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Glioblastoma (GB) is the most aggressive primary malignant brain tumor and is associated with short survival. O-GlcNAcylation is an intracellular glycosylation that regulates protein function, enzymatic activity, protein stability, and subcellular localization. Aberrant O-GlcNAcylation is related to the tumorigenesis of different tumors, and mounting evidence supports O-GlcNAc transferase (OGT) as a potential therapeutic target. Here, we used two human GB cell lines alongside primary human astrocytes as a non-tumoral control to investigate the role of O-GlcNAcylation in cell proliferation, cell cycle, autophagy, and cell death. We observed that hyper O-GlcNAcylation promoted increased cellular proliferation, independent of alterations in the cell cycle, through the activation of autophagy. On the other hand, hypo O-GlcNAcylation inhibited autophagy, promoted cell death by apoptosis, and reduced cell proliferation. In addition, the decrease in O-GlcNAcylation sensitized GB cells to the chemotherapeutic temozolomide (TMZ) without affecting human astrocytes. Combined, these results indicated a role for O-GlcNAcylation in governing cell proliferation, autophagy, cell death, and TMZ response, thereby indicating possible therapeutic implications for treating GB. These findings pave the way for further research and the development of novel treatment approaches which may contribute to improved outcomes and increased survival rates for patients facing this challenging disease.
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Cognitive dysfunction is often reported in patients with post-coronavirus disease 2019 (COVID-19) syndrome, but its underlying mechanisms are not completely understood. Evidence suggests that severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) Spike protein or its fragments are released from cells during infection, reaching different tissues, including the CNS, irrespective of the presence of the viral RNA. Here, we demonstrate that brain infusion of Spike protein in mice has a late impact on cognitive function, recapitulating post-COVID-19 syndrome. We also show that neuroinflammation and hippocampal microgliosis mediate Spike-induced memory dysfunction via complement-dependent engulfment of synapses. Genetic or pharmacological blockage of Toll-like receptor 4 (TLR4) signaling protects animals against synapse elimination and memory dysfunction induced by Spike brain infusion. Accordingly, in a cohort of 86 patients who recovered from mild COVID-19, the genotype GG TLR4-2604G>A (rs10759931) is associated with poor cognitive outcome. These results identify TLR4 as a key target to investigate the long-term cognitive dysfunction after COVID-19 infection in humans and rodents.
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COVID-19 , Disfunción Cognitiva , Humanos , Animales , Ratones , COVID-19/complicaciones , Glicoproteína de la Espiga del Coronavirus/genética , SARS-CoV-2/metabolismo , Receptor Toll-Like 4 , Síndrome Post Agudo de COVID-19RESUMEN
The Salmonella enterica bacteriophage P22 is one of the most promising models for the development of virus-like particle (VLP) nanocages. It possesses an icosahedral T = 7 capsid, assembled by the combination of two structural proteins: the coat protein (gp5) and the scaffold protein (gp8). The P22 capsid has the remarkable capability of undergoing structural transition into three morphologies with differing diameters and wall-pore sizes. These varied morphologies can be explored for the design of nanoplatforms, such as for the development of cargo internalization strategies. The capsid proteic nature allows for the extensive modification of its structure, enabling the addition of non-native structures to alter the VLP properties or confer them to diverse ends. Various molecules were added to the P22 VLP through genetic, chemical, and other means to both the capsid and the scaffold protein, permitting the encapsulation or the presentation of cargo. This allows the particle to be exploited for numerous purposes-for example, as a nanocarrier, nanoreactor, and vaccine model, among other applications. Therefore, the present review intends to give an overview of the literature on this amazing particle.
