Ziel des geplanten Forschungsvorhabens ist die Identifikation biologisch effektiver Signale und Mechanismen zur Steuerung des Wachstums von Spiralganglienneuriten (SGN, 1. Neuron der der Hörbahn) in vitro und anschließender Untersuchung der Praktikabilität, Effizienz, und Sicherheit dieser Verfahren in vivo im Säugetierinnenohr. Dabei sollen die unterschiedlichen Regionen der Spiralganglienneurone, die in der Cochlea (Hörschnecke) tonotopisch angeordnet sind, differenziell untersucht werden. Im Einzelnen werden Mitglieder der Neurotrophinfamilie (wie NT-3, BDNF und FGF-1), verschiedene extrazelluläre Matrixproteine (wie Fibronektin, Laminin, Tenaskin) und Ephrin/Eph-Rezeptor- Signalwege (EphA4, EphAl) untersucht. Alle diese Signale werden nachweislich in vivo von Zellen des Innenohres gebildet. Die Induktion und Steuerung eines gerichteten Neuritenwachstums von Spiralganglienneuronen ist im Hinblick auf die Innervation des Corti-Organs sowohl von entwicklungsbiologischem Interesse als auch für zukünftige klinische Anwendungen von Bedeutung. Eine günstigere Interaktion von Spiralganglienneuriten mit Cochlea- Implantatelektroden könnte zu einer höheren Effektivität dieser operativ in die Hörschnecke einsetzbaren elektronischen Innenohrimplantate fuhren, und damit die Hörrehabilitation für Patienten mit hochgradiger Innenohrschädigung verbessern.
The objective of the supported research project was the identification of biologically effective signals for stimulation and guidance of dentrite outgrowth in adult neurons of the inner ear's Spiralganglion (SG), the first neuron in the auditory pathway. Therefore, soluble members of the neurotrophin famil, immobilized extracellular matrix proteins and cell surface signaling mechanism interactions were investigated. Form the developmental point of view, P20 is the earliest time point, the rodent inner ear can be considered fully mature regarding to anatomy and function. At the beginning of the supported research project, very few studies had investigated neurotrophin effects in adult rodent spiral ganglion neurons, while in the vast majority of studies neonatal spiral ganglion neurons had been investigated. However from the applied scientific point of view, therapeutic interventions - like cochlea implants - even in infants do target mature and not neonatal spiral ganglion neurons. Establishment of primary organotypic cultures of adult SG neurons as the basis for the proposed in vitro experiments proved to be difficult due to technically very difficult dissection of the ganglion out of the by then fully calcified Rosenthal's canal. Furthermore, adult SG neuron survival in culture was initially very limited. However, maintenance and sufficient stimulation of neurite outgrowth for successive guidance experiments could be achieved by using neurons from postnatal day 20 (P20) rats in serum-free media supplemented with a combination of glial-cell-line derived neurotrophic factor (GDNF, 25-100 ng/ml), its soluble co-receptor GFRa1 (100 ng/ml) and brain derived neurotrophic factor (BDNF, 10 ng/ml). In parallel to optimization of the actual culture system for adult SG neurons, a 2-dimensional stripe assay setup was optimized for testing the extracellular matrix (ECM) proteins fibronectin and laminin versus the adhaesion factor poly-L-lysine on neonatal SG neurons. Later into the project, adult SG neurons were investigated in this setup to test the hypothesis if mature SG neuntes still can respond to closely patterned ECM proteins. The later might therefore be utilized for neurite guidance in therapeutic interventions. Recently, neuronal cell adhaesion molecule (NCAM) was identified as an alternative receptor for GDNF. In addition, GDNF's co-receptor GFRal had been shown to function as a ligand-induced cell-cell adhaesion molecule itself. Both molecules have been implicated in neuronal synapse formation as well. Having seen GDNF's function as a potent survival and growth factor during optimization of the current cell culture system for mature SG neurons, these recent findings lead to the question if NCAM and GFRal might be functional receptors for GDNF in the SG, linking neurotrophin- and cell adhaesion signaling in guidance of SG neuritis, and possibly synapse formation in the inner ear. Therefore GDNF signaling through its alternative receptors Ret (rearranged during transformation) and NCAM were further investigated in neonatal and mature SG neurons. GFRal's capability to guide SG neuntes or to induce expression of pre- or postsynaptic proteins were investigated by immobilizing GFRal in the above mentioned 2-dimensional stripe assay or on poly-styrene beads added to the cell culture system. NCAM's further reaching functions itself with regard to dentrite targeting and it's possible synaptic effects in sound transmission were investigated in a NCAM knockout mouse model.