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,1,1
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* State Key laboratory of Brain and Cognitive Sciences, Institute of Biophysics, Academia Sinica, Beijing, China;
Graduate School of the Chinese Academy of Sciences, Beijing, China;
Institute of Laboratory Animal Science, Chinese Academy of Medical Science, Beijing, China;
The Salk Institute for Biological Studies, La Jolla, California, USA; and
|| E-Institutes of NO and Inflammation, Shanghai University of Traditional Chinese Medicine. Shanghai, China
2Correspondence: Institute of Biophysics, Academia Sinica, 15 Datun Rd., Chaoyang District, Beijing 100101, China. E-mail: zhaobl{at}sun5.ibp.ac.cn
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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B and c-Myc by inhibiting the activation of MAP kinases (MAPKs). As a result, the activity of inducible NOS and the production of NO are down-regulated. RNA interference experiments show that the above nicotine-mediated process requires 
7 nAChR. Nicotine decreases Aß via the activation of 7nAChRs through MAPK, NF-B, and c-myc pathways. Nicotine also inhibits apoptosis and cell cycle progression in this mouse line. The dissected signaling pathway of nicotine-mediated neuroprotection in the present study provides a mechanistic basis for the potential development of drug targets for treating AD.—Liu, Q., Zhang, J., Zhu, H., Qin, C., Chen, Q., Zhao, B. Dissecting the signaling pathway of nicotine-mediated neuroprotection in a mouse Alzheimer disease model.
Key Words: AD • 7nAChRs • signal pathway
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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2% of the population in industrialized countries (http://www.alz.org). Deficits in cholinergic function contribute to the pathology of AD, affecting cognition, behavior, and activities of daily living. This is exemplified by reductions in choline acetyltransferase (ChAT) activity and acetylcholine (ACh) synthesis, which are strongly correlated with the degree of cognitive impairment in patients with AD (1
, 2)
. Pharmacological intervention directed toward these deficits is based on acetyl cholinesterase (AChE) inhibition, the enzyme responsible for degrading ACh, thereby increasing ACh levels in the brain (3)
. Although AChE therapy slows the AD memory impairments, currently available agents have limited effects on cognitive function and long-term efficacy appears modest.
Deposition of the ß-amyloid peptide (Aß), produced as a cleavage product of the amyloid precursor protein (APP), is a hallmark of AD. Nicotinic ACh receptors (nAChRs) are expressed by at least 12 subunits (2-10 and ß2-ß4) in the mammalian central nervous system (CNS). The density of these receptors appears to be selectively reduced in AD, especially in the region of deposition of Aß (4)
. Nicotine is widely recognized as a pharmacological agent that facilitates cognitive performance in human smokers as well as preclinical models using rodents or nonhuman primates (5)
. Furthermore, epidemiological studies have consistently shown that the incidence of neurodegenerative diseases such as AD and Parkinson’s disease (PD) is lower in cigarette smokers than in age-matched controls (6)
. These findings have prompted speculation that brain nicotinic receptors could be important therapeutic targets for AD. The beneficial effects of nicotine have been attributed to an up-regulation of nicotine receptors that are deficient in the AD brain, or possibly a protection from the Aß-induced neurotoxicity (7
, 8)
or inhibiting ß-amyloidosis in transgenic mice (9
, 10)
. However, the molecular and cellular mechanisms of nicotine-mediated protection are still unknown.
The presence of extracellular Aß plaques in the AD brain appears not to be merely a result of disease progression. Many research groups have provided evidence to show that Aß is centrally involved in the pathogenesis of AD via some distinct but intertwined mechanisms. According to the Aß theory, deposition of the aggregated Aß becomes the initiating factor for multiple neurotoxic pathways, which may include excitotoxicity, Ca2+ homeostatic disruption, free radical production, and neuroinflammation (11
12
13)
. Related to the neurotoxic pathway, the mitogen-activated protein kinase (MAPK) family receives much attention. The MAPK family is comprised of three well-characterized subgroups: extracellular signal-regulated protein kinases (ERKs), c-Jun N-terminal kinases (JNKs), and p38-MAPK; all are involved in mediating cellular responses to extracellular stimuli. The expression of ERKs, JNKs, and p38-MAPK has been investigated in mild to severe cases of AD and is related to the progression of the disease (14
, 15)
. Aß has been reported to induce the phosphorylation of MAPKs. For instance, using acute and organotypic hippocampal slice preparations, Dineley et al. and Chen et al. demonstrated that Aß couples to the MAPK cascade via 7 nicotinic ACh receptors (nAChR) (16
, 17)
.
