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• W2016194769 abstract "A de novo 9q33.3-q34.11 microdeletion involving STXBP1 has been found in one of four individuals (group A) with early-onset West syndrome, severe hypomyelination, poor visual attention, and developmental delay. Although haploinsufficiency of STXBP1 was involved in early infantile epileptic encephalopathy in a previous different cohort study (group B), no mutations of STXBP1 were found in two of the remaining three subjects of group A (one was unavailable). We assumed that another gene within the deletion might contribute to the phenotype of group A. SPTAN1 encoding α-II spectrin, which is essential for proper myelination in zebrafish, turned out to be deleted. In two subjects, an in-frame 3 bp deletion and a 6 bp duplication in SPTAN1 were found at the initial nucleation site of the α/β spectrin heterodimer. SPTAN1 was further screened in six unrelated individuals with WS and hypomyelination, but no mutations were found. Recombinant mutant (mut) and wild-type (WT) α-II spectrin could assemble heterodimers with β-II spectrin, but α-II (mut)/β-II spectrin heterodimers were thermolabile compared with the α-II (WT)/β-II heterodimers. Transient expression in mouse cortical neurons revealed aggregation of α-II (mut)/β-II and α-II (mut)/β-III spectrin heterodimers, which was also observed in lymphoblastoid cells from two subjects with in-frame mutations. Clustering of ankyrinG and voltage-gated sodium channels at axon initial segment (AIS) was disturbed in relation to the aggregates, together with an elevated action potential threshold. These findings suggest that pathological aggregation of α/β spectrin heterodimers and abnormal AIS integrity resulting from SPTAN1 mutations were involved in pathogenesis of infantile epilepsy. A de novo 9q33.3-q34.11 microdeletion involving STXBP1 has been found in one of four individuals (group A) with early-onset West syndrome, severe hypomyelination, poor visual attention, and developmental delay. Although haploinsufficiency of STXBP1 was involved in early infantile epileptic encephalopathy in a previous different cohort study (group B), no mutations of STXBP1 were found in two of the remaining three subjects of group A (one was unavailable). We assumed that another gene within the deletion might contribute to the phenotype of group A. SPTAN1 encoding α-II spectrin, which is essential for proper myelination in zebrafish, turned out to be deleted. In two subjects, an in-frame 3 bp deletion and a 6 bp duplication in SPTAN1 were found at the initial nucleation site of the α/β spectrin heterodimer. SPTAN1 was further screened in six unrelated individuals with WS and hypomyelination, but no mutations were found. Recombinant mutant (mut) and wild-type (WT) α-II spectrin could assemble heterodimers with β-II spectrin, but α-II (mut)/β-II spectrin heterodimers were thermolabile compared with the α-II (WT)/β-II heterodimers. Transient expression in mouse cortical neurons revealed aggregation of α-II (mut)/β-II and α-II (mut)/β-III spectrin heterodimers, which was also observed in lymphoblastoid cells from two subjects with in-frame mutations. Clustering of ankyrinG and voltage-gated sodium channels at axon initial segment (AIS) was disturbed in relation to the aggregates, together with an elevated action potential threshold. These findings suggest that pathological aggregation of α/β spectrin heterodimers and abnormal AIS integrity resulting from SPTAN1 mutations were involved in pathogenesis of infantile epilepsy. West syndrome (WS) is a common infantile epileptic syndrome characterized by brief tonic spasms, an electroencephalogram pattern called hypsarrhythmia, and mental retardation.1Kato M. A new