Mammalian cells contain various kinds of phospholipases that hydrolyze phospholipids to various kinds of products to play important roles in cellular physiology. There are four classes of phospholipases: PLA (PLA1 and PLA2), PLB, PLC, and PLD, that are grouped according to the bond hydrolyzed on phospholipids substrates. Among them, phospholipase A2 hydrolyzes the sn-2 (enriched in polyunsaturated fatty acid) ester bond of glycerophospholipid to release fatty acids and lysophospholipids, and both of which have potent biological activity. Furthermore, subcellular organelles have been known to have different lipid compositions. As a result, the action of phospholipase A2 at specific membrane compartment may affect the physical properties of cellular membrane that may influence cell shape, endocytotic and secretory processes. Phospholipase A2 is classified into four categories: cytosolic PLA2 (cPLA2), Ca2+-independent PLA2 (iPLA2), secretory PLA2 (sPLA2), and platelet-activating factor acetylhydrolase (PAF-AH). So far, at least 20 gene loci for human PLA2 are found.

The cytosolic (group IV) phospholipase A2 family is composed of six intracellular enzymes named cPLA2α, cPLA2β, cPLA2γ, cPLA2δ, cPLA2ε, cPLA2ζ, all of which have different degrees of homology in lipase and C2 domains to each other except cPLA2γ, which doesn't contain C2 domain.

cPLA2α was first identified by purification of the protein, and now is most extensively studied cytosolic PLA2 enzyme. It is widely expressed in mammalian cells and has Ca2+-dependent activity, substrate preference for arachidonoyl phospholipids. Until now we have known about many biochemical and physiological property of cPLA2α such as increased expression of cPLA2α by certain proinflammatory cytokines and growth factors in some cell lines, regulation of cPLA2α membrane binding and localization by its catalytic and C2 domains, and phosphorylation, novel nuclear localization, and some cPLA2α binding proteins. Furthermore, to date, there have been many literatures about functional roles of cPLA2α in various kinds of disease models and physiological processes.

Human cPLA2γ lacks a C2 domain and is constitutively membrane bound by C-terminal CAAX sequence, which has been shown to be farnesylated in mammalian cells. Enzymatic analysis of purified human cPLA2γ has shown that it has approximately 25-100-fold greater lysophospholipase activity than PLA2 activity. It may also function as a transacylase in HEK293 cells when over-expression. In 2006, a principle variant of endogenous human cPLA2β, cPLA2β3, was identified in a human lung epithelial cell line (BEAS-2B). It has calcium dependent PLA2 activity against PE but not PC and low level lysophospholipase activity. It was constitutively associated with membrane in BEAS-2B cells and localized to mitochondria and early endosomes. In 2010, murine cPLA2β was found to have an increased activity on phosphatidylcholine vesicles by anionic phosphoinositides and cardiolipin.

In 2005, our research group identified three novel cytosolic phospholipase A2s, murine cPLA2δ, Σ and ζ by wide database queries. The preliminary data showed that they have distinct enzymatic properties, tissue distribution and subcellular localization and suggested that they may have different roles in cellular function. Human cPLA2δ was found to be expressed in stratified squamous epithelium of the cervix, fetal skin and also associated with psoriasis. In 2010, cPLA2δ was reported to have relatively higher phospholipase A1 activity than PLA2 activity. In 2007, cPLA2ζ was found to be expressed in mouse lung fibroblasts and to have higher lysophospholipase and PLA2 activity than cPLA2β, which could be inhibited by pyrrolidine-2 and Wyeth-1. In addition, EGFP- cPLA2ζ could translocate to membrane ruffles and vesicles in response to ionomycin. Preliminarily cPLA2ε was reported to be associated with lysosomes and had Ca2+-dependent PLA2 activity. In 2010, purified human cPLA2ε protein was found to have low enzymatic activity but tend to increase activity by anionic phosphoinositides on the vesicle membrane. However, its biochemical characteristics and biological role are still largely unknown.

