Bcl-2 phosphorylation in the BH4 domain precedes caspase-3 activation and cell death after neonatal cerebral hypoxic-ischemic injury

To date, there are very few in vivo studies addressing the role of Bcl-2 phosphorylation. In a model of neonatal hypoxic-ischemic (HI) brain injury, we characterized the spatial and temporal phosphorylation of Bcl-2 at serine-24 (PS24-Bcl-2), using a site-specific antibody. Very few cells positive for PS24-Bcl-2 were found in control animals, but the number increased during reperfusion in all investigated brain areas in the ipsilateral hemisphere after HI, particularly in the border region between intact and damaged tissue. The highest numbers were encountered 24 h post-HI. Phosphorylation of Bcl-2 at serine-24 coincided with cytochrome c release after hypoxia-ischemia and preceded caspase-3 activation. Injured neurons displayed a predominantly nuclear, but also mitochondrial, localization of PS24-Bcl-2 immunoreactivity. In conclusion, phosphorylation of Bcl-2 at serine 24 was induced by hypoxia-ischemia, presumably resulting in loss of its anti-apoptotic function.     

FK506 prevents mitochondrial-dependent apoptotic cell death induced by 3-nitropropionic acid in rat primary cortical cultures

The mitochondrial toxin 3-nitropropionic acid (3-NP) has been largely used to study neurodegenerative disorders in which bioenergetic defects are implicated. In the present study, we analyzed the molecular pathways involved in FK506 neuroprotection against cell death induced by 3-NP, using cultured cortical neurons. 3-NP induced cytochrome c release and increased caspases -2, -3, -8, and -9-like activities, although, calpain activity was not significantly affected. FK506 decreased cytochrome c release and caspase-3-like activity induced by 3-NP, without changing the activities of other caspases. FK-506 also decreased the number of apoptotic neurons, determined by Hoechst. Under these conditions, FK506 alone significantly reduced calcineurin activity by about 50%. Our results also showed a decrease in mitochondrial Bax and an increase in mitochondrial Bcl-2 levels upon exposure to FK506 and 3-NP. However, no significant changes occurred in total Bcl-2 and Bax levels. Altogether, the results suggest that FK506 neuroprotection against 3-NP-induced apoptosis is associated with the redistribution of Bcl-2 and Bax in the mitochondrial membrane.     

Impaired mitochondrial energy metabolism and neuronal apoptotic cell death after chronic dichlorvos (OP) exposure in rat brain

The present study elucidates a possible mechanism by which chronic organophosphate exposure (dichlorvos 6 mg/kg bw, s.c. for 12 weeks) causes neuronal degeneration. Mitochondria, as a primary site of cellular energy generation and oxygen consumption represent itself a likely target for organophosphate poisoning. Therefore, the objective of the current study was planned with an aim to investigate the effect of chronic dichlorvos exposure on mitochondrial calcium uptake, oxidative stress generation and its implication in the induction of neuronal apoptosis in rodent model. Mitochondrial preparation from dichlorvos (DDVP) treated rat brain demonstrated significant increase in mitochondrial Ca(2+) uptake (644.2 nmol/mg protein). Our results indicated decreased mitochondrial electron transfer activities of cytochrome oxidase (complex IV) along with altered mitochondrial complex I, and complex II activity, which might have resulted from elevated mitochondrial calcium uptake. The alterations in the mitochondrial calcium uptake and mitochondrial electron transfer enzyme activities in turn might have caused an increase in malondialdehyde, protein carbonyl and 8-hydroxydeoxyguanosine formation as a result of enhanced lipid peroxidation, and as well as protein and mtDNA oxidation. All this could have been because of enhanced oxidative stress, decreased GSH levels and also decreased Mn-SOD activity in the mitochondria isolated from dichlorvos treated rat brain. Thus, chronic organophosphate exposure has the potential to disrupt cellular antioxidant defense system which in turn triggers the release of cytochrome c from mitochondria to cytosol as well as caspase-3 activation in dichlorvos treated rat brain as revealed by immunoblotting experiments. Low-level long-term organophosphate exposure finally resulted in oligonucleosomal DNA fragmentation, a hallmark of apoptosis. These studies provide an evidence of impaired mitochondrial bioenergetics and apoptotic neuronal degeneration after chronic low-level exposure to dichlorvos.     

