MUNC18–1 gene abnormalities are involved in neurodevelopmental disorders through defective cortical architecture during brain development
© The Author(s). 2017
Received: 6 October 2017
Accepted: 19 November 2017
Published: 30 November 2017
While Munc18–1 interacts with Syntaxin1 and controls the formation of soluble N-ethylmaleimide-sensitive factor attachment protein receptors (SNARE) complex to regulate presynaptic vesicle fusion in developed neurons, this molecule is likely to be involved in brain development since its gene abnormalities cause early infantile epileptic encephalopathy with suppression-burst (Ohtahara syndrome), neonatal epileptic encephalopathy and other neurodevelopmental disorders. We thus analyzed physiological significance of Munc18–1 during cortical development. Munc18–1-knockdown impaired cortical neuron positioning during mouse corticogenesis. Time-lapse imaging revealed that the mispositioning was attributable to defects in radial migration in the intermediate zone and cortical plate. Notably, Syntaxin1A was critical for radial migration downstream of Munc18–1. As for the underlying mechanism, Munc18–1-knockdown in cortical neurons hampered post-Golgi vesicle trafficking and subsequent vesicle fusion at the plasma membrane in vivo and in vitro, respectively. Notably, Syntaxin1A-silencing did not affect the post-Golgi vesicle trafficking. Taken together, Munc18–1 was suggested to regulate radial migration by modulating not only vesicle fusion at the plasma membrane to distribute various proteins on the cell surface for interaction with radial fibers, but also preceding vesicle transport from Golgi to the plasma membrane. Although knockdown experiments suggested that Syntaxin1A does not participate in the vesicle trafficking, it was supposed to regulate subsequent vesicle fusion under the control of Munc18–1. These observations may shed light on the mechanism governing radial migration of cortical neurons. Disruption of Munc18–1 function may result in the abnormal corticogenesis, leading to neurodevelopmental disorders with MUNC18–1 gene abnormalities.
Munc18 (mammalian homologs of Caenorhabditis elegans uncoordinated-18) proteins are essential regulators of exocytosis, and mammals express 3 highly homologous isoforms, Munc18–1~3. While Munc18–1 is primarily expressed in neurons and neuroendocrine cells [1, 2], Munc-18-2 and Munc-18-3 are expressed widely including brain [3–5]. As for the function, Munc18–1 regulates neuronal exocytosis by serving as a molecular chaperone for Syntaxin1, and controls SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein receptor) complex formation through modulating Syntaxin1 level at the plasma membrane . On the other hand, Munc18–3, which has been intensively analyzed for the regulation of insulin-mediated GLUT4 (glucose transporter) localization in adipocytes, seems to be involved in corticogenesis .
Based on the reports of gene abnormalities including haploinsufficiency and heterozygous mutations, an essential role of MUNC-18-1 has been predicted in the etiology of early infantile epileptic encephalopathy with suppression-burst (EIEE; Ohtahara syndrome) and other neurodevelopmental disorders such as neonatal epileptic encephalopathy (NEE), intellectual disability (ID) and autism spectrum disorders (ASD) [8–16]. Although molecular mechanism underlying the etiology of disorders with MUNC-18-1 (also known as STXBP1) gene abnormalities remains to be elucidated, the abovementioned reports may indicate that MUNC18–1 regulates excitatory neuron positioning and synapse network formation during brain development to establish the cortical architecture. In this context, abnormal positioning of cortical neurons (a focal cortical dysplasia type 1a) has been observed in an autopsy sample from an epilepsy patient with a MUNC18–1 mutation (c.1631G > T; p.Gly544Val) .
While intensive studies have been performed for the role of Munc18–1 in the neurotransmitter release of differentiated neurons, little is known about its physiological role during corticogenesis. Pathophysiological significance of MUNC18–1 mutations in neurodevelopmental disorders also remains to be enigmatic, although EIEE-causative mutations may render MUNC-18-1 thermolabile with a strong propensity to aggregate . These mutants are assumed to form aggregates with wild type MUNC-18-1, leading to proteasomal degradation, and thus lower the level of functional MUNC-18-1 [17, 18].
