Functional analysis of Attractin-Mahogunin signaling

描述（由申请人提供）：神经退行性疾病，如阿尔茨海默病和帕金森病是常见的破坏性疾病，但导致神经元死亡的机制尚不清楚。在Attractin（Atrn）和Mahogunin（Mgn）基因中具有突变的小鼠发展脑的进行性海绵状变性，因此代表了新认识到的神经变性模型。由于色素细胞特异性途径的缺陷，这些突变体也具有毛色表型。两个不相关的表型Atrn和Mgn突变体之间的相似性表明这些基因的共同功能。虽然Atrn在神经元中的作用尚不清楚，但Mgn编码含有环指的蛋白质，其在体外充当泛素连接酶（E3）。泛素化蛋白聚集体的积累是许多神经退行性疾病的标志，而另一种E3蛋白Parkin的突变导致帕金森病的家族性形式。因此，我们推测，在Atrn和Mgn突变体中，泛素介导的特定靶蛋白降解的缺陷导致神经元死亡。古恩实验室的长期研究目标是确定如何。 短期目标是检验以下假设： 1)通过测试Mgn的RING结构域（体外E3活性所需的）是否是体内正常Mgn功能所必需的，以及通过使用酵母双杂交测定和使用生物化学测定来鉴定Mgn相互作用蛋白以测试这些蛋白是否被Mgn靶向用于泛素介导的衰变并在Mgn突变小鼠的脑中积累，证实了Mgn在体内起E3的作用。 2)为了证实Mgn和Atrn在相同的途径中起作用，使用标记基因表达、蛋白质印迹分析和/或免疫组织化学来检查Atrn突变体中的Mgn水平和定位，并确定被鉴定为用于泛素化的Mgn靶标的蛋白质是否在Atrn突变小鼠的脑中积累。 3)一种新发现的Atrn同源物Lurin通过Mgn通路发出信号， 缺乏Lurin并在正常和Atrn无效背景下检查它们的表型（包括Mgn靶的积累）。由于Mgn突变体显示在Atrn突变体中未观察到的一些表型，Lrn可以补偿一些组织中Atrn的损失，并且Lrn和Atrn两者的损失可以重现完整的Mgn表型。

DESCRIPTION (provided by applicant): Neurodegenerative disorders such as Alzheimer's and Parkinson's disease are common and devastating diseases, but the mechanism leading to neuronal death is not well understood. Mice with mutations in the Attractin (Atrn) and Mahogunin (Mgn) genes develop progressive spongy degeneration of the brain and thus represent newly recognized models of neurodegeneration. These mutants also have a coat color phenotype due to a defect in a pigment-cell specific pathway. The similarity between Atrn and Mgn mutants for two unrelated phenotypes suggests a common function for these genes. While the role of Atrn in neurons is unclear, Mgn encodes a RING-finger containing protein that acts as a ubiquitin ligase (E3) in vitro. Accumulation of ubiquitinated protein aggregates is a hallmark of many neurodegenerative disorders and mutations in another E3, Parkin, cause a familial form of Parkinson's disease. Thus, we hypothesize that defects in ubiquitin-mediated degradation of specific target proteins lead to neuronal death in Atrn and Mgn mutants. The long-term goal of research in the Gunn laboratory is to determine how. The short term goals are to test the following hypotheses: 1) that Mgn functions as an E3 in vivo, by testing whether the RING domain of Mgn (required for E3 activity in vitro) is essential for normal Mgn function in vivo, and by identifying Mgn-interacting proteins using a yeast two hybrid assay and using biochemical assays to test whether these proteins are targeted by Mgn for ubiquitin-mediated decay and accumulate in the brains of Mgn mutant mice. 2) that Mgn and Atrn act in the same pathway, using marker gene expression, western analysis and/or immunohistochemistry to examine Mgn levels and localization in Atrn mutants and determine whether proteins identified as Mgn targets for ubiquitination accumulate in the brains of Atrn mutant mice. 3) that a newly discovered Atrn homolog, Lurin, signals through the Mgn pathway, by generating mice lacking Lurin and examining their phenotype (including accumulation of Mgn targets) on normal and Atrn null backgrounds. As Mgn mutants display some phenotypes not observed in Atrn mutants, Lrn may compensate for loss of Atrn in some tissues and loss of both Lrn and Atrn may recapitulate the full Mgn phenotype.