Skip to main content
SpringerLink
Log in

Changes in cholesterol metabolism are associated with PS1 and PS2 gene regulation in SK-N-BE

  • Original Article
  • Published:
Journal of Molecular Neuroscience Aims and scope Submit manuscript

Abstract

Several lines of evidence suggest that the cholesterol content of neuronal membranes influences amyloid precursor protein (APP) processing; however, its role in transcriptional regulation of the cofactor for γ-secretase, the key enzyme for the production of the Aβ peptide, is poorly understood. This study investigates whether the changes in cellular cholesterol metabolism modulate the expression of genes involved in the γ-secretase complex function. The abundance of mRNA transcripts for presenilin 1 and 2 (PS1 and PS2), APP, and nicastrin were evaluated in neuroblastoma cells exposed either to serum-depleted medium or to low-density lipoproteins (LDL). Cholesterol esterification was markedly inhibited by mevinolin and U18666A; but was not significantly affected by any other of the tested treatments, γ-Secretase genes and cofactors were not co-regulated and were not influenced by statin inhibition of cholesterol synthesis. Nicastrin and the APP isoforms showed constitutive expression. In the absence of exogenous lipids, cell PS1 and PS2 expression was induced by LDL and by lysosomal sequestration of cholesterol. However, a different pattern of induction of presenilin gene expression was observed in the latter condition, suggesting that lysosomal cholesterol levels are strong inducers of PS2 transcription. Taken together, these results indicate that lipid metabolism has a complex influence of γ-secretase transcriptional pathways and, in particular, exogenous cholesterol and compartmentalization in neuroblastoma cells play a relevant role in regulating the transcription of presenilins, while modulation of the cholesterol biosynthesis pathway seems to exert a minor influence on the expression of γ-secretase genes and cofactors.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Similar content being viewed by others

References

  • Armitage P. and Berry G. (1987) Statistical Methods in Medical Research, Second Edition, Blackwell Scientific Publications, Oxford.

    Google Scholar 

  • Bazan N. G. and Lukiw W. J. (2002) Cyclooxygenase-2 and presenilin-1 gene expression induced by interleukin-1beta and amyloid beta 42 peptide is potentiated by hypoxia in primary human neural cells. J. Biol. Chem. 277, 30,359–30,367.

    Article  CAS  Google Scholar 

  • Bodovitz S. and Klein W. L. (1996) Cholesterol modulates alpha-secretase cleavage of amyloid precursor protein. J. Biol. Chem. 271, 4436–4440.

    Article  PubMed  CAS  Google Scholar 

  • Bradford M. (1976) A sensitive method for quantitation of microgram quantities of protein utilizing the principle of dye-binding. Anal. Biochem. 72, 248–252.

    Article  PubMed  CAS  Google Scholar 

  • Brown M. S., Faust J. R., and Goldstein J. L. (1975) Role of the low density lipoprotein receptor in regulating the content of free and esterified cholesterol in human fibroblasts. J. Clin. Invest. 55, 783–793.

    Article  PubMed  CAS  Google Scholar 

  • Brown M. S. and Goldstein J. L. (1983) Lipoprotein metabolism in the macrophages: implications for cholesterol deposition in atherosclerosis. Ann. Rev. Biochem. 52, 223–261.

    Article  PubMed  CAS  Google Scholar 

  • Confaloni A. M., Terreni L., Piscopo P., et al., (2003) Nicastrin gene in familial and sporadic Alzheimer's disease. Neurosci. Lett. 353, 61–65.

    Article  PubMed  CAS  Google Scholar 

  • De Strooper B., Beullens M., Contreras B., et al. (1997) Phosphorylation, subcellular localization, and membrane orientation of the Alzheimer's disease-associated presenilins. J. Biol. Chem. 272, 3590–3598.

    Article  PubMed  Google Scholar 

  • Dehouck B., Fenart L., Dehouck M. P., Pierce A., Torpier, G., and Cecchelli R. (1997) A new function for the LDL receptor: transcytosis of LDL across the blood-brain barrier. J. Cell Biol. 138, 877–889.

