Mapping proteomic composition of excitatory postsynaptic sites in the cerebellar cortex.

Functions of the cerebellar cortex, from motor learning to emotion and cognition, depend on the appropriate molecular composition at diverse synapse types. Glutamate receptor distributions have been partially mapped using immunogold electron microscopy. However, information is lacking on the distribution of many other components, such as Shank2, a postsynaptic scaffolding protein whose cerebellar dysfunction is associated with autism spectrum disorders. Here, we used an adapted Magnified Analysis of the Proteome, an expansion microscopy approach, to map multiple glutamate receptors, scaffolding and signaling proteins at single synapse resolution in the cerebellar cortex. Multiple distinct synapse-selective distribution patterns were observed. For example, AMPA receptors were most concentrated at synapses on molecular layer interneurons and at climbing fiber synapses, Shank1 was most concentrated at parallel fiber synapses on Purkinje cells, and Shank2 at both climbing fiber and parallel fiber synapses on Purkinje cells but little on molecular layer interneurons. Our results are consistent with gene expression data but also reveal input-selective targeting within Purkinje cells. In specialized glomerular structures of the granule cell layer, AMPA receptors as well as most other synaptic components preferentially targeted to synapses. However, NMDA receptors and the synaptic GTPase activating protein SynGAP preferentially targeted to extrasynaptic sites. Thus, glomeruli may be considered integrative signaling units through which mossy fibers differentially activate synaptic AMPA and extrasynaptic NMDA receptor complexes. Furthermore, we observed NMDA receptors and SynGAP at adherens junctions, suggesting a role in structural plasticity of glomeruli. Altogether, these data contribute to mapping the cerebellar 'synaptome'.

Adaptation of Magnified Analysis of the Proteome for Excitatory Synaptic Proteins in Varied Samples and Evaluation of Cell Type-Specific Distributions.

Growing evidence suggests a remarkable diversity and complexity in the molecular composition of synapses, forming the basis for the brain to execute complex behaviors. Hence, there is considerable interest in visualizing the spatial distribution of such molecular diversity at individual synapses within intact brain circuits. Yet this task presents significant technical challenges. Expansion microscopy approaches have revolutionized our view of molecular anatomy. However, their use to study synapse-related questions outside of the labs developing them has been limited. Here we independently adapted a version of Magnified Analysis of the Proteome (MAP) and present a step-by-step protocol for visualizing over 40 synaptic proteins in brain circuits. Surprisingly, our findings show that the advantage of MAP over conventional immunolabeling was primarily due to improved antigen recognition and secondarily physical expansion. Furthermore, we demonstrated the versatile use of MAP in brains perfused with paraformaldehyde or fresh-fixed with formalin and in formalin-fixed paraffin-embedded tissue. These tests expand the potential applications of MAP to combinations with slice electrophysiology or clinical pathology specimens. Using male and female mice expressing YFP-ChR2 exclusively in interneurons, we revealed a distinct composition of AMPA and NMDA receptors and Shank family members at synapses on hippocampal interneurons versus on pyramidal neurons. Quantitative single synapse analyses yielded comprehensive cell type distributions of synaptic proteins and their relationships. These findings exemplify the value of the versatile adapted MAP procedure presented here as an accessible tool for the broad neuroscience community to unravel the complexity of the "synaptome" across brain circuits and disease states.

Alternative splicing and heparan sulfation converge on neurexin-1 to control glutamatergic transmission and autism-related behaviors.

Neurexin synaptic organizing proteins are central to a genetic risk pathway in neuropsychiatric disorders. Neurexins also exemplify molecular diversity in the brain, with over a thousand alternatively spliced forms and further structural heterogeneity contributed by heparan sulfate glycan modification. Yet, interactions between these modes of post-transcriptional and post-translational modification have not been studied. We reveal that these regulatory modes converge on neurexin-1 splice site 5 (S5): the S5 insert increases the number of heparan sulfate chains. This is associated with reduced neurexin-1 protein level and reduced glutamatergic neurotransmitter release. Exclusion of neurexin-1 S5 in mice boosts neurotransmission without altering the AMPA/NMDA ratio and shifts communication and repetitive behavior away from phenotypes associated with autism spectrum disorders. Thus, neurexin-1 S5 acts as a synaptic rheostat to impact behavior through the intersection of RNA processing and glycobiology. These findings position NRXN1 S5 as a potential therapeutic target to restore function in neuropsychiatric disorders.