Gut microbiome and serum metabolome alterations in isolated dystonia

doi:10.21203/rs.3.rs-139606/v1

Download PDF

Research

Gut microbiome and serum metabolome alterations in isolated dystonia

https://doi.org/10.21203/rs.3.rs-139606/v1

This work is licensed under a CC BY 4.0 License

Version 1

posted

You are reading this latest preprint version

Background

Dystonia is a complex neurological movement disorder characterised by involuntary muscle contractions. The relationship between the gut microbiota and isolated dystonia remains poorly explored.

Methods

We collected faeces and blood samples to study the microbiome and the serum metabolome from a cohort of 57 drug-naïve isolated dystonia patients and 27 age- and environment-matched healthy individuals. We first sequenced the V4 regions of the 16S rDNA gene from all faeces samples. Further, we performed metagenomic sequencing of gut microbiome and non-targeted metabolomics profiling of serum from dystonia patients with significant dysbiosis.

Results

Gut microbial β-diversity was significantly different, with a more heterogeneous community structure among dystonia individuals than healthy controls, while no difference in α-diversity was found. Gut microbiota in dystonia patients was enriched with Blautia obeum, Dorea longicatena and Eubacterium hallii, but depleted with Bacteroides vulgatus and Bacteroides plebeius. Metagenomic sequencing revealed that genes related to the citrate cycle, vitamin B6 and glycan metabolism were less abundant in dystonia, while genes linked to purine and tryptophan biosynthesis were more abundant. Serum metabolome analysis revealed altered levels of tyrosine and glutamate. The integrative analysis of the gut microbiome and serum metabolomics identified dystonia-associated gut microbial species linked to changes in serum metabolites, reflecting the effect of the gut microbiome on metabolic activity in isolated dystonia.

Conclusion

This study is the first to reveal gut microbial dysbiosis in dystonia patients. Our findings identified previously unknown links between intestinal microbiota alterations, circulating amino acids and dystonia, providing new insight into the pathogenesis of isolated dystonia.

Neurobiology of Disease

Dystonia

Gut microbiome

Serum metabolomics

Multi-omics analysis

Evidence before this study

Dystonia is a complex neurological movement disorder and its pathogenesis has not been fully elucidated. Emerging evidence has implicated the gut microbiome as an important player in many neurological diseases. One case was reported that anti-clostridial agents were capable of reversing both gastrointestinal and neurological symptoms in one sporadic case of myoclonic-dystonia, which might provide clues to the mechanisms driving dystonia and potentially other neurological conditions with a gut microbial component. However, the relationship between the gut microbiota and isolated dystonia remains poorly explored.

Added value of this study

Our results demonstrated the alterations of gut microbiome in isolated dystonia. The most pronounced high-level correlation was a general overrepresentation of Clostridiales and underrepresentation of Bacteroidetes in dystonia compared with Healthy controls. Furthermore, genes related to the citrate cycle, vitamin B6 and glycan metabolism were less abundant in dystonia, while genes linked to purine and tryptophan biosynthesis were more abundant. Altered levels of several serum metabolites were found to be associated with microbial changes, such as D-tyrosine and glutamate, indicating differences in neurotransmitter metabolism.

Implications of all the available evidence

This is the first study to reveal significant alterations of gut microbiome in isolated dystonia patients. Integrative analysis of microbiome and metabolome suggest that 5-HT system dysfunction may be a possible pathway by which the gut mcirobiome participates in the development of dystonia. The gut microbiome changes provide new insight on the pathogenesis of dystonia, suggesting new potential therapeutic directions.

Dystonia is a complex, highly variable neurological movement disorder characterised by sustained or intermittent muscle contractions¹. It can be the manifestation of many neurological disorders, either in isolation (isolated dystonia) or in combination with additional symptoms (combined dystonia)². Isolated dystonia includes syndromes in which dystonia is the sole phenotypic manifestation, with the exception of tremor ¹. Until now, the etiology and pathogenesis of isolated dystonia had not been fully elucidated. Most patients with common adult-onset dystonia tend to arise sporadically with no clear etiology, though several associated genes were identified as the cause of childhood- and adolescent-onset dystonia³.

