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. 2024 May 18;33(11):991-1000.
doi: 10.1093/hmg/ddae035.

Reduced synaptic depression in human neurons carrying homozygous disease-causing STXBP1 variant L446F

Miriam Öttl  1 , Ruud F Toonen  1 , Matthijs Verhage  1   2
Affiliations

Reduced synaptic depression in human neurons carrying homozygous disease-causing STXBP1 variant L446F

Miriam Öttl et al. Hum Mol Genet. .

Abstract

MUNC18-1 is an essential protein of the regulated secretion machinery. De novo, heterozygous mutations in STXBP1, the human gene encoding this protein, lead to a severe neurodevelopmental disorder. Here, we describe the electrophysiological characteristics of a unique case of STXBP1-related disorder caused by a homozygous mutation (L446F). We engineered this mutation in induced pluripotent stem cells from a healthy donor (STXBP1LF/LF) to establish isogenic cell models. We performed morphological and electrophysiological analyses on single neurons grown on glial micro-islands. Human STXBP1LF/LF neurons displayed normal morphology and normal basal synaptic transmission but increased paired-pulse ratios and charge released, and reduced synaptic depression compared to control neurons. Immunostainings revealed normal expression levels but impaired recognition by a mutation-specific MUNC18-1 antibody. The electrophysiological gain-of-function phenotype is in line with earlier overexpression studies in Stxbp1 null mouse neurons, with some potentially human-specific features. Therefore, the present study highlights important differences between mouse and human neurons critical for the translatability of pre-clinical studies.

Keywords: CRISPR; STXBP1; electrophysiology; epilepsy; induced pluripotent stem cells.

