Establishment of a novel human T-cell leukemia virus type 1 infection model using cell-free virus.

The primary mode of infection by human T-cell leukemia virus type 1 (HTLV-1) is cell-to-cell transmission during contact between infected cells and target cells. Cell-free HTLV-1 infections are known to be less efficient than infections with other retroviruses, and transmission of free HTLV-1 is considered not to occur in vivo. However, it has been demonstrated that cell-free HTLV-1 virions can infect primary lymphocytes and dendritic cells in vitro, and that virions embedded in biofilms on cell membranes can contribute to transmission. The establishment of an efficient cell-free HTLV-1 infection model would be a useful tool for analyzing the replication process of HTLV-1 and the clonal expansion of infected cells. We first succeeded in obtaining supernatants with high-titer cell-free HTLV-1 using a highly efficient virus-producing cell line. The HTLV-1 virions retained the structural characteristics of retroviruses. Using this cell-free infection model, we confirmed that a variety of cell lines and primary cultured cells can be infected with HTLV-1 and demonstrated that the provirus was randomly integrated into all chromosomes in the target cells. The provirus-integrated cell lines were HTLV-1-productive. Furthermore, we demonstrated for the first time that cell-free HTLV-1 is infectious in vivo using a humanized mouse model. These results indicate that this cell-free infection model recapitulates the HTLV-1 life cycle, including entry, reverse transcription, integration into the host genome, viral replication, and secondary infection. The new cell-free HTLV-1 infection model is promising as a practical resource for studying HTLV-1 infection.IMPORTANCECo-culture of infected and target cells is frequently used for studying HTLV-1 infection. Although this method efficiently infects HTLV-1, the cell mixture is complex, and it is extremely difficult to distinguish donor infected cells from target cells. In contrast, cell-free HTLV-1 infection models allow for more strict experimental conditions. In this study, we established a novel and efficient cell-free HTLV-1 infection model. Using this model, we successfully evaluated the infectivity titers of cell-free HTLV-1 as proviral loads (copies per 100 cells) in various cell lines, primary cultured cells, and a humanized mouse model. Interestingly, the HTLV-1-associated viral biofilms played an important role in enhancing the infectivity of the cell-free infection model. This cell-free HTLV-1 infection model reproduces the replication cycle of HTLV-1 and provides a simple, powerful, and alternative tool for researching HTLV-1 infection.

Performance evaluation of Espline HTLV-I/II, a newly developed rapid immunochromatographic antibody test for different diagnostic situations.

IMPORTANCE: The World Health Organization estimated that 5-10 million people are infected with human T-cell leukemia virus type 1 (HTLV-1). This number is likely to be underestimated because reliable endemic data are available for only approximately 1.5 billion people worldwide. The point-of-care test is a powerful tool for the easy and quick detection of infections without the requirement for expensive instruments and laboratory equipment. Espline HTLV-I/II, a newly developed rapid immunochromatographic antibody test that was evaluated in this study, might significantly advance our understanding of the global epidemiology of HTLV-1 infection.

Four conserved amino acids on human papillomavirus E6 predict clinical high-risk types.

Human papillomavirus (HPV) types included in the genus alpha papillomavirus (alpha-HPVs) are subdivided into high- and low-risk HPVs associated with tumorigenicity. According to conventional risk classification, over 30 alpha-HPVs remain unclassified and HPV groups phylogenetically classified using the L1 gene do not exactly correspond to the conventional risk classification groups. Here, we propose a novel cervical lesion progression risk classification strategy. Using four E6 risk distinguishable amino acids (E6-RDAAs), we successfully expanded the conventional classification to encompass alpha-HPVs and resolve discrepancies. We validated our classification system using alpha-HPV-targeted sequence data of 325 cervical swab specimens from participants in Japan. Clinical outcomes significantly correlated with the E6-RDAA classification. Four of five HPV types in the data set that were not conventionally classified (HPV30, 34, 67, and 69) were high-risk according to our classification criteria. This report sheds light on the carcinogenicity of rare genital HPV types using a novel risk classification strategy.

