RESUMO
Mass spectrometry has revolutionized cell signaling research by vastly simplifying the analysis of many thousands of phosphorylation sites in the human proteome. Defining the cellular response to perturbations is crucial for further illuminating the functionality of the phosphoproteome. Here we describe µPhos ('microPhos'), an accessible phosphoproteomics platform that permits phosphopeptide enrichment from 96-well cell culture and small tissue amounts in <8 h total processing time. By greatly minimizing transfer steps and liquid volumes, we demonstrate increased sensitivity, >90% selectivity, and excellent quantitative reproducibility. Employing highly sensitive trapped ion mobility mass spectrometry, we quantify ~17,000 Class I phosphosites in a human cancer cell line using 20 µg starting material, and confidently localize ~6200 phosphosites from 1 µg. This depth covers key signaling pathways, rendering sample-limited applications and perturbation experiments with hundreds of samples viable. We employ µPhos to study drug- and time-dependent response signatures in a leukemia cell line, and by quantifying 30,000 Class I phosphosites in the mouse brain we reveal distinct spatial kinase activities in subregions of the hippocampal formation.
Assuntos
Fosfopeptídeos , Fosfoproteínas , Proteômica , Proteômica/métodos , Humanos , Animais , Camundongos , Fosfoproteínas/metabolismo , Fosforilação , Linhagem Celular Tumoral , Fosfopeptídeos/metabolismo , Fosfopeptídeos/análise , Espectrometria de Massas/métodos , Transdução de Sinais , Proteoma/metabolismo , Reprodutibilidade dos Testes , Hipocampo/metabolismo , Hipocampo/citologiaRESUMO
Sepsis-associated encephalopathy (SAE) is a frequent complication of severe systemic infection resulting in delirium, premature death, and long-term cognitive impairment. We closely mimicked SAE in a murine peritoneal contamination and infection (PCI) model. We found long-lasting synaptic pathology in the hippocampus including defective long-term synaptic plasticity, reduction of mature neuronal dendritic spines, and severely affected excitatory neurotransmission. Genes related to synaptic signaling, including the gene for activity-regulated cytoskeleton-associated protein (Arc/Arg3.1) and members of the transcription-regulatory EGR gene family, were downregulated. At the protein level, ARC expression and mitogen-activated protein kinase signaling in the brain were affected. For targeted rescue we used adeno-associated virus-mediated overexpression of ARC in the hippocampus in vivo. This recovered defective synaptic plasticity and improved memory dysfunction. Using the enriched environment paradigm as a non-invasive rescue intervention, we found improvement of defective long-term potentiation, memory, and anxiety. The beneficial effects of an enriched environment were accompanied by an increase in brain-derived neurotrophic factor (BDNF) and ARC expression in the hippocampus, suggesting that activation of the BDNF-TrkB pathway leads to restoration of the PCI-induced reduction of ARC. Collectively, our findings identify synaptic pathomechanisms underlying SAE and provide a conceptual approach to target SAE-induced synaptic dysfunction with potential therapeutic applications to patients with SAE.
