Protective Effect of Melatonin on Methamphetamine-Induced Apoptosis in Glioma Cell Line

Methamphetamine (METH) is a highly addictive drug causing neurodegenerative diseases. METH has been known to be neurotoxic by inducing oxidative stress, free radical, and pro-inflammatory cytokines. Previous studies have shown that METH could induce neuron and glial cell death, especially inducing glial cell-mediated neurotoxicity that plays a critical role in stress-induced central nervous system damage. Therefore, the aim of the present study is to explore the mechanisms of METH-induced cell death in the glial cell. METH-induced glial cells death is mediated via mitochondrial damage pathway. METH activates the upregulation of the Bax, cytochrome c, cleavage caspase 9 and 3 proteins, and downregulation of Bcl-X_L protein in cascade. Pretreatment with melatonin, a neurohormone secreted by the pineal gland, effectively reduced glial cell death. Moreover, melatonin increased the Bcl-X_L/Bax ratio but reduced the level of cytochrome c, cleavage caspase 9 and 3 proteins. Therefore, these results demonstrated that melatonin could reduce the cytotoxic effect of METH by decreasing the mitochondrial death pathway activation in glial cells. This outcome suggests that melatonin might be beneficial as the neuroprotection in neurodegenerative diseases caused by METH or other pathogens.     

Postnatal apoptosis in cerebellar granule cells of homozygous leaner (<Emphasis Type="Italic">tg</Emphasis><Superscript>la</Superscript>/<Emphasis Type="Italic">tg</Emphasis><Superscript>la</Superscript>) mice

Leaner mice carry a homozygous, autosomal recessive mutation in the mouse CACNA1A gene encoding the α_1A subunit of P/Q-type calcium channels, which results in an out-of-frame splicing event in the carboxy terminus of the α_1A protein. Leaner mice exhibit severe ataxia, paroxysmal dyskinesia and absence seizures. Functional studies have revealed a marked decrease in calcium currents through leaner P/Q-type channels and altered neuronal calcium ion homeostasis in cerebellar Purkinje cells. Histopathological studies of leaner mice have revealed extensive postnatal cerebellar Purkinje and granule cell loss. We examined the temporospatial pattern of cerebellar granule cell death in the leaner mouse between postnatal days (P) 10 and 40. Our observations clearly indicate that leaner cerebellar granule cells die via an apoptotic process and that the peak time of neuronal death is P20. We did not observe a significant increase in microglial and astrocytic responses at P20, suggesting that glial responses are not a cause of neuronal cell death. We propose that the leaner cerebellar granule cell represents anin vivo animal model for low intracellular [Ca²⁺]-induced apoptosis. Since intracellular [Ca²⁺] is critical in the control of gene expression, it is quite likely that reduced intracellular [Ca²⁺] could activate a lethal cascade of altered gene expression leading to the apoptotic granule cell death in the leaner cerebellum.     

Release of mitochondrial Opa1 following oxidative stress in HT22 cells

Cellular mechanisms involved in multiple neurodegenerative diseases converge on mitochondria to induce overproduction of reactive oxygen species, damage to mitochondria, and subsequent cytochrome c release. Little is currently known regarding the contribution mitochondrial dynamics play in cytochrome c release following oxidative stress in neurodegenerative disease. Here we induced oxidative stress in the HT22 cell line with glutamate and investigated key mediators of mitochondrial dynamics to determine the role this process may play in oxidative stress induced neuronal death. We report that glutamate treatment in HT22 cells induces increase in reactive oxygen species (ROS), release of the mitochondrial fusion protein Opa1 into the cytosol, with concomitant release of cytochrome c. Furthermore, following the glutamate treatment alterations in cell signaling coincide with mitochondrial fragmentation which culminates in significant cell death in HT22 cells. Finally, we report that treatment with the antioxidant tocopherol attenuates glutamate induced-ROS increase, release of mitochondrial Opa1 and cytochrome c, and prevents cell death.     

