RESUMO
Acetylcholinergic (ACh) neurotransmission is essential for key organismal functions such as locomotion and cognition. However, the mechanism through which ACh is regulated in the central nervous system is not fully understood. The vesicular acetylcholine transporter (VAChT) mediates the packaging and transport of ACh for exocytotic release and is a critical component of the ACh release machinery. Yet its precise role in the maintenance of cholinergic tone remains a subject of active investigation. Here we use the overexpression of VAChT as a tool to investigate the role of changes in ACh exocytosis on the regulation of synaptic activity and its downstream consequences. We measured the effect of an increase in VAChT expression on locomotion and cognitive performance as well as on organismal survival across the lifespan. We report the surprising finding that increased VAChT expression results in a significantly shorter lifespan in comparison to control flies. Moreover, constructs overexpressing VAChT demonstrate an age-dependent decrease in locomotion performance. Importantly, we report clear deficits in learning and memory which we measured through a courtship conditioning assay. Together, these data provide evidence for the adverse effects of overexpression of the vesicular acetylcholine transporter in the maintenance of normal behavioral abilities in Drosophila and demonstrates for the first time a role for ACh in the regulation of organismal survival.
Assuntos
Fatores Etários , Cognição/fisiologia , Aprendizagem/fisiologia , Proteínas Vesiculares de Transporte de Acetilcolina/metabolismo , Animais , Proteínas de Transporte/metabolismo , Sistema Nervoso Central/metabolismo , Drosophila/metabolismo , Locomoção/fisiologia , Proteínas de Membrana Transportadoras/metabolismo , Transmissão Sináptica/fisiologia , Proteínas de Transporte Vesicular/metabolismoRESUMO
Multiple sclerosis (MS) is an autoimmune disease targeting the central nervous system (CNS) mainly in young adults, and a breakage of immune tolerance to CNS self-antigens has been suggested to initiate CNS autoimmunity. Age and microbial infection are well-known factors involved in the development of autoimmune diseases, including MS. Recent studies have suggested that alterations in the gut microbiota, referred to as dysbiosis, are associated with MS. However, it is still largely unknown how gut dysbiosis affects the onset and progression of CNS autoimmunity. In this study, we investigated the effects of age and gut dysbiosis on the development of CNS autoimmunity in humanized transgenic mice expressing the MS-associated MHC class II (MHC-II) gene, HLA-DR2a, and T-cell receptor (TCR) genes specific for MBP87-99/DR2a that were derived from an MS patient. We show here that the induction of gut dysbiosis triggers the development of spontaneous experimental autoimmune encephalomyelitis (EAE) during adolescence and early young adulthood, while an increase in immunological tolerance with aging suppresses disease onset after late young adulthood in mice. Furthermore, gut dysbiosis induces the expression of complement C3 and production of the anaphylatoxin C3a, and down-regulates the expression of the Foxp3 gene and anergy-related E3 ubiquitin ligase genes. Consequently, gut dysbiosis was able to trigger the development of encephalitogenic T cells and promote the induction of EAE during the age window of young adulthood.
Assuntos
Sistema Nervoso Central/imunologia , Disbiose/imunologia , Microbioma Gastrointestinal/imunologia , Tolerância Imunológica/imunologia , Animais , Autoimunidade/imunologia , Complemento C3a/imunologia , Regulação para Baixo/imunologia , Encefalomielite Autoimune Experimental/imunologia , Fatores de Transcrição Forkhead/imunologia , Genes MHC da Classe II/imunologia , Antígeno HLA-DR2/imunologia , Humanos , Camundongos , Camundongos Transgênicos , Esclerose Múltipla/imunologia , Receptores de Antígenos de Linfócitos T/imunologia , Linfócitos T/imunologia , Ubiquitina-Proteína Ligases/imunologiaRESUMO
The development of the central nervous system (CNS) is a complex process that must be exquisitely controlled at multiple levels to ensure the production of appropriate types and quantity of neurons. RNA alternative polyadenylation (APA) contributes to transcriptome diversity and gene regulation, and has recently been shown to be widespread in the CNS. However, the previous studies have been primarily focused on the tissue specificity of APA and developmental APA change of whole model organisms; a systematic survey of APA usage is lacking during CNS development. Here, we conducted global analysis of APA during mouse retinal development, and identified stage-specific polyadenylation (pA) sites that are enriched for genes critical for retinal development and visual perception. Moreover, we demonstrated 3'UTR (untranslated region) lengthening and increased usage of intronic pA sites over development that would result in gaining many different RBP (RNA-binding protein) and miRNA target sites. Furthermore, we showed that a considerable number of polyadenylated lncRNAs are co-expressed with protein-coding genes involved in retinal development and functions. Together, our data indicate that APA is highly and dynamically regulated during retinal development and maturation, suggesting that APA may serve as a crucial mechanism of gene regulation underlying the delicate process of CNS development.
