Protection of Okadaic Acid-induced Tau Hyperphosphorylation by Bioflavonoids in Neuroblastoma Cells

PAN, Tak Yin

A Thesis Submitted in Partial Fulfilment of the Requirement for the degree of Master of Philosophy in Biochemistry

• The Chinese University of Hong Kong

Nov 2007

• . 紅3 1 m )i| SilTY~ZW/ Ng^BRARY smmyW Thesis Committee Members:

Prof. P.C. Shaw (Chairman)

Prof. David C.C. Wan (Thesis supervisor)

Prof. K.B. Wong (Committee member)

Prof. Tsim Wah Keung (External examiner) Acknowledgments

I would like to express my sincere thank to my supervisor, Dr. C.C.Wan for his kind guidance, advice and preparation of this thesis.

Moreover, I am grateful to all people in the Department of Biochemistry, especially Dennis Ip, for their help, support and friendship.

Finally, I need to express my deepest thanks to my parents. Their understanding and encouragement have enabled me to overcome the hard time during my M.Phil, study.

i Abstract

Alzheimer's disease is a progressive neurodegenerative disease affecting large numbers of population, especially elderly. The disease is characterized by loss of neurons and two pathological hallmarks, amyloid deposits and neurofibrillary tangles (NFT). NFT are composed of abnormally hyperphosphorylated microtubule-associated proteins (MAP) tau. Normally, tau promotes the polymerization of microtubules and stabilizes neurites formation, which is phosphorylated at low level in normal adult brain. However, the imbalance of protein kinases and protein phosphatases activities contribute to the excessive tau phosphorylation and NFT formation. This may hinder microtubule assembly, disrupt axoplasmic and dendroplasmic transports, and cause neuronal death.

Therefore, reducing the level of tau phosphorylation is one of the rational strategies for prevention or treatment of the disease.

Flavonoids are polyphenolic compounds, produced widely in fruit, vegetables and nuts. Flavonoids are normally consumed in diet which is estimated from 6 to 64 mg/day.，They have been shown to have a wide range of beneficial effects to health, including maintenance of capillary wall integrity, anti-inflammatory effect and antitumor. Moreover, flavonoids have also been reported to protect neuronal death by scavenging free radical in the primary neuron from rat but many studies have shown that it is unlikely to be the sole explanation for their neuroprotective function since some molecules (e.g. Vitamin C) with high antioxidant activity can not protect neuronal death.

ii In this study, an assay based on the protection of tau hyperphosphorylation induced by okadaic acid in neuroblastoma cell line was developed. The assay is aimed to search for neuroprotective efficacies of naturally occurring flavanoids.

The differentiated human neuroblastoma SH-SY5Y was treated with okadaic acid, a protein phosphatase inhibitor, to induce the hyperphosphorylation of tau protein and cell death, which were confirmed by western blotting analysis. After characterization, the model was used to search for any flavonoids with the ability to prevent tau hyperphosphorylation and cell death.

iii 摘要

阿尔茨海默病是一种影响很多人尤其是老年人群的进行性神经系统

退化疾病。该病发生大量神经元缺失并伴有两类病理特征：淀粉样沉积和

神经纤维缠结（NFT)�神经纤维缠结由异常的过磷酸化微管相关蛋白tau

组成。正常情况下，tail促进微管形成多聚结构并稳定神经突发生，二者在

正常成人脑中磷酸化水平不高。然而，蛋白激酶和蛋白磷酸酶活化不平衡

会导致tau过度磷酸化和神经纤维缠结形成。这会阻碍微管排列，破坏轴突

和树突介质运输并引起神经元死亡。因此，降低tau磷酸化程度是预防或治

疗阿尔茨海默病的方法之一。

黄酮类和多酷类化合物在水果、蔬菜和坚果中广泛存在。黄酮类物质

在每天的饮食中约含有6到64克不等。它们对人体健康有很多好处，比如

维持血管壁完整性，抗炎症和抗肿瘤。此外，有报道发现黄酮类物质还可

以清除大鼠初级神经元的自由基从而保护神经元。但是这应该不是它们保

护神经元的唯一途径，因为很多研究表明一些具有强抗氧化活性的分子比

如维他命C就不能保护神经元。

本实验在成神经细胞瘤细胞系中对抗岡田酸诱导的tau过度磷酸化。该

实验旨在寻找自然存在的具有神经保护效用的黄酮类物质。用岡田酸，一

种蛋白磷酸酶抑制剂，处理分化的人成神经细胞瘤细胞SY-SY5Y来诱导 tau蛋白过度磷酸化和细胞死亡，并用以蛋白印迹法(Western blotting)方法证

实。之后应用该模型寻找具有抑制tau过度磷酸化和细胞死亡的黄酮类物

质。

iv Content Acknowledgements i Abstract (English) ii Abstract (Chinese) iv Content v Abbreviations x List of Figures xi List of Tables xii

Chapter 1: Introduction 1

1.1 Alzheimer's Disease 1 1.1.1 Cholinergic hypothesis 2 1.1.2 p-amyloid hypothesis 2 1.1.3 Taupathy hypothesis 3 1.1.4 Current therapies 4

1.2 Proteins Involved in Alzhemer's Disease 5 1.2.1 Acetylcholinesterase (AChE) 5 1.2.2 p-amyloid 6 1.2.3 Paired helical filaments (PHF) 7 1.2.4 Protein kinases 8 1.2.4.1 Glycogen synthase kinase-3 (GSK-3) 9 1.2.4.2 Cyclin-dependent kinase-5 (CDK-5) 9 1.2.5 Protein phosphatase (PP) 10 1.2.5.1 Protein phosphatase 1 (PP-1) 11 1.2.5.2 Protein phosphatise 2A (PP-2A) 12 1.2.5.3 Protein phosphatise 2B (PP-2B) 13 1.2.6 Apoptotic and Anti-apoptotic proteins 14 1.2.6.1 Caspase-3 15 1.2.6.2 Bcl-2 15

1.3 Flavonoids 16

V 1.3.1 Biosynthesis of flavonoids 17 1.3.2 Biological functions of flavonoids in plants 18 1.3.3 Beneficial effects of flavonoids on human health 19

Chapter 2: Materials and Methods 20

2.1 Differentiation of SHSY-5Y cells 20 2.1.1 SHSY-5Y cell culture 20 2.1.2 Counting cells 20 2.1.3 Retinoic acid differentiation 21

2.2 Western blot analysis 21 2.2.1 Extraction of proteins from mammalian cells 21 2.2.2 Sodium dodecyl sulfate polyacrylamide gel 22 electrophoresis (SDS-PAGE) 2.2.3 Semi-dry protein transfer to nitrocellulose 23 membrane 2.2.4. Membrane blocking and immunostaining 24

2.3 MTT assay 25

2.4 Hoechst 33342 Nuclei staining 25

2.5 Cell cycle analysis 25 2.5.1 Ethanol fixation 25

2.5.2 Propidium iodide staining 26

2.6 Annexin V-FITC & Propidium iodide staining 26

2.7 DNA fragmentation analysis 26

2.7.1 Phenol/Chloroform extraction of DNA 26 2.7.2 Ethanol precipitation of DNA 27 2.7.3 Agarose gel electrophoresis of DNA 27

vi 2.8 Proteomic analysis 28 2.8.1 First dimension: isoelectric focusing 28 2.8.2 Second dimension: SDS PAGE 29 2.8.3 Gel staining 30 2.8.3.1 Silver staining 30 2.8.3.2 SYBRO Ruby staining 31 2.8.4 Gel scanning and image analysis 31 2.8.5 In-gel digestion 32 2.8.6 Zip Tip for desalting the digested sample 33 2.8.7 Protein identification with mass spectrometry and 33 database search

Chapter 3: Characterization of Okadaic acid-induced 35 tau hyperphosphorylation in SHSY-5Y cells

3.1 Introduction 35

3.2 Objectives 37

3.3 Results 38 3.3.1 Differentiation of SH-SY5Y cell 38 3.3.2 Changes of protein expression after okadaic acid 40 treatment 3.3.3 Neurite Retraction Induced by okadaic acid 42 3.3.4 Okadaic acid-induced Cell Death measured by MTT 44 assay 3.3.5 Hoechst 33342 Nuclei Staining 44 3.3.6 Cell cycle analysis by propidium iodide staining 47 3.3.7 Early Apoptotic cells detection by Annexin V/PI stainii 49 3.3.8 DNA fragmentation 51

vii 3.4 Discussion 53

Chapter 4: Flavonoids screening for protecting 57 neuronal death by preventing tau hyperphosphorylation

4.1 Introduction 57

4.2 Objectives 58

4.3 Tested flavonoids 59

4.4 Results 60 4.4.1 Toxicity of flavonoids 60 4.4.2 Effects of flavonoid pre-treatment on OA-induced neu 62 retractions and cell death 4.4.3 Western blot analysis 65 4.4.4 The effect of different concentrations of hesperidin or 70 OA treatment 4.4.5 Proteomic analysis 74

4.5 Discussion 78

Chapter 5: General Discussion 82

References

viii Abbreviations

ACh Acetylcholine AChE Acetylcholinesterase AD Alzheimer's disease ALS Amyotrophic Lateral Sclerosis APP Amyloid precursor protein AP P-amyloid BCA Bicinchoninic Acid CAD Caspase-Activated DNase CDK-5 Cyclin-dependent kinase-5 CNS Central nervous system DMEM/F-12 Dulbecco ’ s modified Eagle, s medium- F-12 DMSO Dimethyl sulfoxide EGCG Pigallocatechin gallate FBS Fetal bovine serum GSK-3 Glycogen synthase kinase-3 HD Huntington's diseases lAA lodoacetimdie ICAD Inhibitor of Caspase-Activated DNase lEF Isoelectric electrophoresis focusing IPG strips Immobiline™ DryStrips with pH 3-10 MAP Microtubule-associated protein MAPK Mitogen-activated protein kinase NFT Neurofibrillary tangle NFTs Neurofibrillary tangles OA Okadaic acid OD Optical density PD Parkinson disease PHF Paired helical filaments

ix PI Propidium iodide Pin 1 Peptidyl-prolyl isomerase A PKs Protein kinases PP Protein phosphatase PS Posphatidylserine PSl Presenilin 1 PS2 Presenilin 2 RA Retinoic acid ROS Reactive oxygen species SDS-PAGE Sodium dodecyl sulfate polyacrylamide gel electrophoresis STS Staurosporine VOL Spot volume %VOL Relative volume 2-DE Two dimensional electrophresis

V List of figures

Figure 1.1 Diagrammatic representation of the phenylpropanoid metabolic 17 pathway. Source from: (Winkel-Shirley, 2001) Figure 3.1 The human neuroblastoma SH-SY5Y cells were differentiated 39 into neuron like cells with more neurite outgrowth. Figure 3.2 Changes of protein expression after okadaic acid treatment. 41 Figure 3.3 Neurite retraction induced by okadaic acid in a dose dependent 43 manner. Figure 3.4 Cytotoxic effect of Okadaic acid in differentiaed SH-SY 5Y 45 cells. Figure 3.5 Increase of fragmented and condensed nuclei by Okadaic acid in 46 a dose dependent manner. Figure 3.6 Flow cytometry analysis of Okadaic acid-induced apoptosis 48 using propidium iodide (PI) staining. Figure 3.7 Flow cytometry analysis of early apoptotic cells after OA 50 treatment. Figure 3.8 DNA fragmentation in Differentiated SH-SY5Y cells treated 52 with Okadaic acid. Figure 4.1 Cytotoxicity test of the flavonoids on differentiated SH-SY5Y 61 cells. Figure 4.2 Changes of cell morphology after flavonoids and OA treatments. 63 Figure 4.3 Cell viability test after flavonoids and OA treatments. 64 Figure 4.4 Hyperphosphorylation tau protein level after flavonoids and 66 okadaic acid treatments. Figure 4.5 Pro and active forms of caspase 3 protein levels after flavonoids 67 and okadaic acid treatments. Figure 4.6 BCL 2 protein level after flavonoids and okadaic acid 68 treatments. Figure 4.7 Change of neurodegenerative markers after pretreatment of 71 different concentrations of Hesperidin and Hesperetin, and Okadaic acid treatments. Figure 4.8 Flow cytometry analysis of early apoptotic cells after different 72

xi concentrations of hesperidin and OA treatments. Figure 4.9 2-DE pattern of 50nM okadaic acid treatment on differentiated 75 SH-SY5Y cells. Figure 4.10 2-DE pattern of 5\iM hesperidin treatment followed with 50nM 76 okadaic acid treatment on differentiated SH-SY5Y cells. Figure 5.1 Cis-trans isomerization of peptidyl-prolyl bonds catalysed by Pin 86

1

xii List of tables

Table 2.1 Components of lysis buffer for protein extraction 21 Table 2.2 Components of SDS-PAGE running gel and staking gel 23 Table 2.3 The components of lysis buffer of protein extraction used in 2-DE 28 Table 2.4 Components of second dimension SDS-PAGE running gel 30 Table 3.1 Percentage of cells in different cell phases after okadaic acid 48 treatment. Table 3.2 Percentage of cells in different cell stages after okadaic acid 50 treatment. Table 4.1 Summary of the changes of protein levels after flavonoids and 69 okadaic acid treatments. Table 4.2 Percentage of cells in different cell stages after okadaic acid 73 treatment. Table 4.3 Differential expressed proteins in 50nM okadaic acid treatment 77 and hesperidin treatment followed with 50nM okadaic acid treatment.

xiii Chapter 1 Introduction

1.1 Alzheimer's disease

Alzheimer's disease (AD) is the most common neurodegenerative disease that usually affects people over age 65. It is an irreversible and age-related disorder, which causes the patients with AD losing their memory and cognitive ability gradually, and having difficulties in movement and a dramatic change in personality. At the last stage of AD, patients will lose the capacity to take care of themselves and are dependent on other people. In many developed countries, AD is a major health-care problem that puts a heavy economic burden on society because the cost of hospitalization and nursing care is increasing due to the rise in the population over the age 65 and the increase of the AD patients (National

Institute on Aging, 2000).

With the intensive study of AD for over three decades, the mechanism of the pathogenesis of the disease is beginning to be understood. Two distinct hallmarks of this disease were discovered: the deposition of P-amyloid (Ap) plaques and neurofibrillary tangle (NFT) in brain (Alzheimer, 1907). According to these two hallmarks, two hypotheses were proposed. The first hypothesis is known as beta- amyloid hypothesis. In this hypothesis, it states that Ap plaque is formed due to the aggregation of large amount of Ap protein and other molecules, e.g. apolipoprotein, ubiquitin. The major component, Ap protein, is resulting from the cleavage of a plasma membrane protein known as amyloid precursor protein

(APP) (Selkoe, 1994). The other hypothesis is taupathy hypothesis which explains that the neurofibrillary tangle is formed by the aggregation of paired helical filament (PHF) that is composed of hyperphosphorylated tau protein (Lee et al”

1991). Both of these depositions may cause neuronal death and neurodegeneration.

1 Apart from the two hypotheses, many mechanisms have also been proposed to explain the pathology of AD. One of the hypotheses, cholinergic hypothesis, suggested that the loss in cholinergic function was thought to be responsible for the symptom of AD. However, there is still no definite treatment that can stop or reverse the neurodegenerative process.

