Neuroprotective Effects of Temsirolimus in Animal Models of Parkinson’s Disease

  • Rosalba Siracusa
  • Irene Paterniti
  • Marika Cordaro
  • Rosalia Crupi
  • Giuseppe Bruschetta
  • Michela Campolo
  • Salvatore Cuzzocrea
  • Emanuela Esposito


Parkinson’s disease (PD) is a disorder caused by degeneration of dopaminergic neurons. At the moment, there is no cure. Recent studies have shown that autophagy may have a protective function against the advance of a number of neurodegenerative diseases. Temsirolimus is an analogue of rapamycin that induces autophagy by inhibiting mammalian target of rapamycin complex 1. For this purpose, in the present study we investigated the neuroprotective effects of temsirolimus (5 mg/kg intraperitoneal) on 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine-induced (MPTP) neurotoxicity in in vivo model of PD. At the end of the experiment, brain tissues were processed for histological, immunohistochemical, Western blot, and immunofluorescent analysis. Treatment with temsirolimus significantly ameliorated behavioral deficits, increased the expression of specific markers of PD such as tyrosine hydroxylase, dopamine transporter, as well as decreased the upregulation of α-synuclein in the substantia nigra after MPTP induction. Furthermore, Western blot and immunohistochemistry analysis showed that temsirolimus administration significantly increased autophagy process. In fact, treatment with temsirolimus maintained high Beclin-1, p62, and microtubule-associated protein 1A/1B-light chain 3 expression and inhibited the p70S6K expression. In addition, we showed that temsirolimus has also anti-inflammatory properties as assessed by the significant inhibition of the expression of mitogen-activated protein kinases such as p-JNK, p-p38, and p-ERK, and the restored levels of neurotrophic factor expression such as BDNF and NT-3. On the basis of this evidence, we clearly demonstrate that temsirolimus is able to modulate both the autophagic process and the neuroinflammatory pathway involved in PD, actions which may underlie its neuroprotective effect.


Neurodegenerative disease Autophagy Neuroinflammation Neuroprotection Rapamycin 


  1. 1.
    Moore DJ, West AB, Dawson VL, Dawson TM (2005) Molecular pathophysiology of Parkinson’s disease. Annu Rev Neurosci 28:57–87. doi:10.1146/annurev.neuro.28.061604.135718 PubMedCrossRefGoogle Scholar
  2. 2.
    Sulzer D, Surmeier DJ (2013) Neuronal vulnerability, pathogenesis, and Parkinson’s disease. Movement disorders : official journal of the Movement Disorder Society 28(1):41–50. doi:10.1002/mds.25095 CrossRefGoogle Scholar
  3. 3.
    Dauer W, Przedborski S (2003) Parkinson’s disease: mechanisms and models. Neuron 39(6):889–909PubMedCrossRefGoogle Scholar
  4. 4.
    Marras C, Lang A (2008) Invited article: changing concepts in Parkinson disease: moving beyond the decade of the brain. Neurology 70(21):1996–2003. doi:10.1212/01.wnl.0000312515.52545.51 PubMedCrossRefGoogle Scholar
  5. 5.
    Hirsch EC, Hunot S (2009) Neuroinflammation in Parkinson’s disease: a target for neuroprotection? Lancet Neurol 8(4):382–397. doi:10.1016/S1474-4422(09)70062-6 PubMedCrossRefGoogle Scholar
  6. 6.
    Mehta SH, Tanner CM (2016) Role of neuroinflammation in Parkinson disease: the enigma continues. Mayo Clin Proc 91(10):1328–1330. doi:10.1016/j.mayocp.2016.08.010 PubMedCrossRefGoogle Scholar
  7. 7.
    Mitra S, Ghosh N, Sinha P, Chakrabarti N, Bhattacharyya A (2016) Alteration of nuclear factor-kappaB pathway promote neuroinflammation depending on the functions of estrogen receptors in substantia nigra after 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine treatment. Neurosci Lett 616:86–92. doi:10.1016/j.neulet.2016.01.046 PubMedCrossRefGoogle Scholar
  8. 8.
    Lee HJ, Kim C, Lee SJ (2010) Alpha-synuclein stimulation of astrocytes: potential role for neuroinflammation and neuroprotection. Oxidative Med Cell Longev 3(4):283–287. doi:10.4161/oxim.3.4.12809 CrossRefGoogle Scholar
  9. 9.
