Pathogenic variation in SYNGAP1, the gene encoding SynGAP proteins, is a leading cause of sporadic neurodevelopmental disorders (NDDs) defined by impaired cognitive function, seizure, autistic features, and challenging behaviors (Deciphering Developmental Disorders Study, 2015; Deciphering Developmental Disorders Study, 2017; Hamdan et al., 2009; Vlaskamp et al., 2019; Parker et al., 2015; Mignot et al., 2016; Iossifov et al., 2014; Satterstrom et al., 2020). De novo loss-of-function variants leading to SYNGAP1 haploinsufficiency cause a genetically defined developmental encephalopathy (ICD-10 code: F78.A1) that overlaps substantially with diagnoses of generalized epilepsy, global developmental delay, intellectual disability, and autism (Vlaskamp et al., 2019; Parker et al., 2015; Mignot et al., 2016; Holder et al., 1993; Weldon et al., 2018). SYNGAP1 is completely intolerant of loss-of-function (LOF) variants (Llamosas et al., 2020). Thus, the presence of a clear LOF variant in a patient will lead to the diagnosis of a SYNGAP1-mediated developmental encephalopathy. The range of neuropsychiatric disorders causally linked to SYNGAP1 pathogenicity, combined with the complete penetrance of LOF variants in humans, demonstrate the crucial role that this gene plays in the development and function of neural circuits that promote cognitive abilities, behavioral adaptations, and balanced excitability.

SynGAP proteins have diverse cellular functions (Llamosas et al., 2020; Kilinc et al., 2018; Gamache et al., 2020). The best characterized of these is the regulation of excitatory synapse structure and function located on forebrain glutamatergic projection neurons. In these synapses, SynGAP is predominately localized within the postsynaptic density (PSD), where it exists in protein complexes with synapse-associated-protein (SAP) families (Kim et al., 1998; Chen et al., 1998). Within these complexes, SynGAP proteins regulate signaling through NMDARs, where they constrain the activity of various small GTPases through non-canonical activity of a RasGAP domain (Kilinc et al., 2018; Gamache et al., 2020). This regulation of GTPase activity is required for excitatory synapse plasticity (Ozkan et al., 2014; Araki et al., 2015). Reduced expression of SynGAP in both human and rodent neurons causes enhanced excitatory synapse function during early brain development and is a process thought to impair cognitive functioning (Llamosas et al., 2020; Clement et al., 2012; Clement et al., 2013). SynGAP also regulates dendritic arborization. Reduced SynGAP protein expression impairs the development of dendritic arborization in neurons derived from both rodent and human tissues (Llamosas et al., 2020; Aceti et al., 2015; Michaelson et al., 2018), which disrupts the function and excitability of neural networks from both species. While reduced SynGAP expression enhances postsynaptic function regardless of glutamatergic projection neuron subtype, this same perturbation has an unpredictable impact on dendritic arborization, with some neurons undergoing precocious dendritic morphogenesis (Llamosas et al., 2020; Aceti et al., 2015), while others displaying stunted morphogenesis (Michaelson et al., 2018). This is an example of pleiotropy, where Syngap1 gene products have unique functions depending on the neuronal subtype, or possibly within distinct subcellular compartments of the same type of neuron.

How SynGAP performs diverse cellular functions remains unclear. One potential mechanism is through alternative splicing. Indeed, the last three exons of Syngap1 undergo alternative splicing (Araki et al., 2020; Gou et al., 2020; McMahon et al., 2012), which results in four distinct C-termini (a1, a2, b, g). These SynGAP C-terminal protein isoforms are expressed in both rodents and humans, and they are spatially and temporally regulated across mammalian brain development (Araki et al., 2020; Gou et al., 2020). Moreover, protein motifs present within these differentially expressed C-termini impart SynGAP with distinct cellular functions, with α-derived motifs shown to regulate post-synapse structure and function (Rumbaugh et al., 2006; Vazquez et al., 2004), while the β-derived sequences linked to in vitro dendritic morphogenesis (Araki et al., 2020). Absolute abundances of C-terminal isoforms are unclear, though estimates of relative expression of each have been made in adult mice (Araki et al., 2020), with α1 and α2 each contributing ~40%, β contributing ~15%, and γ contributing ~5%. Syngap1 heterozygous null mice, which model the genetic impact of SYNGAP1 haploinsufficiency in humans, express a robust endophenotype characterized by increased horizontal activity, poor learning/memory, and seizure (Kilinc et al., 2018; Ozkan et al., 2014; Clement et al., 2012; Komiyama et al., 2002; Sullivan et al., 2020). Currently, it remains unknown to what extent endogenous in vivo expression of alternatively spliced isoforms contribute to systems-level endophenotypes expressed in animal models.

The last three exons of Syngap1 undergo alternative splicing (Figure 1A), which results in four distinct C-termini (Figure 1B). Exon 19 is spliced into two reading frames (e19b/e19a) (Figure 1C). Because e19b lacks a stop codon, coding sequences from e20 and e21 are also included in mature transcripts. This leads to expression ofα1, α2, or γ C-terminal isoforms (Figure 1C–D). γ isoforms arise from inclusion e20, while α1 and α2 arise from the absence of e20, but inclusion of e21. e21 itself has two reading frames, with one leading to expression of α1 while the other codes for α2 (Figure 1E). SynGAP-β arises from splicing of e19 into the ‘a’ reading frame, which contains an internal stop codon (Figure 1C). To address how expression or function of isoforms contribute to cognitive function, behavior, and seizure latency, we created three distinct mouse lines, each with targeted modifications within the final three exons of the Syngap1 gene. Each line expressed a unique signature with respect to C-terminal SynGAP protein variant expression or function. For example, in the Syngap1^td/td line, αisoform expression was disrupted while β forms were upregulated (Figure 1F–G). In contrast, Syngap1^β*/β* mice were opposite with respect to expression of α and β isoforms, with the former upregulated and the later disrupted (Figure 1H). Finally, the Syngap1^PBM/PBM line, which expressed point mutations that selectively disrupted an essential function of SynGAP-α1 (Figure 1I), was useful for determining to what extent phenotypes in the other two lines may have been driven by upregulated or downregulated isoforms.

