RESUMO
Glycogen-synthase kinase 3 (GSK3) is a kinase mediating phosphorylation on serine and threonine amino acid residues of several target molecules. The enzyme is involved in the regulation of many cellular processes and aberrant activity of GSK3 has been linked to several disease conditions such as fragile X syndrome (FXS). Recent evidences demonstrating an increased activity of GSK3 in murine models of FXS, suggest that dysregulation/hyperactivation of the GSK3 path should contribute to FXS development. A likely possibility could be that in FXS there is a functional impairment of the upstream inhibitory input over GSK3 thus making overactive the kinase. Since GSK3 signaling is a central regulatory node for critical neurodevelopmental pathways, understanding the contribution of GSK3 dysregulation to FXS, may provide novel targets for therapeutic interventions for this disease. In this study we used AF3581, a potent GSK3 inhibitor that we recently discovered, in an in vivo FXS mouse model to elucidate the crucial role of GSK3 in specific behavioral patterns (locomotor activity, sensorimotor gating and social behavior) associated with this disease. All the behavioral alterations manifested by Fmr1 knockout mice were reverted after a chronic treatment with our GSK3 inhibitor, confirming the importance of this pathway as a therapeutic target.
Assuntos
Síndrome do Cromossomo X Frágil , Animais , Modelos Animais de Doenças , Proteína do X Frágil da Deficiência Intelectual/genética , Proteína do X Frágil da Deficiência Intelectual/metabolismo , Síndrome do Cromossomo X Frágil/tratamento farmacológico , Síndrome do Cromossomo X Frágil/genética , Quinase 3 da Glicogênio Sintase/genética , Quinase 3 da Glicogênio Sintase/metabolismo , Camundongos , Camundongos Knockout , FenótipoRESUMO
A pharmacokinetic/pharmacodynamic (PK/PD) model was developed to describe the time course of writhings after intraperitoneal injection of acetic acid in mice. The model was applied to investigate the antinociceptive effect of trazodone and gabapentin alone and in combination. Writhings time course was described by a transit compartment model with the delay due to the transit of the acetic acid being represented by a chain of intermediate compartments. In the drug-treated animals, the number of writhings decreases according to a k2 factor linking drug concentration and antinociceptive effect. Compounds' potency parameters were 10.9 and 0.0459 L/µmoles/min for trazodone and gabapentin, respectively, indicating a much higher in vivo potency of trazodone in the PD writhing test. The PK/PD parameters were used to simulate the expected writhing counts in mice at combined doses without efficacy alone, assuming pharmacological additivity. Simulation results indicated that, at low dose combinations, experimental data were mostly below the simulated writhings median, suggesting possible synergic effect. Such hypothesis was tested by adding the γ parameter in the PK/PD model to represent the deviation from the assumption of no-interaction, leading to a reduction of the objective function compared to the additive model. On this basis, several simulations were performed to identify possible starting dose combinations of trazodone and gabapentin in humans, by selecting doses yielding systemic exposures close to those being synergic in the mouse. Simulations indicated that doses of 50-100 mg trazodone could enhance gabapentin antinociceptive effect in humans, supporting the development of a low dose combination for optimal analgesia treatment.
Assuntos
Neuralgia , Trazodona , Humanos , Camundongos , Animais , Gabapentina , Trazodona/farmacologia , Roedores , Analgésicos/farmacologia , Neuralgia/tratamento farmacológico , Ácido Acético , Modelos BiológicosRESUMO
Here we report an in vitro model system for studying the molecular and cellular mechanisms that underlie the neurodegenerative disease amyotrophic lateral sclerosis (ALS). Embryonic stem cells (ESCs) derived from mice carrying normal or mutant transgenic alleles of the human SOD1 gene were used to generate motor neurons by in vitro differentiation. These motor neurons could be maintained in long-term coculture either with additional cells that arose during differentiation or with primary glial cells. Motor neurons carrying either the nonpathological human SOD1 transgene or the mutant SOD1(G93A) allele showed neurodegenerative properties when cocultured with SOD1(G93A) glial cells. Thus, our studies demonstrate that glial cells carrying a human SOD1(G93A) mutation have a direct, non-cell autonomous effect on motor neuron survival. More generally, our results show that ESC-based models of disease provide a powerful tool for studying the mechanisms of neural degeneration. These phenotypes displayed in culture could provide cell-based assays for the identification of new ALS drugs.
