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BACKGROUND: Cerebral Cavernous Malformations (CCM) is a genetic disease characterized by vascular abnormalities in the brain and spinal cord, affecting 0.4-0.5 % of the population. We identified two novel pathogenic mutations, CCM1/KRIT1 c.811delT (p.Trp271GlyfsTer5) and CCM2/MGC4607 c.613_614insGG p.Glu205GlyfsTer31), which disrupt crucial protein domains and potentially alter disease progression. OBJECTIVE: The study aims to comprehensively analyze a Brazilian cohort of CCM patients, integrating genetic, clinical, and structural aspects. Specifically, we sought to identify novel mutations within the CCM complex, and explore their potential impact on disease progression. METHODS: We conducted a detailed examination of neuroradiological and clinical features in both symptomatic and asymptomatic CCM patients, performing genetic analyses through sequencing of the CCM1/KRIT1, CCM2/MGC4607, and CCM3/PDCD10 genes In silico structural predictions were carried out using PolyPhen-2, SIFT, and Human Genomics Community tools. Protein-protein interactions and docking analyses were explored using the STRING database. RESULTS: Genetic analysis identifies 6 pathogenic mutations, 4 likely pathogenic, 1 variants of uncertain significance, and 7 unclassified mutations, including the novel mutations in CCM1 c.811delT and CCM2 c.613_614insGG. In silico structural analysis revealed significant alterations in protein structure, supporting their pathogenicity. Protein-protein interaction analysis indicated nuanced impacts on cellular processes. Clinically, we observed a broad spectrum of symptoms, including seizures and focal neurological deficits. However, no statistically significant differences were found in lesion burden, age of first symptom onset, or sex between the identified CCM1/KRIT1 and CCM2/MGC4607 mutations among all patients studied. CONCLUSION: This study enhances the understanding of CCM by linking clinical variability, genetic mutations, and structural effects. The identification of these novel mutations opens new avenues for research and potential therapeutic strategies.
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BACKGROUND: Cerebral Cavernous Malformation (CCM) is one of the most common types of vascular malformation of the central nervous system. Intracerebral hemorrhage, seizures, and lesional growth are the main clinical manifestations. Natural history studies have tried to identify many risk factors; however, the clinical course remains highly unpredictable. OBJECTIVE: Here, we have analyzed a multicenter CCM cohort looking for the differential clinical data regarding the patients harboring supra and/or infratentorial cavernous malformations in order to better understand risk factors involved in the anatomical location of the unique neurosurgical disease. METHODS: We have presented a multicenter, Propensity Score Matched (PSM), case-control study including 149 consecutive CCM cases clinically evaluated from May 2017 to December 2022 from three different neurosurgical centers. Epidemiological data were defined at each clinical assessment. Logistic regression was used to identify the independent contribution of each possible risk factor to the bleeding risk. To balance baseline covariates between patients with and without symptoms, and specifically between those with and without symptomatic bleeding, we used a PSM strategy. The Kaplan-Meier curve was drawn to evaluate if patients with infratentorial lesions had a greater chance of bleeding earlier in their life. RESULTS: The presence of infratentorial lesions was a risk factor in the multivariate analysis comparing the bleeding risk with pure asymptomatic individuals (OR: 3.23, 95% CI 1.43 - 7.26, P = 0.005). Also, having an infratentorial CCM was a risk factor after PSM (OR: 4.56, 95% CI 1.47 - 14.10, P = 0.008). The presence of an infratentorial lesion was related to precocity of symptoms when the time to first bleed was compared to all other clinical presentations in the overall cohort (P = 0.0328) and in the PSM group (P = 0.03). CONCLUSION: Here, we have provided some evidence that infratentorial cerebral cavernous malformation may have a more aggressive clinical course, being a risk factor for symptomatic haemorrhage and precocity of bleeding.
