Mice overexpressing human caspase 3 appear phenotypically normal but exhibit increased apoptosis and larger lesion volumes in response to transient focal cerebral ischaemia.

Caspase 3 activation has been implicated in cell death following a number of neurodegenerative insults. To determine whether caspase genes can affect the susceptibility of cells to neurodegeneration, a transgenic mouse line was created, expressing human caspase 3 under control of its own promoter. The human gene was regulated by the murine homeostatic machinery and human procaspase 3 was expressed in the same tissues as mouse caspase 3. These novel transgenic mice appeared phenotypically and developmentally normal and survived in excess of 2 years. Behavioural assessment using the 5-choice serial reaction time task found no differences from wild-type littermates. Caspase activity was found to be tightly regulated under physiological conditions, however, significantly larger lesions were obtained when transgenic mice were subjected to focal cerebral ischaemia/reperfusion injury compared to wild-type littermates. These data demonstrate that mice overexpressing human caspase 3 are essentially normal, however, they have increased susceptibility to degenerative insults.

Serum withdrawal causes apoptosis in SHSY 5Y cells.

A proportion of differentiated SH-SY5Y cells undergo cell death in response to withdrawal of serum. This death manifests the hallmark features of apoptosis including changes in nuclear morphology, processing and activation of caspase 3 and cleavage of the caspase 3 substrates acetyl-Asp-Glu-Val-Asp-aminomethylcoumarin and poly(ADP-ribose) polymerase. These findings represent the first demonstration of serum withdrawal induced apoptosis in SH-SY5Y cells. The reduction in viability induced by serum deprivation and assessed using an inhibitor of mitochondrial respiration can be partially inhibited by FK506, but FK506 does not prevent caspase 3 processing or cleavage of caspase 3 substrates. FK506 is also able to promote the viability of a small proportion of embryonic mouse sensory neurons following nerve growth factor-withdrawal induced apoptosis. FK506 did not promote viability in either cell type in the absence of serum- or nerve growth factor-withdrawal. These observations are consistent with a survival-promoting effect of FK506 in cultured neurons.

Phenotypic characterisation and infection of ovine microglial cells with Maedi-Visna virus.

Maedi-Visna Virus (MVV) infection of the central nervous system (CNS) results in pathological changes, the mechanisms of which are poorly understood. MVV preferentially infects cell of the monocyte/macrophage lineage in vivo. The neuroparenchymal microglial cells are the resident tissue macrophages in the CNS and therefore likely targets for MVV infection. However, no information is currently available on the susceptibility of these cells to MVV infection or their contribution to neuropathological changes as a result of MVV infection. Highly enriched primary ovine microglial cell cultures were set up from brain tissues of lambs. These cells were amoeboid or bipolar with spikes, a morphology consistent with microglial cells of other species, and stained positive for CD1, CD11a, CD11c, CD14, MHC-class I, MHC-class II, and beta-N-acetyl galactose, but not with markers of astrocytes or oligodendrocytes. These sheep microglial cells were permissive for MVV infection. Productive MVV infection resulted in selective transcriptional up-regulation of the pro-inflammatory cytokines TNFalpha and IL-6. In contrast, there was no change in levels of transcripts for TGFbeta1, IL-1beta, GM-CSF, IL-10, or IL-12. These data provide the first evidence that ovine microglial cells can support productive infection with MVV, and that this leads to a selective up-regulation of proinflammatory cytokines. These may contribute to visna neuropathology.

Altruistic cell suicide and the specialized case of the virus-infected nervous system.

Depending on their differentiation state, vertebrate neurones can commit suicide after neurotropic virus infection. Such suicide might be an evolved strategy in multicellular organisms for limiting virus expansion. Regulation of suicide in this context operates by a programme similar to that activated during embryogenesis or in response to nervous-system injury and disease. In contrast to immature neurones that can readily initiate apoptosis following infection, mature neurones are generally highly resistant and can survive for long periods if they remain functional. Mature, infected neurones might gain competence to die owing to the attuned activation of pathways that sensitize the cell to subsequent stress. The consequence of either perturbation of function as a result of viral persistence or a chronic but progressive loss of infected neurones might be a failure of key neural functions.

Caspase 6 activity initiates caspase 3 activation in cerebellar granule cell apoptosis.

