Received: 17 July 2019 | Revised: 4 February 2020 | Accepted: 17 February 2020 DOI: 10.1111/jnc.14988 ORIGINAL ARTICLE SynGAP splice variants display heterogeneous spatio-temporal expression and subcellular distribution in the developing mammalian brain Gemma Gou1,2 | Adriana Roca-Fernandez3 | Murat Kilinc4 | Elena Serrano5 | Rita Reig-Viader1,2 | Yoichi Araki6,7 | Richard L. Huganir6,7 | Cristian de Quintana- Schmidt8 | Gavin Rumbaugh4 | Àlex Bayés1,2 1 Molecular Physiology of the Synapse Laboratory, Biomedical Research Institute Abstract Sant Pau (IIB Sant Pau), Barcelona, Spain The SynGAP protein is a major regulator of synapse biology and neural circuit func- 2 Universitat Autònoma de Barcelona, tion. Genetic variants linked to epilepsy and intellectual disability disrupt synaptic Bellaterra (Cerdanyola del Vallès), Spain 3 Nuffield Department of Clinical function and neural excitability. SynGAP has been involved in multiple signaling path- Neuroscience, University of Oxford, Oxford, ways and can regulate small GTPases with very different roles. Yet, the molecular UK 4 bases behind this pleiotropy are poorly understood. We hypothesize that different Department of Neuroscience, The Scripps Research Institute, Jupiter, FL, USA SynGAP isoforms will mediate different sets of functions and that deciphering their 5 Biobank, Biomedical Research Institute spatio-temporal expression and subcellular localization will accelerate understand- Sant Pau (IIB Sant Pau), Barcelona, Spain 6 ing their multiple functions. Using isoform-specific antibodies recognizing SynGAP Solomon H. Snyder Department of Neuroscience, Johns Hopkins University in mouse and human samples we found distinctive developmental expression pat- School of Medicine, Baltimore, MD, USA terns for all SynGAP isoforms in five mouse brain areas. Particularly noticeable was 7 Kavli Neuroscience Discovery Institute, the delayed expression of SynGAP-α1 isoforms, which directly bind to postsynaptic Johns Hopkins University, Baltimore, MD, USA density-95, in cortex and hippocampus during the first 2 weeks of postnatal develop- 8 Department of Neurosurgery, Hospital de ment. Suggesting that during this period other isoforms would have a more promi- la Santa Creu i Sant Pau, Barcelona, Spain nent role. Furthermore, we observed subcellular localization differences between Correspondence isoforms, particularly throughout postnatal development. Consistent with previous Àlex Bayés, Molecular Physiology of the Synapse Laboratory, IIB Sant Pau, C/Sant reports, SynGAP was enriched in the postsynaptic density in the mature forebrain. Quintí, 77-79, 08041 Barcelona, Spain. However, SynGAP was predominantly found in non-synaptic locations in a period of Email: abayesp@santpau.cat early postnatal development highly sensitive to SynGAP levels. While, α1 isoforms Gavin Rumbaugh, Department of were always found enriched in the postsynaptic density, α2 isoforms changed from Neuroscience, The Scripps Research Institute, Jupiter, FL 33458, USA. a non-synaptic to a mostly postsynaptic density localization with age and β isoforms Email: gavin@scripps.edu were always found enriched in non-synaptic locations. The differential expression Funding information and subcellular distribution of SynGAP isoforms may contribute to isoform-specific MINECO, Grant/Award Number: BFU2012- regulation of small GTPases, explaining SynGAP pleiotropy. 34398, BFU2015-69717-P, RTI2018- Abbreviations: C-term, C-terminal; DIV, days in vitro; DOC, sodium deoxycholate; EGFP, enhanced green fluorescent protein; FDR, false discovery rate; ID, intellectual disability; IP, immunoprecipitation; MS, mass spectometry; MS/MS, tandem mass spectrometry; N, number of technical replicas; N-term, N-terminal; ON, over-night; PBS, phosphate-buffered saline; PH, pleckstrin homology; PND, postnatal day; ppm, parts per million; PSD, postsynaptic density; PVDF, polyvinylidene fluoride; RRID, Research resource identifier; S1, homogenate without nuclei; SDS, sodium dodecyl sulfate; SNP, synaptic non-PSD; TBS, tris-buffered saline; tSynGAP, total SynGAP; ver., version; α1, alpha1; α2, alpha2; β, beta; γ, gamma. This is an open access article under the terms of the Creative Commons Attribution-NonCommercial License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited and is not used for commercial purposes. © 2020 The Authors. Journal of Neurochemistry published by John Wiley & Sons Ltd on behalf of International Society for Neurochemistry 618 |  wileyonlinelibrary.com/journal/jnc Journal of Neurochemistry. 2020;154:618–634. GOU et al. | 619 097037-B-100, IEDI-2017-00822 and KEYWORDS BES-2013-063720; Career Integration Grant, Grant/Award Number: 304111; pleiotropy, postnatal development, protein expression pattern, protein isoforms, subcellular Ramón y Cajal Fellowship, Grant/Award localization, SynGAP Number: RYC-2011-08391p; AGAUR, Grant/Award Number: SGR14-297 and 2017SGR1776; NIH, Grant/Award Number: MH096847, MH108408, NS064079 and RO1 MH112151 1 | I NTRO D U C TI O N Rap1 (Krapivinsky, Medina, Krapivinsky, Gapon, & Clapham, 2004), Rap2 (Walkup et al., 2015), and Rab5 (Tomoda, 2004). Furthermore, De novo mutations in the human SYNGAP1 gene resulting in ge- Syngap1± mice present increased levels of GTP-bound Rac1 in fore- netic haploinsufficiency cause mental retardation type 5 (MRD-5; brain extracts (Carlisle, Manzerra, Marcora, & Kennedy, 2008), indi- OMIM #612621), an autosomal dominant form of intellectual dis- cating that SynGAP also regulates Rac1, either directly or indirectly. ability (ID) with high rates of progressively worsening childhood ep- The GAP activity of SynGAP participates in the regulation of several ilepsy (Agarwal, Johnston, & Stafstrom, 2019; Hamdan et al., 2009; important signaling pathways for synaptic physiology, such as Ras- Mignot et al., 2016; Parker et al., 2015; Vlaskamp et al., 2019). This MAPK (Komiyama et al., 2002), Ras-PI3K (Qin et al., 2005), Rap-p38 debilitating neurodevelopmental disorder is estimated to be respon- (Krapivinsky et al., 2004; Zhu, Qin, Zhao, Aelst, & Malinow, 2002), sible for up to 1% of all cases of ID (Berryer et al., 2013). Studies in and Rac1-PAK (Carlisle et al., 2008). mouse models of this condition indicate that a Syngap1 genetic defi- It remains unclear how SynGAP can have such a broad im- cit during specific developmental stages causes premature synaptic pact on neuronal signaling. Alternative splicing of Syngap1 mRNA, maturation in excitatory neurons that result in enhanced neuronal which results in many protein isoforms, is likely one mechanism. excitability (Aceti et al., 2014; Clement et al., 2012; Clement, Ozkan, In mammals, the Syngap1 gene encodes different protein isoforms Aceti, Miller, & Rumbaugh, 2013; Ozkan et al., 2014). In addition, that differ in their N- and C-terminus (Chen et al., 1998; Kim et al., more recent studies have identified non-developmental functions of 1998; Li et al., 2001; McMahon et al., 2012). The central part of the the Syngap1 gene that contribute to memory expression and seizure protein is thus common to all isoforms and accounts for most of it, threshold (Creson et al., 2019). Together, these findings indicate that extending 1,091 residues (>80% of the longest protein isoform) in Syngap1 is critical for brain cell function. Thus, in depth study of this rat and human. This core region presents a truncated PH domain, gene will provide insights into the molecular and cellular processes lacking the first 24 residues, a C2 domain, a GTPase-activating that contribute to neurological and psychiatric disorders. protein (GAP) domain, a large disordered region of around 600 Syngap1 encodes the synaptic Ras/Rap GTPase-activating pro- residues and, finally, a truncated coiled-coil domain lacking its tein (SynGAP), which was first described as one of the most abun- final 11 residues, which is involved in SynGAP multimerization dant components of the postsynaptic density (Chen, Rojas-Soto, (Zeng et al., 2016). Five N-terminal (A1, A2, B, C, and D) and four Oguni, & Kennedy, 1998; Kim, Liao, Lau, & Huganir, 1998). Indeed, C-terminal (α1, α2, β, and γ) SynGAP variants have been described. this protein regulates the structure and function of excitatory syn- Of the 20 possible combinations of N- and C-termini with the core apses in the mammalian forebrain (Jeyabalan & Clement, 2016; region, 13 have been reported either in NCBI, ENSEMBL, or the Kilinc et al., 2018). SynGAP has a prominent role in the molecular literature (Chen et al., 1998; Kim et al., 1998; Li et al., 2001). In mechanisms governing synaptic plasticity, being involved in the two mouse, SynGAP isoforms will vary in their molecular weight, rang- hallmarks of this process, incorporation of AMPA receptors into the ing between 148.3 kDa (SynGAP/A2-α2, the largest) and 121.4 kDa synaptic plasma membrane (Kim, Lee, Takamiya, & Huganir, 2003; (SynGAP/C-β, the smallest). Isoforms with A1/2, B, and D N-termini Rumbaugh, Adams, Kim, & Huganir, 2006) and dendritic spine en- present an entire PH domain, while isoforms containing the C largement (Aceti et al., 2014; Vazquez, Chen, Sokolova, Knuesel, N-terminal do not include its first 24 residues. At the other end