Cyclin-C-dependent cell-cycle entry is required for activation of non-homologous end joining DNA repair in postmitotic neurons.

It is commonly believed that neurons remain in G(0) phase of the cell cycle indefinitely. Cell-cycle re-entry, however, is known to contribute to neuronal apoptosis. Moreover, recent evidence demonstrates the expression of cell-cycle proteins in differentiated neurons under physiological conditions. The functional roles of such expression remain unclear. Since DNA repair is generally attenuated by differentiation in most cell types, the cell-cycle-associated events in postmitotic cells may reflect the need to re-enter the cell cycle to activate DNA repair. We show that cyclin-C-directed, pRb-dependent G(0) exit activates the non-homologous end joining pathway of DNA repair (NHEJ) in postmitotic neurons. Using RNA interference, we found that abrogation of cyclin-C-mediated exit from G(0) compromised DNA repair but did not initiate apoptosis. Forced G(1) entry combined with prevention of G(1) --> S progression triggered NHEJ activation even in the absence of DNA lesions, but did not induce apoptosis in contrast to unrestricted progression through G(1) --> S. We conclude that G(0) --> G(1) transition is functionally significant for NHEJ repair in postmitotic neurons. These findings reveal the importance of cell-cycle activation for controlling both DNA repair and apoptosis in postmitotic neurons, and underline the particular role of G(1) --> S progression in apoptotic signaling, providing new insights into the mechanisms of DNA damage response (DDR) in postmitotic neurons.

Estrogen protects against the synergistic toxicity by HIV proteins, methamphetamine and cocaine.

BACKGROUND: Human immunodeficiency virus (HIV) infection continues to increase at alarming rates in drug abusers, especially in women. Drugs of abuse can cause long-lasting damage to the brain and HIV infection frequently leads to a dementing illness. To determine how these drugs interact with HIV to cause CNS damage, we used an in vitro human neuronal culture characterized for the presence of dopaminergic receptors, transporters and estrogen receptors. We determined the combined effects of dopaminergic drugs, methamphetamine, or cocaine with neurotoxic HIV proteins, gp120 and Tat. RESULTS: Acute exposure to these substances resulted in synergistic neurotoxic responses as measured by changes in mitochondrial membrane potential and neuronal cell death. Neurotoxicity occurred in a sub-population of neurons. Importantly, the presence of 17beta-estradiol prevented these synergistic neurotoxicities and the neuroprotective effects were partly mediated by estrogen receptors. CONCLUSION: Our observations suggest that methamphetamine and cocaine may affect the course of HIV dementia, and additionally suggest that estrogens modify the HIV-drug interactions.

Homocysteine elicits a DNA damage response in neurons that promotes apoptosis and hypersensitivity to excitotoxicity.

Elevated plasma levels of the sulfur-containing amino acid homocysteine increase the risk for atherosclerosis, stroke, and possibly Alzheimer's disease, but the underlying mechanisms are unknown. We now report that homocysteine induces apoptosis in rat hippocampal neurons. DNA strand breaks and associated activation of poly-ADP-ribose polymerase (PARP) and NAD depletion occur rapidly after exposure to homocysteine and precede mitochondrial dysfunction, oxidative stress, and caspase activation. The PARP inhibitor 3-aminobenzamide (3AB) protects neurons against homocysteine-induced NAD depletion, loss of mitochondrial transmembrane potential, and cell death, demonstrating a requirement for PARP activation and/or NAD depletion in homocysteine-induced apoptosis. Caspase inhibition accelerates the loss of mitochondrial potential and shifts the mode of cell death to necrosis; inhibition of PARP with 3AB attenuates this effect of caspase inhibition. Homocysteine markedly increases the vulnerability of hippocampal neurons to excitotoxic and oxidative injury in cell culture and in vivo, suggesting a mechanism by which homocysteine may contribute to the pathogenesis of neurodegenerative disorders.

The prostate apoptosis response-4 protein participates in motor neuron degeneration in amyotrophic lateral sclerosis.

