環境因子與Abeta1-40加成性傷害空間學習及記憶行為

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(1)國立台灣師範大學生命科學系博士論文. 環境因子與Aβ1-40 加成性傷害空間學習及記憶行為 The synergistic effects of environmental factors and Aβ1-40 impaired the spatial learning and memory in mice. 研究生：黃慧貞 Hei-Jen Huang. 指導教授：謝秀梅博士 Hsiu-Mei Hsieh. 中華民國九十八年十二月.

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(4) 致謝博士班學習過程中在指導教授謝秀梅副教授之庇護及指導下，臨畢業前的感覺竟然是不安的，有如當初要嫁為人妻的感覺，意味著往後所有一切都要自己負起責任的重擔。因此不斷反覆思索著未來之研究該如何規劃才不會枉費博士班訓練之社會資源，期許自己對社會能有所貢獻。我相信會有如此的責任感應該是我的指導教授長期以身教方式讓我有如此之領悟，記憶最深刻的是老師不論面臨多大之困境都仍能放下一切與我討論論文之修改作業。不僅如此在學習過程中修課、文獻報告、研究結果報告、參與研討會、研究架構、實驗態度、論文撰寫及上傳送審等都要衷心感謝指導教授耐心及細心的指導，得以順利完成本論文之撰寫，浩瀚師恩永銘在心。. 論文口試期間，承蒙國立台灣大學心理系. 梁庚辰教授、台北榮. 民總醫院精神部洪成志副教授、國立台灣師範大學生命科學蘇銘燦助理教授及王慈蔚助理教授細心審閱，並惠予諸多指證及寶貴建議，使論文更加完整，令學生至為感激，謹致以最深的敬意與謝忱。尤其更感謝梁庚辰教授實驗室長達 6 年多於動物房及行為房之協助與支持。.

(5) 研究過程中，感謝實驗室全體夥伴如雅津、興杰、君宇、峻緯、振銘、偉毅、智剛、陸泉、彥旭等給予我許多協助與支持。並感謝馬偕醫護管理專科學校學生乙晴、家玲、婉真、宴瑜、家羽及如芳等對動物房小鼠之照顧及相關實驗之協助。. 感謝馬偕醫護管理專科學校給予進修博士班的機會，尤其感謝歷屆護理科科主任李美玉主任、彭碧智主任及田聖芳主任的支持，給我單純之環境及足夠之思考空間面對一切;更要感謝基礎醫學組同仁們分擔了絕大部分之行政工作及好友美玲與均衡在生活上給予協助與鼓勵，不論是開心或是難過時因為有你們的陪伴，使博士生涯不致枯燥乏味。. 感謝台灣師範大學生命科學系負責貴重儀器的負責老師李桂楨教授及方剛教授在儀器使用上的借用與幫忙，及系辦漢英助教在行政上的服務與叮嚀。. 最後，感激我的先生一路無怨無悔全力支持及兩個女兒的貼心鼓勵，更感謝老天爺賜給我一對健康的父母親讓我安心進行實驗，使我完全專注地完成論文，謹以此文獻給您們。.

(6) 摘要阿茲海默氏症(Alzheimer's Disease)於中樞神經系統內主要病變特徵之一為澱粉樣斑塊堆積。Aβ蛋白質（長約39到43個胺基酸）是穿膜本體蛋白質β-amyloid precursor protein (APP)的代謝物，是造成澱粉樣斑塊最主要之原因；大部分Aβ蛋白質為Aβ1-40與Aβ1-42。雖然Aβ1-42於澱粉樣斑塊及認知功能受損中扮演著舉足輕重的角色，但是在阿茲海默氏症早期階段時Aβ1-40的分泌量遠超過Aβ1-42；甚至有研究發現 Aβ1-40在阿茲海默氏症晚期階段病因形成有所相關。因此本論文主要探討環境因子是否會提升Aβ1-40的毒性及如何造成神經退化之機轉。首先，第一個實驗所運用的環境因子為第I型糖尿病所造成的高血糖與Aβ1-40交互作用所產生的影響。結果發現，單獨高血糖或Aβ1-40都無法造成海馬回顯著性地神經退化及空間學習及記憶之損壞；但是高血糖同時併入Aβ1-40會造成Aβ1-40大量堆積、氧化壓力增加及細胞凋亡，最後導致空間學習及記憶之受損。第二個實驗主要探討老化(aged)與 Aβ1-40之間的關係，所使用的老鼠為阿茲海默氏症雙基因 (APP/PS1) 突變的背景鼠種(C57BL/6J × C3H)。雖然不論何種阿茲海默氏症基因轉殖鼠均無法完全模擬人類罹患阿茲海默氏症，然而不同鼠種背景之阿茲海默氏症基因轉殖鼠其致病原因及行為受損程度卻有所差異。因為我們實驗室APP/PS1雙基因突變的基因轉殖鼠其背景鼠種為. I.

(7) (C57BL/6J × C3H)，所以此實驗對象為C57BL/6J (母) 與 C3H (公) 交配所產生的第一子代公鼠。結果發現，老化與Aβ1-40交互作用會產生短暫性體重下降及空間記憶提取之問題；所以推測體重短暫性下降或許是老年癡呆症的早期訊號。最後一個實驗探討之環境因子是目前時下許多人所面臨之狀況—壓力，觀察壓力與Aβ1-40之間的交互作用；結果發現海馬回處出現細胞凋亡及突觸功能受損而導致嚴重的空間學習及記憶之受損。綜合以上的結果，發現Aβ1-40的毒性會受到環境因子之影響而提升而且不論哪一種環境因子與Aβ1-40相互作用都是透過氧化壓力之傷害，因此建議抗氧化壓力之治療或預防措施能延緩阿茲海默氏症病程之發展。. 關鍵字：阿茲海默氏症、Aβ1-40蛋白質、高血糖、老化、壓力、抗氧化治療。. II.

(8) ABSTRACT. The deposition of beta amyloid (Aβ) as soluble or insoluble aggregates in senile plaques has been well characterized in the Alzheimer’s disease (AD) brain. Aβ peptides are composed of 39-43 amino acids derived from the soluble metabolic products of amyloid beta precursor protein (APP). Most of the soluble Aβ species comprise the species Aβ1-40 and Aβ1-42. Many studies focus on the Aβ1-42, which plays an important role in plaques and behavioral deficits in the AD. However, Aβ1-40 receives more attention in recent AD studies. A higher proportion of Aβ1-40 is present in the brain under the general condition, an increase of Aβ1-40 / Aβ1-42 ratio in cerebrospinal fluid was identified at the early stage of AD, and an important role of Aβ1-40 was identified in the pathogenesis of late-onset sporadic AD. Therefore, this dissertation worked on characterization of the effects of the interaction between environmental factors and Aβ1-40. First, we found that the neurotoxicity of Aβ1-40 could be enhanced by hyperglycemia, that enhanced the AD symptoms through the oxidative stress caused by Aβ accumulation. Second, the interaction between Aβ1-40 infusion and aged not only caused transient body-weight loss but also impaired the retrieval of spatial reference memory in the C57BL/6J × C3H hybrid mice which have been used as the common AD transgenic model, the APP/PS1 double mutant mice. Therefore, transient body-weight loss may be an important sign of early dementia with aging. Finally, the combined treatment of the stress and oligomer Aβ1-40 induced severe impairment of spatial learning and memory through apoptosis and synaptic dysfunction in the hippocampus. The above results, we III.

(9) suggested that the interaction between Aβ1-40 and environmental factors induced the cognitive dysfunction through the oxidative stress. Therefore, the antioxidant therapy may be a potential strategy to delay the onset of these devastating pathologies. Keywords: Alzheimer’s disease, Aβ1-40, hyperglycemia, aging, stress, antioxidant therapy.. IV.

(10) CONTENTS ABSTRACT（in Chinese） ...................................................................... I ABSTRACT（in English） .................................................................... III CONTENTS.............................................................................................. V LIST OF TABLES.................................................................................... X LIST OF FIGURES ................................................................................. XI SUPPLEMENTAL DATA ................................................................... XIII LIST OF ABBREVIATIONS............................................................... XIV. CHAPTER 1 Introduction ...................................................................... 1 1.1 Characterization and classification of Alzheimer’s disease ................ 1 1.2 Modification of amyloid cascade hypothesis ...................................... 2 1.3 Nontransgenic as exogenous amyloid model versus the transgenic model of ADs........................................................................................ 3 1.4 Aβ1-40 versus Aβ1-42 .............................................................................. 5. CHAPTER 2 Intrahippocampal administration of Aβ1–40 impairs spatial learning and memory in hyperglycemic mice........................... 9 Abstract ...................................................................................................... 9 2.1 Introduction........................................................................................ 10 2.2 Material and methods......................................................................... 12. V.

