Xiaoli Fang, Sha Liu, Bilal Muhammad, Mingxuan Zheng, Xing Ge, Yan Xu, Shu Kan, Yang Zhang, Yinghua Yu,Kuiyang Zheng, Deqin Geng,*, Chun-Feng Liu

Abstract Parkinson’s disease is a neurodegenerative disease characterized by motor and gastrointestinal dysfunction.Gastrointestinal dysfunction can precede the onset of motor symptoms by several years.Gut microbiota dysbiosis is involved in the pathogenesis of Parkinson’s disease, whether it plays a causal role in motor dysfunction, and the mechanism underlying this potential effect, remain unknown.CCAAT/enhancer binding protein β/asparagine endopeptidase (C/EBPβ/AEP) signaling, activated by bacterial endotoxin, can promote α-synuclein transcription, thereby contributing to Parkinson’s disease pathology.In this study,we aimed to investigate the role of the gut microbiota in C/EBPβ/AEP signaling, α-synuclein-related pathology, and motor symptoms using a rotenone-induced mouse model of Parkinson’s disease combined with antibiotic-induced microbiome depletion and fecal microbiota transplantation.We found that rotenone administration resulted in gut microbiota dysbiosis and perturbation of the intestinal barrier, as well as activation of the C/EBP/AEP pathway, α-synuclein aggregation, and tyrosine hydroxylase-positive neuron loss in the substantia nigra in mice with motor deficits.However, treatment with rotenone did not have any of these adverse effects in mice whose gut microbiota was depleted by pretreatment with antibiotics.Importantly, we found that transplanting gut microbiota derived from mice treated with rotenone induced motor deficits, intestinal inflammation, and endotoxemia.Transplantation of fecal microbiota from healthy control mice alleviated rotenone-induced motor deficits, intestinal inflammation, endotoxemia, and intestinal barrier impairment.These results highlight the vital role that gut microbiota dysbiosis plays in inducing motor deficits, C/EBPβ/AEP signaling activation, and α-synuclein-related pathology in a rotenone-induced mouse model of Parkinson’s disease.Additionally, our findings suggest that supplementing with healthy microbiota may be a safe and effective treatment that could help ameliorate the progression of motor deficits in patients with Parkinson’s disease.

Key Words: C/EBP/AEP signaling pathway; endotoxemia; fecal microbiota transplantation; intestinal barrier; intestinal inflammation; microbiota-gut-brain axis;Parkinson’s disease

Introduction

Parkinson’s disease (PD) is characterized by motor and gastrointestinal(GI) dysfunction, which result from α-synuclein (α-syn) aggregation and dopaminergic (DA) neuron loss within the substantia nigra (SN) (Martinez-Martin, 2011; Claudino Dos Santos et al., 2023).GI deficits, such as gastroparesis and constipation, have been reported to precede the onset of motor symptoms by several years and affect approximately 60–80% of PD patients (Bhattarai et al., 2021).The high prevalence and early onset of GI deficits in PD have led to the proposal of several mechanisms underlying the development of PD, including gut microbiota dysbiosis, disruption of intestinal permeability, and inflammation (Bhattarai and Kashyap, 2020).The bidirectional communication that takes place between the gut microbiota and the central nervous system is known as the gut-brain axis (Morais et al.,2021).Dysbiosis of the gut microbiota triggers chronic inflammation in the intestinal epithelium, disrupting the integrity of the intestinal barrier (Padhi et al., 2022).Pro-inflammatory microbial products, such as lipopolysaccharide(LPS) and cytokines, induce systemic inflammation and disrupt the blood-brain barrier (Buford, 2017).As a result, neuroinflammation and cell death of DA neurons may occur in the SN, inducing motor deficits (Sun and Shen, 2018).Alterations in the abundance of many microbial taxa have been reported in patients with PD (Liu et al., 2021).In addition, the status of the gut microbiota has been correlated with PD clinical phenotype, duration, and severity.However, it remains unclear whether alterations in the gut microbiota cause gut-brain axis damage and motor dysfunction in patients with PD.

Mammalian asparagine endopeptidase (AEP), also known as legumain, is a cysteine protease that is highly activated in the brains of patients with PD (Zhang et al., 2017).AEP cleaves α-syn in the PD brain and promotes its aggregation and neurotoxicity, contributing to DA neuron loss and motor deficits.CCAAT/enhancer binding protein β (C/EBPβ), an inflammationregulated transcription factor, regulates AEP mRNA transcription and protein levels in the brain.Additionally, C/EBPβ acts as a transcription factor to upregulate α-syn and monoamine oxidase B (MAOB), both of which have been implicated in PD pathogenesis (Wu et al., 2021).C/EBPβ has been reported to be activated by LPS and inflammatory cytokines (interleukin [IL]-1β, IL-6, and tumor necrosis factor [TNF]-α) (Magalini et al., 1995; Ejarque-Ortiz et al., 2007).Therefore, gut microbiota dysbiosis and inflammation activation contribute to PD pathology through the C/EBPβ/AEP signaling pathway.

Thus, we hypothesized that gut microbiota dysbiosis, along with leaky gutinduced bacterial endotoxins, activates C/EBPβ/AEP signaling and α-syn pathology, ultimately leading to neurodegeneration in PD.To test this hypothesis, we used a rotenone-induced PD mouse model, combined with antibiotic-induced microbiome depletion and fecal microbiota transplantation,to investigate the role of the gut microbiota in C/EBPβ/AEP signaling, α-synrelated pathology, and motor symptoms.

Methods

Animal and experimental design

Female C57BL/6 mice are characterized by pronounced cyclical fluctuations in physiological indicators and volatile hormone levels (ter Horst et al., 2012;Szoeke et al., 2021), and were thus excluded from the present study.Ninetysix specific pathogen-free-grade male C57BL/6J mice (weighing 20–23 g and aged 7 weeks) were obtained from Nanjing Laboratory Animal Research Center and housed in the Experimental Animal Center of Xuzhou Medical University(license No.SCXK (Su) 2015-0009).All mice were housed four to five to a cage in a climate-controlled room at 24°C with a 12/12-hour dark/light cycle and hadad libitumaccess to food and water.All experiments were carried out in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals (8thed., National Research Council, 2011) and were approved by the Ethics Committee of Laboratory Animals of Xuzhou Medical University (202111A303) on November 16, 2021.In addition, all experiments were designed and reported according to the Animal Research: Reporting ofIn VivoExperiments (ARRIVE) guidelines (Percie du Sert et al., 2020).

