RESUMO
The synapse, which represents the structural and functional basis of neuronal communication, is one of the first elements affected in several neurodegenerative diseases. To better understand the potential role of gene expression in synapse loss, we developed an original high-content screening (HCS) model capable of quantitatively assessing the impact of gene silencing on synaptic density. Our approach is based on a model of primary neuronal cultures (PNCs) from the neonatal rat hippocampus, whose mature synapses are visualized by the relative localization of the presynaptic protein Synaptophysin with the postsynaptic protein Homer1. The heterogeneity of PNCs and the small sizes of the synaptic structures pose technical challenges associated with the level of automation necessary for HCS studies. We overcame these technical challenges, automated the processes of image analysis and data analysis, and carried out tests under real-world conditions to demonstrate the robustness of the model developed. In this article, we describe the screening of a custom library of 198 shRNAs in PNCs in the 384-well plate format. © 2023 The Authors. Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Culture of primary hippocampal rat neurons in 384-well plates Basic Protocol 2: Lentiviral shRNA transduction of primary neuronal culture in 384-well plates Basic Protocol 3: Immunostaining of the neuronal network and synaptic markers in 384-well plates Basic Protocol 4: Image acquisition using a high-throughput reader Basic Protocol 5: Image segmentation and analysis Basic Protocol 6: Synaptic density analysis.
Assuntos
Placas Ósseas , Cultura , Animais , Ratos , Automação , Análise de Dados , Neurônios , RNA Interferente PequenoRESUMO
Apicomplexa phylum includes numerous obligate intracellular protozoan parasites that are life threatening for humans and animals. In this context, Plasmodium falciparum and Toxoplasma gondii are of particular interest, as they are responsible for malaria and toxoplasmosis, respectively, for which efficient vaccines are presently lacking and therapies need to be improved. Apicomplexan parasites have a highly polarized morphology, with their apical end containing specific secretory organelles named rhoptries and micronemes, which depend on the unique receptor and transporter sortilin TgSORT for their biogenesis. In the present study, we took advantage of the subcellular polarity of the parasite to engineer a clonal transgenic Toxoplasma line that expresses simultaneously the green fluorescent protein TgSORT-GFP in the post-Golgi-endosome-like compartment and the red fluorescent protein rhoptry ROP1-mCherry near the apical end. We utilized this fluorescent transgenic T. gondii to develop a miniaturized image-based phenotype assay coupled to an automated image analysis. By applying this methodology to 1,120 compounds, we identified 12 that are capable of disrupting the T. gondii morphology and inhibiting intracellular replication. Analysis of the selected compounds confirmed that all 12 are kinase inhibitors and intramembrane pumps, with some exhibiting potent activity against Plasmodium falciparum. Our findings highlight the advantage of comparative and targeted phenotypic analysis involving two related parasite species as a means of identifying molecules with a conserved mode of action.
Assuntos
Parasitos , Toxoplasma , Animais , Humanos , Toxoplasma/genética , Toxoplasma/metabolismo , Parasitos/metabolismo , Plasmodium falciparum , Proteínas de Protozoários/metabolismo , Endossomos/metabolismo , Proteínas de Fluorescência Verde/genéticaRESUMO
Progression of tuberculosis is tightly linked to a disordered immune balance, resulting in inability of the host to restrict intracellular bacterial replication and its subsequent dissemination. The immune response is mainly characterized by an orchestrated recruitment of inflammatory cells secreting cytokines. This response results from the activation of innate immunity receptors that trigger downstream intracellular signaling pathways involving adaptor proteins such as the TIR-containing adaptor protein (Tirap). In humans, resistance to tuberculosis is associated with a loss-of-function in Tirap. Here, we explore how genetic deficiency in Tirap impacts resistance to Mycobacterium tuberculosis (Mtb) infection in a mouse model and ex vivo. Interestingly, compared to wild type littermates, Tirap heterozygous mice were more resistant to Mtb infection. Upon investigation at the cellular level, we observed that mycobacteria were not able to replicate in Tirap-deficient macrophages compared to wild type counterparts. We next showed that Mtb infection induced Tirap expression which prevented phagosomal acidification and rupture. We further demonstrate that the Tirap-mediated anti-tuberculosis effect occurs through a Cish-dependent signaling pathway. Our findings provide new molecular evidence about how Mtb manipulates innate immune signaling to enable intracellular replication and survival of the pathogen, thus paving the way for host-directed approaches to treat tuberculosis.
