RESUMO
Alzheimer's disease (AD) is the most common form of dementia characterized by cognitive dysfunctions. Pharmacological interventions to slow the progression of AD are intensively studied. A potential direction targets neuronal sigma-1 receptors (S1Rs). S1R ligands are recognized as promising therapeutic agents that may alleviate symptom severity of AD, possibly via preventing amyloid-ß-(Aß-) induced neurotoxicity on the endoplasmic reticulum stress-associated pathways. Furthermore, S1Rs may also modulate adult neurogenesis, and the impairment of this process is reported to be associated with AD. We aimed to investigate the effects of two S1R agonists, dimethyltryptamine (DMT) and PRE084, in an Aß-induced in vivo mouse model characterizing neurogenic and anti-neuroinflammatory symptoms of AD, and the modulatory effects of S1R agonists were analyzed by immunohistochemical methods and western blotting. DMT, binding moderately to S1R but with high affinity to 5-HT receptors, negatively influenced neurogenesis, possibly as a result of activating both receptors differently. In contrast, the highly selective S1R agonist PRE084 stimulated hippocampal cell proliferation and differentiation. Regarding neuroinflammation, DMT and PRE084 significantly reduced Aß1-42-induced astrogliosis, but neither had remarkable effects on microglial activation. In summary, the highly selective S1R agonist PRE084 may be a promising therapeutic agent for AD. Further studies are required to clarify the multifaceted neurogenic and anti-neuroinflammatory roles of these agonists.
Assuntos
Doença de Alzheimer , Receptores sigma , Doença de Alzheimer/tratamento farmacológico , Doença de Alzheimer/metabolismo , Peptídeos beta-Amiloides/metabolismo , Animais , Modelos Animais de Doenças , Camundongos , N,N-Dimetiltriptamina , Neurogênese , Doenças Neuroinflamatórias , Fragmentos de Peptídeos , Receptores sigma/metabolismo , Receptor Sigma-1RESUMO
Neurogenesis plays a crucial role in cognitive processes. During aging and in Alzheimer's disease (AD), altered neurogenesis and neuroinflammation are evident both in C57BL/6J, APPSwe/PS1dE9 (Tg) mice and humans. AD pathology may slow down upon drug treatment, for example, in a previous study of our group P33, a putative neuroprotective agent was found to exert advantageous effects on the elevated levels of APP, Aß, and neuroinflammation. In the present study, we aimed to examine longitudinal alterations in neurogenesis, neuroinflammation and AD pathology in a transgenic (Tg) mouse model, and assessed the putative beneficial effects of long-term P33 treatment on AD-specific neurological alterations. Hippocampal cell proliferation and differentiation were significantly reduced between 8 and 12 months of age. Regarding neuroinflammation, significantly elevated astrogliosis and microglial activation were observed in 6- to 7-month-old Tg animals. The amounts of the molecules involved in the amyloidogenic pathway were altered from 4 months of age in Tg animals. P33-treatment led to significantly increased neurogenesis in 9-month-old animals. Our data support the hypothesis that altered neurogenesis may be a consequence of AD pathology. Based on our findings in the transgenic animal model, early pharmacological treatment before the manifestation of AD symptoms might ameliorate neurological decline.