(11) 2.2.1 Animals ........................................................................................... 12 2.2.2 Experimental timeline..................................................................... 12 2.2.3 Hyperglycemia procedure............................................................... 13 2.2.4 Preparation of oligomer and monomer Aβ1–40 ................................ 13 2.2.5 Animal surgery ............................................................................... 14 2.2.6 Morris water maze (MWM)............................................................ 14 2.2.7 Histology and immunohistochemistry............................................ 16 2.2.8 Statistical analysis........................................................................... 17 2.3 Results and discussion ....................................................................... 18 2.3.1 Hyperglycemia was induced by the STZ........................................ 18 2.3.2 Impaired spatial reference learning and memory of oligomer Aβ1–40-treated hyperglycemic mice ................................................. 18 2.3.3 Increasing Aβ accumulation, apoptotic signal, and oxidative stress in the CA1 regions of hyperglycemic mice injected with oligomer Aβ1–40 ............................................................................................... 21. CHAPTER 3 Continuous Aβ1-40 infusion affects the retrieval of spatial reference memory and body weight in C57BL/6J × C3H hybrid aging mice ................................................................................. 34 Abstract .................................................................................................... 34 3.1 Introduction........................................................................................ 34 3.1.1 The role of the Aβ1-40 in AD ........................................................... 34. VI.

(12) 3.1.2 Aging in C57BL/6J × C3H hybrid mice......................................... 35 3.2 Methods.............................................................................................. 36 3.2.1 Subjects ........................................................................................... 36 3.2.2 Procedures....................................................................................... 36 3.2.3 Morris water maze (MWM)............................................................ 37 3.2.4 Immunohistochemistry ................................................................... 37 3.2.5 Statistical analysis........................................................................... 38 3.3 Results................................................................................................ 38 3.3.1 Transient body weight loss accompanied with apoptosis of the LH region in aging treated Aβ1-40 mice................................................ 38 3.3.2 Impairment of spatial memory........................................................ 39 3.4 Discussion .......................................................................................... 40 3.5 Conclusion ......................................................................................... 43. CHAPTER 4 The interaction between acute oligomer Aβ1-40 and stress severely impaired the spatial learning and memory .............. 51 Abstract .................................................................................................... 51 4.1 Introduction........................................................................................ 52 4.1.1 The role of stress in AD.................................................................. 52 4.1.2 Hippocampus .................................................................................. 53 4.2 Materials and methods ....................................................................... 54 4.2.1 Subjects ........................................................................................... 54 VII.

(13) 4.2.2 Experiment timeline and establishment of the stress model .......... 55 4.2.3 Analysis of corticosterone level...................................................... 56 4.2.4 Preparation of oligomer .................................................................. 56 4.2.5 Animal surgery ............................................................................... 56 4.2.6 Locomotor....................................................................................... 57 4.2.7 Light-dark transition test................................................................. 57 4.2.8 Morris water maze (MWM)............................................................ 58 4.2.9 Immunohistochemistry ................................................................... 58 4.2.10 Western blot analysis .................................................................... 59 4.2.11 Data analysis ................................................................................. 60 4.3 Results................................................................................................ 60 4.3.1 Stress enhanced the plasma corticosterone level of treated animals .................................................................................................................. 60 4.3.2 The avoidance into the dark compartment was increased with aversive experience in stress mice................................................. 61 4.3.3 Motor activity was not affected in stress mice regardless of oligomer. Aβ1-40 or vehicle treatment .......................................... 61. 4.3.4 Alteration of cholinergic, noradrenergic, and serotonergic immunoreactive neurons in stress and oligomer Aβ1-40 treated animals ........................................................................................... 62 4.3.5 Increasing Aβ accumulation, oxidative stress, and apoptotic signal in the stress and oligomer Aβ1–40 treated mice .............................. 63 4.3.6 Reduction of calbindin immunoreactive neurons in stress and VIII.

(14) oligomer Aβ1-40 treated mice.......................................................... 64 4.3.7 Increases in the GR/MR ratio and CRF-1 expression, and decreases in NF-κB and NR 2A/2B expression were observed in the hippocampus of the combined stress and oligomer Aβ1-40-treated mice................................................................................................ 64 4.3.8 Spatial reference learning and memory was severely impaired in stress and oligomer Aβ1-40 treated mice......................................... 64 4.4 Discussion .......................................................................................... 66. CHAPTER 5 Conclusions and future prospect ................................ 81 5.1 The impacts of environmental factors on Aβ1-40................................ 81 5.2 The potential of antioxidant therapy in AD....................................... 81 5.3 The future prospect in AD ................................................................. 81. References ............................................................................................. 83 Autobiographical statement ................................................................ 96. IX.

(15) LIST OF TABLES Table 1 Body weight measurements, Aβ deposition, oxidative stress, and apoptosis of the different treatment group............................................... 44 Table 2 The quantification of the immunohistochemical analyses in the different treatment mice........................................................................... 71. X.

(16) LIST OF FIGURES Figure 1 Senile plaque and neurofibrillary tangle ..................................... 7 Figure 2 Amyloid cascade hypothesis ....................................................... 8 Figure 3 Hyperglycemia of C57BL/6J male mice induced by STZ treatment .................................................................................... 26 Figure 4 Effect of Aβ1–40 peptide on performance of water maze task ... 27 Figure 5 The Aβ1–40 injection sites confirmed by cresyl violet staining . 30 Figure 6 Immunohistochemistry of Aβ1–40 deposition in the CA1 region of hippocampus.......................................................................... 31 Figure 7 Results of caspase-3 signal showing apoptosis in the subregions of the mouse hippocampus ........................................................ 32 Figure 8 Results of MnSOD staining showing oxidative stress occurred in mouse brains .............................................................................. 33 Figure 9 Effects of Aβ1–40 on mouse performance during a water maze task ............................................................................................. 45 Figure 10 Reduction of cholinergic, noradrenergic, and calbindin immunoreactive neurons in treated mice................................. 49 Figure 11 The experimental timeline of this study.................................. 72 Figure 12 Plasma corticosterone levels in stress and nonstress mice...... 73 Figure 13 Anxiety evaluation of stress and nonstress mice after acute CA1 administration with oligomer Aβ1–40 and vehicle ........... 74 Figure 14 The interactive effects of oligomer Aβ1–40 and stress on the motor activity of mice.............................................................. 75 XI.

(17) Figure 15 Reduction of cholinergic, noradrenergic, and serotonergic immunoreacitve neurons in treated mice................................. 76 Figure 16 Immunohistochemistry of Aβ1–40 accumulation, oxidative stress and apoptosis in treated mice................................................... 77 Figure 17 Calbindin expression in the CA1 subregion of hippocampus in treated mice.............................................................................. 78 Figure 18 GR/MR ratio, CRF-1, NF-κB and NR2A/2B expressions in hippocampus of the treated mice ............................................. 79 Figure 19 Interactive effects of oligmer Aβ1–40 peptide and stress on mouse performance during a water maze task......................... 80. Figure 20 The diagram showing the interaction between Aβ1-40 and environmental factors and resulting cognitive dysfunction by oxidative stress ....................................................................... 82. XII.

(18) SUPPLEMENTAL DATA (A)Experimental timeline of examining the role of the Aβ1–40 in different age hybrid mice................................................................................... 50 (B)The structure of Aβ1–40 after incubation at 37℃ for 7 days and 14 days .................................................................................................................. 50. XIII.

