Dexamethasone administration to the neonatal rat results in neurological dysfunction at the juvenile stage even at low doses.

Dexamethasone (DEX), a synthetic glucocorticoid, has been widely used to prevent the development of a variety of poor health conditions in premature infants including chronic lung disease, inflammation, circulatory failure, and shock. Although there are some reports of neurologic complications related to DEX exposure, its full effects on the premature brain have not been examined in detail. To investigate the effects of DEX on neural development, we first administered low doses (0.2 mg/kg bodyweight or less) of the glucocorticoid to neonatal rats on a daily basis during the first postnatal week and examined subsequent behavioral alterations at the juvenile stage. DEX-treated rats exhibited not only a significant reduction in both somatic and brain weights but also learning disabilities as revealed in the shuttle avoidance test. The hippocampi of DEX-treated rats displayed a high apoptotic and a low mitotic cell density compared to control rats on day 7 after birth. In a subsequent experiment, neural stem/progenitor cells were cultured in the presence of DEX for 6 days. The glucocorticoid inhibited cell growth without an increase in cell death. These results suggest that administration of DEX to premature infants induces neurological dysfunction via inhibition of the proliferation of neural stem/progenitor cells.

A highly-sulfated chondroitin sulfate, CS-E, adsorbs specifically to neurons with nuclear condensation.

A highly sulfated chondroitin sulfate, CS-E, prevents excitatory amino acid-induced neuronal cell death by an as yet unknown mechanism. To reveal this mechanism, we pretreated neurons in culture with various inhibitors, and examined whether N-methyl-D-aspartic acid (NMDA)-induced neuronal cell death was reduced in the presence of CS-E. The inhibitors of protein kinase C (PKC) and Ca(2+)/calmodulin-dependent protein kinase II (CaMKII) ameliorated NMDA-induced neuronal cell death, but did not affect the neuroprotective activity of CS-E. Among the growth factors with which CS-E can interact, high concentration of BDNF protected against the NMDA-induced neuronal cell death and strengthened neuroprotection by CS-E. CS-E, but neither CS-A nor CS-C, adsorbed to a subclass of neurons with nuclear condensation, namely pyknosis. Contactin-1 (CNTN-1), a putative receptor for neuritogenic activity of CS-E, was present in cortical neurons, but a neutralizing antibody to CNTN-1 did not block neuroprotective activity of CS-E. The results suggest that CS-E may prevent the progression of cell death at the early stages of excitotoxicity through a signaling pathway different from CNTN-1.

Differential gene expression of multiple chondroitin sulfate modification enzymes among neural stem cells, neurons and astrocytes.

Chondroitin sulfate/dermatan sulfate (CS/DS) polysaccharides have been reported to play a crucial role in the proliferation and maintenance of neural stem cells (NSCs). However, little is known about the structural changes and functional role of CS/DS chains in the differentiation of NSCs. Western blots of NSCs, neurons and astrocytes in culture, with three CS-polysaccharide antibodies of different specificities, revealed marked differences in CS structure among the three cell types. To confirm this finding, we measured gene expression levels of CS sulfotransferases and C5-epimerase in these cell types, as these are responsible for producing the high structural diversity of CS/DS. Expressions of chondroitin 4-O-sulfotransferase, chondroitin 6-O-sulfotransferase, and N-acetylgalactosamine 4-sulfate 6-O-sulfotransferase mRNAs were low in cultures of differentiated neural cells, such as neurons and astrocytes, in comparison to NSCs. In contrast, expressions of uronyl 2-O-sulfotransferase and C5-epimerase mRNAs were higher in the differentiated neural cells than NSCs. Thus, we first provide evidence to support the hypothesis that CS/DS undergoes structural changes during NSC differentiation. The structural changes in CS/DS may be implicated in the regulation of NSC differentiation through interactions with growth/neurotrophic factors and cytokines.

Neuroglycan C, a brain-specific chondroitin sulfate proteoglycan, interacts with pleiotrophin, a heparin-binding growth factor.