Experiment 1: After 1 week of habituation to the laboratory environment, the mice were matched by body weight and randomly divided into two groups:(1) mice in the control (Con) group received vehicle (n= 14), and (2) mice in the rotenone (Rot) group (n= 14) received rotenone (10 mg/kg) daily for 6 weeks by intragastric administration.Both groups underwent interventions for 6 weeks, followed by motor behavior tests (rotarod tests and pole tests)at 7 weeks.At 8 weeks, the mice were euthanized with 0.9% pentobarbitone sodium (FWD Chemicals Ltd., Shanghai, China) (100 mg/kg body weight) via intraperitoneal injection.Blood, colon, colon contents, and brain tissue were collected and stored at –80°C for further analysis (Additional Figure 1).

Experiment 2: After 1 week of habituation to the laboratory environment,the mice were matched by body weight and randomly divided into two groups: (1) Con mice treated with antibiotics (Con + Ab) (n= 10), and (2) Rot mice treated with antibiotics (Rot + Ab) (n= 10).The antibiotics selected and the dosages used were based on a previous study, and included neomycin(1 g/L), ampicillin (1 g/L), metronidazole (1 g/L), and vancomycin (0.25 g/L)(all purchased from MedChemExpress, Monmouth Junction, NJ, USA) added to the drinking water and renewed three times per week for 6 weeks (Zou et al., 2018).Both groups underwent interventions for 6 weeks, followed by motor behavior tests at 7 weeks.At 8 weeks, the mice were euthanized with pentobarbitone sodium (100 mg/kg body weight) via intraperitoneal injection.Colonic length was measured, and blood, colon, colon contents, and brain tissue were collected and stored at –80°C for further analysis (Additional Figure 2).

Experiment 3: After 1 week of habituation to the laboratory environment, the mice were matched by body weight and randomly divided into two groups: (1)Con mice transplanted with microbiota from Con mice (Con + FMTCon) (n= 10),and (2) Con mice transplanted with microbiota from Rot mice (Con + FMTRot;n= 10).Fecal bacteria transplantation was performed as follows: fresh feces from Rot mice were collected from the disinfected anus and placed into new sterile tubes daily to maintain microbial viability.The Con + FMTRotgroup received daily oral gavage for 4 weeks to achieve widespread colonization with the fecal flora.The fecal microbiome was then allowed to colonize in extenso for 2 weeks, and the motor behavior tests were performed at 7 weeks.At 8 weeks, the mice were euthanized with pentobarbitone sodium (100 mg/kg body weight) via intraperitoneal injection.Blood, colon, colon contents, and brain tissue were collected and stored at –80°C for further analysis (Additional Figure 3).

Experiment 4: After 1 week of habituation to the laboratory environment for,the mice were matched by body weight and randomly divided into two groups:(1) Rot mice treated with phosphate-buffered saline (PBS) (0.1 mL/kg/d;Rot + PBS;n= 14), and (2) Rot mice transplanted with microbiota from donor Con mice (Rot + FMTCon;n= 14).Fecal bacteria transplantation was performed as follows: fresh feces from Con mice were collected from the disinfected anus and placed into new sterile tubes daily to maintain microbial viability.Both groups received daily oral gavage for 4 weeks.The fecal microbiome was then allowed to colonize in extension for 2 weeks, and the motor behavior tests were performed at 7 weeks.At 8 weeks the mice were euthanized with pentobarbitone sodium (100 mg/kg body weight) via intraperitoneal injection.Colonic length was measured, and blood, colon, colon contents, and brain tissue were collected and stored at –80°C for further analysis (Additional Figure 4).

Oral gavage

The rotenone solution was prepared by dissolving 10 mg/kg of rotenone(Apexbio, Houston, TX, USA, Cat# B5462) in a vehicle solution containing 4%carboxymethylcellulose (Solarbio, Beijing, China, Cat# IS9000) and 1.25%chloroform.Age- and weight-matched C57BL/6J mice were randomly assigned to receive daily gavage with either freshly prepared rotenone solution(rotenone group) or vehicle solution (control group) for 6 weeks.

Fecal microbiota transplantation

Fecal microbiota transplantation was performed as previously described(Bruce-Keller et al., 2015; Olson et al., 2018; Li et al., 2020).Feces (collected from mice in Experiment 1 after 6 weeks) were rehydrated by soaking in sterile PBS for 15 minutes, followed by thorough homogenization.The resulting mixture was filtered through a 70-μm pore size filter, and the filtrate was centrifuged at 1000 ×gfor 5 minutes.The resulting pellet was resuspended and centrifuged again, resulting in the final bacterial suspension.This suspension was then diluted with an equal volume of sterile PBS to achieve a concentration of approximately 1 × 1011CFU/L of flora.The recipient mice received the bacterial suspension at a dose of 10 mL/kg by oral gavage every day for 4 weeks.

Rotarod test

The motor activity of the mice was assessed by rotarod test.An automated rotarod machine (Anhui Zhenghua Co., Huaibei, China, ZH-600B) equipped with timers and falling sensors was utilized (Liu et al., 2018; McKinley et al., 2019; Zhao et al., 2021).The mice were placed on a 3-cm-diameter cylinder and allowed to adapt by remaining on the stationary cylinder for 5 minutes during the trial sessions.This adaptation period was repeated for 3 consecutive days to allow the mice to become familiar with the rotarod apparatus.Then, the animals were adapted to the rotarod motion by rotating it slowly (10 r/min) without acceleration.Whenever the animal fell from the cylinder, it was promptly placed back on the apparatus, up to five times.After 3 days of repeated training on the slowly spinning apparatus, the experiment was conducted.During the experiment, the rotation speed with accelerated from 4 to 20 r/min over a period of 1 minute, and the latency to fall was automatically recorded by the sensors.

Pole test

The pole climbing test, another classical method for evaluating the motor coordination of mice (Liu et al., 2018; Zhao et al., 2021), was also carried out.Locomotion was assessed by measuring the time it took for the mice to descend from the top to the bottom of the pole, with both forepaws touching the ground.The pole used in this experiment was constructed in-house and consisted of a 75-cm-tall metal rod with a diameter of 9 mm, wrapped with gauze.The mice were positioned near the top of the pole, approximately 7.5 cm from the top, facing upwards.The time taken to reach the base of the pole was recorded.Prior to the actual test, the mice underwent 2 consecutive days of training.On the day of experiment, each mouse was tested twice, and the average time needed to fully descend was calculated.