Several phospholipase A2s are expressed in the central nervous system and play roles in the pathophysiologic aspects. There is increasing evidence for the involvement of cPLA2α in regulation of inflammation, excitatory functions and plasticity in the brain. Cytosolic PLA2α was suggested to regulate the persistent decrease in the expression of AMPA receptors, and may play a role in cerebellar LTD (long term depression) and motor learning. In the brain, the basal expression and activity of group VI iPLA2s are higher than other PLA2. In human astrocyte A172 cells, iPLA2β is present, and takes a part in lipid remodeling process. iPLA2γ is present in endoplasmic reticulum of the brain and may be involved in oxidant-induced cell death and lipid peroxidation. Mice with iPLA2β deficiency developed normally, but showed severe motor dysfunction due to degeneration of axons.

Murine cPLA2ε was identified by mouse genome search and reported that cPLA2ε was expressed in the brain, heart and skeletal muscle detected by northern blotting. To confirm its endogenous protein and investigate its biological role, polyclonal antibody was generated. Endogenous protein was detected in mouse brain, heart, and skeletal muscle by immunoprecipitation. Immunohistochemical study showed that it was expressed in neurons rather than astrocytes, a result compatible with mRNA expression detected by RT-qPCR. In addition, primary neuron culture system was used for analysis and showed that the expression of cPLA2ε was first increased then decreased during neuron/brain maturation. Furthermore, knockdown of cPLA2ε gene was conducted, and revealed that the neurons with cPLA2ε knockdown have longer total dendritic length than those with scrambled control. Therefore, murine cPLA2ε might have a unique role in dendritic development during brain development.

PLA2 enzymes principally catalyze hydrolysis at the sn-2 position to generate a lysophospholipid and a free fatty acid, although they may also possess PLA1, lysophospholipase, transacylase, and lysophospholipid acyltransferase activities. In addition to generation of lipid signaling molecules, in recent years, cytoplasmic PLA2 enzymes are also associated with various intracellular trafficking events directly or indirectly, such as the formation of membrane tubules from the Golgi complex and endosomes, and membrane fusion events in the secretory and endocytic pathways. The discovery and characterization of the SNARE family of proteins has led to a detailed understanding of protein-mediated membrane fusion. It is possible that phospholipid-modifying enzymes also play a role in membrane fusion to achieve maxima fusion efficiencies by modifying phospholipid composition of membranes. PLA2 activity has been reported to play a role in the fusion of endosomes in vivo and in vitro. Cytosolic cPLA2α is required for the formation of the traffic-dependent intercisternal tubules and for intra-Golgi transport but not peri-Golgi vesicles. Recently, some phospholipases A2 were found to locate at some organelles such as early endosomes or cytoplasmic vesicles. But its exact functions on these vesicles are still unknown.

Subcellular localization of cPLA2ε was also examined and showed that cPLA2ε was mostly co-localized with endosomes and lysosomes, not with Golgi apparatus, endoplasmic reticulum and mitochondria. In addition, centrifuged fractionation was also performed and revealed that cPLA2ε was mainly associated with the membrane. As a result, it might imply that cPLA2ε have a part in endosomal fusion and regulation.

Living cells contain various kinds of intracellular organelles, which have distinct structures and functions. They play important roles in numerous cellular events, such as cell proliferation, differentiation, maintenance, and death. However, little is known about these organelles' functions in neurons, which have extremely polarized cellular shapes. The endosomal-lysosomal system is a highly dynamic system of acidified cytoplasmic organelles that have essential roles in cellular functions. In nervous system, endocytic activity is particularly high at nerve terminals, dendritic domains and the retrograde translocation of vesicles to endosome/lysosome related compartments that might be one part of signaling communication. Endosomal pathway to lysosomal degradation is critical for dendrite development in a recent genetic study using Drosophila DA sensory neurons. The functions of recycling endosomes (REs) in neuritogenesis have been investigated by many studies using methods to block RE trafficking. Moreover, overexpression of protrudin induces neurite-like structures through its binding to Rab11, indicating protruding-rab11 system has roles in the membrane recycling required for neurite extension.

Murine cPLA2ε was present at the membranes of endosomal/lysosomal systems with interaction with Rab11 proteins revealed by immunoprecipitation, which suggested that cPLA2ε might be a participant in the endosome-mediated regulation of neuronal development. In addition, the recycling endosomes are also reported to be associated with neuronal plasticity and important for supplying postsynaptic AMPA-type glutamate receptors during LTP (long term potentiation). Therefore, it is possible as well that murine cPLA2ε might associated with the turnover of receptors in neurons through the endosomal system.

