In cultured neurons, β-actin
mRNA is transported into axons and dendrites, while γ-actin
mRNA remains in the cell body (Bassell et al., 1998
; Zheng et al., 2001
; Tiruchinapalli et al., 2003
). This axonal localization is driven by the 3'UTR of β-actin
mRNA through a conserved 'zip code' element (Kislauskis et al., 1994
). We have taken advantage of this zip code element to test for axonal localization in vivo
. For this, we generated transgenic mice expressing a destabilized, myristoylated GFP with the 3'UTR of rat β-actin
mRNAs (). The 54 nt zip code region of rat β-actin
shows 100% identity to the corresponding region of mouse β-actin
mRNA 3'UTR; rat γ-actin 3'UTR shows 94% identity with the mouse γ-actin
mRNA (data not shown). Myristoylation limits GFP diffusion in neuronal processes to provide a reporter for sites of translation (Aakalu et al., 2001
; Willis et al., 2007
; Yudin et al., 2008
). The neuronal-specific Tα1 tubulin promoter, which is activated during periods of axon growth (Gloster et al., 1994
), was used to drive transgene expression.
Transgenic model for axonal protein synthesis
Genotyping by PCR and Southern blotting confirmed transgene integration (). Two β-actin lines and one γ-actin line were chosen for subsequent studies based on robust GFP expression in DRGs. Tα1-GFP-3'γ-actin line has four transgene copies and Tα1-GFP-3'β-actin2a and Tα1-GFP-3'β-actin2b lines have two transgene copies (). The two Tα1-GFP-3'β-actin lines were crossed to generate a line carrying four transgene copies (Tα1-GFP-3'β-actin2ax2b; ). DRG cultures from the Tα1-GFP-3'β-actin lines showed GFP in cell bodies and neurites, but those from the Tα1-GFP-3'γ-actin line showed GFP only in the neuronal cell bodies (). RT-PCR of axonal isolates from DRGs cultured from the transgenic lines also confirmed axonal localization of GFP-3’β-actin but not GFP-3’γ-actin mRNA (data not shown). RT-PCR and immunoblotting showed no evidence for GFP expression in Schwann cells in these mice ().
To determine if the 3'UTR of β-actin
can support in vivo localization of GFP
mRNA in the PNS, sciatic nerve was crushed to activate the Tα1 promoter and L4–5 DRGs and sciatic nerve were analyzed. Both transgenes showed increase in GFP fluorescence in the DRG after injury (). GFP signals were highest in the DRGs over 5–14 d after crush injury, consistent with previous reports for Tα1 activation (Gloster et al., 1994
). Only the nerve sections from the Tα1-GFP-3’β-actin
mice showed GFP signals (). The Tα1-GFP-3'γ-actin
nerves did not show signals above the autofluorescence of wild type nerve.
Tα1-GFP-3’β-actin but not Tα1-GFP-3’γ-actin mice show GFP in PNS axons
Since we saw no transgene expression in non-neuronal cells ( and ), we used RT-qPCR to analyze GFP mRNA levels. DRGs from both transgenic lines showed increase in GFP mRNA peaking 5 d after crush (). Relative transgene induction in the DRGs was overall higher in Tα1-GFP-3'β-actin than in Tα1-GFP-3'γ-actin lines, but the absolute levels of the GFP mRNA were not significantly different prior to injury (). Curiously, the sciatic nerve GFP-3’β-actin mRNA levels showed peak accumulation at 1 d after injury, which precedes the 5 d peak in GFP-3’β-actin mRNA levels in the DRG (). Relative nerve GFP mRNA levels in Tα1-GFP-3'γ-actin mice could not be calculated over 0–28 d samples since threshold values were not reached for this ΔΔCt analyses. Thus, we analyzed raw RT-qPCR amplification plots. DRG and sciatic nerve RT-qPCR plots for Tα1-GFP-3'β-actin mice rose from baseline within a few cycles of one another (). Plots for the DRG RNA from Tα1-GFP-3'γ-actin mice were similar to the Tα1-GFP-3'β-actin mice. However, the sciatic nerve RTqPCR plots from Tα1-GFP-3'γ-actin mice were essentially the same as wild type (). Thus, GFP-3'γ-actin mRNA does not appear to be transported into distal sciatic nerves.
Sciatic nerve injury increases axonal levels of β-actin reporter mRNA
The RT-qPCR amplification plots for the naïve Tα1-GFP-3'β-actin sciatic nerves show higher Ct values, indicative of less GFP mRNA, than the naïve DRG and 5 d crushed nerve RNA samples, but clearly indicate presence of GFP-3'β-actin mRNA in the mature uninjured nerve (). Thus, the neuronally expressed transgene mRNA with the β-actin 3'UTR also localizes into uninjured PNS axons.
As a more rigorous test for axonal localization of the GFP-3'β-actin
mRNA, we transected the CF nerve of transgenic mice and provided a wild type nerve graft as a regeneration substrate (English et al., 2005
). FISH for GFP
mRNA showed signals in 14 d grafts from Tα1-GFP-3'β-actin
mice that were clearly distinct from Schwann cells (). The Tα1-GFP-3'γ-actin
lines only showed background GFP
mRNA signals in grafts (). Axons from transected nerves similarly regenerated into acellular allografts (data not shown); RT-qPCR for acellular grafts also showed Ct
values for Tα1-GFP-3'β-actin
nerves indicative of GFP
mRNA localization while grafts of Tα1-GFP-3'γ-actin
mice showed no evidence for GFP
Axonal mRNA localization has been detected in the developing spinal cord (Brittis et al., 2002
). Thus, we asked if Tα1-GFP-3' β-actin
mice showed axonal GFP fluorescence in their spinal cord axons. Since PNS nerve injury increased axonal GFP signals in the Tα1-GFP-3'β-actin
mice, we reasoned that spinal cord injury might also lead to localization of the GFP-3’ β-actin
mRNA into ascending DRG axons. Scattered GFP positive axons of the sham Tα1-GFP-3'β-actin
mice (). Strong GFP fluorescence was seen in axons of the Tα1-GFP-3'β-actin
mice at 10 d after contusion injury (). By confocal imaging, these GFP signals overlapped with axonal markers but were distinct from glial markers (). No GFP fluorescence was seen in the Tα1-GFP-3'γ-actin
spinal cord (). Lower thoracic DRGs in these animals showed no increase in GFP signals with spinal cord injury ( inset panels), suggesting that this injury did not activate the Tα1 promoter.
Axonal localization of GFP-3'β-actin product in central axons