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.
Authors
Christopher Patzke, Yan Han, Jason Covy, Fei Yi, Stephan Maxeiner, Marius Wernig, Thomas C. Südhof
(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.