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Hoi, Chu Peng. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2007. / Includes bibliographical references (leaves 81-103). / Abstracts in English and Chinese. / Acknowledgements --- p.II / Abstract --- p.III / Abstract (in Chinese) --- p.V / List of Abbreviations --- p.VI / List of Figures --- p.VIII / List of Tables --- p.X / Table of Contents --- p.XI / Chapter Chapter One --- General introduction --- p.1 / Chapter 1.1 --- Alzheimer's disease --- p.1 / Chapter 1.1.1 --- Epidemiology and risk factors --- p.2 / Chapter 1.1.2 --- Clinical manifestation and course --- p.4 / Chapter 1.1.3 --- Clinical diagnosis --- p.5 / Chapter 1.1.4 --- Neuropathology and pathogenesis of AD --- p.8 / Chapter 1.1.5 --- Drug therapy of AD --- p.11 / Chapter 1.1.5.1 --- Drugs for symptomatic treatment --- p.11 / Chapter 1.1.5.2 --- Drugs based on epidemiology --- p.12 / Chapter 1.1.5.3 --- Drugs with potential disease-modifying effects --- p.14 / Chapter 1.1.5.4 --- Herbal supplements --- p.15 / Chapter 1.2 --- Models for drug discovery in Alzheimer Disease --- p.15 / Chapter 1.2.1 --- In vivo (animal) models --- p.16 / Chapter 1.2.2 --- In vitro (cellular) models --- p.18 / Chapter 1.3 --- Chinese herbs for the treatment of AD --- p.20 / Chapter 1.3.1 --- Ginkgo biloba L --- p.21 / Chapter 1.3.2 --- Magnolia officinalis --- p.24 / Chapter 1.3.3 --- Acori graminei Rhizoma (AGR) --- p.26 / Chapter 1.3.4 --- Gastrodia elata (G. elata) --- p.27 / Chapter 1.3.5 --- Rhodiola rosea L.( R. rosea) --- p.29 / Chapter 1.3.6 --- Scutellariae baicalensis --- p.30 / Chapter 1.3.7 --- Curcuma longa L.(Zingiberaceae) --- p.31 / Chapter 1.4 --- Aims of the study --- p.33 / Chapter Chapter Two --- Materials and Methods --- p.34 / Chapter 2.1 --- Materials --- p.34 / Chapter 2.1.1 --- Chemicals and reagents --- p.34 / Chapter 2.1.2 --- Materials for cell culture --- p.35 / Chapter 2.1.3 --- Instruments --- p.35 / Chapter 2.2 --- Methods --- p.36 / Chapter 2.2.1 --- Cell culture --- p.36 / Chapter 2.2.2 --- MTT cell viability assay --- p.38 / Chapter 2.2.3 --- Characterization of the cytotoxicity of Aβ peptide in NGF-differentiated PC 12 cells --- p.38 / Chapter 2.2.4 --- Screening of the neuroprotective effect of major principles from selected herbs on PC 12 cells against Aβ-induced cytotoxicity --- p.39 / Chapter 2.2.5 --- Measurement of reactive oxygen species (ROS) --- p.40 / Chapter 2.2.6 --- Measurement of intracellular calcium levels --- p.41 / Chapter 2.2.7 --- Measurement of caspase-3 activity --- p.42 / Chapter 2.2.8 --- Propidium iodide (PI) staining to evaluate apoptosis and necrosis --- p.43 / Chapter 2.3 --- Statistics --- p.45 / Chapter Chapter Three --- Results --- p.46 / Chapter 3.1 --- NGF-differentiated PC 12 cells --- p.46 / Chapter 3.1.1 --- Determination of an appropriate cell density for the screening experiments --- p.46 / Chapter 3.1.2 --- Characterization of Aβ-induced cytotoxicity in NGF-differentiated PC 12 cells --- p.47 / Chapter 3.1.2.1 --- Cytotoxicity of Aβ-related fragments in NGF-differentiated PC 12 cells --- p.48 / Chapter 3.1.2.2 --- Dose-dependent cytotoxic effect of Aβ on PC 12 cells --- p.48 / Chapter 3.1.2.3 --- Time-dependent effect of Aβ-induced toxicity on PC12 cells --- p.50 / Chapter 3.1.3 --- Protective effect of selected active principles against Aβ1-4-induced toxicity in PC 12 cells --- p.51 / Chapter 3.2 --- Measurement of reactive oxygen species (ROS) --- p.54 / Chapter 3.2.1 --- Measurement of ROS induced by H202 --- p.54 / Chapter 3.2.2 --- Measurement of ROS induced by Aβ --- p.56 / Chapter 3.3 --- Measurement of Intracellular calcium levels --- p.57 / Chapter 3.4 --- Measurement of caspase-3 activity --- p.58 / Chapter 3.4.1 --- AMC reference standard curve --- p.59 / Chapter 3.4.2 --- Measurement of caspase-3 activity --- p.59 / Chapter 3.5 --- PI staining for evaluate apoptosis and necrosis --- p.60 / Chapter Chapter Four --- Discussion --- p.64 / Chapter 4.1 --- Aβ-induced cytotoxicity in NGF-differentiated PC 12 cells as an in vitro model of Alzheimer's disease --- p.64 / Chapter 4.1.1 --- Cell line selection --- p.65 / Chapter 4.1.2 --- Characterization of Aβ-induced cytotoxicity in NGF-differentiated PC 12 cells --- p.66 / Chapter 4.2 --- Screening of the neuroprotective effects of selected active principles against Aβ-induced cytotoxicity in NGF-differentiated PC 12 cells --- p.67 / Chapter 4.3 --- Neuroprotection via inhibition of the ROS generation --- p.71 / Chapter 4.4 --- Neuroprotection via suppression of calcium homeostasis --- p.73 / Chapter 4.5 --- Neuroprotective via inhibition of Aβ-induced apoptosis --- p.75 / Chapter 4.5.1 --- Inhibition of caspase-3 activation --- p.75 / Chapter 4.5.2 --- PI staining for evaluation of apoptosis and necrosis --- p.76 / Chapter Chapter Five --- Conclusion and future work --- p.79 / Chapter 5.1 --- Conclusion --- p.79 / Chapter 5.2 --- Future work --- p.80 / References --- p.81

Un dispositif de stimulation électrique in vitro sur des lignées cellulaires a été optimisé afin de nous permettre d'étudier les mécanismes cellulaires et moléculaires de la SHF. Deux lignées cellulaires (GH3 et PC12) ont été analysées au niveau transcriptomique, protéomique et de la sécrétion hormonale et de neurotransmetteurs.Nous avons comparé les niveaux de sécrétion de PRL des GH3 traitées par SHF, SBF ou par la dopamine; ainsi que les niveaux des catécholamines (DA, AD et NA) des PC12 traitées par SHF, SBF ou par la 6-OHDA. La synthèse protéique des cellules stimulées a été analysée par les techniques d'incorporation de 35S méthionine et de SELDI-TOF-MS. Enfin nous avons recherché les modifications d'expression génique des cellules stimulées en utilisant la technique des microarray à base d'oligonucléotides longs sur nylon et détection radioactive.Les premières expériences montrent une diminution significative de la quantité de prolactine à un niveau comparable à celui obtenu avec l'inhibiteur conventionnel, la dopamine. De la même façon, la production de catécholamines dans le milieu est inhibée. Les données transcriptomiques montrent l'existence des profils d'expression caractéristique de la neurostimulation à haute fréquence, avec environ 100 gènes discriminants impliqués dans la synthèse protéique, la signalisation calcique, l'énergétique cellulaire pour les principaux. Nous avons confirmé l'impact de la neurostimulation sur la synthèse protéique en SELDI-TOF comme en incorporation de méthionine. Un mécanisme original de SHF peut donc être proposé, impliquant la neutralisation de la synthèse protéique dans l'inactivation réactionnelle des structures neuronale

<p>The developing nervous system depends largely on extracellular cues to shape its complex network of neurons. Classically, neurotrophins are known to be important mediators in this process. More recently, Bone Morphogenetic Proteins (BMPs), belonging to the Transforming Growth Factor beta (TGFβ) superfamily of secreted cytokines, have been shown to exert a wide range of effects, such as cellular growth, differentiation, survival and apoptosis, both in the developing and adult nervous system. They signal via serine/threonine kinase receptor essentially to the Smad pathway, which carries the signal to the nucleus where the transcription of target genes is regulated.