レビュー論文：松果体の石灰化、メラトニンの生成、老化、関連する健康への影響、松果体の若返り(Pineal Calcification, Melatonin Production, Aging, Associated Health Consequences and Rejuvenation of the Pineal Gland)

出典：https://www.mdpi.com/1420-3049/23/2/301

© 2018 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/).

© 2018年、著者による。被許諾者 MDPI、Basel、スイス。この記事は、クリエイティブ・コモンズ・アトリビューション（CC BY）ライセンス（http://creativecommons.org/licenses/by/4.0/）の条件に基づいて配布されるオープンアクセス記事です。

Authors
by Dun Xian Tan*, Bing Xu, Xinjia Zhou

Molecules 2018, 23(2), 301; https://doi.org/10.3390/molecules23020301

Submission received: 13 January 2018 / Revised: 24 January 2018 / Accepted: 26 January 2018 / Published: 31 January 2018

(This article belongs to the Special Issue Melatonin as an Antioxidant and a Functionally Pleiotropic Molecule: Synthesis, Metabolism and Activities in Organisms)

Abstract

The pineal gland is a unique organ that synthesizes melatonin as the signaling molecule of natural photoperiodic environment and as a potent neuronal protective antioxidant. An intact and functional pineal gland is necessary for preserving optimal human health. Unfortunately, this gland has the highest calcification rate among all organs and tissues of the human body. Pineal calcification jeopardizes melatonin’s synthetic capacity and is associated with a variety of neuronal diseases. In the current review, we summarized the potential mechanisms of how this process may occur under pathological conditions or during aging. We hypothesized that pineal calcification is an active process and resembles in some respects of bone formation. The mesenchymal stem cells and melatonin participate in this process. Finally, we suggest that preservation of pineal health can be achieved by retarding its premature calcification or even rejuvenating the calcified gland.

松果体は自然な光周期環境のシグナル分子として、また強力な神経保護抗酸化物質としてメラトニンを合成する特異な器官である。完全で機能的な松果体は最適なヒトの健康を維持するために必要である。残念なことに、この腺は人体のすべての器官および組織の中で最も石灰化率が高い。松果体石灰化はメラトニンの合成能を危険にさらし、様々な神経疾患と関連している。今回のレビューでは、この過程が病理学的条件下または老化中にどのように生じるかの潜在的機序を要約した。松果体の石灰化は活発な過程であり、いくつかの点で骨形成に類似していると仮定した。間葉系幹細胞とメラトニンはこの過程に関与する。最後に、松果体の健康の維持は、その早期石灰化を遅らせるか、石灰化腺を若返らせることによって達成できることを示唆する。

Keywords:
pineal gland; calcification; melatonin; aging; neurodegenerative diseases; rejuvenation

1. Introduction

Pineal gland is a unique organ which is localized in the geometric center of the human brain. Its size is individually variable and the average weight of pineal gland in human is around 150 mg [1], the size of a soybean. Pineal glands are present in all vertebrates [2]. Pineal-like organs are also found in non-vertebrate organisms such as insects [3,4,5]. It appears that the sizes of pineal glands in vertebrates are somehow associated with survival in their particular environments and their geographical locations. The more harsh (colder) their habitant, the larger their pineal glands are. A general rule is that the pineal gland increases in size in vertebrates from south to north or from the equator to the poles [6]. It is unknown whether if the same species moved to a different environment this would cause a change in the size of their pineal gland.

松果体は人間の脳の幾何学的中心に局在する特異な器官である。その大きさは個体差があり、人間の松果体の平均重量は約150mg [1]で、大豆の１個分の大きさである。松果体はすべての脊椎動物に存在する [2]。松果体様器官は昆虫などの非脊椎動物にも見られる[3、4、5]。脊椎動物の松果体の大きさは、特定の環境や地理的位置での生存と何らかの関係があるようである。生息環境が厳しい(寒い)ほど、松果体は大きくなる。一般的に、脊椎動物の松果体は南から北へ、あるいは赤道から両極へと大きくなる [6]。同じ種が異なる環境に移動した場合、松果体の大きさに変化が生じるかどうかは不明である。

It was reported that several physiological or pathological conditions indeed alter the morphology of the pineal glands. For example, the pineal gland of obese individuals is usually significantly smaller than that in a lean subject [7]. The pineal volume is also significantly reduced in patients with primary insomnia compared to healthy controls and further studies are needed to clarify whether low pineal volume is the basis or a consequence of a functional sleep disorder [8]. These observations indicate that the phenotype of the pineal gland may be changeable by health status or by environmental factors, even in humans. The largest pineal gland was recorded in new born South Pole seals; it occupies one third of their entire brain [9,10]. The pineal size decreases as they grow. Even in the adult seal, however, the pineal gland is considerably large and its weight can reach up to approximately 4000 mg, 27 times larger than that of a human. This huge pineal gland is attributed to the harsh survival environments these animals experience [11].

いくつかの生理学的または病理学的条件が実際に松果体の形態を変化させることが報告されている。例えば、肥満者の松果体は通常、痩せた被験者よりも有意に小さい [7]。松果体容積も原発性不眠症患者では健常対照者と比較して有意に減少しており、松果体容積の低さが機能性睡眠障害の基礎なのか結果なのかを明らかにするためにはさらなる研究が必要である [8]。これらの観察は、松果体の表現型が、人間においてさえ、健康状態または環境因子によって変化し得ることを示す。最大の松果体は、生まれたばかりの南極アザラシで記録された；それは脳全体の1/3を占めています [9, 10]。松果体の大きさは成長とともに減少する。しかし、アザラシの成体でも松果体はかなり大きく、その重量はヒトの27倍の約4000mgに達する。この巨大な松果体は、これらの動物が経験する過酷な生存環境に起因する [11]。

The human pineal gland has been recognized for more than 2000 years. The father of anatomy, the Greek anatomist, Herophilus (325–280 BC), described the pineal gland as a valve of animal memory. René Descartes (1596–1650), a French philosopher, mathematician, and scientist, regarded the pineal gland as the principal seat of the soul and the place in which all thoughts are formed. A real biological function of pineal gland was not uncovered until 1958 [12], that is, this gland is a secretory organ which mainly produces and releases a chemical, called melatonin, into the blood circulation and into the cerebrospinal fluid (CSF). In addition, it also produces some peptides [13,14] and other methylated molecules, for example, N,N-dimethyltryptamine (DMT or N,N-DMT) [15,16], a potent psychedelic. This chemical was suggested to be exclusively generated by the pineal gland at birth, during dreaming, and/or near death to produce “out of body” experiences [17]. However, the exact biological consequences (if any) of these substances remain to be clarified. Recently, it was reported that pineal gland is an important organ to synthesize neurosteroids from cholesterol. These neurosteroids include testosterone (T), 5α- and 5β-dihydrotestosterone (5α- and 5β-DHT), 7α-hydroxypregnenolone (7α-OH PREG) and estradiol-17β (E2). The machinery for synthesis of these steroids has been identified in the gland. 7α-OH PREG is the major neurosteroid synthesized by the pineal gland. Its synthesis and release from gland exhibits a circadian rhythm and it is regulates the locomote activities of some vertebrates, especially in birds [18]. These observations opened a new avenue for functional research on pineal gland; the observations require further confirmation.

人間の松果体は2000年以上前から認識されている。解剖学の父であるギリシャの解剖学者Herophilus(紀元前325~280年)は、松果体を動物の記憶の弁と表現した。フランスの哲学者、数学者、科学者であったRené Descartes(1596–1650)は、松果体を魂の主要な座であり、すべての思考が形成される場所であるとみなしました。松果体の真の生物学的機能は1958年 [12]まで明らかにされなかった、すなわち、松果体は主にメラトニンと呼ばれる化学物質を産生し、血液循環および脳脊髄液(CSF)に放出する分泌器官である。さらに、いくつかのペプチド[13, 14]や他のメチル化分子、たとえば強力な幻覚剤であるN, N-ジメチルトリプタミン(DMTまたはN, N-DMT) [15, 16]も産生する。この化学物質は、出生時、夢を見ている時、死の間際に松果体でのみ生成され、「幽体離脱」体験をもたらすと示唆されている [17]。しかしながら、これらの物質の正確な生物学的影響(もしあるとすれば)は明らかにされていない。最近、松果体がコレステロールから神経ステロイドを合成する重要な器官であることが報告された。これらの神経ステロイドにはテストステロン(T)、5α‐および5β‐ジヒドロテストステロン(5α‐および5β‐DHT)、7α‐ヒドロキシプレグネノロン(7α‐OH PREG)およびエストラジオール‐17β(E2)が含まれる。これらのステロイドを合成する装置は腺で同定されている。7α-OH PREGは松果体で合成される主要な神経ステロイドである。その合成と腺からの放出は概日リズムを示し、一部の脊椎動物、特に鳥類の移動活動を調節している [18]。これらの観察は松果体の機能研究に新しい道を開いた；観察はさらなる確認を必要とする。

The most widely accepted concept is that melatonin is the recognized major product of the pineal gland. Melatonin is the derivative of tryptophan. It was first isolated from the pineal gland of the cow and it was initially classified as a neuroendocrine-hormone [19]. Subsequently, it was discovered that retina [20,21] and Harderian gland [20,22,23,24] also produced melatonin. Recently, it has been found that almost all organs, tissues and cells tested have the ability to synthesize melatonin using the same pathway and enzymes the pineal uses [25,26]. These include, but not limited to, skin [27], lens [28], ciliary body [29,30], gut [31,32], testis [33], ovary [34,35], uterus [36], bone marrow [37,38], placenta [39,40], oocytes [41], red blood cells [42], plantlets [43], lymphocytes [44], astrocytes, glia cells [45], mast cells [46] and neurons [47]. Not only melatonin but also the melatonin biosynthetic machinery including mRNA and proteins of arylalkylamine N-acetyltransferase (AANAT) and/or N-acetyl-serotonin methyltransferase (ASMT) [formerly hydroxyindoleO-methyltransferase (HIOMT)] have been identified in these organs, tissues and cells. It was calculated that the amounts of extrapineal derived melatonin is much greater than that produced by the pineal [48]. However, the extra pineal-derived melatonin cannot replace/compensate for the role played by the pineal-derived melatonin in terms of circadian rhythm regulation. As we know pineal melatonin exhibits a circadian rhythm in circulation and in the CSF with a secretory peak at night and low level during the day [19]; thus, the primary function of the pineal-derived melatonin is as a chemical signal of darkness for vertebrates [49]. This melatonin signal helps the animals to cope with the light/dark circadian changes to synchronize their daily physiological activities (feeding, metabolism, reproduction, sleep, etc.).

最も広く受け入れられている概念は、メラトニンが松果体の主要産物として認識されているというものである。メラトニンはトリプトファンの誘導体である。最初に牛の松果体から単離され、当初は神経内分泌ホルモンに分類された [19]。その後、網膜[20, 21]とハーダー腺[20, 22, 23, 24]もメラトニンを産生することが発見された。最近、試験されたほとんどすべての器官、組織および細胞は、松果体が使用するのと同じ経路および酵素を使用してメラトニンを合成する能力を有することが見出されている [25, 26]。これらには、皮膚[27]、水晶体[28]、毛様体[29, 30]、腸[31, 32]、精巣[33]、卵巣[34, 35]、子宮 [36]、骨髄[37, 38]、胎盤[39, 40]、卵母細胞[41]、赤血球[42]、小植物[43]、リンパ球[44]、星状細胞、グリア細胞[45]、肥満細胞[46]およびニューロン[47]が含まれるが、これらに限定されない。メラトニンだけでなく、アリルアルキルアミン
N‐アセチルトランスフェラーゼ(AANAT)および/またはN‐アセチル‐セロトニンメチルトランスフェラーゼ(ASMT)[以前はヒドロキシインドールO‐メチルトランスフェラーゼ(HIOMT)]のmRNAおよび蛋白質を含むメラトニン生合成機構もこれらの器官、組織および細胞で同定されている。松果体由来のメラトニンの量は松果体で産生される量よりはるかに多いと計算されている [48]。しかしながら、松果体由来の余分なメラトニンは、概日リズム調節に関して松果体由来のメラトニンが果たす役割を置換/補償できない。私たちが知っているように、松果体メラトニンは循環中およびCSF中で概日リズムを示し、夜間に分泌ピークを示し、日中は低レベルである [19]。したがって、松果体由来のメラトニンの主な機能は、脊椎動物にとって暗闇の化学シグナルである [49]。このメラトニン信号は、動物が日内の明暗の変化に対処し、日々の生理的活動(摂食、代謝、生殖、睡眠など)を同調させるのを助ける。

For the photoperiod sensitive reproductive animals, the melatonin signal regulates their reproductive activities to guide them to give birth during the right seasons [50]. Interestingly, even low ranking species that lack a pineal gland, for example, marine zooplankton, also exhibit a melatonin circadian rhythm which is responsible for their daily physiological activities [51]. While, the extrapineal melatonin in vertebrates does not contribute to the melatonin circadian rhythm and it does not serve as the chemical signal of darkness since pinealectomy in animals distinguishes this rhythm [52,53,54]. This was further confirmed by the recent discovery that the expressions of AANAT and ASMT are present in mitochondria of both pinealocytes and neuron cells and their mitochondria synthesized melatonin. However, the expressions of AANAT and ASMT exhibit a circadian rhythm that matched the fluctuation in melatonin levels only in the mitochondria of pineal gland while this rhythm was absent in the mitochondria of neuronal cells [55]. Thus, the primary function of extrapineal melatonin (except for the retina; retinas not only possess an internal melatonin rhythm [56,57]; retinal melatonin might participate in melatonin circadian rhythm of the general circulation in some species [58,59,60]) is to serve as an antioxidant, autocoid, paracoid and tissue factor locally [49,61].

