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Insulin-like growth factor-1 inhibits STS-induced cell death and increases functional recovery of in vitro differentiated neurons Bruna Pucci,1,* Francesca Romana Bertani,1 Manuela Indelicato,1,3 Patrizio Sale,1,2 Emanuela Lococo,2 Francesca Grassi,4,5 Francesca Pagani,4,5 Valeria Colafrancesco,6 Emanuela Morgante,2 Marco Tafani,1 Massimo Fini1 and Matteo A. Russo1,2 of Cellular and Molecular Pathology; IRCCS San Raffaele Pisana; Rome Italy; 2Department of Experimental Medicine; La Sapienza University; Rome Italy; 3Department of Biomedical Science; University of Catania; Catania Italy; 4Laboratory of Neurophysiology; IRCCS San Raffaele Pisana; Rome Italy; 5Department of Human Physiology and Pharmacology and Excellence Center BEMM; La Sapienza University; Rome Italy; 6IRCCS Fondazione Bietti; Rome Italy
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Abbreviations: STS, staurosporine; NaB, sodium butyrate; IGF-1, insulin growth factor 1; SEM, scanning electron microscopy; TEM, transmission electron microscopy
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of neuronal circuits.2 Unfortunately, many patients experience loss of one or more neuronal populations due to accidents or pathological processes. For example, hippocampal and cortical neuronal death is typical of Alzheimer’s disease;3 in Parkinson’s disease the dopaminergic neurons of the midbrain die by apoptosis;4 Huntington’s disease is characterized by cell death of striated neurons whereas in ALS there is loss of motoneurons;5,6 cell loss occurs after cerebral ischemia during reperfusion and repair. Apoptosis has been implicated in the majority of neurodegenerative diseases. In these cases damage and clinical symptoms proceed slowly over many years. Interestingly, even if individual neurons are damaged they survive for long periods of time only to die rapidly at a later stage. Therefore, the progressive deficit typical of neurodegenerative diseases is a result of cumulative damage to single neurons. Programmed cell death (PCD) is defined by a series of biochemical and morphological alterations genetically controlled, leading to cellular dismantling through ATP-dependent mechanisms. In physiological conditions, PCD is strictly regulated in both development and tissue homeostasis.7 Alterations of PCD regulation have been implicated in several pathologies including neurodegeneration.8 Cell death can be activated by intrinsic stress such as growth factor deprivation or by extrinsic mechanisms such as activation of death receptors.9 The final decision to activate the suicide program is controlled by the Bcl-2 protein family.10 Bcl-2 family members are divided into three sub-families; pro-survival (Bcl-2, Bcl-xL, Bcl-w, Mcl-1 and A1), pro-apoptotic (Bax, Bak and Bok), and BH3 pro-apoptotic (Bad, Bid, Bik, Blk, Hrk, BNIP3 and BimL).11 The pro-apoptotic Bcl-2 proteins are often found in the cytosol where they act as sensors of cellular damage or stress. During apoptosis, they relocate to the surface of the mitochondria thereby inducing the permeabilization of the mitochondrial membrane and the formation of the apoptosome. The apoptosome is a cytosolic complex formed by the interaction between cytochrome c, pro-caspase-9 and APAF-1. Clustering and activation of pro-caspase-9 leads to activation of the effector caspase3.12 This executioner enzyme promotes a number of morphological changes including cell shrinkage, DNA fragmentation and plasma membrane blebbing that are considered hallmarks of apoptosis.13
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NG108-15 cells differentiate into neurons by 1 mM sodium butyrate (NaB) treatment. Differentiated cells resulted more resistant to staurosporine (STS) than proliferating cells. In particular, STS treatment decreased Bcl-2 and Bcl-xL content in mitochondria of proliferating cells, but not in mitochondria of differentiated cells. Bad was phosphorylated and downregulated only in differentiated cells. Bax accumulated in the mitochondria of proliferating but not differentiated cells. Mitochondrial release of cytochrome c was observed in proliferating cells, whereas mitochondria of differentiated cells retained cytochrome c. Proliferating cells treated with STS accumulated Endo G and AIF in the nucleus. By contrast, differentiated cells did not show such nuclear accumulation. Treatment of differentiated cells with Insulin-like Growth Factor-1 (IGF-1) and STS resulted in a 17.1% increase of cell viability. The survival role of IGF-1 was demonstrated by treating differentiated cells with an anti-IGF-1 neutralizing antibody. Such treatment significantly increased STS-induced cell death. Electrophysiology studies showed that in STS-treated cells membrane potential oscillations were reduced in amplitude and did not give rise to spontaneous action potentials (APs). However, the percentage of cells yielding overshooting APs returned to control values after STS removal. It is concluded that neuronal differentiation of NG108-15 cells induces resistance to apoptotic cell death and that IGF-1 plays a central role in sustaining this mechanism.
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Key words: apoptosis, neurons, Bcl-2 family, IGF-1
Introduction
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Proliferating tissues are characterized by rapid cellular turnover. By contrast, neurons generally survive the entire existence of the organism.1 Such an ability is necessary to maintain the functionality
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*Correspondence to: Bruna Pucci; Department of Cellular and Molecular Pathology; IRCCS San Raffaele Pisana; Via dei Bonacolsi snc; Rome 00163 Italy; Tel.: 39.06.66130420; Fax: 39.06.66130407; Email:
[email protected] Submitted: 09/29/08; Accepted: 10/27/08 Previously published online as a Cell Cycle E-publication: http://www.landesbioscience.com/journals/cc/article/7261 www.landesbioscience.com
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Apoptosis is a kinetic event. A study of the apoptotic cell death kinetic has revealed a dependence from both cell types and the inducers used.14 By comparing the apoptotic process in two different cell lines, Wolbers and colleagues concluded that the apoptotic turnover depends on the apoptotic stimulus, while the transition within the apoptotic cascade is determined by the cell type.14 However, further studies have revealed that the kinetic of apoptosis is also influenced by cellular differentiation.15,16 In fact, differentiated PC12 cells were more resistant to apoptosis induced by growth factor withdrawal than not differentiated PC12 cells.16 These observations suggested that additional antiapoptotic controls are acquired during neuronal differentiation and are important for the long term survival of post-mitotic neurons.16 Insulin-like Growth Factor-1 is a peptide hormone produced by the liver. Upon binding to its receptor, IGF-1 activates phosphatidyl inositol 3 kinase (PI3K) that, in turn recruits, phosphorylates and activates the Ser/Thr-kinase B/AKT.17 Activated AKT exerts its survival function by phosphorylating and inactivating the proapoptotic protein Bad. Interestingly, changes in the levels of IGF-1 have been observed in several neurodegenerative disease18 as well as in cognitive decline and dementia during ageing.19 Moreover, IGF-1 has been shown to posses neuroprotective and neurogenic function during ischemic brain injury.20 Following ischemic or traumatic brain injury IGF-1 and its binding proteins and receptors are intensely induced within damaged brain regions, suggesting a possible role for IGF-1 in the recovery process. Exogenous administration of IGF-1 protects both gray and white matter after brain injury.17 In the present work we studied the apoptotic kinetic in differentiated neurons. We demonstrated that regulation of expression of Bcl-2 family members and mitochondrial response to the damage play a major role in delaying the irreversible phase of the apoptotic process in neuron cells. Moreover, we demonstrated that IGF-1 plays an important role in increasing the survival of differentiated neurons. For this purpose we differentiated the NG108-15 cell line in vitro with NaB. To mimic cellular damage the in vitro differentiated neurons were treated with STS. Molecular and electrophysiological studies were performed.
