Oleamide attenuates apoptotic death in cultured rat cerebellar granule neurons

The effect of oleamide on apoptosis was investigated by using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide reduction assay, DNA staining assay with propidium iodide and caspase-3 activity analyses. The present results showed that oleamide significantly attenuated the cell death and nuclear condensation of cultured rat cerebellar granule neurons induced by K(+) deprivation in a dose-dependent manner. The oleamide actions were well parallel with the attenuation of caspase-3 activity in the process of apoptotic death. Moreover, neither elaidic acid nor stearic acid, two fatty acids structurally related to oleamide without the Delta(9)-cis double bond, had similar effects on the cell death, suggesting the selectively structural features of oleamide required for this action. These data provided the first evidence of a protective effect of oleamide against apoptosis in a structurally specific manner.     

Adenosine receptor ligands protect against a combination of apoptotic and necrotic cell death in cerebellar granule neurons

Agonists at A₁ receptors and antagonists at A_2A receptors are known to be neuroprotective against excitotoxicity. We set out to clarify the mechanisms involved by studying interactions between adenosine receptor ligands and endogenous glutamate in cultures of rat cerebellar granule neurons (CGNs). Glutamate and the selective agonist N-methyl-d-aspartate (NMDA), applied to CGNs at 9 div (days in vitro), both induced cell death in a concentration-dependent manner, which was attenuated by treatment with the NMDA receptor antagonists dizocilpine, d-2-amino-5-phosphono-pentanoic acid (d-AP5) or kynurenic acid (KYA), but not by the non-NMDA receptor antagonist 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX). Glutamate toxicity was reduced in the presence of all of the following: cyclosporin A (CsA), a blocker of the membrane permeability transition pore, the caspase-3 inhibitor, benzyloxycarbonyl-Asp(OMe)-Glu(OMe)-Val-Asp(OMe)-fluoromethylketone (Z-DEVD-fmk), the poly (ADP-ribose) polymerase (PARP-1) inhibitor 3,4-dihydro-5-[4-(1-piperidinyl)butoxyl]-1(2H)-isoquinolinone (DPQ), and nicotinamide. This is indicative of involvement of both apoptotic and necrotic processes. The A₁ receptor agonist, N ⁶-cyclopentyladenosine (CPA), and the A_2A receptor antagonist 4-(2-[7-amino-2-[2-furyl][1,2,4]triazolo[2,3-a][1,3,5]triazo-5-yl-amino]ethyl)phenol (ZM241385) afforded significant protection, while the A₁ receptor blocker 8-cyclopentyl-1,3-dipropylxanthine (DPCPX) and the A_2A receptor agonist 2-p-(2-carboxyethyl)phenethylamino-5′-N-ethylcarboxyamidoadenosine (CGS21680) had no effect. These results confirm that glutamate-induced neurotoxicity in CGNs is mainly via the NMDA receptor, but show that a form of cell death which exhibits aspects of both apoptosis and necrosis is involved. The protective activity of A₁ receptor activation or A_2A receptor blockade occurs against this mixed profile of cell death, and appears not to involve the selective inhibition of classical apoptotic or necrotic cascades.     

Effect of transitory glucose deprivation on mitochondrial structure and functions in cultured cerebellar granule neurons

We found that 60-min glucose deprivation leads to progressive decrease in the mitochondrial membrane potential and increase in [Ca²⁺]_i in cultured cerebellar granule neurons. The latter effect was fully reversible, returning to the basal level 60 min after restoration of normal glucose level in the incubation medium, whereas mitochondrial membrane potential remained at 10.0 ± 1.8% below the initial value. Electron microscopy indicated that glucose deprivation induced appearance of mitochondria with local lightening of the matrix and destruction of cristae. This mitochondrial conformation was preserved during the restoration phase after glucose level in the cultivation medium returned to the normal level. Neuronal death within a 24-h period after 60-min glucose deprivation was relatively small, being 14.0 ± 4.4%.     

