Dendritic distribution of CDK5 mRNA and p35 mRNA, and a glutamate-responsive increase of CDK5/p25 complex contribute to tau hyperphosphorylation.

BACKGROUND: In Alzheimer's disease (AD), abnormally phosphorylated tau in the somatodendrite compartment of brain neurons causes synaptic loss, resulting in neuron death. Although the mechanism by which hyperphosphorylated tau appears in dendrites remains unclear, we have previously reported that local translation of tau mRNA and GSK3ß mRNA in response to glutamatergic stimulation triggers an increase of tau protein and initiation of a cycle for amplification of reactivated preexisting GSK3ß, respectively. In this study, we investigated the mechanism responsible for neural excitation-dependent activation of another major tau kinase, CDK5, within dendrites. METHODS: Primary hippocampal neurons were treated with glutamate and examined by in situ hybridization, immunocytochemistry and Western blotting. RESULTS: The mRNAs for both CDK5 and its neural-specific activator, p35, were found to be constitutively distributed in dendrites. Glutamate treatment induced immediate local dendritic translation of these proteins as well as conversion of p35 to p25, which forms the hyper-activated CDK5/p25 complex. This neural excitation-dependent tau phosphorylation by CDK5 was suppressed in the presence of a calpain inhibitor or a NMDA receptor antagonist. CONCLUSION: Our results indicate that in addition to an increase of dendritic tau and reactivation of preexisting GSK3ß, increase and hyper-activation of CDK5 are evoked by translation of dendrite-distributed mRNAs upon NMDA receptor-mediated neural excitation. GENERAL SIGNIFICANCE: Hyperphosphorylated tau with AD epitopes is locally produced in dendrites via translational activation of dendrite-distributed mRNAs in response to glutamatergic stimulation. Therefore, tau hyperphosphorylation may play a crucial role in synaptic transduction.

Glutamate-responsive translation of dendritic GSK3ß mRNA triggers a cycle for amplification of reactivated preexisting GSK3ß that is indispensable for tau hyperphosphorylation.

The molecular mechanism responsible for hyperphosphorylated tau accumulation in dendrites of Alzheimer's disease (AD) neurons has not been fully clarified. Recently, we reported that tau mRNA is distributed into dendrites, and that translation and phosphorylation of tau protein are immediately enhanced in response to glutamatergic stimulation. Here, we focused on dendritic glycogen synthase kinase 3ß (GSK3ß), a key enzyme for tau phosphorylation, and investigated the mechanism responsible for the neural stimulation-induced hyperphosphorylation of the newly translated dendritic tau protein. We found that GSK3ß mRNA was also distributed into dendrites of cultured hippocampal neurons, and that a glutamate-dependent slight increase of translation occurred in a short time. Concomitantly, dephosphorylation at the Ser9 residue of the preexisting GSK3ß, which reactivates this kinase, was strongly induced without an increase of its phosphatase PP1 or a decrease of the PP1 inhibitor I-2. Instead, I-2 phosphorylation was observed, suggesting disinhibition of PP1. This glutamate-dependent phosphorylation of I-2 and the dephosphorylation of preexisting GSK3ß were abolished in the presence of GSK3ß inhibitors. Interestingly, translational obstruction of GSK3ß mRNA also canceled these reactions. These results indicate that dendrites exhibit a glutamate-responsive cycle for amplification of reactivated preexisting GSK3ß operating via PP1 disinhibition, whose activation requires neural activity-dependent translation of dendritic GSK3ß mRNA. This would explain why a slight increase of dendritic GSK3ß is sufficient to trigger hyperphosphorylation of significantly increased tau protein.

Indirubin 3'-oxime inhibits anticancer agent-induced YB-1 nuclear translocation in HepG2 human hepatocellular carcinoma cells.

