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Endometriosis is a chronic estrogen-dependent gynecological disease where endometrial cells implant at inappropriate sites causing significant pelvic pain, decreased quality of life, and often infertility. It affects 10% of women of reproductive age, and there is no minimally invasive diagnostic test. Consequently the time to diagnosis, which occurs during laparoscopic surgery followed by pathological confirmation of disease, is prolonged and exceeds 11 years. During this time, the disease often worsens and women thus experience avoidable morbidity. Additionally, endometriosis is a financial burden on the healthcare system; its annual cost was $69.4 billion (U.S.) and $1.8 billion (Canada) in 2009. For these reasons, identifying a clinical marker remains a top priority. Although multiple putative markers have been identified and reviewed, emerging evidence suggests a relationship between neurotrophins and endometriosis. The neurotrophins are growth factors recognized for promoting neuronal differentiation, growth, and maintenance. Recently, they have been shown to induce pathways central to endometriosis including proliferation, adhesion, angiogenesis and resistance to apoptosis, in cultured neurons, epithelial cells, fibroblasts, and cancer cell lines. Although two studies have suggested elevated concentrations of brain-derived neurotrophic factor (BDNF) in the plasma and eutopic endometrium of women with endometriosis, relatively little is known about uterine BDNF. Herein, we demonstrate the conservation of BDNF and its high affinity receptor in the mammalian uterus, and show the upregulation of BDNF and its low affinity receptor by estradiol in the mouse uterus. Encouraged by our results, we assessed circulating BDNF for its ability to differentiate between women with and without endometriosis, as excess estradiol in endometriotic lesions might increase BDNF in women with disease. Our results revealed that circulating BDNF concentrations were significantly higher in women with endometriosis, particularly those with Stage I and II disease compared to controls. Furthermore, women with endometriosis undergoing ovarian suppression had significantly lower circulating BDNF than women not undergoing treatment, suggesting that BDNF may provide an opportunity to monitor patient response to treatment. Taken together, the data herein advances our limited knowledge of uterine neurotrophins, and supports a link between BDNF and endometriosis. I therefore strongly suggest that BDNF is a useful clinical marker of endometriosis, and encourage additional research to determine its role in the pathophysiology of disease. / Thesis / Doctor of Philosophy (PhD) / Endometriosis is a chronic condition that affects over 10% of women of reproductive age. Women with endometriosis suffer from debilitating pelvic pain and it takes approximately 12 years before they are diagnosed during surgery. This is in part because there is no blood test to diagnose disease. We are interested in using a protein called brain-derived neurotrophic factor (BDNF) that is linked to several of the pathways that are disturbed in women with endometriosis as a means of determining whether or not a woman has endometriosis. The key goals of this thesis are to show that BDNF is a protein which is found in the uterus of many species, that it is controlled by estrogen, and that it might be useful in diagnosing endometriosis and monitoring how well a patient is responding to endometriosis treatment.

During orthodontic tooth movement, stress is applied on the dento-alveolar complex, initiating a biological response. This response results in the remodelling of the periodontal ligament and the alveolar bone. When a force is placed on a tooth, the periodontal ligament is stretched and compressed depending on the direction of force. On the side where the ligament is stretched, a response resulting in bone deposition is initiated. On the opposite side, where the periodontal ligament is compressed, a response resulting in the resorption of bone begins. These responses are believed to be modulated by factors that are derived from the immune or nervous systems. When stress is placed on the periodontal ligament, it is believed that nerve fibres and neuroreceptors within the tissue are distorted, leading to the release of neurotrophins and a common concomitant clinical response of pain and pressure. These neurotrophins may interact with cells within the dentoalveolar complex, including fibroblasts, endothelial and alveolar bone cells, resulting in the initiation of bone resorption via the activation of intracellular secondary messengers, which leads to cellular proliferation and differentiation. Neurotrophin levels may play a role in the modulation of cellular activity in the periodontal ligament during orthodontic movement. They are a family of protein polypeptides which are important in neural cell differentiation and survival. One relatively well studied member of the family, Nerve Growth Factor (NGF), is a polypeptide essential for supporting cholinergic innervation in the brain and sympathetic and sensory innervation in the peripheral tissues. Within the dento-alveolar complex, the function and localization of neurotrophins and their receptors are yet to be determined. Previous studies have shown that there is an increase in NGF expression in tissues in response to injury, suggesting that NGF expression may increase in regions within the dento-alveolar complex where inflammation and bone remodelling are occurring. A study showed elevated levels of NGF mRNA in human periodontal ligament cells in vitro during increased transcription and translation of the bone-related proteins alkaline phosphatase and osteopontin, suggesting NGF facilitates