טכניון מכון טכנולוגי לישראל
הטכניון מכון טכנולוגי לישראל - בית הספר ללימודי מוסמכים  
Ph.D Thesis
Ph.D StudentMandel Ilana
SubjectMechanisms of Immune Dysregulation in Multiple Sclerosis:
The Modulation of Tight Junction Proteins (TJPs),
Apoptotic Processes and the
Ubiquitylation System
DepartmentDepartment of Medicine
Supervisors Professor Ariel Miller
Dr. Tamar Paperna
Full Thesis textFull thesis text - English Version


Abstract

Multiple sclerosis (MS) is a central nervous system (CNS) disease characterized predominantly by inflammation and demyelination. Our study focused the aberrant migratory process of immune cells through the blood brain barrier (BBB), apoptosis related proteins, and regulation by the ubiquitin-proteasome system (UPS) of proteins involved in these processes.

Tight junction protein (TJP) complexes are mainly expressed by endothelial cells (ECs) and are major contributors to the barrier properties of the BBB. We report the expression of the TJPs claudin 1 and 5 in leukocytes, predominantly in T and B lymphocytes. Various T cell activators caused claudin 1 upregulation in leukocytes, as well as in Jurkat T cells and U-937 monocytic lines. Claudin 5 RNA levels were significantly higher in leukocytes from MS patients in relapse compared to patients in remission or healthy individuals. Glucocorticoid (GC) treatment of MS patients led to a significant reduction in the RNA levels of the TJ genes JAM3 and claudin 5 RNA and protein levels. Similarly, GC treatment reduced claudin 1 RNA levels in T and monocytic cell lines and claudin 5 RNA levels in T cells. Interferon-β (IFN-β) treatment of MS patients did not affect TJ expression levels in leukocytes; however, a correlation between claudin 5 RNA levels prior to treatment and response phenotype was detected.

Since claudin 5 levels appeared to be altered in MS leukocytes, we studied the mechanisms regulating its protein levels. Our results indicate that claudin 5 is regulated by UPS and lysosome activities: it can be poly-ubiquitinylated, and proteosome inhibition extended its short half life, and led to its accumulation in the cytoplasm of cells, in co-localization with the RAB8A GTPase. We identified lysine 199 as a possible site for claudin 5 ubiquitination.

T cell survival depends on the balance between pro and anti-apoptotic proteins. We detected a decrease in the levels of the pro-apoptotic protein BAK, concomitant with an increase in the levels and half life of the anti-apoptotic MCL1 protein in activated T cells of MS patients, ,compared to healthy controls. These findings support previous reports suggesting increased resistance to apoptosis in immune cell in MS, and set the stage for further research to determine if these alterations are a consequence of UPS dysregulation.

Our study suggests novel mechanisms in the pathogenesis of MS, which may, based on future research, lead to new directions for development of novel therapeutic strategies for MS and other immune-mediated diseases.