| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
,¶
* State Key Laboratory of Brain and Cognitive Sciences, Institute of Biophysics, Academia Sinica, Beijing, Peoples Republic of China;
Salk Institute for Biological Studies, La Jolla, California, USA;
Laboratory of Nuclear Analytical Techniques, Institute of High Energy Physics, Academia Sinica;
Institute of Laboratory Animal Science, Chinese Academy of Medical Science;
|| Sychrotron Radiation Laboratory, Institute of High Energy Physics, Academia Sinica; and
¶ Graduate School of the Chinese Academy of Sciences, Beijing, Peoples Republic of China
1Correspondence: Institute of Biophysics, Academia Sinica, 15 Datun Rd., Chaoyang District, Beijing 100101, P.R. China. E-mail: zhaobl{at}sun5.ibp.ac.cn
ABSTRACT
Nicotine reduces ß-amyloidosis and has a beneficial effect against Alzheimer’s disease (AD), but the underlying mechanism is not clear. The abnormal interactions of ß-amyloid (Aß) with metal ions such as copper and zinc are implicated in the process of Aß deposition in AD brains. In the present study, we investigated the effect of nicotine on metal homeostasis in the hippocampus and cortex of APPV717I (London mutant form of APP) transgenic mice. A significant reduction in the metal contents of copper and zinc in senile plaques and neuropil is observed after nicotine treatment. The densities of copper and zinc distributions in a subfield of the hippocampus CA1 region are also reduced after nicotine treatment. We further studied the mechanism of nicotine-mediated effect on metal homeostasis by using SH-SY5Y cells overexpressing the Swedish mutant form of human APP (APPsw). Nicotine treatment decreases the intracellular copper concentration and attenuates Aß-mediated neurotoxicity facilitated by the addition of copper, and these effects are independent of the activation of nicotinic acetylcholine-receptor. These data suggest that the effect of nicotine on reducing ß-amyloidosis is partly mediated by regulating metal homeostasis.—Zhang, J., Liu, Q., Chen, Q., Liu, N.-Q., Li, F.-L., Lu, Z.-B., Qin, C., Zhu, H., Huang, Y.-Y., He, W., and Zhao, B.-L. Nicotine attenuates ß-amyloid-induced neurotoxicity by regulating metal homeostasis.
Key Words: Alzheimer’s disease • Aß • copper
ALZHEIMER’S DISEASE (AD) is the most common form of dementia in the elderly. A major hallmark of AD is neuronal degeneration associated with senile plaques involved in ß-amyloid (Aß) accumulation (1)
. Inhibition of Aß accumulation is considered to be an effective therapeutic intervention for AD treatment (2
, 3)
. The mechanism of AD pathogenesis still remains unclear. However, one of the mechanisms of Aß accumulation may be due to the disturbance in metal homeostasis in AD brains (4)
. Aß peptides are the major constituent of the amyloid core of senile plaques, which are derived from APP and are secreted into extracelluar spaces. Both APP and Aß contain a copper binding domain (CuBD; refs 5
, 6
). High concentrations of copper, zinc, and iron have been found within the amyloid deposits in AD brains (7)
. Aß peptides can be rapidly precipitated by Cu2+ under mildly acidic conditions (5)
and by zinc at low physiological (submicromolar) concentrations (8)
. An age-dependent binding between Aß peptides with excess brain metals (copper, iron and zinc) induces Aß peptides to precipitate into metal-enriched plaques (9)
.
The reaction between Aß and metals leads to the generation of reactive oxygen species (ROS), which play a key role in Aß-mediated neurotoxicity (10
, 11)
. Cu2+ serves as cofactors to Aß peptides, facilitating the process of oxidative stress. Double electrons can be transferred to oxygen (O2) to generate H2O2 when Aß binds to copper and reduces Cu2+ to Cu+ (5
, 12
, 13)
. Cu+ then reacts with H2O2 to produce hydroxyl radicals (Fenton-type reaction; refs 14
, 15
). These ROS directly induce oxidative damage in AD brains.
