PuF, An Antimetastatic and Developmental Signaling Protein, Interacts with the Alzheimer’s Amyloid-β Precursor Protein via a Tissue-Specific Proximal Regulatory Element (PRE)

Background: Alzheimer’s disease (AD) is intimately tied to amyloid-β (Aβ) peptide. Extraneuronal brain plaques consisting primarily of Aβ aggregates are a hallmark of AD. Intraneuronal Aβ subunits are strongly implicated in disease progression. Protein sequence mutations of the Aβ precursor protein (APP) account for a small proportion of AD cases, suggesting that regulation of the associated gene (APP) may play a more important role in AD etiology. The APP promoter possesses a novel 30 nucleotide sequence, or “proximal regulatory element” (PRE), at −76/−47, from the +1 transcription start site that confers cell type specificity. This PRE contains sequences that make it vulnerable to epigenetic modification and may present a viable target for drug studies. We examined PRE-nuclear protein interaction by gel electrophoretic mobility shift assay (EMSA) and PRE mutant EMSA. This was followed by functional studies of PRE mutant/reporter gene fusion clones. Results: EMSA probed with the PRE showed DNA-protein interaction in multiple nuclear extracts and in human brain tissue nuclear extract in a tissue-type specific manner. We identified transcription factors that are likely to bind the PRE, using competition gel shift and gel supershift: Activator protein 2 (AP2), nm23 nucleoside diphosphate kinase/metastatic inhibitory protein (PuF), and specificity protein 1 (SP1). These sites crossed a known single nucleotide polymorphism (SNP). EMSA with PRE mutants and promoter/reporter clone transfection analysis further implicated PuF in cells and extracts. Functional assays of mutant/reporter clone transfections were evaluated by ELISA of reporter protein levels. EMSA and ELISA results correlated by meta-analysis. Conclusions: We propose that PuF may regulate the APP gene promoter and that AD risk may be increased by interference with PuF regulation at the PRE. PuF is targeted by calcium/calmodulin-dependent protein kinase II inhibitor 1, which also interacts with the integrins. These proteins are connected to vital cellular and neurological functions. In addition, the transcription factor PuF is a known inhibitor of metastasis and regulates cell growth during development. Given that APP is a known cell adhesion protein and ferroxidase, this suggests biochemical links among cell signaling, the cell cycle, iron metabolism in cancer, and AD in the context of overall aging

Alzheimer\u27s Disease (AD)-Like Pathology in Aged Monkeys after Infantile Exposure to Environmental Metal Lead (Pb): Evidence for a Developmental Origin and Environmental Link for AD

The sporadic nature of Alzheimer\u27s disease (AD) argues for an environmental link that may drive AD pathogenesis; however, the triggering factors and the period of their action are unknown. Recent studies in rodents have shown that exposure to lead (Pb) during brain development predetermined the expression and regulation of the amyloid precursor protein (APP) and its amyloidogenic β-amyloid (Aβ) product in old age. Here, we report that the expression of AD-related genes [APP, BACE1 (β-site APP cleaving enzyme 1)] as well as their transcriptional regulator (Sp1) were elevated in aged (23-year-old) monkeys exposed to Pb as infants. Furthermore, developmental exposure to Pb altered the levels, characteristics, and intracellular distribution of Aβ staining and amyloid plaques in the frontal association cortex. These latent effects were accompanied by a decrease in DNA methyltransferase activity and higher levels of oxidative damage to DNA, indicating that epigenetic imprinting in early life influenced the expression of AD-related genes and promoted DNA damage and pathogenesis. These data suggest that AD pathogenesis is influenced by early life exposures and argue for both an environmental trigger and a developmental origin of AD

Lifespan profiles of Alzheimer's disease–associated genes and their products in monkeys and mice.

Alzheimer's disease (AD) is characterized by plaques of amyloid–beta (Aβ) peptide, cleaved from amyloid–β precursor protein (AβPP). Our hypothesis is that lifespan profiles of AD-associated mRNA and protein levels in monkeys would differ from mice, and that differential lifespan expression profiles would be useful to understand human AD pathogenesis. We compared profiles of AβPP mRNA, AβPP protein, and Aβ levels in rodents and primates. We also tracked a transcriptional regulator of the AβPP gene, specificity protein 1 (SP1), and the β amyloid precursor cleaving enzyme (BACE1). In mice, AβPP and Sp1 mRNA and their protein products were elevated late in life; Aβ levels declined in old age. In monkeys, Sp1, AβPP, and BACE1 mRNA declined in old age, while protein products and Aβ levels rose. Proteolytic processing in both species did not match production of Aβ. In primates, AβPP and Sp1 mRNA levels coordinate, but an inverse relationship exists with corresponding protein products, as well as Aβ levels. Comparison of human DNA and mRNA sequences to monkey and mouse counterparts revealed structural features that may explain differences in transcriptional and translational processing. These findings are important for selecting appropriate models for AD and other age–related diseases

