Nicotine attenuates β-amyloid peptide-induced neurotoxicity, free radical and calcium accumulation in hippocampal neuronal cultures

Abstract

1. Recent studies indicate that neuronal loss in Alzheimer's disease (AD) is accompanied by the deposition of β-amyloid protein (Aβ) in senile plaques. Nicotine as a major component of cigarette smoke has been suggested to have a protective effect for neurons against Aβ neurotoxicity.

2. Our present study demonstrates that nicotine protected cultured hippocampal neurons against the Aβ-induced apoptosis. Nicotine effectively inhibits apoptosis in hippocampal cultures caused by Aβ_25–35 or Aβ_1–40 treatment and increase of caspase activity induced by Aβ_25–35 or Aβ_1–40.

3. Measurements of cellular oxidation and intracellular free Ca²⁺ showed that nicotine suppressed Aβ-induced accumulation of free radical and increase of intracellular free Ca²⁺.

4. Cholinergic antagonist mecamylamine inhibited nicotine-induced protection against Aβ-induced caspase-3 activation and ROS accumulation.

5. The data show that the protection of nicotine is partly via nicotinic receptors. Our results suggest that nicotine may be beneficial in retarding the neurodegenerative diseases such as AD.

Introduction

Alzheimer's disease (AD) is one of the most common forms of dementia, and one of the neuropathological hallmarks of AD is the neuronal degeneration associated to senile plaques (Vickers et al., 2000). Such plaques are composed of compacted amyloid β-peptide (Aβ), which is a 40–43 amino-acid peptide (Soto et al., 1994; 1995; Selkoe, 1998). Aβ is originated by the proteolytic processing of a transmembrane glycoprotein called β-amyloid precursor protein (β-APP), which can be secreted (Saitoh et al., 1989) or cleaved releasing Aβ by the action of β- and γ-secretases (Hass & de Strooper, 1999). The deposition of soluble Aβ produces the aggregation of the peptide-forming amyloid fibrils, which have been reported to be neurotoxic in vitro (Yankner, 1996; Alvarez et al., 1998; Munoz & Inestrosa, 1999) and in vivo (Inestrosa & Reyes, 1998; Soto et al., 1998; Inestrosa & Larrondo, 2000). A shorter hydrophobic fragment of the protein Aβ_25–35, though not present in biological systems, is widely used together with, or instead of, the endogenous fragment Aβ_{1– 42(43)} by a number of investigators, and is found to be at least as toxic as the full-length fragment (Yankner et al., 1989).

It is true that fibrillar materials have toxic effects on a range of cells (Pike et al., 1993; 1997; Cribbs et al., 1997; Lorenzo et al., 1994; Pollard et al., 1995; Mark et al., 1996). The intrinsic toxicity of high levels of fibrils themselves could result from generation of oxygen-free radicals by Aβ fibrils (in the absence of any cellular elements) (Hensley et al., 1994), or their destabilization of membranes, resulting in changed intracellular calcium homeostasis and eventual cell death (Mark et al., 1996).

Numerous epidemiological studies have reported a highly significant negative association between cigarette smoking and neurodegenerative disorders, especially in Parkinson's disease (PD) (Morens et al., 1995; Ramon & Estefanýa et al., 1998) and AD (Brenner et al., 1993; Hillier et al., 1997; Ulrich et al., 1997). Nicotine is a predominant component of cigarette smoke, and is currently being used in pilot clinical studies for the treatment of AD (Wilson et al., 1995; Emilien et al., 2000). α4β2 nicotinic receptor activation plays an important role in neuroprotection against Aβ cytotoxicity (Kihara et al., 1997). Garrido et al. (2000; 2001) reported that nicotine could exert potent neuroprotective effects by inhibiting arachidonic acid-induced apoptotic cascades (caspase-3 activation and cytochrome c release) of spinal cord neurons. The α7 nicotinic acetylcholine receptor subtype mediates nicotine protection against NMDA excitotoxicity in primary hippocampal cultures through a Ca²⁺-dependent mechanism (Dajas-Bailador et al., 2000). Also, we have studied the scavenging effect of nicotine on the oxygen-free radicals and found that it could effectively scavenge superoxide and hydroxyl radicals, and even could scavenge the free radicals in the gas phase of cigarette smoke (Liu et al., 2003).

