Dual-Hit Model of Parkinson's Disease: Impact of Dysbiosis on 6-Hydroxydopamine-Insulted Mice-Neuroprotective and Anti-Inflammatory Effects of Butyrate

Abstract

Recent evidence highlights Parkinson's disease (PD) initiation in the gut as the prodromal phase of neurodegeneration. Gut impairment due to microbial dysbiosis could affect PD pathogenesis and progression. Here, we propose a two-hit model of PD through ceftriaxone (CFX)-induced dysbiosis and gut inflammation before the 6-hydroxydopamine (6-OHDA) intrastriatal injection to mimic dysfunctional gut-associated mechanisms preceding PD onset. Therefore, we showed that dysbiosis and gut damage amplified PD progression, worsening motor deficits induced by 6-OHDA up to 14 days post intrastriatal injection. This effect was accompanied by a significant increase in neuronal dopaminergic loss (reduced tyrosine hydroxylase expression and increased Bcl-2/Bax ratio). Notably, CFX pretreatment also enhanced systemic and colon inflammation of dual-hit subjected mice. The exacerbated inflammatory response ran in tandem with a worsening of colonic architecture and gut microbiota perturbation. Finally, we demonstrated the beneficial effect of post-biotic sodium butyrate in limiting at once motor deficits, neuroinflammation, and colon damage and re-shaping microbiota composition in this novel dual-hit model of PD. Taken together, the bidirectional communication of the microbiota-gut-brain axis and the recapitulation of PD prodromal/pathogenic features make this new paradigm a useful tool for testing or repurposing new multi-target compounds in the treatment of PD.

Keywords: antibiotic-induced intestinal injury; gut microbiota; neurodegenerative disorders; neuroinflammation; short-chain fatty acids.

Figure 1

Grouping and schedule (A). Effects of BuNa (100 mg/kg) on motor coordination in 6-OHDA-lesioned mice pretreated or not with CFX (B–D). CD1 mice were treated with BuNa daily, and time spent on the rod (sec) was assessed and recorded at 3 (A), 7 (C), and 14 (D) days. Values are expressed as mean ± SEM of retention time on the rotating bar. Evaluation of apomorphine-induced rotational behavior (E–G). The number of net ipsilateral and contralateral rotations was counted per minute in all animals from all groups after 3 (E), 7 (F), and 14 (G) days. Values are expressed as mean ± SEM (n = 8–12) of all turns collected during 40 min. Labeled means without a common letter differ, p < 0.05.

Figure 2

Neuroprotective effects of BuNa (100 mg/kg) measured using tyrosine hydroxylase, inflammatory and apoptotic markers in the striatum in mice: (A) TH (B) nNOS, (C) iNOS (D) COX-2, (E) Bcl-2 and Bax expression reported as the ratio of optical densities of their bands. Representative immunoblots of all tissues analyzed were shown. Densitometric analysis of protein bands is reported: the levels are expressed as the density ratio of target to control protein bands (β-actin). Values are expressed as mean ± SEM (n = 4–6). Labeled means without a common letter differ, p < 0.05.

Figure 3

BuNa-modulated systemic parameters of the immune system and inflammation altered by 6-OHDA and CFX. BuNa-limited (A) TNF-α, (B) IL-1β, (C) IFN-γ, and (D) LPS increased in 6-OHDA and/or CFX+6-OHDA mice. All data are shown as mean ± S.E.M (n = 6 per group). Labeled means without a common letter differ, p < 0.05.

Figure 4

Histological analysis of distal colon sections of mice. (A) Haematoxylin & eosin staining (magnification ×400). (B–D) Transcriptional levels of Il1b, Ptgs2, and Il10 were evaluated in colonic tissues. All data are shown as mean ± S.E.M (n = 4–6). Labeled means without a common letter differ, p < 0.05.

Figure 5

Antibiotic-induced dysbiosis in 6-OHDA-lesioned mice. (A) Venn diagram illustrating bacterial genera overlaps among core microbiomes with an attached list of the 26 genera missing in 6-OHDA+CFX to Sham group; in red are the 7 bacterial genera that specifically failed to recover in 6-OHDA+CFX. (B–D) Gut microbiota differences at genus taxonomic level based on linear discriminant analysis (LDA) combined with effect size (LEfSe) algorithm (p > 0.05 for both Kruskal–Wallis and pairwise Wilcoxon tests and a cutoff value of LDA score above 2.0). In each panel, LDA scores, p-values, and relative abundance of key genera discriminating bacterial communities are reported, when possible, and the species taxonomic classification/genus is also reported.

Figure 6

Co-occurrence network of species belonging to key genera discriminating Sham, 6-OHDA, CFX, and 6-OHDA+CFX mice. In the networks: the nodes represent the species, and the edges show nonparametric Spearman correlations with a correlation coefficient >0.5 or ≤0.5 that is statistically significant (p < 0.05 after Bonferroni correction), within green copresence interaction type, while in red mutual exclusion interaction type; node and label sizes are proportional to the relative abundance and its degree (the number of edges and nodes connected to each node), respectively. The colors of nodes represent their classification at the phylum level (green: Actinobacteria; blue: Bacteroidetes; pink: Firmicutes; yellow: Proteobacteria; grey: TM7).

Figure 7

BuNa-restructured gut microbiota in 6-OHDA+CFX mice. (A) Venn diagram illustrating bacterial genera overlaps among core microbiomes with an attached list of the 7 genera shared by Sham and 6-OHDA+CFX+BuNa mice and missing in 6-OHDA+CFX mice. (B) Gut microbiota differences at genus taxonomic level based on linear discriminant analysis (LDA) combined with effect size (LEfSe) algorithm (p > 0.05 for both Kruskal–Wallis and pairwise Wilcoxon tests and a cutoff value of LDA score above 2.0). LDA scores, p-values, and relative abundance of key genera discriminating bacterial communities are reported in each panel.