MPEP reduces seizure severity in Fmr-1 KO mice over expressing human Abeta

Abstract

Metabotropic glutamate receptor 5 (mGluR(5)) regulates the translation of amyloid precursor protein (APP) mRNA. Under resting conditions, mRNA is bound to and translationally repressed by the fragile X mental retardation protein (FMRP). Upon group 1 mGluR activation, FMRP dissociates from the mRNA and translation ensues. APP levels are elevated in the dendrites of primary neuronal cultures as well as in synaptoneurosomes (SN) prepared from embryonic and juvenile fmr-1 knockout (KO) mice, respectively. In order to study the effects of APP and its proteolytic product Abeta on Fragile X syndrome (FXS) phenotypes, we created a novel mouse model (FRAXAD) that over-expresses human APPSwe/Abeta in an fmr-1 KO background. Herein, we assess (1) human APP(Swe) and Abeta levels as a function of age in FRAXAD mice, and (2) seizure susceptibility to pentylenetetrazol (PTZ) after mGluR(5) blockade. PTZ-induced seizure severity is decreased in FRAXAD mice pre-treated with the mGluR(5) antagonist MPEP. These data suggest that Abeta contributes to seizure incidence and may be an appropriate therapeutic target to lessen seizure pathology in FXS, Alzheimer's disease (AD) and Down syndrome (DS) patients.

Keywords: APP; FMRP; FRAXAD; PTZ; Tg2576; seizure; β-amyloid.

Figure 1

WT and fmr-1 KO mice (3.5 months old) have equivalent levels of brain APP and Aβ. (A) APP levels in lysates from hippocampus, cortex and cerebellum brain regions of WT and fmr-1 KO mice (25 μg per lane) were assessed by western blot analyses. Phosphorimager units for APP were normalized to β-Actin levels. All WT samples were set to 100%. There was no statistical difference between WT (n = 3) and fmr-1 KO mice (n = 3 mice). (B) Aβ_1–40 and (C) Aβ_1–42 levels in hippocampus, cortex and cerebellum brain regions of WT (n = 3) and fmr-1 KO (n = 3) mice were assessed by ELISA and presented as a percentage compared to levels in WT hippocampus. The 1.5X increase in Aβ_1–42 levels in fmr-1 KO brain was not statistically significant (p = 0.14).

Figure 2

APP and Aβ Levels in Young Adult Tg2576 and FRAXAD Mice. GnHCl-soluble lysates were prepared from brain hemispheres harvested from 2-month old mice and diluted 1:100 to reduce the GnHCl concentration to below 0.1 M. Lysates were further diluted 1:5 for APP/APPα analyses. (A) APP/APPα and (B) Aβ_1–40 ELISAs were performed as described in the Methods Section and plotted as pg/μg lysate. There were no statistically significant differences between Tg2576 (Tg) [females (F): n = 7, males (M): n = 8] and FRAXAD (FR) [F: n = 7, M: n = 7] mice.

Figure 3

FRAXAD Mice (16–18 months) Exhibit Elevated Brain APP/APPα Compared to Age-Matched Tg2576. GnHCl-soluble lysates were prepared from 16–18-month old Tg2576 (n = 4) and FRAXAD (n = 3) mice and diluted 1:800 (APP/APPα), 1:25,000 (Aβ_1–40) or 1:75,000 (Aβ_1–42) for ELISA analyses of (A) APP/APPα in brain hemispheres and (B) Aβ_1–40 and (C) Aβ_1–42 in hippocampus, cortex and cerebellum brain regions. There was a statistically significant 28% increase in APP/APPα levels in FRAXAD brain (p<0.05). There was a trend for increased Aβ_1–40 and Aβ_1–42 in cortex, hippocampus and cerebellum but the differences were not statistically significant between Tg2576 and FRAXAD.

Figure 4

Tg2576 and FRAXAD Mice Have Equivalent α- and β-Secretase Activity. The enzymatic activities of α-secretase (A) and β-secretase (B) were quantitated by R&D Systems fluorometric assays of whole brain lysates prepared from Tg2576 (n = 3) and FRAXAD (n = 3) mice (9 months old). The proform and mature forms of BACE were assessed by western blot analyses (C) and quantitated with ImageQuant software (Molecular Dynamics, Inc.) (D). Lysates (100 μg per lane) prepared in cell extraction buffer were separated by 12% SDS-PAGE and transferred to 0.45 u,m nitrocellulose. Blocked membranes were hybridized with anti-BACE (dilution, 1 u,g/mL, Zymed catalog #34–4900) and anti-SNAP25 (dilution, 1:2000, Abcam catalog #ab5666) followed by anti-rabbit HRP-conjugated secondary antibody and visualization with ECL+, n = 3 each.

Figure 5

Model of FMRP-Mediated Regulation of APP Translation. APP mRNA is a synaptic target for regulation by FMRP. Through UV crosslinking CLIP assays, we've shown that FMRP binds directly to the coding region of APP mRNA at a guanine-rich region. FMRP also protects a 29-base cis-element in the 3'-UTR from ribonuclease digestion of anti-FMRP immunoprecipitates [1]. RNA binding proteins such as nucleolin, hnRNP C and YB1 bind to cis-elements in the 3'-UTR [–45]. Nucleolin and YB1 are protein binding factors of FMRP [–47], which suggests that that protein/protein interactions bring multiple cis-elements in APP mRNA in close proximity to regulate translation (Repressed Translation State). Stimulation of cortical SN with DHPG, a group 1 mGluR agonist, releases FMRP from APP mRNA while increasing APP translation (Regulated Translation State). In the absence of FMRP (fmr-1 KO SN and primary neuronal cells), basal APP levels are increased and nonresponsive to mGluR₅ signaling (FXS: Constitutive Translation State). mRNA/protein interactions are likewise important for the movement of APP mRNA from the soma to the dendrites as human APP_Swe, which lacks the 3'-UTR, is localized in the soma.