Advertisement

α7 Nicotinic Receptor Transduces Signals to Phosphatidylinositol 3-Kinase to Block A β-Amyloid-induced Neurotoxicity*

Open AccessPublished:April 27, 2001DOI:https://doi.org/10.1074/jbc.M008035200
      Multiple lines of evidence, from molecular and cellular to epidemiological, have implicated nicotinic transmission in the pathogenesis of Alzheimer's disease (AD). Here we show the signal transduction mechanism involved in nicotinic receptor-mediated protection against β-amyloid-enhanced glutamate neurotoxicity. Nicotine-induced protection was suppressed by an α7 nicotinic receptor antagonist (α-bungarotoxin), a phosphatidylinositol 3-kinase (PI3K) inhibitor (LY294002 and wortmannin), and a Src inhibitor (PP2). Levels of phosphorylated Akt, an effector of PI3K, and Bcl-2 were increased by nicotine. The α7 nicotinic receptor was physically associated with the PI3K p85 subunit and Fyn. These findings indicate that the α7 nicotinic receptor transduces signals to PI3K in a cascade, which ultimately contributes to a neuroprotective effect. This might form the basis of a new treatment for AD.
      Alzheimer's disease (AD)1 is one of the most common diseases presenting dementia. There are no definitive treatments or prophylactic agents for this neurodegenerative disease. AD is characterized by the presence of two types of abnormal deposit, senile plaques and neurofibrillary tangles, and by extensive neuronal loss (
      • Giannakopoulos P.
      • Hof P.R.
      • Kovari E.
      • Vallet P.G.
      • Herrmann F.R.
      • Bouras C.
      ). β-Amyloid (Aβ) is a major constituent of senile plaques and one of the candidates for the cause of the neurodegeneration found in AD, because a negative correlation was found between senile plaques and neuron density (
      • Giannakopoulos P.
      • Hof P.R.
      • Kovari E.
      • Vallet P.G.
      • Herrmann F.R.
      • Bouras C.
      ). It has been hypothesized that accumulation of Aβ precedes other pathological changes and causes neurodegeneration or neuronal death in vivo (
      • Yankner B.A.
      • Duffy L.K.
      • Kirschner D.A.
      ). Several mutations of the Aβ precursor protein are found in familial AD, and these mutations are involved in amyloidogenesis (
      • Citron M.
      • Oltersdorf T.
      • Haass C.
      • McConlogue L.
      • Hung A.Y.
      • Seubert P.
      • Vigo P.C.
      • Lieberburg I.
      • Selkoe D.J.
      ). It has also been shown that familial AD mutations of presenilin 1 enhance the generation of Aβ 1–42 (
      • Tomita T.
      • Maruyama K.
      • Saido T.C.
      • Kume H.
      • Shinozaki K.
      • Tokuhiro S.
      • Capell A.
      • Walter J.
      • Grunberg J.
      • Haass C.
      • Iwatsubo T.
      • Obata K.
      ). However, presenilin 1 transgenic mice do not have amyloid plaques in their brains, possibly because presenilin 1 mutations facilitate apoptotic neuronal death without plaque formation (
      • Guo Q.
      • Fu W.
      • Sopher B.L.
      • Miller M.W.
      • Ware C.B.
      • Martin G.M.
      • Mattson M.P.
      ). In addition, it is controversial whether Aβ is directly toxic to neurons or not.
      We have found that Aβ 25–35 is toxic to neurons and that this cytotoxicity is inhibited by MK801, anN-methyl-d-aspartate receptor antagonist.
      T. Kihara, S. Shimohama, H. Sawada, K. Honda, T. Nakamizo, H. Shibasaki, T. Kume, and A. Akaike, unpublished data.
      2T. Kihara, S. Shimohama, H. Sawada, K. Honda, T. Nakamizo, H. Shibasaki, T. Kume, and A. Akaike, unpublished data.