    Article  PubMed  CAS  Google Scholar 

  • Dermaut B., Theuns J., Sleegers K., et al. (2002) The gene encoding nicastrin, a major gamma-secretase component, modifies risk for familial early-onset Alzheimer disease in a Dutch population-based sample. Am. J. Hum. Genet. 70, 1568–1574.

    Article  PubMed  CAS  Google Scholar 

  • Dietschy J. M. and Turley S. D. (2001) Cholesterol metabolism in the brain. Curr. Opin. Lipidol. 12, 105–112.

    Article  PubMed  CAS  Google Scholar 

  • Endermann G., Stanton L. W., Madden K. S., Bryant C. M., White, R. T., and Protter, A. A. (1993) CD36 is a receptor for oxidized low density lipoprotein. J. Biol. Chem. 268, 11,811–11,816.

    Google Scholar 

  • Esler W. P. and Wolfe M. S. (2001) A portrait of Alzheimer secretases-new features and familiar faces. Science 293, 1449–1454.

    Article  PubMed  CAS  Google Scholar 

  • Fassbender K., Simons M., Bergmann C., et al. (2001) Simvastatin strongly reduces levels of Alzheimer's disease beta-amyloid peptides Abeta 42 and Abeta 40 in vitro and in vivo. Proc. Natl. Acad. Sci. U. S. A. 98, 5856–5861.

    Article  PubMed  CAS  Google Scholar 

  • Fernandes M. A., Proenca M. T., Nogueira A. J., et al. (1999) Effects of apolipoprotein E genotype on blood lipid composition and membrane platelet fluidity in Alzheimer's disease. Biochim. Biophys. Acta 1454, 89–96.

    PubMed  CAS  Google Scholar 

  • Francis R., McGrath G., Zhang J., et al. (2002) Aph-1 and pen-2 are required for Notch pathway signaling, gamma-secretase cleavage of betaAPP and presenilin protein accumulation. Dev. Cell 3, 85–97.

    Article  PubMed  CAS  Google Scholar 

  • Frears E. R., Stephens D. J., Walters C. E., Davies H., and Austen B. M. (1999) The role of cholesterol in the biosynthesis of beta-amyloid. Neuroreport 10, 1699–1705.

    Article  PubMed  CAS  Google Scholar 

  • Fukuyama R., Chandrasekaran K., and Rapoport S. I. (1993) Nerve growth factor-induced neuronal differentiation of the amyloid precursor protein (APP) in PC12 cells and variant PC12S cells. Brain Res. Mol. Brain Res. 17, 17–22.

    Article  PubMed  CAS  Google Scholar 

  • Galbete J. L., Martin T. R., Peressini E., Modena P., Bianchi R., and Forloni G. (2000) Cholesterol decreases secretion of the secreted form of amyloid precursor protein by interfering with glycosylation in the protein secretory pathway. Biochem. J. 348, 307–313.

    Article  PubMed  CAS  Google Scholar 

  • Goldstein J. L. and Brown M. S. (1977) The low density lipoprotein pathway and its relation to atherosclerosis. Ann. Rev. Biochem. 46, 897–930.

    Article  PubMed  CAS  Google Scholar 

  • Goparlraj R. K., Zhu H., Kelly J. F., et al. (2005) Genetic association of low density lipoprotein receptor and Alzheimer's disease. Neurobiol. Aging 26, 1–7.

    Article  CAS  Google Scholar 

  • Goutte C., Tsunozaki M., Hale V. A., and Priess J. R. (2002) APH-1 is a multipass membrane protein essential for the Notch signaling pathway in Caenorhabditis elegans embryos. Proc. Natl. Acad. Sci. U. S. A. 99, 775–779.

    Article  PubMed  CAS  Google Scholar 

  • Grundy S. M. amd Bilheimer D. W. (1984) Inhibition of 3-hydroxy-3-methyglutaryl-CoA reductase by mevinolin in familial hypercholesterolemia heterozygotes: effects on cholesterol balance. Proc. Natl. Acad. Sci. U. S. A. 81, 2538–2542.

    Article  PubMed  CAS  Google Scholar 

  • Higgins M. E., Davies J. P., Chen F. W., and Ioannou Y. A. (1999) Niemann-Pick C1 is a late endosome-resident protein that transiently associates with lysosomes and the trans-Golgi network. Mol. Genet. Metab. 68, 1–13.

