ASK1 is essential for endoplasmic reticulum stress-induced neuronal cell death triggered by expanded polyglutamine repeats

Expansion of CAG trinucleotide repeats that encode polyglutamine is the underlying cause of at least nine inherited human neurodegenerative disorders, including Huntington's disease and spinocerebellar ataxias. PolyQ fragments accumulate as aggregates in the cytoplasm and/or in the nucleus, and induce neuronal cell death. However, the molecular mechanism of polyQ-induced cell death is controversial. Here, we show the following: (1) polyQ with pathogenic repeat length triggers ER stress through proteasomal dysfunction; (2) ER stress activates ASK 1 through formation of an IRE1-TRAF2-ASK1 complex; and (3) ASK1(-/-) primary neurons are defective in polyQ-, proteasome inhibitor-, and ER stress-induced JNK activation and cell death. These findings suggest that ASK1 is a key element in ER stress-induced cell death that plays an important role in the neuropathological alterations in polyQ diseases.     

程序性细胞死亡因子4是替代活化巨噬细胞的新分子标志物

背景：程序性细胞死亡因子4的表达水平与经典活化的巨噬细胞的表型负相关,但是程序性细胞死亡因子4的表达水平与巨噬细胞替代活化的关系仍不明确。 目的：观察替代活化的巨噬细胞中程序性细胞死亡因子4的表达水平改变,及其对替代活化的影响。 方法：用白细胞介素4、地塞米松单独刺激或联合刺激诱导NR 8383 细胞的替代活化;采用qPCR检测巨噬细胞替代活化的分子标志物的表达水平,确定诱导巨噬细胞替代活化的最适条件;采用qPCR和Western blot 检测替代活化的巨噬细胞模型中程序性细胞死亡因子4表达的改变;分别用大鼠程序性细胞死亡因子4高表达质粒和程序性细胞死亡因子4干扰质粒转染NR 8383细胞株,采用荧光倒置显微镜观察转染的效率,并检测程序性细胞死亡因子4的表达水平;分别检测程序性细胞死亡因子4 高表达和程序性细胞死亡因子4敲低后 NR 8383 细胞株中巨噬细胞经典活化和替代活化的分子标志。 结果与结论：①白细胞介素4和地塞米松联合刺激比白细胞介素4或地塞米松单独刺激可更有效的诱导巨噬细胞的替代活化;10 μg/L白细胞介素4+50 nmol/ L 地塞米松刺激24 h可有效诱导巨噬细胞的替代活化。②在替代活化的巨噬细胞中程序性细胞死亡因子4的表达水平显著升高,是替代活化的分子标志。③程序性细胞死亡因子4高表达上调替代活化的分子标志的表达(P < 0.05);而敲低程序性细胞死亡因子4可下调 CD206的表达(P < 0.05),并显著上调经典活化的分子标志诱导型一氧化氮合酶的表达(P < 0.05)。结果表明程序性细胞死亡因子4上调是替代活化巨噬细胞的一个重要分子标志物。 中国组织工程研究杂志出版内容重点：组织构建;骨细胞;软骨细胞;细胞培养;成纤维细胞;血管内皮细胞;骨质疏松;组织工程     

TRPV1 is a novel target for omega-3 polyunsaturated fatty acids

Omega-3 ( n -3) fatty acids are essential for proper neuronal function, and they possess prominent analgesic properties, yet their underlying signalling mechanisms are unclear. Here we show that n -3 fatty acids interact directly with TRPV1, an ion channel expressed in nociceptive neurones and brain. These fatty acids activate TRPV1 in a phosphorylation-dependent manner, enhance responses to extracellular protons, and displace binding of the ultrapotent TRPV1 ligand [³H]resiniferatoxin. In contrast to their agonistic properties, n -3 fatty acids competitively inhibit the responses of vanilloid agonists. These actions occur in mammalian cells in the physiological concentration range of 1–10 μ m . Significantly, docosahexaenoic acid exhibits the greatest efficacy as an agonist, whereas eicosapentaenoic acid and linolenic acid are markedly more effective inhibitors. Similarly, eicosapentaenoic acid but not docosahexaenoic acid profoundly reduces capsaicin-evoked pain-related behaviour in mice. These effects are independent of alterations in membrane elasticity because the micelle-forming detergent Triton X-100 only minimally affects TRPV1 properties. Thus, n -3 fatty acids differentially regulate TRPV1 and this form of signalling may contribute to their biological effects. Further, these results suggest that dietary supplementation with selective n -3 fatty acids would be most beneficial for the treatment of pain.     

