Pentameric ligand-gated ion channels (pLGICs) are membrane-embedded receptors found extensively in pre- and post-synaptic membranes throughout the nervous system where they play an important role in neurotransmission. The function of the prototypic pLGIC, the nicotinic acetylcholine receptor (nAChR) is highly sensitive to changes in its lipid environment, while other pLGICs display varying lipid sensitivities. This thesis presents a multidisciplinary investigation into the features of the transmembrane domain (TMD) that determine the unique functional and physical traits of different pLGICs. Using two prokaryotic homologues of the nAChR, ELIC and GLIC, as models, I focus on the outermost, lipid-exposed α-helix, M4, which, despite being distant from the primary allosteric pathway coupling agonist binding to channel gating, exercises significant control over channel function. Here, I present evidence that M4 acts as a lipid sensor, detecting changes in the surrounding lipids and transmitting these changes to the channel pore via contacts with the adjacent TMD α-helices, M1 and M3, and/or with structures in the extracellular domain. Using ELIC and GLIC chimeras, I first show that the TMD is the main driver of pLGIC thermal stability. I then demonstrate that the M4 α-helices in each channel play different roles in channel maturation and function, which suggests a divergent evolutionary path. Following this, I show that the M4 C-terminus is essential to both maturation and function in GLIC, while in ELIC its role is less defined, again showcasing possible evolutionary differences. Building on these findings, I examined the role of aromatic residues at the M4 – M1/M3 interface, and found that they predictably determine the interactions between M4 and M1/M3. Notably, the addition of aromatic residues to enhance M4-M1/M3 interactions in ELIC promotes channel function, while the elimination of aromatic residues at the M4-M1/M3 interface in GLIC is detrimental to channel function. Furthermore, I show that these same aromatics alter the strength of pLGIC lipid sensing and the sensitivity to certain disease-causing mutations, both indicating that aromatic residues are key players in channel function, stability and modulation. Finally, I and my collaborators identified and characterized a novel desensitization-linked lipid binding site in ELIC. Extensive mutagenesis studies coupled with biophysical measurements allowed us to develop a model describing how lipid binding influences the rates of ELIC desensitization to shape the agonist-induced response.

Alcohol, recently named the most dangerous drug in the world, contributes to nearly 40% of violent crimes and fatal traffic accidents, increases risk of roughly 60 different diseases and injuries, and is responsible for 2.5 million deaths each year worldwide. Despite these staggering figures, treatments remain ineffective and riddled with adverse side effects, making successful use of even the most effective treatments unlikely. Moreover, many of the treatments, and the supporting research, have focused only on male subjects, despite sex differences in various alcohol-related behaviors. Human alcohol use is frequently accompanied by nicotine use, and vice versa, suggesting a common mechanism of the two drugs. In fact, alcohol may act through the same family of receptors as nicotine, the nicotinic acetylcholine receptors (nAChRs), eliciting similar activation of the reward pathway as nicotine and other drugs of abuse. Studies have shown that nAChRs containing the α4 and/or α6 subunits are involved in nicotine-induced activation of the reward pathway, leading to the hypothesis that these same receptor subtypes may be important for alcohol effects in the brain as well. Using male and female genetic mouse models and various behavioral assays, we have shown not only that these α4 and/or α6-containing nAChRs are involved in alcohol- related behaviors and activation of the reward pathway, but also show sex differences in this involvement. Uncovering the mechanism of alcohol in the brain, in males as well as in females, is an important step in developing targeted treatments for alcohol abuse.

Indiana University-Purdue University Indianapolis (IUPUI) / Nicotine use among individuals with schizophrenia occurs at extremely high rates. The prevailing theory is that individuals with schizophrenia smoke as a form of self-medication to ameliorate sensory and cognitive deficits. However, these individuals also have enhanced rates of addiction to several drugs of abuse and may therefore smoke as a result of enhanced addiction liability. The experiments described herein explored these two hypotheses by assessing the effect that nicotine has on working memory, addiction vulnerability (locomotor sensitization and self-administration), and nicotinic acetylcholine receptor (nAChR) expression as well as the developmental expression of these characteristics in the neonatal ventral hippocampal (NVHL) neurodevelopmental animal model of schizophrenia. The results from these studies indicate that NVHLs had working memory impairments in both adolescence and adulthood, with nicotine having a negligible effect. Additionally, NVHLs displayed enhanced locomotor sensitization to nicotine which emerged in adulthood as well as an enhanced acquisition of nicotine self-administration, administering more nicotine overall. These behavioral differences cannot be attributed to nAChR expression as nicotine upregulated nAChR to a similar extent between NVHL and SHAM control animals. These data indicate that the enhanced rates of nicotine use among individuals with schizophrenia may occur as a result of an enhanced vulnerability to nicotine addiction.

No description available.

