Proteome analysis of soluble nuclear proteins reveals that HMGB1/2 suppress genotoxic stress in polyglutamine diseases.

Nuclear dysfunction is a key feature of the pathology of polyglutamine (polyQ) diseases. It has been suggested that mutant polyQ proteins impair functions of nuclear factors by interacting with them directly in the nucleus. However, a systematic analysis of quantitative changes in soluble nuclear proteins in neurons expressing mutant polyQ proteins has not been performed. Here, we perform a proteome analysis of soluble nuclear proteins prepared from neurons expressing huntingtin (Htt) or ataxin-1 (AT1) protein, and show that mutant AT1 and Htt similarly reduce the concentration of soluble high mobility group B1/2 (HMGB1/2) proteins. Immunoprecipitation and pulldown assays indicate that HMGBs interact with mutant AT1 and Htt. Immunohistochemistry showed that these proteins were reduced in the nuclear region outside of inclusion bodies in affected neurons. Compensatory expression of HMGBs ameliorated polyQ-induced pathology in primary neurons and in Drosophila polyQ models. Furthermore, HMGBs repressed genotoxic stress signals induced by mutant Htt or transcriptional repression. Thus, HMGBs may be critical regulators of polyQ disease pathology and could be targets for therapy development.

Associations of polymorphisms in the genes of FGFR2, FGF1, and RBFOX2 with breast cancer risk by estrogen/progesterone receptor status.

Genetic polymorphisms of fibroblast growth factor receptor 2 (FGFR2) have been demonstrated to be associated with breast cancer risk, presumably through elevation of FGFR2 expression. Fibroblast growth factor 1 (FGF1) and RNA binding protein fox-1 homolog 2 (RBFOX2), which are functionally related to FGFR2, may also associate with breast cancer risk. We investigated the associations between breast cancer risk and the polymorphisms of FGFR2 rs2981582, FGF1 rs250108, and RBFOX2 rs2051579 among 839 incident breast cancer cases and 863 age-matched controls in the Guangzhou Breast Cancer Study. Stratified odds ratios (ORs) and 95% confidence intervals (CIs) were calculated by estrogen receptor (ER)/progesterone receptor (PR) status using multivariate logistic regression. FGFR2 rs2981582 was confirmed to be significantly associated with the risk of ER-positive but not ER-negative breast cancer. In contrast, FGF1 rs250108 was significantly associated with the risk of ER-negative breast cancer (OR (95% CI) = 1.68 (1.20-2.35) for CT + TT vs. CC genotype) but not ER-positive breast cancer. CA + AA genotypes at RBFOX2 rs2051579 were associated with a reduced risk of ER-negative (0.71 (0.52-0.97)) but not ER-positive breast cancer compared to the CC genotype. Similar results were observed when differentiating breast cancer cases by PR status. Neither of the pairs between the three SNPs had a significant interaction on breast cancer risk. Our findings show a suggestively stronger association between FGFR2 rs2981582 and ER-positive breast cancer risk and suggest a greater association of FGF1 rs250108 and RBFOX2 rs2051579 with ER-negative compared to ER-positive breast cancer.

Transcriptional repression induces a slowly progressive atypical neuronal death associated with changes of YAP isoforms and p73.

Transcriptional disturbance is implicated in the pathology of polyglutamine diseases, including Huntington's disease (HD). However, it is unknown whether transcriptional repression leads to neuronal death or what forms that death might take. We found transcriptional repression-induced atypical death (TRIAD) of neurons to be distinct from apoptosis, necrosis, or autophagy. The progression of TRIAD was extremely slow in comparison with other types of cell death. Gene expression profiling revealed the reduction of full-length yes-associated protein (YAP), a p73 cofactor to promote apoptosis, as specific to TRIAD. Furthermore, novel neuron-specific YAP isoforms (YAPDeltaCs) were sustained during TRIAD to suppress neuronal death in a dominant-negative fashion. YAPDeltaCs and activated p73 were colocalized in the striatal neurons of HD patients and mutant huntingtin (htt) transgenic mice. YAPDeltaCs also markedly attenuated Htt-induced neuronal death in primary neuron and Drosophila melanogaster models. Collectively, transcriptional repression induces a novel prototype of neuronal death associated with the changes of YAP isoforms and p73, which might be relevant to the HD pathology.

The induction levels of heat shock protein 70 differentiate the vulnerabilities to mutant huntingtin among neuronal subtypes.

