The results of the Western blot analysis reveal that

The results of the Western blot analysis reveal that downregulated expression of the examined proteins is observed in all transgenic 5×FAD mice, but is more pronounced for 6-month-old mice (nos. 6 and 8), especially for STIM2. In 4-month-old transgenic 5×FAD mice, STIM2 expression (2 and 4) was downregulated by 20% compared with the control group. A tendency to a decreased level of PSD95 was also found for this group, but it did not reach a statistically significant difference (see Fig. 1a). STIM2 expression in 6-month-old 5×FAD mice was downregulated by more than 75%, while that of PSD95 was downregulated by 50%. Thus, we can conclude that the STIM2→nSOCE pathway was also impaired in 5×FAD mice. The amount of amyloid plaques in the brain of 5×FAD mice. Mice of this line have a strong phenotype, which manifests itself in cognitive function impairment at the age of 4–5 months, with plaque accumulation in the brain observed as early as at 4 months. Immunohistochemical staining of amyloid beta (Aβ) with Aβ-specific rock inhibitor 6E10 (Fig. 2a) was used for analyzing the amount of plaques. The staining was performed in thin sections of the brain of wild-type (WT) and 5×FAD transgenic mice aged 3.5 months. The technical characteristics of the laboratory\'s confocal microscope did not allow to image Aβ-plaques over the entire surface of the coronary section. For this reason, we decided to image the amyloid beta in five different regions of the coronary section of the mouse brain (Fig. 2a). The distribution of Aβ-plaques over the section\'s surface appeared non-uniform. In particular, it was found that the plaques were mainly present in the cerebral cortex and completely absent in the hippocampus of the mice examined (see Fig. 2a). The cerebral cortex was chosen for quantitative analysis of the number of Aβ-plaques; its C1 regions (cortex 1) were taken, with those with the highest plaque density from a 5×FAD mouse (Fig. 2b). We analyzed three sections for each three WT and 5×FAD mice. Fig. 2c shows the mean values ± SEM (p < 0.05). Staining results showed that wild type mice completely lacked amyloid plaques (diagram I in Fig. 2c), while the mean area occupied by amyloid plaques in 5×FAD mice was about 1000 µm2 (diagram II in Fig. 2c). The effect of STIM2 overexpression. Earlier studies proved using the PS1-M146V and APPKI models of familial forms of Alzheimer\'s disease and the low amyloid toxicity model that overexpression of the STIM2 protein restores the STIM2→nSOCE pathway [7–9] and thus prevents synaptic loss and delays the progression of the disease. In order to examine the possibility of delaying AD pathogenesis via STIM2 overexpression in 5×FAD mice, we conducted experiments in which said overexpression in the hippocampus of 5×FAD mice was achieved by stereotactic injection of the AAV2/1CMVSTIM2WThrGFP virus into the CA1 hippocampal region of 2-month-old mice. The AAV2/1CMVNLS-GFP virus was used as control. Like the first virus, it was injected to three transgenic and three WT 5×FAD mice. After six weeks (when the mice reached the age of 3.5 months) the amount of amyloid plaques was analyzed by immunohistochemical staining of thin brain sections (Fig. 3). The results of the experiments shown in Fig. 3 clearly demonstrate that STIM2 overexpression reduces the amount of Aβ-plaques in the cerebral cortex of transgenic 5×FAD mice by 3 times.
Conclusion In this paper, we have studied the peculiarities of the pathogenesis of Alzheimer\'s disease in 5×FAD mice. These mice have five mutations in their genotype, with two of them causing impaired function of the PSEN1 protein, and three encoding mutant human APP. Thus, 5×FAD mice exhibit two toxicities: amyloid and presenilin. Mice of this line display cognitive impairment and memory deficits as early as at 4 months of age, and emergence of amyloid plaques was detected even earlier, at the age of 2 months [10,11]. These plaques are accumulations of insoluble oligomeric forms of amyloid beta A β 42 and are highly toxic to the neurons surrounding them.