Ritsuko Katoh-Semba

Brain development and multiple molecular species of proteoglycan

The occurrence of multiple proteoglycan species is a characteristic of the brain. The structural features of individually characterized proteoglycans in the brain are first introduced in brief, then some examples are shown that suggest a relationship between multiple proteoglycans and the many distinct cell types and neural circuits in the brain. Typical experiments demonstrated the neuronal-activity-dependent expression of neural proteoglycans during the critical developmental period of some functional systems such as the visual and vibrissal barrel systems. In addition, the binding properties of neural proteoglycans to other cell surface molecules are discussed in conjunction with their involvement in cell-cell and cell-substratum interactions. This review also covers other potential functions of proteoglycans not only in the development and maintenance of the brain but also in the pathogenesis of Alzheimers disease. Proteoglycans are really coming of age in neuroscience.

Regulation by Androgen of Levels of the β Subunit of Nerve Growth Factor and Its mRNA in Selected Regions of the Mouse Brain

Abstract: Our previous studies showed that the concentration of the β subunit of nerve growth factor (β‐NGF) in nervous tissues is higher in male than in female mice. To identify the brain regions that are affected by androgens, the amounts of β‐NGF protein and its mRNAs were measured in male, female, and castrated male CD‐1 mice and testicular feminization mice at 3–4 months of age. Among tissues examined, the hypophysis of males contained the highest average concentration of β‐NGF protein. In most regions of the brain, individual levels were more variable in males than in females. However, after the castration, such variations in β‐NGF levels disappeared. Average levels of β‐NGF protein in males were higher in the cerebellum (eightfold higher), olfactory bulb (12‐fold higher), hypothalamus (sixfold higher), and hypophysis (72‐fold higher) than thope in corresponding regions of females. No significant differences were observed in levels of β‐NGF protein in the hippocampus, cerebral cortex, striatum, septum, and brainstem. The castration of male mice caused a reduction in levels of β‐NGF protein in the hypothalamus and hypophysis, but not in the cerebellum and olfactory bulb, to the femgle levels. The concentrations of β‐NGF protein in testicular feminization mice were similar to those in female CD‐1 mice in all regions. The concentrations of mRNA for β‐NGF in the olfactory bulb and hypophysis from males were higher than those from females. By contrast, northern blots showed no remarkable differences in the amounts of brain‐derived neurotrophic factor and neurotrophin‐3 between the two sexes. Thus, in some regions of the brain, the production of β‐NGF appears to be regulated by testosterone, but the regulatory mechanisms do not appear to be simple. Our present results indicate that the binding of testosterone to its receptor is an important step in the regulation of the level of β‐NGF in these region.

Phencyclidine-Induced Decrease of Synaptic Connectivity via Inhibition of BDNF Secretion in Cultured Cortical Neurons

Repeated administration of phencyclidine (PCP), a noncompetitive N-methyl-D-aspartate (NMDA) receptor blocker, produces schizophrenia-like behaviors in humans and rodents. Although impairment of synaptic function has been implicated in the effect of PCP, the molecular mechanisms have not yet been elucidated. Considering that brain-derived neurotrophic factor (BDNF) plays an important role in synaptic plasticity, we examined whether exposure to PCP leads to impaired BDNF function in cultured cortical neurons. We found that PCP caused a transient increase in the level of intracellular BDNF within 3 h. Despite the increased intracellular amount of BDNF, activation of Trk receptors and downstream signaling cascades, including MAPK/ERK1/2 and PI3K/Akt pathways, were decreased. The number of synaptic sites and expression of synaptic proteins were decreased 48 h after PCP application without any impact on cell viability. Both electrophysiological and biochemical analyses revealed that PCP diminished glutamatergic neurotransmission. Furthermore, we found that the secretion of BDNF from cortical neurons was suppressed by PCP. We also confirmed that PCP-caused downregulation of Trk signalings and synaptic proteins were restored by exogenous BDNF application. It is possible that impaired secretion of BDNF and subsequent decreases in Trk signaling are responsible for the loss of synaptic connections caused by PCP.