The reason why vulnerabilities to mutant polyglutamine (polyQ) proteins are different among neuronal subtypes is mostly unknown. In this study, we compared the gene expression profiles of three types of primary neurons expressing huntingtin (htt) or ataxin-1. We found that heat shock protein 70 (hsp70), a well known chaperone molecule protecting neurons in the polyQ pathology, was dramatically upregulated only by mutant htt and selectively in the granule cells of the cerebellum. Granule cells, which are insensitive to degeneration in the human Huntington's disease (HD) pathology, lost their resistance by suppressing hsp70 with siRNA, whereas cortical neurons, affected in human HD, gained resistance by overexpressing hsp70. This indicates that induction levels of hsp70 are a critical factor for determining vulnerabilities to mutant htt among neuronal subtypes. CAT (chloramphenicol acetyltransferase) assays showed that CBF (CCAAT box binding factor, CCAAT/enhancer binding protein zeta) activated, but p53 repressed transcription of the hsp70 gene in granule cells. Basal and mutant htt-induced expression levels of p53 were remarkably lower in granule cells than in cortical neurons, suggesting that different magnitudes of p53 are linked to distinct induction levels of hsp70. Surprisingly, however, heat shock factor 1 was not activated in granule cells by mutant htt. Collectively, different levels of hsp70 among neuronal subtypes might be involved in selective neuronal death in the HD pathology.

Omi / HtrA2 is relevant to the selective vulnerability of striatal neurons in Huntington's disease.

Selective vulnerability of neurons is a critical feature of neurodegenerative diseases, but the underlying molecular mechanisms remain largely unknown. We here report that Omi/HtrA2, a mitochondrial protein regulating survival and apoptosis of cells, decreases selectively in striatal neurons that are most vulnerable to the Huntington's disease (HD) pathology. In microarray analysis, Omi/HtrA2 was decreased under the expression of mutant huntingtin (htt) in striatal neurons but not in cortical or cerebellar neurons. Mutant ataxin-1 (Atx-1) did not affect Omi/HtrA2 in any type of neuron. Western blot analysis of primary neurons expressing mutant htt also confirmed the selective reduction of the Omi/HtrA2 protein. Immunohistochemistry with a mutant htt-transgenic mouse line and human HD brains confirmed reduction of Omi/HtrA2 in striatal neurons. Overexpression of Omi/HtrA2 by adenovirus vector reverted mutant htt-induced cell death in primary neurons. These results collectively suggest that the homeostatic but not proapoptotic function of Omi/HtrA2 is linked to selective vulnerability of striatal neurons in HD pathology.

Expression of human PQBP-1 in Drosophila impairs long-term memory and induces abnormal courtship.

Frame shift mutations of the polyglutamine binding protein-1 (PQBP1) gene lead to total or partial truncation of the C-terminal domain (CTD) and cause mental retardation in human patients. Interestingly, normal Drosophila homologue of PQBP-1 lacks CTD. As a model to analyze the molecular network of PQBP-1 affecting intelligence, we generated transgenic flies expressing human PQBP-1 with CTD. Pavlovian olfactory conditioning revealed that the transgenic flies showed disturbance of long-term memory. In addition, they showed abnormal courtship that male flies follow male flies. Abnormal functions of PQBP-1 or its binding partner might be linked to these symptoms.

Oct-3/4 repression accelerates differentiation of neural progenitor cells in vitro and in vivo.

Oct-3/4 (Oct-3/Oct-4/POU5F1) is a critical regulator of embryonic stem (ES) cell differentiation, though its role in tissue stem cells that persist in differentiated tissues has not been shown. Here, we show that Oct-3/4 is expressed in neurospheres (NS) composed of neural stem cells and neural progenitor cells and that up- or down-regulation of Oct-3/4 by using adenovirus vectors influences cell fate. Oct-3/4 down-regulation accelerates neuronal differentiation of progenitor cells while its sustained expression prevents neuronal differentiation. Transplantation of neurospheres into the adult rat brain shows that down-regulation of Oct-3/4 promotes differentiation of NS cells in vivo. Our findings indicate that Oct-3/4 is an essential regulator of NS cell differentiation and suggest that the modulation of Oct-3/4 could be a useful tool in clinical application of NS cells.

Changes in neurocan expression in the distal spinal cord stump following complete cord transection: a comparison between infant and adult rats.

The distal transected cords of infant rats are more permissive for axon extension than those of adults. To elucidate the biomolecular basis for this phenomenon, we examined the expression pattern of neurocan using semi-quantitative reverse transcription polymerase chain reaction and immunostaining in the distal cord of both adult and infant rats after transection. Neurocan is a chondroitin sulfate proteoglycan with well-documented axon growth-inhibitory properties in the central nervous system. Neurocan mRNA was up-regulated in the distal cord of adult rats shortly after transection, followed by a longer wide distribution of neurocan immunoreactivity (IR) in both neurons and astrocytes; by contrast, upregulation of neurocan mRNA was not seen in infant rats, although transient expression of neurocan IR was seen in neurons. Combined with the different regenerative capacity of infant and adult rats, the present results suggest that neurocan inhibits spinal cord regeneration.

