小胞体ストレスとパーキンソニズム発症の関連に関する研究

パーキンソン病(PD)は黒質ドーパミン神経の選択的変性により、進行性の運動障害をきたす原因不明の神経変性疾患である。本研究において我々は、小胞体ストレス蛋白の一つORP150、若年性遺伝性パーキンソン病の原因遺伝子の一つであり、ユビキチン架橋酵素であるParkin、Parkinの基質として同定され、小胞体ストレスを誘起することが判明している蛋白質Pael受容体(Parkinassociated endothelin like receptor: PaelR)、更に、やはり小胞体を誘起することが判明している分泌系蛋白質Megsinを用いて、パーキンソニズムにおける小胞体ストレスの役割をより明らかにする事を目指した。アデノウイルスを用いて、PaelRをマウス線条体から逆行性に黒質緻密層(SNpc)に発現させた所、SNpc特異的に小胞体ストレス由来の神経細胞死が起こり、それらはGRP78やParkin等の小胞体関連蛋白の強制発現で改善されたが、HSP70など細胞質局在のストレス蛋白の強制発現では改善されなかった。また、小胞体ストレスモデル動物であるメグシン過剰発現ラット(Tg Meg rat)の海馬及びSNpcでは、神経細胞内凝集体の存在、小胞体ストレスの上昇に加えて、神経変性(神経細胞死)が観察された。このことから、SNpcの神経細胞が特に小胞体ストレスに対して脆弱であることが明らかになった。本研究を通じて、小胞体ストレスとパーキンソニズム発症の関連がより明らかになり、今後、小胞体ストレス制御による新しい神経保護法の開発が可能になると期待される。Selective loss of dopaminergic neurons is the final common pathway in Parkinson\u27s disease, the second most common neurodegenerative disorder. Selective neuronal expression of Pael-R(Parkin associated endothelin-like receptor)in mouse brain was achieved by injecting adenoviral vectors carrying a modified neuron-specific promoter and Cre-recombinase into the striatum. Upregulation of Pael-Receptor in the substantia nigra pars compacts (SNpc) of mice by retrograde infection induced endoplasmic reticulum(ER) stress lead to decreased levels of tyrosine hydroxylase and death of dopaminergic neurons. Neuronal cell death was not observed in the other areas of the brain projecting to/from the SNpc. The role of ER stress in dopaminergic neuronal vulnerability was highlighted by their decreased survival in mice deficient in the ubiquitin-protein ligase Parkin and the ER chaperone ORP150(150 kDa oxygen regulated protein), compared with their robust survival consequent to overexpression of either P arkin or an ER chaperone, 78 kDa glucose regulated protein (GRP78). Dopamine-related toxicity was also a key factor, as a dopamine synthetase inhibitor blocked neuronal death in parkin null mice. These data suggest a model in which ER- and dopamine-related slam are major contributors to decreased viability of dopaminergic neurons in a setting relevant to Parkinson\u27s disease.FENIB (familial encephalopathy with neuroserpin inclusion bodies)is caused by intracellular accumulation/polymerization of mutant neuroserpins. Transgenic rats overexpressing megsin (Tg meg), a newly identified serine protease inhibitor (serpin), demonstrated intraneuronal periodic-acid Schiff (PAS)-positive inclusions distributed throughout deeper layers of cerebral cortex, CA1 of the hippocampus, and substantia nigra. Hippocampal extracts from Tg meg rats showed increased expression of ER stress proteins, and activation of caspases-12 & -3, associated with decreased neuronal density. Enhanced ER stress was also observed in dopaminergic neurons in the substantia nigra, in parallel with decreased neuronal viability and motor coordination. In each case, PAS-positive inclusions were also positive for megsin. These data suggest that overexpression of megsin results in ER stress, eventuating in the formation of PAS-positive inclusions. Tg meg rats provide a novel model relevant to FENIB in which accumulation of serpins in the ER induces selective dysfunction/loss of specific neuronal populations.研究課題/領域番号:17500226, 研究期間(年度):2005-2007出典：「小胞体ストレスとパーキンソニズム発症の関連に関する研究」研究成果報告書　課題番号17500226 (KAKEN：科学研究費助成事業データベース（国立情報学研究所）)　　　本文データは著者版報告書より作

