Calcium-Regulated Ubiquitin Signaling at the Centrosome Drives Dendrite Patterning in the Mammalian Brain

The proper formation and morphogenesis of dendrites is fundamental to the establishment of neural circuits in the brain. Following exit from the cell cycle and migration to the appropriate location, postmitotic neurons undergo carefully orchestrated steps in dendrite morphogenesis, including the generation and elaboration of extensive dendrite arbors followed by dendrite retraction and pruning. Although the cell-intrinsic and cell-extrinsic regulators of dendrite morphogenesis have been investigated, the function of the ubiquitin proteasome system in this process and its relationship to extracellular cues such as calcium has remained unknown. In my dissertation, I have discovered an essential role for the major E3 ubiquitin ligase Cdc20-APC in dendrite morphogenesis in postmitotic neurons. Knockdown of Cdc20 in cerebellar slices and in postnatal rats in vivo impairs dendrite elaboration. Strikingly, Cdc20-APC functions at centrosome where it triggers the polyubiquitination and degradation of the HLH protein Id1 to stimulate dendrite development. Notably, the centrosome-associated protein HDAC6 promotes Cdc20 polyubiquitination, stimulates centrosomal Cdc20-APC, and drives dendrite differentiation. These findings define a novel postmitotic function for Cdc20-APC in the morphogenesis of dendrites.;I have also identified the first catalytic function of the major protein kinase CaMKIIbeta in the mammalian brain. CaMKIIbeta operates at the centrosome in a CaMKIIalpha-independent manner to drive dendrite pruning and retraction by inhibiting Cdc20-APC. In particular, CaMKIIbeta phosphorylates Cdc20 at Ser51, inducing Cdc20 dispersion and inhibiting centrosomal Cdc20-APC activity. Thus, in contrast to CaMKIIalpha which promotes dendrite elaboration, CaMKIIbeta triggers a switch from growth to retraction of dendrites. Importantly, I have identified TRPC5 as the source of calcium entry that stimulates the isoform-specific functions of CaMKIIbeta. TRPC5 activates CaMKIIbeta and promotes its interaction with the centrosomal targeting protein PCM1, leading to the activation of centrosomal CaMKIIbeta signaling and dendrite retraction. Accordingly, disruption of TRPC5 by knockdown or knockout approaches impairs dendrite pruning in the cerebellar cortex in vivo, and TRPC5 knockout mice have deficits in motor coordination consistent with these morphological defects. Taken together, my dissertation work elucidates an entire pathway that couples calcium signaling to the ubiquitin proteasome machinery at the centrosome and thereby orchestrates dendrite morphogenesis. These findings have important implications for diverse biological processes including neuronal connectivity in the brain.