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Keywords:

  • amyloid-β;
  • apoptosis inducing factor;
  • neuronal culture;
  • neuroprotection;
  • nicotine;
  • phosphatidylinositol 3-kinase

Abstract

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References
  8. Supporting Information

J. Neurochem. (2011) 119, 848–858.

Abstract

The neurotoxicity of amyloid-β (Aβ) involves caspase-dependent and -independent programmed cell death. The latter is mediated by the nuclear translocation of the mitochondrial flavoprotein apoptosis inducing factor (AIF). Nicotine has been shown to decrease Aβ neurotoxicity via inhibition of caspase-dependent apoptosis, but it is unknown if its neuroprotection is mediated through caspase-independent pathways. In the present study, pre-treatment with nicotine in rat cortical neuronal culture markedly reduced Aβ1–42 induced neuronal death. This effect was accompanied by a significant reduction of mitochondrial AIF release and its subsequent nuclear translocation as well as significant inhibition of cytochrome c release and caspase 3 activation. Pre-treatment with selective α7nicotinic acetylcholine receptor(nAChR) antagonist (methyllycaconitine), but not the α4 nAChR antagonist (dihydro-β-erythroidine), could prevent the neuroprotective effect of nicotine on AIF release/translocation, suggesting that nicotine inhibits the caspase-independent death pathway in a α7 nAChR-dependent fashion. Furthermore, the neuroprotective action of nicotine on AIF release/translocation was suppressed by LY294002, a phosphatidylinositol 3-kinase (PI3K) inhibitor. Pre-treatment with nicotine significantly restored Akt phosphorylation, an effector of PI3K, in Aβ1–42-treated neurons. These findings indicate that the α7 nAChR activation and PI3K/Akt transduction signaling contribute to the neuroprotective effects of nicotine against Aβ-induced cell death by modulating caspase-independent death pathways.

Abbreviations used

amyloid-β

α-BTX

α-bungarotoxin

AD

Alzheimer’s disease

AIF

apoptosis-inducing factor

DAPI

4′,6-diamidino-2-phenylindole

DHβE

dihydro-β-erythroidine

DMSO

dimethylsulfoxide

ERK

extracellular signal-regulated kinase

MLA

methyllycaconitine

MTT

3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide

nAChRs

nicotinic acetylcholine receptors

PCD

programmed cell death

PI3K

phosphatidylinositol 3-kinase

SDS

sodium dodecylsulfate

Programmed cell death (PCD) has been implicated in the pathophysiology of both acute and chronic neurodegenerative disorders. In Alzheimer’s disease (AD), neuronal degeneration is believed to result partly from PCD triggered by the accumulation of β-amyloid (Aβ) (Selkoe 2002; Mattson 2004). Previous studies have shown that above its physiological range (pM-nM), Aβ triggers neuronal PCD (Mattson 2000; Yuan and Yankner 2000). Most studies have focused mainly on Aβ-induced caspase-dependent PCD (Yaar et al. 1997; Giovanni et al. 2000; LeBlanc 2005). However, recent evidence has suggested that Aβ-induced PCD also involves caspase-independent pathways both in vitro (Movsesyan et al. 2004) and in postmortem human AD brains (Yu et al. 2010). The caspase-independent PCDs are mainly mediated by the mitochondrial flavoprotein apoptosis-inducing factor (AIF) (Susin et al. 1999; Joza et al. 2001; Cregan et al. 2004; Krantic et al. 2007). Under normal physiological conditions, AIF is localized in the mitochondrial intermembrane space (Otera et al. 2005). In response to death-inducing signals, AIF is cleaved to a truncated AIF form (57 kDa), which is then released from the mitochondria and translocated into the nucleus, where it participates in high molecular weight DNA fragmentation, chromatin condensation and cell death (Daugas et al. 2000; Susin et al. 2000).

A large number of studies have demonstrated that nicotine can protect against many types of insults leading to neuronal death (Picciotto and Zoli 2008). At low concentrations, nicotine can improve memory functions and reduce amyloid plaque burden in transgenic mouse model of AD (Nordberg et al. 2002; Levin et al. 2006), suggesting its neuroprotective potential. The neuroprotective actions of nicotine in the CNS are mediated by neuronal nicotinic acetylcholine receptors (nAChRs), which are pentameric ligand-gated ion channels (Picciotto and Zoli 2008; Albuquerque et al. 2009). Of particular relevance to AD, the activation of nAChRs by nicotine and nicotinic agonists can prevent cell death from neurotoxic insults such as excess glutamate or Aβ in cultured neurons (Picciotto and Zoli 2008). This neuroprotective effect has been demonstrated in particular for the α4β2 and α7 nAChR receptor subtypes (Daugas et al. 2000; Akaike et al. 2010), the most abundant nAChR subtypes expressed in the brain (Zoli et al. 1998).

