Disease Mechanisms in Human Ubiquilinopathy

描述(申请人提供)：肌萎缩侧索硬化症(ALS)的特征是大脑和脊髓中的大型运动神经元退化，导致随意肌肉的进行性萎缩和瘫痪，呼吸衰竭，最终死亡。家族性ALS(FALS)约占ALS病例的10%，其中大部分(90%)表现为散发性ALS(SALS)。近20年来，ALS基因病因的发现一直是ALS研究的推动力。这些发现为发病机制和动物模型提供了新的见解。然而，尽管做出了这样的努力，ALS的全球治疗仍然是不可能的，因为不同病因下游的共同分子机制并不明显。显然，其他病因和共同下游机制的发现将极大地促进对ALS的分子理解和找到适当的治疗方法。在本申请中，我们建议在实现这些目标的方向上取得进展。我们已经发现UBQLN2突变是X连锁显性ALS和FTD类型的ALS/痴呆的原因。ALS患者的脊髓和皮质中有泛素2阳性包涵体，ALS/FTD患者的海马区有额外的包涵体，这是一种新颖、独特的病理改变。令我们惊讶的是，我们在迄今为止研究的所有SALS、ALS/FTD和FALS病例中都发现了类似的泛素2阳性包涵体。这些病例中没有一例有UBQLN2突变，这证明泛素2在整个ALS中起着翻译后的作用。我们最初的体外和体内数据表明，泛素蛋白酶体综合征(UPS)和自噬在突变的泛蛋白2存在的情况下存在功能障碍。此外，在表达致病UBQLN2突变(P497H)的转基因小鼠中，观察到认知和行为缺陷以及远场记录中长时程增强的降低。在这个项目中，我们提出了四个具体的目标来了解突变的泛素2的致病机制(S)。我们将在我们目前的基础上建立病理、认知和其他行为表型及其电生理相关性，并开发突变泛素2的小鼠模型。我们还将使用我们已经建立的ubqln2基因敲除小鼠模型，并开发ubqln1/ubqln2的ubqln1基因敲除和双基因敲除，以测试突变的ubiquilin2缺陷是否表现为ubiquilin2与UPS和自噬系统相关的功能丧失，或者突变的ubiquilin2是否表现出一种新的毒性特性。该项目的成功完成不仅将为了解X连锁肌萎缩侧索硬化症的致病机制提供深入的见解，而且还将迅速为更广泛的研究界提供有用的试剂，用于未来的研究以及筛选和测试潜在的治疗方法。该项目的成果对了解其他神经退行性疾病的发病机制和治疗也具有重要意义。 公共卫生相关性：肌萎缩侧索硬化症(ALS)是一种致命的神经退行性疾病，全世界估计有350,000人受到影响，其中50%在发病3年内死亡。在疾病的最后几年，这家人每年的费用接近20万美元，这种疾病无法接受任何治疗。在过去的20年里，ALS不同遗传原因的发现一直是ALS研究的推动力，它为ALS的发病机制提供了新的见解。显然，ALS的其他病因的发现和疾病机制的统一将极大地促进对ALS的分子理解和找到适当的治疗方法。我们发现一种名为UBQLN2的基因突变是一种遗传性ALS和ALS/痴呆症的原因，在散发性和家族性ALS以及ALS/痴呆症中也是一种令人兴奋和独特的病理。功能研究表明，在存在突变形式的泛素2的情况下，蛋白质降解途径受到损害。尽管蛋白质降解受损被认为与许多神经退行性疾病发展过程中有害蛋白质包涵体的形成有关，但这一途径突变的直接证据有限。因此，凭借ALS和ALS/痴呆的共同病理，泛素2为研究其突变对细胞蛋白质质量控制的影响提供了一个独特的机会，以期了解UBQLN2突变导致神经退化的致病机制。在这项研究中，我们将开发和分析合适的动物模型，以确定突变泛素2导致神经元蛋白质经济失衡的分子机制，从而导致突触形成障碍，并导致ALS和痴呆的临床症状。

DESCRIPTION (provided by applicant): Amyotrophic lateral sclerosis (ALS) is characterized by degeneration of large motor neurons in the brain and spinal cord, resulting in progressive wasting and paralysis of voluntary muscles, respiratory failure and ultimately death. Familial ALS (FALS) accounts for around 10% of all ALS cases, with the majority (90%) of the disease presenting as sporadic ALS (SALS). Discovery of genetic etiologies of ALS has been the driving force in ALS research for the last 20 years. These discoveries have provided fresh insight into pathogenesis and animal models. However, in spite of this effort, global treatments have not been possible for ALS because a common molecular mechanism downstream from the distinct etiologies has not become apparent. It is clear that discovery of additional etiologies and common downstream mechanisms will greatly advance the goals for a molecular understanding of ALS and in finding appropriate treatments. In this application we propose an advance in direction of those goals. We have discovered mutations in UBQLN2 as cause of X-linked dominant ALS and ALS/dementia of the FTD type. The pathology is novel, distinct and unique with ubiquilin2 positive inclusions in the spinal cord and cortex in ALS patients and additional inclusions in the hippocampus of patients with ALS/FTD. To our surprise we found similar ubiquilin2 positive inclusions in all cases of SALS and ALS/FTD and FALS cases studied thus far. None of those cases had mutations in UBQLN2, arguing for a posttranslational role for ubiquilin2 in ALS as a whole. Our initial in vitro and in vivo data