中草藥及天然或合成化合物對阿茲海默氏症細胞與動物模式的治療效用

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(1)國立臺灣師範大學生命科學系博士論文. 中草藥及天然或合成化合物對阿茲海默氏症細胞與動物模式的治療效用 Therapeutic Benefits of Chinese Herbal Medicine Extract and Natural/Synthetic Compounds on β-amyloid Induced Cell and Animal Models of Alzheimer’s Disease. 研究生：邱雅貞 Ya-Jen Chiu 指導教授：李桂楨博士 Guey-Jen Lee-Chen 中華民國 107 年 8 月.

(2) Index Index ...........................................................................................................i Abstract (Chinese) ...................................................................................vi Abstract ................................................................................................. viii List of tables and figures.......................................................................... x Chapter 1: General Introduction ............................................................ 1 1.1 What is Alzheimer’s disease ................................................................................... 2 1.1.1 Alzheimer’s stages & symptoms .......................................................................... 2 1.1.2 Forms of Alzheimer’s Disease ............................................................................. 4 1.2 How does Alzheimer’s Disease affect the brain ..................................................... 5 1.2.1 The characteristics of the brain with Alzheimer’s Disease ................................ 5 1.2.2 Neurofibrillary tangles ......................................................................................... 6 1.2.3 Amyloid plaques ................................................................................................... 7 1.2.4 Neuroinflammation .............................................................................................. 9 1.2.5 Oxidative stress ................................................................................................... 12 1.2.7 Neurite outgrowth .............................................................................................. 17 1.3 Drug treatments for Alzheimer’s disease ............................................................. 19 1.3.1 Cholinesterase inhibitors ................................................................................... 19 1.3.2 NMDA receptor antagonists .............................................................................. 20 i.

(3) 1.4 Biomarkers for Alzheimer’s disease diagnosis .................................................... 22 1.4.1 Genetic biomarkers ............................................................................................ 22 1.4.2 CSF-derived biomarkers .................................................................................... 23 1.4.3 Blood-derived biomarkers .................................................................................. 25 1.5 Aβ42-GFP expressing cells ................................................................................... 27 1.5.1 Inducible Aβ42-GFP 293 cells .......................................................................... 28 1.5.2 Inducible Aβ42-GFP SH-SY5Y cells ................................................................. 29 1.6 BV-2 microglia ...................................................................................................... 30 1.7 Transgenic mouse models of Alzheimer’s disease ............................................... 31 1.8 Aims ....................................................................................................................... 33. Chapter 2: Materials and Methods....................................................... 35 2.1 Materials ................................................................................................................ 36 2.1.1 G. inflata extract and NC009 compounds ......................................................... 36 2.1.2 Chemicals ........................................................................................................... 37 2.1.3 Solutions ............................................................................................................. 38 2.1.4 Medium for cell culture ..................................................................................... 39 2.1.5 List of primary antibodies .................................................................................. 40 2.2 Methods ................................................................................................................. 41 2.2.1 Thioflavin T binding assay ................................................................................ 41 2.2.2 1,1–diphenyl-2-picryl hydrazyl (DPPH) assay .................................................. 42 2.2.3 Cell culture ......................................................................................................... 42. ii.

(4) 2.2.4 Aβ-GFP293 cells fluorescence assay ................................................................ 44 2.2.5 Reactive oxygen species (ROS) analysis ........................................................... 45 2.2.6 Neurite outgrowth analysis ................................................................................ 45 2.2.7 Acetylcholinesterase (AChE) and SOD activity assays..................................... 46 2.2.8 Nitric oxide (NO) assay ...................................................................................... 47 2.2.9 Enzyme-linked immunosorbent assay (ELISA) ................................................ 47 2.2.10 BV-2 conditioned media (CM) preparation..................................................... 47 2.2.11 MTT cell viability assay ................................................................................... 48 2.2.12 Aβ-GFP SH-SY5Y cells fluorescence assay .................................................... 48 2.2.13 Lactate dehydrogenase (LDH) and caspase 3 assays ..................................... 49 2.2.14 Apoptosis antibody array.................................................................................. 49 2.2.15 Western blot analysis ....................................................................................... 50 2.2.16 AD-related gene expression analysis ............................................................... 50 2.2.17 Real-time PCR analysis ................................................................................... 51 2.2.18 RNA interference ............................................................................................. 52 2.2.19 Animal studies .................................................................................................. 52 2.2.20 Open field test ................................................................................................... 53 2.2.21 Y-maze task....................................................................................................... 54 2.2.22 Morris water maze task .................................................................................... 54 2.2.23 Immunohistochemistry (IHC) and image analysis ......................................... 55 2.2.24 Statistical analysis ............................................................................................ 56. iii.

(5) Chapter 3: G. inflata and its active components .................................. 58 3.1 Introduction ........................................................................................................... 59 3.2 G. inflata extract, active constituents, and IC50 cytotoxicity ............................... 62 3.3 Aβ aggregation inhibition of G. inflata extract/constituents ............................... 62 3.4 Radical-scavenging activity of G. inflata extract/constituents ............................ 63 3.5 Effects of G. inflata extract/constituents on Aβ-GFP 293 cells .......................... 63 3.6 Effects of G. inflata extract/constituents on Aβ-GFP SH-SY5Y cells ................ 65 3.7 Effects of G. inflata extract/constituents on LPS/IFN-γ-activated BV-2 cells ... 66 3.8 Effects of G. inflata extract/constituents on BV-2 CM-inflamed Aβ-GFP SHSY5Y cells .................................................................................................................... 67 3.9 Apoptosis-related gene expression profiles in Aβ-GFP SH-SY5Y cells .............. 68 3.10 Summary.............................................................................................................. 70. Chapter 4: NC009-1 ............................................................................... 72 4.1 Introduction ........................................................................................................... 73 4.2 Identification of aggregate Aβ inhibitors using biochemical assay .................... 75 4.3 Effects of NC009 compounds in Aβ-GFP 293/SH-SY5Y cells ............................ 76 4.4 Expression profiles of AD-related genes in Aβ-GFP SH-SY5Y cells ................. 78 4.5 ABCA1, APOE, CHAT, TRKA and SERPINA3 expressions in peripheral leukocytes of AD patients ............................................................................................ 78 4.6 Effects of NC009-1 on APOE and TRKA expression in Aβ-GFP SH-SY5Y cells ...................................................................................................................................... 79 4.7 APOE and TRKA as therapeutic targets in NC009-1-treated Aβ-GFP SH-SY5Y iv.

(6) cells .............................................................................................................................. 80 4.8 Effects of NC009-1 on spatial learning and memory in STZ-treated 3×Tg-AD mice .............................................................................................................................. 81 4.9 Effects of NC009-1 on NeuN, Aβ and Tau levels in STZ-treated 3×Tg-AD mice ...................................................................................................................................... 84 4.10 Effects of NC009-1 on APOE and TRKA expression in STZ-treated 3×Tg-AD mice .............................................................................................................................. 86 4.11 Summary .............................................................................................................. 86. Chapter 5: General Discussion.............................................................. 88 5.1 Neuropathologic changes in Alzheimer’s disease ............................................... 89 5.2 G. inflata and its active constituents .................................................................... 89 5.3 NC009-1................................................................................................................. 94 5.4 The drugs development process ............................................................................ 97 5.5 Future perspectives ............................................................................................. 100. References ............................................................................................. 102. v.

(7) 中文摘要阿茲海默氏症(AD)是有關漸進性認知衰退和記憶力喪失的疾病，也是最常見的一種老人痴呆症，病理學上病徵之一是由 β-澱粉樣 (Aβ)沉積物組成的老年斑。研究發現，腦中的 Aβ 堆積會造成氧化壓力和發炎損傷，進而導致神經細胞凋亡及認知功能失調。有鑑於此，尋找可能減少 Aβ 聚集的方法，可能有效治療 AD。本研究檢視中藥脹果甘草(G. inflata)及其活性成分甘草查爾酮 A (Licochalcone A)、甘草素(Liquiritigenin)，和本校化學系姚清發老師提供的合成化合物 NC009-1，抑制 Aβ 聚集的情形及神經保護性。誘導性 Aβ-GFP 293 細胞、硫黃素 T (Thioflavin T)或 DPPH 清除自由基試驗結果顯示，脹果甘草/活性成分和 NC009-1，皆可抑制 Aβ 蛋白聚集和相關聯的氧化壓力。此外，LPS/IFN-γ 刺激發炎的小鼠 BV-2 微膠細胞試驗顯示，脹果甘草/活性成分可減少 BV-2 的發炎反應，來達到抗發炎的效果。另外，誘導性 Aβ-GFP SH-SY5Y 細胞試驗結果顯示，脹果甘草/活性成分和 NC009-1 可抑制乙醯膽鹼酶(acetylcholinesterase) 活性、增強 SOD2 表現、及/或促進神經突生長。為瞭解脹果甘草/活性成分和 NC009-1 對細胞保護的分子機制，以抗體或 PCR 矩陣，檢測 Aβ-GFP SH-SY5Y 細胞中，與細胞凋亡相關蛋白或阿茲海默氏症相關基因的表現。結果發現，脹果甘草/活性成分可減緩 BCL2 的下降，並減低 IGFBP2 的上升、凋亡蛋白酶 3 的切割、BAD 及 BAX 的量，來保護 Aβ-GFP SH-SY5Y 細胞免於 BV-2 制約培養液誘導的細胞死亡。然而 NC009-1 在 Aβ-GFP SH-SY5Y 細胞中，可正調控 APOE 和 TRKA 的表現。在鏈脲佐菌素(Streptozocin)誘導高血糖的 PS1M146V、APPSwe 和 TauP301L 三基因(3×Tg) AD 轉殖鼠的實驗中， NC009-1 並可挽救 APOE 和 TRKA 的減少，降低海馬迴及腦皮層中 Aβ 及 Tau 的量，及減緩認知缺失。這些研究結果指出，脹果甘草/ 活性成分與 NC009-1 可能作為 AD 的治療策略。 vi.

