Yuan Wen Ge

The Fetal Basis of Amyloidogenesis: Exposure to Lead and Latent Overexpression of Amyloid Precursor Protein and β-Amyloid in the Aging Brain

The fetal basis of adult disease (FeBAD) hypothesis states that many adult diseases have a fetal origin. According to FeBAD, injury or environmental influences occurring at critical periods of organ development could result in “programmatic” changes via alterations in gene expression or gene imprinting that may result in functional deficits that become apparent later in life. Alzheimers disease (AD) is a progressive neurodegenerative disorder that is characterized by excessive deposits of aggregated β-amyloid (Aβ) peptides, which are snippets of the β-amyloid precursor protein (APP). The predominately sporadic nature of AD suggests that the environment must play a role in neurodegeneration. To examine latent responses to an environmental agent, we exposed rodents to lead and monitored the lifetime expression of the APP gene. We observed that APP mRNA expression was transiently induced in neonates, but exhibited a delayed overexpression 20 months after exposure to Pb had ceased. This upregulation in APP mRNA expression was commensurate with a rise in activity of the transcription factor Sp1, one of the regulators of the APP gene. Furthermore, the increase in APP gene expression in old age was accompanied by an elevation in APP and its amyloidogenic Aβ product. In contrast, APP expression, Sp1 activity, as well as APP and Aβ protein levels were unresponsive to Pb exposure during old age. These data suggested that environmental influences occurring during brain development predetermined the expression and regulation of APP later in life, potentially altering the course of amyloidogenesis.

Phenserine regulates translation of β-amyloid precursor protein mRNA by a putative interleukin-1 responsive element, a target for drug development

The reduction in levels of the potentially toxic amyloid-β peptide (Aβ) has emerged as one of the most important therapeutic goals in Alzheimers disease. Key targets for this goal are factors that affect the expression and processing of the Aβ precursor protein (βAPP). Earlier reports from our laboratory have shown that a novel cholinesterase inhibitor, phenserine, reduces βAPP levels in vivo. Herein, we studied the mechanism of phenserines actions to define the regulatory elements in βAPP processing. Phenserine treatment resulted in decreased secretion of soluble βAPP and Aβ into the conditioned media of human neuroblastoma cells without cellular toxicity. The regulation of βAPP protein expression by phenserine was posttranscriptional as it suppressed βAPP protein expression without altering βAPP mRNA levels. However, phenserines action was neither mediated through classical receptor signaling pathways, involving extracellular signal-regulated kinase or phosphatidylinositol 3-kinase activation, nor was it associated with the anticholinesterase activity of the drug. Furthermore, phenserine reduced expression of a chloramphenicol acetyltransferase reporter fused to the 5′-mRNA leader sequence of βAPP without altering expression of a control chloramphenicol acetyltransferase reporter. These studies suggest that phenserine reduces Aβ levels by regulating βAPP translation via the recently described iron regulatory element in the 5′-untranslated region of βAPP mRNA, which has been shown previously to be up-regulated in the presence of interleukin-1. This study identifies an approach for the regulation of βAPP expression that can result in a substantial reduction in the level of Aβ.

Gene structure and organization of the human β-secretase (BACE) promoter

The first step in the generation of the amyloid‐β peptide (Aβ) deposited in the brains of patients with Alzheimers disease (AD) is the processing of the larger Aβ precursor protein (APP) by an integral membrane aspartyl protease named the β‐site APP‐cleaving enzyme (BACE). We present the genomic organization of the BACE gene. BACE mRNAs are synthesized as nine exons and eight introns from a 30.6 kb region of chromosome 11q23.2–11q23.3. Regulation of BACE may play an important role in regulating the levels of Aβ produced and is therefore likely to play an important role in AD. Herein, we report the cloning and detailed analysis of 3765 nucleotides of the promoter region of BACE and 364 nucleotides of the 5′ untranslated region of the BACE mRNA (5′ UTR). Characteristic “CAAT” and “TATA” boxes are absent within 1.5 kb of the transcription start site (TSS). The promoter region and 5′ UTR contain multiple transcription factor binding sites, such as activator protein (AP)1, AP2, cAMP response element binding protein (CREB), estrogen responsive element (ERE), glucocorticoid responsive element (GRE), “GC” box, nuclear factor (NF)‐κB, signal transducer and activator of transcription (STAT)1, stimulating protein (SP)1, metal‐regulatory elements, and possible Zeste binding sites. Limited interspecies similarity was observed between the human sequence and corresponding genomic DNA from the rat and mouse sequences, but several transcription factor‐binding sites are conserved. Thus, the BACE gene contains basal regulatory elements, inducible features and sites for regulated activity by various transcription factors. These results identify the important regions for functional analysis of the binding domains and neuron‐specific expression (1). Such a study will allow us to further examine the possible role of changes in the promoter of BACE in AD pathogenesis.