bone formation. The NGF may be modulating cellular activity within the periodontal ligament. O’Hara et al. used immunohistochemical staining in the rat dento-alveolar complex to show that there was evidence of an increase in NGF synthesis and release by certain cells or tissues within the region of alveolar bone remodelling during the initial injury response period to orthodontic tooth movement. Ho used a similar model to test the hypothesis that there may be a positive relationship between the presence of osteoclasts and pre-osteoclasts with areas of NGF localisation. He found no relationship between osteoclasts and areas of NGF; however, his findings showed areas of unknown tissue within the periodontal ligament that were associated with NGF. To date, the relationship between NGF and the cellular process of orthodontic tooth movement remains unknown. Further studies are required to describe the distribution of neurotrophins and neurotrophic receptors and cellular interactions within the rat dento-alveolar complex. / Thesis (D.Clin.Dent.) -- University of Adelaide, School of Dentistry, 2010

Inflammation is an ubiquitous response of the body to cellular damage and injury. It not only leads to increased production of inflammatory molecules such as prostaglandins, bradykinins, histamine, and nerve growth factor (NGF) that sensitize the neurons, but is also associated with acidosis due to a local physiological decrease in extracellular pH, to as low as 5.3. The addition of protons to the sensitized area results in increased membrane conductance and depolarization in sensory neurons involving the acid sensing ion channels (ASICs), the capsaicin sensitive transient receptor potential vanilloid type 1 receptor (TRPV1), and blockade of background potassium conductance. Collectively, this combination heightens the pain state. As expression and activation of the NGF receptor tropomyosin-related kinase A (TrkA) are critically linked to inflammation-associated nociceptor sensitization, this led us to hypothesize that decreased pH may contribute to this process by depolarizing the neuron and mobilizing more TrkA to the cell membrane. This hypothesis is premised by previous experiments in other neuronal populations demonstrating that increased neuronal activity mobilizes more of the neurotrophin receptor TrkB to the membrane. We explored this question utilizing an in vitro model of acidosis akin to that observed with inflammation. Primary sensory neurons were grown on coverslips at a physiological pH of 7.4. The pH of the media was decreased to an acidic pH of 6.5 in half of the cultures for 30 minutes, followed by fixation under permeablizing or non-permeablizing conditions. Immunocytochemical analysis revealed a significant increase in the mobilization of TrkA to the plasma membrane in response to acidosis (confirmed using a biotinylation assay) and an enhanced level of TrkA activation in response to brief NGF challenge. This rapid mobilization of TrkA was attenuated with the addition of proton-sensitive channel blockers capsazepine and amiloride, for the TRPV and ASIC channels respectively. Unexpectedly, the amount of activated TrkA was also increased at pH 6.5 in the absence of NGF challenge. Taken together, the data suggests that sensory neurons can be rapidly sensitized to NGF in response to a decrease in pH and as such likely plays a role in the sensitization and hyperalgesia associated with an inflammatory state.

Inflammation is an ubiquitous response of the body to cellular damage and injury. It not only leads to increased production of inflammatory molecules such as prostaglandins, bradykinins, histamine, and nerve growth factor (NGF) that sensitize the neurons, but is also associated with acidosis due to a local physiological decrease in extracellular pH, to as low as 5.3. The addition of protons to the sensitized area results in increased membrane conductance and depolarization in sensory neurons involving the acid sensing ion channels (ASICs), the capsaicin sensitive transient receptor potential vanilloid type 1 receptor (TRPV1), and blockade of background potassium conductance. Collectively, this combination heightens the pain state. As expression and activation of the NGF receptor tropomyosin-related kinase A (TrkA) are critically linked to inflammation-associated nociceptor sensitization, this led us to hypothesize that decreased pH may contribute to this process by depolarizing the neuron and mobilizing more TrkA to the cell membrane. This hypothesis is premised by previous experiments in other neuronal populations demonstrating that increased neuronal activity mobilizes more of the neurotrophin receptor TrkB to the membrane. We explored this question utilizing an in vitro model of acidosis akin to that observed with inflammation. Primary sensory neurons were grown on coverslips at a physiological pH of 7.4. The pH of the media was decreased to an acidic pH of 6.5 in half of the cultures for 30 minutes, followed by fixation under permeablizing or non-permeablizing conditions. Immunocytochemical analysis revealed a significant increase in the mobilization of TrkA to the plasma membrane in response to acidosis (confirmed using a biotinylation assay) and an enhanced level of TrkA activation in response to brief NGF challenge. This rapid mobilization of TrkA was attenuated with the addition of proton-sensitive channel blockers capsazepine and amiloride, for the TRPV and ASIC channels respectively. Unexpectedly, the amount of activated TrkA was also increased at pH 6.5 in the absence of NGF challenge. Taken together, the data suggests that sensory neurons can be rapidly sensitized to NGF in response to a decrease in pH and as such likely plays a role in the sensitization and hyperalgesia associated with an inflammatory state.