光周期に敏感な生殖動物では、メラトニン信号が生殖活動を調節し、適切な季節に出産するように導いている [50]。興味深いことに、松果体を持たない低ランクの種、例えば海洋性動物プランクトンでさえ、日々の生理的活動の原因となるメラトニンの概日リズムを示す [51]。一方、脊椎動物の松果体外メラトニンはメラトニン概日リズムに寄与せず、動物の松果体切除はこのリズムを識別するため、暗さの化学シグナルとしては機能しない [52, 53, 54]。このことは、AANATとASMTの発現が松果体細胞とニューロン細胞の両方のミトコンドリアに存在し、それらのミトコンドリアがメラトニンを合成するという最近の発見によってさらに確認された。しかし、AANATとASMTの発現は、松果体のミトコンドリアでのみメラトニンレベルの変動と一致する概日リズムを示すが、このリズムは神経細胞のミトコンドリアには存在しない [55]。このように、松果体外メラトニンの主な機能(網膜を除く；網膜にはメラトニンリズム[56, 57]があるだけでなく；網膜メラトニンはいくつかの種において全身循環のメラトニン概日リズムに関与するかもしれない[58, 59, 60])は、抗酸化剤、オートコイド、パラコイドおよび組織因子として局所的に働くことである[49, 61]。

In addition to synthesizing the “signaling-melatonin” which differs from extrapineal melatonin, the pineal gland also participates in the CSF production and recycling. The blood filtration rate of this gland is comparable to the kidney [62]; this is, far more than its metabolic requirement. It was hypothesized that pineal gland may function like the kidney as a blood filter to generate CSF; this is similar to the function of choroid plexus to recycle the CSF [63]. Pineal gland and choroid plexus share a similar vasculature structure with the abundance of the vasculature spaces and fenestrated capillaries. A direct morphological connection between pineal gland and choroid plexus has been reported in birds [64]. The functional and vascular structural similarities may explain the high calcification rates of both structures [65,66].

松果体は、松果体外メラトニンとは異なる「シグナル伝達メラトニン」の合成に加えて、CSFの産生とリサイクルにも関与している。この腺の血液濾過率は腎臓に匹敵する [62]；これは、その代謝要求をはるかに超えています。松果体は腎臓と同様にCSFを産生する血液フィルターとして機能しているのではないかという仮説が立てられた；これは脳脊髄液をリサイクルする脈絡叢の機能に似ている [63]。松果体と脈絡叢は、豊富な脈管構造空間と有窓毛細血管を有する類似の脈管構造を共有する。鳥類では、松果体と脈絡叢の直接的な形態学的関連が報告されている [64]。機能的および血管構造的類似性は、両構造の高い石灰化率を説明する可能性がある[65, 66]。

The calcium deposits in the pineal gland were recognized several decades ago in vertebrates [67,68]. Some researchers believe that pineal calcification was associated with certain endocrine diseases such as schizophrenia, and mammary carcinoma [69,70,71,72,73,74,75]. Others feel that it is a natural process and has no consequences for human physiopathology since this process occurs early in childhood [76] and it also may not impact the melatonin synthetic ability of the gland in some animals [77,78]. Recently, additional studies have shown that pineal calcification indeed jeopardizes the melatonin production in humans and it seems to have a direct influence on neurodegenerative diseases and aging [79,80,81]. This review summarizes the current developments in the field and also provides opinions and comments on pineal physiology and pineal gland calcification (PGC).

松果体におけるカルシウム沈着は数十年前に脊椎動物で確認された[67, 68]。一部の研究者は、松果体石灰化が統合失調症や乳癌などの内分泌疾患と関連していると考えている[69, 70, 71, 72, 73, 74, 75]。他の研究者は、これは自然なプロセスであり、このプロセスは小児期の早期に起こるため、ヒトの生理病理学に影響を与えないと感じており[76]、また、一部の動物では腺のメラトニン合成能力に影響を与えない可能性もある[77, 78]。最近、追加の研究は、松果体石灰化がヒトにおけるメラトニン産生を実際に危険にさらし、それが神経変性疾患と老化に直接影響するようであることを示した[79, 80, 81]。このレビューは、この分野における最近の発展を要約し、松果体生理学と松果体石灰化(PGC)に関する意見とコメントも提供する。

2. Pineal Gland and the Melatonin Circadian Rhythm(松果体とメラトニン概日リズム)

The pineal gland is situated in the geometric center of the human brain and it is directly connected to the third ventricle; it is classified as a circumventricular organ (CVO) and participates in the biological rhythm regulation in vertebrates. Herein, we refer to the structures which regulate biorhythms as the suprachiasmatic nucleus (SCN)-melatonin loop. This loop includes melanopsin-containing retinal ganglion cells (MRGC), retino-hypothalamic tract (RHT), SCN, paraventricular nucleus (PVN), Intermediolateral cell column, sympathetic cervical ganglia (SCG), the pineal gland, melatonin rhythm which feedback impacts the SCN (Figure 1).

松果体は人間の脳の幾何学的中心に位置し、第３脳室に直接つながっている。脳室周囲器官(CVO)として分類され、脊椎動物の生体リズム調節に関与している。ここでは、視交叉上核(SCN)‐メラトニンループとして生体リズムを調節する構造を参照する。このループは、メラノプシン含有網膜神経節細胞(MRGC)、網膜視床下部路(RHT)、SCN、室傍核(PVN)、中間外側細胞柱、交感神経頸神経節(SCG)、松果体、フィードバックがSCNに影響を与えるメラトニンリズムを含む(図1)。

Figure 1. Illustration of the SCN-melatonin loop. Solid arrows indicate the neuronal connections and the direction of neuronal projections. Dash arrows indicate the input signals. SCN is the master clock which determines the biological rhythms as well as the melatonin circadian rhythm. Its intrinsic circadian interval is longer than 24 h. The natural photoperiod (photos) serves as an input signal to entrain melatonin circadian rhythm to 24 h; in turn, melatonin functions as a signal of photoperiod to re-entrain the biological rhythm of SCN to 24 h. MRGC: melanopsin-containing retinal ganglion cells; RHT: retino-hypothalamic tract; SCN: suprachasmatic nucleus; PVN: paraventricular nucleus; IMCC: Intermediolateral cell column; SCG: sympathetic cervical ganglion; PG: pineal gland. Mel: melatonin.

図１．SCN-メラトニンループの図。実線の矢印はニューロンの連結とニューロンの投射方向を示す。ダッシュ矢印は入力信号を示します。SCNは生体リズムとメラトニン概日リズムを決定するマスタークロックである。その固有の概日間隔は24時間より長い。自然光周期 (写真) はメラトニン概日リズムを24時間同調させるための入力シグナルとして機能する；次に、メラトニンはSCNの生体リズムを24時間に再同調させる光周期のシグナルとして機能する。MRGC：メラノプシン含有網膜神経節細胞；RHT：網膜視床下部路；SCN：視交叉上核；PVN：室傍核；IMCC：中間外側セルカラム；SCG：交感神経頸神経節；PG：松果体。Mel：メラトニン。

Any defect of the loop results in a diminished melatonin circadian rhythm and the disturbance of chronobiology. For example, SCN or PVN lesions [82,83,84], blockade of the cervical ganglia [85,86] or pinealectomy [52,53] is always accompanied by the loss of the melatonin rhythm in vertebrates. This loop is important to regulate the biological rhythms of vertebrates. SCN is believed to be the master clock or the pacemaker [87]. This pacemaker has its internal circadian timer which is longer than 24 h. It is synchronized to 24 h circadian rhythm by the environmental photoperiod clues. However, melatonin is a major chemical message to synchronize its activity of SCN [88].

ループの欠損はメラトニン概日リズムの減少と時間生物学の障害をもたらす。例えば、SCNまたはPVNの病変[82, 83, 84]、頚神経節の遮断[85, 86]または松果体切除[52, 53]は、脊椎動物では常にメラトニンリズムの消失を伴う。このループは脊椎動物の生体リズムの調節に重要である。SCNはマスタークロックあるいはペースメーカーと考えられている [87]。このペースメーカーは24時間より長い体内概日タイマーを有する。環境の光周期の手がかりにより24時間の概日リズムに同調する。しかし、メラトニンはSCNの活性を同調させる主要な化学メッセージである [88]。

Melatonin membrane receptors have been identified in the SCN of vertebrates [56,89] and the signal transduction pathways seemed to be involved in both MT1 and MT2 to induce an increase in the expression of two clock genes, Period 1 (Per1) and Period 2 (Per2) [89,90,91]. Without the feedback information of melatonin, SCN would not properly interpret the natural photoperiodic changes [92] and would exhibit a free running internal rhythm in which the cycle is longer than 24 h. In this situation the SCN would also instruct the pineal gland to exhibit an unusual melatonin circadian rhythm which is also longer than 24 h. This phenomenon is apparent in completely blind animals and humans whose eyes, specifically the MRGC, do not appropriately receive environmental photoperiodic information [93,94,95]. Importantly, melatonin administration to blind subjects partially re-entrains their biological rhythms close to normal [94,96,97].

メラトニン膜受容体は脊椎動物のSCNで同定されており[56, 89]、シグナル伝達経路はMT1とMT2の両方に関与し、2個の時計遺伝子、Period1(Per1)とPeriod2(Per2)の発現増加を誘導するようであった[89, 90, 91]。メラトニンのフィードバック情報がなければ、SCNは自然の光周期変化を適切に解釈できず[92]、周期が24時間より長い自由走行内部リズムを示す。このような状況では、SCNは松果体に、やはり24時間より長い異常なメラトニン概日リズムを示すように指示する。この現象は、眼、特にMRGCが環境の光周期情報[93, 94, 95]を適切に受け取らない完全に盲目の動物と人間で明白である。重要なことに、盲目の被験者へのメラトニン投与は、正常に近い生体リズムを部分的に再同調させる[94, 96, 97]。

Pineal gland is mainly comprised of pinealocytes, microglia and astrocytes. The lineage of pinealocytes is elusive. Current information suggests that pinealocytes are differentiated from Pax6-expresssing neuroepithelial cells [98]. They are specialized to synthesize and release melatonin (and possible some other substances). This explains why pinealocytes with two special characteristics regarding their mitochondria. First, the pinealocytes contain many more mitochondria than those of neuronal cells. Second, the morphologies of these mitochondria exhibit obvious dynamic alterations related to their fission, fusion and mitophagy activities during a 24 h period [99]. Because of the high density of mitochondria, we speculated the mitochondria are the major sites for melatonin synthesis [100]. Subsequent studies have proven this speculation. Melatonin synthesis was identified in the mitochondria of both animal and plant cells [101,102]. Recently, this was further confirmed by Suofa et al. [55]. They observed that the mitochondria are the exclusive sites of melatonin production in pinelocytes and in neuronal cells. The exact subsite of melatonin synthesis occurred in the matrix of mitochondria. Thus, the numerous mitochondria in pinealocytes relate to their melatonin synthetic function. This does not naturally exclude the extra-mitochondrial melatonin production. In cytosol, melatonin can also be synthesized. For example, red blood cells and platelets which are without mitochondria still produced melatonin [42,43]. Due to the substrate, particularly acetyl coenzyme A availability, melatonin synthesis in the extra-mitochondrial sites would not be as efficient as in the mitochondria since acetyl coenzyme A is concentrated in the mitochondria [99].

