- Reviewing EditorHugo BellenBaylor College of Medicine, Houston, United States of America
- Senior EditorLu ChenStanford University, Stanford, United States of America
Reviewer #1 (Public Review):
The manuscript by Kadkova et al. describes an electrophysiological analysis of 3 neurodevelopmental disease-causing SNAP-25 mutations in hippocampal neuron autaptic cultures. The work expands on a prior study of these 3 mutations, along with several others in SNAP-25, that was performed in acutely dissociated hippocampal cultures by another group (Alten et al, 2021). Most of the physiology defects found are pretty similar for the 3 mutations the two research groups characterized, with differences largely found in the effects on the size of the readily releasable pool (RRP) of SVs. These differences could be due to technical differences in the approach but are also likely to reflect in part differences in autapses as a model that have been previously described. In addition to the physiological analysis in cultured neurons, the current work extends the analysis beyond the prior study by analyzing the effects of these SNAP-25 mutations in in vitro liposome fusion assays with purified proteins, and some modeling of the effects on energy landscapes during priming and fusion.
The authors use lentiviral expression of wildtype or one of the 3 mutants in SNAP-25 autaptic neurons and assay neuronal survival and synaptic output. The authors also combine wildtype with each of the 3 mutants as well, given these diseases manifest as spontaneous mutations in only 1 of the SNAP-25 alleles, suggesting a dominant effect. The authors observe that the V48F and D166Y alleles (that are suggested to disrupt the Syt1-SNAP-25/SNARE interface) result in a very large increase in spontaneous release that exceeds the Syt1 null mutant alone, suggesting an effect on spontaneous SV release beyond a lack of Syt1 regulation of SNARE-mediated fusion. In contrast, Syt1 nulls have a much more severe loss of evoked release, through both V48F and D166Y also have modest decreases in release. They find both mutants also cause a decrease in the RRP. Applying some modeling for these results, the authors suggest V48F and D166Y lowers the energy barrier for fusion, creating the enhanced spontaneous release rates and causing a decrease of the RRP. They also find evidence for reduced SV priming. In contrast, a SNAP-25 I167N disease mutation in the SNARE assembly interaction layer causes dramatic decreases in both evoked and spontaneous release, consistent with a disruption to SNARE assembly/stability. In vitro fusion assays with these mutant SNAP-25 alleles was also done and provided supportive evidence for these interpretations for all 3 alleles. The ability to control calcium, Syt1, PIP2 and Complexin levels in the in vitro assays provided additional information on defining the precise steps of the fusion process these mutations disrupt. Together, the study indicates the I167N mutation acts as a dominant-negative allele to block fusion, while the other two alleles have both loss- and gain-of function properties that cause more complex disruptions that decrease evoked release while dramatically enhancing spontaneous fusion.
Overall, these results build on prior work and shed light on how disruptions to the SNAP-25 t-SNARE alter the process of SV priming and fusion.
Reviewer #2 (Public Review):
Kádková, Murach, Pedersen, and colleagues studied how three disease-causing missense mutations in SNAP25 affect synaptic vesicle exocytosis. These mutations have previously been studied by Alten et al., 2021. The authors observed similar impairments in spontaneous and evoked release as Alten et al., 2021, but the measurement of readily releasable pool (RRP) size differed between the two studies. The authors found that the V48F and D166Y mutations affect the interaction with the Ca2+ sensor synaptotagmin-1 (Syt1), but do not entirely phenocopy Syt1 loss-of-function because they also exhibit a gain-of-function. Thus, these mutations affect multiple aspects of the energy landscape for vesicle priming and fusion. The I67N mutation specifically increases the fusion energy barrier without affecting upstream vesicle priming.
The strength of the study includes careful and technically excellent dissection of the synaptic release process and a combination of electrophysiology, biophysics, and modeling approaches. This study gained a deeper mechanistic understanding of these mutations in vesicle exocytosis than the previous study but did not result in a paradigm shift in our understanding of SNAP25-associated encephalopathy because the same spontaneous and evoked release phenotypes were previously identified.
Comments on revised version:
The authors fully addressed the two previous technical concerns and improved the introduction of the paper.