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Transthyretin (TTR) is an homotetrameric protein involved in the transport of thyroxine. More than 150 different mutations have been described in the TTR gene, several of them associated with familial amyloid cardiomyopathy. Recently, our group described a new variant of TTR in Brazil, namely A39D-TTR, which causes a severe cardiac condition. Position 39 is in the AB loop, a region of the protein that is located within the thyroxine-binding channels and is involved in tetramer formation. In the present study, we solved the structure and characterize the thermodynamic stability of this new variant of TTR using urea and high hydrostatic pressure. Interestingly, during the process of purification, A39D-TTR turned out to be a dimer and not a tetramer, a variation that might be explained by the close contact of the four aspartic acids at position 39, where they face each other inside the thyroxine channel. In the presence of subdenaturing concentrations of urea, bis-ANS binding and dynamic light scattering revealed A39D-TTR in the form of a molten-globule dimer. Co-expression of A39D and WT isoforms in the same bacterial cell did not produce heterodimers or heterotetramers, suggesting that somehow a negative charge at the AB loop precludes tetramer formation. A39D-TTR proved to be highly amyloidogenic, even at mildly acidic pH values where WT-TTR does not aggregate. Interestingly, despite being a dimer, aggregation of A39D-TTR was inhibited by diclofenac, which binds to the thyroxine channel in the tetramer, suggesting the existence of other pockets in A39D-TTR able to accommodate this molecule.
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Nonsteroidal anti-inflammatory drugs (NSAIDs) regulate inflammation, which is associated with their role in preventing neurodegenerative diseases in epidemiological studies. It has sparked interest in their unconventional application for reducing neuroinflammation, opening up new avenues in biomedical research. However, given the pharmacological drawbacks of NSAIDs, the development of formulations with naturally antioxidant/anti-inflammatory dietary fatty acids has been demonstrated to be advantageous for the clinical translation of anti-inflammatory-based therapies. It includes improved blood-brain barrier (BBB) permeability and reduced toxicity. It permits us to speculate about the value of linoleic acid (LA)-isomers in preventing and treating neuroinflammatory diseases compared to NSAIDs. Our research delved into the impact of various factors, such as administration route, dosage, timing of intervention, and BBB permeability, on the efficacy of NSAIDs and LA-isomers in preclinical and clinical settings. We conducted a systematic comparison between NSAIDs and LA-isomers regarding their therapeutic effectiveness, BBB compatibility, and side effects. Additionally, we explored their underlying mechanisms in addressing neuroinflammation. Through our analysis, we've identified challenges and drawn conclusions that could propel advancements in treating neurodegenerative diseases and inform the development of future alternative therapeutic strategies.
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Anti-Inflamatórios não Esteroides , Barreira Hematoencefálica , Suplementos Nutricionais , Ácido Linoleico , Doenças Neuroinflamatórias , Humanos , Animais , Anti-Inflamatórios não Esteroides/farmacologia , Anti-Inflamatórios não Esteroides/administração & dosagem , Doenças Neuroinflamatórias/tratamento farmacológico , Ácido Linoleico/farmacologia , Ácido Linoleico/química , Barreira Hematoencefálica/efeitos dos fármacos , Barreira Hematoencefálica/metabolismo , IsomerismoRESUMO
The involvement of innate immune mediators to the Zika virus (ZIKV)-induced neuroinflammation is not yet well known. Here, we investigated whether neutrophil extracellular traps (NETs), which are scaffolds of DNA associated with proteins, have the potential to injure peripheral nervous. The tissue lesions were evaluated after adding NETs to dorsal root ganglia (DRG) explants and to DRG constituent cells or injecting them into mouse sciatic nerves. Identification of NET harmful components was achieved by pharmacological inhibition of NET constituents. We found that ZIKV inoculation into sciatic nerves recruited neutrophils and elicited the production of the cytokines CXCL1 and IL-1ß, classical NET inducers, but did not trigger NET formation. ZIKV blocked PMA- and CXCL8-induced NET release, but, in contrast, the ZIKV nonstructural protein (NS)-1 induced NET formation. NET-enriched supernatants were toxic to DRG explants, decreasing neurite area, length, and arborization. NETs were toxic to DRG constituent cells and affected myelinating cells. Myeloperoxidase (MPO) and histones were identified as the harmful component of NETs. NS1 injection into mouse sciatic nerves recruited neutrophils and triggered NET release and caspase-3 activation, events that were also elicited by the injection of purified MPO. In summary, we found that ZIKV NS1 protein induces NET formation, which causes nervous tissue damages. Our findings reveal new mechanisms leading to neuroinflammation by ZIKV.