The gut microbiome contains a complex community of microbes that live within the gastrointestinal tract. Gut microbes communicate with the central nervous system through nervous-, endocrine- and immune-signalling mechanisms. The brain-gut-microbiome axis allows the brain to control gut function, but also provides an opportunity for the gut microbiome to influence the brain⁴. Emerging evidence has implicated the gut microbiome as an important player in neurodevelopment, as well as in brain diseases such as PD⁵, Alzheimer’s disease (AD)⁶ and multiple sclerosis⁷. Interestingly, it was reported that anti-clostridial agents were capable of reversing both gastrointestinal and neurological symptoms in one sporadic case of myoclonic-dystonia⁸, which provides clues to the mechanisms driving dystonia and potentially other neurological conditions with a gut microbial component. However, the relationship between the gut microbiota and isolated dystonia remains poorly explored.

In the present study, we first investigated gut microbial characteristics in drug-naïve patients with isolated dystonia using 16S rDNA amplicon sequencing. We also evaluated whether the parameters correlated with clinically relevant features of dystonia, such as age at disease onset, disease duration, direction of rotation, and dystonia severity. Furthermore, we performed shotgun metagenomic sequencing and non-targeted metabolomics profiling of serum from patients with significant gut dysbiosis in 16S rDNA analysis to investigate the microbial functional alteration and the association of the altered gut microbiome with serum metabolites in the host.

Participants

Fifty-seven patients with isolated dystonia (15 male, 42 female) and 27 age- and sex-matched healthy participants (nine male, 18 female) were enrolled. Among the patients, 44 had focal dystonia, 11 had segmental dystonia, and two had general dystonia. All patients, from 12 provinces across the country, admitted to the Movement Disorders and Botulinum Toxin-A Treatment center underwent a standardised neurological examination by a specialist in movement disorders. All dystonia patients were newly diagnosed and did not receive oral medicine or botulinum toxin injection. None of the participants had a family history of dystonia. All patients were negative for the genetic testing of DYT1 (TOR1A), DYT4（TUBB4）,DYT6 (THAP1) ,DYT23 (CIZ1),DYT24（ANO3）,DYT25（GNAL）and COL6A3. Subjects who have no neurological or psychiatric disease were enrolled as Healthy controls (HCs). Subjects were excluded if they had gastrointestinal diseases, malignant tumours, autoimmune disorders, infectious diseases, a history of gastrointestinal surgery, or were administered antibiotics or laxatives or immunosuppressive agents in the past three months. No subjects were or are on any antianxiety or antidepressant medication. No relevant constipation was present in all subjects; dietary and smoking habits did not differ between groups. No detailed dietary plan was requested prior to the feces collection and samples were collected as first bowel movement of the day.

Demographic information (gender, age, age of disease onset, disease duration, height and weight,) and severity evaluation scores, including Global Dystonia Rating Scale (GDS) and Unified Dystonia Rating Scale (UDRS) were collected from all patients. Evaluation for anxiety and depression including Hamilton Anxiety Scale (HAMA) and Hamilton Depression Scale (HAMD), were assessed for all subjects. Written informed consent was obtained from all individuals. The study was approved by the ethics committees of Beijing Tiantan Hospital.

Analysis of Fecal Microbiota by 16s rDNA Amplificon Sequencing

Stool samples freshly collected from each participant were immediately transported to the laboratory and frozen at -80°C. Bacterial DNA was extracted and the V4 region of the 16S rRNA gene was amplified by PCR using barcoded universal fusion primers 515 and 806. PCR products were sequenced and for taxonomic assignment. The number of reads in each sample was standardised to 37,666 for further analysis. Dirichlet multinomial mixtures (DMM) modeling examines taxa frequencies and determines how many microbial community states (MCS) exist within a dataset using the DirichletMultinomial⁹. Please refer to the Supplemental Methods section for further details on the laboratory protocols.

Functional Analysis of Microbiome by Shotgun Metagenomic Sequencing

Total DNA from the 26 fecal samples (13 cases and 13 controls) were subject to shotgun metagenomic sequencing. Sheared DNA (350 bp) was used for Illumina library construction and index codes were added to attribute sequences to each sample. Functional annotation was carried out by BLASTP searches against the Kyoto Encyclopedia of Genes and Genomes (KEGG database (e-value ≤1e-5 and high-scoring segment pair scoring >60). For each functional feature (KO in the KEGG database), we estimated its abundance by accumulating the relative abundance of all genes belonging to that feature. Please refer to the Supplemental Methods for further details.