(8) 關鍵詞：阿茲海默氏症、β-澱粉樣、脹果甘草、甘草查爾酮 A、甘草素、合成化合物. vii.

(9) Abstract Alzheimer’s disease (AD) is the most prevalent form of dementia associated with progressive cognitive decline and memory loss. One of the pathological hallmarks of AD is senile plaques consisting of βamyloid (Aβ) deposits. Studies have proposed that Aβ deposition causes oxidative stress and inflammatory damage which lead to apoptotic cell death and eventual cognitive deficits. Therapeutic approaches to identify novel Aβ aggregate reducers could be an effective treatment for the disease. In this study, Chinese herbal medicine G. inflata and its bioactive constituents licochalcone A and liquiritigenin, and synthetic compound NC009-1 provided by Professor Ching-Fa Yao from Department of Chemistry of NTNU, were examined for Aβ aggregation reduction and neuroprotection. By using inducible Aβ-GFP 293 cells, biochemical thioflavin T or DPPH free radical scavenging assays, G. inflata/constituents and NC009-1 reduced Aβ aggregation and associated oxidative stress. Besides, G. inflata/constituents showed antiinflammatory effect by attenuating the inflammatory response of BV-2 microglia under LPS/IFN-γ stimulation. In addition, G. inflata/constituents and NC009-1 displayed acetylcholinesterase inhibition, SOD2 up-regulation, and/or neurite outgrowth promotion in inducible Aβ-GFP SH-SY5Y cells. To reveal the molecular mechanisms underlying protective effects of G. inflata/constituents and NC009-1, antibody or PCR array was used to assess expression changes of viii.

(10) apoptosis-associated proteins or AD-related genes in the Aβ-GFP SHSY5Y cells. G. inflata/bioactive constituents protected Aβ-GFP SHSY5Y cells from BV-2 conditioned media-induced cell death by ameliorating reduced BCL2 and attenuating increased IGFBP2, cleaved CASP3, BAD and BAX, whereas NC009-1 up-regulated the expression of APOE and TRKA in Aβ-GFP SH-SY5Y cells. NC009-1 further rescued the down-regulated APOE and TRKA and reduced Aβ and Tau levels in hippocampus and cortex, and ameliorated cognitive deficits in streptozocin-induced hyperglycemic PS1M146V, APPSwe, and tauP301L 3×Tg-AD mice. These results indicate that G. inflata/constituents and NC009-1 could be possible treatment strategies for AD.. Keywords: Alzheimer’s disease, β-amyloid, Glycyrrhiza inflata, licochalcone A, liquiritigenin, Indole compound. ix.

(11) List of tables and figures. Tables Table 1. Proteins identified by using human apoptosis antibody array. 138 Table 2. Genes identified in human AD PCR array showing < -1.5-fold expression changes in Aβ-GFP SH-SY5Y cells. ............................ 139 Table 3. Characteristics of the subjects and mRNA expression in AD patients and controls. ...................................................................... 140 Figures Figure 1. Pathological effects of Aβ. ..................................................... 141 Figure 2. The intrinsic apoptosis pathway. ............................................ 142 Figure 3. The extrinsic apoptosis pathway. ........................................... 144 Figure 4. Human apoptosis antibody array map.................................... 145 Figure 5. Human AD RT² Profiler™ PCR Array. .................................. 146 Figure 6. HPLC chromatogram and cytotoxicity of licochalcone A and liquiritigenin in G. inflata extract................................................... 147 Figure 7. Anti-aggregation and anti-oxidative effects of G. inflata extract, licochalcone A and liquiritigenin in biochemical assays. ............... 148 Figure 8. Anti-aggregation and anti-oxidative effects of G. inflata extract, licochalcone A and liquiritigenin in Aβ-GFP 293 cells. ................. 149 Figure 9. Neuroprotective effects of G. inflata extract, licochalcone A and liquiritigenin in Aβ-GFP SH-SY5Y cells. ............................... 151 Figure 10. Anti-inflammatory activities of G. inflata, licochalcone A and x.

(12) liquiritigenin in LPS and IFN-γ-stimulated BV-2 microglia. ......... 154 Figure 11. Cell protection and anti-aggregation effects of G. inflata extract, licochalcone A and liquiritigenin against cytotoxicity of BV2 CM on Aβ-GFP SH-SY5Y cells. ................................................. 156 Figure 12. Therapeutic targets of G. inflata extract, licochalcone A and liquiritigenin against BV-2 CM-induced Aβ-GFP SH-SY5Y cell death. .............................................................................................. 158 Figure 13. A tentative mechanism of G. inflata extract and its constituents, licochalcone A and liquiritigenin in treating Alzheimer’s disease........................................................................ 160 Figure 14. Compound screen by Trx-His-Aβ and Aβ biochemical assay. ........................................................................................................ 161 Figure 15. Protective effects of NC009 compounds in Aβ-GFP 293/SHSY5Y cells. ..................................................................................... 162 Figure 16. Biomarkers identification via RT² Profiler™ PCR array. .... 164 Figure 17. Indole compound NC009-1 augments APOE and TRKA and downstream pERK and pAKT expression in Aβ-GFP SH-SY5Y cells. ............................................................................................... 165 Figure 18. APOE/TRKA gene silence reduced neurite outgrowth in NC009-1-treated Aβ-GFP SH-SY5Y cells. .................................... 167 Figure 19. Indole compound NC009-1 rescues spatial working memory, spatial learning and memory in STZ-treated 3×Tg-AD mice. ....... 169 Figure 20. Indole compound NC009-1 increases NeuN and decreases Aβ xi.

(13) and Tau immunoreactivity in STZ-treated 3×Tg-AD mice. ........... 171 Figure 21. Indole compound NC009-1 augments APOE and TRKA expression in STZ-treated 3×Tg-AD mice. .................................... 173 Figure 22. A tentative mechanism of NC009-1 in treating Alzheimer’s disease. ........................................................................................... 174. xii.

(14) Chapter 1 General Introduction. 1.

(15) Chapter 1: General Introduction. 1.1 What is Alzheimer’s disease Alzheimer’s disease (AD) is the most common type of dementia associated with progressive cognitive decline. The first official report of AD was established by German physician, Alois Alzheimer, in 1907 (Alzheimer et al., 1995). Alois Alzheimer was born in 1864 in Markbreit in Bavaria, Southern Germany. He graduated with a medical degree in 1887. In 1903, He created a new laboratory for brain research in the Munich medical school. When studying the conditions and diseases of the brain, he identified an unusual disease of the cerebral cortex which affected a 50-year-old woman, and caused memory loss, disorientation, hallucinations and eventually death at 55-year-old. The post-mortem showed various abnormalities of the brain, including thinner cerebral cortex, senile plaque, and neurofibrillary tangles. After that, the disease is named after Dr. Alois Alzheimer.. 1.1.1 Alzheimer’s stages & symptoms The most common early symptom of AD is difficulty remembering newly learned information. Alzheimer’s damage typically begins in the hippocampal part of brain, which is essential for forming memories. The symptoms of Alzheimer’s disease worsen over time, including: disorientation, mood and behavior changes; deepening confusion about. 2.

