Human Nek6 is a monomeric mostly globular kinase with an unfolded short N-terminal domain

Background  

The NIMA-related kinases (Neks) are widespread among eukaryotes. In mammalians they represent an evolutionarily conserved family of 11 serine/threonine kinases, with 40-45% amino acid sequence identity to the Aspergillus nidulans mitotic regulator NIMA within their catalytic domains. Neks have cell cycle-related functions and were recently described as related to pathologies, particularly cancer, consisting in potential chemotherapeutic targets. Human Nek6, -7 and -9 are involved in the control of mitotic spindle formation, acting together in a mitotic kinase cascade, but their mechanism of regulation remain elusive.     

MicroRNA-21介导非对称性二甲基精氨酸诱导的内皮细胞衰老

目的：观察非对称性二甲基精氨酸（ADMA）对内皮细胞中microRNA-21（miR-21）表达的影响,探讨microRNA-21在ADMA诱导的内皮细胞衰老中的作用。方法：人脐静脉内皮细胞（HUVEC）与10uM的ADMA孵育48小时后收集细胞提取总RNA及蛋白,荧光定量实时RT—PCR检测miR-21表达,Westernblot检测超氧化物歧化酶2（SOD2）表达,衰老相关半乳糖苷酶（SA-β-gal）染色鉴定衰老的内皮细胞;然后HUVEC与miR-21抑制剂转染6小时后继续与10uM的ADMA孵育48小时留取细胞按上述方法检测相关指标。结果：HUVEC与ADMA孵育后miR-21表达量明显增加（P〈0．01）,同时衰老的内皮细胞数量增多（P〈0．05）,而SOD2表达减少（P〈0．01）;MiR-21抑制剂转染HUVEC后ADMA诱导的miR-21表达明显减少,同时衰老的内皮细胞减少,而SOD2表达明显增加（所有P〈0．01）。结论：ADMA诱导了HUVEC中miR-21表达及细胞衰老,miR-21介导了ADMA诱导的内皮细胞衰老作用,其机制可能与其抑制SOD2表达有关。     

CD44v6 regulates growth of brain tumor stem cells partially through the AKT-mediated pathway

Identification of stem cell-like brain tumor cells (brain tumor stem-like cells; BTSC) has gained substantial attention by scientists and physicians. However, the mechanism of tumor initiation and proliferation is still poorly understood. CD44 is a cell surface protein linked to tumorigenesis in various cancers. In particular, one of its variant isoforms, CD44v6, is associated with several cancer types. To date its expression and function in BTSC is yet to be identified. Here, we demonstrate the presence and function of the variant form 6 of CD44 (CD44v6) in BTSC of a subset of glioblastoma multiforme (GBM). Patients with CD44(high) GBM exhibited significantly poorer prognoses. Among various variant forms, CD44v6 was the only isoform that was detected in BTSC and its knockdown inhibited in vitro growth of BTSC from CD44(high) GBM but not from CD44(low) GBM. In contrast, this siRNA-mediated growth inhibition was not apparent in the matched GBM sample that does not possess stem-like properties. Stimulation with a CD44v6 ligand, osteopontin (OPN), increased expression of phosphorylated AKT in CD44(high) GBM, but not in CD44(low) GBM. Lastly, in a mouse spontaneous intracranial tumor model, CD44v6 was abundantly expressed by tumor precursors, in contrast to no detectable CD44v6 expression in normal neural precursors. Furthermore, overexpression of mouse CD44v6 or OPN, but not its dominant negative form, resulted in enhanced growth of the mouse tumor stem-like cells in vitro. Collectively, these data indicate that a subset of GBM expresses high CD44 in BTSC, and its growth may depend on CD44v6/AKT pathway.     

Concerted action of two novel tRNA mtDNA point mutations in chronic progressive external ophthalmoplegia