Untargeted metabolomics analysis of serum samples

All serum samples were thawed on ice and a quality control (QC) sample, made by blending equal volumes (20 µl) of each serum sample, was used to estimate a mean profile representing all the analytes encountered during analysis. Metabolite extraction was performed by mixing 100 µl thawed serum and 400 µl ice-cold 80% methanol and 0·1% formic acid, vortexing for 30 s, and keeping at −20 °C for one hour. After centrifuged at 14,000 g and 4 °C for 20 min，some of supernatant was diluted to final concentration containing 53% methanol by LC-MS grade water. The samples were subsequently transferred to a fresh Eppendorf tube and then were centrifuged at 14,000 g, 4°C for 20 min. Finally, the supernatants were subjected to metabolomics profiling by high performance liquid chromatograph (HPLC)-MS in both positive and negative ionization modes. The raw data files were processed using the Compound Discoverer 3·1 (CD3·1, ThermoFisher). Peak intensities were normalized to the total spectral intensity. All data were mean-centred and unit variance (UV)-scaled before multivariate statistical analysis, and partial least squares discriminant analysis (PLS-DA) was performed. Significantly different metabolites were determined by a combination of VIP value >1 from the PLS-DA model and p-values <0·05 from two-tailed Student’s t tests. Please refer to the Supplemental Methods for further details.

Spearman’s multi-omics correlation analysis

Spearman’s correlation coefficients were computed for relationships between the relative abundance of identified dystonia-associated species and normalised individual metabolomic features. A scaled heatmap was constructed for the correlation matrix, including cladogram classification of variables, using the default clustering method. Visual presentation of multiple omics correlations was performed using the Corrplot package in R (v3·5 ·3). Significant microbiota-metabolite correlations were determined based on an r value <-0·3 or >0·3 and a false discovery rate (FDR)-adjusted p-value <0·05.

Demographics and clinical data

Fifty-seven isolated dystonia patients and 27 HCs were recruited and an integrative analysis of the gut microbiome and serum metabolome was performed. Clinical details of the study cohort are shown in Table 1.

Data overview

Totally, 4,397,348 high-quality 16S rDNA V4 sequences were obtained, with 52,349 ± 8462 (mean ± SD) per sample after de-multiplexing and quality control (Supplementary Table S1). A total of 997 OTUs (> 97% similarity, excluding singletons and low-abundance taxa with frequency <3 for samples, range = 204–461, median = 306 taxa per sample) were identified, representing 22 taxonomic phyla. The dominant phyla were Firmicutes (66·4%), Bacteroidetes (17·5%), Proteobacteria (9·4%) and Actinobacteria (5·3%). At the genus level, Bacteroides (13%), Faecalibacterium (10%), unidentified Lachnospiraceae (7%), Agathobacter (6%) and Blautia (5%) were the most abundant, of which all but Bacteroides belong to Clostridiales in Firmicutes (Supplementary Figure S1).

Gut microbiota revealed by 16s rDNA amplicon sequencing

Although gut microbial α-diversity was not significantly different as calculated by Shannon and Simpson indices (Figure 1A), NMDS analysis demonstrated a significant difference between isolated dystonia and HCs (Figure 1B, PERMANOVA, R² = 0·157, p = 0·002). We also observed higher β-diversity in the microbiota of dystonia patients based on ANOSIM analysis, indicating a more heterogeneous community structure among dystonia patients compared with HCs (Figure 1C). LEfSe analysis showed that Clostridiales and Bacteroidetes were the key taxa distinguishing isolated dystonia from HCs. Dystonia patients were significantly enriched with Blautia, Bifidobacterium and unidentified Lachnospiraceae, while depleted with Bacteroidetes and unidentified Clostridiales as compared with HCs (Figure 1D). The relative abundance of the most abundant taxa (≥1%) at different taxonomic levels was further compared (Supplementary Table S2 and Figure S2). At the species level, Eubacterium hallii, Blautia obeum and Dorea longicatena were enriched in dystonia subjects, while Bacteroides vulgatus, Bacteroides_plebeius and Fusobacterium varium were enriched in HCs (Figure 1E).

Stratification of bacterial community in dystonia patients

Given more heterogeneity, we applied DMM to our cohort to investigate possible gut microbial subgroups in dystonia, and identified two significantly distinct MCSs (n = 8 and n = 49; R² = 0·138; p <0·001) using a Laplace approximation (Figure 2A). Specific co-occurring bacterial families were characteristically enriched in these groups; the MCS1 (n = 8) microbiota was characteristically enriched by Bacteroidaceae, Ruminococcaceae and Lachnospiraceae. The second larger group, which we designated MCS2A (n = 20) and MCS2B (n = 29), respectively，exhibited a sharp decreased abundance of Bacteroidaceae, especially in MCS2B (Figure 2B). NMDS analysis confirmed a strong and significant relationship between MCS class and bacterial β-diversity (PERMANOVA, R² = 0·29, p <0·001), corroborating the existence of compositionally distinct microbial states (Figure 2C). These distinct microbial states exhibited significant differences in diversity (Shannon, Kruskal-Wallis tests, p = 0·002; Figure 2D), with MCS2B exhibiting the lowest index. We also investigated whether specific factors (illness duration, age at onset, BMI and severity evaluation scores) that was differentially associated with microbial community states. No significant correlation was detected.

