Mol Neurobiol DOI 10.1007/s12035-015-9229-8
Opposite Dysregulation of Fragile-X Mental Retardation Protein and Heteronuclear Ribonucleoprotein C Protein Associates with Enhanced APP Translation in Alzheimer Disease Antonella Borreca 1 & Katia Gironi 2 & Giusy Amadoro 3,4 & Martine Ammassari-Teule 1,5
Received: 24 February 2015 / Accepted: 21 May 2015 # Springer Science+Business Media New York 2015
Abstract Amyloid precursor protein (APP) is overexpressed in familiar and sporadic Alzheimer Disease (AD) patients suggesting that, in addition to abnormalities in APP cleavage, enhanced levels of APP full length might contribute to the pathology. Based on data showing that the two RNA binding proteins (RBPs), Fragile-X Mental Retardation Protein (FMRP) and heteronuclear Ribonucleoprotein C (hnRNP C), exert an opposite control on APP translation, we have analyzed whether expression and translation of these two RBPs vary in relation to changes in APP protein and mRNA levels in the AD brain at 1, 3, and 6 months of age. Here, we show that, as expected, human APP is overexpressed in hippocampal total extract from Tg2576 mice at all age points. APP overexpression, however, is not stable over time but reaches its maximal level in 1-month-old mutants in association with the stronger (i) reduction of FMRP and (ii) augmentation of hnRNP C.
Electronic supplementary material The online version of this article (doi:10.1007/s12035-015-9229-8) contains supplementary material, which is available to authorized users. * Martine Ammassari-Teule
[email protected] 1
Institute of Cellular Biology and Neurobiology (IBCN), Consiglio Nazionale delle Ricerche, Via Del Fosso di Fiorano, 64 00143 Rome, Italy
2
University of Rome, La Sapienza. Piazza Aldo Moro, Rome, Italy
3
Institute of Translational Pharmacology (IFT)-National Research Council (CNR), Via Fosso del Cavaliere, 100-00133 Rome, Italy
4
European Brain Research Institute (EBRI), Via del Fosso di Fiorano, 64-65 00143 Rome, Italy
5
Fondazione Santa Lucia, Via del Fosso di Fiorano, 64 00143 Rome, Italy
APP levels then decrease progressively as a function of age in close relationship with the gradual normalization of FMRP and hnRNP C levels. Consistent with the mouse data, expression of FMRP and hnRNP C are, respectively, decreased and increased in hippocampal synaptosomes from sporadic AD patients. Our findings identify two RBP targets that might be manipulated for reducing abnormally elevated levels of APP in the AD brain, with the hypothesis that acting upstream of amyloidogenic processing might contribute to attenuate the amyloid burden. Keywords Alzheimer disease . RNA binding protein . APP . Translational control . SAD . FMRP . hnRNP C
Introduction Alzheimer disease (AD) is a progressive neurodegenerative disorder with histopathological hallmarks including an excessive accumulation of toxic extracellular proteinaceous deposits (amyloid plaques) and intraneuronal bundles of paired helical filaments (neurofibrillary tangles) in brain regions critical for cognition [1, 2]. The plaques are predominantly composed of β-amyloid (Aβ), a 39–42 amino acid peptide, abnormally cleaved from the amyloid precursor protein (APP). Accordingly, therapeutic strategies against AD have focused on the molecular pathways responsible for abnormal APP cleavage with the objective of correcting it to reduce the levels of this plaque-promoting peptide [3–10] There is however evidence that, due to alterations in APP mRNA translation, APP full length is overexpressed in human platelets [11] and fibroblasts [12] of familiar AD as well as in the temporal cortex of sporadic AD [13], suggesting that AD-related pathological changes might be secondary to enhanced APP expression.
Mol Neurobiol
Importantly, data obtained in vitro [14–16] indicate that APP mRNA is subjected to extensive regulation by two posttranscriptional RNA binding proteins (RBPs): Fragile-X Mental Retardation Protein (FMRP) and heteronuclear Ribonucleoprotein C (hnRNP C). Specifically, FMRP and hnRNP C bind to the same segment of the APP mRNA coding region but FMRP, which is a negative regulator of translation [17], represses it [18] while hnRNP C, which also binds to the 3′UTR region of APP mRNA, stabilizes and enhances it [19, 20]. How these two RBPs are translated and expressed in the AD brain is, however, poorly understood. On the one hand, contrasting findings emerged from two recent studies in which cortical levels of FMRP were measured in APPswe/PS1ΔE9 mutant mice or sporadic AD (SAD) patients. FMRP expression was found either unaltered in both mutant mice and in SAD patients [21] or enhanced in the same mutants [22]. On the other hand, whether hnRNP C expression is modified in the AD brain has, to our knowledge, not yet been examined. To explore the translational control of FMRP and hnRNP C on APP levels in the AD brain, we monitored expression and translation of these three proteins in the hippocampus of Tg2576 mutant mouse overexpressing mutant hsweAPP. Measurements were performed at three age points corresponding to the presymptomatic (1 month), early symptomatic (3 months), and fully symptomatic (6 months) stages of the pathology. Then, to verify the translational value of our observations, we controlled how APP, FMRP, and hnRNP C are expressed in SAD hippocampal synaptosomes.