(16) events, time and place; unfounded suspicions about family, friends and professional caregivers; more serious memory loss and behavior changes; and difficulty speaking, swallowing and walking. On average, AD’s patients live 4 to 8 years after diagnosis, but can live as long as 20 years, depending on other factors (Tarawneh and Holtzman, 2012). Based on Dr. Resiberg’s system, the symptoms of AD can be divided into 7 stages (Reisberg et al., 1982). Stage 1: No impairment. AD is not detectable and no memory problems or other symptoms of dementia are evident. Stage 2: Very mild decline. The patient may notice minor memory problems, but the disease is unlikely to be detected by family members or physicians. Stage 3: Mild decline. The family members and friends of the patient may begin to notice cognitive problems. Physicians can use memory tests to detect impaired cognitive function. The symptoms of patient in stage 3 will have difficulty in many areas including finding the right word during conversations; organizing and planning; and remembering names of new acquaintances and location of valuables. Stage 4: Moderate decline. Patient has difficulty with simple arithmetic and poor short-term memory; inability to manage finance and pay bills; and forget details about their life. Stage 5: Moderately severe decline. Patient in this stage of the disease has difficulty dressing; inability to recall simple details about themselves; and feeling extremely confused. But they usually still know their family members and some detail about their personal histories, especially their childhood and youth. 3.

(17) Stage 6: Severe decline. Symptoms of this stage including confusion or unawareness of environment and surroundings; inability to recognize faces except for the closest friends and relatives; inability to remember most details of personal history; loss of bladder and bowel control; personality changes; and often disoriented. Stages 7: Very severe decline. It is the final stage of AD. Because the disease is a terminal illness, patient in this stage is nearing death. In this stage of the disease, patient loses the ability to communicate or respond to their environment. Patient needs assistance with all activities of daily living.. 1.1.2 Forms of Alzheimer’s Disease AD can be divided into two forms: familial AD (FAD) and sporadic AD (SAD). SAD is also known as late-onset AD because it does not appear to involve genetic factor or family link. The molecular mechanisms of neuropathological changes in SAD includes insulin signaling, energy metabolism, oxidative stress, neuroinflammation, excitotoxicity, neurotransmission abnormalities, and so on (Efthymiou and Goate, 2017). FAD is also known as early-onset AD because it is hereditary and is characterized by AD symptoms that appear at an unusually early age. Mutations in the amyloid precursor protein (APP), presenilin 1 (PSEN1) and presenilin 2 (PSEN2) genes are considered to be the major features of FAD (Hardy, 1997). Both AD forms have very similar symptoms and both form Aβ plaques and neurofibrillary tangles 4.

(18) (Tellechea et al., 2018).. 1.2 How does Alzheimer’s Disease affect the brain The changes in the brain begin long before the first signs of memory loss. In brains of AD, magnetic resonance imaging (MRI) images showed overall volume loss and shape changes in specific brain structures, such as the hippocampus, amygdala, corpus callosum, and other regions (Fields, 2008). The shape change of brain is caused by increasing neurons death, leading to passive enlargement of the brain ventricles. Among them, hippocampus is the first area that is most severely affected by cellular and structural alterations, and the progressive shrinkage of this area is the cause of memory loss (Villain et al., 2008). When the cerebral cortex (the outer layer of the brain) is also beginning to be damaged, judgment declines, emotional outbursts may occur and language is impaired. In the final stage of AD, damage is widespread, and brain tissue has shrunk significantly. Injuries to these areas lead to the development of AD.. 1.2.1 The characteristics of the brain with Alzheimer’s Disease In Dr. Alzheimer’s pioneering article, he found that the patient’s brain has many abnormal clumps (now called amyloid plaques) and tangled bundles of fibers (now called neurofibrillary, or tau tangles) (Alzheimer et al., 1995). These plaques and tangles in the brain are still crucial 5.

(19) morphological criteria for the definite diagnosis of AD. Other features include the loss of connections between neurons and the changes of cellular signaling pathways in brain. Studies are underway to determine which changes may cause AD and which may be a result of the disease.. 1.2.2 Neurofibrillary tangles Neurofibrillary tangles (NFTs) are pathological insoluble aggregates of abnormally phosphorylated tau proteins, which, in their normal state, bind to microtubules and stabilize them to form a functioning cytoskeleton in neurons (Bakota and Brandt, 2016; Grundke-Iqbal et al., 1986). Normal microtubule assembly is tightly regulated by a combination of protein kinases and phosphatases that balance the amount of tau phosphorylation (Whiteman et al., 2009; Iqbal et al., 2009). But when this balance is broken, tau exists in a hyperphosphorylated state, which leads to aberrant secondary structures and loss of function, resulting in reduced ability to bind to microtubules and promote microtubule assembly (Mietelska-Porowska et al., 2014). Tau is rich in serine/threonine and lysine residues, and is known to undergo posttranslational modifications, such as phosphorylation, acetylation, ubiquitination, and sumoylation (Martin et al., 2011). Among them, phosphorylation is the most common type of tau post-translational modification. To date, more than 80 phosphorylation sites have been identified in the tau molecule (Mietelska-Porowska et al., 2014; Kimura 6.

(20) et al., 2016). It has been found that phosphorylation of tau protein at sites serine 396 and serine 404 is associated with abnormal tau processing (Mondragón-Rodríguez et al., 2014; Duka et al., 2013). Additionally, several molecular mechanisms may underlie the tau-induced toxicity, such as mitochondrial dysfunction (Li et al., 2016a), oxidative stress (Stamer et al., 2002), inflammation (Wang et al., 2013) and synaptic damage (Yin et al., 2016), which ultimately leads to neurodegeneration (Wang and Liu, 2008). Emerging evidence suggests that Aβ is upstream of tau in AD pathogenesis and triggers the conversion of tau from a normal to a toxic state (Hurtado et al., 2010; Roberson et al., 2007; Gotz et al., 2001; King et al., 2006), but there is also evidence that toxic tau enhances Aβ toxicity via a feedback loop (Rapoport et al., 2002; Ittner et al., 2010).. 1.2.3 Amyloid plaques Amyloid plaques, which are mainly composed of aggregated amyloid β (Aβ), are one of the neuropathological diagnostic criteria for AD. Research is ongoing to better understand how, and at what stage of the disease, the various forms of Aβ influence AD. As described, mutations in the APP, PSEN1 and PSEN2 genes have been identified as the genetic causes of familial AD (Hardy, 1997). According to the amyloid cascade hypothesis, mutations in these genes were thought to be the initiating events in the Aβ deposition leading to 7.

(21) cell death and dementia in AD (Barage and Sonawane, 2015). APP is a cell surface receptor and transmembrane protein that is proteolytically processed to generate Aβ peptides with 39 to 43 amino acids (Masters et al., 1985). Among them, the 42-residue form Aβ (Aβ42) is produced in small amounts but is the predominant peptide deposited in the amyloid plaques of AD (Roher et al., 1993). APP processing is divided into two pathways: Non-amyloidogenic and amyloidogenic pathways (Chow et al., 2010). In the nonamyloidogenic pathway, APP is cleaved by α- and γ-secretases resulting in the generation of a long-secreted form of APP (sAPPα) and C-terminal fragments (CTF 83, p3 and AICD50). In the amyloidogenic pathway, APP is cleaved by β- and γ-secretases resulting in the generation of a long-secreted form of APP (sAPPβ), C-terminal fragments (CTF 99 and CTF 89) and Aβ. The resulting production of Aβ will oligomerize and fibrillize, leading to AD pathology. Monomeric Aβ folds into aggregated oligomers, protofibrils and insoluble fibrillar aggregates (Koo et al., 1999) that are toxic to neurons (Hensley et al., 1994; Small et al., 2001). Additionally, excess Aβ production, aggregation and deposition have deleterious effects on lipid metabolism, intracellular signaling cascades, autophagy regulation, neurotransmitter release and synaptic function (Jucker and Walker, 2013). As shown in Fig. 1, numerous studies stress that Aβ accumulates intraneuronally (Billings et al., 2005; Casas et al., 2004; Leon et al., 8.

(22) 2010; Oddo et al., 2003; Wirths et al., 2002), and the intraneuronal compartment is also a place to form toxic aggregates (Leon et al., 2010; Takahashi et al., 2004; Walsh et al., 2000) which can impair synaptic function (Jin et al., 2011; Lambert et al., 1998; Shankar et al., 2008; Walsh et al., 2002) and neurite outgrowth (Evans et al., 2008), induce cognitive dysfunction (Cleary et al., 2005; Iulita et al., 2014; Lesne et al., 2006) and oxidative stress (Cheignon et al., 2018), unleash a proinflammatory reaction (Bruno et al., 2009; Leon et al., 2010), and cause neuroinflammation by activating microglia (Cai et al., 2014).. 1.2.4 Neuroinflammation Brain inflammation is another pathological hallmark of AD. In the brains of AD patients, microglial cells are phenotypically activated and closely associated with amyloid deposits (Perlmutter et al., 1990; Wisniewski et al., 1992). Additionally, damaged neurons activate microglia to produce neurotoxic factors, which are toxic to surrounding neurons, resulting in perpetuating toxicity (Shanmuga Sundaram et al., 2012). The activation patterns of microglia have two different phenotypes: M1 (classically activated) and M2 (alternatively activated). M1 microglia induce neurotoxicity due to the release of pro-inflammatory factors and various neurotoxic mediators and often setup a vicious cycle between dying neurons and acute inflammation (Block et al., 2007). It has been 9.