Prostate apoptosis response-4 (Par-4), a protein containing a leucine zipper domain within a death domain, is up-regulated in prostate cancer cells and hippocampal neurons induced to undergo apoptosis. Here, we report higher Par-4 levels in lumbar spinal cord samples from patients with amyotrophic lateral sclerosis (ALS) than in lumbar spinal cord samples from neurologically normal patients. We also compared the levels of Par-4 in lumbar spinal cord samples from wild-type and transgenic mice expressing the human Cu/Zn-superoxide dismutase gene with a familial ALS mutation. Relative to control samples, higher Par-4 levels were observed in lumbar spinal cord samples prepared from the transgenic mice at a time when they had hind-limb paralysis. Immunohistochemical analyses of human and mouse lumbar spinal cord sections revealed that Par-4 is localized to motor neurons in the ventral horn region. In culture studies, exposure of primary mouse spinal cord motor neurons or NSC-19 motor neuron cells to oxidative insults resulted in a rapid and large increase in Par-4 levels that preceded apoptosis. Pretreatment of the motor neuron cells with a Par-4 antisense oligonucleotide prevented oxidative stress-induced apoptosis and reversed oxidative stress-induced mitochondrial dysfunction that preceded apoptosis. Collectively, these data suggest a role for Par-4 in models of motor neuron injury relevant to ALS.

ALS-linked Cu/Zn-SOD mutation impairs cerebral synaptic glucose and glutamate transport and exacerbates ischemic brain injury.

Although degeneration of lower motor neurons is the most striking abnormality in amyotrophic lateral sclerosis (ALS), more subtle alterations may occur in the brain. Mutations in copper/zinc superoxide dismutase (Cu/Zn-SOD) are responsible for some cases of inherited ALS, and expression of mutant Cu/Zn-SOD in transgenic mice results in progressive motor neuron loss and a clinical phenotype similar to that of ALS patients. It is now reported that Cu/Zn-SOD mutant mice exhibit increased vulnerability to focal ischemic brain injury after transient occlusion of the middle cerebral artery. Levels of glucose and glutamate transport in cerebral cortex synaptic terminals were markedly decreased, and levels of membrane lipid peroxidation were increased in Cu/Zn-SOD mutant mice compared to nontransgenic mice. These findings demonstrate that mutant Cu/Zn-SOD may endanger brain neurons by a mechanism involving impairment of glucose and glutamate transporters. Moreover, our data demonstrate a direct adverse effect of the mutant enzyme on synaptic function.

Evidence that Par-4 participates in the pathogenesis of HIV encephalitis.

Progressive neuronal degeneration in brain regions involved in learning and memory processes is a common occurrence in patients infected with human immunodeficiency virus type 1 (HIV-1). We now report that levels of Par-4, a protein recently linked to neuronal apoptosis in Alzheimer's disease, are increased in neurons in hippocampus of human patients with HIV encephalitis and in monkeys infected with a chimeric strain of HIV-1 and simian immunodeficiency virus. Par-4 levels increased rapidly in cultured hippocampal neurons following exposure to the neurotoxic HIV-1 protein Tat, and treatment of the cultures with a Par-4 antisense oligonucleotide protected the neurons against Tat-induced apoptosis. Additional findings show that Par-4 participates at an early stage of Tat-induced neuronal apoptosis before caspase activation, oxidative stress, and mitochondrial dysfunction. Our data suggest that Par-4 may be a mediator of neuronal apoptosis in HIV encephalitis and that therapeutic approaches targeting the Par-4 apoptotic cascade may prove beneficial in preventing neuronal degeneration and associated dementia in patients infected with HIV-1.

Pivotal role of mitochondrial calcium uptake in neural cell apoptosis and necrosis.

