Liquid Biopsy in Alzheimer's Disease Patients Reveals Epigenetic Changes in the PRLHR Gene.

In recent years, new DNA methylation variants have been reported in genes biologically relevant to Alzheimer's disease (AD) in human brain tissue. However, this AD-specific epigenetic information remains brain-locked and unreachable during patients' lifetimes. In a previous methylome performed in the hippocampus of 26 AD patients and 12 controls, we found higher methylation levels in AD patients in the promoter region of PRLHR, a gene involved in energy balance regulation. Our aim was to further characterize PRLHR's role in AD and to evaluate if the liquid biopsy technique would provide life access to this brain information in a non-invasive way. First, we extended the methylation mapping of PRLHR and validated previous methylome results via bisulfite cloning sequencing. Next, we observed a positive correlation between PRLHR methylation levels and AD-related neuropathological changes and a decreased expression of PRLHR in AD hippocampus. Then, we managed to replicate the hippocampal methylation differences in plasma cfDNA from an additional cohort of 35 AD patients and 35 controls. The isolation of cfDNA from the plasma of AD patients may constitute a source of potential epigenetic biomarkers to aid AD clinical management.

Association of Blood-Based DNA Methylation Markers With Late-Onset Alzheimer Disease: A Potential Diagnostic Approach.

BACKGROUND AND OBJECTIVES: There is an urgent need to identify novel noninvasive biomarkers for Alzheimer disease (AD) diagnosis. Recent advances in blood-based measurements of phosphorylated tau (pTau) species are promising but still insufficient to address clinical needs. Epigenetics has been shown to be helpful to better understand AD pathogenesis. Epigenetic biomarkers have been successfully implemented in other medical disciplines, such as oncology. The objective of this study was to explore the diagnostic accuracy of a blood-based DNA methylation marker panel as a noninvasive tool to identify patients with late-onset Alzheimer compared with age-matched controls. METHODS: A case-control study was performed. Blood DNA methylation levels at 46 cytosine-guanine sites (21 genes selected after a comprehensive literature search) were measured by bisulfite pyrosequencing in patients with "probable AD dementia" following National Institute on Aging and the Alzheimer's Association guidelines (2011) and age-matched and sex-matched controls recruited at Neurology Department-University Hospital of Navarre, Spain, selected by convenience sampling. Plasma pTau181 levels were determined by Simoa technology. Multivariable logistic regression analysis was performed to explore the optimal model to discriminate patients with AD from controls. Furthermore, we performed a stratified analysis by sex. RESULTS: The final study cohort consisted of 80 patients with AD (age: median [interquartile range] 79 [11] years; 58.8% female) and 100 cognitively healthy controls (age 77 [10] years; 58% female). A panel including DNA methylation levels at NXN, ABCA7, and HOXA3 genes and plasma pTau181 significantly improved (area under the receiver operating characteristic curve 0.93, 95% CI 0.89-0.97) the diagnostic performance of a single pTau181-based model, adjusted for age, sex, and APOE ɛ4 genotype. The sensitivity and specificity of this panel were 83.30% and 90.00%, respectively. After sex-stratified analysis, HOXA3 DNA methylation levels showed consistent association with AD. DISCUSSION: These results highlight the potential translational value of blood-based DNA methylation biomarkers for noninvasive diagnosis of AD. REGISTRATION INFORMATION: Research Ethics Committee of the University Hospital of Navarre (PI17/02218).

circRNA from APP Gene Changes in Alzheimer's Disease Human Brain.

