Glutamate-Treated Rat Cortical Neuronal Cultures Die in a Way Different from the Classical Apoptosis Induced by Staurosporine

The alkaloid protein kinase inhibitor staurosporine induced neuronal cell death with both the morphological and the biochemical characteristics of apoptosis. The punctate chromatin associated with apoptosis with retention of plasma membrane integrity was observed in neurons identified by colocalization of NeuN staining. Such cells had DNA fragmentation visualized byin situend-labeling which was seen as a laddered pattern upon gel electrophoresis. In contrast cells treated with glutamate did not exhibit either of these morphological or biochemical hallmarks of apoptosis. Instead a much smaller and more compact pyknotic structure was observed associated with smeared DNA fragmentation patterns. A confocal time-lapse study of the appearance of the morphological changes in individual nuclei after staurosporine treatment showed collapse into punctate chromatin over a period of 10 min. In contrast, the collapse into small pyknotic nuclei after glutamate treatment was at least 10 times slower. It is concluded that excitotoxicity produced by glutamate did not induce cell death by an apoptotic mechanism in cultured cortical neurons.     

Non-apoptogenic killing of hela cervical carcinoma cells after short exposure to the alkylating agent N-methyl-N'-nitro-N-nitrosoguanidine (MNNG)

We examined the action of N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) on HeLa cells and compared it with that of cisplatin (CP). MNNG directly killed a substantial number of cells within 1 hour and resulted in strong DNA-damage as evidenced by Comet measurements. Despite appearance of DNA lesions, p53 protein was not activated. Analysis of HeLa cells treated with MNNG for 1h, 3h and 6h by flow cytometry and by Hoechst staining did not reveal any sub-G(1) cell population and chromatin condensation/fragmentation characteristic for apoptosis, respectively. Also, no biochemical changes typical for apoptosis such as activation of caspase-3 or release of cytochrome C from mitochondria were detected. Inactivation of PARP-1 reduced the direct cytotoxicity exerted by MNNG. Our results showing that despite appearance of severe DNA lesions after short exposure of HeLa cells to MNNG neither activation of p53 response nor induction of apoptosis occurred implicate that generation of strong DNA damage is not sufficient to stabilize p53 protein in HeLa cells. Our data unequivocally show that the conscientious determination of the type of cell death induced by genotoxic agents is necessary. The assessment of the changes based on at least a few independent criteria is required to discriminate between apoptosis and necrosis. Since the alkylating agents generate DNA strand breaks, the recruitment of methods based on determination of DNA cleavage such as DNA ladder or TUNEL assay for evaluation of apoptosis is not adequate.     

Neuronal death is an active, caspase-dependent process after moderate but not severe DNA damage

Mild insults to neurons caused by ischemia or glutamate induce apoptosis, whereas severe insults induce non apoptotic death, such as necrosis. The molecular targets that are damaged by these insults and ultimately induce cell death are not fully established. To determine if DNA damage can induce apoptotic or non apoptotic death depending on the severity, neurons were treated with up to 128 Gy of ionizing radiation. Such treatment induced a dose-related increase in DNA single-strand breaks but no immediate membrane disruption or lipid peroxidation. Following moderate doses of < or = 32 Gy, neuronal death had many characteristics of apoptosis including nuclear fragmentation and DNA laddering. Nuclear fragmentation and membrane breakdown after moderate DNA damage could be blocked by inhibition of active protein synthesis with cycloheximide and by inhibition of caspases. In contrast, cell death after doses of > 32 Gy was not blocked by cycloheximide or caspase inhibitors, and membrane breakdown occurred relatively early in the cell death process. These data suggest that cell death after high dose irradiation and severe DNA damage can occur by non apoptotic mechanisms and that blocking apoptotic pathways may not prevent death after severe damage.     

Chk1 has an essential role in the survival of differentiated cortical neurons in the absence of DNA damage

Neuronal death in the central nervous system contributes to the development of age-related neurodegeneration. The ATR/Chk1 pathway appears to function neuroprotectively to prevent DNA damage induced by cytotoxic agents. Here, we examine the function of Chk1 on cell viability of cortical neurons in the absence of additional DNA damaging stimuli. The Chk1-specific inhibitor, UCN-01, and the ATR inhibitor, Caffeine, cause neuronal apoptosis in differentiated neurons in the absence of additional treatment, whereas inhibition of ATM or Chk2, does not. UCN-01 treatment increased the detection of γ-H2AX phosphorylation, DNA strand breaks, and an activated p53-dependent DNA damage response (DDR), suggesting that Chk1 normally helps to maintain genomic stability. UCN-01 treatment also enhanced the apoptosis seen in neurons treated with DNA damaging agents, such as camptothecin (CPT). Our results indicate that Chk1 is essential for neuronal survival, and perturbation of this pathway increases a cell’s sensitivity to naturally accumulating DNA damage.     

