Takekazu Kubo

Brain-derived neurotrophic factor (BDNF) can prevent apoptosis of rat cerebellar granule neurons in culture

Cerebellar granule neurons obtained from 9-day-old rats were grown in vitro for 4 days in high K+ (26 mM) medium. The culture medium was then replaced with that containing low K+ (5 mM) which caused a large number of granule neurons to die. The death of granule neurons has been characterized as apoptosis. In this study, we investigated the effects of various neurotrophins on neuronal survival using the above system. We found that brain-derived neurotrophic factor (BDNF) and neurotrophin-4/5 (NT-4/5) but not nerve growth factor (NGF) can protect these neurons from apoptosis in low K+. Neurotrophin-3 (NT-3) had a small effect on neuronal survival as reported. To determine whether the granule neurons respond directly to BDNF, we analyzed the induction of the Fos protein in these neurons. Individual cells that synthesize Fos protein after exposure to neurotrophin can be recognized using antibodies to Fos. Immunocytochemical staining of the cultures demonstrated that a relatively large number of cerebellar granule neurons showed immunoreactivity in response to BDNF, but few of them were immunoreactive in the absence of BDNF or in the presence of NGF. Our results suggested that BDNF has a direct effect on mature cerebellar granule neurons and can protect these neurons from apoptosis in low K+.

Involvement of phosphatidylinositol-3 kinase in prevention of low K+-induced apoptosis of cerebellar granule neurons

Cerebellar granule neurons obtained from 9-day-old rats die in an apoptotic manner when cultured in serum-free medium containing a low concentration of potassium (5 mM). A high concentration of potassium (26 mM) in the culture medium and BDNF can effectively prevent this apoptosis. The survival effects of high potassium and BDNF were additive, and the effect of high potassium was not blocked by addition of anti-BDNF antibody. These observations indicated that these survival effects were independent. To examine which molecules are involved in the survival pathway induced by BDNF or high K+, we used wortmannin, a specific inhibitor of PI-3 kinase. Wortmannin blocked the survival effects of both BDNF and high K+ on cerebellar granule neurons. Furthermore, in vitro PI-3 kinase assay showed that treatment with BDNF or high K+ induced PI-3 kinase activity, which was diminished by addition of wortmannin. These results indicate that different survival-promoting agents, BDNF and high K+, can prevent apoptosis in cerebellar granule neurons via a common enzyme, PI-3 kinase.

Oxygen Toxicity Induces Apoptosis in Neuronal Cells

Abstract1. A high oxygen atmosphere induced apoptosis in cultured neuronal cells including PC12 cells and rat embryonic cortical, hippocampal, and basal forebrain neurons associated with DNA fragmentation and nuclear condensation.2. The sensitivity of CNS neurons to a high-oxygen atmosphere was the following order; cortex > basal forebrain > hippocampus.3. Cycloheximide and actinomycin-D inhibited the apoptosis, indicating that it depends on new macromolecular synthesis. In contrast, cultured postnatal CNS neurons were resistant to oxidative stress.4. Neurotrophic factors such as nerve growth factor (NGF), fibroblast growth factor (FGF), and epidermal growth factor (EGF) blocked the apoptosis induced by a high-oxygen atmosphere.

Oxygen-induced apoptosis in PC12 cells with special reference to the role of Bcl-2

We previously reported that PC12h cells are killed by a high oxygen atmosphere. In this study, we further characterized this oxygen-induced cell death and found apoptotic features, as follows. Firstly, chromatin condensation was observed in cells cultured in a 50% O2 atmosphere. Secondly, cycloheximide and cordycepin, protein and RNA synthesis inhibitors, respectively, prevented the oxygen-induced cell death in PC12h cells, suggesting that it is mediated by an intracellular death program. Thirdly, NGF, CPT-cAMP and depolarization by high potassium medium also effectively inhibited this apoptotic cell death in PC12h cells. The effect of high K+ is thought to be mediated by the influx of Ca2+ into cells through voltage-dependent Ca2+ channels, because nifedipine, an L-type Ca2+ channel blocker, inhibited the effect of high K+. In addition, since the oxygen-induced apoptosis was blocked by the antioxidant vitamin E, this oxygen toxicity is suggested to be mediated by reactive oxygen species. To further characterize this oxygen-induced apoptosis at the molecular level, we used PC12 cells overexpressing the proto-oncogene bcl-2. Although a large number of PC12 cells transfected with the control vector died in a 50% O2 atmosphere within 6 days, bcl-2-transfected PC12 cells survived and proliferated. These findings suggested that our system using PC12 cells will be a useful model with which to analyze the molecular mechanisms of apoptosis induced by oxidative stress in neuronal cells.