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Reduced α1/2 C-terminal isoform expression is associated with enhanced seizure latency and cognitive impairment

We previously reported the generation of a Syngap1 mouse line with an insertion of an IRES-TdTomato (IRES-TD) cassette within the 3’-UTR to facilitate endogenous reporting of active Syngap1 mRNA translation in cells (Spicer et al., 2018). The cassette was placed within the last Syngap1 exon (e21) between the stop codons of α1 and α2 coding sequences (Figure 1E; Figure 2A). Our prior study reported neuronal expression of fluorescent protein and normal total SynGAP (t-SynGAP) protein expression as measured by antibodies that recognize all splice forms. Due to our interest in understanding how in vivo expression of C-terminal variants impacts brain systems and behavior, we performed an in-depth characterization of behavioral phenotypes and SynGAP isoform expression in IRES-TD mice. Heterozygous (Syngap1^+/td) breeding of IRES-TD animals resulted in offspring of expected mendelian ratios (Figure 2B). However, while all WT (Syngap1^+/+) mice survived during the 100-day observation period, significant post-weaning death occurred in IRES-TD mice, with approximately two-thirds of homozygous mice (Syngap1^td/td) failing to survive past PND 50 (Figure 2B). It is well established that complete loss of t-SynGAP protein stemming from homozygous inclusion of null alleles leads to early postnatal death (Komiyama et al., 2002; Kim et al., 2003). However, ~ 50% t-SynGAP expression, like that occurring in heterozygous KO mice (Figure 2—figure supplement 1A), has no impact on survival (Komiyama et al., 2002; Kim et al., 2003). Given the unexpectedly poor survival of Syngap1^td/td animals, we thoroughly examined SynGAP C-terminal isoform protein expression in this line. At PND21, when all three genotypes are abundant (Figure 2B), t-SynGAP protein in mouse cortex homogenate was reduced in Syngap1^+/td and Syngap1^td/td mice compared to WT controls (Figure 2C; Source data1). Reduced t-SynGAP levels appeared to be largely driven by near-complete disruption of α1/2 protein expression from the targeted allele. Reduced α isoform expression coincided with increased protein levels of β-containing C-terminal isoforms. Even with β compensation, Syngap1^td/td mice expressed only ~50% of t-SynGAP at PND21. Whole exome sequencing was carried out in each genotype. Differential gene expression (DGE) analysis revealed only a single mRNA, Syngap1, was abnormally expressed (Supplementary file 1). There was a ~ 25% reduction in mRNA levels in both Syngap1^+/td and Syngap1^td/td mice (Figure 2—figure supplement 1B). While the IRES-TD cassette destabilized a proportion of Syngap1 mRNAs, the similarity in mRNA levels from both Syngap1^+/td and Syngap1^td/td samples indicated that other mechanisms must also contribute to reduced protein expression of α1/2 isoforms. Indeed, a recent study identified 3’UTR-dependent regulation of α isoform protein expression (Yokoi et al., 2017), suggesting that the IRES-TD cassette is also disrupting translation of these C-terminal variants. We next addressed expression of SynGAP isoforms in adulthood. In this additional experiment, only Syngap1^+/+ and Syngap1^+/td mice were used because of limited survival and poor health of homozygous mice in the post-weaning period (Figure 2B). The general pattern of abnormal SynGAP levels persisted into adulthood, with both α isoforms reduced by ~50% compared to WT levels, while β isoforms were significantly enhanced (Figure 2—figure supplement 1C). However, the effect on t-SynGAP was less pronounced in older animals and did not rise to significance. This finding highlights the importance of measuring the expression of individual isoforms in addition to total levels of SynGAP protein in samples derived from animal or cellular models.

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Syngap1 heterozygous KO mice, which have 50% reduction of t-SynGAP and 50% reduction of all isoforms (Figure 2—figure supplement 1—source data 1), have normal post-weaning survival rates (Komiyama et al., 2002; Kim et al., 2003). However, survival data from Syngap1^td/td mice above, which also expressed a ~ 50% reduction of t-SynGAP, but loss of α isoform expression (Figure 2C; Figure 1G), suggest that expression of these isoforms is required for survival. α isoforms are highly enriched in brain (Araki et al., 2020), suggesting that reduced survival stems from altered brain function. Therefore, we next sought to understand how reduced α1/2 expression (but in the context of β compensation) impacted behaviors known to be sensitive to reduced t-SynGAP expression in mice. We obtained minimal data from adult Syngap1^td/td mice because they exhibit poor health and survival in the post-weaning period. However, two animals were successfully tested in the open field, and they exhibited very high levels of horizontal activity (Figure 2D). A more thorough characterization of behavior was carried out in adult Syngap1^+/td mice, which have significantly reduced α isoforms, enhanced β expression, but relatively normal t-SynGAP levels (Figure 2—figure supplement 1A). Syngap1^+/td mice exhibited significantly elevated open-field activity, seized more quickly in response to flurothyl, and froze less during remote contextual fear memory recall (Figure 2E–G). These phenotypes are all present in conventional Syngap1^+/- +/- (Ozkan et al., 2014; Clement et al., 2012; Aceti et al., 2015; Creson et al., 2019), which again express ~50% reduction of all isoforms (Figure 2—figure supplement 1A). In contrast, Morris water maze acquisition, which is impaired in Syngap1^+/- +/- (Komiyama et al., 2002; Kim et al., 2003), was unchanged in Syngap1^+/td mice (Figure 2H). Thus, certain behaviors, including horizontal activity, freezing in response to conditioned fear, and behavioral seizure, are sensitive to reduced levels of α isoforms, but not necessarily to t-SynGAP levels. Moreover, ~ 50% loss of α1/α2 isoforms appear sufficient to disrupt long-term memory (Figure 2G), but insufficient to disrupt spatial learning (Figure 2H).

