美金刚提高淀粉样β蛋白损伤海马的可塑性

doi:10.3969/j.issn.1673-5374.2013.01.006

摘要/Abstract

摘要：

课题组前期研究证实，低亲和力的N-甲基-D-天氡氨酸受体拮抗剂美金刚对淀粉样β蛋白毒性损伤具有急性保护作用，实验拟进一步揭示其慢性作用和相关机制。结果发现5, 20mg/kg的美金刚连续腹腔注射7d对大鼠海马长时程增强无影响，而40mg/kg的美金刚可显著抑制大鼠海马长时程增强。因此，实验取5, 20mg/kg美金刚作用的大鼠海马脑片进行体外研究，发现20mg/kg的美金刚可改善淀粉样β蛋白对大鼠海马长时程增强的抑制作用。说明适当剂量的美金刚长期干预可拮抗淀粉样β蛋白引起的海马长时程增强抑制，提高突触可塑性。

关键词: 神经再生, 神经退行性疾病, 美金刚, 淀粉样β蛋白, 长时程增强, 突触可塑性, 阿尔茨海默病, 海马, 基金资助文章, N-甲基-D-天氡氨酸受体

Abstract:

Previous studies indicate that memantine, a low-affinity N-methyl-D-aspartate receptor antagonist, exerted acute protective effects against amyloid-β protein-induced neurotoxicity. In the present study, the chronic effects and mechanisms of memantine were investigated further using electrophysiological methods. The results showed that 7-day intraperitoneal application of memantine, at doses of 5 mg/kg or 20 mg/kg, did not alter hippocampal long-term potentiation induction in rats, while 40 mg/kg memantine presented potent long-term potentiation inhibition. Then further in vitro studys were carried out in 5 mg/kg and 20 mg/kg memantine treated rats. We found that 20 mg/kg memantine attenuated the potent long-term potentiation inhibition caused by exposure to amyloid-β protein in the dentate gyrus in vitro. These findings are the first to demonstrate the antagonizing effect of long-term systematic treatment of memantine against amyloid-β protein triggered long-term potentiation inhibition to improve synaptic plasticity.

Key words: neural regeneration, neurodegenerative diseases, memantine, amyloid-&beta, protein, long-term potentiation, synaptic plasticity, N-methyl-D-aspartate receptor, Alzheimer’s disease, hippocampus, grants-supported paper, neuroregeneration

. 美金刚提高淀粉样β蛋白损伤海马的可塑性[J]. 中国神经再生研究(英文版), 2013, 8(1): 49-55.

Fushun Li, Xiaowei Chen, Feiming Wang, Shujun Xu, Lan Chang, Roger Anwyl, Qinwen Wang. Chronic pre-treatment with memantine prevents amyloid-beta protein-mediated long-term potentiation disruption[J]. Neural Regeneration Research, 2013, 8(1): 49-55.

参考文献

[1] Hardy J. A hundred years of Alzheimer's disease research. Neuron. 2006;52(1):3-13.http://www.cell.com/neuron/retrieve/pii/S0896627306007239

[2] Walsh DM, Klyubin I, Fadeeva JV, et al. Naturally secreted oligomers of amyloid beta protein potently inhibit hippocampal long-term potentiation in vivo. Nature. 2002;416(6880):535-539.http://www.nature.com/nature/journal/v416/n6880/full/416535a.html

[3] Selkoe DJ. Soluble oligomers of the Amyloid β-protein impair synaptic plasticity and behavior. Behav Brain Res. 2008;192(1):106-113.http://www.sciencedirect.com/science/article/pii/S0166432808000831

[4] Small DH, Mok SS, Bornstein JC. Alzheimer’s disease and Abeta toxicity: from top to bottom. Nat Rev Neurosci. 2001;2(8):595-598.http://www.nature.com/nrn/journal/v2/n8/full/nrn0801_595a.html

[5] Cleary JP, Walsh DM, Hofmeister JJ, et al. Natural oligomers of the amyloid-beta protein specifically disrupt cognitive function. Nat Neurosci. 2005;8(1):79-84.http://www.nature.com/neuro/journal/v8/n1/full/nn1372.html

[6] Wang Q, Walsh DM, Rowan MJ, et al. Block of long-term potentiation by naturally secreted and synthetic amyloid beta-peptide in hippocampal slices is mediated via activation of c-Jun N-terminal kinase, cyclin-dependent kinase 5, and p38 mitogen-activated protein kinase as well as metabotropic glutamate receptor type 5. J Neurosci. 2004;24(13):3370-3378.http://www.jneurosci.org/content/24/13/3370.long