AMPA Neurotoxicity in Cultured Cerebellar Granule Neurons: Mode of Cell Death

Various forms of cell death induced by the glutamate receptor agonist,

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-amino-3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA), were analyzed by determining the capacity of cultured cerebellar granule cells to metabolize 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) into formazan, by measuring the leakage of lactate dehydrogenase (LDH), by using confocal microscopy to visualize propidium iodide staining of apoptotic nuclei, and by using field inversion gel electrophoresis (FIGE) for the detection of AMPA-produced cleavage of DNA into high molecular-weight fragments (50 kbp). All these measures indicated that stimulation of AMPA receptors may be involved in the neurotoxic effects of glutamate, and that AMPA-induced neurotoxicity in cerebellar granule cells display morphologically distinct features of both necrotic and apoptotic modes of cell death. In agreement with previous observations, a blockade of AMPA receptor desensitization was necessary to unmask AMPA-induced functional responses in cultured cerebellar granule neurons in vitro. Microfluorimetric measurements of free cytoplasmic calcium concentrations ([Ca²⁺]_i) in single cerebellar neurons revealed that AMPA neurotoxicity was accompanied by a pronounced elevation of [Ca²⁺]_i. Our current results add further evidence to the notion that glutamate-induced neurotoxicity in cerebellar granule cells is mediated not only through NMDA receptors but also through a direct activation of AMPA receptor-regulated cation channels.     

Mitochondria-derived reactive oxygen species mediate caspase-dependent and -independent neuronal deaths

Mitochondrial dysfunction and oxidative stress are implicated in many neurodegenerative diseases. Mitochondria-targeted drugs that effectively decrease oxidative stress, protect mitochondrial energetics, and prevent neuronal loss may therefore lend therapeutic benefit to these currently incurable diseases. To investigate the efficacy of such drugs, we examined the effects of mitochondria-targeted antioxidants MitoQ₁₀ and MitoE₂ on neuronal death induced by neurotrophin deficiency. Our results indicate that MitoQ₁₀ blocked apoptosis by preventing increased mitochondria-derived reactive oxygen species (ROS) and subsequent cytochrome c release, caspase activation, and mitochondrial damage in nerve growth factor (NGF)-deprived sympathetic neurons, while MitoE₂ was largely ineffective. In this paradigm, the most proximal point of divergence was the ability of MitoQ₁₀ to scavenge mitochondrial superoxide (O₂⁻). MitoQ₁₀ also prevented caspase-independent neuronal death in these cells demonstrating that the mitochondrial redox state significantly influences both apoptotic and nonapoptotic pathways leading to neuronal death. We suggest that mitochondria-targeted antioxidants may provide tools for delineating the role and significance of mitochondrial ROS in neuronal death and provide a new therapeutic approach for neurodegenerative conditions involving trophic factor deficits and multiple modes of cell death.     

3,4-Dihydroxyphenylacetic acid (DOPAC) modulates the toxicity induced by nitric oxide in PC-12 cells via mitochondrial dysfunctioning

It has been postulated that dihydroxyphenylacetic acid (DOPAC), a major dopamine metabolite, and nitric oxide (NO) induce mitochondrial dysfunction in a synergistic manner. We examined the combined effects of NO and DOPAC on PC-12 cells in terms of cell viability, nuclear morphology, mitochondrial parameters and cell death mechanisms. The apoptotic cell death induced by the NO-donor, S-nitroso-N-acetylpenicillamine (SNAP), was differently modulated by DOPAC as a function of DOPAC/cell ratios. Whereas below 200 nmol/10⁶ cells, DOPAC inhibited a typical apoptotic pathway induced by exposure the cells to the NO donor, above 200 nmol DOPAC/10⁶ cells, the cell death was not only enhanced but encompassed a distinct mechanism. Loading the cells with dopamine mimicked the effects of DOPAC. Specifically, the combination of DOPAC and NO induced an early mitochondrial membrane potential dissipation and ATP depletion followed by loss of cellular membrane integrity. Mitochondrial dysfunction was accompanied by the release of cytochrome c in both cases, NO individually and in combination with DOPAC, but caspase-3 and caspase-9 activation were only observed in the absence of DOPAC. DOPAC alone was ineffective. Thus, our results suggest a role for DOPAC as a modulator of cell fate and point to a pathway of cell death involving DOPAC and NO, via mechanisms that include mitochondrial dysfunction but do not involve the activation of the typical apoptotic caspase cascade. The significance of these results is discussed in connection with the mechanisms of cell death underlying Parkinson's disease.     

