INTRODUÇÃO: A Asma Brônquica é caracterizada por obstrução ao fluxo aéreo, reversível ou não, e processo inflamatório pulmonar, caracterizado principalmente por eosinofilia. A persistência da inflamação pode induzir processo de reparo pulmonar associado à redução progressiva da função pulmonar. A recente descrição do sistema colinérgico anti-inflamatório, um mecanismo neural que suprime a resposta imune inata e controla a inflamação por inibição de citocinas proinflammatórias, e a detecção de alguns de seus componentes em células de vias aéreas sugerem uma importante participação deste sistema na fisiopatologia de doenças pulmonares. O principal mediador deste sistema é a acetilcolina (ACh), que é estocada em vesículas sinápticas pelo transportador vesicular de ACh (VAChT), proteína essencial para sua liberação. OBJETIVOS: Avaliar os efeitos da deficiência colinérgica por redução da VAChT nas alterações pulmonares observadas em modelo experimental de inflamação pulmonar induzida pela exposição crônica a ovoalbumina. METODOLOGIA: A redução colinérgica foi induzida pela modificação genética nos níveis de VAChT. Camundongos machos selvagens e mutantes foram submetidos ao protocolo de sensibilização subcutânea com ovoalbumina ou salina nos dias 0, 7 e 14. Após, foram submetidos a desafios inalatórios com ovalbumina a 1% ou inalações de salina por 20 minutos nos dias 26, 27 e 28. No dia 29, foi realizada a avaliação da mecânica pulmonar, da inflamação no lavado broncoalveolar e no tecido e análise histológica de remodelamento e expressão de MMP-9 e TIMP-1 por imunohistoquímica. Também foi quantificado por ELISA níveis de IL-4, IL-10 e de TNF-a no homogenato pulmonar. A análise estatística considerou um p<0,05 como significativo. RESULTADOS: Animais sensibilizados apresentaram hyperresponsividade brônquica, inflamação e edema peribrônquico e deposição de fibras colágenas e elásticas ao redor das vias aéreas comparado ao grupo salina (p<0,05). Além disso houve aumento de IL-4 no homogenato pulmonar e da expressão de MMP-9 e TIMP-1 nas células inflamatórias. Os animais mutantes, independente de serem ou não sensibilizados, apresentaram aumento de TNF-a no pulmão. Os animais mutantes que foram submetidos ao protocolo de sensibilização mostraram aumento da hiperresponsividade brônquica, do eosinófilos, do edema e da deposição de colágeno comparado aos animais selvagens que também foram sensibilizados com ovoalbumina. Estas alterações podem ser atribuídas ao aumento de IL-4 e de MMP-9/TIMP-1 que foi observado nos animais mutantes e sensibilizada em comparação com o os selvagens sensibilizados. Não houve diferença nos níveis de IL-10 nos grupos experimentais. Conclusão: A deficiência colinérgica piora a hiperresponsividade brônquica, a inflamação eosinofílica e o remodelamento, principalmente por interferir com a citocina pró-inflamatória IL-4 e na proporção de MMP-9 e TIMP-1. Estes dados sugerem que a via colinérgica antiinflamatória está envolvida na fisiopatogenia da asma e necessita ser mais investigada / BACKGROUND: Bronchial asthma is characterized by reversible or not airflow obstruction and pulmonary inflammation, mainly characterized by eosinophilia. The persistence of inflammation can induce lung repair process associated with progressive reduction in lung function. Recent evidence of the cholinergic anti-inflammatory system, a neural mechanism that suppresses the innate immune response and control inflammation by proinflammatory cytokines inhibition, and the detection of some of its components in airway cells suggest an important role of this system in pulmonary physiopathology. The main mediator of this system is acetylcholine (ACh), which is stored in synaptic vesicles by vesicular acetylcholine transporter (VAChT), an essential protein for ACh release. AIMS: To evaluate the effects of cholinergic deficiency by VAChT reduction on pulmonary alterations observed in an experimental model of pulmonary inflammation induced by chronic exposure to ovalbumin. METHODS: The cholinergic deficiency was induced by genetic modification on VAChT levels. Wild-type and mutant male mice were submitted to subcutaneous ovalbumin sensitization or saline protocol on days 0, 7 and 14. After, animals were submitted to inhalation challenge with ovalbumin 1% or saline for 20 minutes on days 26, 27 and 28. On day 29, we evaluated the pulmonary mechanics, inflammation in bronchoalveolar lavage and in airways, histological analysis of airway remodeling and the expression of MMP-9 and TIMP-1 by immunohistochemistry. It was also quantified by the levels of IL-4, IL-10 and TNF-a in lung homogenate. The statistical analysis were performed and a p<0.05 was considered significant. RESULTS: Sensitized