The key pathological change in the brain linked to the emergence and progressive development of dementia is the gradual degeneration of nerve cells and the related loss of specific synaptic connections. Only a highly specific subset of nerve cells shows vulnerability to degeneration, especially in hippocampal and cortical neurons. So, for the purpose of this reason, here we studied the protective effect of nicotine on Aβ-induced apoptosis in hippocampal neuronal cultures. We also try to explore whether changes of Ca²⁺ and reactive oxygen species (ROS) is involved in Aβ-induced neurotoxicity and whether nicotine can reverse this effect.

Methods

Materials

Chemicals were obtained from the following companies: Dulbecco's modified Eagle's medium (DMEM) from GIBCO (Grand Island, NY, U.S.A.); fetal bovine serum (FBS) from Hyclone (logan, UT, U.S.A.); nicotine from Zhengzhou Tobacco Academy of China. 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl (MTT), 2,7-dichlorofluorescein diacetate (DCF-DA), DNase I, ethylenediaminotetraacetic acid (EDTA), trypsin, penicillin, and streptomycin from Sigma Chemical Co. (St Louis, MO, U.S.A.); Fluo-3 acetoxymethyl ester (fluo-3 AM) from Calbiochem (San Diego, CA, U.S.A.); The Cell Death Detection ELISA^plus (CDDE) from Roche Molecular Biochemicals (Germany, Cat # 1 544 675); ApoAlert Caspase Fluorescent Assay Kit from Clontech (U.S.A.).

Aβ_25–35 and Aβ_1–40 from Bachem (Torrance, CA, U.S.A.) was stored dry at –20°C, and was dissolved in double-distilled water at a concentration of 1 mg ml⁻¹ stock solution, and stored at –20°C until use. The stock solution was then stored for 2–4 days at 37°C and used for the aged Aβ condition. The Reverse sequence of Aβ_1–40, Aβ_40–1, was obtained from Sigma Chemical Co. (St Louis, MO, U.S.A.).

Hippocampal cell cultures

Primary neuronal cultures were prepared from the hippocampal tissue of newborn Sprague–Dawley rats, as previously described (Farinelli et al., 1998), with a few modifications. Briefly, the hippocampus of newborn animals was dissected out in DMEM, dissociated mechanically, digested with trypsin and, after stopping the reaction with FBS, filtered through nylon net. After centrifugation, the suspension was distributed in 96-well or 6-well poly-L-lysine-coated tissue culture plates (Costar). Cells were grown in DMEM supplemented with heat-inactivated 10% FBS (HyClone), 20 mM sodium bicarbonate, 1 mM sodium pyruvate, 20 mM HEPES, 100 U ml⁻¹ penicillin, 100 μg ml⁻¹ streptomycin, and 20 mM potassium chloride. The cells were seeded at a density of 2 × 10⁵ cells cm⁻² and maintained at 37°C in humidified 5% CO₂, 95% air at 37°C. After 48 h, nonneuronal cell division was inhibited by a 24-h exposure to 10⁻⁵ M cytosine arabinoside. The culture media was refreshed every 3 days. Most experiments were performed using cultures at 7–9 days.

Mitochondrial function MTT conversion assay (determination of cell survival)

The mitochondrial function of cultured hippocampal neurons was measured by MTT conversion assay. This assay may also serve as a general indicator of cell viability. MTT conversion was performed as described earlier (Mosmann, 1983). This assay takes advantage of the conversion of the yellow MTT to purple formazan crystals by mitochondrial succinate dehydrogenase in viable cells. Briefly, MTT was diluted in water and added to cells grown in 96-well plates at a final concentration of 0.5 mg ml⁻¹. Following a 4-h incubation to allow its conversion into formazan crystals, the media was removed and cells were lysed with DMSO to allow the crystals to dissolve. Absorbance was read at 595 nm using a Bio-RAD 3350 microplate reader, and the results were expressed as a percentage of control (no nicotine and no Aβ_25–35 treated).

Intracellular calcium concentration [Ca²⁺]_i

The concentration of intracellular Ca²⁺ was measured as described elsewhere (Aoshima et al., 1997) with fluo-3 AM, a calcium fluorescent ester chelator. After Aβ_25–35 or nicotine incubation, cells were harvested by gentle scraping, washed, and resuspended in a standard medium (containing 140 mM NaCl, 5 mM KCl, 1 mM MgCl₂, 5.6 mM glucose, 1.5 mM CaCl₂, and 20 mM HEPES-Na (final pH 7.4)), which was used for loading and for the subsequent fluorescence measurements.

