Intracellular Amyloid-ß Detection from Human Brain Sections Using a Microfluidic Immunoassay in Tandem with MALDI-MS.

Alzheimer's disease (AD) currently affects more than 30 million people worldwide. The lack of understanding of AD's physiopathology limits the development of therapeutic and diagnostic tools. Soluble amyloid-ß peptide (Aß) oligomers that appear as intermediates along the Aß aggregation into plaques are considered among the main AD neurotoxic species. Although a wealth of data are available about Aß from in vitro and animal models, there is little known about intracellular Aß in human brain cells, mainly due to the lack of technology to assess the intracellular protein content. The elucidation of the Aß species in specific brain cell subpopulations can provide insight into the role of Aß in AD and the neurotoxic mechanism involved. Here, we report a microfluidic immunoassay for in situ mass spectrometry analysis of intracellular Aß species from archived human brain tissue. This approach comprises the selective laser dissection of individual pyramidal cell bodies from tissues, their transfer to the microfluidic platform for sample processing on-chip, and mass spectrometric characterization. As a proof-of-principle, we demonstrate the detection of intracellular Aß species from as few as 20 human brain cells.

Integrated DNA Methylation/RNA Profiling in Middle Temporal Gyrus of Alzheimer's Disease.

Alzheimer's disease is a neurodegenerative disorder clinically defined by gradual cognitive impairment and alteration in executive function. We conducted an epigenome-wide association study (EWAS) of a clinically and neuropathologically characterized cohort of 296 brains, including Alzheimer's disease (AD) and non-demented controls (ND), exploring the relationship with the RNA expression from matched donors. We detected 5246 CpGs and 832 regions differentially methylated, finding overlap with previous EWAS but also new associations. CpGs previously identified in ANK1, MYOC, and RHBDF2 were differentially methylated, and one of our top hits (GPR56) was not previously detected. ANK1 was differentially methylated at the region level, along with APOE and RHBDF2. Only a small number of genes showed a correlation between DNA methylation and RNA expression statistically significant. Multiblock partial least-squares discriminant analysis showed several CpG sites and RNAs discriminating AD and ND (AUC = 0.908) and strongly correlated with each other. Furthermore, the CpG site cg25038311 was negatively correlated with the expression of 22 genes. Finally, with the functional epigenetic module analysis, we identified a protein-protein network characterized by inverse RNA/DNA methylation correlation and enriched for "Regulation of insulin-like growth factor transport", with IGF1 as the hub gene. Our results confirm and extend the previous EWAS, providing new information about a brain region not previously explored in AD DNA methylation studies. The relationship between DNA methylation and gene expression is not significant for most of the genes in our sample, consistently with the complexities in the gene expression regulation.

Blood RNA transcripts reveal similar and differential alterations in fundamental cellular processes in Alzheimer's disease and other neurodegenerative diseases.

BACKGROUND: Dysfunctional processes in Alzheimer's disease and other neurodegenerative diseases lead to neural degeneration in the central and peripheral nervous system. Research demonstrates that neurodegeneration of any kind is a systemic disease that may even begin outside of the region vulnerable to the disease. Neurodegenerative diseases are defined by the vulnerabilities and pathology occurring in the regions affected. METHOD: A random forest machine learning analysis on whole blood transcriptomes from six neurodegenerative diseases generated unbiased disease-classifying RNA transcripts subsequently subjected to pathway analysis. RESULTS: We report that transcripts of the blood transcriptome selected for each of the neurodegenerative diseases represent fundamental biological cell processes including transcription regulation, degranulation, immune response, protein synthesis, apoptosis, cytoskeletal components, ubiquitylation/proteasome, and mitochondrial complexes that are also affected in the brain and reveal common themes across six neurodegenerative diseases. CONCLUSION: Neurodegenerative diseases share common dysfunctions in fundamental cellular processes. Identifying regional vulnerabilities will reveal unique disease mechanisms. HIGHLIGHTS: Transcriptomics offer information about dysfunctional processes. Comparing multiple diseases will expose unique malfunctions within diseases. Blood RNA can be used ante mortem to track expression changes in neurodegenerative diseases. Protocol standardization will make public datasets compatible.

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.

MIMAS: microfluidic platform in tandem with MALDI mass spectrometry for protein quantification from small cell ensembles.