Assuntos
Doença de Alzheimer , Fármacos Neuroprotetores , Doença de Alzheimer/tratamento farmacológico , Doença de Alzheimer/genética , Doença de Alzheimer/metabolismo , Peptídeos beta-Amiloides/farmacologia , Precursor de Proteína beta-Amiloide/genética , Precursor de Proteína beta-Amiloide/metabolismo , Animais , Modelos Animais de Doenças , Humanos , Lactente , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Neurogênese , Fármacos Neuroprotetores/farmacologia , Fármacos Neuroprotetores/uso terapêutico , Presenilina-1/genética , Presenilina-1/metabolismoRESUMO
BACKGROUND: Heat-shock protein B1 (HSPB1) is among the most well-known and versatile member of the evolutionarily conserved family of small heat-shock proteins. It has been implicated to serve a neuroprotective role against various neurological disorders via its modulatory activity on inflammation, yet its exact role in neuroinflammation is poorly understood. In order to shed light on the exact mechanism of inflammation modulation by HSPB1, we investigated the effect of HSPB1 on neuroinflammatory processes in an in vivo and in vitro model of acute brain injury. METHODS: In this study, we used a transgenic mouse strain overexpressing the human HSPB1 protein. In the in vivo experiments, 7-day-old transgenic and wild-type mice were treated with ethanol. Apoptotic cells were detected using TUNEL assay. The mRNA and protein levels of cytokines and glial cell markers were examined using RT-PCR and immunohistochemistry in the brain. We also established primary neuronal, astrocyte, and microglial cultures which were subjected to cytokine and ethanol treatments. TNFα and hHSPB1 levels were measured from the supernates by ELISA, and intracellular hHSPB1 expression was analyzed using fluorescent immunohistochemistry. RESULTS: Following ethanol treatment, the brains of hHSPB1-overexpressing mice showed a significantly higher mRNA level of pro-inflammatory cytokines (Tnf, Il1b), microglia (Cd68, Arg1), and astrocyte (Gfap) markers compared to wild-type brains. Microglial activation, and 1 week later, reactive astrogliosis was higher in certain brain areas of ethanol-treated transgenic mice compared to those of wild-types. Despite the remarkably high expression of pro-apoptotic Tnf, hHSPB1-overexpressing mice did not exhibit higher level of apoptosis. Our data suggest that intracellular hHSPB1, showing the highest level in primary astrocytes, was responsible for the inflammation-regulating effects. Microglia cells were the main source of TNFα in our model. Microglia isolated from hHSPB1-overexpressing mice showed a significantly higher release of TNFα compared to wild-type cells under inflammatory conditions. CONCLUSIONS: Our work provides novel in vivo evidence that hHSPB1 overexpression has a regulating effect on acute neuroinflammation by intensifying the expression of pro-inflammatory cytokines and enhancing glial cell activation, but not increasing neuronal apoptosis. These results suggest that hHSPB1 may play a complex role in the modulation of the ethanol-induced neuroinflammatory response.
Assuntos
Lesões Encefálicas/induzido quimicamente , Lesões Encefálicas/metabolismo , Etanol/toxicidade , Proteínas de Choque Térmico/biossíntese , Mediadores da Inflamação/metabolismo , Chaperonas Moleculares/biossíntese , Animais , Lesões Encefálicas/genética , Células Cultivadas , Etanol/administração & dosagem , Expressão Gênica , Proteínas de Choque Térmico/genética , Humanos , Injeções Subcutâneas , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Chaperonas Moleculares/genéticaRESUMO
Aging and female sex are the major risk factors for Alzheimer's disease and its associated brain amyloid-ß (Aß) neuropathology, but the mechanisms mediating these risk factors remain uncertain. Evidence indicates that Aß aggregation by Zn2+ released from glutamatergic neurons contributes to amyloid neuropathology, so we tested whether aging and sex adversely influences this neurophysiology. Using acute hippocampal slices, we found that extracellular Zn2+-elevation induced by high K+ stimulation was significantly greater with older (65 weeks vs 10 weeks old) rats, and was exaggerated in females. This was driven by slower reuptake of extracellular Zn2+, which could be recapitulated by mitochondrial intoxication. Zn2+:Aß aggregates were toxic to the slices, but Aß alone was not. Accordingly, high K+ caused synthetic human Aß added to the slices to form soluble oligomers as detected by bis-ANS, attaching to neurons and inducing toxicity, with older slices being more vulnerable. Age-dependent energy failure impairing Zn2+ reuptake, and a higher maximal capacity for Zn2+ release by females, could contribute to age and sex being major risk factors for Alzheimer's disease.
Assuntos
Doença de Alzheimer/metabolismo , Peptídeos beta-Amiloides/metabolismo , Espaço Extracelular/metabolismo , Hipocampo/metabolismo , Agregação Patológica de Proteínas/metabolismo , Zinco/metabolismo , Animais , Feminino , Masculino , Ratos , Ratos Wistar , Fatores de RiscoRESUMO
Anticancer peptides (ACPs) could potentially offer many advantages over other cancer therapies. ACPs often target cell membranes, where their surface mechanism is coupled to a conformational change into helical structures. However, details on their binding are still unclear, which would be crucial to reach progress in connecting structural aspects to ACP action and to therapeutic developments. Here we investigated natural helical ACPs, Lasioglossin LL-III, Macropin 1, Temporin-La, FK-16, and LL-37, on model liposomes, and also on extracellular vesicles (EVs), with an outer leaflet composition similar to cancer cells. The combined simulations and experiments identified three distinct binding modes to the membranes. Firstly, a highly helical structure, lying mainly on the membrane surface; secondly, a similar, yet only partially helical structure with disordered regions; and thirdly, a helical monomeric form with a non-inserted perpendicular orientation relative to the membrane surface. The latter allows large swings of the helix while the N-terminal is anchored to the headgroup region. These results indicate that subtle differences in sequence and charge can result in altered binding modes. The first two modes could be part of the well-known carpet model mechanism, whereas the newly identified third mode could be an intermediate state, existing prior to membrane insertion.
