Yuta Komoike

A functional analysis of GABARAP on 17p13.1 by knockdown zebrafish

Array-based comparative genomic hybridization identified a 2.3-Mb microdeletion of 17p13.2p13.1 in a boy presenting with moderate mental retardation, intractable epilepsy and dysmorphic features. This deletion region was overlapped with the previously proposed shortest region overlapped for microdeletion of 17p13.1 in patients with mental retardation, microcephaly, microretrognathia and abnormal magnetic resonance imaging (MRI) findings of cerebral white matter, in which at least 17 known genes are included. Among them, DLG4/PSD95, GPS2, GABARAP and KCTD11 have a function in neuronal development. Because of the functional importance, we paid attention to DLG4/PSD95 and GABARAP, and analyzed zebrafish in which the zebrafish homolog of human DLG4/PSD95 and GABARAP was knocked down and found that gabarap knockdown resulted in small head and hypoplastic mandible. This finding would be similar to the common findings of the patients with 17p13.1 deletions. Although there were no pathogenic mutations in DLG4/PSD95 or GABARAP in a cohort study with 142 patients with idiopathic developmental delay with/without epilepsy, further studies would be required for genes included in this region.

Zebrafish Gene Knockdowns Imply Roles for Human YWHAG in Infantile Spasms and Cardiomegaly

Williams‐Beuren syndrome (WBS) is a neurodevelopmental disorder presenting with an elfin‐like face, supravalvular aortic stenosis, a specific cognitive‐behavioral profile, and infantile hypercalcemia. We encountered two WBS patients presenting with infantile spasms, which is extremely rare in WBS. Array comparative genomic hybridization (aCGH) and fluorescent in situ hybridization (FISH) analyses revealed atypical 5.7‐Mb and 4.1‐Mb deletions at 7q11.23 in the two patients, including the WBS critical region and expanding into the proximal side and the telomeric side, respectively. On the proximal side, AUTS2 and CALN1 may contribute to the phenotype. On the telomeric side, there are two candidate genes HIP1 and YWHAG. Because detailed information of them was unavailable, we investigated their functions using gene knockdowns of zebrafish. When zebrafish ywhag1 was knocked down, reduced brain size and increased diameter of the heart tube were observed, indicating that the infantile spasms and cardiomegaly seen in the patient with the telomeric deletion may be derived from haploinsufficiency of YWHAG. genesis 48:233–243, 2010.

Experimental Evidence Shows Salubrinal, an eIF2α Dephosphorylation Inhibitor, Reduces Xenotoxicant-Induced Cellular Damage.

Accumulating evidence indicates that endoplasmic reticulum (ER) stress and the subsequent unfolded protein response (UPR) are involved in the pathogenesis of not only the protein misfolding disorders such as certain neurodegenerative and metabolic diseases, but also in the cytotoxicity of environmental pollutants, industrial chemicals, and drugs. Thus, the modulation of ER stress signaling pathways is an important issue for protection against cellular damage induced by xenotoxicants. The substance salubrinal has been shown to prevent dephosphorylation of the eukaryotic translation initiation factor 2 alpha (eIF2α). The phosphorylation of eIF2α appears to be cytoprotective during ER stress, because inhibition of the translation initiation activity of eIF2α reduces global protein synthesis. In addition, the expression of activating transcription factor 4 (ATF4), a transcription factor that induces the expression of UPR target genes, is up-regulated through alternative translation. This review shows that salubrinal can protect cells from the damage induced by a wide range of xenotoxicants, including environmental pollutants and drugs. The canonical and other possible mechanisms of cytoprotection by salubrinal from xenotoxicant-induced ER stress are also discussed.

Characterization of a novel KRAS mutation identified in Noonan syndrome

Noonan syndrome (NS) is the most common non‐chromosomal syndrome seen in children and is characterized by short stature, dysmorphic facial features, chest deformity, a wide range of congenital heart defects and developmental delay of variable degree. Mutations in the Ras/mitogen‐activated protein kinase (MAPK) signaling pathways cause about 70% of NS cases with a KRAS mutation present in about 2%. In a cohort of 65 clinically confirmed NS patients of Japanese origin, we screened for mutations in the RAS genes by direct sequencing. We found a novel mutation in KRAS with an amino acid substitution of asparagine to serine at codon 116 (N116S). We analyzed the biological activity of this mutant by ectopic expression of wild‐type or mutant KRAS. NS‐associated KRAS mutation resulted in Erk activation and active Ras–GTP levels, and exhibited mild cell proliferation. In addition, kras‐targeted morpholino knocked‐down zebrafish embryos caused heart and craniofacial malformations, while the expression of mutated kras resulted in maldevelopment of the heart. Our findings implicate that N116S change in KRAS is a hyperactive mutation which is a causative agent of NS through maldevelopment of the heart.