Enhanced α1/2 C-terminal isoform expression is associated with seizure protection and improved cognitive function

The results in IRES-TD mice suggested that certain core Syngap1-sensitive behavioral phenotypes are caused, at least in part, by reduced α1/2 isoform expression. If α isoforms directly contribute to behavioral phenotypes in mice, then increasing their expression may drive phenotypes in the opposite direction. To test this idea, we created a new mouse line designed to upregulate SynGAP-α expression in vivo. This line, called Syngap1^β*/β*, contained a point mutation that prevented use of the e19a spliced reading frame (Figure 3A–B), the mechanism leading to expression of the SynGAP-β C-terminal variant (Figure 1C). This design was expected to force all mRNAs to use the e19b reading frame, leading to an increase in α variants (and loss of β expression). This line appeared healthy, bred normally, and resulting offspring were of expected Mendelian ratios. The CRISPR-engineered point mutation had the predicted impact on SynGAP isoform expression. While there was no change in t-SynGAP expression, there was a copy-number-dependent decrease in β expression, and a modest, but significant, increase in α2 expression in neonatal mice and α1 in young adult mice (Figure 3C; Figure 1H; Figure 3—source data 1). These animals were then evaluated in behavioral paradigms sensitive to Syngap1 haploinsufficiency. Homozygous Syngap1^β*/β* mice exhibited significantly less horizontal activity in the open field (Figure 3D), and also took longer to express behavioral evidence of seizure (Figure 3E). Further, they expressed no change in freezing levels during remote contextual memory recall (Figure 3F). Unexpectedly, homozygous β* mice exhibited improved learning in the Morris water maze (Figure 3G), with normal memory expression during the probe test. Thus, a significant increase in α isoform expression (in the presence of nearly absent β; Figure 1H) protected against seizure and improved behavioral measures associated with cognitive function, such as learning during spatial navigation.

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Given the observation of seizure protection and improved learning in Syngap1^β*/β* mice, we were curious if the impact of the β allele was penetrant in a Syngap1 heterozygous (Syngap1^+/-) background. This is important given that Syngap1 heterozygous mice, which model genetic impacts of SYNGAP1 haploinsufficiency in humans, have seizures and significant cognitive impairments. To test this idea, we crossed Syngap1^+/β* and Syngap1^−/+ + , which yielded offspring with four distinct genotypes: Syngap1^+/+, Syngap1^+/β*, Syngap1^−/+, Syngap1^−/β* (Figure 4A). We first measured t-SynGAP protein in each of the four genotypes. In general terms, offspring from this cross expressed changes in SynGAP protein levels that were predicted by the known impact of each allele. For example, the effect of the Syngap1 null allele (by comparing Syngap1^+/+ to Syngap1^-/+ samples) was to cause a significant reduction in t-SynGAP, and each of the measured C-terminal isoforms compared to Syngap1^+/+ (WT) animals (; Figure 4—figure supplement 1—source data 1). The effect of the Syngap1β* allele was to increase both α1 and α2 expression, and decrease β expression, whether the Syngap1 null allele was present or absent, and these effects were also present at two developmental time points (Figure 4B–C, Figure 4—figure supplement 1). Given these results, we next performed behavioral analyses on all four genotypes. Results on behavioral endophenotypes were consistent with changes in SynGAP protein. For example, the Syngap1 null allele impaired performance in each of the three behavioral tests performed. Comparing Syngap1^+/+ to Syngap1^-/+ animals revealed an increase in horizontal distance in the open field, faster time to seizure, and reduced freezing during remote contextual fear recall (Figure 4D–F; two-way ANOVA; null (-) allele, p < 0.05). These results replicate many past studies demonstrating the sensitivity of these behaviors to Syngap1 haploinsufficiency in mice (Kilinc et al., 2018; Clement et al., 2012; Aceti et al., 2015; Michaelson et al., 2018; Komiyama et al., 2002; Creson et al., 2019; Guo et al., 2009). Interestingly, for both open field and seizure threshold tests, the presence of β* allele significantly improved measures in both WT (Syngap1^+/+) and Syngap1 heterozygous (Syngap1^-/+) backgrounds (Figure 4D–E; two-way ANOVA; β* allele, p < 0.01; interaction of null and β alleles, p > 0.5). These findings were consistent with behavioral results from homozygous β* mice in the prior study (Figure 3F–G) and demonstrated that these two behavioral tests are sensitive to the presence of a single β* allele. Also consistent with the prior study in Syngap1^β*/β* mice, the β* allele had no impact on freezing during remote contextual fear recall in either WT or Syngap1 heterozygous backgrounds (Figure 4F). Thus, the β* allele partially rescued phenotypes caused by Syngap1 heterozygosity.

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Alpha1 C-terminal isoform function is required for cognitive function and seizure protection

The results obtained from Syngap1 IRES-TD and β* mouse lines indicated that a respective decrease, or increase, in α1/2 isoform expression impaired, or improved, behavioral phenotypes known to be sensitive to Syngap1 heterozygosity. However, it is also possible that compensatory changes in β expression underlies these phenotypes. This alternative is unlikely, given that α and β expression is anticorrelated in both mouse lines. Thus, for β to drive phenotypes, its expression would need to be both anti-cognitive and pro-seizure, which is inconsistent with isoform expression patterns in Syngap1^-/+ mice (Figure 2—figure supplement 1A), where all protein variants are reduced by half. To directly test the hypothesis that behavioral phenotypes are sensitive to the presence of α isoforms, we attempted to create a third mouse line with point mutations that selectively impacted α isoforms, with minimal effect to SynGAP-β. We took advantage of a known molecular function exclusive to SynGAP-α1. This C-terminal variant is the only isoform that expresses a PDZ-binding motif (PBM). Importantly, cell-based studies have shown that the α1-exclusive PBM imparts unique cellular functions to this isoform (Araki et al., 2015; Zeng et al., 2016), such as the ability to become enriched at the post-synaptic density through liquid-liquid phase separation (LLPS). Past studies have shown that mutating the PBM disrupts the ability of SynGAP to regulate synapse structural and functional properties (Rumbaugh et al., 2006; Vazquez et al., 2004), including glutamatergic synapse transmission and dendritic spine size. Before this mouse could be engineered, we had to first identify PBM-disrupting point mutations within the α1 coding sequence that were silent within the open reading frames of the remaining C-terminal isoforms. In silico predictions and prior studies (Rumbaugh et al., 2006; Zeng et al., 2016) suggested that a double point mutation within the α1 PBM could meet these requirements (Figure 5A–B). To test this prediction, we introduced these point mutations into a cDNA that encoded the PBM and then tested how this impacted PDZ binding. Using an established cell-based assay that reports PDZ binding between the SynGAP PBM and PSD95 (Zeng et al., 2016), we found that these point mutations had a large effect on SynGAP-PDZ binding. When expressed individually in HeLa cells, PSD95-tRFP localized to the cytoplasm, while a SynGAP fragment containing the coiled-coil domain and α1 C-tail (EGFP-CCα1) was enriched in the nucleus (Figure 5C–E). The co-expression of these two proteins led to SynGAP localization into the cytoplasm. However, this shift in localization did not occur when PBM point mutations were present (Figure 5D–E), indicating that the selected amino acid substitutions severely impaired binding to the PDZ domains. Moreover, co-immunoprecipitation in heterologous cells indicated that the point mutations in the PBM disrupted the direct association of full-length SynGAP-α1 with PSD95 (Figure 5—figure supplement 1—source data 1). Finally, these point mutations also reduced synaptic enrichment of exogenously expressed SynGAP-α1 fragments in cultured forebrain neurons (Figure 5—figure supplement 1C-E).

