RESUMO
ClC-K/barttin channels are involved in the transepithelial transport of chloride in the kidney and inner ear. Their physiological role is crucial in humans because mutations in CLCNKB or BSND, encoding ClC-Kb and barttin, cause Bartter's syndrome types III and IV, respectively. In vitro experiments have shown that an amino acid change in a proline-tyrosine motif in the C-terminus of barttin stimulates ClC-K currents. The molecular mechanism of this enhancement and whether this potentiation has any in vivo relevance remains unknown. We performed electrophysiological and biochemical experiments in Xenopus oocytes and kidney cells co-expressing ClC-K and barttin constructs. We demonstrated that barttin possesses a YxxØ motif and, when mutated, increases ClC-K plasma membrane stability, resulting in larger currents. To address the impact of mutating this motif in kidney physiology, we generated a knock-in mouse. Comparing wild-type (WT) and knock-in mice under a standard diet, we could not observe any difference in ClC-K and barttin protein levels or localization, either in urinary or plasma parameters. However, under a high-sodium low-potassium diet, known to induce hyperplasia of distal convoluted tubules, knock-in mice exhibit reduced hyperplasia compared to WT mice. In summary, our in vitro and in vivo studies demonstrate that the previously identified PY motif is indeed an endocytic YxxØ motif in which mutations cause a gain of function of the channel. KEY POINTS: It is revealed by mutagenesis and functional experiments that a previously identified proline-tyrosine motif regulating ClC-K plasma membrane levels is indeed an endocytic YxxØ motif. Biochemical characterization of mutants in the YxxØ motif in Xenopus oocytes and human embryonic kidney cells indicates that mutants showed increased plasma membrane levels as a result of an increased stability, resulting in higher function of ClC-K channels. Mutation of this motif does not affect barttin protein expression and subcellular localization in vivo. Knock-in mice with a mutation in this motif, under conditions of a high-sodium low-potassium diet, exhibit less hyperplasia in the distal convoluted tubule than wild-type animals, indicating a gain of function of the channel in vivo.
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Megalencephalic Leukoencephalopathy with subcortical Cysts (MLC) is a type of vacuolating leukodystrophy, which is mainly caused by mutations in MLC1 or GLIALCAM. The two MLC-causing genes encode for membrane proteins of yet unknown function that have been linked to the regulation of different chloride channels such as the ClC-2 and VRAC. To gain insight into the role of MLC proteins, we have determined the brain GlialCAM interacting proteome. The proteome includes different transporters and ion channels known to be involved in the regulation of brain homeostasis, proteins related to adhesion or signaling as several G protein-coupled receptors (GPCRs), including the orphan GPRC5B and the proposed prosaposin receptor GPR37L1. Focusing on these two GPCRs, we could validate that they interact directly with MLC proteins. The inactivation of Gpr37l1 in mice upregulated MLC proteins without altering their localization. Conversely, a reduction of GPRC5B levels in primary astrocytes downregulated MLC proteins, leading to an impaired activation of ClC-2 and VRAC. The interaction between the GPCRs and MLC1 was dynamically regulated upon changes in the osmolarity or potassium concentration. We propose that GlialCAM and MLC1 associate with different integral membrane proteins modulating their functions and acting as a recruitment site for various signaling components as the GPCRs identified here. We hypothesized that the GlialCAM/MLC1 complex is working as an adhesion molecule coupled to a tetraspanin-like molecule performing regulatory effects through direct binding or influencing signal transduction events.
Assuntos
Cistos/genética , Doenças Desmielinizantes Hereditárias do Sistema Nervoso Central/genética , Receptores Acoplados a Proteínas G/genética , Animais , Astrócitos/metabolismo , Encéfalo/metabolismo , Moléculas de Adesão Celular Neurônio-Glia/genética , Moléculas de Adesão Celular Neurônio-Glia/metabolismo , Proteínas de Ciclo Celular/genética , Canais de Cloreto/genética , Cistos/metabolismo , Células HEK293 , Células HeLa , Doenças Desmielinizantes Hereditárias do Sistema Nervoso Central/metabolismo , Humanos , Leucoencefalopatias/genética , Leucoencefalopatias/metabolismo , Proteínas de Membrana/genética , Camundongos , Camundongos Knockout , Mutação , Proteínas do Tecido Nervoso/genética , Proteínas do Tecido Nervoso/metabolismo , Malformações do Sistema Nervoso/metabolismo , Transporte Proteico , Receptores Acoplados a Proteínas G/metabolismoRESUMO
Megalencephalic leukoencephalopathy with subcortical cysts (MLC) is a rare type of leukodystrophy caused by mutations in either MLC1 or GLIALCAM. GlialCAM is necessary for the correct targeting of MLC1, but also for the targeting of the Cl- channel ClC-2. Furthermore, GlialCAM modifies ClC-2 functional properties in vitro. However, in vivo studies in GlialCAM-/- mice have shown that the modification of ClC-2 activity only occurs in oligodendrocytes, despite GlialCAM and ClC-2 being expressed in astrocytes. Thus, the relationship between GlialCAM, MLC1 and ClC-2 in astrocytes is unknown. Here, we show that GlialCAM, ClC-2 and MLC1 can form a ternary complex in cultured astrocytes, but only under depolarizing conditions. We also provide biochemical evidences that this ternary complex exists in vivo. The formation of this complex changes ClC-2 localization in the membrane and its functional properties. ClC-2 association with GlialCAM/MLC1 depends on calcium flux through L-type calcium channels and activation of calcium-dependent calpain proteases. Based on these studies, we propose that the chloride influx mediated by GlialCAM/MLC1/ClC-2 in astrocytes may be needed to compensate an excess of potassium, as occurs in conditions of high neuronal activity. We suggest that a defect in this compensation may contribute to the pathogenesis of MLC disease.
