic acid, prostaglandin E2 and poly-unsaturated fatty acids to elicit microglia overactivation and exacerbate AD symptoms in the brain10,12. Moreover, some gut microbes were capable of directly metabolizing diet-derived cholesterol through bacterial enzymes17–20, which might thereby regulate pathological processes like Aβ deposition, tau hyperphosphorylation and neuroinflammation21,22. Nonetheless, it is unknown how oral intake of capsaicin modulates host metabolism in a gut microbiota-dependent way to affect the AD pathogenesis.

In this work, we seek to investigate the effect and mechanism of oral capsaicin intake in affecting AD pathogenesis, and found in human cohorts that moderate-to-high dietary capsaicin consumption was associated with improved cognitive performance. Similarly, long-term oral capsaicin administration in 5×FAD mice alleviated AD-like pathologies. Unexpectedly, these protective effects of capsaicin persisted following global knock-out of capsaicin receptor TRPV1. Further studies revealed that the gut microbiota, especially the Oscillibacter genus had an important contribution to the protective effects of capsaicin, which modulated lipid metabolism to elevate host 24(S)-hydroxycholesterol (24-HC) level, thereby enhancing microglial phagocytic activity in the brain, and facilitating the liver x receptor β (LXRβ)-mediated transcriptional inhibition of proinflammatory factors production. These findings suggest a way of capsaicin to alleviate AD pathologies through gut microbiome-dependent regulation on 24-HC metabolism, and the potential of capsaicin or capsaicin-rich diets in the prevention or treatment of AD and related diseases.

Results
Capsaicin intake correlates with cognitive improvements in AD patients and 5×FAD mice
Firstly, we accessed the association between the dietary capsaicin intake levels and cognitive function in two Chinese cohorts (N = 151 and 95, respectively). Subject information of sex, age, education level etc. was summarized in the Supplementary Data 1. Cognitive function was assessed using Mini-Mental State Examination (MMSE) and/or Montreal Cognitive Assessment (MoCA). The cohorts consisted of subjects diagnosed to be cognitively normal (CN), with mild cognitive impairments (MCI), moderate to severe cognitive impairment (M/SCI) based on MMSE scores, or definite AD based on comprehensive clinical assessment. Capsaicin intake in diets were evaluated using a self-developed scale (Supplementary Data 2). The results showed that subjects with moderate-to-high levels of chili intake had higher cognitive scores in subgroups with cognitive impairments and AD patients following adjustment of other covariates including sex, age, education years, hypertension, diabetes, coronary heart disease and gastritis in the analysis of covariance (ANCOVA) (Fig. 1a, Supplementary Data 3).

In addition, we also evaluated the preference and tolerance to chili-rich diets, which were both found to tightly correlate with the intake level of chili-rich diets, and slightly correlate with cognitive scores, respectively. Besides, there was lower prevalence of AD in the subgroup with moderate-to-high level of chili intake (Supplementary Fig. S1).

Next, we examined the potential dose-dependent effect of dietary capsaicin (abbreviated as Cap in figures) intake in 3-month-old wild-type (WT) C57BL/6 mice, increasing doses of capsaicin (0.01–1 mg/kg) were administrated intragastrically (i.g.) once every day for a month. We found that capsaicin at 1 mg/kg, but not at lower doses, had the tendency to facilitate object-place recognition, novel-object recognition of mice, and to enhance spatial memory in Morris-water maze test. By contrast, capsaicin at all doses did not affect contextual fear memory. Additionally, capsaicin also increased the time of mice spent in the central zone in open field test, and time in the open arms in elevated-plus maze test, possibly thanks to the decrease in anxiety following capsaicin (Supplementary Fig. S2).

In subsequent, we treated 5-month-old WT and 5×FAD mice, a transgenic line expressing human APP and PSEN1 with a total of five AD-linked mutations23, with capsaicin at the dosage of 1 mg/kg for a month (Fig. 1b). Capsaicin again showed trends in improving object-place recognition and spatial memory Morris-water maze in WT mice, and more importantly, it alleviated cognitive impairments of 5xFAD mice in these behavioral tests (Fig. 1c–i). Moreover, capsaicin also increased the discriminative index in the novel-object recognition test, but did not change conditioned contextual fear memory (Supplementary Fig. S3a-b). Intriguingly, by contrast to WT mice, capsaicin had only limited effects on the anxiety-like behaviors of 5×FAD mice in both open-field and elevated-plus maze tests (Supplementary Fig. S3c-d), which might be attributed to the hyperactivity of this transgenic line at early age24.

Capsaicin reduces Aβ burden and inhibits microglia overactivation in the hippocampus of 5xFAD mice
In consistent with the capsaicin-induced improvements in cognitive functions of 5xFAD mice, we found that capsaicin decreased the density of beta-amyloid (Aβ) plaques stained by 6E10 antibody in the hippocampus of brain, and downregulated both the levels of SDS-insoluble and soluble Aβ40 and Aβ42 fragments (Fig. 1j–l, supplementary Fig. S3e). Of note, capsaicin did not change total protein levels of Aβ precursor protein (APP), β-Site APP cleaving enzyme 1 (BACE1) and presenilin-1 (PSEN1) (Supplementary Fig. S3f-g), suggesting non-significant change in the production of Aβ.

Subsequently, we analyzed the capsaicin-induced transcriptomic changes in the hippocampus of 5×FAD mice by bulk RNA sequencing, and found that differentially expressed genes (DEGs) were predominantly enriched in GO_biological pathways involved in immune and inflammatory regulation (Supplementary Fig. S3h), and the majority of these DEGs were downregulated in the Cap-treated group (Supplementary Data 4). We measured local levels of pro-inflammatory factors interleukin 1β (IL-1β) and tumor necrosis factor alpha (TNF-α) in the hippocampus of 5×FAD mice utilizing enzyme-linked immunosorbent assay (ELISA), and found these two factors were both downregulated by capsaicin (Supplementary Fig. S3i), suggesting an anti-inflammatory effect of capsaicin in the brain.

It has been well-recognized in AD brains that overactivation of microglia, the major type of immune cell in central nervous system, impairs its capacity of Aβ phagocytosis and amplifies neuroinflammatory responses25. Thereby, we examined how capsaicin affected microglia. On one hand, capsaicin decreased the number of plaque-surrounded cell aggregates of microglia, which was consistent with the reduction of plaque numbers (Fig. 1m, n). On the other hand, for separated microglia that were not clustered around plaques, capsaicin intake decreased the volume of soma but increased the branching complexity of cell processes (Fig. 1o, p), these morphologic signs all suggested suppression of microglia overactivation26.

To examine whether the capsaicin-induced suppression of microglia activation and inflammation in 5×FAD mice was a primary outcome or merely a secondary consequence following Aβ plaques reduction, we administrated capsaicin in 3-month-old WT C57BL/6 mice with repeat intraperitoneal (i.p.) injections of lipopolysaccharide (LPS), a model showing central neuroinflammation but no Aβ deposition27. We found in these mice that capsaicin treatment still showed effectiveness in downregulation IL-1β and TNF-α, and in decreasing microglia activation in the hippocampus (Supplementary Fig. S4). These results suggest that the observed anti-inflammatory effects of capsaicin in 5×FAD mice occurred at least partly in prior to the reduction of Aβ plaques.

Knock-out of TRPV1 has limited impact on the protective effects of capsaicin against AD
To investigate mechanisms underlying the protective effects of capsaicin in AD mice, we firstly measured the distribution of capsaicin in 5×FAD mice following repeated oral administration. The results showed that capsaicin was enriched in the contents of stomach and gut, and to relatively less concentration contained in tissues of stomach, intestines, liver and brain (Fig. 2a).

To examine whether the anti-AD effects of capsaicin was dependent on its binding to the well-recognized capsaicin receptor, transient receptor potential vanilloid sub-type 1 (TRPV1), especially in the stomach and intestines, we generated a mouse line of 5×FAD mice with global TRPV1 gene knock-out, termed 5×FAD&TRPV1–/– (5×FAD&KO) (Fig. 2b–d). Unexpectedly, we found both in Veh- and Cap-treated 5xFAD mice at 6-month of age that TRPV1 KO changed none of Aβ plaques (Fig. 2e, f), microglia morphology (Fig. 2g, h), inflammatory factors (Fig. 2i) and cognitive performance in object-place recognition (Fig. 2j, k) and Morris-water maze tests (Fig. 2l–n).

In addition, taken into consideration potential compensatory effect mediated by other receptors that appear to be indirectly regulated by capsaicin, like TRP channels TRPA1, TRPV2 and voltage-gated ion channels Nav1.7, Kv7.428–30, we measured the mRNA levels of these genes, but found significant influence neither by TRPV1 KO nor capsaicin treatment (Supplementary Fig. S5).

Altogether, these results suggest that TRPV1 is not indispensable for the observed protective effects against AD of oral capsaicin treatment.

Gut microbes contribute to the protective effects of capsaicin
Given that gut microbiota has been widely-recognized to be involved in the AD pathogenesis31, we next investigated the impact of capsaicin on gut microbes in 5×FAD mice. Metagenome sequencing of intestinal contents was performed, and non-redundant geneset was acquired with the similarity threshold at 95% and coverage threshold at 90%. Principal-component analysis (PCA) based on the taxonomic species revealed distinct clustering patterns among samples from vehicle and capsaicin-treated 5×FAD mice (Fig. 3a). 5×FAD mice showed reduction in alpha diversity of microbiota taxonomy, which was reversed by capsaicin, as indicated by increase in the abundance-based coverage estimator (ACE) index (Fig. 3b). In specific, capsaicin predominantly increased the abundance of Firmicutes (also known as Bacillota), and decreased the abundance of Bacteroidetes, Proteobacteria and Verrucomicrobia at the phylum level (Fig. 4c). Utilizing the linear discriminant analysis effect size (LEfSe) method, we found many featured species, including several belong to genus of the Alistipes, Oscillibacter, Prevotella, Hungatella, etc. enriched in the capsaicin-treated group (Fig. 3d).

To examine whether the protective effects of capsaicin were mediated through its modulation on gut microbiota, we performed gut microbiota transfer (GMT) from 5-month 5xFAD mice receiving 1-month capsaicin treatment to a new batch of 5-month 5xFAD mice. Contents from both small intestines and colon were collected to maximize gut microbe enrichment and minimize interference from environmental bacteria in the housing conditions and prevent the disruption of anaerobic bacteria by oxygen exposure. Gut contents from Veh- or Cap-fed 5xFAD mice were collected and immediately processed under anaerobic conditions. Given the low water solubility of capsaicin, all samples underwent repeated washing with pre-reduced PBS followed by centrifugation. The supernatant was obtained and further extracted with oil to remove any potential residual capsaicin. It was confirmed that the Cap was virtually undetectable in the final aqueous phase containing the gut microbes (Fig. 3e, f). The final processed solution was used for GMT in 5-month 5xFAD mice (0.5 mL each day, once every other day for a month), equal doses of PBS was delivered as control (Fig. 3g).

We found that GMT from Cap-treated mice, but not from Veh, significantly reduced Aβ plaques density while inhibiting microglia activation (Fig. 3h–k), and downregulated inflammatory factors in the hippocampus (Fig. 3l). Consistently, GMT from Cap-treated mice improved cognitive functions of 5xFAD mice compared with Veh-GMT and PBS controls in both object-place recognition and Morris-water maze tests (Fig. 3m–p). These results supported a pivotal contribution of the gut microbiota in mediating the amelioration of AD phenotypes by oral capsaicin intake.

Depletion of gut microbes diminishes the beneficial effects of capsaicin
To further ascertain the contribution of gut microbes to the capsaicin’s protective effects, we applied high-dose cocktail of antibiotics (ABX) to deplete the gut microbes of either Veh- or Cap-treated 5xFAD mice at 5-month of age. ABX was pretreated through oral gavage every day for 7 days before capsaicin administration and persisted throughout the experiment. The elimination of gut microbes by ABX was confirmed by bacteria culturing (Supplementary Fig. S6a–c). We found in both WT and 5xFAD mice that ABX hindered the cognitive-improving effect of capsaicin (Supplementary Fig. S6d-e). Intriguingly, for 5xFAD mice, ABX alone reduced Aβ plaques and insoluble Aβ42, as well as downregulated levels of microglia activation and inflammatory factors in the hippocampus (Supplementary Fig. S6f–k), potentially thanks to the prevention of gut dysbiosis and reduction of harmful metabolites following pan-elimination of gut microbes32. Notably, the depletion of gut microbes by ABX nearly abolished the beneficial effects of capsaicin in reducing Aβ aggregates in the brain, and eliminated the anti-inflammatory effects of capsaicin by increasing microglia activation and pro-inflammatory factors levels (Supplementary Fig. S6f–k), suggesting a pivotal contribution of gut microbiota in the anti-AD effects of capsaicin.

Gut microbiota regulates 24-hydroxycholesterol level in 5×FAD mice under capsaicin treatment
To investigate how the capsaicin-induced gut microbiota remodeling mediated the observed protective effects against AD, we performed further analysis of differentially expressed metagenomic gene sets in capsaicin-treated 5×FAD mice, and found the top-2 enriched pathways of the gene sets as glycan and lipid metabolism (Fig. 4a, b). Subsequently, we examined metabolic profile in the plasma of 5×FAD mice following repeated capsaicin treatment (Fig. 4c, d, Supplementary Data 5), and found that different metabolites were predominantly involved in lipid metabolism-associated pathways (Fig. 4e, Supplementary Data 6). Specifically, we noted that capsaicin downregulated the level of cholesterol while upregulated its derivate 24(S)-hydroxycholesterol (24-HC, also known as cerebrosterol) (Fig. 4d, f), the latter has been revealed to be capable of alleviating cognitive deficits and reducing Aβ burden33,34, but traditionally considered to be specifically synthesized from cholesterol in central nervous system by CYP46A135.

Thereby, we measured whether pan depletion of gut microbiota ABX affects 24-HC levels by liquid chromatography mass spectrometry (LC-MS) in the gut contents, plasma and hippocampus of WT and 5xFAD mice. Firstly, 5xFAD mice had dominantly lower levels of 24-HC in all those detected samples compared with WT mice. For both WT and 5xFAD mice, capsaicin treatment upregulated 24-HC levels in the intestinal contents, plasma and hippocampus, which were all reversed by the depletion of gut microbes with ABX. Especially, in the intestinal contents, ABX almost brought down the level of 24-HC to nil, suggesting a contribution of gut microbiota in the capsaicin-induced upregulation of 24, especially in the peripheral (supplementary Fig. S7a-b).

Additionally, we examined whether the reduction of 24-HC at different levels were resulted from potential inhibition of 24-HC synthesis in the brain by ABX, both mRNA and protein levels of CYP46A1 in the brain were measured. It was found that CYP46A1 was downregulated in 5xFAD mice compared with WT mice, suggesting that the central production of 24-HC in AD was also downregulated in the brain of AD mice aside from gut. Nonetheless, CYP46A1 expression in the brain was affected neither by capsaicin nor ABX treatment (Supplementary Fig. S7c–e).

Taken together, these data suggest the existence of peripheral production of 24-HC, and a pivotal contribution of gut Oscillibacter as well as modulatory effect of capsaicin in this process.

Gut Oscillibacter is involved in the production of 24-HC
Next, we sought to investigate whether 24-HC can be synthesized in the gut in a microbiome-dependent way. Combinative analysis of metagenomic and metabolomic data revealed that the level of 24-HC metabolism had most predominantly positive correlation with the Oscillibacter genus belonging to the Oscillospiraceae family in Firmicutes phylum (Fig. 4g), which was revealed to be capable of metabolizing cholesterol to generate hydroxycholesterol, presumed to depend on the expression of ismA homologous gene17,20. Thus, we next focused on the regulation of 24-HC by Oscillibacter. Further analysis of metagenomic data revealed reduced abundance of several belonging species and the overall Oscillibacter genus in 5xFAD mice, which was significantly rescued by capsaicin treatment (Fig. 4h, i). Meanwhile, the Oscillibacter abundance correlated negatively with plasma cholesterol level and positively with 24-HC level in liner regression analysis (Fig. 4j).

Furthermore, we examined Oscillibacter changes in AD patients in GMrepo v2, a curated human gut microbiome database36. In one of the three AD datasets included37–39, namely dataset PRJNA770746, a significant enrichment of Oscillibacter in healthy controls was found (LDA score = −3.17). Overall, the abundance of Oscillibacter was significantly downregulated in AD patients compared with healthy controls (Fig 5a-b). Besides, one previous case study found that sustained treatment of in vitro cultured human feces with capsaicin led to a significant increase in Oscillibacter abundance40. Collectively, these findings suggest a potential link between dietary capsaicin intake with increase in gut Oscillibacter abundance.

Next, we isolated gut microbes under anaerobic conditions from Cap-treated 5xFAD mice, and identified Oscillibacter via whole-genome sequencing (Fig. 5c). The isolated Oscillibacter was further validated by prominent expression of preciously reported marker genes including Oscillibacter-specific 16S rRNA (V3-V4 region), ismA and CgT homologous genes17, as measured by qPCR compared with negative controls of E. coli and positive control of J115 (Fig. 5d). Besides, we ascertained the capacity of Oscillibacter to generate 24-HC in cultures. Cholesterol treatment for 24 h significantly increased the 24-HC level in the culture medium, which was further upregulated under capsaicin co-administration (Fig. 5e). Subsequently, we grafted Oscillibacter repeatedly into 5-month 5xFAD mice for a month, equal doses of culture medium were inoculated as control (Fig. 5f). The results showed that Oscillibacter supplement significantly reduced Aβ plaques density (Fig. 5g), inhibited microglia overactivation while downregulating pro-inflammatory factors levels (Fig. 5g–k), and improved cognitive functions of 5xFAD mice (Fig. 5l–o). These outcomes mimicked the protective effects of oral capsaicin intake.

24-HC facilitates microglia phagocytosis of Aβ and suppresses inflammation
To examine whether and how 24-HC affects microglia overactivation and Aβ phagocytosis, increasing doses of 24-HC were administrated in BV2 cells, a mouse-derived microglia cell line. Limited cytotoxicity of 24-HC was observed at concentrations lower than 50 μM in the MTT assay (supplementary Fig. S8a). In subsequent, Rhodamine B (RhB)-conjugated Aβ was added in LPS pre-treated BV2 cell cultures. After wash-out of extracellular Aβ in the medium, we found that 24-HC at 10 μM significantly increased the amount of Aβ phagocytized by BV2 cells at 4.5 h post administration, and upregulated clearance rate of phagocytized Aβ (intracellular Aβ: (4.5 h − 24 h)/4.5 h) (Fig. 6a–c). The facilitation of Aβ phagocytosis in the 24-HC-treated group was also confirmed by increased portion of Aβ-containing cells at 4.5 h (Fig. 6d).

Furthermore, 24-HC at 10 μM also significantly reversed the Aβ-induced upregulation of pro-inflammatory factors IL-1β and TNF-α (Fig. 6e), increased percentage of BV2 cells expressing CD206 that were traditionally recognized to have high phagocytic and low pro-inflammatory activities, as well as decreased percentage of cells expressing CD16/32 and CD86, which are recognized to be highly pro-inflammatory and aggressive41 (Supplementary Fig. S8b–e). Consistently, the effects of 24-HC in facilitating Aβ phagocytosis and downregulating inflammatory factors at 10 μM were also observed in primary cultured mouse microglia (Supplementary Fig. S8f–h).