Understanding cell-to-cell variation at the molecular level provides relevant information about biological phenomena and is critical for clinical and biological research. Proteins carry important information not available from single-cell genomics and transcriptomics studies; however, due to the minute amount of proteins in single cells and the complexity of the proteome, quantitative protein analysis at the single-cell level remains challenging. Here, we report an integrated microfluidic platform in tandem with matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF-MS) for the detection and quantification of targeted proteins from small cell ensembles (> 10 cells). All necessary steps for the assay are integrated on-chip including cell lysis, protein immunocapture, tryptic digestion, and co-crystallization with the matrix solution for MALDI-MS analysis. We demonstrate that our approach is suitable for protein quantification by assessing the apoptotic protein Bcl-2 released from MCF-7 breast cancer cells, ranging from 26 to 223 cells lysed on-chip (8.75 nL wells). A limit of detection (LOD) of 11.22 nM was determined, equivalent to 5.91 × 107 protein molecules per well. Additionally, the microfluidic platform design was further improved, establishing the successful quantification of Bcl-2 protein from MCF-7 cell ensembles ranging from 8 to 19 cells in 4 nL wells. The LOD in the smaller well designs for Bcl-2 resulted in 14.85 nM, equivalent to 3.57 × 107 protein molecules per well. This work shows the capability of our approach to quantitatively assess proteins from cell lysate on the MIMAS platform for the first time. These results demonstrate our approach constitutes a promising tool for quantitative targeted protein analysis from small cell ensembles down to single cells, with the capability for multiplexing through parallelization and automation.

A meta-analysis of epigenome-wide association studies in Alzheimer's disease highlights novel differentially methylated loci across cortex.

Epigenome-wide association studies of Alzheimer's disease have highlighted neuropathology-associated DNA methylation differences, although existing studies have been limited in sample size and utilized different brain regions. Here, we combine data from six DNA methylomic studies of Alzheimer's disease (N = 1453 unique individuals) to identify differential methylation associated with Braak stage in different brain regions and across cortex. We identify 236 CpGs in the prefrontal cortex, 95 CpGs in the temporal gyrus and ten CpGs in the entorhinal cortex at Bonferroni significance, with none in the cerebellum. Our cross-cortex meta-analysis (N = 1408 donors) identifies 220 CpGs associated with neuropathology, annotated to 121 genes, of which 84 genes have not been previously reported at this significance threshold. We have replicated our findings using two further DNA methylomic datasets consisting of a further >600 unique donors. The meta-analysis summary statistics are available in our online data resource ( www.epigenomicslab.com/ad-meta-analysis/ ).

Cell death and survival pathways in Alzheimer's disease: an integrative hypothesis testing approach utilizing -omic data sets.

Whether a cell lives or dies is controlled by an array of intercepting and dynamic molecular pathways. Although there is evidence of neuronal loss in Alzheimer's disease (AD) and multiple programmed cell death (PCD) pathways have been implicated in this process, there has been no comprehensive evaluation of the dominant pathway responsible for cell death in AD. Likewise, the relative dominance of survival and PCD pathways in AD remains unclear. Here, we present the results of hypothesis-driven bioinformatic analysis of PCD and survival pathway activation in paired methylation and expression data from the middle temporal gyrus (MTG) as well as expression from laser-captured cells from the MTG and hippocampus. The results not only indicate activation of cell death pathways in AD-of which apoptosis is responsible for the largest fraction of upregulated genes-but also of cell survival pathways. These results are indicative of a complex balance between survival and death pathways in AD that future studies should work to delineate at a single cell level.

Highly Sensitive and Multiplexed In-Situ Protein Profiling with Cleavable Fluorescent Streptavidin.

The ability to perform highly sensitive and multiplexed in-situ protein analysis is crucial to advance our understanding of normal physiology and disease pathogenesis. To achieve this goal, we here develop an approach using cleavable biotin-conjugated antibodies and cleavable fluorescent streptavidin (CFS). In this approach, protein targets are first recognized by the cleavable biotin-labeled antibodies. Subsequently, CFS is applied to stain the protein targets. Though layer-by-layer signal amplification using cleavable biotin-conjugated orthogonal antibodies and CSF, the protein detection sensitivity can be enhanced at least 10-fold, compared with the current in-situ proteomics methods. After imaging, the fluorophore and the biotin unbound to streptavidin are removed by chemical cleavage. The leftover streptavidin is blocked by biotin. Upon reiterative analysis cycles, a large number of different proteins with a wide range of expression levels can be profiled in individual cells at the optical resolution. Applying this approach, we have demonstrated that multiple proteins are unambiguously detected in the same set of cells, regardless of the protein analysis order. We have also shown that this method can be successfully applied to quantify proteins in formalin-fixed paraffin-embedded (FFPE) tissues.