paradigm for West syndrome based on molecular and cell biology.Epilepsy Res. 2006; 70: S87-S95Abstract Full Text Full Text PDF PubMed Scopus (55) Google Scholar Brain malformations and metabolic disorders can be underlying causes of WS, but many cases remain etiologically unexplained.1Kato M. A new paradigm for West syndrome based on molecular and cell biology.Epilepsy Res. 2006; 70: S87-S95Abstract Full Text Full Text PDF PubMed Scopus (55) Google Scholar Only two causative genes, ARX (MIM ∗300382) and CDKL5 (MIM ∗300203), are mutated in a subset of familial and sporadic X-linked WS cases (ISSX1 and ISSX2 [MIM #308350 and #300672]).2Bahi-Buisson N. Nectoux J. Rosas-Vargas H. Milh M. Boddaert N. Girard B. Cances C. Ville D. Afenjar A. Rio M. et al.Key clinical features to identify girls with CDKL5 mutations.Brain. 2008; 131: 2647-2661Crossref PubMed Scopus (189) Google Scholar, 3Strømme P. Mangelsdorf M.E. Shaw M.A. Lower K.M. Lewis S.M. Bruyere H. Lütcherath V. Gedeon A.K. Wallace R.H. Scheffer I.E. et al.Mutations in the human ortholog of Aristaless cause X-linked mental retardation and epilepsy.Nat. Genet. 2002; 30: 441-445Crossref PubMed Scopus (368) Google Scholar, 4Kato M. Das S. Petras K. Sawaishi Y. Dobyns W.B. Polyalanine expansion of ARX associated with cryptogenic West syndrome.Neurology. 2003; 61: 267-276Crossref PubMed Google Scholar Early infantile epileptic encephalopathy with suppression-burst (EIEE) is the earliest form of infantile epileptic syndrome.5Djukic A. Lado F.A. Shinnar S. Moshé S.L. Are early myoclonic encephalopathy (EME) and the Ohtahara syndrome (EIEE) independent of each other?.Epilepsy Res. 2006; 70: S68-S76Abstract Full Text Full Text PDF PubMed Scopus (52) Google Scholar, 6Ohtahara S. Yamatogi Y. Ohtahara syndrome: With special reference to its developmental aspects for differentiating from early myoclonic encephalopathy.Epilepsy Res. 2006; 70: S58-S67Abstract Full Text Full Text PDF PubMed Scopus (106) Google Scholar The transition from EIEE to WS occurs in 75% of individuals with EIEE, suggesting a common pathological mechanism between these two syndromes.5Djukic A. Lado F.A. Shinnar S. Moshé S.L. Are early myoclonic encephalopathy (EME) and the Ohtahara syndrome (EIEE) independent of each other?.Epilepsy Res. 2006; 70: S68-S76Abstract Full Text Full Text PDF PubMed Scopus (52) Google Scholar, 6Ohtahara S. Yamatogi Y. Ohtahara syndrome: With special reference to its developmental aspects for differentiating from early myoclonic encephalopathy.Epilepsy Res. 2006; 70: S58-S67Abstract Full Text Full Text PDF PubMed Scopus (106) Google Scholar We have recently reported that de novo mutations of STXBP1 (MIM ∗602926) cause EIEE.7Saitsu H. Kato M. Mizuguchi T. Hamada K. Osaka H. Tohyama J. Uruno K. Kumada S. Nishiyama K. Nishimura A. et al.De novo mutations in the gene encoding STXBP1 (MUNC18-1) cause early infantile epileptic encephalopathy.Nat. Genet. 2008; 40: 782-788Crossref PubMed Scopus (389) Google Scholar Spectrins are submembranous scaffolding proteins involved in the stabilization of membrane proteins.8Bennett V. Healy J. Organizing the fluid membrane bilayer: Diseases linked to spectrin and ankyrin.Trends Mol. Med. 2008; 14: 28-36Abstract Full Text Full Text PDF PubMed Scopus (133) Google Scholar, 9Bennett V. Baines A.J. Spectrin and ankyrin-based pathways: Metazoan inventions for integrating cells into tissues.Physiol. Rev. 2001; 81: 1353-1392Crossref PubMed Scopus (755) Google Scholar Spectrins are flexible and long molecules consisting of α and β subunits, which are assembled in an antiparallel side-by-side manner into heterodimers. Heterodimers form by end-to-end tetramers integrating into the membrane cytoskelton.8Bennett V. Healy J. Organizing the