Zusammenfassung
Hintergrund: Der Phosphatidylinositol-3-Kinase (PI3K) wird eine wesentliche Rolle bei der intrazellulären
Übertragung von Neurotrophin-Signalen vom Rezeptor an der Zellmembran zum Zellkern
zugesprochen.
Methode: In dieser Studie wurde die Beteiligung der PI3K bei der Vermittlung von Neurotrophin-Effekten
im Spiralganglion (SG) neugeborener Ratten untersucht. Die SG-Explantate wurden in
vitro mit Neurotrophin-3 (NT-3) stimuliert und zusätzlich mit Wortmannin, einem spezifischen
Inhibitor der PI3K behandelt. Nach Fixierung und immunhistochemischer Färbung der
Explantate erfolgte die Auswertung des Neuritenwachstums.
Ergebnisse: Die Stimulation mit NT-3 führte zu dem bereits bekannten Effekt einer Zunahme sowohl
der Anzahl, als auch der Länge der auswachsenden SG-Neuriten im Vergleich zu Kontrollexplantaten.
Die Behandlung NT-3-stimulierter SG-Explantate mit Wortmannin resultierte in einer
konzentrationsabhängigen Reduktion beider Parameter, während nach der Inkubation von
Kontrollexplantaten mit Wortmannin keine Änderung des Neuritenwachstums beobachtet
wurde.
Schlussfolgerung: Die Ergebnisse der Untersuchung zeigen, dass neurotrophin-induziertes Neuritenwachstum
von SG-Explantaten durch den PI3K-Inhibitor Wortmannin moduliert werden kann und deuten
darauf hin, dass die PI3K ein wichtiges Schlüsselenzym bei der Vermittlung von Effekten
des Wachstumfaktors NT-3 in cochleären Neuronen darstellt. Zusammen mit den Ergebnissen
früherer Untersuchungen deuten die aktuellen Resultate darauf hin, dass sowohl die
Aktivierung der PI3K, als auch des G-Proteins Ras und der mitogen-aktivierten Proteinkinase
Kinase (MEK) für das Neuritenwachstum von SG-Neuronen notwendig sind. Die weitere
Aufklärung dieser, das Wachstum der SG-Neuriten beeinflussenden molekulären Mechanismen
lässt für die Zukunft neue therapeutische Ansätze für Innenohrerkrankungen erwarten.
Abstract
Background: Phosphatidylinositol 3-kinase (PI3K) is considered to be an important enzyme in cell
signaling, mediating certain aspects of neurotrophin signals from the cell surface
receptor to the nucleus.
Methods: The participation of PI3K in the mediation of neurotrophin-induced effects in the
spiralganglion (SG) of neonatal rats was investigated in vitro. SG explants were stimulated
with neurotrophin (NT)-3 and treated with Wortmannin, a specific inhibitor of PI3K.
After fixation and immunhistochemical staining, the growth of the SG neurites was
evaluated.
Results: Stimulation with NT-3 lead to significant increases in number and length of neurites,
when compared to non-stimulated controls. Treatment of NT-3 stimulated SG explants
resulted in a dose-dependent reduction of both parameters, whereas the neurite growth
of non-stimulated control explants was not significantly influenced by the incubation
with Wortmannin.
Conclusions: The results demonstrate that neurotrophin-induced neurite growth from SG explants
can be modulated with the PI3K inhibitor Wortmannin and indicate that PI3K is a key
enzyme in the mediation of NT-3 effects in cochlear neurons. These observations together
with results of previous studies suggest that the activation of PI3K as well as Ras
and MEK are essential for neurite growth in cochlear neurons. Further knowledge of
cell signaling mechanisms influencing SG neurite growth could lead to new therapeutical
strategies for the treatment of inner ear diseases.
Schlüsselwörter
NT-3, Wortmannin - PI3K - Spiralganglion - Neuritenwachstum
Key words
NT-3, Wortmannin - PI3K - Spiralganglion - Neurite growth
Literatur
1
Levi J A, Thomson D, Sandeman T, Tattersall M, Raghavan D, Byrne M, Gill G, Harvey V,
Burns I, Snyder R.
A prospective study of cisplatin-based combination chemotherapy in advanced germ cell
malignancy: role of maintenance and long-term follow-up.
J Clin Oncol.
1988;
6 (7)
1154-1160
2
Kaplan D R, Stephens R M.
Neurotrophin signal transduction by the Trk receptor.
J Neurobiol.
1994;
25 (11)
1404-1417
3
Dazert S, Kim D, Luo L, Aletsee C, Garfunkel S, Maciag T, Baird A, Ryan A F.
Focal delivery of fibroblast growth factor-1 by transfected cells induces spiral ganglion
neurite targeting in vitro.
J Cell Physiol.