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Results
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NaB-induced differentiation in NG108-15 cells. NG108-15 cells were grown with 1 mM NaB for 7 days in 2% FBS and neuronal differentiation was evaluated. As a control, NG108-15 cells were grown in proliferating conditions (10% FBS). Proliferating cells appeared round in shape and without long processes (Fig. 1A). NaB treated cells displayed axon-like long processes (Fig. 1B). At SEM proliferating cells appeared spherical, with an irregular surface and short microvilli (Fig. 1C). NaB differentiated cells showed dendrites and axonal processes apparently interacting with surrounding cells (Fig. 1D). TEM of an axonal process showed many morphological markers of differentiation such as neuropetide granule, neurotubules and neurofilaments (Fig. 1E). Figure 1F shows that only NaB differentiated cells expressed Map-5, a microtubule associated neuronal-specific protein. NaB differentiated NG108-15 cells are more resistant to STS-induced cell death. NaB differentiated and proliferating NG108-15 cells were exposed to 0.5 μM STS for different times to test the hypothesis that in vitro neuronal differentiation decreased sensitivity to cell death. The apoptotic kinetic was evaluated by 3870
Figure 1. NaB induces differentiation in NG108-15 cells. Light microscopy pictures of proliferating (A) and 1 mM NaB differentiated cells (B) (20X). SEM of undifferentiated (C) and NaB differentiated cells (D). Note the change in the shape of the cells, the appearance of well differentiated axons and dendrites which interact with the cell bodies of close or distant cells. Bars indicate 20 μm and 10 μm, respectively. (E) TEM of the cytoplasm of an axon in NaB differentiated cells. Note the abundant neurotubules and neurofilaments well organized along the longitudinal axis of the neurite. In addition, round dense granules of neuropeptides are present close to the plasmamembrane. The bar represents 1 μm. (X 28,000). (F) Western blotting analysis of Map-5 protein on cellular extracts obtained from proliferating (P) and differentiated cells (NaB). Results in each panel are representative of three independent experiments.
flow cytometric analysis (Fig. 2A). The percentage of dead cells in untreated NG108-15 cells was 4% and 9.6% in proliferating and differentiated cells, respectively. After 24 h of STS treatment death increased and 96% of proliferating cells were dead. On the contrary, only 54.1% of differentiated cells was dead (Fig. 2A). Apoptosis was evaluated by the presence of DNA fragmentation and condensation. After 24 h of STS-treatment, Hoechst 33258 staining of nucleic acid revealed typical apoptotic chromatin. Proliferating cells (Fig. 2B) showed a higher number of pycnotic nuclei than differentiated cells (Fig. 2B). Control cells did not show any features of apoptosis (Fig. 2B). Bcl-2 family expression and localization in STS induced apoptosis in proliferating and differentiated cells. STS induces apoptosis
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was observed in the mitochondria of proliferating cells (Fig. 3A). When differentiated cells were treated with STS, mitochondrial expression of the pro-apoptotic protein Bad was suppressed. In addition the remaining Bad was phosphorylated and inactivated (Fig. 3A). By contrast, in proliferating cells Bad expression was increased in both the mitochondrial and cytosolic fraction and was not phosphorylated (Fig. 3A and B). Bax, another pro-apoptotic protein, increased in the mitochondria while decreasing in the cytosol of proliferating cells after STS treatment (Fig. 3A and B). In differentiated cells, STS-treatment did not induce any Bax increase in the mitochondrial fraction (Fig. 3A). Prohibitin and β-actin were used as mitochondrial and cytosolic loading controls respectively. Neuronal differentiation prevents the activation of the mitochondrial apoptotic pathway in NG108-15 cells. The mitochondrial membrane potential was assessed during STS treatment of proliferating and differentiated cells by using the fluorescence dye JC-1. JC-1 is a green fluorescent monomer at low membrane potential. However, at higher potential, i.e., within the mitochondrial matrix, it forms red fluorescent aggregates.22 Both proliferating and differentiated cells were stained with 2.5 μg/ml JC-1. After 10 min cells were washed and treated with 0.5 μM STS. Cells were then observed at confocal microscopy after 30, 60 and 100 min. In proliferating cells, STS induced a loss of mitochondria membrane potential that started Figure 2. NaB differentiated NG108-15 cells are more resistant to STS induced cell death than at 30 min and was almost complete after 90 NG108-15 proliferating cells. (A) Apoptotic kinetic of proliferating and NaB differentiated NG10815 cells was evaluated by flow cytometry after propidium iodide staining. Proliferating or NaB min (Fig. 4A). By contrast, in differentiated differentiated NG108-15 cells were kept in 0.5 μM STS containing media for the indicated time. cells, mitochondria were localized mostly in the Data shown represent the analysis of three different experiments each carried out in duplicate neuronal processes and appeared fully energized with error bars indicated. (B) Hoechst 33258 staining of NG108-15 cells: untreated proliferating after 100 min of STS treatment (Fig. 4A). NG108-15 cells (upper left), proliferating NG108-15 cells treated with 0.5 μM STS for 6 h (lower Figure 4B shows that cytochrome c was left), untreated differentiated NG108-15 cells (upper right), differentiated NG108-15 cells treated with 0.5 μM STS for 24 h (lower right). Results in each panel are representative of three indepen- released from the mitochondria of proliferating cells treated with 0.5 μM STS. By contrast, dent experiments. in differentiated cells, STS treatment caused by activating the intrinsic mitochondrial pathway that is regulated a small release of cytochrome c (Fig. 4B). Caspase-3 activation by Bcl-2 proteins.21 Figure 3A shows that Bcl-2, an anti-apoptotic following cytochrome c release was examined in proliferating cells protein, was more expressed in the mitochondrial fraction of differ- after 2, 4 and 6 h of STS treatment. Active caspase-3 accumulated entiated than proliferating cells. After STS treatment, Bcl-2 protein in proliferating cells after 4 and 6 h of STS treatment (Fig. 4C). By levels decreased in proliferating cells. In differentiated cells, the Bcl-2 contrast, there was only a slight increase in active caspase-3 after 6 h protein level increased in mitochondrial extracts after STS treatment. of STS treatment in differentiated cells (Fig. 4C). Cytosolic Bcl-2 protein levels did not change in proliferating cells Endo G and AIF nuclear accumulation in proliferating and whereas, in differentiated cells cytosolic Bcl-2 increased after 6 h and NaB-differentiated NG108-15 cells. In the intrinsic apoptotic decreased after 16 h of STS treatment (Fig. 3B). pathway, AIF and endonuclease G (Endo G) are among the factors Bcl-xL, another anti-apoptotic protein, showed a pattern similar that are released from mitochondria. AIF and Endo G directly induce to that of Bcl-2. Differentiated cells showed an higher mito- nuclear fragmentation by translocating from the mitochondria to the chondrial Bcl-xL protein level than proliferating cells. Bcl-xL nucleus.23,24 translocation from cytosol to mitochondria during STS treatment Figure 5 shows that in differentiated cells AIF level did not change was observed only in differentiated cells (Fig. 3A and B). No Bcl-xL in the nucleus after STS treatment whereas, in proliferating cells AIF www.landesbioscience.com