The mechanisms of nicotine-mediated neuroprotection are still not well defined and might be associated with modulation of both survival and apoptotic signaling cascades (Buckingham et al. 2009). Indeed, a recent study has suggested that nicotine protects hippocampal neurons from Aβ-induced PCD by inhibiting caspase 3 activation (Liu and Zhao 2004). Other studies have shown that nicotine-mediated neuroprotection may involve activation of several survival signaling pathways such as, extracellular signal-regulated kinase (ERK) and phosphatidylinositol 3-kinase (PI3K) (Kihara et al. 2001; Dajas-Bailador et al. 2002; Jin et al. 2004; Buckingham et al. 2009). However, the neuroprotective action of nicotine in putative inhibition of AIF-mediated caspase-independent apoptosis has not yet been explored.

In the present study, we investigated the involvement of caspase-dependent and -independent PCDs as well as relevant pro-survival pathways in neuroprotective actions of nicotine against Aβ-induced neurotoxicity. We observed that in addition to the caspase-dependent pathway, nicotine can also protect cortical neurons from Aβ-induced PCD by preventing AIF release/translocation, and thus inhibiting caspase-independent cell death. Furthermore, we show that this neuroprotective effect of nicotine is mediated through the activation of α7 nAChRs and PI3K/Akt signaling pro-survival cascades.

Materials and methods

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References
  8. Supporting Information

Chemicals and suppliers

The source of drugs and materials used are as follows: Cell culture materials (Gibco BRL, Burlington, ON, Canada); Aβ1–42,42-1, (−)-nicotine, mecamylamine, dihydro-β-erythroidine-HBr (DHβE) and methyllycaconitine (MLA) (Sigma, St Louis, MO, USA); Antibodies against AIF, β-actin and peroxidase-conjugated secondary antibodies (Santa Cruz Biotechnology, Santa Cruz, CA, USA); Mouse anti-cytochrome c antibody (BD Bioscience, San Diego, CA, USA); LY294002, PD98059, antibodies against active caspase 3, histone H1, Cox IV, phospho- and non-phospho-specific PI3K and Akt (Cell Signaling Technology, Beverly, MA, USA); Mitochondria isolation kit and the nuclear and cytoplasmic extraction kit (Pierce Biotechnology, Rockford, IL, USA).

Primary neuronal cultures

Cortical neuronal cultures were derived from the cerebral cortex of Sprague–Dawley rat embryos (E17-18) as described previously (Bastianetto et al. 1999). Briefly, cells dissociated from the cerebral cortex of embryos were seeded at a density of 5 × 105 cells/mL onto 96-well and 6-well tissue culture plates and 4-well chamber slide system (Corning, Corning, NY, USA) pre-coated with poly-d-lysine and these cells are used for neuronal cytotoxicity, Western blot and confocal image analysis, respectively. Cultures were incubated in neurobasal medium supplemented with 2% B27, 15 mM HEPES, 25 μM l-glutamate, 100 U/mL penicillin and 100 μg/mL streptomycin and maintained at 37°C in a 5% CO2 humidified atmosphere. Experiments were performed at 37°C on the culture day, 8–10 in neurobasal medium without B27 supplement.

All experiments presented in this study were approved by the McGill Animal Care Committee and according to the guidelines of the Canadian Council on Animal Care and the National Institutes of Health Guide for the Care and Use of Laboratory Animals (NIH Publication No. 8023).

Treatment of the cultures

Cultured neurons were exposed to 1, 5, 10, 20 and 30 μM of Aβ1–42 peptides for 24 h to determine the optimal concentration for the following experiments. Prior to use, Aβ1–42 was dissolved in sterilized distilled water containing ammonium hydroxide 0.02% (v/v) at a concentration of 10−3 M and stored at −20°C before use (Bastianetto et al. 2006). The obtained Aβ1–42 contains Aβ dimers, trimers and higher molecular weight Aβ-derived diffusible ligends (Figure S1). To determine the neuroprotective effects of nicotine on Aβ-induced neurotoxicity, neurons were pre-treated with (−)-nicotine alone (0.1, 1, 5, 10 μM) followed by exposure to 10 μM Aβ1–42 for 24 h. The extent to which caspase-dependent cell death pathways contribute to neuroprotective actions of nicotine was studied by using a multi-step pharmacological protocol. According to this protocol, neurons were first pre-treated with pan-caspase inhibitor, Boc-D-fmk (150 μM) together with (−)-nicotine (10 μM) then followed by exposure to 10 μM Aβ1–42 for 24 h. Involvement of nAChRs in neuroprotective effect of nicotine was explored by using mecamylamine (10 μM), a cholinergic antagonist; MLA (10 nM), a selective α7 nAChR antagonist and DHβE (100 nM), a selective α4β2 nAChR antagonist. These antagonist concentrations were chosen on the basis of previous primary neuronal culture studies (Kihara et al. 1998; Liu and Zhao 2004). To determine whether the neuroprotective effect of nicotine was mediated by PI3K and ERK pathways, the PI3K inhibitor, LY294002 (10 μM) and ERK inhibitor, PD98059 (10 μM) were used on the basis of previous primary neuronal culture studies (Kihara et al. 2001; Steiner et al. 2007). Nicotine, nAChR antagonists as well as the pathway inhibitors at above-mentioned concentrations when added alone did not show significant influence on AIF release/translocation in our neuronal culture model (data not shown).