A sepse causa uma alta taxa de mortalidade no mundo. A fisiopatologia da doença envolve uma rede complexa de mediadores inflamatórios que promovem a lesão de diversos tecidos, além de diversas alterações hemodinâmicas e disfunção do sistema nervoso autonômico (SNA). Assim sabe-se que o sistema nervoso cumpre um papel importante no controle da inflamação sistêmica mediante a via colinérgica anti-inflamatória (VCA) através do receptor nicotínico de acetilcolina alfa7 (alfa7nAChR). O uso das células-tronco mesenquimais (CTM) tem mostrado efeitos benéficos em diversos ensaios clínicos de doenças inflamatórias. Neste contexto, as células-tronco mesenquimais derivadas da geleia de Wharton do cordão umbilical (CTM-GW) tornam-se promissórias, uma vez que essas células são reconhecidas pela regulação da resposta imunológica, reparação neural, efeito anti-apoptose, assim como a melhora da sobrevida na sepse, em modelos experimentais. Nossa hipótese foi de que as CTM-GW poderiam cumprir um papel neuroimunomodulador através da VCA e atenuar a disfunção de múltiplos órgãos em um modelo animal de sepse de ligadura e punção do ceco (LPC). Inicialmente células da matriz do cordão umbilical foram isoladas e caracterizadas de acordo com o consenso internacional vigente. Ratos Wistar machos adultos foram subdivididos em grupos: 1) sham (operação simulada); 2) LPC; 3) LPC+CTM-GW (injetado 106 CTM-GW via intraperitoneal, i.p. 6 h após LPC) e 4) LPC+MLA+CTM-GW (MLA: Metillicaconitine, antagonista do alfa7nAChR, i.p., 5:30 h após LPC e 106 CTM-GW 6h após). Às 24 horas após LPC, foram avaliadas a função cardiovascular, hemodinâmica assim como os outros parâmetros. Interessantemente, o tratamento com CTM-GW na sepse atenuou a disfunção diastólica e protegeu a sensibilidade baroreflexa. Além disso, as CTM-GW estimularam a atividade autonômica, simpática e parassimpática no coração. Observamos que o tratamento celular induziu uma regulação da expressão do receptor alfa7nAChR e TLR4 no baço e no coração, assim como a redução da relação p-STAT3TYR705 e STAT3 total no baço. Outros efeitos importantes e adicionais foram a diminuição da infiltração de leucócitos e a regulação das citocinas pró-inflamatórias pelas células. O bloqueio da VCA usando MLA confirmou que o receptor alfa7nAChR pode ser um provável alvo, chave da ação das CTM entre vários outros mecanismos envolvidos na resposta imune. Finalmente, as CTM-GW conseguiram reduzir a apoptose no pulmão e no baço independentemente da VAC reforçando o conceito de que as células-tronco tem efeitos diversos além da imuno-regulação. Em conclusão, as CTM-GW na sepse foram capazes de atenuar a lesão cardiopulmonar assim como modular a atividade autonômica, reduzindo a inflamação sistêmica, pelo menos em parte, através da via colinérgica anti-inflamatória. Indubitavelmente todos estes efeitos anteriormente descritos e em associação se demonstraram fundamentais no mecanismo de reparo e proteção tecidual em resposta a sepse. Mais estudos pré-clínicos e futuros testes clínicos precisam ser realizados para maior compreensão destes mecanismos bem como uma possível validação terapêutica / Sepsis induces organ dysfunction due to overexpression of the inflammatory host response, involving cardiorespiratory and autonomic dysregulation, thus increasing the associated morbidity and mortality. The cholinergic anti-inflammatory pathway (CAP) is mediated by nervous system through alpha7 nicotinic acetylcholine receptor (alpha7nAChR). This receptor has an important role in systemic inflammation control. Wharton\'s jelly-derived mesenchymal stem cells (WJ-MSCs) are known to express genes and secreted factors related to neurological and immunological protection, as well as to improve survival in experimental sepsis. We hypothesized that WJ-MSCs play a modulatory role through the CAP and attenuate sepsis-induced organ injury in a cecal ligation and puncture (CLP) model. Rats were randomly divided into 4 groups: 1) Control (sham-operated); 2) submitted to CLP without treatment; 3) submitted to CLP and treated with 106 WJ-MSCs 6 h later and 4) CLP+MLA+WJ-MSC group (MLA: Methyllycaconitine, alpha7nAChR antagonist). All experiments were performed 24 h post-surgery. Echocardiographic parameters and heart rate variability were assessed. Importantly, treatment with WJ-MSCs attenuated diastolic heart failure and recovered barorreflex sensitivity. Moreover, WJ-MSCs injection increased cardiac sympathetic and cardiovagal activity. In cardiac and splenic tissue, WJ-MSC treatment downregulated TLR4 and alpha7nAChR expression, as well as it reduced p-STAT3/Total STAT3 ratio in the spleen. In addition, WJ-MSC reduced leukocyte infiltration and pro-inflammatory cytokines, which only were abolished by MLA treatment. Finally, WJ-MSC treatment diminished apoptosis in lung and spleen tissue. Together these findings suggest that treatment with WJ-MSCs appears to protect against sepsis-induced organ injury reducing systemic inflammation, at least in part, through cholinergic anti-inflammatory pathway

