Animals and Group Design

Six-week-old male C57BL/6J mice (18 ± 2 g) were acquired from the Zhaoyan New Drug Research Center (Suzhou, China) and acclimatized for 1 week before treatment. The mice were maintained (12 h light/dark cycle) under pathogen-free conditions at 24 ± 2.0 °C and 55 ± 10% humidity and allowed free access to water and standard rodent chow food (four mice/cage) or the fasting mimicking diet (one mouse/cage). All experimental procedures were approved by the Animal Ethics Committee of Jiangnan University.

To verify the neuroprotection of FMD on PD mice, at 7 weeks of age, C57BL/6J mice were randomly divided into four groups: the NS-AL group, which was treated with normal saline by intraperitoneal injection and fed ad libitum; the NS-FMD group, which was treated with normal saline by intraperitoneal injection and fasting mimicking diet; the MPTP-AL group, which was treated with MPTP and ad libitum; and the MPTP-FMD group, which was treated with MPTP and fasting mimicking diet. In a word, mice in this experiment treated with normal saline or MPTP on the last day of the second cycle (on day 21; Fig. 1a). Behavior training was conducted once per day for three consecutive days (on days 26, 27, 28; Fig. 1a), and behavior test was conducted on the day after the last treatment (on day 29; Fig. 1a). The whole brain, striatum and feces were collected after behavior test (on day 29; Fig. 1a).

Fig. 1 FMD treatment retains motor function in PD mice without an overall reduction in calorie intake. (a) Timeline for the experimental procedure, including FMD, MPTP administration, and tissue collection. (b) Body weight changes. NS-AL group (n = 17), NS-FMD group (n = 15), MPTP-AL group (n = 18), MPTP-FMD group (n = 16). (c) Average food intake in per cycle, n = 13 mice per group. (d) Pole descent test: time taken represents degree of bradykinesia, F 3,59 = 48.325, p < 0.001. NS-AL group (n = 16), NS-FMD group (n = 15), MPTP-AL group (n = 18), MPTP-FMD group (n = 14). (e) Traction test: suspension reflex score for evaluation of muscle strength and equilibrium, F 3,62 = 33.929, p < 0.001. NS-AL group (n = 17), NS-FMD group (n = 15), MPTP-AL group (n = 18), MPTP-FMD group (n = 16). Statistical comparison by one-way ANOVA with post hoc comparisons of LSD; data represent the means ± SEM; ***p < 0.001 Full size image

To explore the role of gut microbiota between PD and FMD, mice, pretreated with an antibiotic cocktail and MPTP, were randomly divided into four groups: the PD-PBS/G group, which was treated with 20% glycerol in sterile phosphate-buffered solution (PBS) by gastric gavage; the PD-NA group, which was treated with gut microbiota from NS-AL mice by gastric gavage; the PD-NF group, which was given gut microbiota from NS-FMD mice by gastric gavage; the PD-NF/HK group, which was treated with heat-killed (HK) gut microbiota from NS-FMD mice by gastric gavage. The striatum was collected on the day after the last treatment (on day 25; Fig. 7a). In all of these four groups, levels of striatal DA and serotonin (5-HT) were measured by high-performance liquid chromatography.

FMD Treatment

Mice were fed ad libitum with irradiated LAD0011 rodent chow (Trophic Animal Feed High-tech Co. Ltd., Jiangsu, China), containing 3.4 kcal/g of digestible energy (protein 22%, carbohydrate 65%, fat 13%). On average, mice in the control group consumed 12 kcal/day (or 3.5 g/day) based on pre-experiments. The experimental FMD was based on a nutritional screen that identified ingredients that allowed high nourishment during periods of low calorie consumption. The FMD consisted of two different components designated as the day 1 diet and day 2–3 diet that were fed in this respective order. The day 1 diet contained 50% of the standard daily calorie intake and was designed to adapt the mouse to a period of low caloric intake. The day 2–3 diet contained 10% of the standard daily calorie intake. All mice were supplied with fresh food during the morning hours (8 am–10 am). Mice consumed all the supplied food on each day of the FMD regimen and showed no signs of food aversion. After the end of the 3-day FMD, we supplied LAD0011 rodent chow ad libitum for 4 days of refeeding before starting another FMD cycle. Prior to supplying the FMD, animals were transferred into fresh cages to avoid feeding on residual chow and coprophagy. Food intake was measured daily.