Assuntos
Fator Neurotrófico Derivado do Encéfalo , Disfunção Cognitiva , Proteínas do Citoesqueleto , Modelos Animais de Doenças , Hipocampo , Plasticidade Neuronal , Encefalopatia Associada a Sepse , Animais , Camundongos , Disfunção Cognitiva/etiologia , Disfunção Cognitiva/metabolismo , Disfunção Cognitiva/terapia , Disfunção Cognitiva/genética , Encefalopatia Associada a Sepse/metabolismo , Encefalopatia Associada a Sepse/etiologia , Encefalopatia Associada a Sepse/terapia , Encefalopatia Associada a Sepse/genética , Hipocampo/metabolismo , Fator Neurotrófico Derivado do Encéfalo/metabolismo , Fator Neurotrófico Derivado do Encéfalo/genética , Proteínas do Citoesqueleto/genética , Proteínas do Citoesqueleto/metabolismo , Proteínas do Tecido Nervoso/genética , Proteínas do Tecido Nervoso/metabolismo , Dependovirus/genética , Masculino , Potenciação de Longa Duração , Receptor trkB/metabolismo , Receptor trkB/genética , Vetores Genéticos/administração & dosagem , Vetores Genéticos/genética , Sinapses/metabolismoRESUMO
Microglia are innate immune cells in the brain and show exceptional heterogeneity. They are key players in brain physiological development regulating synaptic plasticity and shaping neuronal networks. In pathological disease states, microglia-induced synaptic pruning mediates synaptic loss and targeting microglia was proposed as a promising therapeutic strategy. However, the effect of microglia depletion and subsequent repopulation on dendritic spine density and neuronal function in the adult brain is largely unknown. In this study, we investigated whether pharmacological microglia depletion affects dendritic spine density after long-term permanent microglia depletion and after short-term microglia depletion with subsequent repopulation. Long-term microglia depletion using colony-stimulating-factor-1 receptor (CSF1-R) inhibitor PLX5622 resulted in increased overall spine density, especially of mushroom spines, and increased excitatory postsynaptic current amplitudes. Short-term PLX5622 treatment with subsequent repopulation of microglia had an opposite effect resulting in activated microglia with increased synaptic phagocytosis and consequently decreased spine density and reduced excitatory neurotransmission, while Barnes maze and elevated plus maze testing was unaffected. Moreover, RNA sequencing data of isolated repopulated microglia showed an activated and proinflammatory phenotype. Long-term microglia depletion might be a promising therapeutic strategy in neurological diseases with pathological microglial activation, synaptic pruning, and synapse loss. However, repopulation after depletion induces activated microglia and results in a decrease of dendritic spines possibly limiting the therapeutic application of microglia depletion. Instead, persistent modulation of pathological microglia activity might be beneficial in controlling synaptic damage.
Assuntos
Encéfalo , Espinhas Dendríticas , Camundongos Endogâmicos C57BL , Microglia , Animais , Microglia/efeitos dos fármacos , Microglia/metabolismo , Espinhas Dendríticas/efeitos dos fármacos , Masculino , Camundongos , Potenciais Pós-Sinápticos Excitadores/efeitos dos fármacos , Potenciais Pós-Sinápticos Excitadores/fisiologia , Fagocitose/fisiologia , Fagocitose/efeitos dos fármacos , Plasticidade Neuronal/fisiologia , Plasticidade Neuronal/efeitos dos fármacos , Camundongos Transgênicos , Receptores de Fator Estimulador das Colônias de Granulócitos e Macrófagos/antagonistas & inibidores , Receptores de Fator Estimulador das Colônias de Granulócitos e Macrófagos/metabolismo , Compostos OrgânicosRESUMO
In comparison to bulk sequencing or single cell sequencing, spatial transcriptomics preserves the spatial information in tissue slices and can even be mapped to immunofluorescent stainings, allowing translation of gene expression information into their spatial context. This enables to unravel complex interactions of neighboring cells or to link cell morphology to transcriptome data. The 10× Genomics Visium platform offers to combine spatial transcriptomics with immunofluorescent staining of cryo-sectioned tissue slices. We applied this technique to fresh frozen mouse brain slices and developed a protocol that still protects RNA quality while improving buffers for immunofluorescent staining. We investigated the impact of various parameters, including fixation time and buffer composition, on RNA quality and antibody binding. Here, we propose an improved version of the manufacturer protocol, which does not alter RNA quality and facilitates the use of multiple additional antibodies that were not compatible with the manufacturer protocol before. Finally, we discuss the influence of various staining parameters, which contribute to the development of application specific staining protocols.