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Based on this evidence, we introduced the PBM-disrupting point mutations into the final exon of the mouse Syngap1 gene through homologous recombination (Figure 5A and F–H). Both heterozygous and homozygous PBM mutant animals (hereafter Syngap1^+/PBM or Syngap1^PBM/PBM) were viable, appeared healthy, and had no obvious dysmorphic features. We observed Mendelian ratios after interbreeding Syngap1^+/PBM animals (Figure 5—figure supplement 1F), demonstrating that disrupting the PBM had no impact on survival. Western blot analysis of forebrain homogenates isolated from Syngap1^+/PBM or Syngap1^PBM/PBM mutant animals demonstrated no difference in t-SynGAP protein levels using antibodies that detect all SynGAP splice variants (Figure 5—source data 1I-J). Moreover, using isoform-selective antibodies (Araki et al., 2020; Gou et al., 2019), we observed normal expression of SynGAP-β and SynGAP-α2 isoforms (Figure 5I–J). A reduced signal of ~60% was observed in samples probed with α1-specific antibodies. However, we also observed a similarly reduced signal in heterologous cells expressing a cDNA encoding the mutant PBM (Figure 5—figure supplement 1—source data 1), indicating that these antibodies have reduced affinity for the mutated α1 motif. Together, these data strongly suggest that the α1 variant is expressed normally in Syngap1^PBM/PBM animals. This interpretation was supported by RNA-seq data, where normal levels of mRNA containing the α1 reading frame were observed in brain samples (Figure 5—figure supplement 1J). These data, combined with the observation of no change in total SynGAP protein expression in Syngap1^PBM/PBM samples (Figure 5I–J), strongly support the conclusion that the PBM-disrupting point mutations do not change the expression levels of the major SynGAP C-terminal splice variants, including those containing the PBM. Thus, this animal model is suitable for understanding the putative biological functions mediated by α1-specific splicing.

Given the disruption to SynGAP-α1 PBM, we sought to understand how altering this functional motif impacted previously defined features of SynGAP at excitatory postsynapses. α1 is believed to be anchored within the PSD in part through PBM binding to PDZ domain containing proteins. However, SynGAP molecules multimerize in vivo and it is currently unknown to what extent this feature contributes to homo- vs. hetero-multimerization. Thus, it is unclear how a functional disruption to one isoform generally impacts native SynGAP complexes at synapses. This is important given that C-terminal isoforms have distinct functions within excitatory neurons (Araki et al., 2020). t-SynGAP levels were reduced in unstimulated PSD fractions prepared from either adult hippocampal homogenates or primary neurons from Syngap1^PBM/PBM mice (). Importantly, a corresponding increase in t-SynGAP was observed in the triton-soluble synaptosomal fraction, further supporting the observation of reduced t-SynGAP levels in the PSD. PSD abundance of SynGAP-β and α2 isoforms were not significantly different in PBM mice compared to WT littermates (Figure 6A), which suggested that α1 may exist in distinct biochemical complexes compared to other C-terminal isoforms (i.e. homomeric SynGAP-α1 complexes). Unfortunately, this could not be tested directly in these samples due to reduced affinity of α1-specific antibodies in Syngap1^PBM/PBM mice (Figure 5—figure supplement 1H). Therefore, we performed an additional experiment to address the potential existence of isoform-specific biochemical complexes. This required culturing neurons, inducing chemical LTP (cLTP), and then measuring how the stimulus impacted PSD abundance of total SynGAP and C-terminal isoforms. First, we found that a typical cLTP paradigm drove extrusion of total SynGAP from the PSD of WT mice (Figure 6—figure supplement 1—source data 1), while a weak cLTP stimulation did not (Figure 6—figure supplement 1B), which are findings consistent with past studies that defined SynGAP dynamics within biochemical fractions or subcellular compartments (Araki et al., 2015; Araki et al., 2020). In contrast, weak cLTP was capable of driving a reduction in total SynGAP from PSDs in Syngap1^PBM/PBM mice (Figure 6—figure supplement 1B). Immunoblotting with isoform-specific antibodies in the weak cLTP condition provided insight into the differential behavior of total SynGAP in PSDs from WT vs. PBM mice. For example, weak cLTP was sufficient to drive reduced PSD abundance for both α2 and β isoforms in both WT and PBM neurons (Figure 6—figure supplement 1B). However, α1 PSD abundance was unchanged in WT mice after weak cLTP, demonstrating that this isoform, when intact, exhibits distinct properties in response to synaptic NMDAR activation. Replicating this approach in PBM neurons revealed that this distinct feature of α1 was due to the existence of an intact PBM motif (Figure 6—figure supplement 1B). These data indicate that reduced PSD abundance of t-SynGAP in Syngap1^PBM/PBM mice is driven by altered biochemical features and dynamics of α1. The other isoforms appear minimally impacted by the PBM mutation. Based on this model, spontaneous activity within Syngap1^PBM/PBM neurons may drive reduced SynGAP PSD abundance, presumably by reducing the stimulus threshold required to drive α1 out of this compartment. To test this, we measured SynGAP PSD abundance and ERK1/2 signaling in WT and PBM neurons with and without activity blockers (Figure 6B). Acute blockade of synaptic activity normalized SynGAP levels in the PSD and ERK1/2 signaling (Figure 6B). Similar treatments also normalized enrichment of SynGAP in dendritic spines and surface expression of GluA1 in neurons derived from Syngap1^PBM/PBM mice (Figure 6C and D). These results indicate that endogenous PBM binding of the α1 isoform regulates an activity-dependent process within excitatory synapses.