1.1.1 Cholinergic hypothesis

The cholinergic hypothesis was initially presented over 20 years ago and suggests that a dysfunction of acetylcholine containing neurons in the brain contributes to the cognitive decline observed in those with advanced age and Alzheimer's disease (Terry and Buccafusco, 2003). This premise has since served as the basis for the majority of treatment strategies and drug development approaches for AD to date (Giacobini, 1991).

1.1.2 p-amyloid hypothesis

The p-amyloid hypothesis suggests that accumulation of Ap plaques in the brain and the associated signaling response may contribute to the cause of AD. This hypothesis is established mainly by the study of mutations in familial patients.

The mutation genes including APP, presenilin 1 (PSl) and presenilin 2 (PS2) were found to be related to the onset of familial AD. Moreover, the cloning of the gene encoding the APP and its localization to chromosome 21 suggested that trisomy 21 (Downs syndrome) leads invariably to the pathology of AD (Lemere

et al., 1996). There are at least three enzymes including a-secretase, p-secretase

and y-secretase, involving in the cleavage of APP to produce secreted form of

amyloid protein. However, the mutations promote the generation of A|3 by

2 favoring proteolytic processing of APP by P-secretase and y-secretase.

Furthermore, APP mutations inside the A|3 sequence promote self-aggregation of

AP. There are two major forms of Ap: Ap40 and Ap42, which have 40 and 42 amino acid residues respectively. Although Ap42 is the minor product, it plays an important role in AD pathogenesis because Ap42 is easy to aggregate into plaques than Ap 40 (Jarrette et al., 1993). The Ap plaques formation can recruit reactive glial cells which produce pro-inflammatory cytokines for inducing inflammatory response. The response generates excessive reactive oxygen species, damaging proteins, lipids and the cells. Moreover, the plaque accumulation can alter ionic homeostasis, particularly calcium ions. The excessive calcium entry eventually causes cell death (Mattson et d., 1992).

1.1.3 Taupathy hypothesis

Paired helical filaments (PHF) are the major building blocks of neurofibrillary tangles. They are composed of hyperphosphorylated tau protein. Tau protein isolated from normal adult brain contains 2-3 moles of phosphate per mole of tau protein while from the brain of AD adult; it contains 5-12 moles (Selden and

Pollard, 1983). Tau is predominantly expressed in axons, which is a group of microtubule-associated protein that normally promotes and stabilizes the assembly of microtubules (Hong et aL, 1997). In adult human brain, there are six isoforms of tau ranging from 352 to 441 amino acids. All six isoforms are phosphoproteins (Wood et aL, 1986). About half of the known phosphorylation sites in tau are serine or threonine residues followed by a proline. Paired helical filament-tau differs from normal tau by its abnormal hyperphosphorylation, which reduces the binding affinity of tau to microtubule. It has been shown that

3 hyperphosphorylation tau does not bind to microtubules unless it is enzymatically dephosphorylated (Sontag et al, 1999). This reduced amount of normal tau may lead to the destabilization of microtubules and result in impairment of axonal transport, neuronal degeneration, and the aggregation of paired helical filaments.

Therefore the hyperphosphorylation of tau is believed to be a key event in the pathogenesis of Alzheimer's disease (Lee et al” 1991).

The phosphorylation of tau is regulated by kinases and phosphatases. Normal tau can be phosphorylated by many kinases, such as glycogen synthase kinase

(GSK)-3a and -3p, cyclin-dependent kinases 5 and mitogen-activated protein kinase (MAPK). The phosporylation state of a protein can also be regulated by protein phosphatase. If the the expression level or the activity of protein phosphatase reduce, tau phosphorylation will increase due to the lack of dephosphorylation by phosphatase. The expression level of two phosphatases, phosphatase 2A and phosphatase 2B, were shown to be reduced in AD brain than normal brain (Gong et al., 1995). This links to the tauopathy hypothesis suggesting that hyperphosphorylation of tau is a key event for the pathology of

AD.

1.1.4 Current therapies

There is currently no effective treatment for Alzheimer's disease (National

Institute on Aging, 2006). Currently available medications only offer symptomatic benefit for some patients but do not slow the progression of the disease. Acetylcholine esterase inhibitors are the only group of medicine being approved by FDA for symptomatic treatment of AD (Enz and Gentsch, 2004).

These drugs do not slow down the disease progression, but they can temporarily

4 stabilize cognitive impairment and maintain body muscle function, often delay the needs for patient placement in nursing homes by several months (Terry and

Buccafiisco, 2003). Discovery of the mutation genes in early onset AD is critical in exploring the role of amyloid plaques in neuronal cell death, and designing mechanism-based drugs. These include inhibitors for A(3-generating proteases, agents for preventing AP oligomerization and immunotherapies to reduce Ap in brain. Discovery of neurofibrillary tangles formed by aggregation of hyperphosphorylated tau proteins in AD brain has led to strategies for identifying selective inhibitors of tau kinases, modulators of phosphatase and brain-permeable drugs that help maintain microtubule integrity (Michaelis, 2002).

1.2 Proteins involved in Alzhemer's disease

1.2.1 Acetylcholinesterase (AChE)

Acetylcholinesterase is the main enzyme involved in cholinergic neurotransmission. It can break down the acetylcholine (ACh), which is a neurotransmitter synthesized in certain neurons by the enzyme choline acetyltransferase, and allow control of muscle contraction (Ellis, 2005). Cognitive deterioration in AD is related to progressive loss of cholinergic neurons and a decline in levels of acetylcholine in the brain, particularly in the temporal and parietal neocortex and hippocampus. Inhibiting the activity of acetylcholinesterase can increase the level of acetylcholine in synapses and enhance the cholinergic function which provides the symptomatic improvements in treated with cholinesterase inhibitors. Several acetylcholinesterase inhibitors have been approved by FDA for symptomatic treatment of AD, including

Donepezil and galantamine (reversible AChE inhibitors), and rivastigmine (a

5 slowly reversible inhibitor) (Ellis, 2005). It has been reported that AChE is co- localized with amyloid plaque and forms a stable complex with the plaque.

Moreover, the neurotoxicity of the plaque is increased by the presence of AChE

(reviewed by Talesa, 2001).

1.2.2 p-amyloid

The amyloid precursor portein is a transmembrane glycoprotein that is proteolytically processed by two competing pathways, the non-amyloidogenic and amyloidogenic (beta amyloid forming) pathways. Three major secretases are involved in the proteolytic cleavage of APP. These include a-secretase, beta APP cleaving enzyme (BACE, formally known as p-secretase) and the y-secretase. The a-secretase cleaves within the Ap domain of APP generating non-amyloidogenic fragments and a secreted form of APP (a-APPs). In the amyloidogenic pathway,

BACE cleaves near the N-terminus of the Ap domain on the APP molecule, generating another soluble form of APP, p-APPs and a C-terminal fragment (C99) containing the whole Ap domain. The final step in the amyloidogenic pathway is the intramembranous cleavage of the C99 fragment by y-secretase, to form the A(3 peptide (reviewed in Verdile, 2004). Excessive production of P-amyloid promotes the formation of neurotoxic amyloid plaque, which can induce neuronal cell death

and recruit reactive glial cells producing pro-inflammatory cytokines. The

inflammatory response generates excessive reactive oxygen species, damaging

proteins, lipids and the cells (Mattson et a\., 1992).

6 1.2.3 Paired helical filaments (PHF)

Microtubules are required for the formation of the neuronal cytoskeleton and axonal transport. Alzheimer's disease (AD) is characterized by existence of paired helical filaments (PHF) in certain selected brain neurons, especially in hippocampus. PHF exist as neurofibrillary tangles in neuronal cell bodies, as neuropil threads in the dystrophic neurites of affected neurons, and in degenerating neurites surrounding the extracellular amyloid in senile plaques.

Microtubule-associated protein tau is the major protein subunit of PHF (Grundke-

Iqbal et al, 1986). Tau in AD brain is abnormally phosphorylated. The abnormally phosphorylated tau from AD brain contains 5-9 mol of phosphate/mol of the protein, which is about three to four times the level of phosphate in normal brain tau (Kopke et al, 1993). PHF-tau is phosphorylated at multiple sites (Iqbal et al., 1989). There are 21 abnormal phosphorylation sites of PHF-tau (all Ser/Thr sites) identified either by phosphorylation-dependent (Kanemaru et al, 1992) or by mass spectrometry (Hasegawa et al., 1992). Most of the phosphates are clustered at two sites, one amino-terminal (Ser-198 to Thr-217) and one carboxyl- terminal (Ser-396 to Ser-422) to the microtubule-binding repeat domains (Gln-

244 to Gly-367). As a result of the abnormal hyperphosphorylation, the molecular weight of PHF-tau on SDS-polyacrylamide gels is higher than that of normal tau

(Lee et al., 1991). Furthermore, PHF-tau does not promote the assembly of microtubule unless it is dephosphorylated before its interaction with microtubule

(Iqbal et al,, 1994).

7 1.2.4 Protein kinases

Protein phosphorylation is believed to be one of the major mechanisms for regulation of cellular function. Tau is a phosphoprotein which was discovered and best known for promoting the assembly of microtubules (Weingarten et al., 1965).

The interaction of tau with other brain proteins appears to be regulated by phosphorylation. The in vitro phosphorylation of tau has been reported to inhibit both the polymerization of tubulin (Lindwall and Cole, 1984) and the interaction of microtubules with actin filaments (Seldon and Pollard, 1983). The abnormal phosphorylation of tau in neurons in AD might take place over a long period of time. The neuronal microtubule-associated protein tau is a target of phosphorylations on a large number of sites, and a substrate for several kinases according to the in vitro studies. Some of these are proline-directed kinases, e.g,

MAP-kinase, GSK3b and Cdk5, while other enzymes including MARK kinase, protein kinase A and others are nonproline directed kinases (Grundke-Iqbal et al,

1984). Studies have claimed that some of tau phosphorylating kinases, such as

GSK-3 beta, MAP-kinase, microtubule-affinity regulating kinase MARK and

Cdk5 phospohorylate tau at sites that are elevated in Alzheimer's paired helical filaments (PHF). Hyperphosphorylations could lead to structural alteration in tau, and decrease in its interaction with microtubules (Binder et al, 1985). It is possible that these kinases could be sensitive to changes in their regulatory patterns or at the structural level, thus participating in the molecular pathway leading to neurodegeneration.

8 1.2.4.1 Glycogen synthase kinase-3 (GSK-3)

Glycogen synthase kinase 3 (GSK-3), a multifunctional serine/threonine kinase, was initially found to be able to phosphorylate and inactivate glycogen synthase which is the final enzyme in glycogen biosynthesis (Hughes et al, 1993). The inactive glycogen synthase then results in dramatic reduction of glycogen synthesis. GSK-3 is also a key regulator of numerous signaling pathways� including cellular response to Wnt, receptor tyrosine kinase and G-protein- coupled receptors and is involved in many cellular processes, including cell cycle regulation, cell proliferation, microtubule dynamics and protein synthesis

(Prickaerts et al, 2006). GSK-3 is unusual in that it is normally active in cells and is primarily regulated through inhibition of its activity. However, if the pathways in which GSK-3 acts as a key regulator is dysregulated, it will lead to development of human disease such as bipolar disorder, diabetes, cancer and AD

(Doble and Woodgett, 2003). V

1.2.4.2 Cyclin-dependent kinase-5 (CDK-5)

Cdk5 is a Ser/Thr protein kinase with postmitotic activity that phosphorylates

KSP protein motifs on MAP lb protein, KSPXK protein motifs on tau (Kobayashi

et al, 1993) or KSPXX on neurofilament proteins, actinbinding protein

caldesmon and proteins of the synaptic vesicles (Sun et al, 1996, Shetty et al,

1993). This kinase was identified and characterized as a brain proline-directed

kinase (Lew et al, 1992). Cdk5 was shown as an active enzyme in postmitotic

cells such as neurons, and as a kinase that phosphorylates tau protein (Paudel et

al” 1993). After these discoveries, protein responsible for activation of brain

Cdk5 was found. The Cdk5/p35 system participates in normal development of

9 neuronal cells and the axonal growth (Paglini et aL, 1998). Therefore, Cdk5 is treated as one of the important kinases phosphorylating tau in neuronal cells.

Cdk5 was identified on the basis of biochemical similarities to Cdc2 protein kinase, a regulator of mitosis. Unlike Cdc2, Cdk5 is not found in dividing cells, and exhibits a preferential distribution in brain differentiating neurons (Kobayashi,

1993). The formation of a stable Cdk5/p35 complex following phosphorylation of

Cdk5 in hippocampal neurons has been postulated as a factor responsible for constitutive activation of this protein kinase. As a consequent� iincreaset s in phosphorylation at sites that have been found in tau pathology (Alvarez, 1999).

1.2.5 Protein phosphatase (PP)

In contrast to the protein kinases that have been discovered, relatively few protein

phosphatases are known (Andreassen et aL, 1998). In this regard protein

phosphatases were often considered as the poorer cousins of kinases. However, it

is acknowledged that the regulation of protein phosphorylation requires the

control of both kinases and phosphatases and that the regulation of phosphatases

is as complex as that of kinases. In many ways, the phosphorylation-

dephosphorylation cycle can be regarded as a molecular "on-off switch although

this is probably an over simplification of the processes involved (Ling et aL,

2004). The end result of these associated pathways is the regulation of many

different cellular processes, i.e. glycogen metabolism, calcium transport, muscle

contraction, gene expression, protein synthesis, intracellular transport,

phototransduction, cell cycle progression and apoptosis (McCluskey and Sakoff,

2001).

10 Several phosphatases like protein phosphatase 1 (PPl), PP2A, PP2B, and PP2C have been implicated in dephosphorylation of tau. However, only PPl, PP2A, and

PP2B have been shown to dephosphorylate abnormally hyperphosphorylated tau

(Gong et al, 1994). Although PP2C can dephosphorylate tau when it is phosphorylated by PKA in vitro, it is not capable of dephosphorylating the abnormally hyperphosphorylated tau isolated from AD brain tissue (Gong et al,

1994). It seems that PP2A is the phosphatase that acts on most phosphorylation sites of tau (Goedert et al, 1995) PP2A is composed of three subunits, A, B, and

C, and the B subunits are mainly involved in substrate recognition. PP2A binds to tau through its tubulin binding region (Goedert et al, 2000). Mutations in this region could decrease the capacity ofPP2A to bind to tau and produce an increase in tau phosphorylation.

1.2.5.1 Protein phosphatase 1 (PP-1)

Protein phosphatase 1 (PPl) is enzyme involved in a wide array of physiological

processes, including glycogen metabolism, muscle contraction, and gene

expression. The role of PPl in these diverse aspects of cellular physiology has

been difficult to understand the philosophy behind by using available inhibitors

because of a lack of pharmacological specificity (Allen et al., 2004). One

approach to identifying specific targets for the enzyme is elaborating on the

control mechanisms that direct PPl activity toward specific substrates. This

strategy leads to characterization of multiple regulatory subunits, and many of

these proteins are shown to either promote, or to inhibit, PPl activity toward

substrates in specific settings (Cohen, 2002).