</p><p>This thesis investigates the functions of BMPs in the nervous system, using a set of different models. Firstly, a targeted deletion of GDF10 (BMP3b) in the mouse was established to evaluate the role of this growth/differentiation factor in the hippocampal formation, a brain area known to be involved in memory processing. Other members of the TGFβ superfamily likely compensate for the lack of GDF10, since no detectable alterations in hippocampal function or gene transcription profile have been found. Secondly, a mouse model was set up, with the aim to study impaired BMP-signalling in dopaminergic neurons. The tyrosine hydroxylase (TH) locus was used to drive the expression of dominant negative BMP receptors by means of bicistronic mRNAs. TH is the rate-limiting enzyme in the biosynthesis of catecholamine and the mice described, show a graded decrease of TH-activity resulting in severe to mild dopamine deficiency. The contribution of the dominant negative BMP receptors to the phenotype is however secondary to the apparent TH hypomorphism. The final theme of this thesis is the potentiating effects of BMPs on neurotrophin-induced neurite outgrowth as studied in explanted ganglia from chick embryos and in the rat phaeochromocytoma cell line PC12. A number of pharmacological inhibitors of intracellular signalling kinases were applied to the cultures in order to reveal the contribution of different pathways to the enhanced neurite outgrowth. We made the unexpected finding that inhibition of MEK signalling mimicked the potentiating effects of BMP stimulation in the chick system. The underlying mechanisms for the synergistic effects, however, are still an enigma.</p>

The developing nervous system depends largely on extracellular cues to shape its complex network of neurons. Classically, neurotrophins are known to be important mediators in this process. More recently, Bone Morphogenetic Proteins (BMPs), belonging to the Transforming Growth Factor beta (TGFβ) superfamily of secreted cytokines, have been shown to exert a wide range of effects, such as cellular growth, differentiation, survival and apoptosis, both in the developing and adult nervous system. They signal via serine/threonine kinase receptor essentially to the Smad pathway, which carries the signal to the nucleus where the transcription of target genes is regulated. This thesis investigates the functions of BMPs in the nervous system, using a set of different models. Firstly, a targeted deletion of GDF10 (BMP3b) in the mouse was established to evaluate the role of this growth/differentiation factor in the hippocampal formation, a brain area known to be involved in memory processing. Other members of the TGFβ superfamily likely compensate for the lack of GDF10, since no detectable alterations in hippocampal function or gene transcription profile have been found. Secondly, a mouse model was set up, with the aim to study impaired BMP-signalling in dopaminergic neurons. The tyrosine hydroxylase (TH) locus was used to drive the expression of dominant negative BMP receptors by means of bicistronic mRNAs. TH is the rate-limiting enzyme in the biosynthesis of catecholamine and the mice described, show a graded decrease of TH-activity resulting in severe to mild dopamine deficiency. The contribution of the dominant negative BMP receptors to the phenotype is however secondary to the apparent TH hypomorphism. The final theme of this thesis is the potentiating effects of BMPs on neurotrophin-induced neurite outgrowth as studied in explanted ganglia from chick embryos and in the rat phaeochromocytoma cell line PC12. A number of pharmacological inhibitors of intracellular signalling kinases were applied to the cultures in order to reveal the contribution of different pathways to the enhanced neurite outgrowth. We made the unexpected finding that inhibition of MEK signalling mimicked the potentiating effects of BMP stimulation in the chick system. The underlying mechanisms for the synergistic effects, however, are still an enigma.