A lesão pulmonar aguda (LPA) é caracterizada por inflamação pulmonar de início súbito com recrutamento de polimorfonucleares e liberação de mediadores próinflamatórios. É uma condição grave que evolui com óbito em aproximadamente 40% dos casos. Diversos estudos que elucidaram a fisiopatologia da LPA, o tratamento ainda é insatisfatório. O sistema colinérgico anti-inflamatório foi descrito no pulmão e está relacionado a um reflexo via nervo vago que inibe a liberação de citocinas inflamatórias por efeitos relacionados a ação da acetilcolina em receptores nicotínicos. Nossa hipótese é de que a redução de VAChT, que está relacionada ao déficit na liberação de ACh, module a resposta inflamatória pulmonar em modelo de LPS. Objetivo: 1. Avaliar se a deficiência de VAChT modula a resposta pulmonar em animais geneticamente modificados; 2. Avaliar se a deficiência colinérgica induzida por redução de VAChT está envolvida na resposta pulmonar ao LPS e elucidar alguns mecanismos envolvidos; 3. Avaliar o potencial terapêutico do PNU, um agonista de alfa7nAChR nas alterações funcionais e histopatológicas em modelo de LPA em animais C57Bl6. Metodologia: Foram utilizados camundongos machos geneticamente modificados mutante (VAChT KDHOM) ou selvagem (WT) e C57BL/6. Inicialmente avaliamos a função pulmonar e a histopatologia pulmonar em animais VAChT KDHOM. Após, animais WT e VAChT KDHOM receberam instilação intranasal de LPS ou salina e a resposta inflamatória foi avaliada de 1,5h até 72 horas após. Ainda, foi avaliado a resposta pulmonar em VAChT KDHOM e WT após a instilação de LPS intraperitoneal. Por fim, animais C57BL/6 instilados com LPS intranasal, receberam tratamento prévio ou após com PNU, agonista do receptor nicotínico alfa7. Resultados: Animais mutante apresentaram maior quantidade de células recuperadas no lavado bronco alveolar (LBA) e aumento de citocinas próinflamatórias, aumento de edema peribrônquico e piora da função pulmonar. Ainda, observamos aumento da expressão de NF-kB e redução de JAK2. A deficiência de VAChT induziu aumento de células inflamatórias em animais que receberam LPS somente em 1.5h após a indução, sendo os valores iguais ao dos animais WT em 24 e 72 horas. Nos animais WT, o estimulo do receptor nicotínico melhora a inflamação, enquanto o estímulo de receptores muscarínicos parece contribuir com a piora da resposta da inflamação pulmonar. Os efeitos do PNU parecem que dependem da via colinérgica intacta, uma vez que esta droga não teve o mesmo efeito em animais mutante. Entretanto, o tratamento com PNU em animais C57BL/6 reduziu a inflamação, a produção de citocinas, a deposição de colágeno no tecido pulmonar e os níveis de MMP-2, MMP-9 e TIMP-1, melhorando a função pulmonar. Estes efeitos parecem estar associados a redução de macrófagos perfil M1, e a inibição de NF-kB. Conclusão: Estes dados claramente demonstram que o sistema colinérgico anti-inflamatório está envolvido no controle da resposta inflamatória pulmonar, seja na manutenção da homeostasia ou ainda nas fases iniciais do desenvolvimento da LPA. Ainda, está claro que o estímulo de receptores nicotínicos tem grande potencial como alvo terapêutico a ser explorado na SDRA / Acute lung injury (ALI) is characterized by acute lung inflammation with recruitment of polymorphonuclear and release of proinflammatory mediators. It is a severe condition since leads to death 40% of the cases. Several studies have elucidated the pathophysiology of ALI, however the treatment is still unsatisfactory. The anti-inflammatory cholinergic system was described in the lung and is related to a vagal nerve reflex that inhibits the release of inflammatory cytokines by the action o ACh on nicotinic receptors. Our hypothesis is that the VAChT reduction, which is related to the deficit in the release of ACh, modulates the pulmonary inflammatory response in a model of LPS. Aim: 1. To assess whether VAChT deficiency modulates the pulmonary response in genetically modified animals; 2. Assess whether cholinergic deficiency induced reduction VAChT is involved in pulmonary response to LPS and elucidate some mechanisms involved; 3. To evaluate the therapeutic potential of PNU, an agonist alfa7nAChR, in functional and