Fas-Mediated Apoptosis in Primary Cultured Mouse Hepatocytes

The Fas antigen is a member of the tumor necrosis factor/nerve growth factor receptor family and is expressed in tissues such as the thymus, liver, and ovary. Agonistic anti-Fas antibodies have cytolytic activity against cell lines expressing the Fas antigen. In this study, we show that anti-Fas antibody can induce the death of mouse hepatocytes in primary culture. Cell death via apoptosis was evidenced by the fact that the dying cells displayed DNA fragmentation, extensive surface bleb formation, and chromatin condensation. Anti-Fas antibody alone induced apoptosis in less than 20% of the cultured hepatocytes, whereas all cells were killed within 24 h by anti-Fas antibody in the presence of actinomycin D, cycloheximide, or protein kinase C (PKC) inhibitors such as H-7 and HA1004. These results indicate that the Fas antigen expressed in mouse hepatocytes functionally transduces the apoptotic signal and suggest that cultured mouse hepatocytes express protective proteins against apoptosis and that phosphorylation by PKC is also involved in protection of the hepatocytes from Fas-mediated apoptosis.     

Ras Homolog Enriched in Brain (Rheb) Enhances Apoptotic Signaling

Rheb is a homolog of Ras GTPase that regulates cell growth, proliferation, and regeneration via mammalian target of rapamycin (mTOR). Because of the well established potential of activated Ras to promote survival, we sought to investigate the ability of Rheb signaling to phenocopy Ras. We found that overexpression of lipid-anchored Rheb enhanced the apoptotic effects induced by UV light, TNFα, or tunicamycin in an mTOR complex 1 (mTORC1)-dependent manner. Knocking down endogenous Rheb or applying rapamycin led to partial protection, identifying Rheb as a mediator of cell death. Ras and c-Raf kinase opposed the apoptotic effects induced by UV light or TNFα but did not prevent Rheb-mediated apoptosis. To gain structural insight into the signaling mechanisms, we determined the structure of Rheb-GDP by NMR. The complex adopts the typical canonical fold of RasGTPases and displays the characteristic GDP-dependent picosecond to nanosecond backbone dynamics of the switch I and switch II regions. NMR revealed Ras effector-like binding of activated Rheb to the c-Raf-Ras-binding domain (RBD), but the affinity was 1000-fold lower than the Ras/RBD interaction, suggesting a lack of functional interaction. shRNA-mediated knockdown of apoptosis signal-regulating kinase 1 (ASK-1) strongly reduced UV or TNFα-induced apoptosis and suppressed enhancement by Rheb overexpression. In conclusion, Rheb-mTOR activation not only promotes normal cell growth but also enhances apoptosis in response to diverse toxic stimuli via an ASK-1-mediated mechanism. Pharmacological regulation of the Rheb/mTORC1 pathway using rapamycin should take the presence of cellular stress into consideration, as this may have clinical implications.     

Phosphoproteomic Analysis of Neurotrophin Receptor TrkB Signaling Pathways in Mouse Brain

SUMMARY 1. The signaling pathways activated by trkB neurotrophin receptor have been studied in detail in cultured neurons, but little is known about the pathways activated by trkB in intact brain. TrkB is a tyrosine kinase and protein phosphorylation is a key regulatory process in the neuronal signal transduction pathways.2. We have investigated trkB signaling in the transgenic mice overexpressing trkB in postnatal neurons (trkB.TK) using phosphoproteomics.3. We found that several proteins are overphosphorylated on tyrosine residues in the brain of trkB.TK mice and identified some of these proteins.4. We demonstrate that the well characterized signaling molecules mitogen-activated protein kinase (MAPK) and cyclic AMP responsive element binding protein (CREB) were phosphorylated at a higher level in the brain of trkB.TK mice when compared to the wild type littermates. Furthermore, we found that β-actin was tyrosine phosphorylated in the brain of the transgenic mice.5. Our results demonstrate that phosphoproteomics is a sensitive approach to investigate signaling pathways activated in mouse brain.     