Mitochondrial dysfunction in a neural cell model of spinal muscular atrophy

Mutations of the survival motor neuron (SMN) gene in spinal muscular atrophy (SMA) lead to anterior horn cell death. The cause is unknown, but motor neurons depend substantially on mitochondrial oxidative phosphorylation (OxPhos) for normal function. Therefore, mitochondrial parameters were analyzed in an SMA cell culture model using small interfering RNA (siRNA) transfection that decreased Smn expression in NSC‐34 cells to disease levels. Smn siRNA knock‐down resulted in 35% and 66% reduced Smn protein levels 48 and 72 hr posttransfection, respectively. ATP levels were reduced by 14% and 26% at 48 and 72 hr posttransfection, respectively, suggesting decreased ATP production or increased energy demand in neural cells. Smn knock‐down resulted in increased mitochondrial membrane potential and increased free radical production. Changes in activity of cytochrome c oxidase (CcO), a key OxPhos component, were observed at 72 hr with a 26% increase in oxygen consumption. This suggests a compensatory activation of the aerobic pathway, resulting in increased mitochondrial membrane potentials, a condition known to lead to the observed increase in free radical production. Further testing suggested that changes in ATP at 24 hr precede observable indices of cell injury at 48 hr. We propose that energy paucity and increased mitochondrial free radical production lead to accumulated cell damage and eventual cell death in Smn‐depleted neural cells. Mitochondrial dysfunction may therefore be important in SMA pathology and may represent a new therapeutic target. © 2009 Wiley‐Liss, Inc.     

Mechanisms of Compartmental Purkinje Cell Death and Survival in the Lurcher Mutant Mouse

The Lurcher mutant mouse is characterized by its ataxic gait and loss of cerebellar Purkinje cells and their afferents, granule cells and olivary neurons, during the first weeks of postnatal development. For the 50 years since its discovery, the heterozygous Lurcher mutant has served as an important model system for studying neuron–target interactions in the developing cerebellum and cerebellar function. The identification of the Lurcher (Lc) gene over 10 years ago as a gain-of-function mutation in the δ2 glutamate receptor (GluRδ2) led to extensive studies of cell death mechanisms in the Lc/+ cerebellum. The advantage of this model system is that GluRδ2⁺ receptors and GluRδ2 ^Lc channels are expressed predominantly in Purkinje cells, making it possible to study the effects of a well-characterized leak current in a well-defined cell type during a critical phase of neuronal development. Yet there is still controversy surrounding the mechanisms of neuronal death in Lc/+ Purkinje cells with competing hypotheses for necrotic, apoptotic, and autophagic cell death pathways as a consequence of the excitotoxic stress caused by the GluRδ2 ^Lc leak current. The goal of this review is to summarize recent studies that critically test the role of various cell death pathways in Lc/+ Purkinje cell degeneration with respect to evidence for the molecular heterogeneity of Purkinje cells. We propose that the expression of putative survival factors, such as heat shock proteins, in a subset of cerebellar Purkinje cells may affect cell death pathways and account for the pattern and diverse mechanisms of Lc/+ Purkinje degeneration.     

The metabotropic glutamate receptor mGlu5 controls the onset of developmental apoptosis in cultured cerebellar neurons

Cultured cerebellar granule cells grown in medium containing 10 mm K⁺ undergo apoptosis after 4–5 days in vitro (DIV), and, at that time, the activity of metabotropic glutamate (mGlu) receptors coupled to polyphosphoinositide (PI) hydrolysis begins to decline. In granule cells at 4 DIV, the mGlu receptor subtype mGlu5 was expressed at high levels. The expression of another PI-coupled mGlu receptor, the mGlu1a, was low at 4 DIV but increased during the following days. In cultures at 4–5 DIV, the few cells that already showed an apoptotic phenotype were devoid of mGlu5 receptors, but they all expressed mGlu1a receptors. The development of apoptosis was accelerated after treating the cultures with: (i) mGlu5 antisense oligonucleotides; (ii) the mixed mGlu receptor antagonist, (+)-α-methyl-4-carboxyphenylglycine; or (iii) the glutamate depleting enzyme, alanine aminotransferase. In contrast, an induced overexpression of mGlu5 receptors protected cultured granule cells against apoptotic death. We suggest that the activity of mGlu5 receptors supports cell survival, and a decline in the expression of mGlu5 receptors gives access to programmed cell death in cerebellar granule cells developing in primary cultures.     