(23) suggested that the M1 microglia responds to lipopolysaccharide (LPS) in combination with interferon-γ (IFN-γ) and produces a massive inflammatory response releasing interleukin-1β (IL-1β), IL-12, tumor necrosis factor-α (TNF-α), and inducible nitric oxide synthase (iNOS) (Tang and Le, 2014; Nakagawa and Chiba, 2014). M2 microglia are the major effector cells with the potential to dampen pro-inflammatory immune responses and promote the repair genes expression. It has been suggested that the M2 microglia responds to IL-4, IL-13, IL-10, and TGF-β to antagonize the pro-inflammatory responses, that finally results in immunosuppression and neuron protection (Butovsky et al., 2005; Zhou et al., 2012). In the case of neuroinflammation, activated glial cells release highly toxic products such as reactive oxygen intermediates, nitric oxide, inflammatory cytokines, proteolytic enzymes, complement factors, or excitatory amino acids (Halliday et al., 2000; Su et al., 2016). Increasing evidence suggests that all of these factors contribute to neuronal dysfunction and cell death, either alone or in concert (Abbas et al., 2002; Bezzi et al., 2001; Brown and Bal-Price, 2003). Among them, the levels of cytokines are altered in AD patients and that cytokines are key components of neuroinflammation. Cytokines are a large and heterogeneous family of proteins that include the interleukins, TNF-α, IFN-γ and transforming growth factor-β (TGF-β). It has been suggested that several pro-inflammatory cytokines 10.

(24) cause neuronal damage in AD, such as IL-1β, IL-6, TNF-α and IFN-γ (Meraz-Ríos et al., 2013; Calsolaro and Edison, 2016). The IL-1 family consists of 11 members, 7 of which have been demonstrated to have broad pro-inflammatory activity (IL-1α, IL-1β, IL-18, IL-33, IL-36α, IL36β and IL-36γ) while the remaining 4 have antagonistic (IL-1Ra, IL36Ra, IL-38) or anti-inflammatory (IL-37) properties. Among them, IL1β is synthesized in pro-form (pro-IL-1β) in the cytoplasm and then cleaved by the protease caspase-1 into a mature and biologically active form. It has been shown that overexpression of IL-1β exacerbates tau phosphorylation and tangle formation via aberrant activation of P38MAPK and glycogen synthase kinase 3 (GSK3), which may affect synaptic plasticity (long-term potentiation) in learning and memory (Kitazawa et al., 2011). Another interleukin, IL-6, has been described as having pleiotropic effects with increased levels in AD brains (Rubio-Perez and MorillasRuiz, 2012; McGeer and McGeer, 1997). IL-6 is produced by activating Toll-like receptor 2 (TLR2)-MyD88 signaling pathways in microglia and the JNK-AP1 pathway in human brain endothelial cells (Vukic et al., 2009; Jana et al., 2008). It has been suggested that under inflammatory conditions, excessive expression of IL-6 may impair cognitive processes by activation of neuronal NADPH-oxidase (Dugan et al., 2009). In addition, IL-6 can also activate the JAK/STATs, NMDA receptor and the MAPK-P38 protein kinases, both of which are involved in 11.

(25) hyperphosphorylation of tau (Spooren et al., 2011). TNF-α is another mediator, actively produced by microglia during inflammation. TNF-α has two cognate transmembrane receptors called TNF receptor 1 (TNFR1) and TNFR2. Signaling via the cognate TNF receptors elicits distinct cellular responses, including cell proliferation, cell migration, and apoptosis mediated through the activation of several downstream signal transduction cascades involving NF-κB, c-Jun Nterminal kinase (JNK), P38, and ceramide-sphingomyelinase pathways (Montgomery et al., 2013). It has been showed that TNF-α stimulates BACE1 expression and enhances amyloidogenic processing in AD animal model (Yamamoto et al., 2007). IFN-γ is another cytokine up-regulated in AD brains (McGeer and McGeer, 1997). Additionally, IFN-γ could enhance Aβ production and deposition in AD animal model, possible via BACE1 expression and suppression of Aβ clearance (Yamamoto et al., 2007). It has been demonstrated that IFN-γ activates JAK2 and ERK1/2 and then phosphorylated STAT1 binds to the putative STAT1 binding sequences in BACE1 promoter region to modulate BACE1 protein expression in astrocytes (Cho et al., 2006). These studies indicate that cytokine released by activated glial cells plays key role in neuroinflammatory-mediated cell death in AD.. 1.2.5 Oxidative stress 12.

(26) Another important feature in AD is the presence of oxidative damages in neuronal lipids and proteins, and the overproduction of reactive oxygen species (ROS), which is considered as a major contribution to the cause of oxidative damage (Cheignon et al., 2018). ROS are broadly defined as oxygen-containing chemicals with reactive properties (Gorrini et al., 2013). The life in aerobic environment and with O2 as a final electron acceptor results in a constant production of ROS in our body. Most of the ROS produced in the cell come from the respiratory chain and are toxic to cell. Thus several enzymes and small compounds exist to control the levels of ROS. Generally, ROS are kept at a low level in cells. However, excessively high levels of ROS are detrimental to cells and are defined as oxidative stress. ROS accumulation can occur either by an overproduction or an insufficient elimination of ROS (Beckhauser et al., 2016; Nickel et al., 2014). Since the mitochondrial respiratory chain is a major site of ROS production in the cell, mitochondria are particularly susceptible to oxidative stress (Grivennikova and Vinogradov, 2006; Tan et al., 1998). Additionally, evidence suggests that the presence of Aβ in mitochondria was associated with impaired mitochondrial metabolism and increased mitochondrial ROS production (Manczak et al., 2006; Caspersen et al., 2005). ROS released from mitochondria (e.g., superoxide anion O2-•) interact with nitric oxide (NO) produced by nitric oxide synthase (NOS) to produce reactive nitrogen species (RNS), such as peroxynitrite 13.

(27) (ONOO-), which covalently modify proteins. O2-• can also directly oxidize proteins, lipids, and carbohydrates. To alleviate O2-•-induced damage, O2-• may also be dismutated to H2O2 by superoxide dismutase (SOD) enzymes. Although H2O2 is not a free radical, it can still cause damage to cell at a relatively low concentration (10 μM). However, some antioxidant enzymes such as catalase, glutathione peroxidase and peroxidase can eliminate H2O2 (Phaniendra et al., 2015; Butterfield et al., 2013). Oxidative stress occurs early in the course of AD, which would support the role of ROS in AD pathogenesis, in relation with the presence of Aβ. Studies have shown that elevated levels of Aβ40 and Aβ42 are associated with increased levels of oxidation products from proteins, lipids and nucleic acids in AD hippocampus and cortex (Wang et al., 2014; Butterfield and Lauderback, 2002). By contrast, brain regions with low Aβ levels (e.g., cerebellum) did not present high concentrations of oxidative stress markers (Sultana et al., 2006; Hensley et al., 1995; Butterfield et al., 1999). Other studies also found that the redox active metal ions can catalyze the production of ROS when bound to the Aβ. Thus, elevated ROS levels may contribute to oxidative damage on both the Aβ peptide itself and on surrounding molecule (proteins, lipids, and so on) (Nakamura et al., 2007; Guilloreau et al., 2007). These studies indicate that Aβ may directly disrupt mitochondrial function and lead to increased oxidative stress as well as deficiency of energy metabolism, 14.

(28) ultimately leading to neuronal death.. 1.2.6 Apoptosis Apoptosis is a genetically mediated programmed cell death, plays a central role in normal development, tissue homeostasis, and the elimination of infected or damaged cells (Hengartner and Bryant, 2000). The morphological changes of apoptosis observed in most cell types include: (1) contraction in cell volume and condensation of the nucleus despite the stable morphology of intracellular organelles (e.g. mitochondria), (2) blebbing of the plasma membrane, (3) fragmentation of nucleus leading to eventual cell fragmentation, and (4) phagocytosis of apoptotic bodies (Kataoka and Tsuruo T, 1996). It has been found that the cells are impaired or influenced by various factors such as B-cell lymphoma-2 (Bcl-2) family members, caspase, Aβ, TNF-α, and ROS, leading to cell death and neurodegenerative diseases like AD (Obulesu and Lakshmi, 2014). Despite the existence of various stimuli, the ultimate phases of apoptosis are implemented by a few common caspases (Alvarez et al., 2011). In addition, balance between the antagonistic actions of the pro-apoptotic and anti-apoptotic members of the Bcl-2 family is involved in the maintenance of the mitochondrial integrity (Zapała et al., 2010; Jahanshahi et al., 2013). The two most important pathways that can initiate apoptosis are: (1) the mitochondria-dependent or intrinsic apoptosis pathway, and (2) the 15.

