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Hedgehog Signaling Modulates the Release of Gliotransmitters from Cultured Cerebellar Astrocytes

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Abstract

Sonic hedgehog (Shh), a member of the Hedgehog (Hh) family, plays essential roles in the development of the central nervous system. Recent studies suggest that the Hh signaling pathway also functions in mature astrocytes under physiological conditions. We first examined the expression of genes encoding Hh signaling molecules in the adult mouse cerebellum by in situ hybridization histochemistry. mRNA for Patched homolog 1 (Ptch1), a receptor for Hh family members, was expressed in S100β-positive astrocytes and Shh mRNA was expressed in HuC/D-positive neurons, implying that the Hh signaling pathway contributes to neuro-glial interactions. To test this hypothesis, we next examined the effects of recombinant SHH N-terminal protein (rSHH-N) on the functions of cultured cerebellar astrocytes. rSHH-N up-regulated Hh signal target genes such as Ptch1 and Gli-1, a key transcription factor of the Hh signaling pathway. Although activation of Hh signaling by rSHH-N or purmorphamine influenced neither glutamate uptake nor gliotransmitters release, inhibition of the Hh signaling pathway by cyclopamine, neutralizing antibody against SHH or intracellular Ca2+ chelation decreased glutamate and ATP release from cultured cerebellar astrocytes. On the other hand, cyclopamine, neutralizing antibody against SHH or Ca2+ chelator hardly affected d-serine secretion. Various kinase inhibitors attenuated glutamate and ATP release, while only U0126 reduced d-serine secretion from the astrocytes. These results suggested that the Hh signaling pathway sustains the release of glutamate and ATP and participates in neuro-glial interactions in the adult mouse brain. We also propose that signaling pathways distinct from the Hh pathway govern d-serine secretion from adult cerebellar astrocytes.

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Abbreviations

AKT:

RAC-alpha serine/threonine-protein kinase

BAPTA-AM:

1,2-Bis-(2-aminophenoxy)ethane-Af,N,N′,N′-tetraacetic acid acetoxymethyl ester

[Ca2+]i :

Intracellular Ca2+ concentration

DMEM:

Dulbecco’s Modified Eagle’s Medium

Gfap:

Glial fibrillary acidic protein

Glast:

Glutamate/aspartate transporter

Gli-1:

GLI-Kruppel family member GLI1

Glt-1:

Glutamate transporter 1

Gs:

Glutamine synthetase

Hh:

Hedgehog

JNK:

c-Jun N-terminal kinase

KRS:

Krebs–Ringer solution

MAPK:

Mitogen-activated protein kinase

MEK:

MAP/extracellular signal-regulated kinase

PI3K:

Phosphatidylinositol 3-kinase

PLL:

Poly-l-lysine

Ptch1:

Patched homolog 1

Shh:

Sonic hedgehog

Smo:

Smoothened homolog

TuJ1:

Beta III tubulin

rSHH-N:

Recombinant SHH N-terminal

References

  1. Ingham PW, McMahon AP (2001) Hedgehog signaling in animal development: paradigms and principles. Genes Dev 15:3059–3087

    Article  CAS  PubMed  Google Scholar 

  2. Petrova R, Joyner AL (2014) Roles for Hedgehog signaling in adult organ homeostasis and repair. Development 141:3445–3457

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  3. Wang Y, Zhou Z, Walsh CT, McMahon AP (2009) Selective translocation of intracellular Smoothened to the primary cilium in response to Hedgehog pathway modulation. Proc Natl Acad Sci USA 106:2623–2628

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  4. Dessaud E, McMahon AP, Briscoe J (2008) Pattern formation in the vertebrate neural tube: a sonic hedgehog morphogen-regulated transcriptional network. Development 135:2489–2503

    Article  CAS  PubMed  Google Scholar 

  5. Chiang C, Litingtung Y, Lee E, Young KE, Corden JL, Westphal H, Beachy PA (1996) Cyclopia and defective axial patterning in mice lacking Sonic hedgehog gene function. Nature 383:407–413