Expression of cyclin A decreases during neuronal apoptosis in cultured rat cerebellar granule neurons.

Cultured cerebellar granule neurons died in an apoptotic manner when the K+ concentration in culture medium was lowered to the normal level (5 mM) after maturation of cells with a high concentration of K+ (26 mM). The changes in expression of 14 cell cycle-related genes in this CNS apoptosis model were analyzed by quantitative RT-PCR. Most of the genes analyzed were stable during apoptosis. The expression of cyclin A mRNA, however, transiently decreased 1 h after the induction of apoptosis, and recovered within 3 h to above the basal level. In this system, the level of cyclin D1, which has been reported to be up-regulated in apoptosis of NGF-deprived cultured sympathetic neurons, did not change. These results suggest that the molecular mechanisms in these two apoptosis models are different. To determine cyclin A protein level, we used an immunostaining method. The number of cyclin A-positive neurons decreased during apoptosis. Moreover, the numbers of MAP2- and cdk2-positive neurons also decreased in a similar manner. Taken together, these results suggest that there is a relationship between apoptosis and cell cycle, and that morphological changes during apoptosis result from cytoskeletal structure degradation.

Flow cytometric analysis of serum deprivation-induced apoptosis of PC12 cells, with special reference to role of bcl-2.

To investigate the mechanism of serum deprivation-induced apoptosis in PC12 cells, we performed flow cytometry with the viable cell-specific fluorogen, fluorescein diacetate (FDA). Intact PC12 cells were positive when stained with FDA, and those cells were identical with the major population which possess larger forward and side scattered light. Apoptotic PC12 cells decreased the forward and side scatter light associated with the loss of FDA-positive cells. Nerve growth factor (NGF) and the bcl-2 protein protected the cells from serum deprivation-induced apoptosis. These data suggested that the process of apoptosis in PC12 cells can be analyzed with flow cytometry.

Oxygen Toxicity Induces Apoptotic Neuronal Death in Cultured Rat Hippocampal Neurons

Oxygen metabolism is the most important event for the aerobic energy metabolism and the redox-based biosynthesis. This means that cells are constantly exposed to oxidative stress during their life. As a consequence of a normal aerobic metabolism, several oxidants such as O 2 − , H2O2 and ·OH are produced by successive additions of electrons to O2. These byproducts cause ubiquitous cell damage and are thought to contribute to aging and to degenerative diseases (Ames, et al., 1993). It is well-known that the brain is one of the most energy consuming organs in mammalian body and exclusively depends on aerobic energy metabolism using oxygen and glucose. Thus, the oxygen molecule is not only an indispensable material for highly organized CNS activities but also a cytotoxic agent which constantly brings oxidative stresses to neurons during their long life without division. In addition, the brain readily undergoes oxidative damage as a result of cerebrovascular injury such as ischemia and this causes neuronal degeneration and death. Since mature neurons can not regenerate, once the survived neurons degenerate, neuronal activity is irreversibly declined. Thus, elucidation of the protection machinery of neuronal cells against oxidative damage is not only an important clue to understand the mechanism(s) of postmitotic neurons to be long-lived but also should provide useful information to design the cure for a number of neurological pathologies.

BIOCHEMICAL CHARACTERISTICS OF OXYGEN-INDUCED AND LOW K+ MEDIUM-INDUCED APOPTOTIC NEURONAL DEATH

Neuronal cell death is thought to be an important phenomenon not only in the developmental but also in the post-matured stages of PNS and CNS. In the developmental stages, massive neuronal death is observed at specific periods, e.g. the immediately after the arrival of axons to the postsynaptic target fields. This phenomenon is precicely programmed in the developing nervous system and called programmed (or apoptotic) neuronal death. Recent studies also suggest that similer apoptotic neuronal death occurs in the matured nervous system, e.g. neurodegenerative diseases, such as Alzheimer’s and Parkinson’s diseases, and aging. Thus, it is expected that the study of apoptosis in the nervous system give us useful clues to know how the neuronal net work are constructed in the development and why the neurodegenerateve diseases and brain aging occur.