    Xilouri M, Brekk OR, Stefanis L (2016) Autophagy and alpha-synuclein: relevance to Parkinson’s disease and related synucleopathies. Movement disorders : official journal of the Movement Disorder Society 31(2):178–192. doi:10.1002/mds.26477 CrossRefGoogle Scholar
  10. 10.
    de Oliveira RM, Vicente Miranda H, Francelle L, Pinho R, Szego EM, Martinho R, Munari F, Lazaro DF et al (2017) The mechanism of sirtuin 2-mediated exacerbation of alpha-synuclein toxicity in models of Parkinson disease. PLoS Biol 15(3):e2000374. doi:10.1371/journal.pbio.2000374 PubMedPubMedCentralCrossRefGoogle Scholar
  11. 11.
    Redmann M, Wani WY, Volpicelli-Daley L, Darley-Usmar V, Zhang J (2017) Trehalose does not improve neuronal survival on exposure to alpha-synuclein pre-formed fibrils. Redox Biol 11:429–437. doi:10.1016/j.redox.2016.12.032 PubMedPubMedCentralCrossRefGoogle Scholar
  12. 12.
    Levine B, Klionsky DJ (2004) Development by self-digestion: molecular mechanisms and biological functions of autophagy. Dev Cell 6(4):463–477PubMedCrossRefGoogle Scholar
  13. 13.
    Wang C, Liang CC, Bian ZC, Zhu Y, Guan JL (2013) FIP200 is required for maintenance and differentiation of postnatal neural stem cells. Nat Neurosci 16(5):532–542. doi:10.1038/nn.3365 PubMedPubMedCentralCrossRefGoogle Scholar
  14. 14.
    Yue Z, Friedman L, Komatsu M, Tanaka K (2009) The cellular pathways of neuronal autophagy and their implication in neurodegenerative diseases. Biochim Biophys Acta 1793(9):1496–1507. doi:10.1016/j.bbamcr.2009.01.016 PubMedPubMedCentralCrossRefGoogle Scholar
  15. 15.
    Hu X, Song Q, Li X, Li D, Zhang Q, Meng W, Zhao Q (2017) Neuroprotective effects of Kukoamine A on neurotoxin-induced Parkinson’s model through apoptosis inhibition and autophagy enhancement. Neuropharmacology 117:352–363. doi:10.1016/j.neuropharm.2017.02.022 PubMedCrossRefGoogle Scholar
  16. 16.
    Webb JL, Ravikumar B, Atkins J, Skepper JN, Rubinsztein DC (2003) Alpha-Synuclein is degraded by both autophagy and the proteasome. J Biol Chem 278(27):25009–25013. doi:10.1074/jbc.M300227200 PubMedCrossRefGoogle Scholar
  17. 17.
    Hu Q, Wang G (2016) Mitochondrial dysfunction in Parkinson’s disease. Translational neurodegeneration 5:14. doi:10.1186/s40035-016-0060-6 PubMedPubMedCentralCrossRefGoogle Scholar
  18. 18.
    Ouyang L, Zhang L, Liu B (2016) Autophagy pathways and key drug targets in Parkinson’s disease. Yao xue xue bao = Acta pharmaceutica Sinica 51(1):9–17PubMedGoogle Scholar
  19. 19.
    Switon K, Kotulska K, Janusz-Kaminska A, Zmorzynska J, Jaworski J (2016) Molecular neurobiology of mTOR. Neuroscience. doi:10.1016/j.neuroscience.2016.11.017 PubMedGoogle Scholar
  20. 20.
    Sabers CJ, Martin MM, Brunn GJ, Williams JM, Dumont FJ, Wiederrecht G, Abraham RT (1995) Isolation of a protein target of the FKBP12-rapamycin complex in mammalian cells. J Biol Chem 270(2):815–822PubMedCrossRefGoogle Scholar
  21. 21.
    Pong K, Zaleska MM (2003) Therapeutic implications for immunophilin ligands in the treatment of neurodegenerative diseases. Curr Drug Targets CNS Neurol Disord 2(6):349–356PubMedCrossRefGoogle Scholar
  22. 22.
    Radad K, Moldzio R, Rausch WD (2015) Rapamycin protects dopaminergic neurons against rotenone-induced cell death in primary mesencephalic cell culture. Folia Neuropathol 53(3):250–261. doi:10.5114/fn.2015.54426 PubMedCrossRefGoogle Scholar
  23. 23.
    Sola E, Lopez V, Burgos D, Cabello M, Gutierrez C, Martin A, Pena M, Gonzalez-Molina M (2006) Pulmonary toxicity associated with sirolimus treatment in kidney transplantation. Transplant Proc 38(8):2438–2440. doi:10.1016/j.transproceed.2006.08.037 PubMedCrossRefGoogle Scholar
  24. 24.