    Article  CAS  PubMed  Google Scholar 

  6. Martí E, Bovolenta P (2002) Sonic hedgehog in CNS development: one signal, multiple outputs. Trends Neurosci 25:89–96

    Article  PubMed  Google Scholar 

  7. Martí E, Bumcrot DA, Takada R, McMahon AP (1995) Requirement of 19 K form of Sonic hedgehog for induction of distinct ventral cell types in CNS explants. Nature 375:322–325

    Article  PubMed  Google Scholar 

  8. Roelink H, Porter JA, Chiang C, Tanabe Y, Chang DT, Beachy PA, Jessell TM (1995) Floor plate and motor neuron induction by different concentrations of the amino-terminal cleavage product of sonic hedgehog autoproteolysis. Cell 81:445–455

    Article  CAS  PubMed  Google Scholar 

  9. Tanabe Y, Roelink H, Jessell T (1995) Induction of motor neurons by sonic hedgehog is independent of floor plate differentiation. Curr Biol 5:651–658

    Article  CAS  PubMed  Google Scholar 

  10. Kohtz JD, Baker DP, Corte G, Fishell G (1998) Regionalization within the mammalian telencephalon is mediated by changes in responsiveness to Sonic Hedgehog. Development 125:5079–5089

    CAS  PubMed  Google Scholar 

  11. Nery S, Wichterle H, Fishell G (2001) Sonic hedgehog contributes to oligodendrocyte specification in the mammalian forebrain. Development 128:527–540

    CAS  PubMed  Google Scholar 

  12. Tekki-Kessaris N, Woodruff R, Hall AC, Gaffield W, Kimura S, Stiles CD, Rowitch DH, Richardson WD (2001) Hedgehog-dependent oligodendrocyte lineage specification in the telencephalon. Development 128:2545–2554

    CAS  PubMed  Google Scholar 

  13. Lai K, Kaspar BK, Gage FH, Schaffer DV (2003) Sonic hedgehog regulates adult neural progenitor proliferation in vitro and in vivo. Nat Neurosci 6:21–27

    Article  CAS  PubMed  Google Scholar 

  14. Machold R, Hayashi S, Rutlin M, Muzumdar MD, Nery S, Corbin JG, Gritli-Linde A, Dellovade T, Porter JA, Rubin LL, Dudek H, McMahon AP, Fishell G (2003) Sonic hedgehog is required for progenitor cell maintenance in telencephalic stem cell niches. Neuron 39:937–950

    Article  CAS  PubMed  Google Scholar 

  15. Ahn S, Joyner AL (2005) In vivo analysis of quiescent adult neural stem cells responding to Sonic hedgehog. Nature 437:894–897

    Article  CAS  PubMed  Google Scholar 

  16. Dahmane N, Ruiz i Altaba A (1999) Sonic hedgehog regulates the growth and patterning of the cerebellum. Development 126:3089–3100

    PubMed  Google Scholar 

  17. Wallace VA (1999) Purkinje-cell-derived Sonic hedgehog regulates granule neuron precursor cell proliferation in the developing mouse cerebellum. Curr Biol 9:445–448

    Article  CAS  PubMed  Google Scholar 

  18. Wechsler-Reya RJ, Scott MP (1999) Control of neuronal precursor proliferation in the cerebellum by Sonic Hedgehog. Neuron 22:103–114

    Article  CAS  PubMed  Google Scholar 

  19. Wallace VA, Raff MC (1999) A role for Sonic hedgehog in axon-to-astrocyte signaling in the rodent optic nerve. Development 126:2901–2909

    CAS  PubMed  Google Scholar 

  20. Garcia AD, Petrova R, Eng L, Joyner AL (2010) Sonic hedgehog regulates discrete populations of astrocytes in the adult mouse forebrain. J Neurosci 30:13597–13608