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Hemorragia Cerebral , Hemangioma Cavernoso del Sistema Nervioso Central , Puntaje de Propensión , Humanos , Hemangioma Cavernoso del Sistema Nervioso Central/complicaciones , Masculino , Femenino , Factores de Riesgo , Estudios de Casos y Controles , Adulto , Persona de Mediana Edad , Hemorragia Cerebral/epidemiología , Hemorragia Cerebral/etiología , Adulto Joven , Adolescente , Niño , AncianoRESUMEN
Cerebral cavernous malformation (CCM) is a vascular disease that affects the central nervous system, which familial form is due to autosomal dominant mutations in the genes KRIT1(CCM1), MGC4607(CCM2), and PDCD10(CCM3). Patients affected by the PDCD10 mutations usually have the onset of symptoms at an early age and a more aggressive phenotype. The aim of this study is to investigate the molecular mechanism involved with CCM3 disease pathogenesis. Herein, we report two typical cases of CCM3 phenotype and compare the clinical and neuroradiological findings with five patients with a familial form of KRIT1 or CCM2 mutations and six patients with a sporadic form. In addition, we evaluated the PDCD10 gene expression by qPCR and developed a bioinformatic pipeline to understand the structural changes of mutations. The two CCM3 patients had an early onset of symptoms and a high lesion burden. Furthermore, the sequencing showed that Patient 1 had a frameshift mutation in c.222delT; p.(Asn75Thrfs*14) that leads to lacking the last 124 C-terminal amino acids on its primary structure and Patient 2 had a variant on the splicing site region c.475-2A > G. The mRNA expression was fourfold lower in both patients with PDCD10 mutation. Using in silico analysis, we identify that the frameshift mutation transcript lacks the C-terminal FAT-homology domain compared to the wild-type PDCD10 and preserves the N-terminal dimerization domain. The two patients studied here allow estimating the potential impact of mutations in clinical interpretation as well as support to better understand the mechanism and pathogenesis of CCM3.
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Hemangioma Cavernoso del Sistema Nervioso Central , Humanos , Sistema Nervioso Central , Hemangioma Cavernoso del Sistema Nervioso Central/diagnóstico por imagen , Hemangioma Cavernoso del Sistema Nervioso Central/genética , Mutación/genética , Fenotipo , Proteínas Proto-Oncogénicas/genéticaRESUMEN
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
BACKGROUND: Cerebral Cavernous Malformations (CCM) predispose patients to a lifetime risk of seizures and symptomatic hemorrhage. Only a small percentage of people affected will develop clinical symptoms and the molecular mechanisms underlying lesional activity remain unclear. We analyzed a panel of Single Nucleotide Polymorphisms (SNPs) in CCM patients. We looked for plasmatic inflammatory cytokines, checking for a pattern of plasma expression heterogeneity and any correlation with genetic variations identified with different CCM clinical phenotypes. METHODS: This was a case-control study from a long-term follow-up cohort including 23 CCM patients, of which 16 were symptomatic, and 7 were asymptomatic. A 200-SNP panel was considered through next-generation sequencing and 18 different plasma molecules were assessed through a suspension array system. RESULTS: Fcγ receptor IIa rs1801274 (FCGR2A) and protein tyrosine phosphatase non-receptor type 2 rs72872125 PTPN2 were statistically different between groups. Patients who had a combination of the presence of FCGR2A and the absence of PTPN2 also had symptoms earlier in life. The combination of genetic polymorphisms and serum level of GM-CSF showed the best diagnostic biomarker to distinguish symptomatic patients as formulated: [0.296*(FCGR2A)] + [-0.788*(PTPN2)] + [-0.107*(GM-CSF)]. CONCLUSION: We have shown that SNPs in inflammation genes might be related to a symptomatic phenotype in CCM. We also demonstrated that a formula based on two of these polymorphisms (FCGR2A+ and PTPN2+) is possibly capable of predicting a symptomatic phenotype during a patient's lifetime.
