Aluminum-induced apoptosis in cultured cortical neurons and its effect on SAPK/JNK signal transduction pathway

Aluminum exposure and apoptotic cell death has been implicated in several neurodegenerative diseases. The mechanisms by which aluminum interacts with the nervous system are only partly understood. In this study, we used cultured cortical neurons to investigate the ability of aluminum to induce the apoptosis of neurons and to explore the role of SAPK/JNK (stress-activated protein kinase or c-jun N-terminal kinase) signal transduction pathway on the apoptosis induced by aluminum. We found that aluminum-induced degeneration of cortical neurons involved the DNA fragmentation characteristic of apoptosis, and staining of aluminum-treated neurons with the DNA-binding fluorochrome Hoechst 33258 revealed the typical apoptotic condensation and fragmentation of chromatin. The rate of apoptosis increased significantly (from 4.9 to 13.1, 21.4, and 59.8%, P<0.01), which was measured by TdT-mediated dUTP nick end labeling. Western blot analysis showed that SAPK/JNK activities of cortical neurons varies when the exposure time of AlCl(3) were different. The phosphorylation levels were 4.2, 3.3, 1.9 and 1.1 times greater compared to control cultures for 6, 12, 24, and 48 h, respectively (P<0.01). Furthermore, a JNK pathway inhibitor, CEP-11004 (KT8138) inhibited the activation of SAPK/JNK to protect cortical neurons from apoptosis induced by aluminum chloride. Our study demonstrates that aluminum can induce the apoptosis of cortical neurons and SAPK/JNK signal transduction pathway may play an important role in the apoptosis.     

Aluminum-induced apoptosis in cultured astrocytes and its effect on calcium homeostasis

Aluminum exposure and apoptotic cell death has been implicated in several neurodegenerative conditions including Alzheimer's disease. In this study, we use cultured astrocytes to investigate the ability of aluminum to induce the apoptosis of astrocytes. The proportion of apoptotic cells and cell cycle distribution were determined by flow cytometric analysis. Our results showed that exposure to aluminum at low levels (100 and 200 microM) for up to 6 days did not result in the apoptosis of astrocytes, and a dramatic blockage of apoptotic cells was found at 200 microM aluminum. However, at 400 microM, aluminum markedly induced the apoptosis of astrocytes, which was associated with a significant change in cell cycle distribution characterized by increase of G2/M phase cells (128%). Measurements of intracellular Ca(2+) concentration using the fluorescent calcium indicator dye Fluo-3 demonstrated a significant increase in the levels of intracellular calcium after aluminum treatment. However, no differences were observed among aluminum-treated groups. These findings suggest that aluminum induce and block selectively the apoptosis of astrocytes, which depend upon the concentrations of aluminum. Increased intracellular Ca(2+) may not be the primary mechanism of aluminum-mediated apoptotic cell death.     

Accumulation of aluminum by primary cultured astrocytes from aluminum amino acid complex and its apoptotic effect

Aluminum salts or doses that are unlikely in the human system have been employed in toxicity studies and much attention had been focused on the secondary target (neurons) of its toxicity rather than the primary target (astroglia). In order to address these issues, we have investigated the uptake and apoptotic effects of aluminum amino acid complex on primary cultured astrocytes because these are fundamental in understanding the mechanism of aluminum neurotoxicity. Aluminum solubilized by various amino acids was differentially internalized by astrocytes (glycine>serine>glutamine>glutamate), but aluminum was not internalized from citrate complex following 24 h of exposure. Inhibition of glutamine synthetase, by methionine sulfoximine (MSO), enhanced the uptake of aluminum from various amino acid complexes within 8 h except from glutamine complex. Blockade of selective GLT-1 (EAAT2) and GlyT1, as well as nonspecific transporters, did not inhibit or had no effect on uptake of aluminum in complex with the corresponding amino acids. Ouabain also failed to inhibit uptake of aluminum complexed with glycine. Pulse exposure to aluminum glycinate in the absence or presence of MSO caused apoptosis in over 25% of primary cultured astrocytes, and apoptotic features such as chromatin condensation and fragmentation became evident as early as 3 days of culture in normal medium. Lower doses (as low as 0.0125 mM) also caused apoptosis. The present findings demonstrate that aluminum solubilized by amino acids, particularly glycine, could serve as better candidate for neurotoxicity studies. Citrate may be a chelator of aluminum rather than a candidate for its cellular uptake. Amino acid transporters may not participate in the uptake of aluminum solubilized by their substrates. Another pathway of aluminum internalization may be implicated in addition to passive diffusion but may not require energy in form of Na+/K+-ATPase. Impaired astrocyes' metabolism can aggravate their accumulation of aluminum and aluminum can compromise astrocytes via apoptosis. Thus, loss of astrocytic regulatory and supportive roles in the central nervous system (CNS) may be responsible for neurodegeneration observed in Alzheimer's disease.     

