Mouse-derived Synaptosomes Trypsin Cleavage Assay to Characterize Synaptic Protein Sub-localization.

Abstract

Neurons communicate through neurotransmission at highly specialized junctions called synapses. Each neuron forms numerous synaptic connections, consisting of presynaptic and postsynaptic terminals. Upon the arrival of an action potential, neurotransmitters are released from the presynaptic site and diffuse across the synaptic cleft to bind specialized receptors at the postsynaptic terminal. This process is tightly regulated by several proteins at both presynaptic and postsynaptic sites. The localization, abundance, and function of these proteins are essential for productive neurotransmission and are often affected in neurological and neurodegenerative disorders. Here, we outline a method for purifying mouse synaptosomes and using limited tryptic digestion to assess the subcellular localization of synaptic proteins. During synaptosomes purification, presynaptic terminals reseal and are protected from proteolysis, while postsynaptic proteins remain susceptible to tryptic cleavage. These changes can easily be evaluated by western blot analysis. This approach offers a straightforward and reliable method to evaluate the subcellular localization of synaptic proteins based on their proteolytic sensitivity, providing valuable insights into synaptic physiology and pathology. Key features • Builds upon the method developed by Boyken et al. [1] and introduces the use of isolated mouse synaptosomes to assess synaptic protein sub-localization. • Limited tryptic digestion differentiates between presynaptic and postsynaptic proteins based on proteolytic sensitivity. • Requires standard biochemical reagents and western blotting equipment and can be completed in two/three days, including synaptosome purification and western blot analysis. Graphical overview Overview of the synaptosomes trypsin cleavage assay. This protocol describes the isolation of synaptosomes from mouse brain tissue, followed by limited trypsin digestion to assess the compartmental localization of synaptic proteins. Synaptosomes, which are isolated via differential centrifugation, consist of resealed presynaptic terminals that are protected from proteolysis, while the exposed postsynaptic compartments are accessible to trypsin and undergo proteolytic cleavage. Following digestion, samples are analyzed using SDS-PAGE and western blotting. Proteins of interest are probed using specific antibodies to determine whether they are presynaptic or postsynaptic. Presynaptic proteins (e.g., synaptophysin, SNAP-25) remain intact, while postsynaptic proteins (e.g., GluA1, GluN2A) are cleaved.