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Factor Estimulante de Colonias de Granulocitos y Macrófagos/genética , Hemangioma Cavernoso del Sistema Nervioso Central/genética , Proteína Tirosina Fosfatasa no Receptora Tipo 2/genética , Receptores de IgG/genética , Adulto , Anciano , Estudios de Casos y Controles , Femenino , Marcadores Genéticos , Secuenciación de Nucleótidos de Alto Rendimiento , Humanos , Masculino , Persona de Mediana Edad , Adulto JovenRESUMEN
BACKGROUND: Intraorbital and intracerebral cavernous malformation (CM) lesions are considered independent entities. Purely cerebral CMs have variable biology with recent evidence depicting inflammation as an important player and a risk factor for aggressiveness. We describe a case of concomitant left intraaxial and extraaxial CMs, linked by the ipsilateral basal vein, where the extraaxial component has developed an aggressive behavior. CASE DESCRIPTION: A 35-year-old female patient presented with a rapid and progressive exophthalmos and loss of vision on the left eye. Cranial magnetic resonance and angiography examinations demonstrated a left craniofacial CM and large intraorbital component. The lesion was connected through a large basal vein to a cerebral intraventricular CM. Transconjunctival resection showed typical findings of CM. A complete histopathology and immunostaining analysis was performed and revealed a clear acute lymphomononuclear reaction with a predominant immune cellular inflammation. CONCLUSIONS: A case of intraorbital and extracranial cavernomatous mass, connected to a cerebral intraventricular CM through a large basal vein, has presented with an aggressive course. A complete histopathologic and immunohistochemical analysis of the orbital mass has pictured a clear immune-cellular inflammatory reaction adding to the amounting evidence of association between inflammation and site aggressiveness in the setting of CMs.
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Venas Cerebrales/diagnóstico por imagen , Neoplasias del Ventrículo Cerebral/diagnóstico por imagen , Hemangioma Cavernoso del Sistema Nervioso Central/diagnóstico por imagen , Neoplasias Primarias Múltiples/diagnóstico por imagen , Neoplasias Orbitales/diagnóstico por imagen , Adulto , Linfocitos B/inmunología , Linfocitos B/patología , Angiografía Cerebral , Femenino , Hemangioma Cavernoso del Sistema Nervioso Central/inmunología , Hemangioma Cavernoso del Sistema Nervioso Central/patología , Hemangioma Cavernoso del Sistema Nervioso Central/cirugía , Humanos , Inflamación/inmunología , Inflamación/patología , Macrófagos/inmunología , Macrófagos/patología , Imagen por Resonancia Magnética , Neoplasias Primarias Múltiples/inmunología , Neoplasias Primarias Múltiples/patología , Neoplasias Primarias Múltiples/cirugía , Neoplasias Orbitales/inmunología , Neoplasias Orbitales/patología , Neoplasias Orbitales/cirugía , Células Plasmáticas/inmunología , Células Plasmáticas/patologíaRESUMEN
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
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
Glioblastoma (GBM) is a grade IV astrocytoma. GBM patients show resistance to chemotherapy such as temozolomide (TMZ), the gold standard treatment. In order to simulate the molecular mechanisms behind the different chemotherapeutic responses in GBM patients we compared the cellular heterogeneity and chemotherapeutic resistance mechanisms in different GBM cell lines. We isolated and characterized a human GBM cell line obtained from a GBM patient, named GBM11. We studied the GBM11 behaviour when treated with Tamoxifen (TMX) that, among other functions, is a protein kinase C (PKC) inhibitor, alone and in combination with TMZ in comparison with the responses of U87 and U118 human GBM cell lines. We evaluated the cell death, cell cycle arrest and cell proliferation, mainly through PKC expression, by flow cytometry and western blot analysis and, ultimately, cell migration capability and f-actin filament disorganization by fluorescence microscopy. We demonstrated that the constitutive activation of p-PKC seems to be one of the main metabolic implicated on GBM malignancy. Despite of its higher resistance, possibly due to the overexpression of P-glycoprotein and stem-like cell markers, GBM11 cells presented a subtle different chemotherapeutic response compared to U87 and U118 cells. The GBM11, U87, U118 cell lines show subtle molecular differences, which clearly indicate the characterization of GBM heterogeneity, one of the main reasons for tumor resistance. The adding of cellular heterogeneity in molecular behaviour constitutes a step closer in the understanding of resistant molecular mechanisms in GBM, and can circumvents the eventual impaired therapy.
