Sequential replacement of PSD95 subunits in postsynaptic supercomplexes is slowest in the cortex

Joint Public Review:

The present study explored the principles that allow cells to maintain complex subcellular proteinaceous structures despite the limited lifetimes of the individual protein components. This is particularly critical in the case of neurons, where the size and protein composition of synapses define synaptic strength and encode memory.

PSD95 is an abundant synapse protein that acts as a scaffold in the recruitment of transmitter receptors and other signaling proteins and is required for proper memory formation. The authors used super-resolution microscopy to study PSD95 super-complexes isolated from the brains of mice expressing tagged PSD variants (Halo-Tag, mEos, GFP). Their results show compellingly that a large fraction (~25%) of super-complexes contains two PSD95 copies about 13 nm apart, that there is substantial turnover of PSD95 proteins in super-complexes over a period of seven days, and that ~5-20% of the super-complexes contain new and old PSD95 molecules. This percentage is higher in synaptic fractions as compared to total brain lysates, and highest in isocortex samples (~20%). These important findings support the notion put forward by Crick that sequential subunit replacement gives synaptic super-complexes long lifetimes and thus aids in memory maintenance. Overall, this is very interesting, providing key insights into how synaptic protein complexes are formed and maintained. On the other hand, the actual role of these PSD95 super-complexes in long-term memory storage remains unknown.

Strengths

(1) The study employed an appropriate and validated methodology.

(2) Large numbers of PSD95 super-complexes from three different mouse models were imaged and analyzed, providing adequately powered sample sizes.

(3) State-of-the-art super-resolution imaging techniques (PALM and MINFLUX) were used, providing a robust, high-quality, cross-validated analysis of PSD95 protein complexes that is useful for the community.

(4) The result that PSD95 proteins in dimeric complexes are on average 12.7 nm apart is useful and has implications for studies on the nanoscale organization of PSD95 at synapses.

(5) The finding that postsynaptic protein complexes can continue to exist while individual components are being renewed is important for our understanding of synapse maintenance and stability.

(6) The data on the turnover rate of PSD95 in super-complexes from different brain regions provide a first indication of potentially meaningful differences in the lifetime of super-complexes between brain regions.

Weaknesses

(1) The manuscript emphasizes the hypothesis that stable super-complexes, maintained through sequential replacement of subunits, might underlie the long-term storage of memory. While an interesting idea, this notion requires considerably more research. The presented experimental data are indeed consistent with this notion, but there is no evidence that these complexes are causally related to memory storage.

(2) Much of the presented work is performed on biochemically isolated protein complexes. The biochemical isolation procedures rely on physical disruption and detergents that are known to alter the composition and structure of complexes in certain cases. Thus, it remains unclear how the protein complexes described in this study relate to PSD95 complexes in intact synapses.

(3) Because not all GFP molecules mature and fold correctly in vitro and the PSD95-mEos mice used were heterozygous, the interpretation of the corresponding quantifications is not straightforward.

(4) It was not tested whether different numbers of PSD95 molecules per super-complex might contribute to different retention times of PSD95, e.g. in synaptic vs. total-forebrain super-complexes.

(5) The conclusion that the population of 'mixed' synapses is higher in the isocortex than in other brain regions is not supported by statistical analysis.

(6) The validity of conclusions regarding PSD95 degradation based on relative changes in the occurrence of SiR-Halo-positive puncta is limited.

Author response:

(1) The manuscript emphasizes the hypothesis that stable super-complexes, maintained through sequential replacement of subunits, might underlie the long-term storage of memory. While an interesting idea, this notion requires considerably more research. The presented experimental data are indeed consistent with this notion, but there is no evidence that these complexes are causally related to memory storage.

We agree with the reviewer that, while our data support the idea that subunit exchange in supercomplexes could underlie long-term memory storage, more research is necessary to conclusively validate this hypothesis. The experimental data presented are consistent with the idea that stable supercomplexes, maintained through sequential replacement of subunits, play a role in memory retention. However, establishing a causal relationship between these supercomplexes and memory storage will require additional experiments and in-depth analyses.

(2) Much of the presented work is performed on biochemically isolated protein complexes. The biochemical isolation procedures rely on physical disruption and detergents that are known to alter the composition and structure of complexes in certain cases. Thus, it remains unclear how the protein complexes described in this study relate to PSD95 complexes in intact synapses.

Whilst it could be the case that biochemical isolation procedures have the potential to alter the composition and structure of protein complexes, we have previously published the protocol used to isolate PSD95-containing supercomplexes (Nat Commun. 2016; 7: 11264). In that study, we demonstrated that the isolated supercomplexes are approximately 1.5 MDa in size and contain multiple proteins, including other scaffolding proteins (e.g., PSD93) and receptors (e.g., NMDARs). Importantly, these supercomplexes remain stable when exposed to detergents and dilution, strongly indicating that they represent the native complexes present in intact synapses.

(3) Because not all GFP molecules mature and fold correctly in vitro and the PSD95-mEos mice used were heterozygous, the interpretation of the corresponding quantifications is not straightforward.

Although genetic tagging ensures a 1:1 labeling stoichiometry, we acknowledge that the presence of unfolded GFP and the use of heterozygous PSD95-mEos mice can complicate the analysis. We have highlighted this limitation in the manuscript. Nonetheless, our results show a high level of consistency across the different genetic fusions used in this study.

(4) It was not tested whether different numbers of PSD95 molecules per super-complex might contribute to different retention times of PSD95, e.g. in synaptic vs. total-forebrain super-complexes.

The potential impact of varying numbers of PSD95 molecules per super-complex on retention times was considered. However, our analysis showed minimal differences in the distribution of molecule numbers per super-complex between the synaptic and forebrain samples.

(5) The conclusion that the population of 'mixed' synapses is higher in the isocortex than in other brain regions is not supported by statistical analysis.

The conclusion that the population of 'mixed' synapses is higher in the isocortex than in other brain regions is indeed supported by statistical analysis. All relevant statistical data are detailed in Table S2, and the finding is statistically significant. We will emphasize this point in the revised manuscript.

(6) The validity of conclusions regarding PSD95 degradation based on relative changes in the occurrence of SiR-Halo-positive puncta is limited.

We recognize that conclusions based solely on the relative changes in SiR-Halo-positive puncta concerning PSD95 degradation have limitations. To address this, we also quantified the “new” PSD95 by analyzing AF488-Halo-positive molecules.