histological changes in C57BL6 mice with LPA. Methods: Mutant genetically modified male mice (VAChT KDHOM) or wild (WT) and C57BL/6 were used. First, we evaluated lung function and lung histopathology in VAChT KDHOM animals. After, WT animals and VAChT KDHOM received intranasal instillation of LPS or saline and the inflammatory response was assessed 1.5 hours to 72 hours. Moreover, the pulmonary response was evaluated in WT and VAChT KDHOM after instillation of LPS intraperitoneally. Finally, C57BL6 instilled with intranasal LPS received prior or post-treatment with PNU, an alfa7 nicotinic receptor agonist. Results: Mutant animals had higher number of cells recovered in brochoalveolar lavage (BAL) and increased pro-inflammatory cytokines, peribronchial edema and worsening of lung function. Still, there was an increase of NF_kB expression and reduction of JAK2. The VAChT deficiency induced increase in inflammatory cells in animals receiving LPS only 1.5h after the LPS instilation, and the values were similar to WT in 24 and 72 hours. In WT mice, the stimulation of the nicotinic receptor improves inflammation, while the stimulation of muscarinic receptors appears to contribute to the worsening of the pulmonary inflammatory response. The effects of PNU seem to depend on the intact cholinergic pathway, since this drug had no effects on mutant animals. However, treatment with PNU in C57BL6 reduced pulmonar inflammation, cytokine production, collagen deposition in lung tissue and the levels of MMP-2, MMP-9 and TIMP-1, improving pulmonary function. These effects appear to be associated with reduced profile M1 macrophages and the inhibition of NF-kB. Conclusion: These data clearly demonstrate that the anti-inflammatory cholinergic system is involved in the control of lung inflammatory response, both to maintain the lung homeostasis or in the early stages of the development of ALI. Finally, it is clear that the stimulation of nicotinic receptors has great potential as a therapeutic target to be explored in ARDS

A lesão pulmonar aguda (LPA) é caracterizada por inflamação pulmonar de início súbito com recrutamento de polimorfonucleares e liberação de mediadores próinflamatórios. É uma condição grave que evolui com óbito em aproximadamente 40% dos casos. Diversos estudos que elucidaram a fisiopatologia da LPA, o tratamento ainda é insatisfatório. O sistema colinérgico anti-inflamatório foi descrito no pulmão e está relacionado a um reflexo via nervo vago que inibe a liberação de citocinas inflamatórias por efeitos relacionados a ação da acetilcolina em receptores nicotínicos. Nossa hipótese é de que a redução de VAChT, que está relacionada ao déficit na liberação de ACh, module a resposta inflamatória pulmonar em modelo de LPS. Objetivo: 1. Avaliar se a deficiência de VAChT modula a resposta pulmonar em animais geneticamente modificados; 2. Avaliar se a deficiência colinérgica induzida por redução de VAChT está envolvida na resposta pulmonar ao LPS e elucidar alguns mecanismos envolvidos; 3. Avaliar o potencial terapêutico do PNU, um agonista de alfa7nAChR nas alterações funcionais e histopatológicas em modelo de LPA em animais C57Bl6. Metodologia: Foram utilizados camundongos machos geneticamente modificados mutante (VAChT KDHOM) ou selvagem (WT) e C57BL/6. Inicialmente avaliamos a função pulmonar e a histopatologia pulmonar em animais VAChT KDHOM. Após, animais WT e VAChT KDHOM receberam instilação intranasal de LPS ou salina e a resposta inflamatória foi avaliada de 1,5h até 72 horas após. Ainda, foi avaliado a resposta pulmonar em VAChT KDHOM e WT após a instilação de LPS intraperitoneal. Por fim, animais C57BL/6 instilados com LPS intranasal, receberam tratamento prévio ou após com PNU, agonista do receptor nicotínico alfa7. Resultados: Animais mutante apresentaram maior quantidade de células recuperadas no lavado bronco alveolar (LBA) e aumento de citocinas próinflamatórias, aumento de edema peribrônquico e piora da função pulmonar. Ainda, observamos aumento da expressão de NF-kB e redução de JAK2. A deficiência de VAChT induziu aumento de células inflamatórias em animais que receberam LPS somente em 1.5h após a indução, sendo os valores iguais ao dos animais WT em 24 e 72 horas. Nos animais WT, o estimulo do receptor nicotínico melhora a inflamação, enquanto o estímulo de receptores muscarínicos parece contribuir com a piora da resposta da inflamação pulmonar. Os efeitos do PNU parecem que dependem da via colinérgica intacta, uma vez que esta droga não teve o mesmo efeito em animais mutante. Entretanto, o tratamento com PNU em animais C57BL/6 reduziu a inflamação, a produção