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Alzheimer’s disease (AD) is a progressive neurodegenerative disorder with loss of cognitive, executive, and other mental functions, and is the most common form of age-related dementia. Amyloid-β peptide (Aβ) contributes to the etiology and progression of the disease. Aβ is derived from the amyloid-β precursor protein (APP). Multiple microRNA (miRNA) species are also implicated in AD. We report that human hsa-miR20b-5p (miR-20b), produced from the MIR20B gene on Chromosome X, may play complex roles in AD pathogenesis, including Aβ regulation. Specifically, miR-20b-5p miRNA levels were altered in association with disease progression in three regions of the human brain: temporal neocortex, cerebellum, and posterior cingulate cortex. In cultured human neuronal cells, miR-20b-5p treatment interfered with calcium homeostasis, neurite outgrowth, and branchpoints. A single-nucleotide polymorphism (SNP) upstream of the MIR20B gene (rs13897515) associated with differences in levels of cerebrospinal fluid (CSF) Aβ1-42 and thickness of the entorhinal cortex. We located a miR-20b-5p binding site in the APP mRNA 3′-untranslated region (UTR), and treatment with miR-20b-5p reduced APP mRNA and protein levels. Network analysis of protein-protein interactions and gene coexpression revealed other important potential miR-20b-5p targets among AD-related proteins/genes. MiR-20b-5p, a miRNA that downregulated APP, was paradoxically associated with an increased risk for AD. However, miR-20b-5p also reduced, and the blockade of APP by siRNA likewise reduced calcium influx. As APP plays vital roles in neuronal health and does not exist solely to be the source of “pathogenic” Aβ, the molecular etiology of AD is likely to not just be a disease of “excess” but a disruption of delicate homeostasis.

Fragile X mental retardation protein (FMRP) binds to and regulates the translation of amyloid-β protein precursor (App) mRNA, but the detailed mechanism remains to be determined. Differential methylation of App mRNA could underlie FMRP binding, message localization and translation efficiency. We sought to determine the role of FMRP and N6-methyladeonsine (m6A) on nuclear export of App mRNA. We utilized the m6A dataset by Hsu and colleagues to identify m6A sites in App mRNA and to determine if the abundance of message in the cytoplasm relative to the nucleus is altered in Fmr1 knockout mouse brain cortex. Given that processing of APP to Aβ and soluble APP alpha (sAPPα) contributes to disease phenotypes, we also investigated whether Fmr1KO associates with nuclear export of the mRNAs for APP protein processing enzymes, including β-site amyloid cleaving enzyme (Bace1), A disintegrin and metalloproteinases (Adams), and presenilins (Psen). Fmr1KO did not alter the nuclear/cytoplasmic abundance of App mRNA. Of 36 validated FMRP targets, 35 messages contained m6A peaks but only Agap2 mRNA was selectively enriched in Fmr1KO nucleus. The abundance of the APP processing enzymes Adam9 and Psen1 mRNA, which code for a minor alpha-secretase and gamma-secretase, respectively, were selectively enriched in wild type cytoplasm.

Alzheimer's disease (AD) is a progressive neurodegenerative disorder with loss of cognitive, executive, and other mental functions, and is the most common form of age-related dementia. Amyloid-β peptide (Aβ) contributes to the etiology and progression of the disease. Aβ is derived from the amyloid-β precursor protein (APP). Multiple microRNA (miRNA) species are also implicated in AD. We report that human hsa-miR20b-5p (miR-20b), produced from the MIR20B gene on Chromosome X, may play complex roles in AD pathogenesis, including Aβ regulation. Specifically, miR-20b-5p miRNA levels were altered in association with disease progression in three regions of the human brain: temporal neocortex, cerebellum, and posterior cingulate cortex. In cultured human neuronal cells, miR-20b-5p treatment interfered with calcium homeostasis, neurite outgrowth, and branchpoints. A single-nucleotide polymorphism (SNP) upstream of the MIR20B gene (rs13897515) associated with differences in levels of cerebrospinal fluid (CSF) Aβ

Late Onset Alzheimer’s Disease is the most common cause of dementia, characterized by extracellular deposition of plaques primarily of amyloid-β (Aβ) peptide and tangles primarily of hyperphosphorylated tau protein. We present data to suggest a noninvasive strategy to decrease potentially toxic Aβ levels, using repeated electromagnetic field stimulation (REMFS) in primary human brain (PHB) cultures. We examined effects of REMFS on Aβ levels (Aβ40 and Aβ42, that are 40 or 42 amino acid residues in length, respectively) in PHB cultures at different frequencies, powers, and specific absorption rates (SAR). PHB cultures at day in vitro 7 (DIV7) treated with 64 MHz, and 1 hour daily for 14 days (DIV 21) had significantly reduced levels of secreted Aβ40 (p = 001) and Aβ42 (p = 0.029) peptides, compared to untreated cultures. PHB cultures (DIV7) treated at 64 MHz, for 1 or 2 hour during 14 days also produced significantly lower Aβ levels. PHB cultures (DIV28) treated with 64 MHz 1 hour/day during 4 or 8 days produced a similar significant reduction in Aβ40 levels. 0.4 W/kg was the minimum SAR required to produce a biological effect. Exposure did not result in cellular toxicity nor significant changes in secreted Aβ precursor protein-α (sAPPα) levels, suggesting the decrease in Aβ did not likely result from redirection toward the α-secretase pathway. EMF frequency and power used in our work is utilized in human magnetic resonance imaging (MRI, thus suggesting REMFS can be further developed in clinical settings to modulate Aβ deposition.