Next, we tested how direct supplement of 24-HC in 5-month-old 5xFAD mice affected microglia function and Aβ deposition in the brain. 24-HC was intraperitoneally (i.p.) injected once every other day (0.01 mg/kg, dissolved in DMSO) for a consecutive month, which significantly elevated the level of 24-HC in the hippocampus (Fig. 6f–g), indicating that peripheral 24-HC can at least partly cross the blood-brain barrier (BBB). To further confirm the 24-HC absorption in gut tracts and its penetrability across the BBB, we conjugated 24-HC with fluorescein BODIPY-FL and administrated the 24-HC-BODIPY-FL (0.02 mg/kg, i.g.) in 5-month-old 5xFAD mice for consecutively 7 days. Prominent fluorescent signals were detected in homogenates of gut contents, intestines, plasma and hippocampus tissue, respectively, though the percentage of labeled 24-HC reaching the brain appeared to remain much lower than those in guts and blood after treatment, which was possibly due to rapid metabolism in the liver and other peripheral organs. Moreover, to rule out potential interference from fluorescein on the membrane-penetrating efficiency of 24-HC, we also

Methods

Peer review rounds
[{"round": 1, "reviewer_comments": {"reviewer_1": "What are the noteworthy results?\ncapsaicin lower amyloid pathology and inflammation in a mouse model of AD and though a gut microbiota dependent\nmechanism\nWill the work be of significance to the field and related fields? How does it compare to the established literature? If the work\nis not original, please provide relevant references.\nIt can add some originality, on the gut microbiota dependent mechanisms. The mechanisms and the claim of this be due to\n24HC need to be better elucidated\nDoes the work support the conclusions and claims, or is additional evidence needed?\nAddidional evidence is needed.\nFigure 1: The human cohort needs further characterization. Did the subject described as healthy have memory complaints\n(subjective cognitive decline)? How many females and males were there? What was the age? Were the data corrected for\nthese variables? There is no mention of this in the results.\nIn general, sometimes WT appears, sometimes not, it is important to see effects of capsaicin on WT and in relation to FAD\nmice. For example, Fig 1, do the capsaicin mice perform as good as wild type mice? this type of data is not possible to get in\nthe present design.\nfig3 would be good to see quantifications of TRPV1 by immuno and also other technique. It would be good to see data on\nthe KO mice, to see the reduction of levels in TRPV1 in different regions.\nfig 5 and 6. here I have several concerns. 24HC should be mostly present in serum and brain. first of all, I would like to see\nlevels of 24HC in wt mice; in fad mice, the concentration in the brain seems quite low (usually around 40-60 ng/mg). a further\nstudy on the levels of cyp46a1 is necessary, in brain, and intestine. The effects of the antibiotic on 24OH levels is missing\nand it is central. The authors need to prove that this is mediated by 24HC. In the present form I dont think there is enough\nproof for this. Fig 6 is not bringing evidence, it is already known that 24HC is protective.\nAre there any flaws in the data analysis, interpretation and conclusions? Do these prohibit publication or require revision?\nsee comment above\nIs the methodology sound? Does the work meet the expected standards in your field?\nsex of mice is not reported\nIs there enough detail provided in the methods for the work to be reproduced?\nsee comment above", "reviewer_2": "This study addresses an important and timely topic by exploring the neuroprotective effects of capsaicin in the context of AD,\nwith a particular focus on the gut–brain axis. Although previous reports have suggested that capsaicin may confer benefits in\nAD models, the underlying mechanisms have remained largely unclear. The authors provide compelling evidence that\ncapsaicin exerts its effects not through its canonical receptor TRPV1 but rather by modulating the gut microbiome and\nassociated metabolites, leading to enhanced microglial phagocytic activity in the brain. This represents a novel and\nintriguing perspective aligned with the growing interest in microbiota-mediated neuroimmune interactions.\nHowever, while the study is conceptually interesting and potentially impactful, several claims would benefit from further\nexperimental support. Specifically:\n1. While the observation that capsaicin intake is associated with cognitive performance in Figure 1A-C is intriguing, the\nanalysis appears to lack adequate control for potential confounding factors. Differences in age, sex, education level,\nphysical activity, social engagement, and overall dietary habits between high and low capsaicin intake groups may have\ninfluenced the results. It is unclear whether these variables were appropriately adjusted for during statistical analysis.\nIncorporating multivariable regression or other methods such as propensity score matching would strengthen the validity of the\nfindings.\nFurthermore, the cross-sectional nature of the survey limits causal inference, as individuals with higher cognitive function\nmay inherently prefer spicy foods, or dietary preferences could shift due to cognitive decline. Finally, the subjective\nperception of spiciness may vary widely among individuals, potentially introducing bias into the data collection process, and\nit is not clear whether this was adequately standardized or accounted for during data collection.\n2. While the observation that capsaicin does not exert protective effects in TRPV1 knockout 5×FAD mice is intriguing, it\nalone may not suffice to conclusively demonstrate TRPV1-independent mechanisms. To strengthen this conclusion,\nadditional data addressing potential compensatory mechanisms should be included. For instance, investigating the\nupregulation or functional involvement of other TRP channels (e.g., TRPA1, TRPV2) or alternative signaling pathways\nactivated by capsaicin would provide critical insights. Such experiments could clarify whether the observed effects are\ngenuinely independent of TRPV1 and further elucidate the molecular basis of capsaicin's protective actions.\n3. The finding that capsaicin’s protective effects are abolished following antibiotic (ABX) treatment in 5×FAD mice suggests\nthat the gut microbiota plays a key role in mediating its actions (Figure4E-K). However, several previous studies AD mouse\nmodel have reported that ABX treatment alone can reduce Aβ plaque burden and ameliorate cognitive decline, potentially\nvia modulation of microglial activation (PMID: 40033338). Given these contrasting findings, it would be important for the\nauthors to discuss how their results relate to the existing literature. In particular, further clarification regarding the\nexperimental conditions (e.g., age at ABX administration, duration, and spectrum of antibiotics), and the role of microglial\nresponses under ABX treatment, would help reconcile the observed discrepancies. Including such a discussion would\nstrengthen the interpretation of the ABX data presented in this figure.\n4. The authors propose that capsaicin increases plasma levels of 24-hydroxycholesterol (24-HC), which in turn enhances\nmicroglial phagocytosis and suppresses inflammatory responses through a gut-brain axis mechanism. While the direct\neffects of 24-HC on microglial function are partially demonstrated using BV2 cells treated with exogenous 24-HC and\nthrough i.p. injection experiments, it remains unclear whether the observed in vivo effects are indeed mediated by\nperipherally derived 24-HC.\nTo substantiate the claim that 24-HC acts as a peripheral metabolite mediating microglial responses in the brain, additional\nexperimental evidence is needed. In particular, it is important to determine whether 24-HC generated in the periphery can\ncross the blood-brain barrier and reach the CNS. Tracer studies using labeled 24-HC would be highly informative in this\nregard. Moreover, while the authors show in Figure 5 that plasma levels of 24-HC are decreased in 5XFAD mice compared\nto WT, they do not provide corresponding data on 24-HC levels in the hippocampus or intestine in these groups. Such\ncomparative quantification would clarify the compartmental dynamics of 24-HC in the disease context.\nFurthermore, since 24-HC is primarily synthesized in the brain by CYP46A1 and released into the circulation, the observed\nincrease in plasma 24-HC could reflect enhanced CNS synthesis rather than peripheral production. In this regard, the data\nin Figure 9A—showing elevated plasma 24-HC levels in human subjects with high capsaicin intake—are intriguing but\ninsufficient to determine the source of 24-HC (i.e., gut vs. brain origin). Therefore, quantitative analysis of 24-HC levels in the\nbrain, plasma, and intestinal contents following capsaicin treatment would provide more convincing support for the proposed\ngut-brain axis mechanism.\n5. In relation to comment 4, in Figure 8B, the authors show the level of 24-HC in the hippocampus of P301S mice; however,\ngiven the central hypothesis that peripheral 24-HC contributes to the gut-brain axis mechanism, the levels of 24-HC in\nplasma and intestinal contents should also be provided in the same model.\n6. The authors report that 24-HC increases phagocytosis at physiologically relevant concentrations (0.01–10 μM), while it\nsuppresses Aβ-induced TNF-α and IL-1β production at a much higher concentration (10 mM) (figure 6). However, the\nconcentration used for inflammation assays is excessively high and likely to induce cytotoxic effects, which could confound\nthe interpretation of the observed anti-inflammatory effects. It is unclear whether these effects are directly mediated by 24-HC\nor are secondary to cell viability loss.\nTo address this issue, the authors should provide data on cell viability following 10 mM 24-HC treatment, such as MTT or\nLDH assays, to rule out cytotoxicity as a confounding factor. Additionally, investigating whether lower concentrations of 24-\nHC (e.g., 1–10 μM) can also exert anti-inflammatory effects would ensure consistency with the phagocytosis assay and\nstrengthen the validity of the findings.\nFurthermore, the authors should justify why such vastly different concentrations were used for the\nphagocytosis and inflammation assays.\n7. Although 3D reconstruction data are included, the current representative image in Figure 6P does not clearly support the\nauthors’ intended interpretation. It remains ambiguous whether the image is meant to highlight an overall reduction in Aβ\nburden or enhanced microglial phagocytosis of Aβ. The colocalization of Iba1 and 6E10 alone does not provide definitive\nevidence of active\nphagocytosis, and the image as presented does not sufficiently convey either outcome. To improve\nclarity, it is recommended that the authors (1) clearly specify the primary message they aim to deliver through this image,\nand (2) replace or supplement the figure with a more representative and interpretable image, ideally one that includes clearer 3D\nvisualization of Aβ engulfment by microglia. Furthermore, accompanying quantitative data would greatly strengthen the\nreliability of this claim.\n8. In Figures 6 and 7, the authors primarily use the BV2 cell line to investigate the protective mechanisms of 24-HC. While\nBV2 cells are widely used as a model for microglial studies, they differ from primary microglia in several aspects, including\ngene expression profiles and functional responses. To strengthen the validity and physiological relevance of the findings,\nit would be valuable to verify key results—such as enhanced phagocytosis and LXRβ sumoylation—in primary microglia.\n9. In Figure 7, the authors investigate the effects of LXRβ knockdown on microglial responses. However, to validate the\ninterpretation of the data, it is necessary to confirm whether the knockdown was specifically achieved in microglia. The\nauthors should provide experimental evidence demonstrating microglia-specific knockdown efficiency, such as\nimmunostaining for LXRβ in Iba1-positive cells or cell-type–specific expression analysis following viral delivery.\n10. While the authors focus primarily on the microglial effects of 24-HC, it is important to recognize that 24-HC can also\ninfluence other brain cell types, including neurons and astrocytes. The current manuscript lacks discussion regarding the\npotential impact of 24-HC on these cell populations. Given that 24-HC has been reported to affect astrocyte cholesterol\nmetabolism and neuronal functon(PMID: 34067119), it is important to consider whether the protective effects observed in the\nP301S tauopathy model may also involve non-microglial mechanisms. The authors should include either experimental\nvalidation or at least a discussion addressing the possible roles of 24-HC in neurons and astrocytes, particularly in the\ncontext of tau pathology.\n11. In lines 377–378, the authors state, \"Elevation of 24-HC in the plasma and brain was found here to protect against AD\nboth in 5×FAD and P301S mice.\" This sentence implies that 24-HC levels were elevated in both the plasma and brain in\nboth models. However, plasma 24-HC data are not presented for the P301S mice. The authors should revise this statement\nto more accurately reflect the data shown in the manuscript.\n12. In lines 191 and 196, the figure is referred to as \"Figure 4,\" but based on the interpretation of the results, it seems that\n\"Figure 5\" is the correct.", "reviewer_3": "The manuscript by Li et al., seeks to identify the mechanism through which capsaicin (Cap) improves memory and reduce\nbrain pathophysiology in mouse models for Alzheimer’s Disease (AD) and other neurodegenerative disorders. Using a\ncombination of mouse behavior, immunohistochemistry, and molecular biology, the authors replicated previous findings that\nCap treatment rescued abnormal behavior and microglial biology in the 5X-FAD mouse model for AD and provide new data\nthat Cap increases levels of a cholesterol metabolite (24-HC), which has also been previously associated with AD. The\nauthors show that antibiotics treatment blocks the Cap-mediated rescue of AD-like phenotypes as well as the increase in 24-\nHC levels. Importantly, the authors extend their findings from the 5xFAD mouse and show that (a) Cap also rescues\nbehavioral deficits and increases 24-HC in the hTau.P301S mouse model for frontotemporal dementia and (b) 24-HC levels\nare correlated to increased cognition in humans with AD.\nThe study of mechanism of action of a therapeutic drug and the role of the gut-microbiota-brain axis in Alzheimer’s disease\nare of interest to the field. The use of orthogonal models for neurodegenerative disorders and data from humans provide\nimportant pieces of data. While the findings are interesting, several important experiments are missing which would better\nsupport the authors’ claim that the mechanism through which Cap rescues deficits is through the microbiome.\nPlease see below for more specific comments/questions:\n1. Would FMT of microbes from 5xFAD mice treated with Cap into untreated 5xFAD mice also rescue the behavioral and\nbrain pathological phenotypes? Are the Cap-mediated changes in the microbiome directly causing the increases in 24-HC\nor is an interaction between the microbes and Cap treatment necessary for Cap to have an effect. In other words, are the\nchanges in the microbiome downstream of Cap or working synergistically with Cap?\n2. The authors state that deletion of Trpv1 has no effect on capsaicin rescue of phenotypes in the 5xFAD mice. While the\ndata support this for the brain pathology (Fig 3E-J), the data are less convincing for the behavior data. Is there a significant\ndifference between the 5xFAD+Veh and 5xFAD&KO + Cap in Fig 3L and 3N? What is the effect of Trpv1 KO on 5xFAD\nmice independent of Cap treatment?\n3. What effects do ABX alone have on the phenotypes in the 5xFAD mice? This important control is missing.\n4. By how much does ABX reduce the amount of 24-HC in plasma (5H)? Does it reduce all the way to the levels seen in\nVehicle treated mice or only partially? This will help to determine how much of a role the microbiome plays in the Cap-\nmediated increase in 24-HC. Please show the absolute amount of 24-HC in plasma (5G) to allow for a more direct\ncomparison.\n5. Does treatment with SOT prevent the beneficial effects on 24-HC on behavior and brain pathology? This experiment will\nhelp to determine whether 24-HC is needed in the brain or whether 24-HC has peripheral effects which lead to the\nphenotype rescue.\n6. Is the memory enhancing effect of Cap in WT mice dependent on changes in the microbiome and 24-HC? Is there a dose-\ndependent effect of Cap on microbiome changes? The authors should treat WT mice with ABX (or use germ-free mice) and\nCap (and with an ABX only group) to determine whether depleting the microbiome blocks this effect in WT mice as in the\n5xFAD mice.\n7. Please include more methodological details regarding the animal experiments:\na. At what age were treatments and experiments performed on the different mouse models? Did treatment begin before or\nafter the onset of different phenotypes (e.g., behavior) in these mice? If treatment began before the onset of phenotypes\n(prevention-type experiment), would treatment after the onset of phenotypes (rescue-type experiment) also be efficacious?\nb. What breeding scheme was used to generate the mice for the experiment? A recent report (DOI:\n10.1016/j.neuron.2024.12.025) suggests that parental origin of the transgene is an important variable\nin disease phenotypes and therefore this information should be included in the methods section.\nc. How were mice housed for these experiments? Were control and transgenic animals housed in the same cage? Were\nmice in different treatment groups housed together or separately?\nd. When sequencing “small intestinal contents,” was the entire small intestines used or just one section (i.e., duodenum,\njejunum, or ileum)?\ne. It is unclear how the data for the fear conditioning test are presented (Supplementary Fig 2). Please define what test/probe\nis referring to. Typically, the % time freezing 24 hours after the foot shock is shown for this behavior."}, "author_response": "Point-by-point responses to the reviewers’ comments\nReviewer #1:\nWhat are the noteworthy results?\ncapsaicin lower amyloid pathology and inflammation in a mouse model of AD and though a gut\nmicrobiota dependent mechanism\nWill the work be of significance to the field and related fields? How does it compare to the established\nliterature? If the work is not original, please provide relevant references.\nIt can add some originality, on the gut microbiota dependent mechanisms. The mechanisms and the\nclaim of this be due to 24HC need to be better elucidated\nDoes the work support the conclusions and claims, or is additional evidence needed?\nAddidional evidence is needed\nComment 1: Figure 1: The human cohort needs further characterization. Did the subject described as\nhealthy have memory complaints (subjective cognitive decline)? How many females and males were\nthere? What was the age? Were the data corrected for these variables? There is no mention of this in\nthe results.\nResponse: Thank you for your constructive comments. In the present study, all subjects in the\ncognitively normal (CN) group did not report any memory complaints or subjective cognitive decline\nduring interviews. Moreover, CN subjects were further identified based on the criteria: 1) MMSE score\n≥ 27 and/or MoCA score ≥ 26, 2) absence of AD-like pathology as confirmed by clinic diagnosis\nsupported by results from MRI imaging and cerebrospinal fluid or plasma biomarkers (e.g. p-tau217,\nAβ42). These details have been added in the revised version of the manuscript (please see the revised\nMethods section - Human cohorts).\nDetailed information of all subjects (including sex, age, education years, etc.) are provided in the\nrevised version of supplementary Table S1. For ease of reference, we have also attached it below.\nSpecifically, for more accurate analysis of capsaicin's subgroup-specific effects in cohort 1, we re-\nstratified subjects based on the MMSE scores in accordance with the widely-accepted criteria:\ncognitive normal (CN), MMSE score ≥ 27; mild cognitive impairment (MCI), MMSE score 20 ~ 26;\nmoderate to serve cognitive impairment (M/SCI), MMSE score < 20.\nSupplementary Table S1. Information of human subjects\nCohort 1 (total N = 149) Cohort 2 (total N = 84)\nCN MCI M/SCI CN MCI AD\nN 46 55 48 30 27 27\nAge (year) 63.39 (14.08) 66.35 (13.25) 75.89 (11.50) 60.32 (15.53) 70.03 (7.15) 72.64 (10.31)\nGender (male, %) 45.65 49.09 47.92 29.03 35.29 21.43\nEducation (year) 10.39 (3.57) 9.32 (2.77) 9.29 (3.39) 13.77 (2.91) 12.29 (3.29) 11.71 (4.54)\nMMSE 28.45 (1.07) 23.91 (1.77) 14.78 (3.45) 28.87 (1.18) 25.2 (2.85) 11.18 (8.11)\nMoCA NA NA NA 26.94 (2.02) 19.46 (3.59) 7.07 (6.01)\nPlasma Aβ42/Aβ40 NA NA NA 0.09 (0.01) 0.08 (0.01) 0.07 (0.01)\nPlasma p-tau217\nNA NA NA 0.10 (0.05) 0.20 (0.13) 0.71 (0.41)\n(pg/mL)\nPlasma p-tau181\nNA NA NA 1.55 (0.56) 2.09 (0.79) 2.81 (0.90)\n(pg/mL)\nPlasma NfL\nNA NA NA 12.73 (7.47) 28.29 (23.49) 35.51 (38.86)\n(pg/mL)\nHypertension (%) 43.48 45.45 47.92 13.33 18.52 48.14\nDiabetes (%) 8.70 12.72 14.58 6.67 7.41 22.22\nCoronary heart disease 2.17 9.09\n8.33 3.33 7.41 3.70\n(%)\nGastritis (%) 17.39 29.09 18.75 3.33 3.70 3.70\nTo examine the influence of capsaicin and other co-factors on cognitive function, we have re-\nanalyzed our data through analysis of covariance (ANCOVA) using sex, age, education years,\nhypertension, diabetes, coronary heart disease and gastritis as covariates. The results showed that\nsubjects with moderate-to-high dietary capsaicin intake tended to have higher MMSE scores in\nsubgroups with cognitive impairment (namely MCI, M/SCI (cohort 1), and AD (cohort 2)), but not in\nthe CN subgroup. Multivariable adjustment showed that all covariates, except the education in cohort\n2 (F = 5.84, p = 0.02), had a significant influence on the capsaicin’s effect on cognitive scores. We\nhave added these results in the revised manuscript Fig.1a, and supplementary Table S3.\nFig.1a. MCI, M/SCI and AD patients with moderate-to-high chili intake in diets generally had higher\nMMSE and/or MoCA scores.\nSupplementary Table S3. Tests of between-subjects effects for different factors on cognitive\nperformance\nCohort 1 - MMSE Cohort 2 - MMSE Cohort 2 - MoCA\nF p F p F p\nCapsaicin intake level 10.29 0.002* 3.80 0.02* 3.22 0.04*\nSex 0.34 0.56 0.02 0.88 0.21 0.65\nAge 1.32 0.25 0.35 0.55 1.48 0.23\nEducation 0.53 0.47 5.84 0.02* 10.38 0.01*\nHypertension 0.03 0.88 0.63 0.43 0.01 0.98\nDiabetes 3.10 0.08 1.15 0.29 0.11 0.74\nCoronary heart disease 0.46 0.50 0.06 0.52 0.01 0.62\nGastritis 1.87 0.17 1.02 0.63 0.02 0.75\nComment 2: In general, sometimes WT appears, sometimes not, it is important to see effects of\ncapsaicin on WT and in relation to FAD mice. For example, Fig 1, do the capsaicin mice perform as\ngood as wild type mice? this type of data is not possible to get in the present design.\nResponse: Thank you for your comments. We have performed additional experiments to test how\ncapsaicin affects WT mice in accompany with 5xFAD mice. In consistent with our previous results,\ncapsaicin stilled reduced AD-related pathologies in 5xFAD mice. By contrast, for WT mice, we found\nthat the same dose of capsaicin did not significantly change Aβ burden, microglia activation and\nbehavioral performance in MWM tests, but merely tended to improve object-place recognition (p =\n0.07, two-way ANOVA followed by Tukey’s multiple comparisons tests). These data have been\nincluded in the revised version of Fig. 1c-p.\nTraining\ne f\nc d WT 5xFAD\nVeh Cap Veh Cap Time Veh\nA A A A ≥30s Cap\nTest\nA\nB\ng h i\nTraining phase Test phase WT 5xFAD\nVeh Cap Veh Cap\nWT+Veh\nn.s.