c-Jun contributes to amyloid beta-induced neuronal apoptosis but is not necessary for amyloid beta-induced c-jun induction

The role of gene expression in neuronal apoptosis may be cell- and apoptotic stimulus-specific. Previously, we and others showed that amyloid beta (Abeta)-induced neuronal apoptosis is accompanied by c-jun induction. Moreover, c-Jun contributes to neuronal death in several apoptosis paradigms involving survival factor withdrawal. To evaluate the role of c-Jun in Abeta toxicity, we compared Abeta-induced apoptosis in neurons from murine fetal littermates that were deficient or wild-type with respect to c-Jun. We report that neurons deficient for c-jun are relatively resistant to Abeta toxicity, suggesting that c-Jun contributes to apoptosis in this model. When changes in gene expression were quantified in neurons treated in parallel, we found that Abeta treatment surprisingly led to an apparent activation of the c-jun promoter in both the c-jun-deficient and wild-type neurons, suggesting that c-Jun is not necessary for activation of the c-jun promoter. Indeed, several genes induced by Abeta in wild-type neurons were also induced in c-jun-deficient neurons, including c-fos, fosB, ngfi-B, and ikappaB. In summary, these results indicate that c-Jun contributes to Abeta-induced neuronal death but that c-Jun is not necessary for c-jun induction.     

Neuroprotective activities of bacopa,lycopene, astaxanthin, and vitamin B12 combination on oxidative stress-dependent neuronal death

Oxidative stress is considered the common effector of the cascade of degenerative events in many neurological conditions. Thus, in this paper we tested different nutraceuticals in H₂O₂ in vitro model to understand if could represent an adjuvant treatment for neurological diseases. In this study, nutraceuticals bacopa, lycopene, astaxanthin, and vitamin B12 were used alone or in combination in human neuronal differentiated SH-SY5Y cells upon hydrogen peroxide-induced injury and neuroprotective, neuronal death pathways were analyzed. The nutraceuticals analyzed were able to protect H₂O₂ cytotoxic effects, through increasing cell viability and proteins involved in neuroprotection pathways and restoring proteins involved in cell death pathways. On this basis, it is possible to propose the use of these compounds as dietary supplement for the prevention or as adjuvant to the only symptomatic treatments so far available for neurodegenerative diseases.     

A monoclonal antibody to amyloid precursor protein induces neuronal apoptosis

Although there is considerable evidence suggesting that altered metabolism of beta-amyloid precursor protein (APP) and accumulation of its beta-amyloid fragment are key features of Alzheimer's disease (AD), the normal physiological function of APP remains elusive. We investigated the potential role of APP in neurons using the monoclonal antibody 22C11, which binds to the extracellular domain of the human, rat, or mouse APP. Exposure of cortical neurons to 22C11 induced morphological changes including neurite degeneration, nuclear condensation, and internucleosomal DNA cleavage that were consistent with neurons dying by apoptosis. Supporting a role for 22C11-mediated apoptosis occurring by binding to APP were data demonstrating that preincubation of 22C11 with either purified APP or a synthetic peptide (APP(66-81)) that contains the epitope for 22C11 significantly attenuated neuronal damage induced by 22C11. The specificity of 22C11 was further supported by data showing no apparent effects of either mouse IgG or the monoclonal antibody P2-1, which is specific for the aminoterminal end of human but not rat APP. In addition, biochemical features indicative of apoptosis were the formation of 120- and 150-kDa breakdown products of fodrin following treatment of cortical neurons with 22C11. Both the morphological and the biochemical changes induced by 22C11 were prevented following pretreatment of neurons with the general caspase inhibitor N-benzyloxycarbonyl-Val-Ala-Asp(O-methyl)-fluoromethyl ketone. Prior incubation of cortical neurons with GSH ethyl ester (GEE), a cell-permeable form of GSH, resulted in complete protection from the 22C11 insult, thus implicating an oxidative pathway in 22C11-mediated neuronal degeneration. This was further supported by the observation that prior treatment of neurons with buthionine sulfoximine, an inhibitor of gamma-glutamylcysteinyl synthetase, potentiated the toxic effects of 22C11. Finally, with use of compartmented cultures of hippocampal neurons, it was also demonstrated that selective application of 22C11 caused local neuritic degeneration that was prevented by the addition of GEE to the neuritic compartment. Thus, the binding of a monoclonal antibody to APP initially triggers neurite degeneration that is followed by caspase-dependent apoptosis in neuronal cultures and illustrates a novel property of this protein in neurons that may contribute to the profound neuronal cell death associated with AD.     