Association of Epstein-Barr virus and passive smoking with the risk of breast cancer among Chinese women.

Reactivation of Epstein-Barr virus (EBV), as indexed by the higher immunoglobulin A (IgA) antibody titers, was reported to be associated with an increased risk of breast cancer. Passive smoking plays a role in host immune responses and may modify the association of EBV with breast cancer. We carried out a case-control study using data from 349 incident breast cancer cases and 500 age-matched controls in the Guangzhou Breast Cancer Study to investigate the interactions of EBV antibodies and passive smoking on breast cancer risk. A higher risk of breast cancer was observed in passive smokers who were seropositive for EBV viral capsid antigen IgA or nuclear antigen-1 IgA in serum compared with those with the seronegativity and no passive smoking [odds ratio 3.13 (95% confidence interval, 1.76-5.56)]. There was a significant linear trend for the risk of breast cancer from IgA seropositivity with passive smoking, only IgA seropositivity, only passive smoking, to seronegativity without passive smoking (P<0.001), but the interaction in either multiplicative or additive models was not significant. No significant association was found between passive smoking and EBV IgA seropositivity. The present study confirmed the associations of EBV IgA antibodies and passive smoking with the risk of breast cancer and suggested that there was no synergic action between passive smoking and EBV IgA seropositivity on the risk of breast cancer.

Headspace single-drop microextraction gas chromatography mass spectrometry for the analysis of volatile compounds from herba asari.

A rapid headspace single-drop microextraction gas chromatography mass spectrometry (SDME-GC-MS) for the analysis of the volatile compounds in Herba Asari was developed in this study. The extraction solvent, extraction temperature and time, sample amount, and particle size were optimized. A mixed solvent of n-tridecane and butyl acetate (1 : 1) was finally used for the extraction with sample amount of 0.750 g and 100-mesh particle size at 70°C for 15 min. Under the determined conditions, the pound samples of Herba Asari were directly applied for the analysis. The result showed that SDME-GC-MS method was a simple, effective, and inexpensive way to measure the volatile compounds in Herba Asari and could be used for the analysis of volatile compounds in Chinese medicine.

Effects of passive smoking on breast cancer risk in pre/post-menopausal women as modified by polymorphisms of PARP1 and ESR1.

OBJECTIVES: The association between passive smoking and breast cancer risk differs in pre- and post-menopausal women. We aimed to explore the modification effects of PARP1 rs1136410 and ESR1 rs2234693 on the association between passive smoking and breast cancer risk among pre- and post-menopausal women. DESIGN AND METHODS: A case-control study of 839 breast cancer cases and 863 controls was conducted. The gene-environment interactions were tested after adjusting for potential breast cancer risk factors with unconditional logistic regression models. RESULTS: We found that the effect of passive smoking was modified by the genotypes in both pre- and post-menopausal women, but in opposite directions. The combination of the TC/CC genotypes of ESR1 rs2234693 and passive smoking significantly increased the risk of breast cancer [OR (95%CI): 2.06 (1.39-3.05)] in pre-menopausal women. A significant association was observed between TT genotype and passive smoking [OR (95%CI): 2.40 (1.27-4.53)] in postmenopausal women. For PARP1 rs1136410, similar differential associations were observed, but the interactions were not significant. CONCLUSIONS: These results imply that the risk of breast cancer from passive smoking may be influenced by genetic factors, and that the association may differ depending on menopausal status.

Hepatoma-derived growth factor, a new trophic factor for motor neurons, is up-regulated in the spinal cord of PQBP-1 transgenic mice before onset of degeneration.

Hepatoma-derived growth factor (HDGF) is a nuclear protein homologous to the high-mobility group B1 family of proteins. It is known to be released from cells and to act as a trophic factor for dividing cells. In this study HDGF was increased in spinal motor neurons of a mouse model of motor neuron degeneration, polyglutamine-tract-binding protein-1 (PQBP-1) transgenic mice, before onset of degeneration. HDGF promoted neurite extension and survival of spinal motor neurons in primary culture. HDGF repressed cell death of motor neurons after facial nerve section in newborn rats in vivo. We also found a significant increase in p53 in spinal motor neurons of the transgenic mice. p53 bound to a sequence in the upstream of the HDGF gene in a gel mobility shift assay, and promoted gene expression through the cis-element in chloramphenicol acetyl transfer (CAT) assay. Finally, we found that HDGF was increased in CSF of PQBP-1 transgenic mice. Collectively, our results show that HDGF is a novel trophic factor for motor neurons and suggest that it might play a protective role against motor neuron degeneration in PQBP-1 transgenic mice.

