Transcription factor regulation of amyloid-beta pathway genes by SP1-Modulating compounds : a novel approach in Alzheimer's Disease

Indiana University-Purdue University Indianapolis (IUPUI)Alzheimer's disease (AD) is a neurodegenerative disorder characterized by the presence of neuritic plaques consisting of extracellular amyloid-beta (Aβ) and neurofibrillary tangles comprised of hyperphosphorylated microtubule associated tau. Aβ is produced following the cleavage of amyloid precursor protein (APP) by the enzyme BACE1. Transcription factors (TFs) are proteins involved in the regulation of gene transcription. Expression levels of some TFs are perturbed in AD. SP1 binding sites on both the APP and BACE1 promoters implicate its potential role in AD. Aβ peptide itself mediates activation of cyclindependent kinase 5 (CDK5), an enzyme which phosphorylates the FOXO (Forkhead Box) TFs. In order to study mechanisms of TF regulation of Aβ production in human models, neuronally differentiated cells as well as a primary human neurosphere culture were used to test the effects of TF-modulating compounds. Our hypothesis is that by targeting relevant TFs via pharmacological inhibitors in human cells, BACE1 activity or APP expression will decrease and Aβ production will be reduced as a result. To test the involvement of TFs in the regulation of APP, we treated several mammalian cells lines and post-mitotic human neuronal cells with roscovitine, mithramycin A (MTM), MTM analogs (MTM-SDK, MTM-SK), and tolfenamic acid (TA). MTM and TA treatment of neurons differentially activated several TFs implicated in AD. Treatment of differentiated neurospheres with MTM led to a significant decrease in APP and SP1 expression along with Aβ40 levels. Epigenetic mechanisms involve alteration of the binding affinity between DNA and transcription factors. We predict that modulation of these TFs may be influenced by epigenetic modifications. To test the effects of drugs on epigenetic markers, histone deacetylase (HDAC) and DNA methyltransferase (DNMT) activity was measured. MTM-SDK significantly decreased DNMT activity in differentiated neuroblastoma cells, this may enhance or decrease the ability of SP1 to bind to target DNA and affect transcription of BACE1 or APP. Targeting TF activity is a novel means to manipulate the amyloid pathway. Compounds modifying TF binding to sites on the BACE1 or APP promoters may provide a means to limit the production of amyloid-beta and slow the symptoms of AD

Latent consequences of early-life lead (Pb) exposure and the future: Addressing the Pb crisis

Background. The lead (Pb) exposure crisis in Flint, Michigan has passed from well-publicized event to a footnote, while its biological and social impact will linger for lifetimes. Interest in the “water crisis” has dropped to pre-event levels, which is neither appropriate nor safe. Flint’s exposure was severe, but it was not unique. Problematic Pb levels have also been found in schools and daycares in 42 states in the USA. The enormity of Pb exposure via municipal water systems requires multiple responses. Herein, we focus on addressing a possible answer to long-term sequelae of Pb exposure. We propose “4R’s” (remediation, renovation, reallocation, and research) against the Pb crisis that goes beyond a short-term fix. Remediation for affected individuals must continue to provide clean water and deal with both short and long-term effects of Pb exposure. Renovation of current water delivery systems, at both system-wide and individual site levels, is necessary. Reallocation of resources is needed to ensure these two responses occur and to get communities ready for potential sequelae of Pb exposure. Finally, properly focused research can track exposed individuals and illuminate latent (presumably epigenetic) results of Pb exposure and inform further resource reallocation. Conclusion. Motivation to act by not only the general public but also by scientific and medical leaders must be maintained beyond initial news cycle spikes and an annual follow-up story. Environmental impact of Pb contamination of drinking water goes beyond one exposure incident in an impoverished and forgotten Michigan city. Population effects must be addressed long-term and nationwide