Newborn cortical neurons generated in the VZ are primarily multipolar, and exhibit slow and irregular movement in the lower IZ for ~24 h. Then, they transform into a bipolar shape with a leading process and an axon in the IZ, and perform radial migration toward the pial surface [19, 20]. Strict spatiotemporal regulation of neuron migration is crucial for brain development and intellectual functioning. In this study, we found that Munc-18-1 as well as Syntaxin1A is indispensable for radial migration of cortical neuron during brain development. Munc18–1 was shown to regulate post-Golgi transport of vesicles to the plasma membrane as well as subsequent vesicle fusion at cell surface for proper distribution of proteins crucial for neuron migration, while Syntaxin1A appeared to be involved in the vesicle fusion as a downstream effector of Munc18–1. Thus, differential roles of Munc18–1 and Syntaxin1A may be critical for cortical neuron migration. Protein kinase C(PKC)-mediated phosphorylation seemed to be required for the migration regulation. Then, epilepsy-related MUNC18–1 gene abnormalities were suggested to induce a loss-of-function which may cause defective neuronal migration, leading to abnormal cytoarchitecture of cerebral cortex associated with the clinical manifestations of patients with the gene abnormalities.
Materials and methods
We followed the Fundamental Guidelines for Proper Conduct of Animal Experiments and Related Activity in Academic Research Institution under the jurisdiction of the Ministry of Education, Culture, Sports, Science and Technology, Japan. All of the protocols for animal handling and treatment were reviewed and approved by the Animal Care and Use Committee of Institute for Developmental Research, Aichi Human Service Center (Approval number, M10).
Mouse (m) Munc18–1 clone (mStxbp1) was kindly provided from Dr. T. Izumi (Gunma Univ., Japan) . pCAG-HA-N-Cadherin was from Dr. T. Kawauchi (Inst. Biomed. Res. Innov., Kobe, japan). mMunc18–2, mMunc18–3, mSyntaxin1A and mSyntaxin1B were amplified by RT-PCR with adult mouse brain RNA pool. These cDNAs were constructed into pCAG-Myc vector (Addgene Inc., Cambridge, MA). The following target sequences were inserted into pSuper-puro RNAi vector (OligoEngine, Seattle, WA); mMunc18–1#1, GGACATTGGCACAAGAATA (1558–1574), mMunc18–1#2, GAGGATGAACACTGGCGAG (942–960) and mStx1A#1, GGACATTGGCACAAGAATA (mSyntaxin1A, 1558–1574). Numbers indicate the positions from translational start sites. It should be noted that we could prepare only one RNAi vector specific for mSyntaxin1A, since Syntaxin1A and 1B are small proteins (288 aa) with high homology (84%). For RNAi-resistant versions of mMunc18–1 and mSyntaxin1A, mMunc18–1R and mSyntaxin1A, respectively, silent mutations were introduced in the target sequences as underlined (ATGGGCACTGGCATAAAAACA in mMunc18–1#1; ATGGGCACTGGCATAAAAACA in mStx1A#1). For the control RNAi experiments, we used pSuper-H1.shLuc designed against luciferase (CGTACGCGGAATACTTCGA) . By the use of pCAG-GFP-mMunc18–1R as a template, site-directed mutagenesis was performed with KOD-Plus Mutagenesis kit (Toyobo, Osaka, Japan) to generate Munc18–1 mutants at PKC- and Cyclin-dependent kinase 5(Cdk5)-phosphorylation sites (Ser306/Ser313 and Thr574, respectively), causative mutants for EIEE (c.539G > A, p.C180Y; c.1217G > A, p.R406H; c.1328 T > G, p.M443R) and NEE (c.1631G > T; p.G544 V). Chimeric cDNA, mSyntaxin1AB, containing N- and C-terminal regions of mSyntaxin1A (aa1–149) and B (aa145–255), respectively, was constructed by PCR. All constructs were verified by DNA sequencing.
