Functional activity of the novel Alzheimer's amyloid �-peptide interacting domain (A�ID) in the APP (original) (raw)
Related papers
Gene, 2011
Amyloid β peptide (Aβ) plaque in the brain is the primary (post mortem) diagnostic criterion of Alzheimer's disease (AD). Any physiological role of Aβ constituent is poorly understood. We have previously determined an Aβ interacting domain (AβID) in the promoters of AD-associated genes (Maloney and Lahiri, 2011). This AβID interacts in a DNA-sequence specific manner with Aβ. We now demonstrate novel Aβ activity as a possible transcription factor. Herein, we demonstrate Aβ-chromatin interaction in cell culture by ChIP assay. We observed that human neuroblastoma (SK-N-SH) cells treated with FITC conjugated Aβ1-40 localized Aβ to the nucleus in the presence of H 2 O 2-mediated oxidative stress. Furthermore, primary rat fetal cerebrocortical cultures were transfected with APP and BACE1 promoter-luciferase fusions, and rat PC12 cultures were transfected with polymorphic APP promoter-CAT fusion clones. Transfected cells were treated with different Aβ peptides and/or H 2 O 2. Aβ treatment of cell cultures produced a DNA sequence-specific response in cells transfected with polymorphic APP clones. Our results suggest the Aβ peptide may regulate its own production through feedback on its precursor protein and BACE1, leading to amyloidogenesis in AD.
Gene, 2011
Deposition of extracellular plaques, consisting of amyloid β peptide (Aβ), in the brain is the confirmatory diagnostic of Alzheimer's disease (AD); however, the physiological and pathological role of Aβ is not fully understood. Herein, we demonstrate novel Aβ activity as a putative transcription factor upon AD-associated genes. We used oligomers from 5'-flanking regions of the apolipoprotein E (APOE), Aβ-precursor protein (APP) and β-amyloid site cleaving enzyme-1 (BACE1) genes for electrophoretic mobility shift assay (EMSA) with different fragments of the Aβ peptide. Our results suggest that Aβ bound to an Aβ-interacting domain (AβID) with a consensus of "KGGRKTGGGG". This peptide-DNA interaction was sequence specific, and mutation of the first "G" of the decamer's terminal "GGGG" eliminated peptide-DNA interaction. Furthermore, the cytotoxic Aβ25-35 fragment had greatest DNA affinity. Such specificity of binding suggests that the AβID is worth of further investigation as a site wherein the Aβ peptide may act as a transcription factor.
2010
Amyloidogenic processing of the amyloid precursor protein (APP) by and ␥-secretases generates several biologically active products, including amyloid- (A) and the APP intracellular domain (AICD). AICD regulates transcription of several neuronal genes, especially the A-degrading enzyme, neprilysin (NEP). APP exists in several alternatively spliced isoforms, APP 695 , APP 751 , and APP 770. We have examined whether each isoform can contribute to AICD generation and hence up-regulation of NEP expression. Using SH-SY5Y neuronal cells stably expressing each of the APP isoforms, we observed that only APP 695 up-regulated nuclear AICD levels (9-fold) and NEP expression (6-fold). Increased NEP expression was abolished by a or ␥-secretase inhibitor but not an ␣-secretase inhibitor. This correlated with a marked increase in both A 1-40 and A 1-42 in APP 695 cells as compared with APP 751 or APP 770 cells. Similar phenomena were observed in Neuro2a but not HEK293 cells. SH-SY5Y cells expressing the Swedish mutant of APP 695 also showed an increase in A levels and NEP expression as compared with wild-type APP 695 cells. Chromatin immunoprecipitation revealed that AICD was associated with the NEP promoter in APP 695 , Neuro2a, and APP Swe cells but not APP 751 nor APP 770 cells where AICD was replaced by histone deacetylase 1 (HDAC1). AICD occupancy of the NEP promoter was replaced by HDAC1 after treatment of the APP 695 cells with a but not an ␣-secretase inhibitor. The increased AICD and NEP levels were significantly reduced in cholesterol-depleted APP 695 cells. In conclusion, A and functional AICD appear to be preferentially synthesized through -secretase action on APP 695 .
