Information for researchers
The department of Functional Genomics (FGA) headed by Professor Matthijs Verhage has over 20 years of experience and published more than 40 peer reviewed papers dissecting the cellular function(s) of STXBP1/MUNC18-1 in the brain. FGA is part of the Center for Neurogenomics and Cognitive Research based at the Vrije Universiteit and VUmc in Amsterdam.
We have identified STXBP1 as a central factor in synaptic transmission. We have dissected at which steps(s) STXBP1/MUNC18-1 acts and provided insight into the molecular and intramolecular mechanisms of STXBP1 function. Furthermore, we have mapped several cellular pathways that regulate MUNC18-1 function, especially phosphorylation by PKC, ERK and neuronal Src family kinases. Initially, these studies were performed using mouse models. Currently, we are expanding our model systems from rodent to human neurons, allowing the analysis of the effect of STXBP1 gene defects in a human (patient) genetic background.
Munc18-1 is a key player in neurotransmission
In 2000, we discovered that in the absence of Munc18-1, synaptic vesicles cannot fuse. Synaptic vesicles are located at the synapse, but no fusion events were observed, no spontaneous events and also no response to action potential stimulation, chemical depolarization or agents that normally trigger fusion. This defect is absolute (not a single event is ever observed) and is observed in different synapses, from the CNS to the neuromuscular junction (Verhage et al., 2000). These findings showed that Munc18-1 essential for synaptic transmission.
In subsequent studies, the synaptic role of Munc18-1 was further characterized. We have exploited cell models that express Munc18-1 endogenously. i.e., primary (post-mitotic) neurons and adrenal chromaffin cells from transgenic mouse models, knockout (Munc18-1 null) or conditional knockout (Munc18-1 lox) mouse lines. In the knockout background, mutant versions of Munc18-1 were introduced to address the role of specific amino acid residues, protein modifications, such as phosphorylation, or protein domains of the protein.
Together with research teams in Göttingen, Germany (the lab of prof. Neher, dr. Klingauf and dr. Moser) and Copenhagen, Denmark (prof. Sörensen), we have provided detailed models of how Munc18-1 regulates secretory vesicle docking (Voets et al., 2001, Toonen et al., 2006, De Wit et al., 2009) and subsequently characterized, also in collaboration with a research team in Heidelberg (prof. Sollner), downstream functions in vesicle priming and in preventing de-priming (Gulyás-Kovács et al., 2007, Munch et al., 2016, He et al., 2017). Our most recent mechanistic work targets sub-molecular configurations of the protein that help vesicles to transit from the initial docking to the primed state to fusion by accommodating sequential interactions with syntaxin, SNAP25 and synaptobrevin/VAMP2 (Munch et al., 2016, Meijer et al., 2017), but maybe not after SNARE-complexes have formed (Meijer et al., 2012).
We have shown that the two known splice variants of Munc18-1 have very similar roles in the synapse, but modulate presynaptic short-term plasticity in a slightly differential manner (Meijer et al., 2015).
The amount of Munc18-1 expressed at the synapse or in a chromaffin cell is a determinant of the size of the readily releasable vesicle pool (RRP). Munc18-1 overexpression leads to a 50% increase in pool size and a concomitant delayed run down during repetitive stimulation, whereas a 50% reduction of Munc18 protein levels leads to early run down (Toonen et al., 2006, Wierda et al., 2007).
Syntaxin-1 is the main interacting partner of Munc18-1 and most of its known functions are in concert with Syntaxin-1. In the absence of Munc18-1, Syntaxin-1 levels are reduced by 70% (Toonen et al., 2005). The dependence between Munc18-1 and Syntaxin-1 is reciprocal, as Munc18-1 levels are also reduced upon Syntaxin-1 deficiency. This interdepency is not observed with other synaptic proteins (Gerber et al., 2008, Vardar et al., 2016).