NHE-1: a molecular target for signalling and cell matrix interactions

The activation of sodium/hydrogen exchanger (NHE) is associated with a variety of cell functions like cell adhesion, migration, proliferation, and apoptosis. Since its discovery, 9 NHE isoforms have been identified, but the most widely spread and the most important for the cellular functions is NHE-1. This ubiquitously expressed sodium/hydrogen exchanger (NHE-1) plays a central housekeeping role in all cells regulating cell volume and internal pH (pHi). At physiological pHi, NHE-1 is essentially inactive but it is extremely sensitive to pHi changes, being rapidly activated by small intracellular hydrogen concentration increases. NHE-1 activity can be stimulated via a series of cell surface receptors, including tyrosine kinase, G-protein-coupled, and integrin receptors. These signals converge, regulating the affinity of the internal hydrogen-binding site. NHE-1 also is a plasma membrane-anchoring protein for the cytoskeleton. Cytoskeleton anchoring of NHE-1 is important for cell adhesion to extracellular matrix proteins and cell migration. Moreover, NHE-1 plays the role of a "scaffold" for the building of various intracellular signaling molecule clusters.     

Calreticulin is a B cell molecular target in some gastrointestinal malignancies

Calreticulin, upon translocation to the cell surface, plays a critical role in the recognition of tumour cells and in experimentally induced cellular anti‐tumour immunity. However, less is known about anti‐calreticulin antibodies and their role in malignancies. Using enzyme‐linked immunosorbent assay (ELISA), we found immunoglobulin (Ig)A and/or IgG anti‐calreticulin antibodies in sera of approximately 63% of patients with hepatocellular carcinoma (HCC), 57% of patients with colorectal adenocarcinoma (CRA) and 47% of patients with pancreatic adenocarcinoma (PACA), while healthy controls, patients with viral hepatitis C and with chronic pancreatitis reached only 2%, 20% and 31% seropositivity, respectively. We found significantly elevated mean levels of IgA anti‐calreticulin antibodies (P < 0·001) in patients with HCC (78·7 ± 52·3 AU, mean ± standard deviation), PACA (66·5 ± 30·9 AU) and CRA (61·8 ± 25·8 AU) when compared to healthy controls (41·4 ± 19·2 AU). Significantly elevated mean levels of IgG anti‐calreticulin antibodies (P < 0·001) were detected in patients with HCC (121·9 ± 94·2 AU), gall bladder adenocarcinoma (118·4 ± 80·0 AU) and PACA (88·7 ± 55·6 AU) when compared to healthy controls (56·7 ± 22·9 AU). Pepscan analysis revealed a large number of antigenic epitopes of calreticulin recognized by both IgA and IgG antibodies of patients with HCC and PACA, indicating robust systemic immune response. Moreover, significantly elevated levels of antibodies against peptide KGEWKPRQIDNP (P < 0·001) in these patients, tested by ELISA, confirmed the distinct character of antibody reactivity against calreticulin. The high occurrence and specificity of serum anti‐calreticulin autoantibodies in the majority of patients with some gastrointestinal malignancies provide the evidence for their possible clinical relevance.     

Oncostatin M receptor is a novel therapeutic target in cervical squamous cell carcinoma

Cervical carcinoma is the second most common cause of cancer deaths in women worldwide. Treatments have not changed for decades and survival rates for advanced disease remain low. An exciting new molecular target for the treatment of cervical squamous cell carcinoma (SCC), and possibly for SCCs at other anatomical sites, is the oncostatin M receptor (OSMR). This cell surface cytokine receptor is commonly copy number gained and overexpressed in advanced cervical SCC, changes that are associated with significantly worse clinical outcomes. OSMR overexpression in cervical SCC cells results in enhanced responsiveness to the major ligand oncostatin M (OSM), which induces several pro‐malignant effects, including a pro‐angiogenic phenotype and increased cell migration and invasiveness. OSMR is a strong candidate for antibody‐mediated inhibition, a strategy that has had a major impact on haematological malignancies and various solid tumours such as HER2‐positive breast cancers. © 2013 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of Pathological Society of Great Britain and Ireland.     

