Non‐Synaptic Function and Localization of Syntaxin‐Binding Protein 1 in a Mouse Model of STXBP1‐Related Epileptic Encephalopathy
Tao Yang, Rajat Banerjee, Yamei Deng, Sheetal Jahagirdar, Joo Hyun Kim, Wu Chen, Mingshan Xue, Alexey I. Nesvizhskii, Michael D. Uhler, Jack M. Parent, Yu Wang
TL;DR
This study explores the non-synaptic roles of STXBP1 in the brain, revealing its involvement in neuronal survival and dendritic growth beyond its known synaptic functions.
Contribution
The study identifies non-synaptic functions of STXBP1 and its role in neuronal membrane cytoskeleton trafficking in a mouse model of epileptic encephalopathy.
Findings
STXBP1 is localized in neuronal soma and processes, and interacts with cytoskeletal and membrane periodic structures.
Sparse Stxbp1 knockout in the mouse forebrain causes cell death, which is rescued by wild-type STXBP1 but not pathogenic mutants.
STXBP1 interacts with alpha II Spectrin and ARPC2, and is essential for their localization on the neuronal membrane.
Abstract
De novo mutations in the syntaxin‐binding protein 1 (STXBP1), encoded by STXBP1, are among the most prevalent causes of variable neurodevelopmental disorders, including epileptic encephalopathy, developmental delay, and movement disorders. Although STXBP1 has been proposed as a critical presynaptic protein controlling synaptic vesicle exocytosis, clinical phenotypes also suggest that its biological function could be more diverse. The expression pattern of STXBP1 was studied using immunostaining in vitro and in vivo. Synaptosome isolation was performed to investigate the synaptic and non‐synaptic localization of STXBP1 in the brain. STXBP1 immunoprecipitation followed by mass spectrometry (MS) was conducted to identify protein complexes interacting with STXBP1. Cre‐in utero electroporation (IUE) was done on Stxbp1 F/F mice to generate an in vivo knockout (KO) cellular model for studying…
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Taxonomy
TopicsCellular transport and secretion · Neuroscience and Neuropharmacology Research · Epilepsy research and treatment
Pathogenic mutations in Munc18‐1/syntaxin‐binding protein 1 (STXBP1) are associated with various severe neurological disorders. Most patients experience severe early‐onset epileptic encephalopathies, severe intellectual disability without epilepsy, and motor dysfunction, including ataxia, spasticity, and dyskinesia.1, 2, 3, 4, 5 Older patients with STXBP1 mutations have shown signs of neurodegeneration and Parkinsonism.6, 7 At the genetic level, disease‐causing STXBP1 mutations, including missense, nonsense, and frameshift, as well as intragenic, whole gene, and multi‐gene deletions, have been identified throughout the protein structure, with no domain either particularly spared or susceptible, and with most missense mutations studied to destabilize the protein, causing aggregation or degradation.5 STXBP1 plays a critical role in neurotransmitter release by binding syntaxin‐1. It prevents syntaxin‐1 from forming ectopic uncontrolled SNARE‐complex while trafficking to the cell surface and facilitates SNAREcomplex formation and neurotransmitter release at the synapse.8, 9, 10, 11, 12, 13, 14, 15, 16, 17 In addition, STXBP1 interacts with other synaptic proteins, such as RAB3, DOC2, and MINT proteins.18, 19 However, fluorescent‐tagged STXBP1 could be seen in cell bodies, not restricted to axons and axon terminals,20 and the function of STXBP1 beyond synapses and its relevance to disease is yet to be fully understood. For example, STXBP1 has been shown to regulate post‐Golgi transport of vesicles to the plasma membrane and vesicle fusion at the cell surface for proper protein (e.g., syntaxin‐1) localization.21, 22, 23 Interestingly, STXBP1‐homolog ROP is a key regulator of dendrite development via interaction with the exocyst subunit SEC6,24 and dendrite defect is the strongest pathological correlate for intellectual disability, a primary phenotype of STXBP1‐related encephalopathy. In this study, we characterized the cellular distribution of endogenous STXBP1 protein in rodent and human neurons and identified its interactors that play important roles in neurodevelopment, dendrite growth, and cell survival.
Methods
In Utero Electroporation
Stxbp1 flox/flox mice were bred to generate homozygous embryos for in utero electroporation (IUE). The IUE procedure was done on embryonic day 15. Briefly, the pCAG‐GFP and/or pCAG‐RFP, or Se‐Cre/FLEx‐GFP vectors were in utero injected into the lateral ventricle, followed by electroporation to transfect plasmids into the neural progenitor cells along the dorsal ventricle. The electroporation was conducted across the mouse embryonic head using 5 electric pulses (35 V, 50 ms duration, 1‐second intervals; Boston apparatus, BTX‐100).25 The pCAG‐eGFP or RFP vector labeled the pyramidal neurons, which are the progeny of the dorsal neural progenitor cells, as control. Se‐Cre and FLEx‐GFP plasmids were co‐transfected into the pyramidal neurons, and Cre actively induced the GFP expression from the FLEx‐GFP construct and simultaneously knocked out Stxbp1. Pathogenic STXBP1 (A406H, R92H, and C180Y) or benign STXBP1 mutant (R305W and V84I) were in utero injected and transfected together with Se‐Cre for the rescue experiments. The protocol for the study was approved by the Institutional Animal Care and Use Committee at the University of Michigan, and all studies were conducted in accordance with the United States Public Health Service's Policy on Humane Care and Use of Laboratory Animals.
