TWI695891B - 早發型帕金森病（ｄ３３１ｙ）ｐｌａ２ｇ６突變基因嵌入模式與藥物篩選平台和方法 - Google Patents
早發型帕金森病（ｄ３３１ｙ）ｐｌａ２ｇ６突變基因嵌入模式與藥物篩選平台和方法 Download PDFInfo
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Abstract
本發明揭露(D331Y) PLA2G6基因嵌入小鼠，其顯現與臨床帕金森病病人相似的臨床症狀，且其於約6個月大時開始出現中腦黑質區多巴胺神經細胞死亡、突觸核蛋白病變及Tau病變，其中該多巴胺神經細胞內的粒線體不僅結構異常且功能缺損。該(D331Y) PLA2G6基因嵌入小鼠對L-多巴胺顯現良好的治療反應。該(D331Y) PLA2G6基因嵌入小鼠可用於開發治療帕金森病之藥物和方法。
Description
本發明關於早發型帕金森病之基因檢測模式和篩選用於治療早發型帕金森病的藥物之平台和方法。
相關揭露
本發明之內容已於2018年8月8日線上發表於期刊Molecular Neurobiology (網址https://doi.org/10.1007/s12035-018-1118-5)。
帕金森病(Parkinson’s disease, PD)是最常見的神經元病變性運動障礙疾病且係僅次於阿茲海默氏症之第二常見的神經退化疾病。臨床表徵為顫抖、動作緩慢、僵硬及平衡障礙，且病理變化為中腦黑質區(substantia nigra)多巴胺神經細胞(dopaminergic neurons)進行性死亡和路易氏體(Lewy body)。
迄今對於帕金森病的致病機轉仍不清楚，且目前帕金森病的治療只能改善症狀而不能延緩神經退化。
大部分帕金森病屬於偶發性，但在約5至10%的家族遺傳性帕金森病患者之基因研究中找到超過10個與帕金森病相關的基因變異，且該等病患的臨床症狀和病理變化與偶發性病患並無差異，其顯示遺傳性與偶發性帕金森病的發病極可能源自於相同之分子機轉所導致的神經退化。
已知磷脂酶A2第VI型(PLA2G6)的基因突變能引起第十四型遺傳型隱性帕金森病(PARK14)且為導致早發型隱性帕金森病常見的異常基因之一。PARK14的病患與常見的偶發性帕金森病病患皆表現類似的臨床特徵。先前研究顯示(D331Y) PLA2G6突變是PARK14患者最為常見的基因變異(Shi CH et al., Neurology, 77(1):75-81)，該患者雖同樣表現典型帕金森病但病程進展惡化較快，且帶有雜型合子(D331Y) PLA2G6突變亦增加帕金森病早發的風險(Lu CS et al., Am J Med Genet 2012, 159B:183-191)。
PLA2G6主要功能為催化脂肪酸代謝且同時參與細胞生長、細胞凋亡及粒線體之生理功能。PLA2G6基因檢測大多應用於幼兒神經軸突性疾病(infantile neuroaxonal dystrophy)但於早發型帕金森病無相關應用。
對於帕金森病之臨床檢測，目前無PLA2G6基因突變的檢測。
另一方面，雖然基因研究大幅增進對帕金森病之病因和致病機轉的了解且提供未來開發新穎神經保護治療的基礎和方向，但是目前帕金森病的小鼠動物模式大多是基因轉殖小鼠(transgenic mice)或是基因剔除小鼠(knockout mice)。
基因轉殖和基因剔除雖皆屬研究基因變異與神經疾病之關連的方法，但仍存在潛在的問題：
(1)無法完全剔除目標基因：
(A)因篩選標記(selection marker)***目標基因內，仍有部分未受影響的基因片段仍能表現部分蛋白；
(B)具有其他啟動子或是起始密碼(AUG)可調控目標基因的表現，使得目標基因仍能被表現；及
(C)異常表現序列(exon)的產生；
(2)其他基因的剔除：為能剔除目標基因，通常會大範圍剔除目標基因的基因表現區域，但同時可能會剔除其他基因而無法真正闡明目標基因的功能；
(3)篩選基因的影響：利用同源重組(homologous recombination)後，存在基因體內的篩選基因會影響表型；
(4)目前帕金森病相關基因轉殖小鼠未能顯現早發性中腦黑質區多巴胺神經細胞死亡：
(A)PLA2G6基因剔除小鼠模式(PLA2G6 knockout mice)中，14個月大的小鼠之中腦黑質區未發現多巴胺神經細胞死亡(Beck G et al., PLoS One. 2016, 11: e0153789)；
(B)12個月大的(G2019S) LRRK2基因嵌入小鼠未發現中腦黑質區多巴胺神經細胞死亡(Longo F et al., Acta Neuropathol Commun., 2017, 5:22)，且LRRK2基因剔除小鼠要至15個月大時方才開始出現中腦黑質區多巴胺神經細胞死亡(Giaime E et al., Neuron. 2017, 96:796-807)；
(C)PINK1基因剔除小鼠在8至12月大時未發現中腦黑質區多巴胺神經細胞死亡(Kitada T et al., PNAS. 2007, 104:11441-6；Madeo G et al., Mov Disord. 2014, 29:41-53；Akundi RS et al., PLoS One. 2011, 6:e16038)；及
(D)12至14月大的Parkin基因剔除小鼠未發現中腦黑質區多巴胺神經細胞死亡(Pickrell AM et al., Neuron. 2015, 87(2):371-81；Dai Y et al., Mitochondrion 2013, 13:282-291；Goldberg MS et al., J Biol Chem. 2003, 278:43628-35)；及