Do nuclear condensation or fragmentation and DNA fragmentation reflect the mode of neuronal death?

It is generally believed that nuclear condensation and fragmentation as well as DNA fragmentation reflect the events related to the neuronal apoptosis. Our report demonstrates that severe oxygen-glucose deprivation (OGD) induced condensation and fragmentation of nuclear chromatin of neurones in primary cultures of cerebellar granule cells without intemucleosomal DNA fragmentation. DNA fragmentation detected by TUNEL assay was seen only after mild OGD or after addition of colchicine but not after severe OGD. Thus, at least in primary cerebellar granule cell cultures, the chromatin condensation and fragmentation cannot be considered as a hallmark of apoptosis but rather reflect the neuronal death despite of its form.     

Depletion of intracellular zinc induces macromolecule synthesis- and caspase-dependent apoptosis of cultured retinal cells

Although zinc deficiency may contribute to age-related macular degeneration (ARMD), the pathogenic mechanism is as yet uncertain. In light of evidence that cellular zinc depletion induces apoptosis in cortical neurons and thymocytes, in the present study, we examined the possibility that the same phenomenon occurs also in retinal cells. Exposure of primary retinal cell cultures to 1-3 microM of a cell membrane-permeant zinc chelator TPEN for 24 h induced concentration-dependent death of neurons, photoreceptor cells, and astrocytes. Addition of zinc or copper reversed TPEN toxicity to all cell components, indicating the particular involvement of zinc chelation in cell death. Consistent with apoptosis, oligonucleosomal DNA fragmentation and chromatin condensation accompanied, and the protein synthesis inhibitor cycloheximide blocked the TPEN-induced retinal cell death. During TPEN-induced retinal cell apoptosis, cleavage/activation of procaspase-1, but little of procaspase-3, was observed. Consistent with this finding, a broad-spectrum caspase inhibitor (zVAD-fmk) was significantly more protective than a caspase-3-selective inhibitor (DEVD-fmk). The present study has demonstrated that depletion of intracellular zinc is sufficient to induce macromolecule synthesis- and caspase-dependent apoptosis of cultured retinal cells. In light of the possibility that zinc depletion may contribute to the pathogenesis of ARMD, the current culture model may be a useful tool for the investigation of the mechanism of zinc depletion-induced retinal cell death.     

Oxidized proteins in astrocytes generated in a hyperbaric atmosphere induce neuronal apoptosis

In the present study, we investigated the influence of the oxidative damage to astrocytes on neuronal cell survival using cultures of rat cerebral astrocytes and neurons. The exposure of astrocytes to hyperbaric oxygen induced a time-dependent apoptotic cell death, as observed by DNA ladder assessment. When astrocytes damaged by oxidative stress were cocultured with normal neurons from the cerebrum of a newborn rat, neuronal cell death was markedly induced, although normal astrocytes not subjected to hyperoxia cocultured with normal neurons showed no neuronal cell apoptosis. It was found that either the supernatant from the homogenate of astrocytes cultured in hyperbaric oxygen atmosphere or a protein mixture extracted from the supernatant induced neuronal cell death. The level of protein carbonyls, an index of protein oxidation analysis, in cultured astrocytes increased significantly with oxidative stress, and vitamin E inhibited the increase in the level of such oxidized proteins in astrocytes. Furthermore, a two-dimensional (2D) electrophoresis of a protein mixture extracted from the supernatant showed several changes in proteins. These results imply that reactive oxygen species (ROS) induced by oxidative stress attack astrocytes to induce oxidatively denatured proteins in the cells that act as a neurotoxic factor, and that vitamin E protects neurons by inhibiting astrocyte apoptosis caused by oxidative stress.     