      Therefore, we hypothesized that Aβ might modulate or enhance glutamate-induced cytotoxicity. Glutamate, one of the excitotoxic neurotransmitters in the CNS, can cause intracellular Ca2+ influx, activation of Ca2+-dependent enzymes such as nitric oxide (NO) synthase, and production of toxic oxygen radicals leading to cell death (
      • Tamura Y.
      • Sato Y.
      • Akaike A.
      • Shiomi H.
      ). In addition, some reports have shown that Aβ causes a reduction in glutamate uptake in cultured astrocytes (
      • Harris M.E.
      • Wang Y.
      • Pedigo N.W.J.
      • Hensley K.
      • Butterfield D.A.
      • Carney J.M.
      ), indicating that Aβ-induced cytotoxicity might be mediated via glutamate cytotoxicity to some extent.
      In our previous reports, we showed that nicotinic acetylcholine receptor agonists exert a protective effect against glutamate- and Aβ-induced neurotoxicity (
      • Akaike A.
      • Tamura Y.
      • Yokota T.
      • Shimohama S.
      • Kimura J.
      ,
      • Shimohama S.
      • Akaike A.
      • Kimura J.
      ,
      • Kaneko S.
      • Maeda T.
      • Kume T.
      • Kochiyama H.
      • Akaike A.
      • Shimohama S.
      • Kimura J.
      ,
      • Kihara T.
      • Shimohama S.
      • Sawada H.
      • Kimura J.
      • Kume T.
      • Kochiyama H.
      • Maeda T.
      • Akaike A.
      ,
      • Kihara T.
      • Shimohama S.
      • Urushitani M.
      • Sawada H.
      • Kimura J.
      • Kume T.
      • Maeda T.
      • Akaike A.
      ). Recently, it has been reported that activated phosphatidylinositol 3-kinase (PI3K) and Akt kinase promote neuron survival (
      • del Peso L.
      • Gonzalez-Garcia M.
      • Page C.
      • Herrera R.
      • Nunez G.
      ). Anti-apoptotic proteins such as Bcl-2, Bcl-x, and Bad were thought to be involved in this survival system. Nicotinic receptors are ionotropic receptors, which allow Ca2+ to enter cells and function physiologically. It has been shown that the PI3K cascade is activated by tyrosine kinase or G protein-mediated signals in neuronal cells (
      • Perkinton M.S.
      • Sihra T.S.
      • Williams R.J.
      ). Conversely, there is no evidence that nicotinic receptors contain a G protein or tyrosine kinase. However, α-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA) receptors are also ionotropic receptors, and it was recently shown that a member of the Src family, Lyn, is physically associated with AMPA receptors and mediates signals to PI3K (
      • Hayashi T.
      • Umemori H.
      • Mishina M.
      • Yamamoto T.
      ). Thus, there is a possibility that ionotropic receptors such as nicotinic receptors could be associated with a tyrosine kinase such as Src.
      In the present study, we showed that, at physiological concentrations, Aβ itself is not neurotoxic but enhances the cell death induced by glutamate. The neuroprotective effect of nicotine was examined, focusing on the involvement of the PI3K cascade. In addition, we investigated whether nicotinic receptors function as metabotropic receptors through any kinase families.

      DISCUSSION

      There is still a controversy about the role of Aβ and glutamate in the pathogenesis of AD. However, amyloid accumulation is one of the earliest changes in AD pathology, and this peptide may cause neuronal death in the central nervous system (
      • Yankner B.A.
      • Duffy L.K.
      • Kirschner D.A.
      ). The precise mechanism of Aβ-induced cytotoxicity remains unknown, although various hypotheses have been suggested. Oxidative stress, or free radical generation, is one of the candidates for the cause of Aβ-induced cytotoxicity. Previous reports have shown that Aβ stimulates NO production through Ca2+ entry triggered by activatedN-methyl-d-aspartate-gated channels (
      • O'Mahony S.
      • Harkany T.
      • Rensink A.A.
      • Abraham I.