Gut microbiota analysis

DNA extraction, polymerase chain reaction (PCR) amplification, and 16S rRNA gene sequencing were performed as follows.After 8 weeks, the cecum contents of the mice were collected.Microbial DNA was extracted using a HiPure Stool DNA Kit (Magen, Guangzhou, China) following the manufacturer’s recommended protocol.The V3–V4 region of 16s rRNA genes was PCR-amplified using the primers 341-F, 5′-CCT ACG GGN GGC WGC AG-3′ and 806-R, 5′-GGA CTA CHV GGG TAT CTA AT-3′ (Guo et al., 2017).The amplification procedure consisted of an initial denaturation step at 94°C for 2 minutes, followed by denaturation at 98°C for 10 seconds, annealing at 65°C for 30 seconds, and extension at 68°C for 30 seconds.These steps were repeated for 30 cycles, with a final extension at 68°C for 5 minutes.PCR reactions were performed in triplicate, and the reaction mix comprised 5 μL of 10 × KOD buffer, 5 μL of 2 mM dNTPs, 3 μL of 25 mM MgSO4, 1.5 μL of each primer (10 mM), 1 μL of KOD polymerase, and 100 ng of template DNA, with a final volume of 50 μL.Following amplification, the products were purified using an AxyPrep DNA Gel Extraction Kit (Axygen Biosciences, Union City, CA, USA) and quantified using an ABI StepOnePlus Real-Time PCR System(Life Technologies, Foster City, USA).The purified products were then pooled in equimolar ratios and subjected to paired-end sequencing (PE250) on an Illumina platform according to standard protocols.

Sequence data processing: High-quality clean reads were obtained as follows.Raw reads containing more than 10% unknown nucleotides (N) and reads with less than 60% of bases with a quality value (Q-value) greater than 20 were removed using FASTP (version 0.18.0; https://github.com/OpenGene/fastp;Chen et al., 2018).Paired-end clean reads were merged as raw tags using FLSAH (version 1.2.11; http://www.cbcb.umd.edu/software/flash; Magoc and Salzberg, 2011, requiring a minimum overlap of 10 bp and allowing a mismatch error rate of 2%.Noisy raw tag sequences were filtered using the QIIME (version 1.9.1; http://qiime.sourceforge.net/; Caporaso et al., 2010)pipeline with specific filtering conditions (Bokulich et al., 2013) to obtain highquality clean tags.The filtering conditions involved breaking raw tags from the first low-quality base site, where the number of bases in the continuous lowquality value reached the set length, and then filtering tags with a continuous high-quality base length of less than 75% of the tag length.The clean tags were checked for chimeras using the UCHIME algorithm against the reference database (http://drive5.com/uchime/uchime_download.html).After removing chimeric tags, the final tag set was used for further analysis.

Bioinformatic analysis: The clean tags were clustered into operational taxonomic units (OTUs) with a similarity of 97% or greater using the UPARSE(version 9.2.64; http://drive5.com/uparse/) pipeline (Edgar, 2013).Next,α-diversity indices, which assess gut microbial community richness (the Ace and Chao1 estimators) and community diversity (the Shannon estimator),were calculated using Mothur (https://www.mothur.org; Schloss et al.,2009).Principal coordinate analysis (PCoA) based on Bray-Curtis distance and permutational multivariate analysis of variance (PERMANOVA) were performed to compare overall microbiota composition after the intervention in each group at the phylum, genus, and OTU levels.Differences in taxon abundance were detected using the linear discriminant analysis (LDA) effect size (LEfSe) algorithm, which focuses on statistical significance, biological consistency, and effect relevance (Segata et al., 2011).Differences with log 10 LDA scores (absolute values) greater than 3.0 andP-values less than 0.05 were considered significant (Sung et al., 2020).The functional potential of the gut microbial communities was estimated using the PICRUSt12 algorithm (Langille et al., 2013).In the univariate analysis of gut microbiota and predicted Kyoto Encyclopedia of Genes and Genomes (KEGG) biochemical pathways in each group, a pairedt-test or Wilcoxon matched-pairs test was applied, andP-values were adjusted for multiple comparisons using the Benjamini-Hochberg false discovery rate.

Lipopolysaccharide determination

Endotoxin concentrations in mouse serum were quantified using a chromogenic endpoint Tachypleus Amebocyte Lysate kit (Xiamen Bioendo Technology Co., Ltd., Xiamen, China) according to the manufacturer’s instructions.The absorbance was measured at 545 nm using a spectrophotometer (Bio-Tek Synergy2, Vermont, USA), and the measurable concentrations ranged from 0.1 to 1.0 EU/mL.All LPS measurements were performed in duplicate.

Measuring the thickness of the colonic mucus layer

Colon samples were fixed in Carnoy’s solution and stored in methanol.Samples were subsequently washed twice in PBS, dehydrated, and then embedded in paraffin for histological analysis.Sections (5 μm) were obtained from paraffin blocks and placed on glass slides.Alcian blue (Abcam,Cambridge, UK, Cat# ab150662) staining was performed according to established protocols (Desai et al., 2016).The thickness of the colonic mucus was measured using ImageJ (version 1.53T; https://imagej.nih.gov/ij/; 10 measurements per section/two sections per animal/three animals per group).The sections were cross-validated with primary antibody anti-mucin-2 (MUC2)(rabbit, 1:500, Abclonal, Cat# A14659) staining at 4°C overnight, followed by incubation with goat-anti-rabbit IgG H&L/AF594 antibody (goat, 1:1000, Bioss,Beijng, China, Cat# bs-0295G-AF594) for 1 hour at room temperature.Finally,the sections were counterstained with DAPI (Sigma-Aldrich, St.Louis, MO,USA, Cat# D9542).

Western blot analysis and immunofluorescence staining

Western blot analysis was performed as we described earlier (Shi et al.,2021; Yang et al., 2021).Briefly, total protein was extracted from colon and brain tissues, and the protein concentration was measured using a BCA protein assay kit (Beyotime Biotechnology, Shanghai, China, Cat#P0010).Subsequently, 25 μg of protein was separated on 7.5% or 10%SDS-polyacrylamide gels and transferred onto polyvinylidene difluoride membranes (Millipore, Burlington, MA, USA, Cat# ISEQ00010).The membranes were then blocked with 5% freshly prepared milk in TBST for 2 hours at room temperature.Next, the membranes were incubated overnight at 4°C with the following primary antibodies: anti-C/EBPβ (rabbit, 1:1000,Abcam, Cat# ab32358, RRID: AB_726796), anti-AEP (rabbit, 1:10,000, Abcam,Cat# ab183028), anti-pS129 α-syn (rabbit, 1:400, Abcam, Cat# ab51253), anti-MAOB (rabbit, 1:1000, Abcam, Cat# ab259928), anti-Occludin (rabbit, 1:1000,Abcam, Cat# ab167161, RRID: AB_2756463), anti-zonula occludens-1 (ZO-1; rabbit, 1:5000, Proteintech, Cat# 21773-1-AP, RRID: AB_10733242), and anti-β-actin (rabbit, 1:50,000, ABclonal, Cat# AC026, RRID: AB_2768234).Afterward, the membranes were washed three times for 10 minutes with TBST and incubated with HRP-conjugated anti-rabbit IgG secondary antibody(goat, 1:10,000, ABclonal, Cat# AS014, RRID: AB_2769854) or HRP-conjugated anti-mouse IgG secondary antibody (goat, 1:10,000, ABclonal Cat# AS003,RRID: AB_2769851) at room temperature for 1 hour.Finally, the protein bands were visualized using enhanced chemiluminescence (Biosharp, Hefei, China,Cat# BL520B), and images were acquired with a ChemiDoc MP System (Bio-Rad, Hercules, CA, USA).Immunoreactive bands were quantified using Image Lab software (version 5.2, Bio-Rad), and the protein concentrations were normalized to β-actin.

