The nervous system represents the most complex part of the body. It is estimated that in the human brain 10¹¹ neuronal cells are connected into networks through 10¹³ synaptic connections, which are assembled with a stunning precision and reproducibility. Understanding the molecular mechanisms underlying neuron-specific functions and selective connectivity represents a major challenge. Disruptions in neuronal circuits are believed to underlie numerous neuropsychiatric disorders such as autism and schizophrenia, therefore, uncovering the mechanisms of synaptic specificity has important implications for human health.

The use of molecular codes for cell and synapse-specific recognition in the nervous system has long been hypothesized to contribute to the functional and morphological diversification of neuronal cells. In fact, several gene families have been identified that encode highly polymorphic neuronal cell surface receptors, which may confer specific neuronal identities and mediate cellular recognition. Remarkable examples of such polymorphic proteins are the neurexins (NRXNs), transmembrane proteins potentially expressed in thousands of distinct variants in the mammalian nervous system. Importantly, NRXNs have emerged as important risk factors in neuropsychiatric disorders such as autism, schizophrenia, and drug abuse [1-7].

The goal of this proposal is to use the molecular diversity of neurexins as an entry point for dissecting functional and structural diversification of neurons. There are three neurexin genes, NRXN1, NRXN2 and NRXN3, each expressed from two alternative promoters. Transcription from the upstream promoter results in larger α-neurexin isoforms, whereas the downstream promoter yields shorter β-neurexins. The majority of NRXN molecular diversity results from extensive alternative splicing at five sites [8, 9], potentially resulting in more than three thousand different isoforms. An attractive model for the function of NRXNs is that they mediate splice isoform-specific biochemical interactions. Support for this idea has come from the analysis of certain alternative splice isoforms where isoform-specific ligands have been discovered (e.g. neuroligins, LRRTMs, Cbln1, and α-DG) [10-14]. However, neurexin variants have only been mapped on the mRNA level as the molecular complexity has hindered a quantitative analysis of the corresponding protein isoforms. In fact, a systematic mapping of any highly polymorphic protein family as not been performed.

In this proposal we will use a systematic proteomics approach for comprehensive mapping of molecular diversity in neuronal cells.

Specifically, we will address the following aims:

Aim 1: We will perform a comprehensive isoform mapping of neurexin variants in developing and adult mouse brain.

Aim 2: We will use the methods established for isoform mapping to probe modification of the neurexin proteome after learning paradigms and ablation of a mRNA splicing factor.

Aim 3: We will probe the logic of neurexin isoform diversity by correlating it with protein levels of regulators of neuronal function and synaptic connectivity.