    Vlahakis NE, Rickman OB, Morgenthaler T (2004) Sirolimus-associated diffuse alveolar hemorrhage. Mayo Clin Proc 79(4):541–545. doi:10.4065/79.4.541 PubMedCrossRefGoogle Scholar
  25. 25.
    Alkhatib AA (2006) Sirolimus-induced intractable chronic diarrhea: a case report. Transplant Proc 38(5):1298–1300. doi:10.1016/j.transproceed.2006.02.123 PubMedCrossRefGoogle Scholar
  26. 26.
    Altomare JF, Smith RE, Potdar S, Mitchell SH (2006) Delayed gastric ulcer healing associated with sirolimus. Transplantation 82(3):437–438. doi:10.1097/ PubMedCrossRefGoogle Scholar
  27. 27.
    Rubinsztein DC, Codogno P, Levine B (2012) Autophagy modulation as a potential therapeutic target for diverse diseases. Nat Rev Drug Discov 11(9):709–730. doi:10.1038/nrd3802 PubMedPubMedCentralCrossRefGoogle Scholar
  28. 28.
    Yazbeck VY, Buglio D, Georgakis GV, Li Y, Iwado E, Romaguera JE, Kondo S, Younes A (2008) Temsirolimus downregulates p21 without altering cyclin D1 expression and induces autophagy and synergizes with vorinostat in mantle cell lymphoma. Exp Hematol 36(4):443–450. doi:10.1016/j.exphem.2007.12.008 PubMedCrossRefGoogle Scholar
  29. 29.
    Cortes CJ, La Spada AR (2014) The many faces of autophagy dysfunction in Huntington’s disease: from mechanism to therapy. Drug Discov Today 19(7):963–971. doi:10.1016/j.drudis.2014.02.014 PubMedPubMedCentralCrossRefGoogle Scholar
  30. 30.
    Jiang T, Yu JT, Zhu XC, Zhang QQ, Cao L, Wang HF, Tan MS, Gao Q et al (2014) Temsirolimus attenuates tauopathy in vitro and in vivo by targeting tau hyperphosphorylation and autophagic clearance. Neuropharmacology 85:121–130. doi:10.1016/j.neuropharm.2014.05.032 PubMedCrossRefGoogle Scholar
  31. 31.
    Graziani EI (2009) Recent advances in the chemistry, biosynthesis and pharmacology of rapamycin analogs. Nat Prod Rep 26(5):602–609. doi:10.1039/b804602f PubMedCrossRefGoogle Scholar
  32. 32.
    Bozec A, Etienne-Grimaldi MC, Fischel JL, Sudaka A, Toussan N, Formento P, Milano G (2011) The mTOR-targeting drug temsirolimus enhances the growth-inhibiting effects of the cetuximab-bevacizumab-irradiation combination on head and neck cancer xenografts. Oral Oncol 47(5):340–344. doi:10.1016/j.oraloncology.2011.02.020 PubMedCrossRefGoogle Scholar
  33. 33.
    Malagelada C, Jin ZH, Jackson-Lewis V, Przedborski S, Greene LA (2010) Rapamycin protects against neuron death in in vitro and in vivo models of Parkinson’s disease. J Neurosci 30(3):1166–1175. doi:10.1523/JNEUROSCI.3944-09.2010 PubMedPubMedCentralCrossRefGoogle Scholar
  34. 34.
    Esposito E, Impellizzeri D, Mazzon E, Paterniti I, Cuzzocrea S (2012) Neuroprotective activities of palmitoylethanolamide in an animal model of Parkinson’s disease. PLoS One 7(8):e41880. doi:10.1371/journal.pone.0041880 PubMedPubMedCentralCrossRefGoogle Scholar
  35. 35.
    Heeneman S, Sluimer JC, Daemen MJ (2007) Angiotensin-converting enzyme and vascular remodeling. Circ Res 101(5):441–454. doi:10.1161/CIRCRESAHA.107.148338 PubMedCrossRefGoogle Scholar
  36. 36.
    Lee KW, Zhao X, Im JY, Grosso H, Jang WH, Chan TW, Sonsalla PK, German DC et al (2012) Apoptosis signal-regulating kinase 1 mediates MPTP toxicity and regulates glial activation. PLoS One 7(1):e29935. doi:10.1371/journal.pone.0029935 PubMedPubMedCentralCrossRefGoogle Scholar
  37. 37.