of & Kennedy, 2004). The activity of SynGAP toward small GTPases the protein, isoforms with C-terminal variants α1, α2, and γ pres- is considered to be its key functional role, with the other domains ent an entire coiled-coil domain, while those with the β variant lack and sequence motifs being involved in regulating it. For instance, its last 11 residues. In support of the idea that Syngap1 alternative the C2 domain is key in the GAP activity toward Rap GTPases (Pena splicing alters protein function, the distinct C-terminal spliced se- et al., 2008) and phosphorylation determines substrate specific- quences have been shown to cause opposing effects on synaptic ity, as CaMK2α promotes RapGAP activity while CDK5 and PLK2 strength, with α1 driving synaptic depression (McMahon et al., stimulate RasGAP activity (Walkup, Sweredoski, Graham, Hess, & 2012; Rumbaugh et al., 2006) and α2 driving synaptic potentiation Kennedy, 2018; Walkup et al., 2015). The exact role of sequences (McMahon et al., 2012). such as the pleckstrin homology (PH) domain, the SH3-binding, Thus, the multitude of available N- and C-termini likely bestows or poly-histidine motifs in the function of SynGAP are not yet un- distinctive functional properties to SynGAP isoforms. However, the derstood. In vitro studies with purified proteins have shown that expression pattern and subcellular localization of distinct SynGAP SynGAP directly modulates the activity of HRas (Kim et al., 1998), isoforms remain largely unexplored, particularly during early 620 | GOU et al. postnatal development, when SynGAP is known to have a strong until weaning (postnatal day [PND] 21), whereas adult mice were fed impact on synaptic (Clement et al., 2012, 2013) or dendritic (Aceti with regular chow (T.2014.12; Envigo). The total number of animals et al., 2014; Michaelson et al., 2018) maturation and neuronal devel- used to conduct mass spectrometry (MS)-based, spatio-temporal ex- opment (Muhia, Yee, Feldon, Markopoulos, & Knuesel, 2010; Agarwal pression, and subcellular localization studies was 135. The specific et al., 2019). Here, we present a systematic study of the expression number of animals used per age was: PND0/1:24, PND4: 28, PND7: of SynGAP isoforms in five different brain regions and four postnatal 6, PND11: 43, PND14: 6, PND21: 13, PND56: 15 (See Figure 1). For developmental stages, identifying specific expression patterns for all ages between PND0 and 21, female and male mice were used at isoforms, both between brain regions and throughout development. equal ratios. PND56 mice were males. Mice culling between PND0 Furthermore, we investigate the differential subcellular localization and 4 was performed by head dissection or by cervical dislocation of SynGAP isoforms and describe how this varies during cortical de- from that age onwards without the use of anesthetic in any case and velopment. Together, our data illustrates the complexity of SynGAP minimizing animal suffering. All experiments were conducted from roles within brain cells, and the key role that C-term variants are likely 9 a.m. to 9 p.m. and those animals that were clearly smaller than the to play in SynGAP biology. We also find that SynGAP C-termini are rest of the littermates were excluded for subsequent analyses. important for its subcellular localization and that SynGAP, generally regarded as almost exclusively found at the synapse, is very abun- dant in the cytosol, specially early in postnatal development, when 2.3 | Mouse brain dissection the brain is most sensitive to Syngap1 haploinsufficiency (Aceti et al., 2014; Clement et al., 2012; Ozkan et al., 2014). Mouse heads were soaked with chilled 1× phosphate-buffered sa- line (PBS, 0.144 M NaCl, 2.683 KCl mM, 10.144 mM Na2HPO4, 0.735 mM KH2PO4, [P5368-10PAK from Sigma]) and dissected 2 | M ATE R I A L S A N D M E TH O DS using scalpel blades while placed onto a glass petri dish with a fil- ter paper (Merck-Millipore). The skull and meninges were removed 2.1 | Ethics statement and procedures on human from brain using Iris scissors (PMD120; Thermo Scientific) and tissue cortical samples forceps 1:2 (PMD023445; Thermo Scientific). For brain dissection of PND0-7 animals a magnifying loupe (Olympus KC 1,500 Ledplus; All surgical procedures were approved by the Ethics Committee on Olympus) was used. Brain areas were dissected as previously de- Clinical Research from the Hospital de la Santa Creu i Sant Pau (ap- scribed (Spijker, 2011). Tissue weight was recorded before snap- proval reference number 16/041). All samples collected originated freezing in liquid nitrogen and stored in a −80°C freezer. from neuro-oncological surgery unit at the Hospital de Sant Pau i la Santa Creu between 2016 and 2018. Adult healthy cortical samples, as determined by pre-surgery nuclear magnetic resonance, were col- 2.4 | Anti-SynGAP-β antibody generation lected in those cases that a corticectomy had to be performed to access subcortical pathological tissue. All patients were informed and signed SynGAP-β antibody was raised against SynGAP aa.1273–1285 at an informed consent. Resected tissue was rapidly wrapped in aluminum the research laboratory of Prof. Richard L. Huganir, Johns Hopkins foil and snap-frozen in liquid nitrogen. Samples were stored in −80°C. University. The antigen peptide with N-terminus Cysteine (NH2- CGGGGAAPGPPRHG-COOH) was coupled with keyhole limpet hemocyanin (77,600; Thermo Fisher). The antigen was injected into 2.2 | Ethics statement on animal research and rabbit and antisera were collected after primary and several booster animal handling injections. Antisera were further purified with affinity column con- taining sulfo-link coupling resin (20,401; Thermo Fisher) coupled All procedures, unless specifically indicated, were done with with same antigen peptide. This antibody will be shared upon rea- C56BL/6J mice (Jackson Laboratories, Research Resource Identifier, sonable request. RRID:MGI:5656552) in accordance with national and European leg- islation (Decret 214/1997 and RD 53/2013). These were approved by the Ethics Committee on Animal Research from the Institut de 2.5 | Total protein extraction, subcellular Recerca de l´Hospital de la Santa Creu i Sant Pau (IR-HSCP) and fractionation and protein quantification the Departament de Territori i Sostenibilitat from the Generalitat de Catalunya (approval reference num. 9,655). Maintenance, treat- For extraction of total proteins, samples were mixed with chilled ment and experimental procedures with mice were conducted at the buffer (50 mM Tris-HCl pH 9 [T1503-1KG], 1% sodium deoxycho- Animal Facility of the IR-HSCP. Mice were housed at a 12 hr light/ late [30970-100G], 50 mM NaF [106449, Merck-Millipore], 20 mM dark cycle with fresh water and food ad libitum. No more than five ZnCl2 [96468-50G], 1 mM sodium orthovanadate [S6508-10G], mice of a given gender were place in the same cage. Special chow 1:2,500 phenyl methane sulfonyl fluoride [P7626-5G], 2 μg/ml (T.2019.12, Envigo) was administered to pregnant mothers and litter aprotinin [616370-10MG, Merck-Millipore], and 2 μg/ml leupeptin GOU et al. | 621 F I G U R E 1 Study timeline for developmental expression and subcellular localization studies. Postnatal day (PND) indicates postnatal day of tissue collection. The total number of animals used for discovery MS-based studies, spatio-temporal expression and subcellular distribution of SynGAP and its isoforms at each PND is also indicated [108976-10MG, all from Sigma-Aldrich unless indicated]) at a 1:17.5 protein concentrations were corrected by silver stain (1610449, tissue:extraction buffer ratio (g/ml). Brain tissue was homogenized Silver Stain Plus™ kit; Bio-Rad). by 30 strokes in 1 or 7-ml borosilicate Dounce homogenizers (357542 & 357544, glass-Teflon tissue grinder; Wheaton) depending on the volume of buffer required. Then, it was incubated on ice for 1 hr 2.6 | Protein dialyzation for detergent exchange and centrifuged at 21,000 g for 30 min at 4°C in 1.5 ml centrifuge tubes (3810x; Eppendorf). The resulting pellet was re-homogenized Dialysis was used to exchange sodium deoxycholate with Triton X-100 twice following the same procedure and resulting supernatants were from total protein extracts prior to immunoprecipitation (IP). Membranes pooled. In the last re-homogenization cycle half of the initial w/v ratio for dialysis (Visking Corporation) were activated according to manufac- was used. Prior to protein quantification, 1% sodium dodecyl sulfate turer's instructions. Samples were dialyzed over-night (ON) against the (SDS) (428029-1EA; Merck-Millipore) was added to all samples. dialysis buffer (50 mM Tris HCl pH7.4 and 1% Triton X-100) at a v/v ratio Subcellular fractions were prepared following previously de- of 1:1,000 in constant agitation at 4°C. After dialysis, Triton X-100 con- scribed procedures (Bayés et al., 2012; Carlin, Grab, Cohen, & centration was adjusted to 1% if required. Finally, samples were soni- Siekevitz, 1980). All centrifugation steps were done at 4°C and sam- cated with an ultrasonic bath Sonicator (Thermo Fisher Scientific) at 5% ples were always kept in ice. Briefly, tissue was homogenized using of its maximum intensity during 45 s with 1.45 s on/off cycles. 