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Bacteriófago P22 , Viroides , Cápside , Proteínas de la Cápside/genética , Núcleo Celular , NanotecnologíaRESUMEN
Cellular prion protein (PrPC) is a highly conserved glycoprotein, present both anchored in the cell membrane and soluble in the extracellular medium. It has a diversity of ligands and is variably expressed in numerous tissues and cell subtypes, most notably in the central nervous system (CNS). Its importance has been brought to light over the years both under physiological conditions, such as embryogenesis and immune system homeostasis, and in pathologies, such as cancer and neurodegenerative diseases. During development, PrPC plays an important role in CNS, participating in axonal growth and guidance and differentiation of glial cells, but also in other organs such as the heart, lung, and digestive system. In diseases, PrPC has been related to several types of tumors, modulating cancer stem cells, enhancing malignant properties, and inducing drug resistance. Also, in non-neoplastic diseases, such as Alzheimer's and Parkinson's diseases, PrPC seems to alter the dynamics of neurotoxic aggregate formation and, consequently, the progression of the disease. In this review, we explore in detail the multiple functions of this protein, which proved to be relevant for understanding the dynamics of organism homeostasis, as well as a promising target in the treatment of both neoplastic and degenerative diseases.
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Neoplasias , Enfermedades Neurodegenerativas , Proteínas PrPC , Sistema Nervioso Central/metabolismo , Humanos , Células Madre Neoplásicas/metabolismo , Proteínas PrPC/genética , Proteínas PrPC/metabolismoRESUMEN
Glioblastoma is the most common and malignant type of primary brain tumor. Previous studies have shown that alterations in centrosome amplification and its components are frequently found in treatment-resistant tumors and may be associated with tumor progression. A centrosome protein essential for centrosome biogenesis is the centromere protein J (CENPJ), known to control the proliferation of neural progenitors and hepatocarcinoma cells, and also neuronal migration. However, it remains unknown the role of CENPJ in glioblastoma. Here we show that CENPJ is overexpressed in human glioblastoma cell lines in comparison to human astrocytes. Using bioinformatics analysis, we find that high Cenpj expression is associated with poor prognosis in glioma patients. Examining Cenpj loss of function in glioblastoma by siRNA transfection, we find impairments in cell proliferation and migration. Using a Cenpj mutant version with the deleted PN2-3 or TCP domain, we found that a conserved PN2-3 region is required for glioblastoma migration. Moreover, Cenpj downregulation modulates glioblastoma morphology resulting in microtubules stabilization and actin filaments depolymerization. Altogether, our findings indicate that CENPJ controls relevant aspects of glioblastoma progression and might be a target for therapeutic intervention and a biomarker for glioma malignancy.
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Neoplasias Encefálicas , Glioblastoma , Glioma , Neoplasias Encefálicas/metabolismo , Línea Celular Tumoral , Movimiento Celular , Proliferación Celular , Centrómero/metabolismo , Centrómero/patología , Regulación Neoplásica de la Expresión Génica , Glioblastoma/metabolismo , Glioma/metabolismo , HumanosRESUMEN
The misfolding and aggregation of the protein α-synuclein (aSyn) into potentially neurotoxic oligomers is believed to play a pivotal role in the neuropathogenesis of Parkinson's disease (PD). Herein, we explore how apomorphine (Apo), a nonselective dopamine D1 and D2 receptor agonist utilized in the therapy for PD, affects the aggregation and toxicity of aSyn in vitro. Our data indicated that Apo inhibits aSyn fibrillation leading to the formation of large oligomeric species (Apo-aSyn-O), which exhibit remarkable toxicity in mesencephalic dopaminergic neurons in primary cultures. Interestingly, purified Apo-aSyn-O, even at very low concentrations, seems to be capable of converting unmodified aSyn monomer into neurotoxic species. Collectively, our findings warn for a possible dangerous effect of Apo on aSyn misfolding/aggregation pathway.
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alfa-SinucleínaRESUMEN
Glioblastoma is the most life-threatening tumour of the central nervous system. Temozolomide (TMZ) is the first-choice oral drug for the treatment of glioblastoma, although it shows low efficacy. Silver nanoparticles (AgNPs) have been shown to exhibit biocidal activity in a variety of microorganisms, including some pathogenic microorganisms. Herein, the antiproliferative effect of AgCl-NPs on glioblastoma cell lines (GBM02 and GBM11) and on astrocytes was evaluated through automated quantitative image-based analysis (HCA) of the cells. The cells were treated with 0.1-5.0 µg/ml AgCl-NPs or with 9.7-48.5 µg/ml TMZ. Cells that received combined treatment were also analysed. At a maximum tested concentration of AgCl-NPs, GBM02 and GBM11, the growth decreased by 93% and 40%, respectively, following 72 h of treatment. TMZ treatment decreased the proliferation of GBM02 and GBM11 cells by 58% and 34%, respectively. Combinations of AgCl-NPs and TMZ showed intermediate antiproliferative effects; the lowest concentrations caused an inhibition similar to that obtained with TMZ, and the highest concentrations caused inhibition similar to that obtained with AgCl-NPs alone. No significant changes in astrocyte proliferation were observed. The authors' findings showed that HCA is a fast and reliable approach that can be used to evaluate the antiproliferative effect of the nanoparticles at the single-cell level and that AgCl-NPs are promising agents for glioblastoma treatment.