Assuntos
Poliadenilação , Retina/embriologia , Retina/metabolismo , Regiões 3' não Traduzidas/genética , Animais , Sequência de Bases , Regulação da Expressão Gênica no Desenvolvimento , Camundongos Endogâmicos C57BL , Motivos de Nucleotídeos/genética , Poliadenilação/genética , RNA Longo não Codificante/genética , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Fatores de TempoRESUMO
BACKGROUND: Transcription factor Pax6 plays an essential role in the expression of other transcription factors, cell adhesion molecules and is crucial for neurogenesis in the developing forebrain. Analysis of gene expression profiles through microarray experiments in Pax6 mutants allowed us to focus on CRALBP, one of the many genes that were downregulated. METHODS: We studied the expression of CRALBP in wt and Pax6-/- mutants through in situ hybridization and immunohistochemistry. ChIP assay and luciferase reporter assay were performed to show the regulatory role of Pax6 on CRALBP promoter. RESULTS: RNA and protein expression data show that CRALBP expression was completely abolished in Pax6 mutants. In vivo binding assays and in vitro reporter assays indicate that Pax6 not only binds the promoter of CRALBP but also positively regulates protein expression. CONCLUSIONS: This work provides evidence supporting that CRALBP is a direct downstream target of Pax6. However, the role of CRALBP in the cortex is yet to be elucidated. GENERAL SIGNIFICANCE: Pax6 is a marker expressed on neural stem cells and progenitor cells. Understanding Pax6-dependent gene regulatory mechanisms unravels signaling cascades that occur early during development.
Assuntos
Proteínas de Transporte/metabolismo , Proteínas do Olho/metabolismo , Proteínas de Homeodomínio/metabolismo , Fatores de Transcrição Box Pareados/metabolismo , Proteínas Repressoras/metabolismo , Animais , Sítios de Ligação , Células CHO , Proteínas de Transporte/genética , Imunoprecipitação da Cromatina , Cricetinae , Cricetulus , Regulação para Baixo , Embrião de Mamíferos/citologia , Embrião de Mamíferos/metabolismo , Olho/citologia , Olho/metabolismo , Proteínas do Olho/genética , Deleção de Genes , Regulação da Expressão Gênica no Desenvolvimento , Proteínas de Homeodomínio/genética , Imuno-Histoquímica , Hibridização In Situ , Camundongos , Fator de Transcrição PAX6 , Fatores de Transcrição Box Pareados/genética , Regiões Promotoras Genéticas/genética , Ligação Proteica , Proteínas Repressoras/genética , TransfecçãoRESUMO
The role of the vesicular acetylcholine transporter (VAChT) in the regulation of cholinergic neurotransmission has not been fully elucidated. Here we sought to develop a tool for studying vesicular acetylcholine transporter function, and we present data on the validation of our new anti-VAChT antibody. We show that the immunoreactivity of the antibody is not due to an artifact of secondary antibody staining, and we present two additional validation data. First, the peptide epitope used to generate the antibody is able to block the binding of the anti-VAChT antibody in vivo. Further, RNA interference (RNAi) -mediated knockdown of VAChT function in cholinergic neurons drastically reduces anti-VAChT staining in those constructs. Additional evidence for the antibody functionality is presented in our research article on the subject (Boppana et al., 2017) [1].