11 In the nervous system, PPl plays a role in the regulation of synaptic plasticity by controlling activity of a broad range of ion channels and signal transduction enzymes (Greengard et ai, 1999 and Lisman and Zhabotinsky, 2001). As in other tissues, the PPl activity toward its various targets in brain likely depends on association with different regulatory subunits. Some of the neuronal regulatory subunits have been well characterized. Within the basal ganglia and cortical regions, multiple neurotransmitters act through convergent biochemical pathways to control PPl activity by the regulatory molecule, dopamine- and cAMP- regulated phosphoprotein-32 kDa (DARPP-32) (Greengard et al, 1999). A protein structurally related to DARPP-32, inhibitor-1, has been implicated in the

PPl inhibition required for the induction of longterm potentiation at hippocampal synapses (Allen et al., 2000).

1.2.5.2 Protein phosphatase 2A (PP-2A)

PP2A is involved in a broad range of cellular processes, including membrane receptor desensitization, signal transduction, transcriptional regulation and control of DNA replication, and mitosis. This diversity of PP2A function is due to a diversity of targeting/regulatory subunits and several levels of post-translational modification (McCright et al, 1996). PP2A exists in the cell as a heterotrimeric complex consisting of a 36-kDa catalytic C subunit, a 65-kDa structural/regulatory A subunit, and a variable regulatory B subunit. The A and C subunits are each encoded by two highly related (85 and 97% identity, respectively) and widely expressed genes. The variable B subunits identified to date are encoded by three unrelated gene families (Veerle and Jozef, 2001). The

most prevalent isoform, named variously PR55, B55, or simply B, is encoded by

12 at least three genes with several splice variants (DePaoli-Roach et cd., 1994). The

PR72/130 regulatory subunit is encoded by a single gene that generates two splice forms that vary in the size of their amino terminus (Hendrix et al, 1993).

1.2.5.3 Protein phosphatise 2B (PP-2B)

PP2B (calcineurin, Ca^^ / calmodulin dependent protein phosphatase) is a heterodimer with A and B subunits that share some overlapping substrate specificity with both PPl and PP2A (Wera and Hemmings, 1995). PP2B A subunit, a 60kDa catalytic subunit, has a 40% homology with the catalytic subunits of PPl and PPA, but contains an additional 170 C-terminal amino acids which is the calmodulin binding domain. The B subunit has a molecular weight of

19kDa with four Ca^"^-binding sites. PP2B is modulated by Ca^^, calmodulin, and possibly by FK506-binding proteins and the cyclophilins (Griffiths et al., 1995).

PP2B shows relatively low activity in the heart, spleen, liver and testes but is expressed highly in brain tissue, particularly in the striatum and hippocampus and localised along dendritic spines. This is consistent with its proposed role in memory by modulating long term potentiation and long term depression of

synaptic efficacy (Mitsuhashi et al, 2000). Over expression of PP2B in transgenic

mice produced deficits in long-term memory and a constraint on long term

potentiation. Other neurological studies have shown PP2B to be highly expressed

in areas of the brain, which are vulnerable to stroke, epilepsy and

neurodegenerative diseases. It has been suggested that high level activity of PP2B

predisposes neuronal cells to apoptotic cell death and that PP2B inhibitors reduce

such susceptibility (Asai et aL, 1999). Some of the neurological and physiological

13 and abnormalities of Downs syndrome may also be attributed to PP2B inhibition

(Fuentes et al, 2000).

1.2.6 Apoptotic and Anti-apoptotic proteins

Neurodegenerative diseases are characterized by dysfunction and death of specific neuronal cells and the presence of intracellular and extracellular aggregates of soluble proteins, a common feature in several disorders of the central nervous system (CNS), such as AD, Parkinson disease (PD) and Huntington's diseases

(HD) and Amyotrophic Lateral Sclerosis (ALS) (Selkoe, 2004).

Neuronal death can occur through necrosis or apoptosis, both processes involving mitochondrial function (Ankarcrona et al., 1995). Necrosis is a rapid type of death resulting from an acute event that leads to complete failure of mitochondrial function and cellular homeostasis. Apoptosis is, instead, a slow and active process triggered by less severe stimulates and this might be due to the mechanisms for neurons to delay death, e.g. various neuroprotective programs activated involving

molecules and genes with anti-apoptotic function (Mattson, 2000). For the

apoptotic process, it is characterized by cell shrinkage, plasma membrane

blebbing, nuclear chromatin condensation, DNA fragmentation and increase of a

number of apoptotic markers, e.g. caspase and Bel protein families. Moreover,

mitochondria play a pivotal role in cell decision, which is supposed to depend on

a balance between apoptotic and anti-apoptotic signals (Lilia and Maria, 2006).

In fact, the initial trigger induces some toxic events and/or synergizes with other

apoptotic cascades, thus causing a persistent imbalance of neuronal homeostasis,

mitochondrial dysfunction and neuronal death.

14 1.2.6.1 Caspase-3

Caspases are a group of cysteine proteases that cleave proteins after aspartic acid residues (Alnemri et al., 1996). To date, there are 14 known caspases that are divided into two main classes: initiators (or activators) and executioners (or effectors). Caspases exist as immature pro-caspases that must first be cleaved in order to become active. It has long been understood that caspases are essential proteins for the induction of apoptosis (Cohen, 1997).

Caspase-3 is considered as the primary executioner caspase because it cleaves other important proteins to induce apoptosis. Caspase-3 induces apoptosis by: cleaving Inhibitor of Caspase-Activated DNase (ICAD)，which then allows

Caspase-Activated DNase (CAD) to cleave DNA into fragments; cleaves nuclear laminins, which allows the nucleus to condense; and it cleaves PAK-2, which plays a role in membrane blebbing; among other things. DNA fragmentation, nuclear condensation, and membrane blebbing are all hallmarks of apoptosis

(Wolfe/ a/., 1999).

Caspase-3 has been extensively studied with respects to its role in apoptosis. It has been shown that active caspase-3 is expressed in neurons, astrocytes, and oligodendrocytes (Mouser et al., 2006). It has been showed that active caspase-3 is present in the brain during the early stages of development, but present in

limited amounts in postmitotic neurons in later stages of development (Lesuisse

and Lee, 2002).

1.2.6.2 Bcl-2

The Bcl-2 protein family has an important role in intracellular apoptotic signal

transduction. This family includes both anti-apoptotic and pro-apoptotic proteins

that contain one or more Bcl-2 homology (BH) domains (Merry and Korsmeyer,

15 1997). The major anti-apoptotic members of the Bcl-2 family, Bcl-2 and Bcl-x, are localized to the mitochondrial outer membrane and to the endoplasmic reticulum and perinuclear membrane. Some studies have shown that Bcl-2 can support the survival of neurons in the absence of NGF and the overexpression of

Bcl-2 can override the death signal induced by the withdrawal of a trophic factor

(Garcia et al.’ 1992). Moreover, transgenic mice expressing Bcl-2 in the nervous system has found to be protected against neuronal cell death during development

(Martinou et al.’ 1994).

The expression of Bcl-2 is high in the central nervous system during development and is down regulated after birth; however the expression of Bcl-2 in the peripheral nervous system is maintained in whole life. Although the development of the nervous system in Bcl-2-knockout mice is normal, there is a subsequent loss of motor, sensory and sympathetic neurons after birth, suggesting that Bcl-2 is crucial for the maintenance of neuronal survival (Veis et al, 1993).

1.3 Flavonoids The flavonoids are a group of polyphenolic compounds widely distributed in the plant kingdom. More than 6400 known flavonoid compounds have been discovered. Flavonoids contribute to the flavor and pigment of the fruits and vegetables and they also have important roles in plant growth, reproduction, and pathogen resistance (Harbome and Williams, 1992). They have a Ce-Cs-Ce flavone skeleton in which the three-carbon bridge between the phenyl groups is commonly cyclized with oxygen. Flavonoids can be divided in several classes based on the degree of unsaturation and oxidation of the three-carbon segment

(Peluso, 2006). Usually, they are present in plants either as aglycones or as

16 glycoside conjugates including glucose, arabinose, glucorhamnose, galactose, and lignin (Ross and kasum, 2002). The contents of flavonoids in common foods may be obtained from United States Department of Agriculture databases.

1.3.1 Biosynthesis of flavonoids

Flavonoids are synthesized by the phenylpropanoid metabolic pathway in which phenylalanine is used to produce 4-coumaroyl-CoA. Then it will be combined with malonyl-CoA to produce the true backbone of flavonoids, i.e. chalcones, which contain two phenyl rings. Conjugate ring-closure of chalcones results in the three-ringed structure of a flavone. The metabolic pathway continues through a series of enzymatic modifications to yield flavanones 一 dihydroflavonols — anthocyanins (Fig.l). Moreover, this pathway can also produce many other flavonoids, including the flavonols, flavan-3-ols and proanthocyanidins (Winkel-

Shirley, 2001).

Phenylalanine I PAL Cinnamic acid I C4H 丁AL Tyrosine • 4-Coumarie acid j 4CL 4-Coumaryl-CoA Malonyl CoA I CHS Tetrahydroxy-chalcone I CHI Flavanone I F3H + F3’H/F3’5’H

Dihydroflavonols I DFR Flavan-3,4-diols j ANS Anthocyanidins I

Anthocyanins

Fig. 1.1: Diagrammatic representation of the phenylpropanoid metabolic pathway. Source from: (Winkel-Shirley, 2001)

17 1.3.2 Biological functions of flavonoids in plants

An important role of flavonoids is to serve as visual signals for animals in attracting pollinators in flowers, and later for animals eating the fruits and helping in seed dispersal. In fruits, flavonoids may also contribute as color, flavor or texture to fruit quality. They are also involved in the formation of undesirable brown colour in fruits after cutting and bruising (Amiot et a!., 1997).

The composition of flavonoids varies greatly in different fruit species (Roberts and Antolovich, 1997). Anthocyanins are pigments that contribute red, violet and blue color to fruits. The main anthocyanins in fruits are glycosides of six anthocyanidins. The other predominant flavonoid group in fruits is proanthocyanidins and their monomer units, catechins or gallocatechins, which are the natural substrates of polyphenol oxidases and are involved in the browning phenomenon of fruits.

In addition to acting as pigments in fruits and flowers, flavonoids are involved in many biological functions in plants. They play an important role in the plant- microbe interaction and in sexual reproduction by promoting the pollen tube development (Koes et aL, 1994). Flavonoids also have apparent roles in plant stress defence, such as protection against damage caused by attack of pathogen and UV-light. Flavonoids, mainly anthocyanins are responsible for the colour changes of plants during autumn, which have been suggested to protect leaf cells from photo-oxidative damage (Feild et aL, 2001).

Flavonoids are strongly UV-absorbing compounds, and accumulate mainly in the epidermal cells of plant tissues after production caused by UV-induction. The epidermal layers of plants can absorb 90-99% of the UV radiation and serve as shields against this harmful radiation (Robberecht and Caldwell, 1983).

18 1.3.3 Beneficial effects of flavonoids on human health Flavonoids are polyphenolic compounds which are rich in many foods in our diet.

For example, tea, coffee, chocolate, wine, and beer have high amount of polyphenols contributing to the in vitro antioxidant capacity. Flavonoids have been investigated actively and they have possible beneficial influence on human health. Flavonoids have been found to have potent antioxidant and free radical scavenging activities in vitro (Halliwell, 2007). Moreover, there is growing evidence from human consumption studies supporting a protective role of flavonoids in cardiovascular diseases and cancer. Nevertheless, many flavonoids have been found to possess antiviral, antibacterial, antifungal or anti-allergenic properties. However, their possible interactions with other substances and their metabolism in the human system are still under detail investigation.

19 Chapter 2: Materials and Methods

2.1 Differentiation of SHSY-5Y cells

2.1.1 SHSY-5Y cell culture

SH-SY5Y human neuroblastoma cell was maintained at 37°C in the presence of

5% CO2 in Dulbecco's modified Eagle's medium- F-12 (DMEM/F-12) (Gibco), supplemented with 10% fetal bovine serum (FBS), 1 OOU/ml penicillin and 100|j.g

/ml streptomycin (Invitrogen). For every 3-4 days, the old medium was dispensed and the cells were washed with phosphate buffered saline (PBS). Trypsin/EDTA solution (Gibco) was added to the dish and the cells were agitated to make them slough off from the surface of the plate. After 2 minutes of incubation in 5% CO2 incubator at 37°C�1m olf DMEM / F12 medium (Gibco) was then added to stop the trypsin reaction. The suspension was transferred into a snap-cap tube and centrifuged at ISOOrpm for 4 minutes at room temperature. The pellet was re- suspended in DMEM / F12 medium (Gibco). After counting the cells, a 2 x 10^ cells suspension was added into new culture dish with 10ml DMEM / F12 medium with 10% FBS (Gibco).

2.1.2 Counting cells

The cells were collected by adding trypsin/EDTA solution and centrifuged at

1 SOOrpm for 4 minutes. The pellet was re-suspended in 1ml DMEM / F12 medium (Gibco). 10|j.l of the re-suspended cells were mixed with 10 |j,1 of 1%

Trypan Blue solution. The solution was pipetting up and down and 10 [x\ of the stained cells were filled into the grip of a hemacytometer. The cells were counted

20 within four comers of counting range and the central counting range. The density of cells was calculated by the following equation:

D = (N-4)x2x 10^

(D = Cell density in cells/ml; N= Total no. of cells counted; Dilution factor = 2.)

2.1.3 Retinoic acid differentiation

The cells were seeded in 60mm and 100 mm culture dishes. The seeding density of SH-SY5Y cells was 1.2x10^ cells/ml and the volumes of medium for 60mm and 100mm culture dishes were 10 ml and 3 ml respectively. From day 1 after plating, cells were differentiated in the presence of 10 iiM all-trans-retinoic acid

(RA) (Sigma) for five days. Cells were maintained at 37°C in an atmosphere containing 95% air and 5% CO2.

2.2 Western blot analysis

2.2.1 Extraction of protein from mammalian cells

The SDS Lysis buffer was prepared freshly on the date of experiment. The components are shown in the following table:

ImM Sodium Orthovanadate 100 pi

ImM NaF

IX Protease inhibitor 80 \x\

10/0 SDS 180 ^il

H2O Add to 2000 yd

Table 2.1: Components of lysis buffer for protein extraction

The culture medium was removed and the plates were washed twice with IX PBS.

For 6-well plate, 200 jil freshly prepared lysis buffer was added to each well to

21 lyse the cells. For 100mm plate, 500 |il freshly prepared lysis buffer was added to lyse the cells. The lysed cell samples were collected into individual Eppendorf tubes. The samples were then vortexed at 4°C for 10 minutes and centrifuged at

HOOOrpm at 4°C for 10 minutes. The supernatant which contained the lysed proteins were transferred to new Eppendorf tubes.