松果体は主に松果体細胞、ミクログリア、星状細胞から成る。松果体細胞の系統は捉えどころがない。現在の情報では、松果体細胞はPax6発現神経上皮細胞から分化することが示唆されている[98]。これらはメラトニン(およびおそらく他の物質)の合成と放出に特化している。これは、松果体細胞がミトコンドリアに関して２個の特別な特徴を有する理由を説明する。第１に、松果体細胞は神経細胞よりも多くのミトコンドリアを含んでいる。第２に、これらのミトコンドリアの形態は、24時間の間に分裂、融合およびマイトファジー活性に関連した明らかな動的変化を示す[99]。ミトコンドリアの密度が高いことから、ミトコンドリアはメラトニン合成の主要な部位であると考えられた[100]。その後の研究はこの推測を証明した。メラトニン合成は動物細胞と植物細胞の両方のミトコンドリアで同定された[101, 102]。最近、これはSuofaら[55]によってさらに確認された。彼らは、ミトコンドリアが松果体細胞および神経細胞におけるメラトニン産生の独占的部位であることを観察した。メラトニン合成の正確なサブサイトはミトコンドリアのマトリックスで起こった。このように、松果体細胞の多数のミトコンドリアはメラトニン合成機能に関連している。これは当然、ミトコンドリア外のメラトニン産生を自然に排除するものではない。細胞質ではメラトニンも合成される。たとえば、ミトコンドリアがなくても赤血球や血小板はメラトニンを産生する[42, 43]。基質、特にアセチル補酵素Aの利用可能性のために、ミトコンドリア外部位でのメラトニン合成は、アセチルコエンザイムAがミトコンドリアに濃縮されているので、ミトコンドリア内ほど効率的ではないであろう [99]。

As to the mitochondrial dynamic alterations, generally, at darkness when melatonin is at its synthetic peak, more mitochondrial fusion was observed and, during the day, more fission was obvious. It was speculated that the mitochondrial dynamic changes were associated with their function, i.e., to produce melatonin [103]. However, current studies have reported that melatonin per se can regulate mitochondrial morphology [104,105]. Melatonin upregulates the levels of mitochondrial fusion proteins mitofusin 1 (Mfn1) and Opa1 to promote mitochondrial fusion [106,107] and inhibits the nuclear translocation of dynamin-related protein 1 (DrP1). The nuclear translocation of DrP1 increases mitochondrial fission and the inhibition of DrP1 nuclear translocation by melatonin results in suppression of mitochondrial fission [108,109,110,111,112]. Thus, the net result of melatonin is to promote the mitochondrial fusion and to reduce mitochondrial fission.

ミトコンドリアの動的変化に関しては、一般的に、メラトニンが合成ピークにある暗闇では、より多くのミトコンドリア融合が観察され、日中ではより多くの分裂が明らかであった。ミトコンドリアの動的変化は、その機能、すなわちメラトニンを産生することと関連していると推測された[103]。しかし、最近の研究では、メラトニン自体がミトコンドリアの形態を調節できることが報告されている[104,105]。メラトニンはミトコンドリア融合蛋白質ミトフシン1(Mfn1)とOpa1のレベルを上方調節してミトコンドリア融合を促進し[106, 107]、ダイナミン関連蛋白質1(DrP1)の核移行を阻害する。DrP1の核移行はミトコンドリア分裂を増加させ、メラトニンによるDrP1の核移行の阻害はミトコンドリア分裂の抑制をもたらす [108, 109, 110, 111, 112]。したがって、メラトニンの最終的な結果は、ミトコンドリア融合を促進し、ミトコンドリア分裂を減少させることである。

The effects of melatonin on mitophagy are still elusive. Some reports document that melatonin inhibits mitophagy and others show that melatonin promotes this process depending on the experimental conditions and cell type [109,113,114,115,116,117,118,119,120,121]. Currently it is not possible to determine whether the mitochondrial dynamic changes in pinealocytes relate to their functional activity which may be controlled by the clock genes, such as perd1, 2 or a result of their melatonin production rhythm. Thus, do the changes in melatonin levels generated by pinealocytes result in the mitochondrial dynamic changes.

マイトファジーに対するメラトニンの効果はまだつかみどころはない。いくつかの報告は、メラトニンがマイトファジーを阻害することを実証し、他の報告は、メラトニンが実験条件と細胞型に依存してこのプロセスを促進することを示している[109, 113, 114, 115, 116, 117, 118, 119, 120, 121]。現在、松果体細胞におけるミトコンドリアの動的変化が、perd1, 2のような時計遺伝子によって制御される機能的活性に関係しているのか、あるいはメラトニン産生リズムの結果なのかを決定することは不可能である。このように、松果体細胞によって生成されたメラトニンレベルの変化はミトコンドリアの動的変化をもたらすのであろうか。

In addition to the pinealocytes, the astrocytes and the microglia in the pineal gland also have the capacity to synthesize melatonin with great efficiency. The melatonin synthetic machinery including AANAT/SNAT and HIOMT/ASMT has been identified and melatonin production has been detected in these cells [45,122]. Markus et al. [123] hypothesized that melatonin synthesis was coordinated by both pinealocytes and macrophages/glia and astrocytes for the immunoresponse. For example, an acute inflammatory response drives the transcription factor, NFκB, to switch melatonin synthesis from pinealocytes to macrophages/microglia and, upon acute inflammatory resolution, back to pinealocytes. A participation of melatonin production by these cells would significantly improve the capacity of pineal gland to generate melatonin as a whole. This is particularly important in the situations in which melatonin is required, for example under the oxidative stress or inflammation. It was reported that CSF melatonin and its oxidative metabolites, AFMK, are elevated by several orders of magnitude in patients with meningitis [124]. This is probably the outcome of a coordinated physiology of all of the cells mentioned above. However, the major function of astrocytes and the microglia in the pineal gland is to regulate the pinealocyte melatonin synthesis under normal conditions. The regulatory mechanisms are well documented, i.e., astrocytes/glia are excited under different conditions which include elevated intracellular calcium concentration, which results in the NF-𝜅B activation. The excited astrocytes/microglia, then, release soluble TNFα which is the signaling molecule to the pinealocytes for inhibition of melatonin synthesis by targeting AANAT [98,125,126]. Other regulatory mechanisms may also be involved. For example, purinergic signaling on melatonin synthesis in pineal gland was reported [127]. ATP binding to its receptor in pinealocytes inhibits melatonin synthesis via suppression of the gene expression as well as the activity of the ASMT rather than the AANAT.

松果体細胞に加えて、松果体の星状膠細胞とミクログリアもメラトニンを効率よく合成する能力をもっている。AANAT/SNATおよびHIOMT/ASMTを含むメラトニン合成装置が同定されており、メラトニン産生がこれらの細胞で検出されている[45,122]。Markusら[123]は、メラトニン合成は松果体細胞、マクロファージ/グリア細胞、星状膠細胞の両者によって協調して免疫応答が起こるという仮説を立てた。例えば、急性炎症反応は転写因子NFκBを駆動し、メラトニン合成を松果体細胞からマクロファージ/ミクログリアに切り替え、急性炎症が消失すると松果体細胞に戻す。これらの細胞によるメラトニン産生の関与は、全体としてメラトニンを産生する松果体の能力を有意に改善するであろう。これは、メラトニンが必要とされる状況、例えば酸化ストレスまたは炎症下で特に重要である。髄膜炎患者では、髄液メラトニンとその酸化代謝物AFMKが数桁上昇することが報告されている[124]。これはおそらく、上述したすべての細胞の協調的な生理作用の結果である。しかし、松果体における星状細胞とミクログリアの主な機能は、正常条件下で松果体細胞のメラトニン合成を調節することである。調節メカニズムは十分に文書化されている、すなわち、星状細胞/グリアは、細胞内カルシウム濃度の上昇を含む異なる条件下で興奮し、NF-κB活性化をもたらす。興奮したアストロサイト/ミクログリアは、AANATを標的としてメラトニン合成を阻害するためのシグナル分子である可溶性TNFαを松果体細胞に放出する[98, 125, 126]。他の調節メカニズムも関与している可能性がある。例えば、松果体におけるメラトニン合成に対するプリン作動性シグナル伝達が報告されている[127]。松果体細胞におけるその受容体へのATP結合は、AANATよりむしろASMTの活性と同様に遺伝子発現の抑制を介してメラトニン合成を阻害する。

Once melatonin is synthesized in the pineal gland, it is rapidly released. The outlets for melatonin release are several. The classic concept is that pineal melatonin is released into the precapillary spaces, enters the capillaries, and then, via surrounding veins and sinuses reaches the general circulation. However, a more important route for melatonin release from pineal gland was uncovered, i.e., melatonin is directly release to the CSF of the third ventricle of the mammals. Compelling evidence supports this secretory route. Anatomically, a portion of pineal gland is nakedly-exposed into the CSF of the third ventricle (bathed by the CSF) [128] and many canaliculi of the pineal gland directly open into the CSF of third ventricle [129,130,131,132]. Pineal melatonin via these canaliculi is directly discharge into the CSF. This results in extremely high melatonin level in the CSF of pineal recess of third ventricle. In the sheep, the melatonin levels in the CSF of pineal recess of third ventricle are several orders of magnitude higher than those in the blood [133,134]. A melatonin concentration gradient in the CSF around the pineal recess of third ventricle was observed [135]. This indicated that the main source of CSF melatonin originated from the pineal recess of the third ventricle. The high melatonin levels in CSF have been reported in different species [136]. It is obvious that there are, at least, two parallel melatonin secretory routes, i.e., the general circulation and the CSF (Figure 3). The key question is which one probably transduces the photoperiodic information of retinas to the SCN-pineal loop, particularly to the SCN. If, as previously predicted, the CSF melatonin was from the blood, there was no doubt that general circulatory melatonin was the signal. However, it is known that the CSF melatonin is not from the blood but it is directly derived from the pineal gland; thus, the blood melatonin circadian rhythm as the signal of natural photoperiodic information is open to question, at least in terms of the major signal. Based on the evidence, it was hypothesized that the CSF melatonin released by pineal gland rather than the blood melatonin served as the signal of the natural photoperiodic information [135,137,138]. SCN is close to the third ventricle and the high levels of CSF melatonin can easily be transported into the SCN via simple diffusion or via tanycytes; these cells possess basal processes for the transport of small molecules including melatonin and this melatonin directly targets the SCN as the signaling molecule [130].

メラトニンは松果体で合成されると速やかに放出される。メラトニン放出の出口はいくつかある。古典的な概念は、松果体メラトニンが前毛細血管腔に放出され、毛細血管に入り、周囲の静脈および洞を経て全身循環に達するというものである。しかしながら、松果体からのメラトニン放出のより重要な経路、すなわちメラトニンはほ乳類の第３脳室のCSFに直接放出されることが明らかになった。この分泌経路を支持する有力な証拠がある。解剖学的には、松果体の一部が第３脳室のCSFにむき出しで露出しており(CSFに浸されている)[128]、松果体の多くの小管が第３脳室のCSFに直接開口している[129, 130, 131, 132]。松果体メラトニンは、これらの小管を介してCSFに直接放出される。この結果、第３脳室松果体陥凹の脳脊髄液中のメラトニン濃度が著しく高くなる。羊では、第で脳室松果体陥凹のCSF中のメラトニン濃度は、血液中の濃度よりも数桁高い[133, 134]。第３脳室の松果体陥凹周辺のCSFにおけるメラトニン濃度勾配が観察された[135]。このことは、CSFメラトニンの主な供給源が第３脳室の松果体陥凹に由来することを示した。脳脊髄液中のメラトニン濃度が高いことは、さまざまな動物種で報告されている[136]。少なくとも２個の平行したメラトニン分泌経路、すなわち全身循環とCSFがあることは明らかである(図3)。重要な問題は、網膜の光周期情報をSCN‐松果体ループ、特にSCNに伝達するのはどちらかということである。以前に予測されたように、脳脊髄液のメラトニンが血液由来であるならば、全身循環のメラトニンがシグナルであることは疑いない。しかし、脳脊髄液のメラトニンは血液由来ではなく、直接松果体由来であることが知られている。したがって、自然の光周期情報のシグナルとしての血中メラトニン概日リズムは、少なくとも主要なシグナルに関しては疑問の余地がある。これらの証拠に基づき、血中メラトニンよりも松果体から放出される脳脊髄液メラトニンが自然光周期情報のシグナルとして機能しているという仮説が立てられた[135, 137, 138] 。SCNは第３脳室に近く、高濃度の脳脊髄液メラトニンは単純拡散またはタニサイトを介してSCNに容易に輸送される。これらの細胞はメラトニンを含む小分子輸送のための基本過程を有し、このメラトニンはシグナル伝達分子としてSCNを直接標的とする[130] 。

It is now a common knowledge that many foods contain melatonin. These include herbs, vegetables, fruits, cereals, beans, eggs, meets, fish, milk, wine, beer and coffee [139,140,141,142,143,144]. Consumption of these foodstuffs increases the circulating melatonin levels [145]. In some cases, the food-derived melatonin could elevate serum melatonin levels as high as the night time peak levels of melatonin [146,147,148,149]. Whether this food-derived melatonin alters the signaling information and produces chronobiological consequences remains unknown. If the melatonin in general circulatory system serves as the photoperiodic signaling; food-derived melatonin may have the chronobiological effects. If the CSF melatonin serves as the exclusive signaling to the SCN, the serum melatonin derived from food would not impact the chronobiology since the food-derived melatonin unlikely reaches night time CSF melatonin levels. The SCN is likely regulated by the high levels and square shape of melatonin rhythm that is completely different from the serum melatonin as to their shapes and it would not response to the low level and other shapes of melatonin rhythm input [63] (Figure 2). Thus, the photoperiod induced melatonin message is a precise trait that would not be influenced by non-CSF melatonin level alterations.

