La maladie d’Alzheimer (MA) est une maladie neurodégénérative du système nerveux central qui se caractérise notamment par l’accumulation de peptide beta-amyloïde (Aβ) dans le tissus nerveux. Dans la première partie de cette thèse nous avons montré que l’interaction des oligomères Aβ1-42 de haut poids moléculaire avec la membrane plasmique des cellules PC12 différenciées ou des cellules nerveuses (neurones et astrocytes primaires) provoque des variations de la [Ca2+]i dépendant de l’activation des récepteurs NMDA. Dans la seconde partie nous avons montré qu’une pré-exposition des cellules PC12 et des cellules nerveuses à de faibles concentrations de peptide Aβ module l’interaction ultérieure des oligomères avec la membrane plasmique. Enfin dans le cadre d’une collaboration avec l’entreprise Innovative Health Diagnostics (IHD) nous avons participé à la caractérisation d’une sonde amyloïde fluorescente développée pour réaliser des tests de détection de la MA à partir d’échantillons sanguins. / Alzheimer’s disease (AD) is a neurodegenerative disease of the central nervous system which is characterized in particular by the accumulation of beta amyloïde peptide (Aβ) in nerve tissues. In the first part of this thesis we showed that the interaction of high molecular weight Aβ1-42 oligomers with the plasma membrane of differenciated PC12 or nerve cells (neurons and astrocytes) triggers variations of their depending on the activation of the NMDA receptors. In the second part we showed that a pre-exposure of PC12 and nerve cells with low concentrations Aβ1-42 of modulates the later interaction of oligomers with the plasma membrane. Finally in collaboration with the company Innovative Health Diagnostics (IHD) we participated in the characterization of a fluorescent amyloid probe developed to realize detection test of AD from blood samples.

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

No description available.

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.

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.

<p>The ß-carbolines norharman and harman structurally resemble the synthetic compound 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) that is known for its ability to damage neuromelanin-containing dopaminergic neurons of the substantia nigra and thereby induce parkinsonism. MPTP is, however, not normally present in the environment whereas the ß-carbolines are present in cooked food and tobacco smoke. </p><p>In this thesis it was demonstrated that norharman and harman had affinity to melanin and were retained in neuromelanin-containing neurons of frogs up to 30 days post-injection (the longest survival time examined). It was also demonstrated that norharman induced neurodegeneration, activation of glia cells and motor impairment in mice. Furthermore, this compound induced ER stress and cell death in PC12 cells. An in vitro model of dopamine melanin-loaded PC12 cells was developed in order to study the effect of melanin on norharman-induced toxicity. In this model, melanin seemed to attenuate toxicity induced by low concentrations of norharman. After exposure to the highest concentration of norharman, melanin clusters were disaggregated and there was an increased expression of stress proteins and caspases-3, known to be involved in apoptosis.</p><p>The polycyclic aromatic hydrocarbon, 7,12-dimethylbenz[<i>a</i>]anthracene was demonstrated to have a CYP1A1-dependent localization in endothelial cells in the choroid plexus, in the veins in the leptomeninges and in the cerebral veins of mice pre-treated with CYP1-inducers. </p><p>These results demonstrate that the distribution of environmental compounds could be influenced by the presence of neuromelanin and expression of CYP enzymes in the brain and that norharman may induce neurotoxic effects in vivo and in vitro.</p>

The ß-carbolines norharman and harman structurally resemble the synthetic compound 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) that is known for its ability to damage neuromelanin-containing dopaminergic neurons of the substantia nigra and thereby induce parkinsonism. MPTP is, however, not normally present in the environment whereas the ß-carbolines are present in cooked food and tobacco smoke. In this thesis it was demonstrated that norharman and harman had affinity to melanin and were retained in neuromelanin-containing neurons of frogs up to 30 days post-injection (the longest survival time examined). It was also demonstrated that norharman induced neurodegeneration, activation of glia cells and motor impairment in mice. Furthermore, this compound induced ER stress and cell death in PC12 cells. An in vitro model of dopamine melanin-loaded PC12 cells was developed in order to study the effect of melanin on norharman-induced toxicity. In this model, melanin seemed to attenuate toxicity induced by low concentrations of norharman. After exposure to the highest concentration of norharman, melanin clusters were disaggregated and there was an increased expression of stress proteins and caspases-3, known to be involved in apoptosis. The polycyclic aromatic hydrocarbon, 7,12-dimethylbenz[a]anthracene was demonstrated to have a CYP1A1-dependent localization in endothelial cells in the choroid plexus, in the veins in the leptomeninges and in the cerebral veins of mice pre-treated with CYP1-inducers. These results demonstrate that the distribution of environmental compounds could be influenced by the presence of neuromelanin and expression of CYP enzymes in the brain and that norharman may induce neurotoxic effects in vivo and in vitro.

No description available.