Profiling cell type-specific ubiquitome in UBE3A mutant mice

University Of Wisconsin-Madison, Madison WI

Investigators

Abstract

Project Summary Title: Profiling cell type-specific ubiquitome in UBE3A mutant mice Angelman Syndrome (AS) is a rare genetic and severe neurodevelopmental disorder with complex symptoms. It is caused by the failure to inherit a typical maternal allele of the UBE3A gene, which encodes a HECT-type ubiquitin protein ligase. This genetic mutation contributes to 85%–90% of AS cases. While UBE3A expression and function suggest that UBE3A may target different proteins in various cell types, at different ages, and in different brain regions, the detailed molecular mechanisms regulated by UBE3A ubiquitination in the brain remain elusive. No study has yet identified UBE3A substrates in a cell-type and age-specific manner, nor is it known whether UBE3A substrates are differentially ubiquitinated in different brain regions in mammalian animals due to the lack of available tools. Identifying cell-type and age-specific UBE3A substrates is critical for understanding the functions of UBE3A and the mechanisms underlying UBE3A deficiency in AS. This knowledge is essential for developing potential cures for AS. We hypothesize that UBE3A targets distinct proteins in a cell type-specific and developmental stage specific manner in the brain. The goal of this project is to systematically identify differentially ubiquitinated proteins, potentially UBE3A substrates, in UBE3A model mice in different cell types (excitatory and inhibitory neurons) at different developmental stages. Additionally, we aim to investigate the spatial ubiquitination of UBE3A substrates by visualizing their ubiquitination patterns. We have successfully engineered a Cre-dependent Ubiquitin Tagging (CUT) mouse line designed to selectively tag endogenously ubiquitinated proteins within specific cell types in the mouse tissue. This innovative mouse line facilitates spatial visualization of protein ubiquitination within specific cell types at the single-cell level and enables purification of ubiquitinated proteins in specific cell types for cell-type-specific ubiquitome profiling. By combining our specific-Cre::CUT tool with Proximity Ligation Assay (PLA) and confocal microscopy, we will visualize cell type-specific and age-specific ubiquitination of UBE3A substrates at the single-cell level in different brain regions. Focusing on the P7 and P56 cortical brain regions, we will generate triple transgenic mice (Nex1- cre::CUT::Ube3am-/p+ for excitatory neurons and Gad2-cre::CUT::Ube3am-/p+ for inhibitory neurons) to profile ubiquitomes using pulldown experiments and mass spectrometry. This will help identify differentially ubiquitinated proteins and potential UBE3A targets across different cell types and ages, allowing for a comprehensive analysis of UBE3A functions. This study will identify differentially ubiquitinated proteins in UBE3A mutant mice in both excitatory and inhibitory neurons at different ages. This will elucidate UBE3A's varying functions across cell types and ages, enhancing our understanding of its role in AS and advancing towards effective treatments. Our data will also serve as a valuable resource for AS research.