Assuntos
Peptídeos Catiônicos Antimicrobianos/química , Antineoplásicos/química , Venenos de Abelha/química , Membrana Celular/metabolismo , Motivos de Aminoácidos , Peptídeos Catiônicos Antimicrobianos/metabolismo , Antineoplásicos/metabolismo , Venenos de Abelha/metabolismo , Vesículas Extracelulares/metabolismo , Humanos , Ligação Proteica , Domínios Proteicos , CatelicidinasRESUMO
Sigma-1 receptor (S1R) is an intracellular, multi-functional, ligand operated protein that also acts as a chaperone. It is considered as a pluripotent drug target in several pathologies. The publication of agonist and antagonist bound receptor structures has paved the way for receptor-based in silico drug design. However, recent studies on this subject payed no attention to the structural differences of agonist and antagonist binding. In this work, we have developed a new ensemble docking-based virtual screening protocol utilizing both agonist and antagonist bound S1R structures. This protocol was used to screen our in-house compound library. The S1R binding affinities of the 40 highest ranked compounds were measured in competitive radioligand binding assays and the sigma-2 receptor (S2R) affinities of the best S1R binders were also determined. This way three novel high affinity S1R ligands were identified and one of them exhibited a notable S1R/S2R selectivity.
Assuntos
Isoxazóis/química , Simulação de Acoplamento Molecular/métodos , Pentazocina/química , Piridinas/química , Receptores sigma/química , Sítios de Ligação , Interações Hidrofóbicas e Hidrofílicas , Isoxazóis/análise , Isoxazóis/farmacologia , Ligantes , Estrutura Molecular , Pentazocina/análise , Pentazocina/farmacologia , Ligação Proteica , Piridinas/análise , Piridinas/farmacologia , Ensaio Radioligante/métodos , Receptores sigma/agonistas , Receptores sigma/análise , Receptores sigma/antagonistas & inibidores , Receptor Sigma-1RESUMO
Regulated intramembrane proteolysis (RIP) of the amyloid precursor protein (APP) leads to the formation of fragments, among which the intracellular domain of APP (AICD) was also identified to be a causative of early pathological events. AICD-counteracting proteins, such as Fe65, may serve as alternative therapeutic targets of Alzheimer's disease (AD). The detection of elevated levels of Fe65 in the brains of both human patients and APP transgenic mice may further strengthen the hypothesis that influencing the interaction between Fe65 and APP may have a beneficial effect on the course of AD. Based on a PXP motif, proven to bind to the WW domain of Fe65, a new pentapeptide was designed and tested. The impedimental effect of P33 on the production of beta amyloid (Aß) (soluble fraction and aggregated plaques) and on the typical features of the AD pathology (decreased dendritic spine density, synaptic markers, elevated inflammatory reactions) was also demonstrated. Significant enhancements of both learning ability and memory function were observed in a Morris water maze paradigm. The results led us to formulate the theory that P33 acts by altering the conformation of Fe65 via binding to its WW domain, consequently hindering any interactions between Fe65 and key members involved in APP processing.
Assuntos
Precursor de Proteína beta-Amiloide/metabolismo , Memória/efeitos dos fármacos , Proteínas do Tecido Nervoso/metabolismo , Plasticidade Neuronal/efeitos dos fármacos , Proteínas Nucleares/metabolismo , Oligopeptídeos/farmacologia , Doença de Alzheimer/metabolismo , Animais , Feminino , Masculino , Camundongos , Camundongos Transgênicos , Proteínas do Tecido Nervoso/química , Proteínas Nucleares/química , Oligopeptídeos/química , Conformação ProteicaRESUMO
Engineering water-soluble stand-alone ß-sandwich mimetics is a current challenge because of the difficulties associated with tailoring long-range interactions. In this work, single cis-(1R,2S)-2-aminocyclohexanecarboxylic acid mutations were introduced into the edge strands of the eight-stranded ß-sandwich mimetic structures from the betabellin family. Temperature-dependent NMR and CD measurements, together with thermodynamic analyses, demonstrated that the modified peripheral strands exhibited an irregular and partially disordered structure but were able to exert sufficient shielding on the hydrophobic core to retain the predominantly ß-sandwich structure. Although the frustrated interactions decreased the free energy of unfolding, the temperature of the maximum stabilities increased to or remained at physiologically relevant temperatures. We found that the irregular peripheral strands were able to prevent edge-to-edge association and fibril formation in the aggregation-prone model. These findings establish a ß-sandwich stabilization and aggregation inhibition approach, which does not interfere with the pillars of the peptide bond or change the net charge of the peptide.
