Hierarchical organization and genetically separable subfamilies of PSD95 postsynaptic supercomplexes

Abstract PSD95 is an abundant postsynaptic scaffold protein in glutamatergic synapses that assembles into supercomplexes composed of over 80 proteins including neurotransmitter receptors, ion channels and adhesion proteins. How these diverse constituents are organized into PSD95 supercomplexes in vivo is poorly understood. Here, we dissected the supercomplexes in mice combining endogenous gene‐tagging, targeted mutations and quantitative biochemical assays. Generating compound heterozygous mice with two different gene‐tags, one on each Psd95 allele, showed that each ~1.5 MDa PSD95‐containing supercomplex contains on average two PSD95 molecules. Gene‐tagging the endogenous GluN1 and PSD95 with identical Flag tags revealed N‐methyl D‐aspartic acid receptors (NMDARs) containing supercomplexes that represent only 3% of the total population of PSD95 supercomplexes, suggesting there are many other subtypes. To determine whether this extended population of different PSD95 supercomplexes use genetically defined mechanisms to specify their assembly, we tested the effect of five targeted mouse mutations on the assembly of known PSD95 interactors, Kir2.3, Arc, IQsec2/BRAG1 and Adam22. Unexpectedly, some mutations were highly selective, whereas others caused widespread disruption, indicating that PSD95 interacting proteins are organized hierarchically into distinct subfamilies of ~1.5 MDa supercomplexes, including a subpopulation of Kir2.3‐NMDAR ion channel‐channel supercomplexes. Kir2.3‐NMDAR ion channel‐channel supercomplexes were found to be anatomically restricted to particular brain regions. These data provide new insight into the mechanisms that govern the constituents of postsynaptic supercomplexes and the diversity of synapse types. Read the Editorial Highlight for this article on page 500. Cover Image for this issue: doi. 10.1111/jnc.13811.

PSD95 is a scaffold protein composed of three PDZ (PSD95, Dlg, ZO-1 homologous region) domains, an SH3 domain and a guanylate kinase domain that mediate interactions with numerous synaptic proteins including neurotransmitter receptors, adhesion and signalling proteins (Husi et al. 2000;Fern andez et al. 2009;Zhu et al. 2016). However, the size, stoichiometry and in what quaternary molecular state PSD95 is assembled in vivo is presently unclear (Hsueh et al. 1997;Christopherson et al. 2003;Zeng et al. 2016). We recently reported that PSD95 resides almost exclusively within~1.5 MDa supercomplexes and that NMDARs are organized into supercomplexes containing both PSD95 and PSD93 (Frank et al. 2016). Here, using an integrated genetic and biochemical strategy, we show that, on average, a dimer of PSD95 hierarchically organizes postsynaptic proteins into multiple distinct~1.5 MDa PSD95 supercomplex subfamilies in the brain.

Antibodies
The following antibodies were used in this study: mAb
Blue native and SDS-PAGE immunoblot Adult (P56-70) mouse forebrains (cortex and hippocampus) were dissected and homogenized in buffer H (1 mM Na HEPES pH7.4, 320 mM sucrose with protease inhibitors). Samples were collected for sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The homogenate pellet was collected by centrifugation with 1168 g. (MLA-80, 5000 rpm) at 2°C for 10 min and rehomogenized (6 strokes) in 2 mL buffer H and centrifuged as before. Pooled first and second 1168 g. supernatants were centrifuged at 16860 g. (MLA80, 19 000 rpm) to pellet the crude membranes. Crude membranes were re-suspended in 2.5 mL buffer H and extracted with 2.5 mL buffer X (1% Na deoxycholate, 100 mM NaCl, 50 mM tris.Cl pH8) for 1 h at 6-10°C. Next, insoluble and non-specifically aggregated proteins were removed from the total extract by centrifugation at 116760 g. (MLA-80, 50 000 rpm) for 40 min at 8°C. Samples were collected for blue native PAGE (BNP) and immediately run according to Schagger (Schagger 2001) followed by immunoblot.

Statistical analysis
All experiments were performed using at least six biological and two technical replicates. Student's t-test was used to compare two experimental groups. p values < 0.05 were considered as statistically significant.

