药学学报

Model	Mutation	Aβ plaques	NTFS	Other pathology	Cognitive deficits
PDAPP	APP^V717F	Both diffuse and cored Aβ plaques since 6-9 months in hippocampus, corpus callosum, and cerebral cortex^[3]	None	Astrocytosis and microgliosisassociated with plaques^[3]. Synaptic loss and altered LTP induction^[3]	Robust deficits in the radial arm maze at 3 months and object recognition at 6 months^[4]
Tg2576	APP^K670N/M671L	Numerous parenchymal Aβ plaques since 9 months^[5]	None	Dendritic spine loss by 4.5 months and decline in LTP in the dentate gyrus after performant path stimulation^[6]	Impairment in contextual fear conditioning by 5 months and Morris water maze after 9 months^{[5, 6]}
APP23	APP^K670N/M671L	Congophillic, dense-core plaques since 6 months^[7]	None	Neuronal loss, microglia activation, and hyperphosphorylated Tau associated with plaques^[7]	Spatial memory defects in Morris Water maze at 3 months and progresses with age^[8]
APP/PS1	APP^K670N/M671L PSEN1^dE9	Abundant plaques in the hippocampus and cortex since 9 months^[11]	None	Neuronal loss and activated astrocytes associated with plaques. Age-dependent loss of synapse and impaired LTP induction^[10]	Impairment in the Morris water maze at 12 months and commit more errors at 13 months^[10]
5×FAD	APP^K670N/M671L APP^I716V APP^V717I PSEN1^M146L PSEN1^L286V	Extracellular Aβ deposition begins around 2 months. Intraneuronal Aβ also accumulates in an aggregated form within the soma and neurites starting at 1.5 months^[12]	None	Neuronal loss in cortical layer V and subiculum. Gliosis begins at 2 months. Synaptic loss and impaired LTP induction^{[12, 13]}	Impaired spatial working memory in Y maze at 4-5 months^[13]
JNPL3	Tau^P301L	None	NFTs develop as early as 4.5 months in homozygotes and 6.5 months in heterozygotes^[14]	Neuronal loss especially in the spinal cord and astrogliosis in brainstem, diencephalon, and basal telencephalon by 10 months^{[14, 15]}	Unknown
rTg4510	Tau^P301L	None	Pretangles as early as 2.5 months. Argyrophilic tangle-like inclusions in cortex by 4 months and in hippocampus by 5.5 months^[16]	Progressive neuronal loss in hippocampal CA1 area and significant loss of dendritic spines at 8-9 months. Impaired LTP at 4.5 months^{[10, 16]}	Impaired of spatial memory demonstrated by Morris water maze from 2.5 to 4 months. Spatial memory improved when transgene suppressed by dox^{[16, 17]}
TAPP	APP^K670N/M671L Tau^P301L	Has Aβ plaques similar in number and distribution to those of comparably aged Tg2576^[20]	NFTs were morphologically similar in TAPP and JNPL3 mice, older female TAPP mice had a marked increase in NFTs inlimbic areas, subiculum, and hippocampus^[20]	Activated astrocytes and microglia as early as 3 months in the hippocampus and increased with age^[20]	Unknown
3xTg	APP^K670N/M671L Tau^P301L PSEN1^M146L	Extracellular Aβ deposits by 6 months in frontal cortex and progress with age^[21]	By 12 months extensive Tau immunoreactivity in CA1 neurons of the hippocampus^[21]	Activated astrocytes at 7 months. Impairment in LTP and basal synaptic transmission by 6 months^[21]	Cognitive impairment manifests at 4 months as a deficit in long-term retention and correlates with the accumulation of intraneuronal Aβ in the hippocampus and amygdala^[22]

Model	Mutation	Aβ plaques	NTFS	Other pathology	Cognitive deficits
PDAPP	APP^V717F	Both diffuse and cored Aβ plaques since 6-9 months in hippocampus, corpus callosum, and cerebral cortex^[3]	None	Astrocytosis and microgliosisassociated with plaques^[3]. Synaptic loss and altered LTP induction^[3]	Robust deficits in the radial arm maze at 3 months and object recognition at 6 months^[4]
Tg2576	APP^K670N/M671L	Numerous parenchymal Aβ plaques since 9 months^[5]	None	Dendritic spine loss by 4.5 months and decline in LTP in the dentate gyrus after performant path stimulation^[6]	Impairment in contextual fear conditioning by 5 months and Morris water maze after 9 months^{[5, 6]}
APP23	APP^K670N/M671L	Congophillic, dense-core plaques since 6 months^[7]	None	Neuronal loss, microglia activation, and hyperphosphorylated Tau associated with plaques^[7]	Spatial memory defects in Morris Water maze at 3 months and progresses with age^[8]
APP/PS1	APP^K670N/M671L PSEN1^dE9	Abundant plaques in the hippocampus and cortex since 9 months^[11]	None	Neuronal loss and activated astrocytes associated with plaques. Age-dependent loss of synapse and impaired LTP induction^[10]	Impairment in the Morris water maze at 12 months and commit more errors at 13 months^[10]
5×FAD	APP^K670N/M671L APP^I716V APP^V717I PSEN1^M146L PSEN1^L286V	Extracellular Aβ deposition begins around 2 months. Intraneuronal Aβ also accumulates in an aggregated form within the soma and neurites starting at 1.5 months^[12]	None	Neuronal loss in cortical layer V and subiculum. Gliosis begins at 2 months. Synaptic loss and impaired LTP induction^{[12, 13]}	Impaired spatial working memory in Y maze at 4-5 months^[13]
JNPL3	Tau^P301L	None	NFTs develop as early as 4.5 months in homozygotes and 6.5 months in heterozygotes^[14]	Neuronal loss especially in the spinal cord and astrogliosis in brainstem, diencephalon, and basal telencephalon by 10 months^{[14, 15]}	Unknown
rTg4510	Tau^P301L	None	Pretangles as early as 2.5 months. Argyrophilic tangle-like inclusions in cortex by 4 months and in hippocampus by 5.5 months^[16]	Progressive neuronal loss in hippocampal CA1 area and significant loss of dendritic spines at 8-9 months. Impaired LTP at 4.5 months^{[10, 16]}	Impaired of spatial memory demonstrated by Morris water maze from 2.5 to 4 months. Spatial memory improved when transgene suppressed by dox^{[16, 17]}
TAPP	APP^K670N/M671L Tau^P301L	Has Aβ plaques similar in number and distribution to those of comparably aged Tg2576^[20]	NFTs were morphologically similar in TAPP and JNPL3 mice, older female TAPP mice had a marked increase in NFTs inlimbic areas, subiculum, and hippocampus^[20]	Activated astrocytes and microglia as early as 3 months in the hippocampus and increased with age^[20]	Unknown
3xTg	APP^K670N/M671L Tau^P301L PSEN1^M146L	Extracellular Aβ deposits by 6 months in frontal cortex and progress with age^[21]	By 12 months extensive Tau immunoreactivity in CA1 neurons of the hippocampus^[21]	Activated astrocytes at 7 months. Impairment in LTP and basal synaptic transmission by 6 months^[21]	Cognitive impairment manifests at 4 months as a deficit in long-term retention and correlates with the accumulation of intraneuronal Aβ in the hippocampus and amygdala^[22]

Compound	Mechanism	Animal model	Evaluation index	Outcome assessment
Donepezil	AChE inhibitor	APP23	Morris water maze	Improve cognitive function^[125]
APP/PS1	¹H-MRS	Improve metabolic profile in cortex and hippocampus^[126]
Intracerebroventricular Aβ injection	Y-maze Fear conditioning test	Improve cognitive function^[127]
Galantamine	AChE inhibitor	APP23	Morris water maze	Improve cognitive function^[125]
Intracerebroventricular Aβ injection	Novel object recognition Fear conditioning test	Improve cognitive function^[127]
Rivastigmine	AChE inhibitor	APP23	Morris water maze	Improve cognitive function^[125]
Memantine	NMDAR antagonist	Tg2576	Aβ plaque density Neuronal morphology Synapse density	Reduce plaque burden, increase synapse density and degenerating axons^[128]
3xTg	Morris water maze Novel object recognition Passive inhibitory avoidance Tau pathology Aβ accumulation LTP induction	Improve cognitive function, reduce Tau phosphorylation and Aβ accumulation, reduce levels of Aβoligomers and reverse oligomeric Aβ-induced deficits in LTP^[129]

Compound	Mechanism	Animal model	Evaluation index	Outcome assessment
Donepezil	AChE inhibitor	APP23	Morris water maze	Improve cognitive function^[125]
APP/PS1	¹H-MRS	Improve metabolic profile in cortex and hippocampus^[126]
Intracerebroventricular Aβ injection	Y-maze Fear conditioning test	Improve cognitive function^[127]
Galantamine	AChE inhibitor	APP23	Morris water maze	Improve cognitive function^[125]
Intracerebroventricular Aβ injection	Novel object recognition Fear conditioning test	Improve cognitive function^[127]
Rivastigmine	AChE inhibitor	APP23	Morris water maze	Improve cognitive function^[125]
Memantine	NMDAR antagonist	Tg2576	Aβ plaque density Neuronal morphology Synapse density	Reduce plaque burden, increase synapse density and degenerating axons^[128]
3xTg	Morris water maze Novel object recognition Passive inhibitory avoidance Tau pathology Aβ accumulation LTP induction	Improve cognitive function, reduce Tau phosphorylation and Aβ accumulation, reduce levels of Aβoligomers and reverse oligomeric Aβ-induced deficits in LTP^[129]

抗阿尔茨海默病药物非临床药效学评价体系的探索

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黄龙舰 ¹ , 赵春阳 ² , 冯新红 ³ , 兰嘉琦 ¹ , 唐婧姝 ¹ , 王庆利 ²^,^* , 彭英 ¹^,^*

药学学报 | 综述 2020,55(5): 789-805

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药学学报 | 综述 2020, 55(5): 789-805

抗阿尔茨海默病药物非临床药效学评价体系的探索

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黄龙舰¹, 赵春阳², 冯新红³, 兰嘉琦¹, 唐婧姝¹, 王庆利²^,^*, 彭英¹^,^*

作者信息

1.中国医学科学院、北京协和医学院药物研究所, 北京 100050

2.国家药品监督管理局药品审评中心, 北京 100022

3.清华大学附属北京清华长庚医院神经内科, 北京 102218

通讯作者:

*王庆利, Tel:86-10-83165742, Fax:86-10-63017757, E-mail:wangql@cde.org.cn;

