Abstract
BackgroundAstrocytes regulate neuronal function, synaptic formation and maintenance partly through secreted extracellular vesicles (EVs). In amyotrophic lateral sclerosis (ALS) astrocytes display a toxic phenotype that contributes to motor neuron (MN) degeneration.MethodsWe used human induced astrocytes (iAstrocytes) from 3 ALS patients carrying C9orf72 mutations and 3 non-affected donors to investigate the role of astrocyte-derived EVs (ADEVs) in ALS astrocyte toxicity. ADEVs were isolated from iAstrocyte conditioned medium via ultracentrifugation and resuspended in fresh astrocyte medium before testing ADEV impact on HB9-GFP+ mouse motor neurons (Hb9-GFP+ MN). We used post-mortem brain and spinal cord tissue from 3 sporadic ALS and 3 non-ALS cases for PCR analysis.FindingsWe report that EV formation and miRNA cargo are dysregulated in C9ORF72-ALS iAstrocytes and this affects neurite network maintenance and MN survival in vitro. In particular, we have identified downregulation of miR-494-3p, a negative regulator of semaphorin 3A (SEMA3A) and other targets involved in axonal maintenance. We show here that by restoring miR-494-3p levels through expression of an engineered miRNA mimic we can downregulate Sema3A levels in MNs and increases MN survival in vitro. Consistently, we also report lower levels of mir-494-3p in cortico-spinal tract tissue isolated from sporadic ALS donors, thus supporting the pathological importance of this pathway in MNs and its therapeutic potential.InterpretationALS ADEVs and their miRNA cargo are involved in MN death in ALS and we have identified miR-494-3p as a potential therapeutic target.Funding: Thierry Latran Fondation and Academy of Medical Sciences.
Highlights
Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease characterized by motor neuron (MN) degeneration
The mechanisms and sequence of events leading to MN death are still widely unknown, but the observation that the first pathophysiological changes observed in patients involve neuromuscular junction (NMJ) disruption have given rise to the theory known as the ‘dying-back’ hypothesis [1]
We previously showed that C9orf72 iAstrocytes derived through direct conversion of fibroblasts into induced neural progenitor cells (iNPCs) induced a decrease in survival in Hb9-GFP+ mouse MNs in co-culture compared to non-ALS astrocytes [16]
Summary
Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease characterized by motor neuron (MN) degeneration. Polymorphic (G4C2)n hexanucleotide repeat expansions within the C9ORF72 gene are the most common genetic cause of ALS and frontotemporal dementia [7,8] They are known to mediate neurotoxicity through multiple mechanisms including alterations of pre-mRNA processing [9,10], along with dysregulations of autophagy, protein homeostasis and vesicle trafficking [11,12,13]. Findings: We report that EV formation and miRNA cargo are dysregulated in C9ORF72-ALS iAstrocytes and this affects neurite network maintenance and MN survival in vitro. Interpretation: ALS ADEVs and their miRNA cargo are involved in MN death in ALS and we have identified miR494-3p as a potential therapeutic target.
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