All pathway inhibitors or nAChR antagonist were added to the cell culture medium 1 h before the treatment with (−)-nicotine (10 μM). Then, the medium was removed and an identical dose of pathway inhibitors or nAChR antagonists were applied for the second time together with (−)-nicotine for another 12 h. Subsequently, the medium was removed and the cell culture was further exposure to 10 μM Aβ1–42 alone for another 24 h.

Neuronal cytotoxicity Assay

Aβ-induced neuronal cytotoxicity was determined using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay, as described previously (Bastianetto et al. 2000). Briefly, MTT was diluted in Hanks’ balanced salt solution at 5 mg/mL and added to cells grown in 96-well plates at a final concentration of 0.5 mg/mL. Following a 3 h incubation to allow its conversion into formazan crystals, the medium was removed and cells were lysed to dissolve the crystals. Absorbance at 570 nm was measured.

Hoechst staining

To reveal the nuclear morphological changes in cultured neurons, cells were stained with nuclear dye Hoechst 33258. Briefly, cultured cells were fixed in 4% paraformaldehyde for 30 min and incubated in 10 μg/mL Hoechst 33258 (Sigma) for 30 min at 24°C. Nuclear condensation and fragmentation were then assessed with a Nikon TE800 fluorescent microscope.

Cell fractionation

The harvested cells were rinsed twice with ice-cold Hanks’ balanced salt solution and lysed in RIPA buffer [20 mM Tris–HCl, pH 8.0, 150 mM NaCl, 1 mM EDTA, 1% IgepalCA-630, 0.1% sodium dodecylsulfate (SDS), 50 mM NaF, 1 mM NaVO3; 2 mM phenylmethylsulfonyl fluoride, 10 μg/mL leupeptin, 50 μg/mL aprotinin] on ice for 30 min. Cell lysates were centrifuged at 15 000 g for 15 min at 4°C twice, and the resulting supernatant, representing the cytosolic fraction, was recovered. The mitochondrial fraction was isolated with the Mitochondria Isolation Kit. Briefly, the harvested cell pellet was suspended in isolation reagent A and incubated on ice for 2 min, next the cell suspension was lysated in Dounce Tissue Grinder on ice, an equal volume of isolation reagent C was added into the cell lysate and was mixed by inverting the tube several times. After centrifugation at 700 g for 10 min at 4°C, the supernatant was further centrifuged at 12 000 g for 15 min at 4°C. The pellet containing the isolated mitochondria was re-suspended in RIPA buffer. The nuclear fraction was isolated by using the Nuclear and Cytoplasmic Extraction kit. In brief, the harvested cells were washed and suspended in Subcell Buffer-I and incubated on ice for 10 min. The cells were then lysated through a narrow opening syringe needle. Subcell Buffer-II was added into the cell lysate and then centrifuged at 700 g for 10 min to pellet the nuclei. The nuclear pellet was cleared by resuspending it in Subcell buffer-III and centrifugation at 700 g for 10 min.

Western blotting

Western blotting was performed as described previously (Reix et al. 2007). In brief, subcellular fractions were resuspended in RIPA buffer. Samples with equal amounts of protein were then separated by 4–20% polyacrylamide gel electrophoresis, and the resolved proteins were electrotransferred onto hybond-C nitrocellulose. Membranes were incubated with 5% non-fat milk in TBST (10 mM Tris–HCl, pH 8.0, 150 mM NaCl and 0.2% Tween-20) for 1 h at 24°C and with appropriate primary antibodies overnight at 4°C. Membranes were then washed twice with TBST and probed with corresponding secondary antibodies conjugated with horseradish peroxidase (anti-goat/mouse-horseradish peroxidase, 1 : 3000) at 24°C for 1 h. Membranes were finally washed several times with TBST and immunoreactive bands were visualized using an ECL detection kit (Amersham, Toronto, ON, Canada). To reprobe with a different antibody, the membranes were first stripped in buffer containing 100 mM β-mercaptoethanol, 2% SDS, and 62.5 mM Tris–HCl (pH 6.7) at 60°C for 30 min, extensively washed, reblocked with 5% non-fat milk in TBST and then incubated with the relevant antibody.

Confocal laser scanning fluorescence microscopy

Primary rat cortical neurons were plated at a density of 5 × 105 cells/mL in 4-well chamber slide system pre-coated with poly-d-lysine. At 8 day in vitro, the primary neurons were pre-treated with 10 μM nicotine and exposed to Aβ for 24 h as described above. Neurons were fixed with 4% paraformaldehyde in phosphate-buffered saline (pH 7.5) for 10 min at 24°C. Cells were washed with phosphate-buffered saline before being incubated for 1 h at 24°C in blocking buffer containing 10% normal goat serum (Vector Laboratories, Burlingame, CA, USA). Cells were then incubated overnight at 4°C with purified rabbit polyclonal anti-AIF antisera (1 : 200) diluted with blocking buffer. Finally, cells were incubated in goat-anti rabbit IgG conjugated with Alexa Fluor 568 (1:200). AIF-immunoreactivity (-IR) was labeled red while nuclei were counter-stained with 4′,6-diamidino-2-phenylindole (DAPI). Finally, sections were cover-slipped with anti-fading mounting medium and examined under a Nikon PCM 2000 laser scanning confocal microscope. Controls included omission of primary antibodies and pre-adsorption of purified AIF antiserum with its blocking peptide. Under these conditions, staining was always completely abolished (not shown).