Mouse monoclonal anti-Munc18–1, anti-Syntaxin1A, anti-nestin and anti-N-Cadherin antibodies were purchased from Abnova Inc. (H00006812-M01; Taipei, Taiwan), Synaptic Systems (110,111; Gottingen, Germany), R&D Systems (MAB2736; Minneapolis, MN) and BD Biosciences (610,920; San Jose, CA), respectively. Mouse monoclonal anti-Myc (#192–3) and polyclonal rabbit anti-GFP (#598) were from MBL Inc. (Nagoya, Japan). Polyclonal rabbit anti-Myc and anti-Sept11 were prepared as previously described [23, 24]. Rabbit polyclonal anti-RFP, anti-HA (hemagglutinin) and anti-N-Cadherin recognizing the extracellular domain were from Rockland Immunochemicals Inc. (#600–401-379; Gilbertsville, PA) and Santa Cruze Biotech. (sc-805 and sc-7939; Santa Cruze, CA), respectively. Polyclonal anti-Tag-1 and monoclonal anti-GM130 were from R&D systems (AF4439; Minneapolis, MN) and BD Biosciences (610,822; San Jose, CA), respectively.
Cell culture, transfection and immunofluorescence
COS7 cells were cultured essentially as previously described  and transfected with Lipofectamine 2000 (Life Technologies Japan, Tokyo) according to the manufacturer’s instruction. Immunofluorescence analyses were done as previously described . Alexa Fluor 488- or 568-labeled IgG (Life Technologies Japan) was used as a secondary antibody. 4′,6-diamidino-2-phenylindole (DAPI) was used for DNA staining. Fluorescent images were obtained using FV-1000 confocal laser microscope (Olympus, Tokyo, Japan).
In utero electroporation
In utero electroporation was performed with pregnant ICR mice essentially as previously described . Briefly, expression plasmids and/or pSuper-RNAi plasmid were injected with pCAG-GFP or pCAG-RFP (red fluorescent protein) into the lateral ventricles of embryos, followed by electroporation using CUY21 electroporator (NEPA Gene, Chiba, Japan) with 50 ms of 35 V electronic pulse for 5 times with 450 ms intervals. As for quantitative analyses of neuronal migration, distribution of GFP- or RFP-positive cells was quantified by calculation of the number of labeled cells in each region of the brain slices [22, 28].
After in utero electroporation, organotypic coronal slices (250 μm thickness) were prepared with a microtome from the anterior third of the forebrain at indicated time points, placed on insert membranes, mounted in collagen gel as previously described [29, 30]. The dishes were then cultured in a CO2 incubator chamber (5% CO2, at 37 °C) fitted onto the confocal laser microscope, and the dorsomedial region of the neocortex was examined. Approximately 8–15 optical Z sections were acquired every 5 to 15 min for 24 h, and about 10 focal planes (50-μm thickness) were merged to visualize the entire shape of the cells.
EdU (5-ethynil-2′-deoxyuridine) incorporation experiments
pCAG-GFP vector was electroporated in utero into embryos with control or shMunc#1 at embryonic day (E)14. Forty h after electroporation, pregnant mice were given an intraperitoneal injection of EdU at 25 mg/kg body weight. One h after injection, brains were fixed with 4% paraformaldehyde and frozen sections were made. GFP and EdU were detected with anti-GFP and Alexa Fluor555 azide (Life Technologies Japan), respectively.
Results were expressed as means ± SD. When data were obtained from only two groups, Student’s and Welch’s t test were used for comparison. For other experiments, the rate of cell scores was initially analyzed using the one-way analysis of variance (ANOVA). Subsequently, Tukey-Kramer least significant difference (LSD) test was applied to absolute values as a post hoc test of multiple comparisons. The level of statistical significance was considered to be p < 0.05. Statistical analysis was performed using Statcel3 software (OMS Inc., Tokorozawa, Japan).
Distribution of Munc18–1 in embryonic mouse brain
Roles of Munc18–1 in excitatory neuron migration during corticogenesis
Since Munc18–3 is also involved in corticogenesis , we asked if the observed migration defects are indeed ascribed to Munc18–1-silencing. Consequently, neither sh-Munc#1 nor #2 silenced mMunc18–3 as well as mMunc18–2 in COS7 cells, strongly suggesting that the observed migration defects were caused by Munc18–1 knockdown (Fig 2f).
At the end of radial migration, the migratory mode changes to the terminal translocation, a crucial step for the completion of neuronal migration, just beneath the marginal zone (MZ) . When we asked if Munc18–1-knockdown affects the terminal translocation, it was completed under the conditions where Munc18–1 was silenced (Additional file 2: Figure S2). The deficient neurons could enter the outermost region of the CP termed primitive cortical zone, and the tip of the leading process was attached to the MZ. These results indicate that Munc18–1 is not involved in the terminal translocation.
