Evaluation of promising miRNA modulation in neural cells and derived secretomes as therapeutic tools in ALS

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease characterized by motor neuron (MN) loss, astrocyte/microglia dysfunction and with lack of effective treatments. Dysregulation of inflammatory-miRNAs in cells and their dissemination via secretome (as free species and encapsulated in sEVs) contribute to ALS pathophysiology and their regulation may constitute a therapeutic approach. Thus, they have temporal/local differences in their expression, determining their differential role in the control of neurodegeneration and gliosis. We previously found that brain cortical astrocytes from SOD1-G93A (mSOD1) mice pups have miR-146a downregulation and an aberrant and neurotoxic phenotype. Thus, in this Thesis, we upregulated miRNA-146a with pre-miR-146a or treated with the immunoregulatory GUDCA and VS to evaluate their potential in rescuing mSOD1 astrocytic aberrancy. We also assessed the benefits of the secretome-derived from treated-mSOD1 astrocytes in recovering MN/microglia homeostasis. Transfection with miRNA-mimic and VS abrogated mSOD1 astrocyte aberrancy (restored GFAP/S100B/HMGB1/Cx43/vimentin levels), while GUDCA only restored Cx43/vimentin genes. The pre-miR-146a modulation also prevented calcium overload and induced the release of miR-146a-enriched sEVs by mSOD1 astrocytes. Both secretome derived from pre-miR-146a- and VS-treated-mSOD1 astrocytes prevented the activation of apoptosis and FGFR in MNs and microglia. They also counteracted the dysregulation of synaptic and axonal markers in MNs as well as microglia activation. Proteomic analysis of the modulated-mSOD1 astrocytes revealed changes in oxidative stress response and sEV transport and their derived secretome drove alterations in mitochondria regulation and inflammation in MNs and microglia. In the second Chapter, we focused on our previous data showing that the transfection of mSOD1 MNs with anti-miR-124 prevented neurodegeneration and its secretome counteracted pathogenicity in spinal cord (SC) organotypic cultures from early symptomatic mSOD1 mice. Therefore, we aimed to assess the therapeutic potential of this preconditioned secretome in the in vivo model. For that, we performed an intrathecal injection of secretome in early symptomatic mSOD1 mice. Our results showed that secretome prevented motor disabilities in mSOD1 mice at the symptomatic stage, prevented muscle atrophy, neuronal/glial dysregulation, astrocyte aberrancy and miR-146a/miR-155/miR-21 upregulation in the SC. Finally, we aimed to evaluate the therapeutic benefits of the secretome derived from pre-miR-146a-modulated-mSOD1 astrocytes in mSOD1 mice, by performing an intracerebroventricular injection of the abovementioned secretome in mSOD1 mice at early symptomatic stage. We confirmed again that mSOD1 mice evidenced motor deficits. Moreover, MN loss, deregulation of mitochondrial dynamics/axonal transport, increased myelination/MBP, non-reactive astrocytes and disease-associated microglia were also detected in the motor cortex of mSOD1 mice. The secretome prevented motor performance, supported the myelination and keep a sustained non-reactive astrocyte phenotype. It also upregulated CX3CR1 levels, suggesting the preservation of the microglia-MN signaling homeostasis, abrogated downregulated miR-146a and reduced miR-21 levels in the motor cortex. Overall, our results support the modulation of miR-146a and miR-124 in mSOD1 astrocytes and MNs, respectively, as efficient strategies to prevent cellular pathologies and produce a secretome with therapeutic properties. The injection of the secretome in the mSOD1 in vivo model precluded motor disabilities and neuronal/glial homeostatic imbalance, reinforcing their potential to be translated into ALS patients as a personalized and autologous treatment.Santa Casa da Misericórdia de LisboaLa Caixa Foundation-Luzón Foundatio