** Veh\nWT+Cap\nCap\n5xFAD+Veh\n**\n5xFAD+Cap\nSDS-insoluble Aβ\nj k l\nVeh Cap\nAβ Aβ\nWT+Veh WT+Cap 5xFAD+Veh 5xFAD+Cap 40 42\n6E10\n200 μm\nm n Cell aggregates o Soma\nWT+Veh WT+Cap 5xFAD+Veh 5xFAD+Cap\nVeh\nCap\n100 μm\np\nCell processes\nIba-1DAPI\nWT+Veh\nn.s.**\nWT+Cap\n5xFAD+Veh\n**\n5xFAD+Cap\n20 μm\nFig. 1c-p. Effects of capsaicin on AD-associated phenotypes in WT and 5xFAD mice\nComment 3: fig3 would be good to see quantifications of TRPV1 by immuno and also other technique.\nIt would be good to see data on the KO mice, to see the reduction of levels in TRPV1 in different\nregions.\nResponse: Thank you for your suggestion. We have performed Western blotting to validate the knock-\nout of TRPV1 in stomach, intestine and brain (hippocampus) tissue of 5xFAD&KO mice. These results\nhave been added in the revised version of Fig. 2d. Additionally, the efficiency of TRPV1 KO in this\ntransgenic line (B6.129X1-Trpv1tm1Jul/J, IMSR_JAX:003770) used here has been extensively\nvalidated in previous studies (eg. Caterina et al., Science. 2000). Prior to all experiments using mice\nfrom this genetic background, each mouse was subjected to PCR-based genotyping confirmation.\nFig. 2d. Validation of TRPV1 knock-out in 5xFAD&KO mice by WB.\nComment 4: fig 5 and 6. here I have several concerns. 24HC should be mostly present in serum and\nbrain. first of all, I would like to see levels of 24HC in wt mice; in fad mice, the concentration in the\nbrain seems quite low (usually around 40-60 ng/mg). a further study on the levels of cyp46a1 is\nnecessary, in brain, and intestine. The effects of the antibiotic on 24OH levels is missing\nand it is central. The authors need to prove that this is mediated by 24HC. In the present form I don’t think\nthere is enough proof for this.\nFig 6 is not bringing evidence, it is already known that 24HC is protective.\nResponse: Thank you for your comments. Firstly, we have performed LC-MS to measure 24-HC\nlevels, as suggested, in intestinal contents, plasma and hippocampus in both WT and 5xFAD mice that\nadministrated with vehicle or capsaicin in combination with either normal saline (NS) or ABX,\nrespectively. The results showed that 5xFAD mice had dominantly lower levels of 24-HC compared\nwith WT mice. In general, for both WT and 5xFAD mice, capsaicin treatment upregulated 24-HC\nlevels, while depletion of gut microbes by ABX downregulated 24-HC levels. Notably, in the intestinal\ncontents, ABX almost brought down the level of 24-HC to nil, suggesting a pivotal role of gut microbes\nin peripheral production of 24-HC. We have added these data in the revised version of Fig. 4h-j.\nFig. 4h-j. Changes in 24-HC levels in WT and 5xFAD mice following capsaicin and/or ABX\ntreatment.\nSecondly, we have evaluated both the mRNA and protein levels of CYP46A1 in the hippocampus\ntissue of WT and 5xFAD mice, respectively. It was found that CYP46A1 mRNA and protein levels\nwere markedly decreased in 5xFAD mice, but remained largely unaffected by capsaicin or ABX\ntreatment (please see the revised version of supplementary Fig. S6f-h). In addition, nearly no\nCYP46A1 expression was detected by RT–qPCR either in the contents or tissues of small intestine\n(data not shown, CT values in RT-qPCR cannot be calculated in most samples). These results were\nconsistent with previous findings that CYP46A1 was a brain-specific enzyme catalyzing 24-HC\nsynthesis from cholesterol (Mast et al., J Biol Chem. 2014).\nSupplementary Fig. S6f-h. Downregulated expression of CYP46A1 in the hippocampus of 5xFAD\nmice, but no influence of capsaicin and ABX on CYP46A1 was measured.\nLastly, we agree with reviewer that there have been several studies revealing protective effects of\n24-HC against AD, such as inhibition on intracellular trafficking of amyloid precursor protein (Urano\net al., FASEB J. 2013) and prevention of Aβ monomers-induced tau hyperphosphorylation (Testa et\nal., Redox Biol. 2018). Nevertheless, to our knowledge, the exact mechanistic role of 24-HC in\nmitigating AD pathology, including its specific effects on microglial activation and Aβ phagocytic\nclearance, has not been fully elucidated. Consequently, we have performed in vitro and in vivo\nexperiments to examine the effects of 24-HC in suppressing microglia overactivation and Aβ clearance.\nThe corresponding results remain presented in the revised version of Fig. 5 (the Fig. 6 in the original\nversion of manuscript).\nReviewer #2:\nThis study addresses an important and timely topic by exploring the neuroprotective effects of\ncapsaicin in the context of AD, with a particular focus on the gut–brain axis. Although previous reports\nhave suggested that capsaicin may confer benefits in AD models, the underlying mechanisms have\nremained largely unclear. The authors provide compelling evidence that capsaicin exerts its effects not\nthrough its canonical receptor TRPV1 but rather by modulating the gut microbiome and\nassociated metabolites, leading to enhanced microglial phagocytic activity in the brain. This represents a novel\nand intriguing perspective aligned with the growing interest in microbiota-mediated neuroimmune\ninteractions.\nHowever, while the study is conceptually interesting and potentially impactful, several claims would\nbenefit from further experimental support. Specifically:\nComment 1: 1. While the observation that capsaicin intake is associated with cognitive performance\nin Figure 1A-C is intriguing, the analysis appears to lack adequate control for potential confounding\nfactors. Differences in age, sex, education level, physical activity, social engagement, and overall\ndietary habits between high and low capsaicin intake groups may have influenced the results. It is\nunclear whether these variables were appropriately adjusted for during statistical analysis.\nIncorporating multivariable regression or other methods such as propensity score matching would\nstrengthen the validity of the findings.\nFurthermore, the cross-sectional nature of the survey limits causal inference, as individuals with higher\ncognitive function may inherently prefer spicy foods, or dietary preferences could shift due to cognitive\ndecline. Finally, the subjective perception of spiciness may vary widely among individuals, potentially\nintroducing bias into the data collection process, and it is not clear whether this was adequately\nstandardized or accounted for during data collection.\nResponse: Thank you for your constructive comments. We have re-analyzed our data by analysis of\ncovariance (ANCOVA) following adjustment of several other covariates including sex, age, education\ndegree, hypertension, diabetes, coronary heart disease and gastritis. The results showed that all\ncovariates, except years of education (F = 5.84, p = 0.02) in cohort 2, had a significant influence on\nthe capsaicin’s effect on cognitive function. We have added these results in the revised supplementary\nTable S3.\nSupplementary Table S3. Tests of between-subjects effects for different factors on cognitive\nperformance\nCohort 1 - MMSE Cohort 2 - MMSE Cohort 2 - MoCA\nF p F p F p\nCapsaicin intake level 10.29 0.002* 3.80 0.02* 3.22 0.04*\nSex 0.34 0.56 0.02 0.88 0.21 0.65\nAge 1.32 0.25 0.35 0.55 1.48 0.23\nEducation 0.53 0.47 5.84 0.02* 10.38 0.01*\nHypertension 0.03 0.88 0.63 0.43 0.01 0.98\nDiabetes 3.10 0.08 1.15 0.29 0.11 0.74\nCoronary heart disease 0.46 0.50 0.06 0.52 0.01 0.62\nGastritis 1.87 0.17 1.02 0.63 0.02 0.75\nFor the causal inference of capsaicin’s effect on cognition, we fully agree with you that the cross-\nsectional survey here has limited us to exclude the interference of inherence and shit of preference to\nchili-rich diets for participants with higher cognitive score. Thus, a longitudinal study in our cohorts\nis currently underway to follow up the association between dietary capsaicin intake with changes in\ncognitive function and AD pathology biomarkers. The results are expected to be reported in the future.\nCurrently, to minimize potential biases in the data collection process, we have recruited two\nindependent Chinese cohorts in our study, cohort 1 was recruited from Guizhou Province, where chili-\nrich diets are prevalent, and cohort 2 was from Beijing, where more balanced and diverse dietary\nculture exist. All applicants fulfilling none of the exclusion criteria were included in this study, with\nno further selective filtering applied (please see the Methods section – Human cohorts in manuscript).\nAs for the concern about whether dietary preferences could shift due to cognitive decline, it can\nonly be definitively answered upon completion of our longitudinal follow-up studies in the future.\nNevertheless, we have re-analyzed our current data, and found that: (1) both capsaicin preference and\ntolerance demonstrated parallel and dose-dependent relationships with chili consumption levels, (2)\nboth chili-rich diets preference and tolerance has slight but statistical correlation with MMSE scores\n(p < 0.05) (please see the revised version of supplementary Fig. S1a-d). While these data are\ninsufficient to completely disentangle the intricate relationship between dietary capsaicin intake and\ncognitive function, they nevertheless suggest a positive association between two variables.\nSupplementary Fig. S1a-d. Positive association between the degree of chili-rich diets tolerance and\npreference with the scale of chili intake level.\nComment 2: While the observation that capsaicin does not exert protective effects in TRPV1 knockout\n5×FAD mice is intriguing, it alone may not suffice to conclusively demonstrate TRPV1-independent\nmechanisms. To strengthen this conclusion, additional data addressing potential compensatory\nmechanisms should be included. For instance, investigating the upregulation or functional involvement\nof other TRP channels (e.g., TRPA1, TRPV2) or alternative signaling pathways activated by capsaicin\nwould provide critical insights. Such experiments could clarify whether the observed effects are\ngenuinely independent of TRPV1 and further elucidate the molecular basis of capsaicin's protective\nactions.\nResponse: Thank you for your comments. Indeed, current evidences are insufficient to rule out the\npotential involvement of other receptors other than TRPV1 in mediating the protective effects of\ncapsaicin against AD. Although previous studies have proved that both TRPA1 and TRPV2 did not\ndirectly respond to capsaicin stimulation (Caterina et al., Nature. 1999; Story et al., Cell. 2003), these\nTRP channels might be still indirectly regulated by capsaicin. Several voltage‐gated ion channels like\nNav1.7, Kv7.4 were also reported to contribute to the action of capsaicin independent of capsaicin\n(reviewed in Oz M et al., Med Res Rev. 2023).\nIn the present study, due to practical limitations in experimental resources, specific evaluation by\nknock-out experiments for each of these candidate targets was not included. Instead, we employed RT-\nqPCR to evaluate expression changes in potential alternative targets, including TRPA1, TRPV2,\nNav1.7, and Kv7.4. Notably, none of these genes exhibited significant transcriptional alterations in\n5xFAD mice, and following TRPV1 knockout or capsaicin treatment (please see the revised\nSupplementary Fig. S5). These results support our main finding that capsaicin alleviates AD-related\npathology primarily through gut microbiota-dependent modulation on 24-HC production.\nSupplementary Fig. S5. Nonsignificant change of TRPA1, TRPV2, Nav1.7 and Kv7.4 gene expression\nin 5xFAD mice, and following TRPV1 KO or capsaicin treatment.\nComment 3: The finding that capsaicin’s protective effects are abolished following antibiotic (ABX)\ntreatment in 5×FAD mice suggests that the gut microbiota plays a key role in mediating its actions\n(Figure4E-K). However, several previous studies AD mouse model have reported that ABX treatment\nalone can reduce Aβ plaque burden and ameliorate cognitive decline, potentially via modulation of\nmicroglial activation (PMID: 40033338). Given these contrasting findings, it would be important for\nthe authors to discuss how their results relate to the existing literature. In particular, further clarification\nregarding the experimental conditions (e.g., age at ABX administration, duration, and spectrum of\nantibiotics), and the role of microglial responses under ABX treatment, would help reconcile the\nobserved discrepancies. Including such a discussion would strengthen the interpretation of the ABX\ndata presented in this figure.\nResponse: Thank you for your insightful comments. Indeed, several previous studies have found that\nboth depletion of gut microbiota by ABX / germ-free housing and fecal microbiota transplantation\nfrom healthy donors alleviated AD pathologies in AD transgenic mice (Schaible et al., J\nNeuroinflammation. 2025; Loh et al., Signal Transduct Target Ther. 2024), potentially thanks to the\nprevention of gut dysbiosis and reduction of harmful metabolites following pan-elimination of gut\nmicrobes. However, this approach inevitably leads to the depletion of gut probiotics, thereby\neliminating the basis of gut microbiota-dependent therapeutics. Consequently, it is mechanistically\nplausible that ABX globally attenuates AD pathological phenotypes, while simultaneously abolishes\ncapsaicin's beneficial effect like the modulation of 24-HC metabolism through microbiota-dependent\nmechanisms.\nTo evaluate the effects of ABX, either administrated alone and in combination with capsaicin, on\nAD-related pathologies, we have performed additional experiments in a new batch of 5-month-old\n5xFAD mice. In consistent with previous findings, we and found that ABX alone reduced Aβ plaques,\nsuppressed microglia activation and inflammatory factors in the hippocampus of 5xFAD mice. Notably,\ncombinative treatment with ABX+capsaicin significantly impaired the protective effects of capsaicin\nin ameliorating AD pathologies, which supported our main conclusion that gut microbiota contributed\npivotally to the anti-AD effects of capsaicin. We have added new results in the revised version of Fig.\n3e-k, and extended discussion accordingly in the revised manuscript.\nBesides, detailed information about spectrum of antibiotics, drugs administration (ABX,\ncapsaicin), animal ages, etc. have been provided in the Methods section – ABX treatment, Drugs\ntreatment in mice, and in experimental scheme in Fig. 3e and supplementary Fig. S6a, respectively.\nFig. 3e-k. ABX alleviated Aβ burden and microglia activation in the hippocampus of 5xFAD mice,\nbut impaired the protective effects of capsaicin.\nComment 4: The authors propose that capsaicin increases plasma levels of 24-hydroxycholesterol\n(24-HC), which in turn enhances microglial phagocytosis and suppresses inflammatory responses\nthrough a gut-brain axis mechanism. While the direct effects of 24-HC on microglial function are\npartially demonstrated using BV2 cells treated with exogenous 24-HC and through i.p. injection\nexperiments, it remains unclear whether the observed in vivo effects are indeed mediated by\nperipherally derived 24-HC.\nTo substantiate the claim that 24-HC acts as a peripheral metabolite mediating microglial responses in\nthe brain, additional experimental evidence is needed. In particular, it is important to determine\nwhether 24-HC generated in the periphery can cross the blood-brain barrier and reach the CNS. Tracer\nstudies using labeled 24-HC would be highly informative in this regard. Moreover, while the authors\nshow in Figure 5 that plasma levels of 24-HC are decreased in 5XFAD mice compared to WT, they\ndo not provide corresponding data on 24-HC levels in the hippocampus or intestine in these groups.\nSuch comparative quantification would clarify the compartmental dynamics of 24-HC in the disease\ncontext.\nFurthermore, since 24-HC is primarily synthesized in the brain by CYP46A1 and released into the\ncirculation, the observed increase in plasma 24-HC could reflect enhanced CNS synthesis rather than\nperipheral production. In this regard, the data in Figure 9A—showing elevated plasma 24-HC levels\nin human subjects with high capsaicin intake—are intriguing but insufficient to determine the source\nof 24-HC (i.e., gut vs. brain origin). Therefore, quantitative analysis of 24-HC levels in the brain,\nplasma, and intestinal contents following capsaicin treatment would provide more convincing support\nfor the proposed gut-brain axis mechanism.\nResponse: Thank you for your comments. To determine whether 24-HC in the intestinal tract can be\nabsorbed and subsequently cross the blood-brain barrier to reach the brain, as suggested, we have\nadministrated 24-HC conjugated with fluorescein BODIPY-FL in 5-month-old 5xFAD mice by\ngavage (0.02 mg/kg of mice weight), once daily for consecutively 7 days. Equal amount of DMSO\nwere administrated as controls. Fluorescent intensity at the 500 nm (ex) / 509 nm (em) were measured\nusing a Biotek Synergy 2 microplate reader for homogenates of intestinal content, plasma and\nhippocampus tissue, respectively. In all these samples, we have detected prominent fluorescent signals\nin the mice treated with 24-HC-BODIPY-FL, but not DMSO. Consistently, in brain slices, we also\nobserved BODIPY-FL signals across various regions (revised version of supplementary Fig. S7a-b).\nNevertheless, given that the conjugation of BODIPY-FL might disturb the membrane-penetrating\nefficiency of natural 24-HC, we also administrated equal dose of unlabeled 24-HC in 5xFAD mice,\nand subsequently measured its level by LC-MS. The results showed that 5xFAD mice administrated\nwith unlabeled 24-HC had significantly higher levels of 24-HC both in intestinal contents, plasma and\nhippocampus (revised version of supplementary Fig. S7c). These results indicated that 24-HC can be\nabsorbed from intestines and enter the brain.\nSupplementary Fig. S7. 24-HC can be absorbed from intestinal tract and enter the brain.\nBesides, we also conducted LC-MS analysis of 24-HC levels across intestinal contents, plasma,\nand hippocampal tissues in both WT and 5xFAD mice under four treatment conditions: vehicle±ABX\nand capsaicin±ABX. The results showed that 5xFAD mice had dominantly lower levels of 24-HC\ncompared with WT mice. In general, for both WT and 5xFAD mice, capsaicin treatment upregulated\n24-HC levels, while depletion of gut microbes by ABX downregulated 24-HC levels. Especially, in\nthe intestinal contents, ABX almost brought down the level of 24-HC to nil, suggesting the modulation\nof capsaicin on the peripheral production of 24-HC and a pivotal contribution of gut microbes in this\nprocess. We have added these data in the revised version of Fig. 4h-j.\nFig. 4h-j. Changes in 24-HC levels in WT and 5xFAD mice following capsaicin and/or ABX\ntreatment.\nComment 5: In relation to comment 4, in Figure 8B, the authors show the level of 24-HC in the\nhippocampus of P301S mice; however, given the central hypothesis that peripheral 24-HC contributes\nto the gut-brain axis mechanism, the levels of 24-HC in plasma and intestinal contents should also be\nprovided in the same model.\nResponse: We have accessed the levels of 24-HC level of P301S mice in intestinal contents, plasma\nand hippocampus, respectively. In similar with 5xFAD mice, P301S mice at 6 months showed reduced\n24-HC levels in all these regions compared with age-match WT control, which were partly revered by\ncapsaicin treatment (the revised Fig. 7b). These data supported our conclusion that capsaicin alleviated\nAD-related tau pathologies in P301S mice through systematically upregulating the 24-HC level.\nFig. 7b. Capsaicin partly reversed the systematic reduction of 24-HC in P301S mice.\nComment 6: The authors report that 24-HC increases phagocytosis at physiologically relevant\nconcentrations (0.01–10 μM), while it suppresses Aβ-induced TNF-α and IL-1β production at a much\nhigher concentration (10 mM) (figure 6). However, the concentration used for inflammation assays is\nexcessively high and likely to induce cytotoxic effects, which could confound the interpretation of the\nobserved anti-inflammatory effects. It is unclear whether these effects are directly mediated by 24-HC\nor are secondary to cell viability loss.\nTo address this issue, the authors should provide data on cell viability following 10 mM 24-HC\ntreatment, such as MTT or LDH assays, to rule out cytotoxicity as a confounding factor. Additionally,\ninvestigating whether lower concentrations of 24-HC (e.g., 1–10 μM) can also exert anti-inflammatory\neffects would ensure consistency with the phagocytosis assay and strengthen the validity of the\nfindings.\nFurthermore, the authors should justify why such vastly different concentrations were used for the\nphagocytosis and inflammation assays.\nResponses: Thank you for catching this important discrepancy. Upon reviewing our original data, we\nconfirmed this was a typo during the preparation of manuscript, where the unit of 24-HC concentration\nhere should be μM (in consistent with tests in Aβ phagocytosis by BV2 cells), but not mM. We have\ncorrected this error in the Result section of revised manuscript. Besides, we agree with the reviewer\nthat it is important to test the potential dose-dependent cytotoxicity of 24-HC, especially at high\nconcentrations. Therefore, we have still performed MTT assay in BV2 cells, and found that 24-HC\nexhibited nonsignificant cytotoxicity at concentrations ≤20 μM, while predominantly impaired cell\nviability at extremely high concentrations ranging 50 μM ~10 mM. We have added these data in the\nrevised version of supplementary Fig. S7a.\nSupplementary Fig. S7a. Dose-dependent effect of 24-HC on BV2 cell viability measured by MTT\nassay.\nComment 7: Although 3D reconstruction data are included, the current representative image in Figure\n6P does not clearly support the authors’ intended interpretation. It remains ambiguous whether the\nimage is meant to highlight an overall reduction in Aβ burden or enhanced microglial phagocytosis of\nAβ. The colocalization of Iba1 and 6E10 alone does not provide definitive evidence of active\nphagocytosis, and the image as presented does not sufficiently convey either outcome. To improve\nclarity, it is recommended that the authors (1) clearly specify the primary message they aim to deliver\nthrough this image, and (2) replace or supplement the figure with a more representative and\ninterpretable image, ideally one that includes clearer 3D visualization of Aβ engulfment by microglia.\nFurthermore, accompanying quantitative data would greatly strengthen the reliability of this claim.\nResponses: Thank you for your suggestion. We have revised the representative images to show more\nclearly the enhancement of microglia phagocytosis of Aβ, and reduction of Aβ plaques following 24-\nHC administration, please see the revised version of Fig. 5l-n (the figure numbering has been updated\nto accommodate data reorganization during manuscript revision).\nFig. 5l-n. Enhancement of Aβ phagocytosis by microglia and reduction of Aβ plaques in the\nhippocampus of 5xFAD mice following 24-HC supplement.\nComment 8: In Figures 6 and 7, the authors primarily use the BV2 cell line to investigate the protective\nmechanisms of 24-HC. While BV2 cells are widely used as a model for microglial studies, they differ\nfrom primary microglia in several aspects, including gene expression profiles and functional responses.\nTo strengthen the validity and physiological relevance of the findings, it would be valuable to verify\nkey results—such as enhanced phagocytosis and LXRβ sumoylation—in primary microglia.\nResponses: As suggested, we have performed additional experiments in primary cultured mouse\nmicroglia to verify key results obtained from the BV2 cell line. In consistent with our previous findings,\n24-HC at 10 μM increased the amount of Aβ phagocytized by primary cultured microglia at both 4.5\nand 24 hours post administration, with an Aβ clearance rate of 57.75% for 24-HC v.s. 37.11% for\nvehicle. Besides, 24-HC also downregulated the level of pro-inflammatory factors IL-1β and TNF-α\n(the revised version of supplementary Fig. S7f-h), and elevated the SUMOylation and total level of\nLXRβ in the nuclear protein component (the revised version of supplementary Fig. S10a)\nVeh 24-HC\nSupplementary Fig. S7f-h. 24-HC promoted Aβ phagocytosis, and downregulated pro-inflammatory\nfactors in primary cultured mouse microglia.