Skip to main content
SpringerLink
Log in

Inhibition of Vesicular Monoamine Transporter-2 Activity in α-Synuclein Stably Transfected SH-SY5Y Cells

Cellular and Molecular Neurobiology Aims and scope Submit manuscript

Abstract

α-Synuclein plays a key role in the pathological neurodegeneration in Parkinson’s disease. Although its contribution to normal physiology remains elusive, the selective degeneration of α-synuclein-containing dopaminergic neurons in Parkinson’s disease may be linked to abnormal α-synuclein induced toxicity. In the present study, a complex of α-synuclein and vesicular monoamine transporter-2 was identified by GST-Pull Down experiment. In wild-type α-synuclein stably transfected SH-SY5Y cell lines, the activity of vesicular monoamine transporter-2 decreased by 31% as determined by [3H] dopamine uptake, and its expression also decreased in both protein and mRNA levels using western and northern blot analysis. Overexpression of wild-type α-synuclein did not induce cell death or apoptosis, but significantly enhanced the intracellular reactive oxygen species level as assayed by flow cytometry. These data suggest that Up-regulated α-synuclein expression inhibits the activity of vesicular monoamine transporter-2, thereby interrupting dopamine homeostasis and resulting in dopaminergic neuron injury in Parkinson’s disease.

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

Access this article

Log in via an institution

Price includes VAT (France)

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5
Fig. 6
Fig. 7

Abbreviations

PD:

Parkinson’s disease

VMAT2:

Vesicular monoamine transporter-2

ROS:

Reactive oxygen species

TH:

Tyrosine hydroxylase

DAT:

Dopamine transporter

DA:

Dopamine

PBS:

Phosphate-buffered saline

PVDF:

Polyvinylidene difluoride

MAO:

Monoamine oxidase

MPTP:

1-Methyl- 4-phenyl-1,2,3,6-tetrahydropyridine

MPP+ :

1-Methyl-4-phenylpyridinium

References

  • Alam ZI, Daniel SE, Lees AJ, Marsden CD, Jenner P, Halliwell B (1997a) A generalized increase in protein carbonyls in the brain in Parkinson’s but not incidental Lewy body disease. J Neurochem 69:1326–1329

    Article  PubMed  CAS  Google Scholar 

  • Alam ZI, Jenner A, Daniel SE, Lees AJ, Cairns N, Marsden CD,Jenner P, Halliwell B (1997b) Oxidative DNA damage in the parkinsonian brain: an apparent selective increase in 8-hydroxyguanine levels in substantia nigra. J Neurochem 69:1196–1203

    Article  PubMed  CAS  Google Scholar 

  • Baptista MJ, O’Farrell C, Daya S, Ahmad R, Miller DW, Hardy J, Farrer MJ, Cookson MR (2003) Co-ordinate transcriptional regulation of dopamine synthesis genes by alpha-synuclein in human neuroblastoma cell lines. J Neurochem 85:957–968

    Article  PubMed  CAS  Google Scholar 

  • Ben-Shachar D, Zuk R, Glinka Y (1995) Dopamine neurotoxicity: inhibition of mitochondrial respiration. J Neurochem 64:718–723

    Article  PubMed  CAS  Google Scholar 

  • Bernheimer H, Birkmayer W, Hornykiewicz O, Jellinger K, Seitelberger F (1973) Brain dopamine and the syndromes of Parkinson and Huntington: clinical, morphological, and neurochemical correlations. J NeurolSci 20:415–455

    Article  CAS  Google Scholar 

  • Chiba-Falek O, Lopez GJ, Nussbaum RL (2006) Levels of alpha-synuclein mRNA in sporadic Parkinson disease patients. Mov Disord 21:1703–1708

    Article  PubMed  Google Scholar 

  • Clayton DF, George JM (1999) Synucleins in synaptic plasticity and neurodegenerative disorders. J Neurosci Res 58:120–129

