The relationship of intramuscular nerve branching and synaptogenesis to motoneuron survival.

The target has been considered for some time to play a major role in allowing neurons to survive the period of naturally occurring cell death. For the motoneurons that innervate the chick limb, evidence is presented that suggests access to target-derived trophic factor via intramuscular nerve branches and synapses may be important in regulating neuronal survival. Alterations in branching and synapse formation produced by activity blockade as well as by alteration of adhesion molecule function are shown to result in changes in motoneuron survival consistent with the proposed hypothesis. The relevance of these observations to the numerical-matching hypothesis of vertebrate neuronal cell death is also considered.     

Mature neurons: equipped for survival

Neurons completely transform how they regulate cell death over the course of their lifetimes. Developing neurons freely activate cell death pathways to fine-tune the number of neurons that are needed during the precise formation of neural networks. However, the regulatory balance between life and death shifts as neurons mature beyond early development. Mature neurons promote survival at all costs by employing multiple, often redundant, strategies to prevent cell death by apoptosis. This dramatic shift from permitting cell death to ensuring cellular survival is critical, as these post-mitotic cells must provide neuronal circuitry for an organism''s entire lifetime. Importantly, as many neurodegenerative diseases afflict adult neuronal populations, the survival mechanisms in mature neurons are likely to be either reversed or circumvented during neurodegeneration. Examining the adaptations for inhibiting apoptosis during neuronal maturation is key to comprehending not just how neurons survive long term, but may also provide insight for understanding how neuronal toxicity in various neurodegenerative diseases may ultimately lead to cell death.     

Mitogen-activated protein kinases and cerebral ischemia

Mitogen-activated protein kinases (MAPKs) have crucial roles in signal transduction from the cell surface to the nucleus and regulate cell death and survival. Recent papers support the hypothesis that neuronal apoptosis and cerebral ischemia induce the robust activation of MAPK cascades. Although extracellular signal-regulated kinases pathways promote cell survival and proliferation, and c-Jun N-terminal protein kinases/p38 pathways induce apoptosis in general, the roles of MAPK cascades in neuronal death and survival seem to be complicated and altered by the type of cells and the magnitude and timing of insults. Some specific inhibitors of MAPK cascades provide important information in clarifying the roles of each molecule in neuronal death and survival, but the results are still controversial. Further studies are necessary to elucidate the activated signal transduction upstream and downstream of the cascades in cerebral ischemia, and to define the crosstalk between the cascades and other signaling pathways, before MAPK cascades can be candidate molecules in the treatment of cerebral ischemia.     

Retrograde transport of neurotrophins: fact and function

Retrograde signals generated by nerve growth factor (NGF) and other neurotrophins promote the survival of appropriately connected neurons during development, and failure to obtain sufficient retrograde signals may contribute to neuronal death occurring in many neurodegenerative diseases. The discovery over 25 years ago that NGF supplied to the axon terminals is retrogradely transported to the cell bodies suggested that NGF must reach the cell body to promote neuronal survival. Research during the intervening decades has produced a refinement of this hypothesis. The current hypothesis is that NGF bound to TrkA at the axon terminal is internalized into signaling endosomes, with NGF in their lumens bound to phosphorylated TrkA in their membranes, which are retrogradely transported to the cell bodies, where TrkA activates downstream signaling molecules that promote neuronal survival and regulate many aspects of neuronal gene expression. This model has been extrapolated to retrograde signaling by all neurotrophins. We consider the evidence for this model, focusing on results of experiments with neurons in compartmented cultures. Results to date indicate that while the transport of signaling endosomes containing NGF bound to TrkA may carry retrograde signals, retrograde survival signals can be carried by another mechanism that is activated by NGF at the axon terminal surface and travels to the cell body unaccompanied by the NGF that initiated it. It is hypothesized that multiple mechanisms of retrograde signaling exist and function under different circumstances. The newly discovered potential for redundancy in retrograde signaling mechanisms can complicate the interpretation of experimental results.     