CPEO (chronic progressive external ophthalmoplegia) is a common mitochondrial disease phenotype in adults which is due to mtDNA (mitochondrial DNA) point mutations in a subset of patients. Attributing pathogenicity to novel tRNA mtDNA mutations still poses a challenge, particularly when several mtDNA sequence variants are present. In the present study we report a CPEO patient for whom sequencing of the mitochondrial genome revealed three novel tRNA mtDNA mutations: G5835A, del4315A, T1658C in tRNATyr, tRNAIle and tRNAVal genes. In skeletal muscle, the tRNAVal and tRNAIle mutations were homoplasmic, whereas the tRNATyr mutation was heteroplasmic. To address the pathogenic relevance, we performed two types of functional tests: (i) single skeletal muscle fibre analysis comparing G5835A mutation loads and biochemical phenotypes of corresponding fibres, and (ii) Northern-blot analyses of mitochondrial tRNATyr, tRNAIle and tRNAVal. We demonstrated that both the G5835A tRNATyr and del4315A tRNAIle mutation have serious functional consequences. Single-fibre analyses displayed a high threshold of the tRNATyr mutation load for biochemical phenotypic expression at the single-cell level, indicating a rather mild pathogenic effect. In contrast, skeletal muscle tissue showed a severe decrease in respiratory-chain activities, a reduced overall COX (cytochrome c oxidase) staining intensity and abundant COX-negative fibres. Northern-blot analyses showed a dramatic reduction of tRNATyr and tRNAIle levels in muscle, with impaired charging of tRNAIle, whereas tRNAVal levels were only slightly decreased, with amino-acylation unaffected. Our findings suggest that the heteroplasmic tRNATyr and homoplasmic tRNAIle mutation act together, resulting in a concerted effect on the biochemical and histological phenotype. Thus homoplasmic mutations may influence the functional consequences of pathogenic heteroplasmic mtDNA mutations.     

Identification of Differentially Expressed Proteins in Murine Embryonic and Postnatal Cortical Neural Progenitors

Background

The central nervous system (CNS) develops from a heterogeneous pool of neural stem and progenitor cells (NSPC), the underlying differences among which are poorly understood. The study of NSPC would be greatly facilitated by the identification of additional proteins that mediate their function and that would distinguish amongst different progenitor populations.

Methodology/Principal Findings

To identify membrane and membrane-associated proteins expressed by NSPC, we used a proteomics approach to profile NSPC cultured as neurospheres (NS) isolated from the murine cortex during a period of neurogenesis (embryonic day 11.5, E11.5), as compared to NSPC isolated at a peak of gliogenesis (postnatal day 1, P0) and to differentiated E11.5 NS. 54 proteins were identified with high expression in E11.5 NS, including the TrkC receptor, several heterotrimeric G proteins, and the Neogenin receptor. 24 proteins were identified with similar expression in E11.5 and P0 NS over differentiated E11.5 NS, and 13 proteins were identified with high expression specifically in P0 NS compared to E11.5 NS. To illustrate the potential relevance of these identified proteins to neural stem cell biology, the function of Neogenin was further studied. Using Fluorescence Activated Cell Sorting (FACS) analysis, expression of Neogenin was associated with a self-renewing population present in both E11.5 and adult subventricular zone (SVZ) NS but not in P0 NS. E11.5 NS expressed a putative Neogenin ligand, RGMa, and underwent apoptosis when exposed to a ligand-blocking antibody.

Conclusions/Significance

There are fundamental differences between the continuously self-renewing and more limited progenitors of the developing cortex. We identified a subset of differentially expressed proteins that serve not only as a set of functionally important proteins, but as a useful set of markers for the subsequent analysis of NSPC. Neogenin is associated with the continuously self-renewing and neurogenic cells present in E11.5 cortical and adult SVZ NS, and the Neogenin/RGMa receptor/ligand pair may regulate cell survival during development.     

Multi-Kinase Inhibitor C1 Triggers Mitotic Catastrophe of Glioma Stem Cells Mainly through MELK Kinase Inhibition

Glioblastoma multiforme (GBM) is a highly lethal brain tumor. Due to resistance to current therapies, patient prognosis remains poor and development of novel and effective GBM therapy is crucial. Glioma stem cells (GSCs) have gained attention as a therapeutic target in GBM due to their relative resistance to current therapies and potent tumor-initiating ability. Previously, we identified that the mitotic kinase maternal embryonic leucine-zipper kinase (MELK) is highly expressed in GBM tissues, specifically in GSCs, and its expression is inversely correlated with the post-surgical survival period of GBM patients. In addition, patient-derived GSCs depend on MELK for their survival and growth both in vitro and in vivo. Here, we demonstrate evidence that the role of MELK in the GSC survival is specifically dependent on its kinase activity. With in silico structure-based analysis for protein-compound interaction, we identified the small molecule Compound 1 (C1) is predicted to bind to the kinase-active site of MELK protein. Elimination of MELK kinase activity was confirmed by in vitro kinase assay in nano-molar concentrations. When patient-derived GSCs were treated with C1, they underwent mitotic arrest and subsequent cellular apoptosis in vitro, a phenotype identical to that observed with shRNA-mediated MELK knockdown. In addition, C1 treatment strongly induced tumor cell apoptosis in slice cultures of GBM surgical specimens and attenuated growth of mouse intracranial tumors derived from GSCs in a dose-dependent manner. Lastly, C1 treatment sensitizes GSCs to radiation treatment. Collectively, these data indicate that targeting MELK kinase activity is a promising approach to attenuate GBM growth by eliminating GSCs in tumors.