There has been intense interest in the role of oxygen free radicals as a contributing factor in AD pathology (11
, 12)
. Immunohistochemical data, for example, have demonstrated many of the hallmark modifications of oxidative damage in AD brain, including the presence of proteins modified with advanced glycation end products, malondialdehyde, 8-hydroxy-deoxyguanosine, 4-hydroxynonenal, and nitrotyrosine, along with increased amounts of lipid peroxidation (12)
. Oxygen free radical-mediated stress not only leads to direct cellular injury; it may also influence neuronal integrity by triggering redox-sensitive, NF-B-mediated transcription of various proinflammatory and/or apoptosis-related genes in surrounding cells (4
, 18
, 19)
.
Neuronal loss and its antecedent reduction in synapses are believed to be the most direct cause of the clinical expression of AD. Some laboratories have documented a correlation between nerve cell loss and the appearance of cell cycle-related proteins (20
21
22
23)
; these results show that cell cycle events play a major role in the loss of neurons in AD. The c-myc proto-oncogene encodes the c-myc transcription factor, and its expression strongly correlates with cell proliferation. A key biological function of c-myc is its ability to promote cell cycle progression (24
25
26)
. In addition, expression of c-myc sensitizes cells to a wide range of proapoptotic stimuli, such as hypoxia, DNA damage, and depleted survival factors, as well as to signaling through the CD95, tumor necrosis factor (TNF), and TNF-related apoptosis-inducing ligand death receptors (27
, 28)
. Given the important role of c-myc in cell proliferation and death, the potential effect of c-myc in AD needs to be investigated.
Nicotine has been suggested to be protective against neurotoxicity in AD, but the molecular mechanism of its protection remains unclear. In this study we sought to define the molecular signaling pathway for nicotine-mediated neuroprotection in a transgenic mouse model of AD.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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7 nAChR, anti-actin, anti-CDK4, anti-cyclinD1, and anti-caspase-3 were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Aß1–42 antibody (Ab) 6E10 was purchased from Signet Labs.(Princeton, NJ, USA). PD169316, ERK activation inhibitor II, isohelenin, and c-myc inhibitor were all purchased from Calbiochem Co. (San Diego, CA, USA). MAPK activators, P38 MAPK activator anisomycin, and ERK1 activator isoproterenol were all purchased from Sigma Chem. Co. (St. Louis, MO, USA).
Nicotine treatment in transgenic mice
APPV717I transgenic mice (C57/BL) express high levels of human APP751 containing the London (V717I) mutation (29)
. Nine-month-old transgenic mice were treated with nicotine plus sucrose (2%) or sucrose alone for 5 months in drinking water. Nicotine was gradually increased from 25 µg/ml (free base) on day 1 to 50 µg/ml on days 2–3, 100 µg/ml on days 4–6, and 200 µg/ml thereafter (30)
. Mice were sacrificed by cervical dislocation after removing the blood by perfusion and brain samples were quickly removed. One cerebral hemisphere of each mouse was fixed in 4% paraformaldehyde in PBS and stored in saline with sodium azide. The remaining cerebral hemisphere was further dissected to isolate the cerebral cortex and hippocampus, respectively. The samples were immediately frozen and stored at –80°C.
Western blot analysis
Cells and brain tissues were lysed on ice in the lysis buffer (20 mM Tris-HCl, pH 7.4, 2.5 mM EDTA, 1% Triton X-100, 1% sodium deoxycholate, 0.1% SDS, 100 mM NaCl, 10 mM NaF, 1 mM Na3VO4, 1 mM Pefabloc SC, 10 mg/ml aprotinin, 10 mg/ml leupeptin, 10 mg/ml pepstatin A). Protein concentrations were determined using a bicinchoninic acid (BCA) kit (Pierce, Rockford, IL, USA). The same amount of protein from each sample was used for SDS-PAGE and immunoblotting analyses. The immunoreactive bands were visualized by enhanced chemiluminescence (ECL). The intensity of immunoreactive bands was quantified using an NIH Image tool.