A Truncated Fragment of Src Protein Kinase Generated by Calpain-mediated Cleavage Is a Mediator of Neuronal Death in Excitotoxicity

Excitotoxicity resulting from overstimulation of glutamate receptors is a major cause of neuronal death in cerebral ischemic stroke. The overstimulated ionotropic glutamate receptors exert their neurotoxic effects in part by overactivation of calpains, which induce neuronal death by catalyzing limited proteolysis of specific cellular proteins. Here, we report that in cultured cortical neurons and in vivo in a rat model of focal ischemic stroke, the tyrosine kinase Src is cleaved by calpains at a site in the N-terminal unique domain. This generates a truncated Src fragment of ∼52 kDa, which we localized predominantly to the cytosol. A cell membrane-permeable fusion peptide derived from the unique domain of Src prevents calpain from cleaving Src in neurons and protects against excitotoxic neuronal death. To explore the role of the truncated Src fragment in neuronal death, we expressed a recombinant truncated Src fragment in cultured neurons and examined how it affects neuronal survival. Expression of this fragment, which lacks the myristoylation motif and unique domain, was sufficient to induce neuronal death. Furthermore, inactivation of the prosurvival kinase Akt is a key step in its neurotoxic signaling pathway. Because Src maintains neuronal survival, our results implicate calpain cleavage as a molecular switch converting Src from a promoter of cell survival to a mediator of neuronal death in excitotoxicity. Besides unveiling a new pathological action of Src, our discovery of the neurotoxic action of the truncated Src fragment suggests new therapeutic strategies with the potential to minimize brain damage in ischemic stroke.     

Excitotoxicity triggered by Neurobasal culture medium

Neurobasal defined culture medium has been optimized for survival of rat embryonic hippocampal neurons and is now widely used for many types of primary neuronal cell culture. Therefore, we were surprised that routine medium exchange with serum- and supplement-free Neurobasal killed as many as 50% of postnatal hippocampal neurons after a 4 h exposure at day in vitro 12–15. Minimal Essential Medium (MEM), in contrast, produced no significant toxicity. Detectable Neurobasal-induced neuronal death occurred with as little as 5 min exposure, measured 24 h later. D-2-Amino-5-phosphonovalerate (D-APV) completely prevented Neurobasal toxicity, implicating direct or indirect N-methyl-D-aspartate (NMDA) receptor-mediated neuronal excitotoxicity. Whole-cell recordings revealed that Neurobasal but not MEM directly activated D-APV-sensitive currents similar in amplitude to those gated by 1 µM glutamate. We hypothesized that L-cysteine likely mediates the excitotoxic effects of Neurobasal incubation. Although the original published formulation of Neurobasal contained only 10 µM L-cysteine, commercial recipes contain 260 µM, a concentration in the range reported to activate NMDA receptors. Consistent with our hypothesis, 260 µM L-cysteine in bicarbonate-buffered saline gated NMDA receptor currents and produced toxicity equivalent to Neurobasal. Although NMDA receptor-mediated depolarization and Ca²⁺ influx may support survival of young neurons, NMDA receptor agonist effects on development and survival should be considered when employing Neurobasal culture medium.     

NeuroD regulates neuronal migration

NeuroD is required for the survival of many subtypes of developing neurons in the vertebrate central nervous system. Because NeuroD-deficient neurons in the hippocampus, cerebellum, and inner ear die prematurely in the early stage of neurogenesis, the role of NeuroD during the later stages of neurogenesis of these cell subtypes is not well understood. In addition, the mechanism of NeuroD-deficient neuronal death has not been investigated. It was hypothesized that NeuroD-dependent neuronal death occurs through a Bax-dependent apoptotic pathway. Based on this hypothesis, this study attempted to rescue neuronal cell death by deleting the Bax gene in NeuroD null mice to investigate the role of NeuroD in surviving neurons. The NeuroD and Bax double null mice displayed a decrease in the number of apoptotic cells in the hippocampus and the cerebellum and the rescue of vestibulocochlear ganglion (VCG) neurons that failed to migrate and innervate. In addition, at E13.5, the NeuroD^−/−Bax^−/− VCG neurons failed to express TrkB and TrkC, which are known to be essential for the survival of those neurons. These data suggest that neuronal death in NeuroD null mice is mediated by Bax-dependent apoptosis and that NeuroD is required for the migration of VCG neurons. Finally, these data show that TrkB and TrkC expression in E13.5 VCG neurons requires NeuroD and that TrkB and TrkC expression may be necessary for the normal migration and innervations of those neurons.     

