Nuclear factor-kappaB-dependent cyclin D1 induction and DNA replication associated with N-methyl-D-aspartate receptor-mediated apoptosis in rat striatum

Cell cycle reentry has been found during apoptosis of postmitotic neurons under certain pathological conditions. To evaluate whether nuclear factor-kappaB (NF-kappaB) activation promotes cell cycle entry and neuronal apoptosis, we studied the relation among NF-kappaB-mediated cyclin induction, bromodeoxyuridine (BrdU) incorporation, and apoptosis initiation in rat striatal neurons following excitotoxic insult. Intrastriatally injected N-methyl-D-aspartate receptor agonist quinolinic acid (QA, 60 nmol) elicited a rise in cyclin D1 mRNA and protein levels (P<0.05). QA-induced NF-kappaB activation occurred in striatal neurons and nonneuronal cells and partially colocalized with elevated cyclin D1 immunoreactivity and TUNEL-positive nuclei. QA triggered DNA replication as evidenced by BrdU incorporation; some striatal BrdU-positive cells were identified as neurons by colocalization with NeuN. Blockade of NF-kappaB nuclear translocation with the recombinant peptide NF-kappaB SN50 attenuated the QA-induced elevation in cyclin D1 and BrdU incorporation. QA-induced internucleosomal DNA fragmentation was blunted by G(1)/S-phase cell cycle inhibitors. These findings suggest that NF-kappaB activation stimulates cyclin D1 expression and triggers DNA replication in striatal neurons. Excitotoxin-induced neuronal apoptosis may thus result from, at least partially, a failed cell cycle attempt.     

Inactivation of aconitase during the apoptosis of mouse cerebellar granule neurons induced by a deprivation of membrane depolarization

During the excitotoxic neuronal cell death which accompanies an overflow of extracellular Ca(2+) into neurons, aconitase, an oxidative stress-sensitive enzyme of the tricarboxylic acid (TCA)-cycle in mitochondria, is inactivated due to the generation of oxidative stress (Patel et al. [1996] Neuron 16:345-355). In this study, we investigated whether aconitase could be inactivated during the apoptosis of mouse cerebellar granule cells (CGCs), which was caused by a deprivation of membrane depolarization followed by a stoppage of Ca(2+) influx into CGCs. Upon lowering the potassium (K(+)) concentration in medium from 25 to 5 mM (low K(+)), aconitase was inactivated in accordance with the decrease in methylthiazoletetrazolium (MTT)-reducing activity although its mRNA expression did not change. The blockade of Ca(2+) influx into CGCs mediated by nicardipine at 25 mM KCl also caused the inactivation of aconitase, accompanying induction of the apoptosis of CGCs. Suppression of the apoptosis of CGCs mediated by the Ca(2+) influx or neurotrophic factors such as brain-derived neurotrophic factor (BDNF) and adenylate cyclase activating polypeptide-38 (PACAP-38) attenuated the aconitase inactivation as well as the lactate dehydrogenase (LDH)-release and the decrease in MTT reduction. On the other hand, the levels of intracellular glutathione and manganese superoxide dismutase-2 mRNA decreased under the low K(+) condition, supporting a cause for oxidative stress at low K(+) due to a loss of anti-oxidant activity. Thus, the inactivation of aconitase is also caused by a deprivation of Ca(2+) influx into neurons, suggesting that aconitase is a key mitochondrial enzyme influencing the viability of neurons in response to oxidative stress.     

Changes of KCl sensitivity of proliferating neural progenitors during in vitro neurogenesis

The effects of KCl-treatment on the survival and proliferation of NE-4C self-renewing neural progenitor cells were investigated during early phases of in vitro induced neurogenesis. NE-4C cells, derived from the anterior brain vesicles of embryonic mouse (E9), divided continuously under non-inducing conditions, but acquired neuronal features within 6 days, if induced by all-trans retinoic acid (RA). During the first 2 days of induction, the cells went on proliferating and did not show signs of morphological differentiation. In this stage, the resting membrane potential of RA-induced cells adopted more negative values in comparison to non-induced ones. Despite the increased membrane polarity and K+ conductance, addition of 20-50 mM KCl failed to elicit inward Na+ currents and did not induce an increase in the intracellular Ca+ level. Long-term treatment with 25 mM KCl, on the other hand, resulted in a selective loss of cells committed to neuronal fate by both decreasing the rate of cell proliferation and increasing the rate of cell death. The data indicate that the viability and proliferation of neural progenitors are influenced by extracellular K+-level in a differentiation stage-dependent manner.     

