BMP4 於神經肌肉系統生理功能之探討

Abstract

骨形成蛋白 (bone morphogenetic proteins, BMPs) 屬於TGF家族的成員，過去的研究指出BMPs對神經系統的發育及維持非常的重要，並且會參與調控突觸的形成。然而，在哺乳類動物的研究中，BMPs在神經肌肉系統中所調控的生理功能仍未完全了解。本實驗室初步的研究資料顯示BMPs的type II受體 (bone morphogenetic protein type II receptor, BMPRII) 會表現在神經與肌肉接合處 (neuromuscular junction, NMJ) ，而從本論文中的免疫染色實驗結果觀察到骨形成蛋白-4 (BMP4) 會表現在肌肉及許旺細胞上，且BMP4與乙醯膽鹼受體 (acetylcholine receptors, AChRs) 有colocalization的現象。由double nerve ligations的實驗觀察到BMP4會堆積在打結處的兩端，顯示BMP4可能是由肌肉或許旺細胞分泌後送進運動神經元之軸突內運輸，其方向為雙向性運輸，而利用Q-PCR mRNA定量實驗發現BMP4 mRNA在double-ligated之坐骨神經中表現量下降，但在肌肉中表現量則顯著增加。\n由上述實驗顯示肌肉細胞為BMP4主要來源之ㄧ，利用NG108-15神經細胞及C2C12肌肉細胞培養，我們發現BMP4 mRNA在C2C12肌小管上有高度表現，相反地在分化後的NG108-15神經細胞上表現量極少，而BMP4的mRNA及protein在C2C12肌肉上的表現量則受到神經衍生蛋白Agrin的調控。此外我們亦發現來自於肌肉的BMP4則會保護分化後的NG108-15神經細胞對抗Glutamate所誘導的細胞死亡反應。綜合這些結果，我們認為BMP4主要來自於運動神經元之周邊的肌肉及許旺細胞，其可能會參與調控運動神經元的存活機制。

Bone morphogenetic proteins (BMPs), members of the TGF superfamily, have been shown to play important roles in the development of nervous system including neuronal survival and synaptogenesis. However, the physiological functions of BMP signaling at the mammalian neuromuscular system are not well understood. Our preliminary data showed that proteins of the type II bone morphogenetic receptors (BMPRII) were specifically expressed in nerve terminals at neuromuscular junctions. In this study, we found that proteins of bone morphogenetic protein-4 (BMP4) were detected at Schwann cells and colocalized with postsynaptic acetylcholine receptors (AChRs) in skeletal muscle fibers. In double-ligated nerves, BMP4 proteins were accumulated at the proximal and distal portions of the axons, suggesting that Schwann cell- and muscle fiber-derived BMP4 proteins were anterogradely and retrogradely transported by motor neurons. Additionally, BMP4 mRNA was significantly up-regulated in the muscle but down-regulated in ligated sciatic nerves. \nThe physiological functions of BMP4 in the neuromuscular system were further examined in vitro. We found that mRNA of BMP4 was highly expressed in differentiated C2C12 muscle cells, but it was barely detectable in NG108-5 neurons. The expression of BMP4 mRNA and protein in C2C12 muscle cells were upregulated when the motor neuron-derived factor, agrin, was presented in the culture. Moreover, muscle-derived BMP4 could protect NG108-5 neurons from glutamate-induced excitotoxicity. These results together suggest that BMP4 is a peripheral-derived factor that may regulate the survival of motor neurons.