RESUMO
The intra-erythrocytic proliferation of the human malaria parasite Plasmodium falciparum requires massive synthesis of PE (phosphatidylethanolamine) that together with phosphatidylcholine constitute the bulk of the malaria membrane lipids. PE is mainly synthesized de novo by the CDP:ethanolamine-dependent Kennedy pathway. We previously showed that inhibition of PE biosynthesis led to parasite death. In the present study we characterized PfECT [P. falciparum CTP:phosphoethanolamine CT (cytidylyltransferase)], which we identified as the rate-limiting step of the PE metabolic pathway in the parasite. The cellular localization and expression of PfECT along the parasite life cycle were studied using polyclonal antibodies. Biochemical analyses showed that the enzyme activity follows Michaelis-Menten kinetics. PfECT is composed of two CT domains separated by a linker region. Activity assays on recombinant enzymes upon site-directed mutagenesis revealed that the N-terminal CT domain was the only catalytically active domain of PfECT. Concordantly, three-dimensional homology modelling of PfECT showed critical amino acid differences between the substrate-binding sites of the two CT domains. PfECT was predicted to fold as an intramolecular dimer suggesting that the inactive C-terminal domain is important for dimer stabilization. Given the absence of PE synthesis in red blood cells, PfECT represents a potential antimalarial target opening the way for a rational conception of bioactive compounds.
Assuntos
Plasmodium falciparum/enzimologia , Proteínas de Protozoários/química , RNA Nucleotidiltransferases/química , Animais , Sítios de Ligação , Feminino , Humanos , Cinética , Camundongos , Camundongos Endogâmicos BALB C , Modelos Moleculares , Fosfatidiletanolaminas/metabolismo , Plasmodium falciparum/genética , Plasmodium falciparum/metabolismo , Conformação Proteica , Estrutura Terciária de Proteína , Proteínas de Protozoários/genética , Proteínas de Protozoários/metabolismo , RNA Nucleotidiltransferases/genética , RNA Nucleotidiltransferases/metabolismoRESUMO
In a constantly changing environment, organisms must track the current relationship between actions and their specific consequences and use this information to guide decision-making. Such goal-directed behaviour relies on circuits involving cortical and subcortical structures. Notably, a functional heterogeneity exists within the medial prefrontal, insular, and orbitofrontal cortices (OFC) in rodents. The role of the latter in goal-directed behaviour has been debated, but recent data indicate that the ventral and lateral subregions of the OFC are needed to integrate changes in the relationships between actions and their outcomes. Neuromodulatory agents are also crucial components of prefrontal functions and behavioural flexibility might depend upon the noradrenergic modulation of the prefrontal cortex. Therefore, we assessed whether noradrenergic innervation of the OFC plays a role in updating action-outcome relationships in male rats. We used an identity-based reversal task and found that depletion or chemogenetic silencing of noradrenergic inputs within the OFC rendered rats unable to associate new outcomes with previously acquired actions. Silencing of noradrenergic inputs in the prelimbic cortex or depletion of dopaminergic inputs in the OFC did not reproduce this deficit. Together, our results suggest that noradrenergic projections to the OFC are required to update goal-directed actions.
Assuntos
Objetivos , Roedores , Ratos , Masculino , Animais , Córtex Pré-Frontal/fisiologia , Motivação , Transdução de SinaisRESUMO
The striatum, the main input structure of the basal ganglia, is critical for action selection and adaptive motor control. To understand the neuronal mechanisms underlying these functions, an analysis of microcircuits that compose the striatum is necessary. Among these, cholinergic interneurons (ChIs) provide intrinsic striatal innervation whose dysfunction is implicated in neuropsychiatric diseases, such as Parkinson's disease and Tourette syndrome. The ability to experimentally manipulate the activity of ChIs is critical to gain insights into their contribution to the normal function of the striatum and the emergence of behavioral abnormalities in pathological states. In this study, we generated and tested CAV-pChAT-GFP, a replication-defective canine adenovirus type 2 (CAV-2) vector carrying the green fluorescent protein (GFP) sequence under the control of the human choline acetyltransferase (ChAT) promoter. We first tested the potential specificity of CAV-pChAT-GFP to label striatal ChIs in a rat before performing experiments on two macaque monkeys. In the vector-injected rat and monkey striatum, we found that GFP expression preferentially colocalized with ChAT-immunoreactivity throughout the striatum, including those from local circuit interneurons. CAV-2 vectors containing transgene driven by the ChAT promoter provide a powerful tool for investigating ChI contributions to circuit function and behavior in nonhuman primates.
