Evaluation of the neuroprotective potential of Lactobacillus acidophilus via the microbiota-gut-brain axis = Lactobacillus acidophilus 의 미생물총-장-뇌 축을 통한 신경 보호 잠재력 평가

다국어 초록 (Multilingual Abstract)

The gut microbiota plays a pivotal role in maintaining host health and is increasingly implicated in the pathogenesis of neurodegenerative diseases via the microbiota-gut-brain axis. Parkinson’s disease (PD), potentially caused by neuroinflammation and specific protein aggregation, mainly for α- synuclein, has been associated with gut microbial dysbiosis Probiotics isolated from human infants could support gut and brain health with their neuroprotective effects, but remain underexplored in integrative models. In this study, we investigated the therapeutic potential of Lactobacillus acidophilus SLAM_LAA02 (LAA02) in mitigating PD-related neuropathology and behavioral impairments via modulation of the gut-brain axis. Following comprehensive safety and functional assessments, LAA02 was evaluated in Caenorhabditis elegans(C. elegans) for its effects on neuroprotection. Importantly, LAA02 supplementation significantly extended lifespan, enhanced antimicrobial defense, improved behavioral responses, and suppressed α-synuclein gene expression in transgenic worms. In addition, under lipopolysaccharide (LPS)-induced inflammatory conditions, LAA02 and its cell-free supernatant restored gut barrier integrity by upregulating tight junction genes (Cldn1, Ocln) and alleviating inflammation through the downregulation of Nos2 and Tnf expression. Moreover, using a rotenone-induced mouse model of Parkinson’s disease (PD), we demonstrated that LAA02 administration ameliorated motor dysfunction, increased expression of neuroprotective genes, restored gut microbial diversity, and improved intestinal barrier function. These effects were accompanied by a notable reduction in α-synuclein expression and pro- inflammatory gene expression, along with an upregulation of brain-derived neurotrophic factor (BDNF). Taken together, our findings suggest that LAA02 exhibits neuroprotective and gut-modulating properties, supporting its potential as a promising probiotic candidate for alleviating PD-related pathologies through the gut-brain axis.

국문 초록 (Abstract)

장내미생물은 숙주의 건강 유지에 중요한 역할을 하며, 미생 물–장–뇌 축(microbiota–gut–brain axis)을 통해 신경퇴행성 질 환의 발병기전과 연관된다는 보고가 점점 증가하고 있다. 특히, α -synuclein 단백질의 비정상적인 응집과 신경염증으로 특징지어 지는 파킨슨병은 장내 미생물 불균형과 밀접한 관련이 있는 것으 로 알려져 있다. 인간 영아로부터 분리된 프로바이오틱스는 신경 보호 효과를 통해 장 및 뇌 건강을 지원할 가능성이 있으나, 통합 적인 모델에서의 연구는 여전히 부족한 실정이다.
본 연구에서는 Lactobacillus acidophilus SLAM_LAA02 (LAA02)의 장–뇌 축 조절을 통한 PD 관련 신경병리 및 행동장 애 개선 효능을 평가하였다. 먼저 LAA02의 안전성 및 기능적 특성을 종합적으로 검증한 뒤, 예쁜꼬마선충(Caenorhabditis elegans) 모델을 이용하여 신경보호 효과를 확인하였다. 그 결과, LAA02 보충군은 수명이 유의하게 연장되었으며, 항균 방어능이 향상되고, 행동 반응이 개선되었으며, 형질전환 선충에서 α- synuclein 발현이 억제되었다. 또한 LPS로 유도된 염증 환경에 서 LAA02 및 그 상등액은 세포 간 밀착연접 단백질 유전자의 발 현을 증가시켜 장 장벽 무결성을 회복시키고, 염증성 매개 인자의 발현을 감소시켜 염증을 완화하였다. 더 나아가 rotenone으로 파 킨슨병이 유도된 마우스 모델에서 LAA02 투여는 운동기능 저하 를 개선하고, 신경보호 관련 유전자의 발현을 증가시키며, 장내 미 생물 다양성을 회복시키고 장 장벽 기능을 향상시켰다. 이러한 효 과는 α-synuclein 발현 및 염증성 유전자 발현의 감소, 그리고 뇌유래신경영양인자(BDNF) 발현 증가와 함께 나타났다.
종합하면, 본 연구 결과는 LAA02가 신경보호 및 장내 환경 조절 특성을 동시에 지니며, 장–뇌 축을 통한 파킨슨병 관련 병리 완화에 기여할 수 있는 유망한 프로바이오틱스 후보임을 제시한다.

