PPARgamma agonists rescue increased phosphorylation of FGF14 at S226 in the Tg2576 mouse model of Alzheimer's disease

Abstract

Background: Cognitive impairment in humans with Alzheimer's disease (AD) and in animal models of Aβ-pathology can be ameliorated by treatments with the nuclear receptor peroxisome proliferator-activated receptor-gamma (PPARγ) agonists, such as rosiglitazone (RSG). Previously, we demonstrated that in the Tg2576 animal model of AD, RSG treatment rescued cognitive deficits and reduced aberrant activity of granule neurons in the dentate gyrus (DG), an area critical for memory formation.

Methods: We used a combination of mass spectrometry, confocal imaging, electrophysiology and split-luciferase assay and in vitro phosphorylation and Ingenuity Pathway Analysis.

Results: Using an unbiased, quantitative nano-LC-MS/MS screening, we searched for potential molecular targets of the RSG-dependent rescue of DG granule neurons. We found that S226 phosphorylation of fibroblast growth factor 14 (FGF14), an accessory protein of the voltage-gated Na⁺ (Nav) channels required for neuronal firing, was reduced in Tg2576 mice upon treatment with RSG. Using confocal microscopy, we confirmed that the Tg2576 condition decreased PanNav channels at the AIS of the DG, and that RSG treatment of Tg2576 mice reversed the reduction in PanNav channels. Analysis from previously published data sets identified correlative changes in action potential kinetics in RSG-treated T2576 compared to untreated and wildtype controls. In vitro phosphorylation and mass spectrometry confirmed that the multifunctional kinase GSK-3β, a downstream target of insulin signaling highly implicated in AD, phosphorylated FGF14 at S226. Assembly of the FGF14:Nav1.6 channel complex and functional regulation of Nav1.6-mediated currents by FGF14 was impaired by a phosphosilent S226A mutation. Bioinformatics pathway analysis of mass spectrometry and biochemistry data revealed a highly interconnected network encompassing PPARγ, FGF14, SCN8A (Nav 1.6), and the kinases GSK-3 β, casein kinase 2β, and ERK1/2.

Conclusions: These results identify FGF14 as a potential PPARγ-sensitive target controlling Aβ-induced dysfunctions of neuronal activity in the DG underlying memory loss in early AD.

Keywords: Alzheimer's disease; Confocal microscopy; Fibroblast growth factor 14; Mass spectrometry; PPARgamma.

Fig. 1

Identification of peptide AGVTPsKSTSASAImNGGK and quantification of phosphorylation of S226 in FGF14 regulated by RSG diet. A) Complete MS spectrum at RT=143.46, m/z = 400–2000. B) MS2 of peptide AGVTPsKSTSASAImNGGK (s denotes phosphorylated serine; m denotes oxidized methionine), RT=143.57 with labeled b and y ion series. * denotes loss of NH₃; o denotes loss of H₂O. Inset, top right: labeled b/y ion series with 19 out of 54 potential ions identified. C) Zoom of A at RT=143.54 showing peptide AGVTPsKSTSASAImNGGK. The area under the 4 peaks of the ¹⁸O isotopic envelope cluster (red, RSG diet) was divided by the area of the 4 peaks of the ¹⁶O isotopic envelope cluster (blue, control diet). A ratio of 0.74 indicated that RSG decreased phosphorylation of S226 in Tg2576 mice. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

Fig. 2

Redistribution of Nav complex-associated proteins at the AIS in DG. Confocal images of the DG of the hippocampus in wild type (WT), Tg2576 (Tg), and Tg2576 + rosiglitazone (Tg +RSG) mice showing immunolabeling with mouse anti-FGF14, rabbit anti-PanNav, and mouse anti-AnkG antibodies. (A) 20× zoom of the WT DG treated with antibodies against AnkG (green), PanNav (red), FGF14 (blue) with scale bars shown. (B) 63× zoom, cropped, of the WT DG in A, showing the FGF14 channel (grayscale). (C) ROI generated by image segmentation and thresholding applied to B by ImageJ Fiji, shown in yellow outline (see methods). (D) 63× zoom, cropped, of WT, Tg, and Tg + RSG, treated with antibodies against FGF14 (green), PanNav (red), AnkG (blue). Far right: composite image of all three channels. (E–G) Raw intensities of AnkG, PanNav, and FGF14 channels for all groups. (H–J) Channel intensity ratios (AnkG/PanNav, PanNav/FGF14, FGF14/AnkG) for all groups. Statistical analysis: One-way ANOVA with Tukey's multiple comparisons test with single-pooled variance. Data mean ± SEM; versus WT: * p < 0.05, ** p < 0.01, *** p < 0.001; versus TG: # p < 0.05, ## p < 0.01, ### p < 0.001. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