Dietary supplementation with melatonin reduces levels of amyloid beta-peptides in the murine cerebral cortex

Abstract:  Melatonin levels decrease with aging in mice. Dietary supplementation with melatonin has recently been shown to result in a significant rise in levels of endogenous melatonin in the serum and all other tissue samples tested. Herein, the effects of dietary melatonin on brain levels of nitric oxide synthase, synaptic proteins and amyloid beta‐peptides (Aβ) were determined in mice. Melatonin supplementation did not significantly change cerebral cortical levels of nitric oxide synthase or synaptic proteins such as synaptophysin and SNAP‐25. Increased brain melatonin concentrations however, led to a significant reduction in levels of toxic cortical Aβ of both short and long forms which are involved in amyloid depositions and plaque formation in Alzheimers diseases. Thus, melatonin supplementation may retard neurodegenerative changes associated with brain aging. Depletion of melatonin in the brain of aging mice may in part account for this adverse change.

Age-related changes in serum melatonin in mice: higher levels of combined melatonin and 6-hydroxymelatonin sulfate in the cerebral cortex than serum, heart, liver and kidney tissues

Abstract:  Age‐related changes in levels of melatonin and 6‐hydroxymelatonin sulfate and effects of dietary melatonin on their levels in different tissues were determined in mice. Levels of melatonin were highest in the serum followed by liver, kidney, cerebral cortex and heart as measured by a quantitative and sensitive enzyme‐labeled immunosorbent assay (ELISA). Serum melatonin levels decreased with age, and were reduced by 80% in 27‐month old mice relative to 12‐month old mice. Levels of 6‐hydroxymelatonin sulfate were measured independently in various tissues. Levels of the melatonin metabolite, 6‐hydroxymelatonin sulfate were significantly higher than free melatonin in all tissues tested. Levels of 6‐hydroxymelatonin sulfate were highest in the cerebral cortex followed by the serum, heart, kidney, and liver. In 12‐month old mice 6‐hydroxymelatonin sulfate concentration was approximately 1000‐fold greater than that of melatonin in the cerebral cortex, it was only 3‐fold greater than melatonin levels in the serum. Thus only 0.1% of total melatonin in the brain was present in the free and unconjugated form but the corresponding value for serum was 27.4%. The cerebral cortex had the highest levels of combined melatonin and 6‐hydroxymelatonin sulfate than other tissue tested in control mice. There was no significant change in 6‐hydroxymelatonin sulfate levels between young and old mice. There was also no age‐dependent change in levels of serotonin or cortisol in the serum samples. Dietary supplementation with melatonin resulted in a significant increase in levels of melatonin in the serum and all other tissue samples tested. Thus, any age‐related decline of tissue melatonin can be reversed by supplementation with dietary melatonin.

Lead (Pb) exposure and its effect on APP proteolysis and Aβ aggregation

Alzheimers disease (AD) is a progressive neurodegenerative disorder with clinical manifestations appearing in old age, however, the initial stages of this disease may begin early in life. AD is characterized by the presence of excessive deposits of aggregated β‐amyloid (Aβ) peptides, which are derived from the β‐amyloid precursor protein (APP) following processing by β‐secretase and γ‐secretase. Recently, we have reported that developmental exposure of rats to Pb resulted in latent elevation of APP mRNA, APP, and Aβ in old age. Here we examined whether latent up‐regulation in APP expression and Aβ levels is exacerbated by concurrent disturbances in APP processing or Aβ aggregation. Among the environmental metals tested, only Aβ solutions containing Pb promoted the formation of Aβ aggregates at nanomolar concentrations. The lifetime profiles of α‐, β‐, and γ‐secretases remained constant in adult and aging animals, and developmental exposure to Pb did not alter them. Furthermore, the addition of various concentrations of Pb (0.1 to 50 µM) to cerebral cortical extracts derived from control animals also did not affect the proteolytic activities of these enzymes. Therefore, we propose that amyloidogenesis is promoted by a latent response to developmental reprogramming of the expression of the APP gene by early exposure to Pb, as well as enhancement of Aβ aggregation in old age. In rodents, these events occur without Pb‐induced disturbances to the enzymatic processing of APP. The aforementioned results provide further evidence for the developmental basis of amyloidogenesis and late‐life disturbances in AD‐associated proteins by environmental agents.