Glutamate Uptake Impairment and Neuronal Damage in Young and Aged Rats In Vivo

Abstract: The extracellular concentration of glutamate increases during hypoxia/ischemia probably due to deficient uptake. Glutamate might contribute to neuronal damage associated with this disorder and to neurodegeneration during aging. In the present study, we have tested the effect of two inhibitors of glutamate transport, l - trans -pyrrolidine-2,4-dicarboxylate and dihydrokainate, on the extracellular levels of glutamate and on neuronal damage, which was quantitatively studied by image analysis of histological brain sections. Drugs were administered by microdialysis and glutamate concentration was determined by HPLC in the striatum and the hippocampus of 3-month-old and 22–24-month-old rats. In both regions studied, the basal concentration of extracellular glutamate was higher in aged than in young rats. Pyrrolidine dicarboxylate induced a substantial elevation of extracellular glutamate in both regions, and although this increase was almost twofold higher in old than in young animals, no neuronal damage was observed. In contrast, dihydrokainate had a poor effect on glutamate levels, but induced clear neuronal damage in the striatum and the hippocampus in both groups of rats. The present results suggest that age appears not to be a significant factor in the sensitivity of neurons to the toxic effect of extracellular glutamate increase via blockade of its transport system.     

Role of DNA-dependent protein kinase in neuronal survival

DNA-dependent protein kinase (DNA-PK) is a DNA repair enzyme composed of a DNA-binding component called Ku70/80 and a catalytic subunit called DNA-PKcs. Many investigators have utilized DNA-PKcs-deficient cells and cell lines derived from severe combined immunodeficiency (scid) mice to study DNA repair and apoptosis. However, little is known about the CNS of these mice. This study was carried out using primary neuronal cultures derived from the cerebral hemispheres of new-born wild-type and scid mice to investigate the effects of loss of DNA-PK function on neuronal maturation and survival. Purified neuronal cultures developed comparably in terms of neurite formation and expression of neuronal markers, but scid cultures showed a significant increase in the percentage of dying cells. Furthermore, when apoptosis was induced by staurosporine, scid neurons died more rapidly and in higher numbers. Apoptotic scid neurons exhibited nuclear condensation, DNA fragmentation and caspase-3 activation, but treatment with the general caspase inhibitor, N-benzyloxycarbonyl-Val-Ala-Asp-(O-methyl) fluoromethyl ketone did not prevent staurosporine-induced apoptosis. We conclude that a DNA-PK deficiency in cultured scid neurons may cause an accumulation of DNA damage and increased susceptibility to caspase-independent forms of programmed cell death.     

Adenovirus-Mediated Gene Transfer of Inhibitors of Apoptosis Proteins Delays Apoptosis in Cerebellar Granule Neurons

Abstract : The inhibitor of apoptosis (IAP) family of anti-apoptotic genes, originally discovered in baculovirus, exists in animals ranging from insects to humans. Here, we investigated the ability of IAPs to suppress cell death in both a neuronal model of apoptosis and excitotoxicity. Cerebellar granule neurons undergo apoptosis when switched from 25 to 5 m M potassium, and excitotoxic cell death in response to glutamate. We examined the endogenous expression of four members of the IAP family, X chromosome-linked IAP (XIAP), rat IAP1 (RIAP1), RIAP2, and neuronal apoptosis inhibitory protein (NAIP), by semiquantitative reverse PCR and immunoblot analysis in cultured cerebellar granule neurons. Cerebellar granule neurons express significant levels of RIAP2 mRNA and protein, but expression of RIAP1, NAIP, and XIAP was not detected. RIAP2 mRNA content and protein levels did not change when cells were switched from 25 to 5 m M potassium. To determine whether ectopic expression of IAP influenced neuronal survival after potassium withdrawal or glutamate exposure, we used recombinant adenoviral vectors to target XIAP, human IAP1 (HIAP1), HIAP2, and NAIP into cerebellar granule neurons. We demonstrate that forced expression of IAPs efficiently blocked potassium withdrawal-induced N -acetly-Asp-Glu-Val-Asp-specific caspase activity and reduced DNA fragmentation. However, neurons were only protected from apoptosis up to 24 h after potassium withdrawal, not at later time points suggesting that IAPS delay but do not block apoptosis in cerebellar granule neurons. In contrast, treatment with 100 μ M or 1 m M glutamate did not induce caspase activity and adenoviral-mediated expression of IAPs had no influence on subsequent excitotoxic cell death.     