彭英, Tel:86-10-68585566, Fax:86-10-68584189, E-mail:ypeng@imm.ac.cn

Exploration of nonclinical pharmacodynamics evaluation system of Alzheimer's disease

Long-jian HUANG¹, Chun-yang ZHAO², Xin-hong FENG³, Jia-qi LAN¹, Jing-shu TANG¹, Qing-li WANG²^,^*, Ying PENG¹^,^*

Affiliations

1. Institute of Materia Medica, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing 100050, China

2. Center for Drug Evaluation, National Medical Product Administration, Beijing 100022, China

3. Department of Neurology, Beijing Tsinghua Changgung Hospital, Beijing 102218, China

出版时间: 2020-05-12 doi: 10.16438/j.0513-4870.2019-0899

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摘要

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阿尔茨海默病（Alzheimer's disease，AD）是引发老年人痴呆的最常见的神经退行性疾病。目前，AD的发病机制尚不明确，已上市的药物全部为对症治疗药物，无法延缓或逆转疾病的进程，同时在研药物面临严峻的临床转化难题。因此，围绕AD展开新药研发，解决未被满足的临床需求具有重大的社会意义和经济价值。本文结合了近年来国内外抗AD药物的研发进展，主要从动物模型和药效评价指标两个方面对抗AD药物非临床药效学评价体系进行归纳总结，以期为抗AD药物的非临床开发提供参考。

关键词

阿尔茨海默病 / 动物模型 / 行为学检测 / 非临床药效学评价体系 / 新药研发

Abstract

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Alzheimer's disease (AD) is the most common neurodegenerative disease that causes dementia among elderly people. The pathogenesis of AD is still unclear, and currently approved drugs only provide symptomatic benefits and do not prevent or delay progressive neurodegeneration. Meanwhile, potential drugs in development are facing great challenges in clinical translation. Therefore, finding effective treatment for the unmet clinical needs of AD is of great economic value and social significance. In this review, we will summarize the current models and pharmacodynamics evaluation methods of anti-AD drug based on the recent studies at home and abroad, and provide reference for drug development in AD at nonclinical stage.

Key words

Alzheimer's disease / animal model / behavioristics / non-clinical pharmacodynamics / drug discovery

引用本文

黄龙舰, 赵春阳, 冯新红, 兰嘉琦, 唐婧姝, 王庆利, 彭英. 抗阿尔茨海默病药物非临床药效学评价体系的探索. 药学学报, 2020 , 55 (5) : 789 -805 . DOI: 10.16438/j.0513-4870.2019-0899

Long-jian HUANG, Chun-yang ZHAO, Xin-hong FENG, Jia-qi LAN, Jing-shu TANG, Qing-li WANG, Ying PENG. Exploration of nonclinical pharmacodynamics evaluation system of Alzheimer's disease[J]. Acta Pharmaceutica Sinica, 2020 , 55 (5) : 789 -805 . DOI: 10.16438/j.0513-4870.2019-0899

正文

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阿尔茨海默病(Alzheimer's disease, AD)又称老年痴呆, 是引发老年人认知障碍的最常见原因, 其主要临床症状为不断减退的记忆功能, 并伴有语言、判断、情感、行为等方面的改变, 严重影响患者的日常生活。随着人口老龄化的不断加剧, 预计到2050年, 全球的AD患者将达到1.15亿, 这将为家庭和社会带来沉重的经济负担^[1]。目前, AD的发病机制尚不明确, 研究认为可能与β-淀粉样蛋白(amyloid β-protein, Aβ)沉积、Tau蛋白异常修饰、神经细胞凋亡、突触丢失、氧化应激和神经炎症等因素密切相关。现阶段, 临床上用于治疗AD的药物多以胆碱酯酶抑制剂和N-甲基-D-天冬氨酸受体(N-methyl-D-aspartic acid receptor, NMDAR)阻断剂为主, 然而这类药物仅能缓解部分症状, 无法改善AD的病理进程。靶向Aβ和Tau的疗法虽然在动物实验中表现出了诱人的开发前景, 却在临床试验中屡屡碰壁, 面临严峻的转化难题。因此, 明确AD的发病机制, 寻找有效的治疗手段解决未被满足的临床需求, 具有重大的社会意义和经济价值, 已成为国内外医学研究的重点课题。

药物非临床研究是新药开发的重要阶段, 是评价受试药物安全性和有效性的基础工作。药物非临床研究结果的准确性和可靠性不仅影响临床试验的获批准入, 更对提高临床转化成功率和降低临床研究风险起到了关键作用。然而, 在当前的药物非临床药效学研究中, 存在诸多动物模型和评价方法学上的缺陷, 为临床转化埋下了隐患。为此, 本文结合了近年来国内外抗AD药物的研发进展, 主要从动物模型和药效评价指标两个方面对抗AD药物非临床药效学评价体系进行归纳总结, 以期为抗AD药物的非临床开发提供参考。

1 抗阿尔茨海默病药物非临床药效学评价模型的建立与选择

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考虑到AD复杂的发病机制和临床表现, 理想的动物模型首先应该贴合临床实际, 其次, 还要兼备稳定、易得、经济、便于评价等特点。当前在抗AD药物的非临床研究中, 常用的动物模型主要包括转基因动物模型、自发性衰老动物模型、人工损伤性动物模型以及低等模式动物模型等。此外, 作为对传统动物模型的重要补充, 近年来出现的体外诱导性多能干细胞(induced pluripotent stem cells, iPSCs)模型也越来越多地受到了人们的关注。

1.1 转基因小鼠模型

1.1.1 表现Aβ病变的转基因小鼠模型

野生型小鼠与人类的淀粉样前体蛋白(amyloid precursor protein, APP)在Aβ内部存在3个氨基酸位点的差异(R5G、Y10F和H13R), 无法自发生成Aβ沉积, 因此只有通过转入人源性APP基因才能表现出Aβ相关的病理改变^[2]。该类小鼠模型最初由APP单转基因小鼠发展而来, 代表品系为PDAPP小鼠、Tg2576小鼠以及APP23小鼠。其中, PDAPP小鼠在PDGF-β启动子的调节下表达人类APP Indiana突变(APP^V717F), 其表达量是小鼠内源性APP的10倍。该模型小鼠在6~9月龄时开始出现Aβ斑块, 并出现神经胶质增生、突触丢失等AD相关病理改变^[3]。其认知障碍常早于Aβ斑块出现, 但无法形成神经原纤维缠结(neurofibrillary tangles, NFTs), 也未见广泛的神经元丢失^[4]。Tg2576小鼠在PrP启动子的调控下表达人类APP Swedish突变(APP^K670N/M671L), 其Aβ的表达量显著提升, 特别是Aβ_1-42/43的增加尤为明显^[5]。该模型小鼠通常于9月龄后出现明显认知障碍, 并伴有额颞叶皮层、海马及小脑等处的Aβ沉积^[5]。此外, 在该模型小鼠中还可观察到突触丢失、海马长时程增强(long-term potentiation, LTP)损伤及神经胶质增生等病理改变, 但依然未见NFTs和广泛的神经元丢失^[6]。APP23小鼠在Thy1启动子的调控下表达人类APP Swedish突变(APP^K670N/M671L), 其表达量是小鼠内源性APP的7倍^[7]。该模型小鼠在3月龄即表现出认知障碍^[8], 6月龄时开始出现Aβ沉积, 在脑组织和脑血管壁中均有表达^[9], 其斑块多呈致密型, 周围可见明显的神经炎症、轴突变性以及Tau过度磷酸化, 但未见NFTs形成, 神经元丢失则主要集中于海马CA1区域^[9]。

继APP单转基因小鼠之后, 又出现了同时表达多个家族性AD (familial AD, fAD)突变的多转基因小鼠。其中包括表达APP Indiana突变和Swedish突变的J20转基因小鼠, 以及最被人们熟知的APP/PS1转基因小鼠。当前, 各种品系的APP/PS1转基因小鼠已广泛应用于AD的非临床研究中, 包括APP_SWE×PS1dE9、APP_SWE×PS1_M146L、APP_SWE×PS1_L166P、APP_SL×PS1_M146L以及5×FAD转基因小鼠等。由于PS1基因的引入, 该类转基因小鼠Aβ沉积和老年斑形成的速度明显加快, 学习记忆出现障碍的时间显著提前。其中APP_SWE×PS1dE9转基因小鼠在3月龄时即出现脑片LTP减弱^[10], 4月龄时可见突触标记蛋白异常丢失, 6月龄时开始出现Aβ沉积, 并随疾病进程不断加重^[11], 12月龄时水迷宫实验提示已出现明显的认知功能障碍^[10]。在APP/PS1转基因小鼠中, 疾病进展最快的模型是5×FAD小鼠, 由于转入了APP^K670N/M671L (Swedish)、APP^I716V (Florida)、APP^V717I (London)、PS1^M146L以及PS1^L286V这5种fAD相关突变, 5×FAD小鼠在1.5月龄时即出现Aβ₄₂在神经元内的聚集, 2月龄时可在海马下托和皮层深处出现Aβ斑块, 并伴有胶质增生^[12], 4月龄时开始出现突触标记蛋白异常丢失和海马LTP损伤^[13], 同期Y迷宫实验显示已出现明显的空间记忆障碍^[13]。

上述各种小鼠模型均通过转入人源性APP或PS1突变基因构建而成, 其共同特征是大量Aβ沉积和老年斑的出现, 并伴有胶质增生和突触丢失等异常改变。然而需要注意的是, 该类模型无法像AD患者一样表现出广泛的神经退行性病变和脑萎缩。此外, 尽管部分研究报道了磷酸化Tau蛋白的聚集, 但该类模型始终无法形成NFTs, 因此主要用于靶向Aβ药物的非临床研究。

