Amyloid Precursor Protein Promoter Research Articles

Histone deacetylase-3 regulates the expression of the amyloid precursor protein and its inhibition promotes neuroregenerative pathways in Alzheimer's disease models.

HDAC3 inhibition has been shown to improve memory and reduce amyloid-β (Aβ) in Alzheimer's disease (AD) models, but the underlying mechanisms are unclear. We investigated the molecular effects of HDAC3 inhibition on AD pathology, using invitro and exvivo models of AD, based on our finding that HDAC3 expression is increased in AD brains. For this purpose, N2a mouse neuroblastoma cells as well as organotypic brain cultures (OBCSs) of 5XFAD and wild-type mice were incubated with various concentrations of the HDAC3 selective inhibitor RGFP966 (0.1-10 μM) for 24 h. Treatment with RGFP966 or HDAC3 knockdown in N2a cells was associated with an increase on amyloid precursor protein (APP) and mRNA expressions, without alterations in Aβ42 secretion. Invitro chromatin immunoprecipitation analysis revealed enriched HDAC3 binding at APP promoter regions. The increase in APP expression was also detected in OBCSs from 5XFAD mice incubated with 1 μM RGFP966, without changes in Aβ. In addition, HDAC3 inhibition resulted in a reduction of activated Iba-1-positive microglia and astrocytes in 5XFAD slices, which was not observed in OBCSs from wild-type mice. mRNA sequencing analysis revealed that HDAC3 inhibition modulated neuronal regenerative pathways related to neurogenesis, differentiation, axonogenesis, and dendritic spine density in OBCSs. Our findings highlight the complexity and diversity of the effects of HDAC3 inhibition on AD models and suggest that HDAC3 may have multiple roles in the regulation of APP expression and processing, as well as in the modulation of neuroinflammatory and neuroprotective genes.

Association of APP gene polymorphisms and promoter methylation with essential hypertension in Guizhou: a case–control study

BackgroundSingle-nucleotide polymorphisms (SNPs) and DNA methylation are crucial regulators of essential hypertension (EH). Amyloid precursor protein (APP) mutations are implicated in hypertension development. Nonetheless, studies on the association of APP gene polymorphism and promoter methylation with hypertension are limited. Therefore, this case–control aims to evaluate the genetic association of APP gene polymorphism and promoter methylation with EH in Guizhou populations.Objective and methodsWe conducted a case–control study on 343 EH patients and 335 healthy controls (including Miao, Buyi, and Han populations) in the Guizhou province of China to analyze 11 single-nucleotide polymorphisms (rs2040273, rs63750921, rs2211772, rs2830077, rs467021, rs368196, rs466433, rs364048, rs364051, rs438031, rs463946) in the APP gene via MassARRAY SNP. The MassARRAY EpiTYPER was employed to detect the methylation levels of the promoters.ResultsIn the Han population, the rs2211772 genotype distribution was significantly different between disease and control groups (χ2 = 6.343, P = 0.039). The CC genotype reduced the risk of hypertension compared to the TT or TC genotype (OR 0.105, 95%CI 0.012–0.914, P = 0.041). For rs2040273 in the Miao population, AG or GG genotype reduced the hypertension risk compared with the AA genotype (OR 0.533, 95%CI 0.294–0.965, P = 0.038). Haplotype TCC (rs364051–rs438031–rs463946) increased the risk of EH in Guizhou (OR 1.427, 95%CI 1.020–1.996, P = 0.037). Each 1% increase in CpG_19 (− 613 bp) methylation level was associated with a 4.1% increase in hypertension risk (OR 1.041, 95%CI 1.002–1.081, P = 0.039). Each 1% increase in CpG_1 (− 296 bp) methylation level was associated with an 8% decrease in hypertension risk in women (OR 0.920, 95%CI 0.860–0.984, P = 0.015). CpG_19 significantly correlated with systolic blood pressure (r = 0.2, P = 0.03). The methylation levels of CpG_19 in hypertensive patients with rs466433, rs364048, and rs364051 minor alleles were lower than that with wild-type alleles (P < 0.05). Moreover, rs467021 and rs364051 showed strong synergistic interaction with EH (χ2 = 7.633, P = 0.006). CpG_11, CpG_19, and rs364051 showed weak synergistic interaction with EH (χ2 = 19.874, P < 0.001).ConclusionIn summary, rs2211772 polymorphism and promoter methylation level of APP gene may be linked to EH in Guizhou populations. Our findings will provide novel insights for genetic research of hypertension and Alzheimer's disease.

