Necdin protects embryonic motoneurons from programmed cell death

NECDIN belongs to the type II Melanoma Associated Antigen Gene Expression gene family and is located in the Prader-Willi Syndrome (PWS) critical region. Necdin-deficient mice develop symptoms of PWS, including a sensory and motor deficit. However, the mechanisms underlying the motor deficit remain elusive. Here, we show that the genetic ablation of Necdin, whose expression is restricted to post-mitotic neurons in the spinal cord during development, leads to a loss of 31% of specified motoneurons. The increased neuronal loss occurs during the period of naturally-occurring cell death and is not confined to specific pools of motoneurons. To better understand the role of Necdin during the period of programmed cell death of motoneurons we used embryonic spinal cord explants and primary motoneuron cultures from Necdin-deficient mice. Interestingly, while Necdin-deficient motoneurons present the same survival response to neurotrophic factors, we demonstrate that deletion of Necdin leads to an increased susceptibility of motoneurons to neurotrophic factor deprivation. We show that by neutralizing TNFα this increased susceptibility of Necdin-deficient motoneurons to trophic factor deprivation can be reduced to the normal level. We propose that Necdin is implicated through the TNF-receptor 1 pathway in the developmental death of motoneurons.     

BCL-2 protects peroxynitrite-treated thymocytes from poly(ADP-ribose) synthase (PARS)-independent apoptotic but not from PARS-mediated necrotic cell death

In thymocytes, peroxynitrite induces poly(ADP-ribose) synthetase (PARS) activation, which results in necrotic cell death. In the absence of PARS, however, peroxynitrite-treated thymocytes die by apoptosis. Because Bcl-2 has been reported to inhibit not only apoptotic but also some forms of necrotic cell death, here we have investigated how Bcl-2 regulates the peroxynitrite-induced apoptotic and necrotic cell death. We have found that Bcl-2 did not provide protection against peroxynitrite-induced necrotic death, as characterized by propidium iodide uptake, mitochondrial membrane potential decrease, secondary superoxide production, and cardiolipin loss. In the presence of a PARS inhibitor, peroxynitrite-treated thymocytes from Bcl-2 transgenic mice showed no caspase activation or DNA fragmentation and displayed smaller mitochondrial membrane potential decrease. These data show that Bcl-2 protects thymocytes from peroxynitrite-induced apoptosis at a step proximal to mitochondrial alterations but fails to prevent PARS-mediated necrotic cell death. Activation of tissue transglutaminase (tTG) occurs in various forms of apoptosis. Peroxynitrite did not induce transglutaminase activity in thymocytes and did not have a direct inhibitory effect on the purified tTG. Basal tTG was not different in Bcl-2 transgenic and wild type cells.     

Identification of mRNAs associated with programmed cell death in immature thymocytes.

Programmed cell death is an essential cellular process that occurs in epithelial turnover, neural development, and regulation of cell populations of the immune system. Thymocytes undergo programmed cell death in response to several inductive stimuli, including exposure to glucocorticoids or radiation. This program can be blocked by inhibitors of RNA or protein synthesis; this implies that new proteins are required to execute the death programs. To search for possible death-associated mRNAs, we directionally cloned cDNA representing mRNA from control and dexamethasone-treated thymocytes. These libraries were used to produce ample amounts of DNA and RNA used in subtractive hybridization for the removal of sequences present in both control and induced cells. The remaining unhybridized sequences were selectively amplified by polymerase chain reaction and cloned to produce a library enriched for sequences expressed in death-induced cells. From this library we isolated cDNAs of death-associated mRNAs. One of these mRNAs, RP-8, appears within 1 h after exposure to gamma radiation, and a second mRNA, RP-2, is observed within 2 h. Both of these mRNAs accumulate during a period when a reference mRNA, actin, is declining. RP-2 and RP-8 are no longer detectable after 6 h postinduction, when apoptosis and mRNA degradation are evident in the culture. Sequence analysis of RP-8 cDNA indicates the presence of a zinc finger domain suggestive of a possible DNA regulatory role for the RP-8 protein. cDNA sequence results on RP-2 classify the corresponding protein as an integral membrane protein. We conclude that RP-2 and RP-8 are death-associated mRNAs that should be functionally evaluated in the context of the death process. As previously suggested, it may be that a family of "death genes" is activated by various stimuli depending on the type of cell, in a manner somewhat analogous to the induction of heat shock (stress) protein genes.     

Genetically programmed cell death (apoptosis)

Extensively and successfully studied problems of programmed cell death are considered. Recent evidence on apoptosis genes is presented, including the bcl-2 family and other genes with similar functions. A scheme of pathways of the main apoptosis mechanism is constructed. Examples of associations of apoptosis and diseases are presented in a special section.     