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did increase after 16 h. Endo G expression increased after 2 h of STS treatment in proliferating cells and its level remained steady through the time course. In differentiated cells, there was an initial decrease of Endo G followed by an increase after 16 and 24 h treatment (Fig. 5). Viability recovery from STS induced apoptosis in NaB differentiated cells by IGF-1 treatment. The slower kinetics of STS-induced apoptosis observed in differentiated cells suggested the possibility that the cells may terminate the death program when exposed to agents able to inhibit the apoptotic mechanism. Therefore, we treated NaB differentiated NG108-15 cells with 0.5 μM STS for 24 h in the presence or in absence of 50 ng/ml IGF-1 and 100 ng/ml NGF. Figure 6A shows that treatment of differentiated cells Figure 3. Bcl-2 homologues protein expression during STS-induced apoptosis in proliferatwith STS and IGF decreased cell death from 55.4 ing and NaB differentiated cells. (A) Proliferating and differentiated NG108-15 cells were to 32.8%. Therefore, IGF treatment increased cell either left untreated or treated with STS for the indicated times. Mitochondria were isolated and the content of Bcl-2, Bcl-x , Bad, Bax and prohibitin analyzed by Western blot. Lane 1, viability by 17.1%. NGF did not show any effect on untreated NaB differentiated Lcells; lane 2, NaB differentiated cells treated with STS for 4 STS-induced apoptosis (Fig. 6A). No effect on cell h; lane 3, NaB differentiated cells treated with STS for 6 h; lane 4, NaB differentiated cells killing by both IGF-1 and NGF was observed in prolif- treated with STS for 16 h; lane 5, untreated proliferating cells; lane 6, proliferating cells erating cells (not shown). The protective role of IGF-1 treated with STS for 2 h; lane 7, proliferating cells treated with STS for 4 h; lane 8, prolifwas demonstrated by treating differentiated cells with erating cells treated with STS for 6 h. (B) Proliferating and differentiated NG108-15 cells were either left untreated or treated with STS for the indicated times. A cytosolic fraction was STS in the presence of an IGF-1 neutralizing antibody. obtained and the content of Bcl-2, Bcl-x , Bad, Bax and β-actin analyzed by Western blot. L Figure 6B shows that addition of 10 and 50 μg/ml of Lane 1, untreated NaB differentiated cells; lane 2, NaB differentiated cells treated with STS neutralizing antibody to the differentiation medium for 4 h; lane 3 NaB differentiated cells treated with STS for 6 h; lane 4, NaB differentiated increased the STS cell death from 55% to 67 and cells treated with STS for 16 h; lane 5, untreated proliferating cells; lane 6, proliferating cells treated with STS for 2 h; lane 7, proliferating cells treated with STS for 4 h; lane 8, 80.7%, respectively. proliferating cells treated with STS for 6 h. Immunoblots in each panel are representative of Excitability of NG108-15 differentiated cells. We three independent experiments. next investigated the effect of the STS and/or IGF treatments on the electrical functionality of differentiated NG108-15 cells. spontaneous APs and were able to generate APs in response to Untreated differentiated cells showed spontaneous oscillations of depolarizing current. Peak and rise slope of both overshooting the membrane potential that reached the threshold and gave rise to and abortive APs were similar to those measured in untreated cells action potentials (APs) (Fig. 7A). In STS treated cells, membrane (Table 1). Differentiated cells co-treated with IGF-1 and STS had a potential oscillations were reduced in amplitude and never gave rise behavior similar to that of STS treated cells. No cells presented spontaneous APs, the percentage of cells with overshooting and abortive to spontaneous APs (Fig. 7B). We further examined cell excitability, by studying the ability APs did not change (44% and 56%, respectively) and the APs (Fig. to generate evoked action potentials, a fundamental characteristic 7E) parameters were similar to those measured in STS treated cells of neuronal cells. To standardize experimental conditions, all the (Table 1). To investigate if STS effect was reversible, we removed STS from cells were hyperpolarized to a holding potential of -74.5 ± 0.5 mV the treated cultures. Cell excitability was tested after 24 hours. The and depolarized with stimuli ranging from 95 to 105 pA. A small percentage of cells yielding overshooting APs was similar for both percentage of cells, varying from 0% to 20% failed to respond to untreated (80%) and STS treated cells (100%). Moreover, the overthe depolarizing protocol. The majority of the control cells tested shooting responses of treated cells not only reached a similar peak, (84%), responded with overshooting, fast rising APs (Fig. 7C left but also showed a not statistically different rise slope (Table 2). trace), while the remaining cells (16%) showed abortive APs (Fig. 7C right trace), which did not invert the membrane potential and had a Discussion slower rise (Table 1). Treatment with STS resulted in a reduced cell excitability: the percentage of cells with overshooting APs decreased This study documents that differentiation of NG108-15 cells into to 60%, while the percentage of cells responding with abortive APs neurons is accompanied by an increased resistance to STS-induced correspondingly increased to 40%. Compared to the untreated cells, apoptosis. Differentiation altered the expression and intracellular the overshooting responses of STS treated cells reached a similar peak redistribution of several members of Bcl-2 family proteins. Bcl-2 and (Fig. 7D) but they showed a significant slower rise phase (Table 1). Bcl-xL increased in differentiated cells during STS treatment (Fig. 3). 300 nM TTX blocked APs in both untreated and STS-treated differ- In differentiated cells, the pro-apoptotic protein Bad was inactivated by phosphorylation and did not accumulate in either the mitoentiated cells (data not shown). We investigated the effect of IGF-1 on cellular excitability. chondrial or cytosolic fraction. Similarly, Bax did not accumulate Similar to untreated differentiated cells, IGF-1 treated cells presented in the mitochondria of differentiated cells after STS treatment. By 3872
