Fractalkine overexpression suppresses tau pathology in a mouse model of tauopathy

Abstract

Alzheimer's disease is characterized by amyloid plaques, neurofibrillary tangles, glial activation, and neurodegeneration. In mouse models, inflammatory activation of microglia accelerates tau pathology. The chemokine fractalkine serves as an endogenous neuronal modulator to quell microglial activation. Experiments with fractalkine receptor null mice suggest that fractalkine signaling diminishes tau pathology, but exacerbates amyloid pathology. Consistent with this outcome, we report here that soluble fractalkine overexpression using adeno-associated viral vectors significantly reduced tau pathology in the rTg4510 mouse model of tau deposition. Furthermore, this treatment reduced microglial activation and appeared to prevent neurodegeneration normally found in this model. However, in contrast to studies with fractalkine receptor null mice, parallel studies in an APP/PS1 model found no effect of increased fractalkine signaling on amyloid deposition. These data argue that agonism at fractalkine receptors might be an excellent target for therapeutic intervention in tauopathies, including those associated with amyloid deposition.

Fig. 1.

Fractalkine ligand (CX3CL1) and receptor(CX3CR1) levels in transgenic mice. (A) Western blot with anti-CX3CR1 antibody using hippocampal cell lysates of APP/PS1, Tg4510, or nontransgenic (NTg) littermate controls and normalizing to GAPDH staining; (B) Enzyme-linked immunosorbent assay for CX3CL1 of hippocampal cell lysates of APP/PS1, Tg4510, or nontransgenic littermate controls.

Fig. 2.

Expression of FKN constructs. (A) Western blot with anti-HA tag antibody using cell lysates of plasmid transfected HEK293 cells. (B) Anti-HA Western blot of the media from transfected HEK293 cells, only sFKN is observed in the media. (C and D) Anti-HA immunohistochemistry of hippocampus of GFP-injected and sFKN-injected mice. Scale = 200 μm. Abbreviations: DG, dentate gyrus; FKN, fractalkine; GFP, green fluorescent protein (control); HA, hemagglutinin; n, native (membrane associated) FKN; sFKN, secreted FKN.

Fig. 3.

Tau pathology is reduced after sFKN transduction. Gallyas staining of rAAV-GFP (A) and rAAV-sFKN (B) -injected mice hippocampi. Staining with anti-phosphotau 396 antibody in rAAV-GFP (C) and rAAV-sFKN (D) -injected mice. Percentage area positive staining for Gallyas (E) and anti-phopsphotau 396 (F). ** P < 0.01; * P < 0.05. Scale = 200 μm. Abbreviations: FKN, fractalkine; GFP, green fluorescent protein; rAAV, recombinant adeno-associated virus serotype; sFKN, secreted FKN.

Fig. 4.

Anti-NeuN immunohistochemistry is increased after rAAV-sFKN treatment. Staining with anti-NeuN antibody in rAAV-GFP (A) and rAAV-sFKN (B) -injected mice. Percentage area positive staining for anti-NeuN in the CA1–CA3 of HPC (C) or DG subfield (D). * P < 0.05. Scale = 200 μm. Abbreviations: DG, dentate gyrus; FKN, fractalkine; GFP, green fluorescent protein; HPC, hippocampus; rAAV, recombinant adeno-associated virus serotype; sFKN, secreted FKN.

Fig. 5.

Stereological analysis demonstrates a reduction in neuron loss in the AAV-sFKN treated group compared with the AAV-GFP treated group. HPC volume (μm³) (A) and granular cell counts of the DG (B). * P < 0.02. Abbreviations: AAV, adeno-associated virus serotype; DG, dentate gyrus; FKN, fractalkine; GFP, green fluorescent protein; HPC, hippocampus; NonTg, nontransgenic; sFKN, secreted FKN.

Fig. 6.

Western blot analysis shows reduced phospho-tau levels. (A) Intensity of staining is represented as fold change over the control GFP treated group after standardizing to GAPDH staining. Total tau (H150 and HT7 antibody), anti-pS199/S202, pS262, pS396, and pS356 tau; anti-GSK3 α;*216/β279, and anti-PHF tau antibodies AT8, AT180, and AT270 are shown. (B) Representative Western blot images of panel A. (C) Fold change in intensity for the formic acid tau fraction. * P < 0.01. Abbreviations: FKN, fractalkine; GAPDH, glyc-eraldehyde-3-phosphate dehydrogenase; GFP, green fluorescent protein; PHF, pair helical filament; sFKN, secreted FKN.

Fig. 7.

Anti-CD45 immunohistochemistry is reduced after rAAV-sFKN treatment. Anti-CD45 staining of rAAV-GFP (A) and rAAV-sFKN (B) -injected mice hippocampi (scale = 200 μm). Morphology of CD45-stained cells in an rAAV-GFP injected mouse (scale = 50 μm) (C). Percentage area positive staining for anti-CD45 (D). * P < 0.02. Abbreviations: AAV, adeno-associated virus serotype; FKN, fractalkine; GFP, green fluorescent protein; rAAV, recombinant AAV; sFKN, secreted FKN.

Fig. 8.

RAWM test demonstrates no phenotype rescue with AAV-sFKN treatment in the hippocampus compared with nonTg and AAV-GFP treated Tg4510 mice. Number of errors is shown as an average of every 3 trials as 1 block. Abbreviations: AAV, adeno-associated virus serotype; FKN, fractalkine; GFP, green fluorescent protein; nonTG, nontransgenic; RAWM, radial arm water maze; sFKN, secreted FKN.

Fig. 9.

Amyloid levels in APP/PS1 mice treated with GFP or sFKN virus are unaltered. Anti-Aβ staining of rAAV-GFP (A) and rAAV-sFKN (B) -injected mice hippocampi. Congo red staining of rAAV-GFP (C) and rAAV-sFKN (D) -injected mice hippocampi. Percentage area positive staining for anti-Aβ (E) and Congo red staining (F). Scale = 200 μm. Abbreviations: Aβ, amyloid beta; AAV, adeno-associated virus serotype; FKN, fractalkine; GFP, green fluorescent protein; rAAV, recombinant AAV; sFKN, secreted FKN.