Motoneuron programmed cell death in response to proBDNF

Motoneurons (MN) as well as most neuronal populations undergo a temporally and spatially specific period of programmed cell death (PCD). Several factors have been considered to regulate the survival of MNs during this period, including availability of muscle-derived trophic support and activity. The possibility that target-derived factors may also negatively regulate MN survival has been considered, but not pursued. Neurotrophin precursors, through their interaction with p75(NTR) and sortilin receptors have been shown to induce cell death during development and following injury in the CNS. In this study, we find that muscle cells produce and secrete proBDNF. ProBDNF through its interaction with p75(NTR) and sortilin, promotes a caspase-dependent death of MNs in culture. We also provide data to suggest that proBDNF regulates MN PCD during development in vivo.     

Prevention of activity-dependent neuronal death: Vasoactive intestinal polypeptide stimulates astrocytes to secrete the thrombin-inhibiting neurotrophic serpin,protease nexin I

Neuronal cell death occurs as a programmed, naturally occurring mechanism and is the primary regressive event in central nervous system development. Death of neurons also occurs on an injury-induced basis after trauma and in human neurodegenerative diseases. Classical neurotrophic factors can reverse this phenomenon in experimental models prompting initiation of clinical trials in conditions such as amyotrophic lateral sclerosis and Alzheimer's disease. The glial-derived protease nexin I (PNI), a known promoter of neurite outgrowth in cell culture and a potent inhibitor of serine proteases, also enhances neuronal cell survival. PNI, in nanomolar concentrations, rescues spinal cord motor neurons from both naturally-occurring programmed cell death in the chick embryo as well as following injury in the neonatal mouse. The potent neuromodulator, vasoactive intestinal polypeptide (VIP), influences neuronal survival through glial-mediated factors and also induces secretion of newly synthesized astrocyte PNI. We now report that subnanomolar amounts of PNI enhance neuronal survival in mixed spinal cord cell culture, especially when neuronal cells were made electrically silent by administration of tetrodotoxin. The mediation of this effect is by inhibition of the multifunctional serine protease, thrombin, because hirudin, a thrombin-specific inhibitor, has the same effect. In addition, spinal cord neurons are exquisitely sensitive to thrombin because picomolar and lower levels of the coagulation factor causes neuronal death. Thus, PNI is an astrocyte-derived, thrombin-inhibiting, activity-dependent neurotrophic agent, enhanced secretion of which by VIP may be one approach to treat neurological disorders. © 1996 John Wiley & Sons, Inc.     

Bcl-2 family regulation of neuronal development and neurodegeneration

Neuronal cell death is a key feature of both normal nervous system development and neuropathological conditions. The Bcl-2 family, via its regulation of both caspase-dependent and caspase-independent cell death pathways, is uniquely positioned to critically control neuronal cell survival. Targeted gene disruptions of specific bcl-2 family members and the generation of transgenic mice overexpressing anti- or pro-apoptotic Bcl-2 family members have confirmed the importance of the Bcl-2 family in the nervous system. Data from studies of human brain tissue and experimental animal models of neuropathological conditions support the hypothesis that the Bcl-2 family regulates cell death in the mature nervous system and suggest that pharmacological manipulation of Bcl-2 family action could prove beneficial in the treatment of human neurological conditions such as stroke and neurodegenerative diseases.     

Persistent mitochondrial dysfunction and oxidative stress hinder neuronal cell recovery from reversible proteasome inhibition

Oxidative stress, proteasome impairment and mitochondrial dysfunction are implicated as contributors to ageing and neurodegeneration. Using mouse neuronal cells, we showed previously that the reversible proteasome inhibitor, [N-benzyloxycarbonyl-Ile-Glu (O-t-bytul)-Ala-leucinal; (PSI)] induced excessive reactive oxygen species (ROS) that mediated mitochondrial damage and a caspase-independent cell death. Herein, we examined whether this insult persists in neuronal cells recovering from inhibitor removal over time. Recovery from proteasome inhibition showed a time and dose-dependent cell death that was accompanied by ROS overproduction, caspase activation and mitochondrial membrane permeabilization with the subcellular relocalizations of the proapoptotic proteins, Bax, cytochrome c and the apoptosis inducing factor (AIF). Caspase inhibition failed to promote survival indicating that cell death was caspase-independent. Treatments with the antioxidant N-acetyl-cysteine (NAC) were needed to promote survival in cell recovering from mild proteasome inhibition while overexpression of the antiapoptotic protein Bcl-xL together with NAC attenuated cell death during recovery from potent inhibition. Whereas inhibitor removal increased proteasome function, cells recovering from potent proteasome inhibition showed excessive levels of ubiquitinated proteins that required the presence of NAC for their removal. Collectively, these results suggest that the oxidative stress and mitochondrial inhibition induced by proteasome inhibition persists to influence neuronal cell survival when proteasome function is restored. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Expression of apoptosis-inducing factor during early neural differentiation in the chick embryo