Aß1–42 immunohistochemistry staining
Frozen sections of tissue were reacted with the corresponding primary antibodies and secondary antibodies. Immunoreactive staining was visualized by immunoperoxidase using an avidin-biotin complex (ABC) kit (Vector Labs, Burlingame, CA, USA) plus 3, 3-diaminobenzidine (3,3'-diaminobenzidine) and hydrogen peroxide. In some cases the immunostaining was performed using a fluorescein-labeled horse anti-mouse secondary Ab and mounted with Vectashield fluorescence mounting medium. Sections were analyzed by standard light microscopy or laser scanning confocal microscopy.
Quantitation of Aß using sandwich ELISA
Samples of mouse cortex or hippocampus were homogenized in ice-cold PBS containing 5M guanidine HCl and protease inhibitor mixture (pH 8.0) (Calbiochem). Homogenates were mixed for 3–4 h at room temperature and centrifuged at 16,000 g for 20 min at 4°C. The supernatant was diluted 10-fold in Dulbecco’s PBS (pH 7.4) containing 5% BSA and 0.03% Tween 20. After treatment with saline, specific inhibitors, and siRNA, the conditioned medium was collected and secreted Aß was measured by a sandwich ELISA as described by Xie et al. (31)
. Levels of Aß1–40 and 1–42 were analyzed by colorimetric sandwich ELISA kits, Signal SelectTM human ß amyloid 1–40 and 1–42 kit, respectively (BioSource International, Inc., Camarillo, CA, USA). The values were calculated by comparison with a standard curve of Aß 1–40 and 1–42. Briefly, 96-well plates were coated with mouse monoclonal antibody (mAb) specific to Aß40 (Ab 266) or Aß42 (Ab 21F12). After blocking, the wells were incubated overnight at 4°C with test samples of the conditioned cell culture medium and horseradish peroxidase-conjugated anti-Aß Ab was added. The plates were then developed with tetramethylbenzidine reagent and absorbance was measured at 450 nm.
siRNA preparation and transfection
RNA oligonucleotides were synthesized by Gene Pharma Co.(Beijing, China). Sequences used were 7 nAChR sense, 5'-CAACCACUCACCGUCUACUTT-3'; 7 nAChR antisense, 5'-AGUAGACGGUGAGUGGUUGCG-3'. siRNA (20 pM) was transfected into SH-SY5Y (APP695) cells plated in 6-well dishes using Lipofectimine 2000 (Invitrogen, Carlsbad, CA, USA) and cells were harvested 48 h later. The level of 7 nAChR protein was examined using an Ab that recognizes 7 nAChR (Santa Cruz).
Real-time reverse transcriptase-polymerase chain reaction (real-time RT-PCR)
Total RNA was isolated from tissues or cells using an SV Total RNA Isolation System (Promega) and subjected to DNase I digestion to remove any contamination of genomic DNA. Total RNA was dissolved in nuclease-free water and stored at –70°C. Reverse transcription was performed using a SuperScript II RNase H-reverse transcriptase (Invitrogen) and the reaction mix was subjected to quantitative real-time PCR to detect levels of the corresponding actin and nicotinic ACh receptor isoform cDNAs using the corresponding primer sets listed in Table 1
. The set of actin primers was used as an internal control for each specific gene amplification. The relative levels of expression were quantified and analyzed using MJ Research OpticonTM 2 software (MJ Research, Watertown, MA, USA). The real-time value for each sample was averaged and compared using the CT method, where the amount of target RNA (2– 
CT) was normalized to the endogenous actin reference (CT) and related to the amount of target gene in tissue cells, which was set as the calibrator at 1.0.
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Electrophoretic mobility shift assays (EMSA)
Nuclear extracts were prepared using the NE-PER nuclear extraction reagent (Pierce). Biotin end-labeled double-stranded oligonucleotides 5'-biotin-AGTTGAGGGGACTTTCCCAGGC-3' and 5'-biotin-GCCTGGGAAAGTCCCCTCAACT-3' as well as nonlabeled oligonucleotide were generated as described previously. The biotin-labeled DNA was detected by LightShift chemiluminescent EMSA kit (Pierce).
