摘要:帕金森氏症 (Parkinson’s disease, PD) 主要是由於黑質緻密帶 (pars compacta of substantia nigra, SNpc) 的神經退化及紋狀體 (striatum) 缺乏多巴胺 (dopamine) 所致。而在PD患者SNpc,已知細胞質內會出現稱為路易氏體 (Lewy bodies) 的蛋白質聚集結構。黑質在生理上與氧化壓力 (oxidative stress)、粒線體功能障礙 (mitochondrial dysfunction)、激活毒性(excitotoxicity) 及炎症反應 (inflammation) 有關;但這些改變與神經退化過程間的關係仍不清楚。再者,從一些線索推測,ubiquitin-proteasome系統對於清除廢棄的蛋白質 (如:遭氧化損傷、突變及摺疊錯誤的蛋白質) 上的缺損是PD產生的主要原因。在一系列的研究中,我們計劃使用proteasome的抑制劑,carbobenzoxy-L-leucul-L-leucyl-L-leucinal (MG-132),以抑制26/20S proteasome的功能,導致α-synuclein/ubiquitin細胞質聚集的產生,而使大鼠出現的PD症狀。本計畫主要的假說是:以顯微注射方法將MG-132打入SNpc,應能引發PD在大鼠模式中產生。第一年我們將首先確認MG-132直接顯微注射入單側SNpc,是否能在大鼠成功產生PD症狀。我們將比較打入MG-132前後的局部活性,包括以酪氨酸水解酶免疫反應(tyrosine hydroxylase-immunoreactive, TH-ir) 檢測SNpc和腹蓋膜區 (ventral tegmental area, VTA) 中神經數目,以及使用穿透式電子顯微鏡 (transmission electron microscopy, TEM) 觀察MG-132處理後,微膠細胞 (microglia) 中粒線體
Abstract: The predominant lesion in Parkinson’s disease (PD) is the degeneration of pigmented neurons in the pars compacta of the substantia nigra (SNpc) and the deficient dopamine in striatum. The formation of intracytoplasmic protein aggregation known as Lewy bodies presents in SNpc in PD patients. Nigral pathology is associated with oxidative stress, mitochondrial dysfunction, excitotoxicity and inflammation; however, the relationship between these changes and the neurodegenerative process remain unclear. More recently, several lines of evidence suggest that failure of ubiquitin-proteasome system to clear unwanted proteins (e.g. oxidatively damaged, mutant and misfolded proteins) plays a major role in the etiopathogenesis of PD. In current study we plan to use a proteasome inhibitor, carbobenzoxy-L-leucul-L-leucyl-L-leucinal (MG-132), to inhibit 26/20S proteasomal function, which may cause subsequent formation of -synuclein/ubiquitin-positive cytoplasmic inclusions, to induce PD features in rats. Our central hypothesis is that microinjection of MG-132 into SNpc would induce Parkinsonism features in a rat model. In first year of proposal we will first determine whether MG-132, directly microinjected into unilateral SNpc, could successfully induced Parkinsonism in rats. We will determine the alterations of locomotion activity before and after administration of MG-132, measure the cell number of tyrosine hydroxylase-immunoreactive (TH-ir) neurons in the SNpc and ventral tegmental area (VTA) after administration of MG-132, and observe whether microglia are activated after administration of MG-132 by observing the ultrastructure of mitochondria of the microglia using transmission electron microscopy (TEM). Our previous observation has suggested that the subsequent increase and release of cytokines, such as interleukin-1 (IL-1) and tumor necrosis factor- (TNF-), after microglial activation contributes to the pathogenesis of PD in a rotenone-, a mitochondrial complex I inhibitor, induced PD rat model. Those results indicate the proinflammatory cytokines play an important role. Based upon this observation, we hypothesize that the increased cytokines in the CNS after microglial activation is involved in the pathogenesis of PD in a MG-132 rat model. We will then determine whether the concentrations of cytokines and the mRNA expressions are altered in several distinct brain regions, including hypothalamus, midbrain (which contains SNpc), striatum and cerebral cortex, after administration of MG-132 into the SNpc by Western blot and ribonucleus protection assay (RPA). TNF- binds to TNF receptor (TNFR) and leads to activate transcription factors NF-B through activation of IB kinase (IKK). Furthermore, IL-1 via IL-1 receptor type 1 activates adenylate cyclase act