Occurrence of Reovirus Infection in Ontario

Reovirus Type 2 was isolated from three patients aged 5, 9 and 10 years. The etiological role of Reovirus in one case was confirmed by demonstration of antibodies in convalescent serum and none in acute serum. Symptoms in this case were suggestive of infectious mononucleosis. In the second case, rhinitis and non-purulent otitis were preceded by two waves of fever, abdominal pain and emesis. Acute serum was not available but convalescent serum had a high titre to a Reovirus, thus supporting a Reovirus etiology. Symptoms in the third case were fever, intermittent headache, neck stiffness, abdominal cramps and weakness of a leg. Because of the unavailability of convalescent serum, evidence of a Reovirus etiology was incomplete. No attempt is made to associate Reovirus with a particular clinical picture. Rather, attention is simply drawn to the existence of Reovirus infections in Ontario.     

MAVS-MKK7-JNK2 Defines a Novel Apoptotic Signaling Pathway during Viral Infection

Viral infection induces innate immunity and apoptosis. Apoptosis is an effective means to sacrifice virus-infected host cells and therefore restrict the spread of pathogens. However, the underlying mechanisms of this process are still poorly understood. Here, we show that the mitochondrial antiviral signaling protein (MAVS/VISA/Cardif/IPS-1) is critical for SeV (Sendai virus)-induced apoptosis. MAVS specifically activates c-Jun N-terminal kinase 2 (JNK2) but not other MAP kinases. Jnk2−/− cells, but not Jnk1−/− cells, are unable to initiate virus-induced apoptosis and SeV further fails to trigger apoptosis in MAPK kinase 7 (MKK7) knockout (Mkk7−/−) cells. Mechanistically, MAVS recruits MKK7 onto mitochondria via its 3D domain, which subsequently phosphorylates JNK2 and thus activates the apoptosis pathway. Consistently, Jnk2−/− mice, but not Jnk1−/− mice, display marked inflammatory injury in lung and liver after viral challenge. Collectively, we have identified a novel signaling pathway, involving MAVS-MKK7-JNK2, which mediates virus-induced apoptosis and highlights the indispensable role of mitochondrial outer membrane in host defenses.     

Abnormal Sperm Parameters in Humans Are Indicative of an Abortive Apoptotic Mechanism Linked to the Fas-Mediated Pathway

The life cycle of many cell types can hinge on the presence of death factors that can control programmed cell death. The Fas-mediated apoptotic pathway has been implicated in controlling apoptosis during spermatogenesis in a number of mammalian species. In the human, the presence of nuclear DNA damage in ejaculated spermatozoa has pointed to a possible role for apoptosis during spermatogenesis. The presence of other molecular markers of apoptosis has, however, not been shown. More importantly, differences in these markers have not been investigated in men with normal and abnormal sperm parameters. In this study we examine for the presence of the cell surface protein Fas in ejaculated human spermatozoa. Ejaculated spermatozoa (55 samples) were labeled with anti-human Fas antibody and the number of spermatozoa displaying Fas were counted using a fluorescence-activated cell sorter (FACS). In 30/31 (96.8%) normal males (>20 million sperm per milliliter), less than 10% of the spermatozoa were Fas positive. In contrast, 14/24 (58.3%) oligozoospermic samples (<20 million sperm per milliliter) contained more than 10% Fas-positive spermatozoa. Similar differences were observed in men whose spermatozoa had poor motility and morphology. These results indicate that apoptosis is a major mechanism in regulating spermatogenesis in the human and that there are clear differences in molecular markers of apoptosis between males with normal and abnormal sperm parameters. We propose that the presence of Fas-labeled spermatozoa in the ejaculate of these men is indicative of an "abortive apoptosis" having taken place, whereby the normal apoptotic mechanisms have misfunctioned, have been overridden, or have not been completed.     

