Parkinson‟s disease (PD) is the most common neurodegenerative movement disorder. It is characterized by the progressive degeneration of dopaminergic neurons in substantia nigra pars compacta (SNpc). Although the etiology of PD remains incompletely understood, emerging evidence suggests that iron homeostasis dysregulation may be involved. A pathological hallmark of PD is the formation of Lewy bodies, intra-cytoplasmic inclusions that are major composed of α-synuclein (α-syn). α-synuclein is encoded by the SNCA gene. It is generally believed that α-synuclein aggregation is a main pathogenic feature and the cause of PD. Previous in vitro studies have provided direct evidence showing that iron could interact with α-synuclein and facilitate its aggregation. Nevertheless, the exact role of iron in the pathogenesis of PD is still inconclusive, and so far no studies have proved the interaction between iron and α-synuclein in vivo. / Here, based on a Drosophila model, we tested the hypothesis that the interaction between iron and α-synuclein accumulation accelerates the pathogenesis of PD, and that restoring brain iron homeostasis provides neuroprotective effects against PD. In our present studies, two groups of Drosophila, including w¹¹¹⁸ control and mutant α-synuclein A53T Drosophila were cultured under normal- (normal medium) and high-iron diet (medium added with 30mM ferric ammonium citrate (FAC)) for up to 30 days. During chronic iron treatment, startle-induced negative geotaxis assay was conducted every ten days to test the locomotor ability in the flies. After that, whole-mount immunostaining was used to assess dopaminergic neuronal survival. These flies were also collected and subjected to the quantification of brain iron content for the characterization of the brain iron content status. Furthermore, quantitative real-time PCR and western-blot analysis were conducted to investigate the amount of various α-synuclein conformations. / In the first part, we observed that α-synuclein A53T fly exhibited age-related increase of brain iron content compared with age-matched control. These were accompanied by shorter life-span, locomotor dysfunction, and TH-positive neuronal loss in PPM1/2 and PPM3 cluster. Meanwhile, we have demonstrated that neuronal toxicity and motor deficits were associated with increased proteinase K resistant, insoluble α-synuclein rather than the total amount of protein level. The insoluble α-synuclein was regarded as α-synuclein aggregation. / In the second part, we found that in α-synuclein A53T fly, excessive iron uptake aggravated locomotion defects and led to specific TH positive neuronal loss in cluster PPM3 after 30 days of iron treatment. Moreover, the excessive iron-induced neurological toxicity and motor dysfunction were also associated with increased α-synuclein aggregation. Overall, these two sets of results suggest that abnormal up-regulation of brain iron content may be associated with α-synuclein, and contribute to the pathogenesis of PD through α-synuclein aggregation-dependent mechanisms. / In the third part, we further explored the potential neuroprotective effect of restoring brain iron homeostasis in PD. We made use of genetic modification to manipulate iron-transport protein DMT1 expression, in turn to identify the protective effect of decreasing brain iron content in α-synuclein Drosophila model. Our present results proved that inactivation of Malvolio in α-synuclein A53T fly can suppress the increase of brain iron contents, and can also prolong