2.2.2 Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE)

The Mini-Protein II electrophoresis cell (Bio-Rad) was used for SDS-PAGE. The gel-mould was assembled according to the manufacturer's instruction. Before adding gel solution, small amount of water was filled into the gel-mould to check for leaking. The 12% running gel solution was firstly prepared as shown in Table

2.2. The running gel solution was poured into the gel-mould to 3/5 full. An amount of 150 [i\ of propanol was added on the top of the running gel solution surface to remove air bubbles and to keep the solution away from air. The gel was allowed to polymerize for 15-30 minutes at room temperature. The 4% stacking gel solution was prepared as shown in Table 2.2. After the stacking gel was poured into the remaining space, a comb was inserted into the layer of stacking gel solution, allowed the stacking gel to polymerize for 15-30 minutes at room temperature. The components of SDS-PAGE running gel and staking gel are shown in the following table:

22 12% Running gel Volume 4% Stacking gel Volume

30% acrylamide solution 4 ml 30% acrylamide solution 1.3 ml

1.5MTris-HCL, pH 8.8 2.5 ml 1.0 M Tris-HCL�p H6.8 2.5 ml~

10% SDS 100 [i\ 10% SDS 100

Distilled H2O 3.4 ml Distilled H2O 6.1 ml

10% APS 10% APS 5o7a

TEMED IM TEMED

Table 2.2: Components of SDS-PAGE running gel and staking gel

The samples were denatured by adding loading dye and boiling at 100°C. The denatured samples and protein marker were loaded into the wells of the prepared

SDS PAGE gel. The gel was run at a constant voltage of lOOV until the dye front reached appropriate distance.

2.2.3 Semi-dry protein transfer to nitrocellulose membrane

After performed SDS-PAGE, the gel was removed from the glass cassette set and removed away any stacking gel. The gel and nitrocellulose membrane were immersed in transfer buffer for 5 minutes, six pieces of filter paper were soaked into the transfer buffer for 30 seconds. In assembling the transfer stack, three pieces of wet filter papers was firstly placed on the cathode electrode plate, then the gel and nitrocellulose membrane was added respectively. Another three pieces of wet filter paper was placed on the top of the membrane. Finally, the anode electrode plate was placed on the top of the stack. The transfer stack was run at

15V for 1.5 hours.

23 2.2.4. Membrane blocking and immunostaining

After the proteins were transferred on the membrane, the membrane was washed in transfer buffer for 5 minutes. For Western Blotting analysis, the membrane was soaked into 10 ml blocking solution (IX TBS-T, 5% dry milk) 1 hour/ovemight, primary antibodies at 1:5000, 1:10000 dilution were added and incubated for 1 hour. Afterwards, the membrane was then incubated with secondary antibodies

(HRP anti-mouse, anti-rabbit antibodies) at 1:5000 dilutions in 10 ml TBS-T with

5% dry milk for 1 hour. Finally, the membrane was washed for three times with

IX TBST for 5 minutes.

The immunoreactive protein bands in the blot were visualized by the ECL detection kit (Amersham). For signal detection, the solution 1 of the kit was mixed with solution 2 of the kit. The solution was added to the membrane that was placed on a piece of gradwrap. After 1-minute incubation, the excess solution was removed by blotting paper. The membrane was then covered with another piece of plastic Gradwrap and then placed in the X-ray film cassette with a X-ray film on the top. The film was usually exposed to the membrane for different time periods. If necessary, the membrane could be stripped and re-probed with other antibodies.

The probes bound to the nitrocellulose membrane could be stripped off by incubating with strip buffer at 60 °C. The membrane is then blocked by 10 ml blocking solution (IX TBS-T, 5% dry milk) 1 hour/ovemight and other antibodies the stripped membrane is ready for probing with other antibodies.

24 2.3 MTT assay

The SHSY-5Y cells were seeded in 96-well tissue culture plate. The number of the cells for each well was 3-5x1 O^ cells. To avoid evaporation, only central part of the plate (60 internal wells) was used. After differentiation, the cells were undergone drug treatment. After treatment, MTT solution (50 1 mg/ml) was added to each well and incubated for 3-4 hours. The formazan crystals were extracted from the cells with 150|al DMSO in each well. The colour was measured with 96-well ELISA plate reader at 550-570 run.

2.4 Hoechst 33342 Nuclei staining

The cells were seeded in 6-well plate with the seeding density 1.2x10^ cells/ml and the volume of medium for 6-well plate was 3ml for each well. After treatment, the old medium was discarded and the cells were washed with PBS twice. 0.5 ml

PBS with lO^M Hoechst 33342 (Molecular probe) was added to the cells and the cells were incubated 15 min in the dark. The PBS with Hoechst 33342 was discarded and the cells were washed with PBS for one time. The stained nuclei were observed using the fluorescence microscope.

2.5 Cell cycle analysis

2.5.1 Ethanol fixation

Adherent cells were trypsinized and collected from 60mm culture dishes. After centrifugation and washes with PBS twice at 4 the cells then finally collected by centrifugation. The cell pellet was then re-suspended in 1 ml ice cold 70% ethanol for overnight fixation at -20

25 2.5.2 Propidium iodide staining

After fixation, the cells were then centrifuged and resuspended in 1 ml PBS. l|al

10 mg/ml RNase (invitrogen) and 20 |LI1 Img/ml propidium iodide were added to the cells incubated at 37 for 30 mins. The DNA contents of the cells were determined in term of fluorescence intensity using FACSort flow cytometer

(Becton Dickinson). Data obtained were analyzed using WinMDI 2.7 software

(http://facs.scripps.edu，J. Trotter).

2.6 Annexin V-FITC & Propidium iodide staining

The adherent cells were trypsinized and collected from 60mm culture dishes.

After centrifugion and washes with PBS twice at 4 the cells were then centrifuged to collect the cell pellet. The cell pellet was then re-suspended in IX binding buffer at 1 x 10^ cells/ml. An aliquot of 100 \x\ of the re-suspended cells

(1 X 10^ cells) was transferred to a 5 ml culture tube. An amount of 5 \x\ 0.2|ag/|il annexin V-FITC (Santa cruz) and 10 |il 1 mg/ml propidum iodide (PI, Invitrogen) were added to the cells and incubated for 15 mins at room temperature in the dark.

An amount of 400 [i\ of Ix binding buffer was added to the tube and the cells were analyzed in term of fluorescence intensity using FACSort flow cytometer

(Becton Dickinson) within 1 hour.

2.7 DNA fragmentation analysis

2.7.1 Phenol/Chloroform extraction of DNA

The cells were washed by PBS and lysed with the lysis buffer containing Tris-

HCL, SDS, EDTA and protease K and incubated at 37 overnight or 45� Cfor

2hours. An aliquot of 0.5 ml lysate was added to 1ml

26 phenol/chloroform/isopropanol mixture with a ratio of 25:24:1 by volume. The mixture was then mixed rapidly by vortexing and shaking at room temp for 10 mins. After centrifugation at 12000rpm for 2 mins, the DNA containing aqueous layer was collected and transferred to a new tube and then ready for ethanol precipitation.

2.7.2 Ethanol precipitation of DNA

Two volumes of -20 absolute ethanol were added to the aqueous solution containing DNA. 50 [x\ 3M sodium acetate was added to the solution. The mixture was incubated at -20 °C overnight or -70 °C for 30 mins. Then the precipitated

DNA was collected by centrifugation at 12000rpm for 10 mins at 4 The DNA pellet was washed by 70% ethanol at room temp. The DNA pellet was dried by air and dissolved in TE buffer.

2.7.3 Agarose gel electrophoresis of DNA

The isolated DNA was separated by 1.5% TBE agarose gel. The DNA sample was mixed with 6x gel loading buffer. After the gel was formed, it was put into the gel tank with TBE buffer just covering the gels. Then, the DNA samples were

loaded into the gel and the electrophoresis was operated at constant V, 90V until

the bromophenol blue dye front was migrated to desired distance. The gel can be

visualized under UV illamination.

27 2.8 Proteomic analysis

2.8.1 First dimension: isoelectric focusing

The lysis buffer of protein extraction used in two dimensional electrophresis (2-DE) was different from western blotting. The components are shown in the following table:

8M Urea 19.2 g

4% CHAPS

2% Pharmalyte (pH 3-10) 800 \i\

Double distilled H2O To 40 ml

Table 2.3: The components of lysis buffer of protein extraction used in 2-DE

The protein extraction followed the steps in 2.3.1. A commercial available

13cm Immobiline™ DryStrips with pH 3-10 (IPG strips) (Amersham Biosciences)

were used for first dimension isoelectric focusing. For 13cm long of IPG strip,

250 |j.l rehydration buffer with 1.25 jal IPG buffer (Amersham Biosciences) and

50 |il protein sample (40 |ag protein) were mixed and slowly added to the centre

point in the slot of the IPGphor strip holder (Amersham Biosciences). Bubbles

were removed in the solution mixture. The protective cover from the IPG strip

was removed from the acidic (pointed) end. The IPG strip was positioned with the

gel side down and was lowered onto the solution. After the IPG strip was placed

in the IPGphor strip holder, the strip was overlaid with DryStrip Cover fluid.

Then, the cover was placed on the strip holder to ensure that the IPG strip

maintains good contact with the electrodes as the gel swells. The strip holder was

properly positioned on the IPGphor platform. The pointed end of the strip holder

was over the anode and the blunt end was over the cathode. The safety lid of the

28 platform was closed. Two of the three pressure pads under the safety lid pressed gently against the cover of strip holder to ensure contact between the electrodes and the electrode areas. lEF was performed using the following parameters: 0-

500V for 1 hour, 500-1000V for 1 hour, 1000-3000V for 1 hour, 3000-5000V for

1 hour, 5000-8000V for 1 hour, and 8000V for 2 hours. After running the lEF, the

strip was taken out and the cover fluid remained on the strip was removed by

blotting on a piece of Scott blotting paper. Then, the strip was equilibrated in the

equilibration solution (composition???) with DTT for 15 minutes. The strip was

further equilibrated in the equilibration solution with iodoacetimdie (lAA) for 15

minutes.

2.8.2 Second dimension; SDS PAGE

The Hoefer SE 600 vertical electrophoresis system (Amersham Pharmacia

biotech) was used for second dimension SDS-PAGE. The gel-mould was

assembled according to the manufacturer's instruction. Before adding gel solution,

small amount of water was filled into the gel-mould to check for leaking. The

12% running gel solution was firstly prepared as shown in Table 2.4. The running

gel solution was poured into the gel-mould nearly full remaining small space for

IPG strip. An amount of 300 \x\ of butanol was added on the top of the running gel

solution surface to remove air bubbles and to keep the solution away from air.

The gel was allowed to polymerize for 15-30 minutes at room temperature. The

components of second dimension SDS-PAGE running gel is shown in the

following table:

29 12% Running gel Volume

30% acrylamide solution 16 ml

1.5MTris-HCL, pH 8.8 ~

10% SDS 400 111~

Distilled H2O 13.6 ml

10% APS 200 III~

TEMED 407ii

Table 2.4: Components of second dimension SDS-PAGE running gel

The equilibrated strip was placed into the top of prepared second dimension SDS-

PAGE running gel. Protein marker was added to the left of the gel. Then agarose sealing solution was added to the remaining space. The gel was run at a constant voltage of 1OOV until the dye front reached appropriate distance.

2.8.3 Gel staining

2.8.3.1 Silver staining

Silver staining kit from Amersham Biosciences was used for detection of proteins on the 2-DE gel. For fixing the gel, 100ml of silver staining fixing solution was added to a glass box with the gel inside for 2hours or overnight. After fixing, the fixing solution was removed and 100ml of sensitizing solution was added to the box for sensitizing the gel. After that, the sensitizing solution was removed and the gel was washed with 100ml distilled water for 5 minutes three times. Then,

100ml silver solution was added to the gel for 30 minutes. Again, the gel was washed with 100ml distilled water for 1 minutes 2 times. 100ml of developing solution was added to the gel and removed until the appearance of most protein spots. To stop the reaction, 100ml stopping solution was added to the box for 10

30 minutes. The gel was preserved in 100 ml preserving solution for gel scanning and protein spots were excised from the gel for further analysis.

2.8.3.2 SYBRO Ruby staining

SYBRO ruby staining from Molecular Probes was the fluorescence dye used for

detection of proteins on the 2-DE gel. For fixing the gel, 100ml of fixing solution

was added to a glass/plastic box with the gel inside for 2 hours or overnight. After

fixing, the fixing solution was removed and 100 ml of SYBRO Ruby dye was

added to the box for staining overnight. After that, the staining solution was

removed and the gel was washed with 100ml distilled water for 30 minutes twice.

2.8.4 Gel scanning and image analysis

The silver-stained 2-DE gels were digitalized using a flatbed scanner at 300dpi

(Amersham Bioscience) while the SYBRO Ruby-stained 2-DE gels were scanned

by Typhoon laser scanner (Amersham Bioscience). The image data were analyzed

by Image master 2D platinum Software (Amersham Bioscience). In order to

eliminate the individual variations on 2-DE patterns, three spots pairs appearing

in same positions on two gels were picked up, yielding landmarks for the gels.

Then the gels were overlapped according to the positions of the landmarks. Spots

on each gel were paired according to their positions. Spot volume (VOL) was

defined as the integration of optical density (OD) over the spot's area, while

relative volume (%VOL) of a given spot was the ratio of VOL to total VOL over

the whole image. Therefore, the effect of the fluctuation in the gel staining density

can be minimized by comparing the %VOL of each spot. The %VOL of the spots

31 was analyzed to detect specific spots showing significant differences between control and after drug treatment.

2.8.5 In-gel digestion

Protein spots were excised and cut into smaller pieces with surgical needle and placed into Eppendrof tubes. For silver stained-protein spots, 10 pi Potassium

Ferricyanide (30 mM) and 10 |il Sodium thiosulphate (lOOmM) were added to each Eppendrof tubes. The Eppendrof tubes were vortexed until the dark stain was removed and the protein spots were washed with autoclaved nano-water. For those SYBRO Ruby-stained protein spots, the de-staining step could be omitted.

Then, the protein spots were equilibrated in 20 jil Ammonium bicarbonate

(200mM) for 10 minutes and then washed with nano-pure water. The washing and equilibration procedures were repeated once. After equilibration, the protein spots were dehydrated with 100 jal acetonitrile for 10 minutes for three times. Then, the protein spots were dried in the speed vacuum for 2-5 minutes until the protein spots became 'dust like'. The protein spots were rehydrated with 10 jil trypsin enzyme in buffer (40ng/|jl trypsin in 50mM Ammonium bicarbonate) and incubated on ice for 30 minutes. After that, enough buffer solution (50mM

Ammonium bicarbonate) was added to cover the hydrated protein spots at 30 overnight. After digestion, the proteins in the protein spots were extracted by adding 20 |j.l 50mM Ammonium bicarbonate to the Eppendrof tubes and the protein digests were sonicated for 10 minutes. The protein spots were spun down and the supematants were transferred to new Eppendrof tubes. An amount of 10

Acetonitrite and 10 \i\ 5% TFA was added to the Eppendrof tubes and the

protein spots was sonicated for 10 minutes. Again, the supematants were

32 transferred to the new Eppendrof tubes. This step was repeated three times. Lastly,

10 pi Acetonitrile was added. After sonication, the supernatant was transferred to the tubes again. The supernatant extract was dried with speedy vacuum to obtain the protein sample.