BACE1 Gene Regulation: A Novel Drug Target in Alzheimer’s Disease

poster abstractAlzheimer’s disease (AD) is the most common form of dementia in the elderly in the United States. AD is characterized by the presence of amyloid-β (Aβ) peptide plaques. Aberrant deposition is believed to result from the misregulation of the production or the clearance of Aβ. The rate-limiting step in Aβ production is the processing of amyloid- β precursor protein (APP) by β-site APP-cleaving enzyme (BACE1). BACE1 could play a critical role in the development of AD and is a promising drug target. In this study, we aim to reduce BACE1 enzyme levels by reducing BACE1 gene expression. We previously analyzed the promoter activity of BACE1 and the 5’ untranslated region of BACE1 mRNA. The BACE1 promoter contains many transcription factor sites including SP1, MEF2, and STAT1, which have been shown to play a role in the regulation of BACE1 gene expression. Mithramycin A (MithA) has been previously shown to selectively inhibit SP1-mediated transcriptional activation. We expect inhibition of SP1 to lead to downregulation of BACE1 and decreased Aβ, providing a novel target for AD. We have tested several BACE1 promoter-deletion constructs by DNA transfection in human neuronal cultures, treatment with MithA, and performed luciferase reporter assays. In a neuroblastoma (NB) cell line, we observed the significant increase in luciferase reporter activity of two BACE1 plasmid constructs after treatment with MithA. Treatment also correlated with an increase in BACE1 protein expression and a decrease in APP expression. This suggests that the mechanism by which MithA influences the BACE1 promoter could be complex and or due to other transcription factor sites as well. Further experiments will include using differentiated NB cells and human primary fetal neurons, along with the use of other Sp1 inhibitors including tolfenamic acid to elucidate the regulation of APP and BACE1 promoters leading to lower Aβ levels

Transgenerational latent early-life associated regulation unites environment and genetics across generations

The origin of idiopathic diseases is still poorly understood. The latent early-life associated regulation (LEARn) model unites environmental exposures and gene expression while providing a mechanistic underpinning for later-occurring disorders. We propose that this process can occur across generations via transgenerational LEARn (tLEARn). In tLEARn, each person is a 'unit' accumulating preclinical or subclinical 'hits' as in the original LEARn model. These changes can then be epigenomically passed along to offspring. Transgenerational accumulation of 'hits' determines a sporadic disease state. Few significant transgenerational hits would accompany conception or gestation of most people, but these may suffice to 'prime' someone to respond to later-life hits. Hits need not produce symptoms or microphenotypes to have a transgenerational effect. Testing tLEARn requires longitudinal approaches. A recently proposed longitudinal epigenome/envirome-wide association study would unite genetic sequence, epigenomic markers, environmental exposures, patient personal history taken at multiple time points and family history

Initial analysis of peripheral lymphocytic extracellular signal related kinase activation in autism

BACKGROUND: Dysregulation of extracellular signal-related kinase (ERK) activity has been potentially implicated in the pathophysiology of autistic disorder (autism). ERK is part of a central intracellular signaling cascade responsible for a myriad of cellular functions. ERK is expressed in peripheral blood lymphocytes, and measurement of activated (phosphorylated) lymphocytic ERK is commonly executed in many areas of medicine. We sought to conduct the first study of ERK activation in humans with autism by utilizing a lymphocytic ERK activation assay. We hypothesized that ERK activation would be enhanced in peripheral blood lymphocytes from persons with autism compared to those of neurotypical control subjects. METHOD: We conducted an initial study of peripheral lymphocyte ERK activation in 45 subjects with autism and 26 age- and gender-matched control subjects (total n = 71). ERK activation was measured using a lymphocyte counting method (primary outcome expressed as lymphocytes staining positive for cytosolic phosphorylated ERK divided by total cells counted) and additional Western blot analysis of whole cell phosphorylated ERK adjusted for total ERK present in the lymphocyte lysate sample. RESULTS: Cytosolic/nuclear localization of pERK activated cells were increased by almost two-fold in the autism subject group compared to matched neurotypical control subjects (cell count ratio of 0.064 ± 0.044 versus 0.034 ± 0.031; p = 0.002). Elevated phosphorylated ERK levels in whole cell lysates also showed increased activated ERK in the autism group compared to controls (n = 54 total) in Western blot analysis. CONCLUSIONS: The results of this first in human ERK activation study are consistent with enhanced peripheral lymphocytic ERK activation in autism, as well as suggesting that cellular compartmentalization of activated ERK may be altered in this disorder. Future work will be required to explore the impact of concomitant medication use and other subject characteristics such as level of cognitive functioning on ERK activation