Molecular Neurobiology

, Volume 55, Issue 1, pp 470–482 | Cite as

Prolyl Oligopeptidase Regulates Dopamine Transporter Phosphorylation in the Nigrostriatal Pathway of Mouse

  • Ulrika H Julku
  • Anne E Panhelainen
  • Saija E Tiilikainen
  • Reinis Svarcbahs
  • Anne E Tammimäki
  • T Petteri Piepponen
  • Mari H Savolainen
  • Timo T Myöhänen


Alpha-synuclein is the main component of Lewy bodies, a histopathological finding of Parkinson’s disease. Prolyl oligopeptidase (PREP) is a serine protease that binds to α-synuclein and accelerates its aggregation in vitro. PREP enzyme inhibitors have been shown to block the α-synuclein aggregation process in vitro and in cellular models, and also to enhance the clearance of α-synuclein aggregates in transgenic mouse models. Moreover, PREP inhibitors have induced alterations in dopamine and metabolite levels, and dopamine transporter immunoreactivity in the nigrostriatal tissue. In this study, we characterized the role of PREP in the nigrostriatal dopaminergic and GABAergic systems of wild-type C57Bl/6 and PREP knockout mice, and the effects of PREP overexpression on these systems. Extracellular concentrations of dopamine and protein levels of phosphorylated dopamine transporter were increased and dopamine reuptake was decreased in the striatum of PREP knockout mice, suggesting increased internalization of dopamine transporter from the presynaptic membrane. Furthermore, PREP overexpression increased the level of dopamine transporters in the nigrostriatal tissue but decreased phosphorylated dopamine transporters in the striatum in wild-type mice. Our results suggest that PREP regulates the function of dopamine transporter, possibly by controlling the phosphorylation and transport of dopamine transporter into the striatum or synaptic membrane.


Prolyl oligopeptidase PREP Dopamine Dopamine transporter DAT Microdialysis 



This work was supported by grants from the Academy of Finland (267788 and 2737991), University of Helsinki research grants, Jane and Aatos Erkko Foundation and from the Sigrid Juselius Foundation to TTM. Authors would like to thank Liisa Lappalainen and Susanna Norrbacka for excellent technical assistance.

Compliance with Ethical Standards

Conflict of Interest

The authors declare no competing financial interests.


This work was supported by grants from the Academy of Finland (267788 and 2737991), University of Helsinki research grants, Jane and Aatos Erkko Foundation and from the Sigrid Juselius Foundation to TT Myöhänen.

Supplementary material

12035_2016_339_MOESM1_ESM.docx (4.5 mb)
ESM 1 (DOCX 4.53 mb)