2.8.6 Zip Tip for desalting the digested sample

TheZipTipClS microcolumn (Millipore) was used to desalt and concentrate the protein samples. The vacuum-dried protein samples were resuspened in 10 |il

0.1% TFA. The ZipTip was wet by aspirated 10 |il 50% acetonitrite into the tip

and dispensed to waste two times. An amount of 10 [ol equilibrium solution (0.1%

TFA) was aspirated into the tip and dispensed to waste two times. The sample

was bound into the ZipTip by aspirated and dispensed 10 cycles in the Eppendrof

tube containing resuspened protein sample. The ZipTip with the protein sample

bound was washed by 10 |il 0.1% TFA by aspirated and dispensed into waste 5

times. The protein sample was eluted with 2 |j.l 50% acetonitrite / 0.1 % TFA in a

new 0.5ml tube by aspirated and dispensed 5 times.

2.8.7 Protein identification with mass spectrometry and database search

The MALDI-TOF mass spectrometry was performed on ABI4700 proteomic

Analyzer equipped with 200Hz solid-state laser (Applied Biosystems). The

MALDI-TOF mass spectrometer was operated in positive-ion, delayed extraction

(150ns delayed time), and reflector mode. Trypsin autocleavage peaks were used

as internal standard for mass calibration. The mass spectrometry peaks were

analyzed with Data Explore software that is based on Mascot search engine to

33 identify the proteins. The masses of all the tryptic peptides were queried on the

SwissProt and NCBInr protein databases.

34 Chapter 3: Characterization of Okadaic acid-induced tau hyperphosphorylation in SH-SY5Y cells

3.1 Introduction

Alzheimer's disease (AD) is characterized by neuronal cell loss and several pathologic hallmarks, e.g. amyloid plaques deposits and neurofibrillary tangles (NFT) (Vickers et al., 2000). It has been shown that the amount of NFT correlates closely with the degree of dementia NFT are composed of abnormally hyperphosphorylated microtubule- associated protein (MAP) tau (Arriagada et al., 1992). Tau, mainly expressing in neuronal axons, is phosphorylated at very low level in normal adult brain

Hyperphosphorylated tau fails to bind microtubules, leading to disassembly of microtubules, disruption of axonal transport, and ultimately leading to neurodegeneration (Iqbal et al., 2000).

Regulation of tau phosphorylation by protein kinases (PKs) and protein phosphatases

(PPs) maintains dynamic balance of phosphorylation state of tau under normal physiological conditions. Normal tau can be phosphorylated by many kinases, such as glycogen synthase kinase (GSK)-3a and -3p，cyclin-dependent kinases 5 and mitogen- activated protein kinase (MAPK). The tau dephosporylation is regulated by protein phosphatise, such as phosphatase 2A and phosphatase 2B. Thus, the dephosphorylation

activity of PP is necessary for keeping tau at low phosphorylated level and with normal

biological functions (Kins et al., 2001). The PP activity has been reported to be reduced

by about 30% in AD brain compared to control (Gong et aL, 1995). This implies that

the defect of PP activity is closely related to the abnormally hyperphosphorylation of

35 tau which may then contribute to the disassembly of microtubules, disruption of axonal transport and neurodegeneration.

Okadaic acid is a complex polyether derivative of a 38-carbon fatty acid. It is synthesized by marine dinoflagellates and accumulates in filter feeding organisms such as shellfish or the black sponge (Tachibana et al., 1981). Okadaic acid is harmful to human health due to its ability to cause diarrhetic shellfish poisoning. It can inhibit certain members of the serine/threonine protein phosphatase family, including PPl,

PP2A and PP2B by binding to the catalytic subunit for enzymatic activity inhibition

(Bialojan and Takai, 1988). Because of its inhibition of phosphatase activity, okadaic acid quickly became a tool to investigate the cellular functions of the respective okadaic acid-sensitive phosphatases (Schonthal, 1992).

36 3.2 Objectives

Since abnormal hyperphosphorylation of the microtubule-associated protein tau is a

key pathologic molecular change correlated to the neurodegeneration in Alzheimer's

disease brain, thus this study is aimed at establishing an okadaic acid-induced apoptosis

cell line model that can be used to search for any potential flavonoids against tau

hyperphosphorylation and cell death. Before flavonoids screening, the cell model was

characterized to examine whether it can mimic some molecular or morphological

changes in Alzheimer's disease affected neurons. The molecular changes included

phosphorylated tau protein level, apoptotic and anti-apoptotic protein level e.g. caspase

3 and Bcl-2. And also the morphological change was observed, e.g. neurite retraction

and nucleus condensation. Moreover, the apoptosis and cell death after OA treatment

was also measured by using flow cytometry.

37 3.3 Results

3.3.1 Differentiation of SH-SY5Y cell

SH-SY5Y is a human neuroblastoma cell line with two cell phenotypes which are neuroblastic phenotype (N—type) and substrate adherent phenotypes (S-type).After the retinoic acid treatment, the N-type cells will become differentiated and the S-type cell will undergo apoptosis. Undifferentiated SH-SY5Y cells exhibited roundish features initially (Figure 3.1 A). Treatment with 10|aM RA for 5 days induced neuronal differentiation resulting in cells that acquired a more neuron-like phenotype and signs of neurite outgrowth (Figure 3.IB).

38 IHBi

Fig. 3.1: The human neuroblastoma SH-SY5Y cells were differentiated into neuron like cells with more neurite outgrowth. (A) Undifferentiated SHSY- 5Y cells exhibited roundish shapes with few neurite outgrowth. (B) Retinoic acid induced neuronal differentiation with the loss of round morphology and signs of neurites outgrowth. Cells were visualized using phase contrast microscopy (200X). Bar = 25 |im.

39 3.3.2 Changes of protein expression after okadaic acid treatment

After differentiation, the cells exhibited an extensive neurite outgrowth. These cells were incubated with DMSO (control), lOnM, 25nM, 50nM, lOOnM okadaic acid (OA) and 0.5|aM staurosporine (STS) for 18 hours. For detecting tau phosphorylation, PHF

ATS antibody was used. This antibody recognizes paired helical filament with phosphorylated tau at site Ser202 which is one of the dephosphorylation site regulated by protein phosphatase 2A. Since it can recognize different tau isomers, several bands were detected due to the different sizes of the isomers. After okadaic acid treatment, tau phosphorylation increased in dose-dependent manner and there was no tau phosphorylation after treatment of another apoptosis inducer-staurosporine (Fig. 3.2A).

Caspase-3 is an apoptotic enzyme, which is first synthesized as inactive pro-enzyme with the size 35-kDa. After activated by the cleavage of caspase-8 or caspase 9, the active fragment of caspase 3 with the sizes 17-kDa can cleave other protein substrates within the cell, resulting in the apoptotic process�e.g DN. A ladder formation. After 18 hours OA treatment, both the expression of pro and active forms of caspase 3 increased with the OA concentration. There was also increase of pro and active forms of caspase

3 after staurosporine treatment (Fig. 3.2B). Bcl-2 is an anti-apoptotic enzyme with the

size 25-kDa, which governs mitochondrial membrane permeabilisation. It can inhibit

the release of cytochrome C to activate caspase cascade. The western blot showed that

the Bcl-2 protein level increased after okadaic acid treatment and peaked at lOOnM OA

treatment (Fig. 3.2C).

40 A B

��‘… n •K..M 10nHH M.. 25noc„iiiMi 50nM100ncn«MHnn«niMi Control O.SijMIOn^^^ M 25nM 50nM lOOnM STS OA OA OA OA STS OA OA OA OA PHF-AT8 “~ -SIP^ 1 一 , Pro- CapaseS 78~150KDa _ _ 35KDa

GAPDH —-- - 17KDa'' 37 Kn= ^.^^^^^mmmtrnK^jmam-jtmrnimmm^^ 5 37KDa

1=3 PHF-AT8 levels Pro Caspase 3 levels 2.0 - 丁 ^4.00 • I 1 Active Caspase 3 levels I I

11.5- r^ i ^ 3.00 - T "§ T CO O •_ T n 0)：^ T • -h

^ CO w ro „„ • if" n^ It iLiir 1 I 。，JI jJlll 0.0 J “ 1 >——1 1——I 1 I 1——I 0.00 J•~I•I~•I•I~••~I™_I c 0.5MM10nM 25nM 50nM lOOnM Control STS OA OA OA OA BCL-2 -W—- •‘ 丨I" “丨 —— ”丨•丨丨丨丨丨••• mmmrn- 25KDa .. GAPDH 37KDa 5 4. 1=1 Bel 2 levels

•E 二 3 r-U

巧 T Si- ^ r^ o:i ^ in 1 • —

0-1 — — — —

Fig. 3.2: Changes of protein expression after okadaic acid treatment. Western blots of SH-SY5Y cell extracts prepared from the cells that had been treated with different concentrations of okadaic acid (OA) and 0.5|iM Staurosporine (STS) for 18 hours were labeled with antibodies to (A) PHF-AT8 tau, (B) Pro- and Active- form of caspase 3，(C) Bcl-2. GAPDH immunoblot was used as constitutive protein control to normal protein loading in each sample. The results were repeated with two independent treatments (n=3).

41 3.3.3 Neurite Retraction Induced by okadaic acid

Since hyperhosphorylated tau fails to bind microtubules, the microtubules will be destabilized and disassembled, thus neurite retraction of the neurons will be observed.

After okadaic acid and staurosporine treatments, the morphologies of SHSY-5Y were shown as Figure 3.3. When the concentration of okadaic acid increased, more cells suffered from neurite retraction and became roundish shapes. At 50nM OA treatment

(Fig. 3.3D), most cells exhibited roundish shapes with few neurite outgrowths and cell death occurred obviously at lOOnM OA treatment (Fig.3.3E). However, compared to another cell death inducer, staurosporine (STS) induced cell death of the cell but the neurite of the cells remained without retraction (Fig.3.3F).

42 A. Control (DMSO) B. 10nM OA

C. 25nM OA D. 50nM OA HHHHSI •• •E. lOOnM OA •F. 0.5pM ST S

Fig. 3.3: Neurite retraction induced by okadaic acid in a dose dependent manner. Differentiated Cells were incubated with different concentrations of (A-E) okadaic acid (OA) and (F) 0.5iaM Staurosporine (STS) for 18 hours. Cells were visualized using phase contrast microscopy (lOOX). Bar = 50|am.

43 3.3.4 Okadaic acid-induced Cell Death measured by MTT assay

The cell death induced by okadaic acid was measured by the MTT assay. The SHSY-

5Y cells were seeded in 96-well culture plate at a density of 4x10"^ cells for each well and then differentiated by lO^iM retinoic acid. Cells were then treated with various (10-

100 nM) concentration of okadaic acid for 18 hours and the MTT assay was performed to measure the cell viability. Figure 3.4A showed that survival percentage greatly decreased to 60% at 50nM okadaic acid and dropped to 11% at lOOnM while there was less cytotoxic effect at lower okadaic acid concentrations. The cell death induced by okadaic acid at different time periods was also investigated. Figure 3.4B showed that survival percentage greatly decreased to 58% at 18 hours treatment and dropped to

13% at 24 hours treatment while there was less cytotoxic effect at 0, 2, 4, 6 hours treatments.

3.3.5 Hoechst 33342 Nuclei Staining

Hoechst 33342 is a fluoresence dye that can bind preferentially to A-T base-pairs of

DNA. When the cell undergoes apoptosis, it will have condensed and fragment nuclei, with higher fluoresence intensity compared to the normal cells. Figure 3.5 showed the

Hoechst 33342 nuclei staining after okadaic and staurosporine treatment. In control, there were few numbers of apoptotic nuclei showing bright blue fluoresence and most cells were attached on the culture plate (Fig.3.5A). However, the number of apoptotic nuclei increased with the OA concentrations and more cells were detached from the plate (Fig.3.5 B-E).

44 A 140 1

g 120 -

f 8� - \ I 60. \ 16 40- \ I 20 • \ CO 0 -

1 1 1 1 1 “ 0 10 25 50 100 B [Okadaic Acid] nM

120 n

g 100 -

I 60_ \

ro 40 - \

I 20- \

0 I I I I I I Ohr 2 hrs 6 hrs 8 hrs 18 hrs24 hrs

50nM Okadaic Acid Treatment

Fig. 3.4; Cytotoxic effect of Okadaic acid in differentiaed SH-SY5Y cells. (A) Cells were incubated with different concentrations of Okadaic Acid for 18 hours. Cell death was quantified by MTT assay measuring the mitochondria activity. (B) Cells were incubated with 50 nM Okadaic Acid in different time periods. Cell death was quantified by MTT assay. Data were mean 土 SEM (bars) values of three independent experiments

45 •A. Control (DMSO) •B. 10nM OA •C. 25nM OA •D. 50nM OA E. lOOnM OA F. 0.5|jM STS I^HB

Fig.3.5: Increase of fragmented and condensed nuclei by Okadaic acid in a dose dependent manner. Differentiated Cells were incubated with different concentrations of okadaic acid (OA) and 0.5|iM Staurosporine (STS) for 18 hours, then the cells were stained with Hoechst 33342 dye and examined by fluorescence microscope (lOOx) for morphological change. Bar = 50|a,m.

46 3.3.6 Cell cycle analysis by propidium iodide staining

To investigate the apoptotic effect of okadaic acid, cell cycle analysis by flow cytometry was studied. Propidium iodide was used as the fluorescence dye for the analysis. PI can bind to the major groove of double strand DNA. The intensity of the fluorescence can reflect the DNA content of cells. During apoptosis, the genomic DNA is cleaved by endonuclease to form DNA fragments. After fixation, the DNA fragments may leak out due to permeabilisation of cell membrane and the cells contain less DNA content and thus fluorescence intensity of the cells decreases. Figure 3.6 showed the histograms representing the number of events (cells) against PI intensity-area (i.e.

DNA contents) after okadaic acid treatment. Table 3.1 summarized the percentage of cells with different DNA contents representing different cell cycle stages of the cells.

Okadaic acid increased apoptotic cells in a dose dependent manner. The apoptotic sub

G1 cells that were sub-diploid rose from 3.4% (Control: DMSO) to 18% (lOOnM OA).

However, the diploid GO/Gl cells decreased from 90% (Control: DMSO) to 80%

(lOOnM OA). For S and G2/M cells, they decreased from 5.41% (Control: DMSO) to

2.38-0/0 (lOOnM OA).

47 A. Con (DMSO) B. 25nM OA Go Gi

Go,Gi

Subcl S,G./M M1Q i10- 10- • ro' 10- I ？0' FL2-A FL2-A C. SOnM OA ^D. lOOnM OA

. r r ” Go，GI I Cji I j

Sub^i^^ S, G2/M^ Sub Gil S，GjM

� _ n I •• , Ma^li^^^ ..... 10= ？0' 10' 10' FL2-A FL2-A

Fig. 3.6: Flow cytometry analysis of Okadaic acid-induced apoptosis using propidium iodide (PI) staining. Cells were incubated with different concentrations of Okadaic Acid for 18 hours and stained with propidium iodide. After staining, the DNA contents of cell populations were analyzed by flow cytometer.

Apoptotic Sub Gi Go, Gi S, G2/M

Con (DMSO) 3.42% 90.82% 5.41%

25nM OA 5.04% 91.58% 3.31%

SOnM OA 12.53% 83.46% 4.36%

lOOnM OA 18.07% 80.12% 2.38%

Table 3.1: Percentage of cells in different cell phases after okadaic acid treatment. The percentages of cells were calculated by the size of areas at different phases in the PI intensity-area histogram plot.

