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. 2015 Jul;134(1):147-55.
doi: 10.1111/jnc.13113. Epub 2015 Apr 27.

Neuronally produced versican V2 renders C-fiber nociceptors IB4 -positive

Oliver Bogen  1   2 , Olaf Bender  1 , Jana Löwe  3 , Wolfgang Blenau  3 , Beatrice Thevis  4 , Wolfgang Schröder  5 , Richard U Margolis  6 , Jon D Levine  2 , Ferdinand Hucho  1
Affiliations

Neuronally produced versican V2 renders C-fiber nociceptors IB4 -positive

Oliver Bogen et al. J Neurochem. 2015 Jul.

Abstract

A subpopulation of nociceptors, the glial cell line-derived neurotrophic factor (GDNF)-dependent, non-peptidergic C-fibers, expresses a cell-surface glycoconjugate that can be selectively labeled with isolectin B4 (IB4 ), a homotetrameric plant lectin from Griffonia simplicifolia. We show that versican is an IB4 -binding molecule in rat dorsal root ganglion neurons. Using reverse transcriptase polymerase chain reaction (RT-PCR), in situ hybridization and immunofluorescence experiments on rat lumbar dorsal root ganglion, we provide the first demonstration that versican is produced by neurons. In addition, by probing Western blots with splice variant-specific antibodies we show that the IB4 -binding versican contains only the glycosaminoglycan alpha domain. Our data support V2 as the versican isoform that renders this subpopulation of nociceptors IB4 -positive (+). A subset of nociceptors, the GDNF-dependent non-peptidergic C-fibers can be characterized by its reactivity for isolectin B4 (IB4), a plant lectin from Griffonia simplicifolia. We have previously demonstrated that versican V2 binds IB4 in a Ca2 + -dependent manner. However, given that versican is thought to be the product of glial cells, it was questionable whether versican V2 can be accountable for the IB4-reactivity of this subset of nociceptors. The results presented here prove - for the first time - a neuronal origin of versican and suggest that versican V2 is the molecule that renders GDNF-dependent non-peptidergic C-fibers IB4-positive.

Keywords: IB4; V2; nociceptors; pain; sensory neurons; versican.

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Figures

Figure 1
Figure 1. IB4-reactivity in rat spinal cord
Rat spinal cord tissue was fractionated according to a synaptosome preparation scheme (Gray and Whittaker 1962). A combined light membrane and synaptosome fraction was treated with hyaluronidase to release hyaluronan-bound proteins, such as versican, into the supernatant. Proteins in the hyaluronidase-extract were immunoprecipitated with the monoclonal anti-versican antibody (12C5), which binds all known versican splice variants (Westling et al. 2004). Different amounts of proteins (see fractionation scheme on the right) from each fraction were separated by SDS-PAGE and electroblotted onto a nitrocellulose membrane. IB4-reactivity (arrow) was visualized using HRP-conjugated IB4 and ECL as the detection system.
Figure 2
Figure 2. Versican transcripts in rat DRG
To analyze for the presence of versican transcripts in rat DRG an RT-PCR for each splice variant was performed. Primer pairs were chosen to determine the presence or absence of a certain splice variant in the DRG [V0: Exon 7_for - Exon 8_rev = 673 bp; V1: Exon 4_for - Exon 8_rev = 925 bp; V2: Exon 7_for -Exon 11_rev = 626 bp; V3: Exon 4_for - Exon 11 _rev = 878 bp; M = Marker]. PCR amplification products were separated on 2% agarose gels and visualized by ethidium bromide intercalation.
Figure 3
Figure 3. Versican mRNAs in rat DRG
In situ hybridization analysis of versican expression in rat L6 DRG. 20 μm thick tissue sections were exposed to digoxigenin (DIG)-labeled riboprobes for the highly conserved N-terminus of versican (exon 4 to exon 6). For detection of hybrids, the sections were incubated with anti-DIG antibody conjugated with alkaline phosphatase and stained through exposure with nitroblue tetrazolium chloride/5-bromo-4-chloro-3-indolyl phosphate, 4-toluidine salt solution.
Figure 4
Figure 4. Co-localization of IB4- and anti-versican immunoreactivity in rat L4 DRG
Dual-labeling immunofluorescence experiments were performed on 20 μm thick cryostat sections. IB4-reactivity (green) was revealed with FITC conjugated IB4 (A), anti-versican immunoreactivity (red) by probing the tissue sections with the mouse monoclonal anti-versican antibody followed by a Cy3-labeled rabbit anti-mouse antibody (B). Subcellular areas in which both immunoreactivities co-localize appear yellow in the merged image (C). Corresponding bright field image captured with differential interference contrast optics (D). Arrows: IB4-binding sensory neurons that also express versican. Arrowheads: Versican immunoreactivity in the extracellular matrix. Scale bar: 50 μm.
Figure 5
Figure 5. Versican V2 is the IB4-binding splice variant
A subcellular fraction of rat spinal cord tissue was treated with hyaluronidase to release hyaluronan-associated proteins such as versican into the supernatant. The extract was then either directly analyzed by Western blotting using anti-GAG alpha or GAG beta specific antibodies (A) or first immunoprecipitated with a monoclonal anti-versican antibody and subsequently analyzed by Western blotting using the anti-GAG alpha selective antibody only (B). A) A hyaluronidase extracted subcellular fraction of rat spinal cord tissue only contains V2. Lane 1: Hyaluronidase extract probed with the anti-GAG alpha antibody; Lane 2: Hyaluronidase extract probed with IB4-HRP; Lane 3: Hyaluronidase extract probed with the anti-GAG beta antibody. B) The IB4-binding molecule is versican V2. Proteins derived from the same subcellular fractions as those that were used to demonstrate the enrichment of the IB4-binding molecule in Figure 1 were separated by SDS-PAGE and analyzed by Western blotting with the anti-GAG alpha specific antibody. See fractionation scheme on the right in Fig. 1 for more detailed information about the different subcellular fractions that were analyzed and the amount of protein that was loaded onto each lane. Arrow: Position of the IB4-binding molecule/anti-GAG alpha immunoreactivity on the Western blot.
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