Dysgerminoma of the Left Ovary in a Patient with Balanced Translocation 46X, t(X:1) (q22;q21): A Case Report.

We report a case of dysgerminoma in a 22-year-old woman diagnosed with chromosomal abnormality, balanced translocation 46X,t(X:1)(q22;q21). She had received hormone replacement therapy for 7 years for primary amenorrhea. She visited a primary care physician because of lower abdominal distension, and a large tumor in the pelvis was discovered. She was admitted to our hospital for further examination of the pelvic tumor. She underwent laparotomy and was diagnosed with stage IIIA1 dysgerminoma (pT3apN0pM0) of the left ovary. Young female patients without the Y chromosome who are treated for primary amenorrhea may also develop malignant germ cell tumors; therefore, gynecologists should provide hormone replacement therapy and periodic pelvic evaluation.

Variant-specific changes in RAC3 function disrupt corticogenesis in neurodevelopmental phenotypes.

Variants in RAC3, encoding a small GTPase RAC3 which is critical for the regulation of actin cytoskeleton and intracellular signal transduction, are associated with a rare neurodevelopmental disorder with structural brain anomalies and facial dysmorphism. We investigated a cohort of 10 unrelated participants presenting with global psychomotor delay, hypotonia, behavioural disturbances, stereotyped movements, dysmorphic features, seizures and musculoskeletal abnormalities. MRI of brain revealed a complex pattern of variable brain malformations, including callosal abnormalities, white matter thinning, grey matter heterotopia, polymicrogyria/dysgyria, brainstem anomalies and cerebellar dysplasia. These patients harboured eight distinct de novo RAC3 variants, including six novel variants (NM_005052.3): c.34G > C p.G12R, c.179G > A p.G60D, c.186_188delGGA p.E62del, c.187G > A p.D63N, c.191A > G p.Y64C and c.348G > C p.K116N. We then examined the pathophysiological significance of these novel and previously reported pathogenic variants p.P29L, p.P34R, p.A59G, p.Q61L and p.E62K. In vitro analyses revealed that all tested RAC3 variants were biochemically and biologically active to variable extent, and exhibited a spectrum of different affinities to downstream effectors including p21-activated kinase 1. We then focused on the four variants p.Q61L, p.E62del, p.D63N and p.Y64C in the Switch II region, which is essential for the biochemical activity of small GTPases and also a variation hot spot common to other Rho family genes, RAC1 and CDC42. Acute expression of the four variants in embryonic mouse brain using in utero electroporation caused defects in cortical neuron morphology and migration ending up with cluster formation during corticogenesis. Notably, defective migration by p.E62del, p.D63N and p.Y64C were rescued by a dominant negative version of p21-activated kinase 1. Our results indicate that RAC3 variants result in morphological and functional defects in cortical neurons during brain development through variant-specific mechanisms, eventually leading to heterogeneous neurodevelopmental phenotypes.

Expression Analyses of Rac3, a Rho Family Small GTPase, during Mouse Brain Development.

Rac3 is a member of Rho family small GTPases which regulate cellular signaling and cytoskeletal dynamics. The RAC3 gene abnormalities have been shown to cause neurodevelopmental disorders with structural brain anomalies, including polymicrogyria/dysgyria, callosal abnormalities, brainstem anomalies, and cerebellar dysplasia. Although this evidence indicates that Rac3 is essential in brain development, not only its molecular mechanism but also the expression profile is yet to be elucidated. In this study, we carried out expression analyses of Rac3 with mouse brain tissues. In immunoblotting, Rac3 exhibited a tissue-dependent expression profile in the young adult mouse and was expressed in a developmental stage-dependent manner in brain. In primary cultured hippocampal neurons, while Rac3 was distributed mainly in the cytoplasm, it was visualized in axon and dendrites with partial localization at synapses, in consistent with the observation in biochemical fractionation analyses. In immunofluorescence analyses with brain slices, Rac3 was distributed strongly and moderately in the axon and cytoplasm, respectively, of cerebral cortex at postnatal day (P) 2 and P18. Similar distribution profile was also observed in hippocampus. Taken together, the results obtained strongly suggest that Rac3 plays an important physiological role in neuronal tissues during corticogenesis, and defects in the Rac3 function induce structural brain anomalies leading to pathogenesis of neurodevelopmental disorders.