Immunohistochemistry
Brains were dissected and fixed in 4% paraformaldehyde in PBS following the transcardial perfusion. After the overnight fixation, brains were sectioned at 50 μm using a Leica VT1000S vibratome and processed for immunohistochemistry as free‐floating sections. For immunostaining, brain sections were permeabilized with 0.2% Triton X‐100 in phosphate‐buffered saline (PBS), blocked with 2% donkey serum in PBS, and incubated overnight at 4°C with primary antibodies. The primary antibodies used were mouse anti‐STXBP1 (1:200; Abnova, Cat #H00006812‐M01), rabbit anti‐Caspase 9 cleaved (1:500, Thermo Fisher Scientific, Cat #PA5‐17913), chicken anti‐GFP (1:500; Aves Labs, Cat #GFP‐1020), rabbit anti‐NEAs (1:200; Abcam, Cat #11755), rabbit anti‐ArPC2 (1:200; Abcam, Cat #ab133315), and chicken anti‐map2 (1:200; Abcam, Cat #Ab5392). Then, the sections were rinsed and probed with secondary antibodies. Fluorescently conjugated secondary antibodies (Alexa Fluor 488, 594, or 647, 1:500) were obtained from Molecular Probes, and neurons were counterstained with Nissl staining (Invitrogen, Cat #N21479).
Synaptosome Preparation
Synaptosome was extracted from CD1 mouse brain cortex at the age of P16. The cortex of each mouse brain was lysed in Syn‐PER reagent (Synaptic Protein Extraction Reagent; Pierce, Cat #87793) at a proportion of 1 ml/gram of tissue supplemented with protease Inhibitor (Complete; Roche, Cat #11836170001) and phosphatase inhibitor (Halt; Invitrogen, Cat #1862495). Tissue was lysed according to the manufacturer's protocol. Lysates were initially centrifuged at 1200 × g, and 20% of the supernatant was stored as homogenate. The remaining supernatant was further centrifuged at 15,000 × g for 20 minutes, and the subsequent supernatant was stored as the cytosolic fraction. The pellet was resuspended in Syn‐PER reagent. All procedures were performed in 4°C.
Western Blot Analysis
Tissues were lysed in RIPA buffer (Sigma; #R0278) supplemented with protease Inhibitor (Complete; Roche, #11836170001) and phosphatase inhibitor (Halt; Invitrogen, #1862495) at a ratio of 1 ml buffer per gram of tissue. Tissues were homogenized in 1.5 ml pestle set (Monarch, NEB, #T3000L) repeatedly for uniform lysis followed by sonication. The lysates were further vortexed and centrifuged at 10,000 g for 10 minutes. The supernatant was collected, quantified for protein concentration, and run on a standard SDS‐PAGE gel at 50 μg per well. The gel was transferred to the membrane and blotted for antibodies to STXBP1 (Abnova; #H00006812‐M01, 1:1000 dilution), β‐Actin (Invitrogen; #2336, 1:5000 dilution), NMDAR2B (Invitrogen; #MA1‐2014, 1:500 dilution), Synaptophysin (Sigma; #S5768, 1:1000 dilution), NEAS/α2‐Spectrin (Abcam; #11755, 1:500 dilution), ARPC2 (Abcam; #133315, 1:1000 dilution), and ARPC3 (PTG Lab; #14652‐1‐AP, 1:2000 dilution). The HRP‐conjugated secondary antibodies from the source species of primary antibodies were used. Signal was acquired with chemiluminescent reagent (Thermo Scientific; #34577) in a Bio‐Rad ChemiDoc imaging system.
Co‐Immunoprecipitation
All process was carried out on ice. Cortex tissues from adult CD‐1 mouse brain were collected, snap frozen in liquid nitrogen, and preserved in −80°C. Tissues were thawed and homogenized in RIPA buffer (Sigma; #R0278) supplemented with protease inhibitor (Complete; Roche #11836170001) and phosphatase inhibitor (Halt; Invitrogen #1862495) at a ratio of 1 ml buffer per gram of tissue. Tissues were homogenized in 1.5 ml pestle set (Monarch; NEB, #T3000L) repeatedly to make a uniform lysate, then vortexed several times. Finally, the resulting lysates were further homogenized by repeated pipetting and passing through 22‐gauge syringe and needle assembly for at least 6 times. Samples were then clarified by centrifuging at 10,000 g for 10 minutes and quantified with Spectrophotometry (AD340; Beckman Coulter).