Perturbed cellular calcium homeostasis has been implicated in both apoptosis and necrosis, but the role of altered mitochondrial calcium handling in the cell death process is unclear. The temporal ordering of changes in cytoplasmic ([Ca2+]C) and intramitochondrial ([Ca2+]M) calcium levels in relation to mitochondrial reactive oxygen species (ROS) accumulation and membrane depolarization (MD) was examined in cultured neural cells exposed to either an apoptotic (staurosporine; STS) or a necrotic (the toxic aldehyde 4-hydroxynonenal; HNE) insult. STS and HNE each induced an early increase of [Ca2+]C followed by delayed increase of [Ca2+]M. Overexpression of Bcl-2 blocked the elevation of [Ca2+]M and the MD in cells exposed to STS but not in cells exposed to HNE. The cytoplasmic calcium chelator BAPTA-AM and the inhibitor of mitochondrial calcium uptake ruthenium red prevented both apoptosis and necrosis. STS and HNE each induced mitochondrial ROS accumulation and MD, which followed the increase of [Ca2+]M. Cyclosporin A prevented both apoptosis and necrosis, indicating critical roles for MD in both forms of cell death. Caspase activation occurred only in cells undergoing apoptosis and preceded increased [Ca2+]M. Collectively, these findings suggest that mitochondrial calcium overload is a critical event in both apoptotic and necrotic cell death.

ALS-linked Cu/Zn-SOD mutation increases vulnerability of motor neurons to excitotoxicity by a mechanism involving increased oxidative stress and perturbed calcium homeostasis.

We employed a mouse model of ALS, in which overexpression of a familial ALS-linked Cu/Zn-SOD mutation leads to progressive MN loss and a clinical phenotype remarkably similar to that of human ALS patients, to directly test the excitotoxicity hypothesis of ALS. Under basal culture conditions, MNs in mixed spinal cord cultures from the Cu/Zn-SOD mutant mice exhibited enhanced oxyradical production, lipid peroxidation, increased intracellular calcium levels, decreased intramitochondrial calcium levels, and mitochondrial dysfunction. MNs from the Cu/Zn-SOD mutant mice exhibited greatly increased vulnerability to glutamate toxicity mediated by alpha-amino-3-hydroxy-5-methylisoxazole-4-propionate receptors. The increased vulnerability of MNs from Cu/Zn-SOD mutant mice to glutamate toxicity was associated with enhanced oxyradical production, sustained elevations of intracellular calcium levels, and mitochondrial dysfunction. Pretreatment of cultures with vitamin E, nitric oxide-suppressing agents, peroxynitrite scavengers, and estrogen protected MNs from Cu/Zn-SOD mutant mice against excitotoxicity. Excitotoxin-induced degeneration of spinal cord MNs in adult mice was more extensive in Cu/Zn-SOD mutant mice than in wild-type mice. The mitochondrial dysfunction associated with Cu/Zn-SOD mutations may play an important role in disturbing calcium homeostasis and increasing oxyradical production, thereby increasing the vulnerability of MNs to excitotoxicity.

Calcium and reactive oxygen species mediate staurosporine-induced mitochondrial dysfunction and apoptosis in PC12 cells.

The bacterial alkaloid staurosporine is widely employed as an inducer of apoptosis in many cell types including neurons. The intracellular cascades that mediate staurosporine-induced apoptosis are largely unknown. Exposure of cultured PC12 cells to staurosporine resulted in a rapid (min) and prolonged (1-6 hr) elevation of intracellular free calcium levels [Ca2+]i, accumulation of mitochondrial reactive oxygen species (ROS), and decreased mitochondrial 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) reduction (1-4 hr). These early events were followed by membrane lipid peroxidation, loss of mitochondrial transmembrane potential, and nuclear apoptotic changes. Treatment of cells with serum or nerve growth factor within 1-2 hr of staurosporine exposure resulted in recovery of [Ca2+]i and ROS levels, and rescued the cells from apoptosis. The increased [Ca2+]i and ROS production were required for staurosporine-induced apoptosis because the intracellular calcium chelator BAPTA and uric acid (an agent that scavenges peroxynitrite) each protected cells against apoptosis. The caspase inhibitor zVAD-fmk and the anti-apoptotic gene product Bcl-2 prevented the sustained [Ca2+]i increase and ROS accumulation induced by staurosporine indicating that caspases act very early in the apoptotic process. Our data indicate that a [Ca2+]i increase is an early and critical event in staurosporine-induced apoptosis that engages a cell death pathway involving ROS production, oxidative stress, and mitochondrial dysfunction.