    Article  PubMed  CAS  Google Scholar 

  • Daniels AJ, Reinhard JF (1988) Energy-driven uptake of the neurotoxin 1-methyl-4-phenylpyridinium into chromaffin granules via the catecholamine transporter. J Biol Chem 263:5034–5036

    PubMed  CAS  Google Scholar 

  • Del Zompo M, Piccardi MP, Ruiu S, Quartu M, Gessa GL, Vaccari A (1993) Selective MPP+ uptake into synaptic dopamine vesicles: possible involvement in MPTP neurotoxicity. Br J Pharmacol 109:411–414

    PubMed  Google Scholar 

  • Eiden LE (2000) The vesicular neurotransmitter transporters: current perspectives and future prospects. FASEB J 14:2396–2400

    Article  PubMed  CAS  Google Scholar 

  • Floor E, Wetzel MG (1998) Increased protein oxidation in human substantia nigra pars compacta in comparison with basal ganglia and prefrontal cortex measured with an improved dinitrophenylhydrazine assay. J Neurochem 70:268–275

    Article  PubMed  CAS  Google Scholar 

  • Fon EA, Pothos EN, Sun BC, Killeen N, Sulzer D, Edwards RH (1997) Vesicular transport regulates monoamine storage and release but is not essential for amphetamine action. Neuron 19:1271–1283

    Article  PubMed  CAS  Google Scholar 

  • Gainetdinov RR, Fumagalli F, Wang YM, Jones SR, Levey AI, Miller GW, Caron MG (1998) Increased MPTP neurotoxicity in vesicular monoamine transporter 2 heterozygote knockout mice. J Neurochem 70:1973–1978

    Article  PubMed  CAS  Google Scholar 

  • George JM (2002) The synucleins. Genome Biol 3:1–6

    Google Scholar 

  • Glinka YY, Youdim MB (1995) Inhibition of mitochondrial complexes I and IV by 6-hydroxydopamine. Eur J Pharmacol 292:329–332

    PubMed  CAS  Google Scholar 

  • Goedert M (2001) α-Synuclein and neurodegenerative diseases. Nat Rev Neurosci 2:492–501

    Article  PubMed  CAS  Google Scholar 

  • Goodall AR, Danks K, Walker JH, Ball SG, Vaughan PF (1997) Occurrence of two types of secretory vesicles in the human neuroblastoma SH-SY5Y. J Neurochem 68:1542–1552

    Article  PubMed  CAS  Google Scholar 

  • Graham DG (1978) Oxidative pathways for catecholamines in the genesis of neuromelanin and cytotoxic quinones. Mol Pharmacol 14:633–643

    PubMed  CAS  Google Scholar 

  • Ichimura T, Isobe T, Okuyama T, Takahashi N, Araki K, Kuwano R,Takahashi Y (1988) Molecular cloning of cDNA coding for brainspecific14-3-3 protein, a protein kinase-dependent activator of tyrosine and tryptophan hydroxylases. Proc Natl Acad Sci USA 85:7084–7088

    Article  PubMed  CAS  Google Scholar 

  • Iwai A, Masliah E, Yoshimoto M, Ge N, Flanagan L, de Silva HA, Kittel A, Saitoh T (1995) The precursor protein of non-Aß component of Alzheimer’s disease amyloid is a presynaptic protein of the central nervous system. Neuron 14:467–475

    Article  PubMed  CAS  Google Scholar 

  • Jenco J, Rawlingson A, Daniels B, Morris A (1998) Regulation of phospholipase D2: selective inhibition of mammalian phospholipase D isoenzymes by α and β synucleins. Biochemistry 37:4901–4909

    Article  PubMed  CAS  Google Scholar 

  • Jenner P (1998) Oxidative mechanisms in nigral cell death in Parkinson’s disease. Mov Disord 13(Suppl 1):24–34