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Armadilhas Extracelulares , Infecção por Zika virus , Zika virus , Animais , Camundongos , Doenças Neuroinflamatórias , Nervo IsquiáticoRESUMO
BACKGROUND: Soluble oligomeric forms of alpha-synuclein (aSyn-O) are believed to be one of the main toxic species in Parkinson's disease (PD) leading to degeneration. aSyn-O can induce Ca2+ influx, over activating downstream pathways leading to PD phenotype. Calcineurin (CN), a phosphatase regulated by Ca2+ levels, activates NFAT transcription factors that are involved in the regulation of neuronal plasticity, growth, and survival. METHODS: Here, using a combination of cell toxicity and gene regulation assays performed in the presence of classical inhibitors of the NFAT/CN pathway, we investigate NFAT's role in neuronal degeneration induced by aSyn-O. RESULTS: aSyn-O are toxic to neurons leading to cell death, loss of neuron ramification and reduction of synaptic proteins which are reversed by CN inhibition with ciclosporin-A or VIVIT, a NFAT specific inhibitor. aSyn-O induce NFAT nuclear translocation and transactivation. We found that aSyn-O modulates the gene involved in the maintenance of synapses, synapsin 1 (Syn 1). Syn1 mRNA and protein and synaptic puncta are drastically reduced in cells treated with aSyn-O which are reversed by NFAT inhibition. CONCLUSIONS: For the first time a direct role of NFAT in aSyn-O-induced toxicity and Syn1 gene regulation was demonstrated, enlarging our understanding of the pathways underpinnings synucleinopathies.
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Doença de Parkinson , alfa-Sinucleína , Humanos , alfa-Sinucleína/genética , Linfócitos T , Homeostase , Apoptose , CalcineurinaRESUMO
Biomolecular condensates, membrane-less entities arising from liquid-liquid phase separation, hold dichotomous roles in health and disease. Alongside their physiological functions, these condensates can transition to a solid phase, producing amyloid-like structures implicated in degenerative diseases and cancer. This review thoroughly examines the dual nature of biomolecular condensates, spotlighting their role in cancer, particularly concerning the p53 tumor suppressor. Given that over half of the malignant tumors possess mutations in the TP53 gene, this topic carries profound implications for future cancer treatment strategies. Notably, p53 not only misfolds but also forms biomolecular condensates and aggregates analogous to other protein-based amyloids, thus significantly influencing cancer progression through loss-of-function, negative dominance, and gain-of-function pathways. The exact molecular mechanisms underpinning the gain-of-function in mutant p53 remain elusive. However, cofactors like nucleic acids and glycosaminoglycans are known to be critical players in this intersection between diseases. Importantly, we reveal that molecules capable of inhibiting mutant p53 aggregation can curtail tumor proliferation and migration. Hence, targeting phase transitions to solid-like amorphous and amyloid-like states of mutant p53 offers a promising direction for innovative cancer diagnostics and therapeutics.
