Protein instability, haploinsufficiency, and cortical hyper-excitability underlie STXBP1 encephalopathy

Abstract

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.

Figure 1

Morphological and electrophysiological characteristics of dissociated hippocampal neurons expressing the human disease variants in Stxbp1 null mouse neurons. (A) Schematic overview of some of the previously discovered STXBP1 truncations, deletions and missense mutations in human patients showing the seven mutations tested in colour-coded boxes. (B) Dissociated cortical neurons were stained for Munc18-1, dendritic marker MAP2 and synaptic marker synaptobrevin (VAMP). Examples represent Stxbp1 null neurons expressing wild-type Munc18-1 (WT), C180Y or M433R. (C–G) Morphological and synaptic characteristics of Munc18-1 wild-type neurons, Stxbp1^+/− neurons and null mutant neurons expressing one of the human disease variants: (C) mean synapse density calculated as the ratio of synapse number and total dendritic length. (D) Mean soma Munc18-1 level. (E) Mean synaptic Munc18-1 level. (F and G) Mean somatic and synaptic syntaxin-1 level. (H) Example traces of evoked release from wild-type neurons, Stxbp1 null neurons expressing C180Y and M433R upon a single action potential stimulation. (I) Mean EPSC amplitude expressed as the ratio of the wild-type value. (J) Paired-pulse ratio (calculated as the ratio of second EPSC and first EPSC) depending on the pulse interval. (K) Frequency of spontaneous release normalized to the wild-type values. (L). Amplitude of spontaneous release normalized to the wild-type values. (M) Example traces of spontaneous release in Stxbp1 null neurons expressing Munc18-1 wild-type, C180Y or M433R human variants. (N) The size of the readily releasable pool derived from back extrapolation of cumulative total charge released during 40 Hz train, 100 APs. The number of analysed cells is indicated in the bars. *P < 0.05, **P < 0.01, ***P < 0.001 versus infected wild-type control. Explanation of statistical analysis is provided in the Supplementary material. mEPSC = mini excitatory postsynaptic current; RRP = readily releasable pool.

Figure 2

Morphological and electrophysiological characteristics of dissociated hippocampal neurons expressing the human disease variants in Stxbp1^+/− mouse neurons. (A) Dissociated cortical neurons were stained for Munc18-1 and synaptic marker synaptobrevin (VAMP). Examples represent Stxbp1^+/− neurons expressing wild-type Munc18-1 (WT), C180Y or M433R human variants. (B–D) Morphological and synaptic characteristics of Stxbp1^+/− neurons expressing wild-type Munc18-1 or one of the human disease variants: (B) mean synapse density: calculated as the ratio of synapse number and total dendritic length. (C and D) Mean somatic and synaptic Munc18-1 level. (E) Example traces of evoked release upon a single action potential stimulation from Stxbp1^+/− neurons expressing wild-type Munc18-1, C180Y, M433R, V84D, G544D or R388X human variants. (F) Mean EPSC amplitude expressed as the ratio of the infected wild-type values. (G) Paired-pulse ratio (second EPSC/first EPSC) depending on the pulse interval. (H) Readily releasable pool estimate derived from back-extrapolation of cumulative total charge released during 40 Hz train, 100 APs (I) Frequency of spontaneous release expressed as the ratio of infected wild-type values. (J) Amplitude of spontaneous release expressed as the ratio of infected wild-type values. (K) Example traces of spontaneous release in Stxbp1^+/− neurons expressing wild-type Munc18-1, C180Y, M433R, G544D, R388X or V84D human variants. The number of analysed cells is indicated in the bars. ***P < 0.001 versus infected wild-type control. mEPSC = mini excitatory postsynaptic current; RRP = readily releasable pool.