Highly Sensitive and Multiplexed Protein Imaging With Cleavable Fluorescent Tyramide Reveals Human Neuronal Heterogeneity.

The ability to comprehensively profile proteins in intact tissues in situ is crucial for our understanding of health and disease. However, the existing methods suffer from low sensitivity and limited sample throughput. To address these issues, here we present a highly sensitive and multiplexed in situ protein analysis approach using cleavable fluorescent tyramide and off-the-shelf antibodies. Compared with the current methods, this approach enhances the detection sensitivity and reduces the imaging time by 1-2 orders of magnitude, and can potentially detect hundreds of proteins in intact tissues at the optical resolution. Applying this approach, we studied protein expression heterogeneity in a population of genetically identical cells, and performed protein expression correlation analysis to identify co-regulated proteins. We also profiled >6,000 neurons in a human formalin-fixed paraffin-embedded (FFPE) hippocampus tissue. By partitioning these neurons into varied cell clusters based on their multiplexed protein expression profiles, we observed different sub-regions of the hippocampus consist of neurons from distinct clusters.

AmpliSeq Transcriptome of Laser Captured Neurons from Alzheimer Brain: Comparison of Single Cell Versus Neuron Pools.

Alzheimer's disease (AD) is the most common cause of dementia in older adults. However, the pathogenesis of AD remains to be fully understood and clinically effective treatments are lacking. Recent advances in single cell RNA sequencing offers an opportunity to characterize the heterogeneity of cell response and explore the molecular mechanism of complex diseases at a single cell level. Here, we present the application of the Ion AmpliSeq transcriptome approach to profile gene expression in single laser captured neurons as well as pooled 10 and 100 neurons from hippocampal CA1 of AD brains versus matching normal aged brains. Our results demonstrated the high sensitivity and high genome coverage of the AmpliSeq transcriptome in single cell sequencing. In addition to capturing the known changes related to AD, our data confirmed the diversity of neuronal profiles in AD brain, which allow the potential identification of single cell response that might be hidden in population analyses. Notably, we also revealed the extensive inhibition of olfactory signaling and confirmed the reduction of neurotransmitter receptors in AD hippocampus. We conclude that although single neuron data show more variance than data from 10 or 100 pooled neurons, single neuron data can be informative. These findings support the utility of the Ion AmpliSeq method for obtaining and analyzing gene expression data from single defined laser captured neurons.

Alzheimer's disease-associated (hydroxy)methylomic changes in the brain and blood.

BACKGROUND: Late-onset Alzheimer's disease (AD) is a complex multifactorial affliction, the pathogenesis of which is thought to involve gene-environment interactions that might be captured in the epigenome. The present study investigated epigenome-wide patterns of DNA methylation (5-methylcytosine, 5mC) and hydroxymethylation (5-hydroxymethylcytosine, 5hmC), as well as the abundance of unmodified cytosine (UC), in relation to AD. RESULTS: We identified epigenetic differences in AD patients (n = 45) as compared to age-matched controls (n = 35) in the middle temporal gyrus, pertaining to genomic regions close to or overlapping with genes such as OXT (- 3.76% 5mC, pSidák = 1.07E-06), CHRNB1 (+ 1.46% 5hmC, pSidák = 4.01E-04), RHBDF2 (- 3.45% UC, pSidák = 4.85E-06), and C3 (- 1.20% UC, pSidák = 1.57E-03). In parallel, in an independent cohort, we compared the blood methylome of converters to AD dementia (n = 54) and non-converters (n = 42), at a preclinical stage. DNA methylation in the same region of the OXT promoter as found in the brain was found to be associated with subsequent conversion to AD dementia in the blood of elderly, non-demented individuals (+ 3.43% 5mC, pSidák = 7.14E-04). CONCLUSIONS: The implication of genome-wide significant differential methylation of OXT, encoding oxytocin, in two independent cohorts indicates it is a promising target for future studies on early biomarkers and novel therapeutic strategies in AD.

Transcriptome Changes in the Alzheimer's Disease Middle Temporal Gyrus: Importance of RNA Metabolism and Mitochondria-Associated Membrane Genes.