\nSupplementary Fig. S10a. 24-HC upregulated SUMOylation and total level of nuclear LXRβ in\nprimary cultured mouse microglia\nComment 9: In Figure 7, the authors investigate the effects of LXRβ knockdown on microglial responses.\nHowever, to validate the interpretation of the data, it is necessary to confirm whether the\nh\n5.4\nh\n42\n4.5 h\n24 h\n20 μm\nAβPhalloidineDAPI\n)2mμ\n/\n.u.a(\nytisned\nβA\nf g h\nIL-1β TNF-α\nknockdown was specifically achieved in microglia. The authors should provide experimental evidence\ndemonstrating microglia-specific knockdown efficiency, such as immunostaining for LXRβ in Iba1-\npositive cells or cell-type–specific expression analysis following viral delivery.\nResponses: Thank you for your comments. We firstly examined the specificity of AAV(MG1.2)-\nmediated EGFP expression in microglia of 5xFAD mice by immunostaining. A total of 98.5% EGFP-\npositive cells were co-labeled with Iba1, and 89.2% Iba1+ cells were EGFP-positive (please see the\nrevised version of Fig. 6n, and supplementary Fig. S10b), suggesting efficient and almost exclusive\ninfection of AAVs in microglia. This were consistent with extensive previous studies that the\ncombinative application of DIO (double-floxed inverse orientation)-containing AAV-MG1.2 and\nCx3cr1-Cre(ERT2) transgenic mice had high specificity in delivering exogenous gene expression in\nmouse microglia (eg. Pan et al., Lin et al., Nat Methods. 2022; Umpierre et al., Neuron. 2024; Nat\nMetab. 2025; Feng et al., Nat Biotechnol. 2025).\nFig. 6n and supplementary Fig.S10b. Co-labeling rate of AAV-expressed EGFP with Iba1.\nFurthermore, to confirm the selectivity of LXRβ knock-down in the microglia, as suggested, we\nperformed immunofluorescent staining of LXRβ, and found that AAV-shLXRβ prominently\ndownregulated the integrated density (IntDen) of LXRβ staining compared with control virus. Besides,\nwe also utilized fluorescence-activated cell sorting to separate EGFP-positive and EGFP-negative cells\nin mouse hippocampus, and subsequently accessed LXRβ mRNA level by RT-qPCR. The results\nshowed that AAV-MG1.2-DIO-shLXRβ-EGFP downregulated the expression of LXRβ only in the\nEGFP-positive cells, but not in the EGFP-negative cells (revised supplementary Fig. S10c-f).\nTaken together, these results demonstrated the high selectivity and efficiency of LXRβ knock-\ndown in the microglia of 5xFAD mice hippocampus.\nSupplementary Fig. S10c-f. Validation of the microglia-specific LXRβ knock-down by AAV-MG1.2 in\nthe hippocampus of 5xFAD mice\nComment 10: While the authors focus primarily on the microglial effects of 24-HC, it is important to\nrecognize that 24-HC can also influence other brain cell types, including neurons and astrocytes. The\ncurrent manuscript lacks discussion regarding the potential impact of 24-HC on these cell populations.\nGiven that 24-HC has been reported to affect astrocyte cholesterol metabolism and neuronal function\n(PMID: 34067119), it is important to consider whether the protective effects observed in the\nP301S tauopathy model may also involve non-microglial mechanisms. The authors should include either\nexperimental validation or at least a discussion addressing the possible roles of 24-HC in neurons and\nastrocytes, particularly in the context of tau pathology.\nResponses: Thank you for your comments. Indeed, 24-HC has been also recognized to affect function\nof neurons and astrocytes. In neurons, 24-HC acted as a positive allosteric modulator of NMDA\nreceptors and activated estrogen receptors-mediated sex hormone signaling, both of which\npotentiated synaptic plasticity and memory of mice (Paul et al., J Neurosci. 2013; Wei et al., Neuropharmacology.\n2019; Latorre-Lea et al., Sci Adv. 2024). Additionally, 24-HC induced proteolysis of tau through the\nactivating the SIRT1/PGC1α/Nrf2 axis (Testa et al., Antioxidants. 2023). In astrocytes, 24-HC\npromoted cholesterol transport via ApoE and LXR-mediated pathways (Abildayeva et al., J Biol Chem.\n2006). However, the roles of 24-HC in AD remains complex and context-dependent. While\nphysiological levels appear neuroprotective, elevated concentrations (25-50 μM) have been reported\nto induce oxidative stress, amplify Aβ toxicity in neurons (Gamba et al., Aging Cell. 2011), and trigger\nastrocyte reactivity (Petrov et al., Neurotherapeutics. 2019). This dose-dependent dichotomy warrants\nfurther systematic investigation. We have extended the discussion accordingly in the revised\nmanuscript.\nComment 11: In lines 377–378, the authors state, \"Elevation of 24-HC in the plasma and brain was\nfound here to protect against AD both in 5×FAD and P301S mice.\" This sentence implies that 24-HC\nlevels were elevated in both the plasma and brain in both models. However, plasma 24-HC data are\nnot presented for the P301S mice. The authors should revise this statement to more accurately reflect\nthe data shown in the manuscript.\nResponses: We sincerely appreciate for identifying this error in our manuscript, and have carefully\nrefined language throughout the manuscript to enhance clarity and precision. Besides, in response to\nyour comment #5, we have performed additional experiments to measure 24-HC levels in intestinal\ncontents, plasma and brain, respectively, in P301S mice compared with WT mice and following\ncapsaicin treatment. These results have been added in the revised version of Fig. 7b.\nComment 12: In lines 191 and 196, the figure is referred to as \"Figure 4,\" but based on the\ninterpretation of the results, it seems that \"Figure 5\" is the correct.\nResponses: The manuscript has been rigorously proofread to correct inaccuracies and strengthen the\nscientific validity of the statements.\nReviewer #3:\nThe manuscript by Li et al., seeks to identify the mechanism through which capsaicin (Cap) improves\nmemory and reduce brain pathophysiology in mouse models for Alzheimer’s Disease (AD) and other\nneurodegenerative disorders. Using a combination of mouse behavior, immunohistochemistry, and\nmolecular biology, the authors replicated previous findings that Cap treatment rescued abnormal\nbehavior and microglial biology in the 5X-FAD mouse model for AD and provide new data that Cap\nincreases levels of a cholesterol metabolite (24-HC), which has also been previously associated with\nAD. The authors show that antibiotics treatment blocks the Cap-mediated rescue of AD-like\nphenotypes as well as the increase in 24-HC levels. Importantly, the authors extend their findings from\nthe 5xFAD mouse and show that (a) Cap also rescues behavioral deficits and increases 24-HC in the\nhTau.P301S mouse model for frontotemporal dementia and (b) 24-HC levels are correlated to\nincreased cognition in humans with AD.\nThe study of mechanism of action of a therapeutic drug and the role of the gut-microbiota-brain axis\nin Alzheimer’s disease are of interest to the field. The use of orthogonal models for neurodegenerative\ndisorders and data from humans provide important pieces of data. While the findings are interesting,\nseveral important experiments are missing which would better support the authors’ claim that the\nmechanism through which Cap rescues deficits is through the microbiome.\nPlease see below for more specific comments/questions:\nComment 1: Would FMT of microbes from 5xFAD mice treated with Cap into untreated 5xFAD mice\nalso rescue the behavioral and brain pathological phenotypes? Are the Cap-mediated changes in the\nmicrobiome directly causing the increases in 24-HC or is an interaction between the microbes and Cap\ntreatment necessary for Cap to have an effect. In other words, are the changes in the microbiome\ndownstream of Cap or working synergistically with Cap?\nResponse: Thank you for your insightful comments. We acknowledge your valuable perspective on\napplying fecal microbiota transplantation (FMT) from capsaicin-treated donors to validate the\ncontribution of gut microbiota remodeling in capsaicin's alleviation of AD pathologies. However, there\nremains a technical constraint that capsaicin was substantially accumulated in both intestinal contents\nand feces following oral administration (please see the revised Fig. 2a). It is quite challenging to\nseparate the residual capsaicin from microbial components in fecal matter. Thus, transplantation of\nsuch homogenates would inherently introduce two confounding therapeutic variables, namely the\nmicrobiota and residual capsaicin. This fundamental limitation prompted our choice of alternative\napproaches for clearer mechanistic interpretation, namely through depletion of gut microbiota by ABX\nto examine its influence on the capsaicin-induced protective effects against AD (please see the revised\nversion of Fig. 3e-k, and supplementary Fig. S6d). These experiments also provided supportive\nevidence to our conclusion.\nFig. 2a. Distribution of capsaicin in digestive tracts and different tissues of 5×FAD mice following\nrepeated administration.\nRegarding the concern of whether the gut microbiota acts as a downstream effector of capsaicin\nor functions synergistically with capsaicin, it might be insufficient to definitively discriminate between\nthese two possibilities based on current evidence. First of all, the composition and gene expression of\ngut microbes were modulated by capsaicin, while the protective effects of capsaicin are attenuated\nupon microbiota depletion, suggesting that gut microbiota serves as at least one of downstream factors\nof capsaicin treatment. Nonetheless, potential extra-intestinal effects may exist. For example, persist\nactivation of TRPV1 by capsaicin in the autonomic nervous system could potentially modulate host’s\nvoluntary preferences for capsaicin-rich foods, affect the absorption of capsaicin, or regulate non-\ninflammatory responses in the brain. We have extended the discussion accordingly in the revised\nmanuscript.\nComment 2: The authors state that deletion of Trpv1 has no effect on capsaicin rescue of phenotypes\nin the 5xFAD mice. While the data support this for the brain pathology (Fig 3E-J), the data are less\nconvincing for the behavior data. Is there a significant difference between the 5xFAD+Veh and\n5xFAD&KO + Cap in Fig 3L and 3N? What is the effect of Trpv1 KO on 5xFAD mice independent\nof Cap treatment?\nResponse: Thank you for your comments. We have performed additional experiments in a new batch\nof mice to examine the influence of TRPV1 knock-out alone and on the protective effect of capsaicin.\nThe results showed that TRPV1 KO per se neither impaired baseline learning and memory of 5xFAD\nmice, nor attenuated the cognitive-enhancing effects of capsaicin in object-place recognition and\nMorris-water maze tests. Similarly, nonsignificant effects of TRPV1 KO were observed on Aβ plaque\ndeposition and microglial activation status. We have exhibited these data in the revised version of Fig.\n2e-n.\nFig. 2e-n. TRPV1 KO neither modified AD-related pathological phenotypes nor affected the\nprotective effects of capsaicin treatment.\nComment 3: What effects do ABX alone have on the phenotypes in the 5xFAD mice? This important\ncontrol is missing.\nResponse: To evaluate the effects of ABX alone on AD-related pathologies, we have performed\nadditional experiments in new groups of mice including Veh+NS, Veh+ABX, Cap+NS and Cap+ABX.\nIt was found that ABX alone significantly reduced Aβ plaques as well as downregulated levels of\nmicroglia activation and inflammatory factors in the hippocampus of 5xFAD mice. These results were\nconsistent with previous studies that depletion of gut microbiota alone in AD transgenic mice\nalleviated AD pathologies, possibly thanks to the prevention of gut dysbiosis and reduction of harmful\nmetabolites (Schaible et al., J Neuroinflammation. 2025; Loh et al., Signal Transduct Target Ther. 2024).\nHowever, ABX might inevitably lead to the depletion of gut probiotics, thereby eliminating the\nbasis of gut microbiota-dependent therapeutics. Consequently, we found ABX co-administration with\ncapsaicin significantly abolished the latter's beneficial effects against AD. We have added these new\nresults in the revised version of Fig. 3e-k, and extended the discussion accordingly.\nFig. 3e-k. ABX alleviated Aβ burden and microglia activation in the hippocampus of 5xFAD mice,\nbut impaired the protective effects of capsaicin.\nComment 4: By how much does ABX reduce the amount of 24-HC in plasma (5H)? Does it reduce\nall the way to the levels seen in Vehicle treated mice or only partially? This will help to determine how\nmuch of a role the microbiome plays in the Cap-mediated increase in 24-HC. Please show the absolute\namount of 24-HC in plasma (5G) to allow for a more direct comparison.\nResponse: We have conducted LC-MS to measure 24-HC levels in the plasma of both WT and 5xFAD\nmice under four treatment conditions: Veh+NS, Veh+ABX, Cap+NS, Cap+ABX. The results showed\nthat for both WT and 5xFAD mice, depletion of gut microbes by ABX downregulated 24-HC levels\nin plasma, and ABX treatment nearly abolished the capsaicin-induced increase in 24-HC levels.\nAdditionally, similar effects of ABX on 24-HC levels were also observed in intestinal contents and\nhippocampus. These results suggest a pivotal contribution of gut microbes to the capsaicin-induced\nelevation in 24-HC. We have added these data in the revised version of Fig. 4h-j.\nFig. 4h-j. Changes in 24-HC levels in WT and 5xFAD mice following capsaicin and/or ABX\ntreatment.\nComment 5: Does treatment with SOT prevent the beneficial effects on 24-HC on behavior and brain\npathology? This experiment will help to determine whether 24-HC is needed in the brain or whether\n24-HC has peripheral effects which lead to the phenotype rescue.\nResponse: To evaluate whether blocking of central 24-HC synthesis affects AD-associated behavior\nand brain pathology, as required, we treated 5xFAD mice with SOT / normal saline (NS) in\ncombination with 24-HC / vehicle for a month. The dose of SOT was 50 mg/kg (i.g.) (Nishi et al., Sci\nRep. 2020). The results showed that SOT significantly reduced 24-HC levels both in hippocampus and\nplasma, but did not change the intestinal 24-HC. Notably, oral supplement of 24-HC was still effective\nin elevating hippocampal 24-HC even under SOT co-administration.\nFor AD-associated phenotypes: (1) SOT alone increased Aβ plaques in the hippocampus, and the\ncombinative treatment of SOT with 24-HC impaired, but not abolished, the latter’s effect in reducing\nAβ burden. (2) SOT did not further aggravate morphological signs of microglia activation in 5xFAD\nmice, possibly thanks to a ceiling effect. (3) Hippocampal level of IL-1β was upregulated by SOT,\nboth when administered alone and in combination. (4) SOT significantly hindered the beneficial effect\nof 24-HC in improving object-place recognition. These results suggest that 24-HC synthesis in both\nthe brain and gut plays a crucial role in mitigating brain pathologies. We have added these data in the\nrevised version of supplementary Fig. S9.\nSupplementary Fig. S9. SOT reduces 24-HC and aggravates AD-related pathologies in the brain of\n5xFAD mice.\nComment 6: Is the memory enhancing effect of Cap in WT mice dependent on changes in the\nmicrobiome and 24-HC? Is there a dose-dependent effect of Cap on microbiome changes? The authors\nshould treat WT mice with ABX (or use germ-free mice) and Cap (and with an ABX only group) to\ndetermine whether depleting the microbiome blocks this effect in WT mice as in the 5xFAD mice.\nResponse: We have performed additional experiments to deplete gut microbiome with ABX in WT\nmice, and subsequently test its effect on the capsaicin-induced cognitive improvement. In consistent\nwith our previously findings, repeat capsaicin administration at 1mg/kg tended to improve object-place\nrecognition of NS-treated WT mice, while this beneficial tendency disappeared in ABX co-treated\nmice. Importantly, similar effects of ABX were also observed in 5-month-old 5xFAD mice (the revised\nversion of supplementary Fig. S6d-e).\nAltogether, these data suggest an important contribution of gut microbiome to the capsaicin-mediated\ncognitive improvement in both WT and 5xFAD mice.\nSupplementary Fig. S6d-e. ABX abolished the cognitive-improving effects of capsaicin in both WT\nand 5xFAD mice.\nAs for the potential dose-dependent effects of capsaicin on gut microbiota regulation, we have\nreviewed existing literature. It has been found that low doses of capsaicin (≤5 mg/kg) enhanced\nbeneficial bacteria like Firmicutes, Lachnospiraceae, and Blautia, which increased butyrate\nproduction and improve gut barrier function. Moderate doses of capsaicin further boosted probiotics\nsuch as Akkermansia and Faecalibacterium prausnitzii, thus enhancing insulin sensitivity and anti-\ninflammatory activity. Conversely, high doses of capsaicin (>10 mg/kg) disrupted microbial balance\nby increasing Bacteroidetes while depleting mucin-degrading bacteria (Akkermansia,\nLachnospiraceae), which may impair intestinal integrity, elevate permeability, promote bacterial\ntranslocation, and create a pro-inflammatory/metastatic environment (Corral-Guerrero et al.,\nMetabolites. 2025; Kang et al., J Clin Endocrinol Metab. 2016; Gong et al., Food Nutr Res. 2022;\nXiang et al., Foods. 2022). These findings suggest a shift in the effect of capsaicin from beneficial at\nlow-to-moderate doses to detrimental at supraphysiological high doses. We have extended the\ndiscussion accordingly in the revised manuscript.\nComment 7: Please include more methodological details regarding the animal experiments:\na. At what age were treatments and experiments performed on the different mouse models? Did\ntreatment begin before or after the onset of different phenotypes (e.g., behavior) in these mice? If\ntreatment began before the onset of phenotypes (prevention-type experiment), would treatment after\nthe onset of phenotypes (rescue-type experiment) also be efficacious?\nResponse: The ages of mice have been added in revised Results / Methods sections, and experimental\nflowcharts in figures, respectively. In the present study, drug treatments (capsaicin, ABX, 24-HC, SOT)\nin AD models and WT littermates begun at 5-month-old, namely after the early onset of pathological\nphenotypes.\nb. What breeding scheme was used to generate the mice for the experiment? A recent report (DOI:\n10.1016/j.neuron.2024.12.025) suggests that parental origin of the transgene is an important variable\nin disease phenotypes and therefore this information should be included in the methods section.\nResponse: The breeding scheme of transgenic mice have been added in the revised Methods section.\nIn our study, 5xFAD, 5xFAD & TRPV1 KO, 5×FAD / Cx3cr1-CreERT2, P301S mice were all from\npaternal origin.\nc. How were mice housed for these experiments? Were control and transgenic animals housed in the\nsame cage? Were mice in different treatment groups housed together or separately?\nResponse: The housing strategy of mice have been added in the revised Methods section. Here, to\ncontrol for potential transfer effects of gut microbiota, transgenic and WT littermates were kept\nseparately in different cages throughout experiments.\nd. When sequencing “small intestinal contents,” was the entire small intestines used or just one section\n(i.e., duodenum, jejunum, or ileum)?\nResponse: Contents from the entire small intestines were used for metagenome sequencing. The\nMethods section has been revised accordingly.\ne. It is unclear how the data for the fear conditioning test are presented (Supplementary Fig 2). Please\ndefine what test/probe is referring to. Typically, the % time freezing 24 hours after the foot shock is\nshown for this behavior.\nResponse: The test/probe ratio referred to the ratio of mouse freezing time at day-2 (test, after foot-\nshock) to day-1 (probe, before foot-shock). To preclude any possible misunderstanding, we have re-\nanalyzed our data and showed the time percentage of mice freezing at day-2 in the revised version of\nsupplementary Fig. S2f and Fig. S3b. The statistical re-examination and updated visualization of the\ndataset did not change our original experimental findings.\nSupplementary Fig. S2f and S3b. Capsaicin treatment did not change conditional fear memory\nindicated by freezing time percentage of WT (S2f) and 5xFAD (S3b) mice at day-2.\nPoint-by-point responses\nReviewer #1:\nComment 1: The authors have addressed most of the previous comments and questions\nthrough several additional experiments. The manuscript is now significantly improved\nand more complete. However, I still have a few concerns that I would like the authors\nto take into account and address before publication:\n- The claim that “capsaicin intake ameliorates cognitive impairments in AD patients”\n(line 92) may be too strong. Given that some correlations did not reach statistical\nsignificance following the new analyses, I recommend softening this statement. A\nmore appropriate phrasing would be “capsaicin use correlates with cognitive performance”\nor a similar wording.\nResponse: We appreciate your insightful comment. As suggested, we have changed the\nsubtitle of this part into “Capsaicin intake correlates with cognitive impairments in AD\npatients and 5×FAD mice” in the revised manuscript.\nComment 2: Although the authors have conducted additional impressive experiments\ninvolving soclestat and labeled 24OH, I believe caution should be taken to conclude\nthat capsaicin reduces AD pathology primarily via 24OH. The data suggest indeed that\n24OH contributes to the observed effects, but it is likely part of complex mechanisms.\nThe title and the conclusions should be revised to reduce the emphasis on 24OH as\nthe principal mediator of capsaicin's effects.\nResponse: Thank you for your comment, we agree with you that the effects of capsaicin\non AD pathology should be complex, though we have revealed in this paper that the gut\nmicrobiota-dependent modulation of 24OH (namely 24-HC) production is of pivotal\nimportance. To enhance the precision and scientific rigor of our manuscript, we have\nrephrased the title into “Gut microbiota-dependent 24-hydroxycholesterol metabolism\ncontributes to capsaicin-induced amelioration of Alzheimer’s disease pathology”, and\nrefined the conclusion accordingly.\nComment 3: The lipidomic analysis reveals changes in several other molecules not\nonly 24OH, including cholesterol. It is worth discussing whether the modulation of\ncholesterol levels itself may contribute to the effects induced by capsaicin. A broader\ninterpretation and discussion would strengthen the manuscript.\nResponse: Indeed, though we focused in the present study the protective effects against\nAD of capsaicin-induced upregulation of 24-HC levels, while potential roles of other\nmetabolites also exist. Especially, multiple cohort studies have revealed a significant\ncorrelation of blood low-density lipoprotein cholesterol (LDL-C) level with the risk of\nAD and dementia1,2, as well as cerebral amyloidosis imaging index3. Excessive uptake\nof peripheral cholesterol drives a major component of chronic inflammation in the brain\nthrough dysregulating glia activation, and promotes Aβ secretion and tau\nhyperphosphorylation4,5. Consistently, reduction of cholesterol level is effective for\nalleviating neuroinflammation and AD pathologies6,7. These findings raise the\npossibility that the effects of capsaicin might be also at least partly mediated by the\ndownregulation of cholesterol levels. Besides, our metabolomic data also suggest an\nalteration of plasma bile acid following the capsaicin treatment, which has been found\nto be associated with the development of AD8,9. Elucidating these alternative pathways\nis of significant scientific interest. We have extended the discussion accordingly in the\nrevised manuscript.