07 Feb 2025

Synaptic dysfunction heralds cognitive and behavioral decline in neurodegenerative conditions such as Alzheimer’s and Parkinson’s diseases. Oligomers of Aβ, tau, and ɑ-synuclein may be to blame, but their minuscule size makes them difficult to study through optical microscopy. Enter SynPull, a new synaptosome imaging method developed by David Klenerman, John Danial, and colleagues at the University of Cambridge, England. SynPull combines single-molecule pull-down of synaptosomes with direct stochastic optical reconstruction microscopy, aka dSTORM, which offers a resolution limit of around 20nm, good enough to characterize single aggregates.

The SynPull imaging protocol isolates synaptosomes for super-resolution microscopy.
It can detect single aggregates of Aβ and tau inside and outside synapses.
Tau aggregates appear inside synapses from human brain samples with Aβ pathology.
The method can be adapted to study other synaptic proteins.

As reported in Cell Chemical Biology on January 23, first authors Shekhar Kedia and Emre Fertan used an antibody targeting the presynaptic protein neurexin 1 to secure synaptosomes to coated slides. Along with an automated image analysis protocol, this single-molecule pull-down technique allowed the authors to examine synaptosomes while reducing nonspecific signals that complicate high-resolution microscopy.

With SynPull, the scientists determined the number, size, and shape of protein clusters inside and outside of synaptosomes derived from postmortem human orbitofrontal cortex tissue from the Cambridge Brain Bank. They compared Aβ and tau clusters in synapses from AD cases, and ɑ-synuclein clusters in synapses from PD cases, with aggregates in synapses from otherwise healthy, age-matched controls. They also compared synapses from the APP^NL-G-F mouse models of AD, the P310S model of tauopathy, and the MI2 model of PD with those from wild-type mice.

Synaptic Assemblies. Pseudo color dSTORM images show α-synuclein aggregates in SynPull synaptosomes from control and PD brain (left two panels, respectively). Likewise for Aβ tau in control and AD brains. [Adapted from Kedia et al, 2025.]

Most aggregates in AD and PD samples appeared outside synaptosomes. A similar number of AD and PD synapses held Aβ and ɑ-synuclein aggregates, respectively, as did synapses from healthy controls. Likewise, the number of aggregates per synapse was not different between healthy and disease brains.

The difference between cases and controls was in size. Synaptic aggregates were larger than extra-synaptic aggregates in all cases. Aβ aggregates in AD averaged 141 nm long, while those from controls were 83 nm, and synuclein inclusions were 160 nm in PD versus 80 nm in control synapses. In mice, the pattern was similar. Transgenic and wild-type synapses held about the same number of aggregates, though again, clusters inside synapses were larger than extra-synaptic ones.

A different scenario emerged for tau deposits. Synaptosomes from AD brain samples were more than eight times as likely to harbor AT8-positive tau aggregates than were control synaptosomes. Over three times as many P310S mouse synaptosomes carried AT8-positive tau aggregates than did wild-type synaptosomes.

Within human AD synaptosomes, clusters of AT8-positive tau were more common than Aβ aggregates, consistent with aggregated tau being intracellular while aggregated Aβ is extracellular. Averaging 100 nm, synaptic tau assemblies were about the same length in control and AD synapses, while extrasynaptic ones were about 50 percent longer.

More AD human synaptosomes contained multiple AT8-positive tau clusters than would be expected by chance. By contrast, tau aggregates in synaptosomes from P310S mice, which do not develop amyloid plaques, followed a more random pattern. This supports the notion that Aβ pathology drives synaptic tau accumulation in AD, the authors claim (Jan 2025 news; April 2014 news).

These preliminary results indicate SynPull could be used to more fully characterize synapse aggregates in disease states. The technique “will be particularly useful for those interested in understanding how brain connections are lost in diseases such as Alzheimer's and Parkinson's,” wrote Pranesh Padmanabhan of the University of Queensland, who was not involved in the research.

The Cambridge scientists plan to collect more data on synaptic protein clusters at various stages of different neurodegenerative diseases, pointing out that while they used the SynPull technique to study aggregates associated with neurodegeneration, the approach could apply more broadly. “Once you immobilize [a synaptosome], you can study any protein of interest,” Kedia told Alzforum. Further, by targeting select different proteins during the “pull down” step, scientists could focus on specific synapses.

Hwan-Ching Tai of Xiamen University would like to see more samples analyzed with this new protocol. “SynPull can offer a standard method for examining synaptic pathologies in postmortem brain tissues, and this should be done on a larger scale,” he said. Previously, Tai led a National Taiwan University effort to immobilize synaptosomes for dSTORM imaging through electrostatic attraction, which they used to study tau from mouse tissue samples (Bhattacharya et al., 2021). He noted the Cambridge team advanced the field of super-resolution synaptosome imaging by introducing automated analysis and examining human tissue.—Lauren Schneider

Single-Molecule Imaging Spies Protein Aggregates in Synapses

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