RESUMO
Vesicular acetylcholine transporter (VAChT) function is essential for organismal survival, mediating the packaging of acetylcholine (ACh) for exocytotic release. However, its expression pattern in the Drosophila brain has not been fully elucidated. To investigate the localization of VAChT, we developed an antibody against the C terminal region of the protein and we show that this antibody recognizes a 65KDa protein corresponding to VAChT on an immunoblot in both Drosophila head homogenates and in Schneider 2 cells. Further, we report for the first time the expression of VAChT in the antennal lobe and ventral nerve cord of Drosophila larva; and we independently confirm the expression of the protein in mushroom bodies and optic lobes of adult Drosophila. Importantly, we show that VAChT co-localizes with a synaptic vesicle marker in vivo, confirming previous reports of the localization of VAChT to synaptic terminals. Together, these findings help establish the vesicular localization of VAChT in cholinergic neurons in Drosophila and present an important molecular tool with which to dissect the function of the transporter in vivo.
Assuntos
Fibras Colinérgicas/metabolismo , Neurônios Colinérgicos/metabolismo , Drosophila melanogaster/metabolismo , Corpos Pedunculados/metabolismo , Vesículas Sinápticas/metabolismo , Proteínas Vesiculares de Transporte de Acetilcolina/metabolismo , Acetilcolina/metabolismo , Envelhecimento , Animais , Proteínas de Transporte/metabolismo , Drosophila melanogaster/crescimento & desenvolvimento , Imuno-Histoquímica/métodos , LarvaRESUMO
Glatiramer acetate (GA) is an FDA-approved efficacious drug for the treatment of relapsing-remitting multiple sclerosis (RRMS). However, this treatment is not effective for all RRMS patients. Therefore, it is important to identify reliable biomarkers that can predict a beneficial clinical response to GA therapy. Since an increase in IL-27 has been demonstrated to suppress autoimmune and allergic diseases of inflammatory origin, we examined the effect of GA on the production of IL-27. We observed that IL-27 production in PBMCs cultured with GA was heterogeneous amongst MS patients and healthy donors (HD), and thus, defined these MS patients as either efficient, weak, or non-IL-27 producers. Interestingly, GA could induce the expression of the IL-27p28 subunit more efficiently than the IL-27 EBI3 subunit, and the production of IL-27 depended on MHC class II binding by GA. In addition, we found that GA could augment Toll-like receptor (TLR)-mediated IL-27 production. Importantly, serum production of IL-27 and IL-10 was significantly increased at 6months during GA therapy in clinical responders to GA, but not in GA non-responders. Altogether, our data suggest that GA-induced IL-27 may represent a therapeutic mechanism of GA-mediated immunomodulation and that GA-mediated IL-27 production in PBMCs is worth exploring as a biomarker to screen for GA responders prior to the initiation of GA treatment.
Assuntos
Acetato de Glatiramer/uso terapêutico , Imunossupressores/uso terapêutico , Interleucinas/sangue , Esclerose Múltipla Recidivante-Remitente/sangue , Esclerose Múltipla Recidivante-Remitente/tratamento farmacológico , Adulto , Biomarcadores/sangue , Células Cultivadas , Feminino , Acetato de Glatiramer/farmacologia , Células HEK293 , Humanos , Imunossupressores/farmacologia , Leucócitos Mononucleares/efeitos dos fármacos , Leucócitos Mononucleares/metabolismo , Masculino , Pessoa de Meia-Idade , Ligação Proteica/fisiologia , Resultado do Tratamento , Adulto JovemRESUMO
Exposure to certain pesticides induces oxidative stress and increases Parkinson's disease (PD) risk. Mitochondrial DNA (mtDNA) damage is found in dopaminergic neurons in idiopathic PD and following pesticide exposure in experimental models thereof. Base excision repair (BER) is the major pathway responsible for repairing oxidative DNA damage in cells. Whether single nucleotide polymorphisms (SNPs) in BER genes alone or in combination with pesticide exposure influence PD risk is unknown. We investigated the contributions of functional SNPs in 2 BER genes (APEX1 and OGG1) and mitochondrial dysfunction- or oxidative stress-related pesticide exposure, including paraquat, to PD risk. We also studied the effect of paraquat on levels of mtDNA damage and mitochondrial bioenergetics. 619 PD patients and 854 population-based controls were analyzed for the 2 SNPs, APEX1 rs1130409 and OGG1 rs1052133. Ambient pesticide exposures were assessed with a geographic information system. Individually, or in combination, the BER SNPs did not influence PD risk. Mitochondrial-inhibiting (OR = 1.79, 95% CI [1.32, 2.42]), oxidative stress-inducing pesticides (OR = 1.61, 95% CI [1.22, 2.11]), and paraquat (OR = 1.54, 95% CI [1.23, 1.93]) were associated with PD. Statistical interactions were detected, including for a genetic risk score based on rs1130409 and rs1052133 and oxidative stress inducing pesticides, where highly exposed carriers of both risk genotypes were at the highest risk of PD (OR = 2.21, 95% CI [1.25, 3.86]); similar interactions were estimated for mitochondrial-inhibiting pesticides and paraquat alone. Additionally, paraquat exposure was found to impair mitochondrial respiration and increase mtDNA damage in in vivo and in vitro systems. Our findings provide insight into possible mechanisms involved in increased PD risk due to pesticide exposure in the context of BER genotype variants.
