Pleiotrophin increases neurite length and number of spiral ganglion neurons in vitro

  • Sebastian Bertram
  • Lars Roll
  • Jacqueline Reinhard
  • Katharina Groß
  • Stefan Dazert
  • Andreas Faissner
  • Stefan VolkensteinEmail author
Research Article


Acoustic trauma, aging, genetic defects or ototoxic drugs are causes for sensorineural hearing loss involving sensory hair cell death and secondary degeneration of spiral ganglion neurons. Auditory implants are the only available therapy for severe to profound sensorineural hearing loss when hearing aids do not provide a sufficient speech discrimination anymore. Neurotrophic factors represent potential therapeutic candidates to improve the performance of cochlear implants (CIs) by the support of spiral ganglion neurons (SGNs). Here, we investigated the effect of pleiotrophin (PTN), a well-described neurotrophic factor for different types of neurons that is expressed in the postnatal mouse cochlea. PTN knockout mice exhibit severe deficits in auditory brainstem responses, which indicates the importance of PTN in inner ear development and function and makes it a promising candidate to support SGNs. Using organotypic explants and dissociated SGN cultures, we investigated the influence of PTN on the number of neurons, neurite number and neurite length. PTN significantly increased the number and neurite length of dissociated SGNs. We further verified the expression of important PTN-associated receptors in the SG. mRNA of anaplastic lymphoma kinase, αv integrin, β3 integrin, receptor protein tyrosine phosphatase β/ζ, neuroglycan C, low-density lipoprotein receptor-related protein 1 and syndecan 3 was detected in the inner ear. These results suggest that PTN may be a novel candidate to improve sensorineural hearing loss treatment in the future.


Cochlea Hearing loss Neurite growth Neurotrophin Organotypic explant 



Anaplastic lymphoma kinase


Brain-derived neurotrophic factor


Basic medium


Bone morphogenetic protein-2


Cochlear implant


Dorsal root ganglion


Glial cell line-derived neurotrophic factor


Heparin affinity regulatory peptide


Heparin-binding growth-associated molecule


Heparin-binding brain mitogen


Heparin-binding growth factor 8


Hair cell


Leukemia inhibitory factor




Low-density lipoprotein receptor-related protein




Neuroglycan C


Neurite growth-promoting factor 1






Postnatal day






Receptor protein tyrosine phosphatase β/ζ




Spiral ganglion


Spiral ganglion neuron


Spinal motor neuron



This study was supported by MED-EL Deutschland GmbH research grant PVBO2012/2. We gratefully acknowledge Susanne Kanabey and Sabine Kindermann for excellent technical assistance.

Compliance with ethical standards

Conflict of interest

The authors declare that they have no conflict of interest.

Supplementary material

221_2019_5644_MOESM1_ESM.docx (25 kb)
Supplementary material 1 (DOCX 25 kb)


