Yasunari Sakai

Protein Interactome Reveals Converging Molecular Pathways Among Autism Disorders

We developed an autism protein interaction network that provides a framework for studying autism pathogenesis and reveals shared molecular pathways among distinct autism spectrum disorders. Building Connections in Autism Autism is a collection of neurodevelopmental disorders characterized by impaired social skills, delayed language development, and repetitive behaviors. In classic (idiopathic) autism, these three characteristics predominate, whereas in syndromic autism disorders, these characteristics are part of a much larger set of symptoms. Clearly, common molecular pathways should underlie these similar disorders but trying to identify these pathways has proved difficult. Genetic studies have implicated genes mutated in syndromic autism disorders such as fragile X syndrome, Angelman syndrome, and Rett syndrome. Meanwhile, recent genome-wide association studies have pinpointed a large number of susceptibility loci that may contribute to autism spectrum disorders. Now, Zoghbi and her team turn their attention from genes associated with autism to the proteins they encode. These authors have compiled a network of protein-protein interactions based on autism-associated genes with the aim of finding common molecular pathways that underpin both classic and syndromic autism. The authors divided autism-associated genes into three groups and then used a yeast two-hybrid screen of a human complementary DNA library to obtain interacting partners for the products of 26 autism-associated genes. They then built a protein-protein interaction network and from it pulled out very highly connected proteins such as those known to be mutated in fragile X syndrome. Their network showed that two proteins, SHANK3 and PSD95, located in the postsynaptic region of neurons and known to be associated with classic autism interact with each other and share nine common protein binding partners. SHANK3 also interacts with the TSC1 protein mutated in a form of syndromic autism called tuberous sclerosis complex, and these two proteins share 21 binding partners. SHANK3 and TSC1 also associate with two other postsynaptic proteins, ACTN1 and HOMER3. With their protein interaction network as a guide, the authors then performed microarray-based comparative genome hybridization for genes encoding 627 proteins in the network using DNA from 288 patients with classic autism. They discovered four new gene mutations including a mutation in the FLNA gene encoding a protein that turns out to bind to SHANK3. This new protein-protein interaction network provides a starting point for elucidating the common molecular pathways that may underlie both classic and syndromic autism. It also provides a framework on which to build a much bigger autism interactome that could be used to develop drugs with potential for treating several different forms of autism. To uncover shared pathogenic mechanisms among the highly heterogeneous autism spectrum disorders (ASDs), we developed a protein interaction network that identified hundreds of new interactions among proteins encoded by ASD-associated genes. We discovered unexpectedly high connectivity between SHANK and TSC1, previously implicated in syndromic autism, suggesting that common molecular pathways underlie autistic phenotypes in distinct syndromes. ASD patients were more likely to harbor copy number variations that encompass network genes than were control subjects. We also identified, in patients with idiopathic ASD, three de novo lesions (deletions in 16q23.3 and 15q22 and one duplication in Xq28) that involve three network genes (NECAB2, PKM2, and FLNA). The protein interaction network thus provides a framework for identifying causes of idiopathic autism and for understanding molecular pathways that underpin both syndromic and idiopathic ASDs.

Oligogenic heterozygosity in individuals with high-functioning autism spectrum disorders

Autism spectrum disorders (ASDs) are a heterogeneous group of neuro-developmental disorders. While significant progress has been made in the identification of genes and copy number variants associated with syndromic autism, little is known to date about the etiology of idiopathic non-syndromic autism. Sanger sequencing of 21 known autism susceptibility genes in 339 individuals with high-functioning, idiopathic ASD revealed de novo mutations in at least one of these genes in 6 of 339 probands (1.8%). Additionally, multiple events of oligogenic heterozygosity were seen, affecting 23 of 339 probands (6.8%). Screening of a control population for novel coding variants in CACNA1C, CDKL5, HOXA1, SHANK3, TSC1, TSC2 and UBE3A by the same sequencing technology revealed that controls were carriers of oligogenic heterozygous events at significantly (P < 0.01) lower rate, suggesting oligogenic heterozygosity as a new potential mechanism in the pathogenesis of ASDs.

An Oxidized Purine Nucleoside Triphosphatase, MTH1, Suppresses Cell Death Caused by Oxidative Stress

MTH1 hydrolyzes oxidized purine nucleoside triphosphates such as 8-oxo-2′-deoxyguanosine 5′-triphosphate (8-oxo-dGTP) and 2-hydroxy-2′-deoxyadenosine 5′-triphosphate (2-OH-dATP) and thus protects cells from damage caused by their misincorporation into DNA. In the present study, we established MTH1-null mouse embryo fibroblasts that were highly susceptible to cell dysfunction and death caused by exposure to H2O2, with morphological features of pyknosis and electron-dense deposits accumulated in mitochondria. The cell death observed was independent of both poly(ADP-ribose) polymerase and caspases. A high performance liquid chromatography tandem mass spectrometry analysis and immunofluorescence microscopy revealed a continuous accumulation of 8-oxo-guanine both in nuclear and mitochondrial DNA after exposure to H2O2. All of the H2O2-induced alterations observed in MTH1-null mouse embryo fibroblasts were effectively suppressed by the expression of wild type human MTH1 (hMTH1), whereas they were only partially suppressed by the expression of mutant hMTH1 defective in either 8-oxo-dGTPase or 2-OH-dATPase activity. Human MTH1 thus protects cells from H2O2-induced cell dysfunction and death by hydrolyzing oxidized purine nucleotides including 8-oxo-dGTP and 2-OH-dATP, and these alterations may be partly attributed to a mitochondrial dysfunction.

