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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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Targeting miR-124 in motor neurons as a therapeutic strategy to prevent neurodegeneration and glial activations in ALS
Publication . Vizinha, Daniela Catarina Ribeiro; Brites, Dora Maria Tuna Oliveira; Botelho, Ana Rita Mendonça Vaz
Amyotrophic Lateral Sclerosis (ALS) is a progressive and fatal neurodegenerative disease characterized by the loss of motor neurons (MNs) from the motor cortex, brainstem and spinal cord (SC). Despite being studied for several years, there are only two available therapies, each one playing a modest impact on disease outcome. Initially, it was thought initially that the disease was only caused by motor neuron degeneration, but today it is known that ALS is a non-cell-autonomous disease involving glial cells, which show several dysfunctionalities considered to be aberrant. Glial cells were shown to lose their neuro-supportive function and to contribute to neurodegeneration and disease progression. However, it is not known which dysregulated mechanisms are affecting cell-to-cell communication, and the molecular mediators of neuronal injury, either existing as soluble factors or mediated by extracellular microvesicles.
MicroRNAs (miRNAs) are non-coding RNAs whose function is to suppress gene expression through their binding to mRNAs. Lately, miRNAs were discovered as diagnostic biomarkers and potential therapeutic targets in ALS. Differentially expressed miRNAs were identified, some with recognized association with known or suggested pathways in ALS pathogenesis. miRNAs can act in a tissue-/cell-type-specific manner and are involved in cellular communication, where they may affect gene expression, either on the cells of origin or in recipient ones.
MiRNA(miR)-124 is one of the most expressed in the central nervous system (CNS) and described as usually associated with neuronal differentiation. It has been found elevated in the cerebrospinal fluid of ALS patients, as well as in the ALS mice with the G93A mutation in the SOD1 gene (mSOD1), namely at active neurodegeneration sites, and identified in our studies as being upregulated in MN NSC-34 cell line with the same mutation. Interestingly, we noticed that miR-124 was upregulated in exosomes derived from such MNs, which showed to induce several activated subtypes once collected by microglia. In physiological conditions, miR-124 is involved in neuronal maturation, as well as in cell cytoskeleton organization, neurite outgrowth and autophagic regulation. Lately, dysregulation of miR-124 has been found in several CNS disorders associated with neurodegeneration, stress, neuro-immune dysregulation, and brain tumors, among others.
Here, we aimed to explore the consequences of miR-124 inhibition and overexpression on the MN function and structure, as well as on microglia and astrocyte immunoregulation. For that, we used the NSC-34-MN-like cell line, either non-mutated (wild type, WT), or expressing the G93A mutation (mSOD1), which is a model that mimics the SOD1 accumulation and the neuronal degeneration observed in ALS. We transfected WT MNs with miR-124 mimic (pre-miR-124), and mSOD1 MNs with the miR-124 antagonist (anti-miR-124), to assess whether the effects produced by their secretome differently affected glia reactivity. In this sense, the collected secretome was incubated with mice microglial N9 cells for 4 h and with SC astrocytes from WT and mSDO1 8-days-old mice for 24 h. MN viability was not changed by both miR-124 modulations and that using anti-miR-124 prevented SOD1 accumulation. This one also prevented the increase in gene and protein expression of High Mobility Group Box 1 (HMGB1), as well as of the inducible Nitric Oxide Synthase (iNOS) protein, and of S100 Calcium-binding Protein B (S100B) mRNA. Overexpression of miR-124 in WT MNs was reflected only in S100B mRNA increase. At miRNAs level, anti-miR-124 in mSOD1 MNs decreased the overexpression of miR-125b and increased that of miR-21 and miR-146a, which were diminished in the disease model. Interestingly, pre-miR-124 in WT MNs led to the profile observed in mutated cells, namely to upregulation of miR-125b and to downregulation of miR-21 and miR-146a, validating miR-124 as a driver of such miRNA dysregulation. Importantly, anti-miR-124 was also capable of diminishing the expression of the pre-synaptic marker synaptophysin and of the retrograde transport marker dynein, both elevated in mSOD1 MNs and in WT-treated pre-miR-124 MNs, toward WT levels, highlighting miR-124 as a major contributor to such increases. In opposite, this miRNA revealed to be associated with the decrease of the post-synaptic marker PSD-95 and the anterograde transport marker kinesin, since anti-miR-124 led to a remarkable increase in the expression of their genes. Thus, we can conclude that anti-miR-124 favors anterograde transport (from soma to the end of the axon), deficient in the mutated cells, in which dynein elevation favors retrograde transport (back to the soma) described to be associated with miR-124 upregulation. Overexpressed miR-124 was also shown to relate with fewer ramifications and number of primary neurites, as well as with their increased length, which were counteracted to values close to control levels with anti-miR-124 in mSOD1 MNs. This indicates the influence of this miRNA in cytoskeleton regulation, cellular morphology, and MN function. In fact, anti-miR-124 was able to recover mitochondrial viability in mSOD1 MNs, whose decrease seems to be related with miR-124 upregulation. It also favored Mitofusin expression (an inducer of mitochondrial fusion), found diminished in mutated cells, and decreased DRP1 (associated with mitochondrial fission), increased in mutated cells. Once again, the overexpression of miR-124 showed to be involved in DRP1 upregulation in the mutated cells. Such alterations suggest the existence of oxidative stress associated with miR-124 overexpression and that anti-miR-124 may have a beneficial action by favoring anti-oxidant mechanisms.