The FASEB Journal, 2005
Alzheimer's disease is characterized by brain deposition of toxic amyloid β-peptide (Aβ), generated from the Aβ precursor protein (APP). APP gene expression is regulated via the proximal promoter region (PPR; −46/−1 in the human sequence; +1 transcription start) and the 5′-untranslated region (5′-UTR; +1/+147). We have recently identified a unique CAGA sequence, "amyloid," (+83/+86) present only in the APP gene from amyloid plaque-forming species, absent in all APP-like-proteins' (APLP1 and APLP2) genes. To assay functional activity of PPR + UTR and 5′-UTR regions that either contain or lack the "amyloid" box, we tested nine constructs in transient transfection studies. We observed significantly high reporter gene activity with −46/144, −46/100, −46/54, and 54/144 constructs. The 54/100 fragment, which contains a transforming growth factor-β/"amyloid"/interleukin-1 acute box cassette, showed different activity depending on cell type. Electrophoretic mobility shift assay (EMSA) showed distinct DNA-nuclear protein interaction in all fragments, differing among both cell types and specific fragment. Reporter gene expression corroborates with the DNA-protein binding pattern. To directly examine the "amyloid" box, we generated oligomers for CAGA mutants or mutated adjacent nucleotides. EMSA results showed that altering "amyloid" or adjacent sequence alters specific DNA-nuclear protein interaction in both mutation-and cell-type-specific manners. Reporter gene assay reveals mutant-specific expression effects. Therefore, the −46/54 region appears to be essential for basal expression of the APP gene, the 54/100 and 100/144 regions may have tissue-specific activity, and the "amyloid" CAGA box plays a role in APP gene regulation.
The coding sequence of amyloid-β precursor protein APP contains a neural-specific promoter element
Brain Research, 2006
The amyloid-β precursor protein APP is generally accepted to be involved in the pathology of Alzheimer's disease. Since its physiological role is still unclear, we decided to study the function of APP via stable transgenesis in the amphibian Xenopus laevis. However, the application of constructs encoding (mutant) APP fused to the C-terminus of the green fluorescent protein GFP (GFP-APP), and harboring a tissue-specific or an inducible gene promoter did not result in transgene expression of APP in neuronal and neuroendocrine cells. Surprisingly, a construct encoding either Xenopus or human APP fused to the Nterminus of GFP (APP-GFP) gave fluorescence throughout the whole brain of the tadpole, despite the fact that a proopiomelanocortin gene promoter was used to target transgene expression specifically to the intermediate pituitary cells. Detailed analysis with deletion mutants revealed the presence of a neural-specific, transcriptionally active DNA element within the 3′-end of the APP-coding sequence that gave rise to an aberrant transcript and protein in the APP-GFP transgenic animals. The DNA element appears to prevent proper APP transgene expression in Xenopus neuronal and neuroendocrine cells. Thus, the coding sequences of Xenopus and human APP contain a neural-specific promoter element, the physiological significance of which is at present unclear.