Assuntos
Moléculas de Adesão Celular Neurônio-Glia/metabolismo , Cistos/metabolismo , Doenças Desmielinizantes Hereditárias do Sistema Nervoso Central/metabolismo , Proteínas de Membrana/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Animais , Astrócitos/metabolismo , Encéfalo/metabolismo , Encefalopatias/patologia , Canais de Cloro CLC-2 , Canais de Cálcio Tipo L/genética , Canais de Cloreto , Cistos/genética , Células HEK293 , Células HeLa , Doenças Desmielinizantes Hereditárias do Sistema Nervoso Central/genética , Humanos , Proteínas de Membrana/genética , Camundongos , Transporte Proteico/genéticaRESUMO
Megalencephalic leukoencephalopathy with subcortical cysts (MLC) is a rare type of leukodystrophy caused by mutations in either MLC1 or GLIALCAM genes. Previous work indicated that chloride currents mediated by the volume-regulated anion channel (VRAC) and ClC-2 channels were affected in astrocytes deficient in either Mlc1 or Glialcam. ClC-2 forms a ternary complex with GlialCAM and MLC1. LRRC8 proteins have been identified recently as the molecular components of VRAC, but the relationship between MLC and LRRC8 proteins is unknown. Here, we first demonstrate that LRRC8 and MLC1 are functionally linked, as MLC1 cannot potentiate VRAC currents when LRRC8A, the main subunit of VRAC, is knocked down. We determine that LRRC8A and MLC1 do not co-localize or interact and, in Xenopus oocytes, MLC1 does not potentiate LRRC8-mediated VRAC currents, indicating that VRAC modulation in astrocytes by MLC1 may be indirect. Investigating the mechanism of modulation, we find that a lack of MLC1 does not influence either mRNA or total and plasma membrane protein levels of LRRC8A; and neither does it affect LRRC8A subcellular localization. In agreement with recent results that indicated that overexpression of MLC1 decreases the phosphorylation of extracellular signal-regulated kinases (ERK), we find that astrocytes lacking MLC1 show an increase in ERK phosphorylation. In astrocytes with reduced or increased levels of MLC1 we observe changes in the phosphorylation state of the VRAC subunit LRRC8C. Our results thus reinforce previous suggestions that indicated that GlialCAM/MLC1 might modify signal transduction pathways that influence the activity of different proteins, such as VRAC.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Astrócitos/metabolismo , Cistos/metabolismo , Doenças Desmielinizantes Hereditárias do Sistema Nervoso Central/metabolismo , Proteínas de Membrana/metabolismo , Proteínas/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/análise , Proteínas Adaptadoras de Transdução de Sinal/genética , Sequência de Aminoácidos , Animais , Astrócitos/química , Astrócitos/patologia , Proteínas de Ciclo Celular , Células Cultivadas , Cistos/patologia , Células HeLa , Doenças Desmielinizantes Hereditárias do Sistema Nervoso Central/patologia , Humanos , Proteínas de Membrana/análise , Proteínas de Membrana/genética , Proteínas/análise , Proteínas/genética , Ratos , XenopusRESUMO
INTRODUCTION: Mutations in CLCN1 cause recessive or dominant forms of myotonia congenita (MC). Some mutations have been found to exhibit both patterns of inheritance but the mechanism explaining this behavior is unknown. METHODS: A known recessive missense mutation, A493E, was identified in a family with dominant MC. The mutant p.A493E alone or in co-expression with wild-type (WT) ClC-1 was expressed in Xenopus oocytes. Currents were measured and biochemical assays were performed. RESULTS: The mutant showed no significant activity and reduced total and plasma membrane (PM) protein levels. Co-expression with the mutant reduced the activity and PM levels of an engineered lower expression variant of ClC-1, whereas no effect was observed on a higher expression variant. DISCUSSION: Our results suggest that the dominant effect of some CLCN1 mutations showing recessive or dominant inheritance patterns may be due to a dose-dependent defect in PM delivery of the WT channel. Muscle Nerve, 2018.
