Introduction

Neurotrophins, including nerve growth factor (NGF) and brain-derived neurotrophic factor (BDNF), are essential regulators of neuronal differentiation, survival, plasticity, and other associated physiological actions of neurons throughout the entire life. By binding to their receptors TrkA (for NGF), TrkB (for BDNF), and p75 (for both), NGF and BDNF activate avariety of signals such as Ras-mitogen-activated protein kinase (MAPK), phosphatidylinositol 3-kinase-Akt, and phospholipase Cγ pathways. Our research group previously demonstrated that secretory phospholipase A2 (sPLA2), a group of enzymes that catalyze the hydrolysis of the sn-2 ester bond of membrane phospholipids to release free fatty acids and lysophospholipids, shows neurotrophin-like activity, i.e. induction of neurite outgrowth in PC12 cells, and protection of cerebellar granule neurons (CGNs) from apoptosis, mimicking the actions of NGF and BDNF. Subsequent studies showed that the neurotrophin-like actions of sPLA2 are mediated through the release of one of the lysophospholipids, lysophosphatidylcholine (LPC). Indeed, LPC added to the cultures of PC12 and CGNs recapitulated the neurotrophin-like activity of sPLA2. In this study, I have demonstrated that LPC promotes neurotrophin-Trk receptor signals in PC12 cells and in CGNs. I have also analyzed the underlying mechanism and confirmed that LPC specifically enhances NGF-induced signals through the extracellular domain of TrkA in PC12 cells, and BNDF-induced signals through TrkB in CGNs. Results from the neurite outgrowth assay in PC12 cells suggested that LPC at least partially potentiates NGF-induced differentiation of PC12 cells. It was unlikely that these processes were mediated by G protein-coupled receptors G2A and GPR4, although they have been implicated in the biological actions of LPC in previous studies. Taken together, the findings obtained in this study might provide important evidences for the investigation of the mechanism by which LPC displays neurotrophin-like effect.

Chapter 1. Lysophosphatidylcholine promotes NGF-induced MAPK and Akt signals by enhancing the activation of TrkA in PC12 cells

LPC is one of the major lysophospholipids generated from the hydrolysis of phosphatidylcholine (PC) by sPLA2. In our previous studies, LPC generated by sPLA2 was found to induce neurite outgrowth in PC12 cells. Since NGF also induces neuronal differentiation, I tested if a cross-talk between NGF- and LPC-induced signals exists in PC12 cells by examining the effect of LPC on NGF-induced MAPK and Akt phosphorylation. I found that LPC significantly enhances NGF-induced MAPK and Akt phosphorylation. Other lysophospholipids, such as lysophosphatidic acid, lysophosphatidylethanolamine, and lysophosphatidylserine, did not display similar effect. Quantitative RT-PCR analysis showed that LPC upregulates the expression level of NGF-induced immediate early genes, c-fos and NGF-IA, which are necessary for the initiation and maintenance of differentiation. Neurite outgrowth assay showed that LPC partially potentiates NGF-induced differentiation of PC12 cells. Next, the signaling pathway by which LPC potentiates NGF-induced MAPK and Akt phosphorylation in PC12 cells was characterized. Phosphorylation of both MEK and TrkA, the upstream cellular components of MAPK, was enhanced by LPC. In contrast, LPC did not show any effect on MAPK phosphorylation induced by other growth factors, epidermal growth factor (EGF) and basic fibroblast growth factor. In accordance, EGF receptor phosphorylation induced by EGF was not increased by LPC. Furthermore, Akt phosphorylation induced by insulin-like growth factor-1 (IGF-1) was not affected by LPC. Collectively, these results indicate that LPC specifically promotes NGF-induced MAPK and Akt phosphorylation through enhancing the activation of TrkA.

Chapter 2. Lysophosphatidylcholine potentiates BDNF-induced MAPK and Akt signals through stimulating the activation of TrkB in cerebellar granule neurons

BDNF plays critical roles in regulating the survival and functions of neurons, particularly those in the brain, through binding to its receptor TrkB. BDNF is known to support the survival of cerebellar granule neurons (CGNs), the most abundant neurons in the brain, through the activation of Ras-MAPK pathway. Cultured CGNs are suitable model for studying the cell survival, since they undergo apoptosis when shifted to the culture medium containing low concentration of potassium (LK). IGF-1 is also known to protect CGNs from LK-induced apoptosis through PI3K-Akt cascade. Previously, our research group showed that LPC protects CGNs from LK-induced apoptosis, mimicking the actions of BDNF and IGF-1, although the mechanism has not been identified. In this Chapter, I studied the effect of LPC on BDNF-induced MAPK and Akt phosphorylation in TrkB-transfected CHO-K1 cells and in CGNs. I first confirmed that MAPK was not phosphorylated upon treatments with BDNF, LPC, or both, in the wild type and vector-transfected CHO-K1 cells. In TrkB-transfected CHO-K1 cells, BDNF slightly induced phosphorylation of MAPK, which was significantly enhanced by LPC. In CGNs, although LPC alone did not induce phosphorylation of MAPK and Akt, it significantly elevated phosphorylation of MAPK and Akt induced by BDNF, but not by IGF-1. Furthermore, BDNF-induced TrkB phosphorylation was increased by LPC, suggesting that LPC promotes BDNF-induced MAPK and Akt signals through enhancing the activation of TrkB. Although LPC failed to further potentiate the effect of BDNF in rescuing CGNs from apoptosis, the results presented here, together with those shown in Chapter 1, indicate that LPC specifically enhances neurotrophin-Trk receptor signaling cascades.