\nComment 4: While 24OH can reach the brain, its concentrations appear to remain\nlower in the brain than other organs after treatment, possibly due to rapid metabolism\nin the liver and other peripheral organs. This should be acknowledged and discussed in\nthe manuscript.\nResponse: Thank you for raising this point. As suggested, we have added clarification\nin the revised manuscript. In addition, while fluorescence tracking of 24-HC revealed\na much lower relative proportion enters the brain than remains in blood, potentially due\nto rapid peripheral metabolism, direct quantification of total unlabeled 24-HC\nconfirmed that the absolute amount in the brain is still significantly greater than in the\nperiphery. This was consistent with previous studies that 24-HC is distributed at\nsignificantly higher levels in the brain than in peripheral tissues10.\nComment 5: A notable limitation of the study is the exclusive use of male mice.\nThis should be clearly acknowledged in the discussion, along with a consideration of how\nsex differences might impact the findings and their interpretation.\nResponse: We have acknowledged this limitation of the current study, and extended\nthe discussion of revised manuscript.\nReviewer #2:\n1. The authors propose that 24-HC modulates microglial inflammation via\nSUMOylation of LXRβ, supported by si-LXRβ knockdown experiments. To more\ndefinitively establish LXRβ as a critical mediator of 24-HC’s anti-inflammatory effects,\nit would be highly informative to demonstrate whether these effects are abolished under\nsi-LXRβ conditions. Showing that 24-HC fails to exert its regulatory effects in the\nabsence of LXRβ would provide a more direct mechanistic link supporting the\nproposed 24-HC → LXRβ → inflammation pathway.\nResponse: Thank you for your insightful comments, we have performed additional\nexperiments to examine whether LXRβ knock-down via RNAi impaired the anti-\ninflammatory effects of 24-HC in cultured BV2 cells. In consistent with our previous\nresults, 24-HC treatment significantly decreased levels of IL-1β and TNF-α,\ndecreased\nratio of CD16/32+ to CD206+ cell portion, and promoted Aβ phagocytosis in Aβ-treated\nBV2 cells transfected with control vectors, while all these effects were nearly abolished\nunder LXRβ knock-down, suggesting a pivotal role of LXRβ in mediating the anti-\ninflammatory effect of 24-HC on microglia. We have added these results in the revised\nFig.6g-j of manuscript.\nFig. 6. (g) si-LXRβ increased IL-1β and TNF-α levels, and abolished the anti-\ninflammatory effect of 24-HC in BV2 cells co-administrated with Aβ. n = 3 wells of cell\nin each group. Data were normalized to the mean value of control group. * P < 0.05,\nn.s. nonsignificant, two-way ANOVA followed by Tukey’s multiple comparisons tests.\n(h-i) si-LXRβ increased the portion of CD16/32-positive BV2 cells while decreased the\nportion of CD206-positive cells, and abolished the effect of 24-HC in suppressing the\npro-inflammatory switch of BV2 cells. n = 3 batches of experiments in each group. * P\n< 0.05, ** P < 0.01, n.s. nonsignificant, two-way ANOVA followed by Tukey’s multiple\ncomparisons tests.\n(j) si-LXRβ decreased the amount of Aβ phagocytized in BV2 cells, and inhibited the\neffect of 24-HC in facilitating Aβ phagocytosis. n = 5 wells of cell in each group. * P <\n0.05, ** P < 0.01, two-way ANOVA followed by Tukey’s multiple comparisons tests.\n2. The authors have made thoughtful revisions and performed additional experiments\nthat strengthen the manuscript. This study provides valuable insights into the gut–brain\naxis underlying capsaicin’s effects. To further enhance the translational relevance of the\nfindings, I suggest discussing whether similar alterations in Oscillospiraceae have been\nobserved in microbiome studies of AD patients. If available, referencing data on\nOscillospiraceae abundance in relation to capsaicin intake in humans would be ideal.\nEven if direct evidence is lacking, incorporating relevant findings into the Discussion\nwould provide broader context and underscore the potential relevance of the findings\nto human disease.\nOverall, the manuscript has improved substantially, and addressing these points would\nfurther enhance its clarity and impact.\nResponse: Thank you for your positive comments on our work. We have conducted in-\ndepth analysis of the gut microbiota data of 5xFAD mice following capsaicin feeding,\nand found a potential association between the elevation of 24-HC level with the increase\nof Oscillibacter genus within the Oscillospiraceae family, which has been previously\nreported to express ismA homologous gene that is involved in metabolizing cholesterol\nto generate hydroxycholesterol11,12. Thus, as suggested, we examined gut Oscillibacter\nchanges in AD patients in GMrepo v2, a curated human gut microbiome database13. In\none of the three AD datasets included (PRJNA770746), a significant enrichment of\nOscillibacter in healthy controls was found (LDA score = -3.17). Overall, the\nabundance of Oscillibacter was significantly downregulated in AD patients compared\nwith healthy controls (n = 218 for AD and 15,780 for healthy, adjusted p < 0.0001).\nThis finding is consistent with our observations in 5xFAD mice. We have added these\ndata in the revised Fig5a-b.\nFig5a-b. (a) Featured enrichment of gut microbes at genus level, including\nOscillibacter, in healthy individuals (N = 15,780) versus AD patients (N = 218)\nrecorded in GMrepo v2 database. Linear Discriminant analysis (LDA) scores were\nacquired using LEfSe analysis. (b) AD patients showed significantly decreased\nOscillibacter abundance compared with healthy group. *** adjusted P = 1.36e-17,\nMann-Whitney U test.\nNext, we attempted to investigate the impact of capsaicin-containing food intake\non human gut microbiota, particularly on Oscillibacter. In consistent with our findings,\none case study found that sustained treatment of in vitro cultured human feces with\ncapsaicin (3 mg/mL, for 14 days) led to a significant increase in Oscillibacter\nabundance14. However, another study involving 29 healthy subjects reported that\nconsuming tomato juice plus capsaicin (2 × 1.8 g per day) for five consecutive days\nresulted in a reduction of Oscillibacter, while this change became non-significant after\nstratification by body mass index15. We attempted to perform more in-depth analysis of\nthe data from these two studies, but neither provided publicly accessible raw sequencing\ndata or detailed lists of gut microbial abundances.\nNonetheless, these evidences suggest that Oscillibacter abundance is significantly\nreduced in the gut microbiota of AD patients and may represent one of the mechanisms\nunderlying the dysregulation of cholesterol metabolism in AD. Dietary intake of\ncapsaicin might influence the abundance of Oscillibacter in human gut microbiota,\nthough its specific effects may vary across populations and be subject to confounding\nfactors. We have extended discussion accordingly in the revised manuscript.\nReviewer #4:\nThere have been interesting relationships observed between capsaicin consumption and\nAlzheimer’s disease (AD) pathologies and cognitive functions, based on both\npreclinical and observational human studies. However, the existing research—\nincluding contradictory findings regarding amyloid processing—indicates a need for\nfurther investigation and human clinical trials. Additionally, there is a lack of evidence\nsupporting the underlying mechanisms involved in these interactions.\nIn the current manuscript, Li et al. claim that capsaicin consumption enhances cognitive\nfunction and mitigates Aβ deposition via the gut microbiome–25-HC–\nneuroinflammation pathway. This is an interesting and potentially important finding for\nmechanistically understanding the interactions between capsaicin (Cap.) and AD\npathogenesis. However, the current version of the manuscript remains correlational, and\nthere is insufficient evidence proving that capsaicin-associated microbiome and 25-HC\nmodulate neuroinflammation and AD-related pathologies. More detailed comments are\nprovided below:\nComments 1~2: 1. As other reviewers—notably Reviewer 3—have pointed out, there\nis a lack of evidence supporting a causal relationship between Cap.-associated\nmicrobiome and AD-related pathologies. As R3 requested, the FMT approach (or an\nalternative approach to probe the Cap.-associated microbiome is really involved in the\ninteractions) is very critical in this study, as the novelty lies in the role of the capsaicin-\nassociated gut microbiome mediating these interactions.\n2. The authors mention the infeasibility of FMT due to high capsaicin levels in the\nintestine, making it difficult to dissociate the effects of capsaicin itself from those of\nthe microbiome. However, the authors could attempt FMT after multi-filtration of the\nfecal material to remove capsaicin molecules. Moreover, in the FMT procedure, FMT\nwould not be administered as frequently as capsaicin treatment. Also, the capsaicin\nlevel would also likely decrease as the fecal material travels through the gut. Therefore,\nthis approach remains worth trying, especially considering its importance to the study.\nResponses: Thank you for your thoughtful comments. We fully agree with you that\nFMT would provide compelling evidence to demonstrate whether the protective effects\nof capsaicin are mediated through its modulation on gut microbiota. As suggested, we\nhave performed additional experiments, while gut microbiota transfer (GMT)\nexperiments were performed in the present study instead of FMT to minimize\ninterference from environmental bacteria in the housing conditions and prevent the\ndisruption of anaerobic bacteria by oxygen exposure. Gut contents from Veh- or Cap-\nfed 5xFAD mice were collected and immediately processed under anaerobic conditions.\nWe used 5-month 5xFAD mice here since it has been reported that FMT from wild-type\nto AD mice per se showed effectiveness in alleviating AD phenotypes16,17.\nGiven the low water solubility of Cap, all samples underwent repeated washing\nwith pre-reduced PBS followed by centrifugation (final solvent at 1 mL per gram of gut\ncontent). The supernatant was obtained and further extracted with corn oil to remove\nany potential residual capsaicin. It was confirmed that the Cap was virtually\nundetectable in the final aqueous phase containing the gut microbes. This processed\nmaterial was stored at -80℃ and used for GMT in 5-month 5xFAD mice (0.5 mL each\nday, once every other day for a month), equal doses of PBS was delivered as control.\nIn consistent with our previous findings, we found that GMT from Cap-, but not Veh-\ntreated mice significantly reduced Aβ plaques, inhibited microglia activation, reduced\ninflammatory factors levels in the hippocampus, and improved cognitive functions of\n5xFAD mice compared with PBS control.\nTogether with our previous findings that ABX elimination of gut microbiota\nabolished the protective effects of capsaicin, our results collectively supported a pivotal\ncontribution of the gut microbiota in mediating the amelioration of AD phenotypes by\noral capsaicin intake. We have added these data in the revised version of Fig. 3e-p of\nmanuscript.\nFig. 3e-p. (e-f) Experimental scheme of gut microbes isolation and validation of\ncapsaicin removal from GMT samples. n = 4 pooled samples in each group. *** P <\n0.001, one-way ANOVA followed by Tukey’s multiple comparisons tests. (g)\nExperimental schedule of GMT. (h-k) GMT from Cap-treated mice decreased density\nof Aβ plaques (i) and microglia soma volume (i), and increased microglia processes\nbranching complexity (k) in the hippocampal CA1 of 5xFAD mice. Arrows in\npresentative images (h) indicate cells showed in reconstructed images (lower panels).\nn = 40 cells from 8 mice in each group. n.s. nonsignificant, * P < 0.05, ** P < 0.01,\none-way (i, j) or two-way (k) ANOVA followed by Tukey’s multiple comparisons tests.\n(l) GMT from Cap-treated mice downregulated pro-inflammatory levels in the\nhippocampus tissue. n = 8 mice in each group. Data were normalized to the mean value\nof PBS group. n.s. nonsignificant, * P < 0.05, ** P < 0.01, two-way ANOVA followed\nby Tukey’s multiple comparisons tests. (m-p) GMT from Cap-treated mice improved\nobject-place recognition (m), and spatial learning (n) and memory (o-p) in the Morris-\nwater maze test of 5xFAD mice. n = 8 mice in each group. n.s. nonsignificant, * P <\n0.05, ** P < 0.01, one-way (m, o) or repeated measures (n) ANOVA followed by Tukey’s\nmultiple comparisons tests.\nComment 3: The authors analyze microbiome data only at a high taxonomic level\n(phylum and family), focusing on broad compositional differences. Since metagenomic\nanalysis was used, the study could and should identify more detailed microbiome\nfeatures—potentially down to the species level—that are involved in the interactions\nbetween capsaicin and pathology. These strains could then be introduced directly into\nmice to determine whether the same effects observed in Cap.-treated mice are replicated.\nThis could be alternative approach as a causal probe.\nResponse: Thank you for your comments. As suggested, we performed more in-depth\nLEfSe analysis of metagenomic data, and found many species showing featured\nenrichment in the group of Cap-treated 5xFAD mice, including\nOscillibacter_sp._CAG_241 and Oscillibacter_sp._KLE_1745 (please see the revised\nFig. 3d).\nFig 3d. Taxonomic cladogram of intestinal bacteria to species level revealed by LEfSe\nanalysis. Cyan area and dots indicate featured bacteria with featured enrichment in\ncapsaicin-treated 5×FAD mice.\nIt has been previously recognized that Oscillibacter is capable of metabolizing\ncholesterol to generate hydroxycholesterol, presumed depending on the expression of\nismA homologous gene11,12. Thus, we next focused on the role of Oscillibacter in\nmediating the protective role of capsaicin. Further analysis revealed the reduction in\nabundance of Oscillibacter in 5xFAD mice, which was significantly rescued by\ncapsaicin treatment. Meanwhile, the Oscillibacter abundance correlated negatively with\nplasma cholesterol level and positively with 24-HC level (revised Fig. 4h-j).\nFig. 4h-j. (h-i) Capsaicin partly rescued the downregulation of gut Oscillibacter\nabundance in 5xFAD mice in many species (h) and at the overall genus level (i). n = 8\nmice in each group. * P < 0.05, ** P < 0.01, two-way (h) or one-way (i) ANOVA\nfollowed by Tukey’s multiple comparisons tests. (j) Oscillibacter abundance negatively\ncorrelated with plasma cholesterol level, while positively correlated with 24-HC level.\nLinear regression analysis.\nFurthermore, we examined Oscillibacter changes in AD patients in GMrepo v2, a\ncurated human gut microbiome database13. In one of the three AD datasets included\n(PRJNA770746), a significant enrichment of Oscillibacter in healthy controls was\nfound (LDA score = -3.17). Overall, the abundance of Oscillibacter was significantly\ndownregulated in AD patients (revised Fig5a-b). Besides, one previous case study\nfound that sustained treatment of in vitro cultured human feces with capsaicin led to a\nsignificant increase in Oscillibacter abundance14, though another study involving 29\nhealthy subjects reported that consuming tomato juice plus capsaicinfor five\nconsecutive days resulted in a reduction of Oscillibacter, while this change became non-\nsignificant after stratification by body mass index15. In general, these data suggest that\nOscillibacter abundance is significantly reduced in the gut microbiota of AD patients,\nand dietary intake of capsaicin might influence the abundance of Oscillibacter in human\ngut microbiota, though its specific effects may vary across populations and be subject\nto confounding factors. We have also extended discussion accordingly in the revised\nmanuscript.\nFig5a-b. (a) Featured enrichment of gut microbes at genus level, including\nOscillibacter, in healthy individuals (N = 15,780) versus AD patients (N = 218)\nrecorded in GMrepo v2 database. Linear Discriminant analysis (LDA) scores were\nacquired using LEfSe analysis. (b) AD patients showed significantly decreased\nOscillibacter abundance compared with healthy group. *** adjusted P = 1.36e-17,\nMann-Whitney U test.\nSubsequently, we isolated gut microbes from Cap-treated 5xFAD mice under\nanaerobic conditions, and identified Oscillibacter via whole-genome sequencing. The\nisolated Oscillibacter was further validated by prominent expression of previously\nidentified marker genes, including Oscillibacter-specific 16S rRNA, ismA and CgT\nhomologous genes11, as measured by qPCR compared with negative controls of E.coli\nand positive control of J115. Furthermore, we ascertained the capacity of Oscillibacter\nto generate 24-HC in cultures. Cholesterol treatment for 24 h significantly increased the\n24-HC level in the culture medium, which was further upregulated under capsaicin co-\nadministration (revised Fig. 5c-e).\nAs suggested, we grafted Oscillibacter repeatedly into 5-month 5xFAD mice for a\nmonth, equal doses of culture medium were inoculated as control. The results showed\nthat Oscillibacter supplement significantly reduced Aβ plaques density, inhibited\nmicroglia overactivation, downregulated pro-inflammatory factors levels, and\nimproved cognitive functions of 5xFAD mice (revised Fig. 5f-o). These outcomes\nmimicked the protective effects of oral capsaicin intake.\nFig. 5c-o. (c) Experimental procedures of Oscillibacter isolation from gut contents of\nCap-treated 5xFAD mice. (d) Isolated Oscillibacter (Osc.) showed high expression\nlevels of Osc.-specific 16S rRNA, ismA and CgT homologous genes relative to the level\nof pan bacterial 16S rRNA. Culture medium and E. coli were used as negative control.\nJ115 was used as positive control. (e) 24-HC level in the culture medium of\nOscillibacter was upregulated following cholesterol treatment, and further increased\nunder Cap co-treatment. n = 5 dishes per group. * P < 0.05, one-way ANOVA followed\nby Tukey’s multiple comparisons tests. (f) Experimental scheme of medium and\nOscillibacter delivery in 5xFAD mice. (g-j) Direct Oscillibacter grafting decreased Aβ\nplaques density (h), decreased the soma volume (i) and increased cell processes\nbranching complexity (j) of microglia in the hippocampal CA1 of 5xFAD mice. Arrows\nin presentative images (g) indicate microglia showed in reconstructed images (lower\npanels). n = 40 cells from 8 mice in each group. * P < 0.05, ** P < 0.01, unpaired t\ntests (h, i) or two-way ANOVA (j). (k) Oscillibacter downregulated pro-inflammatory\nlevels in the hippocampal tissue. n = 8 mice in each group. Data were normalized to\nthe mean value of medium group. * P < 0.05, ** P < 0.01, two-way ANOVA followed\nby Tukey’s multiple comparisons tests. (l-m) Oscillibacter improved object-place\nrecognition of 5xFAD mice. n = 8 mice in each group. * P < 0.05, unpaired t test. (n-o)\nOscillibacter improved spatial learning (n) and memory (o) of 5xFAD mice in the\nMorris-water maze test. n = 8 mice in each group. * P < 0.05, ** P < 0.01, two-way\nANOVA (n) or unpaired t test (o).\nCollectively, these results suggest a role of gut Oscillibacter in generating 24-HC,\nand an important contribution of Oscillibacter increase in mediating the capsaicin-\ninduced amelioration of AD phenotypes.\nComment 4: Related to the point above (also R3 asked about details in methodology):\nwhy do the authors focus only on the intestinal microbiome/content? The colonic/fecal\nmicrobiome is known to be more diverse and is more commonly discussed in the\ncontext of the microbiome–gut–brain axis.\nResponse: Thank you for raising this crucial point regarding the sampling site for gut\nmicrobiota analysis. In the initial phase of this study, we collected small intestinal\ncontents for metagenomic analysis based on the rationale that the small intestine is the\nprimary site for chemical digestion and nutrient absorption, and thus might be more\nsusceptible to the acute effects of capsaicin. However, as the project progressed, we\nrecognized that the colonic microbiota is far more abundant than that of the small\nintestine. Besides, there are numerous seminal studies in both animal and human\nutilizing fresh fecal samples reported alterations of key taxa, including Oscillibacter, in\nthe context of AD. This highlighted a potential limitation of our initial focus solely on\nthe small intestine.\nTo address this limitation, we conducted a preliminary experiment comparing the\nrelative abundance of Oscillibacter between small intestinal and colonic contents via\nqPCR measurement of Oscillibacter-specific 16S rRNA. Indeed, a predominantly\nhigher abundance of Oscillibacter in the colon relative to small intestines was revealed\n(please see the Fig. r1 as follows).\nFig. r1. Higher abundance of Oscillibacter level in the colonic contents compared with\nsmall intestines (SI) in 5-month wild-type mice. Data were normalized by the mRNA\nlevel of conservative bacterial 16S rRNA for each sample. n = 5 mice in each group. *\nP < 0.05, unpaired t test.\nConsequently, we revised our protocol for additional experiments to collect\ncontents from the gut tract including both small intestine and colon for microbiota\nanalysis and transfer. We have updated the revised Materials and Methods section to\nclearly describe this methodological evolution. To prevent potential confusion and\nmaintain consistency of the main text, we have unified the terminology throughout the\nmanuscript and figures by replacing the original \"intestinal contents\" with the more\ngeneral words \"gut contents\".\nComment 5: Regarding Figure 3 (after revisions in response to R3), it remains\nimportant to provide FMT or other direct evidence demonstrating the effect of Cap.-\nassociated microbiota on neuroinflammation and pathology. It is difficult to interpret\nthe meaning of antibiotic (ABX) treatment blocking capsaicin effects. The authors\nspeculate that beneficial microbiota induced by capsaicin are reduced by ABX, but this\nremains pure speculation. Many alternative explanations are possible. For instance,\nlong-term administration of high doses of mixed antibiotics (over approximately 1.5\nmonths) could cause leaky gut or other physiological/anatomical changes. The\nmicrobiome–gut–brain axis is a complex system, and many factors could contribute. It\nis risky to attribute ABX effects solely to microbiome depletion. Although ABX is\ndesigned to kill bacteria broadly, its mechanisms are not fully understood, and there are\nreports indicating that ABX can modulate the immune system being microbiome-\nindependent. Therefore, direct manipulation of Cap.-associated microbiota is needed to\nsubstantiate downstream effects.\nResponse: We agree that ABX treatment is not a specific method for depleting the gut\nmicrobiota. As noted, high-dose ABX may affect many physiological functions beyond\nthe microbiome. Therefore, we performed GMT and Oscillibacter transplantation\nexperiments following the suggestions of the reviewers to more directly examine the\nmediative role of oral capsaicin intake in exerting its protective effects in 5xFAD mice\n(please see our responses to your comments 1~3).\nNevertheless, we believe the results from ABX intervention still hold important\nreference value, since they provide supporting evidence from another perspective that\nindicate the essential contribution of gut microbiota to the phenotypic improvements\ninduced by capsaicin, and to the systematic upregulation of 24-HC level. Accordingly,\nwe have retained this set of data in the revised manuscript, but moved it to the\nsupplementary materials (supplementary Fig. S6 and S7).\nComment 6: Regarding Comment 6 from R3: the improvement in object-place\nrecognition in NS-treated WT mice suggests that the cognitive enhancement from Cap.