Rivastigmine (or Exelon) is a cholinesterase inhibitor, currently used as a symptomatic treatment for mild-to-moderate Alzheimer’s disease (AD). Amyloid-β peptide (Aβ) generated from its precursor protein (APP) by β-secretase (or BACE1) and γ-secretase endoproteolysis. Alternative APP cleavage by α-secretase (a family of membrane-bound metalloproteases– Adamalysins) precludes the generation of toxic Aβ and yields a neuroprotective and neurotrophic secreted sAPPα fragment. Several signal transduction pathways, including protein kinase C and MAP kinase, stimulate α-secretase. We present data to suggest that rivastigmine, in addition to anticholinesterase activity, directs APP processing away from BACE1 and towards α-secretases. We treated rat neuronal PC12 cells and primary human brain (PHB) cultures with rivastigmine and the α-secretase inhibitor TAPI and assayed for levels of APP processing products and α-secretases. We subsequently treated 3×Tg (transgenic) mice with rivastigmine and harvested hippocampi to assay for levels of APP processing products. We also assayed postmortem human control, AD, and AD brains from subjects treated with rivastigmine for levels of APP metabolites. Rivastigmine dose-dependently promoted α-secretase activity by upregulating levels of ADAM-9, -10, and -17 α-secretases in PHB cultures. Co-treatment with TAPI eliminated rivastigmine-induced sAPPα elevation. Rivastigmine treatment elevated levels of sAPPα in 3×Tg mice. Consistent with these results, we also found elevated sAPPα in postmortem brain samples from AD patients treated with rivastigmine. Rivastigmine can modify the levels of several shedding proteins and directs APP processing toward the non-amyloidogenic pathway. This novel property of rivastigmine can be therapeutically exploited for disease-modifying intervention that goes beyond symptomatic treatment for AD.

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.

The multitude of molecular pathways underlying memory impairment in neurological disorders and aging-related disorders has been a major hurdle against therapeutic targeting. Over the years, neuronal growth promoting factors, intracellular kinases, and specific transcription factors, particularly cyclic AMP response element-binding protein (CREB), have emerged as crucial players of memory storage, and their disruption accompanies many cognitive disabilities. However, a molecular link that can influence these major players and can be a potential recovery target has been elusive. Recent reports suggest that extracellular cues at the synapses might evoke an intracellular signaling cascade and regulate memory function. Herein, we report novel function of an extracellular serine protease, kallikrein 8 (KLK8/Neuropsin) in regulating the expression of microtubule associated dendrite growth marker microtubule-associated protein (MAP2)c, dendrite architecture and protein kinase A (PKA)-CREB signaling. Both knockdown of KLK8 via siRNA transfection in mouse primary hippocampal neurons and via intra-hippocampal administration of KLK8 antisense oligonucleotides in vivo reduced expression of MAP2c, dendrite length, dendrite branching and spine density. The KLK8 mediated MAP2c deficiency in turn inactivated PKA and downstream transcription factor phosphorylated CREB (pCREB), leading to downregulation of memory-linked genes and consequent impaired memory consolidation. These findings revealed a protease associated novel pathway of memory impairment in which KLK8 may act as a “regulator of regulators”, suggesting its exploration as an important therapeutic target of memory disorders.

Lithium (Li) is a medication long-used to treat bipolar disorder. It is currently under investigation for multiple nervous system disorders, including Alzheimer’s disease (AD). While perturbation of RNA levels by Li has been previously reported, its effects on the whole transcriptome has been given little attention. We, therefore, sought to determine comprehensive effects of Li treatment on RNA levels. We cultured and differentiated human neuroblastoma (SK-N-SH) cells to neuronal cells with all-trans retinoic acid (ATRA). We exposed cultures for one week to lithium chloride or distilled water, extracted total RNA, depleted ribosomal RNA and performed whole-transcriptome RT-sequencing. We analyzed results by RNA length and type. We further analyzed expression and protein interaction networks between selected Li-altered protein-coding RNAs and common AD-associated gene products. Lithium changed expression of RNAs in both non-specific (inverse to sequence length) and specific (according to RNA type) fashions. The non-coding small nucleolar RNAs (snoRNAs) were subject to the greatest length-adjusted Li influence. When RNA length effects were taken into account, microRNAs as a group were significantly less likely to have had levels altered by Li treatment. Notably, several Li-influenced protein-coding RNAs were co-expressed or produced proteins that interacted with several common AD-associated genes and proteins. Lithium’s modification of RNA levels depends on both RNA length and type. Li activity on snoRNA levels may pertain to bipolar disorders while Li modification of protein coding RNAs may be relevant to AD.

