摘要
目的
阿尔茨海默病(Alzhermer disease, AD)是引起老年痴呆的最常见原因,在AD患者及APP/PS1转基因小鼠中均观察到海马结构的UCH-L1含量及树突棘密度的明显降低。Rember是一种新开发的治疗阿尔茨海默病(Alzheimer disease, AD)的药物,其主要成分是亚甲蓝,在二期临床试验中表现出良好的疗效。亚甲蓝低浓度下可以使神经元纤维缠结(Neurofibrillary tangles, NFTs)溶解,极低浓度下能够增强线粒体复合体Ⅵ功能从而延缓人胚肺成纤维细胞(IMR90)衰老。目前关于亚甲蓝对阿尔茨海默病模型鼠的研究很少,而亚甲蓝对APP／PS1小鼠UCH-L1及树突棘密度的影响尚未见文献报道。
因此,本研究拟通过给予APP/PS1小鼠口服适量亚甲蓝,采用跳台回避实验、Western blotting、免疫荧光、高尔基染色等方法观察亚甲蓝对APP/PS1转基因小鼠学习记忆及UCH-L1表达改变及对树突棘密度变化的的影响。本研究结果将为老年性痴呆的预防和治疗以及亚甲蓝的临床应用提供可靠的实验依据。
材料与方法
1、实验动物及分组：中国医科大学实验动物部提供的20只3月龄APP／PS1转基因小鼠,以及10只同龄相同品系C57bL／6小鼠(野生型),将动物分为3组,每组10只,雌雄不限。(1)模型组：转基因小鼠,自由饮水。(2)给药组：根据小鼠饮水量将亚甲蓝加入日常饮水中,用量约25 mg·kg-1·d-1,连用4个月。(3)对照组：C57bL／6野生小鼠,不施加任何处理因素。
2、跳台回避实验：测试各组小鼠的学习记忆能力,记录每组小鼠的错误次数和潜伏期。
3、噻嗪红染色：形态学鉴定APP/PS1小鼠及观察APP/PS1小鼠海马结构老年斑分布。
4、Elisa方法：检测各组小鼠海马结构Aβ1-40、Aβ1-42的含量。
5、Aβ快速染色：观察3组小鼠海马结构Aβ的表达。
6、免疫荧光及Western blotting方法：观察各组小鼠海马结构UCH-L1的分布和表达情况。
7、高尔基染色：观察各组小鼠海马CA1区树突棘密度的变化。
8、统计学分析：结合图像采集分析系统,所得数据用均数±标准差(x±s)表示,应用SPSS11.0统计软件,用单因素方差分析进行组间比较,P<0.05为有统计学意义,P<0.01为有显著统计学意义。
实验结果
1、跳台回避实验结果表明模型组小鼠各时间段的平均潜伏期较对照组明显延长,错误次数增多(P<0.05),而给药组较模型组各时间段的平均潜伏期明显缩短,错误次数减少(P<0.05)。
2、7月龄APP／PS1小鼠海马结构观察到老年斑,而正常小鼠未观察到。
3、Elisa检测结果显示治疗组与模型组Aβ1-40、Aβ1-42无明显差别(P>0.05),在正常小鼠中未检测到Aβ1-40、Aβ1-42。
4、免疫荧光及免疫印记结果证明了模型组小鼠海马结构UCH-L1的表达量较正常对照组明显降低(P<0.01)。而治疗组较模型组含量明显增加(P<0.01)。
5、高尔基银染结果证实了给药组小鼠海马CA1区的第2级树突的树突棘平均密度较模型组增加28.79%,两组存在明显差异(P<0.01)。而给药组与正常对照组之间无明显统计学差异。
结论
1、跳台回避实验结果表明MB可改善APP/PS1小鼠的学习记忆能力。
2、Elisa方法及Aβ快速染色结果中显示APP/PS1小鼠海马结构均出现Aβ1.40、Aβ1-42表达,但MB对APP/PS1小鼠海马结构Aβ1-40、Aβ1-42无明显影响,而正常对照组未检测到Aβ的表达。
3、免疫印记及免疫荧光(CA1区)结果证实了亚甲蓝可以上调APP/PS1小鼠海马结构可溶性UCH-L1表达。
4、高尔基银染结果证明亚甲蓝可以增加APP/PS1小鼠海马CA1区树突棘密度。
Objective
Alzheimer disease is a degenerative disease of the brain that causes dementia. UCH-L1 is down-regulated in idiopathic AD and APP/PS1 mouse. Rember. the main component of which is MB, the PhaseⅡdata seem to have been impressive. MB delays senescence at nM levels in normal human lung fibroblasts (IMR90) by enhancing mitochondrial function. There is few study on the effect of MB on UCH-L1 and spine density in APP/PS1 mouse.