RESUMO
OBJECTIVES: Identification of CCR5 as an antiretroviral target led to the development of several CCR5 antagonists in clinical trials and the approval of maraviroc. Evaluating the mechanism of drug resistance to CCR5 agents may have implications in the clinical development of this class of agents. We have analysed the resistance profile of two R5 HIV-1 strains [BaL and a clinical isolate (CI)] after long-term passage in cell culture in the presence of TAK-779, the first developed non-peptidic small molecule targeting CCR5. METHODS: Genotypic and phenotypic tests were used to evaluate the resistance of virus isolated from cell culture in the presence of the CCR5 inhibitor TAK-779. RESULTS: Mutations conferring resistance appeared in the gp120 sequence but were not confined to the V3 loop region, and both strains had a different mutation pattern. Recombination of the env gene of the BaL-derived resistant virus into the HIV-1 HXB2 wild-type backbone conferred resistance to TAK-779 and cross-resistance to maraviroc, with 63- and 11-fold changes in their EC(50) (50% effective concentration), respectively, together with an apparent reduction of the maximal plateau inhibition (MPI) of TAK-779 but not of maraviroc. Conversely, the resistant CI viruses showed an approximately 50% reduction in MPI for both TAK-779 and maraviroc. CONCLUSIONS: We confirm that different pathways to the generation of CCR5 drug resistance/cross-resistance may occur that strongly depend on cell culture conditions, CCR5 availability and the genetic background of the HIV strain. Our study provides complementary information to understand the complexity of resistance to CCR5 antagonists.
Assuntos
Amidas/farmacologia , Fármacos Anti-HIV/farmacologia , Antagonistas dos Receptores CCR5 , Farmacorresistência Viral , HIV-1/efeitos dos fármacos , Compostos de Amônio Quaternário/farmacologia , Linhagem Celular , Células Cultivadas , Cicloexanos/farmacologia , Proteína gp120 do Envelope de HIV/genética , HIV-1/genética , Humanos , Concentração Inibidora 50 , Leucócitos Mononucleares/virologia , Maraviroc , Testes de Sensibilidade Microbiana , Triazóis/farmacologiaRESUMO
We have studied the mechanism of action of Arg(*)-Arg-Nal(2)-Cys(1x)-Tyr-Gln-Lys-(d-Pro)-Pro-Tyr-Arg-Cit-Cys(1x)-Arg-Gly-(d-Pro)(*) (POL3026), a novel specific beta-hairpin mimetic CXC chemokine receptor (CXCR)4 antagonist. POL3026 specifically blocked the binding of anti-CXCR4 monoclonal antibody 12G5 and the intracellular Ca(2+) signal induced by CXC chemokine ligand 12. POL3026 consistently blocked the replication of human immunodeficiency virus (HIV), including a wide panel of X4 and dualtropic strains and subtypes in several culture models, with 50% effective concentrations (EC(50)) at the subnanomolar range, making POL3026 the most potent CXCR4 antagonist described to date. However, 1-[[4-(1,4,8,11-tetrazacyclotetradec-1-ylmethyl)phenyl]methyl]-1,4,8,11-tetrazacyclotetradecane (AMD3100)-resistant and stromal cell-derived factor-1alpha-resistant HIV-1 strains were cross-resistant to POL3026. Time of addition experiments and a multiparametric evaluation of HIV envelope function in the presence of test compounds confirmed the activity of POL3026 at an early step of virus replication: interaction with the coreceptor. Generation of HIV-1 resistance to POL3026 led to the selection of viruses 12- and 25-fold less sensitive and with mutations in gp120, including the V3 loop region. However, POL3026 prevented the emergence of CXCR4-using variants from an R5 HIV-1 strain that may occur in the presence of anti-HIV agents targeting CC chemokine receptor 5.