  1. 1.
    Myöhänen TT, García-Horsman JA, Tenorio-Laranga J, Männistö PT (2009) Issues about the physiological functions of prolyl oligopeptidase based on its discordant spatial association with substrates and inconsistencies among mRNA, protein levels, and enzymatic activity. J Histochem & Cytochem 57:831–848CrossRefGoogle Scholar
  2. 2.
    García-Horsman JA, Männistö PT, Venäläinen JI (2007) On the role of prolyl oligopeptidase in health and disease. Neuropept 41:1–24CrossRefGoogle Scholar
  3. 3.
    Mantle D, Falkous G, Ishiura S, Blanchard PJ, Perry EK (1996) Comparison of proline endopeptidase activity in brain tissue from normal cases and cases with Alzheimer’s disease, Lewy body dementia, Parkinson’s disease and Huntington’s disease. Clin Chim Acta 249:129–139CrossRefPubMedGoogle Scholar
  4. 4.
    Männistö PT, Venäläinen J, Jalkanen A, García-Horsman JA (2007) Prolyl oligopeptidase: a potential target for the treatment of cognitive disorders. Drug News Perspect 20:293–305CrossRefPubMedGoogle Scholar
  5. 5.
    Tenorio-Laranga J, Männistö PT, Storvik M, Van der Veken P, García-Horsman JA (2012) Four day inhibition of prolyl oligopeptidase causes significant changes in the peptidome of rat brain, liver and kidney. Biochimie 94:1849–1859CrossRefPubMedGoogle Scholar
  6. 6.
    Jalkanen AJ, Savolainen K, Forsberg MM (2011) Inhibition of prolyl oligopeptidase by KYP-2047 fails to increase the extracellular neurotensin and substance P levels in rat striatum. Neurosci Lett 502:107–111CrossRefPubMedGoogle Scholar
  7. 7.
    Myöhänen TT, Venäläinen JI, Garcia-Horsman JA, Piltonen M, Männistö PT (2008) Cellular and subcellular distribution of rat brain prolyl oligopeptidase and its association with specific neuronal neurotransmitters. J Comp Neurol 507:1694–1708CrossRefPubMedGoogle Scholar
  8. 8.
    Myöhänen TT, Kääriäinen TM, Jalkanen AJ, Piltonen M, Männistö PT (2009) Localization of prolyl oligopeptidase in the thalamic and cortical projection neurons: a retrograde neurotracing study in the rat brain. Neurosci Lett 450:201–205CrossRefPubMedGoogle Scholar
  9. 9.
    Peltonen I, Myöhänen TT, Männistö PT (2012) Different interactions of prolyl oligopeptidase and neurotensin in dopaminergic function of the rat nigrostriatal and mesolimbic pathways. Neurochem Res 37:2033–2041CrossRefPubMedGoogle Scholar
  10. 10.
    Schulz I, Zeitschel U, Rudolph T, Ruiz-Carrillo D, Rahfeld JU, Gerhartz B, Bigl V, Demuth HU et al (2005) Subcellular localization suggests novel functions for prolyl endopeptidase in protein secretion. J Neurochem 94:970–979CrossRefPubMedGoogle Scholar
  11. 11.
    Hannula MJ, Myöhänen TT, Tenorio-Laranga J, Männistö PT, Garcia-Horsman JA (2013) Prolyl oligopeptidase colocalizes with α-synuclein, β-amyloid, tau protein and astroglia in the post-mortem brain samples with Parkinson’s and Alzheimer’s diseases. Neurosci 242:140–150CrossRefGoogle Scholar
  12. 12.
    Myöhänen TT, Hannula MJ, Van Elzen R, Gerard M, Van Der Veken P, García-Horsman JA, Baekelandt V, Männistö PT et al (2012) A prolyl oligopeptidase inhibitor, KYP-2047, reduces α-synuclein protein levels and aggregates in cellular and animal models of Parkinson’s disease. Br J Pharmacol 166:1097–1113CrossRefPubMedPubMedCentralGoogle Scholar
  13. 13.