HIV-1 protein Tat induces apoptosis of hippocampal neurons by a mechanism involving caspase activation, calcium overload, and oxidative stress.

Patients infected with HIV-1 often exhibit cognitive deficits that are related to progressive neuronal degeneration and cell death. The protein Tat, which is released from HIV-1-infected cells, was recently shown to be toxic toward cultured neurons. We now report that Tat induces apoptosis in cultured embryonic rat hippocampal neurons. Tat induced caspase activation, and the caspase inhibitor zVAD-fmk prevented Tat-induced neuronal death. Tat induced a progressive elevation of cytoplasmic-free calcium levels, which was followed by mitochondrial calcium uptake and generation of mitochondrial-reactive oxygen species (ROS). The intracellular calcium chelator BAPTA-AM and the inhibitor of mitochondrial calcium uptake ruthenium red protected neurons against Tat-induced apoptosis. zVAD-fmk suppressed Tat-induced increases of cytoplasmic calcium levels and mitochondrial ROS accumulation, indicating roles for caspases in the perturbed calcium homeostasis and oxidative stress induced by Tat. An inhibitor of nitric oxide synthase, and the peroxynitrite scavenger uric acid, protected neurons against Tat-induced apoptosis, indicating requirements for nitric oxide production and peroxynitrite formation in the cell death process. Finally, Tat caused a delayed and progressive mitochondrial membrane depolarization, and cyclosporin A prevented Tat-induced apoptosis, suggesting an important role for mitochondrial membrane permeability transition in Tat-induced apoptosis. Collectively, our data demonstrate that Tat can induce neuronal apoptosis by a mechanism involving disruption of calcium homeostasis, caspase activation, and mitochondrial calcium uptake and ROS accumulation. Agents that interupt this apoptotic cascade may prove beneficial in preventing neuronal degeneration and associated dementia in AIDS patients.

Acridine orange as an indicator of the cytoplasmic ribosome state.

In hippocampal neurons of ground squirrels and neuroblastoma culture the ribosome state was analyzed by staining with acridine orange (AO), labeling with radioactive amino acids, and electron microscopy. Electron microscopy indicated that the extent to which ribosomes associated in polysomes varied from 25% in brain cells of torpid ground squirrels up to 93% in growing neuroblastoma cells. In control rat neurons, it was 75%. The fluorescence of the AO-rRNA complex in ribosomes changed with the polysome/monosome ratio. The red fluorescence of AO-single-stranded rRNA complex as well as ribosome association in polysomes decreased greatly as a function of the shift from polysomes to monosomes. The green fluorescence of AO intercalated in the double-stranded rRNA changed insignificantly. As a result, the ratio of red to green fluorescence intensity, Kalpha, changed more than 3-fold with the changing polysome/monosome ratio. Protein labeling also showed strong positive correlation with Kalpha. Thus, rRNA showed different accessibility for AO binding in active and inactive ribosomes. A possible mechanism of partial rRNA shielding with proteins is proposed. AO fluorescence in the cytoplasm, i.e., AO binding to rRNA in ribosomes, is presumed to reflect adequately the profound changes in the state of cell protein synthesizing system that can be regulated by both functional activity and stress factors.

Evidence that 4-hydroxynonenal mediates oxidative stress-induced neuronal apoptosis.

Oxidative stress is believed to play important roles in neuronal cell death associated with many different neurodegenerative conditions (e.g., Alzheimer's disease, Parkinson's disease, and cerebral ischemia), and it is believed also that apoptosis is an important mode of cell death in these disorders. Membrane lipid peroxidation has been documented in the brain regions affected in these disorders as well as in cell culture and in vivo models. We now provide evidence that 4-hydroxynonenal (HNE), an aldehydic product of membrane lipid peroxidation, is a key mediator of neuronal apoptosis induced by oxidative stress. HNE induced apoptosis in PC12 cells and primary rat hippocampal neurons. Oxidative insults (FeSO4 and amyloid beta-peptide) induced lipid peroxidation, cellular accumulation of HNE, and apoptosis. Bcl-2 prevented apoptosis of PC12 cells induced by oxidative stress and HNE. Antioxidants that suppress lipid peroxidation protected against apoptosis induced by oxidative insults, but not that induced by HNE. Glutathione, which binds HNE, protected neurons against apoptosis induced by oxidative stress and HNE. PC12 cells expressing Bcl-2 exhibited higher levels of glutathione and lower levels of HNE after oxidative stress. Collectively, the data identify that HNE is a novel nonprotein mediator of oxidative stress-induced neuronal apoptosis and suggest that the antiapoptotic action of glutathione may involve detoxification of HNE.

