Membrane Protein synthesis in axons

National Institute Of Neurological Disorders And Stroke

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Abstract

Structurally, neurons are complex entities. They have a cell body which contains the nucleus and the organelles to synthesize proteins, many of which are transported to axons and dendrites. In some instances, axons can extend for meters from the central protein synthesis machinery of the cell body. Therefore, it is reasonable to imagine that axons possess an autonomous capacity to produce proteins to respond to local challenges. Cumulative evidence suggest that local protein translation is essential for axonal maintenance, development, guidance and synaptic plasticity. Giant axons from the Humboldt squid are larger than 1 mm in diameter and could be easily dissected for as long as 20 cm. From single axons, we have been able to extract enough pure axoplasm to explore their RNA composition and generate polyA and random primers libraries to be sequenced using next generation sequencing. Interestingly, axoplasm contains large amount of ribosomal RNAs, unprecedented proportions of Signal Recognition Particle (SRP) RNA (68% identical to human homolog) and 8000 messenger RNA species, many encoding the translation machinery, membrane proteins, translocon and signal recognition particle (SRP) subunits and endomembrane-associated proteins. These are the elements that form the molecular machinery needed to synthesized membrane proteins, which led us to hypothesized that isolated axons are capable to newly synthesize membrane proteins. Isolated squid giant axons were able to translate the information provided by in vitro-cRNA encoding for Shaker KV channel. In addition to being an exogenous membrane protein for squid, these channels display a distinctive and unique fast inactivation which is absent in squid KV channels expressed natively. Presently, we are beginning to assess whether the squid giant axon system is capable of glycosylate proteins. Because these axons are large, we can directly inject directly in the axoplasm fucose and other sugars containing click chemistry moieties. After injection, axons are sealed at both ends and incubated overnight at 12 C. Applying click chemistry fluorophores from the outside has revealed that isolated axons have have a robust glycosylation capacity. We do not know which are the substrates, but likely they would be proteins and lipids.