Nuclear factor-kappaB p65 and upregulation of interleukin-6 in retinal ischemia/reperfusion injury in rats

We previously demonstrated that endogenous interleukin-6 (IL-6) is upregulated and may be neuroprotective after retinal ischemia. The purpose of this study is to investigate the role of nuclear factor kappa-B (NF-kappaB) in regulating IL-6 expression after ischemia. NF-kappaB p65 mRNA levels were significantly elevated between 2 and 12 h after the insult. A high number of NF-kappaB p65 positive cells were detected in the inner retina at 12 h after ischemia. Activated nuclear NF-kappaB p65 and IL-6 were colocalized in cells, which were also marked by a microglial/phagocytic cell marker (ED1) in the inner retina. Carbobenzoxy-L-leucyl-L-leucyl-L-leucinal (MG-132, a proteasome inhibitor, which inhibits IkappaB degradation and hence prevents the activation and translocation of NF-kappaB into the nucleus) abolished the increase in NF-kappaB p65 mRNA levels after the insult, while there was no effect by helenalin (an inhibitor which inhibits NF-kappaB activity by alkylation of the p65 subunit, thereby blocking its binding to the target DNA). However, MG-132 and/or helenalin significantly diminished the increase in IL-6 mRNA levels after the insult. Small interfering RNAs (siRNAs, inhibit target gene expression through the sequence-specific destruction of the target messenger RNA) against NF-kappaB p65 significantly reduced the increase in NF-kappaB p65 mRNA levels as well as IL-6 mRNA levels after ischemia. The number of retinal ganglion cells (RGCs) was also significantly decreased using the inhibitors of NF-kappaB compared with those of the controls after ischemia. These findings support the hypothesis that upregulation of endogenous retinal IL-6 in retinal I/R injury in microglial/phagocytic cells is controlled predominantly by NF-kappaB p65.     

Protection of adult mouse progenitor cells and human glioma cells by de novo decorin expression in an oxygen- and glucose-deprived cell culture model system.

We employed an in vitro hypoxia cell culture model system and gene transfer technology to examine the effect of the decorin gene on cell survival against oxygen and glucose deprivation (OGD). Ectopic expression of decorin in subventricular zone (SVZ) cells from adult male mouse brain and human glioblastoma U-87 cells kept the cells viable against 24 h of OGD. Fewer than 1% of decorin-synthesizing cells were apoptotic after 12 h of OGD. In contrast, 100% of the control cells were apoptotic even after 4 h of OGD. De novo decorin synthesis in SVZ and U-87 cells induced expression of p21, p27 and Ras, AKT (acutely transforming retrovirus AKT8 in rodent T-cell lymphoma), and phosphorylated AKT. Blocking of phosphoinositide 3-kinase (PI-3K), Ras, and the epidermal growth factor receptor with specific inhibitors had no effect on induction of Ras, p21, and p27 at the messenger RNA level in decorin-synthesizing SVZ and U-87 cells. PI-3K inhibitors significantly increased apoptosis in decorin-expressing cells. Our data indicate that induction of p21, p27, Ras, AKT, and phosphorylated AKT by decorin inhibits apoptosis and protects U-87 and SVZ cells against OGD. Therefore, our data suggest that decorin is a potent trophic factor that protects neuronal progenitor cells and glioma cells from OGD.     

Long-term expression of the c-fos protein during the in vitro differentiation of cerebellar granule cells induced by potassium or NMDA.

Levels of the c-fos protein were assayed in mouse cerebellar granule cells during their in vitro development under different culture conditions. When grown in media favoring both their survival and differentiation, i.e. in the presence of 30 mM K+ or 12.5 mM K+ plus 100 microM N-methyl-D-aspartate (NMDA), the c-fos protein becomes detectable in the nucleus of granule cells on and after 6 days and persists to high levels until the culture begins to decline. The protein c-fos appears therefore after the critical period described for the survival effect of K+ depolarization or NMDA receptor stimulation which corresponds to days 2-5 after plating. The c-fos protein remains however scarcely detectable or undetectable throughout the life-span of cells cultured under conditions providing poor survival and differentiation, i.e. in the presence of low K+ (5 or 12.5 mM) alone or when the effect of NMDA is blocked by the NMDA receptor antagonist MK-801. Interestingly, in cortical and striatal neurons, the survival and differentiation of which being not affected by depolarizing media, no c-fos protein is detected whatever the culture conditions tested at least during the first 18 days in vitro. This suggests that long-term expression of the c-fos gene might be related to some aspect of the late in vitro differentiation process of cerebellar granule cells.     

