RESUMO
Alzheimer's disease (AD) is a progressive neurodegenerative disease characterized by amyloid plaques and cognitive decline, the latter of which is thought to be driven by soluble oligomeric amyloid-ß (oAß). The dysregulation of G protein-gated inwardly rectifying K+ (GIRK; also known as Kir3) channels has been implicated in rodent models of AD. Here, seeking mechanistic insights, we uncovered a sex-dependent facet of GIRK-dependent signaling in AD-related amyloid pathophysiology. Synthetic oAß1-42 suppressed GIRK-dependent signaling in hippocampal neurons from male mice, but not from female mice. This effect required cellular prion protein, the receptor mGluR5, and production of arachidonic acid by the phospholipase PLA2. Although oAß suppressed GIRK channel activity only in male hippocampal neurons, intrahippocampal infusion of oAß or genetic suppression of GIRK channel activity in hippocampal pyramidal neurons impaired performance on a memory test in both male and female mice. Moreover, genetic enhancement of GIRK channel activity in hippocampal pyramidal neurons blocked oAß-induced cognitive impairment in both male and female mice. In APP/PS1 AD model mice, GIRK-dependent signaling was diminished in hippocampal CA1 pyramidal neurons from only male mice before cognitive deficit was detected. However, enhancing GIRK channel activity rescued cognitive deficits in older APP/PS1 mice of both sexes. Thus, whereas diminished GIRK channel activity contributes to cognitive deficits in male mice with increased oAß burden, enhancing its activity may have therapeutic potential for both sexes.
Assuntos
Doença de Alzheimer , Peptídeos beta-Amiloides , Canais de Potássio Corretores do Fluxo de Internalização Acoplados a Proteínas G , Hipocampo , Animais , Canais de Potássio Corretores do Fluxo de Internalização Acoplados a Proteínas G/metabolismo , Canais de Potássio Corretores do Fluxo de Internalização Acoplados a Proteínas G/genética , Peptídeos beta-Amiloides/metabolismo , Feminino , Masculino , Hipocampo/metabolismo , Camundongos , Doença de Alzheimer/metabolismo , Doença de Alzheimer/genética , Neurônios/metabolismo , Camundongos Transgênicos , Camundongos Endogâmicos C57BL , Receptor de Glutamato Metabotrópico 5/metabolismo , Receptor de Glutamato Metabotrópico 5/genética , Fragmentos de Peptídeos/metabolismo , Fragmentos de Peptídeos/farmacologia , Fragmentos de Peptídeos/genética , Caracteres Sexuais , Modelos Animais de Doenças , Fosfolipases A2/metabolismo , HumanosRESUMO
Pseudomonas aeruginosa is a frequent cause of antimicrobial-resistant hospital-acquired pneumonia, especially in critically ill patients. Inflammation triggered by P. aeruginosa infection is necessary for bacterial clearance but must be spatially and temporally regulated to prevent further tissue damage and bacterial dissemination. Emerging data have shed light on the pro-resolving actions of angiotensin-(1-7) [Ang-(1-7)] signaling through the G protein-coupled receptor Mas (MasR) during infections. Herein, we investigated the role of the Ang-(1-7)/Mas axis in pneumonia caused by P. aeruginosa by using genetic and pharmacological approach and found that Mas receptor-deficient animals developed a more severe form of pneumonia showing higher neutrophilic infiltration into the airways, bacterial load, cytokines, and chemokines production and more severe pulmonary damage. Conversely, treatment of pseudomonas-infected mice with Ang-(1-7) was able to decrease neutrophilic infiltration in airways and lungs, local and systemic levels of pro-inflammatory cytokines and chemokines, and increase the efferocytosis rates, mitigating lung damage/dysfunction caused by infection. Notably, the therapeutic association of Ang-(1-7) with antibiotics improved the survival rates of mice subjected to lethal inoculum of P. aeruginosa, extending the therapeutic window for imipenem. Mechanistically, Ang-(1-7) increased phagocytosis of bacteria by neutrophils and macrophages to accelerate pathogen clearance. Altogether, harnessing the Ang-(1-7) pathway during infection is a potential strategy for the development of host-directed therapies to promote mechanisms of resistance and resilience to pneumonia.
Assuntos
Angiotensina I , Antibacterianos , Camundongos Endogâmicos C57BL , Fragmentos de Peptídeos , Proto-Oncogene Mas , Infecções por Pseudomonas , Pseudomonas aeruginosa , Receptores Acoplados a Proteínas G , Animais , Angiotensina I/metabolismo , Pseudomonas aeruginosa/efeitos dos fármacos , Camundongos , Infecções por Pseudomonas/tratamento farmacológico , Infecções por Pseudomonas/metabolismo , Infecções por Pseudomonas/microbiologia , Fragmentos de Peptídeos/metabolismo , Fragmentos de Peptídeos/farmacologia , Receptores Acoplados a Proteínas G/metabolismo , Antibacterianos/farmacologia , Antibacterianos/uso terapêutico , Proteínas Proto-Oncogênicas/metabolismo , Proteínas Proto-Oncogênicas/genética , Pneumonia Bacteriana/tratamento farmacológico , Pneumonia Bacteriana/microbiologia , Pneumonia Bacteriana/patologia , Pneumonia Bacteriana/metabolismo , Citocinas/metabolismo , Camundongos Knockout , Pneumonia/tratamento farmacológico , Pneumonia/metabolismo , Pneumonia/microbiologia , Masculino , Pulmão/microbiologia , Pulmão/metabolismo , Pulmão/patologia , Transdução de Sinais/efeitos dos fármacos , Infiltração de Neutrófilos/efeitos dos fármacosRESUMO
The life cycle of Ebola and Marburg viruses includes a step of the virion envelope fusion with the cell membrane. Here, we analyzed whether the fusion of liposome membranes under the action of fragments of fusion peptides of Ebola and Marburg viruses depends on the composition of lipid vesicles. A fluorescence assay and electron microscopy were used to quantify the fusogenic activity of the virus fusion peptides and to identify the lipid determinants affecting membrane merging. Differential scanning calorimetry of lipid phase transitions revealed alterations in the physical properties of the lipid matrix produced by virus fusion peptides. Additionally, we found that plant polyphenols, quercetin, and myricetin inhibited vesicle fusion induced by the Marburg virus fusion peptide.
