Functional and selective RNA interference in developing axons and growth cones

Abstract

Developing axons and growth cones contain "local" mRNAs that are translated in response to various extracellular signaling molecules and have roles in several processes during axonal development, including axonal pathfinding, orientation of axons in chemotactic gradients, and in the regulation of neurotransmitter release. The molecular mechanisms that regulate mRNA translation within axons and growth cones are unknown. Here we show that proteins involved in RNA interference (RNAi), including argonaute-3 and argonaute-4, Dicer, and the fragile X mental retardation protein, are found in developing axons and growth cones. These proteins assemble into functional RNA-induced silencing complexes as transfection of small interfering RNAs selectively into distal axons results in distal axon-specific mRNA knock-down, without reducing transcript levels in proximal axons or associated diffusion of small interfering RNA into proximal axons or cell bodies. RhoA mRNA is localized to axons and growth cones, and intra-axonal translation of RhoA is required for growth cone collapse elicited by Semaphorin 3A (Sema3A), an axonal guidance cue. Selective knock-down of axonal RhoA mRNA abolishes Sema3A-dependent growth cone collapse. Our results demonstrate functional and potent RNAi in axons and identify an approach to spatially regulate mRNA transcripts at a subcellular level in neurons.

Figure 1.

Localization of RNAi-related proteins in axons and growth cones. A–D, E15 dissociated rat DRG neuronal cultures (DIV 3) were labeled with antibodies directed against argonaute 3 (A), argonaute 4 (B), FMRP (C), and Dicer (D). E, Colocalization of Ago 3 (green), FMRP (red), with 4′,6′-diamidino-2-phenylindole dihydrochloride (DAPI; blue). F, Colocalization of Ago 4 (green), Dicer (red), with DAPI (blue). Scale bar, 10 μm. G, H, Each of these proteins exhibit punctate localization in both axons and growth cones. Scale bar, 10 μm. I, J, High-power magnification of the growth cones boxed in G and H. Scale bar, 5 μm.

Figure 2.

Selective knock-down of mRNA in axons. A, Schematic diagram of compartmentalized siRNA transfection in Campenot culturing chambers. Dissociated E15 DRG neurons were plated in the central compartment of Campenot chambers, in the presence of 10 ng/ml NGF. Axon growth into the side compartments is promoted by inclusion of NGF at 100 ng/ml in the side chambers. Axons cross by DIV 5, at which point NGF is removed from the central compartment to induce the death of neurons whose axons have not yet crossed. B–E, RhoA immunofluorescence staining reveals knock-down of axonal RhoA protein caused by transfection of RhoA-specific siRNA into the side chamber (D) but not by a control siRNA (B). The axons were visualized by GAP-43 immunofluorescence (C, E). F–O, FISH with a scrambled (F, G) or a RhoA mRNA-specific probe (H, J, L, N) of axons in the side (H–K) or the central compartment (L–O). The axons are visualized by labeling with Alexa 488–WGA (G, I, K, M, O). Transfection of RhoA-specific siRNA in the side compartment results in RhoA mRNA knock-down in the axons that have crossed into the side compartment (J, K) but not in contiguous axons in the central compartment (N, O). RhoA-specific siRNA (D, E, J, K, N, O) or control, nontargeting siRNA (B, C, H, I, L, M) were transfected into axons on DIV 5, and RhoA transcripts were detected by FISH on DIV 7. P, Q, No retrograde transport or diffusion was observed 48 h after transfection of FITC-labeled control siRNA into side chamber axons. Dotted line, Border of divider. Scale bar, 10 μm. R, Quantification of the intensity of FISH signals in side and contiguous central chamber axons transfected with RhoA-specific or control siRNA. Axons in the side compartment were transfected on 5 DIV, and FISH with a RhoA-specific probe was performed on 7 DIV. **p < 0.0001, unpaired, two-tailed t test; n ≥ 27 per condition. S, Transfection of RhoA-specific siRNA into the side chamber prevents growth cone collapse in response to Sema3A. Axons in the side compartment were transfected as above, and growth cone collapse assays were performed on 7 DIV. Sema3A (450 ng/ml) induces collapse in 60 min in axons transfected with control siRNA but elicits substantially reduced collapse in axons transfected with RhoA-specific siRNA. **p < 0.0001, unpaired, two-tailed t test; n = 5 optical fields with a total of ≥65 growth cones per condition.