      • De Jong G.I.
      • Varga J.L.
      • Zarandi M.
      • Penke B.
      • Nyakas C.
      • Luiten P.G.
      • Leonard B.E.
      ). Other reports have suggested that Aβ inhibits glutamate uptake and causes extracellular glutamate increase (
      • Harris M.E.
      • Wang Y.
      • Pedigo N.W.J.
      • Hensley K.
      • Butterfield D.A.
      • Carney J.M.
      ). There are also some reports that have proposed that Aβ enhances the toxicity induced by excitotoxins (
      • Dornan W.A.
      • Kang D.E.
      • McCampbell A.
      • Kang E.E.
      ,
      • Morimoto K.
      • Yoshimi K.
      • Tonohiro T.
      • Yamada N.
      • Oda T.
      • Kaneko I.
      ). These reports implied that Aβ-induced cytotoxicity might be, at least in part, mediated via glutamate toxicity. The present study also indicated that Aβ enhances glutamate neurotoxicity.
      The present data show that only a combination of Aβ 1–40 and Aβ 1–42 enhanced glutamate cytotoxicity. The concentration of these peptides used in this study were almost the same level as that detected in the cerebrospinal fluid of AD patients (
      • Jensen M.
      • Schroder J.
      • Blomberg M.
      • Engvall B.
      • Pantel J.
      • Ida N.
      • Basun H.
      • Wahlund L.O.
      • Werle E.
      • Jauss M.
      • Beyreuther K.
      • Lannfelt L.
      • Hartmann T.
      ). Other fragments did not enhance the toxicity, even when administered simultaneously with Aβ 1–40 or Aβ 1–42. Therefore, the enhancing effect appears to be related to the structure of the peptides so that the combination of Aβ 1–40 and Aβ 1–42 might play a specific role in making neurons vulnerable to glutamate. The full length of both peptides appears to be necessary.
      We previously showed that nicotinic receptor stimulation protects neurons from Aβ- and glutamate-induced cell death (
      • Akaike A.
      • Tamura Y.
      • Yokota T.
      • Shimohama S.
      • Kimura J.
      ,
      • Shimohama S.
      • Akaike A.
      • Kimura J.
      ,
      • Kaneko S.
      • Maeda T.
      • Kume T.
      • Kochiyama H.
      • Akaike A.
      • Shimohama S.
      • Kimura J.
      ,
      • Kihara T.
      • Shimohama S.
      • Sawada H.
      • Kimura J.
      • Kume T.
      • Kochiyama H.
      • Maeda T.
      • Akaike A.
      ,
      • Kihara T.
      • Shimohama S.
      • Urushitani M.
      • Sawada H.
      • Kimura J.
      • Kume T.
      • Maeda T.
      • Akaike A.
      ). In the present study, we showed that nicotinic receptor stimulation, especially α7 receptor stimulation, inhibits glutamate toxicity and that PI3K-Akt signal transduction contributes to this effect. In addition, the Bcl-2 family is stimulated downstream of the PI3K-Akt cascade and works as an anti-neuronal death factor. It is proposed that PI3K-Akt activation promotes cell survival, and up-regulation of Bcl-2 is one of the major reasons for cell survival (
      • Matsuzaki H.
      • Tamatani M.
      • Mitsuda N.
      • Namikawa K.
      • Kiyama H.
      • Miyake S.
      • Tohyama M.
      ,
      • Eves E.M.
      • Xiong W.
      • Bellacosa A.
      • Kennedy S.G.
      • Tsichlis P.N.
      • Rosner M.R.
      • Hay N.
      ). Nicotinic receptor stimulation transduces these survival signals besides its role as a transmitter. The β sheet conformation of Aβ might influence its function, such as toxicity or modulation of survival signals. However, in our experiments, nicotine and nicotinic agonists did not influence the β sheet conformation (
      • Kihara T.
      • Shimohama S.
      • Akaike A.
      ). Instead, signal transduction was shown to be important for the protective effect of nicotine.