Functional analysis of the isolated dystonia by metagenomic sequencing

As shown above, 29 of 57 (51%) the isolated dystonia underwent a gut microbial dysbiosis. To further explore microbial functional feature of dystonia, we performed metagenomic sequencing of a subgroup consisting of 13 patients from MCS2B and 13 gender- and age-matched HCs. An average of 10·7 ± 2·1 Gb clean reads were generated per sample，except for one HC sample removed due to low depth of sequencing (Supplementary Table S3).

Consistent with the 16S rDNA analysis, B. obeum, D. longicatena and E. hallii were significantly increased in dystonia, while B. vulgatus and B. plebeius were decreased (Figure 1F and Supplementary Figure S3). We estimated the abundance of metabolic pathways using metagenomic reads mapped to functional orthologs from the KEGG databases to explore differences in the metabolic potential of gut microbiomes between dystonia and HCs. Dystonia communities were functionally different from healthy communities, and less closely clustered together among individuals, suggesting that inter-individual functional variation was higher in dystonia than in HCs (Figure 3A).

A total of 22 metabolic pathways (level 3) were found to be significantly different (p <0·05, q <0·02) in abundance (>0·01%) between dystonia patients and HCs, including those involved in nucleotide, amino acid, carbohydrate and lipid metabolism (Supplementary Table S4). Interestingly, we identified several different pathways that appeared to be more active in the microbiome of dystonia patients, including purine metabolism (ko00230), peptidoglycan biosynthesis (M00550), glycerolipid/glycerophospholipid metabolism (ko00561, ko00564), phenylalanine, tyrosine and tryptophan biosynthesis (ko00350, ko00400) and sulphur metabolism (ko00920) (Figure 3B). While genes related to TCA cycle (ko00020, ko00630, ko00720), glycan biosynthesis and metabolism pathways (ko00511, ko00531, ko00540, ko00600, ko00603) and vitamin B6 metabolism (ko00750) were less abundant in dystonia (Figure 3C). Furthermore, we traced the contributing genes and determined their likely taxonomic origin to determine which bacteria are involved in these pathways (Figure 3B, C).

Associations between gut microbial species and serum metabolites

To investigate the extent to which the altered microbiome in the dystonia patients was associated with serum metabolites in the host, we performed non-targeted metabolomics profiling of serum from the 13 dystonia patients and the 12 HCs in metagenomic analysis, and yielded 1543 features (Supplementary Table S5). A supervised PLS-DA using two components (R²X = 0.525, R²Ycum = 0·99, Qcum² = 0·975, p <0·001) was performed, resulting in some separation tendencies between dystonia cases and HCs (Figure 4A). Based on the PLS-DA models of metabolite profiling data, 242 metabolites were found to be significantly different in abundance between dystonia cases and HCs, with 87 metabolites having a higher concentration and 169 metabolites having a lower concentration in dystonia compared with HCs (p <0·05, VIP >1 and FC >2 or <0·5, Figure 4B, Supplementary Table S5).

Subsequently, spearman’s correlation coefficients were computed for relationships between the relative abundance of the identified dystonia-associated species and the different 242 normalised individual metabolomic features. Metabolites were grouped into two clusters depending on the correlations, and correlation coefficients with significant p-values (<0·01) are shown in Figure 4C. Clostridiales species enriched in dystonia patients correlated positively with the first metabolite cluster, including L-glutamic acid, phenylalanylphenylalanine and taurine. While Bacteroides species with decreased abundance in dystonia patients correlated positively with the second cluster, including D-tyrosine, D-(-)-aspartic acid and N, N-dimethylacetamide (DMAc).