Differential effects of glycosphingolipids on protein kinase C activity in PC12D pheochromocytoma cells

Previous studies have shown that certain glycosphingolipids may function as modulators of protein kinase C (PKC) activity. To study the structure-activity relationship, we examined the effects of 17 gangliosides, 10 neutral glycolipids, as well as sulfatide, psychosine and ceramide on PKC activity in PC12D cells. Using an in vitro assay system, we found that all but one (GQ1b) ganglioside inhibited PKC activity at concentrations between 25 and 100 &mgr;M, and the potency was proportional to the number of sialic acid residues. However, at lower concentrations several gangliosides, including GM1 and LM1 behaved as mild activators of PKC activity. GQ1b had no effect within the range 0.1-10 &mgr;M, but acted as a mild activator of PKC activity at 25 &mgr;M. On the other hand, fucosyl-GM1 and GM1 containing blood group B determinant, which are abundant in PC12 cells, were potent inhibitors of PKC activity. Among the neutral glycosphingolipids tested, LacCer, Gb3, GalGb3, and GA1, all of which have a terminal galactose residue, were found to be ineffective or acted as mild activators of PKC activity. In contrast, GA2, Gb4 and Gb5 which have a terminal N-acetylgalactosamine residue, were potent inhibitors of the PKC activity. Thus, the terminal sugar residue may play a pivotal role in determining the effect of glycosphingolipids in modulating PKC activity. In addition, we also found that GalCer containing normal fatty acids acted as potent activators of PKC activity. Ceramide and GlcCer appeared to be ineffective in modulating PKC activity, wheras psychosine and sulfatides appeared to be inhibitory. We conclude that the carbohydrate head groups and the hydrophobic groups of gangliosides and neutral glycolipids may modulate the PKC system in unique manners, which may in turn affect various biological processes in the cell. Copyright 1994 S. Karger AG, Basel

Interaction of bilirubin with brain capillaries and its toxicity

The interaction of bilirubin with isolated brain capillaries, and the effect of bilirubin on the uptake of 2-deoxyglucose by the capillaries were investigated with 1-month-old Sprague-Dawley rats. The binding of bilirubin to the brain capillaries was observed only at a molar ratio of bilirubin to bovine serum albumin higher than 1.0. An absorption spectrum with a microspectrophotometer of the bilirubin-capillary complex showed a broad absorption maximum from 425 to 440 nm with a shoulder near 490 nm, but no shoulder was observed in the case of the bilirubin emulsion. The bilirubin binding activity was dependent on pH and temperature of the medium, but was not affected by sulfhydryl blocking agents such as p-chloromercuribenzoate and N-ethylmaleimide. Bilirubin saturation kinetics gave an apparent Km for bilirubin of 61.7 microM. Release of bilirubin from the brain capillaries to the medium was observed at 37 degrees C but not at 4 degrees C. The uptake of 2-deoxyglucose by the isolated brain capillaries was inhibited by bilirubin in a noncompetitive manner, giving an apparent Ki for the pigment of 137 microM. These results suggest that bilirubin may be responsible for the decreased 2-deoxyglucose uptake in the brain capillaries by disturbing the membrane structure of the capillary endothelial cells.

Effect of Irradiation on the Erythrocyte Membranes of Homozygous Gunn Rat Sucklings

Erythrocyte membranes were isolated from irradiated Gunns strain of jaundiced rat sucklings, and photodynamic action of bilirubin in vivo was investigated using sodium dodecyl sulfate-polyacrylamide-gel electrophoresis. The effectiveness of irradiation was verified in terms of decrease of plasma bilirubin concentrations and cerebellar bilirubin contents, and also by recovery from the cellbellar hypoplasia, a characteristic feature of the homozygotes (j/j). The cross-linking of erythrocyte membrane proteins by photodynamic action of bilirubin was demonstrated in vitro in rat erythrocytes as reported for human erythrocytes by Girotti (Biochemistry 14: 3377, 1975). The phenomenon, however, was not observed in vivo. Instead, a protein band with a mol. wt. of 32,000 was increased by the irradiation both in the homozygous (j/j) and heterozygous (j/+) Gunn rats.