Materials and Methods Mice Male mice overexpressing the APP695 fragment with the Swedish mutation (TgHuAPP695swe: Tg2576) in a hybrid genetic background (87 % C57BL/6×12.5 % SJL) were subsequently backcrossed to C57BL/6×SJL F1 females. The offspring was genotyped to confirm the presence of human mutant APP DNA sequence by PCR. Each experiment was carried out in transgenic mice and wild-type (WT) mice of 1, 3, and 6 months of age. Mice were maintained on a 12-h light/ dark cycle with ad libitum access to food and water. All experiments were performed in accordance with the guidelines provided by the European Communities Council Directive of 24 November 1986 (86/609/EEC). Western Blot Mice were sacrificed by cervical dislocation, and their brain was rapidly removed for dissection of the hippocampi. The hippocampi were homogenized and extracted in RIPA buffer [10 mM Tris-HCl, pH 7.5, 150 mM NaCl, 2 % Nonidet P-40, 5 mM EDTA, 0.1 mM phenylmethylsulfonyl fluoride, 1 mM β-glycerophosphate, 1 mM sodium orthovanadate, 10 mM sodium fluoride, 0.1 M SDS, 1 % protease inhibitor cocktail—Sigma-Aldrich] for 5 min on ice and
centrifuged for 10 min at 4 °C (8000 rpm). The supernatant was collected, and protein content was quantified by Bradford colorimetric assay (Bio-Rad, Milano, Italy). Homogenates containing 30 μg of total protein were separated by 4–15 % gradient sodium dodecyl sulfate–polyacrylamide gels (BioRad Laboratories, Hercules, CA) and transferred to PVDF membrane (Bio-Rad Laboratories, Hercules, CA). Western blots were blocked in 5 % nonfat dry milk in TBST buffer (0.1 % Tween 20 in Tris–borate saline) then incubated with FMRP antibody (1:1000; Abcam; 1:1000 Cell signaling), hnRNP C (1:1000; Santa Cruz and Abcam) APP (1:2000; Sigma), Aβ-42 (1:1000; Cell signaling), and Presenilin 1 (1:500; Millipore). Blots were then incubated with appropriate conjugated to horseradish peroxidase (Chemicon) and developed by ECL Western Blotting Analysis System (GE Healthcare). Anti-GAPDH antibody (1:1000; Millipore) and β-actin (1:1000, Sigma) anti-GAPDH and β-actin antibody were used as a loading control for all experiments. Optical densities of the bands were analyzed using NIH Image software. RNA Extraction The hippocampi from Tg2576 and WT mice of different age were used for the analysis. The RNA extraction was conducted according to the procedure based on Trizol reagent (Invitrogen). One microliter of RNA extract was used for retrotranscription followed by PCR. RT-Quantitative-PCR Analysis First-strand synthesis was achieved using p (dN) 6 and 100 U of M-MLV RT (Invitrogen). An aliquot (1 μl) was used in a PCR reaction with Taq polymerase using gene-specific primers for APP human, APP mouse, fmr1, hnRNP C, and GAPDH mRNAs (see Table 1 for primers sequences). Immunofluorescence Hippocampal slices of 40 μm obtained from Tg2576 and WT mice of each age group were used for immunofluorescence staining. After three washes with PBS 1X, the slices were treated with NH4CL for 30 min and then with blocking solution (PBS 1X with 3 % normal goat serum). Table 1
List of primer sequences used for RT-PCR experiments
Gene
Sequence (5′–3′)
Human APP
FOR-GCCAAAGAGACATGCAGTGA REV-AGTCATCCTCCTCCGCATC FOR-GACAAGAAGGCCGTTATCC REV-GTCTCTCATTGGCTGCTTTCC FOR-GAAGCCAGTAAACAACTGGAG REV-CACTGCATCTTGATCCTCTC FOR-GGAAAATTGAAAGGTGATGAC REV-GAGAAGGACAAGTCTGCTTG FOR-GTGAACGGATTTGGCCGTAT REV-GAATTTGCCGTGAGTGGAGT
Mouse APP fmr1 hnRNPC GAPDH
Mol Neurobiol
The incubation with primary antibody (anti-FMRP; 1:200 Cell signaling and hnRNP C; 1:1000 Abcam) in PBS 1X plus triton 0.3 % will be conducted overnight at 4 °C. Then, slices were washed three times with PBS 1X and incubated with Cy3-conjugated goat anti-rabbit secondary antibody for 2 h at room temperature avoiding light. DAPI staining (1:1000, Enzo Life Science) was performed at the last wash with PBS 1X. At least the sections were mounted with fluoromount (sigma) and coverslipped. The staining was visualized with a confocal microscope (Zeiss LSM700; objective ×10). Crude Synaptosomal Fraction from Patient Donor Samples Hippocampus samples obtained from brains of one sporadic AD patient and an age-matched control patient were homogenized in 3 ml of homogenization buffer (320 mM sucrose, 4 mM Hepes ph 7.4; 1 mM EGTA; 0.4 M PMSF; 1 mM NaOVa; 10 mM NaF; 1 mM β-glicerophosphate and 1× of protease inhibitor cocktail (Sigma) with 10 strokes in a right fitting glass dounce tissue grinder. The homogenate was centrifuged at 1000g for 10 min. The supernatant was collected and centrifuged at 12,000g for 15 min, and the second pellet was re-suspended in 2.5 mL of homogenization buffer and centrifuged at 13,000g for 15 min. The resulting pellet representing the crude synaptosomal fraction (CSF) was resuspended in 0.5 ml of homogenization buffer.
Statistical Analyses
Fig. 1 Analysis of APP expression and translation in Tg2576 and WT mice during development and adulthood. a Quantitative analysis of APP levels in hippocampal extracts and representative immunoblots used for quantification. b Quantitative analysis of APP mRNA levels measured on RNA extracted from Tg2576 and WT hippocampi. The analysis of mRNA was carried out according to the ΔΔct analysis. Data were normalized with GAPDH and expressed as mean percentage±s.e.m. (*P
