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

A Doença de Parkinson (DP) é um distúrbio neurodegenerativo, caracterizada em parte pela perda de neurônios dopaminérgicos da via nigroestriatal, originada na substância negra com projeções para o estriado, causando vários déficits motores. Atualmente, o tratamento mais utilizado é a administração de L-DOPA, um análogo da dopamina. Porém, essa droga apresenta eficácia limitada e induz diversos efeitos colaterais. A exploração dos efeitos neuroprotetores, proliferativos e neuroregenerativos da bradicinina (BK) em modelo animal de DP pode conduzir à substituição celular do tecido lesionado pela 6-hidroxidopamina (6-OHDA). De fato, a BK e seus receptores possuem um grande espectro de ações fisiológicas, estando classicamente envolvida no controle da homeostase cardiovascular e inflamação, além de exercer efeitos protetores em fisiopatologias do sistema nervoso, como em modelos de acidente vascular cerebral. Vários tipos celulares têm suas vias de sinalização associadas à ativação do receptor B2 de cininas (B2BKR). Trabalhos anteriores de nosso grupo mostraram que a BK está envolvida na diferenciação neural de células progenitoras neurais por um loop autócrino que resulta em ativação do B2BKR. Os resultados apresentados neste trabalho mostram a eficácia do tratamento com BK, um agonista de B2BKR, em animais submetidos à lesão da via nigro-estriatal induzida por 6-OHDA. Além disso, há uma recuperação comportamental e histológica desses animais quando tratados com Captopril®, um potencializador dos efeitos farmacológicos da BK, e com [Phe8Ψ(CH-NH)Arg9]-Bradicinina, agonista estável do receptor B2BKR. Assim, concluímos que a ativação de B2BKR pela BK desencadeiaum processo de neuroregeneração dopaminérgica de animais submetidos à lesão por 6-OHDA. Trabalhos recentes mostram que o receptor B2BKR desempenha um importante papel neuroprotetor em modelo animal da Doença de Alzheimer, o que corrobora nossos achados. Juntos, esses resultados contribuem para o estabelecimento da ação neuroprotetora e neurorregenerativa da BK no modelo de animal de neurodegeneração dopaminérgica, tornando-a uma excelente candidata para aplicação em terapias de reparo neuronal. / Parkinson\'s disease (PD) is a neurodegenerative disorder partially characterized by the loss of dopaminergic neurons from the nigrostriatal pathway, originated in the substantia nigra with projections to the striatum, which causes several motor deficits. Currently, the most commonly used drug for PD treatment is levodopa. However, it has limited efficacy and induces several side effects. Elucidation of the neuroprotective, proliferative and neuroregenerative effects of bradykinin (BK) in animal models of PD can culminate in cellular replacement of the tissue damaged by 6-hydroxydopamine (6-OHDA). In fact, BK and its receptor have several physiological effects, being classically involved in the control of cardiovascular homeostasis and inflammation. Besides, BK exerts protective effects on nervous system pathophysiology, as observed in stroke models. Several cell types have their signaling pathways associated with the B2 kinin receptor (B2BKR) activation. Previous work from our group showed that BK is involved in differentiation of neural progenitor cells by an autocrine loop that results in activation of B2BKR. The results presented in this thesis show the efficacy of treatment with BK, through B2BKR activation, in animals submitted to nigrostriatal pathway injury induced by 6-OH dopamine. Furthermore, behavioral and histological recoveries of these animals were observed when treated with Captopril®, a potentiator of BK pharmacological effects, and with [Phe8Ψ (CH-NH) Arg9] -BK, a stable agonist of the B2BKR receptor. Thus, we conclude that BK activation of B2BKR triggers neuroregenerative processes in animals submitted to 6- OHDA injury. Recent studies showed that the B2BKR receptor plays an important neuroprotective role in an animal model of Alzheimer\'s disease, which corroboratesour findings. Together, these results contribute to the establishment of the neuroprotective and neuroregenerative actions of BK - an excellent candidate for neural repair therapies.