\ntreatment may be independent of its effects on AD pathology.\nResponse: Our results showed that capsaicin improved cognitive function in NS-\ntreated WT mice. One of potential explanations is our finding that capsaicin treatment\nalso showed a trend toward increasing 24-HC levels in WT mice (p = 0.06 ~ 0.09,\nsupplementary Fig. S7a). Beyond the role of 24-HC in mitigating AD pathology via the\nmicroglia-inflammation-Aβ phagocytosis axis revealed in the present study, 24-HC has\nbeen also known to function as a positive allosteric modulator of NMDA receptors and\nactivated estrogen receptors-mediated sex hormone signaling, both of which\npotentiated synaptic plasticity and memory in mice18-20. Moreover, capsaicin has been\nalso reported to act as modulators of voltage-gated Na+, K+, and Ca2+ channels, all of\nwhich are pivotal in regulating neuronal excitability and plasticity21. Thus, we cannot\nexclude the possibility that additional mechanisms, yet unidentified, may also\ncontribute to the cognitive-enhancing effects of capsaicin. We have extended the\ndiscussion accordingly in the revised manuscript.\nComment 7: The authors mentioned to R3’s question: “As for the potential dose-\ndependent effects of capsaicin on gut microbiota regulation, we have reviewed existing\nliterature. It has been found that low doses of capsaicin (≤5 mg/kg) enhanced beneficial\nbacteria like Firmicutes, Lachnospiraceae, and Blautia, which increased butyrate\nproduction and improved gut barrier function. Moderate doses of capsaicin further\nboosted probiotics such as Akkermansia and Faecalibacterium prausnitzii, thus\nenhancing insulin sensitivity and anti-inflammatory activity.” Are these strains related\nto 25-HC production?, which the authors propose as a key mediator?\nResponse: There is currently no direct evidence demonstrating that these gut bacteria\ncan produce 24-HC, the oxysterol of interest in this study, nor for 25-HC. Nevertheless,\nit has been revealed that certain species among them can metabolize cholesterol. In\nspecific, sp. Faecalibacterium prausnitzii, belonging to the Oscillospiraceae family of\nFirmicutes phylum, express microbial cholesterol dehydrogenases encoded by ismA\nhomologous genes and can catabolize cholesterol12. sp. Akkermansia muciniphila\nbelonging to the Verrucomicrobiota phylum can reduce the activity of genes involved\nin mucins-based cholesterol production22. Moreover, Akkermansia muciniphila and\nLachnospiraceae supplement in healthy human were both found to associate with\nreduction in plasma total cholesterol level23,24. In type 2 diabetes mellitus (T2DM)\npatients, the increase of Blautia, also belonging to the Firmicutes phylum, induced by\nmetformin and AMC treatment was significantly correlated with the improvements in\nlipid homeostasis25. Collectively, these studies have provided direct or correlative\nevidence suggesting the involvement of these gut bacteria in metabolizing cholesterol\nto generate hydroxycholesterols.\nAs for which species is more pivotal for the 24-HC production, our data from\nadditional experiments has directly demonstrated that Oscillibacter, belonging to the\nOscillospiraceae family of Firmicutes phylum, can metabolize cholesterol and\nproducing 24-HC, and this process is positively regulated by capsaicin (please see the\nresponse to your comment 3), which is consistent with previous findings that\nOscillibacter expresses ismA homologous genes and catabolizes cholesterol in the gut11.\n8. The experiment using P301S mice is somewhat distracting in the current manuscript,\nunless the authors perform a systematic analysis comparable to that used in the\namyloidosis models, including microbiome manipulation. Additionally, the age of the\nP301S mice used is too young to draw meaningful conclusions regarding pathology. In\nthis mouse model, substantial tau pathology is typically observed after 6–7 months of\nage (with only weak p-tau expression), and neurodegeneration generally becomes\nevident after 9 months. Prior studies also have reported microbiome effects on\npathology only after 9 months of age (PMID: 36634180).\nResponse: We examined here the effects of capsaicin in the P301S mouse model only\non several key phenotypes like microglial activation, 24-HC levels, and tau-related\nneuropathology. We propose that these results provide key clues suggesting that\ncapsaicin may alleviate AD-related tau pathology by modulating 24-HC metabolism.\nNevertheless, we agree with the reviewer that more systematic and in-depth\ninvestigations, in similar to what was performed in the 5xFAD model, are required to\nestablish a definitive causal relationship. We sincerely thank you for the valuable\nsuggestions provided here. However, in terms of workload, this would require\nsubstantial time and resources, and could constitute a standalone research project in\nitself. We are currently planning to undertake this line of investigation. To maintain\nfocus in the main text, we have moved all P301S mice-related results to the\nsupplementary materials (please see the supplementary Fig. S12) of the revised\nmanuscript.\nRegarding the age of P301S mice, we initiated capsaicin administration at 5 months\nof age and continued until phenotype assessment at 6 months. As shown in our results,\nP301S mice already exhibit significant microglial abnormalities and tau pathology at\n5~6 months of age. This is consistent with previous literatures reporting that\nneuroinflammation generally emerges in this strain as early as 2~3 months, followed\nby tau pathology and cognitive impairment at 4~6 months of age26,27 (summarized in\nhttps://www.alzforum.org/research-models/tau-p301s-line-ps19#overview). Therefore,\nwe consider it reasonable to initiate the intervention at 5 months of age and assess the\noutcomes at 6 months.\nTo our knowledge, direct evidence of how the gut microbiota changes in P301S\nmice at 5~6 months of age and how it correlates with tau-related pathology is lacking\nto date. In another study using ADLPAPT mice, a line exhibiting both Aβ and tau\npathologies in the brain, researchers observed significant alterations in gut microbiota\ncomposition as early as 2 months of age, while transplanting gut microbiota from\nhealthy mice starting at this early stage for a duration of 4 months effectively reduced\nboth Aβ- and tau-related pathological phenotypes and cognitive deficits17. In TE4\nmouse line, it was noted in the mentioned study28 that though ABX treatment did not\nchange AT8-staining signal of PHF-tau, TE4 mice reared in a germ-free environment,\nfor less invasively depleting gut microbiota, tended to exhibit reduced microglial\nactivation as indicated by increased length and complexity of cell processes in\nmorphology, and lower gene expression levels of reactive microglia biomarker Axl and\nFgr at 12 weeks of age. The absence of observed significant changes of tau pathology\nin this line may be multifactorial: the 12-week timepoint may still be too early, the AT8\nantibody may have lower sensitivity in immunohistochemistry compared to other\nphospho-tau antibodies, the influence of ApoE4 gene expression and the inherent\nsubstantial variability between different transgenic mouse lines. We have extended the\ndiscussion accordingly in the revised manuscript.\n9. The protective effect of peripheral administration of 25-HC is interesting, particularly\ngiven that there are reports claiming 25-HC is promoting neuroinflammation and AD-\nrelated pathologies (PMID: 32552741; PMID: 40001197).\nResponse: Thank you for your insightful comment. Indeed, 25-HC has been found to\npromoted proinflammatory cytokines production and impair microglial phagocytic\ncapacity29-31. These effects might collectively exacerbate neuroinflammation and\namyloid pathology in AD. In contrast, the effects of 24-HC on neuroinflammation and\nAD appear to be more complex32. 24-HC treatment at 50 μM for 16 h in cultured\nmacrophages increased the TNF-α level33, while upregulation of local 24-HC in the\nbrain of 5xFAD mice via CYP46A1 overexpression was found to ameliorate\nneuroinflammation, amyloidosis and improved cognition functions34. Similar\nprotective effects of 24-HC were also widely observed under treatment of efavirenz, a\npositive allosteric modulator of CYP46A135-37. Moreover, 24-HC prevented tau\nhyperphosphorylation induced by Aβ monomers in h-tau transgenic mice38, and\ncerebrospinal fluid levels of 24-HC was found to negatively correlate with\nneurodegeneration markers in a clinical cohort of AD patients18. The complexity of 24-\nHC actions may be attributed to factors such as its concentration-dependent effects and\nthe duration of exposure.\nCollectively, these findings present compelling evidence for an intriguing\ndichotomy that, despite of high similarity in structure, 24-HC, 25-HC, and potentially\nother hydroxycholesterols may exert divergent regulatory effects on neuroinflammation\nand AD-related phenotypes. The underlying mechanisms responsible for these\nfunctional differences deserve in-depth investigation in future studies. We have\nextended the discussion accordingly in the revised manuscript.\nReferences\n1. Ferguson, E. L. et al. Independent associations of high-density lipoprotein\ncholesterol and triglyceride levels with Alzheimer's disease and related dementias.\nAlzheimers. Dement. 21, e14575 (2025).\n2. Luo, J. et al. Genetic Associations Between Modifiable Risk Factors and\nAlzheimer Disease. JAMA Netw. Open. 6, e2313734 (2023).\n3. Reed, B. et al. Associations between serum cholesterol levels and cerebral\namyloidosis. JAMA Neurol. 71, 195-200 (2014).\n4. Tcw, J. et al. Cholesterol and matrisome pathways dysregulated in astrocytes and\nmicroglia. Cell. 185, 2213-2233 (2022).\n5. Ahmed, H. et al. Brain cholesterol and Alzheimer's disease: challenges and\nopportunities in probe and drug development. Brain. 147, 1622-1635 (2024).\n6. van der Kant, R. et al. Cholesterol Metabolism Is a Druggable Axis that\nIndependently Regulates Tau and Amyloid-beta in iPSC-Derived Alzheimer's\nDisease Neurons. Cell Stem Cell. 24, 363-375 (2019).\n7. Ding, J. et al. APOE4 Exacerbates Cerebral Tau Pathology Through Cholesterol-\nInduced Degradation of Phosphatase in Atherosclerosis. CNS Neurosci. Ther. 31,\ne70536 (2025).\n8. Ren, Z. et al. Increased intestinal bile acid absorption contributes to age-related\ncognitive impairment. Cell Rep. Med., 101543 (2024).\n9. Chen, T. et al. Metabolic phenotyping reveals an emerging role of ammonia\nabnormality in Alzheimer's disease. Nat. Commun. 15, 3796 (2024).\n10. Lutjohann, D. et al. Cholesterol homeostasis in human brain: evidence for an age-\ndependent flux of 24S-hydroxycholesterol from the brain into the circulation. Proc.\nNatl. Acad. Sci. U. S. A. 93, 9799-9804 (1996).\n11. Li, C. et al. Gut microbiome and metabolome profiling in Framingham heart study\nreveals cholesterol-metabolizing bacteria. Cell. 187, 1834-1852 (2024).\n12. Kenny, D. J. et al. Cholesterol Metabolism by Uncultured Human Gut Bacteria\nInfluences Host Cholesterol Level. Cell Host Microbe. 28, 245-257 (2020).\n13. Dai, D. et al. GMrepo v2: a curated human gut microbiome database with special\nfocus on disease markers and cross-dataset comparison. Nucleic. Acids. Res. 50,\nD777-D784 (2022).\n14. Mahalak, K. K. et al. Analysis of the Ability of Capsaicin to Modulate the Human\nGut Microbiota In Vitro. Nutrients. 14, 1283 (2022).\n15. Bu, S., Kar, W., Tucker, R. M. & Comstock, S. S. Minimal Influence of Cayenne\nPepper on the Human Gastrointestinal Microbiota and Intestinal Inflammation in\nHealthy Adult Humans-A Pilot Study. Life-Basel. 12, 1849 (2022).\n16. Zhang, Y. et al. Transmission of Alzheimer's disease-associated microbiota\ndysbiosis and its impact on cognitive function: evidence from mice and patients.\nMol. Psychiatry. 28, 4421-4437 (2023).\n17. Kim, M. S. et al. Transfer of a healthy microbiota reduces amyloid and tau\npathology in an Alzheimer's disease animal model. Gut. 69, 283-294 (2020).\n18. Latorre-Leal, M. et al. CYP46A1-mediated cholesterol turnover induces sex-\nspecific changes in cognition and counteracts memory loss in ovariectomized mice.\nSci. Adv. 10, eadj1354 (2024).\n19. Wei, X., Nishi, T., Kondou, S., Kimura, H. & Mody, I. Preferential enhancement\nof GluN2B-containing native NMDA receptors by the endogenous modulator 24S-\nhydroxycholesterol in hippocampal neurons. Neuropharmacology. 148, 11-20\n(2019).\n20. Paul, S. M. et al. The major brain cholesterol metabolite 24(S)-hydroxycholesterol\nis a potent allosteric modulator of N-methyl-D-aspartate receptors. J. Neurosci. 33,\n17290-17300 (2013).\n21. Oz, M., Lorke, D. E. & Howarth, F. C. Transient receptor potential vanilloid 1\n(TRPV1)-independent actions of capsaicin on cellular excitability and ion transport.\nMed. Res. Rev. 43, 1038-1067 (2023).\n22. Davey, L. E. et al. A genetic system for Akkermansia muciniphila reveals a role\nfor mucin foraging in gut colonization and host sterol biosynthesis gene expression.\nNat. Microbiol. 8, 1450-1467 (2023).\n23. Depommier, C. et al. Supplementation with Akkermansia muciniphila in\noverweight and obese human volunteers: a proof-of-concept exploratory study. Nat.\nMed. 25, 1096-1103 (2019).\n24. Telle-Hansen, V. H. et al. Replacing saturated fatty acids with polyunsaturated\nfatty acids increases the abundance of Lachnospiraceae and is associated with\nreduced total cholesterol levels-a randomized controlled trial in healthy individuals.\nLipids Health Dis. 21, 92 (2022).\n25. Tong, X. et al. Structural Alteration of Gut Microbiota during the Amelioration of\nHuman Type 2 Diabetes with Hyperlipidemia by Metformin and a Traditional\nChinese Herbal Formula: a Multicenter, Randomized, Open Label Clinical Trial.\nMbio. 9, e02317-e02392 (2018).\n26. Wenger, K. et al. Common mouse models of tauopathy reflect early but not late\nhuman disease. Mol. Neurodegener. 18, 10 (2023).\n27. Billings, L. M., Oddo, S., Green, K. N., McGaugh, J. L. & LaFerla, F. M.\nIntraneuronal Abeta causes the onset of early Alzheimer's disease-related cognitive\ndeficits in transgenic mice. Neuron. 45, 675-688 (2005).\n28. Seo, D. et al. ApoE isoform- and microbiota-dependent progression of\nneurodegeneration in a mouse model of tauopathy. Science (New York, N.Y.). 379,\neadd1236 (2023).\n29. Choi, H. et al. 25-Hydroxycholesterol modulates microglial function and\nexacerbates Alzheimer's disease pathology: mechanistic insights and therapeutic\npotential of cholesterol esterification inhibition. J. Neuroinflammation. 22, 50\n(2025).\n30. Romero, J., Toral-Rios, D., Yu, J., Paul, S. M. & Cashikar, A. G. 25-\nhydroxycholesterol promotes brain cytokine production and leukocyte infiltration\nin a mouse model of lipopolysaccharide-induced neuroinflammation. J.\nNeuroinflammation. 21, 251 (2024).\n31. Wong, M. Y. et al. 25-Hydroxycholesterol amplifies microglial IL-1β production\nin an apoE isoform-dependent manner. J. Neuroinflammation. 17, 192 (2020).\n32. Sodero, A. O. 24S-hydroxycholesterol: Cellular effects and variations in brain\ndiseases. J. Neurochem. 157, 899-918 (2021).\n33. Gc, J. B. et al. Molecular basis for the recognition of 24-(S)-hydroxycholesterol by\nintegrin αvβ3. Sci. Rep. 13, 9166 (2023).\n34. Tsitsou-Kampeli, A. et al. Cholesterol 24-hydroxylase at the choroid plexus\ncontributes to brain immune homeostasis. Cell Rep. Med., 101278 (2023).\n35. Mast, N., Li, Y. & Pikuleva, I. A. Increased Acetylcholine Levels and Other Brain\nEffects in 5XFAD Mice after Treatment with 8,14-Dihydroxy Metabolite of\nEfavirenz. Int. J. Mol. Sci. 23, (2022).\n36. Petrov, A. M. et al. CYP46A1 Activation by Efavirenz Leads to Behavioral\nImprovement without Significant Changes in Amyloid Plaque Load in the Brain of\n5XFAD Mice. Neurotherapeutics. 16, 710-724 (2019).\n37. Mast, N. et al. Cholesterol-metabolizing enzyme cytochrome P450 46A1 as a\npharmacologic target for Alzheimer's disease. Neuropharmacology. 123, 465-476\n(2017).\n38. Testa, G. et al. A silver lining for 24-hydroxycholesterol in Alzheimer's disease:\nThe involvement of the neuroprotective enzyme sirtuin 1. Redox Biol. 17, 423-431\n(2018)."}, {"round": 2, "reviewer_comments": {"reviewer_1": "The authors have addressed most of the previous comments and questions through several additional experiments. The\nmanuscript is now significantly improved and more complete. However, I still have a few concerns that I would like the\nauthors to take into account and address before publication :\n- The claim that “capsaicin intake ameliorates cognitive impairments in AD patients” (line 92) may be too strong. Given that\nsome correlations did not reach statistical significance following the new analyses, I recommend softening this statement. A\nmore appropriate phrasing would be “capsaicin use correlates with cognitive performance” or a similar wording.\n- Although the authors have conducted additional impressive experiments involving soclestat and labeled 24OH, I believe\ncaution should be taken to conclude that capsaicin reduces AD pathology primarily via 24OH. The data suggest indeed that\n24OH contributes to the observed effects, but it is likely part of complex mechanisms. The title and the conclusions should\nbe revised to reduce the emphasis on 24OH as the principal mediator of capsaicin's effects.\n- The lipidomic analysis reveals changes in several other molecules not only 24OH, including cholesterol. It is worth\ndiscussing whether the modulation of cholesterol levels itself may contribute to the effects induced by capsaicin. A broader\ninterpretation and discussion would strengthen the manuscript.\n- While 24OH can reach the brain, its concentrations appear to remain lower in the brain than other organs after treatment,\npossibly due to rapid metabolism in the liver and other peripheral organs. This should be acknowledged and discussed in\nthe manuscript.\n- A notable limitation of the study is the exclusive use of male mice.\nThis should be clearly acknowledged in the discussion, along with a consideration of how sex differences might impact the\nfindings and their interpretation.", "reviewer_2": "1. The authors propose that 24-HC modulates microglial inflammation via SUMOylation of LXRβ, supported by si-LXRβ\nknockdown experiments. To more definitively establish LXRβ as a critical mediator of 24-HC’s anti-inflammatory effects,\nit would be highly informative to demonstrate whether these effects are abolished under si-LXRβ conditions. Showing that 24-\nHC fails to exert its regulatory effects in the absence of LXRβ would provide a more direct mechanistic link supporting the\nproposed 24-HC → LXRβ → inflammation pathway.\n2. The authors have made thoughtful revisions and performed additional experiments that strengthen the manuscript. This\nstudy provides valuable insights into the gut–brain axis underlying capsaicin’s effects. To further enhance the translational\nrelevance of the findings, I suggest discussing whether similar alterations in Oscillospiraceae have been observed in\nmicrobiome studies of AD patients. If available, referencing data on Oscillospiraceae abundance in relation to capsaicin\nintake in humans would be ideal. Even if direct evidence is lacking, incorporating relevant findings into the Discussion\nwould provide broader context and underscore the potential relevance of the findings to human disease.\nOverall, the manuscript has improved substantially, and addressing these points would further enhance its clarity and\nimpact.", "reviewer_4": "*Sending an anonymised report:\nThere have been interesting relationships observed between capsaicin consumption and Alzheimer’s disease (AD)\npathologies and cognitive functions, based on both preclinical and observational human studies. However, the existing\nresearch—including contradictory findings regarding amyloid processing—indicates a need for further investigation and\nhuman clinical trials. Additionally, there is a lack of evidence supporting the underlying mechanisms involved in these\ninteractions.\nIn the current manuscript, Li et al. claim that capsaicin consumption enhances cognitive function and mitigates Aβ deposition\nvia the gut microbiome–25-HC–neuroinflammation pathway. This is an interesting and potentially important finding for\nmechanistically understanding the interactions between capsaicin (Cap.) and AD pathogenesis. However, the current\nversion of the manuscript remains correlational, and there is insufficient evidence proving that capsaicin-associated\nmicrobiome and 25-HC modulate neuroinflammation and AD-related pathologies. More detailed comments are provided\nbelow:\n1. As other reviewers—notably Reviewer 3—have pointed out, there is a lack of evidence supporting a causal relationship\nbetween Cap.-associated microbiome and AD-related pathologies. As R3 requested, the FMT approach (or an alternative\napproach to probe the Cap.-associated microbiome is really involved in the interactions) is very critical in this study, as the\nnovelty lies in the role of the capsaicin-associated gut microbiome mediating these interactions.\n2. The authors mention the infeasibility of FMT due to high capsaicin levels in the intestine, making it difficult to dissociate\nthe effects of capsaicin itself from those of the microbiome. However, the authors could attempt FMT after multi-filtration of\nthe fecal material to remove capsaicin molecules. Moreover, in the FMT procedure, FMT would not be administered as\nfrequently as capsaicin treatment. Also, the capsaicin level would also likely decrease as the fecal material travels through\nthe gut. Therefore, this approach remains worth trying, especially considering its importance to the study.\n3. The authors analyze microbiome data only at a high taxonomic level (phylum and family), focusing on broad\ncompositional differences. Since metagenomic analysis was used, the study could and should identify more detailed\nmicrobiome features—potentially down to the species level—that are involved in the interactions between capsaicin and\npathology. These strains could then be introduced directly into mice to determine whether the same effects observed in\nCap.-treated mice are replicated. This could be alternative approach as a causal probe.\n4. Related to the point above (also R3 asked about details in methodology): why do the authors focus only on the intestinal\nmicrobiome/content? The colonic/fecal microbiome is known to be more diverse and is more commonly discussed in the\ncontext of the microbiome–gut–brain axis.\n5. Regarding Figure 3 (after revisions in response to R3), it remains important to provide FMT or other direct evidence\ndemonstrating the effect of Cap.-associated microbiota on neuroinflammation and pathology. It is difficult to interpret\nthe meaning of antibiotic (ABX) treatment blocking capsaicin effects. The authors speculate that beneficial microbiota induced\nby capsaicin are reduced by ABX, but this remains pure speculation. Many alternative explanations are possible. For\ninstance, long-term administration of high doses of mixed antibiotics (over approximately 1.5 months) could cause leaky gut\nor other physiological/anatomical changes. The microbiome–gut–brain axis is a complex system, and many factors could\ncontribute. It is risky to attribute ABX effects solely to microbiome depletion. Although ABX is designed to kill bacteria\nbroadly, its mechanisms are not fully understood, and there are reports indicating that ABX can modulate the immune\nsystem being microbiome-independent. Therefore, direct manipulation of Cap.-associated microbiota is needed to\nsubstantiate downstream effects.\n6. Regarding Comment 6 from R3: the improvement in object-place recognition in NS-treated WT mice suggests that the\ncognitive enhancement from Cap. treatment may be independent of its effects on AD pathology.