Detection of NO and NOS activity by ESR
Electron spin resonance (ESR) is a direct and specific method to detect free radicals (32)
. Briefly, the cortex and hippocampus from individual animals were homogenized in 5 volumes of ice-cold suspending buffer (100 mM NaCl, 10 mM Tris-HCl, pH 7.6, 1 mM EDTA, pH 8.0, a protease inhibitor cocktail tablet from Roche, Nutley, NJ, USA) plus the spin trapping reagent Fe2+ and diethyldithiocarbamate. After centrifugation at 20,000 g for 30 min, the supernatant was collected and the total protein content was determined with a BCA Kit (Pierce). NOS enzymatic activity was detected by ESR as the NO production per 1 mg protein in 1 min. ESR spectra were recorded in a quartz tube at room temperature with an ER-200 spectrometer (Bruker, Wiesbaden, Germany) operating at X-band with 100 kHz modulation.
Statistics
Error bars represent SEM. Asterisks indicate a significant difference from control (sucrose treatment), as determined by one-way ANOVA (P<0.05).
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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. To investigate the efficacy of nicotine on inhibiting Aß deposition, the APP (V717I) transgenic mice that overexpress pathological familial AD (flavin adenine dinucleotide) -associated APP (London mutant V717I) (29)
were treated with nicotine in drinking water for 5 months. The levels of Aß in the cortex and hippocampus were measured by immunohistochemistry and Western blot analyses. The nicotine concentration reaches 100 ng/g (30)
in the treated brain and also in transgenic mice (10)
. Nicotine clearly decreased the deposition of Aß in the hippocampus (Fig. 1
A). After nicotine treatment, both insoluble and soluble Aß (Aß1–42 and Aß1–40) in hippocampus and cortex were significantly decreased (Fig. 1B, C
). These observations were consistent with previous studies (10)
.
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Nicotine inhibits NO production, NOS activity and reduced the expression of iNOS
Since aggregation of Aß causes an increase in the expression of iNOS and NO production, which lead to an increase in the generation of oxygen and NO radicals (4
, 17)
, we asked whether nicotine played a role in this process. Using ESR analysis, NO production and NOS activity were measured and quantified in the nicotine-treated or untreated mouse brain. Figure 2
shows that nicotine causes a significant decrease in NO production and NOS activity in the brain, especially in the cortex and hippocampus (P<0.05). We next examined by real-time RT-PCR and Western blot whether the decreased NO production and NOS enzymatic activity are attributable to iNOS gene expression. Both the mRNA (Fig. 2C
) and protein (Fig. 2D
) levels of iNOS are decreased by the nicotine treatment. In contrast, no change was seen in mRNA and protein levels of endothelial NOS (eNOS) and neuronal NOS (nNOS) (data not shown).
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Nicotine inhibits the NF-B expression and activity
Aß–induced overproduction of NO and NO radicals is mediated partly by an inflammatory pathway (18)
. Since the NF-B-MAPK pathway plays an important role in the activation of microglial cells during inflammation (24
25
26)
, we studied the effect of nicotine on the expression of the p65 and p50 subunits of NF-B. Both protein (Fig. 3
A, B) and mRNA (Fig. 3C, D
) levels of p65 and p50 in the cortex and hippocampus are significantly decreased by nicotine, suggesting that nicotine affects the gene expression of p65 and p50 at least partly at the gene transcriptional stage. To further determine the effect of nicotine on the NF-B signaling pathway, we analyzed nuclear extracts from the hippocampus and cortex by EMSA. NF-B binds to a specific DNA site as shown previously (24)
, and the binding is significantly inhibited by nicotine (Fig. 3E
). To determine whether nicotine inhibits the NF-B pathway through the MAPK pathway, the P38MAPK inhibitor PD169316 and ERK activation inhibitor II were used to prevent MAPK pathway activation. PD169316 (10 µM) and ERK activation inhibitor II (100 µM) significantly decreased p65 expression in SH-SY5Y cells; after nicotine and inhibitor treatment, p65 expression was further decreased (Fig. 3F
), which is consistent with the results in transgenic mice. Our data indicate that nicotine regulates the NF-B pathway through the MAPK pathway. However, the MAPK inhibitor cannot completely prevent P65 expression, and nicotine just partially inhibits the expression of P65, so MAPK-mediated inhibition of NF-B independent of nicotine may also be involved as well as nicotine-mediated effects that are independent of MAPK and NF-B.