Assuntos
Mycobacterium tuberculosis , Tuberculose , Humanos , Camundongos , Animais , Receptores de Interleucina-1/genética , Receptores de Interleucina-1/metabolismo , Transdução de Sinais , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Concentração de Íons de Hidrogênio , Glicoproteínas de Membrana/metabolismoRESUMO
Hepatitis E virus (HEV) infection is the most common cause of acute viral hepatitis worldwide. Hepatitis E is usually asymptomatic and self-limiting but it can become chronic in immunocompromised patients and is associated with increased fulminant hepatic failure and mortality rates in pregnant women. HEV genome encodes three proteins including the ORF2 protein that is the viral capsid protein. Interestingly, HEV produces 3 isoforms of the ORF2 capsid protein which are partitioned in different subcellular compartments and perform distinct functions in the HEV lifecycle. Notably, the infectious ORF2 (ORF2i) protein is the structural component of virions, whereas the genome-free secreted and glycosylated ORF2 proteins likely act as a humoral immune decoy. Here, by using a series of ORF2 capsid protein mutants expressed in the infectious genotype 3 p6 HEV strain as well as chimeras between ORF2 and the CD4 glycoprotein, we demonstrated how an Arginine-Rich Motif (ARM) located in the ORF2 N-terminal region controls the fate and functions of ORF2 isoforms. We showed that the ARM controls ORF2 nuclear translocation likely to promote regulation of host antiviral responses. This motif also regulates the dual topology and functionality of ORF2 signal peptide, leading to the production of either cytosolic infectious ORF2i or reticular non-infectious glycosylated ORF2 forms. It serves as maturation site of glycosylated ORF2 by furin, and promotes ORF2-host cell membrane interactions. The identification of ORF2 ARM as a unique central regulator of the HEV lifecycle uncovers how viruses settle strategies to condense their genetic information and hijack cellular processes.
Assuntos
Vírus da Hepatite E , Hepatite E , Motivos de Aminoácidos , Proteínas do Capsídeo/química , Proteínas do Capsídeo/metabolismo , Feminino , Glicosilação , Hepatite E/genética , Hepatite E/metabolismo , Vírus da Hepatite E/crescimento & desenvolvimento , Humanos , GravidezRESUMO
Drug repurposing has the advantage of shortening regulatory preclinical development steps. Here, we screened a library of drug compounds, already registered in one or several geographical areas, to identify those exhibiting antiviral activity against SARS-CoV-2 with relevant potency. Of the 1,942 compounds tested, 21 exhibited a substantial antiviral activity in Vero-81 cells. Among them, clofoctol, an antibacterial drug used for the treatment of bacterial respiratory tract infections, was further investigated due to its favorable safety profile and pharmacokinetic properties. Notably, the peak concentration of clofoctol that can be achieved in human lungs is more than 20 times higher than its IC50 measured against SARS-CoV-2 in human pulmonary cells. This compound inhibits SARS-CoV-2 at a post-entry step. Lastly, therapeutic treatment of human ACE2 receptor transgenic mice decreased viral load, reduced inflammatory gene expression and lowered pulmonary pathology. Altogether, these data strongly support clofoctol as a therapeutic candidate for the treatment of COVID-19 patients.
