Altered sphingolipid function in Alzheimer's disease;:a gene regulatory network approach

Sphingolipids (SLs) are bioactive lipids involved in various important physiological functions. The SL pathway has been shown to be affected in several brain-related disorders, including Alzheimer's disease (AD). Recent evidence suggests that epigenetic dysregulation plays an important role in the pathogenesis of AD as well. Here, we use an integrative approach to better understand the relationship between epigenetic and transcriptomic processes in regulating SL function in the middle temporal gyrus of AD patients. Transcriptomic analysis of 252 SL-related genes, selected based on GO term annotations, from 46 AD patients and 32 healthy age-matched controls, revealed 103 differentially expressed SL-related genes in AD patients. Additionally, methylomic analysis of the same subjects revealed parallel hydroxymethylation changes in PTGIS, GBA, and ITGB2 in AD. Subsequent gene regulatory network-based analysis identified 3 candidate genes, that is, SELPLG, SPHK1 and CAV1 whose alteration holds the potential to revert the gene expression program from a diseased towards a healthy state. Together, this epigenomic and transcriptomic approach highlights the importance of SL-related genes in AD, and may provide novel biomarkers and therapeutic alternatives to traditionally investigated biological pathways in AD.</p

Novel method to ascertain chromatin accessibility at specific genomic loci from frozen brain homogenates and laser capture microdissected defined cells

We describe a novel method for assessing the “open” or “closed” state of chromatin at selected locations within the genome. This method combines the use of Benzonase, which can digest DNA in the presence of actin, with quantitative polymerase chain reaction to define digested regions. We demonstrate the application of this method in brain homogenates and laser captured cells. We also demonstrate application to selected sites within more than 1 gene and multiple sites within 1 gene. We demonstrate the validity of the method by treating cells with valproate, known to render chromatin more permissive, and by comparison with classical digestion with DNase I in an in vitro preparation. Although we demonstrate the use of this method in brain tissue, we also recognize its applicability to other tissue types

Blood Transcript Biomarkers Selected by Machine Learning Algorithm Classify Neurodegenerative Diseases including Alzheimer’s Disease

The clinical diagnosis of neurodegenerative diseases is notoriously inaccurate and current methods are often expensive, time-consuming, or invasive. Simple inexpensive and noninvasive methods of diagnosis could provide valuable support for clinicians when combined with cognitive assessment scores. Biological processes leading to neuropathology progress silently for years and are reflected in both the central nervous system and vascular peripheral system. A blood-based screen to distinguish and classify neurodegenerative diseases is especially interesting having low cost, minimal invasiveness, and accessibility to almost any world clinic. In this study, we set out to discover a small set of blood transcripts that can be used to distinguish healthy individuals from those with Alzheimer’s disease, Parkinson’s disease, Huntington’s disease, amyotrophic lateral sclerosis, Friedreich’s ataxia, or frontotemporal dementia. Using existing public datasets, we developed a machine learning algorithm for application on transcripts present in blood and discovered small sets of transcripts that distinguish a number of neurodegenerative diseases with high sensitivity and specificity. We validated the usefulness of blood RNA transcriptomics for the classification of neurodegenerative diseases. Information about features selected for the classification can direct the development of possible treatment strategies

Differential processing of amyloid precursor protein in brain and in peripheral blood leukocytes

Because amyloid precursor protein (APP) fragments exist in many tissues throughout the body, including the fluid compartments of blood, they have been the focus of numerous investigations into their potential as a biomarker of Alzheimer\u27s disease. Using immunohistochemistry, immunoelectron microscopy, Western blot, and quantitative real-time-polymerase chain reaction (qRT-PCR) analysis we examined whether APP processing in leukocytes is analogous to APP processing in the brain. We show APP immunoreactivity at light and electron microscopic levels in the cytoplasm and nucleus of peripheral blood leukocytes (PBL) yet our Western blot analysis data demonstrated that brain and PBL contain different APP fragments and differentially expressed APP processing enzymes. A Disintegrin and Metalloproteinase domain 10 (ADAM10), nicastrin, and beta-secretase 2 (BACE2) were present in brain but were undetected in PBL. Presenilin 1 and beta-secretase 1 (BACE1) were detected in both tissues but showed different patterns in Western blots. Quantitative PCR results identified Neprilysin as the only processing enzyme we interrogated in which Western and quantitative PCR data coincided. Although our data on differential processing of APP in brain and PBL point to exercising caution when generalizing between blood and brain with regard to mechanisms, they have no implications regarding utility as biomarkers. © 2013 Elsevier Inc

Aberrant intracellular localization of H3k4me3 demonstrates an early epigenetic phenomenon in Alzheimer's disease