Functional characterization of the 5′ flanking region of the BACE gene: identification of a 91 bp fragment involved in basal level of BACE promoter expression

Pathological characteristics of Alzheimers disease (AD) include amyloid‐β (Aβ) plaques. Aβ is derived from the Aβ peptide precursor protein (APP) by γ‐ and β‐secretases, the latter known as β‐site APP‐cleaving enzyme 1 (BACE1, or herein BACE). We have also described potentially important regions in the promoter of BACE, which may regulate its activity (1). Herein, we have functionally dissected the regulatory regions within the BACE promoter into areas containing positive and negative regulatory elements. The 4.1 kb promoter region (−3765/+364, +1 being the transcription start site [TSS]) includes positive regulatory element in the ‐2975 to ‐2062 region flanked on either side by negative regulatory elements at −3764/−2975 and −2062/−1056. This is separated from the minimal promoter and 5′ UTR by a neutral region of roughly 700 base pairs (bp). A 91 bp fragment (224/314) is the shortest region with a significant reporter gene activity and constitutes the minimal promoter element for BACE. Neuronal preference of the promoter was apparent in the 141 bp fragment (224/364) that contained the 91 bp fragment. Gel shift results also suggest the strongest signal of DNA‐protein interaction with the 91 bp fragment in neuronal nuclear extracts. This interaction was strongly blocked by an activator protein (AP)2 binding oligomer. Super gel shift assays suggest that both the AP2‐binding oligomer and the 91 bp fragment interfered with each others binding capacity. An AP2 sequence is predicted to occur within the 91 bp fragment. We also found stimulating protein (SP)1‐binding sites in this region of the promoter. Thus, functional and gel shift analysis indicate that the 91 bp fragment containing the AP2 and SP1 binding sites constitutes the core promoter region of BACE. Changes in the activity of this region could play an important role in regulating BACE activity in neurons.

FUNCTIONAL ACTIVITY OF THE NOVEL ALZHEIMER’S AMYLOID β–PEPTIDE INTERACTING DOMAIN (AβID) IN THE APP AND BACE1 PROMOTER SEQUENCES AND IMPLICATIONS IN ACTIVATING APOPTOTIC GENES AND IN AMYLOIDOGENESIS

Amyloid-β peptide (Aβ) plaque in the brain is the primary (post mortem) diagnostic criterion of Alzheimers disease (AD). The physiological role(s) of Aβ are poorly understood. We have previously determined an Aβ interacting domain (AβID) in the promoters of AD-associated genes (Maloney and Lahiri, 2011. Gene. 15,doi:10.1016/j.gene.2011.06.004. epub ahead of print.). This AβID interacts in a DNA sequence-specific manner with Aβ. We now demonstrate novel Aβ activity as a possible transcription factor. Herein, we detected 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 H2O2-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 H2O2. 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.

Age‐related changes in murine CNS mRNA gene expression are modulated by dietary melatonin

Abstract:  Brain cellular functions decline with normal aging, accompanied by a changing profile of gene expression. Gene array analysis was used to quantitatively estimate messenger RNA (mRNA) expression levels in the cerebral cortex of both young (4‐month) and old (27‐month) B6C3F1 male mice. A stringent degree of significance was obtained by using multiple gene chips. Out of 12,423 mRNA levels, only 25 changed significantly with age. Nine of these genes coded for inflammatory proteins, all of which were elevated in aged, relative to younger mice. Melatonin (200 p.p.m.) included in the diet of aged animals for 8 wk elevated serum and cortical melatonin and reversed 13 of the 25 genes altered with age. In no case did melatonin potentiate age‐related changes in gene expression. The restoration of a more youthful gene profile to brains of aged animals by melatonin, to a large extent, involves reversal of age‐induced elevation of basal inflammatory parameters.

Presence of a "CAGA box" in the APP gene unique to amyloid plaque-forming species and absent in all APLP-1/2 genes: Implications in Alzheimer's disease

Potentially toxic amyloid β‐peptide (Aβ) in Alzheimers disease (AD) is generated from a family of Aβ‐containing precursor proteins (APP), which is regulated via the 5′‐untranslated region (5′‐ UTR) of its mRNA. We analyzed 5′‐UTRs of the APP superfamily, including amyloid plaque‐ forming and non‐amyloid plaque‐forming species, and of prions (27 different DNA sequences). A “CAGA” sequence proximal to the “ATG” start codon was present in a location unique to APP genes of amyloid plaque‐forming species and absent in all other genes surveyed. This CAGA box is immediately upstream of an interleukin‐1‐responsive element (acute box). In addition, the proximal CAGA box is predicted to appear on a stem‐loop structure in both human and guinea pig APP mRNA. This stem‐loop is part of a predicted bulge‐loop that encompasses a known iron regulatory element (IRE). Electrophoretic mobility shift with segments of the APP 5′‐UTR showed that a region with the proximal CAGA sequence binds nuclear proteins, and this UTR fragment is active in a reporter gene functional assay. Thus, the 5′‐UTR in the human APP but not those of APP‐like proteins contains a specific region that may participate in APP regulation and may determine a more general model for amyloid generation as seen in AD. The 5′‐UTR of human APP contains several interesting control elements, such as an acute box element, a CAGA box, an IRE, and a transforming growth factor‐β‐responsive element, that could control APP expression and provide suitable and specific drug targets for AD.