fluid membrane bilayer: Diseases linked to spectrin and ankyrin.Trends Mol. Med. 2008; 14: 28-36Abstract Full Text Full Text PDF PubMed Scopus (133) Google Scholar, 9Bennett V. Baines A.J. Spectrin and ankyrin-based pathways: Metazoan inventions for integrating cells into tissues.Physiol. Rev. 2001; 81: 1353-1392Crossref PubMed Scopus (755) Google Scholar The spectrin repertoire in humans includes two α subunits and five β subunits. Defects of erythroid α-I and β-I spectrins and neuronal β-III spectrin are associated with hereditary spherocytosis (SPH3 and SPH2 [MIM #270970 and +182870]) and spinocerebellar ataxia type 5 (SCA5 [MIM #600224]), respectively.8Bennett V. Healy J. Organizing the fluid membrane bilayer: Diseases linked to spectrin and ankyrin.Trends Mol. Med. 2008; 14: 28-36Abstract Full Text Full Text PDF PubMed Scopus (133) Google Scholar, 10Ikeda Y. Dick K.A. Weatherspoon M.R. Gincel D. Armbrust K.R. Dalton J.C. Stevanin G. Dürr A. Zühlke C. Bürk K. et al.Spectrin mutations cause spinocerebellar ataxia type 5.Nat. Genet. 2006; 38: 184-190Crossref PubMed Scopus (263) Google Scholar, 11Perrotta S. Gallagher P.G. Mohandas N. Hereditary spherocytosis.Lancet. 2008; 372: 1411-1426Abstract Full Text Full Text PDF PubMed Scopus (365) Google Scholar The α-II spectrin is considered as the major α spectrin expressed in nonerythroid cells, and α-II/β-II spectrin heterodimers are the predominant species in these cells.9Bennett V. Baines A.J. Spectrin and ankyrin-based pathways: Metazoan inventions for integrating cells into tissues.Physiol. Rev. 2001; 81: 1353-1392Crossref PubMed Scopus (755) Google Scholar, 12Meary F. Metral S. Ferreira C. Eladari D. Colin Y. Lecomte M.-C. Nicolas G. A mutant alphaII-spectrin designed to resist calpain and caspase cleavage questions the functional importance of this process in vivo.J. Biol. Chem. 2007; 282: 14226-14237Crossref PubMed Scopus (18) Google Scholar Abnormal development of nodes of Ranvier and destabilizing initial clusters of voltage-gated sodium channels (VGSC) were observed in zebrafish α-II spectrin mutants harboring a nonsense mutation. The mutants also showed impaired myelination in motor nerves and in the dorsal spinal cord, suggesting that α-II spectrin plays important roles in the maintenance of the integrity of myelinated axons.13Voas M.G. Lyons D.A. Naylor S.G. Arana N. Rasband M.N. Talbot W.S. alphaII-spectrin is essential for assembly of the nodes of Ranvier in myelinated axons.Curr. Biol. 2007; 17: 562-568Abstract Full Text Full Text PDF PubMed Scopus (70) Google Scholar Here, we describe three cases of early-onset WS with cerebral hypomyelination harboring SPTAN1 (MIM ∗182810) aberrations. Two individuals with in-frame mutations showed more severe phenotypes than one individual with SPTAN1 and STXBP1 deletion. In-frame mutations of SPTAN1 result in aggregation of α-II (mut)/β-II and α-II (mut)/β-III spectrin heterodimers, suggesting dominant-negative effects of the mutations. Spectrin aggregation is associated with disturbed clustering of VGSC and an elevated action potential threshold. Our findings revealed essential roles of α-II spectrin in human brain development and suggest that abnormal AIS is possibly involved in pathogenesis of infantile epilepsy. Subjects 1, 2, and 3 have been originally reported as three of four individuals with early onset WS, severe hypomyelination, reduced white matter, and developmental delay (group A: subjects 1, 2, and 3 were previously named as No. 2, No. 1, and No. 3, respectively, and No. 4 was unavailable for this study).14Tohyama J. Akasaka N. Osaka H. Maegaki Y. Kato M. Saito N. Yamashita S. Ohno K. Early onset West syndrome with cerebral hypomyelination and reduced cerebral white matter.Brain Dev. 2008; 30: 349-355Abstract Full Text Full Text PDF PubMed Scopus (32) Google Scholar Subject 1 has been shown to possess a 9q33.3-q34.11 microdeletion including STXBP1.7Saitsu H. Kato M. Mizuguchi T. Hamada K. Osaka H. Tohyama J. Uruno K. Kumada S. Nishiyama K. Nishimura A. et al.De novo mutations in the gene encoding STXBP1 (MUNC18-1) cause early infantile epileptic encephalopathy.Nat. Genet. 