animals presented bronchial hyperresponsividade, airway inflammation and edema and collagen and elastic fibers deposition of collagen and elastic fibers around the airways compared to saline group (p <0.05). Furthermore, there was an increase of IL-4 in lung homogenate and the expression of MMP-9 and TIMP-1 in inflammatory cells. The mutant animals, regardless the sensitization, showed an increase in lung content of TNF-a. The mutant and sensitized animals showed an increase in bronchial hyperresponsiveness, in eosinophils, edema and collagen deposition in airways compared to the wild type and sensitized animals. These changes can be attributed to increased IL-4 and MMP-9/TIMP-1 that were observed in mutant and sensitized animals. There was no difference in levels of IL-10 in the experimental groups. Conclusion: The cholinergic deficiency worsens bronchial hyperresponsiveness, eosinophilic inflammation, and airway remodeling mainly by interfering with the pro-inflammatory cytokine IL-4 and in MMP-9/TIMP-1 ratio. These data suggest that anti-inflammatory cholinergic pathway is involved in the asthma pathogenesis deserves further investigation

Introdução: O enfisema pulmonar é o maior componente da doença pulmonar obstrutiva crônica (DPOC), é caracterizado pelo alargamento, destruição alveolar e inflamação do parênquima e vias aéreas pulmonares. A recente descrição do sistema colinérgico anti-inflamatório, um mecanismo neural que controla a inflamação por inibição de citocinas pró-inflamatórias, sugere uma importante participação deste sistema na fisiopatologia das doenças pulmonares. A acetilcolina (ACh), principal mediador deste sistema, é estocada em vesículas sinápticas pelo transportador vesicular de ACh (VAChT), proteína essencial para sua liberação na fenda sináptica. Objetivos: Avaliar se os efeitos da hipofunção colinérgica, por redução da expressão do VAChT, interferem com as alterações pulmonares em modelo experimental de enfisema pulmonar. Metodologia: Camundongos machos selvagens e mutantes, estes últimos com redução da função colinérgica por modificação genética nos níveis do VAChT, foram submetidos ao protocolo de elastase (PPE instilação nasal) ou salina. No dia 28, foi avaliado a função pulmonar, a inflamação e o remodelamento pulmonar. Por imunohistoquímica, avaliou-se a expressão de macrófago, NF-kB e isoprostano no pulmão. Algumas citocinas pró-inflamatórias foram avaliadas no homogenato pulmonar pelo Bioplex. Resultados: Animais selvagem que receberam elastase tiveram redução de elastância de tecido, aumento da inflamação no LBA e no tecido, aumento de citocinas pró-inflamatórias e IL-10, aumento do remodelamento pulmonar, e da expressão de NF-kB e de isoprostano. A deficiência colinérgica nestes animais submetidos ao mesmo protocolo de elastase amplificou a resposta inflamatória (macrófago e neutrófilo) no pulmão, níveis de MCP-1 e também aumento células positivas para NF-kB e isoprostano na região do eixo broncovascular. Conclusão: A ACh parece ter um papel protetor da inflamação neste modelo de enfisema pulmonar, pelo menos em parte pelo controle do NF-kB e do estresse oxidativo. Estes resultados sugerem ainda que o remodelamento e a função pulmonar no enfisema experimental não dependem totalmente do grau de inflamação pulmonar / Banckground: Pulmonary emphysema is a major component of chronic obstructive pulmonary disease (COPD), is characterized by enlargement, alveolar destruction and inflammation of the airways and lung tissue. The recent description of the cholinergic anti-inflammatory, a neural mechanism that controls inflammation by inhibition of proinflammatory cytokines, suggests an important role of this system in the pathophysiology of lung disease. The main mediator of this system is acetylcholine (ACh), which is stored in synaptic vesicles by vesicular acetylcholine transporter (VAChT) protein, which is essential for ACh release into the synaptic cleft. Aim: To evaluate whether the effects of cholinergic hypofunction by reduction on VAChT expression, interferes with pulmonary alterations in an experimental model of pulmonary emphysema. Methods: Male mice wild-type and mutant, the last one with reduced cholinergic function by genetic modification in the levels of VAChT, were submitted to the protocol of elastase (PPE intranasally) or saline. On day 28, pulmonary mechanics, inflammation in bronchoalveolar lavage fluid and tissue remodeling were analyzed. By immunohistochemistry, the expression of macrophage, NF-kB and isoprostane in lung was evaluated. Some proinflammatory cytokines were measured in lung homogenate by Bio Plex. Results: Wild-Type animals that received elastase presented a reduction in tissue elastance, an increase in