Hepatocellular carcinoma (HCC) is a disease with poor prognosis. Nuclear accumulation of YB-1 is closely related to the malignancy of HCC. Treatment with anticancer agents often induces translocation of YB-1 from cytoplasm to nucleus and activates the expression of multidrug resistance gene 1 (MDR1). Therefore, any effective inhibitor of this phenomenon would be useful for cancer treatment. Here we examined various indirubin derivatives and found that indirubin 3'-oxime inhibits actinomycin D-induced nuclear transport of YB-1 and suppresses the activation of MDR1 gene expression in the human hepatocellular carcinoma cell line HepG2. Furthermore, use of both indirubin 3'-oxime and actinomycin D in combination increased the anticancer effect on HepG2 cells. Indirubin 3'-oxime is a novel and efficient inhibitor of anticancer agent-induced YB-1 nuclear translocation.

Indirubin derivatives protect against endoplasmic reticulum stress-induced cytotoxicity and down-regulate CHOP levels in HT22 cells.

Indirubin and its derivatives have been reported to exhibit anti-cancer and anti-inflammatory activities. Recently, some of its derived analogs have been shown to have neuroprotective potential. Endoplasmic reticulum (ER) stress has been demonstrated to contribute to the pathogenesis of various neurodegenerative diseases, whereas the effects of indirubin derivatives on ER stress-induced cell death have not been addressed. In the present study, a series of 44 derivatives of indirubin was prepared to search for a novel class of neuroprotective agents against ER stress-induced neuronal death. The MTT reduction assay indicated that tunicamycin (TM), an inducer of ER stress, significantly decreased the viability of hippocampal neuronal HT22 cells. Among the compounds tested, eight showed significant inhibitory activity against TM-induced cell death. Western blot analysis showed that application of these analogs to the cells simultaneously with TM reduced the TM-induced expression of CHOP, an established mediator of ER stress. Our results suggest that the preventive effect of these indirubin derivatives against ER stress-induced neuronal death may be due, at least in part, to attenuation of the CHOP-dependent signaling system.

Four nucleocytoplasmic-shuttling proteins and p53 interact specifically with the YB-NLS and are involved in anticancer reagent-induced nuclear localization of YB-1.

In cancer cells, anticancer reagents often trigger nuclear accumulation of YB-1, which participates in the progression of cancer malignancy. YB-1 has a non-canonical nuclear localization signal (YB-NLS). Here we found that four nucleocytoplasmic-shuttling RNA-binding proteins and p53 interact specifically with the YB-NLS and co-accumulate with YB-1 in the nucleus of actinomycin D-treated cells. To elucidate the roles of these YB-NLS-binding proteins, we performed a dominant-negative experiment in which a large excess of YB-NLS interacts with the YB-NLS-binding proteins, and showed inhibitory effects on actinomycin D-induced nuclear transport of endogenous YB-1 and subsequent MDR1 gene expression. Furthermore, the YB-NLS-expressing cells were also found to show increased drug sensitivity. Our results suggest that these YB-NLS-associating proteins are key factors for nuclear translocation/accumulation of YB-1 in cancer cells.

YB-1 gene expression is kept constant during myocyte differentiation through replacement of different transcription factors and then falls gradually under the control of neural activity.

We have previously reported that translation of acetylcholine receptor α-subunit (AChR α) mRNA in skeletal muscle cells is regulated by Y-box binding protein 1 (YB-1) in response to neural activity, and that in the postnatal mouse developmental changes in the amount of YB-1 mRNA are similar to those of AChR α mRNA, which is known to be regulated by myogenic transcription factors. Here, we examined transcriptional regulation of the YB-1 gene in mouse skeletal muscle and differentiating C2C12 myocytes. Although neither YB-1 nor AChR α was detected at either the mRNA or protein level in adult hind limb muscle, YB-1 expression was transiently activated in response to denervation of the sciatic nerve and completely paralleled that of AChR α, suggesting that these genes are regulated by the same transcription factors. However, during differentiation of C2C12 cells to myotubes, the level of YB-1 remained constant even though the level of AChR α increased markedly. Reporter gene, gel mobility shift and ChIP assays revealed that in the initial stage of myocyte differentiation, transcription of the YB-1 gene was regulated by E2F1 and Sp1, and was then gradually replaced under the control of both MyoD and myogenin through an E-box sequence in the proximal region of the YB-1 gene promoter. These results suggest that transcription factors for the YB-1 gene are exchanged during skeletal muscle cell differentiation, perhaps playing a role in translational control of mRNAs by YB-1 in both myotube formation and the response of skeletal muscle tissues to neural stimulation.