[7] Hock C, Konietzko U, Streffer JR, et al. Antibodies against beta-amyloid slow cognitive decline in Alzheimer's disease. Neuron. 2003;38(4):547-554.http://www.cell.com/neuron/retrieve/pii/S0896627303002940

[8] Klyubin I, Walsh DM, Lemere CA, et al. Amyloid beta protein immunotherapy neutralizes Abeta oligomers that disrupt synaptic plasticity in vivo. Nat Med. 2005;11(5): 556-561.http://www.nature.com/nm/journal/v11/n5/full/nm1234.html

[9] Schenk DB, Seubert P, Grundman M, et al. Abeta immunotherapy: lessons learned for potential treatment of Alzheimer's disease. Neurodegener Dis. 2005;2(5): 255-260.http://www.ncbi.nlm.nih.gov/pubmed?term=Abeta%20immunotherapy%3A%20lessons%20learned%20for%20potential%20treatment%20of%20Alzheimer's%20disease

[10] Lipton SA. Failures and successes of NMDA receptor antagonists: molecular basis for the use of open-channel blockers like Memantine in the treatment of acute and chronic neurologic insults. NeuroRx. 2004;1(1):101-110.http://link.springer.com/article/10.1602%2Fneurorx.1.1.101

[11] Seow D, Gauthier S. Pharmacotherapy of Alzheimer disease. Can J Psychiatry. 2007;52(10):620-629. http://www.ncbi.nlm.nih.gov/pubmed/18020110

[12] Lipton SA. Pathologically activated therapeutics for neuroprotection. Nat Rev Neurosci. 2007;8(10):803-808.http://www.nature.com/nrn/journal/v8/n10/full/nrn2229.html

[13] Parsons CG, Stöffler A, Danysz W. Memantine: a NMDA receptor antagonist that improves memory by restoration of homeostasis in the glutamatergic system-too little activation is bad, too much is even worse. Neuropharmacology. 2007;53(6):699-723.http://www.sciencedirect.com/science/article/pii/S0028390807002298

[14] Raina P, Santaguida P, Ismaila A, et al. Effectiveness of cholinesterase inhibitors and memantine for treating dementia: evidence review for a clinical practice guideline. Ann Intern Med. 2008;148(5):379-397.http://annals.org/article.aspx?articleid=739930

[15] Miguel-Hidalgo JJ, Alvarez XA, Cacabelos R, et al. Neuroprotection by memantine against neurodegeneration induced by beta-amyloid(1-40). Brain Res. 2002;958(1):210-221.http://www.sciencedirect.com/science/article/pii/S0006899302037319

[16] Alley GM, Bailey JA, Chen D, et al. Memantine lowers amyloid-beta peptide levels in neuronal cultures and in APP/PS1 transgenic mice. J Neurosci Res. 2010;88(1): 143-154. http://dx.doi.org/10.1002/jnr.22172

[17] Chipana C, Torres I, Camarasa J, et al. Memantine protects against amphetamine derivatives-induced neurotoxic damage in rodents. Neuropharmacology. 2008;54(8):1254-1263.http://www.sciencedirect.com/science/article/pii/S002839080800097X

[18] Babu CS, Ramanathan M. Pre-ischemic treatment with memantine reversed the neurochemical and behavioural parameters but not energy metabolites in middle cerebral artery occluded rats. Pharmacol Biochem Behav. 2009; 92(3):424-432.http://www.sciencedirect.com/science/article/pii/S0091305709000252

[19] Hare WA, Wheeler L. Experimental glutamatergic excitotoxicity in rabbit retinal ganglion cells: block by memantine. Invest Ophthalmol Vis Sci. 2009;50(6): 2940-2948. http://www.iovs.org/content/50/6/2940.long

[20] Barber TA, Haggarty MK. Memantine ameliorates scopolamine-induced amnesia in chicks trained on taste-avoidance learning. Neurobiol Learn Mem. 2010;93(4):540-545.http://www.sciencedirect.com/science/article/pii/S1074742710000304

[21] Barnes CA, Danysz W, Parsons CG.. Effects of the uncompetitive NMDA receptor antagonist memantine on hippocampal long-term potentiation, short-term exploratory modulation and spatial memory in awake, freely moving rats. Eur J Neurosci. 1996;8(3):565-571.http://www.ncbi.nlm.nih.gov/pubmed?term=Effects%20of%20the%20uncompetitive%20NMDA%20receptor%20antagonist%20memantine%20on%20hippocampal%20long-term%20potentiation%2C%20short-term%20exploratory%20modulation%20and%20spatial%20memory%20in%20awake%2C%20freely%20moving%20rats