      Our hypothesis for the survival signal transduction is shown in Fig.8. It is not clear from our experiments whether other Src family members besides Fyn are associated with α7 receptors. However, a relationship between nicotinic receptors and Fyn was implicated because catecholamine release induced by nicotine is dependent upon the presence of Fyn and extracellular Ca2+, and no other Src member was detected (
      • Allen C.M.
      • Ely C.M.
      • Juaneza M.A.
      • Parsons S.J.
      ). In our preliminary data, removal of extracellular Ca2+ suppressed Akt phosphorylation induced by nicotine (data not shown). We showed that an inhibitor of Src tyrosine kinase reduced Akt phosphorylation. In addition, PI3K and Fyn are physically associated with α7 nicotinic receptors. Therefore, nicotinic receptor stimulation might phosphorylate Akt by signal transduction through Fyn to PI3K, and extracellular Ca2+ might contribute to this process.
      Figure thumbnail gr8
      Figure 8A schematic model of the neuroprotection induced by α7 nicotinic receptor stimulation.
      In the brain, nicotinic receptors include several subtypes with differing properties and functions. The abundant presence of α7 receptors in the hippocampus, neocortex, and basal ganglia (
      • Clarke P.B.
      • Schwartz R.D.
      • Paul S.M.
      • Pert C.B.
      • Pert A.
      ), in conjunction with the memory-enhancing activity of selective α7 nicotinic agonists such as DMXB (
      • Meyer E.M.
      • Tay E.T.
      • Papke R.L.
      • Meyers C.
      • Huang G.L.
      • de Fiebre C.M.
      ), suggests a significant role for α7 receptors in learning and memory. In addition, the protective action of nicotine is mediated, at least partially, through α7 receptors. Recently it was reported that Aβ 1–42 binds to α7 receptors (
      • Wang H.Y.
      • Lee D.H.
      • D'Andrea M.R.
      • Peterson P.A.
      • Shank R.P.
      • Reitz A.B.
      ), and this might inhibit α7 nicotinic receptor-dependent learning and memory. The reduction of α7 receptor activation might cause neurons vulnerable to various toxic insults such as glutamate. In our study, however, the lysate immunoprecipitated with anti-α7 antibody did not contain Aβ 1–42 (data not shown). This might be because the antibody we used was different from that used in the report (
      • Wang H.Y.
      • Lee D.H.
      • D'Andrea M.R.
      • Peterson P.A.
      • Shank R.P.
      • Reitz A.B.
      ), but we could not prove that enhancement of glutamate toxicity depends upon the reduction of α7 nicotinic receptors. In addition, Aβ 12–28, which suppresses the formation of the α7-Aβ complex (
      • Wang H.Y.
      • Lee D.H.
      • D'Andrea M.R.
      • Peterson P.A.
      • Shank R.P.
      • Reitz A.B.
      ), did not inhibit the enhanced glutamate toxicity induced by the combined exposure to of Aβ 1–40 and Aβ 1–42 (Fig. 2b). Therefore, it is unlikely that the protective effect of nicotine depends upon the displacement of α7-Aβ binding.
      Recently, it was shown that ionotropic receptors have properties similar to metabotropic receptors. AMPA receptors are physically associated with a member of the Src family, Lyn (
      • Hayashi T.
      • Umemori H.
      • Mishina M.
      • Yamamoto T.
      ). The AMPA receptor activates Lyn, which then activates MAPK. Through the Lyn-MAPK pathway, AMPA receptors generate intracellular signals and transmit them from the cell surface to the nucleus. Nicotinic receptors are known to be ionotropic receptors. The present study indicated that nicotinic receptors also have metabotropic properties, which contribute to neuronal survival. It is likely, however, that many unrecognized receptor functions still remain.
      The cholinergic system is affected in dementia-causing diseases, AD among others, and a reduction in the number of nicotinic receptors in these diseases has been reported (
      • Shimohama S.