(19) LIST OF ABBREVIATIONS APP AD ApoE ACSF BLA D28K ChAT CRF-1 DM DAB FAD GR GABA HPA LH LC MS/DB MR MWM NR2A/2B NF-κB PS1 PS2 ROS 5-HT SR SAD STZ TH Aβ. Amyloid precursor protein Alzheimer’s disease Apolipoprotein E Artificial cerebrospinal fluid Basolateral part of amygdala Calbindin Choline acetyltransferase Corticotrophin releasing factor 1 receptor Diabetes mellitus Diaminobenzidine Familial Alzheimer’s disease Glucocorticoid receptor Glutamate decarboxylase 65/67 Hypothalamic-pituitary-adrenal axis Lateral hypothalamus Locus coeruleus Medial septum/diagnoid band Mineralocorticoid receptor Morris water maze NMDA receptor 2A/2B Nuclear transcription factor Presenilin 1 Presenilin 2 Reactive oxygen species Serotonergic Situation reminder Sporadiac Alzheimer’s disease Streptozotocin Tyrosine hydroxylase β-amyloid. XIV.

(20) CHAPTER I Introduction 1.1 Characterization and Classification of Alzheimer’s disease Alzheimer’s disease (AD), first described by Alois Alzheimer in 1906, shows the neuropathological characterizations of intraneuronal tangles and extracellular “amyloid” plaques in the brain (Fig. 1A & B) and followed by progressive loss of memory and general cognitive decline. However, Alzheimer could not decipher whether the tangles or plaques were causative or merely markers of the disease.. AD is multifactor, with both genetic and environmental factors implicated in its pathogenesis and is classified into familial early-onset type (FAD) or sporadic late-onset type (SAD). The majority of cases (90-95%) are SAD, and the remainders are FAD. Generally, the dementia onset is 40-60 years for the FAD, and usually over 60 years for the SAD. To date, major mutations of the FAD are localized on four genes, presenilin 1 (PS1) gene on chromosome 14, the presenilin 2 gene (PS2) on chromosome 1, the amyloid precursor protein gene (APP) on chromosome 21, and apolipoprotein E (ApoE ε4 variant) on chromosome 19—all serve to transmit AD via autosomal-dominant inheritance. There are several other genes are considered susceptible or risk factors for AD, including α2-macroglobulin [1], the K-variant of butyryl-cholinesterase [2], and several mitochondrial genes [3]. Epidemiological studies have suggested several risk factors might be involved in the SAD, such as age, gender, previous head injury, and cardiovascular diseases [3]. Although 1.

(21) both SAD and FAD have the same brain lesions and distribution pattern, much work remains to be done to elucidate environmental factors that can influence both the onset and progression of AD.. 1.2 Modification of amyloid cascade hypothesis The hypothesis that mismetabolism of APP leads to the aggregation of Aβ is proposed by Selkoe [4]. The aggregation of Aβ induces the formation of plaque or neurofibrillary tangles, and then disruption of the synaptic connections, death of neurons in the hippocampal related regions, which finally results in dementia (Fig. 2 ) [5]. An early study already found that Aβ peptide is the main component of plaques [6]. Much evidence also suggests that the Aβ was the primary neuropathological insult in the AD [7,8]. However, recent studies found that soluble, oligomeric rather than insoluble form of Aβ might play a critical role in AD [9-11]. Therefore, the aggregation of the soluble Aβ seems to be determinants in inducing several molecular cascades, leading to dramatic cognitive alterations. In addition, non-transgenic models have confirmed an involvement of the diffusible forms of Aβ in a rapid process of synaptic dysfunction [12]. Convergent data have also shown that secondary processes such as inflammatory and oxidative cascades induced by the fibrillar Aβ might play a major role in the long-term effect of amyloid pathology on synaptic and cellular functions [13-15]. Taken together, these data suggest that the accumulation of Aβ in the brain is the primary influence driving AD pathogenesis. However, there are also some puzzling observations which hint that this hypothesis is not complete. For example, whereas the transgenic mouse models bearing the 2.

(22) FAD mutations do not show evidence of significant neuronal loss [16-18], little tau phosphorylation, and no tangle formation [19]. A relatively high concentration (two or three orders of magnitude) of Aβ was needed to exert toxicity, and some studies still failed to demonstrate Aβ toxicity in vivo [19-21]. Furthermore, it was reported that under certain culture conditions, Aβ promoted neurite outgrowth [22,23] instead of exerting toxic action. The most important is that Aβ deposition has been observed in various brain areas without accompanying neurodegeneration [24,25], whereas neurodegeneration can occur in areas with no Aβ deposition [26]. Thus, Aβ may not be the sole active fragment in AD, and some other factors could be involved in inducing neuronal loss.. 1.3 Nontransgenic versus transgenic models of ADs Normal rodents that receive exogenous administration of Aβ in different areas of their brain present several neuropathological characteristics and behavioral features of AD and provide a valuable experimental model for studying the specific pathogenesis induced by the amyloid component of the disease. In parallel, the creation of transgenic mice overexpressing specific genes which cause familial forms of the disease has proven to have great value for studying the pathogenesis of this disorder at the molecular, cellular and behavioral levels. Some of these transgenic mice display neuropathological and behavioral features of AD, including an increase in the soluble form of Aβ with age, amyloid deposits, neuritic plaques, gliosis, synaptic alterations and memory impairment [27,28]. The creation of double- or triple-cross transgenic mice [29], by crossing several factors that either cause or contribute to the 3.

(23) risk of developing AD, has increased the neuropathological similarities in the phenotype of these mice (e.g. tangles and amyloid burden) with the human disease. However, important differences between transgenic mouse models and AD have also been observed in neuronal loss. Previous evidence further suggests that Tg2576 mice do not exhibit widespread, profound cognitive impairment, even into old age [30]. This may reflect their predominant C57BL/6 background and an apparent inability of the mutant transgene to profoundly alter performance therein. Importantly, previous study further suggests that cerebral amyloid angiopathy in aging APP23 mice had striking similarities to that observed in human aging and Alzheimer's disease [31]. This suggests that contributory factors that lower the threshold of neuronal death may be present in AD or that compensatory factors may also be triggered by the introduction of a human mutant, APP or PS1, gene to these mice. Although the model of exogenous administration of Aβ to normal brain tissue does not reproduce the full spectrum of the human pathology, many studies have reported that neurodegeneration and extensive microglial activation close to Aβ deposits in the parenchyma [32,33]. The rationale for focusing on the specific effect of amyloid is to isolate the neuropathological events during the successive structural and biochemical changes in AD, to identify the events that lead to neuronal dysfunction. Important advantages of the current approach include the ability to administer defined amounts of Aβ of known sequence or length or to introduce controlled cofactors. Another advantage lies in the ability to accelerate the experimental timeline by optimizing the parameters in short period experimentory time, rather than waiting for the rodents to 4.

(24) age. We certainly aware that the intracerebral Aβ injection has its own limitations as compared a model for natural amyloid-induced pathology. The injection/infusion approach involves inevitable injury associated with the invasive procedure required to dispense Aβ into the brain. Moreover, the vehicle in which Aβ is dissolved can itself affect Aβ neurotoxicity in the brain. This could be a major contributor to the inflammatory processes induced by this procedure. However, these limitations can be overcome to a certain degree by adjusting the infusion rate, the vehicle, the volume of injection and the resting time before the examination of the animal to minimize the confounding effect of the procedure involved in administering Aβ.. 1.4. Aβ1-40 versus Aβ1-42 Aβ is a metabolic product of the transmembrane protein amyloid precursor protein (APP) via proteolytic cleavage by β- and γ-secretases [34]. The two key peptides Aβ1–40 and Aβ1–42 are the central players in the neuropathology of AD. These peptides are normally cleaved from the precursor APP molecule in a roughly 9:1 ratio [19]. Aβ1–40 is the major species secreted from cultured cells and found in the cerebrospinal fluid, whereas Aβ1–42 is the major component of amyloid deposits in the brain with AD [35]. Although Aβ1-40 is likely a physiologically relevant molecule and tends to be less amyloidogenic, Aβ1-42 is highly amyloidogenic and seems to be a major component of the amyloid plaques. Recent study further suggests that Aβ1-40 and Aβ1–42 have similar neurotoxic properties even though their amyloidogenic properties are significantly different [36]. Therefore, in this study, we tried to explore 5.

(25) some fundamental mechanistic ideas on Aβ1-40 pathophysiology in the differential environmental factors paradigm as hyperglycemia, aged and chronic stress condition.. 6.

