Identifying Pathogenic Protein Aggregates in ALS through Autocatalytic Misfolding

描述（由申请人提供）：肌萎缩性侧索硬化症（ALS）是一种无法治愈的疾病，其特征是运动神经元不断死亡，导致瘫痪和死亡。90%的病例是零星发生的，这些病例的原因尚不清楚。强有力的证据表明，一些细胞蛋白或蛋白质的异常聚集导致散发性ALS，但这些蛋白质尚未得到最终鉴定，也没有确定聚集的原因。我们的建议背后的假设是，ALS中的蛋白质聚集是由自我繁殖或“自催化”蛋白质聚集过程驱动的，该过程类似于导致朊病毒疾病的聚集过程，但涉及的蛋白质不同于朊病毒蛋白质。我们的目标是利用最近开发的方法，即蛋白质错误折叠环扩增（PMCA），在体外有效地传播聚集的朊病毒蛋白，以鉴定这种聚集导致ALS的假定蛋白质。在大纲中，我们建议的程序如下：从散发性ALS死亡的人身上提取少量（“种子”）均质脊髓组织与大量正常脊髓组织混合。同时，准备正常脐带种子的匀浆。这两种均质混合物将采用修改后的PMCA程序。来自ALS脊髓的自催化聚集蛋白将导致相同的蛋白特异性地聚集在正常脊髓中。我们将比较两个样品中聚集的蛋白质。那些只有在ALS脊髓“播种”时才聚集的蛋白质很可能是自催化聚集体，并且可能是散发性ALS的原因。PMCA程序不是为我们提出的蛋白质组研究设计的，因此我们将系统地修改程序，以尽量减少非自催化聚集，同时保持自催化聚集，使用朊病毒蛋白作为假定的als引起蛋白的替代物。我们还将使用PMCA寻找TDP-43的自催化聚集，TDP-43是一种最近被发现在散发性ALS患者的脊髓神经元中聚集的蛋白质。这些研究有可能确定散发性肌萎缩侧索硬化症的病因，这种疾病每年导致近6000名美国人死亡，而且没有有效的治疗方法。可以确定治疗治疗的特定靶蛋白和致病过程。此外，如果成功，该方法可以很容易地修改，以用作潜在治疗化合物的高通量筛选工具。

DESCRIPTION (provided by applicant): Amyotrophic lateral sclerosis (ALS) is an incurable disease marked by a relentless dying-off of motor neurons that causes paralysis then death. Ninety percent of cases occur sporadically and the cause of these cases is not known. Strong evidence indicates that abnormal aggregation of some cell protein or proteins causes sporadic ALS, but these proteins have not been conclusively identified, nor has the cause of the aggregation been determined. The hypothesis behind our proposal is that protein aggregation in ALS is driven by a self-propagating, or "autocatalytic" process of protein aggregation that is analogous to the process of aggregation that causes prion diseases, but involves a protein different from the prion protein. We aim to exploit recently developed methods, known as protein misfolding cyclic amplification (PMCA), that efficiently propagate aggregated prion protein in vitro to identify this putative protein that aggregates to cause ALS. In outline, the procedure we propose is as follows: A small amount ("seed") of homogenized spinal cord tissue from persons who died of sporadic ALS will be mixed with a larger amount of normal spinal cord tissue. Simultaneously, a homogenate of normal cord seeded with normal cord will be prepared. The two homogenate mixes will be subjected to a modified PMCA procedure. Putative autocatalytically aggregating proteins from the ALS spinal cord will cause the same protein to specifically aggregate in the normal cord. We will compare the aggregated proteins in the two samples. Those proteins that aggregate only when "seeded" by ALS cord are likely to be autocatalytic aggregates and potentially the cause of sporadic ALS. PMCA procedures were not designed for the proteome-wide studies we proposed, so we will systematically modify the procedures to minimize non-autocatalytic aggregation while maintaining autocatalytic aggregation, using the prion protein as a surrogate for the putative ALS-causing protein. We will also use PMCA to look for autocatalytic aggregation of TDP-43, a protein recently identified to aggregate in spinal cord neurons of persons with sporadic ALS. These studies have the potential to determine the cause of sporadic ALS, a disease that kills almost 6000 Americans annually, and for which there is no effective treatment. A specific target protein and a pathogenic process for curative therapies may be identified. Further, if successful, the approach can be readily modified to use as an tool for high-throughput screening of potentially therapeutic compounds.