Amyloid β induces interneuron-specific changes in the hippocampus of APPNL-F mice.

Alzheimer’s disease (AD) is a neurodegenerative disorder characterized by cognitive decline and amyloid-beta (Aβ) depositions generated by the proteolysis of amyloid precursor protein (APP) in the brain. In APPNL-F mice, APP gene was humanized and contains two familial AD mutations, and APP–unlike other mouse models of AD–is driven by the endogenous mouse APP promoter. Similar to people without apparent cognitive dysfunction but with heavy Aβ plaque load, we found no significant decline in the working memory of adult APPNL-F mice, but these mice showed decline in the expression of normal anxiety. Using immunohistochemistry and 3D block-face scanning electron microscopy, we found no changes in GABAA receptor positivity and size of somatic and dendritic synapses of hippocampal interneurons. We did not find alterations in the level of expression of perineuronal nets around parvalbumin (PV) interneurons or in the density of PV- or somatostatin-positive hippocampal interneurons. However, in contrast to other investigated cell types, PV interneuron axons were occasionally mildly dystrophic around Aβ plaques, and the synapses of PV-positive axon initial segment (AIS)-targeting interneurons were significantly enlarged. Our results suggest that PV interneurons are highly resistant to amyloidosis in APPNL-F mice and amyloid-induced increase in hippocampal pyramidal cell excitability may be compensated by PV-positive AIS-targeting cells. Mechanisms that make PV neurons more resilient could therefore be exploited in the treatment of AD for mitigating Aβ-related inflammatory effects on neurons.

Interaction of Sp1 and APP promoter elucidates a mechanism for Pb2+ caused neurodegeneration

A ubiquitously expressed transcription factor, specificity protein 1 (Sp1), interacts with the amyloid precursor protein (APP) promoter and likely mediates APP expression. Promoter-interaction strengths variably regulate the level of APP expression. Here, we examined the interactions of finger 3 of Sp1 (Sp1–f3) with a DNA fragment containing the APP promoter in different ionic solutions using atomic force microscope (AFM) spectroscopy. Sp1–f3 molecules immobilized on an Si substrate were bound to the APP promoter, which was linked to the AFM tips via covalent bonds. The interactions were strongly influenced by Pb2+, considering that substituting Zn2+ with Pb2+ increased the binding affinity of Sp1 for the APP promoter. The results revealed that the enhanced interaction force facilitated APP expression and that APP overexpression could confer a high-risk for disease incidence. An increased interaction force between Sp1–f3 and the APP promoter in Pb2+ solutions was consistent with a lower binding free energy, as determined by computer-assisted analysis. The impact of Pb2+ on cell morphology and related mechanical properties were also detected by AFM. The overexpression of APP caused by the enhanced interaction force triggered actin reorganization and further resulted in an increased Young's modulus and viscosity. The correlation with single-force measurements revealed that altered cellular activities could result from alternation of Sp1–APP promoter interaction. Our AFM findings offer a new approach in understanding Pb2+ associated neurodegeneration.

Interplay between Pur? and Egr-1 in the Transcriptional Regula-tion of Amyloid Precursor Protein Gene Expression