1998;
177 (1)
123-129
4
Aletsee C, Mullen L, Kim D, Pak K, Brors D, Dazert S, Ryan A F.
The disintegrin kistrin inhibits neurite extension from spiral ganglion explants cultured
on laminin.
Audiol Neurootol.
2001;
6 (2)
57-65
5
Ylikoski J, Pirvola U, Moshnyakov M, Palgi J, Arumae U, Saarma M.
Expression patterns of neurotrophin and their receptor mRNAs in the rat inner ear.
Hear Res.
1993;
65 (1-2)
69-78
6
Ernfors P, Rosario C M, Merlio J P, Grant G, Aldskogius H, Persson H.
Expression of mRNAs for neurotrophin receptors in the dorsal root ganglion and spinal
cord during development and following peripheral or central axotomy.
Brain Res Mol Brain Res.
1993;
17 (3-4)
217-226
7
Pirvola U, Arumae U, Moshnyakov M, Palgi J, Saarma M, Ylikoski J.
Coordinated expression and function of neurotrophins and their receptors in the rat
inner ear during target innervation.
Hear Res.
1994;
75 (1-2)
131-144
8
Pirvola U, Ylikoski J, Palgi J, Lehtonen E, Arumae U, Saarma M.
Brain-derived neurotrophic factor and neurotrophin 3 mRNAs in the peripheral target
fields of developing inner ear ganglia.
Proc Natl Acad Sci USA.
1992;
89 (20)
9915-9919
9
Ernfors P, Duan M L, ElShamy W M, Canlon B.
Protection of auditory neurons from aminoglycoside toxicity by neurotrophin-3.
Nat Med.
1996;
2 (4)
463-467
10
Malgrange B, Lefebvre P P, Martin D, Staecker H, van de Water T R, Moonen G.
NT-3 has a tropic effect on process outgrowth by postnatal auditory neurones in vitro.
Neuroreport.
1996;
7 (15-17)
2495-2499
11
Farinas I, Jones K R, Backus C, Wang X Y, Reichardt L F.
Severe sensory and sympathetic deficits in mice lacking neurotrophin-3.
Nature.
1994;
369 (6482)
658-661
12
Staecker H, Kopke R, Malgrange B, Lefebvre P, van de Water T R.
NT-3 and/or BDNF therapy prevents loss of auditory neurons following loss of hair
cells.
Neuroreport.
1996;
7 (4)
889-894
13
Kopke R, Staecker H, Lefebvre P, Malgrange B, Moonen G, Ruben R J, van de Water T R.
Effect of neurotrophic factors on the inner ear: clinical implications.
Acta Otolaryngol.
1996;
116 (2)
248-252
14
Knipper M.
NT-3 and BDNF for potential inner ear therapy.
HNO.
1997;
45 (4)
181-183
15
Fritzsch B, Pirvola U, Ylikoski J.
Making and breaking the innervation of the ear: neurotrophic support during ear development
and its clinical implications.
Cell Tissue Res.
1999;
295 (3)
369-382
16
Chao M V.
Neurotrophin receptors: a window into neuronal differentiation.
Neuron.
1992;
9 (4)
583-593
17
Lamballe F, Klein R, Barbacid M.
trkC, a new member of the trk family of tyrosine protein kinases, is a receptor for
neurotrophin-3.
Cell.
1991;
66 (5)
967-979
18
Merlio J P, Ernfors P, Jaber M, Persson H.
Molecular cloning of rat trkC and distribution of cells expressing messenger RNAs
for members of the trk family in the rat central nervous system.
Neuroscience.
1992;
51 (3)
513-532
19
Carter B D, Dechant G, Frade J M, Kaltschmidt C, Barde Y A.
Neurotrophins and their p75 receptor.
Cold Spring Harb Symp Quant Biol.
1996;
61
407-415
20
Greene L A, Kaplan D R.
Early events in neurotrophin signaling via Trk and p75 receptors.
Curr Opin Neurobiol.
1995;
5 (5)
579-587
21
Rodriguez-Viciana P, Warne P H, Khwaja A, Marte B M, Pappin D, Das P, Waterfield M D,
Ridley A, Downward J.
Role of phosphoinositide 3-OH kinase in cell transformation and control of the actin
cytoskeleton by Ras.
Cell.
1997;
89 (3)
457-467
22
Aletsee C, Beros A, Mullen L M, Palacios S, Kwang Pak K, Dazert S, Ryan A F.
Ras/MEK but not p38 signaling mediates NT-3 induced neurite extension from spiral
ganglion neurons.
Journal of the ARO.
im Druck 2001;
23
Yao R, Cooper G M.
Requirement for phosphatidylinositol-3 kinase in the prevention of apoptosis by nerve
growth factor.
Science.
1995;
267 (5206)
2003-2006
24
Franke T F, Kaplan D R, Cantley L C.