Indirubin derivatives alter DNA binding activity of the transcription factor NF-Y and inhibit MDR1 gene promoter.

Indirubin derivatives exert antitumor activity. However, their effects on the expression of multidrug resistance gene 1 (MDR1) have not been investigated. Here we found three derivatives that inhibit the MDR1 gene promoter. To investigate the effects of indirubins on the DNA binding of NF-Y, a major MDR1 gene transcription factor that recognizes an inverted CCAAT element in the promoter, gel mobility shift assay was performed using the element as a probe with nuclear extracts from NG108-15, MCF7, HepG2, C2C12, and SK-N-SH cells. Among 17 compounds, 5-methoxyindirubin inhibited the DNA binding of NF-Y significantly, whereas indirubin-3'-oxime and 7-methoxyindirubin 3'-oxime increased the binding considerably. After evaluating a suitable concentration of each compound for transcription analysis using living tumor cells, we performed a reporter gene assay using a reporter DNA plasmid containing EGFP cDNA fused to the MDR1 gene promoter region. Indirubin-3'-oxime exerted a significant inhibitory effect on the MDR1 promoter activity in MCF7 and HepG2 cells, and 5-methoxyindirubin inhibited the activity only in MCF7 cells; 7-methoxyindirubin 3'-oxime suppressed the activity in all of the cell lines. We further confirmed that the compounds reduced endogenous MDR1 transcription without any inhibitory effect on NF-Y expression. Moreover, each compound increased the doxorubicin sensitivity of MCF7 cells. These results indicate that each indirubin derivative acts on the DNA binding of NF-Y and represses the MDR1 gene promoter with tumor cell-type specificity.

Roles of YB-1 under arsenite-induced stress: translational activation of HSP70 mRNA and control of the number of stress granules.

BACKGROUND: When cells become stressed, they form stress granules (SGs) and show an increase of the molecular chaperone HSP70. The translational regulator YB-1 is a component of SGs, but it is unclear whether it contributes to the translational induction of HSP70 mRNA. Here we examined the roles of YB-1 in SG assembly and translational regulation of HSP70 mRNA under arsenite-induced stress. METHOD: Using arsenite-treated NG108-15 cells, we examined whether YB-1 was included in SGs with GluR2 mRNA, a target of YB-1, and investigated the interaction of YB-1 with HSP70 mRNA and its effect on translation of the mRNA. We also investigated the distribution of these mRNAs to SGs or polysomes, and evaluated the role of YB-1 in SG assembly. RESULTS: Arsenite treatment reduced the translation level of GluR2 mRNA; concomitantly, YB-1-bound HSP70 mRNA was increased and its translation was induced. Sucrose gradient analysis revealed that the distribution of GluR2 mRNA was shifted from heavy-sedimenting to much lighter fractions, and also to SG-containing non-polysomal fractions. Conversely, HSP70 mRNA was shifted from the non-polysomal to polysome fractions. YB-1 depletion abrogated the arsenite-responsive activation of HSP70 synthesis, but SGs harboring both mRNAs were still assembled. The number of SGs was increased by YB-1 depletion and decreased by its overexpression. CONCLUSION: In arsenite-treated cells, YB-1 mediates the translational activation of HSP70 mRNA and also controls the number of SGs through inhibition of their assembly. GENERAL SIGNIFICANCE: Under stress conditions, YB-1 exerts simultaneous but opposing actions on the regulation of translation via SGs and polysomes.