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Glioblastoma , Nanopartículas del Metal , Línea Celular Tumoral , Cloruros , Glioblastoma/tratamiento farmacológico , Humanos , Plata/farmacología , Compuestos de PlataRESUMEN
Arboviruses pose a major threat throughout the world and represent a great burden in tropical countries of South America. Although generally associated with moderate febrile illness, in more severe cases they can lead to neurological outcomes, such as encephalitis, Guillain-Barré syndrome, and Congenital Syndromes. In this context astrocytes play a central role in production of inflammatory cytokines, regulation of extracellular matrix, and control of glutamate driven neurotoxicity in the central nervous system. Here, we presented a comprehensive genome-wide transcriptome analysis of human primary astrocytes infected with Chikungunya, Mayaro, Oropouche, or Zika viruses. Analyses of differentially expressed genes (DEGs), pathway enrichment, and interactomes have shown that Alphaviruses up-regulated genes related to elastic fiber formation and N-glycosylation of glycoproteins, with down-regulation of cell cycle and DNA stability and chromosome maintenance genes. In contrast, Oropouche virus up-regulated cell cycle and DNA maintenance and condensation pathways while down-regulated extracellular matrix, collagen metabolism, glutamate and ion transporters pathways. Zika virus infection only up-regulated eukaryotic translation machinery while down-regulated interferon pathways. Reactome and integration analysis revealed a common signature in down-regulation of innate immune response, antiviral response, and inflammatory cytokines associated to interferon pathway for all arboviruses tested. Validation of interferon stimulated genes by reverse transcriptase quantitative polymerase chain reaction (RT-qPCR) corroborated our transcriptome findings. Altogether, our results showed a co-evolution in the mechanisms involved in the escape of arboviruses to antiviral immune response mediated by the interferon (IFN) pathway.
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Fiebre Chikungunya , Infección por el Virus Zika , Virus Zika , Astrocitos , Humanos , Inmunidad InnataRESUMEN
Neural precursor cells differentiate into several cell types that display distinct functions. However, little is known about how cell surface mechanics vary during the differentiation process. Here, by precisely measuring membrane tension and bending modulus, we map their variations and correlate them with changes in neural precursor cell morphology along their distinct differentiation fates. Both cells maintained in culture as neural precursors as well as those plated in neurobasal medium reveal a decrease in membrane tension over the first hours of culture followed by stabilization, with no change in bending modulus. During astrocyte differentiation, membrane tension initially decreases and then increases after 72 h, accompanied by consolidation of glial fibrillary acidic protein expression and striking actin reorganization, while bending modulus increases following observed alterations. For oligodendrocytes, the changes in membrane tension are less abrupt over the first hours, but their values subsequently decrease, correlating with a shift from oligodendrocyte marker O4 to myelin basic protein expressions and a remarkable actin reorganization, while bending modulus remains constant. Oligodendrocytes at later differentiation stages show membrane vesicles with similar membrane tension but higher bending modulus as compared to the cell surface. Altogether, our results display an entire spectrum of how membrane elastic properties are varying, thus contributing to a better understanding of neural differentiation from a mechanobiological perspective.
