Glycogen synthase kinase-3β regulates fractalkine production by altering its trafficking from Golgi to plasma membrane: implications for Alzheimer's disease

Abstract

Glycogen synthase kinase-3β (GSK-3β) is a serine-threonine kinase implicated in multiple processes and signaling pathways. Its dysregulation is associated with different pathological conditions including Alzheimer's disease (AD). Here we demonstrate how changes in GSK-3β activity and/or levels regulate the production and subsequent secretion of fractalkine, a chemokine involved in the immune response that has been linked to AD and to other different neurological disorders. Treatment of primary cultured neurons with GSK-3β inhibitors such as lithium and AR-A014418 decreased full-length fractalkine in total cell extracts. Opposite effects were observed after neuron transduction with a lentiviral vector overexpressing the kinase. Biotinylation assays showed that those changes mainly affect the plasma membrane-associated form of the protein, an observation that positively correlates with changes in the levels of its soluble form. These effects were confirmed in lithium-treated wild type (wt) mice and in GSK-3β transgenic animals, as well as in brain samples from AD patients, evident as age-dependent (animals) or Braak stage dependent changes (humans) in both the membrane-bound and the soluble forms of the protein. Further immunohistochemical analyses demonstrated how GSK-3β exerts these effects by affecting the trafficking of the chemokine from the Golgi to the plasma membrane, in different and opposite ways when the levels/activity of the kinase are increased or decreased. This work provides for the first time a mechanism linking GSK-3β and fractalkine both in vitro and in vivo, with important implications for neurological disorders and especially for AD, in which levels of this chemokine might be useful as a diagnostic tool.

Keywords: Alzheimer’s disease; Fractalkine; GSK-3β; Golgi network; Rab8.

Fig. 1

Effects of GSK-3β on soluble and transmembrane fractalkine levels in vitro. Inhibition with lithium (20 mM) (a) or AR-A014418 (33 μM) (b) decreases total levels of fractalkine in neuronal lysates. GSK-3β-increased activity by pharmacological approaches (Akt inhibitor VIII (5 μM)) (c) or genetic approaches (lentiviral transduction) (d) increases total fractalkine levels in neuronal lysates. Biotinylation assays show how GSK-3β-induced fractalkine changes mainly affect the membrane-bound form of the protein (e–h). Western blot detection of fractalkine in concentrated media derived from transfected HEK293 cells treated with lithium (20 mM) or AR-A014418 (33 μM) (i) or in co-transfection experiments (j) showing a correlation with the observed changes in the membrane-anchored form. FKN, Fractalkine; β-tub, β-tubulin; Li, lithium; AR, AR-A014418; Akt Inhib. VIII, Akt Inhibitor VIII. *P < 0.05; **P < 0.01; ***P < 0.001 versus control/GFP/pcDNA3-FKN samples. N ≥ 3 biological replicates. Data are expressed as mean ± SEM

Fig. 2

Effects of GSK-3β on fractalkine localization in the trans Golgi network. Immunostaining with trans Golgi marker TGN38 and fractalkine antibody showing an increased colocalization between both markers in neurons where the kinase was inhibited either by lithium (20 mM) or AR-014418 (33 μM) (a) and in neurons transduced with control GFP or GSK-3β-GFP lentiviruses (b). Graphs representing the results of the colocalization analysis in treated (c, d) or transduced neurons (e). Right column in a and b shows merge images. *P < 0.05; **P < 0.01 versus control. Scale bar 3 μm. N ≥ 3 biological replicates. Data are expressed as mean ± SEM

Fig. 3

GSK-3β overexpression affects fractalkine colocalization in Rab8⁺ vesicles. Immunostaining with Rab8 and fractalkine antibodies in GSK-3β-GFP transduced neurons. Arrows show colocalization between both markers (a). GSK-3β decreases fractalkine colocalization with Rab8⁺ vesicles (b). Total Rab8⁺ vesicle pool is affected by GSK-3β upregulation (c). *P < 0.05 versus GFP transduced neurons. Scale bar 3 μm. N ≥ 3 biological replicates. Data are expressed as mean ± SEM

Fig. 4

Effects of GSK-3β on soluble and transmembrane fractalkine levels in vivo. GSK-3β inhibition also decreases fractalkine levels in membrane pellet extracts from wt animals treated with lithium (a). Tet/GSK-3β transgenic animals show increased fractalkine levels in membrane pellet extracts from 3-month-old animals (c) and 13-month-old mice (e). Graphs showing quantification of soluble fractalkine measured by ELISA in soluble enriched brain samples from wt animals treated with lithium (b) or 3-month-old (d) and 13-month-old (f) Tet/GSK-3β animals. A correlation between the soluble fractalkine and the membrane-bound form levels was found in lithium-treated animals and young transgenic mice. FKN fractalkine. *P < 0.05; **P < 0.01; ***P < 0.001 versus control. Lithium-treated mice, N = 9 per group; 3-month-old transgenic animals, wt N = 13, Tet/GSK-3β N = 10; 13-month-old animals, N = 6 per group. Data are expressed as mean ± SEM

Fig. 5

Alzheimer’s disease human samples also show altered transmembrane and soluble fractalkine levels. Fractalkine changes differ according to AD progression with increased levels in membrane pellet samples from II to III Braak stage (a) and no changes in V–VI Braak stage (c). Graphs representing the quantification of soluble fractalkine measured by ELISA in soluble enriched brain samples showing increased fractalkine levels in II–III Braak stage samples (b) and no changes in V–VI Braak stage Alzheimer’s disease patients (d). FKN fractalkine. *P < 0.05; **P < 0.01; ***P < 0.001 versus control. Total N = 19 (see Fig. S7). Data are expressed as mean ± SEM