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Notes: Structural changes in proteoglycans (PGs) were examined during the neuritogenesis of PC12 cells induced by nerve growth factor (NGF). (1) A heparan sulfate (HS) PG and a chondroitin sulfate (CS) PG were synthesized by PC 12 cells, irrespective of the presence of NGF or the duration of culture. PGs released from PC12 cells into the culture medium were mostly CSPGs. (2) In the absence of NGF, the apparent molecular mass of HSPG prepared from PC 12 cells after 3 days of culture was in the range of 90–190 kDa for the intact form (Kav= 0.38 on Sepharose CL-6B), 12 kDa for HS, and 61 kDa for the core protein. In the presence of NGF, these values were 90–190 kDa, 10 kDa, and 51 kDa and 61 kDa, respectively. The intact forms of cell-associated CSPG had apparent molecular mass ranges of 120–150 kDa and 120–190 kDa (Kav= 0.38 and 0.34), with CSs of 15 kDa and 20 kDa in the presence and absence of NGF, respectively. The apparent molecular mass of the core protein of cell-associated CSPG was 92 kDa, irrespective of the presence of NGF. The molecular sizes of cell-associated PGs and their glycosaminoglycans remained unchanged during culture. (3) CSPGs released by PC12 cells into the culture medium were separated into two peaks (I and II) by column chromatography on DEAE-cellulose. The peak II fraction prepared from the medium with NGF after 3 days of culture consisted of CSPG with Kav= 0.22 on Sephacryl S-300 [40–84 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE)]. The CS and core protein of this CSPG had apparent molecular masses of 26 kDa and 15–17 kDa, respectively. The peak I fraction was separated further into three fractions of different molecular sizes on Sephadex G-200 (I-1,I-2,I-3). In the presence of NGF, peak I-1 prepared after 3 days of culture consisted of mostly CSPG (130–170 kDa by SDS-PAGE; Kav= 0.13 on Sephacryl S-300) with CS of 32 kDa and a core protein of 105 kDa. Peaks 1–2 and 1–3 contained, respectively, CSPGs of 38–74 kDa and 31–56 kDa for their intact forms, with CSs of 25 kDa and 15 kDa. The apparent molecular masses of the core proteins of their CSPGs were 15–17 kDa. These CSPGs had similar molecular sizes, irrespective of the presence of NGF or the duration of culture. (4) These results indicate that structural changes in PGs during the neuritogenesis in PC12 cells induced by NGF occurred mostly in cell-associated PGs and preceded elongation of neurites.

Notes: Abstract: PC12D cells, a new subline of conventional PC12 cells, respond not only to nerve growth factor but also to cyclic AMP by extending their neurites. These cells are flat in shape and are similar in appearance to PC12 cells that have been treated with nerve growth factor for a few days. In both cell lines, we have characterized the glycosaminoglycans, the polysaccharide moieties of proteoglycans, which are believed to play an important role in cell adhesion and in cell morphology. Under the present culture conditions, only chondroitin sulfate was detected in the media from PC12 and PC12D cells, whereas both chondroitin sulfate and heparan sulfate were found in the cell layers. The levels of cell-associated heparan sulfate and chondroitin sulfate were about twofold and fourfold higher in PCI 2D cells than in PC12 cells, respectively. Compared to PC12 cells, the amounts of [35S]sulfate incorporated for 48 h into chondroitin sulfate were twofold lower but those into heparan sulfate were 35% higher in PC12D cells. The amount of chondroitin sulfate released by PC12D cells into the medium was about a half of that released by PC12 cells. The ratio of [35S]sulfate-labeled heparan sulfate to chondroitin sulfate was 6.2 in PC12D cells and 2.2 in PC12 cells. These results suggest that there may be some correlation between the increase in content of glycosaminoglycans and the change in cell morphology, which is followed by neurite outgrowth.

