Relevance of motoneuron specification and programmed cell death in embryos to therapy of ALS

The molecular cues that generate spinal motoneurons in early embryonic development are well defined. Motoneurons are generated in excess and consequently undergo a natural period of programmed cell death. Although it is not known exactly how motoneurons compete for survival in embryonic development, it is hypothesized that they rely on the ability to access limited amounts of trophic factors from peripheral tissues, a process that is tightly regulated by skeletal muscle activity. Attempts to elucidate the molecular mechanisms that underlie motoneuron generation and programmed cell death in embryos have led to various effective strategies for treating injury and disease in animal models. Such studies provide great hope for the amelioration of human amyotrophic lateral sclerosis (ALS), a devastating progressive motoneuron degenerative disease. Here we review the clinical relevance of studying motoneuron specification and death during embryonic development.     

Nitric oxide synthase activity is required for postsynaptic differentiation of the embryonic neuromuscular junction

Agrin, a synapse-organizing protein externalized by motor axons at the neuromuscular junction (NMJ), initiates a signaling cascade in muscle cells leading to aggregation of postsynaptic proteins, including acetylcholine receptors (AChRs). We examined whether nitric oxide synthase (NOS) activity is required for agrin-induced aggregation of postsynaptic AChRs at the embryonic NMJ in vivo and in cultured muscle cells. Inhibition of NOS reduced AChR aggregation at embryonic Xenopus NMJs by 50-90%, whereas overexpression of NOS increased AChR aggregate area 2- to 3-fold at these synapses. NOS inhibitors completely blocked agrin-induced AChR aggregation in cultured embryonic muscle cells. Application of NO donors to muscle cells induced AChR clustering in the absence of agrin. Our results indicate that NOS activity is necessary for postsynaptic differentiation of embryonic NMJs and that NOS is a likely participant in the agrin-MuSK signaling pathway of skeletal muscle cells.     

DAP kinase and DRP-1 mediate membrane blebbing and the formation of autophagic vesicles during programmed cell death

Death-associated protein kinase (DAPk) and DAPk-related protein kinase (DRP)-1 proteins are Ca+2/calmodulin-regulated Ser/Thr death kinases whose precise roles in programmed cell death are still mostly unknown. In this study, we dissected the subcellular events in which these kinases are involved during cell death. Expression of each of these DAPk subfamily members in their activated forms triggered two major cytoplasmic events: membrane blebbing, characteristic of several types of cell death, and extensive autophagy, which is typical of autophagic (type II) programmed cell death. These two different cellular outcomes were totally independent of caspase activity. It was also found that dominant negative mutants of DAPk or DRP-1 reduced membrane blebbing during the p55/tumor necrosis factor receptor 1-induced type I apoptosis but did not prevent nuclear fragmentation. In addition, expression of the dominant negative mutant of DRP-1 or of DAPk antisense mRNA reduced autophagy induced by antiestrogens, amino acid starvation, or administration of interferon-gamma. Thus, both endogenous DAPk and DRP-1 possess rate-limiting functions in these two distinct cytoplasmic events. Finally, immunogold staining showed that DRP-1 is localized inside the autophagic vesicles, suggesting a direct involvement of this kinase in the process of autophagy.     

Input and output synapses on identified motor neurones of a locust revealed by the intracellular injection of horseradish peroxidase

Summary Physiologically characterised motor neurones in the thoracic ganglia of the locust were injected with horseradish peroxidase in order that the spatial relationship between their input and output synapses could be observed with the electron microscope. A modification in the development procedure for the peroxidase ensured that the internal fine structure of the stained neurones was not obscured by the diaminobenzidine reaction product. Input and output synapses may occur within 1 m of each other on the neuropilar processes of the motor neurones. This supports physiological evidence that motor neurones may be involved in local circuit interactions within the thoracic ganglia.     