Chapter 3. Analyses of the role of lysophosphatidylcholine on NGF-induced TrkA signal

Results shown in Chapter 1 demonstrated that TrkA, but not EGFR, is responsive to the effect of LPC. To further understand the mode of action of LPC on TrkA, I aimed to analyze the domain(s) of TrkA involved in the effect of LPC. To this end, I first analyzed receptor phosphorylation in TrkA-, EGFR-, or TrkA/EGFR chimera-transfected cells. However, spontaneous phosphorylation of TrkA occurred in transfected cells, and this was not further increased by NGF and/or LPC treatments, which hampered further analysis.

I next tested the effect of NGF, EGF, and LPC on the downstream signal, MAPK phosphorylation, in the same experimental system. I found that the wild type and vector-transfected CHO-K1 cells do not respond to NGF and EGF, i.e. MAPK was not phosphorylated upon NGF and EGF treatments, since no functional TrkA and EGFR were expressed in CHO-K1 cells. In TrkA-transfected cells, NGF weakly induced MAPK phosphorylation, and it was significantly increased by LPC. In EGFR-transfected cells, EGF induced MAPK phosphorylation, but this was not affected by LPC. These results indicate that transfected TrkA and EGFR behaved as those in PC12 cells. Next, the domain(s) of TrkA involved in the effect of LPC was analyzed by examining MAPK phosphorylation in TrkA/EGFR chimera-transfected CHO-K1 cells. TrkA/EGFR chimeras C1, C2, C3, and C4 were constructed by swapping the extracellular (ED), transmembrane (TMD), and intracellular (ID) domains between TrkA and EGFR. In C1 (TrkA ED/EGFR TMD+ID chimera)- or C3 (TrkA ED+TMD/EGFR ID chimera)-transfected cells, LPC enhanced MAPK phosphorylation triggered by NGF, as was seen in the TrkA-transfected cells. In C2 (EGFR ED/TrkA TMD+ID chimera)- or C4 (EGFR ED+TMD/TrkA ID chimera)-transfected cells, MAPK was strongly phosphorylated upon EGF treatment, but this was not further enhanced by LPC. These results indicate that the ED, but not TMD and ID, of TrkA is responsible for the effect of LPC in enhancing NGF-induced MAPK phosphorylation.

As described in Introduction, LPC is generated through the hydrolysis of PC by sPLA2. I next examined the effect of sPLA2 addition on NGF-induced MAPK phosphorylation. I found that exogenously-added sPLA2 enhances NGF-induced MAPK phosphorylation at a comparable level to LPC. This suggests that LPC generated in situ by the hydrolysis of plasma membrane PC acts synergistically with NGF.

Accumulating evidence suggests the involvement of G protein-coupled receptor, G2A and GPR4, in the biological actions of LPC. In addition, our research group previously found that sPLA2-induced neuritogenesis in PC12 cells was mediated by G2A. I therefore examined if G2A and GPR4 regulate the effect of LPC on NGF-induced MAPK phosphorylation in PC12 cells. However, overexpression of neither G2A nor GPR4 affected the enhancement of NGF-induced MAPK phosphorylation by LPC, indicating that the action of LPC on NGF-induced MAPK phosphorylation is not mediated by G2A and GPR4.

It is well accepted that NGF induces dimerization and autophosphorylation of TrkA, thereby activating the downstream signaling events. Recently studies have shown, however, that the majority of TrkA preforms dimers in the endoplasmic reticulum before reaching to the cell surface; NGF activates the preformed, yet inactive, TrkA dimer on the cell surface. To examine if LPC regulates the dimerization state of TrkA, I performed crosslinking experiment using the divalent crosslinker bis[sulfosuccinimidyl] suberate In accordance with the study, I succeeded in detecting TrkA dimer in TrkA-transfected PC12 cells irrespective of NGF addition. No increase in the amount of TrkA dimer was detected by LPC addition, indicating that LPC does not affect dimerization of TrkA.

Conclusion

Although various biological activities have been attributed to LPC, the precise mechanism is not fully understood. Results in this study have demonstrated that LPC enhances NGF-induced MAPK and Akt signaling pathways via the extracellular domain of TrkA. Similar effect was observed in BDNF-TrkB signaling. Further analyses suggested that LPC might display neurotrophin-like effect either by promoting NGF-induced MAPK and Akt signaling cascades, or through activating additional signaling pathway independently of NGF-TrkA. Deciphering the molecular mechanism of action of LPC might provide an important clue for the development of a new therapeutic method for neurodegenerative diseases.