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(28) CHAPTER 2 Intrahippocampal administration of Aβ1–40 impairs spatial learning and memory in hyperglycemic mice. Abstract Age-related neurodegenerative dementia, particularly Alzheimer’s disease (AD), may be exacerbated by several interacting risk factors including genetic predisposition, beta amyloid (Aβ) protein accumulation, environmental toxins, head trauma, and abnormal glycolytic metabolism. We examined the spatial learning and memory effects of Aβ1–40 administration on hyperglycemic mice by their performance in the Morris water maze. Chronic hyperglycemia was induced in male C57BL/6J mice to mimic diabetes mellitus by intraperitoneal injection of streptozotocin (STZ), which specifically destroys pancreatic β-islet cells. Ten days after STZ treatment, intrahippocampal infusion of vehicle, monomer, or oligomer Aβ1–40 was given to these hyperglycemic mice. Our results demonstrate that in comparison with vehicle or monomer Aβ1–40, oligomer Aβ1–40 induced significant deficits in spatial learning and memory in hyperglycemic mice. Apoptotic signals were identified in the CA1 and dentate gyrus of the hippocampus in hyperglycemic mice. Aβ accumulation, oxidative stress, and apoptosis in the CA1 region were more intensive in hyperglycemic mice than that in normoglycemic mice after acute treatment with oligomer Aβ1–40 peptide treatment. These results indicate that CA1 apoptosis was enhanced by oxidative stress resulting from accumulation of Aβ. Taken together, these findings suggest that hyperglycemic mice are more vulnerable to the 9.

(29) Aβ-induced-oxidative stress than normal subjects. We therefore propose that Aβ accumulation would be enhanced by hyperglycemia, and the oxidative stress caused by Aβ accumulation would in turn enhance the AD symptoms.. 2.1 Introduction Alzheimer’s disease (AD) is a slowly progressive neurodegenerative disease accompanied with dementia. The beta amyloid (Aβ), a peptide of 39–43 amino acids resulting from proteolytic cleavage of amyloid precursor protein (APP), plays an important role in the AD brain. Deposition of Aβ is an early and critical event in the pathogenesis of AD [37], first forming in temporal cortical regions including the hippocampus [38-40], a region implicated in memory formation [41-43]. It was proposed that Aβ aggregates to form neurotoxic plaques, which leads to neurodegeneration accompanied by dementia. Studies in APP transgenic mice have supported the hypothesis that memory loss is related to Aβ [17,44-47]. However, there is no consensus about which form of Aβ is responsible for AD. Various forms of Aβ, characterized by their aggregation states, are found in the brains of APP transgenic mice of different ages [48]. Recent data show that the more harmful physical forms of Aβ are small, still soluble and diffusible aggregates of low molecular weight [11,49-52]. Therefore, the mechanism underlying Aβ neuronal toxicity is still poorly understood despite several recent proposals [53-57]. Mild to moderate impairments of cognitive functioning have been reported in patients with diabetes mellitus (DM) [58]. Previous evidence suggests that the hyperglycemia is the major 10.

(30) “toxic” effect in the development of diabetic end-organ damage to the brain in Type I and Type II diabetes [59]. Diabetes can be induced experimentally in mice by streptozotocin (STZ) administration, which develops a delayed and/or progressive hyperglycemia, insulitis, and severe destruction of β cells [60-62]. However, the mechanism of any correlation between hyperglycemia and Aβ as a cause of Alzheimer’s disease in DM patient is still unknown. The risk for developing a neurodegenerative disorder increases with age and may be associated with an excessive generation of reactive oxygen species (ROS) and oxidative stress [63]. As a main pathway of neuronal death, apoptosis is an active form of cell degeneration and is executed by caspase proteins. There is a growing consensus that ROS is a potent inducer of apoptosis, and apoptosis contributes to the loss of neurons during normal aging [64]. To evaluate the interactions between abnormal glucose metabolism and Aβ in vivo, we have created an animal model to evaluate the vulnerability of chronically hyperglycemic subjects to the damage of intrahippocampal Aβ1–40 administration. From the observation of neuropathology, we found that Aβ accumulation, oxidative stress, and apoptosis in the CA1 region were more intense in hyperglycemic mice than in normoglycemic mice after acute oligomer Aβ1–40 treatment. The STZ-induced hyperglycemia group showed induction of apoptosis in the dentate gyrus of the hippocampus. We also found the impairment of spatial reference learning and memory was only present in hyperglycemic mice treated with oligomer Aβ1–40. These results suggest that cell loss in the dentate gyrus and CA1 regions of the hippocampus by the hyperglycemia with oligomer Aβ1–40-treated mice induced an impairment of spatial reference 11.

(31) learning and memory. Furthermore, we also suggest that Aβ accumulation, oxidative stress, and apoptosis in the CA1 region of the hippocampus is enhanced by hyperglycemia which then leads to increased impairment of spatial reference learning and memory.. 2.2 Materials and methods 2.2.1 Animals Male C57BL/6J mice (6–8 weeks) were purchased from the National Breeding Center for Laboratory Animals (Nankang, Taiwan) and group housed in a vivarium maintained at 20–25 °C with 60% relative humidity. Food and water were provided ad libitum. A light/dark cycle of 12/12 h was installed with lights on at 7:00 AM. The procedure adhered to Guidelines for Care and Use of Experimental Animals and was approved by the Institutional Animal Care and Use Committee of National Taiwan University, Taipei, Taiwan. Subjects were divided into six groups: (1) normoglycemic mice treated with double-distilled water, (2) normoglycemic mice treated with monomer Aβ1–40, (3) normoglycemic mice treated with oligomer Aβ1–40, (4) hyperglycemic mice treated with double-distilled water, (5) hyperglycemic mice treated with monomer Aβ1–40, and (6) hyperglycemic mice treated with oligomer Aβ1–40. Each group had 9–12 mice.. 2.2.2 Experiment timeline After one week of adaptation to the home cage, mouse body weight and blood glucose were measured on days 1, 10, 15, and 23. Streptozotocin (STZ) or vehicle control was injected into mice on day 1. 12.

(32) Oligomer Aβ1–40, monomer Aβ1–40, and vehicle were injected into the hippocampal CA1 region of mice on day 11. The water maze pretraining, training, testing, and probe trials were performed on days 16–22. Mice were killed for immunohistochemical analyses on day 23.. 2.2.3 Hyperglycemia procedure After animals were weighed, blood samples were obtained by tail prick and glucose levels were measured by a commercial glucometer (Accu-Check Active; Roche) before any treatment [61]. STZ (Sigma; 200mg/kg in 0.1ml of sodium citrate buffer, pH 4.5) was intraperitoneally injected into non-fasting mice within 15min to induce chronic hyperglycemia. The normoglycemic groups were injected with an equivalent volume of the citrate buffer. Ten days later, blood glucose levels and body weight were measured. The STZ-treated subjects who failed to develop hyperglycemia (defined as blood glucose concentrations > 200 mg/dl) were not used. Untreated normoglycemic and STZ-treated hyperglycemic subjects were infused with oligomer Aβ1–40, monomer Aβ1–40, or vehicle double-distilled water) in the CA1 region of the hippocampus.. 2.2.4 Preparation of oligomer and monomer Aβ1–40 The Aβ1–40 was dissolved in 1 ml of double-distilled water to a concentration of 0.23 mM and incubated at 37 °C for 7 days to allow oligomer formation as previously described [65]. Monomer Aβ1–40 was freshly prepared in 1ml of double-distilled water to a concentration of 0.23 mM. 13.

(33) 2.2.5 Animal surgery Mice were weighed and blood glucose measured before surgery. They were anesthetized with pentobarbital (50 mg/kg; MTC Pharmaceuticals, Cambridge) and placed in a stereotaxic instrument (DKI-900, David Kopf Instruments, CA). An incision was made in scalp and hole was drilled in the skull over the injection site. The 30-gauge-needle was lowered into the dorsal hippocampus. Coordinates for the anterior–posterior (from bregma), medial–lateral (from midline), and dorsal–ventral (from surface of the skull) axes were -2.3, ±2.5, and -1.5 mm, respectively. Bilateral intrahippocampal infusion was administrated via a 10.0 μl Hamilton microsyringe with a 30-gauge needle fitted to the arm of the stereotaxic instrument. Double-distilled water as vehicle for peptides was used in the study as a control infusion. A 0.6 μl volume of oligomer Aβ1–40, freshly made Aβ1–40 peptide solution, or double-distilled water alone (as a vehicle control) was slowly infused at a rate of 0.2 μl/min. After an additional 5 min, to assure adequate diffusion, the needle was slowly retracted. Four days post-surgery, all mice were weighed and blood glucose measured. On the fifth day, mice were trained and tested on the Morris water maze.. 2.2.6 Morris water maze (MWM) Spatial memory was evaluated with a conventional MWM, commonly adopted for studying cognitive deficits in APP transgenic mice [66]. The water maze apparatus consisted of a circular pool (1.0 m in 14.

