Translationally Controlled Tumor Protein Is a Novel Biological Target for Neurofibromatosis Type 1-associated Tumors

Neurofibromatosis type 1 (NF1) is an autosomal dominant disease that predisposes individuals to develop benign neurofibromas and malignant peripheral nerve sheath tumors (MPNSTs). Due to the lack of information on the molecular mechanism of NF1-associated tumor pathogenesis or biomarkers/therapeutic targets, an effective treatment for NF1 tumors has not been established. In this study, the novel NF1-associated protein, translationally controlled tumor protein (TCTP), was identified by integrated proteomics and found to be up-regulated via activated MAPK/PI3K-AKT signaling in response to growth factors in NF1-deficient Schwann cells. Immunohistochemical analysis of NF1-associated tumors revealed that the TCTP expression level correlated with tumorigenicity. In NF1-deficient MPNST cells, TCTP protein but not mRNA was down-regulated by NF1 GTPase-activating protein-related domain or MAPK/PI3K inhibitors, and this correlated with suppression of mammalian target of rapamycin (mTOR) signaling. mTOR inhibition by rapamycin also down-regulated TCTP protein expression, whereas knockdown or overexpression of TCTP suppressed or activated mTOR signaling, respectively, and affected cell viability. These results suggest that a positive feedback loop between TCTP and mTOR contributes to NF1-associated tumor formation. Last, the anti-tumor effect of artesunate, which binds to and degrades TCTP, was evaluated. Artesunate significantly suppressed the viability of MPNST cells but not normal Schwann cells, and the TCTP level inversely correlated with artesunate sensitivity. Moreover, combinational use of artesunate and rapamycin enhanced the cytotoxic effect on MPNST cells. These findings suggest that TCTP is functionally implicated in the progression of NF1-associated tumors and could serve as a biological target for their therapy.     

Age‐dependent song changes in a closed‐ended vocal learner: elevation of song performance after song crystallization

Birdsong is a sexual signal that serves as an indicator of male quality. There is already abundant evidence that song elaboration reflects early life‐history because early developmental stress affects neural development of song control systems, and leaves irreversible adverse effects on song phenotypes. Especially in closed‐ended vocal learners, song features crystallized early in life are less subject to changes in adulthood. This is why less attention has been paid to lifelong song changes in closed‐ended learners. However, in the eyes of female birds that gain benefits from choosing mates based on male songs, not only past but also current conditions encoded in songs would be meaningful, given that even crystallized songs in closed‐ended learners would not be identical in the long term. In this study, we examine within‐individual song changes in the Java sparrow Lonchura oryzivora, with the aim of shedding light on the relationship between song and long‐term life history. Specifically, we compared song length, tempo, and song complexity measures between the point just after song crystallization and around 1 yr later, and also compared those traits between fathers and sons to clarify the effect of vocal learning. While it is not surprising that song complexity did not differ depending on age or between fathers and sons, we found that song length and tempo increased with age. Follow‐up analyses have revealed that frequency bandwidth and peak frequency of song notes also elevated with age. Our results show that song performance related to motor skills can be improved even after song crystallization. We also suggest that song performance in closed‐ended vocal learners gives a reliable clue for mate choice by reflecting male quality with aging.     

Devising Assisted Reproductive Technologies for Wild-Derived Strains of Mice: 37 Strains from Five Subspecies of Mus musculus

Wild-derived mice have long offered invaluable experimental models for mouse genetics because of their high evolutionary divergence from laboratory mice. A number of wild-derived strains are available from the RIKEN BioResource Center (BRC), but they have been maintained as living stocks because of the unavailability of assisted reproductive technology (ART). In this study, we sought to devise ART for 37 wild-derived strains from five subspecies of Mus musculus maintained at the BRC. Superovulation of females was effective (more than 15 oocytes per female) for 34 out of 37 strains by treatment with either equine chorionic gonadotropin or anti-inhibin serum, depending on their genetic background (subspecies). The collected oocytes could be fertilized in vitro at mean rates of 79.0% and 54.6% by the optimized protocol using fresh or frozen-thawed spermatozoa, respectively. They were cryopreserved at the 2-cell stage by vitrification with an ethylene glycol-based solution. In total, 94.6% of cryopreserved embryos survived the vitrification procedure and restored their normal morphology after warming. A conventional embryo transfer protocol could be applied to 25 out of the 35 strains tested. In the remaining 10 strains, live offspring could be obtained by a modified embryo transfer protocol using cyclosporin A treatment and co-transfer of ICR (laboratory mouse strain) embryos. Thus, ART for 37 wild-derived strains was devised successfully and is now routinely used for their preservation and transportation. The information provided here might facilitate broader use and wider distribution of wild-derived mice for biomedical research.     