(5)MPTP神經毒素誘導之帕金森病小鼠模式需要長達21天投予25 mg/kg L-多巴胺(L-DOPA)藥物治療方才顯現明顯的治療效果(Zhao TT et al., BMC Complement Altern Med. 2017, 17:449；Zhao TT et al., Neuroscience. 2016, 339:644-654)。
相對地，基因嵌入(knockin mice)小鼠能精確表現基因突變的位點並能模擬突變基因造成之神經退化的機轉且不影響其他基因。
目前帕金森病之動物模式無早期出現神經退化的症狀。對於PARK14之研究，目前亦無PLA2G6基因嵌入小鼠。
因此，需要利用僅含基因點突變的基因嵌入動物模式，其除了不會影響其他基因的表現且亦大幅減少篩選基因的影響。
為提早診斷早發型PARK14，對於PLA2G6基因突變的檢測，自懷疑罹患早發型PARK14病患之血液檢體抽取基因組DNA，針對PLA2G6基因的編碼區，利用特異性引子(primer)對進行聚合酶連鎖反應(PCR)與基因定序分析以檢測該懷疑罹患早發型PARK14病患所顯現的PLA2G6基因突變型態。
進行該PCR使用之材料和試劑包括：懷疑罹患早發型PARK14病患之基因組DNA、特異性引子對、緩衝液、PCR聚合酶及去離子水。
該特異性引子對包括正向(F)引子和反向(R)引子，該F引子和R引子可選自表1：
表1
若該懷疑罹患早發型PARK14病患之PLA2G6基因的編碼區存在G991T之核苷酸突變，即對應D331Y之胺基酸突變，則表示該病患確實罹患早發型PARK14。
因此，本發明提供一種診斷早發型PARK14之方法，其包含下述步驟：
(1) 自懷疑罹患早發型PARK14病患之血液檢體抽取基因組DNA；
(2) 針對該基因組DNA之PLA2G6基因的編碼區，利用如表1所示之特異性引子對進行PCR擴增；及
(3) 對經該PCR擴增之DNA片段進行定序並與正常DNA片段進行比對，
其中若該懷疑罹患早發型PARK14病患之PLA2G6基因的編碼區存在G991T之核苷酸突變，即表示該病患確實罹患早發型PARK14。
本發明亦提供一種診斷早發型PARK14之套組，其包括：如表1所示之特異性引子對、緩衝液及PCR聚合酶以及使用指示說明。
進一步，基於PLA2G6 D331Y之胺基酸突變，本案發明人建立同型合子(D331Y) PLA2G6
D331Y/D331Y基因嵌入小鼠模式以模擬PARK14病患常見的(D331Y) PLA2G6基因變異，該近似PARK14病患的PLA2G6
D331Y/D331Y基因嵌入小鼠模式表現之PLA2G6蛋白活性明顯降低且能顯現早發性多巴胺神經死亡和神經退化。
於病理生理上，該PLA2G6
D331Y/D331Y基因嵌入小鼠顯現：
(1)早發性中腦黑質區神經退化：目前帕金森病小鼠模式皆顯現晚發性神經退化或是於約12個月大方才出現神經退化症狀。該PLA2G6
D331Y/D331Y基因嵌入小鼠於約6個月大時即顯現神經退化現象，其中該小鼠之中腦黑質區出現多巴胺神經細胞死亡和內質網壓力(ER stress)相關蛋白的大量表現。
(2)突觸核蛋白病變(synucleinopathy)和Tau病變(tau pathology)：目前帕金森病基因轉殖或基因剔除小鼠模式皆於約12個月大方才於腦部出現路易氏體。PLA2G6
D331Y/D331Y基因嵌入小鼠同時顯現突觸核蛋白病變和Tau病變且於約9個月大時中腦黑質區即出現路易氏體堆積，且亦發現大量磷酸化Tau蛋白表現。
(3)早發型帕金森病症狀：目前帕金森病小鼠模式通常於約12個月大方才出現運動失調或動作障礙。PLA2G6
D331Y/D331Y基因嵌入小鼠於約6至9個月大時即出現明顯的運動機能障礙。
(4)粒線體結構和功能異常：約9個月大之
PLA2G6
D331Y/D331Y基因嵌入小鼠的多巴胺神經細胞之外鞘(myelin)結構異常、粒線體嵴(cristae)減少且粒線體型態改變。同時，PLA2G6
D331Y/D331Y基因嵌入小鼠的粒線體複合體I (mitochondrial complex I)之活性降低而無法產生足夠的ATP，且過多自由基(reactive oxygen species；ROS)的產生使得粒線體產生過多的脂質過氧化與代謝物且促進細胞色素c釋放到細胞質中，進而使多巴胺神經細胞走向細胞凋亡。此外，亦發現PLA2G6
D331Y/D331Y基因嵌入小鼠之粒線體自噬(mitophagy)功能異常，表現較少的粒線體自噬相關蛋白，即表示(D331Y) PLA2G6突變會損害粒線體自噬功能。
(5)對L-Dopa之藥物治療反應良好：L-Dopa是臨床上最常用於治療帕金森病的藥物。約9個月大的PLA2G6
D331Y/D331Y基因嵌入小鼠會出現運動機能障礙。投與較低劑量L-DOPA (1.5 mg/kg)可明顯改善PLA2G6
D331Y/D331Y基因嵌入小鼠之運動機能障礙。因此，PLA2G6
D331Y/D331Y基因嵌入小鼠可作為治療藥物篩選的動物模式和平台。
(6)特異性訊號傳遞路徑：利用微陣列(microarray)分析PLA2G6
D331Y/D331Y基因嵌入小鼠之轉錄體(transcriptome)，發現PLA2G6
D331Y/D331Y基因嵌入小鼠顯現多個特異性基因的改變，諸如調控神經細胞存活、神經細胞分化、細胞凋亡訊號傳遞路徑的改變。PLA2G6
D331Y/D331Y基因嵌入小鼠之CTNNB1基因(轉譯蛋白為Catenin beta-1)的表現明顯下降，即表示PLA2G6調控CTNNB1基因之表現。