(34) diameter and 0.47 m high) made of white plastic. The pool was filled to a depth of 40 cm with water (24–25 °C) made opaque by the addition of non-toxic white paint. During conventional MWM training, an escape platform (10 cm in diameter) made of white plastic, with a grooved surface for better grip, was submerged 1.0 cm underneath the water surface. Cues of various types provided distant landmarks in the testing area of the room. The swim path of a mouse during each trial was recorded by a video camera suspended 2.5 m above the center of the pool and connected to a video tracking system. One day before the spatial training commenced, all mice underwent pretraining (day 16) to familiarize them with the requirements of the test. Each mouse was first placed on a visible platform located in the center of the pool and allowed to remain there for 20 s. In the following three 60-s trials, mice were released into the water facing the wall of the pool from semirandomly chosen cardinal compass points. If a mouse failed to swim to the platform or stay on it for 20 sec, it was placed on the platform by an experimenter. The mice were given a 4-day training (days 17–20) with four 60-s training trials (inter-trial interval: 20–30 min) per day. The hidden platform was always placed in the same location of the pool (Northeast quadrant) throughout the training period. During each trial, from quasi-randomly chosen cardinal compass points, the mouse was released into the water facing the pool wall. After climbing onto the platform, the mouse was allowed to rest on it for 20 s. On day 21, 24 h after the last training trial, all mice were given three testing trials to measure the time to climb onto the hidden platform. On day 22, all mice were given one probe trial to evaluate their spatial memory for the platform. 15.

(35) 2.2.7 Histology and immunohistochemistry Immediately after the behavioral test, mice were overdosed with pentobarbital and transcardially perfused with 4% paraformaldehyde in phosphate-buffered saline. Brains were removed and post-fixed with 4%paraformaldehyde overnight and then placed in 30% sucrose in phosphate-buffered saline for 2 days. Brains were serially sectioned at 30 μm on a cryostat. Histology and immunohistochemistry were performed to assess the location of the infusion needle tip and to identify any anatomical and neurochemical abnormalities induced by the STZ and oligomer Aβ1–40 treatments. A rabbit anti- β-amyloid1–40 polyclonal antibody (Chemicon, CA, 1:200 dilution) staining was used to detect the presence of Aβ, using standard procedures. Specific antibodies were used to assess oxidative stress (MnSOD, Upstate, CA, 1:200 dilution) and caspase 3 expression (Chemicon, 1:40 dilution).. For immunohistochemistry, free-floating sections were immunostained. In brief, sections were rinsed in 0.1M phosphate-buffered saline (PBS) three times (10 min/wash). Endogenous peroxidase activity was blocked by incubation with 3% H2O2 for 30min, and sections were then washed in PBS three times (10 min/wash). Non-specific epitopes were then blocked by incubation in 5% normal goat serum and 0.1% Triton X-100 in PBS for 2 h. Sections were incubated in primary antibodies overnight at room temperature and then washed three times in phosphate-buffered saline for 10 min/wash. Secondary antibodies were applied to the sections by a linking reagent 16.

(36) (DAKO, CA) for 1 h. Immunostaining was highlighted using substrate-chromogen solution and DAB oxidation. All sections were mounted on coated slides and coverslipped for light microscopy. The extent of Aβ1–40 accumulation, oxidative stress, and apoptosis in the CA1 region of the hippocampus were quantities by two experimenters who were unaware of the experimental condition for these slices. Images were digitized with a CCD camera. A stage micrometer was used to calibrate the pixel-to-μm conversion and a double-blind paradigm was used to calculate all areas. The percentage of staining area of the Aβ1–40 accumulation, oxidative stress, and apoptosis in the CA1 region of the hippocampus was analyzed by research-based digital image analysis software (Image Pro Plus, Media Cybernetics, Silver Spring, MD, USA).. 2.2.8 Statistical analysis To determine the effects of STZ, body weight and blood glucose were analyzed by one-way ANOVA test followed by post hoc LSD multiple range tests for comparison among different time point in each treatment. The same analysis was used to evaluate statistical differences for the timing of the escape latencies among groups of different treatments. Furthermore, acquisition of spatial navigation response and searching the target quadrant in water maze pool were analyzed by mixed two-way ANOVA tests followed by post hoc LSD multiple range tests for comparison among treatments in different training days. For the immunostainings, we used two-tailed Student’s t-tests to assess the staining area of different groups. The Statistical results are expressed as 17.

(37) means ± SEM.. 2.3 Results and discussion 2.3.1 Hyperglycemia was induced by the STZ treatment The results of body weight and blood glucose measurements are shown in Fig. 3. As expected, the body weights were decreased in the 3 STZ-treated groups (p < .05) but not in the sodium citrate-treated control groups (Fig. 3A). Blood glucose levels were significantly increased at day 10, 15, and 23 compared to the level of day 1 when mice were not yet treated with STZ (p < .05; Fig. 3B). These results were consistent with previous studies [60,61]. Therefore, we confirmed that diabetes mellitus was successfully induced in these mice by a single high dose injection of STZ. Mouse blood glucose levels and body weights were monitored throughout the study.. 2.3.2 Impaired spatial reference learning and memory of oligomer Aβ1–40-treated hyperglycemic mice Hyperglycemic and normoglycemic mice that were naïve to the water maze showed no deficits in swimming abilities or climbing onto a visible platform during pretraining (data not shown). In subsequent tests on whether treatment of Aβ1–40 to hyperglycemic mice resulted in a functional deficit in spatial learning and memory, Aβ1–40 (freshly prepared monomer or 7 day-incubated oligomer) or vehicle was infused into the bilateral intrahippocampal CA1 of mice with hyperglycemia or normoglycemia. In addition, the preparation of monomer and the 18.

(38) oligomer Aβ1–40 was confirmed with the Western blot in this study (data not shown). We found that only hyperglycemic mice given oligomer Aβ1–40 had significantly impaired performance in spatial reference learning and memory compared to the other groups (F (5, 65)＝2.399, p ＝0.047; Fig. 4A). We also evaluated the effects of oligomer Aβ1–40 on acquisition of the spatial navigation response. The statistical analysis showed significant difference between groups of hyperglycemia and normoglycemia (F (1,19)＝6.2192, p＝0.022; Fig. 4B) and training days (F (3,19)＝4.9450, p＝0.0042), and insignificant interaction effect in group × training days (F (3, 19)＝0.2922, p＝0.8308). Therefore, the data suggested that hyperglycemic mice with oligomer Aβ1–40 injection were slower in learning ability than normoglycemic mice injected with oligomer Aβ1–40. We further identified that the speed and extent of learning were significantly different between hyperglycemic mice treated with different Aβ1–40. Statistical analysis by mixed two-way ANOVA reveals significant effects among different Aβ1–40 treatments (F (2, 6)＝ 5.0137, p < 0.05) and training days F (3,97)＝3.5490, p < 0.05), and insignificant interaction effect of group × training days (F (6, 97)＝ 0.8319, p > 0.05). In fact, the escape latency of the oligomer Aβ1–40-treated hyperglycemic mice did not reach the same asymptotic level as the monomer- or vehicle-treated mice (Fig. 4C). The average swim velocity during the 4 training days was similar among all hyperglycemic mice (Fig. 4D; p > 0.05), indicating that infusion of Aβ 19.

