Análise dos efeitos biológicos da lectina extraída de Canavalia villosa (Cvill) em linhagem de glioma C6 (Rattus norvegicus)

TCC(graduação) - Universidade Federal de Santa Catarina. Centro de Ciências Biológicas.Biologia.Os gliomas representam tumores cerebrais que têm origem nas células gliais ou precursores gliais. Estes tumores apresentam rápida proliferação e grande resistência aos mecanismos de morte celular, causando elevada mortalidade. Os tratamentos disponíveis ainda são muito limitados, invasivos e pouco seletivos. Para tanto, novas estratégias de tratamento têm sido investigadas. Lectinas são proteínas que reconhecem e se ligam a carboidratos com alta especificidade, desencadeando diversos efeitos biológicos, incluindo ações antitumorais. Notavelmente, células tumorais podem apresentar alteração no perfil de glicanos expressos na sua superfície, evento que pode torna-las alvo seletivo para ação de lectinas. No presente estudo foi avaliado o potencial citotóxico da lectina Cvill, com afinidade por glicose/manose, purificada a partir das sementes de Canavalia villosa. Para esta abordagem foram utilizadas células tumorais da linhagem C6 de ratos (Rattus norvegicus) como modelo in vitro de glioma. As células foram incubadas por períodos de 1, 3, 6, 12, 24 e 48h com a lectina Cvill nas concentrações de 10, 30, 50 e 100μg/mL. Os resultados indicaram dois efeitos antitumorais importantes induzidos pelo tratamento com Cvill: morte celular e inibição parcial da migração/proliferação. Neste sentido foi observado diminuição da viabilidade celular de maneira tempo e concentração-dependente, iniciando a partir de 6h, na concentração de 30μg/mL. Esta ação foi dependente do domínio de reconhecimento a carboidratos (CRD) e da estrutura terciária da lectina. Da mesma forma a morfologia celular mostrou alterações evidentes como perda de prolongamentos citoplasmáticos e degradação da matriz extracelular. O tratamento intermitente com a lectina revelou resistência celular na dose de 30μg/mL. Em relação aos possíveis mecanismos de morte celular induzido por Cvill foi observado aumento precoce (6-12h) de organelas vesiculares ácidas e de LC3II, indicando autofagia. Além disso, um aumento da marcação de fosfatidilserina por anexina V em 6h de tratamento, indica a indução de apoptose. Nesse mesmo período, também foi verificado diminuição do potencial da membrana mitocondrial comprometendo a maquinaria energética da célula. Embora mais estudos sejam necessários para elucidar os mecanismos moleculares e alvos da lectina Cvill sobre as células de glioma, os resultados apontam um potencial para aplicação desta lectina com vista a terapia antitumoral.Gliomas represent brain tumors originate in glial cells or glial precursors. These tumors present rapid proliferation and high resistance to mechanisms of cell death, causing high mortality. Available treatments are still very limited, invasive and poorly selective. Therefore, new treatment strategies have been investigated. Lectins are proteins that recognize and bind to carbohydrates with high specificity, triggering various biological effects, including antitumor actions. Notably, tumor cells may have a change in the profile of glycans expressed on their surface, an event that may make them a selective target for lectin action. In the present study the cytotoxicity potential of the Cvill, a mannose/glucose binding lectin, purified from Canavalia villosa seeds was evaluated. For this approach, rat C6 line (Rattus norvegicus) tumor cells were used as the in vitro model of glioma. Cells were incubated for periods of 1, 3, 6, 12, 24 and 48h with Cvill lectin at concentrations of 10, 30, 50 and 100μg/mL. The results indicated two important antitumor effects induced by Cvill treatment: cell death and partial inhibition of migration/proliferation. In this sense, a decrease in cell viability was observed in a time and concentration-dependent manner, starting at 6 h at a concentration of 30μg/mL. This action was dependent on the carbohydrate recognition domain (CRD) and the lectin tertiary structure. In the same way the cellular morphology showed evident alterations like loss of cytoplasmic prolongations and degradation of the extracellular matrix. Intermittent treatment with the lectin revealed cell resistance at the dose of 30μg/mL. Regarding the possible mechanisms of cell death induced by Cvill, early enlargement (6-12h) of acidic vesicle organelles and LC3II, indicating autophagy, was observed. In addition, an increase in the labeling of phosphatidylserine by annexin V within 6 h of treatment indicates the induction of apoptosis. In the same period, it was also observed a decrease in the mitochondrial membrane potential compromising the energetic machinery of the cell. Although more studies are needed to elucidate the molecular mechanisms and targets of Cvill lectin on glioma cells, the results point to a potential for the application of this lectin for antitumor therapy