R-spondin1 is a novel hormone mediator for mammary stem cell self-renewal

Signals from the niche play pivotal roles in regulating adult stem cell self-renewal. Previous studies indicated that the steroid hormones can expand mammary stem cells (MaSCs) in vivo. However, the facilitating local niche factors that directly contribute to the MaSC expansion remain unclear. Here we identify R-spondin1 (Rspo1) as a novel hormonal mediator in the mammary gland. Pregnancy and hormonal treatment up-regulate Rspo1 expression. Rspo1 cooperates with another hormonal mediator, Wnt4, to promote MaSC self-renewal through Wnt/β-catenin signaling. Knockdown of Rspo1 and Wnt4 simultaneously abolishes the stem cell reconstitution ability. In culture, hormonal treatment that stimulates the expression of both Rspo1 and Wnt4 can completely substitute for exogenous Wnt proteins, potently expand MaSCs, and maintain their full development potential in transplantation. Our data unveil the intriguing concept that hormones induce a collaborative local niche environment for stem cells.     

Regulation of p27Kip1 during gentamicin mediated hair cell death

The INK4 and Kip/Cip families of Cyclin Dependent Kinase inhibitors (CKIs) are regulators of the cell cycle. In addition, CKIS including p27(Kip1) can protect cells from apoptosis in vitro. However, little is known about protective effect of p27(Kip1) in vivo. We used systemic treatment with aminoglycosides to induce hair-cell death in the basilar papilla (BP), the auditory organ of the avian inner ear, and characterised the expression of p27(Kip1) with confocal and immunofluorescence microscopy. In contrast to the adult mammalian cochlea where p27(Kip1) is expressed only in supporting cells, p27(Kip1) is found in the nuclei of both hair cells and supporting cells in the BP of the normal, mature bird. Forty-eight hours after gentamicin treatment, hair cells with TUNEL positive nuclei and hair cells with pyknotic nuclei were both detected, suggesting many hair cells die by apoptosis. When the BP was double labelled for p27(Kip1) and myosin VIIa, a hair-cell specific protein, all dying hair cells that had been ejected from the epithelium were found to be myosin VIIa positive but negative for p27(Kip1) even though nuclear remnants were still visible. In the transition zone where partial hair-cell loss occurs, freshly ejected hair cells lying immediately above the surface of the BP no longer expressed p27(Kip1). Damaged hair cells within the epithelium in the transition zone contained p27(Kip1) in their cytoplasm but not in their nuclei. These data support recent in vitro findings suggesting that p27(Kip1) protects cells from apoptosis and that its downregulation may be a general feature of programmed cell death.     

Paraptosis——一种新的细胞死亡方式

人们一直认为细胞死亡有两种方式—细胞坏死和凋亡。但最新研究表明还存在一种非凋亡性细胞程序性死亡 ,命名为paraptosis。Paraptosis在形态学上与凋亡不同 ,不具有凋亡的典型特征 ,即缺少染色质的新月形凝集和凋亡小体。它是以胞浆空泡的形成为特征 ,在整个过程中胞浆和胞核密度增加 ,线粒体肿胀 ,但细胞膜和细胞器仍保持完整 ;无“阶梯状DNA区带” ,ISEL ,TUNEL标记为阴性。paraptosis过程中有RNA及蛋白质合成 ,并为caspase非依赖性的。目前已知 paraptosis与许多生理和病理现象相关。     

Prohibitin——肿瘤与代谢性疾病治疗的新靶标

Prohibitin(PHB)是一种高度保守、分布广泛的蛋白质。PHB不仅是一类新型的分子伴侣蛋白，而且参与调节细胞周期、维持线粒体结构和功能，具有抗细胞凋亡、衰老和增殖的作用。PHB的多种功能和不同定位，以及在多种疾病中存在表达差异的现象提示，PHB可能参与疾病的发生与发展过程。因此，PHB在多种疾病如：肿瘤、糖尿病和肥胖等的治疗方面将会成为有用的药物靶标。     

Myristoyltransferase and calcineurin: novel molecular therapeutic target for epilepsy

