Enzyme mediated labeling of synaptic proteins for proteomics and imaging

Abstract

The neuronal synapse is one of the most important cellular structures in neuroscience. It is the junction through which signaling occurs between neurons that in aggregate, mediates complex physiological brain function. At a molecular level, this is the site where neurotransmitters are released and recognized, and where remodeling of its protein components occurs to tune the connection strength between neurons, processes crucial to brain functions such as learning and memory. Despite its importance to brain function, its small size makes synapses difficult to identify by conventional microscopy. Particularly, there remains an incomplete knowledge of the molecular components of the synaptic cleft, the gap space between the pre and postsynaptic neuron, hindering a complete mechanistic understanding of this structure. This is because the cleft is extracellular and non-membrane bound, and hence intractable to biochemical fractionation. Herein, we describe chemical genetic methodologies that allow for the enzymatic labeling of synaptic proteins either for the discovery of new synaptic components, or to elucidate specific trans-synaptic interactions at this inter-cellular junction. The first part of this thesis describes the application of peroxidase mediated, proximity-dependent biotinylation to map the proteomes of the excitatory and inhibitory synaptic clefts, which mediate distinctively different intercellular signaling between neurons; the former propagates neuronal signals while the latter inhibits them. This work identified numerous novel synaptic proteins and allowed for a comprehensive molecular differentiation of the two synaptic cleft types. Using the proteomes, we identified a specificity factor that mediates accurate matching of inhibitory presynaptic terminals to their post-synaptic cognate receptors. The second half of this thesis describe enzymatic probe ligation methodologies for synaptic proteins, 1) to identify the trans-synaptic interaction of the neurexin-neuroligin complex across the synaptic cleft, and 2) an "eraser" for LpIA based probe ligation.