RESUMO
A series of novel mimetic peptides were designed, synthesised and biologically evaluated as inhibitors of Aß42 aggregation. One of the synthesised peptidic compounds, termed compound 7 modulated Aß42 aggregation as demonstrated by thioflavin T fluorescence, acting also as an inhibitor of the cytotoxicity exerted by Aß42 aggregates. The early stage interaction between compound 7 and the Aß42 monomer was investigated by replica exchange molecular dynamics (REMD) simulations and docking studies. Our theoretical results revealed that compound 7 can elongate the helical conformation state of an early stage Aß42 monomer and it helps preventing the formation of ß-sheet structures by interacting with key residues in the central hydrophobic cluster (CHC). This strategy where early "on-pathway" events are monitored by small molecules will help the development of new therapeutic strategies for Alzheimer's disease.
Assuntos
Peptídeos beta-Amiloides/antagonistas & inibidores , Oligopeptídeos/farmacologia , Fragmentos de Peptídeos/antagonistas & inibidores , Peptidomiméticos/farmacologia , Conformação Proteica em alfa-Hélice/efeitos dos fármacos , Peptídeos beta-Amiloides/química , Peptídeos beta-Amiloides/metabolismo , Linhagem Celular Tumoral , Humanos , Simulação de Acoplamento Molecular , Oligopeptídeos/síntese química , Oligopeptídeos/metabolismo , Oligopeptídeos/toxicidade , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/metabolismo , Peptidomiméticos/síntese química , Peptidomiméticos/metabolismo , Peptidomiméticos/toxicidade , Ligação ProteicaRESUMO
Alzheimer's disease is one of the most common chronic neurodegenerative disorders. Despite several in vivo and clinical studies, the cause of the disease is poorly understood. Currently, amyloid ß (Aß) peptide and its tendency to assemble into soluble oligomers are known as a main pathogenic event leading to the interruption of synapses and brain degeneration. Targeting neurotoxic Aß oligomers can help recognize the disease at an early stage or it can be a potential therapeutic approach. Unnatural ß-peptidic foldamers are successfully used against many different protein targets due to their favorable structural and pharmacokinetic properties compared to small molecule or protein-like drug candidates. We have previously reported a tetravalent foldamer-dendrimer conjugate which can selectively bind Aß oligomers. Taking advantage of multivalency and foldamers, we synthesized different multivalent foldamer-based conjugates to optimize the geometry of the ligand. Isothermal titration calorimetry (ITC) was used to measure binding affinity to Aß, thereafter 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) based tissue viability assay and impedance-based viability assay on SH-SY5Y cells were applied to monitor Aß toxicity and protective effects of the compounds. Important factors for high binding affinity were determined and a good correlation was found between influencing the valence and the capability of the conjugates for Aß binding.
Assuntos
Doença de Alzheimer/tratamento farmacológico , Peptídeos beta-Amiloides/química , Dendrímeros/química , Fragmentos de Peptídeos/química , Doença de Alzheimer/genética , Peptídeos beta-Amiloides/antagonistas & inibidores , Precursor de Proteína beta-Amiloide/antagonistas & inibidores , Precursor de Proteína beta-Amiloide/química , Animais , Calorimetria , Dendrímeros/uso terapêutico , Humanos , Ligantes , Neurônios/química , Neurônios/efeitos dos fármacos , Fragmentos de Peptídeos/uso terapêutico , Ligação Proteica , Conformação Proteica/efeitos dos fármacos , Dobramento de Proteína/efeitos dos fármacosRESUMO
Protein dyshomeostasis is the common mechanism of neurodegenerative diseases such as Alzheimer's disease (AD). Aging is the key risk factor, as the capacity of the proteostasis network declines during aging. Different cellular stress conditions result in the up-regulation of the neurotrophic, neuroprotective amyloid precursor protein (APP). Enzymatic processing of APP may result in formation of toxic Aß aggregates (ß-amyloids). Protein folding is the basis of life and death. Intracellular Aß affects the function of subcellular organelles by disturbing the endoplasmic reticulum-mitochondria cross-talk and causing severe Ca2+-dysregulation and lipid dyshomeostasis. The extensive and complex network of proteostasis declines during aging and is not able to maintain the balance between production and disposal of proteins. The effectivity of cellular pathways that safeguard cells against proteotoxic stress (molecular chaperones, aggresomes, the ubiquitin-proteasome system, autophagy) declines with age. Chronic cerebral hypoperfusion causes dysfunction of the blood-brain barrier (BBB), and thus the Aß-clearance from brain-to-blood decreases. Microglia-mediated clearance of Aß also declines, Aß accumulates in the brain and causes neuroinflammation. Recognition of the above mentioned complex pathogenesis pathway resulted in novel drug targets in AD research.