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Diferenciación Celular , Membrana Celular/fisiología , Elasticidad , Células-Madre Neurales/citología , Animales , Astrocitos/citología , Biomarcadores/metabolismo , Fenómenos Biomecánicos , Células Cultivadas , Medios de Cultivo , Citoesqueleto/metabolismo , Ratones , Pinzas ÓpticasRESUMEN
Diosmin is a flavone glycoside clinically used as the main component of Daflon for the treatment of venous diseases. Several studies demonstrated that this natural compound can induce apoptosis in different tumors. However, isolated diosmin has not been studied regarding its effects on glioblastoma so far. Since glioblastoma is a highly lethal and fast-growing brain tumor, new therapeutic strategies are urgently needed. Herein, we evaluated the role of this flavonoid against glioblastoma cells using in vitro assays. Diosmin significantly reduced the viability of GBM95, GBM02, and U87MG glioblastoma cells, but not of healthy human astrocytes, as verified by MTT assay. Vimentin immunostaining showed that diosmin induced morphological changes in GBM95 and GBM02 cells, making them smaller and more polygonal. Diosmin did not inhibit GBM95 and GBM02 cell proliferation, but it caused DNA fragmentation, as verified by the TUNEL assay, and increased cleaved caspase-3 expression in these cells. In summary, diosmin is able to induce caspase-dependent apoptosis specifically in tumor cells and, therefore, could be considered a promising therapeutic compound against glioblastoma.
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Apoptosis/efectos de los fármacos , Diosmina/farmacología , Neoplasias Encefálicas/patología , Línea Celular Tumoral , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Glioblastoma/patología , Humanos , Transducción de Señal/efectos de los fármacosRESUMEN
The guanine-based purines (GBPs) have essential extracellular functions such as modulation of glutamatergic transmission and trophic effects on neurons and astrocytes. We previously showed that GBPs, such as guanosine-5'-monophosphate (GMP) or guanosine (GUO), promote the reorganization of extracellular matrix proteins in astrocytes, and increase the number of neurons in a neuron-astrocyte co-culture protocol. To delineate the molecular basis underlying these effects, we isolated cerebellar neurons in culture and treated them with a conditioned medium derived from astrocytes previously exposed to GUO or GMP (GBPs-ACM) or, directly, with GUO or GMP. Agreeing with the previous studies, there was an increase in the number of ß-tubulin III-positive neurons in both conditions, compared with controls. Interestingly, the increase in the number of neurons in the neuronal cultures treated directly with GUO or GMP was more prominent, suggesting a direct interaction of GBPs on cerebellar neurons. To investigate this issue, we assessed the role of adenosine and glutamate receptors and related intracellular signaling pathways after GUO or GMP treatment. We found an involvement of A2A adenosine receptors, ionotropic glutamate N-methyl-D-aspartate (NMDA), and non-NMDA receptors in the increased number of cerebellar neurons. The signaling pathways extracellular-regulated kinase (ERK), calcium-calmodulin-dependent kinase-II (CaMKII), protein kinase C (PKC), phosphatidilinositol-3'-kinase (PI3-K), and protein kinase A (PKA) are also potentially involved with GMP and GUO effect. Such results suggest that GMP and GUO, and molecules released in GBPs-ACM promote the survival or maturation of primary cerebellar neurons or both via interaction with adenosine and glutamate receptors.
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Adenosina/metabolismo , Guanosina/metabolismo , Neuronas/metabolismo , Receptores de Glutamato/metabolismo , Animales , Astrocitos/metabolismo , Sistema Nervioso Central/metabolismo , Ácido Glutámico/metabolismo , Guanosina Monofosfato/metabolismo , Receptores Purinérgicos P1/metabolismoRESUMEN
Parkinson's disease (PD) is characterized by selective death of dopaminergic neurons in the substantia nigra, degeneration of the nigrostriatal pathway, increases in glutamatergic synapses in the striatum and aggregation of α-synuclein. Evidence suggests that oligomeric species of α-synuclein (αSO) are the genuine neurotoxins of PD. Although several studies have supported the direct neurotoxic effects of αSO on neurons, their effects on astrocytes have not been directly addressed. Astrocytes are essential to several steps of synapse formation and function, including secretion of synaptogenic factors, control of synaptic elimination and stabilization, secretion of neural/glial modulators, and modulation of extracellular ions, and neurotransmitter levels in the synaptic cleft. Here, we show that αSO induced the astrocyte reactivity and enhanced the synaptogenic capacity of human and murine astrocytes by increasing the levels of the known synaptogenic molecule transforming growth factor beta 1 (TGF-ß1). Moreover, intracerebroventricular injection of αSO in mice increased the number of astrocytes, the density of excitatory synapses, and the levels of TGF-ß1 in the striatum of injected animals. Inhibition of TGF-ß1 signaling impaired the effect of the astrocyte-conditioned medium on glutamatergic synapse formation in vitro and on striatal synapse formation in vivo, whereas addition of TGF-ß1 protected mesencephalic neurons against synapse loss triggered by αSO. Together, our data suggest that αSO have important effects on astrocytic functions and describe TGF-ß1 as a new endogenous astrocyte-derived molecule involved in the increase in striatal glutamatergic synaptic density present in early stages of PD. OPEN SCIENCE BADGES: This article has received a badge for *Open Materials* because it provided all relevant information to reproduce the study in the manuscript. The complete Open Science Disclosure form for this article can be found at the end of the article. More information about the Open Practices badges can be found at https://cos.io/our-services/open-science-badges/. Cover Image for this issue: doi: 10.1111/jnc.14514.
