Article Figures & Data
Figures
- Figure 1.
Cell type-specific transcripts related to synapse function, immune system, lipid metabolism, and actin organization are changed in Stxbp1 KO hippocampi. A, Cartoon of experimental design. E18 hippocampi were dissected from WT and Stxbp1 KO brains, RNA was isolated, and RNAseq was performed. Typical examples of WT and KO E18 hippocampi are shown in Extended Data Figure 1-1. B, PCA of transcript abundance levels showing PC1 (78% variance explained) and PC2 (10% variance explained) of control and Stxbp1 KO samples. N = 4 independent replicates. Two outliers were excluded from analysis, see Extended Data Figure 1-2. C, Volcano plot showing 2123 transcripts (906 downregulated, 1217 upregulated) significantly dysregulated in Stxbp1 KO hippocampi from controls. D, Several significant transcripts were validated using qPCR. Shown are Log2 Fold changes of transcripts using RNAseq (x-axis) and qPCR (y-axis). Green dotted line indicates correlation of 1. N = 6 independent replicates. Effect sizes were highly comparable for the included transcripts, except for Stxbp1. This is explained by the fact that despite the deletion of exons 2–6 in Stxbp1, a (nonfunctional) transcript is transcribed that is not detected by the qPCR primers (targeted within the deleted region). qPCR primers can be found in Extended Data Table 1-1. E, Triangle plot showing cell specificity using the Barres RNAseq database. Downregulated transcripts are depicted in blue, upregulated transcripts in orange. F, Bar graphs showing cell specificity using BrainRich. Upregulated transcripts are shown left, downregulated transcripts right. G, GO enrichment analysis of the significant hits. Shown are the Bonferroni correct p-values and the number of transcripts associated with every GO term. H, Triangle plots showing cell specificity of transcripts associated with GO term groups using the Barres RNAseq database.
- Figure 2.
Proteins related to synapse function and neuron development are most severely affected in primary Stxbp1 KO neurons. A, Cartoon of experimental design. Primary neuronal cultures were generated from E18 WT and Stxbp1 KO cortices. Neurons were harvested at DIV2 and DIV3 and analyzed using mass spectrometry. Quality control measures are shown in Extended Data Figure 2-1. B, PCA of peptide abundance levels showing PC1 (19.7% variance explained) and PC2 (16.5% variance explained). N = 6 independent replicates. C, Volcano plots showing significantly dysregulated proteins in Stxbp1 KO neurons at DIV2 (142 proteins, 82 downregulated, 60 upregulated) and DIV3 (399 proteins, 233 downregulated, 166 upregulated). Volcano plots of regulated proteins between DIV2 and DIV3 are shown in Extended Data Figure 2-2. D, Triangle plot showing cell specificity using the Barres RNAseq database. Downregulated transcripts are depicted in blue, upregulated transcripts in orange. E, Overlap of significant proteins between DIV2 and DIV3. A total of 28 proteins were only significant at DIV2, 114 proteins significant at DIV2 and DIV3, and 285 only significant at DIV3. F, FDR p-values at DIV2 and DIV3. For 379 proteins, p-values increased at DIV3, for 48 proteins p-values decreased. G, Log2 fold changes at DIV2 and DIV3. Downregulated proteins are depicted in blue, upregulated in orange. H, GO enrichment analysis of the significant hits. Shown are the Bonferroni correct p-values and the number of transcripts associated with every GO term. The proteins in significant GO terms can be found in Extended Data Table 2-1. I, Overlap of proteins involved in enriched GO terms between DIV2 and DIV3.
- Figure 3.