Results
Using targeted genetic tags to quantify 1:17 molar ratio of NMDAR to PSD95 in the mouse forebrain We recently reported that NMDARs were partitioned betweeñ 0.8 MDa tetrameric ion channel complexes and~1.5 MDa supercomplexes (hereafter referred to as 0.8-NR and 1.5-NR respectively), whereas almost all forebrain PSD95 was retained within~1.5 MDa supercomplexes (hereafter referred to as 1.5-PSD95) (Frank et al. 2016). To quantify the molar ratio of PSD95 and NMDARs, we used two knockin mouse lines where the PSD95 and the obligatory subunit of NMDARs, GluN1, were tagged with an identical 3xFlag tag targeted to the genes encoding these proteins (Glun1 TAP/TAP and Psd95 TAP/TAP , respectively) (Frank et al. 2016). In dotblots and SDS-PAGE immunoblots, no Flag was detected in wildtype mouse total forebrain, whereas that in Glun1 TAP/TAP and Psd95 TAP/TAP mice indicated the amount of PSD95 and GluN1, respectively (Fig. 1a). The intensity measured by densitometry of immuno-dotblots from Glun1 TAP/TAP mouse forebrains is 6 AE 1% that of Psd95 TAP/TAP , which corresponds to a 17 AE 3 fold (mean AE SD) molar excess of PSD95 over GluN1 ( Fig. 1a and b). Since~50% of GluN1 subunits are assembled with PSD95 (Frank et al. 2016), 1.5-NRs represent a~3% subset of an extended family of~1.5 MDa supercomplexes containing PSD95.
Using mouse genetics to measure the oligomeric state of PSD95 in~1.5 MDa supercomplexes It is possible that multiple copies or oligomers of PSD95 are found in each 1.5-PSD95 (Hsueh et al. 1997;Christopherson et al. 2003;Zhu et al. 2016). To measure the average number of PSD95 molecules in each~1.5 MDa supercomplex in the mouse forebrain, we targeted two different tags, 3xFlag (Fern andez et al. 2009) and GFP (Broadhead et al. 2016); one to each allele of the gene encoding PSD95 (Psd95) to produce a compound heterozygous knockin line, Psd95 TAP/EGFP (Fig. 2a). Since equal expression of both alleles is expected in each cell, observing the ratio of coassembly of PSD95-TAP and PSD95-GFP in forebrain extracts gave a direct measure of the average number of PSD95 molecules in each complex. If only a single molecule of PSD95 were required in each complex, immuno-capture of PSD95-GFP with anti-GFP antibody would co-purify none of the TAP-tagged PSD95. If on average two, three or four molecules of PSD95 assemble in each complex, 50%, 75% or 87.5% PSD95-TAP would be co-captured with PSD95-GFP respectively (Fig. 2b). As seen in Fig. 2c-f when all PSD95-GFP was immuno-captured, 49 AE 3% (mean AE SD) PSD95-TAP was co-purified, indicating that each complex contains on average two molecules of PSD95.
1.5 MDa NMDAR supercomplexes contain both PSD95 and PSD93 (Frank et al. 2016). In accordance, serial purification of 39Flag and GFP from Psd95 TAP/EGFP compound heterozygous mice showed that dimers of PSD95 also contain NMDAR and PSD93 (Fig 2f). The mass of a dimer of PSD95 is~170 kDa, thus the remaining mass of 1.5-PSD95 must be occupied by other proteins.