BALF and tissue inflammation as well as in proinflammatory cytokines, IL-10, pulmonary remodeling, and expression of NF-kB and isoprostane. Cholinergic deficient in these animals submitted to the same elastase-induced emphysema protocol amplified the inflammatory response (macrophage and neutrophils) in the lungs, the levels of MCP-1 and the number of positive cells to NF-kB and isoprostane in bronchovascular axis. Conclusions: The ACh seems to have a protective role inflammation in this experimental model of emphysema, at least in part by controlling NF-kB and oxidative stress. These results further suggest that the remodeling and lung function in experimental emphysema does not depend entirely on the degree of lung inflammation

INTRODUÇÃO: A Asma Brônquica é caracterizada por obstrução ao fluxo aéreo, reversível ou não, e processo inflamatório pulmonar, caracterizado principalmente por eosinofilia. A persistência da inflamação pode induzir processo de reparo pulmonar associado à redução progressiva da função pulmonar. A recente descrição do sistema colinérgico anti-inflamatório, um mecanismo neural que suprime a resposta imune inata e controla a inflamação por inibição de citocinas proinflammatórias, e a detecção de alguns de seus componentes em células de vias aéreas sugerem uma importante participação deste sistema na fisiopatologia de doenças pulmonares. O principal mediador deste sistema é a acetilcolina (ACh), que é estocada em vesículas sinápticas pelo transportador vesicular de ACh (VAChT), proteína essencial para sua liberação. OBJETIVOS: Avaliar os efeitos da deficiência colinérgica por redução da VAChT nas alterações pulmonares observadas em modelo experimental de inflamação pulmonar induzida pela exposição crônica a ovoalbumina. METODOLOGIA: A redução colinérgica foi induzida pela modificação genética nos níveis de VAChT. Camundongos machos selvagens e mutantes foram submetidos ao protocolo de sensibilização subcutânea com ovoalbumina ou salina nos dias 0, 7 e 14. Após, foram submetidos a desafios inalatórios com ovalbumina a 1% ou inalações de salina por 20 minutos nos dias 26, 27 e 28. No dia 29, foi realizada a avaliação da mecânica pulmonar, da inflamação no lavado broncoalveolar e no tecido e análise histológica de remodelamento e expressão de MMP-9 e TIMP-1 por imunohistoquímica. Também foi quantificado por ELISA níveis de IL-4, IL-10 e de TNF-a no homogenato pulmonar. A análise estatística considerou um p<0,05 como significativo. RESULTADOS: Animais sensibilizados apresentaram hyperresponsividade brônquica, inflamação e edema peribrônquico e deposição de fibras colágenas e elásticas ao redor das vias aéreas comparado ao grupo salina (p<0,05). Além disso houve aumento de IL-4 no homogenato pulmonar e da expressão de MMP-9 e TIMP-1 nas células inflamatórias. Os animais mutantes, independente de serem ou não sensibilizados, apresentaram aumento de TNF-a no pulmão. Os animais mutantes que foram submetidos ao protocolo de sensibilização mostraram aumento da hiperresponsividade brônquica, do eosinófilos, do edema e da deposição de colágeno comparado aos animais selvagens que também foram sensibilizados com ovoalbumina. Estas alterações podem ser atribuídas ao aumento de IL-4 e de MMP-9/TIMP-1 que foi observado nos animais mutantes e sensibilizada em comparação com o os selvagens sensibilizados. Não houve diferença nos níveis de IL-10 nos grupos experimentais. Conclusão: A deficiência colinérgica piora a hiperresponsividade brônquica, a inflamação eosinofílica e o remodelamento, principalmente por interferir com a citocina pró-inflamatória IL-4 e na proporção de MMP-9 e TIMP-1. Estes dados sugerem que a via colinérgica antiinflamatória está envolvida na fisiopatogenia da asma e necessita ser mais investigada / BACKGROUND: Bronchial asthma is characterized by reversible or not airflow obstruction and pulmonary inflammation, mainly characterized by eosinophilia. The persistence of inflammation can induce lung repair process associated with progressive reduction in lung function. Recent evidence of the cholinergic anti-inflammatory system, a neural mechanism that suppresses the innate immune response and control inflammation by proinflammatory cytokines inhibition, and the detection of some of its components in airway cells suggest an important role of this system in pulmonary physiopathology. The main mediator of this system is acetylcholine (ACh), which is stored in synaptic vesicles by vesicular acetylcholine transporter (VAChT), an essential protein for ACh release. AIMS: To evaluate the effects of cholinergic deficiency by VAChT reduction on pulmonary alterations observed in an experimental model of pulmonary inflammation induced by chronic exposure to