Neuromelanin selectively induces apoptosis in dopaminergic SH-SY5Y cells by deglutathionylation in mitochondria: involvement of the protein and melanin component

Parkinson's disease (PD) is characterized by selective depletion of nigral dopamine (DA) neurons containing neuromelanin (NM), suggesting the involvement of NM in the pathogenesis. This study reports induction of apoptosis by NM in SH-SY5Y cells, whereas protease-K-treated NM, synthesized DA- and cysteinyl dopamine melanin showed much less cytotoxicity. Cell death was mediated by mitochondria-mediated apoptotic pathway, namely collapse of mitochondrial membrane potential, release of cytochrome c , and activation of caspase 3, but Bcl-2 over-expression did not suppress apoptosis. NM increased sulfhydryl content in mitochondria, and a major part of it was identified as GSH, whereas dopamine melanin significantly reduced sulfhydryl levels. Western blot analysis for protein-bound GSH demonstrated that only NM reduced S -glutathionylated proteins in mitochondria and dissociated macromolecular structure of complex I. Reactive oxygen and nitrogen species were required for the deglutathionylation by NM, which antioxidants reduced significantly with prevention of apoptosis. These results suggest that NM may be related to cell death of DA neurons in PD and aging through regulation of mitochondrial redox state and S -glutathionylation, for which NM-associated protein is absolutely required. The novel function of NM is discussed in relation to the pathogenesis of PD.     

An Integrated Approach of Differential Mass Spectrometry and Gene Ontology Analysis Identified Novel Proteins Regulating Neuronal Differentiation and Survival