Memory loss is one of the most tragic symptoms of Alzheimer’s disease. Our laboratory has recently demonstrated that ‘i-Extract’ of Ashwagandha (Withania somnifera) restores memory loss in scopolamine (SC)-induced mice. The prime target of i-Extract is obscure. We hypothesize that i-Extract may primarily target muscarinic subtype acetylcholine receptors that regulate memory processes. The present study elucidates key target(s) of i-Extract via cellular, biochemical, and molecular techniques in a relevant amnesia mouse model and primary hippocampal neuronal cultures. Wild type Swiss albino mice were fed i-Extract, and hippocampal cells from naïve mice were treated with i-Extract, followed by muscarinic antagonist (dicyclomine) and agonist (pilocarpine) treatments. We measured dendritic formation and growth by immunocytochemistry, kallikrein 8 (KLK8) mRNA by reverse transcription polymerase chain reaction (RT-PCR), and levels of KLK8 and microtubule-associated protein 2, c isoform (MAP2c) proteins by western blotting. We performed muscarinic receptor radioligand binding. i-Extract stimulated an increase in dendrite growth markers, KLK8 and MAP2. Scopolamine-mediated reduction was significantly reversed by i-Extract in mouse cerebral cortex and hippocampus. Our study identified muscarinic receptor as a key target of i-Extract, providing mechanistic evidence for its clinical application in neurodegenerative cognitive disorders.

Alzheimer’s disease (AD) is the most common age-related form of dementia, associated with deposition of intracellular neuronal tangles consisting primarily of hyperphosphorylated microtubule-associated protein tau (p-tau) and extracellular plaques primarily comprising amyloid- β (Aβ) peptide. The p-tau tangle unit is a posttranslational modification of normal tau protein. Aβ is a neurotoxic peptide excised from the amyloid-β precursor protein (APP) by β-site APP-cleaving enzyme 1 (BACE1) and the γ-secretase complex. MicroRNAs (miRNAs) are short, single-stranded RNAs that modulate protein expression as part of the RNA-induced silencing complex (RISC). We identified miR-298 as a repressor of APP, BACE1, and the two primary forms of Aβ (Aβ40 and Aβ42) in a primary human cell culture model. Further, we discovered a novel effect of miR-298 on posttranslational levels of two specific tau moieties. Notably, miR-298 significantly reduced levels of ~55 and 50 kDa forms of the tau protein without significant alterations of total tau or other forms. In vivo overexpression of human miR-298 resulted in nonsignificant reduction of APP, BACE1, and tau in mice. Moreover, we identified two miR-298 SNPs associated with higher cerebrospinal fluid (CSF) p-tau and lower CSF Aβ42 levels in a cohort of human AD patients. Finally, levels of miR-298 varied in postmortem human temporal lobe between AD patients and age-matched non-AD controls. Our results suggest that miR-298 may be a suitable target for AD therapy.

In addition to the devastating symptoms of dementia, Alzheimer’s disease (AD) is characterized by accumulation of the processing products of the amyloid-β (Aβ) peptide precursor protein (APP). APP’s non-pathogenic functions include regulating intracellular iron (Fe) homeostasis. MicroRNAs are small (~ 20 nucleotides) RNA species that instill specificity to the RNA-induced silencing complex (RISC). In most cases, RISC inhibits mRNA translation through the 3′-untranslated region (UTR) sequence. By contrast, we report a novel activity of miR-346: specifically, that it targets the APP mRNA 5′-UTR to upregulate APP translation and Aβ production. This upregulation is reduced but not eliminated by knockdown of argonaute 2. The target site for miR-346 overlaps with active sites for an iron-responsive element (IRE) and an interleukin-1 (IL-1) acute box element. IREs interact with iron response protein1 (IRP1), an iron-dependent translational repressor. In primary human brain cultures, miR-346 activity required chelation of Fe. In addition, miR-346 levels are altered in late-Braak stage AD. Thus, miR-346 plays a role in upregulation of APP in the CNS and participates in maintaining APP regulation of Fe, which is disrupted in late stages of AD. Further work will be necessary to integrate other metals, and IL-1 into the Fe-miR-346 activity network. We, thus, propose a “FeAR” (Fe, APP, RNA) nexus in the APP 5′-UTR that includes an overlapping miR-346-binding site and the APP IRE. When a “healthy FeAR” exists, activities of miR-346 and IRP/Fe interact to maintain APP homeostasis. Disruption of an element that targets the FeAR nexus would lead to pathogenic disruption of APP translation and protein production.