Time-lapse imaging of migration of Munc18–1-deficient neurons in cortical slices
In the control experiment, GFP-positive neurons normally transformed from multipolar to bipolar in the upper IZ and moved into the CP. Then, typical radial migration was visualized toward the pial surface (Fig. 4b upper panels, C left panel and Additional file 3: Video 1). On the contrary, Munc18–1-deficient cells frequently remained stranded in the upper IZ - lower CP during the imaging time period (~24 h) and the migration was significantly prevented during the analyses (Fig. 4b, lower panels, c right panel and Additional file 4: Video 2). Although the multipolar-bipolar shape transition was normal, the deficient cells could not pass the IZ-CP boundary efficiently.
On the other hand, considerable population of Munc18–1-deficient cells crossed the IZ-CP border. They, however, frequently showed an abnormal migration phenotype in the CP. Although the deficient cells were morphologically indistinguishable from control cells and maintained normal bipolar polarity from the beginning of the imaging (E17.5) (Fig. 4d and e), they displayed a migration delay with a reduced speed after entering the CP (Fig. 4e, lower panels, f and Additional files 5 and 6: Videos 3 and 4).
As for the aberrant migration phenotype, it was notably less characteristic than knockdown phenotypes of other genes. For example, SIL1 (a chaperone-related protein)-deficient neurons exhibited abnormal bipolar-multipolar shape turnover during radial migration, while Rbfox1(an RNA-splicing factor)-deficient neurons showed a stepwise migration profile [22, 37]. Meanwhile, Nr1d1 (a circadian clock protein)-knockdown neurons displayed characteristic phenotypes such as jump in a tangential direction and reverse-directed migration .
Collectively, Munc18–1 was supposed to be involved in two steps of excitatory neuron migration during corticogenesis; crossing of the IZ-CP boundary and radial migration in the CP. While migration defects may occur at the IZ-CP boundary when the RNAi effect is strong, delayed locomotion in the CP may occur when the RNAi effect is relatively weak.
Munc18–1 does not regulate neuronal progenitor proliferation
Since prolonged cell cycle results in a migration delay of newly generated neurons , we looked into the effect of Munc18–1-silencing on the cell cycle of progenitor and stem cells in the VZ/SVZ. When S-phase cells were labeled with EdU to detect DNA synthesis, Munc18–1-deficient cells entered S-phase to a similar extent as the control cells (Additional file 7: Figure S3). These results indicate that the cell cycle G1-progression rate was not statistically different between the control and the deficient cells, and that Munc18–1-silencing did not affect cell proliferation in the VZ/SVZ. Also, positioning of EdU/GFP-double positive cells in the VZ/SVZ was not affected by the knockdown (Additional file 7: Figure S3a). Taken together with the data that Munc18–1 was barely detected in the VZ/SVZ (Fig 1b), we conclude that the neuronal positioning defects by Munc18–1-knockdown were most likely to be caused by aberrant migration.
Effects of PKC- and Cdk5-mediated phosphorylation of Munc18–1 on cortical neuron migration
Cdk5 is known to play a central role in neuronal migration during corticogenesis  and phosphorylate Munc18–1 at Thr574 to regulate neuronal secretion through the modulation of interaction with Syntaxin 1A [41, 42]. Meanwhile, PKC-mediated phosphorylation of Munc18–1 at Ser306 and Ser313 has been reported to be crucial for its localization in nerve terminals , interaction with Syntaxin1A  and neurotransmitter release . Analyses with chemical inhibitors suggest possible involvement of PKC in radial migration . We thus analyzed the role(s) of PKC as well as Cdk5 in Munc18–1-mediated cortical neuron migration by examining whether Munc18–1 mutants at the phosphorylation sites by these kinases rescue the knockdown phenotype. As for Cdk5, both Munc18–1-T574D and -T574A mimicking phosphorylated and unphosphorylated states, respectively, rescued the migration defects (Additional file 8: Figure S4a). In contrast, no mutations at the PKC sites mimicking phosphorylated and unphosphorylated states could rescue the abnormal migration phenotype (Additional file 8: Figure S4b). Expression of each mutant was confirmed immunohistochemically (Additional file 8: Figure S4c).