  1. Afratis NA, Nikitovic D, Multhaupt HA, Theocharis AD, Couchman JR, Karamanos NK (2017) Syndecans-key regulators of cell signaling and biological functions. FEBS J 284:27–41CrossRefGoogle Scholar
  2. Aletsee C, Mullen L, Kim D, Pak K, Brors D, Dazert S, Ryan AF (2001) The disintegrin kistrin inhibits neurite extension from spiral ganglion explants cultured on laminin. Audiol Neurootol 6:57–65CrossRefGoogle Scholar
  3. Asai H, Yokoyama S, Morita S, Maeda N, Miyata S (2009) Functional difference of receptor-type protein tyrosine phosphatase zeta/beta isoforms in neurogenesis of hippocampal neurons. Neuroscience 164:1020–1030CrossRefGoogle Scholar
  4. Bodmer D, Gloddek B, Ryan AF, Huverstuhl J, Brors D (2002) Inhibition of the c-Jun N-terminal kinase signaling pathway influences neurite outgrowth of spiral ganglion neurons in vitro. Laryngoscope 112:2057–2061CrossRefGoogle Scholar
  5. Budenz CL, Pfingst BE, Raphael Y (2012) The use of neurotrophin therapy in the inner ear to augment cochlear implantation outcomes. Anat Rec (Hoboken) 295:1896–1908CrossRefGoogle Scholar
  6. Clement AM, Nadanaka S, Masayama K, Mandl C, Sugahara K, Faissner A (1998) The DSD-1 carbohydrate epitope depends on sulfation, correlates with chondroitin sulfate D motifs, and is sufficient to promote neurite outgrowth. J Biol Chem 273:28444–28453CrossRefGoogle Scholar
  7. Dodson HC, Mohuiddin A (2000) Response of spiral ganglion neurones to cochlear hair cell destruction in the guinea pig. J Neurocytol 29:525–537CrossRefGoogle Scholar
  8. DuBridge RB, Tang P, Hsia HC, Leong PM, Miller JH, Calos MP (1987) Analysis of mutation in human cells by using an Epstein-Barr virus shuttle system. Mol Cell Biol 1:379–387CrossRefGoogle Scholar
  9. Eshraghi AA, Nazarian R, Telischi FF, Rajguru SM, Truy E, Gupta C (2012) The cochlear implant: historical aspects and future prospects. Anat Rec (Hoboken) 295:1967–1980CrossRefGoogle Scholar
  10. Euteneuer S, Yang KH, Chavez E, Leichtle A, Loers G, Olshansky A, Pak K, Schachner M et al (2013) Glial cell line-derived neurotrophic factor (GDNF) induces neuritogenesis in the cochlear spiral ganglion via neural cell adhesion molecule (NCAM). Mol Cell Neurosci 54:30–43CrossRefGoogle Scholar
  11. Faissner A, Clement A, Lochter A, Streit A, Mandl C, Schachner M (1994) Isolation of a neural chondroitin sulfate proteoglycan with neurite outgrowth promoting properties. J Cell Biol 126:783–799CrossRefGoogle Scholar
  12. Furness DN (2015) Molecular basis of hair cell loss. Cell Tissue Res 361:387–399CrossRefGoogle Scholar
  13. Geleoc GS, Holt JR (2014) Sound strategies for hearing restoration. Science 344:1241062CrossRefGoogle Scholar
  14. Gillespie LN, Clark GM, Bartlett PF, Marzella PL (2001) LIF is more potent than BDNF in promoting neurite outgrowth of mammalian auditory neurons in vitro. NeuroReport 12:275–279CrossRefGoogle Scholar
  15. Hansen MR, Vijapurkar U, Koland JG, Green SH (2001a) Reciprocal signaling between spiral ganglion neurons and Schwann cells involves neuregulin and neurotrophins. Hear Res 161:87–98CrossRefGoogle Scholar
  16. Hansen MR, Zha XM, Bok J, Green SH (2001b) Multiple distinct signal pathways, including an autocrine neurotrophic mechanism, contribute to the survival-promoting effect of depolarization on spiral ganglion neurons in vitro. J Neurosci 21:2256–2267CrossRefGoogle Scholar
  17. Hida H, Jung CG, Wu CZ, Kim HJ, Kodama Y, Masuda T, Nishino H (2003) Pleiotrophin exhibits a trophic effect on survival of dopaminergic neurons in vitro. Eur J Neurosci 17:2127–2134CrossRefGoogle Scholar
  18. Hida H, Masuda T, Sato T, Kim TS, Misumi S, Nishino H (2007) Pleiotrophin promotes functional recovery after neural transplantation in rats. NeuroReport 18:179–183CrossRefGoogle Scholar
  19. Ichihara-Tanaka K, Oohira A, Rumsby M, Muramatsu T (2006) Neuroglycan C is a novel midkine receptor involved in process elongation of oligodendroglial precursor-like cells. J Biol Chem 281:30857–30864CrossRefGoogle Scholar
