第一型血基質氧化酵素及細胞自噬在神經退化性疾病保護機轉之探討

Neuroprotective mechanism of HO-1 and autophagy in neurodegenerative diseases

洪詩雅(Shih-Ya Hung)

指導教授 ： 符文美

國立臺灣大學/醫學院/藥理學研究所/博士(2009年)

https://doi.org/10.6342/NTU.2009.01355

摘要

Heme oxygenase-1 (HO-1) 在細胞遭受氧化壓力時會被誘發表現，用以催化血氧基質 (pro-oxidant heme) 裂解、轉換成一氧化碳 (CO)，鐵離子 (Fe2+) 及膽紅素 (bilirubin)。目前已有研究指出，HO-1蛋白在巴金森氏症 (Parkinson`s disease) 病人腦部有較高表現，顯示HO-1可能參與巴金森氏症病程發展。在本研究中我們將攜有人類HO-1基因的反轉錄病毒 (adenovirus-HO-1, Ad-HO-1) 與能誘導多巴胺神經元(dopaminergic neuron)死亡的神經毒物1-methyl-4-phenylpyridinium (MPP+) 一同打入大鼠中腦黑核區 (substantia nigra)。在打入大鼠中腦七天之後，以免疫染色 (immunohistochemistry)、高效率液態層析 (HPLC) 及免疫酵素分析 (ELISA) 這些方法去研究，大量表現HO-1對於黑核區多巴胺神經元數目、多巴胺 (dopamine) 含量及發炎反應物質(inflammatory cytokine) 含量是否有影響。本研究結果顯示，利用Ad-HO-1大量表現HO-1對於MPP+所誘發的黑核區多巴胺神經元的退化及死亡具有顯著抑制效果，並同時抑制發炎反應物質TNF-α及IL-1β的增加，提高多巴胺在損傷部位之含量。另外，我們也發現大量表現HO-1，可以促進黑核區神經滋養因子BDNF及GDNF之表現。據先前報導已知，Apomorphine可以在MPP+損傷的巴金森氏症動物模式中，誘發動物自發性旋轉，本研究中我們也發現HO-1可大幅抑制此自發性旋轉動作。另一方面，我們也發現HO-1酵素抑制劑ZnPPIX會促進MPP+所誘發的大鼠朝損傷側旋轉動作，同時也大幅降低損傷側多巴胺含量。HO-1在離體培養的大鼠胚胎中腦組織 (in vitro midbrain neuron-glia co-culture) 中具有類似的保護作用，而經由大量表現HO-1會在星狀細胞 (astrocyte) 中增加BDNF及GDNF表現；在多巴胺神經元中增加BDNF表現。我們的研究顯示，大量表現HO-1不管在in vitro 與 in vivo中對MPP+所誘發的多巴胺神經元死亡模式都具有相當良好的保護效果。內生性HO-1的產生在巴金森氏症的病程扮演相當重要角色，抑制HO-1的產生會加重病情。

由於已知HO-1在細胞遭受氧化壓力時，會被誘導表現以保護細胞、避免損傷。且HO-1及HO-2會分解相同受質 (substrate)，也就是血氧基質去產生一氧化碳 (CO)，鐵離子 (Fe2+) 及膽紅素 (bilirubin)。目前研究指出，膽紅素是一個極強的抗氧化與神經保護劑。而BDNF及GDNF這兩種神經滋養因子在多巴胺神經元的存活及神經元的型態、分化則扮演極重要角色。據我們先前研究結果顯示，將Ad-HO-1打入大鼠中腦黑核部位會增加此區BDNF、GDNF表現。接著，我們將就HO-1如何增加神經滋養因子表現之機轉加以探討。在離體培養的大鼠胚胎中腦組織，加入BDNF、GDNF抗體去中和細胞所分泌的BDNF、GNDF，發現會增加多巴胺神經元死亡率；但如果同時給予Ad-HO-1則可降低此種抗體中和所造成的神經元死亡。在Ad-HO-1打入大鼠中腦黑核區二十四小時後，由共軛焦雷射顯微影像數據顯示Ad-HO-1所產生的HO-1表現在多巴胺神經元、星狀細胞 (astrocyte) 及microglia。另外，相較於Ad打入側，Ad-HO-1打入側會誘導產生26與21倍的BDNF、GDNF mRNA。HO下游產物之一 『膽紅素』 在glia-enriched culture中會透過活化ERK、PI3K-Akt及增加NFkB入核這些訊息傳遞路徑增加GDNF表現。另外，膽紅素也可在cortical neuron-enriched cultures以相似方式增加BDNF表現。同時，我們利用CO donor來研究HO另一個下游產物 『一氧化碳』 在增加神經滋養因子方面的機轉。我們發現 [Ru(CO)3Cl2]2 在glia及神經細胞則是透過活化sGC-PKG路徑增加神經滋養因子表現。本研究結果顯示HO-1及其下游產物 『膽紅素』 及 『一氧化碳』 會分別經由不同訊息傳遞路徑在神經及glia調控BDNF及GDNF表現。