多くの食品にメラトニンが含まれていることは今や常識となっている。これらには、ハーブ、野菜、果物、シリアル、豆、卵、ミート、魚、牛乳、ワイン、ビール、コーヒーが含まれる[139, 140, 141, 142, 143, 144]。これらの食品の摂取は血中メラトニン濃度を上昇させる[145]。場合によっては、食物由来のメラトニンは、血清メラトニン濃度を夜間のメラトニンのピーク濃度まで上昇させることができる[146, 147, 148, 149]。この食物由来メラトニンがシグナル伝達情報を変化させ、時間生物学的結果をもたらすかどうかは不明である。一般循環系のメラトニンが光周期性シグナル伝達として働く場合；食物由来のメラトニンは時間生物学的効果を有する可能性がある。もしCSFメラトニンがSCNへの独占的なシグナル伝達として働くならば、食物由来のメラトニンが夜間CSFメラトニンレベルに達する可能性は低いので、食物由来の血清メラトニンは時間生物学に影響しないであろう。SCNは、その形状が血清メラトニンとは全く異なる高レベルおよび正方形のメラトニンリズムによって制御されている可能性が高く、低レベルおよび他の形状のメラトニンリズム入力には応答しない[63](図2)。したがって、光周期誘導メラトニンメッセージは、非CSFメラトニンレベルの変化に影響されない正確な特性である。

Figure 2. The different levels and shapes of melatonin circadian rhythms in the CSF of the third ventricle and in the peripheral blood. The nightly melatonin levels in CSF of the third ventricle are more robust than those in the peripheral plasma and also exhibit sharp rises and falls (square wave). The pattern of the melatonin circadian rhythm in CSF of the third ventricle is similar to that of the pineal gland rather than in the plasma (see the insert part which illustrates the melatonin synthetic pattern in pineal gland of rat). The data was obtained from the long term (5 days) pineal gland dialysis in a free running rat. Extrapineal-generated melatonin and the diet-derived melatonin may increase peripheral plasma melatonin levels; however, they do not mimic the pattern and reach the high level of the melatonin circadian rhythm in the CSF of the third ventricle to impact the function of bio-clock. From Tan et al. [63].

図２．第３脳室の脳脊髄液と末梢血におけるメラトニンの概日リズムの異なるレベルと形状。第３脳室の脳脊髄液中の夜間メラトニンレベルは末梢血漿中のメラトニンレベルよりも強く、また急激な上昇と下降(方形波)を示す。第３脳室のCSFにおけるメラトニンの概日リズムのパターンは、血漿よりもむしろ松果体のパターンに類似している(ラットの松果体におけるメラトニン合成パターンを示す挿入部分を参照)。データは、自由走行ラットにおける長期(5日間)松果体透析から得た。松果体外産生メラトニンおよび食事由来メラトニンは末梢血漿メラトニン濃度を上昇させる可能性がある；しかしながら、このパターンを模倣することはなく、第３脳室のCSFでメラトニン概日リズムの高レベルに達し、体内時計の機能に影響を与えない。Tanら[63]より。

The high levels of CNS melatonin also exhibit protective effects on the brain tissue [130]. Pinealectomy results in the accelerated neurodegenerative changes and evidence of premature aging in animals [150,151,152,153]. Pineal grafts in brain protected the brain from oxidative damage induced by the ischemia/reperfusion in mice [154]. These effects are mainly attributed to the antioxidative, anti-inflammatory and anti-apoptotic effects of CSF melatonin directly released by pineal gland.

高濃度の中枢神経系メラトニンは脳組織に対しても保護作用を示す[130]。松果体切除は動物の神経変性変化の加速と早期老化の証拠をもたらす[150, 151, 152, 153]。脳の松果体移植片は、マウスの虚血/再灌流により誘発される酸化的損傷から脳を保護した[154]。これらの効果は主に、松果体から直接放出されるCSFメラトニンの抗酸化、抗炎症および抗アポトーシス効果に起因する。

3. Pineal Gland Calcification (PGC), Melatonin Production, Neurodegenerative Diseases and Aging(松果体石灰化 (PGC)、メラトニン産生、神経変性疾患、老化)

Pineal calcification (synonyms include corpora arenacea, acervuli, brain sand, psammoma bodies and pineal concretions) was observed as early as in 1653 in humans [155]. Its presence was identified in a wide range of species including human, ox, sheep, horse, donkey, monkey, cow, gerbil, rat, guinea pig, chicken and turkey [156]. Even through the concretions were not found in the pineal organs of fish, amphibians, reptiles the high calcium content was detected in their pineal by ultrastructural calcium histochemistry [157]. Thus, pineal calcium metabolism and pineal calcification are wide spread phenomenon across species. Its rate increases with aging and in some species the pineal calcification rates are as high as 100% with age [158,159]. Ironically, calcification also occurs in neonatal humans [160,161]. It was reported that the pineal calcification in humans failed to impact the melatonin production and its circadian rhythm [77,78]. As a result, some believed that the pineal calcification might be a physiological process and not associated with pathological or aging changes. Rather, it might be related to the metabolic activity of pineal gland per se. In gerbils, the accumulation of pineal calcium deposits is blocked by superior cervical ganglionectomy which is believed to shut down completely the function of the pineal gland [162,163]. Gerbils exposed to short photoperiod (LD 10:14) exhibited significantly higher numbers of pineal concretions than those that were exposed to long photoperiod (LD 14:10) [164]. In addition, pineal calcification was enhanced in the gerbils with bilateral optic enucleation in which the animals are completely devoid a photoperiodic influence [165] with the generation of more melatonin. This evidence supported the metabolic theory of pineal calcification.

松果体石灰化(同義語には、脳砂、分生子層、脳砂、砂粒体、松果体結石がある)は、早くも1653年に人間で観察されている[155] 。その存在は、人間、ウシ、ヒツジ、ウマ、ロバ、サル、ウシ、アレチネズミ、ラット、モルモット、ニワトリおよび七面鳥を含む広範囲の種で確認された[156]。魚類、両生類、爬虫類の松果体器官には結石は認められなかったが、超微細構造カルシウム組織化学によって松果体に高カルシウム含量が検出された[157]。このように、松果体カルシウム代謝と松果体石灰化は種を越えて広く広がった現象である。その割合は加齢とともに増加し、ある種では松果体石灰化率は加齢とともに100%にも達する[158, 159]。皮肉なことに、石灰化は新生児でも起こる [160, 161] 。人間の松果体石灰化はメラトニン産生とその概日リズムに影響しないことが報告されている [77, 78]。その結果、松果体石灰化は生理学的プロセスであり、病理学的変化や加齢変化とは関連しないと考えられた。むしろ、それは松果体自体の代謝活性に関連している可能性がある。アレチネズミでは、松果体のカルシウム沈着の蓄積は、上頸神経節切除によって封鎖され、松果体の機能を完全に停止させると考えられている[162, 163]。短光周期(LD 10:14)に曝露されたスナネズミは、長光周期(LD 14:10)に曝露されたスナネズミよりも有意に多数の松果体結石を示した[164]。加えて、松果体石灰化は、動物がより多くのメラトニンの生成を伴う光周性の影響を完全に欠いている両側性眼球摘出を伴うアレチネズミで増強された[165]。この証拠は松果体石灰化の代謝説を支持した。

Large amounts of evidence, however, also suggest that the pineal calcification was indeed associated with human pathological disorders and aging. Decades ago several studies pointed out the relationship between the pineal calcification and schizophrenia [73,166,167,168]. The highest pineal calcium content was detected in the pineal gland of patients who died of renal disease associated with hypertension among other diseases [169]. Currently, additional studies have reported the strong association of PGC and neurodegenerative diseases, particularly Alzheimer’s disease [170]. This association is connected with the melatonin levels synthesized by this gland. It is well established that melatonin is a neuroprotector with its potent antioxidant function and anti-inflammatory activity [171,172,173,174,175,176]. The brain is rich in lipid, lacks the antioxidative enzyme, catalase, and consumes large quantity of oxygen (roughly 20% of the total oxygen consumed by the brain with 1% of the total body weight). This makes the brain more vulnerable to the oxidative stress than other organs. Decrease of endogenous melatonin will result in the neurons being less resistance to the oxidative stress or brain inflammation. Several studies have reported the negative association between the Alzheimer’s disease and serum or CSF melatonin levels [177,178]. The mechanistic investigations uncovered that in addition to its antioxidant and anti-inflammatory activities, melatonin directly inhibits the secretion and deposition of the β amyloid protein (AD plague) [179,180] which is the hallmark of this disease; it also suppresses tau protein hyperphosphorylation thereby reducing intracellular neurotangles [181,182,183], another biomarker of AD. The majority of the small scale clinical trials support that melatonin application improved the symptoms of sundowning syndrome and retarded the progress of AD [184,185,186,187,188,189,190].

しかし、多くの証拠は、松果体石灰化が実際に人間の病理学的疾患および老化と関連していることも示唆している。数十年前、いくつかの研究が松果体石灰化と統合失調症の関係を指摘した[73, 166, 167, 168]。松果体カルシウム含量が最も高かったのは、高血圧を伴う腎疾患で死亡した患者の松果体であった[169]。現在、PGCと神経変性疾患、特にアルツハイマー病との強い関連性が追加研究で報告されている[170]。この関連は、この腺で合成されるメラトニン濃度と関連している。メラトニンが強力な抗酸化機能と抗炎症活性を有する神経保護物質であることは十分に確立されている[171, 172, 173, 174, 175, 176]。脳は脂質が豊富で、抗酸化酵素であるカタラーゼを欠き、大量の酸素を消費する(体重の1%で脳が消費する全酸素の約20%)。これは脳を他の臓器より酸化ストレスに対して脆弱にする。内因性メラトニンの減少は、酸化ストレスまたは脳炎症に対するニューロンの抵抗性を低下させる。いくつかの研究では、アルツハイマー病と血清または脳脊髄液中のメラトニン濃度との間に負の相関があることが報告されている[177, 178]。メカニズム研究は、抗酸化および抗炎症活性に加えて、メラトニンがこの疾患の特徴であるβアミロイド蛋白質(ADペスト)[179, 180]の分泌および沈着を直接阻害することを明らかにした；また、タウタンパク質の過剰リン酸化を抑制することにより、ADのもう１個のバイオマーカーである細胞内神経原線維変化を減少させる[181, 182, 183]。大部分の小規模臨床試験は、メラトニン投与が日没症候群の症状を改善し、ADの進行を遅らせることを支持している[184, 185, 186, 187, 188, 189, 190]。

The most suggestive results come from the animal studies. In single, double or triple gene mutated AD animal models large doses of melatonin (100 mg/L drinking water or 10 mg/kg body weight/day) prolonged their life span, positively modulated the biochemical and morphological alterations and improved their cognitive performance [191,192,193,194,195,196]. To date, the large doses of melatonin used in animal studies have not been applied in clinical trials of Alzheimer’s disease. Considering the unique safety margin of melatonin, larges dose of melatonin can be used in AD patients and it may achieve its maximum treatment effects on this devastating disease. Recently, it was found that melatonin treatment for the sporadic AD animal model (OXYS rats) also produced impressive results. Very interestingly, OXYS rats exhibit significantly lower endogenous melatonin levels during night compared to their controls (Wistar rats). Melatonin treatment was especially effective in preserving the microstructures of hippocampal neurons and their mitochondrial distribution and integrity in this pathological animal model [197,198,199,200]. The sporadic AD includes roughly 95% of the clinical AD cases. These observations provide solid evidence suggesting the use of relatively large doses of melatonin to treat AD clinically. In a few cases, melatonin treatment did not result in expected results in AD patients [201] or in animals [202]; however, there were, at least, no serious adverse effects of the treatment. For other neurodegenerative diseases including Parkinson’s disease, amyotrophic lateral sclerosis (ALS), multiple Sclerosis (MS) and Huntington’s disease, melatonin applications also achieved positive results in patients and animal models [203,204,205,206,207,208]. For example, in MS patients their biochemical markers and some of the symptoms were improved after melatonin supplementation [209,210,211,212,213,214].