Distinct signaling pathways of precursor BDNF and mature BDNF in cultured cerebellar granule neurons

Recent studies have focused on a distinctive contrast between bioactivities of precursor brain-derived neurotrophic factor (proBDNF) and mature BDNF (matBDNF). In this study, using a proteolytic cleavage-resistant proBDNF mutant (CR-proBDNF), signaling mechanisms underlying the proapoptotic effect of proBDNF and antiapoptotic effect of matBDNF on the low potassium (LK)-inducing cell death of cultured cerebellar granule neurons (CGNs) were analyzed. A time course study demonstrated that unlike matBDNF, CR-proBDNF failed to induce TrkB phosphorylation for up to 360 min. CR-proBDNF did not activate ERK-1, ERK-2 and Akt, which are involved in TrkB-induced cell survival signaling, while matBDNF activated these kinases. On the other hand treatment of CGNs with CR-proBDNF led to a rapid activation of Rac-GTPase and phosphorylation of JNK which are involved in p75^NTR-induced apoptosis. In addition, a JNK-specific inhibitor, SP600125, inhibited the CR-proBDNF-induced apoptosis but did not affect the antiapoptotic effect of matBDNF. CR-proBDNF treatment led to an earlier appearance of active caspase-3. In contrast, matBDNF dramatically postponed the appearance of active caspase-3. Not like other signaling molecules, activation of caspase-3 was conversely regulated by both CR-proBDNF and matBDNF. These results thus suggest that in CGNs proBDNF elicits apoptosis via activation of p75^NTR, Rac-GTPase, JNK, and caspase-3, while matBDNF signals cell survival via activation of TrkB, ERKs and Akt, and deactivation of caspase-3.     

Activation of Fas receptor is required for the increased formation of the disialoganglioside GD3 in cultured cerebellar granule cells committed to apoptotic death

Apoptosis was induced in cultured cerebellar granule cells by lowering extracellular K+ concentrations (usually from 25 to 10 mM). The apoptotic phenotype was preceded by an early and transient increase in the intracellular levels of the disialoganglioside, GD3, which behaves as a putative pro-apoptotic factor. We examined whether activation of Fas receptor mediates the increase in GD3 formation in granule cells committed to die. Degenerating granule cells showed increased expression of both Fas receptor and its ligand (Fas-L), at times that coincided with the increase in GD3 levels and the induction of GD3 synthase mRNA. Addition of neutralizing anti-Fas-L antibodies reduced the extent of 'low-K+'-induced apoptosis and abolished the increase in GD3 levels and GD3 synthase mRNA. Similar reductions were observed in cultures prepared from gld or lpr mice, which harbor loss-of-function mutations of Fas-L and Fas receptor, respectively. In addition, exogenous application of soluble Fas-L further enhanced both the increase in GD3 formation and cell death in cultured granule cells switched from 25 into 10 mM K+. We conclude that activation of Fas receptor is entirely responsible for the increase in GD3 levels and contributes to the development of apoptosis by trophic deprivation in cultured cerebellar granule cells.     

Roles of cyclin-dependent kinase 4 and p53 in neuronal cell death induced by doxorubicin on cerebellar granule neurons in mouse

Cell cycle regulators such as cyclin-dependent kinases (Cdks) and their inhibitors (Ckis) have been reported to be involved in neuronal cell death (NCD) induced by a variety of insults such as ischemia, UV-irradiation, nerve growth factor (NGF)-withdrawal, and anticancer therapeutics. But their precise interactive regulation has still to be unveiled. In the present study, we focused on cell cycle regulators such as Cdk4, p21(WAF1) and p53 to clarify their regulatory mechanisms, using NCD induced by doxorubicin (D-NCD) in mouse cerebellar granule neurons as a model. Doxorubicin induced NCD in a dose-dependent manner, a typical feature of apoptosis as determined by TUNEL assay. Doxorubicin increased the protein expression of p53 in time- and dose-dependent manners. The protein expression of p21(WAF1), a Cki of Cdk4, was stimulated by doxorubicin at low concentrations, but it disappeared at high concentrations. Doxorubicin activated the kinase activity of Cdk4 without the enhancement of Cdk4 protein. 3-Amino-9-thio(10H)-acridone (3-ATA), the specific inhibitor of Cdk4, prevented D-NCD in a dose-dependent manner. Wortmannin, an inhibitor of ATM (ataxia telangiectasia, mutated) that has high homology with the phosphatidyl-inositol-3-kinase (PI3K) family and has protein kinase activity for the induction of p53 with specificity for serine and threonine residues, inhibited the activation of Cdk4 without the induction of p53 in D-NCD. These data suggest that (1) Cdk4 is one of the essential components for inducing NCD, that (2) p53 may prevent D-NCD through the induction of p21(WAF1) at low concentrations of doxorubicin, and that (3) Cdk4 might be activated by the same signal-molecules, like ATM, that are necessary for the activation of p53 in D-NCD.     