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.

Late-onset Alzheimer's disease (LOAD) is the most common neurodegenerative disorder in older adults, affecting over 50% of those over age 85. Aging is the most important risk factor for the development of LOAD. Aging is associated with the decrease in the ability of cells to cope with cellular stress, especially protein aggregation. Here we describe how the process of aging affects pathways that control the processing and degradation of abnormal proteins including amyloid-β (Aβ). Genetic association studies in LOAD have successfully identified a large number of genetic variants involved in the development of the disease. However, there is a gap in understanding the interconnections between these pathomolecular events that prevent us from discovering therapeutic targets. We propose novel, pertinent links to elucidate how the biology of aging affects the sequence of events in the development of LOAD. Furthermore we analyze and synthesize the molecular-pathologic-clinical correlations of the aging process, involving the HSF1 and FOXO family pathways, Aβ metabolic pathway, and the different clinical stages of LOAD. Our new model postulates that the aging process would precede Aβ accumulation, and attenuation of HSF1 is an "upstream" event in the cascade that results in excess Aβ and synaptic dysfunction, which may lead to cognitive impairment and/or trigger "downstream" neurodegeneration and synaptic loss. Specific host factors, such as the activity of FOXO family pathways, would mediate the response to Aβ toxicity and the pace of progression toward the clinical manifestations of AD.

Autism spectrum disorder (ASD) and Fragile X syndrome (FXS) are developmental disorders. No validated blood-based biomarkers exist for either, which impedes bench-to-bedside approaches. Amyloid-β (Aβ) precursor protein (APP) and metabolites are usually associated with Alzheimer’s disease (AD). APP cleavage by α-secretase produces potentially neurotrophic secreted APPα (sAPPα) and the P3 peptide fragment. β-site APP cleaving enzyme (BACE1) cleavage produces secreted APPβ (sAPPβ) and intact Aβ. Excess Aβ is potentially neurotoxic and can lead to atrophy of brain regions such as amygdala in AD. By contrast, amygdala is enlarged in ASD but not FXS. We previously reported elevated levels of sAPPα in ASD and FXS vs. controls. We now report elevated plasma Aβ and total APP levels in FXS compared to both ASD and typically developing controls, and elevated levels of sAPPα in ASD and FXS vs. controls. By contrast, plasma and brain sAPPβ and Aβ were lower in ASD vs. controls but elevated in FXS plasma vs. controls. We also detected age-dependent increase in an α-secretase in ASD brains. We report a novel mechanistic difference in APP pathways between ASD (processing) and FXS (expression) leading to distinct APP metabolite profiles in these two disorders. These novel, distinctive biochemical differences between ASD and FXS pave the way for blood-based biomarkers for ASD and FXS.

Fragile X syndrome (FXS) is the most common form of inherited intellectual disability and is associated with up to 5% of autism cases. Several promising drugs are in preclinical testing for FXS; however, bench-to-bedside plans for the clinic are severely limited due to lack of validated biomarkers and outcome measures. Published work from our laboratories has demonstrated altered levels of amyloid-beta (Aβ) precursor protein (APP) and its metabolites in FXS and idiopathic autism. Westmark and colleagues have focused on β-secretase (amyloidogenic) processing and the accumulation of Aβ peptides in adult FXS models, whereas Lahiri and Sokol have studied α-secretase (non-amyloidogenic or anabolic) processing and altered levels of sAPPα and Aβ in pediatric autism and FXS. Thus, our groups have hypothesized a pivotal role for these Alzheimer's disease (AD)-related proteins in the neurodevelopmental disorders of FXS and autism. In this review, we discuss the contribution of APP metabolites to FXS and autism pathogenesis as well as the potential use of these metabolites as blood-based biomarkers and therapeutic targets. Our future focus is to identify key underlying mechanisms through which APP metabolites contribute to FXS and autism condition-to-disease pathology. Positive outcomes will support utilizing APP metabolites as blood-based biomarkers in clinical trials as well as testing drugs that modulate APP processing as potential disease therapeutics. Our studies to understand the role of APP metabolites in developmental conditions such as FXS and autism are a quantum leap for the neuroscience field, which has traditionally restricted any role of APP to AD and aging.