Pro-apoptotic signaling in neuronal cells following iron and amyloid beta peptide neurotoxicity

In a previous report, we characterized several oxidative stress parameters during the course of amyloid beta (Abeta) peptide/Fe2+-induced apoptotic death in neuronal cells. In extending these findings, we now report a marked decrease in protein kinase C (PKC) isoforms, reduced Akt serine/threonine kinase activity, Bcl 2-associated death promoter (BAD) phosphorylation and enhanced p38 mitogen-activated protein kinase (MAPK) and caspase-9 and -3 activation, 12 h after addition of both 5 micro m Abeta and 5 micro m Fe2+. These activities reminiscent for a pro-apoptotic cellular course were blocked in the presence of the iron chelator deferroxamine. Abeta alone, increased PKC isoform levels between three- and four-fold after 12 h, enhanced Akt activity approximately eight-fold and Ser136 BAD phosphorylation two-fold, suggesting that by itself is not toxic. Fe2+ alone transiently enhanced p38 MAPK and caspase-9 and -3 enzymes indicative for cell damage, but was not sufficient to cause cell death as previously indicated. GF, a PKC inhibitor or wortmannin, a blocker of the Akt pathway enhanced Abeta/Fe2+-induced toxicity, while SB, a p38 MAPK inhibitor, prevented cell damage and apoptosis. These findings further support the hypothesis that metal ion chelation and inhibitors of pro-apoptotic kinase cascades may be beneficial for Alzheimer's disease therapy.     

Nitric oxide induces oxidative stress and apoptosis in neuronal cells

Within the central nervous system and under normal conditions, nitric oxide (NO) is an important physiological signaling molecule. When produced in large excess, NO also displays neurotoxicity. In our previous report, we have demonstrated that the exposure of neuronal cells to NO donors induced apoptotic cell death, while pretreatment with free radical scavengers L-ascorbic acid 2-[3, 4-dihydro-2,5,7,8-tetramethyl-2-(4,8, 12-trimethyltridecyl)-2H-1-benzopyran-6-yl-hydrogen phosphate] potassium salt (EPC-K1) or superoxide dismutase attenuated apoptosis effectively, suggesting that reactive oxygen species (ROS) may be involved in the cascade of events leading to apoptosis. In the present investigation, we directly studied the kinetic generation of ROS in NO-treated neuronal cells by flow cytometry using 2', 7'-dichloro-fluorescein diacetate and dihydrorhodamine 123 as redox-sensitive fluorescence probes. The results indicated that exposure of cerebellar granule cells to the NO donor S-nitroso-N-acetylpenicillamine (SNAP) induced oxidative stress, which was characterized by the accumulation of cytosolic and mitochondrial ROS, the increase in the extracellular hydrogen peroxide level, and the formation of lipid peroxidation products. SNAP treatment also induced apoptotic cell death as confirmed by the formation of cytosolic mono- and oligonucleosomes. Pretreating cells with the novel antioxidant EPC-K1 effectively prevented oxidative stress induced by SNAP, and attenuated cells from apoptosis.     

癫痫发作后神经元凋亡的多重调控机制

癫痫是一种慢性脑部疾病。以脑部神经元过度同步化放电所致的突然、反复和短暂的中枢神经系统功能失常为特征。细胞凋亡(apoptosis)是由基因控制的由细胞内部程序激活而发生的自杀性死亡,它在细胞分化、促进体内正常细胞的更新和调节机体发育等方面都起到重要作用。细胞凋亡是癫痫发作后神经元丢失的重要形式。目前认为,癫痫发作后细胞凋亡分子水平的多重调控主要集中在三个方面:(1)细胞凋亡的相关基因及其调控方面;(2)caspase(cysteine proteinases with specificity for aspartic acidresidues,半胱天冬氨酸酶)在细胞凋亡中的作用;(3)线粒体途径在细胞凋亡中的作用。     

癫痫发作后神经元凋亡的多重调控机制

癫痫是一种慢性脑部疾病。以脑部神经元过度同步化放电所致的突然、反复和短暂的中枢神经系统功能失常为特征。细胞凋亡（apoptosis）是由基因控制的由细胞内部程序激活而发生的自杀性死亡,它在细胞分化、促进体内正常细胞的更新和调节机体发育等方面都起到重要作用。细胞凋亡是癫痫发作后神经元丢失的重要形式。目前认为,癫痫发作后细胞凋亡分子水平的多重调控主要集中在三个方面：（1）细胞凋亡的相关基因及其调控方面;（2）caspase（cysteine proteinases with specificity for aspartic acid residues,半胱天冬氨酸酶）在细胞凋亡中的作用;（3）线粒体途径在细胞凋亡中的作用。     