Antibiotic Treatment

Antibiotic-pretreated animals were provided a cocktail consisting of 1 mg/ml bacitracin, 0.5 mg/ml gentamycin, 0.2 mg/ml ciprofloxacin, 1 mg/ml neomycin, 1 mg/ml penicillin, 1 mg/ml metronidazole, 0.5 mg/ml ceftazidime, 0.5 mg/ml vancomycin, and 2 mg/ml streptomycin [23,24,25] and were administered daily by gastric gavage at a dose of 200 μl [26]. Antibiotics were administered daily for 7 consecutive days prior to MPTP treatment and fecal microbiota transplantation (Fig. 7a). Two days after cessation of antibiotic treatment, mice were administered MPTP [24].

MPTP Treatment

MPTP is known to cause a destruction of the dopaminergic nigrostriatal pathway and elicit symptoms of PD [27]. C57BL/6J mice were administered intraperitoneal with MPTP (20 mg/kg dissolved in sterilized saline) in a volume of 10 ml/kg of body weight on four occasions, with each administration separated by a 2-h interval [28]. In experiments involving FMD, MPTP was administered on the last day of the second FMD cycle (Fig. 1a). The control mice were treated with NS.

Transplantation of Fecal Microbiota

Fresh fecal pellets were collected from NS-AL and NS-FMD mice, then diluted immediately with sterile PBS (1 fecal pellet/ml). For each experiment, several fecal pellets from different mice in the corresponding group were resuspended together in PBS [24]. Briefly, the feces were steeped in sterile PBS for about 15 min, shaken, and then centrifuged at 1000×g, 4 °C for 5 min. The suspension was centrifuged at 8000×g, 4 °C for 5 min to get total microbiota, then washed twice in PBS. The final microbial suspension was mixed with an equal volume of 40% sterile glycerol to a final concentration of 20%, then stored at − 80 °C. In addition, heat-killed microbiota from NS-FMD mice was boiled for 45 min after washing twice in PBS [19]. For each mouse, 200 μl of microbial suspension (108 CFU/ml) was transplanted to gut microbiota-depleted mice after antibiotic treatment by gastric gavage each day over consecutive 7 days. The dilution coating method was used for determination of microbial concentrations from mice by calculating colonies on solid medium under anaerobic incubation as previously described [29].

Behavioral Test

On the 5th day after MPTP treatment, the mice began behavioral training once per day for 3 days. Protocols of behavioral tests (pole descent test and traction test) have been described in previous studies [30, 31].

The pole test is a useful method for evaluating bradykinesia in mouse PD models. In the pole descent test, a 0.5-m-long pole, 1 cm in diameter and wrapped with nonadhesive gauze to facilitate gripping, and with a spherical protuberance, 2 cm in diameter on top, was placed in the home cage. Mice were placed head-down on the top of the pole and their descent back into the home cage was timed. Timing began when the experimenter released the animal and ended when one hind-limb reached the home cage base. The test was performed three times for each animal, the beginning at the day after the last treatment, and the average of the three trials was calculated for statistical analyses.

The traction test measures muscle strength and equilibrium. In the traction test, the forepaws of the mice were placed on a horizontal rope (diameter 5 mm) and observed for 10 s, whereas the hind limb placements were scored from 1 to 4, with the lowest score indicating the most severe deficit. Animals were assigned a score of 4 for gripping the rope with both hind paws, 3 for gripping the rope with one hind paw, 2 for gripping the rope with both front paws, and 1 for gripping the rope with one front paw. The test was performed three times for each animal, the beginning on the day after the last treatment, and the average of the three trials was calculated for statistical analyses.

Sample Collection and Tissue Preparation

For collecting feces, mice were placed individually in empty autoclaved cages and allowed to defecate freely in the morning after the day of last treatment. Once feces were formed of each mouse, feces were collected immediately in individual sterile EP tubes on ice and then stored at − 80 °C. For obtaining fresh striatum tissue, mice were deeply anesthetized with isoflurane and then received a transcardiac perfusion of ice-cold sterilized saline. The tissues were immediately stored at − 80 °C.