Background: One of the pathological hallmarks of Alzheimer’s disease is the presence of fibrillary amyloid-β deposits, which result from cleavage of the amyloid precursor protein. Understanding the regulatory mechanism of the amyloid precursor protein gene expression is crucial for comprehending the genesis and development of Alzheimer’s disease. The nucleic acid binding protein, Purα, is best characterized as a transcriptional factor (TF) with affinity to singlestrand G/C-rich regions. In a previous study, we demonstrated that the Purα protein can downregulate amyloid precursor protein (APP) promoter activity, but the mechanism underlying this downregulation requires further investigation. To better understand this mechanism, we analyzed the characteristic of the APP promoter and found that another transcriptional factor, namely Egr-1, can bind the APP promoter and may exert transcriptional regulatory effects on APP gene expression. Therefore, the interaction between these two transcriptional factors may explain the mechanism in regulating APP gene expression. Methodology/Principal Findings: The binding sites of Purα and Egr-1 on the APP promoter 5’-UTR were identified, and reporter plasmids in which the binding sites for Purα and Egr-1 were deleted have been constructed. A luciferase assay was performed, and the results demonstrated that both Purα and Egr-1 lost their regulatory effects when these binding sites were deleted. The luciferase results also demonstrated that Purα can suppress the effects of Egr-1 on APP promoter activities. The electrophoresis mobility shift assay (EMSA) and chromatin immunoprecipitation (ChIP) assay results demonstrated that both Purα and Egr- 1 can competently bind to the APP promoter. The endogenous Egr-1 expression was disturbed with the HDAC inhibitor and suramin, and the Egr-1 expression level affected the APP promoter activities and APP gene expression. Purα can also suppress the endogenous expression of Egr-1. Conclusion/Significance: The mechanism through which Purα regulates AβPP gene expression may involve its interaction with Egr-1, which is a positive regulator of the APP promoter. Because both transcriptional factors possess the binding sites in the APP promoter 5’-UTR and the position of these sites are overlapped, there may exist a displacement mechanism for these two transcriptional factors. In addition, Purα also suppresses the endogenous Egr-1 expression. All of these findings explain the mechanism through which Purα regulates APP gene expression.

Epigenetic regulation in amyloid precursor protein and the Lesch-Nyhan syndrome

Lesch-Nyhan syndrome (LNS) is a neurogenetic disorder of purine metabolism in which the enzyme, hypoxanthine–guanine phosphoribosyltransferase (HPRT) is defective. A major unsolved question is how the loss of HPRT enzyme function affects the brain to cause the neurobehavioural syndrome in LNS and its attenuated variants (LNVs). To address this issue, a search for a link between LNS and the amyloid precursor protein (APP) is developed. Here, I identified, for the first time in fibroblasts from normal subjects as well as from LNS and LNV patients: (a) several APP-mRNA isoforms encoding divers APP protein isoforms ranging from 120 to 770 amino acids (with or without mutations and/or deletions) accounted for epigenetic mechanisms in the regulation of alternative APP pre-mRNA splicing and (b) five novel independent polymorphisms in the APP promoter: −956A>G, −1023T>C, −1161A>G, −2224G>A, −2335C>T relative to the transcription start site. A role for epistasis between mutated HPRT and APP genes affecting the regulation of alternative APP pre-mRNA splicing in LNS is suggested. An accurate quantification of various APP isoforms in brain tissues for detection of initial pathological changes or pathology development is needed. My findings may provide new directions not only for investigating the role of APP in neuropathology associated with HPRT-deficiency in LNS but also for the research in neurodevelopmental and neurodegenerative disorders by which various APP isoforms involved in the pathogenesis of the diseases such as Alzheimer’s disease.