Alzheimer's disease (AD) is the most common cause of age-related dementia. Amyloid precursor protein (APP) is the precursor of Aß peptides, and its role in AD has been widely investigated. Recently, it has been reported that a circular RNA (circRNA) originated from APP gene can serve as a template for Aß synthesis, postulating it as an alternative pathway for the Aß biogenesis. Moreover, circRNAs play important roles in brain development and in neurological diseases. Therefore, our aim was to study the expression of a circAPP (hsa_circ_0007556) and its linear cognate in AD human entorhinal cortex, a brain region most vulnerable to AD pathology. First, we confirmed the presence of circAPP (hsa_circ_0007556) in human entorhinal cortex samples using RT-PCR and Sanger sequencing of PCR products. Next, a 0.49-fold decrease in circAPP (hsa_circ_0007556) levels was observed in entorhinal cortex of AD cases compared to controls (p-value < 0.05) by qPCR. In contrast, APP mRNA expression did not show changes in the entorhinal cortex between AD cases and controls (Fold-change = 1.06; p-value = 0.81). A negative correlation was found between Aß deposits and circAPP (hsa_circ_0007556) and APP expression levels (Rho Spearman = -0.56, p-value < 0.001 and Rho Spearman = -0.44, p-values < 0.001, respectively). Finally, by using bioinformatics tools, 17 miRNAs were predicted to bind circAPP (hsa_circ_0007556), and the functional analysis predicted that they were involved in some pathways, such as the Wnt-signaling pathway (p = 3.32 × 10-6). Long-term potentiation (p = 2.86 × 10-5), among others, is known to be altered in AD. To sum up, we show that circAPP (hsa_circ_0007556) is deregulated in the entorhinal cortex of AD patients. These results add to the notion that circAPP (hsa_circ_0007556) could be playing a role in the pathogenesis of AD disease.

Epigenetic clock indicates accelerated aging in glial cells of progressive multiple sclerosis patients.

Background: Multiple sclerosis (MS) is a chronic inflammatory neurodegenerative disease of the central nervous system (CNS) characterized by irreversible disability at later progressive stages. A growing body of evidence suggests that disease progression depends on age and inflammation within the CNS. We aimed to investigate epigenetic aging in bulk brain tissue and sorted nuclei from MS patients using DNA methylation-based epigenetic clocks. Methods: We applied Horvath's multi-tissue and Shireby's brain-specific Cortical clock on bulk brain tissue (n = 46), sorted neuronal (n = 54), and glial nuclei (n = 66) from post-mortem brain tissue of progressive MS patients and controls. Results: We found a significant increase in age acceleration residuals, corresponding to 3.6 years, in glial cells of MS patients compared to controls (P = 0.0024) using the Cortical clock, which held after adjustment for covariates (P adj = 0.0263). The 4.8-year age acceleration found in MS neurons (P = 0.0054) did not withstand adjustment for covariates and no significant difference in age acceleration residuals was observed in bulk brain tissue between MS patients and controls. Conclusion: While the findings warrant replication in larger cohorts, our study suggests that glial cells of progressive MS patients exhibit accelerated biological aging.

Profile of TREM2-Derived circRNA and mRNA Variants in the Entorhinal Cortex of Alzheimer's Disease Patients.

Genetic variants in TREM2, a microglia-related gene, are well-known risk factors for Alzheimer's disease (AD). Here, we report that TREM2 originates from circular RNAs (circRNAs), a novel class of non-coding RNAs characterized by a covalent and stable closed-loop structure. First, divergent primers were designed to amplify circRNAs by RT-PCR, which were further assessed by Sanger sequencing. Then, additional primer sets were used to confirm back-splicing junctions. In addition, HMC3 cells were used to assess the microglial expression of circTREM2s. Three candidate circTREM2s were identified in control and AD human entorhinal samples. One of the circRNAs, circTREM2_1, was consistently amplified by all divergent primer sets in control and AD entorhinal cortex samples as well as in HMC3 cells. In AD cases, a moderate negative correlation (r = -0.434) was found between the global average area of Aß deposits in the entorhinal cortex and circTREM2_1 expression level. In addition, by bioinformatics tools, a total of 16 miRNAs were predicted to join with circTREM2s. Finally, TREM2 mRNA corresponding to four isoforms was profiled by RT-qPCR. TREM2 mRNA levels were found elevated in entorhinal samples of AD patients with low or intermediate ABC scores compared to controls. To sum up, a novel circRNA derived from the TREM2 gene, circTREM2_1, has been identified in the human entorhinal cortex and TREM2 mRNA expression has been detected to increase in AD compared to controls. Unraveling the molecular genetics of the TREM2 gene may help to better know the innate immune response in AD.

NXN Gene Epigenetic Changes in an Adult Neurogenesis Model of Alzheimer's Disease.