神経成長因子(Nerve growth factor; NGF)や脳由来神経栄養因子(Brain-derived neurotrophic factor; BDNF)などに代表されるニューロトロフィン類は神経細胞の生存や分化に必須の役割を果たしている。それらはニューロトロフィン受容体と呼ばれる一群の膜貫通型チロシンキナーゼを介して作用する。例えばNGFがその受容体であるTrkAに結合すると細胞内領域のチロシンキナーゼが活性化し、自己リン酸化が誘導される。自己リン酸化部位には様々なアダプタータンパク質がリクルートされ、それらを介して下流のRas-MAPキナーゼ(MAPK)経路等が活性化する。ニューロトロフィン類はこれらの経路を通じて神経系における機能を発揮している。

申請者の所属する研究室では、以前、微生物代謝産物を対象にニューロトロフィン様の活性を示す物質のスクリーニングが行われ、その結果、分泌型ホスホリパーゼA2(secretory PLA2; 以下sPLA2)が神経栄養因子と類似の作用を示すことが見出された。sPLA2はリン脂質のsn-2位を切断し脂肪酸とリゾリン脂質を遊離する酵素であるが、神経栄養因子作用を示すことは全く知られていなかった。さらに解析が進められた結果、sPLA2の神経栄養因子作用がリゾリン脂質の一種であるリゾホスファチジルコリン(LPC)の産生を介したものであることがわかった。興味深いことに、このような作用が見られるのはLPCのみであり、リゾホスファチジン酸(LPA)等は全く神経栄養因子作用を示さなかった。

このような背景の下、本論文はNGF-TrkA経路とsPLA2-LPC経路との間にcross-talkがあること、およびその機構を様々な実験系を用いて示したものであり、序章、結果を述べた3つの章、および総括と展望を記した終章からなる。

第一章ではラット副腎褐色細胞種由来のPC12細胞を用いた解析を行っている。PC12細胞はTrkAを発現し、NGFによってMAPK経路の活性化などを介して交感神経様に分化する。申請者はLPCが0.1および1 μMという低濃度でNGFによるMAPKの活性化(リン酸化)を数倍上昇させることを見出した。また、NGF存在下、MAPKの下流で転写誘導される最初期遺伝子c-fosなどの発現もLPCによって有意に上昇した。

NGF刺激からMAPK活性化に至る過程のどの段階をLPCが増強しているのかを明らかにするため、MAPKの上流因子であるMEK、およびNGF受容体であるTrkAの活性化を調べた。その結果、LPCはどちらの活性化も増強することが分かり、LPCが最上流に位置するTrkAの活性化を促進することで下流のシグナルも増強することが分かった。一方、LPCは上皮成長因子(Epidermal growth factor; EGF)等によるMAPK活性化は促進しなかった。従ってLPCはNGF-TrkA経路特異的に作用するものと考えられた。

第二章ではもう一つのニューロトロフィンであるBDNFとその受容体であるTrkBに対するLPCの効果を検討している。内在的にTrkBを発現する小脳顆粒細胞の初代培養を用いて検討を行ったところ、BDNFによるMAPKおよびTrkBのリン酸化がLPCによって促進されることがわかった。以上の結果から、LPCがBDNF-TrkB経路を特異的に活性化すること、即ちLPCによるシグナル増強効果はニューロトロフィン-ニューロトロフィン受容体の経路に特異的に認められることが強く示唆された。

第三章ではLPCがどのような機構でNGF-TrkA経路を活性化するかを解析している。この目的のため、CHO-K1細胞にTrkAをトランスフェクトし、NGF/LPCによるMAPKのリン酸化を調べた。その結果、TrkA発現細胞ではNGFによってMAPKが活性化し、それがLPCによって増強されることが分かった。一方、EGFもEGFR発現細胞においてMAPKの活性化を誘導したが、LPCによる増強は認められなかった。このことから、トランスフェクトした両受容体からMAPKに至る情報伝達過程がPC12細胞におけるそれを再現できることが分かった。次にTrkAのどの領域がLPCに応答するのかを調べるため、TrkAとEGFRを細胞外・膜貫通・および細胞内領域に分割してそれぞれの領域を交換することでキメラ受容体を作製し、トランスフェクトした細胞の応答を調べた。その結果、TrkAの細胞外領域を持つキメラ受容体を発現させた細胞ではNGFによるMAPKのリン酸化が誘導され、それはLPCによって増強された。一方、EGFRの細胞外領域とTrkAの膜貫通・細胞内領域のキメラ受容体を発現する細胞ではEGFによるMAPKのリン酸化は誘導されたものの、LPCによる増強は認められなかった。以上の結果から、LPCの作用にはTrkAの細胞外領域が関与すること、膜貫通や細胞内領域はLPCによる受容体の活性化促進には関与しないことが明らかとなった。

以上、本論文はLPCがニューロトロフィン-ニューロトロフィン受容体の経路を特異的に活性化する現象の発見、およびその分子機構に関する解析を行ったものであり、学術的・応用的に貢献するところが少なくない。よって審査委員一同は本論文が博士(農学)の学位論文として価値あるものと認めた。