Assuntos
Tratamento Farmacológico da COVID-19 , SARS-CoV-2 , Animais , Antivirais/farmacologia , Clorobenzenos , Chlorocebus aethiops , Cresóis , Humanos , Pulmão , Camundongos , Células VeroRESUMO
A rare but severe complication of curative-intent radiation therapy is the induction of second primary cancers. These cancers preferentially develop not inside the planning target volume (PTV) but around, over several centimeters, after a latency period of 1-40 years. We show here that normal human or mouse dermal fibroblasts submitted to the out-of-field dose scattering at the margin of a PTV receiving a mimicked patient's treatment do not die but enter in a long-lived senescent state resulting from the accumulation of unrepaired DNA single-strand breaks, in the almost absence of double-strand breaks. Importantly, a few of these senescent cells systematically and spontaneously escape from the cell cycle arrest after a while to generate daughter cells harboring mutations and invasive capacities. These findings highlight single-strand break-induced senescence as the mechanism of second primary cancer initiation, with clinically relevant spatiotemporal specificities. Senescence being pharmacologically targetable, they open the avenue for second primary cancer prevention.
Assuntos
Reparo do DNA , Segunda Neoplasia Primária , Animais , Carcinogênese , Transformação Celular Neoplásica , Senescência Celular , Quebras de DNA de Cadeia Simples , Dano ao DNA , CamundongosRESUMO
Mycobacterium tuberculosis is able to colonize, persist, and massively replicate in host cells, such as phagocytes and epithelial cells. The intracellular stage of the bacteria is critical to the development of tuberculosis pathogenesis. The detailed mechanisms of intracellular trafficking of the bacillus are not fully understood and require further investigations. Therefore, increasing the knowledge of this process will help to develop therapeutic tools that will lower the burden of tuberculosis. M. tuberculosis is genetically tractable and tolerates the expression of heterologous fluorescent proteins. Thus, the intracellular distribution of the bacteria expressing fluorescent tracers can be easily defined using confocal microscopy. Advances in imaging techniques and images-based analysis allow the rapid quantification of biological objects in complex environments. In this chapter, we detailed high-content / high-throughput imaging methods to track the bacillus within host cell settings.
Assuntos
Células Dendríticas/microbiologia , Células Epiteliais/microbiologia , Ensaios de Triagem em Larga Escala/métodos , Macrófagos/microbiologia , Mycobacterium tuberculosis/crescimento & desenvolvimento , Fagócitos/microbiologia , Tuberculose/microbiologia , Animais , Células Dendríticas/metabolismo , Testes Diagnósticos de Rotina , Células Epiteliais/metabolismo , Humanos , Macrófagos/metabolismo , Camundongos , Mycobacterium tuberculosis/patogenicidade , Estresse Oxidativo , Fagócitos/metabolismo , Espécies Reativas de Oxigênio , Tuberculose/metabolismoRESUMO
Mycobacterium tuberculosis (M.tb), the etiologic agent of tuberculosis, remains the leading cause of death from a single infectious agent worldwide. The emergence of drug-resistant M.tb strains stresses the need for drugs acting on new targets. Mycolic acids are very long chain fatty acids playing an essential role in the architecture and permeability of the mycobacterial cell wall. Their biosynthesis involves two fatty acid synthase (FAS) systems. Among the four enzymes (MabA, HadAB/BC, InhA and KasA/B) of the FAS-II cycle, MabA (FabG1) remains the only one for which specific inhibitors have not been reported yet. The development of a new LC-MS/MS based enzymatic assay allowed the screening of a 1280 fragment-library and led to the discovery of the first small molecules that inhibit MabA activity. A fragment from the anthranilic acid series was optimized into more potent inhibitors and their binding to MabA was confirmed by 19F ligand-observed NMR experiments.