Diabetes, cardiovascular disease, hypertension, and other disorders have been unified within the metabolic syndrome. Recently, it has been proposed that Alzheimer's disease (AD) and other degenerative, age-related neurological disorders may also be etiologically linked to the metabolic syndrome in a metabolic-cognitive syndrome. We review current evidence in the field for this unification. In addition, we describe how the latent early-life associated regulation (LEARn) model provides specific mechanisms to predict genetic targets for both metabolic disorders, e.g., diabetes, and neurodegenerative disorders, e.g., AD. The LEARn model is based on environmental induction of latent epigenetic misregulation, which develops into disease upon suffering additional environmental insults. We review structural differences between gene sequences that are and are not susceptible to LEARn misregulation. In addition to suggesting research targets such as the IDE and SORCS1 genes, which are implicated in both AD and diabetes, LEARn suggests specific mechanisms for pre-disease remediation, based on nutritional adjustment of aberrant DNA methylation and oxidation. The possibility of a single metabolic-cognitive disorder opens up the possibility of unified preventative treatments that reduce monetary and social costs of disease. LEARn suggests specific, testable pathways within the large theory.

Alzheimer's disease (AD) is the leading cause of dementia in the elderly. Extraneuronal plaque comprising mostly the amyloid β peptide and intraneuronal tangles of hyperphosphorylated microtubule-associated τ protein (τ, gene MAPT) are typical of AD. Misfolded τ is also implicated in Parkinson's disease and frontotemporal dementia. We aim to understand the regulation of the human MAPT promoter by mapping its functional domains. We subcloned a 4868 base pair (bp) fragment from human BAC RPCI-11 100C5. Sequence analysis revealed an H2 haplotype MAPT promoter, 5′-UTR, and intronal fragment. Database analysis of the fragment showed 50%–75% homology with mouse and > 90% with rhesus monkey. Comparison with human H1 sequences revealed differences that crossed predicted transcription factor sites. DNA-protein interaction studies by electrophoretic mobility shift assay suggested hypoxia response and an active specificity protein 1 (SP1) site in the 5′-untranslated region. Transfection of a series of MAPT promoter deletions revealed unique functional domains. The distal-most had different activities in neuronal vs. non-neuronal cells. We have cloned, sequenced, and functionally characterized a 4868 bp fragment of the human MAPT 5′-flanking region, including the core promoter region (− 302/+4), neurospecific domains (− 4364/−1992 and + 293/+504, relative to + 1 TSS), and a hypoxia-inducible element (+ 60/+84). Our work extended functional analysis of the MAPT sequence further upstream, and explores cell-type specificity of MAPT promoter activity. Finally, we provided direct comparison of likely transcription factor binding sites, which are useful to understand differences between H1/H2 pathogenic associations.

Alzheimer's disease (AD) is a leading cause of aging related dementia and has been extensively studied by several groups around the world. A general consensus, based on neuropathology, genetics and cellular and animal models, is that the 4 kDa amyloid β protein (Aβ) triggers a toxic cascade that induces microtubule-associated protein τ (MAPT) hyperphosphorylation and deposition. Together, these lesions lead to neuronal dysfunction and neurodegeneration, modeled in animals, that ultimately causes dementia. Genetic studies show that a simple duplication of the Aβ precursor (APP) gene, as occurs in Down syndrome (trisomy 21), with a 1.5-fold increase in expression, can cause dementia with the complete AD associated neuropathology. The most fully characterized form of AD is early onset familial AD (FAD). Unfortunately, by far the most common form of AD is late onset AD (LOAD). FAD has well-identified autosomally dominant genetic causes, absent in LOAD. It is reasonable to hypothesize that environmental influences play a much stronger role in etiology of LOAD than of FAD. Since AD pathology in LOAD closely resembles FAD with accumulation of both Aβ and MAPT, it is likely that the environmental factors foster accumulation of these proteins in a manner similar to FAD mutations. Therefore, it is important to identify environmentally driven changes that "phenocopy" FAD in order to find ways to prevent LOAD. Epigenetic changes in expression are complex but stable determinants of many complex traits. Some aspects are regulated by prenatal and early post-natal development, others punctuate specific periods of maturation, and still others occur throughout life, mediating predictable changes that take place during various developmental stages. Environmental agents such as mercury, lead, and pesticides can disrupt the natural epigenetic program and lead to developmental deficits, mental retardation, feminization, and other complex syndromes. In this review we discuss latent early- life associated regulation (LEARn), where apparently temporary changes, induced by environmental agents, become latent and present themselves again at maturity or senescence to increase production of Aβ that may cause AD. The model provides us with a novel direction for identifying potentially harmful agents that may induce neurodegeneration and dementia later in life and provides hope that we may be able to prevent age-related neurodegenerative disease by "detoxifying" our environment.