Important role of Munc18–1-Syntaxin1A interaction in cortical neuron migration
Regulation of cortical neuron migration by Syntaxin1A
Role of Munc18–1 in Syntaxin1A localization in migrating neurons
Munc18–1 regulates intracellular trafficking of N-Cadherin
Since N-Cadherin exerts its function at cell surface, we next analyzed the involvement of Munc18–1 in vesicle fusion at cell surface. To this end, we used primary cultured cortical neurons to detect cell surface N-Cadherin as neurons were too closely packed in cortical slices to dissect. E14 mouse brain was electroporated in utero with pCAG-GFP together with the control RNAi vector or shMunc#1. After isolation at E16, neurons were cultured for 48 h and then stained for cell-surface N-Cadherin without permeabilization, followed by permeabilization for staining total N-Cadherin. Endogenous N-Cadherin was distributed on the surface of control cell bodies, whereas such distribution was suppressed when Munc18–1 was silenced (Fig. 8d and e). Cell surface distribution of N-Cadherin was also decreased in neurites of the deficient neurons as in the case of cell body (Fig. 8f).
Although transport of Syntaxin1A from Golgi to the plasma membrane region was dependent on Munc18–1, Syntaxin1A per se was not required for the post-Golgi vesicle transport when N-Cadherin was used as an indicator (Additional file 9: Figure S5a, b). On the other hand, Syntaxin1A-knockdown suppressed N-Cadherin distribution on the cell surface as in the case of Munc18–1-silencing (Additional file 9: Figure S5c). Syntaxin1A was thus supposed to regulate the vesicle fusion process to ensure proper interaction of migrating neurons with glial fibers during corticogeneis.
Collectively, while Munc18–1 is known to be essential for the vesicle priming/fusion process in the neurotransmitter release, we here clarified that 1) Munc18–1 likely regulates post-Golgi transport of vesicles containing N-Cadherin, a crucial process for radial migration of excitatory neurons during corticogenesis, and 2) subsequent vesicle fusion and distribution of N-Cadherin to migrating cell surface.
It is well accepted that Munc18–1 is a presynaptic chaperone for Syntaxin1 and regulates neurotransmitter release through the modulation of SNARE complex formation in developed neurons . Although Munc18–1-knockout mice showed complete loss of neurotransmitter secretion from synaptic vesicles, corticogenesis was morphologically normal; cortical layer structure, fiber pathways and synapse formation were completed normally . These observations suggest that cortical development including synaptic connectivity does not depend on the Munc18–1 function.
On the other hand, expression of Munc18–1 in developing cerebral cortex suggests its role in cortical development. Critical function of Munc18–1 during brain development also should be approved by MUNC18–1 gene abnormalities that cause neurodevelopmental disorders such as EIEE, NEE, ID and ASD. Little is, however, known about the significance of MUNC18–1 during brain development and in the above neurodevelopmental disorders.
In the present study, we show that Munc18–1 is involved in excitatory neuron migration during corticogenesis, based on acute knockdown experiments with in utero electroporation. The results obtained may indicate a novel role of Munc18–1 in the embryonic stage where functional synapses were not detected by electron microscopic analyses . The discrepancy between the knockout mice and acute knockdown experiments may be explained by functional redundancy by Munc18 isoforms. Considering that Munc18–2 and Munc18–3 are expressed widely, they may compensate for the loss of Munc18–1 function in the knockout mouse. Notably, Munc18–3 appeared to be essential for brain development since poorly formed axon fibers and mispositioned neurons were observed in the IZ of the null mouse . We assume that acute conditional knockdown of Munc18–1 may circumvent the compensatory effects of general gene-knockout approaches.
We here focused on the pyramidal neurons generated at ~E14.5 which form layer II/III of cerebral cortex. When Munc18–1 was silenced in utero at E14.5 and neuronal migration was monitored by time-lapse imaging from E16.5 for ~24 h, characteristic radial migration delay was observed. Given the absence of functional synapses in E17 mouse neocortex , it is plausible that Munc18–1 plays a yet unidentified role in radial migration. The basic molecular mechanism of Munc18–1 function in neuronal migration, however, might be at least partially common to that of the vesicle fusion process in neurotransmitter release of adult neurons, because intracellular vesicle trafficking is required to add new membrane and a variety of molecules to specific regions of migrating cortical neurons [40, 52]. Our results suggest that, when Syntaxin1A or N-Cadherin was used as a tracer, Munc18–1 regulates post-Golgi vesicle trafficking to the plasma membrane and subsequent vesicle fusion at cell surface in migrating neurons during corticogenesis.