For immunoprecipitation (IP)‐mass spectrometry (MS), 25 μg of Stxbp1 antibody (PTG Lab; #20562‐1‐AP) was coupled with 1.5 mg of Dynabeads magnetic beads using the Dynabeads Co‐Immunoprecipitation kit (Invitrogen; #14321D) following the manufacturer's instructions. Beads were allowed to couple with antibody for 16 hours at 37°C in a rotary shaker, followed by a series of washes according to the manufacturer's instructions. Each 1.5 mg of antibody‐coupled beads was used to precipitate 1.25 mg of clarified cortex lysate in a volume of 400 μl diluted with IP lysis buffer (provided with the kit) supplemented with protease and phosphatase inhibitors. This ratio was maintained throughout all co‐immunoprecipitation (co‐IP) experiments with antibodies to STXBP1, NEAS (alpha II‐Spectrin), ARPC2, and ARPC3 and scaled up accordingly whenever needed for STXBP1 MS. Corresponding rabbit or mouse IgG was used as a control. Antibody‐bound, pulled‐down protein was washed and eluted with elution buffer provided with the kit according to the manufacturer's protocol and stored at −80°C until use.
Mass Spectrometry
The MS experiments were performed on a hybrid ion trap‐Orbitrap mass spectrometer (Orbitrap Velos; Thermo Fisher Scientific) for all analytical runs. The instrument was set up to run the TOP 20 method for tandem MS/MS in an ion trap. The acquired MS data were analyzed using FragPipe (version 22.0) with the default LFQ‐MBR workflow. The raw data were converted into mzML format using MSConvert from ProteoWizard, and MS2 spectra were searched using MSFragger against the Uniprot Mouse proteome database appended with decoy sequences and common contaminants. The search was restricted to tryptic peptides, allowing up to 2 cleavage sites. Acetylation of the protein N‐terminus and oxidation of methionine were considered as variable modifications, and carboxymethylation of cysteine was set as a fixed modification. The search results were further processed using MSBooster for rescoring, Percolater for PSM validation, ProteinProphet for protein grouping, and Philosopher for false discovery rate (FDR) filtering, where 1% PSM, peptide, and protein FDR was applied. The PSM outputs from Philosopher were further subjected into IonQuant for protein and peptide quantification.
Spectral counts are commonly used to measure the relative changes of protein amounts across different samples in IP‐MS studies; however, they can be biased when peptides are shared by multiple proteins. To address this, we perform protein quantification by adopting the distributed Normalized Spectral Abundance Factor (dNSAF) method, which is considered a gold standard for achieving unbiased data for label‐free quantitative measure of protein by MS analysis.26
We used the combined_peptide.tsv and combined_protein.tsv files from IonQuant to extract necessary information, such as the peptide spectral counts and their protein assignments. For each protein, we classified its peptides into 2 groups: those uniquely mapped to that protein (unique peptides) and those shared with other proteins (shared peptides). We calculated a uSpC value for each protein by summing the spectral counts of its unique peptides. For shared peptides, we distributed the shared peptide spectral count proportionally among the proteins it mapped, and these distribution factors are determined based on the uSpC values of these proteins. Proteins with higher uSpC got more share of the spectral count. We then added up these distributed spectral counts for each protein's shared peptides to get an sSpC value. By combining the uSpC and sSpC, we got an adjusted total spectral count (tSpC) for each protein. We then normalized the tSpC by protein length to produce a dSAF value and normalized these values across all proteins to derive the final dNSAF values, which corrects for shared peptide bias. The custom script for protein dNSAF calculation is available as described in the Data and Code availability.
Differential analysis was performed using the linear model from the Limma R package. Log2 fold change (log2FC) was represented by a moderated t‐statistic, and a moderated p value of 0.05 was used to identify up‐ and downregulated proteins.
Gene Ontology Term Enrichment Analyses
The Gene Ontology (GO) term enrichment and clustering analyses were performed using the Database for Annotation, Visualization, and Integrated Discovery (https://davidbioinformatics.nih.gov/home.jsp). The official gene symbols were converted through the conversion tool provided in the database for mouse, and candidate STXBP1‐interacting proteins were subsequently submitted to DAVID. The enriched GO terms with p values ≤ 0.05 were considered. For GO term enrichment and clustering analyses of the biological process (BP) category, GO BP terms at level 3 (GO_BP_3) were used.
In Vitro Neuronal Culture
Glass‐bottom plates (35 mm, CellVis, #D35‐20‐1.5N) were pretreated by incubation overnigh with 10 μg/ml Poly‐D‐Lysine (Millipore#A‐003‐E) and 5 μg/ml Laminin (Sigma; #L2020) in 500 μl sterile water. The plates were washed twice in sterile water and used for neuronal culture.