A Doença de Parkinson (DP) é um distúrbio neurodegenerativo, caracterizada em parte pela perda de neurônios dopaminérgicos da via nigroestriatal, originada na substância negra com projeções para o estriado, causando vários déficits motores. Atualmente, o tratamento mais utilizado é a administração de L-DOPA, um análogo da dopamina. Porém, essa droga apresenta eficácia limitada e induz diversos efeitos colaterais. A exploração dos efeitos neuroprotetores, proliferativos e neuroregenerativos da bradicinina (BK) em modelo animal de DP pode conduzir à substituição celular do tecido lesionado pela 6-hidroxidopamina (6-OHDA). De fato, a BK e seus receptores possuem um grande espectro de ações fisiológicas, estando classicamente envolvida no controle da homeostase cardiovascular e inflamação, além de exercer efeitos protetores em fisiopatologias do sistema nervoso, como em modelos de acidente vascular cerebral. Vários tipos celulares têm suas vias de sinalização associadas à ativação do receptor B2 de cininas (B2BKR). Trabalhos anteriores de nosso grupo mostraram que a BK está envolvida na diferenciação neural de células progenitoras neurais por um loop autócrino que resulta em ativação do B2BKR. Os resultados apresentados neste trabalho mostram a eficácia do tratamento com BK, um agonista de B2BKR, em animais submetidos à lesão da via nigro-estriatal induzida por 6-OHDA. Além disso, há uma recuperação comportamental e histológica desses animais quando tratados com Captopril®, um potencializador dos efeitos farmacológicos da BK, e com [Phe8Ψ(CH-NH)Arg9]-Bradicinina, agonista estável do receptor B2BKR. Assim, concluímos que a ativação de B2BKR pela BK desencadeiaum processo de neuroregeneração dopaminérgica de animais submetidos à lesão por 6-OHDA. Trabalhos recentes mostram que o receptor B2BKR desempenha um importante papel neuroprotetor em modelo animal da Doença de Alzheimer, o que corrobora nossos achados. Juntos, esses resultados contribuem para o estabelecimento da ação neuroprotetora e neurorregenerativa da BK no modelo de animal de neurodegeneração dopaminérgica, tornando-a uma excelente candidata para aplicação em terapias de reparo neuronal. / Parkinson\'s disease (PD) is a neurodegenerative disorder partially characterized by the loss of dopaminergic neurons from the nigrostriatal pathway, originated in the substantia nigra with projections to the striatum, which causes several motor deficits. Currently, the most commonly used drug for PD treatment is levodopa. However, it has limited efficacy and induces several side effects. Elucidation of the neuroprotective, proliferative and neuroregenerative effects of bradykinin (BK) in animal models of PD can culminate in cellular replacement of the tissue damaged by 6-hydroxydopamine (6-OHDA). In fact, BK and its receptor have several physiological effects, being classically involved in the control of cardiovascular homeostasis and inflammation. Besides, BK exerts protective effects on nervous system pathophysiology, as observed in stroke models. Several cell types have their signaling pathways associated with the B2 kinin receptor (B2BKR) activation. Previous work from our group showed that BK is involved in differentiation of neural progenitor cells by an autocrine loop that results in activation of B2BKR. The results presented in this thesis show the efficacy of treatment with BK, through B2BKR activation, in animals submitted to nigrostriatal pathway injury induced by 6-OH dopamine. Furthermore, behavioral and histological recoveries of these animals were observed when treated with Captopril®, a potentiator of BK pharmacological effects, and with [Phe8Ψ (CH-NH) Arg9] -BK, a stable agonist of the B2BKR receptor. Thus, we conclude that BK activation of B2BKR triggers neuroregenerative processes in animals submitted to 6- OHDA injury. Recent studies showed that the B2BKR receptor plays an important neuroprotective role in an animal model of Alzheimer\'s disease, which corroboratesour findings. Together, these results contribute to the establishment of the neuroprotective and neuroregenerative actions of BK - an excellent candidate for neural repair therapies.

