Targeted downregulation of N-acetylgalactosamine 4-sulfate 6-O-sulfotransferase significantly mitigates chondroitin sulfate proteoglycan-mediated inhibition.

Chondroitin sulfate-4,6 (CS-E) glycosaminoglycan (GAG) upregulation in astroglial scars is a major contributor to chondroitin sulfate proteoglycan (CSPG)-mediated inhibition [Gilbert et al. (2005) Mol Cell Neurosci 29:545­558]. However, the role of N-acetylgalactosamine 4-sulfate 6-O-sulfotransferase (GalNAc4S6ST) catalyzed sulfation of CS-E, and its contribution to CSPG-mediated inhibition of CNS regeneration remains to be fully elucidated. Here, we used in situ hybridization to show localized upregulation of GalNAc4S6ST mRNA after CNS injury. Using in vitro spot assays with immobilized CS-E, we demonstrate dose-dependent inhibition of rat embryonic day 18 (E18) cortical neurons. To determine whether selective downregulation of CS-E affected the overall inhibitory character of extracellular matrix produced by reactive astrocytes, single [against (chondroitin 4) sulfotransferase 11 (C4ST1) or GalNAc4S6ST mRNA] or double [against C4ST1 and GalNAc4S6ST mRNA] siRNA treatments were conducted and assayed using quantitative real-time polymerase chain reaction and high-performance liquid chromatography to confirm the specific downregulation of CS-4S GAG (CS-A) and CS-E. Spot and Bonhoeffer stripe assays using astrocyte-conditioned media from siRNA-treated rat astrocytes showed a significant decrease in inhibition of neuronal attachment and neurite extensions when compared with untreated and TGF-treated astrocytes. These findings reveal that selective attenuation of CS-E via siRNA targeting of GalNAc4S6ST significantly mitigates CSPG-mediated inhibition of neurons, potentially offering a novel intervention strategy for CNS injury.

Polyglycylation domain of beta-tubulin maintains axonemal architecture and affects cytokinesis in Tetrahymena.

Polyglycylation occurs through the post-translational addition of a polyglycine peptide to the gamma-carboxyl group of glutamic acids near the C terminus of alpha- and beta-tubulin, and has been found only in cells with axonemes, from protists to humans. In Tetrahymena thermophila, multiple sites of polyglycylation on alpha-tubulin are dispensable. By contrast, mutating similar sites on beta-tubulin has site-specific effects, affecting cell motility and cytokinesis, or resulting in cell death. Here, we address the lethality of a polyglycylation deficiency in T. thermophila using heterokaryons. Cells with a lethal mutation in the polyglycylation domain of beta-tubulin assembled axonemes that lack the central pair, B-subfibres and the transitional zone of outer microtubules (MTs). Furthermore, an arrest in cytokinesis occurred, and was associated with incomplete severing of cortical MTs positioned near the cleavage furrow. Thus, tubulin polyglycylation is required for the maintenance of some stable microtubular organelles that are all known to be polyglycylated in vivo, but its effects on MTs appear to be organelle-specific.

Hypoxia regulates assembly of cilia in suppressors of Tetrahymena lacking an intraflagellar transport subunit gene.

We cloned a Tetrahymena thermophila gene, IFT52, encoding a homolog of the Chlamydomonas intraflagellar transport protein, IFT52. Disruption of IFT52 led to loss of cilia and incomplete cytokinesis, a phenotype indistinguishable from that of mutants lacking kinesin-II, a known ciliary assembly transporter. The cytokinesis failures seem to result from lack of cell movement rather than from direct involvement of ciliary assembly pathway components in cytokinesis. Spontaneous partial suppressors of the IFT52 null mutants occurred, which assembled cilia at high cell density and resorbed cilia at low cell density. The stimulating effect of high cell density on cilia formation is based on the creation of pericellular hypoxia. Thus, at least under certain conditions, ciliary assembly is affected by an extracellular signal and the Ift52p function may be integrated into signaling pathways that regulate ciliogenesis.

Cell context-specific effects of the beta-tubulin glycylation domain on assembly and size of microtubular organelles.

Tubulin glycylation is a posttranslational modification found in cells with cilia or flagella. The ciliate Tetrahymena has glycylation on ciliary and cortical microtubules. We showed previously that mutating three glycylation sites on beta-tubulin produces immotile 9 + 0 axonemes and inhibits cytokinesis. Here, we use an inducible glycylation domain mutation and epitope tagging to evaluate the potential of glycylation-deficient tubulin for assembly and maintenance of microtubular systems. In axonemes, the major defects, including lack of the central pair, occurred during assembly, and newly made cilia were abnormally short. The glycylation domain also was required for maintenance of the length of already assembled cilia. In contrast to the aberrant assembly of cilia, several types of cortical organelles showed an abnormally high number of microtubules in the same mutant cells. Thus, the consequences of deficiency in tubulin glycylation are organelle type specific and lead to either insufficient assembly (cilia) or excessive assembly (basal bodies and cortical microtubules). We suggest that the diverse functions of the beta-tubulin glycylation domain are executed by spatially restricted microtubule-associated proteins.

Cysteine proteinase inhibitors in murine cysticercosis.

We explored the prophylactic efficacies of two novel protease inhibitors in murine cysticercosis. Our results demonstrated a 95% and 80% reduction in parasite burden for mice injected with Z-LLL-FMK and Z-LLY-FMK, respectively. Further studies are merited on the role of cysteine proteinase inhibitors in treatment of cysticercosis.