    Savolainen MH, Yan X, Myöhänen TT, Huttunen HJ (2015) Prolyl oligopeptidase enhances alpha-synuclein dimerization via direct protein-protein interaction. J Biol Chem 290:5117–5126CrossRefPubMedPubMedCentralGoogle Scholar
  14. 14.
    Savolainen MH, Richie CT, Harvey BK, Männistö PT, Maguire-Zeiss KA, Myöhänen TT (2014) The beneficial effect of a prolyl oligopeptidase inhibitor, KYP-2047, on alpha-synuclein clearance and autophagy in A30P transgenic mouse. Neurobiol Dis 68:1–15CrossRefPubMedGoogle Scholar
  15. 15.
    Jalkanen AJ, Piepponen TP, Hakkarainen JJ, De Meester I, Lambeir A-M, Forsberg MM (2012) The effect of prolyl oligopeptidase inhibition on extracellular acetylcholine and dopamine levels in the rat striatum. Neurochem Int 60:301–309CrossRefPubMedGoogle Scholar
  16. 16.
    Rice ME, Cragg SJ (2008) Dopamine spillover after quantal release: rethinking dopamine transmission in the nigrostriatal pathway. Brain Res Rev 58:303–313CrossRefPubMedPubMedCentralGoogle Scholar
  17. 17.
    Melikian HE, Buckley KM (1999) Membrane trafficking regulates the activity of the human dopamine transporter. J Neurosci 19:7699–7710PubMedGoogle Scholar
  18. 18.
    Vaughan RA, Huff RA, Uhl GR, Kuhar MJ (1997) Protein kinase C-mediated phosphorylation and functional regulation of dopamine transporters in striatal synaptosomes. J Biol Chem 272:15541–15546CrossRefPubMedGoogle Scholar
  19. 19.
    Daniels GM, Amara SG (1999) Regulated trafficking of the human dopamine transporter clathrin-mediated internalization and lysosomal degradation in response to phorbol esters. J Biol Chem 274:35794–35801CrossRefPubMedGoogle Scholar
  20. 20.
    Ishibashi K, Oda K, Ishiwata K, Ishii K (2014) Comparison of dopamine transporter decline in a patient with Parkinson’s disease and normal aging effect. J Neurol Sci 339:207–209CrossRefPubMedGoogle Scholar
  21. 21.
    Chen L, Ding Y, Cagniard B, Van Laar AD, Mortimer A, Chi W, Hastings TG, Kang UJ et al (2008) Unregulated cytosolic dopamine causes neurodegeneration associated with oxidative stress in mice. J Neurosci 28:425–433CrossRefPubMedGoogle Scholar
  22. 22.
    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 15:916–926CrossRefPubMedGoogle Scholar
  23. 23.
    Butler B, Saha K, Rana T, Becker JP, Sambo D, Davari P, Goodwin JS, Khoshbouei H (2015) Dopamine transporter activity is modulated by α-synuclein. J Biol Chem 290:29542–29554CrossRefPubMedPubMedCentralGoogle Scholar
  24. 24.
    Fountaine TM, Wade-Martins R (2007) RNA interference-mediated knockdown of α-synuclein protects human dopaminergic neuroblastoma cells from MPP+ toxicity and reduces dopamine transport. J Neurosci Res 85:351–363CrossRefPubMedGoogle Scholar
  25. 25.
    Lam HA, Wu N, Cely I, Kelly RL, Hean S, Richter F, Magen I, Cepeda C et al (2011) Elevated tonic extracellular dopamine concentration and altered dopamine modulation of synaptic activity precede dopamine loss in the striatum of mice overexpressing human α-synuclein. J Neurosci Res 89:1091–1102CrossRefPubMedPubMedCentralGoogle Scholar
  26. 26.
    Abeliovich A, Schmitz Y, Fariñas I, Choi-Lundberg D, Ho W-H, Castillo PE, Shinsky N, Verdugo JMG et al (2000) Mice lacking α-synuclein display functional deficits in the nigrostriatal dopamine system. Neuron 25:239–252CrossRefPubMedGoogle Scholar
  27. 27.
    Chadchankar H, Ihalainen J, Tanila H, Yavich L (2011) Decreased reuptake of dopamine in the dorsal striatum in the absence of alpha-synuclein. Brain Res 1382:37–44CrossRefPubMedGoogle Scholar
