RESUMO
Chickens are a species with a high number of γδ T cells in various tissues. Despite their abundance, γδ T cells are poorly characterized in chickens, partially due to a lack of specific reagents to characterize these cells. Up until now, the TCR1 clone has been the only γδ T cell-specific monoclonal antibody (mAb) in chickens and additional reagents for γδ T cell subsets are needed. In order to address this issue, new mAb were generated in our laboratory by immunizing mice with in vitro cultured γδ T cells. In an initial flow cytometric screen a new mAb, clone "8D2", displayed an interesting staining pattern that mirrored γδ TCR up- and downregulation in the γδ T cell line D4 over time, prompting us to characterize this antibody further. We compared the expression of the unknown 8D2 epitope in combination with TCR1 staining across various primary cells. In splenocytes, peripheral blood lymphocytes and intestinal epithelial cells, 8D2 consistently labeled a subset of TCR1+ cells. To determine, whether specific γδ T cell receptors were recognized by 8D2, we sorted γδ T cells according to their 8D2 and TCR1 expression and analyzed their TCR V(D)J gene usage by TCR profiling. Strikingly, sorted 8D2+ cells preferentially expressed Vγ3 genes, whereas the TCR Vγ genes used by TCR1+ 8D2- cells were more variable. γδ TCR in 8D2+ cells were most frequently comprised of gamma chain VJ genes TRGV3-8 and TRGJ3, and delta chain VDJ genes TRDV1-2, TRDD2, TRDJ1. To confirm binding of 8D2 to specific γδ TCR, the preferentially utilized combination of TRG and TRD was expressed in HEK293 cells in combination with CD3, demonstrating surface binding of the 8D2 mAb to this Vγ3 γδ TCR-expressing cell line. Conversely, HEK293 cells expressing either Vγ1 or Vγ2 TCR did not react with 8D2. In conclusion, 8D2 is a novel tool for identifying specific Vγ3 bearing γδ T cells.
RESUMO
In the central nervous system of vertebrates, glutamate serves as the primary excitatory neurotransmitter. However, in the retina, glutamate released from photoreceptors causes hyperpolarization in post-synaptic ON-bipolar cells through a glutamate-gated chloride current, which seems paradoxical. Our research reveals that this current is modulated by two excitatory glutamate transporters, EAAT5b and EAAT7. In the zebrafish retina, these transporters are located at the dendritic tips of ON-bipolar cells and interact with all four types of cone photoreceptors. The absence of these transporters leads to a decrease in ON-bipolar cell responses, with eaat5b mutants being less severely affected than eaat5b/eaat7 double mutants, which also exhibit altered response kinetics. Biophysical investigations establish that EAAT7 is an active glutamate transporter with a predominant anion conductance. Our study is the first to demonstrate the direct involvement of post-synaptic glutamate transporters in inhibitory direct synaptic transmission at a central nervous system synapse.
Assuntos
Sistema X-AG de Transporte de Aminoácidos , Retina , Transmissão Sináptica , Proteínas de Peixe-Zebra , Peixe-Zebra , Animais , Peixe-Zebra/metabolismo , Retina/metabolismo , Sistema X-AG de Transporte de Aminoácidos/metabolismo , Sistema X-AG de Transporte de Aminoácidos/genética , Proteínas de Peixe-Zebra/metabolismo , Proteínas de Peixe-Zebra/genética , Ácido Glutâmico/metabolismo , Mutação , Células Bipolares da Retina/metabolismoRESUMO
Adherens junction-associated protein 1 (AJAP1) has been implicated in brain diseases; however, a pathogenic mechanism has not been identified. AJAP1 is widely expressed in neurons and binds to γ-aminobutyric acid type B receptors (GBRs), which inhibit neurotransmitter release at most synapses in the brain. Here, we show that AJAP1 is selectively expressed in dendrites and trans-synaptically recruits GBRs to presynaptic sites of neurons expressing AJAP1. We have identified several monoallelic AJAP1 variants in individuals with epilepsy and/or neurodevelopmental disorders. Specifically, we show that the variant p.(W183C) lacks binding to GBRs, resulting in the inability to recruit them. Ultrastructural analysis revealed significantly decreased presynaptic GBR levels in Ajap1-/- and Ajap1W183C/+ mice. Consequently, these mice exhibited reduced GBR-mediated presynaptic inhibition at excitatory and inhibitory synapses, along with impaired synaptic plasticity. Our study reveals that AJAP1 enables the postsynaptic neuron to regulate the level of presynaptic GBR-mediated inhibition, supporting the clinical relevance of loss-of-function AJAP1 variants.