The distribution of apoptosis-inducing factor (AIF) immunoreactivity has been studied in the developing somites and nervous system of the chick embryo at embryonic day 4. AIF was found to be expressed primarily in the cytoplasm of cells of the ventral motor roots, at the points of their insertion into the neural tube. Co-localization of mitochondrial AIF immunoreactivity with the epitopes recognized by the monoclonal antibodies HNK-1 and 1E8 suggests that the AIF may be present in Schwann cell precursors as well as in nerve fibres. AIF immunoreactivity was not observed in either cell bodies in the neural tube, or in the somitic tissue surrounding the ventral roots. The results are consistent with the hypothesis that AIF may be involved in neuronal cell death during development, and that target-derived neuronal survival factors may act by controlling AIF activity.     

Peg3/Pw1 is a mediator between p53 and Bax in DNA damage-induced neuronal death

Neuronal cell death after DNA damage requires p53 and Bax, but the mechanism by which p53 activation leads to Bax translocation and cell death in neurons is not known. We report here that Peg3/Pw1 is up-regulated after DNA damage in cortical neurons in a p53-dependent manner. Overexpression of Peg3/Pw1 leads to decreased neuronal viability. The deleterious effect of Peg3/Pw1 on neuronal survival is abrogated by deletion of either p53 or Bax, indicating an essential role for both in Peg3/Pw1-mediated neuronal death. Moreover, overexpression of a Peg3/Pw1 dominant negative protein inhibits Bax translocation and neuronal cell death after DNA damage. These findings implicate Peg3/Pw1 as a mediator between p53 and Bax in a neuronal cell death pathway activated by DNA damage.     

Fibroblast growth factor-2 stimulates cell proliferation and decreases sexually dimorphic cell death in an avian song control nucleus

The neural system controlling song in birds has proven a useful model for investigating how neuronal growth and survival are regulated by sexual differentiation. The present study focused on one song control area, the robust nucleus of the archistriatum (RA), and explored how sex differences in the proliferation of putative glia cells in this region influence sexually dimorphic cell survival. In zebra finches (Poephila guttata), RA neuron death is much greater in young females than in males, resulting in marked sex differences in RA neuron number. An earlier study indicated that just prior to this sexually dimorphic neuron death the proliferation of putative glia cells within the RA is significantly lower in females than in males and remains so throughout the peak of neuron death. This suggests that sex differences in glia (or glia-derived molecules) might regulate neuron survival during sexual differentiation of the RA. To determine whether increased cell proliferation within the RA favors increased cell survival, we infused the potent glia mitogen fibroblast growth factor-2 (FGF-2) into the RA unilaterally in young females. We find that FGF-2 infusions increase RA cell proliferation and concurrently decrease the incidence of degenerating RA cells, results consistent with the hypothesis that glia exert neurotrophic effects on RA neurons during sexual differentiation. © 1998 John Wiley & Sons, Inc. J Neurobiol 37: 573–581, 1998     

