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Vanishing white matter disease (VWM) is an autosomal recessive neurological disorder caused by mutation(s) in any subunit of eukaryotic translation initiation factor 2B (eIF2B), an activator of translation initiation factor eIF2. VWM occurs with mutation of the genes encoding eIF2B subunits (EIF2B1, EIF2B2, EIF2B3, EIF2B4, and EIF2B5). However, little is known regarding the underlying pathogenetic mechanisms or how to treat patients with VWM. Here we describe the identification and detailed analysis of a new spontaneous mutant mouse harboring a point mutation in the Eif2b5 gene (p.Ile98Met). Homozygous Eif2b5I98M mutant mice exhibited a small body, abnormal gait, male and female infertility, epileptic seizures, and a shortened lifespan. Biochemical analyses indicated that the mutant eIF2B protein with the Eif2b5I98M mutation decreased guanine nucleotide exchange activity on eIF2, and the level of the endoplasmic reticulum stress marker activating transcription factor 4 was elevated in the 1-month-old Eif2b5I98M brain. Histological analyses indicated up-regulated glial fibrillary acidic protein immunoreactivity in the astrocytes of the Eif2b5I98M forebrain and translocation of Bergmann glia in the Eif2b5I98M cerebellum, as well as increased mRNA expression of an endoplasmic reticulum stress marker, C/EBP homologous protein. Disruption of myelin and clustering of oligodendrocyte progenitor cells were also indicated in the white matter of the Eif2b5I98M spinal cord at 8 months old. Our data show that Eif2b5I98M mutants are a good model for understanding VWM pathogenesis and therapy development. Cover Image for this issue: doi: 10.1111/jnc.14751.

DOI： 10.1111/jnc.14887

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Loss-of-function mutations in dystonin (DST) can cause hereditary sensory and autonomic neuropathy type 6 (HSAN-VI) or epidermolysis bullosa simplex (EBS). Recently, DST-related diseases were recognized to be more complex than previously thought because a patient exhibited both neurological and skin manifestations, whereas others display only one or the other. A single DST locus produces at least three major DST isoforms: DST-a (neuronal isoform), DST-b (muscular isoform) and DST-e (epithelial isoform). Dystonia musculorum (dt) mice, which have mutations in Dst, were originally identified as spontaneous mutants displaying neurological phenotypes. To reveal the mechanisms underlying the phenotypic heterogeneity of DST-related diseases, we investigated two mutant strains with different mutations: a spontaneous Dst mutant (Dstdt-23Rbrc mice) and a gene-trap mutant (DstGt mice). The Dstdt-23Rbrc allele possesses a nonsense mutation in an exon shared by all Dst isoforms. The DstGt allele is predicted to inactivate Dst-a and Dst-b isoforms but not Dst-e There was a decrease in the levels of Dst-a mRNA in the neural tissue of both Dstdt-23Rbrc and DstGt homozygotes. Loss of sensory and autonomic nerve ends in the skin was observed in both Dstdt-23Rbrc and DstGt mice at postnatal stages. In contrast, Dst-e mRNA expression was reduced in the skin of Dstdt-23Rbrc mice but not in DstGt mice. Expression levels of Dst proteins in neural and cutaneous tissues correlated with Dst mRNAs. Because Dst-e encodes a structural protein in hemidesmosomes (HDs), we performed transmission electron microscopy. Lack of inner plaques and loss of keratin filament invasions underneath the HDs were observed in the basal keratinocytes of Dstdt-23Rbrc mice but not in those of DstGt mice; thus, the distinct phenotype of the skin of Dstdt-23Rbrc mice could be because of failure of Dst-e expression. These results indicate that distinct mutations within the Dst locus can cause different loss-of-function patterns among Dst isoforms, which accounts for the heterogeneous neural and skin phenotypes in dt mice and DST-related diseases.