Assuntos
Neurotransmissores , Sinapses , Transmissão Sináptica , Animais , Feminino , Humanos , Masculino , Camundongos , Alelos , Epilepsia/metabolismo , Epilepsia/genética , Epilepsia/patologia , Mutação com Perda de Função , Camundongos Knockout , Transtornos do Neurodesenvolvimento/metabolismo , Transtornos do Neurodesenvolvimento/genética , Transtornos do Neurodesenvolvimento/patologia , Plasticidade Neuronal , Neurônios/metabolismo , Neurotransmissores/metabolismo , Sinapses/metabolismo , Moléculas de Adesão Celular/genética , Moléculas de Adesão Celular/metabolismoRESUMO
GABAB receptors (GBRs), the G protein-coupled receptors for GABA, regulate synaptic transmission throughout the brain. A main synaptic function of GBRs is the gating of Cav2.2-type Ca2+ channels. However, the cellular compartment where stable GBR/Cav2.2 signaling complexes form remains unknown. In this study, we demonstrate that the vesicular protein synaptotagmin-11 (Syt11) binds to both the auxiliary GBR subunit KCTD16 and Cav2.2 channels. Through these dual interactions, Syt11 recruits GBRs and Cav2.2 channels to post-Golgi vesicles, thus facilitating assembly of GBR/Cav2.2 signaling complexes. In addition, Syt11 stabilizes GBRs and Cav2.2 channels at the neuronal plasma membrane by inhibiting constitutive internalization. Neurons of Syt11 knockout mice exhibit deficits in presynaptic GBRs and Cav2.2 channels, reduced neurotransmitter release, and decreased GBR-mediated presynaptic inhibition, highlighting the critical role of Syt11 in the assembly and stable expression of GBR/Cav2.2 complexes. These findings support that Syt11 acts as a vesicular scaffold protein, aiding in the assembly of signaling complexes from low-abundance components within transport vesicles. This mechanism enables insertion of pre-assembled functional signaling units into the synaptic membrane.
Assuntos
Camundongos Knockout , Transdução de Sinais , Sinaptotagminas , Animais , Sinaptotagminas/metabolismo , Sinaptotagminas/genética , Camundongos , Humanos , Neurônios/metabolismo , Transmissão Sináptica , Receptores de GABA-B/metabolismo , Receptores de GABA-B/genética , Terminações Pré-Sinápticas/metabolismo , Canais de Cálcio Tipo N/metabolismo , Canais de Cálcio Tipo N/genética , Complexo de Golgi/metabolismo , Ligação Proteica , Células HEK293RESUMO
T cell receptor (TCR) repertoire sequencing has emerged as a powerful tool for understanding the diversity and functionality of T cells within the host immune system. Yet, the chicken TCR repertoire remains poorly understood due to incomplete genome annotation of the TCR loci, despite the importance of chickens in agriculture and as an immunological model. Here, we addressed this critical issue by employing 5' rapid amplification of complementary DNA ends (5'RACE) TCR repertoire sequencing with molecular barcoding of complementary DNA (cDNA) molecules. Simultaneously, we enhanced the genome annotation of TCR Variable (V), Diversity (D, only present in ß and δ loci) and Joining (J) genes in the chicken genome. To enhance the efficiency of TCR annotations, we developed VJ-gene-finder, an algorithm designed to extract VJ gene candidates from deoxyribonucleic acid (DNA) sequences. Using this tool, we achieved a comprehensive annotation of all known chicken TCR loci, including the α/δ locus on chromosome 27. Evolutionary analysis revealed that each locus evolved separately by duplication of long homology units. To define the baseline TCR diversity in healthy chickens and to demonstrate the feasibility of the approach, we characterized the splenic α/ß/γ/δ TCR repertoire. Analysis of the repertoires revealed preferential usage of specific V and J combinations in all chains, while the overall features were characteristic of unbiased repertoires. We observed moderate levels of shared complementarity-determining region 3 (CDR3) clonotypes among individual birds within the α and γ chain repertoires, including the most frequently occurring clonotypes. However, the ß and δ repertoires were predominantly unique to each bird. Taken together, our TCR repertoire analysis allowed us to decipher the composition, diversity, and functionality of T cells in chickens. This work not only represents a significant step towards understanding avian T cell biology, but will also shed light on host-pathogen interactions, vaccine development, and the evolutionary history of avian immunology.