Radiation-induced interphase death of rat thymocytes is internally programmed (apoptosis)

Thymocytes are highly radiosensitive and show 'interphase death' within a few hours after low doses of irradiation. However, the mechanisms responsible for this type of death remain ill-defined. Separation of the dead thymocyte fraction from irradiated thymocyte suspensions by centrifugation on Percoll gradients provided homogeneous populations of dead cells suitable for detailed study. Using this method, radiation-induced interphase death of thymocytes was found to involve a sharp but transient increase in buoyant density, concomitant with the appearance of distinctive morphologic changes which included disappearance of microvilli and blistering of the cell surface. The chromatin in the dead cells had a molecular weight sufficiently low to resist sedimentation, and consisted of short oligonucleosome chains. We were unable to detect populations of cells intermediate between the dead and normal in the above characteristics. Interphase death thus involves a discrete, abrupt transition from the normal state and is not merely the consequence of progressive and degenerative changes. Furthermore, immediate cessation of development of interphase death by cycloheximide suggested a possible involvement of protein synthesis on this transition step.     

IL-2 protects against anti-CD3-induced cell death in human medullary thymocytes

In recent years, several studies have confirmed the clonal elimination of thymocytes with receptors that recognize Ag and MHC molecules present on the membrane of thymic stromal cells, a process that may be relevant to the establishment of self-tolerance. In our work, we show that anti-CD3 treatment of single positive CD4+ or CD8+ human medullary thymocytes (obtained by anti-CD1a plus C) induces their apoptotic death. Some events commonly associated with the early steps of normal activation (IL-2R expression, increase in cytoplasmic Ca2+) are also induced after anti-CD3 treatment. Nevertheless, IL-2 is not secreted by these activated cells. The addition of exogenous IL-2 inhibits the apoptosis induced by anti-CD3. We suggest that the lack of secretion of IL-2 by medullary thymocytes may be a physiologic mechanism implicated in the process of negative selection that leads to tolerance.     

动物细胞培养过程中的细胞自然凋亡

细胞培养过程中的细胞自然凋亡是细胞受环境压力的影响而发生的现象。随着细胞自然凋亡的分子生物学和生物化学研究的深入,对以动物细胞产品生产为目的的细胞培养产业将产生极有价值的影响。采用DNA重组技术把预防细胞自然凋亡的基因导入细胞和在培基中加入具有抗细胞自然凋亡的化合物等手段已用于预防或减缓细胞培养过程中的细胞自然凋亡。这些技术将大大延长细胞达到饱和密度后的培养时间,从而使细胞培养系统的生产效率得以显著提高。     

Induction of bcl-2 expression by Epstein-Barr virus latent membrane protein 1 protects infected B cells from programmed cell death.

Epstein-Barr virus (EBV) not only induces growth transformation in human B lymphocytes, but has more recently been shown to enhance B cell survival under suboptimal conditions where growth is inhibited; both effects are mediated through the coordinate action of eight virus-coded latent proteins. The effect upon cell survival is best recognized in EBV-positive Burkitt's lymphoma cell lines where activation of full virus latent gene expression protects the cells from programmed cell death (apoptosis). Here we show by DNA transfection into human B cells that protection from apoptosis is conferred through expression of a single EBV latent protein, the latent membrane protein LMP 1. Furthermore, we demonstrate that LMP 1 mediates this effect by up-regulating expression of the cellular oncogene bcl-2. The interplay between EBV infection and expression of this cellular oncogene has important implications for virus persistence and for the pathogenesis of virus-associated malignant disease.     

Synthesis of cyclic peptides from unprotected precursors using removable N alpha-(1-(4-methoxyphenyl)-2-mercaptoethyl) auxiliary.

A new method to cyclize unprotected peptides is presented. The method involves the use of a 1-phenyl-2-mercaptoethyl derivative on the N-terminal glycine. This template acts as an auxiliary thiol-containing group in order to drive cyclization with a counterpart thioester moiety on the same molecule. Subsequent facile removal of the derivative generates products with only native peptide structure. The successful, high-yield cyclization of the peptide GSPYSSDTTPA is described.     

Measurement of both protein-bound and total S-2-(3-aminopropylamino)ethanethiol (WR-1065) in blood by high-performance liquid chromatography

A high-performance liquid chromatographic method for automated analysis of both protein-bound and total S-2-(3-aminopropylamino)ethanethiol (WR-1065) in blood has been developed in our laboratory. WR-1065 is the active thiol metabolite of the radio- and chemo-protector drug amifostine (WR-2721). Using WR-1065 quality control levels over the experimental range: 7.0, 45.0 and 85.0 μmol/l spiked into plasma, method validation for total WR-1065 included between-run assessment of imprecision (SD/C.V.%: 1.11/16.7%, 6.58/15.5% and 9.24/11.3%, respectively) and % accuracy (94.7, 106.0 and 97.2%).     