A recent article in Gene highlighted potential function of miR-346 in human breast cancer (Yang et al., 2017). We request an explanation or correction of the report. In its current state, the text will certainly create confusion in the field and lead to incorrect assumptions. The authors made several critical errors. The abstract stated "we found that the expression of miR-346 was higher in breast cancer tissues than in their paired corresponding non-cancerous tissues" and the main text and legend for Fig. 1A stated "miR-346 expression was significantly higher in breast cancer tissues than in their paired corresponding non-cancerous tissues (Fig. 1A, Yang et al., 2017)" and "miR-346 was upregulated in breast cancer tissues and cell lines. (A)", respectively. It was also stated that "SRCIN1 expression levels were significantly down-regulated in breast cancer compared to the adjacent normal tissues (Fig. 5B, Yang et al., 2017)". The problem with these statements is that they contradict the actual data presented in the paper! This misrepresentation of the effects of miR-346 in breast cancer could prove harmful by sidetracking future research. Further, clinical trials may be incorrectly directed towards lowering miR-346 without a complete and fair assessment of the internal contradictions in the data. Inaccurately-presented data impede progress of biomedical research, deplete scientific resources and compromise public trust.

Deposition of extracellular plaques, primarily consisting of amyloid β peptide (Aβ), in the brain is the confirmatory diagnostic of Alzheimer's disease (AD); however, the physiological and pathological role of Aβ is not fully understood. Herein, we demonstrate novel Aβ activity as a putative transcription factor upon AD-associated genes. We used oligomers from 5'-flanking regions of the apolipoprotein E (APOE), Aβ-precursor protein (APP) and β-amyloid site cleaving enzyme-1 (BACE1) genes for electrophoretic mobility shift assay (EMSA) with different fragments of the Aβ peptide. Our results suggest that Aβ bound to an Aβ-interacting domain (AβID) with a consensus of "KGGRKTGGGG". This peptide-DNA interaction was sequence specific, and mutation of the first "G" of the decamer's terminal "GGGG" eliminated peptide-DNA interaction. Furthermore, the cytotoxic Aβ25-35 fragment had greatest DNA affinity. Such specificity of binding suggests that the AβID is worth of further investigation as a site wherein the Aβ peptide may act as a transcription factor.

The effects of amyloid-β are extremely complex. Current work in the field of Alzheimer disease is focusing on discerning the impact between the physiological signaling effects of soluble low molecular weight amyloid-β species, and the more global cellular damage that could derive from highly concentrated and/or aggregated amyloid. Being able to dissect the specific signaling events, to understand how soluble amyloid-β induces its own production by upregulating BACE1 expression, could lead to new tools to interrupt the distinctive feedback cycle with potential therapeutic consequences. Here we describe a positive loop that exists between the secretases that are responsible for the generation of the amyloid-β component of Alzheimer disease. According to our hypothesis, in familial Alzheimer disease, the primary overproduction of amyloid-β can induce BACE1 transcription and drive a further increase of amyloid-β precursor protein processing and resultant amyloid-β production. In sporadic Alzheimer disease, many factors, among them oxidative stress and inflammation, with consequent induction of presenilins and BACE1, would activate a loop and proceed with the generation of amyloid-β and its signaling role onto BACE1 transcription. This concept of a signaling effect by and feedback on the amyloid-β precursor protein will likely shed light on how amyloid-β generation, oxidative stress, and secretase functions are intimately related in sporadic Alzheimer disease.

The neurodegenerative disorder Alzheimer's disease (AD) is the 6th leading cause of death in the USA. In addition to neurological and psychiatric symptoms, AD is characterized by deficiencies in S-adenylmethionine (SAM), vitamin B12, and folate. Deficiency in these nutrients has been shown to result in gene promoter methylation with upregulation of AD-associated genes. While some cases of AD are due to specific mutations in genes such as presenilin 1 (PSEN) and the amyloid-beta peptide precursor protein (APP), these familial AD (FAD) cases account for a minority of cases. The majority of genetic contribution consists of risk factors with incomplete penetrance. Several environmental risk factors, such as cholesterol and diet, head trauma, and reduced levels of exercise, have also been determined for AD. Nevertheless, the majority of risk for AD appears to be established early in life. We propose to explain this via the LEARn (Latent Early-life Associated Regulation) model. LEARn-AD (LAD) would be a "two-hit" disorder, wherein the first hit would occur due to environmental stress within the regulatory sequences of AD-associated genes, maintained by epigenetic changes such as in DNA methylation. This hit would most likely come in early childhood. The second hit could consist of further stress, such as head trauma, poor mid-life diet, or even general changes in expression of genes that occur later in life independent of any pathogenesis. Given that the primary risk for LAD would be maintained by DNA (hypo)methylation, we propose that long-term nutritional remediation based on the LEARn model, or LEARn-based nutritional gain (LEARnING), beginning early in life, would significantly reduce risk for AD late in life.

Neurobiological disorders have diverse manifestations and symptomology. Neurodegenerative disorders, such as Alzheimer’s 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. Molecular Psychiatry (2009) 14, 992–1003; doi:10.1038/mp.2009.82

SummaryCyclin-dependent kinase 5 (cdk5) has been implicated in Alzheimer's 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.