    Article  PubMed  CAS  Google Scholar 

  • Hiramatsu K. H., Rosen H., Heinecke J. W., Wolbfbouer G., and Chait A. (1987) Superoxide initiates oxidation of low density lipoprotein by human monocytes. Arteriosclerosis 7, 55–60.

    PubMed  CAS  Google Scholar 

  • Hofman A., Ott A., Breteler M. M., et al. (1997) Atherosclerosis, apoliprotein E, and prevalence of dementia and Alzheimer's disease in the Rotterdam study. Lancet 349, 151–154.

    Article  PubMed  CAS  Google Scholar 

  • Howland D. S., Trusko S. P., Savage M. J., et al. (1998) Modulation of secreted-amyloid precursor protein and amyloid beta-peptide in brain by cholesterol. J. Biol. Chem. 273, 16,576–16,582.

    Article  CAS  Google Scholar 

  • Hu Y. and Fortini M. E. (2003) Different cofactor activities in γ-secretase assembly: Evidence for a nicastrin-Aph-1 subcomplex. J. Cell Biol. 161, 685–690.

    Article  PubMed  CAS  Google Scholar 

  • Hung A. Y., Koo E. H., Haass C., and Selkoe D. J. (1992) Increased expression of beta-amyloid precursor protein during neuronal differentiation is not accompanied by secretory cleavage. Proc. Natl. Acad. Sci. U. S. A. 89, 9439–9443.

    Article  PubMed  CAS  Google Scholar 

  • Ilaya N. T., Evin G., Masters C. L., and Culvenor J. G. (2004) Nicastrin expression in mouse peripheral tissues is not co-ordinated with presenilin and is high in muscle. J. Neurochem. 91, 230–237.

    Article  PubMed  CAS  Google Scholar 

  • Iwatsubo T. (2004) The gamma-secretase complex machinery for intramembrane proteolysis. Curr. Opin. Neurobiol. 14, 379–383.

    Article  PubMed  CAS  Google Scholar 

  • Jarvik G. P., Wijsman E. M., Kukull W. A., Schellenberg G. D., Yu C., and Larson E. B. (1995) Interaction of apolipoprotein E genotype, total cholesterol level, and sex in prediction of Alzheimer disease in a case-control study. Neurology 45, 1092–1096.

    PubMed  CAS  Google Scholar 

  • Jick H., Zornberg G. L., Jick S. S., Seshadri S., and Drachman D. A. (2000) Statins and the risk of dementia. Lancet 356, 1627–1631.

    Article  PubMed  CAS  Google Scholar 

  • Jin L. W., Meezawa I., Vincent I., and Bird T. (2004) Intracellular accumulation of amyloidogenic fragments of amyloid-β precursor protein in neurons with Niemann Pick type c defects is associated with endosomal abnormalities. Am. J. Pathol. 164, 975–985.

    PubMed  CAS  Google Scholar 

  • Kang J., Lemaire H. G., Unterbeck A., et al. (1987) The precursor of Alzheimer's disease amyloid A4 protein resembles a cell-surface receptor. Nature 325, 733–736.

    Article  PubMed  CAS  Google Scholar 

  • Keller J. N., Hanni K. B., Gabbita S. P., Friebe V., Mattson M. P., and Kindy M. S. (1999) Oxidized lipoproteins increase reactive oxygen species formation in microglia and astrocyte cell lines. Brain Res. 830, 10–15.

    Article  PubMed  CAS  Google Scholar 

  • Kim H. S., Lee S. H., Kim S. S., et al. (1998) Post-ischemic changes in the expression of Alzheimer's APPisoforms in rat cerebral cortex. Neuroreport 9, 533–537.

    PubMed  CAS  Google Scholar 

  • Kitaguchi N., Takahashi Y., Tokushima Y., Shiojiri S., and Ito H. (1988) Novel precursor of Alzheimer's disease amyloid protein shows protease inhibitory activity. Nature 331, 530–532.

    Article  PubMed  CAS  Google Scholar 

  • Konig G., Masters C. L., and Beyreuther K. (1990) Retinoic acid induced differentiated neuroblastoma cells show increased expression of the beta A4 amyloid gene of Alzheimer's disease and an altered splicing pattern. FEBS Lett. 269, 305–310.