N-myristoylation is a co-translational, irreversible addition of a fatty acyl moiety to the amino terminus of many eukaryotic cellular proteins. These myristoylated proteins in the cell have diverse biological functions such as signal transduction, cellular transformation and oncogensis. Known myristoylated proteins [Src family kinases, the catalytic subunit of cAMP-dependent protein kinase and calcineurin (CaN)] are either protein kinases or a protein phosphatases which modulate various cellular metabolic processes. Myristoylation is catalyzed by N-myristoyltransferase (NMT) and is recognized to be a widespread and functionally important modification of proteins. The main objective of this review is to focus on the potential role of NMT and CaN in epileptic brain and its involvement in neuronal apoptosis. The findings on the interaction of NMT and CaN with various signaling molecules in epileptic chickens adds to our understanding of the mechanism of CaN signaling in neuronal apoptosis. Understanding the regulation of NMT by specific inhibitors may help us to control the action of this enzyme on its specific substrates and may lead to improvements in the management of various neurological disorders like Alzheimer's disease, ischemia and epilepsy.     

The major cell surface glycoprotein procyclin is a receptor for induction of a novel form of cell death in African trypanosomes in vitro

Bloodstream forms (BSF) and procyclic culture forms (PCF) of African trypanosomes were incubated with a variety of lectins in vitro. Cessation of cell division and profound morphological changes were seen in procyclic forms but not in BSF after incubation with concanavalin A (Con A), wheat germ agglutinin and Ricinus communis agglutinin. These lectins caused the trypanosomes to cease division, become round and increase dramatically in size, the latter being partially attributable to the formation of what appeared to be a large 'vacuole-like structure' or an expanded flagellar pocket. Con A was used in all further experiments. Spectrophotometric quantitation of extracted DNA and flow cytometry using the DNA intercalating dye propidium iodide showed that the DNA content of Con A-treated trypanosomes increased dramatically when compared to untreated parasites. Examination of these cells by fluorescence microscopy showed that many of the Con A-treated cells were multinucleate whereas the kinetoplasts were mostly present as single copies, indicating a disequilibrium between nuclear and kinetoplast replication. Immunofluorescence experiments using monoclonal antibodies (mAb) specific for paraflagellar rod proteins and for kinetoplastid membrane protein-11 (KMP-11), showed that the Con A-treated parasites had begun to duplicate the flagellum but that this had only proceeded along part of the length of the cells, suggesting that the cell division process was initiated but that cytokinesis was subsequently inhibited. Tunicamycin-treated wild-type trypanosomes and mutant trypanosomes expressing both high levels of non-glycosylated procyclins and procyclin isoforms with truncated N-linked sugars were resistant to the effects of Con A, suggesting that N-linked carbohydrates on the procyclin surface coat were the ligands for Con A binding. This was supported by data obtained using mutant parasites created by deletion of all three EP procyclin isoforms, two of which contain N-glycosylation sites, by homologous recombination. The knockout mutants showed reduced binding of fluorescein-labelled Con A as determined by flow cytometry and were resistant to the effects of Con A. Taken together the results show that Con A induces multinucleation, a disequilibrium between nuclear and kinetoplast replication and a unique form of cell death in procyclic African trypanosomes and that the ligands for Con A binding are carbohydrates on the EP forms of procyclin. The possible significance of these findings for the life cycle of the trypanosomes in the tsetse fly vector is discussed.     

Threonine 66 in the death domain of IRAK-1 is critical for interaction with signaling molecules but is not a target site for autophosphorylation

Ligand binding in the TLR/IL-1R family results in the transient formation of an intracellular signaling complex, which contains, amongst others, the serine/threonine-specific kinase IL-1R-associated kinase 1 (IRAK-1). Concomitantly, the kinase function of IRAK-1 becomes activated, resulting in massive autophosphorylation and finally in the dissociation of the initially constituted signaling complex. The death domain (DD) of IRAK-1 mediates the interaction with other molecules of the signaling complex, e.g., the adaptor MyD88, the silencer Tollip, and the activator kinase IRAK-4. The conserved threonine at position 66 (T66), located within the DD, is a putative autophosphorylation target site. Here, we provide evidence that T66 critically impacts the secondary structure of the IRAK-1 DD. Thereby, it ensures the transient manner of interactions between IRAK-1 and the other signaling molecules. This essential role, however, is not regulated by phosphorylation of T66 itself.     