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Astrocitoma/tratamiento farmacológico , Heterogeneidad Genética , Glioblastoma/tratamiento farmacológico , Proteína Quinasa C/genética , Miembro 1 de la Subfamilia B de Casetes de Unión a ATP/genética , Apoptosis/efectos de los fármacos , Astrocitoma/genética , Astrocitoma/patología , Puntos de Control del Ciclo Celular/efectos de los fármacos , Línea Celular Tumoral , Movimiento Celular/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Dacarbazina/administración & dosificación , Dacarbazina/análogos & derivados , Resistencia a Antineoplásicos/genética , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Glioblastoma/genética , Glioblastoma/patología , Humanos , Clasificación del Tumor , Transducción de Señal/efectos de los fármacos , Tamoxifeno/administración & dosificación , TemozolomidaRESUMEN
Astrocytes play a critical role in the development and homeostasis of the central nervous system (CNS). Astrocyte dysfunction results in several neurological and degenerative diseases. However, a major challenge to our understanding of astrocyte physiology and pathology is the restriction of studies to animal models, human post-mortem brain tissues, or samples obtained from invasive surgical procedures. Here, we report a protocol to generate human functional astrocytes from cerebral organoids derived from human pluripotent stem cells. The cellular isolation of cerebral organoids yielded cells that were morphologically and functionally like astrocytes. Immunolabelling and proteomic assays revealed that human organoid-derived astrocytes express the main astrocytic molecular markers, including glutamate transporters, specific enzymes and cytoskeletal proteins. We found that organoid-derived astrocytes strongly supported neuronal survival and neurite outgrowth and responded to ATP through transient calcium wave elevations, which are hallmarks of astrocyte physiology. Additionally, these astrocytes presented similar functional pathways to those isolated from adult human cortex by surgical procedures. This is the first study to provide proteomic and functional analyses of astrocytes isolated from human cerebral organoids. The isolation of these astrocytes holds great potential for the investigation of developmental and evolutionary features of the human brain and provides a useful approach to drug screening and neurodegenerative disease modelling.
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Astrocitos/citología , Corteza Cerebral/citología , Proyección Neuronal , Organoides/citología , Animales , Astrocitos/metabolismo , Señalización del Calcio , Células Cultivadas , Proteínas del Citoesqueleto/genética , Proteínas del Citoesqueleto/metabolismo , Ácido Glutámico/metabolismo , Humanos , Ratones , Células Madre Pluripotentes/citología , Células Madre Pluripotentes/metabolismo , Proteoma/genética , Proteoma/metabolismoRESUMEN
BACKGROUND: Cavernous malformation (CM) is a vascular malformation found in the encephalic parenchyma, spinal cord, nerve roots, and extraneural tissue. CM in the trigeminal distribution is exquisitely uncommon and its biological behavior not completely understood. The clinical picture might be diverse, depending on the affected sector of the trigeminal architecture, and literature debating its pathobiology is scarce. CASE DESCRIPTION: We describe a case of 56-year-old woman who presented with left trigeminal neuralgia and a rapidly growing cavernous malformation of the entire distribution of the fifth nerve. The clinical picture evolved to a progressive gait ataxia and follow-up neuroimaging showed a large intracranial mass leading to a brainstem compression. After microsurgical resection, the mass proved to be a typical CM of the trigeminal root. CONCLUSION: We present an uncommonly aggressive progression of a CM of the trigeminal root, Gasserian ganglion, and cavernous sinus evolving to severe brainstem compression. The documentation of this unique case as well as its management is presented is discussed.
