Early-Onset Molecular Derangements in the Olfactory Bulb of Tg2576 Mice: Novel Insights Into the Stress-Responsive Olfactory Kinase Dynamics in Alzheimer's Disease

Abstract

The olfactory bulb (OB) is the first processing station in the olfactory pathway. Despite smell impairment, which is considered an early event in Alzheimer's disease (AD), little is known about the initial molecular disturbances that accompany the AD development at olfactory level. We have interrogated the time-dependent OB molecular landscape in Tg2576 AD mice prior to the appearance of neuropathological amyloid plaques (2-, and 6-month-old), using combinatorial omics analysis. The metabolic modulation induced by overproduction of human mutated amyloid precursor protein (APP) clearly differs between both time points. Besides the progressive perturbation of the APP interactome, functional network analysis unveiled an inverse regulation of downstream extracellular signal-regulated kinase (ERK1/2), and p38 mitogen-activated protein kinase (MAPK) routes in 2-month-old Tg2576 mice with respect to wild-type (WT) mice. In contrast, Akt and MAPK kinase 4 (SEK1)/ stress-activated protein kinase (SAPK) axis were parallel activated in the OB of 6-months-old-Tg2576 mice. Furthermore, a survival kinome profiling performed during the aging process (2-, 6-, and 18-month-old) revealed that olfactory APP overexpression leads to changes in the activation dynamics of protein kinase A (PKA), and SEK1/MKK4-SAPK/JNK between 6 and 18 months of age, when memory deficits appear and AD pathology is well established in transgenic mice. Interestingly, both olfactory pathways were differentially activated in a stage-dependent manner in human sporadic AD subjects with different neuropathological grading. Taken together, our data reflect the early impact of mutated APP on the OB molecular homeostasis, highlighting the progressive modulation of specific signaling pathways during the olfactory amyloidogenic pathology.

Keywords: Alzheimer’s disease; Tg2576 mice; network biology; olfactory bulb; proteomics; transcriptomics.

Figure 1

Olfactory β-Amyloid Aβpathology increases with the disease progression in TG2576 mice. Olfactory bulbs (OBs) were harvested from 2-(A–C), 6-(D–F) and 14-(G–J) month-old Tg2576 mice. Intraneuronal Aβ immunoreactivity can be observed in 2-month-old mice (arrow heads; panel B and more detailed in panel C). OB samples from 6-month-old animals (panels D–F) shows moderate Aβ deposition in form of diffuse plaques (asterisk). By contrast, mature plaques (asterisk in panels G,H and insert J) and vascular Aβ (insert I) is evident in 14-month-old Tg2576. Scale bars (500 μm for panels A,D,G, 100 μm for panels B,E,H) or 10 μm (C,F,I,J).

Figure 2

Dual-omic approach to characterize the OB molecular homeostasis between 2-month-old and 6-month-old Tg2576 mice. (A) An overview of the experimental workflow used in this study. (B) Differential molecular profiling detected by-omics in Tg2576 OBs (2 and 6 months of age). The OB protein expression levels of amyloid precursor protein (APP) at early Alzheimer’s disease (AD) stages in Tg2576 mice is shown. (C) Heat maps representing the degree of change for the differentially expressed proteins (Supplementary Table S2) between 2-and 6-month-old Tg2576 mice respect to wild-type (WT) littermates. Red and green, up- and down-regulated proteins, respectively. (D) Venn diagram for the differentially expressed genes detected in Tg2576 mice at both time points (Supplementary Table S1).

Figure 3

Profiling of molecular biofunctions potentially altered in the OB of Tg2576 mice. Functional analysis was performed with IPA software using exclusively the database information of experimental and predictive origin regarding central nervous system to be confident about the potential affected signaling pathways (see Supplementary Table S3 for details).

Figure 4

Modulation of the APP functional interactome in Tg2576 mice at the level of OB. Adaptation of APP functional network in Tg2576 OBs at 2 months of age (A) and 6 months of age (B). Relationships between differential expressed genes/proteins and APP functional interactors are represented with blue lines. Continuous lines represent direct interactions, while discontinuous lines correspond to indirect functional interactions. Up-regulated molecules in red, and down-regulated molecules in green (See complete legend at: http://qiagen.force.com/KnowledgeBase/KnowledgeNavigatorPage#).

Figure 5

Protein synthesis, and mitochondrial homeostasis are early compromised in Tg2576 at olfactory level. (A) Molecular network representing the up-regulation of ribosomal proteins (blue circles) in the OB of 2-month-old Tg2576. (B) Molecular network highlighting the dysregulation of specific components of the mitochondrial respiratory chain (Complex I, and Complex VI subunits) in the OB of 2-month-old Tg2576. (C) Time-dependent disruption of the olfactory Phb1 in Tg2576 mice. Phb expression was monitored by Western-blotting. (D) Time-dependent disruption of the olfactory Phb2 in Tg2576 mice. Phb expression was monitored by Western-blotting. Equal loading of the gels was assessed by stain-free digitalization. Panels show histograms of band densities. Data are presented as mean ± standard error of the mean (SEM) from three independent OB samples per group (*P < 0.05 vs. control group; **P < 0.01 vs. control group). Right graphs represent the expression of both Phb subunits during the aging process in WT and Tg2576 mice (2-, 6-, and 18-month-old).

Figure 6

APP overproduction affects the activation state of olfactory ERK1/2, Akt, and p38 mitogen-activated protein kinase (MAPK) at early AD stages in Tg2576 mice. Levels and residue-specific phosphorylation of MEK1/2 (A), ERK1/2 (B), Akt (C), and p38 MAPK-ATF2 axis (D). Equal loading of the gels was assessed by stain-free digitalization. Panels show histograms of band densities. Data are presented as mean ± SEM from three independent OB samples per group. *P < 0.05 vs. control group; **P < 0.01 vs. control group.

Figure 7

APP overproduction specifically modulates the activation of the SEK1/MKK4-stress-activated protein kinase (SAPK)/JNK axis in 6-month-old Tg2576 mice. Time-dependent expression of total and phosphorylated levels of SEK1 (A), SAPK/JNK (B), and protein kinase A (PKA; C). Equal loading of the gels was assessed by stain-free digitalization. Panels show histograms of band densities. Data are presented as mean ± SEM from three independent OB samples per group. *P < 0.05 vs. control group.

Figure 8

Monitorization of survival kinases during the aging process in WT and Tg2576 OBs. Western-blotting were performed for the kinase panel (total and phosphorylated levels) in the OB from WT and Tg2576 mice of 2, 6, and 18 months of age. Quantitation data were referred to the observed levels in 2-month-old mice for each condition. *P < 0.05 vs. 2-month-old mice; **P < 0.01 vs. 2-month-old mice.

Figure 9

Olfactory SEK1/MKK4 and PKA are differentially activated across Braak stages in human AD. Levels and residue-specific phosphorylation of SEK1/MKK4 (A), and PKA (B) in the OB across AD phenotypes. Equal loading of the gels was assessed by Ponceau staining and hybridization with a GAPDH specific antibody. Right panels show histograms of band densities. Data are presented as mean ± SEM from five independent OB samples per group. *P < 0.05 vs. control group; ***P < 0.001 vs. control group. Representative Western blot gels are shown.