Analysis of conditional heterozygous STXBP1 mutations in human neurons

Abstract

Heterozygous mutations in the syntaxin-binding protein 1 (STXBP1) gene, which encodes Munc18-1, a core component of the presynaptic membrane-fusion machinery, cause infantile early epileptic encephalopathy (Ohtahara syndrome), but it is unclear how a partial loss of Munc18-1 produces this severe clinical presentation. Here, we generated human ES cells designed to conditionally express heterozygous and homozygous STXBP1 loss-of-function mutations and studied isogenic WT and STXBP1-mutant human neurons derived from these conditionally mutant ES cells. We demonstrated that heterozygous STXBP1 mutations lower the levels of Munc18-1 protein and its binding partner, the t-SNARE-protein Syntaxin-1, by approximately 30% and decrease spontaneous and evoked neurotransmitter release by nearly 50%. Thus, our results confirm that using engineered human embryonic stem (ES) cells is a viable approach to studying disease-associated mutations in human neurons on a controlled genetic background, demonstrate that partial STXBP1 loss of function robustly impairs neurotransmitter release in human neurons, and suggest that heterozygous STXBP1 mutations cause early epileptic encephalopathy specifically through a presynaptic impairment.

Figure 7. Heterozygous STXBP1 mutations decrease presynaptic neurotransmitter release at synapses formed by iN cells onto cocultured mouse cortical neurons as revealed by optogenetic analysis of unitary synaptic connections.

(A) Flow diagram of optogenetic iN cell experiments using cocultured mouse neurons. Heterozygous STXBP1-mutant or WT control neurons were generated as described for Figure 1, but with coexpression of tdTomato-CHiEF. iN cells were cocultured with a large excess of mouse cortical neurons at day 7 and analyzed by patch-clamping at day 21. (B) Representative micrographs of tdTomato-CHiEF–transduced human neurons (red) that were cocultured with primary cortical mouse neurons on day 7 and analyzed on day 21. All neurons were labeled with MAP2 (green), synapsin (pink), and DAPI (blue). Scale bar: 100 μm. (C) Sample traces of light-evoked EPSCs recorded from mouse neurons. (D) Summary graphs of the amplitude of optogenetically evoked EPSCs (left) and of the coefficient of variation (right). Summary graphs show mean ± SEM; numbers of cells/independent cultures analyzed are indicated in the bars. *P < 0.05; **P < 0.01; ***P < 0.001, Student’s t test comparing heterozygous STXBP1 mutants to controls.

Figure 6. Heterozygous STXBP1 mutations decrease presynaptic neurotransmitter release at iN cell synapses as revealed by optogenetic analysis of unitary synaptic connections.

(A) Flow diagram of optogenetic iN cell experiments using sparse channelrhodopsin transfections. Heterozygous STXBP1-mutant or WT control iN cells were generated as described for Figure 1, sparsely transfected at day 21 with tdTomato-CHiEF (a derivative of channelrhodopsin-2), and analyzed by patch-clamping at day 26. For rescue experiments, rat Munc18-1 was cotransfected with channelrhodopsin at day 21. (B–D) Representative confocal micrographs of transfected iN cells expressing tdTomato-CHiEF (red); iN cells were counterstained for MAP2 (green) and synapsin (pink). Higher magnification images of the boxed areas are shown on the right (B1, WT control; B2, heterozygous STXBP1-mutant without rescue; B3, heterozygous STXBP1-mutant with rescue). Scale bars: 100 μm. (E) Schematic diagram of optogenetic analyses of unitary synaptic connections. tdTomato-CHiEF–positive presynaptic neurons were activated by short light pulses, and EPSCs were recorded from tdTomato-CHiEF–negative postsynaptic neurons. (F) Sample traces of light-evoked EPSCs. Black bar above the traces illustrates the 2-ms light pulse. (G) Summary graphs showing EPSC amplitudes (left) and their coefficient of variation (right). Graphs display mean ± SEM; number of cells/independent cultures analyzed are indicated in the bars. *P < 0.05; **P < 0.01, Student’s t test.

Figure 5. Heterozygous STXBP1 mutations impair evoked neurotransmitter release.

(A and B) Representative traces (A) and amplitude summary graphs (B) of EPSCs evoked by isolated action potentials in control and STXBP1-mutant neurons derived from 2 different ES cell clones. (C) Representative traces of EPSCs evoked by 10 Hz/10 second stimulus trains in control and heterozygous STXBP1-mutant human neurons. (D and E) Quantitative analyses of EPSCs evoked by 10 Hz stimulus trains as absolute (D) or relative amplitudes normalized to the first response (E). The amplitudes over the entire 10-second train (left panels) and over the first 10 stimuli (middle panels) are plotted as a function of stimulus number, while the average amplitudes evoked by the last 10 stimuli are shown in the right panels. Note that while heterozygous STXBP1-mutant neurons exhibit uniformly reduced absolute amplitudes, synaptic plasticity as reflected by relative amplitudes is normal. (F and G) Measurements of release induced by hypertonic sucrose to assess the size of the RRP of vesicles. Panels show representative traces (F) and summary graphs of the cumulative charge transfer as a function of time (G, left) or of the total mean charge transfer (G, right). Summary graphs show mean ± SEM; numbers of cells/independent cultures analyzed are indicated in the bars. Statistical comparisons were made by Student’s t test comparing heterozygous STXBP1 mutants to controls. *P < 0.05; **P < 0.01; ***P < 0.001.