Nicotine inhibits amyloid formation, restrains Aß deposition (16)
, and attenuates Aß peptide-induced neurotoxicity in hippocampal neurons (17)
. Chronic nicotine treatment reduces Aß plaque burdens in AD transgenic mice. Previous evidence has shown that nicotine might exert its neuroprotective effect by interacting with Aß peptides (16
, 18
19
20)
. However, the precise mechanism is unclear. Since nicotine has shown its capacity to act as an iron chelator via its pyridine nitrogen group (21
, 22)
, nicotine may also contain the potential chelating capacity on other metals such as zinc or copper.
In the present study, we investigated the effect of nicotine on amyloidosis and its relation to metal homeostasis. The effect of nicotine on amyloidosis was investigated by using a human APPV717I transgenic mouse model. To study the mechanism of nicotine-mediated effect involved in metal homeostasis, we developed a SH-SY5Y cell line stably overexpressing the Swedish mutant form of human APP (APPsw). Aß was overproduced in the cell line but was not toxic to the cells when cultured in the absence of Cu2+. In contrast, Aß-mediated neurotoxicity was shown in the presence of Cu2+, suggesting the neurotoxicity of Aß is mediated by AßCu2+. This is a novel in vitro cell model for inducing Aß-mediated neurotoxicity in which copper acts as a stimulator for Aß when supplemented in culture medium.
MATERIALS AND METHODS
Reagents
Dulbecco’s modified Eagle’s medium (DMEM), fetal calf serum, HEPES, and 3-(4, 5-dimethylthiazol-2-yl)-2,5-diphenyltetrazoliumbromide (MTT) were purchased from Gibco BRL (Grand Island, NY). Nicotine, G418, mecamylamine, 
-Bungarotoxin (-BTX), ethidium bromide, agarose, proteinase K, and DNA marker were purchased from Sigma (St. Louis, MO). Rabbit polyclonal antibodies against APP and Aß were purchased from Cell Signaling Technology (Cell Signaling Technology, MA); antibodies against superoxide dismutase (SOD)-1, copper chaperone for SOD (CCS), and actin were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). The immunogens of the antibodies were as follows: APP, a synthetic peptide (KLH-coupled) corresponding to residues surrounding Thr668 of human APP695; Aß, a synthetic peptide (KLH-coupled) corresponding to residues at the amino terminus of human ß-amyloid peptides; SOD-1, the C terminus (17 residues) of SOD-1 of human origin; CCS, an internal region (20 residues) of CCS of human origin; and actin, the C terminus (11 residues) of actin of human origin. All other chemicals made in China were analytical grade.
APP transgenic mice, nicotine treatment, and sample preparation
APPV717I transgenic mice in the C57/BL genetic background express high levels of human APP751 containing the London (V717I) mutant form of APP, which markedly increase the generation of Aß42 (23)
. Nine-month-old mice were treated with nicotine plus sucrose (2% w/v) or sucrose alone for 5 months in their drinking water. Nicotine was gradually increased from 25 µg/ml (free base) on day 1, to 50 µg/ml on days 2–3, to 100 µg/ml on days 4–6, and to 200 µg/ml thereafter (24)
. Fresh solutions were made every second day. Forty-eight hours before death, the nicotine solution was replaced with sucrose alone.
Mice were killed by cervical dislocation, and the brains were quickly removed. One hemisphere of the brain was fixed in 4% paraformaldehyde in PBS and stored in saline buffer plus sodium azide. The other hemisphere was used for further isolation of the cerebral cortex and hippocampus. The samples were immediately frozen and stored at –80°C.
Cell cultures, transfection, and treatments
Human neuroblastoma SH-SY5Y cells were grown in DMEM supplemented with 10% heat-inactivated fetal calf serum, 100 IU/ml penicillin, and 100 µg/ml streptomycin at 37°C in humidified 5% CO2 air. Stably transfected SH-SY5Y cell lines expressing human APP and APPsw or empty vector (neo) were made by using lipofectamine 2000 (Invitrogen) and selected by G418 resistance. To investigate the effect of copper on Aß-induced neurotoxicity, 10 and 100 µM copper (Cupric Chloride) were added to cells, respectively. Twenty-four hours later, the cells were treated with or without 20 µM nicotine or 2 µM mecamylamine and 100 nM -BTX [nicotinic Ach-receptor (nAChR) antagonists] for another 24 h. Then, the cells and media were collected for further proceeding.