Dysregulated proteins in Stxbp1 KO neurons at DIV3 show minimal overlap with cell death. A, Overlap of significant proteins at DIV3 and proteins significantly regulated during apoptosis (Arntzen and Thiede, 2012). A total of 94 proteins were unique for apoptosis (38 not detected in Stxbp1 KO dataset), five proteins shared between the two datasets, and 379 unique for Stxbp1 KO neurons. B, Overlap of significant proteins at DIV3 and proteins annotated to apoptosis in the KEGG pathway database. Of the 109 proteins annotated to apoptosis, 13 were detected in Stxbp1 KO neurons. Two proteins were significantly downregulated. C, Overlap of significant proteins at DIV3 and dysregulated proteins on excitotoxicity (Hoque et al., 2019). A total of 147 proteins were unique for excitotoxicity neuronal cell death (35 not detected in Stxbp1 KO dataset), 35 proteins shared between the two datasets, and 349 unique for Stxbp1 KO neurons. As for this analysis, only first gene names are used, resulting in 15 duplicates, the total number of significant Stxbp1 KO proteins is lower (384 vs 399 in Fig. 2C). D, GO enrichment analysis of significant dysregulated proteins on excitotoxicity. Shown are the Bonferroni correct p-values and number of proteins associated with each GO term. E, Overlap of significant proteins at DIV3 and proteins significantly regulated during cell death (Arntzen and Thiede, 2012). A total of 985 proteins were unique for cell death (268 not detected in Stxbp1 KO dataset), 115 proteins shared between the two datasets, and 269 unique for Stxbp1 KO neurons. F, GO enrichment analysis of significant dysregulated proteins on excitotoxicity. Shown are the Bonferroni correct p-values and number of proteins associated with each GO term. Detailed information on the overlap can be found in Extended Data Table 3-1.
- Figure 4.
Depletion of MUNC18-1 leads to downregulation of 114 synaptic proteins enriched in presynaptic vesicle release. A, A total of 114 of the significant proteins in Stxbp1 KO neurons were annotated in SynGO. Eight synaptic proteins were only significant at DIV2, 43 synaptic proteins significant at DIV2 and DIV3, and 63 synaptic proteins only significant at DIV3. Overlap to the RNAseq SynGO genes is shown in Extended Data Figure 4-1. B, FDR p-values of significant synaptic proteins at DIV2 and DIV3. For 106 synaptic proteins p-values increased at DIV3, for eight synaptic proteins p-values decreased. C, A total of 85 synaptic proteins were categorized in SynGO biological processes. Shown is a sunburst plot with color-coded enrichment significance of every GO term. Left, SynGO annotation of MUNC18-1 in blue (including its parent terms). Right, Significant proteins associated with synaptic vesicle exocytosis. The SynGO protein list can be found in Extended Data Table 4-1. D, Log2 fold changes of synaptic proteins within SynGO terms. Downregulated proteins are depicted in blue, upregulated proteins in orange. E, Visual representation of affected STXBP1 interactors identified by string database. Only high confidence interactors, derived from experiments and databases, were included. Affected interactors are depicted in red, unaffected interactors in green and undetected interactors in gray. F, Visual representation of affected STXBP1 interactors identified by co-IP experiments. Co-IP correlation strength to MUNC18-1 is depicted in purple gradient. Undetected interactors are depicted in gray, unaffected interactors in white, and the log2 Fold Change of affected interactors is depicted in blue-orange gradient. G, Overlap of significant proteins at DIV3 and proteins significantly regulated during synaptic silence (24-h TTX treatment) or overactivation (24-h bicuculine treatment; Schanzenbächer et al., 2018). Nine proteins are regulated on TTX treatment as well as on depletion of MUNC18-1, whereas bicuculine treatment shares one protein with Stxbp1 KO neurons. As for this analysis only first gene names are used, resulting in 15 duplicates, the total number of significant Stxbp1 KO proteins is lower (384 vs 399 in Fig. 2C).
- Figure 5.