48 3.3.7 Early Apoptotic cells detection by Annexin V/PI staining

For annexin V/PI staining, it can monitor the early stage of apoptosis. When the cell begins apoptosis, the membrane protein: phosphatidylserine (PS) will translocate from inner to outer membrane and become available for annexin V. However, if the cell is at late apoptosis or necrosis, apart from annexin binding, the PI can also penetrate into the cell and bind to nuclei acid.

Figure 3.7 showed that after OA treatment, the normal cells shifted from left bottom region (I) to right bottom region II (Pl-negative/Annexin-V positive) and right upper region III (Pl-positive/Annexin-V positive). The percentage of normal cells decreased from 90% (Control: DMSO) to 58% (lOOnM) while the apoptotic cells increased from

3.7% (Control: DMSO) to 32% (lOOnM). The number of late and necrotic cells slightly increased from 4.75% (Control: DMSO) to 8.67% (lOOnM).

49 ^ A. Con (DMSO) ^B. 10nM OA ^ C. 25nM OA 1 n.’: Ill 1 .Ill 1 m

[：象 10" 10' 10= 10) 10* 10� 10' 10- 10) 1 10� 10' 10- 10' 10* FU-H FL1-H Empty

^D. 50nM OA ^ E. lOOnM OA 1 ； :iii 1 xm

10。 10' 10= 10' 10' 0° 10' 10: 10' 10' Empty Empty

Fig. 3.7: Flow cytometry analysis of early apoptotic cells after OA treatment. Cells were incubated with different concentrations of Okadaic Acid for 18 hours and stained with Annexin V with propidium iodide. After staining, the cell populations were analyzed by flow cytometer. Data from 10,000 cells were analyzed for the experiment. Region I: Pl-negative/annexinV-negative, Region II: Pl-negative/ annexinV-positive, III: Pl-positive/ annexin V-positive

I. Normal II. Apoptotic III. Necrotic

Con (DMSO) 90.06% 3.75% 4.75%

lOnM OA 78.95% 15.67% 5.85%

25nM OA 88.16% 4.59% 3.87%

50nM OA 90.06% 6.12% 3.08%

lOOnM OA 58.15% 32.85% 8.67%

Table 3.2: Percentage of cells in different cell stages after okadaic acid treatment. The cells were quantifed as the percentage of the total cell population at different regions.

50 3.3.8 DNA fragmentation

At the late stage of apoptosis, the inhibitor of caspase-activated DNase (ICAD) will be cleaved by Caspase-3 to become caspase-activated DNase (CAD), which can cleave the genomic DNA into about 200 base pairs DNA fragments. Figure 3.8 showed that the DNA from the differentiated SH-SY5Y cells treated with lOOnM OA resulted in a ladder-like pattern of DNA fragmentation with characteristic fragments separated in size by approximately 200 base pair while the Control, lOnM OA, 25nM OA, 50nM

OA treatments did not showed DNA fragmentation. For 0.1 jxM staurosporine and

O.SjiM staurosporine treatment, they represented the positive controls of this experiment, which showed a clear DNA ladder pattern.

51 // 均,/ /•人cT//身 ////德身々 r 1 •IIIjji

Fig. 3.8: DNA fragmentation in Differentiated SH-SY5Y cells treated with Okadaic acid. DNA was extracted from DMSO- or OA-treated cells and subjected to agarose gel electrophoresis. DNA ladder formation was observed in 100 nM OA treatment for 18 hours. The cells treated with Staurosporine (STS) served as positive control.

52 3.4 Discussion

Neurodegeneration characterized by accumulation of hyperphosphorylated tau and formation of neurofibrillary tangles (NFTs) is a hallmark in Alzheimer's disease (AD)

(Grundke-Iqbal et al., 1986). Studies on AD brains have demonstrated that abnormally hyperphosphorylated tau is the major protein subunit of NFT (Lee et al, 1991), which suggests that hyperphosphorylation of tau may play a role in leading the neuronal cells to undergo apoptosis. Tau is a microtubule-associated protein, which the major function of tau is to promote microtubule assembly and stabilize microtubules formation. The accumulation of tau hyperphosphorylation in the development of neurofibrillary degeneration was observed in the AD brains (Grundke-Iqbal et al., 1986).

It has been shown that during Alzheimer's disease, some neurons in the AD brain showed characteristics of programmed cell death, which contributed to neuronal cell loss observed during the progression of the disease (Su et al., 1994). The characteristics of programmed cell death or apoptosis include changes of apoptotic and anti-apoptotic protein level e.g. caspase 3 and Bcl-2. Moreover, cell shrinkage, plasma membrane blebbing, nuclear chromatin condensation and DNA fragmentation morphological change were also found during apoptosis.

Tau hyperphosphorylation is due to the imbalance of kinase and phosphatase activites.

In this study, okadaic acid (OA), a protein phosphatase inhibitor, was chosen to induce tau hyperphosphorylation by reducing the phosphatase activities. Okadaic acid can

inhibit different phosphatase but the IC50 varies greatly among the phosphatase. When

cells were treated with okadaic acid at nanomolar concentration, the major cellular

effects are contributed to the inhibition of PP2A. However, if the concentration

53 increases to micromolar level, several protein phosphatases can be inhibited effectively, e.g. PPl and PP2A.

From the western analysis, PHF-tau phosphorylation was increased in a dose dependent manner after OA treatment, which is detected by PHF-tau ATS antibody. This antibody recognizes paired helical filament with phosphorylated tau at site Ser202 which is one of the dephosphorylation site regulated by protein phosphatase 2A. This showed that

OA can inhibit phosphatase 2A to induce tau phosphorylation. Moreover, both the pro and active forms of caspase 3 were also increased. This showed that OA can induce the expression of pro-caspase 3 and the level of active form of caspase 3 to promote the apoptotic process. Anti-apoptotic protein Bcl-2 was also increased significantly at

50nM and lOOnM OA treatment, suggesting that the toxicity and stress of OA may

induce a protective mechanism of the cells, which was observed at the neurons in

Alzheimers disease patients. Apart from okadaic acid, staurosporine, an apoptosis

inducer, was used to serve as a positive control. Unlike okadaic acid, treatment of cells

with staurosporine did not show an increase in tau phosphorylation but showed a

slightly increase of pro and active forms of caspase3 and Bcl-2 levels. This data

suggested that the cell death or apoptosis could be induced with or without tau

phosphorylation.

Okadaic acid inhibits PP2A activity and induces tau hyperhosphorylation which leads

to neurite retraction of the neurons due to the destabilization and disassembly of

microtubules. As demonstrated in this study, okadaic acid caused neurite retraction

and cell death whereas staurosporine didn't induce the neurite retraction but exhibited

cytotoxic effect on the cells. These findings indicated that the neurite retraction was not

54 a phenomenon of cell death but probably caused by destabilization of microtubules due to the tau hyperhosphorylation induced by okadaic acid. A measure of cell viability by

MTT assay based on mitochondrial activity showed that OA can induce cell death at high concentrations and long incubation times. Moreover, the condensed and fragmented nuclei were observed by the Hoechst 33342 staining after OA treatment, suggesting that OA induced cell death through apoptosis.

For the cell cycle analysis by PI staining, the apoptotic effect of OA on the SH-SY5Y cells was measured by analyzing the DNA content of the cells which represent different cell stages. The results showed that the apoptotic sub G1 cells increased gradually when the concentration of OA rose up. However, the Gl/GO� Sand G2/M cells were reduced after OA treatments. For the annexin V/ PI staining, it is a sensitive method to measure the percentages of cells undergoing apoptosis or necrosis. The results showed that the apoptotic cells increased after OA treatment while the necrotic cells didn't

change. And the DNA fragmentation assay also showed that the DNA ladder pattern

was observed at lOOnM OA treatment. These results suggested that the cytotoxic effect

of OA was through the apoptosis mechanism, which is also the main mechanism of cell

dying in Alzheimer disease.

In summary, OA treatment of neuroblastoma cells appears to mimic the characteristics

of cellular and biochemical changes associated with tau hyperphosphorylation-induced

neuronal cell death of Alzheimer's disease. OA treatment caused an increase in PHF

tau protein formation, leading to the activation of capase-3 mediated apoptotic cell

death. This cell model was being exploited to examine the neuroprotective effects of

flavonoids.

55 Chapter 4: Flavonoids screening for protecting neuronal death by preventing tau hyperphosphorylation

4.1 Introduction

The flavonoids are a group of polyphenolic compounds widely distributed in the plant kingdom. More than 6400 flavonoid compounds have been discovered contributing to the flavor and pigment of the fruits and vegetables. Flavonoids have important roles in plant growth, reproduction, and pathogen resistance

(Harbome and Williams, 1992). Flavonoids are polyphenolic compounds which are rich in many foods in our diet, for example, tea, coffee, chocolate, and wine.

A well-balanced diet with the recommended daily servings of fruits and vegetables and moderate amounts of tea, coffee, wine, beer, or chocolate can provide well over 1000 mg of total polyphenols and flavonoid per day (Lotito and

Frei, 2006).

Many studies have found that flavonoids can protect brain aging and some neurodegenerative disease. Normal brain aging is associated with elevated levels of reactive oxygen species (ROS) and neuroinflammation. ROS are the by- product of normal metabolism, which can give rise to oxidative cellular damage.

Our brain is especially susceptible to oxidative stress because the brain utilizes

20% of the total oxygen consumption, causing a large production of ROS. It has been found that inflammation of the brain results in the activation of the protecting microglial cells to produce high level of ROS and cytokines. There is evidence that increased oxidative stress plays an important role in the pathogenesis of neurodegenerative diseases such as Alzheimer's disease (AD) and

56 Parkinson disease (PD). Flavonoids have been found to have potent antioxidant and free radical scavenging activities in vitro (Halliwell, 2007). Thus, it has been proposed that flavonoids can relieve oxidative stress and neuroinflammation, which may protect the brain from developing neurodegenerative diseases. Apart from reducing oxidative stress and neuroinflammation, flavonoids have also

involved in many mechanisms against beta-amyloid-induced toxicity (Han et al,

2004) and in promoting neurogenesis (Casadesus et al, 2004).

4.2 Objectives

In the previous study (Chapter 3), an okadaic acid (OA)-induced neuronal apoptosis

cell model has been established and characterized. In light of the great potential of

flavonoids in protecting neuronal cells from apoptosis which can be further exploited

as therapeutic agents to treat neurodegenerative diseases, the effects of flovonoids to

protect against OA-induced neuronal cell apoptosis are being examined. OA is found to

induce tau protein hyperphossphorylation, neurite retraction, capase-3 activation and

nuclear condensation of SH-SY5Y cells leading to apoptotic cell death. In this study,

approximately 32 flovonoids and pure compounds have been screened for their ability

to protect OA-induced tau hyperphosphorylattion, caspase 3 activation, neurite

retraction, nucleus condensation.

57 4.3 Tested flavonoids

Morin, chyrsin, quercetin, rutin, baicalein, (+)-catechin, huperzine, hesperidin and (-)- epicatechin were purchased from Invitrogen. Naringin, baicalin, silybin, fererol, luteolin, scutellarin, wogonin, apigenin, tangeretin, icariin, kaempferol, casticin, daidzein, daidzin, formononetin, genistein, genistin, glycitein, glycitin, puerarin, cardamonin, phloretin and sulphuretin were generous gift from Dr. Tsim Lab. (The

Honk Kong University of Science and Technology). The purities of these flavonoids were over 90% and they were dissolved in dimethyl sulfoxide (DMSO) to give stock solutions at concentration of 1 OOmM.

58 4.4 Results

4.4.1 Toxicity of flavonoids

In view of some flavonoids have previously been reported to have cytoxicity in cancer cells, the effects of these compounds were tested by MTT assay Figure 4.1 showed that at a concentration of 5 [iM, these flavonoids did not produce significant cytotoxicity to the SH-SY5Y cells.

59 120 —

1��-n…--士TT'fr.nn-r;-云 汗--…sfl-p^-;：…Hfr fe-：;…FH. 咨 90 - .5-0.-0.….…m••…再 口-再-�…口-百 aifl……. 0) n D： 80 - �c 70 -

^ 50 - CO > 40 - i 30 - 20 -

10 -

0 ~丨• 11 •• I •丨丨• I I 11 I •丨 I • I I •丨丨 I" I 丨丨• 111" I 丨 I • I 丨• 11 •丨 11111111 丨 11 丨 111 I •丨 11 丨 1111 丨丨 111 111 I • I I • 111 丨 I _ I 11 丨 CCCCCCCCD^CCC-oCCCCCCoCCCCCCCCCCCCC 111 ^ 1 s ^ 111 i 12 i 111 …pf}1 塌|i购pt|

Fig. 4.1: Cytotoxicity test of the flavonoids on differentiated SH-SY5Y cells. Differentiated Cells were incubated with SjiM Flavonoids 24 hours. Cell death was quantified by MTT assay measuring the mitochondria activity. Percentage of cell survival compared to control was calculated. Data were mean 土 SEM (bars) values of three independent experiments.

60 4.4.2 Effects of flavonoid pre-treatment on OA-induced neurite retractions and cell death

As shown in previous study, one of the observable effects of OA on neumal cells is the retraction of neurite outgrowth, suggesting OA disrupts the cytoskeletal structure of neuronal cells (Fig. 4.2B). In order to examine whether flavonoid pre-treatment could prevent neurite retraction, cells were treated with 5}iM flavonoids for 24 hours followed by the induction of cell apoptosis by OA (50 nM) treatment. The representative results were shown in Fig 4.2 C-F. None of the 32 flavonoids examined here is able to reverse the neurite retraction induced by OA

The ability of flavonoid to protect OA-induced cell death was also examined. The cell viability was measured by the MTT assay. Cell cultures treated with 50 nM OA for 18 hours caused approximately 37.5% cell death compared to the control cultures. Pre- treatment of the cells with various flavonoids failed to show any protection of the cell death induced by okadaic acid. Casticin, for example, was found to enhance slightly the cytotoxic effect of OA, causing a significant 60% cell death in the cultures.

61 A. Control (DMSO) B. 50nM OA

C. 5|jM Quercetin + SOnM OA D. SPM Rutin + 50nM OA

• ： ：： r. . •• ” ——'：、�、、：：.，>!

E. 5mM (+)-Catechin + SOnM OA F. 5jjM Hesperidin + SOnM OA 、’•，. i ' }§•• I .‘ • _ • : .、 ？，...：’.... •、- , -” ''jH^'Ci t .

Fig. 4.2: Pre-treatment of flavonoids on OA induced neurite retractions in SH- SY5Y cells. Differentiated SH-SY5Y cells were pre-incubated with flavonoids for 24 hours, followed by the inclusion of 50nM OA in the cultures, After 18 hours of incubation, cells were examined using phase contrast microscopy (lOOX). The representative figures pretreated with quercetin (C), rutin (D), (+)-Catechin (E) and hesperidin (F). Bar = 50 \im

62 110 T

100 -： ri

90 -：

系 80 (D 70 T T f ： xs nfli . s J} ifi ns s F^n ^ g 60 n n r^s n t f^i工 n 门Tn?^ 门 ft g nnfi nnnn niH Hnn H fi I 门Pi 门： ^ 50 -： 门 "to -r I 40-； n

5 30

20 -：

10 -： 0 I I I I I I I I I I I I I I I 1 I I I I I I i I I I I I I I I I I I 0

。营。！ •虔晏IP忍〜」i虐•晏|一|站。P結。, i'工 0 0 LL T

Fig. 4.3: Effects of pre-treatment of flavonoids on OA-induced cell death. Differentiated Cells were incubated with 5|iM Flavonoids 24 hours in addition to 50nM OA 1 Shours. Cell death was quantified by MTT assay measuring the mitochondria activity. Percentage of cell survival compared to control was calculated. Data were mean 土 SEM (bars) values of three independent experiments.