Data analysis

Results are expressed as percentage of control values obtained from cultures not exposed to nicotine or Aβ. Differences were analyzed for statistical significance using one-way anova, followed by Newman-Keuls or Dunnett’s post hoc comparisons. Significance was set at < 0.05.

Results

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References
  8. Supporting Information

Nicotine inhibits Aβ-induced neurotoxicity

The MTT assay allowed to monitor mitochondrial redox function and was used as a general indicator of neurotoxicity (Liu et al. 1997). A significant decline in MTT reduction to formazan was observed in neurons treated for 24 h with different concentrations of Aβ1–42 (1, 5, 10, 20, 30 μM). The effect was concentration-dependent and reached a plateau at 10 μM of Aβ1–42 (Fig. 1a), consequently this concentration was chosen for the subsequent assays. The inverse peptides Aβ42-1, used as negative control, had no neurotoxicity effect (Fig. 1a). To investigate the effect of nicotine on Aβ1–42 neurotoxicity, cortical cells were pre-treated with several concentrations of nicotine (0.1, 1, 5 and 10 μM) for 12 h followed by 10 μM Aβ1–42 administration for 24 h. Pre-treatment with nicotine (10 μM) significantly attenuated Aβ-induced neurotoxicity. The similar effect was observed following an application of 1 and 5 μM nicotine, albeit to a lesser, non-significant level (Fig. 1b). When applied alone, both doses of nicotine had no neurotoxic effect (data not shown).

image

Figure 1.  Effect of nicotine on Aβ1–42 induced MTT reduction in cultured cortical neuronal cells. (a) Cultures were incubated for 24 h with Aβ1–42 or Aβ42–1 at indicated concentrations and MTT assay was performed. Aβ1–42 induced a concentration-dependent MTT reduction. (b) Cultures were incubated with 10 uM Aβ1–42 for 24 h with or without pre-treatment with indicated concentrations of nicotine and MTT assay was performed. Pre-treatment with 10 μM nicotine significantly attenuated Aβ-induced neurotoxicity. Each data point in panels (a) and (b) (±SD; bars) is the mean of eight independent trials. Data are expressed as percentage of control group (no Aβs and no nicotine; defined as 100%). *< 0.05 compared with control group. #< 0.05 compared with Aβ1–42 treated neurons. One-way analysis of variance (anova) followed by the Dunnett’s test.

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To further confirm the neuroprotective effect of nicotine, cell death was quantified based on apoptotic morphology as revealed by Hoechst 33258 staining. In accordance with MTT assay results, a significant increase in the number of cells displaying nuclear condensation or fragmentation was observed following 24 h exposure to 10 μM Aβ1–42. Conversely, treatment with 10 μM nicotine significantly decreased the number of Aβ-induced apoptotic cells (Fig. 2a and b). In addition, administration of the pan-caspase inhibitor, Boc-D-fmk (150 μM) or the selective caspase 3 inhibitor, z-DEVD-fmk (150 μM) significantly reduced Aβ-induced cell death without, however, blocking it completely (Fig. 2c).

image

Figure 2.  Effect of nicotine on Aβ-induced apoptotic death in cultured cortical neuronal cells. (a) Representative images of Hoechst 33258 staining in Aβ1–42-treated rat cortical cell cultures with or without pre-treatment with nicotine. At 10 DIV, cells were treated with 10 μM Aβ1–42 for 24 h with or without pre-treatment with nicotine, and then fixed in 4% formaldehyde, stained and visualized by fluorescent microscopy. Arrows indicate cells displaying nuclear condensation or fragmentation. (b) Pre-treatment with 10 μM nicotine significantly decreased the number of cells with apoptotic morphology in Aβ1–42-treated neuronal cultures. After treatment and Hoechst staining, the number of cells with apoptotic morphology for each treatment was counted under fluorescence microscope in 5–7 randomly chosen fields (at least 200 cells per treatment). (c) Pre-treatment with the pan-caspase inhibitor, Boc-D-fmk, caspase 3 inhibitor, z-DEVD-fmk significantly attenuated Aβ-induced apoptotic death in rat cortical neuronal cultures. At 10 DIV, cells were treated with 10 μM Aβ1–42 for 24 h with or without 1 h pre-treatment with Boc-D-fmk or z-DEVD-fmk (each at 150 μM). After treatment and Hoechst staining, the number of cells with apoptotic morphology for each treatment was counted under fluorescence microscope in 5–7 randomly chosen fields (at least 200 cells per treatment). Each data point in panels (b) and (c) (±SD; bars) is the mean of eight independent trials. Histograms represent the number of apoptotic cells as a percentage of control group (no Aβs and no nicotine; defined as 100%); *< 0.01 compared with control cells; #< 0.05 compared with Aβ-treated neurons. One-way analysis of variance (anova) followed by the Dunnett’s test.