suggest a dysfunction of the ubiquilin proteasome syndrome (UPS) and autophagy in the presence of mutant ubiquilin2. In addition, cognitive and behavioral deficits and decreased long term potentiation in far field recordings were observed in transgenic mice expressing a pathogenic UBQLN2 mutation (P497H). In this project, we propose four specific aims to understand the disease causing mechanism(s) of mutant ubiquilin2. We will establish pathological, cognitive and other behavioral phenotypes and their electrophysiological correlates in our current and to be developed mouse models of mutant ubiquiln2. We will also use our already established ubqln2 knockout mouse model and develop ubqln1 knockout and double knockouts of ubqln1/ubqln2 to test if mutant ubiquilin2 defects are manifestation of a loss of function of ubiquilin2 in relationto the UPS and autophagy systems or whether mutant ubiquilin2 exhibits a novel toxic property. Successful completion of this project will not only provide insight into understanding the pathogenic mechanism of X-linked ALS, but also rapidly provide the wider research community with useful reagents for future studies and for screening and testing potential therapies. The outcome of this project will also have important implications in the understanding of the pathogenesis and treatment of other neurodegenerative diseases. PUBLIC HEALTH RELEVANCE: Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disorder affecting an estimated 350,000 individuals worldwide, with 50% dying within 3 years of onset. The cost to the family approaches $200,000 per year in the final years of illness and the disease defies all treatment. Discovery of diverse genetic causes of ALS has been the driving force in ALS research for the last 20 years by providing fresh insight into mechanisms of disease. It is clear that discovery of additional causes of ALS and unifying mechanisms of disease will greatly advance the goals for a molecular understanding of ALS and in finding appropriate treatments. We have discovered mutations in a gene called UBQLN2 as a cause of a form of inherited ALS and ALS/dementia and an exciting and unique pathology in sporadic and familial ALS as well as ALS/dementia. Functional studies have revealed an impairment of protein degradation pathways in the presence of mutant form of ubiquilin2. Though impaired protein degradation is thought to be associated with the formation of detrimental protein inclusions in the development of many neurodegenerative disorders, direct evidence of mutations in this pathway is limited. Hence, ubiquilin2, by virtue of a common pathology across ALS and ALS/dementia presents a unique opportunity to study the effect of its mutations on cellular protein quality control with a view to understand the pathogenic mechanism by which the UBQLN2 mutations cause neurodegeneration. We will develop and analyze appropriate animal models in this study to identify molecular mechanisms by which mutant ubiquilin2 causes imbalance in the protein economy of neurons which result in impaired synapse formation and leads to clinical syndromes of ALS and dementia.