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Astrocitos/metabolismo , Trastornos Parkinsonianos/metabolismo , Sinapsis/metabolismo , Factor de Crecimiento Transformador beta1/metabolismo , alfa-Sinucleína/metabolismo , Animales , Modelos Animales de Enfermedad , Humanos , Ratones , Neurogénesis/fisiología , Transducción de Señal/fisiologíaRESUMEN
A new series of 1,4-disubstituted-1,2,3-triazole derivatives were synthesized through the copper-catalyzed azide-alkyne 1,3-dipolar cycloaddition (Click chemistry) and their inhibitory activities were evaluated against different human glioblastoma (GBM) cell lines, including highly drug-resistant human cell lines GBM02, GBM95. The most effective compounds were 9d, containing the methylenoxy moiety linked to triazole and the tosyl-hydrazone group, and the symmetrical bis-triazole 10a, also containing methylenoxy moiety linked to triazole. Single crystal X-ray diffraction analysis was employed for structural elucidation of compound 9d. In silico analyses of physicochemical, pharmacokinetic, and toxicological properties suggest that compounds 8a, 8b, 8c, 9d, and 10a are potential candidates for central nervous system-acting drugs.
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Antineoplásicos/farmacología , Diseño de Fármacos , Glioblastoma/tratamiento farmacológico , Triazoles/farmacología , Animales , Antineoplásicos/síntesis química , Antineoplásicos/química , Proliferación Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Relación Dosis-Respuesta a Droga , Ensayos de Selección de Medicamentos Antitumorales , Glioblastoma/patología , Humanos , Estructura Molecular , Ratas , Relación Estructura-Actividad , Triazoles/síntesis química , Triazoles/química , Células Tumorales CultivadasRESUMEN
Glioblastoma is an extremely aggressive and deadly brain tumor known for its striking cellular heterogeneity and capability to communicate with microenvironment components, such as microglia. Microglia-glioblastoma interaction contributes to an increase in tumor invasiveness, and Wnt signaling pathway is one of the main cascades related to tumor progression through changes in cell migration and invasion. However, very little is known about the role of canonical Wnt signaling during microglia-glioblastoma crosstalk. Here, we show for the first time that Wnt3a is one of the factors that regulate interactions between microglia and glioblastoma cells. Wnt3a activates the Wnt/ß-catenin signaling of both glioblastoma and microglial cells. Glioblastoma-conditioned medium not only induces nuclear translocation of microglial ß-catenin but also increases microglia viability and proliferation as well as Wnt3a, cyclin-D1, and c-myc expression. Moreover, glioblastoma-derived Wnt3a increases microglial ARG-1 and STI1 expression, followed by an upregulation of IL-10 mRNA levels, and a decrease in IL1ß gene expression. The presence of Wnt3a in microglia-glioblastoma co-cultures increases the formation of membrane nanotubes accompanied by changes in migration capability. In vivo, tumors formed from Wnt3a-stimulated glioblastoma cells presented greater microglial infiltration and more aggressive characteristics such as growth rate than untreated tumors. Thus, we propose that Wnt3a belongs to the arsenal of factors capable of stimulating the induction of M2-like phenotype on microglial cells, which contributes to the poor prognostic of glioblastoma, reinforcing that Wnt/ß-catenin pathway can be a potential therapeutic target to attenuate glioblastoma progression.
