Endogenous Syngap1 alpha splice forms promote cognitive function and seizure protection
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Evaluation Summary:
Given the well-known importance of the SYNGAP1 mutations in the pathophysiology of neurodevelopmental disorders and the key regulatory roles of SynGAP1 for excitatory synaptic functions, this study provides timely and comprehensive sets of data supporting the in vivo functions of individual SynGAP1 splice variants, including the alpha-1/2 variants, and suggests the therapeutic potential of increasing specific SynGAP1-alpha variants.
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Abstract
Loss-of-function variants in SYNGAP1 cause a developmental encephalopathy defined by cognitive impairment, autistic features, and epilepsy. SYNGAP1 splicing leads to expression of distinct functional protein isoforms. Splicing imparts multiple cellular functions of SynGAP proteins through coding of distinct C-terminal motifs. However, it remains unknown how these different splice sequences function in vivo to regulate neuronal function and behavior. Reduced expression of SynGAP-α1/2 C-terminal splice variants in mice caused severe phenotypes, including reduced survival, impaired learning, and reduced seizure latency. In contrast, upregulation of α1/2 expression improved learning and increased seizure latency. Mice expressing α1-specific mutations, which disrupted SynGAP cellular functions without altering protein expression, promoted seizure, disrupted synapse plasticity, and impaired learning. These findings demonstrate that endogenous SynGAP isoforms with α1/2 spliced sequences promote cognitive function and impart seizure protection. Regulation of SynGAP-αexpression or function may be a viable therapeutic strategy to broadly improve cognitive function and mitigate seizure.
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Evaluation Summary:
Given the well-known importance of the SYNGAP1 mutations in the pathophysiology of neurodevelopmental disorders and the key regulatory roles of SynGAP1 for excitatory synaptic functions, this study provides timely and comprehensive sets of data supporting the in vivo functions of individual SynGAP1 splice variants, including the alpha-1/2 variants, and suggests the therapeutic potential of increasing specific SynGAP1-alpha variants.
(This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. The reviewers remained anonymous to the authors.)
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Reviewer #1 (Public Review):
This study reports that different splice variants of SynGAP1 differentially regulate cognitive function and seizure protection in mice. The authors used three different mouse lines and systematically analyzed the expression patterns of different SynGAP1 splice variants in the brain and their impacts on cognitive function and seizure protection. The authors also analyzed excitatory synaptic transmission and synaptic plasticity in these mice and attempted proteomic analyses to better understand the underlying mechanisms. The overall conclusion of the study is that the SynGAP1-alpha variants are more important than the SynGAP1-beta variants for the regulation of cognitive function and seizure protection in mice.
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Reviewer #2 (Public Review):
Murat Kilinc et al. studied behavioral phenotypes of three Syngap1 mutant mice in order to identify the roles of distinct SYNGAP C-terminal variants. The first mutant mouse (IRES-TD mouse) with insertion of IRES-TDtomato cassette in the last exon (exon21) showed elevated open field activity, reduced time in the induction of seizure by flurothyl, less freezing behavior in remote contextual fear memory recall, and increase in frequency and amplitude of mEPSCs in L2/3 somatosensory neurons. In addition, IRES-TD mice showed a reduction in total SynGAP protein, alpha1, and alpha2 isoforms and increased beta isoform in the forebrain. The second mutant mouse (beta* mouse) with a mutation in the early splice acceptor of exon19 showed a slight reduction in open field activity, increase in time for seizure induction, …
Reviewer #2 (Public Review):
Murat Kilinc et al. studied behavioral phenotypes of three Syngap1 mutant mice in order to identify the roles of distinct SYNGAP C-terminal variants. The first mutant mouse (IRES-TD mouse) with insertion of IRES-TDtomato cassette in the last exon (exon21) showed elevated open field activity, reduced time in the induction of seizure by flurothyl, less freezing behavior in remote contextual fear memory recall, and increase in frequency and amplitude of mEPSCs in L2/3 somatosensory neurons. In addition, IRES-TD mice showed a reduction in total SynGAP protein, alpha1, and alpha2 isoforms and increased beta isoform in the forebrain. The second mutant mouse (beta* mouse) with a mutation in the early splice acceptor of exon19 showed a slight reduction in open field activity, increase in time for seizure induction, no change in freezing behavior in remote contextual memory recall, and decrease in frequency and amplitude of mEPSCs in L2/3 somatosensory neurons. Beta* mice showed no change in total SynGAP protein, a modest increase in alpha2, and a reduction in beta in the forebrain. The third mutant mouse (PBM mouse) has point mutations in exon21 for disrupting the function of the PDZ-binding motif (PBM) of SynGAP to interact with PSD95. This mutant showed elevated open field activity, reduced time in the induction of seizure, less freezing behavior in remote contextual memory recall, and increased frequency and amplitude of mEPSCs in L2/3 somatosensory neurons. PBM mice showed no change in total SynGAP, alpha2, and beta proteins but reduced immunoblot signal of alpha1 in the forebrain. This reduced immunoblot signal was proposed to be related to less sensitivity of the alpha1 specific antibody to the mutated isoform. By thorough behavioral and physiological analyses of three independent mutant mouse lines, the authors conclude that the level of alpha isoforms, rather than the total SynGAP, is critical in several neural circuit functions related to cognition and seizure protections. Previous studies revealed multiple roles of SynGAP in neural circuit development and activity-dependent regulations. This new study provides important information about the isoform-specific functions of SynGAP, especially the critical role of alpha isoform content for proper synapse and circuit activity.