[22] Zajaczkowski W, Frankiewicz T, Parsons CG, et al. Uncompetitive NMDA receptor antagonists attenuate NMDA-induced impairment of passive avoidance learning and LTP. Neuropharmacology. 1997;36(7):961-971.http://www.sciencedirect.com/science/article/pii/S0028390897000701

[23] Klyubin I, Wang Q, Reed MN, et al. Protection against Aβ-mediated apid disruption of synaptic plasticity and memory by memantine. Neurobiol Aging. 2009;32(4): 614-623.http://www.neurobiologyofaging.org/article/S0197-4580(09)00120-1/abstract

[24] Kos T, Popik P. A comparison of the predictive therapeutic and undesired side-effects of the NMDA receptor antagonist, memantine, in mice. Behav Pharmacol. 2005; 16(3):155-161.http://www.ncbi.nlm.nih.gov/pubmed?term=A%20comparison%20of%20the%20predictive%20therapeutic%20and%20undesired%20side-effects%20of%20the%20NMDA%20receptor%20antagonist%2C%20memantine%2C%20in%20mice

[25] Creeley C, Wozniak DF, Labruyere J, et al. Low doses of memantine disrupt memory in adult rats. J Neurosci. 2006; 26(15):3923-2932.http://www.jneurosci.org/cgi/pmidlookup?view=long&pmid=16611808

[26] Minkeviciene R, Banerjee P, Tanila H. Memantine improves spatial learning in a transgenic mouse model of Alzheimer’s disease. J Pharmacol Exp Ther. 2004;311(2): 677-682. http://jpet.aspetjournals.org/content/311/2/677.long

[27] Minkeviciene R, Banerjee P, Tanila H. Cognition- enhancing and anxiolytic effects of memantine. Neuropharmacology. 2008;54(7):1079-1085.http://www.sciencedirect.com/science/article/pii/S002839080800052X

[28] Peskind ER, Potkin SG, Pomara N, et al. Memantine treatment in mild to moderate Alzheimer disease: a 24-week randomized, controlled trial. Am J Geriatr Psychiatry. 2006;14(8):704-715.http://linkinghub.elsevier.com/retrieve/pii/14/8/704

[29] Schulz JB, Rainer M, Klünemann HH, et al. Sustained effects of once-daily memantine treatment on cognition and functional communication skills in patients with moderate to severe Alzheimer's disease: results of a 16-week open-label trial. J Alzheimers Dis. 2011;25(3): 463-475.http://iospress.metapress.com/content/86ug68625k136376/?genre=article&issn=1387-2877&volume=25&issue=3&spage=463

[30] Pietá Dias C, Martins de Lima MN, Presti-Torres J, et al. Memantine reduces oxidative damage and enhances long-term recognition memory in aged rats. Neuroscience. 2007;146(4):1719-1725.http://www.sciencedirect.com/science/article/pii/S0306452207003387

[31] Quan MN, Zhang N, Wang YY, et al. Possible antidepressant effects and mechanisms of memantine in behaviors and synaptic plasticity of a depression rat model. Neuroscience. 2011;182:88-97.http://www.sciencedirect.com/science/article/pii/S0306452211002843

[32] Zorumski CF, Izumi Y. Modulation of LTP induction by NMDA receptor activation and nitric oxide release. Prog Brain Res. 1998;118:173-182.http://www.ncbi.nlm.nih.gov/pubmed?term=Modulation%20of%20LTP%20induction%20by%20NMDA%20receptor%20activation%20and%20nitric%20oxide%20release

[33] Witt A, Macdonald N, Kirkpatrick P. Memantine hydrochloride. Nat Rev Drug Discov. 2004;3(2):109-110.http://www.nature.com/nrd/journal/v3/n2/full/nrd1311.html

[34] Zoladz PR, Campbell AM, Park CR, et al. Enhancement of long-term spatial memory in adult rats by the noncompetitive NMDA-receptor antagonists, memantine and neramexane. Pharmacol Biochem Behav. 2006;85(2): 298-306.http://www.sciencedirect.com/science/article/pii/S0091305706002826

[35] Lockrow J, Boger H, Bimonte-Nelson H, et al. Effects of long-term memantine on memory and neuropathology in Ts65Dn mice, a model for Down syndrome. Behav Brain Res. 2011;221(2):610-622.http://www.sciencedirect.com/science/article/pii/S0166432810002226