Assuntos
Ebolavirus , Flavonoides , Marburgvirus , Ebolavirus/efeitos dos fármacos , Marburgvirus/efeitos dos fármacos , Marburgvirus/química , Flavonoides/química , Flavonoides/farmacologia , Fusão de Membrana/efeitos dos fármacos , Lipossomos/química , Quercetina/química , Quercetina/farmacologia , Internalização do Vírus/efeitos dos fármacos , Doença pelo Vírus Ebola/virologia , Polifenóis/química , Polifenóis/farmacologia , Proteínas Virais de Fusão/química , Proteínas Virais de Fusão/metabolismo , Humanos , Membrana Celular/metabolismo , Peptídeos/química , Peptídeos/farmacologia , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/farmacologiaRESUMO
Background: Impairment of synaptic plasticity along with the formation of amyloid-ß (Aß) plaques and tau-protein neurofibrillary tangles have been associated with Alzheimer's disease (AD). Earlier studies with rat and mouse hippocampal slices have revealed the association of AD with the absence of synthesis of memory related proteins leading to impairment in cognitive functions. The role of hydrogen sulfide (H2S), a gaseous neurotransmitter, has been gaining attention as a neuroprotective agent. However, its role in AD-like conditions has not been studied so far. Objective: To study the neuroprotective role of H2S in AD conditions using rat hippocampal slices and the organic molecule GYY4137, a slow releasing H2S donor. Methods: Electrophysiological recordings were carried out in rat hippocampal slices to look into the impairment of LTP, a cellular correlate of memory. The Aß42 peptide was bath-applied to mimic AD-like conditions and checked for both late-LTP and synaptic tagging and capture (STC) mechanisms of the synapses. GYY4137 was applied to look into its neuroprotective role at different stages during the recording of fEPSP. Results: There has been a steady decline in the plasticity properties of the synapses, in the form of late-LTP and STC, after the application of Aß42 peptide in the hippocampal slices. However, application of GYY4137 rescued these conditions in vitro. Conclusions: GYY4137, with its slow release of H2S, could possibly act as a therapeutic agent in cognitive dysfunctions of the brain, mainly AD.
Assuntos
Doença de Alzheimer , Peptídeos beta-Amiloides , Região CA1 Hipocampal , Sulfeto de Hidrogênio , Morfolinas , Compostos Organotiofosforados , Fragmentos de Peptídeos , Animais , Sulfeto de Hidrogênio/farmacologia , Sulfeto de Hidrogênio/metabolismo , Peptídeos beta-Amiloides/metabolismo , Doença de Alzheimer/metabolismo , Doença de Alzheimer/patologia , Região CA1 Hipocampal/efeitos dos fármacos , Região CA1 Hipocampal/metabolismo , Masculino , Ratos , Fragmentos de Peptídeos/farmacologia , Fragmentos de Peptídeos/metabolismo , Morfolinas/farmacologia , Compostos Organotiofosforados/farmacologia , Modelos Animais de Doenças , Plasticidade Neuronal/efeitos dos fármacos , Plasticidade Neuronal/fisiologia , Neurônios/efeitos dos fármacos , Neurônios/metabolismo , Ratos Wistar , Potenciação de Longa Duração/efeitos dos fármacos , Potenciação de Longa Duração/fisiologia , Fármacos Neuroprotetores/farmacologia , Potenciais Pós-Sinápticos Excitadores/efeitos dos fármacos , Potenciais Pós-Sinápticos Excitadores/fisiologiaRESUMO
Neurotransmission is critical for brain function, allowing neurons to communicate through neurotransmitters and neuropeptides. RVD-hemopressin (RVD-Hp), a novel peptide identified in noradrenergic neurons, modulates cannabinoid receptors CB1 and CB2. Unlike hemopressin (Hp), which induces anxiogenic behaviors via transient receptor potential vanilloid 1 (TRPV1) activation, RVD-Hp counteracts these effects, suggesting that it may block TRPV1. This study investigates RVD-Hp's role as a TRPV1 channel blocker using HEK293 cells expressing TRPV1-GFP. Calcium imaging and patch-clamp recordings demonstrated that RVD-Hp reduces TRPV1-mediated calcium influx and TRPV1 ion currents. Molecular docking and dynamics simulations indicated that RVD-Hp interacts with TRPV1's selectivity filter, forming stable hydrogen bonds and van der Waals contacts, thus preventing ion permeation. These findings highlight RVD-Hp's potential as a therapeutic agent for conditions involving TRPV1 activation, such as pain and anxiety.