Neuroprotection by selective neuronal deletion of Atg7 in neonatal brain injury

Perinatal asphyxia induces neuronal cell death and brain injury, and is often associated with irreversible neurological deficits in children. There is an urgent need to elucidate the neuronal death mechanisms occurring after neonatal hypoxia-ischemia (HI). We here investigated the selective neuronal deletion of the Atg7 (autophagy related 7) gene on neuronal cell death and brain injury in a mouse model of severe neonatal hypoxia-ischemia. Neuronal deletion of Atg7 prevented HI-induced autophagy, resulted in 42% decrease of tissue loss compared to wild-type mice after the insult, and reduced cell death in multiple brain regions, including apoptosis, as shown by decreased caspase-dependent and -independent cell death. Moreover, we investigated the lentiform nucleus of human newborns who died after severe perinatal asphyxia and found increased neuronal autophagy after severe hypoxic-ischemic encephalopathy compared to control uninjured brains, as indicated by the numbers of MAP1LC3B/LC3B (microtubule-associated protein 1 light chain 3)-, LAMP1 (lysosomal-associated membrane protein 1)-, and CTSD (cathepsin D)-positive cells. These findings reveal that selective neuronal deletion of Atg7 is strongly protective against neuronal death and overall brain injury occurring after HI and suggest that inhibition of HI-enhanced autophagy should be considered as a potential therapeutic target for the treatment of human newborns developing severe hypoxic-ischemic encephalopathy.     

Neurogenesis and cell death in olfactory epithelium

The olfactory epithelium (OE) of the mammal is uniquely suited as a model system for studying how neurogenesis and cell death interact to regulate neuron number during development and regeneration. To identify factors regulating neurogenesis and neuronal death in the OE, and to determine the mechanisms by which these factors act, investigators studied OE using two major experimental paradigms: tissue culture of OE; and ablation of the olfactory bulb or severing the olfactory nerve in adult animals, procedures that induce cell death and a subsequent surge of neurogenesis in the OE in vivo. These studies characterized the cellular stages in the olfactory receptor neuron (ORN) lineage, leading to the realization that at least three distinct stages of proliferating neuronal precursor cells are employed in generating ORNs. The identification of a number of factors that act to regulate proliferation and survival of ORNs and their precursors suggests that these multiple developmental stages may serve as control points at which cell number is regulated by extrinsic factors. In vivo surgical studies, which have shown that all cell types in the neuronal lineage of the OE undergo apoptotic cell death, support this idea. These studies, and the possible coregulation of neuronal birth and apoptosis in the OE, are discussed. © 1996 John Wiley & Sons, Inc.     

<Emphasis Type="Italic">S</Emphasis>-Nitrosylation Regulates Cell Survival and Death in the Central Nervous System

Nitric oxide (NO), which is produced from nitric oxide synthase, is an important cell signaling molecule that is crucial for many physiological functions such as neuronal death, neuronal survival, synaptic plasticity, and vascular homeostasis. This diffusible gaseous compound functions as an effector or second messenger in many intercellular communications and/or cell signaling pathways. Protein S-nitrosylation is a posttranslational modification that involves the covalent attachment of an NO group to the thiol side chain of select cysteine residues on target proteins. This process is thought to be very important for the regulation of cell death, cell survival, and gene expression in the central nervous system (CNS). However, there have been few reports on the role of protein S-nitrosylation in CNS disorders. Here, we briefly review specific examples of S-nitrosylation, with particular emphasis on its functions in neuronal cell death and survival. An understanding of the role and mechanisms underlying the effects of protein S-nitrosylation on neurodegenerative/neuroprotective events may reveal a novel therapeutic strategy for rescuing neurons in neurodegenerative diseases.     

Taxol protects against calcium-mediated death of differentiated rat pheochromocytoma cells

Elevated levels of intraneuronal calcium may contribute to neuronal death in both Alzheimer's disease and stroke. In part, this neuronal death may be due to calcium-induced disruption of microtubules and inhibition of axonal transport. Taxol stabilizes microtubules to disaggregation. To determine whether taxol could protect against calcium-mediated neuron cell death, a test system was established using a nerve growth factor-differentiated rat pheochromocytoma cell line (PC12 cells). PC12 cells were cultured with nerve growth factor to induce a neuronal phenotype. After 15 days, the cells were exposed to taxol, the calcium ionophore, A23187, or taxol plus ionophore for up to 24 h. Taxol alone reduced cell survival in a concentration dependent manner. At a concentration of 50 nM survival was reduced to between 63% and 84% of control after 4 h of exposure. The ionophore (1 μM) variably reduced cell survival to between 10 and 55% at 4h. However, when tacol was added to the ionophore the cell survival was significantly increased by 1.5 to 4-fold. The protective effect of taxol lasted up to 24h. We conclude that taxol has a protective effect on calcium-mediated neurotoxicity. Drugs targeting underlying cellular mechanisms involved in calcium-mediated neuronal death may lead to successful therapy for Alzheimer's disease and stroke.