2008; 40: 782-788Crossref PubMed Scopus (389) Google Scholar Clinical information of these three subjects with SPTAN1 aberrations is updated in Table S1 available online. We screened for SPTAN1 mutations in a total of eight unrelated individuals with WS accompanied by severe hypomyelination without episodes of prenatal incidents or neonatal asphyxia (six males and two females, including subjects 2 and 3 of group A). Individuals with these two distinctive features (WS and severe hypomyelination) are relatively rare. These eight patients were totally different from the previously investigated 13 EIEE patients (group B).7Saitsu H. Kato M. Mizuguchi T. Hamada K. Osaka H. Tohyama J. Uruno K. Kumada S. Nishiyama K. Nishimura A. et al.De novo mutations in the gene encoding STXBP1 (MUNC18-1) cause early infantile epileptic encephalopathy.Nat. Genet. 2008; 40: 782-788Crossref PubMed Scopus (389) Google Scholar Screening tests for metabolic disorders (lactate, amino acids, and uric organic acids) were normal in all subjects. ARX and CDKL5 were not mutated in the six male and two female patients, respectively. The diagnosis was made on the basis of clinical features, including tonic spasms with clustering, arrest of psychomotor development, and hypsarrhythmia on electroencephalogram, as well as brain magnetic resonance imaging (MRI) findings. Experimental protocols were approved by the Committee for Ethical Issues at Yokohama City University School of Medicine. Informed consent was obtained from all individuals included in this study, in agreement with the requirements of Japanese regulations. Genomic DNA was obtained from peripheral blood leukocytes by standard methods, amplified by GenomiPhi version 2 (GE Healthcare, Buckinghamshire, UK), and used for mutational screening. Exons 2 to 57, covering the SPTAN1 coding region (of transcript variant 1, GenBank accession number NM_001130438), were screened by high-resolution melting curve (HRM) analysis as previously described.7Saitsu H. Kato M. Mizuguchi T. Hamada K. Osaka H. Tohyama J. Uruno K. Kumada S. Nishiyama K. Nishimura A. et al.De novo mutations in the gene encoding STXBP1 (MUNC18-1) cause early infantile epileptic encephalopathy.Nat. Genet. 2008; 40: 782-788Crossref PubMed Scopus (389) Google Scholar In transcript variant 2 (GenBank accession number NM_003127), the only difference is that exon 37 of variant 1 was missing. PCR conditions and primer sequences are shown in Table S2. If a sample showed an aberrant melting curve shift, the PCR product was sequenced. All mutations were also verified on PCR products directly via genomic DNA (not amplified by GenomiPhi) as a template. DNAs from 250 Japanese normal controls were screened for the two in-frame SPTAN1 mutations by HRM analysis. Normal controls which showed aberrant melting curve shift were sequenced. For all families showing de novo mutations, parentage was confirmed by microsatellite analysis as previously described.7Saitsu H. Kato M. Mizuguchi T. Hamada K. Osaka H. Tohyama J. Uruno K. Kumada S. Nishiyama K. Nishimura A. et al.De novo mutations in the gene encoding STXBP1 (MUNC18-1) cause early infantile epileptic encephalopathy.Nat. Genet. 