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) Immunoblots and Ponceau-stained blots of control and heterozygous and homozygous mutant iN cells. (B) Protein levels in matched control and independent clones of heterozygous STXBP1-mutant iN cells, determined by quantitative immunoblotting (see also Supplemental Figure 2, A and B). *P < 0.05, Student’s t test. (C) Plot of the fraction of surviving neurons compared with controls (dotted line) as a function of culture time. Tested conditions: heterozygous cells from 2 independent ES cell clones (red); homozygous cells generated with standard conditions cultured alone (green) or cocultured with WT iN cells (blue); homozygous STXBP1-mutant iN cells in which the mutation was induced 1 week after iN cell induction (black). Degeneration of homozygous STXBP1-mutant neurons is statistically highly significant under all conditions. P < 0.01, 2-way ANOVA. (D) Heterozygous STXBP1-mutant iN cells stained for the dendritic marker MAP2. Scale bar: 100 μm. (E) Total dendritic length (left), number of branches (middle), and soma size (right) quantified with control and mutant iN cells derived from 2 separate mutant ES cell clones. (F) Dendrites from control and heterozygous STXBP1-mutant iN cells stained for MAP2 and synapsin to visualize presynaptic terminals. Scale bar: 10 μm. (G) Density (left) and size (right) of synapsin-positive puncta along dendrites in heterozygous STXBP1-mutant iN cells derived from 2 independent ES cell clones. Error bars represent mean ± SEM. Numbers of independent experiments performed are indicated in the graphs.