An integrative analysis of the gut microbiome and serum metabolome was employed to explore the relationship between the gut microbiome and isolated dystonia. We found that more than half of the isolated patients (29/57, 51%) got a gut microbial dysbiosis in 16S rRNA analysis, showing a general overrepresentation of Clostridiales and underrepresentation of Bacteroidetes in dystonia compared with HCs. Further analysis thus for this subgroup using shotgun metagenomic sequencing and non-targeted metabolomics profiling of serum was performed to gain an insight into the links between gut dysbiosis and dystonia, and indicated alterations in several genes and serum metabolites. Genes related to the citrate cycle, vitamin B6 and glycan metabolism were less abundant in dystonia, while genes linked to purine and tryptophan biosynthesis were more abundant. Altered levels of several serum metabolites associated with microbial changes, such as D-tyrosine and glutamate, were found, indicating differences in neurotransmitter metabolism.

Evidence is accumulating for a potential role of the microbiota in the pathogenesis of neurological disorders^5–8. One myoclonic dystonia case was reported to present a 90% improvement in tremor and abnormal posture after taking anti-clostridial antibiotics, indicating a possible correlation between the pathogenesis of dystonia and the gut microbiome, underlying the potential role of Clostridial species⁸. In this study, intestinal dysbiosis was characterised by significantly increased Clostridiales and decreased Bacteroidetes in dystonia patients, which might explain the neurological improvement in the abovementioned case after taking anti-clostridial agents, suggesting that Clostridiales may have a potential pathogenic role in the pathogenesis of dystonia.

Blautia and unidentified Lachnospiraceae, belonging to the Lachnospiraceae family, contributed principally to the increase in Clostridiales in dystonia patients. The Lachnospiraceae family is known to participate in the breakdown of carbohydrates into short-chain fatty acids (SCFAs)¹⁰, which are believed to play a key role in microbiota-gut-brain crosstalk^{11, 12}. SCFAs, with acetate, propionate and butyrate being the most abundant components, may influence the brain by crossing the blood-brain barrier (BBB) ¹³, modulating neurotransmission, influencing levels of neurotrophic factors, and promoting serotonin (5hydroxytryptamine, 5HT) biosynthesis¹². In a cell model, acetate and butyrate promoted serotonin biosynthesis by inducing the expression of tryptophan 5-hydroxylase 1 (the rate-limiting enzyme for 5HT synthesis) transcription ¹⁴. Another study found that butyrate and propionate play a critical role in promoting host 5HT biosynthesis, and regulating levels of 5HT in both colon and serum ¹⁵. Furthermore, they also induce tyrosine hydroxylase gene transcription¹⁶, which catalyses the ratelimiting step in the biosynthesis of dopamine, noradrenaline and adrenaline¹⁷. A decrease in SCFA production can cause intestinal barrier dysfunction, but whether an increased dose of SCFAs could directly lead to adverse effects on locomotor behaviour has not been elucidated and requires animal studies in the future. In our current study, the increased abundance of Clostridiales may have led to elevated SCFA levels, and a consequent increase in 5HT.

The abundance of Bacteroides, Parabacteroides and Alistipes was significantly decreased in isolated dystonia compared with HCs. A decrease or low level of Bacteroides is believed to be associated with metabolic diseases such as obesity¹⁸ and diabetes¹⁹, and psychiatric disorders such as depression²⁰. Given that no subjects were associated with obesity or diabetes, and there were no differences in body-mass index (BMI) between the two groups, an association between Bacteroides and metabolic factors can be ruled out in our study. Growing evidence suggests that non-motor symptoms are essential components of dystonia, with psychiatric co-morbidity being most prevalent ²¹, and a link between dystonia and depression has been confirmed ²². In our cohort, patients were newly diagnosed and there was no statistical difference of HAMD between the two groups, it is unlikely that depression was a confounding factor in our study. Alistipes species are indole-positive and may thus influence tryptophan availability²³. As tryptophan is also the precursor of serotonin, decreased abundance of Alistipes might therefore disrupt the balance of the intestinal serotonergic system.

Until now, the pathophysiological basis of dystonia remains poorly understood. Evidence indicates that the pathophysiological basis of dystonia involves dysfunction of the basal ganglia and cortico-striatal-thalamo-cortical circuits²⁴, hence neurotransmitter systems are likely involved. In addition to dopamine, a significant neurotransmitter in motor control, serotonin and serotonin-dopamine interactions have received attention²⁵. Disturbance of the serotonergic system is known to be involved in extrapyramidal movement and psychiatric disorders. A [¹¹c]DASB positron emission tomography study of cervical dystonia patients revealed a significant correlation between serotonin transporter (SERT) binding in the dorsal raphe nucleus and motor and non-motor symptoms²⁶. Higher non-displaceable binding potential (BPND) was statistically correlated with motor severity, pain and sleep disturbance, with motor symptom severity being the most important predictor of SERT binding²⁶. Since Lachnospiraceae and Alistipes are involved in the production and regulation of 5-HT biosynthesis, the increase in the Lachnospiraceae family and the decrease in Alistipes species observed in our study may lead to imbalance of peripheral levels of 5-HT, which might, in turn, modulate brain function by affecting the immune system²⁷ or signalling to the brain via 5HT receptors on vagal afferent fibres²⁸.