Immunohistochemical staining was used to detect the characteristic pathological changes in the SN of midbrain in mice.Anti-pS129 α-syn (rabbit,1:400, Abcam, Cat# ab51253), anti-tyrosine hydroxylase (TH; mouse, 1:400,Santa Cruz Biotechnology, Santa Cruz, CA, USA, Cat# sc-25269).

Quantitative reverse transcription-polymerase chain reaction

Total RNA was extracted from colon tissues using RNA Isolator Total RNA Extraction Reagent (Vazyme, Nanjing, China, Cat# R401).The extracted RNA was then reverse-transcribed to cDNA using a HiScript II Q RT SuperMix for qPCR (+gDNA wiper) kit (Vazyme, Cat# R223).For quantitative reverse transcription-PCR (qRT-PCR), ChamQ Universal SYBR qPCR Master Mix(Vazyme, Cat# Q712) was used, and the reactions were performed on a real-time PCR detection system (Roche LightCycler480, Penzberg, Germany).The qRT-PCR conditions were as follows: 95°C for 5 minutes of denaturation,followed by 45 cycles of 95°C for 30 seconds, 60°C for 30 seconds, and 72°C for 30 seconds.The mRNA levels for specific genes were determined using the 2(–ΔΔCt) method and normalized to β-actin mRNA levels.The primer sequences used for the qRT-PCR reactions are listed in Additional Table 1.

Statistical analysis

No statistical methods were used to predetermine sample sizes; however, our sample sizes are similar to those reported in previous publications (Sampson et al., 2016; Zhao et al., 2021).Data collection and analysis were performed in a blinded manner.The data were analyzed using the statistical package SPSS(Version 24.0).Data distribution normality was assessed using the Shapiro-Wilk normality test.Student’st-test was employed to compare two groups after confirming the normality of the data distribution.Spearman’s correlation analysis was performed to assess the relationship between specific gut microbiota components and variables such as colonic length, ZO-1 expression level, occludin expression level, colonic mucus thickness, serum LPS level,CEBP/β expression level, AEP expression level, p-α-syn expression level, MAOB expression level, and behavior test results.Statistical significance was set atP< 0.05.The results are presented as the mean ± standard error of mean.

Results

Rotenone administration results in gut microbiota dysbiosis

Alterations in the fecal microbiota have been reported to be associated with PD phenotypes and clinical symptoms in clinical studies (Scheperjans et al.,2015).In this study, we established a mouse model of PD with rotenone and examined the cecal microbiota by 16S rRNA sequencing.α-Diversity analysis showed no significant difference in richness and diversity between the Rot group and the Con group (P> 0.05; Additional Figure 5A–F).However, betadiversity analysis, as assess by PCoA, demonstrated a significant separation of the gut microbiota community in the Rot group from that in the Con group (Figure 1A).The relative abundances of phyla were altered in the Rot group, with an increase in Firmicutes, Patescibacteria, and Cyanobacteria,and a decrease in Actinobacteria, Proteobacteria, and Verrucomicrobia(Figure 1B and C).Furthermore, at the family level, the Rot group showed significantly increased abundance of Lachnospiraceae, Ruminococcaceae,and Saccharimonadaceae, while Bifidobacteriaceae, Erysipelotrichaceae,Desulfovibrionaceae, and Atopobiaceae were significantly reduced compared with the Con group (P< 0.05; Figure 1D and E).At the genus level, rotenone administration increased the relative abundance of the bacterial genus Lachnospiraceae_NK4A136_group and decreased Turicibacter, Faecalibacterium, Dubosiella, Bifidobacterium, and Desulfovibrio(Figure 1F and G).Furthermore, the LEfSe results indicated that bacteria belonging to class Clostridia, order Clostridiales, family Lachnospiraceae,phylum Firmicutes, genus Eubacterium_xylanophilum_group, and genus Eubacterium_coprostanoligenes_group were differentially enriched in the gut bacterial communities (LDA score > 4) of the Rot and Con groups (Figure 1H).Additionally, using KEGG annotation and functional enrichment, we identified 12 functional categories that exhibited different enrichment levels between the Rot and Con groups.These categories included carbohydrate metabolism,amino acid metabolism, metabolism of cofactors and vitamins, lipid metabolism, glycan biosynthesis and metabolism, nucleotide metabolism,replication and repair, transcription and folding, sorting, and degradation pathway, all of which were significantly upregulated in Rot mice compared with Con mice (Additional Figure 5G).

Rotenone administration disrupts the intestinal barrier in a gut microbiotadependent manner

Given that rotenone appeared to alter the gut microbiota, we next asked whether rotenone also perturbed intestinal homeostasis, specifically intestinal barrier integrity, permeability, and inflammation.Our findings showed that rotenone administration resulted in a significant reduction in colonic mucus thickness compared with the Con group (P< 0.001; Figure 2A), as demonstrated by Alcian blue staining (Figure 2A and B) and MUC2 immunofluorescence staining (Figure 2C) of colon tissue.Additionally,rotenone administration significantly decreased the expression levels of tight junction proteins, namely ZO-1 and occludin, in the colon (bothP< 0.05,Figure 2D).Consequently, rotenone administration resulted in elevated serum LPS levels, suggesting that rotenone disrupted the integrity of the gut barrier and increased gut permeability to endotoxins (Figure 2E).Furthermore, we observed higher IL-6, TNF-α, and inducible nitric oxide synthase (iNOS) mRNA levels in the colon tissue of mice in the Rot group compared with the Con group (P< 0.05; Figure 2F–H), indicating increased intestinal inflammation.Consistently, we found that a shorter colon length was associated with intestinal inflammation in response to rotenone administration (Figure 2I).

To investigate the role of the gut microbiota in rotenone-induced intestinal barrier disruption and intestinal inflammation, we used a cocktail of oral antibiotics to deplete the effects of the gut microbiota.Interestingly, we found no significant difference in colonic mucus thickness or inMUC2 immunofluorescence staining in the colon between the Con + Ab group and the Rot + Ab group (P> 0.05; Figure 2J–L).Moreover, there were no significant differences between the Con + Ab group and the Rot + Ab group in terms of serum LPS levels, the expression levels of tight junction proteins (ZO-1 and occludin), mRNA expression levels of inflammatory factor (IL-6, TNF-α,and iNOS), and colon length (P> 0.05, Figure 2M–R).Taken together, these findings suggest that the disruption of intestinal barrier integrity and intestinal inflammation that we observed following chronic rotenone administration were dependent on the presence of the gut microbiota.

