星狀膠細胞之神經生長相關蛋白-43調節神經發炎與神經塑性之研究

The Role of Astrocytic GAP43 in Neuroinflammation and Neuronal Plasticity

洪家琪(Chia-Chi Hung)

指導教授 ： 周志銘

共同指導教授 ： 李怡萱(Yi-Hsuan Lee)

臺北醫學大學/醫學院/醫學科學研究所/博士(2016年)

摘要

星狀膠細胞 (astrocytes）在中樞神經系統 (central nervous system, CNS)中主要負責維持環境的恆定；在CNS 受到傷害的情況下，星狀膠細胞會形成活化態並伴隨著型態的改變，此過程稱為星狀膠細胞增生 (astrogliosis)。然而，已知根據不同的訊息來源，星狀膠細胞增生會伴隨產生有益及有害兩種不同的影響。神經生長相關蛋白-43 (growth-associated protein-43; GAP43) 常被做為神經塑性及軸突再生的標記蛋白，其活化機轉主要是經由蛋白磷酸酶 C (protein kinase C) 對其進行磷酸化，進而促進肌動蛋白之聚合纖維(filamentous-actin,F-actin) 的穩定，使得神經軸突生長。因為在我們的初期研究中，意外發現不論細胞或動物實驗皆可觀察到內毒素脂多醣 (lipopolysaccharide, LPS)能夠造成大鼠腦內星狀膠細胞中 GAP43 的增加。本論文研究之目標在於進一步闡明此現象之上游機轉及其對於星狀膠細胞功能的影響。我們的研究結果發現，LPS能夠透過其受體 toll-like receptor 4 (TLR4) 誘導具星狀型態（stellate shape）的星狀膠細胞中GAP43增加，且會伴隨著磷酸化 GAP43 的同步增加，但在扁平型態 (flat shape) 的星狀膠細胞則沒有觀察到此現象，因此，我們以星狀型態之星狀膠細胞為我們研究中主要的實驗系統。我們進一步在動物實驗中確認， LPS 投予的大鼠其腦內扣帶迴（cingulum）及胼肢體（corpus callosum）區域，GAP43 表現量的確會增加於活化的星狀膠細胞中。我們發現 LPS 增加 GAP43 主要透過 TLR4 下游活化 NFkB 與 IL6/STAT3 的訊息路徑，造成其基因轉錄增加，因此，當投予NFkB 的抑制胜肽 TAT-NBD時，亦觀察到 LPS 誘導星狀膠細胞的 GAP43 增加及觸角分佈 (astrocytic arborization) 有被抑制的情形。此外，根據 PKC調控GAP43 磷酸化而會影響 F-actin 的特性，我們以兩種 GAP43 胺基酸序列變異之基因構築轉染至星狀膠細胞中表現:其一是以aspartic acid (D) 取代serine 41 (S41)模擬持續磷酸化之GAP43 (GAP43S41D)，另一為以alanine (A) 取代S41模擬持續去磷酸化狀態之GAP43（GAP43 S41A），結果顯示表現GAP43S41D之星狀膠細胞會有相似於 LPS 誘導之觸角分佈增加及增長之效果，當表現GAP43S41A時，則會抑制此現象；反之，在扁平型態的星狀膠細胞中表達GAP43S41D則是造成細胞肥大（hypertrophy），並不會引起細胞觸角形成而使之星狀化。進而利用小片段干擾核醣核酸 (siRNA) 降低星狀膠細胞內 GAP43表現時 (GAP43 knockdown)，則會減少 LPS 所增加的星狀膠細胞觸角分佈情形，得到與GAP43S41A 類似的結果。重要的是，我們發現GAP43 knockdown 與 LPS 處理星狀膠細胞後所收集的條件培養液(conditioned medium) 中，促發炎性細胞激素 (proinflammatory cytokines) IL6 與 TNFα 的分泌及 iNOS 的基因表現會被更加提升；將此條件培養液投予至微小膠細胞 (microglia)，結果發現其前趨發炎指標 COX-2 及活化指標 OX42 皆會被提高。再者，經LPS處理活化之星狀膠細胞在 GAP43 knockdown 的情形下，由其所收集到之培養液會抑制初代培養的大鼠大腦皮質神經元 (cortical neurons) 中 GAP43 的磷酸化及軸突生長，並會增加神經元的損傷。進一步探討GAP43 knockdown可能造成神經毒性的原因，發現 GAP43 knockdown 的星狀膠細胞其興奮性胺基酸轉運蛋白-2 (excitatory amino acid transporter-2; EAAT2) 的基因表現會被降低，伴隨著細胞外興奮性胺基酸-麩胺酸（glutamate）回收效率亦被降低，我們進而釐清此機轉主要是經由 GAP43 在 LPS 誘導下會透過穩定 F-actin， 進而降低 G-actin，使得受其抑制的transcriptional coactivator megakaryoblastic leukemia 1 (MKL1) 活化，進入細胞核與 serum response factor (SRF) 形成複合體而結合至EAAT2 基因Slc1a2的啟動子上增加其表現。另外，我們在缺血性中風（middle cerebral artery occlusion, MCAo）的動物模式中，也觀察到大鼠大腦皮質與扣帶迴的 GAP43 在被誘導活化之星狀膠細胞中有增加的情形。綜合上述，我們證實了 GAP43 在星狀膠細胞的活化中扮演了有益於免疫調節與維持神經元存活及可塑性的重要角色，而誘發其表現的機轉除了本論文中所闡明的 TLR4-NFκB-STAT3 訊息傳遞路徑外，缺血性中風動物模式的結果亦表示有其它調控機轉有待後續探討，而此研究結果在未來以GAP43 做為神經塑性指標的基礎與臨床研究，將提供重要的判讀與治療策略評估依據。