1.1.2 表现Tau病变的转基因小鼠模型

　JNPL3小鼠是最早利用P301L突变构建的Tau转基因小鼠模型^[14]。在PrP启动子的调控下, 该模型小鼠可在小脑、海马、丘脑以及脊髓等处表达人源性Tau^P301L蛋白, 其表达量是小鼠内源性Tau蛋白的1~2倍。随着疾病进展, JNPL3小鼠在4.5~6.5月龄时会出现NFTs、胶质细胞增生、神经元丢失以及进行性脊髓白质病变, 在10月龄之后, 绝大部分小鼠会表现出认知功能和运动功能障碍^{[14, 15]}。另外一种常用的Tau^P301L转基因小鼠模型是rTg4510小鼠, 在前脑特异性CaMKⅡ启动子和Tet-off系统的调控下, 人源性Tau^P301L蛋白可集中表达于小鼠海马和大脑新皮层, 并受到多西环素等分子开关的诱导。正常情况下, rTg4510小鼠Tau^P301L蛋白的表达量是其内源性Tau蛋白的13倍, 在4~5.5月龄时即可出现NFTs, 并伴有海马CA1区神经元丢失、前脑萎缩、树突棘密度降低以及LTP损伤等病理改变^{[16, 17]}。水迷宫实验结果显示, rTg4510小鼠在4月龄时即出现明显的空间记忆障碍, 并随着疾病进展不断加重。此外, 相比于其他类型的Tau转基因小鼠, rTg4510小鼠不会表现出明显的脊髓病变和肌肉萎缩, 其早期的运动功能也未受到明显影响^[16]。

除P301L突变之外, P301S、R406W、V337M等位点的突变也已用于Tau转基因小鼠的构建。这些小鼠均表现出随病程不断加重的NFTs和认知功能障碍, 部分模型还会出现神经元丢失、胶质增生以及突触损伤等病理表型^[18]。此外, 亦有研究通过转入野生型人源Tau蛋白构建小鼠模型, 但只有在敲除小鼠内源性Tau蛋白的基础上才能表现出Tau相关表型, 提示小鼠内源性Tau可能对野生型人源Tau的聚集起到抑制作用^[19]。需要注意的是, 以上转基因小鼠引入的Tau突变均来自额颞叶痴呆(frontotemporal dementia, FTD)患者, 与AD的发病没有直接关联, 且除rTg4510小鼠外, 其他小鼠模型大多会表现出运动功能障碍, 影响其在行为学检测中的表现。此外, 该类模型缺乏Aβ聚集和斑块沉积, 无法全面模拟AD的病理改变, 因而主要用于靶向Tau药物的非临床研究。

1.1.3 同时表现Aβ和Tau病变的转基因小鼠模型

为了更加全面地模拟AD临床上的病理改变, 一些研究构建出了能够同时表达Aβ斑块和NFTs的小鼠模型, 其中最早报道的是TAPP小鼠, 由携带APP^K670N/M671L突变的Tg2576小鼠和携带Tau^P301L突变的JNPL3小鼠杂交而得^[20]。研究显示, 该模型小鼠在6月龄时开始出现Aβ沉积, 9月龄时可在内嗅皮层、海马、杏仁核等部位出现明显的Aβ斑块, 且在形态、密度和分布范围上与Tg2576小鼠保持了较好的一致性。在Tau病变方面, TAPP小鼠的NFTs最早在3月龄出现, 多见于脑桥和脊髓, 且形态与JNPL3小鼠相近。然而, 随着病程的发展, 雌性TAPP小鼠的NFTs可迅速扩展至内嗅皮层、杏仁核等脑区, 其表达量可上升至JNPL3小鼠的7倍^[20]。

除TAPP小鼠外, 另一种公认的能够较为全面地模拟AD病理改变的模型是3xTg小鼠。该模型小鼠同时携带APP ^K670N/M671L、Tau^P301L以及PS1^M146L三种fAD突变, 在小鼠Thy1.2启动子的调控下, 转入的突变型APP和Tau仅表达于皮层、海马及杏仁核等脑区。研究显示, 3xTg小鼠在3~4月龄时出现胞内Aβ聚集, 并伴有突触丢失、LTP损伤等病理改变。6月龄时在额叶皮层深处可见Aβ斑块, 并随疾病进展不断扩散至其他脑区。12月龄时开始形成NFTs, 初见于海马CA1区, 之后可扩展至皮层^[21]。水迷宫实验结果显示, 该模型小鼠在4月龄时即可出现记忆能力下降, 且与海马和杏仁核等脑区胞内Aβ的聚集程度呈正相关关系^[22]。

综上, 各种转基因小鼠的共同特点是均采用过表达人源性APP、PS1、Tau等突变基因的方法构建, 具体病理表型见表 1。这虽然能够较好地模拟AD临床上最主要的病理改变, 但一直存在两大缺陷。第一, 该类模型中的Aβ和Tau并非正常的生理性表达, 因此很难推断其各种病理表型的出现, 究竟是和Aβ、Tau等致病蛋白本身相关, 还是仅为APP、Tau等突变基因过表达的结果。此外, APP的过表达不仅会导致Aβ的生成增加, 还会产生包括APP氨基末端片段、羧基末端片段(carboxy terminal fragment, CTF)以及APP内部区(APP intracellular domain, AICD)在内的许多其他剪切产物, 而这些片段可能作为独立因素引发和Aβ类似的神经毒性作用, 使得该类转基因小鼠的病理机制变得更加复杂^[23]。第二, 该类模型所转入的突变基因均来自fAD患者或FTD患者, 而占临床发病95%以上的散发性AD (sporadic AD, sAD)患者并未检测到上述突变的存在。考虑到fAD和sAD患者在发病机制和病理表型方面存在的诸多差异, 这些通过fAD动物模型筛选出来的药物进入临床后, 可能无法在sAD患者中观察到同样的治疗效果。

1.2 自发性衰老动物模型

1.2.1 非人灵长类动物模型

在所有表现衰老相关认知障碍的动物模型中, 非人灵长类动物与人类的遗传背景最为接近。常见的非人灵长类动物包括大猩猩、恒河猴和松鼠猴等。研究显示, 大猩猩的Aβ序列与人类完全相同, 可在脑内自发聚集形成Aβ斑块, 或以淀粉样脑血管病(cerebral amyloid angiopathy, CAA)的形式沉积于脑血管壁。但其胞外的Aβ斑块多呈弥散状分布, 缺少AD患者临床上常见的致密斑, 且受累程度较轻^{[24, 25]}。在Tau病变方面, 尽管大猩猩和人类的Tau序列同源性超过99.5%, 部分研究也曾报道在衰老大猩猩脑内发现了过度磷酸化的Tau蛋白, 但较少出现NFTs^[25]。此外, 衰老大猩猩的认知障碍多呈轻度改变, 主要表现为增龄性的记忆减退, 不及AD临床患者严重^[26]。目前, 大猩猩作为模型在抗AD药物的非临床研究中并不多见, 主要由于其较长的寿命以及科学伦理方面的考虑。

相比于大猩猩, 更多的AD非临床研究选用恒河猴和松鼠猴进行。作为旧大陆猴的代表, 恒河猴的Aβ序列与人类完全相同, 其脑部Aβ受累程度也与AD临床患者更加接近^[27]。研究显示, 恒河猴通常在25岁之后出现脑部Aβ斑块, 多见于皮层, 以弥散斑为主, 致密斑大约占到20%^[28]。在Tau病变方面, 尽管恒河猴的Tau序列与人类高度同源, 但其同样无法形成NFTs, 反而是旧大陆猴的另一类代表狒狒, 能够表现出随年龄增长不断加重的Tau病变。研究显示, 在26岁以上的老年狒狒中, 超过90%会出现NFTs, 但其表达仅限于海马, 在其他脑区未见分布^[29]。

松鼠猴是新大陆猴中研究最多的动物模型。该模型的一大特点是其脑内的Aβ大多沉积于小动脉及毛细血管壁, 因此特别适合用于CAA的研究^[30]。此外, 其Aβ亦可在脑内沉积形成斑块, 但多见于皮层和杏仁核, 较少出现在海马, 且斑块面积相较于AD临床患者要小很多。部分松鼠猴神经元中可出现少量磷酸化的Tau蛋白, 但始终未见NFTs的形成^[30]。

1.2.2 其他自发性衰老动物模型

非人灵长类动物虽然是最接近人类的动物模型, 但其价格昂贵, 寿命较长, 因此在实际应用中亦可考虑其他动物。研究显示, 老年犬可自发形成Aβ斑块和CAA, 其斑块多以弥散斑为主, 在分布范围上与AD患者较为接近^[31]。此外, 老年犬还可出现皮质萎缩、脑脊液Aβ_42/40比例降低以及Tau磷酸化等病理表现, 但少有NFTs形成^[32]。另一方面, 由于长期的驯化, 犬类的生活环境和饮食习惯与人类更加接近, 在研究中也表现出更好的依从性。行为学实验结果表明, 犬的认知功能表现出和AD患者一样复杂的退行性变化, 包括在学习、记忆、执行以及空间定位等各个方面的障碍。此外, 老年犬的疾病进程与AD患者表现出了较好的一致性。其从出现AD相关病变到最终死亡的时间, 大概持续3 ~ 4年, 因此非常适合需要对干预手段进行长期观测的研究^[33]。

啮齿类动物的Aβ序列与人类存在差异, 无法自发形成Aβ斑块, 仅表现出与衰老相关的认知及病理改变。然而, 由AKR/J自然变异小鼠近交培养得到的快速老化小鼠(senescence-accelerated mouse, SAM)却表现出AD相关的病理表型。研究显示, SAMP8小鼠的APP表达水平显著升高, 并大量生成一种类似人类Aβ的蛋白, 该蛋白同样能够聚集形成淀粉样斑块, 且主要沉积于海马等脑区^[34]。此外, SAMP8小鼠还表现出Tau蛋白过度磷酸化、氧化损伤、乙酰胆碱转移酶活力降低等AD相关的病理改变, 以及学习记忆能力快速减退、情绪失调、昼夜节律紊乱、免疫功能损伤、寿命缩短等特征^[35], 因此广泛应用于各类衰老相关疾病的研究和新药开发中。

综上, 相比于转基因动物, 自发性衰老动物无需借助外来基因即可表现出AD相关的病理表型, 这更加接近临床上AD的病程变化。其作为药效学评价模型, 在抗AD药物, 尤其是以sAD为主的非临床研究中, 具有更大的应用价值。然而这类模型普遍存在费用昂贵, 寿命较长, 均一性差等问题, 此外, 自发性衰老动物很少出现Tau相关的病理表型, 因此无法应用于以Tau为靶点的新药研发。

1.3 人工诱发性动物模型

1.3.1 中枢胆碱系统损伤性动物模型

中枢胆碱能系统参与调解哺乳动物的神经元兴奋性、皮质可塑性以及学习记忆过程, 与脑认知功能密切相关^[36]。作为非选择性M胆碱受体拮抗剂, 东莨菪碱可以通过血脑屏障进入中枢, 阻断脑内乙酰胆碱与M胆碱受体的结合, 导致中枢胆碱能神经传导受阻, 引发动物短期的学习记忆障碍^[36]。该模型在造模时多选用啮齿类动物, 单次或多次腹腔注射给药, 剂量多在0.5~2 mg·kg^-1之间。单次给药30 min后进行行为学检测, 即可出现明显的学习记忆障碍^[37]。然而由于东莨菪碱对中枢M受体的阻断并非特异性, 该模型会出现焦虑、运动迟缓、自主活动增多等影响行为学评价的不良反应^[38]。此外, 该模型造成的痴呆症状仅是一可逆的过程, 且缺乏Aβ和Tau等AD特征性病理改变。