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Nicotine inhibits the c-Myc expression and its activity
Since c-Myc plays a major role in the transcriptional regulation of apoptosis and the cell cycle (2
, 3)
, we next examined protein expression and mRNA levels of c-Myc in nicotine or non-nicotine-treated transgenic mice. Nuclear binding activity of c-Myc was measured by EMSA. Both mRNA and protein levels of c-Myc are reduced by nicotine in the cortex and hippocampus (Fig. 4
A, B), and nicotine significantly inhibits the nuclear binding activity of c-Myc in the hippocampus (Fig. 4C
). These data suggest that c-Myc is involved in transcriptional regulation of the nicotine-mediated signal pathway in AD.
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However, the MAPK inhibitor cannot completely prevent c-myc expression, and nicotine just partially inhibits the expression of c-myc, so MAPK-mediated inhibition of NF-B independent of nicotine may be involved as well as nicotine-mediated effects that are independent of MAPK and NF-B.
To further identify whether nicotine regulates c-Myc through NF-B and MAPK pathways, PD169316, ERK activation inhibitor II and isohelenin (inhibitor of the NF-B pathway) were used to inhibit the MAPK and NF-B pathways. PD169316 (10 µM), ERK activation inhibitor II (100 µM), and isohelenin (40 µM) significantly decreased phosphorylated c-Myc (p-c-Myc) expression in SH-SY5Y cells with stably transfected with APPsw; after nicotine was added to this system, c-Myc expression was decreased much more (Fig. 4D
). These results further demonstrate nicotine’s role of down-regulation c-myc expression in transgenic mice. Our data suggest that NF-B and MAPK pathways are involved in the process.
To further demonstrate that the activation of c-myc was induced by MAPK, MAPK activators were used to activate the MAPK pathway in SH-SY5Y cells stably transfected with APPsw. The P38 MAPK activator anisomycin and the ERK1 activator isoproterenol both increased the expression of P65 and P-c-myc (Fig. 5
). After nicotine treatment, P65 and P-c-myc were both significantly decreased. After using of MAPK activators, the level of inhibition observed with MAPK activators and nicotine is similar to or even greater than that seen with nicotine alone.
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Nicotine prevents the MAPK pathway and inhibits apoptosis and cell cycle progression
The effect of nicotine on the expression of three major MAP kinases (ERK, c-Jun NH2-terminal kinase (JNK), p38 MAPK) was also studied. The levels of B-Raf, p-ERK1, and phosphorylated-p38 MAPK (p-p38 MAPK) are significantly decreased in the cortex and hippocampus of nicotine-treated mice (Fig. 6
A, C, G), where the level of JNK is not changed (data not shown). These data suggest that long-term nicotine treatment causes the alternation of the MAP kinase pathway. The mRNA levels of B-Raf, ERK1, and p38 MAPK (ß and 
isoforms) are also significantly decreased by nicotine (Fig. 6A
), suggesting that the effect of nicotine on the expression of the MAPK pathway occurs at the transcriptional stage. Taken together, our data suggest that nicotine inhibited the phosphorylation events of two distinct MAPK pathways (ERK and p38 MAPK) as well as the NF-B pathway.
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Other regulatory factors including Bcl-2, Bax, caspase-3, cyclin D1, and CDK4 are also involved in apoptosis and the cell cycle in AD brains (34
, 35)
. To ask whether nicotine affected expression of these regulatory factors in apoptosis and cell cycle control, the effect of nicotine on the expression of Bcl-2, Bax, caspase-3, cyclin D1, and CDK4 was examined. In the cortex and hippocampus of transgenic mice, mRNA and protein levels of two proapoptotic factors Bax and caspase-3 are decreased by nicotine (Fig. 6B, F, H
). Furthermore, mRNA and protein levels of cyclin D1 and CDK4 are significantly decreased by nicotine in the cortex and hippocampus (Fig. 6B, D, E
). These results suggest that nicotine regulates apoptosis and cell cycle progression in a manner that favors its neuroprotective effect. These data are consistent with our previous observation that nicotine inhibited the apoptosis caused by Aß (1–40) by regulating the activity of Bcl-2 and caspase-3 (36)
.
