5 Species microbiome isolated from schizophrenia patients induces schizophrenia-like behaviors in mice

다국어 초록 (Multilingual Abstract)

Schizophrenia (SCZ) is a neuropsychiatric disorder, and the symptoms are characterized by positive symptoms, negative symptoms, and cognitive dysfunction. Although a lot of research has been conducted on the pathogenetic studies of SCZ, no one has been able to clarify a definitive cause of SCZ. As the gut-brain axis hypothesis has recently been suggested as a possible explanation for the pathogenesis of SCZ, I tried to identify the gut microbiome associated with the pathogenesis of SCZ. A comparative analysis of the gut microbiome of healthy controls and SCZ patients was performed, and 5 species microbiome (5-SM; Escherichia fergusonii, Shigella sonnei, Morganella morganii, Citrobacter freundii, and Enterococcus faecalis) that were enriched in SCZ patients were isolated. Several behavioral tests were used to determine whether 5-SM administration induces SCZ-like behaviors. After the behavioral tests, the Western blot analysis was performed to investigate the mechanism of 5-SM on SCZ- like behaviors. Administration of 5-SM could induce SCZ-like behaviors, and these behaviors were ameliorated by treatment with aripiprazole. In addition, the administration of 5-SM decreased synaptic plasticity, altered the N-methyl-D-aspartate (NMDA) receptor expression levels and Ca2+ influx level, and increased inflammation, proline and glutamate levels. These results suggest that the administration of 5-SM induces SCZ-like behaviors with dysfunction of the NMDA receptor signaling pathway similar to those observed in SCZ patients and ameliorated by antipsychotic administration. In addition, these changes could be explained as face validity, constructive validity, and predictive validity of the SCZ animal model, and suggested that this animal model would be a new SCZ-like behavior animal model. Key words: Schizophrenia; Gut-brain axis; Fecal microbiota transplantation; Microbiome; N- methyl-D-aspartate receptor

국문 초록 (Abstract)

조현병은 신경정신질환으로 양성증상과 음성증상, 인지기능 장애 등의 증상을 동반하는 질병이다. 조현병의 발병원인에 대하여 많은 연구가 진행되어 왔지만 확실한 발병원인의 정의를 내리지 못하고 있다. 장-뇌 축 가설이 최근 들어 조현병의 발병원인으로 제시되고 있는 가운데, 본 연구에서는 조현병과 관련이 있는 장내미생물을 발굴하고자 하였다. 정상인과 조현병 환자의 비교 연구를 통해 조현병 환자의 분변에 많이 존재하는 5종의 장내미생물 (5-SM; Escherichia fergusonii, Shigella sonnei, Morganella morganii, Citrobacter freundii, Enterococcus faecalis)을 발굴하였다. 행동실험들을 통해 정상 마우스에게 5-SM 의 투여가 조현병 유사 행동을 나타내는지 확인하고자 하였고, Western blot 분석을 통해 5-SM 의 조현병 유사 행동에 대한 기전을 밝히고자 하였다. 그 결과, 5-SM 의 투여가 조현병 유사 행동을 유발하였고, 이러한 조현병 유사 행동이 아리피프라졸의 투여를 통해 개선이 됨을 확인할 수 있었다. 추가로 5-SM 의 투여가 뇌에서 시냅스 가소성을 감소시키고, N-methylD-aspartate(NMDA)수용체 발현량의 이상을 유발하였으며, 칼슘 이온의 유입량과 염증반응을 증가시켰으며, proline 과 glutamate 의 양을 증가시켰다. 이러한 결과들은 5-SM 의 투여가 조현병 환자에게서 관찰되는 NMDA 수용체를 경유한 신호전달체계의 이상을 유발할 수 있고, 항정신병약의 투여를 통해 개선될 수 있음을 제시한다. 이러한 변화를 통해 5-SM 을 투여한 본 동물 모델이 표면 타당성, 구조 타당성, 예측 타당성에 모두 부합하여 새로운 조현병 동물모델로 제시할 수 있다는 점을 시사하고 있다.키워드: 조현병; 뇌-장축; 장내미생물 이식; 장내미생물; N-methyl-D-aspartate 수용체

목차 (Table of Contents)