除东莨菪碱外, 对中枢胆碱能系统的破坏还可以通过脑部定点注射鹅膏蕈氨酸(ibotenic acid, IBO)、红藻氨酸等神经毒性物质以及免疫毒素192-IgG-saporin的方法来实现。其中IBO是一种神经兴奋毒性剂, 能够与神经元胞体或树突上的NMDA受体结合, 导致神经元钙超载和坏死^[39]。造模时, 借助脑立体定位仪将IBO注射入动物单/双侧的海马或内嗅皮层等脑区, 即可引发动物学习记忆能力下降等拟痴呆症状^[40]。192-IgG-saporin是一种选择性破坏胆碱能神经元的免疫毒素, 由神经生长因子(nerve growth factor, NGF)受体的单克隆抗体IgG与核糖体失活蛋白Saporin结合而成。通过与胆碱能神经元上的NGF受体特异性结合后, saporin与192-IgG发生分离, 进入胞浆, 使核糖体大亚基失去活性, 从而阻止了蛋白质的合成, 导致细胞死亡^[41]。研究发现, 192-IgG-saporin能剂量依赖性地损伤基底前脑胆碱能神经元, 并造成认知功能障碍, 该损伤特异性强, 持续时间久, 且对动物自主活动等方面的不良影响较轻^[42]。

1.3.2 　Aβ注射损伤性动物模型

在非临床药效学研究中, 亦可通过向动物脑内注射Aβ肽段的方法, 来模拟AD临床上的病理改变。目前, 常用于造模的Aβ肽段有Aβ_1-40、Aβ_1-42和Aβ_25-35, 处理办法包括将这些片段制备成为Aβ寡聚体或Aβ纤维, 通过立体定位注入动物侧脑室或者单/双侧海马等区域, 剂量多在5~10 μg之间^{[43, 44]}。此外, 为更好地模拟AD在自然状态下的发病进程, 也可以采用埋管并使用微渗透泵、微透析法以实现Aβ缓慢、持续的灌注效果^[45]。研究显示, 该模型可在短时间内显著诱发学习记忆障碍, 且在注射位点附近还会出现大量胶质细胞活化、神经炎症、氧化应激、神经元丢失以及胆碱能系统损伤等AD相关的病理变化^{[46, 47]}。

相比于APP转基因动物模型, 该模型的优点在于能够引入确定类型的Aβ片段, 避免了其他非Aβ片段过量生成带来的复杂影响。此外, 该模型成模时间短, 无需经历较长的病理进展期, 且可以通过调整Aβ的注射量来控制模型的病变程度。然而需要注意的是, 在造模时, 由于Aβ注射方法(微量注射器一次性注射或微泵渗透)、注射类型(Aβ_1-40、Aβ_1-42、Aβ_25-35等)、Aβ聚集状态(寡聚体或纤维)、稀释溶剂以及注射部位等因素的差异, 可能会导致造模后出现的病理变化及病程进展不尽相同^[48]。

1.4 低等模式动物模型

以低等模式动物构建的AD模型包括果蝇、线虫、斑马鱼等。该类模型凭借遗传背景清晰、生命周期短、易于繁殖和观察等特点, 在研究AD相关基因功能、寻找药物潜在靶点及化合物的高通量筛选上具有一定优势。研究显示, 野生型果蝇的Aβ序列与人类存在差异, 且不具备人类β-分泌酶1 (beta-secretase 1, BACE1)的同源蛋白, 因此需要同时转入人源性APP及BACE1基因, 或将Aβ序列直接与信号肽融合, 才能实现Aβ在果蝇体内的过表达和分泌^[49]。此外, 也可通过转入人类野生型或突变型的Tau蛋白, 构建Tau病变的果蝇模型^[50]。在Gal/UAS系统的调控下, 转入的外源性基因可在果蝇眼部、大脑等特定组织中表达, 并出现视网膜粗糙、翅脉发育异常、寿命缩短、嗅觉记忆损伤及运动障碍等症状, 为靶向Aβ和Tau等抗AD药物的筛选提供了易于观察和检测的指标^[51]。

和果蝇一样, 当前用于药物筛选的线虫模型大多通过转入外源性Aβ或Tau基因构建而成^{[52, 53]}。在不同启动子的调控下, 这些外源性基因可在线虫的特定组织中表达, 呈现出不同的病理表型。例如, unc-54启动子能使Aβ特异性表达于线虫的肌肉组织中, 并导致线虫的进行性瘫痪以及幼虫体内的空泡样病变^[53]。Snb-1启动子则调控Aβ在线虫神经元中的表达, 并影响其对特定化学刺激的感受功能^[54]。而Tau转基因线虫则多表现出神经元丢失、轴突损伤、运动障碍以及寿命缩短等症状, 其中突变型Tau导致的病理变化更加明显^[52]。

相比于果蝇、线虫等无脊椎动物, 斑马鱼的神经系统在结构和功能上与人类更加接近, 能够表达更多AD致病基因的同源基因, 包括APP、microtubule-associated protein tau (MAPT)、presenilin-1 (PSEN1)和PSEN2^[55]。此外, 斑马鱼身体透明, 易于观察, 可以在微孔板中直接培养, 并通过皮肤吸收溶解在水中的药物, 特别适合非临床研究中药效和毒理学方面的评价。目前用于AD研究的斑马鱼模型大多通过转入外源性基因、Aβ胚胎注射或暴露于铝、东莨菪碱、冈田酸等环境中得到。常用的行为学评价方法包括惊恐反应、电击躲避反应以及对游泳路径、速度进行分析, 以反映其学习记忆能力和运动能力方面的变化^[55]。此外, 亦有研究借助斑马鱼色素沉淀深浅、体节发育形状及尾部弯曲程度等表型, 用于GSK-3β抑制剂和γ-分泌酶抑制剂等抗AD化合物的筛选^{[56, 57]}。

1.5 细胞筛选模型

尽管上述动物模型在某些方面能够较好地模拟AD的病理改变, 但其与人类之间的种属差异始终存在, 这为临床转化埋下了诸多隐患。而临床来源的样本虽不存在种属差异, 但其多取自尸体组织, 可及性较差, 且质量难以保证, 因此在使用上一直受到限制。近年来, iPSC的出现有望将上述问题一举解决。首先, iPSC可由包括成纤维细胞、血细胞以及尿路上皮细胞在内的多种人源细胞制备, 其来源不受组织部位的限制。其次, iPSC保留了原供体细胞的全部遗传和突变信息, 且具备无限增殖和分化成为各种体细胞的潜能, 因此能够在体外制备大量特定组织类型的细胞, 并与细胞提供者在病理表型上高度一致^[58]。

现阶段, 许多AD患者和正常老年人来源的iPSC细胞系已被建立。相比于正常老年人, AD患者来源的iPSC可表现出明显的Aβ生成增多和Tau蛋白过度磷酸化^{[59, 60]}。此外, 部分iPSC细胞系还表现出GSK-3β活性升高、内涵体数目增多以及胞内Aβ寡聚体异常聚集等病理表型^[61]。这为Aβ抗体、APP生成调节剂以及Tau聚集抑制剂等抗AD药物提供了新的体外筛选方法^[62-64]。另一方面, 作为传统动物模型的重要补充, iPSC模型对临床转化结果有很好的预判作用。例如, 吲哚美辛、布洛芬、双氯芬酸钠等非甾体抗炎药物具有γ-分泌酶抑制剂的活性, 在APP转基因小鼠模型及APP-CHO细胞模型中已被证实能够降低Aβ₄₂的水平^[65]。然而在iPSC模型中, 相同浓度的药物却不能发挥类似的作用, 提示人源性γ-分泌酶可能不受到该类药物的调控, 与临床结果有较好的一致性^[66]。

目前, iPSC模型正朝着构建3D细胞培养系统的方向发展。该系统利用水凝胶或人工基底膜作为支架, 可以为各种类型的神经元和胶质细胞提供接触空间, 进而更好地模拟大脑的复杂环境^[67]。研究显示, 利用转染了APP^{K670N/M671L/V717I}和PS1^ΔE9的人神经前体细胞构建的3D细胞模型可以在胞外基质中聚集形成Aβ斑块, 并在胞体和变性轴突中出现Tau聚集。这也是首次在体外构建出能够同时表达Aβ和Tau相关病变的细胞模型^[67]。

尽管iPSC模型在抗AD新药的非临床开发中表现出了诱人的应用前景, 但需要注意的是, 当前iPSC模型大多采用fAD患者的细胞构建, 而sAD患者来源的iPSC细胞系则较为少见, 因此无法体现AD患者在临床表型上高度异质性的特点。另一方面, 目前尚未建立iPSC细胞系构建和维持的标准化流程, 其在新药非临床开发中需要的细胞系数量、来源等方面的要求也缺乏相关指南的指导。这些因素为iPSC模型的广泛应用带来了挑战^[68]。

1.6 选择抗AD药物非临床研究模型时应注意的问题

上述各种模型的出现为抗AD药物非临床研究提供了丰富的选择。然而需要注意的是, 这些模型均为对AD的部分模拟, 目前尚未有模型能够全面反映AD复杂的病理改变和临床症状, 因此, 采用单一模型得到的研究结果外推至临床时, 其转化价值将大打折扣。此外, 非生理性动物模型在引入外来基因或手术损伤时带来的复杂影响, sAD和fAD在发病机制和病理表型上的诸多差异, 以及动物模型始终存在的种属差异, 亦是在非临床研究中应该重点考虑的问题。另一方面, 作为AD最主要的临床症状, 认知障碍通常在患者脑内Aβ斑块等病理改变发展数十年之后才会出现。而在大多数AD动物模型, 尤其是各种转基因动物模型中, 认知障碍的出现往往与病理改变同步, 甚至更早, 因此何时给予药物干预治疗, 也是非临床研究中应该考虑的重要问题。