(39) peptides did not grossly affect their sensory or locomotor activities. To confirm the lack of spatial memory in the hyperglycemia group with oligomer Aβ1–40, we conducted probe test and measured the time spent in different quadrants. An animal that has learned the location of the platform should spend more time in the target quadrant than any other quadrants. Compared to the other groups, mice in the hyperglycemic group treated with oligomer Aβ1–40-treated mice spent less time in the target quadrant but more time in the opposite quadrant (F (3, 36)＝4.162, p＝0.012; Fig. 4E). The results of behavioral analysis showed that acute intrahippocampal CA1 administration of oligomer Aβ1–40 induced pronounced impairment of spatial memory in the hyperglycemic mice compared to monomer Aβ1–40 or vehicle treatment. These results were consistent with previous studies that glucose metabolism is decreased in AD patients [67,68]. Several studies also suggest that metabolic dysfunction may increase susceptibility to neurodegeneration or contribute to amyloid neurotoxicity in AD pathogenesis [62,69,70]. In normoglycemic mice, however, injection of oligomer Aβ1–40 resulted in a non-statistically significant loss of spatial memory compared to the mice injected with vehicle or monomer Aβ1–40. The result shows that the acute oligomer Aβ1–40 treatment only partially affected spatial memory but not spatial learning in the normoglycemic mice. The soluble Aβ oligomers, but not fibrils or monomers, have recently been considered responsible for cognitive dysfunction prior to the formation of senile plaques in transgenic mouse overexpressing human APP [17,50,71]. However, the dose and the structure of the Aβ 20.

(40) peptides still are unclear in these transgenic studies. A recent study suggests that specific assemblies, particular trimers, are selective inhibitors of certain forms of hippocampal long-term potentiation [72]. In addition, Ishibashi et al. (2006) also suggested that accumulated oligomer Aβ, but not fibrillar Aβ, is closely associated with synaptic failure, which is the major cause of cognitive dysfunction [73]. Townsend et al. (2006) also suggests that long-term potentiation in juvenile mice is resistant to the effects of Aβ oligomers [72]. Therefore, we suggest that acute single oligomer Aβ treatment might not result in significant damage to spatial learning and memory in younger normoglycemic mice. Furthermore, hyperglycemia could enhance the impairment of the acute oligomer Aβ1–40 in spatial learning and memory. In addition, we also found that STZ alone did not affect spatial learning and memory. Previous study has suggested that hyperglycemia is unlikely to be the only factor in the development of cognitive impairment in Type II DM [74]. Furthermore, another piece of experimental evidence indicates that the behavioral and neurophysiological consequences of DM are accentuated by aging [75]. Previous studies also suggest that mice with diabetes induced for one week of STZ treatment had no nerve alterations, as total fiber number and myelinated fiber size were compatible between hyperglycemic and control mice [61,76]. Rats treated with STZ for 8 weeks showed increased cognitive deficits [77]. Therefore, our results suggest that acute oligomer Aβ1–40 enhances the impairment of spatial learning and memory in the hyperglycemic mice, but not in the normoglycemic mice.. 2.3.3 Increasing Aβ accumulation, apoptotic signal, and oxidative stress 21.

(41) in the CA1 regions of hyperglycemic mice injected with oligomer Aβ1–40 At first, we confirmed that the infusion site in each mouse was exactly in the CA1 region of the hippocampus by cresyl violet staining of mouse brains (Fig. 5). Immunohistochemical analyses showed that Aβ depositions were identified in both of the hyperglycemic and normoglycemic mice at the CA1 region after infusion with oligomer Aβ1–40 (Figs. 6A-1 and A-2). However, the extent of the Aβ1–40 accumulation was more evident in the hyperglycemic mice than in the normoglycemic mice (p < 0.001; Fig. 6B). No positive staining of Aβ accumulation was observed in vehicle-treated mice (Figs. 6A-3 and A-4). Therefore, we suggested that acute oligomer Aβ1–40 might result in increased accumulation of Aβ1–40 around CA1 region in hyperglycemic mice compared to normoglycemic mice. Because a common feature of AD pathology is neuron loss, we used caspase-3 immunostaining to detect the presence of apoptosis in mice with impaired spatial reference memory. Although caspase-3 immunopositive reactions were identified in the CA1 regions of both hyperglycemic and normoglycemic mice treated with oligomer Aβ1–40, the apoptotic CA1 region in hyperglycemic mice was greater than that in normoglycemic mice (p < 0.001; Figs. 7A-1, A-2, and B). Apoptotic signals in the dentate gyrus subregion of hippocampus were also observed in the hyperglycemic mice treated with oligomer Aβ1–40 or vehicle (Figs. 7A-1 and A-3). No signal, however, was observed in the normalglycemic mice infused with vehicle even though there was some cell loss at the infusion site (Fig. 7A-4). Furthermore, in examining the correlation between apoptosis and oxidative stress, we found MnSOD 22.

(42) immunopositive reaction in the CA1 region of hyperglycemic and normoglycemic mice injected with oligomer Aβ1–40 (Figs. 8A and B). The oxidative stress signal in the CA1 region is correlated to the result of the Aβ1–40 accumulation and caspase-3 staining. No signal was identified in the dentate gyrus subregion of the hyperglycemic mice (Figs. 8A-1 and A-3), although apoptotic signal was previously observed in this region. There was no staining observed in CA1 region of hyperglycemic or normoglycemic mice injected with vehicle (Figs. 8A-3 and A-4). Our findings from the serial immunostaining for Aβ1–40, caspase-3, and MnSOD suggest that the increasing Aβ1–40 accumulation induced more apoptotic CA1 neuron in hyperglycemic mice than that in the normoglycemic mice. Previous evidence also indicates that around 70% of neurons in the CA1 region of the hippocampus die during the progression of AD [78]. Therefore, we propose that the interaction between the hyperglycemia and oligomer Aβ1–40 enhanced the damage of the CA1 subregion over the “safe threshold”. In addition, we found the oxidative stress of the CA1 region was also more evident in hyperglycemic mice than in normoglycemic mice. Therefore, these data suggested that acute intrahippocampal CA1 administration of oligomer Aβ1–40 induced more severe damage in hyperglycemic mice than in normoglycemic mice through increasing Aβ accumulation, oxidative stress, and apoptotic neuron. One previous study also suggests a positive feedback loop between the oxidative damage and glucose metabolic dysfunction [79]. A previous dose response experiment doses ranging from 0.01 to 5 μM confirmed the selective vulnerability of CA1 to soluble oligomeric Aβ [80]. Evidence showed that brain tissues from AD 23.

(43) patients have more nerve cells with activated caspase-3 than do those from people who died of other causes [81]. Su et al. (2001) reported that neurons with caspase-3 are found in brains of AD mice or cultured nerve cells [82]. Aβ, in ways that are inhibited by free radical antioxidants like vitamin E, causes brain cell protein oxidation, lipid peroxidation, reactive oxygen species formation, and other oxidative stress responses, suggesting that this peptide is a source of oxidative stress in brain [83]. Furthermore, we also found cell apoptosis in the dentate gyrus of hyperglycemic mice injected with oligomer Aβ1–40 or vehicle. These results are consistent with previous pathological studies in humans and animals showing hyperglycemia preferentially induces neuronal death in the CA1, subiculum, and dentate gyrus of the hippocampus, as well as in the superficial layers of the cortex, or in the striatum [84]. We did not, however, observe any MnSOD immunoreactivity in the dentate gyrus or CA1 region of these mice. A study of 3- and 12-month old rats showing nerve conduction deficits after STZ treatment revealed no changes in antioxidant enzymes except for increased catalase in 12-month-old rats [85]. Therefore, we suspect that the apoptosis in the dentate gyrus was induced by STZ treatment through another signaling pathway instead of from oxidative stress in CA1 region arising from accumulation of Aβ. The anatomical and behavioral data from our study suggest that hyperglycemia itself induced the apoptosis in the dentate gyrus of the hippocampus, but had no effect on spatial learning and memory. However, hyperglycemia group with oligomer Aβ1–40 treated mice induced more accumulation of Aβ1–40, oxidative stress, and apoptosis in the hippocampal CA1 neurons. Many studies have suggested that lesions in 24.

(44) the CA1 and dentate gyrus of the hippocampus induce the impairment of spatial learning and memory. The hippocampus has been well known to play a critical role in certain types of learning, including spatial learning [86-88]. Specific cells within the hippocampus become selectively activated when an animal is replaced in particular locations within its environment [89]. Within the hippocampus, cells of the dentate gyrus serve to restrict or amplify signals that originate in extra-hippocampal sites and propagate into the hippocampus proper [90]. The CA1 region of hippocampus plays significant roles in associational memories [91,92]. Therefore, hyperglycemia in oligomer Aβ1–40-treated mice induced the impairment of spatial learning and memory. In summary, our results not only provide the animal model to evaluate in detail the behavioral and neuroanatomical effects of the interaction between abnormal glucose metabolism and Aβ1–40 in vivo but also provide experimental support for the epidemiological literature indicating that the amyloid accumulation and metabolic dysfunction may interact to exacerbate the pathogenesis of AD.. 25.

