The nuclear kinase Dyrk1a is essential for cell cycle regulation during T cell development [electronic resource]

Dyrk1a is a nuclear kinase within the Down syndrome critical region that is triplicated in Down syndrome (DS). Since respiratory infections are the most common cause of death in DS patients, studying the function of Dyrk1a in the immune system would shed light on the prevalence of infection in DS patients. This dissertation describes for the first time the generation of a Dyrk1a conditional knockout mouse that we used to study the effects of Dyrk1a-deficiency in the immune system. Chapter 1 introduces the concept of thymocyte development, including the signaling pathways and cellular processes critical for this process. We will also review the biochemistry of Dyrk1a and the implications of its kinase activity in T cells. This will provide the foundation for understanding Dyrk1a function and serve as a primer for our observations on the role of Dyrk1a in thymocyte development and peripheral T cell function. Chapter 2 illustrates the generation of a Dyrk1a conditional knockout targeting allele and the subsequent crossing of Dyrk1a fl/fl mice to transgenic mice expressing Cre-recombinase under the control of the immune-specific promoters, Lck and CD4. Using these immune-specific Cre transgenic mice, we observed defects in thymocyte development in Dyk1a-deficient thymocytes, as evidenced by decreased total thymic cellularity and decreased proportion and cellularity of double positive (DP) thymocytes. The Dyrk1a-deficient thymocytes were unable to upregulate activation markers characteristic of DP thymocytes that have undergone selection, and were unable to respond to T cell receptor-induced signaling. Together, these data identify Dyrk1a as a critical regulator for thymocyte development. Chapter 3 describes RNA sequencing data from Dyrk1a-deficient DP thymocytes. RNA sequencing revealed increased expression of cell cycle related genes in Dyrk1a-deficient DP thymocytes as compared to wildtype controls. We demonstrate that Dyrk1a-deficiency leads to increased BrdU incorporation, indicating greater proportions of cycling cells. Many cell cycle checkpoint genes were upregulated as shown by RNA sequencing and confirmed by Western immunoblotting, suggesting that the cell cycles were unproductive in Dyrk1a-deficient DP thymocytes. As a result, DP thymocytes were more susceptible to apoptosis as evidenced by the increase in cleaved-caspase 3 expression throughout different stages of the cell cycle. These data implicate a role for Dyrk1a in regulating mitotic exit in DP thymocytes during thymocyte development. Chapter 4 describes our characterization of Dyrk1a function in peripheral T cells utilizing Dyrk1a fl/fl; CD4-Cre mice. We observed severe lymphopenia in these mice, based on the decreased numbers of CD4+ and CD8+ cells in the spleen and lymph nodes. Within the CD4 and CD8 compartments, a greater proportion of cells expressed CD44 and ICOS, indicative of an activated phenotype. Dyrk1a-deficient T cells proliferated less than wildtype T cells, expressed increased levels of Fas ligand, and secreted more IFN-[gamma] and IL-2 in the presence of stimulation; all of which are calcineurin/NFAT-targets. These data suggest that in peripheral T cells, Dyrk1a-deficiency results in NFAT hyperactivation that ultimately results in increased cell death. Chapter 5 reviews and discusses the results presented in this dissertation, emphasizing the essential role that Dyrk1a plays in regulating cell cycle during thymocyte development. Lastly, we propose future experiments to identify the Dryk1a substrate(s) involved in the thymocyte development phenotype we observed.

Advisor: Crabtree Gerald R. primary advisor. Thesis advisor
Davis Mark M. advisor. Thesis advisor
Utz Paul advisor. Thesis advisor
Weiss Arthur