Deposition of the NG2 proteoglycan at nodes of Ranvier in the peripheral nervous system

Abstract

The node of Ranvier is a complex macromolecular assembly of ion channels and other proteins that is specialized for the rapid propagation of the action potential. A full understanding of the processes responsible for the assembly and maintenance of the node requires first the identification and characterization of the proteins found there. Here we show that NG2, a structurally unique chondroitin sulfate proteoglycan, is a molecular component of the node of Ranvier in the peripheral nervous system. In adult sciatic nerve, NG2 is (1) associated with thin, elongated fibroblast-like cells, (2) on some but not all basal laminae, and (3) at nodes of Ranvier. At the nodes, NG2 is restricted to the nodal gap and is absent from the paranodal or juxtaparanodal region. In dissociated cell cultures of adult sciatic nerve, perineurial fibroblasts but not Schwann cells express NG2 on their surfaces. Approximately 45% of the total NG2 in peripheral nerves is in a soluble, rather than particulate, subcellular compartment. NG2 is also present in membrane fractions that also contain high levels of voltage-dependent sodium channels, caspr, and neuron-glia related cell adhesion molecule. These medium-density membranes likely correspond to the nodal and paranodal region of the axon-Schwann cell unit. These results suggest a model in which perineurial fibroblasts secrete or shed NG2, which subsequently associates with nodes of Ranvier. The growth-inhibitory and anti-adhesive properties of NG2 may limit the lateral extension of myelinating Schwann cells as nodes mature. NG2 may also participate in the barrier functions of the perineurial linings of the nerve.

Fig. 1.

Localization of NG2 in adult sciatic nerve. NG2 was localized in cryostat sections of adult sciatic nerve prepared as described in Materials and Methods. A, Low-power overview of NG2 immunoreactivity in adult nerve. NG2 is associated with the extracellular matrix surrounding the nerve (small-headed arrow), linear cellular elements within the nerve (wide-headed arrow), large blood vessels (large arrowhead), and numerous small puncta (small thin arrows). Scale bar, 100 μm. B, C, High-power views of NG2-positive cells from adult sciatic nerve. These cells, which are stained at their plasma membrane with the anti-NG2 antibodies, have elongated, thin processes that lie between the nerve fibers. D, E, Hoechst 33258-stained nuclei of the cells shown in B and C. Scale bar (E), 50 μm.

Fig. 2.

Comparison of the localization of NG2, MBP, MAG, S100β, and p75 in adult sciatic nerve. Tissue sections were stained as described in Materials and Methods. Each horizontal row shows staining with the individual antibodies and a colorized and digitally merged image showing both fluorochromes.A, Monoclonal anti-NG2; B, rabbit anti-MAG; C, A and Bcolorized and merged. D, Rabbit anti-NG2;E, mouse anti-MBP; F, Dand E colorized and merged. G, Rabbit anti-NG2; H, mouse anti S100β; I,G and H colorized and merged.J, Mouse anti-NG2; K, rabbit anti-p75;L, J and K colorized and merged. Neither the Schwann cells nor the myelin they make are stained with the anti-NG2 antibodies. Scale bar, 20 μm.

Fig. 3.

Lack of colocalization of NG2 and p75 in dissociated cultures of adult rat sciatic nerve. Dissociated cell cultures of rat sciatic nerve were prepared and immunofluorescently stained as described in Materials and Methods. A, Anti-NG2 stain; B, anti-p75 stain. The large, flat NG2-positive cells are p75-negative, whereas the spindle-shaped cells are p75-positive. C, Phase-contrast view of the cultures. D, Anti-NG2 stain; E, anti-thy1.1 stain; F, phase contrast view. The NG2-positive cells are also stained with the anti-Thy1.1 antibodies.G, Anti-p75 stain; H, anti-thy1.1 stain;I, phase contrast view of the culture. The p75-positive Schwann cells do not bind the anti-thy1.1 antibodies. Thearrows in C, F, andI point to spindle-shaped Schwann cells. Scale bar, 20 μm.