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Blocking synaptic activity in Syngap1^PBM/PBM neurons prevented alterations in SynGAP levels at postsynapses (Figure 6A–D). This suggested that the PBM regulates SynGAP-specific functions in excitatory synapses, such as activity-dependent extrusion of α1 from the PSD. However, SynGAP-α1 undergoes LLPS and this mechanism is thought to facilitate the organization of the PSD (Zeng et al., 2016). Thus, disrupted SynGAP post-synaptic levels could also be attributable to altered structural organization of the PSD. To determine if the PBM contributes to the organization of macromolecular complexes within excitatory synapses, we immunoprecipitated PSD95 from neurons obtained from either WT or Syngap1^PBM/PBM mutant neurons. These neurons were treated with APV to avoid the confounds of elevated NMDAR signaling. These samples were then analyzed by mass spectrometry to determine how disrupting SynGAP-PDZ binding impacted the composition of PSD95 macromolecular complexes. In general, we found only minor differences in the abundance of proteins that comprise PSD95 complexes when comparing samples from each genotype (Figure 7—source data 1A). Only 1 out of ~161 proteins (from 133 distinct genes) known to be present within PSD95 complexes (Li et al., 2017) met our threshold for significance, although there were modest changes in proteins with structurally homologous PBMs (Type-1 PDZ ligands), such as Iqseq2 and Dlgap3 (Figure 7B). However, the vast majority of related PBM-containing proteins were not different in mutant neurons, including NMDAR subunits and TARPs (Figure 7C). Consistent with the mass spectrometry analysis, immunoblot analyses found no changes in TARPs or LRRTM2 in isolated PSDs from Syngap1^PBM/PBM mice (Figure 7—source data 2). Although PDZ binding was disrupted, SynGAP protein levels were also unchanged within PSD95 complexes, a result consistent with PSD and synapse localization measurements in APV-treated neurons derived from Syngap1^PBM/PBM mice (Figure 6B–C). These results indicate that SynGAP interacts with PSD95 in a non-PDZ-dependent manner. In support of this interpretation, there is significant overlap between the interactomes of PSD95 (Li et al., 2017) and SynGAP (Wilkinson et al., 2017) macromolecular complexes (Figure 7H). Thus, within intact postsynapses, SynGAP and PSD95 interact, as part of a macromolecular complex, through binding to common protein intermediaries. Together, these data suggest that SynGAP PBM binding to PDZ domains is not a major factor promoting the organization of PSD95 macromolecular complexes or the PSD. Rather, the PBM appears to regulate SynGAP-specific mechanisms that control signaling through NMDARs.

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Given that altering the SynGAP PBM disrupts signaling through NMDARs, we hypothesized that hippocampal CA1 LTP would be disrupted in Syngap1^PBM/PBM mice. The within-train facilitation of responses across the seven theta bursts used to induce LTP did not differ between genotypes (Figure 8A), indicating that standard measures of induction, including NMDAR channel activation, were not impacted by PBM mutations. However, short-term plasticity (STP; Figure 8C and D) and LTP (Figure 8B and E) were both reduced in Syngap1^PBM/PBM mice. The ratio of LTP/STP was no different between genotypes (Figure 8F). Blocking NMDAR channel function is known to disrupt both STP and LTP (Volianskis et al., 2013). However, a key measure of NMDA channel function was normal in PBM mutant mice (Figure 8A). Thus, these data are consistent with the idea that disrupting SynGAP-PDZ binding impairs signaling normally induced downstream of synaptic NMDAR activation. Synaptic plasticity, such as LTP, is thought to contribute importantly to multiple forms of learning and memory. As such, we next measured performance of WT and Syngap1^PBM/PBM mice in a variety of learning and memory paradigms that have previously shown sensitivity in Syngap1 mouse models, including IRES-TD and β* lines. Behavioral analysis in this line revealed a significant increase in horizontal locomotion in the open-field test (Figure 8G), a significantly reduced seizure threshold (Figure 8H), and significantly reduced freezing during retrieval of a remote contextual fear memory (Figure 8I). Moreover, we also observed impaired acquisition during Morris water maze learning (Figure 8J). Together, these behavioral data indicate that the PBM within SynGAP-α1 splice forms is critical for learning and memory, as well as protecting against seizure.

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Alpha1/2 C-terminal isoform expression or function predicts changes in excitatory synapse function

Behavioral results from IRES-TD and PBM mice were consistent with each other, and also consistent with a reduction in all SynGAP isoforms occurring in Syngap1 conventional heterozygous null mice. These three mouse lines share a common molecular feature – reduced expression or function of SynGAP-α1 isoforms (Figure 1F–I; Supplementary file 1). Prior studies have shown that exogenously expressed SynGAP-α1 is a negative regulator of excitatory synapse function (Rumbaugh et al., 2006; Wang et al., 2013). Thus, we hypothesized that IRES-TD and PBM mouse lines would express elevated excitatory synapse function, while Syngap1^β*/β* mice, which have enhanced α1 expression, would express reduced synapse function. To test this idea, we performed whole-cell voltage clamp recordings in acute somatosensory cortex slices derived from all three of these lines because these neurons have been shown to be sensitive to Syngap1 heterozygosity in ex vivo slice preparations (Michaelson et al., 2018). PBM mice exhibited a modest increase in mEPSCs amplitude and a more substantial increase in mEPSC frequency, two measures consistent with enhanced postsynaptic function (Figure 9A–C). We also observed increased excitatory synapse function (both mEPSC amplitude and frequency distributions) in IRES-TD mice (Figure 9D–F). The sample size for PBM mEPSC analysis is somewhat underpowered, although these significant effects agree with independent mEPSC observations from the IRES-TD mice. Moreover, effects on mEPSC amplitude in L2/3 SSC neurons observed in both lines are similar to what has been reported previously in Syngap1^+/- +/- (Michaelson et al., 2018). In contrast, Syngap1^β*/β* mice, which have significantly elevated α1/α2 expression, expressed reduced mEPSC amplitude and frequency measurements relative to littermate control slices (Figure 9G–I), a phenotype consistent with SynGAP-α1 overexpression in excitatory neurons (Rumbaugh et al., 2006; Wang et al., 2013).