7-ml glass-Teflon tissue grinders (357,544, borosilicate Dounce ho- mogenizer; Wheaton). A 1:9 ratio was used and ~40 strokes were applied. Next, a 10 min centrifugation (Epp 5417R; Eppendorf) at 2.7 | Immunoprecipitation 1,400 g was conducted. The resulting supernatant was conserved and the pellet was subjected to two re-homogenizations in the same IPs were performed on total protein extracts from cortical samples conditions. The three pooled supernatants were centrifuged at 700 g at different ages. All IPs were performed at a protein concentration for 10 min, this sample corresponds with the S1 fraction. This was of 8 mg/ml. All steps were performed at 4°C in an orbital agitator centrifuged 30 min at 21,000 g. The resulting soluble fraction was (Stuart). The following amounts of protein were used for each IP: considered the cytosolic fraction, whereas the pellet obtained con- 9 mg for PND0/1 (n of mice = 24), 16 mg for PND11 (n of mice = 15), tained all membranes. This was resuspended with sucrose 0.32 M 8 mg for PND21 (n of mice = 3), and 7 mg for PND56 (n of mice = 3) and 50 mM Tris pH 7.4. A sucrose gradient was prepared with 1 ml of samples. IPs were performed as described by the kit manufacturer (top to bottom): sample, 0.85 M sucrose and Tris 50 mM pH 7.4; 1 M (26,147, Pierce® Direct IP Kit; Thermo Fisher Scientific). A sepharose sucrose and Tris 50 mM pH 7.4, and 1.2 M sucrose and Tris 50 mM resin (Sigma P3391-250MG) was washed four times with condition- pH 7.4. Then, this gradient was centrifuged with a SW60 Ti rotor ing buffer (50 mM Tris pH 7.4). Next a sample pre-clearing step was (Beckman Coulter) at 82,500 g for 2 hr. The interphase between su- performed by mixing it with washed resin for 2 hr at 4°C. Pre-cleared crose 1 and 1.2 M was recovered to obtain the synaptosome fraction. sample was mixed with an anti-SynGAP antibody recognizing an The rest of the gradient was centrifuged at 50,000 g 30 min in a fixed epitope common to all its isoforms (5540S; Cell Signaling Technology rotor and the resulting pellet, containing the non-synaptic membrane [RRID:AB_10695900]) at a 1:15 (v:v) ratio ON. Each 200 μL of pre- (NSM) fraction, was resuspended with 1% SDS and 50 mM Tris pH cleared sample were incubated with 7.5 μL of A sepharose resin dur- 7,4. The synaptosome fraction was diluted to reach a final concentra- ing 3 hr. A 100 g centrifugation step in a column was performed to tion of 10% sucrose with Tris 50 mM pH 7.4 and centrifuged in an Epp recover the resin. Resin was washed three times with dialysis buffer 5117R centrifuge (Eppendorf) at 21,000 g during 30 min using 1.5 ml and once with conditioning buffer. Bound protein was eluted with 15 tubes. The resulting pellet was resuspended in Tris 50 mM pH 7.4, 1% μL of the acidic elution buffer from the kit during 10 min. Triton X-100 (93443-100Ml; Sigma-Aldrich) and maintained in ice for 10 min. Finally, samples were centrifuged at 21,000 g during 30 min. As a result, Triton X-100 soluble fraction, referred as synaptic non- 2.8 | Protein electrophoresis PSD (SNP), and the Triton X-100 insoluble fraction enriched in post- synaptic densities (PSDs), were obtained. Fraction protein yield was Protein samples for electrophoresis were prepared with 1× Laemmli defined as the ratio of total protein amount (µg) by tissue weight (mg). loading sample buffer (50 mM Tris-HCl, pH 6.8; 2% SDS; 1% Protein concentration was determined using a micro-BCA pro- β-mercaptoethanol [M6250-100 ml], and 0.04% bromophenol blue tein assay kit (10249133; Thermo-Fisher Scientific). Prior to IB [B5525-5g, all from Sigma-Aldrich]) and 10% glycerol (Sigma-Aldrich) 622 | GOU et al. and heated at 95°C for 5 min. TGX Stain-Free™ gels (161-0181 & on the antibody. Images were acquired with an Odissey Scanner 161-0185, SF gels; Bio-Rad) were prepared and activated accord- (LI-COR Biosciences) and protein bands were analyzed with Image ing to manufacturer's instructions. All blue or kaleidoscope preci- Studio Lite ver. 3.1 software (LI-COR Biosciences). Membranes sion plus protein standards (Bio-Rad) were used as well as a vertical transferred from TGX Stain-Free™ gels were imaged and quantified MiniProtean system kit (Bio-rad) and 1× running buffer (0.025 M for posterior normalization steps prior to blocking with a ChemiDoc TRIS pH 8.4; 0.187 M glycine [G8898-1KG; Sigma-Aldrich] and 0.1% XRS+ (Bio-Rad) using the Image Lab software (Bio-Rad). SDS). Electrophoretic conditions were 25 mAmp per each 0.75 mm wide gel or 50 mAmp per each 1.5 mm wide gel. Proteins resolved in SDS–PAGE gels were stained ON at 22°C 2.10 | Normalization of immunoblot data with Coomassie solution (B8522-1EA; Sigma-Aldrich). Gels were washed with 2.5% acetic acid (45740-1L-F; Sigma-Aldrich) and 20% In spatio-temporal protein expression studies, band intensity units methanol during 10 min in a rocking platform shaker (Stuart) and (IUs) were first corrected for immunoblot technical variability using later with subsequent washes of 20% methanol, until protein bands the value of total protein transferred to PVDF membranes ob- were clearly visible. Gel images were acquired with ChemiDoc XRS+ tained from the TGX Stain-FreeTM quantification (IU/protein inten- (Bio-Rad) and quantified with Image Studio Lite ver. 3.1 (LI-COR sity). Corrected IUs were then normalized using the average IU of Biosciences). all bands in a blot. This normalization removed the technical vari- ability between blots allowing to accumulate data from immunoblot replicates. 2.9 | Immunoblot In subcellular localization studies, we first corrected band IU (e.g. tSynGAP in PSD) per amount of total protein used for immunoblot- Protein transference was conducted using the MiniProtean kit ting (e.g. tSynGAP IU in PSD/µg PSD protein). These values were (Bio-Rad), and 1× chilled transference buffer (20% methanol next multiplied by protein yield (with units: µg protein/mg tissue) [A3493.5000; Panreac]; 39 mM Glycine; 48 mM TRIS; 0.04% SDS). of their corresponding subcellular fraction, which retrieved a value Proteins were transferred into methanol pre-activated polyvi- of specific protein abundance per fraction (e.g. tSynGAP IU in PSD/ nylidene fluoride (PVDF) membranes (IPFL00010, Immobilon-P; mg tissue). Finally, these values were normalized by the abundance Merck-Millipore). After transference, PVDF membranes were in the starting homogenate (S1 fraction; e.g. tSynGAP IU in PSD/ blocked with 5 ml Odissey blocking solution (927-50000; LI-COR) tSynGAP IU in S1). This normalization step allowed accumulating prepared with 1× tris-buffered saline (TBS) [50 mM Tris·HCl pH7.4; data from immunoblot replicas and compare subcellular distribution 1,5 M NaCl [443824T]) and 0.1% sodic azide [S2002-100G, all between antibodies. from Sigma-Aldrich]) and incubated in a roller mixer (Stuart) with primary antibody solution ON at 4°C or 2 hr at 22°C. Commercial primary antibodies were: total SynGAP (tSynGAP) (NBP2-27541; 2.11 | Sample preparation and mass spectrometry- Novus Biologicals, [RRID:AB_2810282] and Thermo PA1-046, based proteomics [RRID:AB_2287112], only in Figure S2) at 1:2,500 and 1:2,000 di- lution, SynGAP-α1 (06-900; EMD Millipore, [RRID:AB 1163503]) Proteins were separated by SDS-PAGE and were stained with at 1:1,000, SynGAP-α2 (04-1071 [EPR2883Y]; Merck-Millipore, Coomassie (Bio-Rad). Bands between ~120–200 kDa were excised [RRID:AB_1977520]) used at 1:2,000 dilution, PSD-95 (3,450; Cell from acrylamide gels in a transilluminator (22V; Cultex) using scalpel Signaling, [RRID:AB_2292883]) at 1:1,000, Gephyrin (ab32206; blades. Excised gel bands were subjected to an in-gel digestion pro- Abcam, [RRID:AB_2112628]) at 1:500, CaMK2α (05-532; Merck- tocol being first reduced with 10 mM dithiothreitol (Sigma-Aldrich) Millipore, [AB_309787]) at 1:1,000, GAD67 (MAB5406 [1G10.2]; and alkylated with 55 mM iodoacetamide (8.04744.0025; Sigma- Merck-Millipore, [RRID:AB_2278725]) at 1:500, Synaptophysin Aldrich), and later digested with trypsin (V5111; Promega Biotech (Ab8049; Abcam [SY38], [RRID:AB_2198854]) at 1:1,000 and Ibérica). Tryptic peptides were eluted from acrylamide and around GAPDH (ab9484; Abcam, [RRID:AB_307274]) at 1:500 dilution. 80% of each trypsin-digested sample was injected in a linear trap Membranes were washed four times with 1× T-TBS for 5 min before quadrupole (LTQ) Orbitrap VelosPro with a short chromatographic incubation for 1 hr at 22°C protected from light with 5 ml of the fol- method (40 min gradient) in a 25 cm 1.9 µm column. To avoid carry lowing secondary antibodies. Secondary antibodies were prepared over, BSA runs were added between samples. BSA controls were in- with T-TBS (50 mM Tris·HCl pH7.4, 1.5 M NaCl, 0.1% Tween20, cluded both in the digestion and LC-MS/MS analyses for quality con- all from Sigma-Aldrich) at 1:7,500 dilution: anti-rabbit (926- trol. This experiment was done twice. The data were searched using 68073, IRDye 680CW, [AB_10954442]), anti-mouse (926-32212, an internal version of the search algorithm Mascot (Matrix Science) IRDye 800CW [RRID:AB_621847] or 925-68072, IRDye 680RD, against a SynGAP (May 2014) homemade database. The Mascot da- [RRID:AB_2814912]) and anti-goat (926-32214, IRDye 800CW, tabase server search was done with Protein Discoverer ver. 1.4.1.14 [RRID:AB_621846]). Membranes were re-blotted without prior (DBVer.:79) using the following search parameters: mass precision of stripping by an ON incubation at 4°C or 2 hr at 22°C, depending 2 ppm; precursor mass range of 250 Da to 5,000 Da; Trypsin with a GOU et al. | 623 maximum of three miss-cleavages; the peptide cut-off score was set for outliers was done, no data points were excluded and no blinding at 10 and peptide without protein cut-off was set at 5. Peptides were was performed. No statistical method was used to determine sam- filtered based on IonScore >20. The precursor mass tolerance (MS) ple size, which was determined based on the previous experience of was set at 7 ppm and fragment mass tolerance (MS/MS) was set at the group with the goal to minimize the number of animals required. 