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
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.