Caspase-3 is required for apoptosis-associated DNA fragmentation but not for cell death in neurons deprived of potassium

Caspases are crucial effectors of the cell death pathway activated by virtually all apoptosis-inducing stimuli within neurons and nonneuronal cells. Among the caspases, caspase-3 (CPP32) appears to play a pivotal role and has been found to be necessary for developmentally regulated cell death in the brain. We have used mice lacking caspase-3 (-/-CPP32) to examine its involvement in cultured cerebellar granule neurons induced to undergo apoptosis by potassium deprivation (K+). We find that, following K+ deprivation, neurons from -/-CPP32 mice die to the same extent as those from normal (+/+) mice. Although a small delay in the induction of cell death is observed in -/-CPP32 neurons, the rate of cell death is generally comparable to that of +/+ cultures. Though not critical for neuronal death, caspase-3 is required for DNA fragmentation and chromatin condensation as judged by the absence of these apoptotic features in -/-CPP32 neurons. Boc.Asp.fmk, a pan caspase inhibitor, partially protects +/+ neurons from low-K+-mediated cell death and does so to the same extent in -/-CPP32 cultures, suggesting the involvement of a caspase other than caspase-3 in cell death. However, the protective effect of boc.Asp.fmk is not seen beyond 24 hr, suggesting that the effect of caspase inhibition is one of delaying rather than preventing apoptosis. The more selective caspase inhibitors DEVD.fmk, IETD.fmk, and VEID.fmk fail to affect cell death, indicating that members inhibited by these agents (such as caspases - 6 ,7, 8, 9 and 10) are also not involved in low-K+-mediated apoptosis.     

Apoptosis in cultured hNT neurons

Programmed cell death (apoptosis) is an important mechanism shaping the size of different cell populations within the developing nervous system. In our study we used the NT2/D1 clone originally established from the Ntera 2 cell line to investigate the baseline levels of apoptosis in cultured postmitotic hNT (NT2-N) neurons previously treated for 3, 4 or 5 weeks with retinoic acid (RA) and compared it with apoptosis in NT2 precursors unexposed to RA. First, we examined whether different lengths of exposure to RA might affect baseline apoptotic rate in differentiating hNT neurons. Second, we investigated whether cultured hNT neurons, previously shown to possess dopaminergic characteristics, would be preferentially affected by apoptosis. Using the terminal deoxynucleotidyl transferase (tdt)-labeling technique we found that the postmitotic hNT neuronal cells exposed to RA demonstrated significantly higher numbers of apoptotic cells (12.5-15.8%) in comparison to rapidly dividing NT2 precursor cell line (3.6-4.4%) at both studied (1 and 5 days in vitro, DIV) time points. Similar apoptotic nuclear morphology, including a variable extent of nuclear fragmentation was observed in all examined hNT cultures. On the other hand, the incidence of apoptotic nuclei was rare in cultures of NT2 precursors not subjected to RA treatment. Combined immunocytochemistry for tyrosine hydroxylase (TH) and Hoechst staining revealed dopaminergic hNT neurons destined to die. Our double-labeling studies have demonstrated that only a subset of TH-positive hNT cells had condensed chromatin after 1 (approx. 15%) and 5 (approx. 20%) DIV. NT2 precursors were not TH-positive. Collectively, our results demonstrated that exposure to differentiating agent RA triggers an apoptotic commitment in a subset of postmitotic hNT neurons. These results suggest that this cell line may serve as a model of neuronal development to test various pathogenic factors implicated in the etiology of Parkinson's disease (PD), as well as to screen numerous pharmacological treatments that may slow or prevent dopaminergic deterioration.     

