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This dissertation, Evaluation of Using All-trans-retinoic Acid to Differentiate Human Neuroblastoma SH-SY5Y Cells in Neurodegeneration Research by Kwok-wai, Lau, 劉國威, was obtained from The University of Hong Kong (Pokfulam, Hong Kong) and is being sold pursuant to Creative Commons: Attribution 3.0 Hong Kong License. The content of this dissertation has not been altered in any way. We have altered the formatting in order to facilitate the ease of printing and reading of the dissertation. All rights not granted by the above license are retained by the author. Abstract: Abstract of thesis entitled Evaluation of using all-trans-retinoic acid to differentiate human neuroblastoma SH-SY5Y cells in neurodegeneration research Submitted by LAU Kwok Wai For the degree of Master of Philosophy At The University of Hong Kong In August 2007 All-trans-Retinoic acid (RA) has long been used as a cell differentiation agent for numerous cell lines. RA-differentiated human neuroblastoma SH-SY5Y cells have been reported to be more neuronal like and used as an in vitro model for studying neurodegeneration diseases, such as Parkinson's disease (PD) and Alzheimer's disease (AD), neurotoxicity and screening for potential neuroprotective agents. However, neuroscience studies are also carried out in undifferentiated SH-SY5Y cells by different research groups, which raise a question on the necessity of RA- differentiation. Although RA has been reported to differentiate SH-SY5Y cells to become more neuronal like, its influence in the changes of neuronal properties as well as cell susceptibility to neurotoxins after differentiation is still unclear. Therefore, I aim to re-evaluate the effects of RA on the changes of neuronal properties as well as cells susceptibility to Parkinsonism mimetic toxins and beta amyloid peptide in RA- mediated differentiation. Firstly, I compared the protein expression of neuronal cell markers between RA-differentiated and undifferentiated SH-SY5Y cells by Western-blot and immunocytochemical analysis. Secondly, I examined the changes in intracellular signaling involved in RA-mediated differentiation by Western-blot analysis. Finally, I compared their differential responses to 6-hydroxydopamine (6- OHDA), 1-methyl-4-phenylpyridinium (MPP ) and beta amyloid peptide (Aβ). Neurotoxicity was determined by the release of lactate dehydrogenase (LDH), cell proliferation (MTT) assay and activity of caspase-3. The differentiation properties of RA are confirmed. However, whether RA- differentiated SH-SY5Y cells can mimic mature neurons is still questionable. Although there was an increase in expression of neuron specific enolase, synaptophysin and SAP97, expression of NeuN was decreased after differentiation. Besides, immunocytochemical staining of synaptophysin and SAP97 were not clearly shown as puntated form, which is different from the staining carried on actual neurons by other research groups. RA affected cell susceptibility to Parkinsonism mimetic + toxins. While 6-OHDA and MPP induced massive cell death of undifferentiated cells, RA-differentiated SH-SY5Y cells were less sensitive to the challenge of these toxins. That may be explained by activation of Akt by RA. On the other hand, Akt did not protect SH-SY5Y cells from Aβ neurotoxicity. A significant increase of caspase-3- like activity in response to Aβ was observed after differentiation, indicating that RA- differentiated SH-SY5Y cells were more susceptible to Aβ neurotoxicity. Taken together, our results have demonstrated that undifferentiated neuroblastoma cells maintain neuronal properties, and they are more susceptible to Parkinsonism mimetic toxins, which may attribute to activation of Akt survival pathway by RA-differentiation. On the other hand, RA-differentiated cells are more susceptible to Aβ neurotoxicity which may be explained by the increased neuronal property of the cells. Differentiation of

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