AMYLOID PRECURSOR PROTEIN PROCESSING IN ALZHEIMER'S DISEASE

How to Cite this Article: Bhadbhade A, Cheng DW. Amyloid Precursor Protein Processing in Alzheimer’s Disease. Iranian Journal of Child Neurology2012;6(1):1-5. Alzheimer’s disease (AD) is a progressive neurodegenerative disorder and a leading cause of dementia. The AD is characterized by presence of intraneuronal tangles and extracellular plaques in the brain. The plaques are composed of dense and mostly insoluble deposits of amyloid beta peptide (Aβ), formed by sequential cleavage of the Amyloid Precursor Protein (APP), by two pathways amyloidogenic and non-amyloidogenic. Tangles are composed of paired helical fragments, which aggregate to form, microtubular protein tau. Although Aβ plaques are established to be the cause of the disease, there exist genetic factors and other pathological identifications in addition to these which are an integral part of the disease. This article gives an overview into the mechanism of APP action, genetic factors and other pathological identifications contributing to Alzheimer’s disease formation. References Brookmeyer R, Gray S, Kawas C. Projections of Alzheimer’s disease in the United States and the public health impact of delaying disease onset. American Journal of Public Health 1998;88(9):1337. Hebert LE, Scherr PA, Bienias JL, Bennett DA, Evans DA. Alzheimer disease in the US population. Arch Neurol 2003;60(8):1119-22. Moller HJ, Graeber M. The case described by Alois Alzheimer in 1911. European Archives of Psychiatry and Clinical Neuroscience 1998:248(3):111-122. Selkoe D J. (2002). Deciphering the genesis and fate of amyloid beta-protein yields novel therapies for Alzheimer disease. J Clinic Investigat 2002;110(10): 1375-82. Wolfe MS. Tau mutations in neurodegenerative diseases. J Biolog Chem 2009;284(10):6021. Selkoe DJ. Alzheimer’s disease: genes, proteins, and therapy. Physiological reviews 2001;81(2):741. Selkoe DJ. The cell biology of [beta]-amyloid precursor protein and presenilin in Alzheimer’s disease. Trends in Cell Biology 1998;8(11):447-453. Thinakaran G, Koo EH. Amyloid precursor protein trafficking, processing, and function. Journal of Biological Chemistry 2008;283(44):29615. Zhang YW, Thompson R, Zhang H, Xu H. APP processing in Alzheimer’s disease. Mol Brain 2011;4:3. doi: 1756- 6606-4-3 [pii] 10.1186/1756-6606-4-3. Nunan J, Small DH. Regulation of APP cleavage by α-,β- and γ-secretases. J Biolog Chem 2000:483(1):6-10. Pearson HA, Peers C. Physiological roles for amyloid peptides. J Physiology 2006;575(1):5-10. Wang Y, Ha Y. The X-ray structure of an antiparallel dimer of the human amyloid precursor protein E2 domain. Molecular Cell 2004;15(3):343-353. Quitschke WW, Goldgaber D. The amyloid b-protein precursor promoter. Journal Biolog Chem 1992;267(24):17362-17368. Vostrov AA, Taheny MJ, Izkhakov N, Quitschke WW. A nuclear factor-binding domain in the 5’-untranslated region of the amyloid precursor protein promoter: implications for the regulation of gene expression. BMC Research Notes 2010;3:4. Ghosala K,Vogta D, Lianga M, Shenb Y, Lamba BT, Sanjay W, Pimplikara SW. Alzheimer’s disease-like pathological features in transgenic mice expressing the APP intracellular domain. Proceedings of National Academy of Sciences 2009;106(43):18367-77.

Lead exposure in pheochromocytoma cells induces persistent changes in amyloid precursor protein gene methylation patterns

It has been suggested that lead (Pb) exposure in early life may increase amyloid precursor protein (APP) expression and promote the pathogenesis of Alzheimer's disease in old age. The current study examined whether the DNA methylation patterns of APP gene in rat pheochromocytoma (PC12) cells changed after Pb acetate exposure. Undifferentiated PC12 cells were exposed to three doses of Pb acetate (50, 250, and 500 nM) and one control for 2 days or 1 week. The methylation patterns of APP promoter and global DNA methylation were analyzed. The DNA methyltransferase 1 (DNMT1) expression and the level of amyloid β peptide (Aβ) were also investigated. The results showed that the exposure of the three concentrations of Pb acetate could make the APP promoter hypomethylated. The global DNA methylation level and the expression of DNMT1 were changed in the 500 nM group after 2 days exposure and in the 250 and 500 nM group after 7 days exposure. Thus, Pb may exert neurotoxic effects through mechanisms that alter the global and promoter methylation patterns of APP gene. © 2010 Wiley Periodicals, Inc. Environ Toxicol, 2012.