Expression analyses of PLEKHG2, a Rho family-specific guanine nucleotide exchange factor, during mouse brain development.

Abnormalities of PLEKHG2 gene, encoding a Rho family-specific guanine nucleotide exchange factor, are involved in microcephaly with intellectual disability. However, not only the role of PLEKHG2 in the developmental process but also its expression profile is unknown. In this study, we prepared a specific antibody against PLEKHG2 and carried out expression analyses with mouse tissues. In western blotting, PLEKHG2 exhibited a tissue-dependent expression profile in adult mouse and was expressed in a developmental stage-dependent manner in brain. Then, in immunohistochemical analyses, while PLEKHG2 was observed in the cortical plate and ventricular zone surface of the cerebral cortex at embryonic day 14, it came to be distributed throughout the cerebral cortex in layer II/III and V during corticogenesis. PLEKHG2 was also detected mainly in the nucleus of neurons in the hippocampal CA regions and dentate gyrus at P7. Notably, the nuclear accumulation disappeared at P30 and PLEKHG2 came to be located at the axons and/or dendrites at this time point. Moreover, in vitro immunofluorescence revealed that PLEKHG2 was at least partially localized at both excitatory and inhibitory synapses in primary cultured hippocampal neurons. These results suggest roles of PLEKHG2 in the development of the central nervous tissue and synaptic function.

MYCN de novo gain-of-function mutation in a patient with a novel megalencephaly syndrome.

BACKGROUND: In this study, we aimed to identify the gene abnormality responsible for pathogenicity in an individual with an undiagnosed neurodevelopmental disorder with megalencephaly, ventriculomegaly, hypoplastic corpus callosum, intellectual disability, polydactyly and neuroblastoma. We then explored the underlying molecular mechanism. METHODS: Trio-based, whole-exome sequencing was performed to identify disease-causing gene mutation. Biochemical and cell biological analyses were carried out to elucidate the pathophysiological significance of the identified gene mutation. RESULTS: We identified a heterozygous missense mutation (c.173C>T; p.Thr58Met) in the MYCN gene, at the Thr58 phosphorylation site essential for ubiquitination and subsequent MYCN degradation. The mutant MYCN (MYCN-T58M) was non-phosphorylatable at Thr58 and subsequently accumulated in cells and appeared to induce CCND1 and CCND2 expression in neuronal progenitor and stem cells in vitro. Overexpression of Mycn mimicking the p.Thr58Met mutation also promoted neuronal cell proliferation, and affected neuronal cell migration during corticogenesis in mouse embryos. CONCLUSIONS: We identified a de novo c.173C>T mutation in MYCN which leads to stabilisation and accumulation of the MYCN protein, leading to prolonged CCND1 and CCND2 expression. This may promote neurogenesis in the developing cerebral cortex, leading to megalencephaly. While loss-of-function mutations in MYCN are known to cause Feingold syndrome, this is the first report of a germline gain-of-function mutation in MYCN identified in a patient with a novel megalencephaly syndrome similar to, but distinct from, CCND2-related megalencephaly-polymicrogyria-polydactyly-hydrocephalus syndrome. The data obtained here provide new insight into the critical role of MYCN in brain development, as well as the consequences of MYCN defects.

Rho family GTPases, Rac and Cdc42, control the localization of neonatal dentate granule cells during brain development.

The hippocampus is generally considered as a brain center for learning and memory. We have recently established an electroporation-mediated gene transfer method to investigate the development of neonatal dentate granule cells in vivo. Using this new technique, we introduced knockdown vectors against Rac1 small GTPase into precursors for dentate granule cells at postnatal day 0. After 21 days, Rac1-deficient cells were frequently mispositioned between the granule cell layer (GCL) and hilus. About 60% of these mislocalized cells expressed a dentate granule cell marker, Prox1. Both the dendritic spine density and the ratio of mature spine were reduced when Rac1 was silenced. Notably, the deficient cells have immature thin processes during migrating in the early neonatal period. Knockdown of another Rac isoform, Rac3, also resulted in mislocalization of neonatally born dentate granule cells. In addition, knockdown of Cdc42, another Rho family protein, also caused mislocalization of the cell, although the effects were moderate compared to Rac1 and 3. Despite the ectopic localization, Rac3- or Cdc42-disrupted mispositioned cells expressed Prox1. These results indicate that Rho signaling pathways differentially regulate the proper localization and differentiation of dentate granule cells.