RESUMO
A defining feature of sleep is reduced responsiveness to external stimuli, but the mechanisms mediating sensory-evoked arousal remain unclear. We hypothesized that reduced locus coeruleus (LC) norepinephrine (NE) activity during sleep mediates unresponsiveness, and its action promotes sensory-evoked awakenings. We tested this using electrophysiological, behavioral, pharmacological, and optogenetic techniques alongside auditory stimulation in freely behaving rats. We found that systemic reduction in NE signaling lowered probability of sound-evoked awakenings (SEAs). The level of tonic LC activity during sleep anticipated SEAs. Optogenetic LC activation promoted arousal as evident in sleep-wake transitions, EEG desynchronization, and pupil dilation. Minimal LC excitation before sound presentation increased SEA probability. Optogenetic LC silencing using a soma-targeted anion-conducting channelrhodopsin (stGtACR2) suppressed LC spiking and constricted pupils. Brief periods of LC opto-silencing reduced the probability of SEAs. Thus, LC-NE activity determines the likelihood of sensory-evoked awakenings, and its reduction during sleep constitutes a key factor mediating behavioral unresponsiveness.
Assuntos
Nível de Alerta , Locus Cerúleo/fisiologia , Norepinefrina/metabolismo , Sono , Fenômenos Eletrofisiológicos , Neurônios/fisiologia , Optogenética , Transdução de Sinais , Fases do Sono , SomRESUMO
The options available for genetic modification of cells of the central nervous system (CNS) have greatly increased in the last decade. The current panoply of viral and nonviral vectors provides multifunctional platforms to deliver expression cassettes to many structures and nuclei. These cassettes can replace defective genes, modify a given pathway perturbed by diseases, or express proteins that can be selectively activated by drugs or light to extinguish or excite neurons. This review focuses on the use of canine adenovirus type 2 (CAV-2) vectors for gene transfer to neurons in the brain, spinal cord, and peripheral nervous system. We discuss (1) recent advances in vector production, (2) why CAV-2 vectors preferentially transduce neurons, (3) the mechanism underlying their widespread distribution via retrograde axonal transport, (4) how CAV-2 vectors have been used to address structure/function, and (5) their therapeutic applications.
RESUMO
Mucopolysaccharidosis type VII (MPS VII) is a lysosomal storage disease caused by deficient ß-glucuronidase (ß-gluc) activity. Significantly reduced ß-gluc activity leads to accumulation of glycosaminoglycans (GAGs) in many tissues, including the brain. Numerous combinations of mutations in GUSB (the gene that codes for ß-gluc) cause a range of neurological features that make disease prognosis and treatment challenging. Currently, there is little understanding of the molecular basis for MPS VII brain anomalies. To identify a neuronal phenotype that could be used to complement genetic analyses, we generated two iPSC clones derived from skin fibroblasts of an MPS VII patient. We found that MPS VII neurons exhibited reduced ß-gluc activity and showed previously established disease-associated phenotypes, including GAGs accumulation, expanded endocytic compartments, accumulation of lipofuscin granules, more autophagosomes, and altered lysosome function. Addition of recombinant ß-gluc to MPS VII neurons, which mimics enzyme replacement therapy, restored disease-associated phenotypes to levels similar to the healthy control. MPS VII neural cells cultured as 3D neurospheroids showed upregulated GFAP gene expression, which was associated with astrocyte reactivity, and downregulation of GABAergic neuron markers. Spontaneous calcium imaging analysis of MPS VII neurospheroids showed reduced neuronal activity and altered network connectivity in patient-derived neurospheroids compared to a healthy control. These results demonstrate the interplay between reduced ß-gluc activity, GAG accumulation and alterations in neuronal activity, and provide a human experimental model for elucidating the bases of MPS VII-associated cognitive defects.