Oxidative stress, mitochondrial permeability transition and activation of caspases in calcium ionophore A23187-induced death of cultured striatal neurons

Disruption of intracellular calcium homeostasis is thought to play a role in neurodegenerative disorders such as Huntington's disease (HD). To study different aspects of putative pathogenic mechanisms in HD, we aimed to establish an in vitro model of calcium-induced toxicity in striatal neurons. The calcium ionophore A23187 induced a concentration- and time-dependent cell death in cultures of embryonic striatal neurons, causing both apoptosis and necrosis. Cell death was significantly reduced by the cell-permeant antioxidant manganese(III)tetrakis(4-benzoic acid) porphyrin (MnTBAP). Cyclosporin A and its analogue N-MeVal-4-cyclosporin also reduced the incidence of cell death, suggesting the participation of mitochondrial permeability transition in this process. Furthermore, addition of either of two types of caspase inhibitors, Ac-YVAD-CHO (acetyl-Tyr-Val-Ala-Asp-aldehyde) and Ac-DEVD-CHO (acetyl-Asp-Glu-Val-Asp-aldehyde), to the striatal cells blocked A23187-induced striatal cell death in a concentration-dependent manner. These results suggest that oxidative stress, opening of the mitochondrial permeability transition pore and activation of caspases are important steps in A23187-induced cell death.     

Neurotransmitters and neuronal apoptotic cell death of chronically aluminum intoxicated Nile catfish (Clarias gariepinus) in response to ascorbic acid supplementation

Few studies have been carried out to assess the neurotoxic effect of aluminum (Al) on the aquatic creatures. This study aims to evaluate the neurotoxic effects of long term Al exposure on the Nile catfish (Clarias gariepinus) and the potential ameliorative influence of ascorbic acid (ASA) over a 180 days exposure period. Forty eight Nile catfish were divided into four groups: control group, placed in clean water, ASA exposed group (5 mg/l), AlCl₃ received group (28.96 μg/l; 1/20 LC₅₀), and group received AlCl₃ concomitantly with ASA. Brain tissue was examined by using flow cytometry to monitor the apoptotic cell population, HPLC analysis for the quantitative estimation of brain monoamine neurotransmitters [serotonin (5-HT), dopamine (DA), norepinephrine (NE)]. The amino acid neurotransmitters [serum taurine, glycine, aspartate and glutamine and brain gamma aminobutyric acid (GABA)] levels were assessed, plus changes in brain tissue structure using light microscopy. The concentration of Al in both brain tissue and serum was determined by using atomic absorption spectrophotometery. The Al content in serum and brain tissue were both elevated and Al exposure induced an increase in the number of apoptotic cells, a marked reduction of the monoamine and amino acids neurotransmitters levels and changes in tissue morphology. ASA supplementation partially abolished the effects of AL on the reduced neurotransmitter, the degree of apoptosis and restored the morphological changes to the brain. Overall, our results indicate that, ASA is a promising neuroprotective agent against for Al-induced neurotoxicity in the Nile catfish.     

Posttreatment with the Ca(2+)-Mg(2+)-dependent endonuclease inhibitor aurintricarboxylic acid abolishes genotoxic agent-induced nuclear condensation and DNA fragmentation and decreases death of astrocytes

DNA fragmentation and nuclear condensation are important nuclear changes in apoptosis. In this study we determined whether DNA fragmentation and nuclear condensation occur in astrocytes treated with 100-200 microM of the genotoxic agent M-nitroso-N-nitroguanidine (MNNG). Our study also investigated the roles of Ca(2+)-Mg(2+)-dependent endonuclease (CME) in the MNNG-induced nuclear changes. We found that MNNG induced profound ATP depletion as well as marked nuclear condensation and DNA fragmentation in the cells. Both the nuclear condensation and the DNA fragmentation were abolished by posttreatment of the cells with the CME inhibitor aurintricarboxylic acid (ATA). The ATA posttreatment also significantly, but only partially, decreased MNNG-induced cell death. In contrast, pretreatment plus cotreatment with ATA did not affect either MNNG-induced nuclear condensation or cell death. Our study further suggests that ATA does not decrease the cytotoxicity of MNNG by directly inhibiting poly(ADP-ribose) polymerases. Collectively, our observations suggest that MNNG can induce both DNA fragmentation and nuclear condensation in astrocytes by a CME-dependent mechanism, which partially contributes to the genotoxic agent-induced cell death. Published 2008 Wiley-Liss, Inc.     