Avoidance of Apoptosis in Alzheimer's Disease

Internucleosomal DNA fragmentation, which is a well-known feature of apoptosis but not an absolute criterion for identifying apoptosis (1), has often been observed in the brain tissue of Alzheimer's disease (AD). However, classical apoptotic morphology such as nuclear condensation, membrane blebbing and apoptotic bodies are seldom seen in AD brain. In this issue of Journal of Alzheimer's Disease, Velez-Pardo et al. have reported the DNA fragmentation using terminal dUTP labeling (TUNEL) in postmortem brains of familial AD with presenilin-1 [E280A] mutation. Importantly, no classical apoptotic morphology has been observed also in the brains of presenilin-1 familial AD. Furthermore, Velez-Pardo et al. have shown that there is no obvious correlation between DNA fragmentation and the severity of amyloid deposition as well as between DNA fragmentation and the severity of neurofibrillary tangle formation. An apoptotic pathway only takes several hours or at most a few days for completion. In the development of the lateral motor column of the chick embryo, 8,000 cells out of 20,000 cells die, i.e. loss of 40% of the population occurs within 3 days (2). The fact that only 5% of the population in the lateral motor column is undergoing apoptosis at any particular time in this period (2) indicates that apoptotic pathway requires about 10 hours for completion. In a striking contrast with the physiologically programmed cell death, loss of 40% of the population occurs (3,000 neurons out of 7,000 neurons per 50 micron-thick section are lost) within 10 years in the temporal cortex neurons of AD (3). If we suppose that 20-40% of neurons of the temporal cortex are undergoing degeneration at any given time in the course of AD, an individual neuron in the temporal cortex of AD requires 5-10 years to die. Indeed, we can observe neurons displaying many of the features of apoptosis in AD. This fact argues that neurons in AD have mounted an effective defense to apoptotic death (an avoidance of apoptosis) rather than actual completion of apoptosis (4,5). It is noteworthy that nucleic acid oxidation occurs widely in vulnerable neuronal populations in AD (6) and oxidative damage can directly cause DNA fragmentation (7). Therefore, DNA damage possibly resulting from oxidative stress involves vulnerable neurons in AD beyond the distribution of amyloid deposition or neurofibrillary tangles, which may be related to neuronal cell death occurring independently of the classical AD pathology (3).     

Ultrastructural and MRI study of the substantia nigra evolving exofocal post‐ischemic neuronal death in the rat

To clarify the morphological characteristics of exofocal post‐ischemic neuronal death (EPND) in the substantia nigra (SN), we investigated the course of light‐ and electron‐microscopic changes of the SN of rats subjected to occlusion of the left middle cerebral artery (MCA) for 1, 2, 4, 7 and 12 days. To assess cellular edema, sequential magnetic resonance (MR) mapping of the apparent diffusion coefficient (ADC) and the T₂ value test was performed. Histological and electron‐microscopic examination on day 1 showed dotted chromatin clumps in the nuclei of some neurons and mild swelling of the perivascular endfeet of astrocytes in the ipsilateral SN. On day 2, a few cells of the ipsilateral SN pars reticulata (SNr) revealed key morphological signs of apoptosis – apoptotic body‐like condensation and segregation of the chromatin and DNA fragmentation‐like nuclear remnants. On day 4, 38% of neurons became swollen (pale neurons) with cytoplasmic microvacuoles, which appeared to originate from rough endoplasmic reticulum (rER), mitochondria and Golgi apparatus. Twenty percent of neurons showed massive proliferation of the cisternae of the rER, some of which were fragmented or had lost their normal parallel arrangement. In addition, MR mapping revealed a transient ADC decrease with a T₂ increase (signifying a phase of cellular edema), which coordinated with the phase of ultrastructural cellular swelling. Further, the total number of neurons started to decrease gradually, the perivascular endfeet of astrocytes were markedly swollen, and the neuropil became loose on day 4. On day 7, reactive astrocytes and dark neurons occurred most frequently. These results suggest that the EPND in the SN after occlusion of the MCA in adult rats is due to both apoptosis and necrosis, although necrosis seems to be the dominant mechanism of the EPND. However, the morphologic resemblances of EPND to delayed neuronal death suggest these processes have a common pathomechanism.     

