Cell‐type‐specific visualisation and biochemical isolation of endogenous synaptic proteins in mice

Abstract

In recent years, the remarkable molecular complexity of synapses has been revealed, with over 1,000 proteins identified in the synapse proteome. Although it is known that different receptors and other synaptic proteins are present in different types of neurons, the extent of synapse diversity across the brain is largely unknown. This is mainly due to the limitations of current techniques. Here, we report an efficient method for the purification of synaptic protein complexes, fusing a high‐affinity tag to endogenous PSD95 in specific cell types. We also developed a strategy, which enables the visualisation of endogenous PSD95 with fluorescent‐protein tag in Cre‐recombinase‐expressing cells. We demonstrate the feasibility of proteomic analysis of synaptic protein complexes and visualisation of these in specific cell types. We find that the composition of PSD95 complexes purified from specific cell types differs from those extracted from tissues with diverse cellular composition. The results suggest that there might be differential interactions in the PSD95 complexes in different brain regions. We have detected differentially interacting proteins by comparing data sets from the whole hippocampus and the CA3 subfield of the hippocampus. Therefore, these novel conditional PSD95 tagging lines will not only serve as powerful tools for precisely dissecting synapse diversity in specific brain regions and subsets of neuronal cells, but also provide an opportunity to better understand brain region‐ and cell‐type‐specific alterations associated with various psychiatric/neurological diseases. These newly developed conditional gene tagging methods can be applied to many different synaptic proteins and will facilitate research on the molecular complexity of synapses.

Engineering paralog-specific PSD-95 synthetic binders as potent and minimally invasive imaging probes

“…However, the absence of defined epitopes for both PF11 and PSD95.FingR does not allow to convincingly rule out possible perturbations of some of PSD-95 functions when any of the two probes is bound. PSD-95 is indeed a multidomain scaffold protein with a long list of identified partners (Dosemeci et al, 2007;Won et al, 2017;Zhu et al, 2020) as well as numerous post-translational regulation sites (Vallejo et al, 2017), which complicate evaluation of the impact resulting from a synthetic binder interaction. In addition, recent studies support the idea that the protein should not be viewed as a passive scaffolding element of the synapse but rather as an active actor with a capacity to respond to partners binding (Rademacher et al, 2019;Zeng et al, 2018).…”

“…Besides the use of evolved synthetic binders as a strategy to label endogenous PSD-95 in live conditions, a number of genetic approaches have been reported. They all rely on gene editing methods and are typically used to generate PSD-95 fluorescent protein (Broadhead et al, 2016;Fortin et al, 2014;Willems et al, 2020;Zhu et al, 2020) or engineered self-labeling enzyme fusions (Masch et al, 2018). Comparatively, their main advantages over expressed exogenous probes are the ideal stoichiometric labeling (one fluorophore per target protein, to be tempered by the notion of effective labeling efficiency of the fluorophore (Thevathasan et al, 2019)) together, for the knock-in approaches, with the possibility to achieve global labeling.…”

Btbd11 is an inhibitory interneuron specific synaptic scaffolding protein that supports excitatory synapse structure and function

“…To the best of our knowledge, this is the first attempt to uncover differences in PSD composition between INs and ENs using proteomics. A similar method was used to examine differences in PSDs from different subpopulations of ENs (Zhu et al, 2020), highlighting the usefulness of the approach. Initially we focused our effort into a thorough characterizing of Btbd11.…”

Cell type-specific biotin labeling in vivo resolves regional neuronal and astrocyte proteomic differences in mouse brain

“…To date, in vivo BONCAT has been successfully applied to characterizing the nascent neuronal proteome and is well-suited to study turnover of newly synthesized proteins, potentially in non-neuronal cells as well 16 , 17 . Synapse-specific proteomics in vivo has also been achieved using the conditional Cre-lox PSD95 tandem affinity purification (cPSD95-TAP) approach 20 .…”