Recent study reported that an epilepsy-causative C180Y mutation in MUNC18–1 destabilizes protein structure and induces protein degradation through the proteasome in vitro . Since Munc18–1 mutants analyzed here were severely degradated in cells, these mutations appeared to have loss-of-function effects. On the other hand, since it has been reported that Munc18–1 controls aggregative propensity of α-synuclein and that C180Y mutation induces abnormal aggregation of α-synuclein, the migration defects observed in this study might be attributable to the level of toxic α-synuclein aggregation . In this context, disrupted function of Munc18–1 has been shown to trigger neuronal degeneration [53, 54]. Given that clinical symptoms are different in respective patients with MUNC18–1 gene abnormalities, additional environmental or genetic factors affecting neurodegeneration are suggested in each patient.
Previous works using knockout mice and dominant-negative mutants revealed that Cdk5 plays an essential role in cortical neuron migration through regulation of cell morphology and polarity [55, 56]. On the other hand, experiments using chemical inhibitors suggest a role of PKC in the migration, although underlying molecular mechanism has been obscure . The results obtained here suggest that phsophorylation by PKC is required for Munc18–1 function in cortical neuron migration. Since the Munc18–1 mutations at the PKC-phosphorylation sites that mimic phosphorylated or unphosphorylated state both demonstrated similar effects on cortical neuron migration, balance between the phosphorylation and unphosphorylation states and/or dynamic changes of the phosphorylation status might be essential for the regulation of Munc18–1 function during the migration. Given that PKC-mediated phosphorylation is crucial for Munc18–1 localization in nerve terminals in developed neurons , PKC may be involved in the localization of Munc18–1 at specific intracellular sites, where Munc18–1 exerts its functions for proper radial migration.
Accumulating evidence supports the hypothesis that Syntaxin1A and B play important roles in brain development. As for Syntaxin1B, mutations such as truncation, in-frame insertion and deletion have been reported to cause fever-associated epilepsy syndromes with a wide phenotypic spectrum, ranging from simple febrile seizures to severe epileptic encephalopathies . Since these syndromes usually emerge from age of ~6-months, functional defects in the neurotransmitter release are possible to be the major cause of the syndromes. Meanwhile, Syntaxin1A is also related to neuronal development since its single nucleotide polymorphisms (SNPs) are significantly associated with ASD [58, 59]. Based on these observations, both Syntaxin1A and B seemingly take part in cortical development. Considering different implication of Syntaxin1A and B in neurodevelopmental disorders, they might have temporally different roles during brain development and impaired Syntaxin1A function might be related to the clinical features common to those of MUNC18–1 gene abnormalities. In this study, only Syntaxin1A showed rescue effects on Munc18–1-silencing, implying specific functional relationship of Syntaxin1A with Munc18–1 during radial migration. However, it should be noted that other mechanism(s) could contribute to the neurodevelopmental defects observed in this study, since the rescue effect on Munc18–1-silencing by Syntaxin1A expression were inefficient compared to that of Munc18–1R expression.
During corticogenesis, Munc18-1 (Stxbp1) regulates radial migration by modulating vesicle transport from Golgi to the plasma membrane and subsequent fusion, in order to distribute various proteins on the cell surface. Syntaxin1A may participate in the fusion step under the control of Munc18-1. Gene abnormalities in MUNC18-1 may result in abnormal corticogenesis, leading to neonatal epileptic encephalopathy and other neurodevelopmental disorders.
This work was supported in part by JSPS KAKENHI Grant (grant no. 16 J06511, 23590124 and 16 K07211), a grant-in-aid of the Practical Research Project for Rare/Intractable Diseases from Japan Agency for Medical Research and Development (AMED) (15ek0109040h0002), a grant-in-aid of the 24th General Assembly of the Japanese Association of Medical Science, Novartis Foundation and Takeda Science Foundation.
All authors read and approved the final manuscript.
The authors declare that they have no competing interests.
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