Tricellulin is expressed in autotypic tight junctions of peripheral myelinating Schwann cells

Abstract

Autotypic tight junctions are formed by tight junction-like structures in three regions of myelinating Schwann cells, the paranodal loops, Schmidt-Lanterman incisures, and outer/inner mesaxons, and various tight junction molecules, including claudin-19 and junctional adhesion molecule (JAM)-C. Our findings demonstrate the identification and subcellular distribution of a novel tricellular tight junction protein, tricellulin (TRIC), in the autotypic tight junctions of mouse myelinating Schwann cells, compared with the autotypic adherens junction protein E-cadherin and the autotypic tight junction protein JAM-C, which are expressed in the paranodal loops, Schmidt-Lanterman incisures, and mesaxons. In real-time RT-PCR, the expression level of TRIC mRNA was about 10-fold higher in the sciatic nerve than in the spinal cord or cerebrum. In immunostaining, TRIC signals were completely restricted to the peripheral nervous system (PNS) and strongly concentrated at the paranodal loops, Schmidt-Lanterman incisures, and mesaxons of myelinating Schwann cells. In addition, TRIC was expressed in the thin region of the paranode and there was a gap between TRIC and the Na+ channel. Furthermore, TRIC was more distally located from the node than E-cadherin and was colocalized with JAM-C. It is possible that TRIC may be a component to maintain the integrity for PNS myelin function and morphology. This manuscript contains online supplemental material at http://www.jhc.org. Please visit this article online to view these materials.

Figure 1

Tricellulin (TRIC) expression is abundant in peripheral nerves. (A) TRIC mRNA expression levels in mouse sciatic nerve, spinal cord, and cerebrum revealed by RT-PCR. Glyceraldehyde-3-phosphate dehydrogenase is used as an internal control. (B) Quantitative real-time RT-PCR shows expression levels of mRNA in mouse sciatic nerve, spinal cord, and cerebrum as percentage of sciatic nerve (% of SN). (C) The spinal root (peripheral nervous system)–spinal cord (central nervous system) transitional zone is shown by longitudinal sections of mouse dorsal roots at the distal (D) and proximal (P) sides. The left panels show immunoreactivities for TRIC (green), claudin-11 (Cldn-11; green), or myelin protein zero (MPZ; green). The right panels represent merged images with neurofilament proteins (NFP; red) or glial fibrillary acidic protein (GFAP; red) in the same line. N.C., negative control. Bar = 50 μm.

Figure 2

(A) Panels show teased mouse sciatic nerve fibers stained with anti-TRIC antibodies (TRIC) and phase contrast (PhC). The marginal image is shown in bottom (Merge). TRIC immunoreactivities are localized to the paranode (double arrowheads), mesaxon (arrows), and Schmidt–Lanterman incisures (arrowheads). (B) Panels show teased sciatic nerve fibers stained with anti-TRIC (TRIC) and anti-MBP (MBP) antibodies. The bottom panel is marginal image of TRIC and MBP (Merge). TRIC immunoreactivities are localized to mesaxons (arrows) and Schmidt–Lanterman incisures (arrowheads). (C) Transverse sections of adult mouse sciatic nerve fibers are stained with antibody against TRIC (green) and FluoroMyelin Red (red). TRIC immunoreactivities are localized to the outside of myelin sheaths (cytoplasm of Schwann cells), the inner mesaxons (arrows), and paranodal regions or Schmidt–Lanterman incisures (arrowheads). The distinction between the spots of outer mesaxon and cytoplasm of Schwann cells is difficult. Bars: A,B = 50 μm; C = 10 μm.

Figure 3

(A) Paranodal regions of teased mouse sciatic nerves were stained for anti-TRIC (TRIC) and anti-pan Na⁺ channel (NaCh), anti-E-cadherin (E-cad), or anti-junctional adhesion molecule (JAM)-C antibodies. The right panels represent merged images of the left and center panels in the same row (Merge). (B) Schmidt–Lanterman incisures of teased mouse sciatic nerves were stained for anti-TRIC (TRIC) and anti-E-cadherin (E-cad) or anti-JAM-C (JAM-C) antibodies. The right panels represent merged images of the left and center panels in the same row (Merge). Higher magnification images of merged images are presented in the inset in the right panels. Arrowheads indicate Schmidt–Lanterman incisures. Bar = 5 μm.