A nuclear factor-binding domain in the 5'-untranslated region of the amyloid precursor protein promoter: Implications for the regulation of gene expression

BackgroundThe extracellular deposition of aggregated amyloid β-protein is a neuropathological manifestation of Alzheimer disease and Down syndrome. The Amyloid β-protein is derived from a group of larger differentially spliced proteins, the amyloid protein precursors (APP). Data suggests that the level of APP gene expression could contribute to the pathological processes leading to amyloid depositions.FindingsThe 5' untranslated region (UTR) of the APP gene, encompassing 147 base pairs between the transcriptional (+1) and the translational start site, was examined for its role in APP expression. Deletions close to the transcriptional start site reduced expression from the APP promoter in part by transcriptional mechanisms. However, deletions between position +50 and +104 had no effect on transcriptional activity while significantly reducing overall expression from the promoter. A nuclear factor-binding domain designated as DAPB was identified between position +72 and +115 of the 5'-APP-UTR. The binding-recognition sequence was localized between position +96 and +105. The same mutations that eliminated factor-binding also reduced expression from the APP promoter while having no effect on in vitro transcription or the RNA levels transcribed from transfected constructs.ConclusionsA nuclear factor-binding domain designated as DAPB was identified in the 5'-UTR of the APP gene. Elimination of factor-binding correlated with an overall decline in expression from the APP promoter while in vitro transcription and the total amount of in vivo transcribed RNA remained unaffected. This suggests that the binding-factor may have a function in post-transcriptional regulation, including nuclear export of mRNA.

Rac1 inhibition negatively regulates transcriptional activity of the amyloid precursor protein gene

Rac1, a member of the Rho family GTPases, participates in a variety of cellular functions including lamellipodia formation, actin cytoskeleton organization, cell growth, apoptosis, and neuronal development. Recent studies have implicated Rac1 in cytoskeletal abnormalities, production of reactive oxygen species, and generation of the amyloid beta-peptide (Abeta) observed in Alzheimer's disease. In this study, we examined the relationship between Rac1 and amyloid precursor protein (APP), because the abnormal proteolytic processing of APP is a pathologic feature of Alzheimer's disease. In primary hippocampal neurons, the Rac1-specific inhibitor NSC23766 decreased both Rac1 activity and APP protein levels in a concentration-dependent manner. To elucidate how NSC23766 decreases APP protein levels, we examined the effects of NSC23766 on APP processing, degradation, and biosynthesis. NSC23766 did not increase the levels of the proteolytic products of APP, sAPPalpha, Abeta40, and Abeta42. The proteasome inhibitor lactacystin did not reverse the NSC23766-induced decrease in APP protein levels. NSC23766 did, however, decrease the levels of both APP mRNA and APP protein. Decreased levels of APP mRNA and protein were also observed when HEK293 cells were transfected with an expression vector containing a dominant-negative Rac1 mutant or with siRNA targeting Rac1. By overexpressing progressively deleted fragments of the APP promoter in HEK293 cells, we identified a Rac1 response site at positions -233 to -41 bp in the APP promoter. Taken together, our results suggest that Rac1 regulates transcription of the APP gene in primary hippocampal neurons.

The tumour suppressor p53 regulates the expression of amyloid precursor protein (APP)

The expression of the APP (amyloid precursor protein), which plays a key role in the development of AD (Alzheimer's disease), is regulated by a variety of cellular mediators in a cell-dependent manner. In this study, we present evidence that p53 regulates the expression of the APP gene in neuroblastoma cells. Transient expression of ectopic p53, activation of endogenous p53 by the DNA-damaging drug camptothecin or Mdm2 (murine double minute 2) depletion decreases the intracellular levels of APP in murine N2abeta neuroblastoma cells. This effect was also observed in primary cultures of rat neurons as well as in SH-SY5Y cells, a human neuroblastoma cell line. Transient transfection studies using plasmids that contain progressive deletions of the 5' region of the gene demonstrate that p53 represses APP promoter activity through a mechanism that is mediated by DNA sequences located downstream of the transcription start site (+55/+101). Accordingly, expression of a dominant-negative p53 mutant significantly increases the transcriptional activity of the APP promoter. In addition, results obtained in gel mobility-shift assays show that p53 does not bind to the +55/+101 APP region, although it reduces binding of the transcription factor Sp1 (stimulating protein 1). Reduction of Sp1 binding after activation of p53 with camptothecin was also observed in chromatin immunoprecipitation assays. Altogether, our results strongly suggest a mechanism by which p53 precludes binding of Sp1 to DNA, and therefore the stimulation of the APP promoter by this transcription factor.