2008; 40: 782-788Crossref PubMed Scopus (389) Google Scholar Biological parentage was judged if more than four informative markers were compatible and other uninformative markers showed no discrepancies. A full-length human SPTAN1 cDNA was prepared by PCR with first-strand cDNA derived from a human lymphoblastoid cells (LCL) and an IMAGE clone (clone ID 5211391) as a template. The obtained SPTAN1 cDNA was sequenced and confirmed to be identical to a RefSeq mRNA (amino acids 1–2477, GenBank accession number NM_001130438) except for two synonymous base substitutions that have been registered in dbSNP as rs2227864 and rs2227862. Site-directed mutagenesis via a KOD-Plus-Mutagenesis kit (Toyobo, Osaka, Japan) was used to generate SPTAN1 mutants including c.6619_6621 del (p.E2207 del) and c.6923_6928 dup (p.R2308_M2309 dup). A C-terminal Flag-tag was introduced by PCR. All variant cDNAs were verified by sequencing. C-terminal Flag-tagged WT and mutant SPTAN1 cDNAs were cloned into the pCIG vector15Megason S.G. McMahon A.P. A mitogen gradient of dorsal midline Wnts organizes growth in the CNS.Development. 2002; 129: 2087-2098Crossref PubMed Google Scholar, 16Niwa H. Yamamura K. Miyazaki J. Efficient selection for high-expression transfectants with a novel eukaryotic vector.Gene. 1991; 108: 193-199Crossref PubMed Scopus (4431) Google Scholar to express C-terminal Flag-tagged α-II spectrin as well as nuclear-localized EGFP. WT and mutant SPTAN1 cDNAs were also cloned into the pCAG-EGFP-C1 vector, in which EGFP gene and multiple cloning sites of pEGFP-C1 vector (Clontech, Mountain View, CA) are introduced into a CAG-promoter vector,15Megason S.G. McMahon A.P. A mitogen gradient of dorsal midline Wnts organizes growth in the CNS.Development. 2002; 129: 2087-2098Crossref PubMed Google Scholar, 16Niwa H. Yamamura K. Miyazaki J. Efficient selection for high-expression transfectants with a novel eukaryotic vector.Gene. 1991; 108: 193-199Crossref PubMed Scopus (4431) Google Scholar to express N-terminal EGFP-tagged α-II spectrin. For protein expression in Escherichia coli, WT and mutant SPTAN1 cDNAs (amino acids 1445–2477, the last eight spectrin repeats and the EF hand domain) were cloned into pGEX6P-3 (GE Healthcare) to generate glutathione S-transferase (GST) fusion proteins. Human SPTBN1 cDNAs (amino acids 1–1139, GenBank accession number NM_003128, including the actin binding domain and eight spectrin repeats) were prepared by PCR via first-strand cDNA derived from a human LCL, and were cloned into pET-24a (Merck, Darmstadt, Germany) to generate His-tag fusion proteins. Proteins were expressed in Escherichia coli BL21 (DE3). Bacteria were grown at 37°C in Lysogeny Broth media with 300 μg/ml ampicillin to a density yielding an absorbance at 600 nm of 0.8. Protein expression was then induced with 1 mM isopropyl-β-d-thiogalactoside (IPTG) at 20°C overnight. Cells were collected by centrifugation and lysed by sonication. Proteins were purified by affinity chromatography with Glutathione Sepharose High Performance (GE Healthcare) for GST-α-II spectrin or HisTrap HP (GE Healthcare) for β-II spectrin-His. α-II spectrins were further purified by HiTrap Q HP (GE Healthcare) and Superdex-200 (GE Healthcare) columns in a buffer containing 150 mM NaCl, 20 mM sodium phosphate buffer (pH 7.5), and 2 mM dithiothreitol (DTT). β-II spectrin was further purified by Superdex-200 (GE Healthcare) columns in a buffer containing 1 M NaCl, 20 mM sodium phosphate buffer (pH 7.5), and 2 mM DTT. For the GST pull-down assay to examine the assembly of α-II/β-II heterodimers, 0.5 μM GST-α-II spectrin (WT, del mut, or dup mut) or 1 μM GST were preincubated with 1 μM β-II spectrin-His for 1 hr at 4°C with gentle agitation in binding buffer containing 150 mM NaCl, 20 mM sodium phosphate buffer (pH 7.5), and 2 mM DTT. The reaction mixture (100 μl) was transferred onto an Ultrafree-MC (Millipore, Billerica, MA), containing 