Assuntos
Proteínas de Bactérias/antagonistas & inibidores , Descoberta de Drogas , Inibidores Enzimáticos/farmacologia , Ácido Graxo Sintases/antagonistas & inibidores , Mycobacterium tuberculosis/enzimologia , ortoaminobenzoatos/farmacologia , Proteínas de Bactérias/metabolismo , Relação Dose-Resposta a Droga , Avaliação Pré-Clínica de Medicamentos , Inibidores Enzimáticos/química , Ácido Graxo Sintases/metabolismo , Estrutura Molecular , Relação Estrutura-Atividade , ortoaminobenzoatos/químicaRESUMO
The imidazo[2,1-b]thiazole-5-carboxamides (ITAs) are a promising class of anti-tuberculosis agents shown to have potent activity in vitro and to target QcrB, a key component of the mycobacterial cytochrome bcc-aa3 super complex critical for the electron transport chain. Herein we report the intracellular macrophage potency of nine diverse ITA analogs with MIC values ranging from 0.0625-2.5 µM and mono-drug resistant potency ranging from 0.0017 to 7 µM. The in vitro ADME properties (protein binding, CaCo-2, human microsomal stability and CYP450 inhibition) were determined for an outstanding compound of the series, ND-11543. ND-11543 was tolerable at >500 mg/kg in mice and at a dose of 200 mg/kg displayed good drug exposure in mice with an AUC(0-24h) >11,700 ng·hr/mL and a >24 hr half-life. Consistent with the phenotype observed with other QcrB inhibitors, compound ND-11543 showed efficacy in a chronic murine TB infection model when dosed at 200 mg/kg for 4 weeks. The efficacy was not dependent upon exposure, as pre-treatment with a known CYP450-inhibitor did not substantially improve efficacy. The ITAs are an interesting scaffold for the development of new anti-TB drugs especially in combination therapy based on their favorable properties and novel mechanism of action.
Assuntos
Antituberculosos/uso terapêutico , Imidazóis/uso terapêutico , Mycobacterium tuberculosis/efeitos dos fármacos , Tiazóis/uso terapêutico , Tuberculose/tratamento farmacológico , Animais , Antituberculosos/química , Antituberculosos/farmacologia , Células CACO-2 , Chlorocebus aethiops , Inibidores das Enzimas do Citocromo P-450/química , Inibidores das Enzimas do Citocromo P-450/farmacologia , Inibidores das Enzimas do Citocromo P-450/uso terapêutico , Humanos , Imidazóis/química , Imidazóis/farmacologia , Camundongos , Testes de Sensibilidade Microbiana , Mycobacterium tuberculosis/crescimento & desenvolvimento , Células RAW 264.7 , Tiazóis/química , Tiazóis/farmacologia , Células VeroRESUMO
Recent emergence of direct-acting antivirals (DAAs) targeting hepatitis C virus (HCV) proteins has considerably enhanced the success of antiviral therapy. However, the appearance of DAA-resistant-associated variants is a cause of treatment failure, and the high cost of DAAs renders the therapy not accessible in countries with inadequate medical infrastructures. Therefore, the search for new inhibitors with a lower cost of production should be pursued. In this context, the crude extract of Juncus maritimus Lam. was shown to exhibit high antiviral activity against HCV in cell culture. Bio-guided fractionation allowed the isolation and identification of the active compound, dehydrojuncusol. A time-of-addition assay showed that dehydrojuncusol significantly inhibited HCV infection when added after virus inoculation of HCV genotype 2a (50% effective concentration [EC50] = 1.35 µM). This antiviral activity was confirmed with an HCV subgenomic replicon, and no effect on HCV pseudoparticle entry was observed. Antiviral activity of dehydrojuncusol was also demonstrated in primary human hepatocytes. No in vitro toxicity was observed at active concentrations. Dehydrojuncusol is also efficient on HCV genotype 3a and can be used in combination with sofosbuvir. Interestingly, dehydrojuncusol was able to inhibit RNA replication of two frequent daclatasvir-resistant mutants (L31M or Y93H in NS5A). Finally, mutants resistant to dehydrojuncusol were obtained and showed that the HCV NS5A protein is the target of the molecule. In conclusion, dehydrojuncusol, a natural compound extracted from J. maritimus, inhibits infection of different HCV genotypes by targeting the NS5A protein and is active against resistant HCV variants frequently found in patients with treatment failure.IMPORTANCE Tens of millions of people are infected with hepatitis C virus (HCV) worldwide. Recently marketed direct-acting antivirals (DAAs) targeting HCV proteins have enhanced the efficacy of treatment. However, due to its high cost, this new therapy is not accessible to the vast majority of infected patients. Furthermore, treatment failures have also been reported due to the appearance of viral resistance. Here, we report on the identification of a new HCV inhibitor, dehydrojuncusol, that targets HCV NS5A and is able to inhibit RNA replication of replicons harboring resistance mutations to anti-NS5A DAAs used in current therapy. Dehydrojuncusol is a natural compound isolated from Juncus maritimus, a halophilic plant species that is very common in coastlines worldwide. This molecule might serve as a lead for the development of a new therapy that is more accessible to hepatitis C patients in the future.