Estrogen blocks 3-nitropropionic acid-induced Ca2+i increase and cell damage in cultured rat cerebral endothelial cells

Systemic administration of 3-nitropropionic acid (3-NPA, a mycotoxin) induces brain damage accompanied by disturbance in the blood-brain barrier (BBB). Since the endothelial cells are important components of the BBB and the first target of a systemic intoxication, in the present study, the effect of 3-NPA on primary cultured rat brain endothelial cells (rBECs) was examined by studying intracellular Ca(2+) ([Ca(2+)](i)) response using imaging techniques with fura-2. rBECs were prepared using a method of Kis et al. [Eur. J. Pharmacol. 368 (1999) 35-42] and Szabo et al. [Neurobiology 5 (1997) 1-16]. Almost all cells were immunoreactive to antibody against the factor VIII-related antigen (von-Willebrand factor). They showed a typical dose-dependent increase of [Ca(2+)](i) in response to ATP or bradykinin. Low concentrations of 3-NPA (1.7 mM, 3.4 mM) caused no changes, and a medium concentration (6.8 mM) increased the [Ca(2+)](i) gradually and progressively, and the increase was reversed incompletely back to the resting level after washing. A high concentration (13.6 mM) increased the [Ca(2+)](i) irreversibly. These elevations of [Ca(2+)](i) were absent in a Ca(2+)-free medium. In endothelial cells treated with 17beta-estradiol (above 10(-5) M) or with a selective estrogen receptor modulator, tamoxifen (5 x 10(-7) M), no elevation of [Ca(2+)](i) was observed with 3-NPA treatment. The response to ATP was impaired after application of 3-NPA, but it was preserved by cotreatment with 17beta-estradiol or tamoxifen. An estrogen receptor antagonist ICI 182,780 inhibited these effects by 17beta-estradiol or tamoxifen. Lysosomal neutral red uptake and TUNEL experiments revealed the necrotic but not apoptotic cell death at least in this acute stage. Data indicate that a medium to high concentration of 3-NPA induces damage on rBECs as revealed by an accumulation of [Ca(2+)](i), but the damage was protected by cotreatment with 17beta-estradiol or tamoxifen, suggesting that estrogen may be protective for the brain vascular damage via estrogen receptor.     

Caspase-3 dependent proteolytic activation of protein kinase C delta mediates and regulates 1-methyl-4-phenylpyridinium (MPP+)-induced apoptotic cell death in dopaminergic cells: relevance to oxidative stress in dopaminergic degeneration

1-Methyl-4-phenylpyridinium (MPP+), the neurotoxic metabolite of MPTP (1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine), induces apoptosis in dopaminergic neurons; however, the cellular mechanisms underlying the degenerative process are not well understood. In the present study, we demonstrate that caspase-3 mediated proteolytic activation of protein kinase C delta (PKC delta) is critical in MPP+-induced oxidative stress and apoptosis. MPP+ exposure in rat dopaminergic neuronal cells resulted in time-dependent increases in reactive oxygen species generation, cytochrome c release, and caspase-9 and caspase-3 activation. Interestingly, MPP+ induced proteolytic cleavage of PKC delta (72-74 kDa) into a 41-kDa catalytic and a 38-kDa regulatory subunit, resulting in persistently increased kinase activity. The caspase-3 inhibitor Z-DEVD-fmk effectively blocked MPP+-induced PKC delta cleavage and kinase activity, suggesting that the proteolytic activation is caspase-3 mediated. Similar results were seen in MPP+-treated rat midbrain slices. Z-DEVD-fmk and the PKC delta specific inhibitor rottlerin almost completely blocked MPP+-induced DNA fragmentation. The superoxide dismutase mimetic, MnTBAP also effectively attenuated MPP+-induced caspase-3 activation, PKC delta cleavage, and DNA fragmentation. Furthermore, rottlerin attenuated MPP+-induced caspase-3 activity without affecting basal activity, suggesting positive feedback activation of caspase-3 by PKC delta. Intracellular delivery of catalytically active recombinant PKC delta significantly increased caspase-3 activity, further indicating that PKC delta regulates caspase-3 activity. Finally, over-expression of a kinase inactive PKC delta K376R mutant prevented MPP+-induced caspase activation and DNA fragmentation, confirming the pro-apoptotic function of PKC delta in dopaminergic cell death. Together, we demonstrate for the first time that MPP+-induced oxidative stress proteolytically activates PKC delta in a caspase-3-dependent manner to induce apoptosis and up-regulate the caspase cascade in dopaminergic neuronal cells.     