Functions of Rhotekin, an Effector of Rho GTPase, and Its Binding Partners in Mammals.

Rhotekin is an effector protein for small GTPase Rho. This protein consists of a Rho binding domain (RBD), a pleckstrin homology (PH) domain, two proline-rich regions and a C-terminal PDZ (PSD-95, Discs-large, and ZO-1)-binding motif. We, and other groups, have identified various binding partners for Rhotekin and carried out biochemical and cell biological characterization. However, the physiological functions of Rhotekin, per se, are as of yet largely unknown. In this review, we summarize known features of Rhotekin and its binding partners in neuronal tissues and cancer cells.

Dnmt1-dependent Chk1 pathway suppression is protective against neuron division.

Neuronal differentiation and cell-cycle exit are tightly coordinated, even in pathological situations. When pathological neurons re-enter the cell cycle and progress through the S phase, they undergo cell death instead of division. However, the mechanisms underlying mitotic resistance are mostly unknown. Here, we have found that acute inactivation of retinoblastoma (Rb) family proteins (Rb, p107 and p130) in mouse postmitotic neurons leads to cell death after S-phase progression. Checkpoint kinase 1 (Chk1) pathway activation during the S phase prevented the cell death, and allowed the division of cortical neurons that had undergone acute Rb family inactivation, oxygen-glucose deprivation (OGD) or in vivo hypoxia-ischemia. During neurogenesis, cortical neurons became protected from S-phase Chk1 pathway activation by the DNA methyltransferase Dnmt1, and underwent cell death after S-phase progression. Our results indicate that Chk1 pathway activation overrides mitotic safeguards and uncouples neuronal differentiation from mitotic resistance.

Schizophrenia susceptibility gene product dysbindin-1 regulates the homeostasis of cyclin D1.

Dysbindin-1 (dystrobrevin binding protein-1, DTNBP1) is now widely accepted as a potential schizophrenia susceptibility gene and accumulating evidence indicates its functions in the neural development. In this study, we tried to identify new binding partners for dysbindin-1 to clarify the novel function of this molecule. When consulted with BioGRID protein interaction database, cyclin D3 was found to be a possible binding partner for dysbindin-1. We then examined the interaction between various dysbindin-1 isoforms (dysbindin-1A, -1B and -1C) and all three D-type cyclins (cyclin D1, D2, and D3) by immunoprecipitation with the COS7 cell expression system, and found that dysbindin-1A preferentially interacts with cyclin D1. The mode of interaction between these molecules was considered as direct binding since recombinant dysbindin-1A and cyclin D1 formed a complex in vitro. Mapping analyses revealed that the C-terminal region of dysbindin-1A binds to the C-terminal of cyclin D1. Consistent with the results of the biochemical analyses, endogenous dysbindin-1was partially colocalized with cyclin D1 in NIH3T3 fibroblast cells and in neuronal stem and/or progenitor cells in embryonic mouse brain. While co-expression of dysbindin-1A with cyclin D1 changed the localization of the latter from the nucleus to cytosol, cyclin D1-binding partner CDK4 inhibited the dysbindin-cyclin D1 interaction. Meanwhile, depletion of endogenous dysbindin-1A increased cyclin D1 expression. These results indicate that dysbindin-1A may control the cyclin D1 function spatiotemporally and might contribute to better understanding of the pathophysiology of dysbindin-1-associated disorders.

Circadian-relevant genes are highly polymorphic in autism spectrum disorder patients.