Efeitos do ambiente enriquecido nos déficits motores, comportamentais e nos níveis de monoaminas no modelo YAC128 da Doença de Huntington

Dissertação (mestrado) - Universidade Federal de Santa Catarina, Centro de Ciências Biológicas, Programa de Pós-Graduação em Neurociências, Florianópolis, 2021.A Doença de Huntington (DH) é um distúrbio genético neurodegenerativo caracterizado por déficits motores, psiquiátricos, cognitivos e periféricos. O estriado é a região encefálica mais afetada, no entanto outras regiões como o córtex pré-frontal e o hipocampo também estão comprometidas nessa condição. Estudos em humanos e modelos animais têm observado que mesmo antes da degeneração ocorrer, alguns sintomas já estão presentes. Isso implica que outras disfunções devem estar ocorrendo para justificar um fenótipo anterior a degeneração. Apesar de ser uma doença de natureza genética, evidências mostram que o estilo de vida é capaz de modificar a idade de início dos sintomas na DH e a taxa de sua progressão. Neste trabalho, buscou-se avaliar se o ambiente enriquecido (AE), uma analogia para intervenções benéficas ao estilo de vida, poderia atuar de maneira preventiva sobre déficits motores e comportamentais e se os efeitos proporcionados pelo AE no início da vida persistem em idades posteriores. Também avaliamos se o AE apresentava algum efeito sobre os níveis de monoaminas. Para tanto, camundongos machos e fêmeas do tipo selvagem (WT) e YAC128 foram expostos a condições padrão de habitação (AC) ou AE por 39 dias (do dia 21 ao dia 60 pós-natal). Aos 2 meses de idade, uma bateria de testes comportamentais foi aplicada. Após o último teste, o hipocampo, o córtex pré-frontal e o estriado foram dissecados para medir os níveis de monoaminas por cromatografia líquida de alta eficiência (HPLC). Em uma segunda coorte de animais, o AE foi aplicado durante o mesmo período, mas só foram avaliados aos 4 meses quando novamente uma bateria de testes comportamentais foi realizada. Diferenças entre os camundongos YAC128 e WT alojados no AC foram encontradas no peso corporal de machos, no comportamento do tipo-ansioso e no desempenho motor aos 2 meses de idade. O AE foi capaz de prevenir o ganho de peso corporal nos machos, teve efeito ansiolítico nos machos e preveniu os déficits motores. A avaliação dos níveis de monoaminas não mostrou alterações em relação ao genótipo aos 2 meses de idade. No entanto, foi observado que o ambiente foi capaz de diminuir os níveis de monoaminas nas três regiões avaliadas. Interessantemente uma correlação significativa e negativa foi encontrada entre melhor performance motora e menores níveis de monoaminas principalmente no estriado. Em relação aos camundongos analisados aos 4 meses, diferenças genotípicas foram encontradas no comportamento anedônico e o AE intermitente trouxe benefícios parciais. Nossos resultados confirmaram os déficits motores, comportamento do tipo-ansioso, anedônico e ganho de peso corporal em camundongos YAC128 e o AE preveniu ou trouxe benefícios parciais nestas avaliações. Alguns benefícios proporcionados pelo AE podem ter ocorrido, pelo menos em parte, pela modulação dos níveis de monoaminas. Portanto, nossos achados apoiam as intervenções ambientais como uma abordagem terapêutica benéfica para atrasar o início e a sintomatologia da DH.Abstract: Huntington's disease (HD) is a neurodegenerative genetic disorder characterized by motor, psychiatric, cognitive, and peripheral deficits. The striatum is the most affected brain region; however, other areas such as the prefrontal cortex and hippocampus are also compromised in this condition. Studies in humans and animal models have observed that some symptoms are already present even before degeneration occurs, implying that other dysfunctions must be happening to justify a phenotype before degeneration. Despite being a disease of genetic nature, evidence shows that lifestyle can modify the age of onset of symptoms in HD and the rate of its progression. In this work, we sought to assess whether the enriched environment (EE), an analogy for beneficial interventions to lifestyle, could act preventively on motor and behavioral deficits and whether the effects provided by EE in early life persist in later ages. We also assessed whether EE had any effect on monoamine levels. For this purpose, wild-type (WT) and YAC128 male and female mice were exposed to standard housing conditions (CE, control environmental) or EE for 39 days (from postnatal day 21 to postnatal day 60). At 2 months of age, a battery of behavioral tests was applied. After the last test, the hippocampus, prefrontal cortex, and striatum were dissected to measure monoamine levels by high-performance liquid chromatography (HPLC). In the second cohort of animals, the EE was applied during the same period. However, they were only behaviorally evaluated at 4 months of age. Differences between YAC128 and WT mice housed in the CE were found in male body weight, anxiety-like behavior, and motor performance at 2 months of age. The EE was able to prevent body weight gain in males, had anxiolytic effect in males and prevented motor deficits. The evaluation of monoamine levels showed no changes concerning genotype at 2 months of age. However, it was observed that the EE reduced the levels of monoamines in the three regions evaluated. Interestingly, a significant and negative correlation was found between better motor performance and lower levels of monoamines, mainly in the striatum. Genotypic differences were found in anhedonic behavior in mice analyzed at 4 months of age, and intermittent EE exposure showed partial benefits. Our results confirmed the motor deficits, anxiety-like behavior, anhedonia and body weight gain in YAC128 mice and the AE prevented or brought partial benefits to these evaluations. Some benefits provided by EE may have occurred, at least in part, by modulating the levels of monoamines. Therefore, our findings support environmental interventions as a beneficial therapeutic approach to delay the onset and symptomatology of HD

