探討一氧化氮於缺氧環境中對神經細胞的保護作用

NO-mediated Neuroprotection under Hypoxic Conditions

劉昀宗(Yun-Chung Liu)

指導教授 ： 孟子青

國立臺灣大學/生命科學院/生化科學研究所/碩士(2011年)

https://doi.org/10.6342/NTU.2011.01883

摘要

摘要

神經細胞於大腦功能扮演重要角色，神經細胞軸突(Axon)可以傳遞訊息，控制生物行為。神經訊息傳導受阻於病理情況導致腦部功能嚴重喪失。中風，腦部疾病之一，是全世界致死疾病前三名，其中缺氧型中風(Ischemic stroke)是中風的主要類型。缺氧型中風經由血管阻塞使腦部缺氧，並引起腦部傷害。缺氧型中風對腦部造成傷害和引起行為活動失常，但是目前並不清楚基本的機制。Tau蛋白大量表現於軸突中，且可穩定細胞骨架中微管(Microtubule)結構，因此我們推測Tau蛋白於缺氧環境下失去其功能。本篇論文主旨是針對神經細胞Tau蛋白調控於缺氧環境扮演的角色功能探討。
我們於缺氧環境中觀察分化神經細胞Neuro-2a的神經纖維出現嚴重的縮減情形並伴隨著微管結構的崩解。此外我們也觀察到有趣的現象，缺氧刺激內生性的硫胱胺酸蛋白-3(Caspase-3)活化。而硫胱胺酸蛋白酶基質之一，Tau蛋白可能經由缺氧活化硫胱胺酸蛋白-3進行切割調控並失去穩固微管結構的功能。我們將以皮層神經細胞(Cortical neuron)進一步探討此假說。如同Neuro-2a，皮層神經細胞也經由缺氧引起神經纖維的縮減與微管崩解。我們將大量表現Tau蛋白的神經纖維認定為類軸突，並經缺氧處理造成此結構長度縮減。於類軸突結構我們可以觀察到Tau蛋白與微管結構的關聯性。我們得到重要的結果，當皮層神經細胞加入硫胱胺酸蛋白酶抑制劑Z-DEVD抑制硫胱胺酸蛋白酶，可以防止缺氧引起的Tau瓦解、微管崩解與神經纖維縮減。因此我們假設一氧化氮(Nitric oxide, NO)可以防止硫胱胺酸蛋白酶的活性。無論是Neuro-2a還是皮質神經細胞加入S-亞硝基半胱胺酸(S-nitrosocysteine, CSNO)於缺氧環境的確有效保持微管與軸突結構的完整性。此外我們利用共同培養皮層神經細胞與內皮細胞也證實一氧化氮的功效，內皮細胞釋放一氧化氮保護皮層神經細胞的軸突與神經纖維。於此處我們提出經由一氧化氮保持神經功能的新奇機制。

關鍵字

神經細胞 ； 缺氧 ； 微管 ； 神經纖維 ； 硫胱胺酸蛋白-3 ； Tau蛋白 ； 一氧化氮

並列摘要

Abstract
Neurons play an important role in brain function and transport signals through axon to control behavior. Pathological conditions that impair signal transduction of neurons lead to severe dysfunction of brain. Among such conditions, stroke is the third cause of death worldwide. Ischemic stroke is the major type of stroke and the brain damage is caused by blocking blood flow and result in low level of oxygen supply to tissue. Ischemic stroke-induced brain damage and motor dysfunction may result from neuron death and axon injury. However, the underlying mechanism remains elusive. We proposed that Tau, which is highly enriched in axon and stabilizes microtubule, may lose its function under low-oxygen conditions. Therefore, in this study we have focused on the regulatory role of Tau under hypoxia in cultured neuronal cells. When well-differentiated Neuro-2a (N2a) cells were cultured in hypoxia condition, we observed that neurites were undergoing significant retraction in conjugation with microtubule collapse. Interestingly, endogenous expression of Caspase-3 in N2a cells was activated in response to hypoxia, suggesting that Tau, which has been shown as a substrate of caspases, might be cleaved thus losing its protective role in maintenance of microtubule integrity. We tested this hypothesis in primary rat cortical neurons. Consistently, hypoxia insults in cortical neurons led to neurite retraction and microtubule collapse. The Tau-enriched neurite, which was regarded as an axon-like structure, also underwent profound shortening during the course of hypoxic treatment. Focusing on this axon-like structure, we observed that Tau was regarded in association with fragmentation of microtubule. Importantly, when caspases were inactivated by a specific inhibitor, hypoxia-induced degradation of Tau, fragmentation of microtubule and retraction of axon were prevented in cortical neurons. We then hypothesized that nitric oxide (NO) might inactivate caspase, thus exerting a protective role. Indeed, exposure of N2a cells and cortical neurons to S-nitrosocysteine (CSNO) resulted in significant preservation of microtubule integrity and axon structure against hypoxic insults. The protective effect of NO was illustrated by co-culturing cortical neurons together with endothelia under hypoxia. We showed that NO released from endothelia prevented axons and neurites of cortical neurons from hypoxia-mediated disruption. Taken together, we propose a novel NO-dependent mechanism for protecting neuronal functions, against hypoxia-induced insult.

並列關鍵字

neuron ； hypoxia ； microtubule ； neurite ； caspase-3 ； Tau ； nitric oxide

參考文獻

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