In view of the proven link between adult hippocampal neurogenesis (AHN) and learning and memory impairment, we generated a straightforward adult neurogenesis in vitro model to recapitulate DNA methylation marks in the context of Alzheimer's disease (AD). Neural progenitor cells (NPCs) were differentiated for 29 days and Aß peptide 1-42 was added. mRNA expression of Neuronal Differentiation 1 (NEUROD1), Neural Cell Adhesion Molecule 1 (NCAM1), Tubulin Beta 3 Class III (TUBB3), RNA Binding Fox-1 Homolog 3 (RBFOX3), Calbindin 1 (CALB1), and Glial Fibrillary Acidic Protein (GFAP) was determined by RT-qPCR to characterize the culture and framed within the multistep process of AHN. Hippocampal DNA methylation marks previously identified in Contactin-Associated Protein 1 (CNTNAP1), SEPT5-GP1BB Readthrough (SEPT5-GP1BB), T-Box Transcription Factor 5 (TBX5), and Nucleoredoxin (NXN) genes were profiled by bisulfite pyrosequencing or bisulfite cloning sequencing; mRNA expression was also measured. NXN outlined a peak of DNA methylation overlapping type 3 neuroblasts. Aß-treated NPCs showed transient decreases of mRNA expression for SEPT5-GP1BB and NXN on day 9 or 19 and an increase in DNA methylation on day 29 for NXN. NXN and SEPT5-GP1BB may reflect alterations detected in the brain of AD human patients, broadening our understanding of this disease.

Gender-Dependent Deregulation of Linear and Circular RNA Variants of HOMER1 in the Entorhinal Cortex of Alzheimer's Disease.

The HOMER1 gene is involved in synaptic plasticity, learning and memory. Recent studies show that circular RNA derived from HOMER1 (circHOMER1) expression is altered in some Alzheimer's disease (AD) brain regions. In addition, HOMER1 messenger (mRNA) levels have been associated with ß-Amyloid (Aß) deposits in brain cortical regions. Our aim was to measure the expression levels of HOMER1 circRNAs and their linear forms in the human AD entorhinal cortex. First, we showed downregulation of HOMER1B/C and HOMER1A mRNA and hsa_circ_0006916 and hsa_circ_0073127 levels in AD female cases compared to controls by RT-qPCR. A positive correlation was observed between HOMER1B/C, HOMER1A mRNA, and hsa_circ_0073128 with HOMER1B/C protein only in females. Global average area of Aß deposits in entorhinal cortex samples was negatively correlated with HOMER1B/C, HOMER1A mRNA, and hsa_circ_0073127 in both genders. Furthermore, no differences in DNA methylation were found in two regions of HOMER1 promoter between AD cases and controls. To sum up, we demonstrate that linear and circular RNA variants of HOMER1 are downregulated in the entorhinal cortex of female patients with AD. These results add to the notion that HOMER1 and its circular forms could be playing a female-specific role in the pathogenesis of AD.

Early epigenetic changes of Alzheimer's disease in the human hippocampus.

The discovery of new biomarkers would be very valuable to improve the detection of early Alzheimer's disease (AD). DNA methylation marks may serve as epigenetic biomarkers of early AD. Here we identified epigenetic marks that are present in the human hippocampus from the earliest stages of AD. A previous methylome dataset of the human AD hippocampus was used to select a set of eight differentially methylated positions (DMPs) since early AD stages. Next, bisulphite pyrosequencing was performed in an expanded homogeneous cohort of 18 pure controls and 35 hippocampal samples with neuropathological changes of pure AD. Correlation between DNA methylation levels in DMPs and phospho-tau protein burden assessed by immunohistochemistry in the hippocampus was also determined. We found four DMPs showing higher levels of DNA methylation at early AD stages compared to controls, involving ELOVL2, GIT1/TP53I13 and the histone gene locus at chromosome 6. DNA methylation levels assessed by bisulphite pyrosequencing correlated with phospho-tau protein burden for ELOVL2 and HIST1H3E/HIST1H3 F genes. In this discovery study, a set of four epigenetic marks of early AD stages have been identified in the human hippocampus. It would be worth studying in-depth the specific pathways related to these epigenetic marks. These early alterations in DNA methylation in the AD hippocampus could be regarded as candidate biomarkers to be explored in future translational studies. ABBREVIATIONS: AD: Alzheimer's disease; DMPs: Differentially methylated positions; CSF: Cerebrospinal fluid; ßA42: ß-amyloid 42; PET: positron emission tomography; 5mC: 5-methyl cytosine; CpG: cytosine-guanine dinucleotides; ANK1: ankyrin-1; BIN1: amphiphysin II; p-tau: hyperphosphorylated tau; CERAD: Consortium to Establish A Registry for Alzheimer's Disease; SD: standard deviation; ANOVA: one-way analysis of variance; VLCFAs: very long-chain fatty acids; DHA: docosahexaenoic acid; mTOR: mechanistic target of rapamycin.