In the present study, we examined the effect of oral MB treatment on the neurodegenerative pathology and memory deficitsβ-amyoid precursor protein (APP) and presenilin 1 (PS1) double-transgenic mice, a well established AD mouse model. When systemically administered to 4-month-old APP/PS1 mice for 3 months, MB effectively improves cognition of APP/PS1 transgenic (Tg) mice not by altering AB burden but by up-regulating the level of UCH-L1 and increasing the density of dendritic spine.
Materials and Methods
1. Experimental animal grouping:Animal studies followed protocols approved by the Laboratory Animal Centre of China Medical University. We utilized either C57/BL6 mice (wild type) or APP/PS1 mice. APP/PS1 transgenic mice and wild-type littermates were assigned into three groups:MB-treated APP/PS1 mice, untreated APP/PS 1 mice, wild-type mice. Treated groups received MB (25 mg/kg per day) Untreated groups received normal drinking water. Treatment was started when the mice were 4 months old and was continued for 3 months.
2. Step-down Avoidance Test for testing learning and remembrance ability of mice was used. Stepdown latency and the number of error were recorded.
3. Thiazine red stainning was performed to observe the SPs of APP/PS1 mouse.
4. Aβ1-40 and Aβ1-42 Elisas and Aβstainning was performed.
5. Immunohistochemistry and Western blotting for UCH-L1 were performed.
6. Golgi stainning of spine density was performed.
7. Image collection analysis system was applied. The data were expressed as Mean±SD and were analyzed by SPSS 11.0 statistical package. The differences among group were compared with one-way analysis of variance. P＜0.05 was regarded as the significant difference and P＜0.01 was regarded as the extremely significant difference.
Results
1. Results of step down avoidence test:APP/PS1 mice presented lower step-down latency than mice WT groups. APP/PS1 mice treated with MB showed higher values of step-down latency than APP/PS1 mice treated with vehicle (P＜0.01). MB treated transgenic mice showed no statistical difference with WT mice (P＞0.05).
2. SPs was observed in the hippocampus of APP/PS1 mouse aged 7 years.
3. Aβ1-40 and Aβ1-42 Elisas:MB had no effect on the hippocampal soluble or insoluble Aβ1-40 or Aβ1-42 levels as analyzed by Elisa.
4. immunofluorescence and western blotting of UCH-L1:4 months of treatment with MB reestablished normal levels of UCH-L1 in 7-month-old transgenic animals. (P＜0.01, WT versus APP/PS1; P＜0.01, APP/PS1 versus APP/PS1 plus MB; P=0.05, WT versus APP/PS1 plus MB; n=10). Consistent with these results, immunohistological analysis of UCH-Ll revealed a decrease in the transgenic mice, which was returned to control levels by MB
5. Results of Golgi stainning:Compared with model group, the spine density of MB treated group was increased (P＜0.01). There is no statistical difference between treated transgenic mice and WT mice.
Conclusions
1. MB can delay the natural progression of cognitive abnormalities of APP/PS1 mouse.
2. MB had no effect on Aβ1-40 or Aβ1-42 levels.
3. MB up regulats the expression of UCH-L1 and increases the spine density of APP/PS 1 mouse.
4. MB increases the spine density of APP/PS1 mouse.
引文
1 Wiskchik, C M, Edwards P C, Lair Y K, Selective inhibition of Alzheimer disease-like tau aggregation by phenothiazines. Proc Natl Acad Sci.1996; 93:11213-11218.