(45) Figure 3. Hyperglycemia of C57BL/6J male mice induced by STZ treatment. (A) Body weights examined before (day 1) and after STZ injection (days 10, 15, and 23) and (B) Blood glucose levels examined before (day 1) and after STZ injection (days 10, 15, and 23). Reduced body weight and significantly increased blood glucose revealed the effectiveness of hyperglycemic induction by STZ. Error bars indicate standard error of the means. *p < 0.05. 26.

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(48) Figure 4. Effect of Aβ1-40 peptide on performance of water maze task. The 4 training days were scheduled on days 17–20. The 3 testing trials were carried out on day 21. (A) Hyperglycemic mice injected with oligomer Aβ1-40 showed significant impairment in the spatial reference memory compared to mice treated with other treatments. (B) The escape latency of oligomer-treated mice in the water maze during the 4 training days. The oligomer Aβ1-40 injected hyperglycemic mice showed a slower learning ability than the normoglycemic mice (p < 0.05). (C) The escape latency of hyperglycemic mice in the water maze during the 4 training days. The oligomer Aβ1-40 injected hyperglycemic mice showed a slower learning ability than the monomer or vehicle-treated hyperglycemic mice. (D) Swim velocity of hyperglycemic mice in the water maze during the 4 training days. No significant difference was identified among the 3 groups of mice with different Aβ treatments. (E) The performance of mice during probe trial conducted on day 23. The hyperglycemic mice with oligomer Aβ1-40 injection spent less time in the target quadrant than in the other 3 quadrants. These results show that hyperglycemia accelerated the impairment of the spatial reference learning and memory with oligomer Aβ1-40 treatment. Double-distilled water was used as vehicle treatment in the study. Error bars indicate standard error of the means. *p < 0.05.. 29.

(49) Figure 5. The Aβ1-40 injection sites confirmed by cresyl violet staining. (A) Oligomer Aβ1-40 injection into hyperglycemic mice. (B) Oligomer Aβ1-40 injection into normoglycemic mice. (C) Vehicle injection into hyperglycemic mice. (D) Vehicle injection into normoglycemic mice. Cresyl violet staining reveals that injection sites were in the CA1 region of the hippocampus. Arrows indicate the injection site. Scale bar＝100 μm.. 30.

(50) Figure 6. Immunohistochemistry of Aβ1-40 deposition in the CA1 region of hippocampus. (A) The Aβ1-40 immunoreactive granules by DAB staining in the CA1 region of the hippocampus of the mice. (1) Oligomer Aβ1-40 injection into hyperglycemic mice, (2) oligomer Aβ1-40 injection into normoglycemic mice, (3) vehicle injection into hyperglycemic mice, (4) vehicle injection into normoglycemic mice. Scale bar ＝ 100 μm. (B) The percentage of staining area of the Aβ1-40 deposition in the CA1 region. Compared to the normoglycemic mice treated with oligomer Aβ1-40 (n = 3), there was a significantly increased area of Aβ1-40 deposition in the CA1 of hyperglycemic mice treated with oligomer Aβ1-40. *p < 0.001. 31.

(51) Figure 7. Results of caspase-3 signal showing apoptosis in the subregions of the mouse hippocampus. (A) Caspase-3 immunoreactivity in the hippocampus of the mice. (1) Oligomer Aβ1-40 injection into hyperglycemic mice, (2) oligomer Aβ1-40 injection into normoglycemic mice, (3) vehicle injection into hyperglycemic mice, (4) vehicle injection into normoglycemic mice. Caspase-3 positive signals are observed in the CA1 and the dentate gyrus (DG) subregions of the hippocampus in the hyperglycemic mice. Normoglycemia show the caspase-3 positive signals only in the CA1 subregion of the hippocampus with oligomer Aβ1-40-treated mice. Scale bar ＝ 100 μm. (B) The percentage of relative area in the CA1 of the mice. Compared to the normoglycemia group treated with oligomer Aβ1-40 (n = 3), hyperglycemia group treated with oligomer Aβ1-40 (n = 3) showed a significant cell loss in CA1 region. *p < 0.001. 32.

(52) Figure 8. Results of MnSOD staining showing oxidative stress occurred in mouse brains. (A) MnSOD immuno reactivity in the CA1 region of the hippocampus. (1) oligomer Aβ1-40 injection into hyperglycemic mice, (2) oligomer Aβ1-40 injection into normoglycemic mice, (3) vehicle injection into hyperglycemic mice, (4) vehicle injection into normoglycemic mice. Positive oxidative stress signal is observed in the CA1 subregion of the hippocampus only in oligomer Aβ1-40 treated hyperglycemic or normoglycemic mice. Scale bar＝ 100 μm. (B) The percentage of relative area of the normoglycemia and hyperglycemia-treated oligomer Aβ1-40 mice in the CA1 region. Compared to the normoglycemia group-treated oligomer Aβ1-40 (n = 3), hyperglycemia-group treated with oligomer Aβ1-40 (n = 3) show a significant oxidative stress in CA1 region. *p < 0.001. 33.

(53) CHAPTER 3 Continuous Aβ1-40 infusion affects the retrieval of spatial reference memory and body weight in C57BL/6J × C3H hybrid aging mice. Abstract This study elucidated the effects of age, Aβ1-40, and the interaction between these two factors in widely-used Alzheimer’s disease (AD) C57BL/6J × C3H background mice. Morris water maze performance, body weight and immunohistochemistry were analyzed during continuous intracerebroventricular Aβ1-40 or vehicle infusion in young (8-week-old) and age (24-month-old) mice. We found that the interaction between Aβ1-40 infusion and age not only caused transient body-weight loss but also impaired the retrieval of spatial reference memory. Therefore, we suggested that transient body-weight loss may be an important sign of early dementia with age. Furthermore, the interaction between Aβ1-40 infusion and age causes a reduction in cholinergic, noradrenergic and calcium signaling in the hippocampus, which may further result in deficiency in the retrieval of spatial reference memory.. 3.1 Introduction 3.1.1 The role of the Aβ1-40 in AD Alzheimer’s disease (AD) is the most common cause of age-related dementia. Studies suggest that aging reduces central cholinergic and noradrenergic neuron function and calcium-binding protein in hippocampus, which could result in a decline in memory acquisition and 34.

(54) retention [93,94]. A recent study found that brain beta-amyloid (Aβ) deposition is an extremely common sign of aged in many species [95]. Extensive data supporting a central pathogenic role of Aβ in AD remain controversial, in part because a specific neurotoxic species of Aβ has not been defined in vivo. There are two common isoforms of Aβ, Aβ1-40 and Aβ1-42. Early evidence has shown that Aβ1-42 plays an important role in plaques and behavioral deficits in patients with AD [96]. However, under general conditions, a greater proportion of Aβ is produced in the form of Aβ1-40 than Aβ1-42, and in addition, an increase in the Aβ1-40/Aβ1-42 ratio in cerebrospinal fluid has been identified during the early stages of AD [97]. Evidence also suggests that Aβ1-40 may play a key role in the pathogenesis of the late-onset sporadic AD [98]. In our previous study, we found that the neurotoxicity of Aβ1-40 can be enhanced by hyperglycemia [99]. Recent findings further indicate that Aβ1-40 level is associated with cognitive function in healthy older adults in a pattern similar to that of early AD, independent of deleterious effects on cerebrovascular dynamics [100].. 3.1.2 Aging in C57BL/6J × C3H hybrid mice Previous study suggests that the neurobiology of aged mice can vary across strains, as different strains have different life spans and susceptibility to diseases [101]. F1 mice from the C57BL/6J × C3H hybrid are commonly used in a common AD transgenic model, the APP/PS1 double mutant mice model [102]. APP/PS1 transgenic mice show age-dependent increases of Aβ1-40 and Aβ1-42 and amyloid plaques in the brain [103]. However, the aging process in mice of this hybrid genetic 35.

