A neurotherapeutic approach with Lacticaseibacillus rhamnosus E9 on gut microbiota and intestinal barrier in MPTP-induced mouse model of Parkinson’s disease

A previously described Lacticaseibacillus rhamnosus strain isolated from healthy infant feces (L. rhamnosus E9) was used in this study^43,53. L. rhamnosus E9 was maintained at − 30 °C in MRS broth (Merck, Germany) with 10% (v/v) glycerol. Working cultures were prepared from frozen stocks by two sequential transfers in MRS broth and incubations were conducted statically at 37 °C for 24 h and 18 h, respectively. The culture at early stationary phase was harvested by centrifugation at 5000 rpm for 10 min at room temperature. The pellet was re-suspended in 0.85% NaCl (w/v) and the optical density at 600 nm (OD600) was determined to obtain a final concentration of 10⁹ CFU/ml³². The final culture solution that was kept at 4 °C until the administration was enumerated daily on MRS agar to confirm the dose administered to the mice which is10⁸ CFU/day.

After preadaptation and training, behavioral tests for motor functions were performed until the sacrifice. Tests were applied at the same time in the same order.

After sacrification, brain and intestinal tissues were dissected intact and fixed in 10% neutral buffered formaldehyde. Sections were taken from the brain tissues in the coronal plane and from the intestinal tissues in the luminal plane. After tissue processing, paraffin embedding was performed and 4 micron sections were obtained for immunohistochemistry. Histopathological sections were kept in an oven at 70 °C for 30 min. and deparaffinized. The sections were then incubated in a graded alcohol series (100%, 100%, and 95%) for 5 min each and then rinsed under running tap water for 5 min. Antigen retrieval was performed in pH 6 citrate solution or pH 9 tris/EDTA solution depending on the antigen. The Leica Bond Max Autostainer (Shandon, Frankfurt, Germany) was used with the appropriate Leica brand secondary antibody kit. Primary antibodies against tyrosine hydroxylase (TH) (1/100, Biolegend, San Diego, CA, USA), SOX-10 (1/200, Biolegend, San Diego, CA, USA), GFAP (prediluted, Boster Lab, Pleasanton, CA, USA), Zonula Occluden-1 (ZO-1) (1/200, Boster Lab, Pleasanton, CA, USA), and Occludin (1/100, Booster Lab, Pleasanton, CA, USA) were stained using Leica Bond Max Autostainer (Frankfurt, Germany). The evaluation of immunohistochemistry was performed using different methods according to the tissue characteristics and the staining pattern. The detail of the evaluation for each antibody as follows; TH, from each brain tissue, 100 × microphotographs were obtained (Olympus, cellSens software, Tokyo, Japan) from the demonstrative striatum region and pixels in these photographs were converted into numerical values in the Image J software ("color threshold" tool) developed by the NIH (US Department of Health). SOX-10, the luminal sections were examined under a light microscope and positive nuclear reactions in submucosal and myenteric plexus were manually counted. GFAP, expression in the submucosal and the myenteric plexus localization representing the luminal neuronal axis was examined. While microphotographs were taken, mucosa was removed from the sections to rule out cross-reactions and to obtain a more definite result (to reduce the error of digital analysis). For the staining intensities on the wall outside the mucosa, pixel calculation was performed using the image J software. Occludin and ZO-1, evaluation of the epithelial tight junction proteins was performed on the mucosa. The staining intensity in the photographs were scored from 1 to 4 (score 1: barely visible staining, score 2: faint staining, score 3: neither strong nor faint staining, score 4: strong positive reaction).

Weighed brain and distal small intestine tissues were homogenized in ultra-pure-guanidine isothiocyanate (Sigma-Aldrich) using innuSPEED Lysis Tubes P in SpeedMill Plus (Analytik Jena, Germany). Total RNA was isolated and DNA contamination was removed using PureLink™ DNase Set (Invitrogen). Total RNA was converted into cDNA using High-Capacity cDNA Reverse Transcription Kit (Applied Biosystems) according to supplier’s protocol. qPCR was performed using the primers shown at Table S1. AMPIGENE qPCR Green Mix Hi-ROX (Enzo Life Sciences) was used under the following conditions: 95 °C for 2 min for initial denaturation, followed by 5 s (40 cycles) at 95 °C and 30 s at 57 °C. Data were generated in the final step at 95 °C for 15 s and melting curves (65 to 95 °C) were acquired at the end for each primer set. Relative gene expression was calculated by 2^−ΔΔCt method by normalizing gene expression to β-actin⁶⁰.

Brain tissues for detection of dopamine and ROS level were homogenized in T-PER™ Tissue Protein Extraction Reagent and Halt Protease Inhibitor Cocktail, EDTA-Free (Thermo Scientific) using innuSPEED Lysis Tubes E in SpeedMill Plus (Analytik Jena, Germany)^61–63. After centrifugation at max. speed for 5 min, the supernatant was used to carry out the experimental procedure as recommended by the manufacturer and the optical densities were measured at 450 nm.

After weighing, the cecum content was homogenized in PBS and total DNA was extracted using the QIAamp Fast DNA Stool-Mini Kit (Qiagen Sciences, MD) as described previously³². Briefly, a mechanical cell-disruption step was included by 0.1 mm glass-beads (Sigma-Aldrich) and specimens were beaten at maximum speed for six 1-min using the SpeedMill Plus (Analytik Jena, Germany) with intervals of 2-min on ice. Subsequently, a heat treatment step was carried out at 95 °C for 5 min and DNA was extracted. After DNA was quantified by NanoDrop spectrophotometer (Epoch, BioTek), 16S rRNA sequencing was performed using the Illumina NovaSeq 6000 at BMlabosis (Ankara, Turkey).