Assuntos
Galinhas , Linfócitos T , Animais , Galinhas/genética , Receptores de Antígenos de Linfócitos T alfa-beta/genética , DNA Complementar , GenomaRESUMO
In chickens, γδ T cells represent a large fraction of peripheral T cells; however, their function remains largely unknown. Here, we describe the selective in vitro expansion of γδ T cells from total splenocytes by stimulation with the cytokines IL-2 and IL-12. Under these conditions, γδ T cells proliferated preferentially and reached frequencies of >95% within three weeks. Although IL-2 alone also triggered proliferation, an increased proliferation rate was observed in combination with IL-12. Most of the expanded cells were γδ TCR and CD8 double-positive. Splenocytes sorted into TCR1+CD8+, TCR1highCD8-, and TCR1lowCD8- subsets proliferated well upon dual stimulation with IL-2/IL-12, indicating that none of the three γδ T cell subsets require bystander activation for proliferation. TCR1+CD8+ cells maintained CD8 surface expression during stimulation, whereas CD8- subpopulations showed varied levels of CD8 upregulation, with the highest upregulation observed in the TCR1high subset. Changes in the γδ T-cell receptor repertoire during cell culture from day 0 to day 21 were analyzed by next-generation sequencing of the γδ variable regions. Overall, long-term culture led to a restricted γ and δ chain repertoire, characterized by a reduced number of unique variable region clonotypes, and specific V genes were enriched at day 21. On day 0, the δ chain repertoire was highly diverse, and the predominant clonotypes differed between animals, while the most frequent γ-chain clonotypes were shared between animals. However, on day 21, the most frequent clonotypes in both the γ and δ chain repertoires were different between animals, indicating that selective expansion of dominant clonotypes during stimulation seems to be an individual outcome. In conclusion, IL-2 and IL-12 were sufficient to stimulate the in vitro outgrowth of γδ T cells. Analyses of the TCR repertoire indicate that the culture leads to an expansion of individual T cell clones, which may reflect previous in vivo activation. This system will be instrumental in studying γδ T cell function.
Assuntos
Galinhas , Interleucina-2 , Animais , Interleucina-2/farmacologia , Interleucina-12 , Receptores de Antígenos de Linfócitos T gama-delta/genética , Técnicas de Cultura de CélulasRESUMO
Canine parvovirus (CPV) is an important pathogen that emerged by cross-species transmission to cause severe disease in dogs. To understand the host immune response to vaccination, sera from dogs immunized with parvovirus are obtained, the polyclonal antibodies are purified and used to solve the high resolution cryo EM structures of the polyclonal Fab-virus complexes. We use a custom software, Icosahedral Subparticle Extraction and Correlated Classification (ISECC) to perform subparticle analysis and reconstruct polyclonal Fab-virus complexes from two different dogs eight and twelve weeks post vaccination. In the resulting polyclonal Fab-virus complexes there are a total of five distinct Fabs identified. In both cases, any of the five antibodies identified would interfere with receptor binding. This polyclonal mapping approach identifies a specific, limited immune response to the live vaccine virus and allows us to investigate the binding of multiple different antibodies or ligands to virus capsids.
Assuntos
Parvovirus Canino , Animais , Cães , Formação de Anticorpos , Microscopia Crioeletrônica , Anticorpos , Vacinação/veterináriaRESUMO
B-cell cloning methods enable the analysis of antibody responses against target antigens and can be used to reveal the host antibody repertoire, antigenic sites (epitopes), and details of protective immunity against pathogens. Here, we describe improved methods for isolation of canine peripheral blood B cells producing antibodies against canine parvovirus (CPV) capsids by fluorescence-activated cell sorting, followed by cell cloning. We cultured sorted B cells from an immunized dog in vitro and screened for CPV-specific antibody production. Updated canine-specific primer sets were used to amplify and clone the heavy and light chain immunoglobulin sequences directly from the B cells by reverse transcription and PCR. Monoclonal canine IgGs were produced by cloning heavy and light chain sequences into antibody expression vectors, which were screened for CPV binding. Three different canine monoclonal antibodies were analyzed, including two that shared the same heavy chain, and one that had distinct heavy and light chains. The antibodies showed broad binding to CPV variants, and epitopes were mapped to antigenic sites on the capsid. The methods described here are applicable for the isolation of canine B cells and monoclonal antibodies against many antigens.