ovalbumin. METHODS: The cholinergic deficiency was induced by genetic modification on VAChT levels. Wild-type and mutant male mice were submitted to subcutaneous ovalbumin sensitization or saline protocol on days 0, 7 and 14. After, animals were submitted to inhalation challenge with ovalbumin 1% or saline for 20 minutes on days 26, 27 and 28. On day 29, we evaluated the pulmonary mechanics, inflammation in bronchoalveolar lavage and in airways, histological analysis of airway remodeling and the expression of MMP-9 and TIMP-1 by immunohistochemistry. It was also quantified by the levels of IL-4, IL-10 and TNF-a in lung homogenate. The statistical analysis were performed and a p<0.05 was considered significant. RESULTS: Sensitized animals presented bronchial hyperresponsividade, airway inflammation and edema and collagen and elastic fibers deposition of collagen and elastic fibers around the airways compared to saline group (p <0.05). Furthermore, there was an increase of IL-4 in lung homogenate and the expression of MMP-9 and TIMP-1 in inflammatory cells. The mutant animals, regardless the sensitization, showed an increase in lung content of TNF-a. The mutant and sensitized animals showed an increase in bronchial hyperresponsiveness, in eosinophils, edema and collagen deposition in airways compared to the wild type and sensitized animals. These changes can be attributed to increased IL-4 and MMP-9/TIMP-1 that were observed in mutant and sensitized animals. There was no difference in levels of IL-10 in the experimental groups. Conclusion: The cholinergic deficiency worsens bronchial hyperresponsiveness, eosinophilic inflammation, and airway remodeling mainly by interfering with the pro-inflammatory cytokine IL-4 and in MMP-9/TIMP-1 ratio. These data suggest that anti-inflammatory cholinergic pathway is involved in the asthma pathogenesis deserves further investigation

Introdução: O enfisema pulmonar é o maior componente da doença pulmonar obstrutiva crônica (DPOC), é caracterizado pelo alargamento, destruição alveolar e inflamação do parênquima e vias aéreas pulmonares. A recente descrição do sistema colinérgico anti-inflamatório, um mecanismo neural que controla a inflamação por inibição de citocinas pró-inflamatórias, sugere uma importante participação deste sistema na fisiopatologia das doenças pulmonares. A acetilcolina (ACh), principal mediador deste sistema, é estocada em vesículas sinápticas pelo transportador vesicular de ACh (VAChT), proteína essencial para sua liberação na fenda sináptica. Objetivos: Avaliar se os efeitos da hipofunção colinérgica, por redução da expressão do VAChT, interferem com as alterações pulmonares em modelo experimental de enfisema pulmonar. Metodologia: Camundongos machos selvagens e mutantes, estes últimos com redução da função colinérgica por modificação genética nos níveis do VAChT, foram submetidos ao protocolo de elastase (PPE instilação nasal) ou salina. No dia 28, foi avaliado a função pulmonar, a inflamação e o remodelamento pulmonar. Por imunohistoquímica, avaliou-se a expressão de macrófago, NF-kB e isoprostano no pulmão. Algumas citocinas pró-inflamatórias foram avaliadas no homogenato pulmonar pelo Bioplex. Resultados: Animais selvagem que receberam elastase tiveram redução de elastância de tecido, aumento da inflamação no LBA e no tecido, aumento de citocinas pró-inflamatórias e IL-10, aumento do remodelamento pulmonar, e da expressão de NF-kB e de isoprostano. A deficiência colinérgica nestes animais submetidos ao mesmo protocolo de elastase amplificou a resposta inflamatória (macrófago e neutrófilo) no pulmão, níveis de MCP-1 e também aumento células positivas para NF-kB e isoprostano na região do eixo broncovascular. Conclusão: A ACh parece ter um papel protetor da inflamação neste modelo de enfisema pulmonar, pelo menos em parte pelo controle do NF-kB e do estresse oxidativo. Estes resultados sugerem ainda que o remodelamento e a função pulmonar no enfisema experimental não dependem totalmente do grau de inflamação pulmonar / Banckground: Pulmonary emphysema is a major component of chronic obstructive pulmonary disease (COPD), is characterized by enlargement, alveolar destruction and inflammation of the airways and lung tissue. The recent description of the cholinergic anti-inflammatory, a neural mechanism that controls inflammation by inhibition of proinflammatory cytokines, suggests an important role of this system in the pathophysiology of lung disease. The main mediator of this system is acetylcholine (ACh), which is stored in synaptic vesicles by vesicular acetylcholine transporter (VAChT) protein, which is essential for ACh release into the synaptic cleft. Aim: To evaluate whether the effects of cholinergic