Fingolimod modulates multiple neuroinflammatory markers in a mouse model of Alzheimer's disease

Abstract

Sphingosine 1-phosphate (SP1) receptors may be attractive targets for modulation of inflammatory processes in neurodegenerative diseases. Recently fingolimod, a functional S1P1 receptor antagonist, was introduced for treatment of multiple sclerosis. We postulated that anti-inflammatory mechanisms of fingolimod might also be protective in Alzheimer's disease (AD). Therefore, we treated a mouse model of AD, the 5xFAD model, with two doses of fingolimod (1 and 5 mg/kg/day) and measured the response of numerous markers of Aβ pathology as well as inflammatory markers and neurochemistry using biochemical, immunohistochemistry and high resolution magic angle spinning magnetic resonance spectroscopy (MRS). In mice at 3 months of age, we found that fingolimod decreased plaque density as well as soluble plus insoluble Aβ measured by ELISA. Fingolimod also decreased GFAP staining and the number of activated microglia. Taurine has been demonstrated to play a role as an endogenous anti-inflammatory molecule. Taurine levels, measured using MRS, showed a very strong inverse correlation with GFAP levels and ELISA measurements of Aβ, but not with plaque density or activated microglia levels. MRS also showed an effect of fingolimod on glutamate levels. Fingolimod at 1 mg/kg/day provided better neuroprotection than 5 mg/kg/day. Together, these data suggest a potential therapeutic role for fingolimod in AD.

Figure 1

(A) Representative pictures showing Aβ40 and Aβ42 immunostained brain sections of 3 months old 5xFAD mice from untreated and 1 mg/kg/day and 5 mg/kg/day of fingolimod treatment groups (Magnification x40). (B) Effects of fingolimod treatments on Aβ plaque burden. Brain sections of representative groups were stained for Aβ plaque using Aβ40 and Aβ42 antibodies. 1 and 5 mg/kg/day of fingolimod treatment significantly lowered the Aβ42 plaque burden after 2 months of treatment in the 3 month-old mice compared to the regular diet as measured by the percent of cortical area. Decreased Aβ40 plaque burden was detected in the fingolimod-treated groups however the decreases were not significant. *p < 0.05, (n = 8–10 mice/group). The Aβ40 at 1 mg/kg/day almost reached significance (omnibus ANOVA F = 3.22, p = 0.058).

Figure 2. Levels of total (soluble and insoluble) Aβ40 and Aβ42 were quantified by ELISA in the frontal cortex.

1 mg/kg/day of fingolimod treatment significantly decreased the levels of Aβ42 and Aβ40 however 5 mg/kg/day of fingolimod treatment did not reach significant decrease. *p < 0.05, (n = 8–10 mice/group).

Figure 3

(A) Immunohistochemical staining for the astrocytic marker glial fibrillary acidic protein (GFAP) in 5xFAD mouse brain sections from the hippocampus of untreated 5xFAD and fingolimod-treated 5xFAD mouse brains. (B) Analysis of GFAP-positive astrocytes in the hippocampus of fingolimod-treated and untreated 5xFAD mice at 3 months of age. 1 and 5 mg/kg/day of fingolimod treatment were significantly decreased the presence of reactive astrocytes in the hippocampus of 5xFAD mice compared to untreated mice. (*p < 0.05), (n = 8–10 mice/group).

Figure 4

(A) Sections of hippocampus and subiculum immunostained for Iba1 in 5xFAD mice untreated and treated with 1 mg/kg/day, and 5 mg/kg/day of fingolimod. Untreated 3 months old 5xFAD mice showed significant increase in the number of activated microglial cells. (B) Quantitation of total, active and resting variants of microglia in the hippocampus of 5xFAD untreated and fingolimod-treated groups. The number of activated Iba1-positive microglia significantly decreased in the hippocampus of 1 mg/kg/day, and 5 mg/kg/day fingolimod-treated groups compared with untreated group (*p < 0.05), (n = 8–10 mice/group).

Figure 5. Evaluating the power of the various markers to assess protection with fingolimod.

(A) Left) Rankings determined from a relief-f algorithm. GFAP has the highest weighting followed by Iba1 quantified by either activated/total (A/T) or activated/resting (A/R), Aβ42 plaque density, Iba1 activated microglia, Aβ42 ELISA, Aβ40 ELISA, Iba1 (total) or Iba1 (resting). (B) Right) We then used the top four markers (GFAP, Iba1 (A/T), Aβ42 plaque density and Aβ42 ELISA to perform a linear discriminant analysis (Wilk’s lambda = 0.122; p < 0.0001 for function 1, not significant for function 2). This analysis shows a larger average distance from regular diet for the 1 mg/kg/day fingolimod treatment (Lo) than for the 5 mg/kg/day dose (Hi).

Figure 6. HRMAS of fingolimod treatment in 3 months old mice.

(A) Representative HRMAS spectra from hippocampus in 5xFAD normal diet and fingolimod-treated (1 mg/kg/day) a few molecules including N-acetylaspartate (NAA), glutamate, glutamine, creatine, taurine and myo-inositol are labeled. (B) Effect of fingolimod treatment as a function of dose on taurine levels. There is a very significant effect on raising taurine levels in the fingolimod-treated mice. (C) Correlation between taurine levels and GFAP staining in the mice that had both measured. There is a very strong correlation R = 0.83; p < 0.0001.

Figure 7. Effects of age on inflammatory markers in hippocampus in 5xFAD mice.

Left) GFAP staining shows a large increase from WT to 3 months and then 8 months old mice (P < 0.0001 for differences between all groups using ANOVA with a Tukey-HSD post-hoc test). Right) Increased activated/total microglia ratio for 3 and 8 months old animals (p < 0.0001 for difference between groups. There were no activated microglia noted in the WT mice.