0.5 Da with two variable modifications: oxidation (M) and acetyla- Also, no randomization was performed to allocate subjects in this tion (protein N-term), and one fixed modification (C): carbamidome- study and this study was not pre-registered. thyl. False discovery rates determined by reverse database searches and empirical analyses of the distributions of mass deviation and Mascot Ion Scores were used to establish score and mass accuracy 3 | R E S U LT S filters. Application of these filters to this dataset was below 1% false discovery rates as assessed by reverse database searching. 3.1 | Total SynGAP protein expression is different between brain regions and changes throughout postnatal development 2.12 | Primary neuronal and cell culture, transfection, fluorescent immunostaining, and imaging Using an antibody that recognizes a sequence common to all SynGAP isoforms we have analyzed by immunoblot the abundance of all of Hippocampal neurons from PND0 Syngap1flox/flox (Clement them together, what we have called total SynGAP (tSynGAP). We have et al., 2012, 2013) mice were plated on poly-D-lysine (P6407; investigated tSynGAP expression in five mouse brain regions (cortex, Sigma-Aldrich) coated coverslips and infected with AAV.CaMK2α. hippocampus, striatum, olfactory bulb, and cerebellum) at 4, 11, 21, Cre (RRID:Addgene_105558) to obtain Syngap1-/-cells. Cultures were and 56 postnatal days (PND) of life (Figure 2). Depending on the tis- maintained in Neurobasal-A media (12348–017) containing 10 µg/ml sue, tSynGAP presents three patterns of developmental expression Gentamycin (15750060), 2 mM Glutamax (35050061), and 2% B27 (Figure 2a). In cortex and hippocampus, tSynGAP increases sharply, (17504044, all from GIBCO). At days in vitro (DIV) 4, cells were treated reaching its maximum at PND21, and remaining at this level until with 1 µM Ara-C to prevent excessive glial proliferation. At DIV18, PND56. Between PND4 and PND21, tSynGAP levels increase over six cells were transfected with plasmids encoding enhanced green fluo- times in both tissues. Striatum presents a different pattern: tSynGAP rescent protein-tagged full-length SynGAP C-terminal isoforms using expression is maintained constant between PND11 and PND21, and NeuroMag (NM50500, OZBiosciences) in accordance with manufac- its maximum level is not reached until PND56. Yet tSynGAP levels also turer's instructions. Following an ON incubation, neurons were fixed increase notably, also around six times, between PND4 and 56. Finally, for 5 min at 22°C in PBS containing 4% paraformaldehyde (PFA)/4% both the olfactory bulb (OB) and the cerebellum present a very mod- sucrose and thoroughly washed with PBS. Neurons were permeabi- est, albeit significant, increase in tSynGAP levels. Between PNDs 4 and lized with 0.2% Triton-X for 10 min and blocked with 10% normal goat 56, tSynGAP increases 1.7 times in OB and 1.2 in cerebellum. serum in PBS for 1 hr. Samples were then incubated with Alexa Fluor We have also investigated how tSynGAP levels compare between 488 conjugated anti-enhanced green fluorescent protein antibody (A- tissues at each of these four developmental stages (Figure 2b). Early in 21311; Thermo Fisher Scientific, [RRID:AB_2214]) in PBS with 5% goat postnatal development tSynGAP levels are very similar in cortex, hip- serum at 4°C ON. Coverslips were washed multiple times with PBS pocampus, striatum, and OB, while cerebellum already presents the and mounted onto glass slides using Prolong Glass mounting medium. lowest levels. At PND4 there is approximately 2.5 times more tSyn- Images were obtained using a FV1000 Olympus laser scanning confo- GAP in forebrain regions than in cerebellum. This difference becomes cal microscope. For anti-SynGAP-beta antibody validation, HEK293T larger with age, reaching a maximum difference of 30 times when Cells (Kind gift of Joseph Kissil) were cultured in DMEM media con- comparing hippocampal and cerebellar expression at PND21/56 or taining 10% fetal bovine serum and penicillin/streptomycin. Pools of cortical and cerebellar expression at PND21. As mice develop, the these cells were transfected with GFP-tagged SynGAP cDNAs con- levels of tSynGAP in OB also lag behind those of the other forebrain taining one of the four known C-term spliced sequences. areas (Figure 2b), being the maximum difference at PND21, when cortex and hippocampus express seven times more tSynGAP than OB. At PND11, cortex and striatum display similar levels of tSynGAP, 2.13 | Data statistical analyses while hippocampus presents a slight, but significantly higher abun- dance, being the tissue with the highest tSynGAP levels at this age. Statistical tests used are indicated in figure legends, together with At PND21, cortical and hippocampal tSynGAP have similar levels, the exact number of biological and technical replicates. GraphPAD presenting almost twice as much tSynGAP than striatum. This is in Prism ver. 6.0 (GraphPad) was used to conduct statistical analyses. agreement with the sustained tSynGAP levels previously observed in When required data were assessed for normal distribution by de- striatum between PND11 and 21 (Figure 2a). Finally, at PND56, the scriptive statistic measures (mean and median) and applying the abundance profile of tSynGAP at cortex, hippocampus, and striatum is Shapiro-Wilk and Kolmogorov–Smirnov tests. All statistical analyses very similar to that found at PND11. Cortex and striatum have similar were conducted with a significance level of α = 0.05 (p ≤ .05). No test abundance, while hippocampus presents significantly more tSynGAP. 624 | GOU et al. (a) (a1) (a2) (b) (b1) (b2) F I G U R E 2 Abundance of total SynGAP (tSynGAP) in five different brain regions and four postnatal stages. (a) Developmental changes in tSynGAP abundance in five different brain regions (cortex, hippocampus, striatum, olfactory bulb, and cerebellum). Ages investigated were postnatal day (PND) 4, 11, 21, and 56. (a1) Representative immunoblots showing tSynGAP abundance in each of the five tissues. (a2) Box and whiskers plots depict the mean of normalized protein abundance data derived from immunoblot intensity (N: cortex 14–20, hippocampus 12–16, striatum 6–7, olfactory bulb 11–16, and cerebellum 23–24). N indicates total number of technical replicates from a pool of a given brain area coming from of a minimum of two mice. The standard error of the mean (SEM) is also shown. Mean differences were analyzed by one-way ANOVA followed by Tukey's post-hoc test, ***p < .001, **p < .01, and *p < .05. (b) Brain region changes in tSynGAP abundance in four life stages, including three postnatal development stages (PND4, 11, and 21) and adulthood (PND56). (b1) Representative immunoblots showing tSynGAP abundance in each life stage. (b2) box and whiskers plots depict the mean of normalized protein abundance data derived from immunoblot intensities (N: cortex 6–15, hippocampus 6–15, striatum 6–15, olfactory bulb 6–15, and cerebellum 6–15). N indicates total number of technical replicates from a pool of a given brain area coming from of a minimum of two mice. The standard error of the mean (SEM) is also shown. Mean differences were analyzed by one-way ANOVA followed by Tukey's post-hoc test, ***p < .001, **p < .01 and *p < .05 MS-based proteomics. However, we could not identify unique pep- 3.2 | In silico identification and experimental tides for any of these variants. Instead, we identified a unique pep- validation of novel Syngap1 splice variants tide corresponding to the first residues of the D N-terminus (Table S2), which had only been reported at the RNA level (Li et al., 2001). ENSEMBL, NCBI-Gene, and UniProt (as of 02 May 2019), together Importantly, this peptide presented an acetylated initial methionine, with the previous literature (Chen et al., 1998; Kim et al., 1998), re- which is a common post-translational modification of the N-terminus port a total of 15, 9, and 7 Syngap1 transcripts in mouse, rat, and (Varland, Osberg, & Arnesen, 2015). The rat D variant was originally human respectively (Table S1). Remarkably, there is still little overlap submitted (Li et al., 2001) to NCBI as an artifactual sequence resulting between these databases. For instance, in mouse, only two proteins from the fusion of transcripts from two different genes, as reported