Date of Award
synapses, bacterial artificial chromosome (BAC), synaptic protein profiling, Purkinje cell synapse, synaptic specificity
The mammalian central nervous system (CNS) contains billions of neurons each receiving thousands of synaptic inputs. Synapses are specified in part through the precise localization of synaptic proteins, yet it has not previously been possible to analyze the protein content of an individual class of synapses. In order to achieve this, we have used the BAC (bacterial artificial chromosome) transgenic approach to target particular neurons for expression of a given neurotransmitter receptor fused to an affinity tag. Immunohistochemistry of fixed brain tissue confirmed the correct localization of each synaptic fusion protein to the appropriate cell type and morphological structure. In order to isolate the synaptic proteins of interest, we developed a novel method, in which a classically purified crude synaptosome fraction was subject to size exclusion chromatography to enrich for synaptic protein complexes. The tagged synaptic protein complexes were then purified by immobilization with antibody-coated magnetic beads and the eluate analyzed by mass spectrometry. This novel method was used to profile proteins at two classes of synapses. First, we purified the parallel fiber to Purkinje cell synapse of the cerebellum. We identified ~60 post-synaptic proteins, including those involved in phospholipid metabolism and signaling, which are major unrecognized components of this synapse type. Second, we analyzed inhibitory synapses of layer V pyramidal cells of the cerebral cortex, thereby accomplishing the first successful in vivo purification of an inhibitory synaptic protein complex. We identified ~12 proteins, many of which have been implicated in inhibitory synapse structure and function in vitro, such as the scaffolding protein, gephyrin. The result of this work provides a novel approach for detailed investigations of the biochemical complexity of CNS synapse types.
Heller, Elizabeth, "Synaptic Protein Profiling in the Mammalian Brain" (2009). Student Theses and Dissertations. 116.