Neuroglycan C (NGC) is a transmembrane-type chondroitin sulfate proteoglycan that promotes neurite outgrowth. To identify the ligand of NGC, we applied a detergent-solubilized membrane fraction of fetal rat brains to an NGC-immobilized affinity column. Several proteins were eluted from the column including an 18 kDa-band protein recognized by an anti-pleiotrophin antibody. The binding of pleiotrophin (PTN) to NGC was confirmed by a quartz crystal microbalance method and had a Kd of 8.7 nM. PTN bound to the acidic amino acid cluster of the NGC extracellular domain. In addition, PTN bound to both chondroitin sulfate-bearing NGC and chondroitinase-treated NGC prepared from the neonatal rat brain. These results suggest that NGC interacts with PTN.

Chondroitin sulfate, a major niche substance of neural stem cells, and cell transplantation therapy of neurodegeneration combined with niche modification.

Chondroitin sulfate (CS), a polysaccharide moiety of proteoglycans, is one of the major components of the extracellular matrix in the central nervous system and is involved in various cellular events in the formation and maintenance of the neural network. In the developing brain, CS in the milieu of neural stem/progenitor cells (NSPCs) is believed to participate in the regulation of their functions such as proliferation and differentiation. NSPCs are expected to act as a potent cell type in cell replacement therapy for neurodegeneration in various neurological diseases. Recently, it has been shown that transplantation of NSPCs combined with removal of extracellular CS from the host nervous tissues gives a satisfactory outcome in some animal models of nervous tissue injuries including neonatal hypoxic-ischemic injury and adult spinal cord injury. The combination of cell transplantation with modification of the extracellular matrix of the host tissue could be a novel strategy for the treatment of incurable neurodegenerative diseases.

Behavioral abnormalities of fetal growth retardation model rats with reduced amounts of brain proteoglycans.

Fetal growth retardation (FGR) is a critical problem in the neonatal period, because a substantial population of infants born with FGR go on to develop various developmental disorders. In the present study, we produced FGR model rats by continuous administration of a synthetic thromboxane A2 analogue (STA2) to pregnant rats. The FGR pups exhibited a significant delay in postnatal neurological development. Moreover, behavioral analyses revealed the presence of a learning disability in juvenile FGR male rats. To investigate the mechanism underlying the neurological disorders, histological and biochemical analyses of the brain of FGR rats were performed. The density of neurons in the cortical plate of an FGR brain was low compared with the brains of a similarly aged, healthy rat. Consistent with this finding, the density of TUNEL-positive cells was higher in the cortical plate of FGR brains. Western blot analyses showed that the levels of three brain-specific chondroitin sulfate proteoglycans (CSPGs), neurocan, phosphacan, and neuroglycan C, were all significantly reduced in the brain of neonatal FGR rats compared with those of the control. The reduction of CSPG-levels and morphological changes in the brain may be relevant to neurological dysfunction in FGR.

Expression of chondroitin sulfate proteoglycans in barrel field of mouse and rat somatosensory cortex.

Chondroitin sulfate proteoglycans (CSPGs) consist of chondroitin sulfate (CS) glycosaminoglycans (GAGs) and core protein and regulate the migration, axonal outgrowth, and synaptogenesis in mammalian brains. In the present study, we investigated the localization of CSPGs, the effects of sensory deprivation on the density of perineuronal nets (PNNs), and the effects of chondroitinase ABC (Chase) on the formation of barrel structures in the posterior medial barrel subfield (PMBSF). In developing mouse and rat brains, the immunoreactivity of chondroitin-6-sulfate containing proteoglycan (CS-6-PG), phosphacan, and neurocan was stronger at barrel septa as compared with barrel hollows and surrounding cortex, while the labeling of Wisteria floribunda agglutinin (WFA) was observed at barrel hollows. In adult brains, CS-6-PG-immunoreactive and WFA-labeled PNNs were observed mainly at barrel hollows of mouse, but they were seen chiefly at barrel septa of rats. Sensory deprivation of facial vibrissae reduced the number of WFA-labeled PNNs at barrel hollows but not at barrel septa. Intracerebral injection of Chase did not affect the formation of barrel structures in the PMBSF. These data indicate species-dependent heterogeneity of CSPG expression and activity-dependent formation of PNNs in the PMBSF, but CS GAGs have no crucial function in constructing the barrel structures during early postnatal development.

