Proteomic Studies of a Single CNS Synapse Type: The Parallel Fiber/Purkinje Cell Synapse

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Abstract
Precise neuronal networks underlie normal brain function and require distinct classes of synaptic connections. Although it has been shown that certain individual proteins can localize to different classes of synapses, the biochemical composition of specific synapse types is not known. Here, we have used a combination of genetically engineered mice, affinity purification, and mass spectrometry to profile proteins at parallel fiber/Purkinje cell synapses. We identify approximately 60 candidate postsynaptic proteins that can be classified into 11 functional categories. Proteins involved in phospholipid metabolism and signaling, such as the protein kinase MRCKγ, are major unrecognized components of this synapse type. We demonstrate that MRCKγ can modulate maturation of dendritic spines in cultured cortical neurons, and that it is localized specifically to parallel fiber/Purkinje cell synapses in vivo. Our data identify a novel synapse-specific signaling pathway, and provide an approach for detailed investigations of the biochemical complexity of central nervous system synapse types. The brain is composed of many different types of neurons that form very specific connections: synapses are formed with specific cellular partners and on precise subcellular domains. It has been proposed that different combinations of molecules encode the specificity of neuronal connections, implying the existence of a “molecular synaptic code.” To test this hypothesis, we describe a new experimental strategy that allows systematic identification of the protein composition for individual synapse types. We start with mice that are genetically engineered to facilitate the purification of one type of synapse from a given neuronal population in the central nervous system, the parallel fiber/Purkinje cell synapse. The purification is performed using a combination of biochemical fractionation and affinity purification. Subsequent mass spectrometry allows us to identify approximately 60 different proteins present in the resulting sample. We have further analyzed some of the 60 proteins and show that MRCKγ, a newly identified kinase, is localized in the dendritic spines where the parallel fiber/Purkinje cell synapses are formed and that it can modulate the morphogenesis of dendritic spines. The use of this experimental strategy opens up the ability to provide insights into the underlying “molecular code” for the diverse types of synapses in the brain.