Therapeutic approaches to preventing cell death in Huntington disease

Neurodegenerative diseases affect the lives of millions of patients and their families. Due to the complexity of these diseases and our limited understanding of their pathogenesis, the design of therapeutic agents that can effectively treat these diseases has been challenging. Huntington disease (HD) is one of several neurological disorders with few therapeutic options. HD, like numerous other neurodegenerative diseases, involves extensive neuronal cell loss. One potential strategy to combat HD and other neurodegenerative disorders is to intervene in the execution of neuronal cell death. Inhibiting neuronal cell death pathways may slow the development of neurodegeneration. However, discovering small molecule inhibitors of neuronal cell death remains a significant challenge. Here, we review candidate therapeutic targets controlling cell death mechanisms that have been the focus of research in HD, as well as an emerging strategy that has been applied to developing small molecule inhibitors—fragment-based drug discovery (FBDD). FBDD has been successfully used in both industry and academia to identify selective and potent small molecule inhibitors, with a focus on challenging proteins that are not amenable to traditional high-throughput screening approaches. FBDD has been used to generate potent leads, pre-clinical candidates, and has led to the development of an FDA approved drug. This approach can be valuable for identifying modulators of cell-death-regulating proteins; such compounds may prove to be the key to halting the progression of HD and other neurodegenerative disorders.     

EPCAM–A novel molecular target for the treatment of pediatric and adult germ cell tumors

Germ cell tumors (GCTs) are thought to develop from totipotent primordial germ cells. Although the epithelial cell adhesion molecule (EPCAM) is expressed on embryonic stem cells as well as different tumor cells, it has not yet been extensively studied in GCTs. We analyzed EPCAM expression by quantitative RT‐PCR in 48 fresh‐frozen GCT specimens of different histology (10 mature teratoma, MT; 6 immature teratoma, IT; 7 dysgerminoma; 6 mixed malignant GCTs; 19 yolk sac tumor, YST) and in the GCT cell lines NCCIT, TE76.T, JAR and 2102Ep, and correlated its expression with AFP and hCG protein levels, histologic differentiation, and clinical follow‐up data. EPCAM protein was visualized by immunohistochemistry of selected corresponding paraffin embedded tumor tissues. EPCAM was expressed in malignant but not in benign GCTs irrespective of age, sex, site and clinical stage of tumor (P = 0.001). In primary teratomas, EPCAM expression increased with their grade of immaturity (mean 2^?ΔCt values: MT 0.23, IT 1.61, P = 0.007) and significantly correlated with serum AFP (P = 0.03) and hCG (P = 0.03) levels in malignant GCTs. Particularly high EPCAM levels were found in nonseminomatous GCTs such as YSTs (8.49) and choriocarcinoma (13.54). Immunohistochemical analysis verified gene expression data showing a distinct EPCAM staining in YST. Similarly in vitro, highest EPCAM expression was measured in GCT cell lines comprising yolk sac (2102Ep: 5.59) or choriocarcinoma (JAR: 10.65) components. This first comprehensive analysis of EPCAM in GCTs revealed high EPCAM expression in YSTs and choriocarcinomas. Thus, these nonseminomatous GCTs may be interesting targets for EPCAM immunotherapy, which has to be evaluated in further studies. © 2012 Wiley Periodicals, Inc.     

Metabolomics reveals that carnitine palmitoyltransferase‐1 is a novel target for oxidative inactivation in human cells

Oxidative dysfunction in the metabolism has long been implicated in diverse biological disorders. Although a substantial number of metabolic enzymes are targeted for inactivation by oxidative stress, identifying those targets remains difficult due to a lack of comprehensive observations of the metabolism acting through the stress response. We herein developed a metabolomics strategy using integrative liquid chromatography‐mass spectrometry (LC‐MS) and observing rapid metabolomic changes in response to hydrogen peroxide (H₂O₂)‐induced oxidative stress in HeLa cells. Among the many metabolite changes detected, the most characteristic metabolites uniquely indicated carnitine palmitoyltransferase‐1 (CPT1), the critical enzyme for mitochondrial β‐oxidation of long‐chain fatty acids, to be a target for oxidative inactivation. We showed that the enzymatic activity of CPT1 significantly declined by H₂O₂ in several human cells. Interestingly, the inactivation was shown to be a direct effect of H₂O₂ in vitro, but substantially occurred when cells were cultured with some reagents that generate reactive oxygen species (ROS). Thus, our results suggest the generality of CPT1 inhibition under various stress conditions associated with ROS generation, providing an insight into a mechanism for oxidative dysfunction in mitochondrial metabolism. Our metabolome data additionally suggest that certain methyltransferase(s) may be targets of oxidative stress as well.