Abstract
Brill J, Clarke B, Hong I, Huganir R. Proc Natl Acad Sci USA. 2025;122(18): e2427288122. SYNGAP1 is a key Ras-GAP protein enriched at excitatory synapses, with mutations causing intellectual disability and epilepsy in humans. Recent studies have revealed that in addition to its role as a negative regulator of G-protein signaling through its GAP enzymatic activity, SYNGAP1 plays an important structural role through its interaction with postsynaptic density proteins. Here, we reveal that intrinsic excitability deficits and seizure phenotypes in heterozygous Syngap1 knockout (KO) mice are differentially dependent on Syngap1 GAP activity. Cortical excitatory neurons in heterozygous KO mice displayed reduced intrinsic excitability, including lower input resistance, and increased rheobase, a phenotype recapitulated in GAP-deficient Syngap1 mutants. However, seizure severity and susceptibility to pentylenetetrazol (PTZ)-induced seizures were significantly elevated in heterozygous KO mice but unaffected in GAP-deficient mutants, implicating the structural rather than enzymatic role of Syngap1 in seizure regulation. These findings highlight the complex interplay between SYNGAP1 structural and catalytic functions in neuronal physiology and disease.
Commentary
Epilepsies can arise from a wide variety of aberrations in excitation-inhibition imbalance. These are regulated at many checkpoints. One place where this balance is regulated is at the synapse. The synapse is a complex, dynamic structure permitting cells to communicate with one another. It is comprised of hundreds of molecules that subserve many different functions. One of the most abundant proteins in excitatory synapses is the synaptic GTPase-activating protein (SYNGAP1). SYNGAP1 is known to have both enzymatic activity and a structural synapse-stabilizing role in the synapse. There is a seemingly ever-growing list of genes that can be mutated to lead to neurodevelopmental disorders and developmental epileptic encephalopathies. These occur early in life and tend to be associated with exceptionally challenging cognitive and behavior dysfunction and hard to control seizures. Syngap1 is one of these genes. Mutations in Syngap1 lead to neurodevelopmental disorders characterized by intellectual disability, autism, behavioral abnormalities, and epilepsy. 1 Which of the functions of the protein are responsible for which of the phenotypes is not known. Better discerning this would importantly inform potential therapeutics.
Previous work attempting to understand the role of SYNGAP1 employed Syngap1 knockout mice. Since the whole gene is deleted in this model, both the enzymatic and structural functions of SYNGAP1 are impacted. Homozygous knockout mice die early in the perinatal period. 2 Patients with Syngap1 dysfunction display behavioral phenotypes and spontaneous seizures. Heterozygous mutant mice show similar behavioral phenotypes and reduced seizure threshold. 2 In an attempt to evaluate the enzymatic and structural components of SYNGAP1, the authors developed a mouse model with a 2-amino acid substitution in the GTPase domain-encoding region of Syngap1. This impairs the enzymatic function, but not the structural role of SYNGAP1. 3
Using this mouse model, they performed whole-cell recording from somatosensory cortex slices and demonstrated that selective impairment of the GTPase activity led to reduced excitability of layer 2/3 glutamatergic pyramidal neurons in somatosensory cortex slices compared to those from phenotypically wildtype littermates. 4 This was evidenced by reduced input resistance and an increase in the lowest current injection required to elicit spiking, or rheobase, and was consistent with what had been shown for haploinsufficient Syngap1 mice. This suggests that the enzymatic activity of SYNGAP1 drives the intrinsic excitability of cortical neurons. Given that neuronal excitability evolves considerably throughout development the authors were careful to age-match their experimental and control animals.
Previously, this group demonstrated that the structural role of SYNGAP1 dictates the cognitive and behavioral phenotypes seen in these mice. 3 To examine the effect on seizure susceptibility, mice were subjected to seizure induction with the inhibitory gamma-aminobutyric receptor A (GABAA) antagonist, pentylenetetrazol. Syngap1 haploinsufficient mice were robustly sensitive to seizure-induction with pentylenetetrazol, confirming previous findings in these mice. Mice with selective-impairment of Syngap1's GTPase activity did not demonstrate heightened seizure-susceptibility, suggesting that this is not subserved by enzymatic activity, but is more likely subserved by the synapse-stabilizing structural function of SYNGAP1. Neither the haploinsufficient Syngap1 mutant nor the specific mutant with impaired GTPase activity displayed spontaneous seizures as many of the patients with Syngap1 mutations do. Spontaneous seizures and reduced seizure thresholds, however, can be difficult to establish in genetic mouse models of epilepsy. 5 It might prove useful to evaluate additional seizure-induction methods that target alternative mechanisms. Also, in this study seizures were assessed behaviorally, and not via electroencephalogram, which could be more sensitive for detecting spontaneous electrographic seizures.
The authors discuss that it is somewhat counterintuitive that Syngap1 haploinsufficient mice that have reduced cortical neuron excitability should also have heightened seizure susceptibility. They attribute this to seizures being a network phenomenon, and that while the cortical pyramidal neurons they examined demonstrated reduced intrinsic excitability, the overall effect in the circuits comprising the network could be to enhance seizure susceptibility. More work examining neurons in other cell types and anatomical loci involved in the seizure-genic network would be needed to determine where the specific effect was being manifested.
Differentiating the distinct means by which a protein can affect a given system is important when considering therapeutics. In the specific case of SYNGAP1, there are drugs that could target the small regulatory proteins (eg, Ras) that are the target of SYNGAP1's GTPase activity. This work clearly demonstrates that while such an approach might improve the intrinsic excitability of cortical neurons, it would be unlikely to alter seizure susceptibility. More targeted therapies, such as genetic therapies would be needed for this. 6 Given the prior work of this group, such a genetic approach would also be expected to improve cognitive and behavioral consequences of the gene mutation 3 and thus could have a much more profound impact on individuals with the disease.
The structural role of SYNGAP1 is mediated through the PDZ domain of postsynaptic density 95 (PSD-95). If the seizure-susceptibility phenotype is due to the structural roles of SYNGAP1 then perhaps developing pharmaceuticals to target the PDZ domain of PSD-95, and stabilizing the synapse would be beneficial. This approach of targeting one or more of the many PDZ domain proteins is employed in experimental treatment of several cancers. 7 However, in this study, the authors only speculate that since eliminating the GTPase-activating activity of SYNGAP1 did not result in increased seizure susceptibility, that enhanced proclivity to seizures is likely subserved by the structural properties of SYNGAP1. It would potentially be valuable to manipulate the structural components of SYNGAP1 activity and determine whether this would itself lead to increased seizure susceptibility.
PSD-95 is important in proper function of the excitatory α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors. Indeed, drugs such as perampanel, which work through inhibition of AMPA receptors, have been used in Syngap1 mutant mouse models 8 and in some Syngap1 patients. 9 This suggests that modulating upstream targets such as the PDZ domain with genetic therapies could have antiepileptogenic effects. This could potentially be done pharmacologically or genetically. Genetic therapy would potentially have the added benefit of not requiring regular daily dosing with antiseizure or other medications.
Additional work would be needed to understand if there would be other pharmaceutically reachable therapeutics that would affect the underlying epileptic network in these mice. Similar approaches couple be taken in the wide variety of additional genetic epilepsies to try to determine alternate therapies.