PC12 rat pheochromocytoma cells respond to nerve growth factor (NGF) by neuronal differentiation and partial growth arrest. Mouse c-myc and adenovirus E1A genes were introduced into PC12 cells to study the influence of these nuclear oncogenes on neuronal differentiation. Expression of myc and E1A blocked morphological differentiation and caused NGF to stimulate rather than inhibit cell proliferation. NGF binding to cell surface receptors, activation of ribosomal S6 kinase, and ornithine decarboxylase induction were similar in myc and E1A expressing clones compared with wild-type PC12 cells, suggesting that changes in the cellular response to NGF were at a post-receptor level. The ability of myc and E1A expression to block the transcription-dependent induction of microtubule associated proteins by NGF further suggested that these genes may inhibit differentiation by interfering with NGP's ability to regulate transcription. These results illustrate that NGF can promote either growth or differentiation of PC12 cells, and that myc or E1A alter the phenotypic responses to growth factors.

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神經系統裡，神經滋養因子在調控細胞分化與生存作用中扮演非常重要的角色，近年來的研究顯示 BDNF 的神經保護效果是透過細胞外訊息調控激酶 (extracellular signal-regulated kinase, ERK) 及磷脂酰肌醇3-激酶 (phosphatidylinositol-3 kinase, PI3K) 訊息傳遞路徑調控，然而，還有許多其他的細胞信號傳遞路徑可能參與 BDNF 的保護作用機制中。而蛋白激酶 CK2 (casein kinase 2) 是一種普遍存在於細胞且具有高度保留序列的絲胺酸/蘇胺酸蛋白質激酶，在細胞中具有非常重要的地位。近來研究有非常多證據支持 CK2 是細胞凋亡的抑制者。此外，血清反應因子 (serum response factor, SRF) 是一種轉錄因子，會與保留序列 SRE (即 CArG box) 相結合，而此段序列過去曾在早期即時表現基因 (如 c-fos、Egr ) ，或是抗細胞凋亡基因－Mcl-1－上的啟動子被發現， SRF 調控著基因的活化，進而與細胞增生、存活、突觸活性相關聯，然而調節 SRE 調控之基因的作用機制尚未十分明瞭。因此，本論文研究主要探討在 PC12 細胞中， BDNF調節 SRE 調控轉錄作用機轉 CK2 是否參與其中? 由冷光酶活性試驗結果顯示 BDNF 會顯著地促進 SRE 的轉錄活性，並且當 CK2α 過度表現亦會促進 SRE 調控的轉錄活性，而利用小干擾 RNA 抑制內生性 CK2α 生成，則會降低 SRE 的轉錄活性，更進一步證明 CK2α siRNA 會降低 BDNF 促進 SRE 調控的轉錄活性。此外，將 CK2α 與 SRF S99A 質體一同轉染至細胞中，會減緩 CK2 促進的 SRE 啟動子轉錄活性。為了探討 CK2 調控 SRE 的轉錄活性在神經保護作用裡扮演的角色，因此，將 CK2 蛋白表現量增加是否會保護 PC12 細胞對抗Rotenone所誘發的細胞凋亡傷害?結果顯示 CK2 表現量增加會保護細胞對抗Rotenone誘導的細胞凋亡，並減緩 Rotenone 對 SRE 調控的轉錄活性降低，但是，突變型 SRF S99A 蛋白會降低 CK2α 的影響作用。這些結果顯示 BDNF 促進 SRE 調控的基因表現是會透過 CK2 訊息傳遞路徑。 / The neurotrophins play an important role in cell differentiation and survival of the nervous system. Among them, the neuroprotective effects of brain-derived neurotrophic factor (BDNF) is showed to be mediated by extracellular signal-regulated kinase (ERK) and phosphatidylinositol-3 kinase (PI3K) signaling pathway in the recent studies. However, other cellular signaling pathways might be involved in these effects of BDNF. Protein kinase CK2 (casein kinase 2) is a ubiquitous and highly conserved serine/threonine protein kinase and is indicated as a vital cellular role. In recent years, evidences have been mounted in support of the importance of CK2 in the suppression of apoptosis. Serum response factor (SRF) is a transcription factor binding to a consensus DNA sequence SRE (known as a CArG box) which was found in the promoters of some immediately early genes (such as c-fos, Egr) and