Abstract
Kovacevic J, Maroteaux G, Schut D, Loos M, Dubey M, Pitsch J, Remmelink E, Koopmans B, Crowley J, Cornelisse LN, Sullivan PF, Schoch S, Toonen RF, Stiedl O, Verhage M. Brain 2018;141:1350–1374.
De novo heterozygous mutations in STXBP1/Munc18-1 cause early infantile epileptic encephalopathies (EIEE4, OMIM #612164) characterized by infantile epilepsy, developmental delay, intellectual disability, and can include autistic features. We characterized the cellular deficits for an allelic series of seven STXBP1 mutations and developed four mouse models that recapitulate the abnormal EEG activity and cognitive aspects of human STXBP1-encephalopathy. Disease-causing STXBP1 variants supported synaptic transmission to a variable extent on a null background, but had no effect when overexpressed on a heterozygous background. All disease variants had severely decreased protein levels. Together, these cellular studies suggest that impaired protein stability and STXBP1 haploinsufficiency explain STXBP1-encephalopathy and that, therefore, Stxbp1+/− mice provide a valid mouse model. Simultaneous video and EEG recordings revealed that Stxbp1+/− mice with different genomic backgrounds recapitulate the seizure/spasm phenotype observed in humans, characterized by myoclonic jerks and spike-wave discharges that were suppressed by the antiepileptic drug levetiracetam. Mice heterozygous for Stxbp1 in GABAergic neurons only, showed impaired viability, 50% died within 2–3 weeks, and the rest showed stronger epileptic activity. c-Fos staining implicated neocortical areas, but not other brain regions, as the seizure foci. Stxbp1+/− mice showed impaired cognitive performance, hyperactivity and anxiety-like behaviour, without altered social behaviour. Taken together, these data demonstrate the construct, face and predictive validity of Stxbp1+/− mice and point to protein instability, haploinsufficiency and imbalanced excitation in neocortex, as the underlying mechanism of STXBP1-encephalopathy. The mouse models reported here are valid models for development of therapeutic interventions targeting STXBP1-encephalopathy.
Commentary
Variants in genes encoding for proteins that perform essential presynaptic functions have recently emerged as causes of a range of human epilepsies. Of these genes, STXBP1 variants have a particularly strong impact on human health, because 1) they can cause several of the most severe and early-onset forms of epileptic encephalopathies; 2) they are one of the top three causes of epileptic encephalopathies, accounting for approximately 1.4% of the incidence; and 3) almost all patients have severe to profound intellectual disability (1). In rodent models, Stxbp1, also known as Munc18-1, is essential for neurotransmitter release and is a core component of the synaptic vesicle fusion machinery, and animals with biallelic deletion of Stxbp1 do not survive (2, 3). Because of its essential role in synaptic physiology, the molecular and cellular functions of Stxbp1 have been extensively studied, and its protein interactions and role in vesicle docking and fusion are well documented (4). Despite this extensive effort, how STXBP1 variants cause disease phenotypes is not well understood, which is mainly because a thorough description of a mouse model of STXBP1 encephalopathy has not been reported. This is somewhat surprising, because a mouse carrying an Stxbp1-null allele was published in 2000 (2); however, it was not until 2008 that variants in STXBP1 were first reported to cause Ohtahara syndrome in five individuals (5). Then, 5 years later, several larger scale human genetic studies showed that STXBP1 variants were responsible for a range of neurodevelopmental diseases (6–8); thus, elevating its importance and providing a strong motivation to develop an appropriate mouse model to study STXBP1-related encephalopathy.
Because the majority of STXBP1 variants are truncating mutations, it has been assumed that haploinsufficiency is the genetic mechanism, as opposed to gain-of-function or dominant-negative. To confirm this assumption and strengthen the claim that Stxbp1+/− mice are an appropriate disease model, the authors of this paper first performed a series of experiments in which they expressed several missense human variants in cultured neurons from either Stxbp1+/− or Stxbp1−/− mice. These variants had no effect on synaptic transmission or morphology in Stxbp1+/− neurons, suggesting that gain-of-function or dominant-negative mechanisms are unlikely, and were generally unable to restore wild-type levels of synaptic performance to Stxbp1−/− neurons, suggesting that they are loss-of-function variants. Further experiments in heterologous cells then showed that the protein levels of the STXBP1 variants were lower than that of wild-type STXBP1, even when controlling for mRNA expression, suggesting that the mutant proteins are not as stable as wild type. These experiments confirmed that haploinsufficiency is the genetic mechanism, and that Stxbp1+/− mice are a construct-valid disease model.
