Differential regulation of motor neuron survival and choline acetyltransferase expression following axotomy

Although it is well known that motor neuron survival following axotomy is enhanced with maturation, the ability of surviving neurons to express the cholinergic enzyme choline acetyltransferase (ChAT) following axotomy has not been closely examined. Moreover, the utility of the facial nucleus in studies of motoneuron response to injury and to trophic factors, coupled with the increasing importance of the mouse in gene targeting, compelled us to investigate the age dependence of neuronal survival and ChAT expression in the mouse facial nucleus following axotomy. We cut the facial nerve at postnatal day (P)4, 7, 14, 21, and 28 or in the adult and used Nissl staining and ChAT immunocytochemistry to quantitate survival and ChAT expression, respectively, following 1, 2, or 3 weeks' survival at each age. We confirm in this model that the rate and extent of motor neuron death following axotomy is reduced with increasing maturity. The surviving neurons maintain a high ChAT content through P21; however, axotomy from P28 through adulthood results in a striking reduction in ChAT immunoreactivity. That is, although axotomy at P21 results in 61% motor neuron survival, with virtually all of the surviving neurons being ChAT positive, axotomy in the adult results in 72% survival but only 9% of the neurons are ChAT positive. Thus, surviving motor neurons in the adult animals are only weakly cholinergic. These results indicate that a change in the regulation of ChAT expression occurs following P21 so that cell survival and enzyme levels are uncoupled. We suggest that the putative factor or factors that enhances motor neuron survival in maturity is not capable of maintaining ChAT expression. © 1995 John Wiley & Sons, Inc.     

Caspases in ischaemic brain injury and neurodegenerative disease

The importance of caspases in developmental neuronal death is well-established. Recent data provide compelling evidence of caspase activation after ischaemic brain injury. Caspase inhibitors reduce cell death in several models of ischaemic injury. This review summarizes our current understanding of caspase function in ischaemic brain injury and examines the accumulating evidence of caspase participation in several neurodegenerative diseases. The therapeutic consequences of caspase inhibitor treatment in reducing cell death after such injury are also discussed.     

TrkA mediates developmental sympathetic neuron survival in vivo by silencing an ongoing p75NTR-mediated death signal.

Developmental sympathetic neuron death is determined by functional interactions between the TrkA/NGF receptor and the p75 neurotrophin receptor (p75NTR). A key question is whether p75NTR promotes apoptosis by directly inhibiting or modulating TrkA activity, or by stimulating cell death independently of TrkA. Here we provide evidence for the latter model. Specifically, experiments presented here demonstrate that the presence or absence of p75NTR does not alter Trk activity or NGF- and NT-3-mediated downstream survival signaling in primary neurons. Crosses of p75NTR-/- and TrkA-/- mice indicate that the coincident absence of p75NTR substantially rescues TrkA-/- sympathetic neurons from developmental death in vivo. Thus, p75NTR induces death regardless of the presence or absence of TrkA expression. These data therefore support a model where developing sympathetic neurons are "destined to die" by an ongoing p75NTR-mediated apoptotic signal, and one of the major ways that TrkA promotes neuronal survival is by silencing this ongoing death signal.     

Neural activity and survival in the developing nervous system

Recent evidence suggests that blockade of normal excitation in the immature nervous system may have profound effects on neuronal survival during the period of natural cell death. Cell loss following depression of electrical activity in the central nervous system (CNS) may explain the neuropsychiatric deficits in humans exposed to alcohol or other CNS depressants during development. Thus, understanding the role of electrical activity in the survival of young neurons is an important goal of modern basic and clinical neuroscience. Here we review the evidence from in vivo and in vitro model systems that electrical activity participates in promoting neuronal survival. We discuss the potential role of moderate elevations of intracellular calcium in promoting survival, and we address the possible ways in which activity and conventional trophic factors may interact.     

自噬在细胞存活和死亡中的作用

自噬是亚细胞膜结构发生动态变化并经溶酶体介导对细胞内蛋白质和细胞器降解的过程.通过平衡细胞合成和分解代谢,自噬稳定细胞内环境,维持细胞的存活.然而,过度自噬可导致细胞发生Ⅱ型程序性细胞死亡.自噬与凋亡在细胞死亡过程中的关系十分密切.本文对自噬的过程及其在细胞存活和死亡中的作用作一综述.     