life span, partially ameliorate locomotion deficits, and attenuate TH positive neuronal loss in α-synuclein A53T fly. In addition, these beneficial effects might occur through the inhibition of α-synuclein aggregation in α-synuclein A53T fly. Consequently, this result implicates that reducing brain iron by inactivation of iron up-take protein DMT1 can inhibit α-synuclein aggregation and provide beneficial effect on DA neuronal survival in PD model. / In conclusion, we demonstrated that : (1) abnormal up-regulation of brain iron content may be associated with α-synuclein and contributes to the pathogenesis of PD through α-synuclein aggregation-dependent mechanisms; (2) iron uptake protein DMT1 may serve as a potential therapeutic target for alleviating aberrant iron accumulation and retards the progression of neurodegeneration in PD. / 帕金森氏病（PD）是最常見的神經退行性疾病之一，其病理學特徵是黑質緻密部（SNpc）的多巴胺能神經元退行性變性。和，其中α-突觸核蛋白（α-synuclein）是Lewy小體的主要成分。雖然帕金森氏病的發病機制仍不十分清楚，隨著研究的進展，越來越多的證據表明鐵穩態失調可能是其中一個重要的致病因素。細胞內Lewy小體聚集物的形成是帕金森病的一個病理標誌物，其主要的成分是α-synuclein蛋白。α-synuclein是由SNCA基因編碼的蛋白。其聚集通常被認為是帕金森病的一個主要的病理特徵，同時也是導致帕金森病的一個原因。之前的研究證據表明，在體外實驗中，鐵能夠與α-synuclein相互作用並促進α-synuclein的聚集。然而，鐵和α-synuclein的相互關係在帕金森病的發病機制中的確切作用仍不十分確定。 / 我們的課題是基於果蠅模型來驗證腦鐵增加導致的α-synuclein聚集加速了帕金森病的病理進程，且恢復腦鐵平衡可以保護帕金森病人多巴胺能神經元的假說。在我們目前的研究中使用了兩組不同基因型的果蠅，其中包括w¹¹¹⁸對照組和突變體α-synA53T果蠅。兩組果蠅分別同時餵養正常食物（正常培養基）和高鐵食物（30mM檸檬酸鐵銨（FAC））。經過30天的慢性鐵處理，分別收集不同組的果蠅並測定其腦鐵含量用於對不同組果蠅的腦鐵含量進行定量分析。在進行鐵處理的30天期間，每隔10天進行一次趨地性行為學實驗用於評價不同組果蠅的運動能力。行為學實驗過後，可使用整腦免疫染色法來觀察果蠅多巴胺能神經元的存活情況。此外，還运用实时定量PCR和蛋白免疫印跡分析法來檢測不同組果蠅體內α-synuclein mRNA和蛋白的表達情況。 / 在第一部分的實驗中我们發現，與年齡相當的對照組相比，α-synA53T果蠅表現為明顯增高的腦鐵含量，並伴隨著壽命短，運動功能障礙以及PPM1/2和PPM3多巴胺能神經元簇的TH-陽性神經元丟失。同時我們發現，α-synA53T果蠅的神經元的毒性和運動障礙與α-synuclein的蛋白總量無關，而可能與在蛋白酶K中穩定的不溶性α-synuclein蛋白的增加相關，這部分α-synuclein的增加被認為與α-synuclein聚集有關。 / 此外在第二部分實驗中我們還發現，α-synA53T果蠅經過30天鐵處理後會加劇其運動功能障礙，並導致更嚴重的PPM3多巴胺能神經元簇的TH-陽性神經元丟失。並且這種由腦鐵含量增加而導致的神經毒性和運動功能障礙也與α-synuclein聚集增加有關。這兩部分的實驗結果表明，腦鐵含量異常增加調節α-synuclein聚集可能與帕金森病的發病機制有關。 / 在第三部分實驗中，我們進一步探討維持腦鐵穩態在PD中潛在的神經元保護作用。我們利用遺傳學手段調節鐵轉運蛋白DMT1的表達來研究腦鐵含量減少對α-synuclein果蠅的保護作用。目前的結果表明，降低α-synA53T果蠅中DMT1同源基因-Malvolio的表達可以抑制隨著年齡增加而顯著增加的腦鐵含量，並且可以延長果蠅的壽命，部分改善α-synA53T果蠅的運動功能障礙，以及完全減輕了TH陽性神經元的丟失。這些保護作用可能是由於抑制了α-synA53T果蠅中α-synuclein的聚集。因此，這些結果顯示，通過降低DMT1的表達來減少腦鐵含量DMT1可以抑制α-synuclein蛋白聚集並對神經元有保護作用。 / 綜上所述，我們的實驗結果證明了：（1）腦鐵含量異常增加調節α-synuclein聚集可能與帕金森病的發病機制有關; （2）鐵攝取蛋白DMT1可作為一個潛在的選擇性鐵螯合劑治療靶標來減緩帕金森病神經退行性進程。 / Zhu, Zhoujing. / Thesis (Ph.D.)--Chinese University of Hong Kong, 2014. / Includes bibliographical references (leaves 155-181). / Abstracts also in Chinese. / Title from PDF title page (viewed on 03, October, 2016). / Zhu, Zhoujing. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only.
| Identifer | oai:union.ndltd.org:cuhk.edu.hk/oai:cuhk-dr:cuhk_1290675 |
| Date | January 2014 |
| Contributors | Zhu, Zhoujing (author.), Ke, Ya (thesis advisor.), Chinese University of Hong Kong Graduate School. Division of Biomedical Sciences. (degree granting institution.) |
| Source Sets | The Chinese University of Hong Kong |
| Language | English, Chinese |
| Detected Language | English |
| Type | Text, bibliography, text |
| Format | electronic resource], electronic resource, remote, 1 online resource (xix, 181 leaves) : illustrations (some color), computer, online resource |
| Rights | Use of this resource is governed by the terms and conditions of the Creative Commons "Attribution-NonCommercial-NoDerivatives 4.0 International" License (http://creativecommons.org/licenses/by-nc-nd/4.0/) |
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