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Nicotine attenuates cytochrome c release, down-regulates active caspase 3 level and decreases AIF release/translocation in Aβ-treated rat cortical neuronal culture

Since both caspase-dependent and -independent cell death pathways have been previously involved in Aβ-induced cell death in primary neuronal cultures and human postmortem brains (Movsesyan et al. 2004; LeBlanc 2005; Yu et al. 2010), we examined whether the neuroprotective action of nicotine also involves inhibition of both caspase-dependent and -independent cell death pathways. To this end, neuronal cultures were first treated with pan-caspase inhibitor, Boc-D-fmk (150 μM) together with (−)-nicotine (10 μM) followed by exposure to 10 μM Aβ1–42 for 24 h. Whilst nicotine and Boc-D-fmk, added alone reduced Aβ-induced cell death to a similar extent, their combined application resulted in an additive effectiveness (Fig. 3). This synergy strongly indicated that, indeed, nicotine exerts its actions along both caspase-dependent and -independent cell death pathways. This possibility was then further explored by western blot assessment of nicotine actions on the mediators of both cell death pathways in neuronal cultures exposed to Aβ1–42. Treatment of 10 μM Aβ1–42 induced a significant increase of cytochrome c release and active caspase 3 level (Fig. 4a and b) in primary neuronal cultures. Pre-treatment with 10 μM nicotine abolished the increase of cytochrome c release and caspase 3 activation, while application of 0.1, 1 and 5 μM nicotine showed no significant effect on Aβ-induced up-regulation of cytochrome c release and active caspase 3 level (Fig. 4a and b). Moreover, treatment of the cultures with 10 μM Aβ1–42 for 24 h caused significant AIF release from mitochondria and subsequent nuclear translocation. Pre-treatment with 10 μM nicotine could prevent the Aβ-induced AIF release/translocation whereas no significant effect was observed following application of 0.1, 1 and 5 μM nicotine on Aβ-induced AIF release/translocation (Fig. 4c–f). In addition, the Aβ-induced increase of cytochrome c release and active caspase 3 level was significantly blocked by the pan-caspase inhibitor, Boc-D-fmk and the selective caspase 3 inhibitor, z-DEVD-fmk (Fig. 5). In contrast, the Aβ-induced AIF release/translocation was not inhibited by either the pan-caspase inhibitor, Boc-D-fmk or the selective caspase 3 inhibitor, z-DEVD-fmk (Fig. 5). The protective effect of nicotine on AIF release/translocation was further confirmed by fluorescence microscopy. AIF was found to be primarily cytoplasmic with a punctate pattern of localization in control cells whereas it appeared to be co-localized with DAPI in cells treated with Aβ alone. Nicotine treatment reversed the co-localization of AIF with DAPI (Fig. 6).

image

Figure 3.  Effects of nicotine and caspase-dependent pathway inhibitor on Aβ1–42 induced cell death in cultured cortical neuronal cells. Pre-treatment of neuronal culture with nicotine in the presence of pan-caspase inhibitor, Boc-D-fmk (150 μM) followed by exposure to 10 μM Aβ1–42 for 24 h. The pan-caspase inhibitor (Boc-D-fmk, 150 μM) significantly attenuated Aβ-induced cell death. Co-treatment with nicotine and Boc-D-fmk had an additive effect against Aβ-induced cell death in neuronal culture. After treatment and Hoechst staining, the number of cells with apoptotic morphology for each treatment was counted under fluorescence microscope in 5–7 randomly chosen fields (at least 200 cells per treatment).Each data point (±SD; bars) is the mean of eight independent trials. Data are expressed as percentage of control group (no Aβs and no nicotine; defined as 100%). *< 0.05 compared with control group. #< 0.05, ##< 0.01 compared with Aβ1–42-treated neurons. ¤< 0.05 compared with nicotine-treated neurons. One-way analysis of variance (anova) followed by the Dunnett’s test.

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image

Figure 4.  Effect of nicotine on cytochrome c release, caspase 3 activation and AIF release/translocation in Aβ-treated rat cortical neuronal culture. Primary cortical neurons (8–10 DIV) were pre-treated with indicated concentrations of nicotine for 12 h followed by the treatment with 10 μM Aβ1–42 alone for another 24 h. After indicated periods of time, cells were harvested and subjected to immunoblot analysis (a, c, e). Pre-treatment with 10 μM nicotine abolished the up-regulations of cytochrome c release and caspase 3 activation in Aβ-treated rat cortical neuronal culture immunoblot analysis (a, b). Pre-treatment with 10 μM nicotine significantly inhibited the release of AIF from mitochondria and its nuclear translocation induced by Aβ1–42 in primary rat cortical neurons. Changes in AIF protein levels in the mitochondrial (c, d) and nuclear (e, f) compartments following treatment with 10 μM Aβ1–42 with or without pre-treatment with indicated concentrations of nicotine. Each data point in panels (b), (d) and (f) (±SD; bars) is the mean of eight independent experiments. *< 0.05 compared with controls (no Aβs and no nicotine; defined as 100%). #< 0.05 compared with Aβ-treated neurons. One-way anova with Newman-Keuls post hoc comparisons.