RESUMO
Sepsis-associated encephalopathy (SAE) is a severe and frequent complication of sepsis causing delirium, coma, and long-term cognitive dysfunction. We identified microglia and C1q complement activation in hippocampal autopsy tissue of patients with sepsis and increased C1q-mediated synaptic pruning in a murine polymicrobial sepsis model. Unbiased transcriptomics of hippocampal tissue and isolated microglia derived from septic mice revealed an involvement of the innate immune system, complement activation, and up-regulation of lysosomal pathways during SAE in parallel to neuronal and synaptic damage. Microglial engulfment of C1q-tagged synapses could be prevented by stereotactic intrahippocampal injection of a specific C1q-blocking antibody. Pharmacologically targeting microglia by PLX5622, a CSF1-R inhibitor, reduced C1q levels and the number of C1q-tagged synapses, protected from neuronal damage and synapse loss, and improved neurocognitive outcome. Thus, we identified complement-dependent synaptic pruning by microglia as a crucial pathomechanism for the development of neuronal defects during SAE.
Assuntos
Encefalopatia Associada a Sepse , Sepse , Camundongos , Animais , Microglia/metabolismo , Complemento C1q/metabolismo , Encefalopatia Associada a Sepse/etiologia , Encefalopatia Associada a Sepse/metabolismo , Sinapses/metabolismo , Sepse/complicações , Sepse/metabolismoRESUMO
An outbreak of mousepox in a research institution was caused by Ectromelia-contaminated mouse serum that had been used for bone marrow cell culture and the cells subsequently injected into the footpads of mice. The disease initially was diagnosed by identification of gross and microscopic lesions typical for Ectromelia infection, including foci of necrosis in the liver and spleen and eosinophilic intracytoplasmic inclusion bodies in the skin. The source of infection was determined by PCR analysis to be serum obtained from a commercial vendor. To determine whether viral growth in tissue culture was required to induce viral infection, 36 mice (BALB/cJ, C57BL/6J) were experimentally exposed intraperitoneally, intradermally (footpad), or intranasally to contaminated serum or bone marrow cell cultures using the contaminated serum in the culture medium. Mice were euthanized when clinical signs developed or after 12 wk. Necropsy, PCR of spleen, and serum ELISA were performed on all mice. Mice injected with cell cultures and their cage contacts developed mousepox, antibodies to Ectromelia, and lesions, whereas mice injected with serum without cells did not. Mouse antibody production, a tool commonly used to screen biologic materials for viral contamination, failed to detect active Ectromelia contamination in mouse serum.
Assuntos
Vírus da Ectromelia/metabolismo , Ectromelia Infecciosa , Abrigo para Animais , Doenças dos Roedores , Animais , Células da Medula Óssea/citologia , Células da Medula Óssea/virologia , Transplante de Medula Óssea , Células Cultivadas/virologia , Surtos de Doenças , Ectromelia Infecciosa/sangue , Ectromelia Infecciosa/diagnóstico , Ectromelia Infecciosa/epidemiologia , Feminino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Distribuição Aleatória , Doenças dos Roedores/sangue , Doenças dos Roedores/diagnóstico , Doenças dos Roedores/epidemiologia , VacinaçãoRESUMO
The orphan cytokine receptor-like factor 3 (CRLF3) was identified as a neuroprotective erythropoietin receptor in locust neurons and emerged with the evolution of the eumetazoan nervous system. Human CRLF3 belongs to class I helical cytokine receptors that mediate pleiotropic cellular reactions to injury and diverse physiological challenges. It is expressed in various tissues including the central nervous system but its ligand remains unidentified. A CRLF3 ortholog in the holometabolous beetle Tribolium castaneum was recently shown to induce anti-apoptotic mechanisms upon stimulation with human recombinant erythropoietin. To test the hypothesis that CRLF3 represents an ancient cell-protective receptor for erythropoietin-like cytokines, we investigated its presence across metazoan species. Furthermore, we examined CRLF3 expression and function in the hemimetabolous insect Locusta migratoria. Phylogenetic analysis of CRLF3 sequences indicated that CRLF3 is absent in Porifera, Placozoa and Ctenophora, all lacking the traditional nervous system. However, it is present in all major eumetazoan groups ranging from cnidarians over protostomians to mammals. The CRLF3 sequence is highly conserved and abundant amongst vertebrates. In contrast, relatively few invertebrates express CRLF3 and these sequences show greater variability, suggesting frequent loss due to low functional importance. In L. migratoria, we identified the transcript Lm-crlf3 by RACE-PCR and detected its expression in locust brain, skeletal muscle and hemocytes. These findings correspond to the ubiquitous expression of crlf3 in mammalian tissues. We demonstrate that the sole addition of double-stranded RNA to the culture medium (called soaking RNA interference) specifically interferes with protein expression in locust primary brain cell cultures. This technique was used to knock down Lm-crlf3 expression and to abolish its physiological function. We confirmed that recombinant human erythropoietin rescues locust brain neurons from hypoxia-induced apoptosis and showed that this neuroprotective effect is absent after knocking down Lm-crlf3. Our results affirm the erythropoietin-induced neuroprotective function of CRLF3 in a second insect species from a different taxonomic group. They suggest that the phylogenetically conserved CRLF3 receptor may function as a cell protective receptor for erythropoietin or a structurally related cytokine also in other animals including vertebrate and mammalian species.