[36] Arif M, Chikuma T, Ahmed MM, et al. Effects of memantine on soluble Αβ25-35-induced changes in peptidergic and glial cells in Alzheimer's disease model rat brain regions. Neuroscience. 2009;164(3):1199-1209.http://www.sciencedirect.com/science/article/pii/S0306452209014237

[37] Kotermanski SE, Johnson JW. Mg2+ imparts NMDA receptor subtype selectivity to the Alzheimer’s drug memantine. J Neurosci. 2009;29(9):2774-2779.http://www.jneurosci.org/content/29/9/2774.long

[38] Okamoto S, Pouladi MA, Talantova M, et al. Balance between synaptic versus extrasynaptic NMDA receptor activity influences inclusions and neurotoxicity of mutant huntingtin. Nat Med. 2009;15(12):1407-1413.http://www.nature.com/nm/journal/v15/n12/full/nm.2056.html

[39] Xia P, Chen HS, Zhang D, et al. Memantine preferentially blocks extrasynaptic over synaptic NMDA receptor currents in hippocampal autapses. J Neurosci. 2010; 30(33):11246-11250. http://www.jneurosci.org/content/30/33/11246.long

[40] Lu CW, Lin TY, Wang SJ. Memantine depresses glutamate release through inhibition of voltage-dependent Ca2+ entry and protein kinase C in rat cerebral cortex nerve terminals: An NMDA receptor-independent mechanism. Neurochem Int. 2010;57(2):168-176.http://www.sciencedirect.com/science/article/pii/S0197018610001713

[41] Anwyl R. Metabotropic glutamate receptor-dependent long-term potentiation. Neuropharmacology. 2009;56(4): 735-740.http://www.ncbi.nlm.nih.gov/pubmed/19705571

[42] The Ministry of Science and Technology of the People’s Republic of China. Guidance Suggestions for the Care and Use of Laboratory Animals. 2006-09-30.http://www.most.cn/fggw/zfwj/zfwj2006/200609/t20060930_54389.htm

引言

材料方法

Design

A randomized, controlled animal study and in vitro electrophysiological observation.

Time and setting

The experiment was performed at Ningbo University, China and Trinity College Dublin, Ireland, between 2009 and 2010.

Materials

A total of 42 male Wistar rats, weighing 40–80 g and aged 3–4 weeks, were obtained from the Animal House of Trinity College, Ireland and Shanghai Silaike Experimental Animal Co., Ltd. (certificate No. SCXK (Hu) 2007-0005). The use of animals for experimental procedures was conducted in accordance with the Guidance Suggestions for the Care and Use of Laboratory Animals, issued by the Ministry of Science and Technology of China^[42].

Methods

Drug treatment

Memantine hydrochloride was purchased from Sigma (St. Louis, MO, USA). For the experiments, memantine (5, 20, and 40 mg/kg) was prepared for i.p. administration in normal saline (0.9%). Memantine was administered i.p. for 7 days at doses of 5, 20 or 40 mg/kg per day. The control group received the same volume of i.p. dosing of normal saline.

Preparation of hippocampal slices

Transverse slices of the hippocampus were prepared from memantine- or saline-treated rats. The brains were rapidly removed after the rats were sacrificed and placed in cold oxygenated (95% O₂/5% CO₂) physiological media. Slices were cut at a thickness of 350 μM using an Intracell Plus 1000 vibratome (Vibratome Co., St. Louis, MO, USA) and placed in a storage container containing oxygenated medium (self made) at 20–22°C for 1 hour. The slices were then transferred to a recording chamber for submerged slices and continuously superfused at a rate of 5–6 mL/min at 30–32°C. The control media contained NaCl 120 mM; KCl 2.5 mM, NaH₂PO₄ 1.25 mM; NaHCO₃ 26 mM; MgSO₄ 2.0 mM; CaCl₂ 2.0 mM; and D-glucose 10 mM. All solutions contained 100 μM picrotoxin (Sigma) to block GABA_A-mediated activity.

Aβ treatment

In experiments involving the application of Aβ_1-42, Aβ was perfused for 40 minutes before HFS. Synthetic Aβ_1-42 (Bachem, Bubendorf, Switzerland) was prepared as a stock solution of 50 μM in ammonium hydroxide (0.1%), stored at –20°C, and then added to the physiological medium immediately prior to each experiment. Control (0.9% saline-injected) and experimental levels of LTP were measured on slices prepared from the same hippocampus.

