Development of mouse models for autoinflammatory rare diseases

 描述（由申请人提供）：免疫蛋白酶体（i-蛋白酶体）是一种诱导型蛋白酶体，具有不同于标准蛋白酶体的蛋白水解活性。它们不仅在抗原呈递中起重要作用，而且在清除应激中积累的氧化蛋白中也起重要作用。它们还调节多种细胞类型中的细胞信号传导、炎症和细胞因子分泌。最近的纯合性作图和外显子组测序分析显示，i-蛋白酶体β亚基之一LMP 7（Psmb 8基因产物）在多种自身炎性罕见疾病中发生突变，如Nakajo-Nishimura综合征（肌萎缩-脂肪组织异常）、CANDLE综合征和常染色体隐性自身炎性综合征，其特征为关节挛缩、肌肉萎缩、小细胞性贫血和脂膜炎诱导的脂肪营养不良。在欧洲、美国和亚洲人群中发现了LMP 7的多个点突变，显示出相似的疾病表型。这些疾病的一个共同特征是脂肪组织的变形和功能障碍沿着其他组织如骨骼和肌肉的问题。虽然这样的点突变可能会导致问题，但使用小鼠模型来概括疾病状况以解决突变是否或如何导致疾病状态是至关重要的。我们的初步研究表明，LMP 7缺乏抑制体内脂肪积累和响应于禁食的脂肪动员，并损害葡萄糖稳态。我们还发现LMP 7敲低抑制3 T3-L1细胞中的脂肪形成。虽然初步结果极大地促进了我们对LMP 7在疾病相关表型中的功能的理解，但KO小鼠并不理想地重现人类疾病状况。该基金的目的是建立小鼠模型，可以密切重演LMP 7突变引起的自身炎症罕见综合征。复制疾病突变的遗传小鼠模型对于解释疾病机制和开发治疗干预措施将是重要的。我们将产生在LMP 7中携带点突变Thr 75 Met的小鼠。我们将使用TALEN辅助的基因打靶技术结合供体寡核苷酸将点突变引入小鼠基因组。我们证明了T75 M突变对于脂肪细胞分化和3 T3-L1脂肪细胞中的i-蛋白酶体活性是关键的，表明T75 M突变在小鼠细胞中具有功能和表型影响。为了进一步解释疾病的病理生理学，我们还将使用组织特异性小鼠模型，并确定巨噬细胞和脂肪细胞（脂肪组织中的两种主要细胞类型）中i-蛋白酶体在疾病表型中的作用。为此，我们已经制作了LMP 7 flox小鼠。小鼠模型是独特的资源，将大大有助于了解罕见疾病的病理生理学。它们也将有助于探索属于自身免疫性疾病以及代谢性疾病、癌症和神经退行性疾病类别的许多人类疾病。它们对于罕见疾病的治疗药物开发至关重要。

 DESCRIPTION (provided by applicant): Immunoproteasomes (i-proteasomes) are an inducible type of proteasomes that have proteolytic activities different from standard proteasomes. They play important roles not only in antigen presentation but also in removal of oxidized proteins that accumulate in stress. They also regulate cell signaling, inflammation and cytokine secretion in a variety of cell types. Recent homozygosity mapping and exome sequencing analysis revealed that one of the i-proteasomal beta subunits, LMP7 (Psmb8 gene product), is mutated in multiple autoinflammatory rare diseases, such as Nakajo-Nishimura syndrome (amyotrophy-fat tissue anomaly), CANDLE syndrome, and an autosomal-recessive autoinflammatory syndrome characterized by joint contractures, muscle atrophy, microcytic anemia, and panniculitis-induced lipodystrophy. There are multiple point mutations of LMP7 identified in European, United States and Asian populations that show similar disease phenotypes. A common feature of the diseases is deformation and dys-function of fat tissue along with problems in other tissues, such as bone and muscle. Although such a point mutation could cause problems, it is critical to recapitulate the disease conditions using mouse models to address whether or how the mutations cause the disease states. Our preliminary study showed that LMP7 deficiency suppresses fat accumulation in the body and fat mobilization in response to fasting and impairs glucose homeostasis. We also found that LMP7 knockdown suppresses adipogenesis in 3T3-L1 cells. Although the preliminary results greatly contributed to our understanding of the function of LMP7 in the disease-related phenotypes, the KO mice are not seen ideal to recapitulate the human disease conditions. The objective of this grant is to establish mouse models that can closely recapitulate the autoinflammatory rare syndromes caused by LMP7 mutation. The genetic mouse models reproducing the disease mutation will be important to decipher disease mechanisms and for development of therapeutic interventions. We will generate mice that harbor a point mutation Thr75Met in LMP7. We will use TALEN-assisted gene targeting technique combined with a donor oligonucleotide to introduce the point mutation into the mouse genome. We demonstrated that T75M mutation is critical for adipocyte differentiation and the i-proteasomal activity in 3T3-L1 adipocytes, indicating that T75M mutation has functional and phenotypic impact in murine cells. To further decipher the pathophysiology of the diseases, we will also use a tissue-specific mouse model and determine the roles of i-proteasomes in macrophages and adipocytes, the two major cell types residing in adipose tissue, in the disease phenotypes. For this, we have already made LMP7 flox mice. The mouse models are unique resources that will greatly contribute to understanding the pathophysiology of the rare diseases. They will also be useful to explore many human diseases belonging to the categories of autoimmune diseases as well as metabolic diseases, cancer and neurodegenerative diseases. They will be essential for therapeutic drug development for the rare diseases.