de citocinas, a deposição de colágeno no tecido pulmonar e os níveis de MMP-2, MMP-9 e TIMP-1, melhorando a função pulmonar. Estes efeitos parecem estar associados a redução de macrófagos perfil M1, e a inibição de NF-kB. Conclusão: Estes dados claramente demonstram que o sistema colinérgico anti-inflamatório está envolvido no controle da resposta inflamatória pulmonar, seja na manutenção da homeostasia ou ainda nas fases iniciais do desenvolvimento da LPA. Ainda, está claro que o estímulo de receptores nicotínicos tem grande potencial como alvo terapêutico a ser explorado na SDRA / Acute lung injury (ALI) is characterized by acute lung inflammation with recruitment of polymorphonuclear and release of proinflammatory mediators. It is a severe condition since leads to death 40% of the cases. Several studies have elucidated the pathophysiology of ALI, however the treatment is still unsatisfactory. The anti-inflammatory cholinergic system was described in the lung and is related to a vagal nerve reflex that inhibits the release of inflammatory cytokines by the action o ACh on nicotinic receptors. Our hypothesis is that the VAChT reduction, which is related to the deficit in the release of ACh, modulates the pulmonary inflammatory response in a model of LPS. Aim: 1. To assess whether VAChT deficiency modulates the pulmonary response in genetically modified animals; 2. Assess whether cholinergic deficiency induced reduction VAChT is involved in pulmonary response to LPS and elucidate some mechanisms involved; 3. To evaluate the therapeutic potential of PNU, an agonist alfa7nAChR, in functional and histological changes in C57BL6 mice with LPA. Methods: Mutant genetically modified male mice (VAChT KDHOM) or wild (WT) and C57BL/6 were used. First, we evaluated lung function and lung histopathology in VAChT KDHOM animals. After, WT animals and VAChT KDHOM received intranasal instillation of LPS or saline and the inflammatory response was assessed 1.5 hours to 72 hours. Moreover, the pulmonary response was evaluated in WT and VAChT KDHOM after instillation of LPS intraperitoneally. Finally, C57BL6 instilled with intranasal LPS received prior or post-treatment with PNU, an alfa7 nicotinic receptor agonist. Results: Mutant animals had higher number of cells recovered in brochoalveolar lavage (BAL) and increased pro-inflammatory cytokines, peribronchial edema and worsening of lung function. Still, there was an increase of NF_kB expression and reduction of JAK2. The VAChT deficiency induced increase in inflammatory cells in animals receiving LPS only 1.5h after the LPS instilation, and the values were similar to WT in 24 and 72 hours. In WT mice, the stimulation of the nicotinic receptor improves inflammation, while the stimulation of muscarinic receptors appears to contribute to the worsening of the pulmonary inflammatory response. The effects of PNU seem to depend on the intact cholinergic pathway, since this drug had no effects on mutant animals. However, treatment with PNU in C57BL6 reduced pulmonar inflammation, cytokine production, collagen deposition in lung tissue and the levels of MMP-2, MMP-9 and TIMP-1, improving pulmonary function. These effects appear to be associated with reduced profile M1 macrophages and the inhibition of NF-kB. Conclusion: These data clearly demonstrate that the anti-inflammatory cholinergic system is involved in the control of lung inflammatory response, both to maintain the lung homeostasis or in the early stages of the development of ALI. Finally, it is clear that the stimulation of nicotinic receptors has great potential as a therapeutic target to be explored in ARDS

Οι νικοτινικοί υποδοχείς της ακετυλοχολίνης (nAChRs) ανήκουν στην υπερ-οικογένεια των ιοντικών καναλιών που ενεργοποιούνται από τη δέσμευση ενός προσδέτη (LGICs) και αποτελούνται από πέντε ομόλογες υπομονάδες. Κάθε μονομερής υπομονάδα αποτελείται από μία Ν-τελική εξωκυττάρια περιοχή (ΕΚΠ), από τέσσερεις διαμεμβρανικές α-έλικες και από μία κυτταροπλασματική περιοχή. Στην ΕΚΠ βρίσκεται η χαρακτηριστική Cys-θηλιά της υπερ-οικογένειας, καθώς και οι θέσεις πρόσδεσης αγωνιστών και ανταγωνιστών του υποδοχέα. Οι γνώσεις μας γύρω από τη δομή των nAChRs προέρχονται κυρίως από κρυσταλλογραφικές δομές ομολόγων πρωτεϊνών δέσμευσης της ACh (AChBP) μαλακίων, από μια δομή του nAChR από ιχθείς του γένους Torpedo που προέρχεται από ηλεκτρονική μικροσκοπία, από την κρυσταλλογραφική δομή της α1-ΕΚΠ ποντικού σε σύμπλοκο με α-μπουγκαροτοξίνη (α-Btx) και από κρυσταλλογραφικές δομές δύο προκαρυωτικών LGICs. Παρά τη μεγάλη πρόοδο που πραγματοποιήθηκε με τα παραπάνω επιτεύγματα, ακόμη δεν έχει επιλυθεί