| 摘要: | 二價金屬離子參與很多重要的生理機制，近年研究顯示二價金屬離子的失調與神經退化性疾病成因有關。在臨床上，有研究中發現ALS病人腦脊髓液中含有較高的金屬離子濃度；在實驗中，也有文獻指出過量的二價金屬離子會促進ALS病理相關蛋白TDP-43的錯位(Mislocalization)及堆疊。另外，在其他神經退化性疾病的研究中發現二價金屬離子的刺激會促進致病蛋白的傳遞，但二價金屬離子刺激是否會促使TDP-43蛋白釋出則尚未被探討。由於致病蛋白的傳遞，常常被視為細胞的一種保護機制，因此在本篇研究中，我們想要探討二價金屬離子的刺激是否會促使TDP-43蛋白產生致病的蛋白片段(C-TDP-43)，以及這些片段是否會藉由細胞間的傳遞，影響其他受體細胞。本篇研究發現二價金屬離子(鋅、鎂、鉛)會造成神經細胞內TDP-43錯位，其中以鉛離子最為顯著。接著，我們觀察到金屬離子刺激細胞後所收集的培養基，也會造成受體細胞內TDP-43的錯位，顯示培養基中可能含有致病蛋白。我們成功的從培養基收集到不同大小的囊泡，發現到其中包含不少的外泌體(Exosome)，而這些外泌體裡含有一定的TDP-43致病蛋白片段。藉由奈米粒子追蹤分析儀，我們進一步發現到鋅和鉛離子會促進細胞釋出更多的外泌體，配合電顯的結果，我們發現鉛離子刺激出的外泌體尺寸較小。而這些經由金屬離子刺激細胞後所釋出的外泌體除了會造成受體細胞內TDP-43蛋白錯位，也會降低其在細胞核內的流動性，甚至會促使免疫細胞發生發炎反應，顯示含有TDP-43致病蛋白片段的外泌體可能是造成細胞出現疾病相關表現型(Phenotype)的原因。進一步探討外泌體裡發現在鎂和鉛離子的刺激下會增加TDP-43致病蛋白片段的含量，並且鋅和鉛離子的刺激會促使其有較高的聚集程度。總結來說，我們的研究顯示二價金屬離子(鋅、鎂、鉛)的刺激，不只會造成神經供體細胞內TDP-43蛋白錯位，還會增進細胞釋放出更多的外泌體，其中以鉛離子最為顯著。而在鉛離子的刺激下，藉由外泌體釋放出的TDP-43致病蛋白片段顯示出較多的含量及較高的聚集程度，我們推測這是造成受體細胞產生較嚴重的疾病相關表現型的原因。因此我們調查的二價金屬離子中，鉛離子可能是TDP-43致病蛋白片段在細胞間傳遞的重要影響因子。;Divalent metal ions are participated in many physiological mechanisms. Current literature has shown the cause of neurodegenerative diseases is correlated with disrupted metal homeostasis. In clinical study, it was found that the concentration of metal ions in cerebrospinal fluid (CSF) in amyotrophic lateral sclerosis (ALS) patients is higher. In addition, divalent metal ions induce pathological TDP-43 formation in vivo. Though it is shown divalent metal ions enhance the transmission of pathological proteins, however, there is no direct evidence showing TDP-43 following the similar scenario. Since the cell-to-cell transmission of pathological TDP-43 is considered as a defensive mechanism, we would like to investigate whether if the stimulation of divalent metal ion enhanced the formation of pathological TDP-43 fragments (C-TDP-43) and transmitted C-TDP-43 into recipient cells. In this study, we found three divalent metal ions (Zn2+, Mg2+, Pb2+) cause TDP-43 mislocalization in neuronal cells. By characterizing the vesicles in cultured medium, we found that pathological TDP-43 fragments were included in exosomes. Furthermore, we found Zn2+ and Pb2+ induced more exosomes by nanoparticle tracking analysis. With the help of transmission electron microscopy, we also notice Pb2+ induced smaller exosomes than all the other cases. In addition, metal ion-induced exosomes not only cause TDP-43 mislocalization in recipient cells, but also decrease TDP-43 fluidity in nucleus. Even more, these exosomes induced inflammation response in microglia cells. Finally, we have shown that the stimulation Mg2+ and Pb2+ enhanced the content of C-TDP-43 in exosomes, while Zn2+ and Pb2+ enhanced the compactness of C-TDP-43 inside exosomes. Conclusively, we demonstrated three divalent metal ions (Zn2+, Mg2+, Pb2+) were able to cause TDP-43 mislocalization and induce exosome secretion. Within these cases, Pb2+ particularly induced significant C-TDP-43 fragments accumulation and more cell-to-cell transmission. Therefore, Pb2+ could play a dominate role on cell-to-cell transmission of pathological TDP-43 fragments. |