MS-based quantitative proteomics is widely used for large scale identification of proteins. However, an integrated approach that offers comprehensive proteome coverage, a tool for the quick categorization of the identified proteins, and a standardized biological study method is needed for helping the researcher focus on investigating the proteins with biologically important functions. In this study, we utilized isobaric tagging for relative and absolute quantification (iTRAQ)-based quantitative differential LC/MS/MS, functional annotation with a proprietary gene ontology tool (Molecular Annotation by Gene Ontology (MANGO)), and standard biochemical methods to identify proteins related to neuronal differentiation in nerve growth factor-treated rat pheochromocytoma (PC12) cells, which serve as a representative model system for studying neuronal biological processes. We performed MS analysis by using both nano-LC-MALDI-MS/MS and nano-LC-ESI-MS/MS for maximal proteome coverage. Of 1,482 non-redundant proteins semiquantitatively identified, 72 were differentially expressed with 39 up- and 33 down-regulated, including 64 novel nerve growth factor-responsive PC12 proteins. Gene ontology analysis of the differentially expressed proteins by MANGO indicated with statistical significance that the up-regulated proteins were mostly related to the biological processes of cell morphogenesis, apoptosis/survival, and cell differentiation. Some of the up-regulated proteins of unknown function, such as PAIRBP1, translationally controlled tumor protein, prothymosin α, and MAGED1, were further analyzed to validate their significant functions in neuronal differentiation by immunoblotting and immunocytochemistry using each antibody combined with a specific short interfering RNA technique. Knockdown of these proteins caused abnormal cell morphological changes, inhibition of neurite formation, and cell death during each course of the differentiation, confirming their important roles in neurite formation and survival of PC12 cells. These results show that our iTRAQ-MANGO-biological analysis framework, which integrates a number of standard proteomics strategies, is effective for targeting and elucidating the functions of proteins involved in the cellular biological process being studied.MS-based quantitative proteomics strategies such as iTRAQ¹ (1) and stable isotope labeling with amino acids in cell culture (2) are powerfully effective for the comprehensive characterization of biological phenomena (1–5). Although these methods have been applied for cancer biomarker (6, 7) and drug target (8) discovery, their use in the elucidation of biological and functional processes has been limited because of certain technical problems that arise when attempting to meaningfully process the immense amount of data obtained from such experiments. The following four main issues are typically the sources of such difficulties. 1) Quantitative identification by one type of MS system may fail to cover the total proteome because of ionization efficiency differences, such as those between ESI and MALDI, for certain peptides, leading to theoretical limitations in proteome coverage. 2) The public protein databases are often insufficient for searching non-human species because of the limited available genomic information. 3) The identification of the functions and biological processes of thousands of proteins is a formidable task because of the lack of simple and user-friendly software to automate gene ontology (GO) annotation. Furthermore it is difficult to convert large lists of taxonomically diverse proteins into their human orthologs to obtain the richest GO information available. 4) Lastly biological validation strategies for identified proteins have not been standardized. Therefore, we believe an analysis framework that provides (a) comprehensive proteome data; (b) a simple and quick tool for organizing, enriching, and sorting those data to reveal candidate molecules for relation to certain processes; and (c) a standardized biological validation technique would greatly benefit this field. We therefore designed a concise, three-step, sequential proteomics strategy that addresses the above concerns and utilized it successfully in studying the mechanism of neuronal differentiation in PC12 cells.PC12 cells (9) have been widely used as a model of neurons because of their unique advantages, such as stability, homogeneity, strong nerve growth factor (NGF) responsiveness, high differentiation potential, and a wealth of accessible background material, which help to facilitate their manipulation (10). This cell line has also been used for studying the mechanisms of neuronal disorders such as Alzheimer (11), Huntington (12), and Parkinson diseases (13) and neurofibromatosis type 1 (14–16). Here we used PC12 cells as a model for characterizing the mechanisms of neuronal differentiation and neurodegenerative disorders by means of MS-based quantitative proteomics.NGF is one member of a family of structurally and functionally related dimeric polypeptides, neurotrophins, that are essential for the development and maintenance of distinct neuronal populations in the central and peripheral nervous systems (17). The initial signaling cascades in the neuronal cells right after NGF stimulation have been subjected to thorough investigation and characterization by using PC12 cells. After binding of extracellular NGF to the cell membrane-localized tropomyosin-related kinase A (TrkA) receptor, TrkA receptors dimerize and subsequently autophosphorylate each other. Then the phosphorylated receptors recruit a complex of signaling molecules and induce a number of intracellular signaling cascades involving the signaling molecules, such as phosphoinositide 3-kinase, phospholipase C-γ, and Ras (18). The posttranslational modifications, such as phosphorylation cascades, triggered by NGF stimulation play important roles in PC12 cell differentiation. However, knowledge of the precise dynamic molecular events of protein expression in response to NGF signaling in PC12 cells after an interval that allows the stimulation to take full effect and produce morphological changes remains far from complete.Several reported studies have applied such methods as expressed sequence tag (19), restriction landmark cDNA scanning (20), targeted display (21), serial analysis of gene expression (22), and cDNA microarray (23) to survey the global change of differentially expressed genes in PC12 cells before and after NGF treatment (19–23). However, the genes and underlying mechanisms associated with the acquisition of a neuronal phenotype in these cells have not been clarified. Also a few proteomics approaches have been used for identifying the proteins related to NGF-inducible neurite formation in PC12 cells. For example, 2-D electrophoresis was applied in whole-cell extract separation to study the NGF modulation of protein synthesis (24); however, only two peptides were identified (25). Even currently available PC12 cell 2-D databases include merely a few proteins related to NGF stimulation (26–29). There is thus a paucity of functional proteomic information related to PC12 cell biological processes that may be attributed to technical limitations such as those listed above.In this study, we performed the first proteomics survey of proteins differentially expressed in PC12 cells during NGF treatment by using a semiquantitative differential LC shotgun method, namely isobaric tagging for relative and absolute quantitation (iTRAQ) coupled with concurrent use of two tandem MS/MS systems, namely nano-LC-MALDI-TOF-TOF and nano-LC-ESI-Quadrupole/quadrupole/time-of-flight mass spectrometers. The total list of proteins identified was converted into a new file linked to the GO database by our proprietary GO analysis tool for proteomes (MANGO) and categorized by biological process and function using specific classification methods. Thereafter we classified the subset of proteins that were up- or down-regulated during neurite formation into specific molecular categories by combining the differential data obtained by iTRAQ with the proteomic GO analysis results. We then attempted to characterize the functional mechanism of NGF-induced PC12 cell neuronal differentiation. Interestingly the specific up-regulated groups classified in this study were related to apoptosis/cell survival in addition to cell motility, differentiation, stress stimulation, and morphogenesis. To investigate the molecular functions of the up-regulated proteins in relation to both PC12 cell differentiation and apoptosis/survival during neurite formation, some of them were further analyzed with a biochemical and cellular biological strategy using a combined antibody and siRNA technique. Lastly we demonstrated the advantages that our concise, sequential proteomics strategy offers for studying the molecular mechanisms of cellular biological events such as cell differentiation and survival/apoptosis.     