The role of apoptotic volume decrease and ionic homeostasis in the activation and repression of apoptosis

The mechanism of activation and repression of apoptosis has been a central focus of many studies examining the role of programmed cell death in both normal and pathological conditions. Despite intensive research efforts, the precise cellular and molecular mechanisms that trigger and/or prevent apoptosis remain undefined. A universal characteristic of apoptosis is the loss of cell volume or cell shrinkage, recently termed apoptotic volume decrease. While cell shrinkage has traditionally been viewed as a passive event during apoptosis, recent work from several laboratories has shown that the loss of cell volume, or more specifically the flux of ions associated with the change in cell size, play a critical role in the regulation of the cell death machinery. On going studies continue to support the hypothesis that the change in intracellular ions can alter a cells decision to die by apoptosis.     

大鼠严重烫伤淋巴组织细胞凋亡的原位TUNEL及电镜观察

目的：观察严重烧伤后大鼠淋巴组织内细胞凋亡状况。方法：采用大鼠３０％体表面积Ⅲ度烫伤模型，应用原位组织ＴＵＮＥＬ技术及透射电镜，分别观察烫伤后３、６、１２ｈ３个时相点大鼠胸腺、脾、肠系膜淋巴结凋亡细胞的分布、定位和形态特征。结果：烫伤后６、１２ｈ，在胸腺、脾、肠系膜淋巴结出现明显的细胞凋亡，并见其呈散在分布于胸腺的皮质，脾的白髓及肠系膜淋巴结的皮质淋巴小结之间。结论；严重烫伤可致大鼠淋巴组织的细胞     

Effects of urea on K+ fluxes and cell volume in perfused rat liver

Exposure of the perfused rat liver to a perfusate made hyperosmotic by the presence of 200 mmol l^–1glucose led, as expected, to marked, transient hepatocellular shrinkage followed by volume-regulatory net K⁺ uptake. However, even after this volume-regulatory K⁺ uptake had ceased, the liver cells are still slightly shrunken. Withdrawal of glucose from the perfusate resulted in marked transient cell swelling, net K⁺ release from the liver and restoration of cell volume. However, when the Krebs-Henseleit perfusate was made hyperosmotic by the presence of urea (20–300 mM), there was no immediate decrease in liver mass, yet a slight and persistent cell shrinkage developing 2 min after the onset of exposure to urea. Surprisingly, urea induced concentration-dependent net K⁺ efflux from the liver and removal of urea net K⁺ reuptake from the inflowing perfusate. The urea (200 mM)-induced net K⁺ release resembled that observed following a lowering of the influent [NaCl]: making the perfusate hypoosmotic (245 mosmol l^–1, by reducing influent [NaCl] by 30 mM) gave roughly the same K⁺ response as hyperosmotic exposure (505 mosmol/l) resulting from the presence of 200 mM urea. The urea-induced K⁺ efflux was not inhibited in the presence of ouabain (1 mM), or in Ca⁺⁺-free perfusion, but was modified in the presence of quinidine (1 mM) or Ba⁺⁺ (1 mM). The direction in which the liver was perfused had no effect on the urea-induced net K⁺ release. Electrophysiological studies showed that urea led to quinidine-sensitive hyperpolarization and increase in K⁺ selectivity of plasma membranes, suggesting opening of K⁺ channels in the hepatocyte plasma membrane in response to urea. The data suggest that urea, but not glucose, enters the hepatocyte as quickly as water. Furthermore, urea at high concentrations apparently leads to K⁺ efflux from the hepatocyte and cell shrinkage, possibly due to opening of K⁺ channels in the hepatocyte plasma membrane.     