使用本發明揭露之PLA2G6
D331Y/D331Y基因嵌入小鼠可解決下述問題：
(1)了解早發型PARK14的致病分子機轉：多巴胺神經細胞死亡會造成帕金森病症狀的產生。若能早期預防性治療以防止神經退化，則能維持病患的良好生活機能與品質。PLA2G6
D331Y/D331Y基因嵌入小鼠是目前所沒有的早發型帕金森病動物模式。PLA2G6
D331Y/D331Y基因嵌入小鼠在早期(約6個月大)時即顯現神經退化和帕金森病症狀。PLA2G6
D331Y/D331Y基因嵌入小鼠能提供研究早發型帕金森病的疾病模式並有助於了解早期帕金森病的致病機轉。
(2)疾病治療標的與生物標記開發：基於
PLA2G6
D331Y/D331Y基因嵌入小鼠的多巴胺神經細胞之粒線體結構與功能的異常，該異常之粒線體可作為藥物治療的標的，因此可利用PLA2G6
D331Y/D331Y基因嵌入小鼠作為開發粒線體保護藥物的動物模式。PLA2G6
D331Y/D331Y基因嵌入小鼠是目前所沒有的早發型帕金森病動物模式，因此可用於開發生物標記，觀察該生物標記在不同疾病階段的變化，以有助於帕金森病早期的診斷與治療。
(3)神經保護藥物開發與藥物篩選平台：
PLA2G6
D331Y/D331Y基因嵌入小鼠可作為神經保護藥物的開發平台並可研究在神經退化的早期階段所產生的分子變化。PLA2G6
D331Y/D331Y基因嵌入小鼠可作為治療突觸核蛋白病變與Tau病變的動物模式。
因此，本發明提供一種篩選用於治療神經元病變性運動障礙疾病(特別是帕金森病，尤其是PARK14)的藥物之平台，該平台包含PLA2G6
D331Y/D331Y基因嵌入小鼠，該PLA2G6
D331Y/D331Y基因嵌入小鼠顯現早發性黑質區神經退化、突觸核蛋白病變及Tau病變。
本發明亦提供一種使用(D331Y) PLA2G6突變基因嵌入小鼠以篩選用於治療神經元病變性運動障礙疾病(特別是帕金森病，尤其是PARK14)的藥物之方法，該方法包含(1)投與候選藥物至PLA2G6
D331Y/D331Y基因嵌入小鼠和(2)評估該候選藥物改善該PLA2G6
D331Y/D331Y基因嵌入小鼠的運動機能障礙之功效。該功效包含PLA2G6蛋白活性增加、多巴胺神經細胞死亡減少、突觸核蛋白病變與Tau病變減少、路易氏體堆積降低、粒線體複合體I活性增加、ATP合成增加、自由基產生減少、神經細胞生長/神經保護相關基因表現增加、神經細胞凋亡相關基因表現減少及/或Catenin beta-1表現增加。該神經細胞生長/神經保護相關基因包括選自Bmp6、Ccnd2、Ctnnb1、Hspa1b、Kidins220、Mapk1、Psap及Sdc2中一或多者，且該神經細胞凋亡相關基因係Mark4或Xaf1或該二者。
| 編碼區 | 寡核苷酸 | 退火溫度(℃) | 退火時間(秒) |
| 1F | gacagggccaccagtgattg | 55 | 30 |
| 1R | agttcgagatgagacacgggc | 55 | 30 |
| 2F | caggatctggggacaacgc | 55 | 30 |
| 2R | gccaataagacctccaatcc | 55 | 30 |
| 3F | gggaccttctgattccagc | 55 | 30 |
| 3R | gcccacacaagcaggtacac | 55 | 30 |
| 4F | aaagtccgagtttccgagtg | 60 | 30 |
| 4R | aggcctgagagtgacacctg | 60 | 30 |
| 5F | cccggcctctttacgttc | 55 | 30 |
| 5R | ctcaggcacgggacagg | 55 | 30 |
| 6F | cttcatcccacgccacg | 60 | 30 |
| 6R | gaacctgcttcctgaggg | 60 | 30 |
| 7F | cagtgcccacgtgtccc | 55 | 30 |
| 7R | gacagccctcctgcattc | 55 | 30 |
| 8F | ctttgttcttcacttccccg | 58 | 30 |
| 8R | ctcggtccctgtatccacc | 58 | 30 |
| 9F | agctgcttgggatgtaccagc | 55 | 30 |
| 9R | cggcttcctttagtgacttccg | 55 | 30 |
| 10F | ctagggacctctggggtagc | 58 | 30 |
| 10R | gtgaggggcaggaaagc | 58 | 30 |
| 11F | aaagtactgggctgtggcag | 55 | 30 |
| 11R | gcaaagccctgaagacaaac | 55 | 30 |
| 12F | aatttgggtttgcttaggcctc | 55 | 30 |
| 12R | gttccctctgctcccctcaag | 55 | 30 |
| 13F | aattgtggggaaagggaaag | 60 | 30 |
| 13R | accaccccacagcctctc | 60 | 30 |
| 14F | catgggttttatgccagtcc | 58 | 30 |
| 14R | gtccctagcatggtttgctg | 58 | 30 |
| 15F | ccccagagcccagtcttg | 55 | 30 |