Mechanism of YB-1-mediated translational induction of GluR2 mRNA in response to neural activity through nAChR.

BACKGROUND: We have reported previously that YB-1 induces translation of GluR2 mRNA in response to neural activity, and that HSP60 affects the association of YB-1 with polysomes. Here we examined the mechanism of YB-1-mediated translational activation of GluR2 mRNA through the nAChR. METHODS: Expression of nAChRs in NG108-15 cells was verified. Translation of GluR2 mRNA and YB-1/HSP60 interaction were examined in nicotine-treated NG108-15 cells. Effects of inhibition of α7-nAChR and the PI3K/Akt pathway were investigated. The ratios of YB-1 to GluR2 mRNA and to HSP60 were explored in polysomal and non-polysomal fractions, respectively, and the role of HSP60 in cytoplasmic retention of YB-1 was evaluated. RESULTS: Nicotine treatment transiently induced translation of GluR2 mRNA and Akt phosphorylation with a concomitant increase of YB-1/HSP60 interaction. Both α-bungarotoxin and LY294002 abolished the effects of nicotine. On a sucrose gradient, nicotine treatment shifted the distribution of YB-1 to much heavier-sedimenting polysome fractions. In these fractions, the ratio of YB-1 to its binding GluR2 mRNA was decreased, and ribosome association with the YB-1-bound GluR2 mRNA was increased. HSP60 was distributed only in the non-polysomal fractions as its binding to YB-1 increased. In HSP60-depleted cells, nicotine treatment induced nuclear localization of YB-1. CONCLUSION: YB-1 is released from GluR2 mRNA during α7-nAChR-mediated neurotransmission, causing the PI3K/Akt pathway to recruit ribosomes into the translational machinery, and HSP60 is involved in cytoplasmic retention of polysome-free YB-1. GENERAL SIGNIFICANCE: Activation of the PI3K/Akt pathway through the α7-nAChR and YB-1/HSP60 interaction are important for YB-1-mediated translational activation of GluR2 mRNA.

Translational level of acetylcholine receptor α mRNA in mouse skeletal muscle is regulated by YB-1 in response to neural activity.

Y-box-binding protein 1 (YB-1) binds to mRNAs and affects translation. In this study, we focused on skeletal muscle, in which YB-1 expression is restricted to the early postnatal period, and found that YB-1 binds to acetylcholine receptor α-subunit (AChR α) mRNA. Although transcription of the AChR α gene is known to be regulated by myogenic transcription factors, translational control of the mRNA in response to neuromuscular transmission has not been examined. In undifferentiated C2C12 myoblasts, expression of AChR α remained at a low level. However, translation of the mRNA was increased by knockdown of YB-1. Continued overexpression of YB-1 prevented the cells from differentiating. In myotubes, which show clustering of AChRs, translation of the mRNA was induced within 3h after treatment with nicotine. The effect of nicotine was inhibited by α-bungarotoxin, and in the presence of cycloheximide the level of AChR α was reduced, even after nicotine treatment. Sucrose gradient analysis revealed that in nicotine-treated myotubes, YB-1-containing polysomes were shifted to the heavier-sedimenting fractions, and showed an apparent decrease in the amount of YB-1 bound to AChR α mRNA. These results suggest that in skeletal muscle cells, neural activity reduces the molar ratio of YB-1 relative to its binding AChR α mRNA, leading to an increase of ribosome binding to the mRNA, and thus activating translation. Furthermore, in postnatal growing mice, as has already been shown, the level of AChR α mRNA declined during the early period with maturation of neuromuscular synapses, but the translation level was found to increase transiently at postnatal day 10, when the level of YB-1 was markedly reduced. It is suggested that although the level of AChR α mRNA is reduced, the translation can be induced by alteration of the ratio of YB-1 protein to the mRNA.

