Reduced MUNC18-1 Levels, Synaptic Proteome Changes, and Altered Network Activity in STXBP1-Related Disorder Patient Neurons

Abstract

Background: STXBP1-related disorder (STXBP1-RD) is a neurodevelopmental disorder caused by pathogenic variants in the STXBP1 gene. Its gene product MUNC18-1 organizes synaptic vesicle exocytosis and is essential for synaptic transmission. Patients present with developmental delay, intellectual disability, and/or epileptic seizures, with high clinical heterogeneity. To date, the cellular deficits of neurons of patients with STXBP1-RD are unknown.

Methods: We combined live-cell imaging, electrophysiology, confocal microscopy, and mass spectrometry proteomics to characterize cellular phenotypes of induced pluripotent stem cell-derived neurons from 6 patients with STXBP1-RD, capturing shared features as well as phenotypic diversity among patients.

Results: Neurons from all patients showed normal in vitro development, morphology, and synapse formation, but reduced MUNC18-1 RNA and protein levels. In addition, a proteome-wide screen identified dysregulation of proteins related to synapse function and RNA processes. Neuronal networks showed shared as well as patient-specific phenotypes in activity frequency, network irregularity, and synchronicity, especially when networks were challenged by increasing excitability. No shared effects were observed in synapse physiology of single neurons except for a few patient-specific phenotypes. Similarities between functional and proteome phenotypes suggested 2 patient clusters, not explained by gene variant type.

Conclusions: Together, these data show that decreased MUNC18-1 levels, dysregulation of synaptic proteins, and altered network activity are shared cellular phenotypes of STXBP1-RD. The 2 patient clusters suggest distinctive pathobiology among subgroups of patients, providing a plausible explanation for the clinical heterogeneity. This phenotypic spectrum provides a framework for future validation studies and therapy design for STXBP1-RD.

Keywords: MUNC18-1; Neurodevelopmental disorder; SNAREopathy; STXBP1-related disorder; Synaptic transmission; iPSC.

Figure 1

Study design to discover cellular phenotypes in induced neurons derived from patients with STXBP1-related disorder (STXBP1-RD). (A) Overview of STXBP1-RD variants. The STXBP1 gene is depicted; black stripes represent published disease variants, and red stripes show variants included in this study: 2 missense variants D207G and D262V, 2 nonsense variants Arg235X and Lys308X, one frameshift S241fs, and one intronic variant c.1359+5G>C (hereinafter referred to as intronic). Exons and introns are not depicted for clarity. (B) Study design. iPSCs were generated from 6 STXBP1-RD patients and 3 healthy control individuals. (C) Summary table of the clinical phenotypes of the included STXBP1-RD patients. Severity of clinical symptoms is indicated by shading (black = severe; gray = intermediate; white = mild). Detailed information is provided in Table S1 in Supplement 1. (D) Schematic presentation of the experimental design. iPSCs were induced to glutamatergic induced neurons via forced expression of NGN2. After 39–42 days in culture, induced neurons were examined by confocal microscopy, liquid chromatography–tandem mass spectrometry proteomics, patch clamp electrophysiology, and calcium imaging. Sporadically, technical failures prevented analyses of individual patient lines, as indicated by a red X (see main text). For each parameter measured in this study, overarching effects between control individuals and patients were assessed by comparing pooled measurements from all control and all patient lines, while accounting for the multilevel (nested) data structure (see Methods and Materials). In addition, patient-specific differences were assessed by comparing measurements from each patient line separately with the pooled measurements from the control lines, again accounting for the multilevel structure. C, control; iPSC, induced pluripotent stem cell; N/A, not applicable; P, patient; qPCR, quantitative polymerase chain reaction; Spec., spectrometry.

