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Detecting BDNF Protein Forms by ELISA, Western Blot, and Immunofluorescence

  • Stefano Donegà
  • Enrico TongiorgiEmail author
Protocol
Part of the Neuromethods book series (NM, volume 143)

Abstract

The neurotrophin brain-derived neurotrophic factor (BDNF) is extensively expressed in neural cells and other cell types. BDNF displays a support function in both central CNS and peripheral PNS where it is involved in most neuronal mechanisms, from development to neurodegeneration, cellular growth, neuroprotection, survival, and network signaling. In the last decades, decreased levels of BDNF were directly linked to Parkinson’s, Alzheimer’s, and Huntington’s disease, while BDNF upregulation was found in patients affected by epilepsy, autism, and multiple sclerosis. Accordingly, determination of BDNF expression can contribute to gain insight in several pathological models both in vivo and in vitro. In this manuscript, we provide detailed description of the main techniques for BDNF detection in cultured neurons and in brain slices, using different methods such as ELISA, Western blot, and immunofluorescence.

Keywords

BDNF pro-peptide Brain-derived neurotrophic factor Mature BDNF Neurotrophins proBDNF Proteolytic processing Truncated BDNF 

Notes

Acknowledgments

The authors thank Dr. Elsa Fabbretti for critically reading the manuscript. This work has been supported by the University of Trieste internal funds FRA 2016 to E.T.

