Localization and transport of ribosomes in axons of the mammalian PNS and CNS
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Abstract
In recent years, many studies have indicated that some neuronal cell types produce proteins not only in the cell body, but also locally in dendrites, especially at synaptic sites, and in the axon. Local axonal translation seems to be important for the maintenance and growth of the axon, for retrograde signaling, and for the regeneration of peripheral nervous system (PNS) axons after injury. It was shown that messenger RNAs (mRNAs) are transported in ribonucleoprotein particles (RNPs) and there is increasing evidence that their axonal localization is selective for specific mRNAs. Additionally, the presence of ribosomes in the axonal compartment has been reported and many studies assumed that they are likewise transported in RNPs from the neuronal cell body. Recently, however, glial cells were proposed as an alternative source of axonal ribosomes. Such a glia-to-axon transfer is believed to account for the increased ribosomal levels observed in PNS axons after injury and to support local protein synthesis for regeneration.
The aim of the present study was to show that axonally localized ribosomes originate from the neuronal cell body in vivo, also upon injury. To visualize ribosomes, the ribosomal protein L4 (RPL4), a component of the large 60S subunit of the ribosomal complex, was labeled in a transgenic mouse line. ‘RiboTracker’ mice contained the fusion construct of RPL4 tagged with tandem dimer protein Tomato (tdTomato) downstream of a floxed stop cassette in the ROSA26 locus. Crossbreeding with mice expressing Cre recombinase under the calcium/calmodulin-dependent protein kinase II alpha (CamKIIα) or Advillin promoter led to the neuron-specific expression of L4-tdTomato as shown for central nervous system (CNS) hippocampal and cortical neurons (CamKIIα), as well as dorsal root ganglia (DRG) neurons (CamKIIα, Advillin). However, when focusing on axons, L4-tdTomato was neither detected in vitro nor in vivo, even though the staining with an antibody against the ribosomal protein L26 (RPL26) confirmed the presence of axonal ribosomes. In the case of PNS injury, increased axonal levels of RPL26-stained ribosomes were observed, but again, L4-tdTomato signals were not found in the sciatic nerve fibers. These findings led to the conclusion that ribosomes are not transported from the neuronal cell body to the axon. Paralleling experiments with recombinant adeno-associated virus (rAAV) that express RPL4 fused to enhanced green fluorescence protein (eGFP) in a neuron-specific way supported the in vitro data from neuronal RiboTracker-Cre mice. Based on the prior in vivo results, however, studies with rAAVs were not continued.
Next, glial cells were reconsidered as an alternative source of axonal ribosomes. To investigate this hypothesis, RiboTracker mice were crossed with a glial Cre line driven by the 2',3'-cyclic-nucleotide 3'-phosphodiesterase (CNP) promoter. Satellite glia cells and Schwann cells in the PNS showed strong expression of the transgene, but some DRG neurons also contained L4-tdTomato. These neuronal subpopulations were similar to those in the neuron-specific RiboTracker-Cre mice, therefore, axonal ribosomes were unlikely to originate from the neuronal cell body. In the sciatic nerve, occasional L4-tdTomato signals were detected in uninjured axons of the RiboTracker-CNP-Cre mice. Upon injury, a massive increase of axonally localized tagged ribosomes was observed, paralleling the findings of immunostaining with the anti-RPL26 antibody. Since increased axonal levels of L4-tdTomato were also observed distally to the lesion site, glial cells represent the only possible source of axonal ribosomes. This is the first study investigating the origin of axonal ribosomes in transgenic mice in vivo.
Taken together, the results from the present work strongly support the hypothesis of a glia-to-axon transfer of ribosomes, and go even beyond this by providing evidence for Schwann cells being the exclusive source of axonal ribosomes in the peripheral nervous system.