Beta-amyloid activated microglia induce cell cycling and cell death in cultured cortical neurons

Immunocytochemical studies of postmortem human tissue have shown that the neurons at risk for degeneration in Alzheimer’s are marked by the ectopic expression of several cell cycle components. The current work investigates the roles that β-amyloid activated microglia might play in leading neurons to re-express cell cycle components. Stable cultures of E16.5 mouse cortical neurons were exposed to β-amyloid alone, microglial cells alone, or microglial cells activated by β-amyloid. Increased cell death was found in response to each of these treatments, however, only the amyloid activated microglial treatment increased the number of neurons that were positive for cell cycle markers such as PCNA or cyclin D and incorporation of BrdU. Double labeling with BrdU and TUNEL techniques verified that the ‘dividing’ neurons were dying, most likely through an apoptotic mechanism. The identity of the soluble factor(s) elaborated by the microglia remains unknown, but FGF2, a suspected neuronal mitogen, was ruled out. These results further support a model in which microglial activation by β-amyloid is a key event in the progression in Alzheimer’s disease.     

Neuroprotection of microglia conditioned media from apoptotic death induced by staurosporine and glutamate in cultures of rat cerebellar granule cells

Microglia, the immune cells of the mammalian CNS, have often been indicated as dangerous effector cells for their activation in response to traumatic CNS injuries or immunological stimuli and for their involvement in many chronic neurodegenerative diseases. Recently, several in vitro and in vivo studies have emphasized that microglial activity is essential in promoting neuronal survival. We have tested the efficacy of media directly conditioned by microglia or conditioned by microglia after having been exposed to apoptotic neurons, towards neuroprotection of rat cerebellar granule cells (CGCs) challenged with staurosporine or glutamate. Apoptotic death of CGC caused by staurosporine, as well as by a mild excitotoxic stimulus delivered through sub-chronic glutamate treatment, was significantly counteracted by microglia conditioned media. On the other hand, an acute excitotoxic insult delivered through a short pulse of glutamate exposure in the absence of magnesium and resulting in a mix of apoptotic and necrotic death was only marginally counteracted by microglia conditioned media. The present results extend the available information regarding the neuroprotective role of microglia and support the usefulness of employing the culture approach for perspective identification of neuroprotective factors released by these cells. Furthermore, the use of media previously exposed to apoptotic neurons to elicit the neuroprotective response of microglia, indicate the feasibility to re-create also in the isolated culture conditions, at least some of the elements at the basis of neuron/microglia cross-talk.     

Role of programmed cell death in normal neuronal development and function

The consequences of eliminating the process of programmed cell death during the development of the nervous system is examined by reviewing studies in the genetic model organisms Caenorhabditis elegans, Drosophila melanogaster, Danio rerio and Mus musculus, where mutations of cell death genes have eliminated or reduced programmed cell death in the nervous system. In many cases, genetic elimination of cell death leads to embryonic mortality or gross anatomical malformations; however, there are cases where animals develop normally but with excess neurons and glia in the nervous system. Undead cells either differentiate and function as working neurons, in some instances being of smaller size, or fail to differentiate and lack normal connections with their targets. Changes in motor control and sensory processing are generally not observed, except for during the most complex of behaviors. Examination of organisms where death genes have been genetically eliminated reveals that programmed cell death may play an important role in sculpting gross brain structure during early development of the neural tube. In contrast, the consequences of preventing neuronal cell death at later developmental stages (e.g. during vertebrate synapse formation) are just beginning to be understood.     

Comparative analysis of the effects of resveratrol in two apoptotic models: inhibition of complex I and potassium deprivation in cerebellar neurons