Figure 1 ｜Rotenone administration results in the microbiota dysbiosis.(A) Principal coordinate analysis plot of weighted UniFrac distances.(B) Composition of the microbiota at the phylum level.(C) Comparison of representative taxonomic abundance at the phylum level.(D) Composition of the microbiota at the family level.(E)Comparison of representative taxonomic abundance at the family level.(F) Composition of the microbiota at the genus level.(G) Comparison of representative taxonomic abundance at the genus level.(H) Linear discriminant analysis (LDA) effect size (LDA >4.0) showing the most abundant taxa that were significantly differentially enriched in the microbiota from Con and Rot mice.Values are presented as mean ± standard error of the mean (n = 6).*P < 0.05, **P < 0.01, vs. Con.Con: control; Rot: rotenone.c: Class; f:family; g: genus; o: order; p: phylum.

Mice treated with rotenone exhibit C/EBP/AEP pathway activation,α-syn aggregation, and tyrosine hydroxylase-positive neuron loss in the substantia nigra, and these effects are dependent on the gut microbiota

It has been reported that C/EBPβ is activated by LPS (Ejarque-Ortiz et al.,2007) and pro-inflammatory cytokines (Cox et al., 2013; Pulido-Salgado et al.,2015).Given our finding that LPS and inflammatory cytokine levels increased after rotenone administration, we assessed the expression levels of C/EBPβ/AEP signaling molecules in the SN of mice treated with rotenone by western blot.Both C/EBPβ and AEP levels were upregulated in the SN of the Rot group compared with the Con group (P< 0.05; Figure 3A and B).This upregulation in C/EBPβ/AEP signaling was gut microbiota-dependent, since the levels of C/EBPβ and AEP did not differ between the Rot + Ab and Con + Ab groups (Figure 3E and F).Previously, it was reported that the C/EBPβ/AEP signaling pathway regulates transcription and proteolytic cleavage of α-syn and MAOB (Wu et al.,2021).In this study, we found that both α-syn and MAOB protein levels were significantly increased in the Rot group compared with the Con group (P< 0.05,Figure 3C and D), but not in the Rot + Ab and Con + Ab groups (Figure 3G and H).Immunofluorescence staining revealed abnormal α-syn aggregation around tyrosine hydroxylase-positive neurons along with decreased α-syn expression in the SN in the Rot group (Figure 3I), but not in the Rot + Ab group (Figure 3J).As a consequence, mice in the Rot group exhibited motor impairment, a main symptom of PD, as assessed by the rotarod test and pole test (Figure 3K and L).However, this rotenone-induced motor impairment was eliminated by using antibiotics to ablate the gut microbiota (Figure 3M and N).Taken together, these findings suggest that the gut microbiota plays an important role in triggering the CEBP/β-AEP signaling pathway to regulate transcription and proteolytic cleavage of α-syn and MAOB, thereby leading to PD pathogenesis.Spearman’s correlation analysis was used to investigate the relationship between gut bacterial abundance and colonic mucosa thickness, serum LPS, the C/EBPβ/AEP signaling pathway, and motor behavior(Figure 4).We found that Firmicutes abundance positively correlated with motor deficits (r= 0.6979,P< 0.05) and negatively correlated with colonic mucosa thickness (r= –0.8191,P< 0.01) and ZO-1 (r= –5975,P< 0.05).Actinobacteria abundance positively correlated with colonic mucosa thickness(r= 0.7951,P< 0.01).Proteobacteria abundance negatively correlated with motor deficits (r= –0.6594,P< 0.05).Cyanobacteria abundance negatively correlated with motor deficits (r= –0.639,P< 0.05) and ZO-1 (r= –0.6038,P< 0.05) and positively correlated with serum LPS levels (r= 0.6966,P< 0.05).Lachnospiraceae abundance positively correlated with AEP expression level (r= 0.6622,P< 0.05) and negatively correlated with colonic mucosa thickness(r= –0.7957,P< 0.01).Ruminococcaceae abundance positively correlated with motor deficits (r= 0.6976,P< 0.05) and serum LPS levels (r= 0.7899,P< 0.01) and negatively correlated with colonic mucosa thickness (r= –0.7227,P< 0.05).Saccharimonadaceae abundance positively correlated with AEP expression level (r= 0.7972,P< 0.05) and negatively correlated with colonic length (r= –0.9595,P< 0.01).Erysipelotrichaceae abundance negatively correlated with motor deficits (r= –0.6458,P< 0.05) and α-syn expression levels (r= –0.7930,P< 0.01) and positively correlated with colonic mucosa thickness (r=0.8073,P< 0.01).Bifidobacteriaceae and Bifidobacterium abundance positively correlated with colonic mucosa thickness (r= 0.7791,P< 0.01).Desulfovibrionaceae (r= –0.7970,P< 0.01) and Desulfovibrio (r= –0.6683, P < 0.05) abundance negatively correlated with motor deficits.Turicibacter abundance positively correlated with colonic mucosa thickness(r= 0.6046,P< 0.05).Faecalibacterium abundance negatively correlated with motor deficits (r= –0.6214,P< 0.05) and MAOB expression levels (r=–0.7188,P< 0.05) and positively correlated with colonic mucosa thickness(r= 7365,P< 0.01).Dubosiella abundance negatively correlated with α-syn expression levels (r= –0.7182,P< 0.05) and positively correlated with colonic mucosa thickness (r= 0.6047,P< 0.05).These findings suggest that rotenone-induced motor impairment and other elements of PD pathology are associated with disruption of the gut microbiota.

Transplantation of gut microbiota from mice treated with rotenone induces motor deficits, intestinal inflammation, and endotoxemia

To further investigate whether the changes in the gut microbiota associated with rotenone administration are intrinsically capable of impairing motor behavior in mice, we performed fecal microbiota transplantation via oral gavage from Rot and Con mice to C57BL/6J mice pretreated with antibiotics.Transplantation from Rot mice induced motor impairment, as indicated by poorer performance of mice from the Con + FMTRotgroup in the rotarod test and pole test (P< 0.05; Figure 5A and B) compared with mice from the Con + FMTCongroup.Furthermore, the Con + FMTRotgroup exhibited shorter colon length (P< 0.05, Figure 5C) and higher levels of IL-6 and TNF-α mRNA expression in the colon (P< 0.05; Figure 5D and E) compared with the Con + FMTCongroup.Additionally, we found elevated serum LPS levels in mice from the Con + FMTRotgroup compared with mice from the Con + FMTCongroup (P< 0.05; Figure 5F).These findings provide further evidence for an association between gut dysbiosis and neurological dysfunction and suggest that manipulating the gut microbiota could attenuate the neurological complications induced by rotenone.