Downregulated proteins in Stxbp1 KO neurons show high overlap to proteins normally upregulated during this developmental stage. A, A total of 96 proteins annotated to development are dysregulated in Stxbp1 KO neurons. Three developmental proteins were only significant at DIV2, 30 developmental proteins significant at DIV2 and DIV3, and 63 development proteins only significant at DIV3. B, Log2 fold changes of proteins associated with development. Downregulated proteins are depicted in blue, upregulated proteins in orange. C, Overlap in proteins annotated in SynGO and GO development. A total of 49 proteins were only annotated to development, 47 proteins to both development and SynGO, and 74 proteins were only annotated in SynGO. D, All individual GO terms that were included in the neuron development cluster. Shown are the Bonferroni correct p-values. E, GO enrichment analysis of significant upregulated proteins between DIV1 and DIV5 (Frese et al., 2017). Shown are the Bonferroni correct p-values. GO term clusters chemical synaptic transmission and neuron development were shared with Stxbp1 KO neurons. F, Overlap of significant proteins at DIV3 and proteins significantly upregulated between DIV1 and DIV5 (i.e., Frese dataset). A total of 653 proteins were unique for the Frese dataset, 108 proteins shared between the two datasets, and 114 unique for Stxbp1 KO neurons. Of the 108 proteins shared, 32 were annotated to GO development and 61 in SynGO. As for this analysis, only first gene names are used, resulting in 15 duplicates, the total number of significant Stxbp1 KO proteins is lower (384 vs 399 in Fig. 2C). G, 167 proteins in the Frese dataset were categorized in SynGO biological processes. Shown is a sunburst plot with color-coded enrichment significance of every GO term. Right are depicted all proteins in the Frese dataset associated with synaptic vesicle exocytosis, color-coded to dysregulation in Stxbp1 KO neurons. Significantly regulated transcription factors are shown in Extended Data Figure 5-1.
Extended Data
Extended Data Figure 1-2
RNAseq outlier exclusion. A, Dendrogram of WT and KO samples. WT3 and KO5 separated from other samples. B, PCA of transcript abundance levels showing PC1 (51% variance explained) and PC2 (25% variance explained) of control and Stxbp1 KO samples. N = 5 independent replicates. WT and KO showed clear clusters, except for WT3 and KO5. Download Figure 1-2, EPS file.
Extended Data Figure 1-1
Cleaved-Caspase 3 reactivity in E18 hippocampi of WT and Stxbp1 KO mice. A, Typical examples of E18 hippocampal slices from WT and Stxbp1 KO mice. Download Figure 1-1, EPS file.
Extended Data Table 1-1
qPCR primers. Download Table 1-1, DOCX file.
Extended Data Figure 2-1
QC mass spectrometry proteomics. A, Q-values for peptides detected in at least one sample. B, CoV values for detected peptides for every experimental group. C, Dendrogram of WT and KO samples in Euclidean distances. Cluster confidence is shown in red. Download Figure 2-1, EPS file.
Extended Data Figure 2-2
Significant proteins between DIV2 and DIV3 in WT and KO. A, Volcano plots showing significantly dysregulated proteins between DIV2 and DIV3. For WT cultures, one protein was significantly upregulated at DIV3. For KO cultures, 24 proteins were significantly upregulated at DIV3. Download Figure 2-2, EPS file.
Extended Data Table 2-1
Protein lists Gene Ontology analysis. Download Table 2-1, XLS file.
Extended Data Table 3-1
Protein lists overlap other proteomic studies. Download Table 3-1, XLS file.
Extended Data Table 4-1
Protein list SynGO analysis. Download Table 4-1, XLS file.
Extended Data Figure 4-1
Overlap in significant hits proteomic and transcriptomic analysis. A, Overlap of significant SynGO-annotated transcripts in mass spectrometry analysis. B, Overlap of significant SynGO-annotated proteins in RNAseq analysis. C, Functional annotation of the 36 overlapping genes found both to be regulated at transcript and protein level. Right, All genes significantly dysregulated on protein level in the mass spectrometry analysis. Red highlights overlapping significant transcripts. Download Figure 4-1, EPS file.
Extended Data Figure 5-1
Regulated transcription factors in Stxbp1 KO neurons. A, Among the 399 significantly dysregulated proteins at DIV3, seven were annotated as TF (Lambert et al., 2018). Five TFs (underscore) are involved in neuron development. Download Figure 5-1, EPS file.
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