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Figure 4. Mitochondrial membrane potential, cytochrome c release and caspase-3 cleavage in proliferating and NaB differentiated NG108-15 cells after STS treatment. (A) Differentiated NG108-15 cells were loaded with 2.5 μg/ml JC-1 and then either left untreated or treated with STS for 30 min, 60 min and 100 min. Similarly, proliferating NG108-15 cells were loaded with JC-1 and then either left untreated or treated with STS for 30 min, 60 min and 90 min. The green fluorescence from JC-1 monomers and the red fluorescence from JC-1 aggregates was analysed by confocal microscopy. (B) Proliferating and differentiated NG108-15 cells were treated with 0.5 μM STS up to 6 h. Cytosolic fractions were obtained and assayed for cytochrome c by Western blot as described in Materials and Methods. Lane 1, untreated proliferating cells; lane 2, proliferating cells treated with STS for 4 h; lane 3 proliferating cells treated with STS for 6 h; lane 4, untreated NaB differentiated cells; lane 5, NaB differentiated cells treated with STS for 4 h; lane 6, NaB differentiated cells treated with STS for 6 h. (C) Western blot analysis of active caspase-3 in proliferating and NaB differentiated cells exposed to 0.5 μM STS up to 6 h. Lane 1, untreated proliferating cells; lane 2, proliferating cells treated with STS for 4 h; lane 3 proliferating cells treated with STS for 6 h; lane 4, untreated NaB differentiated cells; lane 5, NaB differentiated cells treated with STS for 4 h; lane 6, NaB differentiated cells treated with STS for 6 h. Results in each panel are representative of three independent experiments.
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contrast, STS treatment of proliferating cells decreased the mitochondrial localization of both Bcl-2 and Bcl-xL, whereas Bad and Bax accumulated in the mitochondria (Fig. 3). Moreover, STS induced mitochondrial damage in proliferating cells after 30 minutes. By contrast, in differentiated cells mitochondria were still energized after 100 minutes of STS treatment (Fig. 4A). Accordingly, cytochrome c release and caspase-3 cleavage were significantly delayed in differentiated cells (Fig. 4B and C). At nuclear level, in proliferating cells STS induced AIF and Endo G accumulation in the nucleus. By contrast AIF did not accumulated and Endo G increase was delayed in differentiated cells (Fig. 5). IGF-1 treatment rescued differentiated cells from STS-induced apoptosis and treatment with a IGF-1 neutralizing antibody significantly increased STS-induced apoptosis (Fig. 6). Electrical functionality was also recovered in these cells after removing STS (Fig. 7). A previous study demonstrated that STS treatment of proliferating NG108-15 cells induced an increase of Bax expression whereas Bcl-2 www.landesbioscience.com
levels decreased.25 Moreover, Bcl-2 protein level has been demonstrated to be upregulated in brain of patients with Alzheimer’s disease (AD).26 Such upregulation might protect remaining neurons from subsequent apoptosis. Furthermore, brain from patients with AD also showed a significant increase in Bak and Bad protein levels.26 However, a detailed study of the molecular aspects at the basis of the slow kinetics of apoptosis observed in neurons after damage is still lacking. In fact, such a study could suggest strategies to increase the survival rate of a neuronal population. In line with these considerations, our results show that neuronal differentiation of NG108-15 cells increases the resistance to STS-induced apoptosis. The correlation between differentiation and apoptosis has been shown in several studies.15,16 In particular, activation of Akt and Erk survival pathways in differentiated neuroblastoma SH-SY5Y cells induced resistance to apoptosis by geldanamycin.27 In our system, apoptotic kinetics in differentiated cells is influenced by a altered expression and localization of Bcl-2
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family members. In particular, the anti-apoptotic proteins Bcl-2 and Bcl-xL are upregulated in differentiated but not in proliferating cells. The pro-apoptotic mitochondrial protein Bad is inactivated in differentiated cells by phosphorylation. Bax is localized in the mitochondria of proliferating cells more than differentiated cells. These observations indicate that in differentiated cells the mitochondrial ratio among pro- and anti-apoptotic members of the Bcl-2 family decreases because of a reduction of Bad and Bax and a concomitantly increase in Bcl-2 and Bcl-xL content. A fundamental consequence of this shift in the balance between pro- and anti-apoptotic proteins is that mitochondria of differentiated cells are more resistant to the apoptotic stimulus. In fact, loss of membrane potential and cytochrome c release were significantly delayed in NG108-15 cells after neuronal differentiation (Fig. 4). Furthermore, AIF and Endo G did not accumulate in the nuclear fraction of the differentiated cells after STS treatment (Fig. 5). These results suggest that neuronal differentiation is accompanied by mitochondrial alterations responsible for the increased resistance of the differentiated cells to apoptosis. The central role of mitochondria in neurodegenerative diseases has been underlined by several studies. In amyotrophic lateral sclerosis (ALS), changes occur in mitochondrial respiratory chain enzymes and in mitochondrial cell death proteins, indicative of an activation of programmed cell death pathways.28 In Alzheimer’s disease there is evidence that both beta-amyloid and the amyloid precursor protein may directly interact with mitochondria, increasing free radical production.29 In the case of Huntington’s disease (HD), the coactivator PGC1alpha, a key regulator of mitochondrial biogenesis in respiration, is downregulated in patients with HD and in several animal models of this neurodegenerative disorder.30,31 The slower onset and kinetics of the apoptotic process that we observed when NG108-15 cells acquired a neuronal phenotype, when extended to the whole cell population could explain the slow progression typical of neurodegenerative diseases. Our study showed that only IGF-1 successfully increased the survival of a significant percentage of differentiated cells (Fig. 6A). Furthermore, using an IGF-1 neutralizing antibody approach, we showed that in our system the increased resistance to STS-induced apoptosis observed in the differentiated neurons was acquired through the activation of the IGF-1 survival pathway. IGF-1 has been shown to provide trophic support and to possess anti-apoptotic activity in many cellular systems mainly through the activation of the Akt/MAPK pathway.32,33 The protective effect of IGF-1 in PC12 cells has been linked to an upregulation of Bcl-xL.34 Furthermore,