Bifunctional apoptosis inhibitor (BAR) protects neurons from diverse cell death pathways

The bifunctional apoptosis regulator (BAR) is a multidomain protein that was originally identified as an inhibitor of Bax-induced apoptosis. Immunoblot analysis of normal human tissues demonstrated high BAR expression in the brain, compared to low or absent expression in other organs. Immunohistochemical staining of human adult tissues revealed that the BAR protein is predominantly expressed by neurons in the central nervous system. Immunofluorescence microscopy indicated that BAR localizes mainly to the endoplasmic reticulum (ER) of cells. Overexpression of BAR in CSM 14.1 neuronal cells resulted in significant protection from a broad range of cell death stimuli, including agents that activate apoptotic pathways involving mitochondria, TNF-family death receptors, and ER stress. Downregulation of BAR by antisense oligonucleotides sensitized neuronal cells to induction of apoptosis. Moreover, the search for novel interaction partners of BAR identified several candidate proteins that might contribute to the regulation of neuronal apoptosis (HIP1, Hippi, and Bap31). Taken together, the expression pattern and functional data suggest that the BAR protein is involved in the regulation of neuronal survival.     

Poly(ADP-ribose) glycohydrolase silencing protects against H2O2-induced cell death

PAR [poly(ADP-ribose)] is a structural and regulatory component of multiprotein complexes in eukaryotic cells. PAR catabolism is accelerated under genotoxic stress conditions and this is largely attributable to the activity of a PARG (PAR glycohydrolase). To overcome the early embryonic lethality of parg-knockout mice and gain more insights into the biological functions of PARG, we used an RNA interference approach. We found that as little as 10% of PARG protein is sufficient to ensure basic cellular functions: PARG-silenced murine and human cells proliferated normally through several subculturing rounds and they were able to repair DNA damage induced by sublethal doses of H2O2. However, cell survival following treatment with higher concentrations of H2O2 (0.05-1 mM) was increased. In fact, PARG-silenced cells were more resistant than their wild-type counterparts to oxidant-induced apoptosis while exhibiting delayed PAR degradation and transient accumulation of ADP-ribose polymers longer than 15-mers at early stages of drug treatment. No difference was observed in response to the DNA alkylating agent N-methyl-N'-nitro-N-nitrosoguanidine, suggesting a specific involvement of PARG in the cellular response to oxidative DNA damage.     

Induction of programmed cell death (apoptosis) in mature lymphocytes

The apoptosis of human peripheral blood lymphocytes was analyzed by the breakdown of DNA into oligonucleosome-sized fragments. The mature lymphocytes were rendered sensitive to apoptosis by either the omission of fetal bovine serum in the culture medium or the addition of polymyxin B. In the first case it was counteracted by phorbol myristate acetate. The possible involvement of protein kinase C in cell survival is pointed out.     

Genetic control of programmed cell death (apoptosis) in Drosophila

Programmed cell death, or apoptosis, is a highly conserved cellular process that has been intensively investigated in nematodes, flies, and mammals. The genetic conservation, the low redundancy, the feasibility for high-throughput genetic screens and the identification of temporally and spatially regulated apoptotic responses make Drosophila melanogaster a great model for the study of apoptosis. Here, we review the key players of the cell death pathway in Drosophila and discuss their roles in apoptotic and non-apoptotic processes.     

IL-4 enhances programmed cell death (apoptosis) in stimulated human monocytes.

Because IL-4 down-regulates several proinflammatory functions associated with human monocytes/macrophages, we explored the possibility that IL-4 also decreases monocyte survival. IL-4 caused a concentration-dependent decrease in viability of IL-1 or LPS stimulated, but not unstimulated, monocytes. Nonviable cells demonstrated classic features of programmed cell death or apoptosis, in that they were condensed and contained oligonucleosome-sized (200 bp) DNA fragments. When compared with several other cytokines commonly associated with inflammatory lesions, IL-4 was uniquely effective in enhancing cell death. We found that IL-4 enhanced death more quickly in IL-1-stimulated cells than in LPS-stimulated cells, that stimulated monocytes did not become resistant to the effects of IL-4 during culture, and that the effects of IL-4 on viability were antagonized by IFN-gamma. Enhanced cell death was stimulus-specific in that monocyte viability maintained by certain activating agents, such as Con A or CSF, was unaffected by IL-4. These findings represent the first evidence of cytokine-enhanced programmed cell death in monocytes and suggest that the antiinflammatory effects of IL-4 are mediated in part by reducing survival of stimulated monocytes in chronic lesions.     