Using a well documented ex vivo system consisting of rodent cerebellar granule cells (CGCs) the activation of caspases 3 and 6 during apoptosis induced by withdrawal of trophic support was analyzed. At the time of deprivation, the addition of the irreversible, broad-spectrum caspase inhibitor zVADfmk or the cell permeable, caspase 6 inhibitor CP-VEID-cho can transiently suppress the appearance of apoptosis, including the early appearance of DNA fragmentation. Using immunoblotting and fluorogenic peptide assays we observe deprivation-induced activation of caspases 3 and 6, but not caspase 9. Furthermore, active caspase 6 is capable of processing and activating procaspase 3 in cellular extracts prepared from non-apoptotic CGCs, whereas caspase 3 failed to activate caspase 6. In consonant with this, the cell permeable caspase 6 inhibitor prevented deprivation-induced caspase 3 activation whereas a cell permeable caspase 3 inhibitor, CP-DEVD-cho, had no effect on caspase 6 activation. This would indicate that caspase 6 is a significant inducer of the early caspase 3 activity in apoptotic CGCs.

Virus infection induces neuronal apoptosis: A comparison with trophic factor withdrawal.

Multicellular organisms can employ a number of defences to combat viral replication, the most dramatic being implementation of a cell autonomous apoptotic process. The overall cost to the viability of an organism of losing infected cells by apoptosis may be small if the dying cells can be substituted. In contrast, suicide of irreplaceable cells such as highly specialised neurons may have a more dramatic, even fatal consequence. Previous in vitro approaches to understanding whether neurotropic viruses cause neurons to apoptose have utilised transformed cell lines. These are not in the appropriate state of differentiation to provide an accurate indication of events in vivo. We have chosen to characterise the ability of a model CNS disease-causing virus, Semliki Forest virus (SFV), to infect and trigger apoptosis in primary cultures of nerve growth factor (NGF)-dependent sensory neurons. These cells are known to die when deprived of NGF and constitute a useful indicator of apoptosis. We observe that infection causes cell death which bears the morphological hallmarks of apoptosis, this occurs even in the present of survival promoting NGF and is concomitant with new virus production. Using the TUNEL (transferase dUTP nick end labelling) technique we show that SFV-induced apoptosis involves DNA fragmentation and requires caspase (CED-3/ICE cysteine protease) activation, as does apoptosis induced by NGF-deprivation. Extensive areas of apoptosis, as defined using a combination of ultrastructural analysis and TUNEL occur in infected neonatal mouse brains. The novel evidence that infection of primary neurons with SFV induces apoptosis with activation of one or more caspases defines a system for the further anlaysis of apoptosis regulation in physiologically relevant neurons.

bcl-2 acts early to restrict Semliki Forest virus replication and delays virus-induced programmed cell death.

As characterized by morphological assessment and terminal deoxynucleotidyltransferase-mediated dUTP nick end labeling, Semliki Forest virus (SFV) infection of rat prostatic adenocarcinoma cells triggers an apoptotic cell response. Cell death proceeded more rapidly following infection with the neurovirulent L10 strain of SFV than with the avirulent A7 strain. Overexpression of the antiapoptotic proto-oncogene bcl-2 allowed survival of cultures infected with either strain of virus. bcl-2 overexpression drastically reduced the numbers of productively infected cells within the cultures. In situ hybridization for viral message-sense RNA coupled with immunostaining for viral protein indicated that bcl-2 functions at an early stage of the virus life cycle, at entry, pretranscriptional events or at transcription, to inhibit virus replication. Double-immunofluorescent labeling for bcl-2 and viral glycoproteins revealed double-positive cells, demonstrating that with time, this early block in replication can be overcome. These productively infected bcl-2-expressing cells do, with time, undergo apoptosis. As a result of changing the balance between cell death and cell division by restricting productive virus replication and delaying virus-induced cell death, bcl-2 expression led to the establishment of chronically infected cell lines which could be passaged.

Role of Bcl-2 in the brain-derived neurotrophic factor survival response.

Developing neurons die if they fail to obtain an adequate supply of neurotrophins from their targets but how neurotrophins suppress cell death is not known. Although over-expression of exogenous Bcl-2 can prevent the death of cultured neurons deprived of members of the nerve growth factor family of neurotrophins it is not known if this effect is physiologically relevant. To determine if Bcl-2 participates in the neurotrophin survival response we used antisense bcl-2 RNA to inhibit endogenous Bcl-2 expression. Here we show that brain-derived neurotrophic factor (BDNF)-dependent neurons are killed by antisense bcl-2 RNA in the presence of BDNF. However, when these neurons were supported with ciliary neurotrophic factor (CNTF) their survival was not affected by antisense bcl-2 RNA. Likewise, the survival of CNTF-dependent ciliary neurons was not affected by antisense bcl-2 RNA. Our findings suggest that Bcl-2 is required for the BDNF survival response and that alternative, Bcl-2-independent survival mechanisms operate in sensory and parasympathetic neurons exposed to CNTF.

TrkA mediates an NGF survival response in NGF-independent sensory neurons but not in parasympathetic neurons.