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Figure 5. AIF and EndoG expression in the nuclear extracts of proliferating and NaB differentiated cells after STS treatment. Western blot analysis of AIF and EndoG in nuclear extracts obtained from proliferating and NaB differentiated cells exposed to 0.5 μM STS. Lane 1, untreated NaB differentiated cells; lane 2, NaB differentiated cells treated with STS for 2 h; lane 3, NaB differentiated cells treated with STS for 4 h; lane 4, NaB differentiated cells treated with STS for 6 h; lane 5, NaB differentiated cells treated with STS for 16 h; lane 6, NaB differentiated cells treated with STS for 24 h; lane 7, untreated proliferating cells; lane 8, proliferating cells treated with STS for 2 h; lane 9, proliferating cells treated with STS for 4 h; lane 10, proliferating cells treated with STS for 6 h; lane 11, proliferating cells treated with STS for 16 h. SP1 was used as loading control for the nuclear fraction. Results in each panel are representative of three independent experiments.
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Figure 6. IGF-1 rescues NaB differentiated cells from STS-induced apoptosis. (A) NaB differentiated NG108-15 cells were kept in 0.5 μM STS containing media for 24 h in presence or absence of IGF-1 50 ng/ml or NGF 100 ng/ml. The percentage of dead NaB differentiated NG108-15 cells was evaluated by flow cytometry after propidium iodide staining. Data shown represent the analysis of three different experiments each carried out in duplicate with error bars indicated. * = significantly different from STS 24 h (p < 0.05). (B) NaB differentiated NG108-15 cells were kept in 0.5 μM STS containing media for 24 h in presence or absence of an anti IGF-1 neutralizing antibody as indicated. The percentage of dead NaB differentiated NG108-15 cells was evaluated by flow cytometry after propidium iodide staining. Data shown represent the analysis of three different experiments each carried out in duplicate with error bars indicated. * = significantly different from STS 24 h (p < 0.05).
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grant the maintenance of a functional neural network. This fact is particularly interesting, as even a small fraction of surviving neurons can adequately grant the function of particular brain structures. For instance, the symptoms of Parkinson’s disease become clinically relevant only after the death of over 80% of the dopaminergic neurons in the substantia nigra.37 These results demonstrate that a significant number of NG108-15 differentiated cells can survive a 24 h STS treatment and that these surviving cells posses neuronal electrical characteristics. Such an observation and the fact that IGF-1 treatment improved cell survival, suggests the possibility to increase the neuronal population generating APs in our in vitro system. In conclusion, this study details the molecular mechanisms that cause apoptotic resistance in NG108-15 cells when they undergo differentiation into neurons. Moreover, we suggest possible strategies to increase cell survival and to recover electrical activity in neuronal populations characterized by sub-lethal damage. Further experiments performed on this in vitro system could be useful to test new molecules (i.e., antioxidants and/or a combination of growth and survival factors) and new strategies for the treatment of neurodegenerative disease.
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Materials. Unless stated otherwise reagents were purchased from Sigma Aldrich (St. Louis, MO). STS was dissolved in dimethyl sulfoxide (DMSO) as 1mM stock solution. IGF-1 was dissolved as 50 μg/ml solution in H2O. Hoechst 33258 was dissolved as 10 mg/ ml solution in H2O. Sodium butyrate (NaB) was dissolved as 0.5 M solution in H20. Propidium Iodide (PI) was dissolved as 100 μg/ml in H2O. Collagen I was dissolved as 100 μg/ml solution in H2O. JC-1 was purchased from Invitrogen Life Technologies and dissolved as 5 mg/ml solution in DMSO. Dulbecco’s modified Eagle’s (DMEM) without pyruvate and HAT supplement were purchased from Invitrogen Life Technologies (Carlsbad, CA, USA). FBS was purchased from Lonza (CH). Anti MAP51B-MAP5 monoclonal antibody was purchased from GENETEX (San Antonio, TX, USA). Anti-caspase-3 polyclonal and anti-cytochrome c monoclonal antibodies were purchased from Chemicon International (Temecula, CA, USA). Anti BAD polyclonal antibody was purchased from ABcam (Cambridge, UK) and monoclonal anti-mouse Bax antibodies were purchased from Santa Cruz Biotechnology, Inc., (Santa Cruz, CA, USA). AIF polyclonal antibody was purchased from Cell Signaling Technology, Inc., (Danvers MA, USA). Anti-rat prohibitin monoclonal antibody, anti-human β-actin and anti EndoG polyclonal antibodies were purchased from Novus Biologicals (Littleton, CO, 80160 USA). Anti-human Bcl-2 and anti-human Bcl-xL polyclonal antibodies were purchased from Lab Vision (Fremont, CA 94539 USA). Anti-mouse IGF-1 neutralizing antibody was purchased from R&D Systems (Minneapolis MN, USA). An enhanced chemiluminescent detection system (ECL kit) was purchased from (Sigma Aldrich (St. Louis, MO). Cell culture. The cell line NG108-15 (mouse neuroblastoma/ rat glioma hybrid cell line) was purchased from ATCC (Manassas, VA) and grown in Dulbecco’s modified Eagle’s (DMEM) medium supplemented with 10% (v/v) FBS and HAT supplement. For differentiation experiments, cells were plated on collagen I on plastic
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Figure 7. The excitability of NaB-differentiated cells is impaired by STS. Spontaneous membrane potential oscillations trigger action potentials in untreated NaB differentiated cells (A), but not in cells treated with STS (0.5 μM) (B). Depolarizing stimuli (ranging from 95 to 105 pA) evoke overshooting, fast-rising action potentials (APs) (left trace) in 84% of the untreated cells and abortive APs (right trace) in 16% of the untreated cells (C); slow APs in cells treated with STS 0.5 μM (D) or STS 0.5 μM and IGF 50 ng/ml (E). Results in each panel are representative of four independent experiments.