In vitro electrophysiological recording

Standard electrophysiological techniques were used to record field potentials^[6]. Presynaptic stimulation was applied to the medial perforant pathway of the dentate gyrus using a bipolar insulated tungsten wire electrode (Sutter Instrument Company, Novato, CA, USA), and field excitatory postsynaptic potentials (EPSPs) were recorded at a control test frequency of 0.033 Hz from the middle one-third of the molecular layer of the dentate gyrus with a glass microelectrode (Sutter Instrument Company). The inner blade of the dentate gyrus was used in all studies. In each experiment, an input-output curve (afferent stimulus intensity versus EPSP amplitude) was plotted at the test frequency. For all experiments, the amplitude of the test EPSP was adjusted to one-third of maximum (–1.2 mV). LTP was evoked by HFS consisting of eight trains, each consisting of eight stimuli at 200 Hz, with inter-train intervals of 2 seconds. The stimulation voltage was increased during the HFS to evoke an initial EPSP of the train of double the normal test EPSP amplitude. Control (0.9% saline-treated) and experimental levels of LTP were measured on slices prepared from the same hippocampus.

Statistical analysis

Recordings were analyzed using p-CLAMP (Axon Instruments, Sunnyvale, CA, USA). Data were expressed as mean ± SEM. Analysis of variance was used for statistical comparison. P < 0.05 was considered statistically significant.

讨论

文章快阅

延伸阅读

1 实验设计构思介绍：
课题组前期研究已经证明美金刚对Abetad毒性的急性保护作用（Neurobiology of Aging, 2009,32(4): 614-623），文章拟进一步对美金刚的本身毒性及慢性作用进行研究，深入地探讨美金刚烷胺的作用机理，探讨其安全作用剂量，对临床治疗提供指导。

2 国内外同类研究水平的介绍：
作为首个治疗晚期阿尔茨海默病的药物，美金刚烷胺的临床试验表明能显著改善阿尔茨海默病和血管性痴呆患者认知障碍、精神运动驱动缺乏、抑郁程度、运动障碍，同时还显示日常生活能力和社交活动改善。美金刚烷胺能增强正常的突触递质传递，取代镁离子对N-甲基-D-天氡氨酸受体的阻滞作用。它对谷氨酸递质的作用取决于谷氨酸能神经突触前后神经元功能状态以及突触间隙谷氨酸浓度。在持续性病理缺血、缺氧状态下，谷氨酸释放增加时，美金刚烷胺能阻断N-甲基-D-天氡氨酸受体，防止大量钙离子内流，因此具有保护神经元免受谷氨酸兴奋性毒性作用。

3 与其他同类研究的比较：
此前FDA已批准了数种用于治疗轻度阿尔茨海默病的药物，多为胆碱酯酶抑制剂，但都未能从根本上治愈，且基本上是针对中早期患者。多数药物置只起到部分改善延缓症状作用，有些甚至有很严重的副作用。研究表明，美金刚烷胺可用于治疗晚期阿尔茨海默病患者。通常在病者在患病后3年半左右病情就会发展到该阶段，这也正是给社会和家庭带来负担最重的时期。美金刚烷胺是第一个在阿尔茨海默病和血管性痴呆方面有显著疗效的N-甲基-D-天氡氨酸受体拮抗剂。文章在研究了其急性作用基础上，进一步研究了其本身毒性及慢性作用，探讨其安全作用剂量，对临床治疗提供指导。

4 查新报告：
所检国内文献中，有关美金刚治疗阿尔茨海默病的研究有较多的相关文献报道，但未见有关美金刚治疗阿尔茨海默病的机制的研究，未见有关美金刚对长时程增强的影响的相关文献报道，未见有关美金刚对β淀粉样蛋白抑制长时程增强的调制机理的相关文献报道。

所检国外文献中，有关美金刚治疗阿尔茨海默病的研究有较多的相关文献报道，有关提及美金刚对β淀粉样蛋白抑制的相关文献报道有10余篇，文献也可见关于美金刚抑制β淀粉样蛋白机制的研究报道，有关提及美金刚对长时程增强的影响的相关文献报道有1篇，但未见有关美金刚对β淀粉样蛋白抑制长时程增强的调制机理的相关文献报道。

美金刚提高淀粉样β蛋白损伤海马的可塑性

Chronic pre-treatment with memantine prevents amyloid-beta protein-mediated long-term potentiation disruption

Fushun Li¹, Xiaowei Chen¹, Feiming Wang², Shujun Xu¹, Lan Chang¹, Roger Anwyl³, Qinwen Wang¹

PDF