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Neoplasias , Ácidos Nucleicos , Humanos , Proteína Supressora de Tumor p53/genética , Proteína Supressora de Tumor p53/química , Proteína Supressora de Tumor p53/metabolismo , Agregados Proteicos , Neoplasias/metabolismo , Amiloide/químicaRESUMO
The aggregation of epitopes that are also able to bind major histocompatibility complex (MHC) alleles raises questions around the potential connection between the formation of epitope aggregates and their affinities to MHC receptors. We first performed a general bioinformatic assessment over a public dataset of MHC class II epitopes, finding that higher experimental binding correlates with higher aggregation-propensity predictors. We then focused on the case of P10, an epitope used as a vaccine candidate against Paracoccidioides brasiliensis that aggregates into amyloid fibrils. We used a computational protocol to design variants of the P10 epitope to study the connection between the binding stabilities towards human MHC class II alleles and their aggregation propensities. The binding of the designed variants was tested experimentally, as well as their aggregation capacity. High-affinity MHC class II binders in vitro were more disposed to aggregate forming amyloid fibrils capable of binding Thioflavin T and congo red, while low affinity MHC class II binders remained soluble or formed rare amorphous aggregates. This study shows a possible connection between the aggregation propensity of an epitope and its affinity for the MHC class II cleft.
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Resumo O artigo apresenta resultados de pesquisas sobre percepção pública da ciência na pandemia no Brasil, realizadas no Centro de Estudos SoU_Ciência, sediado na Universidade Federal de São Paulo (Unifesp). Com o intuito de responder à pergunta: "A pandemia da COVID-19 alterou a percepção da sociedade brasileira sobre ciência, cientistas e universidades?", realizamos estudos quantitativos e qualitativos entre agosto/2021 e julho/2022. Em levantamentos quantitativos nacionais de opinião pública, coletamos dados exclusivos de série histórica de enquetes sobre o tema no Brasil, e em grupos focais, aprofundamos estudos sobre percepção e posicionamento político de diferentes segmentos sociais. Em meio ao quadro de crescimento do negacionismo científico, retrocessos políticos e sociais, desmonte de políticas públicas, especificamente científicas e tecnológicas, decorrentes do impeachment de 2016 e da eleição de Bolsonaro em 2018, as pesquisas indicam, aparentemente contrariando a tendência política obscurantista, uma expressiva ampliação do interesse público pela ciência na pandemia no país. Este trabalho analisa a emergência de uma "onda pró-ciência" na opinião pública no Brasil, os fatores que propiciaram seu surgimento na pandemia e suas perspectivas na atualidade.
Abstract This article presents the results of our study on the public perception of science during the COVID-19 pandemic in Brazil, carried out at the Centro de Estudos SoU_Ciência, from Universidade Federal de São Paulo (UNIFESP). To answer the question: "Has the COVID-19 pandemic changed the perception of Brazilian society about science, scientists, and universities?", quantitative and qualitative studies were conducted between August 2021 and July 2022. In national quantitative public opinion surveys, we collected exclusive data from a historical series of polls on the subject in Brazil, and in focus groups, we deepened studies on the perception and political position of different social segments. Amid the growth of scientific denialism; political and social setbacks; and the dismantling of public policies, specifically scientific and technological, resulting from the impeachment of 2016 and the election of Bolsonaro in 2018, research indicates, apparently contrary to an obscurantist political tendency, a significant expansion of public interest in science during the pandemic in the country. This paper analyzes the emergence of a "pro-science wave" in public opinion in Brazil, the factors that led to its emergence during the pandemic, and its current prospects.
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Studies showed that JM-20, a benzodiazepine-dihydropyridine hybrid molecule, protects against rotenone and 6-hydroxydopamine neurotoxicity. However, its protective effects against cytotoxicity induced by endogenous neurotoxins involved in Parkinson's disease (PD) pathogenesis have never been investigated. In this study, we evaluated the ability of JM-20 to inhibit alpha-synuclein (aSyn) aggregation. We also evaluated the interactions of JM-20 with aSyn by molecular docking and molecular dynamics and assessed the protective effect of JM-20 against aminochrome cytotoxicity. We demonstrated that JM-20 induced the formation of heterogeneous amyloid fibrils, which were innocuous to primary cultures of mesencephalic cells. Moreover, JM-20 reduced the average size of aSyn positive inclusions in H4 cells transfected with SynT wild-type and synphilin-1-V5, but not in HEK cells transfected with synphilin-1-GFP. In silico studies showed the interaction between JM-20 and the aSyn-binding site. Additionally, we showed that JM-20 protects SH-SY5Y cells against aminochrome cytotoxicity. These results reinforce the potential of JM-20 as a neuroprotective compound for PD and suggest aSyn as a molecular target for JM-20.