Aß1–42 immunohistochemistry staining
Coronal sections (50 µm) of the brain were cut, and the first section located at about –4.30 mM from bregma was selected (25)
. Ten consecutive sections were cut from every hemisphere and numbered as (A1, A2, ... , A10; B1, B2, ... , B10; ... ; J1, J2, ... , J10). These sections from each brain were selected to constitute 10 serials (A1, B1,... , J1; A2, B2,... , J2; ... ; A10, B10,... , J10). Nine serials of the sections formed nine units were stained for Aß. Senile plaques were visualized immunohistochemically by reacting with the Aß antibody (Ab) using an avidin-biotin-horseradish peroxidase kit (Vector Laboratories). Briefly, brain sections were blocked with nonfat milk and immunoreacted with Aß Ab (1:400 dilution) for 40 min at 40°C. After reacting with the biotinylated secondary Ab (horse antimouse IgG), the sections were treated with avidin-horseradish peroxidase solution for 60 min at 37°C; 3,3'-diaminobenzidine was used as chromogen. One serial selected from the nine stained serial sections and nonstained sections was mounted on polycarbonate film. The metal contents in any reagents used in the immunohistochemistry staining were also measured to test the external metal contamination.
Immunoblotting for Aß and other proteins
The cortex and hippocampus tissues were homogenized in 1:5 (w:v) ice-cold lysis buffer (50 mM Tris-Cl, 150 mM NaCl, 100 µg/ml PMSF, 1 µg/ml aprotinin, and 1%Triton X-100). SH-SY5Y cells were washed with ice-cold PBS once and lysed with lysis buffer as above. The samples were centrifuged at 12,000 g for 20 min at 4°C. The supernatant was collected and total protein levels were measured by a micro bicinchoninic acid (BCA) protein assay kit (Pierce Biotechnology).
Forty micrograms of protein were separated on SDS-polyacrylamide gels. Then, the proteins were transferred to nitrocellulose membranes. After being boiled in PBS for 5 min (only used in Aß detection), the membrane was incubated in 20 mM Tris pH 7.5, 150 mM NaCl, 0.1% Tween-20, and 5% horse serum overnight at 4°C. The membrane was incubated for 2 h with the Ab of Aß and APP in 1:200 dilution; Ab of SOD-1, CCS, and actin in 1:400 dilution followed by incubating with peroxidase-conjugated secondary Ab for 1 h with constant agitation; and then washed and reacted with the supersignal chemiluminescent substrate (Pierce Biotechnology, IL) and visualized by exposed to Kodak-XAR film. The film was digitized and analyzed by a National Institutes of Health imaging software.
Synchrotron radiation X-ray fluorescence analysis
The concentration of trace elements in brain sections and the intracellular copper in SH-SY5Y cells were measured by synchrotron radiation X-ray fluorescence analysis (SRXRF). SRXRF is characterized by its simultaneous determination of multi-elements, which is a highly sensitive microanalysis without damage of the irradiated samples. This nondestructive analytical technique is used to detect and image the distribution of metal elements (26
, 27)
. SRXRF was performed at beam line 4W1B at Beijing Synchrotron Radiation Facility. Each point was taken every 40 s. The electron beam energy in the storage ring was 2.0 GeV, with a maximum current of 100 mA. The cross-section of the beam focused by a Kirkpatrik-Baze (KB) mirror was 15 x 15 µm2 on the sample. The methods for measurement and quantification were described previously (28)
. An Al absorber-foil was used to reduce the lower energy intensity of white light. The Aß-stained brain sections and the nonstained brain sections were both used in this study.
For senile plaque and neuropil metal content measurement, Aß-stained sections were used to measure the contents of copper and zinc in senile plaques and the adjacent neuropil (27)
. The beam line was equipped with a CCD camera. The image from this camera gave visual information on the measuring points. In these Aß-stained sections (Fig. 1
), the senile plaque was visualized. Therefore, the X-ray beam can be localized on the senile plaque to get its metal concentration. We also selected some points near the senile plaque to take as neuropil to detect its metal content. Four senile plaques and four neuropil were probed from each section in the same serial.
|
For scan measurement, nonstained sections were used to test the positional distribution of metals in the subfield of hippocampal CA1 region. The scan area is shown in Results. The scan region, a matrix with 30 x 40 points, was identified also by the CCD camera image.
