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

硫酸软骨素（CS）蛋白多糖在神经元的发育和再生中起重要作用。根据二糖单元的硫酸化模式，CS分为四个亚类，称为CS-A、CS-C、CS-D和CS-E。在多巴胺能神经元中，已知CS调节发育过程中的轴突生长和损伤后的细胞粘附。虽然CS的生物学重要性是公认的，但由于CS的复杂性和异质性，其对多巴胺能神经元影响的确切结构和分子机制知之甚少。在这项研究中，我们研究了合成CS以及CS多糖的生物活性，以促进中脑多巴胺能神经元的轴突生长和CS的作用的细胞内机制。CS-E多糖促进多巴胺能神经元突起生长，而CS-A、CS-C和CS-D多糖则无此作用。CS-E对神经突起生长的促进作用与其对神经突起生长的抑制作用一致。 ...更多信息 通过用软骨素酶ABC将CS-E多糖消化成二糖单位而完全消除。与CS-E多糖类似，合成CS-E四糖刺激多巴胺能神经元突起生长，但合成CS-E二糖或未硫酸化四糖不影响突起生长。细胞内机制的分析揭示了磷脂酶C（PLC）信号转导参与CS-E四糖的作用。CS-E四糖酯的作用可被PLC、蛋白激酶C（PKC）、肌醇1，4，5-三磷酸（IP_3）受体、钙调蛋白依赖性激酶II（CaMKII）或钙调磷酸酶抑制剂所阻断。这些结果表明，CS-E的硫酸化基序和CS-E四糖单元是CS在多巴胺能神经元中促轴突生长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CaMK Ⅱ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