Human N-methyl D-aspartate receptor antibodies alter memory and behaviour in mice
Human N-methyl D-aspartate receptor antibodies alter memory and behaviour in mice
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DOI：
10.1093/brain/awu310
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发表时间：
2015-01-01
期刊：
影响因子：
14.5
通讯作者：
Dalmau, Josep
中科院分区：
文献类型：
--
作者：
Planaguma, Jesus;Leypoldt, Frank;Dalmau, Josep
抗 N-甲基 D-天冬氨酸受体 (NMDAR) 脑炎是一种严重的神经精神疾病，与显着的记忆和行为缺陷有关。患者的抗体与 NMDAR 的 GluN1（以前称为 NR1）亚基的 N 端结构域发生反应，导致培养的神经元选择性且可逆地内化细胞表面受体。这些效应和对免疫治疗的频繁反应表明抗体介导的发病机制，但迄今为止，还没有动物模型表明患者的抗体会导致记忆和行为缺陷。为了开发这样的模型，C57BL6/J 小鼠接受了与渗透泵连接的心室导管的放置，该渗透泵持续输注患者或对照脑脊液（流速 0.25 A mu l/h，14 天）。在输注期间和之后应用标准化测试，包括评估记忆的任务（开放场和V型迷宫范式中的新物体识别）、快感缺失行为（蔗糖偏好测试）、抑郁样行为（悬尾、强迫游泳测试）、焦虑（黑白、高架十字迷宫测试）、攻击性（居民-入侵者测试）和运动活动（水平和垂直）。使用共聚焦显微镜和免疫印迹分析，检查在第 5、13、18、26 和 46 天处死的动物的脑结合抗体以及抗体对总 NMDAR 簇和突触 NMDAR 簇以及蛋白质浓度的影响。这些实验表明，注入患者脑脊液但不控制脑脊液的动物会出现进行性记忆缺陷以及快感缺失和抑郁样行为，但不会影响其他行为或运动任务。记忆缺陷逐渐恶化，直到第 18 天（输注停止后 4 天），所有症状在下周内消失。伴随的脑组织研究显示，脑结合的人类抗体逐渐增加，主要是在海马体中（第 13-18 天达到最大），经过酸提取和用表达 GluN1 的人胚胎肾细胞表征后，证实该抗体具有抗 NMDAR 的能力。海马体的共聚焦显微镜和免疫印迹分析显示，总 NMDAR 簇和突触簇的密度以及总 NMDAR 蛋白浓度（第 18 天达到最大值）逐渐降低，但不影响突触后密度蛋白 95 (PSD95) 和 α-氨基-3-羟基-5-甲基-4-异恶唑丙酸 (AMPA) 受体。这些影响与记忆和其他行为缺陷同时发生，并在第 18 天后逐渐改善，症状可逆，伴随着脑结合抗体的减少和 NMDAR 水平的恢复。总体而言，这些发现建立了记忆和行为缺陷与抗体介导的 NMDAR 减少之间的联系，为去除抗体和抗体产生细胞改善神经功能提供了生物学基础，并为测试这种疾病和类似疾病的实验疗法提供了模型。
Anti-N-methyl D-aspartate receptor (NMDAR) encephalitis is a severe neuropsychiatric disorder that associates with prominent memory and behavioural deficits. Patients' antibodies react with the N-terminal domain of the GluN1 (previously known as NR1) subunit of NMDAR causing in cultured neurons a selective and reversible internalization of cell-surface receptors. These effects and the frequent response to immunotherapy have suggested an antibody-mediated pathogenesis, but to date there is no animal model showing that patients' antibodies cause memory and behavioural deficits. To develop such a model, C57BL6/J mice underwent placement of ventricular catheters connected to osmotic pumps that delivered a continuous infusion of patients' or control cerebrospinal fluid (flow rate 0.25 A mu l/h, 14 days). During and after the infusion period standardized tests were applied, including tasks to assess memory (novel object recognition in open field and V-maze paradigms), anhedonic behaviours (sucrose preference test), depressive-like behaviours (tail suspension, forced swimming tests), anxiety (black and white, elevated plus maze tests), aggressiveness (resident-intruder test), and locomotor activity (horizontal and vertical). Animals sacrificed at Days 5, 13, 18, 26 and 46 were examined for brain-bound antibodies and the antibody effects on total and synaptic NMDAR clusters and protein concentration using confocal microscopy and immunoblot analysis. These experiments showed that animals infused with patients' cerebrospinal fluid, but not control cerebrospinal fluid, developed progressive memory deficits, and anhedonic and depressive-like behaviours, without affecting other behavioural or locomotor tasks. Memory deficits gradually worsened until Day 18 (4 days after the infusion stopped) and all symptoms resolved over the next week. Accompanying brain tissue studies showed progressive increase of brain-bound human antibodies, predominantly in the hippocampus (maximal on Days 13-18), that after acid extraction and characterization with GluN1-expressing human embryonic kidney cells were confirmed to be against the NMDAR. Confocal microscopy and immunoblot analysis of the hippocampus showed progressive decrease of the density of total and synaptic NMDAR clusters and total NMDAR protein concentration (maximal on Day 18), without affecting the post-synaptic density protein 95 (PSD95) and alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors. These effects occurred in parallel with memory and other behavioural deficits and gradually improved after Day 18, with reversibility of symptoms accompanied by a decrease of brain-bound antibodies and restoration of NMDAR levels. Overall, these findings establish a link between memory and behavioural deficits and antibody-mediated reduction of NMDAR, provide the biological basis by which removal of antibodies and antibody-producing cells improve neurological function, and offer a model for testing experimental therapies in this and similar disorders.