DGIST Scholar: Region-specific accumulation of soluble amyloid-β in the olfactory system in Alzheimer’s disease

Department of Brain Sciences Theses Ph.D.

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Region-specific accumulation of soluble amyloid-β in the olfactory system in Alzheimer’s disease

Title: Region-specific accumulation of soluble amyloid-β in the olfactory system in Alzheimer’s disease

Alternative Title: 알츠하이머 병의 후각 신경계 내부 영역 특이적 수용성 베타-아밀로이드 축적에 관한 연구

Author(s): Gowoon Son

DGIST Authors: Son, Gowoon ; Moon, Cheil ; Steinbusch, Harry W.M.

Advisor: 문제일

Co-Advisor(s): Harry W.M. Steinbusch

Issued Date: 2021

Awarded Date: 2021/08

Type: Thesis

Subject: Olfactory dysfunction, Alzheimer's disease, The primary olfactory pathway, Amyloid-β, Nasal discharge

Description: Olfactory dysfunction, Alzheimer's disease, The primary olfactory pathway, Amyloid-β, Nasal discharge

Abstract: Alzheimer’s disease (AD) is an incurable neurodegenerative disease, the precise cause of which remains unclear. Identifying the characteristic symptoms at the early stages of disease can provide clues to the cause of AD. Olfactory dysfunction, which is reported beginning in the mild cognitive impairment stage, is one of the most prevalent early symptoms of AD. However, precisely how the olfactory nervous system plays a role in the progression of olfactory dysfunction in AD has been overlooked. I have investigated the accumulation of soluble amyloid-β (Aβ) proteins in the primary olfactory system and the effect thereof, given that Aβ accumulation is the most typical early pathological symptom in AD patients. Considering that the primary olfactory system is the first neural component of olfactory signaling, I hypothesized that the features of the primary olfactory system may be involved in the progression of AD pathology, which may induce olfactory dysfunction in the early stages of disease. First, I identified that olfactory dysfunction result in partial anosmia early in the pathogenesis of AD. The abnormality was indicated by region-specific Aβ accumulation in the primary olfactory system. Next, I illustrated that multimodal damage using postmortem tissue from AD patients, including morphological alterations with Aβ accumulation concomitant with microgliosis and neurotransmitter deficits, was influenced by region-specific degeneration, even in the human olfactory glomerulus. Finally, based on these findings, I identified soluble Aβ from the olfactory epithelium in the nasal discharge, which could be used to predict cognitive decline in AD patients. Taken together, the results show that soluble Aβ, which accumulates in a region-specific manner in the primary olfactory nervous system, can be measured as a sign of olfactory neurodegeneration, which induces olfactory dysfunction in AD. Furthermore, I suggest that the primary olfactory nervous system can mirror and predict pathological mechanisms in the onset stage of AD.|본 논문은 알츠하이머 병의 초기에 흔히 나타나는 증상인 후각 장애의 특징과 원인에 대한 연구이다. 알츠하이머 병은 원인이 명확하지 않고 치료약이 전무하여, 병의 시작단계에서 나타나는 증상의 특징과 원인을 파악하는 것은 알츠하이머 병의 치료와 원인 규명에 단서를 제공할 수 있다. 후각 장애는 알츠하이머 발병 이전의 경도 인지 장애 단계서부터 보고되지만, 알츠하이머 병의 진행이 후각 손상에 미치는 영향과 그 특성에 대하여 후각 신경계가 어떤 역할을 하는지는 명확하지 않다. 본 논문은 수용성 베타-아밀로이드의 축적이 알츠하이머 환자에게서 관찰할 수 있는 일반적인 초기 병리학적 증상이라는 점에 착안하여 후각 신호처리의 첫번째 회로인 일차 후각 경로 내부의 베타-아밀로이드 단백질의 축적과, 그것이 후각 장애에 미치는 영향을 조사했다. 첫번째로, 후각장애가 알츠하이머 병의 초기 병리발생 단계에서 후각저하증으로 나타나는 것을 확인했다. 또한 이 증상은 일부 향에 특이적임을 관찰했다. 이러한 부분 후각 저하증은 일차 후각 신경계 내부에서 신경재생-사멸의 재구성이 활발한 영역-특이적으로 축적된 베타-아밀로이드와 연관성이 있음을 확인했다. 다음으로, 알츠하이머 병 환자 사후 조직 내부에서도 일차 후각신경계의 신경 접합부에서 영역 특이적 베타-아밀로이드의 축적과 베타-아밀로이드의 영향을 받는 마이크로글리아의 활성화 및 신경전달물질 결손을 포함한 조직 내 병변과 구조 손상을 확인했다. 더 나아가, 후각 상피에서 관찰할 수 있는 수용성 베타-아밀로이드의 존재를 바탕으로 알츠하이머 병 환자의 인지 저하를 예측할 수 있는 콧물 내 베타-아밀로이드의 유형을 확인함과 더불어 콧물 내 단백질 조성의 변화를 동정했다. 본 논문의 결과를 바탕으로 일차 후각 신경계 내부 영역 특이적으로 축적되는 수용성 베타-아밀로이드는 후각 신경계에 의존적인 신경퇴화의 징후임을 나타내며, 이는 알츠하이머 병의 후각 기능 장애를 유도한다. 더불어 일차 후각 신경계는 알츠하이머 병 시작 단계의 병리학적 기전을 반영할 수 있음을 시사한다.

