Expanding Axonal Transcriptome Brings New Functions for Axonally Synthesized Proteins in Health and Disease

Abstract

Intra-axonal protein synthesis has been shown to play critical roles in both development and repair of axons. Axons provide long-range connectivity in the nervous system, and disruption of their function and/or structure is seen in several neurological diseases and disorders. Axonally synthesized proteins or losses in axonally synthesized proteins contribute to neurodegenerative diseases, neuropathic pain, viral transport, and survival of axons. Increasing sensitivity of RNA detection and quantitation coupled with methods to isolate axons to purity has shown that a surprisingly complex transcriptome exists in axons. This extends across different species, neuronal populations, and physiological conditions. These studies have helped define the repertoire of neuronal mRNAs that can localize into axons and imply previously unrecognized functions for local translation in neurons. Here, we review the current state of transcriptomics studies of isolated axons, contrast axonal mRNA profiles between different neuronal types and growth states, and discuss how mRNA transport into and translation within axons contribute to neurological disorders.

Keywords: RNA binding protein; axon; mRNA transport; ribonucleoprotein particle; translation.

Figure 1. Mechanisms of axonally mRNA transport

Schematic of neuron showing transport of multiple mRNAs into axons with translation at branch points and within growth cones. Note that translation also likely occurs along axon shaft based on studies from the Holt lab (Zivraj and others 2010). Some of the known functions for axonally translated proteins are noted in green text and examples of altered RNA transport and/or translation in specific neurological disorders is noted in red text.

Figure 2. Comparison of analytical methods for axonal RNA-Seq studies

A-B, Axonal mRNAs of embryonic DRG neurons (A) and motor neurons (B) are displayed by relative enrichment in axons over cell bodies (y-axis) and its counts in axons (x-axis) (Briese and others 2016; Minis and others 2014). Raw datasets from the Minis et al. (2014) and Briese et al. (2016) data sets were mapped to Ensembl mouse genome (mm10) using STAR application (Dobin and Gingeras 2015) and counts for each gene was calculated with HTSeq software (Anders and others 2015). FKPM counts were normalized by EdgeR (Robinson and others 2010). These normalized counts were used to calculate the relative enrichment in axons vs. cell bodies, and values for the top 2000 most enriched axonal mRNAs were graphed using R software. Axonal mRNAs with biological roles previously in axon pathfinding (red), injury signaling and regeneration (orange), axon maintenance (green), and neurodegeneration (blue) are shown. The dashed blue line shows cutoff for axonal enrichment. C-D, In contrast to A-B, the top 2000 most abundant axonal mRNAs from normalized FKPM of the raw data from Minis et al. (2014) and Briese et al. (2016) studies are graphed relative to their cell body normalized FKPM values. Embryonic DRG neurons (C) and motor neurons (D) are shown with dashed blue line representing enrichment cutoff. Axonal mRNAs with previously proven biological functions are annotated as above. E, Venn diagram of overlap for top 2000 axonally enriched mRNAs from embryonic DRG vs. motor neurons from A-B is shown. Less than one quarter of the mRNAs are shared by this analytical method. F, In contrast, Venn diagram of overlap between sensory and motor axons for 2000 most abundant axonal mRNAs from C-D, shows much greater overlap between the Minis et al. (2014) and Briese et al. (2016) datasets.

Figure 3. Unresolved mechanisms to regulate axonal mRNA dynamics

A, Where are mRNAs stored in axons? There is clear evidence for storage of mRNAs in axons, where they are somehow sequestered away from the translational machinery and kept dormant until needed. The mechanisms underlying this are not known. How does an RNP disengage from the motor proteins used for transport into axons and avoid translation of its mRNA? Binding of additional proteins to the RNP (1) or replacement of RNP proteins with other RBPs (2) are possible mechanisms. Additionally, there must be stimulus-dependent mechanisms to recruit axonal mRNAs from their storage site (3). B, Does local translation impart unique function(s) to an axonal protein? For some proteins the axon has a dual source – cell body-synthesized protein that is transported down the axon (1) and the protein derived from the axonally translated mRNA. It is not clear how or if these two protein sources (3,4) are functionally distinct. C, Does axonal translation impart unique function(s) to retrogradely transported proteins? Several transcription factors are known to be translated in axons (1,2) and retrogradely transported to the neuronal cell body (3). For all identified to date, there is already a cell body (or nuclear) pool of the transcription factor resident in the neuron that is derived from protein synthesis in the cell body (4). It is not clear how or if the neuron distinguishes these two sources of protein. Do they have distinct functional roles, perhaps regulating different gene cohorts? For legend refer to Figure 1.