Fig. 3

RSG affects time to rise and time to decay half-amplitude in Tg2576 dentate gyrus granule cells restoring them to the control level. (A) Representative traces of evoked action potential at current threshold for wild type, Tg2576 and RSG-treated Tg2576 dentate gyrus granule cells. (B) Bar graphs summarizing the effect of RSG diet on the time to rise half-amplitude of granule cells evoked action potential for wild type, Tg2576 and RSG-treated Tg2576 mice. (C) Bar graphs summarizing the effect of RSG diet on the time to decay half-amplitude of granule cells evoked action potential for wild type, Tg2576 and RSG-treated Tg2576 mice. “n” represents total number of neurons obtained for each group of animals: 27 neurons were recorded from 15 wild type, 31 from 16 Tg2576 and 37 from 21 Tg2576 on RSG diet mice, respectively. *p < 0.05, **p < 0.01, ***p < 0.005; Kruskal-Wallis with post hoc Dunn's multiple comparison tests.

Fig. 4

Mass spectrometry identifying in vitro phosphorylation of FGF14 at S226. (A) Full sequence of FGF14-1b (H. sapiens), highlighting the GSK–3 consensus phosphorylation motif (SXXXS) beginning at S226. (B) Phosphorylation of peptide KPGVTPsKSTSASAIMNGGK-NH2 at S226 by GSK–3β. Main figure: MS/MS fragmentation spectra showing the b and y ion series and the dominant peak [M+3H]³⁺ (theoretical mass: 666.34; experimental mass: 666.34; ppm < 15). Top: The b and y ion series for KPGVTPsKSTSASAIMNGGK, with the site-determining ions b5, b8²⁺, and y12. Inset: high resolution MS spectra showing the parent [M+3H]³⁺ peak (666.334).

Fig. 5

Using the split-luciferase assay to assess the phenotype of a S226A phosphosilent mutant. (A) The split-luciferase assay produces a luminescence readout based on the complementation of N-terminal and C-terminal fragments of Photinus luciferase enzyme, which are attached to two interacting proteins (in this case, FGF14 and a CD4-Nav1.6-C-tail construct). Upon interaction of FGF14 and the C-tail of Nav1.6, the C-terminal and N-terminal fragments reconstitute into functional luciferase enzyme, producing a robust luminescence response when luciferin substrate is added. (B) Lack of a serine/threonine site, as in the FGF14^S226A mutation, is projected to weaken the interaction between FGF14 and Nav1.6, resulting in a diminished luminescence response compared to wild type FGF14. (C) Real-time raw luminescence response of CLuc-FGF14/CD4-Nav1.6-C-tail-NLuc (black) vs CLuc-FGF14^S226A/CD4-Nav1.6-C-tail-NLuc (orange). (D) Quantification of maximum observed intensity over the course of (C). (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

Fig. 6

Comparison of the effects of FGF14-GFP and FGF14^S226A-GFP on sodium currents in HEK293-Nav1.6 cells. (A, B) Representative traces of fast transient sodium currents recorded from HEK293-Nav1.6 cells transiently transfected with (A) FGF14-GFP or (B) FGF14^S226A-GFP. (C) Current-voltage relationship of peak Na⁺ current densities recorded from HEK293-Nav1.6 transiently expressing either FGF14-GFP (black) or FGF14^S226A-GFP (orange). (D) Bar graphs represents peak Na⁺ current density at voltage step of –10 mV recorded from HEK293-Nav1.6 transiently expressing either FGF14-GFP (black) or FGF14^S226A-GFP (orange). (E) Voltage-dependence of Nav current activation and (F) steady-state inactivation plotted against membrane potential (mV) and fitted using the Boltzmann equation were obtained from HEK293-Nav1.6 transiently expressing either FGF14-GFP (black) or FGF14^S226A-GFP (orange). **p < 0.01; Student t-test. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

Fig. 7

Bioinformatics using Ingenuity Pathways Analysis™ of proteins central to RSG regulation of a key network connecting FGF14, SCN8A, GSK, PPARG and ERK1/2. Key: FGF14, fibroblast growth factor 14; SCN8A, voltage-gated sodium channel Na1.6; GSK–3, glycogen synthase kinase 3; CSNK2B, casein kinase 2 beta; PPARG, peroxisome proliferator-activated receptor gamma; ERK1/2, p42/p44 mitogen activated protein kinase; NFκB, nuclear factor kappa B. (See www.ingenuity.com for a more detailed description of network statistical calculations, molecule naming and symbol descriptions).