Student Theses and Dissertations

Yun Zhong

Date of Award

2009

Document Type

Thesis

Degree Name

Doctor of Philosophy (PhD)

RU Laboratory

Heintz Laboratory

Abstract

Beclin 1 is a mammalian autophagy protein and has important functions in development, tumor suppression and neurodegeneration. Beclin 1 exists in a protein complex with Vps34, which is a class III phosphatidylinositol (PtdIns) 3-kinase and mediates multiple vesicle trafficking pathways including autophagy and endocytosis. However the precise role of Beclin 1 in autophagy regulation is not well understood and information is lacking regarding the function of Beclin 1 in neuronal development and degeneration. In a study that combines mouse genetics and biochemistry, three novel Beclin 1 interaction proteins Atg14L (yeast Atg14-like), Rubicon (RUN domain and cysteine-rich domain containing, Beclin 1-interacting protein) and Nrbf2 were identified from mouse tissues. Gel filtration and co-immunoprecipitation show that these proteins co-exist in one large in vivo protein complex, from which multiple sub-complexes may be generated. Functional assays reveal that Atg14L positively regulates Vps34/PtdIns3K kinase activity and autophagy while Rubicon shows negative regulatory effects. Moreover, Beclin 1 and Atg14L synergistically promote autophagosome biogenesis while over-expression of Rubicon causes formation of aberrant late endosomes/early lysosomes and blocks autophagosome maturation. Therefore Atg14L and Rubicon regulate autophagy in opposite directions and at different steps, possibly through forming distinctive complexes with Beclin 1 and mediating Vps34/PtdIns3K kinase activity. Meanwhile, to study the function of Beclin 1 in neuronal development and degeneration, targeted deletion of beclin 1 was performed in pyramidal cells of the hippocampus and the cerebral cortex and in cerebellar Purkinje cells. Consequently, the deletion leads to rapid and severe degeneration in these cells. In degenerating pyramidal cells, there is intracellular protein accumulation that may have resulted from impaired autophagy, and activation of apoptotic pathway is also observed. In Purkinje cells, electron microscopy studies show a large number of aberrant electron dense structures and they are closely associated with intracellular membranes. Meanwhile, immuno-staining shows that deletion of beclin 1 induces localization change of PtdIns(3)P, the product of Vps34/PtdIns3K. Therefore it is suggested that Beclin 1 is critical for normal neuronal development and that its deletion not only results in impaired autophagy but also may induce abnormal membrane and vesicle trafficking, possibly due to disrupted regulation of Vps34/PtdIns3K.

Comments

A Thesis Presented to The Faculty of The Rockefeller University in Partial Fulfillment of the Requirements for The degree of Doctor of Philosophy

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