(29) death receptor-mediated or extrinsic apoptosis pathway (Raychaudhuri, 2010). In the intrinsic and extrinsic pathways, the cell senses cell stress from itself or from other cells respectively, and kills itself by activating initiator and executioner caspases to induce cell death. The intrinsic pathway of apoptosis is regulated by mitochondrial parameters (Fig. 2). When mitochondrial dysfunction, cytochrome c (cyt-c) is released from mitochondria into the cytoplasm. Mitochondrial anti-apoptotic proteins Bcl-2 and Bcl-XL inhibit cyt-c release, while pro-apoptotic proteins Bcl2-associated X protein (Bax), Bcl-2 homologous antagonist (Bak) and BH3 interacting domain death agonist (Bid) promote cyt-c release from mitochondria. Cyt-c and deoxyadenosine triphosphate (dATP) interact with apoptotic protease activating factor (Apaf-1) and caspase 9 to form a heptameric complex known as the apoptosome (Acehan et al., 2002). Caspase 9 in the apoptosome activates effector caspases 3, 6, and 7 to lead to the widespread cleavage of apoptotic substrates and the irreversible morphological changes and DNA cleavage that characterize apoptosis (Siegel, 2006; Fuentes-Prior and Salvesen, 2004). The extrinsic apoptosis pathway is initiated by oligomerization of death receptors like Fas, TNFR, DR3, TRAIL-R4, and TRAIL-R5 after associating with their corresponding ligands (Fig. 3, Fas is used here as an example). This oligomerization further stimulates initiator caspases 8 and 10 to initiate apoptosis. Caspases 8 and 10 triggers apoptosis in two ways: cleave and activate caspases 3, 6, 7 directly, or cleave Bid to tBid (truncated Bid), 16.

(30) which then translocates to the mitochondrial outer membrane and results in an intrinsic apoptotic pathway, to lead to DNA fragmentation and cell death (Siegel, 2006). As mentioned earlier, activated glial cells may release cytokine to cause apoptosis. It has been found that increasing levels of IL-1β and TNF-α provoke neuronal death and apoptosis (Ye et al., 2013). IL-1β induces mitochondria-mediated apoptosis by reducing the Bcl-2/Bax ratio and enhancing release of Cyt-c from mitochondria to cytosol (Shen et al., 2017; Wang et al., 2012). In addition, growing body of evidence suggests that TNF-α initiates FasL expression through nuclear factor of activated T cells (NFAT) activation in neuroblastoma cells, thus contributing to apoptosis (Alvarez et al., 2011). Binding of FasL to its receptor Fas, initiates the intracellular machinery associated with the death receptor Fas. The Fas-FasL interaction recruits the Fas-associated death domain adaptor protein (FADD) via death domain binding, and ultimately promotes apoptosis by caspase activation and subsequent DNA cleavage (Alvarez et al., 2011; Jayanthi et al., 2005). Moreover, TNF-α also stimulates release of Cyt-c from mitochondria, which leads to caspase 9 activation and promotes cell death (Alvarez et al., 2011).. 1.2.7 Neurite outgrowth Inhibition of neurite growth is also associated with Aβ deposition. Recently studies have demonstrated that nerve growth factor (NGF) 17.

(31) treatment can ameliorate the changes of Aβ pathologies and inhibit memory impairment in AD animal models (Zhang et al., 2013). NGF is the first well-characterized neurotrophic factor in the family of neurotrophins which promotes neuronal survival and differentiation and regulates neurite outgrowth, synaptic function and plasticity (Allen et al., 2013). Additionally, NGF binds at the plasma membrane to two distinct NGF receptors, the high-affinity TrkA receptor (tyrosine kinase receptor) and the low-affinity p75NTR (a member of the tumor necrosis factor receptor superfamily), to mediate its biological activity (Aloe and Rocco, 2015). Among them, NGF promotes cell survival and differentiation during neurodevelopment through activation of TrkA receptor (Huang and Reichardt, 2003). Activated Trk receptor recruits and increases the phosphorylation of phospolipase C-γ (PLC-γ) and Src homology 2containing protein (Shc), leading to activation of extracellular signalregulated-kinases (ERK) 1/2 and phosphoinositide-3-kinase (PI3K)/AKT pathways (Kaplan and Miller, 2000; Arévalo et al., 2004). Studies have indicated that both these two pathways are important for neuronal survival and differentiation (Zhao et al., 2015; Zhuang et al., 2011). In addition, apolipoprotein E (APOE) can also activate ERK and PI3K/AKT signaling pathways by binding to lipoprotein receptors (LRP) (Hoe et al., 2005; Qiu et al., 2004). In sum, these two signaling pathways may be a potential candidate in the pathogenesis of AD and a therapy that may increase neuronal survival and differentiation. 18.

(32) 1.3 Drug treatments for Alzheimer’s disease Although there are no medical treatments that can cure AD, some medicines have been developed for AD that can temporarily relieve symptoms or slow the progression of the disease. So far two types of drugs are currently used to treat cognitive symptoms: cholinesterase inhibitors and N-methyl-D-aspartate receptor (NMDAR) receptor antagonists.. 1.3.1 Cholinesterase inhibitors Cholinesterase is a family of enzymes that catalyzes the hydrolysis of the neurotransmitter acetylcholine (Ach) into choline and acetic acid. Cholinesterase inhibitors inhibit the cholinesterase enzyme from breaking down ACh, increasing both the level and duration of the neurotransmitter action (Giacobini, 2004). The brain of mammals contains two major forms of cholinesterases: acetylcholinesterase (AChE) and butyrylcholinesterase (BuChE). The two forms differ genetically, structurally and for their kinetics. In AD, AChE activity decreases progressively in the brain of AD patients, while BuChE activity shows some increase (Lane et al., 2006). In the peripheral nervous system, ACh is the neurotransmitter at the neuromuscular junction between the motor and skeletal muscle. In the central nervous system (CNS), ACh is mainly present in interneurons and involves in a few important long-axon 19.

(33) cholinergic pathways. The pathway associated with the AD is the cholinergic projection from the nucleus basalis of Meynert (in the basal forebrain) to the forebrain neocortex and associated limbic structures. Degeneration of this pathway has been found to be one of the pathologies associated with AD (Perry et al., 1999). Tacrine was the first AChE inhibitor approved for the AD treatment in 1993, but its use has been abandoned due to the high incidence of side effects including hepatotoxicity (Watkins et al., 1994). Donepezil is indicated for the symptomatic treatment of mild to moderate AD. It is a selective, reversible AChE inhibitor that also delays the deposition of amyloid plaques (Arce et al., 2009; Castro and Martinez, 2006). Cholinesterase inhibitor rivastigmine shows superior properties in terms of specificity of action and lower risk of adverse effects (Birks and Grimley Evans, 2015). Treatment of mild to moderate AD with rivastigmine can produce modest improvements in cognitive function and slow down cognitive decline (Onor et al., 2007). Galantamine, another reversible inhibitor of acetylcholinesterase, enhances the intrinsic action of acetylcholine on its receptors, leading to increased cholinergic neurotransmission in the CNS (Razay and Wilcock, 2008).. 1.3.2 NMDA receptor antagonists Memantine is a low to moderate affinity NMDAR antagonist that was approved as a therapeutic drug in moderate to severe AD in 2003 by the 20.

(34) USA Food and Drug Administration (FDA) (van Marum, 2009). NMDAR is a glutamate-gated cation channel with high calcium permeability, and is involved in the development of the CNS, the generation of rhythms for breathing and locomotion, and the processes underlying learning, memory, and synaptic plasticity (Dingledine et al., 1999). Glutamate is the main excitant neurotransmitter in the brain released from the presynaptic terminal of neurons in the brain. Since NMDAR has the highest affinity for glutamate among the glutamate receptors, numerous studies have shown that NMDAR is the primary agent of glutamate-mediated neurotoxicity. Indeed, blockade of NMDAR activation prevents neuronal death in excitotoxicity assays (Choi et al., 1988). In addition, emerging evidence supports the role of dysregulated glutamate in the pathophysiology of neurodegenerative diseases (Hynd et al., 2004). Therefore, glutamate NMDAR has emerged as key therapeutic target for AD. Memantine uncompetitively blocks the NMDAR, and thus may have potent neuroprotective effects to prevent neuronal loss and restore the function of damaged neurons. Addition of memantine to donepezil monotherapy may be beneficial in patients with cognitively deteriorating (Atri et al., 2013). Although these drugs represent the best current available pharmacological treatments in AD, they have relatively small average overall effect and do not alter the course of neurodegenerative process. It is likely that the down-regulation of cholinergic transmission occurs 21.