氧化修饰在调控细胞凋亡信号转导中的作用

氧化修饰是细胞内的活性氧诱导生物大分子发生氧化反应引起的结构及构象改变,发挥调控信号转导和对应激作出反应的功能。氧化修饰发生在凋亡信号转导中的多个生物大分子,包括凋亡相关蛋白质的氧化,如caspase-9、线粒体通透性转变孔及电压依赖的阴离子通道(voltagedependent anion channel,VDAC),同时也包括膜磷脂的氧化修饰,如磷脂酰丝氨酸及线粒体特异的心磷脂。氧化修饰作用也涉及凋亡诱导因子、促凋亡的凋亡信号调控激酶1(apoptosis signalregulatin gkinasel,ASK1)信号转导途径及抗凋亡的转录因子NF—kB的激活和活性。所以氧化修饰可能是调控凋亡信号转导机制中除磷酸化、泛素化外的另一个新的分子机制。     

Blocking Apoptotic Signaling Rescues Axon Guidance in Netrin Mutants

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Monocytes Regulate the Mechanism of T-cell Death by Inducing Fas-Mediated Apoptosis during Bacterial Infection

Monocytes and T-cells are critical to the host response to acute bacterial infection but monocytes are primarily viewed as amplifying the inflammatory signal. The mechanisms of cell death regulating T-cell numbers at sites of infection are incompletely characterized. T-cell death in cultures of peripheral blood mononuclear cells (PBMC) showed ‘classic’ features of apoptosis following exposure to pneumococci. Conversely, purified CD3⁺ T-cells cultured with pneumococci demonstrated necrosis with membrane permeabilization. The death of purified CD3⁺ T-cells was not inhibited by necrostatin, but required the bacterial toxin pneumolysin. Apoptosis of CD3⁺ T-cells in PBMC cultures required ‘classical’ CD14⁺ monocytes, which enhanced T-cell activation. CD3⁺ T-cell death was enhanced in HIV-seropositive individuals. Monocyte-mediated CD3⁺ T-cell apoptotic death was Fas-dependent both in vitro and in vivo. In the early stages of the T-cell dependent host response to pneumococci reduced Fas ligand mediated T-cell apoptosis was associated with decreased bacterial clearance in the lung and increased bacteremia. In summary monocytes converted pathogen-associated necrosis into Fas-dependent apoptosis and regulated levels of activated T-cells at sites of acute bacterial infection. These changes were associated with enhanced bacterial clearance in the lung and reduced levels of invasive pneumococcal disease.     

Modafinil Abrogates Methamphetamine-Induced Neuroinflammation and Apoptotic Effects in the Mouse Striatum

Methamphetamine is a drug of abuse that can cause neurotoxic damage in humans and animals. Modafinil, a wake-promoting compound approved for the treatment of sleeping disorders, is being prescribed off label for the treatment of methamphetamine dependence. The aim of the present study was to investigate if modafinil could counteract methamphetamine-induced neuroinflammatory processes, which occur in conjunction with degeneration of dopaminergic terminals in the mouse striatum. We evaluated the effect of a toxic methamphetamine binge in female C57BL/6 mice (4×5 mg/kg, i.p., 2 h apart) and modafinil co-administration (2×90 mg/kg, i.p., 1 h before the first and fourth methamphetamine injections) on glial cells (microglia and astroglia). We also evaluated the striatal expression of the pro-apoptotic BAX and anti-apoptotic Bcl-2 proteins, which are known to mediate methamphetamine-induced apoptotic effects. Modafinil by itself did not cause reactive gliosis and counteracted methamphetamine-induced microglial and astroglial activation. Modafinil also counteracted the decrease in tyrosine hydroxylase and dopamine transporter levels and prevented methamphetamine-induced increases in the pro-apoptotic BAX and decreases in the anti-apoptotic Bcl-2 protein expression. Our results indicate that modafinil can interfere with methamphetamine actions and provide protection against dopamine toxicity, cell death, and neuroinflammation in the mouse striatum.     