Postmitotic neurons develop a p21‐dependent senescence‐like phenotype driven by a DNA damage response

In senescent cells, a DNA damage response drives not only irreversible loss of replicative capacity but also production and secretion of reactive oxygen species (ROS) and bioactive peptides including pro‐inflammatory cytokines. This makes senescent cells a potential cause of tissue functional decline in aging. To our knowledge, we show here for the first time evidence suggesting that DNA damage induces a senescence‐like state in mature postmitotic neurons in vivo. About 40–80% of Purkinje neurons and 20–40% of cortical, hippocampal and peripheral neurons in the myenteric plexus from old C57Bl/6 mice showed severe DNA damage, activated p38MAPkinase, high ROS production and oxidative damage, interleukin IL‐6 production, heterochromatinization and senescence‐associated β‐galactosidase activity. Frequencies of these senescence‐like neurons increased with age. Short‐term caloric restriction tended to decrease frequencies of positive cells. The phenotype was aggravated in brains of late‐generation TERC?/? mice with dysfunctional telomeres. It was fully rescued by loss of p21(CDKN1A) function in late‐generation TERC?/?CDKN1A?/? mice, indicating p21 as the necessary signal transducer between DNA damage response and senescence‐like phenotype in neurons, as in senescing fibroblasts and other proliferation‐competent cells. We conclude that a senescence‐like phenotype is possibly not restricted to proliferation‐competent cells. Rather, dysfunctional telomeres and/or accumulated DNA damage can induce a DNA damage response leading to a phenotype in postmitotic neurons that resembles cell senescence in multiple features. Senescence‐like neurons might be a source of oxidative and inflammatory stress and a contributor to brain aging.     

Long-term culture of mouse cortical neurons as a model for neuronal development, aging, and death

A long-term cell culture system was used to study maturation, aging, and death of cortical neurons. Mouse cortical neurons were maintained in culture in serum-free medium (Neurobasal supplemented with B27) for 60 days in vitro (DIV). The levels of several proteins were evaluated by immunoblotting to demonstrate that these neurons matured by developing dendrites and synapses and remained continuously healthy for 60 DIV. During their maturation, cortical neurons showed increased or stable protein expression of glycolytic enzyme, synaptophysin, synapsin IIa, alpha and beta synucleins, and glutamate receptors. Synaptogenesis was prominent during the first 15 days and then synaptic markers remained stable through DIV60. Very early during dendritic development at DIV3, beta-synuclein (but not alpha-synuclein) was localized at the base of dendritic growth cones identified by MAP2 and alpha-amino-3-hydroxy-5-methyl-4-isoxazole (AMPA) receptor GluR1. In mature neurons, alpha and beta synucleins colocalized in presynaptic axon terminals. Expression of N-methyl-D-aspartate (NMDA) and AMPA receptors preceded the formation of synapses. Glutamate receptors continued to be expressed strongly through DIV60. Cortical neurons aging in vitro displayed a complex profile of protein damage as identified by protein nitration. During cortical neuron aging, some proteins showed increased nitration, while other proteins showed decreased nitration. After exposure to DNA damaging agent, young (DIV5) and old (DIV60) cortical neurons activated apoptosis mechanisms, including caspase-3 cleavage and poly(ADP)-ribose polymerase inactivation. We show that cultured mouse cortical neurons can be maintained for long term. Cortical neurons display compartmental changes in the localization of synucleins during maturation in vitro. These neurons sustain protein nitration during aging and exhibit age-related variations in the biochemistry of neuronal apoptosis.     

Survival and proliferation of cells expressing caspase-uncleavable Poly(ADP-ribose) polymerase in response to death-inducing DNA damage by an alkylating agent

To determine whether caspase-3-induced cleavage of poly(ADP-ribose) polymerase (PARP), a DNA damage-sensitive enzyme, alters the balance between survival and death of the cells following DNA damage, we created stable cell lines that express either caspase-uncleavable mutant or wild type PARP in the background of PARP (-/-) fibroblasts. The survival and apoptotic responses of these cells were compared after exposure to N-methyl-N'-nitro-N-nitrosoguanidine (MNNG), a DNA-damaging agent that activates PARP, or to tumor necrosis factor-alpha, which causes apoptosis without initial DNA damage. In response to MNNG, the cells with caspase-uncleavable PARP were very resistant to loss of viability or induction of apoptosis. Most significantly, approximately 25% of these cells survived and retained clonogenicity at a level of DNA damage that eliminated the cells with wild type PARP or PARP (-/-) cells. Expression of caspase-uncleavable PARP could not protect the cells from death induced by tumor necrosis factor, although there was a slower progression of apoptotic events in these cells. Therefore, one of the functions for cleavage of PARP during apoptosis induced by alkylating agents is to prevent survival of the extensively damaged cells.     

