In this study, the authors show that NgR1 and NgR3 can act as functional receptors for chondroitin sulfate proteoglycans (CSPGs), mediating inhibition of axonal growth and regeneration. This suggests a convergent mechanism for CSPG- and myelin-associated inhibitor activities after axonal injury in the CNS. In the adult mammalian CNS, chondroitin sulfate proteoglycans (CSPGs) and myelin-associated inhibitors (MAIs) stabilize neuronal structure and restrict compensatory sprouting following injury. The Nogo receptor family members NgR1 and NgR2 bind to MAIs and have been implicated in neuronal inhibition. We found that NgR1 and NgR3 bind with high affinity to the glycosaminoglycan moiety of proteoglycans and participate in CSPG inhibition in cultured neurons. Nogo receptor triple mutants (Ngr1−/−; Ngr2−/−; Ngr3−/−; which are also known as Rtn4r, Rtn4rl2 and Rtn4rl1, respectively), but not single mutants, showed enhanced axonal regeneration following retro-orbital optic nerve crush injury. The combined loss of Ngr1 and Ngr3 (Ngr1−/−; Ngr3−/−), but not Ngr1 and Ngr2 (Ngr1−/−; Ngr2−/−), was sufficient to mimic the triple mutant regeneration phenotype. Regeneration in Ngr1−/−; Ngr3−/− mice was further enhanced by simultaneous ablation of Rptpσ (also known as Ptprs), a known CSPG receptor. Collectively, our results identify NgR1 and NgR3 as CSPG receptors, suggest that there is functional redundancy among CSPG receptors, and provide evidence for shared mechanisms of MAI and CSPG inhibition.

Camptothecin is an S-phase-specific anticancer agent that inhibits the activity of the enzyme DNA topoisomerase-I (topo-I). Irreversible DNA double-strand breaks are produced during DNA synthesis in the presence of camptothecin, suggesting that this agent should not be toxic to nondividing cells, such as neurons. Unexpectedly, camptothecin induced significant, dose-dependent cell death of postmitotic rat cortical neurons in vitro; astrocytes were more resistant. Aphidicolin, an inhibitor of DNA polymerase alpha, did not prevent camptothecin-induced neuronal death, while death was prevented by actinomycin D and 5,6-dichloro-1-beta-D-ribofuranosyl benzimidazole as well as cycloheximide and anisomycin, inhibitors of RNA and protein synthesis, respectively. Camptothecin-induced neuronal death was apoptotic, as characterized by chromatin condensation, cytoplasmic shrinking, plasma membrane blebbing, and fragmentation of neurites. DNA fragmentation was also confirmed by the use of the in situ DNA end labeling assay. In addition, aurintricarboxylic acid, an inhibitor of the apoptotic endonuclease, partially protected against camptothecin-induced neuronal death. The toxicity of stereoisomers of a camptothecin analogue was stereospecific, demonstrating that toxicity was a result of inhibition of topo-I. The difference in sensitivity to camptothecin between neurons and astrocytes correlated with their transcriptional activity and level of topo-I protein expression. These data indicate important roles for topo-I in postmitotic neurons and suggest that topo-I inhibitors can induce apoptosis independent of DNA synthesis. We suggest a model based on transcriptionally mediated DNA damage, a novel mechanism of action of topo-I poisons.

Glycosaminoglycan (GAG) side chains endow extracellular matrix proteoglycans with diversity and complexity based upon the length, composition and charge distribution of the polysaccharide chain. Using cultured primary neurons, we show that specific sulfation in the GAG chains of chondroitin sulfate mediates neuronal guidance cues and axonal growth inhibition. Chondroitin-4-sulfate (CS-A), but not chondroitin-6-sulfate (CS-C), exhibits a strong negative guidance cue to mouse cerebellar granule neurons. Enzymatic and gene-based manipulations of 4-sulfation in the GAG side chains alter their ability to direct growing axons. Furthermore, 4-sulfated chondroitin sulfate GAG chains are rapidly and significantly increased in regions that do not support axonal regeneration proximal to spinal cord lesions in mice. Thus, our findings show that specific sulfation along the carbohydrate backbone carries instructions to regulate neuronal function.

Previous studies have demonstrated that G1/S cell cycle blockers and inhibitors of cyclin-dependent kinases (CDKs) prevent the death of nerve growth factor (NGF)-deprived PC12 cells and sympathetic neurons, suggesting that proteins normally involved in the cell cycle may also serve to regulate neuronal apoptosis. Past findings additionally demonstrate that DNA-damaging agents, such as the DNA topoisomerase (topo-I) inhibitor camptothecin, also induce neuronal apoptosis. In the present study, we show that camptothecin-induced apoptosis of PC12 cells, sympathetic neurons, and cerebral cortical neurons is suppressed by the G1/S blockers deferoxamine and mimosine, as well as by the CDK-inhibitors flavopiridol and olomoucine. In each case, the IC₅₀values were similar to those reported for inhibition of death induced by NGF-deprivation. In contrast, other agents that arrest DNA synthesis, such as aphidicolin andN-acetylcysteine, failed to block death. This suggests that the inhibition of DNA synthesis per se is insufficient to provide protection from camptothecin. We find additionally that the cysteine aspartase family protease inhibitor zVAD-fmk inhibits apoptosis evoked by NGF-deprivation but not camptothecin treatment. Thus, despite their shared sensitivity to G1/S blockers and CDK inhibitors, the apoptotic pathways triggered by these two causes of death diverge at the level of the cysteine aspartase. In summary, neuronal apoptosis induced by the DNA-damaging agent camptothecin appears to involve signaling pathways that normally control the cell cycle. The consequent death signals of such deregulation, however, are different from those that result from trophic factor deprivation.

Astrocytes, oligodendrocytes, and oligodendrocyte/type 2 astrocyte progenitors (O2A cells) can all produce molecules that inhibit axon regeneration. We have shown previously that inhibition of axon growth by astrocytes involves proteoglycans. To identify inhibitory mechanisms, we created astrocyte cell lines that are permissive or nonpermissive and showed that nonpermissive cells produce inhibitory chondroitin sulfate proteoglycans (CS-PGs). We have now tested these cell lines for the production and inhibitory function of known large CS-PGs. The most inhibitory line, Neu7, produces three CS-PGs in much greater amounts than the other cell lines: NG2, versican, and the CS-56 antigen. The contribution of NG2 to inhibition by the cells was tested using a function-blocking antibody. This allowed increased growth of dorsal root ganglion (DRG) axons over Neu7 cells and matrix and greatly increased the proportion of cortical axons able to cross from permissive A7 cells onto inhibitory Neu7 cells; CS-56 antibody had a similar effect. Inhibitory fractions of conditioned medium contained NG2 coupled to CS glycosaminoglycan chains, whereas noninhibitory fractions contained NG2 without CS chains. Enzyme preparations that facilitated axon growth in Neu7 cultures were shown to either degrade the NG2 core protein or remove CS chains. Versican is present as patches on Neu7 monolayers, but DRG axons do not avoid these patches. Therefore, NG2 appears to be the major axon-inhibitory factor made by Neu7 astrocytes. In the CNS, NG2 is expressed by O2A cells, which react rapidly after injury to produce a dense NG2-rich network, and by some reactive astrocytes. Our results suggest that NG2 may be a major obstacle to axon regeneration.