Figure 2

Neuronal morphology and synapse formation are normal in patient-derived induced neurons. (A) Typical examples of single induced neurons from each control and STXBP1-related disorder (STXBP1-RD) line stained for dendritic marker (MAP2), presynapse marker (synaptophysin) and postsynaptic PSD-95. Scale bar = 100 μm; scale bar zoom = 20 μm. (B) Dendritic length was not different between control lines and STXBP1-RD induced neuron lines. n/N = 57–96/3–6. (C) Soma area was significantly smaller for STXBP1-RD induced neurons at group level. n/N = 57–96/3–6. (D) Density of presynaptic puncta, labeled by synaptophysin immunostaining, was not different between control and STXBP1-RD induced neuron lines. n/N = 53–99/3–6. (E) Density of postsynaptic puncta, labeled by PSD-95 immunostaining, was not different between control and STXBP1-RD induced neuron lines. n/N = 53–99/3–6. Data in panels (B–E) are presented in Tukey plots, where data outside of 1.5 times the interquartile range are plotted individually. Statistical details are listed in Table S2 in Supplement 1. ∗∗p < .01. n/N: number of neurons/number of independent replicates. C, control; n.s., not significant.

Figure 3

MUNC18-1 protein and RNA levels are reduced in STXBP1-related disorder (STXBP1-RD) induced neurons. (A) Typical examples of induced neurons stained for MAP2 (dendritic marker), synaptophysin (synaptic marker), and MUNC18-1. Scale bar = 100 μm. (B) (Left panel) Neuronal MUNC18-1 levels were reduced in STXBP1-RD induced neurons compared with control induced neurons. (Right panel) Significant reductions of MUNC18-1 levels were observed for D207G and S241fs induced neurons. All intensities were acquired with the same detection settings. Afterward average intensities were normalized to the average of the 3 control lines. (C) (Left panel) STXBP1-RD induced neurons showed reduced levels of synaptic MUNC18-1. (Right panel) At the individual patient level, no significant effects were observed. (D) MUNC18-1 levels were quantified by immunoblotting and normalized to the levels of gamma-tubulin. STXBP1-RD cultures showed reduced total MUNC18-1 levels. (E) (Left panel) STXBP1 RNA levels (using STXBP1 3′ primers) were lower in STXBP1-RD induced neurons compared with control induced neurons. (Right panel) Significantly reduced levels were observed for S241fs and R235Q. (F) Syntaxin-1A RNA levels were not different in the group-level comparison between STXBP1-RD and control induced neurons. (G)STXBP1-RD induced neurons did not show lower syntaxin-1B RNA levels. (H) Group-level SNAP25 RNA levels were not different in STXBP1-RD induced neurons. Data on the left of panels (B–E) and all of panel (F) are presented in Tukey plots, where data outside of 1.5 times the interquartile range are plotted individually. Data on the right of panels (B) and (C) are presented in Tukey plots with dots representing individual neurons; in panels (D) and (E) individual data points are shown. Immunocytochemistry n/N = 98–130/5; Western blot N: 2–10; quantitative polymerase chain reaction N: 4–5. ∗p < .05, ∗∗p < .01, ∗∗∗p < .001, #p < .1. Statistical details are listed in Table S2 in Supplement 1. n/N: number of neurons/number of independent replicates. A.U., arbitrary unit; C, control; Norm., normal; n.s., not significant; SYP, synaptophysin.

Figure 4

Proteins related to synaptic and RNA biological processes are most severely affected in STXBP1-related disorder (STXBP1-RD) induced neurons. (A) PCA of peptide abundance levels performed on all detected proteins showing PC1 (18.3% of variance explained) and PC2 (16.1% of variance explained) of STXBP1-RD and control induced neurons. N = 3 independent replicates. (B) Volcano plot showing 176 (92 downregulated, 84 upregulated) proteins significantly regulated in STXBP1-RD induced neurons compared with control lines. STXBP1/MUNC18-1 was one among the top regulated proteins. (C) Heatmap visualizing patient-specific log₂-FC of the 176 significant proteins. Directionality and effect sizes of regulated proteins were comparable across patients. Fold changes were capped at −1.5 and 1.5 for visualization. Hierarchical clustering was used to visualize patient subgroups based on similarity in log₂-FC. (D) Functional annotation of the significant hits (minimum 5 proteins per term, minimum 5% of the GO term) covered 58 proteins. The number of proteins associated with every GO term are shown. GO terms related to the synapse and RNA processes were most prominent. (E) Of the significant proteins, 44 were annotated in SynGO, of which 32 were categorized in SynGO biological processes. A sunburst plot with color-coded gene counts of every GO term (including child terms) is shown. (F) Group-level log₂-FC for the 44 SynGO proteins. (G) Top row shows the number of significantly regulated proteins in 5 STXBP1-RD induced neuron lines. Functional GO enrichment of patient-level contrasts is shown below. The number of proteins associated with significantly enriched GO biological processes is depicted for every individual line, color coded by the percentage of all regulated proteins per line. (H) Proteomic similarity matrix. Coefficients of determination (R²) were calculated between every STXBP1-RD pair, based on significantly regulated proteins in at least one of the 2 lines. The upper part of graph shows R² values, and lower part shows scatterplots of the log₂-FC of the included proteins. Red line indicates a robust regression fit. (I) Heatmap visualizing log₂-FC of proteins that were in the top 100 significant proteins in at least one of the STXBP1-RD induced neuron lines. Fold changes were capped at −1 and 1 for visualization. Hierarchical clustering was used to visualize patient subgroups based on similarity in log₂-FC. C, control; cotranslat., cotranslation; ER, endoplasmic reticulum; FC, fold change; FDR, false discovery rate; GO, Gene Ontology; mRNA, messenger RNA; Neg, negative; PCA, principal component analysis; presyn, presynaptic; SRP-dep, signal recognition particle–dependent.