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Di-Hidropiridinas , Neuroblastoma , Doença de Parkinson , Humanos , alfa-Sinucleína , Benzodiazepinas , Simulação de Acoplamento Molecular , Doença de Parkinson/tratamento farmacológicoRESUMO
Synucleinopathies are a group of progressive neurodegenerative diseases known for the accumulation of insoluble aggregates containing the protein alpha-synuclein (aSyn). Recently, it has been assumed that pathology spreads in the brain during disease progression, implying that, at some point in the process, aSyn may exist outside of cells. In this context, extracellular-aSyn (e-aSyn) might transduce signals to the inside of the cells it interacts with, and/or be internalized by different types of cells through the extracellular matrix. Both negatively charged lipids and membrane receptors have been hypothesized as modulators of the loss of cellular homeostasis and cytotoxicity, and of the internalization of e-aSyn. Internalized e-aSyn causes the disruption of multiple cellular processes such as the autophagy lysosomal pathway (ALP), mitochondrial function, endoplasmic reticulum (ER)-stress, UPR activation, or vesicular transport. These processes happen not only in neurons but also in glial cells, activating inflammatory or anti-inflammatory pathways that can affect both neuronal function and survival, thereby affecting disease progression. In this review, we explore possible effects e-aSyn, all the way from the extracellular matrix to the nucleus. In particular, we highlight the glial-neuronal relationship as this is particularly relevant in the context of the spreading of aSyn pathology in synucleinopathies.
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Sinucleinopatias , alfa-Sinucleína , Progressão da Doença , Humanos , Lisossomos/metabolismo , Neurônios/metabolismo , alfa-Sinucleína/metabolismoRESUMO
Aged and photoaged skin exhibit fine wrinkles that are signs of epidermal inflammation and degeneration. It has been shown that healthy elderly skin expresses amyloidogenic proteins, including α-Synuclein, which are known to oligomerize and trigger inflammation and neurodegeneration. However, little is known about their putative role in skin physiology and sensitivity. To unravel this possible role, we investigated the impact of oligomeric α-Synuclein (Oα-Syn) in 2D and 3D keratinocyte human models. Exogenous Oα-Syn caused degeneration of reconstructed human epidermis (RHE) by diminishing proliferation and thickness of the stratum basale. Oα-Syn also increased NF-kB nuclear translocation in keratinocytes and triggered inflammation in the RHE, by increasing expression of interleukin-1ß and tumor necrosis factor-alpha, and the release of tumor necrosis factor-alpha in a time-dependent manner. Dexamethasone and an IL-1ß inhibitor partially diminished RHE degeneration caused by Oα-Syn. These findings suggest that Oα-Syn induces epidermal inflammation and decreases keratinocyte proliferation, and therefore might contribute to epidermal degeneration observed in human skin aging.
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Fator de Necrose Tumoral alfa , alfa-Sinucleína , Idoso , Epiderme/metabolismo , Epiderme/patologia , Humanos , Inflamação/metabolismo , Queratinócitos/metabolismo , Fator de Necrose Tumoral alfa/metabolismo , alfa-Sinucleína/metabolismoRESUMO
Fungal infection is an important health problem in Latin America, and in Brazil in particular. Paracoccidioides (mainly P. brasiliensis and P. lutzii) is responsible for paracoccidioidomycosis, a disease that affects mainly the lungs. The glycoprotein gp43 is involved in fungi adhesion to epithelial cells, which makes this protein an interesting target of study. A specific stretch of 15 amino acids that spans the region 181-195 (named P10) of gp43 is an important epitope of gp43 that is being envisioned as a vaccine candidate. Here we show that synthetic P10 forms typical amyloid aggregates in solution in very short times, a property that could hamper vaccine development. Seeds obtained by fragmentation of P10 fibrils were able to induce the aggregation of P4, but not P23, two other peptides derived from gp43. In silico analysis revealed several regions within the P10 sequence that can form amyloid with steric zipper architecture. Besides, in-silico proteolysis studies with gp43 revealed that aggregation-prone, P10-like peptides could be generated by several proteases, which suggests that P10 could be formed under physiological conditions. Considering our data in the context of a potential vaccine development, we redesigned the sequence of P10, maintaining the antigenic region (HTLAIR), but drastically reducing its aggregation propensity.