Nature and distribution of brain lesions in rats intoxicated with 3-nitropropionic acid: A type of hypoxic (energy deficient) brain damage

Summary The clinical signs and morphological brain lesions associated with histotoxic hypoxia induced by subcutaneous injection of 3-nitropropionic acid (NPA) in rats are described, and compared to hypoxic brain damage from other causes including ischemia and hypoglycemia. The brains were perfusion-fixed with paraformaldehyde/glutaraldehyde fixative, and examined by light and electron microscopy. Intoxicated rats developed severe neurological disease characterized by somnolence, uncoordinated gait with stereotypical paddling movements, and ventral or lateral recumbency. Recumbent rats had a selective, bilaterally symmetrical pattern of severe morphological injury in the caudate-putamen, hippocampus, and thalamus. Recumbency was a consistent indicator of the development of morphological brain lesions. In contrast to reports describing rat models of ischemia and hypoglycemia, morphological injury was not seen in the cerebral and cerebellar cortices of NPA-intoxicated rats. Ultrastructurally, neuronal alterations ranged from chromatin clumping with increased cytoplasmic lucency to severe cellular shrinkage or swelling with marked mitochondrial swelling (high amplitude swelling). White matter alterations included axonal swelling and adaxonal splitting of myelin lamellae. Vascular changes included perivascular deposits of proteinaceous material presumably from leakage of serum proteins, variable electron lucency of endothelial cell cytoplasm, an apparent increase in pinocytotic vesicles, rare platelet thrombosis of capillaries, and rare intravascular blebs of luminal plasma membrane. As a model of brain damage following energy deficiency, NPA intoxication has the advantages of producing morphological brain injury in a highly predictable anatomical pattern, and at a time paralleling the onset of clinical recumbency.Supported by US Public Health Service grant numbers 5 T15 CA09408 and 5 T32 ES07152 awarded by the National Cancer Institute and the National Institute of Environmental Health Sciences, and by the CSU Agricultural Experiment Station/USDA Animal Health and Disease Research Program     

Possible involvement of cytosolic phospholipase A(2) in cell death induced by 1-methyl-4-phenylpyridinium ion, a dopaminergic neurotoxin, in GH3 cells

Previously we reported that 1-methyl-4-phenylpyridinium ion (MPP(+)), a dopaminergic neurotoxin, induced apoptosis of GH3 cells established from rat anterior pituitary. In the present study, the role of MPP(+) along with that of other apoptotic factors such as Ca(2+) and H(2)O(2) in cell death was examined. Ionomycin induced DNA fragmentation and lactate dehydrogenase (LDH) leakage in GH3 cells. H(2)O(2) also induced LDH leakage. Co-addition of MPP(+), in conditions where MPP(+) had no effect by itself, enhanced ionomycin- and H(2)O(2)-induced cell death. Because the stimulation of phospholipase A(2) (PLA(2)) causing arachidonic acid (AA) release has been proposed to be involved in neuronal cell death, the effect of MPP(+) on AA release in GH3 cells was investigated. MPP(+) treatment for 8 h enhanced ionomycin- and H(2)O(2)-stimulated AA release mediated by activation of cytosolic PLA(2) in a concentration-dependent manner, although MPP(+) by itself had no effect on AA release. An inhibitor of cytosolic PLA(2) inhibited MPP(+)-induced cell death. These findings suggest a synergistic effect of MPP(+) on Ca(2+)- and H(2)O(2)-induced cell death, and the involvement of cytosolic PLA(2) activation in MPP(+)-induced cell death in GH3 cells. Pretreatment with a caspase inhibitor or EGF did not modify the ionomycin- or H(2)O(2)-induced AA release, or enhancement by MPP(+), but the pretreatment inhibited the cell death in the presence and absence of MPP(+). The involvement of caspase(s) on activation of PLA(2) by MPP(+) was excluded, and EGF inhibited MPP(+)-induced cell death downstream of the AA release.