Assuntos
Doença de Alzheimer/metabolismo , Peptídeos beta-Amiloides/metabolismo , Encéfalo/metabolismo , Encéfalo/patologia , Doença de Alzheimer/tratamento farmacológico , Doença de Alzheimer/patologia , Secretases da Proteína Precursora do Amiloide/antagonistas & inibidores , Secretases da Proteína Precursora do Amiloide/metabolismo , Precursor de Proteína beta-Amiloide/metabolismo , Animais , Ácido Aspártico Endopeptidases/antagonistas & inibidores , Ácido Aspártico Endopeptidases/metabolismo , Barreira Hematoencefálica/metabolismo , Retículo Endoplasmático/metabolismo , Humanos , Mitocôndrias/metabolismo , Agregação Patológica de Proteínas/metabolismo , Transdução de Sinais , Regulação para CimaRESUMO
During the past 15 years, several genetically altered mouse models of human Alzheimer's disease (AD) have been developed. These costly models have greatly facilitated the evaluation of novel therapeutic approaches. Injecting synthetic ß-amyloid (Aß) 1-42 species into different parts of the brain of non-transgenic rodents frequently provided unreliable results, owing to a lack of a genuine characterization of the administered Aß aggregates. Previously, we have published a new rat AD-model in which protofibrillar-fibrillar Aß1-42 was administered into rat entorhinal cortex (Sipos 2007). In order to develop a more reliable model, we have injected well-characterized toxic soluble Aß1-42 species (oligomers, protofibrils and fibrils) intracerebroventricularly (icv) into rat brain. Studies of the distribution of fluorescent-labeled Aß1-42 in the brain showed that soluble Aß-species diffused into all parts of the rat brain. After seven days, the Aß-treated animals showed a significant decrease of spatial memory in Morris water maze test and impairment of synaptic plasticity (LTP) measured in acute hippocampal slices. The results of histological studies (decreased number of viable neurons, increased tau levels and decreased number of dendritic spines) also supported that icv administration of well-characterized toxic soluble Aß species into rat brain provides a reliable rat AD-model.
Assuntos
Doença de Alzheimer/tratamento farmacológico , Peptídeos beta-Amiloides/uso terapêutico , Memória Espacial/efeitos dos fármacos , Doença de Alzheimer/fisiopatologia , Peptídeos beta-Amiloides/administração & dosagem , Animais , Modelos Animais de Doenças , Masculino , Aprendizagem em Labirinto/efeitos dos fármacos , RatosRESUMO
Alzheimer's disease (AD) is characterized by extracellular amyloid-ß (Aß) deposits and microglia-dominated inflammatory activation. Innate immune signaling controls microglial inflammatory activities and Aß clearance. However, studies examining innate immunity in Aß pathology and neuronal degeneration have produced conflicting results. In this study, we investigated the pathogenic role of innate immunity in AD by ablating a key signaling molecule, IKKß, specifically in the myeloid cells of TgCRND8 APP-transgenic mice. Deficiency of IKKß in myeloid cells, especially microglia, simultaneously reduced inflammatory activation and Aß load in the brain and these effects were associated with reduction of cognitive deficits and preservation of synaptic structure proteins. IKKß deficiency enhanced microglial recruitment to Aß deposits and facilitated Aß internalization, perhaps by inhibiting TGF-ß-SMAD2/3 signaling, but did not affect Aß production and efflux. Therefore, inhibition of IKKß signaling in myeloid cells improves cognitive functions in AD mice by reducing inflammatory activation and enhancing Aß clearance. These results contribute to a better understanding of AD pathogenesis and could offer a new therapeutic option for delaying AD progression.