The alpha-synuclein 5'untranslated region targeted translation blockers: anti-alpha synuclein efficacy of cardiac glycosides and Posiphen

Increased brain α-synuclein (SNCA) protein expression resulting from gene duplication and triplication can cause a familial form of Parkinson's disease (PD). Dopaminergic neurons exhibit elevated iron levels that can accelerate toxic SNCA fibril formation. Examinations of human post mortem brain have shown that while mRNA levels for SNCA in PD have been shown to be either unchanged or decreased with respect to healthy controls, higher levels of insoluble protein occurs during PD progression. We show evidence that SNCA can be regulated via the 5'untranslated region (5'UTR) of its transcript, which we modeled to fold into a unique RNA stem loop with a CAGUGN apical loop similar to that encoded in the canonical iron-responsive element (IRE) of L- and H-ferritin mRNAs. The SNCA IRE-like stem loop spans the two exons that encode its 5'UTR, whereas, by contrast, the H-ferritin 5'UTR is encoded by a single first exon. We screened a library of 720 natural products (NPs) for their capacity to inhibit SNCA 5'UTR driven luciferase expression. This screen identified several classes of NPs, including the plant cardiac glycosides, mycophenolic acid (an immunosuppressant and Fe chelator), and, additionally, posiphen was identified to repress SNCA 5'UTR conferred translation. Western blotting confirmed that Posiphen and the cardiac glycoside, strophanthidine, selectively blocked SNCA expression (~1 μM IC(50)) in neural cells. For Posiphen this inhibition was accelerated in the presence of iron, thus providing a known APP-directed lead with potential for use as a SNCA blocker for PD therapy. These are candidate drugs with the potential to limit toxic SNCA expression in the brains of PD patients and animal models in vivo

Epigenetics, oxidative stress, and Alzheimer disease

Alzheimer disease (AD) is a progressive neurodegenerative disorder whose clinical manifestations appear in old age. The sporadic nature of 90% of AD cases, the differential susceptibility to and course of the illness, as well as the late age onset of the disease suggest that epigenetic and environmental components play a role in the etiology of late-onset AD. Animal exposure studies demonstrated that AD may begin early in life and may involve an interplay between the environment, epigenetics, and oxidative stress. Early life exposure of rodents and primates to the xenobiotic metal lead (Pb) enhanced the expression of genes associated with AD, repressed the expression of others, and increased the burden of oxidative DNA damage in the aged brain. Epigenetic mechanisms that control gene expression and promote the accumulation of oxidative DNA damage are mediated through alterations in the methylation or oxidation of CpG dinucleotides. We found that environmental influences occurring during brain development inhibit DNA-methyltransferases, thus hypomethylating promoters of genes associated with AD such as the β-amyloid precursor protein (APP). This early life imprint was sustained and triggered later in life to increase the levels of APP and amyloid-β (Aβ). Increased Aβ levels promoted the production of reactive oxygen species, which damage DNA and accelerate neurodegenerative events. Whereas AD-associated genes were overexpressed late in life, others were repressed, suggesting that these early life perturbations result in hypomethylation as well as hypermethylation of genes. The hypermethylated genes are rendered susceptible to Aβ-enhanced oxidative DNA damage because methylcytosines restrict repair of adjacent hydroxyguanosines. Although the conditions leading to early life hypo- or hypermethylation of specific genes are not known, these changes can have an impact on gene expression and imprint susceptibility to oxidative DNA damage in the aged brain