    PubMed  Google Scholar 

  • Junn M, Mouradian MM (2002) Human α-synuclein over-expression increases intracellular reactive oxygen species levels and susceptibility to dopamine. Neurosci Lett 320:146–150

    Article  PubMed  CAS  Google Scholar 

  • Kruger R, Kuhn W, Muller T, Woitalla D, Graeber M, Kosel S, Przuntek H, Epplen JT, Schols L, Riess O (1998) Ala30Pro mutation in the gene encoding α-synuclein in Parkinson’s disease. Nat Genet 18:106–108

    Article  PubMed  CAS  Google Scholar 

  • Lavedan C (1998) The synuclein family. Genome Res 8:871–880

    PubMed  CAS  Google Scholar 

  • Lee CS, Samii A, Sossi V, Ruth TJ, Schulzer M, Holden JE, Wudel J, Pal PK, de la Fuente-Fernandez R, Calne DB, Stoessl AJ (2000) In vivo positron emission tomographic evidence for compensatory changes in presynaptic dopaminergic nerve terminals in Parkinson’s disease. Ann Neurol 47:493–503

    Article  PubMed  CAS  Google Scholar 

  • Lee FJ, Liu F, Pristupa ZB, Niznik HB (2001) Direct binding and functional coupling of α-synuclein to the dopamine transporters accelerate dopamine-induced apoptosis. FASEB J 15:916–926

    Article  PubMed  CAS  Google Scholar 

  • Lotharius J, Barg S, Wiekop P, Lundberg C, Raymon HK, Brundin P (2002) Effect of mutant α-synuclein on dopamine homeostasis in a new human mesencephalic cell Line. J Biol Chem 277:38884–38894

    Article  PubMed  CAS  Google Scholar 

  • Maker HS, Weiss C, Silides DJ, Cohen G (1981) Couple of dopamine oxidation monoamine oxidation via the generation of hydrogen peroxide in rat brain homogenates. J Neurochem 36:589–593

    Article  PubMed  CAS  Google Scholar 

  • Markopoulou K, Wszolek ZK, Pfeiffer RF, Chase BA (1999) Reduced expression of the G209A alpha-synuclein allele in familial Parkinsonism. Ann Neurol 46:374–381

    Article  PubMed  CAS  Google Scholar 

  • Maroteaux L, Campanelli JT, Scheller RH (1988) Synuclein: a neuron-specific protein localized to the nucleus and presynaptic nerve terminal. J Neurosci 8:2804–2815

    PubMed  CAS  Google Scholar 

  • Mizuno Y, Suzuki K, Sone N, Saitoh T (1987) Inhibition of ATP synthesis by 1-methyl-4-phenylpyridinium ion (MPP+) in isolated mitochondria from mouse brains. Neurosci Lett 81:204–208

    Article  PubMed  CAS  Google Scholar 

  • Mooslehner KA, Chan PM, Xu W, Liu L, Smadja C, Humby T, Allen ND, Wilkinson LS, Emson PC (2001) Mice with very low expression of the vesicular monoamine transporter 2 gene survive into adulthood:potential mouse model for parkinsonism. Mol Cell Biol 21:5321–5331

    Article  PubMed  CAS  Google Scholar 

  • Nicklas WJ, Vyas I, Heikkila R .E (1985) Inhibition of NADH linked oxidation in brain mitochondria by 1-methyl-4-phenylpyridine, a metabolite of the neurotoxin, 1-methyl-4-phenyl-1,2,5,6-tetrahydropyridine. Life Sci 36:2503–2508

    Article  PubMed  CAS  Google Scholar 

  • Ostrerova N, Petrucelli L, Farrer M, Mehta N, Choi P, Hardy J, Wolozin B (1999) α-Synuclein shares physical and functional homology with 14-3-3. J Neurosci 19:5782–5791

    PubMed  CAS  Google Scholar 

  • Parsons SM (2000) Transport mechanisms in acetylcholine and monoamine storage. FASEB J 14:2423–2434