Assuntos
Doença de Alzheimer/metabolismo , Quinase I-kappa B/metabolismo , Células Mieloides/metabolismo , Placa Amiloide/metabolismo , Doença de Alzheimer/patologia , Peptídeos beta-Amiloides/metabolismo , Animais , Células Cultivadas , Deleção de Genes , Humanos , Quinase I-kappa B/deficiência , Quinase I-kappa B/genética , Camundongos , Camundongos Endogâmicos C57BL , Microglia/metabolismo , Transporte ProteicoRESUMO
Tetrameric H10/12 helix stabilization was achieved by the application of aromatic side-chains in ß-peptide oligomers by intramolecular backbone-side chain CH-π interactions. Because of the enlarged hydrophobic surface of the oligomers, a further aim was the investigation of the self-assembly in a polar medium for the ß-peptide H10/12 helices. NMR, ECD, and molecular modeling results indicated that the oligomers formed by cis-[1S,2S]- or cis-[1R,2R]-1-amino-1,2,3,4-tetrahydronaphthalene-2-carboxylic acid (ATENAC) and cis-[1R,2S]- or cis-[1S,2R]-2-aminocyclohex-3-enecarboxylic acid (ACHEC) residues promote stable H10/12 helix formation with an alternating backbone configuration even at the tetrameric chain length. These results support the view that aromatic side-chains can be applied for helical structure stabilization. Importantly, this is the first observation of a stable H10/12 helix with tetrameric chain-length. The hydrophobically driven self-assembly was achieved for the helix-forming oligomers, seen as vesicles in transmission electron microscopy images. The self-association phenomenon, which supports the helical secondary structure of these oligomers, depends on the hydrophobic surface area, because a higher number of aromatic side-chains yielded larger vesicles. These results serve as an essential element for the design of helices relating to the H10/12 helix. Moreover, they open up a novel area for bioactive foldamer construction, while the hydrophobic area gained through the aromatic side-chains may yield important receptor-ligand interaction surfaces, which can provide amplified binding strength.
Assuntos
Peptídeos/química , Dicroísmo Circular , Medição da Troca de Deutério , Interações Hidrofóbicas e Hidrofílicas , Peptídeos/síntese química , Dobramento de Proteína , Estrutura Secundária de ProteínaRESUMO
The involvement of B cells, complement activation and subsequent immune complex deposition has all been implicated in the pathogenesis of rheumatoid arthritis (RA). Although the reduced expression of complement receptor 1 (CR1, CD35) and 2 (CR2, CD21) on the B cells of RA patients has been known for a long time, their exact role in B-cell tolerance and autoimmunity is not yet fully understood. To get a deeper insight into the possible mechanisms, we studied the expression and function of CR1 and CR2 on various subsets of B cells of healthy donors and RA patients at various stages of the disease by FACS analysis, (3)H-thymidine incorporation and ELISA. We found that CD19(+)CD27(-) naive B cells up-regulate the expression of the inhibitory CR1 during differentiation to CD19(+)CD27(+) memory B cells both in healthy donors and in RA patients, whereas the expression of the activatory CR2 is down-regulated. This clearly demonstrates that the expression of these two antagonistic complement receptors is regulated differentially during the development of human B cells, a phenomenon which may influence the maintenance of peripheral B-cell tolerance. Our functional studies show that after clustering CR1 both by its natural ligand and To5 mAb, the inhibitory function of CD35 is maintained in RA patients, despite its significantly reduced expression compared with healthy individuals. Besides blocking B-cell receptor-induced proliferation, CR1 inhibits the differentiation of B cells to plasmablasts and their immunoglobulin production. Since the reduced expression of CR1 in RA patients does not affect its inhibitory function, this receptor might serve as a new target for therapeutical interventions.
Assuntos
Artrite Reumatoide/imunologia , Linfócitos B/imunologia , Imunidade Humoral , Receptores de Complemento 3b/imunologia , Receptores de Complemento 3d/imunologia , Adulto , Idoso , Antígenos CD19/genética , Antígenos CD19/imunologia , Artrite Reumatoide/patologia , Autoimunidade , Linfócitos B/patologia , Estudos de Casos e Controles , Diferenciação Celular , Ensaio de Imunoadsorção Enzimática , Feminino , Citometria de Fluxo , Regulação da Expressão Gênica/imunologia , Humanos , Memória Imunológica , Masculino , Pessoa de Meia-Idade , Tolerância Periférica , Receptores de Antígenos de Linfócitos B/genética , Receptores de Antígenos de Linfócitos B/imunologia , Receptores de Complemento 3b/genética , Receptores de Complemento 3d/genética , Transdução de Sinais , Membro 7 da Superfamília de Receptores de Fatores de Necrose Tumoral/genética , Membro 7 da Superfamília de Receptores de Fatores de Necrose Tumoral/imunologiaRESUMO
Neuronal hyperexcitability is a phenomenon associated with early Alzheimer's disease. The underlying mechanism is considered to involve excessive activation of glutamate receptors; however, the exact molecular pathway remains to be determined. Extracellular recording from the CA1 of hippocampal slices is a long-standing standard for a range of studies both in basic research and in neuropharmacology. Evoked field potentials (fEPSPs) are regarded as the input, while spiking rate is regarded as the output of the neuronal network; however, the relationship between these two phenomena is not fully clear. We investigated the relationship between spontaneous spiking and evoked fEPSPs using mouse hippocampal slices. Blocking AMPA receptors (AMPARs) with CNQX abolished fEPSPs, but left firing rate unchanged. NMDA receptor (NMDAR) blockade with MK801 decreased neuronal spiking dose dependently without altering fEPSPs. Activating NMDARs by small concentration of NMDA induced a trend of increased firing. These results suggest that fEPSPs are mediated by synaptic activation of AMPARs, while spontaneous firing is regulated by the activation of extrasynaptic NMDARs. Synaptotoxic Abeta(1-42) increased firing activity without modifying evoked fEPSPs. This hyperexcitation was prevented by ifenprodil, an antagonist of the NR2B NMDARs. Overall, these results suggest that Abeta(1-42) induced neuronal overactivity is not dependent on AMPARs but requires NR2B.