Relatively to the immunoregulatory action exerted by the MN secretome, we verified that the increased area, perimeter and Ferret’s diameter, as well the circularity reduction in microglia treated with secretome derived from mutated MNs, was not manifested upon incubation with the secretome from mSOD1 MNs treated with anti-miR-124. These parameters showed a direct correlation with the miR-124 overexpression. Microglia phagocytic ability was also affected by the secretome from WT MNs treated with pre-miR-124, as well as with that from mSOD1 modulated or not with anti-miR-124, indicating that miR-124 inhibition is not enough to recover such microglial property. However, anti-miR-124 modulation effectiveness was again manifested when the secretome from mSOD1 MNs acquired the capability to decrease the gene expression of iNOS, arginase1, Tumor Necrosis Factor alpha (TNF-α) and Interleukin 1 beta (IL-1β). All these inflammatory genes were found upregulated in microglia treated with both mSOD1 and pre-miR-124 WT MNs. Additionally, it led to a decrease of HMGB1, highly enhanced after incubation with the secretome from mSOD1 MNs, and to an elevation of the mannose receptor CD206 expression, associated with macrophage endocytic and phagocytic properties. Interestingly, here we found an indirect correlation in that overexpressed miR-124 triggered a CD-206 gene expression decrease. In what concerns the consequences of mSOD1 MN secretome after miR-124 modulation on astrocytes, it was interesting to observe that GLT-1 levels were sustained in this condition, contrasting with the reduction found with the secretome from WT MNs treated with pre-miR-124, as well as with that from mSOD1 MNs. Similar effect was noticed on GFAP levels, whose expression increased relatively to WT and mSOD1 astrocytes treated with the secretome from mSOD1 MNs as well as the WT astrocytes exposed to the secretome from WT MNs modulated with pre-miR-124. Therefore, we can hypothesize that, at least in part, miR-124 elevation in mSOD1 MNs is associated with the production of a secretome determining GFAP and GLT-1 reduced expression, features that relate with the aberrancy of astrocytes in ALS.
Overall, our results indicate that miR-124 overexpression in mSOD1 MNs has a key role in the degeneration and dysfunction of these cells, remarkably contributing to ALS pathogenesis and progression by compromising cellular homeostasis, due to a secretome with immunostimulatory and inductive aberrant features in either WT or mSOD1 astrocytes. Anti-miR-124 showed beneficial effects in preventing SOD1 accumulation in mutated MNs, while exerting synaptic transmission regulation (increase of the neuritic tree, kinesin and PSD-95), anti-oxidant effect (increased mitochondrial viability and Mitofusin and diminished DRP1) and anti-inflammatory action (increase of miR-21 and miR-146a, with decrease of miR-125b). Furthermore, secretome from mSOD1 MNs modulated with anti-miR-124 acquire properties that support microglia functionality (normal morphological characteristics and levels of iNOS, Arginase1, TNF-α, IL-1β and HMGB1 similar to those observed for the secretome from non-modulated WT MNs) and that of astrocytes (increase of GLT-1 and GFAP expression), beyond the elevation of microglial receptor CD206, associated with endocytic and phagocytic abilities. Thus, we can conclude that the decrease of miR-124 expression toward basal levels in mSOD1 MNs backup their capacity to sustain their appropriate function, while preventing a reactive glial response triggered to a secretome with immunostimulatory properties, thus counteracting neurodegeneration and disease progression.
In conclusion, this work supports that the selective targeting of miR-124 in mSOD1 MNs may turn in a promising and broad therapeutic strategy for ALS treatment, by allowing the recovery of MN function and by preventing secretome-mediated glial reactivity, thus acting in multiple targets as usually intended with a combined therapy.