Signaling events in amyloid beta-peptide-induced neuronal death and insulin-like growth factor I protection

Amyloid beta-peptide (Abeta) is implicated as the toxic agent in Alzheimer's disease and is the major component of brain amyloid plaques. In vitro, Abeta causes cell death, but the molecular mechanisms are unclear. We analyzed the early signaling mechanisms involved in Abeta toxicity using the SH-SY5Y neuroblastoma cell line. Abeta caused cell death and induced a 2- to 3-fold activation of JNK. JNK activation and cell death were inhibited by overexpression of a dominant-negative SEK1 (SEK1-AL) construct. Butyrolactone I, a cdk5 inhibitor, had an additional protective effect against Abeta toxicity in these SEK1-AL-expressing cells suggesting that cdk5 and JNK activation independently contributed to this toxicity. Abeta also weakly activated ERK and Akt but had no effect on p38 kinase. Inhibitors of ERK and phosphoinositide 3-kinase (PI3K) pathways did not affect Abeta-induced cell death, suggesting that these pathways were not important in Abeta toxicity. Insulin-like growth factor I protected against Abeta toxicity by strongly activating ERK and Akt and blocking JNK activation in a PI3K-dependent manner. Pertussis toxin also blocked Abeta-induced cell death and JNK activation suggesting that G(i/o) proteins were upstream activators of JNK. The results suggest that activation of the JNK pathway and cdk5 may be initial signaling cascades in Abeta-induced cell death.     

Ceramide in apoptosis signaling: relationship with oxidative stress

Ceramide is one of the major sphingosine-based lipid second messengers that is generated in response to various extracellular agents. However, while widespread attention has focused on ceramide as a second messenger involved in the induction of apoptosis, important issues with regard to the mechanisms of ceramide formation and mode of action remain to be addressed. Several lines of evidence suggest that ceramide and oxidative stress are intimately related in cell death induction. This review focuses on the putative relationships between oxidative stress and sphingolipid metabolism in the apoptotic process and discusses the potential mechanisms that connect and regulate the two phenomena.     

Aging and amyloid beta-induced oxidative DNA damage and mitochondrial dysfunction in Alzheimer's disease: implications for early intervention and therapeutics

Alzheimer's disease (AD) is an age-related progressive neurodegenerative disease affecting thousands of people in the world and effective treatment is still not available. Over two decades of intense research using AD postmortem brains, transgenic mouse and cell models of amyloid precursor protein and tau revealed that amyloid beta (Aβ) and hyperphosphorylated tau are synergistically involved in triggering disease progression. Accumulating evidence also revealed that aging and amyloid beta-induced oxidative DNA damage and mitochondrial dysfunction initiate and contributes to the development and progression of the disease. The purpose of this article is to summarize the latest progress in aging and AD, with a special emphasis on the mitochondria, oxidative DNA damage including methods of its measurement. It also discusses the therapeutic approaches against oxidative DNA damage and treatment strategies in AD.     

Early events of target deprivation/axotomy-induced neuronal apoptosis in vivo: oxidative stress,DNA damage,p53 phosphorylation and subcellular redistribution of death proteins

The mechanisms of injury- and disease-associated apoptosis of neurons within the CNS are not understood. We used a model of cortical injury in rat and mouse to induce retrograde neuronal apoptosis in thalamus. In this animal model, unilateral ablation of the occipital cortex induces apoptosis of corticopetal projection neurons in the dorsal lateral geniculate nucleus (LGN), by 7 days post-lesion, that is p53 modulated and Bax dependent. We tested the hypothesis that this degenerative process is initiated by oxidative stress and early formation of DNA damage and is accompanied by changes in the levels of pro-apoptotic mediators of cell death. Immunoblotting revealed that the protein profiles of Bax, Bak and Bad were different during the progression of neuronal apoptosis in the LGN. Bax underwent a subcellular redistribution by 1 day post-lesion, while Bak increased later. Bad showed an early sustained increase. Cleaved caspase-3 was elevated maximally at 5 and 6 days. Active caspase-3 underwent a subcellular translocation to the nucleus. A dramatic phosphorylation of p53 was detected at 4 days post-lesion. DNA damage was assessed immunocytochemically as hydroxyl radical adducts (8-hydroxy-2-deoxyguanosine) and single-stranded DNA. Both forms of DNA damage accumulated early in target-deprived LGN neurons. Transgenic overexpression of superoxide dismutase-1 provided significant protection against the apoptosis but antioxidant pharmacotreatments with trolox and ascorbate were ineffective. We conclude that overlapping and sequential signaling pathways are involved in the apoptosis of adult brain neurons and that DNA damage generated by superoxide derivatives is an upstream mechanism for p53-regulated, Bax-dependent apoptosis of target-deprived neurons.     

