Author's School

Graduate School of Arts & Sciences

Author's Department/Program

Biology and Biomedical Sciences: Immunology

Language

English (en)

Date of Award

January 2011

Degree Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Chair and Committee

Herbert Virgin

Abstract

Macroautophagy: herein autophagy) is a process by which cells degrade long-lived proteins and organelles. The autophagy pathway and autophagy genes have been implicated in many functions in the cell such as protecting against metabolic stress, degrading damaged organelles, and regulating vesicular trafficking. To study the role of autophagy in primary cells with important physiologic functions, we generated mice lacking essential autophagy genes in B lymphocytes, T lymphocytes, and osteoclasts. We found that the essential autophagy gene Atg5 was important for B cell development and for the maintenance of B-1a B cell numbers but not peripheral B-2 B cell numbers. In T cells, deletion of the essential autophagy genes Atg5 or Atg7 resulted in decreased thymocyte and peripheral T cell numbers in vivo and a decrease in cell proliferation in vitro. Autophagy genes play a critical role in T cell homeostasis, but do not appear important for peripheral B-2 B cell homeostasis in vivo. Whole-genome transcriptional profiling of Atg5-deficient and wild-type thymocytes suggested abnormalities in mitochondria in the absence of Atg5. We confirmed this observation by demonstrating that peripheral Atg5-deficient T cells had an increase in mitochondrial mass that correlated with increased Annexin-V staining in these cells. We speculate that autophagy is required in T cells for the removal of damaged or aged mitochondria and that excess mitochondria contribute to increased cell death in autophagy-deficient T cells. In contrast to lymphocytes, deletion of autophagy genes in osteoclasts did not result in dramatic abnormalities in cell development. However, the biochemical pathway necessary for autophagy was critical for directional secretion in osteoclasts. We found that the autophagosome marker LC3 localized to the resorptive microenvironment in osteoclasts. Deleting Atg5 or Atg7 or overexpressing a dominant negative mutant of ATG4B to inhibit LC3 conjugation reduced localization of lysosomal markers at the resorptive surface and decreased bone resorption in vitro. Furthermore, mice lacking Atg5 in osteoclasts and other myeloid-lineage cells were protected from ovarectomy-induced bone loss, a mouse model of osteoporosis. Together, these studies demonstrate that autophagy genes are important in cell development, survival, mitochondrial maintenance, and directional secretion in physiologically important, primary mammalian cells.

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Permanent URL: http://dx.doi.org/10.7936/K7M043DZ

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