Aluminum pretreatment impairs the ability of astrocytes to protect neurons from glutamate mediated toxicity

A number of laboratories have shown that astrocytes protect neurons from glutamate excitotoxicity. The experiments described in this paper were designed to address the question whether prior exposure of astrocytes to aluminum (in the form of aluminum citrate) interfered with the ability of astrocytes to protect neurons from glutamate excitotoxicity. Our culture paradigm consisted of highly enriched cultures of neurons and astrocytes grown on separate coverslips; this design enables one to subject either the neurons or the astrocytes to specific treatments and recombine the cells into the same petri dish simply by moving ceverslips from dish to dish. We have confirmed findings of other laboratories that astrocytes could protect from glutamate-induced death when glutamate (100 μM) is added to the culture medium. We have also demonstrated that prior treatment of astrocytes with 100 μM aluminum citrate impairs this ability of astrocytes to promote neuronal survival. No differences, however, were observed in the ability of control and aluminum-treated astrocytes to take up glutamate. These findings suggest that aluminum may cause astrocytes to: (i) secrete a factor that makes neurons more susceptible to glutamate-induced toxicity; (ii) secrete a neuronotoxic factor in the presence of glutamate; or (iii) reduce secretion of a factor that protects neurons from glutamate excitotoxicity.     

NF-kappaB-dependent production of nitric oxide by astrocytes mediates apoptosis in differentiated PC12 neurons following exposure to manganese and cytokines

Neuronal injury in manganism is accompanied by activation of astroglia within the basal ganglia that is thought to increase production of inflammatory mediators such as nitric oxide (NO). The present studies postulated that astroglial-derived NO mediates neuronal apoptosis induced by manganese (Mn) and pro-inflammatory cytokines. Pheochromocytoma (PC12) cells differentiated with nerve growth factor (NGF) were co-cultured with primary astrocytes and exposed to Mn and tumor necrosis factor-alpha (TNF-alpha) plus interferon-gamma (IFN-gamma). Mn enhanced cytokine-induced expression of inducible nitric oxide synthase (NOS2, EC 1.14.13.39) and production of NO in astrocytes that correlated with apoptosis in co-cultured neurons, as determined by caspase activity, terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick-end labeling (TUNEL), and nuclear morphology. Apoptosis in PC12 neurons required the presence of astrocytes and was blocked by overexpression of a phosphorylation-deficient mutant of IkappaBalpha (S32/36A) in astrocytes that prevented induction of NOS2. Pharmacologic inhibition of NOS2 with (+/-)-2-amino-5,6-dihydro-6-methyl-4H-1,3-thiazine (AMT) significantly reduced neuronal apoptosis, and the addition of low concentrations of the NO donor, S-nitroso-N-acetylpenicillamine (SNAP), to neurons cultured without astrocytes was sufficient to recover the apoptotic phenotype following exposure to Mn and TNF-alpha/IFN-gamma. It is concluded that Mn- and cytokine-dependent apoptosis in PC12 neurons requires astroglial-derived NO and NF-kappaB-dependent expression of NOS2.     

Effect of Gas6 on secretory phospholipase A(2)-IIA-induced apoptosis in cortical neurons

Gas6, a product of the growth-arrest-specific gene 6, protects cortical neurons from amyloid beta protein (Abeta)-induced apoptosis. Neuronal apoptosis is also caused by human group IIA secretory phospholipase A(2) (sPLA(2)-IIA), which is expressed in the cerebral cortex after brain ischemia. sPLA(2)-IIA induces Ca(2+) influx via L-type voltage-sensitive calcium channels (L-VSCCs), leading to its neurotoxicity. In the present study, we investigated effects of Gas6 on sPLA(2)-IIA-induced cell death in primary cultures of rat cortical neurons. sPLA(2)-IIA caused neuronal cell death in a concentration- and time-dependent manner. Gas6 significantly prevented neurons from sPLA(2)-IIA-induced cell death. Gas6 suppressed sPLA(2)-IIA-induced apoptotic features such as the condensation of chromatin and the fragmentation of DNA. Prior to cell death, sPLA(2)-IIA increased the influx of Ca(2+) into neurons through L-VSCCs. Gas6 significantly inhibited the sPLA(2)-IIA-induced Ca(2+) influx. The blocker of L-VSCCs also suppressed sPLA(2)-IIA-induced neuronal cell death. The cortical cultures contained few non-neuronal cells, indicating that Gas6 affected the survival of neurons directly, but not indirectly via non-neuronal cells. In conclusion, we demonstrate that Gas6 rescues cortical neurons from sPLA(2)-IIA-induced apoptosis. Furthermore, the present study indicates that inhibition of L-VSCC contributes to the neuroprotective effect of Gas6.     