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Neoplasias Encefálicas/complicaciones , Neoplasias Encefálicas/diagnóstico , Hemangioma Cavernoso del Sistema Nervioso Central/complicaciones , Hemangioma Cavernoso del Sistema Nervioso Central/diagnóstico , Neuralgia del Trigémino/etiología , Neoplasias Encefálicas/cirugía , Femenino , Hemangioma Cavernoso del Sistema Nervioso Central/cirugía , Humanos , Persona de Mediana Edad , Neuralgia del Trigémino/diagnóstico , Neuralgia del Trigémino/terapiaRESUMEN
PURPOSE: The phenotypic manifestations of cerebral cavernous malformation disease caused by rare PDCD10 mutations have not been systematically examined, and a mechanistic link to Rho kinase-mediated hyperpermeability, a potential therapeutic target, has not been established. METHODS: We analyzed PDCD10 small interfering RNA-treated endothelial cells for stress fibers, Rho kinase activity, and permeability. Rho kinase activity was assessed in cerebral cavernous malformation lesions. Brain permeability and cerebral cavernous malformation lesion burden were quantified, and clinical manifestations were assessed in prospectively enrolled subjects with PDCD10 mutations. RESULTS: We determined that PDCD10 protein suppresses endothelial stress fibers, Rho kinase activity, and permeability in vitro. Pdcd10 heterozygous mice have greater lesion burden than other Ccm genotypes. We demonstrated robust Rho kinase activity in murine and human cerebral cavernous malformation vasculature and increased brain vascular permeability in humans with PDCD10 mutation. Clinical phenotype is exceptionally aggressive compared with the more common KRIT1 and CCM2 familial and sporadic cerebral cavernous malformation, with greater lesion burden and more frequent hemorrhages earlier in life. We first report other phenotypic features, including scoliosis, cognitive disability, and skin lesions, unrelated to lesion burden or bleeding. CONCLUSION: These findings define a unique cerebral cavernous malformation disease with exceptional aggressiveness, and they inform preclinical therapeutic testing, clinical counseling, and the design of trials.Genet Med 17 3, 188-196.
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Proteínas Reguladoras de la Apoptosis/genética , Neoplasias del Sistema Nervioso Central/patología , Hemangioma Cavernoso del Sistema Nervioso Central/patología , Péptidos y Proteínas de Señalización Intracelular/genética , Proteínas de la Membrana/genética , Mutación , Proteínas Proto-Oncogénicas/genética , Quinasas Asociadas a rho/metabolismo , 1-(5-Isoquinolinesulfonil)-2-Metilpiperazina/análogos & derivados , 1-(5-Isoquinolinesulfonil)-2-Metilpiperazina/farmacología , Adolescente , Adulto , Animales , Proteínas Reguladoras de la Apoptosis/metabolismo , Proteínas Portadoras/genética , Células Cultivadas , Neoplasias del Sistema Nervioso Central/enzimología , Neoplasias del Sistema Nervioso Central/genética , Niño , Preescolar , Modelos Animales de Enfermedad , Hemangioma Cavernoso del Sistema Nervioso Central/enzimología , Hemangioma Cavernoso del Sistema Nervioso Central/genética , Células Endoteliales de la Vena Umbilical Humana , Humanos , Lactante , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Queratina-1/genética , Proteínas de la Membrana/metabolismo , Ratones , Persona de Mediana Edad , Estudios Prospectivos , Proteínas Proto-Oncogénicas/metabolismo , Fibras de Estrés/efectos de los fármacos , Fibras de Estrés/metabolismo , Adulto Joven , Quinasas Asociadas a rho/antagonistas & inhibidoresRESUMEN
BACKGROUND: Glioblastoma (GBM) is the most common primary brain tumor and the most aggressive glial tumor. This tumor is highly heterogeneous, angiogenic, and insensitive to radio- and chemotherapy. Here we have investigated the progression of GBM produced by the injection of human GBM cells into the brain parenchyma of immunocompetent mice. METHODS: Xenotransplanted animals were submitted to magnetic resonance imaging (MRI) and histopathological analyses. RESULTS: Our data show that two weeks after injection, the produced tumor presents histopathological characteristics recommended by World Health Organization for the diagnosis of GBM in humans. The tumor was able to produce reactive gliosis in the adjacent parenchyma, angiogenesis, an intense recruitment of macrophage and microglial cells, and presence of necrosis regions. Besides, MRI showed that tumor mass had enhanced contrast, suggesting a blood-brain barrier disruption. CONCLUSIONS: This study demonstrated that the xenografted tumor in mouse brain parenchyma develops in a very similar manner to those found in patients affected by GBM and can be used to better understand the biology of GBM as well as testing potential therapies.