The mechanism involved in neuronal apoptosis is largely unknown. Studies performed on neuronal cell cultures provide information about the pathways which orchestrate the process of neuronal loss and potential drugs for the treatment of neurological disorders. In the present study we select resveratrol, a natural antioxidant, as a potential drug for the treatment of neurodegenerative diseases. We evaluate the neuroprotective effects of resveratrol in two apoptotic models in rat cerebellar granule neurons (CGNs): the inhibition of mitochondrial complex I using 1-methyl-4-phenylpyridinium (MPP(+)) (an in vitro model of Parkinson's disease) and serum potassium withdrawal. We study the role of the mammalian silent information regulator 2 (SIRT1) in the process of neuroprotection mediated by resveratrol. Because recent studies have demonstrated that SIRT1 is involved in cell survival and has antiaging properties, we also measured changes in the expression of this protein after the addition of these two apoptotic stimuli. MPP(+)--induced loss of cell viability and apoptosis in CGNs was prevented by the addition of RESV (1 microM to 100 microM). However, the neuroprotective effects were not mediated by the activation of SIRT1, since sirtinol-an inhibitor of this enzyme--did not attenuate them. Furthermore MPP(+) decreases the protein expression of SIRT1. RESV did not prevent serum potassium withdrawal-induced apoptosis although it did completely attenuate oxidative stress production by these apoptotic stimuli. Furthermore, serum potassium withdrawal increases the expression of SIRT1. Our results indicate that the antiapoptotic effects of RESV in MPP(+) are independent of the stimulation of SIRT1 and depend on its antioxidant properties. Furthermore, because SIRT1 is involved in neuronal survival depending on the apoptotic stimuli, changes in the expression of SIRT1 could be involved in the regulation of the apoptotic route.     

Massive cell death of cerebellar granule neurons accompanied with caspase-3-like protease activation and subsequent motor discoordination after intracerebroventricular injection of vincristine in mice

Vincristine, a microtubule-depolymerizing agent, is known to induce neuronal cell damage. Biochemical, histological and behavioral alterations were investigated after intracerebroventricular injection of vincristine in mice. Intracerebroventricular injection of vincristine caused caspase-3-like protease activation followed by nucleosomal release in the cerebellum. Histological examinations showed that vincristine-induced damage was relatively specific to granule cells in the cerebellum, and terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick-end labeling-positive cells were observed among these cells. Chromatin condensation, one of the criteria for apoptosis, was seen on electron microscopy. Behavioral changes, namely head movements, pivoting and backward walking, were observed in parallel with the increase of caspase-3-like protease activity and nucleosomal release. Furthermore, motor function tests (bulb balance test and rotating rod test) showed deficits of motor coordination ability. These observations suggest that intracerebroventricular vincristine causes massive apoptosis of cerebellar granule cells accompanied with caspase-3-like protease activation, leading to motor dysfunction, in this model. These vincristine-treated mice should be a useful in vivo model for examination of neuronal apoptosis, which might be involved in a variety of neurodegenerative diseases.     

Cedrol restricts the growth of colorectal cancer in vitro and in vivo by inducing cell cycle arrest and caspase-dependent apoptotic cell death

Background: Cedrol is a natural sesquiterpene alcohol found in Cedrus atlantica, which has been proven to have a broad spectrum of biological activities, such as antimicrobial, anti-inflammatory, analgesic, anxiolytic, and anti-cancer effects. However, the underlying anticancer mechanisms and in vivo inhibitory effects of cedrol on colorectal cancer (CRC) have not been elucidated. In the present study, we investigated the anti-CRC potential of cedrol using in vitro and in vivo models.Methods: The effects of cedrol on cell viability, cell cycle progression, and apoptosis of HT-29 and CT-26 cells were detected by MTT, flow cytometry, and TUNEL assays. Western blotting was used to measure protein expression for molecular signaling analyses.Results: Cedrol inhibited HT-29 and CT-26 cell proliferation in a time- and dose-dependent manner, with IC₅₀ values of 138.91 and 92.46 µM, respectively. Furthermore, cedrol induced cell cycle arrest at the G0/G1 phase by regulating the expression of cell cycle regulators, such as CDK4 and cyclin D1, and triggered apoptosis through extrinsic (FasL/caspase-8) and intrinsic (Bax/caspase-9) pathways. In addition, cedrol in combination with the clinical drug 5-fluorouracil exhibited synergistic inhibitory effects on CRC cell growth. Importantly, cedrol treatment suppressed the progression of CRC and improved the survival rate of animals at a well-tolerated dose.Conclusion: These results suggest that cedrol has an anti-cancer potential via induction of cell cycle arrest and apoptosis, and it could be considered as an effective agent for CRC therapy.     