The V3-V4 region was amplified for library preparation, using specific primers (F-5ʹTCGTCGGCAGCGTCAGATGTGTATAAGAGACAGCCTACGGGNGGCWGCAG-3ʹ, R-5ʹGTCTCGTGGGCTCGGAGATGTGTATAAGAGACAGGACTACHVGGGTATCTAATCC-3ʹ). The Nextera XT index kit (Illumina, San Diego, CA) was used to attach unique identifiers to both 5′ and 3′ ends. The amplicons with equal quantities were pooled after PCR products were cleaned up using AMPure XP magnetic beads. Paired end reads sequenced on NovaSeq 6000 system underwent a quality-filtering and were trimmed with quality cut-off of Phred score of 20 by DADA2. OTUs were generated using DADA2 and data analysis was assessed in QIIME2 framework. The sequences were submitted to the Sequence Read Archive (SRA) database with the accession number of PRJNA1015441.

The sample size was estimated to be five/group with 80% power (unpaired t-test; α = 0.05) to detect differences between the control and the treatment groups^21,64. For microbiome, the statistical difference between groups was analyzed using the Mont Carlo test, a non-parametric test based on random permutations, in package ade4 generated by R 3.6.1 as described by de Carcer et al.^65–68. The statistical difference of the between groups was evaluated with the randtest.between function in package ade4 of R 3.6.1. The zero (0) values were replaced with the limit of detection, which is assessed by the ratio of one to the lowest number of read in the data set. The Benjamini Hochberg procedure was applied to manage the false-discovery rate. The dominant genera that raised or diminished in abundance were identified by correspondence analysis in package ade4 of R 3.6.1⁶⁸. Statistical difference for the rest of the analyses was performed with the Student’s t test or the Wilcoxon rank sum test using JMP Pro (SAS Institute Inc., Cary, NC) and introduced as mean ± SD. Statistical difference was ascertained at a P value of ≤ 0.05.

Factors affecting the neural degeneration in sporadic PD patients include an increase in the production of free radicals and oxidative stress^7,8. Reactive oxygen and nitrogen are mainly produced in mitochondria as a result of aerobic metabolism. Incomplete induction of oxygen and nitrogen causes the formation of radicals such as superoxide radical (O2-), hydrogen peroxide (H2O2), and nitric oxide (NO). These radicals can cause oxidative damage to proteins, membranes, and DNA⁸⁵. Oxidative stress has found to be high in the brain tissues of PD patients⁸⁶. Reactive oxygen species (ROS) in the brain was determined to evaluate oxidative damage resulting from inflammation. In the MPTP group, a significant (p < 0.05) increase was observed in the ROS level compared to the control group. It was not significantly different in mice administered E9 from the control. E9 reduced the level of ROS formed in the striatal tissue by MPTP injection (Fig. 4). ROS has been shown to correlate positively with the inflammation in the brain of PD mice⁸⁷. Additionally, studies suggest that production of ROS leads to misfolding of α-syn in the brain and contributes to development of synucleinopathies including PD⁸⁸. Lactobacilli administration has been previously shown to prevent α-syn aggregation in substantia nigra of MPTP-induced mice by inhibiting the oxidative damage and the inflammation in brain⁸⁷. Further investigation of the impact of E9 on inflammation in MPTP-induced mouse model of PD would provide more insight into the mechanism of action by E9 on oxidative stress and neuroprotection.

Chronic inflammatory state, an imbalance between pro-inflammatories and anti-inflammatories has been thought to be the main cause of PD, which accelerates aging and damages neural cells^85,103. Gastrointestinal microbiota is one of the main factors affecting and even regulating the immune system in the host^104,105. Gut microbiota is also known to communicate with the brain and this communication maintains the gastrointestinal and the nervous system homeostasis along with the immune system^13–15. A disruption in the communication within the gut-brain axis is associated with various neurodegenerative diseases including PD¹⁶. As a matter of fact, clinical studies have revealed dysbiosis in the intestinal microbiota of PD patients^19–21. Recent research shows that histological and behavioral findings in PD are associated with dysbiosis in the gut microbiome and the potential of probiotics to prevent PD progression is one of the latest innovations in the field of neurodegenerative diseases^70,106,107. However, studies unrevealing the mechanism of probiotic action on PD pathogenesis with the association of the brain-gut axis is limited. In our study, we demonstrated the changes in the microbial composition in MPTP-induced mouse model of PD and impact of E9 administration on the altered cecal microbiota by MPTP injection using 16S rRNA sequencing. As a result of sequencing, a total of 1,776,677 filtered reads were obtained from the cecal content samples of 20 mice. The number of reads per sample ranged from 47,182–168,617 with an average number of 88,833.85 reads/sample. After assigning the each read to a taxonomic level, 18 phyla, 26 classes, 54 orders, 108 families and 257 genera were identified. To assess whether sufficient sequence reads had been obtained to accurately determine the diversity of organisms present, Chao1 and Shannon index were computed (Fig. S1). Additionally, a principal coordinate analysis (PCoA) plots using weighted Unifrac distances for beta diversity were generated.

E9 strain used in this study is known to be a high EPS producer^43–45. EPSs are shown to have immunomodulatory effect and regulate the gut microbiota and the dopaminergic system in mice^46–50. Modulating the gut microbiota, taking role in the neurodegenerative diseases, by E9 could be associated with the ability of E9 to produce high amount of EPS known to have biological activities such as anti-proliferative and anti-inflammatory activity, and neuroprotective effect^43–45,51 Since the impact of EPSs on PD has not been studied yet, further investigation on E9 EPSs for their impact on immunomodulation and PD development could be significant to understand the mechanism of probiotic action.

Supplementary Information.