The role of neurodegeneration-associated proteins in ALS and medulloblastoma

Amyotrophic Lateral Sclerosis (ALS) is a neurodegenerative disease for which there is currently no effective treatment. Therefore, it is essential to investigate the mechanisms leading to motor neuron death to find potential therapeutic targets. In this thesis, we deepen our understanding of 3 proteins involved in the pathogenesis of ALS (Fused in Sarcoma -FUS, Vesicle-associated Membrane Protein B -VAPB, and the ephrin receptor A4-EPHA4) by analyzing their effects when mutated, absent, or when their activity is inhibited. There is increasing evidence that suggests that overlapping mechanisms play a role in neurodegeneration and cell transformation. Therefore, we investigated these ALS -related proteins in neurodegeneration and medulloblastoma development. We describe that protein translation –a well-known mechanism that cells upregulate when transformed- is one of the first and most fundamental pathways decreased in cells with ALS -related mutations. We also show that interferon-gamma (IFN-y) upregulates translation-associated genes, specifically in ALS MNs. Regarding the function of ALS-associated proteins in medulloblastoma, we reveal novel roles for VAPB and EPHA4 in tumor progression. In summary, we provide evidence to support the hypothesis that neurodegeneration and tumorigenesis are the results of the same deregulated signaling pathways, which could lead to a better understanding of these devastating diseases and generate new intervention strategies to improve the lives of patients in the future

IFNγ protects motor neurons from oxidative stress via enhanced global protein synthesis in FUS-associated amyotrophic lateral sclerosis

Amyotrophic lateral sclerosis type 6 (ALS6) is a familial subtype of ALS linked to Fused in Sarcoma (FUS) gene mutation. FUS mutations lead to decreased global protein synthesis, but the mechanism that drives this has not been established. Here, we used ALS6 patient-derived induced pluripotent stem cells (hIPSCs) to study the effect of the ALS6 FUSR521H mutation on the translation machinery in motor neurons (MNs). We find, in agreement with findings of others, that protein synthesis is decreased in FUSR521H MNs. Furthermore, FUSR521H MNs are more sensitive to oxidative stress and display reduced expression of TGF-β and mTORC gene pathways when stressed. Finally, we show that IFNγ treatment reduces apoptosis of FUSR521H MNs exposed to oxidative stress and partially restores the translation rates in FUSR521H MNs. Overall, these findings suggest that a functional IFNγ response is important for FUS-mediated protein synthesis, possibly by FUS nuclear translocation in ALS6.</p

Neurodegeneration-associated protein VAPB regulates proliferation in medulloblastoma

VAMP (Vesicle-associated membrane protein)-associated protein B and C (VAPB) has been widely studied in neurodegenerative diseases such as ALS, but little is known about its role in cancer. Medulloblastoma is a common brain malignancy in children and arises from undifferentiated cells during neuronal development. Therefore, medulloblastoma is an interesting model to investigate the possible relationship between VAPB and tumorigenesis. Here we demonstrate that high VAPB expression in medulloblastoma correlates with decreased overall patient survival. Consistent with this clinical correlation, we find that VAPB is required for normal proliferation rates of medulloblastoma cells in vitro and in vivo. Knockout of VAPB (VAPB KO) delayed cell cycle progression. Furthermore, transcript levels of WNT-related proteins were decreased in the VAPB KO. We conclude that VAPB is required for proliferation of medulloblastoma cells, thus revealing VAPB as a potential therapeutic target for medulloblastoma treatment. </p

Characterization of the secretome of multipotent mesenchymal stromal cells from various tissues

