Cerebral Aβ deposition precedes reduced cerebrospinal fluid and serum Aβ42/Aβ40 ratios in the AppNL-F/NL-F knock-in mouse model of Alzheimer's disease

Abstract

Background: Aβ42/Aβ40 ratios in cerebrospinal fluid (CSF) and blood are reduced in preclinical Alzheimer's disease (AD), but their temporal and correlative relationship with cerebral Aβ pathology at this early disease stage is not well understood. In the present study, we aim to investigate such relationships using App knock-in mouse models of preclinical AD.

Methods: CSF, serum, and brain tissue were collected from 3- to 18-month-old App^NL-F/NL-F knock-in mice (n = 48) and 2-18-month-old App^NL/NL knock-in mice (n = 35). The concentrations of Aβ42 and Aβ40 in CSF and serum were measured using Single molecule array (Simoa) immunoassays. Cerebral Aβ plaque burden was assessed in brain tissue sections by immunohistochemistry and thioflavin S staining. Furthermore, the concentrations of Aβ42 in soluble and insoluble fractions prepared from cortical tissue homogenates were measured using an electrochemiluminescence immunoassay.

Results: In App^NL-F/NL-F knock-in mice, Aβ42/Aβ40 ratios in CSF and serum were significantly reduced from 12 and 16 months of age, respectively. The initial reduction of these biomarkers coincided with cerebral Aβ pathology, in which a more widespread Aβ plaque burden and increased levels of Aβ42 in the brain were observed from approximately 12 months of age. Accordingly, in the whole study population, Aβ42/Aβ40 ratios in CSF and serum showed a negative hyperbolic association with cerebral Aβ plaque burden as well as the levels of both soluble and insoluble Aβ42 in the brain. These associations tended to be stronger for the measures in CSF compared with serum. In contrast, no alterations in the investigated fluid biomarkers or apparent cerebral Aβ plaque pathology were found in App^NL/NL knock-in mice during the observation time.

Conclusions: Our findings suggest a temporal sequence of events in App^NL-F/NL-F knock-in mice, in which initial deposition of Aβ aggregates in the brain is followed by a decline of the Aβ42/Aβ40 ratio in CSF and serum once the cerebral Aβ pathology becomes significant. Our results also indicate that the investigated biomarkers were somewhat more strongly associated with measures of cerebral Aβ pathology when assessed in CSF compared with serum.

Keywords: Alzheimer’s disease; Beta-amyloid; Biomarker; Blood; Cerebrospinal fluid.

Fig. 1

CSF and serum Aβ42/Aβ40 ratios in App^{NL−F/NL−F} knock-in mice. CSF and serum Aβ42/Aβ40 ratios were measured in 3 (n = 9)-, 6 (n = 7)-, 9 (n = 8)-, 12 (n = 7)-, 16 (n = 8)-, and 18 (n = 9)-month-old App^{NL−F/NL−F} knock-in mice. A The Aβ42/Aβ40 ratio in CSF showed a significant reduction from 12 months of age while B the  Aβ42/Aβ40 ratio in serum was significantly declined from 16 months of age. C The Aβ42/Aβ40 ratio in serum correlated significantly positive with the corresponding ratio in CSF. Data is presented as median and IQR. Whiskers represent data within 1.5IQR of the lower and upper quartiles. For comparison between groups, statistical analyses were performed using the Kruskal–Wallis H test followed by the Mann–Whitney U test for post hoc group comparisons (*p < 0.05, **p < 0.01, ***p < 0.001). Correlation analysis was performed using Spearman’s rank-ordered correlation coefficient. Abbreviations: Aβ, amyloid beta; CSF, cerebrospinal fluid; IQR, interquartile range

Fig. 2

Cerebral Aβ plaque burden in App^{NL−F/NL−F} knock-in mice. Cerebral Aβ42 immunoreactivity and thioflavin S-positive fibrillar dense-core plaques were measured in 3 (n = 9)-, 6 (n = 7)-, 9 (n = 8)-, 12 (n = 7)-, 16 (n = 8)-, and 18 (n = 9)-month-old App^{NL−F/NL−F} knock-in mice. Minor initial deposition of extracellular Aβ plaques in cortical brain regions was observed from 6 months of age. The burden of cortical and hippocampal A Aβ42 immunoreactivity and B thioflavin S-positive fibrillar dense-core plaques was significantly increased in an age-dependent manner. Data is presented as median and IQR. Whiskers represent data within 1.5IQR of the lower and upper quartiles. For comparison between groups, statistical analyses were performed using the Kruskal–Wallis H test followed by the Mann–Whitney U test for post hoc group comparisons. Scale bars: 500 μm. Abbreviations: Aβ, amyloid beta; IQR, interquartile range

Fig. 3

CSF and serum Aβ42/Aβ40 ratios and their associations with cerebral Aβ plaque burden in App^{NL−F/NL−F} knock-in mice. In the whole study population, the Aβ42/Aβ40 ratio in CSF and serum inversely correlated with A–D Aβ42 immunoreactivity and E–H thioflavin S-positive fibrillar dense-core plaques in the cortex and hippocampus. Correlation analyses were performed using Spearman’s rank-ordered correlation coefficient. Abbreviations: Aβ, amyloid beta; CSF, cerebrospinal fluid

Fig. 4

Cortical TBS- and FA-soluble Aβ42 and their associations with CSF and serum Aβ42/Aβ40 ratios in App^{NL−F/NL−F} knock-in mice. Cortical TBS- and FA-soluble Aβ42 was measured in 3 (n = 9)-, 6 (n = 7)-, 9 (n = 8)-, 12 (n = 7)-, 16 (n = 8)-, and 18 (n = 9)-month-old App^{NL−F/NL−F} knock-in mice. A TBS-soluble and D FA-soluble Aβ42 increased in an age-dependent manner with significant changes from 12 and 9 months, respectively. In the whole study population, the Aβ42/Aβ40 ratio in CSF and serum inversely correlated with Aβ42 in the B-C TBS-soluble fraction and the E-F FA-soluble fraction prepared from cortical brain tissue homogenates. Data is presented as median and IQR. Whiskers represent data within 1.5IQR of the lower and upper quartiles. For comparison between groups, statistical analyses were performed using the Kruskal–Wallis H test followed by the Mann–Whitney U test for post hoc group comparisons (**p < 0.01, ***p < 0.001). Correlation analyses were performed using Spearman’s rank-ordered correlation coefficient. Abbreviations: Aβ, amyloid beta; CSF, cerebrospinal fluid; FA, formic acid; IQR, interquartile range; TBS, tris-buffered saline