Neurons were dissociated from the brain cortex tissue of E19 Stxbp1 ^F/F^ (C57BL/6) mice pups in HBSS (Invitrogen; #14175095) supplemented with 10 mM HEPES (Invitrogen; #15630080) in a 15 ml conical tube (Falcon; #352196) on ice. Collected tissues were washed once in Hank's Balanced Salt Solution (HBSS) with 10 mM HEPES, and approximately 1 ml tissue was dissociated in 200 μl of TrypLE (Invitrogen; #12604013) and 40 μl (80 units) of DNAse I (NEB; #M0303S) by incubation at 37°C for 10 minutes. After incubation, the trypsin media was removed and washed once with plating media containing Neurobasal media (Invitrogen; #21103049) supplemented with 5% fetal bovine serum (FBS; Sigma; # F2442), 1% Glutamax (Invitrogen; #35050061), 1% Amphotericin B (Invitrogen; #15290018), 1% PenStrep (Sigma; #P0781), and 2% B27 supplement (Invitrogen; #17504044). Tissue was then further homogenized by repeated pipetting through fire‐polished Pasteur pipettes 6 times and was passed through a 100 μm cell strainer (Corning; #431752). Cells were then counted, and 250,000 cells in 400 μl of plating media were plated at the center of the 35‐mm glass‐bottom plates and mixed well. After 16 hours, the plating media was removed, and the addition of 3 ml of growth media containing Neurobasal media (Invitrogen; #21103049) supplemented with 0.5% FBS, 1% Glutamax, 1% Amphotericin B, 1% PenStrep, and 2% B27 supplement. The media was changed every 2 days by removing 1.5 ml old media with fresh 1.5 ml of growth media. Neurons were allowed to grow and were used at DIV 6.
Image Acquisition, Soma Size, Cortical Thickness Measurement, and Statistical Analyses
Multi‐channel imaging was performed using a Leica SP5 confocal microscope. All the images were further processed in Adobe Photoshop software. The primary dendrite number was counted in the somatosensory cortex with ImageJ software. The circulation was measured by ImageJ software. The average number of primary dendrites and circularity/roundness was quantified. Statistical analysis was performed using GraphPad 10.
Quantification and Statistical Analysis
All Western blot band intensities were quantified by ImageJ software (FIJI), normalized to their corresponding Actin/GAPDH internal control bands, and expressed as arbitrary densitometric units (DU) and plotted in GraphPad Prism software. Statistical analyses were performed using GraphPad Prism 10 software, which utilized inbuilt programs, either ANOVA or t test, as mentioned in the figure legends. The p values were determined by the Student's t test and 1‐way ANOVA. All data were shown as mean ± SEM. A p value less than 0.05 was considered to be statistically significant. Convention for significance is shown as: p < 0.05 (), p < 0.01 (), and p < 0.0001 ().
Results
STXBP1 Is a Synaptic and Non‐Synaptic Protein
To study the function of STXBP1 in vivo at the cellular level, we took advantage of the floxed Stxbp1 transgenic mouse line and applied Cre‐IUE to knock out Stxbp1 in cortical excitatory neurons27, 28, 29 (Fig 1A). We first confirmed the efficiency of Cre‐IUE‐mediated gene recombination by co‐electroporating self‐excised(se) Cre, RFP, and Flex‐GFP plasmids at E15 and examined the brain at P14. The Cre‐Flex cassette combines inversion and excision, utilizing 2 different lox sites, loxP and lox2272, in opposite directions (see Fig 1A). Without Cre expression, transfected cells could only express RFP (insets of Fig 1B–B″). Upon Cre expression, the same lox sites (loxP or lox2272) will be in the same direction, and the other type of lox site in between will be deleted, and the transfected neurons will express both RFP and GFP. Nearly all RFP‐expressing cells also expressed GFP (see Fig 1B–B″), suggesting a high efficiency of IUE‐mediated gene recombination. We then bred female mice with male mice homozygous for floxed Stxbp1 and performed Cre‐IUE at E15. Immunohistochemistry on the P1 brain with Cre‐IUE showed that transfected neurons were negative for STXBP1 staining (Fig 1C–C″). This result indicates that Cre‐IUE efficiently mediates Stxbp1 knockout in cortical excitatory neurons. We then performed Cre‐IUE followed by acute neuronal culture to confirm the specificity of the STXBP1 staining. Immunocytochemistry at DIV3 showed that most transfected neurons were negative for STXBP1 antibody staining (Fig 1D–D″). In contrast, nearly all control plasmids transfected neurons were positive for STXBP1 (insets of Fig 1D–D″). Interestingly, STXBP1 was most abundant in neuronal soma and processes, consistent with its reported localization when tagged with a fluorescence protein.20 Western blotting of the whole mouse brain displayed a clean and single band at approximately 68 kD (Fig 2A), further supporting the specificity of the STXBP1 antibody. Interestingly, STXBP1 expression in the brain increased gradually throughout the developmental stages from E15 to P28. We then isolated synaptosomes from P16 mouse cortices to provide biochemical evidence for the non‐synaptic localization of STXBP1. We showed the presence of STXBP1 in both cytosolic and synaptic fractions (Fig 2B). In contrast, NMDAR2B, a subunit of N‐methyl D‐aspartate (NMDA) receptor that binds to the excitatory neurotransmitter, glutamate, was only seen in the synaptosome.30 Our data further showed that the expression of STXBP1 was visible in the subplate region in the embryonic brains, where the earliest thalamocortical projections form30 (Fig 2C). In postnatal brains, STXBP1 expression became more diffuse, with higher expression in cortical layers IV/V, which receive abundant thalamocortical projections. STXBP1 staining was ubiquitous and intense in layer I, which contains a thick and intricate web of apical dendrites and axonal projections.31 Finally, STXBP1 immunocytochemistry revealed that it was present in both the soma and neuronal processes, as marked by MAP1, in cultured mouse cortical neurons (Fig 2D) and human iPSC‐derived neurons (Fig 2E). Collectively, our data clearly demonstrate that STXBP1 is located throughout the entire cell body and its processes.