Células multipotentes mesenquimais estromais (CTM) são células adultas multipotentes que podem ser isoladas a partir de diferentes tecidos e são capazes de atingir sítios danificados, exercer papéis na regeneração tecidual e modular a resposta imune. Estas células demonstraram resultados discrepantes em estudos in vivo dependentes de sua fonte de obtenção. Há na literatura hipóteses de que o mecanismo predominante pelo qual as CTMs atuam no reparo tecidual estaria relacionado à sua atividade parácrina, criando um microambiente com sinais tróficos. Nesse sentido, a avaliação do conteúdo do secretoma destas células é de grande interesse. Portanto, este projeto teve como objetivo analisar o meio condicionado de CTMs obtidas de diferentes fontes (tecido adiposo, músculo esquelético e tubas uterinas) de mesmos indivíduos. A abordagem experimental consistiu em proteômica shotgun (nanocromatografia líquida acoplada a espectrometria de massas em tandem) com o intuito de identificar alvos diferentemente expressos entre as culturas que possam sugerir funções específicas de cada linhagem celular. Os dados espectrais foram obtidos pelo modo de aquisição dependente de dados (Top15). Os dados adquiridos foram processados pelas plataformas MaxQuant e TPP (Trans-Proteomic Pipeline). Foi realizada análise qualitativa de vias enriquecidas por meio do programa Ingenuity utilizando as proteínas em comum nos secretoma de todas as CTMs analisadas. Essa análise permitiu observar vias enriquecidas de proliferação celular, migração celular e desenvolvimento do sistema cardiovascular, demonstrando que as proteínas secretadas por quaisquer das CTMs analisadas podem ser relacionadas a resultados encontrados na literatura utilizando estas células para terapias para patologias. As análises estatísticas para determinar se haveria dependência da composição do secretoma em função do indivíduo doador ou tecido fonte das CTMs revelaram proteínas diferencialmente expressas entre todos os grupos. Estas proteínas diferencialmente expressas são relacionadas à proliferação, sinalização e interação celular, além de modulação do sistema imune e da angiogênese. Neste contexto, podemos concluir que o secretoma das CTMs é muito semelhante, que as CTMs isoladas de quaisquer tecidos ou indivíduos são capazes de secretar moléculas que possivelmente exercem benefícios em determinado tratamento. Entretanto, estes benefícios podem ser exacerbados ou suprimidos pelas moléculas diferencialmente expressas, as quais são dependentes tanto dos tecidos quanto dos indivíduos dos quais as CTMs foram obtidasMultipotent Mesenchymal Stromal Cells (MSCs) are multipotent adult cells that can be isolated from different tissues and are able to reach damaged sites, play a role in tissue regeneration and modulate immune response. These cells showed conflicting results in studies in vivo depending on their tissue origin. It is hypothesised that the predominant mechanism by which MSCs function could be related to its paracrine activity, creating a microenvironment with trophic signals. Accordingly, the evaluation of the content of the secretome of these cells is of great interest. Towards this end, this project analyzed the proteins of conditioned medium of MSCs obtained from different sources from the same donors (adipose tissue, uterine tubes and skeletal muscle). The MSCs were characterized by flow cytometry for the presence of membrane markers and by differentiation in vitro into adipocytes, chondrocytes, and osteoblasts. The conditioned media were obtained and the protein profile was analysed by liquid nanochromatography coupled to tandem mass spectrometry. Spectral data were obtained by full-acquisition mode MS / dd-MS2 (Top15). The acquired data were processed by MaxQuant software and TPP (Trans-Proteomic Pipeline). Qualitative analysis of enriched pathways through the Ingenuity program using the shared proteins between the cell lineages was performed.It showed enriched pathways related to cell proliferation, cell migration and development of the cardiovascular system. This allows considering that the secreted proteins from the analyzed MSCs might be related to findings in the literature using these cells for therapies. After this, the proteins were analyzed for differential expression by comparing the MSCs into groups of different sources or different donors. In which were observed differentially expressed proteins related to proliferation, cell signaling and interaction, modulation of the immune system and angiogenesis. In this context, we can conclude that MSC\'s secretome is very similar in the analyzed lineages, and that any MSCs are able to secrete molecules which potentially exert for certain treatment benefits. However, these benefits can be exacerbated or annulled by differentially expressed molecules, which are dependent both as the individual and tissues from which MSCs were obtaine