We used Illumina Human HT-12 v4 arrays to compare RNA expression of middle temporal gyrus (MTG; BA21) in Alzheimer's disease (AD = 97) and non-demented controls (ND = 98). A total of 938 transcripts were highly differentially expressed (adj p < 0.01; log2 FC ≥ |0.500|, with 411 overexpressed and 527 underexpressed in AD. Our results correlated with expression profiling in neurons from AD and ND obtained by laser capture microscopy in MTG from an independent dataset (log2 FC correlation: r = 0.504; p = 2.2e-16). Additionally, selected effects were validated by qPCR. ANOVA analysis yielded no difference between genders in response to AD, but some gender specific genes were detected (e.g., IL8 and AGRN in males, and HSPH1 and GRM1 in females). Several transcripts were associated with Braak staging (e.g., AEBP1 and DNALI1), antemortem MMSE (e.g., AEBP1 and GFAP), and tangle density (e.g., RNU1G2, and DNALI1). At the pathway level, we detected enrichment of synaptic vesicle processes and GABAergic transmission genes. Finally, applying the Weighted Correlation Network Analysis, we identified four expression modules enriched for neuronal and synaptic genes, mitochondria-associated membrane, chemical stimulus and olfactory receptor and non-coding RNA metabolism genes. Our results represent an extensive description of MTG mRNA profiling in a large sample of AD and ND. These data provide a list of genes associated with AD, and correlated to neurofibrillary tangles density. In addition, these data emphasize the importance of mitochondrial membranes and transcripts related to olfactory receptors in AD.

Association of AEBP1 and NRN1 RNA expression with Alzheimer's disease and neurofibrillary tangle density in middle temporal gyrus.

We explored RNA expression changes in the middle temporal gyrus (MTG) of Alzheimer's Disease patients (AD) by RNA sequencing the whole transcriptome of 8 AD and 8 Non-Demented (ND) controls. We used three additional expression datasets from related brain regions to validate the findings. The results highlighted the upregulation of AEBP1 and downregulation of NRN1 in AD, as well as their association with Braak staging and neurofibrillary tangles density. Furthermore, more than 400 protein-coding RNAs enriched for "Clathrin-mediated endocytosis" were validated in independent datasets from the same brain region. Finally, using in silico prediction analysis we found a signature of 52 non-protein coding RNAs that perturb key pathways involved in GABAergic transmission and peptide chain elongation. The association of AEBP1 in our data confirmed other published work examining gene expression in the hippocampus of AD patients. NRN1 is involved in neurite outgrowth, and in previous studies it has been shown to reverse synaptic defects and cognitive function impairment in Tg2576 mice. Finally, our results on non-protein coding RNAs suggest a role of these transcripts in altering synaptic and amyloid-ß associated pathways.

Gradual hypertension induction in middle-aged Cyp1a1-Ren2 transgenic rats produces significant impairments in spatial learning.

Hypertension is a major health concern in the developed world, and its prevalence increases with advancing age. The impact of hypertension on the function of the renal and cardiovascular systems is well studied; however, its influence on the brain regions important for cognition has garnered less attention. We utilized the Cyp1a1-Ren2 xenobiotic-inducible transgenic rat model to mimic both the age of onset and rate of induction of hypertension observed in humans. Male, 15-month-old transgenic rats were fed 0.15% indole-3-carbinol (I3C) chow to slowly induce renin-dependent hypertension over a 6-week period. Systolic blood pressure significantly increased, eventually reaching 200 mmHg by the end of the study period. In contrast, transgenic rats fed a control diet without I3C did not show significant changes in blood pressure (145 mmHg at the end of study). Hypertension was associated with cardiac, aortic, and renal hypertrophy as well as increased collagen deposition in the left ventricle and kidney of the I3C-treated rats. Additionally, rats with hypertension showed reduced savings from prior spatial memory training when tested on the hippocampus-dependent Morris swim task. Motor and sensory functions were found to be unaffected by induction of hypertension. Taken together, these data indicate a profound effect of hypertension not only on the cardiovascular-renal axis but also on brain systems critically important for learning and memory. Future use of this model and approach may empower a more accurate investigation of the influence of aging on the systems responsible for cardiovascular, renal, and neurological health.

Upregulation of histone deacetylase 2 in laser capture nigral microglia in Parkinson's disease.

