Micro-RNAs secreted through astrocyte-derived extracellular vesicles cause neuronal network degeneration in C9orf72 ALS

Background: Astrocytes 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. Methods: We 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. 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. In particular, we have identified downregulation of miR-494-3p, a negative regulator of semaphorin 3A (SEMA3A). 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 and higher levels of SEMA3A in cortico-spinal tract tissue isolated from sporadic ALS donors, thus demonstrating the pathological importance of this pathway in MNs and its therapeutic potential. Interpretation: ALS ADEVs and their miRNA cargo are involved in MN death in ALS and we have identified miR-494-3p as a potential therapeutic target. Gene expression profiling utilised total RNA extracted from extracellular vesicles derived from astrocytes directly reprogrammed from human C9ORF72 ALS patient (n=3) and control fibroblasts (n=3).