| 15R | gtctcctccaacaccaaagg | 55 | 30 |
| 16+17F | gctccgagagtgcaggg | 60 | 30 |
| 16+17R | gcaggggctgaatggac | 60 | 30 |
基因嵌入
利用分子技術與基因轉殖的方法，將(D331Y) PLA2G6基因變異以同源染色體互換(homologous recombination)的方法在小鼠胚胎進行基因改造以產生能表現(D331Y) PLA2G6突變基因的PLA2G6
D331Y/D331Y基因嵌入小鼠。
粒線體型態與功能分析
利用電子顯微鏡觀察多巴胺神經細胞內粒線體的細微結構，並利用實驗試劑分析粒線體功能：粒線體複合物I活性、ATP產生、自由基生成及粒線體脂質過氧化情況。
小鼠帕金森病症狀與小鼠行為分析
在小鼠於不同月大時，分別記錄運動能力與協調性，出現帕金森病症狀時間，分別在L-Dopa給藥前後紀錄並分析小鼠活動力與運動的變化。藉由下列方法，紀錄並分析小鼠行為與運動能力：
A. 利用小鼠影像系統(TopScan video tracking system)紀錄並分析PLA2G6
D331Y/D331Y基因嵌入小鼠之運動與行為。
B. 小鼠爬竿實驗(pole test)分析小鼠平衡感與運動協調性。
C. 圓筒測試(cylinder test)檢測小鼠前肢與旋轉的對稱性。
D. 滾筒式跑步機平衡(Rotarod test)分析小鼠運動協調能力。
多巴胺神經細胞存活率
A. 利用動物影像系統(microPET)確認黑質區多巴胺神經狀態。
B. 利用免疫組織染色(immunohistochemistry)確認多巴胺神經細胞存活情況並利用分析軟體定量多巴胺神經數量。
訊號傳遞路徑分析
利用微陣列分析轉錄體的變化並進一步利用即時定量聚合酶連鎖反應(Real-time quantitative PCR)確認基因表現。利用西方墨點法(Western blot)確認蛋白質表現量。
實施例1. PLA2G6基因檢測
自懷疑罹患早發型PARK14病患之血液檢體抽取基因組DNA，針對PLA2G6基因的編碼區，利用如表1所示之包含正向(F)引子和反向(R)引子之特異性引子對進行PCR擴增與基因定序分析以檢測該懷疑罹患早發型PARK14病患所顯現的PLA2G6基因突變型態。
材料和試劑
懷疑罹患早發型PARK14病患之基因組DNA (100 μg/μl) 1 μl
正向(F)引子(10 μM) 1 μl
反向(R)引子(10 μM) 1 μl
10X PCR緩衝液 2.5 μl
Qiagen HotStarTaq DNA聚合酶 0.5 μl
去離子水 加至25 μl
PCR擴增
定序分析
該懷疑罹患早發型PARK14病患之PLA2G6基因的編碼區存在G991T之核苷酸突變，即對應D331Y之胺基酸突變，表示該病患確實罹患早發型PARK14。
實施例2. (D331Y) PLA2G6基因變異載體製備與(D331Y) PLA2G6基因嵌入小鼠(PLA2G6
D331Y/D331Y基因嵌入小鼠)的建立
利用分子生物技術將帶有(D331Y) PLA2G6基因變異的DNA片段(9.3 kb)接入pBluescript SK (+)載體裡，再利用限制酶Not I與Xho I將帶有(D331Y) PLA2G6變異的DNA片段切下，以顯微注射的方式注入129/Sv小鼠胚胎細胞(129/Sv embryonic stem cell)中，進行同源染色體置換(圖1A)，再將經染色體置換的胚胎細胞注入C57BL/6J小鼠的囊胚(blastocyst)中，產生的小鼠子代與C57BL/6J小鼠配種繁衍，建立對照組、雜型合子(Heterozygous PLA2G6
D331Y/D331Y)及同型合子(Homozygous PLA2G6
WT/D331Y)的基因嵌入小鼠(圖1B, 1C)。相較於對照組小鼠，PLA2G6
D331Y/D331Y基因嵌入小鼠黑質區神經細胞內PLA2G6蛋白質的磷脂酶活性明顯降低(圖1D)。(D331Y) PLA2G6基因嵌入小鼠帶有突變的(D331Y) PLA2G6基因型與降低的蛋白活性，可做為模擬早發型PARK14的動物模型。
實施例3. PLA2G6
D331Y/D331Y基因嵌入小鼠顯現早發性多巴胺神經細胞死亡與多巴胺神經末梢退化
利用免疫組織法分析對照組與(D331Y) PLA2G6基因嵌入小鼠的中腦黑質區(SN：substantia nigra；圖2A)與紋狀體(ST：striatum；圖2B)多巴胺神經細胞退化情形。經過酪胺酸羥化酶專一性抗體(Anti-tyrosine hydroxylase antibody)染色後，6個月大的PLA2G6
D331Y/D331Y基因嵌入小鼠顯現中腦黑質區神經退化，且9個月大的PLA2G6
D331Y/D331Y基因嵌入小鼠神經退化情況更加明顯(圖2A, 2C, 2D)。但在紋狀體部分，對照組和PLA2G6
D331Y/D331Y基因嵌入小鼠皆沒有神經退化的情況(圖2B)，結果顯示(D331Y) PLA2G6突變基因造成黑質區內神經細胞死亡(圖2A, 2C, 2D)。(D331Y) PLA2G6突變基因不會影響紋狀體的神經細胞死亡(圖2E)，利用動物影像系統(microPET imaging)分析紋狀體多巴胺神經末梢分布(nigrostriatal dopaminergic terminals)，發現PLA2G6
D331Y/D331Y基因嵌入小鼠顯現紋狀體多巴胺神經末梢退化(圖2F)。利用免疫組織染色確認紋狀體多巴胺神經末梢情況，(D331Y) PLA2G6基因嵌入小鼠具有紋狀體多巴胺神經末梢退化(圖2G)。PLA2G6