Functional analyses revealed that phenylalanine, tyrosine and tryptophan biosynthesis tend to be more active in dystonia, as reported previously for PD²⁹. These aromatic amino acids are important as precursors of 5-HT and catecholamines. Correspondingly, metabolome analysis found that the serum concentration of D-tyrosine, the precursor for serotonin, was significantly elevated in dystonia cases. Composition analysis, functional analysis of the gut microbiome, serum metabolome analysis, and imaging all point to a potential role of serotonergic system dysfunction in the pathogenesis of isolated dystonia.

Additionally, the sulphur metabolism pathway appeared to be more active in the microbiome of dystonia patients. In the brain, sulphur metabolism is intimately involved in metabolic cooperation, and provides cells with four major reagents critical for methylation reactions (S-adenosylmethionine), antioxidant capacity (glutathione), signalling (H2S), and cell volume regulation (taurine). An important intermediate in sulphur metabolism is homocysteine (HCY), a neuronal excitotoxic amino acid, and hyperhomocysteinaemia is considered an independent vascular risk factor. Increased HCY has also been observed in movement disorders such as PD³⁰, Huntington’s disease (HD)³¹ and dystonia^{32, 33}. In patients with isolated dystonia, hyperhomocysteinaemia was observed independently from immune activation³³. Our study indicates that hyperhomocysteinaemia might, to some extent, be related to an increase in sulphur metabolism caused by disturbance of the gut microbiome.

It should be noted that the diversity of the gut microbiome is influenced by several factors, including health status, age, diet, and antibiotics³⁴. One highlight of our study is the enrollment of drug-naïve patients. Evidence is accumulating on effects that drugs can have on the composition and function of the gut microbiome³⁵. A case-control study on cause-effect relationships could be performed in the future by analysing drug-naïve dystonia patients.

Despite being, to our knowledge, the first study to explore the alterations of gut microbiome in dystonia, the present study has some limitations that should be acknowledged. First, this study was performed at one single centre (Tiantan Hospital). Selection bias and unknown confounding factors cannot be excluded. However, our hospital has a big center for neurological diseases and appeals to the patients across the country. Patients in our corhort came from 12 provinces and are to some extent, representative. Second, our study is an exploratory study and the number of our patients is relatively small. More studies including larger cohort from different regions and ethics will be needed to confirm the role of gut microbiome in isolated dystonia.

This is the first study to reveal a significant gut microbial dysbiosis in isolated dystonia patients, which may be associated with altered serum metabolites. Integrative analysis of microbiome and metabolome suggest that 5-HT system dysfunction may be a possible pathway by which the gut mcirobiome participates in the development of dystonia. The findings provide new insight on the pathogenesis of dystonia, indicating the potential involvement of the enteric neurotransmitter system in dystonic patients. Further validation is needed in animal experiments and in large populations to explore clearer role of gut microbiome in pathogenesis of dystonia.

HC: Healthy control; AD: Alzheimer’s disease; PD: Parkinson's disease; GDS: Global Dystonia Rating Scale；UDRS：Unified Dystonia Rating Scale; HAMA: Hamilton Anxiety Scale; HAMD: Hamilton Depression Scale; 16S rRNA: 16S ribosomal RNA; OTUs: Operational taxonomic units; MCS: Microbial community states; DMM: Dirichlet multinomial mixtures; LC-MS: Liquid Chromatography Mass Spectrometry (LC-MS); PLS-DA: partial least squares discriminant analysis; KEGG: Kyoto Encyclopedia of Genes and Genomes; SCFA: short-chain fatty acids; 5HT: 5hydroxytryptamine.

Acknowledgments

We appreciated all the participants in our study.

Authors’ contributions

LY Ma, M Chen and YF Hu participated in the design of the study, collected medical data of the subjects, undertook laboratory work, analyzed the data and drafted the manuscript. Hua Pan collected medical data of the patients. Bo Liu and Jian Yang performed bioinformatic analysis. YF Hu, Tao Feng and Qi Jin carried out the conceptualization of the study.

Funding

This research was supported by the Beijing Natural Science Foundation (No.7164254), the CAMS Innovation Fund for Medical Sciences (2017-I2M-3-017).