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  21. Cahoy JD, Emery B, Kaushal A, Foo LC, Zamanian JL, Christopherson KS, Xing Y, Lubischer JL, Krieg PA, Krupenko SA, Thompson WJ, Barres BA (2008) A transcriptome database for astrocytes, neurons, and oligodendrocytes: a new resource for understanding brain development and function. J Neurosci 28:264–278

    Article  CAS  PubMed  Google Scholar 

  22. Newman EA (2003) New roles for astrocytes: regulation of synaptic transmission. Trends Neurosci 26:536–542

    Article  CAS  PubMed  Google Scholar 

  23. Hamilton NB, Attwell D (2010) Do astrocytes really exocytose neurotransmitters? Nat Rev Neurosci 11:227–238

    Article  CAS  PubMed  Google Scholar 

  24. Araque A, Carmignoto G, Haydon PG, Oliet SH, Robitaille R, Volterra A (2014) Gliotransmitters travel in time and space. Neuron 81:728–739

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  25. Araque A, Li N, Doyle RT, Haydon PG (2000) SNARE protein-dependent glutamate release from astrocytes. J Neurosci 20:666–673

    CAS  PubMed  Google Scholar 

  26. Pasti L, Zonta M, Pozzan T, Vicini S, Carmignoto G (2001) Cytosolic calcium oscillations in astrocytes may regulate exocytotic release of glutamate. J Neurosci 21:477–484

    CAS  PubMed  Google Scholar 

  27. Pascual O, Traiffort E, Baker DP, Galdes A, Ruat M, Champagnat J (2005) Sonic hedgehog signalling in neurons of adult ventrolateral nucleus tractus solitarius. Eur J Neurosci 22:389–396

    Article  PubMed  Google Scholar 

  28. Jourdain P, Bergersen LH, Bhaukaurally K, Bezzi P, Santello M, Domercq M, Matute C, Tonello F, Gundersen V, Volterra A (2007) Glutamate exocytosis from astrocytes controls synaptic strength. Nat Neurosci 10:331–339

    Article  CAS  PubMed  Google Scholar 

  29. Henneberger C, Papouin T, Oliet SH, Rusakov DA (2010) Long-term potentiation depends on release of d-serine from astrocytes. Nature 463:232–236

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  30. Halassa MM, Florian C, Fellin T, Munoz JR, Lee SY, Abel T, Haydon PG, Frank MG (2009) Astrocytic modulation of sleep homeostasis and cognitive consequences of sleep loss. Neuron 61:213–219

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  31. Araque A, Parpura V, Sanzgiri RP, Haydon PG (1999) Tripartite synapses: glia, the unacknowledged partner. Trends Neurosci 22:208–215

    Article  CAS  PubMed  Google Scholar 

  32. Halassa MM, Fellin T, Haydon PG (2009) Tripartite synapses: roles for astrocytic purines in the control of synaptic physiology and behavior. Neuropharmacology 57:343–346

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  33. Perea G, Navarrete M, Araque A (2009) Tripartite synapses: astrocytes process and control synaptic information. Trends Neurosci 32:421–431

    Article  CAS  PubMed  Google Scholar 

  34. Okuda H, Tatsumi K, Morita S, Shibukawa Y, Korekane H, Horii-Hayashi N, Wada Y, Taniguchi N, Wanaka A (2014) Chondroitin sulfate proteoglycan tenascin-R regulates glutamate uptake by adult brain astrocytes. J Biol Chem 289:2620–2631

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  35. Okuda H, Tatsumi K, Horii-Hayashi N, Morita S, Okuda-Yamamoto A, Imaizumi K, Wanaka A (2014) OASIS regulates chondroitin 6-O-sulfotransferase 1 gene transcription in the injured adult mouse cerebral cortex. J Neurochem 130:612–625