Nitric oxide synthase inhibitors modulate nerve growth factor‐mediated regulation of amyloid precursor protein expression in PC12 cells

Nerve growth factor (NGF) can regulate nitric oxide synthase (NOS) expression and nitric oxide (NO) can modulate NGF-mediated neurotrophic responses. In this study, the role of NO in NGF-stimulated amyloid precursor protein (APP) levels was studied. PC12 cells were treated with either the non-selective NOS inhibitor N(omega)-nitro-L-arginine methylester (L-NAME) or the inducible NOS selective inhibitor s-methylisothiourea (S-MIU), and the effect on NGF-mediated increases in APP expression was determined. NGF significantly increased total APP protein levels following 96 h of treatment and this increase was prevented in cells pre-treated with S-MIU. Pre-treatment of cells with actinomycin D also blocked this NGF-mediated induction of APP, indicating de novo protein synthesis is necessary. Treatment with NGF increased APP promoter activity; however, this increase was only partially inhibited by pre-treatment with S-MIU and was increased in the presence of L-NAME. This suggests that NO may be modulating other aspects of APP expression in addition to transcription. Inhibition of NGF signaling pathways was also investigated using inhibitors of mitogen-activated protein (MAP) kinase (U0126), Akt (LY294002) and protein kinase C (PKC; U73122 and bisindolylmaleimide 1 (BIS-1)) activation. Inhibition of each of these pathways prevented NGF-mediated increases in APP protein expression; however, only BIS-1 attenuated NGF-mediated increases in promoter activation. This study indicates that NO is involved in the NGF-mediated regulation of APP, in part at the level of APP transcription and could involve the modulation of NGF signal transduction pathways.

Genetic risk and transcriptional variability of amyloid precursor protein in Alzheimer's disease

It is well established that Alzheimer's disease causing mutations in APP, PSEN1 and PSEN2 lead to a relative increased production of Abeta42, thereby fostering its deposition in plaques. Recently others and we showed that amyloid precursor protein (APP) overproduction, either as a result of genomic locus duplication or altered regulatory sequences in the APP promoter region, leads to early-onset disease. Here, we have expanded our study of genetic variability in the APP promoter to a large group of well-documented Belgian patients (n = 750, mean onset age = 75.0 +/- 8.6, range = 37-96). We identified three different APP promoter mutations (-369C-->G, -534G-->A and -479C-->T) in seven patients. In patients with onset < or =70 years (n = 204), we identified one patient carrying the London APP V717I mutation while no patients carried an APP locus duplication, indicating that APP promoter mutations (n = 2) were more frequently associated with increased risk for early-onset Alzheimer's disease. The two mutations (-369C-->G and -534G-->A) increasing APP promoter activity by nearly 2-fold and mimicking an APP duplication, appeared in probands of families with multiple patients with dementia. The -479C-->T mutation that increased APP expression only mildly (1.2-fold), was observed in four patients with onset ages ranging from 62 to 79 years (mean 71.5 years), suggesting that its contribution to disease risk is more pronounced at later age due to modulating factors. In conclusion, we provided evidence that mutations in APP regulatory sequences are more frequent than APP coding mutations, and that increased APP transcriptional activity constitutes a risk factor for Alzheimer's disease with onset ages inversely correlated with levels of APP expression.

The 5′‐untranslated sequence of the amyloid precursor protein transcript exerts post‐transcriptional control of gene expression by variable mechanisms

The promoter of the human amyloid precursor protein (APP) gene directs cell-type specific expression with about 121 base pairs 5′ to the main transcriptional start site (+1). Transcription from this region is activated by transcription factors such as CTCF and USF. Downstream from the transcriptional start site the APP transcriptional unit contains an untranslated region (UTR) of 147 base pairs. We have here investigated the role of the APP-UTR in regulating expression from the APP promoter. For this purpose, deletions and transverse block mutations were introduced within the UTR. The constructs were analyzed by transient transfection, in vitro transcription, in vitro translation, DNase I footprinting, and mobility shift electrophoresis. The results showed that mutating sequences close to the transcriptional start site (position +1 to +25) interfered with transcriptional activity. In contrast, mutations between position +61 and +105 reduced expression in vivo in a transcriptionally independent manner. Within this domain two subdomains were identified. Mutations within one domain reduced translational activity in vitro. In contrast, mutations within a second domain eliminated a nuclear factor binding site while having no effect on translational activity. The protein that binds to the APP-UTR has not been identified but it is distinct from the thyroxin response element. In summary, two domains within the APP-UTR (position +61 to +105) affect expression from the APP promoter by independent post-transcriptional mechanisms. [Supported by NS30994].