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Amiloide/química , Antígenos de Neoplasias/química , Antígenos/química , Paracoccidioides/imunologia , Paracoccidioidomicose/prevenção & controle , Algoritmos , Animais , Antígenos de Fungos/imunologia , Dicroísmo Circular , Biologia Computacional/métodos , Simulação por Computador , Epitopos , Proteínas Fúngicas/química , Vacinas Fúngicas/imunologia , Glicoproteínas/química , Humanos , Técnicas In Vitro , Paracoccidioidomicose/imunologia , Peptídeos/química , Conformação Proteica , Dobramento de Proteína , Software , Solventes/química , Desenvolvimento de VacinasRESUMO
Despite evidence of health benefits from kefir administration, a systematic review with meta-analysis on bioactive compounds associated with these benefits is still absent in the literature. Kefir is fermented milk resulting from the metabolism of a complex microbiota in symbiosis. Recent researches have investigated the bioactive compounds responsible for the preventive and therapeutic effects attributed to kefir. However, differences in functional potential between industrial and artisanal kefir are still controversial. Firstly, we identified differences in the microbial composition among both types of kefir. Available evidence concerning the action of different bioactive compounds from kefir on health, both from in vitro and in vivo studies, was subsequently summarized to draw a primary conclusion of the dose and the intervention time for effect, the producer microorganisms, the precursor in the milk, and the action mechanism. Meta-analysis was performed to investigate the statistically significant differences (P < 0.05) between intervention and control and between both types of kefir for each health effect studied. In summary, the bioactive compounds more commonly reported were exopolysaccharides, including kefiran, bioactive peptides, and organic acids, especially lactic acid. Kefir bioactive compounds presented antimicrobial, anticancer, and immune-modulatory activities corroborated by the meta-analysis. However, clinical evidence is urgently needed to strengthen the practical applicability of these bioactive compounds. The mechanisms of their action were diverse, indicating that they can act by different signaling pathways. Still, industrial and artisanal kefir may differ regarding functional potential-OR of 8.56 (95% CI: 2.27-32.21, P ≤ .001)-according to the observed health effect, which can be associated with differences in the microbial composition between both types of kefir.
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Anti-Infecciosos/farmacologia , Antineoplásicos/farmacologia , Produtos Biológicos/farmacologia , Agentes de Imunomodulação/farmacologia , Kefir , Leite/química , Animais , Fermentação , HumanosRESUMO
The accumulation of protein aggregates in human tissues is a hallmark of more than 40 diseases called amyloidoses. In seven of these disorders, the aggregation is associated with neurodegenerative processes in the central nervous system such as Alzheimer's disease (AD), Parkinson's disease (PD), and Huntington's disease (HD). The aggregation occurs when certain soluble proteins lose their physiological function and become toxic amyloid species. The amyloid assembly consists of protein filament interactions, which can form fibrillar structures rich in ß-sheets. Despite the frequent incidence of these diseases among the elderly, the available treatments are limited and at best palliative, and new therapeutic approaches are needed. Among the many natural compounds that have been evaluated for their ability to prevent or delay the amyloidogenic process is epigallocatechin-3-gallate (EGCG), an abundant and potent polyphenolic molecule present in green tea that has extensive biological activity. There is evidence for EGCG's ability to inhibit the aggregation of α-synuclein, amyloid-ß, and huntingtin proteins, respectively associated with PD, AD, and HD. It prevents fibrillogenesis (in vitro and in vivo), reduces amyloid cytotoxicity, and remodels fibrils to form non-toxic amorphous species that lack seed propagation. Although it is an antioxidant, EGCG in an oxidized state can promote fibrils' remodeling through formation of Schiff bases and crosslinking the fibrils. Moreover, microparticles to drug delivery were synthesized from oxidized EGCG and loaded with a second anti-amyloidogenic molecule, obtaining a synergistic therapeutic effect. Here, we describe several pre-clinical and clinical studies involving EGCG and neurodegenerative diseases and their related mechanisms.