Basic FGF attenuates amyloid beta-peptide-induced oxidative stress, mitochondrial dysfunction, and impairment of Na+/K+-ATPase activity in hippocampal neurons.

Basic fibroblast growth factor (bFGF) exhibits trophic activity for many populations of neurons in the brain, and can protect those neurons against excitotoxic, metabolic and oxidative insults. In Alzheimer's disease (AD), amyloid beta-peptide (A beta) fibrils accumulate in plaques which are associated with degenerating neurons. A beta can be neurotoxic by a mechanism that appears to involve induction of oxidative stress and disruption of calcium homeostasis. Plaques in AD brain contain high levels of bFGF suggesting a possible modulatory role for bFGF in the neurodegenerative process. We now report that bFGF can protect cultured hippocampal neurons against A beta25-35 toxicity by a mechanism that involves suppression of reactive oxygen species (ROS) accumulation and maintenance of Na+/K+-ATPase activity. A beta25-35 induced lipid peroxidation, accumulation of H2O2, mitochondrial ROS accumulation, and a decrease in mitochondrial transmembrane potential; each of these effects of A beta25-35 was abrogated in cultures pre-treated with bFGF. Na+/K+-ATPase activity was significantly reduced following exposure to A beta25-35 in control cultures, but not in cultures pre-treated with bFGF. bFGF did not protect neurons from death induced by ouabain (a specific inhibitor of the Na+/K+-ATPase) or 4-hydroxynonenal (an aldehydic product of lipid peroxidation) consistent with a site of action of bFGF prior to induction of oxidative stress and impairment of ion-motive ATPases. By suppressing accumulation of oxyradicals, bFGF may slow A beta-induced neurodegenerative cascades.

A role for T cell CD4 in contact mediated T dependent B cell activation.

Using a monoclonal antibody to CD4 we have shown that occupation of CD4 on T cells induces a strong dose dependent inhibition of in vitro IgM plaque forming cell (PFC) response of spleen cells to the T dependent antigen (Ag), sheep red blood cells (SRBC), in Mishell-Dutton cultures. This inhibitory effect is not due simply to nonspecific perturbation or Fc binding, since F(ab) fragments of anti-CD4 are as potent as the intact antibodies, whereas antibodies to class I molecules or T cell CD5 have no effect. The anti-CD4 antibody appears to block contact dependent interaction between T and B cells and this inhibitory effect cannot be overcome by cytokines. Anti-CD4 did not inhibit the PFC response to the T independent antigen, trinitrophenylated lipopolysaccharide. The anti-CD4 antibody prevented the interaction of preactivated fixed SRBC specific T helper cells with B cells, suggesting that CD4 had a role in contact mediated interactions between T cells and B cells. Surprisingly, antibodies to CD40L failed to inhibit the SRBC specific PFC response. Thus CD4 appears to be an important molecule required for cognate interactions between T and B cells that are needed to generate an Ag specific PFC response.

Differentiation and apoptosis of murine neuroblastoma cells N1E115.

The mode and the kinetics of differentiation and death of murine N1E115 neuroblastoma cells induced by dimethyl sulfoxide and other nonspecific factors in vitro were investigated. After morphological differentiation neuroblastoma cells die by apoptosis which is indicated by characteristic morphological features and by internucleosomal DNA fragmentation. Durations of both differentiation and apoptosis are dependent on the nature of stimuli used. Protein synthesis inhibitor cycloheximide does not prevent differentiation and apoptosis of neuroblastoma cells induced by dimethyl sulfoxide and even accelerates both processes. The relationship between cell death and differentiation is discussed.