Differential neuroglycan C expression during retinal degeneration in Rpe65-/- mice.

PURPOSE: An increased mRNA expression of the genes coding for the extracellular matrix proteins neuroglycan C (NGC), interphotoreceptor matrix proteoglycan 2 (IMPG2), and CD44 antigen (CD44) has been observed during retinal degeneration in mice with a targeted disruption of the Rpe65 gene (Rpe65-/- mouse). To validate these data, we analyzed this differential expression in more detail by characterizing retinal NGC mRNA isoform and protein expression during disease progression. METHODS: Retinas from C57/Bl6 wild-type and Rpe65-/- mice, ranging 2 to 18 months of age, were used. NGC, IMPG2, and CD44 mRNA expression was assessed by oligonucleotide microarray, quantitative PCR, and in situ hybridization. Retinal NGC protein expression was analyzed by western blot and immunohistochemistry. RESULTS: As measured by quantitative PCR, mRNA expression of NGC and CD44 was induced by about 2 fold to 3 fold at all time points in Rpe65-/- retinas, whereas initially 4 fold elevated IMPG2 mRNA levels progressively declined. NGC and IMPG2 mRNAs were expressed in the ganglion cell layer, the inner nuclear layer, and at the outer limiting membrane. NGC mRNA was also detected in retinal pigment epithelium cells (RPE), where its mRNA expression was not induced during retinal degeneration. NGC-I was the major isoform detected in the retina and the RPE, whereas NGC-III was barely detected and NGC-II could not be assessed. NGC protein expression was at its highest levels on the apical membrane of the RPE. NGC protein levels were induced in retinas from 2- and 4-month-old Rpe65-/- mice, and an increased amount of the activity-cleaved NGC ectodomain containing an epidermal growth factor (EGF)-like domain was detected. CONCLUSIONS: During retinal degeneration in Rpe65-/- mice, NGC expression is induced in the neural retina, but not in the RPE, where NGC is expressed at highest levels.

Reduction of brain injury in neonatal hypoxic-ischemic rats by intracerebroventricular injection of neural stem/progenitor cells together with chondroitinase ABC.

Perinatal hypoxia-ischemia (HI) remains a critical issue. Cell transplantation therapy could be a potent treatment for many neurodegenerative diseases, but limited works on this kind of therapy have been reported for perinatal HI. In this study, the therapeutic effect of transplantation with neural stem/ progenitor cells (NSPCs) and chondrotinase ABC (ChABC) in a neonatal HI rat model is evaluated. Histological studies showed that the unaffected area of the brain in animals treated with NSPCs together with ChABC was significantly larger than that in the animals treated with vehicle or NSPCs alone. The wet weight of the brain that received the combined treatment was also significantly higher than those of the vehicle and their individual treatments. These results indicate that intracerebroventricular injection of NSPCs with ChABC reduces brain injury in a rat neonatal HI model.

BMP signaling through BMPRIA in astrocytes is essential for proper cerebral angiogenesis and formation of the blood-brain-barrier.

Bone morphogenetic protein (BMP) signaling is involved in differentiation of neural precursor cells into astrocytes, but its contribution to angiogenesis is not well characterized. This study examines the role of BMP signaling through BMP type IA receptor (BMPRIA) in early neural development using a conditional knockout mouse model, in which Bmpr1a is selectively disrupted in telencephalic neural stem cells. The conditional mutant mice show a significant increase in the number of cerebral blood vessels and the level of vascular endothelial growth factor (VEGF) is significantly upregulated in the mutant astrocytes. The mutant mice also show leakage of immunoglobulin around cerebral microvessels in neonatal mice, suggesting a defect in formation of the blood-brain-barrier. In addition, astrocytic endfeet fail to encircle cortical blood vessels in the mutant mice. These results suggest that BMPRIA signaling in astrocytes regulates the expression of VEGF for proper cerebrovascular angiogenesis and has a role on in the formation of the blood-brain-barrier.

Reduced expression of MAb6B4 epitopes on chondroitin sulfate proteoglycan aggrecan in perineuronal nets from cerebral cortices of SAMP10 mice: a model for age-dependent neurodegeneration.