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A molecular hallmark in Parkinson's disease (PD) pathogenesis are α-synuclein aggregates. Cerebral dopamine neurotrophic factor (CDNF) is an atypical growth factor that is mostly resident in the endoplasmic reticulum but exerts its effects both intracellularly and extracellularly. One of the beneficial effects of CDNF can be protecting neurons from the toxic effects of α-synuclein. Here, we investigated the effects of CDNF on α-synuclein aggregation in vitro and in vivo. We found that CDNF directly interacts with α-synuclein with a KD = 23 ± 6 nM and reduces its auto-association. Using nuclear magnetic resonance (NMR) spectroscopy, we identified interaction sites on the CDNF protein. Remarkably, CDNF reduces the neuronal internalization of α-synuclein fibrils and induces the formation of insoluble phosphorylated α-synuclein inclusions. Intra-striatal CDNF administration alleviates motor deficits in rodents challenged with α-synuclein fibrils, though it did not reduce the number of phosphorylated α-synuclein inclusions in the substantia nigra. CDNF's beneficial effects on rodent behavior appear not to be related to the number of inclusions formed in the current context, and further study of its effects on the aggregation mechanism in vivo are needed. Nonetheless, the interaction of CDNF with α-synuclein, modifying its aggregation, spreading, and associated behavioral alterations, provides novel insights into the potential of CDNF as a therapeutic strategy in PD and other synucleinopathies.
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Fatores de Crescimento Neural/química , Fatores de Crescimento Neural/metabolismo , Doença de Parkinson/fisiopatologia , Substância Negra/metabolismo , alfa-Sinucleína/química , alfa-Sinucleína/metabolismo , Animais , Sítios de Ligação , Linhagem Celular , Modelos Animais de Doenças , Dopamina/metabolismo , Humanos , Espectroscopia de Ressonância Magnética , Masculino , Camundongos , Modelos Moleculares , Doença de Parkinson/metabolismo , Fosforilação , Cultura Primária de Células , Agregados Proteicos , Ligação Proteica , Conformação Proteica , RatosRESUMO
Background CDNF (cerebral dopamine neurotrophic factor) belongs to a new family of neurotrophic factors that exert systemic beneficial effects beyond the brain. Little is known about the role of CDNF in the cardiac context. Herein we investigated the effects of CDNF under endoplasmic reticulum-stress conditions using cardiomyocytes (humans and mice) and isolated rat hearts, as well as in rats subjected to ischemia/reperfusion (I/R). Methods and Results We showed that CDNF is secreted by cardiomyocytes stressed by thapsigargin and by isolated hearts subjected to I/R. Recombinant CDNF (exoCDNF) protected human and mouse cardiomyocytes against endoplasmic reticulum stress and restored the calcium transient. In isolated hearts subjected to I/R, exoCDNF avoided mitochondrial impairment and reduced the infarct area to 19% when administered before ischemia and to 25% when administered at the beginning of reperfusion, compared with an infarct area of 42% in the untreated I/R group. This protection was completely abrogated by AKT (protein kinase B) inhibitor. Heptapeptides containing the KDEL sequence, which binds to the KDEL-R (KDEL receptor), abolished exoCDNF beneficial effects, suggesting the participation of KDEL-R in this cardioprotection. CDNF administered intraperitoneally to rats decreased the infarct area in an in vivo model of I/R (from an infarct area of ≈44% in the I/R group to an infarct area of ≈27%). Moreover, a shorter version of CDNF, which lacks the last 4 residues (CDNF-ΔKTEL) and thus allows CDNF binding to KDEL-R, presented no cardioprotective activity in isolated hearts. Conclusions This is the first study to propose CDNF as a new cardiomyokine that induces cardioprotection via KDEL receptor binding and PI3K/AKT activation.
