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 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.