遺伝子導入による興奮性アミノ酸誘導性神経細胞死の抑制

1)虚血性神経細胞死ORP150が小胞体に局在するストレス蛋白として、神経細胞を虚血ストレスから守りうるメカニズムを明らかにするため、本研究では海馬に対するカイニン酸負荷をパラダイムとして用い、ORP150の神経細胞保護効果を明らかにした。すなわち、興奮性アミノ酸によって引き起こされる細胞内カルシウム負荷が、小胞体に強制発現させたORP150によって緩衝され、致死的な細胞内カルシウムの上昇を抑え、神経細胞を虚血による細胞死から救済していることを明らかにした。さらに、小胞体の機能を保持することによって、虚血による神経細胞死を抑制しうることを示し、ORP150遺伝子導入による虚血性神経細胞死の制御の可能性に関しても検索した。2)マウスにおける小脳発生での検討。ORP150を過剰発現させたトランスジェニックマウス(TG)では、プルキンエ細胞に強いORP150の発現を認めるとともに、ORP150ノックアウトヘテロ接合体(KO)では、その発現は明らかに減弱していた。野生型マウスでは生後4日をピークにプルキンエ細胞層で活性化型Caspase-3の免疫陽性細胞が見られたが、TGでは陽性細胞数が有意に減少していた。Calbindin染色で評価したプルキンエ細胞数も生後4-20日にかけてTGで多く、KOで減少していた。小脳発生過程における神経細胞死にも小胞体を介する神経細胞死の関与を明らかにした。3)コンフォーメーショナル病モデルとしてmegsin transgenic rat(Meg Tg)の解析Megsinは東海大学の宮田らによって見いだされた新規serine proteinase inhibitorであり、Meg Tgのヘテロ接合体は、生後4-6ヶ月をピークに、海馬および黒質(SNpc)において神経細胞死が進む。このMeg Tgの黒質における病変が小胞体依存性の神経細胞死であることを示した。ORP150 is a novel stress protein localized in the endoplasmic reticulum (ER). To investigate the role of ORP150 in delayed neuronal cell death, we have examined its expression in the gerbil brain after the ischemic insult. The expression of ORP150 antigen, as well as its transcripts, was observed in the CA1 region after the occlusion of the common carotid altety, and this was enhanced by the preconditioning. In cultured neurons, exposure to either hypoxia or glutamate induced the expression of ORP150, and this was also observed by treating the culture with either thapsigargin or breferdin-A, indicating that both glutamate and hypoxia can cause shess in the ER (ER stress). Neurons became more vulnerable to these stresses following treatment of either cyclcheximide or the infection with an adenovirus carrying ORP150 antisense structure. In cantrast, the overexpression of ORP150 by adenovinus suppressed the neuronal cell death, and this was accompanied by the suppression of the Ca2+ eleva tion and proteolytic activity induced by glutamate. Further, overexpressicn of ORP150 in CA1 neurons by the adenovirus carrying ORP150-sense struciture suppressed delayed neuronal cell death after ischemia. These data suggest a possible function of ORP150 as an intracellular apparatus, which participates in a protective response in ischemic tolerance.A series of events initiated by glutamate-receptor interaction perturbs cellular homeostasis resulting in elevation of intracellular free calcium and cell death. Cells subject to such environmental change express stress proteins, which contribute importantly to maintenance of metabolic homeostasis and viability. We show that an inducible chaperone present in endoplasmic reticulum (ER), the 150 kDa oxygen-regulated protein (ORP150) is expressed both in human brain after seizure attack and in mice hippocampus after kainite administration. Using mice heterozygous for ORP150 deficiency, exposure to excitatory stimuli caused hippocampal neurons to display exaggerated elevation of cytosolic calcium accompanied by activation of μ-calpain and cathepsin B, as well as increased vulnerability to glutamate-induced cell death in vitro and decreased survival to kainate in vivo. In contrast, targeted neuronal overexpression of ORP150 suppressed each of these events, and enhanced neuronal and animal survival in parallel with diminished seizure intensity. Studies using cultured hippocampal neurons showed that ORP150 regulates cytosolic free calcium and activation of proteolytic pathways causing cell death in neurons subject to excitatory stress. Our data underscore a pivotal role for ER stress in glutamate toxicity, and pinpoint a key ER chaperone, ORP150, which orchestrates the protective stress response critical for neuronal survival.研究課題/領域番号:14580725, 研究期間(年度):2002-2004出典：「遺伝子導入による興奮性アミノ酸誘導性神経細胞死の抑制」研究成果報告書　課題番号14580725(KAKEN：科学研究費助成事業データベース（国立情報学研究所）)　　　本文データは著者版報告書より作

Deletion of N-myc downstream-regulated gene 2 attenuates reactive astrogliosis and inflammatory response in a mouse model of cortical stab injury