Assuntos
Esclerose Lateral Amiotrófica/patologia , Neurônios Motores/fisiologia , Neuroglia/fisiologia , Células-Tronco/fisiologia , Análise de Variância , Animais , Diferenciação Celular/fisiologia , Sobrevivência Celular/fisiologia , Células Cultivadas , Técnicas de Cocultura/métodos , Modelos Animais de Doenças , Embrião de Mamíferos , Citometria de Fluxo/métodos , Proteínas de Fluorescência Verde/genética , Humanos , Camundongos , Camundongos Transgênicos , Proteínas do Tecido Nervoso/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa/métodos , Superóxido Dismutase/genética , Fatores de TempoRESUMO
Glycogen synthase kinase 3 (GSK3) is a proline-directed serine-threonine kinase that is associated with several neurological disorders, including Alzheimer's disease and fragile X syndrome (FXS). We tested the efficacy of a novel GSK3 inhibitor AFC03127, which was developed by Angelini Pharma, in comparison to the metabotropic glutamate receptor 5 inhibitor 2-Methyl-6-(phenylethynyl)pyridine hydrochloride (MPEP) and the GSK3 inhibitor SB216763 in in vivo and in vitro assays in Fmr1 KO mice, a mouse model useful for the study of FXS. The in vivo assay tested susceptibility to audiogenic-induced seizures (AGS) whereas the in vitro assays assessed biomarker expression and dendritic spine length and density in cultured primary neurons as a function of drug dose. MPEP and SB216763 attenuated AGS in Fmr1 KO mice, whereas AFC03127 did not. MPEP and AFC03127 significantly reduced dendritic expression of amyloid-beta protein precursor (APP). All drugs rescued spine length and the ratio of mature dendritic spines. Spine density was not statistically different between vehicle and GSK3 inhibitor-treated cells. The drugs were tested over a wide concentration range in the in vitro assays to determine dose responses. A bell-shaped dose response decrease in APP expression was observed in response to AFC03127, which was more effective than SB216763. These findings confirm previous studies demonstrating differential effects of various GSK3 inhibitors on AGS propensity in Fmr1 KO mice and confirm APP as a downstream biomarker that is responsive to GSK3 activity.
RESUMO
Neuropathic pain is a chronic debilitating condition caused by injury or disease of the nerves of the somatosensory system. Although several therapeutic approaches are recommended, none has emerged as an optimal treatment leaving a need for developing more effective therapies. Given the small number of approved drugs and their limited clinical efficacy, combining drugs with different mechanisms of action is frequently used to yield greater efficacy. We demonstrate that the combination of trazodone, a multifunctional drug for the treatment of major depressive disorders, and gabapentin, a GABA analogue approved for neuropathic pain relief, results in a synergistic antinociceptive effect in the mice writhing test. To explore the potential relevance of this finding in chronic neuropathic pain, pharmacodynamic interactions between low doses of trazodone (0.3 mg/kg) and gabapentin (3 mg/kg) were evaluated in the chronic constriction injury (CCI) rat model, measuring the effects of the two drugs both on evoked and spontaneous nociception and on general well being components. Two innate behaviors, burrowing and nest building, were used to assess these aspects. Besides exerting a significant antinociceptive effect on hyperalgesia and on spontaneous pain, combined inactive doses of trazodone and gabapentin restored in CCI rats innate behaviors that are strongly reduced or even abolished during persistent nociception, suggesting that the combination may have an impact also on pain components different from somatosensory perception. Our results support the development of a trazodone and gabapentin low doses combination product for optimal multimodal analgesia treatment.