Assuntos
Endocanabinoides , Simulação de Acoplamento Molecular , Canais de Cátion TRPV , Canais de Cátion TRPV/metabolismo , Canais de Cátion TRPV/antagonistas & inibidores , Humanos , Células HEK293 , Endocanabinoides/farmacologia , Endocanabinoides/metabolismo , Endocanabinoides/química , Cálcio/metabolismo , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/farmacologia , Fragmentos de Peptídeos/metabolismo , Simulação de Dinâmica Molecular , HemoglobinasRESUMO
Angiotensin-converting enzyme 2 (ACE2), a crucial element of the renin-angiotensin system (RAS), metabolizes angiotensin II into Ang (1-7), which then combines with the Mas receptor (MasR) to fulfill its protective role in various diseases. Nevertheless, the involvement of ACE2 in sepsis-induced cardiomyopathy (SIC) is still unexplored. In this study, our results revealed that CLP surgery dramatically impaired cardiac function accompanied with disruption of the balance between ACE2-Ang (1-7) and ACE-Ang II axis in septic heart tissues. Moreover, ACE2 knockin markedly alleviated sepsis induced RAS disorder, cardiac dysfunction and improved survival rate in mice, while ACE2 knockout significantly exacerbates these outcomes. Adoptive transfer of bone marrow cells and in vitro experiments showed the positive role of myeloid ACE2 by mitigating oxidative stress, inflammatory response, macrophage polarization and cardiomyocyte apoptosis by blocking NF-κB and STAT1 signals. However, the beneficial impacts were nullified by MasR antagonist A779. Collectively, these findings showed that ACE2 alleviated SIC by inhibiting M1 macrophage via activating the Ang (1-7)-MasR axis, highlight that ACE2 might be a promising target for the management of sepsis and SIC patients.
Assuntos
Enzima de Conversão de Angiotensina 2 , Cardiomiopatias , Macrófagos , NF-kappa B , Fator de Transcrição STAT1 , Sepse , Transdução de Sinais , Animais , Enzima de Conversão de Angiotensina 2/metabolismo , Enzima de Conversão de Angiotensina 2/genética , Sepse/complicações , Sepse/metabolismo , NF-kappa B/metabolismo , Cardiomiopatias/metabolismo , Camundongos , Fator de Transcrição STAT1/metabolismo , Macrófagos/metabolismo , Masculino , Camundongos Endogâmicos C57BL , Camundongos Knockout , Apoptose/efeitos dos fármacos , Sistema Renina-Angiotensina/efeitos dos fármacos , Receptores Acoplados a Proteínas G/metabolismo , Miócitos Cardíacos/metabolismo , Miócitos Cardíacos/efeitos dos fármacos , Miócitos Cardíacos/patologia , Estresse Oxidativo/efeitos dos fármacos , Angiotensina I/metabolismo , Angiotensina I/farmacologia , Proto-Oncogene Mas , Fragmentos de Peptídeos/metabolismo , Fragmentos de Peptídeos/farmacologia , Peptidil Dipeptidase A/metabolismo , Peptidil Dipeptidase A/genéticaRESUMO
Background: Aggregated forms of the amyloid-ß (Aß) peptides which form protofibrils and fibrils in the brain are signatures of Alzheimer's disease (AD). Aggregates are also recognized by microglia, which in early phases may be protective and in later phases contribute to the pathology. We have identified several small molecules, decoys which interfere with Aß oligomerization and induce other aggregation trajectories leading to aggregated macrostructures which are non-toxic. Objective: This study investigates whether the small-molecule decoys affect microglial activation in terms of cytokine secretion and phagocytosis of Aß peptide. Methods: The effects of the decoys (NSC 69318, NSC 100873, NSC 16224) were analyzed in a model of human THP-1 monocytes differentiated to microglia-like cells. The cells were activated by Aß40 and Aß42 peptides, respectively, and after treatment with each decoy the secreted levels of pro-inflammatory cytokines and the Aß phagocytosis were analyzed. Results: NSC16224, which generates a double-stranded aggregate of thin protofibrils, was found to block Aß40- and Aß42-induced increase in microglial secretion of pro-inflammatory cytokines. NSC 69318, selective for neurotoxicity of Aß42, and NSC 100873 did not significantly reduce the microglial activation in terms of cytokine secretion. The uptake of Aß42 was not affected by anyone of the decoys. Conclusions: Our findings open the possibility that the molecular decoys of Aß aggregation may block microglial activation by Aß40 and Aß42 in addition to blocking neurotoxicity as shown previously.
Assuntos
Peptídeos beta-Amiloides , Microglia , Fragmentos de Peptídeos , Fagocitose , Peptídeos beta-Amiloides/metabolismo , Peptídeos beta-Amiloides/toxicidade , Microglia/efeitos dos fármacos , Microglia/metabolismo , Humanos , Fagocitose/efeitos dos fármacos , Fragmentos de Peptídeos/toxicidade , Fragmentos de Peptídeos/farmacologia , Citocinas/metabolismo , Células THP-1RESUMO
Amyloid-beta peptide (Aß) is a neurotoxic constituent of senile plaques in the brains of Alzheimer's disease (AD) patients. The detailed mechanisms by which protein kinase C-delta (PKCδ) contributes to Aß toxicity is not yet entirely understood. Using fully differentiated primary rat cortical neurons, we found that inhibition of Aß25-35-induced PKCδ increased cell viability with restoration of neuronal morphology. Using cyclin D1, proliferating cell nuclear antigen (PCNA), and histone H3 phosphorylated at Ser-10 (p-Histone H3) as the respective markers for the G1-, S-, and G2/M-phases, PKCδ inhibition mitigated cell cycle reentry (CCR) and subsequent caspase-3 cleavage induced by both Aß25-35 and Aß1-42 in the post-mitotic cortical neurons. Upstream of PKCδ, signal transducers and activators of transcription (STAT)-3 mediated PKCδ induction, CCR, and caspase-3 cleavage upon Aß exposure. Downstream of PKCδ, aberrant neuronal CCR was triggered by overactivating cyclin-dependent kinase-5 (CDK5) via calpain2-dependent p35 cleavage into p25. Finally, PKCδ and CDK5 also contributed to Aß25-35 induction of p53-upregulated modulator of apoptosis (PUMA) in cortical neurons. Together, we demonstrated that, in the post-mitotic neurons exposed to Aßs, STAT3-dependent PKCδ expression triggers calpain2-mediated p35 cleavage into p25 to overactivate CDK5, thus leading to aberrant CCR, PUMA induction, caspase-3 cleavage, and ultimately apoptosis.