Next, they tested three different Stxbp1+/− mouse lines to determine whether they displayed phenotypes consistent with STXBP1 encephalopathy. One line was a floxed allele on a C57BL/6J background, the second was a deletion allele on a C57BL/6J background that retained a very small portion of 129S1/Sv genomic DNA flanking the deletion area, and the third was a deletion allele on a mixed 129S1/SvImJ, C57BL/6J background. All three lines were viable and fertile, and, although Stxbp1+/− mice weighed slightly less, showed no general abnormalities in motor performance, spontaneous activity, or diurnal behavior.
Epileptiform activity, of course, was a big question, so the authors performed video EEG recordings. A single electrode placed over the motor cortex showed spike wave discharges that lasted 1.5 to 2 seconds in all three lines, which were significantly correlated with twitches from the video. The authors point out that this abnormal motor and EEG activity in the Stxbp1+/− mouse lines resembled tonic spasms and myoclonic jerks previously reported in human patients, as does the fact that they occurred predominantly during rest/sleep. They also treated one of the lines with levetiracetam, a drug to which a small number of STXBP1 patients have responded (1), and observed a significant reduction in the number of spike wave discharges. Finally, the authors performed extensive behavioral analyses of their models. The Stxbp1+/− models showed altered behavioral flexibility and increased spontaneous motor activity and anxiety, but not altered spatial learning or impairment of sociability. While there were some minor behavioral differences in the three lines, they all showed the core features of epileptiform activity, altered behavioral flexibility and increased spontaneous motor activity, providing strong evidence that these are direct effects of Stxbp1 loss and not due to strain differences.
One of the most interesting results of this study was that when the floxed allele was crossed to a Gad2-Cre line to delete one copy of Stxbp1 in GABAergic neurons, many animals died between P14 and P21 and had more severe epileptiform EEG activity than the globally Stxbp1 haploinsufficient mice. This finding is similar to a previously published report in an Scn1a model, where deletion of Scn1a in GABAergic neurons driven by Vgat-Cre caused earlier lethality and more severe seizures than global deletion (9). Thus, although a reduction in Stxbp1 levels impairs neurotransmitter release in both GABAergic and glutamatergic neurons, the most damaging effects to the organism are those in GABAergic neurons, and importantly, the reduction in release at glutamatergic neurons in the global animals is protective against the more severe excitatory-inhibitory imbalance seen in the Gad2-Cre line.
How well do the Stxbp1+/− mouse lines model STXBP1-related epileptic encephalopathy? The presence of analogous epileptiform activity on the EEG in a heterozygous animal is encouraging, as is the presence of behavioral abnormalities that are consistent with some patient symptoms. On the other hand, STXBP1 patients tend to have severe intellectual disability, whereas the mouse cognitive phenotype is on the mild side. STXBP1 encephalopathy in humans is known to exhibit an unusually large phenotypic spectrum, so it is not surprising that these mouse models recapitulate some, but not all, of the key features of the human disease. Future studies of heterozygous mice on different genetic backgrounds, or of mice carrying genomic alterations that are analogous to those found in human patients, may prove valuable in modeling other facets of the disease.
Although this study presents a thorough characterization of the behavioral effects of Stxbp1 haploinsufficiency, and investigates the neurophysiological effects via EEG, there is still a long way to go to understand the mechanisms through which STXBP1 variants cause seizures and cognitive impairments, and to devise new treatment strategies. Based on the nature of the Stxbp1 protein, and data from this study and a previous study on synaptic transmission in cultured Stxbp1+/− neurons (10), a good starting point will be a comprehensive assessment of the dysfunction at GABAergic synapses induced by Stxbp1 loss-of-function. The physiological nature of this dysfunction, and the types of GABAergic neurons and circuits affected, are all open questions. Moreover, although STXBP1 encephalopathies are thought to manifest early in life, this study used adult mice for most of their experiments. What the developmental effects of these gene variants are, and whether they are reversible later in life, are key questions to understanding and treating STXBP1-associated diseases.