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) 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.