Alzheimer's disease is characterized by amyloid-beta peptide (Abeta)-loaded plaques in the brain. Abeta is a cleavage fragment of amyloid-beta protein precursor (APP) and over production of APP may lead to amyloidogenesis. The regulatory region of the APP gene contains consensus sites recognized by the transcription factor, specificity protein 1 (SP1), which has been shown to be required for the regulation of APP and Abeta. To understand the role of SP1 in APP biogenesis, herein we have characterized the relative distribution and localization of SP1, APP, and Abeta in various brain regions of rodent and primate models using immunohistochemistry. We observed that overall distribution and cellular localization of SP1, APP, and Abeta are similar and neuronal in origin. Their distribution is abundant in various layers of neocortex, but restricted to the Purkinje cell layer of the cerebellum, and the pyramidal cell layer of hippocampus. These findings suggest that overproduction of Abeta in vivo may be associated with transcriptional pathways involving SP1 and the APP gene.

Apolipoprotein E (ApoE), encoded by the apolipoprotein E gene (APOE), plays an important role in the pathogenesis of Alzheimer's disease (AD). The APOE epsilon4 variant is strongly associated with AD. APOE promoter polymorphisms have also been reported to associate with higher AD risk. Mouse models of APOE expression have long been used to study the pathogenesis of AD. Elucidating the role of the APOE gene in AD requires understanding of how its regulation differs between mouse and human APOE genes, and how the differences influence AD risk. We compared the structure and function of both the human APOE gene promoter (hAPOEP) and mouse APOE gene promoter (mAPOEP) regions. Homology is less than 40% at 180 bp or more upstream of the two species' transcription start site (TSS, +1). Functional analysis revealed both similarities and important differences between the two sequences, significantly affected by human versus rodent cell line origin. We likewise probed nuclear extracts from several cell lines of different origins (astrocytic, glial, and neuronal) and mouse brain with specific hAPOEP and mAPOEP fragments. Each fragment shared DNA-protein interactions with the other but, notably, also bound distinct factors, demonstrated by gel shift and southwestern analyses. We determined possible identities for these distinct factors. These results suggest that regulation of mouse and human APOE genes may be sufficiently unique to justify the use of both the human APOE promoter sequence in transgenic rodent models and non-rodent AD models for studying factors involved in AD pathogenesis.

Early-emerging, temperamental differences in fear-related traits may be a heritable vulnerability factor for anxiety disorders. Previous research indicates that the serotonin transporter promoter region polymorphism is a candidate gene for such traits.Associations between 5-HTTLPR genotype and indices of fearful child temperament, derived from maternal report and standardized laboratory observations, were examined in a community sample of 95 preschool-aged children.Children with one or more long alleles of the 5-HTTLPR gene were rated as significantly more nervous during standardized laboratory tasks than children who were homozygous for the short alleles. Children homozygous for the short alleles were also rated as significantly shyer, by maternal report, than those with at least one copy of the long allele of the 5-HTTLPR gene.This study extends the literature linking the short alleles of the serotonin transporter promoter region polymorphism to fear and anxiety-related traits in early childhood and adulthood, and is one of very few studies to examine the molecular genetics of preschoolers' temperament using multiple measures of traits in a normative sample.

Alzheimers disease (AD) is currently the most prominent form of dementia among the elderly. Although AD manifests in late adult life, it is not clear when the disease actually starts and how long the neuropathological processes take to develop AD. The major unresolved question is the timing and the nature of triggering leading to AD. Is it an early or developmental and/or late phenomenon and what are the factors that trigger the cascade of pathobiochemical processes? To explain the etiology of AD one should consider the neuropathological features, such as neuronal cell death,θ tangles, and amyloid plaque, and environmental factors associated with AD, such as diet, toxicological exposure, and hormonal factors. Current dominant theories of AD etiology are “protein-only”, they attribute the cause of the disease directly to the activities of associated proteins once they have been produced; the major limitation is that protein aggregations occur “late in the game”. Development and progression of AD has not been explained by protein-only models. In view of this limitation, we propose a “Latent Early-Life Associated Regulation” (LEARn) model, which postulates a latent expression of specific genes triggered at the developmental stage. According to this model, environmental agents (e.g., heavy metals), intrinsic factors (e.g., cytokines), and dietary factors (e.g., cholesterol) perturb gene regulation in a long-term fashion, beginning at early developmental stages; however, these perturbations do not have pathological results until significantly later in life. For example, such actions would perturb APP gene regulation at very early stage via its transcriptional machinery, leading to delayed overexpression of APP and subsequently of Aβ deposition. This model operates on the regulatory region (promoter) of the gene and by the effect of methylation at certain sites within the promoter of specific genes. Promoters tend to have both positive and negative regulatory elements, and promoter activity can be altered by changes in the primary DNA sequence and by epigenetic changes through mechanisms such as DNA methylation at CpG dinucleotides or oxidation of guanosine residues. The basis of the LEARn model is that environmental factors, including metals and dietary factors, operate by interfering the interaction of methylated CpG clusters with binding proteins, such as MeCP2 and SP1. The LEARn model may explain the etiology of AD and other neuropsychiatric and developmental disorders.