D331Y/D331Y基因嵌入小鼠在早期即出現多巴胺神經死亡與多巴胺神經末梢退化。
實施例4. (D331Y) PLA2G6基因嵌入模式同時顯現突觸核蛋白病變與Tau病變
目前仍無動物模式同時顯現突觸核蛋白病變與Tau病變。路易氏體是造成帕金森病突觸核蛋白病變的蛋白沉澱物質。路易氏體內主要由α-突觸核蛋白(α-synuclein)與磷酸化α-突觸核蛋白(phosphorylated α-synuclein)所組成。此外，帕金森病的另外一病理特徵是Tau病變，其會造成神經纖維纏繞(neurofibrillary tangles)，其中主要的組成蛋白即是Tau蛋白。利用辨認路易氏體專一性抗體(anti-phospho-α-synuclein
Ser129antibody)和磷酸化Tau抗體(anti-phospho-Tau
Ser202/Thr205antibody)，偵測中腦黑質區內路易氏體和神經纖維纏繞產生情形。9個月大正常對照組小鼠並未發現路易氏體堆積(圖3A, 3B)；但在9個月大(D331Y) PLA2G6基因嵌入小鼠的中腦黑質區內可發現神經細胞內路易氏體堆積(圖3C)。進一步利用西方墨點法確認路易氏體內α-突觸核蛋白的表現，在9個月大PLA2G6
D331Y/D331Y基因嵌入小鼠中腦黑質區內有大量的α-突觸核蛋白與磷酸化α-突觸核蛋白表現(圖3D)。此外，(D331Y) PLA2G6基因嵌入小鼠的中腦質區內可發現大量磷酸化Tau蛋白(phosphor-Tau; p-Tau)的表現(圖3E)。所建立的(D331Y) PLA2G6基因嵌入小鼠顯現特殊的帕金森病理特徵，即突觸核蛋白病變與Tau病變。
實施例5. (D331Y) PLA2G6基因嵌入模式顯現早發性帕金森病症狀
目前帕金森病的動物模式通常都需到至晚期(12個月以上)才顯現帕金森病症狀或動作障礙，並無法解釋早發性帕金森病之機轉或發展治療方法。(D331Y) PLA2G6基因嵌入小鼠在早期(約6個月大)即顯現早發性帕金森病的動作障礙。為測試對照組小鼠與PLA2G6
D331Y/D331Y基因嵌入小鼠的動作協調性，利用各項小鼠行為方法分析小鼠的行為表現。分析方法包含：活動量測試(open field test)、圓筒試驗(cylinder test)、滾筒式跑步機(rotarod test)及爬竿測試(pole test)。相較於正常對照組小鼠，約6個月大的(D331Y) PLA2G6基因嵌入小鼠在活動量測試中，顯現活動力(locomotor activity)降低(圖4A, 4B)，且於約12個月大時活動力大幅減少(圖4A, 4B)。在圓筒試驗中，約6個月大的PLA2G6
D331Y/D331Y基因嵌入小鼠開始出現運動協調異常(圖4C)，於約12月個大時顯現明顯的肢體不對稱性(圖4C)。利用滾筒式跑步機分析小鼠的運動協調性，(D331Y) PLA2G6基因嵌入小鼠在約6個月大時開始出現運動協調性異常，且於約12個月大時顯現嚴重的運動失調(圖4D)。利用爬竿測試檢測小鼠的平衡感與運動協調性，約6至12個月大的正常對照組小鼠無任何動作上的異常，PLA2G6
D331Y/D331Y基因嵌入小鼠在約6個月大時開始顯現運動功能不平衡與異常(圖4E)，且於12個月大時顯現嚴重的運動失調(圖4E)。L-多巴胺(Levodopa)是目前最主要用於治療帕金森病的藥物。因此，藉由利用L-多巴胺治療是否能改善運動失調情況，能更加確定是否小鼠為帕金森病小鼠模式。約9個月大的正常對照組小鼠沒有任何運動功能的異常，因此給予L-多巴胺對於運動功能上未顯現任何改變(圖4F, 4G)。約9個月大的(D331Y) PLA2G6基因嵌入小鼠經給予L-多巴胺後，能明顯改善該小鼠之動作活動力(圖4F, 4G)且能增加該小鼠運動的距離和速率(圖4F, 4G)。PLA2G6
D331Y/D331Y基因嵌入小鼠在約6個月大即出現運動功能失調與障礙，因此顯現早發性帕金森病症狀。
實施例6. (D331Y) PLA2G6基因嵌入小鼠顯現異常之粒線體結構與功能
粒線體是神經細胞內提供能量(ATP)的胞器。粒線體異常會導致神經細胞死亡。利用電子顯微鏡檢測中腦黑質區內多巴胺神經細胞粒線體的型態。正常對照組小鼠中腦黑質區內多巴胺神經細胞的粒線體具有完整外觀與結構，粒線體嵴(mitochondrial cristae)排列整齊且完整(圖5A, 5B)。在PLA2G6
D331Y/D331Y基因嵌入小鼠中腦黑質區多巴胺神經細胞的粒線體形態變短且粒線體嵴明顯缺少(圖5C)。進一步分析粒線體能量代謝功能，正常對照小鼠中腦黑質區內神經細胞顯現良好的粒線體複合體I (mitochondrial complex I)活性和ATP產生能力(圖5D, 5E)，且粒線體其他相關功能良好(圖5F至5H)。但在(D331Y) PLA2G6基因嵌入小鼠中腦黑質區內多巴胺神經細胞的粒線體複合體I活性明顯下降(圖5D)，ATP合成明顯減少(圖5E)，大量自由基產生(圖5F)與粒線體脂質過氧化(圖5G)，且有大量細胞色素c (cytochrome c)蛋白由粒線體釋放至細胞質中(圖5H)，進而引起細胞凋亡。PLA2G6
D331Y/D331Y基因嵌入小鼠中腦黑質區內多巴胺神經細胞粒線體在早期即出現功能失調與異常。
實施例7. PLA2G6
D331Y/D331Y基因嵌入小鼠在中腦黑質區內顯現細胞凋亡(apoptosis)的活化、內質網壓力(ER stress)的增加及粒線體自噬失調(mitophagy impairment)