Munc18-1 is a key element in adaptation and modulation of synaptic transmission
Some of the major mechanisms to modulate synaptic transmission target Munc18-1 as one of the main molecular targets for modulation. For instance the diacylglycerol (DAG)/protein kinase C (PKC) pathway known to enhance synaptic transmission and inhibition by activation of presynaptic metabotropic glutamate receptors (mGluR) or cannabinoid receptors (CB1R). Firstly, we have shown that Munc18-1 is rapidly phosphorylated by PKC in response to strong stimulation (De Vries et al., 2000). This leads to phosphorylation-dependent translocation of Munc18-1 (Cijsouw et al., 2014). In addition, PKC-dependent phosphorylation of Munc18-1 is required for DAG-dependent enhancement of synaptic transmission, probably in conjunction with activation of Munc13 (Wierda et al., 2007, Genc et al., 2014).
Secondly, we have shown that ERK-dependent phosphorylation of Munc18-1 is an essential downstream event in mGluR and CB1R activated synaptic inhibition (Schmitz et al., 2016).
Finally, we have recently shown that tyrosine phosphorylation of Munc18-1 by Src-kinases and possibly other (unknown) kinases is a powerful way to inhibit synaptic transmission (Meijer et al., 2017).
Munc18-1 is involved in many regulated secretory pathways
The evidence for the role of Munc18-1 in synaptic transmission and regulated secretion in chromaffin cells is robust and undisputed (see Toonen and Verhage., 2003, Toonen and Verhage., 2007). In addition, we have shown that Munc18-1 is required for regulated secretion from neurosecretory terminals in the anterior pituitary, resulting in a dramatic reduction in the levels of circulating pituitary hormones (Korteweg et al. 2005). The expression of the neuropeptide genes is reduced in the neocortex of Munc18-1 null mice, as revealed by microarray analysis and qPCR (Bouwman et al., 2006). In collaboration with a research team in the US (prof. Thurmond) we have shown that Munc18-1 regulates the 1st phase of insulin secretion in the pancreas (Oh et al., 2012). Hence, we conclude that Munc18-1 is important for most regulated secretory pathways in our body.
Munc18-3 (also called Munc18c) supports regulated exocytosis of glucose transporter vesicles in muscle and fat cells (Thurmond et al., 1998).
Munc18-1 plays a role in synaptic development
While membrane incorporation via exocytosis is generally considered to contribute to neurite outgrowth, we showed that Munc18-1 is not essential for outgrowth and initial synapse development, but that filopodia of outgrowing growth cones are hypermotile and the base of the cone progresses slower in the absence of Munc18-1 (Broeke et al., 2010). Munc18-1 does play a crucial role in synapse maintenance (Heeroma et al., 2003, Bouwman et al., 2004, Korteweg et al., 2004), but this may be an indirect effect, related to the role of Munc18-1 in cell viability (see below).
An separate role for Munc18-1 in cell viability
In addition to its well-described role in synaptic transmission, we have discovered that Munc18-1 plays a crucial role in neuronal survival (Verhage et al., 2000, Heeroma et al., 2004, Santos et al., 2017). Neurons that cannot express Munc18-1 die in a cell-intrinsic manner. Their survival can be extended, but not rescued by trophic factors such as insulin or BDNF (Heeroma et al., 2004). Recently, it was shown that neuronal survival in Munc18-1 null neurons is rescued by over-expressing other, non-cognate Munc18 genes, Munc18-2 and Munc18-3, that are normally expressed only at low levels in the brain. Expression of either of these genes rescues neuronal viability, but not synaptic transmission (Santos et al., 2017). This indicates that Munc18-1 has an essential, yet unknown, role in neuronal viability which is unrelated to synaptic transmission. Ongoing research in the lab is dedicated to find this essential function.
Translational & clinical research
In addition to our research on the fundamental role of STXBP1/MUNC18-1 in neurons, we also dedicate our work to understand how mutations in STXBP1 lead to STXBP1-encephalopathy. On our page for patients, you can find more information on STXBP1-E. Here, we briefly outline our current research lines concerning STXBP1-E.
