High-throughput 16S rRNA gene sequencing reveals gut microbial changes in 6-hydroxydopamine-induced Parkinson’s disease mice

Recently, there has been a rapid increase in studies on the relationship between brain diseases and gut microbiota, and clinical evidence on gut microbial changes in Parkinson’s disease (PD) has accumulated. 6-hydroxydopamine (6-OHDA) is a widely used neurotoxin that leads to PD pathogenesis, but whether the alterations of gut microbial community in 6-OHDA-treated mice has not been investigated. Here we performed the 16S rRNA gene sequencing to analyze changes in gut microbial community of mice. We found that there were no significant changes in species richness and its diversity in the 6-OHDA-lesioned mice. The relative abundance of Lactobacillus gasseri and L. reuteri probiotic species in feces of 6-OHDA-lesioned mice was significantly decreased compared with those of sham-operated mice, while the commensal bacterium Bacteroides acidifaciens in 6-OHDA-treated mice was remarkably higher than sham-operated mice. These results provides a baseline for understanding the microbial communities of 6-OHDA-induced PD model to investigate the role of gut microbiota in the pathogenesis of PD.

triggered by gut microbiota changes could enhance the inflammatory reactions that induce 48 misfolded α-synuclein, which is a pathological hallmark of PD (12, 13). 49 Scheperjans and his colleagues explored the relationship between gut microbiota changes 50 and clinical phenotypes of PD with fecal microbiome analysis, which showed a reduction in 51 beneficial Prevotellaceae and an elevation of pathogenic Enterobacteriaceae in PD patients 52 with severe gait disturbance (14). In addition, Keshavarzian et al (15) reported that anti-53 inflammatory bacterial genera such as Blautia, Coprococcus, and Roseburia were less 54 abundant in feces of PD patients whereas Ralstonia known as a pro-inflammatory bacterial 55 genus was more abundant in the mucosa of PD patients. These studies suggest that changes in 56 gut microbiota composition are closely associated with PD pathogenesis, however, whether gut 57 microbial changes could occur by intracerebral injection of chemical neurotoxins that cause 4 58 the pathogenesis of PD has not been studied yet. 59 This study aimed to investigate whether unilateral brain lesions induced by intracerebral 60 injection of 6-hydroxydopamine (6-OHDA) neurotoxin, which not only causes the death of 61 nigrostriatal dopaminergic neurons in brain but also GI dysfunctions such as gastroparesis (16,62 17), affects the alteration of gut microbiota composition. To do this, we administrated 6-OHDA 63 directly to the striatum of mouse brain and performed high-throughput sequencing of 16S

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The condition of secondary amplification is equal to the former one except the amplification 107 cycle set to 8. The PCR product was confirmed by using 2% agarose gel electrophoresis and

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The alteration of bacterial species richness in 6-OHDA-lesioned mice 147 All analyzed sequences contained at least 2 of the V3 and V4 16S rRNA gene regions (30).

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Each read was taxonomically assigned according to the EzTaxon database. When each 149 phylotype at species level was defined using a baseline of 97% nucleotide sequence similarity The alteration of bacterial diversity in 6-OHDA-lesioned mice 161 To investigate the alterations of bacterial diversity in mice feces followed by 6-OHDA 162 lesions, we estimated several diversity indexes such as Shannon's diversity index, Simpson's 163 diversity index, and phylogenic diversity at species level. We found that there was no 164 difference between the groups (Fig. 2). These data show that bacterial species diversity was 165 unaffected by 6-OHDA lesion. Ruminococcaceae, Prevotellaceae, Lachnospiraceae, and Erysipelotrichaceae was not 181 significant between the groups (Fig 3). Lachnospiraceae_uc, Prevotella, and Ruminococcaceae JN713389_g did not show any 192 significant difference between the groups (Fig 4). In Fig. 5  ABC transporters metabolism pathway genes were enriched in sham-operated group (Fig 6). The composition of gut microbiota can have a remarkable impact on disease status as well 240 as normal physiology in brain (31, 32). Emerging evidence supports that gut dysbiosis by 241 altered gut microbiota has the potential to be closely linked with neurodegenerative diseases,

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including PD (33-36). In this study, we found the changes in gut microbiota composition in a 243 PD mouse model by unilateral 6-OHDA-lesion using the high-throughput 16S rRNA gene 244 sequencing method.

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First, we confirmed the well-established PD mouse model induced by intracerebral injection 246 of 6-OHDA as shown in severe motor deficits and dopaminergic neuronal damage (Fig S1). In 247 this model, we found little difference between the sham-operated and 6-OHDA-lesioned 248 groups on species richness, bacterial diversity (Fig 1, 2). We also found no significance 249 between two groups on a principal coordinates analysis evaluated by Permutational 250 multivariate analysis of variance (p=0.153, Fig S2). It has been reported that microbial species 251 richness and its diversity indicate whether gut microbiota impacts biological entities, but the 252 ecological meaning of this index simply shows the number of bacterial species, and is not an 253 indicator of gut dysbiosis (37-39).

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Next, we observed a significant differentiation in gut microbiota composition at the 255 phylogenic family, genus, and species levels between the sham-operated and 6-OHDA-256 lesioned groups (Fig. 3-5). We showed a relative lower abundance of Lactobacillaceae and 257 Lactobacillus at the family and genus level, respectively, thereby the relative abundance of L.