Lithium-pilocarpine-induced status epilepticus produces necrotic neurons with internucleosomal DNA fragmentation in adult rats

Prolonged and continuous epileptic seizures [status epilepticus (SE)] produce a widespread pattern of neuronal death, primarily in limbic brain regions. Because it has been suggested that seizure-induced neuronal death may be apoptotic in nature, we tested the hypothesis that lithium-pilocarpine-induced status epilepticus (LPCSE) produces apoptotic neurons. LPCSE lasting 3 h was induced in male Wistar rats which were allowed to recover for 24 or 72 h before perfusion-fixation. Neuronal death was assessed by light microscopy with the haematoxylin-and-eosin stain (H&E), with in situ DNA nick-end labelling (TUNEL stain), by electron microscopy, and by agarose gel electrophoresis of DNA extracted from vulnerable brain regions. Ultrastructurally, acidophilic neurons identified with H&E were dark, shrunken and necrotic in appearance, exhibiting pyknotic nuclei, irregular, dispersed chromatin clumps and cytoplasmic vacuolization. No cells with apoptotic features were seen. Acidophilic neurons were found in 21 out of 23 brain regions examined, and comprised 26-45% of the total number of neurons examined. A subset of these neurons (< 10% of the total number of neurons) were TUNEL-positive at 72 h, but not 24 h, after SE. Internucleosomal DNA cleavage (DNA 'laddering') was found in the six brain regions examined ultrastructurally 24 and 72 h after SE. These results indicate that, in adult rats, LPCSE produces neuronal injury with the appearance of necrosis rather than apoptosis. The necrotic neurons show nuclear pyknosis, chromatin condensation and internucleosomal DNA fragmentation, confirming the nonspecificity of these nuclear changes. Internucleosomal DNA cleavage and other programmed cell death mechanisms can be activated by SE in neurons which become necrotic.     

Activity-dependent survival of neurons in culture: a model of slow neurodegeneration

Central neurons express persistent spontaneous electrical network activity both in the developing brain in vivo as well as in dissociated cultures. This electrical activity is important for the formation of connections among neurons, and for their survival. Prolonged suppression of the spontaneous activity using the sodium channel blocker tetrodotoxin (TTX) causes the death of the cultured neurons. In the present study, we investigated molecular mechanisms that may underlie the activity-suppressed slow degeneration of cortical neurons in culture. Already after 6–7 days of exposure to TTX, neurons begin to express apoptotic vacuoles and shrunken dendrites. Eventually, neurons activate p53, caspase-3 and BAX, hallmarks of neuronal apoptosis, before they die. This death is restricted to neurons, and no parallel process is seen in glial cells that co-exist in the culture. These experiments may lead to a better understanding of slow neuronal death, akin to that found in neurodegenerative diseases of the brain.     

Acetaldehyde cytotoxicity in cultured rat astrocytes

The effect of acetaldehyde on astrocytes have been investigated because not only do they play an important role in brain maturation but also recent reports have shown their delayed proliferation following both 'in vivo' and 'in vitro' ethanol exposure. Biochemical parameters related to apoptotic and necrotic processes were examined in primary cultures of rat astrocytes exposed for 4 days to acetaldehyde generated from ethanol by co-cultured alcohol dehydrogenase-transfected Chinese hamster ovary cells. Acetaldehyde levels in the culture media attained concentrations of approximately 450 microM. To study ethanol effects, alcohol oxidation was inhibited by 4-methylpyrazole (an inhibitor of alcohol dehydrogenase). Acetaldehyde but not ethanol increased intracellular calcium levels by 155%. Moreover, significant DNA fragmentation was detected using a random oligonucleotide primed synthesis assay, by flow cytometry and when using agar gel electrophoresis. Transglutaminase activity was elevated in the cells treated with acetaldehyde but when acetaldehyde formation was inhibited by 4-methylpyrazole the enzyme activity was unaffected. Nitrate levels in the culture media were unchanged. Additionally, microscopic examination of cell nuclei revealed chromatin condensation in astrocytes exposed to acetaldehyde. It can be concluded, that in 'in vitro' acetaldehyde exposed rat astrocytes apoptotic pathways are activated.     