Genetic susceptibility to simple febrile seizures: Interleukin-1β promoter polymorphisms are associated with sporadic cases

Febrile seizures (FSs) are the commonest form of convulsions. A genetic predisposition to FSs is known, based on family studies, twin studies, and complex segregation analysis. Simple FSs may be more homogenous in their clinical manifestations, and show better agreement with the multifactorial inheritance theory than the complex type. Interleukin-1 (IL-1) beta is one of the pro-inflammatory cytokines that are postulated to be involved in the development of FSs. To determine whether or not function-related polymorphisms of the IL-1beta (IL1B) gene are associated with susceptibility to simple FSs, the genotypes for two biallelic polymorphisms in the promoter region at positions -31 and -511 of the IL1B gene were determined by means of PCR-restriction fragment length polymorphism in 229 FS patients (108 sporadic and 60 familial simple FS, and 61 complex FS patients) and 158 controls. IL1B -31C/T, a TATA box polymorphism, has been found to be in complete linkage disequilibrium with the IL1B -511C/T polymorphism. Sporadic simple FS patients exhibited significantly higher frequencies of IL1B -31C/-511T alleles and homozygotes than controls (uncorrected p = 0.0094 and 0.0029, corrected p = 0.038 and 0.035, respectively), while no differences were observed in patients with all or familial simple FSs versus controls. There were no significant differences in the frequencies of -31C/T and -511C/T in the IL-1beta promoter gene between complex FS patients and controls. The present study suggests that the IL-1beta gene contributes to a genetic susceptibility to the development of simple FSs of sporadic occurrence.

Induction of cells with cancer stem cell properties from nontumorigenic human mammary epithelial cells by defined reprogramming factors

Cancer stem cells (CSCs), a small and elusive population of undifferentiated cancer cells within tumors that drive tumor growth and recurrence, are believed to resemble normal stem cells. Although surrogate markers have been identified and compelling CSC theoretical models abound, actual proof for the existence of CSCs can only be had retrospectively. Hence, great store has come to be placed in isolating CSCs from cancers for in-depth analysis. On the other hand, although induced pluripotent stem cells (iPSCs) hold great promise for regenerative medicine, concern exists over the inadvertent co-transplantation of partially or undifferentiated stem cells with tumorigenic capacity. Here we demonstrate that the introduction of defined reprogramming factors (OCT4, SOX2, Klf4 and c-Myc) into MCF-10A nontumorigenic mammary epithelial cells, followed by partial differentiation, transforms the bulk of cells into tumorigenic CD44+/CD24low cells with CSC properties, termed here as induced CSC-like-10A or iCSCL-10A cells. These reprogrammed cells display a malignant phenotype in culture and form tumors of multiple lineages when injected into immunocompromised mice. Compared with other transformed cell lines, cultured iCSCL-10A cells exhibit increased resistance to the chemotherapeutic compounds, Taxol and Actinomycin D, but higher susceptibility to the CSC-selective agent Salinomycin and the Pin1 inhibitor Juglone. Restored expression of the cyclin-dependent kinase inhibitor p16INK4a abrogated the CSC properties of iCSCL-10A cells, by inducing cellular senescence. This study provides some insight into the potential oncogenicity that may arise via cellular reprogramming, and could represent a valuable in vitro model for studying the phenotypic traits of CSCs per se.

Functional MxA promoter polymorphism associated with subacute sclerosing panencephalitis

Background: The antivirally active MxA protein is induced by interferon (IFN) α/β and inhibits the replication of single-stranded RNA viruses including measles virus (MV). The authors investigated whether the MxA gene contributed to the development of subacute sclerosing panencephalitis (SSPE) in Japanese individuals. Methods: Single-nucleotide polymorphisms (SNP) in the promoter region of the MxA gene were screened, association studies were performed between two SNP and SSPE, and then a functional difference in the promoter activities of the two SNP was investigated by a dual luciferase reporter assay. Results: Four SNP were found (−88 G/T, −123 C/A, −200 T/C, and −213 G/T), and SSPE patients exhibited a higher frequency of both the −88T allele and the −88TT genotype than controls (p = 0.040 and 0.003). The IFN-induced up-regulation of the MxA promoter activity of the sequence with −88T was found to be significantly higher than that with G. Conclusions: MxA promoter −88 G/T SNP may confer host genetic susceptibility to SSPE in Japanese individuals. The finding that homozygotes of the MxA −88T allele with a high MxA-producing capability were more frequently seen in SSPE patients suggests that the MxA protein promotes the establishment of persistent MV infection of neural cells.