Fluo-3 AM (5 mM) was added to the cell suspension, which was subsequently incubated for 30 min at 37°C. The cells were then washed three times, resuspended in the standard medium and transferred to thermostatically controlled cuvette equipped with a magnetic stirrer. The fluorescence intensity of fluo-3 was quantified by a fluorescence spectrophotometer (Hitachi F-4500), with a single excitation wavelength set at 490 nm and an emission wavelength monitored at 526 nm. [Ca²⁺] was calculated from the fluo-3 fluoresce intensity using the equation

For the purpose of calculation of [Ca²⁺]_i, the Kd was assumed to remain constant between 10 and 25°C, and increase linearly up to 42°C and Kd=400 nmol l⁻¹ at 25°C.

The maximal Fluo-3 fluorescence intensity (F_max) was determined by adding 0.1% Triton X-100, and the minimal fluorescence (F_min) was determined by quenching Fluo-3 fluorescence with 5 mM EGTA. F is the fluorescence measured without adding Triton-X-100 or EGTA.

Measurement of intracellular ROS

The level of intracellular ROS was quantified by fluorescence with 2′, 7′-dichlorofluorescein diacetate (2′, 7′-DCF-DA). DCF-DA, a nonfluorescent compound, is deacetylated by viable cells to 2′, 7′-dichlorofluorescin (DCF) by H₂O₂. Hanks balanced salt solution (HBSS) (0.25 ml) and 10 ml of 0.5 mM DCF-DA dissolved in N,N-dimethyl-formamide were added to six-well plates. Cells were loaded with DCF-DA by incubating them for 50 min in the presence of 100 μM DCF-DA. At the end of the incubation period, cells were washed once and the relative levels of fluorescence were quantified using a fluorescence spectrophotometer (485-nm excitation and 535-nm emission, Blanc et al., 1997). The measured fluorescence values were expressed as a percentage of fluorescence in control cultures (no nicotine and no Aβ_25–35 treated).

Measurement of caspase-3 proteolytic activity

Caspase-3 activity was quantified by means of the ApoAlert Caspase Fluorescent Assay Kits (BD Biosciences Clontech), according to the manufacturer's instructions. This assay is based on the release of the fluorochrome 7-amino-4-trifluoromethyl coumarin (AFC), when the provided pseudosubstrate acetyl-Asp-Glu-Val-Asp-aldehyde-AFC is cleaved by caspase-3. Free AFC produces fluorescence that can be monitored and quantified to estimate caspase-3 activity. Briefly, the cultured hippocampal cells were lysed and cell extracts were centrifuged to eliminate cellular debris. Aliquots (50 μl) of the cell extracts were incubated for 1 h at 37°C in the presence of the substrate. Generation of free AFC was quantified using a fluorescence spectrophotometer (400-nm excitation and 505-nm emission).

Cellular DNA fragmentation measured by ELISA

This assay is based on assessment of accumulation of DNA fragments in the cytoplasm of apoptotic cells. The enrichment of mono- and oligo-nucleosomes in the cytoplasm is due to the fact that DNA degradation occurs several hours before plasma membrane breakdown (Duke et al., 1986). In this assay, the accumulation of cytosolic histone-bound DNA fragments was quantified using a commercial ELISA kit (Cat #: 1 544 675, Roche Molecular Biochemicals). The measurement of apoptosis by this assay is sensitive and consistent with other morphometric indices of apoptosis (Ye et al., 1999). Briefly, the hippocampal cells were plated into poly-L-lysine-coated 96-well plates at a density of 3.2 × 10⁴ cm⁻². Cells were exposed to aged Aβ_25–35 or nicotine. After aged Aβ_25–35 or nicotine treatment, the cultures were washed twice with 0.01 M PBS, and cultured cells were lysed. A volume of 20 μl of cell lysate from each well was mixed with 80 μl of antibody solution in the coated wells. The loaded wells were incubated at room temperature for 2 h. The substrate was added to each well after it was washed three times in incubation buffer. After incubation at room temperature for 10–20 min, the optical density was measured using a Bio-RAD 3350 microplate reader with a light filter of 405 nm. The readings were used to measure the degree of apoptosis. The DNA fragmentation was expressed in the enrichment of histone-associated mono- and oligonucleosomes released into the cytoplasm. The enrichment factor was calculated according to absorption at 405 nm, which represented the enrichment of histone-associated DNA fragmentation and accounted for apoptosis of hippocampal cells.