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Figures

Figure 1
Figure 1
Selection and quality control of CRISPR/Cas9 edited iPSCs. (A) Selection workflow: After the CRISPR reaction and single-cell picking, clones were sequenced and selected based on successful edit. Then, high-quality DNA was extracted from the selected clones and qPCR on genomic DNA was performed as well as copy number variant (CNV) analysis, on which the final selection of clones was based. The inset shows the principle of genomic qPCR where, in a hemizygous clone, a loss of a gene fragment abolishes the binding of a primer, which leads to amplification of the intact gene only. This results in an allele number of 1 in the analysis, as opposed to a number of 2 when both alleles are intact. (B) Electropherograms from example clones showing the site of the mutation. The arrows point to position c.1336 which is mutated C > T, appearing homozygous in clones #2 and #5 and heterozygous in clone #6. (C) Number of alleles as calculated from qPCR on genomic DNA. Most clones show allele numbers of 2 except for #5 which is hemizygous due to an allele number of 1. Note that this clone appears homozygous from sequencing shown in B. Dots show data from 2 technical replicates. Lines show means of replicates.
Figure 2
Figure 2
STXBP1LF/LF neurons have normal morphology. (A) Example images of human single isolated glutamatergic neurons from STXBP1WT/WT, the STXBP1LF/LF clone #2 (as in Fig. 1) and the STXBP1WT/LF clone. Neurons were stained for MAP2 and synaptophysin-1 to analyze dendritic length and synapse density, respectively. Scale bar = 50 μm. (B) Total dendrite length was based on a MAP2 mask for each cell. (C) Synapse density per mm dendrite was based on the number of synaptophysin-1-positive puncta within the MAP2 mask.
Figure 3
Figure 3
The L446F mutation leads to impaired recognizability by an antibody. (A) Scheme of the MUNC18-1 protein (Uniprot ID: P61764-1) showing its functional domains. The two antibodies used for detection of MUNC18-1 are indicated with the amino acid sequences they were raised against. Antibody A is specific for the last 14 amino acids of the isoform mostly expressed in NGN2 neurons. The L446 residue (marked by a line within domain 3) lies within the epitope of antibody B which recognizes all isoforms. (B) Example images of neurons from STXBP1WT/WT, the STXBP1LF/LF clone #2 and the STXBP1WT/LF clone stained for MUNC18-1 using an antibody specific for the short isoform of MUNC18-1 (antibody A). Scale bar = 50 μm. (C) MUNC18-1 staining intensity using antibody A was measured within the soma (based on the MAP2 mask) and normalized to wildtype (WT) per experimental week. (D) MUNC18-1 staining intensity using antibody A was measured within synapses (based on the synaptophysin-1 mask) and normalized to wildtype (WT) per experimental week. (E) Example images of neurons stained for MUNC18-1 using an antibody with an epitope that covers the mutation site (antibody B). Scale bar = 50 μm. (F) MUNC18-1 staining intensity using antibody B was measured within the soma (based on the MAP2 mask) and normalized to wildtype (WT) per experimental week. (G) MUNC18-1 staining intensity using antibody B was measured within synapses (based on the synaptophysin-1 mask) and normalized to wildtype (WT). This antibody cannot detect MUNC18-1 with the L446F mutation. (H) Western blot performed using antibody A. MUNC18-1 bands were normalized to corresponding tubulin bands and then the percent of STXBP1WT/WT level was calculated per experimental week. (I) Western blot performed using antibody B. MUNC18-1 bands were normalized to corresponding tubulin bands and then the percent of STXBP1WT/WT level was calculated per experimental week. Boxplots show medians with interquartile range with whiskers extending to the most extreme data points within 1.5 times the interquartile range. One dot represents one cell, separated horizontally by experimental week. Outliers are shown as rings. Crosses show means. *P < 0.05, **P < 0.005, ***P < 0.0005.
Figure 4
Figure 4
STXBP1LF/LF neurons display normal basal synaptic transmission. (A) Example traces of gap-free recordings showing mEPSCs of the control (STXBP1WT/WT) and mutated clones (STXBP1LF/LF #2 and #3). (B) mEPSC frequency within 30 s recordings is not different between STXBP1LF/LF clones and STXBP1WT/WT. (C) mEPSC amplitudes are not different between STXBP1LF/LF clones and STXBP1WT/WT. (D) Example traces of single evoked EPSCs. (E) EPSC amplitudes of single stimulations of 1 ms (+30 mV) are not different between STXBP1LF/LF clones and STXBP1WT/WT. (F) EPSC charge of single stimulations of 1 ms (+30 mV) is not different between STXBP1LF/LF clones and STXBP1WT/WT. Boxplots show medians with interquartile range with whiskers extending to the most extreme data points within 1.5 times the interquartile range. One dot represents one cell, separated horizontally by experimental week. Outliers are shown as rings. Crosses show means.
Figure 5
Figure 5
Less synaptic depression in STXBP1LF/LF neurons. (A) Example traces of paired stimulations with 50 ms inter-pulse intervals. Dotted lines level at the peaks of each first amplitude. (B) The paired-pulse ratio (PPR) with 50 ms interval was calculated by dividing the second amplitude by the first. Both STXBP1LF/LF clones show significantly higher PPRs compared to STXBP1WT/WT. (C) Summary of paired-pulse ratios with different inter-pulse intervals (IPIs). (D) Example traces of recordings with 100 stimulations (1 ms to +30 mV) at 40 Hz. The first 10 pulses were additionally magnified (magnified part marked by a dashed box). (E) Absolute EPSC amplitudes from recordings exemplified in D were plotted by pulse number to visualize the rundown of release over time. The inset shows the same plot only for the first 10 pulses as marked by the dashed box. (F) Normalized EPSC amplitudes from recordings exemplified in D were plotted by pulse number to visualize the rundown of release over time. The inset shows the same plot only for the first 10 pulses as marked by the dashed box. (G) Total amount of charge released within the first 10 pulses of the 40 Hz stimulation as exemplified in D. In B and G, boxplots show medians with interquartile range with whiskers extending to the most extreme data points within 1.5 times the interquartile range. One dot represents one cell, separated horizontally by experimental week. Outliers are shown as rings. Crosses show means. In C, E and F, data are presented as means ± SEM. *P < 0.025, **P < 0.005.
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References

    1. Rizo J. Molecular mechanisms underlying neurotransmitter release. Annu Rev Biophys 2022;51:377–408. - PMC - PubMed
    1. Toonen RFG, Verhage M. Munc18-1 in secretion: lonely Munc joins SNARE team and takes control. Trends Neurosci 2007;30:564–72. - PubMed
    1. Dudok JJ, Groffen AJA, Toonen RFT. et al. . Deletion of Munc18-1 in 5-HT neurons results in rapid degeneration of the 5-HT system and early postnatal lethality. PLoS One 2011;6:e28137. - PMC - PubMed
    1. Verhage M, Maia AS, Plomp JJ. et al. . Synaptic assembly of the brain in the absence of neurotransmitter secretion. Science 2000;287:864–9. - PubMed
    1. Toonen RFG, Wierda K, Sons MS. et al. . Munc18-1 expression levels control synapse recovery by regulating readily releasable pool size. Proc Natl Acad Sci USA 2006;103:18332–7. - PMC - PubMed

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