Characterization of canonical and non-canonical functions of KCTD7 and BCL-xL in autophagy and apoptosis

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Johns Hopkins University
Cells have evolved intricate regulatory mechanisms to ensure that the biochemical processes essential to sustain life occur efficiently. These processes continuously generate byproducts that need to be degraded or recycled, byproducts that would otherwise interfere with the functions of critical cellular components such as DNA and proteins. Autophagy is a key intracellular degradation/recycling pathway that removes protein aggregates and whole organelles in both a non-selective and selective manner, ensuring that cellular waste is efficiently removed. Defects in the autophagy pathway are now thought to underlie many human diseases including neurodegeneration and cancer, and autophagy is manipulated to the advantage of many infectious pathogens. Failed autophagy leads to programmed cell death. Apoptotic cell death, a form of programmed cell death, also enables the disposal of dead and dying cells by facilitating their engulfment and degradation by neighboring cells, a process that utilizes some of the autophagy machinery. Defective apoptosis can result in the accumulation of damaged cells that compromise tissue integrity. Thus, the induction of apoptosis is an efficient way to eliminate harmful or defective cells, which is essential for normal development and tissue homeostasis, and defective apoptosis underlies disease pathology. Induction of apoptosis is tightly controlled, largely by members of the BCL-2 protein family. Improper regulation of BCL-2 family members is known to cause degenerative disorders (too much cell death) and cancer progression (too little cell death). The BCL-2 family protein BCL-xL, which is expressed in neurons and developing T-cells, inhibits apoptotic cell death but also regulates other cellular processes including metabolism and autophagy. This thesis describes investigations into different aspects of both autophagy and apoptotosis pathways in two divergent projects. In Chapter 1, the roles of caspase cleavage BCL-xL in the development of T-cell progenitors in vitro and in the thymus are investigated. In Chapters 2 and 3, the uncharacterized Kctd7 molecule is investigated for its ability to regulate autophagy in vitro and to cause epilepsy in vivo. BCL-xL, a classic pro-survival BCL-2 family member, can be inactivated and converted into a pro-death molecule by caspase cleavage in the N-terminal unstructured loop. To determine if caspase cleavage of BCL-xL has physiological significance in thymocyte development, I utilized genetically modified mice expressing caspase-uncleavable BCL-xL (D61A/D76A). Development of thymocytes was assessed in these mice as these cells express high levels of BCL-xL at specific stages of development in the thymus where they undergo several developmental bottlenecks that eliminate a large portion of the thymocyte population through apoptotic cell death pathways. The results did not uncover any major phenotypes, but suggest that mice expressing uncleavable BCL-xL may had delayed thymic atrophy and altered T-cell receptor signaling based on transiently decreased expression of the surface glycoprotein CD5, a marker for TCR signaling strength, at 1-2 months, suggesting protection from failed positive selection. Thymocytes expressing caspase-uncleavable BCL-xL remained sensitive to both in vitro and in vivo death stimuli and did not exhibit proliferative defects. However, the lifespan of thymocytes expressing caspase-resistant BCL-xL was detectably shorter, suggesting possible accumulation of defective cells. However, these are small changes that do not persist in mice at ages beyond four months and up to 24 months of age. Therefore, cleavage of BCL-xL may not represent a prominent death pathway during normal thymocyte development. The second project defines a role for KCTD7 in autophagy. Bi-allelic mutations in KCTD7 cause a rare neurological disorder, progressive myoclonic epilepsy 3 (EPM3). A Kctd7 knockout-first reporter mouse model revealed that Kctd7 is prominently expressed in the hippocampus as well as discrete layers of the brain cortex. Mice deficient in Kctd7 were susceptible to seizures at an early age, mimicking the unusually early age of onset in human patients. On a cellular level, KCTD7 displayed functional interactions with VPS34 and ATG14L, two components of the PI3KC3-C1 complex, which is required for autophagosome biogenesis and is suggested to be regulated by BCL-xL. Patient mutations induced aggregation of KCTD7 proteins within cells, and these aggregates compromised VPS34 and ATG14L localization. Therefore, mutations in KCTD7 that manifest in EPM3 may have underlying autophagy defects due to compromised VPS34 and ATG14L activity.
apoptosis, cell death, caspase, BCL-2, BCL-xL, thymocytes, T-cells, KCTD, EPM3, epilepsy, autophagy, PI3K-C1, VPS34, ATG14L, Cullin-3