Assuntos
Hepacivirus/efeitos dos fármacos , Fenantrenos/farmacologia , Replicação Viral/efeitos dos fármacos , Antivirais/farmacologia , Linhagem Celular , Farmacorresistência Viral/genética , Genótipo , Células HEK293 , Hepacivirus/genética , Hepatite C/genética , Hepatite C/virologia , Hepatite C Crônica/virologia , Hepatócitos/virologia , Humanos , Fenantrenos/metabolismo , Fenetilaminas/farmacologia , Extratos Vegetais/farmacologia , Inibidores de Proteases/farmacologia , Replicon/efeitos dos fármacos , RizomaRESUMO
The treatment of hepatitis C virus (HCV) infection by combination of direct acting antivirals (DAA), with different mode of action, has made substantial progress in the past few years. However, appearance of resistance and high cost of the therapy is still an obstacle in the achievement of the therapy, more specifically in developing countries. In this context, search for affordable antivirals with new mechanisms of action is still needed. Tea, after water, is the most popular drink worldwide. Polyphenols extracted from green tea have already shown anti-HCV activity as entry inhibitors. Here, three different theaflavins, theaflavin (TF1), theaflavin-3'-monogallate (TF2), and theaflavin-3-3'-digallate (TF3), which are major polyphenols from black tea, were tested against HCV in cell culture. The results showed that all theaflavins inhibit HCV infection in a dose-dependent manner in an early step of infection. Results obtained with HCV pseudotyped virions confirmed their activity on HCV entry and demonstrated their pan-genotypic action. No effect on HCV replication was observed by using HCV replicon. Investigation on the mechanism of action of black tea theaflavins showed that they act directly on the virus particle and are able to inhibit cell-to-cell spread. Combination study with inhibitors most widely used in anti-HCV treatment regimen demonstrated that TF3 exerts additive effect. In conclusion, theaflavins, that are present in high quantity in black tea, are new inhibitors of HCV entry and hold promise for developing in therapeutic arsenal for HCV infection.