Assuntos
Analgésicos/uso terapêutico , Ansiolíticos/uso terapêutico , Gabapentina/uso terapêutico , Neuralgia/tratamento farmacológico , Trazodona/uso terapêutico , Analgésicos/farmacologia , Animais , Ansiolíticos/farmacologia , Transtorno Depressivo Maior/tratamento farmacológico , Modelos Animais de Doenças , Sinergismo Farmacológico , Gabapentina/farmacologia , Masculino , Camundongos , Nociceptividade/efeitos dos fármacos , Trazodona/farmacologiaRESUMO
The major cause of bacterial resistance to ß-lactams is the production of hydrolytic ß-lactamase enzymes. Nowadays, the combination of ß-lactam antibiotics with ß-lactamase inhibitors (BLIs) is the main strategy for overcoming such issues. Nevertheless, particularly challenging ß-lactamases, such as OXA-48, pose the need for novel and effective treatments. Herein, we describe the screening of a proprietary compound collection against Klebsiella pneumoniae OXA-48, leading to the identification of several chemotypes, like the 4-ideneamino-4H-1,2,4-triazole (SC_2) and pyrazolo[3,4-b]pyridine (SC_7) cores as potential inhibitors. Importantly, the most potent representative of the latter series (ID2, AC50 = 0.99 µM) inhibited OXA-48 via a reversible and competitive mechanism of action, as demonstrated by biochemical and X-ray studies; furthermore, it slightly improved imipenem's activity in Escherichia coli ATCC BAA-2523 ß-lactam resistant strain. Also, ID2 showed good solubility and no sign of toxicity up to the highest tested concentration, resulting in a promising starting point for further optimization programs toward novel and effective non-ß-lactam BLIs.
RESUMO
We investigated whether chronic sciatic ligation modifies the glutamate release in spinal cord nerve endings (synaptosomes) as well as the expression and the function of presynaptic release-regulating mGlu2/3 autoreceptors and 5-HT2A heteroreceptors in these particles. Synaptosomes were from the spinal cord of animals suffering from the sciatic ligation that developed on day 6 post-surgery a significant decrease of the force inducing paw-withdrawal in the lesioned paw. The exocytosis of glutamate (quantified as release of preloaded [3H]D-aspartate, [3H]D-Asp) elicited by a mild depolarizing stimulus (15 mM KCl) was significantly increased in synaptosomes from injured rats when compared to controls (uninjured rats). The mGlu2/3 agonist LY379268 (1000 pM) significantly inhibited the 15 mM KCl-evoked [3H]D-Asp overflow from control synaptosomes, but not in terminals isolated from injured animals. Differently, a low concentration (10 nM) of (±) DOI, unable to modify the 15 mM KCl-evoked [3H]D-Asp overflow in control spinal cord synaptosomes, significantly reduced the glutamate exocytosis in nerve endings isolated from the injured rats. Acute oral trazodone (TZD, 0.3 mg/kg on day 7 post-surgery) efficiently recovered glutamate exocytosis as well as the efficiency of LY379268 in inhibiting this event in spinal cord synaptosomes from injured animals. The sciatic ligation significantly reduced the expression of mGlu2/3, but not of 5-HT2A, receptor proteins in spinal cord synaptosomal lysates. Acute TZD recovered this parameter. Our results support the use of 5-HT2A antagonists for restoring altered spinal cord glutamate plasticity in rats suffering from sciatic ligation.
RESUMO
Staphylococcus aureus and Staphylococcus epidermidis are leading pathogens of biofilm-related infections and represent the most common cause of osteomyelitis and biomedical implants infections. Biofilm-related infections usually require long-term antibiotic treatment, often associated to surgical interventions. Dalbavancin is a newer lipoglycopeptide approved for the treatment of acute skin and skin-structure infections caused by Gram-positive pathogens. In addition, dalbavancin has recently been considered as a potential option for the treatment of staphylococcal osteomyelitis and orthopedic implant infections. In this study, time-kill kinetics of dalbavancin against S. aureus and S. epidermidis biofilms were determined over prolonged exposure times (up to 7â¯days), using both a standardized biofilm susceptibility model and biofilms grown onto relevant orthopedic biomaterials (i.e. titanium and cobalt-chrome disks). Dalbavancin (at concentrations achievable in bone and articular tissue) showed a potent activity against established staphylococcal biofilms in both tested models, and was overall superior to the comparator vancomycin.