For immunofluorescence, mice were deeply anesthetized with isoflurane and then transcardially perfused with PBS followed by 4% paraformaldehyde in 0.01 M PBS, pH 7.4. Tissues were postfixed in 4% paraformaldehyde at 4 °C overnight, kept in 20% sucrose at 4 °C for 24 h, then transferred into 30% sucrose at 4 °C for 24 h, and then embedded in optimal cutting temperature compound (O.C.T. Compound, Tissue-Tek, Torrance, CA, USA). Ten micrometer-thick cross sections were cut using a cryostat microtome (CM1950, Leica, Wetzlar, Germany).

Measurement of Neurotransmitters

High-performance liquid chromatography with a fluorescence detector (Waters 2475, Milford, MA, USA) was used to measure the levels of striatal DA, 5-HT, and their metabolites, including 3, 4-dihydroxyphenylacetic (DOPAC), homovanillic acid (HVA), and 5-hydroxyindoleacetic acid (5-HIAA), following previous methods with moderate modifications [32]. The separation system (Waters 2695) was used with an Atlantis T3 column (150 mm × 4.6 mm, 5 l μm, Waters). The mobile phases were composed of water, acetonitrile, and 0.01 M PBS (pH 4.0). Briefly, the striatum was homogenized in 0.1 M perchloric acid (10 μl/mg of striatum tissue) by sonication and the homogenate was centrifuged at 13,000×g, 4 °C for 10 min. Then, the supernatants were collected and filtered through a 0.22-μm filter and 25 μl of sample was injected into the column. In parallel, DA, 5-HT and their metabolites, and a hydrochloride standard (Sigma-Aldrich, St. Louis, MO, USA) solution were prepared freshly by diluting the stock solution in the mobile phase. The linearity ranges of neurotransmitters were determined by serial concentrations of standard solution before detection.

Western Blot Analysis and Enzyme-Linked Immunosorbent Assay

Total protein was extracted by homogenizing 20 mg tissue in 200 μl radio immunoprecipitation assay lysis buffer (Beyotime, Shanghai, China) with 2 μl phenylmethanesulfonyl fluoride (Solarbio, Beijing, China). After centrifugation of the homogenate at 13,000×g, 4 °C for 5 min, the supernatant was collected and measured with a BCA protein assay kit (Biosharp, Anhui, China). Forty micrograms of total protein was separated by sodium dodecyl sulfate-polyacrylamide electrophoresis-polyacrylamide gel electrophoresis, blotted onto polyvinylidene difluoride membranes (Millipore, Billerica, MA, USA), and then probed with the following antibodies: mouse anti-tyrosine hydroxylase (TH, 1:1000, MAB318, Millipore), rabbit anti-BDNF (1:1000, ab108319, Abcam, Cambridge, MA, USA), rabbit anti-β tubulin (1:1000, 10,068-1-AP, Proteintech, Wuhan, China) and mouse anti-GAPDH (1:8000, 60,001-1-Ig, Proteintech). Goat anti-rabbit IgG (1:1000, BA1054, Boster, Pleasanton, CA, USA) and goat anti-mouse IgG (1:1000, BA1050, Boster), both conjugated to horseradish peroxidase, were used as secondary antibodies. Protein bands were visualized by incubation with BeyoECL Plus (P0018, Beyotime) for 1 min and imaged by a Gel Image System (Bio-Rad, Hercules, CA, USA). Densitometry was performed by using the ImageJ software (NIH, Bethesda, MA, USA).

TNF-α and IL-1β concentrations of the striatum were detected using a commercial enzyme-linked immunosorbent assay kit (EK0527/EK0394, Boster). The limit of detection is 15.6–1000 pg/ml and 12.5–800 pg/ml respectively. All experimental procedures were performed according to the manufacturer’s instructions.