N-myc downstream-regulated gene 2 (Ndrg2) is a differentiation- and stress-associated molecule predominantly expressed in astrocytes in the CNS. In this study, we examined the expression and the role of Ndrg2 after cortical stab injury. We observed that Ndrg2 expression was elevated in astrocytes surrounding the wounded area as early as day 1 after injury in wild-type mice. Deletion of Ndrg2 resulted in lower induction of reactive astroglial and microglial markers in the injured cortex. Histological analysis showed reduced levels of hypertrophic changes in astrocytes, accumulation of microglia, and neuronal death in Ndrg2-/- mice after injury. Furthermore, activation of the IL-6/signal transducer and activator of transcription 3 (STAT3) pathway, including the expression of IL-6 family cytokines and phosphorylation of STAT3, was markedly reduced in Ndrg2-/- mice after injury. In a culture system, both of Il6 and Gfap were up-regulated in wild-type astrocytes treated with forskolin. Deletion of Ndrg2 attenuated induction of these genes, but did not alter proliferation or migration of astrocytes. Adenovirus-mediated reexpression of Ndrg2 rescued the reduction of IL-6 expression after forskolin stimulation. These findings suggest that Ndrg2 plays a key role in reactive astrogliosis after cortical stab injury through a mechanism involving the positive regulation of IL-6/STAT3 signaling. © 2014 International Society for Neurochemistry

Expression of the oxygen-regulated protein ORP150 accelerates wound healing by modulating intracellular VEGF transport

金沢大学医薬保健研究域医学系Expression of angiogenic factors such as VEGF under conditions of hypoxia or other kinds of cell stress contributes to neovascularization during wound healing. The inducible endoplasmic reticulum chaperone oxygen-regulated protein 150 (ORP150) is expressed in human wounds along with VEGF. Colocalization of these two molecules was observed in macrophages in the neovasculature, suggesting a role of ORP150 in the promotion of angiogenesis. Local administration of ORP150 sense adenovirus to wounds of diabetic mice, a treatment that efficiently targeted this gene product to the macrophages of wound beds, increased VEGF antigen in wounds and accelerated repair and neovascularization. In cultured human macrophages, inhibition of ORP150 expression caused retention of VEGF antigen within the endoplasmic reticulum (ER), while overexpression of ORP150 promoted the secretion of VEGF into hypoxic culture supernatants. Taken together, these data suggest an important role for ORP150 in the setting of impaired wound repair and identify a key, inducible chaperone-like molecule in the ER. This novel facet of the angiogenic response may be amenable to therapeutic manipulation

Transmission of cell stress from endoplasmic reticulum to mitochondria: enhanced expression of Lon protease

The rat homologue of a mitochondrial ATP-dependent protease Lon was cloned from cultured astrocytes exposed to hypoxia. Expression of Lon was enhanced in vitro by hypoxia or ER stress, and in vivo by brain ischemia. These observations suggested that changes in nuclear gene expression (Lon) triggered by ER stress had the potential to impact important mitochondrial processes such as assembly and/or degradation of cytochrome c oxidase (COX). In fact, steady-state levels of nuclear-encoded COX IV and V were reduced, and mitochondrial-encoded subunit II was rapidly degraded under ER stress. Treatment of cells with cycloheximide caused a similar imbalance in the accumulation of COX subunits, and enhanced mRNA for Lon and Yme1, the latter another mitochondrial ATP-dependent protease. Furthermore, induction of Lon or GRP75/mtHSP70 by ER stress was inhibited in PERK (−/−) cells. Transfection studies revealed that overexpression of wild-type or proteolytically inactive Lon promoted assembly of COX II into a COX I–containing complex, and partially prevented mitochondrial dysfunction caused by brefeldin A or hypoxia. These observations demonstrated that suppression of protein synthesis due to ER stress has a complex effect on the synthesis of mitochondrial-associated proteins, both COX subunits and ATP-dependent proteases and/or chaperones contributing to assembly of the COX complex

A rat model of human FENIB (familial encephalopathy with neuroserpin inclusion bodies)

金沢大学大学院医学系研究科脳細胞分子学FENIB (familial encephalopathy with neuroserpin inclusion bodies) is caused by intracellular accumulation/polymerization of mutant neuroserpins in the endoplasmic reticulum (ER). Transgenic rats overexpressing megsin (Tg meg), a newly identified serine protease inhibitor (serpin), demonstrated intraneuronal periodic-acid Schiff (PAS)-positive inclusions distributed throughout deeper layers of cerebral cortex, CA1 of the hippocampus, and substantia nigra. Hippocampal extracts from Tg meg rats showed increased expression of ER stress proteins, and activation of caspases-12 and -3, associated with decreased neuronal density. Enhanced ER stress was also observed in dopaminergic neurons in the substantia nigra, in parallel with decreased neuronal viability and motor coordination. In each case, PAS-positive inclusions were also positive for megsin. These data suggest that overexpression of megsin results in ER stress, eventuating in the formation of PAS-positive inclusions. Tg meg rats provide a novel model of FENIB, where accumulation of serpins in the ER induces selective dysfunction/loss of specific neuronal populations. © 2006 Elsevier Inc. All rights reserved