Reduced programmed cell death in the retina and defects in lens and cornea of Tgfbeta2(-/-) Tgfbeta3(-/-) double-deficient mice

We have previously shown that immunoneutralization of transforming growth factor- (TGF-) in the chick embryo significantly reduces programmed cell death (PCD) in peripheral neurons, spinal cord, and retina. In order to validate these results we have begun to analyze PCD in mice with targeted ablations of the TGF-2 and TGF-3 genes. Recent analyses of mice lacking TGF-3 had failed to reveal an overt eye phenotype, while retinae of TGF-2-deficient mice showed retinal hypercellularity. We report now that eyes of Tgf2/Tgf3 double-deficient mice display severe alterations in the morphology of the retina, lens, and cornea. The inner neural retina—the region where TGF- receptor (TR) I and II immunoreactivities are most prominent—is significantly thickened, and numbers of TUNEL-positive cells are significantly reduced compared to wild-type littermates. In Tgf2^–/–Tgf3^–/– and Tgf2^–/–Tgf3^+/– littermates the retina was consistently detached from the underlying pigment epithelium. Cornea, corneal stroma, and lens epithelium were significantly thinner in these mutants. In contrast, retinal morphology in Tgf2^+/–Tgf3^–/– mutant littermates resembles the situation observed in wild-type retinae except for the retinal detachment. Thus, regression in the thickness of cornea and corneal stroma seems to be TGF- isoform and gene dose dependent. Our results substantiate the notion based on previous analyses of chick embryos with reduced levels of endogenous TGF- that TGF-, most notably TGF-2, is required to mediate PCD in developing retinal cells in vivo. Moreover, our data indicate that TGF-s play essential roles in cornea and lens development.This work was supported by grants from the Deutsche Forschungsgemeinschaft.     

Tauroursodeoxycholic acid (TUDCA) protects photoreceptors from cell death after experimental retinal detachment

Background

Detachment of photoreceptors from the underlying retinal pigment epithelium is seen in various retinal disorders such as retinal detachment and age-related macular degeneration and leads to loss of photoreceptors and vision. Pharmacologic inhibition of photoreceptor cell death may prevent this outcome. This study tests whether systemic administration of tauroursodeoxycholic acid (TUDCA) can protect photoreceptors from cell death after experimental retinal detachment in rodents.

Methodology/Principal Findings

Retinal detachment was created in rats by subretinal injection of hyaluronic acid. The animals were treated daily with vehicle or TUDCA (500 mg/kg). TUNEL staining was used to evaluate cell death. Photoreceptor loss was evaluated by measuring the relative thickness of the outer nuclear layer (ONL). Macrophage recruitment, oxidative stress, cytokine levels, and caspase levels were also quantified. Three days after detachment, TUDCA decreased the number of TUNEL-positive cells compared to vehicle (651±68/mm² vs. 1314±68/mm², P = 0.001) and prevented the reduction of ONL thickness ratio (0.84±0.03 vs. 0.65±0.03, P = 0.002). Similar results were obtained after 5 days of retinal detachment. Macrophage recruitment and expression levels of TNF-a and MCP-1 after retinal detachment were not affected by TUDCA treatment, whereas increases in activity of caspases 3 and 9 as well as carbonyl-protein adducts were almost completely inhibited by TUDCA treatment.

Conclusions/Significance

Systemic administration of TUDCA preserved photoreceptors after retinal detachment, and was associated with decreased oxidative stress and caspase activity. TUDCA may be used as a novel therapeutic agent for preventing vision loss in diseases that are characterized by photoreceptor detachment.     

Production of (R)-3-Chloro-1,2-Propanediol from Prochiral 1,3-Dichloro-2-Propanol by Corynebacterium sp. Strain N-1074

The production of (R)-3-chloro-1,2-propanediol [(R)-MCP] from prochiral 1,3-dichloro-2-propanol (DCP) was examined with a bacterial strain identified as a Corynebacterium strain. The addition of glycerol as a carbon source or some chlorinated alcohols to a medium was effective for the induction of activity catalyzing the transformation of DCP into MCP. The optimum pH for (R)-MCP production by the resting cell reaction was around 8.0. The optical purity of (R)-MCP formed was improved by keeping the level of DCP in the reaction mixture at a low concentration. (R)-MCP was obtained from 77.5 mM DCP with a 97.3% molar conversion yield and an 83.8% enantiomeric excess of its optical purity by periodic feeding of the substrate.