CLEM and Volume EM to study synapse architecture in Angelman Syndrome

Angelman Syndrome (AS) is a neurogenetic disorder characterized by severe intellectual disability, speech impairment, seizure disorder, abnormal sleep patterns, motor dysfunction and unusually happy demeanor. AS is caused by the loss of the maternal copy of the UBE3A gene, which is biallelically expressed in all cell types, but only maternally expressed in neurons due to epigenetic silencing of the paternal allele. Studies in AS models suggest that the UBE3A gene, encoding for the ubiquitin-protein ligase E3A, is crucial for synaptic formation and maintenance, neuronal plasticity, learning and memory formation and, because of having only the maternal copy of UBE3A gene, neurons are quite vulnerable to its loss. During development, the synapse is subjected to fine modification ranging from activity-dependent dendritic spine enlargement to changes in size and shape of the postsynaptic density. Similar modifications are at the bases of synaptic plasticity. To study the effect of UBE3A inactivation on the functional organization of synapses and neuronal connectivity in AS, we will combine sparse in utero electroporation of layer 2/3 pyramidal neurons of the mouse somatosensory cortex with CRISPR/Cas9-mediated genome editing to inactivate endogenous Ube3a. The fluorescent protein tdTomato will be used as a filler to identify and select electroporated neurons. We will use Serial Block Face Scanning Electron Microscopy and CLEM to generate volumetric dataset to reconstruct electroporated neurons and to quantitatively study synapses in their environment, finally connecting the geometry of synapses with their functional state.