Intra-axonal translation and retrograde trafficking of CREB promotes neuronal survival.

During development of the nervous system, axons and growth cones contain mRNAs such as beta-actin, cofilin and RhoA, which are locally translated in response to guidance cues. Intra-axonal translation of these mRNAs results in local morphological responses; however, other functions of intra-axonal mRNA translation remain unknown. Here, we show that axons of developing mammalian neurons contain mRNA encoding the cAMP-responsive element (CRE)-binding protein (CREB). CREB is translated within axons in response to nerve growth factor (NGF) and is retrogradely trafficked to the cell body. In neurons that are selectively deficient in axonal CREB transcripts, increases in nuclear pCREB, CRE-mediated transcription and neuronal survival elicited by axonal application of NGF are abolished, indicating a signalling function for axonally synthesized CREB. These studies identify a signalling role for axonally derived CREB, and indicate that signal-dependent synthesis and retrograde trafficking of transcription factors enables specific transcriptional responses to signalling events at distal axons.

Functional and selective RNA interference in developing axons and growth cones.

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.

Local translation of RhoA regulates growth cone collapse.

Neuronal development requires highly coordinated regulation of the cytoskeleton within the developing axon. This dynamic regulation manifests itself in axonal branching, turning and pathfinding, presynaptic differentiation, and growth cone collapse and extension. Semaphorin 3A (Sema3A), a secreted guidance cue that primarily functions to repel axons from inappropriate targets, induces cytoskeletal rearrangements that result in growth cone collapse. These effects require intra-axonal messenger RNA translation. Here we show that transcripts for RhoA, a small guanosine triphosphatase (GTPase) that regulates the actin cytoskeleton, are localized to developing axons and growth cones, and this localization is mediated by an axonal targeting element located in the RhoA 3' untranslated region (UTR). Sema3A induces intra-axonal translation of RhoA mRNA, and this local translation of RhoA is necessary and sufficient for Sema3A-mediated growth cone collapse. These studies indicate that local RhoA translation regulates the neuronal cytoskeleton and identify a new mechanism for the regulation of RhoA signalling.

PLC zeta: a sperm-specific trigger of Ca(2+) oscillations in eggs and embryo development.

Upon fertilisation by sperm, mammalian eggs are activated by a series of intracellular Ca(2+) oscillations that are essential for embryo development. The mechanism by which sperm induces this complex signalling phenomenon is unknown. One proposal is that the sperm introduces an exclusive cytosolic factor into the egg that elicits serial Ca(2+) release. The 'sperm factor' hypothesis has not been ratified because a sperm-specific protein that generates repetitive Ca(2+) transients and egg activation has not been found. We identify a novel, sperm-specific phospholipase C, PLC zeta, that triggers Ca(2+) oscillations in mouse eggs indistinguishable from those at fertilisation. PLC zeta removal from sperm extracts abolishes Ca(2+) release in eggs. Moreover, the PLC zeta content of a single sperm was sufficient to produce Ca(2+) oscillations as well as normal embryo development to blastocyst. Our results are consistent with sperm PLC zeta as the molecular trigger for development of a fertilised egg into an embryo.