RESUMO
The genome of Drosophila melanogaster includes homologs to approximately one-third of the currently known human disease genes. Flies and humans share many biological processes, including the principles of information processing by excitable neurons, synaptic transmission, and the chemical signals involved in intercellular communication. Studies on the molecular and behavioral impact of genetic risk factors of human neuro-developmental disorders [autism spectrum disorders (ASDs), schizophrenia, attention deficit hyperactivity disorders, and Tourette syndrome] increasingly use the well-studied social behavior of D. melanogaster, an organism that is amenable to a large variety of genetic manipulations. Neuroligins (Nlgs) are a family of phylogenetically conserved postsynaptic adhesion molecules present (among others) in nematodes, insects, and mammals. Impaired function of Nlgs (particularly of Nlg 3 and 4) has been associated with ASDs in humans and impaired social and communication behavior in mice. Making use of a set of behavioral and social assays, we, here, analyzed the impact of two Drosophila Nlgs, Dnlg2 and Dnlg4, which are differentially expressed at excitatory and inhibitory central nervous synapses, respectively. Both Nlgs seem to be associated with diurnal activity and social behavior. Even though deficiencies in Dnlg2 and Dnlg4 appeared to have no effects on sensory or motor systems, they differentially impacted on social interactions, suggesting that social behavior is distinctly regulated by these Nlgs.
RESUMO
The cytokine erythropoietin (Epo) mediates various cell homeostatic responses to environmental challenges and pathological insults. While stimulation of vertebrate erythrocyte production is mediated by homodimeric "classical" Epo receptors, alternative receptors are involved in neuroprotection. However, their identity remains enigmatic due to complex cytokine ligand and receptor interactions and conflicting experimental results. Besides the classical Epo receptor, the family of type I cytokine receptors also includes the poorly characterized orphan cytokine receptor-like factor 3 (CRLF3) present in vertebrates including human and various insect species. By making use of the more simple genetic makeup of insect model systems, we studied whether CRLF3 is a neuroprotective Epo receptor in animals. We identified a single ortholog of CRLF3 in the beetle Tribolium castaneum, and established protocols for primary neuronal cell cultures from Tribolium brains and efficient in vitro RNA interference. Recombinant human Epo as well as the non-erythropoietic Epo splice variant EV-3 increased the survival of serum-deprived brain neurons, confirming the previously described neuroprotective effect of Epo in insects. Moreover, Epo completely prevented hypoxia-induced apoptotic cell death of primary neuronal cultures. Knockdown of CRLF3 expression by RNA interference with two different double stranded RNA (dsRNA) fragments abolished the neuroprotective effect of Epo, indicating that CRLF3 is a crucial component of the insect Epo-responsive receptor. This suggests that a common urbilaterian ancestor of the orphan human and insect cytokine receptor CRLF3 served as a neuroprotective receptor for an Epo-like cytokine. Our work also suggests that vertebrate CRLF3, like its insect ortholog, might represent a tissue protection-mediating receptor.
RESUMO
Field studies often necessitate immobilization of animal subjects, but field conditions may complicate the induction of anesthesia. Likewise, researchers must ensure that animals are fully recovered before releasing them. The authors successfully tested an anesthesia induction and reversal regimen under field conditions.