Biochemical characterization of programmed cell death in NGF-deprived sympathetic neurons

Young sympathetic neurons die when deprived of nerve growth factor (NGF). Under such circumstances, cell death is appropriate to the developing nervous system and requires RNA and protein synthesis. We have hypothesized the existence of an endogenous death program within neurons that is suppressed by trophic factors. The extent and timing of required changes in the synthetic events that comprise the death program are unknown. In an effort to characterize the biochemical events that mediate the death program further, we performed several experiments on embryonic rat sympathetic neurons in vitro. The death program was blocked with cycloheximide when total protein synthesis was inhibited ≥80%. When protein synthesis was inhibited within 22 ± 4 h of NGF deprivation, death was prevented in half the neurons. Hence, we define the commitment point for protein synthesis to be 22 ± 4 h. Analogously, the commitment point for RNA synthesis was 26 ± 4 h and that for NGF rescue, 24 ± 4 h. We tested the ability of a wide variety of chemicals to interfere with the death program. Most compounds tested were unable to prevent neuronal death. Some treatments, however, did save NGF-deprived neurons and were subsequently characterized. These included ultraviolet light and agents that raise intracellular concentrations of cAMP. Finally, we looked for the neuronal expression in vitro and in vivo of genes that have been associated with programmed death in other cell types, including TRPM-2/SGP-2, polyubiquitin, TGFβ-1, c-fos, and c-myc. None of these genes showed significant activation associated with neuronal death. © 1992 John Wiley & Sons, Inc.     

CEP-1347/KT7515 prevents motor neuronal programmed cell death and injury-induced dedifferentiation in vivo

CEP-1347, also known as KT7515, a derivative of a natural product indolocarbazole, inhibited motor neuronal death in vitro, inhibited activation of the stress-activated kinase JNK1 (c-jun NH terminal kinase) in cultured spinal motor neurons, but had no effect on the mitogen-activated protein kinase ERK1 in these cells. Results reported here profile the functional activity of CEP-1347/KT7515 in vivo in models of motor neuronal death or dedifferentiation. Application of CEP-1347/KT7515 to the chorioallantoic membrane of embryonic chicks rescued 40% of the lumbar motor neurons that normally die during the developmental period assessed. Peripheral administration of low doses (0.5 and 1 mg/kg daily) of CEP-1347/KT7515 reduced death of motor neurons of the spinal nucleus of the bulbocavernosus in postnatal female rats, with efficacy comparable to testosterone. Strikingly, daily administration of CEP-1347/KT7515 during the 4-day postnatal window of motor neuronal death resulted in persistent long-term motor neuronal survival in adult animals that received no additional CEP-1347/KT7515. In a model of adult motor neuronal dedifferentiation following axotomy, local application of CEP-1347/KT7515 to the transected hypoglossal nerve substantially reduced the loss of choline acetyl transferase immunoreactivity observed 7 days postaxotomy compared to untreated animals. Results from these experiments demonstrate that a small organic molecule that inhibits a signaling pathway associated with stress and injury also reduces neuronal death and degeneration in vivo. © 1998 John Wiley & Sons, Inc. J Neurobiol 35: 361–370, 1998     

Human eosinophils: Apoptosis versus survival in the mediation of inflammation

Eosinophil-derived mediators are thought to make a major contribution to the inflammation underlying a number of allergic diseases, most notably asthma. The toxic potential of eosinophils at tissue sites of inflammation might be limited if they were cleared by the process of programmed cell death or apoptosis. In this review we have examined the relationship between the signal transduction pathways important in controlling cytokine-induced prolonged survival and the mechanisms responsible for the induction and control of apoptosis in the eosinophil. A greater understanding of these processes might result in the development of novel therapeutic agents which would promote the safe and rapid removal by apoptosis of this important pro-inflammatory cell.     

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.     