Assuntos
Infecções por Parvoviridae , Parvovirus Canino , Parvovirus , Cães , Animais , Anticorpos Antivirais , Parvovirus Canino/genética , Anticorpos Monoclonais , Epitopos , Clonagem Molecular , Infecções por Parvoviridae/veterináriaRESUMO
Intestinal helminth infection promotes a Type 2 inflammatory response in resistant C57BL/6 mice that is essential for worm clearance. The study of inbred mouse strains has revealed factors that are critical for parasite resistance and delineated the role of Type 1 versus Type 2 immune responses in worm clearance. In C57BL/6 mice, basophils are key innate immune cells that promote Type 2 inflammation and are programmed via the Notch signalling pathway during infection with the helminth Trichuris muris. However, how the host genetic background influences basophil responses and basophil expression of Notch receptors remains unclear. Here we use genetically susceptible inbred AKR/J mice that have a Type 1-skewed immune response during T. muris infection to investigate basophil responses in a susceptible host. Basophil population expansion occurred in AKR/J mice even in the absence of fulminant Type 2 inflammation during T. muris infection. However, basophils in AKR/J mice did not robustly upregulate expression of the Notch2 receptor in response to infection as occurred in C57BL/6 mice. Blockade of the Type 1 cytokine interferon-γ in infected AKR/J mice was not sufficient to elicit infection-induced basophil expression of the Notch2 receptor. These data suggest that the host genetic background, outside of the Type 1 skew, is important in regulating basophil responses during T. muris infection in susceptible AKR/J mice.
Assuntos
Parasitos , Tricuríase , Animais , Camundongos , Camundongos Endogâmicos AKR , Trichuris , Basófilos , Receptor Notch2 , Camundongos Endogâmicos C57BL , Suscetibilidade a Doenças , InflamaçãoRESUMO
Type 2 inflammation is associated with epithelial cell responses, including goblet cell hyperplasia, that promote worm expulsion during intestinal helminth infection. How these epithelial responses are regulated remains incompletely understood. Here, we show that mice deficient in the prostaglandin D2 (PGD2) receptor CRTH2 and mice with CRTH2 deficiency only in nonhematopoietic cells exhibited enhanced worm clearance and intestinal goblet cell hyperplasia following infection with the helminth Nippostrongylus brasiliensis. Small intestinal stem, goblet, and tuft cells expressed CRTH2. CRTH2-deficient small intestinal organoids showed enhanced budding and terminal differentiation to the goblet cell lineage. During helminth infection or in organoids, PGD2 and CRTH2 down-regulated intestinal epithelial Il13ra1 expression and reversed Type 2 cytokine-mediated suppression of epithelial cell proliferation and promotion of goblet cell accumulation. These data show that the PGD2-CRTH2 pathway negatively regulates the Type 2 cytokine-driven epithelial program, revealing a mechanism that can temper the highly inflammatory effects of the anti-helminth response.