    Article  CAS  PubMed  Google Scholar 

  36. Nakamura K, Sasajima J, Mizukami Y, Sugiyama Y, Yamazaki M, Fujii R, Kawamoto T, Koizumi K, Sato K, Fujiya M, Sasaki K, Tanno S, Okumura T, Shimizu N, Kawabe J, Karasaki H, Kono T, Ii M, Bardeesy N, Chung DC, Kohgo Y (2010) Hedgehog promotes neovascularization in pancreatic cancers by regulating Ang-1 and IGF-1 expression in bone-marrow derived pro-angiogenic cells. PLoS ONE 5:e8824

    Article  PubMed  PubMed Central  Google Scholar 

  37. Ribes V, Balaskas N, Sasai N, Cruz C, Dessaud E, Cayuso J, Tozer S, Yang LL, Novitch B, Marti E, Briscoe J (2010) Distinct Sonic Hedgehog signaling dynamics specify floor plate and ventral neuronal progenitors in the vertebrate neural tube. Genes Dev 24:1186–1200

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  38. Atkinson PJ, Dellovade T, Albers D, Von Schack D, Saraf K, Needle E, Reinhart PH, Hirst WD (2009) Sonic Hedgehog signaling in astrocytes is dependent on p38 mitogen-activated protein kinase and G-protein receptor kinase 2. J Neurochem 108:1539–1549

    Article  CAS  PubMed  Google Scholar 

  39. Sinha S, Chen JK (2006) Purmorphamine activates the Hedgehog pathway by targeting Smoothened. Nat Chem Biol 2:29–30

    Article  CAS  PubMed  Google Scholar 

  40. Incardona JP, Gaffield W, Kapur RP, Roelink H (1998) The teratogenic Veratrum alkaloid cyclopamine inhibits sonic hedgehog signal transduction. Development 125:3553–3562

    CAS  PubMed  Google Scholar 

  41. Bezzi P, Carmignoto G, Pasti L, Vesce S, Rossi D, Rizzini BL, Pozzan T, Volterra A (1998) Prostaglandins stimulate calcium-dependent glutamate release in astrocytes. Nature 391:281–285

    Article  CAS  PubMed  Google Scholar 

  42. Coco S, Calegari F, Pravettoni E, Pozzi D, Taverna E, Rosa P, Matteoli M, Verderio C (2003) Storage and release of ATP from astrocytes in culture. J Biol Chem 278:1354–1362

    Article  CAS  PubMed  Google Scholar 

  43. Osawa H, Ohnishi H, Takano K, Noguti T, Mashima H, Hoshino H, Kita H, Sato K, Matsui H, Sugano K (2006) Sonic hedgehog stimulates the proliferation of rat gastric mucosal cells through ERK activation by elevating intracellular calcium concentration. Biochem Biophys Res Commun 344:680–687

    Article  CAS  PubMed  Google Scholar 

  44. Innocenti B, Parpura V, Haydon PG (2000) Imaging extracellular waves of glutamate during calcium signaling in cultured astrocytes. J Neurosci 20:1800–1808

    CAS  PubMed  Google Scholar 

  45. Elia D, Madhala D, Ardon E, Reshef R, Halevy O (2007) Sonic hedgehog promotes proliferation and differentiation of adult muscle cells: involvement of MAPK/ERK and PI3K/Akt pathways. Biochim Biophys Acta 1773:1438–1446

    Article  CAS  PubMed  Google Scholar 

  46. Madhala-Levy D, Williams VC, Hughes SM, Reshef R, Halevy O (2012) Cooperation between Shh and IGF-I in promoting myogenic proliferation and differentiation via the MAPK/ERK and PI3K/Akt pathways requires smo activity. J Cell Physiol 227:1455–1464

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  47. Takeuchi H, Jin S, Wang J, Zhang G, Kawanokuchi J, Kuno R, Sonobe Y, Mizuno T, Suzumura A (2006) Tumor necrosis factor-alpha induces neurotoxicity via glutamate release from hemichannels of activated microglia in an autocrine manner. J Biol Chem 281:21362–21368