    Article  PubMed  CAS  Google Scholar 

  • Perez RG, Waymire JC, Lin E, Liu JJ, Guo F, Zigmond MJ (2002) A role for α-synuclein in the regulation of dopamine biosynthesis. J Neurosci 22:3090–3099

    PubMed  CAS  Google Scholar 

  • Polymeropoulos MH, Lavedan C, Leroy E, Ide SE, Dehejia A, Dutra, A, Pike B, Root H, Rubenstein J, Boyer R, Stenroos ES, Chandrasekharappa S, Athanassiadou A, Papapetropoulos T, Johnson WG, Lazzarini AM, Duvoisin RC, Di Iorio G, Golbe LI, Nussbaum RL (1997) Mutation in the α-synuclein gene identified in families with Parkinson’s disease. Science 276:2045–2047

    Article  PubMed  CAS  Google Scholar 

  • Pothos EN, Larsen KE, Krantz DE, Liu Y, Haycock JW, Setlik W, Gershon MD, Edwards RH, Sulzer D (2000) Synaptic vesicle transporter expression regulates vesicle phenotype and quantal size. J Neurosci 20:7297–7306

    PubMed  CAS  Google Scholar 

  • Pothos EN, Sun BC, Killeen N, Sulzer D, Edwards RH (1997) Vesicular transport regulates monoamine storage and release but is not essential for amphetamine action. Neuron 19:1271–1283

    Article  PubMed  Google Scholar 

  • Ramsay RR, Salach JI, Dadgar J, Singer TP (1986) Inhibition of mitochondrial NADH dehydrogenase by pyridine derivatives and its possible relation to experimental and idiopathic parkinsonism. Biochem Biophys Res 135:269–275

    Article  CAS  Google Scholar 

  • Reinhard JF Jr, Diliberto EJ Jr, Viveros OH, Daniels AJ (1987) Subcellular compartmentalization of 1-methyl-4-phenylpyridinium with catecholamines in adrenal medullary chromaffin vesicles may explain the lack of toxicity to adrenal chromaffin cells. Proc Natl Acad Sci USA 84:8160–8164

    Article  PubMed  CAS  Google Scholar 

  • Yu S, Zuo X, Li Y, Zhang C, Zhou M, Zhang YA, Uéeda K, Chan P (2004) Inhibition of tyrosine hydroxylase expression in α-synuclein-transfected dopaminergic neuronal cells. Neurosci Lett 367:34–39

    Article  PubMed  CAS  Google Scholar 

  • Spillantini MG, Schmidt ML, Lee VM, Trojanowski JQ, Jakes R, Goedert M (1997) α-synuclein in Lewy bodies. Nature 388:839–840

    Article  PubMed  CAS  Google Scholar 

  • Steiner-Murdoch S, Shirvan A, Schuldiner S. (1996) .Modification of the pH profile and tetrabenazine sensitivity of rat VMAT1 by replacement of aspartate 404 with glutamate. J Biol Chem 271:13048–13054

    Article  Google Scholar 

  • Suzuki K, Mizuno Y, Yamauchi Y, Nagatsu T, Mitsuo Y (1992) Selective inhibition of complex I by N-methylisoquinolinium ion and N-methyl-1,2,3,4-tetrahydroisoquinoline in isolated mitochondria prepared from mouse brain. J Neurol Sci 109:219–223

    Article  PubMed  CAS  Google Scholar 

  • Tabrizi SJ, Orth M, Wilkinson JM, Taanman J, Warner W TT, Cooper JM, Schapira AHV (2000) Expression of mutant α-synuclein causes increased susceptibility to dopamine toxicity. Hum Mol Genet 9:2683–2689