Brief exposure to NMDA produces long-term protection of cerebellar granule cells from apoptosis

Cerebellar granule cells (CGCs) require excitatory inputs to survive during their postnatal migration from the external to the internal granule cell layers. The lack of innervation of mossy fibres induces CGC death by apoptosis. In vitro, CGCs die by apoptosis in the presence of physiological concentrations of KCl (5 mm or K5) but they survive in the presence of depolarizing concentrations of KCl (25 mm or K25) or N-methyl-d-aspartate (NMDA) by a mechanism dependent on calcium influx. The addition of NMDA or K25, for only 24 h, to immature CGCs cultures [2 days in vitro (DIV)] was able to produce a remarkable and long-term protection until 8 DIV. Moreover, our data show that NMDA and K25-mediated long-lasting protection was related to an inhibition of caspase-3 activity. By using different protein kinase inhibitors, we have shown that the inhibition of caspase-3 activation by NMDA was dependent on the activation of tyrosine kinases and phosphatidylinositol 3-kinase (PI3-kinase). Moreover, an impairment in NMDA-mediated neuroprotection and caspase-3 inhibition was observed when the action of brain-derived neurotrophic factor (BDNF) was blocked. By contrast, K25-mediated neuroprotection was BDNF-independent and was mediated by a mitogen-activated protein kinase- and PI3-kinase-dependent inhibition of caspase-3.     

NOX2 mediates apoptotic death induced by staurosporine but not by potassium deprivation in cerebellar granule neurons

Neuronal apoptotic death involves the participation of reactive oxygen species (ROS), but their sources have not been completely elucidated. Previous studies have demonstrated that the ROS‐producing enzyme NADPH oxidase is present in neuronal cells and that this enzyme could participate in the apoptotic neuronal death. Cerebellar granule neurons (CGN) undergo apoptosis when cells are transferred from a medium with 25 mM KCl (K25) to a 5 mM KCl (K5) medium or when they are treated with staurosporine (ST). Under these conditions, apoptotic death of CGN is dependent on ROS production. In this study, we evaluated the role of NOX2, an NADPH oxidase homolog, in the apoptotic death of CGN induced by two different conditions. In CGN from NOX2‐deficient (ko) mice, a significantly lower rate of apoptotic death occurs compared with wild‐type (wt) CGN. Also, caspase‐3 activation, NADPH oxidase activity, and superoxide anion production induced by ST were markedly lower in ko neurons than in wt CGN. In contrast to the case with ST, when CGN were treated with K5, no differences were observed between ko and wt cells in any of the parameters measured. However, all NADPH oxidase inhibitors tested noticeably reduced cell death and apoptotic parameters induced by K5 in both wt and ko CGN. These results suggest that NOX2 could be necessary for apoptotic death induced by ST, but not by K5, which could require other member of the NOX family in the apoptotic process. © 2009 Wiley‐Liss, Inc.     

Inhibition of 5-HT1A receptor-dependent cell survival by cAMP/protein kinase A: role of protein phosphatase 2A and Bax

Serotonergic 5-HT(1A) receptor signaling leading to nuclear factor-kappaB (NF-kappaB) activation appears to be critical for cell survival. Adenylyl cyclase and protein kinase A (AC/PKA) are effectors of the 5-HT(1A) receptor that are inhibited by Galpha(i) subunits. Conversely, Gbetagamma(i) subunits downstream from the 5-HT(1A) receptor participate in the activation of extracellular signal-regulated kinases (ERK1/2), phosphatidylinositol 3-kinase (PI3K), Akt, and NF-kappaB. To model the contribution of pro- and antiapoptotic signaling cascades downstream of activated 5-HT(1A) receptor in cell survival, Chinese hamster ovarian (CHO) cells were employed that exogenously overexpress 5-HT(1A) receptors. Stimulation with the 5-HT(1A) receptor agonist 8-OH-DPAT and pharmacological agonists of AC induced PKA and protein phosphatase 2A (PP2A) activity, which in turn inhibited: Akt activity, IkappaBalpha degradation, nuclear translocation of NF-kappaB, and expression of X-linked inhibitor of apoptosis protein (XIAP/BIRC4). Pharmacological inhibition of PP2A with calyculin A potentiated Akt activity while attenuating ERK1/2 signaling via increased inhibitory phosphorylation of Raf (pSer259). In contrast, increased cAMP levels enhanced Bax translocation to the mitochondria, resulting in the release of cytochrome c, caspase-3 activation, and apoptosis induction. Our data suggest a central role of cAMP/PKA-dependent PP2A in shifting the homeostasis of intracellular signaling downstream of activated 5-HT(1A) receptor toward cell death in biological systems linked to neuropsychiatric disorders.     