Increases in tumor necrosis factor-alpha following transient global cerebral ischemia do not contribute to neuron death in mouse hippocampus

The actions of tumor necrosis factor-alpha (TNF-alpha) produced by resident brain cells and bone marrow-derived cells in brain following a transient global ischemia were evaluated. In wild-type mice (C57Bl/6J) following 20 min ischemia with bilateral common carotid artery occlusion (BCCAo), TNF-alpha mRNA expression levels in the hippocampus were significantly increased at 3 h and 36 h and exhibited a biphasic expression pattern. There were no hippocampal TNF-alpha mRNA expression levels at early time points in either wild-type mice bone marrow transplanted (BMT)-chimeric-TNF-alpha gene-deficient (T/W) or TNF-alpha gene-deficient mice BMT-TNF-alpha gene-deficient mice (T/T), although TNF-alpha mRNA levels were detectable in T/W BMT mice at 36 h. Histopathological findings showed no intergroup differences between wild-type and TNF-alpha gene-deficient mice at 4 and 7 days after transient ischemia. In addition, nuclear factor-kappaB (NF-kappaB) was activated within 12 h after global cerebral ischemia, but electrophoretic mobility shift assays (EMSA) showed no intergroup differences between wild type and TNF-alpha gene-deficient mice. In summary, early hippocampal TNF-alpha mRNA expression may not be related to bone marrow-derived cells, and secondary TNF-alpha expression as early as 36 h after ischemia probably resulted mainly from endogenous brain cells and possibly a few bone marrow-derived cells. Although we cannot exclude the possibility of the TNF-alpha contribution to the physiologic changes of hippocampus after transient global ischemia, these results indicate that TNF-alpha does not influence the morphological changes of the hippocampal neurons under our study condition.     

Isoform- and subcellular fraction-specific differences in hippocampal 14-3-3 levels following experimentally evoked seizures and in human temporal lobe epilepsy

14-3-3 proteins are a family of signaling molecules involved in diverse cellular functions, which can mediate anti-apoptotic effects. Seizure-induced neuronal death may involve programmed (apoptotic) cell death pathways and is associated with a decline in brain 14-3-3 levels. Presently, we investigated the subcellular localization and effects of seizures on isoforms of 14-3-3 in rat hippocampus, and contrasted these to findings in human temporal lobe epilepsy (TLE). All brain isoforms of 14-3-3 were detected in the cytoplasmic compartment of rat hippocampus, while 14-3-3gamma and -zeta were also present in mitochondrial and microsome-enriched fractions. Focally evoked seizures in rats significantly reduced 14-3-3gamma levels within the microsome-enriched compartment at 4 h, with similar responses for 14-3-3zeta, while cytoplasm-localized 14-3-3beta, -epsilon and -eta remained unchanged. Analysis of human autopsy control hippocampus revealed similar 14-3-3 isoform expression profiles. In TLE samples, the microsome-enriched fraction also showed differences, but here 14-3-3epsilon and -zeta levels were higher than controls. TLE sample 14-3-3 isoform abundance within the cytoplasmic fraction was not different to controls. This study defines the subcellular localization of 14-3-3 isoforms in rat and human hippocampus and identifies the microsome-enriched fraction as the main site of altered 14-3-3 levels in response to acute prolonged and chronic recurrent seizures.     

Sexually dimorphic neuron addition to an avian song-control region is not accounted for by sex differences in cell death

Only male zebra finches sing, and several brain regions implicated in song behavior exhibit marked sex differences in neuron number. In one region, the high vocal center (HVC), this dimorphism develops because the incorporation of new neurons is greater in males than in females during the first several weeks after hatching. Although estrogen (E₂) exposure stimulates neuron addition in females, it is not known where (E₂) acts, or to what extent sexual differentiation influences the production, specification, or survival of HVC neurons. In the present study we first reassessed sex and (E₂)-induced differences in cell degeneration within the HVC using the TUNEL technique to identify cells undergoing DNA fragmentation indicative of apoptosis. HVC neuron number, as well as the density and number of TUNEL-labeled and pyknotic cells within the HVC were measured in normal 20- and 30-day-old males and females, and in 30-day-old females implanted with E₂ on posthatch day 18. Although HVC neuron number was greater in males than in females, and was masculinized in E₂ females, no group differences were evident in the absolute number of dying cells. These results indicate that sex differences in cell survival within the HVC do not entirely account for sexually dimorphic neuron addition to this region. Rather, sexual differentiation acts on some HVC neurons before they complete their migration and/or early differentiation. Although the migratory route of HVC neurons is not known, a large number of E₂ receptor-containing cells (ER cells) reside just ventromedial to the HVC and adjacent to the proliferative ventricular zone. Next, we investigated whether these ER cells contribute to early-arising sex differences in HVC neuron addition. By combining [³H] thymidine autoradiography with immunocytochemistry for ERs, we first established that ER-expressing cells are not generated during posthatch sexually dimorphic HVC neuron addition, and thus are not young HVC neurons that transiently express ERs during their migration. Furthermore, in 25-day-old birds we found no sex difference in the density of pyknotic cells among this group of ER cells, suggesting that these cells do not promote the differential survival of HVC neuronal precursors migrating through this region. Rather, ER cells or other cell populations may establish sex differences in HVC neuron number by creating dimorphisms in cellular specification. © 1997 John Wiley & Sons, Inc. J Neurobiol 33: 61–71, 1997     