63 4.4.3 Western blot analysis

In order to investigate whether flavonoids exhibits neuroprotective effect on the dying cells induced by OA or not, the Western blot analysis was performed to measure the change of several protein expressions, i.e. phosphorylated tau at site Ser202, pro and active forms of caspase 3 and Bcl-2 proteins. After OA treatment, tau phosphorylation increased since OA inhibited the PP2A dephosphorylation activity. When the cells were treated with 5|aM flavonoids in addition to SOnM OA treatment, several flavonoids showed the ability to reduce the tau hyperphosphorylation at site ser202.

These flavonoids included hesperidin (8)，fererol (12), genistin (25) and glycitein (26)

(Fig. 4.4). Many flavonoids were found to reduce the levels of both pro and active forms of caspase 3. These flavonoids included (+)-catechin (6), huperzine (7), hesperidin (8), fererol (12)，luteolin (13)，wogonin (15), apigenin (16)，tangeretin (17) and icariin (18) (Fig. 4.5). The anti-apoptotic protein- Bcl-2 was found significantly increased after OA treatment, probably due to a feedback protective regulation in response to OA toxicity. Many flavonoids were found to increase the levels of Bcl-2.

These included (+)-catechin (6), huperzine (7), hesperidin (8), luteolin (13), scutellarin

(14), tangeretin (17).

64 5|jM Flavonoids + 50nM OA 1 Morin 50nM0.5|jM ： c。n OA STS 1 2 3 4 5 6 7 8 2 Chyrsin PHF-AT8 — Quercetin = 78 �150KDa,r B _ ftHMBJI 霧 _ 4 Rutin GAPDH 5 Baicalein 37 KDa — •____ _垂 � 6 (+)-Catechin 7 Huperzi 门 e 5pM Flavonoids + 50nM OA 8 Hesperidin

con^r 9 10 1112 13 14 15 16 17 18 工 __ : 10 Baicalin

GAPDH ‘ "T^ r—T- 37 KDa ,睡-漏 _. _ •眷雄一 • 眷 _ 14 Scutellarin 15 Wogonin 5|jM Flavonoids + 50nM OA ^piger^ Con^OA^19 20 21 22 23 24 25 26 27 28 "Tz Tangeretin PHF-AT8 WmSISSiSSmSW] ~ Icariin “ 78�150KD a^nVS^^HBSmS B 19 Kaempferol GAPDH 職 Casticin “ 37 KDa _ _.__._丨_.<«»^ ~ Daidzein “ 22 Daidzin 23 Formononetin Flavonoids Genistein + 50nM OA ^ sonM^ ^^ ^^ �� 25 Genistin Con OA 29 30 31 32 PHF-AT8 as 26 Glycitein_ 78~150KDa .SIIMfl 27 Glycitin GAPDH "C .. � IT Puerarin~ 37 KDa 29 Cardamonin ~30 Phloretin 31 (-)-Epicatechin 32 Sulphuretin

Fig. 4.4: Effects of flavonoids pre-treatment on OA-induced hyperphosphorylated tau expressions. The differentiated SH-SY5Y cells were pretreated with 5uM flavonoid for 24 hours, followed by 50nM okadaic acid for 18 hours. Cell lysates were prepared for Western blots against antibodies to PHF- AT8 tau (Ser202). GAPDH immunoblot was used as constitutive protein control to normal protein loading in each sample. The results were repeated with one independent treatment (n=2).

65 1 Morin 5[JM Flavonoids + 50nM OA _2 Chyrsin c� n 1 2 3 4 5 6 7 8 工 Quercetin Pro- Capase-3 �� _ 4 Rutin 35KDa 麵雜•藝麵^^^眷•春 —Baicalein •

_7 Huperzine ^APDH ______8 Hesperid~

9 Naringin 5pM Flavonoids + 50nM OA

Con^OA^Q 10 11 12 13 14 15 16 17 18 "Ti sii^ Pro- Capase-3 .. f� � "li Fererol 35KDa 雄 _ _ _ I 雖•擎 13 Luteolin Active- Capase-3 . , — ^ , „ . 17KDa - •讀.« . 14 Scutellarin GAPDH J!__W�_n 37KDa 看靜眷雜 16 Apigenin 17 Tangeretin 5|JM Flavonoids + 50nM OA "Is icariin Con^OA^9 20 21 22 23 24 25 26 27 28 19 Kaempferol Pro- Capase-3 c —i "TT； �—�� 35KDa •麵一轉••藝.雄—雄^!* ^ 20 Casticin Active- Capase-3 t 誦.’fillilgli—1 21 Daidzein 17KDa ..'饭/WFNWlHIillWI 22 Daidzin

GAPDH � ..".. “ , _ 睡塵睡I 23 Formononetin 37KDa {-^-mmmmmmmmmrnn^ — Genistein —

5|JM Flavonoids 25 Genistin + 50nM OA "26 Glycitein A oOnM : Con OA 29 30 31 32 27 Glycitin

Pro- Capase-3 吻.“ 28 Puerarin 35KDa 29 Cardamonin Active- Capase-3 � _ ^ M\ lo Phloretin 17KDa ..條— —,�[ • ‘ k — • . • • � 31 (-)-Epicatechin , ~32 Sulphuretin 37KDa L •丨 I

Fig. 4.5: Effects of flavonoids pre-treatment on OA-induced Pro and Active forms of caspase 3 expressions. The Differentiated SH-SY5Y cells were pretreated with 5uM flavonoid for 24 hours, followed by 50nM okadaic acid for 18 hours. Cell lysates were prepared for Western blots against antibodies to Pro- and Active-form of caspase 3. GAPDH immunoblot was used as constitutive protein control to normal protein loading in each sample. The results were repeated with one independent treatment (n=2).

66 1 Morin _2 Chyrsin 5|JM Flavonoids + 50nM OA "3 Quercetin

Con5Sr 二 2 3 4 5 6 7 8 ~ Rutin ^s'k'Da • •————5 Baicalein wua ^ .toM_ 6 (+)-Catechin 广 ADPiU _ •^HirCT^iW^j^ 炉— ^^MMMHMMMMM^ Huperzine 8 Hespendin 9 Naringin

5mM Flavonoids + 50nM OA Baicalin

Con^T 9 10 11 12 13 14 15 16 17 18 ——^~ BCL-2 12 Fererol 25KDa …一眷.•等着•餐•驗着 "Is Luteolin GAPDH ‘ ‘ ‘“ 14 Scutellarin 37KDa 一 •等善•麵華春華等雄等j ~15 Wogonin 16 Apigenin 17 Tangeretin 5ijM Flavonoids + 50nM OA 18 Icariin

Con^oTlQ 20 21 22 23 24 25 26 27 28 19 Kaempferol BCL-2 20 Casticin 々氏 ua 21 Daidzein GAPDH 一 22 Daidzin 37KDa “ 23 Formononetin 24 Genistein

5mM Flavonoids ！ Genistin “ + 50nM OA 26 Glycitein 口。I�3 0 31 32 Glycitin 一 25KDa . • ~P—rin GAPDH 一 ]丨 _ _ _Carda國in 37KDa . ^ — — 30 Phloretin 31 (-)-Epicatechin 32 Sulphuretin

Fig. 4.6: Effects of flavonoids pre-treatment on OA-induced Bcl-2 expression. The differentiated SH-SY5Y cells were pretreated with 5uM flavonoid for 24 hours, followed by 50nM okadaic acid for 18 hours. Cell lysates were prepared for Western blots against an antibody to Bcl-2. GAPDH immunoblot was used as constitutive protein control to normal protein loading in each sample. The results were repeated with one independent treatment (n=2).

67 1 Morin 9 Naringin 17 Tangeretin 25 Genistin 2 Chyrsin ‘ "To Baicalin "TF Icariin Iq Glycitein 3 Quercetin ~ Silybin lo""Kaempferol Glycitin ~ Rutin I2 Fererol Casticin Iq Puerarin 5 Baicalein 13 Luteolin TT Daidzein W Cardamonin ‘ T "(+)-Catechin 1a Scutellaria ~Daidzin lo Phloretin~ 7 Huperzine 15 Wogonin "23" Formononetin'~ (-)-Epicatechin 8 I Hesperidin |l6| Apigenin 1241 Genistein | 32 [ Sulphuretin

I 1 I 2 I 3 I 4 丨 5 丨 6 I 7 I 8 I 9 丨10丨11丨12丨13|14丨15|16 Tau Hyperphosphorylation y Y Reduction Caspase-3 Reduction �" ^ ^"y ^� BCL-2 Increase "y "y

Tau Hyperphosphorylation ^��“7 Reduction Caspase-3 Reduction Y Y BCL-2 Increase "y

Table 4.1: Summary of the protective effects of flavonoids against OA—induced tau PHF hyperphosphorylation, caspase-3 and Bcl-2 changes in differentiated SH-SY5Y cells. The label Y indicated the presence of changes.

68 4.4.4 The effect of different concentrations of hesperidin on OA treatment

From the preliminarly results, it was found that hesperidin could significantly reverse the tau phosphorylation at site ser202 and the activation of active forms of caspase 3 induced by OA. On the other hand, hesperidin could enchance the induction of Bc-2 level. Taken these together, it appears that hesperidin may have beneficial protective effect against the tau induced cell toxicity. To confirm this，different concentrations of hesperidin and hesperitin, an aglycone of hesperidin, were examined, Figures 4.7 showed that hersperidin at 1-10 uM is effective in reducing the PFF tau phosphoryation, whereas hesperitin failed to reverse the PHF phosphorylation. The active form of caspase 3 was also reduced by hesperidin. Moreover, hesperidin treatments induced the

Bcl-2 protein level. In contrast, hesperitin, could not show an obvious reduction of tau phosphorylation.

Figures 4.8 showed the flow cytometry analysis of early apoptotic cells after different concentrations of hesperidin in addition to OA treatments. After okadaic acid (50 nM) treatment, the percentage of normal cells decreased from 96.59% (control: DMSO) to

83.86% (50 nM OA) while the apoptotic cells increased from 1.2% (Control: DMSO) to 4.56% (50 nM OA). For the late apoptotic and necrotic cells, they showed an

increase from 1.55% (Control: DMSO) to 11.02% (50 nM OA) (Table 4.2). Hesperdin

pre-treatment was however unable to protect apoptotic cell death. A slightly decrease

on the percentage of necrotic cells from 11.02% (50 nM OA) to 8.77% (1 |iM

Hesperidin + 50 nMOA) was observed.

69 Hesperidin Hesperitin +50nM OA +50nM OA r^rmtr…50nM <^ontroi OA 1|JM 5PM LOPM 1|JM 5|JM

Pro- CapaseS 35KDa L^HHHi^HHHHHIHHHiiK

CapaseS i7KDa HHHHHHHIHHHHHH

HH^BimiiiiiiiiimiB 25KDa mm^m^nniiiiiiimim

GAPDH 37 KDa HHHHHH^HK

Fig. 4.7: Effects of of different concentrations of hesperidin and hesperetin, on okadaic acid (OA) induced PHF phosphorylation, caspase-3 activation and Bcl-2 expression. The differentiated SH-SY5Y cells were pretreated with different concentrations of hesperidin and hesperetin for 24 hours, followed by 50 nM okadaic acid for 18 hours. Cell lysates were subjected to Western blots against antibodies to PHF-AT8, caspase-3, and Bcl-2. GAPDH immunoblot was used as a constituitive positive control. The results were repeated with one independent treatment (n=2).

70 A. Con (DMSO) B. 50nM OA C.1 pM hesperidin o , +50nM OA

::.“:::礙.___ r ：暴

10° 10' 10= 10' 10 ‘ lo' 10' W To' lO^ 10'

D.5|iM hesperidin E. 10|iM hesperidin o +50nM OA o +50nM OA

10» ““10= lO' 10' 10° 10' 10= 10' 10' E_y

Fig. 4.8: Flow cytometry analysis after pre-treatment with different concentrations of hesperidin on OA-induced apoptosis. Differentiated SH-SY5Y cells were pretreated with different concentrations of hesperidin for 24 hours, followed by 50 nM okadaic acid for 18 hours. Cells were stained with Annexin V with propidium iodide as described in Section 2.6. After staining, the cell populations were analyzed by flow cytometer. Region I: Pl-negative/annexinV-negative, Region II: Pl-negative/ annexinV-positive, III: Pl-positive/ annexin V-positive

71 I. Normal II. Apoptotic III. Necrotic Con (DMSO) 96.59% “ 1.20% 一 1.55% SOnMOA 83.86% “ 4.56% 11.02% luM Hesperidin 86.73% 4.11% 8.77% + SOnMOA 5\iM Hesperidin 85.41% 4.19% 9.96% + SOnMOA lOuM Hesperidin 85.35% 4.35% 9.86% + SOnMOA

Table 4.2: Percentage of cells in different cell stages after okadaic acid treatment. The percentages of cells were calculated by the size of areas at different phases in the PI intensity-area histogram plot.

72 4.4.5 Proteomic analysis

To investigate the mechanism of the effect of hesperdin on okadaic acid-induced treatment, proteomic analysis was performed to identify the differences between pairs of sample in different conditions i.e. 50nM OA treatment and 5\iM hesperidin treatment followed with 50nM OA treatment. After gel electrophoresis and staining, the two-dimensional gels were scanned to obtain digital photographs which were then analyzed by Image master 2D platinum Software (Fig. 4.9 and 4.10). Spots on each gel were paired according to their positions. If the volume of one spot was increased or decreased more than fifty percent compared to its pair spot，it was distinguished as differentially expressed proteins and was then excised for in gel digestion and MALDI-

TOF mass spectrometry.

There were a total nineteen spots identified (Table 4.3). Spots 1 to 9 were excised from the 2 DE gel with 50nM OA-treatment, showing that the volume of the spots increased more than fifty percent compared to that of their pair spots. Spots 10-18 were excised from the 2 DE gel with 5|iM hesperidin treatment followed by 50nM OA treatment.

Table 5 showed the protein identity as determined by MALDI-TOF tandem mass

spectrometry. The protein name, accession, observed and theoretical MW, observed

and theoretical pi are shown.

73 pi 3 4 5 6 789 10 丨 I I I I I 丨 I I I I _ r ,1 •! — — • I'

115.5kD — ^

82.2kD 一 , ，• 、 2 =- ^^^^ i -

參 19.4kD — fi / 14.8kD 一 ^ • 參

m I

Fig. 4.9: 2-DE pattern of SOnM okadaic acid treatment on differentiated SH- SY5Y cells. Sample were resolved by 2-DE on pH 3-10 IPG strips followed by separation on a 12% SDS-PAGE gel in the second dimension. Proteins were visualized by SYBRO Ruby staining.

