Impaired axonal regeneration by isolectin B4-binding dorsal root ganglion neurons in vitro

Abstract

The subpopulation of dorsal root ganglion (DRG) neurons recognized by Griffonia simplicifolia isolectin B4 (IB4) differ from other neurons by expressing receptors for glial cell line-derived neurotrophic factor (GDNF) rather than neurotrophins. Additionally, IB4-labeled neurons do not express the laminin receptor, alpha7-integrin (Gardiner et al., 2005), necessary for optimal axonal regeneration in the peripheral nervous system. In cultures of dissociated DRG neurons of adult mice on laminin, robust spontaneous neurite outgrowth from IB4-negative neurons occurs and is strongly enhanced by previous axotomy. In contrast, IB4-labeled neurons show little neurite outgrowth and do not express GAP 43, even after axotomy or culture with GDNF. Moreover, growth of their axons through collagen gels is impaired compared with other DRG neurons. To determine whether the sparse neurite outgrowth of IB4-labeled neurons is attributable to lack of integrin expression, DRG cultures were infected with a herpes simplex 1 vector encoding alpha7-integrin, but its forced expression failed to promote neurite outgrowth in either IB4-labeled or other DRG neurons or in cultured adult retinal ganglion cells. Forced coexpression of both alpha7-integrin and GAP 43 also failed to promote neurite outgrowth in IB4-labeled neurons. In addition, cultured sciatic nerve segments were found to release much lower levels of GDNF, demonstrated by ELISA, than nerve growth factor. These findings together with their impaired intrinsic axonal regeneration capacity may contribute to the known vulnerability of the IB4-labeled population of DRG neurons to peripheral nerve injury.

Figure 1.

BHK cells infected with HSV-1–α7-integrin. Normal BHK cells viewed under fluorescence (A, C) and phase contrast (B, D) after labeling with antibody to α7-integrin. In uninfected cells (A), low background fluorescence is visible, whereas in cultures infected with HSV-1–α7-integrin (C), many cells show strong immunofluorescence. Scale bar, 100 μm.

Figure 2.

Spontaneous neurite outgrowth from IB4-labeled neurons is shorter than that from RT97-labeled neurons. Dissociated DRG cells cultured on laminin for 1 d, labeled with IB4 (B, C, E) or mAb RT97 (A, D) showing neurite outgrowth from normal (A, B), primed (D, E), or GDNF-treated neurons (C). Scale bars, 100 μm (A applies to A, B; C applies to C–E).

Figure 3.

IB4-labeled dissociated DRGs do not express GAP 43. Dissociated cells from normal DRGs cultured on laminin for 1 d labeled with IB4 (A) or antibody to GAP 43 (B). Neurons labeled by IB4 (asterisk) do not express GAP 43, whereas GAP 43-expressing neurons (arrow) are not labeled by IB4. Scale bar, 100 μm.

Figure 4.

IB4-labeled DRGs express SPRR1A after axotomy in vivo. Dissociated cells from normal (A, B) and conditioned (C, D) DRGs cultured on laminin for 1 d labeled with IB4 (A, C) or antibody to SPRR1A. Dissociated neurons from normal DRGs do not express SPRR1A (B), but, in cultures from conditioned DRGs, it is expressed by both IB4-labeled (arrow) and IB4-negative (arrowhead) neurons (D). Scale bar, 100 μm.

Figure 5.

IB4-labeled neurons express lower levels of βIII-tubulin. Dissociated cells from normal DRGs, cultured for 1 d on laminin, labeled using IB4 (A, C), and antibodies to βIII-tubulin (B) or PGP 9.5 (D). In A and B, IB4-labeled neurons show fainter βIII-tubulin immunofluorescence (arrow) than IB4-negative neurons (arrowhead), whereas PGP 9.5 immunofluorescence intensities for IB4-labeled (arrow) and IB4-negative (arrowhead) neurons are similar (C, D). Scale bar, 100 μm.

Figure 6.

Infection of IB4-labeled DRGs with HSV-1–α7-integrin does not enhance axonal regeneration. Dissociated DRG cells cultured on laminin for 3 d after infection with the HSV-1–GFP (A, B, E, F) or HSV-1– α7-integrin (C, D, G, H), labeled green using antibodies to GFP (A, E) or α7-integrin (C, G) or red using an antibody to βIII-tubulin (B, D) or IB4 (F, H). Many neurons expressing GFP (A) or α7-integrin (C) show extensive neurite outgrowth (arrow), as do non-infected neurons visualized by βIII-tubulin immunoreactivity (arrowheads). However, neurons labeled by IB4 (arrowhead in F, H) expressing GFP (E) or α7-integrin (G) seldom show neurite outgrowth. Scale bars, 100 μm.

Figure 7.

Infection of RGCs with HSV-1–α7-integrin does not increase axonal regeneration. Dissociated retinal cells cultured on laminin-2 for 3 d after infection with HSV-1–α7-integrin or HVS-1–GFP, labeled green with antibodies to GFP (A) or α7-integrin (B) and red using an antibody to βIII-tubulin to visualize neurons. E and F show the nuclei of all cells labeled with DAPI. Retinal neurons expressing GFP occasionally extend neurites (arrow), but very few of those expressing α7-integrin (arrowheads) showed neurite outgrowth. Scale bar, 100 μm.

Figure 8.

Forced expression of α7-integrin and GAP 43 fails to promote neurite outgrowth from IB4-labeled neurons. In cultures of DRG cells electroporated with cDNAs encoding α7-integrin, GAP 43 (together with GFP as a marker), GFP-positive neurons labeled by IB4 (arrows in A, B) did not usually show neurite outgrowth. In contrast, GFP-positive neurons not labeled by IB4 (arrowheads in C, D) often showed extensive neurite outgrowth, although neurite lengths did not appear greater in transfected neurons than in GAP 43-positive neurons in normal DRG cultures and were therefore not quantified. Some GFP-negative IB4-labeled neurons (*) without neurites are also visible in D. Scale bar, 50 μm.

Figure 9.

Conditioned medium enhances axonal outgrowth from PN-DRGs. PN-DRGs after culture in collagen gels for 3 d, labeled with an antibody to PGP 9.5 (visualized with HRP-conjugated avidin) taken using dark-ground illumination to make it easier to see fine axons at low magnification. In a control preparation (A) cultured in RPMI medium, only a few axons have grown out of the cut end of the peripheral nerve, whereas extensive axonal outgrowth has occurred from a preparation cultured in medium conditioned by lesioned sciatic nerve segments (B). Scale bar, 200 μm.

Figure 10.

Regeneration of IB4-labeled and GAP 43-positive axons after peripheral nerve crush. PN-DRGs in collagen gels after 3 d in culture with NGF and GDNF, labeled green using antibody to GAP 43 immunoreactivity and red to visualize IB4 binding. In contralateral control preparations (A, B) and preparations conditioned by intercostal nerve crush 3 weeks earlier but trimmed proximal to the original crush site (C, D), numerous GAP 43-positive axons extend from cut ends of the peripheral nerve (A, C). Small numbers of IB4-labeled axons (arrowheads) are also visible (B, D), but these are usually also GAP 43 positive and are invariably much shorter than the GAP 43-positive/IB4-negative axons, indicating impaired growth in collagen gels. In cultured conditioned preparations that include the original crush site and ∼5 mm of distal nerve (E, F), GAP 43-positive axons and IB4-labeled (arrowheads) axons extend from the cut nerve ends into the gels, indicating that both classes of axons have regenerated through the initial crush site, although IB4-labeled axons were invariably shorter than IB4-negative ones. Scale bar, 100 μm.