hypofunction by reduction on VAChT expression, interferes with pulmonary alterations in an experimental model of pulmonary emphysema. Methods: Male mice wild-type and mutant, the last one with reduced cholinergic function by genetic modification in the levels of VAChT, were submitted to the protocol of elastase (PPE intranasally) or saline. On day 28, pulmonary mechanics, inflammation in bronchoalveolar lavage fluid and tissue remodeling were analyzed. By immunohistochemistry, the expression of macrophage, NF-kB and isoprostane in lung was evaluated. Some proinflammatory cytokines were measured in lung homogenate by Bio Plex. Results: Wild-Type animals that received elastase presented a reduction in tissue elastance, an increase in BALF and tissue inflammation as well as in proinflammatory cytokines, IL-10, pulmonary remodeling, and expression of NF-kB and isoprostane. Cholinergic deficient in these animals submitted to the same elastase-induced emphysema protocol amplified the inflammatory response (macrophage and neutrophils) in the lungs, the levels of MCP-1 and the number of positive cells to NF-kB and isoprostane in bronchovascular axis. Conclusions: The ACh seems to have a protective role inflammation in this experimental model of emphysema, at least in part by controlling NF-kB and oxidative stress. These results further suggest that the remodeling and lung function in experimental emphysema does not depend entirely on the degree of lung inflammation

The brain of Alzheimer???s disease (AD) is characterized by accumulations of ??-amyloid peptide aggregates which promote neurodegentartive dysfunction. Comprehensive understanding of the interaction between ??-amyloid aggregates and acetylcholine (ACh) neurocycle is required to uncover the physiological processes related to AD and might result in improving therapeutic approaches for AD. Pharmacokinetics (PK) and pharmacodynamics (PD) techniques were applied to allow predicting the extent of the interaction of certain doses of AD drugs and ??-amyloid inhibitors and levels of ACh as well. Although many researchers focused on the ??-amyloid interactions, the mechanisms by which ??-amyloid affects cholinergic neurons and reduction of ACh are still unclear. The prediction of ACh and drug concentrations in the tissues and body needs an understanding of the physiology and mechanisms of ??-amyloid aggregates processes and their compilation into a mechanistic model In this work, two hypotheses are proposed to investigate the dynamic behavior of the interaction between ??-amyloid peptide aggregates and cholinergic neurocycle and the possible therapeutic approaches through proposing pharmacokinetic/pharmacodynamics (PK/PD) models to represent the impact of ??-amyloid aggregates in AD. The effect of ??-amyloid peptide aggregates is formulated through incorporating ??- amyloid aggregates into non-linear model for the neurocycle of ACh where the presynaptic neuron is considered as compartment 1 and both synaptic cleft and postsynaptic neurons are considered as compartment 2. In the first hypothesis which is choline leakage hypothesis, ??-amyloid peptide aggregates are considered to be located in the membrane of the presynaptic neuron and create pathways inside the membrane to allow for the intracellular choline to leak outside the cholinergic system. It is observed that ??-amyloid aggregates via the choline leakage hypothesis could cause significant reductions of ACh and choline levels in both compartments. Furthermore, the process rates of ACh synthesis and hydrolysis have been affected negatively by a wide range of ??-amyloid aggregate concentrations. It is found that as the input rate of ??-amyloid aggregates to compartment 1 increases, the loss of choline from compartment 1 increases leading to an increase in the intracellular concentration of ??-amyloid. In the second hypothesis, ??-amyloid peptide aggregates are proposed to interact with the enzyme ChAT which is responsible for the synthesis of ACh in compartment 1; three different kinetic mechanisms are suggested to account for the interaction between ??-amyloid aggregates and ChAT activity. In the first and second kinetic mechanisms, ??-amyloid aggregate is supposed to attack different species in the enzyme. It is found that there is a significant decrease in the rate of ACh synthesis in compartment 1 and ACh concentrations in both compartments. However, it is observed that there is no effect on choline levels in both compartments, the rate of ACh hydrolysis in compartment 2, pH, and ACh levels in compartment 2. In the third kinetic mechanism, all species in ChAT are attacked by ??-amyloid aggregates; it is observed that at very high input rates of ??