Figure 3

Cellular stability of wild-type and human disease variants of Munc18-1 in HEK293 cells and neurons. (A) Immunochemistry of HEK293 cells infected with wild-type Munc18-1 (WT), C180Y, M443R, C522R and T574P constructs stained for Munc18-1, EGFP and Golgi marker (GM130). (B) Normalized Munc18-1 levels in HEK293 cells after viral infection with wild-type, C180Y, M443R, C522R and T574P constructs. The inset shows representative western blot of HEK293 cells after viral infection; n = 5, 5, 5, 2 and 2, respectively. (C) Western blot analysis of Munc18-1 protein levels 0, 6, 12, 24 and 30 h after block of protein synthesis with cycloheximide for HEK293 cells infected with wild-type, C180Y, M433R, C522R or T574P constructs. The infection with wild-type construct was used as a control for all performed western blot analysis. (D) Quantitative analysis of the Munc18-1 protein expression from western blots in HEK cells represented in C. (E) Western blot analysis of Munc18-1 protein levels 0, 12, 24 and 36 h after block of protein synthesis with cycloheximide for wild-type, C180Y, M433R, C522R or T574P constructs in Stxbp1 null neurons. The infection with wild-type construct was used as a control for all performed western blot analysis. (F) Quantitative analysis of the Munc18-1 protein expression from western blots in neurons represented in E. (G–I) Normalized Munc18-1 protein levels from three constructs expressed in HEK cells treated with MG132, Leupeptin or Pepstatin; n = 3, 2 and 2, respectively.

Figure 4

Generation, genomic analysis, routine behavioural observation and spontaneous activity of Stxbp1^+/− mice. (A–D) Generations of four lines of Stxbp1^+/− mice: conditional, congenic BL6, reverse 129Sv and Gad2- Stxbp1 line. Chromosomes bearing Stxbp1 mutation originating from the C57BL/6J genetic background are shown in grey, and those from the 129/SvJ genetic background are shown in orange. Stxbp1 mutation is represented with grey triangle; LoxP sites are represented with black rectangles and Cre-deleter lines are represented with scissors (red scissors: Cre expressed in all neuron, blue scissors: Gad2tm2-Cre mice with Cre-recombinase expressed in only GABAergic neurons). The flanking gene region is represented as orange region in the grey chromosome in the congenic BL6 line and vice versa in the reverse 129Sv line (adapted from Wolfer et al., 2002). (E and F) High-density genomic analysis showed the size and position of flanking gene region in Stxbp1^+/− samples from congenic BL6 and reverse 129Sv lines. ‘Me-PaMuFind-It’ web tool (http://me-pamufind-it.org/) revealed three genes with passenger mutations from 129Sv genetic background within flanking genes region in congenic BL6 Stxbp1^+/− samples. (G) Stxbp1^+/− mice showed lower body weight compared to their controls. (H) Grip strength was normal in Stxbp1^+/− mice. (I) Motor coordination and motor learning in Stxbp1^+/− mice was normal, as assessed on the rotarod. (J and K) Circadian rhythm assessed by changes in the activity in anticipation of (filled bars), and response to (open bars), day/night transitions and proportion of time spent outside the shelter was normal in Stxbp1^+/− mice. (L) Proportion of activity duration during the first 3 h of the dark phase days in the home-cage environment (PhenoTyper) showed increased activity of Stxbp1^+/− mice from all three lines during the first dark phase. (M) Proportion of activity duration in the PhenoTyper during the light phases was overall lower for Stxbp1^+/− mice (conditional mice, congenic BL6 and reverse 129Sv). The number of animals assigned is shown in the graphs. *P < 0.05; ***P < 0.001.