BACKGROUND: The genetic background of autism spectrum disorder (ASD) is considered a multi-genetic disorder with high heritability. Autistic children present with a higher prevalence of sleep disorders than has been observed in children with normal development. Some circadian-relevant genes have been associated with ASD (e.g., PER1, PER2, NPAS2, MTNR1A, and MTNR1B). METHODS: We analyzed 28 ASD patients (14 with sleep disorders and 14 without) and 23 control subjects of Japanese descent. The coding regions of 18 canonical clock genes and clock-controlled genes were sequenced. Detected mutations were verified by direct sequencing analysis, and additional control individuals were screened. RESULTS: Thirty-six base changes with amino acid changes were detected in 11 genes. Six missense changes were detected only in individuals with ASD with sleep disturbance: p.F498S in TIMELESS, p.S20R in NR1D1, p.R493C in PER3, p.H542R in CLOCK, p.L473S in ARNTL2, and p.A325V in MTNR1B. Six missense changes were detected only in individuals with ASD without sleep disturbance: p.S1241N in PER1, p.A325T in TIMELESS, p.S13T in ARNTL, p.G24E in MTNR1B, p.G24E in PER2, and p.T1177A in PER3. The p.R493C mutation in PER3 was detected in both groups. One missense change, p.P932L in PER2, was detected only in the control group. Mutations in NR1D1, CLOCK, and ARNTL2 were detected only in individuals with ASD with sleep disorder. The prevalence of the mutations detected only single time differed significantly among all ASD patients and controls (p=0.003). Two kinds of mutations detected only in individuals with ASD with sleep disorder, p.F498S in TIMELESS and p.R366Q in PER3, were considered to affect gene function by three different methods: PolyPhen-2, scale-invariant feature transform (SIFT) prediction, and Mutation Taster (www.mutationtaster.org). The mutations p.S20R in NR1D1, p.H542R in CLOCK, p.L473S in ARNTL2, p.A325T in TIMELESS, p.S13T in ARNTL, and p.G24E in PER2 were diagnosed to negatively affect gene function by more than one of these methods. CONCLUSION: Mutations in circadian-relevant genes affecting gene function are more frequent in patients with ASD than in controls. Circadian-relevant genes may be involved in the psychopathology of ASD.

Expression of drebrin, an actin binding protein, in basal cell carcinoma, trichoblastoma and trichoepithelioma.

Drebrin, an F-actin binding protein, is known to play important roles in cell migration, synaptogenesis and neural plasticity. Although drebrin was long thought to be specific for neuronal cells, its expression has recently been reported in non-neuronal cells. As for skin-derived cells, drebrin was shown to be enriched at adhering junctions (AJs) in cultured primary keratinocytes and also be highly expressed in basal cell carcinoma (BCC) cells. Since BCC and two types of benign neoplasm, trichoblastoma and trichoepithelioma, are considered to derive from the same origin, follicular germinative cells, it is sometimes difficult to morphologically distinguish BCC from trichoblastoma and trichoepithelioma. In this study, we performed immunohistochemical staining of drebrin in BCC, trichoblastoma and trichoepithelioma, to examine whether drebrin could serve as a biomarker for BCC diagnosis. In western blotting, drebrin was detected highly and moderately in the lysates from a squamous cell carcinoma cell line, DJM-1, and normal human epidermis, respectively. In immunofluorescence analyses, drebrin was colocalized with markers of AJs and tight junctions in DJM-1 cells and detected at cell-cell junction areas of human normal epidermis tissue. We then examined the distribution patterns of drebrin in BCC, trichoblastoma and trichoepithelioma. In BCC tissues, intense and homogeneous drebrin expression was observed mainly at tumor cell-cell boundaries. In contrast, drebrin was stained only weakly and non-homogeneously in trichoblastoma and trichoepthelioma tissue samples. For differential diagnosis of BCC, drebrin may be a novel and useful marker.

MAGI-1 acts as a scaffolding molecule for NGF receptor-mediated signaling pathway.

