Browsing by Author "Ehlers, Michael D"

Ube3a is a HECT domain E3 ubiquitin ligase originally recognized for its role in degrading p53 in the presence of the human papilloma virus protein E6. Loss of maternal Ube3a expression causes Angelman syndrome, a severe neurodevelopmental disorder characterized by mental retardation, ataxia, epilepsy, lack of speech, and a unique behavioral phenotype that includes a happy demeanor and frequent laughing. However, characterization of the endogenous properties and cellular role for Ube3a has been limited. Over the last few years, an interesting cohort of Ube3a interacting partners and putative substrates were named, though the consequences of these interactions were not thoroughly investigated. These include two Golgi localized proteins - PIST and Golgin-160 - as well as several proteins that can regulate trafficking of proteins at the Golgi apparatus: Src family kinases, ubiquilin, and tuberin. Therefore, we decided to focus on whether Ube3a could regulate Golgi structure or function.

In this dissertation, I will describe a new role for Ube3a at the Golgi apparatus in the regulation of intralumenal ion homeostasis. First, I characterized the expression pattern of endogenous Ube3a and overexpressed Ube3a isoforms by immunostaining and fractionation and demonstrated that although Ube3a has diffuse nuclear/cytoplasmic localization, it also associates with membrane fractions. I also confirmed that Ube3a interacts endogenously with both PIST and Golgin-160. Next, I demonstrated that Golgi morphology is perturbed in a cell line with stable knockdown of Ube3a. I found that the Golgi apparatus in Ube3a knockdown cells is under-acidifed, and that this is the primary defect underlying the disrupted Golgi morphology. Finally, I extended these findings in vivo and examined the morphology of the Golgi apparatus in the brains of Angelman syndrome model mice. The Golgi structures in the visual cortex of these mice appeared disorganized by immunohistochemistry and individual cisternae were significantly distended by electron microscopy, consistent with a defect in ion homeostasis at the Golgi apparatus. These findings define new cellular role for Ube3a at the Golgi apparatus and provide insight into the pathogenesis of Angelman syndrome.

The proper modification and trafficking of plasma membrane proteins are essential for normal neuronal function, such as dendrite morphogenesis, spine formation and synaptic plasticity. The secretory organelles including endoplasmic reticulum and Golgi apparatus are critical for the trafficking of these molecules as shown in fibroblasts. Although these secretory organelles have been observed in neurons including dendritic branches, their spatial organization and function in protein trafficking, neuronal development and plasticity are not clear yet. Here, I used photobleaching and photoactivation approaches combined with electron microscopy to show that although rapidly diffusing within the continuous network of the somato-dendritic ER, membrane proteins such as nascent AMPA receptors are confined by ER spatial complexity. The spatial range of ER membrane protein mobility becomes progressively confined over neuronal development and is rapidly restricted by synaptic activity. Thus, constrained lateral mobility within the ER provides a novel mechanism for compartmentalized trafficking of nascent receptors throughout dendrites. I also identified an ER protein as a novel microtubule-associated protein regulating dendritic ER spatial complexity, neuronal dendrite elongation and spine formation. Together, these results describe the spatial organization of dendritic ER and its role in regulating membrane protein trafficking, neuronal morphogenesis and postsynaptic functions.

The specification of a single axon and multiple dendrites is the first observable event during neuronal morphogenesis and such structural specialization underlies neural connectivity and nervous system function. Numerous intracellular signaling components that are required for axon specification have been described but how such signaling paradigms are initiated by extracellular factor(s) within the embryonic milieu is poorly understood. Here, I describe how transforming growth factor-β (TGF-β), an embryonic morphogen that directs structural plasticity and growth in various cell types, initiates signaling pathways both in vivo and in vitro to fate naïve neurites into axons. Using conditional knockout strategies, I found that cortical neurons lacking the type II TGF-β receptor (TβR2) fail to initiate axons during development, and interestingly, fail to engage radial migration. In cultured neurons, exogenous TGF-β is sufficient to direct the rapid growth and differentiation of an axon and genetic enhancement of receptor activity promotes the formation of multiple axons. The cellular polarization of receptor activity occurs through the interaction of the type-I TGF-β receptor with Par6, a component of the axon-specifying Par3/Par6 polarity complex. Receptor distribution is restricted to axons, and downstream signaling events required for axon specification are triggered when Par6 is phosphorylated by TβR2. Together, these results indicate that TGF-β is the extrinsic cue for neuronal polarity in vivo and directs neuronal polarity by controlling Par6 activity and cellular migration during axon generation.

At excitatory synapses, NMDAR-mediated synaptic plasticity occurs in response to activity inputs by modifying synaptic strength. While comprehensive studies have been focused on the induction and expression mechanisms underlying synaptic plasticity, it is less clear whether and how synaptic plasticity itself can be subjected to regulations. The presence of "plasticity of plasticity", or meta-plasticity, has been proposed as an essential mechanism to ensure a proper working range of plasticity, which may also offer an additional layer of information storage capacity. However, it remains elusive whether and how meta-plasticity occurs at single synapses and what molecular substrates are locally utilized. Here, I develop systems allowing for sustained alterations of individual synaptic inputs. By implementing a history of inactivity at single synapses, I demonstrate that individual synaptic inputs control synaptic molecular composition homosynaptically, while allowing heterosynaptic integration along dendrites. Furthermore, I report that subunit-specific regulation of NMDARs at single synapses mediates a novel form of input-specific metaplasticity. Prolonged suppression of synaptic releases at single synapses enhances synaptic NMDAR-mediated currents and increases the number of functional NMDARs containing NR2B. Interestingly, synaptic NMDAR composition is adjusted by spontaneous glutamate release rather than evoked activity. I also demonstrate that inactivated synapses with more NMDARs containing NR2B acquire a lower induction threshold for long-term synaptic potentiation. Together, these results suggest that at single synapses, spontaneous release primes the synapse by modifying its synaptic state with specific molecular compositions, which in turn determine the synaptic gain in an input-specific manner.