2 抗阿尔茨海默病药物非临床药效学评价方法

收起

2.1 行为学检测

2.1.1 　Morris水迷宫实验

　Morris水迷宫实验是神经科学中最经典的行为学研究方法, 广泛用于评价受试药物对啮齿类动物模型认知障碍的改善作用^[69]。Morris水迷宫实验的装置由圆形水池、隐蔽平台、摄像系统以及轨迹分析系统组成。水池大小因所选动物而异, 通常大鼠直径多为150~200 cm, 小鼠直径多为90~120 cm, 水池高约0.5 m, 其内部表面为非反光材料。水池被池壁上的4个等距点分为4个象限, 隐蔽平台放置于其中一个象限的中央, 其直径多为10~12 cm, 位于液面下1~2 cm处^[70]。实验过程中水池的温度维持在19~22 ℃, 液面颜色应和实验动物的毛色形成鲜明对比, 方便摄像头对动物的追踪定位。此外, 水池四周应设置一定数量的标志物, 以为动物的空间定位提供参照^[70]。

Morris水迷宫实验包括定位航行和空间探索两个阶段, 其中定位航行实验的目的是让动物在连续多日的训练中借助各种标志线索找到并记住隐藏在液面下的平台位置。每次训练时, 把动物从任意一个象限面朝池壁放入水中并开始计时, 动物由于求生本能, 会在水池内游泳直到找到隐藏在水面下的平台。每次训练时间一般为1~2 min, 如果动物在规定时间内成功找到平台, 则让其在平台上停留15~30 s。否则, 需要实验人员指引其找到平台并停留相同的时间。定位航行实验一般持续5~6天, 期间需要记录动物每次训练的逃避潜伏期(动物入水到找到平台所用时间)、游泳总路程、游泳速度、游泳轨迹以及搜索策略等指标进行分析。空间探索实验通常在最后一次训练结束的24 h后进行。此时撤去隐蔽平台, 将动物由原平台所在象限的对侧放入水池中, 记录动物在60或90 s内穿越原平台所在位置的次数, 以及在原平台所在象限的停留时间等指标进行分析。在空间探索实验结束后, 通常还需要对动物进行线索提示实验, 方法是将隐蔽平台放回水池中, 并在上面固定一面小旗子, 观察动物是否能在小旗子的提示下直接定位到平台所在的位置。如果动物无法完成这一任务, 则说明其可能存在视觉、运动能力以及逃避动机等方面的问题, 应予以剔除^[71]。

2.1.2 避暗实验

避暗实验又称被动躲避实验, 是根据动物趋暗避明的天性设计而成的行为学实验, 是除水迷宫之外另外一种评价动物认知能力的经典方法。该实验基本原理是给予动物电击等厌恶型刺激, 动物必须在训练中形成记忆, 克服天性并采取被动躲避的方法, 才能避免受到伤害^[72]。避暗实验的实验装置由大小相同的明暗箱、穿梭门、电栅和电刺激器组成。其中明暗箱和穿梭门的尺寸因动物大小而异, 箱体长宽多在15~30 cm之间, 暗箱底面由直径为3~5 mm的不锈钢电栅组成, 其间隔多为1~1.5 cm, 可供动物脚掌同时跨列站立^{[73, 74]}。避暗实验分为适应期、训练期和测试期3个阶段。适应期的目的是让动物熟悉明暗箱环境, 将动物从明箱放入, 5~10 s之后穿梭门打开, 让动物在明暗箱之间自由穿梭探索3~5 min。适应期结束30 min后进入训练期, 此时将动物再次放入明箱, 当动物出于天性进入暗箱之后, 穿梭门关闭, 同时电栅通电, 给予动物一定强度的电流刺激。持续大约20 s后, 将动物从暗箱中取出, 放回至笼中饲养。训练期结束24 h之后进入测试期。测试期通常持续5 min, 此时将动物再次放入明箱, 5~10 s之后穿梭门开启, 记录动物的步入潜伏期(第1次进入暗箱的时间)、进入暗箱的穿梭次数和在暗箱中停留时间, 以供后续分析^{[73, 74]}。

需要注意的是, 在避暗实验中, 电击刺激的强弱至关重要。较弱的电击刺激无法使动物形成有效的记忆, 而过高的刺激则有可能对动物造成损伤。通常电击刺激以刚好能引起小鼠退缩或发声的强度为最佳, 常用的参数包括1 mA, 50~60 Hz, 持续时间1.5~2 s^{[73, 75]}。此外, 部分实验还会采取对动物进行2次训练的方法, 以达到更好的记忆效果^[73]。

2.1.3 新物体识别实验

新物体识别实验是利用啮齿类动物天生喜欢探索新奇物体的本能而设计出的行为学实验, 在动物完全自发的条件下进行^[76]。其装置由开口向上的立方体盒子、形状或尺寸不同的新旧物体、摄像系统以及轨迹分析系统组成。其中立方体盒子的底面长宽尺寸多为40~60 cm, 侧壁多用深色材料围成, 以降低动物在实验中的紧张情绪。新物体实验分为适应期、熟悉期和测试期3个阶段。适应期时将动物放入没有任何物体的立方体盒子中, 使其自由探索并适应实验环境。熟悉期时在底板上平行于一边放置两个完全相同的物体, 将动物背对两物体从另一边中点位置放入立方体盒子中, 令其自由探索并熟悉两个完全相同的物体, 一段时间后将动物取出, 放回至原饲养环境。熟悉期结束一定时间之后进入测试期。此时用一个形状或尺寸不同的新物体代替其中一个旧物体, 并再次将动物放入立方体盒子中, 分别记录其对新旧两个物体的探索时间。动物对新旧物体的探索活动通常规定为其鼻尖在≤2 cm内指向物体或直接接触物体时进行的行为活动, 而当动物坐卧在或倚靠在物体上时则不认为是对物体的探索^[76]。将动物对新物体的探索时间用N表示, 对旧物体的探索时间用F表示, 最终动物对新物体的偏好程度可以用新旧物体的探索时间之差(N-F)和对新物体的辨别指数N/(N+F)等指标表示。

需要注意的是, 在新物体识别实验中, 熟悉期和测试期的持续时长均会影响动物最终对新物体的探索。有研究显示, 当熟悉期为2 min时, 多数大鼠不足以对旧物体充分熟悉, 因此不会表现出对新物体的特殊偏好。而当熟悉期延长至3 min时, 大鼠对新物体的探索将明显增加^[77]。另一方面, 在测试期时, 随着时间的推移, 动物对新物体的存在由最初的新奇逐渐变为熟悉, 因此不会对新物体表现出持续的偏好。当前, 多数实验的熟悉期和测试期多选在3~5 min之间, 可得到较为可靠的实验结果^{[78, 79]}。除此之外, 熟悉期和测试期之间的时间间隔也应根据实验情况合理选择。对于东莨菪碱诱发的记忆损伤模型, 通常在熟悉期结束1 h之后进行测试, 而对于非药物因素引起的记忆自然损伤模型, 则多选用24 h作为间隔^[80]。

2.2 　Aβ检测

2.2.1 　Aβ的生化测定

现阶段, 酶联免疫吸附法(enzyme-linked immunosorbent assay, ELISA)是测定组织匀浆、血浆、脑脊液中Aβ含量的最常用手段。该方法快速、精准、灵敏度高, 可以在数小时至一天的时间内完成测定^[81]。其基本原理多为双抗体夹心法, 主要由检测抗体和捕获抗体构成。根据抗体识别的Aβ羧基末端抗原表位的不同, 可区分Aβ₄₀与Aβ₄₂, 具有良好的特异性。此外, 在提取分离样品时, 根据Aβ构型及存在部位的差异, 可分为水溶性Aβ、去垢剂溶解性Aβ以及甲酸溶解性Aβ, 分别对应细胞外可溶性Aβ单体或寡聚体, 细胞内或跨膜可溶性Aβ单体或寡聚体以及细胞外不溶性Aβ斑块^{[82, 83]}。因此, 在进行检测时, 应该充分考虑到Aβ类型、聚集状态及存在部位, 选择识别特定表位的抗体和样品处理方法, 以便更加全面地评价受试药物对动物模型Aβ含量的影响。

2.2.2 老年斑的组织染色

除ELISA方法外, 还可通过对老年斑进行染色, 观察其在组织中的含量及定位。目前, 老年斑(senileplaque, SP)的染色方法主要有银染、刚果红染色、硫磺素染色(thioflavine)以及免疫组化染色等。其中银染法(如改良Bielschowsky和Gallya's银染法等)是SP染色的经典方法, 该方法可以显示Aβ沉积形成的初级斑和神经突斑, 但不能显示弥漫斑和终末斑, 这可能和这两种斑内缺乏嗜银结构有关^[84]。此外银染法价格昂贵, 耗时长, 并且染色条件不易控制。刚果红是一种可以选择性标记SP中β折叠的物质, 与淀粉样物质结合后能够在偏光镜下和普通光镜下分别呈现特异的苹果绿色荧光信号和桔红色信号。然而, 刚果红染色法只能识别紧密的、纤维外观的Aβ沉积, 无法检测松散、弥漫的Aβ单体或寡聚体^[85]。硫磺素T (thioflavin T, Th-T)是一种广泛用于组织学染色及蛋白聚集检测的荧光染料, 与刚果红类似, Th-T可以特异性地识别淀粉样沉淀中的β折叠结构。与其结合之后, Th-T的发射波长由445 nm增加至482 nm, 荧光强度增加至原先的5~50倍, 因此非常适合于脑组织中SP的标记与测定^[86]。除上述三种方法外, 脑组织中的SP染色还可以通过免疫组化方法实现。目前常用的抗Aβ抗体包括6E10、4G8等, 由于该方法是对Aβ的特征性序列进行识别, 因而能够显示出更多数量的Aβ沉积, 包括微小的弥散斑和终末斑, 是一种更加精准的Aβ组织染色方法。