Assuntos
Antibacterianos/farmacologia , Biofilmes/efeitos dos fármacos , Staphylococcus/efeitos dos fármacos , Staphylococcus/crescimento & desenvolvimento , Teicoplanina/análogos & derivados , Humanos , Cinética , Testes de Sensibilidade Microbiana/métodos , Infecções Estafilocócicas/tratamento farmacológico , Infecções Estafilocócicas/microbiologia , Teicoplanina/farmacologia , Fatores de Tempo , Vancomicina/farmacologiaRESUMO
In this study, a drug discovery programme that sought to identify novel dual bacterial topoisomerase II inhibitors (NBTIs) led to the selection of six optimized compounds. In enzymatic assays, the molecules showed equivalent dual-targeting activity against the DNA gyrase and topoisomerase IV enzymes of Staphylococcus aureus and Escherichia coli. Consistently, the compounds demonstrated potent activity in susceptibility tests against various Gram-positive and Gram-negative reference species, including ciprofloxacin-resistant strains. The activity of the compounds against clinical multidrug-resistant isolates of S. aureus, Clostridium difficile, Acinetobacter baumannii, Neisseria gonorrhoeae, E. coli and vancomycin-resistant Enterococcus spp. was also confirmed. Two compounds (1 and 2) were tested in time-kill and post-antibiotic effect (PAE) assays. Compound 1 was bactericidal against all tested reference strains and showed higher activity than ciprofloxacin, and compound 2 showed a prolonged PAE, even against the ciprofloxacin-resistant S. aureus BAA-1720 strain. Spontaneous development of resistance to both compounds was selected for in S. aureus at frequencies comparable to those obtained for quinolones and other NBTIs. S. aureus BAA-1720 mutants resistant to compounds 1 and 2 had single point mutations in gyrA or gyrB outside of the quinolone resistance-determining region (QRDR), confirming the distinct site of action of these NBTIs compared to that of quinolones. Overall, the very good antibacterial activity of the compounds and their optimizable in vitro safety and physicochemical profile may have relevant implications for the development of new broad-spectrum antibiotics.
Assuntos
Antibacterianos/farmacologia , DNA Girase/efeitos dos fármacos , DNA Topoisomerases Tipo II/efeitos dos fármacos , Inibidores da Topoisomerase II/farmacologia , Animais , Células CHO , Ciprofloxacina/farmacologia , Cricetulus , DNA Topoisomerases Tipo II/metabolismo , DNA Bacteriano/efeitos dos fármacos , Farmacorresistência Bacteriana/efeitos dos fármacos , Escherichia coli/efeitos dos fármacos , Células Hep G2 , Humanos , Staphylococcus aureus Resistente à Meticilina/efeitos dos fármacos , Testes de Sensibilidade Microbiana , Staphylococcus aureus/efeitos dos fármacos , Testes de ToxicidadeRESUMO
Bipolar disorders still represent a global unmet medical need and pose a requirement for novel effective treatments. In this respect, glycogen synthase kinase 3ß (GSK-3ß) aberrant activity has been linked to the pathophysiology of several disease conditions, including mood disorders. Therefore, the development of GSK-3ß inhibitors with good in vivo efficacy and safety profile associated with high brain exposure is required. Accordingly, we have previously reported the selective indazole-based GSK-3 inhibitor 1, which showed excellent efficacy in a mouse model of mania. Despite the favorable preclinical profile, analog 1 suffered from activity at the hERG ion channel, which prevented its further progression. Herein, we describe our strategy to improve this off-target liability through modulation of physicochemical properties, such as lipophilicity and basicity. These efforts led to the potent inhibitor 14, which possessed reduced hERG affinity, promising in vitro ADME properties, and was very effective in a mood stabilizer in vivo model.
