Selenoprotein W expression and regulation in mouse brain and neurons

Abstract

Background Selenoprotein W (Sepw1) is a selenium-containing protein that is abundant in brain and muscle of vertebrate animals. Muscular expression of Sepw1 is reduced by dietary selenium (Se) deficiency in mammals, whereas brain expression is maintained. However, expression of Sepw1 depends on the Se transporter selenoprotein P (Sepp1). Methods We assessed the regional and cellular expression of Sepw1 in the mouse brain and neuronal cultures. Results We found that Sepw1 is widespread in neurons and neuropil of mouse brain and appears in both the soma and processes of neurons in culture. Pyramidal neurons of cortex and hippocampus express high levels of Sepw1. It is also abundant in Purkinje neurons and their dendritic arbors in the cerebellum. Analysis of synaptosome fractions prepared from mice brains indicated that Sepw1 is present at synapses, as were several proteins involved in selenoprotein synthesis. Synaptic expression of Sepw1 expression is reduced in mice lacking Sepp1 compared with control mice, although selenoprotein synthesis factors were similarly expressed in both genotypes. Lastly, Sepw1 mRNA coimmunoprecipitates with Staufen 2 protein in a human neuronal cell line. Conclusions Our results suggest that Sepw1 may be locally synthesized in distal compartments of neurons including synapses.

Keywords: Selenium; selenocysteine; selenoprotein; selenoprotein P; selenoprotein W.

Figure 1

Expression of Sepw1 in cell bodies and processes of pyramidal neurons in hippocampus is reduced in Sepp1−/− mice. Brain sections containing hippocampus were immunolabeled for Sepw1 and combined with a fluorescent Nissl stain in wild-type Sepp1+/+ mice (A) and Sepp1−/− mice (B). Large pyramidal neurons in both CA1 and CA3 displayed Sepw1 immunoreactivity, which prominently extended into the apical dendrites of Sepp1+/+ mice. Sepp1−/− mice had drastically reduced immunoreactivity toward Sepw1. Scale bar = 20 microns.

Figure 2

Regional expression of Selenoprotein W (Sepw1) in neurons of mouse brain. (A) Barrel field of somatosensory cortex displayed Sepw1 staining in cell bodies, which extended into processes (B), and was visible in barrels (C). Photomicrographs of cingulate cortex (D), piriform cortex (E), and cerebellum (F) show Sepw1 expression in neurons. Pyramidal neurons and Purkinje neurons display high Sepw1 immunoreactivity in apical dendrites. (G) Imaging of Sepw1 (green, left) and Tuj1 (magenta, center) in somatosensory cortex demonstrates neuronal localization of Sepw1 and some colocalization (white, right) with Tuj1. Scale bar = 100 microns in A; Scale bar = 20 microns in B–G.

Figure 3

Selenoprotein W (Sepw1) is expressed in cell bodies and processes of neurons in culture. Primary cultures derived from neonatal mouse cortex (A) and (B), and cerebellum (C) were grown on coverslips for 3 weeks and subsequently double immunolabeled for Sepw1 (green, left) and a neuron-specific tubulin, Tuj1 (magenta, center). Merged images show sporadic colocalization between Sepw1 and Tuj1 (white, right) in neuronal soma and neurites in cultures. Scale bar = 20 microns.

Figure 4

Synaptic expression of selenoprotein W (Sepw1) is reduced in mice lacking selenoprotein P (Sepp1). Synaptosome fractions were prepared from Sepp1−/− and Sepp1+/+ littermate mice. Synaptosome fractions (+Syn.) were analyzed in comparison to S1 fractions (−Syn.). Western blotting for Sepw1 (A) and Gpx4 (B) revealed the presence of both selenoproteins, with beta-actin used as a loading control. Quantitation of synaptosomes revealed that Sepw1 expression (C) was significantly decreased (**P < 0.01) in Sepp1−/− mice (n = 3) compared with Sepp1+/+ littermates (n = 4). However, Gpx4 expression in synaptosomes (D) was not significantly different between Sepp1+/+ and Sepp1−/− mice.

Figure 5

Several selenoprotein synthesis factors are present in synaptosomes. (A) To check for contaminating nuclear proteins, TBP was analyzed. TBP was clearly present in the S1 fractions (−Syn.) but not in the synaptosome fractions (+Syn.). Both EFSec (B) and Sbp2 (C), which are required for insertion of Sec during selenoprotein translation, were present in synaptic fractions. Scly (D), which recycles Se from Sec, and Sps2 (E), which generates selenophosphate for Sec biosynthesis, were both found in synaptic fractions. (F) SecP43, a protein associated with the Sec-specific tRNA, was found only in S1 fractions.

Figure 6

Selenoprotein W (Sepw1) mRNA associates with Stau2 in SH-SY5Y neuroblastoma cells. (A) RT-qPCR was performed on Stau1- and Stau2-RNA immunoprecipitation (RIP) samples and Total RNA samples (n = 3) harvested and processed in parallel and normalized to a synthetic mRNA spiked into the samples. Stau2 coimmunoprecipitated more Sepw1 mRNA than the Stau1 immunoprecipitation samples, while also showing greater efficiency compared with Gpx4 mRNA. (B) The qPCR data was normalized to endogenous HPRT mRNA, a putative Stau2-target mRNA. Stau2-RIP coimmunoprecipitates with more Sepw1 mRNA than HPRT mRNA. In contrast, less Gpx4 mRNA is observed in the Stau2-RIP samples when compared with HPRT mRNA. selenoprotein P (Sepp1) mRNA is undetectable in the RIP samples. **P < 0.005, ***P < 0.001.