Statistical analysis

Treatment groups were compared using one-way analysis of variance (ANOVA). Differences in which P<0.05 were considered statistically significant. In cases where significant differences were detected, specific post hoc comparisons between treatment groups were examined using the Student–Newman–Keuls tests.

Result

Nicotine prevents Aβ (Aβ_25–35, Aβ_1–40 or Aβ_40–1)-induced loss of hippocampal neuronal cell viability

The effects of different concentrations of nicotine on Aβ (Aβ_25–35, Aβ_1–40 or Aβ_40–1)-induced alterations of mitochondrial function of cultured hippocampal neurons are shown in Figure 1. Mitochondrial function was measured by the MTT conversion assay, which may also serve as a general indicator of cell viability. As indicated in Figure 1, treatment with 50 μM Aβ_25–35 or 20 μM Aβ_1–40 for 24 h markedly decreased MTT conversion in cultured hippocampal neuronal cells. In addition, treatment with 10 μM of nicotine significantly attenuated Aβ_25–35 or Aβ_1–40-induced decrease in MTT conversion. Lower doses of nicotine, such as 1 μM, also decreased the alterations in cell viability induced by aged Aβ_25–35. Even for low concentrations of Aβ_25–35, this trend could be found. It can be found that there is no any effect on the cell viability for nicotine alone at 1 or 10 μM. Since marked protection against aged Aβ_25–35-induced neurotoxicity was observed with 10 μM nicotine, nicotine at this concentration was used in the remaining experiments. Aβ_40–1 has no effect on the cell viability in the presence or absence of nicotine.

Figure 1.

Effect of nicotine on Aβ (Aβ_25–35, Aβ_1–40 or Aβ_40–1)-induced MTT reduction in hippocampal neuronal cells. Cultures were incubated for 24 h with nicotine or Aβ (Aβ_25–35, Aβ_1–40 or Aβ_40–1) as the indicated concentration. The MTT reduction assay was carried out as described in ‘Method'. Data shown are expressed as percentages of control (no nicotine and no Aβ (Aβ_25–35, Aβ_1–40 or Aβ_40–1) treated) values and each data point (±s.e.m.; bars) is the mean of five independent trials.*P<0.05 significantly different from control cells. A1–40: the concentration of Aβ_1–40; A40–1: the concentration of Aβ_40–1; A25–35: the concentration of Aβ_25–35.

Assessment of hippocampal neuronal cell apoptosis

DNA fragmentation was a marker of cell apoptosis in most cases (Bortner et al., 1995; Earnshaw, 1995; Khodarev et al., 1998) In order to evaluate the effects of nicotine on DNA fragmentation in Aβ_25–35- or Aβ_1–40-induced apoptosis, ELISA kit was used to analyze DNA degradation quantitatively. With anti-histone monoclonal antibody, the histone-associated DNA fragmentation (mono- and oligonucleosomes) was detected in this method. As shown in Figure 2, when the cells were incubated with Aβ (Aβ_25–35 or Aβ_1–40) for 48 h, the enrichment factor (EF) values of DNA fragmentation were concentration-dependently increased significantly (10 and 50 μM). Also, if nicotine was added to the culture with Aβ_25–35 or Aβ_1–40, DNA fragmentation was significantly decreased, especially when the concentration of nicotine was 10 μM. Aβ_40–1 has no effect on DNA fragmentation in the presence or absence of nicotine. It can also be found that there is no any effect on EF for nicotine alone at 1 or 10 μM.

Figure 2.

Quantification of apoptosis of hippocampal cells with a cellular DNA fragmentation ELISA. Cells were plated at a density of 3.2 × 10⁴ cm⁻², and exposed to Aβ (Aβ_25–35, Aβ_1–40 or Aβ_40–1) for 48 h. Apoptosis was measured with a cellular DNA fragmentation ELISA, as described in the Methods. Each data point (±s.e.m.; bars) is the mean of five independent trials.*P<0.05 denotes a statistically significant difference from control (no nicotine and no Aβ (Aβ_25–35, Aβ_1–40 or Aβ_40–1) treated). A1–40: the concentration of Aβ_1–40; A40-1: the concentration of Aβ_40–1; A25–35: the concentration of Aβ_25–35.

