Local Synthesis of Actin-Binding Protein β-Thymosin Regulates Neurite Outgrowth

Abstract

Local protein synthesis plays an essential role in the regulation of various aspects of axonal and dendritic function in adult neurons. At present, however, there is no direct evidence that local protein translation is functionally contributing to neuronal outgrowth. Here, we identified the mRNA encoding the actin-binding protein ␤-thymosin as one of the most abundant transcripts in neurites of outgrowing neurons in culture. ␤-Thymosin mRNA is not evenly distributed in neurites, but appears to accumulate at distinct sites such as turning points and growth cones. Using double-stranded RNA knockdown, we show that reducing ␤-thymosin mRNA levels results in a significant increase in neurite outgrowth, both in neurites of intact cells and in isolated neurites. Together, our data demonstrate that local synthesis of ␤-thymosin is functionally involved in regulating neuronal outgrowth.

Caltubin, a Novel Molluscan Tubulin-Interacting Protein, Promotes Axonal Growth and Attenuates Axonal Degeneration of Rodent Neurons

“…Whereas the exact downstream mechanisms by which local protein synthesis regulates axon growth remain unclear, most mRNAs that are identified in axonal growth cones appear to be structural proteins in both invertebrates (Plunet et al, 2002;Gioio et al, 2004) and in vertebrates (Willis et al, 2005;Hengst et al, 2009), and translational regulation of these proteins occurs during axon regeneration (Twiss et al, 2000). For instance, stimulus-driven axonal growth is dependent on local translation of the cytoskeleton regulator PAR (Hengst et al, 2009), and inhibition of local translation of the actin regulating protein ␤-thymosin enhances rates of neurite outgrowth (van Kesteren et al, 2006). In this study, we provided multiple lines of evidence that local protein synthesis of caltubin is required for neurite regeneration through regulation of ␤-tubulin level.…”

“…1 E2) and mRNA was extracted from the cell bodies and from neurites separately. Realtime qPCR analysis was performed to measure the transcript levels of caltubin and two other known neuritic transcripts, ␤-thymosin and ␤-tubulin (van Kesteren et al, 2006), as positive controls. All mRNA levels were first normalized to the level of 16S rRNA, and neuritic mRNA levels were then divided by somatic mRNA levels.…”

“…Approximately 100 -200 local transcripts have been reported in isolated neurites of various neuronal preparations [e.g., in Aplysia sensory neurons (Moccia et al, 2003;Moroz et al, 2006), in squid axoplasm (Capano et al, 1987;Gioio et al, 1994), and in vertebrate axons (Hengst and Jaffrey, 2007;Willis et al, 2007)]. Many of the local transcripts encode cytoskeletal proteins (e.g., microtubule, microfilament, and intermediate filament proteins) (Kaplan et al, 1992;Eng et al, 1999;Vogelaar et al, 2009) Kesteren et al, 2006), suggesting that regulation of the cytoskeleton through local protein synthesis potentially serves as a conserved mechanism underlying axonal outgrowth and regeneration. Previously, we identified 15 axonally localized mRNAs in Lymnaea pedal A (PeA) neurons (van Kesteren et al, 2006).…”

“…Many of the local transcripts encode cytoskeletal proteins (e.g., microtubule, microfilament, and intermediate filament proteins) (Kaplan et al, 1992;Eng et al, 1999;Vogelaar et al, 2009) Kesteren et al, 2006), suggesting that regulation of the cytoskeleton through local protein synthesis potentially serves as a conserved mechanism underlying axonal outgrowth and regeneration. Previously, we identified 15 axonally localized mRNAs in Lymnaea pedal A (PeA) neurons (van Kesteren et al, 2006). Of these transcripts, one encoded a novel putative calcium-binding protein, caltubin, with no apparent mammalian orthologs or homologs.…”

Slow axoplasmic transport under scrutiny

“…The repertoire of proteins locally synthesized in axons is extensive in terms of type and functions, e.g., cytoskeletal proteins, chaperones, resident endoplasmic reticulum proteins, anti-oxidant and metabolic proteins, membrane receptor proteins and many more. In addition, protein synthesis in axons has been described in different sub-compartments and as being responsible for diverse local functions, including translation at synapses or sensory receptors, synthesis in elongating and regenerating axons and growth cones, protein synthesized at the site of injury to be transported to the perikaryon as signals (Crispino et al, 1997;Gaete et al, 1998;Martin et al, 1998;Sotelo et al, 2000;Brittis et al, 2002;Calliari et al, 2002;Zhang et al, 2002;Hanz et al, 2003;Piper et al, 2005;Verma et al, 2005;Willis et al, 2005;Wu et al, 2005;van Kesteren et al, 2006;Jiménez-Díaz et al, 2008;Dubacq et al, 2009;Hengst et al, 2009;Toth et al, 2009); for reviews, see (Álvarez et al, 2000;Campenot and Eng, 2000;Martin, 2004;Lin and Holt, 2008;Giuditta et al, 2008).…”