2 Atamna H. Nguyen A, Schultz C, et al. Methylene blue delays cellular senescence and enhances key mitochondrial biochemical pathways. FASEB J.2008:22(3):703-12.]
3 Gura T. Hope in Alzheimer's fight emerges from unexpected places. Nat Med.2008; 14:894.
4 Arendash GW, King D L. Gordon M N, et al. Progressive, age-related behavioral impairments in transgenic mice carrying both mutant amyloid precursor protein and presenilin-1 transgenes. Brain Res.2001; 891:42-53.
5 Gong B, Cao Z, Zheng P, et al. Ubiquitin hydrolase Uch-L1 rescues beta-amyloid-induced decreases in synaptic function and contextual memory. Cell.2006; 126:775-88.
6 Donna L, Smith J P, Bing G, et al. Reversal of long-term dendritic spine alterations in Alzheimer disease models. PNAS.2009; 106:16877-16882.
7 Torres-Fernandez. La technica de impregnacion argentica de Golgi. Conmemoracion delcentenario de premio Nobel de Medicina (1906), Compartido por Camillo Golgi y Santiago Ramon y Cajal. Biomedica.2006; 26:498-508.
8 Tarja M. M, Henna I, Gundars G, et al. Pyrrolidine Dithiocarbamate Activates Akt and I mproves Spatial Learning in APP/PS1 Mice without Affecting β-Amyloid Burden. The Journal of Neuroscience.2007; 27:3712-3721.
9 Yoko U, Hiroshi T. Rapid detection of Aβ deposits in APP transgenic mice by Hoechst 33342. Neuroscience Letters.2008; 448:279-281.
10 Riha P D, Bruchey A K, Echevarria D J, et al. Memory facilitation by methylene blue: dose-dependent effect on behavior and brain oxygen consumption. Eur J Pharmacol.2005; 511:151-8.
11 Layfield R, Cavey JR, Iowe J. Role of ubiquitin-mediated proteolysis in the pathogenesis of neurodegenerativ Disorders. Ageing Res Rev.2003; 2:343-356.
12 Nishikawa K, Sun YJ, Sakurai M, et al. Ubiquitin carboxy-terminal hydmlase L1 binds to and stabilizes monoubiquitin in neuron. Hum Mol Genet.2003; 12:1945-1958.
1 layfield R. Cavey JR, Iowe J. Role of ubiquitin-mediated proteolysis in the pathogenesis of neurodegenerative disorders. Ageing Res Rev.2003.2(4):343-356.
2 FW, Gerez I, Benne R. et al. Proteins and aging. Int J Biochem cell Biol.2002; 34(11):1502-1505.
3 Kim TW, Pettingell W H. Hallmark DJ. et al. Endomteolylic cleavage and proteasomal degradation of presenilin 2 in transfected cells. J Biol Chem.1997; 272:11006-11010.
4 Steiner H, Capell A, Pesold B, et al. Expression of Alzheimefs disease-associated prcsenilin-1 is controlled by proteolytie degradation and complex formation. J Biol Chem.1998; 273: 32322-32331.
5 Maraganore DM, Farrer MJ, Hardy JA, et al. Case—conl rol study of the ubiquitin carboxylerminal hydrolase L1 gene in Parkinson's disease. Neurology.1999; 53:1858-1860.
6 W intermeyer P, Kruger R. Kuhn W. et al. M u ralion analysis and association studies of the UCHI 1 gene in German Parkinson disease patients[J]. Neuroreport.2000; 11:2079-2082
7 Gasser T. Pilz P. Omasmeier. et al. Ubiquitin hydrolase L1 in sporadic Parkinson s disease. M ov Disord.2000; 16:201-203.
8 Fernandez-Funez P, Nino-Rosales ML. Deouyon B. et al. Identification of genes that modify ataxin 1 induced neurod generafi. Nature.2000; 408:101-106.