-amyloid aggregates, the oscillatory behavior dominates all components of the neurocycle of ACh. The disturbance observed in ACh levels in both compartments explains the harmful effect of the full attack of ??-amyloid aggregates to all species of ChAT. It is found that to contribute significantly in ACh neurocycle, choline leakage hypothesis needs concentration of ??-amyloid aggregates lower than that needed in ChAT activity hypothesis which is in agreement with experimental observations. The significant decrease in ACh levels observed in both choline leakage and loss of ChAT activity hypotheses leads to cognitive loss and memory impairment which were observed in individuals with AD. A one-compartment drug PK/PD model is proposed to investigate a therapeutic approach for inhibiting ??-amyloid aggregation via choline leakage hypothesis where the maximum feed rate of ??-amyloid (KL2 = 1) is considered. The drug is assumed to interact with the tissues of the presynaptic neurons where ??-amyloid aggregates are located. The PK/PD model is built based on the effect of ??-amyloid aggregates via choline leakage hypothesis where the maximum feed rate of ??-amyloid aggregates is considered. The dynamic behavior of all concentrations of ??-amyloid aggregates, choline, ACh, acetate, and pH in both compartments in addition to the rate of ACh synthesis in compartment 1 and ACh hydrolysis are investigated by monitoring the impacts of the drug on ??-amyloid aggregates and cholinergic neurocycle over a wide range of the input drug dosage. The PK/PD model is able to predict the reduction in levels of ??-amyloid aggregates and the increase in choline and ACh, in both compartments as well as both rates of ACh synthesis and hydrolysis catalyzed. The parameters of the PK/PD model such as maximum concentration (Cmax), maximum time (Tmax), area under the curve (AUC), and maximum effect (Emax) were investigated. It was found that it takes a longer time (Tmax) (3-5 h) to reach Emax as the drug dose increases. Furthermore, AUC was found to increase with increasing drug dosage. The results of the current work show that drugs / therapeutic agents inhibiting ??- amyloid aggregation in the brain represent a likely successful therapeutic approach to give systematic highlights to develop future trials, new diagnostic techniques, and medications for AD. This study is helpful in designing PK and PD and developing experimental animal models to support AD drug development and therapy in the future.

Three-finger toxins (3FTX) are a group of peptides that affect multiple receptor types. One group of proteins affected by 3FTX are nicotinic acetylcholine receptors (nAChR). Structural information on how neurotoxins interact with nAChR is limited and is confined to a small group of neurotoxins. Therefore, in silico methods are valuable in understanding the interactions between 3FTX and different nAChR subtypes, but there are no established protocols to model 3FTX–nAChR interactions. We followed a homology modeling and protein docking protocol to address this issue and tested its success on three different systems. First, neurotoxin peptides co-crystallized with acetylcholine binding protein (AChBP) were re-docked to assess whether Rosetta protein–protein docking can reproduce the native poses. Second, experimental data on peptide binding to AChBP was used to test whether the docking protocol can qualitatively distinguish AChBP-binders from non-binders. Finally, we docked eight peptides with known α7 and muscle-type nAChR binding properties to test whether the protocol can explain the differential activities of the peptides at the two receptor subtypes. Overall, the docking protocol predicted the qualitative and some specific aspects of 3FTX binding to nAChR with reasonable success and shed light on unknown aspects of 3FTX binding to different receptor subtypes.

Myasthenia gravis (MG) is an autoimmune disease whose name means "grave muscular weakness". MG is a rare disease affecting only 200-400 persons per million and the characteristic symptoms include muscle weakness, particularly in highly active voluntary muscles. MG affects the neuromuscular junction in an antibody-mediated manner, resulting in impaired nerve-muscle cell communication in affected individuals. Specifically, two main proteins are targeted: nicotinic acetylcholine receptors (ACh receptors) and a muscle-specific tyrosine kinase (MuSK). Previous studies have discovered the mechanism of MG pathogenesis but the exact mechanisms which cause the failure to maintain self-tolerance have not been discovered. Based on current knowledge of MG, this paper will explore potential causes of the disease and provide numerous hypotheses directed at future research opportunities.