Development and topography of the lateral olfactory tract in the mouse: imaging by genetically encoded and injected fluorescent markers

In mammals, conventional odorants are detected by OSNs located in the main olfactory epithelium of the nose. These neurons project their axons to glomeruli, which are specialized structures of neuropil in the olfactory bulb. Within glomeruli, axons synapse onto dendrites of projection neurons, the mitral and tufted (M/T) cells. Genetic approaches to visualize axons of OSNs expressing a given odorant receptor have proven very useful in elucidating the organization of these projections to the olfactory bulb. Much less is known about the development and connectivity of the lateral olfactory tract (LOT), which is formed by axons of M/T cells connecting the olfactory bulb to central neural regions. Here, we have extended our genetic approach to mark M/T cells of the main olfactory bulb and their axons in the mouse, by targeted insertion of IRES-tauGFP in the neurotensin locus. In NT-GFP mice, we find that M/T cells of the main olfactory bulb mature and project axons as early as embryonic day 11.5. Final innervation of central areas is accomplished before the end of the second postnatal week. M/T cell axons that originate from small defined areas within the main olfactory bulb, as visualized by localized injections of fluorescent tracers in wild-type mice at postnatal days 1 to 3, follow a dual trajectory: a branch of tightly packed axons along the dorsal aspect of the LOT, and a more diffuse branch along the ventral aspect. The dorsal, but not the ventral, subdivision of the LOT exhibits a topographical segregation of axons coming from the dorsal versus ventral main olfactory bulb. The NT-GFP mouse strain should prove useful in further studies of development and topography of the LOT, from E11.5 until 2 weeks after birth.     

Osteogenic properties of human myogenic progenitor cells

Here, we identified human myogenic progenitor cells coexpressing Pax7, a marker of muscle satellite cells and bone-specific alkaline phosphatase, a marker of osteoblasts, in regenerating muscle. To determine whether human myogenic progenitor cells are able to act as osteoprogenitor cells, we cultured both primary and immortalized progenitor cells derived from the healthy muscle of a nondystrophic woman. The undifferentiated myogenic progenitors spontaneously expressed two osteoblast-specific proteins, bone-specific alkaline phosphatase and Runx2, and were able to undergo terminal osteogenic differentiation without exposure to an exogenous inductive agent such as bone morphogenetic proteins. They also expressed the muscle lineage-specific proteins Pax7 and MyoD, and lost their osteogenic characteristics in association with terminal muscle differentiation. Both myoblastic and osteoblastic properties are thus simultaneously expressed in the human myogenic cell lineage prior to commitment to muscle differentiation. In addition, C3 transferase, a specific inhibitor of Rho GTPase, blocked myogenic but not osteogenic differentiation of human myogenic progenitor cells. These data suggest that human myogenic progenitor cells retain the capacity to act as osteoprogenitor cells that form ectopic bone spontaneously, and that Rho signaling is involved in a critical switch between myogenesis and osteogenesis in the human myogenic cell lineage.     

Sorting signals within the Saccharomyces cerevisiae sporulation-specific dityrosine transporter, Dtr1p, C terminus promote Golgi-to-prospore membrane transport

During sporulation in Saccharomyces cerevisiae, the dityrosine transporter Dtr1p, which is required for formation of the outermost layer of the spore wall, is specifically expressed and transported to the prospore membrane, a novel double-lipid-bilayer membrane. Dtr1p consists of 572 amino acids with predicted N- and C-terminal cytoplasmic extensions and 12 transmembrane domains. Dtr1p missing the largest internal cytoplasmic loop was trapped in the endoplasmic reticulum in both mitotically dividing cells and cells induced to sporulate. Deletion of the carboxyl 15 amino acids, but not the N-terminal extension of Dtr1p, resulted in a protein that failed to localize to the prospore membrane and was instead observed in cytoplasmic puncta. The puncta colocalized with a cis-Golgi marker, suggesting that Dtr1p missing the last 15 amino acids was trapped in an early Golgi compartment. Deletion of the C-terminal 10 amino acids resulted in a protein that localized to the prospore membrane with a delay and accumulated in cytoplasmic puncta that partially colocalized with a trans-Golgi marker. Both full-length Dtr1p and Dtr1p missing the last 10 amino acids expressed in vegetative cells localized to the plasma membrane and vacuoles, while Dtr1p deleted for the carboxyl-terminal 15 amino acids was observed only at vacuoles, suggesting that transport to the prospore membrane is mediated by distinct signals from those that specify plasma membrane localization. Transfer-of-function experiments revealed that both the carboxyl transmembrane domain and the C-terminal tail are important for Golgi complex-to-prospore membrane transport.