PI3K: downstream AKTion blocks apoptosis.
Cell.
1997;
88 (4)
435-437
25
Kimura K, Hattori S, Kabuyama Y, Shizawa Y, Takayanagi J, Nakamura S, Toki S, Matsuda Y,
Onodera K, Fukui Y.
Neurite outgrowth of PC12 cells is suppressed by wortmannin, a specific inhibitor
of phosphatidylinositol 3-kinase.
J Biol Chem.
1994;
269 (29)
18 961-18 967
26
Jackson T R, Blader I J, Hammonds-Odie L P, Burga C R, Cooke F, Hawkins P T, Wolf A G,
Heldman K A, Theibert A B.
Initiation and maintenance of NGF-stimulated neurite outgrowth requires activation
of a phosphoinositide 3-kinase.
J Cell Sci.
1996;
109 (Pt 2)
289-300
27
Crowder R J, Freeman R S.
Phosphatidylinositol 3-kinase and Akt protein kinase are necessary and sufficient
for the survival of nerve growth factor-dependent sympathetic neurons.
J Neurosci.
1998;
18 (8)
2933-2943
28
Dolcet X, Egea J, Soler R M, Martin-Zanca D, Comella J X.
Activation of phosphatidylinositol 3-kinase, but not extracellular-regulated kinases,
is necessary to mediate brain-derived neurotrophic factor-induced motoneuron survival.
J Neurochem.
1999;
73 (2)
521-531
29
Hetman M, Kanning K, Cavanaugh J E, Xia Z.
Neuroprotection by brain-derived neurotrophic factor is mediated by extracellular
signal-regulated kinase and phosphatidylinositol 3-kinase.
J Biol Chem.
1999;
274 (32)
22 569-22 580
30
Brain P W, Curtis P J, Hemming H G, Noris G LF.
Wortmannin an antibiotic produced by Penicillum wortmannii.
.
Trans Br Mycol Soc.
1957;
40
365-368
31
Nakanishi S, Kakita S, Takahashi I, Kawahara K, Tsukuda E, Sano T, Yamada K, Yoshida M,
Kase H, Matsuda Y et al.
Wortmannin, a microbial product inhibitor of myosin light chain kinase.
J Biol Chem.
1992;
267 (4)
2157-2163
32
Yano H, Nakanishi S, Kimura K, Hanai N, Saitoh Y, Fukui Y, Nonomura Y, Matsuda Y.
Inhibition of histamine secretion by wortmannin through the blockade of phosphatidylinositol
3-kinase in RBL-2H3 cells.
J Biol Chem.
1993;
268 (34)
25 846-25 856
33
Okada T, Sakuma L, Fukui Y, Hazeki O, Ui M.
Blockage of chemotactic peptide-induced stimulation of neutrophils by wortmannin as
a result of selective inhibition of phosphatidylinositol 3-kinase.
J Biol Chem.
1994;
269 (5)
3563-3567
34
Okada T, Kawano Y, Sakakibara T, Hazeki O, Ui M.
Essential role of phosphatidylinositol 3-kinase in insulin-induced glucose transport
and antilipolysis in rat adipocytes. Studies with a selective inhibitor wortmannin.
J Biol Chem.
1994;
269 (5)
3568-3573
35
Nonomura T, Kubo T, Oka T, Shimoke K, Yamada M, Enokido Y, Hatanaka H.
Signaling pathways and survival effects of BDNF and NT-3 on cultured cerebellar granule
cells.
Brain Res Dev Brain Res.
1996;
97 (1)
42-50
36
Kotani K, Yonezawa K, Hara K, Ueda H, Kitamura Y, Sakaue H, Ando A, Chavanieu A, Calas B,
Grigorescu F et al.
Involvement of phosphoinositide 3-kinase in insulin- or IGF-1-induced membrane ruffling.
Embo J.
1994;
13 (10)
2313-2321
37
Wymann M, Arcaro A.
Platelet-derived growth factor-induced phosphatidylinositol 3-kinase activation mediates
actin rearrangements in fibroblasts.
Biochem J.
1994;
298 Pt 3
517-520
38
Lavie Y, Dybowski J, Agranoff B W.
Wortmannin blocks goldfish retinal phosphatidylinositol 3-kinase and neurite outgrowth.
Neurochem Res.
1997;
22 (4)
373-378
39
Hetman M, Xia Z.
Signaling pathways mediating anti-apoptotic action of neurotrophins.
Acta Neurobiol Exp.
2000;
60 (4)
531-545
PD Dr. Stefan Dazert
Klinik und Poliklinik für Hals-, Nasen- und Ohrenkranke · Bayerische Julius-Maximilians-Universität
Würzburg
Josef-Schneider-Straße 11 · 97080 Würzburg
eMail: s.dazert@mail.uni-wuerzburg.de