Oxidative stress induces neuronal death by recruiting a protease and phosphatase-gated mechanism.

Reactive oxygen species (ROS) cause death of cerebellar granule neurons. Here, a 15-min pulse of H(2)O(2) (100 microm) induced an active process of neuronal death distinct from apoptosis. Oxidative stress activated a caspase-independent but calpain-dependent decline of calcium/calmodulin-dependent protein kinase IV and cAMP- responsive element-binding protein (CREB). Calpain inhibitors restored calcium/calmodulin-dependent protein kinase IV and CREB but did not influence phosphorylated CREB levels or survival, indicating recruitment of an additional dephosphorylation process. Co-treatment with calpain and serine/threonine phosphatase inhibitors restored pCREB levels and rescued neurons. This phosphatase-activated signaling pathway was shown to be dependent on de novo protein synthesis. Further, gene transfer studies revealed that CREB is a common final effector of both apoptosis and ROS-induced death. Our data indicate that dephosphorylation and proteolytic signaling mechanisms underlie ROS-induced programmed cell death.     

Neuroprotective effects of bcl-2 overexpression in hippocampal cultures: interactions with pathways of oxidative damage

Overexpression of bcl-2protects neurons from numerous necrotic insults, both in vitro and in vivo. While the bulk of such protection is thought to arise from Bcl-2 blocking cytochrome c release from mitochondria, thereby blocking apoptosis, the protein can target other steps in apoptosis, and can protect against necrotic cell death as well. There is evidence that these additional actions may be antioxidant in nature, in that Bcl-2 has been reported to protect against generators of reactive oxygen species (ROS), to increase antioxidant defenses and to decrease levels of ROS and of oxidative damage. Despite this, there are also reports arguing against either the occurrence, or the importance of these antioxidant actions. We have examined these issues in neuron-enriched primary hippocampal cultures, with overexpression of bcl-2 driven by a herpes simplex virus amplicon: (i) Bcl-2 protected strongly against glutamate, whose toxicity is at least partially ROS-dependent. Such protection involved reduction in mitochondrially derived superoxide. Despite that, Bcl-2 had no effect on levels of lipid peroxidation, which is thought to be the primary locus of glutamate-induced oxidative damage; (ii) Bcl-2 was also mildly protective against the pro-oxidant adriamycin. However, it did so without reducing levels of superoxide, hydrogen peroxide or lipid peroxidation; (iii) Bcl-2 protected against permanent anoxia, an insult likely to involve little to no ROS generation. These findings suggest that Bcl-2 can have antioxidant actions that may nonetheless not be central to its protective effects, can protect against an ROS generator without targeting steps specific to oxidative biochemistry, and can protect in the absence of ROS generation. Thus, the antioxidant actions of Bcl-2 are neither necessary nor sufficient to explain its protective actions against these insults in hippocampal neurons.     

多巴胺D1和D2受体激动剂和拮抗剂对脑缺血/再灌注损伤的影响

实验应用开阔法、组织病理学方法、原位末端标记(in situ terminal deoxynucleotidyl transferase-metliated de-oxy-UTP mick end labeling,TUNEL)法及免疫组织化学等方法,探讨多巴胺D1、D2受体激动剂和拮抗剂对沙土鼠前脑缺血／再灌注损伤海马CA1区神经元凋亡及凋亡相关基因bcl-2、bax表达的影响。结果显示：前脑缺血5min可引起沙土鼠探索活动增加;再灌注3d,海马CA1区约95％的锥体细胞凋亡;再灌注7d,海马CA1区仅残存约2％—7％的存活锥体细胞;前脑缺血5min可抑制bcl-2的表达并诱导bax表达增高;预先应用D2受体激动剂培高利特可减轻缺血后沙土鼠行为学异常、抑制海马CA1区锥体细胞凋亡、提高锥体细胞存活数、显著诱导bcl-2的表达并抑制bax的表达。预先应用SKF38393、SCH23390及螺哌隆对以上结果无明显影响。实验结果提示,培高利特具有确切的脑保护作用,诱导bcl-2并抑制bax的表达可能是其脑保护作用机制之一。     

Retracted: Downregulation of long noncoding RNA H19 rescues hippocampal neurons from apoptosis and oxidative stress by inhibiting IGF2 methylation in mice with streptozotocin-induced diabetes mellitus

