Bryan Maloney

Alzheimer's 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 Alzheimers 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.

Transcriptional Regulation of β-Secretase by p25/cdk5 Leads to Enhanced Amyloidogenic Processing

Cyclin-dependent kinase 5 (cdk5) has been implicated in Alzheimers disease (AD) pathogenesis. Here, we demonstrate that overexpression of p25, an activator of cdk5, led to increased levels of BACE1 mRNA and protein in vitro and in vivo. A p25/cdk5 responsive region containing multiple sites for signal transducer and activator of transcription (STAT1/3) was identified in the BACE1 promoter. STAT3 interacts with the BACE1 promoter, and p25-overexpressing mice had elevated levels of pSTAT3 and BACE1, whereas cdk5-deficient mice had reduced levels. Furthermore, mice with a targeted mutation in the STAT3 cdk5 responsive site had lower levels of BACE1. Increased BACE levels in p25 overexpressing mice correlated with enhanced amyloidogenic processing that could be reversed by a cdk5 inhibitor. These data demonstrate a pathway by which p25/cdk5 increases the amyloidogenic processing of APP through STAT3-mediated transcriptional control of BACE1 that could have implications for AD pathogenesis.

The LEARn model: an epigenetic explanation for idiopathic neurobiological diseases

Neurobiological disorders have diverse manifestations and symptomology. Neurodegenerative disorders, such as Alzheimers disease, manifest late in life and are characterized by, among other symptoms, progressive loss of synaptic markers. Developmental disorders, such as autism spectrum, appear in childhood. Neuropsychiatric and affective disorders, such as schizophrenia and major depressive disorder, respectively, have broad ranges of age of onset and symptoms. However, all share uncertain etiologies, with opaque relationships between genes and environment. We propose a ‘Latent Early-life Associated Regulation’ (LEARn) model, positing latent changes in expression of specific genes initially primed at the developmental stage of life. In this model, environmental agents epigenetically disturb gene regulation in a long-term manner, beginning at early developmental stages, but these perturbations might not have pathological results until significantly later in life. The LEARn model operates through the regulatory region (promoter) of the gene, specifically through changes in methylation and oxidation status within the promoter of specific genes. The LEARn model combines genetic and environmental risk factors in an epigenetic pathway to explain the etiology of the most common, that is, sporadic, forms of neurobiological disorders.

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.

The Experimental Alzheimer's Disease Drug Posiphen [(+)-Phenserine] Lowers Amyloid-β Peptide Levels in Cell Culture and Mice

Major characteristics of Alzheimers disease (AD) are synaptic loss, cholinergic dysfunction, and abnormal protein depositions in the brain. The amyloid β-peptide (Aβ), a proteolytic fragment of amyloid β precursor protein (APP), aggregates to form neuritic plaques and has a causative role in AD. A present focus of AD research is to develop safe Aβ-lowering drugs. A selective acetylcholinesterase inhibitor, phenserine, in current human trials lowers both APP and Aβ. Phenserine is dose-limited in animals by its cholinergic actions; its cholinergically inactive enantiomer, posiphen (+)-[phenserine], was assessed. In cultured human neuroblastoma cells, posiphen, like phenserine, dose- and time-dependently lowered APP and Aβ levels by reducing the APP synthesis rate. This action translated to an in vivo system. Posiphen administration to mice (7.5–75 mg/kg daily, 21 consecutive days) significantly decreased levels of total APP (tissue mass-adjusted) in a dose-dependent manner. Aβ40 and Aβ42 levels were significantly lowered by posiphen (≥15 mg/kg) compared with controls. The activities of α-, β-, and γ-secretases were assessed in the same brain samples, and β-secretase activity was significantly reduced. Posiphen, like phenserine, can lower Aβ via multiple mechanisms and represents an interesting drug candidate for AD treatment.

Autism, Alzheimer disease, and fragile X: APP, FMRP, and mGluR5 are molecular links

The present review highlights an association between autism, Alzheimer disease (AD), and fragile X syndrome (FXS). We propose a conceptual framework involving the amyloid-β peptide (Aβ), Aβ precursor protein (APP), and fragile X mental retardation protein (FMRP) based on experimental evidence. The anabolic (growth-promoting) effect of the secreted α form of the amyloid-β precursor protein (sAPPα) may contribute to the state of brain overgrowth implicated in autism and FXS. Our previous report demonstrated that higher plasma sAPPα levels associate with more severe symptoms of autism, including aggression. This molecular effect could contribute to intellectual disability due to repression of cell–cell adhesion, promotion of dense, long, thin dendritic spines, and the potential for disorganized brain structure as a result of disrupted neurogenesis and migration. At the molecular level, APP and FMRP are linked via the metabotropic glutamate receptor 5 (mGluR5). Specifically, mGluR5 activation releases FMRP repression of APP mRNA translation and stimulates sAPP secretion. The relatively lower sAPPα level in AD may contribute to AD symptoms that significantly contrast with those of FXS and autism. Low sAPPα and production of insoluble Aβ would favor a degenerative process, with the brain atrophy seen in AD. Treatment with mGluR antagonists may help repress APP mRNA translation and reduce secretion of sAPP in FXS and perhaps autism.

High levels of Alzheimer beta-amyloid precursor protein (APP) in children with severely autistic behavior and aggression

Autism is characterized by restricted, repetitive behaviors and impairment in socialization and communication. Although no neuropathologic substrate underlying autism has been found, the findings of brain overgrowth via neuroimaging studies and increased levels of brain-derived neurotrophic factor (BDNF) in neuropathologic and blood studies favor an anabolic state. We examined acetylcholinesterase, plasma neuronal proteins, secreted beta-amyloid precursor protein (APP), and amyloid-beta 40 and amyloid-beta 42 peptides in children with and without autism. Children with severe autism and aggression expressed secreted beta-amyloid precursor protein at two or more times the levels of children without autism and up to four times more than children with mild autism. There was a trend for children with autism to show higher levels of secreted beta-amyloid precursor protein and nonamyloidogenic secreted beta-amyloid precursor protein and lower levels of amyloid-beta 40 compared with controls. This favors an increased α-secretase pathway in autism (anabolic), opposite to what is seen in Alzheimer disease. Additionally, a complex relationship between age, acetylcholinesterase, and plasma neuronal markers was found. (J Child Neurol 2006;21:444—449; DOI 10.2310/7010.2006.00130).

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.

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.