Análise da expressão de neurotrofinas durante a regeneração de nervo periférico de rato por enxerto venoso Enxertos de veias têm sido empregados para preencher lacunas em nervos periféricos transeccionados para melhor recuperação funcional. No entanto, vários inconvenientes, como a constrição do enxerto secundário foram observados. Uma nova alternativa para esta técnica foi desenvolvida. Simplesmente invertendo a veia de dentro para fora, chamado do Inside- out vein graft. As neurotrofinas são uma família de fatores neurotróficos conhecidos por desempenhar um papel significativo na regeneração de nervos periféricos. A família da neurotrofina é constituído por fator de crescimento nervoso (NGF), fator neurotrófico derivado do cérebro (BDNF), Neurotrofina-3 (NT-3) e Neurotrofina-4 (NT-4). No campo da neurobiologia, vários autores têm utilizado a técnica de PCR a fim de obter mais informações sobre os nervos regenerados. Neste estudo, foi utilizada a técnica de biologia molecular para explorar o papel e o nível das neurotrofinas durante a regeneração de nervos periféricos com enxerto de veia. O nervo isquiático de ratos foi seccionado e reparado com enxerto de veia invertida (IOVG) e técnicas de enxerto de veia padrão (SVG). No grupo controle, os ratos foram operados e o nervo isquiático foi mantido intacto. Os animais foram sacrificados após 6 e 12 semanas e os enxertos foram colhidos para observar o nível das neurotrofinas. Músculos EDL e Sóleo foram excisados e pesados para determinar a diferença de peso entre os grupos. Um pequeno segmento dos cotos distais de ambos os grupos SVG e IOVG também foram excisados e foram processados histologicamente para examinar a quantidade de axónios regenerados. Além disso, um outro pequeno segmento do coto distal foi processado para RT-PCR para analisar o nível das neurotrofinas nesta área.A tecnica do walk track analysis foi realizada para determinar o índice funcional do nervo isquiático nos grupos. Em 6 semanas, não ocorreu crescimento neuronal significativo no coto distal dos dois tipos de enxertos, porém um crescimento foi observado em 12 semanas. Não houve diferença significativa na massa muscular entre IOVG e SVG em ambos os períodos de tempo. No entanto, um aumento significativo na massa muscular foi observado a partir de 6 a 12 semanas nos grupos IOVG e SVG. Um aumento significativo na produção de NT-3 foi observado no grupo de SVG em ambos, enxerto e o coto distal quando comparados a partir de 6 a 12 semanas, no entanto, não houve aumento observado no nível de neurotrofinas dos outros tipos (NGF e NT-4) . Surpreendentemente, não houve aumento significativo da NT-3 no grupo IOVG. Conclui-se que, entre as neurotrofinas avaliadas neste estudo, não há nenhuma diferença significativa no seu nível de RNAm entre os dois grupos, exceto NT-3. Finalmente, uma vez que o nível de RNAm de NT-3 aumenta significativamente entre 6 e 12 semanas no grupo SVG e não no IOVG, observado por estas duas técnicas de nível molecular, estudos adicionais necessitam serem feitos para decifrar o mecanismo exato. / Vein grafts have been employed to bridge the gap in transected peripheral nerves to produce better functional recovery. However several disadvantages such as secondary graft constriction were observed and a new alternative to this technique was developed by simply reversing the vein inside out. Both inside out and standard vein grafts were successfully used in recovering the sensory segmental defect in humans. Neurotrophins are a family of eurotrophic factors known to play an important role in the regeneration of peripheral nerves. The neurotrophin family consists of Nerve Growth Factor (NGF), Brain Derived Neurotrophic Factor (BDNF), Neurotrophin-3 (NT-3) and Neurotropinh-4 (NT-4). In the neurobiology field, several authors have been using PCR technique in order to gain more information regarding regenerated nerves. In this study, we employed this molecular biology technique to explore the role and level of the neurotrophins during the peripheral nerve regeneration with vein graft. The sciatic nerve of rats were sectioned and repaired with Inside out vein graft (IOVG) and standard vein graft techniques (SVG). In the control group the rats were sham operated wherein the sciatic nerve was kept intact. The animals were euthanized at 6 and 12 weeks and the grafts were harvested to observe the level the neurotrophins. EDL and Sol muscles were excised and measured to determine any weight difference between the groups. A small segment of the distal stumps from both the SVG and IOVG groups were also excised and were subjected to histological process to examine the amount of regenerated axon. In addition, another small segment of the distal stump was processed for RT-PCR to further examine the level of the