We have recently found that the membrane-associated guanylate kinase with inverted organization-1 (MAGI-1) was enriched in rat nervous tissues such as the glomeruli in olfactory bulb of adult rats and dorsal root entry zone in spinal cord of embryonic rats. In addition, we revealed the localization of MAGI-1 in the growth cone of the primary cultured rat dorsal root ganglion cells. These results point out the possibility that MAGI-1 is involved in the regulation of neurite extension or guidance. In this study, we attempted to reveal the physiological role(s) of MAGI-1 in neurite extension. We found that RNA interference (RNAi)-mediated knockdown of MAGI-1 caused inhibition of nerve growth factor (NGF)-induced neurite outgrowth in PC12 rat pheochromocytoma cells. To clarify the involvement of MAGI-1 in NGF-mediated signal pathway, we tried to identify binding partners for MAGI-1 and identified p75 neurotrophin receptor (p75NTR), a low affinity NGF receptor, and Shc, a phosphotyrosine-binding adaptor. These three proteins formed an immunocomplex in PC12 cells. Knockdown as well as overexpression of MAGI-1 caused suppression of NGF-stimulated activation of the Shc-ERK pathway, which is supposed to play important roles in neurite outgrowth of PC12 cells. These results indicate that MAGI-1 may act as a scaffolding molecule for NGF receptor-mediated signaling pathway.

Possible role of a septin, SEPT1, in spreading in squamous cell carcinoma DJM-1 cells.

We performed biochemical, histochemical and cell biological characterization of septins by focusing on SEPT1 in human skin tissues and a squamous cell carcinoma (SCC) cell line DJM-1. In immunoblotting, SEPT1, together with other septins, was detected in normal human epidermis, SCC and DJM-1. In immunohistochemical analyses, SEPT1 was detected diffusely in the cytoplasm of human epidermal cells and eccrine gland epithelial cells, and the protein level was increased in some skin tumors. In DJM-1 cells, SEPT1 together with other members of SEPT2-subgroup, SEPT4 and SEPT5, was enriched in lamellipodia and the localization was dependent on the cortical actin structure. SEPT1 distribution at lamellipodia was also observed in melanoma B16 cells. SEPT9, SEPT11 and SEPT14, in contrast, were localized along with microtubules in DJM-1 cells. In immunoprecipitation assays, SEPT1 and SEPT5 were found to form a complex in DJM-1 cells, whereas SEPT9, SEPT11 and SEPT14 formed a distinct complex with other septins including SEPT7, SEPT8 and SEPT10, in which SEPT5 was not included. When SEPT1 was silenced in DJM-1 cells, cell spreading was inhibited. These results suggest that SEPT1 may participate in cell-cell and/or cell-substrate interaction in DJM-1 and exert its function in a coordinated manner with other septins.

Cell biological characterization of a multidomain adaptor protein, ArgBP2, in epithelial NMuMG cells, and identification of a novel short isoform.

ArgBP2 is a member of the SoHo (sorbin-homology) family of adaptor proteins believed to play roles in cell adhesion, cytoskeletal organization, and signaling. We show here a novel splicing isoform of ArgBP2, i.e., ArgBP2™, composed of only three SH3 (src-homology 3) domains and structurally similar to vinexinß. We then characterized the biochemical and cell biological properties of ArgBP2 to compare these with vinexin. Similar to vinexin, ArgBP2 was enriched at focal adhesions in REF52 fibroblast cells and induced anchorage-dependent extracellular signal-regulated kinase activation in NIH3T3 fibroblast cells. In epithelial NMuMG cells, immunofluorescence analyses revealed localization of ArgBP2 at tight junctions (TJs), whereas vinexin was distributed in cytoplasm as well as cell-cell boundaries. During TJ formation, recruitment of ZO-1 to TJs was followed by ArgBP2. Based on mutation analyses, a second SH3 domain was found to be important for ArgBP2 localization to the cell-cell contact sites. These data suggest some role of ArgBP2 in NMuMG cells at TJs that may be distinct from the function of vinexin.

Phase advance of the light-dark cycle perturbs diurnal rhythms of brain-derived neurotrophic factor and neurotrophin-3 protein levels, which reduces synaptophysin-positive presynaptic terminals in the cortex of juvenile rats.