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IGF-1 protected neuroblastoma cells from growth retardation and decreased survival due to low glucose by decreasing mRNA levels of apoptosis-related genes and increasing the expression of GLUT-1 transporter.35 Therefore, multiple survival pathways can be activated by IGF-1 that increase the survival of NG108-15 cells treated with STS. Further experiments will be performed to detail the molecular mechanisms activated by IGF-1 in our system. However, IGF-1 alone had no effect on the excitability of differentiated cells and did not protect the cells from STS-induced impairment of AP generation. By contrast, a full functional recovery was achieved in differentiated cells 24 hrs after removing STS. These data argue against a direct effect of STS on voltage-gated channels. STS, being a potent blocker of protein kinase C, likely lowers the basal phosphorylation of voltage-gated Na+ channels by that kinase, if any. Since PKC-induced phosphorylation is known to reduce the activity of TTX-sensitive Na+ channels and action potential firing,36 it is unlikely that the action of STS on cell excitability is uniquely mediated by its effects on channel phosphorylation. APs generation remained strongly TTX-sensitive in STS-treated differentiated cells, indicating that the treatment did not induce the expression of TTX-resistant Na+ channels. Altogether, our data indicate that chronic exposure to STS activates pathways leading to a marked but reversible reduction of cell excitability, alongside with cell death. This implies that, at least in vitro, neurons surviving STS-induced apoptosis can regain full physiologic function. Neuroprotective treatments that raise the number of neurons surviving to insults may therefore www.landesbioscience.com
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Table 1 Action potential characteristics in control and treated NG108-15 cells Overshooting Abortive Treatments Peak (mV) Rise slope (mV/ms) Peak (mV) STS IGF STS + IGF
11.7 ± 1.5
1
16.7 ± 4.2
1
9.6 ± 2.1
1
28.2 ± 3.0
(17) (10) (10)
-7.7 ± 1.5 (5)
18.5 ± 2.9
2
27.9 ± 6.6
1
16.6 ± 2.8
2
Rise slope (mV/ms) 5.2 ± 0.5
-9.9 ± 2.2
1
(12)
-4.3 ± 1.7
1
(4)
-9.5 ± 3.7
1
(8)
E.
12.8 ± 1.6 (27)
BU T
Control
4.8 ± 0.7
1
5.8 ± 1.4
1
6.3 ± 1.2
1
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The maximal voltage reached and the maximal slope of the rising phase were evaluated in NG108-15 cells after a 24 h exposure to the indicated drugs. Overshooting and abortive action potentials are considered separately. Values are given as mean ± SEM (number of cells tested). All the values are not statistically different (p > 0.1). 1: values not statistically different from control values (p > 0.1); 2: values statistically different from control (p < 0.02), but not for the two treatments.
Table 2 Action potential characteristics in control and treated NG108-15 cells after 24 hours of culture wash Overshooting Abortive Treatment Peak (mV) Rise slope (mV/ms) Peak (mV) 16.0 ± 2.7
STS/wash
8.3 ± 2.2 (13) 1
12.4 ± 2.6 1
STS + IGF/wash
9.6 ± 2.1 (10) 1
15.4 ± 2.6 1
Rise slope (mV/ms)
-4.6, -3.6
3.6, 6.1
-6.1, -8.3
3.7, 4.4
T
10.7 ± 2.4 (8)
1
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Control/wash
1
-10.2, -1.3
4.7, 6.6
(0.1 μg/ml).The coverslips then were washed three with PBS and then examined under a fluorescent microscope (NIKON Eclipse TE2000U). Pictures were taken a digital camera (NIKON DS5N). Western blotting. Cells were harvested, washed twice in PBS and resuspended in a volume of lysis buffer (50 mM Tris pH 7.4, 5 mM EDTA, 250 mM NaCl, 50 mM NaF, 0.1% Triton X-100, 10 μg/ml leupeptin and 1 mM phenylmethylsulfonyl fluoride). After 30 min on ice, lysates were centrifuged and protein collected. To isolate cytosolic and mitochondrial fractions, cells were collected by centrifugation (750 xg) for 10 min at 4°C. The cell pellets were resuspended in 1 ml of 20 mM HEPES-KOH (pH 7.5), 10 mM KCl, 1.5 mM MgCl2, 1 mM EDTA, 1 mM EGTA, 1 mM phenylmethylsulfonyl fluoride, 10 μg/ml aprotinin, 10 μg/ml leupeptin and 250 mM sucrose. The cells were lysed by aspirating 6 times through a 26-gauge needle. The homogenate was centrifuged at 1000 xg for 5 min at 4°C to remove nuclei and unbroken cells. The resulting supernatant was centrifuged at 10,000 xg for 15 min at 4°C and concentrated using a Microcon Centrifugal Filter Device (Millipore, Billerica, MA, USA). The supernatant (cytosol) was collected while the mitochondrial pellet was lysed in 20 mM Tris pH 7.5, 100 mM NaCl, 50 mM NaF, 1% Triton X-100, 10 μg/ml leupeptin and 1 mM phenylmethylsulfonyl fluoride.40 Cytoplasmic and nuclear proteins were fractionated using the Nuclear Extract Kit from Active Motif (Carlsbad CA, USA) according to the manufacturer’s instructions. Protein concentrations were measured by the Bradford assay (Bio-Rad laboratories, Hercules, CA). Proteins were normalized to 60 μg/lane and applied to SDS-polyacrylamide gels. The gels were blotted (2 h at 300 mA) onto a Hybond-ECL nitrocellulose filter (Amersham Life science Inc., Arlington Heights, IL). A Kaleidoscope prestained protein solution (Bio-Rad laboratories, Hercules, CA) was used as a molecular weight standard. The filter was washed twice with TBS-0.1% Tween-20 buffer (TBS-T), before blocking non-specific binding sites with 5% milk/TBS-T for 1 h. The filter