(35) downstream in the pathological process, so that cholinesterase inhibitors are difficult to show overall therapeutic effects (Mufson et al., 2008). With this in mind, targeting the upstream of the pathological process has also been the therapeutic direction of great concern. For instance, neurotransmission is vulnerable to the neurotoxic effects of Aβ and hyperphosphorylated tau (Kar et al., 2002; Anggono et al., 2016). Therefore, anti-amyloid and tau targeted therapies may show more comprehensive therapeutic effects.. 1.4 Biomarkers for Alzheimer’s disease diagnosis Since current therapeutic approaches have been largely toward addressing the earliest clinical symptoms of AD, it is important to identify better biomarkers for early detection of AD with the hope that an effective therapeutic window will emerge. Recent studies have focused on detecting AD biomarkers from the genome, cerebrospinal fluid (CSF) or blood because of relatively inexpensive (compared to neuroimaging modalities) and less invasive sources (Humpel, 2011; Demarin et al., 2011).. 1.4.1 Genetic biomarkers Some researchers have investigated the genetic makeup of those with AD compared to healthy individuals. Traditionally, screening for diseaserelated genes via targeted sequencing methods, whether through Sanger 22.

(36) or next-generation sequencing (NGS) sequencing, is more commonly used than whole-exome sequencing because of lower cost and faster analysis (Foo et al., 2012). Especially through the refined NGS method, it has become a more widely used tool for genetic screening (Beck et al., 2014; Nicolas et al., 2016). It is found that FAD is associated with APP, PSEN1 and PSEN2 genes (Baird et al., 2015). Moreover, SAD is associated with other genes including apolipoprotein E-ε4 (APOE ε4), bridging integrator 1 (BIN1) region, clusterin (CLU), phosphatidylinositol clathrin assembly lymphoid-myeloid (PICALM), and complement receptor 1 (Huynh and Mohan, 2017). The identification of genetic mutations is not a certain predictor of disease or onset age, given that these mutations can vary in terms of penetrance and gene expression (Van Cauwenberghe et al., 2016). However, many studies have suggested that the benefits of disclosing Alzheimer’s diagnosis early since it allows the patient to plan for their future with better access to good medical care and support services (Alzheimer’s association, 2016). For example, APP, PSEN1 and PSEN2 tests are now recommended for patients with FAD who have at least one affected family member (Loy et al., 2014).. 1.4.2 CSF-derived biomarkers Due to the direct contact of CSF with the CNS, it can be used to assess pathological alterations occurring within the CNS (Fiandaca et al., 23.

(37) 2014). It is known that senile amyloid plaques, composed largely of Aβ peptides, accumulate in the cerebral cortex and hippocampus during the early stages of AD (Tapiola et al., 2009; Bateman et al., 2006). The main Aβ species deposited is Aβ42 because it is more hydrophobic and most prone to aggregation (Taylor et al., 2002). It is hypothesized that this accumulation of Aβ42 in the cerebral cortex is the result of overproduction of Aβ42 and/or reduced efflux of Aβ42 across the blood– brain barrier into the CSF. Indeed, post-mortem studies have shown an inverse correlation between the concentration of Aβ42 in CSF and the number of amyloid plaques (Fagan et al., 2006). A clinical study of 20,000 dementia patients and controls showed a 50% reduction in Aβ42 in the CSF of AD patients compared with controls; the diagnostic sensitivity and specificity ranged between 80% and 90% (Sjögren et al., 2001). Compared with other types of dementia, the specificity was approximately 60%. Although CSF Aβ42 is the most widely studied CSF Aβ peptide in AD, some studies have also investigated the biomarker potential of Aβ40, another component of amyloid plaque (Gravina et al., 1995). Emerging studies have also found the possibility of using the ratio of Aβ42 to Aβ40 as an AD biomarker (Dumurgier et al., 2015). Overall, the Aβ42/Aβ40 ratio and Aβ42 levels were found to have similar diagnostic accuracy in terms of discriminating AD from non-AD subjects with a sensitivity/specificity of 73/78% and 78/79%, respectively. These results 24.

(38) raise the possibility that the combination of CSF Aβ42/Aβ40 ratio and Aβ42 levels may better determine the brain amyloid production. Along with Aβ senile plaques, NFTs are also present within the hippocampus and cerebral cortex in the early stages of AD. NFTs are composed of hyperphosphorylated tau protein, and its total concentration is increased in CSF of AD patients (Humpel, 2011; Karch et al., 2012). Although the increase in CSF total tau is not specific for AD, it is indeed associated with clinical disease severity. A clinical study on AD patients and controls showed that CSF phosphorylated tau at threonine 231 correlates with neocortical neurofibrillary pathology in AD (Buerger et al., 2006). However, a second study showed no correlation between CSF phosphorylated tau at threonine 181 with neocortical neurofibrillary pathology in AD (Buerger et al., 2007). Thus various phosphorylated tau proteins might behave differently with regard to neurofibrillary pathology. Besides Aβ42 and tau proteins, proteins such as visinin-like-protein-1 (Tarawneh et al., 2011), chitinase-3 like-1 (Wennström et al., 2015), neurogranin (Kester et al., 2015), β-site amyloid precursor protein cleaving enzyme 1 (De Vos et al., 2016) and APOE ε4 (Shaw et al., 2009) have also been addressed to be used as neurochemical biomarkers in CSF.. 1.4.3 Blood-derived biomarkers 25.

(39) Since obtaining a blood sample is relatively painless and inexpensive, the potential blood-based biomarkers are more advantageous than the CSF-based biomarkers. It has been found that some blood-based biomarkers such as complement factor H (CFH) precursor and α-2macroglobulin (α-2M) appear to be just as diagnostically accurate as the CSF-based and genetic biomarkers (Hye et al., 2006). In 2014, Kiddle et al. reviewed 21 published findings or panel-based proteomics studies aimed to identify blood-based protein biomarkers in AD. They confirmed the potential of α-1-antitrypsin, α-2-macroglobulin, APOE, and complement C3 as blood protein biomarkers for AD. In addition, the predictive potential can be improved by combining certain covariates such as the age of disease onset, gender, and the presence of the APOE ε4 allele (Kiddle et al., 2014). This study indicates that combining proteomic and genomic approaches may improve predictive performance of AD biomarkers. Some studies have examined plasma Aβ levels as predictive ability of AD biomarkers. Positron emission tomography (PET) measurements of brain amyloid burden showed an association between reduced plasma Aβ42/Aβ40 ratio and increased brain amyloid load (Tzen et al., 2014; Rembach et al., 2014). In a recent study examining the levels of plasma Aβ42 and Aβ40 in 2,189 dementia-free individuals over an 8-year period, it was found that a lower plasma Aβ42/Aβ40 ratio was associated with an increased risk of developing AD (Chouraki et al., 2015). Another study 26.

(40) found that plasma Aβ42/Aβ40 ratios exhibited a sensitivity of 85.7% and specificity of 69.7% for prediction of AD among the MCI patients (Fei et al., 2011). These studies provide evidence on the diagnostic value of plasma Aβ42/Aβ40 ratio. Although the AD biomarkers currently found from the genome, CSF or blood exhibit diagnostic value, these results warrant further validation in multiple independent patient cohorts.. 1.5 Aβ42-GFP expressing cells Intracellular accumulation of Aβ plays a key role in the progression of AD and can be the potential target of AD therapy. In order to directly observe the molecular dynamics of Aβ in vivo for drug discovery research, many studies have carried out experiments using Aβ-GFP fusion protein. In the Aβ-GFP fusion construct, Aβ42 is separated from the N-terminus of GFP by a linker encoding the sequence GSAGSAAGSGEF, which effectively couples the aggregation state of Aβ with the fluorescence of GFP (Waldo et al., 1999; Wurth et al., 2002; Kim and Hecht, 2005). Misfolding and aggregation of the Aβ42 interferes with the correct folding of the GFP and thus results in a decrease of fluorescence associated with the native GFP protein. Therefore, the fluorescence of the Aβ42-GFP fusion protein depends on the folding status of the fused Aβ42. Inhibitors that delay or block Aβ42 aggregation enable GFP to fold into its native structure, and can be 27.