Assuntos
Antioxidantes/farmacologia , Antivirais/farmacologia , Biflavonoides/farmacologia , Catequina/farmacologia , Hepacivirus/efeitos dos fármacos , Fígado/virologia , Polifenóis/farmacologia , Chá , Camellia sinensis , Linhagem Celular Tumoral , Relação Dose-Resposta a Droga , Humanos , Fígado/efeitos dos fármacosRESUMO
The search for compounds with biological activity for many diseases is turning increasingly to drug repurposing. In this study, we have focused on the European Union-approved antimalarial pyronaridine which was found to have in vitro activity against Mycobacterium tuberculosis (MIC 5⯵g/mL). In macromolecular synthesis assays, pyronaridine resulted in a severe decrease in incorporation of 14C-uracil and 14C-leucine similar to the effect of rifampicin, a known inhibitor of M. tuberculosis RNA polymerase. Surprisingly, the co-administration of pyronaridine (2.5⯵g/ml) and rifampicin resulted in in vitro synergy with an MIC 0.0019-0.0009⯵g/mL. This was mirrored in a THP-1 macrophage infection model, with a 16-fold MIC reduction for rifampicin when the two compounds were co-administered versus rifampicin alone. Docking pyronaridine in M. tuberculosis RNA polymerase suggested the potential for it to bind outside of the RNA polymerase rifampicin binding pocket. Pyronaridine was also found to have activity against a M. tuberculosis clinical isolate resistant to rifampicin, and when combined with rifampicin (10% MIC) was able to inhibit M. tuberculosis RNA polymerase in vitro. All these findings, and in particular the synergistic behavior with the antitubercular rifampicin, inhibition of RNA polymerase in combination in vitro and its current use as a treatment for malaria, may suggest that pyronaridine could also be used as an adjunct for treatment against M. tuberculosis infection. Future studies will test potential for in vivo synergy, clinical utility and attempt to develop pyronaridine analogs with improved potency against M. tuberculosis RNA polymerase when combined with rifampicin.
Assuntos
Antibióticos Antituberculose/farmacologia , Antimaláricos/farmacologia , Antituberculosos/farmacologia , Proteínas de Bactérias/antagonistas & inibidores , RNA Polimerases Dirigidas por DNA/antagonistas & inibidores , Mycobacterium tuberculosis/efeitos dos fármacos , Naftiridinas/farmacologia , Rifampina/farmacologia , Antimaláricos/química , Antituberculosos/química , Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Sítios de Ligação , RNA Polimerases Dirigidas por DNA/química , RNA Polimerases Dirigidas por DNA/metabolismo , Reposicionamento de Medicamentos , Farmacorresistência Bacteriana , Sinergismo Farmacológico , Quimioterapia Combinada , Humanos , Testes de Sensibilidade Microbiana , Simulação de Acoplamento Molecular , Mycobacterium tuberculosis/enzimologia , Mycobacterium tuberculosis/crescimento & desenvolvimento , Naftiridinas/química , Ligação Proteica , Conformação Proteica , Relação Estrutura-Atividade , Células THP-1RESUMO
Hepatitis C virus (HCV) infection causes 500,000 deaths annually, in association with end-stage liver diseases. Investigations of the HCV life cycle have widened the knowledge of virology, and here we discovered that two piperazinylbenzenesulfonamides inhibit HCV entry into liver cells. The entry of HCV into host cells is a complex process that is not fully understood but is characterized by multiple spatially and temporally regulated steps involving several known host factors. Through a high-content virus infection screening analysis with a library of 1,120 biologically active chemical compounds, we identified SB258585, an antagonist of serotonin receptor 6 (5-HT6), as a new inhibitor of HCV entry in liver-derived cell lines as well as primary hepatocytes. A functional characterization suggested a role for this compound and the compound SB399885, which share similar structures, as inhibitors of a late HCV entry step, modulating the localization of the coreceptor tight junction protein claudin-1 (CLDN1) in a 5-HT6-independent manner. Both chemical compounds induced an intracellular accumulation of CLDN1, reflecting export impairment. This regulation correlated with the modulation of protein kinase A (PKA) activity. The PKA inhibitor H89 fully reproduced these phenotypes. Furthermore, PKA activation resulted in increased CLDN1 accumulation at the cell surface. Interestingly, an increase of CLDN1 recycling did not correlate with an increased interaction with CD81 or HCV entry. These findings reinforce the hypothesis of a common pathway, shared by several viruses, which involves G-protein-coupled receptor-dependent signaling in late steps of viral entry.IMPORTANCE The HCV entry process is highly complex, and important details of this structured event are poorly understood. By screening a library of biologically active chemical compounds, we identified two piperazinylbenzenesulfonamides as inhibitors of HCV entry. The mechanism of inhibition was not through the previously described activity of these inhibitors as antagonists of serotonin receptor 6 but instead through modulation of PKA activity in a 5-HT6-independent manner, as proven by the lack of 5-HT6 in the liver. We thus highlighted the involvement of the PKA pathway in modulating HCV entry at a postbinding step and in the recycling of the tight junction protein claudin-1 (CLDN1) toward the cell surface. Our work underscores once more the complexity of HCV entry steps and suggests a role for the PKA pathway as a regulator of CLDN1 recycling, with impacts on both cell biology and virology.