Nicotine attenuates Aβ (Aβ_25–35, Aβ_1–40 or Aβ_40–1)-induced increase in caspase-3 activity

Aβ_25–35- or Aβ_1–40-induced decrease in viability of hippocampal cells may be caused by induction of apoptosis. To clarify the interaction of nicotine with the intracellular downstream signaling cascade of Aβ_25–35 and Aβ_1–40, the activity of caspase-3, which was an enzyme involved in the executive phase of apoptosis, was measured in hippocampal cultures exposed to Aβ (Aβ_25–35, Aβ_1–40 or Aβ_40–1) and nicotine. As shown in Figure 3, treatment with 50 μM Aβ_25–35 or 20 μM Aβ_1–40 for 24 h markedly increased caspase-3 activity. However, treatment with 10 μM nicotine decreased the Aβ_25–35 and Aβ_1–40-induced elevation of caspase-3 activity to the control levels. Aβ_40–1 has no effect on the caspase-3 activity in the presence or absence of nicotine. Treatment with nicotine alone did not affect caspase-3 activity.

Figure 3.

Inhibitory effect of nicotine on Aβ (Aβ_25–35, Aβ_1–40 or Aβ_40–1)- induced caspase-3 activation. Hippocampal neuronal cells were treated with the indicated concentrations of Aβ (Aβ_25–35, Aβ_1–40 or Aβ_40–1) for 24 h in the presence or absence of nicotine. Each data point (±s.e.m.; bars) is the mean of four independent trials. *P<0.05 compared with control (no nicotine and no Aβ (Aβ_25–35, Aβ_1–40 or Aβ_40–1) treated) denotes a statistically significant difference. A1–40: the concentration of Aβ_1–40; A40–1: the concentration of Aβ_40–1; A25–35: the concentration of Aβ_25–35.

Nicotine attenuates Aβ (Aβ_25–35, Aβ_1–40 or Aβ_40–1)-induced increase of ROS level

Some researchers suggested the involvement of oxidative stress in the pathogenesis of hippocampal neuronal cell death in AD (Miranda et al., 2000a, 2000b). By using ROS fluorescent dye, DCF, it was found that exposure of culture hippocampal cells to 50 μM Aβ_25–35 or 20 μM Aβ_1–40 for 24 h resulted in a highly significant 100% increase in DCF fluorescence in the cells (Figure 4). The increase in DCF fluorescence in culture cells was essentially eliminated by cotreatment with nicotine (10 μM). Aβ_25–35-induced concentration-dependent ROS increase and Aβ_1–40 (20 μM)-induced ROS increase was almost completely inhibited by 10 μM nicotine. Aβ_40–1 has no effect on the generation of ROS in the presence or absence of nicotine. It can be found that 10 μM nicotine has no effect on the generation of ROS in the cells.

Figure 4.

Nicotine attenuates Aβ (Aβ_25–35, Aβ_1–40 or Aβ_40–1)-induced accumulation of ROS. Culture cells were exposed to nicotine or Aβ (Aβ_25–35, Aβ_1–40 or Aβ_40–1) for 24 h, as indicated concentration. DCF-DA was then added to a final concentration of 2 μg ml⁻¹ and DCF fluorescence was determined 40 min later. Each data point (±s.e.m.; bars) is the mean of five independent trials. *P<0.05 compared with control (no nicotine and no Aβ (Aβ_25–35, Aβ_1–40 or Aβ_40–1) treated) denotes a statistically significant difference. A1–40: the concentration of Aβ_1–40; A40-1: the concentration of Aβ_40–1; A25–35: the concentration of Aβ_25–35.

