Elucidating the structure and function of chondrotin sulfate proteoglycan in differentitation, development and regeneration of dopaminergic neurons

硫酸软骨素（CS）蛋白聚糖在神经元发育和再生中起重要作用。根据双糖单位的硫酸化模式，CS可分为CS- a、CS- c、CS- d和CS- e四个亚类。在多巴胺能神经元中，已知CS在发育过程中调节神经突的生长和损伤后的细胞粘附。虽然CS的生物学重要性已得到证实，但由于CS的复杂性和异质性，其影响多巴胺能神经元的精确结构和分子机制尚不清楚。在这项研究中，我们研究了合成的CS和CS多糖促进中脑多巴胺能神经元神经突生长的生物活性，以及CS作用的细胞内机制。CS-E多糖对多巴胺能神经元的突起生长有促进作用，而CS-A、c和d多糖对多巴胺能神经元的突起生长无促进作用。用软骨素酶ABC将CS-E多糖分解成双糖单位，可以完全消除CS-E对神经突生长的刺激作用。与CS-E多糖类似，合成CS-E四糖刺激多巴胺能神经元的神经突生长，但合成CS-E二糖或未酸化的四糖对神经突生长没有影响。胞内机制分析揭示了磷脂酶C （PLC）信号参与CS-E四糖的作用。CS-E四糖的作用被PLC、蛋白激酶C （PKC）、肌醇1,4,5 -三磷酸（IP_3）受体、Ca^<2+>/钙调素依赖性激酶II （CaMKII）或钙调神经磷酸酶抑制剂所消除。这些结果表明CS- e的特定磺化基序和CS- e四糖单元是CS在多巴胺能神经元中神经生成活性的结构决定因素，CS- e的作用是通过PLC/PKC和PLC/IP_3/IP_3受体信号传导介导的，导致CaMKII和钙调磷酸酶的[Ca^<2+>]_i依赖性激活。少

Chondroitin sulfate (CS) proteoglycans play important roles in neuronal development and regeneration. CS is divided into four subclasses, known as CS-A, CS-C, CS-D and CS-E, on the basis of the sulfation patterns of the disaccharide unit. In dopaminergic neurons, CS is known to modulate neurite outgrowth during development and cell adhesion after injury. Although the biological importance of CS is well established, the precise structure and the molecular mechanisms underlying its influence on dopaminergic neurons are poorly understood due to the complexity and heterogeneity of CS. In this study, we investigated the biological activity of synthetic CS as well as CS polysaccharides to promote the neurite outgrowth of mesencephalic dopaminergic neurons and the intracellular mechanisms underlying the action of CS. CS-E polysaccharide, but not CS-A, -C or -D polysaccharide, facilitated the neurite outgrowth of dopaminergic neurons. The stimulatory effect of CS-E on neurite outgrowth was com … More pletely abolished by digestion of CS-E polysaccharide into disaccharide units with chondroitinase ABC. Similarly to CS-E polysaccharide, synthetic CS-E tetrasaccharide stimulated the neurite outgrowth of dopaminergic neurons, but synthetic CS-E disaccharide or unsulfated tetrasaccharide did not affect the neurite outgrowth. Analysis of intracellular mechanisms revealed the involvement of phospholipase C (PLC) signaling in the action of CS-E tetrasaccharide. The effect of CS-E tetrasccharide was abolished by the inhibitor of PLC, protein kinase C (PKC), inositol 1, 4, 5-triphosphate (IP_3) receptors, Ca^<2+>/calmodulin-dependent kinase II (CaMKII) or calcineurin. These results indicate that the specific sulfation motif of CS-E and the CS-E tetrasaccharide unit represent the structural determinants for the neuritogenic activity of CS in dopaminergic neurons, and that the action of CS-E is mediated through PLC/PKC and PLC/IP_3/IP_3 receptor signaling, leading to [Ca^<2+>]_i-dependent activation of CaMKII and calcineurin. Less