Studies on Proteolytic Processing of Amyloid Precursor protein (APP) in Olfactory Epithelium using AD Transgenic Mice

Olfactory impairment is a well-documented abnormality in Alzheimer’s disease (AD). AD is known to begin with abnormal processing of amyloid precursor protein (APP), through sequen-tial cleavages first by β-secretase and then by γ-secretase complex which leads to excess produc-tion of β-amyloid (Aβ) in the cortex. While olfactory dysfunction occurs in the incipient stages of AD even before Aβ deposition and plaque formation in the CNS, the functional correlation of olfac-tory deficit in relation to AD is not well understood. It may be critical to know the process under-lying AD-related olfactory sensory loss to find some novel biomarkers. To this end, two different types of transgenic mice models were used including Tg2576, which overexpresses human APP and Tg6799 (also called 5xFAD), which expresses human APP and Presenilin1 both mutations to-gether. It was found unique APP processing in OE that has significance in providing not only pos-sible biomarkers that can be used for screening and detection of AD before plaque formation but also for treatment purposes. This data demonstrates that the abnormal processing of APP in the OE provides APP fragments including 25 kDa, 55kDa and 80 kDa that can be a potential biomarker in the very early and critical period in the stage of mild cognitive impairment, that is, the critical stage of AD occurrence (before Aβ plaque formation in the CNS). Such biomarkers can be accessed via biopsy and can be used for establishing improved early diagnostic procedure for the AD. Additionally, PS2 increased level was found in OE that possibly involved in unique APP processing and might also be crucial for under-standing the γ-secretase role controlling AD through γ-secretase as a therapeutic target. ⓒ 2014 DGISTINTRODUCTION 7 -- MATERIAL AND METHODS 11 -- 1.1 Animals 12 -- 1.1.1 Transgenic Alzheimer’s disease model Tg2576 12 -- 1.1.2 Transgenic Alzheimer’s disease model 5XFAD 12 -- 1.2 Olfactory behavior test 13 -- 1.3 RT-PCR 14 -- 1.4 Western blot 15 -- RESULTS 17 -- Chapter 1: Olfactory dysfunction behavior 18 -- Chapter 2: Secretases in olfactory (peripheral) and central nervous system 26 -- Chapter 3: Biomarkers for early Alzheimer’s detection through olfactory system 49 -- DISCUSSION AND CONCLUSION 60 -- REFERENCES 65 -- ABSTRACT IN KOREAN 70 -- ACKNOWLEGMENTS 71 -- CURRICULUM VITAE 73알츠하이머병(Alzheimer’s disease, AD)에서의 후각 이상은 잘 알려져 있다. 알츠하이머병은 비정상적인 아밀로이드 전구단백질(APP)의 대사로 인해 발병한다고 알려져 있는데, 이런 아밀로이드 전구단백질의 공정(APP processing)은 먼저 베타-세크레타아제(β-secretase)로 절단되고 연이어 감마-세크레타아제(γ-secretase) 작용으로 절단되어 과량의 베타-아밀로이드(β-amyloid, Aβ)를 생성되는 과정으로 알려져 있다. 하지만 후각 상실이 알츠하이머병의 초기 단계, 특히 중추 신경계 안에서의 베타 아밀로이드 침착도 일어나기 전에 일어나지만 알츠하이머병과 어떤 연관관계를 가지는 지는 잘 알려져 있지 않다. 이런 연관관계에 대한 연구를 한다면 알츠하이머병 관련 후각 감각 상실에 대한 이해와 더불어 이를 통한 새로운 생체지표(biomarker)를 찾을 수 있는데 중요하게 작용 할 수도 있을 것이다. 따라서 이를 위해 형질전환마우스(transgenic mouse)인 인간 APP를 과 발현 시킨 Tg2576과 인간 아밀로이드 전구단백질, 프리세닐린 1(Presenilin1)를 모두 발현 시킨 Tg6799(5xFAD라고도 불림)를 연구에 사용하였다. 또한 각각의 형질전환마우스에서 인지 감소가 나타난다고 알려진 10개월(Tg2576)과 2개월(Tg6799)시점에서 실험을 수행하였다. 정상마우스에서 중추 신경계로 대변되는 대뇌 피질이나 후구에 비해 말초 신경계인 후각 상피에서는 베타-세크레타아제, 감마-세크레타아제1, 2의 발현량과 그 작용에서 차이를 보였다. 더욱이 후각시스템은 말초 신경계로서 중추 신경계에서와 다른 형태의 아밀로이드 전구단백질의 공정이 나타남을 확인하였다. 알츠하이머병 모델인 두 가지 형질전환마우스에서 후각 시스템 내에서는 감마-세크레타아제2의 발현에서 정상과 유의성 있는 차이를 보였다. 아밀로이드 전구단백질의 공정에서도 차이가 나타났고 특이적으로 단백질 크기가 25KDa, 55KDa와 80KDa에서 특이적인 패턴이 보였고 이는 후각 상피에서 얻을 수 있는 알츠하이머병의 생체지표 후보가 될 수 있는 새로운 펩타이드 단편이 될 수 있다. 이러한 결과는 알츠하이머병 환자의 후각 상피에서 비정상적인 APP 공정이 진단 지표가 되는 새로운 펩타이드 단편을 생성함을 증명한다. 또한 AD 발병에 결정적이며 아주 초기에 해당되는 시기로 알려진 경도인지장애(Mild cognitive impairment, MCI) 시기에서 이 펩타이드 단편이 AD를 진단 할 수 있는 생체지표 후보가 될 수 있음을 증명한다. 이러한 생체지표를 이용한다면 AD 조기 진단의 향상을 가져올 것이며 이를 위해 생검(biopsy)을 이용할 수 있을 것이다. ⓒ 2014 DGISTMasterdCollectio