Assuntos
Fármacos Anti-HIV/farmacologia , Farmacorresistência Viral/efeitos dos fármacos , Peptídeos Cíclicos/farmacologia , Receptores CXCR4/antagonistas & inibidores , Fármacos Anti-HIV/química , Anticorpos Monoclonais , Benzilaminas , Sinalização do Cálcio/efeitos dos fármacos , Morte Celular/efeitos dos fármacos , Linhagem Celular , Quimiocina CXCL12/farmacologia , Quimiotaxia/efeitos dos fármacos , Ciclamos , HIV/efeitos dos fármacos , HIV/fisiologia , Compostos Heterocíclicos/farmacologia , Humanos , Leucócitos Mononucleares/efeitos dos fármacos , Leucócitos Mononucleares/virologia , Tecido Linfoide/efeitos dos fármacos , Tecido Linfoide/virologia , Macrófagos/efeitos dos fármacos , Macrófagos/virologia , Peptídeos Cíclicos/química , Fatores de Tempo , Proteínas do Envelope Viral/metabolismo , Replicação Viral/efeitos dos fármacosRESUMO
A small family of S-DABO cytosine analogs (S-DABOCs) has been synthesized and biologically evaluated as HIV-1 inhibitor both on wild type (wt) and drug-resistant mutants leading to the identification of an interesting compound (5d). Molecular modeling studies have been finally performed in order to rationalize the results.
Assuntos
Fármacos Anti-HIV/síntese química , Fármacos Anti-HIV/farmacologia , Citosina/análogos & derivados , Fármacos Anti-HIV/química , Citosina/síntese química , Citosina/química , Citosina/farmacologia , HIV-1/efeitos dos fármacos , Testes de Sensibilidade Microbiana , Modelos MolecularesRESUMO
Megalencephalic leukoencephalopathy with subcortical cysts (MLC) is a rare type of leukodystrophy characterized by dysfunction of the role of glial cells in controlling brain fluid and ion homeostasis. Patients affected by MLC present macrocephaly, cysts and white matter vacuolation, which lead to motor and cognitive impairments. To date, there is no treatment for MLC, only supportive care. MLC is caused by mutations in the MLC1 and GLIALCAM genes. MLC1 is a membrane protein with low identity to the Kv1.1 potassium channel and GlialCAM belongs to an adhesion molecule family. Both proteins form a complex with an as-yet-unknown function that is expressed mainly in the astrocytes surrounding the blood-brain barrier and in Bergmann glia. GlialCAM also acts as an auxiliary subunit of the chloride channel ClC-2, thus regulating its localization at cell-cell junctions and modifying its functional properties by affecting the common gate of ClC-2. Recent studies in Mlc1-, GlialCAM- and Clcn2-knockout mice or Mlc1-knockout zebrafish have provided fresh insight into the pathophysiology of MLC and further details about the molecular interactions between these three proteins. Additional studies have shown that GlialCAM/MLC1 also regulates other ion channels (TRPV4, VRAC) or transporters (Na+/K+-ATPase) in a not-understood manner. Furthermore, it has been shown that GlialCAM/MLC1 may influence signal transduction mechanisms, thereby affecting other proteins not related with transport such as the EGF receptor. Here, we offer a personal biochemical retrospective of the work that has been performed to gain knowledge of the pathophysiology of MLC, and we discuss future strategies that may be used to identify therapeutic solutions for MLC patients.
Assuntos
Cistos/genética , Doenças Desmielinizantes Hereditárias do Sistema Nervoso Central/genética , Proteínas/genética , Animais , Encéfalo/metabolismo , Proteínas de Ciclo Celular , Cistos/patologia , Doenças Desmielinizantes Hereditárias do Sistema Nervoso Central/patologia , Humanos , Proteínas de Membrana/metabolismo , Ligação Proteica , Proteínas/química , Proteínas/metabolismoRESUMO
Following the disclosure of dihydro-alkoxy-, dihydro-alkylthio-, and dihydro-alkylamino-benzyl-oxopyrimidines (DABOs, S-DABOs, and NH-DABOs) as potent and selective anti-HIV-1 agents belonging to the non-nucleoside reverse transcriptase inhibitor (NNRTI) class, we report here the synthesis and biological evaluation of a novel series of DABOs bearing a N,N-disubstituted amino group or a cyclic amine at the pyrimidine-C2 position, a hydrogen atom or a small alkyl group at C5 and/or at the benzylic position, and the favorable 2,6-difluorobenzyl moiety at the C6 position (F2-N,N-DABOs). The new compounds were highly active up to the subnanomolar level against both wt HIV-1 and the Y181C mutant and at the submicromolar to nanomolar range against the K103N and Y188L mutant strains. Such derivatives were more potent than S-DABOs, NH-DABOs, and nevirapine and efavirenz were chosen as reference drugs. The higher inhibitor adaptability to the HIV-1 RT non-nucleoside binding site (NNBS) may account for the higher inhibitory effect exerted by the new molecules against the mutated RTs.