Lipid Composition in Scrapie-Infected Mouse Brain: Prion Infection Increases the Levels of Dolichyl Phosphate and Ubiquinone

Abstract: The neutral and phospholipid composition of mouse brain infected with scrapie prions was investigated. During the later stages of this disease, the level of dolichol decreased by 30% whereas the level of dolichyl phosphate increased by 30%. In terminally ill mice, there was also a 2.5-fold increase in both total ubiquinone and its reduced form. Furthermore, α-tocopherol was elevated at this stage by 50%. In contrast, no changes were observed in phospholipid amount, in phospholipid composition, and in phosphatidylethanolamine plasmalogen content during the entire disease process. The fatty acid and aldehyde composition of individual phospholipids remained unaltered as well. No modifications could be detected in cholesterol content. Thus, the majority of membrane lipids in scrapie-infected mouse brain are modified in neither quantity nor structure, but specific changes occur to a few polyisoprenoid lipids. This specificity indicates that, although prions accumulate in lysosomes, the infection process is not associated with a general membrane destruction caused by lysosomal enzyme leakage.     

Homeobox Genes in the Developing Mouse Brain

Abstract: Any list of past and recent findings on vertebrate brain prenatal development would have to include the fundamental roles of homeobox genes, the genes encoding the nuclear regulatory homeodomain proteins. The discovery of homeobox genes and their involvement as master regulatory elements in programing the development of an embryo into a complete adult organism has provided a key to our understanding of ontogenesis. Also, the correlation of mouse developmental mutants and their corresponding human syndromes with mutations in homeobox genes has provided further evidence for the fundamental role of homeobox genes during the vertebrate brain embryonic development. Here, we review the expression patterns and the phenotypes of gene mutations that implicate a large repertoire of mouse homeobox genes in the specification of neuronal functions during brain embryogenesis.     

Inhibition of the Initiation of Cellular DNA Synthesis After Reovirus Infection

The synthesis of cellular DNA was measured in synchronized L cells after reovirus infection. Initiation of the synthetic phase of the cell cycle was completely inhibited in cells infected 8 h before the beginning of DNA synthesis.     

The Vasculome of the Mouse Brain

The blood vessel is no longer viewed as passive plumbing for the brain. Increasingly, experimental and clinical findings suggest that cerebral endothelium may possess endocrine and paracrine properties – actively releasing signals into and receiving signals from the neuronal parenchyma. Hence, metabolically perturbed microvessels may contribute to central nervous system (CNS) injury and disease. Furthermore, cerebral endothelium can serve as sensors and integrators of CNS dysfunction, releasing measurable biomarkers into the circulating bloodstream. Here, we define and analyze the concept of a brain vasculome, i.e. a database of gene expression patterns in cerebral endothelium that can be linked to other databases and systems of CNS mediators and markers. Endothelial cells were purified from mouse brain, heart and kidney glomeruli. Total RNA were extracted and profiled on Affymetrix mouse 430 2.0 micro-arrays. Gene expression analysis confirmed that these brain, heart and glomerular preparations were not contaminated by brain cells (astrocytes, oligodendrocytes, or neurons), cardiomyocytes or kidney tubular cells respectively. Comparison of the vasculome between brain, heart and kidney glomeruli showed that endothelial gene expression patterns were highly organ-dependent. Analysis of the brain vasculome demonstrated that many functionally active networks were present, including cell adhesion, transporter activity, plasma membrane, leukocyte transmigration, Wnt signaling pathways and angiogenesis. Analysis of representative genome-wide-association-studies showed that genes linked with Alzheimer’s disease, Parkinson’s disease and stroke were detected in the brain vasculome. Finally, comparison of our mouse brain vasculome with representative plasma protein databases demonstrated significant overlap, suggesting that the vasculome may be an important source of circulating signals in blood. Perturbations in cerebral endothelial function may profoundly affect CNS homeostasis. Mapping and dissecting the vasculome of the brain in health and disease may provide a novel database for investigating disease mechanisms, assessing therapeutic targets and exploring new biomarkers for the CNS.     