For single cell measurement, SH-SY5Y cells were washed with 0.1 M Tris-Cl and put on the maylar film. The cross-section of beam was localized on a single cell, and the fluorescence signal was collected. Ten cells in every treatment group in neo, APP, and APPsw cells were detected. The spectra were analyzed by AXIL program to obtain the quasiquantitative metal contents (29
, 30)
.
Assessment of cell viability
Cell viability was measured by quantitative colorimetric MTT assay. Briefly, at the indicated time after treatment, 500 µg/ml MTT (final concentration) were added into the medium and continued to incubate at 37°C for 3h. The MTT solution was removed, and the purple formazan crystal was dissolved in dimethyl sulfoxide (DMSO). The absorbance at 595 nM of each aliquot was determined using a Bio-Rad 3350 microplate reader. Cell viability was expressed as the ratio of the signal obtained from treated cultures and the signal from control cultures.
Measurement of intracellular ROS
Production of ROS including mainly hydrogen peroxide but also other ROS (31)
was monitored spectrofluorimetrically using dichlorofluorescein (DCF)-DA. Oxidation of DCF-DA by peroxides yielded the fluorescent derivative DCF. Two micromoles of DC-DA (final concentration) in N,N-dimethyl-formamide were incubated with cells for 50 min. Loaded cells were washed three times, and the fluorescence intensity of DCF was determined using a CytoFlour 4000 fluorescence spectrophotometer with the excitation wavelength at 485 nM and the emission wavelength at 535 nM.
Aggregation assay
Aggregation assay was carried out as described previously (5)
. Stock solutions were prepared by dissolving lyophilized aliquots of Aß1–40 (2.5 µM) in 150 mM NaCl and 20 mM HEPES (pH 7.4) and incubated for 30 min at 37°C with Cu2+ (Cupric Chloride; 20 µM) in the absence or presence of different concentrations of nicotine, and then the reaction mixtures were centrifuged at 10,000 g for 20 min to sediment copper-induced Aß aggregation. The soluble protein was measured in the supernatant by Micro BCA protein assay.
Conditioned medium preparation and Aß detection by a sandwich ELISA
After cells were treated with copper and nicotine, the culture medium was harvested, centrifuged, and concentrated to one-tenth of volume in the presence of protease inhibitors (10 µM leupeptin, 1 µM peptastin, and 1 mM PMSF). The conditioned media were stored at –70°C until sandwich ELISA analysis. Aß1–40 production was measured by a sensitive fluorescence-based sandwich ELISA assay using a kit from Biosource International (Camarillo, CA) according to the manufacturer’s instructions.
Reverse transcriptase-polymerase chain reaction assay
Total RNA was extracted from cells using a TRIzol reagent (Invitrogen, CA). Two microliters of total RNA was reverse transcribed into cDNA using a SuperScript first-strand synthesis system (Invitrogen, CA). The polymerase chain reaction was performed in the following conditions: 95°C for 45s, 30°C for 60s, 72°C for 45s, for 30 cycles with TaKaRa Ex Taq polymerase (Takara Japan) in MJ Research PTC-200 Peltier Thermor Cycler. Ten microliters of the PCR reaction were electrophoresed on a 2% agarose gel. The primer sets used were as follows: for APP, sense 5'-CTACCACAACTACCACTGAG-3', antisense 5'-TCATCTCCGGGGGTCTCCAG-3'; for actin, sense 5'-CATCTCTTGCTCGAA GTCCA-3', antisense 5'-ATCATGTTTGAGACCTTCAACA-3'.
Statistical analysis
Values in figures are mean ± SE. Data analysis was performed by one-way ANOVA, and P < 0.05 was considered significant.
