SynPull: An advanced method for studying neurodegeneration-related aggregates in synaptosomes using super-resolution microscopy

SIGNIFICANCE Synaptic dysfunction and loss are key pathologies in neurodegenerative disorders such as Alzheimer's disease and Parkinson's disease. The rate of synaptic decline correlates strongly with other changes such as cognitive loss and behavioral abnormalities. The protein aggregation associated with these diseases causes synaptic pathology. However, the current methodologies to study these proteins in the synapse are limited. We have developed and validated SynPull as a method to characterize the protein aggregates inside the synapse at great resolution and detail. By using specific targets to capture the synaptosomes, we have improved the specificity of the method. This will enable the study of sub-populations of synapses selectively affected in various diseases. SynPull is therefore a useful method for researchers studying synaptic proteins in health and disease. SUMMARY Synaptic dysfunction is a primary hallmark of both Alzheimer's and Parkinson's disease, leading to cognitive and behavioral decline. While alpha-synuclein, beta-amyloid, and tau are involved in the physiological functioning of synapses, their pathological aggregation has been linked to synaptopathology. The methodology for studying the small-soluble protein aggregates formed by these proteins is limited. Here we describe SynPull, a method combining single-molecule pull-down, super-resolution microscopy, and advanced computational analyses to characterize the protein aggregates in human and mouse synaptosomes. We show that AT8-positive tau aggregates are the predominant aggregate type in synaptosomes from postmortem Alzheimer's disease brain, although the aggregate size does not change in disease. Meanwhile, the relatively smaller amount of alpha-synuclein and beta-amyloid aggregates found in the synapses are larger than the extra-synaptic ones. Collectively, these results show the utility of SynPull to study pathological aggregates in neurodegeneration, elucidating the disease mechanisms causing synaptic dysfunction.