The role of ALS-related proteins in different cellular contexts

A Esclerose Lateral Amiotrófica (ELA) é uma doença neurodegenerativa para a qual não existe atualmente um tratamento eficaz. Portanto, é de grande importância investigar mais profundamente os mecanismos que levam à morte do neurônio motor para encontrar novos alvos terapêuticos. Nesta tese, aprofundamos nossa compreensão sobre três proteínas envolvidas na patogênese da ELA (Fused in Sarcoma -FUS, Vesicle-associated Membrane Protein B -VAPB e o receptor de efrina A4-EPHA4), analisando seus efeitos quando mutadas, ausentes, ou quando sua atividade é inibida. Além disso, há evidências de mecanismos em comum na neurodegeneração e transformação celular. Portanto, investigamos essas proteínas relacionadas com ELA em diferentes contextos celulares (neurodegeneração e câncer). Para este fim, usamos os modelos celulares de iPSCs de pacientes com ELA tipo 6, bem como um tumor do sistema nervoso central, meduloblastoma. Aqui, nós descrevemos que a tradução de proteínas é uma das primeiras e mais importantes vias afetadas por mutações relacionadas com ELA. Todos os mecanismos que são interrompidos nas células com ELA, levam à regulação negativa da síntese de proteínas. Consistente com a literatura, nossos resultados mostram que a síntese de proteínas está diminuída em neurônios motores (NMs) derivados de iPSC de pacientes com ELA6, e isso se correlaciona com a presença da proteína FUS (normalmente nuclear) no citoplasma. Também mostramos que interferon-gama (IFN-y) - uma citocina multifuncional que, entre outras atividades, auxilia na resposta antiviral - regula positivamente genes associados à tradução especificamente em NMs com ELA6 quando esses neurônios motores são tratados na presença de estresse oxidativo, e que a localização citoplasmática de FUS é reduzida em ELA6-NMs após o tratamento com IFN-y. Este tratamento evita a apoptose de ELA6-NMs. Portanto, a diminuição da síntese de proteínas pode ser característica da patogênese de ELA, e o aumento da tradução pela administração de IFN-y deveria ser estudado como um possível tratamento para esses pacientes. Em relação à função das proteínas associadas a ELA no meduloblastoma, revelamos novas funções para VAPB e EPHA4 na progressão tumoral. Descobrimos que a VAPB - que tem níveis mais baixos de mRNA no líquido cefalorraquidiano de casos esporádicos de ELA - se correlaciona com menor sobrevida geral de pacientes quando expresso em níveis mais altos no meduloblastoma. Além disso, o nocaute de VAPB causa parada do ciclo celular em G1 / 0 e altera os níveis de transcrição de genes relacionados à via do WNT, incluindo CTNNB1. Também mostramos que a regulação negativa de EPHA4 parece ser benéfica para a proliferação celular em meduloblastoma. Além disso, demonstramos que a VAPB se liga a EPHA4 em tecidos neuronais não transformados, mas não em células de meduloblastoma. No entanto, a remoção de VAPB no meduloblastoma aumentou a fosforilação de EPHA4, enquanto a inibição da fosforilação da EPHA4 aumentou a proliferação das células VAPB-KO, destacando a interação entre estas diferentes vias de sinalização. Em resumo, aqui fornecemos evidências para apoiar a hipótese de que a neurodegeneração e a tumorigênese são o resultado da desregulação das mesmas vias de sinalização, embora em direções diferentes. Portanto, é de extrema importância conectar o conhecimento das pesquisas em câncer e em doenças neurodegenerativas. O que pode levar a uma melhor compreensão dessas doenças devastadoras e gerar novas estratégias de intervenção para melhorar a vida dos pacientes no futuro.Amyotrophic Lateral Sclerosis (ALS) is a neurodegenerative disease for which there is currently no effective treatment. Therefore, it is of great importance to further investigate the mechanisms leading to motor neuron death to find new potential therapeutic targets. In this thesis, we deepen our understanding of 3 proteins involved in the pathogenesis of ALS (Fused in Sarcoma -FUS, Vesicle-associated Membrane Protein B -VAPB, and the ephrin receptor A4-EPHA4) by analyzing their effects when mutated, absent, or when their activity is inhibited. In addition, there is evidence for overlapping mechanisms in neurodegeneration and cell transformation. Therefore, we further investigated these ALS -related proteins in different cellular contexts (neurodegeneration and cancer). To this end, we used the cellular models of iPSCs from ALS type 6 patients as well as a central nervous system tumor, medulloblastoma. Herein, we describe that protein translation is one of the first and most important pathways affected by ALS -related mutations. Indeed, all mechanisms that are disrupted in ALS -cells ultimately lead to downregulation of protein synthesis rates. Consistent with the literature, our results show that protein synthesis rates are decreased in iPSC-derived motor neurons from ALS6 patients, and this correlates with the presence of the generally nuclear localized FUS protein in the cytoplasm. We also show that interferon-gamma (IFN-y) - a multifunctional cytokine that, among other things, aids in antiviral response - upregulates translation-associated genes specifically in ALS MNs when these iPSC-derived motor neurons from ALS patients are treated in the presence of oxidative stress, and that the cytoplasmic localization of FUS is reduced in ALS6 MNs after IFN-y treatment. This treatment prevents apoptosis of ALS6 MNs. Therefore, decreased protein synthesis may be a hallmark of ALS pathogenesis, and increasing translation with IFN-y is a potential treatment for these patients. Regarding the function of ALS associated proteins in medulloblastoma, we reveal novel roles for VAPB and EPHA4 in tumor progression. We find that VAPB - which has lower mRNA levels in the cerebrospinal fluid of sporadic ALS cases - correlates with lower overall patient survival when expressed at higher levels in medulloblastoma. Moreover, VAPB knockout arrests cells in G1/0 and alters transcript levels of many WNT-related proteins, including CTNNB1. We also show that downregulation of EPHA4 appears to be beneficial for cell proliferation in medulloblastoma. Furthermore, we found that VAPB binds to EPHA4 in nontransformed neuronal tissues but not in medulloblastoma cells. However, removal of VAPB in medulloblastoma increased EPHA4 phosphorylation, whereas inhibition of EPHA4 phosphorylation increased the cycling of VAPB-KO cells, highlighting the interplay between different signaling pathways. In summary, here we provide evidence to support the hypothesis that neurodegeneration and tumorigenesis are the result of the same deregulated signaling pathways, albeit in different directions. Therefore, it is of utmost importance to connect knowledge of cancer research and neurodegenerative diseases. This will not only lead to a better understanding of these devastating diseases, but could generate new intervention strategies to improve the lives of patients in the future