Nicotine prevents the MAPK, NF-B, and c-Myc pathways through activation of 7 nAChR
The above data show a transcriptional regulation of various genes by long-term nicotine treatment, suggesting that activation of the nicotinic ACh receptors may be involved. Nicotinic ACh receptors (nAChRs) are widely distributed in the cortex and hippocampus, and degeneration of acetylcholinergic neurons of the basal forebrain is one of the earliest pathological signs in AD (37)
. To study which subtype(s) of ACh receptor is involved in the neuroprotective effect in response to long-term nicotine treatment in APP (V717I) transgenic mice, we examined nicotine’s effect on the mRNA expression of various ACh receptors by real-time RT-PCR. The mRNA level of 7 subunit is largely increased (Fig. 7
A) in the hippocampus and cortex of transgenic mice treated with nicotine for 5 months relative to untreated mice. The mRNA level of 3 subunit is also increased in the hippocampus of transgenic mice treated with nicotine, and the mRNA level of the 4 subunit is decreased in the hippocampus of transgenic mice treated with nicotine. In contrast, the mRNA levels of other ACh receptor subunits are not affected as much as the 7 subunit (Fig. 7A
). These data suggest that the ACh receptor 7 subunit may be involved in the nicotine-mediated neuroprotective effect.
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To further investigate whether the 7 subunit is involved in regulating the MAPK pathway in response to nicotine treatment, a specific double-stranded RNA (small interference RNA, siRNA) was used to reduce AchR 7 subunit expression in the SH-SY5Y cell line stably transfected with APPsw. The activity of MAP kinases was then tested. The specific siRNA duplex significantly reduces expression of the 7 subunit in SH-SY5Y cells stably transfected with APPsw (Fig. 7B
). When SH-SY5Y cells stably transfected with APPsw were not transfected with siRNA, pERK1 and p-p38MAPK levels are significantly reduced by nicotine (Fig. 7D, E
). The levels of pERK1 and p-p38MAPK remain unchanged by nicotine after siRNA treatment (Fig. 7D, E
). p65 and p-c-Myc were also detected to determine whether 7 subunit was involved in the regulation of the NF-B pathway and c-Myc. Similarly, after siRNA treatment, p65 and p-c-Myc were not changed by nicotine (Fig. 7C, F
). In contrast, without siRNA treatment, p65 and p-c-Myc were reduced by nicotine (Fig. 7C, F
). The above data suggest that the 7 subunit is the major target for long-term nicotine treatment.
Nicotine decreases the Aß secretion via 7nAChRs
To determine whether Aß secretion is dependent on the 7nAChRs and MAPK/NF-B/c-myc pathways, we used the ELISA assay to quantitate the level of Aß in the medium. As shown in Fig. 8
A, B, after treatment with PD169316, p38 MAPK inhibitor, ERK activation inhibitor II, isohelenin, an inhibitor of nuclear factor kappa B or c-myc inhibitor, Aß1–40 and Aß1–42 secretion was significantly decreased in SH-SY5Y cells. The data suggest that nicotine decreased the level of Aß through the MAPK/NF-B/c-myc pathway. However, when SH-SY5Y cells stably transfected with APPsw were transfected with siRNA of 7nAChRs, Aß1–40 and Aß1–42 secretion was not decreased significantly. Similarly, nicotine did not decrease Aß1–40 and Aß1–42 secretion after cells were transfected with siRNA of 7nAChRs. Furthermore, if the cells were treated with inhibitors of MAPK, NF-B, c-myc, and siRNA of 7nAChRs, Aß1–40 and Aß1–42 secretion was not significantly different from treatment with inhibitors and siRNA plus nicotine. The data suggest that nicotine decreased Aß1–40 and Aß1–42 secretion via the 7nAChRs.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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. A mechanism involving neuronal nAChRs may therefore be linked to the reduced Aß deposition observed in the present investigation. The selective reduction in Aß deposition after chronic nicotine treatment observed in the present study supports an effect of nicotine on Aß aggregation. Consistent with these results, Nordberg et al. (10)
showed that chronic nicotine administration lowered the plaque load in APPsw transgenic mice with extensive Aß plaque pathology. In addition, in vitro nicotine has shown a direct effect on amyloid fibril formation by preventing and breaking down amyloid fibrils (39)
.
4ß2 and 7 nAChRs are two major isoforms widely expressed in the hippocampus and cortex of mammalian CNS and are involved in neurotransmission and cognition. Immunocytochemical studies of postmortem AD brains have revealed a significant reduction in the 7 subunit of the hippocampus (40)
. Yu et al. recently showed that 7 subunits are decreased in the neurons of AD brains whereas the 7 subunits are increased in astrocytes close to the amyloid plaques in AD brains (41)
. Our results show that 7 subunit mRNA expression was significantly increased after chronic nicotine treatment in the APP transgenic mouse. Moreover, binding studies show that significantly higher 125I-BTX binding was found in the cortex of nicotine-treated transgenic (APPsw) mice at 14.5 months age compared with nontransgenic mice (9)
. All these data may indicate the involvement of this receptor in the nicotine’s effect on Aß reduction.