Figure 5

No overarching synaptic changes in single STXBP1-related disorder (STXBP1-RD) induced neurons. (A) Single induced neurons are grown on micro-islands of pregrown glial cells so that the induced neuron forms synapses onto itself. Thus, a single patch pipette can be used to simultaneously stimulate presynaptically and record the postsynaptic response. (B) Typical example traces of spontaneous synaptic activity in control and STXBP1-RD induced neurons. (C) No group-level differences were observed in mEPSC amplitude (for graphs showing the data per patient line, see Figure S5C in Supplement 1). n/N = 10–52/3–6. (D) No group-level differences were observed in mEPSC frequency (for graphs showing the data per patient line, see Figure S5B in Supplement 1). n/N = 8–35/3–6. (E) Typical traces of evoked EPSCs of STXBP1-RD and control induced neuron lines. (F) (Left panel) EPSC amplitude was not different in STXBP1-RD induced neurons at the group level. (Right panel) At the patient-specific level, intronic induced neurons had a significantly higher EPSC amplitude. n/N = 26–61/3–6. (G) (Left panel) No group-level differences were observed in paired-pulse ratio. (Right panel) R235X induced neurons showed a significantly higher paired-pulse ratio compared with control induced neurons. n/N = 27–57/3–6. (H) Typical traces of a stimulation paradigm of 5 action potentials at 5 Hz, followed by a single pulse 2 seconds following the end of the train. Examples for control line C3 (gray) and patient line R235X (red) are shown. (I) (Left panel) No group-level differences were observed between patient and control lines in synaptic depression in response to a 5 Hz train. However, induced neurons with R235X variant showed a significantly higher synaptic depression ratio compared with control neurons. n/N = 21–55/3–6. (J) Linear discriminant analysis using all electrophysiological parameters had an accuracy of 55% [n.s. according to (40)] to discriminate between STXBP1-RD and control induced neurons. (K) Linear discriminant analysis using all electrophysiological parameters had an accuracy of 22% [n.s. according to (40)] to predict line identity. Linear discriminants 1 and 2 are shown. Data in panels (C) and (D) and the left of panels (F), (G), and (I) are presented in Tukey plots, where data outside of 1.5 times the interquartile range are plotted individually. Data on the right of panels (F), (G), and (I) are presented in Tukey plots with dots representing individual neurons. ∗p < .05, #p < .1. Statistical details are listed in Table S2 in Supplement 1. n/N: number of neurons/number of independent replicates. C, control; LDA, linear discriminant analysis; mEPSC, miniature excitatory postsynaptic current; n.s., not significant.