Assuntos
Potenciais de Ação/fisiologia , Peptídeos beta-Amiloides/farmacologia , Região CA1 Hipocampal/fisiologia , Potenciais Pós-Sinápticos Excitadores/fisiologia , Neurônios/fisiologia , Fragmentos de Peptídeos/farmacologia , Receptores de N-Metil-D-Aspartato/fisiologia , Potenciais de Ação/efeitos dos fármacos , Animais , Região CA1 Hipocampal/efeitos dos fármacos , Potenciais Pós-Sinápticos Excitadores/efeitos dos fármacos , Potenciação de Longa Duração/efeitos dos fármacos , Camundongos , Camundongos Endogâmicos BALB C , Neurônios/efeitos dos fármacos , Receptores de AMPA/fisiologiaRESUMO
Deficient energy metabolism and network hyperactivity are the early symptoms of Alzheimer's disease (AD). In this study, we show that administration of exogenous oxidative energy substrates (OES) corrects neuronal energy supply deficiency that reduces the amyloid-beta-induced abnormal neuronal activity in vitro and the epileptic phenotype in AD model in vivo. In vitro, acute application of protofibrillar amyloid-ß1-42 (Aß1-42) induced aberrant network activity in wild-type hippocampal slices that was underlain by depolarization of both the neuronal resting membrane potential and GABA-mediated current reversal potential. Aß1-42 also impaired synaptic function and long-term potentiation. These changes were paralleled by clear indications of impaired energy metabolism, as indicated by abnormal NAD(P)H signaling induced by network activity. However, when glucose was supplemented with OES pyruvate and 3-beta-hydroxybutyrate, Aß1-42 failed to induce detrimental changes in any of the above parameters. We administered the same OES as chronic supplementation to a standard diet to APPswe/PS1dE9 transgenic mice displaying AD-related epilepsy phenotype. In the ex-vivo slices, we found neuronal subpopulations with significantly depolarized resting and GABA-mediated current reversal potentials, mirroring abnormalities we observed under acute Aß1-42 application. Ex-vivo cortex of transgenic mice fed with standard diet displayed signs of impaired energy metabolism, such as abnormal NAD(P)H signaling and strongly reduced tolerance to hypoglycemia. Transgenic mice also possessed brain glycogen levels twofold lower than those of wild-type mice. However, none of the above neuronal and metabolic dysfunctions were observed in transgenic mice fed with the OES-enriched diet. In vivo, dietary OES supplementation abated neuronal hyperexcitability, as the frequency of both epileptiform discharges and spikes was strongly decreased in the APPswe/PS1dE9 mice placed on the diet. Altogether, our results suggest that early AD-related neuronal malfunctions underlying hyperexcitability and energy metabolism deficiency can be prevented by dietary supplementation with native energy substrates.
