Experimental menopause in 3xTg-AD mice triggers metabolic and cognitive dysfunction aligned with Alzheimer's disease processes. Experimental menopause in 3xTg-AD mice triggers metabolic and cognitive dysfunction aligned with Alzheimer's disease processes

There is a compelling link between menopause and Alzheimer's disease (AD), with few established molecular mechanisms. A lack of experimental models limits validation of causal drivers of these interactions. Here, we characterize the accelerated ovarian failure (OF) model of menopause in the triple-transgenic AD (3xTg-AD) mouse. Ovotoxin, 4-vinylcyclohexene diepoxide accelerated follicular loss, ablating circulating progesterone. OF resulted in decreased performance in the Y-maze, Novel Object Recognition, and Barnes Maze, consistent with accelerated AD. OF aggravated age-related impaired glucose tolerance and caused insulin resistance and these alterations correlated with the degree of cognitive impairment. Transcriptomic analyses of the mouse hippocampus identified 19 differentially regulated genes in OF mice, including some linked to AD, including C4b, Ifit3b, Cxcl13 and Gabrg2. We produced new transcriptomic profiles of dementia-free human entorhinal cortex (n=99) and remodeled entorhinal cortex profiles from patients (n=64; Braak scores 0-6). Analyses of our 19 OF genes, identified that C4B expression was upregulated with both age and Braak score, while several revealed novel co-expression relationships, including between Shootin (SHTN1) and GABA receptor subunit (GABRG2). In summary, accelerated ovarian failure presents key cognitive, metabolic and molecular changes associated with age-related decline in cognitive function, indicating that it can be useful for the identification of novel therapeutic targets Overall design: Female triple-transgenic AD (3xTg-AD)(26) mice were purchased through The Jackson Laboratory and the NIH supported Mutant Mouse Resource and Research Center (MMRRC). Mice were housed four animals per cage in an AAALAC-accredited animal facility at the University of Miami Miller School of Medicine. Ovotoxin vinylcyclohexene dioxide (VCD; Sigma, cat. #94956) was diluted in sesame oil (Fisher Scientific, cat #18-606-186). Animals were administered 160 mg/kg VCD (n = 19) to induce OF or sesame oil vehicle (n = 20) at a final volume of 100µL intraperitoneal (i.p.) starting at 4 months of age. Mice were injected 5 days a week for 3 weeks, for a total of 15 days. Mice representing peri-OF (Ctrl n = 9; VCD n = 8) were euthanized 90-100 d.p.i., when mice were 30-32 weeks old. Mice representing post-OF (Ctrl n = 10; VCD n = 10) were euthanized 217-224 d.p.i., approximately 48 weeks old. 28 RNA samples were successfully processed to completed CEL files passing QC.

绝经与阿尔茨海默病（Alzheimer's Disease, AD）之间存在显著关联，但已明确的分子机制寥寥无几。实验模型的匮乏限制了对二者相互作用因果驱动因素的验证。本研究对三转基因阿尔茨海默病（triple-transgenic AD, 3xTg-AD）小鼠的加速卵巢衰竭（ovarian failure, OF）绝经模型开展了系统表征。
卵泡毒素4-乙烯基环己烯二环氧化物（vinylcyclohexene dioxide, VCD）可加速卵泡丢失，消除循环孕酮水平。卵巢衰竭模型小鼠在Y迷宫、新物体识别以及巴恩斯迷宫实验中表现出认知能力下降，这与阿尔茨海默病的加速进展表型一致。卵巢衰竭还加重了年龄相关性糖耐量受损，并引发胰岛素抵抗，且上述代谢改变与认知障碍程度呈正相关。
对小鼠海马的转录组分析鉴定出19个差异调控基因，其中包括部分与阿尔茨海默病相关的基因，如C4b、Ifit3b、Cxcl13及Gabrg2。本研究生成了99份无痴呆人类内嗅皮层样本的转录组图谱，并重新分析了64份来自阿尔茨海默病患者的内嗅皮层转录组数据（Braak分期0-6）。针对上述19个卵巢衰竭相关基因的分析显示，C4B的表达随年龄及Braak分期均显著上调，另有多个基因展现出全新的共表达关联，包括Shootin（SHTN1）与γ-氨基丁酸受体亚基（GABRG2）之间的共调控关系。
综上，加速卵巢衰竭模型呈现出与年龄相关性认知功能下降相关的关键认知、代谢及分子特征，表明该模型可用于筛选全新的阿尔茨海默病治疗靶点。

总体实验设计：
雌性三转基因阿尔茨海默病（3xTg-AD）小鼠（文献标注26）购自杰克逊实验室（The Jackson Laboratory）以及美国国立卫生研究院（NIH）资助的突变小鼠资源与研究中心（Mutant Mouse Resource and Research Center, MMRRC）。小鼠以每笼4只的密度饲养于迈阿密大学米勒医学院AAALAC认证的动物设施中。
将卵泡毒素VCD（Sigma，货号#94956）用芝麻油（Fisher Scientific，货号#18-606-186）稀释，实验组小鼠以160 mg/kg的剂量腹腔注射VCD（n=19）以诱导卵巢衰竭，对照组注射等体积芝麻油溶剂（n=20），单次注射容积为100μL，给药始于小鼠4月龄时。小鼠每周注射5天，连续3周，总给药时长为15天。
围卵巢衰竭期小鼠（对照组n=9；VCD处理组n=8）于注射后90-100天（d.p.i.）实施安乐死，此时小鼠年龄为30-32周龄。卵巢衰竭后期小鼠（对照组n=10；VCD处理组n=10）于注射后217-224天（d.p.i.）实施安乐死，此时小鼠约为48周龄。最终成功制备28份RNA样本，得到了通过质量控制（QC）的CEL文件。