Transplantation of healthy microbiota alleviates rotenone-induced motor deficits, intestinal inflammation, endotoxemia, and intestinal barrier disruption

Next, we investigated whether transplanting fecal microbiota from donor Con mice could rescue the motor deficits and intestinal barrier disruption observed in the Rot mice.The motor deficits observed in the rotarod test and pole test were alleviated in Rot mice transplanted with microbiota from donor Con mice compared with those that received PBS (P< 0.05; Figure 6A and B).The rotenone-induced colon shortening (Figure 6C) and increase in colonic IL-6, TNF-α, and iNOS mRNA expression levels (Figure 6D–F) were rescued by gut microbiota transplantation from donor Con mice.Serum LPS levels were decreased in the Rot + FMTConmice compared with the Rot + PBS mice (P<0.05; Figure 6G), indicating an improvement in endotoxemia and intestinal mucosal barrier function.Moreover, we observed an increase in colonic mucus thickness, as demonstrated by Alcian blue staining (P< 0.05; Figure 6H and I) and MUC2 immunofluorescence staining (Figure 6J), in the Rot + FMTConmice compared with the Rot + PBS mice.Therefore, a healthy gut microbiota can alleviate rotenone-induced motor deficits, intestinal inflammation,endotoxemia, and intestinal barrier disruption.

Discussion

Figure 2 ｜Rotenone administration induces intestinal barrier disruption in a gut microbiota-dependent manner.(A) Colonic mucus layer thickness (per section/two sections per animal, n = 3).(B) Alcian blue-stained colonic sections showing the mucus layer (arrows).The paired black arrows indicate where the mucus layer was measured.Scale bars: 50 μm (n = 3).(C) Immunofluorescence images of colonic sections stained with anti-MUC2 antibody and 4′,6-diamidino-2-phenylindole (DAPI).Paired white arrows indicate the mucus layer.Scale bars: 50 μm.(D) Protein levels of occludin and zonula occludens-1 (ZO-1) in the colon (n = 5).(E) Serum lipopolysaccharide (LPS) endotoxin levels (n = 10).(F–H) mRNA expression levels of interleukin (IL-6; F), tumor necrosis factor-α (TNF-α; G), and inducible nitric oxide synthase (iNOS;H) in the colon (n = 5).(I) Colon length (n = 9), with representative images shown.(J) Colonic mucus layer thickness (per section/two sections per animal, n = 3).(K) Alcian blue-stained colonic sections showing the mucus layer (arrows).Paired black arrows indicate where the mucus layer was measured (scale bars: 50 μm).(L) Immunofluorescence images of colonic sections stained with anti-MUC2 antibody and DAPI.Paired white arrows indicate the mucus layer.Scale bars: 50 μm.(M) Protein levels of occludin and ZO-1 in the colon (n = 5).(N)Serum LPS endotoxin levels (n = 10).(O–Q) mRNA expression levels of IL-6, TNF-α, and iNOS in the colon (n = 5).(R) Colon length (n = 9), with representative images shown.Values are presented as mean ± standard error of the mean.*P < 0.05, **P < 0.01, ***P < 0.001, vs.Con.Con: control; Rot: rotenone; Con + Ab: Con mice treated with antibiotics; Rot + Ab: Rot mice treated with antibiotics.

Figure 3 ｜Mice treated with rotenone exhibit C/EBP/AEP pathway activation, α-syn aggregation, and TH-positive neuron loss in the SN, and these effects are dependent on the gut microbiota.(A–D) Protein levels of C/EBPβ, AEP, p-α-syn, and MAOB in the midbrain containing the SN in the Con and Rot groups.(E–H) Protein levels of C/EBPβ, AEP, p-α-syn, and MAOB in the midbrain containing the SN in the Con + Ab and Rot + Ab groups.(I, J) Representative immunofluorescence images of the SN showing nuclei (DAPI, blue), total TH-positive neurons (TH,green), and α-syn (p-α-syn Ser129, red).Scale bars: 100 μm.(K) Rotarod test results for the Con and Rot groups.The Y-axis shows the time spent on the rotarod.(L) Pole test results for the Con and Rot groups.The Y-axis shows the time of descent from the top of the pole to the ground.(M) Rotarod test results for the Con + Ab and Rot + Ab groups.The Y-axis shows the time spent on the rotarod.(N) Pole test results for the Con + Ab and Rot + Ab groups.The Y-axis shows the time of descent from the top of the pole to the ground.Values are presented as mean ± standard error of the mean (n = 6).*P < 0.05, **P < 0.01, ***P < 0.001, vs.Con.Con: control; Rot: rotenone; Con + Ab: Con mice treated with antibiotics;Rot+Ab: Rot mice treated with antibiotics.AEP: Mammalian asparagine endopeptidase; C/EBPβ: CCAAT/enhancer binding protein β; DAPI: 4′,6-diamidino-2-phenylindole; MAOB:monoamine oxidase B; SN: substantia nigra; TH: tyrosine hydroxylase; α-syn: α-synuclein.

Figure 4 ｜ Rotenone-induced motor impairment, endotoxemia, and C/EBP/AEP signaling pathway alteration are associated with disruption of the gut microbiota.Spearman’s correlation analysis of the association between specific gut microbiota and colonic length, zonula occludens-1 (ZO-1), occluding, CCAAT/enhancer binding protein β, mammalian asparagine endopeptidase (AEP), p-α-synuclein (p-α-syn), monoamine oxidase B (MAOB) expression levels, colonic mucosa thickness, serum lipopolysaccharide(LPS) levels, and behavior test results.*P < 0.05, **P < 0.01.

Figure 5 ｜ Transplantation of the gut microbiota from mice treated with rotenone impairs motor behavioral performance and induces intestinal inflammation and endotoxemia in mice.(A) Rotarod test.The Y-axis shows the time spent on the rotarod.(B) Pole test.The Y-axis shows the time of descent from the top of the pole to the ground.(C) Colon length (n = 9),with representative images shown.(D, E) mRNA expression levels of interleukin (IL)-6 (D)and tumor necrosis factor (TNF)-α (E) in the colon (n = 5).(F) Serum lipopolysaccharide(LPS) level (n = 10).Values are mean ± standard error of the mean.*P < 0.05, **P < 0.01,vs.Con + FMTCon.Con + FMTCon: Mice transplanted with gut microbiota from control mice; Con+FMTRot: control mice transplanted with gut microbiota from mice treated with rotenone.