Assuntos
Fármacos Anti-HIV/síntese química , Transcriptase Reversa do HIV/metabolismo , HIV-1/efeitos dos fármacos , Pirimidinas/síntese química , Inibidores da Transcriptase Reversa/síntese química , Fármacos Anti-HIV/química , Fármacos Anti-HIV/farmacologia , Linhagem Celular , Farmacorresistência Viral , Transcriptase Reversa do HIV/genética , HIV-1/genética , HIV-1/isolamento & purificação , Humanos , Modelos Moleculares , Mutação , Ligação Proteica , Pirimidinas/química , Pirimidinas/farmacologia , Inibidores da Transcriptase Reversa/química , Inibidores da Transcriptase Reversa/farmacologia , Relação Estrutura-AtividadeRESUMO
A series of novel S-DABO analogues, characterized by different substitution patterns at positions 2, 5, and 6 of the heterocyclic ring, were synthesized in a straightforward fashion by means of parallel synthesis and evaluated as inhibitors of human immunodeficiency virus type-1 (HIV-1). Most of the compounds proved to be highly active on the wild-type enzyme both in enzymatic and cellular assays, with one of them emerging as the most active reverse transcriptase inhibitor reported so far (EC50wt=25 pM). The general loss of potency displayed by the compounds toward clinically relevant mutant strains was deeply studied through a molecular modeling approach, leading to the evidence that the dynamic of the entrance in the non-nucleoside binding pocket could represent the basis of the inhibitory activity of the molecules.
Assuntos
Fármacos Anti-HIV/síntese química , Transcriptase Reversa do HIV/metabolismo , HIV-1/efeitos dos fármacos , Pirimidinas/síntese química , Inibidores da Transcriptase Reversa/síntese química , Sulfetos/síntese química , Fármacos Anti-HIV/química , Fármacos Anti-HIV/farmacologia , Linhagem Celular , Transcriptase Reversa do HIV/química , Transcriptase Reversa do HIV/genética , HIV-1/enzimologia , Humanos , Modelos Moleculares , Mutação , Pirimidinas/química , Pirimidinas/farmacologia , Relação Quantitativa Estrutura-Atividade , Proteínas Recombinantes/química , Inibidores da Transcriptase Reversa/química , Inibidores da Transcriptase Reversa/farmacologia , Sulfetos/química , Sulfetos/farmacologiaRESUMO
BACKGROUND: It is well established that mitochondrial damage plays a role in the pathophysiology of Alzheimer's disease (AD). However, studies carried out in humans barely contemplate regional differences with disease progression. OBJECTIVE: To study the expression of selected nuclear genes encoding subunits of the mitochondrial complexes and the activity of mitochondrial complexes in AD, in two regions: the entorhinal cortex (EC) and frontal cortex area 8 (FC). METHODS: Frozen samples from 148 cases processed for gene expression by qRT-PCR and determination of individual activities of mitochondrial complexes I, II, IV and V using commercial kits and home-made assays. RESULTS: Decreased expression of NDUFA2, NDUFB3, UQCR11, COX7C, ATPD, ATP5L and ATP50, covering subunits of complex I, II, IV and V, occurs in total homogenates of the EC in AD stages V-VI when compared with stages I-II. However reduced activity of complexes I, II and V of isolated mitochondria occurs as early as stages I-II when compared with middle-aged individuals in the EC. In contrast, no alterations in the expression of the same genes and no alterations in the activity of mitochondrial complexes are found in the FC in the same series. CONCLUSION: Different mechanisms of impaired energy metabolism may occur in AD, one of them, represented by the EC, is the result of primary and early alteration of mitochondria; the other one is probably the result, at least in part, of decreased functional input and is represented by hypometabolism in the FC in AD patients aged 86 or younger.
Assuntos
Doença de Alzheimer/patologia , Córtex Entorrinal/metabolismo , Córtex Entorrinal/ultraestrutura , Mitocôndrias/metabolismo , Proteínas Mitocondriais/metabolismo , Feminino , Lobo Frontal/metabolismo , Humanos , Masculino , Proteínas Mitocondriais/genética , Complexos Multienzimáticos/genética , Complexos Multienzimáticos/metabolismo , RNA Mensageiro/metabolismoRESUMO
A simple and efficient methodology for the parallel solution-phase synthesis has been set up to obtain a series of thiouracils, in turn selectively S-benzylated under microwave irradiation to give new S-DABOs. Biological screening led to the identification of compounds with nanomolar activity toward both the highly purified recombinant human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (RT) enzyme (wild-type and mutants) and wild-type (wt) and mutant HIV-1 strains. In particular, 20 was found to be the most potent S-DABO reported so far (ID50 = 26 nM toward the isolated wt enzyme) with subnanomolar activity toward both the wt and the pluriresistant virus (IRLL98) HIV-1 strain (EC50 < 0.14 nM and EC50 = 0.22 nM, respectively). Molecular modeling calculations were also performed to investigate the binding mode of such compounds onto the non-nucleoside reverse transcriptase inhibitor binding site and to rationalize the relationships between their chemical structure and activity values toward wt RT.