最も示唆的な結果は動物実験から得られた。単一、二重または三重遺伝子変異を持つAD動物モデルにおいて、大量のメラトニン(飲料水100mg/Lまたは１日あたり10mg/体重1kg)は寿命を延長し、生化学的および形態学的変化を正に調節し、認知能力を改善した[191, 192, 193, 194, 195, 196]。これまでのところ、動物実験で使用された大量のメラトニンは、アルツハイマー病の臨床試験には適用されていない。メラトニンの特異な安全域を考慮すると、大量のメラトニンはAD患者に使用でき、この壊滅的な疾患に対する最大の治療効果を達成する可能性がある。最近、散発性AD動物モデル(OXYSラット)に対するメラトニン治療も印象的な結果をもたらすことが見出された。非常に興味深いことに、OXYSラットは対照(Wistarラット)と比較して夜間に有意に低い内因性メラトニンレベルを示した。メラトニン処置は、この病理学的動物モデルにおいて、海馬ニューロンの微細構造とそれらのミトコンドリア分布および完全性の維持に特に有効であった[197, 198, 199, 200]。散発性ADは臨床AD症例の約95%を含む。これらの観察は、ADを臨床的に治療するための比較的高用量のメラトニンの使用を示唆する確固たる証拠を提供する。少数の症例では、メラトニン治療はAD患者[201]または動物[202]において期待された結果をもたらさなかった；しかしながら、少なくとも治療の重篤な副作用は全くなかった。パーキンソン病、筋萎縮性側索硬化症(ALS)、多発性硬化症(MS)およびハンチントン病を含む他の神経変性疾患に対しても、メラトニンの投与は患者および動物モデルにおいて肯定的な結果を達成した[203, 204, 205, 206, 207, 208]。例えば、多発性硬化症患者では、生化学的マーカーといくつかの症状がメラトニン補充後に改善された[209, 210, 211, 212, 213, 214]。

As to the association between the aging and melatonin production, in most vertebrates, melatonin production wanes with aging. The reasons for this may be two-fold. Melatonin synthetic capacity is dampened during aging due to the reduced density of β-adrenergic receptors in the pineal gland [215,216] and the downregulation of gene expression or phosphorylation of AANAT/SNAT [217]. A second reason is the increased consumption of melatonin. This is due to the metabolic alterations. For example, more ROS are generated by the aged cells than in the young cells and melatonin as the endogenous antioxidant is used to neutralize the overproduced ROS in aging organisms. Both of these effects may cause its low levels in the aged vertebrates. Low melatonin level is considered as a biomarker of aging [218,219,220]. When melatonin production was depressed by pinealectomy in rats, accumulation of oxidatively-damaged products accelerated their aging process [221]. In contrast, when young pineal glands were grafted to the old animals or exogenous melatonin was supplemented, both significantly increased the life span of experimental animals [222].

老化とメラトニン産生との関連では、ほとんどの脊椎動物でメラトニン産生は加齢とともに減少する。その理由は２個考えられる。メラトニン合成能は、松果体におけるβアドレナリン受容体の密度の減少[215, 216]およびAANAT/SNATの遺伝子発現またはリン酸化のダウンレギュレーション[217]により、加齢により低下する。２番目の理由は、メラトニンの消費量の増加です。これは代謝変化によるものである。例えば、より多くのROSが若い細胞よりも老化した細胞によって生成され、内因性抗酸化物質としてのメラトニンは、老化した生物において過剰生産されたROSを中和するために使用される。これらの影響の両方が、老齢脊椎動物における低レベルの原因となる。低メラトニンレベルは老化のバイオマーカーと考えられている[218, 219, 220]。ラットの松果体切除によりメラトニン産生が抑制されると、酸化的損傷を受けた産物の蓄積が老化プロセスを加速させた[221]。対照的に、若い松果体を老齢動物に移植した場合、または外因性メラトニンを補充した場合、いずれも実験動物の寿命を有意に延長させた[222]。

A great deal of attention has recently been given to the relationship of decreased melatonin levels in neurodegenerative diseases and aging associated pineal calcification. With the increased use of the PET scan, susceptibility-weighted magnetic resonance imaging (SWMR) or other advanced technologies, even very small pineal concretions can be identified in patients or animals, which could not be seen previously. It was found that the rates of pineal calcification have been significantly underestimated previously. For example, in non-specifically targeted patients with the average age of 58.7 ± 17.4 years, 214 out of 346 showed PGC on CT scans (62%) [223]; the data of 12,000 healthy subjects from Turkey indicated that the highest intracranial calcifications occurred in the pineal gland with an incidence of 71.6% [65]. PGC appears to occur without significant differences among countries, regions and races. For example, in Iran the PGC incidence is around 71% [224] and in African (Ethiopia), it is roughly 72% [225] and in black people in the US it is 70% [226]. With such a high incidence of PGC in humans and considering the functions of pineal gland, the PGC should not be considered a normal physiological process.

近年、神経変性疾患におけるメラトニン濃度の低下と加齢に伴う松果体石灰化との関係が注目されている。PETスキャン、磁化率強調磁気共鳴画像法(SWMR)または他の先進技術の使用の増加により、以前は見ることができなかった非常に小さな松果体結石でさえ、患者または動物で特定できる。松果体石灰化率は以前は有意に過小評価されていたことが判明した。例えば、平均年齢58.7±17.4歳の非特異的標的患者では、346人中214人(62%)がCTスキャンでPGCを示した[223]。トルコの健常被験者12,000名のデータでは、頭蓋内石灰化の発生率が最も高かったのは松果体であり、その発生率は71.6%であった[65]。PGCは、国、地域および人種間で有意差なく発生するようである。例えば、イランでのPGC発生率は約71%[224]、アフリカ(エチオピア)では約72%[225]、アメリカの黒人では70%[226]である。人間におけるPGCのこのような高い発生率と松果体の機能を考慮すると、PGCは正常な生理学的プロセスと考慮されるべきではない。

PGC is often related to the decreased melatonin levels and several pathological alterations including neurodegenerative diseases (Alzheimer’s, MS), migraine, symptomatic intracerebral hemorrhage, symptomatic cerebral infarction, sleep disorders, defective sense of direction and pediatric primary brain tumor [73,170,227,228,229,230,231]. Interestingly, PGC is mainly associated with brain-related disorders but not few with other organ pathophysiologies while the decreased melatonin levels were detected in the blood which supplies all the tissues. This observation further supports our hypothesis that high levels of melatonin released directly into CSF from pineal gland serve as the biological circadian rhythm regulator and the neuronal antioxidant while the blood melatonin is the residue of the pineal melatonin [63,130]. This residual melatonin only resembles the CSF melatonin rhythm and may be without significant biological functions.

PGCはしばしばメラトニン濃度の低下および神経変性疾患(アルツハイマー病、多発性硬化症)、片頭痛、症候性脳内出血、症候性脳梗塞、睡眠障害、方向感覚の欠損および小児原発性脳腫瘍を含むいくつかの病理学的変化と関連している[73, 170, 227, 228, 229, 230, 231]。興味深いことに、PGCは主に脳関連障害と関連しているが、他の臓器の病態生理とは少なからず関連しており、メラトニンレベルの減少は全ての組織に供給する血液中で検出された。この観察結果は、松果体からCSFに直接放出される高レベルのメラトニンが生物学的概日リズム調節因子および神経抗酸化物質として機能する一方、血中メラトニンは松果体メラトニンの残留物であるという仮説をさらに支持している[63, 130]。この残留メラトニンは脳脊髄液のメラトニンリズムに類似しているだけであり、重要な生物学的機能を有していない可能性がある。

PGC reduces CSF melatonin levels and dampens its rhythm resulting in chronological disturbance including insomnia and migraine. The low levels of CSF melatonin also elevate neuronal damage from ROS, thus, accelerating the neurodegenerative disorders.

PGCはCSFメラトニンレベルを低下させ、そのリズムを減衰させ、不眠症や片頭痛を含む時系列的な乱れをもたらす。CSFメラトニンの低レベルもROSからの神経損傷を高め、神経変性疾患を加速する。

It also has been reported that the serum and salivary melatonin and its urine metabolite are negatively related to the size of the pineal calcification and positively related to the uncalcified portion of the gland [232,233,234,235]. In Alzheimer’s disease, the patients had a higher portion of calcified pineal glands and lower portion of uncalcified glands than patients with other dementias [170]. As mentioned, that the serum melatonin rhythm resembles the CSF melatonin; thus, it can be deduced that the CSF melatonin levels in Alzheimer’s patients would also be significantly reduced. It is difficult to obtain CSF from the Alzheimer’s patients to test melatonin levels. However, the postmortem CSF from these patients indeed proved the low levels of melatonin. Their CSF melatonin level was only 20% of that in their non-Alzheimer’s controls. The authors suggested that the reduction in CSF melatonin levels might be an early event in the development of AD possibly occurring even before the clinical symptoms [177,178]. If these patients had PGC, their CFS melatonin level may further decrease and it would accelerate the process of the disease. The PGC is also associated with aging [236,237] even through the PGC has been detected in the neonatal or in children. It was reported that the incidence of the visible PGC increases with age, i.e., 2% at 0–9, 32% at 10–19, 53% at 20–29 and 83% in over-30 age groups, respectively [238]; clearly the degree of PGC increased with aging [239]. In turkeys and rats the incidence of PGC reaches 100% in advanced age animals. It seems that PGC is an inevitable process of aging in vertebrates. If so, slowdown of this process may retard the aging process. This is discussed later.

また、血清および唾液中のメラトニンとその尿中代謝物は松果体石灰化の大きさと負の相関があり、腺の非石灰化部分と正の相関があることが報告されている[232, 233, 234, 235]。アルツハイマー病では、他の認知症患者に比べて松果体の石灰化が多く、非石灰化が少なかった[170]。前述したように、血清メラトニンリズムは脳脊髄液メラトニンに似ている；したがって、アルツハイマー病患者の脳脊髄液メラトニン濃度も有意に低下していると推測できる。アルツハイマー病患者から脳脊髄液を採取してメラトニン値を検査することは困難である。しかし、これらの患者の死後CSFはメラトニンの低レベルを証明した。彼らの脳脊髄液のメラトニン値は、アルツハイマー病ではない対照群のわずか20%であった。著者らは、脳脊髄液中のメラトニン濃度の低下は、AD発症の初期の事象であり、臨床症状が現れる前に起こる可能性があることを示唆している[177, 178]。もしこれらの患者がPGCを有する場合、CFSメラトニン濃度はさらに低下するかもしれないし、それは疾患の進行を加速するだろう。PGCはまた、新生児または小児でPGCが検出されても、加齢と関連している[236, 237]。目に見えるPGCの発現率は年齢とともに増加し、0~9歳で2%、10~19歳で32%、20~29歳で53%、30代以上で83%と報告されている[238]；PGCの程度は明らかに加齢とともに増加した[239]。七面鳥とラットでは、PGCの発生率は高齢動物で100%に達する。PGCは脊椎動物の老化の避けられない過程であると思われる。もしそうなら、このプロセスの減速は老化プロセスを遅らせるかもしれない。これについては後述します。

4. Potential Mechanisms for PGC Formation(PGC形成の潜在的メカニズム)

Even through PGC is a widespread phenomenon in vertebrates, its importance has been ignored and little attention has been given to this important issue for decades. To date, little is known about its exact formation processes and mechanisms. Here, we summarize several opinions and speculations on the potential mechanisms of PGC formation and also discuss our hypothesis regarding these enigmatic structures. It seems that there are two origins of PGC, that is, in association with pinealocytes or with non-pinealocytes. Some studies found that PGC was restricted to the connective tissue. The mechanisms involved the formation of calcareous deposits within the connective tissue stroma of the gland [240]. These deposits represent the aging-related calcium accumulation within the connective tissue. This type of calcification is similar to that found in the habenular commissure and choroid plexus [238]. The connective tissue derived PGC is predominant in the rat and Pirbright white guinea pig [241,242]. In analysis of the specimens of human pineal gland, Maslinska et al. [160] reported that the initiation of PGC was associated with the tryptase-containing mast cells. During the systemic or local pathological conditions, the tryptase-containing mast cells infiltrate into the pineal gland where they release biologically active substances including tryptase which participates in calcification. This process is pathological but not age related since it also occurs in the children.