In this study, we observed that rotenone administration led to gut microbiota dysbiosis, disruption of intestinal barrier function, and activation of the C/EBP/AEP pathway, resulting in α-syn aggregation and loss of TH-positive neurons in the SN of mice, accompanied by motor deficits.However, these adverse effects were not observed in mice whose gut microbiota was depleted by pretreatment with antibiotics.Furthermore, we demonstrated that the gut microbiota derived from rotenone-treated mice impaired motor performance,induced intestinal inflammation, and caused endotoxemia in recipient mice.Conversely, transplantation of fecal microbes from healthy control mice ameliorated PD symptoms in mice treated with rotenone.Collectively, these findings suggest that the gut microbiota may play a critical role in mediating the effects of the environmental toxin rotenone on intestinal permeability,as well as activation of the C/EBP/AEP pathway in the brain, leading to the development of motor symptoms and contributing to the pathogenesis of PD.A significant difference in the beta diversity, but not the α diversity, of the gut microbiota has been reported in patients with PD (Keshavarzian et al., 2015; Scheperjans et al., 2015; Hopfner et al., 2017; Cirstea et al.,2020; Cosma-Grigorov et al., 2020).Consistent with this, in our rotenoneinduced mouse model of PD we observed alterations in the beta diversity,but not the α-diversity, of the gut microbiota.Moreover, we found that the relative abundance of theFirmicutesphylum was significantly increased and enriched in Rot mice compared with Con mice.This finding is consistent with a clinical study that reported enrichment ofFirmicutesin patients with PD experiencing constipation (Heinzel et al., 2021).In our study, we also found a significant correlation betweenFirmicutesabundance and motor deficits,as well as decreased levels of colonic ZO-1 expression and mucosa thickness.These results suggest that the increased abundance ofFirmicutesin Rot mice may be associated with damage to the intestinal mucosal barrier and the development of PD.Furthermore, we observed an increased abundance of theCyanobacteriaphylum in Rot mice.Cyanobacteriaare known to produce the neurotoxin cyanotoxin β-N-methylamino-L-alanine, which has been reported to induce protein misfolding, mitochondrial dysfunction,innate immune response activation, and chronic gut inflammation (Nunes-Costa et al., 2020).In our study, we found a negative correlation betweenCyanobacteriaabundance and motor deficits and colonic ZO-1 expression levels, as well as a positive correlation with serum endotoxin LPS levels.Therefore, the increased abundance ofCyanobacteriain response to treatment with rotenone may lead to the production of cyanotoxin β-Nmethylamino-L-alanine, ultimately disrupting the gut-brain axis and contributing to the neurodegenerative features observed in PD.Additionally,in line with a previous study (Zhu et al., 2022), we observed a significant increase in the relative abundance of thePatescibacteriaphylum in Rot mice.It has also been reported thatPatescibacterialevels are increased in mice with impaired locomotor activity induced by treatment with silicon dioxide(Diao et al., 2021).Patescibacteriaare anaerobic bacteria that are not yet widely understood and lack a tricarboxylic acid cycle (Wrighton et al., 2012).The alteration inPatescibacteriaabundance that we observed may contribute to the abnormal tricarboxylic acid cycle function and carbon metabolism that have been reported in PD (Kanamatsu et al., 2007).Therefore, the alteration in beta diversity and phylum-level composition of the gut microbiota induced by rotenone may contribute to gut damage, motor dysfunction, and the development of PD pathology.

Figure 6 ｜ Transplantation of healthy gut microbiota alleviates rotenone-induced motor deficits, intestinal inflammation, endotoxemia, and intestinal barrier impairment.(A) Rotarod test.The Y-axis shows the time spent on the rotarod.(B) Pole test.The Y-axis shows the time of descent from the top of the pole to the ground.(C) Colon length (n =9), with representative images shown.(D–F) mRNA expression levels of interleukin (IL)-6,tumor necrosis factor (TNF)-α, and inducible nitric oxide synthase (iNOS) in the colon (n =5).(G) Serum lipopolysaccharide (LPS) endotoxin levels (n = 10).(H) Colonic mucus layer thickness (per section/two sections per animal, n = 3).(I) Alcian blue-stained colonic sections showing the mucus layer (arrows).Paired black arrows show where the mucus layer was measured (scale bars: 50 μm).(J) Immunofluorescence images of colonic sections stained with anti-mucin-2 (MUC2) antibody and 4′,6-diamidino-2-phenylindole(DAPI).Paired white arrows indicate the mucus layer.Scale bars: 50 μm.Values are mean± standard error of the mean.*P < 0.05, **P < 0.01, ***P < 0.001, vs. Rot + PBS.Rot:Rotenone; Rot + FMTCon: Rot mice transplanted with gut microbiota from donor control mice; Rot + PBS: Rot mice treated with PBS.PBS: Phosphate-buffered saline.

In the present study, we observed significant alterations in the composition of the gut microbiota in Rot-treated mice.Specifically, we found changes in the abundance of various bacterial families and genera,includingLachnospiraceae,Ruminococcaceae,Saccharimonadaceae,Bifidobacteriaceae,Erysipelotrichaceae,Desulfovibrionaceae, andAtopobiaceaeat the family level, as well asLachnospiraceae_ NK4A136_group,Turicibacter,Faecalibacterium,Dubosiella,Bifidobacterium, andDesulfovibrioat the genus level.These findings are consistent with previous reports of altered gut microbiota in patients with PD.For instance, the abundance ofLachnospiraceaehas been observed to decrease in patients with PD as the disease progresses (Barichella et al., 2019; Pietrucci et al., 2019).In our study, we also found changes in the abundance ofLachnospiraceaeandLachnospiraceae_ NK4A136_group in Rot-treated mice.These findings suggest that the increased abundance ofLachnospiraceaeand its specific subgroup may occur as a result of exposure to rotenone, particularly in the early stages of PD.Furthermore, we found a significant increase in the abundance ofRuminococcaceaein Rot-treated mice.Consistent with this,clinical studies have reported an increase inRuminococcaceaein patients PD, and this increase has been found to positively correlate with both the duration and the severity of the disease (Hill-Burns et al., 2017; Hegelmaier et al., 2020).Importantly, our study revealed a significant negative correlation betweenRuminococcaceaeabundance and colonic mucosa thickness, as well as a positive correlation with motor deficits.These findings suggest thatRuminococcaceaemay play a crucial role in gut damage and the development of motor symptoms in PD.The familyBifidobacteriaceaeand genus Bifidobacterium, which are important dominant probiotics, play a significant role in various physiological functions, such as inhibiting the overgrowth of harmful gut bacteria, improving the gut ecological environment, and regulating the immune system.Several clinical studies have demonstrated that probiotic interventions includingBifidobacteriumcan alleviate motor impairment in patients PD (Georgescu et al., 2016; Fang, 2019; Hsieh et al.,2020).Furthermore, a clinical study with 2-year follow-up indicated that decreasedBifidobacteriumabundance in patients with PD correlates with worsening of PD symptoms (Minato et al., 2017).In our study, we observed a decrease in the abundance ofBifidobacteriaceaeandBifidobacteriumin Rot-treated mice that correlated positively with colonic mucosa thickness.Therefore, the reduction inBifidobacteriaceaefamily andBifidobacteriumgenus abundance induced by rotenone may contribute to mucosal deficits and subsequent alterations in the gut-brain axis.Faecalibacterium, a dominant bacterium that produces short-chain fatty acids, plays a crucial role in maintaining gut health through its anti-inflammatory and antioxidant properties (Ferreira-Halder et al., 2017).Clinical studies have consistently reported a negative correlation betweenFaecalibacteriumabundance and PD severity and duration (Li et al., 2017).In line with these previous findings (Keshavarzian et al., 2015; Li et al., 2017), we observed a decrease inFaecalibacteriumabundance in Rot-treated mice, a significant positive correlation betweenFaecalibacteriumabundance and colonic mucosa thickness, and a negative correlation betweenFaecalibacteriumabundance and motor deficits and MAOB.These results collectively suggest that rotenone-related gut microbiota dysbiosis might lead to alterations in metabolites (such as short-chain fatty acid deficits), disruption of the mucosal barrier, increased intestinal permeability, and inflammation, all of which contribute to PD pathogenesis.