Figure 6

Altered burst activity and reduced synchronicity in networks of STXBP1-related disorder (STXBP1-RD) induced neurons. (A) Induced neuron networks were incubated with Fluo-4 AM and imaged at 8 Hz for 5 minutes at baseline, and 5 minutes after 4-AP administration to study network activity dynamics. (B) Typical example of induced neuron network loaded with Fluo-4 AM dye. Fluorescence increases on calcium influx. Scale bar = 100 μm. (C) Activity matrix of induced neuron network at baseline and after 4-AP incubation. Fluorescent signal traces of single induced neurons (gray) and average (black) over time (x-axis) are shown. Highly synchronous activity between induced neurons was observed. 4-AP increases event frequency. (D) Typical examples of control line C1, patient line D207G, and intronic induced neuron networks at baseline conditions. (Top panel) Black dots represent the start of events in single neurons (y-axis) over time (x-axis). (Bottom panel) Summation of events in individual neurons (y-axis) across one induced neuron network over time (x-axis). Peaks above the green threshold represent synchronous network events. (E) Burst frequency was not different at the group level. In patient-level comparisons, an increase in baseline burst frequency was observed for D207G, R235X, S241fs, and R235Q induced neuron networks compared with control networks. Intronic networks showed significantly reduced burst frequency (for group-level graph, see Figure S7A in Supplement 1). (F) CoV of interburst intervals was significantly higher in STXBP1-RD induced neuron networks. In patient-level comparisons, no significant effects were found (for group-level graph, see Figure S7C in Supplement 1). (G) No significant difference was found for mean participation between STXBP1-RD and control networks in group-level comparison. Mean participation at baseline was significantly reduced in R235Q and intronic induced neurons (for group-level graph, see Figure S8A in Supplement 1). (H) CoV of participation at baseline was significantly increased in STXBP1-RD induced neuron networks, which was also significant in D207G, S241fs, R235Q, and intronic induced neuron networks in patient-level comparisons (for group-level graph, see Figure S8C in Supplement 1). (I) Typical examples of control line C1, patient line S241fs, and intronic induced neuron networks after 4-AP application. (Top panel) Black dots represent the start of events in single neurons (y-axis) over time (x-axis). (Bottom panel) Summation of events in individual neurons (y-axis) across one induced neuron network over time (x-axis). Peaks above the green threshold represent synchronous network events. (J) Group-level comparison revealed increased burst frequency for STXBP1-RD induced neuron networks. In patient-level comparisons, R235Q and intronic induced neuron networks showed increased burst frequency compared with control induced neuron networks in 4-AP conditions (for group-level graph, see Figure S7I in Supplement 1). (K) Fold change of burst frequency from baseline to 4-AP was not different in group-level comparison. Patient-level comparison showed significant increases for R235Q and intronic induced neuron networks compared with control networks. (L) CoV of interburst intervals in 4-AP conditions was significantly increased in STXBP1-RD induced neuron networks. In patient-level comparisons, significance was reached for R235X, S241fs, and R235Q induced neuron networks (for group-level graph, see Figure S7L in Supplement 1). (M) Mean participation was reduced in STXBP1-RD induced neuron networks. In patient-level comparisons, D207G, R235X, S241fs, and R235Q networks showed significantly reduced mean participation (for group-level graph, see Figure S8E in Supplement 1). (N) The fraction of neurons participating in all network events (participation index = 1) was significantly lower in STXBP1-RD induced neuron networks (for patient-level graph, see Figure S8F in Supplement 1). (O) CoV of participation in 4-AP was significantly increased in D207G, R235X, S241fs, R235Q, and intronic induced neuron networks. (P) Linear discriminant analysis using all network parameters (including all in Figures S7 and S8 in Supplement 1) had an accuracy of 100% to discriminate between STXBP1-RD and control induced neurons. (Q) Linear discriminant analysis using all network parameters (including all in Figures S7 and S8 in Supplement 1) had an accuracy of 55% to predict line identity. Shown are linear discriminants 1 and 2. (R) Coefficients of determination (R²) were calculated between every STXBP1-RD pair, based on all network parameters. R² values ranged between 0.07 and 0.86. Data in panels (E–H) and (J–O) are presented in Tukey plots with dots representing individual networks. Group-level significance (i.e., overarching effects between control and patient groups) are indicated below graph title. Baseline n/N = 16–19/5. 4-AP n/N = 12–18/5. ∗p < .05, ∗∗p < .01, ∗∗∗p < .001, #p < .1. Statistical details are listed in Table S2 in Supplement 1. n/N: number of neurons/number of independent replicates. 4-AP, 4-aminopyridine; C, control; CoV, coefficient of variation; LDA, linear discriminant analysis; n.s., not significant.