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. 2020 Jan 22;10(1):25.
doi: 10.1038/s41398-020-0719-8.

Long-term probiotic intervention mitigates memory dysfunction through a novel H3K27me3-based mechanism in lead-exposed rats

Jie Xiao #  1   2 , Tian Wang #  1   2 , Yi Xu #  3   4 , Xiaozhen Gu  1   2 , Danyang Li  1   2 , Kang Niu  1   2 , Tiandong Wang  1   2 , Jing Zhao  1   2 , Ruiqing Zhou  1   2 , Hui-Li Wang  5   6
Affiliations

Long-term probiotic intervention mitigates memory dysfunction through a novel H3K27me3-based mechanism in lead-exposed rats

Jie Xiao et al. Transl Psychiatry. .

Abstract

Chronic lead exposure is associated with the development of neurodegenerative diseases, characterized by the long-term memory decline. However, whether this pathogenesis could be prevented through adjusting gut microbiota is not yet understood. To address the issue, pregnant rats and their female offspring were treated with lead (125 ppm) or separately the extra probiotics (1010 organisms/rat/day) till adulthood. For results, memory dysfunction was alleviated by the treatment of multispecies probiotics. Meanwhile, the gut microbiota composition was partially normalized against lead-exposed rats, which in turn mediated the memory repairment via fecal transplantation trials. In the molecular aspect, the decreased H3K27me3 (trimethylation of histone H3 Lys 27) in the adult hippocampus was restored with probiotic intervention, an epigenetic event mediated by EZH2 (enhancer of zeste homolog 2) at early developmental stage. In a neural cellular model, EZH2 overexpression showed the similar rescue effect with probiotics, whereas its blockade led to the neural re-damages. Regarding the gut-brain inflammatory mediators, the disrupted IL-6 (interleukin 6) expression was resumed by probiotic treatment. Intraperitoneal injection of tocilizumab, an IL-6 receptor antagonist, upregulated the hippocampal EZH2 level and consequently alleviated the memory injuries. In conclusion, reshaping gut microbiota could mitigate memory dysfunction caused by chronic lead exposure, wherein the inflammation-hippocampal epigenetic pathway of IL-6-EZH2-H3K27me3, was first proposed to mediate the studied gut-brain communication. These findings provided insight with epigenetic mechanisms underlying a unique gut-brain interaction, shedding light on the safe and non-invasive treatment of neurodegenerative disorders with environmental etiology.

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Conflict of interest statement

The authors declare that they have no conflict of interest.