2.3 　Tau蛋白检测

2.3.1 　Tau蛋白及相关激酶的生化测定

研究显示, Tau蛋白不同位点的磷酸化对其功能改变有很好的提示作用。其中, Tau蛋白微管结合区残基(244~368位点)的磷酸化, 对调节微管稳定性至关重要, 特别是Ser262、Ser356位点的磷酸化, 可以改变Tau蛋白与微管结合部位的构象, 导致微管解体, 细胞骨架破坏。此外, Ser214、Thr231等微管结合区以外的位点同样参与了细胞骨架稳定的调节^[87]。另一方面, Tau在不同位点的磷酸化情况可以反映疾病的进程。在疾病早期阶段, NFTs尚未形成, Tau的磷酸化位点主要发生在Ser199、Ser202、Ser409和Ser422等位点^[88], 随着疾病的进展, Ser202和Thr205位点的磷酸化将不断增强, 促进纤维的进一步生成, 而Ser396、Ser404和Thr231位点的磷酸化, 则标志着有更多的成熟的p-Tau组装形成NFTs, 推动疾病进入晚期阶段^[89]。Tau蛋白的磷酸化受到蛋白激酶和蛋白酯酶的活性调控。目前已经鉴定出十几种参与Tau蛋白异常磷酸化的激酶, 其中研究最为深入的是糖原合成激酶-3β (glycogen synthase kinase-3 beta, GSK-3β)和周期蛋白依赖性激酶-5 (cyclin dependent kinase 5, CDK-5)^[90]。研究表明, GSK-3β在AD的发病中起到关键作用, 其活性的异常升高可能与记忆减退、Aβ生成及Tau蛋白过度磷酸化均有重要关系^[90]。作为一种高度保守的丝/苏氨酸蛋白激酶, GSK-3β拥有众多作用位点, 参与了Tau蛋白Ser199/202、Thr231、Ser396和Ser404等位点的磷酸化。与GSK-3β类似, CDK-5在生物体内多条信号通路中都发挥重要作用, 调控的Tau蛋白磷酸化位点包括Ser202、Thr205、Ser235和Ser404^[90]。因此, 应该根据动物模型的发病阶段及受试药物的潜在作用机制, 选择适当的磷酸化位点进行检测。

2.3.2 　NFT的组织染色

　NFTs由双螺旋细丝(paired helical filament, PHF)和直丝两种纤维组成, 可以利用银染、thioflavin-S以及免疫组化等多种方法对NFTs进行染色观察。一些早期的文献曾对不同的染色方法进行过比较^[91-93], 并总结了一系列常见问题, 包括特异性差、容易造成组织损伤以及染色过程费时等。Vallet等^[92]曾对传统的thioflavin-S染色方法做出改良, 极大地提高了染色信号强度。之后, Sun等^[94]对该方法进一步优化, 并在同一张切片上对再次优化后的thioflavin-S染色法、Gallyas银染法和免疫组化染色法进行了比较。结果显示, 再次优化后的thioflavin-S染色法可以显著改善切片破损、脱落等现象, 并降低了切片背景, 减少了荧光猝灭现象的发生, 因而更加适用于常规的实验操作。相比之下, Gallyas银染法虽展现出了相同的灵敏度和特异性, 但其稳定性较差, 且无法进行多重染色。而免疫组化染色虽然可以区分不同亚型的神经纤维缠结, 也可以进行多重标记, 但是该方法灵敏度较差, 且对不同脑区的染色结果存在很大差异。这可能是由于免疫组化所用抗体是对特殊位点进行识别, 而NFTs在形态学和免疫化学上具有较高的异质性, 且在后期加工过程中经过水解, 折叠等多重处理, 造成较多抗原位点丢失所致。

2.4 神经炎症检测

除了异常聚集的Aβ和过度磷酸化的Tau之外, 持续加重的神经炎症反应也是AD的病理特征之一。近年来研究发现, 在AD患者和动物模型脑组织中的Aβ斑块附近, 均存在着大量激活态的小胶质细胞^[95]。在疾病早期, 激活的小胶质细胞可以摄取并清除Aβ, 减少斑块的形成和聚集。然而随着疾病的进展, 过度激活的小胶质细胞将由M2向M1型转化, 释放大量肿瘤坏死因子α (tumor necrosis factor alpha, TNF-α)、白介素1β (interleukin 1 beta, IL-1β)等神经炎症因子, 造成组织损伤, 并影响相关受体的表达, 阻碍其对Aβ的识别和吞噬, 进而加重Aβ的聚集^[96]。此外, 这些持续释放的神经炎症因子将进一步激活未活化的小胶质细胞, 连同活性氧(reactive oxygen species, ROS)、一氧化氮、前列腺素、补体分子等炎症介质, 不断放大神经炎症反应, 造成恶性循环。由于神经炎症反应贯穿AD的整个病理进程, 许多非甾体类抗炎药物, 如布洛芬、罗非昔布、塞来昔布等都曾用于AD的临床研究^[97]。我国传统中医药中的许多天然产物对神经炎症都有很好的抑制作用, 也逐渐成为当前研究的热点。在非临床药效学研究中, 常常利用免疫荧光共染的方法观察Aβ沉积和胶质细胞的共定位, 此外也可以通过Western blot、ELISA或流式细胞术等方法对脑组织中炎症因子、补体因子及各种免疫细胞的表达水平进行分析, 或研究TLR4-p38、TLR4-NF-κB、C1q-C3等通路中相关信号分子的表达, 以更好地揭示受试药物对神经炎症的调控作用。

2.5 氧化损伤检测

氧化损伤是指机体的氧化还原平衡失调, 代谢产生的ROS和活性氮簇(reactive nitrogen species, RNS)等分子大量堆积, 进而对机体造成的损伤, 在AD等神经退行性疾病中发挥着重要的作用。研究显示, AD患者和动物模型脑内氧化应激平衡的改变要早于Aβ斑块形成, 而Aβ则可以与锌、铜、铁等金属离子结合, 产生更多的ROS, 进一步扩大氧化损伤^[98]。在转基因动物模型中, 自由基清除剂依达拉奉能够显著减少Aβ沉积, 减轻Tau磷酸化, 并抑制神经炎症的发生^[99]。而部分天然产物来源的抗氧化剂, 包括姜黄素、槲皮素、鱼肝油等也曾用于AD的临床研究^[100]。

在非临床研究中, 反映组织氧化损伤的标志物主要包括脂质过氧化产物丙二醛(3, 4-methylenedioxyamphetamine, MDA)、4-羟基壬烯醛(4-hydroxynonenal, 4-HNE)、蛋白质羰基化和硝基化产物、以及DNA过氧化产物8-羟化脱氧鸟苷(8-hydroxy-deoxyguanosine, 8-OHdG)等。此外, 亦有研究通过检测超氧化物歧化酶(superoxide dismutase, SOD)、过氧化氢酶(catalase, CAT)、谷胱甘肽-过氧化氢酶(glutathione peroxidase, GSH-PX)、谷胱甘肽(glutathione, GSH)以及维生素E等抗氧化酶或抗氧化物质的含量, 来反映受试药物对于机体氧化还原平衡的改善作用^[101]。近年来, Nrf2-ARE信号通路在抗氧化损伤方面的调控作用被逐渐揭示, 为基于该靶点的抗氧化剂研发和机制研究提供了新的思路^[102]。

2.6 神经递质检测

研究显示, AD的发病与多种神经递质的代谢紊乱相关, 包括乙酰胆碱、谷氨酸、多巴胺、5-羟色胺和γ-氨基丁酸等。此外, 甘氨酸、肾上腺素、去甲肾上腺素、褪黑素等神经递质则可能通过影响体内氧化应激状态, 参与到AD的发病进程中^[103]。研究显示, 给予AD转基因动物胆碱酯酶抑制剂或直接注射5-羟色胺能够抑制Aβ的聚集, 并改善其认知障碍^{[104, 105]}。实际上, 目前被FDA批准用于治疗AD的5个药物均以神经递质为靶点研发, 分别是胆碱酯酶抑制剂他克林、多奈哌齐、加兰他敏、利凡斯的明以及NMDA受体拮抗剂美金刚。鉴于各种神经递质对认知功能的调控以及脑损伤的保护作用, 目前以其为靶点的抗AD药物研发依然是主要策略之一, 临床上进展较快的包括α7-nAChR烟碱乙酰胆碱受体激动剂(nicotinic acetylcholine receptors, nAChR) ABT-126、组胺H3受体拮抗剂ABT-288以及5-羟色胺5-HT4R受体激动剂PRX-3140等^[103]。

考虑到神经递质在生物组织样品中含量低、荧光信号和质谱信号弱以及众多内源性物质的干扰, 建立快速高效的样品前处理方法和灵敏的检测手段对于神经递质的直接测量至关重要。目前, 常用的样品前处理方法包括固相微萃取(solid phase microextraction, SPME)、液相微萃取(liquid-phase microextraction, LPME)以及分散液-液微萃取(dispersive liquid-liquid microextraction, DLLME)技术等, 常用的检测手段包括高效液相色谱法(high-performance liquid chromatography, HPLC)、高效毛细管电泳法(high-performance capillary electrophoresis, HPCE)、气相色谱-质谱联用法(gas chromatography-mass spectrometry, GC-MS)以及液相色谱-质谱联用法(liquid chromatography-mass spectrometry, LC-MS)^[106]。此外, 亦有研究采用电化学检测方法以及基于光学传感器和生物传感器的检测方法对生物组织内的神经递质进行检测, 具有低检出限和实时测量的优点^{[107, 108]}。另一方面, 对于靶向神经递质受体和相关代谢酶的抗AD药物, 则需要考虑其靶点在中枢或外周组织中的特异性问题及不同亚型之间的选择性问题, 以尽可能减少药物脱靶等不良反应的发生。

2.7 神经再生与突触可塑性的检测

在AD的病理学改变中, 神经元和突触丢失被认为与认知障碍最为相关。近年来研究显示, AD转基因小鼠海马齿状回神经再生减少, 前体细胞向神经元的分化比例降低, 同时新生神经元的存活率降低并出现突触异常^[109]。此外, Aβ可抑制体外培养的神经干细胞(neural stem cell, NSCs)增殖分化, 并诱导其凋亡, 而Aβ免疫治疗或NSCs移植治疗可促进转基因小鼠脑内的神经再生, 并改善海马代谢^{[110, 111]}。因此, 以促进神经再生和突触可塑性为靶点, 寻找药物调节海马神经干细胞的增殖、迁移、存活和向神经元的分化, 可能在治疗AD方面存在重大意义。