9 Naze P, Vuillaume I, Destee A, et al. Mutation arialysis and associ ation studies of ubiquitin carboxy terminal hydrolase L1 gene in Huntington s disease. Neurosci lett.2002; 328:1-4.
10 薛素芳贾建平李丹.泛素羧基末端水解酶L1基因S18 Y多态性与散发性阿尔茨海默病的相关性分析[J].脑与神经疾病杂志.2006；14：51-53.
11 Liu Y, Fallon L, Lashuel HA, et al. The UCH-L1 gene an-codes two opposing enzymatic activities that afect alpha-synucle-in degradation and Parkinson'S disease susceptibility. Cell. 2002;111:209-218.
12 Nishikawa K, Sun YJ, Sakurai M, et al. Ubiquitin carboxy-terminal hydmlase L1 binds to and stabilizes monoubiquitin in neuron. Hum Mol Genet.2003; 12:1945-1958.
13 Butterfield D.A, Gnjec A., Poon H.F, et al. Redox proteomics identification of oxidatively modified brain proteins in inherited Alzheimer's disease:an initial assessment. Alzheimers Dis.2006; 10:391-397.
14 Choi J, Levey A I. Weintraub, et al. Oxidative modifications and down-regulation of ubiquitin carboxyl-terminal hydrolase L1 associated with idiopathic Parkinson's and Alzheimer's diseases. J. Biol. Chem.2004.279:13256-13264.
15 Nishikawa K. Li H. Kawamura R. et al. Alterations of structure and hydrolase activity of parkinsonismassociated human ubiquitin carboxyl-terminal hydrolase L1 variants. Biochem.Biophys Res Commun.2003:304:176-183.
16 Castellani R.J. Perry G. Siedlak S L. et al. Hydroxynonenal adducts indicate a role for lipid peroxidation in neocortical and brainstem Lewy bodies in humans. Neurosci Lett.2002:319: 25-28.
17 Montine K S, Olson S J. Amarnath V. et al. Immunohistochemical detection of 4-hydroxy-2-nonenal adducts in Alzheimer's disease is associated with inheritance of APOE4. Am J Pathol.1997; 150:437-443.
18 Nishikawa K. Li H, Kawamura R. et al. Alterations of structure and hydrolase activity of parkinsonismassociated human ubiquitin carboxyl-terminal hydrolase L1 variants. Biochem. Biophys Res Commun.2003; 304:176-183.
19 Halliwell B. Oxidative stress and neurodegeneration:where are we now? Neurochem.2006; 97: 1634-1658.
20 Lin M T, Beal M F. Mitochondrial dysfunction and oxidative stress in neurodegenerative diseases. Nature.2006; 443:787-795.
21 Lowe J, McDermott H. Landon M. et al. Ubiquitin carboxyl-terminal hydrolase (PGP9.5) is selectively present in ubiquitinated inclusion bodies characteristic of human neurodegenerative diseases. Pathol.1990; 161:153-160.
22 Choi, J., Levey. A.I., Weintraub. S.T.et al. Oxidative modifications and down-regulation of ubiquitin carboxyl-terminal hydrolase L1 associated with idiopathic Parkinson's and Alzheimer's diseases. Biol Chem.2004; 279:13256-13264.
23 Ichihara N, Wu J, Chui D H, et al. Axonal degeneration promotes abnormal accumulation of amyloid beta-protein in ascending gracile tract of gracile axonal dystrophy (GAD) mouse. Brain Res.1995; 695:173-178.
24 Gong B, Cao Z, Zheng P, et al. Ubiquitin hydrolase Uch-L1 rescues beta-amyloid-induced decreases in synaptic function and contextual memory. Cell.2006; 126:775-788.
25 Choi J, Evey A I, Weintraubl S T, et al. Oxidative Modifications and Down-regulated of Ubiquitin Carboxyl-terminal Hydrolase L1 associated with Idiopathic Parkinson s and Alzheimer's disease. The Journal of Biological Chemistry.2004; 279:13256-13264.