Genetic and biochemical dissection of NMDAR and PSD95 supercomplex subfamilies Mass spectrometric analysis of 1.5-NR and 1.5-PSD95 supercomplexes identified 55 and 79 different proteins, respectively (Fern andez et al. 2009;Frank et al. 2016), with 89% identity. To determine whether the constituents of 1.5-NR and 1.5-PSD95 are in overlapping or separable populations of supercomplexes, we focussed on four constituents of 1.5-NR and 1.5-PSD95 that were readily detected by blue native PAGE (BNP) immunoblot in wildtype forebrain extract (Frank et al. 2016): the inward-rectifying potassium channel, Kir2.3, an ARF-GEF signalling cofactor, IQsec2/ Brag1, an immediate early gene product, Arc/Arg3.1, and a trans-synaptic adhesion protein, Adam22 (Fig. 3a, left most two lanes). Immunoblots show each of these proteins were partitioned into multiple distinct assemblies that all included a discrete band migrating with masses that ranged from 1.2 to The molar ratio of Flag from Psd95 TAP/TAP and Glun1 TAP/TAP was quantified densitometrically using a dilution series, in which Psd95 TAP/TAP forebrain extracts were diluted with that of wildtype. Densitometry of dilution series indicated TAP-PSD95 was 17 AE 3-fold (mean AE SD) more concentrated than TAP-GluN1. and flow-through (unbound) in a GFP immunoprecipitation (IP) from Psd95 TAP/GFP hybrid mutant forebrain extract. Only PSD95-TAP assemblies containing at least one PSD95-GFP will be captured and the expected distribution of PSD95-TAP and PSD95-GFP subunits between captured and flow-through samples is depicted for different homooligomeric states of 1.5-PSD95 containing on average: 1 (monomer), 2 (dimer), 3 (trimer) or 4 (tetramer) PSD95 molecules. This partitioning, indicated as the percentage split for PSD95-TAP captured and in the flow-through, is dependent on the stoichiometry of PSD95 molecules in each complex. Green and cyan ellipses correspond to PSD95-GFP and PSD95-TAP subunits, respectively. For each oligomeric state, all possible assemblies containing different combinations of GFP-and TAP-tagged PDS95 are shown. (c) Dilution series of Psd95 TAP/GFP forebrain extract into that of wildtype indicated sensitivity of quantification. These data show the dynamic range of sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) Flag immunoblot detection. (d) PSD95-GFP was immunoprecipitated (IP) from Psd95 TAP/GFP and negative control (Psd95 TAP/WT ) forebrain extract supernatant (Input) with GFP antibody. Top panel, SDS-PAGE GFP immunoblot of IP shows near complete immunoprecipitation of all PSD95-GFP (Captured) from Psd95 TAP/GFP hybrid double mutant forebrain extracts. Second panel, Flag immunoblot shows half PSD95-TAP co-precipitated with PSD95-GFP, the remaining unbound PSD95-TAP was detected in the flowthrough. Lower panel, Flag immunoblot of control IP (Psd95 TAP/WT ) shows no PSD95-TAP was captured in the absence of GFP-tagged PSD95. The total protein loaded for SDS immunoblot was normalized across Input, Flow-through and Captured lanes by supplementing with non-tagged (wildtype)samples. Representative data from triplicate experiments shown. (e) Densitometric immunoblot quantification of PSD95-GFP (GFP) in GFP IP. The band intensities from triplicate samples were measured and normalized to that of input. Error bars indicate 1 SD. These data show essentially all the PSD95-GFP was captured by GFP IP. (f) Densitometric immunoblot quantification of PSD95-TAP (Flag) in GFP IP (g, middle panel). The band intensities from triplicate samples were measured and normalized to that of input. Error bars indicate 1 SD. These data show half the PSD95-TAP was cocaptured by PSD95-GFP immunoprecipitation. Thus, each 1.5-PSD95 supercomplex contains on average a dimer of PSD95 molecules. (g) 1.5-PSD95 each containing two molecules of PSD95 were isolated in two sequential steps: Flag immunoaffinity purification ('IAP') followed by GFP immunoprecipitation ('IP') from Psd95 TAP/GFP hybrid double mutant mice. Psd95 TAP/WT mice were used as a negative control. PSD93 and GluN1 were detected by SDS-PAGE immunoblot. These data show that the subset of 1.5-PSD95 containing PSD93 and GluN1 each also contain a dimer of PSD95.    