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image

Figure 5.  Effect of caspase inhibitors on cytochrome c release, caspase 3 activation and AIF release/translocation in Aβ-treated rat cortical neuronal culture. Pre-treatment with pancaspase inhibitor, Boc-D-fmk or caspase 3 inhibitor, z-DEVD-fmk reduced cytochrome c release and caspase 3 activation, but did not alter AIF release/translocation in cultures treated with Aβ1–42. At 8–10 DIV, 10 μM Aβ1–42 with or without 1 h pre-treatment with Boc-D-fmk or z-DEVD-fmk (each at 150 μM) was added to rat cortical neuronal cultures. After 24 h of treatment, cells were harvested and the cytosolic fraction was subjected to immunoblot analysis as described in Materials and methods section. Each data point (±SD; bars) is the mean of eight independent experiments. *< 0.05 compared with controls (no Aβs and no nicotine; defined as 100%). #< 0.05 compared with Aβ-treated neurons. One-way anova with Newman-Keuls post hoc comparisons.

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image

Figure 6.  Effect of nicotine on Aβ-induced AIF translocation into the nucleus. At 8 DIV, 10 μM Aβ1–42 was added to primary rat cortical neuron cultures with or without pre-treatment with 10 μM nicotine. After 24 h, cells were subjected to AIF-immunolabeling. Figures represent immuostaining with anti-AIF (red) and DAPI nuclear staining (blue). Scale bar: 30 μM.

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Involvement of α7 nAChRs in the neuroprotective effects of nicotine on mitochondrial release and nuclear translocation of AIF

Previous studies have shown that the neuroprotective effect of nicotine on inhibition of caspase-dependent PCD pathway was mediated by α7 nAChRs (Liu and Zhao 2004). We therefore investigated whether these receptors also mediate the neuroprotective effect of nicotine on caspase-independent PCD pathway. To this end, experiments were performed with three different nicotinic antagonists, mecamylamine (10 μM), a non-selective cholinergic antagonist; MLA (10 nM), a selective α7 nAChR antagonist as well as DHβE (100 nM), a selective α4β2 nAChR antagonist. As shown in Fig. 7, when 10 μM mecamylamine was added concomitantly with nicotine, the nicotine-mediated inhibition of mitochondrial release (Fig. 7a) and nuclear translocation (Fig. 7b) of AIF were significantly antagonized, suggesting the involvement of nAChRs in the neuroprotective actions of nicotine. Next, we evaluated the effect of α7 and α4 nAChR antagonists, MLA and DHβE, respectively, on the neuroprotective effect of nicotine. When 10 nM MLA was added together with nicotine, the nicotine-mediated inhibition of mitochondrial release and nuclear translocation of AIF was significantly antagonized (Fig. 7a and b). However, the neuroprotective effect of nicotine was not affected by the 100 nM α4 nAChR antagonist, DHβE (Fig. 7a and b).

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Figure 7.  Effect of cholinergic antagonists on nicotine-mediated neuroprotection against Aβ-induced AIF release/translocation. The nicotinic antagonists mecamylamine (1 μM), α-BTX (1 nM) and DHβE (100 nM) were added to the culture medium 1 h before treatment with nicotine (10 μM). A second dose of each antagonist was applied together with nicotine for another 12 h followed by exposure to Aβ peptides alone for 24 h. After indicated periods of time, cells were harvested and subjected to immunoblot analysis. Changes in AIF protein level in the mitochondrial (a) and nuclear (b) compartments following treatment with the indicated concentrations of Aβ1–42. Each data point (±SD; bars) is the mean of six independent trials. *< 0.05 compared with controls (no Aβs and no nicotine; defined as 100%) denotes a statistically significant difference. #< 0.05 compared with Aβ + nicotine-treated neurons. One-way analysis of variance (anova) followed by the Dunnett’s test.

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Neuroprotective effect of nicotine on mitochondrial release and nuclear translocation of AIF is mediated through the activation of the PI3K pathway

