Time- and Sex-Dependent Effects of Fingolimod Treatment in a Mouse Model of Alzheimer's Disease

Abstract

Alzheimer's disease (AD) is the most common cause of dementia. Fingolimod has previously shown beneficial effects in different animal models of AD. However, it has shown contradictory effects when it has been applied at early disease stages. Our objective was to evaluate fingolimod in two different treatment paradigms. To address this aim, we treated male and female APP-transgenic mice for 50 days, starting either before plaque deposition at 50 days of age (early) or at 125 days of age (late). To evaluate the effects, we investigated the neuroinflammatory and glial markers, the Aβ load, and the concentration of the brain-derived neurotrophic factor (BDNF). We found a reduced Aβ load only in male animals in the late treatment paradigm. These animals also showed reduced microglia activation and reduced IL-1β. No other treatment group showed any difference in comparison to the controls. On the other hand, we detected a linear correlation between BDNF and the brain Aβ concentrations. The fingolimod treatment has shown beneficial effects in AD models, but the outcome depends on the neuroinflammatory state at the start of the treatment. Thus, according to our data, a fingolimod treatment would be effective after the onset of the first AD symptoms, mainly affecting the neuroinflammatory reaction to the ongoing Aβ deposition.

Keywords: APPPS1; Alzheimer’s disease; FTY720; Gilenya; amyloid beta; fingolimod; treatment.

Figure A1

Assessment of the circadian activity during a 24 h period revealed no differences between VEH-treated (blue triangles) and FTY-treated (red triangles) males. Animals were treated according to the early treatment paradigm. (A) Mean hourly count of movements of animal groups (grey indicates dark hours; white indicates light hours of the day). (B) Relative distribution of the activity and (C) number of detected movements for each experimental group during light and dark hours. Significance in activity between treatment groups was calculated using the Student’s t-test. Data are presented as mean ± SD, VEH n = 11, FTY n = 12.

Figure A2

Spatial orientation testing using water maze revealed no significant differences between VEH-treated (blue circles) and FTY-treated (red circles) females. Animals were treated according to the early treatment paradigm. (A) Time latency [s] average of each experimental group to find the platform during training trials. (B) Proximity average [cm] during training. (C) Proximity to target and (D) number of target crossings (n ∈ N) during the probe trial. Significance between treatment groups was calculated using the Student’s t-test (C) or Wilcoxon–Mann–Whitney test (D). Data are presented as mean (A,B) and mean ± SD (C,D), VEH n = 11, and FTY n = 11.

Figure 1

The graph shows the weight progression of early treated (left) and late treated animals (right) for all experimental groups. FTY—fingolimod (FTY720)-treated animals; VEH—vehicle-treated control animals. Data are presented as mean ± SD; n = 8 in all groups, except for the late treatment males (n = 13). Statistical analysis was performed using two-way ANOVA (mixed-effects model). No significant differences were found.

Figure 2

Quantification of insoluble (A,C) and soluble (B,D) Aβ42 for late treated males (A,B) and females (C,D) APPtg mice. Data are presented as mean ± SD; n = 8 (except for males VEH, n = 13; FTY, n = 12). Significance was calculated using Wilcoxon–Mann–Whitney test, and it is given as *: p ≤ 0.05, **: p ≤ 0.01.

Figure 3

Quantification of insoluble (A,C) and soluble (B,D) Aβ42 for early treated males (A,B) and females (C,D). Data are presented as mean ± SD; n = 8 (except for FTY treatment females, n = 6). Significance was calculated using Wilcoxon–Mann–Whitney test.

Figure 4

Evaluation of micro- and astrogliosis in mice with late treatment paradigm (at 175 days of age). (A,B) Representative images of Iba1 immunostaining of male (A) and female (B) mice. Microglia activation was determined as density of IBA1-positive cells in males (C) and females (D). (E,F) Representative images of GFAP immunostaining in males (E) and females (F). Astrocyte density in males (G) and females (H). Data are presented as mean ± SD; VEH males n = 13 for IBA1, n = 5 for GFAP, FTY males n = 12 for IBA1, n = 6 for GFAP, females n = 8. Significance was calculated using Student’s t-test, and it is given as ***: p ≤ 0.001. Scale bars: 1 mm.

Figure 5

Levels of selected cytokines in brain tissue in late and early treated animals. (A,B) Quantification of IL-1β, IL-1, IL-6, MCP-1, and TNFα in late treated males (A) and females (B). (C,D) Quantification of the cytokines in early treated males (C) and females (D). Data are presented as mean ± SD; n = 8 except late treatment males (VEH n = 13; FTY n = 12) and early FTY treatment females (n = 6). Significance was calculated using Student’s t-test, and it is given as **: p ≤ 0.01.

Figure 6

Amount of BDNF in brain tissues. (A–D) Graphs show the quantification of BDNF in brain tissues of late treated male (A) and female (B) and early treated male (C) and female (D) APPtg mice. (E) Linear regression analysis between brain BDNF and Aβ concentrations show a positive correlation between both of the proteins. Data are presented as mean ± SD; n = 8, except for late treated males VEH, n = 13; FTY, n = 12) and early treated females (n = 6). Significance between experimental groups was calculated using the Wilcoxon–Mann–Whitney test. Correlation coefficient was analysed using Pearson’s correlation test.

Figure 7

Experimental design. Animals were treated with fingolimod in two treatment paradigms: early treatment (between 50 and 100 days of age) or late treatment (between 125 and 175 days of age). The treatment duration (50 days) was the same in all treatment groups.

Figure 8

Machine learning-assisted morphological analyses of cortex pathology. (A) Schematic presentation of the analysed cortex region (from Figure 4A, left). The brain tissue was automatically detected within the manually marked ROI. (B) Staining of IBA1+ cells in the cortex. The microglia nicely accumulate near-insoluble Aβ deposits and delineate amyloid plaques. (C) Staining of GFAP+ cells in the cortex. Astrocytes show a more diffuse pattern with localised perivascular pronunciation (white triangles). Scale bars: (A) 1 mm, (B,C) 50 µm.