粒線體功能異常除了會引起細胞凋亡路徑活化與內質網壓力增加，同時也會讓原本要清除損傷粒線體的粒線體自噬清除系統失去功能。相較於正常對照組小鼠，約9個月大的(D331Y) PLA2G6基因嵌入小鼠的中腦黑質區內，多巴胺神經細胞大量表現細胞色素c、活化型凋亡蛋白酶(caspase) 9 (active caspase 9)與活化型凋亡蛋白酶3 (active caspase 3)，即表示細胞凋亡路徑活化(圖6A)。PLA2G6
D331Y/D331Y基因嵌入小鼠的神經細胞內Grp78、IRE1α、PERK、CHOP等內質網壓力相關蛋白大量表現，顯現經活化的內質網壓力訊號傳遞路徑(圖6B)。在清除受損粒線體的粒線體自噬路徑，(D331Y) PLA2G6基因嵌入小鼠的中腦黑質區神經細胞之Atg7、TOM20、p62、LC3II等粒線體自噬蛋白明顯減少(圖6C)，表示粒線體自噬功能失去正常的調控。PLA2G6
D331Y/D331Y基因嵌入小鼠的多巴胺神經細胞的細胞凋亡路徑活化、內質網壓力增加與粒線體自噬失調。
實施例8. (D331Y) PLA2G6基因嵌入小鼠中腦黑質區神經細胞內轉錄體的失調(transcriptional dysregulation)
目前PLA2G6基因突變對於神經退化造成的分子病理機轉並不清楚。PLA2G6
D331Y/D331Y基因嵌入小鼠可作為研究神經退化機轉的動物模式。利用微陣列(microarray analysis)分析基因嵌入小鼠中腦黑質區內神經細胞轉錄體(transcriptome)的變化(圖7A)，顯示神經細胞內確實有許多基因發生變化，其中分析10個有顯著統計意義差異的基因。該10個基因包含與神經細胞生長/神經保護相關之基因(Bmp6、Ccnd2、Ctnnb1、Hspa1b、Kidins220、Mapk1、Psap與Sdc2基因)和與神經細胞凋亡相關之基因(Mark4與Xaf1基因)。利用即時聚合酶鏈鎖反應(real-time polymerase chain reaction ; real-time PCR)確認該10個基因的表現。相較於正常對照組小鼠，(D331Y) PLA2G6基因嵌入小鼠中腦黑質區神經細胞的神經細胞生長/神經保護相關基因(Bmp6、Ccnd2、Ctnnb1、Hspa1b、Kidins220、Mapk1、Psap與Sdc2)表現明顯減少，且細胞凋亡相關基因(Mark4與Xaf1)表現大幅增加(圖7B)。在多巴胺神經細胞尚未大量死亡前，與神經細胞生長/神經保護相關之基因和與細胞凋亡相關之基因在約5個月大的PLA2G6
D331Y/D331Y基因嵌入小鼠中即可發現改變(圖7C)。在蛋白質表現方面，利用西方墨點法確認該10個基因之蛋白表現，並利用Image J分析軟體定量該蛋白表現。(D331Y) PLA2G6基因嵌入小鼠中腦黑質區神經細胞可觀察到神經細胞生長/神經保護相關蛋白(BMP6、CCND2、CTNNB1、HSPA1B、KIDINS220、MAPK1、PSAP與SDC2)顯著降低，且細胞凋亡相關蛋白(MARK4與XAF1)明顯增加(圖7D, 7E)。(D331Y) PLA2G6基因嵌入小鼠中腦黑質區異常的基因與蛋白表現極適合作為觀察轉錄體和蛋白質體改變的動物模式。
圖1顯示(D331Y) PLA2G6基因變異載體製備與(D331Y) PLA2G6基因嵌入小鼠(PLA2G6
D331Y/D331Y)的建立。(A) (D331Y) PLA2G6突變基因載體的製備。(B)利用聚合酶連鎖反應(PCR)確認基因嵌入小鼠基因型。(C)以定序方法確認對照組小鼠、雜型合子(PLA2G6
WT/D331Y)及同型合子(PLA2G6
D331Y/D331Y)基因嵌入小鼠的基因表現。(D)檢測對照組小鼠與基因嵌入小鼠PLA2G6蛋白質的磷脂酶活性。相較於對照組小鼠，PLA2G6
D331Y/D331Y基因嵌入小鼠的磷脂酶活性明顯降低。
圖2顯示PLA2G6
D331Y/D331Y基因嵌入小鼠出現早發性多巴胺神經細胞死亡與多巴胺神經末梢退化。(A,C)利用酪胺酸羥化酶(tyrosine hydroxylase)(TH)免疫組織染色分析多巴胺神經退化情況，並以Stereo Investigator軟體定量多巴胺神經數量。PLA2G6
D331Y/D331Y基因嵌入小鼠在6個月大時出現黑質區神經退化，9個月大時黑質區多巴胺神經細胞大量死亡。(D)利用Nissl
+組織免疫染色分析黑質區內神經元的數量，(D331Y) PLA2G6基因嵌入小鼠黑質區內Nissl
+神經細胞明顯減少。(B,E) Stereo Investigator軟體定量紋狀體中NeuN
+神經細胞，對照組小鼠與PLA2G6
D331Y/D331Y基因嵌入小鼠NeuN
+神經細胞數量相近。(F)利用微型正子放射電腦斷層造影(
18F-FP-DTBZ microPET imaging)分析紋狀體多巴胺的神經末梢(nigrostriatal dopaminergic terminals)的退化，9個月大的(D331Y) PLA2G6基因嵌入小鼠具有明顯紋狀體多巴胺神經末梢退化。(G)以TH免疫組織染色分析紋狀體多巴胺的神經末梢，9個月大的PLA2G6
D331Y/D331Y基因嵌入小鼠紋狀體顯現多巴胺神經末梢的退化。
圖3顯示PLA2G6
D331Y/D331Y基因嵌入小鼠中腦黑質區的突觸核蛋白病變與Tau病變。(A至C)利用路易氏體專一性抗體(anti-phospho-α-synuclein
Ser129antibody)進行路易氏體免疫組織染色。(A,B)對照組小鼠的黑質區神經細胞並沒有發現路易氏體。(C)在(D331Y) PLA2G6基因嵌入小鼠黑質區多巴胺神經細胞內可發現大量路易氏體形成。(D)進一步利用西方墨點法分析路易氏體相關蛋白質的表現，PLA2G6