Figure 9

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In this study, we created three distinct mouse lines, each regulating the expression or function of SynGAP protein isoforms (Figure 1F–I), without appreciable change in total SynGAP expression levels. A summary of all measured phenotypes in these lines can be found in Supplementary file 1. The overall conclusion from this study is that α-containing SynGAP isoforms promote cognitive functions that support learning/memory, while also protecting against seizure. It is important to understand the relationship between SynGAP isoform function and systems-level manifestations of the different isoforms, such as behavioral expression related to cognitive function and seizure. It has been shown previously that Syngap1 C-terminal splicing imparts distinct cellular functions of SynGAP proteins (Araki et al., 2020; McMahon et al., 2012; Rumbaugh et al., 2006; Vazquez et al., 2004). Thus, targeting endogenous isoform expression in animal models presents an opportunity to determine to what extent distinct cellular functions of SynGAP could contribute to various intermediate phenotypes present in Syngap1 mouse models. Given that SYNGAP1 is a well-established NDD gene and LOF mutations are highly penetrant in the human population (Deciphering Developmental Disorders Study, 2015; Deciphering Developmental Disorders Study, 2017; Hamdan et al., 2009; Parker et al., 2015; Mignot et al., 2016; Satterstrom et al., 2020; Hamdan et al., 2011; Berryer et al., 2013), studying these relationships have the potential to provide much needed insight into the neurobiology underlying human cognitive and behavioral disorders that first manifest during development. Second, there is increasing interest in targeted treatments for patients with SYNGAP1 disorders due to the penetrance of LOF variants, the relatively homogenous manifestations of the disorder (e.g. cognitive impairment and epilepsy), and the growing number of patients identified with this disorder (Lim et al., 2020). Restoring SynGAP protein expression in brain cells is the most logical targeted treatment for this disorder because most known patients have de novo variants that cause genetic haploinsufficiency (Holder et al., 1993). The most logical therapeutic approach would be to reactivate native expression of the endogenous gene. However, the findings from this study indicate that targeted therapies for SYNGAP1 disorders that enhance expression of α isoforms may be sufficient to provide a benefit to patients. Indeed, only a modest upregulation of α1/2 expression within a Syngap1 heterozygous background was sufficient to improve behavioral deficits commonly observed in that mouse line (Figure 4). Third, the discovery that SynGAP-α1/2 expression/function is pro-cognitive and provides protection from seizure suggests that these isoforms, and the cellular mechanisms that they regulate, could be harnessed to intervene in idiopathic cognitive and excitability disorders, such as neurodegenerative disorders and/or epilepsies with unknown etiology.

Several lines of evidence from this study support the conclusion that SynGAP-α isoform expression or function promotes cognition and seizure protection. IRES-TD and PBM mouse lines each had similar learning/memory and seizure threshold phenotypes, with both mouse lines exhibiting impaired phenotypes related to these two types of behavioral analyses. Indeed, these two mouse lines also shared a common molecular perturbation - reduced expression or function of alpha isoform(s). For example, IRES-TD homozygous mice lacked expression of both α1 and α2 isoforms and these animals exhibited severe phenotypes, including reduced post-weaning survival and dramatically elevated horizontal activity in the open field. Additional phenotypes were also present in heterozygous IRES-TD mice, which underwent more comprehensive testing because of better survival in the post-weaning period. These additional phenotypes included reduced seizure threshold and impaired freezing during a remote contextual fear expression test. PBM homozygous mice had normal expression of SynGAP protein, but lacked a functional domain present exclusively in α1 isoforms, a type-1 PDZ-binding domain. PBM homozygous mice shared phenotypes with IRES-TD mice, including impaired remote contextual fear expression, elevated horizontal activity in the open field, and a reduced seizure threshold. These mice also expressed impaired learning during Morris water maze acquisition. Importantly, these behavioral phenotypes are well established in Syngap1 heterozygous mice (Ozkan et al., 2014; Clement et al., 2012; Aceti et al., 2015; Creson et al., 2019; Guo et al., 2009), indicating that SynGAP protein loss-of-function underlies these abnormalities. Thus, it reasonable to speculate that α isoform LOF is one potential mechanism underlying these behavioral abnormalities. Dysregulation of excitatory synapse function in cortical circuits is one of many possible cellular mechanisms underlying common phenotypes in IRES-TD and PBM mutant mice lines. Whole cell electrophysiology experiments from developing cortical neurons in situ from each line revealed evidence of elevated excitatory synapse strength during the known Syngap1 mouse critical period. Indeed, elevated excitatory synapse strength in developing forebrain glutamatergic neurons is a major cellular outcome present in Syngap1 heterozygous knockout mice (Ozkan et al., 2014; Clement et al., 2012; Clement et al., 2013; Michaelson et al., 2018). Moreover, elevated excitatory synapse strength is consistent with impaired cognitive function and reduced seizure threshold.