In adult rat brains, brain-derived neurotrophic factor (BDNF) rhythmically oscillates according to the light-dark cycle and exhibits unique functions in particular brain regions. However, little is known of this subject in juvenile rats. Here, we examined diurnal variation in BDNF and neurotrophin-3 (NT-3) levels in 14-day-old rats. BDNF levels were high in the dark phase and low in the light phase in a majority of brain regions. In contrast, NT-3 levels demonstrated an inverse phase relationship that was limited to the cerebral neocortex, including the visual cortex, and was most prominent on postnatal day 14. An 8-h phase advance of the light-dark cycle and sleep deprivation induced an increase in BDNF levels and a decrease in NT-3 levels in the neocortex, and the former treatment reduced synaptophysin expression and the numbers of synaptophysin-positive presynaptic terminals in cortical layer IV and caused abnormal BDNF and NT-3 rhythms 1 week after treatment. A similar reduction of synaptophysin expression was observed in the cortices of Bdnf gene-deficient mice and Ca(2+)-dependent activator protein for secretion 2 gene-deficient mice with abnormal free-running rhythm and autistic-like phenotypes. In the latter mice, no diurnal variation in BDNF levels was observed. These results indicate that regular rhythms of BDNF and NT-3 are essential for correct cortical network formation in juvenile rodents.

TDP-43 depletion induces neuronal cell damage through dysregulation of Rho family GTPases.

The 43-kDa TAR DNA-binding protein (TDP-43) is known to be a major component of the ubiquitinated inclusions characteristic of amyotrophic lateral sclerosis and frontotemporal lobar degeneration with ubiquitin-positive inclusions. Although TDP-43 is a nuclear protein, it disappears from the nucleus of affected neurons and glial cells, implicating TDP-43 loss of function in the pathogenesis of neurodegeneration. Here we show that the knockdown of TDP-43 in differentiated Neuro-2a cells inhibited neurite outgrowth and induced cell death. In knockdown cells, the Rho family members RhoA, Rac1, and Cdc42 GTPases were inactivated, and membrane localization of these molecules was reduced. In addition, TDP-43 depletion significantly suppressed protein geranylgeranylation, a key regulating factor of Rho family activity and intracellular localization. In contrast, overexpression of TDP-43 mitigated the cellular damage caused by pharmacological inhibition of geranylgeranylation. Furthermore administration of geranylgeranyl pyrophosphate partially restored cell viability and neurite outgrowth in TDP-43 knockdown cells. In summary, our data suggest that TDP-43 plays a key role in the maintenance of neuronal cell morphology and survival possibly through protein geranylgeranylation of Rho family GTPases.

Possible roles of vinexinbeta in growth and paclitaxel sensitivity in human prostate cancer PC-3 cells.

PURPOSE: Vinexinbeta is an adaptor protein supposed to play pivotal roles in cell adhesion, cytoskeletal organization and signaling. Vinexinbeta is reported to be phosphorylated by extracelluler signal-regulated kinase (ERK) and the phosphorylation has been shown to be involved in cell adhesion, migration and growth. However, physiological function as well as pathophysiological relevance of vinexinbeta in cancer cells is almost unknown. METHODS: By use of biochemical and cell biological techniques, we analyzed the effects of overexpression or RNAi-mediated silencing of vinexinbeta on the growth and paclitaxel sensitivity in a prostate cancer cell line PC-3. RESULTS: Vinexinbeta was highly expressed in androgen-independent prostate cancer cell lines, PC-3 and DU145, but not in androgen-dependent LNCaP cells. We established two PC-3 cell lines, PC-3/Vinbeta#1 and #2, overexpressing GFP-tagged vinexinbeta and found that growth rate of these lines was significantly increased compared to a mock-transfected cell line. In addition, we found that PC-3/Vinbeta#1 and #2 became resistant to the treatment with 100 nM paclitaxel for 48 h. On the other hand, when siRNA-mediated vinexinbeta gene silencing was performed, PC-3 cell growth was suppressed. In addition, by vinexinbeta silencing, PC-3 cells became significantly sensitized to 10 nM paclitaxel treatment for 48 h. CONCLUSIONS: Vinexinbeta plays an important role in PC-3 cell growth, and abrogation of vinexinbeta may be effective for therapeutic cell death and enhanced chemotherapy sensitization in androgen-independent prostate cancer cells.