Progression of Parkinson's disease is associated with gut dysbiosis: Two-year follow-up study

<div><p>Background</p><p>We previously reported gut dysbiosis in patients with Parkinson’s disease (PD).</p><p>Objective</p><p>The aim of this study is to examine whether gut dysbiosis correlates with the progression of PD.</p><p>Methods</p><p>We examined changes in gut microbiota and demographic features in 2 years in 36 PD patients.</p><p>Results</p><p>A change of total UPDRS scores in 2 years was predicted by the counts of <i>Bifidobacterium</i> and <i>Atopobium</i> cluster at year 0 with a correlation coefficient of 0.52. Correlation analysis additionally revealed that low counts of <i>Bifidobacterium</i> and <i>Bacteroides fragilis</i> at year 0 were associated with worsening of UPDRS I scores in 2 years. In addition, low counts of <i>Bifidobacterium</i> at year 0 were associated with worsening of hallucinations/delusions in 2 years. Similarly, low counts of <i>B</i>. <i>fragilis</i> at year 0 were associated with worsening of motivation/initiative in 2 years. The patients were evenly divided into the deteriorated and stable groups based on the degree of worsening of total UPDRS scores. The deteriorated group had lower counts of <i>Bifidobacterium</i>, <i>B</i>. <i>fragilis</i>, and <i>Clostridium leptium</i> than the stable group at year 0 but not at year 2, suggesting that the deteriorated group may demonstrate accelerated lowering of these bacteria at year 0.</p><p>Conclusions</p><p>The total counts of intestinal bacterial decrease in the course of PD progression. Temporal profiles of lowering of bacterial counts are likely to be different from bacteria to bacteria, and also between the deteriorating and stable groups, which may be able to be exploited to differentiate patients with rapidly and slowly progressive PD pathology.</p></div

Difference of bacterial counts between years 0 and 2.

<p>Difference of bacterial counts between years 0 and 2.</p

Prediction of worsening of UPDRS scores using gut microbiota at year 0.

<p><b>(A)</b> Geometric plot of the counts of <i>Bifidobacterium</i> at year 0 in the deteriorated and stable groups. <i>P</i>-value was corrected by the Benjamini and Hochberg method to calculate the false discovery rate (FDR) (<i>q</i>-value). <b>(B)</b> A stepwise linear regression analysis for predicting a change of UPDRS scores in 2 years using the counts of 10 bacterial groups/genera/species at year 0 yielded the plotted prediction model, which is comprised of the counts of <i>Bifidobacterium</i> (standardized β = -0.45, <i>p</i> < 0.05) and <i>Atopobium</i> cluster (standardized β = 0.50, <i>p</i> < 0.01). <b>(C, D)</b> Correlation between a change of UPDRS I scores in 2 years and the count of <i>Bifidobacterium</i> at year 0 <b>(C)</b> and the count of <i>B</i>. <i>fragilis</i> group at year 0 <b>(D)</b>. <b>(E)</b> Correlation between a change of thought disorder scores (Item 2 in UPDRS I) in 2 years and the count of <i>Bifidobacterium</i> at year 0. <b>(F)</b> Correlation between a change of motivation/initiative scores (Item 4 in UPDRS I) in 2 years and the count of <i>B</i>. <i>fragilis</i> group at year 0. <b>(G, H)</b> Correlation between a change of LBP concentrations in 2 years and the count of <i>L</i>. <i>brevis</i> at year 0 <b>(G)</b> and the count of <i>L</i>. <i>plantarum</i> at year 0 <b>(H)</b>. <b>(B, C, D, E, and F)</b> Pearson’s correlation coefficients (r) are indicated with respective <i>p</i>-values.</p

Comparison of demographic features between the deteriorated and stable groups.

<p>Comparison of demographic features between the deteriorated and stable groups.</p

Schematic diagram of <i>L. casei</i> AsnH and glutaminase activity of AsnH proteins.