can be directly related between ENSEMBL and NCBI. Interestingly, later (McMahon et al., 2012), and thus considered nonexistent. Yet, the NCBI Gene database identifies unpublished variants in mice our proteomics experiments identify the acetylated N-terminus of (four N-terminal and one C-terminal). We refer to these unreported the D variant in cortical samples from all ages investigated. The re- N-terminals as A3, A4, E, and F, whereas the C-terminal one as α3 current identification of this acetylated N-terminal peptide provides (Table S1 and Figure S1). The first two N-term variants are shorter strong evidence for the expression of this variant in mouse cortex. versions of A1/2, E presents a unique N-terminus, and F starts at resi- due 430 inside the core of SynGAP. If the F variant is expressed at the protein level, it would lack the PH, C2, and GAP domains, which could 3.3 | SynGAP isoforms present different be functionally relevant to neuronal biology. In order to investigate developmental expression patterns if any of these predicted variants is expressed at the protein level, we immunoprecipitated tSynGAP from mouse cortex at four postna- SynGAP isoforms present four different C-terminal variants tal stages (PND0/1, 11, 21, and 56) and performed high-throughput that have been identified at the protein level, which are named GOU et al. | 625 alpha1 (α1), alpha2 (α2), beta (β), and gamma (γ). Commercial an- particularly for β isoforms, which do not present any difference tibodies are available for two (α1 and α2) and we raised a new in their expression along the postnatal period investigated. In OB, antibody that recognizes the β sequence. We confirmed that both α1 and α2 isoforms present a moderate increase in their ex- these three antibodies are selective by showing that they do pression level, showing a maximum at PND56. Finally, in cerebel- not cross-react (Figure S2). In our experimental conditions, as lum, α1 levels are constant from PND11 onwards, while α2 present in previous works (Kim et al., 1998; Li et al., 2001; McMahon a biphasic increase in expression, with a period of latency between et al., 2012; Yang, Tao-Cheng, Bayer, Reese, & Dosemeci, 2013), PND11 and PND21. these C-terminal specific antibodies distinguish two major Overall, we observed a much better correlation in the develop- bands (Figures 3–4). These two bands correspond with at least mental expression of α1 and α2 isoforms, as this is statistically signif- two different isoforms, which will necessarily present different icant in all brain areas but OB and cerebellum, than between α1 and N-terminus. Notably, we have not found statistically significant β isoforms, which were never found significantly correlated (Table 1). abundance differences between the top and bottom bands in β isoforms present a better expression correlation with α2 isoforms, any of the experiments performed. This indicates that isoforms reaching statistical significance in hippocampus, what suggests that with the same C-terminus display equivalent expression pat- α2 isoforms would present an intermediate expression pattern be- terns along development in the five brain regions investigated. tween those of SynGAP α1 and β-containing isoforms. For this reason, we considered both bands together for subse- quent analysis. In cortex (Figure 3a), α1-containing SynGAP isoforms remain 3.4 | Developmental expression pattern of at very low levels until PND11 as compared with their maximum SynGAP-α1/α2 isoforms follows the formation of expression. Between PND11 and PND21, α1 expression increases excitatory and inhibitory synapses fivefold, to reach over 60% of their adult (PND56) levels. In con- trast, isoforms containing α2 and β C-term variants already present We also sought to explore if the developmental expression pro- around 50% of their maximum abundance at PND11. Interestingly, file of SynGAP isoforms could be linked to the temporal acquisition α1-, α2-, and β-containing isoforms vary in their pattern of cortical of excitatory and inhibitory synapses and/or neurons. To achieve expression. Namely, α1 isoforms do not reach their maximum until this purpose we compared the expression of SynGAP isoforms to PND56, while α2 and β isoforms peak at PND21. Furthermore, that of PSD-95, marker of excitatory synapses; Gephyrin, marker while α2 isoforms maintain their maximum expression level be- of inhibitory synapses; CaMK2α, marker of excitatory neurons; tween PND21 and 56, those of β isoforms decrease significantly and GAD-67, marker of inhibitory neurons (Table 1 and Figure S3). after PND21, presenting 70% of their maximum expression at Interestingly, we found better correlations when comparing the PND56. expression patterns of SynGAP isoforms with both synaptic mark- The hippocampal expression pattern (Figure 3b) of the iso- ers than when comparing them with neuronal markers (Table 1). forms investigated is quite similar to that of cortex. α1 isoforms Indicating that, overall, the developmental expression of SynGAP reach their maximum expression at PND56, while α2 does it at isoforms parallels the formation of excitatory and inhibitory syn- PND21 and β isoforms peak at PND21. Here, we also observed apses rather than the differentiation of excitatory and inhibitory a decrease in β isoforms between PND21 and 56, although it did neurons. Indeed, only a few significant correlations where found not reach statistical significance. Also, the abundance of α1 iso- between the expression levels of SynGAP isoforms and CaMK2α forms does increase between PND4 and 11, as opposed to what or GAD-67 (Table 1). we observed in cortex. Still, α1 isoforms expression fold change Nevertheless, we again observed a marked difference be- between PND11 and PND56 is higher (4-fold) than that observed tween the expression pattern of α1/α2 and β isoforms. While the between PND4 and 11 (2-fold). former correlate very well with the expression of PSD-95 and In striatum (Figure 3c), α1 and α2 isoforms present a biphasic Gephyrin in almost all tissues investigated, β isoforms present a expression pattern that we have not observed in any other tissue. more tissue-restricted correlation with synaptic markers (Table 1). Expression increases from PND4 to PND11 and then again between Actually, β isoforms only present significant correlation with the PND21 and PN56, but during the second and third weeks (PND11- expression of PSD-95 in hippocampus and striatum and with 21) the expression of these isoforms remains constant. Striatal lev- Gephyrin in hippocampus. Importantly, we observed very high els of β isoforms suggest a similar pattern, as PND56 expression levels of correlation between the developmental expression pat- is higher than PND11 and there is no difference between PND11 tern of PSD-95 and Gephyrin in cortex, hippocampus, and striatum and PND21. Yet, the difference between PND21 and PND56 does (Table 1), indicating that the incorporation of excitatory and inhib- not reach statistical significance. Thus, our data could also be inter- itory synapses occurs more or less simultaneously in these tissues preted as that β isoforms reach their maximum level at PND21 and (Oh, Lutzu, Castillo, & Kwon, 2016). Thus, it is not surprising that then this is maintained. the expression of SynGAP isoforms correlates with PSD-95 but In the OB and cerebellum (Figure 3d,e) we observed less de- also with Gephyrin. Despite PSD-95 proteins may only interact di- velopmental variation in the abundance of SynGAP isoforms, rectly with α1 isoforms (Kim et al., 1998), we also observed good 626 | GOU et al. (a) (a1) (a2) (b) (b1) (b2) (c) (c1) (c2) (d) (d1) (d2) (e) (e1) (e2) F I G U R E 3 Compared protein abundance of SynGAP isoforms along postnatal development in five different brain regions. (a–e) data from cortex, hippocampus, striatum, olfactory bulb, and cerebellum respectively. (a1–e1) representative immunoblots for SynGAP isoforms containing each of the three C-terminal variants (α1, α2 and β). (a2–e2) dot plots for each brain region depicting mean normalized protein abundance data from each isoform derived from immunoblot intensities (N: cortex 4–19, hippocampus 6–12, striatum 3–9, olfactory bulb 6–14, and cerebellum 8–15). N indicates total number of technical replicates from a pool of a given brain area coming from a minimum of two mice. The standard error of the mean (SEM) is also shown. Mean differences were analyzed by one-way ANOVA followed by Tukey's post- hoc test, ***p < .001, **p < .01, and *p < .05 expression correlation with PSD-95 and α2 isoforms (Table 1). statistical significance in almost all brain areas investigated, while Actually, the co-expression of PSD-95 with α1 and α2 isoforms the correlation of PSD-95 and α1 isoforms was only significant in presented a higher correlation with α2 isoforms, as these reached cortex and OB. GOU et al. | 627 (a) (a1) (a2) (b) (b1) (b2) (c) (c1) (c2) (d) (d1) (d2) F I G U R E 4 Compared protein abundance of SynGAP isoforms in five different brain areas along postnatal development and in adulthood (a–d) data from postnatal day (PND) 4, 11, 21, and 56, respectively. (a1–d1), representative immunoblots for SynGAP isoforms containing each of the three C-terminal variants (α1, α2, and β). (a2–d2), Dot plots for each life stage depicting normalized protein abundance data from each isoform derived from immunoblot intensities (N: cortex 6, hippocampus 6, striatum 6, olfactory bulb 6, and cerebellum 6). N indicates total number of technical replicates from a pool of a given brain area coming from of a minimum of two mice. The standard error of the mean (SEM) is also shown. Mean differences were analyzed by one-way ANOVA followed by Tukey's post-hoc test, ***p < .001, **p < .01, and *p < .05. 