Spinal cord injury is a very significant clinical as well as social problem with extensive consequences, affecting the patient and also his/her family. Great efforts have been devoted to searching for an effective treatment, which would improve their situation. This thesis evaluated the effects of the natural compound curcumin on spinal cord injury using an experimental balloon compression model. Male Wistar rats were randomized into two groups following the lesion induction, namely vehicle- or curcumin-treated. The behavioral recovery was evaluated using a set of locomotor and sensory tests and a histological and imunohistochemical analysis was performed. The qPCR method was used to observe the expression of some genes related to regeneration and immune response. It was demonstrated that curcumin improved locomotor recovery after the spinal cord injury, particularly in the early stages. Morphometric analysis of the gray and white matter sparing didn't confirm any differences between the two groups. Nevertheless, the glial scar formation was significantly reduced around the central part of the lesion in the curcumin treated group and also the NF-κB activity was substantially inhibited. The gene expression analysis demontrated downregulation of Gfap and Rantes genes and upregulation of the Irf5 gene...

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A sinalização da resposta Imune Inata exerce um papel fundamental na eliminação de patógenos bem como no recrutamento de progenitoras para recuperação de tecidos nervosos lesionados. Nossos estudos iniciais que buscavam a compreensão desta participação da atividade inflamatória na neurorregeneração e degenerção do OE constataram que a administração tópica de lipopolissacarídeo (LPS) gera perda de seus neurônios olfatórios (OSNs), enquanto que camundongos deficientes do gene Myd88 apresentam uma taxa de reposição neuronal pós-lesão mais elevada que a de selvagens e o uso consecutivo de DEX retarda a neurorregeneração olfatória em curto prazo. Prosseguimos agora no esclarecimento destes resultados em três vertentes: A primeira busca descrever as respostas do OE de animais selvagens e trangênicos Tlr4-/-, Myd88-/-, Ticam1-/-, Il1r1-/-, Casp1-/-/Casp4-/- e Casp1-/-/Casp4-/-/Casp4tg a estímulos inflamatórios provenientes de infusões intranasais de ligantes e citocinas recombinantes envolvidas na sinalização dos TLRs. Isto através de análises histológicas por marcação nuclear e análise da espessura e alterações anatômicas do OE, e da expressão de IL1b e Nf&#954bia por hibridização in situ (ISH), ou ainda pela contagem de suas células TUNEL-positivas pós-tratamento. Neste estudo propomos ainda um modelo de lesão para estudos de neurorregenereração através da análise por imunofluorescência (IF) de OSNs em função do tempo após uma infusão intranasal (IN) de LPS. A segunda analisa por microarranjos de oligonucleotídeos (microarray) as alterações de expressão gênicas associadas à ausência do gene Myd88 no OE pós-lesão seguida de análises histológicas de sua regeneração em camundongos com deleções em genes selecionados. Por fim o terceiro estudo investiga a degeneração e a neurorregeneração do OE sob o efeito do uso tópico consecutivo de DEX. O corticoide é co-aplicado topicamente com um insulto inflamatório para avaliar o seu efeito preventivo e consecutivamente por três dias em dois modelos de lesão para avaliar sua interferência na neurorregeneração 14 dias após o tratamento. Foram realizadas IF para quantificação dos volumes neuronais, espessura do OE, proliferação celular, síntese proteica e morte celular por TUNEL. O uso tópico de LPS promove a degeneração do OE via TLR4 a partir de regiões com expressão de Il1b e Nf&#954bia e número de células TUNEL elevada. Este efeito é via MyD88-dependente sem a participação da TRIF-dependente. O gene Myd88 é tão crucial nesta degeneração do epitélio quanto na gerada por rmIL-1β e rmTNFα. Sua ausência não promove citoproteção contra o gás NO. Possivelmente, CASP1 e IL-1R estejam também