Novel therapeutic approach to amyotrophic lateral sclerosis : targeting neurotoxic astrocytes before transplantation

Tese de doutoramento, Farmácia (Biologia Celular e Molecular), Universidade de Lisboa, Faculdade de Farmácia, 2019Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease characterized by the progressive loss of motor neurons (MNs). However, astrocytes are also major contributors in ALS pathogenesis, though the underlying mechanisms are still unclear. Using the brain cortex of the symptomatic ALS mouse model SOD1G93A (mSOD1), we identified markers of astrocyte reactivity and dysfunction, as well as neuronal loss and SOD1 accumulation. Such abnormal features were early manifested in the primary cortical astrocytes isolated from mSOD1 mice 7 day-old pups and cultured for 13 days in vitro (DIV). These astrocytes, showing neurotoxic potential, revealed a specific aberrant phenotype characterized by SOD1high/GFAPlow/GLT-1low/S100Bhigh/Cx43high/Ki-67high. Remarkably, in contrast with spinal astrocytes that revealed elevated miRNA(miR)-155, neonatal cortical astrocytes exhibited decreased miR-146a levels, also present in brain cortex homogenates of mSOD1 mice at either pre- or symptomatic stages. This finding highlights low miR-146a as a potential therapeutic target and ALS biomarker. To further assess astrocyte dysfunctional heterogeneity, spinal and cortical astrocytes were cultured for 5 and 13 DIV. We found that mSOD1 cortical astrocytes exhibited early and sustained aberrancies, as well as deregulated signaling pathways than their age-matched spinal astrocytes. Moreover, with only 5 DIV in culture, cortical astrocytes were able to reduce neurite outgrowth of mSOD1 NSC-34 MNs, a feature only replicated by 13 DIV spinal ones. Among miRNAs known to play a critical role in CNS inflammation, miR-155/miR-21/miR-146a showed discriminatory potential in being upregulated in spinal astrocytes and downregulated in cortical cells. However, when evaluated in extracellular vesicles (EVs) from 13 DIV spinal and cortical mSOD1 astrocytes, depleted miR-155/miR-21/miR-146a was a common feature in both. These data in the mSOD1 mouse model identify regional different therapeutic candidates in astrocytes, depending on their cortical or spinal origin, thus suggesting specific and combined pharmacological approaches to restore their neuroprotective role. To validate such results in human cells, we then used induced astrocytes (iAstrocytes) directly converted from familial and sporadic ALS male and female patients, as well as from matched non-diseased individuals (controls). All ALS patientiAstrocyes were neurotoxic, with reactive and inflammatory markers differently distributed among ALS iAstrocytes, which may have then discriminatory potential in patient stratification. Up-/down-regulation disparities were noticed for miR-146a and replicated in their derived small EVs. Its upregulation in samples showing decreased miR-146a levels recovered diverse iAstrocytes aberrancies and induced synaptic/axonal gene expression in NSC-34 MNs, reinforcing its relevance for developing novel medicines. Overall, our studies reveal that cortical and spinal astrocytes distinctively acquire ALS malfunction properties. Importantly, iAstrocytes are powerful tools to stratify patients, reflecting the ALS phenotypic variability. Results highlight miR-146a as a potential target to recover the neurosupportive role of astrocytes and evidence the requirement of personalized medicine in ALS.Fundação para a Ciência e a Tecnologia (FCT), SFRH/BD/102718/2014, projeto PTDC/MED-NEU/31395/2017; Programa de Investigação Científica da Santa Casa da Misericórdia de Lisboa em ELA, projeto ELA-2015-002; Fundo Europeu de Desenvolvimento Regional (FEDER), Programa Operacional Competitividade e Internacionalização - COMPETE 2020, Programa Operacional Regional de Lisboa, projeto LISBOA-01-0145-FEDER-03139