Assuntos
Peptídeos beta-Amiloides , Apoptose , Ciclo Celular , Córtex Cerebral , Neurônios , Proteína Quinase C-delta , Peptídeos beta-Amiloides/metabolismo , Animais , Neurônios/metabolismo , Neurônios/efeitos dos fármacos , Apoptose/efeitos dos fármacos , Ratos , Proteína Quinase C-delta/metabolismo , Córtex Cerebral/metabolismo , Córtex Cerebral/citologia , Ciclo Celular/efeitos dos fármacos , Quinase 5 Dependente de Ciclina/metabolismo , Fragmentos de Peptídeos/farmacologia , Fragmentos de Peptídeos/metabolismo , Caspase 3/metabolismo , Ratos Sprague-Dawley , Células Cultivadas , Transdução de Sinais/efeitos dos fármacosRESUMO
Autophagy is an evolutionarily ancient catabolic pathway and has recently emerged as an integral part of the innate immune system. While the core machinery of autophagy is well defined, the physiological regulation of autophagy is less understood. Here, we identify a C-terminal fragment of human hemoglobin A (HBA1, amino acids 111-132) in human bone marrow as a fast-acting non-inflammatory inhibitor of autophagy initiation. It is proteolytically released from full-length HBA1 by cathepsin E, trypsin or pepsin. Biochemical characterization revealed that HBA1(111-132) has an in vitro stability of 52 min in human plasma and adopts a flexible monomeric conformation in solution. Structure-activity relationship studies revealed that the C-terminal 13 amino acids of HBA1(120-132) are sufficient to inhibit autophagy, two charged amino acids (D127, K128) mediate solubility, and two serines (S125, S132) are required for function. Successful viruses like human immunodeficiency virus 1 (HIV-1) evolved strategies to subvert autophagy for virion production. Our results show that HBA1(120-132) reduced virus yields of lab-adapted and primary HIV-1. Summarizing, our data identifies naturally occurring HBA1(111-132) as a physiological, non-inflammatory antagonist of autophagy. Optimized derivatives of HBA1(111-132) may offer perspectives to restrict autophagy-dependent viruses.
Assuntos
Autofagia , HIV-1 , Humanos , HIV-1/metabolismo , HIV-1/fisiologia , Relação Estrutura-Atividade , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/metabolismo , Fragmentos de Peptídeos/farmacologia , Sequência de AminoácidosRESUMO
Treatment with melatonin is routinely prescribed for its potent antioxidant and cognitive-promoting effects, nevertheless, it has yet to find neuromodulatory effects in normal and disease conditions. Therefore, to investigate its neuromodulatory mechanisms, melatonin was systemically administered over 10 consecutive days to both intracortical normal saline- and amyloid-ß 1-42 (Aß) peptide-injected rats. At the behavioral level, treatment with melatonin was associated with reduced efficacy in restoring Aß-induced deficit in passive-avoidance memory. Whole-cell patch-clamp recordings from CA1 pyramidal neurons revealed that melatonin treatment reduced spontaneous and evoked intrinsic excitability in control rats while exerting a reduction of spontaneous, but not evoked activity, in the Aß-injected group. Interestingly, treatment with melatonin enhances after-hyperpolarization in control, but not Aß-injected rats. In contrast, our voltage-clamp study showed that Ih current is significantly enhanced by Aß injection, and this effect is further strengthened by treatment with melatonin in Aß-injected rats. Finally, we discovered that the transcription of melatonin receptors 1 (MT1) and 2 (MT2) is significantly upregulated in the hippocampi of Aß-injected rats. Collectively, our study demonstrates that systemic treatment with melatonin has differential neuromodulation on CA1 neuronal excitability, at least in part, via differential effects on after-hyperpolarization and Ih currents due to Aß-induced neurotoxicity.