*Cre‐IUE generates Stxbp1 knockout neurons in Stxbp1
F/F transgenic mice. (A) a diagram of Cre‐IUE to induce GFP expression from a FLEx (flip‐excision)‐GFP construct and Stxbp1 deletion from the floxed allele. (B–B″) Representative P14 cortex from mice that were electroporated with seCre, FLEx‐GFP, and RFP at E15. (B) Constitutive RFP expression; (B′) conditional GFP expression induced by seCre; (B″) merged RFP and GFP images show a high co‐expression pattern. Blue, counterstaining with DAPI. Inserts in the right corner of (B–B″) show that the cerebral cortex without seCre only expresses RFP. (C–C″) A representative P1 Stxbp1
F/F cerebral cortex with seCre/FLEx‐GFP/CAG‐RFP IUE at E15. (C) Constitutive RFP expression suggests the success of IUE, and GFP expression suggests Cre‐mediated gene recombination. (C′) STXBP1 staining shows the lack of STXBP1 expression in the transfected area (arrows). (C″) The merged image of C and C′. Scale bar = 200 μm. (D–D″) Primary culture of cortical neurons isolated from Stxbp1 F/F mouse cortex with Cre‐IUE. (D) GFP expression induced by Cre; (D′) STXBP1 immunostaining shows the lack of STXBP1 immunostaining in neurons transfected with seCre (arrowheads); (D″) a merged image of D and D′. The inserts in the upper right corner show the control neurons without seCre. Blue, DAPI counterstaining. Scale bar = 10 μm. IUE = in utero electroporation. [Color figure can be viewed at www.annalsofneurology.org]*
STXBP1 expression during cortical development. (A) Tissue lysates from the cortex of mouse brain at different ages were run on an SDS‐PAGE gel and blotted with antibodies to STXBP1 (top panel), Actin, as a loading control, was run on the second panel from the top. Band intensities for each protein signal were normalized to their corresponding Actin bands and expressed as arbitrary DU. Band intensities for STXBP1 signal were quantified and plotted (For each mouse, a different color label is used) and significance depicted (∗∗p < 0.01). One‐way ANOVA. The relative STXBP1 expression levels are quantified with a bar chart on the right. (B) Synaptosome fraction, tissue homogenate and cytosolic fraction from cortex of 3 individual mice at age P16 were ran on an SDS‐Page gel and blotted with antibodies to STXBP11 (top panel), Actin (second panel from the top), and synaptic markers NMDAR2b (third panel from the top) as well as Synaptophysin (fourth panel from the top). (C) STXBP1 immunohistochemistry at different developmental stages shows its expression in the cortex. Scale bar = 350 μm. (D–D″) MAP2 and STXBP1 immunostaining of primary cultured neurons and (E–E″) human iPSC induced iNeurons show their overlapping expression pattern in neuronal soma, dendrites, and axon. Scale bar = 100 μm. DU = densitometric unit; P = postnatal day. [Color figure can be viewed at www.annalsofneurology.org]
Pathogenic STXBP1 Mutants Rescue Cell Death
The above findings suggested that STXBP1 is involved in a range of diverse biological functions beyond synaptic transmission. To investigate whether STXBP1 is involved in neural proliferation and migration, we performed seCre‐IUE on E15 Stxbp1 ^F/F^ mice and examined the neuronal migration at E17 and P1 (Fig 3A). In contrast to the previous study using the shRNA approach to knockdown Stxbp1,32 our data showed that GFP‐positive neurons (Cre‐mediated Stxbp1 KO neurons) in both control and seCre‐IUE experiments reached the superficial layer (data not shown). The neural proliferation assays, using Ki67 staining, showed no difference between control and Stxbp1 knockout conditions (data not shown).