RESULTS
Effects of nicotine on Aß plaque density, Aß peptide concentration, Aß peptide secretion, and APP expression
The density of senile plaques in the hippocampus of transgenic mice is decreased by 
60% after nicotine treatment for 5 months compared with sucrose-treated controls (Fig 1, A and B
). The density of senile plaques in sucrose control is 3.5% of the tested area, whereas it was only 1.4% in nicotine-treated mice. The Western blotting results show that nicotine reduces the concentration of Aß peptides by 38.2% in the hippocampus and 40.3% in the cortex relative to controls, respectively (Fig. 1, C and D
).
To examine the effect of nicotine on APP expression and Aß secretion, APPsw and neo cells were used. Figure 2
A shows APP expression in APPsw cells; copper treatment significantly increases APP expression in a dose-dependent manner, and nicotine treatment decreases APP expression. We also tested endogenous APP expression in neo cells and found a similar effect of copper and nicotine on APP expression (Fig. 2C
). To further investigate the mechanism of copper and nicotine effects on APP expression, we examined the mRNA concentration of APP in these cells. The data show that the effects of copper and nicotine on APP695 expression (1 major APP-spliced forms) are regulated at the transcriptional concentration (Fig. 3
). Copper treatment increases and nicotine treatment lowers the mRNA concentration of APP. We next examined Aß1–40 levels by sandwich ELISA assay, and the data are shown in Fig. 4
. Aß1–40 secretion is undetectable in the neo cells but expressed at 100 pg/ml of medium in APPsw cells. However, secreted Aß1–40 levels are in parallel to APP levels in response to copper and/or nicotine treatment in APPsw cells; 10 and 100 µM copper treatment increase Aß1–40 secretion by 20.2 and 28.4%, respectively, and the effects are reverted by 20 µM nicotine treatment.
|
|
|
Effect of nicotine on the expression levels of CCS and SOD-1
To further confirm the effect of nicotine on the copper concentration, we tested the expression of CCS and SOD-1 (copper/zinc SOD) in the brain and APPsw cells by Western blotting. Activation of SOD-1 in eukaryotic cells occurs post-translationally and is generally dependent on CCS, which inserts the catalytic copper cofactor and catalyzes the oxidation of a conserved disulfide bond that is essential for activity. Since CCS expresses at a high concentration and SOD-1 at a low concentration in response to copper deficiency, the CCS protein concentration is used as a marker for evaluating the copper status in tissues (32)
. In the presence of nicotine, the expression of CCS was increased and the expression of SOD-1 was decreased (Fig 2A
) compared with noncopper or 10 or 100 µM copper treatment in APPsw cells, respectively. Figure 2B
shows the relative levels of CCS and SOD-1 in the hippocampus and cortex of the transgenic mice. Nicotine treatment increases the expression of CCS by 25 and 67% in the hippocampus and cortex of transgenic mice, respectively, whereas the concentration of SOD-1 is lowered by 28 and 26% in the hippocampus and cortex, respectively.
Nicotine regulates the APP concentration independent of the activation of nAchR
Figure 2, D and E
, shows APP expression in APPsw cells treated by nAChR antagonists. Mecamylamine is one of the antagonists for most of the subunits of nAChR, while -BTX is an antagonist only for nAChR subunit 7. Figure 2A
shows that nicotine decreases the expression of APP by 57%. Nicotine reduces the levels of APP expression by 53.8 and 44% in the presence of mecamylamine and -BTX, respectively (Fig 2, D and E
), and there is no significant difference compared with the data obtained by nicotine treatment alone (P>0.05). These data suggest that the effect of nicotine on APP expression is not suppressed by nAChR antagonists.
Effect of nicotine on metal levels
Since nicotine reduces the formation of senile plaques and metals are involved in Aß precipitation, we measured the effect of nicotine on metal levels in the brain by using SRXRF analysis. The X-ray spectra collected by SRXRF and the distribution patterns of copper and zinc are shown in Fig. 5
. The copper and zinc concentrations in senile plaques and neuropil were quantified. In Fig. 5, B and C
, the data show that the levels of copper and zinc in senile plaques are higher than that in neuropil of both sucrose- and nicotine-treated groups. Furthermore, the levels of copper and zinc are significantly reduced by 10–20% in senile plaques and neuropil in nicotine-treated mice relative to sucrose-treated controls. In addition, the general distribution of copper and zinc in the subfield of hippocampal CA1 region was also examined (Fig. 5E
). Copper is enriched on the pyramidal neuron layer (arrows), while the distribution of zinc is relatively spread out. The metal content of either copper or zinc is significantly decreased after nicotine treatment and particularly in the pyramidal neuron layer (arrows).