(41) identified by the resulting fluorescent signal (Hussein et al., 2015; Kim et al., 2006). These studies indicate that the Aβ-GFP fusion protein can be used to study the physiological function of Aβ aggregation in living cells, and to perform drug screening by analyzing the impact of Aβ induction.. 1.5.1 Inducible Aβ42-GFP 293 cells The 293 cell line (the original name designates the number of successful transformation experiments; more widely known as human embryonic kidney 293 cells, HEK-293) was derived in 1973 from human primary embryonic kidney cell culture of an aborted embryo (Graham et al., 1977). The 293 cell line and its derivatives are the most used cells after HeLa in cell biology studies and after CHO in biotechnology as a vehicle for the production of adenovirus vaccines and recombinant proteins (Lin et al., 2014; Kovesdi and Hedley, 2010). In 2015, Hussein et al. investigated the ability of DNAJB6 heat shock protein to prevent aggregation of Aβ peptides by transfecting 293 cells with Aβ-GFP fusion construct and performing western blotting and immunofluorescence techniques (Hussein et al., 2015). Moreover, in 2014, Wu et al. found that the inhibitory effect of water-soluble secondary metabolites from Phomopsis occulta on Aβ Aggregation in Aβ42-GFP expressing 293 cells (Wu et al., 2014). These studies demonstrate the ability of Aβ-GFP fusion protein as a model for Aβ peptide-mediated aggregation in HEK-293 cells. 28.

(42) Our laboratory previously established a Flp-In 293 cell line with inducible Aβ42-GFP expression by co-transfection of pcDNA5/FRT/TO/Aβ42-GFP and pOG44 (including Flp recombinase) plasmids into Flp-In T-REx-293 host cells and selection of stablytransfected cells using hygromycin and blasticidin (Huang, 2013). The Flp-In T-REx-293 host cells stably express the tetracycline repressor, and the expression of Aβ42-GFP can be induced by the addition of tetracycline or doxycycline to the cell culture medium. Therefore, the established Tet-On Aβ42-GFP 293 cells were used as a drug screening platform to examine the ability of Chinese herbal medicines and natural or synthetic compounds inhibiting Aβ aggregation.. 1.5.2 Inducible Aβ42-GFP SH-SY5Y cells To further investigate the neuroprotective effect of the selected Chinese herbal medicines and natural/synthetic compounds inhibiting Aβ aggregation, our laboratory also established a Flp-In SH-SY5Y cell line with inducible Aβ42-GFP expression. SH-SY5Y neuroblast-like cells are a subclone of the SK-N-SH neuroblastoma cell line generated in 1970 from bone marrow biopsy containing neuroblast-like and epithelioid cells (Biedler et al., 1973). SHSY5Y cells can be differentiated from a neuroblast-like state into adrenergic, cholinergic or dopaminergic neurons through a variety of different methods (Påhlman et al., 1984; Xicoy et al., 2017; Shipley et al., 29.

(43) 2016). Additionally, retinoic acid (RA)-induced differentiation of SHSY5Y cells has been used to produce neurons with dopaminergic, cholinergic and adrenergic phenotypes (Påhlman et al., 1984; Korecka et al., 2013; Presgraves et al., 2004). Alterations in dopaminergic, cholinergic or adrenergic receptors have been shown to contribute to cognitive impairments and/or deterioration in AD (Xu et al., 2012). Therefore, differentiation of SH-SY5Y cells into these neurons can be used in a multitude of neurobiology experiments to investigate the complex mechanisms and intracellular signaling pathways involved in the cognitive dysfunction associated with the AD.. 1.6 BV-2 microglia Microglia, a resident macrophage-like cell of the CNS, has a broad role in the brain’s innate immunity and in inflammatory neuropathology (Nelson et al., 2002). In AD, microglia are chronically activated and promote the release of pro-inflammatory cytokines which further disrupt normal CNS activity (Navarro et al., 2018). Therefore, it is important to study the role of activated glial cells and glial cytokines in the pathogenesis of AD. A murine cell line (BV-2) was derived from bone marrow via the vraf/v-myc recombinant murine retrovirus, which allows cells to adhere and proliferate (Blasi et al., 1985, 1990). BV-2 cells have been used to explore modulators of microglial inflammation (Lund et al., 2005). In 30.

(44) addition, BV-2 cells have similar functions to human primary microglia, as 90% of the genes induced by LPS-stimulated BV-2 cells were also induced in primary human microglia (Henn et al., 2009). The cells have also shown normal regulation of NO production and functional response to IFN-γ and LPS (Sheng et al., 2011). Conditioned medium (CM) from activated BV-2 cells has been shown to contain pro-inflammatory cytokines and neuron-damaging factors (Dai et al., 2015; Sheng et al., 2011). By co-cultured PC12 cells with BV-2 CM, LPS-stimulated BV-2 activation triggers the TLR4/MyD88/NF-κB signaling pathway, which induces IL-1β release to involve in neural injury (Dai et al., 2015). In addition, using CM from LPS-stimilated BV2, protease-activated receptor 1 (PAR-1) antagonist inhibits the death of PC12 neurons from the cytotoxicity of activated BV-2 cells by activating the PI3K/AKT pathway (Li et al., 2016b). These studies indicate that BV2 CM may be useful for studying the pathological mechanisms of neuroinflammation in AD.. 1.7 Transgenic mouse models of Alzheimer’s disease Transgenic technology exists for many organisms, including mice, rats, fish, flies and worms. Among vertebrates, mice are by far the most commonly used species and have a relatively short life span, and the techniques for performing genetic modifications in mice are well developed. Since AD has the well-recognized pathologies, such as senile 31.

(45) plaques, NFTs, etc., as well as the well-defined behavioral phenotype that can be modeled in mice, AD may be regarded as an ideal disease for modeling in transgenic animals (Elder et al., 2010). Many mouse models have successfully replicated amyloid plaque deposition, generally by deriving mice with APP overexpression along eith expression of a mutant PS1 allele to accelerate the deposition rate and exacerbate the pathological severity (Hsiao et al., 1996; Games et al., 1995). In addition, mutations in the tau gene have been found to promote the development of tauopathies in transgenic mice (Gotz et al., 2001; Higuchi et al., 2002; Lewis et al., 2001). To combine both pathological hallmarks in mouse model, multiple transgenes were introduced into the same mouse by co-injecting human APPSwe and TauP301L into single-cell embryos harvested from the homozygous PS1M146V knock-in mice, and the accompanying manifestations of plaques and tangles in the mice were found (Oddo et al., 2003). The established 3×Tg-AD mouse model is commonly used to study FAD. These mice not only show intraneuronal Aβ staining and cognitive impairment manifesting from 4 months of age, but also display extracellular Aβ plaque at 12 months and progressing with age in the cortex, hippocampus, and amygdala. However, it was not until 12-15 months of age that the changes in hyperphosphorylated and conformationally altered Tau proteins were detected in the hippocampus (Billings et al., 2005; Oddo et al., 2003). Since aberrant brain insulin signaling is associated with increased 32.

(46) accumulation of Aβ, phosphorylated Tau, reactive oxygen/nitrogen species, pro-inflammatory and pro-apoptosis molecules, and cognitive deficits (de la Monte, 2012a, 2012b, 2014; de la Monte et al., 2009), impaired insulin signaling can be seen as a key pathology in the pathogenesis of SAD (Bedse et al., 2015; Liao and Xu, 2009). Streptozotocin (STZ) is a diabetogenic compound that is often used to induce hyperglycemia in animals when administered in the periphery due to its activity of damaging the pancreatic β cells to induce insulin deficiency (Szkudelski, 2001). The intracerebroventricular injection of STZ is found to induce neuroinflammation, brain insulin resistance, cholinergic deficits, accumulation of Aβ and Tau proteins, oxidative stress as well as memory and learning impairment in animals, resembling those found in SAD patients (Salkovic-Petrisic et al., 2009). To establish a mouse model with a broader AD type, Chen et al. used STZ injection of 3×Tg-AD mice and found that mice were able to exacerbate AD-related symptoms at 6 months of age, such as tau phosphorylation, Aβ accumulation, and 20 AD-related genes expression (Chen et al., 2014b). Therefore, in my study, STZ injection of 3×Tg-AD mice was used to test the therapeutic effects of the test compound.. 1.8 Aims Previously Chinese herbal medicine (CHM) extract Glycyrrhiza inflata (G. inflata) (family FabaceaeLeguminosae) was shown to 33.

(47) effectively reduce protein misfolding and ROS production, up-regulate PGC-1α, NRF2, UPR pathways, and down-regulate pro-apoptotic genes in ATXN3/Q75 or ΔK280 tauRD cell models (Chen et al., 2014a; Chang et al., 2016). In addition, a C-alkylated indole compound NC009-1 (C19H16N2O3) (Ramesh et al., 2009) was found with protein misfoldinginhibitory and neuroprotective effects on ΔK280 tauRD cell or TBP/Q79 cell/mouse models (Chang et al., 2017; Chen et al., 2018). However, the effect of G. inflata extract and NC009-1 on Aβ-induced neurotoxicity has not been investigated. In the light of this, the potential of G. inflata and its active components and NC009-1 for the treatment of AD deserves a closer look. The aim of this thesis is devided into the following two parts: (1) G. inflata and its active components, and (2) NC009-1. The specific aims of each part are: (1) G. inflata and its active components: To investigate anti-aggregation, anti-oxidation, anti-inflammatory and neuroprotective effects using Aβ-expressing 293/SH-SY5Y cells To elucidate the underlying mechanisms of these effects 2. NC009-1: To investigate anti-aggregation, anti-oxidation and neuroprotective effects using Aβ-expressing 293/SH-SY5Y cells To identify and validate possible AD biomarkers using clinical casecontrol study, Aβ-expressing cells and AD animal model 34.