Previous studies have shown that the nicotine-mediated neuroprotection via α7 nAChR stimulation involves the activation of the PI3K and ERK pro-survival pathways in hippocampal neurons (Kihara et al. 2001; Dajas-Bailador et al. 2002). We therefore investigated whether the nicotine effects on Aβ-induced toxicity in cortical neurons observed here could also involve the triggering of these pro-survival pathways. PI3 kinase inhibitor LY294002 (10 μM) and MEK1/2 inhibitor PD98059 (10 μM) were co-incubated with 10 μM nicotine prior to the administration of 10 μM Aβ1–42. Co-treatment with LY294002 (10 μM) and nicotine significantly abolished the nicotine-mediated attenuation of Aβ-induced AIF release/translocation in cultured neurons (Fig. 8a and b). In contrast, PD98059 did not significantly affect the protective effect imparted by nicotine against Aβ-induced AIF release/translocation (Fig. 8a and b). The involvement of PI3K pathway in the neuroprotective effect of nicotine was further confirmed by western blot analysis showing that nicotine indeed restored Akt phosphorylation in Aβ1–42-treated neurons via α7 nAChRs (Fig. 9). Compared with non-treated cells, the immunoreactivity of phosphorylated Akt was significantly decreased in Aβ1–42-treated neurons (Fig. 9). Pre-treatment nicotine significantly reversed the decrease of phosphorylated Akt in Aβ1–42-treated neurons. Furthermore, the effect of nicotine on Akt phosphorylation was significantly reduced by nAChR antagonist, mecamylamine (10 μM) and the α7 nAChR antagonist, MLA (10 nM), but not by α4 nAChR antogonist, DHβE (100 nM) (Fig. 9).

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Figure 8.  Involvement of PI3K pathway in nicotine-mediated neuroprotection against Aβ-induced AIF release/translocation. Nicotine (10 μM), LY294002 (10 μM), a PI3K inhibitor, and PD98059 (10 μM), a MEK1/2 blocker were added to primary rat cortical neuron cultures for 12 h followed by exposure to Aβ peptides alone for 24 h. The AIF protein levels in mitochondrial and nuclear compartments were measured with western blotting. (a) Neuroprotective effects of nicotine on the mitochondrial release of AIF induced by Aβ1–42 were selectively suppressed by LY294002 (10 μM). (b) Neuroprotective effects of nicotine on the nuclear translocation of AIF induced by Aβ1–42 were selectively suppressed by LY294002 (10 μM), a PI3K inhibitor. Each data point in panels (a) and (b) (±SD; bars) is the mean of six independent trials. *< 0.05 compared with controls (no nicotine and no Aβ1–42; defined as 100%); #< 0.05 compared with Aβ + nicotine-treated neurons. One-way analysis of variance (anova) followed by the Dunnett’s test.

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Figure 9.  Pre-treatment of nicotine restored Akt phosphorylation via α7 nAChRs in Aβ1–42 treated neurons. Cultured neurons were pre-treated with 10 μM nicotine for 12 h followed by Aβ1–42 exposure for 6 h. Nicotine plus three different nAChR antagonists, mecamylamine (10 μM), MLA (10 nM) and DHβE (100 nM), were added 12 h before Aβ1–42 treatment. The level of phosphorylated Akt (Ser 473) was measured by western blot. Each data point (±SD; bars) is the mean of six independent trials. *< 0.05 compared with controls (no nicotine and no Aβ1–42; defined as 100%); #< 0.05 compared with Aβ + nicotine-treated neurons. One-way analysis of variance (anova) followed by the Dunnett’s test.

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Discussion

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References
  8. Supporting Information

We report here for the first time that neuroprotective actions of nicotine against Aβ-induced neurotoxicity along caspase-independent pathway involve the inhibition of mitochondrial release and nuclear translocation of AIF. These neuroprotective effects of nicotine on Aβ-induced AIF release/translocation are mediated by α7 nAChRs and PI3K/Akt signal transduction.

Aβ accumulation is one of the early pathological changes observed in AD and has been related to the neuronal loss by PCD (Loo et al. 1993; Yuan and Yankner 2000). Previous reports have shown that Aβ induces neuronal PCD through the caspase-dependent pathway (Loo et al. 1993; Yaar et al. 1997; Giovanni et al. 2000; Wellington and Hayden 2000). Exposure of primary neuronal cultures to Aβs causes apoptotic cell death and activation of multiple caspases, namely caspase 2, -3, and -6 (Allen et al. 2001). Activation of caspase 8 by Aβ in primary hippocampal cultures and SH-SY5Y cell lines has also been reported (Wei et al. 2002). However, little attention has been paid to the possible involvement of caspase-independent pathway in Aβ-induced neuronal PCD. One study has suggested that in addition to the caspase-dependent apoptosis pathway, Aβ also induces neuronal PCD through caspase-independent pathway associated with significant increase in the cytosolic levels of AIF (Movsesyan et al. 2004). In the present study, exposure of primary neuronal cultures to Aβ1–42 causes apoptotic cell death via release of cytochrome c, activation of caspase 3, as well as via mitochondrial AIF release and subsequent nuclear translocation. Importantly, co-treatment of cultures with nicotine and caspase-dependent PCD pathway inhibitor such as Boc-D-fmk had an additive effect to nicotine-mediated neuroprotection. This additivity strongly indicates that nicotine acts against the Aβ-induced neurotoxicity through both caspase-dependent and -independent PCD pathways. In line with these results, pre-treatment by caspase inhibitors could partially attenuate Aβ-induced neuronal apoptosis (as reflected by the inhibition of cytochrome c release and activation of caspase 3) but could neither block neuronal apoptosis completely nor prevent AIF release/translocation, further suggesting an involvement of caspase-independent apoptosis in Aβ-induced neuronal death. Our results are thus in agreement with the previous studies indicating that the caspase-independent apoptotic pathways can be triggered when the caspase cascade is blocked in the neuronal culture model (Cregan et al. 2002; Cheung et al. 2005). The functional relevance of caspase-independent PCD pathway in neuronal death observed here in neuronal culture model is further supported by our previous finding in postmortem AD brains, in which AIF nuclear translocation was observed in several regions of AD brain (Yu et al. 2010).

