Richard C. Krueger

CD133+ neural stem cells in the ependyma of mammalian postnatal forebrain

The postnatal forebrain subventricular zone (SVZ) harbors stem cells that give rise to olfactory bulb interneurons throughout life. The identity of stem cells in the adult SVZ has been extensively debated. Although, ependymal cells were once suggested to have stem cell characteristics, subsequent studies have challenged the initial report and postulated that subependymal GFAP+ cells were the stem cells. Here, we report that, in the adult mouse forebrain, immunoreactivity for a neural stem cell marker, prominin-1/CD133, is exclusively localized to the ependyma, although not all ependymal cells are CD133+. Using transplantation and genetic lineage tracing approaches, we demonstrate that CD133+ ependymal cells continuously produce new neurons destined to olfactory bulb. Collectively, our data indicate that, compared with GFAP expressing adult neural stem cells, CD133+ ependymal cells represent an additional—perhaps more quiescent—stem cell population in the mammalian forebrain.

Completion of the mouse aggrecan gene structure and identification of the defect in the cmd-Bc mouse as a near complete deletion of the murine aggrecan gene

Abstract. Mouse cartilage matrix deficiency (cmd), an autosomal recessive phenotype caused by absence of aggrecan, maps to Chromosome (Chr) 7 and is caused by a 7-bp deletion in exon 5 generating a premature stop codon (Watanabe et al. 1994). Another spontaneous mutation with the same locus and phenotype, cmd-Bc, has now been defined as the complete loss of exons 2 to 18, resulting in a significantly shortened mRNA (1.2 kb). The upstream breakpoint is in intron 1, 18.8 kb 3′ of exon 1; the downstream breakpoint lies 10.5 kb past the final aggrecan exon 18. The deletion is flanked by sequences homologous to topoisomerase I and II cleavage sites and a 7-bp direct repeat, suggesting the defect resulted from a nonhomologous recombination event. Additionally, the size of the first intron and the intron-exon structure between exons 12 and 14 were determined, establishing the length of the murine aggrecan gene as 68.6 kb. This report completes the structural analysis of the murine aggrecan gene, defines a second null mutation, and reinforces the importance of aggrecan in development.

THE NANOMELIC MUTATION IN THE AGGRECAN GENE IS EXPRESSED IN CHICK CHONDROCYTES AND NEURONS

We have established the presence of at least two large chondroitin sulfate proteoglycans in the developing chick brain, one that reacts exclusively with HNK‐1, a carbohydrate epitope found on several neural specific molecules, and one that reacts with S103L, a defined peptide epitope in the CS‐2 domain of the cartilage‐specific chondroitin sulfate proteoglycan (CSPG), aggrecan. In order to determine the relationships between the two distinct S103L‐reactive CSPGs from cartilage (chondrocytes) and brain (neurons), as well as among the three large CSPGs expressed in brain, S103L, HNK‐1 and versican, we studied the expression of these multiple proteoglycan species in the brain of nanomelic chicks. We have previously shown that homozygous embryos expressing the nanomelic phenotype exhibit a single point mutation in the aggrecan gene. In the present study, the S103L CSPG is not accumulated or synthesized by embryonic chick CNS tissue or E8CH neuronal cultures derived from nanomelic chick embryo cerebral hemispheres. In contrast, expression of both versican and the HNK‐1 CSPG was normal in the mutant embryo CNS. Pulse chase experiments demonstrated the presence of the 380 kDa precursor in normal neurons and the 300 kDa truncated precursor in nanomelic neurons. Northern blot analysis revealed normal‐sized mRNA but reduced levels of expression of the S103L CSPG message in nanomelic neurons, while expression of the versican message was comparable in normal and nanomelic neurons. Most conclusively, the point mutation previously identified in nanomelic cartilage mRNA was also identified in nanomelic brain mRNA. Together these results provide evidence that a single aggrecan gene is expressed in both cartilage and CNS tissue leading to the production of identical core proteins which then undergo differential and tissue‐specific post‐translation processing, resulting in the characteristic tissue‐specific proteoglycans. Furthermore, versican and the HNK‐1 CSPG, although structurally and chemically similar to the S103L CSPG, are the products of separate genes.

Deglycosylation of chondroitin sulfate proteoglycan by hydrogen fluoride in pyridine.

