A PIN1 polymorphism that prevents its suppression by AP4 associates with delayed onset of Alzheimer's disease

Abstract

Alzheimer's disease (AD), the most common form of dementia, is characterized by the presence of neurofibrillary tangles composed of tau and senile plaques of amyloid-beta peptides (Aβ) derived from amyloid precursor protein (APP). Pin1 is a unique prolyl isomerase that has been shown to protect against age-dependent neurodegeneration by acting on phosphorylated tau and APP to suppress tangle formation and amyloidogenic APP processing. Here we report a functional polymorphism, rs2287839, in the Pin1 promoter that is significantly associated with a 3-year delay in the average age at onset (AAO) of late-onset AD in a Chinese population. More significantly, the Pin1 polymorphism rs2287839 is located within the consensus binding motif for the brain-selective transcription factor, AP4 (CAGCTG) and almost completely abolishes the ability of AP4 to bind and suppress the Pin1 promoter, as shown by chromatin immunoprecipitation, electrophoretic mobility shift assay, and promoter luciferase assay. Moreover, overexpression or knockdown of AP4 resulted in an 80% reduction or 2-fold increase in endogenous Pin1 levels, respectively. Thus, AP4 is a novel transcriptional repressor of Pin1 expression and the Pin1 promoter single nucleotide polymorphism (SNP) identified in this study that prevents such suppression is associated with delayed onset of AD. These results indicate that regulation of Pin1 by AP4 plays a critical role in determining age at onset of AD and might be a novel therapeutic target to delay the onset of AD.

Figure 1. Kaplan–Meier survival plots for rs2287839

256 AD patients diagnosed as probable or possible AD according to NINCDS-ADRAD criteria were recruited and genotyped in this study. The plot showing that AD patients with Pin1 SNP rs2287839 (CG genotype) had a 3-year delay in the age of AD onset compared to those with the wild-type GG genotypes.

Figure 2. Pin1 SNP rs2287839 severely impairs binding of AP4 to the Pin1 promoter

(A) Chromatin immunoprecipitation (ChIP) assay on H4 neuronal cells showing AP4 binds to the AP4-binding site on the Pin1 promoter. (B) Allele-specific binding of AP4. EMSA analysis of allele-specific effect of G (wild-type) and C (mutant) on the interaction of nuclear protein complexes extracted from H4 cells. Wild type G allele (Lane 2 and 3) formed strong complexes with AP4, while C variant (Lane 4 and 5) showed 75% reduction of binding to AP4. Competition experiments were performed with increasing concentrations (100–1000 fold) of unlabeled oligonucleotides (Lane 6–9, 11–14). (C) The images were quantified from three independent experiments using Image J analysis, with the wild-type binding being defined as 100% (Lane 1). Values are mean ± SEM from three independent experiments.

Figure 3. Pin1 SNP rs2287839 completely abolishes the ability of AP4 to repress the Pin1 promoter

(A) Graph showing transcriptional activity of wild-type G or mutant C Pin1 promoter (5.8 kb) luciferase reporter cotransfected with increasing doses of AP4 DNA. AP4 suppresses wild-type Pin1 promoter activity in a dose-dependent manner, whereas the Pin1 mutant promoter showed significantly higher activity and its activity is not affected by AP4, even at high doses. (B) Graph showing transcriptional activity of long (5.8 kb) promoter containing the AP4 binding site or short (2.3 kb) promoter without the AP4 binding site. Note, the dose of AP4 differs between panels A and B. Results showed the AP4 binding site in the long promoter is important for the response to AP4. Values are mean ±SEM from three independent experiments. *p<0.05; ** p<0.001.

Figure 4. AP4 overexpression or knockdown suppresses or enhances Pin1 protein levels, respectively

(A) Overexpression of AP4 in H4 neuronal cells significantly suppressed the expression on Pin1 whereas AP4 knockdown by shRNA increased the expression of Pin1. (B) Graph obtained from three independent experiments showing overexpression of AP4 resulted in an 80% reduction of Pin1 expression but AP4 knockdown increased the expression of Pin1 by two fold.