Exposure to tributyltin induces endoplasmic reticulum stress and the unfolded protein response in zebrafish

Tributyltin (TBT) is a major marine contaminant and causes endocrine disruption, hepatotoxicity, immunotoxicity, and neurotoxicity. However, the molecular mechanisms underlying the toxicity of TBT have not been fully elucidated. We examined whether exposure to TBT induces the endoplasmic reticulum (ER) stress response in zebrafish, a model organism. Zebrafish-derived BRF41 fibroblast cells were exposed to 0.5 or 1 μM TBT for 0.5-16 h and subsequently lysed and immunoblotted to detect ER stress-related proteins. Zebrafish embryos, grown until 32 h post fertilization (hpf), were exposed to 1 μM TBT for 16 h and used in whole mount in situ hybridization and immunohistochemistry to visualize the expression of ER chaperones and an ER stress-related apoptosis factor. Exposure of the BRF41 cells to TBT caused phosphorylation of the zebrafish homolog of protein kinase RNA-activated-like ER kinase (PERK), eukaryotic translation initiation factor 2 alpha (eIF2α), and inositol-requiring enzyme 1 (IRE1), characteristic splicing of X-box binding protein 1 (XBP1) mRNA, and enhanced expression of activating transcription factor 4 (ATF4) protein. In TBT-exposed zebrafish embryos, ectopic expression of the gene encoding zebrafish homolog of the 78 kDa glucose-regulating protein (GRP78) and gene encoding CCAAT/enhancer-binding protein homologous protein (CHOP) was detected in the precursors of the neuromast, which is a sensory organ for detecting water flow and vibration. Our in vitro and in vivo studies revealed that exposure of zebrafish to TBT induces the ER stress response via activation of both the PERK-eIF2α and IRE1-XBP1 pathways of the unfolded protein response (UPR) in an organ-specific manner.

Reduced PLP1 expression in induced pluripotent stem cells derived from a Pelizaeus-Merzbacher disease patient with a partial PLP1 duplication.

Pelizaeus–Merzbacher disease (PMD) is an X-linked recessive disorder characterized by dysmyelination of the central nervous system (CNS). We identified a rare partial duplication of the proteolipid protein 1 gene (PLP1) in a patient with PMD. To assess the underlying effect of this duplication, we examined PLP1 expression in induced pluripotent stem (iPS) cells generated from the patient’s fibroblasts. Disease-specific iPS cells were generated from skin fibroblasts obtained from the indicated PMD patient and two other PMD patients having a 637-kb chromosomal duplication including entire PLP1 and a novel missense mutation (W212C) of PLP1, by transfections of OCT3/4, C-MYC, KLF4 and SOX2 using retro-virus vectors. PLP1 expressions in the generated iPS cells were examined by northern blot analysis. Although PLP1 expression was confirmed in iPS cells generated from two patients with the entire PLP1 duplication and the missense mutation of PLP1, iPS cells generated from the patient with the partial PLP1 duplication manifesting a milder form of PMD showed null expression. This indicated that the underlying effect of the partial PLP1 duplication identified in this study was different from other PLP1 alterations including a typical duplication and a missense mutation.

Potential Teratogenicity of Methimazole: Exposure of Zebrafish Embryos to Methimazole Causes Similar Developmental Anomalies to Human Methimazole Embryopathy

While methimazole (MMI) is widely used in the therapy for hyperthyroidism, several groups have reported that maternal exposure to MMI results in a variety of congenital anomalies, including choanal and esophageal atresia, iridic and retinal coloboma, and delayed neurodevelopment. Thus, adverse effects of maternal exposure to MMI on fetal development have long been suggested; however, direct evidence for the teratogenicity of MMI has not been presented. Therefore, we studied the effects of MMI on early development by using zebrafish as a model organism. The fertilized eggs of zebrafish were collected immediately after spawning and grown in egg culture water containing MMI at various concentrations. External observation of the embryos revealed that exposure to high concentrations of MMI resulted in loss of pigmentation, hypoplastic hindbrain, turbid tissue in the forebrain, swelling of the notochord, and curly trunk. Furthermore, these effects occurred in a dose-dependent manner. Precise observation of the serial cross-sections of MMI-exposed embryos elucidated delayed development and hypoplasia of the whole brain and spinal cord, narrowing of the pharynx and esophagus, severe disruption of the retina, and aberrant structure of the notochord. These neuronal, pharyngeal, esophageal, and retinal anomalous morphologies have a direct analogy to the congenital anomalies observed in children exposed to MMI in utero. Here, we show the teratogenic effects of MMI on the development of zebrafish and provide the first experimental evidence for the connection between exposure to MMI and human MMI embryopathy.