3.5 | SynGAP isoforms present different regional in cerebellum. Actually, α1 isoforms present similarly low expres- expression patterns sion levels in cerebellum and OB in all ages investigated. At PND11, α2 and β-containing isoforms still present higher levels in OB when We next compared the abundance of SynGAP isoforms between compared with cerebellum, yet this difference disappears at PND21 brain regions in three postnatal developmental time points, PND4, and 56, where both tissues express equally low levels of all isoforms. 11, 21, and in young adults (PND56). At PND4 (Figure 4a), the ex- After PND4, hippocampus was the region where SynGAP iso- pression of all isoforms investigated presented equivalent levels in forms presented the highest levels (Figure 4b–d). This was already cortex, hippocampus, and striatum, while cerebellar expression was noticeable at PND11, although not all comparisons reached statisti- always the lowest. In the OB, levels of α2 and β isoforms were un- cal significance, and very clear at PND56. Nevertheless, at PND21 distinguishable from those in the other forebrain areas, but α1 abun- cortical expression of α1 and α2 isoforms becomes more promi- dance was significantly reduced, presenting the same levels found nent. This phenomenon is particularly noticeable for the cortical 628 | GOU et al. TA B L E 1 Postnatal development expression correlation between SynGAP isoforms and synaptic and neuronal markers Pearson R correlation coefficient Cortex Hippocampus Striatum Olfactory bulb Cerebellum Expression correlation between SynGAP isoforms α1 versus α2 0.95* (0.048) 0.96* (0.040) 0.99* (0.007) 0.865 0.89 α1 versus β 0.75 0.84 0.88 0.40 −0.68 α2 versus β 0.90 0.96* (0.041) 0.93 (0.074) 0.36 −0.31 Expression correlation between PSD-95 and Gephyrin PSD-95 versus Gephyrin 0.98* (0.019) 0.99* (0.003) 0.99* (0.014) 0.12 0.90 Expression correlation between PSD-95 and SynGAP isoforms PSD-95 versus α1 0.98* (0.025) 0.95 (0.054) 0.94 (0.063) 0.98* (0.023) 0.89 PSD-95 versusα2 0.99* (0.005) 0.99* (0.004) 0.97* (0.030) 0.94 (0.056) 0.99* (4.7e5) PSD-95 versus β 0.87 0.97* (0.029) 0.96* (0.043) 0.42 −0.31 Expression correlation between Gephyrin and SynGAP isoforms Gephyrin versus α1 0.93 (0.067) 0.97* (0.033) 0.93 (0.067) 0.18 0.61 Gephyrin versus α2 0.98* (0.023) 0.99* (0.003) 0.96* (0.042) 0.06 0.90 Gephyrin versus β 0.93 (0.067) 0.95* (0.049) 0.90 −0.81 0.13 Expression correlation between CaMK2α and SynGAP isoforms CaMK2α versus α1 0.78 0.93 (0.066) 0.49 0.76 −0.95* (0.049) CaMK2α versus α2 0.89 0.99* (0.006) 0.59 0.97* (0.026) −0.88 CaMK2α versus β 0.97* (0.03) 0.98* (0.021) 0.77 0.17 0.49 Expression correlation between GAD-67 and SynGAP isoforms GAD-67 versus α1 0.93 (0.075) N/A 0.89 0.93 (0.067) 0.87 GAD-67 versus α2 0.99* (0.006) N/A 0.94 (0.064) 0.97* (0.03) 0.95* (0.048) GAD-67 versus β 0.90 N/A 0.97* (0.032) 0.74 −0.23 Abbreviations: N/A, not analyzed; PSD, postsynaptic density. *Significant correlation, p-value between parenthesis (p ≤ 0.075 also included). expression of α1 isoforms, which present a cortex to hippocampus 3.7 | Differential subcellular distribution of expression ratio close to 2 at PND21, and around 0.7 at PND11 and tSynGAP along cortical postnatal development and in PND56. Instead the cortical increase of α2 isoforms at PND21 only adult hippocampus brings them to the same levels found in hippocampus. This sharp increase in α1 abundance specific to cortical samples from PND21 Next, we investigated to what extent tSynGAP presented a differ- animals exemplifies the specific regulation at which the expression ential subcellular distribution throughout postnatal development. of SynGAP isoforms can be. This study was performed with mouse cortical samples. Five sub- cellular fractions were prepared: homogenate without nuclei (S1), cytosol, non-synaptic membranes (NSM), SNP and PSD (Figure S5). 3.6 | All SynGAP isoforms investigated are This fractionation protocol was applied to three postnatal devel- expressed in the young and old human cortex opment stages PND7, 14 and 21 and young adult (PND56). Protein yield (i.e., protein amount/ tissue weight) was calculated for all Immunoblots of total cortical extracts from two human individuals subcellular fractions generated (Figure S5a) and used, together with 19 and 67 years of age were performed to elucidate if SynGAP with immunoblot intensity data (Figure S5b), to obtain a measure isoforms are expressed in this human tissue. We found expression of protein abundance in each fraction. Interestingly, we observed of tSynGAP and all C-terminal variants in both samples (Figure S4). a decrease in the SNP yield between PND14 and 21, simultane- In line with the lower neuronal density found in the human cortex, ous to an increase in the PSD yield, likely reflecting the increased as compared with the mouse one (Defelipe, Alonso-Nanclares, & maturation of synapses during this week (Figure S5c). Subcellular Arellano, 2002), human immunoblots present a clear reduction per protein markers (GAPDH, Synaptophysin, Gephyrin and PSD-95) unit of protein of tSynGAP and all its isoforms. Interestingly, human were used to validate the fractionation protocol (Figure S5d). samples presented the same two bands found in mice when investi- The abundance of tSynGAP in different subcellular fractions gated SynGAP isoforms by the same technique. was analyzed by immunoblot together with that of PSD-95 (Figures GOU et al. | 629 (a) (b) (c) (d) (e) (f) (g) (h) (i) F I G U R E 5 Subcellular distribution of total SynGAP (tSynGAP) and PSD-95 in cortex from four life stages and adult hippocampus. (a–d) box and whiskers plots representing the mean of normalized immunoblot intensity data (see Figure S5b) from different subcellular fractions. White boxes present tSynGAP data and gray boxes PSD-95 data. (N: postnatal day [PND]7 6–18, PND14 6–21, PND21 9–18, PND56 6–15). N indicates total number of technical replicates performed using cortical samples coming from six different mice. The standard error of the mean (SEM) is also shown. Mean differences were analyzed by two-way ANOVA followed by Fisher's LSD post-hoc test, ***p < .001, **p < .01, and *p < .05. Subcellular fractions correspond with: cytosol; NSM, non-synaptic membranes; SNP, synaptic non-PSD and PSD, postsynaptic density. Life stages investigated are: PND7 (a), PND14 (b), PND 21 (c), and PND56 (d). (e) Immunoblots presenting subcellular distribution of total SynGAP (tSynGAP), its isoforms, and PSD-95 in adult (PND56) hippocampus. Subcellular fractions investigated: Homogenate without the nuclear fraction (S1); cytosol; SNP, synaptic non-PSD and PSD, postsynaptic density. (f) Dot plot with the mean of normalized immunoblot intensity data of tSynGAP (white dots) and PSD-95 (grey dots) in the subcellular fractions obtained from adult hippocampus (N: PND63 6–9 technical replicates using six biological replicates resulting from a pool of two mouse hippocampus per replica). (g) Dot plot with mean of normalized immunoblot intensity data of SynGAP isoforms containing α1, α2, and β C-terminal variants in the subcellular fractions obtained from adult hippocampus. N: PND63 6–8 technical replicates using six biological replicates resulting from a pool of the hippocampus from two mice per replica. The standard error of the mean (SEM) is also shown. Mean differences were analyzed by one-way ANOVA followed by Tukey's post-hoc test ***p < .001, **p < .01, and *p < .05. (h) Mouse Syngap1 KO hippocampal neurons (DIV17) transfected with constructs expressing enhanced green fluorescent protein (EGFP) tagged SynGAP C-terminal isoforms (α1, α2, and β) along with mRuby2 (cell-fill). (i) Synaptic enrichment is calculated as the ratio of EGFP signal present in dendritic spines versus dendritic shaft. n = 30 spines form 9–11 neurons for each isoform analysis. Error bars indicate SEM. Mean differences were assessed by one-way ANOVA followed by Tukey's multiple comparison test, ***p < .0001 630 | GOU et al. (a) F I G U R E 6 Subcellular distribution of SynGAP isoforms along three postnatal developmental stages and adulthood. (a–d) Dot plots representing the mean of normalized immunoblot intensity data (see Figure S5b) from different subcellular fractions for SynGAP isoforms presenting three different C-terminal variants and PSD-95. (N: PND7 6, PND14 6–9, PND21 6–9, PND56 6–11). N indicates total number of technical replicates performed using six different mouse cortical samples. The standard error of the mean (SEM) is also shown. Mean differences were analyzed by one-way ANOVA followed by Tukey's post-hoc test, ***p < .001, **p < .01, and *p < .05. Subcellular fractions correspond