The accelerated senescence-prone SAMP10 mouse strain is a model for age-dependent neurodegeneration and is characterized by brain atrophy and deficits in learning and memory. Because perineuronal nets play an important role in the synaptic plasticity of adult brains, we examined the distributions of molecules that constitute perineuronal nets in SAMP10 mouse brain samples and compared them with those in control SAMR1 mouse samples. Proteoglycan-related monoclonal antibody 6B4 (MAb6B4) clearly immunostained perineuronal nets in SAMR1 mice cortices, but the corresponding immunostaining in SAMP10 mice was very faint. MAb6B4 recognizes phosphacan/PTPzeta in immature brains. However, this antibody recognized several protein bands, including a 400-kDa core glycoprotein from chondroitin sulfate proteoglycan in homogenates of mature cortices from SAMR1 mice. The 400-kDa band was also recognized by antiaggrecan antibodies. The aggrecan core glycoprotein band was also detectable in samples from SAMP10 mice, but this glycoprotein was faintly immunostained by MAb6B4. Because MAb6B4 recognized the same set of protein bands that the monoclonal antibody Cat-315 recognized in mature cerebral cortices of SAMR1 mice, the MAb6B4 epitope appears to be closely related to that of Cat-315 and presumably represents a novel type of oligosaccharide that attaches to aggrecans. The Cat-315 epitope colocalized with aggrecan in perineuronal nets from SAMR1 mouse brain samples, whereas its expression was prominently reduced in SAMP10 mouse brain samples. The biological significance of the MAb6B4/Cat-315 epitope in brain function and its relationship to the neurodegeneration and learning disabilities observed in SAMP10 mice remain to be elucidated.

A highly sulfated chondroitin sulfate preparation, CS-E, prevents excitatory amino acid-induced neuronal cell death.

Chondroitin sulfate (CS) is a major microenvironmental molecule in the CNS, and there have been few reports about its neuroprotective activity. As neuronal cell death by excitotoxicity is a crucial phase in many neuronal diseases, we examined the effect of various CS preparations on neuronal cell death induced by the excitotoxicity of glutamate analogs. CS preparations were added to cultured neurons before and after the administration of glutamate analogs. Then, the extents of both neuronal cell death and survival were estimated. Pre-administration of a highly sulfated CS preparation, CS-E, significantly reduced neuronal cell death induced by not only NMDA but also (S)-alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid or kainate. Neither CS preparations other than CS-E nor other highly sulfated polysaccharides such as heparin and dextran sulfate exerted any neuroprotective effects. NMDA-induced current in neurons was not changed by pre-administration of CS-E, but the pattern of protein-tyrosine phosphorylation was changed. In addition, the elevation of caspase 3 activity was significantly suppressed in CS-E-treated neurons. These results indicate that CS-E prevents neuronal cell death mediated by various glutamate receptors, and suggest that phosphorylation-related intracellular signals and the suppression of caspase 3 activation are implicated in neuroprotection by CS-E.

Ectodomain shedding of neuroglycan C, a brain-specific chondroitin sulfate proteoglycan, by TIMP-2- and TIMP-3-sensitive proteolysis.

Neuroglycan C (NGC) is a transmembrane-type of chondroitin sulfate proteoglycan with an epidermal growth factor (EGF)-like module that is exclusively expressed in the CNS. Because ectodomain shedding is a common processing step for many transmembrane proteins, we examined whether NGC was subjected to proteolytic cleavage. Western blotting demonstrated the occurrence of a soluble form of NGC with a 75 kDa core glycoprotein in the soluble fraction of the young rat cerebrum. In contrast, full-length NGC with a 120 kDa core glycoprotein and its cytoplasmic fragment with a molecular size of 35 kDa could be detected in the membrane fraction. The soluble form of NGC was also detectable in culture media of fetal rat neurons, and the full-length form existed in cell layers. The amount of the soluble form in culture media was decreased by adding a physiological protease inhibitor such as a tissue inhibitor of metalloproteinase (TIMP)-2 or TIMP-3, but not by adding TIMP-1. Both EGF-like and neurite outgrowth-promoting activity of the NGC ectodomain may be regulated by this proteolytic processing.

NKCC1 activity modulates formation of functional inhibitory synapses in cultured neocortical neurons.