細胞自噬 (autophagy) 是一個細胞內的分解系統，主要是分解細胞內失去功能的胞器以及易糾結 (aggregation-prone)、不易分解的蛋白分子。目前研究已指出，阿滋海默症 (Alzheimer’s disease) 的主要產生原因之一是β-amyloid (Aβ; β類澱粉蛋白) 這種易糾結的蛋白片段沈積在神經細胞外部，造成神經死亡。而研究也發現阿滋海默症患者腦部有大量細胞自噬小體 (autophagosome) 聚積。直至目前為止，細胞自噬這個負責細胞內清除分解系統與阿滋海默症病發是否相關？以及細胞自噬是否影響Aβ所造成的神經元死亡？亦或是細胞外的Aβ能否被運送至胞內被代謝？仍然是未知。為了研究細胞自噬機制是否與Aβ所造成的神經元死亡有關， 我們在SH-SY5Y 細胞內大量表現攜帶EGFP 的LC3 基(SH-SY5Y/EGFP-LC3)。結果顯示，外加Aβ25-35、Aβ1-42或是血清缺乏的情形下皆可引發SH-SY5Y/EGFP-LC3 強烈細胞自噬反應。共軛焦顯微影像更進一步證實，培養液中外加的Aβ1-42會進入SH-SY5Y/EGFP-LC3 細胞內並與EGFP-LC3-II
autophagosome 共存 (colocalization) 。如果用α-bungarotoxin (α-BTX;α7nAChR高度專一性阻斷劑) 阻斷α7nAChR 功能，發現α-BTX 會大幅增加Aβ所造成
SH-SY5Y 細胞死亡。另一方面，我們也發現nicotine (nAChR 致效劑) 會增強SH-SY5Y/pEGFP-LC3 的細胞自噬反應，且對Aβ所造成的神經死亡具有保護作
用。在初級培養的大鼠海馬迴神經元中，我們也證實nicotine 對A 所造成的海馬
迴神經元死亡具有良好的保護作用；而α-BTX 則是促進神經元死亡。運用siRNA
技術去阻斷Atg7 形成 (可經由細胞自噬機制之上游步驟抑制autophagosome 形成)
或是抑制細胞中α7nAChR 表現量，皆可明顯提升Aβ所造成的SH-SY5Y 細胞死
亡。共軛焦顯微影像數據也顯示，nicotine 會促進Aβ與autophagosome 共存
(colocalization)。以上結果顯示，α7AChR 可能扮演一個負責攜帶並鍵結細胞外Aβ
(eAβ) 的角色，藉此把細胞外Aβ攜入神經元內進行細胞自噬之分解作用，進而降
低A 所造成的神經元死亡。我們的研究結果證實，細胞自噬機制在Aβ所造成的
神經元死亡扮演『神經保護』作用。如果細胞內細胞自噬機制發生缺損，這些細
胞外糾結、聚積的A 可能無法被清除而引發神經元死亡。

關鍵字

第一型血基質氧化酵素 ； 細胞自噬 ； 神經退化性疾病 ； 神經保護

並列摘要

Heme oxygenase-1 (HO-1) is up-regulated in response to oxidative stress and catalyzes the degradation of pro-oxidant heme to carbon monoxide (CO), Fe2+ and bilirubin. Intense HO-1 immunostaining in the brain of Parkinsonism is demonstrated, indicating that HO-1 may be involved in the pathogenesis of Parkinsonism. We here locally injected adenovirus containing human HO-1 gene (Ad-HO-1) into rat substantia nigra concomitantly with 1-methyl-4-phenylpyridinium (MPP+). Seven days after injection of MPP+ and Ad-HO-1, the brain was isolated for immunostaining, measurement of dopamine content and inflammatory cytokines. It was found that over-expression of HO-1 significantly increased the survival rate of dopaminergic neuron; reduced the production of TNF-α and IL-1β in substantia nigra; antagonized the reduction of striatal dopamine content in MPP+-lesioned side and also up-regulated BDNF and GDNF expression in substantia nigra. Apomorphine-induced rotation following MPP+-treatment was also inhibited by Ad-HO-1. On the other hand, inhibition of HO enzymatic activity by ZnPPIX facilitated the MPP+-induced rotatory behavior and enhanced the reduction of dopamine content. HO-1 over-expression also exerted protection of dopaminergic neurons against MPP+-induced neurotoxicity in midbrain neuron-glia co-cultures. Over-expression of HO-1 increased the expression of BDNF and GDNF in astrocytes and BDNF in neurons. Our results indicate that HO-1 induction exerts neuroprotection both in vitro and in vivo. Endogenous induction of HO-1 is involved in the neuroprotection in Parkinsonism.