Immunofluorescence and Image Analysis

Briefly, each mouse brain was cut into coronal slices and slices containing the substantia nigra pars compacta were collected. Brain sections were immersed in 0.01 M sodium citrate buffer (pH 6.0) for antigen retrieval and washed in PBS. Then, brain sections were incubated in PBS containing 0.3% v/v Triton X-100 and 5% v/v goat serum for 1 h at 37 °C. Primary antibodies—mouse anti-TH (1:1000, MAB318, Millipore), rabbit anti-glial fibrillary acid protein (GFAP, 1:2000; Z033429, Dako, Glostrup, Denmark), and anti-ionized calcium-binding adaptor molecule 1 (Iba-1, 1:1000, 019-19741, Wako, Osaka, Japan)—were incubated 1 h at 37 °C. For detection of the primary antibodies, appropriate secondary antibodies, coupled to fluorescein isothiocyanate (FITC)-conjugated goat anti-mouse IgG (1:1000, A0568, Beyotime) and CY3-conjugated goat anti-rabbit IgG (1:1000, A0516, Beyotime) were used. Samples were covered with mounting medium (P0126, Beyotime) and examined with an epifluorescence microscope (Nikon Eclipse 80i, Nikon, Tokyo, Japan). For each animal, brain slices containing the major portion of the SN from bregma − 2.92 mm to − 3.52 mm, 10 representative sections were chosen to be doubly stained with TH and NeuN in the both sides of SN, four representative sections were chosen to be doubly stained with TH and GFAP/Iba-1 in the SN. Cells positive for TH, NeuN, GFAP, and Iba-1 were counted in the SN by the ImageJ analyzer (NIH). The abovementioned sections were quantified for each animal, and each group contained 4–6 animals.

Gut Microbiota Profiling

Total genomic DNA from fecal samples was extracted using a PowerSoil DNA Isolation Kit (MOBIO, Carlsbad, CA, USA) according to the manufacturer’s instructions. DNA quality and concentration were monitored by gel electrophoresis and spectrophotometry (Nanodrop 1000, NanoDrop Technologies, Wilmington, DE, USA). The samples for sequencing 16S and 18S rRNA gene V4 regions were prepared following the Illumina protocol for 16S and 18S metagenomic sequencing library preparation. To generate amplicons, the hypervariable V4 regions of 16S [33] and 18S rRNA [34] were amplified using primers (forward primer, 5′-AYTGGGYDTAAAGNG-3′ and 5′-CCAGCASCYGCGGTAATTCC-3′ respectively; reverse primer, 5′-TACNVGGGTATCTAATCC-3′ and 5′-ACTTTCGTTCTTGATYRA-3′ respectively) with a denaturation step at 95 °C for 3 min, followed by 30 cycles of denaturation at 95 °C for 30 s, primer annealing at 55 °C for 30 s, and primer extension at 72 °C for 30s. Upon completion of cycling, a final extension at 72 °C for 5 min was done and then the reaction was held at 4 °C. The PCR products were then purified with magnetic beads (Agencourt AMPure XP, Beckman, CA, USA), and then a second round of PCR amplification was implemented to introduce dual indices and sequencing adapters. The purified products were quantified using a fluorometric quantification method that uses dsDNA binding dyes (Qubit® dsDNA HS Assay Kits, Life Technologies, Carlsbad, CA, USA) and pooled into a library after normalization. RNA sequencing was performed on an Illumina HiSeq 2500 to generate pair-end 250 bp reads.

The data analysis was performed by the QIIME v1.9.1 platform [35]. Raw sequencing data was filtered using FASTQC according to the phred scores, and the reads (110149.1 ± 2708.5 reads per sample for 16S rRNA analysis and 137167.3 ± 5176.9 reads per sample for 18S rRNA analysis) were trimmed if the average phred score in the window (5 bp in size, 1 bp step length) was less than 20. Reads containing ambiguous “N” or with length < 150 bp were discarded. Paired reads were merged into a tag sequence according to their overlap. By aligning with Sliva, Unite, and RDP database, the chimeras were removed using the algorithm of usearch61. High-quality sequences without chimeras were clustered into OTUs (operational taxonomic units), with a similarity of 97% [36]. OTU picking was performed using Uclust on the software platform QIIME v1.9.1. Nonrepetitive ones were extracted from optimized sequences to reduce the redundant computation. Sequences without repeats were removed. OTUs with 97% similarity were clustered. Chimeras were removed during the clustering process and representative sequences of OTU were obtained. All of the optimized sequences were mapped to the representative OTU and those with were selected to generate OTU table. OTUs with low abundance (the threshold value was 0.00001) were screened and filtered. The longest sequence of each OTU was selected as a representative sequence which was annotated by comparing to the Greengenes database (Release 13.5, http://greengenes.secondgenome.com/) by the RDP-classifier method [37, 38].

