The amyloid precursor protein (APP) intracellular domain regulates translation of p44, a short isoform of p53, through an IRES-dependent mechanism

Abstract

p44 is a short isoform of the tumor suppressor protein p53 that is regulated in an age-dependent manner. When overexpressed in the mouse, it causes a progeroid phenotype that includes premature cognitive decline, synaptic defects, and hyperphosphorylation of tau. The hyperphosphorylation of tau has recently been linked to the ability of p44 to regulate transcription of relevant tau kinases. Here, we report that the amyloid precursor protein (APP) intracellular domain (AICD), which results from the processing of the APP, regulates translation of p44 through a cap-independent mechanism that requires direct binding to the second internal ribosome entry site (IRES) of the p53 mRNA. We also report that AICD associates with nucleolin, an already known IRES-specific trans-acting factor that binds with p53 IRES elements and regulates translation of p53 isoforms. The potential biological impact of our findings was assessed in a mouse model of Alzheimer's disease. In conclusion, our study reveals a novel aspect of AICD and p53/p44 biology and provides a possible molecular link between APP, p44, and tau.

Keywords: AICD; Aging; Alzheimer's disease; IRES; p44; p53.

Fig. 1. APP can induce the expression of p44

(A) Quantitative real-time PCR determination of indicated kinases in the hippocampal formation. Animals (males) were 2.5-month-old when analyzed. All values are mean (n=5) ± sd. *p < 0.05; **p < 0.005. (B) Schematic view showing APP upstream of p44. (C) Western blot analysis showing induction of p44 as a result of APP overexpression. A longer exposure of the membrane (high exp.) is included to show the down-regulation of Delta133p53α. The nuclear marker H3 was used as loading control. (D) Schematic view of p53 isoforms showing the domains targeted by D01 and PAb240 antibodies. (E) The nuclear fraction probed with PAb240 in (C) was probed again with D01. (F) Western blot showing migration profile of p53 and p44 following transfection in SH-SY5Y cells. Lane 1, empty vector; lane 2, p53 cDNA; lane 3, p44 cDNA.

Fig. 2. p44 levels in the brain of late-onset AD patients, human trisomy 21 neurons, and brain of APP_695/swe and AICDTg mice

(A) Western blot showing levels of p53 and p44 in the brain of AD patients (mean age: 71 yr; range: 67–91 yr; sex: males) and age-matched controls (mean age: 70 yr; range: 66–78 yr; sex: males). Only the nuclear fraction was analyzed. Representative images are shown in the left panel while quantification is shown in the right panel. (B) Neurons differentiated from DS and control iPSCs. (C) Western blot showing increased levels of p44 in neurons differentiated from DS and control iPSCs. Representative images are shown in the left panel while quantification is shown in the right panel. Two independent lines are shown. (D) Western blot showing increased levels of p44 in the brain of APP_695/swe mice. Representative images are shown in the left panel while quantification is shown in the right panel. NTG, non-transgenic/wild-type. (E) Western blot showing increased levels of p44 in the brain of AICDTg mice. Representative images are shown in the left panel while quantification is shown in the right panel. NTG, non-transgenic/wild-type. All values are mean ± sd. **p < 0.005.

Fig. 3. AICD binds to IRES elements of the p53 mRNA and can induce cap-independent translation of p44

(A) Schematic view of the IRES elements in the p53 mRNA. (B) Quantitative real-time PCR showing that the levels of p44-specific mRNA are not affected by APP overexpression. Results are mean (n=6) ± sd. (C) Western blot showing the presence of AICD in the polysome fraction. N, nucleus; P, polysome; C, cytosol; Ctrl, control (non-transfected) cells; Empty, cells transfected with empty plasmid; APP, cells transfected with APP. (D) RNA-binding protein immunoprecipitation showing that AICD binds to the p53 mRNA in vivo. Input (lane 5) and negative controls (lanes 1–2) are also shown. (E–F) mRNA-protein pull-down showing that AICD binds to the IRES elements of the p53 mRNA in vitro. The pull-down was done with DS fibroblasts (E) and SH-SY5Y cells overexpressing APP (SH-SY5Y_APP; F). A fragment corresponding to the 3′ end of the p53 mRNA was used as negative control. (G) Schematic view of the bicistronic mRNA construct used in (H-J). The GFP was placed upstream of a hairpin structure (shown as a black circle) that impedes ribosomal read-through. The p44-specific IRES element without the first AUG site was placed after the hairpin structure and immediately upstream of the luciferase reporter system. For comparison, see a scheme of the normal p53 mRNA in (A). H-J, Overexpression of APP can induce cap-independent translation of p44. Expression of GFP (H and I) served as control. The luciferase reporter assay (J) was normalized to Renilla and expressed as mean (n=4) + sd. **p < 0.005.

Fig. 4. AICD interacts with nucleolin

(A–B) mRNA-protein pull-down showing that nucleolin (A) and PTBP1 (B) bind to the IRES elements of the p53 mRNA in vitro. The pull-down was done with DS fibroblasts. A fragment corresponding to the 3′ end of the p53 mRNA was used as negative control. (C) Immunoprecipitation of AICD from the cytosolic fraction of DS fibroblasts was able to pull-down nucleolin but not PTBP1. Immunoprecipitation was done with an antibody against the AICD peptide and Protein A (negative control). (D) Western blot showing levels of NCL in the brain of wild-type/non-transgenic mice. Representative Western blots are shown in the upper panel while quantification is shown in lower panel. Values are mean (n=3) ± sd. *p < 0.05.

Fig. 5. Haploinsufficiency of p53/p44 can rescue some of the phenotypic features caused by the overexpression of APP in the mouse

(A) Quantitative real-time PCR determination of indicated kinases in the hippocampal formation. Animals (males) were 2.5-month-old when analyzed. All values are mean (n=12) ± sd. *p < 0.05; **p < 0.005. (B) Immunostaining with anti-phospho-tau antibodies shows reduced labeling in APP_695/swe;p53^+/− mice. Two different antibodies were used: AT8 (against pSer202 and pThr205) and S356 (against pSer356). Mice (males) were ~1-year-old when analyzed. (C) Long-term potentiation (LTP) induction in hippocampal slices. Mice (males) were 2.5-month-old when analyzed. APP_695/swe mice display deficits in the late component of LTP. These deficits are rescued by p53/p44 haploinsufficiency. **p < 0.005.