Notes: Abstract: Chondroitin sulfate proteoglycan (CS-PG) was purified from rat brain and examined for its effect on neurite outgrowth in primary cultures of embryonic rat neocortical neurons. Neurite outgrowth was increased in culture wells coated with CS-PG. The core protein and glycosaminoglycan (GAG) prepared from the CS-PG were also examined for neurite-promoting activity. The activity was observed in culture wells coated with the core protein but not with GAG. These results suggest that CS-PG stimulates neurite outgrowth from the cultured neurons via its core protein.

Notes: Abstract: Glycosaminoglycans (GAGs) were isolated from the brains of pre- and postnatal rats. The GAG content of the brain, based on the amount of DNA, was constant during the period from day 13 to day 15 of gestation. After day 15, the GAG content began to increase and reached a plateau by 10 days after birth. Hyaluronate (HA) was the main GAG (〉 60% of the total) in the fetal rat brain, and the relative amount of HA decreased after birth. Conversely, the relative amount of chondroitin sulfate increased with development and reached the adult level by 20 days after birth. Heparan sulfate (HS) was the major sulfated GAG in the fetal rat brain at early developmental stages, but HS accounted for approximately 10% of the total GAG in the postnatal brains. In addition to these GAGs, a polysialosyl glycoconjugate was isolated from rapidly growing brains of the rat. These three GAGs could be isolated either from the cerebellum, cerebrum, or brainstem of the newborn rat. A closely similar age-related change in the GAG composition was observed in each of these different regions of the brain. The developmental change could be implicated in morphogenesis or maturation of the brain.

Notes: Abstract: Previously, we had suggested that heparan sulfate (HS) makes some contribution to a flat-shaped morphology of PC12D cells. Therefore, we carried out quantitative and qualitative analyses of glycosaminoglycans (GAGs), the polysaccharide moiety of proteoglycans, during neuritogenesis in PC12 cells that is induced by nerve growth factor (NGF). (a) In PC12 cells, NGF induced a flat-shaped morphology with a few short processes after 3 days of culture, and then it elicited short and long neurites after 6 (in ∼30% of cells) and 9 (in 60–70%) days of culture, respectively, (b) HS and chondroitin sulfate (CS) were detected in the cell layer at all times. Only CS was found in the medium at 3 and 6 days, whereas a low level of HS, in addition to CS, was detectable on day 9. (c) In the NGF-treated cultures, the amounts of cell-associated HS per cell were two to three times as high as those in the respective nontreated cultures at all times, whereas the amount based on phospholipid was about twofold higher after 3 days of culture. (d) The levels of HS labeled with [35S]sulfate during the last 48 h of the culture were 1.5-to twofold higher in the NGF-treated cultures than in the respective controls at any time. (e) The amount of cell-associated CS per cell (or per unit of phospholipid), but not of labeled CS per cell, was transiently enhanced at 3 days in culture with or without NGF. At all times, NGF treatment caused an increase in the levels of total and [35S]sulfate-labeled CS associated with the cells and released into the medium, (f) NGF enhanced the amount of N-sulfation of glucosamine residues of HS at all times, but it did not change the ratio of 4-sulfate units to 6-sulfate units in CS. (g) At 3 days in culture, the uptake of [35S]sulfate by PC12 cells was lower in the NGF-treated culture than in the nontreated control. (h) In chase experiments, the percentage of unrecovered CS was about twofold higher in the NGF-treated culture than in the non-treated control. These results suggest that the enhanced synthetic activity and the accumulation of GAGs as well as the structural change of HS induced by NGF occur preceding the neurite elongation from PC12 cells. Also, it is suggested that the increase in content of HS is closely correlated with the morphological change from round to flat in PC12 cells.

Notes: Abstract: Polysialosyl glycopeptides were coisolated with glycosaminoglycans by Pronase digestion of the whole brains of perinatal rats and could be separated from known glycosaminoglycans by two-dimensional electrophoresis on cellulose acetate film. The polysialosyl glycopeptides could not be obtained from fetal rat brain on day 13 of gestation, but began to be detected on day 14. The amount of polysialosyl glycopeptides was estimated from the dye concentration of the Alcian blue-stained spot in the electrophoreto-gram. The glycopeptide content increased almost linearly, on the basis of brain DNA, up to 10 days after birth. Thereafter, the content decreased rapidly, and hardly any polysialosyl glycopeptides could be isolated from the brain at ∼30 days. This developmental change may be involved in morphogenesis and maturation of the brain. The polysialosyl glycopeptides could be isolated from the cerebellum, from the cerebrum, or from the brainstem of the neonatal rat. However, each region of the brain had a postnatal developmental change in glycopeptide content different from those of the other regions.