We then examined the action of downstream products of HO-1, bilirubin and CO. Bilirubin is a potent antioxidant and neuroprotectant. Neurotrophic factors of BDNF and GDNF also play important roles in survival and morphological differentiation of dopaminergic neurons. We have previously found that HO-1 induction by adenovirus containing human HO-1 gene (Ad-HO-1) in substantia nigra of rat increases BDNF and GDNF expression. As mentioned above, HO-1 in the enhancement of neurotrophic factor expression. Treatment of anti-BDNF/GDNF antibody significantly enhanced dopaminergic neuronal death and Ad-HO-1 co-treatment was able to antagonize the apoptosis effect. Injection Ad-HO-1 into substantia nigra of adult rat for 24 h, the confocal imaging shows that HO-1 induction appeared in dopaminergic neuron, astrocyte and microglia. HO-1 induced-BDNF/GDNF mRNA expression in substantia nigra was 26/21 folds of contralateral Ad-injected side. The downstream product of bilirubin also increased GDNF expression in glia-enriched cultures through ERK and PI3K-Akt pathways, which further enhanced NF-kB (p65) nuclear translocation. In addition, bilirubin also enhanced BDNF expression through similar pathway in cortical neuron-enriched cultures. We also examined the effect of another HO product of CO by using CO donor. [Ru(CO)3Cl2]2 induced neurotrophic factor expression via sGC-PKG pathway in both neuron and glia. Our results indicate that the downstream products of HO-1, bilirubin and CO, modulate BDNF and GDNF expression in neuron and astrocyte.

Autophagy is a degradation pathway for the turnover of dysfunctional organelles or aggregated proteins in cells. Extracellular accumulation of β-amyloid peptide has been reported to be a major cause of Alzheimer's disease (AD) and large numbers of autophagic vacuoles accumulate in the brain of AD patient. However, how autophagic process is involved in Aβ-induced neurotoxicity and how Aβ peptide is transported into neuron and metabolized is still unknown. In order to study the role of autophagic process in Aβ-induced neurotoxicity, EGFP-LC3 was over-expressed in SH-SY5Y cells (SH-SY5Y/pEGFP-LC3). It was found that treatment with Aβ25-35, Aβ1-42 or serum-starvation induced strong autophagy response in SH-SY5Y/pEGFP-LC3. Confocal double-staining image showed that exogenous application of Aβ1-42 in medium caused the co-localization of Aβ1-42 with LC3 in neuronal cells. Concomitant treatment of Aβ with a selective α7nAChR antagonist, α-bungarotoxin (α-BTX), enhanced Aβ-induced neurotoxicity in SH-SY5Y cells. On the other hand, nicotine (nAChR agonist) enhanced the autophagic process and also inhibited cell death following Aβ application. In addition, nicotine but not α-BTX increased primary hippocampal neuronal survival following Aβ treatment. Furthermore, using Atg7 siRNA to inhibit autophagosome formation in an early step or α7nAChR siRNA to knockdown α7nAChR significantly enhanced Aβ-induced neurotoxicity. Confocal double-staining image shows that nicotine treatment in the presence of Aβ enhanced the co-localization of α7nAChR with autophagosomes. These results suggest that α7nAChR may act as a carrier to bind with eAβ and internalize into cytoplasm and further inhibit Aβ-induced neurotoxicity via autophagic degradation pathway. Our results suggest that autophagy process plays a neuroprotective role against Aβ-induced neurotoxicity. Defect in autophagic regulation or Aβ-α7nAChR transport system may impair the clearance of Aβ and enhance the neuronal death.

並列關鍵字

HO-1 ； autophagy ； neurodegenerative diseases ； neuroprotection

參考文獻

Amersi F, Buelow R, Kato H, Ke B, Coito AJ, Shen XD, Zhao D, Zaky J, Melinek J, Lassman CR, Kolls JK, Alam J, Ritter T, Volk HD, Farmer DG, Ghobrial RM, Busuttil RW and Kupiec-Weglinski JW (1999) Upregulation of heme oxygenase-1 protects genetically fat Zucker rat livers from ischemia/reperfusion injury. J Clin Invest 104(11):1631-1639.

Baranano DE, Rao M, Ferris CD and Snyder SH (2002) Biliverdin reductase: a major physiologic cytoprotectant. Proc Natl Acad Sci U S A 99(25):16093-16098.

Barnham KJ, Masters CL and Bush AI (2004) Neurodegenerative diseases and oxidative stress. Nat Rev Drug Discov 3(3):205-214.

Beal MF (2003) Mitochondria, oxidative damage, and inflammation in Parkinson's disease. Ann N Y Acad Sci 991:120-131.

Blommaart EF, Krause U, Schellens JP, Vreeling-Sindelarova H and Meijer AJ (1997) The phosphatidylinositol 3-kinase inhibitors wortmannin and LY294002 inhibit autophagy in isolated rat hepatocytes. Eur J Biochem 243(1-2):240-246.