The RNA binding and transport proteins staufen and fragile X mental retardation protein are expressed by rat primary afferent neurons and localize to peripheral and central axons

Abstract

Neuronal proteins have been traditionally viewed as being derived solely from the soma; however, accumulating evidence indicates that dendritic and axonal sites are capable of a more autonomous role in terms of new protein synthesis. Such extra-somal translation allows for more rapid, on-demand regulation of neuronal structure and function than would otherwise be possible. While mechanisms of dendritic RNA transport have been elucidated, it remains unclear how RNA is trafficked into the axon for this purpose. Primary afferent neurons of the dorsal root (DRG) and trigeminal (TG) ganglia have among the longest axons in the neuraxis and such axonal protein synthesis would be advantageous, given the greater time involved for protein trafficking to occur via axonal transport. Therefore, we hypothesized that these primary sensory neurons might express proteins involved in RNA transport. Rat DRG and TG neurons expressed staufen (stau) 1 and 2 (detected at the mRNA level) and stau2 and fragile x mental retardation protein (FMRP; detected at the protein level). Stau2 mRNA was also detected in human TG neurons. Stau2 and FMRP protein were localized to the sciatic nerve and dorsal roots by immunohistochemistry and to dorsal roots by Western blot. Stau2 and FMRP immunoreactivities colocalized with transient receptor potential channel type 1 immunoreactivity in sensory axons of the sciatic nerve and dorsal root, suggesting that these proteins are being transported into the peripheral and central terminals of nociceptive sensory axons. Based on these findings, we propose that stau2 and FMRP proteins are attractive candidates to subserve RNA transport in sensory neurons, linking somal transcriptional events to axonal translation.

Figures

Fig. 1

Stau expression in rat DRG and human TG. Both Stau1 (A) and stau2 (B) mRNAs were expressed by all DRG neurons. Human TG neurons also express stau2 (C) as shown by silver grains from isotopic ISH. Matching image of neurofilament 200 protein (D) is shown to demonstrate the location of neurons in the ganglia. Scale bars=200 μm in all panels.

Fig. 2

Stau2 and FMRP protein expression in the DRG and dorsal horn. Stau2 protein was expressed by all DRG neurons (A), while expression of TRPV1 (B) demonstrates colocalization in small nociceptive neurons (C). FMRP protein was also expressed by all DRG neurons (D), while expression of TRPV1 (E) demonstrates colocalization in small nociceptive neurons (F). Negative control for FMRP (G) with TRPV1 immunoreactivity (H and merged in I). Scale bars=200 μm (A–I) or 20 μm (J–K). High magnification images for stau2 (J) and FMRP (K) show the neuron-specific expression of these proteins in the DRG.

Fig. 3

Assessment of stau2 and FMRP protein and mRNA expression in the dorsal horn (DH), dorsal root (DR) and DRG by Western blot and RT-PCR. (A) Stau2 protein Western blot illustrating that the 62, 59 and 52 kDa isoforms of the protein are present in the DH and DRG, whereas only the 59 and 52 kDa isoforms are present in the DR. βIII-Tubulin (tub) was used as a loading control. (B) Stau1 mRNA was present in the DH, DR and DRG by RT-PCR, whereas stau2 mRNA was only present in the DH and DRG. GAPDH was used as a loading and positive control. (C) FMRP protein, detected as an ~80 kDa protein band, was present in DH, DR and DRG by Western blot. Cortex (Cor) protein was used as a positive control for the known size of FMRP. Tub was used as a loading control. (D) FMRP mRNA was detected by RT-PCR in the DH, DR and DRG. GAPDH was used as a loading and positive control. All Western blot and RT-PCR experiments are representative of samples run from three different animals per tissue.

Fig. 4

Stau2 and FMRP immunoreactivities in the sciatic nerve. Stau2 (A) and TRPV1 (B and merge in C) immunoreactivities observed in the sciatic nerve. Examples of fibers that colocalized stau2 and TRPV1 are illustrated with arrows. High magnification image of a single TRPV1-immunoreactive fiber (E) that colocalized stau2 (D and merge in F). FMRP (G) and peripherin (H and merge in I) immunoreactivities observed in the sciatic nerve. Examples of fibers that colocalized FMRP and peripherin are illustrated with arrows. High magnification image of a single TRPV1-immunoreactive fiber (K) that colocalized FMRP (J and merge in L). Scale bars=10 μm (A–C, G–I and J–L) or 5 μm (D–F).

Fig. 5

Stau2 and FMRP immunoreactivities in the dorsal root and spinal dorsal horn. Stau2 (A) and TRPV1 (B and merge in C) immunoreactivities in the dorsal root. Stau2 (D) and TRPV1 (E and merge in F) immunoreactivities in the spinal dorsal horn. FMRP (G) and TRPV1 (H and merge in I) immunoreactivities in the dorsal root. FMRP (J) and TRPV1 (K and merge in L) immunoreactivities in the spinal dorsal horn. Examples of colocalization are illustrated with sidewise arrows. Examples of TRPV1-positive axons that did not colocalize stau2 or FMRP are illustrated with upward arrows. Scale bars=20 μm (A–C and G–I) or 200 μm (D–F and J–L).