Metabolic inhibition potentiates AMPA-induced Ca fluxes and neurotoxicity in rat cerebellar granule cells

The effects of partial metabolic inhibition (induced by 2 h exposure to low concentrations of cyanide (NaCN)) on the glutamate receptor agonist α-amino-3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA)-induced excitotoxicity and elevation of free cytoplasmic Ca²⁺ levels ([Ca²⁺]_i) were studied in glucose-deprived primary cultures of cerebellar granule cells. Co-application of AMPA plus NaCN caused a marked increase of cell death, with morphological features of both necrotic and apoptotic cell death as estimated by the capacity of cultured cerebellar granule cells to metabolize 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide into formazan (MTT method), and by measuring the amount of DNA fragmentation in neurons using an ELISA test for histone-bound DNA fragments, respectively. Cell morphology was assessed by confocal microscopy of propidium iodide-stained cultures. No toxic effects were observed when AMPA or a low concentration of NaCN (0.1–0.3 mM; in the presence of NMDA receptor antagonist MK-801; 10 μM) were applied alone. The neurotoxic actions induced by AMPA plus NaCN were preceded and accompanied by a significant elevation of [Ca²⁺]_i, as well as by depletion of neuronal ATP stores. The marked enhancement in the functional responsiveness of AMPA receptors in energetically compromised neurons suggests that at least under certain conditions AMPA receptors may play an important role in excitotoxic processes which might be of relevance for the slowly developing neuronal death seen in several neurodegenerative diseases.     

Kainate-induced apoptosis correlates with c-Jun activation in cultured cerebellar granule cells

We have investigated the involvement of c-Jun in cell death induced by exposure of primary cultures of murine cerebellar granule cells to the glutamate receptor agonist kainate (KA) and evaluated its possible use as a marker for apoptosis. Using cerebellar granule cell neurones from postnatal day 7 mice, we found that 1 hr exposure to KA (1–1000 μM) induced a concentration-dependent neuronal cell death with characteristic apoptotic morphology, including cell shrinkage, neurite blebbing and DNA fragmentation. In addition KA-induced a concentration-dependent expression of c-Jun mRNA and protein as determined by in situ hybridization and immunocytochemistry respectively. DNA fragmentation was detected using terminal transferase-mediated nick-end (TUNEL) labelling and agarose gel electrophoresis. KA-induced cell death was significantly attenuated by the non-NMDA receptor antagonist 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX; 50 μM), which shifted the concentration-response curve significantly rightward. The number of apoptotic cell bodies, determined by TUNEL, was also reduced by CNQX (50 μM), with only 15–20% of neurones staining positive after exposure to 1mM KA. In addition, the number of positively stained cells for c-Jun protein and mRNA was substantially reduced by CNQX (50 μM) as determined by random and representative cell counts. These results show for the first time that KA induced apoptotic neuronal death in cultured murine cerebellar granule cells involves the induction of c-Jun mRNA and protein, suggesting the involvement of this immediate early gene in excitotoxic receptor-mediated apoptosis and its potential use as a marker for apoptotic cell death. J. Neurosci. Res. 52:69–82, 1998. © 1998 Wiley-Liss, Inc.     

Menadione reduces rotenone-induced cell death in cerebellar granule neurons

Oxidative stress has been implicated in neuronal death caused by cerebral ischemia or some neurologic disorders. Chemical hypoxia (term defining the simulation by using respiratory inhibitors) chosen as in vitro ischemic model, was induced in primary cultures of rat cerebellar granule neurons by inhibitors of mitochondrial electron transport such as rotenone or paraquat (complex I), 3-nitropropionic acid (3-NPA, complex II), antimycin A (complex III), or sodium azide (complex IV). All compounds caused neuronal death determined by trypan blue staining and MTT-test. On the other hand, neurotoxicity of rotenone and paraquat but not of 3-NPA, antimycin or azide was significantly abolished by menadione (vitamin K3, 2-methyl-1,4-naphthoquinone). This neuroprotective effect of menadione was associated with a decrease of rotenone-induced free radical production.     