|
We further examined the intracellular copper concentration in neo, APP, and APPsw cells, and the intracellular copper concentration in single cells is shown in Fig. 6
. The intracellular copper concentration is increased by either 10 or 100 µM copper treatment. The intracellular copper concentration in APP and APPsw cells is higher than that in neo cells. Nicotine treatment lowers the copper concentration significantly.
|
Effects of copper, nicotine, and both on cell viability
The effects of copper on cell viability in the neo, APP, and APPsw cells were measured by MTT analysis, and the data are shown in Fig. 7
. No significant difference on cell viability among the three groups is seen in the absence of copper and nicotine. However, cell viability is decreased by 11 and 19% in the presence of 10 µM copper in APP and APPsw cells, respectively, whereas no effect is seen in neo cells by the same treatment. When the copper concentration is increased to 100 µM, cell viability is decreased by 25, 38, and 55% in neo, APP, and APPsw cells (Fig 7A
). We further examined the effect of nicotine on cell viability in APPsw cells in the presence of copper (Fig 7B
). Cell viability is increased by 41.5 and 75% by 20 µM of nicotine treatment in the presence of 10 and 100 µM copper relative to controls, respectively.
|
Nicotine attenuates the formation of Aß aggregation induced by copper
Since nicotine decreases Aß deposition, we examined the effect of nicotine on Aß aggregation in vitro, and the data are shown in Fig. 8
. In the absence of copper and nicotine, the self-aggregation of Aß-peptides is only 8%. Twenty micromoles of Cu2+ significantly stimulate Aß aggregation to 71%. Both 20 and 200 µM nicotine inhibit copper-induced Aß aggregation by 20.8 and 81.9%, respectively. These data suggest that nicotine may suppress Aß aggregation induced by copper.
|
Nicotine decreases copper-exacerbated accumulation of ROS
Since oxidative stress is closely related to Aß-induced neurotoxicity and metal homeostasis and copper-induced Aß aggregation may lead to ROS generation (2
, 3)
, we examined ROS levels in neo, APP, and APPsw cells after copper and nicotine treatment by a fluorescence-mediated method, and the data are shown in Fig. 9
. There is no significant difference among neo, APP, and APPsw cells in the absence of copper and nicotine. Ten micromoles of copper increase ROS generation by 27.8% in APP cells and 58.6% in APPsw cells relative to that in neo cells, respectively. One hundred micromoles of copper increase ROS generation by 35.4 and 58.2% in APP and APPsw cells relative to that in neo cells, respectively. The production of ROS in APPsw cells is decreased by 30.3 and 32.8% in the presence of nicotine compared with 10 and 100 µM copper alone. In APP cells, the concentration of ROS is also reduced by nicotine treatment by 16.5 and 16.8% in the presence of 10 and 100 µM copper, respectively.
|
DISCUSSION
In the present study, we show that copper is closely related to Aß neurotoxicity and nicotine attenuates Aß neurotoxicity partly by regulating copper homeostasis in both APPV717I transgenic mice and APPsw overexpressed cells.
Figure 5
shows that metal levels in senile plaques of APPV717I transgenic mice are significantly higher than those in the surrounding neuropil, which is consistent with the notion that copper is involved in the accumulation of senile plaques in AD brains. In our cell model system, the copper concentration in APPsw cells is also higher than that in neo cells. Since CuBD of APP increases Cu2+ uptake (33)
, APP may retain copper and cause an increase in the intracellular copper concentration, which may play a critical role for the induction of AD pathogenesis.