Region-specific amyloid-β accumulation in the olfactory system influences olfactory sensory neuronal dysfunction in 5xFAD mice

Background: Hyposmia in Alzheimer’s disease (AD) is a typical early symptom according to numerous previous clinical studies. Although amyloid-β (Aβ), which is one of the toxic factors upregulated early in AD, has been identified in many studies, even in the peripheral areas of the olfactory system, the pathology involving olfactory sensory neurons (OSNs) remains poorly understood. Methods: Here, we focused on peripheral olfactory sensory neurons (OSNs) and delved deeper into the direct relationship between pathophysiological and behavioral results using odorants. We also confirmed histologically the pathological changes in 3-month-old 5xFAD mouse models, which recapitulates AD pathology. We introduced a numeric scale histologically to compare physiological phenomenon and local tissue lesions regardless of the anatomical plane. Results: We observed the odorant group that the 5xFAD mice showed reduced responses to odorants. These also did not physiologically activate OSNs that propagate their axons to the ventral olfactory bulb. Interestingly, the amount of accumulated amyloid-β (Aβ) was high in the OSNs located in the olfactory epithelial ectoturbinate and the ventral olfactory bulb glomeruli. We also observed irreversible damage to the ectoturbinate of the olfactory epithelium by measuring the impaired neuronal turnover ratio from the basal cells to the matured OSNs. Conclusions: Our results showed that partial and asymmetrical accumulation of Aβ coincided with physiologically and structurally damaged areas in the peripheral olfactory system, which evoked hyporeactivity to some odorants. Taken together, partial olfactory dysfunction closely associated with peripheral OSN’s loss could be a leading cause of AD-related hyposmia, a characteristic of early AD. © 2021, The Author(s).1

The Human SCN10AG1662S Point Mutation Established in Mice Impacts on Mechanical, Heat, and Cool Sensitivity

The voltage-gated sodium channel NAV1.8 is expressed in primary nociceptive neurons and is involved in pain transmission. Mutations in the SCN10A gene (encoding NAV1.8 channel) have been identified in patients with idiopathic painful small fiber neuropathy (SFN) including the SCN10A(G1662S) gain-of-function mutation. However, the role of this mutation in pain sensation remains unknown. We have generated the first mouse model for the G1662S mutation by using homologous recombination in embryonic stem cells. The corresponding Scn10a(G1663S) mouse line has been analyzed for Scn10a expression, intraepidermal nerve fiber density (IENFD), and nociception using behavioral tests for thermal and mechanical sensitivity. The Scn10a(G1663S) mutants had a similar Scn10a expression level in dorsal root ganglia (DRG) to their wild-type littermates and showed normal IENFD in hindpaw skin. Mutant mice were more sensitive to touch than wild types in the von Frey test. In addition, sexual dimorphism was observed for several pain tests, pointing to the relevance of performing the phenotypical assessment in both sexes. Female homozygous mutants tended to be more sensitive to cooling stimuli in the acetone test. For heat sensitivity, male homozygous mutants showed shorter latencies to radiant heat in the Hargreaves test while homozygous females had longer latencies in the tail flick test. In addition, mutant males displayed a shorter reaction latency on the 54°C hot plate. Collectively, Scn10a(G1663S) mutant mice show a moderate but consistent increased sensitivity in behavioral tests of nociception. This altered nociception found in Scn10a(G1663S) mice demonstrates that the corresponding G1662 mutation of SCN10A found in SFN patients with pain contributes to their pain symptoms

Distinct amyloid precursor protein processing machineries of the olfactory system

Processing of amyloid precursor protein (APP) occurs through sequential cleavages first by β-secretase and then by the γ-secretase complex. However, abnormal processing of APP leads to excessive production of β-amyloid (Aβ) in the central nervous system (CNS), an event which is regarded as a primary cause of Alzheimer's disease (AD). In particular, gene mutations of the γ-secretase complex—which contains presenilin 1 or 2 as the catalytic core—could trigger marked Aβ accumulation. Olfactory dysfunction usually occurs before the onset of typical AD-related symptoms (eg, memory loss or muscle retardation), suggesting that the olfactory system may be one of the most vulnerable regions to AD. To date however, little is known about why the olfactory system is affected so early by AD prior to other regions. Thus, we examined the distribution of secretases and levels of APP processing in the olfactory system under either healthy or pathological conditions. Here, we show that the olfactory system has distinct APP processing machineries. In particular, we identified higher expressions levels and activity of γ-secretase in the olfactory epithelium (OE) than other regions of the brain. Moreover, APP c-terminal fragments (CTF) are markedly detected. During AD progression, we note increased expression of presenilin2 of γ-secretases in the OE, not in the OB, and show that neurotoxic Aβ*56 accumulates more quickly in the OE. Taken together, these results suggest that the olfactory system has distinct APP processing machineries under healthy and pathological conditions. This finding may provide a crucial understanding of the unique APP-processing mechanisms in the olfactory system, and further highlights the correlation between olfactory deficits and AD symptoms. © 2017 Elsevier Inc.1