THE CELLULAR NUCLEIC ACID BINDING PROTEIN REGULATES THE ALZHEIMER’S DISEASE β-SECRETASE PROTEIN BACE1

Alzheimer’s disease (AD) is the most common neurodegenerative disease affecting the elderly population and is believed to be caused by the overproduction and accumulation of the toxic amyloid beta (Aβ) peptide in the brain. Aβ is produced by two separate enzymatic cleavage events of the larger membrane bound amyloid precursor protein, APP. The first, and rate-limiting, cleavage event is made by beta-secretase, or BACE1, and is thus an attractive therapeutic target. Our lab, as well as many others, has shown that BACE1 protein and activity are increased in late-stage sporadic AD. We have extended these findings to show that BACE1 is increased in the earliest stages of AD before the onset of significant Aβ accumulation, indicating a potential causal role in the disease. Interestingly, BACE1 mRNA levels are unchanged in AD, leading to reason that a post-transcriptional method of BACE1 regulation is altered in disease. To date, the mechanism for this aberrant post-transcriptional regulation has not been elucidated. This study has implicated the cellular nucleic acid binding protein (CNBP), a highly conserved RNA binding protein, as a positive regulator of BACE1 translation, with implications for the etiology of sporadic AD. CNBP overexpression in cultured cells or spiked into a cell-free in vitro translation system increased BACE1 protein expression without affecting BACE1 mRNA levels. Knockdown of CNBP reduced BACE1 protein and mRNA slightly. Furthermore, CNBP associated with BACE1 mRNA in cell lysates and bound directly to the BACE1 5’ UTR in vitro, which confers most of the regulatory activity. Importantly, CNBP was increased in the progression of AD and correlated with BACE1 expression. Cellular stressors (such as glucose deprivation and oxidative stress) that occur in the AD brain increase BACE1 translation and we have found that these stressors increased CNBP expression as well. Early experimental evidence suggests that CNBP may enhance BACE1 translation through a cap-independent mechanism, which is an alternative translational pathway activated by cell stress. These studies indicate that the RNA binding protein CNBP is a novel trans-acting factor important for the regulation of BACE1 protein production and may be a viable therapeutic target for AD

Alpha-Secretase ADAM10 Regulation: Insights into Alzheimer's Disease Treatment

ADAM (a disintegrin and metalloproteinase) is a family of widely expressed, transmembrane and secreted proteins of approximately 750 amino acids in length with functions in cell adhesion and proteolytic processing of the ectodomains of diverse cell-surface receptors and signaling molecules. ADAM10 is the main α-secretase that cleaves APP (amyloid precursor protein) in the non-amyloidogenic pathway inhibiting the formation of β-amyloid peptide, whose accumulation and aggregation leads to neuronal degeneration in Alzheimer's disease (AD). ADAM10 is a membrane-anchored metalloprotease that sheds, besides APP, the ectodomain of a large variety of cell-surface proteins including cytokines, adhesion molecules and notch. APP cleavage by ADAM10 results in the production of an APP-derived fragment, sAPPα, which is neuroprotective. As increased ADAM10 activity protects the brain from β-amyloid deposition in AD, this strategy has been proved to be effective in treating neurodegenerative diseases, including AD. Here, we describe the physiological mechanisms regulating ADAM10 expression at different levels, aiming to propose strategies for AD treatment. We report in this review on the physiological regulation of ADAM10 at the transcriptional level, by epigenetic factors, miRNAs and/or translational and post-translational levels. In addition, we describe the conditions that can change ADAM10 expression in vitro and in vivo, and discuss how this knowledge may help in AD treatment. Regulation of ADAM10 is achieved by multiple mechanisms that include transcriptional, translational and post-translational strategies, which we will summarize in this review

Transcriptional regulatory mechanisms involved in CYP46A1 up-regulation by histone deacetylase inhibitors:from chromatin structure to transcription factors