Intracellular Cl(-) concentration ([Cl(-)](i)) in immature neurons is higher than that expected for a passive distribution, therefore the equilibrium potential for chloride is more positive than the resting membrane potential, and the resulting GABA renders immature neurons depolarization. The higher [Cl(-)](i) in immature neurons is thought to be attributed to the uptake of Cl(-) mediated by NKCC1 (Na(+), K(+)-2Cl(-) cotransporter). Thus, a dysfunction of this transporter could affect synaptic development through a GABA(A) receptor-mediated pathway. To test this possibility, we examined the effects of a Cl(-)-uptake inhibitor on the development of synaptic activities of rat neocortical neurons in culture. Chronic treatment with bumetanide at 10 microM during the culture diminished the amplitude of synaptically-driven rhythmic depolarizing potentials (RDPs) in neurons and also decreased the frequency of spontaneous inhibitory postsynaptic currents (sIPSCs) but not of spontaneous excitatory postsynaptic currents (sEPSCs). Chronic treatment with bumetanide decreased vesicular GABA transporter (VGAT)-immunopositive particles without affecting paired-pulse ratio of evoked IPSCs (eIPSCs), indicating decrease in the number of functional GABAergic synapses. Acute treatment with bumetanide (10 microM) decreased neuronal [Cl(-)](i), the amplitude of RDPs, and neuronal excitability, while bumetanide had no effect on RDPs and neuronal excitability in the presence of bicuculline. These results suggest that the uptake of Cl(-) by NKCC1 affects the development of inhibitory synapses by promoting a depolarizing GABA-mediated response.

Neuroprotective effect of nipradilol, an NO donor, on hypoxic-ischemic brain injury of neonatal rats.

Hypoxia-ischemia is a common cause of neonatal brain injuries. Nitric oxide (NO) is upregulated in the brain after hypoxia-ischemia and generally believed to exert a paradoxical effect on neurons, neurodestruction and neuroprotection, but it has not been demonstrated that NO is actually neuroprotective in neonatal hypoxic-ischemic encephalopathy. We evaluated the effect of intracerebroventricular administration of nipradilol (3,4-dihydro-8-(2-hydroxy-3-isopropylamino)-propoxy-3-nitroxy-2H-1-benzopyran), a potent NO donor, at various concentrations (0.1 muM to 1 mM in 5 mul PBS/brain) to neonatal rats with hypoxic-ischemic treatment. The extent of the infarct area in the brain was significantly reduced by injection of the 1 muM nipradilol solution. However, denitro-nipradilol (3,4-dihydro-8-(2-hydroxy-3-isopropylamino)-propoxy-3-hydroxy-2H-1-benzopyran), that does not release NO, did not show the neuroprotective effect, suggesting that NO released from nipradilol exerts a neuroprotective effect on neonatal neurons.

Neuroglycan C is a novel midkine receptor involved in process elongation of oligodendroglial precursor-like cells.

Midkine is a heparin-binding growth factor that promotes cell attachment and process extension in undifferentiated bipolar CG-4 cells, an oligodendroglial precursor cell line. We found that CG-4 cells expressed a non-proteoglycan form of neuroglycan C, known as a part-time transmembrane proteoglycan. We demonstrated that neuroglycan C before or after chondroitinase ABC treatment bound to a midkine affinity column. Neuroglycan C lacking chondroitin sulfate chains was eluted with 0.5 m NaCl as a major fraction from the column. We confirmed that CG-4 cells expressed two isoforms of neuroglycan C, I, and III, by isolating cDNA. Among three functional domains of the extracellular part of neuroglycan C, the chondroitin sulfate attachment domain and acidic amino acid cluster box domain showed affinity for midkine, but the epidermal growth factor domain did not. Furthermore, cell surface neuroglycan C could be cross-linked with soluble midkine. Process extension on midkine-coated dishes was inhibited by either a monoclonal anti-neuroglycan C antibody C1 or a glutathione S-transferase-neuroglycan C fusion protein. Finally, stable transfectants of B104 neuroblastoma cells overexpressing neuroglycan C-I or neuroglycan C-III attached to the midkine substrate, spread well, and gave rise to cytoskeletal changes. Based on these results, we conclude that neuroglycan C is a novel component of midkine receptors involved in process elongation.