Unveiling aberrancies and neurotoxic properties of ALS astrocytes using unspecific and microglia-specific activation models
Publication . Maurício, Isabel Maria dos Santos; Brites, Dora Maria Tuna de Oliveira; Botelho, Ana Rita Mendonça Vaz
A Esclerose Lateral Amiotrófica (ELA) é uma doença neurodegenerativa caraterizada pela perda progressiva dos neurónios motores (NMs) superiores e inferiores no Sistema Nervoso Central, nomeadamente, no cérebro, tronco cerebral e medula espinal. A perda de NMs resulta numa paralisia extensa que começa localmente na musculatura dos membros superiores, inferiores ou bulbar. A ELA é uma doença fatal, com uma sobrevida normalmente de 2 e 5 anos após o diagnóstico. Os doentes sucumbem à doença devido a atrofia muscular grave, paralisia e por fim a desenervação dos músculos respiratórios. É uma doença extremamente heterogénea na sua sintomatologia, e com uma patogénese pouco clara. Atualmente, existem apenas duas abordagens terapêuticas muito limitadas quanto à sua eficácia, tornando-se premente compreender as causas subjacentes e os fatores de risco, de forma a encontrar novos alvos terapêuticos para a ELA.
Apesar do envolvimento dos NMs, é bem conhecida a importância da disfuncionalidade das células gliais na doença. Na ELA, os astrócitos alteram a sua forma e padrões de expressão molecular e são referidos como astrócitos reativos ou ativados, perdendo as suas funções benéficas e adquirindo papéis prejudiciais. No entanto, não se sabe qual a causa de tal reatividade e do fenótipo aberrante dos astrócitos na ELA, ou seja, se são intrínsecos à patologia, se são potenciados pela libertação de citocinas da microglia ativada, ou se pelo ambiente neuroinflamatório e seus mediadores.
Esta tese teve a possibilidade única de explorar a morfologia e assinatura inflamatória de astrócitos diretamente convertidos de fibroblastos (iAstrócitos) de uma doente com forma esporádica da ELA, em comparação com uma amostra controlo obtida da mesma forma, de uma pessoa não doente, do mesmo sexo e idade (controlo). Para tornar mais evidente as reações de patogenicidade dos astrócitos ELA, foram usados dois estímulos inflamatórios diferentes, ambos descritos na literatura e envolvendo a ação de citoquinas pró-inflamatórias, para atestar qual o mais eficaz na indução do fenótipo aberrante dos astrócitos na ELA.
Usámos células induzidas percursoras neurais geradas de fibroblastos da doente e respetivo controlo, cedidas por Kathrin Meyer no âmbito da nossa colaboração, que foram diferenciados em astrócitos induzidos (iAstrócitos), durante 7 dias in vitro. Os astrócitos foram de seguida incubados com as citocinas da microglia ativada (TNF-α/IL-1α/C1q) e as associadas a qualquer processo inflamatório (TNF-α/IL-1β) durante 48 horas. Após este período, recolheram-se as células e o seu secretoma.
Os astrócitos da doente com ELA exibiram menos células polarizadas, maior aparência de fibroblastos e menor soma, quando comparados com a amostra controlo, se bem que sem evidência estatística. Esta tendência manteve-se após estimulação com TNF-α/IL-1α/C1q, mas não totalmente com TNF-α/IL-1β, dado que neste caso apenas se verificou a presença de menor soma. A avaliação das propriedades dinâmicas da mitocôndria nos astrócitos, permitiu evidenciar uma diminuição marcada nos astrócitos ELA que se tornaram menos evidentes com TNF-α/IL-1α/C1q e ausentes com TNF-α/IL-1β. Contrariamente ao descrito para os genes associados ao fenótipo aberrante dos astrócitos na ELA, não observámos nos astrócitos ELA o aumento de Cx43, nem de S100B, os quais se apresentaram diminuídos (p<0.01 para ambos), nem qualquer alteração para o transcrito GFAP, habitualmente reduzido. Contudo, identificámos um aumento do marcador de proliferação celular, o Ki-67 (p<0.05). Na presença de TNF-α/IL-1α/C1q, tanto o GFAP como o S100B foram encontrados diminuídos (p<0.05) nos astrócitos ELA. O S100B foi igualmente encontrado diminuído na presença de TNF-α/IL-1β que levou a um aumento marcado da proliferação nos astrócitos ELA. A avaliação de S100B por imunocitoquímica, confirmou a diminuição de S100B nos astrócitos ELA na presença tanto de TNF-α/IL-1α/C1q, como de TNF-α/IL-1β (p<0.05), mas não na ausência destes estímulos. Quanto aos marcadores inflamatórios associados à ativação do inflamassoma, NLRP3, IL1R1 e IL-1β, há a salientar a diminuição dos genes IL-1β e NLRP3 nos astrócitos ELA não estimulados. A redução do NLRP3 foi sustentada na presença de qualquer um dos modelos de estimulação.
