Assessment of phenotypic modulation of ALS-­ASTRO-­TOX cells and their secretome toward motor neuron survival using new advanced experimental models

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Recovery of depleted miR-146a in ALS cortical astrocytes reverts cell aberrancies and prevents paracrine pathogenicity on microglia and motor neurons

Publication . Barbosa, Marta; Gomes, Cátia; Sequeira, Catarina; Gonçalves-Ribeiro, Joana; Pina, Carolina Campos; Carvalho, Luís A.; Moreira, Rui; Vaz, Sandra H.; Vaz, Ana Rita; Brites, Dora

Reactive astrocytes in Amyotrophic Lateral Sclerosis (ALS) change their molecular expression pattern and release toxic factors that contribute to neurodegeneration and microglial activation. We and others identified a dysregulated inflammatory miRNA profile in ALS patients and in mice models suggesting that they represent potential targets for therapeutic intervention. Such cellular miRNAs are known to be released into the secretome and to be carried by small extracellular vesicles (sEVs), which may be harmful to recipient cells. Thus, ALS astrocyte secretome may disrupt cell homeostasis and impact on ALS pathogenesis. Previously, we identified a specific aberrant signature in the cortical brain of symptomatic SOD1-G93A (mSOD1) mice, as well as in astrocytes isolated from the same region of 7-day-old mSOD1 mice, with upregulated S100B/HMGB1/Cx43/vimentin and downregulated GFAP. The presence of downregulated miR-146a on both cases suggests that it can be a promising target for modulation in ALS. Here, we upregulated miR-146a with pre-miR-146a, and tested glycoursodeoxycholic acid (GUDCA) and dipeptidyl vinyl sulfone (VS) for their immunoregulatory properties. VS was more effective in restoring astrocytic miR-146a, GFAP, S100B, HMGB1, Cx43, and vimentin levels than GUDCA, which only recovered Cx43 and vimentin mRNA. The miR-146a inhibitor generated typical ALS aberrancies in wild type astrocytes that were abolished by VS. Similarly, pre-miR-146a transfection into the mSOD1 astrocytes abrogated aberrant markers and intracellular Ca2+ overload. Such treatment counteracted miR-146a depletion in sEVs and led to secretome-mediated miR-146a enhancement in NSC-34-motor neurons (MNs) and N9-microglia. Secretome from mSOD1 astrocytes increased early/late apoptosis and FGFR3 mRNA in MNs and microglia, but not when derived from pre-miR-146a or VS-treated cells. These last strategies prevented the impairment of axonal transport and synaptic dynamics by the pathological secretome, while also averted microglia activation through either secretome, or their isolated sEVs. Proteomic analysis of the target cells indicated that pre-miR-146a regulates mitochondria and inflammation via paracrine signaling. We demonstrate that replenishment of miR-146a in mSOD1 cortical astrocytes with pre-miR-146a or by VS abrogates their phenotypic aberrancies and paracrine deleterious consequences to MNs and microglia. These results propose miR-146a as a new causal and emerging therapeutic target for astrocyte pathogenic processes in ALS.

2021Artigo científico

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

Publication . Barbosa, Marta; Brites, Dora Maria Tuna de Oliveira; Botelho, Ana Rita Mendonça Vaz; Rubin, Lee L.

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.

2022-12Tese de doutoramento

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Entidade financiadora

Fundação para a Ciência e a Tecnologia

Programa de financiamento

OE

Número da atribuição

SFRH/BD/129586/2017