Apolipoprotein E4 potentiates amyloid beta peptide-induced lysosomal leakage and apoptosis in neuronal cells

We assessed the isoform-specific effects of apolipoprotein (apo) E on the response of Neuro-2a cells to the amyloid beta peptide (Abeta1-42). As determined by the intracellular staining pattern and the release of beta-hexosaminidase into the cytosol, apoE4-transfected cells treated with aggregated Abeta1-42 showed a greater tendency toward lysosomal leakage than neo- or apoE3-transfected cells. Abeta1-42 caused significantly greater cell death and more than 2-fold greater DNA fragmentation in apoE4-secreting than in apoE3-secreting or control cells. H2O2 or staurosporine enhanced cell death and apoptosis in apoE4-transfected cells but not in apoE3-transfected cells. A caspase-9 inhibitor abolished the potentiation of Abeta1-42-induced apoptosis by apoE4. Similar results were obtained with conditioned medium from cells secreting apoE3 or apoE4. Cells preincubated for 4 h with a source of apoE3 or apoE4, followed by removal of apoE from the medium and from the cell surface, still exhibited the isoform-specific response to Abeta1-42, indicating that the potentiation of apoptosis required intracellular apoE, presumably in the endosomes or lysosomes. Studies of phospholipid (dimyristoylphosphatidylcholine) bilayer vesicles encapsulating 5-(and-6)-carboxyfluorescein dye showed that apoE4 remodeled and disrupted the phospholipid vesicles to a greater extent than apoE3 or apoE2. In response to Abeta1-42, vesicles containing apoE4 were disrupted to a greater extent than those containing apoE3. These findings are consistent with apoE4 forming a reactive molecular intermediate that avidly binds phospholipid and may insert into the lysosomal membrane, destabilizing it and causing lysosomal leakage and apoptosis in response to Abeta1-42.     

Taurine ameliorates alloxan-induced diabetic renal injury, oxidative stress-related signaling pathways and apoptosis in rats

Hyperglycemia-induced oxidative stress plays a vital role in the progression of diabetic nephropathy. The renoprotective nature of taurine has also been reported earlier; but little is known about the mechanism of this beneficial action. The present study has, therefore, been carried out to explore in detail the mechanism of the renoprotective effect of taurine under diabetic conditions. Diabetes was induced in rats by alloxan (single i.p. dose of 120?mg/kg body weight) administration. Taurine was administered orally for 3?weeks (1% w/v in drinking water) either from the day on which alloxan was injected or after the onset of diabetes. Alloxan-induced diabetic rats showed a significant increase in plasma glucose, enhanced the levels of renal damage markers, plasma creatinine, urea nitrogen and urinary albumin. Diabetic renal injury was associated with increased kidney weight to body weight ratio and glomerular hypertrophy. Moreover, it increased the productions of reactive oxygen species, enhanced lipid peroxidation and protein carbonylation in association with decreased intracellular antioxidant defense in the kidney tissue. In addition, hyperglycemia enhanced the levels of proinflammatory cytokins (TNF-α, IL-6, IL-1β) and Na⁺–K⁺-ATPase activity with a concomitant reduction in NO content and eNOS expression in diabetic kidney. Investigation of the oxidative stress-responsive signaling cascades showed the upregulation of PKCα, PKCβ, PKCε and MAPkinases in the renal tissue of the diabetic animals. However, taurine administration decreased the elevated blood glucose and proinflammatory cytokine levels, reduced renal oxidative stress (via decrease in xanthine oxidase activity, AGEs formation and inhibition of p47phox/CYP2E1 pathways), improved renal function and protected renal tissue from alloxan-induced apoptosis via the regulation of Bcl-2 family and caspase-9/3 proteins. Taurine supplementation in regular diet could, therefore, be beneficial to regulate diabetes-associated renal complications.     

Dysfunction of amyloid precursor protein signaling in neurons leads to DNA synthesis and apoptosis

The classic neuropathological diagnostic markers for AD are amyloid plaques and neurofibrillary tangles, but their role in the etiology and progression of the disease remains incompletely defined. Research over the last decade has revealed that cell cycle abnormalities also represent a major neuropathological feature of AD. These abnormalities appear very early in the disease process, prior to the appearance of plaques and tangles; and it has been suggested that neuronal cell cycle regulatory failure may be a significant component of the pathogenesis of AD. The amyloid precursor protein (APP) is most commonly known as the source of the beta-amyloid (Abeta) peptides that accumulate in the brains of patients with AD. However, a large body of work supports the idea that APP is also a signaling receptor. Most recently, it has been shown that familial AD (FAD) mutations in APP or simple overexpression of wild type APP cause dysfunction of APP signaling, resulting in initiation of DNA synthesis in neurons and consequent apoptosis. In this article, we review the evidence that APP has the potential to activate aberrant neuronal cell cycle re-entry in AD, and we describe a signal transduction pathway that may mediate this abnormal activation of the cell cycle.     