\n7. The authors mentioned to R3’s question: “As for the potential dose-dependent effects of capsaicin on gut microbiota\nregulation, we have reviewed existing literature. It has been found that low doses of capsaicin (≤5 mg/kg) enhanced\nbeneficial bacteria like Firmicutes, Lachnospiraceae, and Blautia, which increased butyrate production and improved gut\nbarrier function. Moderate doses of capsaicin further boosted probiotics such as Akkermansia and Faecalibacterium\nprausnitzii, thus enhancing insulin sensitivity and anti-inflammatory activity.” Are these strains related to 25-HC production?,\nwhich the authors propose as a key mediator?\n8. The experiment using P301S mice is somewhat distracting in the current manuscript, unless the authors perform a\nsystematic analysis comparable to that used in the amyloidosis models, including microbiome manipulation. Additionally,\nthe age of the P301S mice used is too young to draw meaningful conclusions regarding pathology. In this mouse model,\nsubstantial tau pathology is typically observed after 6–7 months of age (with only weak p-tau expression), and\nneurodegeneration generally becomes evident after 9 months. Prior studies also have reported microbiome effects on\npathology only after 9 months of age (PMID: 36634180).\n9. The protective effect of peripheral administration of 25-HC is interesting, particularly given that there are reports claiming\n25-HC is promoting neuroinflammation and AD-related pathologies (PMID: 32552741; PMID: 40001197)."}, "author_response": "Point-by-point responses\nReviewer #1:\nComment 1: The authors have addressed most of the previous comments and questions\nthrough several additional experiments. The manuscript is now significantly improved\nand more complete. However, I still have a few concerns that I would like the authors\nto take into account and address before publication:\n- The claim that “capsaicin intake ameliorates cognitive impairments in AD patients”\n(line 92) may be too strong. Given that some correlations did not reach statistical\nsignificance following the new analyses, I recommend softening this statement. A\nmore appropriate phrasing would be “capsaicin use correlates with cognitive performance”\nor a similar wording.\nResponse: We appreciate your insightful comment. As suggested, we have changed the\nsubtitle of this part into “Capsaicin intake correlates with cognitive impairments in AD\npatients and 5×FAD mice” in the revised manuscript.\nComment 2: Although the authors have conducted additional impressive experiments\ninvolving soclestat and labeled 24OH, I believe caution should be taken to conclude\nthat capsaicin reduces AD pathology primarily via 24OH. The data suggest indeed that\n24OH contributes to the observed effects, but it is likely part of complex mechanisms.\nThe title and the conclusions should be revised to reduce the emphasis on 24OH as\nthe principal mediator of capsaicin's effects.\nResponse: Thank you for your comment, we agree with you that the effects of capsaicin\non AD pathology should be complex, though we have revealed in this paper that the gut\nmicrobiota-dependent modulation of 24OH (namely 24-HC) production is of pivotal\nimportance. To enhance the precision and scientific rigor of our manuscript, we have\nrephrased the title into “Gut microbiota-dependent 24-hydroxycholesterol metabolism\ncontributes to capsaicin-induced amelioration of Alzheimer’s disease pathology”, and\nrefined the conclusion accordingly.\nComment 3: The lipidomic analysis reveals changes in several other molecules not\nonly 24OH, including cholesterol. It is worth discussing whether the modulation of\ncholesterol levels itself may contribute to the effects induced by capsaicin. A broader\ninterpretation and discussion would strengthen the manuscript.\nResponse: Indeed, though we focused in the present study the protective effects against\nAD of capsaicin-induced upregulation of 24-HC levels, while potential roles of other\nmetabolites also exist. Especially, multiple cohort studies have revealed a significant\ncorrelation of blood low-density lipoprotein cholesterol (LDL-C) level with the risk of\nAD and dementia1,2, as well as cerebral amyloidosis imaging index3. Excessive uptake\nof peripheral cholesterol drives a major component of chronic inflammation in the brain\nthrough dysregulating glia activation, and promotes Aβ secretion and tau\nhyperphosphorylation4,5. Consistently, reduction of cholesterol level is effective for\nalleviating neuroinflammation and AD pathologies6,7. These findings raise the\npossibility that the effects of capsaicin might be also at least partly mediated by the\ndownregulation of cholesterol levels. Besides, our metabolomic data also suggest an\nalteration of plasma bile acid following the capsaicin treatment, which has been found\nto be associated with the development of AD8,9. Elucidating these alternative pathways\nis of significant scientific interest. We have extended the discussion accordingly in the\nrevised manuscript.\nComment 4: While 24OH can reach the brain, its concentrations appear to remain\nlower in the brain than other organs after treatment, possibly due to rapid metabolism\nin the liver and other peripheral organs. This should be acknowledged and discussed in\nthe manuscript.\nResponse: Thank you for raising this point. As suggested, we have added clarification\nin the revised manuscript. In addition, while fluorescence tracking of 24-HC revealed\na much lower relative proportion enters the brain than remains in blood, potentially due\nto rapid peripheral metabolism, direct quantification of total unlabeled 24-HC\nconfirmed that the absolute amount in the brain is still significantly greater than in the\nperiphery. This was consistent with previous studies that 24-HC is distributed at\nsignificantly higher levels in the brain than in peripheral tissues10.\nComment 5: A notable limitation of the study is the exclusive use of male mice.\nThis should be clearly acknowledged in the discussion, along with a consideration of how\nsex differences might impact the findings and their interpretation.\nResponse: We have acknowledged this limitation of the current study, and extended\nthe discussion of revised manuscript.\nReviewer #2:\n1. The authors propose that 24-HC modulates microglial inflammation via\nSUMOylation of LXRβ, supported by si-LXRβ knockdown experiments. To more\ndefinitively establish LXRβ as a critical mediator of 24-HC’s anti-inflammatory effects,\nit would be highly informative to demonstrate whether these effects are abolished under\nsi-LXRβ conditions. Showing that 24-HC fails to exert its regulatory effects in the\nabsence of LXRβ would provide a more direct mechanistic link supporting the\nproposed 24-HC → LXRβ → inflammation pathway.\nResponse: Thank you for your insightful comments, we have performed additional\nexperiments to examine whether LXRβ knock-down via RNAi impaired the anti-\ninflammatory effects of 24-HC in cultured BV2 cells. In consistent with our previous\nresults, 24-HC treatment significantly decreased levels of IL-1β and TNF-α,\ndecreased\nratio of CD16/32+ to CD206+ cell portion, and promoted Aβ phagocytosis in Aβ-treated\nBV2 cells transfected with control vectors, while all these effects were nearly abolished\nunder LXRβ knock-down, suggesting a pivotal role of LXRβ in mediating the anti-\ninflammatory effect of 24-HC on microglia. We have added these results in the revised\nFig.6g-j of manuscript.\nFig. 6. (g) si-LXRβ increased IL-1β and TNF-α levels, and abolished the anti-\ninflammatory effect of 24-HC in BV2 cells co-administrated with Aβ. n = 3 wells of cell\nin each group. Data were normalized to the mean value of control group. * P < 0.05,\nn.s. nonsignificant, two-way ANOVA followed by Tukey’s multiple comparisons tests.\n(h-i) si-LXRβ increased the portion of CD16/32-positive BV2 cells while decreased the\nportion of CD206-positive cells, and abolished the effect of 24-HC in suppressing the\npro-inflammatory switch of BV2 cells. n = 3 batches of experiments in each group. * P\n< 0.05, ** P < 0.01, n.s. nonsignificant, two-way ANOVA followed by Tukey’s multiple\ncomparisons tests.\n(j) si-LXRβ decreased the amount of Aβ phagocytized in BV2 cells, and inhibited the\neffect of 24-HC in facilitating Aβ phagocytosis. n = 5 wells of cell in each group. * P <\n0.05, ** P < 0.01, two-way ANOVA followed by Tukey’s multiple comparisons tests.\n2. The authors have made thoughtful revisions and performed additional experiments\nthat strengthen the manuscript. This study provides valuable insights into the gut–brain\naxis underlying capsaicin’s effects. To further enhance the translational relevance of the\nfindings, I suggest discussing whether similar alterations in Oscillospiraceae have been\nobserved in microbiome studies of AD patients. If available, referencing data on\nOscillospiraceae abundance in relation to capsaicin intake in humans would be ideal.\nEven if direct evidence is lacking, incorporating relevant findings into the Discussion\nwould provide broader context and underscore the potential relevance of the findings\nto human disease.\nOverall, the manuscript has improved substantially, and addressing these points would\nfurther enhance its clarity and impact.\nResponse: Thank you for your positive comments on our work. We have conducted in-\ndepth analysis of the gut microbiota data of 5xFAD mice following capsaicin feeding,\nand found a potential association between the elevation of 24-HC level with the increase\nof Oscillibacter genus within the Oscillospiraceae family, which has been previously\nreported to express ismA homologous gene that is involved in metabolizing cholesterol\nto generate hydroxycholesterol11,12. Thus, as suggested, we examined gut Oscillibacter\nchanges in AD patients in GMrepo v2, a curated human gut microbiome database13. In\none of the three AD datasets included (PRJNA770746), a significant enrichment of\nOscillibacter in healthy controls was found (LDA score = -3.17). Overall, the\nabundance of Oscillibacter was significantly downregulated in AD patients compared\nwith healthy controls (n = 218 for AD and 15,780 for healthy, adjusted p < 0.0001).\nThis finding is consistent with our observations in 5xFAD mice. We have added these\ndata in the revised Fig5a-b.\nFig5a-b. (a) Featured enrichment of gut microbes at genus level, including\nOscillibacter, in healthy individuals (N = 15,780) versus AD patients (N = 218)\nrecorded in GMrepo v2 database. Linear Discriminant analysis (LDA) scores were\nacquired using LEfSe analysis. (b) AD patients showed significantly decreased\nOscillibacter abundance compared with healthy group. *** adjusted P = 1.36e-17,\nMann-Whitney U test.\nNext, we attempted to investigate the impact of capsaicin-containing food intake\non human gut microbiota, particularly on Oscillibacter. In consistent with our findings,\none case study found that sustained treatment of in vitro cultured human feces with\ncapsaicin (3 mg/mL, for 14 days) led to a significant increase in Oscillibacter\nabundance14. However, another study involving 29 healthy subjects reported that\nconsuming tomato juice plus capsaicin (2 × 1.8 g per day) for five consecutive days\nresulted in a reduction of Oscillibacter, while this change became non-significant after\nstratification by body mass index15. We attempted to perform more in-depth analysis of\nthe data from these two studies, but neither provided publicly accessible raw sequencing\ndata or detailed lists of gut microbial abundances.\nNonetheless, these evidences suggest that Oscillibacter abundance is significantly\nreduced in the gut microbiota of AD patients and may represent one of the mechanisms\nunderlying the dysregulation of cholesterol metabolism in AD. Dietary intake of\ncapsaicin might influence the abundance of Oscillibacter in human gut microbiota,\nthough its specific effects may vary across populations and be subject to confounding\nfactors. We have extended discussion accordingly in the revised manuscript.\nReviewer #4:\nThere have been interesting relationships observed between capsaicin consumption and\nAlzheimer’s disease (AD) pathologies and cognitive functions, based on both\npreclinical and observational human studies. However, the existing research—\nincluding contradictory findings regarding amyloid processing—indicates a need for\nfurther investigation and human clinical trials. Additionally, there is a lack of evidence\nsupporting the underlying mechanisms involved in these interactions.\nIn the current manuscript, Li et al. claim that capsaicin consumption enhances cognitive\nfunction and mitigates Aβ deposition via the gut microbiome–25-HC–\nneuroinflammation pathway. This is an interesting and potentially important finding for\nmechanistically understanding the interactions between capsaicin (Cap.) and AD\npathogenesis. However, the current version of the manuscript remains correlational, and\nthere is insufficient evidence proving that capsaicin-associated microbiome and 25-HC\nmodulate neuroinflammation and AD-related pathologies. More detailed comments are\nprovided below:\nComments 1~2: 1. As other reviewers—notably Reviewer 3—have pointed out, there\nis a lack of evidence supporting a causal relationship between Cap.-associated\nmicrobiome and AD-related pathologies. As R3 requested, the FMT approach (or an\nalternative approach to probe the Cap.-associated microbiome is really involved in the\ninteractions) is very critical in this study, as the novelty lies in the role of the capsaicin-\nassociated gut microbiome mediating these interactions.\n2. The authors mention the infeasibility of FMT due to high capsaicin levels in the\nintestine, making it difficult to dissociate the effects of capsaicin itself from those of\nthe microbiome. However, the authors could attempt FMT after multi-filtration of the\nfecal material to remove capsaicin molecules. Moreover, in the FMT procedure, FMT\nwould not be administered as frequently as capsaicin treatment. Also, the capsaicin\nlevel would also likely decrease as the fecal material travels through the gut. Therefore,\nthis approach remains worth trying, especially considering its importance to the study.\nResponses: Thank you for your thoughtful comments. We fully agree with you that\nFMT would provide compelling evidence to demonstrate whether the protective effects\nof capsaicin are mediated through its modulation on gut microbiota. As suggested, we\nhave performed additional experiments, while gut microbiota transfer (GMT)\nexperiments were performed in the present study instead of FMT to minimize\ninterference from environmental bacteria in the housing conditions and prevent the\ndisruption of anaerobic bacteria by oxygen exposure. Gut contents from Veh- or Cap-\nfed 5xFAD mice were collected and immediately processed under anaerobic conditions.\nWe used 5-month 5xFAD mice here since it has been reported that FMT from wild-type\nto AD mice per se showed effectiveness in alleviating AD phenotypes16,17.\nGiven the low water solubility of Cap, all samples underwent repeated washing\nwith pre-reduced PBS followed by centrifugation (final solvent at 1 mL per gram of gut\ncontent). The supernatant was obtained and further extracted with corn oil to remove\nany potential residual capsaicin. It was confirmed that the Cap was virtually\nundetectable in the final aqueous phase containing the gut microbes. This processed\nmaterial was stored at -80℃ and used for GMT in 5-month 5xFAD mice (0.5 mL each\nday, once every other day for a month), equal doses of PBS was delivered as control.\nIn consistent with our previous findings, we found that GMT from Cap-, but not Veh-\ntreated mice significantly reduced Aβ plaques, inhibited microglia activation, reduced\ninflammatory factors levels in the hippocampus, and improved cognitive functions of\n5xFAD mice compared with PBS control.\nTogether with our previous findings that ABX elimination of gut microbiota\nabolished the protective effects of capsaicin, our results collectively supported a pivotal\ncontribution of the gut microbiota in mediating the amelioration of AD phenotypes by\noral capsaicin intake. We have added these data in the revised version of Fig. 3e-p of\nmanuscript.\nFig. 3e-p. (e-f) Experimental scheme of gut microbes isolation and validation of\ncapsaicin removal from GMT samples. n = 4 pooled samples in each group. *** P <\n0.001, one-way ANOVA followed by Tukey’s multiple comparisons tests. (g)\nExperimental schedule of GMT. (h-k) GMT from Cap-treated mice decreased density\nof Aβ plaques (i) and microglia soma volume (i), and increased microglia processes\nbranching complexity (k) in the hippocampal CA1 of 5xFAD mice. Arrows in\npresentative images (h) indicate cells showed in reconstructed images (lower panels).\nn = 40 cells from 8 mice in each group. n.s. nonsignificant, * P < 0.05, ** P < 0.01,\none-way (i, j) or two-way (k) ANOVA followed by Tukey’s multiple comparisons tests.\n(l) GMT from Cap-treated mice downregulated pro-inflammatory levels in the\nhippocampus tissue. n = 8 mice in each group. Data were normalized to the mean value\nof PBS group. n.s. nonsignificant, * P < 0.05, ** P < 0.01, two-way ANOVA followed\nby Tukey’s multiple comparisons tests. (m-p) GMT from Cap-treated mice improved\nobject-place recognition (m), and spatial learning (n) and memory (o-p) in the Morris-\nwater maze test of 5xFAD mice. n = 8 mice in each group. n.s. nonsignificant, * P <\n0.05, ** P < 0.01, one-way (m, o) or repeated measures (n) ANOVA followed by Tukey’s\nmultiple comparisons tests.\nComment 3: The authors analyze microbiome data only at a high taxonomic level\n(phylum and family), focusing on broad compositional differences. Since metagenomic\nanalysis was used, the study could and should identify more detailed microbiome\nfeatures—potentially down to the species level—that are involved in the interactions\nbetween capsaicin and pathology. These strains could then be introduced directly into\nmice to determine whether the same effects observed in Cap.-treated mice are replicated.\nThis could be alternative approach as a causal probe.\nResponse: Thank you for your comments. As suggested, we performed more in-depth\nLEfSe analysis of metagenomic data, and found many species showing featured\nenrichment in the group of Cap-treated 5xFAD mice, including\nOscillibacter_sp._CAG_241 and Oscillibacter_sp._KLE_1745 (please see the revised\nFig. 3d).\nFig 3d. Taxonomic cladogram of intestinal bacteria to species level revealed by LEfSe\nanalysis. Cyan area and dots indicate featured bacteria with featured enrichment in\ncapsaicin-treated 5×FAD mice.\nIt has been previously recognized that Oscillibacter is capable of metabolizing\ncholesterol to generate hydroxycholesterol, presumed depending on the expression of\nismA homologous gene11,12. Thus, we next focused on the role of Oscillibacter in\nmediating the protective role of capsaicin. Further analysis revealed the reduction in\nabundance of Oscillibacter in 5xFAD mice, which was significantly rescued by\ncapsaicin treatment. Meanwhile, the Oscillibacter abundance correlated negatively with\nplasma cholesterol level and positively with 24-HC level (revised Fig. 4h-j).\nFig. 4h-j. (h-i) Capsaicin partly rescued the downregulation of gut Oscillibacter\nabundance in 5xFAD mice in many species (h) and at the overall genus level (i). n = 8\nmice in each group. * P < 0.05, ** P < 0.01, two-way (h) or one-way (i) ANOVA\nfollowed by Tukey’s multiple comparisons tests. (j) Oscillibacter abundance negatively\ncorrelated with plasma cholesterol level, while positively correlated with 24-HC level.\nLinear regression analysis.\nFurthermore, we examined Oscillibacter changes in AD patients in GMrepo v2, a\ncurated human gut microbiome database13. In one of the three AD datasets included\n(PRJNA770746), a significant enrichment of Oscillibacter in healthy controls was\nfound (LDA score = -3.17). Overall, the abundance of Oscillibacter was significantly\ndownregulated in AD patients (revised Fig5a-b). Besides, one previous case study\nfound that sustained treatment of in vitro cultured human feces with capsaicin led to a\nsignificant increase in Oscillibacter abundance14, though another study involving 29\nhealthy subjects reported that consuming tomato juice plus capsaicinfor five\nconsecutive days resulted in a reduction of Oscillibacter, while this change became non-\nsignificant after stratification by body mass index15. In general, these data suggest that\nOscillibacter abundance is significantly reduced in the gut microbiota of AD patients,\nand dietary intake of capsaicin might influence the abundance of Oscillibacter in human\ngut microbiota, though its specific effects may vary across populations and be subject\nto confounding factors. We have also extended discussion accordingly in the revised\nmanuscript.\nFig5a-b. (a) Featured enrichment of gut microbes at genus level, including\nOscillibacter, in healthy individuals (N = 15,780) versus AD patients (N = 218)\nrecorded in GMrepo v2 database. Linear Discriminant analysis (LDA) scores were\nacquired using LEfSe analysis. (b) AD patients showed significantly decreased\nOscillibacter abundance compared with healthy group. *** adjusted P = 1.36e-17,\nMann-Whitney U test.\nSubsequently, we isolated gut microbes from Cap-treated 5xFAD mice under\nanaerobic conditions, and identified Oscillibacter via whole-genome sequencing. The\nisolated Oscillibacter was further validated by prominent expression of previously\nidentified marker genes, including Oscillibacter-specific 16S rRNA, ismA and CgT\nhomologous genes11, as measured by qPCR compared with negative controls of E.coli\nand positive control of J115. Furthermore, we ascertained the capacity of Oscillibacter\nto generate 24-HC in cultures. Cholesterol treatment for 24 h significantly increased the\n24-HC level in the culture medium, which was further upregulated under capsaicin co-\nadministration (revised Fig. 5c-e).\nAs suggested, we grafted Oscillibacter repeatedly into 5-month 5xFAD mice for a\nmonth, equal doses of culture medium were inoculated as control. The results showed\nthat Oscillibacter supplement significantly reduced Aβ plaques density, inhibited\nmicroglia overactivation, downregulated pro-inflammatory factors levels, and\nimproved cognitive functions of 5xFAD mice (revised Fig. 5f-o). These outcomes\nmimicked the protective effects of oral capsaicin intake.\nFig. 5c-o. (c) Experimental procedures of Oscillibacter isolation from gut contents of\nCap-treated 5xFAD mice. (d) Isolated Oscillibacter (Osc.) showed high expression\nlevels of Osc.-specific 16S rRNA, ismA and CgT homologous genes relative to the level\nof pan bacterial 16S rRNA. Culture medium and E. coli were used as negative control.\nJ115 was used as positive control. (e) 24-HC level in the culture medium of\nOscillibacter was upregulated following cholesterol treatment, and further increased\nunder Cap co-treatment. n = 5 dishes per group. * P < 0.05, one-way ANOVA followed\nby Tukey’s multiple comparisons tests. (f) Experimental scheme of medium and\nOscillibacter delivery in 5xFAD mice. (g-j) Direct Oscillibacter grafting decreased Aβ\nplaques density (h), decreased the soma volume (i) and increased cell processes\nbranching complexity (j) of microglia in the hippocampal CA1 of 5xFAD mice. Arrows\nin presentative images (g) indicate microglia showed in reconstructed images (lower\npanels). n = 40 cells from 8 mice in each group. * P < 0.05, ** P < 0.01, unpaired t\ntests (h, i) or two-way ANOVA (j). (k) Oscillibacter downregulated pro-inflammatory\nlevels in the hippocampal tissue. n = 8 mice in each group. Data were normalized to\nthe mean value of medium group. * P < 0.05, ** P < 0.01, two-way ANOVA followed\nby Tukey’s multiple comparisons tests. (l-m) Oscillibacter improved object-place\nrecognition of 5xFAD mice. n = 8 mice in each group. * P < 0.05, unpaired t test. (n-o)\nOscillibacter improved spatial learning (n) and memory (o) of 5xFAD mice in the\nMorris-water maze test. n = 8 mice in each group. * P < 0.05, ** P < 0.01, two-way\nANOVA (n) or unpaired t test (o).