Assuntos
Peptídeos beta-Amiloides , Hipocampo , Melatonina , Animais , Melatonina/farmacologia , Peptídeos beta-Amiloides/metabolismo , Ratos , Masculino , Hipocampo/metabolismo , Hipocampo/efeitos dos fármacos , Receptores de Melatonina/metabolismo , Fragmentos de Peptídeos/farmacologia , Receptor MT2 de Melatonina/metabolismo , Receptor MT1 de Melatonina/metabolismo , Ratos Sprague-Dawley , Técnicas de Patch-ClampRESUMO
BACKGROUND: Ventilator-induced lung injury (VILI) is a consequence of inflammation and increased alveolar-capillary membrane permeability due to alveolar hyperdistention or elevated intrapulmonary pressure, but the precise mechanisms remain unclear. The aim of the study was to analyze the mechanism by which angiotensin converting enzyme 2 (ACE2) alleviates endoplasmic reticulum stress (ERS) and protects alveolar cells from pyroptosis in VILI by regulating angiotensin (Ang)1-7/Mas. METHODS: VILI was induced in mice by mechanical ventilation by regulating the tidal volume. The alveolar cell line, A549, mimics VILI in vitro by cyclic stretch (CS). Ang (1-7) (100 nmol/L) was added to the medium. ERS was induced in cells by stimulating with tunicamycin (TM, 2 µg/mL). ERS was inhibited by tracheal instillation of 4-phenylbutyric acid (4-PBA) (1 mg/kg). ACE2's enzymatic function was activated or inhibited by subcutaneous injection of resorcinolnaphthalein (RES, 20 µg/kg) or MLN-4760 (20 µg/kg). pGLV-EF1a-GFP-ACE2 was instilled into the trachea to increase the protein expression of ACE2. The Ang (1-7) receptor, Mas, was antagonized by injecting A779 subcutaneously (80 µg/kg). RESULTS: ACE2 protein levels decreased after modeling. Ang (1-7) level was decreased and Ang II was accumulated. ERS was significantly induced in VILI mice, and pyroptosis was observed in cells. When ERS was inhibited, pyroptosis under the VILI condition was significantly inhibited. Ang (1-7) alleviated ERS and pyroptosis under CS. When ERS was continuously activated, the function of Ang (1-7) in inhibiting pyroptosis was blocked. Resorcinolnaphthalein (RES) effectively promoted Ang II conversion, alleviated the Ang (1-7) level in VILI, ameliorated lung injury, and inhibited ERS and cell pyroptosis. Inhibiting ACE2's function in VILI hindered the production of Ang (1-7), promoted the accumulation of Ang II, and exacerbated ERS and pyroptosis, along with lung injury. The Mas antagonist significantly blocked the inhibitory effects of ACE2 on ERS and pyroptosis in VILI. CONCLUSIONS: Reduced ACE2 expression in VILI is involved in ERS and pyroptosis-related injury. ACE2 can alleviate ERS in alveolar cells by catalyzing the production of Ang (1-7), thus inhibiting pyroptosis in VILI.
Assuntos
Angiotensina I , Enzima de Conversão de Angiotensina 2 , Estresse do Retículo Endoplasmático , Camundongos Endogâmicos C57BL , Fragmentos de Peptídeos , Proto-Oncogene Mas , Piroptose , Lesão Pulmonar Induzida por Ventilação Mecânica , Animais , Piroptose/efeitos dos fármacos , Lesão Pulmonar Induzida por Ventilação Mecânica/metabolismo , Lesão Pulmonar Induzida por Ventilação Mecânica/prevenção & controle , Estresse do Retículo Endoplasmático/efeitos dos fármacos , Enzima de Conversão de Angiotensina 2/metabolismo , Enzima de Conversão de Angiotensina 2/genética , Angiotensina I/metabolismo , Angiotensina I/farmacologia , Fragmentos de Peptídeos/metabolismo , Fragmentos de Peptídeos/farmacologia , Masculino , Camundongos , Peptidil Dipeptidase A/metabolismo , Peptidil Dipeptidase A/genética , Proteínas Proto-Oncogênicas/metabolismo , Receptores Acoplados a Proteínas G/metabolismo , HumanosRESUMO
Chromogranin A (CgA), a â¼ 49 kDa acidic secretory protein, is ubiquitously distributed in endocrine and neuroendocrine cells and neurons. As a propeptide, CgA is proteolytically cleaved to generate several peptides of biological importance, including pancreastatin (PST: hCgA250-301), Vasostatin 1 (VS1: hCgA1-76), and catestatin (CST: CgA 352-372). VS1 represents the most conserved fragment of CgA. A 20 amino acid domain within VS1 (CgA 47-66) exhibits potent antimicrobial and anti-inflammatory activities. Autism is known to be associated with inflammation. Therefore, we seek to test the hypothesis that VS1 modulates autism behaviors by reducing inflammation in the hippocampus. Treatment of C57BL/6 (B6) and BTBR (a mouse model of idiopathic autism) mice with VS1 revealed the following: BTBR mice showed a significant decrease in chamber time in the presence of a stranger or a novel object. Treatment with VS1 significantly increased chamber time in both cases, underscoring a crucial role for VS1 in improving behavioral deficits in BTBR mice. In contrast to chamber time, sniffing time in BTBR mice in the presence of a stranger was less compared to B6 control mice. VS1 did not improve this latter parameter. Surprisingly, sniffing time in BTBR mice in the presence of a novel object was comparable with B6 mice. Proinflammatory cytokines such as IL-6 and IL-1b, as well as other inflammatory markers, were elevated in BTBR mice, which were dramatically reduced after supplementation with VS1. Interestingly, even Beclin-1/p62, pAKT/AKT, and p-p70-S6K/p70-S6K ratios were notably reduced by VS1. We conclude that VS1 plays a crucial role in restoring autistic spectrum disorders (ASD) plausibly by attenuating neuroinflammation.