Intranuclear localization of apoptosis-inducing factor (AIF) and large scale DNA fragmentation after traumatic brain injury in rats and in neuronal cultures exposed to peroxynitrite

Programmed cell death occurs after ischemic, excitotoxic, and traumatic brain injury (TBI). Recently, a caspase-independent pathway involving intranuclear translocation of mitochondrial apoptosis-inducing factor (AIF) has been reported in vitro; but whether this occurs after acute brain injury was unknown. To address this question adult rats were sacrificed at various times after TBI. Western blot analysis on subcellular protein fractions demonstrated intranuclear localization of AIF in ipsilateral cortex and hippocampus at 2-72 h. Immunocytochemical analysis showed AIF labeling in neuronal nuclei with DNA fragmentation in the ipsilateral cortex and hippocampus. Immunoelectronmicroscopy verified intranuclear localization of AIF in hippocampal neurons after TBI, primarily in regions of euchromatin. Large-scale DNA fragmentation ( approximately 50 kbp), a signature event in AIF-mediated cell death, was detected in ipsilateral cortex and hippocampi by 6 h. Neuron-enriched cultures exposed to peroxynitrite also demonstrated intranuclear AIF and large-scale DNA fragmentation concurrent with impaired mitochondrial respiration and cell death, events that are inhibited by treatment with a peroxynitrite decomposition catalyst. Intranuclear localization of AIF and large-scale DNA fragmentation occurs after TBI and in neurons under conditions of oxidative/nitrosative stress, providing the first evidence of this alternative mechanism by which programmed cell death may proceed in neurons after brain injury.     

Merosin (laminin-2/4)-driven survival signaling: complex modulations of Bcl-2 homologs

We have shown previously that the promotion of myofiber survival by the basement membrane component merosin (laminin-2 [alpha2beta1gamma1]/laminin-4 [alpha2beta2gamma1]) is dependent on the activity of the tyrosine kinase Fyn, whereas myofiber anoikis induced by merosin deficiency is dependent on the stress-activated protein kinase p38alpha. To further understand such merosin-driven survival signaling, we analyzed the expression of five Bcl-2 homologs (Bcl-2, Bcl-X(L), Bax, Bak, Bad) and one non-homologous associated molecule (Bag-1) in normal and merosin-deficient myotubes, with or without pharmacological inhibitors for Fyn and p38. Herein, we report that (1) merosin deficiency induces anoikis and causes decreased Bcl-2, Bcl-X(L), and Bag-1 levels, increased Bax and Bak levels, and decreased Bad phosphorylation; (2) Bcl-2, Bcl-X(L), Bag-1, and Bad phosphorylation are also decreased in anoikis-dying, Fyn-inhibited myotubes; (3) the inhibition of p38alpha in Fyn-inhibited and/or merosin-deficient myotubes protects against anoikis and increases Bcl-2 levels above normal, in addition to restoring Bad phosphorylation and Bag-1 levels to normal; (4) the overexpression of merosin in deficient myotubes also rescues from anoikis and increases Bcl-2 levels and Bad phosphorylation above normal, in addition to restoring Bcl-X(L), Bag-1, Bax, and Bak levels to normal; and (5) Bcl-2 overexpression is sufficient to rescue merosin-deficient myotubes from anoikis, even though the expression/phosphorylation levels of the other homologs analyzed are not restored to normal. These results indicate that merosin-driven myofiber survival signaling affects complex, differential modulations of individual Bcl-2 homologs. These further suggest that Bcl-2 can play a major role in suppressing myofiber anoikis.     