with: cytosol; NSM, non-synaptic membranes; PND, postnatal day; SNP, synaptic non- PSD and PSD, postsynaptic density. Life stages investigated are: (b) PND7 (a), PND14 (b), PND21 (c), and PND56 (d) At PND14 and 21, the abundance of tSynGAP at the PSD was sig- nificantly higher than that found in the SNP fraction, yet tSynGAP was still more abundant in the cytosol. At PND21, the PSD:cytosol ratio of tSynGAP is still 0.6. Even at PND56, the remaining frac- tion of tSynGAP at the cytosol is quite large, as this ratio is 1.7. This developmentally regulated subcellular expression pattern of tSynGAP is in stark contrast with that displayed by PSD-95, as this scaffolding protein is predominantly expressed at the PSD in all (c) life stages investigated, presenting almost negligible levels in the cytosolic fraction. We also investigated adult (PND56) subcellular localization of tSynGAP and its isoforms in the hippocampus (Figure 5e–g). Here, we could not find a significantly different expression level of tSyn- GAP between cytosol and PSD, while PSD-95 presented a very restricted PSD localization. When considering the adult hippo- campal expression of SynGAP isoforms we observed that α1 and α2 isoforms were enriched at the PSD, while β isoforms were very much enriched in the cytosolic fraction (Figure 5g). This is clearly (d) indicated by the PSD:cytosol ratios found for these proteins: 3.2 for α1, 2.5 for α2, and 0.3 for β isoforms. We next aimed to validate the different subcellular localization of SynGAP isoforms observed in adult hippocampus using an alternative experimental approach. In this case we transfected primary cultures of hippocampal Syngap1-/- neurons with GFP-tagged forms of SynGAP presenting one of the three C-terminal variants, finally, we quantified GFP signal in den- dritic spines and shafts to obtain a ratio of spine:shaft GFP signal (Figure 5h,i). Notably, we did found a significant difference in the localization of α1 and the other two isoforms, being the later more prominently expressed at dendritic spines, in accordance with the immunoblot data collected from both cortical and hippocampal 5–6). Of the four subcellular fractions investigated, tSynGAP was samples. essentially found in two (Figure 5a–f), cytosol and PSD. For this reason we generated a PSD:cytosol expression ratio to analyze this data. The absence or minute amount of tSynGAP in NSM and 3.8 | Differential subcellular distribution of SynGAP SNP fractions indicated that it is not associated to extra-synaptic isoforms along cortical postnatal development membranes and, furthermore, that within the synaptosome it is essentially found at the PSD. Interestingly, at PND7 tSynGAP was We also investigated the subcellular distribution of SynGAP isoforms largely found in the cytosol (PSD:cytosol ratio 0.1) and it is not until along cortical postnatal development (Figure 6 & Figure S5b). As ex- PND56 that we observed significantly more tSynGAP in the PSD pected, in this study we also localize all isoforms at two main subcel- than in the cytosolic fraction from mouse cortices (Figure 5a–d). lular locations, the cytosol and the PSD. We found very low levels of GOU et al. | 631 α1 isoforms early in postnatal development (PND7 & 14), as a con- we compared isoform expression levels in different mouse brain sequence their subcellular location could not be confidently estab- regions along postnatal development and in adult, as well as their lished at these ages (Figure 6a,b). At later ages, α1 isoforms presented cortical and hippocampal subcellular distribution. We have also a very restricted localization at the PSD (Figure 6c,d, PSD:cytosol shown that isoforms presenting these three C-termini are also ex- ratio at PND21 and PND56 >4). Alpha2- and β-containing isoforms pressed in the cortex of two human individuals aged 19 and 67. were expressed at higher levels early in development and could thus As previously reported (Kim et al., 1998; Li et al., 2001; McMahon be localized at specific subcellular locations from PND7 to 56. These et al., 2012; Yang et al., 2013), the three antibodies identify two isoforms presented an almost exclusive cytosolic location during major bands in all conditions investigated, including in human sam- the first two postnatal weeks (Figure 6a,b, PSD:cytosol <0.2 in all ples. This indicates that each C-terminus will at least be expressed cases), and a much-increased PSD localization between PND21 and with two different N-termini. Importantly, we did not observe PND56. Nevertheless, β isoforms were always found significantly significantly different expression patterns between the top and more expressed in the cytosol than in the PSD, even at PND56 bottom bands, indicating that the C-termini would determine the where the PSD:cytosol ratio is 0.4. In contrast, α2 isoforms present differential expression observed between isoforms. similar expression level in cytosol and PSD at both PND21 and 56, Developmental expression in each of the five tissues analyzed re- as indicated by the PSD:cytosol ratio, which is of 1.5 at PND21 and vealed differences between SynGAP isoforms, although, overall, α1 1.9 at PND56, although PSD levels were found significantly higher. and α2 isoforms presented a more similar expression pattern between Therefore, subcellular localization of SynGAP isoforms in adult hip- each other than with β isoforms. Developmental expression differ- pocampus recapitulate the findings found in adult cortex. ences were most noticeable in cortex, hippocampus, and striatum. As in cerebellum and olfactory bulb PND4 levels remained unaltered, for β isoforms, or just increased moderately, for α1 and α2 isoforms. In 4 | D I S CU S S I O N cortex and hippocampus, the temporal expression of α1 isoforms is importantly delayed relative to that of α2 and β, as α1 isoforms reach It is established that mammals express multiple protein isoforms their maximum at PND56, while α2 and β at PND21. Thus, α1 isoforms from the Syngap1 gene, yet the complete set of SynGAP isoforms is would present very low levels at PND11, during the critical period of still to be defined. Evidence from multiple sources, including tran- Syngap1 deficiency (Clement et al., 2012, 2013), suggesting that other scriptomics data, suggest that human, mouse, and rat SynGAP iso- SynGAP isoforms may play a more relevant role early in this period. forms would at least have 5 N-termini (A1, A2, B, C, and D) and 4 This is in agreement with our identification of unique ion peptide pre- C-termini (α1, α2, β, and γ). Nevertheless, C and D N-termini have not cursors through discovery MS studies for α2 and β from PND0 until been reported in humans and NCBI adds four extra N-termini (A3, PND56 and for α1 only in adulthood as well as with the observation A4, E, and F), and another C-terminus (α3), to the mouse set of vari- that the PDZ binding motif found in α1 isoforms is not key for mediat- ants. To the best of our knowledge, only the D N-terminus has been ing SynGAP function in barrel cortex formation (Barnett et al., 2006). unambiguously identified at the protein level, which we report here Interestingly, the developmental expression observed in striatum pres- for the first time. The fact that MS-based methods have not been ents a bi-phasic pattern, which is unique to this brain region. Protein able to identify unique peptides from the other isoforms, beyond expression increases between PND4 and PND11, remains constant peptides common to A1 and A2 (McMahon et al., 2012), suggests during the critical period, between PND11 and 21, and increases again that their N-termini could be proteolyzed. between PND21 and PND56. This pattern is very obvious for α1 and Different SynGAP isoforms have shown opposed effects in α2 isoforms, while the second stage of increase, between PND21 and the control of synaptic strength (McMahon et al., 2012), to local- PND56, does not reach statistical significance for β isoforms. ize to different subcellular compartments (Li et al., 2001; Moon, Although SynGAP is a well-known component of excitatory syn- Sakagami, Nakayama, & Suzuki, 2008; Tomoda, 2004) or to be able apses from principal neurons, it is also expressed in inhibitory neu- to bind to different proteins (Kim et al., 1998; Li et al., 2001), partic- rons (Berryer et al., 2016) and it has been found co-localizing with ipating in different protein complexes and, by extension, in differ- Gephyrin and GAD-67 in primary neuronal cultures, especially β iso- ent molecular functions. SynGAP-β has even been described at the forms (Moon et al., 2008). We thus wanted to investigate if we could nucleus of cortical and hippocampal neurons (Moon et al., 2008), correlate the developmental expression pattern of SynGAP isoforms which would be in agreement with the signal that the Database of with the expression of markers of excitatory and inhibitory synapses Nuclear Localization Signals (Bernhofer et al., 2017) identifies in and neurons, to investigate if different isoforms presented specific the SynGAP core region (KKRKKD). This motif would be located expression patterns relating them to the developmental processes toward the end of the C2 domain, as it occurs in the Doc2g pro- of