Assuntos
Transtorno Autístico , Cromogranina A , Modelos Animais de Doenças , Hipocampo , Doenças Neuroinflamatórias , Fragmentos de Peptídeos , Animais , Masculino , Camundongos , Transtorno Autístico/tratamento farmacológico , Transtorno Autístico/metabolismo , Cromogranina A/farmacologia , Cromogranina A/metabolismo , Hipocampo/metabolismo , Hipocampo/efeitos dos fármacos , Camundongos Endogâmicos C57BL , Doenças Neuroinflamatórias/tratamento farmacológico , Fragmentos de Peptídeos/farmacologiaRESUMO
Amyloid plaque formation in the brain is mainly responsible for the onset of Alzheimer's disease (AD). Structure-based peptides have gained importance in recent years, and rational design of the peptide sequences for the prevention of Aß-aggregation and related toxicity is imperative. In this study, we investigate the structural modification of tetrapeptides derived from the hydrophobic C-terminal region of Aß42 "VVIA-NH2" and its retro-sequence "AIVV-NH2." A preliminary screening of synthesized peptides through an MTT cell viability assay followed by a ThT fluorescence assay revealed a peptide 13 (Ala-Ile-Aib-Val-NH2) that showed protection against Aß-aggregation and associated neurotoxicity. The presence of the α-helix inducer "Aib" in peptide 13 manifested the conformational transition from cross-ß-sheets to α-helical content in Aß42. The absence of fibrils in electron microscopic analysis suggested the inhibitory potential of peptide 13. The HRMS, DLS, and ANS studies further confirmed the inhibitory activity of 13, and no cytotoxicity was observed. The structure-based peptide described herein is a promising amyloid-ß inhibitor and provides a new lead for the development of AD therapeutics.
Assuntos
Doença de Alzheimer , Peptídeos beta-Amiloides , Fragmentos de Peptídeos , Peptídeos beta-Amiloides/antagonistas & inibidores , Peptídeos beta-Amiloides/metabolismo , Humanos , Doença de Alzheimer/tratamento farmacológico , Doença de Alzheimer/metabolismo , Fragmentos de Peptídeos/antagonistas & inibidores , Fragmentos de Peptídeos/farmacologia , Fragmentos de Peptídeos/metabolismo , Sobrevivência Celular/efeitos dos fármacos , Relação Estrutura-Atividade , Oligopeptídeos/química , Oligopeptídeos/farmacologia , Oligopeptídeos/síntese química , Agregados Proteicos/efeitos dos fármacos , Estrutura Molecular , Relação Dose-Resposta a Droga , Fármacos Neuroprotetores/farmacologia , Fármacos Neuroprotetores/química , Fármacos Neuroprotetores/síntese químicaRESUMO
Ghrelin modulates several biological functions via selective activation of the growth hormone secretagogue receptor (GHSR). GHSR agonists may be useful for the treatment of anorexia and cachexia, while antagonists and inverse agonists may represent new drugs for the treatment of metabolic and substance use disorders. Thus, the identification and pharmacodynamic characterization of new GHSR ligands is of high interest. In the present work the label-free dynamic mass redistribution (DMR) assay has been used to evaluate the pharmacological activity of a panel of GHSR ligands. This includes the endogenous peptides ghrelin, desacyl-ghrelin and LEAP2(1-14). Among synthetic compounds, the agonists anamorelin and HM01, the antagonists HM04 and YIL-781, and the inverse agonist PF-05190457 have been tested, together with HM03, R011, and H1498 from patent literature. The DMR results have been compared to those obtained in parallel experiments with the calcium mobilization assay. Ghrelin, anamorelin, HM01, and HM03 behaved as potent full GHSR agonists. YIL-781 behaved as a partial GHSR agonist and R011 as antagonist in both the assays. LEAP2(1-14) resulted a GHSR inverse agonist in DMR but not in calcium mobilization assay. PF-05190457, HM04, and H1498 behaved as GHSR inverse agonists in DMR experiments, while they acted as antagonists in calcium mobilization studies. In conclusion, this study provided a systematic pharmacodynamic characterization of several GHSR ligands in two different pharmacological assays. It demonstrated that the DMR assay can be successfully used particularly to discriminate between antagonists and inverse agonists. This study may be useful for the selection of the most appropriate compounds to be used in future studies.
Assuntos
Cálcio , Grelina , Receptores de Grelina , Receptores de Grelina/agonistas , Receptores de Grelina/metabolismo , Receptores de Grelina/antagonistas & inibidores , Ligantes , Grelina/farmacologia , Grelina/metabolismo , Cálcio/metabolismo , Humanos , Animais , Cricetulus , Células CHO , Oligopeptídeos/farmacologia , Fragmentos de Peptídeos/farmacologia , Fragmentos de Peptídeos/metabolismo , Hidrazinas , Piperidinas , QuinazolinonasRESUMO
Amyloid ß peptide (Aß) aggregation and deposition are considered the main causes of Alzheimer's disease. In a previous study, we demonstrated that anionic Zn-phthalocyanine (ZnPc) can interact with the Aß peptide and inhibit the fibril-formation process. However, due to the inability of anionic ZnPc to cross the intact blood-brain barrier, we decided to explore the interaction of cationic methylated Zn-phthalocyanine (cZnPc) with the peptide. Using a ThT fluorescence assay, we observed that cZnPc dose-dependently and time-dependently inhibited Aß1-42 fibril levels under in vitro fibril-formation conditions. Electron microscopy revealed that it caused Aß1-42 peptides to form small aggregates. Western blotting and dot immunoblot oligomer experiments demonstrated that cZnPc increased rather than decreased the levels of oligomers from the very early stages of incubation. A binding assay confirmed that cZnPc could bind with the peptide. Docking simulations indicated that the oligomer species of Aß1-42 had a higher ability to interact with cZnPc. ANS fluorescence assay results indicated that cZnPc did not affect the hydrophobicity of the peptide. However, cZnPc significantly increased intrinsic tyrosine fluorescence of the peptide after 8 h of incubation in fibril-formation conditions. Importantly, cell culture experiments demonstrated that cZnPc did not exhibit any toxicity up to a concentration of 10 µM. Instead, it protected a neuronal cell line from Aß1-42-induced toxicity. Thus, our results suggest that cZnPc can affect the aggregation process of Aß1-42, rendering it non-toxic, which could be crucial for the therapy of Alzheimer's disease.