πειραματικά η δομή ανθρώπινου υποδοχέα. Επίσης λίγα είναι γνωστά για την επίδραση της γλυκοζυλίωσης των ΕΚΠ στη λειτουργία του nAChR. Χρησιμοποιώντας ως εκμαγείο την κρυσταλλογραφική δομή του συμπλόκου α1-ΕΚΠ ποντικού/α-Btx δημιουργήθηκαν υπολογιστικά μοντέλα της ανθρώπινης α1-ΕΚΠ προσδεμένης στις τοξίνες α-μπουγκαροτοξίνη (α-Btx), α-κομπρατοξίνη (α-Cbtx), α-κωνοτοξίνη (α-Ctx) ImI και α-κωνοτοξίνη GI. Στα σύμπλοκα με α-Btx και α-Cbtx προστέθηκε η υδατανθρακική αλυσίδα, συνδεδεμένη με το κατάλοιπο Asn141, που συγκρυσταλλώθηκε μαζί με την α1-ΕΚΠ ποντικού. Για να μελετηθεί η δυναμική συμπεριφορά της αλληλεπίδρασης υποδοχέα-τοξίνης καθώς και η συνεισφορά των σακχάρων σε αυτήν πραγματοποιήθηκαν προσομοιώσεις Μοριακής Δυναμικής σε υδατικό περιβάλλον. Με τη χρήση υπολογιστικών εργαλείων για τη μελέτη των συμπλόκων προσδιορίστηκαν σε ατομικό επίπεδο οι αλληλεπιδράσεις που καθοδηγούν την πρόσδεση τοξινών στην α1-ΕΚΠ. Βρέθηκε ότι η υδατανθρακική αλυσίδα συμμετέχει δυναμικά στη δέσμευση της τοξίνης στον υποδοχέα. Τα σάκχαρα συγκλίνουν προς την προσδεμένη τοξίνη στηριζόμενα στα κατάλοιπα Ser187 και Trp184 της α1 υπομονάδας. Η τοξίνη επίσης μετακινείται φέρνοντας τη θηλιά Ι σε επαφή με τα σάκχαρα. Αναγνωρίστηκαν σημαντικές αλληλεπιδράσεις των σακχάρων με τα τοξινικά κατάλοιπα Thr6, Ser9, και Th15 της α-Btx και Thr6 και Pro7 της α-Cbtx. Επίσης επιβεβαιώθηκε η ύπαρξη μιας υδρόφιλης κοιλότητας στο εσωτερικό του υδρόφοβου πυρήνα της α1-ΕΚΠ, η οποία πιθανόν εμπλέκεται στο άνοιγμα του ιοντικού καναλιού του nAChR. Τα αποτελέσματα αυτά παρέχουν σημαντικά δεδομένα για την κατανόηση της επίδρασης της υδατανθρακικής αλυσίδας στη λειτουργία του υποδοχέα, η οποία μπορεί να αξιοποιηθεί στην αντιμετώπιση των πολλών παθολογικών καταστάσεων στις οποίες εμπλέκονται οι nAChRs. / Nicotinic acetylcholine receptors (nAChRs) belong to the superfamily of ligand-gated ion channels (LGICs). LGICs form homo- or hetero-pentamers of related subunits, and each of them consists of a N-terminal extracellular ligand-binding domain (ECD), four transmembrane α-helixes and an intracellular region. The characteristic Cys-loop of the superfamily is found in the ECD of each subunit. The ECD also contains binding sites for agonists and competitive antagonists. Our knowledge regarding the nAChR structure mainly derives from the X-ray crystal structures of the molluscan ACh-binding proteins (AChBPs), the electron microscopy structure of the Torpedo nAChR, the X-ray crystal structure of the mouse nAChR α1-ECD bound to α-bungarotoxin (α-Btx), and the X-ray crystal structures of two prokaryotic LGICs. Despite the progress made by these achievements, the determination of any human nAChR structure has not yet been accomplished. Furthermore, the effect of glycosylation on nAChR function has not yet been explored. Based on the crystal structure of the extracellular domain of the mouse nAChR α1 subunit bound to α-Btx we have generated in silico models of the human nAChR α1-ECD bound to the toxins α-bungarotoxin (α-Btx), α-cobratoxin (α-Cbtx), α-conotoxin (α-Ctx) ImI and α-conotoxin GI. In the case of the α1-ECD/α-Btx and α-Cbtx complexes, a Asn141-linked carbohydrate chain was modeled, its coordinates taken from the crystal structure of the mouse α1-ECD. To gain further insight into the structural role of glycosylation molecular dynamics (MD) simulations were carried out in explicit solvent so as to compare the conformational dynamics of the binding interface between nAChR α1 and the two toxins. The use of computational methods allowed the monitoring of the interactions that govern toxin binding. The MD simulations revealed the strengthening of the receptor-toxin interaction in the presence of the carbohydrate chain. A shift in the position of the sugars towards the bound toxin was observed. Residues Ser187 and Trp184 of nAChR act as critical anchor points for the stabilization of the sugar chain in a close position to the toxin. Toxin Finger I shifts closer to the mannoses, forming important toxin-sugar interactions that implicate residues Thr6, Ser9, and Thr15 of α-Btx, as well as Thr6 and Pro7 of α-Cbtx. Additionally the MD simulations of the human α1 ECD–toxin complexes confirmed the possible accommodation of two water molecules into a hydration cavity inside the hydrophobic core of the subunit, which may contribute to the gating mechanism of the receptor. These findings provide additional structural data that are intended to inspire biophysical studies on the functional role of glycosylation in the gating mechanism of nAChR and also guide the development of novel therapeutic agents for the treatment of nAChR-associated diseases.