Mitigative effects of epigallocatechin gallate in terms of diminishing apoptosis and oxidative stress generated by the combination of lead and amyloid peptides in human neuronal cells

Environmental exposure to lead (Pb) is reported to associate with the development of Alzheimer's disease, where the formation of β‐amyloid peptides (APs) of (1‐40), (1‐42), and (25‐35) is considered as the major risk factor. In this context, we aimed at investigating the effect of epigallocatechin gallate (EGCG), a major flavonoid polyphenol available in green tea, in mitigating the individual and combined toxicity generated by Pb and β‐APs in terms of oxidative stress and apoptosis in human neuronal cells. SH‐SY5Y cells were exposed to Pb and β‐APs of (1‐40) and (25‐35) individually and in different combinations in the presence and absence of EGCG. The results indicated that EGCG mitigated both Pb‐ and β‐AP‐induced oxidative stress in scavenging reactive oxygen species and apoptosis by improving the expression levels of Bax and bcl2 and inhibiting annexin V and caspase‐3. Thus, our study shows that EGCG protects SH‐SY5Y cells against the cytotoxicity induced by Pb and β‐APs by decreasing oxidative stress and inhibiting apoptosis.     

Thioredoxin-1 attenuates post-ischemic neuronal apoptosis via reducing oxidative/nitrative stress

Recent studies show that Thioredoxin (Trx) possesses a neuronal protective effect and that Trx inactivation is closely related to cerebral ischemia injury. Peroxynitrite (ONOO⁻) formation may trigger oxidative/nitrative stress and represent a major cytotoxic effect in cerebral ischemia. The present study was conducted to validate whether treatment with recombinant human Trx-1 (rhTrx-1) would attenuate ONOO⁻ generation and oxidative/nitrative stress in focal transient cerebral ischemia. The results showed that intravenously administered rhTrx-1 (10 mg/kg) significantly improved neurological functions and reduced cerebral infarction and apoptotic cell death following cerebral ischemia. Neuronal ONOO⁻ formation was significantly attenuated after rhTrx-1 treatment. Moreover, rhTrx-1 resulted in a significant decrease in antioxidant capacity and p38 mitogen activated protein kinase (MAPK) activity in ischemic brain tissue. Furthermore, the suppression on ONOO⁻ formation by either rhTrx-1 or an ONOO⁻ scavenger uric acid reduced cerebral infarct size in mice subjected to cerebral ischemia. Peroxynitrite donor SIN-1 not only blocked the neuronal protection of rhTrx-1 but also markedly attenuated rhTrx-1-induced antioxidative/antinitrative effect. We concluded that rhTrx-1 exerts an antioxidative/antinitrative effect against cerebral ischemia injury by blocking ONOO⁻ and superoxide anion formation. These results provide the information that thioredoxin is much more likely to succeed as a therapeutic approach to diminish oxidative/nitrative stress-induced neuronal apoptotic cell death in the ischemic brain.     

Lactoferrin inhibits neutrophil apoptosis via blockade of proximal apoptotic signaling events

Neutrophils are the most abundant leukocyte and have a short lifespan, dying by apoptosis approximately five days after leaving the bone marrow. Their apoptosis can be delayed at sites of inflammation to extend their functional lifespan, but inappropriate inhibition of apoptosis contributes to chronic inflammatory disease. Levels of the physiological iron chelator lactoferrin are raised at sites of inflammation and we have shown previously that iron-unsaturated lactoferrin inhibited human neutrophil apoptosis, but the mechanisms involved were not determined. Here we report that the anti-apoptotic effect of lactoferrin is dependent upon its iron saturation status as iron-saturated lactoferrin did not affect neutrophil apoptosis. We also show that the effect of lactoferrin is mediated at an early stage in apoptosis as it inhibited activation of sphingomyelinase, generation of ceramide, activation of caspase 8 and Bax and cleavage of Bid. Lactoferrin did not inhibit apoptosis induced by exogenous ceramide, supporting the proposal that it acts upstream of ceramide generation. We therefore conclude that raised lactoferrin levels are likely to contribute to chronic inflammation by delaying neutrophil apoptosis and that this is achieved by inhibiting proximal apoptotic signaling events.