The diabetes mellitus (DM)-induced reduction of neurogenesis in the hippocampus is consequently accompanied by cognitive decline. The present study set out to define the critical role played by long noncoding RNA H19 (lncRNA H19) in the apoptosis of hippocampal neurons, as well as oxidative stress (OS) in streptozotocin (STZ)-induced DM mice through regulation of insulin-like growth factor 2 (IGF2) methylation. The expression of lncRNA H19 in the hippocampal neurons and surviving neurons were detected. Hippocampal neurons were cultured and transfected with oe-H19, sh-H19, oe-IGF2, or sh-IGF2, followed by detection of the expressions of IGF2 and apoptosis-related genes. Determination of the lipid peroxide and glutathione levels was conducted, while antioxidant enzyme activity was identified. The IGF2 methylation, the binding of lncRNA H19 to DNA methyltransferase, and the binding of lncRNA H19 to IGF2 promoter region were detected. DM mice exhibited high expressions of H19, as well as a decreased hippocampal neurons survival rate. Higher lncRNA H19 expression was found in DM. Upregulated lncRNA H19 significantly increased the expression of Bax and caspase-3 but decreased that of Bcl-2, thus promoting the apoptosis of hippocampal neuron. Besides, upregulation of lncRNA H19 induced OS. LncRNA H19 was observed to bind specifically to the IGF2 gene promoter region and promote IGF2 methylation by enriching DNA methyltransferase, thereby silencing IGF2 expression. Taken together, downregulated lncRNA H19 reduces IGF2 methylation and enhances its expression, thereby suppressing hippocampal neuron apoptosis and OS in STZ-induced (DM) mice.     

Apoptosis Induced by Differentiation or Serum Deprivation in an Immortalized Central Nervous System Neuronal Cell Line

Abstract: To characterize the nature of programmed cell death (PCD) induced in neuronal cells during development, three regulators of apoptosis were investigated: one, the bcl-2-related genes, modulate cell survival, and the other two, the interleukin-1β converting enzyme (ICE)-related enzymes and the tumor suppressor protein p53, have been implicated as mediators of apoptosis. These regulators were studied in H19-7 cells, an SV40 T^ts-immortalized rat hippocampal neuronal cell line that can be differentiated with basic fibroblast growth factor at the nonpermissive temperature, resulting in a rapid attrition of cells by apoptosis. PCD occurred by two mechanisms in H19-7 cells: The first was initiated by removal of serum from undifferentiated cells, and the second was a consequence of neuronal differentiation. In differentiated H19-7 cells, the survival time was increased by both human bcl-2 and bcl-x_L, and this could be reversed by bcl-x_S.Addition of a peptide inhibitor of the ICE enzyme family to H19-7 cells resulted in a transient protection against differentiation-associated apoptosis, whereas no further protection was observed in the BCL-2- or BCL-X_L-expressing cells. Shifting the differentiated cells to 33°C to inactivate p53 did not significantly affect the apoptotic process, indicating that apoptosis induced by neuronal differentiation is not dependent on the continued presence of p53. By contrast, in undifferentiated cells, cell loss induced by transfer to serum-free media occurred more rapidly on inactivation of large T, consistent with p53 involvement. This medium-induced decrease in cell survival could not be rescued by the ICE inhibitor but was partially rescued by BCL-2 or BCL-X_L. Furthermore, studies involving expression of BCL-2 and BCL-X_L alone or together revealed differences in the survival dependent on the cellular environment. These results suggest that apoptosis of neuronal cellsoccurs by at least two processes: one in undifferentiated cells initiated by removal of serum and one linked to differentiation. The data implicate the ICE enzyme family but not p53 in apoptosis induced by differentiation and demonstrate that either BCL-2 or BCL-X_L can prolong the survival of differentiated neuronal cells.     

Reactive oxygen species, dietary restriction and neurotrophic factors in age-related loss of myenteric neurons

We have studied the mechanisms underlying nonpathological age-related neuronal cell death. Fifty per cent of neurons in the rat enteric nervous system are lost between 12 and 18 months of age in ad libitum (AL) fed rats. Caloric restriction (CR) protects almost entirely against this neuron loss. Using the ROS-sensitive dyes, dihydrorhodamine (DHR) and 2-[6-(4'-hydroxy)phenoxy-3H-xanthen-3-on-9-yl]benzoic acid (HPF) in vitro, we show that the onset of cell death is linked with elevated intraneuronal levels of reactive oxygen species (ROS). Treatment with the neurotrophic factors NT3 and GDNF enhances neuronal antioxidant defence in CR rats at 12-15 months and 24 months but not in adult or aged AL-fed animals. To examine the link between elevated ROS and neuronal cell death, we assessed apoptotic cell death following in vitro treatment with the redox-cycling drug, menadione. Menadione fails to increase apoptosis in 6-month neurons. However, in 12-15mAL fed rats, when age-related cell death begins, menadione induces a 7- to 15-fold increase in the proportion of apoptotic neurons. CR protects age-matched neurons against ROS-induced apoptosis. Treatment with neurotrophic factors, in particular GDNF, rescues neurons from menadione-induced cell death, but only in 12-15mCR animals. We hypothesize that CR enhances antioxidant defence through neurotrophic factor signalling, thereby reducing age-related increases in neuronal ROS levels and in ROS-induced cell death.     