Cre‐IUE mediated Stxbp1 knockout causes neuronal death. (A) Shown is the experimental design (1) the control IUE experiment without seCre; (2) the IUE‐Cre experiment to induce Stxbp1 knockout and GFP expression. (B) P1 IUE brains with or without Cre display bright RFP signal. (C) P7 control IUE brains show less intense RFP signal as compared to P1 likely becuase of increased brain size and cortical thickness. (D) However, P7 Cre‐IUE brains show the lack of RFP signal under the dissection stereoscope. Scale bar = 300 μm. (C′) Abundant RFP‐labeled neurons are seen in the cortex from control‐IUE brains; (D′) Few RFP‐labeled neurons are seen in P7 Cre‐IUE brainss. (E–E″) P1 cortical neurons labeled by Cre‐induced GFP are positive for cleaved Caspase 3 staining (arrowheads). Scale bar = 300 μm. (F) The contralateral hemisphere of the Cre‐IUE cortex displays no cleaved Caspase 3 positive cells. (G–L) Cell death induced by Stxp1 KO is rescued not only by wild‐type STXBP1 (G) and benign STXBP1 variants (H: R305W, I: V84I) but also by pathogenic variants. Scale bar = 30 μm. (J: R406H, K: R292H, L: C180Y). (M–N) Morphological analysis shows that pathogenic variant rescued neurons have fewer primary dendrite numbers (M) and less triangular (N). Each dot in the bars represents one brain sample. For primary dendrite analysis, 45 neurons from WT, 38 neurons from R305W, 40 neurons from V84I, 39 neurons from R406H, 33 neurons from R292H, 51 neurons from C180Y rescue experiments. For the circularity analysis, 45 neurons from WT, 37 neurons from R305W, 39 neurons from V84I, 37 neurons fom R406H and r292H, 36 neurons from C180Y rescue experiments, The white bar, WT; the light grey bars, the benign mutants; the dark grey bars, the pathogenic mutants. KO = knock out; P = postnatal day; IUE = in utero electroporation; WT = wild type. [Color figure can be viewed at www.annalsofneurology.org]
Consistent with the massive neuronal cell death in Stxbp1 germline KO mice, nearly all GFP+ Cre‐IUE KO neurons disappeared at P7 (Fig 3B–D), which was presumably due to cell death because many neurons at P1 were positive for cleaved Caspas‐3, a programmed cell death marker (Fig 3E). Next, we performed rescue experiments by overexpressing human wild‐type STXBP1. When seCre was co‐electroporated with wild‐type STXBP1, cell death was rescued as expected (Fig 3G). Interestingly, 2 benign variants (V84I and R305W; Fig 3H, I) and 3 pathogenic variants (C180Y, R292H, and R406H; Fig 3J–L) also rescued the cell death phenotype due to Stxbp1 deletion. Upon further analysis, Stxbp1 KO neurons rescued by benign variants showed a typical pyramidal soma with an average of approximately 6 primary dendrites, whereas KO neurons rescued by pathogenic variants were dysmorphic with a round soma and approximately 2 primary dendrites (Fig 3M, N).
STXBP1 Interacts With Membrane Periodic Structural Proteins
The morphological phenotypes observed in neurons rescued by pathogenic variants led us to hypothesize that STXBP1, despite its importance in neurotransmitter release via SNARE proteins (e.g., Syntaxin), together with its other cellular interacting partners, plays diverse roles in other cellular functions. Recently, 2 other structural proteins were identified as STXBP1 interacting partners: Myosin Va, a motor protein linked to exocytosis of secretory vesicles and insertion of neurotransmitter receptors into post‐synaptic membranes,33 and SLP4‐A, a RAB27A effector to promote Weibel‐Palade body exocytosis in endothelial cells.34 However, the list of STXBP1 interacting partners is likely far from complete. We used co‐IP to pull down proteins associated with STXBP1 from adult mouse brains, followed by mass spectrometry (MS) to identify interacting partners at the proteomic scale.
Dynabeads coated with anti‐STXBP1 antibody, as well as IgG as a control, were incubated with the adult mouse brain lysates to allow capturing of the proteins that bind directly or indirectly to STXBP1 (Fig 4A). The co‐IP protein mixtures were subsequently analyzed using SDS‐PAGE to assess the extent of purification and showed notable enrichment of STXBP1 at the expected molecular weight in the SDS‐PAGE gel image. In contrast, co‐IP using beads coated with a control IgG hardly pulled down these proteins (Fig 4B). Biological replicates (n = 4) of STXBP1 and control IgG co‐IP products were processed through MS to identify the co‐IP proteins. To reduce potential false positives, we only considered proteins identified in all 4 biological samples, although this criterion may filter out some true STXBP1‐interacting proteins. This resulted in a list of 114 proteins, and a gene ontology (GO) term analysis of the biological process (BP) category was performed based on the DAVID platform (Fig 4C and Supplementary Data 1). In addition to its well‐known function in pre‐synaptic processes, data showed STXBP1 was involved in many other BPs with high significance, including post‐synapses, myelin sheath formation, cytoskeletons, and actin filament‐based processes, organelle organization, and protein localization (see Fig 4C). Because the lysates used in the co‐IP experiments were not fractionated, the identified candidate proteins could include interacting partners in axons, dendrites, and soma from all neuronal sub‐compartments. It should be noted that proteomic analysis by co‐IP and MS may yield false positive results due to nonspecific protein binding to antibody‐coated beads. Negative controls with IgG‐coated beads may not completely remove these false‐positive results. In addition, run‐to‐run variations in chromatography‐MS experiments and weak interactions between proteins can generate false‐negative results. We then performed further validation for a subset of these candidates using reciprocal IP assays (Fig 4D–G) for further confirmation. Among these proteins, we focused on alpha II‐spectrin and ARPC2/3, 2 membrane‐associated proteins that play essential roles in neurodevelopment, synaptogenesis, and dendritic growth.35, 36 Previous studies showed that a lack of STXBP1 in various cells impairs the localization of Syntaxin1A, a canonical synaptic protein. The biochemical interactions between STXBP1 and spectrins, as well as ARPC2/3, led us to hypothesize that STXBP1 is also required for trafficking alpha II‐spectrin and ARPC2/3 to the plasma membrane. To check this hypothesis, we first performed Cre‐IUE at E15 Stxbp1 ^ F/F ^ mouse brains, followed by primary neuronal culture to assess the impact of Stxbp1 KO on the cellular localization of alpha II‐spectrin (Fig 5A–D) and ARPC2 (images not shown here). The percentage of the number of fluorescence puncta of alpha II‐spectrin at the plasma membrane to that at the cytoplasm was measured. In Stxbp1 knockout neurons (see Fig 5B), alpha‐II spectrin was less distributed at the membrane and more aggregated in the cytoplasm than in control neurons (see Fig 5A), in which alpha II‐spectrin (see Fig 5C, D) and ARPC2 (Fig 5E, F) were mainly located on the cell surface. In addition, rescue experiments confirmed that re‐expression of human STXBP1 WT and the benign variant (R305W), not the pathogenic variant C180Y, restored the localization of alpha‐II spectrin in mouse Stxbp1 knockout neurons (Fig 5G–I).