Assuntos
Fármacos Anti-HIV/síntese química , HIV-1/efeitos dos fármacos , Micro-Ondas , Pirimidinas/síntese química , Pirimidinonas/síntese química , Inibidores da Transcriptase Reversa/síntese química , Fármacos Anti-HIV/química , Fármacos Anti-HIV/farmacologia , Linhagem Celular , Farmacorresistência Viral Múltipla , Transcriptase Reversa do HIV/metabolismo , HIV-1/enzimologia , HIV-1/genética , Humanos , Modelos Moleculares , Mutação , Pirimidinas/química , Pirimidinas/farmacologia , Pirimidinonas/química , Pirimidinonas/farmacologia , Inibidores da Transcriptase Reversa/química , Inibidores da Transcriptase Reversa/farmacologia , Soluções , Relação Estrutura-AtividadeRESUMO
Neuroprotection of erythropoietin (EPO) following long-term administration is hampered by the associated undesirable effects on hematopoiesis and body weight. For this reason, we tested carbamylated-EPO (CEPO), which has no effect on erythropoiesis, and compared it with EPO in the AßPP/PS1 mouse model of familial Alzheimer's disease. Groups of 5-month old wild type (WT) and transgenic mice received chronic treatment consisting of CEPO (2,500 or 5,000 UI/kg) or EPO (2,500 U I/kg) 3 days/week for 4 weeks. Memory at the end of treatment was assessed with the object recognition test. Microarray analysis and quantitative-PCR were used for gene expression studies. No alterations in erythropoiesis were observed in CEPO-treated WT and AßPP/PS1 transgenic mice. EPO and CEPO improved memory in AßPP/PS1 animals. However, only EPO decreased amyloid-ß (Aß)plaque burden and soluble Aß(40). Microarray analysis of gene expression revealed a limited number of common genes modulated by EPO and CEPO. CEPO but not EPO significantly increased gene expression of dopamine receptors 1 and 2, and adenosine receptor 2a, and significantly down-regulated adrenergic receptor 1D and gastrin releasing peptide. CEPO treatment resulted in higher protein levels of dopamine receptors 1 and 2 in WT and AßPP/PS1 animals, whereas the adenosine receptor 2a was reduced in WT animals. The present results suggest that the improved behavior observed in AßPP/PS1 transgenic mice after CEPO treatment may be mediated, at least in part, by the observed modulation of the expression of molecules involved in neurotransmission.
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Doença de Alzheimer/complicações , Eritropoetina/análogos & derivados , Regulação da Expressão Gênica/efeitos dos fármacos , Transtornos da Memória/tratamento farmacológico , Transtornos da Memória/etiologia , Sinapses/metabolismo , Doença de Alzheimer/genética , Peptídeos beta-Amiloides/metabolismo , Precursor de Proteína beta-Amiloide/genética , Animais , Peso Corporal/efeitos dos fármacos , Peso Corporal/genética , Modelos Animais de Doenças , Eritropoetina/uso terapêutico , Peptídeo Liberador de Gastrina/metabolismo , Regulação da Expressão Gênica/genética , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Mutação/genética , Fragmentos de Peptídeos/metabolismo , Presenilina-1/genética , Receptores de Catecolaminas/metabolismo , Sinapses/genética , Fatores de TempoRESUMO
Tau P301S transgenic mice (PS19 line) are used as a model of frontotemporal lobar degeneration (FTLD)-tau. Behavioral alterations in these mice begin at approximately 4 months of age. We analyzed molecular changes related to disease progression in these mice. Hyperphosphorylated 4Rtau increased in neurons from 1 month of age in entorhinal and piriform cortices to the neocortex and other regions. A small percentage of neurons developed an abnormal tau conformation, tau truncation, and ubiquitination only at 9/10 months of age. Astrocytosis, microgliosis, and increased inflammatory cytokine and immune mediator expression also occurred at this late stage; hippocampi were the most markedly affected. Altered mitochondrial function, increased reactive oxygen species production, and limited protein oxidative damage were observed in advanced disease. Tau oligomers were only present in P301S mice, they were found in somatosensory cortex and hippocampi at the age of 3 months, and they increased across time in the somatosensory cortex and were higher and sustained in hippocampi. Age-related modifications in lipid composition occurred in both P301S and wild-type mice with regional and phenotypic differences; however, changes of total lipids did not seem to have pathogenic implications. Apoptosis only occurred in restricted regions in late disease. The complex tau pathology, mitochondrial alterations, oxidative stress damage, glial reactions, neuroinflammation, and cell death in P301S mice likely parallel those in FTLD-tau. Thus, therapies should focus first on abnormal tau rather than secondary events that appear late in the course of FTLD-tau.