DOI： 10.1242/dmm.041608

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：CELL PRESS  

The ubiquitin fold modifier 1 (UFM1) cascade is a recently identified evolutionarily conserved ubiquitin-like modification system whose function and link to human disease have remained largely uncharacterized. By using exome sequencing in Finnish individuals with severe epileptic syndromes, we identified pathogenic compound heterozygous variants in UBA5, encoding an activating enzyme for UFM1, in two unrelated families. Two additional individuals with biallelic UBA5 variants were identified from the UK-based Deciphering Developmental Disorders study and one from the Northern Finland Intellectual Disability cohort. The affected individuals (n = 9) presented in early infancy with severe irritability, followed by dystonia and stagnation of development. Furthermore, the majority of individuals display postnatal microcephaly and epilepsy and develop spasticity. The affected individuals were compound heterozygous for a missense substitution, c.1111G>A (p.Ala371Thr; allele frequency of 0.28% in Europeans), and a nonsense variant or c.164G>A that encodes an amino acid substitution p.Arg55His, but also affects splicing by facilitating exon 2 skipping, thus also being in effect a loss-of-function allele. Using an in vitro thioester formation assay and cellular analyses, we show that the p.Ala371Thr variant is hypomorphic with attenuated ability to transfer the activated UFM1 to UFC1. Finally, we show that the CNS-specific knockout of Ufm1 in mice causes neonatal death accompanied by microcephaly and apoptosis in specific neurons, further suggesting that the UFM1 system is essential for CNS development and function. Taken together, our data imply that the combination of a hypomorphic p.Ala371Thr variant in trans with a loss-of-function allele in UBA5 underlies a severe infantile-onset encephalopathy.

DOI： 10.1016/j.ajhg.2016.06.020

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：WILEY-BLACKWELL  

Cancer stem cells (CSCs) are believed to be maintained within a microenvironmental niche. Here we used polymer microarrays for the rapid and efficient identification of glioma CSC (GSC) niche mimicries and identified a urethane-based synthetic polymer, upon which two groups of niche components, namely extracellular matrices (ECMs) and iron are revealed. In cultures, side population (SP) cells, defined as GSCs in the rat C6 glioma cell line, are more efficiently sustained in the presence of their differentiated progenies expressing higher levels of ECMs and transferrin, while in xenografts, ECMs are supplied by the vascular endothelial cells (VECs), including SP cell-derived ones with distinctively greater ability to retain xenobiotics than host VECs. Iron is stored in tumor infiltrating host macrophages (Mφs), whose protumoral activity is potently enhanced by SP cell-secreted soluble factor(s). Finally, coexpression of ECM-, iron-, and Mφ-related genes is found to be predictive of glioma patients' outcome. Our polymer-based approach reveals the intrinsic capacities of GSCs, to adapt the environment to organize a self-advantageous microenvironment niche, for their maintenance and expansion, which redefines the current concept of anti-CSC niche therapy and has the potential to accelerate cancer therapy development. Stem Cells 2016;34:1151-1162.

DOI： 10.1002/stem.2299

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：PUBLIC LIBRARY SCIENCE  

The endoplasmic reticulum (ER), including the nuclear envelope, is a continuous and intricate membrane-bound organelle responsible for various cellular functions. In neurons, the ER network is found in cell bodies, axons, and dendrites. Recent studies indicate the involvement of the ER network in neuronal development, such as neuronal migration and axonal outgrowth. However, the regulation of neural development by ER-localized proteins is not fully understood. We previously reported that the multi-transmembrane protein Dpy19L1 is required for neuronal migration in the developing mouse cerebral cortex. A Dpy19L family member, Dpy19L2, which is a causative gene for human Globozoospermia, is suggested to act as an anchor of the acrosome to the nuclear envelope. In this study, we found that the patterns of exogenous Dpy19L1 were partially coincident with the ER, including the nuclear envelope in COS-7 cells at the level of the light microscope. The reticular distribution of Dpy19L1 was disrupted by microtubule depolymerization that induces retraction of the ER. Furthermore, Dpy19L1 showed a similar distribution pattern with a ER marker protein in embryonic mouse cortical neurons. Finally, we showed that Dpy19L1 knockdown mediated by siRNA resulted in decreased neurite outgrowth in cultured neurons. These results indicate that transmembrane protein Dpy19L1 is localized to the ER membrane and regulates neurite extension during development.