Myogenic differentiation in atrium-derived adult cardiac pluripotent cells and the transcriptional regulation of GATA4 and myogenin on ANP promoter.

We established cardiac pluripotent stem‐like cells from the left atrium (LA‐PCs) of adult rat hearts. These cells could differentiate not only into beating myocytes but also into cells of other lineages, including adipocytes and endothelial cells in the methylcellulose‐based medium containing interleukin‐3 (IL‐3), interleukin‐6 (IL‐6), and stem cell factor (SCF). In particular, IL‐3 and SCF contributed to the differentiation into cardiac troponin I‐positive cells. Notably, small population of LA‐PCs coexpressed GATA4 and myogenin, which are markers specific to cardiomyocytes and skeletal myocytes, respectively, and could differentiate into both cardiac and skeletal myocytes. Therefore, we investigated the involvement of these two tissue‐specific transcription factors in the cardiac transcriptional activity. Coexpression of GATA4 and myogenin synergistically activated GATA4‐specific promoter of the atrial natriuretic peptide gene. This combinatorial function was shown to be dependant on the GATA site, but independent of the E‐box. The results of chromatin immunoprecipitation and electrophoretic mobility shift assays suggested that myogenin bound to GATA4 on the GATA elements and the C‐terminal Zn‐finger domain of GATA4 and the N‐terminal region of myogenin were required for this synergistic activation of transcription. Taken together, these two transcription factors could be involved in the myogenesis of LA‐PCs.

Novel compound heterozygous mutations of POLR3A revealed by whole-exome sequencing in a patient with hypomyelination.

OBJECTIVE Congenital white matter disorders are a heterogeneous group of hypomyelination disorders affecting the white matter of the brain. Recently, mutations in the genes encoding the subunits of RNA polymerase III (Pol III), POLR3A and POLR3B, have been identified as new genetic causes for hypomyelinating disorders. METHOD Whole-exome sequencing was applied to identify responsible gene mutations in a 29-year-old female patient showing hypomyelination of unknown cause. To investigate the pathological mechanism underlying the hypomyelination in this patient, the expression level of 7SL RNA, a transcriptional target of Pol III, was analyzed in cultured skin fibroblasts derived from the patient with POLR3A mutations. RESULTS Novel compound heterozygous mutations of POLR3A were identified in the patient, who started to show cerebellar signs at 3 years, lost ambulation at 7 years, and became bedridden at 18 years. Brain magnetic resonance imaging showed severe volume loss in the brainstem, the cerebellum, and the white matter associated with hypomyelination. In addition to hypodontia and hypogonadism, she showed many pituitary hormone-related deficiencies. The expression level of 7SL RNA in cultured skin fibroblasts derived from this patient showed no significant abnormality. CONCLUSION The many pituitary hormone-related deficiencies identified in this patient may be an essential finding for the Pol III-related leukodystrophies spectrum. Further investigation is needed for a better understanding of the disease mechanism.

Single Nucleotide Variations in CLCN6 Identified in Patients with Benign Partial Epilepsies in Infancy and/or Febrile Seizures

Nucleotide alterations in the gene encoding proline-rich transmembrane protein 2 (PRRT2) have been identified in most patients with benign partial epilepsies in infancy (BPEI)/benign familial infantile epilepsy (BFIE). However, not all patients harbor these PRRT2 mutations, indicating the involvement of genes other than PRRT2. In this study, we performed whole exome sequencing analysis for a large family affected with PRRT2-unrelated BPEI. We identified a non-synonymous single nucleotide variation (SNV) in the voltage-sensitive chloride channel 6 gene (CLCN6). A cohort study of 48 BPEI patients without PRRT2 mutations revealed a different CLCN6 SNV in a patient, his sibling and his father who had a history of febrile seizures (FS) but not BPEI. Another study of 48 patients with FS identified an additional SNV in CLCN6. Chloride channels (CLCs) are involved in a multitude of physiologic processes and some members of the CLC family have been linked to inherited diseases. However, a phenotypic correlation has not been confirmed for CLCN6. Although we could not detect significant biological effects linked to the identified CLCN6 SNVs, further studies should investigate potential CLCN6 variants that may underlie the genetic susceptibility to convulsive disorders.