50 μl of a 75% slurry of Glutathione Sepharose 4B equilibrated in binding buffer, and incubated overnight at 4°C. Unbound proteins were recovered by centrifugation at 500 × g for 2 min. The beads were washed three times with the binding buffer. The bound molecules were eluted with a buffer containing 100 mM NaCl, 20 mM sodium phosphate buffer (pH 7.5), 5 mM DTT, 1 mM EDTA, and 50 mM reduced glutathione. The eluted fractions were analyzed by SDS-PAGE, and protein bands were visualized by staining with Coomassie brilliant blue. For the analytical gel filtration experiments, 3.3 μM GST-α-II spectrin (WT, del mut, or dup mut) were preincubated with or without 3.3 μM β-II spectrin-His for 3 hr at 4°C with gentle agitation in a binding buffer containing 150 mM NaCl, 20 mM sodium phosphate buffer (pH 7.5), and 2 mM DTT. The samples were analyzed by Superdex-200 column equilibrated in binding buffer. The eluted fractions were analyzed by SDS-PAGE and protein bands were visualized by staining with Coomassie brilliant blue. The structure of human α-II spectrin was predicted by homology modeling with Phyre,17Kelley L.A. Sternberg M.J. Protein structure prediction on the Web: A case study using the Phyre server.Nat. Protoc. 2009; 4: 363-371Crossref PubMed Scopus (3451) Google Scholar based on sequence homology between human α-II spectrin (1981–2315 aa) and chicken brain alpha spectrin (1662–1982 aa) (Protein data bank ID, 1U4Q).18Kusunoki H. Minasov G. Macdonald R.I. Mondragón A. Independent movement, dimerization and stability of tandem repeats of chicken brain α-spectrin.J. Mol. Biol. 2004; 344: 495-511Crossref PubMed Scopus (87) Google Scholar The structure and positions of mutations were illustrated by PyMOL with the crystal structure of 1U4Q. For circular dichroism (CD) measurements, GST-α-II spectrin were digested with human rhinovirus 3C protease at 4°C, and then the GST-tag was removed by affinity chromatography with glutathione sepharose 4B (GE Healthcare). We measured far-UV CD spectra and estimated the secondary structure as previously described.7Saitsu H. Kato M. Mizuguchi T. Hamada K. Osaka H. Tohyama J. Uruno K. Kumada S. Nishiyama K. Nishimura A. et al.De novo mutations in the gene encoding STXBP1 (MUNC18-1) cause early infantile epileptic encephalopathy.Nat. Genet. 2008; 40: 782-788Crossref PubMed Scopus (389) Google Scholar In brief, the experiments were performed in 20 mM sodium phosphate buffer (pH 7.5) containing 150 mM NaCl, 2 mM DTT with or without 1 mM CaCl2, which stabilizes the structure of the EF hand domain. α-II and β-II spectrin concentration was adjusted to 1.7 μM (without CaCl2) and 1.5 μM (with CaCl2). Melting (transition midpoint) temperature (Tm) was calculated by fitting a sigmoid-function equation with KaleidaGraph (Synergy Software, Reading, PA). The data from three independent experiments were averaged and the SD was calculated. Similar results were obtained in the presence or absence of 1 mM CaCl2. For primary neuronal cultures for immunofluorescence, cortexes dissected from mice (embryonic days 14 to 15) were dissociated in 0.05% trypsin-EDTA solution (Invitrogen, Carlsbad, CA), and triturated with a Pasteur pipette. The dissociated cells were plated on 200 μg/ml poly-D-lysine (Millipore)/20 μg/ml laminin (Invitrogen)-coated glass coverslips at a density of 15,000 cells/cm2. Expression vectors were introduced at the time of dissociation by electroporation, with the Amaxa Mouse Neuron Nucleofector kit (Lonza, Tokyo, Japan) according to the manufacturer's protocol (Program O-005), and 2 μg plasmid DNA per condition. After cortical neurons attached to coverslips, the medium was changed from normal medium (10% FBS in DMEM) to maintaining medium (2% B27 and 1 × penicillin-streptomycin-glutamine in Neurobasal [Invitrogen]). Half of the medium was replaced with an equal volume of maintaining medium every 4 days. LCLs were grown in RPMI 1640 medium supplemented with 10% FBS, 1 × antibiotic-antimycotic (Invitrogen), and 8 μg/ml tylosin (Sigma, Tokyo, Japan) at 37°C in a 5% CO2 incubator. For the immunofluorescence imaging study, LCLs were plated on coated coverslips as described above for 3–6 hr. Neurons and LCLs were fixed with 2% paraformaldehyde in PBS for 15 min and permeabilized with 0.1% Triton X-100 for 5 min. For detection of VGSCs, cells were fixed with methanol at −20°C for 10 min. Cells were then blocked with 10% normal goat serum for 30 min. Primary antibodies used for the study were shown in figure legends. Secondary antibodies, highly purified to minimize cross-reactivity, were used: Alexa-488-conjugated goat anti-mouse, anti-rabbit, and anti-chicken (Invitrogen), and Cy3-conjugated goat anti-mouse, anti-rabbit, and anti-chicken (Jackson ImmunoResearch, West Grove, PA). Coverslips were mounted with Vectashield (Vector Laboratories, Burlingame, CA) that contained 4,6-diamidino-2-phenylindole (DAPI) and visualized with an AxioCam MR CCD fitted to Axioplan2 fluorescence microscope (Carl Zeiss, Oberkochen, Germany). We captured images with Axio Vision 4.6 software (Carl Zeiss). Immunofluorescence of aggregated mutant α/β spectrins was much brighter than WT α/β spectrins, leading to constant short exposure time compared with the WT. For detection of ankyrinG and VGSCs, the exposure time was fixed in a series of experiments in order to enable direct comparison between different samples. For evaluation of ankyrinG and VGSC expression, 50 isolated transfected neurons were analyzed in each experiment, and representative cells were photographed. The results were confirmed at least in three independent experiments. Mouse neocortices at embryonic day 15 were dissociated and plated on poly-L-lysine-coated plastic coverslips (Cell desk LF, MS-0113L; Sumitomo Bakelite, Tokyo, Japan) at a density of about 100,000 cells/cm2. 1 μg of expression vector for either WT, del mut, or dup mut α-II spectrin was introduced at the time of dissociation by electroporation with an Amaxa Mouse Neuron Nucleofector kit (Lonza). Primary cortical neurons were cultured in neurobasal medium supplemented with B27 and penicillin-streptomycin-glutamine (Invitrogen). During the culture period, one-half of the medium was changed every day. Whole-cell patch-clamp recordings were obtained from mice neocortical neurons at 9 days in vitro (DIV) neuronal culture. A coverslip was assembled to recording chambers on the stages of upright microscopes (Olympus, Tokyo, Japan) and continuously perfused with oxygenated, standard artificial cerebrospinal fluid (ACSF) at a flow rate of 2 ml/min and a temperature of 30°C. The standard ACSF solution contained the following (mM): 126 NaCl, 2.5 KCl, 1.25 NaH2PO4, 2.0 MgSO4, 2.0 CaCl2, 26.0 NaHCO3, and 20.0 glucose. The images of cells were acquired with Olympus BX51 or BX61 microscopes equipped with electron-multiplying CCD (EMCCD) cameras, #C9100-13 or #C9100-02 (Hamamatsu Photonics, Hamamatsu, Japan), respectively. Transfected cells were selected for recordings by their fluorescence in the nucleus via a 40× water-immersion objective lens (UMPlanFI, Olympus). Membrane currents and membrane potentials were recorded with an Axopatch 700A and 200B amplifier, respectively. Signals were low-pass filtered at 10 kHz and digitized at 50 kHz by means of Digidata 1332A data-acquisition system (Molecular Devices, Tokyo, Japan). Passive membrane properties and action potentials were recorded with patch pipettes (5.8–8.0 MΩ) filled with the following intercellular