在目前的非临床研究中, 常通过免疫组化或免疫荧光的方法检测bromodeoxyuridine (BrdU)、Ki-67、doublecortin (DCX)等细胞增殖标志物的含量, 来反映药物对神经再生的影响。其中BrdU是尿嘧啶核苷的衍生物, 可在DNA复制时取代胸腺嘧啶核苷整合入细胞的DNA中, 标记处于S增殖期的细胞。Ki67作为一种增殖细胞相关的核抗原, 与有丝分裂密切相关, 在细胞增殖的G1、S、G2、M期均可表达^[112]。DCX作为一种微管相关蛋白, 在成熟神经元中无表达, 在干细胞及神经元前体细胞中高度表达, 是研究神经元迁移、分化的重要标志物^[113]。在突触可塑性方面, LTP一直以来被认为是学习记忆的基础, 是非临床研究中评价突触功能可塑性的重要指标^[114]。高尔基染色可以观察到神经元形态上的微小改变, 对树突的分枝、结构以及树突棘的形态和数量进行定量分析, 是研究突触形态可塑性的经典方法^[115]。此外, 诸如突触后密度蛋白95 (postsynaptic density protein 95, PSD-95)、生长相关因子43 (growth associated protein 43, GAP-43)、synaptophysin等突触相关蛋白的含量也是非临床研究的重点, 其变化可以很好地反映轴突生长、突触重建及神经递质释放的过程, 也是评价调节突触可塑性的重要指标。

2.8 肠道菌群检测

近年来, 肠道菌群失调与AD发病之间的关系成为了研究的热点。肠道菌群构成了人体微生物总量的95%以上, 可以通过分泌细胞因子、激素以及神经递质等信号物质与肠道和大脑进行双向交流, 影响着机体免疫、大脑发育以及行为认知^[116]。研究显示, AD患者肠道菌群中起促炎作用的埃希氏杆菌和志贺氏菌的比例明显上调, 而起抗炎作用的直肠真杆菌比例降低^[117]。此外, AD患者粪便中菌群的多样性减少, 并伴有厚壁菌和双歧杆菌比例的降低, 以及拟杆菌比例的升高^[118]。这些变化会引起有害代谢产物在外周血中的积累, 促进神经炎症发生和外周免疫细胞浸润, 造成认知功能损伤^[116]。而给予AD转基因小鼠益生菌治疗则能够恢复其肠道菌群的种类和数量, 降低其血清中炎症因子的表达水平, 减少其脑内Aβ的聚集^[119]。直接给予AD转基因小鼠同窝野生型小鼠的粪菌能够改善其脑部的Aβ和Tau相关病变, 逆转突触损伤并改善认知功能障碍^[120]。

目前, 以调控肠道菌群为靶点的干预手段主要包括益生菌治疗、抗生素治疗、粪便转移治疗以及饮食治疗等^[121]。在非临床实验中, 除了从改善神经炎症、氧化应激、Aβ损伤和认知功能障碍等角度考察上述治疗的干预作用外, 通常还会检测动物模型肠道菌群的种类和各自的丰度, 其中最常用到的方法包括基于蛋白质分析的质谱检测技术, 以及近年来不断兴起的基于16S rRNA的高通量测序技术^[122]。后者通过将测序得到的结果与Silva、RDP、Greengene等已知的数据库信息进行比对, 可以对不同来源的微生物样本进行多样性分析, 得到微生物种类、相对丰度、不同组分间的物种差异以及系统进化等信息。此外, 亦有研究采用宏基因组测序手段, 对样品中全部微生物的总DNA进行高通量扩增测序, 配合16S rRNA的测序结果, 更加高效、准确地研究微生物群落组成的多样性及功能^[123]。

3 抗AD药物非临床研究的合理化建议

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为提高抗AD药物的临床转化效率, 降低新药研发成本, 作者认为在当前的非临床研究中, 至少可以从以下三个方面进行改善: ①实验动物和动物模型的选择。在临床研究中, 患者的招募有严格的准入和排除标准。通常需要在国际工作组织(International Work Group, IWG)或美国国立老化研究所与阿尔茨海默病协会(National Institute on Aging-Alzheimer's Association workgroup, NIA-AA)提出的AD诊断标准基础上, 结合简易智力状态评估量表(mini-mental state examination, MMSE)、正电子发射断层扫描(position emission computed tomography, PET)、脑脊液生物标志物检测等手段对患者的病情进行区分, 并排除诸如血管性痴呆、帕金森痴呆、路易体痴呆等其他可能因素的影响, 以最大程度地减少受试者的异质性。因此在非临床研究中, 同样应该充分了解各个动物模型的特点, 并尽可能控制动物年龄、体重、身体状况、饲养条件等无关变量, 以确保样本的均一性。此外, 在选择转基因动物进行研究时, 还应严格把控动物的发病月龄, 若条件允许, 还可以对转入基因的表达含量进行检测, 以对动物的发病程度进行区分。另一方面, 考虑到AD复杂的病理机制及当前各种动物模型的局限性, 在非临床研究中, 建议采用3种以上动物型进行评价, 以更加全面地反映受试药物的治疗效果和潜在作用机制。②研究方案设计。在临床研究中, 主要终点一般为患者认知功能的改善, 选择的评价指标通常为老年痴呆评定量表-认知分量表(Alzheimer's disease assessment scale-cognitive section, ADAS-cog)、临床痴呆评分总和量表(clinical dementia rating-sum of boxes, CDR-SB)、阿尔茨海默病协作研究日常生活能力量表(Alzheimer's disease cooperative study-activities of daily living, ADCS-ADL)、MMSE量表中的一种。而次要终点通常包括主要终点以外的其他评定量表的基线改变, 以及血浆或脑脊液中Aβ_1-40/42、Tau蛋白含量, Aβ PET扫描, 磁共振成像(magnetic resonamc imaging, MRI)检测等指标。与之对应, 在非临床研究中, 对受试药物的评价依然以认知改善作用为主, 因此作者建议至少选择3种行为学实验进行认知评估, 包括水迷宫、T迷宫、放射状迷宫、条件性恐惧、新物体识别、跳台实验、避暗实验等。对于认知以外的部分, 可选用受试药物的潜在靶标或AD公认的特征病理改变进行检测, 作为非临床研究的次要终点。此外, 非临床研究也可以借鉴临床研究中的多中心方案, 在2个以上临床前研究中心进行。临床前多中心试验应当制定并遵循统一的标准, 以保证各中心研究的同质性, 这是对样本进行加和统计的前体条件。虽然多中心临床前试验会增加一定的成本, 但能为后期临床试验的成功提供更大的保障。③数据分析与统计。在临床研究中, 各项独立研究结果常常出现不一致或无统计学差异的情况, 此时采用Meta-分析对数据进行汇总, 可以达到增大样本量, 提高结果可信度的目的。同样地, 非临床研究大多在单个中心进行, 且每组动物数目少, 可重复性差, 在出现相互矛盾的实验结果时, 也可借助Meta-分析方法对多个研究结果的异质性进行分析, 找出差异原因, 判断受试药物是否在临床前研究中有效, 以及是否有必要进入临床研究。另一方面, 与Meta-分析对同质试验进行汇总的思路不同, 亚组分析通常对部分研究对象(亚组)进行分析, 以确定最有可能从治疗中获益的人群。近期, 百健公司正是通过对EMERGE临床试验中接受高剂量(10 mg·kg^-1) aducanumab治疗患者的CDR-SB、MMSE、ADAS-Cog13等评分量表和Aβ PET扫描结果进行亚组分析, 才得出显著性结论, 决定重新启动对aducanumab的申请程序。在非临床研究中, 这一思路同样适用, 在统计结果未出现显著性差异时, 可进一步结合动物模型相关生物标志物的表达水平、药物暴露程度、或药物在动物体内的代谢情况进行亚组分析, 以得到更加科学合理的实验结果。但需要注意的是, 亚组分析一定要正确运用, 且不应对结果过分解读, 否则会出现假阳性实验结果, 给临床资源带来更大的浪费。

4 我国抗AD药物的研发现状

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我国在抗AD药物开发中具有许多独特的优势。一方面, 我国动植物资源丰富, 许多具有抗炎、抗氧化以及神经营养作用的天然产物在AD模型中表现出了认知改善作用, 已成为当前抗AD新药研发的热点。另一方面, 我国老龄化问题日益严重, 为临床试验提供了丰富的患者资源。近期, 国家食品监督管理局有条件批准了甘露特钠胶囊GV-971的上市注册申请, 用于轻度至中度阿尔茨海默病的治疗。临床前研究表示, GV-971能够通过重塑肠道菌群平衡, 降低外周相关代谢产物苯丙氨酸/异亮氨酸的积累, 抑制促炎性辅助T细胞(Th1)和M1型小胶质细胞的活化, 减轻脑内神经炎症, 进而改善认知障碍^[124]。此外, 部分处于临床阶段的1类抗AD新药还包括琥珀八氢吖啶、芬克罗酮、左黄皮酰胺以及AD-35等。

5 小结

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当前, 抗AD新药研发面临严重的考验, 其失败率高达99.6%。许多受试药物在动物模型中表现出的治疗效果无法向临床成功转化, 表明在药物非临床药效学评价中, 依然存在诸多动物模型和评价方法学上的缺陷。因此, 在新药研发的非临床阶段, 建立标准化的药效学评价体系至关重要, 可以提高临床研究的转化效率, 避免不必要的损失。鉴于目前AD的发病机制尚不明确, 许多靶向药物尚未通过临床试验的验证, 因此, 以检测认知功能为主的行为学实验依然是最直接的药效学评价方法, 也是目前为止最安全、常用的检测手段。而动物模型上的缺陷则需要研究人员对各个模型的特点充分了解, 合理选择并结合多种模型的结果对药效学进行全面的评价。表 2^[125-129]对当前FDA批准的用于AD治疗的药物在非临床研究中采用的动物模型及评价指标进行了汇总, 以期为各类在研抗AD药物的非临床开发提供参考。

基金

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国家自然科学基金资助项目(81673420)
国家“重大新药创制”科技重大专项(2018ZX09711001-003-005)
国家“重大新药创制”科技重大专项(2018ZX09711001-003-009)
北京市医院管理局青苗计划专项经费资助(QML20170902)
中国医学科学院医学与健康科技创新工程(2017-I2M-2-004)

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2020年第55卷第5期

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doi: 10.16438/j.0513-4870.2019-0899

接收时间：2019-11-11
首发时间：2026-01-21
出版时间：2020-05-12

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收稿日期：2019-11-11
修回日期：2019-12-06

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国家自然科学基金资助项目(81673420)

国家“重大新药创制”科技重大专项(2018ZX09711001-003-005)

国家“重大新药创制”科技重大专项(2018ZX09711001-003-009)

北京市医院管理局青苗计划专项经费资助(QML20170902)

中国医学科学院医学与健康科技创新工程(2017-I2M-2-004)

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1.中国医学科学院、北京协和医学院药物研究所, 北京 100050

2.国家药品监督管理局药品审评中心, 北京 100022

3.清华大学附属北京清华长庚医院神经内科, 北京 102218

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*王庆利, Tel:86-10-83165742, Fax:86-10-63017757, E-mail:wangql@cde.org.cn;