PGCは脊椎動物で広く見られる現象であるが、その重要性は無視され、この重要な問題には何十年もの間ほとんど注意が払われてこなかった。現在まで、その正確な形成プロセスとメカニズムについてはほとんど知られていない。ここでは、PGC形成の潜在的機構に関するいくつかの意見と推測を要約し、これらの謎めいた構造に関する仮説についても議論する。PGCには松果体細胞との関連と非松果体細胞との関連の２個の起源があると思われる。いくつかの研究はPGCが結合組織に限定されることを明らかにした。そのメカニズムには、腺の結合組織間質における石灰沈着の形成が関与していた[240]。これらの沈着物は、結合組織内の加齢に伴うカルシウムの蓄積を示す。このタイプの石灰化は、手綱交連および脈絡叢にみられるものと類似している[238]。結合組織由来PGCは、ラットおよびピルブライト種白色モルモットで優勢である[241, 242]。人間の松果体標本の分析において、Maslinskaら[160]はPGCの開始がトリプターゼ含有肥満細胞と関連していることを報告した。全身または局所の病理学的状態の間、トリプターゼ含有肥満細胞は松果体に浸潤し、そこで石灰化に関与するトリプターゼを含む生物活性物質を放出する。このプロセスは病的であるが、小児でも起こるので年齢とは関係がない。

As to the PGC of pinealocyte-origin, two speculations should be mentioned. One is proposed by Lukaszyk and Reiter [13,243]. They reported that the pinealocytes extruded polypeptides into the extracellular space in conjunction with their hypothetic carrier protein, neuroepiphysin. The pineal polypeptides of exocytotic microvesicles were actively exchanged for the calcium. The calcium-carrier complex then is formed and deposited on the surface of adjacent mutilayed concretions. Thus, the concretion formation is related to the secretory function of pineal gland. For example, in the gerbil following the superior cervical ganglionectomy, the PGC are completely inhibited; this was attributed to a decrease in the functional activity of the gland [163]. However, this cannot explain the observation of intracellular calcification of the pinealocytes [244,245]. Krstić [246,247] proposed another mechanism to explain the origin of PGC from pinealocytes. He speculated that the cytoplasmic matrix, vacuoles, mitochondria and the endoplasmic reticulum of large clear pinealocytes were the initial intracellular calcification sites. These loci, and particularly those within the cytoplasmic matrix, transformed into acervuli by a further addition of hydroxyapatite crystals. The cells gradually degenerated, died, broke down, and the acervuli reached the extracellular space. High intracellular calcium levels could be a situation that is responsible for eliminating calcium from the cell, with the hypercalcemic intracellular milieu promoting the initial crystallization. The failure of Ca2+-ATPase could be a natural process of aging or pathological conditions [248]. Hence, PGC does not occur under normal conditions and it is a result of altered molecular processes in vertebrates. These speculations; however, cannot completely explain the mechanisms of the PGC formation. Here, we provide an additional speculation which is a complementary of the previous suggestions. It seems that the PGC in some cases is an active rather than a passive process. We previously hypothesized that the pineal gland may have a blood filtration function like the kidney since its vascular structures as well as its blood flow rate are similar to the kidney [63]. The question is whether they share a similarity to the calcification this is observed in both organs. It is well documented that the compositions of PGC is totally different from the kidney stone. Kidney stones are primarily composed of calcium oxalate and its formation is simply a sedimentary process caused by high concentrations of both calcium and oxalate [249]. A main component of a PGC is hydroxyapatite [Ca10(PO4)6(OH)2] [248,250,251] which is the chief structural element of vertebrate bone. The Ca/P molar ratio in pineal concretions is similar to the enamel and dentine [252] and these authors pointed out that the nature and crystallinity of the inorganic tissue of the pineal concretions lead one to think of a physiological rather than pathological ossification type with characteristics between enamel and dentine. It is not very clear how the hydroxyapatite is formed in the bone but there is little doubt that its formation involves the collaboration of bone cells and it is a programmed process. In addition, the concentric laminated pineal concretions are frequent observed [157,239] to be structurally similar the osteons (Figure 3), the major unit of compact bone.

松果体細胞由来のPGCについては、２個の推測を述べる必要がある。１個はLukaszykとReiter[13, 243]によって提案されている。彼らは、松果体細胞が、その仮説上の運搬体タンパク質であるニューロエピフィシンとともにポリペプチドを細胞外空間に押し出したことを報告した。エキソサイトーシス性微小小胞の松果体ポリペプチドはカルシウムと活発に交換された。次いで、カルシウム運搬体複合体が形成され、隣接する切断された結石の表面に沈着する。このように、結石形成は松果体の分泌機能と関連している。例えば、上頸神経節切除後のスナネズミでは、PGCは完全に阻害される；これは腺の機能活性の低下によるものである[163]。しかし、これは松果体細胞の細胞内石灰化の観察を説明できない[244, 245]。Krstić[246, 247]は、PGCが松果体細胞に由来することを説明する別のメカニズムを提唱した。彼は、大きな透明な松果体細胞の細胞質マトリックス、液胞、ミトコンドリアおよび小胞体が最初の細胞内石灰化部位であると推測した。これらの地点、特に細胞質マトリックス内の地点は、ヒドロキシアパタイト結晶のさらなる追加により分生子層に姿を変えた。細胞は徐々に変性し、死滅し、崩壊し、分生子層は細胞外空間に達した。高い細胞内カルシウム濃度は、高カルシウム血症の細胞内環境が初期結晶化を促進し、細胞からカルシウムを除去する状況である可能性がある。Ca2+-ATPaseの機能不全は、老化または病的状態の自然な過程である可能性がある[248]。したがって、PGCは通常の条件下では起こらず、脊椎動物における分子プロセスの変化の結果である。これらの推測；しかしながら、PGC形成のメカニズムを完全に説明することはできない。ここでは、以前の提案を補足する付加的な推測を提供した。PGCは場合によっては受動的なプロセスではなく能動的なプロセスである。私たちは以前、松果体の血管構造や血流速度が腎臓と類似していることから、松果体には腎臓のような血液濾過機能があるのではないかという仮説を立てた[63]。問題は両方の臓器で観察される石灰化と類似しているかどうかである。PGCの組成は腎結石とは全く異なることがよく知られている。腎結石は主にシュウ酸カルシウムからなり、その形成は高濃度のカルシウムとシュウ酸塩の両方によって引き起こされる単純な堆積プロセスである[249]。PGCの主成分は、脊椎動物の骨の主要構造要素であるヒドロキシアパタイト [Ca10(PO4)6(OH)2] [248, 250, 251]である。松果体結石のCa/Pモル比はエナメル質と象牙質に類似しており[252]、これらの著者らは松果体結石の無機組織の性質と結晶性から、エナメル質と象牙質の間の特性を持つ病理学的というよりは生理学的な骨化タイプと考えられることを指摘した。ヒドロキシアパタイトが骨の中でどのように形成されるかはあまり明らかではないが、その形成には骨細胞の協力が関与しており、それがプログラムされたプロセスであることはほとんど疑いがない。加えて、同心円状に積層された松果体結石は、緻密骨の主要な単位である骨単位(図3)と構造的に類似していることがしばしば観察される[157, 239]。

Figure 3. The laminated pineal gland calcification at different ages and their similarity to the osteons of compact bone. (A) Pineal calcification in 14 year old subject; (B) 47 year old; (C) 62 year old; (D) osteons; (A–C) were modified from Hermann et al. [239].

図３．異なる年齢での積層された松果体石灰化とそれらの緻密骨の骨単位との類似性。(A)14歳における松果体石灰化について；(B)47歳；(C)62歳以上；(D)骨単位；(A~C)はHermannら[239] を基に修正した。

The laminated pineal stone indicates its formation is not random but organized and programmed. For example, in humans, laminated pineal stones are associated with aging. The older the individual, the larger number of lamellae (Figure 3) [239]. Our hypothesis is that the pineal calcification, at least partially, may be similar to the bone formation that is, the pineal calcium deposit may be formed by differentiated bone cells under certain conditions. Recently, numerous studies have reported that melatonin facilitates the capacity of mesenchymal stem cells (MSCs) to differentiate into osteoblast-like cells under in vivo or in vitro conditions [253,254,255,256,257]. Mesenchymal cells are found in the early stage of pineal development in birds and in rats [258,259]. Mesenchymal cells have an important role in pineal follicular formation later during development of the gland. It was also documented that the striated muscle fibers are present in the pig and rat pineal gland [260,261]. These striated muscle fibers are of mesenchymal rather than ectodermal origin [261]. These observations indicate that the MSCs are present in the pineal gland and they have the capacity to differentiate into different cell types including muscle as well as probably the osteoblasts and even the osteocytes. The MSCs in the pineal gland may be retained from its early embryonic stage of mesenchymal tissue and/or they may be of vasculature origin. The differentiation from MSCs into osteoblasts/osteocytes seems to be melatonin dependent. The signal transduction pathway of this transition is probably mediated by melatonin membrane receptor 2 (MT2) [261]. The detailed mechanism was proposed by Maria and Witt-Enderby [262]. Simply, melatonin binds to the MT2 of MSCs to promote them to differentiate into pre-osteoblasts. At the same time melatonin increases the levels of parathyroid hormone (PTH); type I collagen and alkaline phosphatase (ALP) and these factors further promote pre-osteoblasts to form osteoblasts. Finally, melatonin upregulates the gene expression of the osteopontin (OSP), bone morphogenetic protein 2 (BMP-2), osteocalcin (OCN) and ALP and facilitates the osteoblast proliferation, osteocyte formation, mineralization and bone formation (Figure 4.)

積層の松果体結石は、その形成がランダムではなく、組織化され、プログラムされていることを示した。例えば、人間では、積層の松果体結石は加齢と関連している。年齢が高い個体ほど薄層の数が多い(図3)[239]。私たちの仮説は、松果体石灰化は、少なくとも部分的には骨形成と類似しているかもしれない、すなわち、松果体カルシウム沈着は、特定の条件下で分化した骨細胞によって形成されるかもしれないということである。最近、多くの研究が、メラトニンがin vivoまたはin vitro条件下で骨芽細胞様細胞に分化する間葉系幹細胞(MSC)の能力を促進することを報告している [253, 254, 255, 256, 257]。間葉細胞は、鳥類とラットの松果体発生の初期段階に見られる[258, 259]。間葉細胞は、腺の発生の後期における松果体濾胞形成に重要な役割を果たす。横紋筋線維がブタとラットの松果体に存在することも報告されている[260, 261]。これらの横紋筋線維は外胚葉由来ではなく間葉由来である [261]。これらの観察は、MSCが松果体に存在し、筋肉だけでなくおそらく骨芽細胞や骨細胞さえも含む異なる細胞型に分化する能力を有することを示す。松果体におけるMSCは間葉組織の初期胚期から保持され、および/またはそれらは血管系起源である可能性がある。MSCから骨芽細胞/骨細胞への分化はメラトニン依存性のようである。この移行のシグナル伝達経路はおそらくメラトニン膜受容体2(MT2)によって媒介される[261]。詳細なメカニズムはMariaとWit-Enderby[262]によって提唱された。単純に、メラトニンはMSCのMT2に結合し、前骨芽細胞への分化を促進する。同時にメラトニンは副甲状腺ホルモン(PTH)のレベルを増加させる。I型コラーゲンとアルカリホスファターゼ(ALP)とこれらの因子は前骨芽細胞の骨芽細胞形成をさらに促進する。最後に、メラトニンはオステオポンチン(OSP)、骨形成蛋白質2(BMP‐2)、オステオカルシン(OCN)およびALPの遺伝子発現をアップレギュレートし、骨芽細胞増殖、骨細胞形成、石灰化および骨形成を促進する(図4)。

Figure 4. The proposed mechanisms underlying melatonin’s actions on bone formation. (A) melatonin induces MSCs differentiation into osteoblasts via MT2; (B) it promotes osteoprotegerin (OPG) expression in preosteoblasts which would inactive RANKL, leading to a suppression of osteoclastogenesis; and (C) through melatonin’s free-radical scavenging and antioxidant properties, protecting against radical induced loss of osteoblasts and osteoclasts. PTH (parathyroid hormone); Type I col (type I collagen); OSP (osteopontin); BMP-2 (bone morphogenetic protein 2); ALP (alkaline phosphatase); OCN (osteocalcin); TRAP (tartrate-resistant acid phosphatase); RANKL (receptor activator of NFĸB ligand); OPG (osteoprotegerin). From Maria and Witt-Enderby [262].