Heterozygous deletion of STXBP1 in mice recapitulates symptoms shown in STXBP1-E
No correlation is apparent between the predicted detrimental effect of mutations in STXBP1 and symptom severity. One explanation for this is that all disease-causing mutations lead to reduced protein stability, resulting in haploinsufficiency (Stamberger et al., 2016). Several lines of evidence from our lab supporting this hypothesis, which are recently published in the peer-reviewed journal Brain (Kovacevic et al., 2018). In this study, in silico analyses of publically available genomics data of hundreds of thousands of humans show that pathogenic mutations in STXBP1 gene are ultra-rare, probably due to extremely strong consequences of STXBP1 mutations for normal brain development and function. Expression of STXBP1 mutations in cell culture system (in vitro) show that all mutations lead to a decreased cellular level of the STXBP1 protein, implicating protein instability as the underlying factor to explain the disease. Stxbp1 heterozygous (Stxbp1+/-) mice have one functional copy of the gene and therefore provide a valid animal model for STXBP1 haploinsufficiency. Three lines of Stxbp1+/- mice were characterized, on different genetic background or by deleting expression in GABA-ergic neurons only, and report spontaneous epilepsy-like behavior and abnormal EEG activity, remarkably similar to human patients. Interestingly, excessive neural activity was detected not in the brain regions most typically involved in seizure activity, but in the more superficial neocortical regions of the brain. This suggests that increased cortical excitability is the underlying cause of disease at least in Stxbp1+/- mice. New EEG analysis in STXBP1-encephalopathy patients will elucidate if this neocortical hyper-excitability is indeed an underlying cause in patients. In Stxbp1+/- mice the abnormal EEG activity were suppressed by antiepileptic drug levetiracetam, again similar to the situation in humans. In addition, Stxbp1+/- mice showed cognitive deficits, especially impaired behavioral flexibility: an impaired ability to suppress previously learned responses and to learn new. Taken together these mouse models are valuable tools for future therapy design because they model the situation in patients closely (construct validity), they show strikingly similar deficits (face validity) and respond in the same way to the same intervention (predictive validity).
Quantification of occurrence of symptoms and mutation sites in STXBP1-E patients
Mutations have been found in all regions of the gene, without a clear focus on particular ‘hotspots’ of the gene. STXBP1-mutations are relatively rare and most literature reports on a single or a few STXBP1-patients at a time. We aim to chart the genotype (specific mutations) and symptoms experienced by patients in a standardized manner, in order to assess whether occurrence of any particular symptoms or manifestations correlate to mutations to e.g. a specific region of the gene and corresponding protein. This could provide insight into the disease mechanisms at the cellular/molecular level and it could aid early diagnosis of future patients and prognosis.
Investigation of Excitation/Inhibition balance in patients with STXBP1-E
For optimal brain function, a delicate balance is required between excitatory and inhibitory drive in brain circuitry. Excess excitatory drive can lead to epileptic activity, whereas excess inhibition can suppress the levels of brain activity. Our work in mouse neurons that have a heterozygous deletion in Munc18-1 has shown that GABAergic (inhibitory) neurons exhibit significantly increased synaptic depression which is not observed in glutamatergic neurons (Toonen et al., 2006). This has led to the hypothesis that STXBP1 mutations, through causing haploinsufficiency, lead to a shift in E/I-balance that could lead to epileptic seizure activity in patients. Using advanced EEG technology developed at our institute, we are now able to measure E/I balance in humans. We aim to assess the E/I balance in patients with STXBP1-E and compare these to healthy controls.
Investigate pathophysiology of STXBP1-E at the cellular/molecular level
As described above, haploinsufficiency is the most likely explanation for STXBP1-E. Nevertheless, this cannot explain the wide range in observed symptoms between patients. Probably, other genetic factors have an additional effect. Therefore, we are investigating iPSC-derived induced human neurons from patients with STXBP1-E. This provides us the the unique opportunity to study the pathophysiology of STXBP1-mutations in vitro using neurons with the exact genetic background of STXBP1-E patients. We may be able to identify cellular processes that are altered as a consequence of a mutation in STXBP1, in order to gain a more thorough understanding of pathological mechanisms underlying the symptoms that patients suffer from.