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The effect of cyanotoxins on fibroblasts with amyotrophic lateral sclerosis genetic mutations
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Abstract(s)
A esclerose lateral amiotrófica é uma doença neurodegenerativa progressiva que afeta quer os
neurónios motores inferiores no tronco cerebral e na medula espinhal como os neurónios
motores superiores no córtex motor. Apesar da investigação em curso, as causas subjacentes a
esta doença permanecem mal compreendidas, não existindo, atualmente, uma cura nem um
teste de diagnóstico específico. A maioria, cerca de 90-95% dos casos, é de natureza
esporádica, sendo os restantes 5-10% de origem familiar. A sua neuropatologia é complexa e
envolve múltiplos mecanismos celulares e moleculares que podem causar a morte dos
neurónios motores através de diferentes processos, como o stress oxidativo, disfunções
mitocondriais, formação de agregados proteicos, toxicidade do glutamato e neuroinflamação.
A genética desempenha um papel importante, tendo sido identificados vários genes
envolvidos nos seus mecanismos patogénicos. No entanto, nem todos os casos podem ser
explicados apenas pela mesma. As toxinas criadas naturalmente no ambiente, como as
cianotoxinas produzidas por cianobactérias, são extremamente neurotóxicas e podem atuar
por várias vias, desempenhando um papel no desenvolvimento de doenças
neurodegenerativas, embora as suas funções na neuropatia sejam ainda discutíveis.
Para investigar a ligação entre a exposição a cianotoxinas e a neurodegeneração,
concentrámo-nos na neurotoxina β-N-metilamino-L-alanina e em extratos brutos de uma
estirpe de cianobactérias isolada na Sardenha (Microcystis aeruginosa), utilizando fibroblastos
cutâneos primários de dadores saudáveis e de pacientes com esclerose lateral amiotrófica,
tanto familiar (com uma mutação A382T no gene TARDBP) como esporádica. O efeito das
cianotoxinas foi avaliado utilizando vários métodos (western blot, imunofluorescência e
citometria de fluxo) e possíveis biomarcadores de neurodegeneração (agregados proteicos,
marcadores apoptóticos e citocinas inflamatórias).
Nos fibroblastos expostos às cianotoxinas, observámos: que não ocorreu morte celular por
apoptose; a deslocalização e agregação de proteínas no citoplasma; e um aumento particular
de citocinas nos modelos esporádicos. Os resultados do presente trabalho sugerem uma
ligação entre a exposição a cianotoxinas e alterações a nível genético e molecular que podem
levar à neurodegeneração, reforçando o papel das cianotoxinas neste processo.
Amyotrophic lateral sclerosis is a progressive neurodegenerative disorder impacting both the lower motor neurons in the brainstem and spinal cord and the upper motor neurons in the motor cortex. Despite ongoing research, underlying causes of this disease remain poorly understood, and there is currently neither a cure nor a specific diagnostic test available. The majority, approximately 90-95% of cases, are sporadic in nature, with the remaining 5-10% being familial in origin. Its neuropathology is complex and involves multiple cellular and molecular mechanisms that can cause motor neurons death through different processes such as oxidative stress, mitochondrial dysfunctions, protein aggregates formation, glutamate toxicity and neuroinflammation. Genetics play a significant role and several genes involved in its pathomechanisms have been identified. However, not all cases can be explained solely by human genetics. Toxins created naturally in the environment, such as cyanotoxins produced by cyanobacteria, are extremely neurotoxic and may act through a number of routes, playing a role in the development of neurodegenerative diseases, although their functions in neuropathy are still debatable. To investigate the link between cyanotoxin exposure and neurodegeneration, we focused on the neurotoxin β-N-methylamino-L-alanine and on crude extracts from a cyanobacteria strain isolated in Sardinia (Microcystis aeruginosa), using primary cutaneous fibroblasts from healthy donors and patients with amyotrophic lateral sclerosis, both familial (with a A382T mutation in the TARDBP gene) and sporadic. The effect of cyanotoxins was evaluated using various methods (western blot, immunofluorescence and flow cytometry) and possible neurodegeneration biomarkers (protein aggregates, apoptotic markers and inflammatory cytokines). In the fibroblasts exposed to cyanotoxins, we observed: no cell death by apoptosis; protein delocalization and aggregation in the cytoplasm; and a particular cytokine increase in the sporadic models. The results of the present work suggest a connection between exposure to cyanotoxins and genetic and molecular alterations that could lead to neurodegeneration, reinforcing the role of cyanotoxins in this process.
Amyotrophic lateral sclerosis is a progressive neurodegenerative disorder impacting both the lower motor neurons in the brainstem and spinal cord and the upper motor neurons in the motor cortex. Despite ongoing research, underlying causes of this disease remain poorly understood, and there is currently neither a cure nor a specific diagnostic test available. The majority, approximately 90-95% of cases, are sporadic in nature, with the remaining 5-10% being familial in origin. Its neuropathology is complex and involves multiple cellular and molecular mechanisms that can cause motor neurons death through different processes such as oxidative stress, mitochondrial dysfunctions, protein aggregates formation, glutamate toxicity and neuroinflammation. Genetics play a significant role and several genes involved in its pathomechanisms have been identified. However, not all cases can be explained solely by human genetics. Toxins created naturally in the environment, such as cyanotoxins produced by cyanobacteria, are extremely neurotoxic and may act through a number of routes, playing a role in the development of neurodegenerative diseases, although their functions in neuropathy are still debatable. To investigate the link between cyanotoxin exposure and neurodegeneration, we focused on the neurotoxin β-N-methylamino-L-alanine and on crude extracts from a cyanobacteria strain isolated in Sardinia (Microcystis aeruginosa), using primary cutaneous fibroblasts from healthy donors and patients with amyotrophic lateral sclerosis, both familial (with a A382T mutation in the TARDBP gene) and sporadic. The effect of cyanotoxins was evaluated using various methods (western blot, immunofluorescence and flow cytometry) and possible neurodegeneration biomarkers (protein aggregates, apoptotic markers and inflammatory cytokines). In the fibroblasts exposed to cyanotoxins, we observed: no cell death by apoptosis; protein delocalization and aggregation in the cytoplasm; and a particular cytokine increase in the sporadic models. The results of the present work suggest a connection between exposure to cyanotoxins and genetic and molecular alterations that could lead to neurodegeneration, reinforcing the role of cyanotoxins in this process.
Description
Trabalho Final de Mestrado Integrado, Ciências Farmacêuticas, 2023, Universidade de Lisboa, Faculdade de Farmácia.
Keywords
Amyotrophic lateral sclerosis Cyanotoxins Neurodegeneration Mestrado Integrado - 2023