Upregulation of nicotinic acetylcholine receptors (nAChRs) is a well-documented response to chronic nicotine exposure. Nicotinic acetylcholine receptors are pentameric ligand-gated ion channels consisting of alpha (α2-10) and beta (β2-4) subunits. Nicotine, an agonist of nAChRs, alters trafficking and assembly of some subtypes of nAChRs, leading to an increase in expression of high sensitivity receptors on the plasma membrane. These physiological changes in nAChRs are believed to contribute to nicotine addiction, although the mechanism of these processes has not been resolved. Recently, many studies have converged on the idea that nicotine induces upregulation by an intracellular mechanism. In this dissertation, expression levels of nAChRs were quantified upon exposure to nicotine and its primary metabolite, cotinine. A pH sensitive variant of GFP, super ecliptic pHluorin (SEP), was integrated with a nAChR subunit to study expression and trafficking of nAChRs by differentiating intracellular and plasma membrane inserted receptors. In this work, cotinine is shown to increase the number of α4β2 nAChRs within a cell. Cotinine also affects trafficking of α4β2, evident by a redistribution of intracellular receptors and an increase in single vesicle insertion events on the plasma membrane. This work shows both nicotine and cotinine alter the overall assembly of α4β2 to favor the high sensitivity (α4)2(β2)3 version. Since cotinine and nicotine induce similar physiological changes in nAChRs, the metabolite potentially plays a role in the mechanism of nicotine addiction. Although an intracellular mechanism for upregulation has been supported, a shift in assembly to the high sensitivity (α4)2(β2)3 version exclusively in the endoplasmic reticulum has not previously been detected. In order to study organelle specific changes in stoichiometry, a novel method was developed to isolate single nAChRs in nanovesicles derived from native cell membranes. Separation of nanovesicles originating from the endoplasmic reticulum and plasma membrane, encompassing isolated nAChRs, allows precise changes in stoichiometry to be monitored in subcellular regions. In this work, single molecule bleaching steps of green fluorescent protein (GFP) encoded in each alpha subunit of the pentamer are detected. The number of bleaching steps, or transitions to a nonfluorescent state upon continuous excitation, corresponds to the number of GFP-labeled alpha subunits present. Therefore, the stoichiometry can be deduced by detection of two bleaching steps, as in (α4)2(β2)3, or three bleaching steps, seen in (α4)3(β2)2. Using this method on isolated nAChRs, a shift to assembly of high sensitivity (α4)2(β2)3 receptors is detected definitively within the endoplasmic reticulum. In addition, an increase in (α4)2(β2)3 receptors located on the plasma membrane is shown when nicotine is present. This work provides convincing evidence that nicotine acts intracellularly, within the endoplasmic reticulum, to alter stoichiometry of nAChRs.

Background: Tendinosis is a painful chronic, degenerative condition characterized by objective changes in the tissue structure of a tendon. Hallmark features in tendinosis tendons include increased number of cells (hypercellularity), extracellular matrix (ECM) degradation and disorganized collagen. The progression of these pathological changes seen in tendinosis is neither well characterized nor fully understood. Studies have suggested that there are biochemical and mechanical elements involved in tendinosis. From a biochemical perspective, studies have shown that the tendon cells, tenocytes, produce a number of neuronal signal substances/neuromodulators, such as substance P (SP) and acetylcholine (ACh), traditionally thought to be confined to the nervous system. Furthermore, it has been shown that the expression of these neuromodulators is elevated in tendinosis tendons as compared to normal healthy tendons. Interestingly, studies on other tissue types have revealed that both SP and ACh can induce tissue changes seen in tendinosis, such as hypercellularity and collagen disorganization. From a mechanical angle, it has been suggested that overload of tendons, including extensive strain on the primary tendon cells (tenocytes), causes the degenerative processes associated with tendinosis. In vivo studies have shown that in overloaded tendons, the presence of neuromodulators is elevated, not least SP, which also precedes the development of the tissue changes seen in tendinosis. This further supports the importance of combining biochemical factors and mechanical factors in the pathogenesis of tendinosis. Hypotheses: In this thesis project, we hypothesize: 1) that neuromodulators, such as SP and ACh when stimulating their preferred receptors, the neurokinin 1 (NK-1 R) and muscarinic receptors (mAChRs), respectively, can cause increased tenocyte proliferation; 