The long-term goals of the proposed research are to elucidate the mechanisms regulating axonal growth and regeneration in the mammalian brain. We recently discovered that the ubiquitin ligase, the anaphase promoting complex (Cdh1-APC), plays a critical role in the control of axonal growth and patterning in the mammalian cerebellum. Cdh1 knockdown by RNAi in primary rat cerebellar granule neurons robustly promoted axonal growth. In cerebellar slice overlay assays and by in vivo knockdown in the postnatal rat cerebellum, we found that Cdh1 cell-autonomously controls the layer-specific growth of granule neuron axons and parallel fiber patterning. In other experiments, Cdh1 knockdown was found to remarkably override myelin-inhibition of axonal growth. Thus, Cdh1-APC may also contribute to the inability of injured neurons to extend axons in the mammalian central nervous system (CNS). Our findings have raised several fundamental questions. How is Cdh1-APC function regulated in neurons? What are the mechanisms by which Cdh1-APC controls axonal growth? How does Cdh1-APC contribute to the negative influence of myelin inhibitory factors on axonal growth? To address these questions, we propose the following specific aims: (1) characterize mechanisms regulating Cdh1-APC function in neurons. We will perform structure/function analyses of Cdh1 and characterize the role of Cdh1 phosphorylation and Cdh1-interacting proteins in Cdh1-APC's control of axonal growth. (2) Determine the mechanism by which Cdh1-APC controls axonal growth. We will identify the substrates of Cdh1-APC that regulate axonal growth. (3) Characterize cell intrinsic role of Cdh1-APC in limiting axonal growth. We will characterize the activity and role of Cdh1-APC in mammalian CNS neurons beyond the cerebellum, and determine how Cdh1-APC contributes to the myelin-inhibition of axonal growth. The proposed research represents an important set of experiments that should address a major gap in our understanding of the cell-intrinsic mechanisms controlling axonal growth in the mammalian brain. In addition, the proposed research should provide the foundation for the development of drugs that might ultimately be used to stimulate axonal regeneration following injury and disease. ? ?
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