Role of caspases and mitochondria in the steroid-induced programmed cell death of a motoneuron during metamorphosis

Accessory planta retractor (APR) motoneurons of the hawk moth, Manduca sexta, undergo a segment-specific pattern of programmed cell death (PCD) 24 to 48 h after pupal ecdysis (PE). Cell culture experiments show that the PCD of APRs in abdominal segment 6 [APR(6)s] is a cell-autonomous response to the steroid hormone 20-hydroxyecdysone (20E) and involves mitochondrial demise and cell shrinkage. Twenty-four hours before PE, at stage W3-noon, APR(6)s require further 20E exposure and protein synthesis (as tested with cycloheximide) to undergo PCD, and death can be blocked by a broad-spectrum caspase inhibitor. By PE, death is 20E- and protein synthesis-independent and the caspase inhibitor blocks cell shrinkage but not loss of mitochondrial function. Thus, the commitment to mitochondrial demise precedes the commitment to execution events. The phenotype of necrotic cell death induced by a mitochondrial electron transfer inhibitor differs unambiguously from 20E-induced PCD. By inducing PCD pharmacologically, the readiness of APR(6)s to execute PCD was found to increase during the final larval instar. These data suggest that the 20E-induced PCD of APR(6)s includes a premitochondrial phase which includes 20E-induced synthetic events and apical caspase activity, a mitochondrial phase which culminates in loss of mitochondrial function, and a postmitochondrial phase during which effector caspases are activated and APR(6) is destroyed.     

Balance of apoptotic cell death and survival in allergic diseases

Allergic diseases result from over-reaction of the immune system in response to exogenous allergens, where inflammatory cells have constantly extended longevity and contribute to an on-going immune response in allergic tissues. Here, we review disequilibrium in the death and survival of epithelial cells and inflammatory cells in the pathological processes of asthma, atopic dermatitis, and other allergic diseases.     

Re-organisation of the cytoskeleton during developmental programmed cell death in Picea abies embryos

Cell and tissue patterning in plant embryo development is well documented. Moreover, it has recently been shown that successful embryogenesis is reliant on programmed cell death (PCD). The cytoskeleton governs cell morphogenesis. However, surprisingly little is known about the role of the cytoskeleton in plant embryogenesis and associated PCD. We have used the gymnosperm, Picea abies, somatic embryogenesis model system to address this question. Formation of the apical-basal embryonic pattern in P. abies proceeds through the establishment of three major cell types: the meristematic cells of the embryonal mass on one pole and the terminally differentiated suspensor cells on the other, separated by the embryonal tube cells. The organisation of microtubules and F-actin changes successively from the embryonal mass towards the distal end of the embryo suspensor. The microtubule arrays appear normal in the embryonal mass cells, but the microtubule network is partially disorganised in the embryonal tube cells and the microtubules disrupted in the suspensor cells. In the same embryos, the microtubule-associated protein, MAP-65, is bound only to organised microtubules. In contrast, in a developmentally arrested cell line, which is incapable of normal embryonic pattern formation, MAP-65 does not bind the cortical microtubules and we suggest that this is a criterion for proembryogenic masses (PEMs) to passage into early embryogeny. In embryos, the organisation of F-actin gradually changes from a fine network in the embryonal mass cells to thick cables in the suspensor cells in which the microtubule network is completely degraded. F-actin de-polymerisation drugs abolish normal embryonic pattern formation and associated PCD in the suspensor, strongly suggesting that the actin network is vital in this PCD pathway.     

Simultaneous cell death and desquamation of the embryonic diffusion barrier during epidermal development

The periderm is an epithelial layer covering the emerging epidermis in early embryogenesis of vertebrates. In the chicken embryo, an additional cellular layer, the subperiderm, occurs at later embryonic stages underneath the periderm. The questions arose what is the function of both epithelial layers and, as they are transitory structures, by which mechanism are they removed. By immunocytochemistry, the tight junction (TJ) proteins occludin and claudin-1 were localized in the periderm and in the subperiderm, and sites of close contact between adjacent cells were detected by electron microscopy. Using horseradish peroxidase (HRP) as tracer, these contacts were identified as tight junctions involved in the formation of the embryonic diffusion barrier. This barrier was lost by desquamation at the end of the embryonic period, when the cornified envelope of the emerging epidermis was formed. By TUNEL and DNA ladder assays, we detected simultaneous cell death in the periderm and the subperiderm shortly before hatching. The absence of caspases-3, -6, and -7 activity, key enzymes of apoptosis, and the lack of typical morphological criteria of apoptosis such as cell fragmentation or membrane blebbing point to a special form of programmed cell death (PCD) leading to the desquamation of the embryonic diffusion barrier.     