We have previously reported in Alzheimer’s disease (AD) the mislocalization of epigenetic molecules between the cell nucleus and the cytoplasm. We have extended our finding to include the aberrant localization of histone 3 trimethylation on lysine 4 (H3k4me3), an epigenetic mark associated with actively transcribing genes as well as those poised for transcription. These findings raise the question of where the ectopic localization of H3k4me3 fits within the cascade of cell biological events in the progression of AD. We, therefore, examined the expression and intracellular location of H3k4me3 as a function of Braak stage and also in relation to a series of tau markers that are indicative of disease state. Both lines of evidence showed that ectopic localization of H3k4me3 is early in the course of disease. Because of the known role of H3k4me3 in the expression of synaptic genes, our data suggest an epigenetic role in synaptic deficits early in the course of AD

Correction: ANK1 is up-regulated in laser captured microglia in Alzheimer's brain; the importance of addressing cellular heterogeneity.

[This corrects the article DOI: 10.1371/journal.pone.0177814.]

Nuclear but not mitochondrial‐encoded oxidative phosphorylation genes are altered in aging, mild cognitive impairment, and Alzheimer's disease

IntroductionWe have comprehensively described the expression profiles of mitochondrial DNA and nuclear DNA genes that encode subunits of the respiratory oxidative phosphorylation (OXPHOS) complexes (I-V) in the hippocampus from young controls, age matched, mild cognitively impaired (MCI), and Alzheimer's disease (AD) subjects.MethodsHippocampal tissues from 44 non-AD controls (NC), 10 amnestic MCI, and 18 AD cases were analyzed on Affymetrix Hg-U133 plus 2.0 arrays.ResultsThe microarray data revealed significant down regulation in OXPHOS genes in AD, particularly those encoded in the nucleus. In contrast, there was up regulation of the same gene(s) in MCI subjects compared to AD and ND cases. No significant differences were observed in mtDNA genes identified in the array between AD, ND, and MCI subjects except one mt-ND6.DiscussionOur findings suggest that restoration of the expression of nuclear-encoded OXPHOS genes in aging could be a viable strategy for blunting AD progression

Multivariate analyses of peripheral blood leukocyte transcripts distinguish Alzheimer's, Parkinson's, control, and those at risk for developing Alzheimer's

The need for a reliable, simple, and inexpensive blood test for Alzheimer\u27s disease (AD) suitable for use in a primary care setting is widely recognized. This has led to a large number of publications describing blood tests for AD, which have, for the most part, not been replicable. We have chosen to examine transcripts expressed by the cellular, leukocyte compartment of blood. We have used hypothesis-based cDNA arrays and quantitative PCR to quantify the expression of selected sets of genes followed by multivariate analyses in multiple independent samples. Rather than a single study with no replicates, we chose an experimental design in which there were multiple replicates using different platforms and different sample populations. We have divided 177 blood samples and 27 brain samples into multiple replicates to demonstrate the ability to distinguish early clinical AD (Clinical Dementia Rating scale 0.5), Parkinson\u27s disease (PD), and cognitively unimpaired APOE4 homozygotes, as well as to determine persons at risk for future cognitive impairment with significant accuracy. We assess our methods in a training/test set and also show that the variables we use distinguish AD, PD, and control brain. Importantly, we describe the variability of the weights assigned to individual transcripts in multivariate analyses in repeated studies and suggest that the variability we describe may be the cause of inability to repeat many earlier studies. Our data constitute a proof of principle that multivariate analysis of the transcriptome related to cell stress and inflammation of peripheral blood leukocytes has significant potential as a minimally invasive and inexpensive diagnostic tool for diagnosis and early detection of risk for AD

<i>ANK1</i> mRNA expression levels in hippocampal homogenates, AD CA1 pyramidal neurons AD CA1 astrocytes and AD CA1 microglia.

<p>Bar graph depicts log2 fold change, comparing AD vs. age matched normal controls. No significant difference was detected in homogenates, AD neurons, or AD astrocytes. In stark contrast, a significant four-fold increase (<i>p<0</i>.<i>001</i>) was observed in AD microglia.</p

Significant, Log2 fold change(s) in genes containing ankyrin repeat domains in AD hippocampal homogenates, AD CA1 pyramidal neurons, AD CA1 astrocytes and AD CA1 microglia.

<p>A) Significantly <i>(p <</i> .<i>05)</i> altered Ankyrin repeat containing genes in hippocampal homogenates in AD compared to ND. B) Significantly altered (p < .05) Ankyrin repeat containing genes in LCM neurons and glial cells (C). D) average log2 mRNA fold difference in ankyrin repeat genes in PD microglia from CA1 of the hippocampus compared to the same matched control subjects used in AD comparisons. LCM neurons, astrocytes and microglia were derived from the same human subjects. Detailed expression changes can be found in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0177814#pone.0177814.s002" target="_blank">S1 Fig</a>.</p