Assuntos
Citocinas/metabolismo , Mucosa Intestinal/parasitologia , Prostaglandina D2/metabolismo , Receptores Imunológicos/metabolismo , Receptores de Prostaglandina/metabolismo , Infecções por Strongylida/parasitologia , Animais , Feminino , Gastroenterite/parasitologia , Gastroenterite/patologia , Células Caliciformes/patologia , Interações Hospedeiro-Parasita/fisiologia , Mucosa Intestinal/patologia , Masculino , Camundongos Endogâmicos C57BL , Nippostrongylus/patogenicidade , Organoides , Receptores Imunológicos/genética , Receptores de Prostaglandina/genética , Infecções por Strongylida/patologiaRESUMO
It is of great interest to understand how invading pathogens are sensed within the brain, a tissue with unique challenges to mounting an immune response. The eukaryotic parasite Toxoplasma gondii colonizes the brain of its hosts, and initiates robust immune cell recruitment, but little is known about pattern recognition of T. gondii within brain tissue. The host damage signal IL-33 is one protein that has been implicated in control of chronic T. gondii infection, but, like many other pattern recognition pathways, IL-33 can signal peripherally, and the specific impact of IL-33 signaling within the brain is unclear. Here, we show that IL-33 is expressed by oligodendrocytes and astrocytes during T. gondii infection, is released locally into the cerebrospinal fluid of T. gondii-infected animals, and is required for control of infection. IL-33 signaling promotes chemokine expression within brain tissue and is required for the recruitment and/or maintenance of blood-derived anti-parasitic immune cells, including proliferating, IFN-γ-expressing T cells and iNOS-expressing monocytes. Importantly, we find that the beneficial effects of IL-33 during chronic infection are not a result of signaling on infiltrating immune cells, but rather on radio-resistant responders, and specifically, astrocytes. Mice with IL-33 receptor-deficient astrocytes fail to mount an adequate adaptive immune response in the CNS to control parasite burden-demonstrating, genetically, that astrocytes can directly respond to IL-33 in vivo. Together, these results indicate a brain-specific mechanism by which IL-33 is released locally, and sensed locally, to engage the peripheral immune system in controlling a pathogen.
Assuntos
Astrócitos/imunologia , Interleucina-33/imunologia , Toxoplasmose Cerebral/imunologia , Adulto , Animais , Astrócitos/metabolismo , Astrócitos/fisiologia , Encéfalo/metabolismo , Feminino , Humanos , Imunidade , Interferon gama/imunologia , Interleucina-33/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Monócitos/imunologia , Transdução de Sinais , Toxoplasma/metabolismo , Toxoplasma/parasitologia , Toxoplasmose/metabolismo , Toxoplasmose Cerebral/metabolismoRESUMO
Atopic dermatitis (AD) is an allergic skin disease that causes significant morbidity and affects multiple species. AD is highly prevalent in companion dogs, and the clinical management of the disease remains challenging. An improved understanding of the immunologic and genetic pathways that lead to disease could inform the development of novel treatments. In allergic humans and mouse models of AD, the disease is associated with Th2 and group 2 innate lymphoid cell (ILC2) activation that drives type 2 inflammation. Type 2 inflammation also appears to be associated with AD in dogs, but gaps remain in our understanding of how key type 2-associated cell types such as canine Th2 cells and ILC2s contribute to the pathogenesis of canine AD. Here, we describe previously uncharacterized canine ILC2-like cells and Th2 cells ex vivo that produced type 2 cytokines and expressed the transcription factor Gata3. Increased circulating Th2 cells were associated with chronic canine AD. Single-cell RNA sequencing revealed a unique gene expression signature in T cells in dogs with AD. These findings underline the importance of pro-allergic Th2 cells in orchestrating AD and provide new methods and pathways that can inform the development of improved therapies.
Assuntos
Dermatite Atópica/veterinária , Doenças do Cão/imunologia , Imunidade Inata , Linfócitos/imunologia , Células Th2/imunologia , Animais , Células Sanguíneas/imunologia , Dermatite Atópica/imunologia , Cães , Feminino , Inflamação , Linfócitos/classificação , Masculino , Análise de Sequência de RNA , Análise de Célula ÚnicaRESUMO
Group 2 innate lymphoid cells (ILC2s) are rare innate immune cells that accumulate in tissues during allergy and helminth infection, performing critical effector functions that drive type 2 inflammation. ILC2s express ST2, the receptor for the cytokine IL-33, and chemoattractant receptor-homologous molecule expressed on Th2 cells (CRTH2), a receptor for the bioactive lipid prostaglandin D2 (PGD2). The IL-33-ST2 and the PGD2-CRTH2 pathways have both been implicated in promoting ILC2 accumulation during type 2 inflammation. However, whether these two pathways coordinate to regulate ILC2 population size in the tissue in vivo remains undefined. In this study, we show that ILC2 accumulation in the murine lung in response to systemic IL-33 treatment was partially dependent on CRTH2. This effect was not a result of reduced ILC2 proliferation, increased apoptosis or cell death, or differences in expression of the ST2 receptor in the absence of CRTH2. Rather, data from adoptive transfer studies suggested that defective accumulation of CRTH2-deficient ILC2s in response to IL-33 was due to altered ILC2 migration patterns. Whereas donor wild-type ILC2s preferentially accumulated in the lungs compared with CRTH2-deficient ILC2s following transfer into IL-33-treated recipients, wild-type and CRTH2-deficient ILC2s accumulated equally in the recipient mediastinal lymph node. These data suggest that CRTH2-dependent effects lie downstream of IL-33, directly affecting the migration of ILC2s into inflamed lung tissues. A better understanding of the complex interactions between the IL-33 and PGD2-CRTH2 pathways that regulate ILC2 population size will be useful in understanding how these pathways could be targeted to treat diseases associated with type 2 inflammation.