      • Taniguchi T.
      • Fujiwara M.
      • Kameyama M.
      ,
      • Whitehouse P.J.
      • Kalaria R.N.
      ). It is of interest that down-regulation of nicotinic receptors can result in neuronal cell death or neurodegeneration (
      • Zoli M.
      • Picciotto M.R.
      • Ferrari R.
      • Cocchi D.
      • Changeux J.P.
      ). Nicotine might function not only as a cholinergic agonist but also as a neuroprotective agent. Our present study suggests that nicotinic receptor stimulation could protect neurons from Aβ-enhanced glutamate toxicity. Thus, by an early diagnosis of AD and protective therapy with nicotinic receptor stimulation, we could delay the progress of AD.

      Acknowledgments

      We thank the Taiho Pharmaceutical Co., for providing us with DMXB.

      REFERENCES

        • Giannakopoulos P.
        • Hof P.R.
        • Kovari E.
        • Vallet P.G.
        • Herrmann F.R.
        • Bouras C.
        J. Neuropathol. Exp. Neurol. 1996; 55: 1210-1220
        • Yankner B.A.
        • Duffy L.K.
        • Kirschner D.A.
        Science. 1990; 250: 279-282
        • Citron M.
        • Oltersdorf T.
        • Haass C.
        • McConlogue L.
        • Hung A.Y.
        • Seubert P.
        • Vigo P.C.
        • Lieberburg I.
        • Selkoe D.J.
        Nature. 1992; 360: 672-674
        • Tomita T.
        • Maruyama K.
        • Saido T.C.
        • Kume H.
        • Shinozaki K.
        • Tokuhiro S.
        • Capell A.
        • Walter J.
        • Grunberg J.
        • Haass C.
        • Iwatsubo T.
        • Obata K.
        Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 2025-2030
        • Guo Q.
        • Fu W.
        • Sopher B.L.
        • Miller M.W.
        • Ware C.B.
        • Martin G.M.
        • Mattson M.P.
        Nat. Med. 1999; 5: 101-106
        • Tamura Y.
        • Sato Y.
        • Akaike A.
        • Shiomi H.
        Brain Res. 1992; 592: 317-325
        • Harris M.E.
        • Wang Y.
        • Pedigo N.W.J.
        • Hensley K.
        • Butterfield D.A.
        • Carney J.M.
        J. Neurochem. 1996; 67: 277-286
        • Akaike A.
        • Tamura Y.
        • Yokota T.
        • Shimohama S.
        • Kimura J.
        Brain Res. 1994; 644: 181-187
        • Shimohama S.
        • Akaike A.
        • Kimura J.
        Ann. N. Y. Acad. Sci. 1996; 777: 356-361
        • Kaneko S.
        • Maeda T.
        • Kume T.
        • Kochiyama H.
        • Akaike A.
        • Shimohama S.
        • Kimura J.
        Brain Res. 1997; 765: 135-140
        • Kihara T.
        • Shimohama S.
        • Sawada H.
        • Kimura J.
        • Kume T.
        • Kochiyama H.
        • Maeda T.
        • Akaike A.
        Ann. Neurol. 1997; 42: 159-163
        • Kihara T.
        • Shimohama S.
        • Urushitani M.
        • Sawada H.
        • Kimura J.
        • Kume T.
        • Maeda T.
        • Akaike A.
        Brain Res. 1998; 792: 331-334
        • del Peso L.
        • Gonzalez-Garcia M.
        • Page C.
        • Herrera R.
        • Nunez G.
        Science. 1997; 278: 687-689
        • Perkinton M.S.
        • Sihra T.S.
        • Williams R.J.
        J. Neurosci. 1999; 19: 5861-5874
        • Hayashi T.
        • Umemori H.
        • Mishina M.
        • Yamamoto T.
        Nature. 1999; 397: 72-76
        • Shimohama S.
        • Ogawa N.
        • Tamura Y.
        • Akaike A.