New Avenues for the Treatment of Huntington’s Disease

Huntington’s disease (HD) is a neurodegenerative disorder caused by a CAG expansion in the HD gene. The disease is characterized by neurodegeneration, particularly in the striatum and cortex. The first symptoms usually appear in mid-life and include cognitive deficits and motor disturbances that progress over time. Despite being a genetic disorder with a known cause, several mechanisms are thought to contribute to neurodegeneration in HD, and numerous pre-clinical and clinical studies have been conducted and are currently underway to test the efficacy of therapeutic approaches targeting some of these mechanisms with varying degrees of success. Although current clinical trials may lead to the identification or refinement of treatments that are likely to improve the quality of life of those living with HD, major efforts continue to be invested at the pre-clinical level, with numerous studies testing novel approaches that show promise as disease-modifying strategies. This review offers a detailed overview of the currently approved treatment options for HD and the clinical trials for this neurodegenerative disorder that are underway and concludes by discussing potential disease-modifying treatments that have shown promise in pre-clinical studies, including increasing neurotropic support, modulating autophagy, epigenetic and genetic manipulations, and the use of nanocarriers and stem cells.Medicine, Faculty ofOther UBCReviewedOthe

Beyond Motor Deficits: Environmental Enrichment Mitigates Huntington’s Disease Effects in YAC128 Mice