DOI： 10.1371/journal.pone.0167985

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Authorship：Lead author   Language：English   Publishing type：Research paper (scientific journal)   Publisher：WILEY-BLACKWELL  

Self-renewing proliferation of neural stem cells (NSCs) is intimately linked to the inhibition of neuronal and glial differentiation, however, their molecular linkage has been poorly understood. We have proposed a model previously explaining partly this linkage, in which fibroblast growth factor 2 (FGF2) and Wnt signals cooperate to promote NSC self-renewal via β-catenin accumulation, which leads to the promotion of proliferation by lymphoid enhancer factor (LEF)/T-cell factor (TCF)-mediated cyclin D1 expression and at the same time to the inhibition of neuronal differentiation by β-catenin-mediated potentiation of Notch signaling. To fully understand the mechanisms underlying NSC self-renewal, it needs to be clarified how these growth factor signals inhibit glial differentiation as well. Here, we demonstrate that cyclin D1, a NSC growth promoting signaling component and also a common component of FGF2 and Wnt signaling pathways, inhibits astroglial differentiation of NSCs. Interestingly, this effect of cyclin D1 is mediated even though its cell cycle progression activity is blocked. Forced downregulation of cyclin D1 enhances astrogliogenesis of NSCs in culture and in vivo. We further demonstrate that cyclin D1 binds to STAT3, a transcription factor downstream of astrogliogenic cytokines, and suppresses its transcriptional activity on the glial fibrillary acidic protein (Gfap) gene. Taken together with our previous finding, we provide a novel molecular mechanism for NSC self-renewal in which growth promoting signaling components activated by FGF2 and Wnts inhibit neuronal and glial differentiation.

DOI： 10.1002/stem.1613

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Language：Japanese   Publishing type：Research paper (scientific journal)  

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：SPRINGER  

A pentaspan membrane glycoprotein prominin-1 (frequently called CD133 in human) is widely used as a surface marker to identify and isolate normal stem/progenitor cells from various organs, although it is also expressed in some types of differentiated cells. Since CD133 was identified as a universal marker to isolate cancer stem cells (CSCs) in tumors derived from multiple tissues, much attention has been directed toward the relationship between its gene regulation and identity of CSCs (i.e., cancer stemness). Prominin-1 (PROM1) gene possesses five alternative promoters yielding multiple first exons within the 5'-untranslated region (UTR) and also splicing variants affecting the open reading frame (ORF) sequence, implicating the complicated gene regulation in a context-dependent manner. This chapter aims to organize the accumulated findings on prominin-1 with a focus on its altered expression and regulation in normal and cancerous cells and to discuss potential regulatory networks underlying cancer stemness.

DOI： 10.1007/978-1-4614-5894-4_5

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：BIOMED CENTRAL LTD  

BACKGROUND: An increasing number of studies support the presence of stem-like cells in human malignancies. These cells are primarily responsible for tumor initiation and thus considered as a potential target to eradicate tumors. CD133 has been identified as an important cell surface marker to enrich the stem-like population in various human tumors. To reveal the molecular machinery underlying the stem-like features in tumor cells, we analyzed a promoter of CD133 gene using human colon carcinoma Caco-2 and synovial sarcoma Fuji cells, which endogenously express CD133 gene. RESULTS: A reporter analysis revealed that P5 promoter, located far upstream in a human CD133 gene locus, exhibits the highest activity among the five putative promoters (P1 to P5). Deletion and mutation analysis identified two ETS binding sites in the P5 region as being essential for its promoter activity. Electrophoretic mobility shift assays demonstrated the specific binding between nuclear factors and the ETS binding sequence. Overexpression of dominant-negative forms of Ets2 and Elk1 resulted in the significant decrease of P5 activity. Furthermore, treatment of Fuji cells with a specific MEK/ERK inhibitor, U0126, also markedly decreased CD133 expression, but there was no significant effect in Caco-2 cells, suggesting cell type-specific regulation of CD133 expression. Instead, the side population, another hallmark of TSLCs, was dramatically diminished in Caco-2 cells by U0126. Finally, Ras-mediated oncogenic transformation in normal human astrocytes conferred the stem-like capability to form neurosphere-like colonies with the increase of CD133 mRNA expression. CONCLUSIONS: In conclusion, the Ras/ERK pathway at least in part contributes to the maintenance and the acquisition of stem-like hallmarks, although the extent of its contribution is varied in a cell type-specific manner. These findings could help our comprehensive understanding of tumor stemness, and also improve the development of eradicative therapies against human malignancies.

DOI： 10.1186/1476-4598-9-39

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