Development of mouse models for autoinflammatory rare diseases

 说明(申请人提供)：免疫蛋白酶体(I-蛋白酶体)是一种诱导类型的蛋白酶体，具有不同于标准蛋白酶体的蛋白分解活性。它们不仅在抗原呈递方面发挥重要作用，而且在清除在应激中积累的氧化蛋白方面也发挥着重要作用。它们还调节各种细胞类型中的细胞信号、炎症和细胞因子的分泌。最近的纯合性图谱和外显子序列分析显示，I-蛋白酶体β亚基之一LMP7(Psmb8基因产物)在多种罕见的自炎性疾病中发生突变，例如Nakajo-Nishimura综合征(肌萎缩-脂肪组织异常)、蜡烛综合征和一种常染色体-隐性自炎综合征，其特征是关节收缩、肌肉萎缩、小细胞性贫血和脂膜炎诱发的脂肪营养不良。在欧洲、美国和亚洲人群中发现了多个LMP7点突变，表现出相似的疾病表型。这种疾病的一个共同特征是脂肪组织的变形和功能障碍，以及骨骼和肌肉等其他组织的问题。尽管这样的点突变可能会引起问题，但使用小鼠模型来概括疾病状况是至关重要的，以解决突变是否或如何导致疾病状态。我们的初步研究表明，LMP7缺乏抑制了脂肪在体内的积累和对禁食的脂肪动员，并损害了血糖稳态。我们还发现，LMP7基因敲除抑制了3T3-L1细胞的脂肪生成。虽然初步结果有助于我们理解LMP7在疾病相关表型中的功能，但KO小鼠并不能理想地概括人类的疾病状况。这笔赠款的目的是建立能够准确再现LMP7突变引起的自发性炎症性罕见综合征的小鼠模型。复制疾病突变的小鼠遗传模型将对破译疾病机制和开发治疗干预措施具有重要意义。我们将产生在LMP7中含有Thr75Met点突变的小鼠。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T75M突变对脂肪细胞的分化和3T3-L1脂肪细胞的I-蛋白酶体活性至关重要，表明T75M突变对小鼠脂肪细胞的功能和表型有影响。为了进一步破译疾病的病理生理学，我们还将使用组织特异性的小鼠模型，并确定巨噬细胞和脂肪细胞中的I-蛋白酶体在疾病表型中的作用。为此，我们已经制作了LMP7 Flox小鼠。小鼠模型是独特的资源，将极大地有助于了解罕见疾病的病理生理学。它们还将有助于探索属于自身免疫性疾病类别的许多人类疾病，以及代谢性疾病、癌症和神经退行性疾病。它们将对治疗这种罕见疾病的药物开发至关重要。

 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.