Assuntos
Movimento Celular/imunologia , Hipersensibilidade/imunologia , Interleucina-33/imunologia , Linfócitos/imunologia , Receptores Imunológicos/metabolismo , Receptores de Prostaglandina/metabolismo , Infecções por Strongylida/imunologia , Transferência Adotiva , Animais , Proliferação de Células , Células Cultivadas , Modelos Animais de Doenças , Feminino , Humanos , Hipersensibilidade/patologia , Imunidade Inata , Interleucina-33/administração & dosagem , Pulmão/citologia , Pulmão/imunologia , Pulmão/patologia , Linfócitos/metabolismo , Camundongos , Camundongos Knockout , Nippostrongylus/imunologia , Cultura Primária de Células , Prostaglandina D2/imunologia , Prostaglandina D2/metabolismo , Receptores Imunológicos/genética , Receptores Imunológicos/imunologia , Receptores de Prostaglandina/genética , Receptores de Prostaglandina/imunologia , Proteínas Recombinantes/administração & dosagem , Proteínas Recombinantes/imunologia , Infecções por Strongylida/parasitologia , Infecções por Strongylida/patologiaRESUMO
Parasitic helminth infection elicits a type 2 cytokine-mediated inflammatory response. During type 2 inflammation, damaged or stimulated epithelial cells exposed to helminths and their products produce alarmins and cytokines including IL-25, IL-33, and thymic stromal lymphopoietin. These factors promote innate immune cell activation that supports the polarization of CD4+ T helper type 2 (Th2) cells. Activated innate and Th2 cells produce the cytokines IL-4, -5, -9, and -13 that perpetuate immune activation and act back on the epithelium to cause goblet cell hyperplasia and increased epithelial cell turnover. Together, these events facilitate worm expulsion and wound healing processes. While the role of Th2 cells in this context has been heavily studied, recent work has revealed that epithelial cell-derived cytokines are drivers of key innate immune responses that are critical for type 2 anti-helminth responses. Cutting-edge studies have begun to fully assess how other factors and pathways, including lipid mediators, chemokines, Fc receptor signaling, danger-associated molecular pattern molecules, and direct cell-cell interactions, also participate in shaping innate cell-mediated type 2 inflammation. In this review, we discuss how these pathways intersect and synergize with pathways controlled by epithelial cell-derived cytokines to coordinate innate immune responses that drive helminth-induced type 2 inflammation.
Assuntos
Citocinas/imunologia , Helmintíase/imunologia , Helmintos/imunologia , Imunidade Inata/imunologia , Animais , Helmintíase/parasitologia , Humanos , Inflamação/imunologia , Inflamação/parasitologia , Células Th2/imunologiaRESUMO
The hippocampus plays key roles in learning and memory and is a main target of Alzheimer's disease (AD), which causes progressive memory impairments. Despite numerous investigations about the processes required for the normal hippocampal functions, the neurotransmitter receptors involved in the synaptic deficits by which AD disables the hippocampus are not yet characterized. By combining histoblots, western blots, immunohistochemistry and high-resolution immunoelectron microscopic methods for GABAB receptors, this study provides a quantitative description of the expression and the subcellular localization of GABAB1 in the hippocampus in a mouse model of AD at 1, 6 and 12 months of age. Western blots and histoblots showed that the total amount of protein and the laminar expression pattern of GABAB1 were similar in APP/PS1 mice and in age-matched wild-type mice. In contrast, immunoelectron microscopic techniques showed that the subcellular localization of GABAB1 subunit did not change significantly in APP/PS1 mice at 1 month of age, was significantly reduced in the stratum lacunosum-moleculare of CA1 pyramidal cells at 6 months of age and significantly reduced at the membrane surface of CA1 pyramidal cells at 12 months of age. This reduction of plasma membrane GABAB1 was paralleled by a significant increase of the subunit at the intracellular sites. We further observed a decrease of membrane-targeted GABAB receptors in axon terminals contacting CA1 pyramidal cells. Our data demonstrate compartment- and age-dependent reduction of plasma membrane-targeted GABAB receptors in the CA1 region of the hippocampus, suggesting that this decrease might be enough to alter the GABAB -mediated synaptic transmission taking place in AD.