In this study, we observed an increase in serum endotoxin LPS levels in Rot-treated mice, accompanied by elevated inflammation in the colon and a reduction in colonic mucus thickness and the expression of tight junction proteins.Importantly, when we depleted the gut microbiota using antibiotics, the alterations induced by rotenone were reversed, indicating that rotenone-induced microbiota alteration is crucial for the development of hyperendotoxemia and inflammation.We also found a positive correlation betweenCyanobacteriaandRuminococcaceaeabundance and serum LPS levels.LPS is an endotoxin produced by gram-negative bacteria and Cyanobacteria (Durai et al., 2015) that is known for its ability to stimulate microglia, as well as being involved in the inflammatory process underlying PD pathogenesis (Liu and Bing, 2011).LPS induces motor impairment and neuroinflammation through microglial activation (Zhao et al., 2019).Studies have reported LPS-induced microglial activation induced in the SN of patients with PD (Imamura et al., 2003; Hirsch and Hunot, 2009).Additionally,elevated serum LPS levels, indicating increased intestinal permeability,have been observed in some patients with PD (Pal et al., 2015).Thus, LPSinduced neuroinflammation is thought to contribute to PD pathogenesis(Deng and Bobrovskaya, 2022; Zhang et al., 2023).Furthermore, our findings highlight the significant role that rotenone-induced microbiota alteration plays in promoting hyperendotoxemia and inflammation.Therefore, these findings suggest that disruption of the blood-brain barrier triggered by hyperendotoxemia and inflammation could contribute to the effect of gut microbiota dysbiosis on neuronal changes.

In this study we observed an increase in C/EBPβ expression in the SN of Rot mice.It has been reported that LPS and inflammatory cytokines (IL-1β, IL-6, and TNF-α) can activate C/EBPβ (Magalini et al., 1995; Poli, 1998; Ejarque-Ortiz et al., 2007; Cox et al., 2013; Pulido-Salgado et al., 2015).Therefore,the hyperendotoxemia and increase in inflammatory cytokine expression induced by Rot may active C/EBPβ, which in turn promotes transcription of AEP, α-syn, and MAOB.Additionally, we observed upregulation of AEP, α-syn,and MAOB expression in the SN of Rot mice.It has been shown that C/EBPβ overexpression in the SN significantly upregulates AEP, α-syn, and MAOB expression, resulting in extensive loss of TH-positive neurons and motor dysfunction.Conversely, C/EBPβ depletion strongly downregulates AEP, α-syn,and MAOB expression, thereby abolishing rotenone-elicited PD pathology and motor impairment (Wu et al., 2021).Thus, the C/EBPβ/AEP pathway plays a vital role in PD-associated pathology.Importantly, we found that the alterations in C/EBPβ activation and α-syn, and MAOB expression levels in the SN of Rot mice were reversed by pretreatment with antibiotics.This suggests that the gut microbiota plays an important role in C/EBPβ activation in the SN and the development of PD-associated pathology, such as increased α-syn and MAOB expression levels and damage to DA neurons.

This study has some limitations that should be noted.First, although we highlighted the significant role that the gut microbiota plays in PD pathogenesis, we did not identify the specific microbiota species that are involved in PD development.Therefore, metagenomic analysis is needed to identify relevant bacterial species.Second, in addition to the role of the gut microbiota, gut microbiota metabolites may also play a critical role in PD development.Thus, non-targeted metabolomics analysis should be performed to identify other possible mechanisms involved in gut microbiota dysbiosismediated PD development.

In conclusion, our findings demonstrate the crucial role of gut microbiota dysbiosis in the development of motor deficits, activation of C/EBPβ/AEP signaling, and α-syn-related pathology induced by rotenone.Our study shows that rotenone induces gut microbiota dysbiosis, which activates the C/EBPβ/AEP signaling pathway.Moreover, our results suggest that supplementation with healthy microbiota may provide a safe and effective treatment strategy to ameliorate the progression of motor deficits in PD.

Acknowledgments:We thank Qingyuan Wu, Yuying Gong, and Menglu Zhou (Jiangsu Key Laboratory of Immunity and Metabolism, Department of Pathogen Biology and Immunology, Xuzhou Medical University) for their technical support.

Author contributions:XF conducted animal experiments, performed statistical analyses and drafted the manuscript.SL, BM, YX, SK and YZ revised the manuscript.MZ and XG conducted statistical analyses, helped in finalizing the figures and revised the manuscript.YY, KZ and DG, designed the studies, edited the manuscript and provied financial support.CFL, gudied and supervised the experiment.All authors have read and agreed to the published version of the manuscript.

Conflicts of interest:The authors declare no competing financial interests.

Data availability statement:No additional data are available.

Open access statement:This is an open access journal, and articles are distributed under the terms of the Creative Commons AttributionNonCommercial-ShareAlike 4.0 License, which allows others to remix, tweak, and build upon the work non-commercially, as long as appropriate credit is given and the new creations are licensed under the identical terms.

Additional files:

Additional Figure 1: Timeline of oral rotenone administration and outcome measurements in Con and Rot mice.

Additional Figure 2: Timeline of oral rotenone administration and outcome measurements in Con+Ab and Rot+Ab mice.

Additional Figure 3: Timeline of oral rotenone administration and outcome measurements in Con+FMTCon and Con+FMTRot mice.

Additional Figure 4: Timeline of oral rotenone administration and outcome measurements in Rot+PBS and Rot+FMTcon mice.

Additional Figure 5: Gut microbiota dysbiosis was observed in mice treated with Rot.

Additional Table 1: Primers used for quantitative reverse transcriptionpolymerase chain reaction.