Assuntos
Antagonistas Adrenérgicos alfa/farmacologia , Anestesia/veterinária , Anestésicos/farmacologia , Hipnóticos e Sedativos/farmacologia , Imidazóis/farmacologia , Ketamina/farmacologia , Medetomidina/farmacologia , Roedores , Anestesia/métodos , Animais , Interações Medicamentosas , Feminino , Masculino , Fatores de TempoRESUMO
An investigation was conducted to determine the cause of morbidity and mortality in a collection of 55 adult male Xenopus (Silurana) tropicalis at the University of California, Berkeley. More than 80% of affected frogs died during the epizootic. All frogs were anorectic and lethargic, had dark pigmentation and excess skin sloughing, and lacked a slime layer. Histologic examination revealed severe hyperplastic and spongiotic dermatitis associated with colonization of the stratum corneum by large numbers of zoosporangia diagnostic of Batrachochytrium dendrobatidis. Treatment with a commercial formalin/malachite green solution at a dilution of 0.007 ml/L of tank water for 24 h, repeated every other day for four treatments, eliminated the organism and was curative. These findings are indicative of epidermal chytridiomycosis as a primary cause of death in this collection of X. tropicalis.
Assuntos
Quitridiomicetos/isolamento & purificação , Dermatomicoses/veterinária , Xenopus , Criação de Animais Domésticos , Animais , Quitridiomicetos/patogenicidade , Dermatomicoses/diagnóstico , Dermatomicoses/terapia , Formaldeído/uso terapêutico , Masculino , Corantes de Rosanilina/uso terapêutico , Resultado do TratamentoRESUMO
An adult female hyena and her two 4-month-old cubs were found to have multifocal areas of alopecia and dermatitis. Dermatophyte culture of hair and skin samples collected from the lesions yielded Trichophyton mentagrophytes. None of 10 other animals in the colony that were tested were found to have dermatophytes. Lesions were treated twice, at 3-week intervals, with thorough cleansing with chlorhexidine scrub followed by topical application of antifungal agents. Lesions resolved, and dermatophyte culture of samples collected 6 weeks after the initiation of treatment did not yield growth.
Assuntos
Antifúngicos/uso terapêutico , Carnívoros/microbiologia , Tinha/veterinária , Administração Tópica , Animais , Anti-Infecciosos Locais/uso terapêutico , Clorexidina/uso terapêutico , Reservatórios de Doenças/veterinária , Feminino , Masculino , Tinha/tratamento farmacológico , Tinha/microbiologia , Trichophyton/isolamento & purificação , Trichophyton/patogenicidade , ZoonosesRESUMO
Autism spectrum disorders (ASDs) are characterized by deficits in social interactions, language development and repetitive behaviours. Multiple genes involved in the formation, specification and maintenance of synapses have been identified as risk factors for ASDs development. Among these are the neuroligin genes which code for postsynaptic cell adhesion molecules that induce the formation of presynapses, promote their maturation and modulate synaptic functions in both vertebrates and invertebrates. Neuroligin-deficient mice display abnormal social and vocal behaviours that resemble ASDs symptoms. Here we show for the fly Drosophila melanogaster that deletion of the dnl2 gene, coding for one of four Neuroligin isoforms, impairs social interactions, alters acoustic communication signals, and affects the transition between different behaviours. dnl2-Deficient flies maintain larger distances to conspecifics and males perform less female-directed courtship and male-directed aggressive behaviours while the patterns of these behaviours and general locomotor activity were not different from wild type controls. Since tests for olfactory, visual and auditory perception revealed no sensory impairments of dnl2-deficient mutants, reduced social interactions seem to result from altered excitability in central nervous neuropils that initiate social behaviours. Our results demonstrate that Neuroligins are phylogenetically conserved not only regarding their structure and direct function at the synapse but also concerning a shared implication in the regulation of social behaviours that dates back to common ancestors of humans and flies. In addition to previously described mouse models, Drosophila can thus be used to study the contribution of Neuroligins to synaptic function, social interactions and their implication in ASDs.