Mitochondrial dynamics following global cerebral ischemia

Global brain ischemia/reperfusion induces neuronal damage in vulnerable brain regions, leading to mitochondrial dysfunction and subsequent neuronal death. Induction of neuronal death is mediated by release of cytochrome c (cyt c) from the mitochondria though a well-characterized increase in outer mitochondrial membrane permeability. However, for cyt c to be released it is first necessary for cyt c to be liberated from the cristae junctions which are gated by Opa1 oligomers. Opa1 has two known functions: maintenance of the cristae junction and mitochondrial fusion. These roles suggest that Opa1 could play a central role in both controlling cyt c release and mitochondrial fusion/fission processes during ischemia/reperfusion. To investigate this concept, we first utilized in vitro real-time imaging to visualize dynamic changes in mitochondria. Oxygen-glucose deprivation (OGD) of neurons grown in culture induced a dual-phase mitochondrial fragmentation profile: (i) fragmentation during OGD with no apoptosis activation, followed by fusion of mitochondrial networks after reoxygenation and a (ii) subsequent extensive fragmentation and apoptosis activation that preceded cell death. We next evaluated changes in mitochondrial dynamic state during reperfusion in a rat model of global brain ischemia. Evaluation of mitochondrial morphology with confocal and electron microscopy revealed a similar induction of fragmentation following global brain ischemia. Mitochondrial fragmentation aligned temporally with specific apoptotic events, including cyt c release, caspase 3/7 activation, and interestingly, release of the fusion protein Opa1. Moreover, we uncovered evidence of loss of Opa1 complexes during the progression of reperfusion, and electron microscopy micrographs revealed a loss of cristae architecture following global brain ischemia. These data provide novel evidence implicating a temporal connection between Opa1 alterations and dysfunctional mitochondrial dynamics following global brain ischemia.     

Neuroprotective effect of melatonin against ischemia/reperfusion-induced neuronal apoptosis in mouse cerebellum

BACKGROUND: Some experiments have demonstrated that melatonin (N-aceyl-5-methoxytryptamine, Mel) has antioxidation. However, whether it has neuroprotective effect in the ischemia/reperfusion injury of central nervous system is unclear. OBJECTIVE: To observe the protective effect of Mel on ischemia/reperfusion-induced cerebellar neuronal apoptosis of rats, and the action mechanism. DESIGN: Controlled observation experiment. SETTING: Department of Biochemistry and Molecular Biology, Tongji Medical College, Huazhong University of Science and Technology. MATERIALS: Eight Sprague-Dawley rats aged 7–8 days and weighing 10–12 g were provided by Medical Experimental Animal Center, Tongji Medical College,Huazhong University of Science and Technology. Anti-cytochrome C monoclonal antibody was purchased from R & D Company; 7-dichlorodihydrofluorescein diacetate(DCFH-DA), rhodamine 123 and Mel were purchased from Sigma Company (USA). Lactate dehydrogenase (LDH) kit was purchased from Nanjing Jiancheng Bioengineering Institute. METHODS: This experiment was carried out in the laboratory for Department of Biochemistry and Molecule Biology, Tongji Medical College between October 2002 and March 2004. Cerebellar neurons of rats were cultured in vitro. After oxygen-glucose deprivation (OGD) for 90 minutes, 1×10–4,1×10–6, 1×10–9 mol/L Mel was added, respectively, namely high-, middle-, and low-concentration Mel groups. Cells, which were cultured by OGD, served as model group, and control group, in which OGD intervention was omitted, was set. ①Cytochrome C level of mitochondrial cells in each group was detected by ELISA method. ②LDH activity in the cell culture fluid was measured, and cell membrane permeability change was analyzed. The cells in the Mel group with the lowest LDH activity served as Mel treatment group, i.e. cells were cultured with OGD, and then Mel was added; Meanwhile, Mel prevention group was set, i.e. Mel was added before OGD. Intervention was not changed in the model group and control group. ③ DNA level was analyzed and cell apoptosis was observed by agarose gel electrophoresis(AGE). ④Mitochondrial transmembrane potential of cells, and apoptotic way in each group were analyzed by confocal laser scanning microscopy. MAIN OUTCOME MEASURES: ①Mitochondrial cytochrome C level of cerebellar nerve cells. ②LDH activity of cerebellar nerve cells. ③ DNA AGE results. ④Mitochondrial transmembrane potential change. RESULTS: ①Mitochondrial cytochrome C level of cerebellar nerve cells: cytochrome C was obviously released at 6 hours of OGD-reperfusion. Mel inhibited the release of cytochrome C in dose-dependent manner. ②LDH activity of cerebellar nerve cells: LDH activity (A value) was significantly lower in the high- and middle-concentration Mel groups than in the model group (P < 0.05). LDH activity (A value) in the low-concentration Mel group was 0.415 0±0.012 9, indicating that Mel could decrease LDH activity of OGD-treated cell supernatant and promote membrane stablization in dose-dependent manner. ③AGE results of DNA: 1×10–9 mol/L was considered as the best concentration of melatonin. Cell DNA was extracted for AGE. Results presented typical ladder shape, indicating apoptosis appeared, while apoptosis was lessened in the Mel treatment group and Mel prevention group.④Mitochondrial transmembrane potential change: Experimental results showed that green fluorescein was evenly distributed in cerebellar granule cells cultured normally, and the axons of neurons were very clear. The body of neurons was condensed and the axons disappeared after cerebellar granule cells undergoing OGD injury. Mel could completely reverse the effect of OGD. CONCLUSION: Mel can enhance cerebellar neuronal membrane stabilization of rats in dose-dependent manner, and suppress OGD-induced apoptosis of cerebellar granule cells by preventing against mitochondrial apoptosis.     

