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

Abstract

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) 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.

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.

Funding: Thierry Latran Fondation and Academy of Medical Sciences.

Keywords: Amyotrophic lateral sclerosis; Astrocytes; Axonal growth; Extracelular vesicles; Gene therapy; Neurodegeneration; miRNA.

Fig. 1

Conditioned medium (CM) and extracellular vesicles (EVs) from C9-ALS iAstrocytes induce MN death. Representative images of Hb9-GFP⁺MN monocultures treated for 72 h with astrocyte conditioned medium from control (CTR) or C9-ALS (C9) astrocytes (a and b respectively) or EVs isolated from the same conditioned media (c and d respectively). Quantification of HB9-GFP⁺MNs with axons performed after 3 days of treatment reveals that conditioned medium (solid bars) and EV treatment (checked bars) have similar effects on MN monocultures (e), with C9-ALS astrocyte medium and EVs inducing significantly lower MN survival. Survival of MN monocultures in MN medium (black solid bar) used as comparative reference (n = 3). Two-way ANOVA (p < .0001), 3xControls versus 3xC9-ALS, N = 3 per condition, error bar = SD. Scale bar = 20 μm.

Fig. 2

EV biogenesis is impaired in C9-ALS iAstrocytes. Representative size distribution graph (a) of ADEVs and TEM (b, c-i, c-ii) show that most ADEVs range between 50 and 120 nm. ADEVs are positive for CD63 (arrow heads) with immunogold labeling (c-iii). ADEV direct quantification using the ZetaView Nanoparticle Tracking System (d) shows a significant decrease in the number of particles secreted by C9 iAstrocytes (One-way ANOVA; p < .01). This suggests impairment in vesicle formation, as supported by TaqMan qRT-PCR data showing downregulation in a number of transcripts involved in EV biogenesis in C9 iAstrocytes (e). t-test (**p < .01; *** p < .001), 3xControls versus 3xC9-ALS, N = 3 per condition, error bar = SD.

Fig. 3

MicroRNAs regulating axonal maintenance are selectively dysregulated in C9 ADEVs. MiRNAs secreted by C9 and Control (CTR) iAstrocytes identify two separate groups in the hierarchical clustering analysis (a). The PCA plot (b) shows how EV-secreted miRNAs differentiate iAstrocytes (iA) and fibroblasts (Fib) on one axis and controls (CTR) and C9 patients (C9) on another axis. iA also show more dramatic differences between patients and controls compared to fibroblasts. Pathway analysis of the dysregulated miRNAs identifies axonal growth and maintenance as the most affected pathway (c). Downregulated (in blue) miRNAs target Semaphorins, RhoA and Rock, thus predicting an increase in these proteins, which would lead to growth cone collapse. Upregulated miRNAs (in red) target Ephs and Wwp1, which would lead to their downregulation. Wwp1 inactivates NogoA, thus this would also lead to axonal collapse. TaqMan qRT-PCR confirms significant downregulation of miR-494-3p, which negatively regulates Sema3A (d). N = 3 per group; error bar = SD. Two-tail unpaired t-test (p < .05). (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Fig. 4

MiR-494-3p mimic restores SEMA3A levels and increases MN survival. QRT-PCR results show that HB9-GFP⁺ MNs treated with C9 iAstrocyte conditioned medium supplemented with a scramble miRNA (miR-scr) display elevated levels of Sema3A compared to control iAstrocyte conditioned medium treatment + miR-scr (a). Sema3A levels, however, are downregulated after 48 h treatment with a miR-494-3p mimic (a). N = 3 per condition; Error bars = SD. Decrease in Sema3A corresponds with improvement in MN survival (b), increase in neurite length (c) and number of nodes/intersection in MN monocultures (d) treated with C9 iAstrocyte conditioned medium plus miR-494-3p. MN measurements were performed 72 h post-treatment. In b N = 3 per condition; in c and d 3 N = 9 (independent replicates for each of the 3 patients and 3 controls); error bar = SD. Statistical analysis: Two-way ANOVA with multiple comparison test and Sidak correction (*p < .05; **p < .01; ***p < .001; ****p < .0001). QRT-PCR analysis of lateral cortico-spinal tract tissue from 3 non-ALS and 3 sporadic ALS patients with limb onset showed a significant 30% reduction in miR-494-3p (e) (two-tailed t-test, p = .02), while no significant change was detected in SEMA3A (f) (two-tailed t-test p = .08).

Fig. 5

Graphic summary of the study. Our study shows that iAstrocytes from C9orf72 patients and controls secrete ADEVs containing miRNAs that affect MN survival and neurite growth. In particular ADEVs from C9orf72 iAstrocytes cause neurite and axonal shortening and MN death. This toxic effect is partly caused by lower levels of miR-494-3p secreted by C9orf72 iAstrocytes. A miRNA mimic of this molecule can restore MN survival and axonal maintenance. In addition, miR-494-3p has the ability to inhibit Sema3A expression in MN, which is, in fact, extremely low in healthy neurons.