Inosine partially mimics the effects of glucose on ionic fluxes,electrical activity,and insulin release in mouse pancreatic B-cells

The purine ribonucleoside inosine is known to be metabolized in islet cells (its ribose moiety feeds into the pentose-phosphate cycle) and stimulate insulin release, but the mechanisms of this stimulation have not been established. These were investigated with mouse islets. In the absence of glucose, 5 mM inosine decreased⁸⁶Rb⁺ efflux from islet cells, depolarized the B-cell membrane, induced electrical activity (slow waves of membrane potential with bursts of spikes on the plateau), accelarated⁴⁵Ca²⁺ efflux and stimulated insulin release with the same efficiency as 10 mM glucose. Raising the concentration of inosine to 20 mM only had a slight further effect and, in particular, failed to cause persistent depolarization of the B-cell membrane. The electrical activity triggered by inosine was blocked by cobalt, and the stimulation of⁴⁵Ca²⁺ efflux and insulin release was abolished in a Ca²⁺-free medium. The effects of 10 mM glucose on electrical activity,⁴⁵Ca²⁺ efflux and insulin release were augmented by as little as 0.5 mM inosine. All effects of inosine were abolished by an inhibitor of nucleoside transport (nitrobenzylthioguanosine) and markedly impaired by inhibitors of nucleoside phosphorylase (formycin B) or of glycolysis (iodoacetate). In conclusion, inosine metabolism in B-cells induces insulin release by triggering the same sequence of events as glucose metabolism: a decrease of K⁺ permeability of the B-cell membrane, leading to depolarization and activation of voltage-dependent Ca channels.     

Effects of celecoxib on ionic currents and spontaneous firing in rat retinal neurons

Accumulating evidence suggests that the side effects of celecoxib, widely used to treat muscle and joint pain, may be mediated in part through cyclooxygenase-2 (COX-2) independent mechanisms, such as inhibition of ion channels. In this study we report effects of celecoxib on ionic currents and neuronal activity in isolated rat retinal neurons. We found that celecoxib suppressed voltage-gated potassium currents in retinal bipolar cells with an effective concentration to inhibit 50% of function (EC(50)) of 5.5 muM. In retinal amacrine and ganglion cells, celecoxib inhibited voltage-dependent sodium channels with an EC(50) of 5.2 muM, and voltage-dependent transient and sustained potassium currents with EC(50)s of 16.3 and 9.1 muM, respectively. Notably, the rate of spontaneous spike activity was dramatically suppressed in ganglion and amacrine cells with an EC(50) of 0.76 muM. All actions of celecoxib on ionic currents and action potentials occurred from the extracellular side and were completely reversible. These findings indicate that inhibition of ion channels by celecoxib in the CNS may affect neuronal function at clinically relevant concentrations.     

Prognostic relevance of apoptotic cell death in non-Hodgkin's lymphomas: a multivariate survival analysis including Ki67 and p53 oncoprotein expression

Aims : To evaluate the independent prognostic value of apoptotic versus proliferative fractions in a series of 92 patients with non-Hodgkin's lymphomas (NHL).  

Methods and results

Apoptotic fractions were quantified by use of the TdT (terminal deoxynucleotidyl-transferase)-mediated in-situ end-labelling technique (TUNEL), the percentage of positive cells constituting the apoptotic index (AI). Proliferative rate was expressed as percentage of Ki67 positive cells (Ki67 LI). Tissues were also stained for p53 protein with the DO-1 antibody. Patients were followed up until death ( n  = 33) or for an average of 63 months ( n  = 56). AI increased with malignancy grade and proliferative activity but was not related to location, cell of origin, clinical stage, bone marrow involvement and p53 expression. In multivariate analysis, overall survival was independently influenced by grade, stage, p53 LI and chemotherapy. The independent predictors of disease-free survival were Ki67 LI location and chemotherapy. AI turned out to be the only independent (negative) predictor of post-relapse survival. On the other hand, a low Ki67 LI increased the risk of relapse (logistic regression analysis) whereas a low p53 LI increased the probability of complete response.  

Conclusions

Our results suggest that the combined assessment of apoptotic fraction, proliferative rate and p53 expression may provide important prognostic information independent of other clinicopathological parameters in NHL.