Plant microtubules reorganization under the indirect UV-B exposure and during UV-B-induced programmed cell death

The role of microtubules in cellular pathways of UV-B signaling in plants as well as in related structural cell response become into focus of few last publications. As microtubules in plant cell reorient/reorganize (become randomized, fragmented or depolymerized) in a response to direct UV-B exposure, these cytoskeletal components could be involved into UV-B signaling pathways as highly responsive players. In the current addendum, indirect UV-B-induced microtubules reorganization in cells of shielded Arabidopsis thaliana (GFP-MAP4) primary roots and the correspondence of microtubules depolymerization with the typical hallmarks of the programmed cell death in Nicotiana tabacum BY-2 (GFP-MBD) cells are discussed.     

Depletion of the novel protein PHACTR-1 from human endothelial cells abolishes tube formation and induces cell death receptor apoptosis

Using suppression subtractive hybridisation (SSH), we identified a hitherto unreported gene PHACTR-1 (Phosphatase Actin Regulating Protein-1) in Human Umbilical Vascular Endothelial Cells (HUVECs). PHACTR-1 is an actin and protein phosphatase 1 (PP1) binding protein which is reported to be highly expressed in brain and which controls PP1 activity and F-actin remodelling. We have also reported that its expression is dependent of Vascular Endothelial Growth Factor (VEGF-A₁₆₅). To study its function in endothelial cells, we used a siRNA strategy against PHACTR-1. PHACTR-1 siRNA-treated HUVECs showed a major impairment of tube formation and stabilisation. PHACTR-1 depletion triggered apoptosis through death receptors DR4, DR5 and FAS, which was reversed using death receptor siRNAs or with death receptor-dependent caspase-8 siRNA. Our findings suggest that PHACTR-1 is likely to be a key regulator of endothelial cell function properties. Because of its central role in the control of tube formation and endothelial cell survival, PHACTR-1 may represent a new target for the development of anti-angiogenic therapy.     

Patterning and organization of motor neuron dendrites in the Drosophila larva

Precise patterns of motor neuron connectivity depend on the proper establishment and positioning of the dendritic arbor. However, how different motor neurons orient their dendrites to selectively establish synaptic connectivity is not well understood. The Drosophila neuromuscular system provides a simple model to investigate the underlying organizational principles by which distinct subclasses of motor neurons orient their dendrites within the central neuropil. Here we used genetic mosaic techniques to characterize the diverse dendritic morphologies of individual motor neurons from five main nerve branches (ISN, ISNb, ISNd, SNa, and SNc) in the Drosophila larva. We found that motor neurons from different nerve branches project their dendrites to largely stereotyped mediolateral domains in the dorsal region of the neuropil providing full coverage of the receptive territory. Furthermore, dendrites from different motor neurons overlap extensively, regardless of subclass, suggesting that repulsive dendrite-dendrite interactions between motor neurons do not influence the mediolateral positioning of dendritic fields. The anatomical data in this study provide important information regarding how different subclasses of motor neurons organize their dendrites and establishes a foundation for the investigation of the mechanisms that control synaptic connectivity in the Drosophila motor circuit.     

The CNTF/LIF signaling pathway regulates developmental programmed cell death and differentiation of rod precursor cells in the mouse retina in vivo