Alpha diversity and beta diversity analyses were performed based on rarefied OTUs. For 16S rRNA analysis, the OTU table was rarified to 72,130 reads, whereas for 18S rRNA analysis, the OTU table was rarified to 82,869 reads. The rarefaction analysis based on mothur v.1.21.1 was conducted to reveal the diversity indices, including Chao1 (http://www.mothur.org/wiki/Chao) and Shannon diversity indices (http://www.mothur.org/wiki/Shannon). Beta diversity was used to evaluate differences of samples in species diversity and was characterized by principal coordinate analysis (PCoA) based on Bray–Curtis distance. The Vegan 2.0 package was used to generate a PCoA figure and the differences among the groups were tested by ANOSIM (analysis of similarity). The software package of ggplot2 and pheatmap provided by R language were used to generate histogram and heatmap separately to describe the composition and diversity of the communities with OTU table as an input file.

Fecal Short-Chain Fatty Acids Profiling

Fecal SCFA concentrations were determined using gas chromatography-mass spectrometry. Fecal samples collected before euthanasia were stored at − 80 °C and then thawed to room temperature for processing. Briefly, 50 mg feces were ultrasonicated with 100 μl of water in a bath ultrasonicator for 5 min and vortex-mixed for 2 min and then centrifuged at 4 °C at 16,000×g for 15 min. An aliquot (100 μl) of the supernatant was acidified by adding 50 μl 50% (v/v) sulfuric acid. After vortexing and standing for 2 min, the organic acids were extracted by adding 225 μl of n-hexane [39, 40], and supernatants were measured by gas chromatography-mass spectrometry on a Trace 1300/Exactive GC apparatus (Thermo Fisher Scientific, Waltham, MA, USA) equipped with flame electron impact ionization and an Rtx-WAX column (30 m × 0.25 mm × 0.25 μm, Bellefonte, PA, USA).

Fecal Untargeted Metabolomics

Fecal untargeted metabolomics was based on liquid chromatography-mass spectrometry. Fecal samples collected before euthanization were stored at − 80 °C and then thawed to room temperature for processing. Water, methanol, and acetonitrile, in order, were selected as solvent for extraction. In brief, each fecal sample of 80 mg was extracted 3 times with 200 μl of solvent by ultrasonication in a noncontact ultrasonic cell grinder (Scientz08-I, Zhejiang, China) for 15 min, vortex-mixing for 2 min, and then centrifugation at 4 °C at 16,000×g for 15 min. The supernatants from the three extractions were transferred to a new 1.5 ml centrifuge tube and vortex-mixed [41]. The mixed extracts were dried with CentriVap (Labconco, Kansas City, USA), redissolved with 200 μl of mixed solvent (water/methanol/acetonitrile = 1:1:1), and then centrifuged at 4 °C at 16,000×g for 5 min. The supernatants, containing fecal metabolomics, were measured by liquid chromatography-mass spectrometry Q Exactive Plus (Thermo Fisher Scientific) equipped with a TSKgel Amide–80 5-μm column (0019697, Tosoh Corporation, Tokyo, Japan). For normalization, the spectral counts of each metabolite were divided by the total spectral counts of all metabolites from the same sample. The data transformation is log transformation. The tool used for statistical analysis is Compound Discoverer 2.1. The database used for identification of peaks is mzCloud.

Statistical Analysis

Microbiome population and untargeted metabolomics statistics are described in detail above. Excluding these, the SPSS 22.0 software was used for data analysis. Statistical analysis was conducted by one-way ANOVA with an LSD post hoc assay. Data are presented as mean ± SEM (standard error of the mean), and p < 0.05 was set as the threshold for significance (*p < 0.05, **p < 0.01, ***p < 0.001). All data were displayed using GraphPad Prism version 7 (GraphPad, Inc., La Jolla, CA, USA).