A sepse causa uma alta taxa de mortalidade no mundo. A fisiopatologia da doença envolve uma rede complexa de mediadores inflamatórios que promovem a lesão de diversos tecidos, além de diversas alterações hemodinâmicas e disfunção do sistema nervoso autonômico (SNA). Assim sabe-se que o sistema nervoso cumpre um papel importante no controle da inflamação sistêmica mediante a via colinérgica anti-inflamatória (VCA) através do receptor nicotínico de acetilcolina alfa7 (alfa7nAChR). O uso das células-tronco mesenquimais (CTM) tem mostrado efeitos benéficos em diversos ensaios clínicos de doenças inflamatórias. Neste contexto, as células-tronco mesenquimais derivadas da geleia de Wharton do cordão umbilical (CTM-GW) tornam-se promissórias, uma vez que essas células são reconhecidas pela regulação da resposta imunológica, reparação neural, efeito anti-apoptose, assim como a melhora da sobrevida na sepse, em modelos experimentais. Nossa hipótese foi de que as CTM-GW poderiam cumprir um papel neuroimunomodulador através da VCA e atenuar a disfunção de múltiplos órgãos em um modelo animal de sepse de ligadura e punção do ceco (LPC). Inicialmente células da matriz do cordão umbilical foram isoladas e caracterizadas de acordo com o consenso internacional vigente. Ratos Wistar machos adultos foram subdivididos em grupos: 1) sham (operação simulada); 2) LPC; 3) LPC+CTM-GW (injetado 106 CTM-GW via intraperitoneal, i.p. 6 h após LPC) e 4) LPC+MLA+CTM-GW (MLA: Metillicaconitine, antagonista do alfa7nAChR, i.p., 5:30 h após LPC e 106 CTM-GW 6h após). Às 24 horas após LPC, foram avaliadas a função cardiovascular, hemodinâmica assim como os outros parâmetros. Interessantemente, o tratamento com CTM-GW na sepse atenuou a disfunção diastólica e protegeu a sensibilidade baroreflexa. Além disso, as CTM-GW estimularam a atividade autonômica, simpática e parassimpática no coração. Observamos que o tratamento celular induziu uma regulação da expressão do receptor alfa7nAChR e TLR4 no baço e no coração, assim como a redução da relação p-STAT3TYR705 e STAT3 total no baço. Outros efeitos importantes e adicionais foram a diminuição da infiltração de leucócitos e a regulação das citocinas pró-inflamatórias pelas células. O bloqueio da VCA usando MLA confirmou que o receptor alfa7nAChR pode ser um provável alvo, chave da ação das CTM entre vários outros mecanismos envolvidos na resposta imune. Finalmente, as CTM-GW conseguiram reduzir a apoptose no pulmão e no baço independentemente da VAC reforçando o conceito de que as células-tronco tem efeitos diversos além da imuno-regulação. Em conclusão, as CTM-GW na sepse foram capazes de atenuar a lesão cardiopulmonar assim como modular a atividade autonômica, reduzindo a inflamação sistêmica, pelo menos em parte, através da via colinérgica anti-inflamatória. Indubitavelmente todos estes efeitos anteriormente descritos e em associação se demonstraram fundamentais no mecanismo de reparo e proteção tecidual em resposta a sepse. Mais estudos pré-clínicos e futuros testes clínicos precisam ser realizados para maior compreensão destes mecanismos bem como uma possível validação terapêutica / Sepsis induces organ dysfunction due to overexpression of the inflammatory host response, involving cardiorespiratory and autonomic dysregulation, thus increasing the associated morbidity and mortality. The cholinergic anti-inflammatory pathway (CAP) is mediated by nervous system through alpha7 nicotinic acetylcholine receptor (alpha7nAChR). This receptor has an important role in systemic inflammation control. Wharton\'s jelly-derived mesenchymal stem cells (WJ-MSCs) are known to express genes and secreted factors related to neurological and immunological protection, as well as to improve survival in experimental sepsis. We hypothesized that WJ-MSCs play a modulatory role through the CAP and attenuate sepsis-induced organ injury in a cecal ligation and puncture (CLP) model. Rats were randomly divided into 4 groups: 1) Control (sham-operated); 2) submitted to CLP without treatment; 3) submitted to CLP and treated with 106 WJ-MSCs 6 h later and 4) CLP+MLA+WJ-MSC group (MLA: Methyllycaconitine, alpha7nAChR antagonist). All experiments were performed 24 h post-surgery. Echocardiographic parameters and heart rate variability were assessed. Importantly, treatment with WJ-MSCs attenuated diastolic heart failure and recovered barorreflex sensitivity. Moreover, WJ-MSCs injection increased cardiac sympathetic and cardiovagal activity. In cardiac and splenic tissue, WJ-MSC treatment downregulated TLR4 and alpha7nAChR expression, as well as it reduced p-STAT3/Total STAT3 ratio in the spleen. In addition, WJ-MSC reduced leukocyte infiltration and pro-inflammatory cytokines, which only were abolished by MLA treatment. Finally, WJ-MSC treatment diminished apoptosis in lung and spleen tissue. Together these findings suggest that treatment with WJ-MSCs appears to protect against sepsis-induced organ injury reducing systemic inflammation, at least in part, through cholinergic anti-inflammatory pathway