Akt, a Target of Phosphatidylinositol 3-Kinase, Inhibits Apoptosis in a Differentiating Neuronal Cell Line

Phosphatidylinositol (PI) 3-kinase has been suggested to mediate cell survival. Consistent with this possibility, apoptosis of conditionally (simian virus 40 T^ts) immortalized rat hippocampal H19-7 neuronal cells was increased in response to wortmannin, an inhibitor of PI 3-kinase. Downstream effectors of PI 3-kinase include Rac1, protein kinase C, and the serine-threonine kinase Akt (protein kinase B). Here, we show that activation of Akt is one mechanism by which PI 3-kinase can mediate survival of H19-7 cells during serum deprivation or differentiation. While ectopic expression of wild-type Akt (c-Akt) does not significantly enhance survival in H19-7 cells, expression of activated forms of Akt (v-Akt or myristoylated Akt) results in enhanced survival which can be comparable to that conferred by Bcl-2. Conversely, expression of a dominant-negative mutant of Akt accelerates cell death upon serum deprivation or differentiation. Finally, the results indicate that Akt can transduce a survival signal for differentiating neuronal cells through a mechanism that is independent of induction of Bcl-2 or Bcl-x_L or inhibition of Jun kinase activity.     

Alterations in the cellular distribution of bcl-2, bcl-x and bax in the adult rat substantia nigra following striatal 6-hydroxydopamine lesions

The proteins of the bcl-2 family play an important role during apoptosis and may also regulate cell death in response to oxidative stress, which has been implicated in Parkinson's disease. In this study we examined the localization of the pro-apoptotic protein bax, and the anti-apoptotic proteins bcl-2 and bcl-x_L in the substantia nigra (SN) of the adult rat and their response to oxidative stress caused by striatal injections of 6-hydroxydopamine (6-OHDA). Our data show that bcl-2, bcl-x and bax proteins are present in the SN. Bcl-2 and bax are localized primarily in neurons including all those positive for tyrosine hydroxylase (TH). The intraneuronal distribution of bcl-2 and bax were different. Bcl-2 was diffuse throughout the cell while bax was localized in well-defined structures around the nucleus and within processes. Bcl-x staining in neurons was weak, though it was strongly expressed in GFAP-positive astrocytes. 6-OHDA injections, which resulted in loss of dopamine neurons between 7–14 days post-lesion, altered the distribution of bax, bcl-2 and bcl-x proteins in the SN. Bcl-2 and bax were decreased in the TH-positive cells of the SN from 3 to 14 days post-lesion and many TH-positive neurons were bcl-2 negative. Neuronal bcl-x was initially unchanged after lesion, but increased in astrocytes between 3–7 days post-lesion before the increase in GFAP immunoreactivity, which was detectable at days 10–14. While the neuronal distribution of bcl-2 and bcl-x does not change following lesion, bax became evenly distributed thought the soma. Morphological features of apoptosis, including TUNEL labeling and chromatin condensation was not observed. These data suggest that striatal 6-OHDA lesions do not result in classical apoptosis in the SN of the adult rat, even though there are changes in the content and distribution of members of the bcl-2 family of proteins.     

Development of a human neuronal cell model for human immunodeficiency virus (HIV)-infected macrophage-induced neurotoxicity: apoptosis induced by HIV type 1 primary isolates and evidence for involvement of the Bcl-2/Bcl-xL-sensitive intrinsic apoptosis pathway