    Article  PubMed  CAS  Google Scholar 

  • Krieger M. and Herz J. (1994) Structures and functions of multiligand lipoprotein receptors: Macrophage scavenger receptors and LDL receptor-related protein (LRP). Ann. Rev. Biochem. 63, 601–637.

    PubMed  CAS  Google Scholar 

  • Kuo Y. M., Emmerling M. R., Bisgaier C. L., et al. (1998) Elevated low-density lipoprotein in Alzheimer's disease correlates with brain Abeta 1–42 levels. Biochem. Biophys. Res. Commun. 252, 711–715.

    Article  PubMed  CAS  Google Scholar 

  • Lambert J. C., Luedecking-Zimmer E., Merrot S., et al. (2003) Association of 3′-UTRpolymorphisms of the oxidised LDL receptor 1 (OLR1) gene with Alzheimer's disease. J. Med. Genet. 40, 424–430.

    Article  PubMed  CAS  Google Scholar 

  • Lange Y., Ye, J., and Steck T. L. (1998) Circulation of cholesterol between lysosomes and the plasma membrane. J. Biol. Chem. 273, 18,915–18,922.

    CAS  Google Scholar 

  • La Voie M. J., Fraering P. C., Ostaszewski B. L., et al. (2003) Assembly of the γ-secretase complex involves early formation of an intermediates subcomplex of Aph-1 and nicastrin. J. Biol. Chem. 278, 37,213–37,222.

    Google Scholar 

  • Leppala J., Kallio M., Nikula T., et al. (1995) Accumulation of99mTc-low-density lipoprotein in human malignant glioma. Br. J. Cancer 71, 383–387.

    PubMed  CAS  Google Scholar 

  • Love S., Bridges L. R., and Case C. P. (1995) Neurofibrillary tangles in Niemann-Pick disease type C. Brain 118, 119–129.

    Article  PubMed  Google Scholar 

  • Lukiw W. J., Gordon W. C., Rogaev E. I., Thompson H., and Bazan N. G. (2001) Presenilin-2 (PS2) expression up-regulation in a model of retinopathy of prematurity and pathoangiogenesis. Neuroreport 12, 53–57.

    Article  PubMed  CAS  Google Scholar 

  • Lynch C. and Mobley W. (2000) Comprehensive theory of Alzheimer's disease. The effects of cholesterol on membrane receptor trafficking. Ann. NY Acad. Sci. 924, 104–111.

    Article  PubMed  CAS  Google Scholar 

  • Makarova A., Williams S. E., and Strickland D. K. (2004) Proteases and lipoprotein receptors in Alzheimer's disease. Cell Biochem. Biophys. 41, 139–178.

    Article  PubMed  CAS  Google Scholar 

  • Mohuczy D., Qian K., and Phillips M. I. (2002) Presenilins in the heart: presenilin-2 expression is increased by low glucose and by hypoxia in cardiac cells. Regul. Pept. 110, 1–7.

    Article  PubMed  CAS  Google Scholar 

  • Nakamura K., Kennedy M. A., Baldan A., Bojanic D. D., Lyons K., and Edwards P. A. (2004) Expression and regulation of multiple murine ATP-binding cassette transporter G1 mRNAs/isoforms that stimulate cellular cholesterol efflux to high density lipoprotein. J. Biol. Chem. 279,(44), 45,980–45,989.

    Article  CAS  Google Scholar 

  • Napolitano M. and Bravo E. (2003) Activation of protein Kinase C by phorbol esters in human macrophages reduces the metabolism of modified LDL by down-regulation of scavenger receptor activity. Intern. J. Biochem. Cell. Biol. 1488, 1–17.

    Google Scholar 

  • Newcombe J., Li H., and Cuzner M. L. (1994) Low density lipoprotein uptake by macrophages in multiple sclerosis plaques: implications for pathogenesis. Neuropathol. Appl. Neurobiol. 20, 152–162.

    Article  PubMed  CAS  Google Scholar 

  • Podlisny M. B., Citron M., Amarante P., et al. (1997) Presenilin proteins undergo heterogeneous endoproteolysis between Thr291 and Ala299 and occur as stable N-and C-terminal fragments in normal and Alzheimer brain tissue. Neurobiol. Dis. 3, 325–337.