Expression of the endoplasmic reticulum molecular chaperone (ORP150) rescues hippocampal neurons from glutamate toxicity

金沢大学医薬保健研究域医学系A series of events initiated by glutamate-receptor interaction perturbs cellular homeostasis resulting in elevation of intracellular free calcium and cell death. Cells subject to such environmental change express stress proteins, which contribute importantly to maintenance of metabolic homeostasis and viability. We show that an inducible chaperone present in endoplasmic reticulum (ER), the 150-kDa oxygen-regulated protein (ORP150), is expressed both in the human brain after seizure attack and in mouse hippocampus after kainate administration. Using mice heterozygous for ORP150 deficiency, exposure to excitatory stimuli caused hippocampal neurons to display exaggerated elevation of cytosolic calcium accompanied by activation of μ-calpain and cathepsin B, as well as increased vulnerability to glutamate-induced cell death in vitro and decreased survival to kainate in vivo. In contrast, targeted neuronal overexpression of ORP150 suppressed each of these events and enhanced neuronal and animal survival in parallel with diminished seizure intensity. Studies using cultured hippocampal neurons showed that ORP150 regulates cytosolic free calcium and activation of proteolytic pathways causing cell death in neurons subject to excitatory stress. Our data underscore a possible role for ER stress in glutamate toxicity and pinpoint a key ER chaperone, ORP150, which contributes to the stress response critical for neuronal survival

The effect of Ndrg2 expression on astroglial activation

N-myc downstream-regulated gene 2 (Ndrg2) is a differentiation- and stress-associated molecule predominantly expressed in astrocytes in the central nervous system (CNS). To study the expression and possible role of Ndrg2 in quiescent and activated astrocytes, mice were administrated with 1-methyl-4-phenyl-1,2,3,6-tetrahydropypridine (MPTP), a Parkinson disease (PD)-related neurotoxin which causes both neurodegeneration and glial activation. Immunohistological analysis revealed that Ndrg2 was highly expressed in both types of astrocytes, but less so in astrocytes during the early process of activation. Ndrg2 was also expressed in astrocyte-like cells, but not in neurons, in human brains from PD and Cortico-basal degeneration (CBD) patients. In cultured astrocytes, gene silencing of Ndrg2 significantly enhanced the numbers of 5-bromo-2′-deoxy-uridine (BrdU)-incorporated and proliferating cell nuclear antigen (PCNA)-positive cells, and reduced the length of cell processes and the amount of F-actin. In contrast, adenovirus-mediated overexpression of Ndrg2 significantly reduced the numbers of BrdU-incorporated and PCNA-positive cells, and enhanced the amount of F-actin. Fractionation and immunocytochemical analysis further revealed that Ndrg2 was located in different cellular fractions including the cytosol and cell surface membranes. These results suggest that Ndrg2 may regulate astroglial activation through the suppression of cell proliferation and stabilization of cell morphology. © 2011 Elsevier Ltd. All rights reserved

AAPH ユライ ペルオキシルラジカル ト ルミノール カガク ハッコウ ヲ クミアワセタ ラジカル ホソク カッセイ ソクテイ ジョウケン ノ ソクテイ

様々なラジカル捕捉活性の測定方法が提案されているが、アゾ化合物の一つであるAAPHをラジカル発生剤に用い、発生したペルオキシルラジカルをルミノール化学発光で検出する方法(AAPH-CL法)の精度向上を目的として、ラジカル捕捉剤を共存させないコントロール時の発光量を増大させる条件を検討した。また得られた条件を用いて、雁ヶ音茶の浸出温度と時間によるラジカル捕捉能の変化を調べた。さらに各温度と時間で浸出した茶の官能検査を実施し、「身体を健康に、かつ、美味しい」茶の浸出条件の一助を求めた。 AAPH-CL法のコントロール値を増大させるためには、加温温度の上昇ならびに時間の伸長が各々の条件に依存的に寄与していることが認められた。また、AAPH濃度、ホウ酸緩衝液濃度も高い方が、コントロール値を高めることも判った。ただ、実験の操作性や再現性などを考慮し、加温温度45℃、加温時間4分、初発AAPH濃度80mM、ルミノール試薬中のホウ酸緩衝液濃度100mMを最適測定条件とした。この条件を用いて、雁ヶ音茶の浸出条件とラジカル捕捉能を調べた結果、温度、時間とも増大するにつれて、捕捉能が高くなることが判明した。官能検査の結果、80℃、90℃の浸出温度において、浸出時間が長いほうが好まれる傾向が見いだされたが、ラジカル捕捉活性との明確な相関性は見られなかった