solutions (in mM): 130 K-methanesulfonic acid, 10 KCl, 2 MgCl2, 0.1 EGTA, and 10 HEPES (pH 7.3) with KOH. For current clamp experiments, cells were held at −60 mV by constant current injection as needed, and their firing pattern were recorded in response to sustained depolarizing current injections (500 ms duration, +10 pA increments) to analyze the input-output relationship in each cell. A single action potential was also evoked to determine their firing threshold. The injection current amplitude (10 ms duration) was increased in 2–10 pA increments from a subthreshold to an intensity well beyond threshold. Voltage-clamp studies for sodium currents were carried out with patch pipettes (5.8–8.0 MΩ) filled with the following intercellular solutions (mM): 145 tetraethylammonium-Cl, 15 NaCl, 2 MgCl2, 10 EGTA, 10 HEPES, 3 MgATP, and 0.4 GTP (pH 7.4) with NaOH. Series resistance was usually below 20 MΩ and compensated by 70%–80%. The remaining linear capacitive and leakage currents were subtracted off-line by scaling average traces recorded at hyperpolarized voltages. Voltage-dependent inward sodium currents were elicited by 500 ms depolarizing steps in 5 mV increments from −90 to +10 mV at a holding membrane potential of −90 mV. For measuring the inactivation protocol of sodium currents, 500 ms long prepotentials started at – 90 mV and were incremented by 5 mV steps while the command potential was kept constant at −30 mV. The current elicited during each test pulse was normalized to the maximal current (I/Imax). Statistical analyses were made with two-way repeated-measures ANOVA followed by a Bonferroni post-test for analysis of the input-output relationship and current amplitude at every voltage step. One-way ANOVA followed by Dunnet's posthoc test was applied for threshold, peak current, kinetics of action potentials, and passive membrane properties. The results are given as mean ± SEM, and threshold p value for statistical significance was 0.05. Statistical comparisons were performed with the Prism 4.0 (GraphPad software, La Jolla, CA). We previously reported a de novo 9q33.3-q34.11 microdeletion involving STXBP1 in an individual with EIEE, who transited afterward to WS at the age of 3 months (subject 1).7Saitsu H. Kato M. Mizuguchi T. Hamada K. Osaka H. Tohyama J. Uruno K. Kumada S. Nishiyama K. Nishimura A. et al.De novo mutations in the gene encoding STXBP1 (MUNC18-1) cause early infantile epileptic encephalopathy.Nat. Genet. 2008; 40: 782-788Crossref PubMed Scopus (389) Google Scholar Subject 1 was originally reported as one of four individuals (group A) who showed early onset WS and severe cerebral hypomyelination (as patient No. 2).14Tohyama J. Akasaka N. Osaka H. Maegaki Y. Kato M. Saito N. Yamashita S. Ohno K. Early onset West syndrome with cerebral hypomyelination and reduced cerebral white matter.Brain Dev. 2008; 30: 349-355Abstract Full Text Full Text PDF PubMed Scopus (32) Google Scholar It is likely that haploinsufficiency of STXBP1 caused EIEE and subsequent WS in subject 1;7Saitsu H. Kato M. Mizuguchi T. Hamada K. Osaka H. Tohyama J. Uruno K. Kumada S. Nishiyama K. Nishimura A. et al.De novo mutations in the gene encoding STXBP1 (MUNC18-1) cause early infantile epileptic encephalopathy.Nat. Genet. 2008; 40: 782-788Crossref PubMed Scopus (389) Google Scholar however, no mutations of STXBP1 were found in two of the remaining three individuals of group A (subjects 2 and 3, previously described as No. 1 and No. 3, and No. 4 was unavailable for this study).14Tohyama J. Akasaka N. Osaka H. Maegaki Y. Kato M. Saito N. Yamashita S. Ohno K. Early onset West syndrome with cerebral hypomyelination and reduced cerebral white matter.Brai" @default.
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