Figure 5

Video and EEG recordings revealed epileptic-like events in Stxbp1^+/− mice. (A) Video monitoring revealed sudden jerks referred as twitches in Stxbp1^+/− mice. (B) Video monitoring revealed sudden jumps in Stxbp1^+/− mice, sometimes accompanied by Straub tail responses previously reported as a common phenomenon observed after seizure onset (Wagnon et al., 2015). (C) Distribution of motor effects of epileptic-like neural activity (twitches and jumps) during 3-h video-monitoring in Stxbp1^+/− mice from three lines: floxed, congenic BL6 and reverse 129Sv line. These motor events were never found in control mice (Stxbp1^+/+). (D) Monitoring for 24 h showed that most of the twitches and jumps in congenic BL6 Stxbp1^+/− mouse occurred during the light phase (twitch: 22/34 and jump: 2/3) of the day/night cycle. (E) Average number of behavioural epileptic-like events per hour per line of Stxbp1^+/− mice. (F) Positions of the recording electrodes and ground electrode relative to Bregma. (G) Representative example of ECoG traces in a congenic BL6 Stxbp1^+/− mouse during the slow-wave sleep, awake state and spike-wave discharges. The red trace is an expanded ECoG trace of spike-wave discharge. (H) Power spectrum of slow wave sleep (SWS), spike wave discharge (SWD) and wake state. (I) Occurrence of behavioural epileptic events (twitches and jumps) and spike-wave discharges detected in cortical and hippocampal EEG traces during 3-h recording in Stxbp1^cre/+ and congenic BL6 Stxbp1^+/− mice. (J) Predicted probability of coincidence of 3/13 twitches and 1/7 jumps with spike-wave discharge detected in a representative congenic BL6 Stxbp1^+/− mouse. Probability lower than 5% (grey line) was considered as concurrence. (K) Probability of concurrence of behavioural epileptic events and spike-wave discharges within 10 s presented as a negative logarithm for Stxbp1^cre/+ and congenic BL6 Stxbp1^+/− mice. (L) Total number of detected spike-wave discharges and epileptic events in the 12-h light phase. The number of mice: three Stxbp1^cre/+ mice, four congenic BL6 Stxbp1^+/− mice for cortical recording and two congenic BL6 Stxbp1^+/− mice for hippocampal recording. (M) Average frequency of detected spike-wave discharges during 6 h of recording after administration of saline, first levetiracetam dose (LEV I: 50 mg/kg, i.p.) and fifth levetiracetam dose (LEV V: 5 days, 50 mg/kg per day, i.p.). Different greyscale circles represent individual mice. *P < 0.05, **P < 0.01 compared to saline administration.

Figure 6

c-Fos expression in Stxbp1^+/− mice. (A–R) Representatives of c-Fos expression in prefrontal cortex (PFC), primary motor cortex (mCx) and somatosensory cortex (ssCx) for Stxbp1^+/+ mice (control), Stxbp1^cre/+ and congenic BL6 Stxbp1^+/− mice. (S) Number of c-Fos positive cells per brain region for Stxbp1^+/+, Stxbp1^cre/+, and congenic BL6 Stxbp1^+/− mice. Scale bars and the number of samples are provided in the figure.