neurotrophins in this area. At 6 weeks, no significant neuronal growth was observed in the distal stump of both graft types but a distinct growth was seen at 12 weeks. Walk track analysis showed poor motor function recovery in the experimental groups during both time intervals. Morphometric analysis demonstrated no significant differences in the amount of myelination between both the groups. There was no significant difference in the muscle mass between IOVG and SVG in both time periods. However, a significant increase in both the muscle mass was observed from 6 to 12 weeks in the IOVG and SVG groups. A significant increase in the production of NT-3 was observed in SVG group in both the distal stump and graft segment when compared from 6 to 12 weeks; however there was no observed increase in the level of other neurotrophins (NGF and NT-4). Surprisingly, no significant increase of NT-3 was noticed in the IOVG group. We conclude that amongst the neurotrophins evaluated in this study, there is no significant difference in their mRNA level between both groups except NT-3. Also, since mRNA level of NT-3 increases significantly between 6 and 12 weeks in SVG and not in IOVG, it suggests that the mechanism by which these two techniques operate at a molecular level may differ and further studies need to be done to decipher the exact mechanism.

The major goal of my study is to understand how the nervous system regulates lung function and disease pathophysiology. Asthma, which is a chronic allergic disease in the lung, has been associated with deregulated airway innervation. The two cell types in the lung that are innervated are airway smooth muscle cells and pulmonary neuroendocrine cells (PNECs). Given that asthma often starts in early childhood, prior research established a neonatal mouse model of allergen exposure to facilitate functional studies of nerves in the development of asthma. Our previous findings showed that allergen exposure to developing, postnatal lungs upregulated levels of neurotrophin 4 (NT4), and caused airway hyperinnervation associated with persistent mucus overproduction. In this work, I describe a novel role of the pulmonary neuroendocrine system in promoting mucus overproduction in early life through deregulated GABAergic signaling. PNECs are the only innervated epithelial cells and express a variety of neuropeptides and bioactive amines. However, how neural innervation affects PNEC secretion and function in disease is not known. Here, I demonstrated that PNECs were the only source of gamma-Aminobutyric acid (GABA) in airways and that GABA hypersecretion from PNECs was required for mucus overproduction following early life allergen exposure. Further, mice lacking NT4 were protected from allergen-induced PNEC hyperinnervation, GABA hypersecretion, and thus mucus overproduction, all which could be rescued with addition of GABA. These findings link PNECs and allergen-induced mucus overproduction through NT4-dependent innervation. Notably, like mice, infant nonhuman primates exhibit PNEC hyperinnervation following early life exposure to ozone and allergens. In addition, I demonstrate that GABA acts in concert with interleukin-13 to induce the proliferation of mucus-producing goblet cells in human airway epithelium cell cultures. Lastly, building upon our previous observations that mast cells contributed to the elevated NT4 levels after allergen exposure, I initiated a research project that investigates the function of a discrete, resident mast cell in: NT4 expression, PNEC innervation, and mucus overproduction. Together, my findings address a novel fundamental role of the neuroendocrine system biology in animal models of asthma. Targeting the nerve–PNEC axis may be a valid treatment strategy for mucus overproduction in asthma. / 2020-06-12T00:00:00Z

Neurotrophins are a family of growth factors necessary for the development and maintenance of the nervous system. They produce their effects through receptor mediated signaling mechanisms that are highly regulated by sophisticated intracellular transport networks. The impairment of intracellular trafficking of neurotrophins in motor neurons has been identified as one possible factor in the development of motor neuron diseases, but remains inadequately studied. Aided by advances in imaging technology and the development of more powerful and sensitive detection tools for in-vitro studies, the dynamics of intracellular transport of neurotrophins are beginning to be unraveled. However, a primary limiting factor in the study of neurotrophin-transport dynamics in motor neurons has been the lack of alternative and easily available in-vitro systems able to substitute the often difficult and costly primary motor neuron cultures. The aim of this project was to develop a suitable motor