Assuntos
Claudina-1/metabolismo , Piperazinas/farmacologia , Inibidores de Proteínas Quinases/farmacologia , Transporte Proteico/efeitos dos fármacos , Receptores de Serotonina/metabolismo , Sulfonamidas/farmacologia , Internalização do Vírus/efeitos dos fármacos , Linhagem Celular Tumoral , Proteínas Quinases Dependentes de AMP Cíclico/antagonistas & inibidores , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Hepacivirus/fisiologia , Hepatócitos/virologia , Humanos , Isoquinolinas/farmacologia , Receptores Acoplados a Proteínas G/metabolismo , Tetraspanina 28/metabolismo , Junções Íntimas/metabolismoRESUMO
Mycolactone, a polyketide molecule produced by Mycobacterium ulcerans, is the etiological agent of Buruli ulcer. This lipid toxin is endowed with pleiotropic effects, presents cytotoxic effects at high doses, and notably plays a pivotal role in host response upon colonization by the bacillus. Most remarkably, mycolactone displays intriguing analgesic capabilities: the toxin suppresses or alleviates the pain of the skin lesions it inflicts. We demonstrated that the analgesic capability of mycolactone was not attributable to nerve damage, but instead resulted from the triggering of a cellular pathway targeting AT2 receptors (angiotensin II type 2 receptors; AT2R), and leading to potassium-dependent hyperpolarization. This demonstration paves the way to new nature-inspired analgesic protocols. In this direction, we assess here the hyperpolarizing properties of mycolactone on nociceptive neurons. We developed a dedicated medium-throughput assay based on membrane potential changes, and visualized by confocal microscopy of bis-oxonol-loaded Dorsal Root Ganglion (DRG) neurons. We demonstrate that mycolactone at non-cytotoxic doses triggers the hyperpolarization of DRG neurons through AT2R, with this action being not affected by known ligands of AT2R. This result points towards novel AT2R-dependent signaling pathways in DRG neurons underlying the analgesic effect of mycolactone, with the perspective for the development of new types of nature-inspired analgesics.
Assuntos
Analgésicos/farmacologia , Toxinas Bacterianas/farmacologia , Macrolídeos/farmacologia , Neurônios/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Gânglios Espinais/citologia , Potenciais da Membrana/efeitos dos fármacos , Neurônios/metabolismo , Neurônios/fisiologia , Receptor Tipo 2 de Angiotensina/metabolismoRESUMO
Genome-wide association studies (GWASs) have identified 19 susceptibility loci for Alzheimer's disease (AD). However, understanding how these genes are involved in the pathophysiology of AD is one of the main challenges of the "post-GWAS" era. At least 123 genes are located within the 19 susceptibility loci; hence, a conventional approach (studying the genes one by one) would not be time- and cost-effective. We therefore developed a genome-wide, high-content siRNA screening approach and used it to assess the functional impact of gene under-expression on APP metabolism. We found that 832 genes modulated APP metabolism. Eight of these genes were located within AD susceptibility loci. Only FERMT2 (a ß3-integrin co-activator) was also significantly associated with a variation in cerebrospinal fluid Aß peptide levels in 2886 AD cases. Lastly, we showed that the under-expression of FERMT2 increases Aß peptide production by raising levels of mature APP at the cell surface and facilitating its recycling. Taken as a whole, our data suggest that FERMT2 modulates the AD risk by regulating APP metabolism and Aß peptide production.