In various biological systems, genes involved in copper homeostasis can be regulated by cellular copper levels (34
, 35)
. Previous studies have shown a reciprocal regulation effect between APP and metal levels. APP deficiency causes an increase in brain copper levels (36)
, whereas overexpression of APP and Aß reduces brain copper levels (37)
, which is consistent with the function of copper-binding motifs found in APP and Aß. These data suggest that APP and Aß play a role in regulating metal homeostasis. Conversely, Cu2+ causes a significant increase in the concentration of APP (38)
and copper depletion down-regulates APP expression (39)
. In the present work, the intracellular copper concentration is increased by treating cells with 10 and 100 µM copper (Fig. 6)
. The increase in the intracellular copper concentration enhances the expression of APP and the secretion of Aß peptides (Figs. 2
and 4)
. Because the RNA concentration of APP is increased by copper, the effect is regulated at the transcriptional concentration. Copper may directly affect APP transcription by interacting with the APP gene promoter, affecting the regulatory factors of the APP gene promoter, or stabilizing APP mRNAs in the mRNA processing or posttranscriptional regulation levels.
ROS production is largely catalyzed by transition metals (especially copper), and oxidative stress plays a critical role in AD pathogenesis. In the present study, the association of metal levels and Aß toxicity is demonstrated in three respects: 1) the effect on cell viability by metal alone and in the combination with APP and Aß; 2) Aß-induced neurotoxicity relevant to oxidative stress indicated by ROS production; and 3) the effect of copper in Aß aggregation. The process of Aß aggregation is accelerated by transition metals via metal-catalyzed oxidation of Aß peptides. APP rapidly binds and reduces Cu2+ to Cu+. APP-Cu+ complexes may be particularly vulnerable to the challenge of ROS (10)
. Aß is a product of sequential cleavage of APP by two proteolytic enzymes ß- and 
-secretases that cleave APP at the N and C termini of Aß, respectively (40)
. To better resemble the in vivo Aß-induced neurotoxicity, instead of directly using Aß treatments, we used a cell system overexpressing APP and APPsw. Therefore, it is important to differentiate if the toxicity of ROS is catalyzed by AßCu2+ or APPCu2+. Our data show that ROS levels are increased by copper at the low concentration (10 µM) only when APP and APPsw are expressed (Fig. 9)
. The production of ROS is correlated with cell viability (Fig. 7)
. Copper at the higher concentration (100 µM) potentiates this effect. Since most metals can have a toxic effect on cell viability, copper at the low concentration may better represent the physiological and pathological statuses. Since ROS production in APPsw cells is more than that in APP cells (Fig. 9)
and copper largely increases Aß production and aggregation, we suggest that copper greatly potentiates Aß–indcued neurotoxicity, which is largely cast by AßCu2+. Since the neurotoxicity is also seen in APP cells in the presence of copper, APPCu2+ may also attribute to some toxic effect on cell viability. Alternatively, metals may influence the APP- and Aß-mediated normal functions. However, since the viability of neo cells is not affected by copper at the low concentration, this possibility is less likely. Since copper concentration is increased by aging in the brain (37
, 41)
, the APP and Aß expression in the aged brain may cause the induction of AD pathogenesis. Therefore, our data are in parallel with increased metal levels and Aß-mediated neurotoxicity and have a profound implication in AD pathogenesis.
Previous studies have suggested several mechanisms involved in nicotine-mediated neuroprotective effects against Aß-induced neurotoxicity in vivo and in vitro. For example, nicotine may activate nAchRs-mediated functions (42)
, perform its antioxidant roles (43)
, or inhibit ß-amyloidosis through its direct interaction with the -helix structure of Aß (18)
. In the present study, we studied the effect of nicotine on metal homeostasis and its role in reducing Aß-induced neurotoxicity and suggest a novel mechanism for nicotine effects against AD pathogenesis. We observed a significant reduction in ß-amyloidosis by nicotine (Fig. 1)
, which is consistent with previous observations (16
, 17
, 20
, 44)
, and found that nicotine significantly reduces copper and zinc levels in senile plaques and neuropil in vivo. In our cell model system, the expression of APP and the generation of Aß are both reduced by nicotine and the intracellular copper concentration is also lowered by nicotine treatment. The effects of metal on ROS production, cell viability, and Aß aggregation are also reverted by nicotine treatment, suggesting that nicotine acts directly against Aß-induced neurotoxicity facilitated by metals. Our data show that nicotine may lower the copper concentration in the transgenic mice brain, and it also can be predicted that nicotine may decrease zinc concentration in the cell model system.