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Estresse do Retículo Endoplasmático/efeitos dos fármacos , Traumatismo por Reperfusão Miocárdica , Miócitos Cardíacos , Fatores de Crescimento Neural/metabolismo , Receptores de Peptídeos/metabolismo , Animais , Cardiotônicos/metabolismo , Cardiotônicos/farmacologia , Humanos , Camundongos , Traumatismo por Reperfusão Miocárdica/metabolismo , Traumatismo por Reperfusão Miocárdica/prevenção & controle , Miócitos Cardíacos/efeitos dos fármacos , Miócitos Cardíacos/metabolismo , Fatores de Crescimento Neural/farmacologia , Fosfatidilinositol 3-Quinases/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Ratos , Proteínas Recombinantes/farmacologia , Transdução de Sinais/efeitos dos fármacosRESUMO
α-Synuclein protein (α-syn) is a central player in Parkinson's disease (PD) and in a spectrum of neurodegenerative diseases collectively known as synucleinopathies. These diseases are characterized by abnormal motor symptoms, such as tremor at rest, slowness of movement, rigidity of posture, and bradykinesia. Histopathological features of PD include preferential loss of dopaminergic neurons in the substantia nigra and formation of fibrillar intraneuronal inclusions called Lewy bodies and Lewy neurites, which are composed primarily of the α-syn protein. Currently, it is well accepted that α-syn oligomers (αSO) are the main toxic agent responsible for the etiology of PD. Glutamatergic excitotoxicity is associated with several neurological disorders, including PD. Excess glutamate in the synaptic cleft can be taken up by the astrocytic glutamate transporters GLAST and GLT-1. Although this event is the main defense against glutamatergic excitotoxicity, the molecular mechanisms that regulate this process have not yet been investigated in an early sporadic model of synucleinopathy. Here, using an early sporadic model of synucleinopathy, we demonstrated that the treatment of astrocytes with αSO increased glutamate uptake. This was associated with higher levels of GLAST and GLT-1 in astrocyte cultures and in a mouse model of synucleinopathy 24 h and 45 days after inoculation with αSO, respectively. Pharmacological inhibition of the TGF-ß1 (transforming growth factor beta 1) pathway in vivo reverted GLAST/GLT-1 enhancement induced by αSO injection. Therefore, our study describes a new neuroprotective role of astrocytes in an early sporadic model of synucleinopathy and sheds light on the mechanisms of glutamate transporter regulation for neuroprotection against glutamatergic excitotoxicity in synucleinopathy.
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Sistema X-AG de Transporte de Aminoácidos/metabolismo , Astrócitos/metabolismo , Modelos Animais de Doenças , Sinucleinopatias/metabolismo , alfa-Sinucleína/toxicidade , Animais , Animais Recém-Nascidos , Astrócitos/efeitos dos fármacos , Astrócitos/patologia , Células Cultivadas , Feminino , Camundongos , Gravidez , Sinucleinopatias/induzido quimicamente , Sinucleinopatias/patologia , alfa-Sinucleína/químicaRESUMO
Neutrophil extracellular traps (NETs) emerge from the cell as a DNA scaffold associated with cytoplasmic and granular proteins, able to immobilize and kill pathogens. This association occurs following nuclear and granular membrane disintegration, allowing contact with the decondensed chromatin. Thus, it is reasonable to speculate that the DNA can also mix with miRNAs and carry them in NETs. Here, we report for the first time the presence of the miRNA carriers associated with NETs and miRNAs present in NET-enriched supernatants (NET-miRs), thus adding a novel class of molecules and new proteins that can be released and transported in the NET platform. We observed that the majority of NET-miRs were common to all four stimuli used (PMA, interleukin-8, amyloid fibrils and Leishmania), and that miRNA-142-3p carried by NETs down-modulates protein kinase Cα and regulates TNF-α production in macrophages upon NET interaction with these cells. Our findings unveil a novel role for NETs in the cell communication processes, allowing the conveyance of miRNA from neutrophils to neighboring cells.