D331Y/D331Y基因嵌入小鼠黑質區內有大量突觸核蛋白(α-synuclein; α-Syn)與磷酸化突觸核蛋白(phospho-α-synuclein; p-α-Syn)的表現。(E)磷酸化Tau (anti-phospho-Tau
Ser202/Thr205；p-Tau)用來偵測黑質區內Tau病變，(D331Y) PLA2G6基因嵌入小鼠黑質區有明顯的磷酸化Tau蛋白產生。
圖4顯示PLA2G6
D331Y/D331Y基因嵌入小鼠顯現早發性帕金森病症狀。(A,B)以活動量測試(open field test)分析小鼠活動速率(A)與活動量(B)，相較於對照組小鼠，(D331Y) PLA2G6基因嵌入小鼠隨著年紀越大活動能力明顯下降。(C)利用圓筒試驗(cylinder test)測試小鼠的運動協調性，PLA2G6
D331Y/D331Y基因嵌入小鼠之運動協調性明顯較對照組小鼠差。(D)利用滾筒式跑步機(Rotarod test)檢視小鼠的運動協調能力，PLA2G6
D331Y/D331Y基因嵌入小鼠在6個月開始出現運動不協調，9至12個月大時出現明顯的運動障礙。(E)利用爬竿測試(Pole test)檢測小鼠的平衡感與運動協調力，對照組小鼠具有很好的運動協調性，PLA2G6
D331Y/D331Y基因嵌入小鼠的運動協調力隨著年紀越大協調性明顯變差。(F,G)以L-Dopa藥物測試是否能改善小鼠的動作障礙。對照組小鼠原本就具有很好的運動能力，給予L-Dopa後運動能力沒有受到影響。(D331Y) PLA2G6基因嵌入小鼠給予L-Dopa藥物後能明顯改善原本的動作障礙且能增加運動活動速率與活動量。
圖5顯示(D331Y) PLA2G6基因嵌入小鼠黑質區多巴胺神經細胞內粒線體結構異常與功能失調。(A至C)電子顯微鏡可觀察多巴胺神經細胞內細胞胞器完整結構，對照組小鼠(A)與雜型合子對照組小鼠(B)，粒線體結構完整且具有明顯的粒線體嵴(cristae)。PLA2G6
D331Y/D331Y基因嵌入小鼠(C)粒線體結構明顯變短且粒線體嵴不完整，粒線體結構異常。(D)粒線體複合體I活性(Mitochondrial complex I)試劑檢測小鼠黑質區多巴胺神經細胞的粒線體複合體I活性，(D331Y) PLA2G6基因嵌入小鼠黑質區內的粒線體複合體I活性明顯降低。(E)利用ATP分析試劑檢測小鼠黑質區神經細胞的ATP合成能力，PLA2G6
D331Y/D331Y基因嵌入小鼠合成ATP能力明顯較對照組小鼠差。(F)利用自由基(ROS)試劑分析小鼠黑質區神經細胞自由基產生情況，相較於對照組小鼠，(D331Y) PLA2G6基因嵌入小鼠的多巴胺神經細胞具有較高的自由基。(G)以TBARS試劑分析小鼠黑質區神經細胞內粒線體脂質過氧化(mitochondrial lipid peroxidation)形成狀況，PLA2G6
D331Y/D331Y基因嵌入小鼠黑質區神經細胞具有較高的粒線體脂質過氧化。(H)細胞色素c (cytochrome c)分析試劑用來檢測細胞色素c蛋白質由粒線體釋放到細胞質的量，(D331Y) PLA2G6基因嵌入小鼠多巴胺神經細胞有大量的細胞色素c由粒線體釋放到細胞質，而後會引發細胞凋亡路徑的活化。
圖6顯示PLA2G6
D331Y/D331Y基因嵌入小鼠黑質區內具有細胞凋亡的活化、內質網壓力的增加與粒線體自噬的失調。(A)當粒線體異常或受到破壞時，使粒線體上的細胞色素c會釋放到細胞質中，進而活化粒線體的細胞凋亡路徑，使下游的活化型凋亡蛋白酶(caspase) 9 (Active Cap9)與活化型凋亡蛋白酶3 (Active Cap3)增加。以西方墨點法分析蛋白質表現並利用Image J軟體定量蛋白質表現。相較於對照組小鼠黑質區，(D331Y) PLA2G6基因嵌入小鼠黑質區具有大量表現的細胞色素c、活化型凋亡蛋白酶9與活化型凋亡蛋白酶3。(B)持續的內質網壓力也是造成神經退化的因素之一，當內質網壓力產生時會活化下游蛋白，其包含Grp78、IRE1α、PERK與CHOP蛋白。以西方墨點法分析小鼠黑質區多巴胺神經細胞的內質網壓力，PLA2G6
D331Y/D331Y基因嵌入小鼠具有明顯之內質網壓力蛋白質的形成。(C)粒線體損傷時會經由粒線體自噬(mitophagy)清除受損的粒線體。利用西方墨點法分析Atg7、TOM20、p62、LC3I與LC3II等粒線體自噬相關蛋白質的表現。與對照組小鼠黑質區比較，(D331Y) PLA2G6基因嵌入小鼠黑質區的粒線體自噬系統失去正常功能，粒線體自噬相關蛋白質表現下降。
圖7顯示(D331Y) PLA2G6基因嵌入小鼠黑質區神經細胞的轉錄體失調與蛋白質異常表現。(A)利用微陣列分析對照組與基因嵌入小鼠黑質區內神經細胞表現差異的基因，經比較分析找到10個在統計上具有表現差異的基因，該10個基因分別為8個與神經細胞生長/神經保護相關之基因(Bmp6、Ccnd2、Ctnnb1、Hspa1b、Kidins220、Mapk1、Psap與Sdc2基因)和2個與細胞凋亡相關之基因(Mark4與Xaf1)。(B)以即時聚合酶連鎖反應(real-time PCR)分析對照組與基因嵌入小鼠黑質區內多巴胺神經細胞10個基因的表現。相較於9個月大的對照組小鼠黑質區神經細胞，PLA2G6