彭英, Tel:86-10-68585566, Fax:86-10-68584189, E-mail:ypeng@imm.ac.cn

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2种不同金属材料的力学参数

科 Family	属数 Number of genus	种数 Number of species	占总种数比例 Percentage of total species (%)	属 Genus	种数 Number of species	占总种数比例 Percentage of total species (%)
鹅膏菌科Amanitaceae	2	11	5.26	鹅膏菌属 Amanita	10	4.78
小菇科 Mycenaceae	2	12	5.74	丝盖伞属 Inocybe	5	2.39
多孔菌科 Polyporaceae	8	14	6.70	蜡蘑属 Laccaria	5	2.39
红菇科 Russulaceae	3	23	11.00	小皮伞属 Marasmius	6	2.87
				小菇属 Mycena	11	5.26
				光柄菇属 Pluteus	5	2.39
				红菇属 Russula	17	8.13
				栓菌属 Trametes	5	2.39

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TxT

Model	Mutation	Aβ plaques	NTFS	Other pathology	Cognitive deficits
PDAPP	APP^V717F	Both diffuse and cored Aβ plaques since 6-9 months in hippocampus, corpus callosum, and cerebral cortex^[3]	None	Astrocytosis and microgliosisassociated with plaques^[3]. Synaptic loss and altered LTP induction^[3]	Robust deficits in the radial arm maze at 3 months and object recognition at 6 months^[4]
Tg2576	APP^K670N/M671L	Numerous parenchymal Aβ plaques since 9 months^[5]	None	Dendritic spine loss by 4.5 months and decline in LTP in the dentate gyrus after performant path stimulation^[6]	Impairment in contextual fear conditioning by 5 months and Morris water maze after 9 months^{[5, 6]}
APP23	APP^K670N/M671L	Congophillic, dense-core plaques since 6 months^[7]	None	Neuronal loss, microglia activation, and hyperphosphorylated Tau associated with plaques^[7]	Spatial memory defects in Morris Water maze at 3 months and progresses with age^[8]
APP/PS1	APP^K670N/M671L PSEN1^dE9	Abundant plaques in the hippocampus and cortex since 9 months^[11]	None	Neuronal loss and activated astrocytes associated with plaques. Age-dependent loss of synapse and impaired LTP induction^[10]	Impairment in the Morris water maze at 12 months and commit more errors at 13 months^[10]
5×FAD	APP^K670N/M671L APP^I716V APP^V717I PSEN1^M146L PSEN1^L286V	Extracellular Aβ deposition begins around 2 months. Intraneuronal Aβ also accumulates in an aggregated form within the soma and neurites starting at 1.5 months^[12]	None	Neuronal loss in cortical layer V and subiculum. Gliosis begins at 2 months. Synaptic loss and impaired LTP induction^{[12, 13]}	Impaired spatial working memory in Y maze at 4-5 months^[13]
JNPL3	Tau^P301L	None	NFTs develop as early as 4.5 months in homozygotes and 6.5 months in heterozygotes^[14]	Neuronal loss especially in the spinal cord and astrogliosis in brainstem, diencephalon, and basal telencephalon by 10 months^{[14, 15]}	Unknown
rTg4510	Tau^P301L	None	Pretangles as early as 2.5 months. Argyrophilic tangle-like inclusions in cortex by 4 months and in hippocampus by 5.5 months^[16]	Progressive neuronal loss in hippocampal CA1 area and significant loss of dendritic spines at 8-9 months. Impaired LTP at 4.5 months^{[10, 16]}	Impaired of spatial memory demonstrated by Morris water maze from 2.5 to 4 months. Spatial memory improved when transgene suppressed by dox^{[16, 17]}
TAPP	APP^K670N/M671L Tau^P301L	Has Aβ plaques similar in number and distribution to those of comparably aged Tg2576^[20]	NFTs were morphologically similar in TAPP and JNPL3 mice, older female TAPP mice had a marked increase in NFTs inlimbic areas, subiculum, and hippocampus^[20]	Activated astrocytes and microglia as early as 3 months in the hippocampus and increased with age^[20]	Unknown
3xTg	APP^K670N/M671L Tau^P301L PSEN1^M146L	Extracellular Aβ deposits by 6 months in frontal cortex and progress with age^[21]	By 12 months extensive Tau immunoreactivity in CA1 neurons of the hippocampus^[21]	Activated astrocytes at 7 months. Impairment in LTP and basal synaptic transmission by 6 months^[21]	Cognitive impairment manifests at 4 months as a deficit in long-term retention and correlates with the accumulation of intraneuronal Aβ in the hippocampus and amygdala^[22]

Model

Mutation

Aβ plaques

NTFS

Other pathology

Cognitive deficits

PDAPP

APP^V717F

Both diffuse and cored Aβ plaques since 6-9 months in hippocampus, corpus callosum, and cerebral cortex^[3]

None

Astrocytosis and microgliosisassociated with plaques^[3]. Synaptic loss and altered LTP induction^[3]

Robust deficits in the radial arm maze at 3 months and object recognition at 6 months^[4]

Tg2576

APP^K670N/M671L

Numerous parenchymal Aβ plaques since 9 months^[5]

None

Dendritic spine loss by 4.5 months and decline in LTP in the dentate gyrus after performant path stimulation^[6]

Impairment in contextual fear conditioning by 5 months and Morris water maze after 9 months^{[5, 6]}

APP23

APP^K670N/M671L

Congophillic, dense-core plaques since 6 months^[7]

None

Neuronal loss, microglia activation, and hyperphosphorylated Tau associated with plaques^[7]

Spatial memory defects in Morris Water maze at 3 months and progresses with age^[8]

APP/PS1

APP^K670N/M671L PSEN1^dE9

Abundant plaques in the hippocampus and cortex since 9 months^[11]

None

Neuronal loss and activated astrocytes associated with plaques. Age-dependent loss of synapse and impaired LTP induction^[10]

Impairment in the Morris water maze at 12 months and commit more errors at 13 months^[10]

5×FAD

APP^K670N/M671L APP^I716V APP^V717I PSEN1^M146L PSEN1^L286V

Extracellular Aβ deposition begins around 2 months. Intraneuronal Aβ also accumulates in an aggregated form within the soma and neurites starting at 1.5 months^[12]

None

Neuronal loss in cortical layer V and subiculum. Gliosis begins at 2 months. Synaptic loss and impaired LTP induction^{[12, 13]}

Impaired spatial working memory in Y maze at 4-5 months^[13]

JNPL3

Tau^P301L

None

NFTs develop as early as 4.5 months in homozygotes and 6.5 months in heterozygotes^[14]

Neuronal loss especially in the spinal cord and astrogliosis in brainstem, diencephalon, and basal telencephalon by 10 months^{[14, 15]}

Unknown

rTg4510

Tau^P301L

None

Pretangles as early as 2.5 months. Argyrophilic tangle-like inclusions in cortex by 4 months and in hippocampus by 5.5 months^[16]

Progressive neuronal loss in hippocampal CA1 area and significant loss of dendritic spines at 8-9 months. Impaired LTP at 4.5 months^{[10, 16]}

Impaired of spatial memory demonstrated by Morris water maze from 2.5 to 4 months. Spatial memory improved when transgene suppressed by dox^{[16, 17]}

TAPP

APP^K670N/M671L Tau^P301L

Has Aβ plaques similar in number and distribution to those of comparably aged Tg2576^[20]

NFTs were morphologically similar in TAPP and JNPL3 mice, older female TAPP mice had a marked increase in NFTs inlimbic areas, subiculum, and hippocampus^[20]

Activated astrocytes and microglia as early as 3 months in the hippocampus and increased with age^[20]

Unknown

3xTg

APP^K670N/M671L Tau^P301L PSEN1^M146L

Extracellular Aβ deposits by 6 months in frontal cortex and progress with age^[21]

By 12 months extensive Tau immunoreactivity in CA1 neurons of the hippocampus^[21]

Activated astrocytes at 7 months. Impairment in LTP and basal synaptic transmission by 6 months^[21]

Cognitive impairment manifests at 4 months as a deficit in long-term retention and correlates with the accumulation of intraneuronal Aβ in the hippocampus and amygdala^[22]

Compound	Mechanism	Animal model	Evaluation index	Outcome assessment
Donepezil	AChE inhibitor	APP23	Morris water maze	Improve cognitive function^[125]
APP/PS1	¹H-MRS	Improve metabolic profile in cortex and hippocampus^[126]
Intracerebroventricular Aβ injection	Y-maze Fear conditioning test	Improve cognitive function^[127]
Galantamine	AChE inhibitor	APP23	Morris water maze	Improve cognitive function^[125]
Intracerebroventricular Aβ injection	Novel object recognition Fear conditioning test	Improve cognitive function^[127]
Rivastigmine	AChE inhibitor	APP23	Morris water maze	Improve cognitive function^[125]
Memantine	NMDAR antagonist	Tg2576	Aβ plaque density Neuronal morphology Synapse density	Reduce plaque burden, increase synapse density and degenerating axons^[128]
3xTg	Morris water maze Novel object recognition Passive inhibitory avoidance Tau pathology Aβ accumulation LTP induction	Improve cognitive function, reduce Tau phosphorylation and Aβ accumulation, reduce levels of Aβoligomers and reverse oligomeric Aβ-induced deficits in LTP^[129]

Compound

Mechanism

Animal model

Evaluation index

Outcome assessment

Donepezil

AChE inhibitor

APP23

Morris water maze

Improve cognitive function^[125]

APP/PS1

¹H-MRS

Improve metabolic profile in cortex and hippocampus^[126]

Intracerebroventricular Aβ injection

Y-maze Fear conditioning test

Improve cognitive function^[127]

Galantamine

AChE inhibitor

APP23

Morris water maze

Improve cognitive function^[125]

Intracerebroventricular Aβ injection

Novel object recognition Fear conditioning test

Improve cognitive function^[127]

Rivastigmine

AChE inhibitor

APP23

Morris water maze

Improve cognitive function^[125]

Memantine

NMDAR antagonist

Tg2576

Aβ plaque density Neuronal morphology Synapse density

Reduce plaque burden, increase synapse density and degenerating axons^[128]

3xTg

Morris water maze Novel object recognition Passive inhibitory avoidance Tau pathology Aβ accumulation LTP induction

Improve cognitive function, reduce Tau phosphorylation and Aβ accumulation,
reduce levels of Aβoligomers and reverse oligomeric Aβ-induced deficits in LTP^[129]