Natural cell death is critical for normal development of the nervous system, but the extracellular regulators of developmental cell death remain poorly characterized. Here, we studied the role of the CNTF/LIF signaling pathway during mouse retinal development in vivo. We show that exposure to CNTF during neonatal retinal development in vivo retards rhodopsin expression and results in an important and specific deficit in photoreceptor cells. Detailed analysis revealed that exposure to CNTF during retinal development causes a sharp increase in cell death of postmitotic rod precursor cells. Importantly, we show that blocking the CNTF/LIF signaling pathway during mouse retinal development in vivo results in a significant reduction of naturally occurring cell death. Using retroviral lineage analysis, we demonstrate that exposure to CNTF causes a specific reduction of clones containing only rods without affecting other clone types, whereas blocking the CNTF/LIF receptor complex causes a specific increase of clones containing only rods. In addition, we show that stimulation of the CNTF/LIF pathway positively regulates the expression of the neuronal and endothelial nitric oxide synthase (NOS) genes, and blocking nitric oxide production by pre-treatment with a NOS inhibitor abolishes CNTF-induced cell death. Taken together, these results indicate that the CNTF/LIF signaling pathway acts via regulation of nitric oxide production to modulate developmental programmed cell death of postmitotic rod precursor cells.     

Regulation of cell survival and death during Flavivirus infections

Flaviviruses, ss(+) RNA viruses, include many of mankind's most important pathogens. Their pathogenicity derives from their ability to infect many types of cells including neurons, to replicate, and eventually to kill the cells. Flaviviruses can activate tumor necrosis factor α and both intrinsic(Bax-mediated) and extrinsic pathways to apoptosis. Thus they can use many approaches for activating these pathways. Infection can lead to necrosis if viral load is extremely high or to other types of cell death if routes to apoptosis are blocked. Dengue and Japanese Encephalitis Virus can also activate autophagy. In this case the autophagy temporarily spares the infected cell, allowing a longer period of reproduction for the virus, and the autophagy further protects the cell against other stresses such as those caused by reactive oxygen species. Several of the viral proteins have been shown to induce apoptosis or autophagy on their own, independent of the presence of other viral proteins. Given the versatility of these viruses to adapt to and manipulate the metabolism, and thus to control the survival of, the infected cells, we need to understand much better how the specific viral proteins affect the pathways to apoptosis and autophagy. Only in this manner will we be able to minimize the pathology that they cause.     

Programmed cell death and cell necrosis activity during hyperthermic stress-induced bleaching of the symbiotic sea anemone Aiptasia sp.

Different cell death pathways were investigated during bleaching in the sea anemone Aiptasia sp. in response to hyperthermic treatment. Using a suite of techniques, (haematoxylin and eosin staining of paraffin wax-embedded tissue sections, in-situ end labelling (ISEL) of fragmented DNA, agarose gel electrophoresis electron microscopy) both necrotic and programmed cell death (PCD) activity were indicated. After a treatment period of 4 days, the host endoderm tissues underwent necrotic cell death. This was indicated by widespread cellular degradation, dilation of cell cytoplasm and organelles, cell swelling and rupture, irregular pyknotic condensation of nuclear chromatin, and abundant cell debris. Host cell necrosis was associated with the release of zooxanthellae with a normal, healthy appearance into the coelenteron. Longer periods of hyperthermic treatment (7 days) were correlated with further animal cell degradation and the in-situ degradation of zooxanthellae remaining within the degraded endoderm. Within the same degraded endoderm tissue, the degradation of zooxanthellae resulted from two forms of cell death occurring simultaneously, which were identified as programmed cell death and cell necrosis. Programmed cell death of zooxanthellae was characterised by condensation of the cytoplasm and organelles, cell shrinkage, formation of accumulation bodies at the periphery of the cell wall, and DNA fragmentation. Cell necrosis of zooxanthellae was characterised by dilation of the cytoplasm and organelles, cell swelling and lysis, dispersion of cell component debris, and DNA fragmentation. The existence of a programmed cell death pathway within zooxanthellae is important to the understanding of coral bleaching events, raising interesting questions regarding the evolution of this process and the activation of the cellular trigger mechanisms involved.     