YB-1 binds to GluR2 mRNA and CaM1 mRNA in the brain and regulates their translational levels in an activity-dependent manner.

The translational regulator YB-1 binds to mRNAs. In the brain, YB-1 is prominently expressed from the prenatal stage until the first week after birth, being associated with polysomes and distributed in neuronal dendrites, but its expression declines to a much lower level thereafter. It is therefore of interest to identify the mRNAs whose translation is controlled by YB-1 in the postnatal growing brain. In this study we found that YB-1 interacted with the mRNAs for glutamate receptor subunit 2 (GluR2) and calmodulin1 (CaM1) in both brain and NG108-15 cells. Overexpression or knockdown of YB-1 altered the levels of these proteins significantly in cultured cells without any change in their mRNA levels. When the cells were treated with neurotransmitters, translation of these proteins was induced within a short time, and a change in the amount of YB-1 on its target mRNAs was observed in the heavy-sedimenting polysome fractions on a sucrose gradient. Depletion of YB-1 expression by siRNA abrogated the translational activation. Furthermore, in the brain of kainic acid-treated mice, the distribution of YB-1 was shifted to much heavier fractions associated with polysomes within 30 min to 1 h after the treatment, and the distribution returned to lighter fractions within the following 2 h. The protein levels of GluR2 and CaM1 were also increased transiently when the distribution of YB-1 on the gradient changed. These results suggest that in the brain of growing mice, YB-1 binds to GluR2 and CaM1 mRNAs and regulates their translation in an activity-dependent manner.

HSP60 interacts with YB-1 and affects its polysome association and subcellular localization.

YB-1 is a DNA/RNA-binding protein which, in the cytoplasm, associates with polysomes and regulates translation. However, YB-1 has a novel nuclear localization signal, and its nuclear accumulation is correlated with cancer induction. Here we designated the amino-acid sequence as YB-NLS and demonstrated that YB-NLS is necessary for the nuclear translocation of overexpressed YB-1 in NG108-15 cells. In addition, we found that a heat shock protein, HSP60, binds to YB-NLS in the cytoplasm. Interestingly, when HSP60 expression was repressed, an increase of polysome-associated YB-1 was observed in heavy-sedimenting fractions on a sucrose gradient. Overexpression of HSP60 resulted in a decrease of YB-1 in the heavy-sedimenting fractions and suppression of YB-NLS activity. Furthermore, the NLS-deleted YB-1 was apparently associated with the heavy-sedimenting polysomes. These results suggest that HSP60 interacts with YB-1 at the YB-NLS region and acts as a regulator of polysome association and the subcellular distribution of YB-1.

YB-1 transcription in the postnatal brain is regulated by a bHLH transcription factor Math2 through an E-box sequence in the 5'-UTR of the gene.

YB-1 controls gene expression at both the transcriptional and translational levels. In the brain, a high level of YB-1 is expressed from prior to the first week after birth. However, YB-1 expression substantially declines to the adult level thereafter. Here, we investigated the regulatory mechanism of YB-1 transcription in the postnatal brain. We found that an E-box sequence within the 5'-untranslated region (5'-UTR) of the gene was recognized by Math2 in the 5-day-old brain, whereas the DNA binding was reduced in the 4-week-old brain. In vitro transcription analysis and reporter gene assay revealed that Math2 was necessary for YB-1 transcription. Furthermore, the expression pattern of Math2 protein in the postnatal brain was correlated with that of YB-1 protein. Our results suggest that YB-1 transcription in the postnatal brain is regulated by Math2, which binds to an E-box in the 5'-UTR of the YB-1 gene.

C9orf10 protein, a novel protein component of Puralpha-containing mRNA-protein particles (Puralpha-mRNPs): characterization of developmental and regional expressions in the mouse brain.