Neuroendocrine Signaling Via the Serotonin Transporter Regulates Clearance of Apoptotic Cells

Serotonin (5-hydroxytryptamine; 5-HT) is a CNS neurotransmitter increasingly recognized to exert immunomodulatory effects outside the CNS that contribute to the pathogenesis of autoimmune and chronic inflammatory diseases. 5-HT signals to activate the RhoA/Rho kinase (ROCK) pathway, a pathway known for its ability to regulate phagocytosis. The clearance of apoptotic cells (i.e. efferocytosis) is a key modulator of the immune response that is inhibited by the RhoA/ROCK pathway. Because efferocytosis is defective in many of the same illnesses where 5-HT has been implicated in disease pathogenesis, we hypothesized that 5-HT would suppress efferocytosis via activation of RhoA/ROCK. The effect of 5-HT on efferocytosis was examined in murine peritoneal and human alveolar macrophages, and its mechanisms were investigated using pharmacologic blockade and genetic deletion. 5-HT impaired efferocytosis by murine peritoneal macrophages and human alveolar macrophages. 5-HT increased phosphorylation of myosin phosphatase subunit 1 (Mypt-1), a known ROCK target, and inhibitors of RhoA and ROCK reversed the suppressive effect of 5-HT on efferocytosis. Peritoneal macrophages expressed the 5-HT transporter and 5-HT receptors (R) 2a, 2b, but not 2c. Inhibition of 5-HTR2a and 5-HTR2b had no effect on efferocytosis, but blockade of the 5-HT transporter prevented 5-HT-impaired efferocytosis. Genetic deletion of the 5-HT transporter inhibited 5-HT uptake into peritoneal macrophages, prevented 5-HT-induced phosphorylation of Mypt-1, reversed the inhibitory effect of 5-HT on efferocytosis, and decreased cellular peritoneal inflammation. These results suggest a novel mechanism by which 5-HT might disrupt efferocytosis and contribute to the pathogenesis of autoimmune and chronic inflammatory diseases.     

Impact of Calcium Signaling during Infection of Neisseria meningitidis to Human Brain Microvascular Endothelial Cells

The pili and outer membrane proteins of Neisseria meningitidis (meningococci) facilitate bacterial adhesion and invasion into host cells. In this context expression of meningococcal PilC1 protein has been reported to play a crucial role. Intracellular calcium mobilization has been implicated as an important signaling event during internalization of several bacterial pathogens. Here we employed time lapse calcium-imaging and demonstrated that PilC1 of meningococci triggered a significant increase in cytoplasmic calcium in human brain microvascular endothelial cells, whereas PilC1-deficient meningococci could not initiate this signaling process. The increase in cytosolic calcium in response to PilC1-expressing meningococci was due to efflux of calcium from host intracellular stores as demonstrated by using 2-APB, which inhibits the release of calcium from the endoplasmic reticulum. Moreover, pre-treatment of host cells with {"type":"entrez-nucleotide","attrs":{"text":"U73122","term_id":"4098075","term_text":"U73122"}}U73122 (phospholipase C inhibitor) abolished the cytosolic calcium increase caused by PilC1-expressing meningococci demonstrating that active phospholipase C (PLC) is required to induce calcium transients in host cells. Furthermore, the role of cytosolic calcium on meningococcal adherence and internalization was documented by gentamicin protection assay and double immunofluorescence (DIF) staining. Results indicated that chelation of intracellular calcium by using BAPTA-AM significantly impaired PilC1-mediated meningococcal adherence to and invasion into host endothelial cells. However, buffering of extracellular calcium by BAPTA or EGTA demonstrated no significant effect on meningococcal adherence to and invasion into host cells. Taken together, these results indicate that meningococci induce calcium release from intracellular stores of host endothelial cells via PilC1 and cytoplasmic calcium concentrations play a critical role during PilC1 mediated meningococcal adherence to and subsequent invasion into host endothelial cells.