Assuntos
Doença de Alzheimer/metabolismo , Hipocampo/metabolismo , Neurônios/metabolismo , Receptores de GABA-B/metabolismo , Sinapses/metabolismo , Doença de Alzheimer/patologia , Precursor de Proteína beta-Amiloide/genética , Precursor de Proteína beta-Amiloide/metabolismo , Animais , Membrana Celular/metabolismo , Modelos Animais de Doenças , Hipocampo/patologia , Camundongos , Camundongos Transgênicos , Neurônios/patologia , Presenilina-1/genética , Presenilina-1/metabolismo , Sinapses/patologia , Transmissão Sináptica/fisiologia , Ácido gama-Aminobutírico/metabolismoRESUMO
Pianp (also known as Leda-1) is a type I transmembrane protein with preferential expression in the mammalian CNS. Its processing is characterized by proteolytic cleavage by a range of proteases including Adam10, Adam17, MMPs, and the γ-secretase complex. Pianp can interact with Pilrα and the GB1a subunit of the GABAB receptor (GBR) complex. A recent case description of a boy with global developmental delay and homozygous nonsense variant in PIANP supports the hypothesis that PIANP is involved in the control of behavioral traits in mammals. To investigate the physiological functions of Pianp, constitutive, global knockout mice were generated and comprehensively analyzed. Broad assessment did not indicate malformation or malfunction of internal organs. In the brain, however, decreased sizes and altered cellular compositions of the dentate gyrus as well as the cerebellum, including a lower number of cerebellar Purkinje cells, were identified. Functionally, loss of Pianp led to impaired presynaptic GBR-mediated inhibition of glutamate release and altered gene expression in the cortex, hippocampus, amygdala, and hypothalamus including downregulation of Erdr1, a gene linked to autism-like behavior. Behavioral phenotyping revealed that Pianp deficiency leads to context-dependent enhanced anxiety and spatial learning deficits, an altered stress response, severely impaired social interaction, and enhanced repetitive behavior, which all represent characteristic features of an autism spectrum disorder-like phenotype. Altogether, Pianp represents a novel candidate gene involved in autism-like behavior, cerebellar and hippocampal pathology, and GBR signaling.
Assuntos
Transtorno do Espectro Autista/genética , Transtorno do Espectro Autista/metabolismo , Cerebelo/patologia , Deleção de Genes , Hipocampo/patologia , Proteínas do Tecido Nervoso/deficiência , Receptores de GABA-B/metabolismo , Animais , Transtorno Autístico/genética , Transtorno Autístico/metabolismo , Cerebelo/metabolismo , Modelos Animais de Doenças , Feminino , Hipocampo/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Proteínas do Tecido Nervoso/genética , Proteínas do Tecido Nervoso/metabolismoRESUMO
Type 2 inflammation drives the clearance of gastrointestinal helminth parasites, which infect over two billion people worldwide. Basophils are innate immune cells that support host-protective type 2 inflammation during murine infection with the helminth Trichuris muris However, the mechanisms required for basophil function and gene expression regulation in this context remain unclear. We show that during T. muris infection, basophils localized to the intestine and up-regulated Notch receptor expression, rendering them sensitive to Notch signals that rapidly regulate gene expression programs. In vitro, Notch inhibition limited basophil cytokine production in response to cytokine stimulation. Basophil-intrinsic Notch signaling was required for T. muris-elicited changes in genome-wide basophil transcriptional programs. Mice lacking basophil-intrinsic functional Notch signaling had impaired worm clearance, decreased intestinal type 2 inflammation, altered basophil localization in the intestine, and decreased CD4+ T helper 2 cell responses following infection. These findings demonstrate that Notch is required for basophil gene expression and effector function associated with helminth expulsion during type 2 inflammation.