Studies in the Syngap1β* line also support this interpretation. These mice were devoid of SynGAP-β protein expression, yet we did not observe cellular or behavioral phenotypes consistent with Syngap1 heterozygosity. Rather surprisingly, mice lacking SynGAP-β expression had intermediate phenotypes that opposed what was commonly observed in Syngap1 heterozygous KO mice (and shared by IRES-TD/PBM lines). For example, β* mice exhibited improved spatial learning in the Morris water maze, reduced horizontal activity in the open field, and an elevated seizure threshold (evidence of seizure protection). These phenotypes were modest in effect size, but highly significant. These phenotypes were reproducible because open field and seizure phenotypes were also present in a separate series of experiments performed in the Syngap1 heterozygous background. This demonstrates that the impact of the β* allele is penetrant even when expression of isoforms is reduced by half compared to WT mice. As a result, the β* allele partially rescued open field and seizure phenotypes present in Syngap1^+/- +/-. For impaired β expression to drive phenotypes, expression of this isoform would be anticorrelated with cognitive function and seizure protection. Put another way, reduced β expression would need to enhance phenotypes and increased expression of these isoforms would need to disrupt them. This outcome is unlikely given that it is inconsistent with phenotypes observed in Syngap1^+/- +/-, which have reduced expression of all isoforms, including SynGAP-β.

Phenotypes in β* mice are likely driven by significantly elevated SynGAP-α expression rather than reduced SynGAP-β. Electrophysiological studies in these mice revealed reduced excitatory neuron synaptic strength, a finding consistent with exogenously elevated SynGAP-α1 expression (Rumbaugh et al., 2006; Wang et al., 2013). Moreover, these synapse-level results are consistent with seizure protection observed in β* mice. Phenotypes in PBM mice also support this hypothesis. This model does not have altered t-SynGAP expression, or a change in β expression. Yet, the behavioral- and synapse-level phenotypes are consistent with those observed in IRES-TD and Syngap1^+/- +/-. The observation that α isoforms promote cognitive function and seizure protection are consistent with known molecular functions of these isoforms, at least with respect to regulation of synapse strength and resultant impacts on neural circuit function. For example, α1 imparts SynGAP with the ability to undergo liquid-liquid phase transitions (Zeng et al., 2016). This biophysical process is associated with regulation of Ras signaling in dendritic spines required for AMPA receptor trafficking that supports use-dependent synapse plasticity (Araki et al., 2015; Araki et al., 2020). Input-specific plasticity is crucial during development to sculpt the assembly of neural circuits (Zhang and Poo, 2001), while also being important in mature circuits to promote experience-dependent changes in already-established circuitry (Lynch et al., 2007).

Syngap1 is a potent regulator of forebrain glutamatergic neuron biology and that many phenotypes observed in models of Syngap1 regulation have origins in these excitatory neurons. Single cell transcriptomics data from adult mice indicate that Syngap1 is principally expressed in glutamatergic neurons in the cortex and hippocampus rather than GABAergic interneurons (Figure 10). Single-cell mRNA expression data agree with experimental evidence of SynGAP protein expression in rodent neurons. For example, SynGAP protein expression is enriched in glutamatergic neurons (Kim et al., 1998; Rumbaugh et al., 2006; Kim et al., 2003), with relatively high levels in upper lamina of isocortex (Butko et al., 2013). Other studies show that SynGAP protein is absent from several types of forebrain GABAergic neurons, but was expressed in a subpopulation of morphologically distinct inhibitory cells (Zhang et al., 1999). Expression data agree with prior experimental observations from electrophysiological and behavioral measurements in mice where Syngap1 expression was conditionally regulated in distinct neuronal subtypes. Commonly observed and robust phenotypes observed in Syngap1 heterozygous null mice (Guo et al., 2009; Muhia et al., 2010) were phenocopied in animals where Syngap1 heterozygosity was restricted to excitatory neurons in the forebrain (Ozkan et al., 2014). Moreover, major electrophysiological and behavioral phenotypes in Syngap1 heterozygous mice were also rescued when gene expression was restored in in this same population (Ozkan et al., 2014). In contrast, only minor phenotypes emerged in mice when Syngap1 expression was disrupted in GABAergic neurons. A separate group reported similar results. In that study, most Syngap1 heterozygous mouse behavioral phenotypes were insensitive to selective disruption within a GABAergic neuron population, although one behavioral measure of cognition was mildly affected (Berryer et al., 2016).

Figure 10

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Alpha isoforms, and α1 in particular, exhibit enrichment in dendritic spine synapses (Gou et al., 2020). As a result, baseline synaptic phenotypes related to Syngap1 gene expression appear dominated by the ability of both α1 and α2 isoforms to suppress excitatory synapse function. Studies from several research groups have shown that SynGAP-α1 is a negative regulator of excitatory synapse structure and function (Araki et al., 2015; Araki et al., 2020; Rumbaugh et al., 2006; Vazquez et al., 2004; Wang et al., 2013). In contrast, the role of α2 isoform protein function on excitatory synapse structure/function is less clear. One study suggested that α2 has an opposing function relative to α1 within excitatory synapses, with the former acting as an enhancer, rather than a suppresser, of excitatory synapse function (McMahon et al., 2012). However, a more recent study demonstrated that α2 has a similar, albeit less robust ability to suppress AMPA receptor content within dendritic spines (Araki et al., 2020), indicating that it too can act as a negative regulator of synapse function. Our results here support the view that both α1 and α2 can act as suppressors of excitatory synapse function. In our studies, α1 and α2 were both co-regulated in the IRES-TD and β* lines, with both isoforms downregulated in the former and upregulated in the latter. In both mouse lines, baseline excitatory synapse strength was inversely proportional to expression levels of α1/2 isoforms. If α1 and α2 had opposing functions at the synapse level, then co-regulation of both isoforms would be expected to lead to no significant differences in synapse function. As a result, we hypothesize that improvements in spatial learning and protection from seizure in β* mice arise through changes in excitatory synapse biology mediated by α isoforms. Thus, in-depth study of α isoform biology at both the cell biological and neural systems levels may reveal molecular and cellular approaches to improve cognition and mitigate uncontrolled excitability.