Translocation of apoptosis-inducing factor in cerebellar granule cells exposed to neurotoxic agents inducing oxidative stress

We have previously shown that the neurotoxic compounds colchicine, methylmercury (MeHg) and hydrogen peroxide (H2O2) cause apoptosis in primary cultures of cerebellar granule cells (CGC), characterized by nuclear condensation and high-molecular weight DNA fragmentation. However, only colchicine triggers the activation of caspases, suggesting that factors other than caspase-activated DNase (CAD) are responsible for DNA cleavage in the other two models. Here we report that the two agents that cause oxidative stress, MeHg (1 micro m) and H2O2 (50 micro m), induce translocation of apoptosis-inducing factor (AIF) from the mitochondria to the nucleus in CGC. Our data suggest that, in absence of caspase activity, AIF translocation could be a key event leading to chromatin condensation and DNA degradation in CGC exposed to MeHg and H2O2.     

Apoptotic morphology does not always require caspase activity in rat cerebellar granule neurons

The death of a cell via apoptosis is characterized by morphological changes including cell shrinkage and nuclear condensation. Intracellularly, proteases, including caspases, are activated. In the present article we have compared the ability of three different neurotoxic agents to induce caspase activity in cerebellar granule cells (CGC). These compounds are the micro-tubule-disrupting agent colchicine and the oxidative stress-inducing agents hydrogen peroxide and meth-ylmercury (MeHg). We have previously shown that each of these agents causes nuclear changes that are consistent with apoptosis, i.e., induction of chromatin condensation and DNA cleavage into fragments of regular size (700, 300 and 50 kbp). However, only colchicine causes a large increase in caspase activity, as monitored by the ability of whole cell extracts to cleave the synthetic caspase substrate DEVD-MCA. In contrast, MeHg and hydrogen peroxide do not induce any significant increase of DEVDase activity as compared to control cells. Immunocytochemistry confirms that active caspase-3 is abundant only in colchicine-exposed cells. In agreement with these findings, the pan-caspase inhibitor, z-VAD-fmk, is efficient in protecting CGC against colchicine, but not against hydrogen peroxide or MeHg. These data suggest that in CGC the activation of caspases is not always required to induce morphological changes and pattern of DNA fragmentation consistent with apoptosis.     

Morphine promotes Jurkat cell apoptosis through pro-apoptotic FADD/P53 and anti-apoptotic PI3K/Akt/NF-kappaB pathways

Opiates have been shown to inhibit cell growth and trigger apoptosis, but the underlying molecular mechanisms remain unclear. We have previously shown that morphine induces Fas expression and promotes Fas-mediated apoptosis. Here, we investigated the mechanisms by which morphine modulates apoptosis in human Jurkat cells. Morphine-induced apoptosis was inhibited by transfection with a dominant negative Fas-associated death domain (FADD) plasmid, revealing that morphine-induced apoptosis is dependent on FADD. Furthermore, suppression of endogenous p53 expression by RNA interference technology considerably attenuated the morphine-induced apoptosis. In addition, morphine-induced apoptosis seems to be dependent on the activation of phosphatidylinositol 3-kinase (PI3K), as PI3K inhibition by the PI3K inhibitor LY294002 significantly enhanced morphine-induced apoptosis. Moreover, inhibition of Akt or nuclear factor-kappaB (NF-kappaB) expression by RNA interference technology also dramatically increased morphine-induced apoptosis. Our study thus demonstrates that morphine induces Jurkat cell apoptosis through FADD/p53, anti-apoptotic PI3K/Akt and NF-kappaB pathways.