  20. Jin Y, Kondo K, Ushio M, Kaga K, Ryan AF, Yamasoba T (2013) Developmental changes in the responsiveness of rat spiral ganglion neurons to neurotrophic factors in dissociated culture: differential responses for survival, neuritogenesis and neuronal morphology. Cell Tissue Res 351:15–27CrossRefGoogle Scholar
  21. Kadomatsu K, Kishida S, Tsubota S (2013) The heparin-binding growth factor midkine: the biological activities and candidate receptors. J Biochem 153:511–521CrossRefGoogle Scholar
  22. Kowalik L, Hudspeth AJ (2011) A search for factors specifying tonotopy implicates DNER in hair-cell development in the chick’s cochlea. Dev Biol 354:221–231CrossRefGoogle Scholar
  23. Kwiatkowska M, Reinhard J, Roll L, Kraft N, Dazert S, Faissner A, Volkenstein S (2016) The expression pattern and inhibitory influence of Tenascin-C on the growth of spiral ganglion neurons suggest a regulatory role as boundary formation molecule in the postnatal mouse inner ear. Neuroscience 319:46–58CrossRefGoogle Scholar
  24. Leake PA, Hradek GT, Snyder RL (1999) Chronic electrical stimulation by a cochlear implant promotes survival of spiral ganglion neurons after neonatal deafness. J Comp Neurol 412:543–562CrossRefGoogle Scholar
  25. Lillis AP, Van Duyn LB, Murphy-Ullrich JE, Strickland DK (2008) LDL receptor-related protein 1: unique tissue-specific functions revealed by selective gene knockout studies. Physiol Rev 88:887–918CrossRefGoogle Scholar
  26. Maeda N, Noda M (1998) Involvement of receptor-like protein tyrosine phosphatase zeta/RPTPbeta and its ligand pleiotrophin/heparin-binding growth-associated molecule (HB-GAM) in neuronal migration. J Cell Biol 142:203–216CrossRefGoogle Scholar
  27. Meijering E, Jacob M, Sarria JC, Steiner P, Hirling H, Unser M (2004) Design and validation of a tool for neurite tracing and analysis in fluorescence microscopy images. Cytometry A 58:167–176CrossRefGoogle Scholar
  28. Mi R, Chen W, Hoke A (2007) Pleiotrophin is a neurotrophic factor for spinal motor neurons. Proc Natl Acad Sci USA 104:4664–4669CrossRefGoogle Scholar
  29. Mikelis C, Sfaelou E, Koutsioumpa M, Kieffer N, Papadimitriou E (2009) Integrin alpha(v)beta(3) is a pleiotrophin receptor required for pleiotrophin-induced endothelial cell migration through receptor protein tyrosine phosphatase beta/zeta. FASEB J 23:1459–1469CrossRefGoogle Scholar
  30. Miller JM, Chi DH, O’Keeffe LJ, Kruszka P, Raphael Y, Altschuler RA (1997) Neurotrophins can enhance spiral ganglion cell survival after inner hair cell loss. Int J Dev Neurosci 15:631–643CrossRefGoogle Scholar
  31. Milner PG, Li YS, Hoffman RM, Kodner CM, Siegel NR, Deuel TF (1989) A novel 17 kD heparin-binding growth factor (HBGF-8) in bovine uterus: purification and N-terminal amino acid sequence. Biochem Biophys Res Commun 165:1096–1103CrossRefGoogle Scholar
  32. Muramatsu H, Zou K, Sakaguchi N, Ikematsu S, Sakuma S, Muramatsu T (2000) LDL receptor-related protein as a component of the midkine receptor. Biochem Biophys Res Commun 270:936–941CrossRefGoogle Scholar
  33. Nakanishi K, Aono S, Hirano K, Kuroda Y, Ida M, Tokita Y, Matsui F, Oohira A (2006) Identification of neurite outgrowth-promoting domains of neuroglycan C, a brain-specific chondroitin sulfate proteoglycan, and involvement of phosphatidylinositol 3-kinase and protein kinase C signaling pathways in neuritogenesis. J Biol Chem 281:24970–24978CrossRefGoogle Scholar
  34. Nayagam BA, Muniak MA, Ryugo DK (2011) The spiral ganglion: connecting the peripheral and central auditory systems. Hear Res 278:2–20CrossRefGoogle Scholar
  35. Nguyen TAK, Cavuscens S, Ranieri M, Schwarz K, Guinand N, van de Berg R, van den Boogert T, Lucieer F et al (2017) Characterization of cochlear, vestibular and cochlear-vestibular electrically evoked compound action potentials in patients with a vestibulo-cochlear implant. Front Neurosci 11:645CrossRefGoogle Scholar
  36. Paveliev M, Fenrich KK, Kislin M, Kuja-Panula J, Kulesskiy E, Varjosalo M, Kajander T, Mugantseva E et al (2016) HB-GAM (pleiotrophin) reverses inhibition of neural regeneration by the CNS extracellular matrix. Sci Rep 6:33916CrossRefGoogle Scholar