D331Y/D331Y基因嵌入小鼠黑質區內神經細胞生長/神經保護相關基因(Bmp6、Ccnd2、Ctnnb1、Hspa1b、Kidins220、Mapk1、Psap與Sdc2基因)表現大量降低；而細胞凋亡相關基因(Mark4與Xaf1)表現大量上升。(C)進一步分析尚未出現神經細胞死亡的5個月大對照組小鼠和基因嵌入小鼠黑質區神經細胞，(D331Y) PLA2G6基因嵌入小鼠黑質區內8個神經細胞生長/神經保護相關基因表現明顯減少，而2個細胞凋亡相關基因表現明顯增加。(D,E)利用西方墨點法分析10個基因所對應的蛋白表現並以Image J軟體定量分析，PLA2G6
D331Y/D331Y基因嵌入小鼠黑質區內神經細胞生長/神經保護相關蛋白(BMP6、CCND2、CTNNB1、HSPA1B、KIDINS220、MAPK1、PSAP與SDC2基因)表現顯著減少，而細胞凋亡相關蛋白(MARK4與XAF1)表現顯著增加。
Claims (9)
- 一種(D331Y)PLA2G6突變基因嵌入小鼠於作為篩選用於治療早發型第十四型帕金森病(PARK14)的藥物之平台之用途，該(D331Y)PLA2G6突變基因嵌入小鼠顯現早發性中腦黑質區神經退化、突觸核蛋白病變及Tau病變。
- 如請求項1之用途，其中該中腦黑質區神經退化、突觸核蛋白病變及Tau病變於約6個月內發生。
- 如請求項1之用途，其中該(D331Y)PLA2G6突變基因嵌入小鼠之多巴胺神經細胞內的粒線體嵴減少且粒線體複合體I活性降低。
- 如請求項1之用途，其中該(D331Y)PLA2G6突變基因嵌入小鼠之多巴胺神經細胞表現降低之磷脂酶活性。
- 如請求項1之用途，其中該(D331Y)PLA2G6突變基因嵌入小鼠之多巴胺神經細胞顯現粒線體自噬功能失調。
- 如請求項1之用途，其中該藥物改善該(D331Y)PLA2G6突變基因嵌入小鼠的運動機能障礙。
- 如請求項6之用途，其中該運動機能障礙之改善包含 下述之一或多者：PLA2G6蛋白活性增加、多巴胺神經細胞死亡減少、突觸核蛋白病變與Tau病變減少、路易氏體堆積降低、粒線體複合體I活性增加、ATP合成增加、自由基產生減少、神經細胞生長/神經保護相關基因表現增加、神經細胞凋亡相關基因表現減少及/或Catenin beta-1表現增加。
- 如請求項7之用途，其中該神經細胞生長/神經保護相關基因包括選自Bmp6、Ccnd2、Ctnnb1、Hspa1b、Kidins220、Mapk1、Psap及Sdc2中一或多者。
- 如請求項7之用途，其中該神經細胞凋亡相關基因係Mark4或Xaf1或該二者。
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| US9599605B1 (en) * | 2013-03-15 | 2017-03-21 | Boston Medical Center Corporation | Parkinson's disease model and methods |
| CN107177670A (zh) * | 2017-05-31 | 2017-09-19 | 上海昂朴生物科技有限公司 | 一种高通量检测帕金森病致病基因突变的方法 |
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| CN107177670A (zh) * | 2017-05-31 | 2017-09-19 | 上海昂朴生物科技有限公司 | 一种高通量检测帕金森病致病基因突变的方法 |
Non-Patent Citations (4)
| Title |
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| Chiu, Ching-Chi, et al. "PARK14 PLA2G6 mutants are defective in preventing rotenone-induced mitochondrial dysfunction, ROS generation and activation of mitochondrial apoptotic pathway." Oncotarget 8.45 (2017): 79046-79060. |
| Chiu, Ching-Chi, et al. "PARK14 PLA2G6 mutants are defective in preventing rotenone-induced mitochondrial dysfunction, ROS generation and activation of mitochondrial apoptotic pathway." Oncotarget 8.45 (2017): 79046-79060. Lai, S-C., et al. "The pathogenic mechanism of PLA2G6 mutations in Parkinson's disease: 55." Movement Disorders 29 (2014): 19-20. 葉篤學，探討第十四型帕金森病的分子病理機轉---利用細胞與基因嵌入小鼠模式. 科技部計畫報告，計畫編號M-B182A-035，104年度(10408~10507) 105/10/6. * |
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| 葉篤學，探討第十四型帕金森病的分子病理機轉---利用細胞與基因嵌入小鼠模式. 科技部計畫報告，計畫編號MOST104-2314-B182A-035，104年度(10408~10507) 105/10/6. |
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