Analysis of conditional heterozygous STXBP1 mutations in human neurons
Christopher Patzke, … , Marius Wernig, Thomas C. Südhof
Christopher Patzke, … , Marius Wernig, Thomas C. Südhof
Published August 17, 2015
Citation Information: J Clin Invest. 2015;125(9):3560-3571. https://doi.org/10.1172/JCI78612.
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Research Article Neuroscience

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.

Authors

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

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Figure 5

Heterozygous STXBP1 mutations impair evoked neurotransmitter release.

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Heterozygous STXBP1 mutations impair evoked neurotransmitter release. (A...
(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.