RESUMO
The demonstration that the small synthetic molecule reversine [2-(4-morpholinoanilino)-N6-cyclohexyladenine] promotes the dedifferentiation of committed cells into multipotent progenitor-type cells has raised hopes on the exploitation of this small chemical tool for the generation of stem cells. Here, we show that reversine causes a failure in cytokinesis and induces polyploidization. These effects of reversine are due to the inhibition of Aurora A and B, two related kinases that are implicated in several aspects of mitosis and that are frequently amplified and overexpressed in human tumors. Reversine inhibits the phosphorylation of histone H3, a direct downstream target of Aurora kinases. Similarly to the Aurora kinase inhibitor VX-680, which has recently entered phase II clinical trials for cancer treatment, reversine inhibited colony formation of leukemic cells from patients with acute myeloid leukemia but was significantly less toxic than VX-680 on cells from healthy donors. The crystal structure of the reversine-Aurora B kinase complex shows that reversine is a novel class of ATP-competitive Aurora kinase inhibitors. Thus, although our studies raise serious doubts on the application of reversine in regenerative medicine, they support the paradigm that reversine might be a useful agent in cancer chemotherapy.
Assuntos
Antineoplásicos/farmacologia , Leucemia Mieloide Aguda/enzimologia , Morfolinas/farmacologia , Inibidores de Proteínas Quinases/farmacologia , Proteínas Serina-Treonina Quinases/antagonistas & inibidores , Purinas/farmacologia , Aurora Quinase B , Aurora Quinases , Sítios de Ligação/efeitos dos fármacos , Diferenciação Celular , Linhagem Celular Tumoral , Proliferação de Células , Relação Dose-Resposta a Droga , Células HeLa , Histonas/metabolismo , Humanos , Leucemia Mieloide Aguda/tratamento farmacológico , Modelos Moleculares , Morfolinas/química , Morfolinas/metabolismo , Fosforilação , Poliploidia , Proteínas Serina-Treonina Quinases/química , Proteínas Serina-Treonina Quinases/metabolismo , Purinas/química , Purinas/metabolismoRESUMO
Bacterial resistance is increasing rapidly, requiring urgent identification of new antibacterial drugs that are effective against multidrug-resistant pathogens. Novel bacterial topoisomerase inhibitors (NBTIs) provide a new strategy for investigating the well-validated DNA gyrase and topoisomerase IV targets while preventing cross-resistance issues. On this basis, starting from a virtual screening campaign and subsequent structure-based hit optimization guided by X-ray studies, a novel class of piperazine-like NBTIs with outstanding enzymatic activity against Staphylococcus aureus and Escherichia coli DNA gyrase and topoisomerase IV was identified. Notably, compounds (±)-33, (±)-35, and (±)-36 with potent and balanced multitarget enzymatic profiles exhibited excellent efficacy against selected Gram-positive and Gram-negative pathogens, as well as clinically relevant resistant strains. Overall, the new NBTI chemotype described herein, owing to the broad-spectrum antibacterial activity and favorable in vitro safety profile, might serve as a basis for the development of novel treatments against serious infections.
Assuntos
DNA Topoisomerase IV/antagonistas & inibidores , Escherichia coli/efeitos dos fármacos , Staphylococcus aureus/efeitos dos fármacos , Inibidores da Topoisomerase/farmacologia , Sequência de Aminoácidos , Infecções Bacterianas/microbiologia , Infecções Bacterianas/prevenção & controle , DNA Girase/genética , DNA Girase/metabolismo , DNA Topoisomerase IV/genética , DNA Topoisomerase IV/metabolismo , Escherichia coli/enzimologia , Escherichia coli/genética , Testes de Sensibilidade Microbiana , Modelos Químicos , Estrutura Molecular , Homologia de Sequência de Aminoácidos , Staphylococcus aureus/enzimologia , Staphylococcus aureus/genética , Relação Estrutura-Atividade , Inibidores da Topoisomerase/síntese química , Inibidores da Topoisomerase/químicaRESUMO
The drug/proton antiporter AcrB, which is part of the major efflux pump AcrABZ-TolC in Escherichia coli, is the paradigm transporter of the resistance-nodulation-cell division (RND) superfamily. Despite the impressive ability of AcrB to transport many chemically unrelated compounds, only a few of these ligands have been co-crystallized with the protein. Therefore, the molecular features that distinguish good substrates of the pump from poor ones have remained poorly understood to date. In this work, a thorough in silico protocol was employed to study the interactions of a series of congeneric compounds with AcrB to examine how subtle chemical differences affect the recognition and transport of substrates by this protein. Our analysis allowed us to discriminate among different compounds, mainly in terms of specific interactions with diverse sub-sites within the large distal pocket of AcrB. Our findings could provide valuable information for the design of new antibiotics that can evade the antimicrobial resistance mediated by efflux pump machinery.