Nicotine and other nAChRs agonists are neuroprotective in several models of neuronal death, both in vivo and in vitro (42)
. However, the intracellular steps that mediate the neuroprotective effects of nACh receptor ligands are not solved. MAPK-NF-B pathway activation plays an important role in the activation of microglial cells during inflammation (24
25
26)
, a pathological hallmark of AD. iNOS activation in response to NF-B resulted in NO production, and the footprints of excess NO production are observed in AD tissue as increased amounts of nirtrotyrosine-modified proteins (12)
. Our present studies show that nicotine prevents activation of NF-B by inhibiting the activation of MAPK. As a result, the activity of iNOS and the production of NO are down-regulated. The data suggest nicotine as a cholinergic agonist activates nAChRs, then prevents Aß-induced neurotoxicity via a novel anti-inflammatory mechanism. The mechanism may provide a new way to interfere with inflammation for therapy of AD. In addition, the antioxidant of nicotine has been observed in which nicotine scavenged the free radicals and decreased cellular reactive oxygen species (ROS) in vitro (36)
.
Normally, once a neuron leaves the ventricular zone it can never re-enter a cell cycle. If it does so, whether by force (43)
or by natural processes (44)
, it will die rather than divide. Accumulation of gene products to an extent that goes beyond that observed during regeneration and the aborted attempt of "differentiated" neurons to activate the cell cycle apparently is a critical event in the pathomechanism of AD (20
21
22
23)
. It is therefore suggested that the re-expression of developmentally regulated genes, induction of post-translational modifications, and the accumulation of gene products are due to a loss of differentiation control that normally is involved in regulating neuronal plasticity. A direct link between cell cycle reactivation and cell death is supported by observations of the neuroprotective action of overexpression of the cyclin-dependent kinase (Cdk) inhibitor p16INK4a, which locks neurons in a differentiated stage and prevents cell cycle re-entry. c-Myc plays a major role in transcriptional regulation of apoptosis and the cell cycle (2
, 3)
. A key biological function of c-myc is its ability to promote cell cycle progression (24
25
26)
. In addition, expression of c-myc sensitizes cells to a wide range of proapoptotic stimuli such as hypoxia, DNA damage, and depleted survival factors, as well as to signaling through the CD95, tumor necrosis factor (TNF), and TNF-related, apoptosis-inducing ligand death receptors (27
, 28)
. c-myc not only promotes cell cycle progression but also induces apoptosis. Colocalization of nuclear c-myc and caspase-3 in central neuroblastomas suggested c-myc expression in caspase-3 dependent apoptosis. Furthermore, c-myc expression was strongly increased in the hippocampus of AD brain (28)
. Our present results indicate that nicotine inhibits apoptosis (bax and caspase-3 down-regulation and bcl-2 up-regulation) and cell cycle progression (CDK4 and cyclin D1 down-regulation) in a manner that favors its neuroprotective effect. These data are consistent with our previous observation that nicotine inhibits the apoptosis caused by Aß 1–40 through regulating the activity of Bcl-2 and caspase-3 (36)
. The data suggest that nicotine may influence the process of neurodegeneration of AD via a precise mechanism that needs to be investigated extensively in an AD model.
Previous studies have shown a strong active, phosphorylation-dependent MAPK/ERK expression in neurons and glial cells in AD (28)
. Increased phosphorylated p38 expression has also been demonstrated in dystrophic neurites and neurons with neurofibrillary tangles in AD (45)
. MAP kinases phosphorylate c-Myc at Thr58 and Ser-62 (46)
, suggesting phosphorylation of c-Myc by MAP kinases in AD. Nicotine may facilitate either up-regulation (47)
or phosphorylation (48)
of antiapoptotic bcl-2 proteins, which suppress caspase activation. Nicotine may also inhibit activation of NF-B, which in turn binds to and mediates transcriptional activation of genes (49)
. c-myc expression was strongly increased in the hippocampus of AD brain (28)
. However, the mechanism by which c-myc is involved in AD pathology is unclear. Our results show that nicotine inhibits c-Myc expression and its activity through the MAPK and NF-B pathways in transgenic mice. The data suggest that c-Myc is involved in transcriptional regulation of the nicotine-mediated neuroprotection pathway in AD. In addition, using specific inhibitors of MAPK or NF-B pathway, we demonstrate that MAPK and NF-B pathways are both involved in regulation of c-myc. This may provide a clue in studying the role of c-myc in AD. The present data all indicate that nicotine regulates c-myc expression via a novel kind of anti-inflammation mechanism.