Nicotine attenuates Aβ (Aβ_25–35, Aβ_1–40 or Aβ_40–1)-induced elevation of neuronal [Ca²⁺]_i

Since the mechanism of toxicity of aged Aβ_25–35 is mediated, in part, by elevations of [Ca²⁺]_i (Mattson et al., 1993; Brorson et al., 1995), we determined whether nicotine affected Aβ (Aβ_25–35, Aβ_1–40 or Aβ_40–1)-induced increase of [Ca²⁺]_i. (Figure 5). Exposure of hippocampal neuronal cells to Aβ_25–35 for 24 h resulted in an about four-fold elevation of [Ca²⁺]_i. Similarly, Aβ_1–40 also resulted in an about 3.5-fold elevation of [Ca²⁺]_i. Aβ_40–1 has no effect on the generation of ROS in the presence or absence of nicotine. Nicotine alone had little effect on [Ca²⁺]_i; however, the [Ca²⁺]_i in hippocampal neuronal cells cotreated with nicotine (10 μM) plus 50 μM Aβ_25–35 or 20 μM Aβ_1–40 was significantly less than that in hippocampal neuronal cells treated with 50 μM Aβ_25–35- or 20 μM Aβ_1–40 alone. Thus, nicotine attenuated Aβ_25–35 or Aβ_1–40-induced elevation of free [Ca²⁺]_i.

Figure 5.

Nicotine attenuates Aβ (Aβ_25–35, Aβ_1–40 or Aβ_40–1)-induced elevation of neuronal [Ca²⁺]_i. Cultured cells were exposed to Aβ (Aβ_25–35, Aβ_1–40 or Aβ_40–1) and (or) nicotine and [Ca²⁺]_i was measured 24 h later. Each data point (±s.e.m.; bars) is the mean of five independent trials.*P<0.05 compared with control (no nicotine and no Aβ (Aβ_25–35, Aβ_1–40 or Aβ_40–1) treated) denotes a statistically significant difference. A1–40: the concentration of Aβ_1–40; A40-1: the concentration of Aβ_40–1; A25–35: the concentration of Aβ_25–35.

Effects of mecamylamine on nicotine-induced protection against Aβ-induced caspase-3 activation

To investigate whether nicotine-induced neuroprotection against Aβ-induced caspase-3 activation is due to a specific effect mediated by nicotinic receptors, the effects of the cholinergic antagonist mecamylamine were examined. The concentration of mecamylamine used was same as previous reports (Kihara et al., 1997; Zamani et al., 1997). As shown in Figure 6, treatment with 10 μM nicotine for 24 h significantly decreased the Aβ_25–35- or Aβ_1–40-induced elevation of caspase-3 activity. Aβ_40–1 had no effect on the caspase-3 activity in the presence or absence of nicotine. However, mecamylamine significantly antagonized nicotine-induced protection against Aβ-induced caspase-3 activation. Mecamylamine alone had no effect on Aβ-induced caspase-3 activation.

Figure 6.

Effects of mecamylamine (cholinergic antagonist) on nicotine-induced protection against Aβ-induced caspase-3 activation. Nicotine was added to the medium containing Aβ (Aβ_25–35, Aβ_1–40 or Aβ_40–1). Mecamylamine was added simultaneously with nicotine and (or) Aβ (Aβ_25–35, Aβ_1–40 or Aβ_40–1) and caspase-3 activity was measured 24 h later. Each data point (±s.e.m.; bars) is the mean of five independent trials.*P<0.05 compared with control (no nicotine and no Aβ (Aβ_25–35, Aβ_1–40 or Aβ_40–1) treated) denotes a statistically significant difference. A1–40: the concentration of Aβ_1–40; A40–1: the concentration of Aβ_40–1; A25–35: the concentration of Aβ_25–35.

Effects of mecamylamine on nicotine-induced protection against Aβ-induced accumulation of ROS

To investigate whether nicotine-induced neuroprotection against Aβ-induced accumulation of ROS is due to a specific effect mediated by nicotinic receptors, the effects of the cholinergic antagonist mecamylamine were examined. As shown in Figure 7, treatment with 10 μM nicotine for 24 h significantly decreased the Aβ_25–35- or Aβ_1–40-induced accumulation of ROS. Aβ_40–1 had no effect on the accumulation of ROS in the presence or absence of nicotine. However, mecamylamine significantly antagonized nicotine-induced protection against Aβ-induced accumulation of ROS. Mecamylamine alone had no effect on Aβ-induced accumulation of ROS.

Figure 7.

Effects of mecamylamine (cholinergic antagonist) on nicotine-induced protection against Aβ-induced accumulation of ROS. Nicotine was added to the medium containing Aβ (Aβ_25–35, Aβ_1–40 or Aβ_40–1). Mecamylamine was added simultaneously with nicotine and (or) Aβ (Aβ_25–35, Aβ_1–40 or Aβ_40–1) and caspase-3 activity was measured 24 h later. Each data point (±s.e.m.; bars) is the mean of five independent trials. *P<0.05 compared with control (no nicotine and no Aβ (Aβ_25–35, Aβ_1–40 or Aβ_40–1) treated) denotes a statistically significant difference. A1–40: the concentration of Aβ_1–40; A40–1: the concentration of Aβ_40–1; A25–35: the concentration of Aβ_25–35.