Assuntos
Proteínas de Escherichia coli/metabolismo , Proteínas Associadas à Resistência a Múltiplos Medicamentos/metabolismo , Interações Hidrofóbicas e Hidrofílicas , Testes de Sensibilidade Microbiana , Simulação de Dinâmica Molecular , Especificidade por SubstratoRESUMO
Presynaptic mGlu2/3 autoreceptors exist in rat spinal cord nerve terminals as suggested by the finding that LY379268 inhibited the 15â¯mM KCl-evoked release of [3H]D-aspartate ([3H]D-Asp) in a LY341495-sensitive manner. Spinal cord glutamatergic nerve terminals also possess presynaptic release-regulating 5-HT2A heteroreceptors. Actually, the 15â¯mM KCl-evoked [3H]D-Asp exocytosis from spinal cord synaptosomes was reduced by the 5-HT2A agonist (±)DOI, an effect reversed by the 5-HT2A antagonists MDL11,939, MDL100907, ketanserin and trazodone (TZD). We investigated whether mGlu2/3 and 5-HT2A receptors colocalize and cross-talk in these terminals and if 5-HT2A ligands modulate the mGlu2/3-mediated control of glutamate exocytosis. Western blot analysis and confocal microscopy highlighted the presence of mGlu2/3 and 5-HT2A receptor proteins in spinal cord VGLUT1 positive synaptosomes, where mGlu2/3 and 5-HT2A receptor immunoreactivities largely colocalize. Furthermore, mGlu2/3 immunoprecipitates from spinal cord synaptosomes were also 5-HT2A immunopositive. Interestingly, the 100 pM LY379268-induced reduction of the 15â¯mM KCl-evoked [3H]D-Asp overflow as well as its inhibition by 100â¯nM (±)DOI became undetectable when the two agonists were concomitantly added. Conversely, 5-HT2A antagonists (MDL11,939, MDL100907, ketanserin and TZD) reinforced the release-regulating activity of mGlu2/3 autoreceptors. Increased expression of mGlu2/3 receptor proteins in synaptosomal plasmamembranes paralleled the gain of function of the mGlu2/3 autoreceptors elicited by 5-HT2A antagonists. Based on these results, we propose that in spinal cord glutamatergic terminals i) mGlu2/3 and 5-HT2A receptors colocalize and interact one each other in an antagonist-like manner, ii) 5-HT2A antagonists are indirect positive allosteric modulator of mGlu2/3 autoreceptors controlling glutamate exocytosis.
Assuntos
Exocitose/fisiologia , Ácido Glutâmico/metabolismo , Terminações Nervosas/metabolismo , Receptor 5-HT2A de Serotonina/metabolismo , Receptores de Glutamato Metabotrópico/metabolismo , Transdução de Sinais/fisiologia , Medula Espinal/ultraestrutura , Animais , Biotinilação , Córtex Cerebral/metabolismo , Relação Dose-Resposta a Droga , Fármacos Atuantes sobre Aminoácidos Excitatórios/farmacologia , Exocitose/efeitos dos fármacos , Feminino , Ácido Glutâmico/farmacologia , Imunoprecipitação , Masculino , Microscopia Confocal , Terminações Nervosas/efeitos dos fármacos , Ratos , Serotoninérgicos/farmacologia , Transdução de Sinais/efeitos dos fármacos , Estatísticas não Paramétricas , Sinaptossomos/efeitos dos fármacos , Sinaptossomos/metabolismo , Proteína Vesicular 1 de Transporte de Glutamato/metabolismoRESUMO
In the version of this article initially published, the catalog numbers for BoNT A and B were given in the Methods section as T0195 and T5644; the correct numbers are B8776 and B6403. The error has been corrected in the HTML and PDF versions of the article.
