Development of an autologous exosome-based therapy by engineering microRNAs in microglia and motor-neurons using mice and human models of amyotrophic lateral sclerosis (ALS)

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Development of secretome-based therapy by motor neuron modulation of miRNA-124 in ALS mouse models

Publication . Morais, Hermes Manuel Medina; Brites, Dora; Botelho, Ana Rita; Braga, Margarida

Amyotrophic Lateral Sclerosis (ALS) is a fatal disease characterized by the degeneration of upper (cortical) and lower (spinal cord, SC) motor neurons (MNs) and aberrancy of glial cells. Results from our group point to a close connection between increased levels of miRNA-124 and the acquisition of pathological characteristics in MNs, astrocytes and microglia in ALS. Our main aim was to validate if the downregulation of the elevated levels of miR-124 in hSOD1G93A (mSOD1) MNs toward normal levels was preventive over neurodegeneration, astrocyte aberrancies and microglia activation in the mSOD1 mice at the early onset of the disease (10-12 weeks). Two ALS models were used: the NSC-34 MN-like cell line expressing mSOD1 (transgenic, TG) or not (wild-type, WT); and the SC organotypic cultures (OCs) from WT and TG mice. Pathological differences between TG and WT SCOCs were investigated. Relatively to the MN models, we used the modulation with pre-miR-124 (only in WT) and that of anti-miR-124 (only in the TG). The isolated secretomes were incubated in WT and TG SCOCs to assess harmful and/or neuroprotective properties. In TG SCOCs we observed: (i) increased necrotic cell death; (ii) disturbed inflammatory-associated miRNAs (increase in miR-21/miR-146a); (iii) and dysregulated neuronal and glial genes (increased CX3CR1, IL-1β, IL-10, SYP, DRP1, GLT-1 and downregulation of iNOS, HMGB1, Dlg4, CX3CL1 and GFAP). WT-MN secretome counteracted pathological markers in TG SCOCs. In contrast, TG MN secretome induced deleterious effects in WT SCOCs. Secretome from miR-124-enriched WT MNs incubated in WT SCOCs led to a profile of miRNAs and protein-coding genes similar to that caused by the TG MN secretome. On the contrary, the secretome from TG MNs depleted in miR-124 restored a deactivated profile in TG SCOCs. Our data reveals MN upregulation of miR-124 as a key player in ALS pathological processes.

2021-01-11Dissertação de mestrado

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THERAPEUTIC POTENTIAL OF INTRATHECAL APPLICATION OF miR-124-BASED SECRETOME IN THE SOD1 G93A MOUSE MODEL OF AMYOTROPHIC LATERAL SCLEROSIS

Publication . Santos, Marta Alexandra Marques dos; Brites, Dora; Braga, Margarida

Amyotrophic lateral sclerosis (ALS) is a progressive neurodegenerative disease with no effective cure and a short life expectancy, characterized by the degeneration of motor neurons (MNs) and glial cell dysfunction. Inflammatory(inflamma)-associated microRNAs (miRNAs/miRs) were found dysreg- ulated in SOD1 G93A (mSOD1) mouse models, as well as in mSOD1 MNs and microglia, and their secre- tome. Prior studies from our lab detected upregulated levels of miR-124 in mSOD1 NSC-34-MN-like cells and demonstrated its association with their degeneration, including mitochondrial dysfunction and axonal/synaptic dysregulation, together with glial activation. Interestingly, the treatment with anti-miR- 124 in mSOD1 MNs prevented their impairment. Furthermore, our group showed that the secretome derived from these modulated mSOD1 MNs (preconditioned secretome) counteracted pathological fea- tures and neuro-immune homeostatic imbalance in spinal cord organotypic cultures from early sympto- matic mSOD1 mice. This issue was never explored in the in vivo model of ALS, though recent evidence sustains that cell secretomes may have therapeutic effects. To decipher the potential therapeutic benefits of such preconditioned secretome, our group performed its intrathecal injection in the mSOD1 mice at the early symptomatic stage of the disease (12-week-old). Notably, this preconditioned secretome pre- vented the glial reactivity/dysfunction, neurodegeneration, and the altered inflammatory-dynamic bal- ance of the 15-week-old symptomatic mSOD1 mice. At the molecular level, the injection of this engi- neered secretome enhanced NeuN mRNA/protein expression levels and the Dlg4/Mbp/Plp/Trem2/Arg1/Inos/Il-10 genes, thus precluding the neuronal/glial cell dysregulation that characterizes the ALS mice. The upregulated GFAP/Cx43/S100B/Iba-1 and the inflamma-associated miRNAs (miR-146a/miR-155/miR-21) displayed by the symptomatic mSOD1 mice were also pre- vented. Overall, this study highlights the intrathecal administration of the anti-miR-124-treated mSOD1 MN preconditioned secretome as a promising cell-free based therapeutic strategy to halt/delay disease progression in the ALS mouse model, supporting its translation potential into the ALS patient as a per- sonalized and autologous treatment.