Mutations that affect the survival of selected amacrine cell subpopulations define a new class of genetic defects in the vertebrate retina

Amacrine neurons are among the most diverse cell classes in the vertebrate retina. To gain insight into mechanisms vital to the production and survival of amacrine cell types, we investigated a group of mutations in three zebrafish loci: kleks (kle), chiorny (chy), and bergmann (bgm). Mutants of all three genes display a severe loss of selected amacrine cell subpopulations. The numbers of GABA-expressing amacrine interneurons are sharply reduced in all three mutants, while cell loss in other amacrine cell subpopulations varies and some cells are not affected at all. To investigate how amacrine cell loss affects retinal function, we performed electroretinograms on mutant animals. While the kle mutation mostly influences the function of the inner nuclear layer, unexpectedly the chy mutant phenotype also involves a loss of photoreceptor cell activity. The precise ration and arrangement of amacrine cell subpopulations suggest that cell-cell interactions are involved in the differentiation of this cell class. To test whether defects of such interactions may be, at least in part, responsible for mutant phenotypes, we performed mosaic analysis and demonstrated that the loss of parvalbumin-positive amacrine cells in chy mutants is due to extrinsic (cell-nonautonomous) causes. The phenotype of another amacrine cell subpopulation, the GABA-positive cells, does not display a clear cell-nonautonomy in chy animals. These results indicate that environmental factors, possibly interactions among different subpopulations of amacrine neurons, are involved in the development of the amacrine cell class.     

植物细胞程序化死亡的形态学和生理生化特征

植物细胞程序化死亡(PCD)是一种由基因控制的、主动的细胞死亡过程,它在植物正常生长发育过程中起着重要作用。发生程序化死亡的植物细胞在形态、生理生化方面表现出一些共性特点和个性特点,该文对这些特点进行了综述。     

Aspects of programmed cell death during leaf senescence of mono- and dicotyledonous plants

Summary Leaf senescence is a highly regulated stage in the plant life cycle, leading to cell death, recently examined as a type of the programmed cell death (PCD). One of the basic features of PCD is the condensation of nuclear chromatin which is caused by endonucleolytic degradation of nuclear DNA (nDNA). In our investigations, we applied the technique of the single-cell electrophoresis system (“comet assay”) in order to determine the type of nDNA fragmentation during leaf senescence. The comet assay, a sensitive method revealing nonrandom internucleosomal damage that is specific for PCD, is especially useful for the detection of nDNA degradation in isolated viable cells. Simultaneously, we analyzed the mesophyll cell ultrastructure and the photosynthetic-pigment concentration in the leaves of two species,Ornithogalum virens andNicotiana tabacum, representing mono- and dicotyledonous plants which differ in the pattern of leaf differentiation. These investigations demonstrated that, in both species, the comet assay revealed nDNA degradation in yellow-leaf protoplasts containing chloroplasts that showed already changed ultrastructure (swelled or completely degraded thylakoids) and cell nuclei with a significant condensation of chromatin. There was no nDNA degradation in green-leaf protoplasts containing differentiated chloroplasts with numerous grana stacks and nuclei with dispersed chromatin. The analysis of intermediate developmental stage showed that the degradation of nDNA precedes condensation of nuclear chromatin. Thus the comet assay is a very useful and sensitive method for early detection of PCD. Moreover, results of our studies indicate that leaf senescence involves PCD.     

Guanylate cyclase and cyclic GMP-dependent protein kinase regulate agrin signaling at the developing neuromuscular junction

During formation of the neuromuscular junction (NMJ), agrin secreted by motor axons signals the embryonic muscle cells to organize a postsynaptic apparatus including a dense aggregate of acetylcholine receptors (AChRs). Agrin signaling at the embryonic NMJ requires the activity of nitric oxide synthase (NOS). Common downstream effectors of NOS are guanylate cyclase (GC), which synthesizes cyclic GMP, and cyclic GMP-dependent protein kinase (PKG). Here we show that GC and PKG are important for agrin signaling at the embryonic NMJ of the frog, Xenopus laevis. Inhibitors of both GC and PKG reduced endogenous AChR aggregation in embryonic muscles by 50-85%, and blocked agrin-induced AChR aggregation in cultured embryonic muscle cells. A cyclic GMP analog, 8-bromo-cyclic GMP, increased endogenous AChR aggregation in embryonic muscles to 3- to 4-fold control levels. Overexpression of either GC or PKG in embryos increased AChR aggregate area by 60-170%, whereas expression of a dominant negative form of GC inhibited endogenous aggregation by 50%. These results indicate that agrin signaling in embryonic muscle cells requires the activity of GC and PKG as well as NOS.