(48) Chapter 2 Materials and Methods. 35.

(49) Chapter 2: Materials and Methods. 2.1 Materials Unless otherwise stated, all the chemicals were supplied by SigmaAldrich (St. Louis, MO, USA). The ultrapure water used was prepared by Milli-Q® Reference Water Purification System (Merck Millipore, Burlington, MA, USA) and filtered or autoclaved as needed. All equipment used in Western blot was supplied by Bio-Rad (Hercules, CA, USA). The 3×Tg-AD mice were purchased from the Jackson Laboratory (004807; Bar Harbor, ME, USA). These mice were provided by Professor Hsiu Mei Hsieh-Li from Department of Life Science of NTNU.. 2.1.1 G. inflata extract and NC009 compounds Aqueous extract from G. inflata was provided by Sun-Ten Pharmaceutical Company (Taipei, Taiwan) and dissolved by ultrapure water as described (Chen et al., 2014a). Its bioactive constituents licochalcone A and liquiritigenin were dissolved in DMSO. In-house NC009 compounds were synthesized and characterized by nuclear magnetic resonance (NMR) spectrum (Ramesh et al., 2009) and dissolved in DMSO. These compounds were provided by Professor Ching-Fa Yao from Department of Chemistry of NTNU.. 36.

(50) 2.1.2 Chemicals Chemicals. Tris Base. Supplier. Chemicals. Amresco. Cyrusbioscience. NaN3. (Taipei, Taiwan). (Solon, OH, USA). Triton X-100 Glycine. Supplier. Blasticidin Hygromycin. Bionovas. InvivoGen (San Diego, CA, USA). (Taipei, Taiwan). AnaSpec. BSA. Human Aβ42. (Fremont, CA, USA) Peprotech. DTT. IFN-γ. USBiological. NJ, USA). (Swampscott, Glycerol. (Rocky Hill,. Invitrogen. MA, USA). Trizol. (Carlsbad, CA, USA). Tween 20. Calbiochem. Wako. Trypsin. (Osaka, Japan). (San Diego, CA, USA). 37.

(51) 2.1.3 Solutions . Phosphate buffered saline (PBS) 2.7 mM KCl, 137 mM NaCl, 1.4 mM KH2PO4, 9 mM Na2HPO4˙ 2H2O (pH 7.4). . Lysis buffer 50 mM Tris-HCl (pH 8.0), 150 mM NaCl, 1 mM EDTA (pH 8.0), 1 mM EGTA (pH 8.0), 0.1% (w/v) SDS, 0.5% (w/v) sodium deoxycholate, 1% (v/v) Triton X-100. . 6X Protein loading buffer 0.5 M Tris-HCl (pH6.8), 10% (w/v) SDS, 30% (v/v) Glycerol, 0.6 M DTT, 0.1% (v/v) bromophenol blue. . Running buffer 25 mM Tris base, 192 mM glycine, 3.5 mM SDS. . Transfer buffer 12 mM Tris base, 96 mM glycine, 200 mL methanol. . Wash buffer 10 mM Tris-HCl (pH 8.0), 0.05% (v/v) Tween 20, 0.02% (w/v) NaN3. 38.

(52) . Antibody diluent buffer 1% (w/v) BSA, 0.05% (v/v) Tween 20, 0.02% (w/v) NaN3 in PBS.  Aβ reaction buffer 150 mM NaCl, 20 mM Tris-HCl (pH 8.0). 2.1.4 Medium for cell culture . Tet-On Aβ-GFP 293 cells Dulbecco’s modified Eagle’s medium (DMEM, Invitrogen), 10% (v/v) fetal bovine serum (FBS, Invitrogen), 1 mM sodium pyruvate, 18 mM sodium bicarbonate, 0.1 g/mL streptomycin sulfate, 0.06 g/mL penicillin G, 5 µg/mL blasticidin, 100 µg/mL hygromycin, adjust to pH 7.4. . Tet-On Aβ-GFP SH-SY5Y cells Dulbecco’s modified Eagle’s medium:nutrient mixture F-12 (DMEM-F12, Invitrogen), 10% (v/v) FBS, 1 mM sodium pyruvate, 18 mM sodium bicarbonate, 0.1 g/mL streptomycin sulfate, 0.06 g/mL penicillin G, 5 µg/mL blasticidin, 100 µg/mL hygromycin, adjust to pH 7.4. . BV-2 cells 39.

(53) DMEM, 1% (activated stage) or 10% (resting stage) (v/v) FBS (Biological Industries, Cromwell, CT, USA), 1 mM sodium pyruvate, 44 mM sodium bicarbonate, 0.1 g/mL streptomycin sulfate, 0.06 g/mL penicillin G, adjust to pH 7.4. 2.1.5 List of primary antibodies Antigen. Supplier. Dilution. TUBB3. Covance (Princeton, NJ, USA). 1:1000. Iba1. Wako. 1:500. BCL2. Santa Cruz (Santa Cruz, CA, USA). 1:500. BAD. Santa Cruz. 1:100. CASP3. Novus Biologicals (Littleton, CO, USA). 1:500. IGFBP2. Santa Cruz. 1:200. GFP. Santa Cruz. 1:500. APOE. GeneTex (Irvive, CA, USA). 1:500. TRKA. Santa Cruz. 1:500. ERK1/2. Cell Signaling (Danvers, MA, USA). 1:500. Cell Signaling. 1:500. AKT. Abcam (Cambridge, MA, USA). 1:1000. pAKT (Ser473). Cell Signaling. 1:500. GAPDH. MDBio (Taipei, Taiwan). 1:1000. β-tubulin. Sigma-Aldrich. 1:5000. pERK1/2 (Thr202/Tyr204). 40.

(54) β-actin. Novus Biologicals. 1:5000. NeuN. Bioss (Woburn, MA, USA). 1:100. Aβ. Bioss. 1:100. Tau. Bioss. 1:100. 2.2 Methods. 2.2.1 Thioflavin T binding assay To measure Aβ aggregation, the thioflavin T assay (LeVine, 1999) was performed. Trx-His-Aβ (Huang, 2013) (10 μM final concentration) or Aβ42 (5 μM final concentration) peptide was incubated with curcumin (1–20 μM) (a compound known to bind amyloid to inhibit Aβ aggregation; Yang et al., 2005), G. inflata extract (5–500 µg/mL), licochalcone A, liquiritigenin, or NC009 compounds (5-20 μM) in Aβ reaction buffer at 37°C for 48 h to form aggregates. Following this, thioflavin T (10 μM final concentration) was added and incubated for 5 min at room temperature and fluorescence intensity of samples was recorded by using a microplate reader (Bio-Tek FLx800, Winooski, VT, USA), with excitation 420 nm and emission 485 nm filter combination. The anti-aggregation activity expressed as half maximal effective concentration (EC50) was defined as the concentration of extract/compound required for inhibition of Aβ aggregation by 50%.. 41.

(55) 2.2.2 1,1–diphenyl-2-picryl hydrazyl (DPPH) assay The free radical scavenging activities of G. inflata, licochalcone A and liquiritigenin were determined using the stable DPPH (1,1-diphenyl2-picrylhydrazyl) free radical assay (Li et al., 2008) with some modifications. Briefly, radical scavenging activity was measured in an ethanol mixture containing 100 µM DPPH radical solution by adding G. inflata extract (0.4–50 mg/mL), curcumin, licochalcone A, liquiritigenin or kaempferol (a natural flavonoid with antioxidant activity, CalderonMontano et al., 2011) (0.1–100 µM). The mixture was vortexed for 15 sec and then left to stand at room temperature for 30 min. Following this step, the scavenging capacity was measured by monitoring the decrease in absorbance at 517 nm using a spectrophotometer (MultiskanTM GO, Thermo Fisher Scientific, Waltham, MA, USA). The radical scavenging activity was calculated using the formula: 1 – (absorbance of sample/absorbance of control) × 100%. The anti-oxidative activity EC50 was defined as the concentration of the extract or compound required for inhibition of the formation of DPPH radicals by 50%.. 2.2.3 Cell culture Cells were incubated at 37°C with 5% CO2 in a humidified incubator (Nuaire, South Wales, UK). All solutions and equipment that come in contact with the cells must be sterile. To avoid bacterial and fungal contamination, all cell culture operations were performed in a laminar 42.