Circular RNA expression profile in blood according to ischemic stroke etiology.

BACKGROUND: The discovery of novel biomarkers of stroke etiology would be most helpful in management of acute ischemic stroke patients. Recently, circular RNAs (circRNAs) have been proposed as candidate biomarkers of neurological conditions due to its high stability. circRNAs function as sponges, sequestering miRNAs and are involved in most relevant biological functions. Our aim was to identify differentially expressed circRNAs in acute ischemic stroke patients according to stroke etiology. METHODS: A comprehensive expression profile of blood circRNAs was conducted by Arraystar Human circRNA arrays (13,617 probes) on a discovery cohort of 30 stroke patients with different stroke etiologies by TOAST classification. Real-time quantitative PCR (RT-qPCR) was used to validate array results in a cohort of 50 stroke patients. Functional in silico analysis was performed to identify potential interactions with microRNAs (miRNAs) and pathways underlying deregulated circRNAs. RESULTS: A set of 60 circRNAs were found to be upregulated in atherotrombotic versus cardioembolic strokes (fold-change > = 1.5 and p-value ≤ 0.05). Differential expression of hsa_circRNA_102488, originated from UBA52 gene, was replicated in the validation cohort. RNA-binding proteins (RBPs) sites of hsa_circRNA_102488 clustered around AGO2 and FUS proteins. Further functional analysis revealed interactions between deregulated circRNAs and a set of miRNAs involved in stroke-related pathways, such as fatty acid biogenesis or lysine degradation. CONCLUSION: Different stroke subtypes show specific profiles of circRNAs expression. circRNAs may serve as a new source of biomarkers of stroke etiology in acute ischemic stroke patients.

Telomere length correlates with subtelomeric DNA methylation in long-term mindfulness practitioners.

Mindfulness and meditation techniques have proven successful for the reduction of stress and improvement in general health. In addition, meditation is linked to longevity and longer telomere length, a proposed biomarker of human aging. Interestingly, DNA methylation changes have been described at specific subtelomeric regions in long-term meditators compared to controls. However, the molecular basis underlying these beneficial effects of meditation on human health still remains unclear. Here we show that DNA methylation levels, measured by the Infinium HumanMethylation450 BeadChip (Illumina) array, at specific subtelomeric regions containing GPR31 and SERPINB9 genes were associated with telomere length in long-term meditators with a strong statistical trend when correcting for multiple testing. Notably, age showed no association with telomere length in the group of long-term meditators. These results may suggest that long-term meditation could be related to epigenetic mechanisms, in particular gene-specific DNA methylation changes at distinct subtelomeric regions.

Microglia-Related Gene Triggering Receptor Expressed in Myeloid Cells 2 (TREM2) Is Upregulated in the Substantia Nigra of Progressive Supranuclear Palsy.

BACKGROUND: The role of the microglia-related gene triggering receptor expressed in myeloid cells 2 (TREM2) in primary tauopathies, such as progressive supranuclear palsy (PSP), still remains unclear. OBJECTIVES: The objective of this study was to profile overall and transcript-specific TREM2 expression levels in the substantia nigra (SN) of PSP patients and controls. METHODS: SN samples from neuropathologically confirmed PSP cases (n = 24) and controls (n = 14) were used to measure TREM2 and TREM2-modulating gene Membrane-spanning 4-domains subfamily A member 4A (MS4A4A) mRNA levels by real-time quantitative polymerase chain reaction. Correlation with hyperphosphorylated tau protein burden was assessed. RESULTS: Overall TREM2 and each of the 3 TREM2 transcripts mRNA levels were significantly increased in the SN of PSP cases versus controls. TREM2 mRNA levels positively correlated with hyperphosphorylated tau burden in SN, specifically in neurons. The MS4A4A gene was also upregulated in PSP patients versus controls. CONCLUSIONS: These results add evidence to the involvement of microglia in the disease process of PSP. These findings support the idea that different tauopathies may share common patterns of deregulation in innate immune molecular pathways. © 2020 International Parkinson and Movement Disorder Society.

DNA methylation signature of human hippocampus in Alzheimer's disease is linked to neurogenesis.