        • Tsukahara T.
        • Iwata H.
        • Kimura J.
        Brain Res. 1993; 632: 296-302
        • Sawada H.
        • Ibi M.
        • Kihara T.
        • Urushitani M.
        • Akaike A.
        • Kimura J.
        • Shimohama S.
        Ann. Neurol. 1998; 44: 110-119
        • Kume T.
        • Kochiyama H.
        • Kaneko S.
        • Maeda T.
        • Kaneko S.
        • Akaike A.
        • Shimohama S.
        • Kihara T.
        • Kimura J.
        • Wada K.
        • Koizumi S.
        Brain Res. 1997; 756: 200-204
        • Jensen M.
        • Schroder J.
        • Blomberg M.
        • Engvall B.
        • Pantel J.
        • Ida N.
        • Basun H.
        • Wahlund L.O.
        • Werle E.
        • Jauss M.
        • Beyreuther K.
        • Lannfelt L.
        • Hartmann T.
        Ann. Neurol. 1999; 45: 504-511
        • Kihara T.
        • Shimohama S.
        • Akaike A.
        Jpn. J. Pharmacol. 1999; 79: 393-396
        • Hunter B.E.
        • de Fiebre C.M.
        • Papke R.L.
        • Kem W.R.
        • Meyer E.M.
        Neurosci. Lett. 1994; 168: 130-134
        • Zhong L.T.
        • Kane D.J.
        • Bredesen D.E.
        Mol. Brain Res. 1993; 19: 353-355
        • Matsuzaki H.
        • Tamatani M.
        • Mitsuda N.
        • Namikawa K.
        • Kiyama H.
        • Miyake S.
        • Tohyama M.
        J. Neurochem. 1999; 73: 2037-2046
        • O'Mahony S.
        • Harkany T.
        • Rensink A.A.
        • Abraham I.
        • De Jong G.I.
        • Varga J.L.
        • Zarandi M.
        • Penke B.
        • Nyakas C.
        • Luiten P.G.
        • Leonard B.E.
        Brain Res. Bull. 1998; 45: 405-411
        • Dornan W.A.
        • Kang D.E.
        • McCampbell A.
        • Kang E.E.
        Neuroreport. 1993; 5: 165-168
        • Morimoto K.
        • Yoshimi K.
        • Tonohiro T.
        • Yamada N.
        • Oda T.
        • Kaneko I.
        Neuroscience. 1998; 84: 479-487
        • Eves E.M.
        • Xiong W.
        • Bellacosa A.
        • Kennedy S.G.
        • Tsichlis P.N.
        • Rosner M.R.
        • Hay N.
        Mol. Cell. Biol. 1998; 18: 2143-2152
        • Allen C.M.
        • Ely C.M.
        • Juaneza M.A.
        • Parsons S.J.
        J. Neurosci. Res. 1996; 44: 421-429
        • Clarke P.B.
        • Schwartz R.D.
        • Paul S.M.
        • Pert C.B.
        • Pert A.
        J. Neurosci. 1985; 5: 1307-1315
        • Meyer E.M.
        • Tay E.T.
        • Papke R.L.
        • Meyers C.
        • Huang G.L.
        • de Fiebre C.M.
        Brain Res. 1997; 768: 49-56
        • Wang H.Y.
        • Lee D.H.
        • D'Andrea M.R.
        • Peterson P.A.
        • Shank R.P.
        • Reitz A.B.
        J. Biol. Chem. 2000; 275: 5626-5632
        • Shimohama S.
        • Taniguchi T.
        • Fujiwara M.
        • Kameyama M.
        J. Neurochem. 1986; 46: 288-293
        • Whitehouse P.J.
        • Kalaria R.N.
        Alzheimer Dis. Assoc. Disord. 1995; 9: 3-5
        • Zoli M.
        • Picciotto M.R.
        • Ferrari R.
        • Cocchi D.
        • Changeux J.P.
        EMBO J. 1999; 18: 1235-1244