STXBP1 is associated with membrane periodic structural proteins. (A) a diagram of STXBP1 antibody based in vivo immunoprecipitation and mass‐spectrometry experiment. (B) Coomassie‐stained gel showing purified STXBP1‐IP eluted proteins with rabbit IgG as the control. (C) STXBP1‐associated proteins were identified through MS analysis and passed through strict quality control, and finally, 114 filtered targets were annotated and submitted to DAVID (Database for Annotation, Visualization, and Integrated Discovery) database (https://davidbioinformatics.nih.gov/). Different biological processes involving groups of targets with their adjusted p values were plotted as bar graph along with superposed line graph indicating percentage of target genes involved in each process. (D) Western blot analysis of purified STXBP1‐IP pulldown with anti‐ STXBP1 alphaII Spectrin, APRC2, and ARPC3. (E–G) Reverse IP with antibodies to alphaII Spectrin, (F) ARPC2, (G) and ARPC3. MS = mass spectrometry. [Color figure can be viewed at www.annalsofneurology.org]
STXBP1 regulates the trafficking of alpha II spectrin and ARPC2/3 to the cell membrane. (A) The alpha II spectrin staining (red) in isolated primary cultured neurons shows that αII spectrin is mainly located on the cell surface. GFP labels the individual neuron. Scale bar = 15 μm. (B) The alpha II spectrin staining (red) in isolated Stxbp1 knockout primary cultured neurons shows that alpha II spectrin is distributed within the cytoplasm. GFP labels the STXBP1 knockout neuron. (C) Percentage of alphaII spectrin puncta on the neuronal surface. n=15 neurons in WT and 12 neurons Stxbp1 KO conditions from two diffferent repeats. (D) Bar chart of the alpha II spectrin puncta density on the dendrites. n=32 in WT and 26 in Stxbp1 KO conditons from two different repeats. (E) Percentage of ARPC2 puncta on the neuronal surface. n=12 in WT and Stxbp1 KO conditions. (F) Bar chart of ARPC2 puncta density on the dendrites. n=8 in WT and 10 IN Stxbp1 KO conditions from two different repeats. (G) In Stxbp1 F/F neurons transfected with Cre‐BFP (blue), expression of STXBP1 WT (the left panel) or the benign R305W variant (middle lane) significantly improved localization of alpha II spectrin as compared to KO neurons transfected with GFP (data not shown here). In contrast, Stxbp1 KO neurons with pathogenic variant C180Y expression (right lane) do not show restored localization of alpha II spectrin. Scale bar = 10 μm. (H) Percentage of alpha II spectrin puncta on the cell surface in Stxbp1 KO neurons rescued by STXBP1 WT, benign, and pathogenic variants. n=10 in WT. 10 in R305W, 11 in C180Y rescue experiments from two different repeats. (I) Bar chart of alpha II spectrin puncta density on the dendrites. n=15 in WT, 15 in R305W, 8 in C180Y rescue experiemtns from two different repeats. KO = knock out; WT = wild type. [Color figure can be viewed at www.annalsofneurology.org]
Discussion
Although the non‐synaptic functions of STXBP1 have been suggested, there is no direct evidence to show its synaptic and non‐synaptic localization and interacting network. We demonstrated, for the first time, its presence in both dendrites and axons as well as in the synaptic and cytosolic fractions, suggesting that STXBP1 is a multifunctional protein beyond its role in presynaptic neurotransmitter release. We next showed that Stxbp1 knockout in a small percentage of excitatory neurons in the mouse cortex led to cell‐autonomous neuronal death. Previously, the extensive neuronal death phenotype in the germline knockout animals was attributed to activity‐dependent apoptosis because Stxbp1 null neurons were synaptically silent. However, follow‐up experiments using Purkinje‐cell specific Stxbp1 knockout or in vitro low‐density neuronal culture also led to cell death, suggesting that the role of STXBP1 in neuronal viability is independent from its function in synaptic transmission or vesicle exocytosis.37 In addition, expression of noncognate paralogs38, 39 in Stxbp1 knockout neurons could rescue vesicle exocytosis but not viability. Syntaxin‐1 and SNAP25, two other essential presynaptic proteins for synaptic transmission, are also critical for neuronal viability during early development. On the other hand, deletion of MUNC13, VAMP2, complexin‐1, and several other presynaptic proteins did not result in neuronal death.40, 41 These studies showed that Stxbp1 knockout neurons could initiate outgrowth of neurites but from thereon stopped development with dramatically reduced speed of growth followed by cell death before they developed synapses or showed synaptic activities, suggesting that STXBP1 is essential to advance neural development and is a “checkpoint” for neurons to initiate cell death in its absence.42 However, it remains unclear how neuronal viability critically depends on STXBP1.