Fig. 4.

Comparison of the localization of NG2 with that of the endoneurial extracellular matrix. Sections of adult sciatic nerve were immunofluorescently stained as described in Materials and Methods.A, Anti-NG2 stain; B, anti-laminin B2 stain; C, colorized and digitally merged image ofA and B. The endoneurial matrix is heavily stained with the anti-laminin B2 antibodies but only contains discontinuous and punctate deposits of anti-NG2 immunoreactivity. Scale bar, 10 μm.

Fig. 5.

NG2 is found at nodes of Ranvier. Sections of adult sciatic nerve were stained with the indicated antibodies and digitally merged as described in Materials and Methods. A, B, Rabbit anti-NG2 (green) and mouse anti-sodium channel (red). C, D, Rabbit anti-NG2 (green) and mouse anti-ankyrin G (red). E, F, Mouse anti-NG2 (red) and rabbit anti-NrCaM (green). G, H, Mouse anti-NG2 (red) and rabbit anti-caspr (green). I, J, Mouse anti-NG2 (red) and rabbit anti-p75 (green). NG2 immunoreactivity colocalizes with that of the sodium channel, ankyrin G, and NrCaM but is not found in the paranodal and juxtaparanodal regions that contain caspr. NG2 is also not associated with the Schwann cell microvilli that stain heavily for p75. Scale bar, 5 μm.

Fig. 6.

NG2 is localized at nodes of Ranvier in teased sciatic nerve fibers. Teased sciatic nerve fibers were stained with the indicated antibodies. A, Rabbit anti-NG2;B, mouse anti-sodium channel; C, phase contrast; D, mouse anti-NG2; E, rabbit anti-p75; F, phase contrast. NG2 (arrowhead) is concentrated at nodes of Ranvier, which also stain with the anti-sodium channel and anti-p75 antibodies. Thearrows in E and F point to a p75-positive spindle-shaped cell that is NG2-negative. Scale bar, 10 μm.

Fig. 7.

Biochemical identification of NG2 in sciatic nerve. A, Sciatic nerves and brainstem white matter were homogenized and differentially extracted as described in Materials and Methods. Fifty micrograms of total protein were separated on 6% SDS-polyacrylamide gels, transferred to nitrocellulose, and immunoblotted using rabbit anti-NG2. Top panel, NG2 is a major component present in total soluble extracts of sciatic nerve but only a minor component in soluble extracts of brainstem white matter.S, Soluble extract; P, particulate extract. Bottom panel, Effects of chondroitinase ABC digestion on the electrophoretic behavior of NG2 in total nonionic detergent extracts of either sciatic nerve or brainstem white matter. Chondroitinase digestion increases the apparent amount of the NG2 core protein of 300,000. −, No treatment; +, digestion with chondroitinase ABC. B, NG2 cofractionates with sodium channels. Sciatic nerves were subjected to subcellular fractionation as described in Materials and Methods, and the proteins present in each fraction were identified by Western blotting. The relevant regions of each Western blot are shown, aligned with the other gels. NG2 is detected as a broad band with a molecular weight of 240,000. Voltage-dependent sodium channels also appear heterogeneous, with a molecular weight of 215,000. Two isoforms of NrCam are detected in sciatic nerve, with molecular weights of 135,000 and 100,000. Caspr has an apparent molecular weight of 192,000. For NG2 and NrCaM, 20 μg of total protein was loaded per lane; for caspr, 40 μg of protein was loaded; and for the sodium channel, 35 μg of total protein was loaded. My, Myelin membranes; L, light membranes, i.e., those membranes collected at the 12/22% sucrose interface; M, medium-density membranes, the 22/27% sucrose interface; H, heavy membranes collected at the 27/35% sucrose interface; P, material sedimenting through 35% sucrose; St, protein standards used as positive controls for the Western blots. For NG2, the standard was a total nonionic detergent extract of sciatic nerve; all otherSt lanes were adult rat brain membranes (5–10 μg total protein).