It is important to note that our interpretation that β* mouse phenotypes are most likely driven by changes in αisoforms does not preclude a fundamental role of β in sculpting neural systems, or that reduced expression of this isoform in Syngap1^+/- +/- has no role in disease pathobiology. Rather, our results highlight the importance of endogenous α isoforms in regulating excitatory synapse function and associated behavioral outcomes. What is known about the function of other C-terminal protein variants, such as β and γ? A recent study suggested that β and γ isoforms lack the ability to regulate excitatory synapse function, further strengthening the idea that α isoforms account for Syngap1-dependent regulation of excitatory synapse function (Araki et al., 2020). However, Syngap1 is known to regulate additional cellular process beyond regulation of excitatory synapse function, such as dendritic morphogenesis and patterning in vivo (Clement et al., 2012; Aceti et al., 2015; Michaelson et al., 2018). Evidence suggests that all isoforms can regulate dendritic morphogenesis in vitro, although SynGAP-β was shown to be a stronger regulator of this process relative to the other C-terminal isoforms (Araki et al., 2020). In vivo, β was found to be expressed earlier in development and to be less enriched in the postsynaptic density compared to other variants (Gou et al., 2020). Thus, β is well positioned to regulate non-synapse related neuronal processes. Future studies will be required to elucidate the specific cellular functions of non-alpha isoforms and how they contribute to the development of neural function and behavior. Given the complexities of Syngap1 regulation on dendritic morphogenesis (Aceti et al., 2015; Michaelson et al., 2018), and the direct linkage between dendritic morphogenesis and circuit function in cortex in Syngap1 mutant animals (Michaelson et al., 2018), future studies on the function of individual isoforms would ideally be carried out in vivo in developing animals.

All data generated or analysed during this study are included in the manuscript and supporting file; Source Data files have been provided for western blots and mass spec experiments.

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Author details

1. Murat Kilinc

   1. Graduate School of Chemical and Biological Sciences, The Scripps Research Institute, Jupiter, United States
   2. Departments of Neuroscience and Molecular Medicine, The Scripps Research Institute, Jupiter, United States

   Contribution

   Conceptualization, Data curation, Investigation, Methodology, Validation, Visualization, Writing - original draft, Writing - review and editing

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0000-0003-0928-7573

2. Vineet Arora

   Departments of Neuroscience and Molecular Medicine, The Scripps Research Institute, Jupiter, United States

   Contribution

   Formal analysis, Investigation, Methodology, Visualization

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0000-0001-7856-0401

3. Thomas K Creson

   Departments of Neuroscience and Molecular Medicine, The Scripps Research Institute, Jupiter, United States

   Contribution

   Investigation, Methodology, Supervision

   Competing interests

   No competing interests declared

4. Camilo Rojas

   Departments of Neuroscience and Molecular Medicine, The Scripps Research Institute, Jupiter, United States

   Contribution

   Data curation, Formal analysis, Investigation, Methodology, Visualization

   Competing interests

   No competing interests declared

5. Aliza A Le

   Department of Anatomy and Neurobiology, The University of California, Irvine, United States

   Contribution

   Formal analysis, Investigation, Methodology

   Competing interests

   No competing interests declared

6. Julie Lauterborn

   Department of Anatomy and Neurobiology, The University of California, Irvine, United States

   Contribution

   Formal analysis, Investigation, Methodology, Writing - review and editing

   Competing interests

   No competing interests declared

7. Brent Wilkinson

   Zilkha Neurogenetic Institute, Keck School of Medicine, University of Southern California, Los Angeles, United States

   Contribution

   Conceptualization, Data curation, Formal analysis

   Competing interests

   No competing interests declared

8. Nicolas Hartel

   Mork Family Department of Chemical Engineering and Materials Science, University of Southern California, Los Angeles, United States

   Contribution

   Data curation, Formal analysis, Investigation

   Competing interests

   No competing interests declared

9. Nicholas Graham

   Mork Family Department of Chemical Engineering and Materials Science, University of Southern California, Los Angeles, United States

   Contribution

   Data curation, Investigation, Visualization

   Competing interests

   No competing interests declared

10. Adrian Reich

    Bioinformatics and Statistics Core, The Scripps Research Institute, Jupiter, United States

    Contribution

    Methodology, Visualization

    Competing interests

    No competing interests declared

11. Gemma Gou

    1. Molecular Physiology of the Synapse Laboratory, Institut d'Investigació Biomèdica Sant Pau, Barcelona, Spain
    2. Universitat Autònoma de Barcelona, Bellaterra, Spain

    Contribution

    Formal analysis

    Competing interests

    No competing interests declared

12. Yoichi Araki

    Department of Neuroscience, Johns Hopkins School of Medicine, Baltimore, United States

    Contribution

    Resources, Validation

    Competing interests

    No competing interests declared

    "This ORCID iD identifies the author of this article:" 0000-0002-3455-9377

13. Àlex Bayés

    Molecular Physiology of the Synapse Laboratory, Institut d'Investigació Biomèdica Sant Pau, Barcelona, Spain

    Contribution

    Formal analysis, Investigation, Project administration, Writing - review and editing

    Competing interests

    No competing interests declared

    "This ORCID iD identifies the author of this article:" 0000-0002-5265-6306

14. Marcelo Coba

    Zilkha Neurogenetic Institute, Keck School of Medicine, University of Southern California, Los Angeles, United States

    Contribution

    Data curation, Formal analysis, Investigation, Methodology, Project administration, Visualization, Writing - review and editing

    Competing interests

    No competing interests declared

15. Gary Lynch

    Department of Anatomy and Neurobiology, The University of California, Irvine, United States

    Contribution

    Formal analysis, Investigation, Methodology, Project administration, Writing - review and editing

    Competing interests

    No competing interests declared

16. Courtney A Miller

    1. Graduate School of Chemical and Biological Sciences, The Scripps Research Institute, Jupiter, United States
    2. Departments of Neuroscience and Molecular Medicine, The Scripps Research Institute, Jupiter, United States

    Contribution

    Writing - review and editing

    Competing interests

    No competing interests declared

17. Gavin Rumbaugh

    1. Graduate School of Chemical and Biological Sciences, The Scripps Research Institute, Jupiter, United States
    2. Departments of Neuroscience and Molecular Medicine, The Scripps Research Institute, Jupiter, United States

    Contribution

    Conceptualization, Formal analysis, Funding acquisition, Investigation, Project administration, Supervision, Writing - original draft

    For correspondence

    gavin@scripps.edu

    Competing interests

    No competing interests declared

    "This ORCID iD identifies the author of this article:" 0000-0001-6360-3894

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