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Microglía/metabolismo , Vía de Señalización Wnt/fisiología , Proteína Wnt3A/metabolismo , beta Catenina/metabolismo , Movimiento Celular/fisiología , Proliferación Celular/fisiología , Glioblastoma/genética , Humanos , FenotipoRESUMEN
Glioblastomas (GBM) are aggressive brain tumors with very poor prognosis. While silver nanoparticles represent a potential new strategy for anticancer therapy, the silver/silver chloride nanoparticles (Ag/AgCl-NPs) have microbicidal activity, but had not been tested against tumor cells. Here, we analyzed the effect of biogenically produced Ag/AgCl-NPs (from yeast cultures) on the proliferation of GBM02 glioblastoma cells (and of human astrocytes) by automated, image-based high-content analysis (HCA). We compared the effect of 0.1-5.0 µg mL-1 Ag/AgCl-NPs with that of 9.7-48.5 µg mL-1 temozolomide (TMZ, chemotherapy drug currently used to treat glioblastomas), alone or in combination. At higher concentrations, Ag/AgCl-NPs inhibited GBM02 proliferation more effectively than TMZ (up to 82 and 62% inhibition, respectively), while the opposite occurred at lower concentrations (up to 23 and 53% inhibition, for Ag/AgCl-NPs and TMZ, respectively). The combined treatment (Ag/AgCl-NPs + TMZ) inhibited GBM02 proliferation by 54-83%. Ag/AgCl-NPs had a reduced effect on astrocyte proliferation compared with TMZ, and Ag/AgCl-NPs + TMZ inhibited astrocyte proliferation by 5-42%. The growth rate and population doubling time analyses confirmed that treatment with Ag/AgCl-NPs was more effective against GBM02 cells than TMZ (~ 67-fold), and less aggressive to astrocytes, while Ag/AgCl-NP + TMZ treatment was no more effective against GBM02 cells than Ag/AgCl-NPs monotherapy. Taken together, our data indicate that 2.5 µg mL-1 Ag/AgCl-NPs represents the safest dose tested here, which affects GBM02 proliferation, with limited effect on astrocytes. Our findings show that HCA is a useful approach to evaluate the antiproliferative effect of nanoparticles against tumor cells.
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BACKGROUND: Salsolinol (SALSO), a product from the reaction of dopamine (DA) with acetaldehyde, is found increased in dopaminergic neurons of Parkinson's disease (PD) patients. The administration of SALSO in rats causes myenteric neurodegeneration followed by the formation of deposits of the protein α-synuclein (aS), whose aggregation is intimately associated to PD. METHODS: NMR, isothermal titration calorimetry and MS were used to evaluate the interaction of SALSO with aS. The toxicity of SALSO and in vitro-produced aS-SALSO species was evaluated on mesencephalic primary neurons from mice. RESULTS: SALSO, under oxidative conditions, stabilizes the monomeric state besides a minor population of oligomers of aS, resulting in a strong inhibition of the fibrillation process. SALSO does not promote any chemical modification of the protein. Instead, the interaction of SALSO with aS seems to occur via hydrophobic effect, likely mediated by the NAC (non-amyloid component) domain of the protein. aS-SALSO species were found to be innocuous on primary neurons, while SALSO alone induces apoptosis via caspase-3 activation. Importantly, exogenous aS monomer was capable of protecting neurons against SALSO toxicity irrespective whether the protein was co-administered with SALSO or added until 2â¯h after SALSO, as evidenced by DAPI and cleaved-caspase 3 assays. Similar protective action of aS was found by pre-incubating neurons with aS before the administration of SALSO. CONCLUSIONS: Interaction of SALSO with aS leads to the formation of fibril-incompetent and innocuous adducts. SALSO toxicity is attenuated by aS monomer. SIGNIFICANCE: aS could exhibit a protective role against the neurotoxic effects of SALSO in dopaminergic neuron.