Tese de doutoramento, Farmácia (Biologia Celular e Molecular), Universidade de Lisboa, Faculdade de Farmácia, 2012Apart from being an essential component of cellular membranes and having a role as a signaling molecule, cholesterol is also the precursor of several bioactive molecules that include bile acids, steroid hormones, oxysterols and vitamin D. In the brain constant levels of this sterol are required for normal functioning and the homeostasis is maintained, in part, by an efficient blood-brain barrier that prevents exchanges with lipoprotein cholesterol from circulation. For that reason, de novo and in situ synthesis occur to meet cholesterol needs in the central nervous system (CNS), which despite the low synthesis rate must be excreted at some degree in order to keep its steady state. The conversion of cholesterol into 24(S)-hydroxycholesterol, by the neuronalspecific cytochrome P450 cholesterol 24-hydroxylase (CYP46A1) has been described as the major elimination mechanism. The main goal of this work was to characterize the effect of histone deacetylase (HDAC) inhibition in the transcriptional regulation of CYP46A1 gene and the molecular mechanism underlying such effect. We started by demonstrating that the inhibition of HDAC activity by trichostatin A (TSA), valproic acid and sodium butyrate cause a potent induction of both CYP46A1 promoter activity and endogenous expression. Indeed, we have shown for the first time that TSA induces an overall increase in histone acetylation levels at CYP46A1 proximal promoter, as a result of the detachment of HDACs and recruitment of histone acetyltransferases (HAT), in a process dependent on Sp3 transcription factor decreased binding to particular cis-elements. This change in chromatin structure culminates in the recruitment of RNA polymerase II and CYP46A1 gene activation. Nevertheless, the fact that histone deacetylation was evident at a time point when the HDAC/HAT ratio should still favor acetylation, led us to investigate if mechanisms besides histone hyperacetylation could participate in the TSA mediated derepression of CYP46A1 gene. Interestingly, we identified the participation of the mitogen-activated kinase kinase (MEK)-extracellular signal-regulated kinase (ERK) signaling pathway in the CYP46A1 response to TSA. A decrease in ERK1/2 phosphorylation levels was observed after TSA treatment concomitantly with a decrease in Sp3 binding activity. Inhibition of protein phosphatase activity by pre-treatment with okadaic acid (OA) completely reversed these changes, and impaired the TSA-mediated CYP46A1 activation without affecting promoter histone hyperacetylation. Our results also show that TSA treatment induces the dissociation of phosphorylated ERK1/2 from the CYP46A1 promoter and specifically from the Sp3-containing DNA fragments. This suggests that in the context of the CYP46A1 promoter phosphorylated Sp3 acts as a transcriptional repressor being responsible for the recruitment of co-repressor complexes, with and without HDAC activity. Moreover, our work highlights the importance of the MEK-ERK signaling pathway in the control of brain cholesterol elimination. The importance of CYP46A1 in cholesterol homeostasis and the drastic effect of HDAC inhibitors (HDACi) in its expression, lead us to evaluate if these compounds can affect the expression of other key players in neuronal cholesterol metabolism. In the last part of our work we have identified TSA as a cholesterol-lowering molecule, by modulating the transcription of other genes involved in cholesterol metabolism in human neuroblastoma cells, namely by up-regulating genes that control cholesterol efflux and dow-regulating genes involved in cholesterol synthesis and uptake, thus leading to an overall decrease in total cholesterol content. Moreover, we have shown that TSA is also able to partially reverse the increased cholesterol content and the transcriptional changes, induced by pathological lysosomal accumulation of intracellular cholesterol. Overall, these results clarify the role of HDACi in the modulation of CYP46A1 gene transcription as well as other key genes in cholesterol metabolism, comprising a significant contribution in the elucidation of the molecular mechanism involved in the transcriptional regulation of CYP46A1 gene and emphasizing the idea of HDAC inhibition as a promising therapeutic tool in neurodegenerative disorders with impaired cholesterol metabolismo.O colesterol é uma molécula essencial à vida. Para além de desempenhar um papel crucial na estrutura das membranas celulares, através da regulação da permeabilidade e fluidez das mesmas, o colesterol é também percursor de inúmeras moléculas de extrema relevância biológica tais como os ácidos biliares, os oxisteróis, as hormonas esteroides e a vitamina D, podendo também atuar como uma molécula sinalizadora. No encéfalo, o colesterol está maioritariamente na forma não esterificada e encontra-se associado às bainhas de mielina e às membranas plasmáticas dos neurónios e células da glia. O colesterol é também essencial à formação e propagação de sinapses, ao crescimento dendrítico e à estabilidade dos microtúbulos. Além disso, o sistema nervoso central (SNC) necessita de níveis constantes de colesterol para um correto funcionamento, sendo que a homeostasia é assegurada, em parte, pela barreira hematoencefálica que impede trocas com o colesterol em circulação. Por esta razão, todo o colesterol presente no encéfalo deriva de uma síntese de novo e in situ. Apesar de no SNC do adulto a síntese de colesterol ser reduzida, uma fração necessita de ser excretada, de forma a manter os níveis de colesterol constantes. A conversão do colesterol em 24(S)- hidroxicolesterol, pelo enzima 24(S)-hidroxilase (CYP46A1), foi descrita como o principal mecanismo de eliminação. O produto enzimático (oxisterol), contrariamente ao colesterol, atravessa com facilidade a barreira hemato-encefálica, entra em circulação e é posteriormente eliminado no fígado, completando desta forma o processo de transporte reverso do colesterol. Nos últimos anos, um grande número de estudos aponta para uma relação entre alterações no metabolismo do colesterol e o desenvolvimento de doenças neurodegenerativas, como a doença de Alzheimer, a doença de Huntington ou a doença de Niemann-Pick tipo C. Para além disso, inúmeras evidências sugerem que a indução do CYP46A1 poderá ter consequências benéficas, nomeadamente na doença de Alzheimer. De facto a sobre-expressão do CYP46A1 poderá ter um efeito inibitório direto sobre a produção do péptido