Figure 7

Learning and memory in Stxbp1^+/− mice in classical spatial paradigms and recently developed automated tasks in the PhenoTyper. (A and B) Latency and distance travelled to find the escape hole in the Barnes maze during the learning phase for congenic BL6 Stxbp1^+/− mice. Congenic BL6 Stxbp1^+/− (HZ BL6) showed longer latency to find new escape hole during the learning phase (P = 0.026) and during R1 (P < 0.001) and travelled longer distance to the new escape hole (R1-R3: P = 0.024) compared to their controls (WT BL6). (C) HZ BL6 mice showed narrower distribution of holes visit around the target hole during the first probe trial (P1) compared to wild-type BL6. (D) Probability of hole visits in the old target octant during the P1 and P2 tended to be higher for HZ BL6 mice compared to their controls (P = 0.086 and P = 0.060, respectively) and there were no differences in the probability of hole visits in the new target octant during the P2. (E and F) Latency and distance travelled to find the escape hole in the Barnes maze during the learning phase were similar for Stxbp1^cre/+ mice (HZ cond) and their controls (wild-type cond). (G and H) Escape latency and distance travelled to the platform during the training in the Morris water maze was similar for reverse 129Sv Stxbp1^+/− mice and control mice. (I) Time spent per quadrant during the probe trial was similar for reverse 129Sv Stxbp1^+/− mice and control mice. (J) Schematic overview of the CognitionWall DL/RL task. (K and L) Kaplan-Meier survival curves shows the fraction of congenic BL6 and conditional Stxbp1 mice that reached the 80% criterion as a function of hole entries during the DL and RL phases. (M) Average number of entries made per group to reach 80% criterion during the DL and RL phases. HZ BL6 mice reached the 80% criterion during RL with lower number of entries compared to control (P = 0.004). (N) Schematic overview of the Shelter task protocol in the PhenoTyper to assess avoidance learning. (O and P) The preference index during the dark phases of the avoidance learning task was similar for HZ BL6 and HZ cond mice and their controls. Insets represent the learning effect on the preference index (D5/D6/D7-D4). The insets in graph O shows that congenic BL6 Stxbp1^+/− mice showed a stronger learning effect compared to their controls (P = 0.012). (Q and R) The aversion index during the dark phases of avoidance learning task showed trend toward lower values for HZ BL6 mice compared to their controls (P = 0.101). The aversion index was similar between HZ cond mice and their controls. *P < 0.05; **P < 0.01; ***P < 0.001 compared to respective control. Statistical analysis is explained in the Supplementary material.

Figure 8

Anxiety-related phenotype and social behaviour in Stxbp1^+/− mice. (A) Schematic overview of the PhenoTyper home-cage. (B) On shelter duration during the dark phase was shorter for Stxbp1^cre/+ and congenic BL6 Stxbp1^+/− (HZ cond and HZ BL6) mice compared to their controls. (C–F) Elevated plus maze (EPM) data. (C) Schematic overview of the EPM. (D–F) Latency to enter open arms was longer, time spent on the open arms and number of visits to the open arms were lower for Stxbp1^cre/+ and congenic BL6 Stxbp1^+/− mice compared to their controls. (G–J) Dark-light box (DLB) data. (G) Schematic overview of the DLB. (H) Latency to visit bright compartment (BC) was longer for congenic BL6 Stxbp1^+/− mice compared to their controls. (I–J) Time spent in the bright compartment and number of visits to the bright compartment was lower for Stxbp1^cre/+ and congenic BL6 Stxbp1^+/− mice compared to their controls. (K–O) Open field data. (K) Schematic overview of the open field. (L) Latency to visit centre zone was longer for Stxbp1^cre/+ and congenic BL- Stxbp1^+/− mice compared to their controls. (M and N) Percentage of distance moved in the centre zone and number of visits to centre zone was similar for Stxbp1^cre/+ and congenic BL6 Stxbp1^+/− mice and respective control mice. (O) The total distance moved was longer for reverse 129Sv Stxbp1^+/− mice compared to their control, and it was similar for Stxbp1^cre/+ and congenic BL6 Stxbp1^+/− mice and respective control mice. (P and Q) Novelty induced hypophagia test (NIHP) data. (P) Schematic overview of the NIHP test. (Q) Latency to consume highly palatable food (cracker) was similar for Stxbp1^cre/+ and congenic BL6 Stxbp1^+/− mice and the respective controls. (R) Schematic overview of three-chamber test protocol. (S) During test for sociability (phase III), Stxbp1^cre/+, congenic BL6 Stxbp1^+/− mice and their respective controls spent more time in the mouse zone than in the object containing zone. (T) During test for social novelty (phase IV), Stxbp1^cre/+, congenic BL6 Stxbp1^+/− mice and their respective controls spent more time in the zone with novel mouse than in the zone with familiar mouse. There were no differences between genotypes. *P < 0.05, **P < 0.01, ***P < 0.001 for comparison between zones.