We next explored the possibility that nicotine mediates its neuroprotective effects through the caspase-independent pathway. We observed that in cortical neurons exposed to Aβ, nicotine could not only significantly inhibit the mitochondrial release of AIF, but also its nuclear translocation. These data are the first to suggest that nicotine may prevent Aβ-induced neuronal PCD by attenuating the release/translocation of AIF. These nicotine effects on AIF are associated with the inhibition of caspase-dependent PCD shown here by nicotine-mediated reduction of cytochrome c release and caspase 3 activation and are also in agreement with previous studies (Kihara et al. 2001; Liu and Zhao 2004).

Although the underlying mechanisms remain to be fully identified, several cellular and molecular targets are likely to be involved in nicotine-mediated neuroprotection. For example, nicotine has been proposed to protect neurons through: (i) direct nAChR activation (Kihara et al. 1997; Zanardi et al. 2002); (ii) inhibition of β-amyloidosis through its direct interaction with the α-helix structure of Aβ (Zeng et al. 2001); (iii) interfering with Aβ binding to α7 nAChR (Wang et al. 2010). The activation of α4 nAChRs has been previously implicated in nicotine-mediated neuroprotection (Kihara et al. 1997, 1998). Importantly, both α7- and α4-containing nAChRs have been involved in the neuroprotective effect of nicotine against Aβ- and glutamate-induced cortical neuronal death (Akaike et al. 2010). In the present study, we show that the activation of α7 nAChR mediates specifically and selectively the neuroprotective effects of nicotine along the caspase-independent PCD pathway via inhibition of Aβ-induced release/translocation of AIF.

Previous studies have suggested that the neuroprotective effects of nicotine may also be associated with the modulation of pro-survival signaling cascades (Kihara et al. 2001). We thus assessed the ability of nicotine to modulate PI3K and ERK1/2 pathways. We found that application of the PI3K inhibitor, LY294002, abrogated the protective effect of nicotine against Aβ-induced caspase-independent PCD. Moreover, pre-treatment of neuronal cultures with nicotine subsequently exposed to Aβ1–42 restored the phosphorylation of an effector of phosphatidylinositol 3-kinase (PI3K) such as Akt. These actions of nicotine appear specific since ERK1/2 pathway was not affected. Notably, activated Akt can target multiple downstream transcription factors and apoptosis-associated proteins (Vivanco and Sawyers 2002; Nuutinen et al. 2006). In addition, nicotine shares common downstream targets with the PI3K/Akt pathway, such as B-cell chronic lymphocytic leukemia/lymphoma and B-cell chronic lymphocytic leukemia/lymphoma-antagonist of cell death (Buckingham et al. 2009).

In conclusion, the present study provides evidence that nicotine is effective in protecting cortical neurons from Aβ-induced caspase-independent neuronal PCD via α7 nAChRs activation and PI3K/Akt pathway signaling. These results shed new light on the neuroprotective actions of nicotine and α7 nAChR agonists in vitro and should guide future studies exploring this phenomenon in vivo, particularly in the context of AD models. Indeed, recent pre-clinical works with α7 nAChR agonists such as ABT-107 have shown that such compounds may be efficient in reducing spinal tau phosphorylation in tau/amyloid precursor protein transgenic AD mice (Bitner et al. 2010). In light of the results reported here, further in vivo testing of candidate therapeutic compounds using AD models should now include the evaluation of their capacity to prevent both caspase-dependent and caspase-independent apoptosis.

Acknowledgements

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References
  8. Supporting Information

This study was supported by a grant from Canadian Institutes of Health Research (CIHR) to R.Q, an FRSQ-INSERM grant to R.Q. and S.K., and FRSQ fellowship to W.Y. and a CIHR fellowship to N.M. (who is now an FRSQ scholar). We also greatly appreciate the help from Mira Thakur for editing and proofreading the manuscript. All authors declare that no conflict of interest exists in the present work.

References

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References
  8. Supporting Information

Supporting Information

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References
  8. Supporting Information

Figure S1. Analysis of Aβ1-42 peptide oligomeric composition in neuronal culture medium. Aβ1–42 was dissolved in sterilized distilled water containing ammonium hydroxide 0.02% (v/v) at a concentration of 10−3 M and stored at −20°C before use. The Aβ1–42 stock solution was further diluted in neuronal culture medium to a final concentration of 10 μM. The concentrated medium was subjected to SDS-PAGE and based on the molecular weight, different assembly states of Aβ peptide were detected using monoclonal antibody (6E10) against Aβ.

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