Autophagy is a catabolic pathway utilized by cells to maintain homeostasis. Dysregulation of this pathway often leads to various diseases, such as cancers and neurodegeneration. Therefore, autophagy must be tightly regulated by the extracellular environment or signaling pathways. The class III PI3 kinase complex, a lipid kinase complex functioning in converting phosphatidylinositol to phosphatidylinositol-3-phosphate, is a key regulator of autophagy that functions as a signaling hub where multiple regulatory signals converge. Here, we demonstrate that the class III PI3 kinase complex is negatively regulated by cyclin-dependent kinases (Cdks). The catalytic subunit of the kinase complex, Vps34, is phosphorylated by Cdk1 in mitotic cells and by Cdk5 in postmitotic cells. Phosphorylation on Vps34 results in its dissociation from a regulatory subunit Beclin 1, leading to decreased lipid kinase activity. As a result, autophagy is inhibited in dividing cells and postmitotic neuronal cells with elevated Cdk5 activity. Since dysfunction of autophagy has been shown to be implicated in cancers and neurodegeneration, which are characterized by abnormal activity of Cdk1 and Cdk5, respectively, our study provides a mechanism by which autophagy is modulated in those diseases. To further discover the regulatory mechanisms of autophagy, we used a novel autophagy inhibitor, spautin-1, identified in a small molecule screening. Spautin-1 inhibits autophagy by inhibiting Usp10/Usp13, which deubiquitinate and stabilize the class III PI3 kinase complex. Interestingly, Usp10/Usp13 are also stabilized by the class III PI3 kinase complex, suggesting that they are reciprocally regulated. These results led us to the observation that p53, a substrate of Usp10 is regulated by the class III PI3 kinase complex and spautin-1. We also report that A70, a more potent derivative of spautin-1, leads to the degradation of mutant p53 through the chaperone-mediated autophagy, whereas the wild-type p53 is degraded by the ubiquitin-proteasome system. Our study demonstrates an important regulatory interaction between the class III PI3 kinase complex and p53, suggesting a novel tumor suppressive function of the class III PI3 kinase complex.

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<p>Neurons, with their enormous membrane contents, depend heavily on regulated membrane trafficking processes to maintain their morphology and function. The phosphatidylinositol 3-kinase class III, or PIK3C3, plays a critical role in various membrane trafficking processes including both the endocytic and autophagic pathways. The functions of PIK3C3 in the nervous system in vivo are un-characterized. We reasoned that studying PIK3C3 in neurons would provide us an entry point into understanding the regulations and functions of the neuronal membrane trafficking processes and their roles in neuronal morphogenesis and homeostasis. </p><p>We generated a conditional allele of Pik3c3 and first deleted it specifically in the peripheral sensory neurons. Mutant large-diameter myelinated sensory neurons accumulated numerous enlarged vacuoles and ubiquitin-positive aggregates and underwent rapid degeneration. By contrast, Pik3c3-deficient small-diameter unmyelinated neurons accumulated excessive numbers of lysosome-like organelles and degenerated slower than large-diameter neurons. These differential degenerative phenotypes are unlikely caused by a disruption of the autophagy pathway, because inhibiting autophagy alone by conditional deletion of Atg7 results in a completely distinct subcellular phenotypes and very slow degenerations of all sensory neurons. More surprisingly, a noncanonical PIK3C3-independent LC3-positive autophagosome formation pathway was activated in Pik3c3-deficient small-diameter neurons. This work uncovered unexpected differences of the endo-lysosomal systems in different types of neurons and discovered a novel autophagy initiation pathway in vivo in neurons. </p><p>To examine the role of PIK3C3 in the central nervous system (CNS), we next deleted Pik3c3 in CNS neural progenitor cells using the Nestin-Cre transgenic line. The resulting conditional knockout mice displayed a severe cortical lamination abnormality caused by defective cortical neuron migration. This finding uncovered a previously under-appreciated role of endocytic trafficking in neural migration, which was further confirmed by electron microscopic analyses of the developing cortex. Moreover, overexpressing the dominant negative forms of Dynamin2 or Rab5, two regulators of endocytosis, caused similar migration defects as Pik3c3-deletion. Mechanistically, Pik3c3-deficient cortical neurons drastically reduced surface Reelin binding sites, and showed significantly decreased levels of Dab1 phosphorylation, despite expressing normal total amount of Reelin receptor ApoER2. This work suggests endocytosis and recycling of Reelin receptors are likely to play an important role in cortical migration regulated by the Reelin signaling pathway. </p><p>These studies represent the first in vivo characterization of PIK3C3 functions in mammals, and provide insight into the complexity and functional importance of neuronal endo-lysosomal and autophagic pathways.</p> / Dissertation

Pathological cardiac hypertrophy often leads to heart failure. Activation of autophagy has been shown in pathological hypertrophic hearts. Autophagy is regulated positively by Class III phosphoinositide 3-kinase (PI3K). However, it is unknown whether Class III PI3K plays a role in the transition of cardiac hypertrophy to heart failure. To address this question, we employed a previously established cardiac hypertrophy model in heat shock protein 27 transgenic mice which shares common features with several types of human cardiomyopathy. Age-matched wild-type mice served as control. Firstly, a prolonged activation of autophagy, as reflected by autophagosome accumulation, increased LC3 conversion and decreased p62 protein levels, was detected in hypertrophic hearts from adaptive stage to maladaptive stage. Moreover, morphological abnormalities in myofilaments and mitochondria were presented in the areas accumulated with autophagosomes. Secondly, activation of Class III PI3K Vacuolar protein sorting 34 (Vps34), as demonstrated by upregulation of Vps34 expression, increased interaction of Vps34 with Beclin-1, and deceased Bcl-2 expression, was demonstrated in hypertrophic hearts from adaptive stage to maladaptive stage. Finally, administration with Wortmaninn, a widely used autophagy inhibitor by suppressing Class III PI3K activity, significantly decreased autophagy activity, improved morphologies of intracellular apartments, and most importantly, prevented progressive cardiac dysfunction in hypertrophic hearts. Collectively, we demonstrated that Class III PI3K plays a central role in the transition of cardiac hypertrophy to heart failure via a prolonged activation of autophagy in current study. Class III PI3K may serve as a potential target for the treatment and management of maladaptive cardiac hypertrophy.