Assuntos
Glicosaminoglicanos/metabolismo , Células-Tronco Pluripotentes Induzidas/patologia , Lisossomos/patologia , Mucopolissacaridose VII/patologia , Vias Neurais , Neurônios/patologia , Células-Tronco/patologia , Estudos de Casos e Controles , Diferenciação Celular , Células Cultivadas , Humanos , Células-Tronco Pluripotentes Induzidas/metabolismo , Lisossomos/metabolismo , Mucopolissacaridose VII/metabolismo , Neurônios/metabolismo , Células-Tronco/metabolismoRESUMO
Phosphatidylcholine is the major lipid component of the malaria parasite membranes and is required for parasite multiplication in human erythrocytes. Plasmodium falciparum CTP:phosphocholine cytidylyltransferase (PfCCT) is the rate-limiting enzyme of the phosphatidylcholine biosynthesis pathway and thus considered as a potential antimalarial target. In contrast to its mammalian orthologs, PfCCT contains a duplicated catalytic domain. Here, we show that both domains are catalytically active with similar kinetic parameters. A virtual screening strategy allowed the identification of a drug-size molecule competitively inhibiting the enzyme. This compound also prevented phosphatidylcholine biosynthesis in parasites and exerted an antimalarial effect. This study constitutes the first step towards a rationalized design of future new antimalarial agents targeting PfCCT.
Assuntos
Domínio Catalítico , Colina-Fosfato Citidililtransferase/metabolismo , Citidina Difosfato Colina/análogos & derivados , Plasmodium falciparum/enzimologia , Proteínas de Protozoários/metabolismo , Sequência de Aminoácidos , Antimaláricos/química , Antimaláricos/farmacologia , Vias Biossintéticas/genética , Colina-Fosfato Citidililtransferase/antagonistas & inibidores , Colina-Fosfato Citidililtransferase/genética , Citidina Difosfato Colina/química , Citidina Difosfato Colina/farmacologia , Humanos , Immunoblotting , Cinética , Microscopia de Fluorescência , Modelos Moleculares , Dados de Sequência Molecular , Estrutura Molecular , Fosfatidilcolinas/biossíntese , Fosfatidilcolinas/química , Plasmodium falciparum/genética , Ligação Proteica , Estrutura Terciária de Proteína , Proteínas de Protozoários/química , Proteínas de Protozoários/genética , Homologia de Sequência de AminoácidosRESUMO
The enzyme CTP:phosphocholine cytidylyltransferase (CCT) is essential in the lipid biosynthesis of Plasmodia (Haemosporida), presenting a promising antimalarial target. Here, we identified two independent gene duplication events of CCT within Apicomplexa and characterized a truncated construct of Plasmodium falciparum CCT that forms a dimer resembling the molecular architecture of CCT enzymes from other sources. Based on biophysical and enzyme kinetics methods, our data show that the CDP-choline product of the CCT enzymatic reaction binds to the enzyme considerably stronger than either substrate (CTP or choline phosphate). Interestingly, in the presence of Mg²âº , considered to be a cofactor of the enzyme, the binding of the CTP substrate is attenuated by a factor of 5. The weaker binding of CTP:Mg²âº , similarly to the related enzyme family of aminoacyl tRNA synthetases, suggests that, with lack of Mg²âº , positively charged side chain(s) of CCT may contribute to CTP accommodation. Thermodynamic investigations by isothermal titration calorimetry and fluorescent spectroscopy studies indicate that accommodation of the choline phosphate moiety in the CCT active site is different when it appears on its own as one of the substrates or when it is linked to the CDP-choline product. A tryptophan residue within the active site is identified as a useful internal fluorescence sensor of enzyme-ligand binding. Results indicate that the catalytic mechanism of Plasmodium falciparum CCT may involve conformational changes affecting the choline subsite of the enzyme.