Fig. 3a and recently published (Frank et al. 2016) showing the effect of different mutations (columns) on distinct components of 1.5-PSD95 supercomplexes (rows). X, denotes assembly of supercomplex was blocked by the mutation. -, denotes assembly of the supercomplex was not blocked by the mutation. (c) Subunit-depletion of GluN2B and PSD95 removes 1.5-Kir2.3. Extracts from Glun1 TAP/TAP /Psd95 GFP/GFP double knockin mice were subunit-depleted with antibodies (shown in lanes), then separated on BNP for immunoblotting with Kir2.3 antibody to show 1.5-Kir2.3. Lanes; Input, total extract; immunodepleting antibodies (lanes shown left to right), non-specific IgG, GFP, GluN2A, GluN2B. Arrow indicates 1.5-Kir2.3. Molecular weight in MDa shown on right. IB, immunoblotting antibody. (d) Subunit-depletion of PSD95 removes all 1.5-Arc. Extracts from Glun1 TAP/TAP /Psd95 GFP/GFP double knockin mice were subunit-depleted with antibodies (shown in lanes) then separated on BNP for immunoblotting with Arc antibody to show 1.5-Arc. Lanes; Input, total extract; immunodepleting antibodies (lanes shown left to right), non-specific IgG, GFP, GluN2B. Arrow indicates 1.5-Arc. Molecular weight in MDa shown on right. IB, immunoblotting antibody. (e) Subunit-depletion of PSD95 removes all 1.5-IQsec. Extracts from Glun1 TAP/TAP /Psd95 GFP/GFP double knockin mice were subunit-depleted with antibodies (shown in lanes), then separated on BNP for immunoblotting with IQsec2 antibody to show 1.5-IQsec2. (f) Subunit-depletion of PSD95 does not remove all Adam22. Extracts from Glun1 TAP/TAP /Psd95 GFP/GFP double knockin mice were subunitdepleted with antibodies (shown in lanes) then separated on BNP for immunoblotting with Adam22 antibody to show 1.5-Adam22. (g) Schematic showing extended family tree of~1.5 MDa supercomplexes that contain PSD95 and their relative abundance in the mouse forebrain. The 1.5-PSD95 was divided into 1.5-NR and 1.5-Non-NR subpopulations. PSD95 is 17-fold more abundant than GluN1. Sincẽ 50% N-methyl D-aspartic acid receptors (NMDARs) interact with PSD95 (Frank et al. 2016), 1.5-Non-NR is 34-fold more abundant than 1.5-NR (ratio indicated in blue). Each subpopulation was further subdivided into those containing Kir2.3, IQseq2, Adam22 and Arc. The distribution of 1.5-Kir2.3, 1.5-IQsec2, 1.5-Adam22 and 1.5-Arc (expressed as a ratio in blue) between 1.5-NR and 1.5-Non-NR were estimated by densitometry of supercomplexes immunodepleted with GluN2B and PSD95-EGFP respectively (see Fig. 3c-e). 3 MDa, hereafter referred to for simplicity as 1.5-Kir2.3, 1.5-IQsec2, 1.5-Arc, and 1.5-Adam22, respectively. We next examined these protein complexes in a battery of mutant mice to identify common and distinct genetic requirements of supercomplex assembly.
As shown in Fig. 3g, these findings show a hierarchy of organization of complexes into supercomplexes, in which 1.5-PSD95 can be divided into a Kir2.3-containing subpopulations: those with NMDARs (1.5-NR) and those lacking receptors (1.5-non-NR). To ask whether a similar organization applies to other PSD95 interacting proteins, we quantified the amount of Arc and IQsec2 in 1.5-NR and 1.5non-NR using the immunodepletion strategy. Densitometric quantification of 1.5-Arc and 1.5-IQsec2 BNP immunoblot bands from GluN2B and PSD95 immunodepleted samples indicated that all interact with PSD95 but that 4% and 5% contain NMDARs, respectively (Fig. 3d and e). In contrast, only 14% of 1.5-Adam22 was removed (Fig. 3f) from PSD95-depleted samples, suggesting that only a very small fraction of 1.5-Adam22 contains PSD95, again consistent with genetic findings (Fig. 3a). Densitometric quantification of these data are summarized Fig. 3g (blue annotation) showing the quantitative distribution of supercomplex subtypes.

Discussion
PSD95 is a central component of the postsynaptic terminal of excitatory synapses with important roles in physiology and behaviour. Although it is known to interact with many proteins and form multiprotein complexes, the stoichiometry of subunits and the specific protein interactions that assemble these complexes is poorly understood in the intact animal. We showed that almost all PSD95 resides within~1.5 MDa supercomplexes that on average each contain two molecules of PSD95 in vivo. Our genetic and biochemical dissection of PSD95 and its interactors suggest that subfamilies of synaptic PSD95 supercomplexes are organized according to a combination of genetic requirements that hierarchically specify their composition.