図４．骨形成に対するメラトニンの作用の根底にある提案されたメカニズム。(A)メラトニンはMT2を介して骨芽細胞へのMSC分化を誘導する；(B)RANKLを不活性化する前骨芽細胞におけるオステオプロテゲリン(OPG)発現を促進し、破骨細胞形成を抑制する；(C)メラトニンのフリーラジカル消去作用と抗酸化作用により、ラジカル誘発性の骨芽細胞と破骨細胞の喪失を防ぐ。PTH(副甲状腺ホルモン)；I型結腸(I型コラーゲン)；OSP(オステオポンチン)；BMP-2(骨形成蛋白2)；ALP(アルカリホスファターゼ)；OCN(オステオカルシン)；TRAP(酒石酸耐性酸性ホスファターゼ)；RANKL(NFĸBリガンド受容体活性化剤)；OPG(オステオプロテゲリン)。Maria and Witt-Enderby[262]より。

MT2 has been identified in MSCs using molecular techniques and classical pharmacology [263,264]. Transgenic knockout of the MT2 in mice inhibited the osteoblast proliferation and bone formation [265]. This indicates that the pineal gland has the capacity to form the bone like structure (calcification) by the pathway including MSCs. The promotor is the high levels of melatonin generated by this gland. The process of PGC in bird (turkey) resembles the bone formation which strongly supports our hypothesis. It requires a microenvironment which includes collagen fibrils, phosphate and calcium. The osteocyte-like cells are found in the center of the pineal concretion and the peripheral part contains the osteoblast-like cells and densely packed collagen fibrils [159] (Figure 5). The intermediate portion is the place of mineralization as bone.

MT2は、分子技術と古典的薬理学を用いてMSCで同定されている[263, 264]。マウスにおけるMT2のトランスジェニックノックアウトは、骨芽細胞の増殖と骨形成を阻害した[265]。これは、松果体がMSCを含む経路により骨様構造(石灰化)を形成する能力を有することを示す。促進物質は、この腺によって産生される高濃度のメラトニンである。鳥類(七面鳥)におけるPGCのプロセスは骨形成に類似しており、私たちの仮説を強く支持している。それはコラーゲン原線維、リン酸塩およびカルシウムを含む微小環境を必要とする。松果体結石の中心部には骨細胞様の細胞がみられ、その周辺部には骨芽細胞様の細胞やコラーゲン線維が密に存在している[159](図5)。中間部は骨として石灰化する場所である。

Figure 5. Histology and ultrastructure of cells located in a concretion of the turkey pineal gland. (A) mature concretions identified with Mallory’s stain. Two types of cells are present in the concretion (semi-thin section stained with toluidine blue); (B) polygonal cells; (C) elongated cells; (D) The presence of calcium in the concretions was demonstrated with Alizarin red S; note the characteristic appearance of cells located in the concretion; (E) ultrastructure of cells located in the calcified area (fixation with the PPA method). 1: the osteocyte-like cell surrounded by mineralized collagen fibrils in the central part of the calcification area. Note the “halo” around the cell and large pyroantimonate precipitate located mainly outside the cell membrane, 2: the junction of processes of osteocyte-like cells, 3: a cell showing a fibrocyte-like appearance in the peripheral part of the calcification area. Note the extra-cellular matrix containing collagen and calcium deposits, 4: numerous calcium precipitates in the intercellular spaces in the peripheral part of the calcification area, 5: The cell process with scattered deposits in the middle part of the concretion. Note the adjacent extra-cellular matrix rich in collagen and pyroantimonate precipitates. Modified from Przybylska-Gornowicz et al. [159].

図５．七面鳥の松果体結石に存在する細胞の組織学と超微細構造。(A) マロリー染色で特定された成熟結石。２種類の細胞が結石 (トルイジンブルーで染色された半薄切片) 中に存在する；(B)多角形細胞；(C)伸長細胞；(D)アリザリンレッドSにより結石中のカルシウムの存在が実証された；結石中にある細胞の特徴的な外観に注意する；(E)石灰化部に位置する細胞の超微細構造(PPA法による固定)。１：石灰化領域の中央部にある石灰化コラーゲン線維に囲まれた骨細胞様細胞。細胞周囲の「halo」と主に細胞膜の外側に位置する大きなピロアンチモン酸塩の沈殿に注目、２：骨細胞様細胞の突起の接合部、３：石灰化領域の周辺部に線維細胞様の外観を示す細胞。コラーゲンやカルシウム沈着物を含む細胞外マトリックスに注目、４：石灰化領域の周辺部の細胞間空間に多数のカルシウム沈着物、５：結石の中央部に散在する沈着物を含む細胞突起。隣接する細胞外マトリックスはコラーゲンとピロアンチモン酸塩の沈殿に富んでいることに注目。Przybylska-Gornowicz et al. [159]を修正。

Based on the current knowledge, we speculated that the “osteocyte-like cells” and the “osteoblast-like cells” were osteocytes and osteoblasts which differentiated from the MSCs in the pineal gland under the influence of melatonin. If the pineal microenvironment facilitates the PGC formation, why are the PGC often associated with aging and some pathological conditions?

現在の知見に基づき、「骨細胞様細胞」および「骨芽細胞様細胞」はメラトニンの影響下で松果体のMSCから分化した骨細胞および骨芽細胞であると私たちは推測した。松果体の微小環境がPGC形成を促進するならば、なぜPGCはしばしば加齢やある種の病理学的状態と関連するのか？

Currently, we cannot definitely answer this question, but several clues might indicate the relationship of PGC with aging/pathology:

現在、この質問に明確に答えることはできないが、いくつかの手がかりはPGCと加齢/病理との関係を示すかもしれない：

(1) Chronic vascular inflammation: The pineal gland has a complicated vascular system with abundance of arteries, fenestrated capillaries and veins. Especially the filtration rate of blood in pineal gland is in excess of most organs and it is only second to the kidney in terms of blood flow. These make the gland venerable to the chronic vascular inflammation during aging or certain disorders. The vascular inflammation mobilizes the MSCs migration and adhesion in the gland or promotes the de novo MSCs proliferation due to the increased levels of pro-inflammatory cytokines, TGF-β or TNF-α. The crosstalk between vascular MSCs and inflammatory mediators, especially, interleukin-22, lead to MSCs proliferation, migration and osteogenic differentiation [266,267] under the influence of high levels of pineal melatonin and finally PGC formation.

(1) 慢性血管炎：松果体は、豊富な動脈、有窓性毛細血管および静脈を有する複雑な血管系を有する。特に松果体の血液ろ過率はほとんどの臓器より高く、血流量では腎臓に次ぐ。これらは、腺を老化または特定の疾患の間の慢性血管炎症に対して起こしやすくする。血管炎症は、炎症性サイトカイン、TGF‐βまたはTNF‐αのレベル増加により、腺におけるMSCの移動および接着を動員するか、または新たなMSC増殖を促進する。血管MSCと炎症性メディエーター、特にインターロイキン-22との間のクロストークは、高レベルの松果体メラトニンの影響下でMSCの増殖、移動および骨形成分化[266, 267]をもたらし、最終的にPGC形成をもたらす。

(2) Brain tissue hypoxia: Many pathological conditions cause brain tissue hypoxia including hypertension, sleep apnea, stroke, and even respiratory disorders. Hypoxia-inducible factor (HIF)-1α is an important regulator of MSCs and it promotes the proliferation, migration and adhesion of MSCs in the hypoxic areas [268,269,270] including to the pineal gland. Generally, hypoxia increases bone resorption and suppresses osteoblastic differentiation and bone-formation [271,272]. However, this may not be applied to the pineal gland. During the dark phase, the pineal produces high levels of melatonin. Under the hypoxic condition, melatonin would promote the osteoblast differentiation and mineralization of MSCs via the p38 MAPK and PRKD1 signaling pathways [273]. In addition, melatonin also inhibits the activity of the osteoclast and osteoclatogenesis [274,275], especially under inflammatory conditions [276]. These processes favor PGC formation under hypoxic conditions.

(2) 脳組織低酸素症：高血圧、睡眠時無呼吸、脳卒中、さらには呼吸障害を含む多くの病的状態が脳組織の低酸素症を引き起こす。低酸素誘導因子(HIF)‐1αはMSCの重要な調節因子であり、松果体を含む低酸素領域[268, 269, 270]におけるMSCの増殖、移動および接着を促進する。一般に、低酸素は骨吸収を増加させ、骨芽細胞の分化と骨形成を抑制する[271, 272]。しかしながら、これは松果体には適用できないかもしれない。暗期には松果体は高レベルのメラトニンを産生する。低酸素条件下では、メラトニンはp38 MAPKおよびPRKD1シグナル伝達経路を介して骨芽細胞分化およびMSCの石灰化を促進する[273]。加えて、メラトニンは破骨細胞と破骨細胞形成の活性も阻害し[274, 275]、特に炎症条件下で阻害する[276]。これらのプロセスは低酸素条件下でPGC形成に有利である。

(3) Intracranial pressure: Some cells of the pineal gland are “swimming” in the third ventricle and, as a result, they are influenced by the intracranial pressure. Intracranial pressure usually increases with cerebral disorders such as idiopathic intracranial hypertension, brain trauma and stroke [277], and even Alzheimer’s disease [278]. The high pressure may impede the pineal filtration rate and induce endoepithelial cell damage by chrono-inflammation. The pressure also promotes the bone remodeling and mineralization, thus, PGC formation.

(3) 頭蓋内圧：松果体の一部の細胞は第３脳室で「泳いで」いるため、頭蓋内圧の影響を受けます。頭蓋内圧は通常、特発性頭蓋内圧亢進症、脳外傷および脳卒中[277]、さらにはアルツハイマー病[278]などの脳疾患でも上昇する。高圧は松果体の濾過速度を妨げ、慢性炎症による内皮細胞の損傷を誘発する可能性がある。圧力はまた骨リモデリングと石灰化を促進し、PGC形成を促進する。

5. Rejuvenation of Pineal Gland?(松果体の若返り？)

As mentioned, the pineal gland may be an important organ for maintaining the optimal health of vertebrates. Its malfunctions including its calcification may have associations with the premature aging and aging-related diseases. To answer this question, researchers had tried to rejuvenate the gland by ectopically pineal transplantation. Initially, it was found that the pineal glands which were transplanted into the anterior chamber of the eye in rats were innervated by surrounding sympathetic nerve endings and their normal rhythm in AANAT activity was established similar to the in situ pineal gland [279,280]. To support this observation, more complicated studies have been performed in which pineal glands were transplanted into a variety of sites in pinealectomized rats. These sites included anterior chamber of the eye, third cerebral ventricle, the pineal region (in situ transplantation), intrastriatal, renal capsule and thymus [280,281,282,283,284,285]. The results indicated that in some cases the pineal gland transplantation did increase the melatonin levels in pinealectomized animals; however, except in the site of anterior chamber of the eye, no melatonin circadian rhythm was detected with the pineal gland transplantation and also the melatonin levels could not match those of the in situ pineal gland produced. The lack of melatonin rhythm after pineal transplantation may relate to lack of sympathetic innervation of the grafted gland at other sites as compared to the anterior chamber of the eye in which the sympathetic innervation was obvious [286].