74 pi 3 4 5 6789 10 I |„„丨11丨丨丨III丄I �-丨——I_

115.5kDa- > ‘ ‘ 82.2kDa-« 丄.二- 64.2kDa — •

4 一 •

• ’ • •• u � 19.4kDa- • •

一 T • •參 17 18 I 14.8kDa—, ^ / 嫩 • 16 / •• t .鲁 I

Fig. 4.10: 2-DE pattern of S^M hesperidin treatment followed with 50nM okadaic acid treatment on differentiated SH-SY5Y cells. Sample were resolved by 2-DE on pH 3-10 IPG strips followed by separation on a 12% SDS-PAGE gel in the second dimension. Proteins were visualized by SYBRO Ruby staining.

75 Observed Observed /Theoretical /Theoretical Volume of Volume of Protein Accession MW (kPa) PI (kPa) Spots Paired Spots

1 Myosin Heavy chain 3 P11055 100.2/224.00 7.03/5.64 0.773 0.211

2 Fascin Q16658 61.10/54.50 7.21/6.84 2.106 0.477 26S protease Regulatory 3 Subunit? P35998 37.52/48.60 7.05/5.71 0.753 0.462

4 Adenosylhomocysteinase P23526 56.35/47.68 6.15/6.03 1.561 0.804 Ubiquitin carboxyl-terminal 5 hydroxylase isozyme 1 P09936 25.11/24.81 5.63/5.33 1.365 0.561

6 Antioxidant protein 2 P30041 25.98/24.89 6.58/6.02 1.365 0.915

7 GTP-binding nuclear protein P28746 24.15/24.41 7.19/7.01 0.248 0.279 Nucleoside diphosphate 8 kinase A P15531 18.35/17.14 5.78/5.83 0.420 0.373

9 Cofilin P23528 16.35/18.49 6.15/8.22 1.106 0.682

10 SCAM-1 Protein gi|14603385 43.52/36.72 8.89/8.86 0.242 0.862 Rho gdp-dissociation inhibitor 11 1 P52565 39.11/23.19 8.12/5.02 0.381 1.293 Heterogeneous nuclear 12 ribonucleoproteins A2/B1 P22626 37.22/37.41 9.11/8.97 0.537 2.708 Heat shock cognate 71KD 13 protein P19120 48.45/71.20 4.75/5.49 0.666 2.708 Guanine nucleotide binding 14 protein G(Q) alpha subunit P50148 21.22/41.40 7.02/5.58 0.433 0.734

15 Thioredoxin peroxidase 2 P32119 23.50/22.10 9.02/8.27 0.350 1.503 51 KD FK506-Binding protein 16 (FKBP51) Q13451 12.50/51.00 5.21/5.71 0.270 0.286 Peptidyl-prolyl cis/trans 17 isomerase A P05092 15.36/17.87 8.12/7.82 0.763 0.543 Peptidyl-prolyl cis/trans 18 isomerase A P05092 15.36/17.87 8.93/7.82 0.477 1.064

19 Cofilin P45592 21.35/18.52 9.11/8.22 0.154 0.279

Table 4.3: Differential expressed proteins in 50nM okadaic acid treatment and hesperidin treatment followed with 50nM okadaic acid treatment. Spots on each gel were paired according to their positions. The volume of the spots increased more than fifty percent compared to that of their pair spots.

76 4.5 Discussion

After establishing the okadaic acid-induced apoptosis cell model, the model was characterised and showed that it can mimic some molecular or morphological changes in Alzheimer's disease affected neurons, including increase of phosphorylated tau protein and Bcl-2 levels，increase of pro and active caspase 3 levels, neurite retraction, nucleus condensation and also increase of apoptotic cells. In this study, thirty two flavonoids were tested to search for any potential drugs that can protect against tau hyperphosphorylation and prevent or delay neuronal cell death. Flavonoids are a large group of naturally occurring, low molecular weight, polyphenolic compounds widely distributed in plant and fruit.

They have important roles in plant growth, reproduction, and pathogen resistance

(Harbome and Williams, 1992). In general, flavonoids appear to be non-toxic but some flavonoids have shown to have apoptotic effects on cancer cells when given at high concentration, e.g. 50 |a,M luteolin caused a apoptotic effect in human

LNCaP prostate cancer cell line (Brusselmans et al., 2005).

In this study, flavonoids that were examined here have minimal cell toxicity at concentraton of 5 [iM. Over 90% cell viability was found after 24 hours treatment

of these flavanoids except casticin which has a slight increase in cell death. When

the differentiated SH-SY5Y cells were treated with 50nM OA, the cells appeared

neurite retraction and became roundish. When the cells were treated with 5jiM

flavonoids in addition to 50nM OA treatment, they still showed severe neurite

retraction similar to those treated with 50nM OA only. Moreover, the MTT assay

also indicated that most flavonoids could not reverse the amount of cell death

77 induced by the okadaic acid. The results suggested that the tested flavonoids are not able to offset the cytotoxicity and neurite retraction induced by OA.

After okadaic treatment, the expression levels of phosphorylated tau at site

Ser202 and pro and active forms of caspase 3 were increased, and the anti- apoptotic protein Bcl-2 level was also induced due to the protective mechanism stimulated by the toxicity and stress of OA. When the cells were treated with 5|xM flavonoids in addition to 50nM OA treatment, six flavonoids showed the ability to reduce the tau hyperphosphorylation at site ser202 and nine flavonoids were found to reduce the levels of both pro and active forms of caspase 3. Moreover, six flavonoids showed induction of Bcl-2 levels. Among these flavonoids，only hesperidin could reverse the tau phosphorylation at site ser202 and the upregulation of pro and active forms of caspase 3, also heperidin could induce

Bcl-2 level. However, the protective effects observed in the molecular changes were not enough to terminate the apoptotic mechanisms induced by OA. Since studies reported that okadaic acid could increase the phosphorylation of p53 without changing p53 levels, which in turn activated the apoptotic pathways (Yan et al,, 1997). This data indicate that the reduction of tau hyperphosphorylation is not a sufficient condtion to protect the apoptosis induced by OA. Nevertheless, the findings that hesperidin significantly reduce the PHF formation is interesting, in the light of the fact that of PHF formation is tightly associated with tangly formation which cause Alzheimer's disease progression.

Hesperidin, a bioflavonoid, is abundant and inexpensive by-product of Citrus

cultivation and is the major flavonoid in the sweet orange and lemon. No signs of

78 toxicity have been observed with the normal intake of hesperidin (Pizzomo and

Murray, 1999). Hesperidin has been reported to possess significant anti- inflammatory and analgesic effects. Moreover, when in combination with diosmin, hesperidin shows a marked protective effect against inflammation disorder, possibly through mechanism involving antioxidant free radical scavenger activity

(Jean and Bodinier, 1994). Thus, it may relieve oxidative stress and neuroinflammation, which may protect brain aging and neurodegenerative diseases. Here hesperidin is shown to have selective reduction of tau phosphorylation and caspase 3 levels, and increase of Bel 2 levels mediated by

OA induced apoptosis.

Hesperidin, but not an aglycone hesperitin, was found to have a concentration- dependent protection of PHF-tau phoshorylation. This indicated that the glycoside of hesperidin is necessary for its action. Previous results showed that no flavonoid could protect cell death measured by MTT assay. However, instead of cell death, early apoptotic cells detected by annexin V can also be monitored to validate the neuroprotection effect of different concentrations of hesperidin. However, Figures

4.8 showed hesperdin could not prevent apoptotic cell death but just slightly decrease the percentage of necrotic cells from 11.02% (50nM OA) to 8.77%

(\\iU Hesperidin + 50nMOA).

In conclusion, hesperidin has shown the protective ability on reduction of phosphorylated tau and caspase 3 levelsl as well as the induction of the anti-

79 apoptotic protein Bcl-2 level. However, these changes were not enough to terminate the apoptotic signal induced by OA

80 Chapter 5: General discussion

Alzheimer's disease (AD) is the most common form of neurodegenerative disease characterized by loss of memory and cognitive abilities which interfere with normal activities of daily living. The disease is hallmarked by a decline in cognitive functions such as remembering, reasoning, and planning. The deterioration of cognitive and motor functions is mainly related to the loss of neuron in the basal forebrain of Alzheimer's disease patient. With the intensive study of AD for over three decades, many causes of the disease pathogenesis are proposed. Some of these causes are closely related to each other and observed in

AD patients. This suggests that many causes or factors work together to achieve the ultimate target: neuronal cell death. These causes or factors include protein misfolding and aggregation i.e. (3-amyloid plaque and neurofibrillary tangles, gene mutations i.e. Mutations of presenilin 1 gene, amyloid precursor protein gene and apolipoprotein E gene, loss of cholinergic neurotransmission and also generation of free radical and neuroinflanimation.

Until now, there is no treatment for Alzheimer's disease and the available medications, acetylcholine esterase inhibitors, only offer symptomatic benefit for some patients but can not slow down the progression. Acetylcholine esterase inhibitors reduce the rate at which acetylcholine (ACh) is broken down and hence increase the concentration of ACh in the brain. These drugs can temporarily stabilize cognitive impairment and maintain body muscle functions; however, they cannot reverse or stop the disease progression and may not show the

81 beneficial effects in some patients. Thus, designing other mechanism-based drugs is the direction to alter disease pathology and progression.

In tauopathy hypothesis, the formation of paired helical filaments composed of hyperphosphorylated tau protein is a key pathologic molecular change contributed to the neurodegeneration. Thus, preventing tau phosphorylation is one of the strategies to reverse or stop the disease progression. In this study, we aimed at establishing an okadaic acid-induced apoptosis cell line model that can be used to search for any potential flavonoids against tau hyperphosphorylation and cell death. The first part of this study was to characterize the cell model and examine whether it can mimic some cellular and biochemical changes in Alzheimer's disease affected neurons. The results showed that okadaic acid treatment caused an increase in PHF-tau protein level and activation of caspase-3 mediated apoptotic cell death. Moreover, after treatment, the neuroblastama cells showed neurite retraction, and both the cell death and apoptotic cells increased, measured by several methods including the MTT assay, cell cycle analysis and annexin V

staining. Studies on AD brains also demonstrated tau hyperphosphorylation and

showed characteristic of programmed cell death. The findings indicate that OA

treatment of neuroblastoma cells mimics the characteristics of cellular and

biochemical changes associated with tau hyperphosphorylation-induced neuronal

cell death of Alzheimer's disease.

After characterization, the model was then used to screen for any potential

flavonoids that can prevent tau hyperphosphorylation and neuronal cell death.

The toxicity test of flavonoid showed that most flavonoids in a concentration of 5

82 P-M had less toxicity, except casticin. In the western blot analysis, six flavonoids showed reduction of tau hyperphosphorylation at site ser202 and nine flavonoids were found to suppress the levels of both pro and active forms of caspase 3.

Moreover, six flavonoids induced Bcl-2 levels. However, the results indicated that 32 flavonoids at a concentration of 5 |iM could not protect the cells from neurite retraction and could not show protection of the cell death induced by okadaic acid. This suggested that the protective effects observed in the molecular changes were not enough to terminate the apoptotic mechanisms induced by OA.

Since studies reported that okadaic acid could induce other apoptotic pathways instead of caspase 3 mediated apoptosis induced by tau hyperphosphorylation.

Among the thirty two flavonoids, only hesperidin could reverse the tau phosphorylation at site ser202 and downregulate the levels of pro and active forms of caspase 3, also induce BCL-2 level. Thus, different concentrations of hesperidin on OA treatment were used to confirm the protection effect on molecular changes observed in previous results. The findings indicated that that different concentration of hesperidin could reduce the tau phosphoryation, active form of caspase 3 levels and induced the BCL 2 protein level. And these effects maintained even at low concentration (l|iM) of hesperidin. However, hesperitin, an aglycone of hesperidin, could not show such effects, indicating that the glycoside of heperidin was necessary for its protective action. Again, the annexin

V-PI staining results showed that hesperdin could not prevent apoptotic cell death but just slightly decrease the percentage of necrotic cells from 11.02% (50nM OA) to 8.77% (IjaM Hesperidin + 50nMOA). This may be due to the activation of

other apoptotic pathways induced by okadaic acid, so the reverse of caspase 3

83 mediated pathway induced by tau hyperphosphorylation was not enough to terminate the process.

The study of the proteome, which is the PROTEin complement expressed by a genOME, has developed remarkably over the past few years. It provides information about the state of gene products, such as the expression levels and isoform. Two-dimensional gel electrophoresis (2-DE), which consists of isoelectric electrophoresis focusing (lEF) and sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS PAGE) is an effective method of separating a large number of proteins，providing a new platform for studies of complex biological functions. In order to investigate the mechanism of the protective effect of hesperidin, proteomic analysis was carried out. There were total nineteen differentially expressed protein spots identified. Spots 1 to 9 were excised from the 2 DE gel with 50nM OA-treatment while Spots 10-18 were excised from the 2 DE gel with 5|iM hesperidin treatment followed by 50nM OA treatment. All the spots were screened out by the criteria that the volume of one spot was increased more than fifty percent compared to its pair spot. Among these identified spots, peptidyl-prolyl isomerase A (Pin 1) was found to be upregulated in heperidin treatment in addition to OA treatment. Since Pin 1 can catalyze the cis-trans isomerization of peptidyl-prolyl bonds (Liou et al., 2003).

After isomerization, the spatial arrangement of phosphate on ser/Thr is different

(Figure 5.1).

84 • T trans

cis b •

Fig. 5.1： Cis-trans isomerization of peptidyl-prolyl bonds catalysed by Pin 1

Studies have shown that phosphorylatied tau also contains such motif and is one of the substrate for Pin 1. Pin 1 can catalyze the isomerization of the cis-form of phosphorylated tau to the trans-form of phosphorylated tau, which is more easily to be dephosphorylated by protein phosphatase 2A, so the function of tau can be restored. Moreover, after knocking out the Pin 1 gene of the mice, tau hyperphosphorylation increased and neurodegeneration was observed. All of this suggested that Pin 1 may involve in protecting against AD. Thus, upregulation of

Pin 1 by hesperidin may be involved in the reverse of tau hyperphosporylation and caspase-3 mediated apoptosis pathway. However, proteomic analysis is just a preliminary study to find out the differential expressed protein. The results need to be confirmed by the western blot technique applied on the 2-D gels using antibody against the proteins.

In summary, the okadaic acid induced apoptosis model was characterized, which mimicked the biological and cellular changes observed in AD. Among the tested flavonoids, hesperidin has shown the protective ability on reduction of phosphorylated tau and caspase 3 levels, also induction of BCL-2 level but these

85 molecular changes were not enough to terminate the apoptotic signal induced by

OA. Preliminary proteomic analysis was carried out to find the differential expressed proteins which involved in the protective mechanism of hesperidin. Pin

1 was found to be upregulated and showed the ability to reduce tau hyperphosphorylation, which validate to the findings of the effect of hesperidin.

In the future, confirmation of the differential expressed protein by 2D western blot analysis is needed and the protective mechanisms can be studied by suppression or overexpression of those differential expressed proteins to find the relationship with tau phosphorylation and the intermediate involved in the protective pathway, which might help to understand more about the tauopathy mechanism.

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