We have investigated the role of trkA, the tyrosine kinase NGF receptor in mediating the survival response of embryonic chicken neurons to NGF. Embryonic trigeminal mesencephalic (TMN) neurons, which normally survive in the presence of BDNF but not NGF, become NGF-responsive when microinjected with an expression vector containing rat trkA cDNA. In contrast, microinjection of CNTF-dependent embryonic ciliary neurons with the same construct does not result in the acquisition of NGF-responsiveness. RT-PCR shows that injected TMN and ciliary neurons do not differ in their ability to express rat trkA mRNA or protein. The failure of injected ciliary neurons to show an NGF-promoted survival response is not due to absence of the low-affinity NGF receptor, p75, in these neurons. Quantitative PCR and immunocytochemistry shows that TMN and ciliary neurons both express p75 mRNA and protein. These findings not only provide the first direct experimental demonstration of trkA mediating a physiological response in an appropriate cell type, i.e. NGF-promoted survival of embryonic neurons, but indicate that not all neurons are able to respond to a trkA-mediated signal transduction event.

Ectopic trkA expression mediates a NGF survival response in NGF-independent sensory neurons but not in parasympathetic neurons.

We have investigated the role of trkA, the tyrosine kinase NGF receptor, in mediating the survival response of embryonic neurons to NGF. Embryonic trigeminal mesencephalic (TMN) neurons, which normally survive in the presence of brain-derived neurotrophic factor (BDNF) but not NGF, become NGF-responsive when microinjected with an expression vector containing trkA cDNA. In contrast, microinjection of ciliary neurotrophic factor (CNTF)-dependent embryonic ciliary neurons with the same construct does not result in the acquisition of NGF responsiveness by these neurons despite de novo expression of trkA mRNA and protein. The failure of trkA to result in an NGF-promoted survival response in ciliary neurons is not due to absence of the low-affinity NGF receptor, p75, in these neurons. Quantitative RT/PCR and immunocytochemistry showed that TMN and ciliary neurons both express p75 mRNA and protein. These findings not only provide the first direct experimental demonstration of trkA mediating a physiological response in an appropriate cell type, namely NGF-promoted survival of embryonic neurons, but indicate that not all neurons are able to respond to a trkA-mediated signal transduction event.

The proto-oncogene bcl-2 can selectively rescue neurotrophic factor-dependent neurons from apoptosis.

Apoptosis plays an important role in regulating cell numbers in a wide variety of tissues during development. The product of the bcl-2 gene inhibits apoptosis in certain cells of the myeloid and lymphoid lineages and is expressed in many cells that have an extended life span. To assess the role of bcl-2 in neuronal apoptosis, we microinjected a bcl-2 expression vector into neurotrophic factor-deprived embryonic neurons. Sensory neurons that depend for survival on one or more members of the nerve growth factor family of neurotrophic factors (nerve growth factor, brain-derived neurotrophic factor, and neurotrophin-3) were rescued by bcl-2, whereas ciliary neurotrophic factor (CNTF)-dependent ciliary neurons were not. Sensory neurons, however, became refractory to bcl-2 after exposure to CNTF. These findings indicate that at least two death pathways operate in neurons that are distinguished by their susceptibility to bcl-2. Neurons may die by either pathway, depending on the factors to which they have been exposed.

A common denominator of growth cone guidance and collapse?

Axonal guidance in the retinotectal system and in spinal nerve segmentation is based on repulsion or inhibition. In both systems the membrane glycoprotein responsible for the guiding activity is capable of inducing growth cone collapse. We discuss two models of axonal guidance that correlate axonal guidance and growth cone collapse. The models are applicable to axon guidance by membrane-associated or diffusible stimuli, and are not based on preferential adhesion of axons to certain substrata.

A developmentally regulated chicken neuronal protein associated with the cortical cytoskeleton.

Monoclonal antibody 3D5 recognizes a single component of the neuronal membrane skeleton isolated from the chicken embryo brain. The 3D5 antigen is highly enriched in the CNS, and smaller amounts are found in the PNS. It is also present in non-neural tissues, but this is due to peripheral innervation. The biochemical and molecular properties of the 3D5 antigen are very similar to those of the previously described mammalian protein B-50 (Zwiers et al., 1985)/GAP 43 (Jacobson et al., 1986)/pp46 (Ellis et al., 1985)/F1 (Chan et al., 1986), and include anomalous SDS gel migration, acidic isoelectric point, and extraction from the cytoplasmic side of the plasma membrane only under extremely alkaline conditions. The 3D5 antigen is also developmentally regulated, with maximum expression in brain occurring at E14-E16, after which levels decrease approximately 4-fold in the adult. Immunofluorescence staining of cultured neurons shows that the 3D5 antigen is located in all parts of the cell but is particularly enriched in the growth cone and the growth cone filopodia. As the 3D5 antigen is enriched in the membrane skeleton, we suggest that this protein is involved in an association between the actin-containing cytoskeleton and the plasma membrane.