Tauopathien wie u.a. die Alzheimer-Demenz sind eine Gruppe neurodegenerativer Erkrankungen mit intrazellulärer Ablagerung des hyperphosphorylierten Tau-Proteins. Perineuronale Netze sind eine spezialisierte Form der extrazellulären Matrix im Zentralen Nervensystem. Sie bestehen u.a. aus Chondroitinsulfat-Proteoglykanen und Tenascin-R. Ziel der vorliegenden Arbeit war der Nachweis einer Protektion perineuronaler Netze vor Tau-Protein-Einlagerung. Zu Beginn wurde die Verteilung des Tau-Proteins und die verschiedenen Arten perineuonaler Netze in der Mauslinie C57Bl6 (Wildtyp) und deren Tenascin-R-Knockout beschrieben. Die Darstellung der Komponenten erfolgte mithilfe von Gefrierschnitten und Immunhistochemie. Um die Ausbreitung von Tau-Protein zu untersuchen, wurden organotypische Hirnschnittkulturen angelegt und markiertes Tau-Protein zugesetzt. Es wurden verschiedene Antikörper genutzt, welche bestimmte Seitenketten von Chondroitinsulfat-Proteoglykanen darstellen. Die Chondroitinsulfat-Seitenketten wurden mit dem Enzym Chondroitinase ABC entfernt, um deren Funktion zu untersuchen. Die Ergebnisse der Arbeit zeigten deutliche Unterschiede in der Verteilung von Tau-Protein. Es reicherte sich bei den Wildtypen und den Tenascin-R-Knockout-Mäusen diffus extrazellulär um Neuronen mit perineuronalen Netzen an. Im Unterschied dazu verteilte sich Tau-Protein nach der Abspaltung der Chondroitinsulfat-Seitenketten vom perineuronalen Netz gleichmäßig über die Schnittkultur ohne sich an perineuronalen Netzen anzureichern. Die vorliegende Arbeit stellt die neuroprotektive Funktion der perineuronalen Netze und insbesondere ihrer Chondroitinsulfat-Seitenketten gegenüber zugegebenem Tau-Protein heraus. Weiterhin weist sie den Verlust der neuroprotektiven Funktion gegenüber zugegebenem Tau-Protein nach Behandlung mit Chondroitinase ABC nach.

The development of new therapeutical approaches, devised to foster the regeneration of neuronal circuits after injury and/or in neurodegenerative diseases, is of great importance. The impairment of dopaminergic projections is especially severe, because these projections are involved in crucial brain functions such as motor control, reward and cognition. In the work presented here, organotypic brain slice co-cultures of (a) the mesostriatal and (b) the mesocortical dopaminergic projection systems consisting of tissue sections of the ventral tegmental area/substantia nigra (VTA/SN), in combination with the target regions of (a) the striatum (STR) or (b) the prefrontal cortex (PFC), respectively, were used to evaluate different approaches to stimulate neurite outgrowth: (i) inhibition of cAMP/cGMP turnover with 3’,5’ cyclic nucleotide phosphodiesterase inhibitors (PDE-Is), (ii) blockade of calcium currents with nimodipine, and (iii) the co-cultivation with bone marrow-derived mesenchymal stromal/stem cells (BM-MSCs). The neurite growth-promoting properties of the tested substances and cell populations were analyzed by neurite density quantification in the border region between the two brain slices, using biocytin tracing or tyrosine hydroxylase labeling and automated image processing procedures. In addition, toxicological tests and gene expression analyses were conducted. (i) PDE-Is were applied to VTA/SN+STR rat co-cultures. The quantification of neurite density after both biocytin tracing and tyrosine hydroxylase labeling revealed a growth promoting effect of the PDE2A-Is BAY60-7550 and ND7001. The application of the PDE10-I MP-10 did not alter neurite density in comparison to the vehicle control. (ii) The effects of nimodipine were evaluated in VTA/SN+PFC rat co-cultures. A neurite growth-promoting effect of 0.1 µM and 1 µM nimodipine was demonstrated in a projection system of the CNS. In contrast, the application of 10 µM nimodipine did not alter neurite density, compared to the vehicle control, but induced the activation of the apoptosis marker caspase 3. The expression levels of the investigated genes, including Ca2+ binding proteins (Pvalb, S100b), immediate early genes (Arc, Egr1, Egr2, Egr4, Fos and JunB), glial fibrillary acidic protein, and myelin components (Mal, Mog, Plp1) were not significantly changed (with the exception of Egr4) by the treatment with 0.1 µM and 1 µM nimodipine. (iii) Bulk BM-MSCs that were classically isolated by plastic adhesion were compared to the subpopulation Sca-1+Lin-CD45--derived MSCs (SL45-MSCs). The neurite growth-promoting properties of both MSC populations were quantified in VTA/SN+PFC mouse co-cultures. For this purpose, the MSCs were seeded on glass slides that were placed underneath the co-cultures. A significantly enhanced neurite density within the co-cultures was induced by both bulk BM-MSCs and SL45-MSCs. SL45-MSCs increased neurite density to a higher degree. The characterization of both MSC populations revealed that the frequency of fibroblast colony forming units (CFU-f ) is 105-fold higher in SL45-MSCs. SL45-MSCs were morphologically more homogeneous and expressed higher levels of nestin, BDNF and FGF2 compared to bulk BM-MSCs. Thus, this work emphasizes the vast potential for molecular targeting with respect to the development of therapeutic strategies in the enhancement of neurite regrowth.

