Liver Kinase B1, a genetic risk factor for multiple sclerosis

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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.