Liver Kinase B1, a genetic risk factor for multiple sclerosis

在招募MS患者进行PBMC研究的过程中，一个有5个兄弟姐妹的家庭被诊断为 MS被发现。某些罕见肿瘤的共病存在导致测序 分析肿瘤抑制基因STK 11，和一个变异（小核苷酸多态性，SNP）， 鉴定大量DNA样本的基因分型表明，这种SNP存在于较高的DNA水平。 高加索人和非裔美国人队列中MS患者的水平。STK 11肝脏代码 激酶B1（LKB 1）在调节细胞代谢和炎症反应中起作用， T细胞、神经胶质细胞和少突胶质细胞。由于LKB 1在MS中的作用尚未得到表征， 由于脑星形胶质细胞在维持能量平衡和限制神经元的功能方面起着重要作用， 我们假设，减少星形胶质细胞LKB 1的表达或活性， 加重EAE。我们使用从星形胶质细胞敲除LKB 1的小鼠（“cKO小鼠”）的发现证实了 这一假设，并确定了在星形胶质细胞缺陷小鼠和细胞中改变的途径。 在这个项目中，我们将确定星形胶质细胞中LKB 1缺陷如何影响其代谢特性 （线粒体功能，以及可按需提供给神经元的乳酸盐的产生）。我们 将测试由星形胶质细胞制备的培养基（“条件培养基”，CM）对小胶质细胞的影响。 细胞，看看小胶质细胞活化是否增加。我们将星形胶质细胞CM添加到幼稚T细胞中，看看是否 CM将它们转化为破坏性表型（Th 1或Th 17;产生有毒物质的细胞 在EAE和MS期间）。我们将测试CM对神经元的影响，看看星形胶质细胞是否产生 神经毒性物质;并将CM添加到少突胶质细胞中以观察成熟是否减少。 在我们的研究中，我们发现星形胶质细胞cKO小鼠中增加最多的蛋白质之一是脂质运载蛋白2 LCN 2是一种参与其他疾病炎症反应的蛋白质，在大肠杆菌中高度表达。 星形胶质细胞然而，星形胶质细胞LCN 2在EAE中的作用尚不清楚。我们将产生新的老鼠 在那里星形胶质细胞LCN 2被敲低，看看是否能减少EAE疾病。我们还发现， 星形胶质细胞LKB 1 cKO小鼠，脊髓运动神经元发生广泛损伤， 脊髓，这可能导致MS患者的虚弱。损坏的原因尚不清楚， 然而，在这些神经元中有大量免疫球蛋白的积累表明， 这些细胞的IgG受体表达增加。实验来验证这一点。 最后，虽然我们的研究是使用从细胞中有条件地敲除LKB 1，但我们不知道是否会发生这种情况。 STK 11 SNP不会导致敲除，但会改变LKB 1水平，具有相同的效果。解决 为此，我们将使用一种新的方法从人外周血单核细胞中产生小胶质细胞。 虽然小胶质细胞不同于星形胶质细胞，但我们将能够比较小胶质细胞与星形胶质细胞的反应。 正常的野生型STK 11基因与具有STK 11 SNP的那些相比。 总的来说，这些研究将扩大我们对LKB 1如何调节MS发展的了解， EAE，并确定MS患者治疗干预的新靶点， LKB 1表达。

During the course of recruiting MS patients for a study of PBMCs, a family with 5 siblings diagnosed with MS was identified. The presence of a co-morbidity for certain rare tumors led to sequencing analysis of tumor suppressor gene STK11, and a variant (small nucleotide polymorphism, SNP) was identified. Genotyping of a large number of DNA samples showed that this SNP is present at higher levels in MS patients in both Caucasian and African American cohorts. STK11 codes for the Liver kinase B1 (LKB1) which has roles in regulation of cellular metabolism and inflammatory responses in Tcells, glial cells, and oligodendrocytes. Since the role of LKB1 in MS has not been characterized, and since brain astrocytes play an important role in maintaining energy balance and restricting immune responses, we hypothesized that reducing astrocyte LKB1 expression or activity would worsen EAE. Our findings using mice with LKB1 knocked out from astrocytes (“cKO mice”) confirmed this hypothesis and identified pathways that are altered in the astrocyte deficient mice and cells. In this project, we will determine how LKB1 deficiency in astrocyte effects their metabolic properties (mitochondrial function, and production of lactate which can be provided to neurons on demand). We will test the effects of the media prepared from the astrocytes (“Conditioned media”, CM) on microglial cells to see if microglial cell activation is increased. We will add astrocyte CM to naïve Tcells to see if the CM converts them into a damaging phenotype (Th1 or Th17; cells that produce toxic substances during EAE and MS). We will test effects of the CM on neurons, to see if the astrocytes produce neurotoxic substances; and add CM to oligodendrocytes to see if maturation is reduced. In our studies, we found that one of the proteins most increased in astrocyte cKO mice was lipocalin 2 (LCN2), a protein involved in inflammatory responses in other diseases, and highly expressed in astrocytes. However, roles for astrocyte LCN2 in EAE are not well known. We will generate new mice where astrocyte LCN2 is knocked down, to see if that reduces EAE disease. We also found that in astrocyte LKB1 cKO mice, there was extensive damage occurring to motor neurons in the spinal cord, which could contribute to weakness in MS patients. The cause of that damage is unknown, however findings that there is a large accumulation of immunoglobulins in those neurons suggests that these cells have increased expression of an IgG receptor. Experiments to test this are proposed. Finally, while our studies are using conditional knockout of LKB1 from cells, we do not know if the STK11 SNP, which does not cause knockout but alters LKB1 levels, has the same effect. To address this, we will use a novel method to generate microglial cells from human peripheral blood monocytes. Although microglia differ from astrocytes, we will be able to compare the responses of microglia with the normal, wildtype STK11 gene to those having the STK11 SNP. Overall, these studies will expand our knowledge of how LKB1 regulates the development of MS and EAE, and identify new targets for therapeutic interventions in MS patients who have deficiencies in LKB1 expression.