Genetically enhancing the expression of chemokine domain of CX3CL1 fails to prevent tau pathology in mouse models of tauopathy

Abstract

Background: Fractalkine (CX₃CL1) and its receptor (CX₃CR1) play an important role in regulating microglial function. We have previously shown that Cx₃cr1 deficiency exacerbated tau pathology and led to cognitive impairment. However, it is still unclear if the chemokine domain of the ligand CX₃CL1 is essential in regulating neuronal tau pathology.

Methods: We used transgenic mice lacking endogenous Cx₃cl1 (Cx₃cl1^-/-) and expressing only obligatory soluble form (with only chemokine domain) and lacking the mucin stalk of CX₃CL1 (referred to as Cx₃cl1^105Δ mice) to assess tau pathology and behavioral function in both lipopolysaccharide (LPS) and genetic (hTau) mouse models of tauopathy.

Results: First, increased basal tau levels accompanied microglial activation in Cx₃cl1^105Δ mice compared to control groups. Second, increased CD45⁺ and F4/80⁺ neuroinflammation and tau phosphorylation were observed in LPS, hTau/Cx₃cl1^-/-, and hTau/Cx₃cl1^105Δ mouse models of tau pathology, which correlated with impaired spatial learning. Finally, microglial cell surface expression of CX₃CR1 was reduced in Cx₃cl1^105Δ mice, suggesting enhanced fractalkine receptor internalization (mimicking Cx₃cr1 deletion), which likely contributes to the elevated tau pathology.

Conclusions: Collectively, our data suggest that overexpression of only chemokine domain of CX₃CL1 does not protect against tau pathology.

Keywords: Alzheimer’s disease; CX3CL1; CX3CR1; Microglia; Neuroinflammation; Tau; Tauopathies.

Fig. 1

LPS-induced tau phosphorylation and microglial activation are exacerbated in Cx₃cl1^105Δ mice. a–d Two-month-old fractalkine (Cx₃cl1^−/−)-deficient mice and the mice exclusively expressing the chemokine domain (lacking the mucin-like domain, red) (CX₃CL1^105Δ) with a Myc tag were injected with LPS (3 mg/kg b.w; i.p) or vehicle (VEH, Hank’s balanced salt solution or HBSS) and sacrificed 24 h post-injection. e–f Western blotting of the hippocampi revealed significantly increased total tau (Tau5) (> 1.5-fold) in VEH-treated Cx₃cl1^105Δ vs. Cx₃cl1^−/− mice (mean + SEM; **p < 0.01; n = 3; two-way ANOVA followed by Tukey’s post hoc test). Both AT8/Tau5 and AT180/Tau5 ratios were significantly higher in LPS-treated Cx₃cl1^105Δ compared to LPS-treated Cx₃cl1^−/− or Non-Tg mice (mean + SEM; *p < 0.05; **p < 0.01; n = 3; two-way ANOVA with Tukey’s post hoc test). g Immunohistochemistry (IHC) analysis revealing a modest increase in AT8 (pS199/pS202 tau) among experimental genotypes or between VEH- or LPS-injected mice in the CA3 hippocampal areas. Scale bar, 20 μm. h–k IHC showing elevated Iba1⁺/F4/80⁺ reactive microglia in VEH-treated Cx₃cl1^105Δ mice that is enhanced with LPS treatment. Quantification reveals statistically higher form factor units (higher number means more towards circular contour) for Iba1⁺ microglia in Non-Tg and Cx₃cl1^105Δ mice in LPS-treated groups (mean + SEM; ***p < 0.0001 vs. **p < 0.01 for Non-Tg with LPS; two-way ANOVA with Tukey’s post hoc test; n = 3–6 mice per group). Scale bars (h, j) 25 μm

Fig. 2

Increased tau pathology, IL-1α/IL-1β, and microglial activation in 6-month-old hTau/Cx₃cl1^−/− and hTau/Cx₃cl1^105Δ mice. a, b Western blotting revealed increases in AT8⁺ tau in the hippocampus of hTau/Cx₃cl1^−/− and hTau/Cx₃cl1^105Δ mice compared to hTau controls. c AT8 IHC revealed increased reactivity in the CA3 regions of hTau/Cx₃cl1^−/− and hTau/Cx₃cl1^105Δ groups compared to hTau controls. Scale bar, 30 μm. d Significant increases in both CD45 and F4/80 immunoreactivities were detected and quantified (e) in the cortex of hTau/Cx₃cl1^−/− and hTau/Cx₃cl1^105Δ mice compared to controls. f A significant increase in IL-1α and IL-1β was observed in both hTau/Cx₃cl1^−/− and hTau/Cx₃cl1^105Δ mice via ELISA. n = 6 mice per group except for ELISA (n = 10). Three independent experiments were performed for each analysis. Error bars represent SEM. One-way ANOVA followed by Tukey’s post hoc test: *p < 0.05, **p < 0.01, ***p < 0.001

Fig. 3

Expression of the microglial CX₃CR1 is decreased in Cx₃cl1^105Δ mice. a, c Flow cytometry on isolated brain mononuclear cells revealed no alteration in the total number of resident microglia in Non-Tg, Cx₃cl1^−/−, or Cx₃cl1^105Δ mice (Cd11b⁺/CD45^low). b, d Overall decreased CX₃CR1 expression in the CD11b⁺CD45^low (microglial population) in Cx₃cl1^105Δ mice compared to Non-Tg and Cx₃cl1^−/− mice (mean + SEM; one-way ANOVA followed by Tukey’s post hoc test: *p < 0.05, **p < 0.01; n = 3 mice per group). e Working model of microglial-neuronal fractalkine signaling axis. Note that the neuronal-derived CX₃CL1 either as full length (in case of Non-Tg mice), as complete knockout (in Cx₃cl1^−/− mice), or as the only soluble form with chemokine domain (Cx₃cl1^105Δ mice) differentially regulates the expression of microglial CX₃CR1 (which is a seven transmembrane G protein-coupled receptor) on the cell surface. This in turn may lead to over-activation of microglia in (Cx₃cl1^105Δ mice) and enhanced neuroinflammation and exacerbation of tau pathology in chemical (LPS) or genetic (hTau) model of tauopathy

Fig. 4

Impaired learning in hTau/Cx₃cl1^−/− and hTau/Cx₃cl1^105Δ mice. Morris water maze was performed on 12-month-old mice. Mice were subjected to a 3-day visible training paradigm (a), followed by a 5-day hidden trial period (memory testing). b Where latency to reach the platform was recorded (seconds, sec). c Analysis of the linear regression slope adjusted for each genotype revealed that hTau mice learned the task better than hTau/Cx₃cl1^−/− or hTau/Cx₃cl1^105Δ mice over the 5-day hidden trial period. d hTau mice had a much lower savings index than hTau/Cx₃cl1^−/− or hTau/Cx₃cl1^105Δ mice during the 5-day hidden trial period. e hTau mice show higher acquisition index than hTau/Cx₃cl1^−/− or hTau/Cx₃cl1^105Δ mice during the 5-day testing period. Mean + SEM; one-way ANOVA followed by Tukey’s post hoc test: *p < 0.05, **p < 0.01, ***p < 0.001; n = 10 mice per group