Cellular pathology of Alexander Disease

我们建议检查中间丝(IF)组织和细胞应激反应的几个方面，这可能与理解Alexander病的病理生理学密切相关。因此，IF蛋白的过度表达可能导致细丝的异常组织，从而导致小HSPs、α-β-晶体蛋白和Hsp27的转录和翻译上调。我们还必须考虑到Alexander病是一种疾病，在一种细胞类型(星形胶质细胞)上表达的突变基因会导致另一种细胞类型(少突胶质细胞)的退化和/或异常发育。我们提出了三个具体目标：1)突变型GFAP是如何在细胞中表达的，它们是否导致IF的异常组织？是否存在IF的积累？如果周转，突变型GFAP的表达会改变吗？2.IF的积聚是否导致细胞应激反应，其中部分导致小HSPs表达上调？如果是这样的话，产生这种应激反应的机制(S)是什么？如果积聚诱导了小的热休克蛋白，我们将研究可能支持这种作用的细胞内应激信号通路，重点是HSF1激活、MAP激酶激活(ERK1/2、JNK、p38激酶)、核因子-kappaB激活和蛋白激酶-N激活。3.在中枢神经系统(星形胶质细胞)的一种细胞类型中，突变蛋白的表达如何对另一种细胞类型(少突胶质细胞)产生有害影响？细胞应激反应是这种联系的重要组成部分吗？星形胶质细胞的病理改变是否干扰少突胶质细胞的分化和/或髓鞘形成？我们将研究IF聚集可能产生的进一步后果，重点关注“应激”星形胶质细胞调节细胞因子的可能性。这些实验将集中在一种细胞类型(星形胶质细胞)中蛋白质的积累如何似乎对另一种细胞类型(少突胶质细胞)产生有害影响，这是从细胞因子对少突胶质细胞有毒的实验中获得的理论基础。此外，我们还将检查其他潜在的有毒物质，包括活性氧物种。如果正在构建的转基因小鼠的神经病理显示少突胶质细胞发育和/或髓鞘形成缺陷，那么我们将使用细胞培养系统直接检查表达GFAP突变的星形胶质细胞和少突胶质细胞前体之间的相互作用。

We propose to examine several aspects of intermediate filament (IF) organization and cellular stress reactions that may be germane to an understanding of the pathophysiology of Alexander disease. Thus, the over-expression of IF proteins may lead to an abnormal organization of filaments, which subsequently leads to up-regulating the transcription and translation of the small hsps, alphabeta-crystallin and hsp27. We must also consider that Alexander disease is a disorder in which a mutant gene expressed on one cell type (astrocyte) results in degeneration and/or abnormal development of another cell type (oligodendrocyte). We propose three specific aims: 1) How are mutant GFAPs expressed in cells and do they result in an abnormal organization of IFs? Is there an accumulation of IFs? Does the expression of mutant GFAPs alter IF turnover. 2. Does the accumulation of IFs lead to a cellular stress response, part of which leads to the up-regulation of small hsps? If so, what are the mechanism(s) that produce such a stress response? If IF accumulation induces small hsps, we will investigate intracellular stress signal pathways that may underlie this effect, focusing on HSF1 activation, MAP kinase activation (ERK1/2, JNK, p38 kinase), NF- kappaB activation, and protein kinase-N activation. 3. How does the expression of a mutant protein in one cell type in the CNS (astrocytes) produce deleterious effects on another cell type (oligodendrocytes)? Is the cellular stress response an important part of this link? Do the pathological changes in astrocytes interfere with oligodendrocyte differentiation and/or myelination? We will examine possible further consequences of IF aggregation, focusing on the possibility that "stressed" astrocytes regulate cytokines. These experiments will focus on how the accumulation of a protein in one cell type (Astrocytes) appears to produce deleterious effects on another cell type (oligodendrocytes), the rationale taken from experiments in which cytokines are toxic to oligodendrocytes. In addition, we will examine other potentially toxic substances, including reactive oxygen species. If the neuropathology of the transgenic mice that is constructing suggest a defect in oligodendrocyte development and/or myelination, then we will examine directly the interactions between astrocytes expressing GFAP mutations and oligodendrocyte progenitors using a cell culture system.