  28. 28.
    German CL, Baladi MG, McFadden LM, Hanson GR, Fleckenstein AE (2015) Regulation of the dopamine and vesicular monoamine transporters: pharmacological targets and implications for disease. Pharmacol Rev 67:1005–1024CrossRefPubMedPubMedCentralGoogle Scholar
  29. 29.
    Jarho EM, Venäläinen JI, Huuskonen J, Christiaans JAM, Garcia-Horsman JA, Forsberg MM, Järvinen T, Gynther J et al (2004) A cyclopent-2-enecarbonyl group mimics proline at the P2 position of prolyl oligopeptidase inhibitors. J Medic Chem 47:5605–5607CrossRefGoogle Scholar
  30. 30.
    Jalkanen AJ, Hakkarainen JJ, Lehtonen M, Venäläinen T, Kääriäinen TM, Jarho E, Suhonen M, Forsberg MM (2011) Brain pharmacokinetics of two prolyl oligopeptidase inhibitors, JTP-4819 and KYP-2047, in the rat. Basic Clinical Pharmacol Toxicol 109:443–451CrossRefGoogle Scholar
  31. 31.
    Jalkanen AJ, Leikas JV, Forsberg MM (2014) KYP-2047 penetrates mouse brain and effectively inhibits mouse prolyl oligopeptidase. Basic Clinical Pharmacol Toxicol 114:460–463CrossRefGoogle Scholar
  32. 32.
    Venäläinen JI, Garcia-Horsman JA, Forsberg MM, Jalkanen A, Wallén EAA, Jarho EM, Christiaans JAM, Gynther J et al (2006) Binding kinetics and duration of in vivo action of novel prolyl oligopeptidase inhibitors. Biochem Pharmacol 71:683–692CrossRefPubMedGoogle Scholar
  33. 33.
    Höfling C, Kulesskaya N, Jaako K, Peltonen I, Männistö PT, Nurmi A, Vartiainen N, Morawski M et al (2016) Deficiency of prolyl oligopeptidase in mice disturbs synaptic plasticity and reduces anxiety-like behaviour, body weight, and brain volume. Eur Neuropsychopharmacol 6:1048–1061CrossRefGoogle Scholar
  34. 34.
    Di Daniel E, Glover CP, Grot E, Chan MK, Sanderson TH, White JH, Ellis CL, Gallagher KT et al (2009) Prolyl oligopeptidase binds to GAP-43 and functions without its peptidase activity. Mol Cell Neurosci 41:373–382CrossRefPubMedGoogle Scholar
  35. 35.
    Paxinos GFK (1997) The mouse brain in stereotaxic coordinates. Elsevier Academic Press, San DiegoGoogle Scholar
  36. 36.
    Käenmäki M, Tammimäki A, Myöhänen T, Pakarinen K, Amberg C, Karayiorgou M, Gogos JA, Männistö PT (2010) Quantitative role of COMT in dopamine clearance in the prefrontal cortex of freely moving mice. J Neurochem 114:1745–1755CrossRefPubMedGoogle Scholar
  37. 37.
    Vihavainen T, Relander TRA, Leiviskä R, Airavaara M, Tuominen RK, Ahtee L, Piepponen TP (2008) Chronic nicotine modifies the effects of morphine on extracellular striatal dopamine and ventral tegmental GABA. J Neurochem 107:844–854CrossRefPubMedGoogle Scholar
  38. 38.
    Parsons LH, Justice JB (1992) Extracellular concentration and in vivo recovery of dopamine in the nucleus accumbens using microdialysis. J Neurochem 58:212–218CrossRefPubMedGoogle Scholar
  39. 39.
    Chefer VI, Thompson AC, Zapata A, Shippenberg TS (2009) Overview of brain microdialysis. Curr Protoc Neurosci, chapter 7, unit 7.1:1–28Google Scholar
  40. 40.
    Chefer VI, Zapata A, Shippenberg TS, Bungay PM (2006) Quantitative no-net-flux microdialysis permits detection of increases and decreases in dopamine uptake in mouse nucleus accumbens. J Neurosci Methods 155:187–193CrossRefPubMedGoogle Scholar
  41. 41.
    Smith A, Justice J (1994) The effect of inhibition of synthesis, release, metabolism and uptake on the microdialysis extraction fraction of dopamine. J Neurosci Methods 54:75–82CrossRefPubMedGoogle Scholar