Notes: N-syndecan, a membrane-bound heparan sulphate proteoglycan, is abundantly present in the developing nervous system and thought to play important roles in the neurite outgrowth. In the present study, we examined the distribution of N-syndecan in the migratory route from the rat olfactory placode using immunohistochemistry and in situ hybridization. At embryonic day 15, both heparan sulphate and N-syndecan immunoreactivities were localized in and around the migrating cell clusters, which contained luteinizing hormone-releasing hormone (LHRH) and calbindin D-28k. Immunoreactivity for other glycosaminoglycan chains, such as chondroitin and keratan sulphate, and core proteins of the chondroitin sulphate proteoglycan, neurocan and phosphacan, were barely detected in the migratory pathway from the olfactory placode. By in situ hybridization histochemistry, N-syndecan mRNA was localized in virtually all of migrating neurons as well as in cells of the olfactory epithelium and the vomeronasal organ. N-syndecan immunoreactivity surrounded cells migrating along the vomeronasal nerves that were immunoreactive for neural cell adhesion molecules, NCAM, L1 and TAG-1. Considering that NCAM is implicated in the migratory process of LHRH neurons and specifically binds to heparan sulphate, it is likely that a heterophilic interaction between NCAM and N-syndecan participates in the neuronal migration from the rat olfactory placode.

Notes: Neurocan is a developmentally regulated chondroitin sulphate proteoglycan in the rat brain. In the present study, spatiotemporal patterns of expression of neurocan and the corresponding mRNA were examined in the developing cortical barrel field of the rat brain by using a monoclonal antibody that was highly specific to neurocan and a riboprobe for a portion of the mRNA. Immunohistochemical analysis revealed that neurocan was distributed throughout the cerebral cortex during early postnatal development but was excluded from the centres of cortical barrels at the time of entry and arborization of thalamocortical axons. At this developmental stage, expression of neurocan mRNA was shown by in situ hybridization to be down-regulated in the barrel centres. When a row of whisker follicles was laser-cauterized on postnatal day 1, the pattern of expression of neurocan was disturbed in the row of barrels that corresponded to the lesioned whisker follicles in the contralateral somatosensory cortex. From these observations, it appears that neuronal stimuli through early thalamocortical fibres from the sensory periphery cause reduced expression of neurocan mRNA in neurocan-producing cells in the presumptive barrel centres. Our findings also suggest that the pattern of distribution of neurocan in early postnatal barrel fields may be due mainly to the down-regulation of expression of neurocan mRNA.

Notes: We examined the molecular mechanisms underlying the formation of the thalamocortical pathway in the cerebral neocortex of normal and reeler mutant mice. During normal development of the mouse neocortex, thalamic axons immunoreactive for the neural cell adhesion molecule L1 rarely invaded the cortical plate and ran centered in the subplate which is immunoreactive for neurocan, a brain-specific chondroitin sulfate proteoglycan. On the other hand, in homozygous reeler mutant mice, thalamic axons took an aberrant course to run obliquely through the cortical plate. Injection of bromodeoxyuridine at embryonic day 11 specifically labeled subplate neurons in normal mice, whilst in the reeler neocortex it labeled cells scattered in the cortical plate as well as in the superficial layer (superplate). Neurocan immunoreactivity was associated with the bromodeoxyuridine-positive cells in the superplate, as well as being present in oblique bands within the cortical plate, along which L1-bearing thalamic axons preferentially ran. The present results support our previous hypothesis proposed for normal rats that a heterophilic molecular interaction between L1 and neurocan is involved in determining the thalamocortical pathway within the neocortical anlage [T. Fukuda et al. (1997)Journal of Comparative Neurology, 382, 141–152].