Assuntos
Moléculas de Adesão Celular Neuronais/genética , Proteínas do Tecido Nervoso/genética , Transtornos do Comportamento Social/genética , Agressão/fisiologia , Comunicação Animal , Animais , Animais Geneticamente Modificados , Moléculas de Adesão Celular Neuronais/deficiência , Relógios Circadianos/genética , Corte , Modelos Animais de Doenças , Drosophila melanogaster , Eletrorretinografia , Audição/genética , Locomoção/genética , Masculino , Proteínas do Tecido Nervoso/deficiência , Comportamento Sexual Animal/fisiologiaRESUMO
Two pair-housed, 1-y-old common marmosets (Callithrix jacchus) had intermittent loose feces and weight loss for approximately 2 mo. Cryptosporidum parvum was identified by ELISA in the feces of both animals. CBC and blood chemistry values, including liver enzymes, were within normal range. Both marmosets were treated with the antibiotic paromomycin (15 mg/kg PO) twice daily for 28 d. Resolution of clinical signs coincided with treatment. Three follow-up samples, taken 2 wk apart after treatment was finished, were negative for cryptosporidium ELISA in both animals. Paromomycin should be considered for treatment of cryptosporidiosis in marmosets.
Assuntos
Antibacterianos/uso terapêutico , Callithrix/parasitologia , Criptosporidiose/veterinária , Doenças dos Macacos/tratamento farmacológico , Doenças dos Macacos/parasitologia , Paromomicina/uso terapêutico , Animais , Criptosporidiose/tratamento farmacológico , Criptosporidiose/parasitologia , Cryptosporidium/efeitos dos fármacos , Diarreia/tratamento farmacológico , Ensaio de Imunoadsorção Enzimática/veterinária , Redução de PesoRESUMO
Audiogenic stress is a well-documented phenomenon in laboratory rodents. Despite the recommendation in the Guide for the Care and Use of Laboratory Animals to consider noise a concern in the animal facility, only a small body of literature empirically addresses the effects of facility noise on laboratory rodents, particularly mice. The objective of this study was to determine whether facility noise generated by a vacuum cleaner induces an acute stress response in a commonly used strain of laboratory mouse under common housing conditions. In each of 2 experiments, 10 young adult, female C57BL/6Cr mice were exposed for 1 h to noise produced by a vacuum cleaner, and 10 control mice were not. In the first experiment, fecal samples were collected to measure concentrations of fecal corticosterone metabolites just before and 2, 4, 6, 8, 10, 14, 24, and 32 h after noise exposure. In the second experiment, stress-sensitive behavioral tests were performed 2 d before, immediately after, and 24 h after noise exposure. Physiologic and behavioral measurements indicated that vacuum cleaner noise did not cause an acute stress response in the noise-exposed mice but may have affected the diurnal variation of their corticosterone levels. These findings could contribute to the development of best practices in noise-control protocols for animal facilities.
Assuntos
Abrigo para Animais , Ruído , Estresse Fisiológico , Bem-Estar do Animal , Animais , Comportamento Animal , Ritmo Circadiano , Corticosterona/metabolismo , Feminino , Camundongos , Camundongos Endogâmicos C57BLRESUMO
We investigated the role of the western gray squirrel (Sciurus griseus) as a reservoir host of the Lyme disease spirochete Borrelia burgdorferi. A survey of 222 western gray squirrels in California showed an overall prevalence of B. burgdorferi infection of 30%, although at a county level, prevalence of infection ranged from 0% to 50% by polymerase chain reaction. Laboratory trials with wild-caught western gray squirrels indicated that squirrels were competent reservoir hosts of the Lyme disease bacterium and infected up to 86% of feeding Ixodes pacificus larvae. Infections were long-lasting (up to 14 months), which demonstrated that western gray squirrels can maintain B. burgdorferi trans-seasonally. Non-native eastern gray squirrels (Sciurus carolinensis) and fox squirrels (Sciurus niger) were infrequently infected with B. burgdorferi.