β-amilóide, um efeito indireto relacionado com a diminuição dos níveis de colesterol das membranas neuronais, ou por aumentar a ativação dos genes alvo do receptor nuclear liver X receptor. O principal objectivo deste trabalho foi a caracterização do efeito da inibição das desacetilases de histonas (HDAC) na regulação da transcrição do gene CYP46A1, bem como dos mecanismos moleculares subjacentes a esse mesmo efeito. Na primeira parte deste trabalho, começámos por demonstrar que a inibição da atividade das HDACs através do tratamento com os inibidores tricostatina A (TSA), ácido valpróico e butirato de sódio, causam uma drástica indução da atividade do promotor e da expressão endógena do gene CYP46A1. Observámos também, pela primeira vez, que o tratamento com TSA induz um aumento significativo dos níveis de acetilação das histonas do promotor próximo do gene CYP46A1, sendo que este efeito resulta da diminuição da presença de HDACs e do recrutamento de acetiltransferases de histonas (HAT). Estudos anteriores do nosso grupo identificaram como essenciais na expressão basal do gene CYP46A1 factores de transcrição da família Sp e, neste estudo, demonstrámos que as proteínas Sp1 e Sp4 são também cruciais para a ativação do CYP46A1 pelo TSA. No entanto o efeito do TSA parece depender da diminuição da ligação do factor de transcrição Sp3 a elementos de resposta específicos do promotor, uma vez que uma diminuição de ligação desta proteína está relacionada com a diminuição de HDACs presentes no promotor. De uma forma geral, as modificações na estrutura da cromatina e no complexo proteico na região do promotor próximo induzidas pelo TSA culminam no recrutamento da RNA polimerase II e na consequente ativação do gene CYP46A1. A evidência de que o nível de acetilação das histonas a determinado momento não reflete a razão entre os níveis dos enzimas HDAC/HAT presentes no promotor, conduziu-nos à investigação de mecanismos independentes da hiperacetilação de histonas que pudessem contribuir para a ativação do gene CYP46A1 pelo TSA. De facto, na segunda parte do nosso trabalho identificámos a participação da via de sinalização da cinase de proteínas ativada por mitogénios (MAPK) na resposta do CYP46A1 ao TSA. Através do pré-tratamento com inibidores químicos específicos bem como a utilização de dominantes negativos da cinase terminal desta via, a cinase de proteínas regulada por sinais extracelulares (ERK), concluímos que a inibição da atividade deste enzima potencia o efeito do TSA, contrariamente ao efeito da inibição das fosfatases de proteína, através do pré-tratamento com ácido ocadáico, que bloqueia quase na totalidade a ativação do gene CYP46A1 pelo TSA. A análise dos níveis de fosforilação da ERK1/2 demonstrou que o TSA induz uma diminuição do nível de ativação destas cinases, bem como da atividade de ligação da proteína Sp3 ao DNA, efeitos esses que são revertidos pelo pré-tratamento com ácido ocadáico, sem que o nível de hiperacetilação das histonas do promotor seja afetado. Os nossos resultados demonstram ainda que o tratamento com TSA induz a dissociação da forma fosforilada da ERK1/2 do promotor do CYP46A1, especificamente, dos fragmentos de DNA que contêm o factor de transcrição Sp3, sugerindo que no contexto do promotor do CYP46A1 a proteína Sp3 atua como um repressor da transcrição e é responsável pelo recrutamento de complexos co-repressores, com e/ou sem atividade HDAC. O TSA ao modular a ativação da ERK1/2 regula indiretamente a atividade deste factor de transcrição e consequentemente a expressão do CYP46A1. De facto, a replicação do efeito do ácido ocadáico e da inibição da atividade da ERK1/2 na atividade do promotor e expressão basal do gene CYP46A1 evidencia também a importância desta via de sinalização intracelular no controlo da eliminação de colesterol do encéfalo. A importância do CYP46A1 na homeostasia do colesterol bem como o efeito drástico dos inibidores das HDACs na sua expressão, conduziu à avaliação do efeito destes compostos na expressão de genes chave no metabolismo do colesterol em células neuronais. Como tal, na última parte deste trabalho demonstrámos a capacidade do TSA de diminuir os níveis de colesterol em células de neuroblastoma humano, através da regulação da expressão de genes envolvidos no metabolismo do colesterol, nomeadamente, através da indução de genes envolvidos no controlo do efluxo (transportadores ATP-binding cassette) e catabolismo (CYP46A1), e repressão de genes essenciais para a síntese (3-metilglutaril Coenzima A redutase) e captação de colesterol (receptor de lipoproteínas de baixa densidade), culminado desta forma na diminuição do conteúdo total de colesterol. O tratamento com o composto U18666A, que mimetiza o fenótipo da doença de Niemann-Pick tipo C, caracterizado pela acumulação patológica de colesterol nos lisossomas, resultou no aumento dos níveis de colesterol em células de neuroblastoma humano, bem com no aumento da expressão de genes envolvidos na síntese e captação de colesterol e diminuição dos genes responsáveis pelo efluxo. No entanto, o tratamento com TSA reverteu todos esses efeitos, contribuindo para a correção das perturbações no metabolismo do colesterol. Apesar de já ter sido demonstrado o efeito benéfico da inibição das HDACs em fibroblastos de pacientes com a doença de Niemann-Pick tipo C, este trabalho demonstra pela primeira vez a capacidade destes compostos de reverter, ao nível da transcrição, os efeitos da acumulação de colesterol nos lisossomas. Em conclusão, os resultados apresentados nesta tese identificam os inibidores das HDACs como agentes farmacológicos que poderão eventualmente vir a ser usados na indução da transcrição do gene CYP46A1, bem como na modelação da transcrição de genes que participam de forma relevante no metabolismo do colesterol no encéfalo. Para além disso constitui uma contribuição significativa para a compreensão dos mecanismos moleculares responsáveis pela regulação da transcrição do gene CYP46A1, identificando a via das MAPKs como umas das vias de sinalização responsáveis pelo controlo do catabolismo do colesterol no encéfalo. A compreensão dos mecanismos moleculares controlados pelos inibidores das HDACs, e envolvidos na homeostasia do colesterol no encéfalo, poderá de facto constituir uma plataforma para o desenvolvimento de possíveis intervenções farmacológicas que vão da neurodegenerescência ao cancro.Fundação para a Ciência e a Tecnologia ( FCT, SFRH/BD/41848/2007, projetos PPCDT/SAU-MMO/55919/2004, PTDC/SAU-GMG/64176/2006) e FEDER (Fundo Europeu de Desenvolvimento Regional