Toxic effect of L-2-chloropropionate on cultured rat cerebellar granule cells is ameliorated after inhibition of reactive oxygen species formation.

Oral administration of rats to L-2-chloropropionate (L-CPA) causes selective necrosis to the granule cell layer of the cerebellum in vivo and to cultured rat cerebellar granule cells in vitro. The present study was conducted to characterize the involvement of reactive oxygen species (ROS) in cell death of L-CPA to rat cerebellar granule cells in vitro. Exposure to L-CPA (0.625-10 mM) produced a concentration dependent increase in formation of 2,7-dichlorofluorescein (DCF) as a measure of formation of ROS. The elevation of ROS was inhibited after incubation of the cells with the ERK-type of MAP kinases inhibitor U0126, the mitochondrial permeability transition pore inhibitor cyclosporin A (CSA), the antioxidant vitamin E, and the spin trap N-tert-butyl-alpha-(2-sulfophenyl)-nitrone (S-PBN). Measurements of nitrite (NO(2)) in the cell culture supernatant using the Griess reagent indicate generation of nitric oxide (NO) after exposure to L-CPA. Incubation with L-CPA (10 mM) for 48 hr lead to cell death (90%). When the granule cells were incubated with L-CPA in combination with the inhibitors of free radical production, the cell death was ameliorated. The results show that L-CPA is toxic to granular cells by production of ROS.     

Binding of copper is a mechanism of homocysteine toxicity leading to COX deficiency and apoptosis in primary neurons, PC12 and SHSY-5Y cells

Children with hereditary severe hyperhomocysteinemia present with a variety of neurological impairment, and mild hyperhomocysteinemia has been associated with neurodegeneration in the elderly. The link of hyperhomocysteinemia to neurological dysfunction is unknown. We investigated mitochondrial mechanisms of homocysteine (HCys) neurotoxicity in rat dopaminergic pheochromocytoma cells, human neuroblastoma cells and primary rat cerebellar granule neurons. HCys dose dependently impaired cytochrome c oxidase (COX) activity as well as stability and induced reactive oxygen species and apoptotic cell death. We found that HCys binds the COX cofactor Cu(2+), and Cu(2+) supplementation prior to HCys treatment preserved COX activity and prevented cell death. The Cu(2+) chelating action of HCys and impairement of COX activity represent novel mechanisms of HCys neurotoxicity, which might be preventable by supplementation of Cu(2+).     