envolvidos. O modelo de lesão imunológica para estudos de neurorregeneração é rápido e eficaz. A ausência do gene Myd88 acompanha uma redução na expressão da enzima degradadora de insulina (IDE) no OE pós-lesão. Camundongos que não a expressam apresentam uma reposição celular do epitélio mais rápida. A co-IN de DEX com LPS impede a degeneração do OE. 10µl deste corticoide a 40ng/µl administrada por IN não é tóxica a este epitélio, porém seu uso consecutivo em curto prazo promove aberrações anatômicas nos modelos de lesão imunológica, além de interferir na sua dinâmica de reposição neural, elevação da taxa de síntese proteica e proliferação celular, sem alterar a diferenciação, após lesão induzida por metimazol. / The innate immune response signaling plays a key role in the elimination of pathogens and in the recruitment of new cells to recover the injured nervous tissues. Our preliminary studies on the roles of the inflammatory activity in OE degeneration and neuroregeneration process showed that the topical administration of lipopolysaccharide (LPS) generates the loss of olfactory sensorial neurons (OSNs), Myd88 gene-deficient mice exhibit a higher neuronal replacement rate after injury than wild-type mice, and that the consecutive use of DEX provokes retarding olfactory neuroregeneration over the short term. We seek now to clarify these results in three ways: The first describes the OE response in wild-type animals Tlr4-/-, Myd88-/-, Ticam1-/-, Il1r1-/-, Casp1-/-/Casp4-/- and Casp1-/-/Casp4-/-/Casp4tg animals to inflammation through the intranasal infusion (IN) of ligands and cytokines associated with Toll-Like Receptors (TLR) signaling. This was acomplished through histological analysis of the thickness and anatomical changes in DAPI stained OE, Il1b and Nfκbia expression analysis by in situ hybridization (ISH), and TUNEL-positive cells counting after treatment. In this study we propose an inflammatory lesion for neuroregeneration studies using immunofluorescence (IF) analysis of the OE in function of time after the intranasal infusion of LPS. The second part analyzes the Myd88 gene loss in the OE gene expression of after a lesion using microarray. This is followed by the histological analysis of regeneration using IF in transgenic mice with the deletion of specific genes (microarray versus literature review). Finally, the third study evaluates the OE degeneration and neuroregeneration under the influence of consecutive topical use of the DEX. The corticosteroid is co-administered with the inflammatory stimulus in order to evaluate its protective effect and consecutively for three days in two models of lesion to assess their influence on neuroregeneration 14 days after treatment. The IF analysis was performed in order to quantify the neuronal volumes, the thickness of the OE, cell proliferation, protein synthesis (by incorporation the identification of puromycin) and cell death by TUNEL. The topical use of LPS promotes the degeneration of OE by TLR4 from sites that feature an overexpression of Il1b and Nfκbia and a high number of TUNEL-positive cells. This effect is MyD88-dependent. The Myd88 gene is as crucial in this degeneration of the epithelium as in those generated by rmIL-1β and rmTNFα. Their absence does not promote cytoprotection against the cytotoxic gas nitric oxide (NO). It is possiby that CASP1 and IL-1R are also involved. The immunologic lesion model for neuroregeneration studies is fast and effective. The absence of Myd88 gene reduces the expression of insulin-degrading enzyme (IDE) in the post-lesion OE. Mice without this enzyme show a rapid cellular restoration in the epithelium. The Co-IN of DEX with LPS prevents the degeneration EO. The doses adopted by us are nontoxic; however its short-term consecutive use promotes anatomical aberrations in the immunological lesion model, and interferes with the dynamics of neural replacement by impairing both the rate of protein synthesis and proliferation of the epithelium without halting their differentiation.