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Neuronas Dopaminérgicas/efectos de los fármacos , Isoquinolinas/toxicidad , Sinapsis/metabolismo , alfa-Sinucleína/fisiología , Animales , Apoptosis/efectos de los fármacos , Calorimetría , Caspasa 3/metabolismo , Células Cultivadas , Cromatografía en Gel , Cromatografía Líquida de Alta Presión , Electroforesis en Gel de Poliacrilamida , Activación Enzimática , Humanos , Espectrometría de Masas , Ratones , Oxidación-Reducción , Ratas , Espectrometría de Fluorescencia , alfa-Sinucleína/metabolismoRESUMEN
The dopamine metabolite 3,4-dihydroxyphenylacetaldehyde (DOPAL) is believed to play a central role in Parkinson's disease neurodegeneration by stabilizing potentially toxic oligomers of the presynaptic protein α-Synuclein (aSyn). Besides the formation of covalent DOPAL-Lys adducts, DOPAL promotes the oxidation of Met residues of aSyn, which is also a common oxidative post-translational modification found in the protein in vivo. Herein we set out to address the role of Met residues on the oligomerization and neurotoxic properties of DOPAL-modified aSyn. Our data indicate that DOPAL promotes the formation of two distinct types of aSyn oligomers: large and small (dimer and trimers) oligomers, which seem to be generated by independent mechanisms and cannot be interconverted by using denaturing agents. Interestingly, H2O2-treated aSyn monomer, which exhibits all-four Met residues oxidized to Met-sulfoxide, exhibited a reduced ability to form large oligomers upon treatment with DOPAL, with no effect on the population of small oligomers. In this context, triple Met-Val mutant M5V/M116V/M127V exhibited an increased population of large aSyn-DOPAL oligomers in comparison with the wild-type protein. Interestingly, the stabilization of large rather than small oligomers seems to be associated with an enhanced toxicity of DOPAL-aSyn adducts. Collectively, these findings indicate that Met residues may play an important role in modulating both the oligomerization and the neurotoxic properties of DOPAL-derived aSyn species.
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Ácido 3,4-Dihidroxifenilacético/farmacología , Metionina/química , Neuronas/efectos de los fármacos , Multimerización de Proteína/efectos de los fármacos , alfa-Sinucleína/toxicidad , Ácido 3,4-Dihidroxifenilacético/análogos & derivados , Animales , Supervivencia Celular/efectos de los fármacos , Células Cultivadas , Humanos , Peróxido de Hidrógeno/farmacología , Metionina/genética , Ratones , Mutación , Neuronas/citología , Oxidantes/farmacología , Oxidación-Reducción/efectos de los fármacos , Procesamiento Proteico-Postraduccional/efectos de los fármacos , alfa-Sinucleína/química , alfa-Sinucleína/genéticaRESUMEN
Glioblastoma (GBM) is the most common and aggressive malignant primary brain tumor and still lacks effective therapeutic strategies. It has already been shown that old drugs like sulfasalazine (SAS) and valproic acid (VPA) present antitumoral activities in glioma cell lines. SAS has also been associated with a decrease of intracellular glutathione (GSH) levels through a potent inhibition of xc- glutamate/cystine exchanger leading to an antioxidant deprotection. In the same way, VPA was recently identified as a histone deacetylase (HDAT) inhibitor capable of activating tumor suppression genes. As both drugs are widely used in clinical practice and their profile of adverse effects is well known, the aim of our study was to investigate the effects of the combined treatment with SAS and VPA in GBM cell lines. We observed that both drugs were able to reduce cell viability in a dose-dependent manner and the combined treatment potentiated these effects. Combined treatment also increased cell death and inhibited proliferation of GBM cells, while having no effect on human and rat cultured astrocytes. Also, we observed high protein expression of the catalytic subunit of xc- in all the examined GBM cell lines, and treatment with SAS blocked its activity and decreased intracellular GSH levels. Noteworthy, SAS but not VPA was also able to reduce the [14C]-ascorbate uptake. Together, these data indicate that SAS and VPA exhibit a substantial effect on GBM cell's death related to an intracellular oxidative response imbalance, making this combination of drugs a promising therapeutic strategy.