Assuntos
Reparo do DNA , Doença de Parkinson/genética , Praguicidas/toxicidade , Idoso , Animais , DNA Mitocondrial/efeitos dos fármacos , Drosophila , Feminino , Humanos , Masculino , Estresse Oxidativo , Consumo de Oxigênio , Doença de Parkinson/etiologia , Reação em Cadeia da Polimerase , Polimorfismo de Nucleotídeo Único , Fatores de RiscoRESUMO
Post-transplantation lymphoproliferative disorder is an infrequent complication after hematopoietic stem cell transplantation. It is hypothesized that lack of T-cell surveillance following transplantation permits reactivation of latent EBV leading to polyclonal B-cell expansion and finally outgrowth of a predominant clone. Most cases are of donor origin. Here, we describe an 8-year old boy with early onset post-transplantation lymphoproliferative disorder following matched-unrelated stem cell transplantation for high-risk T-cell leukemia whose disease was unusual for two reasons. First, his B-cell clone was of host origin and, in contrast to the few PTLD of host origin described so far, not associated with autologous reconstitution. Secondly, using clonal analysis, we could retrospectively show that the B-cell clone emerged during consolidation chemotherapy for T-cell leukemia, 3 months before stem cell transplantation.
Assuntos
Linfócitos B/patologia , Células Clonais/patologia , Infecções por Vírus Epstein-Barr/complicações , Leucemia-Linfoma de Células T do Adulto/cirurgia , Transtornos Linfoproliferativos/etiologia , Complicações Pós-Operatórias/etiologia , Anticorpos Monoclonais/uso terapêutico , Anticorpos Monoclonais Murinos , Soro Antilinfocitário/administração & dosagem , Protocolos de Quimioterapia Combinada Antineoplásica/efeitos adversos , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Asparaginase/administração & dosagem , Asparaginase/efeitos adversos , Criança , Terapia Combinada , Ciclofosfamida/administração & dosagem , Ciclosporina/efeitos adversos , Ciclosporina/uso terapêutico , Citarabina/administração & dosagem , Daunorrubicina/administração & dosagem , Daunorrubicina/efeitos adversos , Dexametasona/administração & dosagem , Etoposídeo/administração & dosagem , Etoposídeo/farmacologia , Evolução Fatal , Glucocorticoides/administração & dosagem , Glucocorticoides/efeitos adversos , Doença Enxerto-Hospedeiro/tratamento farmacológico , Herpesvirus Humano 4/isolamento & purificação , Humanos , Imunossupressores/efeitos adversos , Imunossupressores/uso terapêutico , Leucemia-Linfoma de Células T do Adulto/tratamento farmacológico , Leucemia-Linfoma de Células T do Adulto/patologia , Transfusão de Linfócitos , Transtornos Linfoproliferativos/tratamento farmacológico , Transtornos Linfoproliferativos/patologia , Transtornos Linfoproliferativos/virologia , Masculino , Complicações Pós-Operatórias/tratamento farmacológico , Complicações Pós-Operatórias/patologia , Complicações Pós-Operatórias/virologia , Prednisolona/efeitos adversos , Prednisolona/uso terapêutico , Rituximab , Fatores de Tempo , Quimeras de Transplante , Condicionamento Pré-Transplante , Vincristina/administração & dosagem , Vincristina/efeitos adversos , Ativação Viral/efeitos dos fármacos , Irradiação Corporal TotalRESUMO
Interferon (IFN)-ß is a type I IFN commonly produced by the innate immune system in response to viral infection. IFN-ß is also used for the treatment of patients with the relapsing-remitting form of multiple sclerosis (RRMS); however, IFN-ß therapy is unable to confer a significant benefit for primary-progressive MS (PPMS) patients. In this study, we assessed the gene profiles of peripheral blood mononuclear cells (PBMCs) isolated from PPMS, RRMS, and healthy donors (HD) in response to IFN-ß treatment in vitro to examine genetic mechanisms underlying the inadequate response of IFN-ß therapy in PPMS patients. Here, we show that HLA-G was significantly less up-regulated in response to IFN-ß in PBMCs from PPMS compared to those from RRMS. This data suggests HLA-G to be a possible candidate gene found impaired in IFN-ß-mediated immune regulation in PPMS patients.