2) that the effects of SP and ACh on tenocyte proliferation converge mechanistically via a shared signalling pathway; 3) that mechanical loading of tenocytes results in increased production of SP by the tenocytes; and 4) that SP enhances collagen remodelling by tenocytes via NK-1 R. Model system: In vitro studies offer insight into the function of healthy tendon matrix and the etiology of tendinopathy. Using a cell culture model of human primary tendon cells, highly controlled experiments were performed in this thesis project to study a subset of biological and mechanical parameters that are implicated in tendinosis. The FlexCell® Tension System was used to study the influence of mechanical loading on tenocytes. As well, a collagen gel contraction assay was used to examine the intrinsic ability of tenocytes to reorganise type I collagen matrices under the influence of the neuromodulator SP. Results: The studies showed that exogenous administration of SP and ACh results in increased tenocyte proliferation that is mediated via activation of the ERK1/2 mitogenic pathway when the preferred receptors of SP and ACh, the NK-1 R and mAChRs, respectively, are stimulated. Furthermore, the studies resulted in the novel finding that SP and ACh both converge mechanistically via transforming growth factor (TGF)-β1 and that a negative feedback mechanism is present in which TGF-β1 downregulates the expression of mAChRs and NK-1 R. The studies also showed that SP can increase collagen remodelling and upregulate expression of genes related to tendinosis. Finally, it was established that tenocytes are mechanoresponsive by showing that cyclic mechanical loading increases the expression of SP by human tenocytes. Conclusions: This thesis work concludes that stimulation of NK-1 R and mAChRs results in proliferation of human tenocytes, which both involve the ERK1/2 signalling pathway. It also shows that SP and ACh converge mechanistically via TGF-β1 in their contribution to tenocyte proliferation. The role of hypercellularity in tendinosis tissue is unknown. Possibly, it has different roles at different stages of the disease. The findings also show that SP increases collagen remodelling, suggesting that increased SP not only results in hypercellularity but also contributes to the collagen morphology in tendinosis.

The α7 nicotinic acetylcholine receptor (nAChR) subtype is abundantly expressed in the central nervous system (CNS) and in the periphery. Positive allosteric modulators (PAMs) of the α7 increase the response to an agonist and are divided into two types depending on whether they also decrease desensitization of the receptor (type II) or not (type I). Therefore, this study aims to investigate whether the enhancement of endogenous α7 nAChR function will result in a beneficial effect in nociceptive, inflammatory and chronic neuropathic pain models. While NS1738 and PNU-120596 were not active to reduce acute thermal pain, measured by hot-plate and tail-flick tests, only PNU-120596 dose-dependently attenuated paw-licking behavior in the formalin test. Our results with selective (MEK) inhibitor U0126 argues for an important role of extracellular signal-regulated kinase (ERK1/2) pathways activation in PNU-120596’s antinociceptive effects in formalin-induced pain. The α7 antagonist MLA, via intrathecal and intraplantar administration, reversed PNU-120596’s effects, confirming PNU-120596’s action through central and peripheral α7 nAChRs. Tolerance to PNU-120596 was not developed after chronic treatment of the drug. Furthermore, mixtures of PNU-120596 and choline, an endogenous α7 nAChR agonist, synergistically reduced formalin-induced pain, while interactions of non-antinociceptive doses of PNU-120596 and PHA-543613, a selective α7 nAChR agonist, or nicotine resulted in antinociception. In contrast, PNU-120596 failed to enhance nicotine-induced convulsions, -hypomotility and –antinociception in acute pain models. Surprisingly, it enhanced nicotine-induced hypothermia via α7 nAChRs. In the carrageenan inflammatory test both NS1738 and PNU-120596 significantly reduced thermal hyperalgesia, while only PNU-120596 significantly reduced edema. Importantly, PNU-120596 reversed established thermal hyperalgesia and edema induced by carrageenan. In the chronic neuropathic pain (CCI) model, PNU-120596 had long-lasting (up to 6 hrs), dose-dependent anti-hyperalgesic and anti-allodynic effects after a single injection, while NS1738 was inactive. Subcutaneous and intrathecal administration of MLA reversed PNU-120596’s effects, suggesting the involvement of α7 nAChRs. Finally, PNU-120596 enhanced an ineffective dose of selective agonist PHA-543613 to produce anti-allodynic effects in the CCI model. Our results show a fundamental in vivo difference between type I and II α7 nAChR PAMs, and demonstrate type II’s potential for the treatment of chronic inflammatory pain.