Thalamic retrograde degeneration in the congenitally hydrocephalic rat is attributable to apoptotic cell death

Congenitally hydrocephalic HTX rats develop ventricular dilatation with extensive damage of the cerebral white matter. Recently, we have reported that neuronal cell death also occurs in the thalamus of HTX rats. To investigate the mechanism underlying this thalamic degeneration in these animals, we carried out a histopathological study of the brain at different phases of postnatal development. Eosinophilic neurons with condensed chromatin or fragmented nuclei were observed in the thalamus from postnatal day 17 onward. The incidence of cell death in the thalamus increased with the progression of hydrocephalus. Ultrastructurally, thalamic neurons occasionally had apoptotic features including nuclear chromatin condensation and marginalization. Immunohistochemically, single‐stranded DNA‐positive neuronal nuclei were found in the thalamus. They were also positively stained with the TUNEL method. Marked loss of myelin and axons with many TUNEL‐positive oligodendrocytes were found in the cerebral white matter. These findings suggest that the neuronal cell death observed in the thalamus in hydrocephalic HTX rats is retrograde degeneration due to extensive damage of axons in the cerebral white matter and that the thalamic retrograde degeneration is attributable to apoptotic cell death.     

Cyclosporine induces neuronal apoptosis and selective oligodendrocyte death in cortical cultures

Cyclosporine is used clinically as an immunosuppressant, but carries a risk of central nervous system toxicity due to undefined mechanisms. We examined the abiility of cyclosporine expsure to kill cultured mouse cortical neurons and glia. Mixed neuron/glial cultures exposed to 1 to 20 μM cyclosporine for 24 to 48 hours developed concentration-dependent neuronal death, with most neurons destroyed by 20 μM cyclosporine. This Neuronal death was characterized by cell body shrinkage and bleebbing, chromatin condenstation, and internucleosomal DNA fragmentation, consistent with apoptosis. Neuronal death was reduced by addition of cycloheximide, brain-derived neurotrophic factor, or insulin-like growth factor I but not N-methyl-D-aspartate- or AMPA-type glutamate receptor antagonists. Oligodendrocytes were more sensitive to cyclosporine-induced damage than were neurons, but astrocytes were relatively resistant. Oligodendrocyte death was accompanied by positive TUNEL (terminal deoxynucleotidyl transferase-mediated deoxyuridinetriphosphate-biotin nick end-labeling) staining and was attenuatd by application of ciliary neurotrophic factor or insulin-like growth factor I but not glutamate receptro antagonists. Present observations raise the possibility that the central nervous system toxicity syndrome associated with cyclosporine may be caused by the drug-induced death of oligodendrocytes and neurons.     

Susceptibility to apoptosis varies with time in culture for murine neurons and astrocytes: changes in gene expression and activity

Apoptotic pathways in the brain may differ depending on cell type and developmental stage. To understand these differences, we studied several apoptotic proteins in the murine cortex and primary cultures of neurons and astrocytes of various ages in culture. We then induced apoptosis in our cultures using serum deprivation (SD) and observed changes in these apoptotic proteins. When analyzed by nuclear morphology and TUNEL staining, early cultures showed greater apoptotic injury compared with late cultures, and neuronal cultures showed greater apoptosis than astrocyte cultures. The decrease in apoptosis with development correlated best with a down-regulation of procaspase-3 and bax and decreasing caspase activation. Early culture astrocytes had higher caspase-11 levels compared with neurons. Mitogen-activated protein (MAP) kinases were also differentially expressed with activation of extracellular signal-regulated kinase (ERK) and p38 higher in early culture astrocytes and stress-activated protein kinase/C-jun N-terminal kinase (SAPK/JNK) greater in early culture neurons. However, caspase inhibitors, but not MAP kinase inhibitors reduced cell death. Our findings demonstrate that apoptosis regulatory proteins display cell type and developmentally specific expression and activation.