Puralpha has been implicated in mRNA transport and translation in neurons. We previously reported that Puralpha is a component of mRNA/protein complexes (Puralpha-mRNPs) with several other proteins. Among them, we found the C9orf10 (Homo sapiens chromosome 9 open reading frame 10) protein, which was recently characterized as a component of RNA-containing structures. However, C9orf10 itself remains poorly understood. To characterize C9orf10 expression at the protein level, we raised an antibody against C9orf10 and compared the spatial and developmental expressions of this protein and Puralpha in the mouse brain. C9orf10 was expressed as early as embryo stage 12, whereas Puralpha was expressed from 5 days after birth. In adults, C9orf10 expression was most prominent in the hippocampus, caudate putamen, cerebral cortex, and cerebellum, unlike the uniform distribution of Puralpha. C9orf10-positive cells also showed immunoreactivity to Puralpha. C9orf10 expression was restricted to neurons, judging by the immunoreactivity to neuron-specific nuclear protein or CaM kinase II. These observations suggest an accessory role of C9orf10 for Puralpha in a limited brain region in addition to other possible functions that have not yet been determined.

Isolation and characterization of brain Y-box protein: developmentally regulated expression, polyribosomal association and dendritic localization.

Y-box proteins are DNA- and RNA-binding proteins and control specific gene expression at both transcriptional and translational levels. Particularly in germ cells, it has been reported that Y-box proteins bind to paternal or maternal mRNAs to form mRNPs, mask them from translation and control cell maturation. In this study, we cloned cDNA for a Y-box protein from rat brain. A deduced amino acid sequence of the protein was very similar to that of several other Y-box proteins, and we termed the protein rBYB1 (rat brain Y-box protein 1). rBYB1 was found to be considerably expressed in the cytoplasm of pre- and early postnatal brains, and then decreased to adult levels with brain development. Further, we found rBYB1 to be distributed in both polyribosomal and nonpolyribosomal (mRNP) fractions on a sucrose density gradient, and to be associated with polyribosomes via RNA in the higher-density fractions. Moreover, rBYB1 was localized in dendrites of the primary hippocampal neurons. We compared these sucrose gradient and intracellular rBYB1 localization results with those for fragile X mental retardation protein (FMRP), which is known to be an mRNA-binding and polyribosome-associating translational regulator distributed in neuronal dendrites. Our results suggest that in the brain of prenatal and newborn animals, rBYB1 may function in storage and/or translational regulation of mRNAs involved in the rapid progress of the postnatal brain, and in mature neurons, it may also participate in the control of protein synthesis in dendrites.

Identification of mRNA/protein (mRNP) complexes containing Puralpha, mStaufen, fragile X protein, and myosin Va and their association with rough endoplasmic reticulum equipped with a kinesin motor.

Puralpha, which is involved in diverse aspects of cellular functions, is strongly expressed in neuronal cytoplasm. Previously, we have reported that this protein controls BC1 RNA expression and its subsequent distribution within dendrites and that Puralpha is associated with polyribosomes. Here, we report that, following treatment with EDTA, Puralpha was released from polyribosomes in mRNA/protein complexes (mRNPs), which also contained mStaufen, Fragile X Mental Retardation Protein (FMRP), myosin Va, and other proteins with unknown functions. As the coimmunoprecipitation of these proteins by an anti-Puralpha antibody was abolished by RNase treatment, Puralpha may assist mRNP assembly in an RNA-dependent manner and be involved in targeting mRNPs to polyribosomes in cooperation with other RNA-binding proteins. The immunoprecipitation of mStaufen- and FMRP-containing mRNPs provided additional evidence that the anti-Puralpha detected structurally or functionally related mRNA subsets, which are distributed in the somatodendritic compartment. Furthermore, mRNPs appear to reside on rough endoplasmic reticulum equipped with a kinesin motor. Based on our present findings, we propose that this rough endoplasmic reticulum structure may form the molecular machinery that mediates and regulates multistep transport of polyribosomes along microtubules and actin filaments, as well as localized translation in the somatodendritic compartment.