Phenotypic spectrum of GNAO1 variants: epileptic encephalopathy to involuntary movements with severe developmental delay

De novo GNAO1 variants have been found in four patients including three patients with Ohtahara syndrome and one patient with childhood epilepsy. In addition, two patients showed involuntary movements, suggesting that GNAO1 variants can cause various neurological phenotypes. Here we report an additional four patients with de novo missense GNAO1 variants, one of which was identical to that of the previously reported. All the three novel variants were predicted to impair Gαo function by structural evaluation. Two patients showed early-onset epileptic encephalopathy, presenting with migrating or multifocal partial seizures in their clinical course, but the remaining two patients showed no or a few seizures. All the four patients showed severe intellectual disability, motor developmental delay, and involuntary movements. Progressive cerebral atrophy and thin corpus callosum were common features in brain images. Our study demonstrated that GNAO1 variants can cause involuntary movements and severe developmental delay with/without seizures, including various types of early-onset epileptic encephalopathy.

Association of toll-like receptor 3 gene polymorphism with subacute sclerosing panencephalitis

Innate immunity plays an important role in measles virus (MV) infection. MV-derived double-stranded RNA is recognized by toll-like receptor 3 (TLR3), retinoic acid-inducible protein I (RIG-I) and melanoma differentiation—associated gene 5 (MDA5). We investigated whether genes encoding these molecules contributed to the development of subacute sclerosing panencephalitis (SSPE) in Japanese individuals. Four single nucleotide polymorphisms (SNPs) of the three genes (TLR3 rs3775291:Leu412Phe, RIG1 rs277729 and rs9695310, and MDA5 rs4664463) were assessed in 40 SSPE patients and 84 controls. Because the TLR3 SNP showed a positive association with SSPE, three additional SNPs were subjected to haplotype analysis. The frequency of 412Phe allele of TLR3 rs3775291 in SSPE patients was significantly higher than that in controls (P=.03). In haplotype analysis of four SNPs in the TLR3 gene, the frequency of −7C/IVS3 +71C/Phe412/c.1377C haplotype was significantly increased in SSPE patients (P=.006, odds ration [OR]: 2.2). TLR3 gene may confer host genetic susceptibility to SSPE in Japanese individuals.

Moyamoya disease susceptibility gene RNF213 links inflammatory and angiogenic signals in endothelial cells

Moyamoya disease (MMD) is a cerebrovascular disorder characterized by occlusive lesions of the circle of Willis. To date, both environmental and genetic factors have been implicated for pathogenesis of MMD. Allelic variations in RNF213 are known to confer the risk of MMD; however, functional roles of RNF213 remain to be largely elusive. We herein report that pro-inflammatory cytokines, IFNG and TNFA, synergistically activated transcription of RNF213 both in vitro and in vivo. Using various chemical inhibitors, we found that AKT and PKR pathways contributed to the transcriptional activation of RNF213. Transcriptome-wide analysis and subsequent validation with quantitative PCR supported that endogenous expression of cell cycle-promoting genes were significantly decreased with knockdown of RNF213 in cultured endothelial cells. Consistently, these cells showed less proliferative and less angiogenic profiles. Chemical inhibitors for AKT (LY294002) and PKR (C16) disrupted their angiogenic potentials, suggesting that RNF213 and its upstream pathways cooperatively organize the process of angiogenesis. Furthermore, RNF213 down-regulated expressions of matrix metalloproteases in endothelial cells, but not in fibroblasts or other cell types. Altogether, our data illustrate that RNF213 plays unique roles in endothelial cells for proper gene expressions in response to inflammatory signals from environments.

Interferon-α therapy for chronic active Epstein-Barr virus infection : Potential effect on the development of T-lymphoproliferative disease

Purpose: A patient with aggressive chronic active Epstein-Barr virus (CAEBV) infection is described whose disease activity subsided after interferon (IFN)-α therapy. Patient and Methods: The patient had intermittent fever, cytopenia, liver dysfunction, hepatosplenomegaly, abnormal titers of EBV-associated antibodies, and positive EBV genomes. Results: Despite repeated trials of the antiviral agents prednisolone and γ-globulin, his condition deteriorated. The administration of IFN-α (1 x 105 U/kg subcutaneously 3 times per week) led to a dramatic clinical improvement. During the IFN-α therapy, the rearrangement bands of T-cell antigen receptor genes disappeared assessed by Southern blotting with a decrease in the number of activated T cells, although the EBV-genome remained evident. Conclusions: These observations suggest that IFN-α is useful in managing CAEBV, possibly restraining the clonal development of T-lymphoproliferative disease (LPD) and EBV-associated B-LPD, although it does not eradicate the proliferation of EBV.