anti-apoptotic Mcl-1 gene. The activations of SRF-regulated genes were associated with cell proliferation, cell survival and perception of synaptic activity. However, the regulatory mechanism of SRE-mediated genes is not well studied. The SRE-mediated transcription activity through CK2 signaling by BDNF treatment was studied in the PC12 cells in the present study. Results revealed that BDNF significantly increased the SRE promoter activity by luciferase report assay. The SRE-mediated transcription activity was increased by overexpression of CK2α, and the inhibition of endogenous CK2α by small interfering RNA was also shown to reduce this transcription activity. Furthermore, CK2α siRNA treatment antagonized the up-regulation effects of BDNF on SRE-mediated transcription activity. The co-transfection of CK2 and mutant SRF S99A plasmids significantly diminished up-regulatory effects of CK2 on SRE promoter activity. To test this CK2 induction in SRE-mediated transcription plays a role in neuroprotecion, we determined whether over-expression CK2 protects PC12 cells against rotenone-induced apoptosis. The results revealed that the over-expression of CK2α protected cells against rotenone-induced apoptosis and rescued the SRE-mediated transcription activity. Further, these effects of CK2α were blocked by co-transfection of mutant SRF S99A. These above results demonstrate that the up-regulation of BDNF on SRE-mediated genes is through CK2 signaling pathway.

蛋白質激酶 CK2 是一種多功能的絲胺酸／蘇胺酸蛋白激酶，且普遍存在於哺乳類動物細胞中，CK2 受質眾多，對於細胞週期的發展、轉錄作用以及抗細胞凋亡等過程中扮演很重要的角色。SRF 是一種哺乳類動物的轉錄因子，它會結合到血清反應元素 SRE 上進而調控一些促進細胞存活的基因轉錄作用。Mcl-1歸類於抗細胞凋亡 Bcl-2 家族，具有促進細胞存活的能力。過去研究顯示 SRF 的 DNA 結合活性會受到蛋白激酶 CK2 的磷酸化而增加，且 SRF 對 Mcl-1 的活性調控作用也被描述在其他的研究中，然而，對於細胞的訊息目前還沒有更詳細的研究。在本實驗中，我們探討是否可以藉由 CK2 調控 SRF 的路徑來影響 Mcl-1 的表現以作為抗細胞凋亡的機制。利用 CK2 抑制劑 TBB 處理的結果顯示，在 4 hr 後，phospho-SRF 蛋白質表現的降低具有劑量相關性。而相似的降低也可以從 Mcl-1 的 mRNA 和蛋白質表現量觀察到。處理 24 hr 後，phospho-SRF 的蛋白質表現量有顯著降低，而 Mcl-1 的 mRNA 表現量相較 Mcl-1 的蛋白質影響層面微弱。另一方面，轉染野生型 CK2α 會增加 phospho-SRF，相反的，轉染抑制催化活性的突變型 CK2αA156 則會顯著降低 phospho-SRF 的表現。更進一步，野生型 CK2α 同時增加 Mcl-1 的 mRNA 及蛋白質層級，而 CK2αA156 則會降低 Mcl-1 的表現。突變型的 SRF99A 轉染作用降低 Mcl-1 的 mRNA 及蛋白質，並經由共同轉染的實驗顯示具有抵抗上游野生型CK2α 對 Mcl-1 蛋白質的影響。綜合這些結果我們認為 CK2α對 SRF 的訊息調控影響包括對 Mcl-1 的表現。且這條訊息路徑所促進的 Mcl-1 蛋白質表現可能對魚藤酮處理所引發的細胞凋亡作用具有保護的效果。 / Protein kinase CK2 is a multifunctional serine/threonine protein kinase with many protein substrates and is ubiquitously expressed in mammalian cells to play an important role in cell cycle progression, transcription, and anti-apoptosis. The serum response factor (SRF) is a mammalian transcription factor which binds to serum response element (SRE) and mediates some gene transcriptions relevent to promote the cell survival. The Myeloid cell leukemia 1 (Mcl-1) belongs to the anti-apoptotic Bcl-2 family and its effect are involved in promoting cell viability. Previous studies have revealed that the DNA-binding activity of SRF is enhanced when it is phosphorylated by protein kinase CK2. The activation regulation of Mcl-1 by SRF has also been reported