Os resultados obtidos para os astrócitos da doente com a forma esporádica da ELA apresentam alterações fenotípicas relativamente à amostra controlo, sugestivas de latência e menor capacidade reativa a estímulos, mas mantendo um aumento da capacidade proliferativa, na ausência e presença de TNF-α/IL-1β. Quanto aos efeitos dos dois estímulos testados, nem sempre foram comparáveis. Este trabalho foi pioneiro em mostrar déficits de uma amostra de astrócitos obtidos de doente com ELA, os quais necessitam, contudo, de ser reforçados com um maior número de amostras, incluindo formas familiares. Em suma, o processo inflamatório deverá ser tido em conta na avaliação da patologia associada à ELA e os iAstrócitos sobressaem como uma ferramenta importante na medicina personalizada para esta doença.
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.
Potential roles of inflammatory miRNAs and exosomes in Alzheimer’s disease pathogenesis, diagnostics and therapeutics using advanced Human models
Publication . Garcia, Gonçalo; Brites, Dora Maria Tuna de Oliveira; Malm, Tarja Maarit
Alzheimer’s disease (AD) is a complex brain disorder with a heavy socio-economic burden, affecting over 50 million people worldwide. Although aging, genetics and environmental factors constitute major risk factors, alterations in neuron-glia communication and dysregulation of microRNAs (miRNAs) dysregulation were shown to be key players in AD pathogenesis. The neural-specific miRNA(miR)-124 is a prominent player in neuronal function. We identified its consistent upregulation in SH-SY5Y-APP695 Swedish neuroblastoma cells (SH-SWE) and PSEN1 mutant iPSC-derived neurons (iNEU-PSEN), as well as in their respective exosomes. Considering the controversy around miR-124 in AD, we investigated the outcomes of modulating its levels in both neuronal cells, using specific miR-124 inhibitor/mimic. Results demonstrated that the mimic reduced APP gene expression in both models of AD. Moreover, it stimulated neurite outgrowth, mitochondria membrane potential and reduced small Aβ oligomers in SH-SWE neurons, while it inhibited tau phosphorylation in iNEU-PSEN cells. Besides, miR-124 mimic predominantly repressed the inflammatory-associated miR-125b/miR-21/miR-146a/miR-155. Oppositely, miR-124 inhibitor prompted miR-146a in the two neuronal models, while it reduced neurite outgrowth and affected mitochondrial dynamics in SH-SWE cells, and decreased dendritic spine density in iNEU-PSEN. Thereafter, we evaluated the paracrine influence of the neuronal miR-124 modulation on microglial polarization status, under an inflammatory milieu. For that, we co-cultured miR-124 modulated SWE cells with the human CHME3 microglia stimulated with interferon gamma (IFNγ-MG). Results evidenced that besides the benefits for SWE cells, miR-124 mimic reduced microglial activation (downregulated TNF-α/iNOS), inhibited extracellular MMP-2/MMP-9 activity, and led to proteomic downregulation of 72 proteins (e.g., MAP2K6) and upregulation of 21 (e.g., PAWR). In contrast, miR-124 inhibition favored microglial inflammatory signature (upregulated RAGE/HMGB1/iNOS/IL-1β; downregulated IL-10/ARG-1) and led to proteomic changes favoring the IFNγ-induced signature (e.g., TGFB1 downregulation). By blocking miRNA canonical biogenesis through Dicer1-silencing, we confirmed miR-124 uptake by microglia upon coculture with SWE cells, and upon incubation with SWE-derived exosomes. Finally, we analyzed the miRNA expression profile in cerebrospinal fluid (CSF) samples from a small cohort of Mild Cognitive Impairment (MCI) patients and specifically detected miR-21 elevation in samples from patients that progressed to AD, comparatively to those that didn’t. Such miR-21 elevation was confirmed and cross-validated in AD patient iPSC-derived microglia, neurons, and astrocytes. We further developed a new organotypic hippocampal slice model transplanted with SH or SWE cells. Remarkably, transplantation of SWE cells into hippocampal slices boosted the miR-21 expression and induced both microglia activation and astrocyte reactivity, validating the active role of miR-21 in AD. Overall, our studies support that miR-124 is a promising target with pleiotropic effects, from APP processing and maintenance of dendritic homeostasis to the reshaping of microglial plasticity in AD-associated context and should be regulated whenever necessary. Furthermore, we elucidated the potential of miR-21 as a promising disease-modifying miRNA to be explored in AD, considering its involvement in neuronal dysfunction, microglia activation, and astrocyte reactivity. Together, the novel insights provided in this Thesis may pave the way for the development of future personalized neuropharmacological strategies with miRNAs, either in the secretome or as cargo of exosomes.
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.
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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