The development of new therapeutical approaches, devised to foster the regeneration of neuronal circuits after injury and/or in neurodegenerative diseases, is of great importance. The impairment of dopaminergic projections is especially severe, because these projections are involved in crucial brain functions such as motor control, reward and cognition. In the work presented here, organotypic brain slice co-cultures of (a) the mesostriatal and (b) the mesocortical dopaminergic projection systems consisting of tissue sections of the ventral tegmental area/substantia nigra (VTA/SN), in combination with the target regions of (a) the striatum (STR) or (b) the prefrontal cortex (PFC), respectively, were used to evaluate different approaches to stimulate neurite outgrowth: (i) inhibition of cAMP/cGMP turnover with 3’,5’ cyclic nucleotide phosphodiesterase inhibitors (PDE-Is), (ii) blockade of calcium currents with nimodipine, and (iii) the co-cultivation with bone marrow-derived mesenchymal stromal/stem cells (BM-MSCs). The neurite growth-promoting properties of the tested substances and cell populations were analyzed by neurite density quantification in the border region between the two brain slices, using biocytin tracing or tyrosine hydroxylase labeling and automated image processing procedures. In addition, toxicological tests and gene expression analyses were conducted. (i) PDE-Is were applied to VTA/SN+STR rat co-cultures. The quantification of neurite density after both biocytin tracing and tyrosine hydroxylase labeling revealed a growth promoting effect of the PDE2A-Is BAY60-7550 and ND7001. The application of the PDE10-I MP-10 did not alter neurite density in comparison to the vehicle control. (ii) The effects of nimodipine were evaluated in VTA/SN+PFC rat co-cultures. A neurite growth-promoting effect of 0.1 µM and 1 µM nimodipine was demonstrated in a projection system of the CNS. In contrast, the application of 10 µM nimodipine did not alter neurite density, compared to the vehicle control, but induced the activation of the apoptosis marker caspase 3. The expression levels of the investigated genes, including Ca2+ binding proteins (Pvalb, S100b), immediate early genes (Arc, Egr1, Egr2, Egr4, Fos and JunB), glial fibrillary acidic protein, and myelin components (Mal, Mog, Plp1) were not significantly changed (with the exception of Egr4) by the treatment with 0.1 µM and 1 µM nimodipine. (iii) Bulk BM-MSCs that were classically isolated by plastic adhesion were compared to the subpopulation Sca-1+Lin-CD45--derived MSCs (SL45-MSCs). The neurite growth-promoting properties of both MSC populations were quantified in VTA/SN+PFC mouse co-cultures. For this purpose, the MSCs were seeded on glass slides that were placed underneath the co-cultures. A significantly enhanced neurite density within the co-cultures was induced by both bulk BM-MSCs and SL45-MSCs. SL45-MSCs increased neurite density to a higher degree. The characterization of both MSC populations revealed that the frequency of fibroblast colony forming units (CFU-f ) is 105-fold higher in SL45-MSCs. SL45-MSCs were morphologically more homogeneous and expressed higher levels of nestin, BDNF and FGF2 compared to bulk BM-MSCs. Thus, this work emphasizes the vast potential for molecular targeting with respect to the development of therapeutic strategies in the enhancement of neurite regrowth.:Table of contents Abbreviations 1 1. Introduction 2 1.1 The dopaminergic system 2 1.2 Neurite regeneration following mechanical lesions of the CNS 7 1.3 Organotypic brain slice co-cultures 8 1.4 Promising substances and cells to enhance neuroregeneration 10 1.5 The aim of the thesis 14 2. The original research articles 16 2.1 Phosphodiesterase 2 inhibitors promote axonal outgrowth in organotypic slice co-cultures 17 2.2 Nimodipine enhances neurite outgrowth in dopaminergic brain slice co-cultures 35 2.3 Mesenchymal stem cells support neuronal fiber growth in an organotypic brain slice co-culture model 50 3. References 66 Appendices 73 Summary 73 Zusammenfassung 78 Curriculum Vitae 84 Track Record 85 Selbständigkeitserklärung 87 Acknowledgments 88