Assuntos
Colina-Fosfato Citidililtransferase/metabolismo , Evolução Molecular , Modelos Moleculares , Plasmodium falciparum/enzimologia , Proteínas de Protozoários/metabolismo , Sequência de Aminoácidos , Apicomplexa/enzimologia , Apicomplexa/genética , Apicomplexa/metabolismo , Biocatálise , Domínio Catalítico , Colina-Fosfato Citidililtransferase/química , Colina-Fosfato Citidililtransferase/genética , Citidina Difosfato Colina/química , Citidina Difosfato Colina/metabolismo , Citidina Trifosfato/química , Citidina Trifosfato/metabolismo , Dimerização , Estabilidade Enzimática , Deleção de Genes , Duplicação Gênica , Magnésio/metabolismo , Dados de Sequência Molecular , Fosforilcolina/química , Fosforilcolina/metabolismo , Plasmodium falciparum/genética , Plasmodium falciparum/metabolismo , Ligação Proteica , Proteínas de Protozoários/química , Proteínas de Protozoários/genética , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos , Triptofano/químicaRESUMO
OBJECTIVE: To describe the epidemiological, clinical, and laboratory characteristics of patients with group B streptococcal (GBS) prosthetic joint infections, their diagnoses, treatment, and long-term outcomes. METHODS: We conducted a retrospective cohort study including all patients hospitalized from January 1994 through May 2006 for a GBS prosthetic joint infection. RESULTS: The study included 30 patients, aged 35-87 (median 74) years with prosthetic hip (24) or knee (6) infections, 20 with at least one underlying disease. The route of infection was presumed to be hematogenous in 27 patients, and a portal of entry was identified in 9 (genitourinary tract 4, skin 2, gastrointestinal tract 2, oropharynx 1). All patients underwent surgery (6 debridement-synovectomy, 9 1-stage exchange arthroplasty, 8 2-stage exchange arthroplasty, 6 hip resection arthroplasty, and 1 knee arthrodesis) and received prolonged intravenous antibiotics. Four patients relapsed. One patient developed 2 other infections on her knee prosthesis. Two deaths were infection-related, and one was treatment-related. Nineteen patients followed for >/=2 years were cured. One patient was lost to follow-up and 3 died of causes unrelated to infection or treatment within 2 years. CONCLUSION: GBS prosthetic joint infections are mostly acute hematogenous infections that require prompt management for satisfactory outcome. Despite high antibiotic susceptibility, treatment failure is frequent because of the severity of the infection and patients' advanced age, underlying diseases, and relapses.
Assuntos
Prótese Articular/efeitos adversos , Infecções Relacionadas à Prótese/etiologia , Infecções Estreptocócicas/etiologia , Streptococcus agalactiae , Adulto , Idoso , Idoso de 80 Anos ou mais , Estudos de Coortes , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Infecções Relacionadas à Prótese/diagnóstico , Infecções Relacionadas à Prótese/terapia , Estudos Retrospectivos , Infecções Estreptocócicas/diagnóstico , Infecções Estreptocócicas/terapiaRESUMO
BACKGROUND: Outcome of streptococcal prosthetic hip infection is often thought to be better than that caused by other pathogens. That supposition was not confirmed in our experience with group B streptococcal prosthetic joint infection. OBJECTIVE: We compared outcomes of group B streptococcal and other-pathogen prosthetic hip infections. METHODS: One hundred and thirty nine patients, 24 with group B streptococcal and 115 other-pathogen prosthetic hip infections, were included. The primary outcome was the time from surgical treatment to treatment failure, defined as relapse, infection- or treatment-related death. Secondary outcomes were the times from surgical treatment to relapse or any event (event-free survival). The cumulative incidence estimator was used to model primary and secondary outcomes. Multivariable regression analysis was used to determine a set of independent predictors of treatment failure. RESULTS: With a median follow-up of 22 months, treatment failed more frequently in patients with group B streptococcal prosthetic hip infections (hazard ratio, 4.88 [95% CI, 1.4-17], P=.012). Multivariable analysis retained the American Society of Anesthesiologist score and group B streptococcal infection as independent risk factors of treatment failure; event-free survival was lower for these patients (hazard ratio, 2.64 [95% CI, 1.2-6], P=.02). CONCLUSION: Despite high antibiotic susceptibility, outcomes of group B streptococcal and other-pathogen prosthetic hip infection differ.