2022-11Dissertação de mestrado

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Exploring A1 activation and miR-155 modulation in ALS spinal cord astrocytes

Publication . Sequeira, Catarina Antunes; Brites, Dora; Botelho, Ana

Amyotrophic Lateral Sclerosis (ALS) is a neurodegenerative disorder, characterized by motor neurons (MN) loss in the brain and spinal cord (SC). Astrocyte reactivity and microglia activation account for neurodegeneration through the release of neurotoxins. Cytokines from activated microglia were shown to activate astrocytes and the inhibition of microglial microRNA(miRNA)-155 in ALS mice with G93A mutation in SOD1 gene (mSOD1) to recover their steady state phenotype and to attenuate disease progression. This thesis explored the reactive/inflammatory signature of ALS SC mSOD1 astrocytes, their response to microglial cytokines and the efficacy of anti-miR-155 modulation in recovering astrocyte neuroprotective properties. Astrocytes were isolated from the SC of 8-day-old mSOD1 mice pups and after 11 days in vitro, cells were incubated with cytokines that mimic A1-microglial activation (IL-1α/TNF-α/C1q) during 48h. Mutated astrocytes were transfected (or not) with anti-miRNA-155 and their secretome added to mSOD1-NSC-34 MN-like cells for 48h. We demonstrated that mSOD1 astrocytes have an inflammatory profile (elevated miR-21, miR-124, miR-155, IL-1β, Cx43 and HMGB1), further upregulated by A1-stimulation. Though a similar elevation of miR-155, iNOS and TNF-α was observed in both WT and mutated cells upon stimulation, increased levels of miR-21, miR-124, Cx43 and IL-6 mRNA, together with GFAP, S100B and vimentin protein were more evident in mSOD1 astrocytes, while IL-1β and IL-10 mRNAs remained unchanged and lower miR-146a upregulation was found. Downregulation of miR-155 in mSOD1 astrocytes reduced IL-1β, TNF-α and HMGB1, while upregulated IL-6, IL-10, SOCS1 and miR-146a. Importantly, secretome from astrocytes transfected with anti-miR-155 increased cell survival, mitochondria viability and neurite number in degenerating mSOD1-NSC-34 MN-like cells, in part mediated by miR-124 downregulation. Our results highlight the inflammatory/reactive/aberrant phenotype of mSOD1 SC astrocytes and its exacerbation by activated microglia as players in ALS neuroinflammation. Importantly, treatment with anti-miR-155 reduces the pro-inflammatory pattern of ALS astrocytes and restores their neuroprotective properties.

2018-10-29Dissertação de mestrado

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Entidade financiadora

Fundação para a Ciência e a Tecnologia

Programa de financiamento

9471 - RIDTI

Número da atribuição

PTDC/MED-NEU/31395/2017