Figures

Fig. 1
Fig. 1. Probiotics alleviated the lead-led memory impairment.
a–e MWM tests assessing capacities of rats to be trained to find the hidden platform (n = 10). Latency of rats to find the platform was recorded and analyzed during the training days (a). On the test day, based on their respective moving tracks (b), number of times crossing the hidden platform (c) and distance traveled in the target quadrant (d) were counted at PND 61. The total distance traveled was also recorded to evaluate the locomotor movement of rats (e). f Y-maze test assessing capacities of rats to perform the spontaneous alteration (n = 10). Spontaneous alteration percentages of each group, as well as their respective moving tracks are shown. Ctrl non-treated rats, Pb lead-treated rats, Pb+prob lead and probiotics-treated rats, prob probiotics-treated rats. The data are represented as mean ± SEM; ***p < 0.001, *p < 0.05. For a, repeated-measures ANOVA was performed and asterisk (*) refers to the significance of differences between the Pb and Ctrl groups on the indicated training day.
Fig. 2
Fig. 2. Probiotics rescued the morphological abnormalities of dendritic spines.
At adolescence (PND 22), representative images (~50 cells, n = 6) and quantification of spine densities are shown in the region of CA1 (a, b) and DG (c, d). Each point in the graph represents a single cell subjected to analysis. At adulthood (PND 68), representative images and quantification of spine densities are shown in the region of CA1 (e, f) and DG (g, h). To manifest the tested hippocampal regions, typical morphologies of neurons in the regions of CA1 and DG are presented in the overall perspective (i). The scale bar represents 50 μm. j, k Lead concentration in the rat brains (j) and blood (k) of each group (n = 6). Ctrl non-treated rats, Pb lead-treated rats, Pb+prob lead and probiotics-treated rats, prob probiotics-treated rats. The data are represented as mean ± SEM; ***p < 0.001, **p < 0.01, NS p > 0.05.
Fig. 3
Fig. 3. Probiotic treatment restored specific microbiota changes in lead-exposed rats (n = 6).
a Analysis of alpha diversity-predicted diversity of gut microbiota by Chao1, Shannon, and Simpson analysis. b Non-metric multi-dimensional scaling (NMDS) analysis based on unweighted UniFrac metrics of gut microbiota where samples of rats from different groups are highlighted with different colors. The position and distance of data points indicated the degree of similarity in terms of bacterial taxonomies. The Permanova/Adonis analysis was shown on the right-upper corner. c Stacked bar chart shows microbiota composition at the phylum level. d Relative abundance of the major phyla of gut microbiota upon each treatment. e Heatmap shows relative abundance of representative microbiota at the genus level in four groups. f Microbial taxa identified to significantly differ in abundance (LDA score >2.0, P < 0.05) across the tested groups (Ctrl, Pb, Pb+prob) using LEfSe analysis. g A cladogram representation of LEfSe analysis. The plot shows microbial taxa from phylum to genus level, with taxa significantly enriched (P < 0.05) in the Pb group indicated in red and in Pb+prob indicated in blue. Abbreviations for microbial names are listed right to the graph. Ctrl non-treated rats, Pb lead-treated rats, Pb+prob lead and probiotics-treated rats, prob probiotics-treated rats. The data are represented as mean ± SEM; **p < 0.01, *p < 0.05. p Value was FDR corrected in multiple (>3) comparisons.
Fig. 4
Fig. 4. Fecal transplantation alleviated the lead-led memory impairment.
a–e MWM tests assessing the capacities of rats to be trained to find the hidden platform, and the comparisons were performed between recipient rats transplanted with feces from either lead (n = 10) or lead+probiotics (n = 8) treated donors. Latency of rats to find the platform was recorded and analyzed during the training days (a). On the test day, based on their respective moving tracks (b), number of times crossing the hidden platform (c) and distance traveled in the target quadrant (d) were counted at PND 61. The total distance traveled was also recorded to evaluate the locomotor movement of rats (e). f Y-maze test assessing the capacities of rats to perform the spontaneous alteration, and the comparison was performed between recipient rats transplanted with feces from either lead (n = 10) or lead+probiotics (n = 8) treated donors. Spontaneous alteration percentages of each group were shown. Pb lead-treated rats, Pb.feces fecal transplantation from donor rats treated with lead, Pb+prob.feces fecal transplantation from donor rats treated with lead+probiotics. The data are represented as mean ± SEM; *p < 0.05. For a, repeated-measures ANOVA was performed and asterisk (*) refers to the significance of differences between the Pb/Pb.feces and Pb/Pb+prob.feces groups on the indicated training day.
Fig. 5
Fig. 5. Probiotics reversed the H3K27me3 level in the rat hippocampus (n = 4).
a Immunoblots and quantification of H3K27me3, H3K4me2, and H3K4me3 levels in the hippocampus of adult rats. b Immunostaining of H3K27me3 (green) and nucleus (blue) in DG region of adult rats hippocampus. The scale bar represents 50 μm. c Immunoblots and quantification of EZH2 levels in the hippocampus of adolescent rats. Ctrl non-treated rats, Pb lead-treated rats, Pb+prob lead and probiotics-treated rats, prob probiotics-treated rats. The data are represented as mean ± SEM; ***p < 0.001, **p < 0.01, *p < 0.05.
Fig. 6
Fig. 6. EZH2-H3K27me3 mediated the probiotic alleviation of lead neurotoxicity.
Immunoblots (a) and quantification (b) of EZH2 levels in PC-12 cells supplemented with rat serum (n = 3). c Immunoblots of H3K27me3 and EZH2 levels in PC-12 cells transfected with EZH2-overepressing (OE-EZH2), EZH2-knockdown (KD-EZH2), or empty (GFP) plasmid (n = 3), and the quantifications of EZH2 (d) and H3K27me3 (e) are shown. f Representative images of neurite outgrowth in PC-12 cells upon various treatments; the scale bar represents 20 μm. Number of primary branches (g), secondary branches (h), and sholl analysis (i) of neurite outgrowth of PC-12 cells upon various treatments (~50 cells). Ctrl cells treated with serum of normal rats, prob cells treated with serum of probiotic rats, Pb cells treated with lead and normal rat serum, Pb+prob cells treated with lead and probiotic rat serum; the transfection of corresponding plasmids was involved in b–f. The data are represented as mean ± SEM; ***p < 0.001, **p < 0.01, *p < 0.05, p value was FDR corrected in multiple (>3) comparisons.
Fig. 7
Fig. 7. IL-6 mediated EZH2 changes and the resulting memory recovery.
a The concentration of IL-6 in the serum of each group of rats, as detected by ELISA (n = 6). b Immunoblots and quantification of EZH2 and H3K27me3 levels in the hippocampus of adolescent rats, in response to tocilizumab injection (n = 4). c–g MWM tests assessing the capacities of rats to be trained to find the hidden platform (n = 10). Latency of rats to find the platform was recorded and analyzed during the training days (c). On the test day, based on their respective moving tracks (d), number of times crossing the hidden platform (e) and distance traveled in the target quadrant (f) were counted at PND 61. The total distance traveled was also recorded to evaluate the locomotor movement of the tested rats (g). h Y-maze test assessing the capacities of rats to perform the spontaneous alteration. Spontaneous alteration percentage of each group was shown (n = 10). Ctrl non-treated rats, Pb lead-treated rats, Pb+prob lead and probiotics-treated rats, Pb+tocil lead and tocilizumab-treated rats. The data are represented as mean ± SEM; ***p < 0.001, **p < 0.01, *p < 0.05. For c, two-way ANOVA was performed and asterisk (*) refers to the significance of differences between the Pb and Ctrl groups on the indicated training day.
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