Assuntos
Doença de Alzheimer , Peptídeos beta-Amiloides , Indóis , Isoindóis , Compostos Organometálicos , Fragmentos de Peptídeos , Compostos de Zinco , Peptídeos beta-Amiloides/química , Peptídeos beta-Amiloides/metabolismo , Peptídeos beta-Amiloides/toxicidade , Doença de Alzheimer/metabolismo , Doença de Alzheimer/tratamento farmacológico , Doença de Alzheimer/patologia , Indóis/química , Indóis/farmacologia , Humanos , Compostos de Zinco/química , Compostos de Zinco/farmacologia , Compostos Organometálicos/farmacologia , Compostos Organometálicos/química , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/toxicidade , Fragmentos de Peptídeos/farmacologia , Agregados Proteicos/efeitos dos fármacos , Animais , Simulação de Acoplamento Molecular , Neurônios/efeitos dos fármacos , Neurônios/metabolismoRESUMO
The misfolding and aggregation of beta-amyloid (Aß) peptides have been implicated as key pathogenic events in the early stages of Alzheimer's disease (AD). Inhibiting Aß aggregation represents a potential disease-modifying therapeutic approach to AD treatment. Previous studies have identified various molecules that inhibit Aß aggregation, some of which share common chemical substructures (fragments) that may be key to their inhibitory activity. Employing fragment-based drug discovery (FBDD) methods may facilitate the identification of these fragments, which can subsequently be used to screen new inhibitors and provide leads for further drug development. In this study, we used an in silico FBDD approach to identify 17 fragment clusters that are significantly enriched among Aß aggregation inhibitors. These fragments were then used to screen anti-infective agents, a promising drug class for repurposing against amyloid aggregation. This screening process identified 16 anti-infective drugs, 5 of which were chosen for further investigation. Among the 5 candidates, anidulafungin, an antifungal compound, showed high efficacy in inhibiting Aß aggregation in vitro. Kinetic analysis revealed that anidulafungin selectively blocks the primary nucleation step of Aß aggregation, substantially delaying Aß fibril formation. Cell viability assays demonstrated that anidulafungin can reduce the toxicity of oligomeric Aß on BV2 microglia cells. Molecular docking simulations predicted that anidulafungin interacted with various Aß species, including monomers, oligomers, and fibrils, potentially explaining its activity against Aß aggregation and toxicity. This study suggests that anidulafungin is a potential drug to be repurposed for AD, and FBDD is a promising approach for discovering drugs to combat Aß aggregation.
Assuntos
Doença de Alzheimer , Peptídeos beta-Amiloides , Anidulafungina , Descoberta de Drogas , Reposicionamento de Medicamentos , Doença de Alzheimer/tratamento farmacológico , Doença de Alzheimer/metabolismo , Reposicionamento de Medicamentos/métodos , Peptídeos beta-Amiloides/metabolismo , Descoberta de Drogas/métodos , Humanos , Anidulafungina/farmacologia , Animais , Equinocandinas/farmacologia , Equinocandinas/química , Simulação de Acoplamento Molecular/métodos , Fragmentos de Peptídeos/farmacologia , Fragmentos de Peptídeos/metabolismoRESUMO
BACKGROUND: Cardiovascular disease remains one of the leading causes of death globally. Myocardial ischemia and infarction, in particular, frequently cause disturbances in cardiac electrical activity that can trigger ventricular arrhythmias. We aimed to investigate whether catestatin, an endogenous catecholamine-inhibiting peptide, ameliorates myocardial ischemia-induced ventricular arrhythmias in rats and the underlying ionic mechanisms. METHODS AND RESULTS: Adult male Sprague-Dawley rats were randomly divided into control and catestatin groups. Ventricular arrhythmias were induced by ligation of the left anterior descending coronary artery and electrical stimulation. Action potential, transient outward potassium current, delayed rectifier potassium current, inward rectifying potassium current, and L-type calcium current (ICa-L) of rat ventricular myocytes were recorded using a patch-clamp technique. Catestatin notably reduced ventricular arrhythmia caused by myocardial ischemia/reperfusion and electrical stimulation of rats. In ventricular myocytes, catestatin markedly shortened the action potential duration of ventricular myocytes, which was counteracted by potassium channel antagonists TEACl and 4-AP, and ICa-L current channel agonist Bay K8644. In addition, catestatin significantly increased transient outward potassium current, delayed rectifier potassium current, and inward rectifying potassium current density in a concentration-dependent manner. Catestatin accelerated the activation and decelerated the inactivation of the transient outward potassium current channel. Furthermore, catestatin decreased ICa-L current density in a concentration-dependent manner. Catestatin also accelerated the inactivation of the ICa-L channel and slowed down the recovery of ICa-L from inactivation. CONCLUSIONS: Catestatin enhances the activity of transient outward potassium current, delayed rectifier potassium current, and inward rectifying potassium current, while suppressing the ICa-L in ventricular myocytes, leading to shortened action potential duration and ultimately reducing the ventricular arrhythmia in rats.