Availability of data and materials

The sequencing dataset has been deposited in the Genome Sequence Archive in BIG Data Center, Beijing Institute of Genomics, Chinese Academy of Sciences under BioProject accession code PRJCA003075.

Ethics approval and consent to participate

All participants provided informed consent and the study was approved by the ethics board of Beijing Tiantan Hospital.

Consent for publication

The paper has not been published previously and is not under consideration for publication elsewhere. All co-authors have read and approved the submission.

Competing interests

All authors declare that they have no competing interests.

Albanese A, Bhatia K, Bressman SB, et al. Phenomenology and classification of dystonia: a consensus update. Mov Disord. 2013. 28(7): 863-73.
Balint B, Mencacci NE, Valente EM, et al. Dystonia. Nat Rev Dis Primers. 2018. 4(1): 25.
Lohmann K, Klein C. Update on the Genetics of Dystonia. Curr Neurol Neurosci Rep. 2017. 17(3): 26.
Benakis C, Martin-Gallausiaux C, Trezzi JP, Melton P, Liesz A, Wilmes P. The microbiome-gut-brain axis in acute and chronic brain diseases. Curr Opin Neurobiol. 2019. 61: 1-9.
Wood H. Parkinson disease. Gut reactions--can changes in the intestinal microbiome provide new insights into Parkinson disease. Nat Rev Neurol. 2015. 11(2): 66.
Sun M, Ma K, Wen J, et al. A Review of the Brain-Gut-Microbiome Axis and the Potential Role of Microbiota in Alzheimer's Disease. J Alzheimers Dis. 2020. 73(3): 849-865.
Schepici G, Silvestro S, Bramanti P, Mazzon E. The Gut Microbiota in Multiple Sclerosis: An Overview of Clinical Trials. Cell Transplant. 2019. 28(12): 1507-1527.
Tomas Borody DR, Torres M. Myoclonus-dystonia affected by GI Microbiota. Am J Gastroenterol. 2011. 106(4): S351-352.
Holmes I, Harris K, Quince C. Dirichlet multinomial mixtures: generative models for microbial metagenomics. PLoS One. 2012. 7(2): e30126.
Meehan CJ, Beiko RG. A phylogenomic view of ecological specialization in the Lachnospiraceae, a family of digestive tract-associated bacteria. Genome Biol Evol. 2014. 6(3): 703-13.
Silva YP, Bernardi A, Frozza RL. The Role of Short-Chain Fatty Acids From Gut Microbiota in Gut-Brain Communication. Front Endocrinol (Lausanne). 2020. 11: 25.
Dalile B, Van Oudenhove L, Vervliet B, Verbeke K. The role of short-chain fatty acids in microbiota-gut-brain communication. Nat Rev Gastroenterol Hepatol. 2019. 16(8): 461-478.
Mitchell RW, On NH, Del Bigio MR, Miller DW, Hatch GM. Fatty acid transport protein expression in human brain and potential role in fatty acid transport across human brain microvessel endothelial cells. J Neurochem. 2011. 117(4): 735-46.
Reigstad CS, Salmonson CE, Rainey JF 3rd, et al. Gut microbes promote colonic serotonin production through an effect of short-chain fatty acids on enterochromaffin cells. FASEB J. 2015. 29(4): 1395-403.
Yano JM, Yu K, Donaldson GP, et al. Indigenous bacteria from the gut microbiota regulate host serotonin biosynthesis. Cell. 2015. 161(2): 264-76.
Nankova BB, Agarwal R, MacFabe DF, La Gamma EF. Enteric bacterial metabolites propionic and butyric acid modulate gene expression, including CREB-dependent catecholaminergic neurotransmission, in PC12 cells--possible relevance to autism spectrum disorders. PLoS One. 2014. 9(8): e103740.
Nagatsu T. Tyrosine hydroxylase: human isoforms, structure and regulation in physiology and pathology. Essays Biochem. 1995. 30: 15-35.
Liu R, Hong J, Xu X, et al. Gut microbiome and serum metabolome alterations in obesity and after weight-loss intervention. Nat Med. 2017. 23(7): 859-868.
Zhang X, Shen D, Fang Z, et al. Human gut microbiota changes reveal the progression of glucose intolerance. PLoS One. 2013. 8(8): e71108.
Winter G, Hart RA, Charlesworth R, Sharpley CF. Gut microbiome and depression: what we know and what we need to know. Rev Neurosci. 2018. 29(6): 629-643.
Zurowski M, McDonald WM, Fox S, Marsh L. Psychiatric comorbidities in dystonia: emerging concepts. Mov Disord. 2013. 28(7): 914-20.
Yang J, Shao N, Song W, et al. Nonmotor symptoms in primary adult-onset cervical dystonia and blepharospasm. Brain Behav. 2017. 7(2): e00592.
Song Y, Könönen E, Rautio M, et al. Alistipes onderdonkii sp. nov. and Alistipes shahii sp. nov., of human origin. Int J Syst Evol Microbiol. 2006. 56(Pt 8): 1985-90.
Schirinzi T, Sciamanna G, Mercuri NB, Pisani A. Dystonia as a network disorder: a concept in evolution. Curr Opin Neurol. 2018. 31(4): 498-503.
Smit M, Bartels AL, van Faassen M, et al. Serotonergic perturbations in dystonia disorders-a systematic review. Neurosci Biobehav Rev. 2016. 65: 264-75.
Smit M, Vállez García D, de Jong BM, et al. Relationships between Serotonin Transporter Binding in the Raphe Nuclei, Basal Ganglia, and Hippocampus with Clinical Symptoms in Cervical Dystonia: A [11C]DASB Positron Emission Tomography Study. Front Neurol. 2018. 9: 88.
Stasi C, Bellini M, Bassotti G, Blandizzi C, Milani S. Serotonin receptors and their role in the pathophysiology and therapy of irritable bowel syndrome. Tech Coloproctol. 2014. 18(7): 613-21.
Browning KN. Role of central vagal 5-HT3 receptors in gastrointestinal physiology and pathophysiology. Front Neurosci. 2015. 9: 413.
Bedarf JR, Hildebrand F, Coelho LP, et al. Functional implications of microbial and viral gut metagenome changes in early stage L-DOPA-naïve Parkinson's disease patients. Genome Med. 2017. 9(1): 39.
Saadat P, Ahmadi Ahangar A, Samaei SE, et al. Serum Homocysteine Level in Parkinson's Disease and Its Association with Duration, Cardinal Manifestation, and Severity of Disease. Parkinsons Dis. 2018. 2018: 5813084.
Andrich J, Saft C, Arz A, et al. Hyperhomocysteinaemia in treated patients with Huntington's disease homocysteine in HD. Mov Disord. 2004. 19(2): 226-8.
Müller T, Woitalla D, Hunsdiek A, Kuhn W. Elevated plasma levels of homocysteine in dystonia. Acta Neurol Scand. 2000. 101(6): 388-90.
Muller UJ, Frick B, Winkler C, et al. Homocysteine and serum markers of immune activation in primary dystonia. Mov Disord. 2005. 20(12): 1663-7.
Lozupone CA, Stombaugh JI, Gordon JI, Jansson JK, Knight R. Diversity, stability and resilience of the human gut microbiota. Nature. 2012. 489(7415): 220-30.
Walsh J, Griffin BT, Clarke G, Hyland NP. Drug-gut microbiota interactions: implications for neuropharmacology. Br J Pharmacol. 2018. 175(24): 4415-4429.

