Assessing the putative role of mesenchymal stromal cells’ effectors in Machado-Joseph disease

Abstract

Na última década, as abordagens cell-free tornaram-se naturalmente um motivo de interesse em relação a doenças neurológicas, uma vez que constituem uma alternativa à terapia celular.No presente estudo, tentamos avaliar se os componentes do meio condicionado derivado das MSC (CM-MSC), como fatores secretados e vesículas extracelulares (EVs), poderiam substituir as MSC para fins terapêuticos na MJD.Em primeiro lugar, avaliamos a sobrevivência das MSC in vivo após administração sistêmica e observamos que, apesar de atingirem o parênquima cerebral, desaparecem do cérebro nas primeiras 48 horas e do resto do corpo (principalmente pulmões) em menos de uma semana.Seguidamente, avaliamos o papel putativo de produtos derivados de MSC no efeito neuroprotetor destas células em modelos in vitro de MJD. Começamos por esclarecer que o contexto neuronal e a presença da MJD não são necessários para melhorar as ações terapêuticas do CM-MSC. Além disso, os nossos resultados sugerem que as EVs não são os principais responsáveis pela redução dos níveis de ataxina-3 mutante promovida pelas MSC. Como prova de conceito do potencial neuroprotector do CM-MSC, mostramos que o tratamento pode diminuir a expressão da ataxina-3 mutante em células MJD.Finalmente, investigamos o impacto das MSC e do CM-MSC em diversos mecanismos e vias celulares essenciais para a homeostasia celular. Encontramos algumas pistas de que as MSC podem provavelmente modular de forma parácrina vias relacionadas com a autofagia e a sobrevivência, nomeadamente as vias Pi3K/AKT e ERK/MAPK.Em conclusão, demonstramos que o completo CM-MSC e MSC pode promover a neuroproteção através da modulação de múltiplos mecanismos celulares envolvidos na sobrevivência celular. Assim, os nossos resultados sugerem que o CM-MSC pode ser uma abordagem terapêutica cell-free viável para o tratamento da MJD.

Machado-Joseph disease (MJD), or spinocerebellar ataxia type 3 (SCA3) is the most common autosomal dominantly-inherited spinocerebellar ataxia in the world. This fatal disorder is caused by an expansion of the CAG repeat in the MJD1/ATXN3 gene, which encodes an abnormally long polyglutamine tract in the disease protein, ataxin-3. The mutation leads to the dysfunction of important cellular mechanisms, including quality control pathways, which results in the widespread neuronal degeneration, thus affecting numerous neuronal systems. Currently, there is no cure or therapy that can efficiently delay MJD progression. Over the last decades, mesenchymal stromal cells (MSC) have demonstrated to be a promising therapeutic tool for the treatment of several neurodegenerative diseases, including SCAs. These cells exert a neuroprotective effect mainly through their paracrine activity. However, repeated treatment with MSC is required to get sustained results in vivo. In the last decade, cell-free approaches have naturally become a motif of interest regarding neurological disorders, as they constitute an alternative to cell therapies.In the present study, we aimed at evaluating whether the components of MSC conditioned medium (MSC-CM), such as secreted factors and extracellular vesicles (EVs), could replace MSC for therapeutical purposes in MJD.Firstly, we assessed for MSC survival in vivo upon systemic delivery and observed that besides the fact they can reach the brain parenchyma, they disappear from the brain in the first 48h and from the rest of the body (lungs mainly) in less than a week.Then we evaluated the putative role of MSC-derived products in the neuroprotective effect of MSC in MJD in vitro models. We began by clarifying that neuronal context and MJD insult is not required to enhance the therapeutic actions of the MSC-CM. Plus, our results suggest that EVs are not the main effector responsible for the reduction of mutant ataxin-3 levels promoted by MSC. As a proof-of-concept of MSC-CM neuroprotective potential, we show that treatment can decrease mutant ataxin-3 expression in MJD cells.Finally, we investigated the impact of MSC and MSC-CM in several cellular mechanisms and pathways essential for cell homeostasis. We found some clues that MSC can probably modulate autophagy and pro-survival related pathways, namely Pi3K/AKT and ERK/MAPK pathways, in a paracrine manner.In conclusion, we demonstrated that both MSC and MSC-CM medium can promote neuroprotection by modulating a multitude of cellular mechanisms involved in cell survival. Thus, our findings suggest that MSC-CM may be a feasible cell-free therapeutic approach for MJD.