neuron model using the NSC-34 cell line for the study of receptor mediated trafficking events through endosomal transport pathways. Successful evaluation and characterization of NSC-34 cells for motor neuron specific markers would result in the investigation of the p75 neurotrophin receptor (p75NTR) trafficking pathways in the presence of exogenous neurotrophins, with a variety of confocal imaging techniques. Chapter 3 describes the optimisation of NSC-34 cell culture conditions through media modification and the development of a suitable growth substrate matrix, which significantly improved cell adhesion, differentiation and the ability to culture the cells for extended time periods in serum free conditions. Quantitative measurements of cell proliferation, culture viability, cell-body size and neurite length are described to highlight the increased value of the cell line for long-term culture and experiments examining a broad range of issues relevant to motor neurons. In Chapter 4, multiple experimental approaches were used to extensively screen the NSC-34 cell line for the presence of motor neuron-specific markers, neurotrophin receptors and proteins involved in regulation of endosomal transport. This characterization established the presence of a developing motor neuron-like neurotrophin receptor profile (p75NTR, TrkB and TrkC), a genetic marker of developing motor neurons, cholinergic markers, proteins regulating transport within the endosomal pathway, and additional proteins previously shown to directly interact with neurotrophin receptors, including sortilin, and the lipid raft associated ganglioside GT1b. Furthermore, evidence is provided that NSC-34 cells undergo apoptosis in response to exogenous nerve growth factor (NGF) or neurotrophin-3 (NT-3), but not brain derived neurotrophic factor (BDNF) or neurotrophin-4 (NT-4). In addition characterization of mouse specific p75NTR antibodies is presented to establish their suitability for internalization studies without altering the binding of exogenous neurotrophins to the receptor. Subsequent confocal microscopy examination focusing on p75NTR trafficking in Chapter 5 revealed that internalization and intracellular transport of this receptor is regulated by exogenous neurotrophins at the cell surface where ligand binding and internalization occur, and in endosomal compartments where the bulk of receptors and ligands are targeted to their specific destinations. Evidence is provided showing that p75NTR internalization is altered in the presence of NGF, NT-3, or NT-4, but not BDNF, and the receptor is diverted into non-clathrin mediated endosomal pathways in response to NGF but not BDNF. Immunofluorescence confocal microscopy suggests that p75NTR recycles to the plasma membrane in a Rab4 GTPase dependent manner in the absence of neurotrophins. Addition of neurotrophins diverted p75NTR from the recycling Rab4 positive pathway, into EEA-1 positive sorting endosomes in the presence of NGF or NT-3, or lysosomal degradation in the presence of BDNF or NT-4. This study clearly demonstrates the suitability of the NSC-34 cell line as an alternate in-vitro system for the study of motor neuron biology, particularly the study of neurotrophin receptor trafficking. Taken together the results represented in this study suggest for the first time, that the fate of the p75NTR receptor depends on which neurotrophin is bound. These findings have important implications for understanding the dynamic mechanisms of action of p75NTR in normal neuronal function, and may also offer further insight into the potential role of neurotrophins in the treatment of neurodegenerative diseases.

Over the years, the Burgess group has been focusing on the preparation and testing of small molecules that mimic protein secondary structures for protein-protein interactions. The most successful compounds made are C10 peptide macrocycles that effectively mimic β-turns and have given promising results from biological testing. These peptide macrocycles have also been dimerized to give even more effective ligands for protein-protein interaction. The successes of the peptide macrocycles have enabled us to look into increasing the chemical diversity of our libraries. This we believe will not only improve our ability to obtain high affinity ligands for the receptors of interest, but will also allow us to investigate other receptors. To achieve this, peptoids were incorporated into the C10 system to replace the peptides in the i+1 and i+2 positions. With the help of Microwave irradiation, semi-peptoid macrocycles were synthesized with a total reaction time of less than 2 h. These compounds were characterized and found to mimic β-turn, and show promising biological activity towards the Insulin-like growth factor 1 receptor (IGF-IR).