探討巴金森氏症致病基因PINK1變異對於蛋白酶體抑制劑誘導之抗氧化酶表現之影響

Abstract

巴金森氏症是一種慢性、漸進性發展的動作失調症，也是目前最常見的神經退化性疾病之一。它的主要特徵為大腦黑質緻密部中有大量多巴胺神經元受損的情形。目前在臨床上，僅施予症狀性的治療，而對於神經的死亡則尚未能有效抑制。雖然巴金森氏症的病因尚不明確，目前的許多研究中指出，粒線體功能缺失、氧化壓力、泛素-蛋白酶體系統的異常及不正常的蛋白積聚等，都可能是導致巴金森氏症的重要致病因素。 PINK1是一種粒線體上的膜蛋白，PINK1基因的突變已被證實會造成隱性遺傳的早發性巴金森氏症。在過去的文獻中指出，PINK1可能具有神經保護的功能，然而，PINK1的明確作用，目前仍在研究當中。 在本篇論文中，我們欲探討在蛋白酶體受到抑制的情況下，PINK1的突變對於神經細胞的影響及調控。因此，我們利用人類神經細胞瘤SH-SY5Y細胞株，使其表現PINK1基因G309D變異，並外給蛋白酶體抑制劑MG132。 在研究中，我們發現在給予MG132 (1 μM) 24小時後，會誘導抗氧化酵素HO-1及SOD2在神經細胞的表現量。然而，此現象在帶有PINK1G309D基因變異的神經細胞中，卻受到了抑制。為了進一步研究此情形背後的調控機轉，我們接著探討了調控抗氧化酵素表現的重要轉錄因子-Nrf2，是否同樣會受到PINK1G309D變異影響。研究結果顯示，MG132確實會誘導Nrf2在神經細胞內的mRNA及蛋白合成量；且藉由西方墨點法分析核內蛋白、以及透過免疫螢光染色實驗，我們更進一步觀察到Nrf2表現量除了被誘導而增加外，外給MG132會促進Nrf2轉移到細胞核內，而上述情形皆同樣受到PINK1G309D基因變異而抑制。此外，MG132誘導HO-1表現的作用會被p38-MAPK抑制劑、PI3K/Akt抑制劑、及自由基清除劑所拮抗；我們也觀察到外給MG132會促進細胞中p38-MAPK的磷酸化、而PINK1G309D變異則同樣會抑制此現象。最後，我們發現PINK1G309D的基因變異會加劇MG132所導致的神經死亡。 總結以上結果，本篇研究指出在蛋白酶體功能異常的情況下，PINK1的基因變異會損害PINK1原有的神經保護作用，顯示這可能為巴金森氏症相關基因PINK1的致病機轉之ㄧ。

Parkinson’s disease (PD), a chronic progressive movement disorder, is one of the most common neurodegenerative diseases. It is characterized by prominent loss of dopaminergic neurons in the substantia nigra pars compacta. No treatment is available to suppress the progression of neuron death, and the current medications only provide symptomatic relief. Although the etiology of PD is still not fully clarified, mitochondrial dysfunction, oxidative stress, ubiquitin proteasome system (UPS) impairment and abnormal protein accumulation have been proposed to play a major part in the pathogenesis of PD. PTEN-induced putative kinase 1 (PINK1) is a mitochondrial membrane integral protein. Mutations in PINK1 gene have been identified to cause early-onset autosomal recessive familial PD. Previous studies have suggested that PINK1 may provide a neuroprotective effect. However, the precise function of PINK1 still remains unclear. Here, we have addressed the question whether mutation of PINK1 affects cellular stress response to proteasome inhibition. To evaluate the issue, SH-SY5Y neuronal cells expressing PINK1 G309D mutant were challenged with MG132 (Z-Leu-Leu-Leu-al), the peptide aldehyde proteasome inhibitor. It was found that treatment with 1 μM MG132 for 24 h significantly induced the expression of the antioxidants, HO-1 and SOD2 in control SH-SY5Y cells. However, the antioxidants induction in response to proteasome inhibition was impaired by the expression of recombinant PINK1 G309D mutation. To elucidate the underlying mechanisms for the induction of antioxidative enzymes by proteasome inhibition, we further examined the influence of PINK1 G309D mutation on Nrf2, the critical factor in the regulation of inducible expression of antioxidants. It was demonstrated that MG132 induced the expression of Nrf2 both on the protein and mRNA levels, and the effect was also abrogated by the PINK1 G309D mutation. By using nucleus fraction and Immunofluorescent staining, it was observed that Nrf2 accumulated in the nucleus after proteasome inhibition, which was reduced by the PINK1 G309D mutation. In addition, the MG132-induced HO-1 induction was Akt- and p38 MAPK-dependent, and was inhibited by the application of the free radical scavenger, N-acetyl-L-cysteine (NAC). The phosphorylation of p38 MAPK following treatment of MG132 was inhibited in cells expressing the PINK1 G309D mutant. We also found that the MG132-induced neuronal death was enhanced by PINK1 G309D mutation. These results reveal a novel pathway by which the G309D mutation impairs the neuroprotective function of PINK1 under the stress of proteasome inhibition, which may be related to the pathogenesis of Parkinson’s disease.