<p>(A) Protein sequence alignment was performed using the GENETYX program. Solid boxes indicate highly conserved regions. Regions containing residues important for enzymatic activities are shown in detail (identical residues are indicated by boldface type). The first methionine was consistently included in the numbering and was designated Met-1. Asterisks indicate mutated residues in this study that were critical to glutaminase or synthetase activities. (B) Recombinant AsnH_WT, mutant AsnH_C2S, and mutant AsnH_D265N (0.1 µg) were examined using the glutaminase activity test. Five microliters of a reaction mixture (initial volume, 50 µl) were quenched at 30 min to estimate glutamic acid concentration. Glutaminase activity was monitored in the presence of aspartic acid or cell wall extracts of ATCC 27139, ATP or AMP–PNP plus cell wall extracts. Results are depicted as the means ± standard deviations (SD). Statistical significance was calculated using the Student's <i>t</i>-test. Significant differences indicated between the control and treated groups. ***, <i>p</i><0.001.</p

Partial sequence alignment of <i>L. casei</i> AsnH and orthologs.

<p>Partial sequences were selected from highly conserved regions of the following <i>L. casei</i> AsnH orthologs: <i>L. lactis</i>, <i>R. erythropolis</i>, <i>M. smegmatis</i>, and <i>E. coli</i>. A black background indicates identical residues in all bacteria.</p

Micrographs of <i>L. casei</i> cells.

<p>(A) Gram staining was performed using Fiber G. The cells were observed under a light microscope (×1000). (B) Representative micrographs indicating cell morphologies in several different fields. Digitally magnified (×3) views of typical cell surfaces are shown in the insets. Scale bars = 100 nm. PS, polysaccharide; PG, peptidoglycan; CP, cytoplasm. (C) Peptidoglycan thickness of <i>L. casei</i> cells are presented as the means ± standard error of the mean (SEM) (n = 5). Statistical significance was calculated using the Student's <i>t</i>-test. *, <i>p</i><0.05; **, <i>p</i><0.01; ***, <i>p</i><0.001.</p

Summary of relative sensitivities to J1 phage infection and predicted transposon insertion loci of J1 phage-resistant mutant.

<p>^a The cells of <i>L. casei</i> ATCC 27139 and isogenic Tn<i>5</i> insertion mutants (1.0×10⁷ CFU/ml) were combined with J1 phage (5.0×10⁶ PFU/ml) in ILS broth at an moi of 0.5, and PFU were counted after incubation at 37°C for 24 h. The relative sensitivities to J1 phage were assessed as the ratio of PFU for transposon insertion mutants to that of the wild-type strain.</p><p>^b The genes into which transposons were inserted were identified using BLAST.</p

Induction of cytokine in spleen after <i>L. casei</i> administration.

<p>Heat-killed preparations of <i>L. casei</i> ATCC 27139 or isogenic Tn<i>5</i> insertion mutants (800 µg) were injected i.v. into BALB/c mice. Six mice per group were sacrificed 8 h after injection, and IL-12 (A), TNF-α (B), and IFN-γ (C) proteins were measured from spleen homogenates by ELISA. Columns: white, saline; black, <i>L. casei</i> wild-type; slashed, <i>L. casei</i> J1 phage-resistant mutant; gray, <i>L. casei asnH</i> mutant complemented with the cloned <i>asnH</i>. Results are depicted as the means ± standard deviations (SD). Statistical significance was calculated using the Student's <i>t</i>-test. Significant differences indicated between the control and treated groups. ***, <i>p</i><0.001.</p

Anti-listerial activity of J1 phage-resistant Tn<i>5</i> insertion mutants of <i>L. casei</i>.

<p>Heat-killed preparations of <i>L. casei</i> ATCC 27139 or isogenic Tn<i>5</i> insertion mutants (800 µg) were injected i.v. into BALB/c mice 6 days before an i.v. challenge with <i>L. monocytogenes</i> (2.0×10⁶ CFU/mouse). Six mice per group were dissected 24 h after the challenge, and viable <i>Listeria</i> were detected in spleens. Columns: white, saline; black, <i>L. casei</i> wild-type; slashed, <i>L. casei</i> J1 phage-resistant mutant; gray, <i>L. casei asnH</i> mutant complemented with the cloned <i>asnH</i>. Results are depicted as the means ± standard deviations (SD). Statistical significance was calculated using the Student's <i>t</i>-test. Significant differences indicated between the control and treated groups. ***, <i>p</i><0.001.</p