    Article  PubMed  CAS  Google Scholar 

  • Takahashi N, Miner LL, Sora I, Ujike H, Revay RS, Kostic V, Jackson-Lewis V, Przedborski S, Uhl GR (1997) VMAT2 knockout mice: heterozygotes display reduced amphetamine-conditioned reward, enhanced amphetamine locomotion, and enhanced MPTP toxicity. Proc Natl Acad Sci USA 94:9938–9943

    Article  PubMed  CAS  Google Scholar 

  • Wang YM, Gainetdinov RR, Fumagalli F, Xu F, Jones SR, Bock CB, Miller GW, Wightman RM, Caron MG (1997) Knockout of the vesicular monoamine transporter 2 gene results in neonatal death and supersensitivity to cocaine and amphetamine. Neuron 19:1285–1296

    Article  PubMed  CAS  Google Scholar 

  • Weihe E, Eiden LE (2000) Chemical neuroanatomy of the vesicular amine transporters. FASEB J 14:2435–2449

    Article  PubMed  CAS  Google Scholar 

  • Wersinger C, Sidhu A (2003) Attenuation of dopamine transporter activity by α-synuclein. Neurosci Lett 340:189–192

    Article  PubMed  CAS  Google Scholar 

  • Wersinger C, Prou D, Vernier P, Niznik HB, Sidhu A (2003) Mutations in the lipid-binding domain of α-synuclein confer overlapping, yet distinct, functional properties in the regulation of dopamine transporter activity. Mol Cell Neurosci 24:91–105

    Article  PubMed  CAS  Google Scholar 

  • Xu J, Kao SY, Lee FJ, Song W, Jin LW, Yankner BA (2002) Dopaminedependent neurotoxicity of α-synuclein: a mechanism for selective neurodegeneration in Parkinson’s disease. Nat Med 8:600–606

    Article  PubMed  CAS  Google Scholar 

  • Yoritaka A, Hattori N, Uchida K, Tanaka M, Stadtman ER, Mizuno Y (1996) Immunohistochemical detection of 4-hydroxynonenal protein adducts in Parkinson disease. Proc Natl Acad Sci USA 93:2696–2701

    Article  PubMed  CAS  Google Scholar 

  • Zhou W, Schaack. J, Zawada WM, Freed CR (2002) Overexpression of Human α-synuclein causes dopamine neuron death in primary human mesencephalic culture. Brain Res 926:42–50

    Article  PubMed  CAS  Google Scholar 

Download references

Acknowledgments

We thank Pro. Biao Chen (Xuanwu hospital, Beijing China) for providing the wild-type α-synuclein cDNA and Yu Hu (Peking Union Medical College, Being, China) for her excellent technical support in the radioactive experiment.

Author information

Authors and Affiliations

  1. Institute of Laboratory Animal Sciences, Chinese Academy of Medical Sciences (CAMS), Peking Union Medical College (PUMC), 5 Panjiayuannanli, Beijing, 100021, China

    Jun Tang Guo, An Qi Chen, Qi Kong, Hua Zhu, Chun Mei Ma & Chuan Qin

  2. Department of Pathophysiology, Weifang Medical College, 288 Shenglidong Street, Weifang, Shandong, 261042, China

    Jun Tang Guo

  3. Department of Pathology, Weifang Medical College, 288 Shenglidong Street, Weifang, Shandong, 261042, China

    An Qi Chen

Authors
  1. Jun Tang Guo
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. An Qi Chen
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Qi Kong
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Hua Zhu
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Chun Mei Ma
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Chuan Qin
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Chuan Qin.

Rights and permissions

Reprints and permissions

About this article

Cite this article

Guo, J.T., Chen, A.Q., Kong, Q. et al. Inhibition of Vesicular Monoamine Transporter-2 Activity in α-Synuclein Stably Transfected SH-SY5Y Cells. Cell Mol Neurobiol 28, 35–47 (2008). https://doi.org/10.1007/s10571-007-9227-0

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s10571-007-9227-0

Keywords

search

Navigation