neurogenesis or synaptogenesis. Interestingly, we found that α1 tein, which is known to translocate to the nucleus through this sig- and α2 isoforms better followed the expression of synaptic markers nal (Fukuda, Saegusa, Kanno, & Mikoshiba, 2001). For this reason, than neuronal ones, while β isoforms did not systematically correlate it is essential to find out where and when these isoforms are ex- with neither synaptic nor neuronal markers. Noticeably, β isoforms pressed to have a correct understanding of SynGAP biology. Using only present significant expression correlation with Gephyrin in three highly specific antibodies /against α1, α2, and β C-termini hippocampus, which is in agreement for a role of these isoforms 632 | GOU et al. in hippocampal inhibitory synapses, as previously reported (Moon low amounts of this protein at the SNP fraction, which contains all et al., 2008). synaptic proteins that are not in the PSD. Different SynGAP iso- Isoform abundance comparison between tissues revealed that forms presented a clearly distinctive subcellular localization pattern cerebellum and olfactory bulb presented the lowest expression for between the cytosol and the PSD. Being α1 and β isoforms the ones all SynGAP isoforms, with the exception of α2 and β isoforms in with the most opposed patterns. Alpha1 isoforms were always found the olfactory bulb at PND4. Isoform abundance comparison be- highly restricted to the PSD, while β ones were always enriched in tween cortex, hippocampus, and striatum revealed that at PND11 the cytosolic fraction and expressed with low levels at PSDs until and adulthood the highest expression was found in the hippo- PND21. Isoforms with the α2 C-termini presented an intermediate campus. Yet, at PND21, toward the closure of the critical period, behavior, enriched in the cytosol in PND7 and 14 and enriched at clear differences were observed between isoforms. At this stage, the PSD at PND21 and 56. This differential distribution of SynGAP α1 isoforms presented the highest expression levels at cortex, α2 isoforms was also observed in hippocampal samples by two orthog- isoforms presented equivalent levels in cortex and hippocampus, onal methods, indicating that the same localization pattern could be while hippocampus remained the tissue with the highest expres- found in other brain regions. sion of β isoforms. We therefore observed an increase in the corti- Interestingly, at PND14, when PSDs are already formed in the cal expression of α1 and α2 isoforms relative to their hippocampal mouse cortex (Chandrasekaran et al., 2015; Swulius, Kubota, Forest, levels, this being particularly striking for α1 isoforms. FMRP levels & Waxham, 2010), as indicated by the clear enrichment of PSD-95 were found decreased specifically during PND21-23 in hippocam- in this fraction, α2 and β-containing isoforms present very low PSD ± pus from Syngap1 mice to compensate the necessary increase levels. However, 1 week later (PND21), coinciding with the rise of α1 in the expression of SynGAP mRNA at this point of development isoforms expression and localization to the PSD, the presence of α2 (Paul et al., 2019). Taken all these data together, PND21 is a key and β isoforms in this location clearly increases. Suggesting a coop- age for SynGAP neurobiology. erative mechanism of SynGAP isoforms localization to the PSD. An As SynGAP was first identified in the PSD, it has mainly been alternative explanation would be that between PND14 and PND21 studied in the context of adult synaptic function. Yet, Syngap1 ex- the primary, and yet unknown, interacting point for SynGAP at the pression starts early in embryogenesis (Porter, Komiyama, Vitalis, PSD becomes much more abundant, driving the increase in α2 and β Kind, & Grant, 2005), before the onset of synaptogenesis, indicating isoforms at this location. that SynGAP must have non-synaptic functions. For this reason, we Taking into consideration that the interaction between decided to investigate the distribution of SynGAP into subcellular SynGAP-α1 and PSD-95 is not required for SynGAP localization to compartments and if this changed along postnatal development. We the PSD (Barnett et al., 2006; Rumbaugh et al., 2006; Vazquez et al., looked for SynGAP distribution in four major cellular compartments, 2004) we propose a model in which all SynGAP isoforms would be (a) cytosol, containing soluble proteins not bound to membranes able to locate to the PSD through primary interaction(s) with un- such as GAPDH or Gephyrin, (b) non-synaptic membranes (NSM), (c) identified protein(s). These putative interaction(s) would occur via synapse excluding the PSD (SNP) which would include presynaptic a sequence within the core region of SynGAP. Furthermore, α1 iso- proteins like Synaptophysin and (d) the PSD, and we compared it to forms would present a secondary PSD anchoring point at their PDZ the distribution of the PSD marker PSD-95. Although we detected binding motif, which would result in their increased stability at the SynGAP and its isoforms in all subcellular fractions investigated, PSD. This increased stabilization would not go in detriment of the when normalizing their abundance by the amount of protein found in well-described dispersion of α1 and α2 isoforms from the PSD upon each fraction, we observed that SynGAP largely partitions between synaptic activation, as dispersed isoforms rapidly return to the PSD two locations, the PSD and the cytosol. Indicating that SynGAP has a (Araki, Zeng, Zhang, & Huganir, 2015; Yang et al., 2013; Yang, Tao- cytosolic function, which is essentially uncharacterized, and that this Cheng, Reese, & Dosemeci, 2011). Furthermore, the low abundance is the predominant function early in postnatal development. of α1 isoforms in the cytosol suggests that at any given time the tSynGAP subcellular distribution was remarkably different from proportion of α1 isoforms outside the PSD is relatively small. that of PSD-95, which at PND7 was weakly expressed, but from In summary, we have identified clear developmental expression PND14 onwards was almost exclusively found at the PSD. Instead, pattern differences between SynGAP isoforms, particularly during tSynGAP presented a clear cytosolic localization at all ages investi- the critical period of Syngap1 haploinsufficiency, which could have gated, even in adult cortex and hippocampus. Actually, during the relevance to brain development and mental illness. Furthermore, first two postnatal weeks, tSynGAP was almost exclusively found at we provide strong evidence showing that SynGAP, generally re- the cytosol. The fraction of tSynGAP localized to the PSD, progres- garded as a protein exclusive to the PSD, is also found in the cyto- sively increased along postnatal development but tSynGAP was only sol, where it is most abundant during postnatal brain development. found significantly enriched in the PSD compared to cytosol in adult Different isoforms present clearly distinctive subcellular distribu- cortical samples. Even at PND21, when synaptogenesis is largely tion, being α1 isoforms highly restricted to the PSD, β ones mainly finished (Harris, 1999), tSynGAP was still found more abundant in cytosolic and α2 isoforms presenting an intermediate behavior. the cytosol. It is also interesting to note that within synaptosomes, We have observed that the presence of α2 and β isoforms in the tSynGAP is almost exclusively found at the PSD, as we detected very PSD is developmentally regulated and coincides with the increased GOU et al. | 633 expression of α1 isoforms, while a non-synaptic role could be attri- Berryer, M. H., Chattopadhyaya, B., Xing, P., Riebe, I., Bosoi, C., Sanon, N., … Di Cristo, G. (2016). Decrease of SYNGAP1 in GABAergic cells impairs in- bute to these less known isoforms. Understanding the functional hibitory synapse connectivity, synaptic inhibition and cognitive function. differences between these isoforms will be key to disentangle the Nature Communications, 7, 1–14. https://doi.org/10.1038/ncomm​s13340 multiple functions performed by the SynGAP protein. Berryer, M. H., Hamdan, F. F., Klitten, L. L., Møller, R. S., Carmant, L., Schwartzentruber, J., Patry, L. et al. (2013). Mutations in SYNGAP1 cause intellectual disability, autism and a specific form of epilepsy by AC K N OW L E D G M E N T S inducing haploinsufficiency. Human Mutation, n/a–n/a. Financial support for this work was provided by: BFU2012-34398, Carlin, R. K., Grab, D. J., Cohen, R. S., & Siekevitz, P. (1980). Isolation and BFU2015-69717-P, and RTI2018-097037-B-100 (MINECO), Career characterization of postsynaptic densities from various brain regions: Integration Grant (ref. 304111), Ramón y Cajal Fellowship (RYC-2011- Enrichment of different types of postsynaptic densities. Journal of 08391p) IEDI-2017-00822; AGAUR (SGR14-297 and 2017SGR1776) Cell Biology, 86, 831–845. https://doi.org/10.1083/jcb.86.3.831 Carlisle, H. J., Manzerra, P., Marcora, E., & Kennedy, M. B. (2008). to AB; BES-2013-063720 (MINECO) to GG; MH096847 (NIH), SynGAP regulates steady-state and activity-dependent phosphory- MH108408 (NIH) and NS064079 (NIH) to GR and RO1 MH112151 lation of cofilin. 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