Spatially restricted proteomic mapping of excitatory and inhibitory post-synaptic termini through enzymatic labeling

Abstract

The neuronal synapse is the seat of cognitive computation in the brain, where the minor adjustments in electrical activity between various neuronal circuits results from the sophisticated orchestration of molecules at the synapse. The molecular interplay between the diverse range of synapses and circuits in the brain makes comprehending the full scope of cognition a major challenge for modem neuroscience, one that is limited by crude fractionation schemes for picking apart selective portions of the neuron. While we currently have a strong foundation in our basic understanding of synaptic biology, the challenges we currently face in characterizing precise mechanisms of learning, memory, and cognitive decline will require the ability to finely dissect neurons and synapses into their own distinct subtypes, and understand them from a detailed molecular standpoint. The goal of this thesis is to introduce a novel chemical genetic method, called APEX, into the synapse to enable specific proximity dependent biotinylation of the proteomes at the glutamatergic (excitatory) and GABAergic (inhibitory) postsynaptic densities. In doing so, we provide the first selective and comprehensive survey of the inhibitory postsynaptic density, which has eluded purification by conventional biochemical methods. Furthermore, we demonstrate the use of APEX in state dependent studies of the synapse by exploring the excitatory postsynaptic proteome at early ages and mature ages. Finally, we demonstrate the application of APEX in vivo and illustrate the challenges inherent in such a venture. Thus, we hope to provide the foundation for spatially restricted proteomic mapping of synapses, and demonstrate the utility of such tools designed to enable deeper exploration of neuronal and synaptic diversity.