Alzheimer's disease and other idiopathic dementias are associated with epigenetic transformations. These transformations connect the environment and genes to pathogenesis, and have led to the investigation of epigenetic-based therapeutic targes for the treatment of these diseases. Epigenetic changes occur over time in response to environmental effects. The epigenome-based latent early-life associated regulation (LEARn) hypothetical model indicates that accumulated environmental hits produce latent epigenetic changes. These hits can alter biochemical pathways until a pathological threshold is reached, which appears clinically as the onset of dementia. The hypotheses posed by LEARn are testable via longitudinal epigenome-wide, envirome-wide, and exposome-wide association studies (LEWAS) of the genome, epigenome, and environment. We posit that the LEWAS design could lead to effective prevention and treatments by identifying potential therapeutic strategies. Epigenetic evidence suggests that dementia is not a suddenly occurring and sharply delineated state, but rather a gradual change in crucial cellular pathways, that transforms an otherwise healthy state, as a result of neurodegeneration, to a dysfunctional state. Evidence from epigenetics could lead to ways to detect, prevent, and reverse such processes before clinical dementia.

Major hallmarks of Alzheimer's disease (AD) include brain deposition of the amyloid-beta peptide (Abeta), which is proteolytically cleaved from a large Abeta precursor protein (APP) by beta and gamma- secretases. A transmembrane aspartyl protease, beta-APP cleaving enzyme (BACE1), has been recognized as the beta-secretase. We review the structure and function of the BACE1 protein, and of 4129 bp of the 5'-flanking region sequence of the BACE1 gene and its interaction with various transcription factors involved in cell signaling. The promoter region and 5'-untranslated region (UTR) contain multiple transcription factor binding sites, such as AP-1, CREB and MEF2. A 91 bp fragment is the shortest region with significant reporter gene activity and constitutes the minimal promoter element for BACE1. The BACE1 promoter contains six unique functional domains and three structural domains of increasing sequence complexity as the "ATG" start codon is approached. Notably, the BACE1 gene promoter contains basal regulatory elements, inducible features and sites for regulation by various important transcription factors. Herein, we also discuss and speculate how the interaction of these transcription factors with the BACE1 promoter can modulate synaptic plasticity, neuronal apoptosis and oxidative stress, which are pertinent to the pathogenesis and progression of AD.

Major characteristics of Alzheimer's disease (AD) are synaptic loss, cholinergic dysfunction, and abnormal protein depositions in the brain. The amyloid beta-peptide (Abeta), a proteolytic fragment of amyloid beta 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 Abeta-lowering drugs. A selective acetylcholinesterase inhibitor, phenserine, in current human trials lowers both APP and Abeta. 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 Abeta 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. Abeta40 and Abeta42 levels were significantly lowered by posiphen (> or =15 mg/kg) compared with controls. The activities of alpha-, beta-, and gamma-secretases were assessed in the same brain samples, and beta-secretase activity was significantly reduced. Posiphen, like phenserine, can lower Abeta via multiple mechanisms and represents an interesting drug candidate for AD treatment.

Expression of vaccine antigens in plants and delivery via ingestion of transgenic plant material has shown promise in numerous pre-clinical animal studies and in a few clinical trials. A number of different viral antigens have been tested, and among the most promising are those that can assemble virus-like particles (VLP), which mimic the form of authentic virions and display neutralizing antibody epitopes. We have extensively studied plant expression, VLP assembly, and immunogenicity of hepatitis B surface antigen (HBsAg) and Norwalk virus capsid protein (NVCP). The HBsAg small protein (S protein) was found by TEM to assemble tubular membrane complexes derived from endoplasmic reticulum in suspension cultured cells of tobacco and soybean, and in potato leaf and tuber tissues. The potato material was immunogenic in mice upon delivery by ingestion. Here we describe the plant expression and immunogenicity of HBsAg middle protein (M protein or pre-S2 + S) which contains additional 55 amino acid pre-S2 region at N-terminus of the S protein. Plant-derived recombinant M protein provoked stronger serum antibody responses against HBsAg than did S protein when injected systemically in mice. We discuss implications for use of fusion proteins for enhanced immunogenicity and mucosal targeting of HBsAg, as well as delivery of heterologous fused antigens. NVCP expressed in plants assembled 38 nm virion-size icosahedral (T = 3) VLP, similar to those produced in insect cells. The VLP stimulated serum IgG and IgA responses in mice and humans when they were delivered by ingestion of fresh potato tuber. Here we show that freeze-drying of transgenic NVCP tomato fruit yielded stable preparations that stimulated excellent IgG and IgA responses against NVCP when fed to mice. However, the predominant VLP form in tomato fruit was the small 23 nm particle also observed in insect cell-derived NVCP.