Thy-1抗體導致大鼠背根神經元胞突生長之訊息傳遞機制探討

Abstract

Thy-1又稱CD90，是一種位於非囊泡性脂筏（non-caveolar lipid raft microdomains）的小型表面醣蛋白，與Src家族激酶有高度的關連性，主要出現在胸腺細胞、T淋巴球及神經細胞等細胞膜上，並呈現點狀的分佈。過去學者們的研究支持，Thy-1對於神經胞突生長扮演了抑制的角色，若干擾膜上Thy-1的功能，的確能促使視網膜神經元及PC12神經瘤母細胞胞突的生長、維持及再生。首先，我們想了解以Thy-1抗體阻斷背根神經元膜上Thy-1之正常功能，是否能促使其胞突生長？接著，Thy-1與抗體結合後，活化了哪些訊息路徑進而影響胞突生長？而這些訊息路徑的傳遞是否與脂筏（lipid rafts）有關？ 我們在培養兩天的背根神經元，進行相關的實驗。我們發現，以Thy-1抗體處理六小時後，無論是大型或小型背根神經元，神經胞突長度及其分枝複雜程度均明顯增加。若以methyl-beta-cyclodextrin（MbCD）破壞脂筏（lipid rafts）的結構後，則抑制了Thy-1抗體所誘導的胞突生長，證明Thy-1抗體須經由神經細胞膜上完整且功能正常的脂筏才能達到其作用。而在此過程中，Thy-1抗體除了會導致Thy-1產生內質化（internalization）的情形，可能也趨使了與Thy-1連結的Src家族激酶之訊息活化。於是我們以西方墨點分析來證明，Thy-1抗體處理0、15、30及60分鐘後，的確能使得c-Src、MEK1/2、CaMKII、VASP（PKA受質）及CREB的磷酸化表現增加。接著分別使用MEK的抑制劑PD98059、CaMKII的抑制劑KN-93及PKA的抑制劑Protein kinase inhibitor（PKI）預先處理30分鐘，再加入Thy-1抗體處理六小時後，均可抑制大型及小型背根神經元的胞突生長。進一步以PP2抑制Src活化，可抑制Thy-1抗體處理所引起的MEK磷酸化，足見Src為MEK的上游調控分子。除此之外，實驗也証明以Thy-1抗體處理後，活化了MEK及PKA，進而造成CREB的磷酸化。根據學者們的研究，CREB磷酸化可以促進與胞突生長有關的基因，如neurofilament light chain subunit（NF-L）、brain-derived neurotrophic factor（BDNF）及nerve growth factor（NGF）等表現。所以，我們推論Thy-1抗體的作用，可能經由Thy-1-enriched microdomains，啟動和Thy-1結合的c-Src激酶，進行一連串的訊息傳導：c-Src-MEK-ERK-CREB-neurite outgrowth，同時也有可能活化了PKA-CREB，CaMKII及其下游標的分子，而造成胞突生長的反應。

Thy-1 (CD90), a glycosylphosphatidylinositol (GPI) protein, is located at non-caveolar lipid raft and often colocalized with Src-family kinases within these domains, and is expressed mainly in thymus epithelial cells, T-lymphocytes and neurons. Previous studies suggest an inhibitory role of Thy-1 in neurite growth in retinal ganglion neurons and neuroblastoma cell lines. Using cultured dorsal root ganglion (DRG) neurons, we investigated the underlying signaling mechanism responsible for the effect of Thy-1 antibody on promoting neurite outgrowth. Thy-1 was enriched in microdomain-like punctates on the cell membrane by immunofluorescence observation. Treatment with Thy-1 antibody stimulated neurite outgrowth and increased branching complexity of the neurites in both small and large neurons. This effect of Thy-1 antibody was dependent on the integrity of Thy-1-enriched microdomains, since disruption of these domains by methyl-beta-cyclodextrin (MbCD) prevented Thy-1 antibody-induced neurite outgrowth. Possibly via binding to membranous Thy-1, Thy-1 antibodies induced internalization of Thy-1 molecules, followed by triggering subsequent signaling involving activation of Src kinases. This transient activation of c-Src kinase resulted in increased phosphorylation of mitogen activated kinase kinase (MEK) and cyclic AMP response-element binding protein (CREB), since pretreatment with PP2 abolished Thy-1 antibody-induced neurite outgrowth in DRG neurons and the phosphorylation of MEK and CREB. Phosphorylation of CREB may guide to upregulation of some neurite outgrowth-related proteins, such as neurofilament light chain subunit (NF-L), brain-derived neurotrophic factor (BDNF) and nerve growth factor (NGF), as reported in other neuronal cells. We conclude that Thy-1 antibody activates c-Src kinase-MEK -CREB by targeting on Thy-1-enriched microdomains in the cell membrane of DRG neuron.