Cytosolic phospholipase A2s (except cPLA2γ), have a C2 domain that mediate protein translocation from cytosol to membranes while stimulation with Ca2+ ion in the buffer such as cPLA2α, cPLA2β3, cPLA2δ, cPLA2ζ. However, some of them may be constitutively associated with membrane regardless of calcium ion presence or not, for instance, cPLA2β1.

We found that murine cPLA2ε was mainly associated with the membrane fraction and showed no significant difference between the presence and absence of Ca2+ in the homogenizing buffer. This result was consistent with the phenomenon that murine cPLA2ε only had enzymatic activity when Triton X-100 was added in the assay buffer, and increased enzymatic activity when the membrane of vesicles contained anionic phosphoinositides. The differences of cellular localization and enzymatic activity among PLA2 isoforms may be due to the several different amino acid residues in the C2 and catalytic domains. It implies that murine cPLA2ε may have another unique way for its localization via protein-protein or protein-lipid interaction in addition to Ca2+ mediated membranous binding.

In summary, the endogenous cPLA2ε protein is confirmed in mouse tissues such as the brain, heart and skeletal muscle. In the mouse brain, it is expressed in neurons with characteristic endosomal/lysosomal subcellular localization and might serve as a factor associated with the dendritic development. Moreover, it might also possess a distinct mechanism (Ca2+-independent) to regulate its membranous association. Since the neurodegenerative disease is often associated with endosomal functions, cPLA2ε might be suspected to have some roles in neuronal degeneration.

ホスホリパーゼA2はリン脂質のsn-2位を加水分解することにより遊離脂肪酸とリゾリン脂質を生じる反応を触媒する酵素である。現在、細胞質型ホスホリパーゼA2(cPLA2)として知られる酵素にはcPLA2α~cPLA2ζの6種類が存在し、それらのうちcPLA2δ、cPLA2ε、cPLA2ζはゲノムデータベース検索により推定された新規酵素であり、その機能の詳細については殆ど知られていない。本研究は、マウス細胞質型ホスホリパーゼA2εの生体内における役割を明らかにすることを目的として、同酵素に対するポリクローナル抗体の作成、抗体を用いたマウス組織や細胞等における酵素タンパク質発現分布の解析、および、神経細胞における同酵素の細胞内局在解析、RNAi法による同酵素遺伝子の神経細胞における機能解析等を試みたものであり、下記の結果を得ている。

1.ゲノムから推定された酵素であるマウスcPLA2εが、マウス脳、心臓、骨格筋において、実際にタンパク質として内在的に発現している事が確認された。

2.マウス脳において、マウスcPLA2εはアストロサイトに比べニューロンに比較的高発現していることが示された。

3.脳もしくはニューロンの成熟過程において、マウスcPLA2εは初期に増加し、その後一定のレベルにまで減少するという特徴的な発現パターンを示した。

4.細胞内局在解析の結果、マウスcPLA2εはエンドソームに局在することが示された。

5.遠心分画法による解析の結果、マウスcPLA2εは主として沈殿画分に含まれたことから、同酵素が膜と相互作用している可能性が示された。

6.マウスcPLA2εはRab11と相互作用したことから、細胞内において小胞輸送に関わる可能性が示された。

7.初代培養ニューロンにおけるcPLA2ε遺伝子のノックダウン実験において、cPLA2εをRNAiによりノックダウンすると対照RNAiで処理されたニューロンに比べ、樹状突起の長さの総和、および分枝数が大きいことが見出され、これにより、マウスcPLA2εは樹状突起の発達に関与する可能性が示された。

以上、本論文は発現解析および機能解析により、マウスcPLA2εがニューロンにおいてエンドソームに発現し樹状突起の発達に関わる可能性を示した。本研究は、ゲノムから推定されていたがその機能は全く知られていなかったマウスcPLA2εについて、同酵素が神経系において機能している可能性を示したものであり、リン脂質加水分解酵素の神経系における機能の解明に大きく貢献すると考えられ、学位の授与に値するものと考えられる。