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Neoplasias Encefálicas/patología , Encéfalo/patología , Modelos Animales de Enfermedad , Glioblastoma/patología , Microambiente Tumoral , Animales , Encéfalo/irrigación sanguínea , Neoplasias Encefálicas/complicaciones , Glioblastoma/complicaciones , Glioblastoma/fisiopatología , Gliosis/etiología , Humanos , Inmunocompetencia , Activación de Macrófagos , Imagen por Resonancia Magnética , Masculino , Ratones , Microglía/fisiología , Necrosis/etiología , Neovascularización Patológica/etiología , Trasplante HeterólogoRESUMEN
Inflammation influences the pathogenesis of seizures by boosting neuronal degeneration of temporal lobe epilepsy with hippocampal sclerosis (TLE-HS). This work aimed to determine the activity of metalloproteases (MMPs) in brain tissue fragments of TLE-HS patients and the effect of lobectomy on circulating inflammatory biomarkers. Surgical fragments (n=4) from epileptogenic focus (EF) e perilesion area (PL), and control hippocampus from autopsy (n=5) were processed for glial protein (GFAP), activated microglia (IB4) immunohistochemistry, and metalloprotease activity (MMP-2, -9). Perilesional area showed GFAP positive cells with morphology of activate astrocyte and reactive gliosis nearby the lesion. In the lesion foci, astrocytes had altered cytoarchitecture with disorganized stroma suggestive of necrosis, and numerous mononuclear cells with few projections and morphological characteristics of activate microglia. Analysis of MMP-9 and MMP-2 in the sera before and after hippocampectomy confirmed the inflammatory pattern of TLE-HS, with high MMP-9 activity; high MMP-9/TIMP-1 and urokinase uPAR plasma levels before lobectomy but low after surgery. Maintenance of MMP-2 activity indicates persistent tissue remodeling in both groups. The present work shows that patients with chronic and medically intractable TLE-HS that undergone amigdalo-hippocampectomy for removal of epileptogenic lesion had a clinical enduring benefit of lack seizure recurrence for up to a year, and consistent reduction of proteases (MMP-9 and uPAR) activation that participate as important inflammatory epileptogenic inducers.
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Epilepsia del Lóbulo Temporal/metabolismo , Epilepsia del Lóbulo Temporal/patología , Hipocampo/metabolismo , Hipocampo/patología , Metaloproteasas/sangre , Receptores del Activador de Plasminógeno Tipo Uroquinasa/sangre , Adolescente , Adulto , Lobectomía Temporal Anterior , Citocinas/sangre , Encefalitis/metabolismo , Epilepsia del Lóbulo Temporal/sangre , Epilepsia del Lóbulo Temporal/enzimología , Femenino , Humanos , Masculino , Persona de Mediana Edad , Inhibidores Tisulares de Metaloproteinasas/sangre , Adulto JovenRESUMEN
Connective-tissue growth factor (CTGF/CCN2) is a matricellular-secreted protein involved in complex processes such as wound healing, angiogenesis, fibrosis and metastasis, in the regulation of cell proliferation, migration and extracellular matrix remodeling. Glioblastoma (GBM) is the major malignant primary brain tumor and its adaptation to the central nervous system microenvironment requires the production and remodeling of the extracellular matrix. Previously, we published an in vitro approach to test if neurons can influence the expression of the GBM extracellular matrix. We demonstrated that neurons remodeled glioma cell laminin. The present study shows that neurons are also able to modulate CTGF expression in GBM. CTGF immnoreactivity and mRNA levels in GBM cells are dramatically decreased when these cells are co-cultured with neonatal neurons. As proof of particular neuron effects, neonatal neurons co-cultured onto GBM cells also inhibit the reporter luciferase activity under control of the CTGF promoter, suggesting inhibition at the transcription level. This inhibition seems to be contact-mediated, since conditioned media from embryonic or neonatal neurons do not affect CTGF expression in GBM cells. Furthermore, the inhibition of CTGF expression in GBM/neuronal co-cultures seems to affect the two main signaling pathways related to CTGF. We observed inhibition of TGFß luciferase reporter assay; however phopho-SMAD2 levels did not change in these co-cultures. In addition levels of phospho-p44/42 MAPK were decreased in co-cultured GBM cells. Finally, in transwell migration assay, CTGF siRNA transfected GBM cells or GBM cells co-cultured with neurons showed a decrease in the migration rate compared to controls. Previous data regarding laminin and these results demonstrating that CTGF is down-regulated in GBM cells co-cultured with neonatal neurons points out an interesting view in the understanding of the tumor and cerebral microenvironment interactions and could open up new strategies as well as suggest a new target in GBM control.