Calcium/calmodulin-dependent protein kinase II (CaMKII) inhibition induces neurotoxicity via dysregulation of glutamate/calcium signaling and hyperexcitability

Aberrant glutamate and calcium signalings are neurotoxic to specific neuronal populations. Calcium/calmodulin-dependent kinase II (CaMKII), a multifunctional serine/threonine protein kinase in neurons, is believed to regulate neurotransmission and synaptic plasticity in response to calcium signaling produced by neuronal activity. Importantly, several CaMKII substrates control neuronal structure, excitability, and plasticity. Here, we demonstrate that CaMKII inhibition for >4 h using small molecule and peptide inhibitors induces apoptosis in cultured cortical neurons. The neuronal death produced by prolonged CaMKII inhibition is associated with an increase in TUNEL staining and caspase-3 cleavage and is blocked with the translation inhibitor cycloheximide. Thus, this neurotoxicity is consistent with apoptotic mechanisms, a conclusion that is further supported by dysregulated calcium signaling with CaMKII inhibition. CaMKII inhibitory peptides also enhance the number of action potentials generated by a ramp depolarization, suggesting increased neuronal excitability with a loss of CaMKII activity. Extracellular glutamate concentrations are augmented with prolonged inhibition of CaMKII. Enzymatic buffering of extracellular glutamate and antagonism of the NMDA subtype of glutamate receptors prevent the calcium dysregulation and neurotoxicity associated with prolonged CaMKII inhibition. However, in the absence of CaMKII inhibition, elevated glutamate levels do not induce neurotoxicity, suggesting that a combination of CaMKII inhibition and elevated extracellular glutamate levels results in neuronal death. In sum, the loss of CaMKII observed with multiple pathological states in the central nervous system, including epilepsy, brain trauma, and ischemia, likely exacerbates programmed cell death by sensitizing vulnerable neuronal populations to excitotoxic glutamate signaling and inducing an excitotoxic insult itself.     

Long‐term culture of mouse cortical neurons as a model for neuronal development,aging, and death

A long‐term cell culture system was used to study maturation, aging, and death of cortical neurons. Mouse cortical neurons were maintained in culture in serum‐free medium (Neurobasal supplemented with B27) for 60 days in vitro (DIV). The levels of several proteins were evaluated by immunoblotting to demonstrate that these neurons matured by developing dendrites and synapses and remained continuously healthy for 60 DIV. During their maturation, cortical neurons showed increased or stable protein expression of glycolytic enzyme, synaptophysin, synapsin IIa, α and β synucleins, and glutamate receptors. Synaptogenesis was prominent during the first 15 days and then synaptic markers remained stable through DIV60. Very early during dendritic development at DIV3, β‐synuclein (but not α‐synuclein) was localized at the base of dendritic growth cones identified by MAP2 and α‐amino‐3‐hydroxy‐5‐methyl‐4‐isoxazole (AMPA) receptor GluR1. In mature neurons, α and β synucleins colocalized in presynaptic axon terminals. Expression of N‐methyl‐D ‐aspartate (NMDA) and AMPA receptors preceded the formation of synapses. Glutamate receptors continued to be expressed strongly through DIV60. Cortical neurons aging in vitro displayed a complex profile of protein damage as identified by protein nitration. During cortical neuron aging, some proteins showed increased nitration, while other proteins showed decreased nitration. After exposure to DNA damaging agent, young (DIV5) and old (DIV60) cortical neurons activated apoptosis mechanisms, including caspase‐3 cleavage and poly(ADP)‐ribose polymerase inactivation. We show that cultured mouse cortical neurons can be maintained for long term. Cortical neurons display compartmental changes in the localization of synucleins during maturation in vitro. These neurons sustain protein nitration during aging and exhibit age‐related variations in the biochemistry of neuronal apoptosis. © 2002 Wiley Periodicals, Inc. J Neurobiol 51: 9–23, 2002     