  42. 42.
    Justice J (1993) Quantitative microdialysis of neurotransmitters. J Neurosci Methods 48:263–276CrossRefPubMedGoogle Scholar
  43. 43.
    Airavaara M, Mijatovic J, Vihavainen T, Piepponen TP, Saarma M, Ahtee L (2006) In heterozygous GDNF knockout mice the response of striatal dopaminergic system to acute morphine is altered. Synapse 59:321–329CrossRefPubMedGoogle Scholar
  44. 44.
    Kumar A, Kopra J, Varendi K, Porokuokka LL, Panhelainen A, Kuure S, Marshall P, Karalija N et al (2015) GDNF overexpression from the native locus reveals its role in the nigrostriatal dopaminergic system function. PLoS Genet 11:e1005710CrossRefPubMedPubMedCentralGoogle Scholar
  45. 45.
    Fleming SM, Ekhator OR, Ghisays V (2013) Assessment of sensorimotor function in mouse models of Parkinson’s disease. J Visual Exp, JoVEGoogle Scholar
  46. 46.
    Brooks SP, Dunnett SB (2009) Tests to assess motor phenotype in mice: a user’s guide. Nat Rev Neurosci 10:519–529CrossRefPubMedGoogle Scholar
  47. 47.
    Yorgason JT, España RA, Jones SR (2011) Demon voltammetry and analysis software: analysis of cocaine-induced alterations in dopamine signaling using multiple kinetic measures. J Neurosci Methods 202:158–164CrossRefPubMedPubMedCentralGoogle Scholar
  48. 48.
    Robertson S, Matthies H, Galli A (2009) A closer look at amphetamine-induced reverse transport and trafficking of the dopamine and norepinephrine transporters. Mol Neurobiol 39:73–80CrossRefPubMedPubMedCentralGoogle Scholar
  49. 49.
    Levitt M, Spector S, Sjoerdsma A, Udenfriend S (1965) Elucidation of the rate-limiting step in norepinephrine biosynthesis in the perfused guinea-pig heart. J Pharmacol Exp Therap 148:1–8Google Scholar
  50. 50.
    Peltonen I, Jalkanen AJ, Sinervä V, Puttonen KA, Männistö PT (2010) Different effects of scopolamine and inhibition of prolyl oligopeptidase on mnemonic and motility functions of young and 8-to 9-month-old rats in the radial-arm maze. Basic Clin Pharmacol Toxicol 106:280–287CrossRefPubMedGoogle Scholar
  51. 51.
    Volkow N, Fowler J, Wang G, Logan J, Schlyer D, MacGregor R, Hitzemann R, Wolf A (1994) Decreased dopamine transporters with age in healthy human subjects. Ann Neurol 36:237–239CrossRefPubMedGoogle Scholar
  52. 52.
    Volkow N, Wang G-J, Fowler J, Ding Y-S, Gur R, Gatley J, Logan J, Moberg P et al (1998) Parallel loss of presynaptic and postsynaptic dopamine markers in normal aging. Ann Neurol 44:143–147CrossRefPubMedGoogle Scholar
  53. 53.
    Ishibashi K, Ishii K, Oda K, Kawasaki K, Mizusawa H, Ishiwata K (2009) Regional analysis of age-related decline in dopamine transporters and dopamine D2-like receptors in human striatum. Synapse 63:282–290CrossRefPubMedGoogle Scholar

Copyright information

© Springer Science+Business Media New York 2016

Authors and Affiliations

  • Ulrika H Julku
    • 1
  • Anne E Panhelainen
    • 2
  • Saija E Tiilikainen
    • 1
  • Reinis Svarcbahs
    • 1
  • Anne E Tammimäki
    • 1
  • T Petteri Piepponen
    • 1
  • Mari H Savolainen
    • 1
  • Timo T Myöhänen
    • 1
  1. 1.Division of Pharmacology and PharmacotherapyUniversity of HelsinkiHelsinkiFinland
  2. 2.Institute of BiotechnologyUniversity of HelsinkiHelsinkiFinland

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