Table_4_The Roles of Long-Term Hyperhomocysteinemia and Micronutrient Supplementation in the AppNL–G–F Model of Alzheimer’s Disease.xlsx

A causal contribution of hyperhomocysteinemia to cognitive decline and Alzheimer’s disease (AD), as well as potential prevention or mitigation of the pathology by dietary intervention, have frequently been subjects of controversy. In the present in vivo study, we attempted to further elucidate the impact of elevated homocysteine (HCys) and homocysteic acid (HCA) levels, induced by dietary B-vitamin deficiency, and micronutrient supplementation on AD-like pathology, which was simulated using the amyloid-based AppNL–G–F knock-in mouse model. For this purpose, cognitive assessment was complemented by analyses of ex vivo parameters in whole blood, serum, CSF, and brain tissues from the mice. Furthermore, neurotoxicity of HCys and HCA was assessed in a separate in vitro assay. In confirmation of our previous study, older AppNL–G–F mice also exhibited subtle phenotypic impairment and extensive cerebral amyloidosis, whereas dietary manipulations did not result in significant effects. As revealed by proximity extension assay-based proteome analysis, the AppNL–G–F genotype led to an upregulation of AD-characteristic neuronal markers. Hyperhomocysteinemia, in contrast, indicated mainly vascular effects. Overall, since there was an absence of a distinct phenotype despite both a significant amyloid-β burden and serum HCys elevation, the results in this study did not corroborate the pathological role of amyloid-β according to the “amyloid hypothesis,” nor of hyperhomocysteinemia on cognitive performance. Nevertheless, this study aided in further characterizing the AppNL–G–F model and in elucidating the role of HCys in diverse biological processes. The idea of AD prevention with the investigated micronutrients, however, was not supported, at least in this mouse model of the disease.

高同型半胱氨酸血症对认知衰退与阿尔茨海默病（Alzheimer’s Disease, AD）的因果贡献，以及膳食干预潜在预防或缓解该病理进程的可能性，历来是颇具争议的研究议题。在本项体内研究中，我们旨在进一步阐明由膳食B族维生素缺乏诱导的同型半胱氨酸（homocysteine, HCys）与同型半胱氨酸酸（homocysteic acid, HCA）水平升高，以及微量营养素补充对类阿尔茨海默病病理的影响——该类病理通过基于淀粉样蛋白的AppNL–G–F敲入小鼠模型进行模拟。为此，我们通过对小鼠全血、血清、脑脊液（cerebrospinal fluid, CSF）及脑组织的离体参数分析，补充完善了认知评估的相关数据。此外，我们通过独立的体外实验评估了HCys与HCA的神经毒性。与此前的研究结果一致，老年AppNL–G–F小鼠同样表现出轻微的表型损伤与广泛的脑内淀粉样蛋白沉积，但膳食干预未产生显著影响。基于邻近延伸测定（proximity extension assay, PEA）的蛋白质组分析结果显示，AppNL–G–F基因型会上调阿尔茨海默病特征性神经元标志物的表达。相较之下，高同型半胱氨酸血症主要表现为血管相关效应。总体而言，尽管小鼠同时存在显著的β淀粉样蛋白负荷与血清HCys水平升高，但未观察到明确的特征性表型；因此本研究结果既未支持"淀粉样蛋白假说"中β淀粉样蛋白的病理作用，也未证实高同型半胱氨酸血症对认知功能的负面影响。尽管如此，本研究仍有助于进一步完善AppNL–G–F模型的特征表征，并阐明了HCys在多种生物学过程中的作用。然而，至少在本疾病小鼠模型中，通过本次研究所考察的微量营养素预防阿尔茨海默病的设想并未得到验证。