Assuntos
Interferon beta/metabolismo , Interleucina-1beta/farmacologia , Leucócitos Mononucleares/efeitos dos fármacos , Esclerose Múltipla Crônica Progressiva/patologia , Esclerose Múltipla Recidivante-Remitente/patologia , Regulação para Cima/efeitos dos fármacos , Antígenos HLA-G/genética , Antígenos HLA-G/metabolismo , Humanos , Interferon beta/genética , Leucócitos Mononucleares/metabolismoRESUMO
Multiple sclerosis is an inflammatory and degenerative disease of the central nervous system characterized by demyelination and axonal loss. Genetic and environmental factors contribute to the risk of immune dysregulation in multiple sclerosis. The review focuses on the immunopathogenic role played by various lymphocyte subsets and their cytokine products in the onset and disease progression, and the role of regulatory immune cells in disease remission. The mechanism of action of approved and experimental therapies, and how these mechanisms support the immunopathogenesis paradigm, is also reviewed.
Assuntos
Imunidade , Fatores Imunológicos , Inflamação , Esclerose Múltipla , Axônios/imunologia , Axônios/patologia , Progressão da Doença , Meio Ambiente , Predisposição Genética para Doença , Humanos , Imunidade/efeitos dos fármacos , Imunidade/genética , Fatores Imunológicos/imunologia , Fatores Imunológicos/uso terapêutico , Inflamação/imunologia , Inflamação/fisiopatologia , Esclerose Múltipla/tratamento farmacológico , Esclerose Múltipla/genética , Esclerose Múltipla/imunologia , Esclerose Múltipla/patologia , Esclerose Múltipla/fisiopatologia , Bainha de Mielina/imunologia , Bainha de Mielina/patologia , Degeneração Neural/genética , Degeneração Neural/imunologia , Degeneração Neural/patologia , Degeneração Neural/fisiopatologia , Terapias em EstudoRESUMO
Smad-interacting protein-1 (Sip1) [Zinc finger homeobox (Zfhx1b)] is a transcription factor implicated in the genesis of Mowat-Wilson syndrome in humans. Sip1 expression in the dorsal telencephalon of mouse embryos was documented from E12.5. We inactivated the gene specifically in cortical precursors. This resulted in the lack of the entire hippocampal formation. Sip1 mutant mice exhibited death of differentiating cells and decreased proliferation in the region of the prospective hippocampus and dentate gyrus. The expression of the Wnt antagonist Sfrp1 was ectopically activated, whereas the activity of the noncanonical Wnt effector, JNK, was down-regulated in the embryonic hippocampus of mutant mice. In cortical cells, Sip1 protein was detected on the promoter of Sfrp1 gene and both genes showed a mutually exclusive pattern of expression suggesting that Sfrp1 expression is negatively regulated by Sip1. Sip1 is therefore essential to the development of the hippocampus and dentate gyrus, and is able to modulate Wnt signaling in these regions.