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dishes (100 x 20 mm) and cultured in DMEM without pyruvate supplemented with 2% FBS for 7 days in presence of 1 mM NaB. Medium was changed every other day. Cultures were maintained at 37°C in a humidified atmosphere of 5% CO2 and 95% air. Microscopy. Phase contrast microscopy. Morphology was evaluated in proliferating and differentiated cells by phase contrast microscopy without preliminary fixation. Pictures were produced using an inverted microscope (NIKON Eclipse TE2000U) and a digital camera (NIKON DS5Mc). Confocal microscopy. Measurement of mitochondrial membrane potential. The mitochondrial membrane potential was assessed using the fluorescence dye JC-1 (5,5',6,6'-tetrachloro-1,1',3,3'-tetraethylbenzimidazole carbocyanide iodide).22 Briefly, NG108-15 cells were grown on 35 mm glass dishes previously coated with collagen I for 4 hours and washed twice with PBS. The day of the treatment cells were stained with 2.5 μg/ml of the fluorescent probe JC-1, for 15 min at 37°C. Then cells were then washed with medium and treated with 0.5 μM STS as indicated in the text. After treatment, fluorescence intensity was visualized with a confocal microscope (Nikon Eclipse TE 2000-E). Electron microscopy. Proliferating and NaB differentiated cells were fixed in 2.5% glutaraldehyde in phosphate buffer for 2 hours and then processed for SEM and TEM following standard procedures.38,39 Hoechst 33258 staining. Cells were plated on collagen pretreated sterile 13 mm coverslips placed in 100 x 20 mm plastic dishes. Cells were then grown for 24 h for proliferative experiments or 7 days in 2% FBS e 1 mM NaB for differentiation experiments. After 24 h of 0.5 μM STS treatment the coverslips were removed from the dishes and placed in methanol:acid acetic (3:1 v/v) for 15 min at room temperature to be fixed. After rinsing with PBS, the coverslips were incubated for 10 min at room temperature in HOECST 33258
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The maximal voltage reached and the maximal slope of the rising phase were evaluated in NG108-15 cells after a 24 h wash from the indicated drugs. Values are given as mean ± SEM (number of cells tested) or as individual values when N = 2. 1: values not statistically different (p > 0.1).
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12. Breckenridge DG, Xue D. Regulation of mitochondrial membrane permeabilization by BCL-2 family proteins and caspases. Curr Opin Cell Biol 2004; 16:647-52. 13. Orrenius S. Mitochondrial regulation of apoptotic cell death. Toxicol Lett 2004; 149:19-23. 14. Wolbers F, Buijtenhuijs P, Haanen C, Vermes I. Apoptotic cell death kinetics in vitro depend on the cell types and the inducers used. Apoptosis 2004; 9:385-92. 15. Vondrácek J, Sheard MA, Krejcí P, Minksová K, Hofmanová J, Kozubík A. J Leukoc Biol 2001; 69:794-802. 16. Vyas S, Juin P, Hancock D, Suzuki Y, Takahashi R, Triller A, Evan G. Differentiation-dependent sensitivity to apoptogenic factors in PC12 cells. J Biol Chem 2004; 279:30983-93. 17. Kurmasheva RT, Houghton PJ. IGF-1 mediated survival pathways in normal and malignant cells. Biochim Biophys Acta 2006; 1766:1-22. 18. Trejo JL, Carro E, Garcia-Galloway E, Torres-Aleman I. Role of insulin-like growth factor I signalling in neurodegenerative diseases. J Mol Med 2004; 82:156-62. 19. Ceda GP, Dall’Aglio E, Maggio M, Lauretani F, Bandinelli S, Falzoi C, et al. Clinical implications of the reduced activity of the GH-IGF-I axis in older men. J Endocrinol Invest 2005; 28:96-100. 20. Johnsen SP, Hundborg HH, Sørensen HT, Orskov H, Tjønneland A, Overvad K, et al. Insulin-like factor (IGF)I,-II and IGF binding protein-3 and risk of ischemic stroke. J Clin Endocrinol Metab 2005; 90:5937-41. 21. Tafani M, Minchenko DA, Serroni A, Farber JL. Induction of the mitochondrial permeability transition mediates the killing of HeLa cells by staurosporine. Cancer Res 2001; 61:2459-66. 22. Reers M, Smiley ST, Mottola-Hartshorn C, Chen A, Lin M, Chen LB. Mitochondrial membrane potential monitored by JC-1 dye. Methods Enzymol 1995; 260:405-17. 23. Krantic S, Mechawar N, Reix S, Quirion R. Apoptosis-inducing factor: a matter of neuron life and death. Prog Neurobiol 2007; 81:179-96. 24. Mohamad N, Gutiérrez A, Núñez M, Cocca C, Martín G, Cricco G, et al. Mitochondrial apoptotic pathways. Biocell 2005 29:149-61. 25. Zhang BF, Peng FF, Zhang JZ, Wu DC. Staurosporine induces apoptosis in NG108-15 cells. Acta Pharmacol Sin 2003; 24:663-9. 26. Kitamura Y, Shimohama S, Kamoshima W, Ota T, Matsuoka Y, Nomura Y, et al. Alteration of proteins regulating apoptosis, Bcl-2, Bcl-x, Bax, Bak, Bad, ICH-1 and CPP32, in Alzheimer’s disease. Brain Res 1998; 780:260-9. 27. Shen JH, Zhang Y, Wu NH, Shen YF. Resistance to geldanamycin-induced apoptosis in differentiated neuroblastoma SH-SY5Y cells. Neurosci Lett 2007; 414:110-4. 28. Martin LJ. Mitochondriopathy in Parkinson disease and amyotrophic lateral sclerosis. J Neuropathol Exp Neurol 2006; 65:1103-10. 29. Anandatheerthavarada HK, Devi L. Amyloid precursor protein and mitochondrial dysfunction in Alzheimer’s disease. Neuroscientist 2007; 13:626-38. 30. Beal MF. Mitochondria and neurodegeneration. Novartis Found Symp 2007; 287:183-92. 31. Lin MT, Beal MF. Mitochondrial dysfunction and oxidative stress in neurodegenerative diseases. Nature 2006; 443:787-95. 32. Duarte AI, Santos P, Oliveira CR, Santos MS, Rego AC. Insulin neuroprotection against oxidative stress is mediated by Akt and GSK-3beta signaling pathways and changes in protein expression. Biochim Biophys Acta 2008; 1783:994-1002. 