Assuntos
Doença de Alzheimer/metabolismo , Doença de Alzheimer/fisiopatologia , Precursor de Proteína beta-Amiloide/genética , Neurônios/fisiologia , Presenilina-1/genética , Ácido 3-Hidroxibutírico/administração & dosagem , Ácido 3-Hidroxibutírico/farmacologia , Peptídeos beta-Amiloides/metabolismo , Animais , Encéfalo/efeitos dos fármacos , Encéfalo/metabolismo , Líquido Cefalorraquidiano , Dieta , Metabolismo Energético , Feminino , Hipocampo/metabolismo , Hipocampo/fisiopatologia , Técnicas In Vitro , Masculino , Camundongos , Camundongos Transgênicos , NADP/metabolismo , Ácido Pirúvico/administração & dosagem , Ácido Pirúvico/farmacologia , Transdução de Sinais , Sinapses/fisiologiaRESUMO
In the last years, extracellular vesicles (EVs), secreted by various cells and body fluids have shown extreme potential in biomedical applications. Increasing number of studies suggest that a protein corona could adhere to the surface of EVs which can have a fundamental effect on their function, targeting and therapeutical efficacy. However, removing and identifying these corona members is currently a challenging task to achieve. In this study we have employed red blood cell-derived extracellular vesicles (REVs) as a model system and three membrane active antimicrobial peptides (AMPs), LL-37, FK-16 and CM15, to test whether they can be used to remove protein corona members from the surface of vesicles. These AMPs were reported to preferentially exert their membrane-related activity via one of the common helical surface-covering models and do not significantly affect the interior of lipid bilayer bodies. The interaction between the peptides and the REVs was followed by biophysical techniques, such as flow-linear dichroism spectroscopy which provided the effective applicable peptide concentration for protein removal. REV samples were then subjected to subsequent size exclusion chromatography and to proteomics analysis. Based on the comparison of control REVs with the peptide treated samples, seventeen proteins were identified as external protein corona members. From the three investigated AMPs, FK-16 can be considered as the best candidate to further optimize EV-related applicability of AMPs. Our results on the REV model system envisage that membrane active peptides may become a useful set of tools in engineering and modifying surfaces of EVs and other lipid-based natural particles.
RESUMO
The proline-, glutamate-, valine-, and lysine-rich (PEVK) domain of the giant muscle protein titin is thought to be an intrinsically unstructured random-coil segment. Various observations suggest, however, that the domain may not be completely devoid of internal interactions and structural features. To test the validity of random polymer models for PEVK, we determined the mean end-to-end distances of an 11- and a 21-residue synthetic PEVK peptide, calculated from the efficiency of the fluorescence resonance energy transfer (FRET) between an N-terminal intrinsic tryptophan donor and a synthetically added C-terminal IAEDANS acceptor obtained in steady-state and time-resolved experiments. We find that the contour-length scaling of mean end-to-end distance deviates from predictions of a purely statistical polymer chain. Furthermore, the addition of guanidine hydrochloride decreased, whereas the addition of salt increased the FRET efficiency, pointing at the disruption of structure-stabilizing interactions. Increasing temperature between 10 and 50°C increased the normalized FRET efficiency in both peptides but with different trajectories, indicating that their elasticity and conformational stability are different. Simulations suggest that whereas the short PEVK peptide displays an overall random structure, the long PEVK peptide retains residual, loose helical configurations. Transitions in the local structure and dynamics of the PEVK domain may play a role in the modulation of passive muscle mechanics.
Assuntos
Transferência Ressonante de Energia de Fluorescência , Proteínas Musculares/química , Proteínas Quinases/química , Sequência de Aminoácidos , Conectina , Guanidina/farmacologia , Simulação de Dinâmica Molecular , Dados de Sequência Molecular , Concentração Osmolar , Desnaturação Proteica/efeitos dos fármacos , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , TemperaturaRESUMO
Neurodegenerative diseases (NDDs) are characterized by progressive deterioration of the structure and function of cells and their networks in the nervous system. There are currently no drugs or other treatments that can stop the progression of NDDs. NDDs have many similarities and common pathways, e.g., formation of misfolded amyloid proteins, intra- and extracellular amyloid deposits, and chronic inflammation. Initially, the inflammation process has a cytoprotective function; however, an elevated and prolonged immune response has damaging effects and causes cell death. Neuroinflammation has been a target of drug development for treating and curing NDDs. Treatment of different NDDs with non-steroid anti-inflammatory drugs (NSAIDs) has failed or has given inconsistent results. The use of NSAIDs in diagnosed Alzheimer's disease is currently not recommended. Sigma-1 receptor (Sig-1R) is a novel target for NDD drug development. Sig-1R plays a key role in cellular stress signaling, and it regulates endoplasmic reticulum stress and unfolded protein response. Activation of Sig-1R provides neuroprotection in cell cultures and animal studies. Clinical trials demonstrated that several Sig-1R agonists (pridopidine, ANAVEX3-71, fluvoxamine, dextrometorphan) and their combinations have a neuroprotective effect and slow down the progression of distinct NDDs.