목차 (Table of Contents)

• Chapter 1. Literature review 19
• 1.1. Probiotics 19
• 1.1.1. Definition of probiotics 19
• 1.1.2. Application of probiotics 20
• 1.2. Microbiome and gut-brain axis 21
• 1.2.1. Definition and application of the microbiome 21
• 1.2.2. Microbiome-gut-brain communication 22
• 1.3. Health benefits of probiotics in the host 24
• 1.3.1. Experimental model for probiotic-host interaction 24
• 1.3.2. Probiotics and neurodegenerative disease 25
• 1.3.3. Modulatory effects of probiotics in Parkinson’s disease 26
• Chapter 2. Safety and functional evaluation of Lactobacillus acidophilus using Caenorhabditis elegans and in vitro models 29
• 2.1. Introduction 29
• 2.2. Materials and methods 31
• 2.2.1. Isolation and Identification of lactic acid bacteria 31
• 2.2.2. Bacteria culture condition 31
• 2.2.3. Acid and bile acid tolerance 32
• 2.2.4. Hemolytic activity 32
• 2.2.5. Bile salt hydrolase activity 33
• 2.2.6. D-lactate analysis 33
• 2.2.7. Cell culture 34
• 2.2.8. Lactate dehydrogenase (LDH) cytotoxicity analysis 34
• 2.2.9. Adhesion ability assay 35
• 2.2.10. Antimicrobial activity test 35
• 2.2.11. Antibiotic susceptibility test 36
• 2.2.12. Total DNA extraction and whole genome sequence analysis 36
• 2.2.13. Functional experiment using C. elegans 38
• 2.2.13.1. Bacterial strains and culture conditions 38
• 2.2.13.2. C. elegans strains and maintenance 38
• 2.2.13.3. C. elegans lifespan and killing assays 39
• 2.2.13.4. C. elegans behavioral assay and learning assay 39
• 2.2.13.5. C. elegans learning assay 41
• 2.2.14. Total RNA extraction and RT-qPCR analysis 41
• 2.2.15. LPS treatment in Caco-2 cells 42
• 2.2.16. Statistical analysis 43
• 2.3. Results 45
• 2.3.1. Safety assessment of Lactobacillus acidophilus SLAM_LAA02 45
• 2.3.1.1. Acid and Bile acid tolerance test 45
• 2.3.1.2. BSH activity test 47
• 2.3.1.3. Hemolysis assay 49
• 2.3.1.4. D-lactate assay 51
• 2.3.1.5. LDH assay 53
• 2.3.1.6. Antimicrobial ability test 55
• 2.3.1.7. Adhesion ability test 57
• 2.3.1.8. Measurement of the Minimum Inhibitory Concentration 59
• 2.3.1.9. Genomic analysis of Lactobacillus acidophilus SLAM_LAA02 61
• 2.3.2. Functional evaluation of Lactobacillus acidophilus SLAM_LAA02 using C. elegans 65
• 2.3.2.1. C. elegans lifespan assay 65
• 2.3.2.2. C. elegans killing assay 67
• 2.3.2.3. C. elegans behavior and learning assays 69
• 2.3.2.4. RT-qPCR analysis 74
• 2.3.3. In vitro modulatory effects of Lactobacillus acidophilus SLAM_LAA02 76
• 2.3.3.1. RT-qPCR analysis using Caco-2 cell line 76
• 2.4. Discussion 79
• Chapter 3. Evaluation of Lactobacillus acidophilus improving brain and gut health in Parkinson’s disease induced murine animal model 84
• 3.1. Introduction 84
• 3.2. Materials and methods 88
• 3.2.1. Animals and experimental designs 88
• 3.2.2. Motor function and behavioral tests 88
• 3.2.2.1. Rota-rod test 89
• 3.2.2.2. Pole test 89
• 3.2.2.3. Grip strength test 91
• 3.2.2.4. Open field test (OFT) 91
• 3.2.2.5. Spontaneous alternation Y-maze test (Y-maze) 91
• 3.2.3. Colon length measurement 92
• 3.2.4. Fecal pellet output frequency 92
• 3.2.5. Histological analysis 92
• 3.2.6. Serum analysis 92
• 3.2.7. Total RNA isolation and RT‑qPCR analysis 93
• 3.2.8. Metagenomic analysis 94
• 3.2.9. Statistical analysis 95
• 3.3. Results 98
• 3.3.1. Body weight change 98
• 3.3.2. Motor function and behavioral analysis 100
• 3.3.2.1. Rota-rod test 100
• 3.3.2.2. Pole test 101
• 3.3.2.3. Grip strength test 102
• 3.3.2.4. Anxiety-like behavior 103
• 3.3.2.5. Learning and memory 104
• 3.3.3. Colon length 107
• 3.3.4. Fecal pellet output frequency 109
• 3.3.5. Histological analysis 111
• 3.3.6. Serum analysis 113
• 3.3.7. RT-qPCR analysis 115
• 3.3.8. Metagenomic analysis 121
• 3.3.9. Functional prediction and correlation analysis 128
• 3.4. Discussion 133
• Reference 144
• Summary in Korean 161