Cultured astrocytes express biglycan, a chondroitin/dermatan sulfate proteoglycan supporting the survival of neocortical neurons.
Astrocyte-conditioned medium (ACM) supports the survival of rat E15 neocortical neurons. Using a microtiter assay for neuronal survival, we demonstrated that part of the survival activity is associated with a proteoglycan fraction obtained after two chromatographic steps: (1) preparative Q-Sepharose anion-exchange chromatography under non-denaturating conditions and (2) MonoQ chromatography in the presence of 8 M urea. Analytical SDS-polyacrylamide gradient gel electrophoresis of pooled active MonoQ-fractions (MQ-pool) revealed a broad proteoglycan band migrating with an apparent M(r) in the range of 150-400 kDa. Digestion of the MQ-pool with chondroitin-ABC-lyase yielded a major core protein of 50 kDa. In Western blots the high molecular weight (150-400 kDa) material as well as the 50 kDa core protein band were immunoreactive to chicken polyclonal antibodies raised against purified biglycan from rat meningeal fibroblasts. Northern blot analysis of total RNA prepared from highly enriched astrocyte cultures revealed a single 2.9 kb biglycan transcript. By using in situ hybridization we demonstrated that essentially all cells in these cultures expressed biglycan mRNA. Furthermore, highly purified biglycan from bovine cartilage was shown to markedly enhance survival of rat neocortical neurons. In conclusion, we have shown that astrocytes synthesize and release the small chondroitin/dermatan sulfate proteoglycan (CS/DSPG) biglycan, a molecule that was found to support survival of neocortical neurons in vitro.
Koops
A
A
Department of Neurology, Heinrich Heine University at Düsseldorf, Germany.
Kappler
J
J
Junghans
U
U
Kuhn
G
G
Kresse
H
H
Müller
H W
HW
D000818:Animals; D000831:Animals, Newborn; D000918:Antibody Specificity; D001253:Astrocytes / Q000378:metabolism*; D058578:Biglycan; D015153:Blotting, Western; D002417:Cattle; D002470:Cell Survival; D002478:Cells, Cultured; D002645:Chickens; D002851:Chromatography, High Pressure Liquid; D002852:Chromatography, Ion Exchange; D017077:Culture Media, Conditioned / Q000494:pharmacology; D004591:Electrophoresis, Polyacrylamide Gel; D016326:Extracellular Matrix Proteins; D017403:In Situ Hybridization; D008578:Meninges / Q000166:cytology; D009419:Nerve Tissue Proteins / Q000096:biosynthesis* / Q000235:genetics / Q000276:immunology / Q000302:isolation & purification / Q000494:pharmacology; D009474:Neurons / Q000166:cytology* / Q000187:drug effects; D016133:Polymerase Chain Reaction; D011489:Protein Denaturation; D011509:Proteoglycans / Q000096:biosynthesis* / Q000235:genetics / Q000276:immunology / Q000302:isolation & purification / Q000494:pharmacology; D012333:RNA, Messenger / Q000096:biosynthesis; D051381:Rats; D017208:Rats, Wistar
C546886:Bgn protein, rat; D058578:Biglycan; D017077:Culture Media, Conditioned; D016326:Extracellular Matrix Proteins; D009419:Nerve Tissue Proteins; D011509:Proteoglycans; D012333:RNA, Messenger