RESUMO
In fragile X syndrome (FXS), CGG repeat expansion greater than 200 triplets is believed to trigger FMR1 gene silencing and disease etiology. However, FXS siblings have been identified with more than 200 CGGs, termed unmethylated full mutation (UFM) carriers, without gene silencing and disease symptoms. Here, we show that hypomethylation of the FMR1 promoter is maintained in induced pluripotent stem cells (iPSCs) derived from two UFM individuals. However, a subset of iPSC clones with large CGG expansions carries silenced FMR1. Furthermore, we demonstrate de novo silencing upon expansion of the CGG repeat size. FMR1 does not undergo silencing during neuronal differentiation of UFM iPSCs, and expression of large unmethylated CGG repeats has phenotypic consequences resulting in neurodegenerative features. Our data suggest that UFM individuals do not lack the cell-intrinsic ability to silence FMR1 and that inter-individual variability in the CGG repeat size required for silencing exists in the FXS population.
Assuntos
Metilação de DNA/genética , Proteína do X Frágil da Deficiência Intelectual/genética , Inativação Gênica , Células-Tronco Pluripotentes Induzidas/metabolismo , Mutação/genética , Neurônios/metabolismo , Expansão das Repetições de Trinucleotídeos/genética , Diferenciação Celular/genética , Células Clonais , Epigênese Genética , Feminino , Síndrome do Cromossomo X Frágil/genética , Loci Gênicos , Humanos , Células-Tronco Pluripotentes Induzidas/citologia , Masculino , LinhagemRESUMO
Inositide-specific phospholipase C (PLC) signaling constitutes a central intermediate in a number of cellular functions among which the control of cell growth raises a particular interest. Indeed, we have previously shown that nuclear phospholipase C beta1 (PLC beta1) is central for the regulation of mitogen-induced cell growth. We have also assigned by fluorescence in situ hybridization (FISH) analysis the PLC beta1 to human chromosome 20p12. In this study, we have carried out a detailed analysis of the human gene, showing the existence of alternative splicing, which gives rise, besides the two forms (1a and 1b) already shown in rodents, to a new 600 bp smaller form coding for a 110 kDa protein. We have also identified a new exon at the 5', showing no homology with the rodent sequence. Here we provide the complete determination of the exon/intron structure of the gene spanning 250 kb of DNA. We found that the exons are quite small, ranging from 49 to 222 bp, while the introns vary between 108 bp and 34,400 bp. The availability of the understanding of the genome organization of this inositide-specific PLC, which represents a key step of the cell cycle related signaling, could actually pave the way for further genetic analysis of p12 region of human chromosome 20 in diseases involving alterations of the control of cell growth such as malignancies.