  37. Pfingst BE, Colesa DJ, Swiderski DL, Hughes AP, Strahl SB, Sinan M, Raphael Y (2017) neurotrophin gene therapy in deafened ears with cochlear implants: long-term effects on nerve survival and functional measures. J Assoc Res Otolaryngol 18:731–750CrossRefGoogle Scholar
  38. Raulo E, Chernousov MA, Carey DJ, Nolo R, Rauvala H (1994) Isolation of a neuronal cell surface receptor of heparin binding growth-associated molecule (HB-GAM). Identification as N-syndecan (syndecan-3). J Biol Chem 269:12999–13004Google Scholar
  39. Rauvala H (1989) An 18-kd heparin-binding protein of developing brain that is distinct from fibroblast growth factors. EMBO J 8:2933–2941CrossRefGoogle Scholar
  40. Rauvala H, Vanhala A, Castren E, Nolo R, Raulo E, Merenmies J, Panula P (1994) Expression of HB-GAM (heparin-binding growth-associated molecules) in the pathways of developing axonal processes in vivo and neurite outgrowth in vitro induced by HB-GAM. Brain Res Dev Brain Res 79:157–176CrossRefGoogle Scholar
  41. Sone M, Muramatsu H, Muramatsu T, Nakashima T (2011) Morphological observation of the stria vascularis in midkine and pleiotrophin knockout mice. Auris Nasus Larynx 38:41–45CrossRefGoogle Scholar
  42. Volkenstein S, Brors D, Hansen S, Minovi A, Laub M, Jennissen HP, Dazert S, Neumann A (2009) Influence of bone morphogenetic protein-2 on spiral ganglion neurite growth in vitro. Eur Arch Otorhinolaryngol 266:1381–1389CrossRefGoogle Scholar
  43. Volkenstein S, Kirkwood JE, Lai E, Dazert S, Fuller GG, Heller S (2012) Oriented collagen as a potential cochlear implant electrode surface coating to achieve directed neurite outgrowth. Eur Arch Otorhinolaryngol 269:1111–1116CrossRefGoogle Scholar
  44. Wang Y, Qiu B, Liu J, Zhu WG, Zhu S (2014) Cocaine- and amphetamine-regulated transcript facilitates the neurite outgrowth in cortical neurons after oxygen and glucose deprivation through PTN-dependent pathway. Neuroscience 277:103–110CrossRefGoogle Scholar
  45. Whitlon DS, Grover M, Tristano J, Williams T, Coulson MT (2007) Culture conditions determine the prevalence of bipolar and monopolar neurons in cultures of dissociated spiral ganglion. Neuroscience 146:833–840CrossRefGoogle Scholar
  46. Xu C, Zhu S, Wu M, Han W, Yu Y (2014) Functional receptors and intracellular signal pathways of midkine (MK) and pleiotrophin (PTN). Biol Pharm Bull 37:511–520CrossRefGoogle Scholar
  47. Yamauchi K, Yamauchi T, Mantuano E, Murakami K, Henry K, Takahashi K, Campana WM (2013) Low-density lipoprotein receptor related protein-1 (LRP1)-dependent cell signaling promotes neurotrophic activity in embryonic sensory neurons. PLoS One 8:e75497CrossRefGoogle Scholar
  48. Yao J, Zhang M, Ma QY, Wang Z, Wang LC, Zhang D (2011) PAd-shRNA-PTN reduces pleiotrophin of pancreatic cancer cells and inhibits neurite outgrowth of DRG. World J Gastroenterol 17:2667–2673CrossRefGoogle Scholar
  49. Yao J, Li WY, Li SG, Feng XS, Gao SG (2014) Recombinant lentivirus targeting the pleotrophin gene reduces pleotrophin protein expression in pancreatic cancer cells and inhibits neurite outgrowth of dorsal root ganglion neurons. Mol Med Rep 9:999–1004CrossRefGoogle Scholar
  50. Zhang KD, Coate TM (2017) Recent advances in the development and function of type II spiral ganglion neurons in the mammalian inner ear. Semin Cell Dev Biol 65:80–87CrossRefGoogle Scholar
  51. Zheng QY, Johnson KR, Erway LC (1999) Assessment of hearing in 80 inbred strains of mice by ABR threshold analyses. Hear Res 130:94–107CrossRefGoogle Scholar
  52. Zou P, Muramatsu H, Sone M, Hayashi H, Nakashima T, Muramatsu T (2006) Mice doubly deficient in the midkine and pleiotrophin genes exhibit deficits in the expression of beta-tectorin gene and in auditory response. Lab Invest 86:645–653CrossRefGoogle Scholar

Copyright information

© Springer-Verlag GmbH Germany, part of Springer Nature 2019

Authors and Affiliations

  1. 1.Department of Otorhinolaryngology, Head and Neck Surgery, St. Elisabeth-HospitalRuhr-University BochumBochumGermany
  2. 2.Department of Cell Morphology and Molecular Neurobiology, Faculty of Biology and BiotechnologyRuhr-University BochumBochumGermany

Personalised recommendations