Minocycline inhibits apoptotic cell death via attenuation of TNF-alpha expression following iNOS/NO induction by lipopolysaccharide in neuron/glia co-cultures

We attempted to ascertain the neuroprotective effects and mechanisms of minocycline in inflammatory-mediated neurotoxicity using primary neuron/glia co-cultures treated with lipopolysaccharide (LPS). Neuronal cell death was induced by treatment with LPS for 48 h, and the cell damage was assessed using lactate dehydrogenase (LDH) assays and by counting microtubule-associated protein-2 (MAP-2) positive cells. Through terminal transferase deoxyuridine triphosphate-biotin nick end labeling (TUNEL)-staining and by measuring caspase-3 activity, we found that LPS-induced neuronal cell death was mediated by apoptosis. We determined that pre-treatment with minocycline significantly inhibited LPS-induced neuronal cell death. In addition, LPS induced inducible nitric oxide synthase (iNOS) expression significantly, resulting in nitric oxide (NO) production within glial cells, but not in neurons. Both nitric oxide synthase (NOS) inhibitors (N(G)-monomethyl-L-arginine monoacetate (L-NMMA) and S-methylisothiourea sulfate (SMT)) and minocycline inhibited iNOS expression and NO release, and increased neuronal survival in neuron/glia co-cultures. Pre-treatment with minocycline significantly inhibited the rapid and extensive production of tumor necrosis factor-alpha (TNF-alpha) mediated by LPS in glial cells. We also determined that the signaling cascade of LPS-mediated iNOS induction and NO production was mediated by TNF-alpha by using neutralizing antibodies to TNF-alpha. Consequently, our results show that the neuroprotective effect of minocycline is associated with inhibition of iNOS induction and NO production in glial cells, which is mediated by the LPS-induced production of TNF-alpha.     

Protocol for Culturing Sympathetic Neurons from Rat Superior Cervical Ganglia (SCG)

The superior cervical ganglia (SCG) in rats are small, glossy, almond-shaped structures that contain sympathetic neurons. These neurons provide sympathetic innervations for the head and neck regions and they constitute a well-characterized and relatively homogeneous population (4). Sympathetic neurons are dependent on nerve growth factor (NGF) for survival, differentiation and axonal growth and the wide-spread availability of NGF facilitates their culture and experimental manipulation (2, 3, 6). For these reasons, cultured sympathetic neurons have been used in a wide variety of studies including neuronal development and differentiation, mechanisms of programmed and pathological cell death, and signal transduction (1, 2, 5, and 6). Dissecting out the SCG from newborn rats and culturing sympathetic neurons is not very complicated and can be mastered fairly quickly. In this article, we will describe in detail how to dissect out the SCG from newborn rat pups and to use them to establish cultures of sympathetic neurons. The article will also describe the preparatory steps and the various reagents and equipment that are needed to achieve this.     

A Role for phosphoinositide 3-kinase in the control of cell division and survival during retinal development

Neurogenesis in the retina requires the concerted action of three different cellular processes: proliferation, differentiation, and apoptosis. Class IA phosphoinositide 3-kinase (PI3K) is a heterodimer composed of a p85 regulatory and a p110 catalytic subunit. p110alpha has been shown to regulate cell division and survival. Little is known of its function in development, however, as p110alpha knockout mice exhibit CNS defects, but death at early embryonic stages impairs further study. Here, we examine the role of PI3K in mouse retina development by expressing an activating form of PI3K regulatory subunit, p65(PI3K), as a transgene in the retina. Mice expressing p65(PI3K) showed severely disrupted retina morphogenesis, with ectopic cell masses in the neuroepithelium that evolved into infoldings of adult retinal cell layers. These changes correlated with an altered cell proliferation/cell death balance at early developmental stages. Nonetheless, the most affected cell layer in adult retina was that of photoreceptors, which correlated with selectively increased survival of these cells at developmental stages at which cell division has ceased. These results demonstrate the relevance of accurate PI3K regulation for normal retinal development, supporting class IA PI3K involvement in induction of cell division at early stages of neurogenesis. These data also show that, even after cell division decline, PI3K activation mediates survival of differentiated neurons in vivo.