References

  1. 1.
    Barde YA, Edgar D, Thoenen H (1982) Purification of a new neurotrophic factor from mammalian brain. EMBO J 1(5):549–553CrossRefGoogle Scholar
  2. 2.
    Mizui T, Washikawa Y, Kumanogoh H, Lume M, Matsumoto T, Hara T, Yamawaki S, Takahashi M, Shiosaka S, Itami C, Uega ki K, Saarma M, Kojima M (2015) BDNF pro-peptide actions facilitate hippocampal LTD and are altered by the common BDNF polymorphism Val66Met. Proc Natl Acad Sci U S A 112(23):E3067–E3074CrossRefGoogle Scholar
  3. 3.
    Ibanez CF (1998) Emerging themes in structural biology of neurotrophic factors. Trends Neurosci 21:438–444CrossRefGoogle Scholar
  4. 4.
    Mowla SJ, Farhadi HF, Pareek S, Atwal JK, Morris SJ, Seidah NG, Murphy RA (2001) Biosynthesis and post-translational processing of the precursor to brain-derived neurotrophic factor. J Biol Chem 276(16):12660–12666CrossRefGoogle Scholar
  5. 5.
    Lee R, Kermani P, Teng KK, Hempstead BL (2001) Regulation of cell survival by secreted proneurotrophins. Science 294(5548):1945–1948CrossRefGoogle Scholar
  6. 6.
    Seidah NG, Mowla SJ, Hamelin J, Mamarbachi AM, Benjannet S, Touré BB, Basak A, Munzer JS, Marcinkiewicz J, Zhong M, Barale JC, Lazure C, Murphy RA, Chrétien M, Marcinkiewicz M (1999) Mammalian subtilisin/kexin isozyme SKI-1: a widely expressed proprotein convertase with a unique cleavage specificity and cellular localization. Proc Natl Acad Sci U S A 96(4):1321–1326CrossRefGoogle Scholar
  7. 7.
    Greenberg ME, Xu B, Lu B, Hempstead BL (2009) New insights in the biology of BDNF synthesis and release: implications in CNS function. J Neurosci 29(41):12764–12767CrossRefGoogle Scholar
  8. 8.
    Koshimizu H, Kiyosue K, Hara T, Hazama S, Suzuki S, Uegaki K, Nagappan G, Zaitsev E, Hirokawa T, Tatsu Y, Ogura A, Lu B, Kojima M (2009) Multiple functions of precursor BDNF to CNS neurons: negative regulation of neurite growth, spine formation and cell survival. Mol Brain 2:27CrossRefGoogle Scholar
  9. 9.
    Teng HK, Teng KK, Lee R, Wright S, Tevar S, Almeida RD, Kermani P, Torkin R, Chen ZY, Lee FS, Kraemer RT, Nykjaer A, Hempstead BL (2005) ProBDNF induces neuronal apoptosis via activation of a receptor complex of p75NTR and sortilin. J Neurosci 25(22):5455–5463CrossRefGoogle Scholar
  10. 10.
    Woo NH, Teng HK, Siao CJ, Chiaruttini C, Pang PT, Milner TA, Hempstead BL, Lu B (2005) Activation of p75NTR by proBDNF facilitates hippocampal long-term depression. Nat Neurosci 8(8):1069–1077CrossRefGoogle Scholar
  11. 11.
    Sermasi E, Margotti E, Cattaneo A, Domenici L (2000) Trk B signalling controls LTP but not LTD expression in the developing rat visual cortex. Eur J Neurosci 12(4):1411–1419CrossRefGoogle Scholar
  12. 12.
    Nagappan G, Zaitsev E, Senatorov VV Jr, Yang J, Hempstead BL, Lu B (2009) Control of extracellular cleavage of ProBDNF by high frequency neuronal activity. Proc Natl Acad Sci U S A 106(4):1267–1272CrossRefGoogle Scholar
  13. 13.
    Barnes P, Thomas KL (2008) Proteolysis of proBDNF is a key regulator in the formation of memory. PLoS One 3(9):e3248CrossRefGoogle Scholar
  14. 14.
    Mizui T, Ohira K, Kojima M (2017) BDNF pro-peptide: a novel synaptic modulator generated as an N-terminal fragment from the BDNF precursor by proteolytic processing. Neural Regen Res 12(7):1024–1027CrossRefGoogle Scholar
  15. 15.
    Carlino D, Leone E, Di Cola F, Baj G, Marin R, Dinelli G, Tongiorgi E, De Vanna M (2011) Low serum truncated-BDNF isoform correlates with higher cognitive impairment in schizophrenia. J Psychiatr Res 45(2):273–279CrossRefGoogle Scholar
  16. 16.
    Garcia KL, Yu G, Nicolini C, Michalski B, Garzon DJ, Chiu VS, Tongiorgi E, Szatmari P, Fahnestock M (2012) Altered balance of proteolytic isoforms of pro-brain-derived neurotrophic factor in autism. J Neuropathol Exp Neurol 71(4):289–297CrossRefGoogle Scholar
  17. 17.
    Carlino D, De Vanna M, Tongiorgi E (2012) Is altered BDNF biosynthesis a general feature in patients with cognitive dysfunctions? Neuroscientist 19(4):345–353CrossRefGoogle Scholar
  18. 18.
    Aid T, Kazantseva A, Piirsoo M, Palm K, Timmusk T (2007) Mouse and rat BDNF gene structure and expression revisited. J Neurosci Res 85(3):525–535CrossRefGoogle Scholar
  19. 19.
    Pruunsild P, Kazantseva A, Aid T, Palm K, Timmusk T (2007) Dissecting the human BDNF locus: bidirectional transcription, complex splicing, and multiple promoters. Genomics 90(3):397–406CrossRefGoogle Scholar
  20. 20.
    Matsuda N, Lu H, Fukata Y, Noritake J, Gao H, Mukherjee S, Nemoto T, Fukata M, Poo MM (2009) Differential activity-dependent secretion of brain-derived neurotrophic factor from axon and dendrite. J Neurosci 29(45):14185–14198CrossRefGoogle Scholar
  21. 21.
    Dieni S, Matsumoto T, Dekkers M, Rauskolb S, Ionescu MS, Deogracias R, Gundelfinger ED, Kojima M, Nestel S, Frotscher M, Barde YA (2012) BDNF and its pro-peptide are stored in presynaptic dense core vesicles in brain neurons. J Cell Biol 196(6):775–788CrossRefGoogle Scholar
  22. 22.
    Simonato M, Bregola G, Armellin M, Del Piccolo P, Rodi D, Zucchini S, Tongiorgi E (2002) Dendritic targeting of mRNAs for plasticity genes in experimental models of temporal lobe epilepsy. Epilepsia 43(Suppl 5):153–158CrossRefGoogle Scholar
  23. 23.
    Tongiorgi E, Armellin M, Giulianini PG, Bregola G, Zucchini S, Paradiso B, Steward O, Cattaneo A, Simonato M (2004) Brain-derived neurotrophic factor mRNA and protein are targeted to discrete dendritic laminas by events that trigger epileptogenesis. J Neurosci 24(30):6842–6852CrossRefGoogle Scholar
  24. 24.
    Edelmann E, Lessmann V, Brigadski T (2014) Pre- and postsynaptic twists in BDNF secretion and action in synaptic plasticity. Neuropharmacology 76 Pt C:610–627CrossRefGoogle Scholar
  25. 25.
    Polacchini A, Metelli G, Francavilla R, Baj G, Florean M, Mascaretti LG, Tongiorgi E (2015) A method for reproducible measurements of serum BDNF: comparison of the performance of six commercial assays. Sci Rep 5:17989CrossRefGoogle Scholar
  26. 26.
    Baj G, Patrizio A, Montalbano A, Sciancalepore M, Tongiorgi E (2014) Developmental and maintenance defects in Rett syndrome neurons identified by a new mouse staging system in vitro. Front Cell Neurosci 8:18CrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media New York 2018

Authors and Affiliations

  1. 1.Department of Life Sciences, BRAIN Center for NeuroscienceUniversity of TriesteTriesteItaly

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