BACKGROUND: Drawing the epigenome landscape of Alzheimer's disease (AD) still remains a challenge. To characterize the epigenetic molecular basis of the human hippocampus in AD, we profiled genome-wide DNA methylation levels in hippocampal samples from a cohort of pure AD patients and controls by using the Illumina 450K methylation arrays. RESULTS: Up to 118 AD-related differentially methylated positions (DMPs) were identified in the AD hippocampus, and extended mapping of specific regions was obtained by bisulfite cloning sequencing. AD-related DMPs were significantly correlated with phosphorylated tau burden. Functional analysis highlighted that AD-related DMPs were enriched in poised promoters that were not generally maintained in committed neural progenitor cells, as shown by ChiP-qPCR experiments. Interestingly, AD-related DMPs preferentially involved neurodevelopmental and neurogenesis-related genes. Finally, InterPro ontology analysis revealed enrichment in homeobox-containing transcription factors in the set of AD-related DMPs. CONCLUSIONS: These results suggest that altered DNA methylation in the AD hippocampus occurs at specific regulatory regions crucial for neural differentiation supporting the notion that adult hippocampal neurogenesis may play a role in AD through epigenetic mechanisms.

Hippocampal LMNA Gene Expression is Increased in Late-Stage Alzheimer's Disease.

Lamins are fibrillary proteins that are crucial in maintaining nuclear shape and function. Recently, B-type lamin dysfunction has been linked to tauopathies. However, the role of A-type lamin in neurodegeneration is still obscure. Here, we examined A-type and B-type lamin expression levels by RT-qPCR in Alzheimer's disease (AD) patients and controls in the hippocampus, the core of tau pathology in the brain. LMNA, LMNB1, and LMNB2 genes showed moderate mRNA levels in the human hippocampus with highest expression for the LMNA gene. Moreover, LMNA mRNA levels were increased at the late stage of AD (1.8-fold increase; p-value < 0.05). In addition, a moderate positive correlation was found between age and LMNA mRNA levels (Pearson's r = 0.581, p-value = 0.018) within the control hippocampal samples that was not present in the hippocampal samples affected by AD. A-type and B-type lamin genes are expressed in the human hippocampus at the transcript level. LMNA mRNA levels are up-regulated in the hippocampal tissue in late stages of AD. The effect of age on increasing LMNA expression levels in control samples seems to be disrupted by the development of AD pathology.

PLD3 epigenetic changes in the hippocampus of Alzheimer's disease.

BACKGROUND: Whole-exome sequencing has revealed a rare missense variant in PLD3 gene (rs145999145) to be associated with late onset Alzheimer's disease (AD). Nevertheless, the association remains controversial and little is known about the role of PLD3 in AD. Interestingly, PLD3 encodes a phospholipase that may be involved in amyloid precursor protein (APP) processing. Our aim was to gain insight into the epigenetic mechanisms regulating PLD3 gene expression in the human hippocampus affected by AD. RESULTS: We assessed PLD3 mRNA expression by qPCR and protein levels by Western blot in frozen hippocampal samples from a cohort of neuropathologically confirmed pure AD cases and controls. Next, we profiled DNA methylation at cytosine-phosphate-guanine dinucleotide (CpG) site resolution by pyrosequencing and further validated results by bisulfite cloning sequencing in two promoter regions of the PLD3 gene. A 1.67-fold decrease in PLD3 mRNA levels (p value < 0.001) was observed in the hippocampus of AD cases compared to controls, and a slight decrease was also found by Western blot at protein level. Moreover, PLD3 mRNA levels inversely correlated with the average area of ß-amyloid burden (tau-b = - 0,331; p value < 0.01) in the hippocampus. A differentially methylated region was identified within the alternative promoter of PLD3 gene showing higher DNA methylation levels in the AD hippocampus compared to controls (21.7 ± 4.7% vs. 18.3 ± 4.8%; p value < 0.05). CONCLUSIONS: PLD3 gene is downregulated in the human hippocampus in AD cases compared to controls. Altered epigenetic mechanisms, such as differential DNA methylation within an alternative promoter of PLD3 gene, may be involved in the pathological processes of AD. Moreover, PLD3 mRNA expression inversely correlates with hippocampal ß-amyloid burden, which adds evidence to the hypothesis that PLD3 protein may contribute to AD development by modifying APP processing.