Assuntos
Degeneração Lobar Frontotemporal/metabolismo , Degeneração Lobar Frontotemporal/patologia , Mitocôndrias/patologia , Estresse Oxidativo/fisiologia , Proteínas tau/metabolismo , Animais , Western Blotting , Modelos Animais de Doenças , Progressão da Doença , Regulação da Expressão Gênica , Humanos , Imuno-Histoquímica , Inflamação/metabolismo , Inflamação/patologia , Camundongos , Camundongos Transgênicos , Reação em Cadeia da Polimerase , Proteínas tau/genéticaRESUMO
OBJECTIVES: Duplexes of 21 base pair RNA, known as short-interfering RNA (siRNA), have been shown to inhibit gene expression by a sequence-specific RNA degradation mechanism termed RNA interference (RNAi). The objective of our study was to evaluate the effect of chemokine receptor gene suppression by RNAi on the entry and replication of HIV-1. METHODS: A flow cytometry and microscopy evaluation of HIV co-receptor expression of cells transfected with siRNA. An evaluation of the effect of siRNA on HIV entry and replication by intracellular p24 antigen detection, and virus production by infected cells, respectively. RESULTS: siRNA that target CXCR4 and CCR5 could effectively impede cell surface protein expression and their consequent function as HIV co-receptors. The inhibitory effect of RNAi directed to CXCR4 was detected 48 h after transfection of CXCR4+ U87-CD4+ cells. The expression of CXCR4 and CCR5 was blocked in 63 and 48% of positive cells by the corresponding siRNA. However, siRNA directed to CXCR4 or CCR5 did not have an effect on CD4 cells or green fluorescence protein expression. siRNA directed to CXCR4 did not suppress CCR5 expression or vice versa. The suppression of HIV-1 co-receptor expression effectively blocked the acute infection of CXCR4+ or CCR5+ U87-CD4+ cells by X4 (NL4-3) or R5 (BaL) HIV-1 strains. Inhibition of virus replication occurred regardless of the multiplicity of infection employed. CONCLUSION: Our results demonstrate that RNAi may be used to block HIV entry and replication through the blockade of cellular gene expression. Gene silencing by siRNA may become a valid alternative for HIV intervention.
Assuntos
Antagonistas dos Receptores CCR5 , Infecções por HIV/virologia , HIV-1/metabolismo , Interferência de RNA/fisiologia , Receptores CXCR4/antagonistas & inibidores , Replicação Viral/fisiologia , Linfócitos T CD4-Positivos/virologia , Células Cultivadas , Citometria de Fluxo , Inativação Gênica/fisiologia , Humanos , RNA Interferente Pequeno/fisiologiaRESUMO
Infection by human immunodeficiency virus type 1 (HIV-1) has been associated with increased cell death of both infected and bystander cells. The envelope glycoprotein complex appears to play an active role in HIV-induced death of bystander cells. We quantified cell-to-cell fusion, single cell death and membrane lipid mixing in cocultures of effector, HIV-1 envelope-expressing cells with peripheral blood mononuclear cells or purified CD4 T lymphocytes from HIV-negative donors, in the presence or the absence of the fusion inhibitor enfuvirtide (T-20, pentafuside, Fuzeon). T-20, which blocks gp41-dependent virus-cell fusion, showed a complete and dose-dependent inhibition of syncytium formation in cocultures of envelope-expressing cells with uninfected cells. Similarly, T-20 totally abrogated death of single bystander CD4 T cells with an IC50 of 0.04 microg/ml. Membrane lipid mixing, as a measure of interaction between envelope-expressing cells and CD4 cells, was also dose-dependently inhibited by T-20. Moreover, effector cells chronically infected with a T-20-resistant virus recovered the ability to induce bystander cell death in the presence of the drug, supporting the role of gp41 in single cell death. In conclusion, T-20 is able to protect CD4 T cells from envelope presentation with a dual effect: inhibition of virus replication and blockade of HIV-1 envelope-induced cell death of bystander CD4 T cells. Protection of cells prior to infection from HIV envelope-dependent bystander effect could lead to a better immune restoration of HIV-1-infected patients that are treated with T-20.
