Antibiotic-Induced Gut Dysbiosis Modulates Alzheimer's Disease-Associated Gene Expression and Protein Aggregation in 3xTg-AD Mice via the Gut-Brain Axis

Abstract

Introduction: Alzheimer's disease (AD) is a progressive neurodegenerative disorder that poses a major global health challenge due to its increasing prevalence and lack of effective treatments. Emerging evidence suggests the gut-brain axis may play a pivotal role in AD pathogenesis. However, causal links between dysbiosis and late-stage AD pathology remain unclear.

Methods: This study evaluated the effects of antibiotic-induced gut dysbiosis in aged 3xTg-AD mice (46-48 weeks). Female mice were randomly assigned to control or treatment groups and administered a broad-spectrum antibiotic cocktail (ampicillin, vancomycin, and neomycin) for 14 days. Behavioral tests (Y-maze, elevated plus maze) were performed to assess cognitive and anxiety-like behaviors. Gut microbiota composition was assessed via 16S rRNA qPCR. Gene expression of Acetylcholinesterase (AChE), Butyrylcholinesterase (BChE), and Tumor Necrosis Factor-Alpha (TNF-α) was analyzed via qRT-PCR, and cerebral amyloid-β_1-42 and tau protein levels were quantified by ELISA.

Results: Antibiotic treatment induced significant dysbiosis, with > 90% reduction in Firmicutes and Bacteroidetes. Dysbiotic mice displayed impaired spatial working memory, heightened anxiety-like behavior, and reduced locomotor activity. Molecular analyses revealed region-specific dysregulation of cholinergic genes: AChE was upregulated in the hippocampus but downregulated in the cortex, while BChE showed the opposite trend. TNF-α was significantly elevated in both regions, indicating neuroinflammation. Dysbiosis also led to increased brain levels of amyloid-β_1-42 and tau.

Conclusion: Gut microbiome disruption exacerbates late-stage AD pathology, driving cognitive deficits, neuroinflammation, and hallmark protein aggregation. These findings support the gut-brain axis as a critical modulator of AD and highlight the microbiome as a potential therapeutic target.

Keywords: 3xTg‐AD mice; cholinergic dysfunction; gut–brain axis; neurodegenerative.

FIGURE 1

Illustrates cognitive and anxiety‐related behavioral impairments in dysbiotic mice. (A) Antibiotic treatment significantly impairs spontaneous alternation behavior in the Y‐maze test compared to controls. Dysbiotic mice show reduced spatial working memory, with data presented as mean ± SEM. Statistical analysis (unpaired t‐test, n = 5 per group) reveals a highly significant difference (p < 0.0001). (B) Assessment of anxiety‐related behavior on the Elevated Plus Maze shows that dysbiotic animals spend significantly less time exploring open arms compared to controls, indicating increased anxiety‐like behavior (n = 5 per group; p < 0.001). (C) Dysbiotic mice demonstrate a marked preference for closed arms, spending more time in protected areas than controls, further supporting an anxiety‐like phenotype (n = 5 per group; p < 0.0002).

FIGURE 2

Demonstrates reduced locomotor activity and exploration in dysbiotic mice on the Elevated Plus Maze. (A) Total distance traveled on the Elevated Plus Maze. Control mice showed significantly greater locomotor activity, covering about four times the distance of dysbiotic mice during the 5‐min test (n = 5/group; mean ± SEM; unpaired t‐test, p < 0.0001). (B) Representative exploration pattern of the control group. Control animals displayed distributed movement throughout all maze compartments, indicating normal exploratory behavior. (C) Exploration pattern of the dysbiotic group. Dysbiotic mice exhibited restricted movement and reduced spatial investigation, reflecting behavioral changes due to gut dysbiosis. (D) Spatial exploration patterns across EPM areas. Dysbiotic mice consistently explored less across all maze regions compared to controls, demonstrating a pronounced anxiety‐like behavioral phenotype (n = 5/group; p = 0.0001).

FIGURE 3

Shows region‐specific cholinergic and inflammatory gene dysregulation in dysbiotic mice. (A) Region‐specific regulation of AChE. Dysbiotic mice exhibit significant downregulation of cortical AChE, while hippocampal AChE is strongly upregulated, implying distinct compensatory responses to gut microbiome disruption. (B) Reciprocal regulation of BChE. Cortical BChE is markedly upregulated, whereas hippocampal BChE is downregulated, highlighting selective cholinergic remodeling after dysbiosis. (C) TNF‐α upregulation. Dysbiotic animals show a 3.1‐fold increase in cortical TNF‐α and an even greater 6.8‐fold increase in hippocampal TNF‐α, indicating heightened susceptibility of the hippocampus to dysbiosis‐induced neuroinflammation.

FIGURE 4

Demonstrates increased amyloid‐β and phosphorylated tau levels in dysbiotic mice. (A) Antibiotic treatment results in a modest but statistically significant increase in amyloid‐β_1–42 (Aβ_1–42) levels compared to controls (n = 5/group), as measured by ELISA (absorbance at 450 nm; mean ± SEM; unpaired t‐test, p = 0.0332). (B) Antibiotic treatment significantly elevates phosphorylated tau (pTau) levels relative to controls (n = 5/group), as measured by ELISA (absorbance at 450 nm; mean ± SEM; unpaired t‐test, p < 0.0001).