Table 1 Demographics of subjects

	Dystonia (n = 57)	Control (n = 27)	P value
Male (n) (%)	15 (26.32)	9 (33.33)	0.506
Female (n) (%)	42 (73.68)	18 (66.67)
BMI (means ± SD)	22.71 ± 2.04	23.61 ± 1.80	0.341
Age (year; means ± SD; range)	45.25 ± 13.00	41.1 ± 11.15	0.158
Age at onset (year; means ± SD)	45.05 ± 12.86	-
Duration (month; means ± SD)	5.32 ± 3.77	-
UDRS (means ± SD)	9.91 ± 14.43	-
GDS (means ± SD)	6.54 ± 4.06	-
With family history (n; %)	0	-
HAMA (means ± SD)	8.51± 4.21	6.78± 3.11	0.060
HAMD (means ± SD)	8.14± 4.13	6.52± 2.67	0.066

UDRS, Unified Dystonia Rating Scale; GDS, Global Dystonia Rating Scale; HAMA, Hamilton anxiety scale; HAMD, Hamilton depression scale

Download PDF

Version 1

posted

You are reading this latest preprint version

Gut microbiome and serum metabolome alterations in isolated dystonia

Status:

Version 1

Abstract

Background

Methods

Results

Conclusion

Figures

Research In Context

Introduction

Methods

Results

Discussion

Conclusions

Abbreviations

Declarations

References

Tables

Supplementary Files

Status:

Version 1