Membrane protein dynamics is of great importance for living organisms. The precise localization of proteins composing a synapse on the membrane facing a nerve terminus is essential for proper functioning of the nervous system. In muscle fibers, the nicotinic acetylcholine is densely packed under the motor nerve termini. A receptor associated protein, rapsyn, acts as a linker between the receptor and the other components of the synaptic suramolecular assembly. Advances in fluorescence microscopy have allowed to measure the behavior of a single receptor in the cell membrane. In this work single-molecule microscopy was used to track the motion of ionotropic acetylcholine (nAChR) and serotonin (5HT3R) receptors in the plasma membrane of cells. We present methods for measuring single-molecule diffusion and their analysis. Single molecule tracking has shown a high dependence of acetylcholine receptors diffusion on its associated protein rapsyn. Comparing muscle cells that either express rapsyn or are devoid of it, we found that rapsyn plays an important role on receptor immobilization. A three-fold increase of receptor mobility was observed in muscle cells devoid of rapsyn. However, in these cells, a certain fraction of immobilized receptors was also found immobile. Furthermore, nAChR were strongly confined in membrane domains of few tens of nanometers. This showed that membrane composition and membrane associated proteins influence on receptor localization. During muscle cell differentiation, the fraction of immobile nAChR diminished along with the decreasing nAChR and stable rapsyn expression levels. The importance of rapsyn in nAChR immobilization has been further confirmed by measurements in HEK 293 cells, where co-expression of rapsyn increased immobilization of the receptor. nAChR is a ligand-gated ion-channel of the Cys-loop family. In mammals, members of this receptor family share general structural and functional features. They are homo- or hetero-pentamers and form a membrane-spanning ion channel. Subunits have three major regions, an extracellular ligand binding domain, a transmembrane channel and a large intracellular loop. 5HT3R was used as a model to study the effect of this loop on receptor mobility. Single-molecule tracking experiments on receptors with progressively larger deletions in the intracellular loop did not show a dependence of the size of the loop on the diffusion coefficient of mobile receptors. However, two regions were identified to play a role in receptor mobility by changing the fractions of immobile and directed receptors. Interestingly, a prokaryotic homologue of cys-loop receptors, ELIC, devoid of a large cytoplasmic loop was found to be immobile or to show directed diffusion similar as the wild-type 5HT3R. The scaffolding protein rapsyn stabilizes nAChR clusters in a concentration dependent manner. We have measured the density and self-interactions of rapsyn using FRET microscopy. Point-mutations of rapsyn, known to provoke myopathies, destabilized rapsyn self-interactions. Rapsyn-N88K, and R91L were found at high concentration in the cytoplasm suggesting that this modification disturbs membrane association of rapsyn. A25V was found to accumulate in the endoplasmic reticulum. Fluorescent tools to measure intracellular concentration of calcium ions are of great value to study the function of neurons. Rapsyn is highly abundant at the neuromuscular junction and thus is a genuine synaptic marker. A fusion protein of rapsyn with a genetically encoded ratiometric calcium sensor has been made to probe synapse activity. This thesis has shown that the combined use of biologically relevant system and modern fluorescence microscopy techniques deliver important information on pLGIC behaviour in the cell membrane. / <p>QC 20151217</p>