Sotogaku Naoki, Tully Sarah E, Gama Cristal I, Higashi Hideho, Tanaka Masatoshi, Hsieh-Wilson Linda C, Nishi Akinori
Department of Pharmacology, Kurume University School of Medicine, Kurume, Fukuoka, Japan.
J Neurochem. 2007 Oct;103(2):749-60. doi: 10.1111/j.1471-4159.2007.04849.x. Epub 2007 Aug 6.
In dopaminergic neurons, chondroitin sulfate (CS) proteoglycans play important roles in neuronal development and regeneration. However, due to the complexity and heterogeneity of CS, the precise structure of CS with biological activity and the molecular mechanisms underlying its influence on dopaminergic neurons are poorly understood. In this study, we investigated the ability of synthetic CS oligosaccharides and natural polysaccharides to promote the neurite outgrowth of mesencephalic dopaminergic neurons and the signaling pathways activated by CS. CS-E polysaccharide, but not CS-A, -C or -D polysaccharide, facilitated the neurite outgrowth of dopaminergic neurons at CS concentrations within the physiological range. The stimulatory effect of CS-E polysaccharide on neurite outgrowth was completely abolished by its digestion into disaccharide units with chondroitinase ABC. Similarly to CS-E polysaccharide, a synthetic tetrasaccharide displaying only the CS-E sulfation motif stimulated the neurite outgrowth of dopaminergic neurons, whereas a CS-E disaccharide or unsulfated tetrasaccharide had no effect. Analysis of the molecular mechanisms revealed that the action of the CS-E tetrasaccharide was mediated through midkine-pleiotrophin/protein tyrosine phosphatase zeta and brain-derived neurotrophic factor/tyrosine kinase B receptor pathways, followed by activation of the two intracellular phospholipase C (PLC) signaling cascades: PLC/protein kinase C and PLC/inositol 1,4,5-triphosphate/inositol 1,4,5-triphosphate receptor signaling leading to intracellular Ca(2+) concentration-dependent activation of Ca(2+)/calmodulin-dependent kinase II and calcineurin. These results indicate that a specific sulfation motif, in particular the CS-E tetrasaccharide unit, represents a key structural determinant for activation of midkine, pleiotrophin and brain-derived neurotrophic factor-mediated signaling, and is required for the neuritogenic activity of CS in dopaminergic neurons.
在多巴胺能神经元中，硫酸软骨素（CS）蛋白聚糖在神经元发育和再生中发挥重要作用。然而，由于CS的复杂性和异质性，具有生物活性的CS的精确结构及其影响多巴胺能神经元的分子机制尚不清楚。在本研究中，我们研究了合成CS寡糖和天然多糖促进中脑多巴胺能神经元神经突生长的能力以及CS激活的信号通路。CS-E多糖而非CS-A、-C或-D多糖在生理范围内的CS浓度下促进了多巴胺能神经元的神经突生长。用软骨素酶ABC将CS-E多糖消化成二糖单位后，其对神经突生长的刺激作用完全消除。与CS-E多糖类似，仅显示CS-E硫酸化基序的合成四糖刺激了多巴胺能神经元的神经突生长，而CS-E二糖或未硫酸化的四糖则没有作用。分子机制分析表明，CS-E四糖的作用是通过中期因子-多效营养蛋白/蛋白酪氨酸磷酸酶ζ和脑源性神经营养因子/酪氨酸激酶B受体途径介导的，随后激活两个细胞内磷脂酶C（PLC）信号级联反应：PLC/蛋白激酶C和PLC/肌醇1,4,5-三磷酸/肌醇1,4,5-三磷酸受体信号传导，导致细胞内Ca(2+)浓度依赖性激活Ca(2+)/钙调蛋白依赖性激酶II和钙调神经磷酸酶。这些结果表明，特定的硫酸化基序，特别是CS-E四糖单位，是激活中期因子、多效营养蛋白和脑源性神经营养因子介导的信号传导的关键结构决定因素，并且是CS在多巴胺能神经元中促神经突生长活性所必需的。
