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 iN cell synapses as revealed by optogenetic analysis of unitary synaptic connections.
(A) Flow diagram of optogenetic iN cell experiments using sparse channelrhodopsin transfections. Heterozygous STXBP1-mutant or WT control iN cells were generated as described for Figure 1, sparsely transfected at day 21 with tdTomato-CHiEF (a derivative of channelrhodopsin-2), and analyzed by patch-clamping at day 26. For rescue experiments, rat Munc18-1 was cotransfected with channelrhodopsin at day 21. (B–D) Representative confocal micrographs of transfected iN cells expressing tdTomato-CHiEF (red); iN cells were counterstained for MAP2 (green) and synapsin (pink). Higher magnification images of the boxed areas are shown on the right (B1, WT control; B2, heterozygous STXBP1-mutant without rescue; B3, heterozygous STXBP1-mutant with rescue). Scale bars: 100 μm. (E) Schematic diagram of optogenetic analyses of unitary synaptic connections. tdTomato-CHiEF–positive presynaptic neurons were activated by short light pulses, and EPSCs were recorded from tdTomato-CHiEF–negative postsynaptic neurons. (F) Sample traces of light-evoked EPSCs. Black bar above the traces illustrates the 2-ms light pulse. (G) Summary graphs showing EPSC amplitudes (left) and their coefficient of variation (right). Graphs display mean ± SEM; number of cells/independent cultures analyzed are indicated in the bars. *P < 0.05; **P < 0.01, Student’s t test.