    Article  CAS  PubMed  Google Scholar 

  48. Harwell CC, Parker PR, Gee SM, Okada A, McConnell SK, Kreitzer AC, Kriegstein AR (2012) Sonic hedgehog expression in corticofugal projection neurons directs cortical microcircuit formation. Neuron 73:1116–1126

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  49. Amankulor NM, Hambardzumyan D, Pyonteck SM, Becher OJ, Joyce JA, Holland EC (2009) Sonic hedgehog pathway activation is induced by acute brain injury and regulated by injury-related inflammation. J Neurosci 29:10299–10308

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  50. Pitter KL, Tamagno I, Feng X, Ghosal K, Amankulor N, Holland EC, Hambardzumyan D (2014) The SHH/Gli pathway is reactivated in reactive glia and drives proliferation in response to neurodegeneration-induced lesions. Glia 62:1595–1607

    Article  PubMed  PubMed Central  Google Scholar 

  51. Jin Y, Raviv N, Barnett A, Bambakidis NC, Filichia E, Luo Y (2015) The shh signaling pathway is upregulated in multiple cell types in cortical ischemia and influences the outcome of stroke in an animal model. PLoS ONE 10:e0124657

    Article  PubMed  PubMed Central  Google Scholar 

  52. Foo LC, Allen NJ, Bushong EA, Ventura PB, Chung WS, Zhou L, Cahoy JD, Daneman R, Zong H, Ellisman MH, Barres BA (2011) Development of a method for the purification and culture of rodent astrocytes. Neuron 71:799–811

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  53. Pascual O, Casper KB, Kubera C, Zhang J, Revilla-Sanchez R, Sul JY, Takano H, Moss SJ, McCarthy K, Haydon PG (2005) Astrocytic purinergic signaling coordinates synaptic networks. Science 310:113–116

    Article  CAS  PubMed  Google Scholar 

  54. Bezard E, Baufreton J, Owens G, Crossman AR, Dudek H, Taupignon A, Brotchie JM (2003) Sonic hedgehog is a neuromodulator in the adult subthalamic nucleus. FASEB J 17:2337–2338

    CAS  PubMed  Google Scholar 

  55. Montana V, Malarkey EB, Verderio C, Matteoli M, Parpura V (2006) Vesicular transmitter release from astrocytes. Glia 54:700–715

    Article  PubMed  Google Scholar 

  56. Ruiz-Gómez A, Molnar C, Holguín H, Mayor F Jr, de Celis JF (2007) The cell biology of Smo signalling and its relationships with GPCRs. Biochim Biophys Acta 1768:901–912

    Article  PubMed  Google Scholar 

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Acknowledgments

This work was supported by the Japan Society for the Promotion of Science (Grant Numbers 26293039 and 15K14354 to AW), and by a research fund kindly provided by the Takeda Science Foundation to AW. We thank Dr. Ian Smith (Elite Scientific Editing, UK) for his thorough manuscript editing.

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Authors and Affiliations

  1. Department of Anatomy and Neuroscience, Faculty of Medicine, Nara Medical University, Kashihara City, Nara, 634-8521, Japan

    Hiroaki Okuda, Kouko Tatsumi, Shoko Morita-Takemura, Kazuki Nakahara & Akio Wanaka

  2. Department of Ophthalmology, Faculty of Medicine, Nara Medical University, Kashihara City, Nara, 634-8521, Japan

    Katsunori Nochioka

  3. Department of Anesthesiology, Faculty of Medicine, Nara Medical University, Kashihara City, Nara, 634-8521, Japan

    Takeaki Shinjo & Yuki Terada

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  1. Hiroaki Okuda
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Correspondence to Hiroaki Okuda.

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Okuda, H., Tatsumi, K., Morita-Takemura, S. et al. Hedgehog Signaling Modulates the Release of Gliotransmitters from Cultured Cerebellar Astrocytes. Neurochem Res 41, 278–289 (2016). https://doi.org/10.1007/s11064-015-1791-y

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