Recent studies have shown that Aß1–42 coimmunoprecipitates with the 7 nAChRs in samples from postmortem AD hippocampus (50)
, and Aß peptide has significant effects on a7nAChR function that lead to an impairment in presynaptic release and LTP (16)
. Because of the involvement of the 7 subunit receptor in nicotine’s effect on Aß reduction, whether nicotine-mediated MAPK pathway activation and downstream signaling pathway are dependent on the 7 subunit needs to be elucidated. Our results indicate that nicotine as a cholinergic agonist stimulates 7 subunit of nAChRs to inhibit activation of the MAPK/NF-B/c-myc pathway and results in Aß reduction (Fig. 8)
. These results also imply that the 7 subunit can be used to control the NF-B pathway, a critical step in the control of the immune response and a pharmacological target for several inflammatory disorders. Although the changes in the 3 and 4 subunits are not as great as in the 7 subunit, 3 and 4 subunits may also be involved in regulating nicotine’s effect. The potential effect of 3 and 4 subunits needs to be further elucidated. It is possible that nicotine has an anti-inflammatory effect in APPV717I mice, which may be involved in nicotine’s reduction of Aß.
However, the MAPK inhibitor cannot completely prevent NF-B and c-myc expression, and nicotine just partially inhibit the expression of MAPK, NF-B, and c-myc (Fig. 5)
, so MAPK-mediated inhibition of NF-B independent of nicotine may also be involved as well as nicotine-mediated effects that are independent of MAPK and NF-B. Nicotine can activate microglial alpha7 nicotinic ACh receptors, drive a phospholipase C/IP3 pathway, and modulate cell activation toward a neuroprotective role (51)
. It has been shown that nicotine protects cortical neurons through neuronal a7 nAChRs that transduce signals to the PI3K/Akt pathway in a cascade (52)
. Nicotine’s protection correlated with the induction of XIAP and survivin in a panel of human NSCLC cell lines, and the antiapoptotic effects of nicotine were mediated by dihydro ß-erythroidine-sensitive 3-containing nicotinic ACh receptors and required the Akt pathway (53)
.
Aß1–42 binds with exceptionally high affinity to the 7 nicotinic ACh receptor and accumulates intracellularly in neurons of AD brains (54)
. Nicotine may affect the interaction between Aß and 7 subunit and further decrease the transport of Aß into neurons. Aß binds to several cell surface proteins and initiates numerous signaling cascades. Aß-induced endocytosis of NMDA receptors involves activation of the 7 nicotinic receptor (55)
. Nicotine may also inhibit Aß-induced endocytosis of NMDA receptors. These need to be further investigated in vitro and in vivo.
The schematic drawing (Fig. 9
) shows that nicotine inhibits neurodegeneration progress pathways. It is well known that signaling transduction in the cell is a network wherein there is no single change that does not affect other proteins. Here we talked about one possible pathway by which nicotine may inhibit neurodegeneration progress via activation of 7nAChRs through MAPK/NF-B/c-myc, which is marked by a solid line. Another possible pathway in this 7nAChRs through MAPK/NF-B/c-myc is labeled by a broken line. Nicotine may also inhibit neurodegeneration through other pathways supported by our earlier results (56
, 57)
. We show here that long-term nicotine treatment activates the 7 subunit of the nAChRs-mediated pathway and inhibits accumulation and deposition of Aß in the hippocampus.
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The novel finding presented in this study provides an alternative mechanism for the action of nicotine in neuroprotection. The dissected signaling pathway of nicotine-mediated neuroprotection in our present study provides a mechanistic basis for the potential development of drug target for treating AD.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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4 but not 3 and 7 nicotinic acetylcholine receptor subunits are lost from the temporal cortex in Alzheimer’s disease. J. Neurochem. 73,1635-1640[CrossRef][Medline]