Discussion

Multiple lines of evidence have implicated that Aβ is neurotoxic and neuronal cells enter the death machinery via an apoptotic process. The neurotoxicity of Aβ has been reported to be mediated with ROS and attenuated by antioxidants and free radical scavengers (Behl et al., 1992; Cribbs et al., 1997; Pike et al., 1997; Daniels et al., 1998; Miranda et al., 2000a, 2000b). The present study shows the neuroprotective effect of nicotine against Aβ-induced hippocampal neuronal apoptosis. Nicotine has been demonstrated to display a potent antioxidant property (Linert et al., 1999). There are several studies of neuroprotective effect of nicotine against neuronal injury in vitro and in vivo. There has been extensive evidence indicating that nicotine modulates the neurotoxic effect of Aβ (Zamani et al., 1997; Linert et al., 1999). However, there is no previous report on the protective effects of nicotine against Aβ-induced neuronal injury in hippocampal cultures. This study is the first to report the protective effects of nicotine against hippocampal neuronal apoptosis induced by Aβ. In the present study, nicotine significantly reduced hippocampal neuronal cell death induced by Aβ in comparison with its vehicle (Figure 1).

In normal development and tissue homeostasis, most of the cells die through physiological or programmed cell death to remove excessive or damaged cells (Vaux et al., 1999). Imbalance in apoptosis leads to many pathological states including cancers and neurodegenerative disorders. DNA fragmentation, the cleavage of chromosomal DNA into oliogonuclesome-sized fragments, is a very important hallmark in apoptosis. Also, we use cell death Elisa assay, detection of DNA fragmentation, to quantitate apoptosis. As shown in Figure 2, Aβ_25–35 or Aβ_1–40 treatment induces apoptosis in hippocampal cultures and nicotine effectively inhibits it. As to the intracellular death effectors, the most important family in the process of apoptosis is caspase (Pettmann et al., 1998). Caspase activation plays a critical role in the apoptosis of neurons (Marin et al., 2000; Masumura et al., 2000). In this study, we found that caspase activity was activated in hippocampal neurons by Aβ treatment (Figure 3). Therefore, caspases might be one of the main effector proteins in Aβ-induced neuronal cell death. Interestingly, nicotine inhibited the increase of caspase activity induced by Aβ_25–35 and Aβ_1–40. These results suggest that nicotine can attenuate Aβ-induced apoptosis in neuronal cells through caspase 3.

Previous studies showed that aggregated Aβ accumulates at the plasma membrane of cultured neurons (Mattson et al., 1993) and that free radical peptides are produced during the process of Aβ aggregation (Hensley et al., 1994). Furthermore, Aβ-derived radicals damage cellular enzymes (Hensley et al., 1994) and induce the oxidation of membrane component, which may be responsible for the disruption of Ca²⁺ homeostasis caused by Aβ (Cribbs et al., 1997; Pike et al., 1997; Miranda et al., 2000a, 2000b). Marked elevation in calcium triggers the activation of different degradative processes, such as ROS formation (Kihara et al., 1997; Janus et al., 2000), impaired energy production (Janus et al., 2000), and activation of several hydrolytic enzymes (Castillo et al., 1998; Kruman et al., 1999). Increased intracellular Ca²⁺ stimulates phospholipase A₂ activity and increases the levels of arachidonic acid, which lead to increased free radical production (Bonventre, 1992; Verity, 1993). Also, it was reported that oxidative stress caused rising Ca²⁺ concentrations in the cytoplasm and the nucleus. Ca²⁺ activates calmodulin binding to calcineurin and enhances calcineurin's phosphatase activity. Calcineurin causes cytochrome c release and caspase -3 activation, leading to apoptosis (Ermakv et al., 2001). It suggests that ROS mediates Aβ-induced toxicity. Nicotine increases intracellular calcium (Barrantes et al., 1995), but from Figure 5 it can be found that this increase of calcium was much smaller than Aβ_25–35- and Aβ_1–40-induced increase, and did not induce the apoptosis of hippocampal neuronal cells. When nicotine and Aβ_25–35 or Aβ_1–40 were both added to the culture, calcium level was much smaller than that added Aβ_25–35 alone (Figure 5). The antiamyloidogenic effect of nicotine may be used to explain the decrease (Zeng et al., 2001). We found that intracellular ROS and Ca²⁺ levels increase when Aβ_25–35 or Aβ_1–40 are added to the culture. Also, our present studies demonstrate that nicotine stabilized Ca²⁺ homeostasis and decrease ROS level in hippocampal neuronal cultures exposed to Aβ (Figures 4, 5), which might contribute to neuroprotection. These data implicate that keeping Ca²⁺ homeostasis and ROS level may be responsible for the inhibition of hippocampal neuronal cell apoptosis.

