Alteration of Golgi apparatus ion homeostasis in cellular and mouse models of Angelman syndrome

Ube3a is a HECT domain E3 ubiquitin ligase originally discovered as the protein factor required for the degradation of the oncogene p53 by the high-risk human papillomaviruses and later identified as the critical protein for the neurodevelopmental disorder Angelman syndrome. However, characterization of the endogenous properties of Ube3a has been limited and the mechanism underlying the pathogenesis of Angelman syndrome has not been elucidated. Given ubiquitous expression of Ube3a, we hypothesized Ube3a may regulate a general process required for proper cellular function. Over the last few years, an interesting cohort of Ube3a interacting partners and putative substrates have been named, including Golgi-localized proteins and proteins known to regulate secretory trafficking. Therefore, we hypothesized that Ube3a may regulate Golgi structure or function.;In this dissertation I define a new role for Ube3a function at the Golgi apparatus in the regulation of intralumenal ion homeostasis. A disruption in Golgi morphology by Ube3a overexpression in Clone 9 cells first suggested that Ube3a could regulate the structure of the Golgi apparatus. Cells with stable knockdown of Ube3a have altered Golgi morphology as shown by electron microscopy, with the Golgi appearing disorganized, overly fenestrated, and swollen. Next, we determined that the pH of the Golgi apparatus in Ube3a knockdown cells is significantly elevated, implying that the elevated Golgi pH is primary cause of the altered Golgi morphology in the Ube3a knockdown cells. Finally, we wanted to determine whether these results were applicable to the brain and Angelman syndrome. We found that the morphology of the Golgi structures in Ube3a(m-/p+) visual cortical neurons were greatly perturbed, and could be seen as swollen cisternae by electron microscopy and disorganized Golgi structures by immunohistochemistry. Thus, these results suggest a new role for Ube3a in the regulation of ion homeostasis at the Golgi apparatus, and this function is relevant for both non-neuronal and neuronal cell populations. This is predicted to lead to deficits in Golgi function, particularily in the glycosylation and proteolytic processing of nascent cargo. These findings provide new insight into the function of Ube3a and into the pathogenesis of Angelman syndrome.