Transcriptional regulation of PEN-2, a key component of the γ-secretase complex, by CREB

Gamma-secretase, which is responsible for the intramembranous cleavage of Alzheimer's P-amyloid precursor protein (APP), the signaling receptor Notch, and many other substrates, is a multiprotein complex consisting of at least four components: presenilin (PS), nicastrin, APH-1, and PEN-2. Despite the fact that PEN-2 is known to mediate endoproteolytic cleavage of full-length PS and APH-1 and nicastrin are required for maintaining the stability of the complex, the detailed physiological function of each component remain elusive. Unlike that of PS, the transcriptional regulation of PEN-2, APH-1, and nicastrin has not been investigated. Here, we characterized the upstream regions of the human PEN-2 gene and identified a 238-bp fragment located 353 bp upstream of the translational start codon as the key region necessary for the promoter activity. Further analysis revealed a CREB binding site located in the 238-bp region that is essential for the transcriptional activity of the PEN-2 promoter. Mutation of the CREB site abolished the transcriptional activity of the PEN-2 promoter. Electrophoretic mobility shift assays and chromatin immunoprecipitation analysis showed the binding of CREB to the PEN-2 promoter region both in vitro and in vivo. Activation of the CREB transcriptional factor by forskolin dramatically promoted the expression of PEN-2 mRNA and protein, whereas the other components of the gamma-secretase complex remained unaffected. Forskolin treatment slightly increases the secretion of soluble APP alpha and A beta without affecting Notch cleavage. These results demonstrate that expression of PEN-2 is regulated by CREB and suggest that the specific control of PEN-2 expression may imply additional physiological functions uniquely assigned to PEN-2