The original deglycosylation procedure using HF/pyridine has been modified for maximal removal of carbohydrate from chondroitin sulfate proteoglycan, with minimal alteration of the core protein. Gas-liquid chromatography analysis after treatment for various times showed that 95% of xylose and mannose and 70-85% of other sugars were removed within 30 min, indicating that almost all chondroitin sulfate chains and about 80% of N- and O-linked oligosaccharides were removed. In contrast to the loss of carbohydrate, no change in amino acid composition or loss of immunoreactivity occurred. Longer treatment of up to 16 h resulted in little additional removal of carbohydrate, but did cause a significant decrease in solubility and recovery of the deglycosylated product. Optimal removal of xylose residues after about 1 h was also shown by maximal acceptor activity of the product in a xylosyltransferase assay. Rapid removal of the HF reagent by vacuum evacuation and ion-exchange chromatography, coupled with the reduced time of treatment allowed recovery of an intact, homogenous protein core that is amenable to structural and sequence studies.

Isolation of a new member of the ADP-ribosylation like factor gene family, ARL8, from a cartilage cDNA library

ADP-ribosylation factors (ARFs) and ARF-like proteins (ARLs) are part of the ARF family within the RAS superfamily of regulatory GTPases. Guanine nucleotide binding proteins or GTPases are involved in a diverse spectrum of cellular activities, including regulating cell growth and signal transduction, organization of the cytoskeleton and regulating membrane trafficking along the exocytic and endocytic pathways. ARL proteins share 40-60% sequence identity with the ARF proteins, but ARLs can be distinguished from ARFs based on expression patterns and biological functions. We have identified a new ARL, ARL8, from a fetal cartilage cDNA library. ARL8 contains six exons and five introns, and encodes a 179 amino acid protein that shares homology to the other ARL proteins, especially ARL5. It also shows significant homology with orthologous proteins found in Mus musculus and Drosophila melanogaster. The expression pattern of the mouse ortholog revealed differential tissue expression and an alternate transcript was seen in brain that was age-dependent. ARL8 is an additional member of a family of closely related proteins that are conserved both within the family and across species.

Increased Extracellular Magnesium Increases Cellular Viability and Cellular Proliferation in Primary Cell Culture of Embryonic Chick Telencephalon: Mechanisms of Magnesium's Action

Increased Extracellular Magnesium Increases Cellular Viability and Cellular Proliferation in Primary Cell Culture of Embryonic Chick Telencephalon: Mechanisms of Magnesiums Action

CHANGES IN BRAIN AGGRECAN CSPG SYNTHESIS AFTER GLUTAMATE RECEPTOR STIMULATION, HYPOXIA, OR INDUCTION OF APOPTOSIS 1736

Glutamate (GLU) “excitotoxicity” stimulates B-aggrecan chondroitin sulfate proteoglycan (CSPG) synthesis in chick neurons at the level of transcription (Krueger and Schwartz (1995), Ped. Res. 37:381A). We hypothesize that B-aggrecan synthesis may be stimulated as a defense against neuronal injury or death. B-aggrecan synthesis in neurons was measured after exposure to specific GLU ionotropic agonists, to hypoxia induced by cyanide, or to agents which induce apoptosis. E6C5 chick neurons were incubated for 30 min with 50 mcM of either GLU, NMDA, or kainate (KA), or 1mM NaCN, returned to the 35S-sulfate containing media, and analyzed at 24 hours. Alternatively, cells were exposed to either 2 mcM Wortmannin or 200 nM Staurosporin, agents which induce apoptosis in this system, for 24 hours in the presence of label. Viability was determined at 24 hr by MTT assay. B-aggrecan and the HNK-1 CSPG were assayed by immunoprecipitation, and radioactivity assayed. Glycosaminoglyacans (GAG) were quantified by CPC precipitation. GLU, NMDA, and KA exposure increased B-aggrecan synthesis in these cultures (KA>GLU>NMDA; p 70%, despite only 25% neuronal death. B-aggrecan synthesis is upregulated after GLU-receptor stimulation or hypoxia more than GAG or HNK-1 CSPG synthesis. Conversely, B-aggrecan is downregulated in apoptosis more than GAG or HNK-1 CSPG synthesis. Finally, apoptosis turns off B-aggrecan synthesis, whereas GLU-receptor stimulation and hypoxia increase synthesis. These data suggest that it is injury and/or ionotropic activation, not neuronal dying, which stimulate CSPG/B-aggrecan synthesis. These data are consistent with the hypothesis that B-aggrecan may have a special role in CNS protection and/or healing. Supported by HD-09402 and Wyeth Neonatal Research fund.