Cellular, molecular and functional characterization of proteasomes in EAE

描述（由申请人提供）：我们实验室之前的研究表明，多发性硬化症（MS）及其动物模型实验性自身免疫性脑脊髓炎（EAE）的特征是严重的氧化应激和氧化蛋白在中枢神经系统中的积累，从而降低细胞活力并导致组织损伤。氧化/错误折叠蛋白质的积累通常是蛋白酶体缺陷的结果，蛋白酶体是负责蛋白质水解去除的酶机制。我们还发现，在多发性硬化症和慢性EAE中，蛋白酶体的肽解活性都显著降低，这可能解释了在这些疾病的神经退行性阶段观察到的氧化蛋白积累。虽然非常重要，但所有这些研究都是在使用荧光肽底物的组织匀浆中进行的，并且仍有待确定的关键问题是这些缺陷是否局限于特定细胞类型或影响所有中枢神经系统细胞的程度相同。此外，标准(s)和免疫(i)蛋白酶体核心颗粒（即s- 20s和i- 20s）的蛋白水解活性受到调节帽（19S和11S）和抑制剂结合的极大影响，在疾病过程中，它们在各种中枢神经系统细胞中的表达可能存在差异。在这个建议中，我们假设由于疾病活动神经元，少突胶质细胞，星形胶质细胞和小胶质细胞表达蛋白酶体复合物的独特模式，使得一些中枢神经系统细胞比其他细胞更不具备清除异常蛋白质的能力，从而更容易受到压力。为了验证这一观点，我们将首先通过双免疫组化测定EAE过程中蛋白酶体核心（s-20S和i-20S）和蛋白酶体帽（19S和11S）表达的时空格局及其与炎症和氧化应激程度的关系。然后，我们将从对照组和EAE小鼠的中枢神经系统分离神经元、少突胶质细胞、星形胶质细胞和小胶质细胞，并通过蓝色原生聚丙烯酰胺凝胶电泳和定量western blot分析检测每种复合物（20S、11S- 20S-11S、19S-20S-19S和19S-20S-11S）的水平。最后，我们将比较从对照和EAE组织中分离的各种蛋白酶体复合物消化氧化和泛素化蛋白的能力。这些探索性研究对于理解随着EAE从炎症阶段发展到神经退行性阶段，各种CNS细胞如何调节功能性蛋白酶体复合物的表达和组装，以及确定慢性EAE中蛋白酶体损伤的分子基础至关重要。鉴定蛋白酶体功能改变的细胞最终将有助于设计一种治疗方法，以减少EAE和MS中氧化负担的毒性后果。

DESCRIPTION (provided by applicant): Our laboratory has previously shown that multiple sclerosis (MS) and its animal model experimental autoimmune encephalomyelitis (EAE) are characterized by severe oxidative stress and the accumulation of oxidized proteins in the CNS, which in turn reduces cell viability and leads to tissue damage. Build-up of oxidized/misfolded proteins often occurs as a consequence of deficiencies in the proteasome, the enzymatic machinery responsible for their proteolytic removal. We have also discovered that in both MS and chronic EAE there is a significant reduction in the peptidolytic activities of the proteasome, which may explain the accumulation of oxidized proteins observed in the neurodegenerative stages of these diseases. While very important, all of these studies were carried out in tissue homogenates using fluorescent peptide substrates, and a critical issue that remains to be established is whether these deficits are localized to specific cell types or affect all CNS cells o the same extent. Furthermore, the proteolytic activity of the standard (s) and immuno (i) proteasome core particles (i.e. s-20S and i-20S) are greatly affected by the binding of regulatory caps (19S and 11S) and inhibitors, which may be expressed differentially in the various CNS cells during the course of the disease. In this proposal, we hypothesize that as a result of disease activity neurons, oligodendrocytes, astrocytes and microglia express unique patterns of proteasome complexes, making some CNS cells less equipped than others to remove abnormal proteins and thereby more susceptible to stress. To test this idea, we will first determine by double-immunohistochemistry the temporal/spatial pattern of proteasome core (s-20S and i-20S) and proteasome cap (19S and 11S) expression and their relationship to the extent of inflammation and oxidative stress during the course of EAE. We will then isolate neurons, oligodendrocytes, astrocytes and microglial cells from the CNS of control and EAE mice and measure the levels of each complex (20S, 11S- 20S-11S, 19S-20S-19S and 19S-20S-11S) by blue native-polyacrylamide gel electrophoresis and quantitative western blot analysis. Finally, we will compare the ability of the various proteasome complexes isolated from control and EAE tissues to digest oxidized and ubiquitinated proteins. These exploratory studies are essential for understanding how the various CNS cells regulate the expression and assembly of functional proteasome complexes as EAE progresses from the inflammatory to the neurodegenerative stages, and for determining the molecular basis for proteasome impairment in chronic EAE. Identification of the cell(s) with altered proteasome function will ultimately serve to design a therapeutic approach geared to decrease the toxic consequence of oxidative burden in EAE and MS.