Amyotrophic Lateral Sclerosis, FUS and Protein Synthesis Defects

Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease that mainly affects the motor system. It is a very heterogeneous disorder, so far more than 40 genes have been described as responsible for ALS. The cause of motor neuron degeneration is not yet fully understood, but there is consensus in the literature that it is the result of a complex interplay of several pathogenic processes, which include alterations in nucleocytoplasmic transport, defects in transcription and splicing, altered formation and/or disassembly of stress granules and impaired proteostasis. These defects result in protein aggregation, impaired DNA repair, mitochondrial dysfunction and oxidative stress, neuroinflammation, impaired axonal transport, impaired vesicular transport, excitotoxicity, as well as impaired calcium influx. We argue here that all the above functions ultimately lead to defects in protein synthesis. Fused in Sarcoma (FUS) is one of the genes associated with ALS. It causes ALS type 6 when mutated and is found mislocalized to the cytoplasm in the motor neurons of sporadic ALS patients (without FUS mutations). In addition, FUS plays a role in all cellular functions that are impaired in degenerating motor neurons. Moreover, ALS patients with FUS mutations present the first symptoms significantly earlier than in other forms of the disease. Therefore, the aim of this review is to further discuss ALS6, detail the cellular functions of FUS, and suggest that the localization of FUS, as well as protein synthesis rates, could be hallmarks of the ALS phenotype and thus good therapeutic targets. Graphical Abstract: [Figure not available: see fulltext.]