Table Of Contents: I. Introduction 1
1.1 Preface 1
1.1.1 Olfaction: Alpha et Omega 1
1.2 Research background 1
1.2.1 Olfactory dysfunction in Alzheimer’s disease: early and partial deficits 1
1.2.2 Subzonal organization of the olfactory system 2
1.2.3 The olfactory glomerulus: the first synaptic interface in the primary olfactory system 6
1.2.4 Neuropathology of Alzheimer’s disease 7
1.2.5 Generation and physiology of amyloid-β protein in the nervous system 12
1.2.6 Amyloidopathy in the mouse olfactory system 13
1.2.7 Amyloidopathy in the human olfactory system 14
1.2.8 The amyloid hypothesis: a mouse model 15
1.2.9 Validation in human subjects: amyloid-β in the olfactory system 16
1.3 Research hypothesis 16
1.4 Overview of this thesis 17
II. Materials & Methods 18
2.1 Materials 18
2.1.1 Experimental animals 18
2.1.2 Human nasal discharge 18
2.1.2.1 Subject categorizing criteria 18
2.1.2.2 Nasal discharge collection 20
2.1.3 Human specimen 20
2.1.4 Antibody & Reagent 20
2.2 Methods 23
2.2.1 Animal behavioral tests 23
2.1.1.1 Morris water maze 23
2.1.1.2 Y-maze test 23
2.1.1.3 Food-seeking test 24
2.1.1.4 Odor detection (nose poke) test 25
2.2.2 Histological experiments 26
2.2.2.1 Tissue preparation 26
2.2.2.1.1 Mouse 26
2.2.2.1.2 Human 27
2.2.2.2 Immunohistochemistry 31
2.2.2.3 Immunofluorescence 31
2.2.2.3.1 Mouse 31
2.2.2.3.2 Human 32
2.2.2.4 TUNEL assay 32
2.2.2.5 DAB intensity measurement 33
2.2.2.6 Fluorescence density measurement 33
2.2.2.7 Subregional analysis of the olfactory glomerulus 34
2.2.2.7.1 Mouse 34
2.2.2.7.2 Human 34
2.2.2.8 Three-dimensional reconstruction 36
2.2.2.9 Evaluation of microglia morphology 36
2.2.3 Nasal discharge pre-treatment 37
2.2.4 Biochemical experiments 37
2.2.4.1 Immunoblot 37
2.2.4.2 Immunoprecipitation 38
2.2.4.3 BrdU assay 38
2.2.5 Olfactory sensory neuronal calcium imaging 39
2.2.5.1 Mouse preparation 39
2.2.5.2 Odor stimulation 39
2.2.5.3 Optical recording 40
2.2.5.4 Data analysis 40
2.2.6 Electron microscopy 41
2.2.7 Proteomics 42
2.2.7.1 Liquid chromatography-mass spectrometry/mass spectrometry (LC-MS/MS) 42
2.2.7.2 Mass spectrometry‐based quantitative proteomic analysis (TMT-LC-MS3) 44
2.2.8 Statistical analysis 45
III. Results 46
3.1 Region-specific olfactory dysfunction revealed by transgenic mouse research 46
3.1.1 hAPP-mutated transgenic mice exhibit olfactory dysfunction before se-vere cerebral dysfunction 46
3.1.2 hAPP transgenic mice exhibit high levels of Aβ oligomers in the olfactory epithelium 48
3.1.3 Aβ accumulation in specific regions of the olfactory system in the early stages of disease in Tg mice 50
3.1.4 Evidence of active amyloidosis in the olfactory sensory neurons project-ing to the ventral olfactory glomeruli in Tg mouse 52
3.1.5 hAPP/PS1-mutated Aβ overexpressing mice exhibit olfactory dysfunction before severe cerebral dysfunction 54
3.1.6 Atypical olfactory dysfunction in Tg mice 55
3.1.7 Aβ accumulation in a specific region of the olfactory system in the early stages of disease in of Tg mice 58
3.1.8 Impaired signal transduction with Aβ accumulation in the olfactory glomerulus 61
3.1.9 Region-specific structural damage by disrupted turnover of olfactory sen-sory neurons in Tg mice 73
3.2 Region-specific human olfactory neuropathology 83
3.2.1 Gross morphology of the human olfactory glomerulus in non-demented controls 83
3.2.2 Ventral region-specific alteration of the olfactory glomerulus in AD 83
3.2.3 Aβ accumulation in the ventral olfactory glomeruli in AD patients 87
3.2.4 Increased microgliosis in the ventral olfactory glomeruli in AD patients 87
3.2.5 Impaired signal transduction in the ventral olfactory glomeruli in AD pa-tients 93
3.2.6 Impaired structure of the olfactory glomerulus in AD patients 95
3.3 Identification of soluble Aβ in nasal-derived body fluid 98
3.3.1 Detection of soluble Aβ in nasal discharge in AD patients 98
3.3.2 Positive correlation between detection of soluble Aβ in human nasal dis-charge and cognitive decline in AD patients 105
3.3.3 Profiling of the high-throughput proteome in nasal discharge and associa-tion with soluble Aβ protein in AD progression 110
IV. Discussion 112
4.1 Summary 112
4.2 Olfactory dysfunction in early-stage AD 112
4.3 Features of olfactory neurodegeneration 117
4.4 Impact of region-specific Aβ accumulation in the olfactory system in AD 119
4.5 Features of β-amyloidosis in the olfactory system 123
4.6 Toxicity of oligomeric Aβ in the olfactory nervous system 124
4.7 Biomonitoring of AD progression using soluble Aβ in body fluid from the ol-factory system 127
4.8 Contributions of the olfactory system to AD research: the olfactory system, a window to the brain 131
V. Conclusion 132
5.1 Conclusion 132
References
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