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Comunicação Celular/imunologia , Armadilhas Extracelulares/imunologia , MicroRNAs/genética , Neutrófilos/imunologia , Fator de Necrose Tumoral alfa/genética , Amiloide/farmacologia , Antagomirs/genética , Antagomirs/metabolismo , Meios de Cultivo Condicionados/farmacologia , Armadilhas Extracelulares/metabolismo , Regulação da Expressão Gênica , Humanos , Interleucina-8/farmacologia , Leishmania braziliensis , MicroRNAs/antagonistas & inibidores , MicroRNAs/imunologia , Neutrófilos/efeitos dos fármacos , Neutrófilos/microbiologia , Cultura Primária de Células , Proteína Quinase C-alfa/genética , Proteína Quinase C-alfa/imunologia , Transdução de Sinais , Células THP-1 , Acetato de Tetradecanoilforbol/farmacologia , Fator de Necrose Tumoral alfa/imunologiaRESUMO
The accumulation of cross-ß-sheet amyloid fibrils is a hallmark of all human amyloid diseases. The compound epigallocatechin-3-gallate (EGCG), the main polyphenol present in green tea, has been described to have beneficial effects in several pathologies, including amyloidogenic diseases. This polyphenol blocks amyloidogenesis and disaggregates a broad range of amyloidogenic peptides comprising amyloid fibrils in vitro. The mechanism by which EGCG acts in the context of amyloid aggregation is not clear. Most of the biological effects of EGCG are attributable to its antioxidant activity. However, EGCG-oxidized products appear to be sufficient for the majority of EGCG amyloid remodeling observed against some polypeptides. If controlled, EGCG oxidation can afford homogenous microparticles (MPs) and can serve as drug delivery agents. Herein, we produced EGCG MPs by oxidative coupling and analyzed their activity during the aggregation of the protein α-synuclein (α-syn), the main protein related to Parkinson's disease. The MPs modestly remodeled mature amyloid fibrils and efficiently inhibited the amyloidogenic aggregation of α-syn. The MPs showed low cytotoxicity against both dopaminergic cells and microglial cells. The MPs reduced the cytotoxic effects of α-syn oligomers. Interestingly, the MPs were loaded with another antiamyloidogenic compound, increasing their activity against amyloid aggregation. We propose the use of EGCG MPs as a bifunctional strategy, blocking amyloid aggregation directly and carrying a molecule that can act synergistically to alleviate the symptoms caused by the amyloidogenic pathway.
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Amiloide , Preparações Farmacêuticas , Catequina/análogos & derivados , Humanos , Acoplamento Oxidativo , Polifenóis/farmacologia , CháAssuntos
Neuropatias Amiloides Familiares/epidemiologia , Neuropatias Amiloides Familiares/genética , Pré-Albumina/genética , Adulto , Idade de Início , Idoso , Idoso de 80 Anos ou mais , Neuropatias Amiloides Familiares/patologia , Brasil/epidemiologia , Etnicidade/genética , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , MutaçãoRESUMO
BACKGROUND: Familial amyloid polyneuropathy (FAP) or ATTRv (amyloid TTR variant) amyloidosis is a fatal hereditary disease characterized by the deposition of amyloid fibrils composed of transthyretin (TTR). The current diagnosis of ATTRv relies on genetic identification of TTR mutations and on Congo Red-positive amyloid deposits, which are absent in most ATTRv patients that are asymptomatic or early symptomatic, supporting the need for novel biomarkers to identify patients in earlier disease phases allowing disease control. METHODS: In an effort to search for new markers for ATTRv, our group searched for nine inflammation markers in ATTRv serum from a cohort of 28 Brazilian ATTRv patients. RESULTS: We found that the levels of six markers were increased (TNF-α, IL-1ß, IL-8, IL-33, IFN-ß and IL-10), one had decreased levels (IL-12) and two of them were unchanged (IL-6 and cortisol). Interestingly, asymptomatic patients already presented high levels of IL-33, IL-1ß and IL-10, suggesting that inflammation may take place before fibril deposition. CONCLUSIONS: Our findings shed light on a new, previously unidentified aspect of ATTRv, which might help define new criteria for disease management, as well as provide additional understanding of ATTRv aggressiveness.