In the present of nicotine, the expression of CCS was increased (Fig 2A
) and the intracellular copper concentration decreased (Fig. 6)
compared with noncopper, 10 µM copper, or 100 µM copper treatment in APPsw cells, respectively, which was consistent with the data in the animal experiment. These data suggest that nicotine may act as a chelator to lower the copper concentration in brain and cell to decrease the neurotoxicity of Cu with APP and Aß. One thing that should be pointed is that the expression of CCS was also increased by copper treatment in APPsw cells; one reasonable induction is the intracellular copper concentration was still lower than the extracellular copper concentration in copper treatment condition, which may stimulate the expression of CCS.
CCS directly inserts copper into SOD-1 and prevents the accumulation of free copper ions in cells (45)
. CCS knockout mice are shown to markedly increase tissue total copper levels (46
, 47)
. In the transgenic mice brain, our data suggest that an increase of CCS expression by nicotine causes a decrease in free intracellular copper levels, since more expression of CCS transfers more free intracellular copper ions into proteins. Therefore, the copper ions that can be transported to the extracellular space are reduced, which results in an inhibition of Aß aggregation mediated by free copper ions.
To further clarify, if the effect of nicotine against the neurotoxicity induced by Aß and metals is mediated by activation of nAChRs, then we used antagonists of nAChRs in our cell model experiments. Our data show that the inhibitory effects of nicotine on ß-amyloidosis and Aß-mediated neurotoxicity in the presence of copper are not suppressed by nAChR antagonists, suggesting that nicotine may act in a receptor-independent pathway. It has been shown that maternal nicotine exposure resulted in a reduction of the copper content in a neonatal lung (48)
and that nicotine may chelate metals through pyridine nitrogen (21
, 22)
, and our theory-calculated results suggest that pyrrolidine nitrogen may also combined with metal (data not show). Indeed, nicotine reduces the levels of copper and zinc in senile plaques and neuropil (Fig. 5)
, which counteracts the morbid metal accumulation. Taken together, our data suggest that nicotine may reduce ß-amyloidosis by regulating metal homeostasis. Metal chelating agents have been considered as a potential therapeutic measure for treating AD (8)
. However, the current development has raised some major concerns for these chelating reagents for that: 1) the target metals including copper, zinc, or iron are the essential trace-elements to the human body and discharging metals may cause many side-effects; and 2) the general metal chelators such as triene or penicillamine cannot pass across the blood-brain barrier, which limit their applications. Since nicotine contains a potential metal chelating capability and now we show that this could be a novel mechanism for the inhibitory effect of nicotine against amyloidosis, nicotine may be used as an alternative chelating agent that can overcome the problems mentioned above.
In summary, copper and zinc modulate Aß aggregation and deposition, and therefore, the levels of copper and zinc are crucial to the pathogenesis of AD. We suggest that nicotine reduces ß-amyloidosis in AD transgenic mice and APPsw-overexpressed cells partly by regulating metal homeostasis since nicotine may regulate metal homeostasis through the following processes: 1
) nicotine chelates copper and zinc to decrease ROS generation and Aß aggregation; 2
) nicotine affects the copper homeostasis to down-regulate APP expression; and 3
) increased expression of CCS reduces the free copper ions. This result may help us better understand the mechanism of nicotine that reduces ß-amyloidosis.
ACKNOWLEDGMENTS
This work was supported by a grant from the National Natural Science Foundation of China (10490180; 10175077, Major Project 10490181); a grant 973 (2006CB500700) from the Department of National Science and Technology of China; and the Wang Kang-Cheng foundation. We thank Xue-Fei Wang and Yun-Feng Li for assistance on data analysis.
Received for publication September 28, 2005. Accepted for publication January 19, 2006.
REFERENCES
4 expression are modified by long-term nicotine administration. J. Neurosci. 18,4825-4832This article has been cited by other articles:
|
|
 |
|
  Q. Liu, J. Zhang, H. Zhu, C. Qin, Q. Chen, and B. Zhao Dissecting the signaling pathway of nicotine-mediated neuroprotection in a mouse Alzheimer disease model FASEB J, January 1, 2007; 21(1): 61 - 73. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