Endogenous XIAP,but not other members of the inhibitory apoptosis protein family modulates cerebellar granule neurons survival

Programmed cell death plays a critical role during cerebellar development. In particular, it has been shown in vivo and in vitro that developing cerebellar granule neurons (CGN) die apoptotically. Apoptosis involves a series of morphological changes and the activation of caspases. Inhibitor of apoptosis proteins (IAPs) is implicated in negative regulation of caspase activation and apoptotic cell death. Although apoptotic death of CGN has been extensively studied, there is no information about the role of IAPs in the developing cerebellum. Here, we studied the participation of some members of IAPs in the survival of the developing rat CGN in culture and under physiological conditions. Under these conditions, we found a differential expression pattern of cIAP-1, cIAP-2, XIAP and survivin during cerebellar development in an age-dependent manner, highlighting the significant increase of XIAP levels. We also detected an interaction between XIAP and caspase 3 at postnatal day (P) 12 and 16. On the other hand, we found a significant decrease of XIAP levels in cultured CGN maintained in chronic potassium deprivation, an apoptotic condition, suggesting a possible relationship between XIAP levels and neuronal viability. Under these conditions, we also detected the interaction of XIAP with active caspase-3. The down-regulation of XIAP in CGN cultured under survival conditions (chronic potassium depolarization) induced a reduction of cell viability and an increment of apoptotic cells. These findings support the idea that IAPs could be involved in the survival of CGN and that XIAP might be critical for neuronal survival in cerebellar development and during chronic depolarization in cultured CGN through a mechanism involving caspase inhibition.     

Cytoplasmic cytochrome c immunolabelling in dystrophic neurites in Alzheimer’s disease

Cytochrome c has a well-established role in electron transfer and as a mediator of apoptotic cell death. The cortical and intracellular localisation of cytochrome c immunoreactivity was examined in Alzheimer’s disease and control cases. No differences in the cortical labelling pattern or the density of cytochrome c-positive cells in neocortical layer V were present between control and Alzheimer’s disease cases. Punctate cytochrome c labelling was present in a subset of neocortical neurons, including clusters of intensely labelled pyramidal neurons that were not specifically associated with β-amyloid plaques. With respect to Alzheimer’s disease associated pathology, only 6.7 ± 1.4% of neurons showing neurofibrillary tangle formation demonstrated punctate cytochrome c immunoreactivity. These results suggest that cytochrome c may label a subset of pyramidal neurons that is susceptible, yet relatively resistant, to Alzheimer’s disease pathology. A low percentage of neurofilament triplet protein medium, tau and chromogranin A labelled dystrophic neurites were also cytochrome c-positive. There was also a trend towards an increase in the percentage of cytochrome c immunoreactive dystrophic neurites in pathologically aged control cases compared to Alzheimer’s disease cases, suggesting that cytochrome c may be an early and transient epitope within dystrophic neurites. In contrast to the punctate cytochrome c labelling observed in cortical cells, cytoplasmic cytochrome c labelling was observed within dystrophic neurites. Although cytochrome c release is indicative of the activation of the intrinsic apoptotic pathway, cytoplasmic cytochrome c may also indicate mitochondrial damage or dysfunction.     

Oxidative stress-induced apoptosis is associated with alterations in mitochondrial caspase activity and Bcl-2-dependent alterations in mitochondrial pH (pHm)

Oxidative stress, the result of cellular production of reactive oxygen species (ROS), has been implicated in a number of diseases of the eye. Exposure of eye tissues (e.g. the cornea and retina) to oxidative stress over time has been hypothesized to underlie the development of age-related macular degeneration (AMD) and maturity onset cataract formation. Light-induced free radicals can damage the eye, and alterations in the antioxidant defenses of the eye have been suggested to play a role in the etiology of glaucoma. Mitochondria are both a major endogenous source and target of ROS, and oxidative stress has been shown to induce apoptotic cell death by targeting the mitochondria directly. Mitochondrial-dependent apoptosis has been shown to require release of cytochrome c from mitochondria and subsequent activation of a specific class of cytoplasmic proteases known as caspases. Bcl-2, an anti-apoptotic protein localized to mitochondria, has been shown to inhibit cytochrome c release and protect against oxidative stress-induced apoptosis. Here we demonstrate that oxidative stress causes activation of mitochondrial matrix caspase-2 and -9 activity that is associated with Bcl-2-inhibitable acidification of mitochondrial pH (pH_m). In conjunction with recent reports that caspase activation is maximal at acidic pH, these findings have led us to hypothesize that Bcl-2 may modulate cytochrome c release following oxidative stress by modifying the pH-dependent activation of mitochondrial caspase activity. These studies provide an increased understanding of the mechanism(s) by which oxidative stress damages tissues, and may have important therapeutic implications for treatment of opthamological diseases.