    Article  PubMed  CAS  Google Scholar 

  • Ponte P., Gonzalez-DeWhitt P., Schilling J., et al. (1988) A new A4 amyloid mRNA contains a domain homologous to serine proteinase inhibitors. Nature 331, 525–527.

    Article  PubMed  CAS  Google Scholar 

  • Posse De Chaves E. I., Vance D. E., CampenotR. B., Kiss R. S., and Vance J. E. (2000) Uptake of lipoproteins for axonal growth of sympathetic neurons. J. Biol. Chem. 275, 19,883–19,890.

    Article  CAS  Google Scholar 

  • Raffai R. L. and Weisgraber K. H. (2003) Cholesterol: from heart attacks to Alzheimer's disease. J. Lipid. Res. 44, 1423–1430.

    Article  PubMed  CAS  Google Scholar 

  • Refolo L. M., Malester B., LaFrancois J., et al. (2000) Hypercholesterolemia accelerates the Alzheimer's amyloid pathology in a transgenic mouse model. Neurobiol. Dis. 7, 321–331.

    Article  PubMed  CAS  Google Scholar 

  • Refolo L. M., Pappolla M. A., LaFrancois J., et al. (2001) A cholesterol-lowering drug reduces beta-amyloid pathology in a transgenic mouse model of Alzheimer's disease. Neurobiol. Dis. 8, 890–899.

    Article  PubMed  CAS  Google Scholar 

  • Ricceri L., Minghetti L., Moles A., et al. (2004) Cognitive and neurological deficits induced by early and prolonged basal forebrain cholinergic hypofunction in rats. Exp. Neurol. 189, 162–172.

    Article  PubMed  CAS  Google Scholar 

  • Rockwood K., Kirkland S., Hogan D. B., et al. (2002) Use of lipid-lowering agents, indication bias and the risk of dementia in community-dwelling elderly people. Arch. Neurol. 59, 223–227.

    Article  PubMed  Google Scholar 

  • Sabbagh M., Zahiri H. R., Ceimo J., et al. (2004) Is there a characteristic lipid profile in Alzheimer's disease? J. Alzheimers Dis. 6, 585–589.

    PubMed  CAS  Google Scholar 

  • Sato N., Hori O., Yamaguchi A., et al. (1999) A novel presenilin-2 splice variant in human Alzheimer's disease brain tissue. J. Neurochem. 72, 2498–2505.

    Article  PubMed  CAS  Google Scholar 

  • Satoh J. and Kuroda Y. (2001) Nicastrin, a key regulator of presenilin function, is expressed constitutively in human neural cell lines. Neuropathology 21, 115–122.

    Article  PubMed  CAS  Google Scholar 

  • Scherzer C. R., Offe K., Gearing M., et al. (2004) Loss of apolipoprotein E receptor LR11 in Alzheimer disease. Arch. Neurol. 61, 1200–1205.

    Article  PubMed  Google Scholar 

  • Schmitz G. and Orso E. (2001) Intracellular cholesterol and phospholipid trafficking: comparable mechanisms in macrophages and neuronal cells. Neurochem. Res. 26, 1045–1068.

    Article  PubMed  CAS  Google Scholar 

  • Simons M., Keller P., De Strooper B., Beyreuther K., Dotti C. G., and Simons K. (1998) Cholesterol depletion inhibits the generation of beta-amyloid in hippocampal neurons. Proc. Natl. Acad. Sci. U. S. A. 95, 6460–6464.

    Article  PubMed  CAS  Google Scholar 

  • Simons M., Keller P., Dichgans J., and Schulz J. B. (2001) Cholesterol and Alzheimer's disease: is there a link? Neurology 57, 1089–1093.

    PubMed  CAS  Google Scholar 

  • Simons M., Schwärzler F., Lütjohann D., et al. (2002) Treatment with simvastatin in normocholesterol lemic patients with Alzheimer's disease: a 26-week randomized, placebo-controlled, double-blind trial. Ann. Neurol. 52, 346–350.

