Characterizing Tissue Specific Regulation of Mutant Lamin Protein Degradation

项目摘要 椎板病症是一种广泛的疾病，包括多系统疾病，如衰老 Hutchinson-Guilford早衰症和组织特异性疾病，如Emery-Dreifuss肌肉疾病 营养不良与神经病变--夏科-玛丽-图斯病。所有这些不同的疾病都可以由 然而，LMNA基因的突变是如何导致如此不同的疾病的 不完全理解。已经发现突变形式的层蛋白可以聚集在一起，这种聚集是 与细胞功能受损和疾病表型有关。因此，我们假设某些 由于无法进行组织特异性质量控制，组织容易受到特定的层粘连蛋白突变的影响 降解这些突变形式的机制，导致蛋白质聚集和细胞毒性。我们将测试 这一假设使用果蝇作为我们的模型系统。果蝇允许我们 操纵特定组织中的基因表达，使实验能够梳理出不同的层蛋白 突变会在特定的组织中降解。果蝇有两个人类LMNA基因的同源物，LAMC和 LAM Dm0，这些基因的突变模仿人类疾病。此外，引入等价物 导致人类疾病进入LAMC的突变导致层蛋白聚集和类疾病 表型。此外，在患者肌肉活检中下调的AMPK信号已经被 发现可以减少突变的LAMC在苍蝇肌肉中的聚集。另一方面，我们发现LAM Dm0 衰老过程中肌肉中的聚集，p38MAPK(P38Kb)和CASA复合体调节 LAM Dm0的降解。LAMC和LAM Dm0的一个不同之处在于，两个椎板都是 在肌肉中表达，只有LAM Dm0在神经元中表达。因为LAMC和LAM Dm0有不同的组织 我们还可以评估特定的蛋白质质量控制机制是否能够针对特定的突变体 每个组织中都有不同形式的层粘连蛋白。这将使我们能够确定疾病组织特异性是否是由于 蛋白质质量控制机制，降解某些突变形式的层蛋白，导致肌肉 例如，营养不良而不是神经病。因此，我们将1)确定p38Kb和CASA 复合体调节特定形式的突变LAMC蛋白的聚集，以及这如何有助于 肌肉缺陷，2)表征LAM Dm0中的特定疾病突变如何影响肌肉和 神经元，以及3)确定AMPK和p38Kb是在相同还是不同的途径上调节突变的Lamin 聚合。我们期望我们拟议的研究将提供新的见解，了解突变形式的层粘连蛋白是如何导致 在疾病状态下，如果激活不同的蛋白质质量控制机制可能被证明是有效的 治疗机制。

Project Summary Laminopathies are a wide range of disorders that include both multisystem disorders such as the aging disorder Hutchinson Guilford Progeria and tissue specific disorders such as Emery-Dreifuss Muscular Dystrophy and the neuropathy Charcot-Marie-Tooth disease. All of these different disorders can be caused by mutations in the LMNA gene, however, how mutations in one gene give rise to such different disorders is incompletely understood. Mutant forms of lamin protein have been found to aggregate, and this aggregation is associated with impaired cellular function and disease phenotypes. Therefore, we hypothesize that certain tissues are susceptible to specific lamin mutations due to the inability of tissue specific quality control mechanisms to degrade those mutant forms, leading to protein aggregation and cellular toxicity. We will test this hypothesis using the fruit fly Drosophila melanogaster as our model system. Drosophila allow us to manipulate gene expression in specific tissues, enabling experiments to tease apart how different lamin protein mutations are degraded in specific tissues. Flies have two homologues of the human LMNA gene, LamC and Lam Dm0, and mutations in these genes mimic human disorders. In addition, introducing the equivalent mutations that cause human disease into LamC results in lamin protein aggregation and disease-like phenotypes. Furthermore, AMPK signaling, which is downregulated in muscle biopsies from patients, has been found to reduce mutant LamC aggregation in the fly muscle. On the other hand, we find that Lam Dm0 aggregates in the muscle during aging, and that p38 MAPK (p38Kb) and the CASA complex regulate the degradation of Lam Dm0. One difference between LamC and Lam Dm0 is that while with both lamins are expressed in muscle, only Lam Dm0 is expressed in neurons. As LamC and Lam Dm0 have different tissues specificities, we can also assess if specific protein quality control mechanisms are able to target certain mutant forms of lamin in each tissue. This will allow us to determine if disease tissue specificity is due to the failure of protein quality control machinery to degrade certain mutant forms of lamin protein, resulting in muscular dystrophy rather than neuropathy, for example. Therefore, we will 1) determine if p38Kb and the CASA complex regulate the aggregation of specific forms of mutant LamC proteins and how this contributes to muscle defects, 2) characterize how specific disease mutations in Lam Dm0 affect aggregation in muscles and neurons, and 3) determine if AMPK and p38Kb act in the same or different pathways to regulate mutant lamin aggregation. We expect that our proposed study will provide new insights into how mutant forms of lamin result in a disease state and if activating different protein quality control mechanisms could prove to be an effective therapeutic mechanism.