In the cerebral cortex of female and male mice, amyloid precursor protein (APP) promoter methylation is higher in females and differentially regulated by sex steroids

The over-expression of amyloid precursor protein (APP) gene in certain areas of the brain indicates abnormalities in gene regulation as an important factor for the development of Alzheimer's disease (AD). We have reported recently that APP mRNA expression is lower in female as compared to male and is regulated by sex steroids. As methylation of promoter is crucial for such regulation, we have used isoschizomeric restriction enzymes MspI and HpaII to analyze the pattern of APP promoter methylation in the cerebral cortex of intact, gonadectomized, testosterone- and estradiol-treated adult and old mice of both sexes. Southern blots of DNA digested with HindIII/MspI or HindIII/HpaII from different groups of mice were probed with a 1.27-kb DIG-11-dUTP labeled APP promoter fragment. The results revealed four distinct bands of 315 bp, 596 bp, 911 bp, and 2.6 kb by MspI, a single band of 2.6 kb by HpaII in all groups and an additional 2.9 kb in intact and estradiol treated old male and testosterone-treated adult female mice. The intensity of 2.6-kb band was relatively lower in intact female as compared to male and varied with different treatments in both ages, indicating the modulation of methylation. Thus, these findings showed that APP promoter methylation is higher in female and differentially regulated by sex steroids in the mice cerebral cortex, suggesting a strong correlation between promoter methylation and transcriptional silencing of APP.

Increased processing of APLP2 and APP with concomitant formation of APP intracellular domains in BDNF and retinoic acid‐differentiated human neuroblastoma cells

The amyloid precursor protein (APP) belongs to a conserved gene family, also including the amyloid precursor-like proteins, APLP1 and APLP2. We have previously shown that all members of the APP protein family are up-regulated upon retinoic acid (RA)-induced neuronal differentiation of SH-SY5Y neuroblastoma cells. Here, we demonstrate that RA also affects the processing of APLP2 and APP, as shown by increased shedding of both sAPLP2 and sAPPalpha, as well as elevated levels of the APP intracellular domains (AICDs). Brain-derived neurotrophic factor (BDNF) has been reported to induce APP promoter activity and RA induces expression of the tyrosine kinase receptor B (TrkB) in neuroblastoma cells. We show that the increase in shedding of both APLP2 and APP in response to RA is not mediated through the TrkB receptor. However, BDNF concomitant with RA increased the expression of APP even further. In addition, the secretion of sAPLP2 and sAPPalpha as well as the levels of AICDs were increased in response to BDNF. In contrast, the levels of membrane-bound APP C-terminal fragment C99 significantly decreased. Our results suggest that RA and BDNF shifts APP processing towards the alpha-secretase pathway. In addition, we show that RA and BDNF regulate N-linked glycosylation of APLP1.

Induction of tyrosine kinase receptor b by retinoic acid allows brain-derived neurotrophic factor-induced amyloid precursor protein gene expression in human sh-sy5y neuroblastoma cells

Retinoic acid (RA) is a potent regulator of morphogenesis, growth and cell differentiation. Incubation with RA causes arrest of proliferation and neurite extension in SH-SY5Y cells, a neuroblastoma cell line of human origin. In these cells, RA regulates the expression of the β-amyloid precursor protein. The retinoid increases the levels of intracellular and secreted forms of APP (amyloid precursor protein), APP–mRNA levels and the activity of the APP promoter in transient transfection studies. These responses require long periods of exposition to the ligand, thus suggesting a nondirect effect of the RA receptors on the APP gene. Also in these cells, RA induces the expression of TrkB, the tyrosine kinase receptor for brain-derived neurotrophic factor (BDNF), and 4 days of pretreatment with retinoic acid confers BDNF responsiveness to the APP promoter.