Polyglutamine tract-binding protein-1 dysfunction induces cell death of neurons through mitochondrial stress.

Polyglutamine tract-binding protein-1 (PQBP-1) is a nuclear protein that interacts and colocalizes with mutant polyglutamine proteins. We previously reported that PQBP-1 transgenic mice show a late-onset motor neuron disease-like phenotype and cell death of motor neurons analogous to human neurodegeneration. To investigate the molecular mechanisms underlying the motor neuron death, we performed microarray analyses using the anterior horn tissues of the spinal cord and compared gene expression profiles between pre-symptomatic transgenic and age-matched control mice. Surprisingly, half of the spots changed more than 1.5-fold turned out to be genes transcribed from the mitochondrial genome. Northern and western analyses confirmed up-regulation of representative mitochondrial genes, cytochrome c oxidase (COX) subunit 1 and 2. Immunohistochemistry revealed that COX1 and COX2 proteins are increased in spinal motor neurons. Electron microscopic analyses revealed morphological abnormalities of mitochondria in the motor neurons. PQBP-1 overexpression in primary neurons by adenovirus vector induced abnormalities of mitochondrial membrane potential from day 5, while cytochrome c release and caspase 3 activation were observed on day 9. An increase of cell death by PQBP-1 was also confirmed on day 9. Collectively, these results indicate that dysfunction of PQBP-1 induces mitochondrial stress, a key molecular pathomechanism that is shared among human neurodegenerative disorders.

Present situation and future aspects of spinal cord regeneration.

The central nervous system (CNS) has a limited capacity for regeneration after injury. In spinal cord injury (SCI) patients, total loss of all motor and sensory function occurs below the level of injury. Advances in treatment are expected for orthopedic and spinal surgeons. Recently, evidence of axonal regeneration and functional recovery has been reported in animal spinal cord injury models. Our studies on the roles of inhibitory molecules with a comparison between neonatal and adult animals may help serve as therapeutic targets to enhance axonal regeneration for the injured spinal cord. Also, our cell replacement study indicates the possibility of transplanting neural stem cells to supply the cell source for immature oligodendrocytes, which are thought to be essential for both the myelination and trophic support of regenerating axons in the spinal cord. Administration of neurotrophic factors, prevention of inhibitory factors, and stem cell technology have clinical applications in SCI patients. However, spinal cord regeneration involves a multistep process, and several factors have to be controlled after injury. A combination of several treatments could overcome a nonpermissive environment for spinal cord regeneration. Further understanding of the mechanisms and finding optimal targets of spinal cord regeneration are necessary to obtain successful therapies for SCI patients.

Changes in FGF-2 expression in the distal spinal cord stump after complete cord transection: a comparison between infant and adult rats.

STUDY DESIGN: Expression patterns of fibroblast growth factor-2 (FGF-2) in distal transected spinal cord in infant and adult rats were determined by reverse-transcription polymerase chain reaction (RT-PCR) and immunostaining. OBJECTIVE: To reveal the expression pattern of FGF-2 in distal transected cord of infant and adult rats. SUMMARY OF BACKGROUND DATA: Descending fibers in the spinal cord of infant and adult rats show different regenerative capacity. One explanation is that different levels of FGF-2, an important neurotrophic factor for promoting neurite outgrowth and repair, are expressed in the distal transected cords of the rats, providing different levels of support for severed axons. MATERIALS AND METHODS: Spinal cords of infant and adult rats were completely transected. At 12, 24, and 72 hours and at 1 week, segments of distal spinal cord tissues were removed and expression of FGF-2 mRNA was evaluated by RT-PCR. The distribution of FGF-2 and the phenotype of FGF-2-positive cells were determined by immunostaining. RESULTS: Expression of FGF-2 mRNA was shown to be up-regulated in the distal cord of infant rats but not adult rats. Immunohistochemical analysis showed that neurons in distal cord of infant rats were rich in FGF-2 immunoreactivity (IR), whereas in adult rat neurons FGF-2 IR was hardly observed at all, although a few FGF-2-positive astrocytes were observed in the white matter. CONCLUSION: After complete spinal cord transection, the expression of FGF-2 in the distal cord of infant rats was high compared with that of adults. This may provide neurotrophic support for axonal extension and functional recovery.