Figure 4. Decreased spontaneous neurotransmitter release in heterozygous STXBP1-mutant human neurons.

(A and B) Impaired spontaneous neurotransmitter release in heterozygous STXBP1-mutant human neurons. Representative traces of mEPSCs recorded in 1 μM tetrodotoxin (TTX) from 2 different clones are shown on top. Summary graphs of the mEPSC parameters are shown below. Left, cumulative plot of the mEPSC interevent interval (inset: mean mEPSC frequency); right, cumulative plot of the mEPSC amplitude (inset: mean mini-amplitude). **P < 0.01, unpaired, 1-tailed Student’s t test for comparison of the means; ***P < 0.001, Kolmogorov-Smirnov test for comparison of cumulative distributions. Summary graphs exhibit mean ± SEM; numbers of cells/independent cultures analyzed are indicated in the bars.

Figure 3. Normal intrinsic electrical properties in heterozygous STXBP1-mutant neurons.

(A) Heterozygous STXBP1-mutant iN cells exhibit no changes in input resistance (left) or capacitance (right). Neurons derived from 2 different mutant ES cell clones were analyzed as indicated. (B) Representative traces of analyses of the action potential firing properties of control and heterozygous STXBP1-mutant iN cells. Neurons held in current-clamp mode were injected with increasing current pulses (10 pA increments). Experimental protocol is shown at the bottom. (C) Summary graph of the action potential firing thresholds determined in control and heterozygous mutant iN cells derived from 2 different ES cell clones. Summary graphs show mean ± SEM; numbers of cells/independent cultures analyzed are indicated in the bars.

Figure 2. Protein composition, survival, and neuronal differentiation of STXBP1-mutant human neurons.

(A) Immunoblots and Ponceau-stained blots of control and heterozygous and homozygous mutant iN cells. (B) Protein levels in matched control and independent clones of heterozygous STXBP1-mutant iN cells, determined by quantitative immunoblotting (see also Supplemental Figure 2, A and B). *P < 0.05, Student’s t test. (C) Plot of the fraction of surviving neurons compared with controls (dotted line) as a function of culture time. Tested conditions: heterozygous cells from 2 independent ES cell clones (red); homozygous cells generated with standard conditions cultured alone (green) or cocultured with WT iN cells (blue); homozygous STXBP1-mutant iN cells in which the mutation was induced 1 week after iN cell induction (black). Degeneration of homozygous STXBP1-mutant neurons is statistically highly significant under all conditions. P < 0.01, 2-way ANOVA. (D) Heterozygous STXBP1-mutant iN cells stained for the dendritic marker MAP2. Scale bar: 100 μm. (E) Total dendritic length (left), number of branches (middle), and soma size (right) quantified with control and mutant iN cells derived from 2 separate mutant ES cell clones. (F) Dendrites from control and heterozygous STXBP1-mutant iN cells stained for MAP2 and synapsin to visualize presynaptic terminals. Scale bar: 10 μm. (G) Density (left) and size (right) of synapsin-positive puncta along dendrites in heterozygous STXBP1-mutant iN cells derived from 2 independent ES cell clones. Error bars represent mean ± SEM. Numbers of independent experiments performed are indicated in the graphs.

Figure 1. Genetic engineering of conditional STXBP1 gene mutations in human ES cells and generation of iN cells from conditionally mutant ES cells.

(A) Targeting strategy. The STXBP1 gene was mutated by homologous recombination in H1 ES cells using AAVs containing the indicated sequences. Drug-resistant clones were confirmed by PCR using the primers no. 1 to no. 3. Ex 2, exon 2; red ovals, loxP sites; blue triangles, frt sites. (B) PCR analysis of WT ES cells and 2 independent heterozygous and homozygous ES cell clones. PCRs were performed with the indicated primers (see A). In this panel, Munc18-1^+/+ refers to untargeted ES cells. (C) Design of lentiviral vectors for rapid Ngn2-mediated directed differentiation of ES cells into iN cells. (D) Flow diagram of iN cell experiments. Conditionally mutant ES cells were coinfected at day –1 with the lentiviruses used for iN cell generation (shown in C) plus a lentivirus expressing either Flp-recombinase (which removes the resistance cassette and reactivates STXBP1 expression, resulting in Munc18-1^+/+ neurons) or Cre-recombinase (which deletes exon 2 of the STXBP1 gene, resulting in Munc18-1^–/+ or Munc18-1^–/– neurons). (E) Representative fluorescence images of control and mutant iN cells derived from heterozygous (top) or homozygous conditionally STXBP1-mutant ES cells (bottom). ES cells were coinfected at the day of iN cell induction with an EGFP-expressing lentivirus for visualizing neurons; pictures were taken at day 23. Scale bar: 200 μm. For additional data on the selection of neurons and more representative images, see Supplemental Figure 1.