The results obtained from multiple mutant mouse lines support the key claims of the paper. Still, there are several points that may require more experimental data or otherwise changing the interpretation of the data.
1. One of the key messages of this manuscript is the amount of alpha isoforms, but not the total amount of SynGAP, is critical in multiple behavioral phenotypes in mutant mice. In order to discuss the importance of isoforms, essential information is a relative abundance of each isoform in both wild-type and mutant mice. Throughout this manuscript, the comparisons within isoforms were performed, but no information about relative abundance between isoforms was provided. It is also unclear there are any differences in the relative abundance of isoforms in different brain regions studied in this manuscript, including the forebrain, hippocampus, and somatosensory cortex. The estimate of relative abundance should be provided, or any technical difficulty in this estimation should be explained.
2. The phenotypic similarity between IRES-TD mice and PBM mutant mice suggests critical roles of alpha isoforms in multiple behavioral phenotypes. However, the effect of PBM mutant still needs to be carefully evaluated. If alpha1 is the only isoform expressed in the brain, the interpretation of introducing PSD95-nonbinding mutant is straightforward, but in the presence of other isoforms, dominant-negative effects of PBM mutant should be considered. It is possible that PBM mutant complexed with different isoforms impair localization or function of other isoforms, leading to an effect similar to the reduction of total SynGAP protein. Clarification of this point by additional experimental data is necessary. For example, in Figure 6A, did the PSD fraction from the PBM mutant contain a similar amount of alpha2 and beta isoforms? Also, in Figure 7, native PSD95 complexes from Syngap1PBM mutants were characterized. In the PSD95 complexes, was the amount of other SynGAP isoforms unchanged?
3. The authors selected remote contextual fear conditioning coupled with protein analysis in the forebrain. This selection may be based on the motivation of finding deficits in forebrain-related long-lasting memory in mutant mice. It is unclear why the authors selected Morris water maze as the second behavioral test, which is not designed to evaluate long-lasting memory. The rationale behind selecting two distinct memory-related behavioral tests, remote contextual memory test and water maze-based training performance, should be explained. In addition, the phenotype in the Morris water maze was difficult to interpret. SynGAP heterozygote mice show impairment, but IRES-TD mice showed no change. This is inconsistent with other behavioral tests. Beta* mice showed improvement in the training session, but the 24 h probe test did not show a difference. It is difficult to connect the data with the result of contextual fear memory. This point should be discussed.
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Reviewer #3 (Public Review):
SYNGAP1 is one of the most prominent risk genes for a number of neurodevelopmental disorders, including autism, intellectual disability, epilepsy and schizophrenia. However, its biological roles are still being uncovered. One aspect of this complexity is the role of its many splice variants. In this manuscript, the authors have created and analyzed several genetically modified mouse lines in which they altered the expression levels, and protein interaction sites in a subset of the isoforms of the Syngap1 protein. They then performed an extensive analysis of the mRNA and protein levels of all isoforms, behavioral and physiological phenotypes in mice, assessed seizure susceptibility, and examined cellular and molecular alterations in synapses in these mutant mice. They found that reduced expression of the …
Reviewer #3 (Public Review):
SYNGAP1 is one of the most prominent risk genes for a number of neurodevelopmental disorders, including autism, intellectual disability, epilepsy and schizophrenia. However, its biological roles are still being uncovered. One aspect of this complexity is the role of its many splice variants. In this manuscript, the authors have created and analyzed several genetically modified mouse lines in which they altered the expression levels, and protein interaction sites in a subset of the isoforms of the Syngap1 protein. They then performed an extensive analysis of the mRNA and protein levels of all isoforms, behavioral and physiological phenotypes in mice, assessed seizure susceptibility, and examined cellular and molecular alterations in synapses in these mutant mice. They found that reduced expression of the alpha forms was damaging, while their increased expression was protective in mice.
This work is likely to be very impactful on the field, because it makes an important step forward in clarifying the roles of specific splice variants in physiological and behavioral processes relevant for neurodevelopmental disorders. One important implication of this work could be that increasing the expression of the alpha form using genetic tools, such as gene therapy, could have beneficial therapeutic effects in human patients.
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