Effect of Nerve Growth Factor and Forskolin on Glycosyltransferase Activities and Expression of a Globo‐Series Glycosphingolipid in PC12D Pheochromocytoma Cells

Abstract: The glycosphingolipid (GSL) composition of cells changes dramatically during cellular differentiation. Nerve growth factor (NGF) or forskolin (FRK) are known to induce cellular differentiation including process formation in PC12 pheochromocytoma cells. In this respect, we present the NGF/FRK‐dependent regulation of glycosyltransferase activities and the corresponding GSL expression in PC12D cells. After treatment of PC12D cells with NGF or FRK, the cell processes, including varicoses and growth cones, became strongly immunoreactive with an antibody against a unique globo‐series neutral GSL, Galα1‐3Galα1‐4Galβ1‐4Glcβ1‐1′Cer (GalGb3), and the activity of GalGb3‐synthase increased significantly. Other glycosyltransferase activities, including GM1 containing blood group B determinant (BGM1)‐, GM3‐, GD1a‐, and GM2‐synthases, also increased significantly upon NGF treatment, but the immunoreactivity against BGM1 did not show any appreciable change. For the parent PC12 cells, NGF/FRK treatment significantly increased the percentage of anti‐GalGb3 positive cells and induced some immunoreactive cell processes. Because the parent PC12 cells do not express appreciable amounts of GalGb3, and because PC12D cells are considered to be more differentiated than the parent PC12 cells, the expression of GalGb3 and the increase of GalGb3‐synthase activity may be closely related to the cellular differentiation process in this cell line.

Activation of cultured astrocytes by amphotericin B: stimulation of NO and cytokines production and changes in neurotrophic factors production.

Amphotericin B (AmB) is a polyene antibiotic and reported to be one of a few reagents having therapeutic effects on prion diseases, such as the delay in the appearing of the clinical signs and the prolongation of the survival time. In prion diseases, glial cells have been suggested to play important roles by proliferating and producing various factors such as nitric oxide, proinflammatory cytokines, and neurotrophic factors. However, the therapeutic mechanism of AmB on prion diseases remains elusive. We have previously reported that AmB changed the expression of neurotoxic and neurotrophic factors in microglia (Motoyoshi et al., 2008, Neurochem. Int. 52, 1290-1296). In the present study, we examined the effects of AmB on cellular functions of rat cultured astrocytes. We found that AmB could activate astrocytes to produce nitric oxide via inducible nitric oxide synthase induction. AmB also induced mRNA expression of interleukin-1β and tumor necrosis factor-α, and productions of their proteins in astrocytes. Moreover, AmB changed levels of neurotrophic factor mRNAs and proteins. Among three neurotrophic factors examined here, neurotrophin-3 mRNA expression and its protein production in the cells were down-regulated by AmB stimulation. On the other hand, AmB significantly enhanced the amounts of glial cell line-derived neurotrophic factor and brain-derived neurotrophic factor proteins in the cells and the medium. These results suggest that AmB might show therapeutic effects on prion diseases by controlling the expression and production of such mediators in astrocytes.

An acceleration of age-related increases in levels of the β-subunit of nerve growth factor in selected tissues from senescence-accelerated mice (SAM-P/8)

An investigation was made of age-related changes in levels of the β-subunit of nerve growth factor (β-NGF) in selected tissues and of testosterone in serum in senescence-accelerated mice (SAM-P/8) and in the control mice (senesence-resistant mice; SAM-R/1). The concentrations of testosterone in serum were higher in SAM-P/8 than in SAM-R/1 at ages 2 and 4 mo. The level of β-NGF in the thymus from SAM-R/1 increased with age, resulting in a statistically significant difference in its level between mice at ages 2 and 12 mo. By contrast, there was a transient increase in SAM-P/8 at around age 4 mo with a subsequent decrease. Consequently, significant differences were apparent in levels of β-NGF between the two types of mouse at ages 2 and 4 mo. Similar results were obtained in the adrenal gland and testis. Compared to SAM-R/1 at age 2 mo, the average concentrations of β-NGF in the hypophysis were higher in SAM-R/1 at ages 4 and 8 mo and in SAM-P/8 at all ages. In other tissues tested, no remarkable differences were detected. Our present results indicate that, in SAM-P/8, the elevation in levels of β-NGF in the thymus, adrenal gland, testis, and hypophysis occurs in the early period of life compared to the control mice. Possible dysfunction of the disorder of hypophysis is discussed.