Assuntos
Basófilos/imunologia , Inflamação/patologia , Receptores Notch/metabolismo , Transdução de Sinais , Animais , Ceco/parasitologia , Feminino , Regulação da Expressão Gênica , Inflamação/complicações , Interleucinas/metabolismo , Masculino , Camundongos Endogâmicos C57BL , Trichuris/fisiologia , Regulação para CimaRESUMO
GABAB receptors (GBRs) are key regulators of synaptic release but little is known about trafficking mechanisms that control their presynaptic abundance. We now show that sequence-related epitopes in APP, AJAP-1 and PIANP bind with nanomolar affinities to the N-terminal sushi-domain of presynaptic GBRs. Of the three interacting proteins, selectively the genetic loss of APP impaired GBR-mediated presynaptic inhibition and axonal GBR expression. Proteomic and functional analyses revealed that APP associates with JIP and calsyntenin proteins that link the APP/GBR complex in cargo vesicles to the axonal trafficking motor. Complex formation with GBRs stabilizes APP at the cell surface and reduces proteolysis of APP to Aß, a component of senile plaques in Alzheimer's disease patients. Thus, APP/GBR complex formation links presynaptic GBR trafficking to Aß formation. Our findings support that dysfunctional axonal trafficking and reduced GBR expression in Alzheimer's disease increases Aß formation.
Assuntos
Peptídeos beta-Amiloides/metabolismo , Amiloide/metabolismo , Transporte Axonal , Receptores de GABA-B/metabolismo , Sequência de Aminoácidos , Peptídeos beta-Amiloides/química , Animais , Axônios/metabolismo , Moléculas de Adesão Celular/química , Moléculas de Adesão Celular/metabolismo , Membrana Celular/metabolismo , Dendritos/metabolismo , Epitopos/metabolismo , Proteínas de Ligação ao GTP/metabolismo , Células HEK293 , Humanos , Cinesinas/metabolismo , Camundongos Endogâmicos C57BL , Proteínas do Tecido Nervoso/química , Proteínas do Tecido Nervoso/metabolismo , Ligação Proteica , Estabilidade Proteica , Proteômica , Transdução de Sinais , Sinapses/metabolismoRESUMO
In dissociated neuronal cultures the absence of spatial and temporal cues causes the emergence of mismatched synapses, where post-synaptic proteins of GABAergic synapses are in part apposed to glutamatergic pre-synaptic terminals and vice versa. This mismatch offers an opportunity to study the mechanisms that regulate correct apposition of pre- and post-synaptic elements. We report here that the IQ motif and Sec7 domain-containing protein 3 (IQSEC3; BRAG3; synArfGEF) specifically regulates the mislocalization of GABAergic post-synaptic density (PSD) proteins. Over-expression of IQSEC3 constructs harboring mutations that ablate Sec7 domain or IQ motif function revealed that IQSEC3 catalytic activity is involved in the control of apposition between the GABAergic PSD and glutamatergic terminals. Neurons co-expressing eGFP-gephyrin with IQSEC3 Sec7 mutant displayed a drastically increased fraction of mismatched eGFP-gephyrin clusters compared to other IQSEC3 constructs. Along with eGFP-gephyrin, endogenous GABAA receptor cluster mismatching was increased by IQSEC3 Sec7 mutant over-expression. Conversely, GFP-PSD-95 clusters were unaffected by over-expression of any IQSEC3 construct. The GABAergic PSD mismatch phenotype was recapitulated by Arf6 dominant-negative mutant over-expression, suggesting that Arf6 activation by IQSEC3 is an essential step in this pathway. In addition, we provide biochemical evidence to confirm gephyrin/IQSEC3 interaction near the IQSEC3 IQ motif, which in turn binds calmodulin at low Ca2+ concentrations. Taken together, our findings identify a post-synaptic protein which specifically regulates correct apposition of the GABAergic PSD to pre-synaptic terminals.