    Fleming SM, Mulligan CK, Richter F, Mortazavi F, Lemesre V, Frias C, Zhu C, Stewart A et al (2011) A pilot trial of the microtubule-interacting peptide (NAP) in mice overexpressing alpha-synuclein shows improvement in motor function and reduction of alpha-synuclein inclusions. Mol Cell Neurosci 46(3):597–606. doi:10.1016/j.mcn.2010.12.011 PubMedCrossRefGoogle Scholar
  38. 38.
    Araki T, Kumagai T, Tanaka K, Matsubara M, Kato H, Itoyama Y, Imai Y (2001) Neuroprotective effect of riluzole in MPTP-treated mice. Brain Res 918(1–2):176–181PubMedCrossRefGoogle Scholar
  39. 39.
    Porsolt RD, Bertin A, Blavet N, Deniel M, Jalfre M (1979) Immobility induced by forced swimming in rats: effects of agents which modify central catecholamine and serotonin activity. Eur J Pharmacol 57(2–3):201–210PubMedCrossRefGoogle Scholar
  40. 40.
    Bortolato M, Godar SC, Davarian S, Chen K, Shih JC (2009) Behavioral disinhibition and reduced anxiety-like behaviors in monoamine oxidase B-deficient mice. Neuropsychopharmacology : official publication of the American College of Neuropsychopharmacology 34(13):2746–2757. doi:10.1038/npp.2009.118 CrossRefGoogle Scholar
  41. 41.
    Pellow S, Chopin P, File SE, Briley M (1985) Validation of open:closed arm entries in an elevated plus-maze as a measure of anxiety in the rat. J Neurosci Methods 14(3):149–167PubMedCrossRefGoogle Scholar
  42. 42.
    Cui J, Zhang M, Zhang YQ, Xu ZH (2007) JNK pathway: diseases and therapeutic potential. Acta Pharmacol Sin 28(5):601–608. doi:10.1111/j.1745-7254.2007.00579.x PubMedCrossRefGoogle Scholar
  43. 43.
    Anglade P, Vyas S, Javoy-Agid F, Herrero MT, Michel PP, Marquez J, Mouatt-Prigent A, Ruberg M et al (1997) Apoptosis and autophagy in nigral neurons of patients with Parkinson’s disease. Histol Histopathol 12(1):25–31PubMedGoogle Scholar
  44. 44.
    Mizushima N, Hara T (2006) Intracellular quality control by autophagy: how does autophagy prevent neurodegeneration? Autophagy 2(4):302–304PubMedCrossRefGoogle Scholar
  45. 45.
    Komatsu M, Kominami E, Tanaka K (2006) Autophagy and neurodegeneration. Autophagy 2(4):315–317PubMedCrossRefGoogle Scholar
  46. 46.
    Li J, Li S, Zhang L, Ouyang L, Liu B (2015) Deconvoluting the complexity of autophagy and Parkinson’s disease for potential therapeutic purpose. Oncotarget Google Scholar
  47. 47.
    Siracusa R, Paterniti I, Impellizzeri D, Cordaro M, Crupi R, Navarra M, Cuzzocrea S, Esposito E (2015) The association of palmitoylethanolamide with luteolin decreases neuroinflammation and stimulates autophagy in Parkinson’s disease model. CNS Neurol Disord Drug TargetsGoogle Scholar
  48. 48.
    Asakawa T, Fang H, Sugiyama K, Nozaki T, Hong Z, Yang Y, Hua F, Ding G et al (2016) Animal behavioral assessments in current research of Parkinson’s disease. Neurosci Biobehav Rev 65:63–94. doi:10.1016/j.neubiorev.2016.03.016 PubMedCrossRefGoogle Scholar
  49. 49.
    Nakatogawa H, Suzuki K, Kamada Y, Ohsumi Y (2009) Dynamics and diversity in autophagy mechanisms: lessons from yeast. Nat Rev Mol Cell Biol 10(7):458–467. doi:10.1038/nrm2708 PubMedCrossRefGoogle Scholar
  50. 50.
    Spilman P, Podlutskaya N, Hart MJ, Debnath J, Gorostiza O, Bredesen D, Richardson A, Strong R et al (2010) Inhibition of mTOR by rapamycin abolishes cognitive deficits and reduces amyloid-beta levels in a mouse model of Alzheimer’s disease. PLoS One 5(4):e9979. doi:10.1371/journal.pone.0009979 PubMedPubMedCentralCrossRefGoogle Scholar
  51. 51.
    Hsieh CF, Lai YC, Pan RP, Pan CL (2008) Polarizing terahertz waves with nematic liquid crystals. Opt Lett 33(11):1174–1176PubMedCrossRefGoogle Scholar
  52. 52.