Both benign and pathogenic mutations rescued cell death in vivo in our study, suggesting STXBP1 pathogenic variants with decreased protein stability affect synaptic function, a delicate and more evolved biological function, but not neuronal viability that depends on a more fundamental and conserved process. It is noteworthy that pathogenic variants rescued neurons still displayed neurite growth defects. Dendritic defect is a strong pathological feature of intellectual disability, one prominent clinical feature of STXBP1 encephalopathy.43 Mutations in the Drosophila Rop gene, the Sec1/Munc18 homolog, showed a progressive loss of terminal dendrites and blebbing and/or fragmentation of primary dendrites during later stages of larval development.24 Consistent with our findings, Yamashita et al showed the mislocalization of syntaxin‐1 and impaired neurite growth in a human iPSC model of STXBP1‐related epileptic encephalopathy.44 Therefore, it is conceivable that STXBP1 is important for both presynaptic and postsynaptic membrane fusion with differential requirements for the exocytic machinery in the developing neuron. Indeed, SNARE proteins are essentially involved in nearly all forms of eukaryotic membrane fusion that includes vesicle exocytosis (e.g., neurotransmitter release), constitutive exocytosis/secretion, and plasma membrane recycling.45 For example, cleavage of syntaxin 1/SNAP‐25 by botulinum caused neurodegeneration and neuronal death by blocking plasma membrane recycling processes, independent of synaptic vesicle exocytosis.46 In addition, it has been shown that STXBP1 also regulates post‐Golgi transport of vesicles and subsequent vesicle fusion at the cell surface.21, 22, 23 Altogether, these data suggest that STXBP1 is involved in diverse molecular functions beyond synaptic transmission.
STXBP1 and several other synaptic proteins were identified as membrane periodic structures‐interacting partners in a recent in vitro and in vivo IP‐MS study using anti‐alpha II spectrin or anti‐beta II spectrin antibodies. In this study, membrane‐associated periodic skeleton (MPS)‐interacting proteins included structural components of the MPS that bind to actin filaments, non‐muscle myosin II (NMII) motor proteins, cell‐adhesion molecules, ion channels, and other signal transduction‐related proteins.47 Furthermore, stochastic optical reconstruction microscopy (STORM), a super‐resolution imaging method, has observed that these novel MPS‐interacting proteins have periodic distribution characteristics. Another study also showed that Myosin Va, a member of a family of nonconventional motor proteins that mediate F‐actin‐based vesicular transport toward the plasma membrane, forms a ternary complex with STXBP1 and Syntaxin1A, which is required for Syntaxin1A trafficking to the plasma membrane.33 It is also well‐established that molecular motors and cytoskeletons, including actin and myosin, that play critical roles in post‐synaptic function by carrying the major scaffolding proteins (eg, PSD‐95, SAPAP1/GKAP, Shank, and Homer) to the dendritic spines. Previously, we already showed that CRISPR‐mediated deletion of alpha II spectrin or overexpression of it pathogenic mutants caused clear abnormalities in dendritic, axonal, and synaptic development.29 Here, we showed STXBP1 is essential for the proper membrane localization of alpha II spectrin and other MPS‐associated proteins that are required for dendritic growth and maintenance, providing mechanistic insights into the non‐synaptic function of STXBP1 in the nervous system. However, it is important to note that the functional significance of the interaction between STXBP1 and MPS in the pathogenesis of STXBP1‐related epileptic encephalopathy is unclear. We have not provided direct evidence to conclude whether and how pathogenic STXBP1 variants disrupt the intricate protein–protein interaction. If yes, it is also unclear whether the impaired interaction could cause MPS mislocalization, leading to altered neurodevelopment and excitability. Nonetheless, our data showed that STXBP1 is both a synaptic and non‐synaptic protein, has diverse non‐synaptic functions in cell survival and dendritic growth, and interacts directly or indirectly with MPS, paving the way to developing a more mechanistic understanding of STXBP1‐related epileptic encephalopathy.
Author Contributions
Y.W. contributed to the conception and design of the study; T.Y., R.B., D.Y., A.N., M.U., and J.P. contributed to the acquisition and analysis of data; Y.W., T.Y., R.B., Y.D., A.N., M.U., and J.P. contributed to drafting the text and preparing the figures. [Correction added on 25 February 2026, after first online publication: Author contribution text has been revised in this version.]
Potential Conflicts of Interest
All authors declared no potential conflicts of interest.
Supporting information
Data S1 Supporting Information.
The reference list from the paper itself. Each links out to its DOI / PubMed record.
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