in other studies. However, the detailed cellular signaling has not been studied well. In the present study, we investigate whether the regulation of Mcl-1 expression through CK2-mediated SRF pathway is involved in its anti-apoptotic effects. The results from CK2 inhibitor TBB revealed that the phosphorylated SRF were reduced in a dose-dependent manner after 4 hr of TBB treatments in PC12 cells. The similar decreases were also observed in the mRNA and protein levels of Mcl-1. After a 24 hr exposure of PC12 cells to TBB, a decreased in phosphorylated SRF and Mcl-1 mRNA were observed; a decreased in Mcl-1 protein level was also detected, albeit to a lesser extent. On the other hand, transfection of the wildtype CK2α increased, whereas transfection of the catalytically inactive CK2αA156 mutant decreased phosphorylated SRF. Further, wildtype CK2α increased, whereas CK2αA156 mutant decreased the mRNA and protein levels of Mcl-1. Furthermore, the mutant SRF99A transfection decreased, the mRNA and protein levels of Mcl-1 and antagonized the up-regulatory effects of wildtype CK2α on Mcl-1 protein level in the co-transfection experiments. These results together suggest that CK2α-mediated SRF signaling is involved in the regulation of Mcl-1 expression, and this signaling pathway may involves the anti-apoptotic effects of Mcl-1 against rotenone treatment.

Les paramètres de surface contrôlent les fonctions des cellules, en coopération avec leurs codes génétiques. Des études récentes soulignent l'impact combiné des signaux chimiques, topographiques et mécaniques des substrats d'adhésion sur les processus de différenciation. Cette étude se focalise sur le paramètre énergétique, et plus spécialement, sur l'influence exercée par la distribution spatiale des énergies de surface sur la différenciation des cellules neuronales. Le modèle étudié est constitué par les cellules de la lignée PC12, capables de se différencier en neurones suite au traitement par le facteur de croissance nerveux (NGF). Les cellules sont cultivées sur des surfaces de verre modifiées par auto-assemblage de monocouches d'alkylsiloxanes ou de biopolymères. La modification de la nature chimique et du degré d'organisation des monocouches module la distribution des composantes dispersives et polaires de l'énergie de surface, à une échelle inférieure au micron. Sur des substrats très homogènes (dotés de terminaisons CH3, NH2, ou OH), l'adhésion des cellules PC12 est modulée par le degré d'affinité chimique, et peu de cellules initient des neurites. Inversement, sur des substrats localement très hétérogènes, les cellules adhèrent quel que soit le couple chimique produisant les hétérogénéités (NH2/OH ou CH3/OH), et elles génèrent un nombre important de neurites en moins de 48 h, sans traitement au NGF. Ce travail démontre que les hétérogénéités chimiques de surface exercent une influence critique sur les processus de régénérescence des cellules nerveuses, en induisant des gradients dans les énergies d'adhésion aux échelles nanométriques.