Asunto(s)
Comunicación Celular , Factor de Crecimiento del Tejido Conjuntivo/metabolismo , Glioblastoma/metabolismo , Neuronas/metabolismo , Animales , Línea Celular Tumoral , Movimiento Celular , Técnicas de Cocultivo , Factor de Crecimiento del Tejido Conjuntivo/genética , Expresión Génica , Regulación Neoplásica de la Expresión Génica , Glioblastoma/genética , Proteína Quinasa 3 Activada por Mitógenos/metabolismo , Fosforilación , Cultivo Primario de Células , Regiones Promotoras Genéticas , Ratas , Transducción de Señal , Proteína Smad2/metabolismo , Proteína smad3/metabolismo , Activación Transcripcional , Factor de Crecimiento Transformador beta/metabolismoRESUMEN
Glioblastoma (GBM) is considered incurable due to its resistance to current cancer treatments. So far, all clinically available alternatives for treating GBM are limited, evoking the development of novel treatment strategies that can more effectively manage these tumors. Extensive effort is being dedicated to characterize the molecular basis of GBM resistance to chemotherapy and to explore novel therapeutic procedures that may improve overall survival. Cytolysins are toxins that form pores in target cell membranes, modifying ion homeostasis and leading to cell death. These pore-forming toxins might be used, therefore, to enhance the efficiency of conventional chemotherapeutic drugs, facilitating their entrance into the cell. In this study, we show that a non-cytotoxic concentration of equinatoxin II (EqTx-II), a pore-forming toxin from the sea anemone Actinia equina, potentiates the cytotoxicity induced by temozolomide (TMZ), a first-line GBM treatment, and by etoposide (VP-16), a second- or third-line GBM treatment. We also suggest that this effect is selective to GBM cells and occurs via PI3K/Akt pathway inhibition. Finally, Magnetic resonance imaging (MRI) revealed that a non-cytotoxic concentration of EqTx-II potentiates the VP-16-induced inhibition of GBM growth in vivo. These combined therapies constitute a new and potentially valuable tool for GBM treatment, leading to the requirement of lower concentrations of chemotherapeutic drugs and possibly reducing, therefore, the adverse effects of chemotherapy.
Asunto(s)
Antineoplásicos/farmacología , Apoptosis/efectos de los fármacos , Neoplasias Encefálicas/patología , Venenos de Cnidarios/farmacología , Dacarbazina/análogos & derivados , Etopósido/farmacología , Glioblastoma/patología , Animales , Western Blotting , Línea Celular Tumoral , Dacarbazina/farmacología , Sinergismo Farmacológico , Humanos , Inmunohistoquímica , Imagen por Resonancia Magnética , Masculino , Ratones , Ratones Endogámicos C57BL , TemozolomidaRESUMEN
Assembly of synapses requires proper coordination between pre- and postsynaptic elements. Identification of cellular and molecular events in synapse formation and maintenance is a key step to understand human perception, learning, memory, and cognition. A key role for astrocytes in synapse formation and function has been proposed. Here, we show that transforming growth factor ß (TGF-ß) signaling is a novel synaptogenic pathway for cortical neurons induced by murine and human astrocytes. By combining gain and loss of function approaches, we show that TGF-ß1 induces the formation of functional synapses in mice. Further, TGF-ß1-induced synaptogenesis involves neuronal activity and secretion of the co-agonist of the NMDA receptor, D-serine. Manipulation of D-serine signaling, by either genetic or pharmacological inhibition, prevented the TGF-ß1 synaptogenic effect. Our data show a novel molecular mechanism that might impact synaptic function and emphasize the evolutionary aspect of the synaptogenic property of astrocytes, thus shedding light on new potential therapeutic targets for synaptic deficit diseases.