\nCollectively, these results suggest a role of gut Oscillibacter in generating 24-HC,\nand an important contribution of Oscillibacter increase in mediating the capsaicin-\ninduced amelioration of AD phenotypes.\nComment 4: Related to the point above (also R3 asked about details in methodology):\nwhy do the authors focus only on the intestinal microbiome/content? The colonic/fecal\nmicrobiome is known to be more diverse and is more commonly discussed in the\ncontext of the microbiome–gut–brain axis.\nResponse: Thank you for raising this crucial point regarding the sampling site for gut\nmicrobiota analysis. In the initial phase of this study, we collected small intestinal\ncontents for metagenomic analysis based on the rationale that the small intestine is the\nprimary site for chemical digestion and nutrient absorption, and thus might be more\nsusceptible to the acute effects of capsaicin. However, as the project progressed, we\nrecognized that the colonic microbiota is far more abundant than that of the small\nintestine. Besides, there are numerous seminal studies in both animal and human\nutilizing fresh fecal samples reported alterations of key taxa, including Oscillibacter, in\nthe context of AD. This highlighted a potential limitation of our initial focus solely on\nthe small intestine.\nTo address this limitation, we conducted a preliminary experiment comparing the\nrelative abundance of Oscillibacter between small intestinal and colonic contents via\nqPCR measurement of Oscillibacter-specific 16S rRNA. Indeed, a predominantly\nhigher abundance of Oscillibacter in the colon relative to small intestines was revealed\n(please see the Fig. r1 as follows).\nFig. r1. Higher abundance of Oscillibacter level in the colonic contents compared with\nsmall intestines (SI) in 5-month wild-type mice. Data were normalized by the mRNA\nlevel of conservative bacterial 16S rRNA for each sample. n = 5 mice in each group. *\nP < 0.05, unpaired t test.\nConsequently, we revised our protocol for additional experiments to collect\ncontents from the gut tract including both small intestine and colon for microbiota\nanalysis and transfer. We have updated the revised Materials and Methods section to\nclearly describe this methodological evolution. To prevent potential confusion and\nmaintain consistency of the main text, we have unified the terminology throughout the\nmanuscript and figures by replacing the original \"intestinal contents\" with the more\ngeneral words \"gut contents\".\nComment 5: Regarding Figure 3 (after revisions in response to R3), it remains\nimportant to provide FMT or other direct evidence demonstrating the effect of Cap.-\nassociated microbiota on neuroinflammation and pathology. It is difficult to interpret\nthe meaning of antibiotic (ABX) treatment blocking capsaicin effects. The authors\nspeculate that beneficial microbiota induced by capsaicin are reduced by ABX, but this\nremains pure speculation. Many alternative explanations are possible. For instance,\nlong-term administration of high doses of mixed antibiotics (over approximately 1.5\nmonths) could cause leaky gut or other physiological/anatomical changes. The\nmicrobiome–gut–brain axis is a complex system, and many factors could contribute. It\nis risky to attribute ABX effects solely to microbiome depletion. Although ABX is\ndesigned to kill bacteria broadly, its mechanisms are not fully understood, and there are\nreports indicating that ABX can modulate the immune system being microbiome-\nindependent. Therefore, direct manipulation of Cap.-associated microbiota is needed to\nsubstantiate downstream effects.\nResponse: We agree that ABX treatment is not a specific method for depleting the gut\nmicrobiota. As noted, high-dose ABX may affect many physiological functions beyond\nthe microbiome. Therefore, we performed GMT and Oscillibacter transplantation\nexperiments following the suggestions of the reviewers to more directly examine the\nmediative role of oral capsaicin intake in exerting its protective effects in 5xFAD mice\n(please see our responses to your comments 1~3).\nNevertheless, we believe the results from ABX intervention still hold important\nreference value, since they provide supporting evidence from another perspective that\nindicate the essential contribution of gut microbiota to the phenotypic improvements\ninduced by capsaicin, and to the systematic upregulation of 24-HC level. Accordingly,\nwe have retained this set of data in the revised manuscript, but moved it to the\nsupplementary materials (supplementary Fig. S6 and S7).\nComment 6: Regarding Comment 6 from R3: the improvement in object-place\nrecognition in NS-treated WT mice suggests that the cognitive enhancement from Cap.\ntreatment may be independent of its effects on AD pathology.\nResponse: Our results showed that capsaicin improved cognitive function in NS-\ntreated WT mice. One of potential explanations is our finding that capsaicin treatment\nalso showed a trend toward increasing 24-HC levels in WT mice (p = 0.06 ~ 0.09,\nsupplementary Fig. S7a). Beyond the role of 24-HC in mitigating AD pathology via the\nmicroglia-inflammation-Aβ phagocytosis axis revealed in the present study, 24-HC has\nbeen also known to function as a positive allosteric modulator of NMDA receptors and\nactivated estrogen receptors-mediated sex hormone signaling, both of which\npotentiated synaptic plasticity and memory in mice18-20. Moreover, capsaicin has been\nalso reported to act as modulators of voltage-gated Na+, K+, and Ca2+ channels, all of\nwhich are pivotal in regulating neuronal excitability and plasticity21. Thus, we cannot\nexclude the possibility that additional mechanisms, yet unidentified, may also\ncontribute to the cognitive-enhancing effects of capsaicin. We have extended the\ndiscussion accordingly in the revised manuscript.\nComment 7: The authors mentioned to R3’s question: “As for the potential dose-\ndependent effects of capsaicin on gut microbiota regulation, we have reviewed existing\nliterature. It has been found that low doses of capsaicin (≤5 mg/kg) enhanced beneficial\nbacteria like Firmicutes, Lachnospiraceae, and Blautia, which increased butyrate\nproduction and improved gut barrier function. Moderate doses of capsaicin further\nboosted probiotics such as Akkermansia and Faecalibacterium prausnitzii, thus\nenhancing insulin sensitivity and anti-inflammatory activity.” Are these strains related\nto 25-HC production?, which the authors propose as a key mediator?\nResponse: There is currently no direct evidence demonstrating that these gut bacteria\ncan produce 24-HC, the oxysterol of interest in this study, nor for 25-HC. Nevertheless,\nit has been revealed that certain species among them can metabolize cholesterol. In\nspecific, sp. Faecalibacterium prausnitzii, belonging to the Oscillospiraceae family of\nFirmicutes phylum, express microbial cholesterol dehydrogenases encoded by ismA\nhomologous genes and can catabolize cholesterol12. sp. Akkermansia muciniphila\nbelonging to the Verrucomicrobiota phylum can reduce the activity of genes involved\nin mucins-based cholesterol production22. Moreover, Akkermansia muciniphila and\nLachnospiraceae supplement in healthy human were both found to associate with\nreduction in plasma total cholesterol level23,24. In type 2 diabetes mellitus (T2DM)\npatients, the increase of Blautia, also belonging to the Firmicutes phylum, induced by\nmetformin and AMC treatment was significantly correlated with the improvements in\nlipid homeostasis25. Collectively, these studies have provided direct or correlative\nevidence suggesting the involvement of these gut bacteria in metabolizing cholesterol\nto generate hydroxycholesterols.\nAs for which species is more pivotal for the 24-HC production, our data from\nadditional experiments has directly demonstrated that Oscillibacter, belonging to the\nOscillospiraceae family of Firmicutes phylum, can metabolize cholesterol and\nproducing 24-HC, and this process is positively regulated by capsaicin (please see the\nresponse to your comment 3), which is consistent with previous findings that\nOscillibacter expresses ismA homologous genes and catabolizes cholesterol in the gut11.\n8. The experiment using P301S mice is somewhat distracting in the current manuscript,\nunless the authors perform a systematic analysis comparable to that used in the\namyloidosis models, including microbiome manipulation. Additionally, the age of the\nP301S mice used is too young to draw meaningful conclusions regarding pathology. In\nthis mouse model, substantial tau pathology is typically observed after 6–7 months of\nage (with only weak p-tau expression), and neurodegeneration generally becomes\nevident after 9 months. Prior studies also have reported microbiome effects on\npathology only after 9 months of age (PMID: 36634180).\nResponse: We examined here the effects of capsaicin in the P301S mouse model only\non several key phenotypes like microglial activation, 24-HC levels, and tau-related\nneuropathology. We propose that these results provide key clues suggesting that\ncapsaicin may alleviate AD-related tau pathology by modulating 24-HC metabolism.\nNevertheless, we agree with the reviewer that more systematic and in-depth\ninvestigations, in similar to what was performed in the 5xFAD model, are required to\nestablish a definitive causal relationship. We sincerely thank you for the valuable\nsuggestions provided here. However, in terms of workload, this would require\nsubstantial time and resources, and could constitute a standalone research project in\nitself. We are currently planning to undertake this line of investigation. To maintain\nfocus in the main text, we have moved all P301S mice-related results to the\nsupplementary materials (please see the supplementary Fig. S12) of the revised\nmanuscript.\nRegarding the age of P301S mice, we initiated capsaicin administration at 5 months\nof age and continued until phenotype assessment at 6 months. As shown in our results,\nP301S mice already exhibit significant microglial abnormalities and tau pathology at\n5~6 months of age. This is consistent with previous literatures reporting that\nneuroinflammation generally emerges in this strain as early as 2~3 months, followed\nby tau pathology and cognitive impairment at 4~6 months of age26,27 (summarized in\nhttps://www.alzforum.org/research-models/tau-p301s-line-ps19#overview). Therefore,\nwe consider it reasonable to initiate the intervention at 5 months of age and assess the\noutcomes at 6 months.\nTo our knowledge, direct evidence of how the gut microbiota changes in P301S\nmice at 5~6 months of age and how it correlates with tau-related pathology is lacking\nto date. In another study using ADLPAPT mice, a line exhibiting both Aβ and tau\npathologies in the brain, researchers observed significant alterations in gut microbiota\ncomposition as early as 2 months of age, while transplanting gut microbiota from\nhealthy mice starting at this early stage for a duration of 4 months effectively reduced\nboth Aβ- and tau-related pathological phenotypes and cognitive deficits17. In TE4\nmouse line, it was noted in the mentioned study28 that though ABX treatment did not\nchange AT8-staining signal of PHF-tau, TE4 mice reared in a germ-free environment,\nfor less invasively depleting gut microbiota, tended to exhibit reduced microglial\nactivation as indicated by increased length and complexity of cell processes in\nmorphology, and lower gene expression levels of reactive microglia biomarker Axl and\nFgr at 12 weeks of age. The absence of observed significant changes of tau pathology\nin this line may be multifactorial: the 12-week timepoint may still be too early, the AT8\nantibody may have lower sensitivity in immunohistochemistry compared to other\nphospho-tau antibodies, the influence of ApoE4 gene expression and the inherent\nsubstantial variability between different transgenic mouse lines. We have extended the\ndiscussion accordingly in the revised manuscript.\n9. The protective effect of peripheral administration of 25-HC is interesting, particularly\ngiven that there are reports claiming 25-HC is promoting neuroinflammation and AD-\nrelated pathologies (PMID: 32552741; PMID: 40001197).\nResponse: Thank you for your insightful comment. Indeed, 25-HC has been found to\npromoted proinflammatory cytokines production and impair microglial phagocytic\ncapacity29-31. These effects might collectively exacerbate neuroinflammation and\namyloid pathology in AD. In contrast, the effects of 24-HC on neuroinflammation and\nAD appear to be more complex32. 24-HC treatment at 50 μM for 16 h in cultured\nmacrophages increased the TNF-α level33, while upregulation of local 24-HC in the\nbrain of 5xFAD mice via CYP46A1 overexpression was found to ameliorate\nneuroinflammation, amyloidosis and improved cognition functions34. Similar\nprotective effects of 24-HC were also widely observed under treatment of efavirenz, a\npositive allosteric modulator of CYP46A135-37. Moreover, 24-HC prevented tau\nhyperphosphorylation induced by Aβ monomers in h-tau transgenic mice38, and\ncerebrospinal fluid levels of 24-HC was found to negatively correlate with\nneurodegeneration markers in a clinical cohort of AD patients18. The complexity of 24-\nHC actions may be attributed to factors such as its concentration-dependent effects and\nthe duration of exposure.\nCollectively, these findings present compelling evidence for an intriguing\ndichotomy that, despite of high similarity in structure, 24-HC, 25-HC, and potentially\nother hydroxycholesterols may exert divergent regulatory effects on neuroinflammation\nand AD-related phenotypes. The underlying mechanisms responsible for these\nfunctional differences deserve in-depth investigation in future studies. We have\nextended the discussion accordingly in the revised manuscript.\nReferences\n1. Ferguson, E. L. et al. Independent associations of high-density lipoprotein\ncholesterol and triglyceride levels with Alzheimer's disease and related dementias.\nAlzheimers. Dement. 21, e14575 (2025).\n2. Luo, J. et al. Genetic Associations Between Modifiable Risk Factors and\nAlzheimer Disease. JAMA Netw. Open. 6, e2313734 (2023).\n3. Reed, B. et al. Associations between serum cholesterol levels and cerebral\namyloidosis. JAMA Neurol. 71, 195-200 (2014).\n4. Tcw, J. et al. Cholesterol and matrisome pathways dysregulated in astrocytes and\nmicroglia. Cell. 185, 2213-2233 (2022).\n5. Ahmed, H. et al. Brain cholesterol and Alzheimer's disease: challenges and\nopportunities in probe and drug development. Brain. 147, 1622-1635 (2024).\n6. van der Kant, R. et al. Cholesterol Metabolism Is a Druggable Axis that\nIndependently Regulates Tau and Amyloid-beta in iPSC-Derived Alzheimer's\nDisease Neurons. Cell Stem Cell. 24, 363-375 (2019).\n7. Ding, J. et al. APOE4 Exacerbates Cerebral Tau Pathology Through Cholesterol-\nInduced Degradation of Phosphatase in Atherosclerosis. CNS Neurosci. Ther. 31,\ne70536 (2025).\n8. Ren, Z. et al. Increased intestinal bile acid absorption contributes to age-related\ncognitive impairment. Cell Rep. Med., 101543 (2024).\n9. Chen, T. et al. Metabolic phenotyping reveals an emerging role of ammonia\nabnormality in Alzheimer's disease. Nat. Commun. 15, 3796 (2024).\n10. Lutjohann, D. et al. Cholesterol homeostasis in human brain: evidence for an age-\ndependent flux of 24S-hydroxycholesterol from the brain into the circulation. Proc.\nNatl. Acad. Sci. U. S. A. 93, 9799-9804 (1996).\n11. Li, C. et al. Gut microbiome and metabolome profiling in Framingham heart study\nreveals cholesterol-metabolizing bacteria. Cell. 187, 1834-1852 (2024).\n12. Kenny, D. J. et al. Cholesterol Metabolism by Uncultured Human Gut Bacteria\nInfluences Host Cholesterol Level. Cell Host Microbe. 28, 245-257 (2020).\n13. Dai, D. et al. GMrepo v2: a curated human gut microbiome database with special\nfocus on disease markers and cross-dataset comparison. Nucleic. Acids. Res. 50,\nD777-D784 (2022).\n14. Mahalak, K. K. et al. Analysis of the Ability of Capsaicin to Modulate the Human\nGut Microbiota In Vitro. Nutrients. 14, 1283 (2022).\n15. Bu, S., Kar, W., Tucker, R. M. & Comstock, S. S. Minimal Influence of Cayenne\nPepper on the Human Gastrointestinal Microbiota and Intestinal Inflammation in\nHealthy Adult Humans-A Pilot Study. Life-Basel. 12, 1849 (2022).\n16. Zhang, Y. et al. Transmission of Alzheimer's disease-associated microbiota\ndysbiosis and its impact on cognitive function: evidence from mice and patients.\nMol. Psychiatry. 28, 4421-4437 (2023).\n17. Kim, M. S. et al. Transfer of a healthy microbiota reduces amyloid and tau\npathology in an Alzheimer's disease animal model. Gut. 69, 283-294 (2020).\n18. Latorre-Leal, M. et al. CYP46A1-mediated cholesterol turnover induces sex-\nspecific changes in cognition and counteracts memory loss in ovariectomized mice.\nSci. Adv. 10, eadj1354 (2024).\n19. Wei, X., Nishi, T., Kondou, S., Kimura, H. & Mody, I. Preferential enhancement\nof GluN2B-containing native NMDA receptors by the endogenous modulator 24S-\nhydroxycholesterol in hippocampal neurons. Neuropharmacology. 148, 11-20\n(2019).\n20. Paul, S. M. et al. The major brain cholesterol metabolite 24(S)-hydroxycholesterol\nis a potent allosteric modulator of N-methyl-D-aspartate receptors. J. Neurosci. 33,\n17290-17300 (2013).\n21. Oz, M., Lorke, D. E. & Howarth, F. C. Transient receptor potential vanilloid 1\n(TRPV1)-independent actions of capsaicin on cellular excitability and ion transport.\nMed. Res. Rev. 43, 1038-1067 (2023).\n22. Davey, L. E. et al. A genetic system for Akkermansia muciniphila reveals a role\nfor mucin foraging in gut colonization and host sterol biosynthesis gene expression.\nNat. Microbiol. 8, 1450-1467 (2023).\n23. Depommier, C. et al. Supplementation with Akkermansia muciniphila in\noverweight and obese human volunteers: a proof-of-concept exploratory study. Nat.\nMed. 25, 1096-1103 (2019).\n24. Telle-Hansen, V. H. et al. Replacing saturated fatty acids with polyunsaturated\nfatty acids increases the abundance of Lachnospiraceae and is associated with\nreduced total cholesterol levels-a randomized controlled trial in healthy individuals.\nLipids Health Dis. 21, 92 (2022).\n25. Tong, X. et al. Structural Alteration of Gut Microbiota during the Amelioration of\nHuman Type 2 Diabetes with Hyperlipidemia by Metformin and a Traditional\nChinese Herbal Formula: a Multicenter, Randomized, Open Label Clinical Trial.\nMbio. 9, e02317-e02392 (2018).\n26. Wenger, K. et al. Common mouse models of tauopathy reflect early but not late\nhuman disease. Mol. Neurodegener. 18, 10 (2023).\n27. Billings, L. M., Oddo, S., Green, K. N., McGaugh, J. L. & LaFerla, F. M.\nIntraneuronal Abeta causes the onset of early Alzheimer's disease-related cognitive\ndeficits in transgenic mice. Neuron. 45, 675-688 (2005).\n28. Seo, D. et al. ApoE isoform- and microbiota-dependent progression of\nneurodegeneration in a mouse model of tauopathy. Science (New York, N.Y.). 379,\neadd1236 (2023).\n29. Choi, H. et al. 25-Hydroxycholesterol modulates microglial function and\nexacerbates Alzheimer's disease pathology: mechanistic insights and therapeutic\npotential of cholesterol esterification inhibition. J. Neuroinflammation. 22, 50\n(2025).\n30. Romero, J., Toral-Rios, D., Yu, J., Paul, S. M. & Cashikar, A. G. 25-\nhydroxycholesterol promotes brain cytokine production and leukocyte infiltration\nin a mouse model of lipopolysaccharide-induced neuroinflammation. J.\nNeuroinflammation. 21, 251 (2024).\n31. Wong, M. Y. et al. 25-Hydroxycholesterol amplifies microglial IL-1β production\nin an apoE isoform-dependent manner. J. Neuroinflammation. 17, 192 (2020).\n32. Sodero, A. O. 24S-hydroxycholesterol: Cellular effects and variations in brain\ndiseases. J. Neurochem. 157, 899-918 (2021).\n33. Gc, J. B. et al. Molecular basis for the recognition of 24-(S)-hydroxycholesterol by\nintegrin αvβ3. Sci. Rep. 13, 9166 (2023).\n34. Tsitsou-Kampeli, A. et al. Cholesterol 24-hydroxylase at the choroid plexus\ncontributes to brain immune homeostasis. Cell Rep. Med., 101278 (2023).\n35. Mast, N., Li, Y. & Pikuleva, I. A. Increased Acetylcholine Levels and Other Brain\nEffects in 5XFAD Mice after Treatment with 8,14-Dihydroxy Metabolite of\nEfavirenz. Int. J. Mol. Sci. 23, (2022).\n36. Petrov, A. M. et al. CYP46A1 Activation by Efavirenz Leads to Behavioral\nImprovement without Significant Changes in Amyloid Plaque Load in the Brain of\n5XFAD Mice. Neurotherapeutics. 16, 710-724 (2019).\n37. Mast, N. et al. Cholesterol-metabolizing enzyme cytochrome P450 46A1 as a\npharmacologic target for Alzheimer's disease. Neuropharmacology. 123, 465-476\n(2017).\n38. Testa, G. et al. A silver lining for 24-hydroxycholesterol in Alzheimer's disease:\nThe involvement of the neuroprotective enzyme sirtuin 1. Redox Biol. 17, 423-431\n(2018)."}, {"round": 3, "reviewer_comments": {"reviewer_1": "The authors have performed a thorough work to revise the manuscript and it can be ready for publication", "reviewer_2": "In this revision, the authors have made substantial efforts to address the remaining concerns raised during the review\nprocess. Importantly, they have incorporated analyses of publicly available human microbiome datasets, performed GMT\nexperiments, and conducted bacterial isolation and reconstitution studies, which together provide strong support for both\ncausality and translational relevance.\nWhile certain limitations are inherent to the scope of the study, the authors have taken appropriate and reasonable\napproaches to address key mechanistic and translational questions, and the additional experiments significantly strengthen the\nmanuscript. Overall, the authors have responded thoroughly and constructively, and I believe they have done their best to\nresolve the outstanding concerns. I do not have further major concerns regarding publication at this stage.", "reviewer_4": "Authors well addressed to all my points with impressive additional experiments. Interesting study.\n*Regarding P301S mouse experiments, I did not meant to request further analysis, rather expected to remove the\nexperiments from the manuscript or move to the supplementary section."}, "author_response": "Point-by-point responses\nReviewer #1:\nComment: The authors have performed a thorough work to revise the manuscript and\nit can be ready for publication\nResponse: We are grateful for the insightful feedback provided by the reviewers. Their\ncomments have been instrumental in greatly enhancing the manuscript.\nReviewer #2:\nComment: In this revision, the authors have made substantial efforts to address the\nremaining concerns raised during the review process. Importantly, they have\nincorporated analyses of publicly available human microbiome datasets, performed\nGMT experiments, and conducted bacterial isolation and reconstitution studies, which\ntogether provide strong support for both causality and translational relevance.\nWhile certain limitations are inherent to the scope of the study, the authors have taken\nappropriate and reasonable approaches to address key mechanistic and translational\nquestions, and the additional experiments significantly strengthen the manuscript.\nOverall, the authors have responded thoroughly and constructively, and I believe they\nhave done their best to resolve the outstanding concerns. I do not have further major\nconcerns regarding publication at this stage.\nResponse: We appreciate your careful reading of our paper. Your expert guidance has\nbeen invaluable in refining our work.\nReviewer #4:\nComment: Authors well addressed to all my points with impressive additional\nexperiments. Interesting study.\n*Regarding P301S mouse experiments, I did not meant to request further analysis,\nrather expected to remove the experiments from the manuscript or move to the\nsupplementary section.\nResponse: We are deeply grateful for your insightful comments and your significant\neffort invested in evaluating our manuscript. We have moved the results of P301S\nmouse experiments to the supplementary material."}]

Decision letter