Assuntos
Isoenzimas/genética , Fosfolipases Tipo C/genética , Regiões 3' não Traduzidas , Regiões 5' não Traduzidas , Adulto , Processamento Alternativo , Sequência de Bases , Northern Blotting , Western Blotting , Clonagem Molecular , DNA Complementar/biossíntese , DNA Complementar/química , Feto , Amplificação de Genes , Humanos , Isoenzimas/química , Dados de Sequência Molecular , Fosfolipase C beta , Regiões Promotoras Genéticas , Transfecção , Fosfolipases Tipo C/químicaRESUMO
The availability of human pluripotent stem cells (hPSCs) offers the opportunity to generate lineage-specific cells to investigate mechanisms of human diseases specific to brain regions. Here, we report a differentiation paradigm for hPSCs that enriches for hippocampal dentate gyrus (DG) granule neurons. This differentiation paradigm recapitulates the expression patterns of key developmental genes during hippocampal neurogenesis, exhibits characteristics of neuronal network maturation, and produces PROX1+ neurons that functionally integrate into the DG. Because hippocampal neurogenesis has been implicated in schizophrenia (SCZD), we applied our protocol to SCZD patient-derived human induced pluripotent stem cells (hiPSCs). We found deficits in the generation of DG granule neurons from SCZD hiPSC-derived hippocampal NPCs with lowered levels of NEUROD1, PROX1, and TBR1, reduced neuronal activity, and reduced levels of spontaneous neurotransmitter release. Our approach offers important insights into the neurodevelopmental aspects of SCZD and may be a promising tool for drug screening and personalized medicine.
Assuntos
Hipocampo/citologia , Hipocampo/metabolismo , Neurogênese , Células-Tronco Pluripotentes/citologia , Potenciais de Ação , Diferenciação Celular , Giro Denteado/citologia , Giro Denteado/metabolismo , Fenômenos Eletrofisiológicos , Corpos Embrioides/citologia , Corpos Embrioides/metabolismo , Expressão Gênica , Genes Reporter , Proteínas de Homeodomínio/metabolismo , Humanos , Rede Nervosa , Células-Tronco Neurais/citologia , Células-Tronco Neurais/metabolismo , Neurônios/citologia , Neurônios/metabolismo , Neurotransmissores/biossíntese , Células Piramidais/citologia , Células Piramidais/metabolismo , Esquizofrenia/metabolismo , Esquizofrenia/fisiopatologia , Proteínas Supressoras de Tumor/metabolismoRESUMO
In Huntington's disease (HD), whether transneuronal spreading of mutant huntingtin (mHTT) occurs and its contribution to non-cell autonomous damage in brain networks is largely unknown. We found mHTT spreading in three different neural network models: human neurons integrated in the neural network of organotypic brain slices of HD mouse model, an ex vivo corticostriatal slice model and the corticostriatal pathway in vivo. Transneuronal propagation of mHTT was blocked by two different botulinum neurotoxins, each known for specifically inactivating a single critical component of the synaptic vesicle fusion machinery. Moreover, healthy human neurons in HD mouse model brain slices displayed non-cell autonomous changes in morphological integrity that were more pronounced when these neurons bore mHTT aggregates. Altogether, our findings suggest that transneuronal propagation of mHTT might be an important and underestimated contributor to the pathophysiology of HD.
Assuntos
Doença de Huntington/metabolismo , Doença de Huntington/patologia , Proteínas do Tecido Nervoso/biossíntese , Proteínas do Tecido Nervoso/genética , Neurônios/patologia , Animais , Linhagem Celular , Técnicas de Cocultura , Modelos Animais de Doenças , Células-Tronco Embrionárias , Feminino , Genótipo , Humanos , Proteína Huntingtina , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos CBA , Camundongos Transgênicos , Mutação/genética , Rede Nervosa/citologia , Rede Nervosa/patologia , Proteínas do Tecido Nervoso/fisiologia , Neurônios/metabolismo , Neurônios/fisiologiaRESUMO
It has been proposed that human embryonic stem cells could be used to provide an inexhaustible supply of differentiated cell types for the study of disease processes. Although methods for differentiating embryonic stem cells into specific cell types have become increasingly sophisticated, the utility of the resulting cells for modeling disease has not been determined. We have asked whether specific neuronal subtypes produced from human embryonic stem cells can be used to investigate the mechanisms leading to neural degeneration in amyotrophic lateral sclerosis (ALS). We show that human spinal motor neurons, but not interneurons, are selectively sensitive to the toxic effect of glial cells carrying an ALS-causing mutation in the SOD1 gene. Our findings demonstrate the relevance of these non-cell-autonomous effects to human motor neurons and more broadly demonstrate the utility of human embryonic stem cells for studying disease and identifying potential therapeutics.