Many studies indicate that nicotine-induced protection against Aβ-peptide toxicity is primarily mediated by the α4β2 (Kihara et al., 1998) and α7 nicotinic receptors (Kihara et al., 2000). However, previous study also reported that cytosine (a selective α4β2 nicotinic receptor agonist) and 3-(2,4)-dimethoxybenzylidene anabaseine (DMXB, a selective α7 nicotinic receptor agonist) only partially reversed nicotine-mediated protective effects on Aβ-induced neurotoxicity in cultured neuronal cells (Kihara et al., 1997; 1998; 2000). Therefore, it appears that nicotine exerted effects on cultured neuronal cell viability can also be mediated in part by other nicotinic receptors, or even by yet undefined nonreceptor mechanisms. Recently, the antiamyloidogenic effect of nicotine is reported, that nicotine inhibits fibril Aβ formation probably by binding to a small, soluble β-sheet aggregate (Zeng et al., 2001) and nicotine can also exert its effect by the disruption of preformed fibril Aβ (Ono et al., 2002). The antiamyloidogenic effect of nicotine may be used to explain the decrease of calcium. In addition, some previous studies implied that Aβ-induced cytotoxicity is mediated by glutamate toxicity (Brorson et al., 1995; Le et al., 1995) and nicotine protects neurons form glutamate-induced cytotoxicity (Kaneko et al., 1997; Shimohama et al., 1998). The protective effect of nicotine against Aβ-induced cytotoxicity is probably mediated by its effect on glutamate toxicity (Shimohama et al., 2001). Although the precise mechanism of nicotine neuroprotection remains unclear, all of the above proposals may be involved in the neuroprotective effect of nicotine. Application of antioxidant and nicotinic agonists may be better than just one of them. As mentioned above, nicotine not only has antioxidant property (Linert et al., 1999, Liu et al., 2003), but also has agonistic property.

To further understand the effect of nicotine in caspase activation and accumulation of ROS, we used cholinergic antagonist mecamylamine to study whether nicotinic receptors are involved in protective effect of nicotine. We found that the effect of the nicotine on Aβ-mediated caspase-3 activation and accumulation of ROS is via nicotinic receptors. But mecamylamine (even in higher concentration, data unshown) did not completely inhibit nicotine-induced protection against Aβ-induced caspase-3 activation and accumulation of ROS. As mentioned above, the protection of nicotine is partly via nicotinic receptors.

Increasing data for understanding Aβ-driven pathogenesis come from transgenic mice models for AD in which transgenes for human APP provide elevated brain levels of Aβ. Multiple strains show specific AD-like neurological deficits and strongly support a role for Aβ in the pathogenesis in AD (Hsiao et al., 1996; Janus et al., 2000). The present data indicate that nicotine can protect hippocampal neurons against Aβ toxicity and suggest that nicotine may be beneficial in retarding the neurodegenerative diseases such as AD.

Abbreviations

Aβ

amyloid β-peptide

AD

Alzheimer's disease

AFC

fluorochrome 7-amino-4-trifluoromethyl coumarin

β-APP

β-amyloid precursor protein

CDDE

The cell death detection ELISA^plus

DCF-DA

2,7-dichlorofluorescein diacetate

DMEM

Dulbecco's modified Eagle's medium

EDTA

ethylenediaminotetraacetic acid

FBS

fetal bovine serum

fluo-3 AM

fluo-3 acetoxymethyl ester

HBSS

Hanks balanced salt solution

MTT

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

PD

Parkinson's disease

ROS

reactive oxygen species