Assuntos
Potenciais de Ação , Cromogranina A , Miócitos Cardíacos , Fragmentos de Peptídeos , Ratos Sprague-Dawley , Animais , Masculino , Miócitos Cardíacos/efeitos dos fármacos , Miócitos Cardíacos/metabolismo , Cromogranina A/farmacologia , Cromogranina A/metabolismo , Potenciais de Ação/efeitos dos fármacos , Fragmentos de Peptídeos/farmacologia , Canais de Cálcio Tipo L/metabolismo , Canais de Cálcio Tipo L/efeitos dos fármacos , Arritmias Cardíacas/fisiopatologia , Arritmias Cardíacas/prevenção & controle , Arritmias Cardíacas/metabolismo , Antiarrítmicos/farmacologia , Ventrículos do Coração/efeitos dos fármacos , Ventrículos do Coração/metabolismo , Ventrículos do Coração/fisiopatologia , Canais de Potássio Corretores do Fluxo de Internalização/metabolismo , Canais de Potássio Corretores do Fluxo de Internalização/efeitos dos fármacos , Modelos Animais de Doenças , Bloqueadores dos Canais de Potássio/farmacologia , Ratos , Técnicas de Patch-Clamp , Canais de Potássio de Retificação Tardia/metabolismo , Canais de Potássio de Retificação Tardia/efeitos dos fármacos , Canais de Potássio/metabolismo , Canais de Potássio/efeitos dos fármacosRESUMO
Amyloid beta (Aß) aggregation is one of the distinctive pathological hallmarks of Alzheimer's disease (AD). Therefore, the development of effective inhibitors against Aß aggregate formation offers great promise for the treatment of AD. In this study, we designed a novel negatively charged functionalized conjugate aimed at inhibiting Aß42 aggregation and attenuating neurotoxicity by grafting polysialic acid with mannuronate oligosaccharide, a biocompatible glycan extracted from seaweeds, designated as polysialic acid-mannan conjugate (PSA-MOS). ThT, biological microscopy, TEM and CD confirmed the inhibition of Aß42 aggregation by PSA-MOS, as well as its ability to inhibit the conformational transition of Aß42 to ß-sheet. CCK-8 assay demonstrated that PSA-MOS was not cytotoxic to SH-SY5Y (p < 0.05) and promoted cell proliferation. In the Aß42-induced SH-SY5Y injury models, PSA-MOS dose-dependently ameliorated cytotoxicity (p < 0.0001) and significantly reduced the levels of inflammatory factors of IL-1ß (p < 0.0001), IL-6 (p < 0.0001) and TNF-α (p < 0.05). MD simulations demonstrated that PSA-MOS effectively impeded the α-helix to ß-sheet transition of the Aß42 monomer via electrostatic interactions with its CTR and NTR regions. These findings demonstrate the therapeutic potential of PSA-MOS as promising glycoconjugate for the treatment of AD.
Assuntos
Peptídeos beta-Amiloides , Inflamação , Ácidos Siálicos , Peptídeos beta-Amiloides/metabolismo , Peptídeos beta-Amiloides/química , Humanos , Ácidos Siálicos/química , Ácidos Siálicos/farmacologia , Inflamação/tratamento farmacológico , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/farmacologia , Neurônios/efeitos dos fármacos , Neurônios/metabolismo , Linhagem Celular Tumoral , Mananas/farmacologia , Mananas/química , Proliferação de Células/efeitos dos fármacos , Doença de Alzheimer/tratamento farmacológico , Doença de Alzheimer/metabolismo , Simulação de Dinâmica Molecular , Ácidos HexurônicosRESUMO
Renin-angiotensin-aldosterone system plays a crucial role in the regulation of blood pressure and fluid homeostasis. It is reported to be involved in mediating osteoclastogenesis and bone loss in diseases of inflammatory bone resorption such as osteoporosis. Angiotensin-(1-7), a product of Angiotensin I and II (Ang I, II), is cleaved by Angiotensin-converting enzyme 2 and then binds to Mas receptor to counteract inflammatory effects produced by Ang II. However, the mechanism by which Ang-(1-7) reduces bone resorption remains unclear. Therefore, we aim to elucidate the effects of Ang-(1-7) on lipopolysaccharide (LPS)-induced osteoclastogenesis. In vivo, mice were supracalvarial injected with Ang-(1-7) or LPS ± Ang-(1-7) subcutaneously. Bone resorption and osteoclast formation were compared using micro-computed tomography, tartrate-resistant acid phosphatase (TRAP) stain, and real-time PCR. We found that Ang-(1-7) attenuated tumor necrosis factor (TNF)-α, TRAP, and Cathepsin K expression from calvaria and decreased osteoclast number along with bone resorption at the suture mesenchyme. In vitro, RANKL/TNF-α ± Ang-(1-7) was added to cultures of bone marrow-derived macrophages (BMMs) and osteoclast formation was measured via TRAP staining. The effect of Ang-(1-7) on LPS-induced osteoblasts RANKL expression and peritoneal macrophages TNF-α expression was also investigated. The effect of Ang-(1-7) on the MAPK and NF-κB pathway was studied by Western blotting. As a result, Ang-(1-7) reduced LPS-stimulated macrophages TNF-α expression and inhibited the MAPK and NF-κB pathway activation. However, Ang-(1-7) did not affect osteoclastogenesis induced by RANKL/TNF-α nor reduce osteoblasts RANKL expression in vitro. In conclusion, Ang-(1-7) alleviated LPS-induced osteoclastogenesis and bone resorption in vivo via inhibiting TNF-α expression in macrophages.