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Synaptosome Sorting Using Reversible Fixative—A Versatile Approach for Synaptic Transcriptome Sequencing (SynSURF-Seq)

ABSTRACT: Synaptic plasticity is critically regulated by local protein synthesis, but the mRNAs involved remain largely elusive. It is generally assumed that presynaptic and postsynaptic transcriptomes closely resemble axonal and dendritic transcriptomes, respectively. By developing a high-precision technique to profile synaptic transcriptomes, we demonstrate this assumption to be incorrect. Using the cleavable crosslinker dithiobis(succinimidyl propionate) (DSP), which is unreactive against RNAs and circumvents inter-synaptic aggregation compared to conventional paraformaldehyde fixation, we conducted triple sorting via immunofluorescence markers (synaptophysin and PSD-95) and size-gating to minimize glial contaminations. The sorted synaptosome were reductively de-crosslinked to release intact RNAs for next generation sequencing. Without relying on genetically-encoded tags, our Synaptosome Sorting Using Reversible Fixative (SynSURF) strategy is adaptable to any brain tissue sources. SynSURF-Seq revealed over 1800 mRNAs enriched in excitatory synapses, encoding proteins involved in translation, ubiquitin-proteasome system, RNA regulation, protein transport, and neurodegenerative disorders. The synapse has a unique gene enrichment profile, distinct from those of axons, dendrites, and somas.

ORGANISM(S): Mus musculus

PROVIDER: GSE317091 | GEO | 2026/03/31

REPOSITORIES: GEO

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