Neuronal apoptosis within the central nervous system (CNS) is a characteristic feature of AIDS dementia, and it represents a common mechanism of neuronal death induced by neurotoxins (e.g., glutamate) released from human immunodeficiency virus (HIV)-infected macrophages (HIV/macrophage-induced neurotoxicity). Neuronal apoptosis may result from activation of the intrinsic (mitochondrial/bcl-2 regulated) or extrinsic (death receptor) pathways, although which pathway predominates in CNS HIV infection is unknown. Apoptosis initiated by the intrinsic pathway is typically blocked by antiapoptosis Bcl-2 family proteins, such as Bcl-2 and Bcl-xL, but whether these can block HIV/macrophage-induced neuronal apoptosis is unknown. To determine the potential role of the Bcl-2 family in HIV/macrophage-induced neuronal apoptosis, we developed a unique in vitro model, utilizing the NT2 neuronal cell line, primary astrocytes and macrophages, and primary CNS HIV type 1 (HIV-1) isolates. We validated our model by demonstrating that NT2.N neurons are protected against HIV-infected macrophages by N-methyl-D-aspartate (NMDA) glutamate receptor antagonists, similar to effects seen in primary neurons. We then established stable NT2.N neuronal lines that overexpress Bcl-2 or Bcl-xL (NT2.N/bcl-2 and NT2.N/bcl-xL, respectively) and determined their sensitivity to macrophages infected with primary R5, X4, and R5/X4 HIV-1 isolates. We found that NT2.N/bcl-2 and NT2.N/bcl-xL neurons were resistant to apoptosis induced by either R5, X4, or R5/X4 isolates and that resistance was abrogated by a Bcl-2 antagonist. Thus, the NMDA receptor/bcl-2-regulated apoptotic pathway contributes significantly to HIV/macrophage-induced neuronal apoptosis, and Bcl-2 family proteins protect neurons against the spectrum of primary HIV-1 isolates. Modulation of bcl-2 gene expression may therefore offer adjunctive neuroprotection against development of AIDS dementia.     

Multiple signaling events in amyloid beta-induced,oxidative stress-dependent neuronal apoptosis

Current evidence suggests that amyloid beta peptides (Abeta) may play a major role in the pathogenesis of Alzheimer's disease by eliciting oxidative stress and neuronal apoptosis. In this study we have used differentiated SK-N-BE neurons to investigate molecular mechanisms and regulatory pathways underlying apoptotic neuronal cell death elicited by Abeta(1-40) and Abeta(1-42) peptides as well as the relationships between apoptosis and oxidative stress. Abeta peptides, used at concentrations able to induce oxidative stress, elicit a classic type of neuronal apoptosis involving mitochondrial regulatory proteins and pathways (i.e. affecting Bax and Bcl-2 protein levels as well as release of cytochrome c in the cytosol), poly-ADP rybose polymerase cleavage and activation of caspase 3. This pattern of neuronal apoptosis, that is significantly prevented by alpha-tocopherol and N-acetylcysteine and completely abolished by specific inhibitors of stress-activated protein kinases (SAPK) such as JNKs and p38(MAPK), involved early elevation of p53 protein levels. Pretreatment of neurons with alpha-pifithrin, a specific p53 inhibitor, resulted in a 50-60% prevention of Abeta induced apoptosis. These results suggest that oxidative stress - mediated neuronal apoptosis induced by amyloid beta operates by eliciting a SAPK-dependent multiple regulation of pro-apoptotic mitochondrial pathways involving both p53 and bcl-2.     

CREB Is One Component of the Binding Complex of the Ces-2/E2A-HLF Binding Element and Is an Integral Part of the Interleukin-3 Survival Signal

The Ces-2/E2A-HLF binding element (CBE) is recognized by Caenorhabditis elegans death specification gene product Ces-2 and human acute lymphocytic leukemia oncoprotein E2A-HLF. In an attempt to identify a cellular CBE-binding protein(s) that may be involved in apoptosis regulation in mammals, multiple nuclear binding complexes of CBE were identified in various mammalian cell lines and tissues by electrophoretic mobility shift assay. Cyclic AMP (cAMP)-responsive element (CRE)-binding protein (CREB) was present in one major CBE complex of Ba/F3 and TF-1 cells, and both in vitro-translated and Escherichia coli-synthesized CREB bound to CBE. Activation of CREB by cAMP-elevating chemicals or the catalytic subunit of protein kinase A (PKAc) resulted in induction of the CBE-driven reporter gene. Stimulation of Ba/F3 cells with interleukin-3 (IL-3) promptly induced phosphorylation of CREB at serine(133) partially via a PKA-dependent pathway. Consistently, Ba/F3 cell survival in the absence of IL-3 was prolonged by activation of PKA. Conversely, treatment of cells with a PKA inhibitor or expression of the dominant negative forms of the regulatory subunit type I of PKA and CREB overrode the survival activity of IL-3. Last, the bcl-2 gene was demonstrated to be one candidate cellular target of the CREB-containing CBE complex, as mutations in the CRE and CBE sites significantly reduced the IL-3 inducibility of the bcl-2 promoter. Together, our results suggest that CREB is one cellular counterpart of Ces-2/E2A-HLF and is part of IL-3 dependent apoptosis regulation in hematopoietic cells.