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 performing experiments with pure axoplasm, which can be easily extracted from the squid giant axons. We have developed a protocol that allows us to keep axoplasm sections alive for more than 6 hours, and to subsequently fix and perform molecular characterization experiments. Preliminary results indicate that pure axoplasm does not need Schwann cells to synthesize membrane proteins de novo. Next, we are beginning to corroborate that the fundamental elements needed to synthesize a membrane protein are in the squid axoplasm. We began with the ribosomes because they are indispensable elements in any translational machinery. For immunocytochemistry purposes, ribosomes are a golden system; they are formed by numerous subunits and their structure have been solved with sufficient resolution to locate precisely most of their subunits. With this information, we are actively pursuing ribosomes and other components of the canonical eukaryotic membrane protein translational machinery, like the presence of ER and Golgi organelles-like structures. RNA is believed to be transported to axons within stress granules. The squid genome contains the essential proteins involve in the assembly of human stress granules. We are presently exploring the function of G3BP. In human, there are two redundant isoforms G3BP1 and 2, while in squid there is only one. We are using a battery of in vitro, ex vivo and in vivo approaches to assess the function of this protein in squid.