33. Oh SH, Jin Q, Kim ES, Khuri FR, Lee HY. Insulin-like growth factor-I receptor signaling pathway induces resistance to the apoptotic activities of SCH66336 (lonafarnib) through Akt/mammalian target of rapamycin-mediated increases in survivin expression. Clin Cancer Res 2008; 14:1581-9. 34. Párrizas M, Saltiel AR, LeRoith D. Insulin-like growth factor 1 inhibits apoptosis using the phosphatidylinositol 3'-kinase and mitogen-activated protein kinase pathways. J Biol Chem 1997; 272:154-61. 35. Russo VC, Kobayashi K, Najdovska S, Baker NL, Werther GA. Neuronal protection from glucose deprivation via modulation of glucose transport and inhibition of apoptosis: a role for the insulin-like growth factor system. Brain Res 2004; 1009:40-53. 36. Cantrell AR, Catterall WA. Neuromodulation of Na+ channels: an unexpected form of cellular plasticity. Nat Rev Neurosci 2001; 2:397-407. 37. Sulzer D. Multiple hit hypotheses for dopamine neuron loss in Parkinson’s disease. Trends Neurosci 2007; 30:244-50. 38. Paillart C, Li J, Matthews G, Sterling P. Endocytosis and vesicle recycling at a ribbon synapse. J Neurosci 2003; 23:4092-9. 39. Harris JR. The contribution of microscopy to the study of Alzheimer’s disease, amyloid plaques and Abeta fibrillogenesis. Subcell Biochem 2005; 38:1-44. 40. Tafani M, Karpinich NO, Hurster KA, Pastorino JG, Schneider T, Russo MA, et al. Cytochrome c release upon Fas receptor activation depends on translocation of full-length bid and the induction of the mitochondrial permeability transition. J Biol Chem 2002; 277:10073-82.
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then was incubated for 1 h at room temperature with the specific antibody diluted in 3% milk/TBS-T. The nitrocellulose filter was washed twice and incubated with horseradish peroxidase-conjugated goat anti-mouse or anti-rabbit. Detection was performed at room temperature using the ECL method. Flow cytometry. For proliferative studies, cells (5 x 105) were plated in 10 mm dishes. The following day the cells were treated with 0.5 μM STS. After the treatment the cells were harvested by centrifugation (10 min at 590 xg at 4°C), washed with 5 ml PBS, and resuspended in 500 μl PBS to which 5 ml of cold 70% EtOH was slowly added while stirring. Following overnight incubation at 4°C, cells were centrifuged at 500 xg for 5 min at 4°C and washed once with PBS. The cells were then resuspended in 300 μl of a 20 μg/ml propidium iodide/250 μg/ml RNase A solution and kept at 37°C for 30 min. DNA content was analyzed on a COULTER EPICS XL flow cytometer (Beckman Coulter, Fullerton, CA, USA). Electrophysiology. Patch-clamp recordings. Whole-cell responses were recorded at room temperature (23–26°C), using a Multiclamp 700B amplifier (Axon Instruments, CA, USA) and a gravity-driven perfusion system. For the experiments, culture medium was replaced by external solution containing (mM): 140 NaCl, 2.8 KCl, 2 CaCl2, 2 MgCl2, 10 HEPES/NaOH, 10 glucose, pH 7.3. Patch pipettes of borosilicate glass had a tip resistance of 3–5 MΩ (compensated by 70–80%) when filled with a solution containing (mM): 120 KCl, 10 NaCl, 5 BAPTA, 2 MgCl2, 10 HEPES/KOH, 2 ATP-Mg, at pH 7.3. To block voltage-gated Na+, Tetrodoxin (TTX 300 nM) was added to the extracellular solution). Membrane capacitance (Cm) was evaluated from the whole-cell compensation parameters or by the measurement of capacitive transients evoked by a 10 mV depolarizing step (Vstep). The total charge mobilized by the voltage step (Qstep) was obtained from the transient integral, and then Cm = Qstep/Vstep was calculated. Current clamp condition was used to record spontaneous and evoked action potentials, which were elicited hyperpolarizing membrane potential then injecting depolarizing currents of increasing amplitude at 2-second intervals. Statistical analysis. All experiments were repeated 3–5 times and the mean and the standard error of the mean [SEM] were determined. Significant differences between sets of values for control and test groups were assessed by a one-way ANOVA using a student Newman-Keuls post-hoc test. A p value refers to a comparison of a measured parameter in the experimental group with that of the appropriate control; significance was set at p < 0.05. References
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1. Miller FD and Kaplan DR. To die or not to die: neurons and p63. Cell Cycle 2007; 6:312-7. 2. Morrison JH and Hof PR. Life and death of neurons in the aging cerebral cortex. Int Rev Neurobiol 2007; 81:41-57. 3. Ohm TG. The dentate gyrus in Alzheimer’s disease. Prog Brain Res 2007; 163:723-40. 4. Olanow CW. The pathogenesis of cell death in Parkinson’s disease. Mov Disord 2007; 17:335-42. 5. Stack EC and Ferrante RJ. Huntington’s disease: progress and potential in the field. Expert Opin Investig Drugs 2007; 16:1933-53. 6. Carrì MT, Grignaschi G, Bendotti C. Targets in ALS: designing multidrug therapies. Trends Pharmacol Sci 2006; 27:267-73. 7. Lockshin RA and Zakeri Z. Caspase-independent cell death? Oncogene 2004; 23:2766-73. 8. Leist M and Jäättelä M. Four deaths and a funeral: from caspases to alternative mechanisms. Nat Rev Mol Cell Biol 2001; 2:589-98. 9. Strasser A, O’Connor L, Dixit VM. Apoptosis signaling. Annu Rev Biochem 2000; 69:217-45. 10. Gross A, McDonnell JM, Korsmeyer SJ. BCL-2 family members and the mitochondria in apoptosis. Genes Dev 1999; 13:1899-911. 11. Petros AM, Olejniczak ET, Fesik SW. Structural biology of the Bcl-2 family of proteins. Biochim Biophys Acta 2004; 1644:83-94. www.landesbioscience.com
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