• Contents i
• Table contents iv
• Figure contents v
• Abbreviations vii
• Abstract ix
• 1. Introduction 1
• 1.1. Overview of schizophrenia 1
• 1.2. Causes of schizophrenia 2
• 1.3. Fecal microbiome transplantation 3
• 1.4. Gut microbiome and neurological diseases 4
• 1.5. Gut-brain axis in schizophrenia 5
• 1.6. Proteobacteria 8
• 1.7. Glutamate in schizophrenia 9
• 1.8. Hyperprolinemia 10
• 1.9. Specific aims for the present study 11
• 2. Materials and Methods 12
• 2.1. Collection of feces from SCZ patients and healthy volunteers 12
• 2.2. Animals 14
• 2.3. Materials 14
• 2.4. Microbiota 16S rRNA gene sequencing 15
• 2.5. Culture and isolation of bacteria from fecal donors 16
• 2.6. Drug and microbiome administration 17
• 2.7. Behavioral tests for SCZ-like behaviors 17
• 2.7.1. Open field test (OFT) 17
• 2.7.2. Social interaction test (SIT) 18
• 2.7.3. Novel object recognition test (NORT) 19
• 2.7.4. Acoustic startle response test (ASR) 20
• 2.8. Biological analysis 21
• 2.8.1. Western blot analysis 21
• 2.8.2. DNA extraction from fecal samples 22
• 2.8.3. Quantitative polymerase chain reaction (qPCR) 23
• 2.8.4. Enzyme-linked immunosorbent assay (ELISA) 24
• 2.9. Statistical analysis 24
• 3. Results 25
• 3.1. The effects of FMT from SCZ patients on normal mice 25
• 3.2. Gut microbiome composition of SCZ patients and healthy controls 29
• 3.3. Gut microbiome differences between SCZ patients and healthy controls at the phylum
• level 31
• 3.4. Gut microbiome differences between SCZ patients and healthy controls at the class level
• 33
• 3.5. Differentially enriched gut microbiota in patients with SCZ associated with symptoms
• (delusion, hallucination, and poor concentration) of SCZ 35
• 3.6. Gut microbiome composition of SCZ patients and healthy controls 40
• 3.7. The administration of each microbiome from 5-SM could invoke SCZ-like behaviors in
• mice 44
• 3.8. The administration 5-SM from SCZ patients could invoke SCZ-like behaviors in mice and
• the treatment of aripiprazole could reverse SCZ-like behaviors 49
• 3.9. The changes of expression levels of NR2B, PSD95, iNOS, GFAP and EAAT2 after the
• treatment of 5-SM in the prefrontal cortex (PFC) 53
• 3.10. The changes of expression levels of NR2B, PSD95, iNOS, GFAP and EAAT2 after the
• treatment of 5-SM in the hippocampus 56
• 3.11. The changes of proline and glutamate levels of in the brain after the treatment of fecal
• microbiome and 5-SM 58
• 3.12. The administration with 5-SM reduced the colonic expression of PRODH and induced
• hyperprolinemia in mice 60
• 4. Discussion 63
• References 73
• 국문초록 82
• Table Contents
• Table 1.
• Characterization of healthy control and SCZ patients enrolled for study 13
• Figure Contents
• Figure 1.
• Gut microbiome and schizophrenia connection 7
• Figure 2.
• The effects of FMT from SCZ patients on normal mice 27
• Figure 3.
• The analysis of fecal microbiome from schizophrenia (SCZ) patients and healthy control (HC)
• 30
• Figure 4.
• The analysis of fecal microbiome at the phylum level from schizophrenia (SCZ) patients and
• healthy control (HC) 32
• Figure 5.
• The analysis of fecal microbiome at the class level from schizophrenia (SCZ) patients and
• healthy control (HC) 34
• Figure 6.
• The enriched gut microbiome in SCZ patients, and the relationships between composition of gut
• microbiome and SCZ symptoms 38
• Figure 7.
• The effects of administration of microbiomes from schizophrenia (SCZ) on normal mice ·· 42
• Figure 8.
• The effects of each microbiome from 5-SM treatments on normal mice 47
• Figure 9.
• The effects of aripiprazole on schizophrenia (SCZ)-like behavior mouse model induced by
• administration of 5 species microbiomes (5-SM) from SCZ 51
• Figure 10.
• The effects of administration of 5 species microbiomes (5-SM) from schizophrenia (SCZ) and
• aripiprazole on the expression level of NR2B, PSD95, iNOS, GFAP and EAAT2 in the
• prefrontal cortex 55
• Figure 11.
• The effects of administration of 5 species microbiomes (5-SM) from schizophrenia (SCZ) and
• aripiprazole on the expression level of NR2B, PSD95, iNOS, GFAP and EAAT2 in the
• hippocampus 57
• Figure 12.
• Proline and glutamate levels in the brain of mice receiving administration of 5-SM 59
• Figure 13.
• PRODH expression in the colon and proline levels in the plasma of mice receiving oral
• administration of 5-SM 61