Transforming Growth Factor-β1 Potentiates Amyloid-β Generation in Astrocytes and in Transgenic Mice

Accumulation of the amyloid-beta peptide (Abeta) in the brain is crucial for development of Alzheimer's disease. Expression of transforming growth factor-beta1 (TGF-beta1), an immunosuppressive cytokine, has been correlated in vivo with Abeta accumulation in transgenic mice and recently with Abeta clearance by activated microglia. Here, we demonstrate that TGF-beta1 drives the production of Abeta40/42 by astrocytes leading to Abeta production in TGF-beta1 transgenic mice. First, TGF-beta1 induces the overexpression of the amyloid precursor protein (APP) in astrocytes but not in neurons, involving a highly conserved TGF-beta1-responsive element in the 5'-untranslated region (+54/+74) of the APP promoter. Second, we demonstrated an increased release of soluble APP-beta which led to TGF-beta1-induced Abeta generation in both murine and human astrocytes. These results demonstrate that TGF-beta1 potentiates Abeta production in human astrocytes and may enhance the formation of plaques burden in the brain of Alzheimer's disease patients.

Polymorphisms in the Promoter of the Human APP Gene

Missense mutations in the amyloid precursor protein (APP) gene cause early-onset Alzheimer disease (AD). However, little is known regarding the effects of polymorphisms in regulatory sequences of APP on AD susceptibility. To identify polymorphisms in the APP promoter, to test these for associations with AD, and to assess their influence on APP promoter activity in transfected cells. Community study of 1013 people of white, African American, or Caribbean Hispanic ethnicity, 65 years and older, residing in northern Manhattan. The diagnosis of AD was established by stringent criteria, with multiple follow-up examinations over 7 years. We identified 2 polymorphisms in the APP promoter: a rare G-->C variant at -9 and a frequent G-->C variant at +37 relative to the transcription start site. The +37C allele was most frequent in African American patients (18% frequency), followed by Caribbean Hispanic patients (10%) and white patients of European descent (3%). This allele was overrepresented among patients with AD compared with elderly controls (odds ratio [OR], 1.57; 95% confidence interval [CI], 1.08-2.27 in the combined ethnic groups), but this was not significant after adjusting for age, sex, and education (OR, 1.41; 95% CI, 0.93-2.12). A stronger association was found in participants lacking any apolipoprotein-E epsilon4 allele (OR, 2.12; 95% CI, 1.36-3.32 [univariate analysis]; OR, 2.08; 95% CI, 1.26-3.45 after adjusting for age, sex, and education). The -9C allele was not frequent enough to be evaluated for a disease association. Both variants were tested in promoter-reporter assays in U-87 glioma cells, and no differences in promoter activity were detected. The -9G/C and +37G/C APP promoter polymorphisms are unlikely to contribute strongly to AD susceptibility or to cause major differences in APP expression, but the +37C allele warrants further study for association with AD in larger population samples.

A Region to the N-terminal Side of the CTCF Zinc Finger Domain Is Essential for Activating Transcription from the Amyloid Precursor Protein Promoter

Transcription from the amyloid precursor protein (APP) promoter is largely dependent on a nuclear factor binding site designated as APBbeta. The protein that binds to this site is the multifunctional transcription factor CTCF, which consists of 727 amino acids and contains a domain of 11 zinc finger motifs that is flanked by 267 amino acids on the N-terminal side and 150 amino acids on the C-terminal side. Depleting HeLa cell nuclear extract of endogenous CTCF specifically reduced transcriptional activity from the APP promoter. However, transcriptional activity was restored by replenishing the depleted extract with recombinant CTCF. Deleting 201 amino acids from the C-terminal end of CTCF had no detrimental effect on transcriptional activation, whereas deleting either 248 or 284 amino acids from the N-terminal end abolished transcriptional activation. Competing endogenous CTCF in vivo was accomplished by cotransfecting COS-1 cells with a plasmid overexpressing CTCF constructs and a reporter plasmid containing the APP promoter. Under these conditions, an N-terminal deletion of CTCF reduced expression from the APP promoter, whereas the C-terminal deletion had no effect. These results demonstrate that CTCF activates transcription from the APP promoter and that the activation domain is located on the N-terminal side of the zinc finger domain.