Identification of neurite outgrowth-promoting domains of neuroglycan C, a brain-specific chondroitin sulfate proteoglycan, and involvement of phosphatidylinositol 3-kinase and protein kinase C signaling pathways in neuritogenesis.

Neuroglycan C (NGC) is a transmembrane-type chondroitin sulfate proteoglycan that is exclusively expressed in the central nervous system. We report that the recombinant ectodomain of NGC core protein enhances neurite outgrowth from rat neocortical neurons in culture. Both protein kinase C (PKC) inhibitors and phosphatidylinositol 3-kinase (PI3K) inhibitors attenuated the NGC-mediated neurite outgrowth in a dose-dependent manner, suggesting that NGC promotes neurite outgrowth via PI3K and PKC pathways. The active sites of NGC for neurite outgrowth existed in the epidermal growth factor (EGF)-like domain and acidic amino acid (AA)-domain of the NGC ectodomain. The EGF-domain caused cells to extend preferentially one neurite from a soma, whereas the AA-domain caused several neurites to develop. The EGF-domain also enhanced neurite outgrowth from GABA-positive neurons, but the AA-domain did not. These results suggest that the EGF-domain and AA-domain have distinct functions in terms of neuritogenesis. From these findings, NGC can be considered to be involved in neuritogenesis in the developing central nervous system.

Identification and functions of chondroitin sulfate in the milieu of neural stem cells.

The behavior of cells is generally considered to be regulated by environmental factors, but the molecules in the milieu of neural stem cells have been little studied. We found by immunohistochemistry that chondroitin sulfate (CS) existed in the surroundings of nestin-positive cells or neural stem/progenitor cells in the rat ventricular zone of the telencephalon at embryonic day 14. Brain-specific chondroitin sulfate proteoglycans (CSPGs), including neurocan, phosphacan/receptor-type protein-tyrosine phosphatase beta, and neuroglycan C, were detected in the ventricular zone. Neurospheres formed by cells from the fetal telencephalon also expressed these CSPGs and NG2 proteoglycan. To examine the structural features and functions of CS polysaccharides in the milieu of neural stem cells, we isolated and purified CS from embryonic day 14 telencephalons. The CS preparation consisted of two fractions differing in size and extent of sulfation: small CS polysaccharides with low sulfation and large CS polysaccharides with high sulfation. Interestingly, both CS polysaccharides and commercial preparations of dermatan sulfate CS-B and an E-type of highly sulfated CS promoted the fibroblast growth factor-2-mediated proliferation of neural stem/progenitor cells. None of these CS preparations promoted the epidermal growth factor-mediated neural stem cell proliferation. These results suggest that these CSPGs are involved in the proliferation of neural stem cells as a group of cell microenvironmental factors.

Expression and identification of a new splice variant of neuroglycan C, a transmembrane chondroitin sulfate proteoglycan, in the human brain.

Neuroglycan C (NGC) is a transmembrane chondroitin sulfate proteoglycan with an EGF module. We studied the expression of NGC in the human brain, mainly in the hippocampus, and confirmed some observations by conducting experiments using rat brain. In humans, NGC mRNA was expressed exclusively in the brain, especially in the immature brain. The telencephalon, including the hippocampus and neocortex, showed strong mRNA expression. NGC was immunolocalized to neuropils in the hippocampus and neocortex of the adult rat. RT-PCR experiments showed that four splice variants (NGC-I, -II, -III, and -IV) were expressed in the adult human hippocampus. By Western blotting, the expression as proteins of all splice variants except NGC-II was confirmed in the adult rat hippocampus. NGC-IV, which was first found in the present study, had the shortest cytoplasmic domain among the four variants. NGC-IV mRNA was expressed by neurons, but not by astrocytes, in culture prepared from the fetal rat hippocampus, suggesting that NGC-IV plays a role specific to neurons. In addition, the human NGC gene, which is registered as CSPG5, comprised six exons and was approximately 19 kb in size. In exon 2, a single nucleotide polymorphism resulting in Val188Gly in the NGC ectodomain was observed.