Parkinson’s disease (PD) is a disorder of the nervous system that causes problems with movement (motor symptoms) as well as other problems such as mood disorders, cognitive changes, sleep disorders, constipation, pain, and other non-motor symptoms. The severity of PD symptoms worsens over time as the disease progresses, and while there are treatments for the motor and some non-motor symptoms there is no known cure for PD. Thus there is a high demand for therapies to slow the progressive neurodegeneration observed in PD. Two clinical trials at the University of Kentucky College of Medicine (NCT02369003, NCT01833364) are currently underway that aim to develop a disease-modifying therapy that slows the progression of PD. These clinical trials are evaluating the safety and feasibility of an autologous peripheral nerve graft to the substantia nigra in combination with Deep Brain Stimulation (DBS) for the treatment of PD. By grafting peripheral nerve tissue to the Substantia Nigra, the researchers aim to introduce peripheral nerve tissue, which is capable of functional regeneration after injury, to the degenerating Substantia Nigra of patients with PD. The central hypothesis of these clinical trials is that the grafted tissue will slow degeneration of the target brain region through neural repair actions of Schwann cells as well as other pro-regenerative features of the peripheral nerve tissue. This dissertation details analysis of the peripheral nerve tissue used in the above clinical trials with respect to tissue composition and gene expression, both of injury-naive human peripheral nerve as well as the post-conditioning injury nerve tissue used in the grafting procedure. RNA-seq analysis of sural nerve tissue pre and post-conditioning show significant changes in gene expression corresponding with transdifferentiation of Schwann cells from a myelinating to a repair phenotype, release of growth factors, activation of macrophages and other immune cells, and an increase in anti-apoptotic and neuroprotective gene transcripts. These results reveal in vivo gene expression changes involved in the human peripheral nerve injury repair process, which has relevance beyond this clinical trial to the fields of Schwann cell biology and peripheral nerve repair. To assess the neurobiology of the graft post-implantation we developed an animal model of the grafting procedure, termed Neuro-Avatars, which feature human graft tissue implanted into athymic nude rats. Survival and infiltration of human graft cells into the host brain were shown using immunohistochemistry of Human Nuclear Antigen. Surgical methods and outcomes from the ongoing development of this animal model are reported. To connect the results of these laboratory studies to the clinical trial we compared the severity of motor symptoms before surgery to one year post-surgery in patients who received the analyzed graft tissue. Motor symptom severity was assessed using the Unified Parkinson’s Disease Rating Scale Part III. Finally, the implications and future directions of this research is discussed. In summary, this dissertation advances the translational science cycle by using clinical trial findings and samples to answer basic science questions that will in turn guide future clinical trial design.

Characterization of new pharmacological targets is a promising approach in research of neurorepair mechanisms. The G protein-coupled receptor 17 (GPR17) has recently been proposed as an interesting pharmacological target, e.g., in neuroregenerative processes. Using the well-established ex vivo model of organotypic slice co-cultures of the mesocortical dopaminergic system (prefrontal cortex (PFC) and substantia nigra/ventral tegmental area (SN/VTA) complex), the influence of GPR17 ligands on neurite outgrowth from SN/VTA to the PFC was investigated. The growthpromoting effects of Montelukast (MTK; GPR17- and cysteinyl-leukotriene receptor antagonist), the glial cell line-derived neurotrophic factor (GDNF) and of two potent, selective GPR17 agonists (PSB-16484 and PSB-16282) were characterized. Treatment with MTK resulted in a significant increase in mean neurite density, comparable with the effects of GDNF. The combination of MTK and GPR17 agonist PSB-16484 significantly inhibited neuronal growth. qPCR studies revealed an MTK-induced elevated mRNA-expression of genes relevant for neuronal growth. Immunofluorescence labelling showed a marked expression of GPR17 on NG2-positive glia. Western blot and RT-qPCR analysis of untreated cultures suggest a time-dependent, injury-induced stimulation of GPR17. In conclusion, MTK was identified as a stimulator of neurite fibre outgrowth, mediating its effects through GPR17, highlighting GPR17 as an interesting therapeutic target in neuronal regeneration.

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