Histone deacetylase (HDAC) inhibitors have been widely reported to have considerable therapeutic potential in a host of neurodegenerative diseases. However, HDAC inhibitor selectivity and specificity in specific cell classes have been a source of much debate. To address the role of HDAC2 in specific cell classes, and in disease, we examined glial protein and mRNA levels in the substantia nigra (SN) of Parkinson's disease (PD) and normal controls (NCs) by immunohistochemistry and laser captured microdissection followed by quantitative real time polymerase chain reaction. Differential expression analysis in immunohistochemically defined laser capture microglia revealed significant upregulation of HDAC2 in the PD SN compared to NC subjects. Complementary in vivo evidence reveals significant upregulation of HDAC2 protein levels in PD SN microglia compared to NC subjects. Correspondingly, human immortalized telencephalic/mesencephalic microglial cells reveal significant upregulation of HDAC2 in the presence of the potent microglial activator lipopolysaccharide. These data provide evidence that selective inhibition of HDAC2 in PD SN microglia could be a promising approach to treat microglial-initiated nigral dopaminergic neuronal cell loss in PD.

Oligomeric amyloid ß preferentially targets neuronal and not glial mitochondrial-encoded mRNAs.

INTRODUCTION: Our laboratories have demonstrated that accumulation of oligomeric amyloid ß (OAß) in neurons is an essential step leading to OAß-mediated mitochondrial dysfunction. METHODS: Alzheimer's disease (AD) and matching control hippocampal neurons, astrocytes, and microglia were isolated by laser-captured microdissection from the same subjects, followed by whole-transcriptome sequencing. Complementary in vitro work was performed in OAß-treated differentiated SH-SY5Y, followed by the use of a novel CoQ10 analogue for protection. This compound is believed to be effective both in suppressing reactive oxygen species and also functioning in mitochondrial electron transport. RESULTS: We report decreases in the same mitochondrial-encoded mRNAs in Alzheimer's disease laser-captured CA1 neurons and in OAß-treated SH-SY5Y cells, but not in laser-captured microglia and astrocytes. Pretreatment with a novel CoQ10 analogue, protects neuronal mitochondria from OAß-induced mitochondrial changes. DISCUSSION: Similarity of expression changes in neurons from Alzheimer's disease brain and neuronal cells treated with OAß, and the effect of a CoQ10 analogue on the latter, suggests a pretreatment option to prevent OAß toxicity, long before the damage is apparent.

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.].

Laser-captured microglia in the Alzheimer's and Parkinson's brain reveal unique regional expression profiles and suggest a potential role for hepatitis B in the Alzheimer's brain.

Expression array data from dozens of laboratories, including our own, show significant changes in expression of many genes in Alzheimer's disease (AD) patients compared with normal controls. These data typically rely on brain homogenates, and information about transcripts specific to microglia and other central nervous system (CNS) cell types, which far outnumber microglia-specific transcripts, is lost. We therefore used single-cell laser capture methods to assess the full range of microglia-specific expression changes that occur in different brain regions (substantia nigra and hippocampus CA1) and disease states (AD, Parkinson's disease, and normal controls). Two novel pathways, neuronal repair and viral processing were identified. Based on KEGG analysis (Kyoto Encyclopedia of Genes and Genomes, a collection of biological pathways), one of the most significant viruses was hepatitis B virus (HBV) (false discovery rate < 0.00000001). Immunohistochemical analysis using HBV-core antibody in HBV-positive control, amnestic mild cognitive impairment, and HBV-positive AD cases show increased HBV immunoreactivity as disease pathology increases. These results are the first, to our knowledge, to show regional differences in human microglia. In addition, these data reveal new functions for microglia and suggest a novel risk factor for AD.

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's 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's 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.

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

Recent epigenetic association studies have identified a new gene, ANK1, in the pathogenesis of Alzheimer's disease (AD). Although strong associations were observed, brain homogenates were used to generate the data, introducing complications because of the range of cell types analyzed. In order to address the issue of cellular heterogeneity in homogenate samples we isolated microglial, astrocytes and neurons by laser capture microdissection from CA1 of hippocampus in the same individuals with a clinical and pathological diagnosis of AD and matched control cases. Using this unique RNAseq data set, we show that in the hippocampus, ANK1 is significantly (p<0.0001) up-regulated 4-fold in AD microglia, but not in neurons or astrocytes from the same individuals. These data provide evidence that microglia are the source of ANK1 differential expression previously identified in homogenate samples in AD.