Although NMDAR and PSD95 both co-exist withiñ 1.5 MDa supercomplexes, 1.5-NR is a subset of 1.5-PSD95. Accordingly, we quantified that there is 17-fold more 1.5-PSD95 than GluN1. Because~50% of GluN1 is in 1.5-NR and~50% in 0.8-NR (ion channel complexes alone; lacking PSD95) (Frank et al. 2016) then 1/34 of all PSD95 supercomplexes contain NMDAR in mouse forebrain (Fig. 3g). These molar ratios were from total forebrain and differ somewhat from estimates using mass spectrometric approaches on Triton X-100-resistant fractions of forebrain membranes (Cheng et al. 2006). It is likely each~1.5 MDa supercomplex can accommodate a single tetrameric NMDAR. Since TAP-purified NMDARs contain approximately equal amounts of PSD95 and PSD93 (Frank et al. 2016) and the apparent dimeric oligomeric state of PSD95 in vivo, we suggest 1.5-NR supercomplexes are organized around a core platform containing a dimer of PSD95 and a dimer of PSD93 (Hsueh et al. 1997).
Supercomplexes containing PSD95 with or without NMDARs are further subdivided into 1.5-Arc, 1.5-IQsec2, 1.5-Kir2.3 and 1.5-Adam22 supercomplex subfamilies. This is consistent with the high degree of overlap of proteins identified by mass spectrometry samples purified from Grin1 TAP and Psd95 TAP mice (Frank et al. 2016). While the similarity between these proteomes might suggest that synaptic proteins associate promiscuously or by redundant mechanisms, we show using several mouse mutations that some supercomplexes have strict and selective genetic dependencies for the assembly of their constituent proteins. Interestingly, a similar genetic mechanism has been shown to organize ankyrins, a family of axonal scaffold protein, that cluster ion channels at the nodes of Ranvier (Ho et al. 2014).
The characterization of the Kir2.3-NMDAR ion channelchannel supercomplexes and their neuroanatomical distribution highlights the potential physiological importance of mechanisms controlling the organization of supercomplexes. A functional interaction between inward-rectifying potassium channels and NMDARs has been predicted to be the 'perfect couple' for producing the necessary voltage bi-stability of 'on' and 'off' states within the postsynaptic membrane Sanders et al. 2013). Our finding that a subset of 1.5-Kir2.3 supercomplexes required the cytoplasmic domain of GluN2B suggests that this domain may be directly involved with the mechanism of supercomplex formation between these two channels. Kir2.3-NMDAR ion channel-channel supercomplexes were anatomically enriched within the ventral midbrain regions but absent from the hippocampus and cortex.  Fig. 4b shows synaptic localization of Kir2.3 is disrupted in Psd95 À/À mice. Sections double-stained with Kir2.3 (green, top) and pre-synaptic marker synapsin1 (red, middle) antibodies and merged image (bottom). White arrowheads show large Kir2.3 aggregates in Psd95 À/À mice. Scale bar, 4 lm. Right, histograms quantifying changes in puncta size (upper graph) and density (lower graph) of piriform cortex Kir2.3 quantified from triplicate experiments of Psd95 À/À and WT sections. Error bar, 1 SD. **p ≤ 0.01; ***p ≤ 0.001. Thus, the mechanisms regulating the hierarchical organization of synaptic supercomplex subfamilies appear to specify the diversity of synapse subtypes.
Our results also shed light on the molecular organization of synapses in vertebrate organisms and how their supercomplex diversity arose. Two whole genome duplications occurred 550 million years ago in the vertebrate lineage resulting in the generation of paralogs and an overall expansion of the vertebrate synapse proteome (Emes et al. 2008;Emes and Grant 2012). Our present results show selective effects of paralog mutations in PSD95 and PSD93, as well as GluN2A and GluN2B. Thus, the increase in paralogs has not simply multiplied the number of vertebrate supercomplexes, but rather, the diversification of paralogs has resulted in selective functions that restrict the diversity of supercomplexes.
The hierarchical organization of supercomplexes outlined here is potentially generally relevant to the 66 proteins we have previously reported to be distributed between 220 separable synaptic complexes and supercomplexes (Frank et al. 2016). Indeed, it is possible that the number of distinct complexes and supercomplexes identified using this biochemical approach is underestimated because weakly associated or very low abundance constituents may be refractory to the use of detergents. We propose the hierarchy of genetic requirements that gives rise to the assembly of distinct supercomplex subfamilies may play an important role in defining synaptic function and synaptic subtypes of the brain. Given the diversity of complexes and organization into subfamilies, we suggest there is potential to define a taxonomy based on composition and its genetic determinants.