Soderling, Scott HSpence, Erin2018-09-212020-08-302018https://hdl.handle.net/10161/17451<p>Excitatory synapse formation during development involves the complex orchestration of both structural and functional alterations at the postsynaptic side, beginning with the formation of transient dendritic filopodia. Abnormalities in synapse development are linked to developmental brain disorders such as autism spectrum disorders, schizophrenia, and intellectual disability. However, the molecular mechanisms that underlie excitatory synaptogenesis remain elusive, in part because the internal machinery of developing synapses is largely unknown. Unlike mature excitatory synapses, there is currently no way to biochemically isolate the dendritic filopodia of nascent synapses. This lack of understanding is a critical barrier to our grasp of synapse development as well as the etiology of many neurodevelopmental disorders. This dissertation work focuses on the detection and analysis of proteins which localize to and are critical for spinogenesis and synaptogenesis. Using state-of-the-art in vivo proteomics, we identified a network of proteins which localize to the receiving end of the developing excitatory synapse, the dendritic filopodia. We then used the CRISPR/Cas9 system to identify candidates which drive the formation and maturation of dendritic filopodia. We finally did careful functional analysis of CARMIL3 and the Arp2/3 complex to identify their critical and diverse roles in synaptogenesis. </p><p>In our analysis, we found that CARMIL3 is expressed in the brain predominately during synaptogenesis, localizes to developing dendritic protrusions, and is important for the morphological and functional maturation of synapses, likely through its role in recruiting capping protein to maturing synapses. Loss of CARMIL3 leads to structurally and functionally immature synapses that are capping protein deficient. Further, we found that the Arp2/3 complex, a critical regulator of the actin cytoskeleton which creates branched actin networks, is required for both the functional and morphological maturation of dendritic spines. In the absence of the Arp2/3 complex, dendritic protrusions make presynaptic contact, recruit key proteins such as MAGUKs, and recruit certain receptors such as NMDA receptors, but lack AMPA receptors which are required for synapse unsilencing.</p><p>Together, this work demonstrates that the actin cytoskeleton controls the functional maturation of synapses by altering the cytoskeletal dynamics towards the creation of a branched actin network. CARMIL3 contributes to this process by providing capping protein, which biases actin nucleation towards branched actin networks. Arp2/3 creates the branched actin network. Without this network, there is not a sufficient framework to dock AMPA receptors in the post-synaptic density, and without AMPA receptors, dendritic protrusions remain functionally silent. Together, this work shows that the dynamics of the actin cytoskeleton drive synapse unsilencing.</p>Cellular biologyActinArp2/3 complexcapping proteinCARMIL3dendritic filopodiaNeurodevelopmentDissertation