Molecular Brain Research, 1994
We have investigated protein-DNA interactions in the proximal promotor of the human amyloid precursor protein (APP) gene in temporal lobe neocortical nuclei isolated from control and Alzheimer disease (AD) affected brains. We report that the human APP 5' promotor sequence from -203 to+55 bp, which has been previously reported to contain essential regulatory elements for APP gene transcription, lies in a deoxyribonuclease I, micrococcal nuclease-and restriction endonuclease-sensitive, G+C-rich nucleosome-free gap flanked both 5' and 3' by typical nucleosome structures. As analyzed by electrophoretic mobility shift assay, this extended internucleosomal linker DNA is heavily occupied by nuclear protein factors, and interacts differentially with nuclear protein extracts obtained from HeLa and human brain neocortical nuclei. This suggests that the chromatin conformation of the APP gene promoter may vary in different cell types, and may correlate with differences in APP gene expression. Human recombinant transcription factors AP1, SP1 and TFIID (but not AP2 or brain histones H1, H2B and H4) interact with the -203 to + 55 bp of the human APP promotor sequence. Only minor differences were observed in the chromatin structure of the immediate APP promotor between non-AD and AD affected neocortical nuclei, suggesting either that post-transcriptional processes, or that regulatory
The FASEB Journal, 2005
Alzheimer's disease is characterized by brain deposition of toxic amyloid beta-peptide (Abeta), generated from the Abeta precursor protein (APP). APP gene expression is regulated via the proximal promoter region (PPR; -46/-1 in the human sequence; +1 transcription start) and the 5'-untranslated region (5'-UTR; +1/+147). We have recently identified a unique CAGA sequence, "amyloid" (+83/+86) present only in the APP gene from amyloid plaque-forming species, absent in all APP-like-proteins' (APLP1 and APLP2) genes. To assay functional activity of PPR + UTR and 5'-UTR regions that either contain or lack the "amyloid" box, we tested nine constructs in transient transfection studies. We observed significantly high reporter gene activity with -46/144, -46/100, -46/54, and 54/144 constructs. The 54/100 fragment, which contains a transforming growth factor-beta/ "amyloid"/interleukin-1 acute box cassette, showed different activity depending o...
Signaling effect of amyloid-β42 on the processing of AβPP
Experimental Neurology, 2010
The effects of amyloid-β are extremely complex. Current work in the field of Alzheimer disease is focusing on discerning the impact between the physiological signaling effects of soluble low molecular weight amyloid-β species, and the more global cellular damage that could derive from highly concentrated and/or aggregated amyloid. Being able to dissect the specific signaling events, to understand how soluble amyloid-β induces its own production by upregulating BACE1 expression, could lead to new tools to interrupt the distinctive feedback cycle with potential therapeutic consequences. Here we describe a positive loop that exists between the secretases that are responsible for the generation of the amyloid-β component of Alzheimer disease. According to our hypothesis, in familial Alzheimer disease, the primary overproduction of amyloid-β can induce BACE1 transcription and drive a further increase of amyloid-β precursor protein processing and resultant amyloid-β production. In sporadic Alzheimer disease, many factors, among them oxidative stress and inflammation, with consequent induction of presenilins and BACE1, would activate a loop and proceed with the generation of amyloid-β and its signaling role onto BACE1 transcription. This concept of a signaling effect by and feedback on the amyloid-β precursor protein will likely shed light on how amyloid-β generation, oxidative stress, and secretase functions are intimately related in sporadic Alzheimer disease.
Journal of Alzheimer's Disease
Background: Recent studies suggest a strong association between neuronal DNA damage, elevated levels of amyloid-β (Aβ), and regions of the brain that degenerate in Alzheimer’s disease (AD). Objective: To investigate the nature of this association, we tested the hypothesis that extensive DNA damage leads to an increase in Aβ40 and Aβ42 generation. Methods: We utilized an immortalized human neuronal progenitor cell line (NPCs), ReN VM GA2. NPCs or 20 day differentiated neurons were treated with hydrogen peroxide or etoposide and allowed to recover for designated times. Sandwich ELISA was used to assess secreted Aβ40 and Aβ42. Western blotting, immunostaining, and neutral comet assay were used to evaluate the DNA damage response and processes indicative of AD pathology. Results: We determined that global hydrogen peroxide damage results in increased cellular Aβ40 and Aβ42 secretion 24 h after treatment in ReN GA2 NPCs. Similarly, DNA double strand break (DSB)-specific etoposide damage ...
A putative role of the Amyloid Precursor Protein Intracellular Domain (AICD) in transcription
Acta neurobiologiae experimentalis, 2008
Amyloid Precursor Protein (APP) Intracellular Domain (AICD) is the product of APP processing realized by alpha- or beta-secretases and gamma-secretase. It was shown that AICD is able to interact with several proteins which regulate its stability and cellular localization. The Fe65 adaptor protein translocates AICD into nucleus where the APP-Fe65-Tip60 ternary complex may activate transcription of target genes. In the light of recent studies AICD seems to be another product of APP proteolysis endowed with important biological functions that may contribute to Alzheimer's disease pathology.