(55) background is still unclear. The objective of this study was therefore to elucidate the effects of aged, Aβ1-40, and the combination of these two factors on C57BL/6J × C3H hybrid mice.. 3.2 Methods 3.2.1 Subjects F1 hybrid male mice were obtained from C3H male crossed with C57BL/6J female mice. The procedures involving the animals adhered to the Institutional Guidelines for the Care and Use of Experimental Animals and were approved by the Institutional Animal Care and Use Committee of National Taiwan Normal University. The subjects were divided into four groups: (i) young mice (8-week-old) treated with artificial cerebrospinal fluid (ACSF); (ii) young mice (8-week-old) treated with Aβ1-40; (iii) aged mice (24-month-old) treated with ACSF; and (iv) aged mice (24-month-old) treated with Aβ1-40. Each group contained 13–15 mice. The experimental timeline is presented in Supplementary Fig. 1A.. 3.2.2 Procedures Mice were anesthetized with pentobarbital (50 mg/kg, MTC Pharmaceuticals, Cambridge, UK) and placed on a stereotaxic frame for surgery (DKI-900, David Kopf Instruments, CA, USA) after measurement of their body weight. An infusion cannula was implanted into the lateral ventricle. The coordinates for the anterior–posterior, medial–lateral and dorsal–ventral axes were -2.2 mm from the bregma, ±0.1 mm from the midline, and -2.5 mm from the surface of the skull, 36.

(56) respectively. Continuous infusion of freshly-solubilized Aβ1-40 (300 pmol/day in ACSF) or ACSF was maintained for 2 weeks by attaching the infusion cannula to a mini-osmotic pump (Alzet 1002; Alza, Palo Alto, CA). The oligomeric structure of Aβ1-40 within the pump was confirmed by western blot analysis on days 7 and 14 (Supplementary Fig. 1B).. 3.2.3 Morris water maze (MWM) as above. 3.2.4 Immunohistochemistry Immediately after the MWM test, mice were anesthetized with avertin (0.016 ml/g body weight, Sigma, Germany) and transcardiacally perfused with 4% paraformaldehyde in phosphate-buffered saline (PBS). Brains were removed, post-fixed with 4% paraformaldehyde overnight, then placed in 30% sucrose in PBS for 2 days; they were then serially sectioned (30 µm) on a cryostat, and free-floating sections were immunostained. Rabbit anti-Aβ1-40 polyclonal antibody (Chemicon, 1:200) staining was used to detect the presence of Aβ, and specific antibodies were used to assess oxidative stress (MnSOD, Upstate, 1:200), apoptosis (Caspase3, Chemicon, 1:40), calcium binding protein (Calbindin, Sigma, 1:1000), and neurotransmitters (choline acetyltransferase, ChAT, Chemicon, 1:500; tyrosine hydroxylase, TH, Chemicon, 1:1000; glutamate decarboxylase 65/67, GABA, Chemicon, 1:500). The images were loaded into research-based digital image analysis software (Image Pro Plus, Media Cybernetics, MD, USA) and the DAB pixel counts were measured by setting the threshold to the same value for each section. Pixel count was determined as the average of three adjacent sections for 37.

(57) at least three animals per group.. 3.2.5 Statistical analysis To determine whether age renders the brain more vulnerable to Aβ1-40 toxicity in the hybrid mice, the data were analyzed by mixed two-way ANOVA followed by post hoc LSD multiple range tests. In the water maze task, escape latencies and time spent searching the target quadrant in the water maze pool were analyzed by ANCOVA, with swimming velocity as the covariant. Results are expressed as mean ± SEM.. 3.3 Results 3.3.1 Transient body weight loss accompanied with apoptosis of the LH region in aged treated Aβ1-40 mice Body weight was measured on the 1st, 6th, and 14th day (Table. 1), and the aged mice were found to be about 30% heavier than the young mice (p < 0.05). However, body weight was reduced in the aged mice receiving Aβ1-40 but not vehicle treatment (p < 0.001), and an interaction effect was noted in group × treatment (p < 0.001) only on the 6th day.. Aβ1-40, MnSOD, and caspase-3 were analyzed by immunostaining to estimate the levels of Aβ1-40 deposition, oxidative stress, and apoptosis over the whole brain region. The Aβ1-40, oxidative stress, and apoptosis levels were found to be significantly different only in the lateral hypothalamus (LH) region (Table 1), and not in any other AD-related region, such as the hippocampus or the frontal cortex (data not shown). 38.

(58) We found that aged did not significantly change the level of Aβ1-40 as compared with that of the young mice (p > 0.05). However, continuous Aβ1-40 infusion significantly increased Aβ deposition, oxidative stress, and apoptosis (p < 0.001), and the interactive effect of aged and Aβ1-40 infusion further significantly increased the Aβ, oxidative stress, and apoptosis levels in the LH region (p < 0.001).. 3.3.2 Impairment of spatial memory During the MWM testing phase, the aged mice took longer to reach the hidden platform than the young mice (p < 0.001; Fig. 9A). During the training phase, such a substantial delay in learning was not observed in the Aβ1-40-treated mice (Fig. 9B & C). These results were recapitulated with the distance measurement taken into consideration. On the fourth training day, each mouse swam a significantly shorter distance to the platform than on the first 3 days (p < 0.001; Fig. 9D), and in addition, the young mice swam significantly faster than the aged mice (p < 0.001; Fig. 9E). These results show that the acquisition of spatial learning was not impaired in any group, although the aged mice needed more time to search for the platform than the young mice. The percentage of time spent in the target quadrant in the probe test was calculated (Fig. 9F & G), and it was observed that each group of mice spent significantly more time in the target quadrant (p < 0.05; Fig. 9F & G) than in any other quadrant, with the exception of the Aβ1-40-treated aged mice. In addition, the Aβ1-40-treated aged mice swam along the wall of pool (thigmotaxically) during the probe test period (Fig. 9G).. 39.

(59) The immunopositive neurons of ChAT in the MS/DB, TH in the LC, and calbindin in the CA1 subregion of the hippocampus are shown in Fig. 10A and are quantified in Fig. 10B and C. ChAT, TH, and calbindin staining were more severely reduced in mice affected by both aged and Aβ1-40 than those affected by aged or Aβ1-40 alone (p < 0.001).. 3.4 Discussion Here we present the interactive effects of continuous Aβ1-40 infusion and aged on C3H × C57BL/6J hybrid mice. These data provide the first evidence of an age-dependent interaction with Aβ1-40 in this hybrid strain.. First, we found that hybrid mice undergoing aged or Aβ1-40 transfusion alone exhibited normal spatial reference memory and body weight gain. We further found that aged or Aβ1-40 alone induced limited Aβ deposition, oxidative stress, and apoptosis in the LH region and reduced levels of ChAT in the MS/DB, TH in the LC region, and calbindin in the CA1 region of the hippocampus. Recent study suggests that alterations in neurotransmitters alone cannot induce behavioral impairment [104]. Although it has been previously reported that aged greatly affects memory in C57BL/6 mice [105], and a recent study also pointed out that C57BL/6 and Swiss mice presented similar spatial learning and memory impairment after Aβ1-40 administration [106], our results showed that the limited dysfunction in the LH, MS/DB, and LC regions was not enough to induce impairment of spatial reference memory and body weight in C3H × C57BL/6J hybrid aged mice. Therefore, data suggest that whether aged or Aβ1-40 affects spatial 40.

(60) memory or body weight depends on genetic background.. Second, the interaction between aged and Aβ1-40 infusion induced a transient decrease in body weight gain, which may result from the severe increase in Aβ1-40 deposition, oxidative stress, and apoptosis in the LH region. It has been shown in clinical patients and APP/PS1 transgenic mice that weight loss is a frequent finding with AD [107,108]. Although it has been suggested that weight loss is a general consequence of AD and does not reflect specific brain lesions [109], one study showed an association between the central nervous system pathology and weight loss in AD patients [110]. Some studies have suggested that transient body weight decrease may be a result of adaptive metabolic changes owing to neuroendocrine abnormality [111,112]. The results of our study suggest that continuous Aβ1-40 administration severely induces Aβ1-40 deposition, oxidative stress and apoptosis in the LH, which may result in a transient decrease in body weight gain in aged animals through adaptive metabolic changes. Therefore, the relationship between CNS pathology and observable non-cognitive transient changes in early dementia in aged patients might be useful in screening putative treatments for dementia.. We also found that Aβ1-40 administration in C3H × C57BL/6J hybrid aged mice resulted in a deficit only in spatial reference memory retrieval, and not in acquisition or retention. A previous study proposed that partial damage of the hippocampus impairs memory retrieval in the water maze task [113], and a recent report also pointed out that acquisition and retrieval in the water maze task were two distinct processes [114]. We 41.