Huntington’s disease (HD) is a neurodegenerative genetic disorder characterized by motor, psychiatric, cognitive, and peripheral symptoms without effective therapy. Evidence suggests that lifestyle factors can modulate disease onset and progression, and environmental enrichment (EE) has emerged as a potential approach to mitigate the progression and severity of neurodegenerative processes. Wild-type (WT) and yeast artificial chromosome (YAC) 128 mice were exposed to different EE conditions. Animals from cohort 1 were exposed to EE between postnatal days 21 and 60, and animals from cohort 2 were exposed to EE between postnatal days 60 and 120. Motor and non-motor behavioral tests were employed to evaluate the effects of EE on HD progression. Monoamine levels, hippocampal cell proliferation, neuronal differentiation, and dendritic arborization were also assessed. Here we show that EE had an antidepressant-like effect and slowed the progression of motor deficits in HD mice. It also reduced monoamine levels, which correlated with better motor performance, particularly in the striatum. EE also modulated neuronal differentiation in the YAC128 hippocampus. These results confirm that EE can impact behavior, hippocampal neuroplasticity, and monoamine levels in YAC128 mice, suggesting this could be a therapeutic strategy to modulate neuroplasticity deficits in HD. However, further research is needed to fully understand EE’s mechanisms and long-term effects as an adjuvant therapy for this debilitating condition

Temporal Characterization of Behavioral and Hippocampal Dysfunction in the YAC128 Mouse Model of Huntington’s Disease

Huntington’s disease (HD) is a genetic neurodegenerative disease characterized by motor, psychiatric, and cognitive symptoms. Emerging evidence suggests that emotional and cognitive deficits seen in HD may be related to hippocampal dysfunction. We used the YAC128 HD mouse model to perform a temporal characterization of the behavioral and hippocampal dysfunctions. Early and late symptomatic YAC128 mice exhibited depressive-like behavior, as demonstrated by increased immobility times in the Tail Suspension Test. In addition, YAC128 mice exhibited cognitive deficits in the Swimming T-maze Test during the late symptomatic stage. Except for a reduction in basal mitochondrial respiration, no significant deficits in the mitochondrial respiratory rates were observed in the hippocampus of late symptomatic YAC128 mice. In agreement, YAC128 animals did not present robust alterations in mitochondrial ultrastructural morphology. However, light and electron microscopy analysis revealed the presence of dark neurons characterized by the intense staining of granule cell bodies and shrunken nuclei and cytoplasm in the hippocampal dentate gyrus (DG) of late symptomatic YAC128 mice. Furthermore, structural alterations in the rough endoplasmic reticulum and Golgi apparatus were detected in the hippocampal DG of YAC128 mice by electron microscopy. These results clearly show a degenerative process in the hippocampal DG in late symptomatic YAC128 animals

Structural prediction and characterization of Canavalia grandiflora (ConGF) lectin complexed with MMP1 : unveiling the antiglioma potential of legume lectins

A glioblastoma (GBM) is a highly malignant primary brain tumor with a poor prognosis because of its invasiveness and high resistance to current therapies. In GBMs, abnormal glycosylation patterns are associated with malignancy, which allows for the use of lectins as tools for recognition and therapy. More specifically, lectins can interact with glycan structures found on the malignant cell surface. In this context, the present work aimed to investigate the antiglioma potential of ConGF, a lectin purified from Canavalia grandiflora seeds, against C6 cells. The treatment of C6 cells with ConGF impaired the mitochondrial transmembrane potential, reduced cell viability, and induced morphological changes. ConGF also induced massive autophagy, as evaluated by acridine orange (AO) staining and LC3AB-II expression, but without prominent propidium iodide (PI) labeling. The mechanism of action appears to involve the carbohydrate-binding capacity of ConGF, and in silico studies suggested that the lectin can interact with the glycan structures of matrix metalloproteinase 1 (MMP1), a prominent protein found in malignant cells, likely explaining the observed effects

Characterisation of microbial attack on archaeological bone

As part of an EU funded project to investigate the factors influencing bone preservation in the archaeological record, more than 250 bones from 41 archaeological sites in five countries spanning four climatic regions were studied for diagenetic alteration. Sites were selected to cover a range of environmental conditions and archaeological contexts. Microscopic and physical (mercury intrusion porosimetry) analyses of these bones revealed that the majority (68%) had suffered microbial attack. Furthermore, significant differences were found between animal and human bone in both the state of preservation and the type of microbial attack present. These differences in preservation might result from differences in early taphonomy of the bones. © 2003 Elsevier Science Ltd. All rights reserved