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Neoplasias Encefálicas/patología , Glioblastoma/patología , Espacio Intracelular/metabolismo , Sulfasalazina/farmacología , Ácido Valproico/farmacología , Sistema de Transporte de Aminoácidos y+/metabolismo , Animales , Ácido Ascórbico/metabolismo , Muerte Celular/efectos de los fármacos , Línea Celular Tumoral , Núcleo Celular/efectos de los fármacos , Núcleo Celular/metabolismo , Proliferación Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Quimioterapia Combinada , Glutatión/metabolismo , Humanos , Mesodermo/efectos de los fármacos , Mesodermo/patología , Neuroglía/efectos de los fármacos , Neuroglía/metabolismo , Neuroglía/patología , Oxidación-Reducción , Ratas , Factores de TiempoRESUMEN
Parkinson's disease (PD) is characterized by motor dysfunction, which is preceded by a number of non-motor symptoms including olfactory deficits. Aggregation of α-synuclein (α-syn) gives rise to Lewy bodies in dopaminergic neurons and is thought to play a central role in PD pathology. However, whether amyloid fibrils or soluble oligomers of α-syn are the main neurotoxic species in PD remains controversial. Here, we performed a single intracerebroventricular (i.c.v.) infusion of α-syn oligomers (α-SYOs) in mice and evaluated motor and non-motor symptoms. Familiar bedding and vanillin essence discrimination tasks showed that α-SYOs impaired olfactory performance of mice, and decreased TH and dopamine levels in the olfactory bulb early after infusion. The olfactory deficit persisted until 45days post-infusion (dpi). α- SYO-infused mice behaved normally in the object recognition and forced swim tests, but showed increased anxiety-like behavior in the open field and elevated plus maze tests 20 dpi. Finally, administration of α-SYOs induced late motor impairment in the pole test and rotarod paradigms, along with reduced TH and dopamine content in the caudate putamen, 45 dpi. Reduced number of TH-positive cells was also seen in the substantia nigra of α-SYO-injected mice compared to control. In conclusion, i.c.v. infusion of α-SYOs recapitulated some of PD-associated non-motor symptoms, such as increased anxiety and olfactory dysfunction, but failed to recapitulate memory impairment and depressive-like behavior typical of the disease. Moreover, α-SYOs i.c.v. administration induced motor deficits and loss of TH and dopamine levels, key features of PD. Results point to α-syn oligomers as the proximal neurotoxins responsible for early non-motor and motor deficits in PD and suggest that the i.c.v. infusion model characterized here may comprise a useful tool for identification of PD novel therapeutic targets and drug screening.
Asunto(s)
Síntomas Conductuales/etiología , Encéfalo/efectos de los fármacos , Trastornos del Olfato/etiología , Enfermedad de Parkinson/complicaciones , Enfermedad de Parkinson/etiología , alfa-Sinucleína/toxicidad , Animales , Encéfalo/metabolismo , Células Cultivadas , Discriminación en Psicología/efectos de los fármacos , Modelos Animales de Enfermedad , Embrión de Mamíferos , Humanos , Inyecciones Intraventriculares , Masculino , Aprendizaje por Laberinto/efectos de los fármacos , Mesencéfalo/citología , Ratones , Ratones Transgénicos , Neuronas/efectos de los fármacos , Neuronas/metabolismo , Péptidos/toxicidad , Reconocimiento en Psicología/efectos de los fármacos , Tirosina 3-Monooxigenasa/metabolismo , alfa-Sinucleína/química , alfa-Sinucleína/genética , alfa-Sinucleína/metabolismoRESUMEN
Protein aggregation is a hallmark of several neurodegenerative diseases, including Alzheimer's and Parkinson's diseases. It has been shown that lysine residues play a key role in the formation of these aggregates. Thus, the ability to disrupt aggregate formation by covalently modifying lysine residues could lead to the discovery of therapeutically relevant antiamyloidogenesis compounds. Herein, we demonstrate that an ortho-iminoquinone (IQ) can be utilized to inhibit amyloid aggregation. Using alpha-synuclein and Aß1-40 as model amyloidogenic proteins, we observed that IQ was able to react with lysine residues and reduce amyloid aggregation. We also observed that IQ reacted with free amines within the amyloid fibrils preventing their dissociation and seeding capacity.