前述のように、松果体は脊椎動物の最適な健康を維持するための重要な器官である可能性がある。石灰化を含むその機能不全は、早期老化および老化関連疾患と関連する可能性がある。この疑問に答えるため、研究者らは異所性の松果体移植によって腺を若返らせようと試みた。最初に、ラットの眼の前房に移植された松果体は周囲の交感神経終末によって神経支配されており、AANAT活性の正常なリズムはin situ松果体と同様に確立されていることがわかった[279, 280]。この観察を支持するために、松果体を切除したラットの様々な部位に松果体を移植するより複雑な研究が行われている。これらの部位には、前眼房、第３脳室、松果体部(in situ移植)、線条体内、腎被膜および胸腺が含まれた[280, 281, 282, 283, 284, 285]。結果は、松果体移植が松果体切除動物のメラトニン濃度を増加させる場合があることを示した；しかしながら、前眼房の部位を除いて、松果体移植ではメラトニン概日リズムは検出されず、メラトニン濃度もin situで作製した松果体のものと一致しなかった。松果体移植後のメラトニンリズムの欠如は、交感神経支配が明らかであった眼の前房と比較して、他の部位で移植された腺の交感神経支配の欠如に関連している可能性がある[286]。

In addition to the anatomic and morphological studies, the functional studies of ectopic pineal gland grafts provided promising results. When the pineals of young mice (3–4 months) were grafted into thymus of the old mice (17–18 months), they partially prevented thymic involution in the old animals due to an anti-apoptotic activity [287]. The similar result was observed in the rats. When the pineal glands of young animals were transplanted into the thymus of old rats, they preserved the age-related alterations in erythrocyte membranes by increasing their hemolysis time and decreasing their peroxidation [288]. The young pineal transplants into the thymus of old mice could even prolong the recipients’ life span up to 27% compared to the controls [222]. The authors attributed the life prolongation effects to that the grafted pineal gland might release high level of nocturnal melatonin which acted on the thymus to rejuvenate the gland and preserved the immune responsiveness of these old animals to the levels of young animals. Similarly, the intrastriatal transplantation of pineal tissue significantly reduced the brain infarct size in middle cerebral artery occlusion (MCA) ischemia/reperfusion animal model [154].

解剖学的および形態学的研究に加えて、異所性松果体移植片の機能的研究は有望な結果を提供した。若いマウス(3–4カ月)の松果体を老齢マウス(17–18カ月)の胸腺に移植したところ、抗アポトーシス活性により老齢マウスの胸腺の退縮を部分的に阻止した[287]。同様の結果がラットでも観察された。若い動物の松果体を老齢ラットの胸腺に移植すると、溶血時間の延長と過酸化の減少により赤血球膜の加齢変化が維持された[288]。老齢マウスの胸腺への若い松果体の移植は、レシピエントの寿命を対照に比べて27%まで延ばすことさえできた[222]。著者らはこの延命効果を、移植した松果体が高レベルの夜間メラトニンを放出し、それが胸腺に作用して松果体を若返らせ、これらの老齢動物の免疫応答性を若い動物のレベルに維持したためと考えた。同様に、松果体組織の線条体内移植は、中大脳動脈閉塞(MCA)虚血/再灌流動物モデルにおける脳梗塞のサイズを有意に減少させた[154]。

In general, ectopic pineal gland transplantation appears to be beneficial for health in many aspects. However, it is obvious that it cannot replace the function of the in situ pineal gland. As mentioned previously, the in situ pineal gland produces high level of melatonin, which protects the brain from the oxidative damage after its release into the CSF; secondly, pineal melatonin secretion exhibits a circadian rhythm, especially in the CSF of the third ventricle in which the night time melatonin peak have a sharp rise and fall compared to its serum circadian pattern (Figure 2). This CSF melatonin alteration is believed to serve as the signal of biorhythm of organisms [289]. The ectopic pineal gland transplants lack these two most important aspects. Thus, an improved means to mimic activities of the in situ pineal gland would probably be the pineal transplantation into the pineal region (in situ graft) or into the third ventricle. Some studies have been performed in this regard. The results indicate that the pineal glands which were transplanted into third ventricle or pineal region (in situ transplantation) survived due to re-vascularization and partial re-innervation [7]. They did produce melatonin, but the levels were low and completely without the night time rise [7,283].

一般に、異所性の松果体移植は多くの面で健康に有益であると思われる。しかしながら、in situ松果体の機能を代替できないことは明らかである。すでに述べたように、松果体は高濃度のメラトニンを産生し、脳脊髄液に放出された後の酸化的損傷から脳を保護する；第２に、松果体のメラトニン分泌は概日リズムを示し、特に第３脳室の脳脊髄液では、夜間のメラトニンのピークが血清の概日パターンと比較して急激に増減する(図2)。この脳脊髄液メラトニンの変化は生物のバイオリズムのシグナルと考えられている[289]。異所性松果体移植には、この２個の最も重要な側面が欠けている。したがって、in situ松果体の活性を模倣するための改善された手段は、おそらく松果体領域(in situ移植)または第３脳室への松果体移植であろう。この点に関していくつかの研究が行われている。結果は、第３脳室または松果体領域(in situ移植)に移植された松果体は、再血管新生と部分的神経支配により生存することを示した[7]。それらは確かにメラトニンを分泌したが、そのレベルは低く、夜間の上昇は全くなかった[7, 283] 。

Based on these results, we speculated that a more suitable way to preserve a healthy and functional pineal gland is either to retard its calcification or to recover the functions of the calcified gland. As mentioned, several pathological conditions might promote the premature pineal calcification. However, the environmental biohazards may also contribute to its development. One of them is fluoride. It was reported that the pineal gland in goosander concentrates fluoride which is a water pollutant [290]. The level of fluoride in the pineal gland of goosander was 5-fold higher than that it in the brain of the animal. The similar results were observed in the aged human pineal gland. In addition, the high level of fluoride in the human pineal gland is positively related to its calcium accumulation of the gland [291]. Thus, decrease in environmental fluoride pollution may be helpful in delaying or avoiding premature pineal calcification. It was hypothesized that the lack of calcium salt crystallization inhibitors, such as pyrophosphate and phytate, would favor calcification [292]. Studies indicated that the phytate content in brains of healthy animals was 10-fold higher than that in other tissues [293,294]. Increases in the availability of calcium salt crystallization inhibitors would tend to protect against pathological pineal calcification.

これらの結果から、健康で機能的な松果体を保存するためのより適切な方法は、石灰化を遅らせるか、石灰化した松果体の機能を回復させることであると推測した。上述のように、いくつかの病理学的状態が早発性松果体石灰化を促進する可能性がある。しかしながら、環境バイオハザードもまたその発展に寄与する可能性がある。その１個がフッ化物です。カワアイサの松果体は、水質汚染物質であるフッ化物を濃縮することが報告されている[290]。カワアイサの松果体のフッ化物濃度はその動物の脳の5倍であった。同様の結果が高齢の人間の松果体でも観察された。さらに、人間の松果体におけるフッ化物の高濃度は、松果体のカルシウム蓄積と正の相関がある[291]。このように、環境フッ化物汚染の減少は、松果体の早期石灰化の遅延または回避に役立つ可能性がある。ピロリン酸塩やフィチン酸塩のようなカルシウム塩結晶化阻害剤の欠如が石灰化を促進するという仮説が立てられた[292]。研究は健康な動物の脳のフィチン酸含量は、他の組織の10倍であることが示された[293, 294]。カルシウム塩結晶化阻害剤の利用可能性の増加は、病的松果体石灰化から保護する傾向がある。

Finally, the pineal gland decalcification may not impossible. Currently, pineal microdialysis is frequently used to measure the melatonin production of the pineal gland [295,296,297,298]. This method could also be used to decalcify the gland by use of EDTA or/and acidic solution as the eluent. This solution would have the ability to dissolve the calcium deposits and removed them by dialysis. Also, cells isolated from young pineal gland or engineering-modified stem pinealocytes could be directly injected into the decalcified in situ pineal gland. Such transplanted cells have the high chance of survival in the gland due to the melatonin level generated by the gland. It was frequently reported that elevated levels of melatonin effectively promotes the transplanted stem cell survival and differentiation in different organs and tissues [299,300,301,302,303]. In a preliminary study, we mixed 2 × 105 cells (in 20 μL) collected from pineal gland of one day old chicks were injected into the in situ pineal gland of 4.5–5 year old hens. The results indicated that this procedure improved egg laying rate and the general wellbeing of the old recipients (unpublished observations). This is the first step to rejuvenate the calcified pineal gland to maintain optimal healthy status of humans.

最後に、松果体の脱灰は不可能ではないかもしれない。現在、松果体極小透析は松果体のメラトニン産生を測定するために頻繁に用いられている[295, 296, 297, 298]。この方法はEDTAまたは酸性溶液を溶離液として用いて腺を脱灰するのにも使用できる。この溶液は、カルシウム沈着物を溶解し、透析によってそれらを除去する能力を有する。また、若い松果体または工学的に改変した幹松果体細胞から単離した細胞を、脱灰したin situ松果体に直接注入することができた。このような移植された細胞は、腺によって生成されるメラトニン濃度のために腺内で生存する可能性が高い。メラトニン濃度の上昇は、異なる臓器および組織における移植幹細胞の生存および分化を効果的に促進することがしばしば報告されている[299, 300, 301, 302, 303]。予備的研究として、私たちは生後１日の雛の松果体から採取した2×105個の細胞(20μL)を混合して、4.5–5歳の雌鶏のin situ松果体に注入した。結果は、この手順が産卵率と高齢レシピエントの一般的な健康を改善することを示した(未発表の観察)。これは人間の最適な健康状態を維持するために石灰化した松果体を若返らせる最初のステップである。

6. Conclusions

Accumulating evidence indicates that pineal health is important to preserve the optimal physiological status of animals, including humans. The pineal gland is a unique organ which synthesizes melatonin as the signaling molecule of natural environmental changes and as a potent neuronal protective antioxidant. This gland undergoes calcification due to its anatomic structure (rich in vasculature and blood flow) and functions (melatonin production and CSF generation). The pineal has the highest calcification rate among all organs and tissues. Pineal calcification jeopardizes the melatonin synthetic capacity of this gland and is associated with a variety of neuronal diseases. Although PGC is found in neonates, its occurrence is primarily associated with pathological conditions and aging. The exact mechanisms of how it occurs are currently unknown; however, several theories have been proposed to explain calcium deposit in the gland. We hypothesize that PGC is an active process which is similar to bone formation, that is, the osteocytes (or osteocyte-like cells) and osteoblasts (or osteoblast-like cells) are involved. These cells probably differentiate from MCSc which are the de nova MCSc of the gland; alternatively, they migrated from the vasculature under pathological conditions such as chronic inflammation. High levels of melatonin generated by the gland promote PGC since this molecule enhances the differentiations of MCSc into osteoblasts and osteocytes. To compensate for the functional loss of the pineal gland, pineal grafts have been performed in different organs and tissues. The grafted pineal, however, cannot mimic the functions of the in situ pineal gland, especially since they do not establish the normal melatonin circadian rhythm. Thus, perhaps the best way to preserve a healthy pineal gland is to rejuvenate the in situ pineal gland by decalcification and then stem cell injection into the gland. It is speculated that a healthy pineal gland would be response to high level of melatonin production which benefits to immunomodulation, metabolic balance and anticancer effect generally [304,305,306]. Thus, this process and its outcomes should be investigated with enthusiasm in the future.

蓄積された証拠は、松果体の健康が人間を含む動物の最適な生理学的状態を維持するために重要であることを示している。松果体は自然環境変化のシグナル分子として、また強力な神経保護抗酸化物質としてメラトニンを合成する特異な器官である。この腺はその解剖学的構造(血管系と血流に富む)と機能(メラトニン産生と脳脊髄液産生)のために石灰化する。松果体はすべての器官と組織の中で最も石灰化率が高い。松果体石灰化はこの腺のメラトニン合成能を危険にさらし、様々な神経疾患と関連する。PGCは新生児に見られるが、その発生は主に病理学的状態と加齢と関連している。それがどのように起こるかの正確なメカニズムは現在のところ不明である；しかしながら、腺へのカルシウム沈着を説明するいくつかの理論が提案されている。PGCは骨形成と同様の活性プロセスであり、骨細胞(または骨細胞様細胞)と骨芽細胞(または骨芽細胞様細胞)が関与すると仮定した。これらの細胞はおそらく腺のde nova MCScであるMCScから分化する；あるいは、慢性炎症のような病的な条件下で血管系から移動した。この分子は骨芽細胞と骨細胞へのMCScの分化を促進するので、腺によって生成される高レベルのメラトニンはPGCを促進する。松果体の機能的損失を補うために、松果体移植が様々な器官と組織で行われてきた。しかし、移植された松果体は、特に正常なメラトニン概日リズムを確立しないので、in situ松果体の機能を模倣することはできない。したがって、健康な松果体を維持する最善の方法は、おそらく、脱灰とその後の松果体への幹細胞注入によって、in situ松果体を若返らせることである。健康な松果体は、一般的に免疫調節、代謝バランスおよび抗癌効果に利益をもたらす高レベルのメラトニン産生に反応すると推測される[304, 305, 306]。したがって、このプロセスとその結果は、今後熱意を持って研究されるべきである。

Conflicts of Interest

The authors declare no conflict of interest.

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