Local translation of TC10 is required for membrane expansion during axon outgrowth

Abstract

The surface of developing axons expands in a process mediated by the exocyst complex. The spatial-temporal regulation of the exocyst is only partially understood. Here we report that stimulated membrane enlargement in dorsal root ganglion (DRG) axons is triggered by intra-axonal synthesis of TC10, a small GTPase required for exocyst function. Induced membrane expansion and axon outgrowth are inhibited after axon-specific knockdown of TC10 mRNA. To determine the relationship of intra-axonal TC10 synthesis with the previously described stimulus-induced translation of the cytoskeletal regulator Par3, we investigate the signaling pathways controlling their local translation in response to NGF. Phosphoinositide 3-kinase (PI3K)-dependent activation of the Rheb-mTOR pathway triggers the simultaneous local synthesis of TC10 and Par3. These results reveal the importance of local translation in the control of membrane dynamics and demonstrate that localized, mTOR-dependent protein synthesis triggers the simultaneous activation of parallel pathways.

Targeting Axonal Protein Synthesis in Neuroregeneration and Degeneration

“…The requirement for protein synthesis and degradation is strikingly similar to the requirements for axon elongation and growth cone turning in response to attractive guidance cues [7], and a comparison of the axonal mRNA pools present in uninjured matured and actively regeneration axons of cortical neurons revealed that axotomy triggers the recruitment of transcripts related to axonal targeting [40]. Importantly, while some of the axonally localized mRNAs have been shown to support axon outgrowth and elongation in response to attractive cues, such as β-actin [9,11], Par3 [12], and TC10 [13], the translation of others, including RhoA [8] and cofilin [10], leads to growth cone collapse and axon retraction. Therefore, the axonally localized transcriptome in severed nerves might support the innate regenerative capacity of injured axons but at the same time also mediate the growth preventing effects from the inhibitory molecules in the nerve scar and myelin that ultimately prevent regenerative growth.…”

“…Only at the beginning of this century did work by Holt's group conclusively demonstrate that local protein synthesis is required for the chemotropic responses of axons to attractive and repulsive guidance cues [7]. This work heralded an extremely productive phase of research on local translation in developing axons, and now intra-axonal protein synthesis is recognized to be crucial for growth cone behavior [7][8][9][10][11][12][13][14], axonal pathfinding [15][16][17], axon maintenance [18], and retrograde signaling in developing axons [19,20]. However, far less is known about the extent and importance of localized protein synthesis in axons after the developmental period.…”

“…The coat protein of Sindbis virus has been modified to establish neurotropism [87], and point mutations in its nsP2 gene have been engineered to reduce neurotoxicity [87,88]. In order to allow for direct expression of proteins off the viral genome, the encephalomyocarditis virus internal ribosome entry site (IRES) was introduced into the Sindbis virus expression plasmid SinRep(nsP2S 726 ) [87,89], resulting in efficient transfection and protein expression in transfected axons in culture and in animals [8,12,13,19,90]. With the Sindbis-IRES approach it is possible to transact directly axons and to overexposes proteins locally, without any need for somatic contribution.…”

“…The use of siRNA in axons has several advantages: far fewer mRNAs are found in axons than in whole cells. The lower abundance of the localized mRNAs makes siRNA-mediated knockdown very efficient and quick, often within 24 h [13,45]. Additionally, as there are fewer mRNA species found in axons, the danger of off-target effects is proportionally reduced.…”

“…Presumably, in an in vivo situation many localized transcripts are translated into proteins at any given time and the neuronal or axonal response is the result of the coordinated effect of the temporally and spatially restricted increase in their abundance. In fact, one of the advantages of local translation might be the coordinated activation of seemingly unconnected signaling pathways [13]. Thus, in order to target therapeutically axonal protein synthesis more knowledge is required about the axonal translatome, that is the cohort of actively translated mRNAs in axons under regenerative or degenerative conditions.…”