    Simonsen A, Tooze SA (2009) Coordination of membrane events during autophagy by multiple class III PI3-kinase complexes. J Cell Biol 186(6):773–782. doi:10.1083/jcb.200907014 PubMedPubMedCentralCrossRefGoogle Scholar
  53. 53.
    Kabeya Y, Mizushima N, Ueno T, Yamamoto A, Kirisako T, Noda T, Kominami E, Ohsumi Y et al (2000) LC3, a mammalian homologue of yeast Apg8p, is localized in autophagosome membranes after processing. EMBO J 19(21):5720–5728. doi:10.1093/emboj/19.21.5720 PubMedPubMedCentralCrossRefGoogle Scholar
  54. 54.
    Mogi M, Nagatsu T (1999) Neurotrophins and cytokines in Parkinson’s disease. Adv Neurol 80:135–139PubMedGoogle Scholar
  55. 55.
    Saha AR, Ninkina NN, Hanger DP, Anderton BH, Davies AM, Buchman VL (2000) Induction of neuronal death by alpha-synuclein. Eur J Neurosci 12(8):3073–3077PubMedCrossRefGoogle Scholar
  56. 56.
    Levy OA, Malagelada C, Greene LA (2009) Cell death pathways in Parkinson’s disease: proximal triggers, distal effectors, and final steps. Apoptosis 14(4):478–500. doi:10.1007/s10495-008-0309-3 PubMedPubMedCentralCrossRefGoogle Scholar
  57. 57.
    McGeer PL, McGeer EG (2008) The alpha-synuclein burden hypothesis of Parkinson disease and its relationship to Alzheimer disease. Exp Neurol 212(2):235–238. doi:10.1016/j.expneurol.2008.04.008 PubMedCrossRefGoogle Scholar
  58. 58.
    Pal R, Tiwari PC, Nath R, Pant KK (2016) Role of neuroinflammation and latent transcription factors in pathogenesis of Parkinson’s disease. Neurol Res 38(12):1111–1122. doi:10.1080/01616412.2016.1249997 PubMedCrossRefGoogle Scholar
  59. 59.
    Russo E, Andreozzi F, Iuliano R, Dattilo V, Procopio T, Fiume G, Mimmi S, Perrotti N et al (2014) Early molecular and behavioral response to lipopolysaccharide in the WAG/Rij rat model of absence epilepsy and depressive-like behavior, involves interplay between AMPK, AKT/mTOR pathways and neuroinflammatory cytokine release. Brain Behav Immun 42:157–168. doi:10.1016/j.bbi.2014.06.016 PubMedCrossRefGoogle Scholar
  60. 60.
    Saliba SW, Vieira EL, Santos RP, Candelario-Jalil E, Fiebich BL, Vieira LB, Teixeira AL, de Oliveira AC (2017) Neuroprotective effects of intrastriatal injection of rapamycin in a mouse model of excitotoxicity induced by quinolinic acid. J Neuroinflammation 14(1):25. doi:10.1186/s12974-017-0793-x PubMedPubMedCentralCrossRefGoogle Scholar
  61. 61.
    Citraro R, Leo A, Constanti A, Russo E, De Sarro G (2016) mTOR pathway inhibition as a new therapeutic strategy in epilepsy and epileptogenesis. Pharmacol Res 107:333–343. doi:10.1016/j.phrs.2016.03.039 PubMedCrossRefGoogle Scholar
  62. 62.
    Johnson GL, Lapadat R (2002) Mitogen-activated protein kinase pathways mediated by ERK, JNK, and p38 protein kinases. Science 298(5600):1911–1912. doi:10.1126/science.1072682 PubMedCrossRefGoogle Scholar
  63. 63.
    Gallo KA, Johnson GL (2002) Mixed-lineage kinase control of JNK and p38 MAPK pathways. Nat Rev Mol Cell Biol 3(9):663–672. doi:10.1038/nrm906 PubMedCrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media New York 2017

Authors and Affiliations

  • Rosalba Siracusa
    • 1
  • Irene Paterniti
    • 1
  • Marika Cordaro
    • 1
  • Rosalia Crupi
    • 1
  • Giuseppe Bruschetta
    • 1
  • Michela Campolo
    • 1
  • Salvatore Cuzzocrea
    • 1
    • 2
  • Emanuela Esposito
    • 1
  1. 1.Department of Chemical, Biological, Pharmaceutical and Environmental ScienceUniversity of MessinaMessinaItaly
  2. 2.Department of Pharmacological and Physiological ScienceSaint Louis University School of MedicineSaint LouisUSA

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