    Article  PubMed  CAS  Google Scholar 

  • Smith C. J., Wion D., and Brachet P. (1991) Nerve growth factor-induced neuronal differentiation is accompanied by differential splicing of beta-amyloid precursor mRNAs in the PC12 cell line. Brain Res. Mol. Brain. Res. 10, 351–354.

    Article  PubMed  CAS  Google Scholar 

  • Smith I. F., Boyle J. P., Vaughan P. F., Pearson H. A., and Peers C. (2001) Effects of chronic hypoxia on Ca (2+) stores and capacitative Ca(2+) entry in human neuroblastoma (SH-SY5Y) cells. J. Neurochem. 79, 877–884.

    Article  PubMed  CAS  Google Scholar 

  • Sparrow S. M., Carter J. M., Ridgway N. D., Cook H. W., and Byers D. M. (1999) U18666A inhibits intracellular cholesterol transport and neurotransmitter release in human neuroblastoma cells. Neurochem. Res. 24, 69–77.

    Article  PubMed  CAS  Google Scholar 

  • Steinberg D. (1997) Low density lipoprotein oxidation and its pathobiological significance. J. Biol. Chem. 272, 20,963–20,966.

    Article  CAS  Google Scholar 

  • Sugawa M., Ikeda S., Kushima Y., Takashima Y., and Cynshi O. (1997) Oxidized low density lipoprotein caused CNS neuron cell death. Brain Res. 761, 165–172.

    Article  PubMed  CAS  Google Scholar 

  • Tanzi R. E. and Bertram L. (2001) New frontiers in Alzheimer's disease genetics. Neuron 32, 181–184.

    Article  PubMed  CAS  Google Scholar 

  • Tanzi R. E., McClatchey A. I., Lamperti E. D., Villa-Komaroff L., Gusella J. F., and Neve R. L. (1988) Protease inhibitor domain encoded by an amyloid protein precursor mRNA associated with Alzheimer's disease. Nature 331, 528–530.

    Article  PubMed  CAS  Google Scholar 

  • Thinakaran G., Borchelt D. R., Lee M. K., et al. (1996) Endoproteolysis of presenilin 1 and accumulation of processed derivatives in vivo. Neuron 17, 181–190.

    Article  PubMed  CAS  Google Scholar 

  • Ulery P. G. and Strickland D. K. (2000) LRP in Alzheimer's disease: friend or foe? J. Clin. Invest. 106, 1077–1079.

    Article  PubMed  CAS  Google Scholar 

  • Vance J. E., Hayashi H., and Karten B. (2005) Cholesterol homeostasis in neurons and glial cells. Semin. Cell. Dev. Biol. 16, 193–212.

    Article  PubMed  CAS  Google Scholar 

  • Walter J., Kaether C., Steiner H., and Haass C. (2001) The cell biology of Alzheimer's disease: uncovering the secrets of secretases. Curr. Opin. Neurobiol. 11, 585–590.

    Article  PubMed  CAS  Google Scholar 

  • Yu G., Nishimura M., Arawaka S., et al. (2000) Nicastrin modulates presenilin-mediated notch/glp-1 signal transduction and betaAPP processing. Nature 407, 48–54.

    Article  PubMed  CAS  Google Scholar 

Download references

Author information

Authors and Affiliations

  1. Department of Cellular Biology and Neuroscience, Istituto Superiore di Sanità, Rome, Italy

    A. Crestini, P. Piscopo & A. Confaloni

  2. Department of Hematology, Oncology and Molecular Medicine, Istituto Superiore di Sanità, Rome, Italy

    M. Napolitano & E. Bravo

Authors
  1. A. Crestini
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. M. Napolitano
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. P. Piscopo
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. A. Confaloni
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. E. Bravo
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to A. Confaloni.

Rights and permissions

Reprints and permissions

About this article

Cite this article

Crestini, A., Napolitano, M., Piscopo, P. et al. Changes in cholesterol metabolism are associated with PS1 and PS2 gene regulation in SK-N-BE. J Mol Neurosci 30, 311–322 (2006). https://doi.org/10.1385/JMN:30:3:311

Download citation

  • Received:

  • Accepted:

  • Issue Date:

  • DOI: https://doi.org/10.1385/JMN:30:3:311

Index Entries

Search

Navigation