關鍵字

神經生長相關蛋白-43 ； 星狀膠細胞增生 ； 星狀膠細胞塑性 ； 興奮性胺基酸轉運蛋白-2

並列摘要

Astrocytes are responsible for maintaining the homeostasis of central nervous system (CNS) and are activated with morphological changes, also called astrogliosis, in response to various CNS insults. However, astrogliosis is known to have both beneficial and detrimental effects due to the complex signals. Growth-associated protein 43 (GAP43), a protein kinase C (PKC)-activated phosphoprotein, is often implicated in axonal plasticity and regeneration. In our study, we found that GAP43 can be induced with its S41 phosphorylation in parallel by the endotoxin lipopolysaccharide (LPS) through toll-like receptor 4 (TLR4) in rat primary cultured astrocytes with stellate shape, not flat shape phenotype. We further confirmed that the increase of GAP43 can also be detected in GFAP+ astrocytes in the cingulum and corpus callosum of LPS-infused rat brains. The LPS-induced astrocytic GAP43 was regulated by TLR4 downstream mediator NFkB- and IL6/STAT3-dependent transcriptional activation. In NFkB inhibitory peptide TAT-NBD-treated astrocytes, the LPS-induced GAP43 expression and astrocytic arborization were inhibited. Overexpression of the PKC phosphorylation-mimicking GAP43S41D (constitutive active GAP43) in stellate-shape astrocytes increased the astrocytic process arborizaton and elongation, whereas, GAP43S41A (dominant negative GAP43)-expressing astrocytes blocked LPS-induced process arborization and elongation. In contrast, GAP43S41D only induced cellular hypertrophy, and did not induce process formation or stellation when overexpressed in flat-shape astrocytes. The knockdown of GAP43 with its specific siRNA also decreased the LPS-induced astrocytic arborization, similar to the GAP43S41A effects. Importantly, we found GAP43 knockdown enhanced the proinflammatory cytokines IL6 and TNFα release and iNOS expression in astrocytes, from which the astrocyte-conditioned medium aggravated astrogliosis-induced microglial activation by increasing the expression of proinflammatory indicator COX-2 and the immunoreactivities of microglial activation marker OX42. Moreover, we found that astrocyte-conditioned medium (ACM) can promote axon growth in cortical neurons, and this effect was diminished accompanied with reduction of neuronal survival and neuronal GAP43 phosphorylation when ACM was obtained from GAP43-knockdown astrocytes. For these pro-neurotoxic effects of astrocytic GAP43 knockdown, we further showed astrocytic GAP43 knockdown causes the downregulation of LPS-induced glutamate transporter EAAT2 expression, accompanied by the decrease of LPS-elevated glutamate uptake. The regulation of GAP43 on EAAT2 gene expression was then identified through a disinhibition of actin polymerization-dependent transcriptional activation of the G-actin-associated transcriptional coactivator megakaryoblastic leukemia 1 (MKL1) that binds to serum response elements in the promoter of rat Slc1a2 gene encoding EAAT2 for increasing gene transcription. Finally, we further examined other brain injury model, and found that ischemic brain injury can also induced GAP43 expression in reactive astrocytes in rat brain cortex and corpus callosum. In sum, our study suggests that astrocytic GAP43 mediates glial plasticity during astrogliosis, and provide beneficial effects on neuronal plasticity and survival, and attenuation of microglial activation to dampen neuroinflammatory response during brain injury.

並列關鍵字

growth-associated protein-43 ； astrogliosis ； astrocytic plasticity ； excitatory amino acid transporter-2

參考文獻

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