Assuntos
Fármacos Anti-HIV/farmacologia , Linfócitos T CD4-Positivos/efeitos dos fármacos , Proteína gp41 do Envelope de HIV/farmacologia , HIV-1/efeitos dos fármacos , Fragmentos de Peptídeos/farmacologia , Linfócitos T CD4-Positivos/patologia , Morte Celular/efeitos dos fármacos , Células Cultivadas , Técnicas de Cocultura , Relação Dose-Resposta a Droga , Enfuvirtida , Células Gigantes/efeitos dos fármacos , Proteína gp41 do Envelope de HIV/metabolismo , HIV-1/patogenicidade , Humanos , Virulência/efeitos dos fármacos , Replicação Viral/efeitos dos fármacosRESUMO
HIV-1 strains with a syncytium-inducing phenotype that use CXCR4 (X4 strains) have been associated with faster disease progression and AIDS. Antiviral agents designed to block CXCR4 may prevent the emergence of X4 HIV strains but resistant strains that maintain the X4 phenotype can be raised by sequential passage in cell cultures. We have demonstrated that a laboratory adapted strain (NL4-3) and a cloned clinical isolate (CI-1) of HIV-1 cultured in the presence of the CXCR4 antagonist, AMD3100, became resistant to the compound without a change in co-receptor use. These strains became resistant through divergence with respect to the wild-type virus. Conversely, a clinical isolate made resistant to AMD3100 switched co-receptor use from X4 to R5 through a change in diversity from the original virus population. When dual infection competition/heteroduplex tracking assays were performed, all AMD3100-resistant strains, regardless of co-receptor use showed a significantly diminished fitness compared with the wild-type virus. Single virus infections, at a similar multiplicity of infection, also indicated that the wild-type strains possess better replicative ability than their corresponding resistant strains. Thus, viral resistance development to a CXCR4 antagonist such as AMD3100 is associated with reduced viral fitness.
Assuntos
Fármacos Anti-HIV/farmacologia , Farmacorresistência Viral/genética , HIV-1/efeitos dos fármacos , HIV-1/fisiologia , Compostos Heterocíclicos/farmacologia , Receptores CXCR4/antagonistas & inibidores , Sequência de Aminoácidos , Sequência de Bases , Benzilaminas , Linhagem Celular , Ciclamos , Células Gigantes/fisiologia , Proteína gp120 do Envelope de HIV/química , Proteína gp120 do Envelope de HIV/genética , HIV-1/genética , Humanos , Dados de Sequência Molecular , Mutação , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/genética , Receptores CXCR4/metabolismo , Inoculações Seriadas , Replicação ViralRESUMO
We have studied the binding of biotinylated HIV particles to various cell lines and peripheral blood mononuclear cells (PBMCs). Viruses were harvested from cultures of cell surface-biotinylated cells productively infected with HIV-IIIB. Labeled HIV particles bound to and infected CD4(+) cell lines and PBMCs. The interaction between gp120 and CD4 contributed in part to HIV binding to CD4(+) cells. However, HIV binding was for the most part independent of CD4 expression and sensitive to polyanion inhibition. Polyanion-sensitive interactions involved heparan sulfate in cell lines but not in primary T cells. Interestingly, HIV binding to primary cells was heterogeneous and targeted discrete subsets of CD4(+) and CD4(-) cells. The CD4(+) T cell subset that displayed high HIV-binding capacity contained mostly CD4(+)CD45RO(+) cells, whereas the subset showing undetectable HIV binding contained higher proportions of CD4(+)CD45RO(-) cells. Consistently, purified CD4(+)CD45RO(-) cells or purified CD4(+) T cells with low virus-binding capacity showed lower HIV entry and delayed HIV replication when compared with purified CD4(+)CD45RO(+) or purified CD4(+) T cells with high virus-binding capacity, respectively. Our data suggest that the binding of HIV to cell surface-expressed CD4 might be inefficient in a subset of CD4(+) T cells and that increased binding of HIV to activated and CD4(+)CD45RO(+) cells may contribute to the higher susceptibility of these cells to HIV infection.
Assuntos
Linfócitos T CD4-Positivos/virologia , HIV-1/fisiologia , Biotina , Linfócitos T CD4-Positivos/imunologia , Células Cultivadas , HIV-1/imunologia , Células HeLa , Humanos , Antígenos Comuns de Leucócito/imunologia , Leucócitos Mononucleares/virologia , Ativação Linfocitária , Receptores Virais/imunologia , Replicação ViralRESUMO
The gp41 subunit of HIV-1 has been recently recognized as a target for antiviral therapy. C-34 is a peptide that mimics the heptad repeat 2 in the ectodomain of gp41. Here, we describe two HIV-1 strains selected after 5 and 17 passages in culture with increasing concentrations of C-34 (breakthrough concentration of 10 microg/ml). The HXB2-derived strain was more than 1000-fold resistant and contained a V38E mutation in the gp41 coding DNA sequence. The NL4-3-derived strain was more than 500-fold resistant and contained a L33S mutation in gp41. No cross-resistance to the RT inhibitor AZT, the HIV binding inhibitor dextran sulfate (DS), or the chemokine receptor antagonist ALX-40-4C was detected. These data indicate that HIV-1 can overcome C-34 inhibition through mutations at residues not involved in the formation of the hydrophobic cavity of gp41.