Apoptosis of murine BW 5147 thymoma cells induced by cold shock.

Exposure of thymoma BW 5147 cells to cold (0-2 degrees C) followed by rewarming at 37 degrees C (cold shock) resulted in internucleosomal DNA cleavage. Sensitivity to cold shock-induced cell death was critically dependent on the serum concentration in the medium and limited to serum-deficient medium (2% serum concentration), whereas cells in the complete growth medium (10%) were completely resistant. RNA/protein-synthesis inhibitors (cycloheximide and actinomycin D) had no effect on cold shock-induced DNA cleavage in BW 5147 cells. The DNA fragmentation seems to be independent of increase in the cytosolic Ca2+ level. Moreover, reduction in the calcium content of the external medium by EGTA induced DNA cleavage. Incubation of BW 5147 cells in the presence of colchicine and cytochalasin B led to the apoptosis. The latter suggests that the internucleosomal DNA cleavage induced by cold shock may be concerned with the disruption of some cytoskeletal network caused by cooling. The results are discussed in relation to cell proliferation.

Comparative analysis of cell replacement in hibernators.

1. Cell renewal in hibernators undergoes seasonal rhythm independent of the hibernation state. 2. We propose that seasonal depression of cell renewal in tissues of hibernators is caused by seasonal involution of thymus in these animals. 3. The latter is known to be involved in the control of cell proliferation. 4. The state of hibernation per se has also an effect on cellular proliferation. 5. It induces the block of cells in the permitotic phase. It is suggested that the blockage of cells in renewing tissues of hibernators under natural deep hypothermia throughout a period of torpidity represents the adaptive reaction of the organism.

[The programmed cell death of murine BW5147 thymoma under the action of dexamethasone]. / Programmiruemaia gibel' kletok timomy myshi BW5147 pod deistviem deksametazona.

By flow cytometry, imitation modelling and biochemical analysis, the mode and kinetics of dexamethasone-treated T-lymphoma cell death were studied. The hormone was shown to induce delays in pre- and postsynthetic phases of the cell cycle and the death of part of cells. A short exposure to dexamethasone reveals its cytostatic rather than cytolytic effect. Following G2/M delay and cytokinesis, part of cells dies. A reduced serum concentration (2%) causes shorter delays in the cell cycle and a more rapid cell death. Dexamethasone stimulates apoptosis which is indicated by internucleosomal DNA fragmentation, and by a coincidence in time of the processes of DNA degradation and increase in the other membrane permeability. These results are discussed in relation to the cell death and proliferation.

[The cellular apoptosis of murine BW5147 thymoma during cold shock]. / Apoptoz kletok timomy myshi BW5147 pri kholodovom shoke.

The mode of T-lymphoma cell death induced by cold shock was studied. The rewarming of cells at 37 degrees C following a brief period of cold (0 degrees C) resulted in internucleosomal DNA fragmentation. The cells underwent cold shock-mediated apoptosis only at a reduced (2%) serum concentration. The apoptosis was not blocked by macromolecular synthesis inhibitors such as cycloheximide and antinomycin D, or by Quin-2. EGTA per se was responsible for the initiation of cell death. Colchicine also induced internucleosomal fragmentation of DNA. Our findings suggest that cold shock induced apoptosis is associated with low temperature mediated disruption of microtubules. The role of Ca2+ and growth factors in cold shock induced cell death is discussed.

Apoptosis of murine BW 5147 thymoma cells induced by dexamethasone and gamma-irradiation.

The mode and the kinetics of the death of T-thymoma cells upon dexamethasone treatment and gamma-irradiation (10Gy) have been studied using flow cytometry and biochemical analysis. It has been shown that the hormone and gamma-irradiation induce cell death by apoptosis. In both cases the cells are initially blocked in G2/M and die only after overcoming the blockage and cytokinesis. A short exposure to dexamethasone results in a cytostatic effect, whereas a cytotoxic effect is absent. Reducing serum concentration to 2% causes more rapid death both following gamma-irradiation and dexamethasone. These results are discussed in relation to cell death and proliferation.