Caspase-3 mediated neuronal death after traumatic brain injury in rats

During programmed cell death, activation of caspase-3 leads to proteolysis of DNA repair proteins, cytoskeletal proteins, and the inhibitor of caspase-activated deoxyribonuclease, culminating in morphologic changes and DNA damage defining apoptosis. The participation of caspase-3 activation in the evolution of neuronal death after traumatic brain injury in rats was examined. Cleavage of pro-caspase-3 in cytosolic cellular fractions and an increase in caspase-3-like enzyme activity were seen in injured brain versus control. Cleavage of the caspase-3 substrates DNA-dependent protein kinase and inhibitor of caspase-activated deoxyribonuclease and co-localization of cytosolic caspase-3 in neurons with evidence of DNA fragmentation were also identified. Intracerebral administration of the caspase-3 inhibitor N-benzyloxycarbonyl-Asp-Glu-Val-Asp-fluoromethyl ketone (480 ng) after trauma reduced caspase-3-like activity and DNA fragmentation in injured brain versus vehicle at 24 h. Treatment with N-benzyloxycarbonyl-Asp-Glu-Val-Asp-fluoromethyl ketone for 72 h (480 ng/day) reduced contusion size and ipsilateral dorsal hippocampal tissue loss at 3 weeks but had no effect on functional outcome versus vehicle. These data demonstrate that caspase-3 activation contributes to brain tissue loss and downstream biochemical events that execute programmed cell death after traumatic brain injury. Caspase inhibition may prove efficacious in the treatment of certain types of brain injury where programmed cell death occurs.     

JNK1 is inactivated during thiamine deficiency-induced apoptosis in human neuroblastoma cells

Thiamine deficiency results in selective neuronal damage. A number of mechanisms have been proposed to account for brain damage associated with thiamine deficiency and to account for the focal nature of the loss of neurons. One proposed mechanism is programmed cell death. We found efficient induction of apoptosis in human neuroblastoma cells when the cells were deprived of thiamine. Although extensive mitochondrial damage was seen, the release of cytochrome c was not the triggering mechanism for thiamine deficiency-induced apoptosis. Instead, the activity of the cJun amino terminal kinase Jnk1 was lost, and this loss correlated temporally with induction of apoptosis. The loss was specific for Jnk1; Jnk2/3 activity remained unchanged. Loss of Jnk1 activity was not found in lymphoblasts, a cell type that did not undergo apoptosis when deprived of thiamine. These findings suggest that thiamine deficiency results in a cellular stress that brings about the loss of Jnk1 activity and the loss of its function of protecting cells from programmed cell death. We postulate that focal sensitivity to thiamine deficiency results, in part, from specific neuronal cell types being susceptible to the inactivation of Jnk1 in response to depletion of cellular thiamine.     

缺血诱导体外培养海马神经元损伤机理的探讨

缺血性神经元损伤的机理是神经科学领域极为关注的问题。本实验用体外培养海马神经元建立缺血模型,通过检测神经元死亡率以及观察由缺血引起的神经细胞内生化代谢改变所致的神经元功能障碍,探讨缺血性神经元损伤的机理。结果发现缺血组、缺葡萄糖组神经元死亡率最高,且通过检测细胞膜表面磷脂酰丝氨酸的变化证实此二组神经元凋亡率也最高,进一步研究还发现缺血可引起神经元细胞骨架结构改变,使神经元功能遭到破坏,最终导致神经元不可逆损伤。     

DNA damage response and cellular senescence in tissues of aging mice

The impact of cellular senescence onto aging of organisms is not fully clear, not at least because of the scarcity of reliable data on the mere frequency of senescent cells in aging tissues. Activation of a DNA damage response including formation of DNA damage foci containing activated H2A.X (γ-H2A.X) at either uncapped telomeres or persistent DNA strand breaks is the major trigger of cell senescence. Therefore, γ-H2A.X immunohistochemistry (IHC) was established by us as a reliable quantitative indicator of senescence in fibroblasts in vitro and in hepatocytes in vivo and the age dependency of DNA damage foci accumulation in ten organs of C57Bl6 mice was analysed over an age range from 12 to 42 months. There were significant increases with age in the frequency of foci-containing cells in lung, spleen, dermis, liver and gut epithelium. In liver, foci-positive cells were preferentially found in the centrilobular area, which is exposed to higher levels of oxidative stress. Foci formation in the intestine was restricted to the crypts. It was not associated with either apoptosis or hyperproliferation. That telomeres shortened with age in both crypt and villus enterocytes, but telomeres in the crypt epithelium were longer than those in villi at all ages were confirmed by us. Still, there was no more than random co-localization between γ-H2A.X foci and telomeres even in crypts from very old mice, indicating that senescence in the crypt enterocytes is telomere independent. The results suggest that stress-dependent cell senescence could play a causal role for aging of mice.